Nonadhesive culture system as a model of rapid sphere formation with cancer stem cell properties

Background

Cancer stem cells (CSCs) play an important role in tumor initiation, progression, and metastasis and are responsible for high therapeutic failure rates. Identification and characterization of CSC are crucial for facilitating the monitoring, therapy, or prevention of cancer. Great efforts have been paid to develop a more effective methodology. Nevertheless, the ideal model for CSC research is still evolving. In this study, we created a nonadhesive culture system to enrich CSCs from human oral squamous cell carcinoma cell lines with sphere formation and to characterize their CSC properties further.

Methods

A nonadhesive culture system was designed to generate spheres from the SAS and OECM-1 cell lines. A subsequent investigation of their CSC properties, including stemness, self-renewal, and chemo- and radioresistance in vitro, as well as tumor initiation capacity in vivo, was also performed.

Results

Spheres were formed cost-effectively and time-efficiently within 5 to 7 days. Moreover, we proved that these spheres expressed putative stem cell markers and exhibited chemoradiotherapeutic resistance, in addition to tumor-initiating and self-renewal capabilities.

Conclusions

Using this nonadhesive culture system, we successfully established a rapid and cost-effective model that exhibits the characteristics of CSCs and can be used in cancer research.     

Effect of CD44 Binding Peptide Conjugated to an Engineered Inert Matrix on Maintenance of Breast Cancer Stem Cells and Tumorsphere Formation

Introduction

As cancer cells are affected by many factors in their microenvironment, a major challenge is to isolate the effect of a specific factor on cancer stem cells (CSCs) while keeping other factors unchanged. We have developed a synthetic inert 3D polyethylene glycol diacrylate (PEGDA) gel culture system as a unique tool to study the effect of microenvironmental factors on CSCs response. We have reported that CSCs formed in the inert PEGDA gel by encapsulation of breast cancer cells maintain their stemness within a certain range of gel stiffness. The objective was to investigate the effect of CD44 binding peptide (CD44BP) conjugated to the gel on the maintenance of breast CSCs.

Methods

4T1 or MCF7 breast cancer cells were encapsulated in PEGDA gel with CD44BP conjugation. Control groups included dissolved CD44BP and the gel with mutant CD44BP conjugation. Tumorsphere size and density, and expression of CSC markers were determined after 9 days. For in vivo, cell encapsulated gels were inoculated in syngeneic Balb/C mice and tumor formation was determined after 4 weeks. Effect of CD44BP conjugation on breast CSC maintenance was compared with integrin binding RGD peptide (IBP) and fibronectin-derived heparin binding peptide (FHBP).

Results

Conjugation of CD44BP to the gel inhibited breast tumorsphere formation in vitro and in vivo. The ability of the encapsulated cells to form tumorspheres in the peptide-conjugated gels correlated with the expression of CSC markers. Tumorsphere formation in vitro was enhanced by FHBP while it was abolished by IBP.

Conclusion

CD44BP and IBP conjugated to the gel abolished tumorsphere formation by encapsulated 4T1 cells while FHBP enhanced tumorsphere formation compared to cells in the gel without peptide. The PEGDA hydrogel culture system provides a novel tool to investigate the individual effect of factors in the microenvironment on CSC maintenance without interference of other factors.     

Human Mesenchymal Stem Cells Self-Renew and Differentiate According to a Deterministic Hierarchy

Background

Mesenchymal progenitor cells (MPCs) have been isolated from a variety of connective tissues, and are commonly called “mesenchymal stem cells” (MSCs). A stem cell is defined as having robust clonal self-renewal and multilineage differentiation potential. Accordingly, the term “MSC” has been criticised, as there is little data demonstrating self-renewal of definitive single-cell-derived (SCD) clonal populations from a mesenchymal cell source.

Methodology/Principal Findings

Here we show that a tractable MPC population, human umbilical cord perivascular cells (HUCPVCs), was capable of multilineage differentiation in vitro and, more importantly, contributed to rapid connective tissue healing in vivo by producing bone, cartilage and fibrous stroma. Furthermore, HUCPVCs exhibit a high clonogenic frequency, allowing us to isolate definitive SCD parent and daughter clones from mixed gender suspensions as determined by Y-chromosome fluorescent in situ hybridization.

Conclusions/Significance

Analysis of the multilineage differentiation capacity of SCD parent clones and daughter clones enabled us to formulate a new hierarchical schema for MSC self-renewal and differentiation in which a self-renewing multipotent MSC gives rise to more restricted self-renewing progenitors that gradually lose differentiation potential until a state of complete restriction to the fibroblast is reached.     

A New Strategy Using ALDHhigh-CD8+T Cells to Inhibit Tumorigenesis

Background

Currently, many studies suggest that cancer stem cells (CSCs) are responsible for tumor initiation, tumorigenesis, metastasis and recurrence. CSCs have been identified from various human and murine tumors. The identification of CSCs allows us to develop strategies to target the CSCs.

Methods and Results

In this study, we used ALDEFLUOR as a single marker to isolate the CSCs from the human lung cancer cell line H460. We then characterized the CSCs by testing their sphere formation ability and tumorigenicity. Furthermore, we used CSC lysate-pulsed dendritic cells to stimulate CD8+T cells as a treatment strategy. Our study demonstrated that ALDEFLUOR could be used as a single marker to identify CSCs from the human lung cancer cell line H460. The ALDH^high cells could form more spheres and were more tumorigenic than the ALDH^low cells. Further study demonstrated that ALDH^high-CD8+T cells conferred more significant antitumor effects, resulting in the inhibition of tumor growth and prolonged survival. And the ALDH^high-CD8+T cells-mediated anti-tumor immunity might be due to the directly targeting against ALDH^high cancer stem cells (CSCs).

Conclusions

This study shows that ALDH^high-CD8+T cells mediate anti-tumor immunity by selectively targeting cancer stem cells, which result in inhibiting tumor growth and prolonging the survival of tumor-bearing mice, which provides a new strategy using ALDH^high-CD8+T cells to treat tumors.     

Drug-selected human lung cancer stem cells: cytokine network, tumorigenic and metastatic properties

Background

Cancer stem cells (CSCs) are thought to be responsible for tumor regeneration after chemotherapy, although direct confirmation of this remains forthcoming. We therefore investigated whether drug treatment could enrich and maintain CSCs and whether the high tumorogenic and metastatic abilities of CSCs were based on their marked ability to produce growth and angiogenic factors and express their cognate receptors to stimulate tumor cell proliferation and stroma formation.

Methodology/Findings

Treatment of lung tumor cells with doxorubicin, cisplatin, or etoposide resulted in the selection of drug surviving cells (DSCs). These cells expressed CD133, CD117, SSEA-3, TRA1-81, Oct-4, and nuclear β-catenin and lost expression of the differentiation markers cytokeratins 8/18 (CK 8/18). DSCs were able to grow as tumor spheres, maintain self-renewal capacity, and differentiate. Differentiated progenitors lost expression of CD133, gained CK 8/18 and acquired drug sensitivity. In the presence of drugs, differentiation of DSCs was abrogated allowing propagation of cells with CSC-like characteristics. Lung DSCs demonstrated high tumorogenic and metastatic potential following inoculation into SCID mice, which supported their classification as CSCs. Luminex analysis of human and murine cytokines in sonicated lysates of parental- and CSC-derived tumors revealed that CSC-derived tumors contained two- to three-fold higher levels of human angiogenic and growth factors (VEGF, bFGF, IL-6, IL-8, HGF, PDGF-BB, G-CSF, and SCGF-β). CSCs also showed elevated levels of expression of human VEGFR2, FGFR2, CXCR1, 2 and 4 receptors. Moreover, human CSCs growing in SCID mice stimulated murine stroma to produce elevated levels of angiogenic and growth factors.

Conclusions/Significance

These findings suggest that chemotherapy can lead to propagation of CSCs and prevention of their differentiation. The high tumorigenic and metastatic potentials of CSCs are associated with efficient cytokine network production that may represent a target for increased efficacy of cancer therapy.     

Mesenchymal Stem Cells Promote Mammosphere Formation and Decrease E-Cadherin in Normal and Malignant Breast Cells

Introduction

Normal and malignant breast tissue contains a rare population of multi-potent cells with the capacity to self-renew, referred to as stem cells, or tumor initiating cells (TIC). These cells can be enriched by growth as “mammospheres” in three-dimensional cultures.

Objective

We tested the hypothesis that human bone-marrow derived mesenchymal stem cells (MSC), which are known to support tumor growth and metastasis, increase mammosphere formation.

Results

We found that MSC increased human mammary epithelial cell (HMEC) mammosphere formation in a dose-dependent manner. A similar increase in sphere formation was seen in human inflammatory (SUM149) and non-inflammatory breast cancer cell lines (MCF-7) but not in primary inflammatory breast cancer cells (MDA-IBC-3). We determined that increased mammosphere formation can be mediated by secreted factors as MSC conditioned media from MSC spheroids significantly increased HMEC, MCF-7 and SUM149 mammosphere formation by 6.4 to 21-fold. Mammospheres grown in MSC conditioned media had lower levels of the cell adhesion protein, E-cadherin, and increased expression of N-cadherin in SUM149 and HMEC cells, characteristic of a pro-invasive mesenchymal phenotype. Co-injection with MSC in vivo resulted in a reduced latency time to develop detectable MCF-7 and MDA-IBC-3 tumors and increased the growth of MDA-IBC-3 tumors. Furthermore, E-cadherin expression was decreased in MDA-IBC-3 xenografts with co-injection of MSC.

Conclusions

MSC increase the efficiency of primary mammosphere formation in normal and malignant breast cells and decrease E-cadherin expression, a biologic event associated with breast cancer progression and resistance to therapy.     

Human Bone Marrow Mesenchymal Stem Cells Display Anti-Cancer Activity in SCID Mice Bearing Disseminated Non-Hodgkin's Lymphoma Xenografts

Background

Although multimodality treatment can induce high rate of remission in many subtypes of non-Hodgkin''s lymphoma (NHL), significant proportions of patients relapse with incurable disease. The effect of human bone marrow (BM) mesenchymal stem cells (MSC) on tumor cell growth is controversial, and no specific information is available on the effect of BM-MSC on NHL.

Methodology/Principal Findings

The effect of BM-MSC was analyzed in two in vivo models of disseminated non-Hodgkin''s lymphomas with an indolent (EBV⁻ Burkitt-type BJAB, median survival = 46 days) and an aggressive (EBV⁺ B lymphoblastoid SKW6.4, median survival = 27 days) behavior in nude-SCID mice. Intra-peritoneal (i.p.) injection of MSC (4 days after i.p. injection of lymphoma cells) significantly increased the overall survival at an optimal MSC∶lymphoma ratio of 1∶10 in both xenograft models (BJAB+MSC, median survival = 58.5 days; SKW6.4+MSC, median survival = 40 days). Upon MSC injection, i.p. tumor masses developed more slowly and, at the histopathological observation, exhibited a massive stromal infiltration coupled to extensive intra-tumor necrosis. In in vitro experiments, we found that: i) MSC/lymphoma co-cultures modestly affected lymphoma cell survival and were characterized by increased release of pro-angiogenic cytokines with respect to the MSC, or lymphoma, cultures; ii) MSC induce the migration of endothelial cells in transwell assays, but promoted endothelial cell apoptosis in direct MSC/endothelial cell co-cultures.

Conclusions/Significance

Our data demonstrate that BM-MSC exhibit anti-lymphoma activity in two distinct xenograft SCID mouse models of disseminated NHL.     

EGFR Inhibition Abrogates Leiomyosarcoma Cell Chemoresistance through Inactivation of Survival Pathways and Impairment of CSC Potential

Background

Tumor cells with stem-like phenotype and properties, known as cancer stem cells (CSC), have been identified in most solid tumors and are presumed to be responsible for driving tumor initiation, chemoresistance, relapse, or metastasis. A subpopulation of cells with increased stem-like potential has also been identified within sarcomas. These cells are endowed with increased tumorigenic potential, chemoresistance, expression of embryonic markers, and side population(SP) phenotype. Leiomyosarcomas (LMS) are soft tissue sarcomas presumably arising from undifferentiated cells of mesenchymal origin, the Mesenchymal Stem Cells (MSC). Frequent recurrence of LMS and chemoresistance of relapsed patients may likely result from the failure to target CSC. Therefore, therapeutic cues coming from the cancer stem cell (CSC) field may drastically improve patient outcome.

Methodology/Principal Findings

We expanded LMS stem-like cells from patient samples in vitro and examined the possibility to counteract LMS malignancy through a stem-like cell effective approach. LMS stem-like cells were in vitro expanded both as “tumor spheres” and as “monolayers” in Mesenchymal Stem Cell (MSC) conditions. LMS stem-like cells displayed MSC phenotype, higher SP fraction, and increased drug-extrusion, extended proliferation potential, self-renewal, and multiple differentiation ability. They were chemoresistant, highly tumorigenic, and faithfully reproduced the patient tumor in mice. Such cells displayed activation of EGFR/AKT/MAPK pathways, suggesting a possibility in overcoming their chemoresistance through EGFR blockade. IRESSA plus Vincristine treatment determined pathway inactivation, impairment of SP phenotype, high cytotoxicity in vitro and strong antitumor activity in stem-like cell-generated patient-like xenografts, targeting both stem-like and differentiated cells.

Conclusions/Significance

EGFR blockade combined with vincristine determines stem-like cell effective antitumor activity in vitro and in vivo against LMS, thus providing a potential therapy for LMS patients.     

Resveratrol inhibits pancreatic cancer stem cell characteristics in human and KrasG12D transgenic mice by inhibiting pluripotency maintaining factors and epithelial-mesenchymal transition

Background

Cancer stem cells (CSCs) can proliferate and self-renew extensively due to their ability to express anti-apoptotic and drug resistant proteins, thus sustaining tumor growth. Therefore, the strategy to eradicate CSCs might have significant clinical implications. The objectives of this study were to examine the molecular mechanisms by which resveratrol inhibits stem cell characteristics of pancreatic CSCs derived from human primary tumors and Kras^G12D transgenic mice.

Methodology/Principal Findings

Human pancreatic CSCs (CD133⁺CD44⁺CD24⁺ESA⁺) are highly tumorigenic and form subcutaneous tumors in NOD/SCID mice. Human pancreatic CSCs expressing high levels of CD133, CD24, CD44, ESA, and aldehyde dehydrogenase also express significantly more Nanog, Oct-4, Notch1, MDR1 and ABCG2 than normal pancreatic tissues and primary pancreatic cancer cells. Similarly, CSCs from Kras^G12D mice express significantly higher levels of Nanog and Oct-4 than pancreatic tissues from Pdx-Cre mice. Resveratrol inhibits the growth (size and weight) and development (PanIN lesions) of pancreatic cancer in Kras^G12D mice. Resveratrol inhibits the self-renewal capacity of pancreatic CSCs derived from human primary tumors and Kras^G12D mice. Resveratrol induces apoptosis by activating capase-3/7 and inhibiting the expression of Bcl-2 and XIAP in human CSCs. Resveratrol inhibits pluripotency maintaining factors (Nanog, Sox-2, c-Myc and Oct-4) and drug resistance gene ABCG2 in CSCs. Inhibition of Nanog by shRNA enhances the inhibitory effects of resveratrol on self-renewal capacity of CSCs. Finally, resveratrol inhibits CSC''s migration and invasion and markers of epithelial-mesenchymal transition (Zeb-1, Slug and Snail).

Conclusions/Significance

These data suggest that resveratrol inhibits pancreatic cancer stem cell characteristics in human and Kras^G12D transgenic mice by inhibiting pluripotency maintaining factors and epithelial-mesenchymal transition. In conclusion, resveratrol can be used for the management of pancreatic cancer.     

Recovery of neurological function of ischemic stroke by application of conditioned medium of bone marrow mesenchymal stem cells derived from normal and cerebral ischemia rats

Background

Several lines of evidence have demonstrated that bone marrow-derived mesenchymal stem cells (BM-MSC) release bioactive factors and provide neuroprotection for CNS injury. However, it remains elusive whether BM-MSC derived from healthy donors or stroke patients provides equal therapeutic potential. The present work aims to characterize BM-MSC prepared from normal healthy rats (NormBM-MSC) and cerebral ischemia rats (IschBM-MSC), and examine the effects of their conditioned medium (Cm) on ischemic stroke animal model.

Results

Isolated NormBM-MSC or IschBM-MSC formed fibroblastic like morphology and expressed CD29, CD90 and CD44 but failed to express the hematopoietic marker CD34. The number of colony formation of BM-MSC was more abundant in IschBM-MSC than in NormBM-MSC. This is in contrast to the amount of Ficoll-fractionated mononuclear cells from normal donor and ischemic rats. The effect of cm of BM-MSC was further examined in cultures and in middle cerebral artery occlusion (MCAo) animal model. Both NormBM-MSC Cm and IschBM-MSC Cm effectively increased neuronal connection and survival in mixed neuron-glial cultures. In vivo, intravenous infusion of NormBM-MSC Cm and IschBM-MSC Cm after stroke onset remarkably improved functional recovery. Furthermore, NormBM-MSC Cm and IschBM-MSC Cm increased neurogenesis and attenuated microglia/ macrophage infiltration in MCAo rat brains.

Conclusions

Our data suggest equal effectiveness of BM-MSC Cm derived from ischemic animals or from a normal population. Our results thus revealed the potential of BM-MSC Cm on treatment of ischemic stroke.     

Curcumin Suppresses Crosstalk between Colon Cancer Stem Cells and Stromal Fibroblasts in the Tumor Microenvironment: Potential Role of EMT

Objective

Interaction of stromal and tumor cells plays a dynamic role in initiating and enhancing carcinogenesis. In this study, we investigated the crosstalk between colorectal cancer (CRC) cells with stromal fibroblasts and the anti-cancer effects of curcumin and 5-Fluorouracil (5-FU), especially on cancer stem cell (CSC) survival in a 3D-co-culture model that mimics in vivo tumor microenvironment.

Methods

Colon carcinoma cells HCT116 and MRC-5 fibroblasts were co-cultured in a monolayer or high density tumor microenvironment model in vitro with/without curcumin and/or 5-FU.

Results

Monolayer tumor microenvironment co-cultures supported intensive crosstalk between cancer cells and fibroblasts and enhanced up-regulation of metastatic active adhesion molecules (β1-integrin, ICAM-1), transforming growth factor-β signaling molecules (TGF-β3, p-Smad2), proliferation associated proteins (cyclin D1, Ki-67) and epithelial-to-mesenchymal transition (EMT) factor (vimentin) in HCT116 compared with tumor mono-cultures. High density tumor microenvironment co-cultures synergistically increased tumor-promoting factors (NF-κB, MMP-13), TGF-β3, favored CSC survival (characterized by up-regulation of CD133, CD44, ALDH1) and EMT-factors (increased vimentin and Slug, decreased E-cadherin) in HCT116 compared with high density HCT116 mono-cultures. Interestingly, this synergistic crosstalk was even more pronounced in the presence of 5-FU, but dramatically decreased in the presence of curcumin, inducing biochemical changes to mesenchymal-epithelial transition (MET), thereby sensitizing CSCs to 5-FU treatment.

Conclusion

Enrichment of CSCs, remarkable activation of tumor-promoting factors and EMT in high density co-culture highlights that the crosstalk in the tumor microenvironment plays an essential role in tumor development and progression, and this interaction appears to be mediated at least in part by TGF-β and EMT. Modulation of this synergistic crosstalk by curcumin might be a potential therapy for CRC and suppress metastasis.     

Efficient generation of multipotent mesenchymal stem cells from umbilical cord blood in stroma-free liquid culture

Background

Haematopoiesis is sustained by haematopoietic (HSC) and mesenchymal stem cells (MSC). HSC are the precursors for blood cells, whereas marrow, stroma, bone, cartilage, muscle and connective tissues derive from MSC. The generation of MSC from umbilical cord blood (UCB) is possible, but with low and unpredictable success. Here we describe a novel, robust stroma-free dual cell culture system for long-term expansion of primitive UCB-derived MSC.

Methods and Findings

UCB-derived mononuclear cells (MNC) or selected CD34⁺ cells were grown in liquid culture in the presence of serum and cytokines. Out of 32 different culture conditions that have been tested for the efficient expansion of HSC, we identified one condition (DMEM, pooled human AB serum, Flt-3 ligand, SCF, MGDF and IL-6; further denoted as D7) which, besides supporting HSC expansion, successfully enabled long-term expansion of stromal/MSC from 8 out of 8 UCB units (5 MNC-derived and 3 CD34⁺ selected cells). Expanded MSC displayed a fibroblast-like morphology, expressed several stromal/MSC-related antigens (CD105, CD73, CD29, CD44, CD133 and Nestin) but were negative for haematopoietic cell markers (CD45, CD34 and CD14). MSC stemness phenotype and their differentiation capacity in vitro before and after high dilution were preserved throughout long-term culture. Even at passage 24 cells remained Nestin⁺, CD133⁺ and >95% were positive for CD105, CD73, CD29 and CD44 with the capacity to differentiate into mesodermal lineages. Similarly we show that UCB derived MSC express pluripotency stem cell markers despite differences in cell confluency and culture passages.Further, we generated MSC from peripheral blood (PB) MNC of 8 healthy volunteers. In all cases, the resulting MSC expressed MSC-related antigens and showed the capacity to form CFU-F colonies.

Conclusions

This novel stroma-free liquid culture overcomes the existing limitation in obtaining MSC from UCB and PB enabling so far unmet therapeutic applications, which might substantially affect clinical practice.     

De-Regulated MicroRNAs in Pediatric Cancer Stem Cells Target Pathways Involved in Cell Proliferation,Cell Cycle and Development

Background

microRNAs (miRNAs) have been implicated in the control of many biological processes and their deregulation has been associated with many cancers. In recent years, the cancer stem cell (CSC) concept has been applied to many cancers including pediatric. We hypothesized that a common signature of deregulated miRNAs in the CSCs fraction may explain the disrupted signaling pathways in CSCs.

Methodology/Results

Using a high throughput qPCR approach we identified 26 CSC associated differentially expressed miRNAs (DEmiRs). Using BCmicrO algorithm 865 potential CSC associated DEmiR targets were obtained. These potential targets were subjected to KEGG, Biocarta and Gene Ontology pathway and biological processes analysis. Four annotated pathways were enriched: cell cycle, cell proliferation, p53 and TGF-beta/BMP. Knocking down hsa-miR-21-5p, hsa-miR-181c-5p and hsa-miR-135b-5p using antisense oligonucleotides and small interfering RNA in cell lines led to the depletion of the CSC fraction and impairment of sphere formation (CSC surrogate assays).

Conclusion

Our findings indicated that CSC associated DEmiRs and the putative pathways they regulate may have potential therapeutic applications in pediatric cancers.     

Photodynamic Therapy with 5-Aminolevulinic acid (ALA) Impairs Tumor Initiating and Chemo-Resistance Property in Head and Neck Cancer-Derived Cancer Stem Cells

Background

Head and neck cancer (HNC) ranks the fourth leading malignancy and cancer death in male population in Taiwan. Despite recent therapeutic advances, the prognosis for HNC patients is still dismal. New strategies are urgently needed to improve the chemosensitization to conventional chemotherapeutic drugs and clinical responses of HNC patients. Studies have demonstrated that topical 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) is being used in the treatment of various human premalignant and malignant lesions with some encouraging clinical outcomes. However, the molecular mechanisms of ALA-PDT in the therapeutic effect in HNC tumorigenesis and whether ALA-PDT as chemosensitizer for HNC treatment remain unclear. Accumulating data support cancer stem cells (CSCs) contributes chemo-resistance in HNC. Based on the previous studies, the purpose of the study is to investigate the effect of ALA-PDT on CSCs and chemosensitization property in HNC.

Methodology/Principal Finding

CSCs marker ALDH1 activity of HNC cells with ALA-PDT treatment as assessed by the Aldefluor assay flow cytometry analysis. Secondary Sphere-forming self-renewal, stemness markers expression, and invasiveness of HNC-CSCs with ALA-PDT treatment were presented. We observed that the treatment of ALA-PDT significantly down-regulated the ALDH1 activity and CD44 positivity of HNC-CSCs. Moreover, ALA-PDT reduced self-renewal property and stemness signatures expression (Oct4 and Nanog) in sphere-forming HNC-CSCs. ALA-PDT sensitized highly tumorigenic HNC-CSCs to conventional chemotherapies. Lastly, synergistic effect of ALA-PDT and Cisplatin treatment attenuated invasiveness/colongenicity property in HNC-CSCs.

Conclusion/Significance

Our results provide insights into the clinical prospect of ALA-PDT as a potential chemo-adjuvant therapy against head and neck cancer through eliminating CSCs property.     

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.