Center for Orthopedic

amp

Regenerative Medicine

Science

Michael N Brown MD DC DABPMR-PAIN

Disclosures Corporate Associations

bull Executive with CellPar Tech LLC

ndash Stem cell amp regenerative medicine technology

and research

bull FASER

ndash Force Assisted Soliton Energy Resonance

ndash RampD team of physicist and engineers working

on PEMF use in medicine

ndash Green tech

Notification

Title changehellip

bull If you have the right seeds you do not

need as much fertilizer

ndash Washington is greener than Texas

Texas Washington

A product of repeated clinical

failures

Michael N Brown MD

Where have we beenhellip where are we

going

bull As development proceeds from a

ldquototipotentrdquo zygote cells proliferate and

segregate by lineage-progression resulting

in formation of differentiated phenotypes

bull Processes begin with a totipotent zygote

and continues throughout life

bull As development proceeds from totipotent

zygote cells proliferate and segregated by

lineage-commitment into two pluripotent

layers

ndash TROPHBLAST Extra-embryonic

membranes of placenta and the inner

cell mass which becomes embryo

ndash INNER CELL MASS

bull Hypoblast

bull Epiblast

Hypoblast amp Epiblast

bull Hypoblast and Epiblast

ndash Segregates into pluripotent primary germ layers

bull Ectodermal

bull Mesoderm

bull Endoderm

bull Further segregation through lineage-commitment

-- Multipotent -- Tripotent

-- Bipotent

bull Eventually Unipotent lineages further defines

differentiation pathways to become quiescent reserve

stem cells

bull Fxn maintenance and repair of tissues of postnatal

organism

Precursor Cells in Adults Totipotent SCs Embryo placenta Gamates (sperm amp ovum)

Pluripotent SCs form ectodermal lineage cells mesoderm lineage cells and endoderm lineage cells All cells of the embryo but not the placeta and gamates

Ultra-small nucleated plasma particle (u-NP2)

Small-nucleated plasma particle (s-NP2)

Intermediate-nucleated plasma particle (i-NP2)

Large-nucleated plasma particle (l-NP2)

Germ Layer GL-EctoSCs GL-MesoSCs GL-EndoSCs

Lineage SCs

Progenitor Multipotent PCs hematopoietic

Cells Tripotent PCs chondral-osteo--adipogenic

Bipotent PCs adipo-fibrogenic

Unipotent PCs myoblast fibroblast adipoblast etc

Embryonic Stem Cell Matching

bull Embyronics HLA mismatch

ndash Donor cells are rejected with mismatch

bull Some have got around this with

therapuetic cloning

ndash Cell from recipient nucleus into donor cell and

thus recognized as self

ndash Very expensive

ndash Cells have limited life span and do not have

same plasticity

ndash Can not transplant male to female cells

Holy grail

Unipotent Reserve Stem Cells

bull Examples

ndash Unipotent myosatellite myoblasts of muscle (Mauro

1961 Campion 1984 Grounds et al 1992)

ndash Unipotent adipoblast cells of adipose tissue (Allhaud

et al 1992)

ndash Unipotent chondorgenic cells and osteogeic cells of

perichondrium and periosteum Cruess 1982 Young

et al 1995)

ndash Bipotent reserve mesenchymal stem cells

adipofibroblasts (Vierck et al 1996 Beresford 1989

Caplan et al 1997) and others

Multipotent cells

bull Multipotent stem cells of marrow

ndash Owen 1988 Beresford 1989 Rickard et al

1994 Caplan et al 1997 Prockop 1997)

bull Tripotent preserved mesenchymal stem cells

ndash Cardiogenicosteogenicadipogenic stem cells

of marrow (Pittenger et al 1999)

bull Multipotent hematopoietic stem cells of marrow

(Palis and Segel 1989 Maguire 1998

Ratajczak et al 1998)

Adult pleuripotent cells

bull Pluripotent mesenchymal stem cells initially found in

connective tissues of prenatal avians mice and rats

(Grigoriadis et al 1988 Young et al 1993 1998

Rogers et al 1995)

bull Pluripotent stem cells are not committed to any particular

tissue lineage

ndash Can generate into multiple lineage-committed progenitor

cells from a single clone thus forming any tissue within a

particular germ layer lineage

ndash Isolated from postnatal animals-capable of extended self

renewal as long as they remain uncommitted to a particular

lineage

bull Example PP mesenchymal stem cell clone Myotubes fat cartilage

nodules and bone after 690 cell doublings

Adult pleuripotent cells

bull Unlike progenitor stem cells pluripotent stem cells are

not affected by progression factors such as insulin

IGF-1 IGF-II

bull Differentiation factors such as dexamethasone BMP

muscle morphogenic protein (MMP) does induce lineage

commitment in these cells

bull Once PPSCs commit to a particular lineage they assume

the characteristics of lineage-committed progenitor cells

ndash Their ability to replicate reduces from 600 x to 60 x before

becoming senescent

Adult pleuripotent cells

bull Really Can we do this

bull Studies and postnatal rats rabbits and humans suggest

that resident stem cells consistent both lineage-rdquo

committed cells and lineage non-comitted pluri-

potent stem cells ndash (Pate et al 1993 Lucas et al 1995 1996 Young et al 1999)

bull In addition there are established populations of lineage-

committed progenitor stem cells ndash (Mauro 1961 Cruess 1982 Campion 1984 Owen 1988 Beresford

1989 Ailhaud et al 1992 Grounds et al 1992 Rickard et al 1994

Young et al 1995 Vierck et al 1996 Caplan et al 1997 Prockop

1997 Palis and Segel 1998 Maguire 1998 Parajczak et al 1998

Pittenger et al 1999)

Adult pleuripotent cells bull 2001 Clonogenic Analysis of Reserve Stem Cells in

Postnatal Mamals Isolation from muscle discection

Once isolated and incubated with insulin less than 1 of

the cells displayed phenotypic markers for differentiated

cells of various mesodermal clinic ages

bull Thus the majority of progenitor stem cells were removed

from the population by propagating the cells for more

than 50 cell doublings some prior to cloning

ndash 50 cell doublings causes lineage ndash committed stem cells to

undergo programmed cell senescence and death ( Hayflick

1963 Young et al 1999 Young 2000)

ndash 2001 the larger cells with low ratios of nucleus to cytoplasm

were observed to undergo apoptosis between 40-50 cell

doublings

ndash The remainder of cells that continued to divide were smaller

with low CP Nuc ratio

Totipotent amp pluripotent cultures

vs progenitor cells

bull Regenerative

cells expand to

confluence

bull Toti and Pluri

form layers and

not inhibited by

contact inibition

Precursor cells formed during

embryogensis

bull Remarkably while the vast majority of developing

blastomeres transition through the sequence of

developmental and differentiation for embryogenesis

bull a few cells become reserve precursor cells that provide

for continual maintenance (progenitor cells)and repair

(stem cells)of the organism

bull The totipotent and pluripotent

stem cells can be seen as early

as the morula stage in human

embryonic development

Precursor Cells ESCs

bull Totipotent stem cells equivalent in

differentiation potential to the blastomeres of the

morula and blastocyst

bull Pluripotent stem cells are equivalent in

differentiation potential to the cells of the epiblast

bull Germ layer lineage stem cells

ndash ENDODERM Stem cells gastrointestinal epithelium

hepatocytes and pancreatic cells (α-cells β-cells δ-

cells and ductal cells)

ndash ECTODERM Stem Cells neurons neuroglia

epidermis hair teeth and nails

ndash MESODERM Stem Cells muscle fat cartilage bone

connective tissues endothelial cells and blood cells

Adult pleuripotent cells

bull Unlike progenitor stem cells pluripotent stem cells are

not affected by progression factors such as insulin

IGF-1 IGF-II

bull Differentiation factors such as dexamethasone BMP

muscle morphogenic protein (MMP) does induce lineage

commitment in these cells

bull Once PPSCs commit to a particular lineage they assume

the characteristics of lineage-committed progenitor cells

ndash Their ability to replicate reduces from 600 x to 60 x before

becoming senescent

Adult pleuripotent cells

bull Really Can we do this

bull Studies and postnatal rats rabbits and humans suggest

that resident stem cells consistent both lineage-rdquo

committed cells and lineage non-comitted pluri-

potent stem cells ndash (Pate et al 1993 Lucas et al 1995 1996 Young et al 1999)

bull In addition there are established populations of lineage-

committed progenitor stem cells ndash (Mauro 1961 Cruess 1982 Campion 1984 Owen 1988 Beresford

1989 Ailhaud et al 1992 Grounds et al 1992 Rickard et al 1994

Young et al 1995 Vierck et al 1996 Caplan et al 1997 Prockop

1997 Palis and Segel 1998 Maguire 1998 Parajczak et al 1998

Pittenger et al 1999)

Adult pleuripotent cells bull 2001 Clonogenic Analysis of Reserve Stem Cells in

Postnatal Mamals Isolation from muscle discection

Once isolated and incubated with insulin less than 1 of

the cells displayed phenotypic markers for differentiated

cells of various mesodermal clinic ages

bull Thus the majority of progenitor stem cells were removed

from the population by propagating the cells for more

than 50 cell doublings some prior to cloning

ndash 50 cell doublings causes lineage ndash committed stem cells to

undergo programmed cell senescence and death ( Hayflick

1963 Young et al 1999 Young 2000)

ndash 2001 the larger cells with low ratios of nucleus to cytoplasm

were observed to undergo apoptosis between 40-50 cell

doublings

ndash The remainder of cells that continued to divide were smaller

with low CP Nuc ratio

Totipotent amp pluripotent cultures

vs progenitor cells

bull Regenerative

cells expand to

confluence

bull Toti and Pluri

form layers and

not inhibited by

contact inibition

Precursor cells formed during

embryogensis

bull Remarkably while the vast majority of developing

blastomeres transition through the sequence of

developmental and differentiation for embryogenesis

bull a few cells become reserve precursor cells that provide

for continual maintenance (progenitor cells)and repair

(stem cells)of the organism

bull The totipotent and pluripotent

stem cells can be seen as early

as the morula stage in human

embryonic development

Precursor Cells ESCs

bull Totipotent stem cells equivalent in

differentiation potential to the blastomeres of the

morula and blastocyst

bull Pluripotent stem cells are equivalent in

differentiation potential to the cells of the epiblast

bull Germ layer lineage stem cells

ndash ENDODERM Stem cells gastrointestinal epithelium

hepatocytes and pancreatic cells (α-cells β-cells δ-

cells and ductal cells)

ndash ECTODERM Stem Cells neurons neuroglia

epidermis hair teeth and nails

ndash MESODERM Stem Cells muscle fat cartilage bone

connective tissues endothelial cells and blood cells

Totipotent amp pluripotent cultures

vs progenitor cells

bull Regenerative

cells expand to

confluence

bull Toti and Pluri

form layers and

not inhibited by

contact inibition

Precursor cells formed during

embryogensis

bull Remarkably while the vast majority of developing

blastomeres transition through the sequence of

developmental and differentiation for embryogenesis

bull a few cells become reserve precursor cells that provide

for continual maintenance (progenitor cells)and repair

(stem cells)of the organism

bull The totipotent and pluripotent

stem cells can be seen as early

as the morula stage in human

embryonic development

Precursor Cells ESCs

bull Totipotent stem cells equivalent in

differentiation potential to the blastomeres of the

morula and blastocyst

bull Pluripotent stem cells are equivalent in

differentiation potential to the cells of the epiblast

bull Germ layer lineage stem cells

ndash ENDODERM Stem cells gastrointestinal epithelium

hepatocytes and pancreatic cells (α-cells β-cells δ-

cells and ductal cells)

ndash ECTODERM Stem Cells neurons neuroglia

epidermis hair teeth and nails

ndash MESODERM Stem Cells muscle fat cartilage bone

connective tissues endothelial cells and blood cells

Embryonic stem cell amp

Teratomas bull When ESCs were placed into an animal they can form a

mass of tissue that contained a variety of cell types in a

non-spatially arranged jumbled fashion

bull This configuration is reminiscent of the sacrococcygeal

teratoma that can form during embryogenesis if residual

primitive streak cells remain after gastrulation at the

caudal end of the developing embryo located caudal to

the notochord (the primary inducer of the embryo)

Teratoma in APPSC ATPSC

bull Adult PPSC and TPSC cells once placed in

environment commit to a particular tissuecell

lineage lose the telomerase enzyme

bull Thus assume all attributes of tissue-committed

progenitor cells including a defined biological

lifespan of 8ndash10 population doublings for rodents

and 50ndash70 population doublings for humans

bull Thushellip No Teratomas

1 Rohme D Evidence for a relationship between longevity of mammalian species and life spans of normal

fibroblasts in vitro and erythrocytes in vivo Proc Natl

Acad Sci USA 1981785009ndash5013

2 Hayflick L Moorehead P The serial cultivation of human diploid cell strains Exp Cell Res 196125585ndash621

Embryonic SCs discovered

bull The reports of mouse embryonic stem cells

spurred the development of embryonic stem

cells in other species

bull ESCs utilizing similar technologies have been

verified by other labs in mice and generated

from blastocysts of rat golden hamster rabbit

mink pig sheep cow horse marmoset non-

human primate (Rhesus monkey) human

chicken medaka zebra fish and gilthead sea

bream Alexander A Maximow He was a Russian-

American physician published from 1896 until 1902 first person to use the term ldquostem cellsrdquo

References

bull Young HE Black AC (2005) Adult-derived stem cells Minerva

Biotechnologica 17 55-63

bull Young HE Duplaa C Romero-Ramos M Chesselet MF Vourc h P

et al (2004) Adult reserve stem cells and their potential for tissue

engineering Cell Biochem Biophys 40(1) 1-80

bull Young HE Steele TA Bray RA Hudson J Floyd JA et al (2001)

Human reserve pluripotent mesenchymal stem cells are present in

the connective tissues of skeletal muscle and dermis derived from

fetal adult and geriatric donors Anat Rec 264(1) 51-62

Adult Precursor Cells bull Due to their appearance early in development

and segregation with the developing

blastomeres precursor cells with similar

differentiation potentials as those

described for embryonic cells have been

postulated to occur in most organs and

tissues of the body throughout the lifespan of the

individual

bull Adult precusor cells ndash IDrsquod by isolation from solid organs fluids etc via cyrosection

and immunocytochemical staining for cell precursor cell-specific

antigen markers

ndash IDrsquod in mouse rat rabbit cat dog goat sheep pig cow

horse and human

References

bull Young HE Hyer L Black AC Robinson JS (2013) Adult

stem cells from bench-top to bedside In Qing Liu amp

Hongjun Wang Tissue Regeneration Where

Nanostructure Meets Biology 3DBiotech North

Brunswick NJ Chap 1 pp 1-60

Adult precursor cells

bull These precursor cells were composed of

ndash Progenitor cells

ndash Stem cells

bull The stem cells included

ndash Totipotent stem cells

ndash Pluripotent stem cells

ndash Germ layer lineage

bull ectodermal stem cells

bull mesodermal stem cells

bull endodermal stem cells

References

bull Young HE Black AC (2005) Adult-derived stem cells Minerva Biotechnologica 17

55-63

bull Young HE Duplaa C Romero-Ramos M Chesselet MF Vourc h P et al (2004)

Adult reserve stem cells and their potential for tissue engineering Cell Biochem

Biophys 40(1) 1-80

bull Young HE Steele TA Bray RA Hudson J Floyd JA et al (2001) Human reserve

pluripotent mesenchymal stem cells are present in the connective tissues of skeletal

muscle and dermis derived from fetal adult and geriatric donors Anat Rec 264(1)

51-62

bull Young HE Hyer L Black AC Robinson JS (2013) Adult stem cells from bench-top to

bedside In Qing Liu amp Hongjun Wang Tissue Regeneration Where Nanostructure

Meets Biology 3DBiotech North Brunswick NJ Chap 1 pp 1-60

bull Young HE Steele T Bray RA Detmer K Blake LW et al (1999) Human progenitor

and pluripotent cells display cell surface cluster differentiation markers CD10 CD13

CD56 CD90 and MHC Class-I Proc Soc Exp Biol Med 221(1) 63-71

bull Young HE Duplaa C Yost MJ Henson NL Floyd JA et al (2004) Clonogenic

analysis reveals reserve stem cells in postnatal mammals II Pluripotent epiblastic-

like stem cells Anat Rec A Discov Mol Cell Evol Biol 277(1) 178-203

Blastomere

How did we find the cell

Histology

Energy-dispersive spectral analysis coupled with

scanning electron microscopy

Carbohydrate biochemistry

Qualitative and quantitative enzyme-linked

immunoculture assays

Differential cryopreservation

Repetitive limiting serial dilution clonogenic analysis

Cluster of differentiation (CD) marker analysis

Fluorescent-activated cell sorting

Magnetic bead cell sorting

Molecular analyses for telomerase activity and gene

Expression

Bioactive factor pluripotency expression analyses

Differential centrifugation

Differential isolation and plating

Immunocytochemistry

Carbohydrate histochemistry

Types of TPSCs amp PPSCs

bull 2 Totipotent stem

cell types

bull 6 pluripotent stem

cell types

Clinical application of TPSCs

PPSCs amp MSCs bull 2 Totipotent stem

cell types

bull 6 pluripotent stem

cell types

bull Mesenchymal

Pluri and Toti Isoloation

bull Requires

ndash Agent to expand cell numbers in the patient

(in vivo) 3 x by 30 days

ndash Mobilization agent

bull Reversed vascular diapedesis 2-3 x yield

bull Combination results in isolation of cells

similar to seen in culture and expansion

TPSC amp PPSC Behavior

bull Activation required to use clinically

ndash Isolate the cells

ndash Inject the cells without activation and they

simply reseed the tissues and remain

quiesent

ndash Activation process will meet minimal

manipulation criteria

Cytochemistry ID

Antibodies used for Characterization of Cell Types [9101131-334694]

Antibody Antigen Embryological Origin

CEA-CAM-1

Carcinoembryonic antigen-cell adhesion molecule-1

Totipotent

HCEA Human Carcinoembryonic antigen Totipotent

CEA Carcinoembryonic antigen Totipotent

CD66e Carcinoembryonic antigen Totipotent

DH-TuAg1 Spermatogonia Totipotent

MC-480 SSEA-1 Pluripotent

MC-631 SSEA-3 Pluripotent

MC-813 SSEA-4 Pluripotent

CD10 Neutral endopeptidase Pluripotent

AlkPhos Alkaline Phosphatase Pluripotent

TPSCs

PPSC

bull Transitional Blastomere-Like

Stem Cell

bull Epiblast Like Stem Cells

bull Tr-GLSCs Transitional Germ

Layer

bull Lineage Stem Cells

bull MesoSCs Mesodermal

Germ Layer

bull Lineage Stem Cells Mes

PCs

bull Mesenchymal Progenitor

Cells

Progenitor Cells

bull By definition = lineagetissuecell-committed progenitor cells

ndash No inherent plasticity but rather have both a defined differentiation

potential

ndash Limited replicative lifespan

bull Ie unipotent myosatellite myoblast progenitor cells will only form

skeletal muscle

bull bipotent adipo-fibroblast progenitor cells will only form adipocytes

(fat cells) and fibroblasts

bull tripotent chondroblast-osteoblast-adipoblast progenitor cells will

only form cartilage bone and fat cells

bull while multipotent hematopoietic progenitor cells will form the myriad

of cell types within the hematopoietic lineage but not any cell type

outside that lineage

ndash Progenitor -limited population doublings before pre-programmed

senescence and cell death occurs

ndash 8-10 population doublings for rodents and 50-70 population

doublings for humans

References 1 Ailhaud G Grimaldi P Negrel R (1992) Cellular and molecular aspects of adipose tissue

development Annu Rev Nutr 12 207-233

2 Vierck JL McNamara JP Dodson MV (1996) Proliferation and differentiation of progeny of ovine

unilocular fat cells (adipofibroblasts) In Vitro Cell dev Biol Anim 32(9) 564-572

3 Owen M (1988) Marrow stromal cells J Cell Sci Suppl 10 63-76

4 Prockop DJ (1997) Marrow stromal cells as stem cells for nonhematopoietic tissues Science

276(5309) 71-74

5 Pittenger MF Mackay AM Beck SC Jaiswal RK Douglas R et al (1999) Multilineage potential of

adult human mesenchymal stem cells Science 284(5411) 143-147

6 McGuire WP (1998) High-dose chemotherapy and autologous bone marrow or stem cells

reconstitution for solid tumors Curr Probl Cancer 22(3) 135-177

7 Palis J Segel GB (1998) Developmental biology of erythropoiesis Blood Rev 12(2) 106-114

8 Rohme D (1981) Evidence for a relationship between longevity of mammalian species and life

spans of normal fibroblasts in vitro and erythrocytes in vivo Proc Natl Acad Sci USA 78(8) 5009-

5013

9 Hayflick L Moorehead PS (1961) The serial cultivation of human diploid cell strains Exp Cell Res

25 585-621

Endogenous Stem Cells

bull In contrast to adult-derived lineagetissuescell-

committed progenitor cells adult-derived

lineage-uncommitted stem cells demonstrate ndash inherent plasticity being capable of replicating themselves and

forming their respective downstream cell types

bull Embryonic stem cells preprogrammed to

spontaneously differentiate into tissues of the

embryofetus or lineage-committed progenitor

cells

Endogenous Stem Cells

bull Adult Endogenous Stem cells uncommitted

adult stem cells are not preprogrammed to

form anything

ndash respond to environmental cues (ie inductive agents)

to differentiate into specific cell types based on the

particular activity of an inductive agent

ndash Ie bull nerve growth factor induces totipotent stem cells pluripotent stem

cells and ectodermal stem cells to form neurons

bull bone morphogenetic protein-2 induces totipotent stem cells

pluripotent stem cells and mesodermal stem cells to form bone

bull while hepatocyte growth factor induces totipotent stem cells

pluripotent stem cells and endodermal stem cells to form

hepatocytes

References 1 Young HE Duplaa C Romero-Ramos M Chesselet MF Vourc h P et al (2004) Adult reserve stem cells and their

potential for tissue engineering Cell Biochem Biophys 40(1) 1-80

2 Young HE Duplaa C Yost MJ Henson NL Floyd JA et al (2004) Clonogenic analysis reveals reserve stem cells

in postnatal mammals II Pluripotent epiblastic-like stem cells Anat Rec A Discov Mol Cell Evol Biol 277(1) 178-

203

3 Mauro A (1961) Satellite cell of skeletal muscle fibers J Biophys Biochem Cytol 9 493-495

4 Ailhaud G Grimaldi P Negrel R (1992) Cellular and molecular aspects of adipose tissue development Annu Rev

Nutr 12 207-233

5 Vierck JL McNamara JP Dodson MV (1996) Proliferation and differentiation of progeny of ovine unilocular fat

cells (adipofibroblasts) In Vitro Cell dev Biol Anim 32(9) 564-572

6 Owen M (1988) Marrow stromal cells J Cell Sci Suppl 10 63-76

7 Prockop DJ (1997) Marrow stromal cells as stem cells for nonhematopoietic tissues Science 276(5309) 71-74

8 Pittenger MF Mackay AM Beck SC Jaiswal RK Douglas R et al (1999) Multilineage potential of adult human

mesenchymal stem cells Science 284(5411) 143-147

9 McGuire WP (1998) High-dose chemotherapy and autologous bone marrow or stem cells reconstitution for solid

tumors Curr Probl Cancer 22(3) 135-177

10 Palis J Segel GB (1998) Developmental biology of erythropoiesis Blood Rev 12(2) 106-114

11 Rohme D (1981) Evidence for a relationship between longevity of mammalian species and life spans of normal

fibroblasts in vitro and erythrocytes in vivo Proc Natl Acad Sci USA 78(8) 5009-5013

12 Hayflick L Moorehead PS (1961) The serial cultivation of human diploid cell strains Exp Cell Res 25 585-621

13 Young HE Black AC (2013) Naturally occurring adult pluripotent stem cells In Meyers RA et al (Eds) Stem

Cells From Biology to Therapy Advances in Molecular Biology and Medicine (1st edn) Texas USA p 63-93

Endogenous Stem Cells

bull Unidirectional differentiation

pathway for endogenous

precursor cells ie stem

cells and progenitor cells

BLSCs blastomere-like stem

cells ELSCs epiblast-like

stem cells EctoSCs

ectodermal stem cells

MesoSCs mesodermal stem

cells Endo SCs endodermal

stem cells M multipotent T

Tripotent B Bipotent U

Unipotent

Totipotent

Pluipotent

Histochem

of stem cells

Char1 BLSCs2

Tr-BLSC

ELSCs3

ELSCs4

Tr-GLSCs5 MesoSCs6

MesPCs7

Sizem 02-2 2-6 6-7 7-8 8-10 10-20

Trypan

blue Pos8 PosNeg Neg9 Neg Neg Neg

Days Viab

PM10 30+ 30+ 7+ 5 3 1

Viab T11 4oC 4oC 4oC 4oC 4oC 4oC

Tiss12 Yes Yes Yes Yes Yes Yes

Species13

MRtRb

CtDS

GPCw

HoHu

MRtRb

CtDS

GPCw

HoHu

MRtRb

CtDS

GPCw

HoHu

Hu

MRtRb

CtDS

GPCw

HoHu

MRtR

b

CtDS

GPCw

HoHu

Clones14

BLSC Scl-

44 BLSC

Scl-9

NYD ELSC Scl-

40 NYD

MSC Scl-

2PG

Rt-

My15

Rt-

Adip16

Rt-

Chon1

7

Rt-

Os18

Con Hib19 No No No No Yes Yes

Growth Sus20Adh

21 Adherent Adherent Adherent

Adheren

t

Adhere

nt

No GF22 Quiescent Quiescen

t

Quiescen

t

Quiescen

t

Quiesce

nt

Quiesc

ent

Inhib23 Respond Respond Respond Respond Respon

d

Respon

d

Prolif24 Prolif25 Prolif Prolif Prolif Prolif Prolif

Prolif

Rate26 12-14 hr 12-14 hr 12-14 hr 14-18 hr

18-24

hr

Totipotent cells

Pluripotent cells

Collectively all now

called Nucleated Plasma

Particles NucP2

Endogenous Stem Cells

bull Totipotent stem cells ndash will replicate themselves as well as forming similar downstream

cell types similar to embryonic blastomeres of the morula

stage of development

ndash Will form all somatic tissues within the embryo proper including

spermatogonia

ndash Equivalent in differentiation potential to the blastomeres of the

morula and blastocyst

bull Pluripotent stem cells ndash Will replicate themselves and form similar downstream cell types

as embryonic epiblast cells (all somatic cells of the embryo but

not the gametes)

ndash Equivalent in differentiation potential to the cells of the epiblast

Endogenous Stem Cells

bull Germ layer lineage stem cells (ectodermal

mesodermal and endodermal stem cells) will replicate

themselves as well as form downstream cell types

belonging to their respective germ layer lineages

bull They are thus equivalent in differentiation potential to the

cells comprising the ectodermal mesodermal and

endodermal germ layer lineages respectively

bull Precursor cells that are ldquotruerdquo stem cells and not

misnamed progenitor cells (lineage-uncommitted cells

rather than lineage-committed cells) contain the

telomerase enzyme as long as the cells remain

lineage-uncommitted

MSCshellip

bull ldquoCaplan cellsrdquo ndash CD 105 CD 123 CD 166

ndash Multi potent progenitor cells

ndash Bone cartilage fat

bull Blood derived mesenchymal cells ndash More homogeneous mesenchymal cell population

ndash CD 90 CD 13

ndash Bone marrow fat cartilage blood endothelial cells

connective tissue tendon ligament kidney spleen

or trabeculae

The Telomere

amp Teleomerase

Enzyme

Critical for lineage uncommitted

stem cell replication

Teleomerase

bull Telomerase was discovered by Carol W Greider and Elizabeth

Blackburn in 1984 in the ciliate Tetrahymena[3] Together with Jack

W Szostak Greider and Blackburn were awarded the 2009 Nobel

Prize in Physiology or Medicine for their discovery

bull Telomerase reverse transcriptase (TERT) telomerase RNA (TR or

TERC)

bull TERT has a mitten structure that allows it to wrap around the

chromosome to add single-stranded telomere repeats

bull In normal circumstances without the presence of telomerase if a

cell divides recursively at some point all the progeny will reach their

Hayflick limit

bull With the presence of telomerase each dividing cell can replace the

lost bit of DNA and any single cell can then divide unbounded

MSCshellip

bull ldquoCaplan cellsrdquo ndash CD 105 CD 123 CD 166

ndash Multi potent progenitor cells

ndash Bone cartilage fat

bull Blood derived mesenchymal cells ndash More homogeneous mesenchymal cell population

ndash CD 90 CD 13

ndash Bone marrow fat cartilage blood endothelial cells

connective tissue tendon ligament kidney spleen

or trabeculae

The Telomere

amp Teleomerase

Enzyme

Critical for lineage uncommitted

stem cell replication

Teleomerase

bull Telomerase was discovered by Carol W Greider and Elizabeth

Blackburn in 1984 in the ciliate Tetrahymena[3] Together with Jack

W Szostak Greider and Blackburn were awarded the 2009 Nobel

Prize in Physiology or Medicine for their discovery

bull Telomerase reverse transcriptase (TERT) telomerase RNA (TR or

TERC)

bull TERT has a mitten structure that allows it to wrap around the

chromosome to add single-stranded telomere repeats

bull In normal circumstances without the presence of telomerase if a

cell divides recursively at some point all the progeny will reach their

Hayflick limit

bull With the presence of telomerase each dividing cell can replace the

lost bit of DNA and any single cell can then divide unbounded

Endogenous Stem Cells

The telomerase enzyme adds telomeres to the ends of the

chromosomes protecting the chromosomes from

degradation due to increased mitotic divisions

Protection from chromosomal degradation gives these

postnatal stem cells the capability for extensive

proliferation

The preprogrammed biological clock for endogenous stem

cells does not begin at birth of the individual but rather

when the stem cells commit to a particular progenitor

tissuecell lineage

The rubhellip

bull It has been reported that endogenous

pluripotent stem cells do not exist Miyanishi M Mori

Y Seita J Chen JY Karten S et al (2013) Do Pluripotent Stem Cells Exist in Adult Mice as Very

Small Embryonic Stem Cells Stem Cell Reports 1(2) 198-208

bull au contraire

ndash sufficient number of separate laboratories

have proven that endogenous pluripotent

stem cells do exist and can be readily

isolated characterized and utilized Young HE Black AC Pluripotent Stem Cells Endogenous versus

Reprogrammed a Review Orthopedics amp Rheumatology Volume 1 Issue 3 - 2014

Amniotic Fluid-Derived

Pluripotent Stem Cells bull The amniotic fluid contains a mixture of predominantly

epithelial cells It is thought that they are sloughed off

from the epidermis gastrointestinal and urinary

epithelia They range from six to fifty microns in size and

express cell surface markers from all three primary germ

layer lineages

bull Small percentage of cells that display the c-kit cell

surface marker by FACS analysis This c-kit-positive

population of cells were expanded to sub-confluence

every 48-72 hours

bull These c-kit-positive cells derived from the amniotic fluid

demonstrate greater than 300 population doublings

which is far greater than Hayflickrsquos limit

bull Sandler M (1981) Amnitoic Fluid and its Clinical Significance Marcel Decker New

York USA

bull Von Koskull H Virtenan I Lehto VP Vartio T Dahl D et al (1981) Glial and neuronal

cells in amniotic fluid of anencephalic pregnancies Prenat Diagn 1(4) 259-267

bull Medina Gomez P Johnston TH (1982) Cell morphology in long-term cultures of

normal and abnormal amniotic fluids Hum Genet 60(4) 310-313

bull Cremer M Schachner M Cremer T Schmidt W Voigtlander T (1981) Demonstration

of asctrocytes in cultured amniotic fluid cells of three cases of neural tube defects

Hum Genet 56(3) 365-370

bull Siddiqui MM Atala A (2004) Amniotic-fluid derived pluripotent cells In R Lanza amp D

Melton (Eds) Handbook of Stem Cells Elsevier Academic Press Burlington USA

pp 175-179

Multipotent Adult Progenitor Cells

(MAPCs) bull Multipotent Adult Progenitor Cells (MAPCs) were isolated from the

bone marrow muscle and brain of mice by Reyes M and colleagues

bull Originally these stem cells were designated as mesenchymal

because they appeared to resemble those described by Caplan

bull MAPCs were later demonstrated to form neurons (ectodermal

germ layer lineage) and hepatocytes (endodermal germ layer

lineage) Thus MAPCs can differentiate into cell types from all three

primary germ layer lineages

bull Thus MAPCs appear to be pluripotent stem cells rather than

mesenchymal stem cells as they were first designated

bull Along the stem cell developmental continuum from totipotent stem

cells (which grow in suspension culture to adherent culture) to

unipotent progenitor cells (which grow as adherent cultures)

MAPCs grow as adherent cultures

Very Small Embryonic-Like Stem Cells

bull Very small three to five micron cells were originally isolated from

adult murine bone marrow (Sca1+Lin- CD45-) and human cord

blood (CD133+Lin-CD45-)

bull These cells were detectable with early developmental markers such

as cell surface SSEA and nuclear Oct4 and Nanog transcription

factors

bull These particular cells have a high nuclear to cytoplasmic ratio and

demonstrate a preponderance of euchromatin rather than

heterochromatin

bull Cells with these same characteristics have been found in

bull skeletal muscle

bull gonads

bull heart

bull brain

Very Small Embryonic-Like Stem Cells

bull In the appropriate in vivo model systems these cells

have been shown to differentiate into long-term

repopulating

ndash mesenchymal stem cells

ndash skeletal muscle

ndash endothelial cells

ndash cardiac myocytes

ndash hematopoietic stem cells

ndash lung epithelial cells

ndash tumor stromal cells

ndash oocytes

Very Small Embryonic-Like Stem Cells

bull Because of their small size embryonic markers and

ability to form multiple primitive cell layers of the

conceptus these cells have been designated as very

small embryonic-like stem cells (VSELs)

bull Very low numbers of VSELs circulate throughout the

vasculature under steady-state conditions

bull Additional VSELs can be mobilized in patients during

myocardial infarction stroke acute burns active

inflammatory bowel disease and cancer and with the

use of granulocyte-colony stimulating factor

Adult Pluripotent Stem Cells

bull Originally isolated from mouse rat and human skeletal

muscle of newborn and adult

bull These cells were detectable with early developmental

markers such as cell surface SSEA (stage specific

embryonic antigen) and CEA-CAM-1 (carcinoembryonic

antigen-cell adhesion molecule-1) nuclear transcription

factors Nanog Nanos Bcl-2 CXCR4 Oct4 and

telomerase

bull These particular cells have a high nuclear to cytoplasmic

ratio

bull These cells display a normal karyotype after population

doublings well in excess Hayflickrsquos limit

bull Young HE Black AC (2005) Adult-derived stem cells Minerva Biotechnologica 17 55-63

bull Young HE Duplaa C Romero-Ramos M Chesselet MF Vourc h P et al (2004) Adult reserve

stem cells and their potential for tissue engineering Cell Biochem Biophys 40(1) 1-80

bull Young HE Hyer L Black AC Robinson JS (2013) Adult stem cells from bench-top to bedside In

Qing Liu amp Hongjun Wang Tissue Regeneration Where Nanostructure Meets Biology 3DBiotech

North Brunswick NJ Chap 1 pp 1-60

bull Young HE Steele T Bray RA Detmer K Blake LW et al (1999) Human progenitor and

pluripotent cells display cell surface cluster differentiation markers CD10 CD13 CD56 CD90 and

MHC Class-I Proc Soc Exp Biol Med 221(1) 63-71

bull Young HE Duplaa C Yost MJ Henson NL Floyd JA et al (2004) Clonogenic analysis reveals

reserve stem cells in postnatal mammals II Pluripotent epiblastic-like stem cells Anat Rec A

Discov Mol Cell Evol Biol 277(1) 178-203

bull Young HE Black AC (2013) Naturally occurring adult pluripotent stem cells In Meyers RA et al

(Eds) Stem Cells From Biology to Therapy Advances in Molecular Biology and Medicine (1st

edn) Texas USA p 63-93

bull Romero-Ramos M Vourch P Young HE Lucas PA Wu Y et al (2002) Neuronal differentiation of

stem cells isolated from adult muscle J Neurosci Res 69(6) 894-907

Adult Pluripotent Stem Cells

bull Six to eight micron cells (SSEA+ CD10+) and 02 to 20 micron cells (CEA-CAM-

1+ CD66e+) were originally isolated from mouse rat and human skeletal muscle of

newborn adolescent sexually mature and geriatric-aged individuals

bull These cells were detectable with early developmental markers such as cell surface

SSEA (stage specific embryonic antigen) and CEA-CAM-1 (carcinoembryonic

antigen-cell adhesion molecule-1) nuclear transcription factors Nanog Nanos Bcl-2

CXCR4 Oct4 and telomerase

bull These particular cells have a high nuclear to cytoplasmic ratio These cells display

a normal karyotype after population doublings well in excess Hayflickrsquos limit

bull Cells with these same characteristics have been found in 37 different tissues and

organs thus far including blood bone marrow brain adipose tissue and skeletal

muscle

bull In the appropriate in vivo and in vitro model systems these cells have been shown

to differentiate and demonstrate phenotypic expression markers for 63 (6-8 microm cells)

and 66 (02-20 microm cells) distinct cell types including neurons glial cells

keratinocytes muscle fat cartilage bone connective tissues hematopoietic cells GI

epithelium liver cells pancreatic cells and spermatogonia

Adult Pluripotent Stem Cells

bull Because of their small size presence of embryonic

markers and their ability to form cells from the three

primary germ layer lineages as well as spermatogonia

these cells were named according to parallel

structures found within the developing zygote ie

epiblast-like stem cells (PPSC 6-8 microm cells) and

blastomere-like stem cells (TPSC 02-20 microm cells)

bull Moderate numbers of these cells circulate throughout the

peripheral vasculature under steady state conditions

bull Additional PPSC and TPSC can be mobilized and

harvested following exercise severe trauma and after

ingestion of a nutraceutical

bull Young HE Black AC (2005) Adult-derived stem cells Minerva Biotechnologica 17 55-63

bull Young HE Duplaa C Romero-Ramos M Chesselet MF Vourc h P et al (2004) Adult reserve

stem cells and their potential for tissue engineering Cell Biochem Biophys 40(1) 1-80

bull Young HE Hyer L Black AC Robinson JS (2013) Adult stem cells from bench-top to bedside In

Qing Liu amp Hongjun Wang Tissue Regeneration Where Nanostructure Meets Biology 3DBiotech

North Brunswick NJ Chap 1 pp 1-60

bull Young HE Steele T Bray RA Detmer K Blake LW et al (1999) Human progenitor and

pluripotent cells display cell surface cluster differentiation markers CD10 CD13 CD56 CD90 and

MHC Class-I Proc Soc Exp Biol Med 221(1) 63-71

bull Young HE Duplaa C Yost MJ Henson NL Floyd JA et al (2004) Clonogenic analysis reveals

reserve stem cells in postnatal mammals II Pluripotent epiblastic-like stem cells Anat Rec A

Discov Mol Cell Evol Biol 277(1) 178-203

bull Young HE Black AC (2013) Naturally occurring adult pluripotent stem cells In Meyers RA et al

(Eds) Stem Cells From Biology to Therapy Advances in Molecular Biology and Medicine (1st

edn) Texas USA p 63-93

bull Romero-Ramos M Vourch P Young HE Lucas PA Wu Y et al (2002) Neuronal differentiation of

stem cells isolated from adult muscle J Neurosci Res 69(6) 894-907

Adult Pluripotent Stem Cells

bull Six to eight micron cells (SSEA+ CD10+) and 02 to 20 micron cells (CEA-CAM-

1+ CD66e+) were originally isolated from mouse rat and human skeletal muscle of

newborn adolescent sexually mature and geriatric-aged individuals

bull These cells were detectable with early developmental markers such as cell surface

SSEA (stage specific embryonic antigen) and CEA-CAM-1 (carcinoembryonic

antigen-cell adhesion molecule-1) nuclear transcription factors Nanog Nanos Bcl-2

CXCR4 Oct4 and telomerase

bull These particular cells have a high nuclear to cytoplasmic ratio These cells display

a normal karyotype after population doublings well in excess Hayflickrsquos limit

bull Cells with these same characteristics have been found in 37 different tissues and

organs thus far including blood bone marrow brain adipose tissue and skeletal

muscle

bull In the appropriate in vivo and in vitro model systems these cells have been shown

to differentiate and demonstrate phenotypic expression markers for 63 (6-8 microm cells)

and 66 (02-20 microm cells) distinct cell types including neurons glial cells

keratinocytes muscle fat cartilage bone connective tissues hematopoietic cells GI

epithelium liver cells pancreatic cells and spermatogonia

Adult Pluripotent Stem Cells

bull Because of their small size presence of embryonic

markers and their ability to form cells from the three

primary germ layer lineages as well as spermatogonia

these cells were named according to parallel

structures found within the developing zygote ie

epiblast-like stem cells (PPSC 6-8 microm cells) and

blastomere-like stem cells (TPSC 02-20 microm cells)

bull Moderate numbers of these cells circulate throughout the

peripheral vasculature under steady state conditions

bull Additional PPSC and TPSC can be mobilized and

harvested following exercise severe trauma and after

ingestion of a nutraceutical

Additional Pluripotent Stem Cells

bull Following an isolation and propagation protocol similar to

that used for the MAPCs a cell termed ldquounrestricted

somatic stem cell (USSC)rdquo was isolated from bone

marrow in 1-3 weeks but with only three cell passages

bull The cells showed the capability to form ectodermal

mesodermal and endodermal phenotypes

bull Marrow-isolated adult multilineage inducible (MIAMI)

cells were also derived in a manner similar to MAPCs

bull Their derivation is based on seeding densities at either

clonal or sparse dilutions

bull MIAMI cells demonstrated differentiation into ectodermal

mesodermal and endodermal phenotypes

Additional Pluripotent Stem Cells

bull Human bone marrow-derived multipotent stem cells

(hBMSCs) have been reported

bull As the name implies these cells were isolated from

human bone marrow and selected from adherent

cultures

bull Differentiation studies showed that these cells form

phenotypes belonging to all three germ layer lineages

Fetal somatic stem cells (FSSCs) have been reported

bull The cells derived from fetal soma demonstrate a wide

range of differentiation potentials

Reprogrammed Pluripotent Stem Cells

bull The idea of cloning animals utilizing nuclear transfer was

first proposed by Spemann

bull The first demonstration that Spemannrsquos proposal was

even possible for cloning adult animals was shown by

Gurdon

bull Indeed the concept of reprogramming a patientrsquos

somatic cells into totipotent pluripotent stem cells was

conceived based on four independent breakthroughs in

the field of developmental embryology in the late 1900rsquos

ie somatic cell nuclear transfer in amphibians success

of cloning of sheep (Dolly) by somatic cell nuclear

transfer assisted reproductive technologies for live

human births (Mary Louise Brown) and derivation of

human embryonic stem cells

Somatic Cell Nuclear Transfer (SCNT)

bull The process of somatic cell nuclear transfer ie reprogramming of

somatic cell nuclei into a totipotent embryonic stem cell capable of

forming an organism was first proposed by Hans Spemann when he

referred to a ldquofantastical experimentrdquo in his book Embryonic

Development and Induction

bull However Spemannrsquos process was first demonstrated by Gurdon

bull He utilized the nuclei from the intestinal epithelial cells of Xenopus

laevistad poles and transferred the nuclei into the cytoplasm of

enucleated eggs to demonstrate the development of an adult frog

bull His work demonstrated that the ova contained maternal factors

within its cytoplasm with the inherent ability to reprogram gene

expression of a differentiated somatic cell nucleus and that that

nucleus had the capability of forming a new organism of the same

genetic makeup as the host organism

bull His experiment represented the first reported example of a somatic

cell being reprogrammed back to a totipotent state by an enucleated

egg and developing into a live viable offspring

Somatic Cell Nuclear Transfer (SCNT)

bull Therapeutic cloning or SCNT begins with the same process used

to create Dolly

bull A diploid donor somatic cell from a body tissue such as a fibroblast

from skin is stripped of its plasma membrane and cytoplasm and

fused with an enucleated unfertilized ovum The cytoplasm within

the ovum reprograms the DNA within the diploid donor nucleus to an

embryonic state

bull The fused cell (modified blastomere) is allowed to rest for a defined

length of time and then induced to proliferate until it reaches the

early blastocyst stage composed of the inner cell mass and

trophoblast The inner cell mass cells are harvested and cultured to

create a stable cell line that is genetically matched to the donor

cells The cells are pluripotent in that they

i Have a capacity for unlimited cellular proliferation

ii Will form cells from all three primary germ layer lineages and

iii Will form teratomas These are the hallmarks of embryonic

pluripotent stem cells

Is it the wave

of the future

Induced Pluripotent Stem

Cells (iPSCs) bull The most widely accepted method to generate induced

pluripotent stem cells (iPSCs) is the retroviral vector

introduction of four genes (Oct4 Sox2 Klf4and c-Myc

aka the Yamanaka factors) into more differentiated

cell types such as fibroblasts

bull Following the initial landmark work for which Yamanaka

received the 2013 Nobel prize iPSCs have since been

generated from differentiated fetal and adult fibroblasts

hepatocytes stomach cells Keratinocytes cord blood

peripheral blood fully differentiated B- and T-

lymphocytes dental pulp cells and kidney cells

Precursor Cell Definitions

Stem

Cells

Progenitor

Cells

1) Stem cells ARE ldquoPlasticrdquo ndash can form any

downstream cell type (unidirectional only)

2) Stem cells are NOT committed to any specific cell

tissue type

3) Stem cells have unlimited proliferation potential

4) Stem cells maintain a biological ldquoclockrdquo of zero

until differentiation begins

1) Progenitor cells are NOT ldquoplasticrdquo ndash have a defined

differentiation potential

2) Progenitor cells ARE committed to a specific cell

tissue type

3) Progenitor cells have a limited life span ie

humans 50-70 population doublings before

senescence and death

4) Progenitor cellsrsquo Biological ldquoclockrdquo starts at birth

Precursor Cell Definitions Totipotent Form embryo placenta amp germ Cells (sperm amp

Stem Cell ovum)

Pluripotent Form all cells of the embryo but not will not form

Stem Cell placenta or germ cells

Germ Layer Form ectodermal lineage cells mesodermal lineage

Lineage SCs cells amp endodermal lineage cells

Progenitor Multipotent (ex hematopoietic)

Cells Tripotent (ex chondro-osteo-adipogenic)

Bipotent (ex adipo-fibrogenic)

Unipotent (ex myoblast fibroblast adipoblast etc)

Precursor Cells in Adults Totipotent SCs Blastomere-Like Stem Cell (BLSC)

Pluripotent SCs Epiblast-Like Stem Cell (ELSC)

Ultra-small nucleated plasma particle (u-NP2)

Small-nucleated plasma particle (s-NP2)

Intermediate-nucleated plasma particle (i-NP2)

Large-nucleated plasma particle (l-NP2)

Germ Layer GL-EctoSCs GL-MesoSCs GL-EndoSCs

Lineage SCs

Progenitor Multipotent PCs Tripotent PCs Bipotent PCs amp

Cells Unipotent PCs

Isolated and repetitively cloned from single cells

Totipotent Adult Stem Cells

Adult Totipotent Stem Cells

s-NP2

i-NP2

s-NP2

u-NP2

Differentiated Phenotypic

Expression Markers

(expression of extracellular amp intracellular markers via

monoclonal antibodies with immunocytochemistry and

selective agents (ie enzymes hydrocarbons etc)

combined with histochemical staining

Nucleated Plasma Particle Characteristics u-NP2 s-NP2 i-NP2 l-NP2

Size (unfixed) lt 2 m 2-6 m 6-8 m 7-9 m

Trypan Blue Positive Pos Neg Negative Negative

Lifespan gt 300 pd gt 400 pd gt 400 pd gt 400 pd

Default Quiescence Quiescence Quiescence Quiescence

Activity Mobilize Mobilize Mobilize Mobilize

In Situ Proliferation Proliferation Proliferation Proliferation

In Sequence Lineage Com Lineage Com Lineage Com Lineage Com

Progression Progression Progression Progression

Differentiation Differentiation Differentiation Differentiation

Cells Formed Somatic Cells Somatic Cells Somatic Cells Somatic Cells

(66) (64) (63) (62)

Functional Parkinson Parkinson Parkinson Parkinson

Activities disease disease disease disease

Seen CIDP MS CIDP MS CIDP MS CIDP MS

ALS AD EP ALS AD EP ALS AD EP ALS AD EP

Myocardial Myocardial Myocardial Myocardial

ischemia ischemia ischemia ischemia

Insulin Insulin Insulin Insulin

secretion secretion secretion secretion

COPD IPF COPD IPF COPD IPF COPD IPF

Rotator cuff Rotator cuff Rotator cuff Rotator cuff

Bone fracture Bone fracture Bone fracture Bone fracture

What we are

doinghellip

Center for Orthopedic

amp

Regenerative Medicine Science

What we are doinghellip

bull With current regulatory burden in the United

States preventing physicians from culture and

expansion of mesenchymal stem cells

bull Physicians have had to focus on

ndash Improved tissue extraction techniques

ndash Laboratory cell processing to improve cell and

viability counts to improve clinical outcomes

bull Example

ndash Adipose tissue ndash Liberase (collagenase)

raquo Designed initially for pancreatic tissue digestion

raquo Not well designed for adipose tissue digestion

Adipose tissue prep

bull Adipose tissue SVF prep

ndash Issues with the presence of trypsin

ndash There are methods to bind trypsin and

improve preparation

ndash There are better enzymes to utilize with

adipose tissue

ndash We will be submitting for publication involving

these techniques in the fall of 2015

What are we doinghellip

bull Stopped using SVF

bull Compressed fat for graphs only

bull Stopped using BMAC

bull MSCs blood ndash homogenous

population

bull TPSCs ndash Neurological

neuropathic pain states

bull PPSCs ndash Orthopedic

bull TPSCs amp PPSCs mixed preps

Somatic Cell Nuclear Transfer (SCNT)

bull The process of somatic cell nuclear transfer ie reprogramming of

somatic cell nuclei into a totipotent embryonic stem cell capable of

forming an organism was first proposed by Hans Spemann when he

referred to a ldquofantastical experimentrdquo in his book Embryonic

Development and Induction

bull However Spemannrsquos process was first demonstrated by Gurdon

bull He utilized the nuclei from the intestinal epithelial cells of Xenopus

laevistad poles and transferred the nuclei into the cytoplasm of

enucleated eggs to demonstrate the development of an adult frog

bull His work demonstrated that the ova contained maternal factors

within its cytoplasm with the inherent ability to reprogram gene

expression of a differentiated somatic cell nucleus and that that

nucleus had the capability of forming a new organism of the same

genetic makeup as the host organism

bull His experiment represented the first reported example of a somatic

cell being reprogrammed back to a totipotent state by an enucleated

egg and developing into a live viable offspring

Somatic Cell Nuclear Transfer (SCNT)

bull Therapeutic cloning or SCNT begins with the same process used

to create Dolly

bull A diploid donor somatic cell from a body tissue such as a fibroblast

from skin is stripped of its plasma membrane and cytoplasm and

fused with an enucleated unfertilized ovum The cytoplasm within

the ovum reprograms the DNA within the diploid donor nucleus to an

embryonic state

bull The fused cell (modified blastomere) is allowed to rest for a defined

length of time and then induced to proliferate until it reaches the

early blastocyst stage composed of the inner cell mass and

trophoblast The inner cell mass cells are harvested and cultured to

create a stable cell line that is genetically matched to the donor

cells The cells are pluripotent in that they

i Have a capacity for unlimited cellular proliferation

ii Will form cells from all three primary germ layer lineages and

iii Will form teratomas These are the hallmarks of embryonic

pluripotent stem cells

Is it the wave

of the future

Induced Pluripotent Stem

Cells (iPSCs) bull The most widely accepted method to generate induced

pluripotent stem cells (iPSCs) is the retroviral vector

introduction of four genes (Oct4 Sox2 Klf4and c-Myc

aka the Yamanaka factors) into more differentiated

cell types such as fibroblasts

bull Following the initial landmark work for which Yamanaka

received the 2013 Nobel prize iPSCs have since been

generated from differentiated fetal and adult fibroblasts

hepatocytes stomach cells Keratinocytes cord blood

peripheral blood fully differentiated B- and T-

lymphocytes dental pulp cells and kidney cells

Precursor Cell Definitions

Stem

Cells

Progenitor

Cells

1) Stem cells ARE ldquoPlasticrdquo ndash can form any

downstream cell type (unidirectional only)

2) Stem cells are NOT committed to any specific cell

tissue type

3) Stem cells have unlimited proliferation potential

4) Stem cells maintain a biological ldquoclockrdquo of zero

until differentiation begins

1) Progenitor cells are NOT ldquoplasticrdquo ndash have a defined

differentiation potential

2) Progenitor cells ARE committed to a specific cell

tissue type

3) Progenitor cells have a limited life span ie

humans 50-70 population doublings before

senescence and death

4) Progenitor cellsrsquo Biological ldquoclockrdquo starts at birth

Precursor Cell Definitions Totipotent Form embryo placenta amp germ Cells (sperm amp

Stem Cell ovum)

Pluripotent Form all cells of the embryo but not will not form

Stem Cell placenta or germ cells

Germ Layer Form ectodermal lineage cells mesodermal lineage

Lineage SCs cells amp endodermal lineage cells

Progenitor Multipotent (ex hematopoietic)

Cells Tripotent (ex chondro-osteo-adipogenic)

Bipotent (ex adipo-fibrogenic)

Unipotent (ex myoblast fibroblast adipoblast etc)

Precursor Cells in Adults Totipotent SCs Blastomere-Like Stem Cell (BLSC)

Pluripotent SCs Epiblast-Like Stem Cell (ELSC)

Ultra-small nucleated plasma particle (u-NP2)

Small-nucleated plasma particle (s-NP2)

Intermediate-nucleated plasma particle (i-NP2)

Large-nucleated plasma particle (l-NP2)

Germ Layer GL-EctoSCs GL-MesoSCs GL-EndoSCs

Lineage SCs

Progenitor Multipotent PCs Tripotent PCs Bipotent PCs amp

Cells Unipotent PCs

Isolated and repetitively cloned from single cells

Totipotent Adult Stem Cells

Adult Totipotent Stem Cells

s-NP2

i-NP2

s-NP2

u-NP2

Differentiated Phenotypic

Expression Markers

(expression of extracellular amp intracellular markers via

monoclonal antibodies with immunocytochemistry and

selective agents (ie enzymes hydrocarbons etc)

combined with histochemical staining

Nucleated Plasma Particle Characteristics u-NP2 s-NP2 i-NP2 l-NP2

Size (unfixed) lt 2 m 2-6 m 6-8 m 7-9 m

Trypan Blue Positive Pos Neg Negative Negative

Lifespan gt 300 pd gt 400 pd gt 400 pd gt 400 pd

Default Quiescence Quiescence Quiescence Quiescence

Activity Mobilize Mobilize Mobilize Mobilize

In Situ Proliferation Proliferation Proliferation Proliferation

In Sequence Lineage Com Lineage Com Lineage Com Lineage Com

Progression Progression Progression Progression

Differentiation Differentiation Differentiation Differentiation

Cells Formed Somatic Cells Somatic Cells Somatic Cells Somatic Cells

(66) (64) (63) (62)

Functional Parkinson Parkinson Parkinson Parkinson

Activities disease disease disease disease

Seen CIDP MS CIDP MS CIDP MS CIDP MS

ALS AD EP ALS AD EP ALS AD EP ALS AD EP

Myocardial Myocardial Myocardial Myocardial

ischemia ischemia ischemia ischemia

Insulin Insulin Insulin Insulin

secretion secretion secretion secretion

COPD IPF COPD IPF COPD IPF COPD IPF

Rotator cuff Rotator cuff Rotator cuff Rotator cuff

Bone fracture Bone fracture Bone fracture Bone fracture

What we are

doinghellip

Center for Orthopedic

amp

Regenerative Medicine Science

What we are doinghellip

bull With current regulatory burden in the United

States preventing physicians from culture and

expansion of mesenchymal stem cells

bull Physicians have had to focus on

ndash Improved tissue extraction techniques

ndash Laboratory cell processing to improve cell and

viability counts to improve clinical outcomes

bull Example

ndash Adipose tissue ndash Liberase (collagenase)

raquo Designed initially for pancreatic tissue digestion

raquo Not well designed for adipose tissue digestion

Adipose tissue prep

bull Adipose tissue SVF prep

ndash Issues with the presence of trypsin

ndash There are methods to bind trypsin and

improve preparation

ndash There are better enzymes to utilize with

adipose tissue

ndash We will be submitting for publication involving

these techniques in the fall of 2015

What are we doinghellip

bull Stopped using SVF

bull Compressed fat for graphs only

bull Stopped using BMAC

bull MSCs blood ndash homogenous

population

bull TPSCs ndash Neurological

neuropathic pain states

bull PPSCs ndash Orthopedic

bull TPSCs amp PPSCs mixed preps

Induced Pluripotent Stem

Cells (iPSCs) bull The most widely accepted method to generate induced

pluripotent stem cells (iPSCs) is the retroviral vector

introduction of four genes (Oct4 Sox2 Klf4and c-Myc

aka the Yamanaka factors) into more differentiated

cell types such as fibroblasts

bull Following the initial landmark work for which Yamanaka

received the 2013 Nobel prize iPSCs have since been

generated from differentiated fetal and adult fibroblasts

hepatocytes stomach cells Keratinocytes cord blood

peripheral blood fully differentiated B- and T-

lymphocytes dental pulp cells and kidney cells

Precursor Cell Definitions

Stem

Cells

Progenitor

Cells

1) Stem cells ARE ldquoPlasticrdquo ndash can form any

downstream cell type (unidirectional only)

2) Stem cells are NOT committed to any specific cell

tissue type

3) Stem cells have unlimited proliferation potential

4) Stem cells maintain a biological ldquoclockrdquo of zero

until differentiation begins

1) Progenitor cells are NOT ldquoplasticrdquo ndash have a defined

differentiation potential

2) Progenitor cells ARE committed to a specific cell

tissue type

3) Progenitor cells have a limited life span ie

humans 50-70 population doublings before

senescence and death

4) Progenitor cellsrsquo Biological ldquoclockrdquo starts at birth

Precursor Cell Definitions Totipotent Form embryo placenta amp germ Cells (sperm amp

Stem Cell ovum)

Pluripotent Form all cells of the embryo but not will not form

Stem Cell placenta or germ cells

Germ Layer Form ectodermal lineage cells mesodermal lineage

Lineage SCs cells amp endodermal lineage cells

Progenitor Multipotent (ex hematopoietic)

Cells Tripotent (ex chondro-osteo-adipogenic)

Bipotent (ex adipo-fibrogenic)

Unipotent (ex myoblast fibroblast adipoblast etc)

Precursor Cells in Adults Totipotent SCs Blastomere-Like Stem Cell (BLSC)

Pluripotent SCs Epiblast-Like Stem Cell (ELSC)

Ultra-small nucleated plasma particle (u-NP2)

Small-nucleated plasma particle (s-NP2)

Intermediate-nucleated plasma particle (i-NP2)

Large-nucleated plasma particle (l-NP2)

Germ Layer GL-EctoSCs GL-MesoSCs GL-EndoSCs

Lineage SCs

Progenitor Multipotent PCs Tripotent PCs Bipotent PCs amp

Cells Unipotent PCs

Isolated and repetitively cloned from single cells

Totipotent Adult Stem Cells

Adult Totipotent Stem Cells

s-NP2

i-NP2

s-NP2

u-NP2

Differentiated Phenotypic

Expression Markers

(expression of extracellular amp intracellular markers via

monoclonal antibodies with immunocytochemistry and

selective agents (ie enzymes hydrocarbons etc)

combined with histochemical staining

Nucleated Plasma Particle Characteristics u-NP2 s-NP2 i-NP2 l-NP2

Size (unfixed) lt 2 m 2-6 m 6-8 m 7-9 m

Trypan Blue Positive Pos Neg Negative Negative

Lifespan gt 300 pd gt 400 pd gt 400 pd gt 400 pd

Default Quiescence Quiescence Quiescence Quiescence

Activity Mobilize Mobilize Mobilize Mobilize

In Situ Proliferation Proliferation Proliferation Proliferation

In Sequence Lineage Com Lineage Com Lineage Com Lineage Com

Progression Progression Progression Progression

Differentiation Differentiation Differentiation Differentiation

Cells Formed Somatic Cells Somatic Cells Somatic Cells Somatic Cells

(66) (64) (63) (62)

Functional Parkinson Parkinson Parkinson Parkinson

Activities disease disease disease disease

Seen CIDP MS CIDP MS CIDP MS CIDP MS

ALS AD EP ALS AD EP ALS AD EP ALS AD EP

Myocardial Myocardial Myocardial Myocardial

ischemia ischemia ischemia ischemia

Insulin Insulin Insulin Insulin

secretion secretion secretion secretion

COPD IPF COPD IPF COPD IPF COPD IPF

Rotator cuff Rotator cuff Rotator cuff Rotator cuff

Bone fracture Bone fracture Bone fracture Bone fracture

What we are

doinghellip

Center for Orthopedic

amp

Regenerative Medicine Science

What we are doinghellip

bull With current regulatory burden in the United

States preventing physicians from culture and

expansion of mesenchymal stem cells

bull Physicians have had to focus on

ndash Improved tissue extraction techniques

ndash Laboratory cell processing to improve cell and

viability counts to improve clinical outcomes

bull Example

ndash Adipose tissue ndash Liberase (collagenase)

raquo Designed initially for pancreatic tissue digestion

raquo Not well designed for adipose tissue digestion

Adipose tissue prep

bull Adipose tissue SVF prep

ndash Issues with the presence of trypsin

ndash There are methods to bind trypsin and

improve preparation

ndash There are better enzymes to utilize with

adipose tissue

ndash We will be submitting for publication involving

these techniques in the fall of 2015

What are we doinghellip

bull Stopped using SVF

bull Compressed fat for graphs only

bull Stopped using BMAC

bull MSCs blood ndash homogenous

population

bull TPSCs ndash Neurological

neuropathic pain states

bull PPSCs ndash Orthopedic

bull TPSCs amp PPSCs mixed preps

Precursor Cells in Adults Totipotent SCs Blastomere-Like Stem Cell (BLSC)

Pluripotent SCs Epiblast-Like Stem Cell (ELSC)

Ultra-small nucleated plasma particle (u-NP2)

Small-nucleated plasma particle (s-NP2)

Intermediate-nucleated plasma particle (i-NP2)

Large-nucleated plasma particle (l-NP2)

Germ Layer GL-EctoSCs GL-MesoSCs GL-EndoSCs

Lineage SCs

Progenitor Multipotent PCs Tripotent PCs Bipotent PCs amp

Cells Unipotent PCs

Isolated and repetitively cloned from single cells

Totipotent Adult Stem Cells

Adult Totipotent Stem Cells

s-NP2

i-NP2

s-NP2

u-NP2

Differentiated Phenotypic

Expression Markers

(expression of extracellular amp intracellular markers via

monoclonal antibodies with immunocytochemistry and

selective agents (ie enzymes hydrocarbons etc)

combined with histochemical staining

Nucleated Plasma Particle Characteristics u-NP2 s-NP2 i-NP2 l-NP2

Size (unfixed) lt 2 m 2-6 m 6-8 m 7-9 m

Trypan Blue Positive Pos Neg Negative Negative

Lifespan gt 300 pd gt 400 pd gt 400 pd gt 400 pd

Default Quiescence Quiescence Quiescence Quiescence

Activity Mobilize Mobilize Mobilize Mobilize

In Situ Proliferation Proliferation Proliferation Proliferation

In Sequence Lineage Com Lineage Com Lineage Com Lineage Com

Progression Progression Progression Progression

Differentiation Differentiation Differentiation Differentiation

Cells Formed Somatic Cells Somatic Cells Somatic Cells Somatic Cells

(66) (64) (63) (62)

Functional Parkinson Parkinson Parkinson Parkinson

Activities disease disease disease disease

Seen CIDP MS CIDP MS CIDP MS CIDP MS

ALS AD EP ALS AD EP ALS AD EP ALS AD EP

Myocardial Myocardial Myocardial Myocardial

ischemia ischemia ischemia ischemia

Insulin Insulin Insulin Insulin

secretion secretion secretion secretion

COPD IPF COPD IPF COPD IPF COPD IPF

Rotator cuff Rotator cuff Rotator cuff Rotator cuff

Bone fracture Bone fracture Bone fracture Bone fracture

What we are

doinghellip

Center for Orthopedic

amp

Regenerative Medicine Science

What we are doinghellip

bull With current regulatory burden in the United

States preventing physicians from culture and

expansion of mesenchymal stem cells

bull Physicians have had to focus on

ndash Improved tissue extraction techniques

ndash Laboratory cell processing to improve cell and

viability counts to improve clinical outcomes

bull Example

ndash Adipose tissue ndash Liberase (collagenase)

raquo Designed initially for pancreatic tissue digestion

raquo Not well designed for adipose tissue digestion

Adipose tissue prep

bull Adipose tissue SVF prep

ndash Issues with the presence of trypsin

ndash There are methods to bind trypsin and

improve preparation

ndash There are better enzymes to utilize with

adipose tissue

ndash We will be submitting for publication involving

these techniques in the fall of 2015

What are we doinghellip

bull Stopped using SVF

bull Compressed fat for graphs only

bull Stopped using BMAC

bull MSCs blood ndash homogenous

population

bull TPSCs ndash Neurological

neuropathic pain states

bull PPSCs ndash Orthopedic

bull TPSCs amp PPSCs mixed preps

Differentiated Phenotypic

Expression Markers

(expression of extracellular amp intracellular markers via

monoclonal antibodies with immunocytochemistry and

selective agents (ie enzymes hydrocarbons etc)

combined with histochemical staining

Nucleated Plasma Particle Characteristics u-NP2 s-NP2 i-NP2 l-NP2

Size (unfixed) lt 2 m 2-6 m 6-8 m 7-9 m

Trypan Blue Positive Pos Neg Negative Negative

Lifespan gt 300 pd gt 400 pd gt 400 pd gt 400 pd

Default Quiescence Quiescence Quiescence Quiescence

Activity Mobilize Mobilize Mobilize Mobilize

In Situ Proliferation Proliferation Proliferation Proliferation

In Sequence Lineage Com Lineage Com Lineage Com Lineage Com

Progression Progression Progression Progression

Differentiation Differentiation Differentiation Differentiation

Cells Formed Somatic Cells Somatic Cells Somatic Cells Somatic Cells

(66) (64) (63) (62)

Functional Parkinson Parkinson Parkinson Parkinson

Activities disease disease disease disease

Seen CIDP MS CIDP MS CIDP MS CIDP MS

ALS AD EP ALS AD EP ALS AD EP ALS AD EP

Myocardial Myocardial Myocardial Myocardial

ischemia ischemia ischemia ischemia

Insulin Insulin Insulin Insulin

secretion secretion secretion secretion

COPD IPF COPD IPF COPD IPF COPD IPF

Rotator cuff Rotator cuff Rotator cuff Rotator cuff

Bone fracture Bone fracture Bone fracture Bone fracture

What we are

doinghellip

Center for Orthopedic

amp

Regenerative Medicine Science

What we are doinghellip

bull With current regulatory burden in the United

States preventing physicians from culture and

expansion of mesenchymal stem cells

bull Physicians have had to focus on

ndash Improved tissue extraction techniques

ndash Laboratory cell processing to improve cell and

viability counts to improve clinical outcomes

bull Example

ndash Adipose tissue ndash Liberase (collagenase)

raquo Designed initially for pancreatic tissue digestion

raquo Not well designed for adipose tissue digestion

Adipose tissue prep

bull Adipose tissue SVF prep

ndash Issues with the presence of trypsin

ndash There are methods to bind trypsin and

improve preparation

ndash There are better enzymes to utilize with

adipose tissue

ndash We will be submitting for publication involving

these techniques in the fall of 2015

What are we doinghellip

bull Stopped using SVF

bull Compressed fat for graphs only

bull Stopped using BMAC

bull MSCs blood ndash homogenous

population

bull TPSCs ndash Neurological

neuropathic pain states

bull PPSCs ndash Orthopedic

bull TPSCs amp PPSCs mixed preps

What we are doinghellip

bull With current regulatory burden in the United

States preventing physicians from culture and

expansion of mesenchymal stem cells

bull Physicians have had to focus on

ndash Improved tissue extraction techniques

ndash Laboratory cell processing to improve cell and

viability counts to improve clinical outcomes

bull Example

ndash Adipose tissue ndash Liberase (collagenase)

raquo Designed initially for pancreatic tissue digestion

raquo Not well designed for adipose tissue digestion

Adipose tissue prep

bull Adipose tissue SVF prep

ndash Issues with the presence of trypsin

ndash There are methods to bind trypsin and

improve preparation

ndash There are better enzymes to utilize with

adipose tissue

ndash We will be submitting for publication involving

these techniques in the fall of 2015

What are we doinghellip

bull Stopped using SVF

bull Compressed fat for graphs only

bull Stopped using BMAC

bull MSCs blood ndash homogenous

population

bull TPSCs ndash Neurological

neuropathic pain states

bull PPSCs ndash Orthopedic

bull TPSCs amp PPSCs mixed preps

What we are doing bull Totirsquos

ndash Neuro neurodeg PD SCI

ndash CIDP Neuropathic pain

ndash Demyenliation etc

ndash Lumbar disc

bull Ortho

ndash Pluri + Toti

ndash Pluri + Mesos

bull IV

ndash Depends on protocol and indiction

bull Pulm cardio neuro orhto autoimmune

bull Intranasal

ndash Toti only

bull Nebulized

ndash Pluri and toti

Lab prep very different

The lumbar disc -DDD

bull Totipotent genome tagged with insect

betagalactosidase

ndash Now progeny will express beta-gal and thus

you can monitor where the go and the

progeny they produce

ndash Retrodiscal

ndash Intradiscal

ndash Scafolding HA

ndash Otherhellip

Cell Type Inductive agents

Neurospheres Neurobasal-A B-27 b-FGF EGF

Neurons Laminin Nb-27 Nb-N2 b-FGF EGF

NT3 BDNF

Neuroectoderm Butylated hydroxyanisole DMSO

KCl Hydrocortisone Forskolin N2

Insulin Valproic acid Nb-A

Fibroblasts TGF- b-FGF

CartilageBone BMP-2

Endothelial cells VEGF -FGF BMP-4

Red Blood Cells SCF IL-3 IL-6 EPO

Pancreatic -Cells KGH HGF Insulin Nicotinomide

All cell types Dexamethasone

Induction of Specific Cell Types

Study Rationale bull NucP2 stem cell isolation and deployment technology

bull NucP2 may offer significant advantage over techniques

currently being utilized in regenerative medicine practice

bull NucP2 may be able to overcome some of the significant

limitations in using these types of cellular interventions

bull Another limitation of utilizing autologous bone marrow

and adipose tissue derived mesenchymal stem cell

preparations is that there are few multipotent stem cells

available within the cellular preparation

Study Rationale

bull The majority of the cells that we are currently using in

common orthopedic procedures today are

predominantly composed of progenitor cells which

are lineage committed and having limited capacity for

replication and plasticity

bull What is desired is a regenerative medicine technique

that can utilize autologous cells in order to remain

compliant with FDA regulation and also fall within the

guidelines of minimal manipulation also a requirement of

FDA regulation

Deployment technique experts

Physicians with a foundation in basic

regenerative medicine techniques

such as prolotherapy have an

advantage

Deployment techniques are highly

specialized skills

Physicians highly skilled interventional

pain technology may have better

outcomes

Case examples 1

bull JM 72 yo CF riding bike to work in Seattle WA Struck

by car

ndash Significant right hip pain unresponsive to conservative care

ndash MRI ndash early degenerative changes and acetabular labrum tear

ndash SP arthroscopic debreedment of labrum tear without improvement and

worsening hip pain

ndash CO of chronic low back pain at LS spine and right SI joint region

ndash Numerous medical subspecialty consultations

ndash University of Washington pain medicine Department consultations

pharmaceutical pain management bull Nonresponsive to epidural injections

ndash Ongoing physical therapy

ndash Patient ambulating with a cane-walking stick

ndash Patient is a store owner having to reduce hours and hire more staff to handle

responsibilities

ndash 4 years in chronic pain without improvement

ndash Anxious lawyers wanting to settle or PI case

Case examples 1

bull JM 72 yo CF riding bike to work in Seattle WA Struck

by car

ndash X-rays of lumbar spine = age-related degenerative changes

ndash MRI lumbar spine = age-related degenerative changes

ndash Patient now seen for consultation after 4 years post MVA in

chronic pain

ndash CO

bull Severe low back pain across the lumbosacral spine

bull Severe SIJ pain

bull Severe hip pain

bull Pain radiation into the anterolateral thigh buttock and posterior thigh

bull Significant sensation of muscle tension in the TFL

ndash Complete relief of hip pain and TFL and anterior thigh pain with

intra-articular local anesthetic injection under ultrasound

guidance

Case examples 1

bull JM 72 yo CF riding bike to work in Seattle WA Struck

by car

ndash Symptomatic relief of back pain from lumbar facet

injectionsmedial branch block

ndash Symptomatic relief from SIJ pain with SIJ block

bull Prolotherapy

3 modest improvements of back pain and

recurrent SIJ pain

bull PRP injection directed to acetabular labrum intra-

articular hip lumbar facet and SIJ with modest

improvement in recurrent symptoms

bull Repeated PRP injectionrsquos resulted in same modest

improvements and recurrent symptoms

Case examples 1 bull JM 72 yo CF riding bike to work in Seattle WA Struck

by car

ndash Adipose tissue SVFdirected to intra-articular hip and acetabular

labrum resolved all hip pain complaints

bull Patient no longer having to ambulate with cane

ndash Despite hip improvements persistent low back pain continued to

create functional and work disability issues

bull NucP-2 stem cell

ndash Lumbar facet injections and right sacroiliac joint injection

ndash IV infusion

ndash 3 days later patient attended market walking the entire market for

business purchases in New York without pain

ndash Patient now reporting complete resolution of chronic low back

pain and SIJ pain

Case example 2 bull 68-year-old CF with tricompartmental

osteoarthritis

ndash Progressive worsening knee pain and disability

ndash Now reporting poor walking tolerance reduced

recreational activity tolerance

ndash Modest response to platelet releasate injection

ndash Status post adipose tissue SVF injection with no

significant improvement after 6 months

ndash Patient now facing consideration of TKA

Case example 2

bull 68-year-old CF with tricompartmental

osteoarthritis

ndash Patient consulted to explore nonoperative

options

ndash NucP-2 stem cell

bull Marked improvement

bull Improved functional activity tolerance

bull Return to some recreational activities

ndash More questions and answers

bull Will this have to be repeated

bull How often

bull Post MRI one year pending WORMS BLOCKS

CaLshellip T3 magnet vs T15 magnet etc

Case example 3

bull 69-year-old CF with long-standing history of

CIDP with severe neuropathic pain especially

right lower extremity

ndash Patient met diagnostic criteria

ndash Patient had an progressive worsening of pain and

neurologic function

ndash Multiple medical consultations

ndash Corticosteroids Imuran IVIG gabapentin

nortriptyline etc

ndash Patient not wanting to have chemotherapy ie

CellCept Cytoxan cyclosporine

Case example 3

bull 69-year-old CF with long-standing history of

CIDP with severe neuropathic pain especially

right lower extremity

ndash Patient seen 4 years prior to participate in pilot study

ndash Isolation of TPSCs deployed via intranasal route

overlying cribriform plate

ndash Isolation of PPSCs deployed via IV infusion

ndash Single administration of intranasal and IV deployment

resulted in remission of neuropathic pain improved

gait and functional activity performance

14 months

ndash Patient then started to regress and requesting tx

Case of Parkinsonrsquos Disease

bull 66 YO CM with PD ndash Movement disorder

ndash Speech dysarthria

ndash Social isolation

ndash On Sinemet (Carbidopa-Levodopa)and Stalevo which is becoming less

effective

bull Pt had totipotent adult autologous stem cell

intranasal infusion and IV pluripotent stem cell

infusion

bull Outcome 15 month ndash patient on frac12 dose of medication

ndash Better movement control

ndash Reduced dysarthria

ndash No more social isolation and more confident

Local anesthetics

bull Marcaine Liocaine Prilocaine

Carbocaine Procaine ndash ALL kills stem

cells

bull Naropin ( Ropivicaine )

ndash Only anesthetic that is non-toxic to stem cells

ndash 100 stem cell viability

bull Suspension of cells in saline in pain

sensitive patients also helps

Use of platelet releaseateshellip

PRP growth factor concentrations

differ by system System Platelets

(x103μL)

WBCs

(x103μL)

PDGF-AB

(ngmL)

PDGF-BB

(ngmL)

TGF-B1

(ngmL)

VEGF

(ngmL)

Cascade

MTF

4438

247

11

02 97

36 148

25 01

008

03

03

GPS III

Biomet

5562

2926

344

136 187

128

231

101

01

008

24

11

Magellan

Arteriocyte

7802

2465

110

82 344

107

330

82 02

01

12

08

P values lt00001 0006 0006 0009 0371 0005

Study objective to investigate effects of leukocytes on PRP

Create

leukocyterich

PRP

(LR-PRP)

Create

Leukocyte poor

PRP

(LP-PRP)

Inject into

healthy

patellar

tendon of New

Zealand White

rabbits

5d amp 14d

Harvest

patellar

tendons and

analyze

histology

Results- Inflammatory Mediators

bull LR-PRP causes significant increases in

pro-inflammatory mediators

bull LP-PRP causes significant increase in anti-inflammatory mediators

IL-1β IL-6 IFN-γ TNF-α IL-4 IL-10

PPP + +

LP-PRP + + +

LR-PRP + + + + +

RBC +

Use of platelet releaseateshellip

bull Activated platelet

ndash Alpha granules

bull PD growth factors

TGF-β

Platelet derived growth factor (IGF)

vascular endothelial growth factors

(VEGF) Vasc Endo Growth Factor

Epidermal Growth Factor (EGF)

Fibroblastic Growth Factor -2 (FGF-2)

This is PRP

PDEGF

PDGF-AB bFGF

VEGF PDFG-AA IGF-3

FGF-1 IGFBP-3

BMP-4

IGFBP-3

TGF-B1

hGH

PDGF-BB

bFGF BMP-2

PDGF-AB

IGF-1

IGFBP-2

EGF

HGF

ECGF

PDEGF

BMP

Pro-

Chondrogenic

TGFB-1

IGF-1

bFGF

BMP-2

Anti-

Chondrogenic

VEGF

IGFBP-2

IGFBP-3

PDGF-AA

PDGF-AB

PDGF-BB

EGF

1st Generation PRP

Pro-

Chondrogenic

TGFB-1

IGF-1

bFGF

BMP-2

Anti-

Chondrogenic

VEGF

IGFBP-2

IGFBP-3

PDGF-AA

PDGF-AB

PDGF-BB

EGF

2nd Generation PRP

ADSC Induction Media

bull IGF-I

bull TGF-β1

bull GH

bull FGF-2

Platelet Rich Plasma

bull IGF-1

bull TGF-β1

bull GH

bull FGF-2

bull PDGF

bull VEGF

bull IGF-BP2

bull IGF-BP3

2nd Generation PRP

Create and

activate

PRP

Neutralize specific

PRP growth factors

with antibodies

bull VEGF

bull IGFBP-2

bull IGFBP-3

bull EGF

bull PDGF

Confirm

absence of

neutralized

factors and

presence of key

growth factors

by ELISA

2nd Generation PRP

Sterile inflammation

caused by DAMPs and

mediated by IL-1β or

TNF in the absence of

infection involves

canonical NFκB

activation and

proinflammatory

signalling In OA

molecules from

cartilage breakdown

and intracellular

components ohellip

We will manipulate growth factor

content for target tissue or disease

Signalling proteins

(including growth

factors

chemokines and

other cytokines)

adhesive proteins

and proteases

together with small

molecules and ions

that are secreted by

platelets are found

at high levels in

PRP and interact

with mhellip

mPRP Task Myostatin and TGF-β1 removal

using antibodies bound

to protein AG agarose in sterile conditions

Protein A beads Incubate

1 hour RT Add Ab-s

PBS buffer

Protein AG

beads

TGFβ1 antibodies myostatin antibodies

Protein G

beads

Disc disease and pathological

changes in the neuroforamen

bull Disc leak

ndash PLA2

ndash Immune mediated responses

ndash Inflammation

bull Ischemia

bull Demeylination

bull Inflamatory membrane

PRP and PR

Orthopedic Spine Applications

bull SI joint instability syndromes

bull Accessory ligaments of SI joint

bull Lumbar facet joints

bull LD fascia

bull IL ligament

bull Platelet releaseate epidural

bull Intradiscal PRP with or without fibrin sealant

bull Never had a Toti-Potent or Pleuri-Potent stem

cell population to use until nowhellip

Adipose derived MSCs Carrioacuten and Figueroa Stem Cell Res Ther 2011 May 112(3)23

bull Promotion of angiogenesis vascular endothelial

growth factor (VEGF) insulin like growth factor 1

(IGF-1) monocyte chemoatractant protein 1

(MCP1) basic fibroblast growth factor (bFGF) and

interleukin 6 (IL6)

bull 2) Stem cell growth and differentiation stem cell

factor (SCF) leukemiainhibitory factor (LIF)

macrophage colonystimulating factor (MCSF)

stromal derived factor 1 (SDF1) angiopoietin1 and

activin A

bull 3) Inhibition of fibrosis hepatocyte growth factor

(HGF) bFGF adrenomedullin (ADM)

Adipose derived MSCs Carrioacuten and Figueroa Stem Cell Res Ther 2011 May 112(3)23

bull 4) Inhibition of apoptosis VEGF HGF IGF1 transforming growth

factor (TGF)p bFGF granulocyte macrophage colonystimulating

factor (GMCSF) activin A and thrombospondin 1 Immune mediated

effects include the following (5 to 8)

bull 5) Suppression of T and B cells human leukocyte antigen G5

(HLAG5) HGF inducible nitric oxide synthase (iNOS)

indoleamine23dioxygenase (IDO) prostaglandin E2 (PGE2) bFGF

and TGFp

bull 6) Induction of regulatory T cells (Treg) differentiation and expansion

by TGFp expression

bull 7) Inhibition of natural killer (NK) cells by secretion of IDO PGE2

and TGFp

bull 8) Inhibition of dendritic cell (DC) maturation by secretion of PGE2

Adipose derived MSCs Carrioacuten and Figueroa Stem Cell Res Ther 2011 May 112(3)23

Adipose has more MSC CFUs

bull The number of CFU-F calculated at the

bull basis of 1 106 initially plated cells was

highest for AT (557 + 673) followed by BM

(83 + 61)

Comparative analysis of mesenchymal stem cells from bone marrow umbilical

cord blood or adipose tissue Kern S1 Eichler H Stoeve J Kluumlter H Bieback K

Stem Cells 2006 May24(5)1294-301 Epub 2006 Jan 12

Cell markers show similar phenotype of

AT and BM MSCs

Table 2 Comparison of the expression of surface proteins of

mesenchymal stem cells derived from BM Umbilical Cord Blood and AT as

analyzed by flow cytometry

Antibody BM () (n 9) UCB () (n 10) AT () (n 9)

CD44 975 + 51 997 +05 998 +02

CD73 900 +200 993 + 13 996 + 05

CD90 991 + 25 978 + 71 999 + 02

CD14 12 + 1 1 08 + 09 24 + 50

CD34 16 + 14 12 + 15 50 + 51

CD45 52 + 37 38 + 36 38 + 51

CD105a 881 + 74 724 + 200 904 + 59

CD133 13 + 11 29 + 55 29 + 35

CD29 990 + 15 998 + 04 995 + 13

HLA I 952 + 60 943 + 68 988 + 28

CD106b 663 + 227 700 + 236 303 + 186

HLA II 42 + 61 08 + 12 44 + 62

CD144 45 93 24 40 24 25

The table shows mean values of the percentage of positive cells standard deviation to the total number of cells analyzed

Proinflammatory licensing of

MSCs

bull MSCs have been dubbed as smart immune modulators since their

suppressive effects require a previous licensing step that occurs in the

presence of an inflammatory environment and is mediated by the secretion

of specific cytokines (Jones et al 2007 Jorgensen 2010) Thus IFN-γ

alone or together with tumor necrosis factor (TNF)-α IL-1α or IL-1β are

required to trigger the expression by MSCs of high levels of soluble factors

involved in immunosuppression such as IDO HGF TGF-β and NO

(Aggarwal and Pittenger 2005 Ryan et al 2007 Krampera et al 2006

Ren et al 2008)

bull The need for this activation step has been confirmed in a model of GVHD

since recipients of IFNγ-- T cells did not respond to MSC treatment

evolving into fatal GVHD (Polchert et al 2008)

bull Others have attributed the immunomodulatory function of MSCs mainly to

IL-6-dependent secretion of prostaglandin E2 (PGE2) (Boufii et al 2010)

Case examples 1

bull JM 72 yo CF riding bike to work in Seattle WA Struck

by car

ndash Significant right hip pain unresponsive to conservative care

ndash MRI ndash early degenerative changes and acetabular labrum tear

ndash SP arthroscopic debreedment of labrum tear without improvement and

worsening hip pain

ndash CO of chronic low back pain at LS spine and right SI joint region

ndash Numerous medical subspecialty consultations

ndash University of Washington pain medicine Department consultations

pharmaceutical pain management bull Nonresponsive to epidural injections

ndash Ongoing physical therapy

ndash Patient ambulating with a cane-walking stick

ndash Patient is a store owner having to reduce hours and hire more staff to handle

responsibilities

ndash 4 years in chronic pain without improvement

ndash Anxious lawyers wanting to settle or PI case

Case examples 1

bull JM 72 yo CF riding bike to work in Seattle WA Struck

by car

ndash X-rays of lumbar spine = age-related degenerative changes

ndash MRI lumbar spine = age-related degenerative changes

ndash Patient now seen for consultation after 4 years post MVA in

chronic pain

ndash CO

bull Severe low back pain across the lumbosacral spine

bull Severe SIJ pain

bull Severe hip pain

bull Pain radiation into the anterolateral thigh buttock and posterior thigh

bull Significant sensation of muscle tension in the TFL

ndash Complete relief of hip pain and TFL and anterior thigh pain with

intra-articular local anesthetic injection under ultrasound

guidance

Case examples 1

bull JM 72 yo CF riding bike to work in Seattle WA Struck

by car

ndash Symptomatic relief of back pain from lumbar facet

injectionsmedial branch block

ndash Symptomatic relief from SIJ pain with SIJ block

bull Prolotherapy

3 modest improvements of back pain and

recurrent SIJ pain

bull PRP injection directed to acetabular labrum intra-

articular hip lumbar facet and SIJ with modest

improvement in recurrent symptoms

bull Repeated PRP injectionrsquos resulted in same modest

improvements and recurrent symptoms

Case examples 1 bull JM 72 yo CF riding bike to work in Seattle WA Struck

by car

ndash Adipose tissue SVFdirected to intra-articular hip and acetabular

labrum resolved all hip pain complaints

bull Patient no longer having to ambulate with cane

ndash Despite hip improvements persistent low back pain continued to

create functional and work disability issues

bull NucP-2 stem cell

ndash Lumbar facet injections and right sacroiliac joint injection

ndash IV infusion

ndash 3 days later patient attended market walking the entire market for

business purchases in New York without pain

ndash Patient now reporting complete resolution of chronic low back

pain and SIJ pain

Case example 2 bull 68-year-old CF with tricompartmental

osteoarthritis

ndash Progressive worsening knee pain and disability

ndash Now reporting poor walking tolerance reduced

recreational activity tolerance

ndash Modest response to platelet releasate injection

ndash Status post adipose tissue SVF injection with no

significant improvement after 6 months

ndash Patient now facing consideration of TKA

Case example 2

bull 68-year-old CF with tricompartmental

osteoarthritis

ndash Patient consulted to explore nonoperative

options

ndash NucP-2 stem cell

bull Marked improvement

bull Improved functional activity tolerance

bull Return to some recreational activities

ndash More questions and answers

bull Will this have to be repeated

bull How often

bull Post MRI one year pending WORMS BLOCKS

CaLshellip T3 magnet vs T15 magnet etc

Case example 3

bull 69-year-old CF with long-standing history of

CIDP with severe neuropathic pain especially

right lower extremity

ndash Patient met diagnostic criteria

ndash Patient had an progressive worsening of pain and

neurologic function

ndash Multiple medical consultations

ndash Corticosteroids Imuran IVIG gabapentin

nortriptyline etc

ndash Patient not wanting to have chemotherapy ie

CellCept Cytoxan cyclosporine

Case example 3

bull 69-year-old CF with long-standing history of

CIDP with severe neuropathic pain especially

right lower extremity

ndash Patient seen 4 years prior to participate in pilot study

ndash Isolation of TPSCs deployed via intranasal route

overlying cribriform plate

ndash Isolation of PPSCs deployed via IV infusion

ndash Single administration of intranasal and IV deployment

resulted in remission of neuropathic pain improved

gait and functional activity performance

14 months

ndash Patient then started to regress and requesting tx

Case of Parkinsonrsquos Disease

bull 66 YO CM with PD ndash Movement disorder

ndash Speech dysarthria

ndash Social isolation

ndash On Sinemet (Carbidopa-Levodopa)and Stalevo which is becoming less

effective

bull Pt had totipotent adult autologous stem cell

intranasal infusion and IV pluripotent stem cell

infusion

bull Outcome 15 month ndash patient on frac12 dose of medication

ndash Better movement control

ndash Reduced dysarthria

ndash No more social isolation and more confident

Local anesthetics

bull Marcaine Liocaine Prilocaine

Carbocaine Procaine ndash ALL kills stem

cells

bull Naropin ( Ropivicaine )

ndash Only anesthetic that is non-toxic to stem cells

ndash 100 stem cell viability

bull Suspension of cells in saline in pain

sensitive patients also helps

Use of platelet releaseateshellip

PRP growth factor concentrations

differ by system System Platelets

(x103μL)

WBCs

(x103μL)

PDGF-AB

(ngmL)

PDGF-BB

(ngmL)

TGF-B1

(ngmL)

VEGF

(ngmL)

Cascade

MTF

4438

247

11

02 97

36 148

25 01

008

03

03

GPS III

Biomet

5562

2926

344

136 187

128

231

101

01

008

24

11

Magellan

Arteriocyte

7802

2465

110

82 344

107

330

82 02

01

12

08

P values lt00001 0006 0006 0009 0371 0005

Study objective to investigate effects of leukocytes on PRP

Create

leukocyterich

PRP

(LR-PRP)

Create

Leukocyte poor

PRP

(LP-PRP)

Inject into

healthy

patellar

tendon of New

Zealand White

rabbits

5d amp 14d

Harvest

patellar

tendons and

analyze

histology

Results- Inflammatory Mediators

bull LR-PRP causes significant increases in

pro-inflammatory mediators

bull LP-PRP causes significant increase in anti-inflammatory mediators

IL-1β IL-6 IFN-γ TNF-α IL-4 IL-10

PPP + +

LP-PRP + + +

LR-PRP + + + + +

RBC +

Use of platelet releaseateshellip

bull Activated platelet

ndash Alpha granules

bull PD growth factors

TGF-β

Platelet derived growth factor (IGF)

vascular endothelial growth factors

(VEGF) Vasc Endo Growth Factor

Epidermal Growth Factor (EGF)

Fibroblastic Growth Factor -2 (FGF-2)

This is PRP

PDEGF

PDGF-AB bFGF

VEGF PDFG-AA IGF-3

FGF-1 IGFBP-3

BMP-4

IGFBP-3

TGF-B1

hGH

PDGF-BB

bFGF BMP-2

PDGF-AB

IGF-1

IGFBP-2

EGF

HGF

ECGF

PDEGF

BMP

Pro-

Chondrogenic

TGFB-1

IGF-1

bFGF

BMP-2

Anti-

Chondrogenic

VEGF

IGFBP-2

IGFBP-3

PDGF-AA

PDGF-AB

PDGF-BB

EGF

1st Generation PRP

Pro-

Chondrogenic

TGFB-1

IGF-1

bFGF

BMP-2

Anti-

Chondrogenic

VEGF

IGFBP-2

IGFBP-3

PDGF-AA

PDGF-AB

PDGF-BB

EGF

2nd Generation PRP

ADSC Induction Media

bull IGF-I

bull TGF-β1

bull GH

bull FGF-2

Platelet Rich Plasma

bull IGF-1

bull TGF-β1

bull GH

bull FGF-2

bull PDGF

bull VEGF

bull IGF-BP2

bull IGF-BP3

2nd Generation PRP

Create and

activate

PRP

Neutralize specific

PRP growth factors

with antibodies

bull VEGF

bull IGFBP-2

bull IGFBP-3

bull EGF

bull PDGF

Confirm

absence of

neutralized

factors and

presence of key

growth factors

by ELISA

2nd Generation PRP

Sterile inflammation

caused by DAMPs and

mediated by IL-1β or

TNF in the absence of

infection involves

canonical NFκB

activation and

proinflammatory

signalling In OA

molecules from

cartilage breakdown

and intracellular

components ohellip

We will manipulate growth factor

content for target tissue or disease

Signalling proteins

(including growth

factors

chemokines and

other cytokines)

adhesive proteins

and proteases

together with small

molecules and ions

that are secreted by

platelets are found

at high levels in

PRP and interact

with mhellip

mPRP Task Myostatin and TGF-β1 removal

using antibodies bound

to protein AG agarose in sterile conditions

Protein A beads Incubate

1 hour RT Add Ab-s

PBS buffer

Protein AG

beads

TGFβ1 antibodies myostatin antibodies

Protein G

beads

Disc disease and pathological

changes in the neuroforamen

bull Disc leak

ndash PLA2

ndash Immune mediated responses

ndash Inflammation

bull Ischemia

bull Demeylination

bull Inflamatory membrane

PRP and PR

Orthopedic Spine Applications

bull SI joint instability syndromes

bull Accessory ligaments of SI joint

bull Lumbar facet joints

bull LD fascia

bull IL ligament

bull Platelet releaseate epidural

bull Intradiscal PRP with or without fibrin sealant

bull Never had a Toti-Potent or Pleuri-Potent stem

cell population to use until nowhellip

Adipose derived MSCs Carrioacuten and Figueroa Stem Cell Res Ther 2011 May 112(3)23

bull Promotion of angiogenesis vascular endothelial

growth factor (VEGF) insulin like growth factor 1

(IGF-1) monocyte chemoatractant protein 1

(MCP1) basic fibroblast growth factor (bFGF) and

interleukin 6 (IL6)

bull 2) Stem cell growth and differentiation stem cell

factor (SCF) leukemiainhibitory factor (LIF)

macrophage colonystimulating factor (MCSF)

stromal derived factor 1 (SDF1) angiopoietin1 and

activin A

bull 3) Inhibition of fibrosis hepatocyte growth factor

(HGF) bFGF adrenomedullin (ADM)

Adipose derived MSCs Carrioacuten and Figueroa Stem Cell Res Ther 2011 May 112(3)23

bull 4) Inhibition of apoptosis VEGF HGF IGF1 transforming growth

factor (TGF)p bFGF granulocyte macrophage colonystimulating

factor (GMCSF) activin A and thrombospondin 1 Immune mediated

effects include the following (5 to 8)

bull 5) Suppression of T and B cells human leukocyte antigen G5

(HLAG5) HGF inducible nitric oxide synthase (iNOS)

indoleamine23dioxygenase (IDO) prostaglandin E2 (PGE2) bFGF

and TGFp

bull 6) Induction of regulatory T cells (Treg) differentiation and expansion

by TGFp expression

bull 7) Inhibition of natural killer (NK) cells by secretion of IDO PGE2

and TGFp

bull 8) Inhibition of dendritic cell (DC) maturation by secretion of PGE2

Adipose derived MSCs Carrioacuten and Figueroa Stem Cell Res Ther 2011 May 112(3)23

Adipose has more MSC CFUs

bull The number of CFU-F calculated at the

bull basis of 1 106 initially plated cells was

highest for AT (557 + 673) followed by BM

(83 + 61)

Comparative analysis of mesenchymal stem cells from bone marrow umbilical

cord blood or adipose tissue Kern S1 Eichler H Stoeve J Kluumlter H Bieback K

Stem Cells 2006 May24(5)1294-301 Epub 2006 Jan 12

Cell markers show similar phenotype of

AT and BM MSCs

Table 2 Comparison of the expression of surface proteins of

mesenchymal stem cells derived from BM Umbilical Cord Blood and AT as

analyzed by flow cytometry

Antibody BM () (n 9) UCB () (n 10) AT () (n 9)

CD44 975 + 51 997 +05 998 +02

CD73 900 +200 993 + 13 996 + 05

CD90 991 + 25 978 + 71 999 + 02

CD14 12 + 1 1 08 + 09 24 + 50

CD34 16 + 14 12 + 15 50 + 51

CD45 52 + 37 38 + 36 38 + 51

CD105a 881 + 74 724 + 200 904 + 59

CD133 13 + 11 29 + 55 29 + 35

CD29 990 + 15 998 + 04 995 + 13

HLA I 952 + 60 943 + 68 988 + 28

CD106b 663 + 227 700 + 236 303 + 186

HLA II 42 + 61 08 + 12 44 + 62

CD144 45 93 24 40 24 25

The table shows mean values of the percentage of positive cells standard deviation to the total number of cells analyzed

Proinflammatory licensing of

MSCs

bull MSCs have been dubbed as smart immune modulators since their

suppressive effects require a previous licensing step that occurs in the

presence of an inflammatory environment and is mediated by the secretion

of specific cytokines (Jones et al 2007 Jorgensen 2010) Thus IFN-γ

alone or together with tumor necrosis factor (TNF)-α IL-1α or IL-1β are

required to trigger the expression by MSCs of high levels of soluble factors

involved in immunosuppression such as IDO HGF TGF-β and NO

(Aggarwal and Pittenger 2005 Ryan et al 2007 Krampera et al 2006

Ren et al 2008)

bull The need for this activation step has been confirmed in a model of GVHD

since recipients of IFNγ-- T cells did not respond to MSC treatment

evolving into fatal GVHD (Polchert et al 2008)

bull Others have attributed the immunomodulatory function of MSCs mainly to

IL-6-dependent secretion of prostaglandin E2 (PGE2) (Boufii et al 2010)

Case examples 1 bull JM 72 yo CF riding bike to work in Seattle WA Struck

by car

ndash Adipose tissue SVFdirected to intra-articular hip and acetabular

labrum resolved all hip pain complaints

bull Patient no longer having to ambulate with cane

ndash Despite hip improvements persistent low back pain continued to

create functional and work disability issues

bull NucP-2 stem cell

ndash Lumbar facet injections and right sacroiliac joint injection

ndash IV infusion

ndash 3 days later patient attended market walking the entire market for

business purchases in New York without pain

ndash Patient now reporting complete resolution of chronic low back

pain and SIJ pain

Case example 2 bull 68-year-old CF with tricompartmental

osteoarthritis

ndash Progressive worsening knee pain and disability

ndash Now reporting poor walking tolerance reduced

recreational activity tolerance

ndash Modest response to platelet releasate injection

ndash Status post adipose tissue SVF injection with no

significant improvement after 6 months

ndash Patient now facing consideration of TKA

Case example 2

bull 68-year-old CF with tricompartmental

osteoarthritis

ndash Patient consulted to explore nonoperative

options

ndash NucP-2 stem cell

bull Marked improvement

bull Improved functional activity tolerance

bull Return to some recreational activities

ndash More questions and answers

bull Will this have to be repeated

bull How often

bull Post MRI one year pending WORMS BLOCKS

CaLshellip T3 magnet vs T15 magnet etc

Case example 3

bull 69-year-old CF with long-standing history of

CIDP with severe neuropathic pain especially

right lower extremity

ndash Patient met diagnostic criteria

ndash Patient had an progressive worsening of pain and

neurologic function

ndash Multiple medical consultations

ndash Corticosteroids Imuran IVIG gabapentin

nortriptyline etc

ndash Patient not wanting to have chemotherapy ie

CellCept Cytoxan cyclosporine

Case example 3

bull 69-year-old CF with long-standing history of

CIDP with severe neuropathic pain especially

right lower extremity

ndash Patient seen 4 years prior to participate in pilot study

ndash Isolation of TPSCs deployed via intranasal route

overlying cribriform plate

ndash Isolation of PPSCs deployed via IV infusion

ndash Single administration of intranasal and IV deployment

resulted in remission of neuropathic pain improved

gait and functional activity performance

14 months

ndash Patient then started to regress and requesting tx

Case of Parkinsonrsquos Disease

bull 66 YO CM with PD ndash Movement disorder

ndash Speech dysarthria

ndash Social isolation

ndash On Sinemet (Carbidopa-Levodopa)and Stalevo which is becoming less

effective

bull Pt had totipotent adult autologous stem cell

intranasal infusion and IV pluripotent stem cell

infusion

bull Outcome 15 month ndash patient on frac12 dose of medication

ndash Better movement control

ndash Reduced dysarthria

ndash No more social isolation and more confident

Local anesthetics

bull Marcaine Liocaine Prilocaine

Carbocaine Procaine ndash ALL kills stem

cells

bull Naropin ( Ropivicaine )

ndash Only anesthetic that is non-toxic to stem cells

ndash 100 stem cell viability

bull Suspension of cells in saline in pain

sensitive patients also helps

Use of platelet releaseateshellip

PRP growth factor concentrations

differ by system System Platelets

(x103μL)

WBCs

(x103μL)

PDGF-AB

(ngmL)

PDGF-BB

(ngmL)

TGF-B1

(ngmL)

VEGF

(ngmL)

Cascade

MTF

4438

247

11

02 97

36 148

25 01

008

03

03

GPS III

Biomet

5562

2926

344

136 187

128

231

101

01

008

24

11

Magellan

Arteriocyte

7802

2465

110

82 344

107

330

82 02

01

12

08

P values lt00001 0006 0006 0009 0371 0005

Study objective to investigate effects of leukocytes on PRP

Create

leukocyterich

PRP

(LR-PRP)

Create

Leukocyte poor

PRP

(LP-PRP)

Inject into

healthy

patellar

tendon of New

Zealand White

rabbits

5d amp 14d

Harvest

patellar

tendons and

analyze

histology

Results- Inflammatory Mediators

bull LR-PRP causes significant increases in

pro-inflammatory mediators

bull LP-PRP causes significant increase in anti-inflammatory mediators

IL-1β IL-6 IFN-γ TNF-α IL-4 IL-10

PPP + +

LP-PRP + + +

LR-PRP + + + + +

RBC +

Use of platelet releaseateshellip

bull Activated platelet

ndash Alpha granules

bull PD growth factors

TGF-β

Platelet derived growth factor (IGF)

vascular endothelial growth factors

(VEGF) Vasc Endo Growth Factor

Epidermal Growth Factor (EGF)

Fibroblastic Growth Factor -2 (FGF-2)

This is PRP

PDEGF

PDGF-AB bFGF

VEGF PDFG-AA IGF-3

FGF-1 IGFBP-3

BMP-4

IGFBP-3

TGF-B1

hGH

PDGF-BB

bFGF BMP-2

PDGF-AB

IGF-1

IGFBP-2

EGF

HGF

ECGF

PDEGF

BMP

Pro-

Chondrogenic

TGFB-1

IGF-1

bFGF

BMP-2

Anti-

Chondrogenic

VEGF

IGFBP-2

IGFBP-3

PDGF-AA

PDGF-AB

PDGF-BB

EGF

1st Generation PRP

Pro-

Chondrogenic

TGFB-1

IGF-1

bFGF

BMP-2

Anti-

Chondrogenic

VEGF

IGFBP-2

IGFBP-3

PDGF-AA

PDGF-AB

PDGF-BB

EGF

2nd Generation PRP

ADSC Induction Media

bull IGF-I

bull TGF-β1

bull GH

bull FGF-2

Platelet Rich Plasma

bull IGF-1

bull TGF-β1

bull GH

bull FGF-2

bull PDGF

bull VEGF

bull IGF-BP2

bull IGF-BP3

2nd Generation PRP

Create and

activate

PRP

Neutralize specific

PRP growth factors

with antibodies

bull VEGF

bull IGFBP-2

bull IGFBP-3

bull EGF

bull PDGF

Confirm

absence of

neutralized

factors and

presence of key

growth factors

by ELISA

2nd Generation PRP

Sterile inflammation

caused by DAMPs and

mediated by IL-1β or

TNF in the absence of

infection involves

canonical NFκB

activation and

proinflammatory

signalling In OA

molecules from

cartilage breakdown

and intracellular

components ohellip

We will manipulate growth factor

content for target tissue or disease

Signalling proteins

(including growth

factors

chemokines and

other cytokines)

adhesive proteins

and proteases

together with small

molecules and ions

that are secreted by

platelets are found

at high levels in

PRP and interact

with mhellip

mPRP Task Myostatin and TGF-β1 removal

using antibodies bound

to protein AG agarose in sterile conditions

Protein A beads Incubate

1 hour RT Add Ab-s

PBS buffer

Protein AG

beads

TGFβ1 antibodies myostatin antibodies

Protein G

beads

Disc disease and pathological

changes in the neuroforamen

bull Disc leak

ndash PLA2

ndash Immune mediated responses

ndash Inflammation

bull Ischemia

bull Demeylination

bull Inflamatory membrane

PRP and PR

Orthopedic Spine Applications

bull SI joint instability syndromes

bull Accessory ligaments of SI joint

bull Lumbar facet joints

bull LD fascia

bull IL ligament

bull Platelet releaseate epidural

bull Intradiscal PRP with or without fibrin sealant

bull Never had a Toti-Potent or Pleuri-Potent stem

cell population to use until nowhellip

Adipose derived MSCs Carrioacuten and Figueroa Stem Cell Res Ther 2011 May 112(3)23

bull Promotion of angiogenesis vascular endothelial

growth factor (VEGF) insulin like growth factor 1

(IGF-1) monocyte chemoatractant protein 1

(MCP1) basic fibroblast growth factor (bFGF) and

interleukin 6 (IL6)

bull 2) Stem cell growth and differentiation stem cell

factor (SCF) leukemiainhibitory factor (LIF)

macrophage colonystimulating factor (MCSF)

stromal derived factor 1 (SDF1) angiopoietin1 and

activin A

bull 3) Inhibition of fibrosis hepatocyte growth factor

(HGF) bFGF adrenomedullin (ADM)

Adipose derived MSCs Carrioacuten and Figueroa Stem Cell Res Ther 2011 May 112(3)23

bull 4) Inhibition of apoptosis VEGF HGF IGF1 transforming growth

factor (TGF)p bFGF granulocyte macrophage colonystimulating

factor (GMCSF) activin A and thrombospondin 1 Immune mediated

effects include the following (5 to 8)

bull 5) Suppression of T and B cells human leukocyte antigen G5

(HLAG5) HGF inducible nitric oxide synthase (iNOS)

indoleamine23dioxygenase (IDO) prostaglandin E2 (PGE2) bFGF

and TGFp

bull 6) Induction of regulatory T cells (Treg) differentiation and expansion

by TGFp expression

bull 7) Inhibition of natural killer (NK) cells by secretion of IDO PGE2

and TGFp

bull 8) Inhibition of dendritic cell (DC) maturation by secretion of PGE2

Adipose derived MSCs Carrioacuten and Figueroa Stem Cell Res Ther 2011 May 112(3)23

Adipose has more MSC CFUs

bull The number of CFU-F calculated at the

bull basis of 1 106 initially plated cells was

highest for AT (557 + 673) followed by BM

(83 + 61)

Comparative analysis of mesenchymal stem cells from bone marrow umbilical

cord blood or adipose tissue Kern S1 Eichler H Stoeve J Kluumlter H Bieback K

Stem Cells 2006 May24(5)1294-301 Epub 2006 Jan 12

Cell markers show similar phenotype of

AT and BM MSCs

Table 2 Comparison of the expression of surface proteins of

mesenchymal stem cells derived from BM Umbilical Cord Blood and AT as

analyzed by flow cytometry

Antibody BM () (n 9) UCB () (n 10) AT () (n 9)

CD44 975 + 51 997 +05 998 +02

CD73 900 +200 993 + 13 996 + 05

CD90 991 + 25 978 + 71 999 + 02

CD14 12 + 1 1 08 + 09 24 + 50

CD34 16 + 14 12 + 15 50 + 51

CD45 52 + 37 38 + 36 38 + 51

CD105a 881 + 74 724 + 200 904 + 59

CD133 13 + 11 29 + 55 29 + 35

CD29 990 + 15 998 + 04 995 + 13

HLA I 952 + 60 943 + 68 988 + 28

CD106b 663 + 227 700 + 236 303 + 186

HLA II 42 + 61 08 + 12 44 + 62

CD144 45 93 24 40 24 25

The table shows mean values of the percentage of positive cells standard deviation to the total number of cells analyzed

Proinflammatory licensing of

MSCs

bull MSCs have been dubbed as smart immune modulators since their

suppressive effects require a previous licensing step that occurs in the

presence of an inflammatory environment and is mediated by the secretion

of specific cytokines (Jones et al 2007 Jorgensen 2010) Thus IFN-γ

alone or together with tumor necrosis factor (TNF)-α IL-1α or IL-1β are

required to trigger the expression by MSCs of high levels of soluble factors

involved in immunosuppression such as IDO HGF TGF-β and NO

(Aggarwal and Pittenger 2005 Ryan et al 2007 Krampera et al 2006

Ren et al 2008)

bull The need for this activation step has been confirmed in a model of GVHD

since recipients of IFNγ-- T cells did not respond to MSC treatment

evolving into fatal GVHD (Polchert et al 2008)

bull Others have attributed the immunomodulatory function of MSCs mainly to

IL-6-dependent secretion of prostaglandin E2 (PGE2) (Boufii et al 2010)

Case example 3

bull 69-year-old CF with long-standing history of

CIDP with severe neuropathic pain especially

right lower extremity

ndash Patient met diagnostic criteria

ndash Patient had an progressive worsening of pain and

neurologic function

ndash Multiple medical consultations

ndash Corticosteroids Imuran IVIG gabapentin

nortriptyline etc

ndash Patient not wanting to have chemotherapy ie

CellCept Cytoxan cyclosporine

Case example 3

bull 69-year-old CF with long-standing history of

CIDP with severe neuropathic pain especially

right lower extremity

ndash Patient seen 4 years prior to participate in pilot study

ndash Isolation of TPSCs deployed via intranasal route

overlying cribriform plate

ndash Isolation of PPSCs deployed via IV infusion

ndash Single administration of intranasal and IV deployment

resulted in remission of neuropathic pain improved

gait and functional activity performance

14 months

ndash Patient then started to regress and requesting tx

Case of Parkinsonrsquos Disease

bull 66 YO CM with PD ndash Movement disorder

ndash Speech dysarthria

ndash Social isolation

ndash On Sinemet (Carbidopa-Levodopa)and Stalevo which is becoming less

effective

bull Pt had totipotent adult autologous stem cell

intranasal infusion and IV pluripotent stem cell

infusion

bull Outcome 15 month ndash patient on frac12 dose of medication

ndash Better movement control

ndash Reduced dysarthria

ndash No more social isolation and more confident

Local anesthetics

bull Marcaine Liocaine Prilocaine

Carbocaine Procaine ndash ALL kills stem

cells

bull Naropin ( Ropivicaine )

ndash Only anesthetic that is non-toxic to stem cells

ndash 100 stem cell viability

bull Suspension of cells in saline in pain

sensitive patients also helps

Use of platelet releaseateshellip

PRP growth factor concentrations

differ by system System Platelets

(x103μL)

WBCs

(x103μL)

PDGF-AB

(ngmL)

PDGF-BB

(ngmL)

TGF-B1

(ngmL)

VEGF

(ngmL)

Cascade

MTF

4438

247

11

02 97

36 148

25 01

008

03

03

GPS III

Biomet

5562

2926

344

136 187

128

231

101

01

008

24

11

Magellan

Arteriocyte

7802

2465

110

82 344

107

330

82 02

01

12

08

P values lt00001 0006 0006 0009 0371 0005

Study objective to investigate effects of leukocytes on PRP

Create

leukocyterich

PRP

(LR-PRP)

Create

Leukocyte poor

PRP

(LP-PRP)

Inject into

healthy

patellar

tendon of New

Zealand White

rabbits

5d amp 14d

Harvest

patellar

tendons and

analyze

histology

Results- Inflammatory Mediators

bull LR-PRP causes significant increases in

pro-inflammatory mediators

bull LP-PRP causes significant increase in anti-inflammatory mediators

IL-1β IL-6 IFN-γ TNF-α IL-4 IL-10

PPP + +

LP-PRP + + +

LR-PRP + + + + +

RBC +

Use of platelet releaseateshellip

bull Activated platelet

ndash Alpha granules

bull PD growth factors

TGF-β

Platelet derived growth factor (IGF)

vascular endothelial growth factors

(VEGF) Vasc Endo Growth Factor

Epidermal Growth Factor (EGF)

Fibroblastic Growth Factor -2 (FGF-2)

This is PRP

PDEGF

PDGF-AB bFGF

VEGF PDFG-AA IGF-3

FGF-1 IGFBP-3

BMP-4

IGFBP-3

TGF-B1

hGH

PDGF-BB

bFGF BMP-2

PDGF-AB

IGF-1

IGFBP-2

EGF

HGF

ECGF

PDEGF

BMP

Pro-

Chondrogenic

TGFB-1

IGF-1

bFGF

BMP-2

Anti-

Chondrogenic

VEGF

IGFBP-2

IGFBP-3

PDGF-AA

PDGF-AB

PDGF-BB

EGF

1st Generation PRP

Pro-

Chondrogenic

TGFB-1

IGF-1

bFGF

BMP-2

Anti-

Chondrogenic

VEGF

IGFBP-2

IGFBP-3

PDGF-AA

PDGF-AB

PDGF-BB

EGF

2nd Generation PRP

ADSC Induction Media

bull IGF-I

bull TGF-β1

bull GH

bull FGF-2

Platelet Rich Plasma

bull IGF-1

bull TGF-β1

bull GH

bull FGF-2

bull PDGF

bull VEGF

bull IGF-BP2

bull IGF-BP3

2nd Generation PRP

Create and

activate

PRP

Neutralize specific

PRP growth factors

with antibodies

bull VEGF

bull IGFBP-2

bull IGFBP-3

bull EGF

bull PDGF

Confirm

absence of

neutralized

factors and

presence of key

growth factors

by ELISA

2nd Generation PRP

Sterile inflammation

caused by DAMPs and

mediated by IL-1β or

TNF in the absence of

infection involves

canonical NFκB

activation and

proinflammatory

signalling In OA

molecules from

cartilage breakdown

and intracellular

components ohellip

We will manipulate growth factor

content for target tissue or disease

Signalling proteins

(including growth

factors

chemokines and

other cytokines)

adhesive proteins

and proteases

together with small

molecules and ions

that are secreted by

platelets are found

at high levels in

PRP and interact

with mhellip

mPRP Task Myostatin and TGF-β1 removal

using antibodies bound

to protein AG agarose in sterile conditions

Protein A beads Incubate

1 hour RT Add Ab-s

PBS buffer

Protein AG

beads

TGFβ1 antibodies myostatin antibodies

Protein G

beads

Disc disease and pathological

changes in the neuroforamen

bull Disc leak

ndash PLA2

ndash Immune mediated responses

ndash Inflammation

bull Ischemia

bull Demeylination

bull Inflamatory membrane

PRP and PR

Orthopedic Spine Applications

bull SI joint instability syndromes

bull Accessory ligaments of SI joint

bull Lumbar facet joints

bull LD fascia

bull IL ligament

bull Platelet releaseate epidural

bull Intradiscal PRP with or without fibrin sealant

bull Never had a Toti-Potent or Pleuri-Potent stem

cell population to use until nowhellip

Adipose derived MSCs Carrioacuten and Figueroa Stem Cell Res Ther 2011 May 112(3)23

bull Promotion of angiogenesis vascular endothelial

growth factor (VEGF) insulin like growth factor 1

(IGF-1) monocyte chemoatractant protein 1

(MCP1) basic fibroblast growth factor (bFGF) and

interleukin 6 (IL6)

bull 2) Stem cell growth and differentiation stem cell

factor (SCF) leukemiainhibitory factor (LIF)

macrophage colonystimulating factor (MCSF)

stromal derived factor 1 (SDF1) angiopoietin1 and

activin A

bull 3) Inhibition of fibrosis hepatocyte growth factor

(HGF) bFGF adrenomedullin (ADM)

Adipose derived MSCs Carrioacuten and Figueroa Stem Cell Res Ther 2011 May 112(3)23

bull 4) Inhibition of apoptosis VEGF HGF IGF1 transforming growth

factor (TGF)p bFGF granulocyte macrophage colonystimulating

factor (GMCSF) activin A and thrombospondin 1 Immune mediated

effects include the following (5 to 8)

bull 5) Suppression of T and B cells human leukocyte antigen G5

(HLAG5) HGF inducible nitric oxide synthase (iNOS)

indoleamine23dioxygenase (IDO) prostaglandin E2 (PGE2) bFGF

and TGFp

bull 6) Induction of regulatory T cells (Treg) differentiation and expansion

by TGFp expression

bull 7) Inhibition of natural killer (NK) cells by secretion of IDO PGE2

and TGFp

bull 8) Inhibition of dendritic cell (DC) maturation by secretion of PGE2

Adipose derived MSCs Carrioacuten and Figueroa Stem Cell Res Ther 2011 May 112(3)23

Adipose has more MSC CFUs

bull The number of CFU-F calculated at the

bull basis of 1 106 initially plated cells was

highest for AT (557 + 673) followed by BM

(83 + 61)

Comparative analysis of mesenchymal stem cells from bone marrow umbilical

cord blood or adipose tissue Kern S1 Eichler H Stoeve J Kluumlter H Bieback K

Stem Cells 2006 May24(5)1294-301 Epub 2006 Jan 12

Cell markers show similar phenotype of

AT and BM MSCs

Table 2 Comparison of the expression of surface proteins of

mesenchymal stem cells derived from BM Umbilical Cord Blood and AT as

analyzed by flow cytometry

Antibody BM () (n 9) UCB () (n 10) AT () (n 9)

CD44 975 + 51 997 +05 998 +02

CD73 900 +200 993 + 13 996 + 05

CD90 991 + 25 978 + 71 999 + 02

CD14 12 + 1 1 08 + 09 24 + 50

CD34 16 + 14 12 + 15 50 + 51

CD45 52 + 37 38 + 36 38 + 51

CD105a 881 + 74 724 + 200 904 + 59

CD133 13 + 11 29 + 55 29 + 35

CD29 990 + 15 998 + 04 995 + 13

HLA I 952 + 60 943 + 68 988 + 28

CD106b 663 + 227 700 + 236 303 + 186

HLA II 42 + 61 08 + 12 44 + 62

CD144 45 93 24 40 24 25

The table shows mean values of the percentage of positive cells standard deviation to the total number of cells analyzed

Proinflammatory licensing of

MSCs

bull MSCs have been dubbed as smart immune modulators since their

suppressive effects require a previous licensing step that occurs in the

presence of an inflammatory environment and is mediated by the secretion

of specific cytokines (Jones et al 2007 Jorgensen 2010) Thus IFN-γ

alone or together with tumor necrosis factor (TNF)-α IL-1α or IL-1β are

required to trigger the expression by MSCs of high levels of soluble factors

involved in immunosuppression such as IDO HGF TGF-β and NO

(Aggarwal and Pittenger 2005 Ryan et al 2007 Krampera et al 2006

Ren et al 2008)

bull The need for this activation step has been confirmed in a model of GVHD

since recipients of IFNγ-- T cells did not respond to MSC treatment

evolving into fatal GVHD (Polchert et al 2008)

bull Others have attributed the immunomodulatory function of MSCs mainly to

IL-6-dependent secretion of prostaglandin E2 (PGE2) (Boufii et al 2010)

Local anesthetics

bull Marcaine Liocaine Prilocaine

Carbocaine Procaine ndash ALL kills stem

cells

bull Naropin ( Ropivicaine )

ndash Only anesthetic that is non-toxic to stem cells

ndash 100 stem cell viability

bull Suspension of cells in saline in pain

sensitive patients also helps

Use of platelet releaseateshellip

PRP growth factor concentrations

differ by system System Platelets

(x103μL)

WBCs

(x103μL)

PDGF-AB

(ngmL)

PDGF-BB

(ngmL)

TGF-B1

(ngmL)

VEGF

(ngmL)

Cascade

MTF

4438

247

11

02 97

36 148

25 01

008

03

03

GPS III

Biomet

5562

2926

344

136 187

128

231

101

01

008

24

11

Magellan

Arteriocyte

7802

2465

110

82 344

107

330

82 02

01

12

08

P values lt00001 0006 0006 0009 0371 0005

Study objective to investigate effects of leukocytes on PRP

Create

leukocyterich

PRP

(LR-PRP)

Create

Leukocyte poor

PRP

(LP-PRP)

Inject into

healthy

patellar

tendon of New

Zealand White

rabbits

5d amp 14d

Harvest

patellar

tendons and

analyze

histology

Results- Inflammatory Mediators

bull LR-PRP causes significant increases in

pro-inflammatory mediators

bull LP-PRP causes significant increase in anti-inflammatory mediators

IL-1β IL-6 IFN-γ TNF-α IL-4 IL-10

PPP + +

LP-PRP + + +

LR-PRP + + + + +

RBC +

Use of platelet releaseateshellip

bull Activated platelet

ndash Alpha granules

bull PD growth factors

TGF-β

Platelet derived growth factor (IGF)

vascular endothelial growth factors

(VEGF) Vasc Endo Growth Factor

Epidermal Growth Factor (EGF)

Fibroblastic Growth Factor -2 (FGF-2)

This is PRP

PDEGF

PDGF-AB bFGF

VEGF PDFG-AA IGF-3

FGF-1 IGFBP-3

BMP-4

IGFBP-3

TGF-B1

hGH

PDGF-BB

bFGF BMP-2

PDGF-AB

IGF-1

IGFBP-2

EGF

HGF

ECGF

PDEGF

BMP

Pro-

Chondrogenic

TGFB-1

IGF-1

bFGF

BMP-2

Anti-

Chondrogenic

VEGF

IGFBP-2

IGFBP-3

PDGF-AA

PDGF-AB

PDGF-BB

EGF

1st Generation PRP

Pro-

Chondrogenic

TGFB-1

IGF-1

bFGF

BMP-2

Anti-

Chondrogenic

VEGF

IGFBP-2

IGFBP-3

PDGF-AA

PDGF-AB

PDGF-BB

EGF

2nd Generation PRP

ADSC Induction Media

bull IGF-I

bull TGF-β1

bull GH

bull FGF-2

Platelet Rich Plasma

bull IGF-1

bull TGF-β1

bull GH

bull FGF-2

bull PDGF

bull VEGF

bull IGF-BP2

bull IGF-BP3

2nd Generation PRP

Create and

activate

PRP

Neutralize specific

PRP growth factors

with antibodies

bull VEGF

bull IGFBP-2

bull IGFBP-3

bull EGF

bull PDGF

Confirm

absence of

neutralized

factors and

presence of key

growth factors

by ELISA

2nd Generation PRP

Sterile inflammation

caused by DAMPs and

mediated by IL-1β or

TNF in the absence of

infection involves

canonical NFκB

activation and

proinflammatory

signalling In OA

molecules from

cartilage breakdown

and intracellular

components ohellip

We will manipulate growth factor

content for target tissue or disease

Signalling proteins

(including growth

factors

chemokines and

other cytokines)

adhesive proteins

and proteases

together with small

molecules and ions

that are secreted by

platelets are found

at high levels in

PRP and interact

with mhellip

mPRP Task Myostatin and TGF-β1 removal

using antibodies bound

to protein AG agarose in sterile conditions

Protein A beads Incubate

1 hour RT Add Ab-s

PBS buffer

Protein AG

beads

TGFβ1 antibodies myostatin antibodies

Protein G

beads

Disc disease and pathological

changes in the neuroforamen

bull Disc leak

ndash PLA2

ndash Immune mediated responses

ndash Inflammation

bull Ischemia

bull Demeylination

bull Inflamatory membrane

PRP and PR

Orthopedic Spine Applications

bull SI joint instability syndromes

bull Accessory ligaments of SI joint

bull Lumbar facet joints

bull LD fascia

bull IL ligament

bull Platelet releaseate epidural

bull Intradiscal PRP with or without fibrin sealant

bull Never had a Toti-Potent or Pleuri-Potent stem

cell population to use until nowhellip

Adipose derived MSCs Carrioacuten and Figueroa Stem Cell Res Ther 2011 May 112(3)23

bull Promotion of angiogenesis vascular endothelial

growth factor (VEGF) insulin like growth factor 1

(IGF-1) monocyte chemoatractant protein 1

(MCP1) basic fibroblast growth factor (bFGF) and

interleukin 6 (IL6)

bull 2) Stem cell growth and differentiation stem cell

factor (SCF) leukemiainhibitory factor (LIF)

macrophage colonystimulating factor (MCSF)

stromal derived factor 1 (SDF1) angiopoietin1 and

activin A

bull 3) Inhibition of fibrosis hepatocyte growth factor

(HGF) bFGF adrenomedullin (ADM)

Adipose derived MSCs Carrioacuten and Figueroa Stem Cell Res Ther 2011 May 112(3)23

bull 4) Inhibition of apoptosis VEGF HGF IGF1 transforming growth

factor (TGF)p bFGF granulocyte macrophage colonystimulating

factor (GMCSF) activin A and thrombospondin 1 Immune mediated

effects include the following (5 to 8)

bull 5) Suppression of T and B cells human leukocyte antigen G5

(HLAG5) HGF inducible nitric oxide synthase (iNOS)

indoleamine23dioxygenase (IDO) prostaglandin E2 (PGE2) bFGF

and TGFp

bull 6) Induction of regulatory T cells (Treg) differentiation and expansion

by TGFp expression

bull 7) Inhibition of natural killer (NK) cells by secretion of IDO PGE2

and TGFp

bull 8) Inhibition of dendritic cell (DC) maturation by secretion of PGE2

Adipose derived MSCs Carrioacuten and Figueroa Stem Cell Res Ther 2011 May 112(3)23

Adipose has more MSC CFUs

bull The number of CFU-F calculated at the

bull basis of 1 106 initially plated cells was

highest for AT (557 + 673) followed by BM

(83 + 61)

Comparative analysis of mesenchymal stem cells from bone marrow umbilical

cord blood or adipose tissue Kern S1 Eichler H Stoeve J Kluumlter H Bieback K

Stem Cells 2006 May24(5)1294-301 Epub 2006 Jan 12

Cell markers show similar phenotype of

AT and BM MSCs

Table 2 Comparison of the expression of surface proteins of

mesenchymal stem cells derived from BM Umbilical Cord Blood and AT as

analyzed by flow cytometry

Antibody BM () (n 9) UCB () (n 10) AT () (n 9)

CD44 975 + 51 997 +05 998 +02

CD73 900 +200 993 + 13 996 + 05

CD90 991 + 25 978 + 71 999 + 02

CD14 12 + 1 1 08 + 09 24 + 50

CD34 16 + 14 12 + 15 50 + 51

CD45 52 + 37 38 + 36 38 + 51

CD105a 881 + 74 724 + 200 904 + 59

CD133 13 + 11 29 + 55 29 + 35

CD29 990 + 15 998 + 04 995 + 13

HLA I 952 + 60 943 + 68 988 + 28

CD106b 663 + 227 700 + 236 303 + 186

HLA II 42 + 61 08 + 12 44 + 62

CD144 45 93 24 40 24 25

The table shows mean values of the percentage of positive cells standard deviation to the total number of cells analyzed

Proinflammatory licensing of

MSCs

bull MSCs have been dubbed as smart immune modulators since their

suppressive effects require a previous licensing step that occurs in the

presence of an inflammatory environment and is mediated by the secretion

of specific cytokines (Jones et al 2007 Jorgensen 2010) Thus IFN-γ

alone or together with tumor necrosis factor (TNF)-α IL-1α or IL-1β are

required to trigger the expression by MSCs of high levels of soluble factors

involved in immunosuppression such as IDO HGF TGF-β and NO

(Aggarwal and Pittenger 2005 Ryan et al 2007 Krampera et al 2006

Ren et al 2008)

bull The need for this activation step has been confirmed in a model of GVHD

since recipients of IFNγ-- T cells did not respond to MSC treatment

evolving into fatal GVHD (Polchert et al 2008)

bull Others have attributed the immunomodulatory function of MSCs mainly to

IL-6-dependent secretion of prostaglandin E2 (PGE2) (Boufii et al 2010)

Study objective to investigate effects of leukocytes on PRP

Create

leukocyterich

PRP

(LR-PRP)

Create

Leukocyte poor

PRP

(LP-PRP)

Inject into

healthy

patellar

tendon of New

Zealand White

rabbits

5d amp 14d

Harvest

patellar

tendons and

analyze

histology

Results- Inflammatory Mediators

bull LR-PRP causes significant increases in

pro-inflammatory mediators

bull LP-PRP causes significant increase in anti-inflammatory mediators

IL-1β IL-6 IFN-γ TNF-α IL-4 IL-10

PPP + +

LP-PRP + + +

LR-PRP + + + + +

RBC +

Use of platelet releaseateshellip

bull Activated platelet

ndash Alpha granules

bull PD growth factors

TGF-β

Platelet derived growth factor (IGF)

vascular endothelial growth factors

(VEGF) Vasc Endo Growth Factor

Epidermal Growth Factor (EGF)

Fibroblastic Growth Factor -2 (FGF-2)

This is PRP

PDEGF

PDGF-AB bFGF

VEGF PDFG-AA IGF-3

FGF-1 IGFBP-3

BMP-4

IGFBP-3

TGF-B1

hGH

PDGF-BB

bFGF BMP-2

PDGF-AB

IGF-1

IGFBP-2

EGF

HGF

ECGF

PDEGF

BMP

Pro-

Chondrogenic

TGFB-1

IGF-1

bFGF

BMP-2

Anti-

Chondrogenic

VEGF

IGFBP-2

IGFBP-3

PDGF-AA

PDGF-AB

PDGF-BB

EGF

1st Generation PRP

Pro-

Chondrogenic

TGFB-1

IGF-1

bFGF

BMP-2

Anti-

Chondrogenic

VEGF

IGFBP-2

IGFBP-3

PDGF-AA

PDGF-AB

PDGF-BB

EGF

2nd Generation PRP

ADSC Induction Media

bull IGF-I

bull TGF-β1

bull GH

bull FGF-2

Platelet Rich Plasma

bull IGF-1

bull TGF-β1

bull GH

bull FGF-2

bull PDGF

bull VEGF

bull IGF-BP2

bull IGF-BP3

2nd Generation PRP

Create and

activate

PRP

Neutralize specific

PRP growth factors

with antibodies

bull VEGF

bull IGFBP-2

bull IGFBP-3

bull EGF

bull PDGF

Confirm

absence of

neutralized

factors and

presence of key

growth factors

by ELISA

2nd Generation PRP

Sterile inflammation

caused by DAMPs and

mediated by IL-1β or

TNF in the absence of

infection involves

canonical NFκB

activation and

proinflammatory

signalling In OA

molecules from

cartilage breakdown

and intracellular

components ohellip

We will manipulate growth factor

content for target tissue or disease

Signalling proteins

(including growth

factors

chemokines and

other cytokines)

adhesive proteins

and proteases

together with small

molecules and ions

that are secreted by

platelets are found

at high levels in

PRP and interact

with mhellip

mPRP Task Myostatin and TGF-β1 removal

using antibodies bound

to protein AG agarose in sterile conditions

Protein A beads Incubate

1 hour RT Add Ab-s

PBS buffer

Protein AG

beads

TGFβ1 antibodies myostatin antibodies

Protein G

beads

Disc disease and pathological

changes in the neuroforamen

bull Disc leak

ndash PLA2

ndash Immune mediated responses

ndash Inflammation

bull Ischemia

bull Demeylination

bull Inflamatory membrane

PRP and PR

Orthopedic Spine Applications

bull SI joint instability syndromes

bull Accessory ligaments of SI joint

bull Lumbar facet joints

bull LD fascia

bull IL ligament

bull Platelet releaseate epidural

bull Intradiscal PRP with or without fibrin sealant

bull Never had a Toti-Potent or Pleuri-Potent stem

cell population to use until nowhellip

Adipose derived MSCs Carrioacuten and Figueroa Stem Cell Res Ther 2011 May 112(3)23

bull Promotion of angiogenesis vascular endothelial

growth factor (VEGF) insulin like growth factor 1

(IGF-1) monocyte chemoatractant protein 1

(MCP1) basic fibroblast growth factor (bFGF) and

interleukin 6 (IL6)

bull 2) Stem cell growth and differentiation stem cell

factor (SCF) leukemiainhibitory factor (LIF)

macrophage colonystimulating factor (MCSF)

stromal derived factor 1 (SDF1) angiopoietin1 and

activin A

bull 3) Inhibition of fibrosis hepatocyte growth factor

(HGF) bFGF adrenomedullin (ADM)

Adipose derived MSCs Carrioacuten and Figueroa Stem Cell Res Ther 2011 May 112(3)23

bull 4) Inhibition of apoptosis VEGF HGF IGF1 transforming growth

factor (TGF)p bFGF granulocyte macrophage colonystimulating

factor (GMCSF) activin A and thrombospondin 1 Immune mediated

effects include the following (5 to 8)

bull 5) Suppression of T and B cells human leukocyte antigen G5

(HLAG5) HGF inducible nitric oxide synthase (iNOS)

indoleamine23dioxygenase (IDO) prostaglandin E2 (PGE2) bFGF

and TGFp

bull 6) Induction of regulatory T cells (Treg) differentiation and expansion

by TGFp expression

bull 7) Inhibition of natural killer (NK) cells by secretion of IDO PGE2

and TGFp

bull 8) Inhibition of dendritic cell (DC) maturation by secretion of PGE2

Adipose derived MSCs Carrioacuten and Figueroa Stem Cell Res Ther 2011 May 112(3)23

Adipose has more MSC CFUs

bull The number of CFU-F calculated at the

bull basis of 1 106 initially plated cells was

highest for AT (557 + 673) followed by BM

(83 + 61)

Comparative analysis of mesenchymal stem cells from bone marrow umbilical

cord blood or adipose tissue Kern S1 Eichler H Stoeve J Kluumlter H Bieback K

Stem Cells 2006 May24(5)1294-301 Epub 2006 Jan 12

Cell markers show similar phenotype of

AT and BM MSCs

Table 2 Comparison of the expression of surface proteins of

mesenchymal stem cells derived from BM Umbilical Cord Blood and AT as

analyzed by flow cytometry

Antibody BM () (n 9) UCB () (n 10) AT () (n 9)

CD44 975 + 51 997 +05 998 +02

CD73 900 +200 993 + 13 996 + 05

CD90 991 + 25 978 + 71 999 + 02

CD14 12 + 1 1 08 + 09 24 + 50

CD34 16 + 14 12 + 15 50 + 51

CD45 52 + 37 38 + 36 38 + 51

CD105a 881 + 74 724 + 200 904 + 59

CD133 13 + 11 29 + 55 29 + 35

CD29 990 + 15 998 + 04 995 + 13

HLA I 952 + 60 943 + 68 988 + 28

CD106b 663 + 227 700 + 236 303 + 186

HLA II 42 + 61 08 + 12 44 + 62

CD144 45 93 24 40 24 25

The table shows mean values of the percentage of positive cells standard deviation to the total number of cells analyzed

Proinflammatory licensing of

MSCs

bull MSCs have been dubbed as smart immune modulators since their

suppressive effects require a previous licensing step that occurs in the

presence of an inflammatory environment and is mediated by the secretion

of specific cytokines (Jones et al 2007 Jorgensen 2010) Thus IFN-γ

alone or together with tumor necrosis factor (TNF)-α IL-1α or IL-1β are

required to trigger the expression by MSCs of high levels of soluble factors

involved in immunosuppression such as IDO HGF TGF-β and NO

(Aggarwal and Pittenger 2005 Ryan et al 2007 Krampera et al 2006

Ren et al 2008)

bull The need for this activation step has been confirmed in a model of GVHD

since recipients of IFNγ-- T cells did not respond to MSC treatment

evolving into fatal GVHD (Polchert et al 2008)

bull Others have attributed the immunomodulatory function of MSCs mainly to

IL-6-dependent secretion of prostaglandin E2 (PGE2) (Boufii et al 2010)

This is PRP

PDEGF

PDGF-AB bFGF

VEGF PDFG-AA IGF-3

FGF-1 IGFBP-3

BMP-4

IGFBP-3

TGF-B1

hGH

PDGF-BB

bFGF BMP-2

PDGF-AB

IGF-1

IGFBP-2

EGF

HGF

ECGF

PDEGF

BMP

Pro-

Chondrogenic

TGFB-1

IGF-1

bFGF

BMP-2

Anti-

Chondrogenic

VEGF

IGFBP-2

IGFBP-3

PDGF-AA

PDGF-AB

PDGF-BB

EGF

1st Generation PRP

Pro-

Chondrogenic

TGFB-1

IGF-1

bFGF

BMP-2

Anti-

Chondrogenic

VEGF

IGFBP-2

IGFBP-3

PDGF-AA

PDGF-AB

PDGF-BB

EGF

2nd Generation PRP

ADSC Induction Media

bull IGF-I

bull TGF-β1

bull GH

bull FGF-2

Platelet Rich Plasma

bull IGF-1

bull TGF-β1

bull GH

bull FGF-2

bull PDGF

bull VEGF

bull IGF-BP2

bull IGF-BP3

2nd Generation PRP

Create and

activate

PRP

Neutralize specific

PRP growth factors

with antibodies

bull VEGF

bull IGFBP-2

bull IGFBP-3

bull EGF

bull PDGF

Confirm

absence of

neutralized

factors and

presence of key

growth factors

by ELISA

2nd Generation PRP

Sterile inflammation

caused by DAMPs and

mediated by IL-1β or

TNF in the absence of

infection involves

canonical NFκB

activation and

proinflammatory

signalling In OA

molecules from

cartilage breakdown

and intracellular

components ohellip

We will manipulate growth factor

content for target tissue or disease

Signalling proteins

(including growth

factors

chemokines and

other cytokines)

adhesive proteins

and proteases

together with small

molecules and ions

that are secreted by

platelets are found

at high levels in

PRP and interact

with mhellip

mPRP Task Myostatin and TGF-β1 removal

using antibodies bound

to protein AG agarose in sterile conditions

Protein A beads Incubate

1 hour RT Add Ab-s

PBS buffer

Protein AG

beads

TGFβ1 antibodies myostatin antibodies

Protein G

beads

Disc disease and pathological

changes in the neuroforamen

bull Disc leak

ndash PLA2

ndash Immune mediated responses

ndash Inflammation

bull Ischemia

bull Demeylination

bull Inflamatory membrane

PRP and PR

Orthopedic Spine Applications

bull SI joint instability syndromes

bull Accessory ligaments of SI joint

bull Lumbar facet joints

bull LD fascia

bull IL ligament

bull Platelet releaseate epidural

bull Intradiscal PRP with or without fibrin sealant

bull Never had a Toti-Potent or Pleuri-Potent stem

cell population to use until nowhellip

Adipose derived MSCs Carrioacuten and Figueroa Stem Cell Res Ther 2011 May 112(3)23

bull Promotion of angiogenesis vascular endothelial

growth factor (VEGF) insulin like growth factor 1

(IGF-1) monocyte chemoatractant protein 1

(MCP1) basic fibroblast growth factor (bFGF) and

interleukin 6 (IL6)

bull 2) Stem cell growth and differentiation stem cell

factor (SCF) leukemiainhibitory factor (LIF)

macrophage colonystimulating factor (MCSF)

stromal derived factor 1 (SDF1) angiopoietin1 and

activin A

bull 3) Inhibition of fibrosis hepatocyte growth factor

(HGF) bFGF adrenomedullin (ADM)

Adipose derived MSCs Carrioacuten and Figueroa Stem Cell Res Ther 2011 May 112(3)23

bull 4) Inhibition of apoptosis VEGF HGF IGF1 transforming growth

factor (TGF)p bFGF granulocyte macrophage colonystimulating

factor (GMCSF) activin A and thrombospondin 1 Immune mediated

effects include the following (5 to 8)

bull 5) Suppression of T and B cells human leukocyte antigen G5

(HLAG5) HGF inducible nitric oxide synthase (iNOS)

indoleamine23dioxygenase (IDO) prostaglandin E2 (PGE2) bFGF

and TGFp

bull 6) Induction of regulatory T cells (Treg) differentiation and expansion

by TGFp expression

bull 7) Inhibition of natural killer (NK) cells by secretion of IDO PGE2

and TGFp

bull 8) Inhibition of dendritic cell (DC) maturation by secretion of PGE2

Adipose derived MSCs Carrioacuten and Figueroa Stem Cell Res Ther 2011 May 112(3)23

Adipose has more MSC CFUs

bull The number of CFU-F calculated at the

bull basis of 1 106 initially plated cells was

highest for AT (557 + 673) followed by BM

(83 + 61)

Comparative analysis of mesenchymal stem cells from bone marrow umbilical

cord blood or adipose tissue Kern S1 Eichler H Stoeve J Kluumlter H Bieback K

Stem Cells 2006 May24(5)1294-301 Epub 2006 Jan 12

Cell markers show similar phenotype of

AT and BM MSCs

Table 2 Comparison of the expression of surface proteins of

mesenchymal stem cells derived from BM Umbilical Cord Blood and AT as

analyzed by flow cytometry

Antibody BM () (n 9) UCB () (n 10) AT () (n 9)

CD44 975 + 51 997 +05 998 +02

CD73 900 +200 993 + 13 996 + 05

CD90 991 + 25 978 + 71 999 + 02

CD14 12 + 1 1 08 + 09 24 + 50

CD34 16 + 14 12 + 15 50 + 51

CD45 52 + 37 38 + 36 38 + 51

CD105a 881 + 74 724 + 200 904 + 59

CD133 13 + 11 29 + 55 29 + 35

CD29 990 + 15 998 + 04 995 + 13

HLA I 952 + 60 943 + 68 988 + 28

CD106b 663 + 227 700 + 236 303 + 186

HLA II 42 + 61 08 + 12 44 + 62

CD144 45 93 24 40 24 25

The table shows mean values of the percentage of positive cells standard deviation to the total number of cells analyzed

Proinflammatory licensing of

MSCs

bull MSCs have been dubbed as smart immune modulators since their

suppressive effects require a previous licensing step that occurs in the

presence of an inflammatory environment and is mediated by the secretion

of specific cytokines (Jones et al 2007 Jorgensen 2010) Thus IFN-γ

alone or together with tumor necrosis factor (TNF)-α IL-1α or IL-1β are

required to trigger the expression by MSCs of high levels of soluble factors

involved in immunosuppression such as IDO HGF TGF-β and NO

(Aggarwal and Pittenger 2005 Ryan et al 2007 Krampera et al 2006

Ren et al 2008)

bull The need for this activation step has been confirmed in a model of GVHD

since recipients of IFNγ-- T cells did not respond to MSC treatment

evolving into fatal GVHD (Polchert et al 2008)

bull Others have attributed the immunomodulatory function of MSCs mainly to

IL-6-dependent secretion of prostaglandin E2 (PGE2) (Boufii et al 2010)

2nd Generation PRP

Create and

activate

PRP

Neutralize specific

PRP growth factors

with antibodies

bull VEGF

bull IGFBP-2

bull IGFBP-3

bull EGF

bull PDGF

Confirm

absence of

neutralized

factors and

presence of key

growth factors

by ELISA

2nd Generation PRP

Sterile inflammation

caused by DAMPs and

mediated by IL-1β or

TNF in the absence of

infection involves

canonical NFκB

activation and

proinflammatory

signalling In OA

molecules from

cartilage breakdown

and intracellular

components ohellip

We will manipulate growth factor

content for target tissue or disease

Signalling proteins

(including growth

factors

chemokines and

other cytokines)

adhesive proteins

and proteases

together with small

molecules and ions

that are secreted by

platelets are found

at high levels in

PRP and interact

with mhellip

mPRP Task Myostatin and TGF-β1 removal

using antibodies bound

to protein AG agarose in sterile conditions

Protein A beads Incubate

1 hour RT Add Ab-s

PBS buffer

Protein AG

beads

TGFβ1 antibodies myostatin antibodies

Protein G

beads

Disc disease and pathological

changes in the neuroforamen

bull Disc leak

ndash PLA2

ndash Immune mediated responses

ndash Inflammation

bull Ischemia

bull Demeylination

bull Inflamatory membrane

PRP and PR

Orthopedic Spine Applications

bull SI joint instability syndromes

bull Accessory ligaments of SI joint

bull Lumbar facet joints

bull LD fascia

bull IL ligament

bull Platelet releaseate epidural

bull Intradiscal PRP with or without fibrin sealant

bull Never had a Toti-Potent or Pleuri-Potent stem

cell population to use until nowhellip

Adipose derived MSCs Carrioacuten and Figueroa Stem Cell Res Ther 2011 May 112(3)23

bull Promotion of angiogenesis vascular endothelial

growth factor (VEGF) insulin like growth factor 1

(IGF-1) monocyte chemoatractant protein 1

(MCP1) basic fibroblast growth factor (bFGF) and

interleukin 6 (IL6)

bull 2) Stem cell growth and differentiation stem cell

factor (SCF) leukemiainhibitory factor (LIF)

macrophage colonystimulating factor (MCSF)

stromal derived factor 1 (SDF1) angiopoietin1 and

activin A

bull 3) Inhibition of fibrosis hepatocyte growth factor

(HGF) bFGF adrenomedullin (ADM)

Adipose derived MSCs Carrioacuten and Figueroa Stem Cell Res Ther 2011 May 112(3)23

bull 4) Inhibition of apoptosis VEGF HGF IGF1 transforming growth

factor (TGF)p bFGF granulocyte macrophage colonystimulating

factor (GMCSF) activin A and thrombospondin 1 Immune mediated

effects include the following (5 to 8)

bull 5) Suppression of T and B cells human leukocyte antigen G5

(HLAG5) HGF inducible nitric oxide synthase (iNOS)

indoleamine23dioxygenase (IDO) prostaglandin E2 (PGE2) bFGF

and TGFp

bull 6) Induction of regulatory T cells (Treg) differentiation and expansion

by TGFp expression

bull 7) Inhibition of natural killer (NK) cells by secretion of IDO PGE2

and TGFp

bull 8) Inhibition of dendritic cell (DC) maturation by secretion of PGE2

Adipose derived MSCs Carrioacuten and Figueroa Stem Cell Res Ther 2011 May 112(3)23

Adipose has more MSC CFUs

bull The number of CFU-F calculated at the

bull basis of 1 106 initially plated cells was

highest for AT (557 + 673) followed by BM

(83 + 61)

Comparative analysis of mesenchymal stem cells from bone marrow umbilical

cord blood or adipose tissue Kern S1 Eichler H Stoeve J Kluumlter H Bieback K

Stem Cells 2006 May24(5)1294-301 Epub 2006 Jan 12

Cell markers show similar phenotype of

AT and BM MSCs

Table 2 Comparison of the expression of surface proteins of

mesenchymal stem cells derived from BM Umbilical Cord Blood and AT as

analyzed by flow cytometry

Antibody BM () (n 9) UCB () (n 10) AT () (n 9)

CD44 975 + 51 997 +05 998 +02

CD73 900 +200 993 + 13 996 + 05

CD90 991 + 25 978 + 71 999 + 02

CD14 12 + 1 1 08 + 09 24 + 50

CD34 16 + 14 12 + 15 50 + 51

CD45 52 + 37 38 + 36 38 + 51

CD105a 881 + 74 724 + 200 904 + 59

CD133 13 + 11 29 + 55 29 + 35

CD29 990 + 15 998 + 04 995 + 13

HLA I 952 + 60 943 + 68 988 + 28

CD106b 663 + 227 700 + 236 303 + 186

HLA II 42 + 61 08 + 12 44 + 62

CD144 45 93 24 40 24 25

The table shows mean values of the percentage of positive cells standard deviation to the total number of cells analyzed

Proinflammatory licensing of

MSCs

bull MSCs have been dubbed as smart immune modulators since their

suppressive effects require a previous licensing step that occurs in the

presence of an inflammatory environment and is mediated by the secretion

of specific cytokines (Jones et al 2007 Jorgensen 2010) Thus IFN-γ

alone or together with tumor necrosis factor (TNF)-α IL-1α or IL-1β are

required to trigger the expression by MSCs of high levels of soluble factors

involved in immunosuppression such as IDO HGF TGF-β and NO

(Aggarwal and Pittenger 2005 Ryan et al 2007 Krampera et al 2006

Ren et al 2008)

bull The need for this activation step has been confirmed in a model of GVHD

since recipients of IFNγ-- T cells did not respond to MSC treatment

evolving into fatal GVHD (Polchert et al 2008)

bull Others have attributed the immunomodulatory function of MSCs mainly to

IL-6-dependent secretion of prostaglandin E2 (PGE2) (Boufii et al 2010)

mPRP Task Myostatin and TGF-β1 removal

using antibodies bound

to protein AG agarose in sterile conditions

Protein A beads Incubate

1 hour RT Add Ab-s

PBS buffer

Protein AG

beads

TGFβ1 antibodies myostatin antibodies

Protein G

beads

Disc disease and pathological

changes in the neuroforamen

bull Disc leak

ndash PLA2

ndash Immune mediated responses

ndash Inflammation

bull Ischemia

bull Demeylination

bull Inflamatory membrane

PRP and PR

Orthopedic Spine Applications

bull SI joint instability syndromes

bull Accessory ligaments of SI joint

bull Lumbar facet joints

bull LD fascia

bull IL ligament

bull Platelet releaseate epidural

bull Intradiscal PRP with or without fibrin sealant

bull Never had a Toti-Potent or Pleuri-Potent stem

cell population to use until nowhellip

Adipose derived MSCs Carrioacuten and Figueroa Stem Cell Res Ther 2011 May 112(3)23

bull Promotion of angiogenesis vascular endothelial

growth factor (VEGF) insulin like growth factor 1

(IGF-1) monocyte chemoatractant protein 1

(MCP1) basic fibroblast growth factor (bFGF) and

interleukin 6 (IL6)

bull 2) Stem cell growth and differentiation stem cell

factor (SCF) leukemiainhibitory factor (LIF)

macrophage colonystimulating factor (MCSF)

stromal derived factor 1 (SDF1) angiopoietin1 and

activin A

bull 3) Inhibition of fibrosis hepatocyte growth factor

(HGF) bFGF adrenomedullin (ADM)

Adipose derived MSCs Carrioacuten and Figueroa Stem Cell Res Ther 2011 May 112(3)23

bull 4) Inhibition of apoptosis VEGF HGF IGF1 transforming growth

factor (TGF)p bFGF granulocyte macrophage colonystimulating

factor (GMCSF) activin A and thrombospondin 1 Immune mediated

effects include the following (5 to 8)

bull 5) Suppression of T and B cells human leukocyte antigen G5

(HLAG5) HGF inducible nitric oxide synthase (iNOS)

indoleamine23dioxygenase (IDO) prostaglandin E2 (PGE2) bFGF

and TGFp

bull 6) Induction of regulatory T cells (Treg) differentiation and expansion

by TGFp expression

bull 7) Inhibition of natural killer (NK) cells by secretion of IDO PGE2

and TGFp

bull 8) Inhibition of dendritic cell (DC) maturation by secretion of PGE2

Adipose derived MSCs Carrioacuten and Figueroa Stem Cell Res Ther 2011 May 112(3)23

Adipose has more MSC CFUs

bull The number of CFU-F calculated at the

bull basis of 1 106 initially plated cells was

highest for AT (557 + 673) followed by BM

(83 + 61)

Comparative analysis of mesenchymal stem cells from bone marrow umbilical

cord blood or adipose tissue Kern S1 Eichler H Stoeve J Kluumlter H Bieback K

Stem Cells 2006 May24(5)1294-301 Epub 2006 Jan 12

Cell markers show similar phenotype of

AT and BM MSCs

Table 2 Comparison of the expression of surface proteins of

mesenchymal stem cells derived from BM Umbilical Cord Blood and AT as

analyzed by flow cytometry

Antibody BM () (n 9) UCB () (n 10) AT () (n 9)

CD44 975 + 51 997 +05 998 +02

CD73 900 +200 993 + 13 996 + 05

CD90 991 + 25 978 + 71 999 + 02

CD14 12 + 1 1 08 + 09 24 + 50

CD34 16 + 14 12 + 15 50 + 51

CD45 52 + 37 38 + 36 38 + 51

CD105a 881 + 74 724 + 200 904 + 59

CD133 13 + 11 29 + 55 29 + 35

CD29 990 + 15 998 + 04 995 + 13

HLA I 952 + 60 943 + 68 988 + 28

CD106b 663 + 227 700 + 236 303 + 186

HLA II 42 + 61 08 + 12 44 + 62

CD144 45 93 24 40 24 25

The table shows mean values of the percentage of positive cells standard deviation to the total number of cells analyzed

Proinflammatory licensing of

MSCs

bull MSCs have been dubbed as smart immune modulators since their

suppressive effects require a previous licensing step that occurs in the

presence of an inflammatory environment and is mediated by the secretion

of specific cytokines (Jones et al 2007 Jorgensen 2010) Thus IFN-γ

alone or together with tumor necrosis factor (TNF)-α IL-1α or IL-1β are

required to trigger the expression by MSCs of high levels of soluble factors

involved in immunosuppression such as IDO HGF TGF-β and NO

(Aggarwal and Pittenger 2005 Ryan et al 2007 Krampera et al 2006

Ren et al 2008)

bull The need for this activation step has been confirmed in a model of GVHD

since recipients of IFNγ-- T cells did not respond to MSC treatment

evolving into fatal GVHD (Polchert et al 2008)

bull Others have attributed the immunomodulatory function of MSCs mainly to

IL-6-dependent secretion of prostaglandin E2 (PGE2) (Boufii et al 2010)

Adipose derived MSCs Carrioacuten and Figueroa Stem Cell Res Ther 2011 May 112(3)23

bull Promotion of angiogenesis vascular endothelial

growth factor (VEGF) insulin like growth factor 1

(IGF-1) monocyte chemoatractant protein 1

(MCP1) basic fibroblast growth factor (bFGF) and

interleukin 6 (IL6)

bull 2) Stem cell growth and differentiation stem cell

factor (SCF) leukemiainhibitory factor (LIF)

macrophage colonystimulating factor (MCSF)

stromal derived factor 1 (SDF1) angiopoietin1 and

activin A

bull 3) Inhibition of fibrosis hepatocyte growth factor

(HGF) bFGF adrenomedullin (ADM)

Adipose derived MSCs Carrioacuten and Figueroa Stem Cell Res Ther 2011 May 112(3)23

bull 4) Inhibition of apoptosis VEGF HGF IGF1 transforming growth

factor (TGF)p bFGF granulocyte macrophage colonystimulating

factor (GMCSF) activin A and thrombospondin 1 Immune mediated

effects include the following (5 to 8)

bull 5) Suppression of T and B cells human leukocyte antigen G5

(HLAG5) HGF inducible nitric oxide synthase (iNOS)

indoleamine23dioxygenase (IDO) prostaglandin E2 (PGE2) bFGF

and TGFp

bull 6) Induction of regulatory T cells (Treg) differentiation and expansion

by TGFp expression

bull 7) Inhibition of natural killer (NK) cells by secretion of IDO PGE2

and TGFp

bull 8) Inhibition of dendritic cell (DC) maturation by secretion of PGE2

Adipose derived MSCs Carrioacuten and Figueroa Stem Cell Res Ther 2011 May 112(3)23

Adipose has more MSC CFUs

bull The number of CFU-F calculated at the

bull basis of 1 106 initially plated cells was

highest for AT (557 + 673) followed by BM

(83 + 61)

Comparative analysis of mesenchymal stem cells from bone marrow umbilical

cord blood or adipose tissue Kern S1 Eichler H Stoeve J Kluumlter H Bieback K

Stem Cells 2006 May24(5)1294-301 Epub 2006 Jan 12

Cell markers show similar phenotype of

AT and BM MSCs

Table 2 Comparison of the expression of surface proteins of

mesenchymal stem cells derived from BM Umbilical Cord Blood and AT as

analyzed by flow cytometry

Antibody BM () (n 9) UCB () (n 10) AT () (n 9)

CD44 975 + 51 997 +05 998 +02

CD73 900 +200 993 + 13 996 + 05

CD90 991 + 25 978 + 71 999 + 02

CD14 12 + 1 1 08 + 09 24 + 50

CD34 16 + 14 12 + 15 50 + 51

CD45 52 + 37 38 + 36 38 + 51

CD105a 881 + 74 724 + 200 904 + 59

CD133 13 + 11 29 + 55 29 + 35

CD29 990 + 15 998 + 04 995 + 13

HLA I 952 + 60 943 + 68 988 + 28

CD106b 663 + 227 700 + 236 303 + 186

HLA II 42 + 61 08 + 12 44 + 62

CD144 45 93 24 40 24 25

The table shows mean values of the percentage of positive cells standard deviation to the total number of cells analyzed

Proinflammatory licensing of

MSCs

bull MSCs have been dubbed as smart immune modulators since their

suppressive effects require a previous licensing step that occurs in the

presence of an inflammatory environment and is mediated by the secretion

of specific cytokines (Jones et al 2007 Jorgensen 2010) Thus IFN-γ

alone or together with tumor necrosis factor (TNF)-α IL-1α or IL-1β are

required to trigger the expression by MSCs of high levels of soluble factors

involved in immunosuppression such as IDO HGF TGF-β and NO

(Aggarwal and Pittenger 2005 Ryan et al 2007 Krampera et al 2006

Ren et al 2008)

bull The need for this activation step has been confirmed in a model of GVHD

since recipients of IFNγ-- T cells did not respond to MSC treatment

evolving into fatal GVHD (Polchert et al 2008)

bull Others have attributed the immunomodulatory function of MSCs mainly to

IL-6-dependent secretion of prostaglandin E2 (PGE2) (Boufii et al 2010)

Adipose has more MSC CFUs

bull The number of CFU-F calculated at the

bull basis of 1 106 initially plated cells was

highest for AT (557 + 673) followed by BM

(83 + 61)

Comparative analysis of mesenchymal stem cells from bone marrow umbilical

cord blood or adipose tissue Kern S1 Eichler H Stoeve J Kluumlter H Bieback K

Stem Cells 2006 May24(5)1294-301 Epub 2006 Jan 12

Cell markers show similar phenotype of

AT and BM MSCs

Table 2 Comparison of the expression of surface proteins of

mesenchymal stem cells derived from BM Umbilical Cord Blood and AT as

analyzed by flow cytometry

Antibody BM () (n 9) UCB () (n 10) AT () (n 9)

CD44 975 + 51 997 +05 998 +02

CD73 900 +200 993 + 13 996 + 05

CD90 991 + 25 978 + 71 999 + 02

CD14 12 + 1 1 08 + 09 24 + 50

CD34 16 + 14 12 + 15 50 + 51

CD45 52 + 37 38 + 36 38 + 51

CD105a 881 + 74 724 + 200 904 + 59

CD133 13 + 11 29 + 55 29 + 35

CD29 990 + 15 998 + 04 995 + 13

HLA I 952 + 60 943 + 68 988 + 28

CD106b 663 + 227 700 + 236 303 + 186

HLA II 42 + 61 08 + 12 44 + 62

CD144 45 93 24 40 24 25

The table shows mean values of the percentage of positive cells standard deviation to the total number of cells analyzed

Proinflammatory licensing of

MSCs

bull MSCs have been dubbed as smart immune modulators since their

suppressive effects require a previous licensing step that occurs in the

presence of an inflammatory environment and is mediated by the secretion

of specific cytokines (Jones et al 2007 Jorgensen 2010) Thus IFN-γ

alone or together with tumor necrosis factor (TNF)-α IL-1α or IL-1β are

required to trigger the expression by MSCs of high levels of soluble factors

involved in immunosuppression such as IDO HGF TGF-β and NO

(Aggarwal and Pittenger 2005 Ryan et al 2007 Krampera et al 2006

Ren et al 2008)

bull The need for this activation step has been confirmed in a model of GVHD

since recipients of IFNγ-- T cells did not respond to MSC treatment

evolving into fatal GVHD (Polchert et al 2008)

bull Others have attributed the immunomodulatory function of MSCs mainly to

IL-6-dependent secretion of prostaglandin E2 (PGE2) (Boufii et al 2010)

Stromal Vascular Fraction

BMAC cellular therapy

bull BMAC

ndash Marrow

aspiration

ndash MSCs

ndash Obtained by

centrifuge

technique similar

to PRP

Bone Marrow Aspirate Concentrate

Cellular Therapy

Use of Adult Nucleated Plasma Particles

For Treatment

Neurological

Cardiovascular

Pain

Respiratory

Musculoskeletal

Diabetes

Where are we goinghellip

Plans for 2015-2016

Cell Par Technologies

Knee Meniscus amp DJD

bull We are now questioning

meniscus

debreedmenthellip

bull Biomechanical effects

ndash Increase contact surface

ndash Increased translational

instability

ndash Rapid progress of OA

Posterior horn set uphellip

bull Biomechanics are at the root

bull Ligaments

ndash Coronary ligaments capsule

Coronary ligaments and capsular

ligaments of posterior horn

bull Researchers have proposed that the small

coronary ligaments previously described

attached to the meniscus ( ligaments of

Humphrey and Wrisburg) may play an

important role in osteoarthritis

progression20

Choi J Chung H Ahn J al e Central hole tear of the discoid

meniscus of the knee in magnetic resonance imaging

mimicking the bucket-handle tear J Comput Assist Tomogr

200933155-159

Osteoarthritis pathophysiology

bull Small molecules of necrotic cell material and fragments

of degenerating cartilage can activate the immune

response resulting in inflammation and thickening of this

membrane and result in pain and swelling1

bull It is this inflammation that is linked to both initiation and

progression of osteoarthritis2

bull Inflammatory amp cytokine souphellip bad for the joint

bull How can we modulate the DISEASE PROCESS

1 McGonagle D Lorie sR Tan A al e The concept of a lsquolsquosynovio-entheseal complexrsquorsquo and its

implications for understanding joint inflammation and damage in psoriatic arthritis and beyond

Arthritis Rheum 200756(24 82-2491)

2 Keen H Wakefield R Grainger A al e An ultrasonographic study of osteoarthritis of the hand

synovitis and its relationship to structural pathology and symptoms Arthritis Rheum 2008591756-

1763

OCDs - microfracture

bull Microfracture some short-term benefit

with no significant long-term results31

Mithoefer K McAdams T Williams R Kreuz P Mandelbaum B Clinical efficacy of the

microfracture technique for articular cartilage repair in the knee an evidence-based

systematic analysis Am J Sports Med 200937(10)2053-2063

MSCs for OA knee

bull Mesenchymal stem cells

ndash potential to become many other types of cells

when exposed to specific growth factors or

environments

ndash Cells ability to differentiate into cartilage

lineage has great potential for cell based

articular cartilage repair1-3

1 Pittenger M al e Multilineage potential of adult human mesenchymal stem cells Science

1990284145-147

2 3Kolf C al e Mesenchymal stromal cells Biology of adult mesenchymal stem cells regulation of

niche self-renewal and differentiation Arthritis Res Ther 20079204

3 Chen F al e Technology insight adult stem cells in cartilage regeneration and tissue engineering

Nat Clin Pract Rheumatol 20062373-382

MSCs developmental plasticity

bull Mesenchymal stem cells

ndash Has properties of ldquodevelopmental plasticityrdquo

which means they can change to other

tissues when placed in the right environment

1-2

1 Oreffo R Cooper C Mason C Clements M ldquoMesenchymal stem cells lineage plasticity and skeletal

therapeutic potentialrdquo Stem Cell Reviews 20051(2)169-178

2 Noumlth U Osyczka A Tuli R Hickok N Danielson K Tuan R ldquoMultilineage mesenchymal differentiation

potential of human trabecular bone-derived cellsrdquo Journal of Orthopaedic Research 200220(5)1060-

1069

Improved red amp white zone cartilage volume

bull Studies emerging demonstrating increased

extracellular matrix the substance that cartilage

cells make that appear to be

restoring a meniscus-like tissue

even in the avascular zone of

the cartilage38-41

1 Izuta Y Ochi M Adachi N Deie M Yamasaki T Shinomiya R ldquoMeniscal repair using bone marrow-derived

mesenchymal stem cells experimental study using green fluorescent protein transgenic ratsrdquo Knee 200512(1)217-

223

2 Steinert A Palmer G Capito R et al ldquoGenetically enhanced engineering of meniscus tissue using ex vivo delivery of

transforming growth factor-β1 complementary deoxyribonucleic acidrdquo Tissue Engineering 200711(9)2227-2232

3 Stone K Rodkey W Webber R McKinney W Steadman J ldquoMeniscal regeneration with copolymeric collagen

scaffolds In vitro and in vivo studies evaluated clinically histologically and biochemicallyrdquo American Journal of

Sports Medicine 199220(2)

4 Zellner J Mueller M Berner A et al ldquoRole of mesenchymal stem cells in tissue engineering of meniscusrdquo Journal

of Biomedical Materials Research 201094(4)1150-1161

Adipose tissue derived MSCs

SVF in OA of the knee

bull Arthroscopic second look after SVF ndash SVF vs NaCl control

bull Almost all patients showed significant improvement in all clinical

outcomes of follow-up

bull All of the patients in this study improved at 2 years compared to

the 12 month follow-up 875 of elderly patients greater than

65 years of age (14 out of 16) improved or maintained cartilage

status at least 2 years postoperatively

bull More importantly none of these patient underwent total knee

arthroplasty (joint replacement) during the 2 year follow-up1

1 Koh Y Choi Y Kwon S Kim Y Yeo J Clinical results and second-look

arthroscopic findings after treatment with adipose-derived stem cells for knee

osteoarthritis Knee Surg Sports Traumatol Arthrosc December 11 2013

Chondromalacia

Patellofemoral disease bull Can we really make a difference

ndash Life style modifications

ndash Patellofemoral knee braces

ndash Patellofemoral straps

ndash VMO and quad strengthening

ndash Improved biomechanics

ndash Orthotics

ndash Physical therapy

ndash Soft tissue mob

SVF in patellofemoral arthritis

bull Pak et al

ndash In a recent study one month after injection of

autologous adipose derived stem cells patientrsquos pain

improved 50-70 and after 3 months patientrsquos

improved 80-90 and continued to improve over 1

year44 More importantly the MRI findings

demonstrated recovery of the articular cartilage 1

ndash MRI changes were documentedhellip

ndash After SVF injection Pain improved 50-70 and after

3 months patientrsquos improved 80-90 and continued to

improve over 1 year

1 Pak J Lee JH SH L A novel biological approach to treat chondromalacia

patellae PLoS One 2013208(5)(8)5

ADSCs have been shown to be OA

protectivehellip

bull Protects cartilage cells in osteoarthritis

against cell death and progression of

degeneration1

bull OA involves increase in cytokines

metalloproteinases reactive oxygen

species which are present in osteoarthritis

joints

bull MSCs downregulate pro-inflammatory

cytokines2

bull BMAC MSCs have similar anti-

inflammatory effects in osteoarthritis3

1 Maumus M Jorgensen C Noeumll D Mesenchymal stem cells in regenerative medicine applied to rheumatic diseases

Role of secretome and exosomes Biochimie 2013952229-2234

2 Manferdini C Gabusi E Maumus M et al Adipose stromal cells exert anti-inflammatory effects on chondrocytes

and synoviocytes from osteoarthritis patients via PGE2 Arthritis Rheum 2013651271-1281

3 van Buul G Eillafuertes E Bos P et al Mesenchymal stem cells secrete factors that inhibit inflammatory processes

in short-term osteoarthritic synovium and cartilage explant culture Osteoarthritis Cartilage 2012201186-1196

The hip

bull Hip is treated with

similar principles as

knee

bull Acetabular labrum

bull FAI

bull Peritrochanteric pain

ndash G medius and hip RTC

Peritrochanteric hip pain

ADSCs have been shown to be OA

protectivehellip

bull Protects cartilage cells in osteoarthritis

against cell death and progression of

degeneration1

bull OA involves increase in cytokines

metalloproteinases reactive oxygen

species which are present in osteoarthritis

joints

bull MSCs downregulate pro-inflammatory

cytokines2

bull BMAC MSCs have similar anti-

inflammatory effects in osteoarthritis3

1 Maumus M Jorgensen C Noeumll D Mesenchymal stem cells in regenerative medicine applied to rheumatic diseases

Role of secretome and exosomes Biochimie 2013952229-2234

2 Manferdini C Gabusi E Maumus M et al Adipose stromal cells exert anti-inflammatory effects on chondrocytes

and synoviocytes from osteoarthritis patients via PGE2 Arthritis Rheum 2013651271-1281

3 van Buul G Eillafuertes E Bos P et al Mesenchymal stem cells secrete factors that inhibit inflammatory processes

in short-term osteoarthritic synovium and cartilage explant culture Osteoarthritis Cartilage 2012201186-1196

The hip

bull Hip is treated with

similar principles as

knee

bull Acetabular labrum

bull FAI

bull Peritrochanteric pain

ndash G medius and hip RTC

Peritrochanteric hip pain

Rotator cuff tears

bull Rotator cuff tears

ndash Impingement

ndash Deg

Tendinopathy

ndash Tendinosis

ndash Insertional tears bull Subarticular

bull Bursal surface

bull Interstitial tears

Image guided deployment

bull Requires detailed understanding of

anatomy

bull Understanding of pathology

bull Understanding of image guided

procedures

bull Cell deployment techniques are

completely different than interventional

pain procedures and require special

training

The search for neuronal

stem cells has ended

Neurodegenerative disease stem

Cell therapy

- Finally a pathway into the brain

Let the Body Induce the Preferred Cell Type

By Injecting Naiumlve Nucleated Plasma Particles

into a Particular Sitehellip

Michael N Brown MD

ParkinsonrsquosDisease

Neuronal Repair

Domapinergic neurons

bull Dopaminergic neurons which have their

cell bodies located in the substantia nigra

pars compacta (SNpc)

Domapinergic neurons bull These neurons send axons to the caudate and putamen

(collectively known as the corpus striatum)

bull The progressive loss of these cells results in the gradual

decrease over time of striatal dopamine levels which in

turn produces a decrease in striatal output to the

thalamus

bull selective dopaminergic neurotoxin 6-hydroxydopamine

into the corpus striatum middle forebrain bundle or

substantia nigra pars compacta

bull 6-hydroxydopamine (6-OHDA) is a selective dopamine

neurotoxin which is taken up by transporter proteins at

the nerve terminals within the corpus striatum and

transported in a retrograde manner to the cell bodies in

the substantia nigra29

Dopaminergic neurotoxin 6-hydroxydopamine

bull Bilateral 6-OHDA lesions of the adult rat brain

result in a partial progressive loss of

dopaminergic terminals within the corpus

striatum ipsilateral to the injection site and the

subsequent death of the dopaminergic neurons

projecting from the substantia nigra to the

corpus striatum25

Dopaminergic neurotoxin 6-hydroxydopamine

bull Because of the similarities between the

neurochemical and neuropathological changes

elicited by the local injection of 6-OHDA to those

found in Parkinsonrsquos disease this particular

experimental animal model has often been used

to anticipate the relevance of a given treatment

in the clinical management of the symptoms of

Parkinson disease in humans

Lee CS Cenci MA Schulzer M et al Embryonic ventral mesencephalic grafts improve levodopa-induced

dyskinesia in a rat model of Parkinsonrsquos disease Brain 20001231365ndash1379

Parati EA Bez A Ponti D et al Neural stem cells Biological features and therapeutic potential in Parkinsonrsquos

disease J Neurosurg Sci 2003478ndash17

Stem cells for PD

bull Lindvall proposed that four different cellular sources

could be used to form dopaminergic neurons for neural

transplantation for Parkinson disease

ndash (a) embryonic stem cells from a fertilized egg

ndash (b) neural stem cells from an embryonic brain

ndash (c) neural stem cells from an adult brain or

ndash (d) stem cells from other tissues

bull The crucial issue is whether the transplanted cells would

form functional dopaminergic neurons regardless of the

source of the stem cells

Lindvall O Stem cells for cell therapy in Parkinsonrsquos disease Pharmacol Res 200347279ndash287

Stem Cells for PD

bull Young et al 1 reported the isolation and single cell

cloning of adult-derived pluripotent stem cells from the

connective tissue stroma of multiple organs in animals

and humans

bull They demonstrated that a clonal population of adult-

derived pluripotent cells was capable of objectively

forming 63 of the 220+ possible cells of the body

including multiple types of neurons oligodendrocytes

astrocytes and capillaries 2

1 Young HE Duplaa C Yost MJ et al Clonogenic analysis reveals reserve stem cells in postnatal

mammals II Pluripotent epiblastic-like stem cells Anat Rec 2004277a178ndash203

2 Young HE Black AC Jr Adult-derived stem cells Minerva Biotechnologica 20051755ndash63

Induce loss of dopaminergic neurons with

stereotactic injection of neurotoxin

6-hydroxydopamine

Young HE Hyer L Black AC Jr Robinson JS Jr Treating Parkinson disease with adult stem cells J Neurological

Disorders 21 2013 httpdxdoi org104172jnd100121

Stereotactic injection site for neurotoxin vehicle control or genomically-labeled allogeneic pluripotent stem cells

Reprinted with permission from Young et al J Cell Molec Med 9753-769 2005 copy 2005 CMM Foundation

stereotactically infused

unilaterally into 6-OHDA

hemi-lesioned out-bred

SpraguendashDawley adult rat

brains on the ipsilateral

side

The contralateral side

received

002 ascorbate-saline

buffer only as the

operational control

Induce loss of dopaminergic neurons with

stereotactic injection of neurotoxin

6-hydroxydopamine

bull The sham control hemi-brain

(receiving an infusion of saline-

ascorbate buffer) at two weeks post

infusion = tyrosine hydroxylase-

positive cells were present throughout

the striatum

bull The 6-OHDA control hemi-brain

(receiving an infusion 6-OHDA) two

weeks post infusion = loss of

tyrosine-hydroxylase positive cells in

a central area within the striatum

Stem Cells for PD

bull Young et al 1 reported the isolation and single cell

cloning of adult-derived pluripotent stem cells from the

connective tissue stroma of multiple organs in animals

and humans

bull They demonstrated that a clonal population of adult-

derived pluripotent cells was capable of objectively

forming 63 of the 220+ possible cells of the body

including multiple types of neurons oligodendrocytes

astrocytes and capillaries 2

1 Young HE Duplaa C Yost MJ et al Clonogenic analysis reveals reserve stem cells in postnatal

mammals II Pluripotent epiblastic-like stem cells Anat Rec 2004277a178ndash203

2 Young HE Black AC Jr Adult-derived stem cells Minerva Biotechnologica 20051755ndash63

Induce loss of dopaminergic neurons with

stereotactic injection of neurotoxin

6-hydroxydopamine

Young HE Hyer L Black AC Jr Robinson JS Jr Treating Parkinson disease with adult stem cells J Neurological

Disorders 21 2013 httpdxdoi org104172jnd100121

Stereotactic injection site for neurotoxin vehicle control or genomically-labeled allogeneic pluripotent stem cells

Reprinted with permission from Young et al J Cell Molec Med 9753-769 2005 copy 2005 CMM Foundation

stereotactically infused

unilaterally into 6-OHDA

hemi-lesioned out-bred

SpraguendashDawley adult rat

brains on the ipsilateral

side

The contralateral side

received

002 ascorbate-saline

buffer only as the

operational control

Induce loss of dopaminergic neurons with

stereotactic injection of neurotoxin

6-hydroxydopamine

bull The sham control hemi-brain

(receiving an infusion of saline-

ascorbate buffer) at two weeks post

infusion = tyrosine hydroxylase-

positive cells were present throughout

the striatum

bull The 6-OHDA control hemi-brain

(receiving an infusion 6-OHDA) two

weeks post infusion = loss of

tyrosine-hydroxylase positive cells in

a central area within the striatum

bull Brightfield microscopy of corpus

bull striatum of adult rat brain stained with antibody

to tyrosine hydroxylase (brown) and

counterstained with methyl green (green)

bull A Normal corpus striatum of adult rat brain

(sham control hemi-brain receiving an infusion

of saline-ascorbate buffer) at two weeksbafter

infusion

bull Note immunoreactivity forbtyrosine hydroxylase

(brown)

bull B Adult rat brain lesioned stereotactically with

6- hydroxydopamine two weeks after infusion

Note loss of immunoreactivity for tyrosine

hydroxylase in the central lesioned area but

retention of immunoreactivity peripheral to the

lesion

bull C Adult rat brain lesioned stereotactically with

6-hydroxydopamine and then injected two

weeks later with control buffer solution Note

needle tract (green arrows) devoid of

immunoreactivity for tyrosine hydroxylase

bull D Adult rat brain lesioned stereotactically with

6-hydroxydopamine and then injected two

weeks later with Scl- 40β Note needle tract

(green) containing cells that express

immunoreactivity for tyrosine hydroxylase

(arrows) as well as the presence of cells

immunoreactive for tyrosine hydroxylase in

adjacent tissue

bull The 6-OHDAoperational control

hemi-brain (receiving an

infusion 6-OHDA followed two

weeks later by an infusion of

saline-ascorbate buffer)

demonstrated only a glial

response (small green cells)

along the infusion needle track

but no appearance of tyrosine

hydroxylase positive cells

Note needle tract (green

outlined in black) devoid of

immunoreactivity for

tyrosine hydroxylase

bull The extent of the 6-hydroxydopamine lesions vs saline-injected

controls was visualized two weeks after injection by staining tissue

sections with an antibody to tyrosine hydroxylase

Bright field microscopy of adult rat brain lesioned stereotactically with 6

hydroxydopamine and then injected two weeks later with saline-ascorbate control

buffer solution Note needle track (denoted by lines and arrows) devoid of

immunoreactivity for tyrosine hydroxylase activity Young et al J Cell Molec Med 9753-769 2005

bull Adult rat brain lesioned

stereotactically with 6-

hydroxydopamine and

bull then injected two weeks later with

Lac-Z transfected clone of adult-

derived pluripotent

bull stem cells Note needle tract

(outlined with black bars)

containing cells that

bull express immunoreactivity for

tyrosine hydroxylase as well as

the presence of cells

bull immunoreactive for tyrosine

hydroxylase in adjacent tissue

Injected Vehicle only Tyrosine Hydroxylase

(negative)

Needle Track

Green Cells belong to Glial Scar

Injected Vehicle amp Naiumlve Pluripotent Stem Cells

Tyrosine Hydroxylase (positive) Cells

Tyrosine Hydroxylase-positive (brown) Cells

Summary Slide

- Injected primitive PSCs formed dopaminergic neruons within substantia nigra

Unexpected results

- Primitive PSCs also migrated along needle track and regenerated normal tissues and cells in cerebral cortex damaged during stereotactic injections

Glial cells Pyramidal neurons amp Capillaries

(containing RBCs)

Stem cells for PD bull Adult rat hemi-brains from Parkinson study

were examined for the presence of cells

immunoreactive for beta-galactosidase The

Lac-Z transfected Scl-40beta pluripotent stem

cell clone derived from adult rats was

identified its stem cell phase and

differentiation phase using an antibody to

beta-galactosidase The tissue harvested was

from needle tracks created by previous

stereotactic injections The tissue was

harvested and stained with antibody to beta-

galactosidase (brown color donor cells) and

counterstained with methyl green (green

color host cells) Immunoreactivity to beta-

galactosidase was expressed within the

cytoplasm of differentiated cells ie glia

pyramidal neurons interneuons and

endothelial cells lining newly formed RBC-

filled capillaries

-Galactosidase expressing cells (brown) = Injected ASC origin

Glial Cells

Capillary

Pyramidal

neurons

(brown cells)

White Matter Gray Matter Gray Matter

Post stem cell for PD

bull Results from the animal study

demonstrated

ndash replacement of dopaminergic neurons in the

area of the 6-OHDA lesion

ndash replacement of damaged neuronal cells

damaged neuronal supportive cells and

damaged vascular structures caused by the

needle injections

The pathway to the brain

Young HE Hyer L Black AC Jr Robinson JS Jr Treating Parkinson disease with adult stem cells J Neurological

Disorders 21 2013 httpdxdoi org104172jnd100121

Parkinsons disease pilot

bull Induction ndash extraction islolation of TPSCs and PPSC intra-nasal

infusion of autologous totipotent stem cells and intravenous infusion

of autologous pluripotent stem cells

bull Serial washing of cells with to remove plasma proteins

bull Tx intra-nasal infusion of TPSCs + IV Inf

bull Intranasal infusion in modified Trendelenberg

position

bull 5 min to insure the deposition of the stem

cells on the olfactory mucosa with migration

between the mucosal cells along the olfactory

processes through the cribriform plate to the

olfactory bulb and posteriorly

along the olfactory nerves to gain entrance

underneath the blood-brain barrier and to the

sub-arachnoid cisterns of the brain

bull Adult rat brain lesioned

stereotactically with 6-

hydroxydopamine and

bull then injected two weeks later with

Lac-Z transfected clone of adult-

derived pluripotent

bull stem cells Note needle tract

(outlined with black bars)

containing cells that

bull express immunoreactivity for

tyrosine hydroxylase as well as

the presence of cells

bull immunoreactive for tyrosine

hydroxylase in adjacent tissue

Injected Vehicle only Tyrosine Hydroxylase

(negative)

Needle Track

Green Cells belong to Glial Scar

Injected Vehicle amp Naiumlve Pluripotent Stem Cells

Tyrosine Hydroxylase (positive) Cells

Tyrosine Hydroxylase-positive (brown) Cells

Summary Slide

- Injected primitive PSCs formed dopaminergic neruons within substantia nigra

Unexpected results

- Primitive PSCs also migrated along needle track and regenerated normal tissues and cells in cerebral cortex damaged during stereotactic injections

Glial cells Pyramidal neurons amp Capillaries

(containing RBCs)

Stem cells for PD bull Adult rat hemi-brains from Parkinson study

were examined for the presence of cells

immunoreactive for beta-galactosidase The

Lac-Z transfected Scl-40beta pluripotent stem

cell clone derived from adult rats was

identified its stem cell phase and

differentiation phase using an antibody to

beta-galactosidase The tissue harvested was

from needle tracks created by previous

stereotactic injections The tissue was

harvested and stained with antibody to beta-

galactosidase (brown color donor cells) and

counterstained with methyl green (green

color host cells) Immunoreactivity to beta-

galactosidase was expressed within the

cytoplasm of differentiated cells ie glia

pyramidal neurons interneuons and

endothelial cells lining newly formed RBC-

filled capillaries

-Galactosidase expressing cells (brown) = Injected ASC origin

Glial Cells

Capillary

Pyramidal

neurons

(brown cells)

White Matter Gray Matter Gray Matter

Post stem cell for PD

bull Results from the animal study

demonstrated

ndash replacement of dopaminergic neurons in the

area of the 6-OHDA lesion

ndash replacement of damaged neuronal cells

damaged neuronal supportive cells and

damaged vascular structures caused by the

needle injections

The pathway to the brain

Young HE Hyer L Black AC Jr Robinson JS Jr Treating Parkinson disease with adult stem cells J Neurological

Disorders 21 2013 httpdxdoi org104172jnd100121

Parkinsons disease pilot

bull Induction ndash extraction islolation of TPSCs and PPSC intra-nasal

infusion of autologous totipotent stem cells and intravenous infusion

of autologous pluripotent stem cells

bull Serial washing of cells with to remove plasma proteins

bull Tx intra-nasal infusion of TPSCs + IV Inf

bull Intranasal infusion in modified Trendelenberg

position

bull 5 min to insure the deposition of the stem

cells on the olfactory mucosa with migration

between the mucosal cells along the olfactory

processes through the cribriform plate to the

olfactory bulb and posteriorly

along the olfactory nerves to gain entrance

underneath the blood-brain barrier and to the

sub-arachnoid cisterns of the brain

Summary Slide

- Injected primitive PSCs formed dopaminergic neruons within substantia nigra

Unexpected results

- Primitive PSCs also migrated along needle track and regenerated normal tissues and cells in cerebral cortex damaged during stereotactic injections

Glial cells Pyramidal neurons amp Capillaries

(containing RBCs)

Stem cells for PD bull Adult rat hemi-brains from Parkinson study

were examined for the presence of cells

immunoreactive for beta-galactosidase The

Lac-Z transfected Scl-40beta pluripotent stem

cell clone derived from adult rats was

identified its stem cell phase and

differentiation phase using an antibody to

beta-galactosidase The tissue harvested was

from needle tracks created by previous

stereotactic injections The tissue was

harvested and stained with antibody to beta-

galactosidase (brown color donor cells) and

counterstained with methyl green (green

color host cells) Immunoreactivity to beta-

galactosidase was expressed within the

cytoplasm of differentiated cells ie glia

pyramidal neurons interneuons and

endothelial cells lining newly formed RBC-

filled capillaries

-Galactosidase expressing cells (brown) = Injected ASC origin

Glial Cells

Capillary

Pyramidal

neurons

(brown cells)

White Matter Gray Matter Gray Matter

Post stem cell for PD

bull Results from the animal study

demonstrated

ndash replacement of dopaminergic neurons in the

area of the 6-OHDA lesion

ndash replacement of damaged neuronal cells

damaged neuronal supportive cells and

damaged vascular structures caused by the

needle injections

The pathway to the brain

Young HE Hyer L Black AC Jr Robinson JS Jr Treating Parkinson disease with adult stem cells J Neurological

Disorders 21 2013 httpdxdoi org104172jnd100121

Parkinsons disease pilot

bull Induction ndash extraction islolation of TPSCs and PPSC intra-nasal

infusion of autologous totipotent stem cells and intravenous infusion

of autologous pluripotent stem cells

bull Serial washing of cells with to remove plasma proteins

bull Tx intra-nasal infusion of TPSCs + IV Inf

bull Intranasal infusion in modified Trendelenberg

position

bull 5 min to insure the deposition of the stem

cells on the olfactory mucosa with migration

between the mucosal cells along the olfactory

processes through the cribriform plate to the

olfactory bulb and posteriorly

along the olfactory nerves to gain entrance

underneath the blood-brain barrier and to the

sub-arachnoid cisterns of the brain

Post stem cell for PD

bull Results from the animal study

demonstrated

ndash replacement of dopaminergic neurons in the

area of the 6-OHDA lesion

ndash replacement of damaged neuronal cells

damaged neuronal supportive cells and

damaged vascular structures caused by the

needle injections

The pathway to the brain

Young HE Hyer L Black AC Jr Robinson JS Jr Treating Parkinson disease with adult stem cells J Neurological

Disorders 21 2013 httpdxdoi org104172jnd100121

Parkinsons disease pilot

bull Induction ndash extraction islolation of TPSCs and PPSC intra-nasal

infusion of autologous totipotent stem cells and intravenous infusion

of autologous pluripotent stem cells

bull Serial washing of cells with to remove plasma proteins

bull Tx intra-nasal infusion of TPSCs + IV Inf

bull Intranasal infusion in modified Trendelenberg

position

bull 5 min to insure the deposition of the stem

cells on the olfactory mucosa with migration

between the mucosal cells along the olfactory

processes through the cribriform plate to the

olfactory bulb and posteriorly

along the olfactory nerves to gain entrance

underneath the blood-brain barrier and to the

sub-arachnoid cisterns of the brain

Outcome measures

bull Motor via UPDRS-III38

bull Cognition via Trail Making Part A and B39

bull Affect via Beck Depression

bull Scale-II (BDI-II)40 Function via Functional Assessment

Questionnaire (FAQ)

bull Schwab and England disability scale42 and Hoehn-Yahr Scale43

bull Sleep Epworth Sleepiness Scale (ESS)

bull Overall clinical improvement with the CIBIC-Plus (Clinicianrsquos

Interview-Based Impression of Change

bull Caregiver Input) and Caregiver burden Zarit Burden Scale

bull Eval amp FU baseline 2 weeks 3 months 4 months and three

months post baseline (prior to the intranasal

1) Parkinson disease (PD)

1) mild cases return of function (MA)

2) More severe cases ndash see below (MA + NP2)

2) Chronic idiopathic demyelinating polyneuropathy (CIDP) ndash return of function and

loss of symptoms (MA + NP2)

3) Multiple sclerosis (MS) ndash return of function (MA + NP2)

4) Amyotrophic lateral sclerosis (ALS) ndash stabilization of disease process (MA + NP2)

5) Neurogenic stroke ndash return of function (MA + NP2)

6) Alzheimer disease (AD) ndash return of function (MA + NP2)

7) Neuropathies ndash loss of symptoms (MA + NP2)

8) Epilepsy ndash those individuals with clinically-defined epilepsy showed a decrease in

epileptic seizures with a concurrent decrease in epileptic medications (MA)

Neurological conditions treated with nucleated

plasma particles (NP2 ASCs) andor mobilizing

agent

PD Stem Cell Pilot Study

bull 10 participants with 8 completing

the study

bull We chose subjects who had a

Modified Hoehn and Yahr Staging

15 to 4 (Severe Disability)

bull allowing for a ldquomiddlerdquo range of the

disease process Participants were

tested at baseline (pre-treatment)

at treatment and at 1 month 7

months and 14 months post-

treatment

bull At one month post-treatment all

participants demonstrated an

increase in their respective Hoehn-

Yahr scores Four showed an

increase to 60-80 while four

showed an increase to 90 on a

100 scale

Neuropathic pain states

bull Intranasal infusion

ndash CIDP

ndash Neuropathic pain states

ndash Neurofascial hydrodistension

Myocardial Infarction

Repair

Let the Body Induce the Preferred Cell Type

By Injecting Pluripotent Stem Cells

Directly into site of lesion

or

Systemically via Tail Vein

Neuropathic pain states

bull Intranasal infusion

ndash CIDP

ndash Neuropathic pain states

ndash Neurofascial hydrodistension

Myocardial Infarction

Repair

Let the Body Induce the Preferred Cell Type

By Injecting Pluripotent Stem Cells

Directly into site of lesion

or

Systemically via Tail Vein

Direct Injection of naiumlve PSCs (green)

Vasculature

Myocardium Connective Tissues

Cells In Vitro

Systemic Tail Vein Injection of naiumlve PSCs

(green)

Connective Tissues Myocardium

PSCs in Coronary Artery

(red)

Creation of

Pancreatic Islet Organoids

for

Type-I amp Type-II

Diabetes

Steps to creating a new pancrease

Build a Pancreatic Islet Organoid

1) Decellularized Matrix

2) TSCs amp PSCs

3) Donor Islets

A B

C D

B

D

A

C

mf mf mf

bv

mf

mf

PSCs

attached cells

TSCs

(small spherical cells)

Pre-Glucose Incubation Post-Glucose Incubation

40x

100x 100x

200x

Islets increase integrity

A

B

C

D

Islets

Islets

Pre-Glucose Incubation ~ 3d Post-Glucose Incubation ~ 14 days

100x

100x

100x

100x Islets slightly increase in integrity

Islets

Islets

A B

C D

Parameters for Glucose

Challenge

Native Pancreatic Islets (alone)

Pancreatic Organoids with Matrix-A

Pancreatic Organoids with Matrix-B

Measured insulin secretion sequentially in

response to a glucose challenge in vitro

5 mM glucose for 24 hr

5 mM glucose for 1 hr

25 mM glucose for 1 hr

(Lumelsky et al Science 2921389-1393 2001)

Measured secretion with Rat-specific

Insulin-RIA

Measured insulin secretion sequentially in

response to a glucose challenge in vitro

5 mM glucose for 24 hr

5 mM glucose for 1 hr

25 mM glucose for 1 hr

(Lumelsky et al Science 2921389-1393 2001)

Measured secretion with Rat-specific

Insulin-RIA

22 07 0332

177

221 236

477

773

0

10

20

30

40

50

60

70

80

90

5 mM 24 hr 5 mM 1 hr 25 mM 1 hr

ug

in

su

lin

per

ng

DN

A

Native Islets (NI) Matrix A + SC + NI Matrix B + SC + NI

Insulin Secretion in Response to a Glucose Challenge (n=6)

g insulin per

ng DNA

Respiratory Diseases

1) Individuals with COPD and having a FEV1 of greater than

50 showed benefit with ingestion of mobilizing agent only in

some instances increasing their FEV1 to greater than 85

2) However for those individuals with an FEV1 of less than

50 no increase in FEV1 was noted after long term

ingestion of mobilizing agent only

3) Individuals with FEV1s less than 50 noted improvement

ie increase in FEV1 after treatment with MA + autologous

NP2

Musculoskeletal

1) Acceleration (2x) of the normal healing process occurred in an individual

with a torsion fracture of the fibula with ingestion of mobilizing agent only and

simple casting of the lower extremity

2) Individuals with rotator cuff injuries necessitating surgery demonstrated

healing of their rotator cuff injuries after long term ingestion of mobilizing

agent

3) Other individuals reported less painful osteoarthritic joints after long term

ingestion of mobilizing agent

Other Conditions

1) Type-I diabetic with brown recluse spider bite completely healed in 2 weeks with

MA + single autologous NP2 transplant

2) Individual with SLE Celiac disease and multiple allergies MA + multiple

autologous amp allogeneic NP2 transplants reverted from SLE-Type-IV to SLE-Type-

II and lost allergies to gluten eggs fowl tree nuts

Regenerative Medicine

Characteristics

ESCs or iPSCs Adult NP2s

Proliferation Unlimited Unlimited

Form cell types All All

Naiumlve in vitro Spontan Differen Quiescence

Naiumlve in vivo Teratomas Repair or Maintain

(Cancerous cells) Quiescence

Implant in vivo Differentiated Cells Naiumlve Particles

HLA mismatch Allogeneic Allogeneic

HLA match Therapeutic cloning Autologous

In summaryhellip bull A new era of orthopedic amp regenerative

medicine

ndash A new technology From autologous cell mix

techniques to true TP PP stem cell therapies

ndash A new subspecialty is emerging created by clinical

hybrids

bull Offers patients with spine and orthopedic difficulties new

hope

bull A new change for the baby boomer population who want to

stay active

bull Vascular disease

bull Neurological disease

bull Cardiac disease

bull Respiratory disease

Center for Orthopedic

amp

Regenerative Medicine

Science

Michael N Brown MD DC DABPMR-PAIN

PSCs in Coronary Artery

(red)

Creation of

Pancreatic Islet Organoids

for

Type-I amp Type-II

Diabetes

Steps to creating a new pancrease

Build a Pancreatic Islet Organoid

1) Decellularized Matrix

2) TSCs amp PSCs

3) Donor Islets

A B

C D

B

D

A

C

mf mf mf

bv

mf

mf

PSCs

attached cells

TSCs

(small spherical cells)

Pre-Glucose Incubation Post-Glucose Incubation

40x

100x 100x

200x

Islets increase integrity

A

B

C

D

Islets

Islets

Pre-Glucose Incubation ~ 3d Post-Glucose Incubation ~ 14 days

100x

100x

100x

100x Islets slightly increase in integrity

Islets

Islets

A B

C D

Parameters for Glucose

Challenge

Native Pancreatic Islets (alone)

Pancreatic Organoids with Matrix-A

Pancreatic Organoids with Matrix-B

Measured insulin secretion sequentially in

response to a glucose challenge in vitro

5 mM glucose for 24 hr

5 mM glucose for 1 hr

25 mM glucose for 1 hr

(Lumelsky et al Science 2921389-1393 2001)

Measured secretion with Rat-specific

Insulin-RIA

Measured insulin secretion sequentially in

response to a glucose challenge in vitro

5 mM glucose for 24 hr

5 mM glucose for 1 hr

25 mM glucose for 1 hr

(Lumelsky et al Science 2921389-1393 2001)

Measured secretion with Rat-specific

Insulin-RIA

22 07 0332

177

221 236

477

773

0

10

20

30

40

50

60

70

80

90

5 mM 24 hr 5 mM 1 hr 25 mM 1 hr

ug

in

su

lin

per

ng

DN

A

Native Islets (NI) Matrix A + SC + NI Matrix B + SC + NI

Insulin Secretion in Response to a Glucose Challenge (n=6)

g insulin per

ng DNA

Respiratory Diseases

1) Individuals with COPD and having a FEV1 of greater than

50 showed benefit with ingestion of mobilizing agent only in

some instances increasing their FEV1 to greater than 85

2) However for those individuals with an FEV1 of less than

50 no increase in FEV1 was noted after long term

ingestion of mobilizing agent only

3) Individuals with FEV1s less than 50 noted improvement

ie increase in FEV1 after treatment with MA + autologous

NP2

Musculoskeletal

1) Acceleration (2x) of the normal healing process occurred in an individual

with a torsion fracture of the fibula with ingestion of mobilizing agent only and

simple casting of the lower extremity

2) Individuals with rotator cuff injuries necessitating surgery demonstrated

healing of their rotator cuff injuries after long term ingestion of mobilizing

agent

3) Other individuals reported less painful osteoarthritic joints after long term

ingestion of mobilizing agent

Other Conditions

1) Type-I diabetic with brown recluse spider bite completely healed in 2 weeks with

MA + single autologous NP2 transplant

2) Individual with SLE Celiac disease and multiple allergies MA + multiple

autologous amp allogeneic NP2 transplants reverted from SLE-Type-IV to SLE-Type-

II and lost allergies to gluten eggs fowl tree nuts

Regenerative Medicine

Characteristics

ESCs or iPSCs Adult NP2s

Proliferation Unlimited Unlimited

Form cell types All All

Naiumlve in vitro Spontan Differen Quiescence

Naiumlve in vivo Teratomas Repair or Maintain

(Cancerous cells) Quiescence

Implant in vivo Differentiated Cells Naiumlve Particles

HLA mismatch Allogeneic Allogeneic

HLA match Therapeutic cloning Autologous

In summaryhellip bull A new era of orthopedic amp regenerative

medicine

ndash A new technology From autologous cell mix

techniques to true TP PP stem cell therapies

ndash A new subspecialty is emerging created by clinical

hybrids

bull Offers patients with spine and orthopedic difficulties new

hope

bull A new change for the baby boomer population who want to

stay active

bull Vascular disease

bull Neurological disease

bull Cardiac disease

bull Respiratory disease

Center for Orthopedic

amp

Regenerative Medicine

Science

Michael N Brown MD DC DABPMR-PAIN

Build a Pancreatic Islet Organoid

1) Decellularized Matrix

2) TSCs amp PSCs

3) Donor Islets

A B

C D

B

D

A

C

mf mf mf

bv

mf

mf

PSCs

attached cells

TSCs

(small spherical cells)

Pre-Glucose Incubation Post-Glucose Incubation

40x

100x 100x

200x

Islets increase integrity

A

B

C

D

Islets

Islets

Pre-Glucose Incubation ~ 3d Post-Glucose Incubation ~ 14 days

100x

100x

100x

100x Islets slightly increase in integrity

Islets

Islets

A B

C D

Parameters for Glucose

Challenge

Native Pancreatic Islets (alone)

Pancreatic Organoids with Matrix-A

Pancreatic Organoids with Matrix-B

Measured insulin secretion sequentially in

response to a glucose challenge in vitro

5 mM glucose for 24 hr

5 mM glucose for 1 hr

25 mM glucose for 1 hr

(Lumelsky et al Science 2921389-1393 2001)

Measured secretion with Rat-specific

Insulin-RIA

Measured insulin secretion sequentially in

response to a glucose challenge in vitro

5 mM glucose for 24 hr

5 mM glucose for 1 hr

25 mM glucose for 1 hr

(Lumelsky et al Science 2921389-1393 2001)

Measured secretion with Rat-specific

Insulin-RIA

22 07 0332

177

221 236

477

773

0

10

20

30

40

50

60

70

80

90

5 mM 24 hr 5 mM 1 hr 25 mM 1 hr

ug

in

su

lin

per

ng

DN

A

Native Islets (NI) Matrix A + SC + NI Matrix B + SC + NI

Insulin Secretion in Response to a Glucose Challenge (n=6)

g insulin per

ng DNA

Respiratory Diseases

1) Individuals with COPD and having a FEV1 of greater than

50 showed benefit with ingestion of mobilizing agent only in

some instances increasing their FEV1 to greater than 85

2) However for those individuals with an FEV1 of less than

50 no increase in FEV1 was noted after long term

ingestion of mobilizing agent only

3) Individuals with FEV1s less than 50 noted improvement

ie increase in FEV1 after treatment with MA + autologous

NP2

Musculoskeletal

1) Acceleration (2x) of the normal healing process occurred in an individual

with a torsion fracture of the fibula with ingestion of mobilizing agent only and

simple casting of the lower extremity

2) Individuals with rotator cuff injuries necessitating surgery demonstrated

healing of their rotator cuff injuries after long term ingestion of mobilizing

agent

3) Other individuals reported less painful osteoarthritic joints after long term

ingestion of mobilizing agent

Other Conditions

1) Type-I diabetic with brown recluse spider bite completely healed in 2 weeks with

MA + single autologous NP2 transplant

2) Individual with SLE Celiac disease and multiple allergies MA + multiple

autologous amp allogeneic NP2 transplants reverted from SLE-Type-IV to SLE-Type-

II and lost allergies to gluten eggs fowl tree nuts

Regenerative Medicine

Characteristics

ESCs or iPSCs Adult NP2s

Proliferation Unlimited Unlimited

Form cell types All All

Naiumlve in vitro Spontan Differen Quiescence

Naiumlve in vivo Teratomas Repair or Maintain

(Cancerous cells) Quiescence

Implant in vivo Differentiated Cells Naiumlve Particles

HLA mismatch Allogeneic Allogeneic

HLA match Therapeutic cloning Autologous

In summaryhellip bull A new era of orthopedic amp regenerative

medicine

ndash A new technology From autologous cell mix

techniques to true TP PP stem cell therapies

ndash A new subspecialty is emerging created by clinical

hybrids

bull Offers patients with spine and orthopedic difficulties new

hope

bull A new change for the baby boomer population who want to

stay active

bull Vascular disease

bull Neurological disease

bull Cardiac disease

bull Respiratory disease

Center for Orthopedic

amp

Regenerative Medicine

Science

Michael N Brown MD DC DABPMR-PAIN

Pre-Glucose Incubation Post-Glucose Incubation

40x

100x 100x

200x

Islets increase integrity

A

B

C

D

Islets

Islets

Pre-Glucose Incubation ~ 3d Post-Glucose Incubation ~ 14 days

100x

100x

100x

100x Islets slightly increase in integrity

Islets

Islets

A B

C D

Parameters for Glucose

Challenge

Native Pancreatic Islets (alone)

Pancreatic Organoids with Matrix-A

Pancreatic Organoids with Matrix-B

Measured insulin secretion sequentially in

response to a glucose challenge in vitro

5 mM glucose for 24 hr

5 mM glucose for 1 hr

25 mM glucose for 1 hr

(Lumelsky et al Science 2921389-1393 2001)

Measured secretion with Rat-specific

Insulin-RIA

Measured insulin secretion sequentially in

response to a glucose challenge in vitro

5 mM glucose for 24 hr

5 mM glucose for 1 hr

25 mM glucose for 1 hr

(Lumelsky et al Science 2921389-1393 2001)

Measured secretion with Rat-specific

Insulin-RIA

22 07 0332

177

221 236

477

773

0

10

20

30

40

50

60

70

80

90

5 mM 24 hr 5 mM 1 hr 25 mM 1 hr

ug

in

su

lin

per

ng

DN

A

Native Islets (NI) Matrix A + SC + NI Matrix B + SC + NI

Insulin Secretion in Response to a Glucose Challenge (n=6)

g insulin per

ng DNA

Respiratory Diseases

1) Individuals with COPD and having a FEV1 of greater than

50 showed benefit with ingestion of mobilizing agent only in

some instances increasing their FEV1 to greater than 85

2) However for those individuals with an FEV1 of less than

50 no increase in FEV1 was noted after long term

ingestion of mobilizing agent only

3) Individuals with FEV1s less than 50 noted improvement

ie increase in FEV1 after treatment with MA + autologous

NP2

Musculoskeletal

1) Acceleration (2x) of the normal healing process occurred in an individual

with a torsion fracture of the fibula with ingestion of mobilizing agent only and

simple casting of the lower extremity

2) Individuals with rotator cuff injuries necessitating surgery demonstrated

healing of their rotator cuff injuries after long term ingestion of mobilizing

agent

3) Other individuals reported less painful osteoarthritic joints after long term

ingestion of mobilizing agent

Other Conditions

1) Type-I diabetic with brown recluse spider bite completely healed in 2 weeks with

MA + single autologous NP2 transplant

2) Individual with SLE Celiac disease and multiple allergies MA + multiple

autologous amp allogeneic NP2 transplants reverted from SLE-Type-IV to SLE-Type-

II and lost allergies to gluten eggs fowl tree nuts

Regenerative Medicine

Characteristics

ESCs or iPSCs Adult NP2s

Proliferation Unlimited Unlimited

Form cell types All All

Naiumlve in vitro Spontan Differen Quiescence

Naiumlve in vivo Teratomas Repair or Maintain

(Cancerous cells) Quiescence

Implant in vivo Differentiated Cells Naiumlve Particles

HLA mismatch Allogeneic Allogeneic

HLA match Therapeutic cloning Autologous

In summaryhellip bull A new era of orthopedic amp regenerative

medicine

ndash A new technology From autologous cell mix

techniques to true TP PP stem cell therapies

ndash A new subspecialty is emerging created by clinical

hybrids

bull Offers patients with spine and orthopedic difficulties new

hope

bull A new change for the baby boomer population who want to

stay active

bull Vascular disease

bull Neurological disease

bull Cardiac disease

bull Respiratory disease

Center for Orthopedic

amp

Regenerative Medicine

Science

Michael N Brown MD DC DABPMR-PAIN

Parameters for Glucose

Challenge

Native Pancreatic Islets (alone)

Pancreatic Organoids with Matrix-A

Pancreatic Organoids with Matrix-B

Measured insulin secretion sequentially in

response to a glucose challenge in vitro

5 mM glucose for 24 hr

5 mM glucose for 1 hr

25 mM glucose for 1 hr

(Lumelsky et al Science 2921389-1393 2001)

Measured secretion with Rat-specific

Insulin-RIA

Measured insulin secretion sequentially in

response to a glucose challenge in vitro

5 mM glucose for 24 hr

5 mM glucose for 1 hr

25 mM glucose for 1 hr

(Lumelsky et al Science 2921389-1393 2001)

Measured secretion with Rat-specific

Insulin-RIA

22 07 0332

177

221 236

477

773

0

10

20

30

40

50

60

70

80

90

5 mM 24 hr 5 mM 1 hr 25 mM 1 hr

ug

in

su

lin

per

ng

DN

A

Native Islets (NI) Matrix A + SC + NI Matrix B + SC + NI

Insulin Secretion in Response to a Glucose Challenge (n=6)

g insulin per

ng DNA

Respiratory Diseases

1) Individuals with COPD and having a FEV1 of greater than

50 showed benefit with ingestion of mobilizing agent only in

some instances increasing their FEV1 to greater than 85

2) However for those individuals with an FEV1 of less than

50 no increase in FEV1 was noted after long term

ingestion of mobilizing agent only

3) Individuals with FEV1s less than 50 noted improvement

ie increase in FEV1 after treatment with MA + autologous

NP2

Musculoskeletal

1) Acceleration (2x) of the normal healing process occurred in an individual

with a torsion fracture of the fibula with ingestion of mobilizing agent only and

simple casting of the lower extremity

2) Individuals with rotator cuff injuries necessitating surgery demonstrated

healing of their rotator cuff injuries after long term ingestion of mobilizing

agent

3) Other individuals reported less painful osteoarthritic joints after long term

ingestion of mobilizing agent

Other Conditions

1) Type-I diabetic with brown recluse spider bite completely healed in 2 weeks with

MA + single autologous NP2 transplant

2) Individual with SLE Celiac disease and multiple allergies MA + multiple

autologous amp allogeneic NP2 transplants reverted from SLE-Type-IV to SLE-Type-

II and lost allergies to gluten eggs fowl tree nuts

Regenerative Medicine

Characteristics

ESCs or iPSCs Adult NP2s

Proliferation Unlimited Unlimited

Form cell types All All

Naiumlve in vitro Spontan Differen Quiescence

Naiumlve in vivo Teratomas Repair or Maintain

(Cancerous cells) Quiescence

Implant in vivo Differentiated Cells Naiumlve Particles

HLA mismatch Allogeneic Allogeneic

HLA match Therapeutic cloning Autologous

In summaryhellip bull A new era of orthopedic amp regenerative

medicine

ndash A new technology From autologous cell mix

techniques to true TP PP stem cell therapies

ndash A new subspecialty is emerging created by clinical

hybrids

bull Offers patients with spine and orthopedic difficulties new

hope

bull A new change for the baby boomer population who want to

stay active

bull Vascular disease

bull Neurological disease

bull Cardiac disease

bull Respiratory disease

Center for Orthopedic

amp

Regenerative Medicine

Science

Michael N Brown MD DC DABPMR-PAIN

Respiratory Diseases

1) Individuals with COPD and having a FEV1 of greater than

50 showed benefit with ingestion of mobilizing agent only in

some instances increasing their FEV1 to greater than 85

2) However for those individuals with an FEV1 of less than

50 no increase in FEV1 was noted after long term

ingestion of mobilizing agent only

3) Individuals with FEV1s less than 50 noted improvement

ie increase in FEV1 after treatment with MA + autologous

NP2

Musculoskeletal

1) Acceleration (2x) of the normal healing process occurred in an individual

with a torsion fracture of the fibula with ingestion of mobilizing agent only and

simple casting of the lower extremity

2) Individuals with rotator cuff injuries necessitating surgery demonstrated

healing of their rotator cuff injuries after long term ingestion of mobilizing

agent

3) Other individuals reported less painful osteoarthritic joints after long term

ingestion of mobilizing agent

Other Conditions

1) Type-I diabetic with brown recluse spider bite completely healed in 2 weeks with

MA + single autologous NP2 transplant

2) Individual with SLE Celiac disease and multiple allergies MA + multiple

autologous amp allogeneic NP2 transplants reverted from SLE-Type-IV to SLE-Type-

II and lost allergies to gluten eggs fowl tree nuts

Regenerative Medicine

Characteristics

ESCs or iPSCs Adult NP2s

Proliferation Unlimited Unlimited

Form cell types All All

Naiumlve in vitro Spontan Differen Quiescence

Naiumlve in vivo Teratomas Repair or Maintain

(Cancerous cells) Quiescence

Implant in vivo Differentiated Cells Naiumlve Particles

HLA mismatch Allogeneic Allogeneic

HLA match Therapeutic cloning Autologous

In summaryhellip bull A new era of orthopedic amp regenerative

medicine

ndash A new technology From autologous cell mix

techniques to true TP PP stem cell therapies

ndash A new subspecialty is emerging created by clinical

hybrids

bull Offers patients with spine and orthopedic difficulties new

hope

bull A new change for the baby boomer population who want to

stay active

bull Vascular disease

bull Neurological disease

bull Cardiac disease

bull Respiratory disease

Center for Orthopedic

amp

Regenerative Medicine

Science

Michael N Brown MD DC DABPMR-PAIN

Regenerative Medicine

Characteristics

ESCs or iPSCs Adult NP2s

Proliferation Unlimited Unlimited

Form cell types All All

Naiumlve in vitro Spontan Differen Quiescence

Naiumlve in vivo Teratomas Repair or Maintain

(Cancerous cells) Quiescence

Implant in vivo Differentiated Cells Naiumlve Particles

HLA mismatch Allogeneic Allogeneic

HLA match Therapeutic cloning Autologous

In summaryhellip bull A new era of orthopedic amp regenerative

medicine

ndash A new technology From autologous cell mix

techniques to true TP PP stem cell therapies

ndash A new subspecialty is emerging created by clinical

hybrids

bull Offers patients with spine and orthopedic difficulties new

hope

bull A new change for the baby boomer population who want to

stay active

bull Vascular disease

bull Neurological disease

bull Cardiac disease

bull Respiratory disease

Center for Orthopedic

amp

Regenerative Medicine

Science

Michael N Brown MD DC DABPMR-PAIN