Protocol detail

Pluripotent Stem Cell Differentiation to Hepatic and Pancreatic Endoderm

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hESCs (H9 from WiCell, Maddison, WI, USA) and hIPSCs (BBHX8, A1ATD-1, JRO1D; University of Cambridge, Cambridge, UK) [16 (link)] were passaged weekly using collagenase IV and maintained in chemically defined medium (CDM) supplemented with activin A (10 ng/ml) and FGF2 (12 ng/ml), as described previously [17 (link)]. Differentiation was carried out as described in Fig. 1 and is explained in detail in the electronic supplementary material (ESM) Methods.Fig. 1

Protocols to generate hepatic and pancreatic endoderm from hESCs and hIPSCs. Successive culture conditions driving differentiation of pluripotent stem cells towards pancreatic endoderm and hepatic endoderm. A, activin; ADV, advanced DMEM; B, BMP; CMRL, Connaught Medical Research Laboratories medium; CYCP, cytochrome P450; DAPT, N-(N-[3,5-diflurophenylacetyl]-l-alanyl)-S-phenylglycine t-butyl ester; F, FGF; HGF, hepatocyte growth factor; Ly, LY294002; Nog, noggin; OSM, oncostatin M

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Cho C.H., Hannan N.R., Docherty F.M., Docherty H.M., Joåo Lima M., Trotter M.W., Docherty K, & Vallier L. (2012). Inhibition of activin/nodal signalling is necessary for pancreatic differentiation of human pluripotent stem cells. Diabetologia, 55(12), 3284-3295.

Publication 2012

A1atd Activin Activin a Collagenase Cytochrome p450 Dapt Endoderm Ester Fgf2 Hepatocyte growth factor Hescs Hipscs Ly294002 Noggin Oncostatin m Pancreatic Pluripotent stem cells

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Variable analysis

independent variables

Culture conditions driving differentiation of pluripotent stem cells towards pancreatic endoderm and hepatic endoderm
Passage of hESCs (H9) and hIPSCs (BBHX8, A1ATD-1, JRO1D) using collagenase IV
Maintenance of hESCs and hIPSCs in chemically defined medium (CDM) supplemented with activin A (10 ng/ml) and FGF2 (12 ng/ml)

dependent variables

Differentiation of pluripotent stem cells towards pancreatic endoderm and hepatic endoderm

control variables

Passage of hESCs and hIPSCs weekly
Culture conditions described previously [17 (link)]

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