title: Interspecific in vitro assay for the chimera-forming ability of human pluripotent stem cells.
abstract: Functional assay limitations are an emerging issue in characterizing human pluripotent stem cells (PSCs). With rodent PSCs, chimera formation using pre-implantation embryos is the gold-standard assay of pluripotency (competence of progeny to differentiate into all three germ layers). In human PSCs (hPSCs), however, this can only be monitored via teratoma formation or in vitro differentiation, as ethical concerns preclude generation of human-human or human-animal chimeras. To circumvent this issue, we developed a functional assay utilizing interspecific blastocyst injection and in vitro culture (interspecies in vitro chimera assay) that enables the development and observation of embryos up to headfold stage. The assay uses mouse pre-implantation embryos and rat, monkey and human PSCs to create interspecies chimeras cultured in vitro to the early egg-cylinder stage. Intra- and interspecific chimera assays with rodent PSC lines were performed to confirm the consistency of results in vitro and in vivo. The behavior of chimeras developed in vitro appeared to recapitulate that of chimeras developed in vivo; that is, PSC-derived cells survived and were integrated into the epiblast of egg-cylinder-stage embryos. This indicates that the interspecific in vitro chimera assay is useful in evaluating the chimera-forming ability of rodent PSCs. However, when human induced PSCs (both conventional and naïve-like types) were injected into mouse embryos and cultured, some human cells survived but were segregated; unlike epiblast-stage rodent PSCs, they never integrated into the epiblast of egg-cylinder-stage embryos. These data suggest that the mouse-human interspecies in vitro chimera assay does not accurately reflect the early developmental potential/process of hPSCs. The use of evolutionarily more closely related species as host embryos might be necessary to evaluate the developmental potency of hPSCs.
authors.affiliation: ERATO Nakauchi Stem Cell and Organ Regeneration Project, Japan Technology and Science Agency, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan hmasaki@ims.u-tokyo.ac.jp nakauchi@ims.u-tokyo.ac.jp.
authors.forename: Hideki
authors.initials: H
authors.lastname: Masaki
authors.affiliation: ERATO Nakauchi Stem Cell and Organ Regeneration Project, Japan Technology and Science Agency, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
authors.forename: Megumi
authors.initials: M
authors.lastname: Kato-Itoh
authors.affiliation: ERATO Nakauchi Stem Cell and Organ Regeneration Project, Japan Technology and Science Agency, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
authors.forename: Ayumi
authors.initials: A
authors.lastname: Umino
authors.affiliation: ERATO Nakauchi Stem Cell and Organ Regeneration Project, Japan Technology and Science Agency, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Sato
authors.affiliation: ERATO Nakauchi Stem Cell and Organ Regeneration Project, Japan Technology and Science Agency, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
authors.forename: Sanae
authors.initials: S
authors.lastname: Hamanaka
authors.affiliation: ERATO Nakauchi Stem Cell and Organ Regeneration Project, Japan Technology and Science Agency, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Kobayashi
authors.affiliation: ERATO Nakauchi Stem Cell and Organ Regeneration Project, Japan Technology and Science Agency, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Yamaguchi
authors.affiliation: Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-11 Higashi, Central 4, Tsukuba, Ibaraki 305-8562, Japan.
authors.forename: Ken
authors.initials: K
authors.lastname: Nishimura
authors.affiliation: Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-11 Higashi, Central 4, Tsukuba, Ibaraki 305-8562, Japan.
authors.forename: Manami
authors.initials: M
authors.lastname: Ohtaka
authors.affiliation: Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-11 Higashi, Central 4, Tsukuba, Ibaraki 305-8562, Japan.
authors.forename: Mahito
authors.initials: M
authors.lastname: Nakanishi
authors.affiliation: ERATO Nakauchi Stem Cell and Organ Regeneration Project, Japan Technology and Science Agency, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, 265 Campus Drive, Stanford, CA 94305-5461, USA hmasaki@ims.u-tokyo.ac.jp nakauchi@ims.u-tokyo.ac.jp.
authors.forename: Hiromitsu
authors.initials: H
authors.lastname: Nakauchi
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D002678:Chimera / Q000196:embryology*; D047108:Embryonic Development / Q000502:physiology*; D000882:Haplorhini; D006801:Humans; D066298:In Vitro Techniques / Q000379:methods*; D051379:Mice; D046228:Microarray Analysis; D008845:Microinjections; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000502:physiology*; D051381:Rats; D013045:Species Specificity
keywords: Chimera; ESC; EpiSC; Pluripotency; iPSC