Scientists claim SIRPA is expressed on cardiomyocytes but not other cell types at all stages of hESC and iPSC differentiation.

Investigators have identified a specific cell surface marker on stem-cell derived cardiomyocytes that allows their isolation from a mixed pool of differentiated human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hIPSCs). The researchers, from Ontario Cancer Institute, the University of Toronto, and Monash University in Australia, used a panel of antibodies to screen for antigens present on differentiating cardiomyocyte precursor cells. They identified SIRPA as a marker that was present at all stages of differentiation from both hESCs and hIPSCs, and could be used to enrich for cardiomyocytes using techniques including FACS or magnetic bead sorting.

Reporting in Nature Biotechnology, the team says its antibody screen also identified a panel of antibodies that marked nonmyocytes in differentiation cultures, which could similarly be used to deplete cell populations of nonmyocyte cells. Using this approach only the unwanted cell types are manipulated.

They say access to highly enriched populations of cardiomyocytes using simple cell-sorting techniques will facilitate the development of drug discovery and toxicology assays, provide a source of cells for research, and allow the derivation of cell populations suitable for human transplantation. Ontario Cancer Institute’s Gordon Keller, M.D., and colleagues report their research in a paper titled “SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells.”

The ability to isolate pure populations of cardiomyocytes from differentiated human pluripotent stem cells (hPSCs) would provide a source of heart cells for both drug testing and potentially regenerative therapies, the researchers note. However, achieving this will require the ability both to reproducibly generate enriched populations of cardiomyocytes from different stem cell lines, and identify and purify the desired cell type from the resulting pool of cardiomyocytes, smooth muscle cells, fibroblasts, and endothelial cells that typically result from the differentiation of hPSCs, even under optimal conditions.

However, although cardiomyocotes may represent up to 70% of the population for any given hPSC line, there is considerable variation between different stem cell lines. To date, an induction technique that results in highly enriched populations of cardiomyocytes from a range of hPSCs has yet to be developed, nor have foolproof nonmanipulative methods been developed to isolate cardiomycotyes from differentiation cultures. To try and address this issue, the researchers developed a broadly applicable strategy for enriching hPSC-derived cardiomyocytes using a high-throughput flow cytometry antibody screen to identify human cardiomyocyte-specific cell surface markers.

They first differentiated an hESC HES2 line, which is known to efficiently and reproducibly generate cardiovascular lineage cells under appropriate conditions, and carried out kinetic analyses of the differentiation cultures to confirm that their protocol resulted in the generation of cardiomyocytes. Then, to monitor cardiomyocyte development in real time, they applied the same protocol to an NKX2-5-GFP reporter cell line derived from HES3 hESC cells, which fluoresces on the expression of the cardiac precursor cell marker NKX2-5. This confirmed that the kinetics of NKX2-5–GFP expression closely paralleled the onset of NKX2-5 expression in the HES2 cultures, and indicated that cardiac specification from both hESC lines takes place between days 6 and 8 of differen­tiation.

In the next stage the researchers used a screen of about 370 antibodies to identify antigens that were specifically present on the NKX2-5+ population. This highlighted SIRPA (also known as SHPS-1 or CD172a) as a potential cardiac-specific marker, as the anti-SIRPA antibody stained the majority of the NKX2-5–GFP+ cells, but virtually none of the NKX2-5–GFP− cells. SIRPA was first detected on emerging GFP-NKX2-5+ cells on day 8 of differentiation (at this point cells are considered to repre­sent the cardiac precursor stage of development), and its expression was maintained in the GFP-NKX2-5+ population throughout the 20-day time course of the experiment. Importantly, no SIRPA+ cells could be detected in undifferentiated hESC popula­tions or in the day 5 cardiac mesoderm population.

Encouragingly, an equivalent staining pattern with the SIRPA antibody was found on analyzing embryoid bodies generated from the nongenetically modified HES2 lines. Western blot and immunoprecipitation studies confirmed that the SIRPA protein was present in day 20 embryoid body-derived populations. Notably, the SIRPA+ cells appear to be sub­stantially larger than those in the SIRPA− population, which further suggests that cardiovascular cell size can be monitored using the antibody, the authors write.

SIRPA was co-expressed with cardiac troponin T (cTNT), indicating that it was specifically expressed on cardiomyocyte lineages in differentiated populations of nonmodified HES2 cells, while expression of the SIRPA ligand, CD47, also occurred in parallel. “Collectively, these kinetics studies show that expression of SIRPA uniquely marks the cardiac lineage in hESC dif­ferentiation cultures, beginning with the emergence of NKX2-5+ pre­cursor cells and persisting through the development and expansion of contracting populations,” the team remarks.

To evaluate whether expression of the SIRPA surface receptor on cardiomyocyte lineages could provide a way of generating enriched populations of these cells, SIRPA+ and SIRPA− fractions were isolated by cell sorting from HES2-derived embryoid bodies on days 8, 12, and 20 of differentiation, and then ana­lyzed for expression of cTNT by intracellular flow cytometry. In the unsorted cell population, cTNT expression closely paralleled that of SIRPA at the three time stages evaluated. After sorting, the SIRPA+ fractions from each stage were highly enriched for cTNT+ cardiomyocytes, whereas the SIRPA− fractions were depleted of these cells.

SIRPA+ cells expressed significantly higher levels of NKX2-5, MYH6, MYH7, and MYL7 than SIRPA- cells, whereas the SIRPA- population expressed nonmyocyte markers. Notably, when plated in monolayer cultures, contracting cells were detected in unsorted and SIRPA+-derived populations, but not among SIRPA- cells. Immunohistochemical analysis revealed broad cTNI expression in the SIRPA+ population, confirming the high proportion of cardio­myocytes in these cultures, but not in the SIRPA- population.

The team moved on to assess whether SIRPA expression represents a cardiac lineage marker in two different hIPSC lines, MSC-iPS1 (also known as Y2-1) and 38-2. They found that efficiency of cardiac differentiation from both lines was low (there was a maximum 26.7% proportion of cTNT+ cells in the resulting populations), and low SIRPA expression was detected in embryoid body populations derived from both cell lines.

Nevertheless FACS of the SIRPA+ cells from both iPSC lines still yielded popula­tions significantly enriched for cTNT+ cardiomyocytes. As with the hESC-derived cardiac cells, the hiPSC-derived SIRPA+ cells expressed significantly higher levels of NKX2-5, MYH6, MYH7, MYL2, and MYL7 than the corresponding SIRPA− cells, which instead expressed nonmyocyte markers, including DDR2, PDGFRB, THY1, and NEUROD.

“These data clearly document the utility of this marker for generating enriched cardiac populations from a range of hPSC lines, including those that do not differentiate efficiently to the cardiac lineage with current protocols,” the researchers note.

It was important to check whether SIRPA is also expressed on primary human cardiomyocytes, so the team analyzed expression patterns in 18–20-week fetal cells and in adult heart tissue using RT-qPCR. SIRPA transcripts were detected in fetal-derived tissue from each region of the developing heart, at similar or higher levels than those found in day 20 hESC-derived cells. Similar to the fetal heart, SIRPA expression was also detected in the adult heart, but wasn’t found in undifferentiated hESCs, or control human embryonic kidney cells. Flow cytometric analyses revealed a high proportion of SIRPA+ cells in all fetal heart tissues at levels that correlated with the percentage of cTNT+ cells in the respective fractions.

In addition to antibodies that recognized cardiomyocytes, the team’s screen also identified a panel of antibodies that marked the nonmyocyte population in differentiation cultures. This set of antibodies included anti-CD90 (also known as THY1, expressed on fibroblast cells), anti-CD31 (also known as PECAM1, expressed on endothelial cells), anti-CD140B (also known as PDGFRB, expressed on smooth muscle cells), and anti-CD49A (also known as ITGA1). All the antibodies recognized different proportions of the SIRPA− population of day 20 HES2-derived embryoid bodies.

Taking an approach that is essentially opposite to using SIRPA to enrich for cardiomyocytes, the team used the set of antibodies to remove nonmyocyte cells, and sort embryoid bodies derived from either hESCs or hIPSCs, into lineage-positive (LIN+) and lineage-negative (LIN-) fractions. “This approach has the advantage of gen­erating enriched populations free of any bound antibody or magnetic beads,” they point out. As expected, the LIN− populations were significantly enriched for SIRPA+ cells, whereas the LIN+ populations were depleted for the cardiomyocytes: gene expression analyses confirmed that nonmyocyte-specific genes were primarily expressed in the LIN+ fraction, while cardiac gene expression was restricted to the LIN- fraction. When plated on gelatin-coated dishes or reaggregated as cell clusters, the LIN− fraction generated popula­tions that contained a high proportion of contracting cardiomyocytes.

“Taken together, these data show that cardiomyocytes can be enriched from hPSC-derived differentiation cultures by depletion of the nonmyocte lineages,” the authors conclude. “This method therefore represents an alterna­tive approach to obtaining highly purified cardiomyocyte cultures and as such can be used when purified cardiomyocyte populations free of bound antibodies are required…Our identification of SIRPA as a cardiomyocyte-specific marker now enables easy and routine access to highly enriched populations of cardiomyocytes from hESCs and hiPSCs,” they assert.

“Such 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 team suggests that although the nonmyocyte populations defined by the panel of markers identified in their study have not been fully characterized, the cell markers suggest that they rep­resent a combination of fibroblasts (CD90)21, vascular smooth muscle cells (CD140B)22, and endothelial cells (CD31). “Access to enriched populations of each of these cell types together with cardiomyocytes will allow detailed investigation of interactions between them,” they continue. “Such interactions may alter the drug sensitivity and responsiveness of cardiomyocytes and are likely to influence the survival and maturation of cardiomyocytes in vitro and following transplantation in vivo.”

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