Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become a powerful tool for human disease modeling and therapeutic testing. However, their use remains limited by their immaturity and heterogeneity. To characterize the source of this heterogeneity, we applied complementary single-cell RNA-seq and bulk RNA-seq technologies over time during hiPSC cardiac differentiation and in the adult heart. Using integrated transcriptomic and splicing analysis, more than half a dozen distinct single-cell populations were observed, several of which were coincident at a single time-point, day 30 of differentiation. To dissect the role of distinct cardiac transcriptional regulators associated with each cell population, we systematically tested the effect of a gain or loss of three transcription factors (NR2F2, TBX5, and HEY2), using CRISPR genome editing and ChIP-seq, in conjunction with patch clamp, calcium imaging, and CyTOF analysis. These targets, data, and integrative genomics analysis methods provide a powerful platform for understanding in vitro cellular heterogeneity.

Being able to self-renew and differentiate into virtually all cell types, both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have exciting therapeutic implications for myocardial infarction, neurodegenerative disease, diabetes, and other disorders involving irreversible cell loss. However, stem cell biology remains incompletely understood despite significant advances in the field. Inefficient stem cell differentiation, difficulty in verifying successful delivery to the target organ, and problems with engraftment all hamper the transition from laboratory animal studies to human clinical trials. Although traditional histopathological techniques have been the primary approach for ex vivo analysis of stem cell behavior, these postmortem examinations are unable to further elucidate the underlying mechanisms in real time and in vivo. Fortunately, the advent of molecular imaging has led to unprecedented progress in understanding the fundamental behavior of stem cells, including their survival, biodistribution, immunogenicity, and tumorigenicity in the targeted tissues of interest. This review summarizes various molecular imaging technologies and how they have advanced the current understanding of stem cell survival, biodistribution, immunogenicity, and tumorigenicity.

The inactivation of the retinoblastoma (Rb) tumor suppressor gene in mice results in ectopic proliferation, apoptosis, and impaired differentiation in extraembryonic, neural, and erythroid lineages, culminating in fetal death by embryonic day 15.5 (E15.5). Here we show that the specific loss of Rb in trophoblast stem (TS) cells, but not in trophoblast derivatives, leads to an overexpansion of trophoblasts, a disruption of placental architecture, and fetal death by E15.5. Despite profound placental abnormalities, fetal tissues appeared remarkably normal, suggesting that the full manifestation of fetal phenotypes requires the loss of Rb in both extraembryonic and fetal tissues. Loss of Rb resulted in an increase of E2f3 expression, and the combined ablation of Rb and E2f3 significantly suppressed Rb mutant phenotypes. This rescue appears to be cell autonomous since the inactivation of Rb and E2f3 in TS cells restored placental development and extended the life of embryos to E17.5. Taken together, these results demonstrate that loss of Rb in TS cells is the defining event causing lethality of Rb −/− embryos and reveal the convergence of extraembryonic and fetal functions of Rb in neural and erythroid development. We conclude that the Rb pathway plays a critical role in the maintenance of a mammalian stem cell population.[Keywords: Rb; development; placenta; stem cells] Supplemental material is available at http://www.genesdev.org. The retinoblastoma (Rb) tumor suppressor gene was identified more than two decades ago as the gene responsible for retinoblastoma, but has since been implicated in most human cancers. In contrast to retinoblastoma patients, inheritance of one deleted copy of Rb in mice did not induce retinoblastoma but did increase risk of pituitary and thyroid cancers (Jacks et al. 1992;Hu et al. 1994;Maandag et al. 1994;Williams et al. 1994;Robanus-Maandag et al. 1998;Yamasaki et al. 1998). Deletion of both copies of Rb in mice resulted in a broad range of severe abnormalities that lead to lethality by embryonic day 15.5 (E15.5) (Clarke et al. 1992;Jacks et al. 1992;Lee et al. 1992;Wu et al. 2003). Because Rb is normally expressed in all tissues of the mouse embryo, it was assumed that these developmental abnormalities were due to the absence of Rb protein in the tissues affected. Subsequent analysis of chimeric embryos suggested that Rb function is likely to be much more complex than initially suspected (Maandag et al. 1994;Lipinski et al. 2001). Indeed, recent findings showed that Rb-deficient embryos supplied with a wild-type placenta could develop to term and suggested a critical function of Rb in the placenta that might underlie many of the fetal developmental abnormalities observed in Rb −/− embryos Wu et al. 2003).Because Rb is involved in so many important pro-