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. 2020 Apr 27;11:2041731420905701. doi: 10.1177/2041731420905701

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© The Author(s) 2020

This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License (https://creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access page (https://us.sagepub.com/en-us/nam/open-access-at-sage).

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Figure 3. — In vitro differentiation of human pluripotent stem cells into enteric neural crest cells. Detailed immunofluorescence staining analysis confirms successful derivation of ENCCs from hPSCs. Neurons (TUJ1+, a, h, i, j, and PGP9.5+, k) and glia (S100β+, b) appear in differentiated D15 ENCCs, but not in pre-differentiation D0 hPSCs. Epithelial marker ECAD is expressed in a small population of D15 neurospheres, but no specific epithelial cell lineage is identified in ECAD+ cells (MUC2-, e; CHGA-, f; LYSO-, g). Specific subtypes of neurons are not detected in D15 cells (CALB-, h; CHAT-, i, NOS1-, j; 5-HT-, k). Markers for mesenchymal cell lineage, CKIT (c) and SMA (d), are absent in ENCCs. Pluripotency marker OCT4 (m) is not expressed in D15 neurospheres. PCNA+ indicates cell proliferation (l) and LAMIN+ (n) marks the human origin of engraft cells. DAPI (blue) stains cell nuclei in all images. TUJ1: class III beta-tubulin; S100β: calcium binding protein B of the S100 family; CKIT: ICC-selective receptor tyrosine kinase; SMA: alpha-smooth muscle actin; ECAD: E-cadherin; MUC2: mucin 2; CHGA: chromagranin A; LYSO: lysozyme; CALB: calbindin; CHAT: choline acetyltransferase; NOS1: neuronal nitric oxide synthase; 5-HT: serotonin/5-hydroxytryptamine; PGP9.5: protein gene product 9.5; PCNA: proliferating cell nuclear antigen; OCT4: octamer-binding transcription factor 4; LAMIN: human-specific nuclear envelope protein lamin. D0: day 0 (pre-differentiation); D15: day 15 (post-differentiation); hPSC: human pluripotent stem cell; ENCC: enteric neural crest cell; ICC: interstitial cells of Cajal.