| Spontaneous Malignant Transformation of Human Mesenchymal Stem Cells Reflects Cross-Contamination: Putting the Research Field on Track - Letter. Cancer Res. 2010 Jul 14; Authors: Torsvik A, Røsland GV, Svendsen A, Molven A, Immervoll H, McCormack E, Lønning PE, Primon M, Sobala E, Tonn JC, Goldbrunner R, Schichor C, Mysliwietz J, Lah TT, Motaln H, Knappskog S, Bjerkvig R The Controversy of Spontaneous Transformation of Human Mesenchymal Stem Cells Several groups, including ours, have published results showing spontaneous transformation of human mesenchymal stem cells (MSC). Recently, we reported in this journal spontaneous transformation of bone marrow-derived human MSC (hMSC), isolated and expanded independently in two laboratories (1). Inspired by the recent focus on misidentification of cell lines in which cell culture repositories indicate that between 18 to 36% of the cell lines contain misidentified species, we did DNA fingerprinting and/or short tandem repeat (STR) analysis comparing the normal MSC with their transformed counterparts. The analysis shows that the transformed mesenchymal stem cells (TMC) in one laboratory were cross-contaminated with human fibrosarcoma or osteosarcoma cell lines, whereas in the other laboratory cross-contamination was due to two glioma cell lines. Our observations underscore the need for extremely stringent cell culture procedures when it comes to the use of primary cell cultures, including MSC, for therapeutic purposes. Further, our results highlight the need for cell line verification prior to scientific publishing. The Role of Mesenchymal Stem Cells in Cancer Initiation: Current Controversies Although stem cells are critical to tissue repair, several studies indicate that they also may have important roles in malignant transformation and cancer initiation (2-4). Experimental evidence indicates that gastric cancer may originate from bone marrow-derived cells (3), and that osteosarcomas may be derived from MSC (4). Furthermore, several reports show that MSC may support tumor growth and development of metastasis in vivo (5, 6). On the other hand, MSC have also been shown to have tumor suppressor activity. For instance, it has been shown that MSC can inhibit the malignant phenotypes of human liver cancer cell lines, which include proliferation, colony-forming ability, and oncogene expression both in vitro and in vivo (7). A suppressor function of MSC has also been observed for tumor cells of hematopoietic origin (8). An issue of controversy is whether hMSC may undergo spontaneous transformation in vitro or not. Several groups have reported no transformation of hMSC after long term culture (9, 10), whereas during the last 5 years other groups, including ours, have published results adding to the list showing spontaneous transformation of hMSC derived from adipose tissue (11) or bone marrow (1, 12). In this context, we reported, in a recent study, spontaneous transformation of hMSC observed in two independent laboratories (1). The Issue of Cell Line Misidentification Recent data indicate that between 18 and 36% of cell lines in general use contain misidentified cell types (13). As a part of our internal laboratory quality control, we did a comprehensive DNA fingerprinting of our MSC and the corresponding TMC. In total, 11 TMC were tested, 9 transformations from 6 donors in laboratory 1 (Laboratory 1) and 2 transformations from 2 donors in laboratory 2 (Laboratory 2). Our results show that, in both laboratories, the MSC were contaminated with established human immortalized cell lines. The DNA fingerprint revealed that none of the TMC in the two laboratories had STR profiles comparable to the original hMSC. Also, as shown in Table 1, several TMC revealed fingerprint profiles consistent with a common origin. Comparisons with DNA fingerprints of commercially available cell lines used in the laboratories revealed cross-contamination with the human HT1080 fibrosarcoma and U-2 OS osteosarcoma cell line in Laboratory 1, whereas in Laboratory 2 cross-contamination was observed with the human glioma cell lines U251 and U373 (Table 1). Table 1.STR profile of TMC and various cell lines Cell NameSTR DesignationAMELD8S1179D21S11D18S51D3S1358vWAFGAD5S818D13S317D7S820TMC#1 (Laboratory 1)*XY131428301218161419222511131214910TMC#2 (Laboratory 1)*XY131428301218161419222511131214910TMC#4-1 (Laboratory 1)*XY131428301218161419222511131214910TMC#4-2 (Laboratory 1)*XY131428301218161419222511131214910TMC#4-3 (Laboratory 1)*XY131428301218161419222511131214910TMC#5-1 (Laboratory 1)*XY131428301218161419222511131214910TMC#5-2 (Laboratory 1)*XY131428301218161419222511131214910PT-TMC#1 (Laboratory 1)*XY131428301218161419222511131214910SCT-TMC#1 (Laboratory 1)(dagger)X121431141614182011131112TMC#1 (Laboratory 2)(double dagger)XY1315293013161716182125111210111012TMC#2 (Laboratory 2)(double dagger)X13152913161716182125111210111012HT1080XY131428301218161419222511131214910U-2 OSX121431141614182011131112U251XY13152913161716182125111210111012U373XY1315293013161716182125111210111012 NOTE: Authentication of TMC was done by DNA fingerprinting using the AmpFlSTR Profiler Plus PCR Amplification kit (Applied Biosystems), which amplifies nine STRs and the gender determination amelogenin gene (AMEL). Prefixes PT (poietics) and SCT (stem cell technology) refer to the source of the original hMSC, when these were obtained from commercial distributors (Lonza and Stem Cell Technology, respectively). Laboratory 1 and Laboratory 2 (in parentheses) indicate the laboratory in which the original hMSC were isolated and expanded. None of the cells were transferred between the two laboratories. Abbreviations: PT, poietics; SCT, stem cell technology. *TMC that match HT1080 fibrosarcoma cells. (dagger)TMC that match U-2 OS osteosarcoma cells. (double dagger)TMC that match U251/U373 glioblastoma cells. On the basis of the findings of Garcia and colleagues (14), that single cells of HT1080 can remain undetectable in MSC cultures for months, we did a thorough examination of our research protocols to map the time points when both normal MSC and the contaminating tumor cell lines were cultured in the laboratories. The results from these time point studies indicate that the contaminating cell may have resided in the MSC culture for up to 3 to 4 weeks (data are not shown). However, we cannot rule out that a small fraction of highly clonogenic tumor cells may have been idle in the MSC cultures for extended periods before they were triggered to divide, nor can we rule out that the contaminating cell outgrew the MSC culture within a much shorter time. Do Mesenchymal Stem Cells Contribute to Carcinogenesis? The issue of stem cell contribution to carcinogenesis is currently a major topic in cancer research, in which experimental results may have considerable implications for our understanding of cancer biology as well as in the development of new cell-based therapeutic strategies. In a MedLine search, it was found that 592 articles are indexed in which MSC have been linked to cancer initiation and development or in which MSC have been considered as therapeutic vehicles (search terms: MSC, cancer, development, therapy). However, a similar search revealed DNA fingerprinting of these cells to be virtually nonexistent. The problem of cross-contamination raises a special challenge for laboratories growing normal cells in long term cultures and concomitantly handling immortalized cancer cell lines. In these laboratories, a detailed and regular characterization of the normal cells is particularly needed because human errors may occur, errors that may have severe consequences when it comes to the interpretation of results obtained. However, many commonly used cell lines are also misidentified. In our case, the U251 and U373 cell lines were found to have a common origin, a misidentification recently recognized by the American Type Culture Collection (ATCC; ref. 15). The consequence of using such misidentified cells varies. If general cellular processes are studied, they can have minor drawbacks, but if the cell lines reflect properties of a particular tissue, cancer, or diseased state, the outcome may have severe consequences, driving research groups onto false tracks. In this context, it is also interesting to learn that with the spontaneous transformation of adipose MSC, reported by Rubio and colleagues (11), the TMC also originate from the fibrosarcoma cell line HT1080 (14); a coincidence that strengthened our interpretations in our previous article (1). Research: A Learning Experience We believe our findings, although dismal for our research program, convey important messages to the scientific community and to scientific journals. First, because human errors may occur in any laboratory despite stringent working procedures, DNA fingerprinting should be compulsory for all experiments involving cell lines. Scientists should verify the cell lines in their possession and use electronic databases of authenticated DNA profiles against which they can compare their results. Second, scientific journals should require that all cell lines used in an article are verified before publication. Third, funders of grant proposals should encourage expense estimates for cell line verification, in recognition that such verification procedures will increase the costs of research. Subsequent to a learning experience like this, it is important to remember how great thinkers in science have recognized this type of event. In his major disclosure on the scientific method in An Introduction to the Study of Experimental Medicine (1865), Claude Bernard describes what makes a scientific theory good and what makes science important (16). He states "it is always instructive to acknowledge an error. The precept, therefore, is excellent for we are all likely to make mistakes, except those of us who do nothing. But the first requirement in acknowledging a mistake is to prove that there is an error. It is not enough to say: I was mistaken; we must say how we were mistaken; the important point is precisely that." He further states: "To sum up, we must maintain the conviction that negative facts are determined like positive facts. We posited the principle that all experiments are successful, in that their conditions are determined; in research into the conditions of each of these determinations lie the lessons that teach us the law of a phenomenon; because in this way we learn the conditions necessary to its existence and its nonexistence." Disclosure of Potential Conflicts of Interest R. Bjerkvig: consultant/advisory board, Siena Biotech. The other authors disclosed no potential conflicts of interest. PMID: 20631079 [PubMed - as supplied by publisher] |
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