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Sourcing Niche Cell PopulationsTechniques for Isolating and Characterizing Progenitor Cells

June 24, 2015

Webinar Series

Sponsored by:

Participating ExpertsBrought to you by the Science/AAAS Custom Publishing Office Donald G. Phinney, Ph.D.

The Scripps Research Institute Jupiter, FL

Stefano Da Sacco, Ph.D.Children’s Hospital of Los AngelesLos Angeles, CA

Webinar Series

Sourcing Niche Cell PopulationsTechniques for Isolating and Characterizing Progenitor Cells

June 24, 2015

Sponsored by:

Donald G. Phinney, Ph.D.Professor

Department of Molecular Therapeutics The Scripps Research Institute

Jupiter, Florida, USA

● Component of the hematopoietic niche in marrow ● Regulate skeletal homeostasis via multi-lineage differentiation● Exhibit potent effector (angiogenic, anti-inflammatory,

immuno-modulatory) functions largely via paracrine action

● MSCs routinely isolated from bone marrow by attachment to tissue culture plastic.

● Plastic adherent cultures from mouse bone marrow contain large numbers of hematopoietic cell types.

● Hematopoietic cells persist in these cultures even after serial passage.

● Adherent cultures reconstitute the hematopoietic system of lethally irradiated mice.

CFU-GMPre-B cells (anti-IgM)Anti-CD11b

Plastic Adherent

Hum

anM

ouse

● Magnetic bead technology● Cellular composition of adherent cultures to determine

appropriate antibody cocktail◦ Anti-CD11b (granulocytes)◦ Anti-CD45 (pan hematopoietic marker)◦ Anti-CD34 (endothelial cells and hematopoietic progenitors)

● Growth kinetics of contaminating populations to optimize time frame for depletion

Culture Expand 8-10

days

Harvest Bone Marrow

Day 3 Day 4 Day 6 Day 8

1.45 x 106 cell/cm2

Remove non-adherent cells after 72h by gentle

aspiration

Plastic Adherent Immuno-depleted MSCs

Successive rounds of depletion using

Dynabeads conjugated to anti-CD11b, anti-CD34

and anti-CD45 antibodies

Kopen et al. PNAS 1999; 96:10711Baddoo et al., J. Cell. Biochem. 2003; 89:1235Phinney DG, Meth. Mol. Biol. 2008; 449:171

MSC yields are ~15-20%

of the plastic adherent

cells with >90% viability.

● Harvest before colonies become too dense or grow together

● Use trypsin and physical scraping to detach cells ◦ Freshly thawed trypsin◦ Do not scrape too vigorously◦ Monitor scraping visually to ensure detachment of cells ◦ Work quickly to ensure pH of the medium does not become

alkaline ● Avoid cell clumping◦ After washing cells, flick pellets to loosen before suspending in

media

● Flow Cytometry◦ CD11b ◦ CD19◦ CD31◦ CD34◦ CD45◦ CD29◦ CD44◦ CD73◦ CD105◦ CD106◦ CD146◦ SCA1◦ SSEA1, SSEA4◦ F4/80

Osteogenesis

Adipogenesis

Chondrogenesis

● Multi-lineage differentiation in vitro using standard assays

● Adipogenesis● Chondrogenesis● Osteogenesis

● Growth rate is progressively diminished as a function of passage irrespective of plating density (cells/cm2)

● Become refractory to growth factor stimulation after a few passages

1000

10000

100000

1000000

P1 P2 P3 P4 P5

Cum

ulat

ive

Cel

l Num

ber

Passage Number

1000 5000 13000

● Exposure to 21% vs. 5% oxygen induces significant mitochondrial ROS production leading to mitochondrial membrane depolarization and reduced ATP production

● Inhibits cell growth and induces cellular apoptosis

Boregowda et al. Stem Cells 2012; 30:975-987

0

1

2

3

4

5

5% 21%

Mito

SoxR

ed (

FU)

0

5

10

15

20

5% 21%

ATP

(nm

ol/m

g)

0

0.5

1

1.5

5% 21%

JC-1

Flu

ores

cenc

e

**#

*p<0.05, #p<0.005

Boregowda et al. Stem Cells 2012; 30:975-987

0

20

40

60

5% 21%

p53+/+

8-O

xogu

anin

e (%

) p<0.0001

PCNA

p53

● Procurement and expansion in a closed system in 5% oxygen affords a 2400-fold increase in cell yield by passage 4

● Low oxygen conditions preserve stem/progenitor functions

1000

10000

100000

1000000

10000000

100000000

P1 P2 P3 P4 P5

Cum

ulat

ive

Cel

l Num

ber

Passage Number

500 1000 5000

p53β-Actin

● MSCs from p53-/- mice are insensitive to oxygen-induced stress as evidenced by lack of ROS generation and changes in mitochondrial function.

0

1

2

3

4

5

5% 21%

Mito

SoxR

ed (F

U)

0

0.5

1

1.5

5% 21%

JC-1

Flu

ores

cenc

e

0

5

10

15

5% 21%

ATP

(nm

ol/m

g)

Boregowda et al. Stem Cells 2012; 30:975-987

1.E+03

1.E+05

1.E+07

1.E+09

1.E+11

1.E+13

P0 P1 P2 P3 P4 P5

Cum

ulat

ive

Cel

l Num

ber

Passage Number

5% 21%

Boregowda et al. Stem Cells 2012; 30:975-987

● Most labs propagate plastic adherent cultures long-term (weeks to months) in 21% oxygen to remove hematopoietic lineages.

● Over time a rapidly dividing subpopulation of cells emerge.

● Cells that are insensitive to oxygen are characterized as “stem cells”.

● Growth restrictive conditions selects for clones that lack a functional p53 protein.

● p53 mutants are insensitive to oxidative stress and escape oxygen-induced growth inhibition.

● Expansion of immortalized clones.

Krishnappa et al. Cytotherapy 2013; 15:536

● p53-/- MSCs are defective in supporting hematopoiesis● p53-/- MSCs showed reduced adipogenic differentiation● p53-/- MSCs showed greatly enhanced osteogenic differentiation

0

40

80

120

160

p53+/+ p53-/-

CA

FC (#

)

0

1

2

3

4

p53+/+ p53-/-

Adi

po-In

duct

ion

0

4

8

12

16

p53+/+ p53-/-

Ost

eo-In

duct

ion

#

#

*

*p<0.05, **p<0.01, #p<0.005, ++, p<0.001

● Over 65% of labs surveyed from North America, Europe and Asia procure mouse MSCs by long-term expansion in atmospheric oxygen.

● Almost 80% of labs routinely culture mouse MSCs in atmospheric (21%) oxygen.

0 20 40 60 80 100

Positive selectionNegative selection

Long-term expansionCell sorting

Mechanical seperationOther

Isolation Method

0 20 40 60 80 100

Atmospheric oxygen

Low oxygenincubator

Closed low oxygensystem

Culture conditions

● Many published studies employ cell types that are erroneously characterized as primary mouse MSCs. ◦ Cell lines derived by selection in oxygen◦ Cell lines from embryonic mesoderm - NIH3T3, PA317,

C3H/10T1/2◦ Cell lines from newborn calvaria - MC3T3

● Careful consideration should be made with respect to the source of mouse MSCs when interpreting experimental findings.

● Phinney lab funded by NIH to distribute primary MSCs from inbred, transgenic and knockout mice to the scientific community

● Request cells via http://www.scripps.edu/phinney/index.html

or E-mail: LTalaia@scripps.edu or dphinney@scripps.edu

Past Lab MembersMaria Dutreil, Melody Baddoo,Wen-Tzu Lai PhD, Irina A Isakova PhD, Gene Kopen, PhD

Sid Boregowda, DVM, PhD

Veena Krishnappa, DVM, PhD

Christopher C Haga, PhD

Jacqueline Strivelli BSc

Funding

Participating ExpertsBrought to you by the Science/AAAS Custom Publishing Office Donald G. Phinney, Ph.D.

The Scripps Research Institute Jupiter, FL

Stefano Da Sacco, Ph.D.Children’s Hospital of Los AngelesLos Angeles, CA

Webinar Series

Sourcing Niche Cell PopulationsTechniques for Isolating and Characterizing Progenitor Cells

June 24, 2015

Sponsored by:

Children’s Hospital Los AngelesDepartment of Urology

Saban Research InstituteUniversity of Southern California

Isolation of nephrogenic progenitors

from fetal kidneys: a new approach

Stefano Da Sacco, PhD

The kidneys perform the essential function

of removing waste products from the

blood and regulating the water fluid levels

The functional unit of the kidney is the

nephron

Renal vesicle

Comma-shapedbody

S-shapedbody

Mature nephron: glomerulus + proximal and distal tubules

Nephrogenesis in mammals

Intermediate mesoderm

Metanephric mesenchyme

Cap mesenchyme Stromalprogenitors

Vascular progenitors

Nephric duct

Commonnephric duct

Ureteric bud

Collecting ductUreteric tipPretubularaggregate

PericytesRenal interstitium

Mesangium

Renal vesicle

Proximal renal vesicle Distal renal vesicle

PodocytesParietal epithelial cells

Proximal tubuleLoop of HenleDistal tubule

Connecting tubule

Osr1

Hox genes

Six2 Cited1 Eya1Osr1 Meox1 Hox genes Foxd1

Hox genes

Why is the nephrogenic zone so important?

In the adult mammals the nephrogenic zone is completely depleted.

In humans this occurs before the 36th week of gestation

A human kidney has between 500,000 to 1 million nephrons at birth. No more nephrons can be generated after birth

CLINICAL RELEVANCE: Damage to nephrons leads to an irreversible loss of kidney functionality

In animal models, it is possible to isolate and study specific renal progenitors populations by using for example Six2+GFP mice

Isolation of nephrogenic progenitorsPROBLEM: nephrogenic markers are intracellular markers

No known surface markers difficult isolation

negative selection for CD326 (Ep-CAM) PDGFRα, CD105 and TER-119’

Transgenic mice

Mechanical Separation

Negative Selection

A “pure” human self renewing nephrogenic cell population has never been isolated

Can we find any alternative approach tailored to study human development?

NCAM+ isolation

What about human nephrogenic progenitors?

Our knowledge on nephrogenic cells builds on the study of mouse renal development

Isolation of live nephrogenic cells

Smartflare RNA probes

Smartflare probes can be used to analyze/sort live cells for transcription factors or intracellular markers using

immunofluorescence and/or flow cytometry

Source: www.emdmillipore.com/smartflare

Isolation of live nephrogenic cells

Smartflarecocktail

Single cell suspension

Positive selection Negative selection

Isolation of live nephrogenic cells

- Digestion of human fetal kidneys: - 0.5mg/ml collagenase I in RPMI media (37C, 2 hours)- cells are strained through a 100 micron strainer to obtain a single cell suspension- Cells are seeded on 100mm tissue culture dishes

- Staining with Smartflare RNA probes:- The Smartflare cocktail (Cy-3 and Cy-5) is added to the 100mm plates- Staining is performed overnight (37C, 5%CO2)

- Fluorescence Activated Cell Sorting (FACS):- Cells are trypsinized and washed twice with PBS- Sorting is performed on a FACSAria- Sorted cells are seeded on 96 well plates for further expansion or processed for RNA extraction

Uptake (–cy3 and cy5) and scramble (–cy3 and –cy5) probes are used as positive and negative controls

Isolation of nephrogenic progenitor cells from human tissue

0.25%0.17%

The nephrogenic progenitor cells represent a rare population

Percentage of isolated cells ranged between 0.11 and 0.3%

Protein expression of RNA markers

Green: antibodyRed: Smartflare‐Cy5Blue: DAPI

Green: antibodyRed: Smartflare‐Cy3Blue: DAPI

(24h after selection)(24h after selection)

Cells positive for the Smartflare probe are also expressing the protein as confirmed by immunofluorescence after selection

Percentage of cells double positive for the cy3 and cy5 Smartflare probes is similar to the percentage found using antibodies against the relative proteins

Are we selecting the right cells?

Gene expression of nephrogenic markers

ACTB

SIX2

1 2 3 4 5 HEK

PCR analysis confirmed the nephrogenic signature of different isolated populations.

HOXA11

EYA1

Samples(isolated nephrogenic progenitors)

Control(whole fetal kidney)

Disaggregation/re-aggregation assay

Single cell suspension

Smartflare selected cells

Disaggregation/re-aggregation assay- Digestion of human fetal kidneys:

- 0.05% collagenase I in RPMI media (37C, 2 hours)- cells are strained through a 100 micron strainer to obtain a single cell suspension

- Labelling of previously isolated and expanded nephrogenic progenitors:- Cells are labeled with CM-DiI or CFSE (either overnight or during a 1 hour procedure) to allow identification in the co-culture

- Co-culture of dissociated fetal kidneys with human nephrogenic progenitors: - labeled nephrogenic progenitors are mixed with the fetal kidney cell suspension (1:10 ratio)- Cells are seeded on a filter at the air-liquid interface for up to 7 days .

- Fixation and immunofluorescence analysis

Conclusions

- Our knowledge of the human nephrogenic progenitors is limited and based on data from animal models

- Isolation of human nephrogenic cells is hampered by the lack of specific surface markers

- Isolation of cells based on expression of transcription factors is made possible by use of RNA probes

- Upon isolation, cells are viable, can be used to test nephrogenic potential and for studies of human kidney cell specification

Laura Perin, PhDRoger De Filippo, MDAstgik Petrosyan, PhD StudentRuby Kim, NIH STEP-UP Student

Brendan Grubbs, MDMatthew Thornton

Thanks to:

Flow Cytometry Core Laboratory, CHLA

Participating ExpertsBrought to you by the Science/AAAS Custom Publishing Office To submit your

questions, click theAsk a Question

button

Webinar Series

Sourcing Niche Cell PopulationsTechniques for Isolating and Characterizing Progenitor Cells

June 24, 2015

Sponsored by:

Donald G. Phinney, Ph.D.The Scripps Research Institute Jupiter, FL

Stefano Da Sacco, Ph.D.Children’s Hospital of Los AngelesLos Angeles, CA

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Sourcing Niche Cell PopulationsTechniques for Isolating and Characterizing Progenitor Cells

June 24, 2015

Sponsored by: