peripheral blood stem cells - · 2018-04-09 · peripheral blood stem cells ... annual symposium...

Peripheral Blood Stem Cells

Charles F. Mahl MD FACS FICS

GenLife Regenerative Medicine

2333 Ponce De Leon Blvd Suite 302

Coral Gables, Miami, Florida 33134

[email protected]

305 332-7234

305 669-0241 FaxCharles F Mahl MD FACS FICS

mailto:[email protected]

Definitions

• Pluripotent Stem Cells-4 subgroups (ie: HaLscs, CoLSCs, ELSCs, GLSCs) based on size and surface markers, capable of forming all somatic cell types within the body, telomerase positive and capable of unlimited proliferation potential

• Totipotent Stem Cells -2 subgroups (ie: BLSCs-sm, BLSCs-lg) based on size and attachment issues, capable of forming all somatic cell types and the germ cells (sperm and ova), telomerase positive and capable of unlimited proliferation as long as cells remain undifferentiated.

• Mesodermal Stem Cells(MesoSCs) capable of forming any cell type from mesodermal germ layer lineage, telomerase positive and capable of unlimited cell proliferation

• Progentior cells –aka maintenance cells- support the functional (aka differentiated) cells on a day to day basis. As functional cells wear out and die, maintenance cells proliferate, differentiate, and become their replacements. They are cells that have committed to a particular tissue lineage, are telomerase negative, have limited proliferation potential (human 50-70 population doublings before pre-programmed senescence and death)

Charles F Mahl MD FACS FICS

Types of Progenitor Cells

• Adipofibroblast – a bipotent pc with the capability to form adipose tissue and fibroblasts.

• Chondroblast - a unipotent pc with capability to form cartilage

• Hematopoietic Stem Cell (HCs) – a multipotent pc with capability to form all 18 + cell lineages within the hematopoietic cell lineage-ie: RBCs, WBCs, NK-cells,

• Mesenchymal Stem Cells (MSCs)- a tripotent pcwith capability to form 3 tissues-adipose, cartilage and bone.

• Myoblast – a unipotent pc – forms muscle• Osteoblast - a unipotent pc that forms bone


Cell Types

• Healing cells-true stem cells; are normally dormant and can be found hibernating within the stromal connective tissue of the body. Their function is to replace functional cells ad maintenance cells lost due to trauma and/or disease. Once healing cells become maintenance cells and/or functional cells, they assume the respective loss of telomerase activity, lifespans, genetic programming, aging and death of these two groups of cells. They are telomerase positive, have unlimited lifespan until they become the other two cell types.

• Maintenance cells-progenitor cells, x-blasts. –

support the functional cells on a day to day basis; as functional cells wear out and die, the maintenance cells proliferate, differentiate, and become their replacem,ents. They can be multi, tri, bi or uni-potent, they are telomerase negative and have limited lifespan as they are programmed to age and die and they decrease in number with aging.

• Functional cells –differentiated cells, x-cytes – composed of parenchyma and stroma and comprise the majority of 220 known cell types in the body. They function on a day to day basis for sensory input, regulation of internal function, gas exchange, nutrition, waste removal, defense, energy storage, reproduction, support, ambnulation, etc. Telomerase negative and have defined lifespan as programmed to age and die.


Distinct Population of Pluripotent Stem Cells in Peripheral Blood

• High numbers in Peripheral Blood• Can use autologous • Can use young healthy donor (allogeneic)• Can combine with PRP (from same sample)-

double whammy• No issue with FDA• Simple Procedure• Less invasive/Less Expensive than BMAC and

ADSC• No large set up• “PRP on Steroids”


Why PBSC ?

• Easier to perform for both physician and patient

• Ability to enhance bone marrow stem cell production before blood draw

• Results in musculoskeletal injuries and diseases?

• Results in TBI ?• Results in Age Frailty and Organ Regeneration?


Cell Type Specifics for Diseases

• Musculoskeletal –TSCs + PSCs + MesoSCs• Brain – use TSCs (0.1-2.0 microns) only• (NOT PSCs because they too large to fit

between the nasal olfactory cells and will clog the spaces between the olfactory cells.

• COPD – nebulize both PSCs (6-8 microns) and TSCs to form the endoderm-derived epithelium that forms the inner lining of alveolar sacs and alveolar ducts


PBSC-PRP Kits

• Combine the well known and documented benefits of PRP, a concentration of platelets in plasma, containing many growth and healing factors, that initiate repair and attract the critical assistance of stem cells with stem cells that are taken from whole blood via IV .


PBSC

• There is a distinct population of pluripotent stem cells in peripheral blood



• Tithon Biotech has discovered an isolated pluripotent stem cell contained in peripheral blood.

• This stem cell exhibits great potential in both regenerative and therapeutic procedures in humans.

• In each case these cells are re-delivered through a cell isolation processing device, employing a simple medical procedure commonly used in PRP therapies.

• There are hundreds of thousands of these cells per ml of plasma in the peripheral blood depending on age.


Henry Young Ph.D PBSC/Intranasal Stem Cells Study• Licensed Investigator• National Clinical Trial• Investigational Review Board Oversight• Fully Funded for Patients (only $100

application fee)• Requires 2 Year Commitment• TBI/Alzheimer’s/Parkinson’s

Disease/Autoimmune/ Musculoskeletal/Osteoarthritis/etc

• 40 Patients with Each Specific Disease


Henry E. Young PhD Study

• A PHASE-IA, NON-BLINDED MULTIPLE CASE STUDY TRIAL INDIVIDUALLY EVALUATING THE SAFETY AND EFFICACY OF USE OF AUTOLOGOUS NUCLEATED PLASMA PARTICLES (NucP2s) FOR THE TREATMENT OF TRAUMATIC BRAIN INJURY, NEURODEGENERATIVE DISEASES AND PULMONARY DISEASES.


Young PBSC Study

• This is a clinical trial assessing the safety and efficacy of autologous totipotent and pluripotent stem cells in the treatment of neurodegenerative and pulmonary diseases.

• It includes the utilization of nutraceutical formulations to optimize circulating stem cell counts in hopes of repairing damaged tissues as seen in neurodegenerative and traumatic brain injured patients.

• A month long pretreatment nutritional protocol is applied followed by isolation of circulating stem cells from the patient’s blood. These cells are then activated and returned to the patient in hopes of repairing damaged tissues. The activation process is thought to synchronize the cells to work in concert to better repair injured tissue.

• Patients with osteoarthritis,multiple sclerosis, chronic inflammatory demyelinating polyneuropathy, copd, traumatic brain injured patients, partial spinal cord injuries ( non-severed cord), Alzheimer’s and Parkinson’s Disease may be candidates for this trial.


Use of Supplements to enhance Stem Cell Population

• If you use certain supplements for 30 days, you can increase the number of stem cells harvested by 3 x.

• There are 1 million stem cells in blood; if you supplement for 30 days, you cause the stem cells in peripheral tissue to start proliferating; then a 2nd treatment of supplements causes these cells to mobilized and go into peripheral blood, yielding 3 million cells.


Publications by Henry Young PhD 1

• Young HE. Pluripotent stem cells. Edited by M.A. Brown and S. Neufield, Cambridge Healthtech Institute Press, Newton Upper Falls, MA. In: Second Annual Symposium on Tissue Engineering / Regenerative Healing / Stem Cell Biology, 469-530, 1999.

• Young HE. Stem cells and tissue engineering. In: Gene Therapy in Orthopaedic and Sports Medicine, J. Huard and F.H. Fu, eds., Springer-Verlag New York, Inc., Chap. 9, pg. 143-173, 2000.

• Young. Existence of reserve quiescent stem cells in adults, from amphibians to humans. Curr Top Microbiol Immunol. 280:71-109, 2004.

• Young and Black. Adult stem cells. Anat. Rec. 276A:75-102, 2004.

• Young and Black. Differentiation potential of adult stem cells. In: Contemporary Endocrinology: Stem Cells in Endocrinology, L.B. Lester, ed., The Humana Press Inc., Totowa, NJ. Chap. 4, p. 67-92, 2005.

• Young and Black. Adult-derived stem cells. Minerva Biotechnologica Cancer Gene Mechanisms and Gene Therapy Reviews 17:55-63, 2005.

• Young and Black. Naturally occurring adult pluripotent stem cells. In: Stem Cells: From Biology to Therapy, Advances in Molecular Biology and Medicine. 1st Ed, R.A. Meyers, Ed, WILEY-BLACKWELL-VCH Verlag GmbH & Co. KGaA. Chap 3, pp. 63-93, 2013.

• Young and Black. Pluripotent Stem Cells, Endogenous versus Reprogrammed, a Review. MOJ Orthop Rheumatol 1(4): 00019, 2014.



• Young et al. Cryopreservation of embryonic chick myogenic lineage-committed stem cells. Journal of Tissue Culture Methods, 13:275-284, 1991.

• Young et al. Enzyme-linked immuno-culture assay. Journal of Tissue Culture Methods, 14:31-36, 1992.

• Young et al. Isolation of embryonic chick myosatellite and pluripotent stem cells. Journal of Tissue Culture Methods, 14:85-92, 1992.

• Young et al. Pluripotent mesenchymal stem cells reside within avian connective tissue matrices. In Vitro Cellular & Developmental Biology, 29A:723-736, 1993.

• Young et al. Mesenchymal stem cells reside within the connective tissues of many organs. Developmental Dynamics 202:137-144, 1995.

• Young et al. Bioactive factors affect proliferation and phenotypic expression in pluripotent and progenitor mesenchymal stem cells. Wound Repair and Regeneration 6(1):65-75, 1998.

• Young et al. Muscle morphogenetic protein induces myogenic gene expression in Swiss-3T3 cells. Wound Rep Reg 6(6):543-554, 1998.



• Young et al. Human progenitor and pluripotent cells display cell surface cluster differentiation markers CD10, CD13, CD56, CD90 and MHC Class-I. Proc. Soc. Exp. Biol. Med. 221:63-71, 1999.

• Young et al. Clonogenic analysis reveals reserve stem cells in postnatal mammals. I. Pluripotent mesenchymal stem cells. Anat. Rec. 263:350-360, 2001.

• Young et al. 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. 264:51-62, 2001.

• Young et al. Adult reserve stem cells and their potential for tissue engineering. Cell Biochem Biophys, 40(1):1-80, 2004.

• Young et al. Clonogenic analysis reveals reserve stem cells in postnatal mammals. II. Pluripotent epiblastic-like stem cells. Anat. Rec. 277A:178-203, 2004.

• Young et al. Adult-derived stem cells and their potential for tissue repair and molecular medicine. J Cell Molec Med 9:753-769, 2005.

• Young et al. Adult stem cells: from bench-top to bedside. In: Tissue Regeneration: Where Nanostructure Meets Biology, 3DBiotech, North Brunswick, NJ Chap 1, pp 1-60, 2013.

• Young et al. Treating Parkinson disease with adult stem cells. J Neurological Disorders, 2:1, 2013.



• oung et al. Environmental conditions prerequisite for complete limb regeneration in the postmetamorphic adult land-phase salamander, Ambystoma. Anatomical Record, 206:289-294, 1983.

• Young et al. Gross morphological analysis of limb regeneration in postmetamorphic adult Ambystoma. Anatomical Record, 206:295-306, 1983.

• Young et al. Identification of hyaluronate within peripheral nervous tissue matrices during limb regeneration. Edited by Coates, P.W., Markwald, R.R., Kenny, A.D., Alan R. Liss, Inc., New York. In: Developing and Regenerating Vertebrate Nervous Systems, Neurology and Neurobiology, 6:175-183, 1983.

• Young et al. Histological analysis of limb regeneration in postmetamorphic adult Ambystoma. Anatomical Record, 212:183-194, 1985.

• Young et al. Initial characterization of small proteoglycans synthesized by embryonic chick leg muscle-associated connective tissues. Connective Tissue Research, 17:99-118, 1988.

• Young et al. Effect of selected denervations on glycoconjugate composition and tissue morphology during the initiation phase of limb regeneration in adult Ambystoma. Anatomical Record, 223:223-230, 1989.

• Young et al. Glycoconjugates in normal wound tissue matrices during the initiation phase of limb regeneration in adult Ambystoma. Anatomical Record, 223:231-241, 1989.

• Young et al. Histochemical analysis of newly synthesized and resident sulfated glycosaminoglycans during musculogenesis in the embryonic chick leg. Journal of Morphology, 201:85-103, 1989.

• Young et al. Changes in synthesis of sulfated glycoconjugates during muscle development, maturation, and aging in embryonic to senescent CBF-1 mouse. Mechanisms of Ageing and Development, 53:179-193, 1990.


PDGF BB Enhances Osteogenesis in Adipose but not BM XFLAWED

• Stem Cells. 2015 Sep;33(9):2773-84.• Platelet-Derived Growth Factor BB Enhances

Osteogenesis of Adipose-Derived But Not Bone Marrow-Derived Mesenchymal

• Stromal/Stem Cells, Hung BP et al.


Allogeneic Donors• The donors need to be SAME gender; the

same blood type or have O-negative blood (O-negative is universal donor to 99.99% of the population) [However, if they have the BOMBAY blood group (0.01% of population) it has to be from another with BOMBAY blood group (individuals from the Bombay area of India)]; do NOT have a family history of ANY genetic disease; do NOT have an infectious disease that can be transmitted to the recipient through body fluids; have a similar personality (personality can change to that of donor and similar hair color - there may be a change in hair color to donor's original hair color


Allogeneic Donors

• Donors will be screened for genetic diseases using gene sequencing (pricey) and infectious disease testing (also pricey)


Their hypothesis may be correct, their method for testing "direct comparisons" has so many

flaws:• 1) The two groups examined were different, both in gender and in age

• MSCs - ALL male, 20's to 32• ASCs - ALL female, 47 to 54

• 2) Life style for either group are unknown - factors known to affect viability of cell types include alcohol, caffeine, smoking, sleep, exercise.

• 3) The composition of cells within bone marrow and adipose tissue are naturally different.

• 4) CD marker analysis was for identification only, not for cell sorting - meaning only 4 CD markers were used for identification. Other cell types might have been present, but without using appropriate CD markers they would never have been recognized. The non-identified cell types may be the ones being tested.

• 5) Different concentrations of FBS were used in osteogenic (10%) versus non-osteogenic (6%) media. Suggest that factor(s) present in serum had either additive or synergistic effects with other media constituents. To be comparative, FBS concentrations need to be the same.


PDGF-BB in Adipose vs BM

• Suggest comparing BM-MSCs versus Adipose-ASCs with PDGF-BB using 5 ng/ml in the SAME individual and see which population is better. Since it is the same patient, you have controlled for everything, i.e., gender, age, lifestyle, composition, cell ID, etc.

• That is the ONLY true comparison between the groups of cells to determine which is the preferred group for osteogenesis.


Diseases

• Osteoarthritis• Joint and Connective tissue Disorders• TBI• Parkinsons• Alzheimer’s Disease• Multiple myeloma • Leukemia • Multiple Sclerosis• Aplastic Anemia • Diabetic foot/Ischemic Limb• Thalassemia


PBSC

• Circulating CD34+ stem cells are present in blood circulation also; however, their number is too small to be used for clinical transplantation.

• With the advent of growth factors and mobilization techniques, peripheral blood stem cells (PBSCs) have witnessed dramatic increase in its usage for treating hematologic malignancies.

• Peripheral blood stem cells (PBSCs) have become a promising alternative to bone marrow grafting in the last two decades and replaced bone marrow transplant to a large extent.

• Korbling M, Freireich EJ. Twenty-five years of peripheral blood stem cell transplantation. Blood. 2011;117(24):6411-6.

•


Increasing The Stem Cell Population

• GM-CSF (Granulocyte-macrophage colony stimulating factor)

• Recombinant human erythropoietin (EPO• Stem cell factor (SCF) • Recombinant human TPO (rhTPO) • Pegfilgrastim (Pegylated G-CSF) • Plerixafor • Collection- apheresis technique• Supplements • PTH


Supplements

• Stem X Cell – vit D3, antioxidants, green tea, etc

• Neo 40 –Nitric Oxide• TA 65 -Telomeres• Vitamin D3• Vitamin C• B Complex• Stem Kine - Lactobacillus Fermentum and the

powerful antioxidants astragalus root, goji berry, and green tea.

• Resveratrol• L Carnosine• NG- Stem-Trition- t AFA (Aphanizomenon Flos-

Aquae-fresh water cyanobacter) –this sti,ulates replacement of cells but does not increase stem cell numbers above what was lost. The commonly available products can contain heavy metals and some others contain microcrystalins which could cause liver damage.

• Flucoidans-polysaccharide found in brown algae and brown seaweed


Mobilizing Stem Cells

• The first mechanism discovered to mobilize stem cells from bone marrow was chemotherapy. 50 fold increase in number of HSC.

• The discovery of G-CSF Recombinant human granulocyte-colony stimulating factor (rhG-CSF) exhibited 1000-fold increase in number of stem cells.


Allogeneic PBSCs

• Transplantation of allogenic PBSCs also exhibited rapid hematologic recovery with low incidence of acute graft versus host disease.

• Careful here…..immune reactions still can occur


G-CSF

• Mobilization with disease specific chemotherapy plus G- CSF is an effective approach for PBSCs mobilization in patients who need salvage therapy.

• Mobilization with chemotherapy plus G-CSF exhibited 2 to 6 fold higher yield of cells in comparison to G-CSF alone.


Recombinant human granulocyte-colony stimulating factor (rhG-

CSF) • The most preferred cytokine for PBSCs

mobilization is rhG-CSF. • G-CSF mobilized peripheral blood stem cells

demonstrated better engraftment, faster neutrophils and platelets recovery, faster lymphocyte reconstitution and lower mortality rate as compared to bone marrow or umbilical cord blood.


Injuries

• Brain• Musculoskeletal System


Parathyroid Hormone (PTH)

• Parathyroid hormone (PTH) is a peptide hormone that is naturally released from the parathyroid glands. It mainly acts on bone and kidney cells and is involved in systemic

calcium homeostasis.

Presently, PTH and PTH equivalents such as Forteo (the first 34 amino acid sequence of PTH), are the only anabolic FDA approved treatments for human patients with osteoporosis.

PTH is believed to enhance stem cell trafficking because it has been found that PTH activates the bone marrow stem cell niche, which regulates osteoblasts]. Osteoblasts produce hematopoietic growth factors and are activated by PTH or the locally produced, PTH-related protein (PTH-rP), through the PTH/PTHrP receptor (PPR).


PBSC


Goals PBSC

• Autologous vs Allogeneic • Allogenic – young and healthy, male to male,

female to female, Blood type compatible, RH factor compatible

• IA, IM, IV, • Unpublished studies from Young's lab suggest

that matching genders as well as ABO groups and Rho-D positive or Rho-D negative must be taken into consideration when transplanting allogeneic stem cells.


Stem Cell Types


PTH

• in vitro, murine calvarial osteoblasts treated with PTH were able to support B-cell differentiation.

• Studies of transgenic mice have demonstrated that disruption of the PTH receptor via Gsα ablation early within the osteoblast lineage leads to a reduction in early B cell lineages . Conversely, constitutive activation of the PTH receptor in osteoblasts increased HSC number and activity

• . Exogenous treatment with PTH in mouse models expands HSC number within the bone marrow , induces HSC mobilization into peripheral circulation, and leads to superior engraftment in mouse models of bone marrow transplantation.


Autologous Stem Cell Transplantation

A curative procedure for many patients with lymphomas, and has been shown to improve survival in patients with multiple myeloma.

Approximately 20% of patients are unable to mobliize sufficient hematopoietic stem cells to proceed safely to autologous stem cell transplantation.

Parathyroid Hormone (PTH) affects osteoblasts and the stem cell niche, and has been shown to improve survival when given posttransplant in a mouse competitive transplant model.


PTH

• Factors that are known to affect osteoblast activity, such as parathyroid hormone (PTH), have also been shown to affect the HSCs. Treatment of mice with PTH has led to beneficial effects on the HSC pool, which have led to clinical trials of PTH treatment to enhance HSC-based therapies.


PTH

• Continuous administration of full-length (84 amino acid) PTH leads to bone loss and can cause osteoporosis. In contrast, intermittent PTH administration leads to bone formation. Furthermore, the 34 aa amino terminus of PTH maintains the anabolic activity of full-length PTH without causing bone loss.

• The PTH amino-terminal fragment, known as teriparatide, has been used in promising clinical trials for treating osteoporosis, and was used in this study.


PTH

• In vitro and animal studies have demonstrated that both hematopoiesis and the HSC niche may be modulated by parathyroid hormone (PTH) activity within osteoblasts



Phase I Trial of Parathyroid Hormone to Facilitate Stem Cell MobilizationKaren K.Ballen1Elizabeth J.Shpall4DavidAvigan3Beow Y.Yeap1David C.Fisher2KathleenMcDermott2Bimalangshu R.Dey1EyalAttar1StevenMcAfee1MarinaKonopleva4Joseph H.Antin2Thomas R.Spitzer1

http://www.sciencedirect.com/science/article/pii/S1083879107002169#!

PTH Study

• In this Phase I study, 20 subjects who had 1 or 2 unsuccessful stem cell mobilization attempts, received PTH in escalating doses of 40 μg, 60 μg, 80 μg, and 100 μg for 14 days. On days 10-14 of treatment, subjects received filgrastim 10 μg/kg. The PTH was tolerated well and there was no dose-limiting toxicity. Forty-seven percent of subjects who had failed 1 prior mobilization attempt met the mobilization criteria of >5 CD 34+ cells/μL in the peripheral blood. Forty percent of subjects who failed to reach adequate CD34+ cell counts in 2 prior mobilization attempts met the mobilization criteria. PTH was well tolerated at doses up to 100 μg in human cancer patients.


PTH

• Cells of the osteoblast lineage play an important role in regulating the hematopoietic stem cell (HSC) niche and early B cell development in animal models, perhaps via parathyroid hormone (PTH) dependent mechanisms


PTH

• We found that teriparatide treatment led to an early increase in circulating HSC number of 40% ± 14% (p=0.004) by month 3, which persisted to month 18 before returning to near baseline by 24 months. There were no significant changes in transitional B cells or total B cells over the course of the study period. In addition, there were no differences in complete blood count profiles as quantified by standard automated flow cytometry


HSC

• The hematopoietic stem cell (HSC) niche is thought to be modulated by both osteoblasts and osteoprogenitors within the endosteal and perivascular stroma.

• Increasing evidence suggests that osteoblasts also regulate the development of the lymphoid, erythroid, and myeloid lineages.

• Nilsson SK, Johnston HM, Coverdale JA. Spatial localization of transplanted hemopoietic stem cells: inferences for the localization of stem cell niches. Blood. 2001 May 15;97(8):2293–9. [PubMed]


https://www.ncbi.nlm.nih.gov/pubmed/11290590

Oncotarget. 2016 Mar 15;7(11):11899-912. doi: 10.18632/oncotarget.7727.Rationale and design of the allogeneic human mesenchymal stem cells (hMSC) in patients with aging fRAilTy via intravenous delivery (CRATUS) study: A phase I/II, randomized, blinded and placebo controlled trial to evaluate the safety and potential efficacy of allogeneic human mesenchymal stem cell infusion in patients with aging frailty.Golpanian S1,2, DiFede DL1, Pujol MV1, Lowery MH3, Levis-Dusseau S3,4, Goldstein BJ1,5, Schulman IH1,3,6, Longsomboon B1, Wolf A1, Khan A1, Heldman AW1,3,7, Goldschmidt-Clermont PJ3,7, Hare JM1,3,7.

Age Frailty Study


https://www.ncbi.nlm.nih.gov/pubmed/?term=Golpanian%20S%5BAuthor%5D&cauthor=true&cauthor_uid=26933813

https://www.ncbi.nlm.nih.gov/pubmed/?term=DiFede%20DL%5BAuthor%5D&cauthor=true&cauthor_uid=26933813

https://www.ncbi.nlm.nih.gov/pubmed/?term=Pujol%20MV%5BAuthor%5D&cauthor=true&cauthor_uid=26933813

https://www.ncbi.nlm.nih.gov/pubmed/?term=Pujol%20MV%5BAuthor%5D&cauthor=true&cauthor_uid=26933813

https://www.ncbi.nlm.nih.gov/pubmed/?term=Lowery%20MH%5BAuthor%5D&cauthor=true&cauthor_uid=26933813

https://www.ncbi.nlm.nih.gov/pubmed/?term=Levis-Dusseau%20S%5BAuthor%5D&cauthor=true&cauthor_uid=26933813

https://www.ncbi.nlm.nih.gov/pubmed/?term=Goldstein%20BJ%5BAuthor%5D&cauthor=true&cauthor_uid=26933813

https://www.ncbi.nlm.nih.gov/pubmed/?term=Goldstein%20BJ%5BAuthor%5D&cauthor=true&cauthor_uid=26933813

https://www.ncbi.nlm.nih.gov/pubmed/?term=Schulman%20IH%5BAuthor%5D&cauthor=true&cauthor_uid=26933813

https://www.ncbi.nlm.nih.gov/pubmed/?term=Longsomboon%20B%5BAuthor%5D&cauthor=true&cauthor_uid=26933813

https://www.ncbi.nlm.nih.gov/pubmed/?term=Wolf%20A%5BAuthor%5D&cauthor=true&cauthor_uid=26933813

https://www.ncbi.nlm.nih.gov/pubmed/?term=Wolf%20A%5BAuthor%5D&cauthor=true&cauthor_uid=26933813

https://www.ncbi.nlm.nih.gov/pubmed/?term=Khan%20A%5BAuthor%5D&cauthor=true&cauthor_uid=26933813

https://www.ncbi.nlm.nih.gov/pubmed/?term=Heldman%20AW%5BAuthor%5D&cauthor=true&cauthor_uid=26933813

https://www.ncbi.nlm.nih.gov/pubmed/?term=Goldschmidt-Clermont%20PJ%5BAuthor%5D&cauthor=true&cauthor_uid=26933813

https://www.ncbi.nlm.nih.gov/pubmed/?term=Goldschmidt-Clermont%20PJ%5BAuthor%5D&cauthor=true&cauthor_uid=26933813

https://www.ncbi.nlm.nih.gov/pubmed/?term=Hare%20JM%5BAuthor%5D&cauthor=true&cauthor_uid=26933813

Allogeneic Human Mesenchymal Stem Cell Infusions for Aging Frailty.Golpanian S, DiFede DL, Khan A, Schulman IH, Landin AM, Tompkins BA, Heldman AW, Miki R, Goldstein BJ, Mushtaq M, Levis-Dusseau S, Byrnes JJ, Lowery M, Natsumeda M, Delgado C, Saltzman R, Vidro-Casiano M, Pujol MV, Da Fonseca M, Oliva AA Jr, Green G, Premer C, Medina A, Valasaki K, Florea V, Anderson E, El-Khorazaty J, Mendizabal A, Goldschmidt-Clermont PJ, Hare JM.J Gerontol A Biol Sci Med Sci. 2017 Oct 12;72(11):1505-1512. doi: 10.1093/gerona/glx056


https://www.ncbi.nlm.nih.gov/pubmed/28444181

Tithon Human Sciences, Australia

• Peripheral Blood Stem Cell Protocol• Licensing Agreements• Investigator Status


PBSC-PRP KITFor the harvesting of a combination of peripheral blood derived

pluripotent stem cells and PRP


• Cell storage Plate x 1• Blunt tip needles x 2• PRP Blood Collection Tubes x 3

packets of four• 0.22µm syringe driven filter x 2• 5µm syringe driven filter x 2


Other Materials Required• Biosafety Cabinet ( Recommended

Thermofisher, 1.2m MSC Advantage Biological Safety Cabinet, with UV sterilisation lamp) or other sterile chamber

• Luer Lock Syringes• Blunt Tip 18G Needles• Alcohol Swab• 15mL Sterile Centrifuge Tubes• 50mL Sterile Centrifuge tubes• Sterile Saline• Swing Bucket, Benchtop Centrifuge• Refrigerator• Ultrasonic Bath 50Hz• Surgical Gloves• Protective Clothing


Collect Peripheral Blood via venepuncture of patient and fill three packets of AdiStem Blood Collection Tubes (12 tubes)


Spin ALL tubes at 200x g for 10 minutes to separate RBC and the plasma layer

200x g10 minutes

Plasma

RBC


Carefully aspirate plasma layer from 4 AdiStem

Collection Tubes into two 15mL centrifuge tubes for the PLATELET RICH LYSATE. Avoid

RBC as much as possible.These are the PLATELET RICH

PLASMA TUBES. Place at 4°C/39.2°F while preparing Peripheral Blood Stem Cell

step.Aspirate the plasma layer

from the remaining 8 tubes into two new 15 ml centrifuge

tubes for Peripheral Blood Stem Cells.

Note: Plasma levels will vary from patient to patient.

Plasma Only


Peripheral Blood Stem Cell

Using the Peripheral Blood Stem Cell tubes, centrifuge at 800x g for 10 minutes.

50cc centrifuge tube

White 5µm filter

Avoid pellet

800x g

10 minutes

Aspirate supernatant avoidingthe pelletand filter via the white 5.0µm


PBSC

Using the PLATELET RICH PLASMA TUBEScentrifuge at 2500x g for 10 minutes.

2500x g

10 minutes

Pellet

In both tubes, aspirate and discard the supernatant leaving 3mL of plasma with the pellet

.

Resuspend the pellet in residual plasma

Combine the two tubes of residual plasma into one

Supernatant


Completely submerge the combined PRP into the water of the Ultrasonic Bath.Ultrasound the PRP for 30 minutes at 50Hz.


Aspirate the 6mL of PRP lysate into a 10mL syringe.

Using a 0.22µm filter (blue filter), filter the PRP lysate into the 50cc sterile centrifuge tube that contains the Peripheral Blood Stem Cell Isolate.

In a new syringe, aspirate the Stem Cell and PRP medium.


Cell Storage Plate Procedure

CELL STORAGE PLATE

Air Filter Chamber

Insertion Pad

Label plate with patient name, date of birth and the date/time on the top right hand corner of the plate

Attach the blunt needle to the syringe with PRP and Stem Cells for insertion into plate.The uniquely designed and manufactured plate allows for positive selection of our cells, as non required cells adhere to plate surface.

Cell Storage Plate must be left flat, facing upward after

cells have been inserted.

The Insertion Pad and Air Filter Chamber should be visible.

BLUNT NEEDLE


Using an alcohol swab, wipe over the insertion pad to sterilize the area. Hold the plate so that the

insertion pad is at the top right hand side. With the hand that is holding the plate, apply a small amount of pressure (squeezing plate). With the other hand, steadily push the blunt needle into the insertion pad.

Inject the medium into the plate.Note: The plate will not be completely full

Rest the plate standing up, while preparing sterile saline.


Re-sterilise the insertion pad. Once again, while lightly squeezing the storage plate, inject the saline to fill the plate.

NOTE: If the plate begins to expand due to the injection of air, turn the plate around so that the insertion pad is at the bottom left hand corner and extract the air using the syringe.Turn the plate around again and continue injecting saline to fill the plate.

Prepare saline to inject into plate. The plates total volume is approximately 25mL

Fill plate

Remove Air


NOTE: Once plate has been filled, air bubbles may begin to accumulate in the plate. As previously conducted, hold the plate so that the insertion pad is at the bottom left hand corner and aspirate the air out using the syringe.

Leave plate with insertion pad and Air Filter Chamber facing upwards at 4°Covernight (8-16hrs), or at room temperature (22°C) for 2-6 hours.

A full plate with Stem Cells, PRP isolate & SalineCharles F Mahl MD FACS FICS

1200 x g

10 minutes

After removing the Stem Cell and PRP medium from the storage plate, divide the medium into two 50cc centrifuge tubes and spin for 10 minutes at 1200 x g.


Carefully aspirate and discard the supernatant not required from both tubes by knowing the volume you would like to administer.

Note: Pellet is not particularly visible, take care not to accidentally remove cells also.

For example:Knee, Shoulder or Elbow;

Resuspend the pellet into PRP lysate to make 1x 7mL syringes

Your stem cell preparation + PRP is now ready for administration.

Bring cells to room temperature before administration to patient.


Pluripotent Stem Cells

• Henry Young (2004) demonstrated that connective tissue (including blood) contains reserve precursor cells.

• Reserve precursor cells consist of tissue specific progenitor cells, germ layer lineage stem cells, and pluripotent stem cells.

• Tissue specific progenitor cells can be unipotent or pluripotent.

• Progenitor cells can only double 50-70 times while germ layer lineage stem cells and pluripotent stem cells have a much greater lifespan.

• Pluripotent stem cells were thought only to exist in embryonic stem cells until Dr Henry Young’s discovery of them in the peripheral blood in the late 1990’s.



Precursor cells can be:

Tissue specific progenitor cells

Lineage committed (ectodermal, mesodermal, endodermal) germ layer lineage stem cells

Lineage uncommitted pluripotent epiblastic stem cells * this is what we are interested in; some researchers call this blastomere like stem cells



• Lineage uncommitted stem cells can be used to form any tissues in the ectoderm, mesoderm or ectoderm.

• Treatment of a wide range of degenerative diseases : diabetes, osteoarthritis, osteoporosis, alzheimer’s, and age frailty to name a few.


Stem Cells

• Intraarticular/Enthesis - musculoskeletal• Intravenous/Intraarterial - organs• Intranasal - brain


Allogeneic transplantspatients receive stem cells from their brother, sister, or parent i.e. a person who is not related to the patient called an unrelated donor; the stem cells come from a donor often a sibling but sometimes another volunteer whose cells are considered a “match” for the patient. The process of finding a match is called tissue typing or human leukocyte antigen typing. HLA is a protein on the surface of blood cells . Basically, the more “HLA markers” a patient and the potential donor have in common, the greater the chance that the transplant will be successful. Advantage of allogeneic stem cell transplant is that the donor stem cells make their own immune cells, which may help destroy any cancer cells that may remain after high-dose treatment. This is called the graft-versus-cancer effect.


Tithon Technology for PBSC

• Primostem-CPS Technology – reagents, proprietary antibody and components to perform extraction, separation, collection and reinjection of peripheral blood derived novel non-immunogenic parathyroid hormone receptor positive pluripotent stem cells for intravenous delivery, intranasal delivery and IM, IA injection.

• Allogeneic vs Autologous• Banking of Extra CellsCharles F Mahl MD FACS FICS

Sources of Stem Cells

• Bone Marrow• Adipose Derived• Peripheral Blood Derived

• PRP with all


PBSC

• Has been demonstrated to work as a regenerative agent in:

• 1. articular cartilage• 2. bone• 3. soft tissue

2013, The British Editorial Society of Bone and Joint Surgery


PBSC

• MRI cartilage imaging in assessment of the regenerative power of autologous peripheral blood stem cell injection in knee osteoarthritis

• Bassem A. Darwish Khaled A. Ahmad a,*, Yosra A. Ibrahim a, Nayera Z. Saber b 2014/6

• The Egyptian Journal of Radiology and Nuclear Medicine Vol 45. 787-794

• 10 pts bilat knee OA -8 ml of IA autologous PBSCMRI showed increased cartilage thickness and improved function clinically


Definitions

• Autologous –own stem cells• Allogeneic – someone else’s stem cells –

unrelated donor or relative• Syngeneic- stem cells from identical twin


Stem Cells

• The numbers of stem cells (totipotent stem cells, pluripotent stem cells, and mesodermal stem cells) vary depending on the health status of the individual. However, for healthy individuals their percentages in the tissues are TSCs = 0.1%, PSCs = 0.9%, and GLSCs = 9%. Taken together (stem cells = TSCs, PSCs, and GLSCs) hundreds of stem cells can be isolated from Umbilical Cord (Wharton’s Jelly); thousands of stem cells can be isolated from bone marrow, amnion, or placenta; and hundreds of thousands of stem cells can be isolated from adipose tissue, skeletal muscle, or blood.

• From Henry Young PhD


Increasing Stem Cell Numbers

• By having the patient ingest Nutra#1 throughout the study and for the first 30 days and take Nutra#2 18 hours before each harvest, we can increase stem cell numbers in the peripheral blood by about three orders of magnitude.

• So instead of isolating hundreds of thousands of stem cells from peripheral blood, we can isolate hundreds of millions of stem cells from peripheral blood.

• And as most understand, the more stem cells that are given the better the response.

• Henry Young PhD


Mesenchymal Stem Cells

• Young does not use MSCs. The original MSCs were mesenchymal stem cells, as originally identified by Arnold Caplan. They are a tripotent progenitor cell already pre-programmed to form cartilage, fat, and bone. They are telomerase negative, they have a limited lifespan, they respond to proliferation agents and progression agents, but do not respond to inductive agents outside their lineage. In other words, they do not respond to agents that induce anything other than cartilage, fat, or bone.


Mesenchymal Stem Cells

• The “MSC” designation has also been used for “marrow stromal cells” and “medicinal secreting cells”. Both sets of cells secrete exosomes containing factors that support the surrounding tissues.


Type of Stem Cells

• Young does NOT use PBSCs, peripheral blood stem cells. PBSCs are hematopoietic in nature and form cell types within the hematopoietic lineage. Young’s stem cells will form the various types of blood cells, and will also form may other cell types not related to blood. He calls this “Mesodermal Stem Cells” (MesoSC)


Mesodermal Stem Cells

• The specific GLSCs that we use are designated as MesoSCs, which stands for mesodermal stem cells. These cells have been shown to form 39 separate and distinct differentiated cell types within the embryonic mesodermal germ layer lineage, they are telomerase positive, they have essentially an unlimited lifespan (we had a human MesoSC that had been expanded through 690 population doublings before experiment was stopped), they respond to proliferation agents and inductive agents, but do not respond to progression agents. The MesoSCs incorporate into mesodermal tissues and repair the damaged tissues.


Intranasal

• Unlike in intravenous infusions, in an intra-arterial approach, pulmonary circulation can be bypassed, which results in superior delivery and sustained presence of stem cells in the ischemic brain. However, an arterial approach may cause arterial occlusion, resulting in stroke; hence, this approach was not found to be superior to an intravenous approach for recovery after stroke. More recently, several studies reported that intranasal delivery of MSCs improved neurovascular regeneration and functional recovery after stroke. Intranasal delivery may provide a simple, non-invasive, and brain-specific mode for cell therapy.


Ex Vivo Manipulation of Stem Cells

• Decreased functional activity of stem/progenitor cells might be mediated by telomerase shortening/decreased telomerase activity, disturbances of the cell secretome, and altered interactions with the microenvironment. Several methods of culture expansion that improved the proliferation, survival, and trophic support, and reduced senescence of MSCs have been reported, including hypoxic/ischemic preconditioning, genetic modification of cells, trophic factor pretreatment, isolation and use of functional subpopulations of stem cells, modulation of intracellular signal cascades, and modification of the cell microenvironment.


BBB

• Molecules larger than 400 Da cannot pass through the BBB, which may affect the efficacy of cell therapy in stroke patients. Intravenous co-administration of stem cells and mannitol, an osmotic agent that can regulate the permeability of the BBB, may improve outcomes in stroke patients. One preclinical study showed that BBB manipulation using intravenous mannitol prior to MSC treatment resulted in increased levels of trophic factors in the infracted brain [60]. As mannitol is already widely used in clinical practice, BBB manipulation using mannitol could be considered in future clinical trials.



• Clinical Studies• Potential for Healing and Regeneration


peripheral blood stem cells - · 2018-04-09 · peripheral blood stem cells ... annual symposium...

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