c-MYC independent nuclear reprogramming favors cardiogenic potential of induced pluripotent stem cells

Abstract

Induced pluripotent stem cell (iPS) technology has launched a new platform in regenerative medicine aimed at deriving unlimited replacement tissue from autologous sources through somatic cell reprogramming using stemness factor sets. In this way, authentic cardiomyocytes have been obtained from iPS and recently demonstrated in proof-of-principle studies to repair infarcted heart. Optimizing the cardiogenic potential of iPS progeny would ensure a maximized yield of bioengineered cardiac tissue. Here, we reprogrammed fibroblasts in the presence or absence of c-MYC to determine if the acquired cardiogenicity is sensitive to the method of nuclear reprogramming. Using lentiviral constructs that expressed stemness factors SOX2, OCT4, and KLF4 with or without c-MYC, iPS clones generated through fibroblast reprogramming demonstrated indistinguishable characteristics for 5 days of differentiation with similar cell morphology, growth rates, and chimeric embryo integration. However, 4-factor c-MYC dependent nuclear reprogramming produced iPS progeny that consistently prolonged the expression of pluripotent Oct-4 and Fgf4 genes and repressed cardiac differentiation. In contrast, 3-factor c-MYC-less iPS clones efficiently up-regulated pre-cardiac (CXCR4, Flk-1, and Mesp1/2) and cardiac (Nkx2.5, Mef2c, and Myocardin) gene expression patterns. In fact, 3-factor iPS progeny demonstrated early and robust cardiogenesis during in vitro differentiation with consistent beating activity, sarcomere maturation, and rhythmical intracellular calcium dynamics. Thus, nuclear reprogramming independent of c-MYC enhances production of pluripotent stem cells with innate cardiogenic potential.

Figure 1. iPS production with and without c-MYC

A, Mouse embryonic fibroblasts were transduced one day after plating with 4 genes (SOX2, OCT4, KLF4 and c-MYC) or 3 genes (SOX2, OCT4 and KLF4) using HIV-derived lentiviruses containing human genes. Round and compact embryonic-stem-cell-like colonies were observed after 2 weeks in 4 factor-induced (4F-iPS) cultures and after 3 weeks in 3 factor-induced (3F-iPS) clones. B, Genomic integration of viral constructs was detected in transduced progeny, with c-MYC absent from the 3 factor-induced lines. Parental fibroblasts demonstrate no viral sequence integration and are shown as a negative control.

Figure 2. 4F-iPS and 3F-iPS show similar growth kinetics and embryoid body morphology

A, Distinct clones of 4F-iPS (left) and 3F-iPS (right) were kept in the undifferentiated state on feeders, and showed indistinguishable morphology consisting of compact colonies. B, Hanging drops were made with multiple clones of 4F-iPS (left) and 3F-iPS (right). Embryoid bodies from each of the clones demonstrated equivalent size and shape within 5 days of differentiation. Bar=500 μm.

Figure 3. Pluripotent capacity and embryonic developmental potential in 4F-iPS and 3F-iPS

A, 4F-iPS and 3F-iPS stain with similar positive patterns for pluripotency markers alkaline phosphatase (left, AP) and SSEA-1 (right, nuclei revealed with DAPI). B, LacZ-labeled iPS coincubated with diploid embryos (left) revealed the ability of 4F-iPS and 3F-iPS to integrate into host blastocyst (right). C, Integration of both 4F-iPS and 3F-iPS was sustained through embryonic development as shown for 9.5 dpc embryos, containing labeled cells that contributed to most of the tissues including the heart (cardiac inflow and outflow tracts showed in insets).

Figure 4. Divergent gene expression profiles for 4F-iPS and 3F-iPS during in vitro differentiation

Differentiation of iPS progenitors was promoted within hanging drops to form embryoid bodies followed by expansion of progeny on gelatinized plates in multi-layer tissues. Cells were sampled from undifferentiated cultures at day 0, floating embryoid bodies at day 5, and differentiating cultures at day 12 for gene expression analysis. A, Pluripotency genes were immediately downregulated in 3F-iPS with initiation of differentiation, whereas expression levels of pluripotent genes was protracted in 4F-iPS. B, Gastrulation markers consistently peaked at day 5 for 3F-iPS clones with inconsistent levels expressed in 4F-iPS. C, Precardiac genes increased at day 5 for all tested clones, However, relative expression was notably higher in 3F-iPS when compared to 4F-iPS clones. D, Upregulation of cardiac transcription factors was observed at day 12 in 3F-iPS with 4F-iPS clones maintaining lower baseline expression levels.

Figure 5. 3F-iPS differentiate into beating areas while 4F-iPS demonstrate inconsistent morphology and viability in vitro

iPS clones were differentiated into embryoid bodies and plated on day 5 onto gelatinized plates. A, Differences between 4F-iPS and 3F-iPS were noticeable in morphology after a total of 8 days as embryoid bodies. 4F-iPS clones showed areas of round and detached cells (arrows, left). 3F-iPS clones had predictable growth kinetics with sustained integrity of the cell layers that spread from the initial embryoid body towards the periphery of the plate. B, After 12 days, extensive cell loss was apparent in 4F-iPS cultures (arrows, left) while compact and continuous masses of 3F-iPS were observed in all studied clones, including areas of spontaneous beating activity (outlined in red dotted lines). Bar=500 μm.

Figure 6. c-MYC independent nuclear reprogramming favors cardiogenic potential of iPS

A, Differentiating cultures of 4F-iPS and 3F-iPS were observed daily for appearance of beating activity. Between day 7 and 11 of differentiation, no spontaneous contractility was detected with 4F-iPS clones. In contrast, robust and sustained beating activity was documented in 3F-iPS. B, Immunostaining of isolated cardiomyocytes derived from 3F-iPS demonstrated presence of contractile proteins alpha actinin (left) and myosin light chain-2v (MLC-2v) in combination with cardiac transcription factor Mef2c (right). C, Live cell imaging with calcium sensitive Fluo-4AM demonstrated rhythmical calcium transients within individual isolated cardiomyocytes. D, Calcium imaging of beating syncytium demonstrated coordinated activity between multiple cardiomyocytes and synchronized calcium transients.