Showing papers on "Transdifferentiation published in 2009"

TGF-beta-induced epithelial to mesenchymal transition.

Abstract: During development and in the context of different morphogenetic events, epithelial cells undergo a process called epithelial to mesenchymal transition or transdifferentiation (EMT). In this process, the cells lose their epithelial characteristics, including their polarity and specialized cell-cell contacts, and acquire a migratory behavior, allowing them to move away from their epithelial cell community and to integrate into surrounding tissue, even at remote locations. EMT illustrates the differentiation plasticity during development and is complemented by another process, called mesenchymal to epithelial transition (MET). While being an integral process during development, EMT is also recapitulated under pathological conditions, prominently in fibrosis and in invasion and metastasis of carcinomas. Accordingly, EMT is considered as an important step in tumor progression. TGF-β signaling has been shown to play an important role in EMT. In fact, adding TGF-β to epithelial cells in culture is a convenient way to induce EMT in various epithelial cells. Although much less characterized, epithelial plasticity can also be regulated by TGF-β-related bone morphogenetic proteins (BMPs), and BMPs have been shown to induce EMT or MET depending on the developmental context. In this review, we will discuss the induction of EMT in response to TGF-β, and focus on the underlying signaling and transcription mechanisms.

Smooth Muscle Cells Give Rise to Osteochondrogenic Precursors and Chondrocytes in Calcifying Arteries

Abstract: Vascular calcification is a major risk factor for cardiovascular morbidity and mortality. To develop appropriate prevention and/or therapeutic strategies for vascular calcification, it is important to understand the origins of the cells that participate in this process. In this report, we used the SM22-Cre recombinase and Rosa26-LacZ alleles to genetically trace cells derived from smooth muscle. We found that smooth muscle cells (SMCs) gave rise to osteochondrogenic precursor- and chondrocyte-like cells in calcified blood vessels of matrix Gla protein deficient (MGP−/−) mice. This lineage reprogramming of SMCs occurred before calcium deposition and was associated with an early onset of Runx2/Cbfa1 expression and the downregulation of myocardin and Msx2. There was no change in the constitutive expression of Sox9 or bone morphogenetic protein 2. Osterix, Wnt3a, and Wnt7a mRNAs were not detected in either calcified MGP−/− or noncalcified wild-type (MGP+/+) vessels. Finally, mechanistic studies in vitro suggest that Erk signaling might be required for SMC transdifferentiation under calcifying conditions. These results provide strong support for the hypothesis that adult SMCs can transdifferentiate and that SMC transdifferentiation is an important process driving vascular calcification and the appearance of skeletal elements in calcified vascular lesions.

Directed transdifferentiation of mouse mesoderm to heart tissue by defined factors.

Abstract: Heart disease is the leading cause of mortality and morbidity in the western world. The heart has little regenerative capacity after damage, leading to much interest in understanding the factors required to produce new cardiac myocytes. Despite a robust understanding of the molecular networks regulating cardiac differentiation, no single transcription factor or combination of factors has been shown to activate the cardiac gene program de novo in mammalian cells or tissues. Here we define the minimal requirements for transdifferentiation of mouse mesoderm to cardiac myocytes. We show that two cardiac transcription factors, Gata4 and Tbx5, and a cardiac-specific subunit of BAF chromatin-remodelling complexes, Baf60c (also called Smarcd3), can direct ectopic differentiation of mouse mesoderm into beating cardiomyocytes, including the normally non-cardiogenic posterior mesoderm and the extraembryonic mesoderm of the amnion. Gata4 with Baf60c initiated ectopic cardiac gene expression. Addition of Tbx5 allowed differentiation into contracting cardiomyocytes and repression of non-cardiac mesodermal genes. Baf60c was essential for the ectopic cardiogenic activity of Gata4 and Tbx5, partly by permitting binding of Gata4 to cardiac genes, indicating a novel instructive role for BAF complexes in tissue-specific regulation. The combined function of these factors establishes a robust mechanism for controlling cellular differentiation, and may allow reprogramming of new cardiomyocytes for regenerative purposes.

Endothelial cells and pulmonary arterial hypertension: apoptosis, proliferation, interaction and transdifferentiation.

Abstract: Severe pulmonary arterial hypertension, whether idiopathic or secondary, is characterized by structural alterations of microscopically small pulmonary arterioles. The vascular lesions in this group of pulmonary hypertensive diseases show actively proliferating endothelial cells without evidence of apoptosis. In this article, we review pathogenetic concepts of severe pulmonary arterial hypertension and explain the term "complex vascular lesion ", commonly named "plexiform lesion", with endothelial cell dysfunction, i.e., apoptosis, proliferation, interaction with smooth muscle cells and transdifferentiation.

β-catenin controls differentiation of the retinal pigment epithelium in the mouse optic cup by regulating Mitf and Otx2 expression

Abstract: The retinal pigment epithelium (RPE) consists of a monolayer of cuboidal, pigmented cells that is located between the retina and the choroid The RPE is vital for growth and function of the vertebrate eye and improper development results in congenital defects, such as microphthalmia or anophthalmia, or a change of cell fate into neural retina called transdifferentiation The transcription factors microphthalmia-associated transcription factor (Mitf) and orthodenticle homolog 2 (Otx2) are crucial for RPE development and function; however, very little is known about their regulation Here, by using a Wnt-responsive reporter, we show that the Wnt/β-catenin pathway is activated in the differentiating mouse RPE Cre-mediated, RPE-specific disruption of β-catenin after the onset of RPE specification causes severe defects, resulting in microphthalmia with coloboma, disturbed lamination, and mislocalization of adherens junction proteins Upon β-catenin deletion, the RPE transforms into a multilayered tissue in which the expression of Mitf and Otx2 is downregulated, while retina-specific gene expression is induced, which results in the transdifferentiation of RPE into retina Chromatin immunoprecipitation (ChIP) and luciferase assays indicate that β-catenin binds near to and activates potential TCF/LEF sites in the Mitf and Otx2 enhancers We conclude that Wnt/β-catenin signaling is required for differentiation of the RPE by directly regulating the expression of Mitf and Otx2 Our study is the first to show that an extracellular signaling pathway directly regulates the expression of RPE-specific genes such as Mitf and Otx2, and elucidates a new role for the Wnt/β-catenin pathway in organ formation and development

Corneal limbal microenvironment can induce transdifferentiation of hair follicle stem cells into corneal epithelial-like cells.

Abstract: The aim of this study was to investigate the transdifferentiation potential of murine vibrissa hair follicle (HF) stem cells into corneal epithelial-like cells through modulation by corneal- or limbus-specific microenvironmental factors. Adult epithelial stem cells were isolated from the HF bulge region by mechanical dissection or fluorescence-activated cell sorting using antibodies to α6 integrin, enriched by clonal expansion, and subcultivated on various extracellular matrices (type IV collagen, laminin-1, laminin-5, fibronectin) and in different conditioned media derived from central and peripheral corneal fibroblasts, limbal stromal fibroblasts, and 3T3 fibroblasts. Cellular phenotype and differentiation were evaluated by light and electron microscopy, real-time reverse transcription-polymerase chain reaction, immunocytochemistry, and Western blotting, using antibodies against putative stem cell markers (K15, α6 integrin) and differentiation markers characteristic for corneal epithelium (K12, Pax6) or epidermis (K10). Using laminin-5, a major component of the corneo-limbal basement membrane zone, and conditioned medium from limbal stromal fibroblasts, clonally enriched HF stem and progenitor cells adhered rapidly and formed regularly arranged stratified cell sheets. Conditioned medium derived from limbal fibroblasts markedly upregulated expression of cornea-specific K12 and Pax6 on the mRNA and protein level, whereas expression of the epidermal keratinocyte marker K10 was strongly downregulated. These findings suggest that adult HF epithelial stem cells are capable of differentiating into corneal epithelial-like cells in vitro when exposed to a limbus-specific microenvironment. Therefore, the HF may be an easily accessible alternative therapeutic source of autologous adult stem cells for replacement of the corneal epithelium and restoration of visual function in patients with ocular surface disorders.

Neural differentiation potential of human bone marrow-derived mesenchymal stromal cells: misleading marker gene expression

Abstract: In contrast to pluripotent embryonic stem cells, adult stem cells have been considered to be multipotent, being somewhat more restricted in their differentiation capacity and only giving rise to cell types related to their tissue of origin. Several studies, however, have reported that bone marrow-derived mesenchymal stromal cells (MSCs) are capable of transdifferentiating to neural cell types, effectively crossing normal lineage restriction boundaries. Such reports have been based on the detection of neural-related proteins by the differentiated MSCs. In order to assess the potential of human adult MSCs to undergo true differentiation to a neural lineage and to determine the degree of homogeneity between donor samples, we have used RT-PCR and immunocytochemistry to investigate the basal expression of a range of neural related mRNAs and proteins in populations of non-differentiated MSCs obtained from 4 donors. The expression analysis revealed that several of the commonly used marker genes from other studies like nestin, Enolase2 and microtubule associated protein 1b (MAP1b) are already expressed by undifferentiated human MSCs. Furthermore, mRNA for some of the neural-related transcription factors, e.g. Engrailed-1 and Nurr1 were also strongly expressed. However, several other neural-related mRNAs (e.g. DRD2, enolase2, NFL and MBP) could be identified, but not in all donor samples. Similarly, synaptic vesicle-related mRNA, STX1A could only be detected in 2 of the 4 undifferentiated donor hMSC samples. More significantly, each donor sample revealed a unique expression pattern, demonstrating a significant variation of marker expression. The present study highlights the existence of an inter-donor variability of expression of neural-related markers in human MSC samples that has not previously been described. This donor-related heterogeneity might influence the reproducibility of transdifferentiation protocols as well as contributing to the ongoing controversy about differentiation capacities of MSCs. Therefore, further studies need to consider the differences between donor samples prior to any treatment as well as the possibility of harvesting donor cells that may be inappropriate for transplantation strategies.

Neural differentiation potential of human bone marrow-derived mesenchymal stromal cells: misleading marker gene expression

Abstract: Background In contrast to pluripotent embryonic stem cells, adult stem cells have been considered to be multipotent, being somewhat more restricted in their differentiation capacity and only giving rise to cell types related to their tissue of origin. Several studies, however, have reported that bone marrow-derived mesenchymal stromal cells (MSCs) are capable of transdifferentiating to neural cell types, effectively crossing normal lineage restriction boundaries. Such reports have been based on the detection of neural-related proteins by the differentiated MSCs. In order to assess the potential of human adult MSCs to undergo true differentiation to a neural lineage and to determine the degree of homogeneity between donor samples, we have used RT-PCR and immunocytochemistry to investigate the basal expression of a range of neural related mRNAs and proteins in populations of non-differentiated MSCs obtained from 4 donors. Results The expression analysis revealed that several of the commonly used marker genes from other studies like nestin, Enolase2 and microtubule associated protein 1b (MAP1b) are already expressed by undifferentiated human MSCs. Furthermore, mRNA for some of the neural-related transcription factors, e.g. Engrailed-1 and Nurr1 were also strongly expressed. However, several other neural-related mRNAs (e.g. DRD2, enolase2, NFL and MBP) could be identified, but not in all donor samples. Similarly, synaptic vesicle-related mRNA, STX1A could only be detected in 2 of the 4 undifferentiated donor hMSC samples. More significantly, each donor sample revealed a unique expression pattern, demonstrating a significant variation of marker expression. Conclusion The present study highlights the existence of an inter-donor variability of expression of neural-related markers in human MSC samples that has not previously been described. This donor-related heterogeneity might influence the reproducibility of transdifferentiation protocols as well as contributing to the ongoing controversy about differentiation capacities of MSCs. Therefore, further studies need to consider the differences between donor samples prior to any treatment as well as the possibility of harvesting donor cells that may be inappropriate for transplantation strategies.

Caveolin-1-/- null mammary stromal fibroblasts share characteristics with human breast cancer-associated fibroblasts

Abstract: Recently, we reported that human breast cancer-associated fibroblasts show functional inactivation of the retinoblastoma (RB) tumor suppressor and down-regulation of caveolin-1 (Cav-1) protein expression. However, it remains unknown whether loss of Cav-1 is sufficient to confer functional RB inactivation in mammary fibroblasts. To establish a direct cause-and-effect relationship, mammary stromal fibroblasts (MSFs) were prepared from Cav-1(-/-) null mice and subjected to phenotypic analysis. Here, we provide evidence that Cav-1(-/-) MSFs share many characteristics with human cancer-associated fibroblasts. The Cav-1(-/-) MSF transcriptome significantly overlaps with human cancer-associated fibroblasts; both show a nearly identical profile of RB/E2F-regulated genes that are up-regulated, which is consistent with RB inactivation. This Cav-1(-/-) MSF gene signature is predictive of poor clinical outcome in breast cancer patients treated with tamoxifen. Consistent with these findings, Cav-1(-/-) MSFs show RB hyperphosphorylation and the up-regulation of estrogen receptor co-activator genes. We also evaluated the paracrine effects of "conditioned media" prepared from Cav-1(-/-) MSFs on wild-type mammary epithelia. Our results indicate that Cav-1(-/-) MSF "conditioned media" is sufficient to induce an epithelial-mesenchymal transition, indicative of an invasive phenotype. Proteomic analysis of this "conditioned media" reveals increased levels of proliferative/angiogenic growth factors. Consistent with these findings, Cav-1(-/-) MSFs are able to undergo endothelial-like transdifferentiation. Thus, these results have important implications for understanding the role of cancer-associated fibroblasts and RB inactivation in promoting tumor angiogenesis.

Introducing transcription factors to multipotent mesenchymal stem cells: making transdifferentiation possible.

Abstract: Multipotent mesenchymal stem cells (MSCs) represent a promising autologous source for regenerative medicine. Because MSCs can be isolated from adult tissues, they represent an attractive cell source for autologous transplantation. A straightforward therapeutic strategy in the field of stem cell-based regenerative medicine is the transplantation of functional differentiated cells as cell replacement for the lost or defective cells affected by disease. However, this strategy requires the capacity to regulate stem cell differentiation toward the desired cell fate. This therapeutic approach assumes the capability to direct MSC differentiation toward diverse cell fates, including those outside the mesenchymal lineage, a process termed transdifferentiation. The capacity of MSCs to undergo functional transdifferentiation has been questioned over the years. Nonetheless, recent studies support that genetic manipulation can serve to promote transdifferentiation. Specifically, forced expression of certain transcription factors can lead to reprogramming and alter cell fate. Using such a method, fully differentiated lymphocytes have been reprogrammed to become macrophages and, remarkably, somatic cells have been reprogrammed to become embryonic stem-like cells. In this review, we discuss the past and current research aimed at transdifferentiating MSCs, a process with applications that could revolutionize regenerative medicine. STEM CELLS 2009;27:2509–2515

Mitogen-activated protein kinase-signaling stimulates Müller glia to proliferate in acutely damaged chicken retina.

Abstract: Muller glia in the mature retina have the capacity to become progenitor-like cells in a many different vertebrate classes The cell-signaling pathways that control the ability of mature Muller glia to become progenitor-like cells remain uncertain The purpose of this study was to investigate the roles of the Mitogen-Activated Protein Kinase (MAPK) pathway in regulating the activity of Muller glia in the chicken retina In response to acute retinal damage, we found that Muller glia accumulated phosphorylated ERK1/2 and phospho-CyclicAMP Response Element Binding-protein (pCREB), and transiently expressed immediate early genes, cFos and Egr1, that are known to be downstream of MAPK-signaling Egr1 and pCREB were normally expressed by retinal progenitors in the circumferential marginal zone (CMZ), whereas cFos and pERK1/2 were not In addition, small molecule inhibitors of MEK (UO126) and the FGF-receptor (SU5402) suppressed the proliferation of Muller glia-derived progenitor-like cells These inhibitors suppressed the accumulation of Egr1 and pCREB, whereas levels of cFos were unaffected in the glial cells These findings suggest that Egr1 and pCREB are downstream of the signaling cascade activated by FGF-receptors and ERK1/2 Further, our findings suggest that Egr1 and pCREB may promote glial proliferation We propose that activation of both the FGF-receptor and ERK1/2-pathway is required for the proliferation and transdifferentiation of Muller glia into progenitor-like cells

Essential Roles for Early Growth Response Transcription Factor Egr-1 in Tissue Fibrosis and Wound Healing

Abstract: The early growth response gene (Egr-1) codes for a zinc finger transcription factor that has important roles in the regulation of cell growth, differentiation, and survival. Aberrant Egr-1 expression is implicated in carcinogenesis, inflammation, atherosclerosis, and ischemic injury. We reported previously that normal fibroblasts stimulated by transforming growth factor-s showed rapid and transient induction of Egr-1. Moreover, we observed that tissue expression of Egr-1 was elevated in patients with scleroderma, which suggests that Egr-1 may be involved in tissue repair and fibrosis. Here, we investigated matrix remodeling and wound healing in mice harboring gain of function or loss of function mutations of Egr-1. Using the model of bleomycin-induced scleroderma, we found that the early influx of inflammatory cells into the skin and lungs, and the subsequent development of fibrosis in these organs, were markedly attenuated in Egr-1 null mice. Furthermore, full-thickness incisional skin wound healing was impaired, and skin fibroblasts lacking Egr-1 showed reduced migration and myofibroblast transdifferentiation in vitro. In contrast, transgenic mice with fibroblast-specific Egr-1 overexpression showed exuberant tissue repair, with enhanced collagen accumulation and increased tensile strength of incisional wounds. Together, these results point to the fundamental role that Egr-1 plays in the regulation of transforming growth factor-s-dependent physiological and pathological matrix remodeling.

Chemically-induced RAT mesenchymal stem cells adopt molecular properties of neuronal-like cells but do not have basic neuronal functional properties.

Abstract: Induction of adult rat bone marrow mesenchymal stem cells (MSC) by means of chemical compounds (beta-mercaptoethanol, dimethyl sulfoxide and butylated hydroxyanizole) has been proposed to lead to neuronal transdifferentiation, and this protocol has been broadly used by several laboratories worldwide. Only a few hours of MSC chemical induction using this protocol is sufficient for the acquisition of neuronal-like morphology and neuronal protein expression. However, given that cell death is abundant, we hypothesize that, rather than true neuronal differentiation, this particular protocol leads to cellular toxic effects. We confirm that the induced cells with neuronal-like morphology positively stained for NF-200, S100, beta-tubulin III, NSE and MAP-2 proteins. However, the morphological and molecular changes after chemical induction are also associated with an increase in the apoptosis of over 50% of the plated cells after 24 h. Moreover, increased intracellular cysteine after treatment indicates an impairment of redox circuitry during chemical induction, and in vitro electrophysiological recordings (patch-clamp) of the chemically induced MSC did not indicate neuronal properties as these cells do not exhibit Na(+) or K(+) currents and do not fire action potentials. Our findings suggest that a disruption of redox circuitry plays an important role in this specific chemical induction protocol, which might result in cytoskeletal alterations and loss of functional ion-gated channels followed by cell death. Despite the neuronal-like morphology and neural protein expression, induced rat bone marrow MSC do not have basic functional neuronal properties, although it is still plausible that other methods of induction and/or sources of MSC can achieve a successful neuronal differentiation in vitro.

Müller cell activation, proliferation and migration following laser injury.

Abstract: Purpose: Muller cells are well known for their critical role in normal retinal structure and function, but their reaction to retinal injury and subsequent role in retinal remodeling is less well characterized. In this study we used a mouse model of retinal laser photocoagulation to examine injury-induced Muller glial reaction, and determine how this reaction was related to injury-induced retinal regeneration and cellular repopulation. Methods: Experiments were performed on 3–4-week-old C57BL/6 mice. Retinal laser photocoagulation was used to induce small, circumscribed injuries; these were principally confined to the outer nuclear layer, and surrounded by apparently healthy retinal tissue. Western blotting and immunohistochemical analyses were used to determine the level and location of protein expression. Live cell imaging of green fluorescent protein (GFP)-infected Muller cells (AAVGFAP-GFP) were used to identify the rate and location of retinal Muller cell nuclear migration. Results: Upon injury, Muller cells directly at the burn site become reactive, as evidenced by increased expression of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and nestin. These reactive cells re-enter the cell cycle as shown by expression of the markers Cyclin D1 and D3, and their nuclei begin to migrate toward the injury site at a rate of approximately 12 μm/hr. However, unlike other reports, evidence for Muller cell transdifferentiation was not identified in this model. Conclusions: Retinal laser photocoagulation is capable of stimulating a significant glial reaction, marked by activation of cell cycle progression and retinal reorganization, but is not capable of stimulating cellular transdifferentiation or neurogenesis.

Bone marrow stromal cells for spinal cord repair: a challenge for contemporary neurobiology.

Abstract: In the last years, it has been reported that bone marrow stromal cells (BMSC) are able to differentiate towards a neuronal phenotype, in vitro as well as in vivo, and consequently, the possible use of these cells for the treatment of neurological diseases has acquired enormous importance. The objective of this review is to discuss the experimental findings that suggested the utility of BMSC for the treatment of paraplegia, and the possibilities of its clinical application in patients. For this reason, we revise our previous experimental findings about neuronal transdifferentiation of BMSC, and the utility of local BMSC transplantation in an experimental model of chronic paraplegia. Our current experience supports that a neural transdifferentiation of BMSC is possible after these mesenchymal stem cells are transplanted into injured spinal cord tissue. Furthermore, this cell therapy achieves a clear functional improvement of paraplegic animals, together with morphological evidence of spinal cord regeneration. Although at present our efforts should be guided to obtain a better knowledge of the mechanisms of nervous regeneration induced by bone-marrow derived stem cells, it is obvious that cell therapy for nervous system repair is beginning, and BMSC transplantation offers new hope for the treatment of traumatic paraplegia in humans.

Reversible physiological transdifferentiation in the adipose organ.

Abstract: All mammals are provided with two distinct adipose cells, white and brown adipocytes. White adipocytes store lipids to provide fuel to the organism, allowing intervals between meals. Brown adipocytes use lipids to produce heat. Previous descriptions have implied their localization in distinct sites of the body; however, it has been demonstrated that they are present together in many depots, which has led to the new concept of the adipose organ. In order to explain their coexistence the hypothesis of reversible physiological transdifferentiation has been developed, i.e. they are contained together because they are able to convert, one into the other. In effect, if needed the brown component of the organ could increase at the expense of the white component and vice versa. This plasticity is important because the brown phenotype of the organ is associated with resistance to obesity and its related disorders. A new example of reversible physiological transdifferentiation of adipocytes is offered by the mammary gland during pregnancy, lactation and post-lactation stages. The gravidic hormonal stimulus seems to trigger a transdifferentiation of adipocytes into milk-producing and secreting epithelial glands. In the post-lactation period some of the epithelial cells of the mammary gland seem to transdifferentiate into adipocytes. Recent unpublished results suggest that explanted adipose tissue, as well as explanted isolated mature adipocytes, is able to transdifferentiate into glands with epithelial markers of milk-secreting mammary glands. These findings, if confirmed, seem to suggest new windows into the cell biology frontiers of adipocytes.

CLIC4 mediates TGF-beta1-induced fibroblast-to-myofibroblast transdifferentiation in ovarian cancer.

Abstract: Stromal myofibroblasts, activated by crosstalk signaling between the tumour and stroma, play a critical role in tumour development and progression. Chloride intracellular channel 4 (CLIC4) may be functionally import for tumour stromal fibroblast-to-myofibroblast transdifferentiaton, but the molecular mechanism of the process has not been addressed. In this study, the expression of CLIC4 in ovarian cancer tissues was analyzed by immunohistochemistry, and we used an indirect co-culture model of ovarian cancer cells and normal fibroblasts to demonstrate the molecular pathway in which CLIC4 participated during the fibroblast-to-myofibroblast transdifferentiation. The results showed that the expression of CLIC4 in 96.7% of ovarian cancer stroma and correlated with the up-regulation of myofibroblast marker alpha-SMA. Conditioned medium from ovarian cancer cells (CM) or transforming growth factor-beta1 (TGF-beta1) increased cellular reactive oxygen species (ROS) levels in fibroblasts, which initiated up-regulation of CLIC4 expression, then resulted in myofibroblast conversion. Moreover, inhibition of CLIC4 significantly reduced the expressions of factors related to the phenotype and functions of myofibroblasts, such as alpha-SMA, VEGF and HGF. These results suggest that ROS-initiated CLIC4 up-regulation is required for TGF-beta1-induced fibroblast-to-myofibroblast transdifferentiaton in ovarian cancer, indicating that inhibiting the CLIC4 might have therapeutic potential targeting tumour stroma.

CXCR4 and matrix metalloproteinase-1 are elevated in breast carcinoma-associated fibroblasts and in normal mammary fibroblasts exposed to factors secreted by breast cancer cells.

Abstract: The complex molecular communications that occur between neoplastic and stromal cells within the tumor microenvironment play an integral role in breast cancer pathogenesis. Carcinoma-associated fibroblasts (CAF) produce tumor-enhancing factors and have been strongly implicated in breast cancer development. Similar to the way in which tumors have been compared with "wounds that never heal," CAFs have been equated to activated fibroblasts, which are present in inflammatory environments, in which they aid in wound healing through tissue remodeling and repair. Matrix metalloproteinase-1 (MMP-1) and G protein-coupled receptor, CXCR4, are elevated in these activated fibroblasts, in which they facilitate angiogenesis and matrix degradation, processes that are also vital to breast cancer metastasis. In this study, we investigated MMP-1 and CXCR4 expression in normal human mammary fibroblasts (HMF) exposed to soluble breast cancer factors. Historically, elevated CXCR4 expression is associated with breast cancer cells. However, we show that soluble factors secreted by SUM102 breast cancer cells stimulated the expression of MMP-1 and CXCR4 in HMFs. As a result, these stromal cells acquired an invasive and migratory phenotype. To confirm the clinical relevancy of our findings, we analyzed CAFs obtained from primary breast cancers. These cells also displayed elevated MMP-1 and CXCR4 levels compared with counterpart fibroblasts, and were more invasive and migratory. Together, our data suggest that soluble breast cancer factors initiate the transdifferentiation of normal HMFs to tumor-promoting CAFs, and that through the induction of MMP-1 and CXCR4 levels, these cells exhibit an invasive and migratory phenotype.

Adult mouse intrahepatic biliary epithelial cells induced in vitro to become insulin-producing cells

Abstract: Transdifferentiation of cells from a patient’s own liver into pancreatic b-cells could be useful for b-cell replacement. We hypothesized that intrahepatic biliary epithelial cells (IHBECs) could become a new source of insulin-producing cells. IHBECs isolated from adult mice were expanded using our novel culture method termed, collagen-embedded floating culture method (CEFCM). With CEFCM, IHBECs formed three-dimensional ductal cysts and rapidly expanded their number by about 15-fold within 2 weeks. Over 90% of cells were positive for cytokeratin 7 and 19. At day 14, IHBECs were transfected with adenoviral (Ad)- pancreas duodenum homeobox 1 (Pdx-1), NeuroD or Pdx-1/VP16. After 7 additional days in serum- and insulin-free differentiation medium (DM), cell phenotypes were determined by RT-PCR, immunostaining and ELISA for insulin. In DM control IHBECs started to express some endocrine progenitor genes (Neurog3, NeuroD, Nkx6.1, and Pdx-1) but lacked insulin gene (Ins) mRNA. Transduced expression of PDX-1, NEUROD or PDX-1/VP16 led to expression of not only INS but also GLUT2 and prohormone convertase 1 and 2. About 3% of 4000 cells counted in PDX-1/VP16 transduced cultures stained strongly for C-peptide suggesting that a subpopulation may have the capacity for differentiation. Transduced cells released insulin (Ad-PDX-1 0 . 08G0 . 05, Ad-NEUROD 0 . 33G0 . 09, Ad-PDX-1/VP16 0 . 37G 0.14 ng/1!10 5 cells after 48 h in culture). IHBECs can be markedly expanded, and then with molecular manipulation a subpopulation of these cells can differentiate towards a b-cell phenotype. This approach may lead to a new source of b-cells that can be used for transplantation in diabetes.

Gene therapy in the inner ear using adenovirus vectors.

Abstract: Therapies for the protection and regeneration of auditory hair cells are of great interest given the significant monetary and lifestyle impact of hearing loss. The past decade has seen tremendous advances in the use of adenoviral vectors to achieve these aims. Preliminary data demonstrated the functional capacity of this technique as adenoviral-induced expression of neurotrophic and growth factors protected hair cells and spiral ganglion neurons from ototoxic insults. Subsequent efforts confirmed the feasibility of adenoviral transfection of cells in the auditory neuroepithelium via cochleostomy into the scala media. Most recently, efforts have focused on regeneration of depleted hair cells. Mammalian hearing loss is generally considered a permanent insult as the auditory epithelium lacks a basal layer capable of producing new hair cells. Recently, the transcription factor Atoh1 has been found to play a critical role in hair cell differentiation. Adenoviral-mediated overexpression of Atoh1 in culture and in vivo have shown the ability to regenerate auditory and vestibular hair cells by causing transdifferentiation of neighboring epithelial-supporting cells. Functional recovery of both the auditory and vestibular systems has been documented following adenoviral induced Atoh1 overexpression.

Characterization of mouse alveolar epithelial cell monolayers

Abstract: We investigated the influence of extracellular matrix on transport properties of mouse alveolar epithelial cell (AEC) monolayers (MAECM) and transdifferentiation of isolated mouse alveolar epitheli...

Transdifferentiation of human adipose tissue-derived stromal cells into insulin-producing clusters.

Abstract: Type 1 diabetes mellitus is caused by autoimmune destruction of insulin-producing beta cells. The major obstacle to transplantation of insulin-producing cells to cure the disease is the limited source of these cells. To overcome this problem, we describe here a multistep protocol for generation of insulin-producing islet-like clusters from human adipose tissue-derived stromal cells (ADSCs). Analysis using reverse transcription polymerase chain reaction detected enhanced expression of various pancreatic genes during the differentiation of ADSCs. Immunofluorescence analysis revealed functional similarities between cells derived from ADSCs and pancreatic islet cells, i.e., the presence of insulin- and C-peptide-coexpressing cells in the clusters and glucagon expression on the cell surface. The glucose challenge tests revealed the production of insulin, and such production was regulated via physiological signaling pathways. Our insulin-producing cells derived from ADSCs could be potentially used for cell therapy of type 1 diabetes mellitus.

In vivo physiological transdifferentiation of adult adipose cells.

Abstract: Grafts of adipose tissue from adult Rosa26 mice from different sites of the body, irrespective of the sex of the donor, share with the mammary fat the property of giving rise to milk-secreting epithelial cells when exposed to the microenvironment of the mammary gland in pregnant and lactating females. To rule out the possibility that the labeled mammary glandular tissue was derived from stem cells associated with the stroma vascular part of the grafts, we injected into the mammary gland a pure suspension of adipocytes obtained by treating a fragment of adipose tissue with collagenase. X-gal-positive cells were inserted into the alveoli of the native gland, and electron microscopy showed that the labeled cells had transformed into milk-secreting glandular cells. At the site of the adipocyte injection, the labeled alveoli contained a mixture of X-gal-positive and X-gal-negative cells, and a single epithelial cell was occasionally stained in an otherwise unlabeled alveolus. This suggests that growing ducts individually recruit adjacent adipocytes that transdifferentiate into secretory epithelial cells as they became part of the glandular alveoli. After dissociation, the isolated adipocytes retained the morphology and protein markers typical of differentiated fat cells but expressed high levels of stem cell genes and the reprogramming transcription factor Klf4. Thus, the well-documented osteogenic, chondrogenic, myogenic, and angiogenic transformation of preadipocytes associated with the stroma vascular component of the adipose tissue may reflect an intrinsic capability of adipocytes to reprogram their gene expression and transform into different cytotypes.