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Patient iPSCs with NEUROG3 mutation reveal pancreatic insufficiency

ABSTRACT: Patient iPSCs with NEUROG3 mutation reveal pancreatic insufficiency

PROVIDER: PRJNA735124 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR14730277_1.fastq.gz	Fastqsanger.gz
	SRR14730277_2.fastq.gz	Fastqsanger.gz
	SRR14730278_1.fastq.gz	Fastqsanger.gz
	SRR14730278_2.fastq.gz	Fastqsanger.gz
	SRR14730279_1.fastq.gz	Fastqsanger.gz

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Patient iPSCs with NEUROG3 mutation reveal pancreatic insufficiency

Project description:Patients with NEUROGENIN3 mutations have enteric endocrinopathy and diabetes mellitus. We generated pluripotent stem cells from a patient’s fibroblasts to investigate if gene editing restores endocrine differentiation. Corrected cell lines differentiated into all pancreatic lineages while native cell lines failed to activate pancreatic progenitor and lineage determination genes, suggesting that the mutation disrupts pancreatic organogenesis and results in endocrine and exocrine dysfunction. Clinical testing revealed the patient has exocrine pancreatic insufficiency. These results expose a novel role for NEUROGENIN3 in human pancreatic differentiation and illustrates how patient-specific stem cells can be used to interrogate disease etiology and affect patient care.

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Project description:Analysis of FACS-sorted intestinal Neurog3+/+ and Neurog3+/- cells from Neurog3 fluorencent reporter mice carrying two-(+/+) or one- (+/-) Neurog3 alleles. In the intestine, Neurog3 is an enteroendocrine (EE) lineage determinating transcription factor that transiently expressed in early EEC progenitors. By comparison the molecular profiles of Neurog3+/+ and Neurog3+/- cells, we hypothesized that Neurog3 gene dosage regulates the allocation of EE progenitor fates towards EEC vs. goblet cells.

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Genome wide profiling of NEUROG3 occupancy in human pancreatic endocrine progenitors [CUT&RUN]

Project description:Despite this critical role in islet cell development, the precise function and downstream genetic programs regulated directedly by NEUROG3 remain elusive. We therefore mapped genome-wide NEUROG3 occupancy in human induced pluripotent stem cell (iPSC)-derived endocrine progenitors and determined NEUROG3 dependency of associated genes to uncover direct targets. To this aim, we generated a novel hiPSC line (NEUROG3-HA-P2A-Venus), where NEUROG3 is HA-tagged and fused to a self-cleaving fluorescent VENUS reporter. We used the CUT&RUN technique, an alternative method to CHIP-seq allowing transcription factor profiling from a low cell number, to map NEUROG3 occupancy and epigenetic marks in pancreatic endocrine progenitors (PEP) differentiated from this hiPSC line. To optimize the stringency and relevance of NEUROG3 binding sites, we focused our analysis on regions identified both with HA and NEUROG3 antibodies and integrated NEUROG3 occupancy data with chromatin status and gene expression in PEPs and their NEUROG3-dependence. Mapping of NEUROG3 genome occupancy in PEPs uncovers an unexpectedly broad, direct control of the endocrine gene regulatory network (GRN) and raises novel hypotheses on how this master regulator controls islet and beta cell differentiation.

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Genes differentially expressed between NEUROG3-Venus positive and negative hiPSC line differentiated to pancreatic endocrine progenitors [RNA-Seq]

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RNA-seq of Human Embryonic Stem Cell derived pancreas progenitor differentiation, Day 13 of wildtype, HES1-/-, NEUROG3-/- and HES1-/-NEUROG3-/- genotypes

Project description:To better understand the mechanism by which HES1 regulates pancreas development we took advantage of recent developments in directed differentiation of human embryonic stem cells (hESCs) to the pancreatic endocrine lineage via a series of progenitor stages (Figure 1 and (Rezania et al., 2014). Using the iCRISPR platform (González et al., 2014), we introduced indels in the HES1 and NEUROG3 genes, either singly or in combination in iCRISPR H1 cells. Using previously described gRNAs (Zhu et al., 2016) to target exon2 of the NEUROG3 gene and exon2 of the HES1 gene we detected by Sanger and RNA-sequencing a 2 bp insertion and a T insertion, respectively, resulting in premature STOP codons in two/multiple clonal cell lines carrying the introduced mutation and the loss of NEUROG3 by immunostaining We then subjected two or more clonal lines of H1-iCRISPR (wildtype), HES1−/−, NEUROG3−/− and HES1−/−NEUROG3−/− (abbreviated H1N3-dKO or H1−/−N3−/−) to differentiation to β-like cells using a modified version of the protocol from Rezania et al. (2014). Marker analysis showed that all cell lines maintained pluripotency (OCT4+) as hESC and were able to differentiate to FOXA2+ and SOX17+ co-positive definitive endoderm (DE, Day 3), PDX1+ cells at the posterior foregut stage (PF, Day 7), to bipotent progenitors marked by PDX1+ and NKX6-1+ at the Pancreatic Endoderm stage (PE, Day 10), and the Endocrine Precursor stage (EP, Day 13)

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