Increased Cardiomyocyte Differentiation from Human Embryonic Stem Cells in Serum-Free Cultures
http://www.100md.com
《干细胞学杂志》
a Hubrecht Laboratory, Utrecht, Netherlands;
b Interuniversity Cardiology Institute of the Netherlands, Utrecht, Netherlands;
c Department of Cardiothoracic Surgery, University Medical Center Utrecht, Utrecht, Netherlands;
d Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands
Key Words. Cardiac ? Embryonic ? Stem cells ? Coculture ? Endoderm
Correspondence: Christine Mummery, Ph.D., Hubrecht Laboratory, Uppsalalaan 8, 3584 CT Utrecht, Netherlands. Telephone: 31-30-2121800; Fax: 31-30-2516464; e-mail: christin@niob.knaw.nl
ABSTRACT
Cardiomyocytes in the adult mammalian heart are essentially terminally differentiated and do not divide. Although a small percentage of the cells may be capable of proliferation , this is not sufficient for regeneration after myocardial injury. Conventional pharmacological therapy for patients with different stages of ischemic heart disease improves cardiac function, survival, and quality of life, but the ensuing failure is still the most life-threatening disease in Western society. Alternative therapies will be necessary to improve the clinical outcome of the increasing number of patients with ischemic heart disease. In recent years, cell replacement therapy has received considerable attention, intensified by the increasing number of potential cell sources for transplantation, which include skeletal myoblasts, adult cardiac stem cells, bone marrow stem cells, and embryonic stem cells (ESCs) .
ESCs can differentiate to all somatic cell types of the adult. Since the first description of the derivation of human ESCs (hESCs) from donor blastocysts , we and others have reported their differentiation to cardiomyocytes in culture . Recently, we demonstrated that coculture of hESCs with a visceral endoderm-like cell line (END-2), derived from mouse P19 embryonal carcinoma (EC) cells , resulted in the appearance of beating areas. Most (85%) of these hESC-derived cardiomyocytes had a ventricle-like phenotype based on morphological and electrophysiological parameters . Others have reported the spontaneous differentiation of hESCs, cultured as aggregates or embryoid bodies and enhancement of differentiation by the demethylating agent 5-aza-deoxycytidine . Between 8% and 70% of the embryoid bodies showed beating areas in these studies, and 2%–70% of the beating areas consisted of cardiomyocytes. This wide variation in cardiomyocyte differentiation and the relative paucity of quantitative data make it difficult to compare these in vitro models.
In this study, we describe a striking enhancement of cardiomyocyte differentiation in serum-free hESC-END-2 coculture conditions compared with our previous standard coculture in 20% fetal calf serum (FCS). Quantification of the number of cardiomyocytes under these coculture conditions showed a significant increase in the yield of cardiomyocytes without genetic manipulation.
MATERIALS AND METHODS
Effect of Serum on Morphology and the Number of Beating Areas During Coculture
The results shown were consistent in all three hESC lines examined (HES-2, -3, and -4). Data presented are from HES-2 cells. To determine the effect of serum on the number of beating areas during coculture of hESCs with END-2 cells, the serum concentration was reduced to 10%, 5%, 2.5%, and 0% from the start of coculture on day 1 until the end at day 12 and compared with the number of beating areas in a 12-well plate in the standard 20% FCS conditions. As shown in Figure 1, examination of hESC morphology after 5 days in coculture with 20% FCS demonstrated three-dimensional structures with cells spreading out from them (Fig. 1A). After 12 days of coculture, this was more evident, and strings of differentiating hESCs were visible (Fig. 1B). In the absence of serum, the edges of the three-dimensional structures were clearer, and less outward spreading of cells was observed (Figs. 1C, 1D). hESCs cultured on MEF feeders for an additional 12 days in the presence or absence of serum resulted in fewer cells on day 5 (Fig. 1E) compared with hESCs on END-2 cells (Figs. 1A, 1C), but not in the formation of three-dimensional structures. After 12 days, hESCs had spread out but remained predominantly as a two-dimensional sheet (Fig. 1F).
Figure 1. Morphology of hESCs during END-2 or MEF cocultures. Morphology of hESCs is shown after coculture with (A–D) END-2 or (E–F) MEF cells for (A, C, E) 5 days and (B, D, F) 12 days in the (A, B) presence or in the (C–F) absence of 20% fetal calf serum; magnifications x5. Abbreviations: hESC, human embryonic stem cell; MEF, mouse embryonic fibroblast.
Besides these morphological differences, a significant increase in the number of beating areas was observed at lower concentrations of serum, with a 24-fold upregulation in its complete absence compared with cultures containing 20% FCS (Fig. 2A). On average, 1.35 ± 0.26 (n = 21) beating areas per plate were observed at day 12 in 20% FCS cocultures, whereas 32.7 ± 2.3 (n = 27) beating areas were observed in 0% FCS cocultures. Beating areas were normally observed from day 7 onward (occasionally as early as day 5 or 6), with an increase in the number of beating areas until day 12 under all culture conditions. From day 12 onward, additional beating areas appeared, but at a much lower rate (Fig. 2B).
Figure 2. Effect of serum or KSR on the number of beating areas in hESC-END-2 cocultures. (A): Cocultures were initiated in 12-well plates in different concentrations of FCS, and beating areas were counted 12 days later or were counted from (B) days 8–18. (C): hESC-END-2 cocultures were performed in 0% FCS for the first 6 days and in 20% FCS for the next 6 days and vice versa . Beating areas were scored on day 12 and compared with 20% FCS and 0% FCS cocultures. The relative increase as fold-induction with respect to 20% FCS cocultures is shown. (D): Different concentration of KSR is added to hESC-END-2 cocultures, and beating areas are scored on day 12 and compared with 0% FCS cocultures. Each culture condition was tested in at least three independent experiments. *p < .05; **p < .01; ap < 10–12 compared with 20%; ###p < .001 compared with 20+0(d6). Abbreviations: FCS, fetal calf serum; hESC, human embryonic stem cell; KSR, knockout serum replacement.
To study whether the absence of serum was important throughout the 12-day coculture period, hESC-END-2 coculture was initiated in 0% FCS and then 20% FCS was added at day 6. Conversely, cocultures were also initiated in the presence of 20% FCS and changed to 0% FCS, at day 6. In cocultures starting in 0% FCS and changed at day 6 for 20% FCS, the number of beating areas decreased to 57% compared with cocultures maintained in 0% FCS continuously. However, in the cocultures in 20% FCS for the first 6 days, the number of beating areas decreased to only 2% compared with those in 0% FCS continuously (Fig. 2C).
An alternative to serum-free culture is the use of KSR. Various concentrations of KSR were added to hESC-END-2 cocultures. As shown in Figure 2D, a significant inverse relationship was found between the concentration of KSR in culture medium and the number of beating areas, just as in the FCS-supplemented medium. The elimination of insulin or ITS from the serum-free medium during coculture did not further affect the number of beating areas compared with serum-free medium alone (data not shown).
Expression of Cardiac Genes and Proteins in 20% and 0% hESC-END-2 Cocultures
To determine whether the increase in the number of beating areas resulted in a comparable increase in the expression of cardiac genes and proteins, reverse transcription (RT)–PCR and Western analysis were performed on hESC-END-2 cocultures in 0% and 20% FCS. A clear increase in the expression for all cardiac genes was observed by RT-PCR in the 0% FCS cocultures compared with those in 20% FCS (Fig. 3A). Nkx2.5, a homeobox-domain transcription factor, which plays an important role in early cardiac development, was slightly upregulated, whereas the cardiac zinc-finger transcription factor GATA-4 was not changed by 0% FCS compared with 20% FCS cocultures.
Figure 3. Effect of serum concentration on the expression of cardiac genes and proteins in hESC-END-2 cocultures. (A): Reverse transcription–polymerase chain reaction on RNA from 12-day hESC-END-2 cocultures in 0% FCS or 20% FCS. (B): Real-time polymerase chain reaction for -actinin in 0% FCS (n = 3) and 20% FCS (n = 2) hESC-END-2 cocultures using HARP mRNA levels as an internal control. (C): Western blot of protein extracts from 12-day hESC-END-2 cocultures in 0% FCS or 20% FCS and from HFCMs using antibodies against TM and Trop. Abbreviations: ANF, atrial natriuretic factor; FCS, fetal calf serum; hESC, human embryonic stem cell; HFCM, human fetal cardiomyocyte; MHC, myosin heavy chain; MLC, myosin light chain; P-Lamban, phospholamban; TM, tropomyosin; Trop, troponin T-C.
To confirm the results of the semiquantitative RT-PCR, mRNA levels for -actinin in 0% and 20% FCS cocultures were accurately measured by real-time RT-PCR. PCR was performed in triplicate for each sample. As an internal control, HARP mRNA levels were determined. Standard deviations were less than 1% for all triplicate reactions. A 27-fold increase in -actinin mRNA levels was observed in the 0% FCS cocultures compared with the 20% FCS cocultures (Fig. 3B), confirming the results of the RT-PCR.
Increased expression of cardiac structural proteins in 0% FCS cocultures was confirmed by Western blot analysis. In cocultures in 20% FCS, both tropomyosin and troponin T-C are not detectable or are at the detection limit of the assay, whereas in cocultures in 0% FCS, clear bands at 36 kDa for tropomyosin and 40 kDa for troponin T-C were observed. As expected, an even stronger band at the same molecular weight was observed in protein extracts from human fetal hearts (Fig. 3C).
Characterization of Beating Areas and the Presence of Cardiac Progenitor Cells
After 12 days, cocultures in 0% FCS were examined for the presence of beating areas and recorded on video (Fig. 4A). The same samples were then fixed and stained for -actinin (Fig. 4B) and the films overlayed. All beating areas were also positive for -actinin and displayed a characteristic cardiomyocyte-like striated pattern (Fig. 4C). No -actinin–positive areas were detected that were not beating before fixation, indicating the high correlation between the number of beating areas and the number of -actinin–positive areas. After dissection of beating areas and subsequent dissociation, cells were plated on gelatin-coated dishes, fixed, and stained for -actinin. Between 5% and 20% of the cells were positive for -actinin (Fig. 4D). Most of the other cells were positive for Troma-1, which recognizes intermediate cytokeratin 8 and is used as a marker for endoderm (Fig. 4E). By doublestaining immunofluorescence, it is clear that -Troma-1–positive and -actinin–positive cells do not colocalize (Fig. 4F)
Figure 4. Relationship between beating areas with -actinin staining and cardiomyocytes after dissociation. (A): Beating hESC-END-2 12-day cocultures from one well are recorded and then fixed and stained for -actinin. (B): Identical areas are indicated by white dashed lines and are labeled a–e; x5 magnification. (C): Magnification x63 of white dashed box of (B). (D): Dissociated cell of beating areas stained for -actinin (green) and Topro-3 (blue) (x40 magnification). (E): Dissociated and replated cells derived from beating areas stained for Troma-1 (green) and Topro-3 (blue) or -actinin (red) (F).
To determine whether cardiac progenitor cells had formed during differentiation in the hESC-END-2 cocultures, as might be expected, we determined the expression of Isl1. By real-time PCR, a 2.5-fold increase in the expression of Isl1 was found in serum-free hESC-END-2 cocultures at day 12 compared with that of 20% FCS cocultures (Fig. 5A). By immunohistochemistry, we confirmed that nuclear Isl1 protein expression is present in tissue sections of 12-day beating areas (Figs. 5B–5D).
Figure 5. Expression of Isl1 in hESC-END-2 cocultures. (A): Real-time polymerase chain reaction for Isl1 in 0% FCS (n = 2) and 20% FCS (n = 2) 12-day hESC-END-2 cocultures using HARP mRNA levels as an internal control; *p < .05. (B–D): Isl1 protein localization by immunohistochemistry in 4-μm sections of 12-day beating areas from serum-free hESC-END-2 cocultures; magnification (B, C) x20 or (D) x40. Abbreviations: FCS, fetal calf serum; hESC, human embryonic stem cell.
Number of Cardiomyocytes in Cocultures
To determine whether the increase in the number of beating areas and the increase in cardiac gene and protein expression was attributable to the increase of the actual number of cardiomyocytes, -actinin–positive cells with striated sarcomeric patterns were counted by confocal Z-series. This was considered more informative than fluorescence-activated cell sorter (FACS) analysis, because cells showing striated -actinin staining could be selectively included. Cells were counted in different optical planes (Fig. 6A). In cocultures in 20% FCS, the average number of cardiomyocytes per beating area was 312 ± 227 (n = 5). The number of cardiomyocytes per beating area in 0% FCS cocultures was 503 ± 179 (n = 15). However, this was not significantly different and reflects the wide variation in the number of cardiomyocytes per beating area (ranging from 1 to 2,500 cells) (Fig. 6B). Based on these numbers, the average number of cardiomyocytes in a 12-well coculture plate is therefore approximately 16,600 cells in 0% FCS cocultures and 450 cells in 20% FCS cocultures, representing a 39-fold increase in the total number of cardiomyocytes in 0% FCS cocultures (Table 1).
Figure 6. Number of cardiomyocytes in 0% and 20% FCS hESC-END-2 cocultures. (A): BA of hESC-END-212-day coculture, stained for -actinin (red) and Topro-3 (nucleus, blue) in different planes after confocal scanning (I and I’). Only nuclei surrounded by -actinin are counted. Examples are given (white arrows); x20 magnification. (B): Numbers of cardiomyocytes from 0% FCS and 20% FCS hESC-END-2 cocultures are counted and pooled from the different confocal planes. (C): Cocultures were initiated in 12-well plates in serum-free hESC-END-2 with or without AA (n = 6). BAs were scored on day 12; *p < .05. Abbreviations: AA, ascorbic acid; BA, beating area; FCS, fetal calf serum; hESC, human embryonic stem cell.
Table 1. Number of cardiomyocytes in 0% and 20% FCS hESC-END-2 cocultures
The serum-free hESC-END-2 coculture condition represents an improved culture model, without inhibitory factors from serum, for testing other factors for their effect on cardiomyocyte differentiation. Addition of 10–4 M ascorbic acid to serum-free hESC-END-2 cultures, for example, resulted in a further robust increase in the number of beating areas at day 12, 40% higher than in the serum-free cocultures alone (Fig. 6C).
DISCUSSION
We demonstrated an improved efficiency of cardiomyocyte differentiation from hESC-END-2 cocultures in serum-free medium. Serum-free hESC-END-2 coculture represents a more defined in vitro model for identifying the cardiomyocyte-inducing activity from END-2 cells and, in addition, a more straightforward experimental system for assessing potential cardiogenic factors such as bone morphogenic proteins, fibroblast growth factors, Wnts, and their inhibitors, apart from ascorbic acid as tested here, because there will be no interference from serum-derived modulatory factors.
After dissociation, between 5% and 20% of the cells were -actinin–positive cardiomyocytes. This variation can be attributed to many different factors such as the size of the beating area, the number of cardiomyocytes per beating area, and the accessibility of the beating area. In addition, cell death and altered attachment during or after dissociation and time between plating and fixation of dissociated cells may play a role in determining the percentage of cardiomyocytes in the replated dissociated cells (higher proliferation rates of noncardiomyocytes reduce the proposition of cardiomyocytes present). Therefore, selection of cardiomyocytes by FACS using cell-surface markers or by genetic manipulation will further stimulate the use of hESC-derived cardiomyocytes for cell-replacement studies.
The higher number of hESC-derived cardiomyocytes in these cultures will not only provide us with a better in vitro model for understanding cardiac development in humans but will also facilitate upscale for transplantation studies to determine whether hESC-derived cardiomyocytes can survive and functionally integrate with host cardiomyocytes and improve cardiac function in animal models of heart failure. With respect to possible future clinical applications, it is of importance that cardiomyocyte differentiation is feasible in serum-free conditions and thus reduces the risk of cross transfer of animal pathogens. On that note, an alternative for serum, KSR, inhibited the number of beating areas, but upon withdrawal, the number of beating areas again increased (data not shown). This suggests that maintenance of undifferentiated hESCs in the presence of KSR (which would be favorable for future clinical applications), followed by serum-free differentiation cultures, would not affect cardiomyocyte differentiation.
ACKNOWLEDGMENTS
Anversa P, Kajstura J. Ventricular myocytes are not terminally differentiated in the adult mammalian heart. Circ Res 1998;83:1–14.
Passier R, Mummery C. Origin and use of embryonic and adult stem cells in differentiation and tissue repair. Cardiovasc Res 2003;58:324–335.
Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145–1147.
Reubinoff BE, Pera MF, Fong CY et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 2000;18:399–404.
Mummery C, Ward-van Oostwaard D, Doevendans P et al. Differentiation of human embryonic stem cells to cardiomyocytes: role of coculture with visceral endoderm-like cells. Circulation 2003;107:2733–2740.
Kehat I, Kenyagin-Karsenti D, Snir M et al. Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. J Clin Invest 2001;108:407–414.
He JQ, Ma Y, Lee Y et al. Human embryonic stem cells develop into multiple types of cardiac myocytes: action potential characterization. Circ Res 2003;93:32–39.
Xu C, Police S, Rao N et al. Characterization and enrichment of cardiomyocytes derived from human embryonic stem cells. Circ Res 2002;91:501–508.
Mummery CL, van Achterberg TA, van den Eijnden-van Raaij AJ et al. Visceral-endoderm-like cell lines induce differentiation of murine P19 embryonal carcinoma cells. Differentiation 1991;46:51–60.
de Sousa Lopes SM, Carvalho RL, van den Driesche S et al. Distribution of phosphorylated Smad2 identifies target tissues of TGF beta ligands in mouse development. Gene Expr Patterns 2003;3:355–360.
Florini JR, Ewton DZ, Magri KA. Hormones, growth factors, and myogenic differentiation. Annu Rev Physiol 1991;53:201–216.
Sekiguchi M, Oota T, Sakakibara K et al. Establishment and characterization of a human neuroblastoma cell line in tissue culture. Jpn J Exp Med 1979;49:67–83.
Sachinidis A, Gissel C, Nierhoff D et al. Identification of plateled-derived growth factor-BB as cardiogenesis-inducing factor in mouse embryonic stem cells under serum-free conditions. Cell Physiol Biochem 2003;13:423–429.
Wobus AM, Wallukat G, Hescheler J. Pluripotent mouse embryonic stem cells are able to differentiate into cardiomyocytes expressing chronotropic responses to adrenergic and cholinergic agents and Ca2+ channel blockers. Differentiation 1991;48:173–182.
Mummery C, Ward D, van den Brink CE et al. Cardiomyocyte differentiation of mouse and human embryonic stem cells. J Anat 2002;200:233–242.
Cai CL, Liang X, Shi Y et al. Isl1 identifies a cardiac progenitor population that proliferates prior to differentiation and contributes a majority of cells to the heart. Dev Cell 2003;5:877–889.
Florini JR, Ewton DZ. Induction of gene expression in muscle by the IGFs. Growth Regul 1992;2:23–29.
Antin PB, Yatskievych T, Dominguez JL et al. Regulation of avian precardiac mesoderm development by insulin and insulin-like growth factors. J Cell Physiol 1996;168:42–50.
Takahashi T, Lord B, Schulze PC et al. Ascorbic acid enhances differentiation of embryonic stem cells into cardiac myocytes. Circulation 2003;107:1912–1916.
Shim WS, Jiang S, Wong P et al. Ex vivo differentiation of human adult bone marrow stem cells into cardiomyocyte-like cells. Biochem Biophys Res Commun 2004;324:481–488.
van den Eijnden-van Raaij AJ, van Achterberg TA, van der Kruijssen CM et al. Differentiation of aggregated murine P19 embryonal carcinoma cells is induced by a novel visceral endoderm-specific FGF-like factor and inhibited by activin A. Mech Dev 1991;33:157–165.
Dyer MA, Farrington SM, Mohn D et al. Indian hedgehog activates hematopoiesis and vasculogenesis and can respecify prospective neurectodermal cell fate in the mouse embryo. Development 2001;128:1717–1730.
Lough J, Sugi Y. Endoderm and heart development. Dev Dyn 2000;217:327–342.(Robert Passiera,b, Dorien)
b Interuniversity Cardiology Institute of the Netherlands, Utrecht, Netherlands;
c Department of Cardiothoracic Surgery, University Medical Center Utrecht, Utrecht, Netherlands;
d Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands
Key Words. Cardiac ? Embryonic ? Stem cells ? Coculture ? Endoderm
Correspondence: Christine Mummery, Ph.D., Hubrecht Laboratory, Uppsalalaan 8, 3584 CT Utrecht, Netherlands. Telephone: 31-30-2121800; Fax: 31-30-2516464; e-mail: christin@niob.knaw.nl
ABSTRACT
Cardiomyocytes in the adult mammalian heart are essentially terminally differentiated and do not divide. Although a small percentage of the cells may be capable of proliferation , this is not sufficient for regeneration after myocardial injury. Conventional pharmacological therapy for patients with different stages of ischemic heart disease improves cardiac function, survival, and quality of life, but the ensuing failure is still the most life-threatening disease in Western society. Alternative therapies will be necessary to improve the clinical outcome of the increasing number of patients with ischemic heart disease. In recent years, cell replacement therapy has received considerable attention, intensified by the increasing number of potential cell sources for transplantation, which include skeletal myoblasts, adult cardiac stem cells, bone marrow stem cells, and embryonic stem cells (ESCs) .
ESCs can differentiate to all somatic cell types of the adult. Since the first description of the derivation of human ESCs (hESCs) from donor blastocysts , we and others have reported their differentiation to cardiomyocytes in culture . Recently, we demonstrated that coculture of hESCs with a visceral endoderm-like cell line (END-2), derived from mouse P19 embryonal carcinoma (EC) cells , resulted in the appearance of beating areas. Most (85%) of these hESC-derived cardiomyocytes had a ventricle-like phenotype based on morphological and electrophysiological parameters . Others have reported the spontaneous differentiation of hESCs, cultured as aggregates or embryoid bodies and enhancement of differentiation by the demethylating agent 5-aza-deoxycytidine . Between 8% and 70% of the embryoid bodies showed beating areas in these studies, and 2%–70% of the beating areas consisted of cardiomyocytes. This wide variation in cardiomyocyte differentiation and the relative paucity of quantitative data make it difficult to compare these in vitro models.
In this study, we describe a striking enhancement of cardiomyocyte differentiation in serum-free hESC-END-2 coculture conditions compared with our previous standard coculture in 20% fetal calf serum (FCS). Quantification of the number of cardiomyocytes under these coculture conditions showed a significant increase in the yield of cardiomyocytes without genetic manipulation.
MATERIALS AND METHODS
Effect of Serum on Morphology and the Number of Beating Areas During Coculture
The results shown were consistent in all three hESC lines examined (HES-2, -3, and -4). Data presented are from HES-2 cells. To determine the effect of serum on the number of beating areas during coculture of hESCs with END-2 cells, the serum concentration was reduced to 10%, 5%, 2.5%, and 0% from the start of coculture on day 1 until the end at day 12 and compared with the number of beating areas in a 12-well plate in the standard 20% FCS conditions. As shown in Figure 1, examination of hESC morphology after 5 days in coculture with 20% FCS demonstrated three-dimensional structures with cells spreading out from them (Fig. 1A). After 12 days of coculture, this was more evident, and strings of differentiating hESCs were visible (Fig. 1B). In the absence of serum, the edges of the three-dimensional structures were clearer, and less outward spreading of cells was observed (Figs. 1C, 1D). hESCs cultured on MEF feeders for an additional 12 days in the presence or absence of serum resulted in fewer cells on day 5 (Fig. 1E) compared with hESCs on END-2 cells (Figs. 1A, 1C), but not in the formation of three-dimensional structures. After 12 days, hESCs had spread out but remained predominantly as a two-dimensional sheet (Fig. 1F).
Figure 1. Morphology of hESCs during END-2 or MEF cocultures. Morphology of hESCs is shown after coculture with (A–D) END-2 or (E–F) MEF cells for (A, C, E) 5 days and (B, D, F) 12 days in the (A, B) presence or in the (C–F) absence of 20% fetal calf serum; magnifications x5. Abbreviations: hESC, human embryonic stem cell; MEF, mouse embryonic fibroblast.
Besides these morphological differences, a significant increase in the number of beating areas was observed at lower concentrations of serum, with a 24-fold upregulation in its complete absence compared with cultures containing 20% FCS (Fig. 2A). On average, 1.35 ± 0.26 (n = 21) beating areas per plate were observed at day 12 in 20% FCS cocultures, whereas 32.7 ± 2.3 (n = 27) beating areas were observed in 0% FCS cocultures. Beating areas were normally observed from day 7 onward (occasionally as early as day 5 or 6), with an increase in the number of beating areas until day 12 under all culture conditions. From day 12 onward, additional beating areas appeared, but at a much lower rate (Fig. 2B).
Figure 2. Effect of serum or KSR on the number of beating areas in hESC-END-2 cocultures. (A): Cocultures were initiated in 12-well plates in different concentrations of FCS, and beating areas were counted 12 days later or were counted from (B) days 8–18. (C): hESC-END-2 cocultures were performed in 0% FCS for the first 6 days and in 20% FCS for the next 6 days and vice versa . Beating areas were scored on day 12 and compared with 20% FCS and 0% FCS cocultures. The relative increase as fold-induction with respect to 20% FCS cocultures is shown. (D): Different concentration of KSR is added to hESC-END-2 cocultures, and beating areas are scored on day 12 and compared with 0% FCS cocultures. Each culture condition was tested in at least three independent experiments. *p < .05; **p < .01; ap < 10–12 compared with 20%; ###p < .001 compared with 20+0(d6). Abbreviations: FCS, fetal calf serum; hESC, human embryonic stem cell; KSR, knockout serum replacement.
To study whether the absence of serum was important throughout the 12-day coculture period, hESC-END-2 coculture was initiated in 0% FCS and then 20% FCS was added at day 6. Conversely, cocultures were also initiated in the presence of 20% FCS and changed to 0% FCS, at day 6. In cocultures starting in 0% FCS and changed at day 6 for 20% FCS, the number of beating areas decreased to 57% compared with cocultures maintained in 0% FCS continuously. However, in the cocultures in 20% FCS for the first 6 days, the number of beating areas decreased to only 2% compared with those in 0% FCS continuously (Fig. 2C).
An alternative to serum-free culture is the use of KSR. Various concentrations of KSR were added to hESC-END-2 cocultures. As shown in Figure 2D, a significant inverse relationship was found between the concentration of KSR in culture medium and the number of beating areas, just as in the FCS-supplemented medium. The elimination of insulin or ITS from the serum-free medium during coculture did not further affect the number of beating areas compared with serum-free medium alone (data not shown).
Expression of Cardiac Genes and Proteins in 20% and 0% hESC-END-2 Cocultures
To determine whether the increase in the number of beating areas resulted in a comparable increase in the expression of cardiac genes and proteins, reverse transcription (RT)–PCR and Western analysis were performed on hESC-END-2 cocultures in 0% and 20% FCS. A clear increase in the expression for all cardiac genes was observed by RT-PCR in the 0% FCS cocultures compared with those in 20% FCS (Fig. 3A). Nkx2.5, a homeobox-domain transcription factor, which plays an important role in early cardiac development, was slightly upregulated, whereas the cardiac zinc-finger transcription factor GATA-4 was not changed by 0% FCS compared with 20% FCS cocultures.
Figure 3. Effect of serum concentration on the expression of cardiac genes and proteins in hESC-END-2 cocultures. (A): Reverse transcription–polymerase chain reaction on RNA from 12-day hESC-END-2 cocultures in 0% FCS or 20% FCS. (B): Real-time polymerase chain reaction for -actinin in 0% FCS (n = 3) and 20% FCS (n = 2) hESC-END-2 cocultures using HARP mRNA levels as an internal control. (C): Western blot of protein extracts from 12-day hESC-END-2 cocultures in 0% FCS or 20% FCS and from HFCMs using antibodies against TM and Trop. Abbreviations: ANF, atrial natriuretic factor; FCS, fetal calf serum; hESC, human embryonic stem cell; HFCM, human fetal cardiomyocyte; MHC, myosin heavy chain; MLC, myosin light chain; P-Lamban, phospholamban; TM, tropomyosin; Trop, troponin T-C.
To confirm the results of the semiquantitative RT-PCR, mRNA levels for -actinin in 0% and 20% FCS cocultures were accurately measured by real-time RT-PCR. PCR was performed in triplicate for each sample. As an internal control, HARP mRNA levels were determined. Standard deviations were less than 1% for all triplicate reactions. A 27-fold increase in -actinin mRNA levels was observed in the 0% FCS cocultures compared with the 20% FCS cocultures (Fig. 3B), confirming the results of the RT-PCR.
Increased expression of cardiac structural proteins in 0% FCS cocultures was confirmed by Western blot analysis. In cocultures in 20% FCS, both tropomyosin and troponin T-C are not detectable or are at the detection limit of the assay, whereas in cocultures in 0% FCS, clear bands at 36 kDa for tropomyosin and 40 kDa for troponin T-C were observed. As expected, an even stronger band at the same molecular weight was observed in protein extracts from human fetal hearts (Fig. 3C).
Characterization of Beating Areas and the Presence of Cardiac Progenitor Cells
After 12 days, cocultures in 0% FCS were examined for the presence of beating areas and recorded on video (Fig. 4A). The same samples were then fixed and stained for -actinin (Fig. 4B) and the films overlayed. All beating areas were also positive for -actinin and displayed a characteristic cardiomyocyte-like striated pattern (Fig. 4C). No -actinin–positive areas were detected that were not beating before fixation, indicating the high correlation between the number of beating areas and the number of -actinin–positive areas. After dissection of beating areas and subsequent dissociation, cells were plated on gelatin-coated dishes, fixed, and stained for -actinin. Between 5% and 20% of the cells were positive for -actinin (Fig. 4D). Most of the other cells were positive for Troma-1, which recognizes intermediate cytokeratin 8 and is used as a marker for endoderm (Fig. 4E). By doublestaining immunofluorescence, it is clear that -Troma-1–positive and -actinin–positive cells do not colocalize (Fig. 4F)
Figure 4. Relationship between beating areas with -actinin staining and cardiomyocytes after dissociation. (A): Beating hESC-END-2 12-day cocultures from one well are recorded and then fixed and stained for -actinin. (B): Identical areas are indicated by white dashed lines and are labeled a–e; x5 magnification. (C): Magnification x63 of white dashed box of (B). (D): Dissociated cell of beating areas stained for -actinin (green) and Topro-3 (blue) (x40 magnification). (E): Dissociated and replated cells derived from beating areas stained for Troma-1 (green) and Topro-3 (blue) or -actinin (red) (F).
To determine whether cardiac progenitor cells had formed during differentiation in the hESC-END-2 cocultures, as might be expected, we determined the expression of Isl1. By real-time PCR, a 2.5-fold increase in the expression of Isl1 was found in serum-free hESC-END-2 cocultures at day 12 compared with that of 20% FCS cocultures (Fig. 5A). By immunohistochemistry, we confirmed that nuclear Isl1 protein expression is present in tissue sections of 12-day beating areas (Figs. 5B–5D).
Figure 5. Expression of Isl1 in hESC-END-2 cocultures. (A): Real-time polymerase chain reaction for Isl1 in 0% FCS (n = 2) and 20% FCS (n = 2) 12-day hESC-END-2 cocultures using HARP mRNA levels as an internal control; *p < .05. (B–D): Isl1 protein localization by immunohistochemistry in 4-μm sections of 12-day beating areas from serum-free hESC-END-2 cocultures; magnification (B, C) x20 or (D) x40. Abbreviations: FCS, fetal calf serum; hESC, human embryonic stem cell.
Number of Cardiomyocytes in Cocultures
To determine whether the increase in the number of beating areas and the increase in cardiac gene and protein expression was attributable to the increase of the actual number of cardiomyocytes, -actinin–positive cells with striated sarcomeric patterns were counted by confocal Z-series. This was considered more informative than fluorescence-activated cell sorter (FACS) analysis, because cells showing striated -actinin staining could be selectively included. Cells were counted in different optical planes (Fig. 6A). In cocultures in 20% FCS, the average number of cardiomyocytes per beating area was 312 ± 227 (n = 5). The number of cardiomyocytes per beating area in 0% FCS cocultures was 503 ± 179 (n = 15). However, this was not significantly different and reflects the wide variation in the number of cardiomyocytes per beating area (ranging from 1 to 2,500 cells) (Fig. 6B). Based on these numbers, the average number of cardiomyocytes in a 12-well coculture plate is therefore approximately 16,600 cells in 0% FCS cocultures and 450 cells in 20% FCS cocultures, representing a 39-fold increase in the total number of cardiomyocytes in 0% FCS cocultures (Table 1).
Figure 6. Number of cardiomyocytes in 0% and 20% FCS hESC-END-2 cocultures. (A): BA of hESC-END-212-day coculture, stained for -actinin (red) and Topro-3 (nucleus, blue) in different planes after confocal scanning (I and I’). Only nuclei surrounded by -actinin are counted. Examples are given (white arrows); x20 magnification. (B): Numbers of cardiomyocytes from 0% FCS and 20% FCS hESC-END-2 cocultures are counted and pooled from the different confocal planes. (C): Cocultures were initiated in 12-well plates in serum-free hESC-END-2 with or without AA (n = 6). BAs were scored on day 12; *p < .05. Abbreviations: AA, ascorbic acid; BA, beating area; FCS, fetal calf serum; hESC, human embryonic stem cell.
Table 1. Number of cardiomyocytes in 0% and 20% FCS hESC-END-2 cocultures
The serum-free hESC-END-2 coculture condition represents an improved culture model, without inhibitory factors from serum, for testing other factors for their effect on cardiomyocyte differentiation. Addition of 10–4 M ascorbic acid to serum-free hESC-END-2 cultures, for example, resulted in a further robust increase in the number of beating areas at day 12, 40% higher than in the serum-free cocultures alone (Fig. 6C).
DISCUSSION
We demonstrated an improved efficiency of cardiomyocyte differentiation from hESC-END-2 cocultures in serum-free medium. Serum-free hESC-END-2 coculture represents a more defined in vitro model for identifying the cardiomyocyte-inducing activity from END-2 cells and, in addition, a more straightforward experimental system for assessing potential cardiogenic factors such as bone morphogenic proteins, fibroblast growth factors, Wnts, and their inhibitors, apart from ascorbic acid as tested here, because there will be no interference from serum-derived modulatory factors.
After dissociation, between 5% and 20% of the cells were -actinin–positive cardiomyocytes. This variation can be attributed to many different factors such as the size of the beating area, the number of cardiomyocytes per beating area, and the accessibility of the beating area. In addition, cell death and altered attachment during or after dissociation and time between plating and fixation of dissociated cells may play a role in determining the percentage of cardiomyocytes in the replated dissociated cells (higher proliferation rates of noncardiomyocytes reduce the proposition of cardiomyocytes present). Therefore, selection of cardiomyocytes by FACS using cell-surface markers or by genetic manipulation will further stimulate the use of hESC-derived cardiomyocytes for cell-replacement studies.
The higher number of hESC-derived cardiomyocytes in these cultures will not only provide us with a better in vitro model for understanding cardiac development in humans but will also facilitate upscale for transplantation studies to determine whether hESC-derived cardiomyocytes can survive and functionally integrate with host cardiomyocytes and improve cardiac function in animal models of heart failure. With respect to possible future clinical applications, it is of importance that cardiomyocyte differentiation is feasible in serum-free conditions and thus reduces the risk of cross transfer of animal pathogens. On that note, an alternative for serum, KSR, inhibited the number of beating areas, but upon withdrawal, the number of beating areas again increased (data not shown). This suggests that maintenance of undifferentiated hESCs in the presence of KSR (which would be favorable for future clinical applications), followed by serum-free differentiation cultures, would not affect cardiomyocyte differentiation.
ACKNOWLEDGMENTS
Anversa P, Kajstura J. Ventricular myocytes are not terminally differentiated in the adult mammalian heart. Circ Res 1998;83:1–14.
Passier R, Mummery C. Origin and use of embryonic and adult stem cells in differentiation and tissue repair. Cardiovasc Res 2003;58:324–335.
Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145–1147.
Reubinoff BE, Pera MF, Fong CY et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 2000;18:399–404.
Mummery C, Ward-van Oostwaard D, Doevendans P et al. Differentiation of human embryonic stem cells to cardiomyocytes: role of coculture with visceral endoderm-like cells. Circulation 2003;107:2733–2740.
Kehat I, Kenyagin-Karsenti D, Snir M et al. Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. J Clin Invest 2001;108:407–414.
He JQ, Ma Y, Lee Y et al. Human embryonic stem cells develop into multiple types of cardiac myocytes: action potential characterization. Circ Res 2003;93:32–39.
Xu C, Police S, Rao N et al. Characterization and enrichment of cardiomyocytes derived from human embryonic stem cells. Circ Res 2002;91:501–508.
Mummery CL, van Achterberg TA, van den Eijnden-van Raaij AJ et al. Visceral-endoderm-like cell lines induce differentiation of murine P19 embryonal carcinoma cells. Differentiation 1991;46:51–60.
de Sousa Lopes SM, Carvalho RL, van den Driesche S et al. Distribution of phosphorylated Smad2 identifies target tissues of TGF beta ligands in mouse development. Gene Expr Patterns 2003;3:355–360.
Florini JR, Ewton DZ, Magri KA. Hormones, growth factors, and myogenic differentiation. Annu Rev Physiol 1991;53:201–216.
Sekiguchi M, Oota T, Sakakibara K et al. Establishment and characterization of a human neuroblastoma cell line in tissue culture. Jpn J Exp Med 1979;49:67–83.
Sachinidis A, Gissel C, Nierhoff D et al. Identification of plateled-derived growth factor-BB as cardiogenesis-inducing factor in mouse embryonic stem cells under serum-free conditions. Cell Physiol Biochem 2003;13:423–429.
Wobus AM, Wallukat G, Hescheler J. Pluripotent mouse embryonic stem cells are able to differentiate into cardiomyocytes expressing chronotropic responses to adrenergic and cholinergic agents and Ca2+ channel blockers. Differentiation 1991;48:173–182.
Mummery C, Ward D, van den Brink CE et al. Cardiomyocyte differentiation of mouse and human embryonic stem cells. J Anat 2002;200:233–242.
Cai CL, Liang X, Shi Y et al. Isl1 identifies a cardiac progenitor population that proliferates prior to differentiation and contributes a majority of cells to the heart. Dev Cell 2003;5:877–889.
Florini JR, Ewton DZ. Induction of gene expression in muscle by the IGFs. Growth Regul 1992;2:23–29.
Antin PB, Yatskievych T, Dominguez JL et al. Regulation of avian precardiac mesoderm development by insulin and insulin-like growth factors. J Cell Physiol 1996;168:42–50.
Takahashi T, Lord B, Schulze PC et al. Ascorbic acid enhances differentiation of embryonic stem cells into cardiac myocytes. Circulation 2003;107:1912–1916.
Shim WS, Jiang S, Wong P et al. Ex vivo differentiation of human adult bone marrow stem cells into cardiomyocyte-like cells. Biochem Biophys Res Commun 2004;324:481–488.
van den Eijnden-van Raaij AJ, van Achterberg TA, van der Kruijssen CM et al. Differentiation of aggregated murine P19 embryonal carcinoma cells is induced by a novel visceral endoderm-specific FGF-like factor and inhibited by activin A. Mech Dev 1991;33:157–165.
Dyer MA, Farrington SM, Mohn D et al. Indian hedgehog activates hematopoiesis and vasculogenesis and can respecify prospective neurectodermal cell fate in the mouse embryo. Development 2001;128:1717–1730.
Lough J, Sugi Y. Endoderm and heart development. Dev Dyn 2000;217:327–342.(Robert Passiera,b, Dorien)