Reprogramming cell fates towards pluripotent stem cells and other cell types has revolutionized our under- standing of cellular plasticity. During the last decade, transcription factors and microRNAs ...have become powerful reprogramming factors for modulating cell fates. Recently, many efforts are focused on repro- gramming cell fates by non-viral and non-integrating chemical approaches. Small molecules not only are useful in generating desired cell types in vitro for vari- ous applications, such as disease modeling and cell- based transplantation, but also hold great promise to be further developed as drugs to stimulate patients＇ endogenous cells to repair and regenerate in vivo. Here we will focus on chemical approaches for generating induced pluripotent stem cells, neurons, cardiomy- ocytes, hepatocytes and pancreatic is cells. Significantly, the rapid and exciting advances in cellular reprogramming by small molecules will help us to achieve the long-term goal of curing devastating diseases, injuries, cancers and aging.
Single-cell analysis is a valuable tool for dissecting cellular heterogeneity in complex systems
. However, a comprehensive single-cell atlas has not been achieved for humans. Here we use single-cell ...mRNA sequencing to determine the cell-type composition of all major human organs and construct a scheme for the human cell landscape (HCL). We have uncovered a single-cell hierarchy for many tissues that have not been well characterized. We established a 'single-cell HCL analysis' pipeline that helps to define human cell identity. Finally, we performed a single-cell comparative analysis of landscapes from human and mouse to identify conserved genetic networks. We found that stem and progenitor cells exhibit strong transcriptomic stochasticity, whereas differentiated cells are more distinct. Our results provide a useful resource for the study of human biology.
Human induced pluripotent stem cells (hiPSCs) have been generated by reprogramming a number of different somatic cell types using a variety of approaches. In addition, direct reprogramming of mature ...cells from one lineage to another has emerged recently as an alternative strategy for generating cell types of interest. Here we show that a combination of a microRNA (miR-124) and two transcription factors (MYT1L and BRN2) is sufficient to directly reprogram postnatal and adult human primary dermal fibroblasts (mesoderm) to functional neurons (ectoderm) under precisely defined conditions. These human induced neurons (hiNs) exhibit typical neuronal morphology and marker gene expression, fire action potentials, and produce functional synapses between each other. Our findings have major implications for cell-replacement strategies in neurodegenerative diseases, disease modeling, and neural developmental studies.
► miR-124 promotes direct neuronal conversion of human fibroblasts ► Reprogramming of both postnatal and fully adult human fibroblasts ► Robust conversion mediated by a cocktail of miR-124, BRN2, and MYT1L ► Formation of functional synapses between adult human fibroblast-derived neurons
Pancreatic beta cells are of great interest for biomedical research and regenerative medicine. Here we show the conversion of human fibroblasts towards an endodermal cell fate by employing ...non-integrative episomal reprogramming factors in combination with specific growth factors and chemical compounds. On initial culture, converted definitive endodermal progenitor cells (cDE cells) are specified into posterior foregut-like progenitor cells (cPF cells). The cPF cells and their derivatives, pancreatic endodermal progenitor cells (cPE cells), can be greatly expanded. A screening approach identified chemical compounds that promote the differentiation and maturation of cPE cells into functional pancreatic beta-like cells (cPB cells) in vitro. Transplanted cPB cells exhibit glucose-stimulated insulin secretion in vivo and protect mice from chemically induced diabetes. In summary, our study has important implications for future strategies aimed at generating high numbers of functional beta cells, which may help restoring normoglycemia in patients suffering from diabetes.
The simple yet powerful technique of induced pluripotency may eventually supply a wide range of differentiated cells for cell therapy and drug development. However, making the appropriate cells via ...induced pluripotent stem cells (iPSCs) requires reprogramming of somatic cells and subsequent redifferentiation. Given how arduous and lengthy this process can be, we sought to determine whether it might be possible to convert somatic cells into lineage-specific stem/progenitor cells of another germ layer in one step, bypassing the intermediate pluripotent stage. Here we show that transient induction of the four reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) can efficiently transdifferentiate fibroblasts into functional neural stem/progenitor cells (NPCs) with appropriate signaling inputs. Compared with induced neurons (or iN cells, which are directly converted from fibroblasts), transdifferentiated NPCs have the distinct advantage of being expandable in vitro and retaining the ability to give rise to multiple neuronal subtypes and glial cells. Our results provide a unique paradigm for iPSC-factor-based reprogramming by demonstrating that it can be readily modified to serve as a general platform for transdifferentiation.
Human pluripotent stem cells (hPSCs) have potential applications in biological studies and regenerative medicine. However, precise genome editing in hPSCs remains time-consuming and labor-intensive. ...Here we demonstrate that the recently identified CRISPR-Cpf1 can be used to efficiently generate knockout and knockin hPSC lines. The unique properties of CRISPR-Cpf1, including shorter crRNA length and low off-target activity, are very attractive for many applications. In particular, we develop an unbiased drug-selection-based platform feasible for high-throughput screening in hPSCs and this screening system enables us to identify small molecules VE-822 and AZD-7762 that can promote CRISPR-Cpf1-mediated precise genome editing. Significantly, the combination of CRISPR-Cpf1 and small molecules provides a simple and efficient strategy for precise genome engineering.
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