Men-Luh Yen

Isolation of multipotent cells from human term placenta.

Current sources of stem cells include embryonic stem cells (ESCs) and adult stem cells (ASCs). However, concerns exist with either source: ESCs, with their significant ethical considerations, tumorigenicity, and paucity of cell lines; and ASCs, which are possibly more limited in potential. Thus, the search continues for an ethically conducive, easily accessible, and high‐yielding source of stem cells. We have isolated a population of multipotent cells from the human term placenta, a temporary organ with fetal contributions that is discarded postpartum. These placenta‐derived multipotent cells (PDMCs) exhibit many markers common to mesenchymal stem cells—including CD105/endoglin/SH‐2, SH‐3, and SH‐4—and they lack hematopoietic‐, endothelial‐, and trophoblastic‐specific cell markers. In addition, PDMCs exhibit ESC surface markers of SSEA‐4, TRA‐1‐61, and TRA‐1‐80. Adipogenic, osteogenic, and neurogenic differentiation were achieved after culturing under the appropriate conditions. PDMCs could provide an ethically uncontroversial and easily accessible source of multipotent cells for future experimental and clinical applications.

Resveratrol promotes osteogenesis of human mesenchymal stem cells by upregulating RUNX2 gene expression via the SIRT1/FOXO3A axis

Reports of the bone‐protective effects of resveratrol, a naturally occurring phytoestrogen and agonist for the longevity gene SIRT1, have highlighted this compound as a candidate for therapy of osteoporosis. Moreover, SIRT1 antagonism enhances adipogenesis. There has been speculation that resveratrol can promote osteogenesis through SIRT1, but the mechanism remains unclear. In this study we investigated the molecular mechanism of how resveratrol can modulate the lineage commitment of human mesenchymal stem cells to osteogenesis other than adipogenesis. We found that resveratrol promoted spontaneous osteogenesis but prevented adipogenesis in human embryonic stem cell–derived mesenchymal progenitors. Resveratrol upregulated the expression of osteo‐lineage genes RUNX2 and osteocalcin while suppressing adipo‐lineage genes PPARγ2 and LEPTIN in adipogenic medium. Furthermore, we found that the osteogenic effect of resveratrol was mediated mainly through SIRT1/FOXO3A with a smaller contribution from the estrogenic pathway. Resveratrol activated SIRT1 activity and enhanced FOXO3A protein expression, a known target of SIRT1, in an independent manner. As a result, resveratrol increased the amount of the SIRT1‐FOXO3A complex and enhanced FOXO3A‐dependent transcriptional activity. Ectopic overexpression or silencing of SIRT1/FOXO3A expression regulated RUNX2 promoter activity, suggesting an important role for SIRT1‐FOXO3A complex in regulating resveratrol‐induced RUNX2 gene transcription. Further mutational RUNX2 promoter analysis and chromatin immunoprecipitation assay revealed that resveratrol‐induced SIRT1‐FOXO3A complex bound to a distal FOXO response element (−1269/−1263), an action that transactivated RUNX2 promoter activity in vivo. Taken together, our results describe a novel mechanism of resveratrol in promoting osteogenesis of human mesenchymal stem cells by upregulating RUNX2 gene expression via the SIRT1/FOXO3A axis.

Immunomodulatory properties of human adult and fetal multipotent mesenchymal stem cells

In recent years, a large number of studies have contributed to our understanding of the immunomodulatory mechanisms used by multipotent mesenchymal stem cells (MSCs). Initially isolated from the bone marrow (BM), MSCs have been found in many tissues but the strong immunomodulatory properties are best studied in BM MSCs. The immunomodulatory effects of BM MSCs are wide, extending to T lymphocytes and dendritic cells, and are therapeutically useful for treatment of immune-related diseases including graft-versus-host disease as well as possibly autoimmune diseases. However, BM MSCs are very rare cells and require an invasive procedure for procurement. Recently, MSCs have also been found in fetal-stage embryo-proper and extra-embryonic tissues, and these human fetal MSCs (F-MSCs) have a higher proliferative profile, and are capable of multilineage differentiation as well as exert strong immunomodulatory effects. As such, these F-MSCs can be viewed as alternative sources of MSCs. We review here the current understanding of the mechanisms behind the immunomodulatory properties of BM MSCs and F-MSCs. An increase in our understanding of MSC suppressor mechanisms will offer insights for prevalent clinical use of these versatile adult stem cells in the near future.

Brief Report—Human Embryonic Stem Cell‐Derived Mesenchymal Progenitors Possess Strong Immunosuppressive Effects Toward Natural Killer Cells as Well as T Lymphocytes

The derivation of mesenchymal progenitors from human embryonic stem cells (hESCs) has recently been reported. We studied the immune characteristics of these hESC‐derived mesenchymal progenitors (EMPs) and their interactions with T lymphocytes and natural killer cells (NKs), two populations of lymphocytes with important roles in transplantation immunology. EMPs express a number of bone marrow mesenchymal stromal cell (BMMSC) markers, as well as the hESC marker SSEA‐4. Immunologically, EMPs do not express HLA‐DR or costimulatory molecules. On the other hand, HLA‐G, a nonclassic MHC I protein involved in mediating maternal‐fetal tolerance, can be found on the surface of EMPs, and its expression is increased after interferon‐γ stimulation. EMPs can suppress CD4+ or CD8+ lymphocyte proliferation, similar to BMMSCs. However, EMPs are more resistant to NK‐mediated lysis than BMMSCs and can suppress the cytotoxic effects of activated NKs, as well as downregulating the NK‐activating receptors NKp30 and NKp46. With their broad immunosuppressive properties, EMPs may represent a new potential cell source for therapeutic use. STEM CELLS 2009;27:451–456

Spontaneous osteogenesis of MSCs cultured on 3D microcarriers through alteration of cytoskeletal tension.

3-dimensional microcarrier (3D-MC) cell culture systems are often used for expansion of stem cells including mesenchymal stem cells (MSCs) for high cell volumes required in clinical applications. However, compared to 2-dimensional (2D) cell culture, effects of 3D-MC systems on MSC differentiation have not been well studied. In this study, the behavior of various sources of MSCs from two species was observed and compared on 3D collagen I-coated-MCs (COL-MC) versus 2D culture. Proliferation of all MSCs cultured on 3D COL-MC was much decreased compared to 2D culture. Unexpectedly, COL-MC-cultured MSCs underwent spontaneous osteogenesis without exogenous addition of biochemical factors, as evidenced by increased osteogenic genes expression, ALP activity, calcium deposition, and collagen I secretion. Furthermore, MSCs cultured on 3D-MC alone without collagen I coating is sufficient to induce osteogenesis. The spontaneous lineage commitment induced by 3D-MC culture was mediated by increased cytoskeletal tension and actomyosin contraction of MSCs, which could be prevented by latrunculin B and blebbistatin, inhibitors of cytoskeletal tension and actomyosin contraction respectively. Our findings show that the combination of bioengineered MC and MSCs alone can induce specific lineage commitment very efficiently. These data have strong implications in simplifying tissue engineering strategies for therapeutic applications.

The H3K9 methyltransferase G9a is a marker of aggressive ovarian cancer that promotes peritoneal metastasis

BackgroundOvarian cancer (OCa) peritoneal metastasis is the leading cause of cancer–related deaths in women with limited therapeutic options available for treating it and poor prognosis, as the underlying mechanism is not fully understood.MethodThe clinicopathological correlation of G9a expression was assessed in tumor specimens of ovarian cancer patients. Knockdown or overexpression of G9a in ovarian cancer cell lines was analysed with regard to its effect on adhesion, migration, invasion and anoikis-resistance. In vivo biological functions of G9a were tested by i.p. xenograft ovarian cancer models. Microarray and quantitative RT-PCR were used to analyze G9a-regulated downstream target genes.ResultsWe found that the expression of histone methyltransferase G9a was highly correlated with late stage, high grade, and serous-type OCa. Higher G9a expression predicted a shorter survival in ovarian cancer patients. Furthermore, G9a expression was higher in metastatic lesions compared with their corresponding ovarian primary tumors. Knockdown of G9a expression suppressed prometastatic cellular activities including adhesion, migration, invasion and anoikis-resistance of ovarian cancer cell lines, while G9a over-expression promoted these cellular properties. G9a depletion significantly attenuated the development of ascites and tumor nodules in a peritoneal dissemination model. Importantly, microarray and quantitative RT-PCR analysis revealed that G9a regulates a cohort of tumor suppressor genes including CDH1, DUSP5, SPRY4, and PPP1R15A in ovarian cancer. Expression of these genes was also inversely correlated with G9a expression in OCa specimens.ConclusionWe propose that G9a contributes to multiple steps of ovarian cancer metastasis and represents a novel target to combat this deadly disease.

Human mesenchymal stem cells (MSCs) for treatment towards immune- and inflammation-mediated diseases: review of current clinical trials

Human mesenchymal stem cells (MSCs) are multilineage somatic progenitor/stem cells that have been shown to possess immunomodulatory properties in recent years. Initially met with much skepticism, MSC immunomodulation has now been well reproduced across tissue sources and species to be clinically relevant. This has opened up the use of these versatile cells for application as 3rd party/allogeneic use in cell replacement/tissue regeneration, as well as for immune- and inflammation-mediated disease entities. Most surprisingly, use of MSCs for in immune-/inflammation-mediated diseases appears to yield more efficacy than for regenerative medicine, since engraftment of the exogenous cell does not appear necessary. In this review, we focus on this non-traditional clinical use of a tissue-specific stem cell, and highlight important findings and trends in this exciting area of stem cell therapy.

Multilineage Differentiation and Characterization of the Human Fetal Osteoblastic 1.19 Cell Line: A Possible In Vitro Model of Human Mesenchymal Progenitors

The in vitro study of human bone marrow mesenchymal stromal cells (BMMSCs) has largely depended on the use of primary cultures. Although these are excellent model systems, their scarcity, heterogeneity, and limited lifespan restrict their usefulness. This has led researchers to look for other sources of MSCs, and recently, such a population of progenitor/stem cells has been found in mesodermal tissues, including bone. We therefore hypothesized that a well‐studied and commercially available clonal human osteoprogenitor cell line, the fetal osteoblastic 1.19 cell line (hFOB), may have multilineage differentiation potential. We found that undifferentiated hFOB cells possess similar cell surface markers as BMMSCs and also express the embryonic stem cell‐related pluripotency gene, Oct‐4, as well as the neural progenitor marker nestin. hFOB cells can also undergo multilineage differentiation into the mesodermal lineages of chondrogenic and adipocytic cell types in addition to its predetermined pathway, the mature osteoblast. Moreover, as with BMMSCs, under neural‐inducing conditions, hFOB cells acquire a neural‐like phenotype. This human cell line has been a widely used model of normal osteoblast differentiation. Our data suggest that hFOB cells may provide for researchers an easily available, homogeneous, and consistent in vitro model for study of human mesenchymal progenitor cells.

Efficient Derivation and Concise Gene Expression Profiling of Human Embryonic Stem Cell-Derived Mesenchymal Progenitors (EMPs):

New potential sources of stem cells for clinical application include bone marrow mesenchymal stem cells (BMMSCs), human embryonic stem cells (hESCs), and induced pluripotent stem cells (iPS). However, each source is not without its own concerns. While research continues in an effort to overcome these problems, the generation of mesenchymal progenitors from existing hESC lines may circumvent many of these issues. We report here a simple and efficient method of generating hESC-derived mesenchymal progenitors (EMPs) and transcriptome profiling using a concise, custom-designed, oligomnucleotide gene expression microarray. Characterization of EMPs shows that these cells are similar to BMMSCs in terms of differentiation capacity as well as cell surface marker expression. In addition, EMPs express several ESC markers and HLA-G, a nonclassical MHC class I molecule with immunomodulatory properties. Morevoer, EMPs possess significantly enhanced proliferative ability over BMMSCs during which karyotypic stability was maintained. Although derived from hESCs, EMPs do not form any tumors in immunocompromised mice. To efficiently profile gene expression in multiple samples, we designed an oligoarray to probe just over 11,000 genes highly expressed in stem cells. We found that the transcriptome of EMPs is more similar to BMMSCs than hESCs. Both cell types highly express genes involved in processes related to the cytoskeleton, extracellular matrix, and cell adhesion, but EMPs show higher expression of genes involved in cell proliferation whereas BMMSCs showed higher expression of immune-related genes. Based on our data, EMPs may be an accessible source of mesenchymal progenitor for therapeutic use.

Current applications of human pluripotent stem cells: Possibilities and challenges

Stem cells are self-renewable cells with the differentiation capacity to develop into somatic cells with biological functions. This ability to sustain a renewable source of multi- and/or pluripotential differentiation has brought new hope to the field of regenerative medicine in terms of cell therapy and tissue engineering. Moreover, stem cells are invaluable tools as in vitro models for studying diverse fields, from basic scientific questions such as developmental processes and lineage commitment, to practical application including drug screening and testing. The stem cells with widest differentiation potential are pluripotent stem cells (PSCs), which are rare cells with the ability to generate somatic cells from all three germ layers. PSCs are considered the most optimal choice for therapeutic potential of stem cells, bringing new impetus to the field of regenerative medicine. In this article, we discuss the therapeutic potential of human PSCs (hPSCs) including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), reviewing the current preclinical and clinical data using these stem cells. We describe the classification of different sources of hPSCs, ongoing research, and currently encountered clinical obstacles of these novel and versatile human stem cells.