Single-Cell Profiling of an In Vitro Model of Human Interneuron Development Reveals Temporal Dynamics of Cell Type Production and Maturation.

GABAergic interneurons are essential for neural circuit function, and their loss or dysfunction is implicated in human neuropsychiatric disease. In vitro methods for interneuron generation hold promise for studying human cellular and functional properties and, ultimately, for therapeutic cell replacement. Here we describe a protocol for generating cortical interneurons from hESCs and analyze the properties and maturation time course of cell types using single-cell RNA-seq. We find that the cell types produced mimic in vivo temporal patterns of neuron and glial production, with immature progenitors and neurons observed early and mature cortical neurons and glial cell types produced late. By comparing the transcriptomes of immature interneurons to those of more mature neurons, we identified genes important for human interneuron differentiation. Many of these genes were previously implicated in neurodevelopmental and neuropsychiatric disorders.

Engineering of human pluripotent stem cells by AAV-mediated gene targeting.

Precise genetic manipulation of human pluripotent stem cells will be required to realize their scientific and therapeutic potential. Here, we show that adeno-associated virus (AAV) gene targeting vectors can be used to genetically engineer human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Different types of sequence-specific changes, including the creation and correction of mutations, were introduced into the human HPRT1 and HMGA1 genes (HPRT1 mutations being responsible for Lesch-Nyhan syndrome). Gene targeting occurred at high frequencies in both ESCs and iPSCs, with over 1% of all colony-forming units (CFUs) undergoing targeting in some experiments. AAV vectors could also be used to target genes in human fibroblasts that were subsequently used to derive iPSCs. Accurate and efficient targeting took place with minimal or no cytotoxicity, and most of the gene-targeted stem cells produced were euploid and pluripotent.

microRNAs regulate human embryonic stem cell division.

microRNAs (miRNAs) regulate numerous physiological processes such as cell division and differentiation in many tissue types including stem cells. To probe the role that miRNAs play in regulating processes relevant to embryonic stem cell biology, we used RNA interference to silence DICER and DROSHA, the two main miRNA processing enzymes. Consistent with a role for miRNAs in maintaining normal stem cell division and renewal, we found that perturbation of miRNA pathway function in human embryonic stem cells (hESCs) attenuates cell proliferation. Normal cell growth can be partially restored by introduction of the mature miRNAs miR-195 and miR-372. These miRNAs regulate two tumor suppressor genes, respectively: WEE1, which encodes a negative G2/M kinase modulator of the CycB/CDK complex and CDKN1A, which encodes p21, a CycE/CDK cyclin dependent kinase inhibitor that regulates the G1/S transition. We show that in wild-type hESCs, WEE 1 levels control the rate of hESC division, whereas p21 levels must be maintained at a low level for hESC division to proceed. These data support a model for hESC cell cycle control in which miRNAs regulate negative cell cycle modulators at two phases of the cell cycle to ensure proper replenishment of the stem cell population.

X-inactivation reveals epigenetic anomalies in most hESC but identifies sublines that initiate as expected.

The clinical and research value of human embryonic stem cells (hESC) depends upon maintaining their epigenetically naïve, fully undifferentiated state. Inactivation of one X chromosome in each cell of mammalian female embryos is a paradigm for one of the earliest steps in cell specialization through formation of facultative heterochromatin. Mouse ES cells are derived from the inner cell mass (ICM) of blastocyst stage embryos prior to X-inactivation, and cultured murine ES cells initiate this process only upon differentiation. Less is known about human X-inactivation during early development. To identify a human ES cell model for X-inactivation and study differences in the epigenetic state of hESC lines, we investigated X-inactivation in all growth competent, karyotypically normal, NIH approved, female hESC lines and several sublines. In the vast majority of undifferentiated cultures of nine lines examined, essentially all cells exhibit hallmarks of X-inactivation. However, subcultures of any hESC line can vary in X-inactivation status, comprising distinct sublines. Importantly, we identified rare sublines that have not yet inactivated Xi and retain competence to undergo X-inactivation upon differentiation. Other sublines exhibit defects in counting or maintenance of XIST expression on Xi. The few hESC sublines identified that have not yet inactivated Xi may reflect the earlier epigenetic state of the human ICM and represent the most promising source of NIH hESC for study of human X-inactivation. The many epigenetic anomalies seen indicate that maintenance of fully unspecialized cells, which have not formed Xi facultative heterochromatin, is a delicate epigenetic balance difficult to maintain in culture.