Javier García-Castro

Spontaneous Human Adult Stem Cell Transformation

Human adult stem cells are being evaluated widely for various therapeutic approaches. Several recent clinical trials have reported their safety, showing them to be highly resistant to transformation. The clear similarities between stem cell and cancer stem cell genetic programs are nonetheless the basis of a recent proposal that some cancer stem cells could derive from human adult stem cells. Here we show that although they can be managed safely during the standard ex vivo expansion period (6-8 weeks), human mesenchymal stem cells can undergo spontaneous transformation following long-term in vitro culture (4-5 months). This is the first report of spontaneous transformation of human adult stem cells, supporting the hypothesis of cancer stem cell origin. Our findings indicate the importance of biosafety studies of mesenchymal stem cell biology to efficiently exploit their full clinical therapeutic potential.

Molecular Characterization of Spontaneous Mesenchymal Stem Cell Transformation

Background We previously reported the in vitro spontaneous transformation of human mesenchymal stem cells (MSC) generating a population with tumorigenic potential, that we termed transformed mesenchymal cells (TMC). Methodology/Principal Findings Here we have characterized the molecular changes associated with TMC generation. Using microarrays techniques we identified a set of altered pathways and a greater number of downregulated than upregulated genes during MSC transformation, in part due to the expression of many untranslated RNAs in MSC. Microarray results were validated by qRT-PCR and protein detection. Conclusions/Significance In our model, the transformation process takes place through two sequential steps; first MSC bypass senescence by upregulating c-myc and repressing p16 levels. The cells then bypass cell crisis with acquisition of telomerase activity, Ink4a/Arf locus deletion and Rb hyperphosphorylation. Other transformation-associated changes include modulation of mitochondrial metabolism, DNA damage-repair proteins and cell cycle regulators. In this work we have characterized the molecular mechanisms implicated in TMC generation and we propose a two-stage model by which a human MSC becomes a tumor cell.

Pitfalls in spontaneous in vitro transformation of human mesenchymal stem cells

Spontaneous in vitro transformation of human primary cells was, and continues to be, a scarcely described phenomenon. Only the description of the generation of the HaCAT cell line [1] is a canonical example and accepted worldwide. More recent examples included the emergence of tumorogenic populations upon in vitro culture of fetal human mesenchymal stem cells (hMSC), induced by GM-CSF and IL-4, [2] and bone marrow hMSC [3]. Other examples have also been reported after very long-term in vitro culture of telomerized hMSC-TerT [4]. In this scenario, our previous results [5–7] were only a new observation

Bone marrow mesenchymal stem cells from infants with MLL-AF4+ acute leukemia harbor and express the MLL-AF4 fusion gene

MLL-AF4 fusion is a hallmark genetic abnormality in infant B-acute lymphoblastic leukemia (B-ALL) known to arise in utero. The cellular origin of leukemic fusion genes during human development is difficult to ascertain. The bone marrow (BM) microenvironment plays an important role in the pathogenesis of several hematological malignances. BM mesenchymal stem cells (BM-MSC) from 38 children diagnosed with cytogenetically different acute leukemias were screened for leukemic fusion genes. Fusion genes were absent in BM-MSCs of childhood leukemias carrying TEL-AML1, BCR-ABL, AML1-ETO, MLL-AF9, MLL-AF10, MLL-ENL or hyperdiploidy. However, MLL-AF4 was detected and expressed in BM-MSCs from all cases of MLL-AF4+ B-ALL. Unlike leukemic blasts, MLL-AF4+ BM-MSCs did not display monoclonal Ig gene rearrangements. Endogenous or ectopic expression of MLL-AF4 exerted no effect on MSC culture homeostasis. These findings suggest that MSCs may be in part tumor-related, highlighting an unrecognized role of the BM milieu on the pathogenesis of MLL-AF4+ B-ALL. MLL-AF4 itself is not sufficient for MSC transformation and the expression of MLL-AF4 in MSCs is compatible with a mesenchymal phenotype, suggesting a differential impact in the hematopoietic system and mesenchyme. The absence of monoclonal rearrangements in MLL-AF4+ BM-MSCs precludes the possibility of cellular plasticity or de-differentiation of B-ALL blasts and suggests that MLL-AF4 might arise in a population of prehematopoietic precursors.

Deficiency in p53 but not Retinoblastoma Induces the Transformation of Mesenchymal Stem Cells In vitro and Initiates Leiomyosarcoma In vivo

Sarcomas have been modeled in mice by the expression of specific fusion genes in mesenchymal stem cells (MSC), supporting the concept that MSCs might be the target initiating cell in sarcoma. In this study, we evaluated the potential oncogenic effects of p53 and/or retinoblastoma (Rb) deficiency in MSC transformation and sarcomagenesis. We derived wild-type, p53(-/-), Rb(-/-), and p53(-/-)Rb(-/-) MSC cultures and fully characterized their in vitro growth properties and in vivo tumorigenesis capabilities. In contrast with wild-type MSCs, Rb(-/-), p53(-/-), and p53(-/-)Rb(-/-) MSCs underwent in vitro transformation and showed severe alterations in culture homeostasis. More importantly, p53(-/-) and p53(-/-)Rb(-/-) MSCs, but not Rb(-/-) MSCs, were capable of tumor development in vivo after injection into immunodeficient mice. p53(-/-) or p53(-/-)Rb(-/-) MSCs originated leiomyosarcoma-like tumors, linking this type of smooth muscle sarcoma to p53 deficiency in fat tissue-derived MSCs. Sca1+ and Sca1 low/- cell populations isolated from ex vivo-established, transformed MSC lines from p53(-/-)Rb(-/-) tumors showed identical sarcomagenesis potential, with 100% tumor penetrance and identical latency, tumor weight, and histologic profile. Our findings define the differential roles of p53 and Rb in MSC transformation and offer proof-of-principle that MSCs could provide useful tools to dissect the sarcoma pathogenesis.

Retraction: Spontaneous human adult stem cell transformation.

thors retract the article titled “Spontaneous Human Adult Stem Cell Transtion,” which was published in the April 15, 2005, issue of Cancer Research (1). review of the data published in this article, the authors have been unable to uce some of the reported spontaneous transformation events and suspect the menon is due to a cross-contamination artifact. Five of the seven authors have to the retraction of this paper.

Feeder-free maintenance of hESCs in mesenchymal stem cell-conditioned media: distinct requirements for TGF-β and IGF-II

A paracrine regulation was recently proposed in human embryonic stem cells (hESCs) grown in mouse embryonic fibroblast (MEF)-conditioned media (MEF-CM), where hESCs spontaneously differentiate into autologous fibroblast-like cells to maintain culture homeostasis by producing TGF-β and insulin-like growth factor-II (IGF-II) in response to basic fibroblast growth factor (bFGF). Although the importance of TGF-β family members in the maintenance of pluripotency of hESCs is widely established, very little is known about the role of IGF-II. In order to ease hESC culture conditions and to reduce xenogenic components, we sought (i) to determine whether hESCs can be maintained stable and pluripotent using CM from human foreskin fibroblasts (HFFs) and human mesenchymal stem cells (hMSCs) rather than MEF-CM, and (ii) to analyze whether the cooperation of bFGF with TGF-β and IGF-II to maintain hESCs in MEF-CM may be extrapolated to hESCs maintained in allogeneic mesenchymal stem cell (MSC)-CM and HFF-CM. We found that MSCs and HFFs express all FGF receptors (FGFR1-4) and specifically produce TGF-β in response to bFGF. However, HFFs but not MSCs secrete IGF-II. Despite the absence of IGF-II in MSC-CM, hESC pluripotency and culture homeostasis were successfully maintained in MSC-CM for over 37 passages. Human ESCs derived on MSCs and hESCs maintained in MSC-CM retained hESC morphology, euploidy, expression of surface markers and transcription factors linked to pluripotency and displayed in vitro and in vivo multilineage developmental potential, suggesting that IGF-II may be dispensable for hESC pluripotency. In fact, IGF-II blocking had no effect on the homeostasis of hESC cultures maintained either on HFF-CM or on MSC-CM. These data indicate that hESCs are successfully maintained feeder-free with IGF-II-lacking MSC-CM, and that the previously proposed paracrine mechanism by which bFGF cooperates with TGF-β and IGF-II in the maintenance of hESCs in MEF-CM may not be fully extrapolated to hESCs maintained in CM from human MSCs.

Human mesenchymal stem cell transformation is associated with a mesenchymal–epithelial transition

Carcinomas are widely thought to derive from epithelial cells with malignant progression often associated with an epithelial-mesenchymal transition (EMT). We have characterized tumors generated by spontaneously transformed human mesenchymal cells (TMC) previously obtained in our laboratory. Immunohistopathological analyses identified these tumors as poorly differentiated carcinomas, suggesting that a mesenchymal-epithelial transition (MET) was involved in the generation of TMC. This was corroborated by microarray and protein expression analysis that showed that almost all mesenchymal-related genes were severely repressed in these TMC. Interestingly, TMC also expressed embryonic antigens and were able to integrate into developing blastocysts with no signs of tumor formation, suggesting a dedifferentiation process was associated with the mesenchymal stem cell (MSC) transformation. These findings support the hypothesis that some carcinomas are derived from mesenchymal rather than from epithelial precursors.

Bone Environment is Essential for Osteosarcoma Development from Transformed Mesenchymal Stem Cells

The cellular microenvironment plays a relevant role in cancer development. We have reported that mesenchymal stromal/stem cells (MSCs) deficient for p53 alone or together with RB (p53−/−RB−/−) originate leiomyosarcoma after subcutaneous (s.c.) inoculation. Here, we show that intrabone or periosteal inoculation of p53−/− or p53−/−RB−/− bone marrow‐ or adipose tissue‐derived MSCs originated metastatic osteoblastic osteosarcoma (OS). To assess the contribution of bone environment factors to OS development, we analyzed the effect of the osteoinductive factor bone morphogenetic protein‐2 (BMP‐2) and calcified substrates on p53−/−RB−/− MSCs. We show that BMP‐2 upregulates the expression of osteogenic markers in a WNT signaling‐dependent manner. In addition, the s.c. coinfusion of p53−/−RB−/− MSCs together with BMP‐2 resulted in appearance of tumoral osteoid areas. Likewise, when p53−/−RB−/− MSCs were inoculated embedded in a calcified ceramic scaffold composed of hydroxyapatite and tricalciumphosphate (HA/TCP), tumoral bone formation was observed in the surroundings of the HA/TCP scaffold. Moreover, the addition of BMP‐2 to the ceramic/MSC implants further increased the tumoral osteoid matrix. Together, these data indicate that bone microenvironment signals are essential to drive OS development. Stem Cells 2014;32:1136–1148

Absence of hematopoiesis from transplanted olfactory bulb neural stem cells

Neural stem cells giving rise to neurons and glia cells have been isolated from the embryonic and adult central nervous system. The extent to which they are able to differentiate into cells of non‐neural lineages, such as the hematopoietic lineage, is nonetheless unclear. We previously reported the isolation of stem cells from the mouse olfactory bulb neuroepithelium. In the present study, we analysed whether olfactory bulb stem cells (OBSC) can generate cells with hematopoietic features. Cells were prepared from the olfactory bulbs of transgenic mice expressing enhanced green fluorescent protein (EGFP). In culture, transgenic cells proliferated with the same kinetics as wild‐type cells. Following mitogen removal, both cell types gave rise to similar numbers of neurons, astrocytes and oligodendrocytes, indicating that EGFP overexpression does not alter OBSC proliferation and differentiation patterns. When these cells were injected into the tail vein of irradiated mice, no hematopoietic cells derived from the OBSC could be recovered in their peripheral blood, spleen or bone marrow. By contrast, when OBSC were transplanted into the adult brain, EGFP‐positive cells were found in the striatum and corpus callosum; differentiated cells expressed antigenic markers of neurons and astrocytes. These results suggest that embryonic olfactory bulb stem cells are not endowed with the potential to produce hematopoiesis.