Directed differentiation of human induced pluripotent stem cells into functional cholangiocyte-like cells - PubMed (original) (raw)

Directed differentiation of human induced pluripotent stem cells into functional cholangiocyte-like cells

Fotios Sampaziotis et al. Nat Protoc. 2017 Apr.

Abstract

The difficulty in isolating and propagating functional primary cholangiocytes is a major limitation in the study of biliary disorders and the testing of novel therapeutic agents. To overcome this problem, we have developed a platform for the differentiation of human pluripotent stem cells (hPSCs) into functional cholangiocyte-like cells (CLCs). We have previously reported that our 26-d protocol closely recapitulates key stages of biliary development, starting with the differentiation of hPSCs into endoderm and subsequently into foregut progenitor (FP) cells, followed by the generation of hepatoblasts (HBs), cholangiocyte progenitors (CPs) expressing early biliary markers and mature CLCs displaying cholangiocyte functionality. Compared with alternative protocols for biliary differentiation of hPSCs, our system does not require coculture with other cell types and relies on chemically defined conditions up to and including the generation of CPs. A complex extracellular matrix is used for the maturation of CLCs; therefore, experience in hPSC culture and 3D organoid systems may be necessary for optimal results. Finally, the capacity of our platform for generating large amounts of disease-specific functional cholangiocytes will have broad applications for cholangiopathies, in disease modeling and for screening of therapeutic compounds.

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Conflict of interest statement

Competing financial interests: LV is a founder and shareholder of DefiniGEN. The remaining authors have nothing to disclose.

Figures

Figure 1

Figure 1

Generation of Cholangiocyte-like Cells (CLCs) from human Pluripotent Stem Cells (hPSCs). (a) Schematic representation of the protocol for the generation of hPSC-derived CLCs. DE: Definitive Endoderm, FP: Foregut progenitors, HB: Hepatoblasts, CP: Cholangiocyte Progenitors; BMP, bone morphogenetic protein; Ly294002 is a phosphatidylinositol-3-OH kinase inhibitor; CDM, chemically defined medium; RPMI, Roswell Park Memorial Institute medium; SB, SB-431542; HGF, hepatocyte growth factor; RA, retinoic acid; EGF, epidermal growth factor; FGF, fibroblast growth factor. Schematic modified from . The procedure steps corresponding to each stage are noted for reference. (b) Light microscopy images of cells at key stages of CLC differentiation. Scale bars for hPSCs, DE, FPs, CPs: 500 μm; HBs: 100 μm; zoomed in images: 50μm. The procedure steps and day numbers corresponding to each image are noted for reference.

Figure 2

Figure 2

Derivation of CLCs from embryonic stem (ES) cells. IF analyses demonstrating the expression of key biliary markers (CK7, CK19) in a CLC organoid generated from ES cells (H9). Scale bars: 100 μm. See table 2 for a detailed list of the antibodies and concentrations used.

Figure 3

Figure 3

Immunofluorescence analyses demonstrating the expression of characteristic markers at key stages of CLC differentiation. Scale bars: 100 μm. See table 2 for a detailed list of the antibodies and concentrations used The method for staining CLC organoids is described in procedure steps 28-39.

Figure 4

Figure 4

Flow cytometry analyses demonstrating the expression of characteristic markers at key stages of CLC differentiation. CLC organoids were harvested as described in procedure steps 40-47. Cells were dissociated into single cells following incubation with TrypLE for 5 minutes at 37°C and fixed with 4% PFA for 20 minutes at 4°C. The cells were stained for IF as previously described, using the antibodies provided in table 2. A standard gating strategy was used demonstrated in Supplementary Fig 2. A minimum of 2x104 gated events were used for analysis. Post sort fractions are indicated in the quadrants of each graph. The average differentiation efficiency from hPSCs to CLCs across three lines (CK7+/Sox9+ organoids) was 77% (s.d. = 6.5%).

Figure 5

Figure 5

Functional properties of CLC organoids. (a) CLC organoids demonstrating characteristic ALP staining. Mouse embryonic feeders are used as a negative control. Scale bars: 100 μm. (b) GGT activity of CLC organoids measured in absorbance units (AU); n=3; Mouse Embryonic Feeders (MEFs) are used as a negative control. Error bars, standard deviation; individual data points are demonstrated; *P<0.05, two tailed student’s t-test; F-test used to compare variances, _P_=0.1218 (no significant difference in variance). GGT and ALP activity were assessed using commercially available kits (MaxDiscovery™ gamma-Glutamyl Transferase (GGT) Enzymatic Assay Kit and BCIP/NBT Color Development Substrate respectively) according to the manufacturer’s instructions.

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