(続き)
After culturing in ACTH medium for 7 days, this growing population of cells, unlike parental STAP cells, could be passaged as single cells (Fig. 5a, bottom, and Fig. 5b), grow in 2i medium (Extended Data Fig. 8a) and expand exponentially, up to at least 120 days of culture (Fig. 5c; no substantial chromosomal abnormality was seen; Extended Data Fig. 8b, c). Hereafter, we refer to the proliferative cells derived from STAP cells as STAP stem cells.
(続き)
STAP stem cells expressed protein and RNA markers for pluripotent cells (Fig. 5d, e), showed low DNA methylation levels at the Oct4 and Nanog loci (Extended Data Fig. 8d), and had a nuclear fine structure similar to that of ES cells (Extended Data Fig. 8e; few electron-dense areas corresponding to heterochromatin). In differentiation culture25, 26, 27, STAP stem cells generated ectodermal, mesodermal and endodermal derivatives in vitro (Fig. 5f–h and Extended Data Fig. 8f, g), including beating cardiac muscles (Supplementary Video 4), and formed teratomas in vivo (Fig.5i and Extended Data Fig. 8h; no teratocarcinomas, n = 40). After blastocyst injection, STAP stem cells efficiently contributed to chimaeric mice (Fig. 5j), in which germline transmission was seen (Extended Data Fig. 8i). Even in tetraploid complementation assays, injected STAP stem cells could generate mice capable of growing to adults and producing offspring (Fig. 5k, l; in all eight independent lines, Extended Data Fig. 8j).
(続き)
In addition to their expandability, we noticed at least two other differences between STAP stem cells and parental STAP cells. First, the expression of the ES cell marker protein Esrrβ, which was undetectable in STAP cells (Extended Data Fig. 5d, e), was clearly seen in STAP stem cells (Fig. 5e). Second, the presence of H3K27me3 foci, which was found in a substantial proportion of female STAP cells, was no longer observed in STAP stem cells (Extended Data Figs 5f and 8k). Thus, STAP cells have the potential to give rise to expandable cell lines that exhibit features similar to those of ES cells.
マテメソは以下だね。
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STAP stem-cell conversion culture
For establishment of STAP stem-cell lines, STAP cell clusters were transferred to ACTH-containing medium36 on MEF feeder cells (several clusters, up to a dozen clusters, per well of 96-well plates). Four to seven days later, the cells were subjected to the first passage using a conventional trypsin method, and suspended cells were plated in ES maintain medium containing 20% FBS. Subsequent passaging was performed at a split ratio of 1:10 every second day before they reached subconfluency. We tested the following three different genetic backgrounds of mice for STAP stem-cell establishment from STAP cell clusters, and observed reproducible data of establishment: C57BL/6 carrying Oct4-gfp (29 of 29), 129/Sv carrying Rosa26-gfp (2 of 2) and 129/Sv × C57BL/6 carrying cag-gfp (12 of 16). STAP stem cells with all these genetic backgrounds showed chimaera-forming activity.
For clonal analysis of STAP stem cells, single STAP stem cells were manually picked by a thin-glass pipette, and plated into 96-well plates at one cell per well. The clonal colonies were cultured in ES medium containing 20% FBS, and expanded for subsequent experiments.
Article Figure 6とExtended Data Figure 8のリジェンドは以下だ。
(Article Figure 6)
a, Growth of STAP stem cells carrying Oct4-gfp. Scale bar, 50 μm. b, Dissociation culture of STAP stem cells to form colonies. Scale bar, 100 μm. c, Robust growth of STAP stem cells in maintenance culture. Similar results were obtained with eight independent lines. In contrast, parental STAP cells decreased in number quickly. d, Immunostaining of STAP stem cells for pluripotency markers (red). Scale bar, 50 μm. e, qPCR analysis of pluripotency marker gene expression. f–h, In vitro differentiation assays into three-germ-layer derivatives.
f, Ectoderm: Rx+/Pax6+ (retinal epithelium; 83%, n = 6). g, Mesoderm: troponin-T+ (cardiac muscle; 50%, n = 6). h, Endoderm: Sox17+/E-cadherin+ (endodermal progenitors; 67%, n = 6). Scale bar, 50 μm. i, Teratoma formation assays. Formation of keratinized epidermis (ectoderm; left), cartilage (mesoderm; middle) and bronchial-like epithelium (endoderm; right) is shown. Scale bar, 100 μm. j, Blastocyst injection assays. These pictures of live animals were taken serially (asterisk indicates the same chimaeric pup). k, l, Tetraploid complementation assay. ‘All-GFP+’ pups were born (k) and germline transmission was observed (l).
(Extended Data Figure 8)
a, Compatibility of 2i conditions with STAP stem-cell derivation from STAP cells and STAP stem-cell maintenance. STAP stem cells could not be established directly from STAP cells in 2i + LIF medium (top). However, once established in ACTH medium, STAP stem cells were able to survive and expand in 2i + LIF medium. Scale bar, 100 μm. b, Q-band analysis (n = 4; all cell lines showed the normal karyotype). c, Multicolour FISH analysis (n = 8; all cell lines showed the normal karyotype) of STAP stem cells. d, Methylation status of the Oct4 and Nanog promoters. e, Electron microscope analysis of STAP stem cells. Scale bar, 1 μm. f, g, Beating cardiac muscle (mesoderm; 38%, n = 8). Red line indicates an analysed region for kymograph (g).
h, Clonability of STAP stem cells. Clonal expansion from single STAP stem cells was performed. Pluripotency of clonal cell lines was confirmed by teratoma formation assay, showing the formation of neuroectoderm (left), muscle tissue (middle) and bronchial-like epithelium (right). Scale bar, 100 μm. i, Production of chimaeric mice from STAP stem-cell lines using diploid embryos. *These STAP stem-cell lines were generated from independent STAP cell clusters. j, Production of mouse chimaeras from STAP stem-cell lines by the tetraploid complementation method. *These STAP stem-cell lines were generated from independent STAP cell clusters. k, No H3K27me3-dense foci are seen in female STAP stem cells (n = 50; the CD45+ cell is a positive control). Scale bar, 10 μm.
これだけのものの移管が行われていて、その後処理として小保方さんが論文同士の整合を取ってるんでしょうね。笹井さんはそこまでは忙しすぎてやってないんじゃないかしら。STAP cells have a limited self-renewal capacity under the conditions used for establishment (Fig. 2g and Extended Data Figs 2e and 5a). のExtended Data Figs 2e ってTCRの説明よね。そこ全部変ね。
(続き)小保方さんは論文では主にリンパ球を使ってSTAP細胞を作っています。TCR再構成の実験をするときだけはCD90でもう一度T細胞だけをFACS選別します。でも厳格な選別ではないから、丹羽さんがヘテロな集団だと言ってるんですね。
マウスの白血球のもともとの構成割合はB細胞:60%、T細胞:30%、マクロファージ:5%、その他、5%です。脾臓の構成細胞全体での細胞の大きさは5から10マイクロメーターです。
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David Aphkhazava
23.19
Size of mouse splenocytes?
Does anyone know what the size of a mouse splenocyte is?
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Joseph michael Cantor
University of California, San Diego
5-10 um on average, with resting B and T cells comprising the lower end of the range and macrophages on the upper end. Adult mouse spleens typically contain 1x10e8 white blood cells: 50-60% B cells, 30% T cells, 5% macrophages, and <5% others.
[連投5]
参考
>while a small number aggregates gave rise to colonies containing small cells with large nuclei, resembling the morphology of embryonic stem cells. However, most of these cells ceased proliferation at day 7 and gradually regressed. <Investigation of the cellular reprogramming phenomenon referred to as stimulus-triggered acquisition of pluripotency (STAP) Hitoshi Niwa>
