Cardiogenesis from human embryonic stem cells

Abstract

Over the past decade, the ability to culture and differentiate human embryonic stem cells (ESCs) has offered researchers a novel therapeutic that may, for the first time, repair regions of the damaged heart. Studies of cardiac development in lower organisms have led to identification of the transforming growth factor-β superfamily (eg, activin A and bone morphogenic protein 4) and the Wnt/β-catenin pathway as key inducers of mesoderm and cardiovascular differentiation. These factors act in a context-specific manner (eg, Wnt/β-catenin is required initially to form mesoderm but must be antagonized thereafter to make cardiac muscle). Different lines of ESCs produce different levels of agonists and antagonists for these pathways, but with careful optimization, highly enriched populations of immature cardiomyocytes can be generated. These cardiomyocytes survive transplantation to infarcted hearts of experimental animals, where they create new human myocardial tissue and improve heart function. The grafts generated by cell transplantation have been small, however, leading to an exploration of tissue engineering as an alternate strategy. Engineered tissue generated from preparations of human cardiomyocytes survives poorly after transplantation, most likely because of ischemia. Creation of pre-organized vascular networks in the tissue markedly enhances survival, with human capillaries anastomosed to the host coronary circulation. Thus, pathways controlling formation of the human cardiovascular system are emerging, yielding the building blocks for tissue regeneration that may address the root causes of heart failure.

Figure 1

Formation of teratomas after transplanting undifferentiated mouse embryonic stem cells (mESCs) into a mouse heart. (A) Uninjured heart 3 weeks after mESC injection. A complex tumor (outlined by dotted lines) has replaced much of the ventricular wall and obliterated the lumen at this apical level (scale bar 200µ m). Tissue representative of all 3 germ layers is present (see B–F). (B) Mesoderm-derived cartilage and bone. (C) Ectoderm-derived keratinizing squamous epithelium. (D) Endodermderived gut epithelium with goblet cells. (Scale bar B–D, 10µ m.) (E) Infarcted heart 3 weeks after mESCs were injected at the time of coronary occlusion. A teratoma is present within the wall and growing endoluminally (scale bar 200µ m). (F) Histomorphometry demonstrates no increase in cardiomyocyte differentiation within heart teratomas when compared with teratomas from the mouse hind limb that were derived from embryoid bodies, indicating the lack of a cardiogenic environment in either the normal or injured heart.

Figure 2

Protocol for directed differentiation of cardiomyocytes from human embryonic stem cells (hESCs) in high-density monolayers. (A) Mesodermal fate is induced by activin A signaling. Once enriched for mesoderm, BMP4 treatment increases the percentage of spontaneously contracting cardiomyocytes. (B–E) Monolayer embryonic stem cells treated with activin A/BMP4. Immunocytochemical analyses demonstrate cardiomyocyte structural proteins: cardiac troponin T (B, red); β-MHC (C, green); α actinin (D and E, green). Panel E is a higher magnification of the insert in panel D, demonstrating sarcomeric structures. Nuclei were stained with DAPI (blue) in all panels. MHC, myosin heavy chain; BMP, bone morphogenic protein.

Figure 3

The Wnt/b-catenin signaling pathway has a biphasic effect on cardiac differentiation in mouse and human embryonic stem cells (ESCs). (A) The addition of Wnt-3A to mouse ESCs from days 2–5 hastens the onset of spontaneous contraction in embryoid bodies (EBs) when compared with a control. (B) Flow cytometry for sarcomeric myosin heavy chain (MF20 antibody, vertical axis) demonstrates that early treatment with Wnt3a enhances the induction of cardiac differentiation by activin A and BMP4. Conversely, blocking endogenous Wnt signaling with Dkk1 markedly inhibits the ability of activin A and BMP4 to induce cardiac differentiation. (C) Quantitative RT-PCR demonstrates enhanced expression of β-MHC mRNA when treated with Activin A/BMP4. This is further increased with the addition of Wnt3a to activin A. If a Wnt inhibitor, Dkk1, is added with BMP4, β-MHC transcription is markedly decreased. (D) Addition of Dkk1 at later time points (days 5–11), post mesoderm induction, enhances β-MHC transcript levels, indicating that endogenous Wnt signals have a biphasic effect, inducing mesoderm early and later inhibiting differentiation of cardiomyocytes from mesoderm. (E) Schematic of Wnt signaling during cardiac differentiation, initially inducing a population of precardiac mesodermal cells. Once mesodermal, further treatment of Wnt will induce other mesodermal fates but will inhibit further cardiac differentiation. The canonical Wnt antagonist, Dkk1, can block Wnt signaling. Dkk1-mediated Wnt inhibition prior to mesoderm formation decreases the population of precardiac mesoderm that will give rise to cardiomyoyctes. Dkk1-mediated Wnt inhibition after mesoderm formation promotes a cardiac phenotype. MHC, myosin heavy chain; BMP, bone morphogenic protein. Dkk1, dikkopf homolog 1; RT-PCR, reverse transcriptase polymerase chain reaction.

Figure 4

Generation of endothelial cells from human embryonic stem cells (hESCs). (A) Embryoid bodies (EBs) treated with multiple factors for the induction of a vascular phenotype demonstrate that VEGF leads to increased levels of CD31, VE-cadherin, and vWF proteins at 14 days. Conversely, coculture with OP9 stromal cells or growth in endothelial growth medium, 2MV, did not consistently enhance endothelial markers. (B) Flow cytormetry analysis demonstrating that VEGF treatment of EBs leads to increased numbers of total VE-cadherin⁺/CD31⁺ cells at days 10 and 14 when compared with untreated EBs. (C,D) hESC-derived endothelial cells purified by CD31 expression, seeded into collagen gel constructs and implanted onto the surface of hearts previously injured by ischemia–reperfusion. The human endothelial cells (human-specific CD31 staining, green) demonstrate vascular networks that are perfused with rat red blood cells (red, autofluorescence). (Scale bars: C, 100 µm; D, 20 µm.) VEGF, vascular endothelial growth factor; vWF, von Willebrand factor.

Figure 5

Human cardiomyocytes engraft in rat infarcts and enhance ventricular function. Human embryonic stem cells (hESCs) were differentiated to a cardiomyocyte cell fate and transplanted to 4-day old infarcts in athymic rats. (A) When assessed at 4 weeks post transplantation, grafts expressed cardiac troponin I (green) and β-MHC (red; yellow in merged image), whereas rat cardiomyocytes were typically β-MHC-negative (scale bar 20µ m). (B) Junctions between host and graft cardiomyocytDense graftses (*) could of transplantedbe demonstrated cell could by be staining determined for cadherin by β-MHC (green (brown);) and the β- nucleiMHC (ofred some) (nuclei β-MHC-positive blue, scale barcells 10 haveµ m). con- (C) densed DNA (arrows), demonstrating mitotic activity post transplantation (scale bar 20µ m). (C) Ongoing proliferation of 4week-old grafts identified by mitotic figures (arrows) in β-MHC-positive graft cells (brown). (D–G) Cardiac MRI used to assess recipient animals for improvement in heart function. Control (pro-survival cocktail (PSC)) only, animals demonstrated a thinned anterior wall infarct (arrow) and dilated chamber in both diastole (D) and systole (E), whereas animals receiving differentiated cardiomyocytes had reduced dilation and increased anterior wall thickening (arrow) in both diastole (F) and systole (G) (scale bar 1 cm). (H) Rats receiving human cardiomyocytes with PSC had a 2.5-fold increase in systolic wall thickening, by MRI, compared with hearts treated with PSC alone, serum-free medium or hESCs differentiated in FBS + PSC, which have low rates of cardiomyocyte production (~0.8%). MHC, myosin heavy chain; FBS, fetal bovine serum; MRI, magnetic resonance imaging.

Figure 6

Human cardiac tissue engineering. Human cardiac patches are created by placing hESC-derived cardiomyocytes in a suspension culture on an orbital shaker. This method reproducibly creates patches of contractile myocardium with a tunable diameter (A), dependent on the input cell number, while its thickness is typically ~400µ m (B). (C) Pretreatment of patches with human umbilical vein endothelial cells (HUVECs) and mouse embryo fibroblasts (MEFs) form vascular-like networks (red, CD31) prior to transplant without compromising a β-MHC phenotype (D, red). These patches are able to be electrically stimulated (E). Prevascularization conveyed a survival advantage over nonvascularized patches when transplanted to rat skeletal muscle, typically demonstrating 11-fold larger graft size (F). The patches also could be sutured to the rat epicardium (G) and after 1 week in vivo, had significant levels of β-MHC-positive human cardiomyocytes (brown) (scale bar 100µ m). (H) Patches also contained robust human CD31 immunofluorescence in vessel-like structures that also held Ter 119+ red blood cells (green, human CD31; scale bar 10µ m) demonstrating connection of the engineered vessels to the vasculature of the rat. MHC, myosin heavy chain; hESC, human embryonic stem cells.