ESC differentiate into different cell types despite efforts for directed differentiation protocols. Sorting specific differentiated or progenitor cell types cannot be circumvented yet. Consequently, there is the necessity to eliminate undesired cell types, before characterization and functional analysis.
For applications such as drug screening as well as pharmacological and/or toxicity predictive studies ESC‐derived cardiomyocytes are considered a valuable tool [1, 101]. Taking into account that differentiation of mESC into spontaneously beating embryoid bodies results in 1‐3% cardiomyocytes [1], it becomes obvious that efficient enrichment and isolation strategies prior to experimentation are critical before manipulation.
If the availability of “pure” cell populations is important for in vitro assays like drug screening, toxicity assays and so forth, this is especially true for cell transplantation experiments, not only in animals but eventually in future clinical settings [102]. A pure and homogenous cell population would thereof demonstrate an invaluable benefit for medicinal applications, like cell replacement therapies for diverse diseases. However, standards on the definition of a “pure” versus “heterogeneous” cell population must be agreed on.
Furthermore is the scale‐up, which should provide a sufficient number of cells, important to address.
As cardiac diseases are the number one cause of death worldwide [103, 104], the possibility of having a source of ESC at hand that develops spontaneously into bona fide cardiac cells, has ever since raised the hope for cell replacement strategies of the damaged heart. So far however, results obtained with cardiac replacement experiments in animal models,
depending on the cell source and the delivery method used, have been modestly successful [14]. Several controversial issues remain to be clarified. Hurdles to overcome include 1) the successful delivery and integration of donor cells in the recipient’s heart, 2) the avoidance of generating tumors or unwanted cell types, and 3) the prevention of arrhythmias deriving from non electrically‐connected cells. Not last, aside from immunological issues that need to be addressed, the overall improved cardiac function, a prolonged life and an improved quality of life would need to be demonstrated [14, 104].
Importantly, for future clinical applications, cell therapy options should become cost effective and sustainable for the society as well as health insurances. While cell therapy probably remains rather a long‐term goal, the use of cardiac cells for drug and toxicity screenings is clearly more nearby [101].
In summary, there is a demand for large amounts of homogenous and enriched cell populations for both investigations into clinical applications as well as toxicity and drug screening assays [3]. Murry et al. calculated one billion cardiomyocytes to be lost during myocardial infarction and for this reason they propose this amount of cells to be used for restoring the infarcted area [2]. However, selection and enrichment strategies are still under investigation and elaboration with variable rate of success. Selection for a specific cell type, like cardiomyocytes, requires a cardiac exclusive marker/phenotype that could be exploited for purification.
For enrichment of cardiac cells, there are different strategies at hand. They will be presented here below. Depending on the method used, enrichment/purification techniques can be applied at different time points of the differentiation process. At early steps of differentiation one can obtain relatively unspecialized and immature cardiac cells that
represent rather cardiac progenitors. The advantage resides in their ability to further proliferate and the possibility of expansion in culture. The isolation of such cardiac progenitor cells would only represent one of several footsteps on the road towards obtaining a pure population of cardiac cells. Careful expansion and subsequent guided differentiation would be the necessary steps for obtaining pure cardiac cell populations.
On the other end, the isolation of cardiac cells at “later” time points of differentiation would result in relatively differentiated cells that could be useful for drug testing since they should resemble cells closer to the adult heart phenotype [105]. The disadvantage resides in their limited or null proliferative potential [106], with the associated drawback of a possible reduced survival potential upon transplantation in vivo [105].
4.1. Non transgenic cardiac enrichment
One great advantage of non‐transgenic cardiac enrichment strategies over transgenic ones is the circumvention of genetic perturbations due to random or directed transgene insertion. This is clearly relevant for clinical applications. Such methods have been quite successful in isolating cardiac progenitor cells thanks to the identification of different cardiac lineage‐specific surface markers. Using antibodies, cardiac cells can be sorted out after dissociation of differentiating EBs and used for subsequent experiments. Very recent markers Flk1 and PDGFR [107] and the signal‐regulatory protein alpha (SIRPA) surface marker (CD172a), have been identified to be specifically expressed on hESC and hiPSC derived cardiomyocytes [108]. Nevertheless, no consensus exists yet on which would be the most relevant and promising marker for sorting.
A less invasive and more subtle strategy for enrichment tries to exploit the molecular pathways occurring during cardiogenesis. Finding additives that trigger and support cardiac differentiation can enhance the cardiac cell outcome. The careful screening for molecules, chemically‐defined supplemented media based on known molecular switches, although time consuming, enables to avoid the use of antibodies and FACS and the related cell stress and damage.
Last but not least, some physical means of enrichment are under development. Rather than being competitive to other known procedures, physical means are presented as promising techniques for the future. Both Raman spectroscopy and deterministic lateral displacement based selection are techniques that constitute at present more proofs of principle rather than real alternatives for cardiac enrichment.
Of interest has been the recently published isolation of differentiated cardiomyocytes using the reversible staining of mitochondria, with a reported cardiac cell purify of 99%. However, cell dissociation and FACS cannot be avoided.
Enrichment strategies will be presented more in detail in the following.
4.1.1. Isolation of cardiac progenitors
Defining the transcriptional profile and isolating cardiac progenitor cells with subsequent expansion and directed differentiation is one promising way of enriching for cardiac cells. In 2006 Wu et al. [109] isolated Nkx‐2.5‐positive cells from mouse differentiating EBs using a
Nkx‐2.5‐enhancer – GFP reporter construct. 28% of these cells were positive also for the surface receptor c‐kit. Nkx‐2.5+, c‐kit+ cells allowed for long‐term culture and differentiation into both beating cardiomyocytes (up to 42%), as well as smooth muscle cells. Authors strengthened their findings with the isolation of Nkx‐2.5+ c‐kit+ cells also from mouse embryos [109].
Alternatively, Moretti et al. proposed a different phenotypic profile to be used for cardiac progenitor isolation, i.e. a “multipotent Isl1+ cardiovascular progenitor cell (MICP) positive for Isl1, Nkx‐2.5 and Flk1 [95].
Hirata et al. used an interesting approach in determining a new cardiac progenitor marker.
Based on the findings that Flk‐1 is not exclusively expressed on cardiac mesoderm but on other cell types derived from mesoderm, like hematopoietic [110] or endothelial [111] cells too, the authors stress the necessity to find another marker that would be more restricted to the cardiac lineage. Authors used microarray results obtained with the EC line P19CL6 that was triggered into the cardiac lineage with DMSO. Hirata et al. related surface molecules to corresponding upregulation of cardiac expression and appearance in the ECs.
The two receptor tyrosine kinases Flk‐1 and PDGFRα were found and validated by flow cytometry with specific antibodies, mRNA expression as well as in situ hybridization in the cardiac crescent and linear heart tube of mouse embryos.
Sorting mESC derived cardiac precursors on day 5 of differentiation resulted in the highest yield of Flk‐1+/PDGFRα+ cells with an outcome of ~50%. After 5 more days of culture on cardiac differentiation supporting OP9 stromal cells, sorted cells showed a 5‐10‐fold higher incidence of beating colonies compared to Flk‐1‐/PDGFRα-, Flk‐1+/PDGFRα‐ and Flk‐1‐
4.1.2. Membrane proteins used for isolation and enrichment
Sorting live populations of cardiomyocytes so far has mainly been possible after transgenic modifications of cells to afford expression of fluorescent reporter genes allowing detection throughout the process of flow cytometry [112]. Targeting and labeling proteins in the cytosol or nucleus with fluorescent antibodies for subsequent FACS is possible, however requires permeabilization, fixation and therefore results in death of targeted cells.
The first paper describing a surface protein exclusively expressed on hESC derived cardiomyocytes was published in 2010 by Van Hoof et al. [102]. The authors selected mechanically cut cardiomyocytes derived from hESC for their analysis. As controls human fetal hearts as well as whole populations of differentiated cells derived from hESC were used. Mass spectrometry (MS) based membrane proteomics were applied in order to find surface proteins being expressed on hESC derived cardiomyocytes but not on the control cells. The authors were able to sort live hESC derived cardiomyocytes from a differentiated EB population by using antibodies against found target membrane protein elastin microfibril interface 2 (EMILIN2) [102]. The dissociation of EBs was performed without trypsin to preserve membrane surface proteins. Authors report to sort 10% live cardiomyocytes. Using fixed cells, the percentage raised to 21%, resembling the 20‐25% cardiomyocytes demonstrated to be expected in beating areas [113]. Obviously, the antibody use on either live or fixed and dead cells influences the outcome of sorted cardiomyocytes.
Dubois et al. report the detection of the cardiac specific surface signal‐regulatory protein alpha (SIRPA) on hESC and hiPSC derived cardiomyocytes [108] but not on mESC derived cardiac cells. SIRPA allowed for cardiac enrichment after targeting with a specific antibody and flow cytometric selection. Examination of sorted cells revealed a 98% co‐staining for
cardiac troponin T staining via flow cytometry. Dubois et al. report that their SIRPA targeting approach is applicable already as early as on day eight of human ESC differentiation.
Generally, detecting membrane proteins in cardiomyocytes and targeting them with fluorescently labeled antibodies prior to FACS represents an elegant solution for isolating cardiomyocytes from differentiating cultures. If antibodies are bound irreversibly or are easily washed out after the sorting procedure, still remains to be investigated. The function of the EMILIN2 and SIPRA protein in the physiological signaling cascade within cardiac cells needs to be addressed in further experiments in order to predict and validate if bound antibodies would have an impact on the functionality of isolated cardiomyocytes.
4.1.3. Stimulation to cardiac commitment via supplements
Another nontransgenic method to enrich for cardiac cells is the supplementation of media with molecules that enhance cardiac differentiation. One of the earliest papers published within this field is presented by Edwards et al. [114]. 0.5 ‐ 1.25% dimethylsulfoxide (DMSO) applied for 48h on the embryonal carcinoma (EC) cell line 01A1 from d8‐d10 increased the % beating during differentiation [114]. 6‐Thioguanine and butyric acid were tested on 01A1 cells and found to lead to an increase in % beating too. The teratocarcinoma cell line 01A1 when cultured in presence of 10‐9 M retinoic acid (RA) showed an increase in cardiac cells [115]. The presence of RA enhanced the differentiation of mESC into cardiac cells [116].
Similarly to the observation made by Edwards, the EC cell line P19 showed an enhanced cardiac outcome upon stimulation with DMSO [117].
Concerning the effect of RA on hESC the reported results are contradictory [118, 119].
Neither for DMSO did Xu et al. find an enhanced number of cardiac cells derived from hESC [119]. Xu et al. carefully analyzed the effect of the DNA demethylating agent 5‐aza‐2’‐
deoxycytidine (5‐aza‐dC). They observed that cardiac differentiation could be enhanced for both hESC lines tested H9 and H1 using the DNA demethylating agent. 10 μM exposure of 5‐
aza‐dC on H9 cells from d6 to d8 led to a 2.5‐fold enrichment in α‐MHC mRNA levels in comparison to untreated controls.
Takahashi et al. presented an interesting approach for the detection of cardiac differentiation stimulating agents during differentiating of mESC. The authors screened a library of over 800 compounds on the mESC line CGR8, stably transduced with an α‐cardiac myosin heavy chain (MHC) promoter driving expression of the transgene eGFP [120]. Using a hanging drop free monolayer protocol, the authors differentiated mESC in the presence of each of the library compounds and evaluated the number of GFP expressing cells. α‐MHC and β‐MHC mRNA levels were demonstrated to be ~10x higher upon ascorbic acid (AA) treatment in comparison to controls. A dose dependent increase of GFP positive cells was demonstrated using PCR, western blot and realtime PCR [120].
The lab of Christine Mummery used a different approach to stimulate cardiogenesis in their culture of hESC. The hESC cell line HES‐2 was cocultured with the END‐2 cell line.
Furthermore, cells were grown in a medium that contained 20% serum. These conditions were shown to lead to enhanced cardiogenesis. Enrichment was assessed by immunocytochemical staining of α‐actinin and counting of positively stained cells in ratio to total cell nuclei. In 2005 the same authors reported a 24‐fold enrichment of beating foci in EBs upon the withdrawal of serum in the culture media. On top, 40% more beating areas
were observed in the presence of 0.1 μM AA in the culture media. These findings were reproduced with two more cell lines, HES‐3 and HES‐4. By these means the total number of cardiomyocytes was enhanced. Nonetheless, the total number of α‐actinin cardiomyocytes per beating area was not different between cardiomyocyte stimulating and conventional culture conditions [113].
Laflamme et al. used hESC derived cardiomyocytes for defining a pro‐survival cocktail that would enhance cardiac survival after transplantation to infarct induced rat hearts [121].
Prior to these experiments they would achieve up to 30% cardiomyocytes by sequential stimulation with activin A and BMP4 on “high‐density undifferentiated monolayer cultures”.
Using these cells for Percoll gradient centrifugation, Laflamme and colleagues reported to routinely obtain between 71% and 95% hESC derived cardiomyocytes.
The group of Morisaki demonstrated the involvement of a MEK‐ERK signaling pathway in cardiogenesis and its potential use for triggering of cardiac differentiation. The authors used the growth factor HRG‐β1 on their previously cloned transgenic Nkx‐2.5 – GFP mESC line [122] to demonstrate phosphorylation of the extracellular signal regulated kinase (ERK) and subsequent enhancement of cardiac differentiation. The pro cardiac effect of HRG‐β1 could be blocked by using an ErbB receptor or a MAP kinase (MEK) 1 inhibitor. Thus they concluded that overexpression of MEK1 lead to enhanced cardiac differentiation [122].
In 2008 Xu and colleagues were able to define a xeno‐free, procardiac medium supporting the differentiation of hESC into the cardiac lineage [123]. The findings of the Mummery lab that END2 coculture was supporting cardiac differentiation of EC cells [123] and of hESC [124], led Xu et al. to further investigate on END2 cells and their influence on conditioned
inhibitor of cardiac differentiation in hESC in serum free conditions. Xu and colleagues demonstrated the END2 and other cells’ capability to clear insulin from media. Because other cells lines were demonstrated to degrade insulin as well but lacked the cardiac supporting effect on hESC cells, Xu and colleagues went on analyzing END2 conditioned media. Using microarray based results the authors found, in contrast to other cell lines with
complete absence of cardiac potential, the gene coding for prostaglandin I2 (PGI2) to be significantly upregulated in END2 cells. The potential of PGI2 to promote cardiac differentiation was demonstrated by the same outcome in cardiomyocytes between END2 conditioned media and PGI2 supplemented media. Due to insulin’s important role in many viable functions in cells, the authors do not suggest a continuous culture in insulin deficient conditions. But they point out that cardiac differentiation was effective until d12 of differentiation in absence of insulin.
Yang et al. induced hESC with various factors (i.e. activin A, BMP4, basic fibroblast growth factor (bFGF) vascular endothelial growth factor (VEGF) and dickkopf homolog 1 (DKK1))
starting on different days of differentiation in serum free media. A KDRlow/ckit‐ population was emerging within the embryoid bodies that would give rise to more than 50%
cardiomyocytes in culture [125].
Graichen et al. achieved to enhance 2.5 fold the cardiomyocyte output for hESC during differentiation. They used conditioned media of END2 cells for the hESC culture and exposed an inhibitor for the p38 Mitogen‐Activated Protein Kinases (MAPK) at a concentration of 10μM [126]. The p38 MAPK inhibitor enhanced mesoderm formation and for this reason exposure of the drug at the beginning of differentiation was sufficient. 80% of all embryoid bodies were reported to beat and over 20% cardiomyocyte outcome was demonstrated by
as early as day 12 of differentiation. In a second paper explaining the molecular background of MAPK inhibition and cardiac enhancement, Kempf et al. revealed the treatment duration to start from day 0 to day 1 of differentiation [127].
Two years later Gaur et al. obtained similar enrichment results as Graichen et al. concerning cardiomyocyte outcome using another p38MAP kinase inhibitor. Gaur report doubled amount of beating of hEBs using the p38MAPK inhibitor SB203580 [128]. hESC were treated from day 4 to day 6 of differentiation with 10 μM inhibitor. Enrichment was demonstrated using FACS and qRT‐PCR.
The Siu lab presented another small molecule for triggering cardiac differentiation on the mouse ESC line D3 [129]. Ouabain is presented as a cardiotonic steroid that signals via the ERK1/2 pathway. After careful examination for finding the best timepoints for Ouabain exposure, differentiating EBs were treated with 20 μmol/l for 7 days. 7 day old EBs were used for analysis. Besides other findings concerning the signaling pathway, enrichment was demonstrated as an increase in % beating as well as via FACS after staining for troponin‐T in comparison to untreated control EBs. The greatest difference in % beating between treated and control cells was detected 4 days after plating. In the untreated control group around 30% of EBs were beating. The treated group revealed around 50% of EBs to be beating.
Quantitative RT‐PCR showed α‐MHC levels to be increased around 3.5 fold in treated cells compared to untreated controls. α‐MHC protein levels were demonstrated to be increased by western blot too. FACS analysis revealed an increase in cardiac cell number.
Kattman et al. used another interesting approach to test a combination of different supplements in the media for the promotion of cardiac differentiation in several pluripotent
population was found not exclusively on cardiac mesoderm [131, 132] and indicate that rather the markers fetal liver kinase 1 (Flk‐1) in combination with the platelet‐derived growth factor receptor alpha (PDGFRα) are proposed for cardiac mesoderm markers [107].
Based on the findings for cardiac induction by Laflamme and Yung [121, 125] who presented cardiac induction with activin A and BMP‐4 on hESC in serum‐free cultures, Kattman et al.
used various concentrations of activin A and BMP‐4 for cardiac stimulation on pluripotent mouse and human cell lines. Concentrations of 5 ng/ml VEGF, 4 ng/ml Activin A and 0.1 ng/ml BMP4 starting from day 2 of differentiation and lasting for 28 hours in serum free
conditions resulted in the highest outcome of mouse derived Flk‐1+/PDGFRα+ progenitors with over 60%. After four days of culture of the sorted Flk‐1+/PDGFRα+ populations, the resulting cells were analyzed on day 7 of differentiation via flow‐cytometry for cTNT expression. Both the sorted and unsorted fraction revealed over 50% positive staining, demonstrating the success of the applied protocol. hESC required a slightly different treatment with cardiac inducers. Differentiating EBs that were treated on day 5 with 3 ng/ml Activin A and 10 ng/ml BMP‐4 revealed a population of 63% positive for Isl‐1 and PDGFRα.
At day 20 both the sorted Flk‐1+/PDGFRα+ population as well as the unsorted population revealed more than 70% cardiac cTNT positively stained cardiomyocytes. Similar results were obtained for induced mESC and induced hESC. The authors present improved protocols for enhanced cardiac differentiation for various cell lines. The advantage of these protocols is that sorting or genetic modification of cardiac cells is circumvented. For these reasons this procedure is interesting for cells that would be used for clinical testing or drug
At day 20 both the sorted Flk‐1+/PDGFRα+ population as well as the unsorted population revealed more than 70% cardiac cTNT positively stained cardiomyocytes. Similar results were obtained for induced mESC and induced hESC. The authors present improved protocols for enhanced cardiac differentiation for various cell lines. The advantage of these protocols is that sorting or genetic modification of cardiac cells is circumvented. For these reasons this procedure is interesting for cells that would be used for clinical testing or drug