Sin-Ting Lau1, Zhixin Li1, Frank Pui-Ling Lai1, Kathy Nga-Chu Lui2, Peng Li2, Jorge O Munera3, Guangjin Pan4, Maxime M Mahe5, Chi-Chung Hui6, James M Wells3, Elly Sau-Wai Ngan7. 1. Department of Surgery, Li Ka Shing Faculty of Medicine, Pokfulam, Hong Kong; Dr Li Dak Sum Research Centre, University of Hong Kong, Pokfulam, Hong Kong. 2. Department of Surgery, Li Ka Shing Faculty of Medicine, Pokfulam, Hong Kong. 3. Division of Developmental Biology, Cincinnati Children's Hospital Research Foundation, Cincinnati, Ohio. 4. CAS Key Laboratory of Regenerative Biology, Guangzhou Institutes of Biomedicine and Health, China. 5. Division of Pediatric General and Thoracic Surgery, Cincinnati Children's Hospital Research Foundation, Cincinnati, Ohio; INSERM UMR 1235-TENS, INSERM, University of Nantes, Nantes, France. 6. Program in Developmental and Stem Cell Biology, Toronto, Ontario, Canada; The Hospital for Sick Children, and Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada. 7. Department of Surgery, Li Ka Shing Faculty of Medicine, Pokfulam, Hong Kong. Electronic address: engan@hku.hk.
Abstract
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.
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.
Authors: Alexander Xi Fu; Kathy Nga-Chu Lui; Clara Sze-Man Tang; Ray Kit Ng; Frank Pui-Ling Lai; Sin-Ting Lau; Zhixin Li; Maria-Mercè Garcia-Barcelo; Pak-Chung Sham; Paul Kwong-Hang Tam; Elly Sau-Wai Ngan; Kevin Y Yip Journal: Genome Res Date: 2020-09-18 Impact factor: 9.043
Authors: Viju Vijayan Pillai; Prasanthi P Koganti; Tiffany G Kei; Shailesh Gurung; W Ronald Butler; Vimal Selvaraj Journal: Biol Open Date: 2021-11-01 Impact factor: 2.422
Authors: Astgik Petrosyan; Filippo Montali; Andrea Peloso; Antonio Citro; Lori N Byers; Catherine La Pointe; Mara Suleiman; Alice Marchetti; Eoin P Mcneill; Allison L Speer; Wai Hoe Ng; Xi Ren; Benedetta Bussolati; Laura Perin; Paolo Di Nardo; Vincenzo Cardinale; Jerome Duisit; Alexandra Rose Monetti; John Richard Savino; Amish Asthana; Giuseppe Orlando Journal: Front Bioeng Biotechnol Date: 2022-09-28