Donor-dependent variations in hepatic differentiation from human-induced pluripotent stem cells

Abstract

Hepatocytes generated from human induced pluripotent stem cells (hiPSCs) are unprecedented resources for pharmaceuticals and cell therapy. However, the in vitro directed differentiation of human pluripotent stem cells into mature hepatocytes remains challenging. Little attention has so far been paid to variations among hiPSC lines in terms of their hepatic differentiation. In the current study, we developed an improved hepatic differentiation protocol and compared 28 hiPSC lines originated from various somatic cells and derived using retroviruses, Sendai viruses, or episomal plasmids. This comparison indicated that the origins, but not the derivation methods, may be a major determinant of variation in hepatic differentiation. The hiPSC clones derived from peripheral blood cells consistently showed good differentiation efficiency, whereas many hiPSC clones from adult dermal fibroblasts showed poor differentiation. However, when we compared hiPSCs from peripheral blood and dermal fibroblasts from the same individuals, we found that variations in hepatic differentiation were largely attributable to donor differences, rather than to the types of the original cells. These data underscore the importance of donor differences when comparing the differentiation propensities of hiPSC clones.

Fig. 1.

Hepatic differentiation of representative hiPS/ESC clones. (A) Schematic representation of the directed hepatic differentiation protocol used in this study. (B) Morphology of hepatic differentiated hiPS/ESCs on day 17. (Scale bar: 100 μm.) (C) Immunostaining analysis of hepatic differentiated hiPS/ESCs (day 10, HNF4A; day 17, AFP, A1AT, and ALBUMIN). (Scale bar: 100 μm.) Arrows indicate mature hepatic binuclear cells.

Fig. 2.

Diverse hepatic differentiation among hiPS/ESC clones. (A) Percentage of albumin-positive cells detected by a flow cytometric analysis after 17 d of hepatic differentiation. (B) Real-time PCR analysis of liver-related and undifferentiated gene expression in the hepatic differentiated hiPS/ESCs on day 17. The graph represents the fold expression of genes relative to KhES1 on day 17. ND, not determined. (C) Albumin secretion potential of the undifferentiated (day 0) and hepatic differentiated hiPS/ESCs on day 17 as determined by an ELISA. (D) Comparison of the ammonia clearance activity of the undifferentiated (day 0) and hepatic differentiated hiPS/ESCs on day 17. Error bars indicate the SD (n = 3).

Fig. 3.

Close comparison of the propensity for hepatic differentiation between sibling hiPSC lines 201B6 and 201B7. (A) Schematic representation of the modified protocol used for hepatic differentiation. In this protocol, hiPS/ESCs were enzymatically digested into single cells and plated on Matrigel-coated dishes. For endodermal cell induction, the cells were cultivated with activin A and Wnt3a for 7 d. A total of 0.5 mM sodium butyrate (NaB) was supplemented from day 1 for various durations (0–6 d). (B) Albumin secretion level after 21 d of hepatic differentiation. The error bars indicate the SD (n = 3). (C) Percentage of CXCR4-positive cells after 7 d of endodermal differentiation as determined by flow cytometry. Error bars indicate the SD (n = 3). (D) Immunostaining of SOX17 and OCT3/4 after 7 d of endodermal differentiation. NaB was added for 3 d. (Scale bar: 100 μm.)

Fig. 4.

Comparison of the propensities for endodermal and hepatic differentiation among various hiPS/ESC lines. (A) Percentage of CXCR4-positive cells on day 7 was determined by flow cytometric analysis. (B) Albumin secretion potential of the hepatic differentiated hiPS/ESCs on day 21 was determined by an ELISA. For each clone, data were obtained after different durations of NaB administration (0, 3, and 6 d during the first 7 d of endodermal differentiation; x axis). Error bars indicate the SD (n = 3). epi, episomal vector; retro, retrovirus vector; SeV, Sendai virus vector. : Data were not obtained due to significant cell death or poor cell growth. (C and D) Statistical analysis of the differences in the albumin secretion potential of hepatic differentiated hiPS/ESCs on day 21. For each clone, the highest level among the different NaB administration periods (0, 3, or 6 d) was used for the analysis. HiPSCs were grouped by the original cell types (C) and derivation methods (D).

Fig. 5.

Comparison of the propensities of hiPSCs from the same individuals for endodermal and hepatic differentiation. Two aHDF-iPSC and PB-iPSC clones derived from the same individual (two Parkinson disease patients, PD-1 and PD-2, and one adult healthy donor, donor91) were differentiated into the hepatic lineage. (A) Percentage of CXCR4-positive cells on day 7 as determined by flow cytometric analysis. (B) Albumin secretion of hepatic differentiated hiPSCs on day 21 was analyzed by an ELISA. For each clone, the data were obtained for different NaB administration periods (0, 3, and 6 d during the first 7 d of endodermal differentiation; x axis). The green and orange bars indicate aHDF-iPSCs and PB-iPSCs, respectively. Error bars indicate the SD (n = 3).