To obtain iPS cells with a cardiomyocyte reporter label, tail tip fibroblasts were isolated from an NKX2-5-GFP transgenic mouse containing GFP in a bacterial artificial chromosome (BAC) containing the human NKX2-5 locus8
. Mouse ES cells containing the same NKX2-5-GFP BAC were previously reported8
. Fibroblasts from the NKX2-5-GFP reporter mice were infected with a combination of four retroviruses encoding factors known to induce pluripotency: Oct-4, Sox-2, Klf4 and c-Myc2
. Three-dimensional colonies were observed from the fifth day after viral infection. By day eight, colonies were large enough to be selected and expanded in a feeder-free culture system (). The cells derived from these colonies expressed SSEA-1 and Oct-4 protein as shown by immunostaining (). To select the best reprogrammed lines, quantitative PCR (qPCR) for the pluripotency genes Nanog and Oct-4 was performed, revealing mRNA levels comparable to those of ES cells in both low (p4) and high (p20–25) passage samples ().
Generation and differentiation of NKX2.5-GFP iPS cells
We also tested whether the cells were able to grow clonally by selecting SSEA-1-expressing cells by FACS () and culturing in 96-well plates at one cell per well. Indeed, new colonies were obtained from single SSEA-1+ cells that could be passaged and expanded while maintaining expression of pluripotency markers. This sorting strategy allowed for purification of potentially heterogenous colonies of iPS cells at the single-cell level. All studies were done with clonally-derived cells. After subcutaneous and intramuscular injection into NOD-SCID mice, undifferentiated iPS cells of each selected line formed teratomas containing derivatives of all germ lines ().
We next evaluated whether the NKX2-5-GFP iPS cells could be differentiated in vitro
, with a special interest in cardiomyocyte differentiation. iPS cells formed beating embryoid bodies (EBs) with high efficiency when induced by the hanging drop system. Spontaneous aggregation in suspension in low-attachment plates is much less labor-intensive and also produced some beating EBs, but with lower efficiency. However, when co-aggregates in suspension were made with the endoderm cell line, END-215
, a high percentage of beating EBs were isolated (). As expected, the beating foci within these EBs were green because of the NKX2-5-GFP reporter (Supplementary Video I, II
). The iPS cell-derived EBs contained derivatives of all three germ layers (), including α-actinin positive cardiomyocytes with typical sarcomeric patterns (). Thus, we concluded that we had derived several independent NKX2-5-GFP iPS cell lines from an adult mouse and that the lines were able to retain their pluripotency under feeder-free culture conditions and responded to guided differentiation in vitro
We quantified the percentage of GFP+ cells in EBs, representing the NKX2-5- expressing population of cardiac progenitor cells and cardiomyocytes at day 8, obtained by several differentiation methods (). Consistent with the count of beating EBs, the hanging drop system yielded the highest percentage of GFP+ cells (). END-2 co-aggregates were not included in this analysis, as the END-2 cells present in the mixture of differentiated cells would confound the result by altering the denominator when calculating percent cardiac progenitors. The expression of a panel of cardiac-enriched genes was higher in sorted GFP+ cells compared to sorted GFP– cells relative to undifferentiated ES or iPS cells (). In addition, the cardiomyocytes derived from the iPS cells after 45 days of differentiation had spontaneous calcium flux and responded to β-adrenergic agonist isoproterenol (Iso) and muscarinic receptor agonist carbachol (CCh) stimulation (). While the NKX2-5-GFP iPS cell lines we generated could efficiently differentiate into cardiomyocytes in vitro, we investigated their potential in vivo in embryos and in the adult. We took advantage of the recent demonstration that injection of ES cells into eight-cell embryos can result in generation of 95–100% chimeras, effectively generating embryos composed almost entirely of the injected ES cells20
. Injection of NKX2-5-GFP iPS cells into eight-cell embryos revealed that the cells could give rise to viable embryos with hearts almost entirely populated by NKX2-5-GFP iPS-derived cardiomyocytes, illustrated by the green fluorescent hearts ().
Cardiac differentiation and quantification from NKX2.5-GFP iPS cells
To determine the similarity of iPS- and ES cell-derived cardiomyocytes at the gene expression level, we took advantage of an NKX2-5-GFP ES cell line generated with exactly the same BAC reporter transgene used to create the NKX2-5-GFP mouse line used for iPS generation8
. Thus, NKX2-5-GFP positive cells isolated from the iPS and ES cell lines should in principle mark very similar populations. Microarray analysis of mRNA from sorted NKX2-5-GFP+ cells after 8 days of differentiation revealed that only 195 out of the 28,853 transcripts represented were significantly different between ES and iPS cell-derived cardiac progenitors, even when applying the least stringent statistical method for defining differential expression (FDR; adjusted p-value to control for false discovery rate using Benjamini-Hochberg method with cut-off p-value set at <= 0.1). Of those, only 38 annotated genes were altered greater than 2-fold, revealing a surprisingly high degree of similarity in the differentiated progeny of ES and iPS cells ( and Supplementary Table I
Gene expression microarray analyses comparing sorted GFP+ iPS or ES-derived cardiomyocyte progenitors (iPS-CM or ES-CM) and undifferentiated ES cells (uES)
Concerns exist regarding the potential variability between iPS cell lines compared to the variability between ES lines, but the degree of variability of gene expression in lineage-specific cells derived from ES and iPS cells has not been interrogated for any cell type. This issue is particularly important for interpreting studies with multiple disease-specific iPS lines. Using the microarray data sets of NKX2-5-GFP-positive ES and iPS cells, we found that the variation between independent iPS cell-derived cardiac progenitors was no larger than that between cardiac progenitors derived from separate ES lines (). The correlation between cell lines was extremely high (R=0.99) (), suggesting that not only were the ES and iPS-derived cardiac progenitors more similar than expected, but the line-to-line variation of the lineage-specific iPS cells was also very low.
To assess the potential of the NKX2-5-GFP iPS cells in regenerative applications, day 8 EBs were dissociated and prepared for injection into acutely infarcted hearts of NOD-SCID mice. Although this approach resulted in large patches of new GFP+ myocardium being formed (), clusters of SSEA-1 and Oct-4 expressing cells were also observed (), indicating that progenitor selection was necessary to avoid tumor formation. We thus sorted for GFP+ cells by flow cytometry to isolate the cardiac progenitor pool prior to intramyocardial transplantation. While the grafts from NKX2-5-GFP+ cells were smaller than those observed after total cell injection, they did not form teratomas and were negative for markers of pluripotency (). These findings suggest that the selected NKX2-5-GFP+ cells may be safe for regenerative approaches, at least with respect to tumor formation.
NKX2.5-GFP iPS cell-derived cardiac progenitors survive in the infarcted mouse heart after transplantation