iMuSCs, #7

Results(part 4)
To clarify the pluripotent potential of the iMuSCs, we performed differentiation assays6,7 in vitro that showed that the iMuSCs were able to form embryoid bodies (EBs) in a petri dish (Fig. 3d,e). After seven days in suspension culture, EBs were expanded and initiated spontaneous differentiation into a variety of ectodermal and mesodermal germ layer derivatives, and after an additional two weeks in culture, attached EBs formed contracting multinucleated myotubes encompassed with neural-like structures (Fig. 3f,g). We further examined the pluripotency of the iMuSCs by teratoma formation in vivo. When grafted into SCID-beige mice (Jackson Lab, USA) for seven weeks, the iMuSCs formed teratomas (90%, n = 7) containing representative tissues of the three germ layers (Fig. 4a). Histological examination revealed that the iMuSCs differentiated into neural, muscle, and adipose tissues, and epithelium. To verify that the teratomas were formed directly from the implanted cells, the iMuSCs were pre-labelled with β-gal before injection, we detected all three germ layer derivatives in the teratomas contained the β-gal+ cells when stained with LacZ (Fig. 4b).

To evaluate whether the iMuSCs could give rise to chimeric mice, a blastocyst injection assay was performed (Fig. 4c). We transferred undifferentiated β-gal+ and GFP-pre-labelled iMuSCs as single cells into BALB/c (Jackson Lab, USA) blastocysts by microinjection following standard procedures8. We obtained eight embryos at E14, six of which developed properly and demonstrated the contribution of GFP+ iMuSCs to the embryo. A high-to-moderate contribution of β-gal and GFP-expressing cells could be seen in these E14 chimeric embryos (Fig. 4c,d and Supplementary Fig. S4a). Histological analysis confirmed that the iMuSCs contributed to all three germ layers (Fig. 4e and Supplementary Fig. S4b). Offspring derived from the iMuSCs-injected blastocysts were born and developed normally. After repeating this experiment 3 times, we obtained 23 pups, all born with a white coat (Supplementary Table S1). Although their hair did not display iMuSCs germline transmission, immunostaining and qPCR analysis revealed the presence of LacZ+ and GFP+ iMuSCs in several tissues of the pups, such as skin, muscle, heart, lung, kidneys, spleen, and brain (Fig. 4f and Supplementary Fig. S4c).

Figure 4: Skeletal muscle injury induced iMuSCs fulfil several in vivo criteria of pluripotency.
iMuSCsの多能性をさらに検討するために、我々はin vivoでの奇形腫形成能を調べた。SCID-ベージュマウス(Jackson Lab, USAより購入)に移植後7週間で、iMuSCsは、90%の確率で奇形腫を形成した(n = 7)。奇形腫は3胚葉の典型的な組織を含んでいた(図4a)。組織学的検索により、iMuSCsは神経組織、筋組織、脂肪織、上皮に分化することが明らかとなった。移植された組織から直接、奇形腫が形成されることを検証するために、iMuSCsをβ-galで予めラベルして置き、静注した。 LacZで染色すると、β-gal陽性細胞を含む奇形腫には3胚葉誘導体が検出された(図4b)。

iMuSCsがキメラマウスになりうるのかどうかを評価するために、次に我々は、胚盤胞注入試験を行なった(図4c)。一般的なマイクロインジェクション手技に従い、 BALB/c(Jackson Lab, USAより購入)の胚盤胞の中に単一細胞として、未分化β-gal陽性のiMuSCsとGFP標識iMuSCsを移注した。E14個体中8体の胎児を得た。8個体のうち6体は正常に発育し、その胎児にGFP陽性iMuSCsの分布を確認できた。β-gal及びGFP-陽性細胞の発現頻度は、高頻度ないし中等度であった(図4c及びd、補足図S4a)。組織学的検討により、iMuSCsは3胚葉すべてに分布していることが分かった(図4e及び図S4b)。iMuSCsを注入して作成した胚盤胞から誘導されたマウスが生まれ、正常に発育した。3回同様の実験を行ない、我々は23匹の子マウスを得、すべて白い体毛に覆われていた(補足テーブルS1)。子マウスの体毛は iMuSCsの生殖細胞系の移入を示さなかったが、免疫染色及び定量リアルタイムPCR(qPCR)による解析から、LacZ陽性及びGFP陽性のiMuSCsは、子マウスの皮膚、筋、心臓、肺、腎臓、脾臓そして脳といったいくつかの組織において存在していることが明らかとなった(図S4c)。