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Pluripotent Stem Cells (pluripotent + stem_cell)
Kinds of Pluripotent Stem Cells Selected AbstractsA Fluorescent Rosamine Compound Selectively Stains Pluripotent Stem Cells,ANGEWANDTE CHEMIE, Issue 41 2010Dr. Chang-Nim Im Ein gutes Image: Die fluoreszierende Verbindung CDy1 färbt selektiv embryonische Stammzellen an (siehe Schema). CDy1 wurde zur Identifizierung von Fibroblasten verwendet, die eine Umprogrammierung zu pluripotenten, GFP exprimierenden Stammzellen eingehen. [source] Pluripotent stem cells: private obsession and public expectationEMBO MOLECULAR MEDICINE, Issue 4 2010Austin Smith First page of article [source] Induced pluripotent stem cells (iPSCs): the emergence of a new champion in stem cell technology-driven biomedical applicationsJOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, Issue 6 2010Anjan Kumar Das Abstract Pluripotent stem cells possess the unique property of differentiating into all other cell types of the human body. Further, the discovery of induced pluripotent stem cells (iPSCs) in 2006 has opened up new avenues in clinical medicine. In simple language, iPSCs are nothing but somatic cells reprogrammed genetically to exhibit pluripotent characteristics. This process utilizes retroviruses/lentiviruses/adenovirus/plasmids to incorporate candidate genes into somatic cells isolated from any part of the human body. It is also possible to develop disease-specific iPSCs which are most likely to revolutionize research in respect to the pathophysiology of most debilitating diseases, as these can be mimicked ex vivo in the laboratory. These models can also be used to study the safety and efficacy of known drugs or potential drug candidates for a particular diseased condition, limiting the need for animal studies and considerably reducing the time and money required to develop new drugs. Recently, functional neurons, cardiomyocytes, pancreatic islet cells, hepatocytes and retinal cells have been derived from human iPSCs, thus re-confirming the pluripotency and differentiation capacity of these cells. These findings further open up the possibility of using iPSCs in cell replacement therapy for various degenerative disorders. In this review we highlight the development of iPSCs by different methods, their biological characteristics and their prospective applications in regenerative medicine and drug screening. We further discuss some practical limitations pertaining to this technology and how they can be averted for the betterment of human life. Copyright © 2010 John Wiley & Sons, Ltd. [source] 2131: Human pluripotent stem cells provide excellent source of functional pigment epithelial cellsACTA OPHTHALMOLOGICA, Issue 2010H SKOTTMAN Purpose Defined differentiation of functional RPE cells from human embryonic (hESC) or induced pluripotent stem cells (iPSC) is a prerequisite for their use in individualised disease modelling, drug discovery and transplantation for retinal diseases. In this study we report differentiation of RPE cells from hESC and iPSC in condition enabling easy translation to clinical quality cell production. Methods Pluripotent stem cells were produced on human fibroblast feeder cells in serum-free medium. The differentiation of the cells was induced using bFGF and feeder cell removal approach under serum-free conditions. The pigmentation and RPE morphology of the cells were analysed and the expression of genes and proteins characteristics for RPE cells were studied. The in vitro functionality of the cells was analysed using ELISA measurements and phagocytosis of photoreceptor outer segments. The integrity of the generated RPE layer was analysed using transepithelial electric resistance (TEER) measurements. Results With our differentiation method, we were able to generate RPE cells with satisfying efficiency. The typical pigmented cobblestone-like morphology and expression of RPE specific markers were confirmed at gene and protein level. The differentiated cells were able to phagocytose and secreted growth factors typical for RPE cells. In addition, cells formed a well polarized epithelium with high integrity, exhibiting good TEER values. Conclusion We have developed progressive differentiation protocol for production of functional RPE cells from hESC and iPSC. The developed production method is currently translated to defined and animal component free conditions enabling clinical grade cell production. [source] Genetic regulation of stem cell origins in the mouse embryoCLINICAL GENETICS, Issue 2 2005A Ralston ,Stem cell' has practically become a household term, but what is a stem cell and where does it come from? Insight into these questions has come from the early mouse embryo, or blastocyst, from which three kinds of stem cells have been derived: embryonic stem (ES) cells, trophoblast stem (TS) cells, and extraembryonic endoderm (XEN) cells. These stem cells appear to derive from three distinct tissue lineages within the blastocyst: the epiblast, the trophectoderm, and the extraembryonic endoderm. Understanding how these lineages arise during development will illuminate efforts to understand the establishment and maintenance of the stem cell state and the mechanisms that restrict stem cell potency. Genetic analysis has enabled the identification of several genes important for lineage decisions in the mouse blastocyst. Among these, Oct4, Nanog, Cdx2, and Gata6 encode transcription factors required for the three lineages of the blastocyst and for the maintenance their respective stem cell types. Interestingly, genetic manipulation of several of these factors can cause lineage switching among these stem cells, suggesting that knowledge of key lineage-determining genes could help control differentiation of stem cells more generally. Pluripotent stem cells have also been isolated from the human blastocyst, but the relationship between these cells and stem cells of the mouse blastocyst remains to be explored. This review describes the genetic regulation of lineage allocation during blastocyst formation and discusses similarities and differences between mouse and human ES cells. [source] Evolution and regeneration of the planarian central nervous systemDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 3 2009Yoshihiko Umesono More than 100 years ago, early workers realized that planarians offer an excellent system for regeneration studies. Another unique aspect of planarians is that they occupy an interesting phylogenetic position with respect to the nervous system in that they possess an evolutionarily primitive brain structure and can regenerate a functional brain from almost any tiny body fragment. Recent molecular studies have revisited planarian regeneration and revealed key information about the cellular and molecular mechanisms underlying brain regeneration in planarians. One of our great advances was identification of a gene, nou-darake, which directs the formation of a proper extrinsic environment for pluripotent stem cells to differentiate into brain cells in the planarian Dugesia japonica. Our recent findings have provided mechanistic insights into stem cell biology and also evolutionary biology. [source] Extrinsic factors derived from mouse embryonal carcinoma cell lines maintain pluripotency of mouse embryonic stem cells through a novel signal pathwayDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 2 2009Shinjirou Kawazoe Embryonic carcinoma (EC) cells, which are malignant stem cells of teratocarcinoma, have numerous morphological and biochemical properties in common with pluripotent stem cells such as embryonic stem (ES) cells. However, three EC cell lines (F9, P19 and PCC3) show different developmental potential and self-renewal capacity from those of ES cells. All three EC cell lines maintain self-renewal capacity in serum containing medium without Leukemia Inhibitory factor (LIF) or feeder layer, and show limited differentiation capacity into restricted lineage and cell types. To reveal the underlying mechanism of these characteristics, we took the approach of characterizing extrinsic factors derived from EC cells on the self-renewal capacity and pluripotency of mouse ES cells. Here we demonstrate that EC cell lines F9 and P19 produce factor(s) maintaining the undifferentiated state of mouse ES cells via an unidentified signal pathway, while P19 and PCC3 cells produce self-renewal factors of ES cells other than LIF that were able to activate the STAT3 signal; however, inhibition of STAT3 activation with Janus kinase inhibitor shows only partial impairment on the maintenance of the undifferentiated state of ES cells. Thus, these factors present in EC cells-derived conditioned medium may be responsible for the self-renewal capacity of EC and ES cells independently of LIF signaling. [source] Transcriptional dynamics of endodermal organ formationDEVELOPMENTAL DYNAMICS, Issue 1 2009Richard I. Sherwood Abstract Although endodermal organs including the liver, pancreas, and intestine are of significant therapeutic interest, the mechanism by which the endoderm is divided into organ domains during embryogenesis is not well understood. To better understand this process, global gene expression profiling was performed on early endodermal organ domains. This global analysis was followed up by dynamic immunofluorescence analysis of key transcription factors, uncovering novel expression patterns as well as cell surface proteins that allow prospective isolation of specific endodermal organ domains. Additionally, a repressive interaction between Cdx2 and Sox2 was found to occur at the prospective stomach,intestine border, with the hepatic and pancreatic domains forming at this boundary, and Hlxb9 was revealed to have graded expression along the dorsal,ventral axis. These results contribute to understanding the mechanism of endodermal organogenesis and should assist efforts to replicate this process using pluripotent stem cells. Developmental Dynamics 238:29,42, 2009. © 2008 Wiley-Liss, Inc. [source] Glucose-responsive insulin-producing cells from stem cellsDIABETES/METABOLISM: RESEARCH AND REVIEWS, Issue 6 2002David J. Kaczorowski Abstract Recent success with immunosuppression following islet cell transplantation offers hope that a cell transplantation treatment for type 1 (juvenile) diabetes may be possible if sufficient quantities of safe and effective cells can be produced. For the treatment of type 1 diabetes, the two therapeutically essential functions are the ability to monitor blood glucose levels and the production of corresponding and sufficient levels of mature insulin to maintain glycemic control. Stem cells can replicate themselves and produce cells that take on more specialized functions. If a source of stem cells capable of yielding glucose-responsive insulin-producing (GRIP) cells can be identified, then transplantation-based treatment for type 1 diabetes may become widely available. Currently, stem cells from embryonic and adult sources are being investigated for their ability to proliferate and differentiate into cells with GRIP function. Human embryonic pluripotent stem cells, commonly referred to as embryonic stem (ES) cells and embryonic germ (EG) cells, have received significant attention owing to their broad capacity to differentiate and ability to proliferate well in culture. Their application to diabetes research is of particular promise, as it has been demonstrated that mouse ES cells are capable of producing cells able to normalize glucose levels of diabetic mice, and human ES cells can differentiate into cells capable of insulin production. Cells with GRIP function have also been derived from stem cells residing in adult organisms, here referred to as endogenous stem cell sources. Independent of source, stem cells capable of producing cells with GRIP function may provide a widely available cell transplantation treatment for type 1 diabetes. Copyright © 2002 John Wiley & Sons, Ltd. [source] Genome-wide DNA methylation profile of tissue-dependent and differentially methylated regions (T-DMRs) residing in mouse pluripotent stem cellsGENES TO CELLS, Issue 6 2010Shinya Sato DNA methylation profile, consisting of tissue-dependent and differentially methylated regions (T-DMRs), has elucidated tissue-specific gene function in mouse tissues. Here, we identified and profiled thousands of T-DMRs in embryonic stem cells (ESCs), embryonic germ cells (EGCs) and induced pluripotent stem cells (iPSCs). T-DMRs of ESCs compared with somatic tissues well illustrated gene function of ESCs, by hypomethylation at genes associated with CpG islands and nuclear events including transcriptional regulation network of ESCs, and by hypermethylation at genes for tissue-specific function. These T-DMRs in EGCs and iPSCs showed DNA methylation similar to ESCs. iPSCs, however, showed hypomethylation at a considerable number of T-DMRs that were hypermethylated in ESCs, suggesting existence of traceable progenitor epigenetic information. Thus, DNA methylation profile of T-DMRs contributes to the mechanism of pluripotency, and can be a feasible solution for identification and evaluation of the pluripotent cells. [source] Generation of endoderm-derived human induced pluripotent stem cells from primary hepatocytes,HEPATOLOGY, Issue 5 2010Hua Liu Recent advances in induced pluripotent stem (iPS) cell research have significantly changed our perspective on regenerative medicine. Patient-specific iPS cells have been derived not only for disease modeling but also as sources for cell replacement therapy. However, there have been insufficient data to prove that iPS cells are functionally equivalent to human embryonic stem (hES) cells or are safer than hES cells. There are several important issues that need to be addressed, and foremost are the safety and efficacy of human iPS cells of different origins. Human iPS cells have been derived mostly from cells originating from mesoderm and in a few cases from ectoderm. So far, there has been no report of endoderm,derived human iPS cells, and this has prevented comprehensive comparative investigations of the quality of human iPS cells of different origins. Here we show for the first time reprogramming of human endoderm-derived cells (i.e., primary hepatocytes) to pluripotency. Hepatocyte-derived iPS cells appear indistinguishable from hES cells with respect to colony morphology, growth properties, expression of pluripotency-associated transcription factors and surface markers, and differentiation potential in embryoid body formation and teratoma assays. In addition, these cells are able to directly differentiate into definitive endoderm, hepatic progenitors, and mature hepatocytes. Conclusion: The technology to develop endoderm,derived human iPS cell lines, together with other established cell lines, will provide a foundation for elucidating the mechanisms of cellular reprogramming and for studying the safety and efficacy of differentially originated human iPS cells for cell therapy. For the study of liver disease pathogenesis, this technology also provides a potentially more amenable system for generating liver disease-specific iPS cells. (HEPATOLOGY 2010;51:1810,1819) [source] Porcine induced pluripotent stem cells may bridge the gap between mouse and human iPSIUBMB LIFE, Issue 4 2010Miguel A. Esteban Abstract Recently, three independent laboratories reported the generation of induced pluripotent stem cells (iPSCs) from pig (Sus scrofa). This finding sums to the growing list of species (mouse, human, monkey, and rat, in this order) for which successful reprogramming using exogenous factors has been achieved, and multiple others are possibly forthcoming. But apart from demonstrating the universality of the network identified by Shinya Yamanaka, what makes the porcine model so special? On one side, pigs are an agricultural commodity and have an easy and affordable maintenance compared with nonhuman primates that normally need to be imported. On the other side, resemblance (for example, size of organs) of porcine and human physiology is striking and because pigs are a regular source of food the ethical concerns that still remain in monkeys are not applicable. Besides, the prolonged lifespan of pigs compared with other domestic species can allow exhaustive follow up of side effects after transplantation. Porcine iPSCs may thus fill the gap between the mouse model, which due to its ease is preferred for mechanistic studies, and the first clinical trials using iPSCs in humans. However, although these studies are relevant and have created significant interest they face analogous problems that we discuss herein together with potential new directions. © 2010 IUBMB IUBMB Life, 62(4): 277,282, 2010 [source] Adult-derived stem cells and their potential for use in tissue repair and molecular medicineJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 3 2005Henry E. Young Abstract This report reviews three categories of precursor cells present within adults. The first category of precursor cell, the epiblast-like stem cell, has the potential of forming cells from all three embryonic germ layer lineages, e.g., ectoderm, mesoderm, and endoderm. The second category of precursor cell, the germ layer lineage stem cell, consists of three separate cells. Each of the three cells is committed to form cells limited to a specific embryonic germ layer lineage. Thus the second category consists of germ layer lineage ectodermal stem cells, germ layer lineage mesodermal stem cells, and germ layer lineage endodermal stem cells. The third category of precursor cells, progenitor cells, contains a multitude of cells. These cells are committed to form specific cell and tissue types and are the immediate precursors to the differentiated cells and tissues of the adult. The three categories of precursor cells can be readily isolated from adult tissues. They can be distinguished from each other based on their size, growth in cell culture, expressed genes, cell surface markers, and potential for differentiation. This report also discusses new findings. These findings include the karyotypic analysis of germ layer lineage stem cells; the appearance of dopaminergic neurons after implantation of naive adult pluripotent stem cells into a 6-hydroxydopamine-lesioned Parkinson's model; and the use of adult stem cells as transport mechanisms for exogenous genetic material. We conclude by discussing the potential roles of adult-derived precursor cells as building blocks for tissue repair and as delivery vehicles for molecular medicine. [source] Retroviral vector silencing during iPS cell induction: An epigenetic beacon that signals distinct pluripotent statesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 4 2008Akitsu Hotta Abstract Retroviral vectors are transcriptionally silent in pluripotent stem cells. This feature has been potently applied in studies that reprogram somatic cells into induced pluripotent stem (iPS) cells. By delivering the four Yamanaka factors in retroviral vectors, high expression is obtained in fibroblasts to induce the pluripotent state. Partial reprogramming generates Class I iPS cells that express the viral transgenes and endogenous pluripotency genes. Full-reprogramming in Class II iPS cells silences the vectors as the endogenous genes maintain the pluripotent state. Thus, retroviral vector silencing serves as a beacon marking the fully reprogrammed pluripotent state. Here we review known silencer elements, and the histone modifying and DNA methylation pathways, that silence retroviral and lentiviral vectors in pluripotent stem cells. Both retroviral and lentiviral vectors are influenced by position effects and often exhibit variegated expression. The best vector designs facilitate full-reprogramming and subsequent retroviral silencing, which is required for directed-differentiation. Current retroviral reprogramming methods can be immediately applied to create patient-specific iPS cell models of human disease, however, future clinical applications will require novel chemical or other reprogramming methods that reduce or eliminate the integrated vector copy number load. Nevertheless, retroviral vectors will continue to play an important role in genetically correcting patient iPS cell models. We anticipate that novel pluripotent-specific reporter vectors will select for isolation of high quality human iPS cell lines, and select against undifferentiated pluripotent cells during regenerative medicine to prevent teratoma formation after transplantation. J. Cell. Biochem. 105: 940,948, 2008. © 2008 Wiley-Liss, Inc. [source] Journal of Cellular Physiology: Volume 225, Number 2, November 2010JOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2010Article first published online: 29 SEP 2010 Cover shows the differential expression of microRNAs (miRs) in human embryonic stem cells (hESCs) vs. human-induced pluripotent stem cells (hiPSCs), revealing 10 highly expressed miRs in hiPSCs with greater than ten-fold difference, which have been shown to be cancer related. (Illustration by Vladimir Galat). Please see article by Collins et al, pages 454,465. [source] Generation of pluripotent stem cells from eggs of aging miceAGING CELL, Issue 2 2010Junjiu Huang Summary Oocytes can reprogram genomes to form embryonic stem (ES) cells. Although ES cells largely escape senescence, oocytes themselves do senesce in the ovaries of most mammals. It remains to be determined whether ES cells can be established using eggs from old females, which exhibit reproductive senescence. We attempted to produce pluripotent stem cell lines from artificial activation of eggs (also called pES) from reproductive aged mice, to determine whether maternal aging affects pES cell production and pluripotency. We show that pES cell lines were generated with high efficiency from reproductive aged (old) mice, although parthenogenetic embryos from these mice produced fewer ES clones by initial two passages. Further, pES cell lines generated from old mice showed telomere length, expression of pluripotency molecular markers (Oct4, Nanog, SSEA1), alkaline phosphatase activity, teratoma formation and chimera production similar to young mice. Notably, DNA damage was reduced in pES cells from old mice compared to their progenitor parthenogenetic blastocysts, and did not differ from that of pES cells from young mice. Also, global gene expression differed only minimally between pES cells from young and old mice, in contrast to marked differences in gene expression in eggs from young and old mice. These data demonstrate that eggs from old mice can generate pluripotent stem cells, and suggest that the isolation and in vitro culture of ES cells must select cells with high levels of DNA and telomere integrity, and/or with capacity to repair DNA and telomeres. [source] Induced pluripotent stem cells (iPSCs): the emergence of a new champion in stem cell technology-driven biomedical applicationsJOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, Issue 6 2010Anjan Kumar Das Abstract Pluripotent stem cells possess the unique property of differentiating into all other cell types of the human body. Further, the discovery of induced pluripotent stem cells (iPSCs) in 2006 has opened up new avenues in clinical medicine. In simple language, iPSCs are nothing but somatic cells reprogrammed genetically to exhibit pluripotent characteristics. This process utilizes retroviruses/lentiviruses/adenovirus/plasmids to incorporate candidate genes into somatic cells isolated from any part of the human body. It is also possible to develop disease-specific iPSCs which are most likely to revolutionize research in respect to the pathophysiology of most debilitating diseases, as these can be mimicked ex vivo in the laboratory. These models can also be used to study the safety and efficacy of known drugs or potential drug candidates for a particular diseased condition, limiting the need for animal studies and considerably reducing the time and money required to develop new drugs. Recently, functional neurons, cardiomyocytes, pancreatic islet cells, hepatocytes and retinal cells have been derived from human iPSCs, thus re-confirming the pluripotency and differentiation capacity of these cells. These findings further open up the possibility of using iPSCs in cell replacement therapy for various degenerative disorders. In this review we highlight the development of iPSCs by different methods, their biological characteristics and their prospective applications in regenerative medicine and drug screening. We further discuss some practical limitations pertaining to this technology and how they can be averted for the betterment of human life. Copyright © 2010 John Wiley & Sons, Ltd. [source] Proteomic analysis of membrane proteins expressed specifically in pluripotent murine embryonic stem cellsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 1 2009Atsushi Intoh Abstract Embryonic stem cells (ESCs) are established from the inner cell mass of preimplantation embryos, are capable of self-renewal, and exhibit pluripotency. Given these unique properties, ESCs are expected to have therapeutic potential in regenerative medicine and as a powerful tool for in vitro differentiation studies of stem cells. Various growth factors and extracellular matrix components regulate the pluripotency and differentiation of ESC progenies. Thus, the cell surface receptors that bind these regulatory factors are crucial for the precise regulation of stem cells. To identify membrane proteins that are involved in the regulation of pluripotent stem cells, the membrane proteins of murine ESCs cultured with or without leukemia inhibitory factor (LIF) were purified and analyzed by quantitative proteomics. 2-D PAGE-based analysis using fluorescently labeled proteins and shotgun-based analysis with isotope-labeled peptides identified 338 proteins, including transmembrane, membrane-binding, and extracellular proteins, which were expressed specifically in pluripotent or differentiated murine ESCs. Functions of the identified proteins revealed cell adhesion molecules, channels, and receptors, which are expected to play important roles in the maintenance of murine ESC pluripotency. Membrane proteins that are expressed in pluripotent ESCs but not in differentiated cells such as Slc16a1 and Bsg could be useful for the selection of the stem cells in vitro. [source] New Insights in Vascular Development: Vasculogenesis and Endothelial Progenitor CellsANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 1 2009S. Käßmeyer Summary In the course of new blood vessel formation, two different processes , vasculogenesis and angiogenesis , have to be distinguished. The term vasculogenesis describes the de novo emergence of a vascular network by endothelial progenitors, whereas angiogenesis corresponds to the generation of vessels by sprouting from pre-existing capillaries. Until recently, it was thought that vasculogenesis is restricted to the prenatal period. During the last decade, one of the most fascinating innovations in the field of vascular biology was the discovery of endothelial progenitor cells and vasculogenesis in the adult. This review aims at introducing the concept of adult vasculogenesis and discusses the efforts to identify and characterize adult endothelial progenitors. The different sources of adult endothelial progenitors like haematopoietic stem cells, myeloid cells, multipotent progenitors of the bone marrow, side population cells and tissue-residing pluripotent stem cells are considered. Moreover, a survey of cellular and molecular control mechanisms of vasculogenesis is presented. Recent advances in research on endothelial progenitors exert a strong impact on many different disciplines and provide the knowledge for functional concepts in basic fields like anatomy, histology as well as embryology. [source] AID in reprogramming: Quick and efficientBIOESSAYS, Issue 5 2010Identification of a key enzyme called AID, its activity in DNA demethylation, may help to overcome a pivotal epigenetic barrier in reprogramming somatic cells toward pluripotency Abstract Current methods of reprogramming differentiated cells into induced pluripotent stem cells remain slow and inefficient. In a recent report published online in Nature, Bhutani et al.1 developed a cell fusion strategy, achieving quick and efficient reprogramming toward pluripotency. Using this assay, they identified an immune system protein called activation-induced cytidine deaminase, or AID, which unexpectedly is actually able to "aid" in reprogramming due to its involvement in DNA demethylation that is required for induction of the two key pluripotency genes, Oct4 and Nanog. More recently, Popp et al.2 also reported online in Nature that AID is important for complete cell reprogramming in mammals. Together, these findings provide new insights into how cells are reprogrammed, identify the specific role of AID in cell fate reversal, and advance the field of regenerative medicine. [source] Cell-free production of transducible transcription factors for nuclear reprogramming,BIOTECHNOLOGY & BIOENGINEERING, Issue 6 2009William C. Yang Abstract Ectopic expression of a defined set of transcription factors chosen from Oct3/4, Sox2, c-Myc, Klf4, Nanog, and Lin28 can directly reprogram somatic cells to pluripotency. These reprogrammed cells are referred to as induced pluripotent stem cells (iPSCs). To date, iPSCs have been successfully generated using lentiviruses, retroviruses, adenoviruses, plasmids, transposons, and recombinant proteins. Nucleic acid-based approaches raise concerns about genomic instability. In contrast, a protein-based approach for iPSC generation can avoid DNA integration concerns as well as provide greater control over the concentration, timing, and sequence of transcription factor stimulation. Researchers recently demonstrated that polyarginine peptide conjugation can deliver recombinant protein reprogramming factor (RF) cargoes into cells and reprogram somatic cells into iPSCs. However, the protein-based approach requires a significant amount of protein for the reprogramming process. Producing fusion RFs in the large amounts required for this approach using traditional heterologous in vivo production methods is difficult and cumbersome since toxicity, product aggregation, and proteolysis by endogenous proteases limit yields. In this work, we show that cell-free protein synthesis (CFPS) is a viable option for producing soluble and functional transducible transcription factors for nuclear reprogramming. We used an E. coli -based CFPS system to express the above set of six human RFs as fusion proteins, each with a nona-arginine (R9) protein transduction domain. Using the flexibility offered by the CFPS platform, we successfully addressed proteolysis and protein solubility problems to produce full-length and soluble R9-RF fusions. We subsequently showed that R9-Oct3/4, R9-Sox2, and R9-Nanog exhibit cognate DNA-binding activities, R9-Nanog translocates across the plasma and nuclear membranes, and R9-Sox2 exerts transcriptional activity on a known downstream gene target. Biotechnol. Bioeng. 2009; 104: 1047,1058. © 2009 Wiley Periodicals, Inc. [source] Special focus: Cell and protein manipulationBIOTECHNOLOGY JOURNAL, Issue 2 2009Article first published online: 18 FEB 200 Synthetic biotechnology: The challenge Screening tool for induced pluripotent stem cells Method to limit oncogene expression to stem cells Intravital two-photon microscopy to solve meningitis mystery Electrostatic forces involved in a packaging motor Simulated Raman scattering microscopy offers high sensitivity in real-time imaging Alternative way to save brain cells after stroke or head trauma [source] 2131: Human pluripotent stem cells provide excellent source of functional pigment epithelial cellsACTA OPHTHALMOLOGICA, Issue 2010H SKOTTMAN Purpose Defined differentiation of functional RPE cells from human embryonic (hESC) or induced pluripotent stem cells (iPSC) is a prerequisite for their use in individualised disease modelling, drug discovery and transplantation for retinal diseases. In this study we report differentiation of RPE cells from hESC and iPSC in condition enabling easy translation to clinical quality cell production. Methods Pluripotent stem cells were produced on human fibroblast feeder cells in serum-free medium. The differentiation of the cells was induced using bFGF and feeder cell removal approach under serum-free conditions. The pigmentation and RPE morphology of the cells were analysed and the expression of genes and proteins characteristics for RPE cells were studied. The in vitro functionality of the cells was analysed using ELISA measurements and phagocytosis of photoreceptor outer segments. The integrity of the generated RPE layer was analysed using transepithelial electric resistance (TEER) measurements. Results With our differentiation method, we were able to generate RPE cells with satisfying efficiency. The typical pigmented cobblestone-like morphology and expression of RPE specific markers were confirmed at gene and protein level. The differentiated cells were able to phagocytose and secreted growth factors typical for RPE cells. In addition, cells formed a well polarized epithelium with high integrity, exhibiting good TEER values. Conclusion We have developed progressive differentiation protocol for production of functional RPE cells from hESC and iPSC. The developed production method is currently translated to defined and animal component free conditions enabling clinical grade cell production. [source] | ||||||||||||||||||||||||||||