Integrin receptors for extracellular matrix receptors are important effectors of cell adhesion, differentiation, and migration in cultured cells and are believed to be critical effectors of these processes during development. To determine when 131 integrins become critical during embryonic development, we generated mutant mice with a targeted disruption of the [31 integrin subunit gene. Heterozygous mutant mice were normal. Homozygous loss of ~1 integrin expression was lethal during early postimplantation development. Homozygous embryos lacking B1 integrins formed normal-looking blastocysts and initiated implantation at E4.5. However, the E4.5 Ill-null embryos in situ had collapsed blastocoeles, and whereas the trophoblast penetrated the uterine epithelium, extensive invasion of the decidua was not observed. Laminin-positive endoderm cells were detected in the inner cell mass area, but endoderm morphogenesis and migration were defective. By E5.5 ~l-null embryos had degenerated extensively. In vitro analysis showed that trophoblast function in Ill-null peri-implantation embryos was largely normal, including expression of tissue-specific markers, and outgrowth on fibronectin-and vitronectin-coated, although not on laminin-coated substrates. In contrast, the inner cell mass region of 131-null blastocyst outgrowths, and inner cell masses isolated from [31-null blastocysts, showed highly retarded growth and defective extraembryonic endoderm morphogenesis and migration. These data suggest that 131 integrins are required for normal morphogenesis of the inner cell mass and are essential mediators of growth and survival of cells of the inner cell mass. Failure of continued trophoblast development in 131-null embryos after inner cell mass failure could be attributable to either an intrinsic requirement for 131 integrins for later stages of trophoblast development, or to the lack of trophic signals from the 131-null inner cell mass.[Key Words: 131 integrins; e~V-integrins; trophoblast; extraembryonic endoderm; survival; migration] Received April 4, 1995; revised version accepted June 15, 1995.Cell--extracellular matrix (ECM) interactions play critical roles in morphogenesis and in the regulation of gene expression (Damsky and Werb 1992;Hynes 1992Hynes , 1994Adams and Watt 1993;Ashkenas et al. 1994;Cross et al. 1994). The integrin family of heterodimeric transmembrane glycoproteins constitutes the major class of receptors mediating cell-ECM interactions. These receptors link the ECM to the internal cytoskeleton and to intracellular signaling pathways. In this role, they mediate cell adhesion and migration, and transduce mechanical and informational signals from the complex extracellular environment, thereby influencing both cytoarchitecture and gene expression. Integrin heterodimeric receptors for ECM can be classified into two major families: {lJ those containing the 131 subunit, and, {2} those containing the oLV subunit. There is extensive apparent redundancy in the ligandbinding preferences of these integrins. For exampl...
Embryonic stem cells promise to provide a well-characterized and reproducible source of replacement tissue for human clinical studies. An early potential application of this technology is the use of retinal pigment epithelium (RPE) for the treatment of retinal degenerative diseases such as macular degeneration. Here we show the reproducible generation of RPE (67 passageable cultures established from 18 different hES cell lines); batches of RPE derived from NIH-approved hES cells (H9) were tested and shown capable of extensive photoreceptor rescue in an animal model of retinal disease, the Royal College of Surgeons (RCS) rat, in which photoreceptor loss is caused by a defect in the adjacent retinal pigment epithelium. Improvement in visual performance was 100% over untreated controls (spatial acuity was approximately 70% that of normal nondystrophic rats) without evidence of untoward pathology. The use of somatic cell nuclear transfer (SCNT) and/or the creation of banks of reduced complexity human leucocyte antigen (HLA) hES-RPE lines could minimize or eliminate the need for immunosuppressive drugs and/or immunomodulatory protocols.
Human induced pluripotent stem cells (hiPSC) have been shown to differentiate into a variety of replacement cell types. Detailed evaluation and comparison with their human embryonic stem cell (hESC) counterparts is critical for assessment of their therapeutic potential. Using established methods, we demonstrate here that hiPSCs are capable of generating hemangioblasts/blast cells (BCs), endothelial cells, and hematopoietic cells with phenotypic and morphologic characteristics similar to those derived from hESCs, but with a dramatic decreased efficiency. Furthermore, in distinct contrast with the hESC derivatives, functional differences were observed in BCs derived from hiPSCs, including significantly increased apoptosis, severely limited growth and expansion capability, and a substantially decreased hematopoietic colony-forming capability. After further differentiation into erythroid cells, >1,000-fold difference in expansion capability was observed in hiPSC-BCs versus hESC-BCs. Although endothelial cells derived from hiPSCs were capable of taking up acetylated low-density lipoprotein and forming capillary-vascular-like structures on Matrigel, these cells also demonstrated early cellular senescence (most of the endothelial cells senesced after one passage). Similarly, retinal pigmented epithelium cells derived from hiPSCs began senescing in the first passage. Before clinical application, it will be necessary to determine the cause and extent of such abnormalities and whether they also occur in hiPSCs generated using different reprogramming methods. STEM CELLS 2010;28:704-712 Disclosure of potential conflicts of interest is found at the end of this article.
Human stem-cell derivatives are likely to play an important role in the future of regenerative medicine. Evaluation and comparison to their in vivo counterparts is critical for assessment of their therapeutic potential. Transcriptomics was used to compare a new differentiation derivative of human embryonic stem (hES) cells--retinal pigment epithelium (RPE)--to human fetal RPE. Several hES cell lines were differentiated into putative RPE, which expressed RPEspecific molecular markers and was capable of phagocytosis, an important RPE function. Isolated hES cell-derived RPE was able to transdifferentiate into cells of neuronal lineage and redifferentiate into RPE-like cells through multiple passages (>30 Population doublings). Gene expression profiling demonstrated their higher similarity to primary RPE tissue than of existing human RPE cell lines D407 and ARPE-19, which has been shown to attenuate loss of visual function in animals. This is the first report of the isolation and characterization of putative RPE cells from hES cells, as well as the first application of transcriptomics to assess embryonic stem-cell derivatives and their in vivo counterparts--a "differentiomics" outlook. We describe for the first time, a differentiation system that does not require coculture with animal cells or factors, thus allowing the production of zoonoses-free RPE cells suitable for subretinal transplantation in patients with retinal degenerative diseases. With the further development of therapeutic cloning, or the creation of the banks of homozygous human leucocyte antigen (HLA) hES cells using parthenogenesis, RPE lines could be generated to overcome the problem of immune rejection and could be one of the nearest term applications of stem-cell technology.
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