Distinct Wnt signaling pathways have opposing roles in appendage regeneration

In contrast to mammals, lower vertebrates have a remarkable capacity to regenerate complex structures damaged by injury or disease. This process, termed epimorphic regeneration, involves progenitor cells created through the reprogramming of differentiated cells or through the activation of resident stem cells. Wnt/beta-catenin signaling regulates progenitor cell fate and proliferation during embryonic development and stem cell function in adults, but its functional involvement in epimorphic regeneration has not been addressed. Using transgenic fish lines, we show that Wnt/beta-catenin signaling is activated in the regenerating zebrafish tail fin and is required for formation and subsequent proliferation of the progenitor cells of the blastema. Wnt/beta-catenin signaling appears to act upstream of FGF signaling, which has recently been found to be essential for fin regeneration. Intriguingly, increased Wnt/beta-catenin signaling is sufficient to augment regeneration, as tail fins regenerate faster in fish heterozygous for a loss-of-function mutation in axin1, a negative regulator of the pathway. Likewise, activation of Wnt/beta-catenin signaling by overexpression of wnt8 increases proliferation of progenitor cells in the regenerating fin. By contrast, overexpression of wnt5b (pipetail) reduces expression of Wnt/beta-catenin target genes, impairs proliferation of progenitors and inhibits fin regeneration. Importantly, fin regeneration is accelerated in wnt5b mutant fish. These data suggest that Wnt/beta-catenin signaling promotes regeneration, whereas a distinct pathway activated by wnt5b acts in a negative-feedback loop to limit regeneration.     

Progenitor cells isolated from the human heart: a potential cell source for regenerative therapy

Background. In recent years, resident cardiac progenitor cells have been identified in, and isolated from the rodent heart. These cells show the potential to form cardiomyocytes, smooth muscle cells, and endothelial cells in vitro and in vivo and could potentially be used as a source for cardiac repair. However, previously described cardiac progenitor cell populations show immature development and need co-culture with neonatal rat cardiomyocytes in order to differentiate in vitro. Here we describe the localisation, isolation, characterisation, and differentiation of cardiomyocyte progenitor cells (CMPCs) isolated from the human heart. Methods. hCMPCs were identified in human hearts based on Sca-1 expression. These cells were isolated, and FACS, RT-PCR and immunocytochemistry were used to determine their baseline characteristics. Cardiomyogenic differentiation was induced by stimulation with 5-azacytidine. Results. hCMPCs were localised within the atria, atrioventricular region, and epicardial layer of the foetal and adult human heart. In vitro, hCMPCs could be induced to differentiate into cardiomyocytes and formed spontaneously beating aggregates, without the need for co-culture with neonatal cardiomyocytes. Conclusion. The human heart harbours a pool of resident cardiomyocyte progenitor cells, which can be expanded and differentiated in vitro. These cells may provide a suitable source for cardiac regeneration cell therapy. (Neth Heart J 2008;16: 163-9.)     

Common origins of blood and blood vessels in adults?

After embryonic development, the vast majority of cells are differentiated and all organs are in place. Growth of the organism then ensues and continues until adulthood, whereupon cell division largely ceases. In some tissues, notably the bone marrow, skin, and gut, cell proliferation continues throughout life to replace cells lost by attrition. This regeneration is fueled by rare, long-lived, and largely quiescent stem cells that give rise to committed progenitors, which in turn generate large numbers of fully differentiated cells. Mounting evidence suggests that such cells can significantly contribute to tissue repair and regeneration in adults and may therefore prove beneficial for autologous cell and gene therapies. This review focuses on the potential of adult stem cells to give rise to hematopoietic and vascular cells. We discuss evidence that a highly purified population of adult stem cells, termed SP cells, serves as a hematopoietic progenitor and can contribute to vascular regeneration after injury. We also discuss the potential relationship of these cells to the embryonic hemangioblast.     

Regeneration of oral siphon pigment organs in the ascidian Ciona intestinalis

Ascidians have powerful capacities for regeneration but the underlying mechanisms are poorly understood. Here we examine oral siphon regeneration in the solitary ascidian Ciona intestinalis. Following amputation, the oral siphon rapidly reforms oral pigment organs (OPO) at its distal margin prior to slower regeneration of proximal siphon parts. The early stages of oral siphon reformation include cell proliferation and re-growth of the siphon nerves, although the neural complex (adult brain and associated organs) is not required for regeneration. Young animals reform OPO more rapidly after amputation than old animals indicating that regeneration is age dependent. UV irradiation, microcautery, and cultured siphon explant experiments indicate that OPOs are replaced as independent units based on local differentiation of progenitor cells within the siphon, rather than by cell migration from a distant source in the body. The typical pattern of eight OPOs and siphon lobes is restored with fidelity after distal amputation of the oral siphon, but as many as 16 OPOs and lobes can be reformed following proximal amputation near the siphon base. Thus, the pattern of OPO regeneration is determined by cues positioned along the proximal distal axis of the oral siphon. A model is presented in which columns of siphon tissue along the proximal-distal axis below pre-existing OPO are responsible for reproducing the normal OPO pattern during regeneration. This study reveals previously unknown principles of oral siphon and OPO regeneration that will be important for developing Ciona as a regeneration model in urochordates, which may be the closest living relatives of vertebrates.     

Aberrant Innate Immune Activation following Tissue Injury Impairs Pancreatic Regeneration

Normal tissue architecture is disrupted following injury, as resident tissue cells become damaged and immune cells are recruited to the site of injury. While injury and inflammation are critical to tissue remodeling, the inability to resolve this response can lead to the destructive complications of chronic inflammation. In the pancreas, acinar cells of the exocrine compartment respond to injury by transiently adopting characteristics of progenitor cells present during embryonic development. This process of de-differentiation creates a window where a mature and stable cell gains flexibility and is potentially permissive to changes in cellular fate. How de-differentiation can turn an acinar cell into another cell type (such as a pancreatic β-cell), or a cell with cancerous potential (as in cases of deregulated Kras activity) is of interest to both the regenerative medicine and cancer communities. While it is known that inflammation and acinar de-differentiation increase following pancreatic injury, it remains unclear which immune cells are involved in this process. We used a combination of genetically modified mice, immunological blockade and cellular characterization to identify the immune cells that impact pancreatic regeneration in an in vivo model of pancreatitis. We identified the innate inflammatory response of macrophages and neutrophils as regulators of pancreatic regeneration. Under normal conditions, mild innate inflammation prompts a transient de-differentiation of acinar cells that readily dissipates to allow normal regeneration. However, non-resolving inflammation developed when elevated pancreatic levels of neutrophils producing interferon-γ increased iNOS levels and the pro-inflammatory response of macrophages. Pancreatic injury improved following in vivo macrophage depletion, iNOS inhibition as well as suppression of iNOS levels in macrophages via interferon-γ blockade, supporting the impairment in regeneration and the development of chronic inflammation arises from aberrant activation of the innate inflammatory response. Collectively these studies identify targetable inflammatory factors that can be used to influence the development of non-resolving inflammation and pancreatic regeneration following injury.     

TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor cells and skeletal muscle regeneration

Inflammation participates in tissue repair through multiple mechanisms including directly regulating the cell fate of resident progenitor cells critical for successful regeneration. Upon surveying target cell types of the TNF ligand TWEAK, we observed that TWEAK binds to all progenitor cells of the mesenchymal lineage and induces NF-kappaB activation and the expression of pro-survival, pro-proliferative and homing receptor genes in the mesenchymal stem cells, suggesting that this pro-inflammatory cytokine may play an important role in controlling progenitor cell biology. We explored this potential using both the established C2C12 cell line and primary mouse muscle myoblasts, and demonstrated that TWEAK promoted their proliferation and inhibited their terminal differentiation. By generating mice deficient in the TWEAK receptor Fn14, we further showed that Fn14-deficient primary myoblasts displayed significantly reduced proliferative capacity and altered myotube formation. Following cardiotoxin injection, a known trigger for satellite cell-driven skeletal muscle regeneration, Fn14-deficient mice exhibited reduced inflammatory response and delayed muscle fiber regeneration compared with wild-type mice. These results indicate that the TWEAK/Fn14 pathway is a novel regulator of skeletal muscle precursor cells and illustrate an important mechanism by which inflammatory cytokines influence tissue regeneration and repair. Coupled with our recent demonstration that TWEAK potentiates liver progenitor cell proliferation, the expression of Fn14 on all mesenchymal lineage progenitor cells supports a broad involvement of this pathway in other tissue injury and disease settings.     

Induction of Retinal Progenitors and Neurons from Mammalian Müller Glia under Defined Conditions

Vision impairment caused by loss of retinal neurons affects millions of people worldwide, and currently, there is no effective treatment. Müller glia of mammalian retina may represent an under-recognized and potential source for regeneration of a wide range of retinal cell types, including retinal ganglion cells and photoreceptors. Here, we demonstrated that mouse Müller glia cells have the capacity to be reprogrammed into the retinal neuronal cell fate and are competent to give rise to photoreceptors under a defined culture condition. Inactivation of p53 released proliferation restriction of Müller glia and significantly enhanced the induction of retinal progenitor from Müller glia in culture. Moreover, following the ocular transplantation, the Müller glia-derived progenitors were differentiated toward the fates of photoreceptors and retinal ganglion cells. Together, these results demonstrate the feasibility of using Müller glia as a potential source for retinal repair and regeneration.     

Expression of regulatory and tissue-specific genes controlling regenerative potencies of the eye tissues in vertebrates

Comparative analysis of the early transformations of differentiated cells of the pigment epithelium, ciliary fold epithelium, and Muller glia in the eye of lower vertebrates and mammals during retina regeneration and cultivation was performed for the first time. Dedifferentiation and proliferation of cells and formation of progenitor multipotent cells, which are a source of retina regeneration in adult newts, were characterized using cell, molecular, and genetic markers. Neurospheres were formed during cultivation of the differentiated cells, in which progenitor multipotent cells were found that transformed into neurons of retina and brain and into glial cells. Comparative analysis of changes in the pigment epithelium cells during retina regeneration and during cultivation of differentiated cells of the pigment and ciliary epithelia and Muller glia suggests similar cell transformations at the early stages of transdifferentiation.     

Expression of regulatory and tissue-specific genes controlling regenerative potencies of eye tissues in vertebrates

Comparative analysis of the early transformations of differentiated cells of the pigment epithelium, ciliary fold epithelium, and Muller glia in the eye of lower vertebrates and mammals during retina regeneration and cultivation was performed for the first time. Dedifferentiation and proliferation of cells and formation of progenitor multipotent cells, which are a source of retina regeneration in adult newts, were characterized using cell, molecular, and genetic markers. Neurospheres were formed during cultivation of the differentiated cells, in which progenitor multipotent cells were found that transformed into neurons of retina and brain and into glial cells. Comparative analysis of changes in the pigment epithelium cells during retina regeneration and during cultivation of differentiated cells of the pigment and ciliary epithelia and Muller glia suggests similar cell transformations at the early stages of transdifferentiation.     

Inducible progenitor-derived Wingless regulates adult midgut regeneration in Drosophila

The ability to regenerate following stress is a hallmark of self-renewing tissues. However, little is known about how regeneration differs from homeostatic tissue maintenance. Here, we study the role and regulation of Wingless (Wg)/Wnt signalling during intestinal regeneration using the Drosophila adult midgut. We show that Wg is produced by the intestinal epithelial compartment upon damage or stress and it is exclusively required for intestinal stem cell (ISC) proliferation during tissue regeneration. Reducing Wg or downstream signalling components from the intestinal epithelium blocked tissue regeneration. Importantly, we demonstrate that Wg from the undifferentiated progenitor cell, the enteroblast, is required for Myc-dependent ISC proliferation during regeneration. Similar to young regenerating tissues, ageing intestines required Wg and Myc for ISC hyperproliferation. Unexpectedly, our results demonstrate that epithelial but not mesenchymal Wg is essential for ISC proliferation in response to damage, while neither source of the ligand is solely responsible for ISC maintenance and tissue self-renewal in unchallenged tissues. Therefore, fine-tuning Wnt results in optimal balance between the ability to respond to stress without negatively affecting organismal viability.     

Left ventricular heart aneurism--a new source of resident cardiac stem cells

In the past few years it has been established that the heart contains a reservoir of stem and progenitor cells that have the ability to differentiate in vitro and in vivo toward vascular and cardiac lineages and that show cardiac regeneration potential in vivo following injection into the infracted myocardium. The aim of the present study was to characterize cardiac stem cells in the tissue of chronic left ventricular aneurism. It was shown that human c-kit positive cells were scattered in fibrous, muscle and adipose parts of aneurism tissue. C-kit positive cells localized mainly in fibrous tissue nearby large vessels, however, c-kit positive cells did not express endothelial, smooth muscle or cardiomyocyte cell markers. Co-localization experiments demonstrated that all c-kit positive cells were of non-hematopoietic origin, since they did not express markers such as CD34 and CD45. Majority of c-kit positive cells expressed MDR1, but showed no proliferation activity (Ki67). It thus appears that aneurism tissue could be an alternative source of autologous cardiac stem cells. However, their regeneration capacity should be further explored.     

Endothelial progenitor cells for regeneration

Endothelial progenitor cells (EPCs) have been recently isolated from peripheral blood and bone marrow (BM), and shown to be incorporated into sites of physiological and pathological neovascularization in vivo. In contrast to differentiated endothelial cells (ECs), transplantation of EPCs successfully enhanced vascular development by in situ differentiation and proliferation within ischemic organs. Based on such a novel concept of closed up function on EPCs in postnatal neovascularization, the beneficial property of EPC is attractive for cell therapy as well as cell-mediated gene therapy applications targeting regeneration of ischemic tissue.     

Vascular wall resident progenitor cells: A review

The vessel wall has usually been thought to be relatively quiescent. But the discovery of progenitor cells in many tissues and in the vasculature itself has led to a reconsideration of the vascular biology. The presence of circulating endothelial and smooth muscle progenitors able to home to the injured vascular wall is a firm acquisition; less known is the notion, coming from embryonic and adult tissue studies, that stem cells able to differentiate into endothelial cells and smooth muscle cells also reside in the arterial wall. Moreover, the existence of a vasculogenic zone has recently been identified in adult human arteries; this niche-like zone is believed to act as a source of progenitors for postnatal vasculogenesis. From the literature it is already apparent that a complex interplay between circulating and resident vascular wall progenitors takes place during embryonal and postnatal life; a structural/functional disarray of these intimate stem cell compartments could hamper appropriate vascular repair, the development of vascular wall disease being the direct clinical consequence in adult life. This review gives an overview of adult large vessel progenitors established in the vascular wall during embryogenesis and their role in the maintenance of wall homeostasis.     

Echinoderm regeneration: an in vitro approach using the crinoid Antedon mediterranea

Among echinoderms, crinoids are well known for their remarkable regenerative potential. Regeneration depends mainly on progenitor cells (undifferentiated or differentiated), which migrate and proliferate in the lesion site. The crucial role of the “progenitor” elements involved in the regenerative processes, in terms of cell recruitment, sources, and fate, is a central problem in view of its topical interest and biological implications. The spectacular regenerative potential of crinoids is used to replace lost internal and external organs. In particular, the process of arm regeneration in the feather star Antedon mediterranea is the regeneration model most extensively explored to date. We have addressed the morphological and functional characterization of the cell phenotypes responsible for the arm regenerative processes by using an in vitro approach. This represents the first successful attempt to culture cells involved in crinoid regeneration. A comparison of these results with others from previous in vivo investigations confirms the diverse cell types contributing to regeneration and underscores their involvement in migration, proliferation, and dedifferentiation processes.     

Regenerative proliferation of differentiated cells by mTORC1‐dependent paligenosis

In 1900, Adami speculated that a sequence of context‐independent energetic and structural changes governed the reversion of differentiated cells to a proliferative, regenerative state. Accordingly, we show here that differentiated cells in diverse organs become proliferative via a shared program. Metaplasia‐inducing injury caused both gastric chief and pancreatic acinar cells to decrease mTORC1 activity and massively upregulate lysosomes/autophagosomes; then increase damage associated metaplastic genes such as Sox9; and finally reactivate mTORC1 and re‐enter the cell cycle. Blocking mTORC1 permitted autophagy and metaplastic gene induction but blocked cell cycle re‐entry at S‐phase. In kidney and liver regeneration and in human gastric metaplasia, mTORC1 also correlated with proliferation. In lysosome‐defective Gnptab^?/? mice, both metaplasia‐associated gene expression changes and mTORC1‐mediated proliferation were deficient in pancreas and stomach. Our findings indicate differentiated cells become proliferative using a sequential program with intervening checkpoints: (i) differentiated cell structure degradation; (ii) metaplasia‐ or progenitor‐associated gene induction; (iii) cell cycle re‐entry. We propose this program, which we term “paligenosis”, is a fundamental process, like apoptosis, available to differentiated cells to fuel regeneration following injury.     

Vascular Endothelial Growth Factor (VEGF) Bioavailability Regulates Angiogenesis and Intestinal Stem and Progenitor Cell Proliferation during Postnatal Small Intestinal Development

Background

Vascular endothelial growth factor (VEGF) is a highly conserved, master regulatory molecule required for endothelial cell proliferation, organization, migration and branching morphogenesis. Podocoryne carnea and drosophila, which lack endothelial cells and a vascular system, express VEGF homologs, indicating potential roles beyond angiogenesis and vasculogenesis. The role of VEGF in the development and homeostasis of the postnatal small intestine is unknown. We hypothesized regulating VEGF bioavailability in the postnatal small intestine would exhibit effects beyond the vasculature and influence epithelial cell stem/progenitor populations.

Methods

VEGF mutant mice were created that overexpressed VEGF in the brush border of epithelium via the villin promotor following doxycycline treatment. To decrease VEGF bioavailability, sFlt-1 mutant mice were generated that overexpressed the soluble VEGF receptor sFlt-1 upon doxycycline administration in the intestinal epithelium. Mice were analyzed after 21 days of doxycycline administration.

Results

Increased VEGF expression was confirmed by RT-qPCR and ELISA in the intestine of the VEGF mutants compared to littermates. The VEGF mutant duodenum demonstrated increased angiogenesis and vascular leak as compared to littermate controls. The VEGF mutant duodenum revealed taller villi and increased Ki-67-positive cells in the transit-amplifying zone with reduced Lgr5 expression. The duodenum of sFlt-1 mutants revealed shorter villi and longer crypts with reduced proliferation in the transit-amplifying zone, reduced expression of Dll1, Bmp4 and VE-cadherin, and increased expression of Sox9 and EphB2.

Conclusions

Manipulating VEGF bioavailability leads to profound effects on not only the intestinal vasculature, but epithelial stem and progenitor cells in the intestinal crypt. Elucidation of the crosstalk between VEGF signaling in the vasculature, mesenchyme and epithelial stem/progenitor cell populations may direct future cell therapies for intestinal dysfunction or disease.     

Resident vascular progenitor cells: An emerging role for non-terminally differentiated vessel-resident cells in vascular biology

Throughout development and adult life the vasculature exhibits a remarkably dynamic capacity for growth and repair. The vasculature also plays a pivotal role in the execution of other diverse biologic processes, such as the provisioning of early hematopoietic stem cells during embryonic development or the regulation of vascular tone and blood pressure. Adding to this importance, from an anatomical perspective, the vasculature is clearly an omnipresent organ, with few areas of the body that it does not penetrate. Given these impressive characteristics, it is perhaps to be expected that the vasculature should require, or at least be associated with, a ready supply of stem and progenitor cells. However, somewhat surprisingly, it is only now just beginning to be broadly appreciated that the vasculature plays host to a range of vessel-resident stem and progenitor cells. The possibility that these vessel-resident cells are implicated in processes as diverse as tumor vascularization and adaptive vascular remodeling appears likely, and several exciting avenues for clinical translation are already under investigation. This review explores the various stem and progenitor cell populations that are resident in the microvasculature, endothelium, and vessel walls and vessel-resident cells capable of phenotypic transformation.     

Dysfunctional stem and progenitor cells impair fracture healing with age

Successful fracture healing requires the simultaneous regeneration of both the bone and vasculature; mesenchymal stem cells(MSCs) are directed to replace the bone tissue, while endothelial progenitor cells(EPCs) form the new vasculature that supplies blood to the fracture site. In the elderly, the healing process is slowed, partly due to decreased regenerative function of these stem and progenitor cells. MSCs from older individuals are impaired with regard to cell number, proliferative capacity, ability to migrate, and osteochondrogenic differentiation potential. The proliferation, migration and function of EPCs are also compromised with advanced age. Although the reasons for cellular dysfunction with age are complex and multidimensional, reduced expression of growth factors, accumulation of oxidative damage from reactive oxygen species,and altered signaling of the Sirtuin-1 pathway are contributing factors to aging at the cellular level of both MSCs and EPCs. Because of these geriatric-specific issues, effective treatment for fracture repair may require new therapeutic techniques to restore cellular function. Some suggested directions for potential treatments include cellular therapies, pharmacological agents, treatments targeting age-related molecular mechanisms, and physical therapeutics.Advanced age is the primary risk factor for a fracture, due to the low bone mass and inferior bone quality associated with aging; a better understanding of the dysfunctional behavior of the aging cell will provide a foundation for new treatments to decrease healing time and reduce the development of complications during the extended recovery from fracture healing in the elderly.     

Cardiac progenitor cells: Potency and control

Stem cell‐based regeneration of the heart has focused much scientific and public attention being cardiac diseases the major cause of disability and death in industrialized countries. Innumerable efforts have been taken to unveil the mechanisms undergoing stem cell proliferation and fate, but much remains to be endeavoured for their application in clinical practice. Nevertheless, the discovery of progenitor cells resident within the cardiac tissue has sparked off enthusiasm about the possibility of efficiently and safely engineering them to repair the injured myocardium. Indeed, the early applications of the cardiac progenitor cells, mostly based on simplistic concepts and techniques, have failed highlighting the prerequisite of expanding the knowledge about progenitor cell features and microenvironmental conditioning. In this review, recent information on resident cardiac progenitor cells has been systematically gathered in order to create a valuable instrument to support investigators in their efforts to establish an efficient cardiac cell therapy. J. Cell. Physiol. 224: 590–600, 2010. © 2010 Wiley‐Liss, Inc.