日本理化研究所公布STAP细胞的详细制作方法

2014年3月5日，日本理化研究所公开了新型万能细胞“STAP细胞”的详细制作方法。

此前很多科研人员表示，根据其论文独立制作STAP细胞，无法再现同样的结果，因此该研究所以此方式做出回应。

STAP 细胞相关论文由位于神户市的理化研究所发生和再生科学综合研究中心的小保方晴子研究组长等人在 1 月底发表于英国《自然》（Nature）杂志。

这一方法比诱导多能干细胞（iPS细胞）和胚胎干细胞（ES细胞）的制作方法简单，使用安全性也较高。此次采用的是小鼠，而若用人类细胞制作成功，该成果则有望应用到再生医疗领域。

把体细胞放进弱酸性溶液加以刺激的制作方法在全球尚属首次。制作出的细胞被命名为“STAP细胞”，分别取自“刺激触发采集多功能”（Stimulus Triggered Acquisition of Pluripotency）的头一字母。

理化研究所表示，本次公开的制作法中加入了许多上述论文中没有提到的详细方法，主要围绕询问较多的地方和容易出错的几点进行了解说。【原文下载】

附STAP细胞的详细制作方法全文

Essential technical tips for STAP cell conversion culture from somatic cells

Introduction

Stimulus-triggered acquisition of pluripotency (STAP) is a cellular reprogramming phenomenon that was recently reported in two papers (Obokata, Nature, 2014a,b). In this reprogramming process, upon strong external stimuli,  neonatal somatic cells are converted into cells that express pluripotency-related genes, such as Oct3/4, and acquire the ability to differentiate into derivatives of all three germ layers in vitro and in vivo. These cells, termed STAP cells, can contribute to chimeric fetuses after blastocyst injection. Moreover, in the blastocyst injection assay, injected STAP cells are  also found in extra-embryonic tissues, such as placenta.STAP cells derived from neonatal somatic cells are thus fully reprogrammed to as state of pluripotency. In the conditions for the establishment of STAP cells, theirproliferative capacity is quite limited, distinct from that of embryonic stem cells (ESCs). STAP cells can be further converted into two types of proliferative cell lines: STAP stem cells and FGF4-induced stem cells (FI stem cells). STAP stem cells, which are converted from STAP cells in ACTH-containing medium (see  Procedure),  lose  the
ability to contribute to extra-embryonic tissues. FI stem cells, which are generated from STAP cells in FGF4-containing medium, in contrast retain the capacity to contribute to both  embryonic and extra-embryonic  lineages  in blastocyst injection assay, although their embryonic contribution is relatively low. The STAP phenomenon induced by external stimuli, thus  potentially sheds new light on our understanding of pluripotency and differentiation in mammalian cells. This unforeseen phenomenon can be triggered in neonatal hematopoietic cells, for instance, by transient exposure to low-pH solution. Despite its seeming simplicity, this procedure requires special care in cell handling and culture conditions, as well as in the choice of  the starting cell population.  The  delivery of the optimal  level of sublethal stress to cells is essential to the process of STAP cell induction. From our experience, STAP conversion is reproducibly seen with culture  conditions  in which most cells survive for one day after low-pH treatment, and in which up to 80% of the initial cell number subsequently die at around days 2–3. Control of the pH of the solution is not the only key factor; the delayed onset of sublethal stress is also critically important. This biological context can also be affected by many other factors. For example, somatic cell preparation and cell handling before and after the exposure to stress must be done with care, as additional damage to the cells may alter the level of stress, causing excessive 3cell death or insufficient triggering. The types of cells used for STAP conversion are
also critical, and the use of cells from other sources (e.g., the use of cultured fibroblasts after passaging)  may also  result  a failure to achieve STAP  conversion. We  have reproducibly observed STAP cell conversion when proper procedures are followed in the correct sequence.To facilitate the broad testing and use of this technique, we are now preparing a full protocol article with step-by-step instructions. However,  as the preparation, submission and publication of a full manuscript takes a significant amount of time, we would like to share a number of technical tips for STAP cell conversion culture (and related experiments) in this Protocol Exchange. We hope that these technical tips may answer many questions frequently asked about the experimental details.Tissue collection and low-pH treatment

1. To isolate CD45+haematopoietic cells, spleens were excised from 1-week-old Oct4-gfp mice (unless specified otherwise), minced by scissors, and mechanically dissociated using a Pasteur pipette.
IMPORTANT
(i) Adherent cells should be dissociated into single cells, either mechanically or enzymatically (by trypsin or collagenase). For the tissues described in Fig. 3a (Obokata et al. Nature, 2014a), muscle, adipose tissue and fibroblasts
were enzymatically dissociated, whereas others were mechanically dissociated.
(ii)  Primary cells should be used.  We have found that it is difficult to reprogram mouse embryonic fibroblasts (MEF) that have been  expanded in
vitro, while fresh MEF are competent.
(iii) For the experiments reported, we used a Oct-3/4-EGFP transgenic mouse line (Ohbo et al, Dev Biol, 2003; Yoshimizu et al, Dev Growth Differ, 1999), which is maintained by the RIKEN Bioresource Center as GOF18-GFP line11 transgenic mouse (B6;B6D2-Tg(GOF18/EGFP)11/Rbrc). Homozygotes of the
transgene were used for the live imaging to obtain the enhanced signal.
(iv) Cells from mice older than one week showed very poor reprogramming efficiency under the current protocol. Cells from male animals showed higher
efficiency than those from female.

2. Dissociated spleen cells were suspended with PBS and strained through a cell strainer (BD Biosciences 352340).

3. After  centrifuging  at 1,000 rpm for 5 min, collected cells were re-suspended in DMEM medium and added to the same volume of lympholyte (Cedarlane),  and then centrifuged at 1,000 g for 20 min.

IMPORTANT
(i) The purity of the starting cells is important for achieving STAP conversion. For  lymphocytes, contamination  with  red blood cells may inhibit the reprogramming event.  When using adherent cells,  the presence of extracellular matrix may interfere with reprogramming.
(ii) Alternatively, red blood cells may be removed by suspension of the cell pellet in 1.8 ml of H2O (Sigma W3500). After 30 seconds, add 0.2 ml of 10×PBS (Gibco 70011-044), followed by 3 ml of 1× PBS (Gibco 10010-023), and strain the cell suspension through a cell strainer.

4. The lymphocyte layer was  isolated  and stained with CD45 antibody (Abcamab25603). CD45+cells were sorted by FACS Aria (BD Biosciences).

IMPORTANT
(i) FACS sorting can be an important step for the confirmation of cell purity, but can affect both cell viability and reprogramming efficiency. Skipping this step may increase reprograming  efficiency,  although this may result in a reduction in confidence in cell identity.

5. After cell sorting, 1 × 106CD45+cells were treated with 500 µl of low-pH HBSS solution (titrated to pH 5.7 by HCl) for 25 min at 37°C, and then centrifuged at 1,000 rpm at room temperature for 5 min.

IMPORTANT
(i) The buffering action of HBSS is weak, so carry-over of the solution may affect pH. Please adjust pH to 5.7 in cell suspension by the following method. First, suspend the cell pellet with 494 µl of HBSS pre-chilled at 4°C, then add
6 µl of diluted HCl (10 µl of 35% HCl in 590 µl of HBSS) to adjust to a final pH of 5.7. Please confirm the final pH in a pilot experiment, and optimize the volume of HCl added, as necessary. Alternatively, suspend the cell pellet in
HBSS-pH 5.4 pre-chilled at 4°C.
(ii) The HBSS we used is Ca2+/Mg2+ free (Gibco 14170-112).(iii) Incubate the cells suspended in HBSS in a CO2 incubator.
(iv) Cell viability is a critical parameter in this step. Under optimal conditions, massive cell death is observed at two days after plating, as shown in Figure 1d(Obokata et al. Nature, 2014a).
(v)  If you find massive cell death at one day after plating, it  may be ameliorated by shortening the incubation period with low-pH HBSS solution
to 15 min.

6. After the supernatant (low-pH solution) was removed, precipitated cells were re-suspended and plated onto non-adhesive culture plates (typically, 1×105cells/ml) in DMEM/F12 medium supplemented with 1,000 U LIF (Sigma) and 2% B27 (Invitrogen).

IMPORTANT
(i) The use of non-adhesive culture plates is recommended, as the formation of cell clusters is  an  important step  for reprogramming, and the adhesive surface may inhibit cell movement needed to form clusters.
(ii) Cell density is critical, and depends on cell viability. Density should be maintained at 1×105~1×106cells per cm2of culture surface.
(iii) B27 (Invitrogen 17504-044) may show variation between batches. Please
check the quality by N2B27-2iLIF culture of ES cells.

7. Cell cluster formation was more sensitive to plating cell density than  to  the percentage of Oct3/4-GFP+cells. The number of surviving cells was sensitive to the age of donor mice, and was low under the treatment conditions described above when adult spleens were used.

IMPORTANT
(i)  The donor mouse should be 1-week old or younger.  Reprogramming efficiency is dramatically reduced using cells from older animals.
(ii)  STAP cells are derived from clusters of multiple cells; they are not monoclonal.8. The addition of LIF during days 2–7 was essential for generating Oct3/4-GFP+STAP cell clusters on day 7, as shown in Extended Data Fig. 1f (Obokata et al. Nature, 2014a). Even in the absence of LIF, Oct3/4-GFP+cells (most of which showed dim signals) appeared transiently in low-pH-treated CD45+cells during days 2–5 of culture, but subsequently disappeared, suggesting that there is a LIF-independent early phase, whereas the subsequent phase is LIF-dependent.

IMPORTANT
(i)  Since LIF is essential for the late step of reprogramming, the reprogramming event may depend on the genetic background. We  mainly
used 129, C57BL6, or their F1 strains, as all of these genetic backgrounds are associated with high responsiveness to LIF.
(ii) The GFP signal is weaker than that of ES cells carrying the same reporter because of the smaller cell volume of STAP cell than ES cell as found in Figure 1g (Obokata et al. Nature, 2014a).STAP stem-cell conversion culture1. To establish STAP  stem-cell lines, STAP cell clusters were transferred to ACTH-containing medium on MEF feeder cells (several clusters, up to a dozen clusters, per well of 96-well plates).

IMPORTANT
(i)  ACTH (1-24) is available from American Peptide  and other companies. We used ACTH synthesized by Kurabo on consignment. The composition of
this medium is GMEM, 15% Knockout Serum ReplacementTM(KSR, Invitrogen), 1 × non-essential amino acids (NEAA), 1 × Sodium Pyruvate, 10-4M 2-mercaptoethanol, 1000 U/ml LIF, and 10 µM ACTH (Ogawa et al, Genes Cells, 2004). The STAP cell cluster was isolated, dissected into small
pieces as in the case of injection into blastocysts as shown in Figure 4a (Obokata et al. Nature, 2014a), and seeded on mouse embryonic fibroblast feeder cells in the ACTH medium.
(ii) ACTH-containing medium was purchased from DS  Pharma Biomedical (Osaka, Japan) as StemMedium@.2. After 4–7 days of culture, the cells were subjected to  a first passage using a conventional trypsin method, and  the  suspended cells were  plated in ESCmaintenance medium containing 20% FBS.

IMPORTANT
(i) ESC maintenance medium consists of KnockoutTMDMEM (Life Technologies), 20% FBS, 1 × NEAA, 1 × Glutamine, 1 × Nucleosides, 10-4M 2-mercaptoethanol, and 1000 U/ml LIF.
(ii) FBS  lots  should be  confirmed  for  suitability for use in  the culture of mouse ES cells.
(iii)  We have established  multiple STAP stem cell lines from  STAP cells derived from CD45+haematopoietic cells.  Of eight clones examined,  none
contained the rearranged TCR allele, suggesting the possibility of negative cell-type-dependent bias (including maturation of the cell of origin) for STAP cells to give rise to STAP stem cells in the conversion process. This may be relevant to the fact that STAP cell conversion  was  less efficient when non-neonatal cells were used as somatic cells of origin in the current protocol.3. Subsequent passaging was performed at a split ratio of 1:10 every second day until reaching subconfluency. We tested the following three different genetic backgrounds of mice for STAP stem-cell establishment from STAP cell clusters, and observed reproducible 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.
FI stem cell conversion culture1. STAP cell clusters were transferred to Fgf4-containing trophoblast stem-cell medium (Tanaka et al, Science, 1998) on MEF feeder cells in 96-well plates(Obokata, Nature, 2014b).IMPORTANT(i) TS medium consists of RPMI 1640 with 20% FBS, 1 mM Sodium Pyruvate, 100 µM 2-mercaptoethanol, 2 mM L-glutamine, 25 ng/ml of recombinant FGF4, and 1 µg/ml of heparin.(ii) Different lots of FBS may  results in significant  differences in  the behavior of cultured cells.
2. In most cases (40 of 50 experiments), colonies grew in 10–50% of wells in 96-well plates. In  a  minority of cases (10 of 50 experiments), no colony growth was observed and/or only fibroblast-like cells appeared.
IMPORTANT
(i) The cells in proliferative colonies also appear similar to fibroblasts,but gradually change morphology, coming to resemble epithelial cells.

3. The cells were subjected to the first passage during days 7–10 using a conventional trypsin method. Subsequent passages were performed at a split ratio of 1:4 every third day before they reached subconfluency.

IMPORTANT
(i) The cells must not be dissociated completely. Partial dissociation  is optimal to maintain viability and self-renewal, as seen in the case of
embryo-derived trophoblast stem cells.

原始链接

STAP細胞作製に関する実験手技解説の発表について

相关阅读

Nature：日本研制新型万能细胞STAP