STAP spherical coloniesを注入したと書いてあるだけです。 When the STAP conversion conditions (low pH) were applied to CD451 lymphocytes, most day-7 clusters that were large and contained more than a few dozen small cells were positive for Oct4 (although the expression level varied). この文章は一旦終わっています。これを入れたとは書いてありません。
続きの文章は以下です。 Therefore, we used only well-formed characteristic clusters (large ones) for this type of study and cut them by microknife to prepare donor cell clusters in a proper size for glass needle injection.
In vitro differentiation assays. For mesodermal differentiation assay, STAP cells were collected at 7 days, and Oct4-GFP-positive cells were collected by cell sorter and subjected to culture in DMEM supplemented with 20% FBS. Medium was exchanged every 3 days. After 7–14 days, muscle cells were stained with an anti-α smooth muscle actin antibody (DAKO). For neural lineage differentiation assay, STAP cells were collected at 7 days and subjected to SDIA or SFEBq culture. For SDIA culture, collected STAP cell clusters were plated on PA6 cell feeder as described previously26. For SFEBq culture, STAP cell clusters (one per well; non-cell-adhesive 96-well plate, PrimeSurface V-bottom, Sumitomo Bakelite) were plated and cultured in suspension as described previously. For endodermal differentiation, STAP cells were collected at 7 days and subjected to suspension culture with inducers in 96-well plates
Apoptosis analysis was performed with flow cytometry using Annexin-V (Biovision) and propidium iodide. Annexin-V analysis by FACS on day 14 showed that most Oct4-GFP1 cells were positive for this apoptotic marker; indeed, the number of surviving cells declined thereafter.
Soft agar assay. Sorted STAP cells (Oct4-GFP-strong or -dim) and control mouse ES cells (1,000 cells per well of 96-well plate) were plated into soft ager medium(0.4% agarose) in LIF-B27 medium. After 7 days of culture, cells were dissociated and their anchorage-independent growth was quantified by fluorescent measurement with the cytoselect 96-well cell transformation assay kit (Cell Biolabs) according to the manufacturer’s protocol.
In vivo differentiation assay. 1 ｘ 107 STAP cells were seeded onto a sheet composed of a non-woven mesh of polyglycolic acid fibres (3 3 3 3 1 mm; 200 mm in pore diameter), cultured for 24 h in DMEM 1 10% FBS, and implanted subcutaneously into the dorsal flanks of 4-week-old mice. In this experiment, to better support tumour formation from slow growing STAP cells by keeping cells in a locally dense manner, we implanted STAP cells with artificial scaffold made of polyglycolic acid fibres. Given the artificial nature of the material, we used NOD/ SCID mice as hosts, to avoid possible enhancement of post-graft inflammation caused by this scaffold even in syngenic mice. STAP stem cells were dissociated into single cells and cell suspension containing 1 x 107 cells was injected into the testis. Six weeks later, the implants were analysed using histochemical techniques.
＞「STAPキメラ・幹細胞作製の手技は論文には書かれていません。」 ← 書いてあります。Nature Aticle 4ページ右コラム下から次のページにかけて幹細胞作製手順が「an adrenocorticotropic hormone (ACTH)1LIF containing medium (hereafter called ACTH medium) supported outgrowth of STAP cell colonies…..Hereafter, we refer to the proliferative cells derived from STAP cells as STAP stem cells.」と、８頁左コラムChimaeric mouse generation and analyses.にキメラ作成手技が書いてあります。
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. ・・・・ 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.
ため息さんです。 ＞Nature Article Extended Data Figure 7 b に培養期間が７日の方が１０日よりキメラへの寄与が大きいというデータがあります。
培養期間というのがどういう期間をさしているのかがあいまいなんです。論文では、STAP細胞を作る時には、generationという言葉を使い、後は、cultureと使いわけているのではないか？と思います。Soft agar assay.は、このあたりで使用するのか？どう関係するのかな？ ため息さんには答えられないと思います。
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 Esrrb, 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.