title: Human induced hepatic lineage-oriented stem cells: autonomous specification of human iPS cells toward hepatocyte-like cells without any exogenous differentiation factors.
abstract: Preparing targeted cells for medical applications from human induced pluripotent stem cells (hiPSCs) using growth factors, compounds, or gene transfer has been challenging. Here, we report that human induced hepatic lineage-oriented stem cells (hiHSCs) were generated and expanded as a new type of hiPSC under non-typical coculture with feeder cells in a chemically defined hiPSC medium at a very high density. Self-renewing hiHSCs expressed markers of both human embryonic stem cells (hESCs) and hepatocytes. Those cells were highly expandable, markedly enhancing gene expression of serum hepatic proteins and cytochrome P450 enzymes with the omission of FGF-2 from an undefined hiPSC medium. The hepatic specification of hiHSCs was not attributable to the genetic and epigenetic backgrounds of the starting cells, as they were established from distinct donors and different types of cells. Approximately 90% of hiHSCs autonomously differentiated to hepatocyte-like cells, even in a defined minimum medium without any of the exogenous growth factors necessary for hepatic specification. After 12 days of this culture, the differentiated cells significantly enhanced gene expression of serum hepatic proteins (ALB, SERPINA1, TTR, TF, FABP1, FGG, AGT, RBP4, and AHSG), conjugating enzymes (UGT2B4, UGT2B7, UGT2B10, GSTA2, and GSTA5), transporters (SULT2A1, SLC13A5, and SLCO2B1), and urea cycle-related enzymes (ARG1 and CPS1). In addition, the hepatocyte-like cells performed key functions of urea synthesis, albumin secretion, glycogen storage, indocyanine green uptake, and low-density lipoprotein uptake. The autonomous hepatic specification of hiHSCs was due to their culture conditions (coculture with feeder cells in a defined hiPSC medium at a very high density) in self-renewal rather than in differentiation. These results suggest the feasibility of preparing large quantities of hepatocytes as a convenient and inexpensive hiPSC differentiation. Our study also suggests the necessity of optimizing culture conditions to generate other specific lineage-oriented hiPSCs, allowing for a very simple differentiation.
authors: Core Facilities for Research and Innovative Medicine, National Cancer Center Research Institute, Chuo-ku, Tokyo, Japan.
authors: Tetsuya
authors: T
authors: Ishikawa
authors: Core Facilities for Research and Innovative Medicine, National Cancer Center Research Institute, Chuo-ku, Tokyo, Japan.
authors: Momoko
authors: M
authors: Kobayashi
authors: Takara Bio, Inc., Otsu, Shiga, Japan.
authors: Satoshi
authors: S
authors: Yanagi
authors: Takara Bio, Inc., Otsu, Shiga, Japan.
authors: Chika
authors: C
authors: Kato
authors: Takara Bio, Inc., Otsu, Shiga, Japan.
authors: Ryokichi
authors: R
authors: Takashima
authors: Takara Bio, Inc., Otsu, Shiga, Japan.
authors: Eiji
authors: E
authors: Kobayashi
authors: Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Chuo-ku, Tokyo, Japan.
authors: Keitaro
authors: K
authors: Hagiwara
authors: Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Chuo-ku, Tokyo, Japan.
authors: Takahiro
authors: T
authors: Ochiya
mesh_terms: D000328:Adult; D000368:Aged; D015415:Biomarkers; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D016000:Cluster Analysis; D003470:Culture Media; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D005260:Female; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D005347:Fibroblasts; D015870:Gene Expression; D020869:Gene Expression Profiling; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D008297:Male; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism
chemical_list: D015415:Biomarkers; D003470:Culture Media; D016222:Fibroblast Growth Factor 2