Transdifferentiation,Metaplasia and Tissue Regeneration

Transdifferentiation is defined as the conversion of one cell type to another. It belongs to a wider class of cell type transformations called metaplasias which also includes cases in which stem cells of one tissue type switch to a completely different stem cell. Numerous examples of transdifferentiation exist within the literature. For example, isolated striated muscle of the invertebrate jellyfish (Anthomedusae) has enormous transdifferentiation potential and even functional organs (e.g. tentacles and the feeding organ (manubrium) can be generated in-vitro. In contrast, the potential for transdifferentiation in vertebrates is much reduced, at least under normal (non-pathological) conditions. But despite these limitations, there are some well-documented cases of transdifferentiation occurring in vertebrates. For example, in the newt, the lens of the eye can be formed from the epithelial cells of the iris. Other examples of transdifferentiation include the appearance of hepatic foci in the pancreas, the development of intestinal tissue at the lower end of the oesophagus and the formation of muscle, chondrocytes and neurons from neural precursor cells. Although controversial, recent results also suggest the ability of adult stem cells from different embryological germlayers to produce differentiated cells e.g. mesodermal stem cells forming ecto- or endodermally-derived cell types. This phenomenon may constitute an example of metaplasia. The current review examines in detail some well-documented examples of transdifferentiation, speculates on the potential molecular and cellular mechanisms that underlie the switches in phenotype, together with their significance to organogenesis and regenerative medicine.     

Transdifferentiation: a cell and molecular reprogramming process

Evidence has emerged recently indicating that differentiation is not entirely a one-way process, and that it is possible to convert one cell type to another, both in vitro and in vivo. This phenomenon is called transdifferentiation, and is generally defined as the stable switch of one cell type to another. Transdifferentiation plays critical roles during development and in regeneration pathways in nature. Although this phenomenon occurs rarely in nature, recent studies have been focused on transdifferentiation and the reprogramming ability of cells to produce specific cells with new phenotypes for use in cell therapy and regenerative medicine. Thus, understanding the principles and the mechanism of this process is important for producing desired cell types. Here some well-documented examples of transdifferentiation, and their significance in development and regeneration are reviewed. In addition, transdifferentiation pathways are considered and their potential molecular mechanisms, especially the role of master switch genes, are considered. Finally, the significance of transdifferentiation in regenerative medicine is discussed.     

Chemical modulation of cell fates: <Emphasis Type="Italic">in situ</Emphasis> regeneration

Chemical modulation of cell fates has been widely used to promote tissue and organ regeneration. Small molecules can target the self-renewal, expansion, differentiation, and survival of endogenous stem cells for enhancing their regenerative power or induce dedifferentiation or transdifferentiation of mature cells into proliferative progenitors or specialized cell types needed for regeneration. Here, we discuss current progress and potential using small molecules to promote in vivo regenerative processes by regulating the cell fate. Current studies of small molecules in regeneration will provide insights into developing safe and efficient chemical approaches for in situ tissue repair and regeneration.     

Pancreatic epithelial plasticity mediated by acinar cell transdifferentiation and generation of nestin-positive intermediates

Epithelial metaplasia occurs when one predominant cell type in a tissue is replaced by another, and is frequently associated with an increased risk of subsequent neoplasia. In both mouse and human pancreas, acinar-to-ductal metaplasia has been implicated in the generation of cancer precursors. We show that pancreatic epithelial explants undergo spontaneous acinar-to-ductal metaplasia in response to EGFR signaling, and that this change in epithelial character is associated with the appearance of nestin-positive transitional cells. Lineage tracing involving Cre/lox-mediated genetic cell labeling reveals that acinar-to-ductal metaplasia represents a true transdifferentiation event, mediated by initial dedifferentiation of mature exocrine cells to generate a population of nestin-positive precursors, similar to those observed during early pancreatic development. These results demonstrate that a latent precursor potential resides within mature exocrine cells, and that this potential is regulated by EGF receptor signaling. In addition, these observations provide a novel example of rigorously documented transdifferentiation within mature mammalian epithelium, and suggest that plasticity of mature cell types may play a role in the generation of neoplastic precursors.     

Transdifferentiation--fact or artifact

Normal development appears to involve a progressive restriction in developmental potential. However, recent evidence suggests that this progressive restriction is not irreversible and can be altered to reveal novel phenotypic potentials of stem, progenitor, and even differentiated cells. While some of these results can be explained by the presence of contaminating cell populations, persistence of pluripotent stem cells, cell fusion, etc., several examples exist that are difficult to explain as anything other than "true transdifferentiation" and/or dedifferentiation. These examples of transdifferentiation are best explained by understanding how the normal process of progressive cell fate restriction occurs during development. We suggest that subversion of epigenetic controls regulating cell type specific gene expression likely underlies the process of transdifferentiation and it may be possible to identify specific factors to control the transdifferentiation process. We predict, however, that transdifferentiation will not be reliable or reproducible and will probably require complex manipulations.     

Chemical modulation of cell fates: in situ regeneration

Chemical modulation of cell fates has been widely used to promote tissue and organ regeneration. Small molecules can target the self-renewal, expansion, differentiation, and survival of endogenous stem cells for enhancing their regenerative power or induce dedifferentiation or transdifferentiation of mature cells into proliferative progenitors or specialized cell types needed for regeneration. Here, we discuss current progress and potential using small molecules to promote in vivo regenerative processes by regulating the cell fate. Current studies of small molecules in regeneration will provide insights into developing safe and efficient chemical approaches for in situ tissue repair and regeneration.     

Adult reserve stem cells and their potential for tissue engineering

Tissue restoration is the process whereby multiple damaged cell types are replaced to restore the histoarchitecture and function to the tissue. Several theories, have been proposed to explain the phenomenon of tissue restoration in amphibians and in animals belonging to higher order. These theories include dedifferentiation of damaged tissues, transdifferentiation of lineage-committed progenitor cells, and activation of reserve, precursor cells. Studies by Young et al. and others demonstrated that connective tissue compartments throughout postnatal individuals contain reserve precursor cells. Subsequent repetitive single cell-cloning and cell-sorting studies revealed that these reserve precursor cells consisted of multiple populations of cells, including, tissue-specific progenitor cells, germ-layer lineage stem cells, and pluripotent stem cells. Tissue-specific progenitor cells display various capacities for differentiation, ranging from unipotency (forming a single cell type) to multipotency (forming multiple cell types). However, all progenitor cells demonstrate a finite life span of 50 to 70 population doublings before programmed cell senescence and cell death occurs. Germ-layer lineage stem cells can form a wider range of cell types than a progenitor cell. An individual germ-layer lineage stem cell can form all cells types within its respective germ-layer lineage (i.e., ectoderm, mesoderm, or endoderm). Pluripotent stem cells can form a wider range of cell types than a single germ-layer lineage stem cell. A single pluripotent stem cell can form cells belonging to all three germ layer lineages. Both germ-layer lineage stem cells and pluripotent stem cells exhibit extended capabilities for self-renewal, far surpassing the limited life span of progenitor cells (50–70 population doublings). The authors propose that the activation of quiescent tissue-specific progenitor cells, germ-layer lineage stem cells, and/or pluripotent stem cells may be a potential explanation, along with dedifferentiation and transdifferentiation, for the process of tissue restoration. Several model systems are currently being investigated to determine the possibilities of using these adult quiescent reserve precursor cells for tissue engineering.     

Loss of stem cell regenerative capacity within aged niches

This work uncovers novel mechanisms of aging within stem cell niches that are evolutionarily conserved between mice and humans and affect both embryonic and adult stem cells. Specifically, we have examined the effects of aged muscle and systemic niches on key molecular identifiers of regenerative potential of human embryonic stem cells (hESCs) and post-natal muscle stem cells (satellite cells). Our results reveal that aged differentiated niches dominantly inhibit the expression of Oct4 in hESCs and Myf-5 in activated satellite cells, and reduce proliferation and myogenic differentiation of both embryonic and tissue-specific adult stem cells (ASCs). Therefore, despite their general neoorganogenesis potential, the ability of hESCs, and the more differentiated myogenic ASCs to contribute to tissue repair in the old will be greatly restricted due to the conserved inhibitory influence of aged differentiated niches. Significantly, this work establishes that hESC-derived factors enhance the regenerative potential of both young and, importantly, aged muscle stem cells in vitro and in vivo; thus, suggesting that the regenerative outcome of stem cell-based replacement therapies will be determined by a balance between negative influences of aged tissues on transplanted cells and positive effects of embryonic cells on the endogenous regenerative capacity. Comprehensively, this work points toward novel venues for in situ restoration of tissue repair in the old and identifies critical determinants of successful cell-replacement therapies for aged degenerating organs.     

The skeletal muscle satellite cell: stem cell or son of stem cell?

The concept of the adult tissue stem cell is fundamental to models of persistent renewal in functionally post-mitotic tissues. Although relatively ignored by stem cell biology, skeletal muscle is a prime example of an adult tissue that can generate terminally differentiated cells uniquely specialized to carry out tissue-specific functions. This capacity is attributed to satellite cells, a population of undifferentiated, quiescent precursors that become activated to divide and differentiate in response to the demands of growth or damage. The aim of this review is to discuss the role of the satellite cell as an adult tissue-specific stem cell. We examine evidence for the presence of behaviourally and phenotypically distinct subpopulations of precursor within the satellite cell pool. Further, we speculate on the possible identity, origins and relevance of multipotent muscle stem cells, a population with both myogenic and hematopoietic potentials that has been isolated from whole muscle. Taken together, current evidence suggests the possibility that the regenerative compartment of adult skeletal muscle may conform to an archetypal stem cell-based hierarchy, maintained within a stem cell niche. It therefore remains to be seen whether all satellite cells are skeletal muscle-specific stem cells, or whether some or all are the progeny of an as yet unidentified muscle stem cell.     

How cells change their phenotype

Recent attention has focused on the remarkable ability of adult stem cells to produce differentiated cells from embryologically unrelated tissues. This phenomenon is an example of metaplasia and shows that embryological commitments can be reversed or erased under certain circumstances. In some cases, even fully differentiated cells can change their phenotype (transdifferentiation). This review examines recently discovered cases of metaplasia, and speculates on the potential molecular and cellular mechanisms that underlie the switches, and their significance to developmental biology and medicine.     

干细胞与再生医学研究进展

干细胞具有分化成为体内所有类型细胞的能力,因此,其在再生医学治疗、体外疾病模拟、药物筛选等方面具有广阔的应用前景。干细胞技术在近些年取得了突飞猛进的发展,特别是诱导多能性干细胞的出现使干细胞领域经历了一场巨大的变革。我国干细胞研究在这场干细胞技术变革中取得了多项重大成果,逐渐成为了世界干细胞研究领域中的重要力量。本综述着重介绍近几年来,主要是诱导多能性干细胞技术出现之后,我国在干细胞和再生医学领域取得的重要进展,主要涵盖诱导多能性干细胞、转分化、单倍体干细胞以及基因修饰动物模型和基因治疗等方面。     

Potential of embryonic and adult stem cells in vitro

Recent developments in the field of stem cell research indicate their enormous potential as a source of tissue for regenerative therapies. The success of such applications will depend on the precise properties and potentials of stem cells isolated either from embryonic, fetal or adult tissues. Embryonic stem cells established from the inner cell mass of early mouse embryos are characterized by nearly unlimited proliferation, and the capacity to differentiate into derivatives of essentially all lineages. The recent isolation and culture of human embryonic stem cell lines presents new opportunities for reconstructive medicine. However, important problems remain; first, the derivation of human embryonic stem cells from in vitro fertilized blastocysts creates ethical problems, and second, the current techniques for the directed differentiation into somatic cell populations yield impure products with tumorigenic potential. Recent studies have also suggested an unexpectedly wide developmental potential of adult tissue-specific stem cells. Here too, many questions remain concerning the nature and status of adult stem cells both in vivo and in vitro and their proliferation and differentiation/transdifferentiation capacity. This review focuses on those issues of embryonic and adult stem cell biology most relevant to their in vitro propagation and differentiation. Questions and problems related to the use of human embryonic and adult stem cells in tissue regeneration and transplantation are discussed.     

Transdifferentiation from striated muscle of medusae in vitro

We have established an in vitro transdifferentiation and regeneration system which is based entirely on mononucleated striated muscle cells. The muscle tissue is isolated from anthomedusae and activated by various means to undergo cell cycles and transdifferentiation to several new cell types. In all cases DNA-replication is initiated and the division products are smooth muscle cells, characterized by their ultrastructure and monoclonal antibodies, and nerve/sensory cells, characterized by their ultrastructure and FMRFamide-staining. Both cell types are found at a 1:1 ratio after the first division. The nerve cells stop to replicate, whereas the smooth muscle cells continue and keep producing in each successive division a smooth muscle cell and a nerve cell. The observed data indicate that smooth muscle cells behave like stem cells. Depending on the destabilization and culturing methods, some isolated muscle tissue will form a bilayered fragment and within only two cell cycles manubria (the feeding and sexual organ) or tentacles will regenerate. In this case six to eight new non-muscle cell types have been formed by transdifferentiation.     

Aging,Stem Cells and Tissue Regeneration: Lessons from Muscle

With age, there is a gradual decline in the regenerative properties of most tissues due to a combination of age-dependent changes in tissue-specific stem cells and in the environmental cues that promote those cells to participate in tissue maintenance and repair. In adult skeletal muscle, where the resident dedicated stem cells (“satellite cells”) are capable of rapid and highly effective regeneration in response to injury, there is just such a loss of regenerative potential with age. Satellite cell activation and cell fate determination are controlled by the Notch signaling pathway that is initiated by the rapid increase in expression of the Notch ligand, Delta, following injury. In old muscle, this upregulation of Delta is blunted and thus satellite cell activation is markedly diminished. However, by indirectly inducing Notch activity, the regenerative potential of aged satellite cells can be restored. Furthermore, exposure of aged satellite cells to serum from young mice, either in vivo by heterochronic parabiotic pairings or in vitro, rejuvenates the satellite cell response. This restorative potential suggests that tissue-specific stem cells do not lose their ability to participate in tissue maintenance and repair. Therefore, it may be that even very old stem cells may be capable of maintaining and repairing aged tissues if provided with optimal environmental cues.     

Sex steroid-mediated reprogramming of vascular smooth muscle cells to stem cells and neurons: Possible utilization of sex steroid combinations for regenerative treatment without utilization of in vitro developed stem cells

Previous work from our laboratory demonstrated that sex steroid combinations, but not individual sex steroids alone, cause transdifferentiation of ovarian epithelial cells - ovarian surface epithelium (OSE) and follicular granulosa cells - into neural stem cells (NSC) and differentiating neurons. In the present study we have chosen primary culture of human vascular smooth muscle cells (SMC), a non-epithelial mesenchymal cells in order to test them as a control cell type regarding their morphology and expression of NSC and neuronal markers. Utilization of estradiol (E2), progesterone (PG) or testosterone (TS) alone did not induce the emergence of neurons from the vascular SMC. However, the treatment with sex steroid combinations (PG+TS or E2+PG+TS) caused transdifferentiation into neural/neuronal type cells. By immunohistochemistry, these cells exhibited strong expression of stem cell markers and neural/neuronal glycoconjugates SSEA-1, SSEA-4, Thy-1, NeuN and NCAM. In the Neurobasal/B27 medium both, the OSE and vascular SMC also transdifferentiated into neuronal cells. Western blot analysis has shown significant increase of NeuN 48-kDa species after E2+PG or PG+TS treatment. Secretion of E2 increased significantly in vascular SMC cultures pretreated with TS, PG or TS+PG. Unlike OSE cells, the vascular SMC accompany as pericytes all vessels, including CNS microvasculature. We also observed that sex steroid combinations could produce SMC stem type cells which differentiated within a few days back to mature vascular SMC. This is of potential interest for the vascular regenerative medicine. Altogether, our observations suggest that sex steroid combinations could induce in vivo improvement of neurodegenerative, traumatic and ischemic neurological disorders and vascular diseases via their effect on resident pluripotent vascular SMC, i.e. without a need of in vitro developed stem cells.     

APC is required for muscle stem cell proliferation and skeletal muscle tissue repair

The tumor suppressor adenomatous polyposis coli (APC) is a crucial regulator of many stem cell types. In constantly cycling stem cells of fast turnover tissues, APC loss results in the constitutive activation of a Wnt target gene program that massively increases proliferation and leads to malignant transformation. However, APC function in skeletal muscle, a tissue with a low turnover rate, has never been investigated. Here we show that conditional genetic disruption of APC in adult muscle stem cells results in the abrogation of adult muscle regenerative potential. We demonstrate that APC removal in adult muscle stem cells abolishes cell cycle entry and leads to cell death. By using double knockout strategies, we further prove that this phenotype is attributable to overactivation of β-catenin signaling. Our results demonstrate that in muscle stem cells, APC dampens canonical Wnt signaling to allow cell cycle progression and radically diverge from previous observations concerning stem cells in actively self-renewing tissues.     

Pax7-expressing satellite cells are indispensable for adult skeletal muscle regeneration

Distinct cell populations with regenerative capacity have been reported to contribute to myofibres after skeletal muscle injury, including non-satellite cells as well as myogenic satellite cells. However, the relative contribution of these distinct cell types to skeletal muscle repair and homeostasis and the identity of adult muscle stem cells remain unknown. We generated a model for the conditional depletion of satellite cells by expressing a human diphtheria toxin receptor under control of the murine Pax7 locus. Intramuscular injection of diphtheria toxin during muscle homeostasis, or combined with muscle injury caused by myotoxins or exercise, led to a marked loss of muscle tissue and failure to regenerate skeletal muscle. Moreover, the muscle tissue became infiltrated by inflammatory cells and adipocytes. This localised loss of satellite cells was not compensated for endogenously by other cell types, but muscle regeneration was rescued after transplantation of adult Pax7(+) satellite cells alone. These findings indicate that other cell types with regenerative potential depend on the presence of the satellite cell population, and these observations have important implications for myopathic conditions and stem cell-based therapeutic approaches.     

Fate restriction in limb muscle precursor cells precedes high-level expression of MyoD family member genes

The mechanisms by which pluripotent embryonic cells generate unipotent tissue progenitor cells during development are unknown. Molecular/genetic experiments in cultured cells have led to the hypothesis that the product of a single member of the MyoD gene family (MDF) is necessary and sufficient to establish the positive aspects of the determined state of myogenic precursor cells: i.e., the ability to initiate and maintain the differentiated state (Weintraub, H., Davis, R., Tapscott, S., Thayer, M., Krause, M., Benezra, R., Blackwell, T. K., Turner, D., Rupp, R., Hollenberg, S. et al. (1991) Science 251, 761-766). Embryonic cell type determination also involves negative regulation, such as the restriction of developmental potential for alternative cell types, that is not directly addressed by the MDF model. In the experiments reported here, phenotypic restriction in myogenic precursor cells is assayed by an in vivo 'notochord challenge' to evaluate their potential to 'choose' between two alternative cell fate endpoints: cartilage and muscle (Williams, B. A. and Ordahl, C. P. (1997) Development 124, 4983-4997). Two separate myogenic precursor cell populations were found to be phenotypically restricted while expressing the Pax3 gene and prior to MDF gene activation. Therefore, while MDF family members act positively during myogenic differentiation, phenotypic restriction, the negative aspect of cell specification, requires cellular and molecular events and interactions that precede MDF expression in myogenic precursor cells. The qualities of muscle formed by the determined myogenic precursor cells in these experiments further indicate that their developmental potential is intermediate between that of myoblastic stem cells taken from fetal or adult tissue (which lack mitotic and morphogenetic potential when tested in vivo) and embryonic stem cells (which are multipotent). We hypothesize that such embryonic myogenic progenitor cells represent a distinct class of determined embryonic cell, one that is responsible for both tissue growth and tissue morphogenesis.     

Stem cells, regenerative medicine, and animal models of disease

The field of stem cell biology and regenerative medicine is rapidly moving toward translation to clinical practice, and in doing so has become even more dependent on animal donors and hosts for generating cellular reagents and assaying their potential therapeutic efficacy in models of human disease. Advances in cell culture technologies have revealed a remarkable plasticity of stem cells from embryonic and adult tissues, and transplantation models are now needed to test the ability of these cells to protect at-risk cells and replace cells lost to injury or disease. With such a mandate, issues related to acceptable sources and controversial (e.g., chimeric) models have challenged the field to provide justification of their potential efficacy before the passage of new restrictions that may curb anticipated breakthroughs. Progress from the use of both in vitro and in vivo regenerative medicine models already offers hope both for the facilitation of stem cell phenotyping in recursive gene expression profile models and for the use of stem cells as powerful new therapeutic reagents for cancer, stroke, Parkinson's, and other challenging human diseases that result in movement disorders. This article describes research in support of the following three objectives: (1) To discover the best stem or progenitor cell in vitro protocols for isolating, expanding, and priming these cells to facilitate their massive propagation into just the right type of neuronal precursor cell for protection or replacement protocols for brain injury or disease, including those that affect movement such as Parkinson's disease and stroke; (2) To discover biogenic factors--compounds that affect stem/progenitor cells (e.g., from high-throughput screening and other bioassay approaches)--that will encourage reactive cell genesis, survival, selected differentiation, and restoration of connectivity in central nervous system movement and other disorders; and (3) To establish the best animal models of human disease and injury, using both small and large animals, for testing new regenerative medicine therapeutics.