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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: [email protected] or [email protected]
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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Brought to you by the Science/AAAS Custom Publishing Office
Webinar Series
www.emdmillipore.com/smartflare
Sourcing Niche Cell PopulationsTechniques for Isolating and Characterizing Progenitor Cells
June 24, 2015
Sponsored by: