Genetic Selection of Cardiomyocytes from Human Embryonic Stem Cells
Authors: Christine Mummery
Journal: Molecular Therapy (2007)

The ability of undifferentiated human embryonic stem cells (hESCs) to undergo indefinite self-renewal while retaining the potential to differentiate to multiple lineages places them among the most attractive cell sources for toxicological screening, in vitromodeling of genetic diseases, and therapeutic cell replacement. However, most differentiation strategies remain suboptimal and yield heterogeneous cell populations at different stages of development. Cells may be required at different stages for each particular purpose. For example, early cardiomyocytes may be best for transplantation because adult cardiomyocytes do not survive, but for drug testing and toxicology screens more mature cardiomyocytes may be required because of their particular electrophysiological responses. Selection of live cells at any stage of development by fluorescence-activated cell sorting (FACS) requires the availability of antibodies against cell-surface antigens on the cell type of interest, but few have so far been identified for lineage progenitors or their early differentiated derivatives. Transgenic selection is then an alternative. The article in this issue by Anderson and colleagues1Anderson D Self T Mellor IR Goh G Hill SJ Denning C Transgenic enrichment of cardiomyocytes from human embryonic stem cells.Mol Ther. 2007; 15: 2027-2036Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar describes just that for cardiac derivatives of hESCs and is one of the first papers to describe successful enrichment of a differentiated cell population for one specific cell type in this way. Cardiomyocytes appear in hESC aggregates typically as spontaneously beating groups of cells that constitute not more than about 20% of the whole cell population unless some enrichment steps have been incorporated (reviewed in ref. 2Passier R Mummery CL Cardiomyocyte differentiation from adult and embryonic stem cells.Curr Opin Biotechnol. 2005; 16: 498-502Crossref PubMed Scopus (49) Google Scholar). These have included physical enrichment by manual dissection of beating areas3Segev H Kenyagin-Karsenti D Fishman B Gerecht-Nir S Ziskind A Amit M et al.Molecular analysis of cardiomyocytes derived from human embryonic stem cells.Dev Growth Differ. 2005; 47: 295-306Crossref PubMed Scopus (39) Google Scholar,4Passier R Ward-van Oostwaard D Snapper J Kuijk E Roelen B Kloots J et al.Increased cardiomyocyte differentiation from human embryonic stem cells in serum-free cultures.Stem Cells. 2005; 23: 772-780Crossref PubMed Scopus (277) Google Scholar or size selection by centrifugation through a Percoll gradient,5Xu C Police S Rao N Carpenter MK Characterization and enrichment of cardiomyocytes derived from human embryonic stem cells.Circ Res. 2002; 91: 501-508Crossref PubMed Scopus (781) Google Scholar but the degree of enrichment may not satisfy regulatory bodies for clinical applications and may even be insufficient for many research purposes, for example, those dependent on gap-junctional coupling between multiple cardiomyocytes. FACS as a method of selection is also not an option, because no appropriate cell-surface markers for cardiomyocytes or their precursors have been identified yet. Transgenic selection would be an alternative route to enrich for a particular cell type, and this has indeed been applied with great success to mouse ESCs. Tissue-specific promoters have been used to identify and select pancreatic β-cells,6Soria B Roche E Berna G Leon-Quinto T Reig JA Martin F Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice.Diabetes. 2000; 49: 157-162Crossref PubMed Scopus (790) Google Scholar neurons,7Andressen C Stocker E Klinz FJ Lenka N Hescheler J Fleischmann B et al.Nestin-specific green fluorescent protein expression in embryonic stem cell-derived neural precursor cells used for transplantation.Stem Cells. 2001; 19: 419-424Crossref PubMed Scopus (71) Google Scholar,8Li M Pevny L Lovell-Badge R Smith A Generation of purified neural precursors from embryonic stem cells by lineage selection.Curr Biol. 1998; 8: 971-974Abstract Full Text Full Text PDF PubMed Google Scholar and cardiomyocytes.9Klug MG Soonpaa MH Koh GY Field LJ Genetically selected cardiomyocytes from differentiating embryonic stem cells form stable intracardiac grafts.J Clin Invest. 1996; 98: 216-224Crossref PubMed Scopus (963) Google Scholar,10Kolossov E Fleischmann BK Liu Q Bloch W Viatchenko-Karpinski S Manzke O et al.Functional characteristics of ES cell-derived cardiac precursor cells identified by tissue specific expression of green fluorescent protein.J Cell Biol. 1998; 143: 2045-2056Crossref PubMed Scopus (162) Google Scholar The mouse ESC model has also been refined to allow the identification of early cardiomyocyte precursor cells11Befar A Zingman LV Hodgson DM Rauzier JM Kane GC Terzic A et al.Stem cell differentiation requires a paracrine pathway in the heart.FASEB J. 2002; 16: 1558-1566Crossref PubMed Scopus (429) Google Scholar,12Hidaka K Lee J Kim HS Ihm CH Iio A Ogawa MT et al.Chamber specific differentiation of Nkx2.5 positive cardiac precursor cells from murine embryonic stem cells.FASEB J. 2003; 17: 740-742PubMed Google Scholar or subpopulations of cardiomyocytes such as ventricular cells,13Muller M Fleischmann BK Selbert S Ji GJ Endl E Middeler G et al.Selection of ventricular like cardiomyocytes from ES cells in vitro.FASEB J. 2000; 14: 2540-2548Crossref PubMed Scopus (250) Google Scholar atrial cells,14Kolossov E Lu Z Drobinskya I Gassanov N Duan Y Sauer H et al.Identification and characterization of embryonic stem cell derived pacemaker and atrial cardiomyocytes.FASEB J. 2005; 19: 577-579PubMed Google Scholar and pacemaker cells.14Kolossov E Lu Z Drobinskya I Gassanov N Duan Y Sauer H et al.Identification and characterization of embryonic stem cell derived pacemaker and atrial cardiomyocytes.FASEB J. 2005; 19: 577-579PubMed Google Scholar,15Gassanov N Er F Zagidullin N Hopp UC Endothelin induces differentiation of ANP-EGFP expressing embryonic stem cells towards a pacemaker phenotype.FASEB J. 2004; 18: 1710-1712Crossref PubMed Google Scholar In each case either ectopic promoters driving selection markers have been introduced into the mouse ESC genome or a reporter has been knocked in to the endogenous gene of interest. hESCs have unfortunately proven to be highly refractory to both approaches—almost a decade after their first derivation16Thomson JA Itskovitch-Eldor J Shapiro SS Waknitz MA Swiergiel JJ Marshall VS et al.Embryonic stem cell lines derived from human blastocysts.Science. 1998; 282: 1145-1147Crossref PubMed Scopus (12363) Google Scholar there are only a handful of reports describing successful genetic modification of hESCs and only two genes have been targeted.17Zwaka TP Thomson JA Homologous recombination in human embryonic stem cells.Nat Biotechnol. 2003; 21: 319-321Crossref PubMed Scopus (565) Google Scholar,18Urbach A Schuldiner M Benvenisty N Modeling for Lesch-Nyhan disease by gene targeting in human embryonic stem cells.Stem Cells. 2004; 22: 635-641Crossref PubMed Scopus (157) Google Scholar This is likely to change in the very near future, and recent reports have addressed the technical challenge of introducing genes into hESCs.19Costa M Dottori M Sourris K Jamshidi P Hatzistavrou T Davis R et al.A method for genetic modification of human embryonic stem cells using electroporation.Nat Protoc. 2007; 2: 792-796Crossref PubMed Scopus (132) Google Scholar,20Vallier L Alexander M Pedersen R Conditional gene expression in human embryonic stem cells.Stem Cells. 2007; 25: 1490-1497Crossref PubMed Scopus (30) Google Scholar,21Liew CG Draper JS Walsh J Moore H Andrews PW Transient and stable transgene expression in human embryonic stem cells.Stem Cells. 2007; 25: 1521-1528Crossref PubMed Scopus (126) Google Scholar,22Denning C Allegrucci C Priddle H Barbadillo-Muñoz MD Anderson D Self T et al.Common culture conditions for maintenance and cardiomyocyte differentiation of the human embryonic stem cell lines, BG01 and HUES-7.Int J Dev Biol. 2006; 50: 27-37Crossref PubMed Scopus (77) Google Scholar Hurdles encountered have included cell-line differences resulting from culture conditions and densities, methods of transfection or infection, nature of promoters used, promoter silencing during propagation and differentiation, and the poor ability of hESCs to survive clonal growth. However, two reports have now been published that describe the establishment of hESC lines stably expressing selectable markers for the cardiac lineage. The first report, by Huber et al.,23Huber I. Itzhaki I. Caspi O. Arbel G. Tzukerman M. Gepstein A. et al.Identification and selection of cardiomyocytes during human embryonic stem cell differentiation.FASEB J. 2007; 21: 2551-2563Crossref PubMed Scopus (233) Google Scholar appeared recently in the FASEB Journal.In that study, a short fragment (250 base pairs) of the human myosin light chain (MLC)-2v promoter homologous to that used previously with success in mouse ESCs was coupled to an enhanced green fluorescent protein (eGFP)-hygr fusion protein, incorporated into a lentiviral vector, and expressed stably in H9.2 cells. H9.2 is a subline of the original H9 hESCs16Thomson JA Itskovitch-Eldor J Shapiro SS Waknitz MA Swiergiel JJ Marshall VS et al.Embryonic stem cell lines derived from human blastocysts.Science. 1998; 282: 1145-1147Crossref PubMed Scopus (12363) Google Scholar and has been described as having high cardiomyogenic potential. MLC-2v is a chamber-specific structural protein expressed in the ventricle of the heart in both humans and rodents.24Chuva de Sousa Lopes SM Hassink RJ Feijen A van Rooijen MA Doevendans PA Tertoolen L et al.Patterning the heart, a template for human cardiomyocyte development.Dev Dyn. 2006; 235: 1994-2002Crossref PubMed Scopus (51) Google Scholar The transgenic line generated displayed eGFP fluorescence, but the authors were for technical reasons unable to use the antibiotic selection strategy effectively. Because of their inability to titrate the dose effectively, high numbers of false-positive clones were obtained. The GFP-expressing cells sorted by FACS were 95% pure, and 85% were viable—which is high, given the relative fragility of human cardiomycytes.4Passier R Ward-van Oostwaard D Snapper J Kuijk E Roelen B Kloots J et al.Increased cardiomyocyte differentiation from human embryonic stem cells in serum-free cultures.Stem Cells. 2005; 23: 772-780Crossref PubMed Scopus (277) Google Scholar In addition, more than 93% expressed cardiac-specific genes, displayed cardiac-specific action potentials, and could form short-term (3-day) grafts in host rat myocardium in vivo. The second report is the one in this issue by Denning's group.1Anderson D Self T Mellor IR Goh G Hill SJ Denning C Transgenic enrichment of cardiomyocytes from human embryonic stem cells.Mol Ther. 2007; 15: 2027-2036Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar Here two approaches were used. One method selected not specifically forcardiomyocytes but rather against proliferating cells using the rationale that it is overgrowth of these cells that results in the gradual decrease in the proportion of cardiomyocytes in the differentiating hESC population. The percentage of hESC-cardiomyocytes engaged in cell-cycle (and marked by Ki67) decreases rapidly from about 50% at 3 weeks of differentiation to almost zero by 5 weeks.25Snir M Kehat I Gepstein A Coleman R Itskovitz-Eldor J Livne E et al.Assessment of the ultrastructural and proliferative properties of human embryonic stem cell-derived cardiomyocytes.Am J Physiol Heart Circ Physiol. 2003; 285: H2355-H2363Crossref PubMed Scopus (272) Google Scholar This selection method, based on the herpes simplex virus thymidine kinase/ganciclovir suicide gene system, resulted in populations containing up to 35% cardiomyocytes, representing an enrichment factor of about sevenfold. Much better selection, however, was achieved using a construct based on the cardiac-specific human α-myosin heavy-chain (MYH6) promoter. This resulted in differentiated hESC populations containing more than 90% cardiomyocytes, corresponding to approximately 14.5-fold enrichment. In this paper the authors were able to use antibiotic selection effectively. These two reports are the first to describe methods for efficient purification of human cardiomyocytes from mixed populations of differentiated hESCs. What can be done with them and what the actual yield of cardiomyocytes is, of course, still determined by the efficiency of the induction method in combination with the ability to scale up the starting population. Purified hESC cardiomyocytes could be used as a basis not only for developing high-throughput screens for testing cardiac drugs but also for identifying noncardiac drugs that might have undesirable side effects on the heart. Targeting these lines to introduce specific mutations found in patients would be an excellent basis for creating specific cardiac disease models. Because multiple genetic diseases in humans are caused by haploinsufficiency, it may be necessary to target only one allele with a mutation. It is quite unlikely, though, that it will be possible to take genetically selectable cells through to clinical application because of the perceived risks associated with random insertion of genes in the genome. However, careful sequencing of the insertion site or the use of homologous recombination could circumvent this problem. Nevertheless, selection of cells may make it possible to identify cell-surface proteins or antigenic determinants on cardiomyocytes or their progenitors, perhaps using the newest developments from proteomics, and facilitate generation of specific antibodies against them. It may be possible to match transcriptional and cell-surface protein profiles to select cardiac progenitor cells at multiple stages of their development that have the potential to expand in culture before their cell-cycle machinery is switched off. Whatever the benefits in the short or long term, these two papers represent a significant step forward in implementing some of the potential applications of cardiomyocytes from hESCs.