U.S. patent application number 15/977505 was filed with the patent office on 2019-10-24 for methods for enriching pluripotent stem cell-derived cardiomyocyte progenitor cells and cardiomyocyte cells based on sirpa expres.
This patent application is currently assigned to UNIVERSITY HEALTH NETWORK. The applicant listed for this patent is UNIVERSITY HEALTH NETWORK. Invention is credited to April M. Craft, Nicole C. Dubois, Gordon Keller.
Application Number | 20190322986 15/977505 |
Document ID | / |
Family ID | 45722784 |
Filed Date | 2019-10-24 |
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United States Patent
Application |
20190322986 |
Kind Code |
A1 |
Keller; Gordon ; et
al. |
October 24, 2019 |
METHODS FOR ENRICHING PLURIPOTENT STEM CELL-DERIVED CARDIOMYOCYTE
PROGENITOR CELLS AND CARDIOMYOCYTE CELLS BASED ON SIRPA
EXPRESSION
Abstract
The present invention relates to in vitro methods of enriching
populations of human pluripotent stem cells that are induced to
differentiate to cardiomyocyte progenitor cells and cardiomyocyte
cells. The cell populations can be enriched by isolating cells that
express SIRPA. The invention also related to in vitro-enriched
populations of cardiomyocyte cells and cardiomyocyte progenitor
cells obtained from populations of pluripotent stem.
Inventors: |
Keller; Gordon; (Toronto,
CA) ; Craft; April M.; (Toronto, CA) ; Dubois;
Nicole C.; (Toronto, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITY HEALTH NETWORK |
Toronto |
|
CA |
|
|
Assignee: |
UNIVERSITY HEALTH NETWORK
Toronto
CA
|
Family ID: |
45722784 |
Appl. No.: |
15/977505 |
Filed: |
May 11, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13819318 |
May 13, 2013 |
9994821 |
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PCT/CA11/00965 |
Aug 26, 2011 |
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15977505 |
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61377665 |
Aug 27, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/5073 20130101;
C12N 2501/16 20130101; C12N 2506/45 20130101; C12N 2501/155
20130101; C12N 5/0657 20130101; C12Q 1/6881 20130101; G01N 33/53
20130101; C12N 2506/02 20130101; C12N 2501/415 20130101; G01N
33/56966 20130101; C12N 2506/00 20130101; C12N 2501/115 20130101;
C12N 2501/165 20130101; C12Q 2600/158 20130101 |
International
Class: |
C12N 5/077 20060101
C12N005/077; C12Q 1/6881 20060101 C12Q001/6881 |
Claims
1-30. (canceled)
31. A composition comprising: a cell population comprising one or
more of human cardiomyocytes and human cardiomyocyte progenitor
cells; and a signal-regulatory protein alpha (SIRPA)-specific
binding member.
32. The composition of claim 31, wherein the cell population is
derived from human pluripotent stem cells.
33. The composition of claim 32, wherein the human pluripotent stem
cells are selected from embryonic stem cells (hESCs) or human
induced pluripotent stem cells (iPSCs).
34. The composition of claim 31, wherein the SIRPA-specific binding
member is selected from the group consisting of an anti-SIRPA
antibody, an anti-SIRPA antibody fragment, and an anti-SIRPA
antibody-like molecule.
35. The composition of claim 31, wherein the cell population
comprises at least 60% cardiomyocyte cells, cardiomyocyte
progenitor cells, or both.
36. The composition of claim 35, wherein the cell population
comprises at least 90% cardiomyocyte cells, cardiomyocyte
progenitor cells, or both.
37. The composition of claim 36, wherein the cell population
comprises at least 98% cardiomyocyte cells, cardiomyocyte
progenitor cells, or both.
38. The composition of claim 31, wherein the cell population is
substantially devoid of cells expressing at least one of the
following cell surface markers: CD90, CD31, CD140B and CD49A.
Description
RELATED APPLICATIONS
[0001] The application claims priority from U.S. Provisional Patent
Application No. 61/377,665 filed on Aug. 27, 2010.
FIELD OF THE INVENTION
[0002] The present invention relates to methods for enriching
pluripotent stem cell-derived cardiomyocyte progenitor cells and
cardiomyocyte cells based on SIRPA expression.
BACKGROUND Of THE INVENTION
[0003] The potential of human embryonic (hESCs) and induced
pluripotent stem cells (hiPSCs) to generate cardiovascular cells in
culture provides a powerful model system for investigating cellular
interactions and molecular regulators that govern the
specification, commitment and maturation of these lineages, as well
as a unique and unlimited source of human cardiomyocytes for drug
testing and regenerative medicine strategies.sup.1-4. Translating
this remarkable potential into practice is, however, dependent on
technologies that enable the reproducible generation of highly
enriched populations of cardiomyocytes, as contaminating cell types
could impact drug responses and other functional properties in
vitro and increase the risk for abnormal growth and teratoma
formation following transplantation in vivo.sup.5. When induced
under optimal cardiac conditions, human pluripotent stem cells
(hPSCs) will efficiently differentiate to generate mixed
cardiovascular populations, including cardiomyocytes, smooth muscle
cells, fibroblasts and endothelial cells.sup.3. While
cardiomyocytes can represent up to 70% of the population for any
given hPSC line, the efficiency of generating this lineage does
vary considerably between different stem cell lines. Further
manipulation of induction conditions has not yet yielded strategies
for the generation of pure populations of cardiomyocytes from a
broad range of hPSC lines.
[0004] To enrich for cardiomyocytes from the differentiation
cultures, cardiomyocyte-specific fluorescent reporters or drug
selectable elements have been introduced into hPSCs.sup.6-8.
Following differentiation, cardiomyocytes can be enriched either by
fluorescent-activated cell sorting (FACS) or the addition of
appropriate selection drugs. Although these strategies do allow for
the generation of enriched cardiomyocyte populations, they suffer
from a major drawback as a reporter vector must be introduced into
each hPSC line used, resulting in genetically modified
cardiomyocytes, thus reducing their utility for clinical
applications. In a more recent study, Hattori et al. demonstrated
that it was possible to isolate cardiomyocytes by FACS, based on
their high mitochondrial content.sup.6. While this approach appears
to be useful for isolating mature cardiomyocytes, cells with fewer
mitochondria, such as immature hPSC-derived cardiomyocytes, may be
more difficult to distinguish from other cell types.
SUMMARY OF THE INVENTION
[0005] In an aspect, there is provided a method of enriching a
population of cells for cardiomyocyte cells and cardiomyocyte
progenitor cells comprising providing the population at cells from
which cardiomyocyte cells and cardiomyocyte progenitor cells are to
be isolated; and isolating from the population, cells expressing
SIRPA; wherein the population of cells comprises a population of
human pluripotent stem cells induced to differentiate into
cardiomyocyte cells and cardiomyocyte progenitor cells.
[0006] In a further aspect, there is provided an enriched
population of cardiomyocyte cells and cardiomyocyte progenitor
cells obtained using any one of the methods described herein.
[0007] In a further aspect, there is provided an isolated
population of cells enriched for cardiomyocyte cells and
cardiomyocyte progenitor cells, wherein the population of cells
comprises at least 60%, preferably at least 90%, cardiomyocyte
cells and cardiomyocyte progenitor cells.
[0008] In a further aspect, there is provided the use of SIRPA for
isolating cardiomyocyte cells and cardiomyocyte progenitor cells
from a population of cells, wherein the population of cells
comprise a population of human pluripotent stem cells induced to
differentiate into cardiomyocyte cells and cardiomyocyte progenitor
cells.
[0009] In a further aspect, there is provided a method of depleting
a population of cells for cardiomyocyte cells and cardiomyocyte
progenitor cells comprising: providing the population of cells from
which cardiomyocyte cells and cardiomyocyte progenitor cells are to
be depleted; and depleting from the population, cells expressing
SIRPA; wherein the population of cells comprises a population of
human pluripotent stem cells induced to differentiate into
cardiomyocyte cells, cardiomyocyte progenitor cells, and
non-cardiomyocytes.
[0010] In a further aspect, there is provided a method of enriching
a population of cells for cardiomyocyte cells and cardiomyocyte
progenitor cells comprising: providing the population of cells from
which cardiomyocyte cells and cardiomyocyte progenitor cells are to
be isolated; and depleting from the population, cells expressing at
least one of CD90, CD31, CD140B and CD49A; wherein the population
of cells comprise a population of human pluripotent stem cells
induced to differentiate into cardiomyocyte cells, and
cardiomyocyte progenitor cells, and non-cardiomyocytes.
DESCRIPTION OF THE DRAWINGS
[0011] Embodiments of the invention may best be understood by
referring to the following description and accompanying drawings.
In the drawings:
[0012] FIG. 1 shows specification of the cardiovascular lineage
from hESCs. (a) Outline of the protocol used to differentiate hESCs
to the cardiac lineage (modified from Yang et al., 2008). (b)
Quantitative PCR (QPCR) analysis of BRACHURY (T), MESP1, ISLET1
(ISL1), NKX2-5, MYH6 (.alpha.MHC), MYH7 (.beta.MHC), MYL2 (MLC2v),
MYL7 (MLC2a), NEUROD1 and FOXA2 in HES2-derived embryoid bodies
(EBs) at different stages during differentiation. Day 0, hES cells;
LV, human fetal left ventricle; LA, human fetal left atria; AH,
human adult heart, Ed, hESC-derived endoderm.sup.13. Bars represent
mean.+-.standard error of the mean, n=3.
[0013] FIG. 2 shows expression of the cell surface receptor SIRPA
during hESC differentiation. (a) Flow cytometric analysis of SIRPA
(SIRPA) on EBs derived from NKX2-5-GFP hESCs. (b) Expression of
SIRPA on HES2-derived EB populations at the indicated times. (c)
RT-qPCR analysis of expression of SIRPA and its ligand CD47 in
HES2-derived EBs at different times of differentiation. Day 0, ES
cells; LV, human fetal left ventricle; LA, human fetal left atrial;
AH, human adult heart. Bars represent mean.+-.standard error of the
mean, n=4. (d) Immunostaining for SIRPA and cardiac Troponin I
(cTNI) on cardiac monolayer cultures. Monolayers were generated
from d20 HES2-derived EBs.
[0014] FIG. 3 shows enrichment of cardiomyocytes from hESC-derived
cultures by cell sorting based on SIRPA expression. (a) Flow
cytometric analysis of SIRPA expression in EBs at d8, d12 and d20
of differentiation. Fluorescent-activated cell sorting (FACS) for
SIRPA was performed at d8, d12 and d20. The presort (PS),
SIRPA.sup.+ and SIRPA.sup.- fractions from each time point were
analyzed for cardiac Troponin T (cTNT) expression by intracellular
flow cytometry. The frequency of cTNT.sup.+ cells at d8, d12 and
d20 was significantly higher in the SIRPA.sup.+ fraction (day8:
95.2%.+-.1.9, day12: 94.4.+-.1.7, day20: 89.6.+-.3.6), compared to
SIRPA.sup.- cells (day8: 13.0.+-.2.1, day12: 14.3.+-.3.9, day20:
15.7.+-.6.0). (b) Average enrichment of cTNT.sup.+ cells from 3
different cell separation experiments. Bars represent standard
error of the mean. Asterisks indicate statistical significance as
determined by student's t-test, *** (p.ltoreq.0.001) (c) QPCR
analysis of PS, SIRPA.sup.+ and SIRPA.sup.- cells. Expression of
SIRPA, NKX2-5, MYH6, MYH7 and MYL7 was significantly higher in the
SIRPA.sup.+ fraction compared to SIRPA.sup.- fraction at all stages
analyzed (d8, d12 and d20). Expression of markers for the
non-cardiac lineages (PECAM and DDR2) segregated to the SIRPA.sup.-
fraction. Bars represent mean.+-.standard error of the mean.
Asterisks indicate statistical significance as determined by
student's t-test, * (p.ltoreq.0.05), ** (p.ltoreq.0.01), ***
(p.ltoreq.0.001), n=3. (d) Immunostaining of cardiac Troponin I
(cTNI) on monolayer cultures generated from PS, SIRPA.sup.+ and
SIRPA.sup.- cells sorted at day20.
[0015] FIG. 4 shows enrichment of cardiomyocytes from hiPSC-derived
cultures by cell sorting based on SIRPA expression. (a) Flow
cytometric analysis of SIRPA expression at d20 of differentiation
on 38-2 and MSC-iPS1 hiPSC-derived cells. Fluorescent-activated
cell sorting (FACS) for SIRPA was performed at d20 and the presort
(PS), SIRPA.sup.+ and SIRPA.sup.- fractions were analyzed for
cardiac Troponin T (cTNT) expression by intracellular flow
cytometry. (b) The frequency of cTNT.sup.+ cells was significantly
higher in the SIRPA.sup.+ fraction of both hiPSC-derived cultures
(MSC-iPS1: 67.0.+-.3.6, 38-2: 71.4.+-.3.8), compared to SIRPA.sup.-
cells (MSC-iPS1: 4.9.+-.2.1, 38-2: 6.2.+-.0.9). Bars represent
mean.+-.standard error of the mean. Asterisks indicate statistical
significance as determined by student's t-test, ** (p.ltoreq.0.01),
*** (p.ltoreq.0.001), n=3. (c) QPCR analysis of PS, SIRPA.sup.+ and
SIRPA.sup.- cells derived form MSC-iPS1 and 38-2 hiPSCs after cell
sorting at d20. Expression of markers specific for the cardiac
lineage (SIRPA, NKX2-5, MYH6, MYH7, MYL2 and MYL7) was
significantly higher in the SIRPA.sup.+ compared to the SIRPA.sup.-
fraction. Expression of markers for the non-cardiac lineages (DDR2,
PDGFRB and NEUROD1) segregated to the SIRPA.sup.- fraction and the
PS cells. Bars represent mean.+-.standard error of the mean.
Asterisks indicate statistical significance as determined by
student's t-test. * (p.ltoreq.0.05). ** (p.ltoreq.0.01), ***
(p.ltoreq.0.001), n=5.
[0016] FIG. 5 shows expression of SIRPA on human fetal
cardiomyocytes and in adult human heart. (a) RT-qPCR analysis for
SIRPA in human fetal heart tissue and adult heart. LV, left
ventricle; RV, right ventricle; AP, Apex; LA, left atria; RA, right
atria, AVJ, atrioventricular junction; HEK, human embryonic kidney
cells; AH, adult heart; day 0, hES cells; d20, day20 of cardiac
differentiation, RT, reverse transcriptase control. Bars represent
mean.+-.standard error of the mean, n=6. (b) Immunostaining for
SIRPA (green) on human fetal ventricular cells and staining with
Mito Tracker Rod (red, accumulates in the mitochondrial matrix) and
DAPI (blue, nuclear dye). (c) Flow cytometric analysis for SIRPA on
human fetal heart tissue. (d) intracellular flow cytometric
analysis for cTNT on human fetal heart tissue.
[0017] FIG. 6 shows the utilization of SIRPA to predict cardiac
differentiation efficiency. (a) Day5 KDR/PDGFRA flow cytometry
profiles of cardiac differentiation cultures induced with varying
combinations of Activin A (ACTA0, 3, 6, 9 ng/ml) and BMP4 (10, 30
ng/ml). The KDR.sup.+PDGFRB.sup.+ population has been shown to
contain the cardiac mesoderm cells.sup.2. (b) Day 9 SIRPA flow
cytometric analysis expression profiles of the cultures described
in (a). (c) Day 20 cTNT profiles (intracellular flow cytometric
analysis) of the cultures described in (a). (d) Quantification of
a-c. Close correlation of expression of SIRPA at day 9 (green dots)
and cTNT expression at day20 (red rhombuses) illustrates the
predictive potential of SIRPA for cardiac differentiation
efficiency.
[0018] FIG. 7 shows enrichment of cardiomyocytes through negative
selection. (a) Flow cytometric analysis of markers specifically
expressed on non-myocyte (SIRPA-negative) cells in day 20
differentiation cultures (HES2). (b) Fluorescent activated cell
sorting for the combination of markers specifically expressed on
non-myocyte cells (in PE: CD31, CD90, CD140B, CD49A). (c) Flow
cytometric analysis of the presort cells, PE-negative (LIN.sup.-)
and PE-positive (LIN.sup.+) samples for SIRPA. (d) Quantification
of non-myocyte markers in at day20 of differentiation (as shown in
(a)), n=4. (e) Quantification of SIRPA-positive cells in PS, LIN-
and LIN+ fractions after cell sorting. Asterisks indicate
statistical significance as determined by student's t-test, ***
(p.ltoreq.0.001), n=3. (f) QPCR analysis of the presort (PS),
LIN.sup.- and LIN.sup.+ samples for non-cardiac markers (PECAM1,
PDGFRB, THY1 and DDR2) and cardiac specific genes (SIRPA, NKX2-5,
MYH6, and MYL7). Bars represent mean.+-.standard error of the mean.
Asterisks indicate statistical significance as determined by
student's t-test, * (p.ltoreq.0.05), ** (p.ltoreq.0.01), ***
(p.ltoreq.0.001), n=3.
[0019] FIG. 8 shows differentiation kinetics of the NKX2.5-GFP HES3
hESC line. Flow cytometric analysis of EBs derived from the
NKX2.5-GFP hESC line at various times during differentiation. GFP
expression is first detected at day8 of differentiation and
increases over time with maximum expression at day20.
[0020] FIG. 9 shows SIRPA expression kinetics of the NKX2.5-GFP
HES3 and the HES2 hESC lines. (a) Analysis and quantification of
SIRPA+/NKX2.5-GFP+ cells by flow cytometric analysis. EBs derived
from the NKX2.5-GFP hESC line were analyzed at various times during
differentiation, n=5. (b) Analysis and quantification of SIRPA+
cells by flow cytometric analysis. EBs derived from the HES2 hESC
line were analyzed at various times during differentiation, n=8.
d0=undifferentiated ES cells, d5-d20=differentiated EBs at
day5-day20.
[0021] FIG. 10 shows flow cytometry analysis strategy and staining
controls. (a) Flow cytometric analysis of day20 EB-derived cells.
All cells were stained with the viability dye DAPI and only
DAPI-negative cells (=viable cells) were analyzed for each
experiment. (b) Viable single cells were further determined by
FSC/SSC (cell size and granularity) in order to exclude debris and
doublets or cell clumps. (c) Unstained control of EB-derived cells
at day20 of differentiation. (d) Flow cytometric analysis of day20
EB-derived cells with the SIRPA-PE-Cy7 antibody and the
corresponding IgG control. (e) Flow cytometric analysis of day20
EB-derived cells with the SIRPA-biotin/Streptavidin-APC
(SIRPA-bio/SA-APC) antibody combination, the corresponding IgG
control and secondary antibody only staining. (f) Comparison of
cell size between SIRPA- and SIRPA+ cell populations (from (e)) by
FSC and SSC.
[0022] FIG. 11 shows Western Blot analysis and confirmation of the
specificity of the SIRPA antibody. (a) Western Blot analysis of 3
samples from day20 (d20) differentiation cultures compared to
undifferentiated ES cells (d0). The SIRPA SE5A5 antibody was used
and Ponceau staining is shown for loading control. (b)
Co-immunoprecipitation with the SIRPA SE5A5a antibody with
controls. SIRPA runs at the predicted size, as previously described
and analyzed in Timms et al., 1999.
[0023] FIG. 12 shows a comparison of SIRPA antibody staining with
mito tracker dye retention labeling. (a) Flow cytometric analysis
of mito tracker dye labelling at day 5, 8, 12 and 20 of
differentiation from HES2 hESCs. (b) Flow cytometric analysis of
SIRPA at day 5, 8, 12 and 20 of differentiation from HES2 hESCs.
(c) Co-staining of SIRPA and mito tracker dye labelling followed by
flow cytometric analysis at day 5, 8, 12 and 20 of differentiation
from HES2 hESCs.
[0024] FIG. 13 shows co-expression of SIRPA and cTNT. Cells were
stained for SIRPA first, then fixed (4 % PFA, 20 min), followed by
intracellular staining for cTNT. Since both primary antibodies have
been raised in mouse, appropriate controls are shown as well. Cells
were stained for anti-SIRPA-biotin/Streptavidin-APC (SIRPA single
stain), anti-SIRPA-biotin/Streptavidin-APC and anti-mouse-PE
(control to demonstrate that the secondary antibody for cTNT does
not recognize SIRPA after fixation),
anti-SIRPA-biotin/Streptavidin-APC and anti-cTNT and anti-mouse-PE
(SIRPA and cTNT co-staining).
[0025] FIG. 14 shows analysis of Sirpa expression in mouse
embryonic stem cell-derived cardiomyocytes and adult mouse tissue
samples. (a) Flow cytometric analysis of mESC-derived cardiac EB
cultures. Cells stained for Sirpa-AP, fixed with 4% PFA and stained
with cTnT/antimouse-PE. Sirpa-expressing cells did not co-stain
with cTnT-expressing cells, suggesting that cardiomyocytes derived
from mES cells do not express Sirpa. (b) Flow cytometric analysis
of mESC-derived cardiac EB cultures. Sirpa-positive cells co-stain
with CD45-PE-Cy7, suggesting that the Sirpa-positive cells present
in these cultures represent hematopoietic cells, which have
previously been described to express Sirpa (ref). (c) QPCR analysis
of Sirpa in adult mouse tissue samples. TA, tibialis anterior
muscle; GA, gastrocnemius muscle; GI, gastrointestinal tract; RT,
reverse transcriptase control; ESCM, mouse embryonic stem cell
derived cardiomyocytes day7 of differentiation (Kattman et al.,
2011). Mouse brain tissue was used as positive control. (d) Western
blot analysis of adult heart, brain and kidney tissue from control
(c) and Sirpa-deficient mice (ko) (Timms et al., 1999) and mouse
ESC-derived cardiomyocytes (d). Sirpa expression was solely
detected in the brain tissue of control mice, but not in any of the
Sirpa-deficient samples or in the control heart, kidney or
mESC-derived samples. Antibodies #16 and #9 (specific for
cytoplasmic domain, common to all Sirpa isoforms, AB#16, AB#9) were
used as described in Timms et al., 1999. ABCAM: anti-Sirpa
antibodies (Abcam, 8120).
[0026] FIG. 15 shows analysis of purity of SIRPA- and SIRPA+
fractions after FACS. (a) Flow cytometric analysis of presort,
SIRPA- and SIRPA+ fraction for SIRPA after cell sorting. (b)
quantification of SIRPA+ cells in presort, SIRPA- and SIRPA+
fraction after cell sorting, n=3.
[0027] FIG. 16 shows enrichment of cardiomyocytes from hESC-derived
cultures by cell sorting based on SIRPA expression. (a) Flow
cytometric analysis of SIRPA expression at day (d)8, d12 and d20 of
differentiation from NKX2.5-GFP HES3 hESCs. Fluorescent-activated
cell sorting (FACS) for SIRPA was performed at d8, d12 and d20 and
the presort (PS), SIRPA+ and SIRPA fractions were analysed for
cardiac TroponinT (cTnT) expression by intracellular flow
cytometry. The frequency of cTnT+ cells at d8, d12 and d20 was
significantly higher in the SIRPA+ fraction (day8: 89.8%.+-.1.9,
day12: 95.0.+-.1.3, day20: 89.4.+-.4.4), compared to SIRPA- cells
(day8: 9.9.+-.1.7, day12: 21.9.+-.2.5, day20: 5.2.+-.0.5), n=3. (b)
QPCR analysis of PS, SIRPA+ and SIRPA- cells after cell sorting.
Expression of markers specific for the cardiac lineage (NKX2.5,
MYH6, MYH7 and MYL7) was significantly higher in the SIRPA+
compared to SIRPA- fraction at all stages analyzed (d8, d12 and
d20). Expression of markers for the non-cardiac lineages (PECAM and
DDR2) segregated to the SIRPA- fraction and the PS cells, n=3.
[0028] FIG. 17 shows isolation of SIRPA+ cardiomyocytes via bead
sorting. (a) Flow cytometric analysis of SIRPA. HES2-derived EBs
were sorted using the Miltenyi magnetic bead sorting system and PS,
SIRPA+ and SIRPA fractions after sorting were analyzed for SIRPA
expression. (b) intracellular cTnT flow cytometric analysis of PS,
SIRPA+ and SIRPA- fractions.
[0029] FIG. 18 shows gene expression analysis of human adult
tissue. (a) QPCR RT analysis of SIRPA. (b) QPCR RT analysis of
CD47.
[0030] FIG. 19 shows expression of non-myocyte markers in
Y2-1-derived differentiation cultures. (a) Flow cytometric analysis
of matters specifically expressed on non-myocyte (SIRPA-) cells in
day 20 differentiation cultures. (b) Quantification of expression
of non-myocyte matters at day 20 of differentiation from Y2-1 iPS
cells.
[0031] FIG. 20 is a table showing the efficiency of
fluorescent-activated cell sorting (FACS) with the SIRPA antibody.
(a) Recovery of SIRPA- cells after FACS of EB-derived cells from
HES2 at day20 of differentiation, n=8. (b) Recovery of SIRPA+ cells
after FACS of EB-derived cells from HES2 at day20 of
differentiation, n=9. Total cell #=total cells passed through the
flow cytometer; SIRPA- (SIRPA+)#=total SIRPA-(SIRPA+) cells
recovered after the sorting procedure; SIRPA-(SIRPA+)% =percentage
of SIRPA-(SIRPA+) cells determined by staining with the SIRPA
antibody; SIRPA-(SIRPA+) exp cell #=cells number of SIRPA-(SIRPA+)
cells expected based on staining with the SIRPA antibody and on
total cell number sorted; Eff SIRPA-(SIRPA+)=efficiency of
SIRPA-(SIRPA+) cell recovery: SIRPA-(SIRPA+) cell #/SIRPA-(SIRPA+)
exp cell #; Eff SIRPA-(SIRPA+)=efficiency of SIRPA-(SIRPA+) cell
recovery in percentage.
[0032] FIG. 21 is a table showing the efficiency of
fluorescent-activated cell sorting (FACS) with the nonmyocyte
markers. (a) Recovery of LIN- cells after FACS of EB-derived cells
from HES2 at day20 of differentiation, n=6. (b) Recovery of LIN+
cells after FACS of EB-derived cells from HES2 at day20 of
differentiation, n=6. Total cell #=total cells passed through the
flow cytometer; LIN-(LIN+)#=total LIN-(LIN+) cells recovered after
the sorting procedure; LIN-(LIN+)% =percentage of LIN-(LIN+) cells
determined by staining with the LIN antibodies; LIN-(LIN+) exp cell
#=cells number of LIN-(LIN+) cells expected based on staining with
the LIN antibodies and on total cell number sorted; Eff
LIN-(LIN+)=efficiency of LIN-(LIN+) cell recovery: LIN-(LIN+) cell
#/LIN-(LIN+) exp cell#; Eff LIN-(LIN+)=efficiency of LIN-(LIN+)
cell recovery in percentage.
DETAILED DESCRIPTION
[0033] There is described herein the use of a high throughput flow
cytometry screen to identify cell surface markers specific for
human cardiomyocytes. Here we report that the cell surface receptor
SIRPA is expressed on hPSC-derived cardiomyocytes as well as on
human fetal cardiomyocytes. Using cell sorting with an antibody
against SIRPA we demonstrate that it is possible to isolate
populations consisting of up to 98% cardiomyocytes from hPSC
differentiation cultures.
[0034] Cell surface antigen, SIRPA (also known as CD172a, BIT,
SHPS1), can be found specifically and exclusively on cardiac
progenitor cells and on troponin T-positive cardiomyocyte cells
generated from human pluripotent stem cells (hPSCs) under
appropriate differentiation conditions.
[0035] Prior to the present application, there was no indication or
evidence in the art that SIRPA is expressed on developing mouse or
human cardiovascular cells. RNA expression of human SIRPA has been
found in different parts of the brain as well as in blood and at
low levels in the lung. However, SIRPA RNA expression has not been
found in the heart (http://biogps.gnf.org). SIRPA protein
expression has been detected in the brain, in blood and lymphoid
tissues and in the colon, and at moderate to weak levels in
placenta, pancreas, spleen, bladder and stomach
(http://www.proteinatlas.org/). However, no protein expression has
been reported for the adult human heart. As such, the discovery
that SIRPA is expressed in hPSC-derived cardiac progenitor cells
and cardiomyocyte cells is both novel and surprising.
[0036] In one example, the use of a SIRPA binding moiety, such as a
SIRPA antibody, provides a simple and novel method to identify,
monitor and isolate cardiomyocyte cells and their progenitor cells
from populations derived from human embryonic stem cells and
induced pluripotent stem cells. Cell isolation is easy and
efficient, yielding populations. In one embodiment, consisting of
greater than 90% cardiomyocyte cells that remain viable and can be
used for the applications disclosed herein.
[0037] SIRPA was identified as a potential cardiac marker in a
screen of over 350 commercially available antibodies supplied by
the Ontario Institute for Cancer Research Antibody Core Facility.
The antibodies were screened against hESC-derived populations
representing different stages of cardiac development generated by
the directed differentiation of the hESCs using a previously
published protocol (Yang et al., 2008)..sup.3 Antibodies that
stained cell populations of similar size to the cardiomyocyte
population in the differentiation cultures (as defined by cardiac
troponin T (cTnT) staining) were investigated further and used for
cell sorting. Of the 350 surface antibodies, one antibody, SIRPA,
specifically and exclusively stained the hESC-derived cardiomyocyte
population.
[0038] Cells isolated based on SIRPA expression represent a novel
source of highly enriched pluripotent stem cell-derived
cardiomyocyte progenitor cells (e.g. at the onset of Nkx2.5
expression but before cell contraction and expression of the
cardiac-specific structural proteins) and cardiomyocyte cells for
various applications, including but not limited to the
establishment of patient-specific disease models as well as
genetic, epigenetic and proteomic analyses of cardiac progenitor
cells and cardiomyocyte cells from normal and patient-specific
pluripotent stem cells.
[0039] The specific expression of SIRPA on cardiac cells and their
precursors suggests a function for this receptor and its downstream
signalling pathways during cardiac development and
differentiation.
[0040] SIRPA can also be used as a negative marker for cell sorting
experiments to enrich for non-cardiogenic PSC-derived lineages such
as including those derived from the somite (progenitor cells of
skeletal muscle, bone, and cartilage/chondrocytes).
[0041] Therefore, in one aspect, there is provided a method of
enriching a population of cells for cardiomyocyte cells and
cardiomyocyte progenitor cells comprising providing the population
of cells from which cardiomyocyte cells and cardiomyocyte
progenitor cells are to be isolated; and isolating from the
population, cells expressing SIRPA; wherein the population of cells
comprises a population of human pluripotent stem cells induced to
differentiate into cardiomyocyte cells and cardiomyocyte progenitor
cells.
[0042] In one embodiment, the human pluripotent stem cells are
embryonic stem cells. In another embodiment, the human pluripotent
stem cells are induced pluripotent stem cells.
[0043] In some embodiments, the human pluripotent stem cells are
exposed to an amount of at least one inducing agent effective to
induce cell differentiation.
[0044] In a preferable embodiment, the at least one inducing agent
comprises a cytokine. The at least one inducing agent may comprise
activin A, preferably at a concentration of up to 40 ng/ml, further
preferably at a concentration of about 6 ng/ml or about 30 ng/ml,
the at least one inducing agent may also independently comprise
bone morphogenetic protein 4, preferably at a concentration of up
to 40 ng/ml, further preferably at a concentration of about 10
ng/ml.
[0045] In some embodiments, the human pluripotent stem cells are
further exposed to a bone morphogenetic protein inhibitor,
preferably selected from the group consisting of Dorsomorphin,
Noggin and soluble bone morphogenetic protein receptors.
[0046] In some embodiments, the human pluripotent stem cells are
further exposed to at least one of VEGF, DKK and bFGF
[0047] In some embodiments, the human pluripotent stem cells are
exposed to the inducing agent for between about 1 and about 5 days,
preferably about 3 days.
[0048] In some embodiments, the time between the initiation of
induction of the human pluripotent stem cells and isolating the
cells expressing SIRPA is between about five days and about
forty-five days, preferably between about 8 and about 25 days.
[0049] In some embodiments, the cells expressing SIRPA are isolated
after the onset of SIRPA expression by the cells, which appears
around the time of onset of Nkx2.5 expression by the cells.
Preferably, the cells having the SIRPA cell surface antigen are
isolated between the time of the onset of Nkx2.5 expression by the
cells and the time of the onset of contraction and expression of
the cardiac-specific structural proteins by the cells.
[0050] In some embodiments, the method further comprises depleting
from the population, cells expressing at least one of CD90, CD31,
CD140B and CD49A, preferably using a corresponding antibody.
[0051] Methods for isolating cells expressing a particular
molecule, in this case SIRPA, are known to a person skilled in the
art. In some embodiments, the presence of SIRPA is directly used to
isolate cells by using a SIRPA-specific ligand, preferably using an
anti-SIRPA antibody or antibody fragment, or antibody-like
molecule, and further preferably an anti-SIRPA antibody. In some
embodiments, the cells are then isolated using magnetic beads
and/or flow cytometry. Alternatively, cells expressing SIRPA may be
indirectly selected. For example, in some embodiments, the cells in
the population comprise a reporter gene operably linked to
regulatory control elements of the SIRPA locus whereby the reporter
gene is expressed in cells that express SIRPA and the step of
isolating the cells expressing SIRPA comprises isolating cells
expressing the reporter gene. In one preferable embodiment, the
reporter gene confers resistance to a cytotoxic agent. In another
preferable embodiment, the reporter gene is a cell surface tag.
[0052] In some embodiments, the enriched population of cells
comprises at least 60%, preferably at least 90%, further preferably
98%, cardiomyocyte cells and cardiomyocyte progenitor cells.
[0053] In a further aspect, there is provided an enriched
population of cardiomyocyte cells and cardiomyocyte progenitor
cells obtained using any one of the methods described herein.
[0054] In a further aspect, there is provided an isolated
population of cells enriched for cardiomyocyte cells and
cardiomyocyte progenitor cells, wherein the population of cells
comprises at least 60%, preferably at least 90%, further preferably
98%, cardiomyocyte cells and cardiomyocyte progenitor cells.
[0055] In a further aspect, there is provided the use of SIRPA for
isolating cardiomyocyte cells and cardiomyocyte progenitor cells
from a population of cells, wherein the population of cells compose
a population of human pluripotent stem cells induced to
differentiate into cardiomyocyte cells and cardiomyocyte progenitor
cells.
[0056] In a further aspect, there is provided a method of depleting
a population of cells for cardiomyocyte cells and cardiomyocyte
progenitor cells comprising: providing the population of cells from
which cardiomyocyte cells and cardiomyocyte progenitor cells are to
be depleted; and depleting from the population, cells expressing
SIRPA; wherein the population of cells comprises a population of
human pluripotent stem cells induced to differentiate into
cardiomyocyte cells, cardiomyocyte progenitor cells, and
non-cardiomyocytes.
[0057] In a further aspect, there is provided a method of enriching
a population of cells for cardiomyocyte cells and cardiomyocyte
progenitor cells comprising: providing the population of cells from
which cardiomyocyte cells and cardiomyocyte progenitor cells are to
be isolated; and depleting from the population, cells expressing at
least one of CD90, CD31, CD140B and CD49A: wherein the population
of cells comprise a population of human pluripotent stem cells
induced to differentiate into cardiomyocyte cells, and
cardiomyocyte progenitor cells, and non-cardiomyocytes.
[0058] The term "enriching", as used in the context of tee present
invention, includes any isolation or sorting process that increases
the relative abundance of a desired cell type, or cell types, in a
population of cells.
[0059] As used herein, the term "cardiomyocyte cells" refers to the
cells that comprise cardiac muscle.
[0060] The term "cardiomyocyte progenitor cells" means progenitor
cells derived from human pluripotent stem cells that have the
capacity to differentiate into cardiomyocyte cells.
[0061] As used herein, the process of "isolating cells" refers to
any method known to those skilled in the art for sorting cells
including, but not limited to, flow cytometry, fluorescence
activated cell sorting, magnetic separation using antibody-coated
magnetic beads, affinity chromatography, and the exploitation of
differences in physical properties (e.g., density gradient
centrifugation).
[0062] "Embryonic stem cells" ("ESC") are pluripotent stem cells
that are derived from early-stage embryos.
[0063] "Induced pluripotent stem cells" ("iPSC"), as used in the
context of the present invention, is a type of pluripotent stem
cell that has been artificially derived from a non-pluripotent cell
by inducing the expression of specific genes.
[0064] The term, "cell surface antigen", refers to antigens on
surfaces of cells that are capable of being recognized by the
immune system and binding significantly to an antibody.
[0065] As used herein, the phrase "induced to differentiate" refers
to any method known in the art used to initiate the differentiation
of human pluripotent stem cells into specialized cell types. These
methods may include exposure of the human pluripotent stem cells to
an inducing agent.
[0066] As used herein, the term "inducing agent" refers to any
agent capable of initiating differentiation of hPSCs into
specialized cell types, including cardiomyocyte cells and
cardiomyocyte progenitor cells. Inducing agent therefore includes
cytokines, including but not limited to activin A, bone
morphogenetic protein 4 (BMP4), basic fibroblast growth factor
(bFGF, also known as FGF2), vascular endothelial growth factor
(VEGF, also known as VEGFA), dickkopf homolog 1 (DKK1), and
combinations therefrom.
[0067] Methods for inducing human pluripotent stem cells to
differentiate into cardiomyocyte cells and cardiomyocyte progenitor
cells are known to a person skilled in the art (for e.g., see Yang
et al..sup.3, and Laflamme et al..sup.19). In some embodiments,
induction conditions (e.g. concentrations of the inducing agents
and timing of their use) can be optimized by measuring SIRPA
concentration in the resulting enriched population.
[0068] The ability to generate cells of the cardiac lineage from
human pluripotent stem cells hPSCs (including embryonic stem cells;
hESCs and induced pluripotent stem cells; hiPSCs) provides a novel
and unlimited supply of human cardiomyocyte cells that will be
useful for: 1) predictive drug toxicology and drug discovery, 2)
transplantation for the treatment of cardiovascular disease and 3)
modeling cardiovascular development and disease in vitro.
[0069] The following examples are illustrative of various aspects
of the invention, and do not limit the broad aspects of the
invention as disclosed herein.
EXAMPLES
Materials and Methods
HPSC Maintenance and Differentiation
[0070] HPSCs were maintained as described.sup.26. Embryoid bodies
(EBs) were differentiated to the cardiovascular lineage as
previously described.sup.2,3 (FIG. 1a). In brief: EBs were
generated on day0 (d0) and BMP4 (1 ng/ml) was added for the first
day of differentiation (d0-d1). At d1, EBs were harvested and
resuspended in induction medium (basic fibroblast growth factor
(bFGF; 2.5 ng/ml), Activin A (6 ng/ml) and BMP-4 (10 ng/ml)). The
medium was changed on d4 and was supplemented with vascular
endothelial growth factor (VEGF; 10 ng/ml) and DKK (150 ng/ml).
Media was changed again on d8 and was supplemented with VEGF (20
ng/ml) and bFGF (10 ng/ml). EBs were cultured in StemPro-34
(Invitrogen) throughout the experiment. Cultures were maintained in
a 5% CO.sub.2, 5% O.sub.2, 90% N.sub.2 environment from d0-d12 and
were then transferred into a 5% CO.sub.2/air environment for the
remainder of the culture period.
[0071] NKX2-5-GFF hESCs were generated by targeting sequences
encoding GFP to the NKX2-5 locus of HES3 cells using previously
described protocols.sup.27 (D.E., A.G.E. and E.G.S., manuscript
submitted).
[0072] Work involving human tissue collection and analysis was
carried out in accordance with and approved through the Human
Ethics Committee at the University Health Network.
Flow Cytometry and Cell Sorting
[0073] Dissociation procedure for day5 to day12 EBs: EBs generated
from hPSC differentiation experiments were dissociated with 0.25%
trypsin/EDTA. Dissociation procedure for day13 and older EBs and
human fetal tissue: EBs generated from hPSC differentiation
cultures were incubated in collagenase type II (1 mg/ml;
Worthington, LS004176) in Hanks solution (NaCl 136 mM, NaHCO3 4.16
mM, NaPO4 0.34 mM, KCl 5.36 mM, KH2PO4 0.44 mM, Dextrose 5.55 mM,
Hepes 5 mM) over night at room temperature with gentle
shaking.sup.25. The following day, the equivalent amount of
dissociation solution (in Hanks solution: taurin, 10 mM, EGTA 0.1
mM, BSA 1 mg/ml, collagenase type II 1 mg/ml) was added to the cell
suspension and the EBs wore pipetted gently for complete
dissociation. Cells were centrifuged (1000 rpm, 5 min) and
filtered. For EBs past day 40 of differentiation, additional
treatment with 0.25% trypsin/EDTA may be required in order to
obtain complete dissociation into single cells.
[0074] Cells were stained at a concentration of 2.5.times.10.sup.6
cells/ml with anti-KDR--allophycocyanin (R&D Systems; 1:10) and
anti-PDGFRA--phycoerythrin (R&D Systems; 1:20),
anti-SIRPA--IgG-phycoerythrin-Cy7 (clone SE5A5; BioLegends;
1:500).sup.10,29, anti-SIRPA--IgG-biotin (clone SE5A5; BioLegends;
1:500).sup.10, anti-cardiac isoform of Troponin T (cTNT) (clone
13-11; NeoMarkers; 1:400), goat anti-mouse IgG--allophycocyanin
(BD; 1:200), Streptavidin--allophycocyanin (BD; 1:200),
anti-IgG1.kappa.--phycoerythrin-Cy7 (clone MOPC-21; BioLegends;
1:500), anti-IgG1.kappa.-biotin (clone MOPC-21; BioLegends;
1:500).
[0075] For cell surface markers, staining was carried out in PBS
with 10% FCS. For intracellular proteins, staining was carried out
on cells fixed with 4% paraformaldehyde (Electron Microscopy
Sciences, Hatfield, Pa., USA) in PBS and stainings were performed
in PBS with 10% FCS and 0.5% saponin (Sigma). Stained cells were
analyzed using an LSRII flow cytometar (BD). For fluorescent
activated cell sorting, the cells were sorted at a concentration of
10.sup.6 cells/ml in IMDM/6% FCS using a FACSAriaTMII (BD) cell
sorter (SickKids-UHN Flow Cytometry Facility, Toronto, ON, Canada).
In order to prevent cell death due to pressure and sheer stress,
all sorts were performed with a 100 micron nozzle. For magnetic
bead sorting, the Miltenyi MACS bead sorting system was used and
the experiments were carried out according to the manufacturer's
guidelines and the sorting conditions for dim markers. For the high
throughput flow cytometry analysis the BD high throughput sampler
(HTS) for the LSRII was used according to the manufacturers
guidelines. Data were analysed using FlowJo software (Treestar,
Ashland, Oreg., USA).
Immunostaining
[0076] Immunostaining was performed as previously described.sup.13
using the following primary antibodies: rabbit anti-cardiac
Troponin I (Abcam; 1:100), mouse anti-SIRPA (BioLegends; 1:100).
Secondary antibodies used were: goat anti-mouse IgGCy3 (Jackson
ImmunoResearch; 1:400), donkey anti-mouse IgG-Alexa 488
(Invitrogen; 1:400). DAPI was used to counterstain nuclei. Mito
Tracker Red (Invitrogen) was used to stain mitochondria. The
stained cells were visualized using a fluorescence microscope
(Leica CTR6000) and images captured using the Leica Application
Suite software.
Quantitiative Real-Time PCR
[0077] Total RNA was prepared with the RNAqueous-Micro Kit (Ambion)
and treated with RNase-free DNase (Ambion). 500 ng to 1 .mu.g of
RNA was reverse transcribed into cDNA using random hexamers and
Oligo (dT) with Superscript III Reverse Transcriptase (Invitrogen).
QPCR was performed on a MasterCycler EP RealPlex (Eppendorf) using
QuantiFast SYBR Green PCR Kit (Qiagen) as described
previously.sup.13. Expression levels were normalized to the
housekeeping gene TATA box binding protein (TBP). In addition to
TBP for normalization across samples, genomic DNA was used as a DNA
standard. The copy number of the target gene present in the genomic
DNA can be directly calculated (Human genome size:
2.7.times.10.sup.9 bp (=1.78.times.10.sup.12 Daltons), corresponds
to 6.022.times.10.sup.23 copies of a single copy gene; 1 .mu.g of
genomic DNA corresponds to 3.4.times.10.sup.5 copies of a single
copy gene). The Y-axis of RT-qPCR graphs represents copy numbers of
the gene of interest divided by copy numbers of TBP, and therefore
represents an arbitrary but absolute unit, that can be compared
between experiments.
[0078] Total human adult heart RNA was purchased from Ambion and a
total human RNA master panel was purchased from Clontech.
Results & Discussion
[0079] Identification of Novel Markers Expressed on hESC-Derived
Cardiomyocytes
[0080] When induced with appropriate concentrations of Activin A
and BMP4 (FIG. 1a), the HES2 hESC line efficiently and reproducibly
differentiates to generate cardiovascular lineage cells.sup.2,3.
Kinetic analyses of the differentiation cultures revealed a
step-wise developmental progression from a primitive streak-like
population defined by BRACHYURY (T) expression (days 2-4) to the
development of the early mesoderm (MESP1; days 3-4) and the
emergence of NKX2-5 and ISLET1 (ISL1) positive cardiac precursors
(days 4-8). Contracting cardiomyocytes were first detected between
days 9 and 12 of differentiation, coincident with the up-regulation
of MYH6 (.alpha.MHC), MYH7 (.beta.MHC) and MYL7 (MLC2a) and later
MYL2 (MLC2v) expression (FIG. 1b). The levels of expression of some
of the cardiac specific genes in the hESC-derived populations were
considerably lower than the levels found in fetal and adult heart
tissue. Low levels of NEUROD1 and FOXA2 expression indicate that
the cultures were not contaminated with substantial numbers of
neuroectoderm or endoderm-derived cells. To be able to monitor
cardiomyocyte development in real time, we applied the above
protocol to an NKX2-5-GFP reporter hESC line that contains the EGFP
cDNA inserted into the NKX2-5 locus of HES3 hESCs (Elliott et al.,
manuscript submitted). The first NKX2-5-GFP.sup.+ cells developed
between days 7 and 8 of differentiation. The size of the
NKX2-5-GFP.sup.+ population increased with time, reaching a maximum
between days 12-20 (FIG. 8). Analysis of NKX2-5-GFP ESC-derived
embryoid bodies (EBs) under epifluorescence confirmed nuclear GFP
expression in the majority of the cells. The kinetics of NKX2-5-GFP
expression closely parallels the onset of NKX2-5 expression in the
HES2 cultures, indicating that cardiac specification from both hESC
lines takes place between days 6 and 8 of differentiation (FIG. 1b,
FIG. 8). The high proportion of NKX2-5-GFP.sup.+ cells in day 20
cultures demonstrates that the differentiation protocol used
efficiently promotes the generation of cardiomyocytes from this
hESC line.
[0081] To determine if the above developmental stages can be
distinguished by cell surface markers, we carried out a screen of
370 known antibodies (http://data.microarrays.ca/AntibodyWeb) using
day 8, 12, and 20 populations generated from the GFP-NKX2-5 cell
line. The initial screen focused on identifying antibodies that
recognized antigens present on the NKX2-5-GFP.sup.+ population.
From this screen, we identified signal-regulatory protein alpha
(SIRPA, also known as SHPS-1, SIRPA) as a potential
cardiac-specific marker, as the anti-SIRPA antibody.sup.10 stained
the majority of the NKX2-5-GFP.sup.+ cells and almost none of the
negative cells (FIG. 2a). From the panel of antibodies analyzed,
SIRPA was the only one that displayed this cardiomyocyte specific
expression pattern. SIRPA was first detected on the emerging
GFP-NKX2-5.sup.+ cells at day 8 of differentiation, a population
considered to represent the cardiac precursor stage of development.
Expression was maintained on GFP-NKX2-5.sup.+ population throughout
the 20-day time course of the experiment (FIG. 2a, FIG. 9a). No
SIRPA.sup.+ cells were detected in undifferentiated hESC
populations or in the day 5 cardiac mesoderm population
characterized by co-expression of KDR and PDGFRA (FIG. 2a and data
not shown).sup.2. Analyses of EBs generated from the
non-genetically modified HES2 line revealed a similar staining
pattern with the anti-SIRPA antibody. SIRPA.sup.+ cells were first
detected at days 7-8 of differentiation and the percentage of
positive cells increased significantly over the next 2-4 days (FIG.
2b, FIG. 9b). Both the directly conjugated (SIRPA-PE-CY7) and the
botinylated (SIRPA-bio) antibodies stained similar portions of the
day 20 EB population (FIG. 10a-e). Interestingly, the SIRPA.sup.+
cells detected in day 20 EBs appear to be substantially larger than
those found in the SIRPA.sup.- population (FIG. 10f), suggesting
that cell size of these populations can be assessed by flow
cytometry. To confirm the specificity of the SIRPA antibody, we
carried out Western Blot analyses and immunoprecipitation followed
by Western Blot analysis (FIG. 11). These experiments demonstrated
the presence of SIRPA protein in 3 independent day 20 EB-derived
populations, but not in undifferentiated hESCs (FIG. 11a).
Immunoprecipitation analyses revealed a band the size of that
previously described for the SIRPA protein (FIG. 11b).sup.11.
[0082] Co-staining of SIRPA and cTNT by flow cytometry displayed
clear co-expression of the two markers (FIG. 12a/b). Indicating
that SIRPA was specifically expressed on the cardiomyocyte lineage
in differentiated populations generated from the non modified HES2
cell line.
[0083] RT-qPCR analyses revealed an expression pattern for SIRPA
that closely mirrored the flow cytometry antibody staining profile,
with an up-regulation of SIRPA mRNA between days 6 and 8 of
differentiation, followed by persistence of expression over the
42-day time course. Expression of CD47, the ligand for SIRPA,
paralleled that observed for SIRPA (FIG. 2c). Flow cytometric
analysis of CD47 reflected the gene expression pattern, showing low
levels of staining on undifferentiated ES cells and on day 5
differentiation cultures, followed by broad staining on the entire
population at days 8 and 20 (data not shown).
[0084] Immunofluorescence analysis of monolayer cultures derived
from day 20 EBs revealed SIRPA surface expression exclusively on
cardiomyocytes, as characterized by co-expression with cardiac
TroponinI (cTNI) (FIG. 2d) The respective controls (IgG and
secondary antibody only) did not show any signal (data not shown).
Collectively, these kinetic studies show that expression of SIRPA
uniquely marks the cardiac lineage in hESC differentiation
cultures, beginning with the emergence of NKX2-5.sup.+ precursor
cells and persisting through the development and expansion of
contracting populations.
[0085] Haitori et al recently demonstrated it was possible to
isolate cardiomyocytes based on mitochondria content, as measured
by retention of a mito tracker dye.sup.9. Comparison of mito
tracker dye labeling with SIRPA staining indicated that both
procedures mark the same cardiomyocyte population in day 20 EBs
(FIG. 13c). The dye retention approach was, however, less useful in
tracking the onset of cardiovascular development, as it marked a
less distinct population at day 12 of differentiation and almost no
cells at day 8 (FIG. 13a/b). In contrast, a substantial SIRP.sup.+
population could be clearly resolved at both these time points
indicating that this surface marker allows one to monitor and
isolate cells from different stages of cardiac development, whereas
labeling with the mito tracker dye can only be used on populations
containing relatively mature cardiomyocytes.
[0086] In contrast to the human cells, Sirpa was not detected on
mouse ESC-derived cardiomyocytes by antibody staining (FIG. 14a).
Sirpa.sup.+ populations in the culture were cardiac Troponin T
(cTnT) negative and CD45 positive, indicating that they represent
hematopoietic cells (FIG. 14a/b). Gene expression analyses
confirmed the flow cytometric data, and showed only low levels of
Sirpa mRNA in the mESC-derived cardiomyocytes as well as in adult
mouse atrial and ventricular tissues, compared to high expression
in the brain (FIG. 14c). Expression of the only other known Sirp
family member in the mouse, Sirpb, could not be detected in any of
these tissues by qPCR (data not shown). Western blot analysis of
control and Sirpa-deficient mouse tissue confirmed high Sirpa
expression in the brain of control mice, but not in any of the
tissues derived from Sirpa-deficient mice (FIG. 14d). Most
importantly, no Sirpa expression was detected in the heart, kidney
or mESC-derived cardiomyocytes from control mice.
[0087] Differences in SIRPA function and protein homology for mouse
and human have been described previously for the interaction of
macrophages and red blood cells.sup.12.
Purification of Cardiomyocytes From hESC-Derived Populations
[0088] To assess whether expression of the SIRPA surface receptor
can be used to generate enriched populations of cardiomyocytes,
SIRPA-positive (SIRPA.sup.+) and SIRPA-negative (SIRPA.sup.-)
fractions were isolated by cell sorting from HES2-derived EBs at
days 8, 12 and 20 of differentiation and analyzed for expression of
cardiac troponin T (cTNT) by intracellular flow cytometry (FIG.
3a). Analyses of the presort (unsorted, PS) populations
demonstrated that cTNT expression closely paralleled that of SIRPA
at the corresponding stages during differentiation (PS: d8, d12,
d20). Following sorting, the SIRPA.sup.+ fractions from each stage
were highly enriched for cTNT.sup.+ cardiomyocytes, whereas the
SIRPA.sup.- fractions were depicted of these cells. It is unclear
if the low numbers of cTNT.sup.+ cells present in the SIRPA.sup.-
fractions are contaminants from the sorting procedure or represent
true SIRPA-negative cardiomyocytes. FACS based separation in
multiple experiments reproducibly yielded significantly enriched
populations of cardiomyocytes (SIRPA.sup.+: day8 (96.2%.+-.1.9),
day12 (94.4%.+-.1.7), day20 (89.6%.+-.3.6); SIRP.sup.-: day8
(13.0%.+-.2.1), day12 (14.3%.+-.3.9), day20 (15.7%.+-.6.0)) (FIG.
3b). The purity of the SIRPA.sup.+0 and SIRPA.sup.- sorted
populations and the efficiency of cell recovery from the sorting
procedure is summarized in FIG. 15 and FIG. 20 (Table 1).
[0089] Molecular analyses revealed that the SIRPA.sup.+ cells
expressed significantly higher levels of NKX2-5, MYH6, MYH7 and
MYL7 than the SIRPA.sup.- population (FIG. 3c), further
demonstrating enrichment of cardiomyocytes. As expected, SIRPA
expression segregated to the SIRPA.sup.+ population in contrast to
the cardiac markers, non-myocyte markers such as the fibroblast
markers DDR2 and THY1 (CD90, data not shown) and the endothelial
marker PECAM (CD31) were expressed at higher levels in the
SIRPA.sup.- population (FIG. 3c).
[0090] When plated in monolayer cultures, cells from both
SIRPA.sup.- and SIRPA.sup.+ fractions formed viable populations
that could easily be maintained for several weeks. Contracting
cells were detected in unsorted (PS) and SIRPA.sup.+-derived
populations, but not in the population generated from the
SIRPA.sup.- cells. Immunohistochemical analysis revealed broad cTNI
expression in the SIRPA.sup.+ population confirming the high
proportion of cardiomyocytes in these cultures. Only few
cTNI-positive cells were detected in the SIRPA.sup.- population
(FIG. 3d)
[0091] As anticipated from the co-expression of SIRPA and
NKX2-5-GFP, it was also possible to isolate populations enriched
for cardiac lineage cells front NKX2-5-GFP HES3-derived cultures by
sorting with the anti-SIRPA antibody. Cardiac precursors (day 8)
and cardiomyocytes (days 12 and 20) defined by gene expression and
cTNT staining, segregated to the SIRPA.sup.+ fraction whereas
non-myocyte cells were enriched in the SIRPA.sup.- population (FIG.
16).
[0092] To enable rapid processing of large numbers of cells, we
also attempted to isolate SIRPA cells by magnetic bead sorting.
Isolation of SIRPA.sup.+ cells from NKX2-5-GFP differentiation
cultures by this approach resulted in populations highly enriched
for cardiomyocytes similar to those derived from FACS experiments
(FIG. 17a-c). However, with current magnetic bead sorting protocols
a substantial amount of cells is lost during the process, resulting
in a lower efficiency of this approach compared to FACS (compare
FIG. 17d to FIG. 20 (Table 1)).
[0093] Taken together, the findings from these cell sorting studies
dearly demonstrate that SIRPA expression marks the cardiac lineage
in hESC-derived differentiation cultures and that cell sorting with
the anti-SIRPA antibody allows for the isolation of populations
highly enriched for cardiomyocytes.
Purification of Cardiomyocytes From Human Induced Pluripotent Stem
Cells
[0094] To determine if SIRPA expression marked the cardiac lineage
in other hPSC-derived populations, we next analyzed EBs generated
from two different hiPSC lines, MSC-iPS1 (also known as Y2-1) and
38-2.sup.13,14. The efficiency of cardiac differentiation from both
lines was low, as demonstrated by the proportion of cTNT.sup.+
cells (MSC-iPS1: 12.2%.+-.5.6, 38-2: 26.7%.+-.5.7; FIG. 4a).
Similar low levels of SIRPA expression were detected in both EB
populations. FACS of the SIRPA.sup.+ cells from both iPSC lines
yielded populations significantly enriched for cTNT.sup.+
cardiomyocytes (SIRPA.sup.+: MSC-iPS1 (67.0%.+-.3.6), 38-2
(71.4%.+-.3.8); SIRPA.sup.-: MSC-iPS1 (4.9%.+-.2.1), 38-2
(6.2%.+-.0.9)) (FIG. 4a,b). These SIRPA.sup.+ populations expressed
significantly higher levels of NKX2-5, MYH6, MYH7, MYL2 and MYL7
than the corresponding SIRPA.sup.- cells. As observed with the
hESC-derived cells, non-myocyte markers including DDR2, PDGFRB,
THY1 and NEUROD segregated to the SIRPA.sup.- fraction (FIG. 4b,c
and data not shown).
[0095] Those data clearly document the utility of this marker for
generating enriched cardiac populations from a range of pluripotent
stem cell lines, including these that do not differentiate
efficiently to the cardiac lineage with the current protocols.
SIRPA Expression in Human Fetal and Adult Heart Cells
[0096] To determine if SIRPA is expressed on primary human
cardiomyocytes, we next analyzed expression patterns in fetal
(18-20 weeks of gestation) and adult heart tissue by RT-qPCR. As
shown in FIG. 5a, SIRPA transcripts were detected in all
fetal-derived heart tissue (left (LA) and right atrial (RA) cells,
left (LV) and right ventricle (RV) cells, apex (AP) and
atrioventricular junction (AVJ)), with comparable levels to those
found in day 20 hESC-derived cells (FIG. 3a). SIRPA was not
expressed in undifferentiated hESCs (d0) or in control HEK (human
embryonic kidney) cells. Similar to the fetal heart, SIRPA
expression was also detected in the adult heart, suggesting that
its expression marks cardiomyocytes at different stages of human
cardiac development. High levels of SIRPA were detected in the
adult human brain and lung (FIG. 18a) with low levels found in many
other tissues. These low levels may reflect the presence of tissue
macrophages that are known to express this receptor.sup.15,16.
CD47, the SIRPA ligand was expressed in most tissues, confirming
the pattern described in previous studies (FIG. 18b).sup.15.
Immunofluorescence staining showed that SIRPA was localized on the
surface membrane of the fetal ventricular cells but was not present
on other membrane fractions such as the mitochondrial membrane, as
indicated by the lack of co-staining with Mito Tracker Red (FIG.
5b). Flow cytometric analyses revealed a high proportion of
SIRPA.sup.+ cells in all fetal heart tissues at levels that
correlated with the percentage of cTNT.sup.+ cells in the
respective fractions (FIG. 5c, d).
[0097] These findings demonstrate clearly that SIRPA is expressed
on fatal cardiomyocytes as well as in adult heart, illustrating
that its cardiac-specific expression is not an artifact of
pluripotent stem cell-derived cultures.
Using SIRPA Expression to Monitor the Efficiency of hPSC
Differentiation
[0098] Recently, we reported that co-expression of KDR and PDGFRA
provides a reliable method to monitor cardiac mesoderm induction
following treatment with BMP4 and Activin A.sup.2 (FIG. 6a). While
this study showed that the induction of a KDR.sup.+PDGFRA.sup.+
population was an essential first step in the generation of the
cardiomyocyte population, not all KDR.sup.+PDGFRA.sup.+ populations
differentiated to give rise to cardiac lineage cells (example of
this type of population: induced with 30 ng/ml BMP4 and no
exogenous Activin A (A0)). To determine if SIRPA would more
accurately predict cardiac potential of differentiating populations
at an early stage, we monitored its expression in day 9 EBs induced
with different concentrations of Activin A and BMP4 (FIG. 6b). The
same populations were evaluated at day 5 for expression of KDR and
PDGFRA (FIG. 6a) and at day 20 for expression of cTNT (FIG. 6c).
While there was little correlation between the size of the
KDR.sup.+PDGFRA.sup.+ population at day 5 and the proportion of
cTNT.sup.+ cells at day 20, the cultures with the largest SIRPA
population at day 9 (Activin A 6 ng/ml, BMP4 10 ng/ml) contained
the highest number of cTNT.sup.+ cells at the later time point.
SIRPA expression correlated well with cTNT output for most
conditions tested and the highest levels of SIRPA predicted the
highest cardiomyocyte development at day 20 (FIG. 6d). These data
demonstrate that expression of SIRPA at day 9 is a reliable
indicator of cardiomyocyte potential, and as such can be used to
monitor and optimize induction protocols for directed
differentiation of hPSCs to the cardiac lineage.
Enrichment of hPSC-Derived Cardiomyocytes Through Depletion of the
Non-Myocyte Lineage Cells
[0099] In addition to antibodies that recognize cardiomyocytes, our
now cytometric screen also identified a panel of antibodies that
marked the non-myocyte population in the differentiation cultures.
This set of antibodies, including anti-CD90 (THY1, expressed on
fibroblast cells), anti-CD31 (PECAM1, expressed on endothelial
cells), anti-CD140B (PDGFRB, expressed on smooth muscle cells) and
anti-CD49A (INTEGRIN1A), all recognized different proportions of
the SIRPA.sup.- population of day 20 HES2-derived EBs (FIG. 7a/d).
The combination of these antibodies marked the majority of
non-myocyte (SIRPA.sup.-) cells in the culture (FIG. 7c. presort).
To determine if it was possible to enrich for cardiomyocytes by
depleting cells expressing the non-myocyte markers, we combined
these antibodies and sorted day 20 EBs into lineage-positive
(LIN.sup.+) and lineage-negative (LIN.sup.-) fractions (FIG. 7b).
This approach has the advantage in generating enriched populations
free of any bound antibody or magnetic beads. As expected, the
LIN.sup.- population was significantly enriched for SIRPA.sup.+
cells, whereas the LIN.sup.+ population was depleted for the
cardiomyocytes (FIG. 7c/e). The efficiency of cell recovery after
FACS for LIN.sup.- and LIN.sup.+ cells is summarized in FIG. 21
(Table 2). Gene expression analyses revealed that non-myocyte
specific genes including PECAM1, PDGFRB, THY1 and DDR2 were
primarily expressed in the LIN.sup.+ fraction, whereas cardiac gene
expression was restricted to the LIN.sup.- fraction (FIG. 7f). When
plated on gelatin coated dishes or re-aggregated as cell clusters,
the LIN.sup.- fraction generated populations that contained a high
proportion of contracting cardiomyocytes (data not shown). The same
lineage cocktail of antibodies also marked the non-myocyte (SIRPA-)
fraction of the iPSC (MSC-iPS1)-derived day 20 EB population (FIG.
19), indicating that this depletion approach can be applied to
different PSC lines with variable differentiation efficiencies.
[0100] Taken together, these data illustrate that cardiomyocytes
can be enriched from hPSC-derived differentiation cultures by
depletion of the non-myocyte lineages. This method therefore
represents an alternative approach to obtaining highly purified
cardiomyocyte cultures and may as such be used for strategies that
require purified cardiomyocyte populations free of any bound
antibodies.
[0101] Advances in our understanding of the signaling pathways that
regulate lineage specification has led to strategies for the
efficient and reproducible directed differentiation of hPSCs to
specific cell types.sup.1. With respect to cardiac lineage
development, protocols have been established that promote the
generation of mixed cardiovascular populations representing the
major cell types found in the human heart including cardiomyocytes,
endothelial cells, vascular smooth muscle cells and fibroblasts.
Cardiomyocytes typically represent between 10% and 70% of such
mixed populations.sup.2,3, depending on the PSC line used. While
such mixed populations have been used to demonstrate the potential
utility of the PSC-derived cells for predictive toxicology.sup.5,
modeling human disease in vitro.sup.17,18 and transplantation based
therapy for heart disease.sup.19, highly enriched and well defined
cell populations will ultimately be required to translate this
potential into practical applications.
[0102] Our identification of SIRPA as a cardiomyocyte-specific
marker now enables, for the first time, easy and routine access to
highly enriched populations of cardiomyocytes from hESCs and
hiPSCs. Those cardiomyocyte enriched populations can be isolated by
FACS or magnetic bead sorting, the latter approach enabling the
isolation of large numbers of cells required for in vivo studies.
Access to highly enriched populations of cardiomyocytes through
simple sorting approaches will enable the development of defined
high throughput drug discovery and toxicology assays, the detailed
phenotypic evaluation of cells generated from patient specific
hPSCs, and the generation of defined populations safe for
transplantation. The fact that SIRPA is expressed on cardiac
lineage cells from the earliest cardiac stage to contracting and
more mature cardiomyocytes will allow for comparisons of the in
vivo potential of the different populations.
[0103] In addition to SIRPA, our screen also identified a panel of
markers defining the non-myocyte fractions of the PSC-derived
cardiovascular population. The markers used suggest that they
represent a combination of fibroblasts (CD90, THY1).sup.20,
vascular smooth muscle cells (CD140B, PDGFRB).sup.21 and
endothelial cells (CD31, PECAM1). Access to enriched populations of
each of these cell types together with cardiomyocytes will allow.
Many of the proposed applications for PSC-derived cardiomyocytes
may require three-dimensional engineered tissue to more accurately
reflect drug responses and function in the adult heart. Recent
studies suggest that appropriate combinations of cardiac cells,
endothelial cells and fibroblasts need to be incorporated into such
tissue constructs in order for them to function best in vitro or in
vivo.sup.22-24. Our ability to generate pure myocyte and
non-myocyte populations will allow for the generation of engineered
constructs consisting of varying proportions of different cell
types, enabling us to determine the optimal proportion of each
required to form heart tissue with structural and functional
properties most similar to that of the human heart.
[0104] The specific expression pattern of SIRPA in the PSC-derived
populations and in the fetal heart tissue suggests that this
receptor plays some functional role in the human cardiomyocyte
lineage, perhaps as early as the precursor stage of development.
The fact that expression of the ligand, CD47, is upregulated in
parallel with SIRPA in the EBs and that CD47 is found on a large
proportion of the cells in the culture further supports the
interpretation that this ligand/receptor pair plays a role in the
human cardiomyocyte development and/or function. One thoroughly
studied role for SIRPA is on macrophages, where it appears to
mediate a signal to eliminate cells from the body that do not
express the ligand CD47.sup.16. The only other suggested function
in human cells is in the smooth muscle lineage, where SIRPA has
been shown to play an important role in mediating IGF-1-induced
mitogenic signaling.sup.26. Given that SIRPA was not detected in
mouse cardiomyocytes, it is possible that its function in human
cells may relate to aspects of cardiomyocyte physiology and/or
function that differ between the two species.
[0105] In summary, the findings reported here demonstrate that
expression of SIRPA uniquely marks the cardiomyocyte lineage in
PSC-differentiation cultures. Isolation of SIRPA.sup.+ cells by
FACS or magnetic bead sorting provides a simple approach for
generating highly enriched populations of cardiomyocytes from a
broad range of PSC lines, including those that do not differentiate
efficiently to the cardiovascular lineage using current
protocols.
[0106] Although preferred embodiments of the invention have been
described herein, it will be understood by those skilled in the art
that variations may be made thereto without departing from the
spirit of the invention or the scope of the appended claims. All
references in this description, including those in the following
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References