Activation of Hedgehog Signaling Promotes Development of Mouse and Human Enteric Neural Crest Cells, Based on Single-Cell Transcriptome Analyses
| Autor: | James M. Wells, Zhixin Li, Guangjin Pan, Sin-Ting Lau, Maxime M. Mahe, Elly Sau-Wai Ngan, Kathy Nga-Chu Lui, Jorge O. Múnera, Chi-chung Hui, Peng Li, Frank Pui-Ling Lai |
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| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Male Cyclopamine Induced Pluripotent Stem Cells Mice Transgenic Biology Enteric Nervous System Cell Line 03 medical and health sciences chemistry.chemical_compound Mice 0302 clinical medicine GLI2 Animals Humans Hedgehog Proteins Intestinal Mucosa Induced pluripotent stem cell Hedgehog Neurons Hepatology Sequence Analysis RNA Gene Expression Profiling Gastroenterology Neural crest Cell Differentiation Hedgehog signaling pathway Cell biology 030104 developmental biology chemistry Neural Crest 030211 gastroenterology & hepatology Enteric nervous system Single-Cell Analysis Smoothened Signal Transduction |
| Zdroj: | Gastroenterology. 157(6) |
| ISSN: | 1528-0012 |
| Popis: | Background & Aims It has been a challenge to develop fully functioning cells from human pluripotent stem cells (hPSCs). We investigated how activation of hedgehog signaling regulates derivation of enteric neural crest (NC) cells from hPSCs. Methods We analyzed transcriptomes of mouse and hPSC-derived enteric NCs using single-cell RNA sequencing (scRNA-seq) to identify the changes in expression associated with lineage differentiation. Intestine tissues were collected from Tg(GBS-GFP), Sufuf/f; Wnt1–cre, Ptch1+/–, and Gli3Δ699/Δ699 mice and analyzed by flow cytometry and immunofluorescence for levels of messenger RNAs encoding factors in the hedgehog signaling pathway during differentiation of enteric NCs. Human NC cells (HNK-1+p75NTR+) were derived from IMR90 and UE02302 hPSC lines. hPSCs were incubated with a hedgehog agonist (smoothened agonist [SAG]) and antagonists (cyclopamine) and analyzed for differentiation. hPSC-based innervated colonic organoids were derived from these hPSC lines and analyzed by immunofluorescence and neuromuscular coupling assay for expression of neuronal subtype markers and assessment of the functional maturity of the hPSC-derived neurons, respectively. Results Single-cell RNA sequencing analysis showed that neural fate acquisition by human and mouse enteric NC cells requires reduced expression of NC- and cell cycle–specific genes and up-regulation of neuronal or glial lineage–specific genes. Activation of the hedgehog pathway was associated with progression of mouse enteric NCs to the more mature state along the neuronal and glial lineage differentiation trajectories. Activation of the hedgehog pathway promoted development of cultured hPSCs into NCs of greater neurogenic potential by activating expression of genes in the neurogenic lineage. The hedgehog agonist increased differentiation of hPSCs into cells of the neuronal lineage by up-regulating expression of GLI2 target genes, including INSM1, NHLH1, and various bHLH family members. The hedgehog agonist increased expression of late neuronal markers and neuronal activities in hPSC-derived neurons. Conclusions In enteric NCs from humans and mice, activation of hedgehog signaling promotes differentiation into neurons by promoting cell-state transition, expression of genes in the neurogenic lineage, and functional maturity of enteric neurons. |
| Databáze: | OpenAIRE |
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