Development of Functional Thyroid C Cell-like Cells from Human Pluripotent Cells in 2D and in 3D Scaffolds.

Medullary thyroid carcinoma contributes to about 3-4% of thyroid cancers and affects C cells rather than follicular cells. Thyroid C cell differentiation from human pluripotent stem cells has not been reported. We report the stepwise differentiation of human embryonic stem cells into thyroid C cell-like cells through definitive endoderm and anterior foregut endoderm and ultimobranchial body-like intermediates in monolayer and 3D Matrigel culture conditions. The protocol involved sequential treatment with interferon/transferrin/selenium/pyruvate, foetal bovine serum, and activin A, then IGF-1 (Insulin-like growth factor 1), on the basis of embryonic thyroid developmental sequence. As well as expressing C cell lineage relative to follicular-lineage markers by qPCR (quantitative polymerase chain reaction) and immunolabelling, these cells by ELISA (enzyme-linked immunoassay) exhibited functional properties in vitro of calcitonin storage and release of calcitonin on calcium challenge. This method will contribute to developmental studies of the human thyroid gland and facilitate in vitro modelling of medullary thyroid carcinoma and provide a valuable platform for drug screening.

In vivo survival and differentiation of Friedreich ataxia iPSC-derived sensory neurons transplanted in the adult dorsal root ganglia.

Friedreich ataxia (FRDA) is an autosomal recessive disease characterized by degeneration of dorsal root ganglia (DRG) sensory neurons, which is due to low levels of the mitochondrial protein Frataxin. To explore cell replacement therapies as a possible approach to treat FRDA, we examined transplantation of sensory neural progenitors derived from human embryonic stem cells (hESC) and FRDA induced pluripotent stem cells (iPSC) into adult rodent DRG regions. Our data showed survival and differentiation of hESC and FRDA iPSC-derived progenitors in the DRG 2 and 8 weeks post-transplantation, respectively. Donor cells expressed neuronal markers, including sensory and glial markers, demonstrating differentiation to these lineages. These results are novel and a highly significant first step in showing the possibility of using stem cells as a cell replacement therapy to treat DRG neurodegeneration in FRDA as well as other peripheral neuropathies.

Programmable Nuclease-Based Integration into Novel Extragenic Genomic Safe Harbor Identified from Korean Population-Based CNV Analysis.

Here, we found two genomic safe harbor (GSH) candidates from chromosomes 3 and 8, based on large-scale population-based cohort data from 4,694 Koreans by CNV analysis. Furthermore, estimated genotype of these CNVRs was validated by quantitative real-time PCR, and epidemiological data examined no significant genetic association between diseases or traits and two CNVRs. After screening the GSH candidates by in silico approaches, we designed TALEN pairs to integrate EGFP expression cassette into human cell lines in order to confirm the functionality of GSH candidates in an in vitro setting. As a result, transgene insertion into one of the two loci using TALEN showed robust transgene expression comparable to that with an AAVS1 site without significantly perturbing neighboring genes. Changing the promoter or cell type did not noticeably disturb this trend. Thus, we could validate two CNVRs as a site for effective and safe transgene insertion in human cells.

Generation of Neural Crest Progenitors from Human Pluripotent Stem Cells.

There are a vast range of diseases and disorders that are neurocristopathic in origin, including Hirschsprung's disease, pheochromocytoma, familial dysautonomia, craniofacial disorders, and melanomas. Having a source of human neural crest cells is highly valuable for investigating potential treatments for such diseases. This chapter describes a robust and well-characterized protocol for deriving neural crest from human pluripotent stem cells (hPSCs), which can then be differentiated to neuronal and non-neuronal lineages. The protocol is adapted to suit hPSC maintenance as a monolayer bulk culture or as manual-passaged colonies, which makes it widely applicable to researchers that may use different systems for hPSC maintenance.

Stochastic clonal expansion of "superstars" enhances the reserve capacity of enteric nervous system precursor cells.

We quantified cell population increase in the quail embryo enteric nervous system (ENS) from E2.5 (about 1500 cells) to E12 (about 8 million cells). We then probed ENS proliferative capacity by grafting to the chorio-allantoic membrane large (600 cells) and small (40 cells) populations of enteric neural crest (ENC) cells with aneural gut. This demonstrated that ENC cells show an extremely high capacity to regulate their proliferation while forming the ENS. Previous mathematical models and clonal label experiments revealed that a few dominant ENS "superstar" cell clones emerge but most clones are small. The model implied that "superstars" arise stochastically, but the same outcome could arise if "superstars" were pre-determined. We investigated these two modes mathematically and by grafting experiments with large and small numbers of ENCs, each including one EGFP-labelled ENC cell. The stochastic model predicts that the frequency of "superstar" detection increases as the ENC population decreases, the pre-determined model does not. Experimentally, as predicted by the stochastic model, the frequency of "superstar" detection increased with small ENC cell number. We conclude that ENS "superstar" clones achieve this status stochastically. Clonal dominance implies that clonal diversity is greatly reduced and in this case, somatic mutations may affect the phenotype. We suggest that somatic mutations coupled with loss of clonal diversity may contribute to variable penetrance and expressivity in individuals with genetically identical ENS pathologies.

Generation of Adrenal Chromaffin-like Cells from Human Pluripotent Stem Cells.

Adrenomedullary chromaffin cells are catecholamine (CA)-producing cells originating from trunk neural crest (NC) via sympathoadrenal progenitors (SAPs). We generated NC and SAPs from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in vitro via BMP2/FGF2 exposure, ascertained by qPCR and immunoexpression of SOX10, ASCL1, TFAP2α, and PHOX2B, and by fluorescence-activated cell sorting selection for p75NTR and GD2, and confirmed their trunk-like HOX gene expression. We showed that continuing BMP4 and curtailing FGF2 in vitro, augmented with corticosteroid mimetic, induced these cells to upregulate the chromaffin cell-specific marker PNMT and other CA synthesis and storage markers, and we demonstrated noradrenaline and adrenaline by Faglu and high-performance liquid chromatography. We showed these human cells' SAP-like property of migration and differentiation into cells expressing chromaffin cell markers by implanting them into avian embryos in vivo and in chorio-allantoic membrane grafts. These cells have the potential for investigating differentiation of human chromaffin cells and for modeling diseases involving this cell type.

CRISPR/Cas9 Targets Chicken Embryonic Somatic Cells In Vitro and In Vivo and generates Phenotypic Abnormalities.

Chickens are an invaluable model for studying human diseases, physiology and especially development, but have lagged in genetic applications. With the advent of Programmable Engineered Nucleases, genetic manipulation has become efficient, specific and rapid. Here, we show that the CRISPR/Cas9 system can precisely edit the chicken genome. We generated HIRA, TYRP1, DICER, MBD3, EZH2, and 6 other gene knockouts in two chicken cell lines using the CRISPR/Cas9 system, with no off-target effects detected. We also showed that very large deletions (>75 kb) could be achieved. We also achieved targeted modification by homology-directed repair (HDR), producing MEN2A and MEN2B mutations of the RET gene. We also targeted DGCR8 in neural cells of the chicken embryo by in vivo electroporation. After FACS isolation of transfected cells, we observed appropriate sequence changes in DGCR8. Wholemount and frozen section antibody labelling showed reduction of DGCR8 levels in transfected cells. In addition, there was reduced expression levels of DGCR8-associated genes DROSHA, YPEL1 and NGN2. We also observed morphological differences in neural tissue and cardiac-related tissues of transfected embryos. These findings demonstrate that precisely targeted genetic manipulation of the genome using the CRISPR/Cas9 system can be extended to the highly adaptable in vivo chicken embryo model.