Research using Mesenchymal Stem/Stromal Cells: quality metric towards developing a reference material.

Mesenchymal stem/stromal cells (MSCs) have been extensively investigated for their regenerative, immune-modulatory, and wound healing properties. While the laboratory studies have suggested that MSC's have a unique potential for modulating the etiopathology of multiple diseases, the results from clinical trials have not been encouraging or reproducible. One of the explanations for such variability is explained by the "art" of isolating and propagating MSCs. Therefore, establishing more than minimal criteria to define MSC would help understand best protocols to isolate, propagate and deliver MSCs. Developing a calibration standard, a database and a set of functional tests would be a better quality metric for MSCs. In this review, we discuss the importance of selecting a standard, issues associated with coming up with such a standard and how these issues can be mitigated.

Somatic expression of LINE-1 elements in human tissues.

LINE-1 expression damages host DNA via insertions and endonuclease-dependent DNA double-strand breaks (DSBs) that are highly toxic and mutagenic. The predominant tissue of LINE-1 expression has been considered to be the germ line. We show that both full-length and processed L1 transcripts are widespread in human somatic tissues and transformed cells, with significant variation in both L1 expression and L1 mRNA processing. This is the first demonstration that RNA processing is a major regulator of L1 activity. Many tissues also produce translatable spliced transcript (SpORF2). An Alu retrotransposition assay, COMET assays and 53BP1 foci staining show that the SpORF2 product can support functional ORF2 protein expression and can induce DNA damage in normal cells. Tests of the senescence-associated beta-galactosidase expression suggest that expression of exogenous full-length L1, or the SpORF2 mRNA alone in human fibroblasts and adult stem cells triggers a senescence-like phenotype, which is one of the reported responses to DNA damage. In contrast to previous assumptions that L1 expression is germ line specific, the increased spectrum of tissues exposed to L1-associated damage suggests a role for L1 as an endogenous mutagen in somatic tissues. These findings have potential consequences for the whole organism in the form of cancer and mammalian aging.

Activation of autophagy in mesenchymal stem cells provides tumor stromal support.

Recent studies have implicated multipotential mesenchymal stem cells (MSCs) as an aid to breast cancer cell proliferation and metastasis, partly as a result of the MSCs secretome. As the tumor gets beyond 2 mm in diameter, the stromal cells could undergo starvation due to the lack of sufficient nutrients in solid tumor microenvironment. In this study, we investigated the survival mechanisms used by stressed stromal cells in breast cancers. We used serum-deprived mesenchymal stem cells (SD-MSCs) and MCF-7 breast cancer cells as model system with a hypothesis that stromal cells in the nutrient-deprived core utilize survival mechanisms for supporting surrounding cells. We tested this hypothesis using in vivo tumor xenografts in immunodeficient mice, which indicated that SD-MSCs supported MCF-7 tumor growth by protection from apoptosis. Histochemical assays showed that SD-MSCs-injected tumors exhibited higher cellularity, decreased apoptosis and decreased differentiation. Beclin-1 staining indicated autophagic areas surrounded by actively proliferating cells. Furthermore, in vitro studies demonstrate that SD-MSCs survive using autophagy and secrete paracrine factors that support tumor cells following nutrient/serum deprivation. Western blot and immunocytochemistry analysis of SD-MSCs demonstrated upregulation and perinuclear relocation of autophagy key regulators such as beclin-1, ATG10, ATG12, MAP-LC3 and lysosomes. Electron microscopic analysis detected a time-dependent increase in autophagosome formation and HDAC6 activity assays indicated the upregulation of autophagy. Taken together, these data suggest that under nutrient-deprived conditions that can occur in solid tumors, stromal cells utilize autophagy for survival and also secrete anti-apoptotic factors that can facilitate solid tumor survival and growth.

Epigenetic reprogramming of IGF1 and leptin genes by serum deprivation in multipotential mesenchymal stromal cells.

Recent studies on the therapeutic effect of multipotential mesenchymal stem cells (MSCs) in various models of injury have shown that paracrine factors secreted by MSCs are responsible for tissue repair with very little engraftment. In this study we tested the hypothesis that MSCs under stress undergo epigenetic modifications that direct secretion of paracrine factors responsible for tissue repair. Microarray assays of MSCs that had been deprived of serum (SD-MSCs), to induce stress, demonstrated an increase in the expression of several angiogenic, prosurvival, and antiapoptotic factors, including insulin-like growth factor 1 (IGF1) and leptin. Real-time polymerase chain reaction assays demonstrated a >200-fold increase in the expression of IGF1 and leptin in SD-MSCs. Chromatin immunoprecipitation of SD-MSCs revealed histone tail modifications consistent with transcriptional activation of IGF1 and leptin promoters in a reversible manner. To identify the functional significance of the epigenetic changes in stressed MSCs, we tested the prosurvival properties of SD-MSCs and the ability of conditioned medium from SD-MSCs to enhance survival of apoptotic cancer cells. First, we showed that SD-MSCs are more resistant to oxidative damage than MSCs using alkaline comet assays. Next, we demonstrated that conditioned medium from SD-MSCs decreased staurosporin-induced cell death in the KHOS osteosarcoma cell line, and that this effect was partially reversed by immunodepletion of IGF1 or leptin from the conditioned medium. In conclusion, we demonstrate that serum deprivation induces epigenetic changes in MSCs to upregulate the expression of the proangiogenic and antiapoptotic factors IGF1 and leptin.

Angiogenic effects of human multipotent stromal cell conditioned medium activate the PI3K-Akt pathway in hypoxic endothelial cells to inhibit apoptosis, increase survival, and stimulate angiogenesis.

Recent reports indicated that vascular remodeling and angiogenesis are promoted by conditioned medium from the cells referred to as multipotent stromal cells (MSCs). However, the molecular events triggered by MSC-conditioned medium (CdM) were not defined. We examined the effects of CdM from human MSCs on cultures of primary human aortic endothelial cells (HAECs). The CdM inhibited hypoxia-induced apoptosis and cell death of HAECs. It also promoted tube formation by HAECs in an assay in vitro. Conditioned medium from multipotent stromal cells incubated under hypoxic conditions in serum-free endothelial basal medium for 2 days (CdM(Hyp)) from hypoxic culture of MSCs was more effective than conditioned medium from MSCs incubated under normoxic conditions in serum-free endothelial basal medium for 2 days from normoxic cultures of MSCs, an observation in part explained by its higher content of antiapoptotic and angiogenic factors, such as interleukin (IL)-6, vascular endothelial growth factor (VEGF), and monocyte chemoattractant protein (MCP)-1. The effects of CdM(Hyp) on hypoxic HAECs were partially duplicated by the addition of IL-6 in a dose-dependent manner; however, anti-IL-6, anti-MCP-1, and anti-VEGF blocking antibodies added independently did not attenuate the effects. Also, addition of CdM(Hyp) activated the PI3K-Akt pathway; the levels of p-Akt and several of its downstream targets were increased by CdM(Hyp), and both the increase in p-Akt and the increase in angiogenesis were blocked by an inhibitor of PI3K-Akt or by expression of a dominant negative gene for PI3K. CdM(Hyp) also increased the levels of p-extracellular signal-regulated kinase (ERK), but there was a minimal effect on p-signal transducer and activator of transcription-3, and an inhibitor of the ERK1/2 pathway had no effect on hypoxia-induced apoptosis of the HAECs. The results are consistent with suggestions that administration of MSCs or factors secreted by MSCs may provide a therapeutic method of decreasing apoptosis and enhancing angiogenesis.

Polycomb group protein Suz12 is regulated by a novel miRNA-like small RNA.

Human mesenchymal stem/stromal cells (hMSCs) provide support for cancer progression, partly through their secretome that includes extracellular vesicles (EVs). Based on deep-sequencing of small RNA from EVs of MSCs, we now report the characterization of novel small RNA, named n-miR-G665, which exhibits typical properties of miRNAs. n-miR-G665 sequence is conserved and expressed in most cell types. Knockdown studies using anti-agomirs and shRNA studies demonstrated that n-miR-G665 plays an important role in cell proliferation. Functional assays to reveal the targets of n-miR-G665 showed that polycomb protein Suz12 is regulated by n-miR-G665, which in turn regulates the expression of n-miR-G665 through feedback loop mechanism. These data shed light on a previously unknown novel feedback regulatory mechanism for controlling Suz12 expression regulated by previously not described miRNA, which may highlight a new therapeutic approach to control the polycomb repressor complex 2 activity in cancers.

Histamine receptor H1 and dermatopontin: new downstream targets of the vitamin D receptor.

UNLABELLED: In this study, we used multipotential MSCs and microarray assays to follow the changing patterns of gene expression as MSCs were differentiated to osteoblasts. We analyzed co-expressed gene groups to identify new targets for known transcription factor VDR during differentiation. The roles of two genes (histamine receptor H1 and dermatopontin) as downstream targets for the VDR were confirmed by gel electromotility shift, siRNA inhibition, and chromatin immunoprecipitation assays. INTRODUCTION: Osteogenesis is stringently controlled by osteoblast-specific signaling proteins and transcription factors. Mesenchymal stem or multipotential stromal cells from bone marrow (MSCs) have been shown to differentiate into osteoblasts in the presence of vitamin D(3). MATERIALS AND METHODS: We used MSCs and microarray assays to follow the changing patterns of gene expression as MSCs were differentiated to osteoblasts. The data were analyzed with a previously developed strategy to identify new downstream targets of the vitamin D receptor (VDR), known osteogenesis transcription factor. Hierarchical clustering of the data identified 15 distinct patterns of gene expression. Three genes were selected that expressed in the same time-dependent pattern as osteocalcin, a known target for the VDR: histamine receptor H1 (HRH1), Spondin 2 (SPN), and dermatopontin (DPT). RT-PCR, electromotility shift, siRNA inhibition assays, and chromatin immunoprecipitation assays were used to analyze the role of VDR in activation of DPT and HRH1 during differentiation. RESULTS AND CONCLUSIONS: RT-PCR assays confirmed that the genes were expressed during differentiation of MSCs. The roles of two genes as downstream targets for the VDR were confirmed by gel electromotility shift and chromatin immunoprecipitation assays that showed the presence of VDR complex binding sequences. Overexpression of VDR in MG-63 osteosarcoma cells induced the expression of HRH1 and DPT. Inhibition studies with siRNA to DPT and HRH1 showed a decrease in MSC differentiation to osteogenic lineage. In addition, osteogenic differentiation of MSCs was inhibited by the HRH1 inhibitor mepyramine but not the HRH2 inhibitor ranitidine. In conclusion, we show that analysis of co-expressed gene groups is a good tool to identify new targets for known transcription factors.

Three dimensional tumor models for cancer studies.

It is well recognized that one of the major drawbacks of using traditional two dimensional cultures to model the living systems is inaccurately reflecting the physiological manner in which modulators, nutrients, oxygen, and metabolites are applied and removed. Moreover, the two dimensional culture system poorly reflects how different cell types interact with each other in the same microenvironment. Since the first global development of three dimensional (3D) cell culture techniques in the late 1960s, this last decade has seen an explosion of studies to promote 3D models in the fields of regenerative medicine and cancer. The recent surge of interest in 3D cell culture in cancer research is attributable to the interest in developing closer to real life models. The ability to include various cell types and extracellular components reflect more the physiological conditions of tumor microenvironment. In this short review, we will discuss different approaches of 3D culture system models and techniques with a focus on the 3D interactions of cancer cells with stromal cells in the goal to reevaluate old and develop new therapeutics.

High CD49f expression is associated with osteosarcoma tumor progression: a study using patient-derived primary cell cultures.

Overall prognosis for osteosarcoma (OS) is poor despite aggressive treatment options. Limited access to primary tumors, technical challenges in processing OS tissues, and the lack of well-characterized primary cell cultures has hindered our ability to fully understand the properties of OS tumor initiation and progression. In this study, we have isolated and characterized cell cultures derived from four central high-grade human OS samples. Furthermore, we used the cell cultures to study the role of CD49f in OS progression. Recent studies have implicated CD49f in stemness and multipotency of both cancer stem cells and mesenchymal stem cells. Therefore, we investigated the role of CD49f in osteosarcomagenesis. First, single cell suspensions of tumor biopsies were subcultured and characterized for cell surface marker expression. Next, we characterized the growth and differentiation properties, sensitivity to chemotherapy drugs, and anchorage-independent growth. Xenograft assays showed that cell populations expressing CD49f(hi) /CD90(lo) cell phenotype produced an aggressive tumor. Multiple lines of evidence demonstrated that inhibiting CD49f decreased the tumor-forming ability. Furthermore, the CD49f(hi) /CD90(lo) cell population is generating more aggressive OS tumor growth and indicating this cell surface marker could be a potential candidate for the isolation of an aggressive cell type in OSs.

Derivation and characterization of an extra-axial chordoma cell line (EACH-1) from a scapular tumor.

BACKGROUND: Extra-axial chordomas are rare low-grade malignant tumors thought to arise from notochordal remnants in the extra-axial skeleton. Few studies have been done on this neoplasm because of its rarity. In addition, there is a lack of a good in vitro model on which to perform more characterization. METHODS: We describe a twenty-eight-year-old man with a mass in the right scapula. Cytomorphology and immunohistochemistry, including brachyury staining, were used to formulate the final diagnosis. A fragment of the tumor was placed in culture, and cells obtained were injected subcutaneously in an immunocompromised mouse. From the tumor developed in mice, a cell line has been derived and characterized by fluorescence-activated cell-sorting analysis, karyotyping, clonogenicity, and cell and tumor growth curves. RESULTS: Cytomorphology on the tumor showed nests of round cells with vacuoles and also physaliferous-like cells with uniform nuclei. Immunochemistry revealed a tumor positive for vimentin, moderately positive for S-100 and cytokeratin AE1/AE3, weakly positive for epithelial membrane antigen, and negative for p63 and cytokeratin (CK)-7. Further analysis revealed the tumor was diffusely and strongly positive for brachyury. The cell line derived from the tumor showed rapid doubling-time, a strong expression of mesenchymal cell surface markers, a karyotype of diploid or hypotetraploid clones with numerous chromosomal aberrations, and the ability to form colonies without attachment and to form tumors in immunocompromised mice. CONCLUSIONS: The diagnosis of the extra-axial chordoma is difficult but can be resolved by the detection of a strong brachyury expression. In addition, the derivation of a human extra-axial chordoma cell line could be a useful tool for the basic research of this rare neoplasm.

CD117 and Stro-1 identify osteosarcoma tumor-initiating cells associated with metastasis and drug resistance.

Emerging evidence indicates the presence of tumor-initiating cells (TIC) or cancer stem cells in osteosarcoma. However, no study has shown specific markers to identify osteosarcoma TICs with in vivo tumor formation ability. Additionally, there has been a lack of investigations gauging the contribution of osteosarcoma TICs to metastatic and drug-resistant properties. In this study, we have identified mouse and human osteosarcoma TICs using mesenchymal stem cell markers CD117 and Stro-1. These markers were preferentially expressed in spheres and doxorubicin-resistant cells. Both mouse and human cells expressing these markers were sorted and analyzed for their abilities of tumor formation with as few as 200 cells, self-renewability, multipotency, drug resistance, metastatic potential, and enrichment of a metastasis-associated marker (CXCR4) and a drug resistance marker (ABCG2). CD117(+)Stro-1(+) cells efficiently formed serially transplantable tumors, whereas CD117(-)Stro-1(-) cells rarely initiated tumors. On orthotopic injections, CD117(+)Stro-1(+ )cell-derived tumors metastasized at a high frequency. Further, CD117(+)Stro-1(+) cells showed high invasive and drug-resistant properties and were efficiently enriched for CXCR4 (20-90%) and ABCG2 (60-90%). These results suggest possible mechanisms for the high metastatic and drug-resistant properties of osteosarcoma TICs. In summary, CD117 and Stro-1 identify osteosarcoma TICs associated with the most lethal characteristics of the disease-metastasis and drug resistance-and these markers offer candidates for TIC-targeted drug delivery aimed at eradicating osteosarcoma.

Short-term exposure of multipotent stromal cells to low oxygen increases their expression of CX3CR1 and CXCR4 and their engraftment in vivo.

The ability of stem/progenitor cells to migrate and engraft into host tissues is key to their potential use in gene and cell therapy. Among the cells of interest are the adherent cells from bone marrow, referred to as mesenchymal stem cells or multipotent stromal cells (MSC). Since the bone marrow environment is hypoxic, with oxygen tensions ranging from 1% to 7%, we decided to test whether hypoxia can upregulate chemokine receptors and enhance the ability of human MSCs to engraft in vivo. Short-term exposure of MSCs to 1% oxygen increased expression of the chemokine receptors CX3CR1and CXCR4, both as mRNA and as protein. After 1-day exposure to low oxygen, MSCs increased in vitro migration in response to the fractalkine and SDF-1alpha in a dose dependent manner. Blocking antibodies for the chemokine receptors significantly decreased the migration. Xenotypic grafting into early chick embryos demonstrated cells from hypoxic cultures engrafted more efficiently than cells from normoxic cultures and generated a variety of cell types in host tissues. The results suggest that short-term culture of MSCs under hypoxic conditions may provide a general method of enhancing their engraftment in vivo into a variety of tissues.

Use of differentiating adult stem cells (marrow stromal cells) to identify new downstream target genes for transcription factors.

We developed a strategy for use of microarray data to rapidly identify new downstream targets of transcription factors known to drive differentiation by following the time courses of gene expression as a relatively homogeneous population of stem/progenitor cells are differentiated to multiple phenotypes. Microarray assays were used to follow the differentiation of human marrow stromal cells (MSCs) into chondrocytes or adipocytes in three different experimental conditions. The steps of the analysis were the following: (a) hierarchical clustering was used to define groups of similarly behaving genes in each experiment, (b) candidates for new downstream targets of transcription factors that drive differentiation were then identified as genes that were consistently co-expressed with known downstream target genes of the transcription factors, and (c) the list of candidate new target genes was refined by identifying genes whose signal intensities showed a highly significant linear regression with the signal intensities of the known targets in all the data sets. Analysis of the data identified multiple new candidates for downstream targets for SOX9, SOX5, CCAAT/enhancer binding protein (C/EBP)-alpha, and peroxisome proliferator-activated receptor (PPAR)-gamma. To validate the analysis, we demonstrated that PPAR-gamma protein specifically bound to the promoters of four new targets identified in the analyses. The same multistep analysis can be used to identify new downstream targets of transcription factors in other systems. Also, the same analysis should make it possible to use MSCs from bone marrow to define new mutations that alter chondogenesis or adipogenesis in patients with a variety of syndromes.

Serum deprivation of human marrow stromal cells (hMSCs) selects for a subpopulation of early progenitor cells with enhanced expression of OCT-4 and other embryonic genes.

Recently there has been interest in developing cell and gene therapies with adult stem cells from human bone marrow referred to as mesenchymal stem cells or marrow stromal cells (hMSCs). We incubated early-passage hMSCs in serum-free medium without cytokines or other supplements for 2 to 4 weeks. Surprisingly, a subpopulation of the cells survived serum deprivation and then began to proliferate in serum-containing medium. The cells selected by serum deprivation had longer telomeres than control cells. Also, the patterns of gene expression revealed by reverse transcriptase-polymerase chain reaction (RT-PCR) assays and microarray data indicated that the cells selected by serum deprivation were a subpopulation of very early progenitor cells with enhanced expression of octomer-binding transcription factor 4 (OCT-4) and several other genes characteristically expressed in embryonic cells.

Rat adult stem cells (marrow stromal cells) engraft and differentiate in chick embryos without evidence of cell fusion.

Cell fusion was recently reported to account for the plasticity of adult stem cells in vivo. Adult stem cells, referred to as mesenchymal stem cells or marrow stromal cells, from rat marrow, were infused into 1.5- to 2-day-old chick embryos. After 4 days, the rat cells had expanded 1.3- to 33-fold in one-third of surviving embryos. The cells engrafted into many tissues, and no multinuclear cells were detected. The most common site of engraftment was the heart, apparently because the cells were infused just above the dorsal aorta. Some of the cells in the heart expressed cardiotin, and alpha-heavy-chain myosin. GFP(+) cells reisolated from the embryos had a rat karyotype. Therefore, the cells engrafted and partially differentiated without evidence of cell fusion.