Showing papers on "Transdifferentiation published in 1999"

Postnatal Sex Reversal of the Ovaries in Mice Lacking Estrogen Receptors α and β

Abstract: Mice lacking estrogen receptors α and β were generated to clarify the roles of each receptor in the physiology of estrogen target tissues. Both sexes of αβ estrogen receptor knockout (αβERKO) mutants exhibit normal reproductive tract development but are infertile. Ovaries of adult αβERKO females exhibit follicle transdifferentiation to structures resembling seminiferous tubules of the testis, including Sertoli-like cells and expression of Mullerian inhibiting substance, sulfated glycoprotein-2, and Sox9. Therefore, loss of both receptors leads to an ovarian phenotype that is distinct from that of the individual ERKO mutants, which indicates that both receptors are required for the maintenance of germ and somatic cells in the postnatal ovary.

Skeletal Myogenic Progenitors Originating from Embryonic Dorsal Aorta Coexpress Endothelial and Myogenic Markers and Contribute to Postnatal Muscle Growth and Regeneration

Abstract: Skeletal muscle in vertebrates is derived from somites, epithelial structures of the paraxial mesoderm, yet many unrelated reports describe the occasional appearance of myogenic cells from tissues of nonsomite origin, suggesting either transdifferentiation or the persistence of a multipotent progenitor. Here, we show that clonable skeletal myogenic cells are present in the embryonic dorsal aorta of mouse embryos. This finding is based on a detailed clonal analysis of different tissue anlagen at various developmental stages. In vitro, these myogenic cells show the same morphology as satellite cells derived from adult skeletal muscle, and express a number of myogenic and endothelial markers. Surprisingly, the latter are also expressed by adult satellite cells. Furthermore, it is possible to clone myogenic cells from limbs of mutant c-Met-/- embryos, which lack appendicular muscles, but have a normal vascular system. Upon transplantation, aorta-derived myogenic cells participate in postnatal muscle growth and regeneration, and fuse with resident satellite cells. The potential of the vascular system to generate skeletal muscle cells may explain observations of nonsomite skeletal myogenesis and raises the possibility that a subset of satellite cells may derive from the vascular system.

TGF-(beta) type I receptor/ALK-5 and Smad proteins mediate epithelial to mesenchymal transdifferentiation in NMuMG breast epithelial cells.

Abstract: The capacities of different transforming growth factor-(beta) (TGF-(beta)) superfamily members to drive epithelial to mesenchymal transdifferentiation of the murine mammary epithelial cell line NMuMG were investigated. TGF-(beta)1, but not activin A or osteogenic protein-1 (OP-1)/bone morphogenetic protein-7 (BMP-7), was able to induce morphological transformation of NMuMG cells as shown by reorganisation of the actin cytoskeleton and relocalisation/downregulation of E-cadherin and (beta)-catenin, an effect that was abrogated by the more general serine/threonine kinase and protein kinase C inhibitor, staurosporine. TGF-(beta)1 bound to TGF-(beta) type I receptor (T(beta)R-I)/ALK-5 and T(beta)R-II, but not to activin type I receptor (ActR-I)/ALK-2. Activin A bound to ActR-IB/ALK-4 and ActR-II, and BMP-7 bound to ActR-I/ALK-2, BMP type I receptor (BMPR-I)/ALK-3, ActR-II and BMPR-II. TGF-(beta)1 and BMP-7 activated the Smad-binding element (SBE)(4) promoter with equal potency, whereas activin A had no effect. Transfection of constitutively active (CA)-ALK-4 activated the 3TP promoter to the same extent as TGF-(beta)1 and CA-ALK-5 indicating that activin signalling downstream of type I receptors was functional in NMuMG cells. In agreement with this, activin A induced low levels of plasminogen activator inhibitor I expression compared to the high induction by TGF-(beta)1. In contrast to activin A and BMP-7, TGF-(beta)1 strongly induced Smad2 phosphorylation. Consistent with these findings, TGF-(beta)1 induced the nuclear accumulation of Smad2 and/or Smad3. In addition, NMuMG cells transiently infected with adenoviral vectors expressing high level CA-ALK-5 exhibited full transdifferentiation. On the other hand, infections with low level CA-ALK-5, which alone did not result in transdifferentiation, together with Smad2 and Smad4, or with Smad3 and Smad4 led to transdifferentiation. In conclusion, TGF-(beta)1 signals potently and passes the activation threshold to evoke NMuMG cell transdifferentiation. The TGF-(beta) type I receptor (ALK-5) and its effector Smad proteins mediate the epithelial to mesenchymal transition. Activin A does not induce mesenchymal transformation, presumably because the number of activin receptors is limited, while BMP-7-initiated signalling cannot mediate transdifferentiation.

Transient upregulation of CBFA1 in response to bone morphogenetic protein-2 and transforming growth factor beta1 in C2C12 myogenic cells coincides with suppression of the myogenic phenotype but is not sufficient for osteoblast differentiation.

Abstract: The bone morphogenetic protein (BMP)-2 is a potent osteoinductive signal, inducing bone formation in vivo and osteoblast differentiation from non-osseous cells in vitro. The runt domain-related protein Cbfa1/PEBP2alphaA/AML-3 is a critical component of bone formation in vivo and transcriptional regulator of osteoblast differentiation. To investigate the relationship between the extracellular BMP-2 signal, Cbfa1, and osteogenesis, we examined expression of Cbfa1 and osteoblastic genes during the BMP-2 induced osteogenic transdifferentiation of the myoblastic cell line C2C12. BMP-2 treatment completely blocked myotube formation and transiently induced expression of Cbfa1 and the bone-related homeodomain protein Msx-2 concomitant with loss of the myoblast phenotype. While induction of collagen type I and alkaline phosphatase (AP) expression coincided with Cbfa1 expression, Cbfa1 mRNA was strikingly downregulated at the onset of expression of osteopontin (OPN) and osteocalcin (OCN) genes, reflecting the mature osteoblast phenotype. TGF-beta1 treatment effectively suppressed myogenesis and induced Cbfa1 expression but was insufficient to support osteoblast differentiation reflected by the absence of ALP, OPN, and OCN. We addressed whether induction of Cbfa1 in response to BMP-2 results in the transcriptional activation of the OC promoter which contains three enhancer Cbfa1 elements. Transfection studies show BMP-2 suppresses OC promoter activity in C2C12, but not in osteoblastic ROS 17/2.8 cells. Maximal suppression of OC promoter activity in response to BMP-2 requires sequences in the proximal promoter (up to nt -365) and may occur independent of the three Cbfa sites. Taken together, our results demonstrate a dissociation of Cbfa1 expression from development of the osteoblast phenotype. Our findings suggest that Cbfal may function transiently to divert a committed myoblast to a potentially osteogenic cell. However, other factors induced by BMP-2 appear to be necessary for complete expression of the osteoblast phenotype.

Myoblast cell grafting into heart muscle: cellular biology and potential applications.

Abstract: ▪ Abstract This review surveys a wide range of cellular and molecular approaches to strengthening the injured or weakened heart, focusing on strategies to replace dysfunctional, necrotic, or apoptotic cardiomyocytes with new cells of mesodermal origin. A variety of cell types, including myogenic cell lines, adult skeletal myoblasts, immortalized atrial cells, embryonic and adult cardiomyocytes, embryonic stem cells, teratoma cells, genetically altered fibroblasts, smooth muscle cells, and bone marrow–derived cells have all been proposed as useful cells in cardiac repair and may have the capacity to perform cardiac work. We focus on the implantation of mesodermally derived cells, the best developed of the options. We review the developmental and cell biology that have stimulated these studies, examine the limitations of current knowledge, and identify challenges for the future, which we believe are considerable.

Glomerular epithelial–myofibroblast transdifferentiation in the evolution of glomerular crescent formation

Abstract: Background. Glomerular cellular crescents consist of epithelial cells and macrophages, which can undergo an irreversible process of fibrous organization. However, the origin of the fibroblast-type cells that mediate this fibrous organization is unclear. Methods. This study examined glomerular epithelial-myofibroblast transdifferentiation (GEMT) in the formation and evolution of glomerular crescents in two distinct rat models of glomerulonephritis: 5/6 nephrectomy and antiglomerular basement membrane (GBM) disease. Results. Early in the course of both disease models, and prior to crescent formation, immunohistochemistry staining and in-situ hybridization demonstrated de novo expression of alpha-smooth-muscle actin (α-SMA), a marker of smooth muscle cells and myofibroblasts, by glomerular parietal epithelial cells (GPEC). The expression of α-SMA by GPEC was accompanied by a loss of E-cadherin staining, a marker of epithelial cells. At this early stage of GEMT, ultrastructural studies identified the presence of characteristic actin microfilaments and dense bodies within GPEC which retained a normal epithelial morphology with apical-basal polarity and microvilli. A late stage of transdifferentiation was seen in fibrocellular crescents. In this case, GPEC attached to intact segments of the capsular basement membrane contained large bundles of actin microfilaments throughout the cell, and this was accompanied by a loss of polarity, microvilli, and tight junctions. There was a significant correlation between the presence of α-SMA + GPEC and glomerular crescent formation. Cellular crescents contained small numbers of α-SMA + myofibroblasts. These cells become the dominant population in fibrocellular crescents, which was associated with marked local proliferation. Relatively few α-SMA + myofibroblasts remained in fibrotic/organizing crescents. Most cells within cellular and fibrocellular crescents expressed transforming growth factor-β (TGF-β) and basic fibroblast growth factor (FGF-2), suggesting that these growth factors may regulate this GEMT process during the evolution of glomerular crescents. Conclusions. This study provides the first phenotypic and morphological evidence that glomerular epithelialmyofibroblast transdifferentiation participates in the formation and evolution of glomerular crescents.

Transdifferentiation of NRP-152 rat prostatic basal epithelial cells toward a luminal phenotype: regulation by glucocorticoid, insulin-like growth factor-I and transforming growth factor-beta

Abstract: The role of basal epithelial cells in prostatic function, development and carcinogenesis is unknown. The ability of basal prostatic epithelial cells to acquire a luminal phenotype was explored in vitro using the NRP-152 rat dorsal-lateral prostate epithelial cell line as a model system. NRP-152, which was spontaneously immortalized and clonally derived, is an androgen-responsive and nontumorigenic cell line that has a basal cell phenotype under normal growth conditions. However, when placed in mitogen-deficient media, these cells undergo a dramatic morphological change to a luminal phenotype. Under these growth-restrictive conditions, immunocytochemical analysis shows that NRP-152 cells acquire the luminal markers Z0-1 (a tight-junction associated protein), occludin (integral tight-junction protein), and cytokeratin 18, and lose the basal markers cytokeratins 5 and 14. Total protein and mRNA levels of cytokeratins 8, 18, c-CAM 105 (the calcium-independent cell adhesion molecule) and Z0-1, as detected by western and/or northern blot analyses, respectively, are induced, while cytokeratin 5 and 15 are lost, and occludin is unchanged. Concomitant with this differentiation, expression of transforming growth factor-beta2 (TGF-beta2), TGF-beta3, and TGF-beta receptor type II (TbetaRII) is induced, while those of TGF-beta1 and TbetaRI remain essentially unchanged. Mitogens, such as insulin-like growth factor-I and dexamethasone inhibit luminal differentiation, while exogenous TGF-beta induces such differentiation. These data together with TGF-beta neutralization experiments using pan-specific antibody implicate an important role for autocrine TGF-beta in the induction of the luminal differentiation.

Conservation of gene expression during embryonic lens formation and cornea-lens transdifferentiation in Xenopus laevis.

Abstract: Few molecular comparisons have been made between the processes of embryogenesis and regeneration or transdifferentiation that lead to the formation of the same structures. In the amphibian, Xenopus laevis, the cornea can undergo transdifferentiation to form a lens when the original lens is removed during tadpole larval stages. Unlike the process of embryonic lens induction, cornea-lens transdifferentiation is elicited via a single inductive interaction involving factors produced by the neural retina. In this study, we compared the expression of a number of genes known to be activated during various phases of embryonic lens formation, during the process of cornea-lens transdifferentiation. mRNA expression was monitored via in situ hybridization using digoxigenin-labeled riboprobes of pax-6, Xotx2, xSOX3, XProx1, and γ6-cry. We found that all of the genes studied are expressed during both embryogenesis and cornea-lens transdifferentiation, though in some cases their relative temporal sequences are not maintained. The reiterated expression of these genes suggests that a large suite of genes activated during embryonic lens formation are also involved in cornea-lens transdifferentiation. Ultimately functional tests will be required to determine whether they actually play similar roles in these processes. It is significant that the single inductive event responsible for initiating cornea-lens transdifferentiation triggers the expression of genes activated during both the early and late phases of embryonic lens induction. These findings have significant implications in terms of our current understanding of the “multistep” process of lens induction. Dev Dyn 1999;215:308–318. © 1999 Wiley-Liss, Inc.

Conjunctival epithelial cells cultured on human amniotic membrane fail to transdifferentiate into corneal epithelial-type cells.

Abstract: PURPOSE Previous studies on the use of human amniotic membrane (HAM) in rabbit stem cell deficiency models have found the new epithelium growing over the HAM to express cornea-specific keratins (K3 and K12) in 40% of the cases, suggesting that HAM may have induced conjunctival epithelial cells to transdifferentiate into cornea-type epithelial cells. The current study was performed to determine whether HAM could induce transdifferentiation of conjunctival epithelia] cells when cultured in vitro. METHODS Conjunctival grafts taken from the fornices of New Zealand white rabbits (6-12 weeks old) were placed over HAMs and lifted to an air-media interface using polypropylene double rings. These cultures were maintained in supplemented hormonal epithelial medium with and without 3T3 feeder cells. Rabbit corneal epithelial cells were cultured similarly using strips of keratolimbal grafts placed over HAM. The cultures were terminated at various times between the 8th and 15th day. The cultured epithelial cells were examined histologically and immunohistochemically using monoclonal antibodies AK-2 (to K12 keratin), AM-3 (to goblet cell mucin), and AE-5 (to K3 keratin). RESULTS Both conjunctival and corneal epithelial cells cultured on HAMs showed multilayered, differentiated epithelial structures. On immunohistochemical examination, both epithelial cells stained positive for AE-5. None of the cultured conjunctival epithelial cells stained positively for AK-2, while the corneal epithelial cells showed positive staining with AK-2. There were no AM-3-positive goblet cells in either epithelial cell culture. There was no difference in the immunohistochemical patterns between cultures with or without 3T3 feeder cells. However, culture without feeder cells seemed to manifest a more degenerative appearance than those with feeders. CONCLUSION HAM does not induce transdifferentiation of conjunctival epithelial cells into corneal-type epithelial cells under the in vitro culture conditions used in this study.

Conversion of non-neuronal cells into neurons: transdifferentiation of epidermal cells

Abstract: The present invention is directed to method of converting, or transdifferentiating the epidermal cells into viable neurons useful in both cell therapy and gene therapy treatment methodologies. The method of transdifferentiating epidermal cells into neuronal cells comprises the following steps: obtaining skin cells from a patient; dedifferentiating these cells with an appropriate medium, neurotrophin or cytokine; transfecting the skin cells with one or more expression vector(s) encoding at least one neurogenic transcription factor or active fragments thereof; expressing at least one of the neurogenic transcription factors; growing the transfected cells in an appropriate medium; and adding to the medium one or more antisense oligonucleotide(s) corresponding to at least one negative regulator of neuronal differentiation, whereby the epidermal cells are transdifferentiated into neuronal cells.

Use of representational difference analysis to study the effect of TGFB on the expression profile of a pancreatic cancer cell line.

Abstract: It has been shown that TGFBs, their receptors, or downstream targets show genetic alterations in pancreatic cancer. This study was designed to identify transcriptional alterations induced by prolonged treatment of pancreatic cancer cell lines with TGFB. The TGFB-responsive PANC-1 cell line was treated with 10-ng/ml TGFB1 for 24 hr. cDNA representational difference analysis was used to generate subtracted hybridization probes enriched for TGFB regulated genes. These probes were hybridized on gridded arrays of cDNA clones containing genes differentially expressed in pancreatic cancer. Twenty-seven distinct cDNA clones were shown to be TGFB target genes. Eleven genes were upregulated by TGFB and were associated with extracellular matrix composition and formation, including genes usually transcribed by cells of mesenchymal origin only. Transcript levels of 16 genes were downregulated by TGFB and could mainly be classified into markers of epithelial differentiation and genes involved in the transcriptional and translational machinery. In conclusion, a 24-hr treatment of PANC-1 cells with TGFB induced a loss of epithelial and a gain of mesenchymal markers. As in other tumors, this epithelial-mesenchymal transdifferentiation may be of general importance during pancreatic carcinogenesis, and may participate, e.g., in the development of the desmoplastic reaction or the acquisition of an invasive phenotype of pancreatic tumor cells. This study demonstrates the usefulness of cDNA RDA and gridded clone libraries to study the effect of signaling cascades on the expression profile of tumor cells. Similar approaches may be helpful in the context of the genome project for the characterization of novel genes. Genes Chromosomes Cancer 26:70-79, 1999.

A transient increase in c‐myc precedes the transdifferentiation of hepatic stellate cells to myofibroblast‐like cells

Abstract: AIMS/BACKGROUND Liver stellate cells are transdifferentiated to collagen-producing myofibroblast-like cells in vivo during liver injury or when placed in culture. The purpose of this study was to determine the presence of retinoids and the expression of the immediate early genes as they relate to the transdifferentiation of liver stellate cells in culture. METHODS Rat liver stellate cells were studied immediately after isolation or sequentially after culture for varying periods of time. RNA was isolated and specific messages were determined by RT-PCR. Cells were also isolated for determination of retinoid autofluorescence and immunofluorescent staining with specific antibodies by laser confocal microscopy. RESULTS c-fos message and immunoprotein were high in the freshly isolated cells prior to culture, while c-myc expression increased markedly after one day of culture. Both c-fos and c-myc gene expression decreased prior to the transdifferentiation of the cells to myofibroblast-like cells and to the increase in alpha 1(I) and alpha 2(I) collagen messages and collagen production. The presence of retinoid autofluorescence and retinoic acid receptor (RAR-alpha and RAR-beta) messages and RAR-beta immunoprotein persisted during initial transdifferentiation of the stellate cells. CONCLUSIONS This study shows a high initial level of c-fos expression and a transient increase in c-myc expression followed by a decrease to lower levels prior to transdifferentiation and collagen production by stellate cells. A total loss of retinoid autofluorescence or a decrease in RAR-alpha or RAR-beta are not required for initial transdifferentiation of stellate cells or collagen production.

Immature peritoneal mast cells in neonatal rats express the CTMC phenotype, as well as functional IgE receptors.

Abstract: The purpose of this study was to investigate the relationship between the differentiation and maturation of mast cells and the expression of IgE receptors on their surface in neonatal animals in vivo. Another aim was to clarify whether connective tissue mast cells (CTMC) undergo a maturation process involving a transdifferentiation from mucosal mast cells (MMC) during this period of time. Mast-cell phenotypes were studied in terms of the profiles of proteinases and proteoglycan. In 1-week-old rats, the mast-cell granules stained with Alcian blue rather than with safranin (AB+/S-) in the Alcian blue/safranin staining sequence, normally regarded as a property of MMC. However, the AB+/S-stained proteoglycan was degradable by nitrous acid and stained with berberine sulphate, thus indicating that it contained heparin rather than chondroitin sulphate. The mast cells expressed rat mast-cell proteinase (RMCP) I rather than RMCP II, which is normally found in MMC. The mast cells of 1-week-old rats expressed functional IgE receptors, by showing a dose-dependent IgE-mediated histamine release of mast cells. About 70% of the IgE receptors on the mast cells were occupied by IgE. In 2- to 3-week-old rats, there was a progressive increase in mast cells stained with both Alcian blue and safranin or with safranin alone, i.e. they gradually changed towards the staining properties of CTMC (AB-/S+). The expression and the degree of IgE occupancy of the receptors increased in 1- to 3-week-old animals. This was paralleled by an increment in cell size and in the content of heparin, histamine and serotonin in the mast cells. The findings thus indicate that the peritoneal mast cells of neonatal rats express the CTMC phenotype and undergo a maturation process at from 1 to 3 weeks of age, without involving a transdifferentiation from MMC. The maturation of the mast cells is accompanied by an increase in the expression of functional IgE receptors on the cell surface. IgE production was detectable as early as in 1-week-old rats.

Gene therapy for renal fibroblasts

Abstract: growth factor b (TGF-b), platelet-derived growth Introduction factor (PDGF), fibroblast growth factor (FGF) and endothelin 1 (ET-1) among others [11,19]. There are The field of gene therapy as applied to the kidney has very few interstitial fibroblasts in the normal kidney great theoretical promise but has been difficult to and this is one of the reasons why the initial events develop into practical strategies [1–3]. As the signalling underlying fibrosis have been difficult to study in detail. processes underlying the pathogenesis of renal disease An intriguing and important question concerns the become better understood, the incentive to target possibility that a proportion of the fibroblasts seen in specific genes and gene products increases, both as the fibrosing kidney may have arisen from the transpotential therapy and in order to test biological hypodifferentiation of other cells particularly tubule epithetheses. Animal model work performed in the last few lial cells [20,21]. In the liver the transdifferentiation of years has demonstrated the potential for gene transfer Ito cells to a myofibroblastic phenotype is an estabinto a range of renal cell types including epithelial lished mode of fibrogenesis. In the kidney epithelial cells, endothelial cells, macrophages and glomerular cells can be induced to express FSP1 by toxic stimuli. cells [4–8]. Two presentations at the American Society In culture tubule epithelial cells can be induced by of Nephrology meeting in Philadelphia in 1998 indigrowth on a 3D matrix to acquire fibroblastic characcated for the first time that it is possible to selectively teristics such as the expression of FSP1 and vimentin transfect renal fibroblasts in vivo [9,10]. This was and to downregulate their expression of cytokeratin accomplished using heamagglutinating virus of Japan [21]. Accordingly, the successful selective transfection (HVJ) and ureteric injection. The interstitial renal of renal interstitial fibroblasts in vivo could be of great fibroblast is a key element in the pathogenesis of renal significance both in the development of anti-fibrotic failure and this work may have far reaching strategies and in the further understanding of the basic implications. biological mechanisms of fibrosis.