STAP Cell Controversy Sets Back Japanese Science

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STAP Cell Controversy Sets Back Japanese Science Mar 25, 2015 13:44:04 GMT

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Post by Admin on Mar 25, 2015 13:44:04 GMT

In her first appearance before the press since her claims of an astounding breakthrough in stem cell research started unraveling, Haruko Obokata, of the RIKEN Center for Developmental Biology in Kobe, Japan, apologized for the trouble she has caused her employer, her colleagues, and the scientific community. But she also firmly maintained that STAP cells, the new type of stem cells she claims to have developed, exist, and said she will not retract the two Nature papers reporting her finding.

“I sincerely apologize to RIKEN, my co-authors, and to many others for the trouble I caused through my insufficient experience and carelessness," Obokata said with a deep bow at the beginning of the press conference, which was held in Osaka. But "STAP cells exist!" she defiantly declared in response to a question. She also pledged to "go anywhere" to help any interested scientist reproduce her results.

Obokata last faced the press when she and colleagues at RIKEN and other institutions in Japan and at Harvard Medical School in Boston published a research article and a letter online in Nature on 29 January. The 30-year-old was lionized in Japan for her unexpected breakthrough, a method to create stem cells that she called "stimulus-triggered acquisition of pluripotency," or STAP. It works by subjecting mature cells to a brief acid bath and then tweaking culture conditions. But soon there were reports of doctored images and plagiarism, and to date, no one has reported replicating the first step in creating STAP cells. One co-author has called for the papers to be retracted.

Within weeks, scientific bloggers identified problematic images. Soon, researchers around the world reported being unable to reproduce the results. RIKEN launched an investigation and by April concluded that Obokata was guilty of research misconduct. Nature retracted the papers in July. Despite numerous problems in the papers, Obokata and co-author Charles Vacanti, a BWH tissue engineer, staunchly maintained that the STAP phenomenon was real and kept releasing tweaks to their published protocols. RIKEN officials decided to settle the issue by methodically redoing the experiments described in the papers. In an interim report released in August, the verification team reported not being able to produce STAP cells. At that time, the team invited Obokata to participate in their efforts to see if there were subtleties in her experimental techniques not defined in her published protocols.

A RIKEN investigating committee announced on 1 April that it had found two instances of research misconduct. RIKEN President Ryoji Noyori said at the time that Obokata would be given a chance to appeal before a disciplinary committee would be convened. Obokata's lawyers filed the appeal with RIKEN yesterday.

At the press conference, Kazuhiko Murotani, one of her attorneys, elaborated on Obokata’s previous claims that there was no intent to deceive, and that the problems in the papers do not affect the results. He did reveal a few new details. For instance, Obokata had previously admitted to using images from her doctoral thesis in the Nature papers by mistake; Murotani explained today that the images did not come directly from the thesis, but rather resulted from a mix-up of PowerPoint slides.

She apologized several times for the errors in the papers, but also repeatedly expressed her faith in her findings. She claimed she has created STAP cells more than 200 times; retracting the Nature papers would indicate that the STAP phenomenon is not real, she added. "If I can continue in research, I want to work to realize the hopes of STAP cells as quickly as possible," she said.

Several critical errors have been found in our Article (http://dx.doi.org/10.1038/nature12968) and Letter, which led to an in-depth investigation by the RIKEN Institute. The RIKEN investigation committee has categorized some of the errors as misconduct (see Supplementary Data 1 and Supplementary Data 2). Additional errors identified by the authors that are not discussed in RIKEN’s report are listed below.

(1) Figure 1a and b in the Letter both show embryos generated from STAP cells, not a comparison of ES- and STAP-derived chimaeric embryos, as indicated in the legend.

(2) Extended Data Fig. 7d in the Article and Extended Data Fig. 1a in the Letter are different images of the same embryo and not, as indicated in the legends, a diploid chimaera embryo and tetraploid chimaera embryo.

(3) There is an erroneous description in Fig. 1a in the Letter. The right panel of Fig. 1a is not a ‘long exposure’ image at the camera level but a digitally enhanced one.

(4) In Fig. 4b of the Letter, STAP cell and ES cell are wrongly labelled in a reverse manner.

(5) In the Article, one group of STAP stem cells (STAP-SCs) was reported as being derived from STAP cells induced from spleens of F1 hybrids from the cross of mouse lines carrying identical cag-gfp insertions in chromosome 18 in the background of 129/Sv and B6, respectively, and that they were maintained in the Wakayama laboratory. However, further analysis of the eight STAP-SC lines indicates that, while sharing the same 129×B6 F1 genetic background, they have a different GFP insertion site. Furthermore, while the mice used for STAP cell induction are homozygous for the GFP transgene, the STAP-SCs are heterozygous. The GFP transgene insertion site matches that of the mice and ES cells kept in the Wakayama laboratory. Thus, there are inexplicable discrepancies in genetic background and transgene insertion sites between the donor mice and the reported STAP-SCs.

We apologize for the mistakes included in the Article and Letter. These multiple errors impair the credibility of the study as a whole and we are unable to say without doubt whether the STAP-SC phenomenon is real. Ongoing studies are investigating this phenomenon afresh, but given the extensive nature of the errors currently found, we consider it appropriate to retract both papers.

Nature 505, 676–680 (2014); doi:10.1038/nature12969

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STAP Cell Controversy Sets Back Japanese Science Mar 27, 2015 14:07:36 GMT

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Post by Admin on Mar 27, 2015 14:07:36 GMT

CD45+ cells were sorted by fluorescence-activated cell sorting (FACS) from the lymphocyte fraction of postnatal spleens (1-week old) of C57BL/6 mice carrying an Oct4-gfp transgene14, and were exposed to various types of strong, transient, physical and chemical stimuli (described below). We examined these cells for activation of the Oct4 promoter after culture for several days in suspension using DMEM/F12 medium supplemented with leukaemia inhibitory factor (LIF) and B27 (hereafter called LIF+B27 medium). Among the various perturbations, we were particularly interested in low-pH perturbations for two reasons. First, as shown below, low-pH treatment turned out to be most effective for the induction of Oct4. Second, classical experimental embryology has shown that a transient low-pH treatment under ‘sublethal’ conditions can alter the differentiation status of tissues. Spontaneous neural conversion from salamander animal caps by soaking the tissues in citrate-based acidic medium below pH 6.0 has been demonstrated previously15, 16, 17.

Without exposure to the stimuli, none of the cells sorted with CD45 expressed Oct4-GFP regardless of the culture period in LIF+B27 medium. In contrast, a 30-min treatment with low-pH medium (25-min incubation followed by 5-min centrifugation; Fig. 1a; the most effective range was pH 5.4–5.8; Extended Data Fig. 1a) caused the emergence of substantial numbers of spherical clusters that expressed Oct4-GFP in day-7 culture (Fig. 1b). Substantial numbers of GFP+ cells appeared in all cases performed with neonatal splenic cells (n = 30 experiments). The emergence of Oct4-GFP+ cells at the expense of CD45+ cells was also observed by flow cytometry (Fig. 1c, top, and Extended Data Fig. 1b, c). We next fractionated CD45+ cells into populations positive and negative for CD90 (T cells), CD19 (B cells) and CD34 (haematopoietic progenitors18), and subjected them to low-pH treatment. Cells of these fractions, including T and B cells, generated Oct4-GFP+ cells at an efficacy comparable to unfractionated CD45+ cells (25–50% of surviving cells on day 7), except for CD34+ haematopoietic progenitors19, which rarely produced Oct4-GFP+ cells (<2%; Extended Data Fig. 1d).

Figure 1: Stimulus-triggered conversion of lymphocytes into Oct4-GFP+ cells.

On day 7, the Oct4-GFP+ spheres expressed pluripotency-related marker proteins22 (Oct4, SSEA1, Nanog and E-cadherin; Fig. 2a) and marker genes (Oct4, Nanog, Sox2, Ecat1 (also called Khdc3), Esg1 (Dppa5a), Dax1 (Nrob1) and Rex1 (Zfp42); Fig. 2b and Extended Data Fig. 3a) in a manner comparable to those seen in ES cells24. Moderate levels of expression of these pluripotency marker genes were observed on day 3 (Fig. 2b and Extended Data Fig. 3b). Notably, the Oct4-GFP+ cells on day 3, but not on day 7, expressed early haematopoietic marker genes such as Flk1 (also called Kdr) and Tal1 (Extended Data Fig. 3c), indicating that Oct4-GFP+ cells on day 3, as judged by their expression pattern at the population level, were still in a dynamic process of conversion.

Figure 2: Low-pH-induced Oct4-GFP+ cells represent pluripotent cells.

On day 7, unlike CD45+ cells and like ES cells, low-pH-induced Oct4-GFP+ cells displayed extensive demethylation at the Oct4 and Nanog promoter areas (Fig. 2c), indicating that these cells underwent a substantial reprogramming of epigenetic status in these key genes for pluripotency. In vitro differentiation assays25, 26, 27 demonstrated that low-pH-induced Oct4-GFP+ cells gave rise to three-germ-layer derivatives (Fig. 2d) as well as visceral endoderm-like epithelium (Extended Data Fig. 3d). When grafted into mice, low-pH-induced Oct4-GFP+ cell clusters formed teratomas (40%, n = 20) (Fig. 2e and Extended Data Fig. 4a–c) but no teratocarcinomas that persistently contained Oct4-GFP+ cells (n = 50). Because some cellular variation was observed in the signal levels of Oct4-GFP within the clusters, we sorted GFP-strong cells (a major population) and GFP-dim cells (a minor population) by FACS on day 7 and separately injected them into mice. In this case, only GFP-strong cells formed teratomas (Extended Data Fig. 4d). In quantitative polymerase chain reaction (qPCR) analysis, the GFP-strong population expressed pluripotency marker genes but not early lineage-specific marker genes, whereas the GFP-dim cells showed substantial expression of some early lineage-specific marker genes (Flk1, Gata2, Gata4, Pax6 and Sox17; Extended Data Fig. 4e) but not Nanog and Rex1. These observations indicate that three-germ-layer derivatives were generated from the GFP-strong cells expressing pluripotency marker genes, rather than from GFP-dim cells that seem to contain partially reprogrammed cells.

Collectively, these findings show that the differentiation state of a committed somatic cell lineage can be converted into a state of pluripotency by strong stimuli given externally. Hereafter, we refer to the fate conversion from somatic cells into pluripotent cells by strong external stimuli such as low pH as ‘stimulus-triggered acquisition of pluripotency’ (STAP) and the resultant cells as STAP cells. Under their establishment conditions, these STAP cells were rarely proliferative (Extended Data Figs 2d and 5a, b). Comparative genomic hybridization array analysis of STAP cells indicated no major global changes in chromosome number (Extended Data Fig. 5c). STAP cells, unlike mouse ES cells, showed a limited capacity for self-renewal in the LIF-containing medium and did not efficiently form colonies in dissociation culture (Fig. 2f, g), even in the presence of the ROCK inhibitor Y-27632, which suppresses dissociation-induced apoptosis28, 29 (Fig. 2h). Also, even under high-density culture conditions after partial dissociation (Fig. 2i), STAP cell numbers started to decline substantially after two passages. Furthermore, expression of the ES cell marker protein Esrrβ was low in STAP cells (Extended Data Fig. 5d, e). In general, female ES cells do not show X-chromosomal inactivation30 and contain no H3K27me3-dense foci (indicative of inactivated X chromosomes), unlike female CD45+ cells and EpiSCs. In contrast, H3K27me3-dense foci were found in ~40% of female STAP cells strongly positive for Oct4-GFP (Extended Data Fig. 5f, g). STAP cells were also dissimilar to mouse EpiSCs, another category of pluripotent stem cell21, 22, 29, 31, and were positive for Klf4 and negative for the epithelial tight junction markers claudin 7 and ZO-1 (Extended Data Fig. 5d, e).

Figure 3: STAP cell conversion from a variety of cells by low-pH treatment.

We next performed similar conversion experiments with somatic cells collected from brain, skin, muscle, fat, bone marrow, lung and liver tissues of 1-week-old Oct4-gfp mice. Although conversion efficacy varied, the low-pH-triggered generation of Oct4-GFP+ cells was observed in day 7 culture of all tissues examined (Fig. 3a and Extended Data Fig. 6a–c), including mesenchymal cells of adipose tissues (Fig. 3a–c) and neonatal cardiac cells that were negatively sorted for CD45 by FACS (Fig. 3d–g; see Extended Data Fig. 6d for suppression of cardiac genes such as Nkx2-5 and cardiac actin).

We next performed a blastocyst injection assay with STAP cells that were generated from CD45+ cells of neonatal mice constitutively expressing GFP (this C57BL/6 line with cag-gfp transgenes is referred to hereafter as B6GFP). We injected STAP cell clusters en bloc that were manually cut into small pieces using a microknife (Fig. 4a). A high-to-moderate contribution of GFP-expressing cells was seen in the chimaeric embryos (Fig. 4b and Extended Data Fig. 7a). These chimaeric mice were born at a substantial rate and all developed normally (Fig. 4c and Extended Data Fig. 7b).

Figure 4: Chimaeric mouse generation from STAP cells.

CD45+ cell-derived STAP cells contributed to all tissues examined (Fig. 4d). Furthermore, offspring derived from STAP cells were born to the chimaeric mice (Fig. 4e and Extended Data Fig. 7c), demonstrating their germline transmission, which is a strict criterion for pluripotency as well as genetic and epigenetic normality32, 33. Furthermore, in a tetraploid (4N) complementation assay, which is considered to be the most rigorous test for developmental potency34, 35 (Fig. 4a, bottom), CD45+ cell-derived STAP cells (from F1 mice of B6GFP × 129/Sv or DBA/2) generated all-GFP+ embryos on embryonic day (E)10.5 (Fig. 4f, Extended Data Fig. 7d and Supplementary Video 3), demonstrating that STAP cells alone are sufficient to construct an entire embryonic structure. Thus, STAP cells have the developmental capacity to differentiate into all somatic-cell lineages as well as germ-cell lineages in vivo.

Figure 5: ES-cell-like stem cells can be derived from STAP cells.

STAP cells have a limited self-renewal capacity under the conditions used for establishment (Fig. 2g and Extended Data Figs 2e and 5a). However, in the context of the embryonic environment, a small fragment of a STAP cell cluster could grow even into a whole embryo (Fig. 4f). With this in mind, we next examined whether STAP cells have the potential to generate expandable pluripotent cell lines in vitro under certain conditions. STAP cells could not be efficiently maintained for additional passages in conventional LIF+FBS-containing medium or 2i medium20 (most STAP cells died in 2i medium within 7 days; Extended Data Fig. 8a). Notably, an adrenocorticotropic hormone (ACTH)+LIF-containing medium (hereafter called ACTH medium) known to facilitate clonal expansion of ES cells36 supported outgrowth of STAP cell colonies. When cultured in this medium on a MEF feeder or gelatin, a portion of STAP cell clusters started to grow (Fig. 5a, bottom; such outgrowth was typically found in 10–20% of wells in single cluster culture using 96-well plates and in >75% when 12 clusters were plated per well). These growing colonies looked similar to those of mouse ES cells and expressed a high level of Oct4-GFP.

Nature 505, 641–647 (30 January 2014) doi:10.1038/nature12968

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STAP Cell Controversy Sets Back Japanese Science Feb 14, 2016 5:39:03 GMT

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Post by Admin on Feb 14, 2016 5:39:03 GMT

Haruko Obokata, the former stem cell scientist dogged by a scandal surrounding her alleged falsification of research papers, will publish a book recounting the controversy that unfolded from early 2014, publisher Kodansha Ltd. said Wednesday.

The 253-page book, titled "Anohi" (That Day), will hit the bookstore shelves Thursday. It will be the first time she has offered a lengthy explanation on the issue of "STAP cells," which she claimed to have made, after quitting the state-backed Riken research institute in December 2014, according to the publisher.

In the introduction of the book written in Japanese, Obokata, 32, apologized over the issue, saying, "I sincerely regret and feel ashamed for lacking knowledge." She also said she decided "to write the truth" in the book, believing that "remaining quiet and waiting for people to forget" the matter could be seen as "cowardly."

Her life took a dramatic turn when a pair of papers lead-authored by the scientist appeared in British journal Nature in January 2014, claiming to demonstrate a new and simpler way to produce stem cells through a process dubbed stimulus-triggered acquisition of pluripotency, or STAP.

What is potentially most contentious about the 253-page book concerns her association with, and then estrangement from, onetime boss Teruhiko Wakayama, a former Riken researcher who now teaches at Yamanashi University. Obokata asserts in the book that crucial parts of the STAP experiments were handled only by Wakayama and alleges that he changed his accounts of how the STAP cells were produced.