Matched Developmental Timing of Donor Cells with the Host Is Crucial for Chimera Formation

Chimeric mice have been generated by injecting pluripotent stem cells into morula-to-blastocyst stage mouse embryo or by introducing more mature cells into later stage embryos that correspond to the differentiation stage of the donor cells. It has not been rigorously tested, however, whether successful chimera formation requires the developmental stage of host embryo and donor cell to be matched. Here, we compared the success of chimera formation following injection of primary neural crest cells (NCCs) into blastocysts or of embryonic stem cells (ESCs) into E8.5 embryos (heterochronic injection) with that of injecting ESCs cells into the blastocyst or NCCs into the E8.5 embryos (isochronic injection). Chimera formation was efficient when donor and host were matched, but no functional chimeric contribution was found in heterochronic injections. This suggests that matching the developmental stage of donor cells with the host embryo is crucial for functional engraftment of donor cells into the developing embryo. Cohen at al. compares the efficiency of chimera formation in heterochronic and isochronic injections of ESCs and NCCs. Using two distinct and well-characterized pre- and post-implantation chimeric platforms, they show that matching of developmental age of donor cells and the host is essential for chimera formation.National Institutes of Health (U.S.) (Grant R37HD045022)National Institutes of Health (U.S.) (Grant R01-NS088538)National Institutes of Health (U.S.) (Grant R01- MH104610

The role of FGF-signaling in early neural specification of human embryonic stem cells

AbstractThe mechanisms that govern human neural specification are not completely characterized. Here we used human embryonic stem cells (hESCs) to study the role of fibroblast growth factor (FGF)-signaling in early human neural specification. Differentiation was obtained by culturing clusters of hESCs in chemically-defined medium. We show that FGF-signaling, which is endogenously active during early differentiation of hESCs, induces early neural specification, while its blockage inhibits neuralization. The early neuralization effect of FGF-signaling is not mediated by promoting the proliferation of existing neural precursors (NPs) or prevention of their apoptosis. The neural instructive effect of FGF-signaling occurs after an initial FGF-independent differentiation into primitive ectoderm-like fate. We further show that FGF-signaling can induce neuralization by a mechanism which is independent of modulating bone morphogenic protein (BMP)-signaling. Still, FGF-signaling is not essential for hESC neuralization which can occur in the absence of FGF and BMP-signaling. Collectively, our data suggest that human neural induction is instructed by FGF-signaling, though neuralization of hESCs can occur in its absence

Molecular Criteria for Defining the Naive Human Pluripotent State.

Recent studies have aimed to convert cultured human pluripotent cells to a naive state, but it remains unclear to what extent the resulting cells recapitulate in vivo naive pluripotency. Here we propose a set of molecular criteria for evaluating the naive human pluripotent state by comparing it to the human embryo. We show that transcription of transposable elements provides a sensitive measure of the concordance between pluripotent stem cells and early human development. We also show that induction of the naive state is accompanied by genome-wide DNA hypomethylation, which is reversible except at imprinted genes, and that the X chromosome status resembles that of the human preimplantation embryo. However, we did not see efficient incorporation of naive human cells into mouse embryos. Overall, the different naive conditions we tested showed varied relationships to human embryonic states based on molecular criteria, providing a backdrop for future analysis of naive human pluripotency.This study was supported by grants from the Simons Foundation (SFLIFE #286977 to R.J) and in part by the NIH (RO1-CA084198) to R.J., from the Swiss National Science Foundation and the European Research Council (KRABnKAP, No. 268721) to D.T. The work in J.R.E’s laboratory was supported by the Howard Hughes Medical Institute and Gordon and Betty Moore Foundation (GBMF3034) and the Mary K. Chapman Foundation. J.R.E is an Investigator of the Howard Hughes Medical Institute. T.W.T. is supported by a Sir Henry Wellcome Postdoctoral Fellowship (098889/Z/12/Z), J.P. by a Foundation Bettencourt Award and by the Association pour la Recherche sur le Cancer (ARC), M.I. by a postdoctoral training grant from the Fonds de la Recherche en Santé du Québec. R.J. is co-founder of Fate Therapeutics and an adviser to Stemgent.This is the final version of the article. It first appeared from Cell Press via http://www.cell.com/cell-stem-cell/abstract/S1934-5909(16)30161-

Enriched Population of PNS Neurons Derived from Human Embryonic Stem Cells as a Platform for Studying Peripheral Neuropathies

BACKGROUND: The absence of a suitable cellular model is a major obstacle for the study of peripheral neuropathies. Human embryonic stem cells hold the potential to be differentiated into peripheral neurons which makes them a suitable candidate for this purpose. However, so far the potential of hESC to differentiate into derivatives of the peripheral nervous system (PNS) was not investigated enough and in particular, the few trials conducted resulted in low yields of PNS neurons. Here we describe a novel hESC differentiation method to produce enriched populations of PNS mature neurons. By plating 8 weeks hESC derived neural progenitors (hESC-NPs) on laminin for two weeks in a defined medium, we demonstrate that over 70% of the resulting neurons express PNS markers and 30% of these cells are sensory neurons. METHODS/FINDINGS: Our method shows that the hNPs express neuronal crest lineage markers in a temporal manner, and by plating 8 weeks hESC-NPs into laminin coated dishes these hNPs were promoted to differentiate and give rise to homogeneous PNS neuronal populations, expressing several PNS lineage-specific markers. Importantly, these cultures produced functional neurons with electrophysiological activities typical of mature neurons. Moreover, supporting this physiological capacity implantation of 8 weeks old hESC-NPs into the neural tube of chick embryos also produced human neurons expressing specific PNS markers in vivo in just a few days. Having the enriched PNS differentiation system in hand, we show for the first time in human PNS neurons the expression of IKAP/hELP1 protein, where a splicing mutation on the gene encoding this protein causes the peripheral neuropathy Familial Dysautonomia. CONCLUSIONS/SIGNIFICANCE: We conclude that this differentiation system to produce high numbers of human PNS neurons will be useful for studying PNS related neuropathies and for developing future drug screening applications for these diseases

The seasonal nitrogen cycle in Wilkinson Basin, Gulf of Maine, as estimated by 1-D biological model optimization

Author Posting. © Elsevier B.V., 2009. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Journal of Marine Systems 78 (2009): 77-93, doi:10.1016/j.jmarsys.2009.04.001.The objective of this study was to fit a simple ecosystem model to climatological nitrogen cycle data in the Gulf of Maine, in order to calibrate the biological model for use in future 3-D modelling studies. First depth-dependent monthly climatologies of nitrate, ammonium, chlorophyll, zooplankton, detritus and primary production data from Wilkinson Basin, Gulf of Maine, were created. A 6-box nitrogen-based ecosystem model was objectively fitted to the data through parameter optimization. Optimization was based on weighted least squares with model-data misfits nondi- mensionalized by assigned uncertainties in the monthly climatological estimates. These uncertainties were estimated as the standard deviations of the raw data from the 6-meter and monthly bin averages. On average the model fits the monthly means almost within their assigned uncertainties. Several statistics are examined to assess model-data misfit. Pattern statistics such as the correlation coefficient lack practical significance when data errors are large relative to the signal, as in this application. Thus Taylor diagrams were not found to be useful. The RMSE and model bias normalized by the data error were found to be the most useful skill metrics as they indicate whether the model fits the data within its estimated error. The optimal simulated nitrogen cycle budgets are presented, as an estimate of the seasonal nitrogen cycle in Wilkinson Basin, and discussed in context of the available data.Wilkinson Basin has spring and fall phytoplankton blooms, and strong summer stratification with a deep chlorophyll maximum near 21 m, just above the nitracline. The mean euphotic zone depth is estimated to be 25 m, relatively constant with season. The model estimates annual primary production as 176 g C m−2 yr−1, annual new production as 71 g C m−2 yr−1 and sinking PON fluxes of 9.7 and 4.7 g N m−2 yr−1 at 24 and 198 m respectively. Areas for improvement in the biological model, the model optimization method, and significant data gaps are identified.This work was supported by ONR, NSF, and NOAA grant to Dennis McGillicuddy

Human neural crest cells contribute to coat pigmentation in interspecies chimeras after in utero injection into mouse embryos

The neural crest (NC) represents multipotent cells that arise at the interphase between ectoderm and prospective epidermis of the neurulating embryo. The NC has major clinical relevance because it is involved in both inherited and acquired developmental abnormalities. The aim of this study was to establish an experimental platform that would allow for the integration of human NC cells (hNCCs) into the gastrulating mouse embryo. NCCs were derived from pluripotent mouse, rat, and human cells and microinjected into embryonic-day-8.5 embryos. To facilitate integration of the NCCs, we used recipient embryos that carried a c-Kit mutation (W[superscript sh]/W[superscript sh]), which leads to a loss of melanoblasts and thus eliminates competition from the endogenous host cells. The donor NCCs migrated along the dorsolateral migration routes in the recipient embryos. Postnatal mice derived from injected embryos displayed pigmented hair, demonstrating differentiation of the NCCs into functional melanocytes. Although the contribution of human cells to pigmentation in the host was lower than that of mouse or rat donor cells, our results indicate that hNCCs, injected in utero, can integrate into the embryo and form mature functional cells in the animal. This mouse–human chimeric platform allows for a new approach to study NC development and diseases.United States. Department of Defense (Grant W81XWH-14-1-0057)Simons Foundation (Grant SFLIFE 286977)National Institutes of Health (U.S.) (Grants HD 045022 and R37-CA084198

Microcephaly Modeling of Kinetochore Mutation Reveals a Brain-Specific Phenotype

Most genes mutated in microcephaly patients are expressed ubiquitously, and yet the brain is the only major organ compromised in most patients. Why the phenotype remains brain specific is poorly understood. In this study, we used in vitro differentiation of human embryonic stem cells to monitor the effect of a point mutation in kinetochore null protein 1 (KNL1; CASC5), identified in microcephaly patients, during in vitro brain development. We found that neural progenitors bearing a patient mutation showed reduced KNL1 levels, aneuploidy, and an abrogated spindle assembly checkpoint. By contrast, no reduction of KNL1 levels or abnormalities was observed in fibroblasts and neural crest cells. We established that the KNL1 patient mutation generates an exonic splicing silencer site, which mainly affects neural progenitors because of their higher levels of splicing proteins. Our results provide insight into the brain-specific phenomenon, consistent with microcephaly being the only major phenotype of patients bearing KNL1 mutation.Simons Foundation. Postdoctoral FellowshipInternational Rett Syndrome Foundation (Postdoctoral Fellowship)Brain & Behavior Research Foundation (Young Investigator Grant)National Institutes of Health (U.S.) (grant HD 045022)National Institutes of Health (U.S.) (grant R37-CA084198)National Institutes of Health (U.S.) (grant 1U19AI131135-01