|  |  |  | | | | | TE-RegenMed-StemCell feed | | | | | | | | | | | | | | | | | | | Gene and cell therapy for primary immunodeficiency diseases. Nihon Rinsho Meneki Gakkai Kaishi. 2010;33(6):312-6 Authors: Otsu M Primary immunodeficiency diseases (PID) represents a group of inherited diseases where mutations in certain gene lead to certain levels of defects in patient immune systems. Among them, several types of PID, including severe combined immunodeficiecny (SCID), warrented development of new types of curative treatment other than allogeneic hematopoietic stem cell transplantation, eventually culiminating in successful stem cell gene therapy tials such as the cases for adenosine deaminase (ADA)-deficiency SCID patients. In this article, I will summarize the current status of stem cell gene therapy for PID, and discuss the problems such clinical trials have in the present forms of treatment, e.g., possible risks of leukemogenesis due to insertional mutagenesis by the use of therapeutic viral vectors. I also try to discuss the future of this type of experimental medicine aiming for the permanent cure of PID, including the one utilizing innovative technologies such as induced pluripotent stem cells. PMID: 21212583 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | An evolutionarily acquired genotoxic response discriminates MyoD from Myf5, and differentially regulates hypaxial and epaxial myogenesis. EMBO Rep. 2011 Jan 7; Authors: Innocenzi A, Latella L, Messina G, Simonatto M, Marullo F, Berghella L, Poizat C, Shu CW, Wang JY, Puri PL, Cossu G Despite having distinct expression patterns and phenotypes in mutant mice, the myogenic regulatory factors Myf5 and MyoD have been considered to be functionally equivalent. Here, we report that these factors have a different response to DNA damage, due to the presence in MyoD and absence in Myf5 of a consensus site for Abl-mediated tyrosine phosphorylation that inhibits MyoD activity in response to DNA damage. Genotoxins failed to repress skeletal myogenesis in MyoD-null embryos; reintroduction of wild-type MyoD, but not mutant Abl phosphorylation-resistant MyoD, restored the DNA-damage-dependent inhibition of muscle differentiation. Conversely, introduction of the Abl-responsive phosphorylation motif converts Myf5 into a DNA-damage-sensitive transcription factor. Gene-dosage-dependent reduction of Abl kinase activity in MyoD-expressing cells attenuated the DNA-damage-dependent inhibition of myogenesis. The presence of a DNA-damage-responsive phosphorylation motif in vertebrate, but not in invertebrate MyoD suggests an evolved response to environmental stress, originated from basic helix-loop-helix gene duplication in vertebrate myogenesis. PMID: 21212806 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Microcomputed tomography-based structural analysis of various bone tissue regeneration models. Nat Protoc. 2011 Jan;6(1):105-10 Authors: Kallai I, Mizrahi O, Tawackoli W, Gazit Z, Pelled G, Gazit D Microcomputed tomography (microCT) analysis is a powerful tool for the evaluation of bone tissue because it provides access to the 3D microarchitecture of the bone. It is invaluable for regenerative medicine as it provides the researcher with the opportunity to explore the skeletal system both in vivo and ex vivo. The quantitative assessment of macrostructural characteristics and microstructural features may improve our ability to estimate the quality of newly formed bone. We have developed a unique procedure for analyzing data from microCT scans to evaluate bone structure and repair. This protocol describes the procedures for microCT analysis of three main types of mouse bone regeneration models (ectopic administration of bone-forming mesenchymal stem cells, and administration of cells after both long bone defects and cranial segmental bone defects) that can be easily adapted for a variety of other models. Precise protocols are crucial because the system is extremely user sensitive and results can be easily biased if standardized methods are not applied. The suggested protocol takes 1.5-3.5 h per sample, depending on bone tissue sample size, the type of equipment used, variables of the scanning protocol and the operator's experience. PMID: 21212786 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | In vivo imaging of transplanted hematopoietic stem and progenitor cells in mouse calvarium bone marrow. Nat Protoc. 2011 Jan;6(1):1-14 Authors: Lo Celso C, Lin CP, Scadden DT In vivo imaging of transplanted hematopoietic stem and progenitor cells (HSPCs) was developed to investigate the relationship between HSPCs and components of their microenvironment in the bone marrow. In particular, it allows a direct observation of the behavior of hematopoietic cells during the first few days after transplantation, when the critical events in homing and early engraftment are occurring. By directly imaging these events in living animals, this method permits a detailed assessment of functions previously evaluated by crude assessments of cell counts (homing) or after prolonged periods (engraftment). This protocol offers a new means of investigating the role of cell-intrinsic and cell-extrinsic molecular regulators of hematopoiesis during the early stages of transplantation, and it is the first to allow the study of cell-cell interactions within the bone marrow in three dimensions and in real time. In this paper, we describe how to isolate, label and inject HSPCs, as well as how to perform calvarium intravital microscopy and analyze the resulting images. A typical experiment can be performed and analyzed in ∼1 week. PMID: 21212779 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Generation of adult human induced pluripotent stem cells using nonviral minicircle DNA vectors. Nat Protoc. 2011 Jan;6(1):78-88 Authors: Narsinh KH, Jia F, Robbins RC, Kay MA, Longaker MT, Wu JC Human induced pluripotent stem cells (hiPSCs) derived from patient samples have tremendous potential for innovative approaches to disease pathology investigation and regenerative medicine therapies. However, most hiPSC derivation techniques use integrating viruses, which may leave residual transgene sequences as part of the host genome, thereby unpredictably altering cell phenotype in downstream applications. In this study, we describe a protocol for hiPSC derivation by transfection of a simple, nonviral minicircle DNA construct into human adipose stromal cells (hASCs). Minicircle DNA vectors are free of bacterial DNA and thus capable of high expression in mammalian cells. Their repeated transfection into hASCs, abundant somatic cell sources that are amenable to efficient reprogramming, results in transgene-free hiPSCs. This protocol requires only readily available molecular biology reagents and expertise, and produces hiPSC colonies from an adipose tissue sample in ∼4 weeks. PMID: 21212777 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Gene and cell therapy for primary immunodeficiency diseases. Nihon Rinsho Meneki Gakkai Kaishi. 2010;33(6):312-6 Authors: Otsu M Primary immunodeficiency diseases (PID) represents a group of inherited diseases where mutations in certain gene lead to certain levels of defects in patient immune systems. Among them, several types of PID, including severe combined immunodeficiecny (SCID), warrented development of new types of curative treatment other than allogeneic hematopoietic stem cell transplantation, eventually culiminating in successful stem cell gene therapy tials such as the cases for adenosine deaminase (ADA)-deficiency SCID patients. In this article, I will summarize the current status of stem cell gene therapy for PID, and discuss the problems such clinical trials have in the present forms of treatment, e.g., possible risks of leukemogenesis due to insertional mutagenesis by the use of therapeutic viral vectors. I also try to discuss the future of this type of experimental medicine aiming for the permanent cure of PID, including the one utilizing innovative technologies such as induced pluripotent stem cells. PMID: 21212583 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | The role of pregnancy associated progenitor cells in the regeneration of injured maternal organs. Nihon Rinsho Meneki Gakkai Kaishi. 2010;33(6):287-92 Authors: Sunami R, Tagaya H, Hirata S Microchimeric fetal cells are present in the maternal body over a long period and thought to have the ability to colonize multiple tissues and organs. They are found in a wide range of maternal tissues affected with variety of diseases, thus, there is a possibility that they may contribute tissue repair and regeneration. To assess their possibility of use in regenerative medicine, we investigated whether fetal cells regenerate infracted maternal organs. We crossbred wild female mice with male transgenic mice, expressing enhanced green fluorescent protein (EGFP), and total hysterectomies were performed at the day 6 of pregnancies. On day 60 after the operations, the mice were injected with streptozotocin (STZ) to induce multiple organs injuries. We also created the ischemic organ injury model ; myocardial infarction model and cerebral infarction model. Detection and quantification of fetal cells were then attempted in a variety of maternal organs via a fluorescent microscope and quantitative PCR amplification of the gfp transgene. Fetal cells were detected only in maternal bone marrow before maternal organs injuries were induced, however, they were detected not only in bone marrow but also in the maternal injured organs. Histological analysis showed that differentiated fetal cells were observed and their morphological appearance was indistinguishable from their maternal counterparts. These results indicate that fetal cells sustained their population in the maternal bone marrow, may have contributed to the maternal tissue regeneration. PMID: 21212580 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Cardiopoietic factors: extracellular signals for cardiac lineage commitment. Circ Res. 2011 Jan 7;108(1):129-52 Authors: Noseda M, Peterkin T, Simões FC, Patient R, Schneider MD Cardiac muscle creation during embryogenesis requires extracellular instructive signals that are regulated precisely in time and space, intersecting with intracellular genetic programs that confer or fashion the ability of the cells to respond. Unmasking the essential signals for cardiac lineage decisions has paramount importance for cardiac development and regenerative medicine, including the directed differentiation of progenitor and stem cells to a cardiac muscle fate. PMID: 21212394 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Dynamic Changes in the Copy Number of Pluripotency and Cell Proliferation Genes in Human ESCs and iPSCs during Reprogramming and Time in Culture. Cell Stem Cell. 2011 Jan 7;8(1):106-18 Authors: Laurent LC, Ulitsky I, Slavin I, Tran H, Schork A, Morey R, Lynch C, Harness JV, Lee S, Barrero MJ, Ku S, Martynova M, Semechkin R, Galat V, Gottesfeld J, Izpisua Belmonte JC, Murry C, Keirstead HS, Park HS, Schmidt U, Laslett AL, Muller FJ, Nievergelt CM, Shamir R, Loring JF Genomic stability is critical for the clinical use of human embryonic and induced pluripotent stem cells. We performed high-resolution SNP (single-nucleotide polymorphism) analysis on 186 pluripotent and 119 nonpluripotent samples. We report a higher frequency of subchromosomal copy number variations in pluripotent samples compared to nonpluripotent samples, with variations enriched in specific genomic regions. The distribution of these variations differed between hESCs and hiPSCs, characterized by large numbers of duplications found in a few hESC samples and moderate numbers of deletions distributed across many hiPSC samples. For hiPSCs, the reprogramming process was associated with deletions of tumor-suppressor genes, whereas time in culture was associated with duplications of oncogenic genes. We also observed duplications that arose during a differentiation protocol. Our results illustrate the dynamic nature of genomic abnormalities in pluripotent stem cells and the need for frequent genomic monitoring to assure phenotypic stability and clinical safety. PMID: 21211785 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | In vitro fertilization, the nobel prize, and human embryonic stem cells. Cell Stem Cell. 2011 Jan 7;8(1):12-5 Authors: Gearhart J, Coutifaris C Robert Edwards was awarded the 2010 Nobel Prize in Physiology or Medicine for the development of human in vitro fertilization. His work not only provided the means to overcome many forms of infertility, but it also enabled research on early stages of human embryos and the derivation of human embryonic stem cells. PMID: 21211779 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Oxidative-reductionist approaches to stem and progenitor cell function. Cell Stem Cell. 2011 Jan 7;8(1):1-2 Authors: Noble M, Pröschel C, Mayer-Pröschel M Redox status is a critical modulator of stem and progenitor cell function. In this issue of Cell Stem Cell, Le Belle et al. (2011) demonstrate that oxidation promotes self-renewal of neuroepithelial stem cells, revealing fascinating differences-and surprising similarities-with how redox pathways regulate glial progenitor cells. PMID: 21211773 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Culture and identification of human amniotic mesenchymal stem cells. Chin Med Sci J. 2010 Dec;29(4):211-4 Authors: Huo SZ, Shi P, Pang XN Objective To establish the method of isolation, purification, and identification ofhuman amniotic mesenchymal stem cells (hAMSCs). Methods hAMSCs were isolatedfrom human amniotic membrane by trypsin-collagenase digestion, and cultured in Dulbecco's modified Eagle's medinm/F12 medium supplemented with 10% fetal bovine serum. Phenotypic characteristics ofthese cells were analyzed by means of immunocytochemistry and flow cytometry. Results The cells successfully isolated from human amniotic membrane expressed representative mesenchymal cell surface markers CD44, CD90, and vimentin, but not CD45.Conclusions This study establishes apotential method for isolation of hAMSCs from human amnion, in vitro culture,and identification. The isolated cells show phenotypic characteristics of mesenchymal stem cells. PMID: 21211173 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Prospects of Induced Pluripotent Stem (iPS) Cell Technology in Regenerative Medicine. Tissue Eng Part B Rev. 2011 Jan 7; Authors: Zhang F, Citra F, Wang DA Induced pluripotent stem (iPS) cells are derived from adult somatic cells via reprogramming with ectopic expression of four transcription factors (Oct3/4, Sox2, c-Myc and Klf4; or, Oct3/4, Sox2, Nanog and Lin28), by which the resultant cells regain pluripotency, namely, the capability exclusively possessed by some embryonic cells to differentiate into any cell lineage under proper conditions. Given the ease in cell sourcing and a waiver of ethical opponency, iPS cells excel embryonic pluripotent cells in the practice of drug discovery and regenerative medicine. With an ex vivo practice in regenerative medicine, many problems involved in conventional medicine dosing, such as immune rejection, could be potentially circumvented. In this article, we briefly summarize the fundamentals and status quo of iPS related applications, and emphasize the prospects of iPS technology in regenerative medicine. PMID: 21210760 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Polyelectrolyte Multilayers in Tissue Engineering. Tissue Eng Part B Rev. 2011 Jan 7; Authors: Detzel CJ, Larkin AL, Rajagopalan P ABSTRACT The layer-by-layer (LbL) assembly of sequentially adsorbed, alternating polyelectrolytes (PEs) has become increasingly important over the past two decades. The ease and versatility in assembling polyelectrolyte multilayers (PEMs), has resulted in numerous wide ranging applications of these materials. More recently, PEMs are being used in biological applications ranging from biomaterials, tissue engineering, regenerative medicine and drug delivery. The ability to manipulate the chemical, physical, surface and topographical properties of these multilayer architectures by simply changing the pH, ionic strength, thickness and post-assembly modifications render them highly suitable to probe the effects of external stimuli on cellular responsiveness. In the field of regenerative medicine, the ability to sequester growth factors and to tether peptides to PEMs has been exploited to direct the lineage of progenitor cells and to subsequently maintain a desired phenotype. Additional novel applications include the use of PEMs in the assembly of three-dimensional layered architectures and as coatings for individual cells to deliver tunable payloads of drugs or bioactive molecules. This review focuses on literature related to the modulation of chemical and physical properties of PEMs for tissue engineering applications and recent research efforts in maintaining and directing cellular phenotype in stem cell differentiation. KEYWORDS: Polyelectrolytes, Multilayers, Tissue Engineering, Stem Cells. PMID: 21210759 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Three-Dimensional Dynamic Perfusion Culture Conditions Promote Hepatic Differentiation of Human Embryonic Stem Cells. Tissue Eng Part C Methods. 2011 Jan 6; Authors: Miki T, Ring A, Gerlach JC The developmental potential of human embryonic stem cells (hESC) holds great promise to provide a source of human hepatocytes for use in drug discovery, toxicology, hepatitis research and extracorporeal bio-artificial liver support. There are, however, limitations to induce fully functional hepatocytes on conventional two-dimensional (2D) static culture. It had been shown that dynamic three-dimensional (3D) perfusion culture is superior to induce maturation in fetal hepatocytes and prolong hepatic functions of primary adult hepatocytes. We investigated the potential of using a four-compartment 3D perfusion culture to induce hepatic differentiation in hESC. Undifferentiated hESC were inoculated into hollow fiber based 3D-perfusion bioreactors with integral oxygenation. Hepatic differentiation was induced with a multistep growth factor cocktail protocol. Parallel controls were operated under equal perfusion conditions without the growth factor supplementations to allow for spontaneous differentiation, as well as in conventional 2D static conditions using growth factors. Metabolism, hepatocyte-specific gene expression, protein expression, and hepatic function were evaluated after 20 days. Significantly up-regulated hepatic gene expression was observed in the hepatic differentiation 3D culture group. Ammonia metabolism activity and albumin production was observed in the 3D directed differentiation culture. Drug induced cytochrome P450 gene expression was increased with rifampicin induction. Using flow cytometry analysis the mature hepatocyte marker asialoglycoprotein receptor (ASGPR) was found on up to 30% of the cells in the 3D system with directed hepatic differentiation. Histological and immunohistochemical analysis revealed structural formation of hepatic and biliary marker positive cells. In contrast to 2D culture, the 3D-perfusion culture induced more functional maturation in hESC-derived hepatic cells. 3D perfusion bioreactor technologies may be useful for further studies on generating hESC-derived hepatic cells. PMID: 21210720 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Treatment of basal cell carcinoma with autogenous growth factors and adipose-derived stem cells. Plast Reconstr Surg. 2010 Dec;126(6):312e-313e Authors: Kleintjes WG PMID: 21124108 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Chondroitin sulphate sulphation motif expression in the ontogeny of the intervertebral disc. Eur Cell Mater. 2011;21:1-14 Authors: Hayes AJ, Hughes CE, Ralphs JR, Caterson B Chondroitin sulphate chains on cell and extracellular matrix proteoglycans play important regulatory roles in developing systems. Specific, developmentally regulated, sulphation motifs within the chondroitin glycosaminoglycan structure may help bind, sequester or present bioactive signalling molecules to cells thus modulating their behaviour. Using monoclonal antibodies 3B3(-), 4C3, 6C3 and 7D4, we have mapped the distribution of different chondroitin sulphation epitopes in a rat intervertebral disc developmental series. The sulphation epitopes had complex, dynamic and specific distributions in the disc and vertebral tissues during their differentiation, growth and ageing. At embryonic day [E]15, prior to disc differentiation, 4C3 and 7D4 occurred within the cellular disc condensations whilst 6C3 was present in the notochordal sheath. At E17, post disc differentiation, 4C3 and 7D4 occurred within the nucleus pulposus, inner annulus and vertebral bodies; 3B3(-) in the nucleus, inner annulus, annulus/vertebral body interface and perichondrium; and 6C3, ventrally, within the perichondrium. At E19, 3B3(-), 4C3 and 7D4 became further restricted to the nucleus, inner annulus, annulus/vertebral body interface and perichondrium. Prior to birth, all four epitopes occurred within the inner annulus and nucleus, with 6C3 and 7D4 also occurring within the future end-plate. Postnatal expression of the sulphation epitopes was more widespread in the disc and also within the growth plate. At 4 months, the epitopes were associated with chondrocyte clusters within the nucleus; and at 24 months, with annular lesions. Overall, our data suggests that differential sulphation of chondroitin correlates with significant events in development, growth and aging of the rat intervertebral disc. PMID: 21213210 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Sintered Microsphere Scaffolds for Controlled Release and Tissue Engineering. Pharm Res. 2011 Jan 7; Authors: Shi X, Su K, Varshney RR, Wang Y, Wang DA PMID: 21213022 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | A phenomenological mixture model for biosynthesis and linking of cartilage extracellular matrix in scaffolds seeded with chondrocytes. Biomech Model Mechanobiol. 2011 Jan 7; Authors: Haider MA, Olander JE, Arnold RF, Marous DR, McLamb AJ, Thompson KC, Woodruff WR, Haugh JM A phenomenological mixture model is presented for interactions between biosynthesis of extracellular matrix (ECM) constituents and ECM linking in a scaffold seeded with chondrocytes. A system of three ordinary differential equations for average apparent densities of unlinked ECM, linked ECM and scaffold is developed along with associated initial conditions for scaffold material properties. Equations for unlinked ECM synthesis and ECM linking include an inhibitory mechanism where associated rates decrease as unlinked ECM concentration in the interstitial fluid increases. Linking rates are proposed to depend on average porosity in the evolving tissue construct. The resulting initial value problem contains nine independent parameters that account for scaffold biomaterial properties and interacting mechanisms in the engineered system. Effects of parameter variations on model variables are analyzed relative to a baseline case with emphasis on the evolution of solid phase apparent density, which is often correlated with the compressive elastic modulus of the tissue construct. The new model provides an additional quantitative framework for assessing and optimizing the design of engineered cell-scaffold systems and guiding strategies for articular cartilage tissue engineering. PMID: 21213013 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Spider silk as a load bearing biomaterial: tailoring mechanical properties via structural modifications. Nanoscale. 2011 Jan 7; Authors: Brown CP, Rosei F, Traversa E, Licoccia S Spider silk shows great potential as a biomaterial: in addition to biocompatibility and biodegradability, its strength and toughness are greater than native biological fibres (e.g. collagen), with toughness exceeding that of synthetic fibres (e.g. nylon). Although the ultimate tensile strength and toughness at failure are unlikely to be limiting factors, its yield strain of 2% is insufficient, particularly for biomedical application because of the inability to mimic the complex ultrastructure of natural tissues with current tissue engineering approaches. To harness the full potential of spider silk as a biomaterial, it is therefore necessary to increase its yield strain. In this paper, we discuss the means by which the mechanical properties of spider silk, particularly the yield strain, can be optimized through structural modifications. PMID: 21212901 [PubMed - as supplied by publisher] | | | | | | | | | |  | |  | |  |
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