Long-term extensive expansion of mouse hepatic stem/progenitor cells in a novel serum-free culture system

Atsunori Tsuchiya, Toshio Heike, Hisanori Fujino, Mitsutaka Shiota, Katsutsugu Umeda, Momoko Yoshimoto, Yasunobu Matsuda, Takafumi Ichida, Yutaka Aoyagi, Tatsutoshi Nakahata

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Abstract

Background & Aims: The liver has high regenerative potential. We attempted to establish a novel culture system for extensive expansion of fetal mouse hepatic stem/progenitor cells and to characterize cultured cells. Methods: Hepatic spheroids collected from 6-day floating cultures were cultured on collagen-coated dishes in serum-free conditions in medium containing growth factors. Cultured cells were mainly characterized by immunocytochemistry and flow cytometry or transplanted into adult mice. Results: Approximately 400 expanding hepatic spheroids were generated from every 1 × 106 fetal liver cells. Subsequently, highly replicative colonies were subcultured with maintaining colony formation on collagen-coated dishes. These colonies consisted of small immature α-fetoprotein-positive cells and hepatocytic and cholangiocytic lineage-committed cells. The immature α-fetoprotein- positive cells could be expanded in a reproducible manner at least 5 × 105-fold (which involved at least 30 passages over >6 months) without losing differentiation potential. Flow cytometric analysis showed that all cultured cells expressed CD49f, but not CD34, Thy-1, c-kit, or CD45. Nearly 15% of the cells expressed Sca-1, and approximately 5%-20% of the cells were side population cells. Both sorted side population cells and Sca-1-positive cells (especially side population cells) produced a large number of α-fetoprotein-positive cells and lineage-committed cells. Expanded cells had bidirectional differentiation potential and improved serum albumin levels in mice with severe liver damage. Conclusions: Long-term extensive expansion of transplantable hepatic stem/progenitor cells was reproducibly achieved in a novel serum-free culture system. Moreover, this culture system yielded side population and Sca-1-positive cell populations that included hepatic stem/progenitor cells with differentiation and proliferation properties.

Original languageEnglish (US)
Pages (from-to)2089-2104
Number of pages16
JournalGastroenterology
Volume128
Issue number7
DOIs
StatePublished - Jun 2005

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ASJC Scopus subject areas

  • Hepatology
  • Gastroenterology

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Long-term extensive expansion of mouse hepatic stem/progenitor cells in a novel serum-free culture system. / Tsuchiya, Atsunori; Heike, Toshio; Fujino, Hisanori; Shiota, Mitsutaka; Umeda, Katsutsugu; Yoshimoto, Momoko; Matsuda, Yasunobu; Ichida, Takafumi; Aoyagi, Yutaka; Nakahata, Tatsutoshi.

In: Gastroenterology, Vol. 128, No. 7, 06.2005, p. 2089-2104.

Research output: Contribution to journalArticle