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| Germline-competent mouse-induced pluripotent stem cell lines generated on human fibroblasts without exogenous leukemia inhibitory factor.
April 1, 2010 at 12:10 PM
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| | Germline-competent mouse-induced pluripotent stem cell lines generated on human fibroblasts without exogenous leukemia inhibitory factor. PLoS One. 2009;4(8):e6724 Authors: Li C, Yu H, Ma Y, Shi G, Jiang J, Gu J, Yang Y, Jin S, Wei Z, Jiang H, Li J, Jin Y Induced pluripotent stem (iPS) cells have attracted enormous attention due to their vast potential in regenerative medicine, pharmaceutical screening and basic research. Most prior established iPS cell lines were derived and maintained on mouse embryonic fibroblast (MEF) cells supplemented with exogenous leukemia inhibitory factor (LIF). Drawbacks of MEF cells impede optimization as well as dissection of reprogramming events and limit the usage of iPS cell derivatives in therapeutic applications. In this study, we develop a reproducible protocol for efficient reprogramming mouse neural progenitor cells (NPCs) on human foreskin fibroblast (HFF) cells via retroviral transfer of human transcriptional factors OCT4/SOX2/KLF4/C-MYC. Two independent iPS cell lines are derived without exogenous LIF. They display typical undifferentiated morphology and express pluripotency markers Oct4 and Sox2. Transgenes are inactivated and the endogenous Oct4 promoter is completely deme! thylated in the established iPS cell lines, indicating a fully reprogrammed state. Moreover, the iPS cells can spontaneously differentiate or be induced into various cell types of three embryonic germ layers in vitro and in vivo when they are injected into immunodeficient mice for teratoma formation. Importantly, iPS cells extensively integrate with various host tissues and contribute to the germline when injected into the blastocysts. Interestingly, these two iPS cell lines, while both pluripotent, exhibit distinctive differentiation tendencies towards different lineages. Taken together, the data describe the first genuine mouse iPS cell lines generated on human feeder cells without exogenous LIF, providing a reliable tool for understanding the molecular mechanisms of nuclear reprogramming. PMID: 19696928 [PubMed - in process] | | |
| Nuclear Receptor Regulation of Stemness and Stem Cell Differentiation.
April 1, 2010 at 12:10 PM
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| | Nuclear Receptor Regulation of Stemness and Stem Cell Differentiation. Exp Mol Med. 2009 Aug 21; Authors: Jeong Y, Mangelsdorf DJ Stem cells include a diverse number of toti-, pluri-, and multi-potent cells that play important roles in cellular genesis and differentiation, tissue development, and organogenesis. Genetic regulation involving various transcription factors results in the self-renewal and differentiation properties of stem cells. The nuclear receptor (NR) superfamily is composed of 48 ligand-activated transcription factors involved in diverse physiological functions such as metabolism, development, and reproduction. Increasing evidence shows that certain NRs function in regulating stemness or differentiation of embryonic stem (ES) cells and tissue-specific adult stem cells. Here, we review the role of the NR superfamily in various aspects of stem cell biology, including their regulation of stemness, forward- and trans-differentiation events; reprogramming of terminally differentiated cells; and interspecies differences. These studies provide insights into the therapeutic potentia! l of the NR superfamily in stem cell therapy and in treating stem cell-associated diseases (e.g., cancer stem cell). PMID: 19696553 [PubMed - as supplied by publisher] | | |
| Transplantation directs oocyte maturation from embryonic stem cells and provides a therapeutic strategy for female infertility.
April 1, 2010 at 12:10 PM
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| | Transplantation directs oocyte maturation from embryonic stem cells and provides a therapeutic strategy for female infertility. Hum Mol Genet. 2009 Aug 20; Authors: Nicholas CR, Haston KM, Grewall AK, Longacre TA, Reijo Pera RA Ten to fifteen percent of couples are infertile with the most common causes being linked to the production of few or no oocytes or sperm. Yet our understanding of human germ cell development is poor, at least in part due to the inaccessibility of early stages to genetic and developmental studies. Embryonic stem cells (ESCs) provide an in vitro system to study oocyte development and potentially treat female infertility. However, most studies of ESC differentiation to oocytes have not documented fundamental properties of endogenous development, making it difficult to determine the physiologic relevance of differentiated germ cells. Here, we sought to establish fundamental parameters of oocyte development during ESC differentiation to explore suitability for basic developmental genetic applications using the mouse as a model prior to translating to the human system. We demonstrate a timeline of definitive germ cell differentiation from ESCs in vitro that initially pa! rallels endogenous oocyte development in vivo by single-cell expression profiling and analysis of functional milestones including responsiveness to defined maturation media, shared genetic requirement of Dazl, and entry into meiosis. However, ESC-derived oocyte maturation ultimately fails in vitro. To overcome this obstacle, we transplant ESC-derived oocytes into an ovarian niche to direct their functional maturation and, thereby, present rigorous evidence of oocyte physiologic relevance and a potential therapeutic strategy for infertility. PMID: 19696121 [PubMed - as supplied by publisher] | | |
| Human microvasculature fabrication using thermal inkjet printing technology.
April 1, 2010 at 12:10 PM
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| | Human microvasculature fabrication using thermal inkjet printing technology. Biomaterials. 2009 Aug 18; Authors: Cui X, Boland T The current tissue engineering paradigm is that successfully engineered thick tissues must include vasculature. As biological approaches alone, such as VEGF, have fallen short of their promises, one may look for an engineering approach to build microvasculature. Layer-by-layer approaches for customized fabrication of cell/scaffold constructs have shown some potential in building complex 3D structures. With the advent of cell printing, one may be able to build precise human microvasculature with suitable bio-ink. Human microvascular endothelial cells (HMVEC) and fibrin were studied as bio-ink for microvasculature construction. Endothelial cells are the only cells to compose the human capillaries and also form the entire inner lining of cardiovascular system. Fibrin has been already widely recognized as tissue engineering scaffold for vasculature and other cells, including skeleton/smooth muscle cells and chondrocytes. In our study, we precisely fabricated micron-si! zed fibrin channels using a drop-on-demand polymerization. This printing technique uses aqueous processes that have been shown to induce little, if any, damage to cells. When printing HMVEC cells in conjunction with the fibrin, we found the cells aligned themselves inside the channels and proliferated to form confluent linings. The 3D tubular structure was also found in the printed patterns. We conclude that a combined simultaneous cell and scaffold printing can promote HMVEC proliferation and microvasculature formation. PMID: 19695697 [PubMed - as supplied by publisher] | | |
| Mobilization of circulating vascular progenitors in cancer patients receiving external beam radiation in response to tissue injury.
April 1, 2010 at 12:10 PM
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| | Mobilization of circulating vascular progenitors in cancer patients receiving external beam radiation in response to tissue injury. Int J Radiat Oncol Biol Phys. 2009 Sep 1;75(1):220-4 Authors: Allan DS, Morgan SC, Birch PE, Yang L, Halpenny MJ, Gunanayagam A, Li Y, Eapen L PURPOSE: Endothelial-like vascular progenitor cells (VPCs) are associated with the repair of ischemic tissue injury in several clinical settings. Because the endothelium is a principal target of radiation injury, VPCs may be important in limiting toxicity associated with radiotherapy (RT) in patients with cancer. METHODS AND MATERIALS: We studied 30 patients undergoing RT for skin cancer (n = 5), head-and-neck cancer (n = 15), and prostate cancer (n = 10) prospectively, representing a wide range of irradiated mucosal volumes. Vascular progenitor cell levels were enumerated from peripheral blood at baseline, midway through RT, at the end of treatment, and 4 weeks after radiation. Acute toxicity was graded at each time point by use of the National Cancer Institute's Common Toxicity Criteria, version 3.0. RESULTS: Significant increases in the proportion of CD34(+)/CD133(+) VPCs were observed after completion of RT, from 0.012% at baseline to 0.048% (p = 0.029), and t! he increase in this subpopulation was most marked in patients with Grade 2 peak toxicity or greater after RT (p = 0.034). Similarly, CD34(+)/vascular endothelial growth factor receptor 2-positive VPCs were increased after the completion of radiation therapy in comparison to baseline (from 0.014% to 0.027%, p = 0.043), and there was a trend toward greater mobilization in patients with more significant toxicity (p = 0.08). The mobilization of CD34(+) hematopoietic stem cells did not increase after treatment (p = 0.58), and there was no relationship with toxicity. CONCLUSIONS: We suggest that VPCs may play an important role in reducing radiation-induced tissue damage. Interventions that increase baseline VPC levels or enhance their mobilization and recruitment in response to RT may prove useful in facilitating more rapid and complete tissue healing. PMID: 19695439 [PubMed - in process] | | |
| Gene targeting in human pluripotent stem cells with adeno-associated virus vectors.
April 1, 2010 at 12:10 PM
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| | Gene targeting in human pluripotent stem cells with adeno-associated virus vectors. Biochem Biophys Res Commun. 2009 Aug 17; Authors: Mitsui K, Suzuki K, Aizawa E, Kawase E, Suemori H, Nakatsuji N, Mitani K Human pluripotent stem cells, such as embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), have the ability to differentiate into various cell types, and will become a potential source of cellular materials for regenerative medicine. To make full use of hESCs or hiPSCs for both basic and clinical research, genetic modification, especially gene targeting via homologous recombination (HR), would be an essential technique. This report describes the successful gene targeting of the hypoxanthine phosphoribosyl transferase 1 (HPRT 1) and the NANOG loci in human pluripotent stem cells with adeno-associated virus (AAV) vectors. At the HPRT 1 locus, up to 1% of stable transformants were targeted via HR with an AAV-HPRT 1 targeting vector, without loss of pluripotency. On the other hand, 20 - 87% of stable transformants were targeted using an AAV-NANOG targeting vector designed for the promoter trap strategy. In the KhES-3 cell line, which shows particu! larly high fragility to experimental manipulation, gene targeting was successful only by using an AAV vector but not by electroporation. In addition to hESC, gene targeting was achieved in hiPSC lines at similar frequencies. These data indicate that AAV vectors may therefore be a useful tool to introduce genetic modifications in hESCs and hiPSCs. PMID: 19695233 [PubMed - as supplied by publisher] | | |
| Adipose tissue mesenchymal stem cell expansion in animal serum-free medium supplemented with autologous human platelet lysate.
April 1, 2010 at 12:10 PM
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| | Adipose tissue mesenchymal stem cell expansion in animal serum-free medium supplemented with autologous human platelet lysate. Transfusion. 2009 Aug 18; Authors: Blande IS, Bassaneze V, Lavini-Ramos C, Fae KC, Kalil J, Miyakawa AA, Schettert IT, Krieger JE BACKGROUND: Mesenchymal stem cells (MSCs) have been considered for human regenerative therapy applications, and safe culture and expansion protocols are needed especially in the context of interspecies contamination. Human platelet lysate (PL) has been proposed as animal serum substitute during in vitro MSC expansion. In this work, a simplified and efficient method to obtain autologous PL to replace animal serum in cell culture applications is described. STUDY DESIGN AND METHODS: PL obtained by freezing and centrifugation procedures was tested as medium supplement for human adipose mesenchymal stem cell (hASC) culture. Differential proliferation, immunophenotypic changes, and differentiation under PL or fetal bovine serum (FBS) were assessed. RESULTS: In contrast to 10% FBS supplementation, cell population doubling time was significantly lower when hASCs were cultured with the same concentration of PL (PL 22.9 +/- 1.5 hr vs. FBS 106.7 +/- 6.5 hr, t test, p < 0.! 05). Furthermore, hASCs maintained with 2.5% PL supplementation also showed satisfactory results. Immunophenotypic analysis revealed no differences between hASCs cultivated with PL or FBS supplementation and both cultures retained the potential to differentiate into adipose cells. These results demonstrate that autologous PL obtained from the same donor can be used as animal serum substitute in hASC culture. CONCLUSIONS: Taken together, evidence is provided that platelets provided by a single donor are sufficient to obtain PL for hASC propagation for clinical-scale applications mitigating the potential untoward side effects associated with the use of animal-derived reagents. PMID: 19694997 [PubMed - as supplied by publisher] | | |
| Epigenetic regulation of X-inactivation in human embryonic stem cells.
April 1, 2010 at 12:10 PM
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| | Epigenetic regulation of X-inactivation in human embryonic stem cells. Epigenetics. 2009 Jan;4(1):19-22 Authors: Dvash T, Fan G X chromosome inactivation (XCI) allows dosage compensation of the expression from sex chromosome in mammalian female cells. Although this mechanism is extensively studied in the mouse model organism, the corresponding mechanism during human development is largely unknown. The generation of human embryonic stem cells (hESCs) provides an invaluable tool to address early embryogenesis in humans. Even though hESCs were supposed to shed light on the XCI process in early human embryogenesis, previous studies largely indicated inconsistency in the status of XCI in these cells. Recently, new data suggested that in vitro culture might affect epigenetic mechanisms such as XCI. In this review we will present the existing data regarding XCI variations in hESC as compared to data from the mouse embryo and embryonic stem cells. We will also suggest possible explanations for the conflicting observations in the literature regarding XCI in hESCs. PMID: 19106643 [PubMed - indexed for MEDLINE] | | |
| Mammary development meets cancer genomics
April 1, 2010 at 12:10 PM
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| Mammary development meets cancer genomics Nature Reports: Stem Cells842 (2009). doi:10.1038/nm0809-842 Authors: Aleix Prat & Charles M. Perou Mammary epithelial cell development is thought to progress from undifferentiated stem cells into at least two differentiated cell types. A new study has now characterized some of these distinct developmental stages and links them to tumor subtypes previously defined by gene expression profiling (pages 907–913). | | | | 
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