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

Multiple endocrine neoplasia type 2B: modelling the disease in human cells and avian embryos

© 2017 Dr Kwaku Dad Abu-BonsrahPaediatric cancer initiation is difficult to study because the early stages are often prenatal. Patient cells at the time of detection already have complex genetic (including epigenetic) changes, and the cancer cell population is heterogeneous. One way to model cancer initiation at the organism level is to engineer the specific initiating mutation into the appropriate cell lineage of an experimental animal embryo. For the human cell context, an ideal cell model would start with the normal human cell of origin and create candidate initiating mutations. Multiple Endocrine Neoplasia type 2B (MEN2B) is an autosomal dominant complex oncologic disease of the neural crest (NC) cell lineage, a so-called neurocristopathy. It presents with i) multiple mucosal ganglioneuromas including hyperplasia of the enteric (gut) nervous system leading to gastrointestinal disorders, ii) pheochromocytoma, with sympathoadrenal (SA) hyperplasia and catecholamine disturbance, and iii) medullary thyroid carcinoma with C-cell hyperplasia, elevated calcitonin and calcium metabolism disturbance. In addition, patients have marfanoid facial features. MEN2B results from de novo germline gain-of-function mutations in the gene RET, most often M918T. RET codes for the signalling receptor for the growth factor ligand GDNF, hence in MEN2B cells, RET signalling is divorced from GDNF availability. MEN2B is rare but it is often misdiagnosed especially early in life. This is due to the nature and diversity of the lineages affected; SA and enteric NC-lineage cells and thyroid C-cells, the latter being of foregut endodermal entero-endocrine lineage. We were the first to successfully use CRISPR/Cas9 to mutate the developing chicken embryo in vivo, showing phenotypic abnormality. This included creating a single point mutation by homology directed repair in vivo in NC cells at the avian MEN2B homologous site (M910T). For the human cell context, we combined the CRISPR/Cas9 technology and knowledge of embryo development and cell differentiation to create MEN2B M918T cells using the human embryonic stem cell (hESC) lines H9, HES3 and MEL2. We modified a hESC differentiation protocol to produce enteric NC-like cells, showing in vitro that these cells upregulated key NC and enteric genes. Functionally we also showed higher proliferation and greater axon production in the MEN2B mutant cells: this is consistent with the ganglioneuroma phenotype. In addition, we developed a new differentiation protocol to produce human SA progenitors and medullary chromaffin-like cells, as marked by expression of key catecholamine genes TH and PNMT, and expression of adrenaline and noradrenaline by HPLC. These cells are affected in MEN2B patient pheochromocytoma. We then developed a novel differentiation protocol for thyroid C-cell-like cells from hESCs via Definitive Endodermal Cells. These cells produce Calcitonin and we validated their functionality by ELISA assay and compared the MEN2B clones with the control hESCs. These cells are affected in MEN2B patients resulting in medullary thyroid carcinoma

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

Summary: 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

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