Tian Sheng Chen

Exosome secreted by MSC reduces myocardial ischemia/reperfusion injury.

Human ESC-derived mesenchymal stem cell (MSC)-conditioned medium (CM) was previously shown to mediate cardioprotection during myocardial ischemia/reperfusion injury through large complexes of 50-100 nm. Here we show that these MSCs secreted 50- to 100-nm particles. These particles could be visualized by electron microscopy and were shown to be phospholipid vesicles consisting of cholesterol, sphingomyelin, and phosphatidylcholine. They contained coimmunoprecipitating exosome-associated proteins, e.g., CD81, CD9, and Alix. These particles were purified as a homogeneous population of particles with a hydrodynamic radius of 55-65 nm by size-exclusion fractionation on a HPLC. Together these observations indicated that these particles are exosomes. These purified exosomes reduced infarct size in a mouse model of myocardial ischemia/reperfusion injury. Therefore, MSC mediated its cardioprotective paracrine effect by secreting exosomes. This novel role of exosomes highlights a new perspective into intercellular mediation of tissue injury and repair, and engenders novel approaches to the development of biologics for tissue repair.

Mesenchymal stem cell exosome: a novel stem cell-based therapy for cardiovascular disease

Cardiovascular disease is a major target for many experimental stem cell-based therapies and mesenchymal stem cells (MSCs) are widely used in these therapies. Transplantation of MSCs to treat cardiac disease has always been predicated on the hypothesis that these cells would engraft, differentiate and replace damaged cardiac tissues. However, experimental or clinical observations so far have failed to demonstrate a therapeutically relevant level of transplanted MSC engraftment or differentiation. Instead, they indicate that transplanted MSCs secrete factors to reduce tissue injury and/or enhance tissue repair. Here we review the evidences supporting this hypothesis including the recent identification of exosome as a therapeutic agent in MSC secretion. In particular, we will discuss the potential and practicality of using this relatively novel entity as a therapeutic modality for the treatment of cardiac disease, particularly acute myocardial infarction.

Derivation and characterization of human fetal MSCs: An alternative cell source for large-scale production of cardioprotective microparticles

The therapeutic effects of mesenchymal stem cells (MSCs) transplantation are increasingly thought to be mediated by MSC secretion. We have previously demonstrated that human ESC-derived MSCs (hESC-MSCs) produce cardioprotective microparticles in pig model of myocardial ischemia/reperfusion (MI/R) injury. As the safety and availability of clinical grade human ESCs remain a concern, MSCs from fetal tissue sources were evaluated as alternatives. Here we derived five MSC cultures from limb, kidney and liver tissues of three first trimester aborted fetuses and like our previously described hESC-derived MSCs; they were highly expandable and had similar telomerase activities. Each line has the potential to generate at least 10(16-19) cells or 10(7-10) doses of cardioprotective secretion for a pig model of MI/R injury. Unlike previously described fetal MSCs, they did not express pluripotency-associated markers such as Oct4, Nanog or Tra1-60. They displayed a typical MSC surface antigen profile and differentiated into adipocytes, osteocytes and chondrocytes in vitro. Global gene expression analysis by microarray and qRT-PCR revealed a typical MSC gene expression profile that was highly correlated among the five fetal MSC cultures and with that of hESC-MSCs (r(2)>0.90). Like hESC-MSCs, they produced secretion that was cardioprotective in a mouse model of MI/R injury. HPLC analysis of the secretion revealed the presence of a population of microparticles with a hydrodynamic radius of 50-65 nm. This purified population of microparticles was cardioprotective at approximately 1/10 dosage of the crude secretion.

Establishment of medakafish as a model for stem cell-based gene therapy: efficient gene delivery and potential chromosomal integration by baculoviral vectors.

Viral vectors hold promise and challenges in gene therapy. Specifically, we have previously shown that baculoviral (BV) vectors have a high efficiency of gene delivery in human embryonic stem (ES) cells. Here we report the development of a complementary system to further our evaluation by utilizing the laboratory fish medaka that has ES cell lines and tools for experimental analyses in vitro and in vivo. We show that BV vectors can give rise to almost 100% of transient gene delivery in the medaka ES cell line MES1. BV-transduced MES1 cells reproducibly (at approximately 10(-5)) produce GFP-expressing colonies that, upon manual isolation, develop into stable clones during 300 days of culture. Surprisingly, BV transduction can also mediate efficient gene integration in the medaka genome, as fluorescent in situ hybridization revealed the presence of the BV-delivered gfp transgene in multiple locations in nuclei and on various chromosomes of metaphase spreads. We show that BV transduction does not compromise the genome stability and pluripotency of MES1 cells. We conclude that BV can efficiently mediate gene delivery and chromosomal integration in medaka ES cells. Therefore, medaka provides a powerful system for analyzing the potential of BV-mediated gene delivery in stem cells and gene therapy.

Measurement of Precursor miRNA in Exosomes from Human ESC-Derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) derived from human embryonic stem cells (ESCs) have been shown to secrete exosomes that are cardioprotective against myocardial ischemia reperfusion injury in a mouse model. To elucidate this cardioprotective mechanism, we have characterized the protein, nucleic acid, and lipid composition of MSC exosomes. Here we describe the isolation and analysis of RNA in MSC exosome. We have previously reported that RNAs in MSC exosome are primarily small RNA molecules of <300 nt and they include many miRNAs. Many of these miRNAs are in the precursor form suggesting that pre-miRNAs, and not mature miRNAs are preferentially loaded into exosomes. The protocols described here include assays to ascertain the presence of pre-miRNAs, profiling of miRNA and pre-miRNA, and quantitative estimation of mature and pre-miRNA.

Mitf is a transcriptional activator of medaka germ genes in culture.

Germ cells express a unique subset of genes called germ genes mostly encoding RNA-binding proteins such as Dazl, Dnd and Vasa. How germ gene expression is controlled remains illusive, because in no organism has a transcription factor been identified that regulate expression of these genes. Microphthalmia-associated transcription factor (Mitf) has been reported to show expression in male mouse germ cells of the adult testis. Here we report in the fish medaka (Oryzias latipes) that Mitf is a transcription activator of germ gene expression. Mitf is a master regulator of melanocyte development, which activates melanogenic genes through binding to the E-box containing consensus CANNTG. The E-box was found to be present in 23-26 copies in the promoters of medaka germ genes dazl, dnd and vasa. Importantly, forced Mitf expression enhanced the transcriptional activity of the three gene promoters by up to more than 10 fold and remarkably increased the level of endogenous dazl, dnd and vasa transcripts in cell culture. Transfection of Mitf expression vectors was sufficient to induce directed differentiation of medaka embryonic stem cells into melanocytes. Fluorescence in situ hybridization revealed the expression of both medaka mitf genes in adult germ cells of male and female gonads. Mitf is well-known as the melanocyte master regulator. Our results offer first evidence that Mitf may act as a transcriptional activator of germ gene expression in medaka.

p53 gene targeting by homologous recombination in fish ES cells.

Background Gene targeting (GT) provides a powerful tool for the generation of precise genetic alterations in embryonic stem (ES) cells to elucidate gene function and create animal models for human diseases. This technology has, however, been limited to mouse and rat. We have previously established ES cell lines and procedures for gene transfer and selection for homologous recombination (HR) events in the fish medaka (Oryzias latipes). Methodology and Principal Findings Here we report HR-mediated GT in this organism. We designed a GT vector to disrupt the tumor suppressor gene p53 (also known as tp53). We show that all the three medaka ES cell lines, MES1∼MES3, are highly proficient for HR, as they produced detectable HR without drug selection. Furthermore, the positive-negative selection (PNS) procedure enhanced HR by ∼12 folds. Out of 39 PNS-resistant colonies analyzed, 19 (48.7%) were positive for GT by PCR genotyping. When 11 of the PCR-positive colonies were further analyzed, 6 (54.5%) were found to be bona fide homologous recombinants by Southern blot analysis, sequencing and fluorescent in situ hybridization. This produces a high efficiency of up to 26.6% for p53 GT under PNS conditions. We show that p53 disruption and long-term propagation under drug selection conditions do not compromise the pluripotency, as p53-targeted ES cells retained stable growth, undifferentiated phenotype, pluripotency gene expression profile and differentiation potential in vitro and in vivo. Conclusions Our results demonstrate that medaka ES cells are proficient for HR-mediated GT, offering a first model organism of lower vertebrates towards the development of full ES cell-based GT technology.

Efficiency of Exosome Production Correlates Inversely with the Developmental Maturity of MSC Donor

Mesenchymal stem cells (MSCs) derived from human embryonic stem cells (ESCs) and fetal tissues have been shown to secrete cardioprotective exosome, a protein- and RNA40 containing vesicle. Since the therapeutic efficacy of MSCs is inversely correlated with developmental stage of the donor, we determine if this correlation extended to the cardioprotective MSC exosomes by examining exosomes secreted by MSCs derived from non-embryonic/fetal tissues e.g. umbilical cord. Unlike ESC- and fetal-MSCs, cord-MSCs have a much smaller proliferative capacity. To circumvent this and produce sufficient MSC exosomes for testing, they were immortalized via MYC over-expression. Like ESC-MSCs, MYC immortalization of cord MSCs expanded their proliferative capacity to bypass senescence, reduced plastic adherence, enhanced growth rate, and eliminated in vitro adipogenic differentiation potential without compromising exosome production. Exosomes produced by immortalized cord-MSCs were cardioprotective, and were equally efficacious in reducing infarct size in a mouse model of myocardial ischemia/reperfusion injury. However, cord MSCs produced the least amount of exosomes followed by fetal- and then ESC-MSC in decreasing order of developmental maturity or youth of the donor tissues, suggesting that the inverse correlation between the therapeutic efficacy of MSC and developmental stage of the donor is underpinned by rate of exosome production.

Nanos3 Gene Targeting in Medaka ES Cells

Gene targeting (GT) by homologous recombination offers the best precision for genome editing in mice. nanos3 is a highly conserved gene and encodes a zinc-finger RNA binding protein essential for germ stem cell maintenance in Drosophila, zebrafish and mouse. Here we report nanos3 GT in embryonic stem (ES) cells of the fish medaka as a lower vertebrate model organism. A vector was designed for GT via homologous recombination on the basis of positive-negative selection (PNS). The ES cell line MES1 after gene transfer and PNS produced 56 colonies that were expanded into ES cell sublines. Nine sublines were GT-positive by PCR genotyping, 4 of which were homologous recombinants as revealed by Southern blot. We show that one of the 4, A15, contains a precisely targeted nanos3 allele without any random events, demonstrating the GT feasibility in medaka ES cells. Importantly, A15 retained all features of undifferentiated ES cells, including stable self-renewal, an undifferentiated phenotype, pluripotency gene expression and differentiation during chimeric embryogenesis. These results provide first evidence that the GT procedure and genuine GT on a chromosomal locus such as nanos3 do not compromise pluripotency in ES cells of a lower vertebrate.

Delineating Biological Pathways Unique to Embryonic Stem Cell-Derived Insulin-Producing Cell Lines from Their Noninsulin-Producing Progenitor Cell Lines

To identify unique biochemical pathways in embryonic stem cell-derived insulin-producing cells as potential therapeutic targets to prevent or delay beta-cell dysfunction or death in diabetic patients, comparative genome-wide gene expression studies of recently derived mouse insulin-producing cell lines and their progenitor cell lines were performed using microarray technology. Differentially expressed genes were functionally clustered to identify important biochemical pathways in these insulin-producing cell lines. Biochemical or cellular assays were then performed to assess the relevance of these pathways to the biology of these cells. A total of 185 genes were highly expressed in the insulin-producing cell lines, and computational analysis predicted the pentose phosphate pathway (PPP), clathrin-mediated endocytosis, and the peroxisome proliferator-activated receptor (PPAR) signaling pathway as important pathways in these cell lines. Insulin-producing ERoSHK cells were more resistant to hydrogen peroxide (H(2)O(2))-induced oxidative stress. Inhibition of PPP by dehydroepiandrosterone and 6-aminonicotinamide abrogated this H(2)O(2) resistance with a concomitant decrease in PPP activity as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Clathrin-mediated endocytosis, which is essential in maintaining membrane homeostasis in secreting cells, was up-regulated by glucose in ERoSHK but not in their progenitor ERoSH cells. Its inhibition by chlorpromazine at high glucose concentration was toxic to the cells. Troglitazone, a PPARG agonist, up-regulated expression of Ins1 and Ins2 but not Glut2. Gene expression analysis has identified the PPP, clathrin-mediated endocytosis, and the PPAR signaling pathway as the major delineating pathways in these insulin-producing cell lines, and their biological relevance was confirmed by biochemical and cellular assays.