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Ken Muneoka - One of the best experts on this subject based on the ideXlab platform.

  • the periosteal requirement and temporal dynamics of bmp2 induced middle phalanx regeneration in the adult mouse
    Regeneration (Oxford England), 2017
    Co-Authors: Lindsay A Dawson, Paula Schanes, Connor P Dolan, Maegan Pela, Britta Petersen, Luis Marrero, Ling Yu, Ken Muneoka
    Abstract:

    Regeneration of mammalian limbs is restricted to amputation of the distal Digit Tip, the terminal phalanx (P3). The adjacent skeletal element, the middle phalanx (P2), has emerged as a model system to investigate regenerative failure and as a site to test approaches aimed at enhancing regeneration. We report that exogenous application of BMP2 stimulates the formation of a transient cartilaginous callus distal to the amputation plane that mediates the regeneration of the amputated P2 bone. BMP2 initiates a significant regeneration response during the periosteal-derived cartilaginous healing phase of P2 bone repair, yet fails to induce regeneration in the absence of periosteal tissue, or after boney callus formation. We provide evidence that a temporal component exists in the induced-regeneration of P2 that we define as the “regeneration window”. In this window, cells are transiently responsive to BMP2 after the amputation injury. Simple re-injury of the healed P2 stump acts to reinitiate endogenous bone repair, complete with periosteal chondrogenesis, thus re-opening the “regeneration window” and thereby recreating a regeneration-permissive environment that is responsive to exogenous BMP2 treatment. This article is protected by copyright. All rights reserved

  • Fibroblast reticular cells engineer a blastema extracellular network during Digit Tip regeneration in mice
    Regeneration, 2017
    Co-Authors: Luis Marrero, Mimi Sammarco, Jennifer Simkin, Ken Muneoka
    Abstract:

    The regeneration blastema which forms following amputation of the mouse Digit Tip is composed of undifferentiated cells bound together by an organized network of fibers. A monoclonal antibody (ER-TR7) that identifies extracellular matrix (ECM) fibers produced by fibroblast reticular cells during lymphoid organogenesis was used to characterize the ECM of the Digit, the blastema, and the regenerate. Digit fibroblast reticular cells produce an ER-TR7(+) ECM network associated with different tissues and represent a subset of loose connective tissue fibroblasts. During blastema formation there is an upregulation of matrix production that returns to its pre-existing level and anatomical pattern in the endpoint regenerate. Co-localization studies demonstrate a strong spatial correlation between the ER-TR7 antigen and collagen type III (COL3) in histological sections. ER-TR7 and COL3 are co-induced in cultured Digit fibroblasts following treatment with tumor necrosis factor alpha and a lymphotoxin beta receptor agonist. These results provide an initial characterization of the ECM during Digit regeneration and identify a subpopulation of fibroblasts involved in producing the blastema provisional matrix that is remodeled during the regeneration response.

  • connective tissue fibroblast properties are position dependent during mouse Digit Tip regeneration
    PLOS ONE, 2013
    Co-Authors: Karen Wang, Jennifer Simkin, Manjong Han, Adrine Karapetyan, Warnakulusuriya Akash Fernando, Elizabeth L Rugg, Ken Muneoka
    Abstract:

    A key factor that contributes to the regenerative ability of regeneration-competent animals such as the salamander is their use of innate positional cues that guide the regeneration process. The limbs of mammals has severe regenerative limitations, however the distal most portion of the terminal phalange is regeneration competent. This regenerative ability of the adult mouse Digit is level dependent: amputation through the distal half of the terminal phalanx (P3) leads to successful regeneration, whereas amputation through a more proximal location, e.g. the subterminal phalangeal element (P2), fails to regenerate. Do the connective tissue cells of the mammalian Digit play a role similar to that of the salamander limb in controlling the regenerative response? To begin to address this question, we isolated and cultured cells of the connective tissue surrounding the phalangeal bones of regeneration competent (P3) and incompetent (P2) levels. Despite their close proximity and localization, these cells show very distinctive profiles when characterized in vitro and in vivo. In vitro studies comparing their proliferation and position-specific interactions reveal that cells isolated from the P3 and P2 are both capable of organizing and differentiating epithelial progenitors, but with different outcomes. The difference in interactions are further characterized with three-dimension cultures, in which P3 regenerative cells are shown to lack a contractile response that is seen in other fibroblast cultures, including the P2 cultures. In in vivo engraftment studies, the difference between these two cell lines is made more apparent. While both P2 and P3 cells participated in the regeneration of the terminal phalanx, their survival and proliferative indices were distinct, thus suggesting a key difference in their ability to interact within a regeneration permissive environment. These studies are the first to demonstrate distinct positional characteristics of connective tissue cells that are associated with their regenerative capabilities.

  • P3 cells are regeneration competent after expansion in vitro.
    2013
    Co-Authors: Karen Wang, Jennifer Simkin, Manjong Han, Adrine Karapetyan, Warnakulusuriya Akash Fernando, Elizabeth L Rugg, Ken Muneoka
    Abstract:

    A) LacZ positive P3 cells were injected into the Digit Tip of SCID mice 1 day prior to amputation and collected at 10 DPA when the regenerate at the blastema stage. LacZ positive cells are present at the injection site in the dorsal connective tissue (*) and are scattered throughout the blastema. B) During the differentiation stage (16 DPA) LacZ positive P3 cells are primarily found in the regenerating connective tissue with small clusters of cells present within the trabeculae of the regenerating bone (arrows). C–E) GFP+ human breast cancer cells injected into P3 connective tissue prior to amputation remained aggregated in the regeneration stump and did not enter the blastema. GFP positive cells (C) shown aggregated in the stump of a 16 DPA regenerate indicating that they survive engraftment. Immunohistochemical co-staining for the endothelial marker vWF (D, E) indicates that these cells differentiate in situ without participating in the regenerative response. A and B, scale bar  = 200 µm; C–E, scale bar  = 100 µm.

  • bmp2 induces segment specific skeletal regeneration from Digit and limb amputations by establishing a new endochondral ossification center
    Developmental Biology, 2012
    Co-Authors: Ling Yu, Ken Muneoka
    Abstract:

    Bone morphogenetic proteins (BMPs) are required for bone development, the repair of damage skeletal tissue, and the regeneration of the mouse Digit Tip. Previously we showed that BMP treatment can induce a regeneration response in mouse Digits amputated at a proximal level of the terminal phalangeal element (P3) (Yu et al., 2010). In this study, we show that the regeneration-inductive ability of BMP2 extends to amputations at the level of the second phalangeal element (P2) of neonatal Digits, and the hindlimb of adult limbs. In these models the induced regenerative response is restricted in a segment-specific manner, thus amputated skeletal elements regenerate distally patterned skeletal structures but does not form joints or more distal skeletal elements. Studies on P2 amputations indicate that BMP2-induced regeneration is associated with a localized proliferative response and the transient expression of established Digit blastema marker genes. This is followed by the formation of a new endochondral ossification center at the distal end of the bone stump. The endochondral ossification center contains proliferating chondrocytes that establish a distal proliferative zone and differentiate proximally into hypertrophic chondrocytes. Skeletal regeneration occurs from proximal to distal with the appearance of osteoblasts that differentiate in continuity with the amputated stump. Using the polarity of the endochondral ossification centers induced by BMP2 at two different amputation levels, we show that BMP2 activates a level-dependent regenerative response indicative of a positional information network. In summary, our studies provide evidence that BMP2 induces the regeneration of mammalian limb structures by stimulating a new endochondral ossification center that utilizes an existing network of positional information to regulate patterning during skeletal regeneration.

Michael T Longaker - One of the best experts on this subject based on the ideXlab platform.

  • skeletal stem cell schwann cell circuitry in mandibular repair
    Cell Reports, 2019
    Co-Authors: Ellen R Jones, Ryan C Ransom, Ankit Salhotra, Kiana S Robertson, Deshka S Foster, Harsh N Shah, Natalina Quarto, Derrick C Wan, Michael T Longaker
    Abstract:

    Summary Regenerative paradigms exhibit nerve dependency, including regeneration of the mouse Digit Tip and salamander limb. Denervation impairs regeneration and produces morphological aberrancy in these contexts, but the direct effect of innervation on the stem and progenitor cells enacting these processes is unknown. We devised a model to examine nerve dependency of the mouse skeletal stem cell (mSSC), the progenitor responsible for skeletal development and repair. We show that after inferior alveolar denervation, mandibular bone repair is compromised because of functional defects in mSSCs. We present mSSC reliance on paracrine factors secreted by Schwann cells as the underlying mechanism, with partial rescue of the denervated phenotype by Schwann cell transplantation and by Schwann-derived growth factors. This work sheds light on the nerve dependency of mSSCs and has implications for clinical treatment of mandibular defects.

  • short hairpin rna silencing of phd 2 improves neovascularization and functional outcomes in diabetic wounds and ischemic limbs
    PLOS ONE, 2016
    Co-Authors: Kevin J Paik, Zeshaan N Maan, Elizabeth R Zielins, Dominik Duscher, Alexander J Whittam, Shane D Morrison, Elizabeth A Brett, Ryan C Ransom, Geoffrey C Gurtner, Michael T Longaker
    Abstract:

    The transcription factor hypoxia-inducible factor 1-alpha (HIF-1α) is responsible for the downstream expression of over 60 genes that regulate cell survival and metabolism in hypoxic conditions as well as those that enhance angiogenesis to alleviate hypoxia. However, under normoxic conditions, HIF-1α is hydroxylated by prolyl hydroxylase 2, and subsequently degraded, with a biological half-life of less than five minutes. Here we investigated the therapeutic potential of inhibiting HIF-1α degradation through short hairpin RNA silencing of PHD-2 in the setting of diabetic wounds and limb ischemia. Treatment of diabetic mouse fibroblasts with shPHD-2 in vitro resulted in decreased levels of PHD-2 transcript demonstrated by qRT-PCR, higher levels of HIF-1α as measured by western blot, and higher expression of the downstream angiogenic genes SDF-1 and VEGFα, as measured by qRT-PCR. In vivo, shPHD-2 accelerated healing of full thickness excisional wounds in diabetic mice compared to shScr control, (14.33 ± 0.45 days vs. 19 ± 0.33 days) and was associated with an increased vascular density. Delivery of shPHD-2 also resulted in improved perfusion of ischemic hind limbs compared to shScr, prevention of distal Digit Tip necrosis, and increased survival of muscle tissue. Knockdown of PHD-2 through shRNA treatment has the potential to stimulate angiogenesis through overexpression of HIF-1α and upregulation of pro-angiogenic genes downstream of HIF-1α, and may represent a viable, non-viral approach to gene therapy for ischemia related applications.

  • clonal analysis reveals nerve dependent and independent roles on mammalian hind limb tissue maintenance and regeneration
    Proceedings of the National Academy of Sciences of the United States of America, 2014
    Co-Authors: Yuval Rinkevich, Daniel T Montoro, Ethan Muhonen, Graham G Walmsley, Masakazu Hasegawa, Michael Januszyk, Andrew J Connolly, Irving L Weissman, Michael T Longaker
    Abstract:

    The requirement and influence of the peripheral nervous system on tissue replacement in mammalian appendages remain largely undefined. To explore this question, we have performed genetic lineage tracing and clonal analysis of individual cells of mouse hind limb tissues devoid of nerve supply during regeneration of the Digit Tip, normal maintenance, and cutaneous wound healing. We show that cellular turnover, replacement, and cellular differentiation from presumed tissue stem/progenitor cells within hind limb tissues remain largely intact independent of nerve and nerve-derived factors. However, regenerated Digit Tips in the absence of nerves displayed patterning defects in bone and nail matrix. These nerve-dependent phenotypes mimic clinical observations of patients with nerve damage resulting from spinal cord injury and are of significant interest for translational medicine aimed at understanding the effects of nerves on etiologies of human injury.

  • germ layer and lineage restricted stem progenitors regenerate the mouse Digit Tip
    Nature, 2011
    Co-Authors: Yuval Rinkevich, Michael T Longaker, Paul Lindau, Hiroo Ueno, Irving L Weissman
    Abstract:

    The regrowth of amputated limbs and the distal Tips of Digits represent models of tissue regeneration in amphibians, fish and mice. For decades it had been assumed that limb regeneration derived from the blastema, an undifferentiated pluripotent cell population thought to be derived from mature cells via dedifferentiation. Here we show that a wide range of tissue stem/progenitor cells contribute towards the restoration of the mouse distal Digit. Genetic fate mapping and clonal analysis of individual cells revealed that these stem cells are lineage restricted, mimicking Digit growth during development. Transplantation of cyan-fluorescent-protein-expressing haematopoietic stem cells, and parabiosis between genetically marked mice, confirmed that the stem/progenitor cells are tissue resident, including the cells involved in angiogenesis. These results, combined with those from appendage regeneration in other vertebrate subphyla, collectively demonstrate that tissue stem cells rather than pluripotent blastema cells are an evolutionarily conserved cellular mode for limb regeneration after amputation.

  • germ layer and lineage restricted stem progenitors regenerate the mouse Digit Tip
    Nature, 2011
    Co-Authors: Yuval Rinkevich, Michael T Longaker, Paul Lindau, Hiroo Ueno, Irving L Weissman
    Abstract:

    The regrowth of amputated limbs and the distal Tips of Digits represent models of tissue regeneration in amphibians, fish and mice. For decades it had been assumed that limb regeneration derived from the blastema, an undifferentiated pluripotent cell population thought to be derived from mature cells via dedifferentiation. Here we show that a wide range of tissue stem/progenitor cells contribute towards the restoration of the mouse distal Digit. Genetic fate mapping and clonal analysis of individual cells revealed that these stem cells are lineage restricted, mimicking Digit growth during development. Transplantation of cyan-fluorescent-protein-expressing haematopoietic stem cells, and parabiosis between genetically marked mice, confirmed that the stem/progenitor cells are tissue resident, including the cells involved in angiogenesis. These results, combined with those from appendage regeneration in other vertebrate subphyla, collectively demonstrate that tissue stem cells rather than pluripotent blastema cells are an evolutionarily conserved cellular mode for limb regeneration after amputation. Newborn and adult mice are able to regrow forelimb and hindlimb Digit Tips after their amputation through the distal interphalangeal joint, but the cellular origin of the regenerated tissue in adult mice has been unclear. Using in vivo genetic fate-mapping techniques, Rinkevich et al. show that a range of tissue-specific stem cells contribute to the restoration of the mouse's distal phalanx. These findings, together with work in chick and salamander limb-regeneration models, suggest that lineage restriction of tissue stem cells — as opposed to dedifferentiation into a mulTipotent cell state — is evolutionarily conserved.

Irving L Weissman - One of the best experts on this subject based on the ideXlab platform.

  • clonal analysis reveals nerve dependent and independent roles on mammalian hind limb tissue maintenance and regeneration
    Proceedings of the National Academy of Sciences of the United States of America, 2014
    Co-Authors: Yuval Rinkevich, Daniel T Montoro, Ethan Muhonen, Graham G Walmsley, Masakazu Hasegawa, Michael Januszyk, Andrew J Connolly, Irving L Weissman, Michael T Longaker
    Abstract:

    The requirement and influence of the peripheral nervous system on tissue replacement in mammalian appendages remain largely undefined. To explore this question, we have performed genetic lineage tracing and clonal analysis of individual cells of mouse hind limb tissues devoid of nerve supply during regeneration of the Digit Tip, normal maintenance, and cutaneous wound healing. We show that cellular turnover, replacement, and cellular differentiation from presumed tissue stem/progenitor cells within hind limb tissues remain largely intact independent of nerve and nerve-derived factors. However, regenerated Digit Tips in the absence of nerves displayed patterning defects in bone and nail matrix. These nerve-dependent phenotypes mimic clinical observations of patients with nerve damage resulting from spinal cord injury and are of significant interest for translational medicine aimed at understanding the effects of nerves on etiologies of human injury.

  • germ layer and lineage restricted stem progenitors regenerate the mouse Digit Tip
    Nature, 2011
    Co-Authors: Yuval Rinkevich, Michael T Longaker, Paul Lindau, Hiroo Ueno, Irving L Weissman
    Abstract:

    The regrowth of amputated limbs and the distal Tips of Digits represent models of tissue regeneration in amphibians, fish and mice. For decades it had been assumed that limb regeneration derived from the blastema, an undifferentiated pluripotent cell population thought to be derived from mature cells via dedifferentiation. Here we show that a wide range of tissue stem/progenitor cells contribute towards the restoration of the mouse distal Digit. Genetic fate mapping and clonal analysis of individual cells revealed that these stem cells are lineage restricted, mimicking Digit growth during development. Transplantation of cyan-fluorescent-protein-expressing haematopoietic stem cells, and parabiosis between genetically marked mice, confirmed that the stem/progenitor cells are tissue resident, including the cells involved in angiogenesis. These results, combined with those from appendage regeneration in other vertebrate subphyla, collectively demonstrate that tissue stem cells rather than pluripotent blastema cells are an evolutionarily conserved cellular mode for limb regeneration after amputation.

  • germ layer and lineage restricted stem progenitors regenerate the mouse Digit Tip
    Nature, 2011
    Co-Authors: Yuval Rinkevich, Michael T Longaker, Paul Lindau, Hiroo Ueno, Irving L Weissman
    Abstract:

    The regrowth of amputated limbs and the distal Tips of Digits represent models of tissue regeneration in amphibians, fish and mice. For decades it had been assumed that limb regeneration derived from the blastema, an undifferentiated pluripotent cell population thought to be derived from mature cells via dedifferentiation. Here we show that a wide range of tissue stem/progenitor cells contribute towards the restoration of the mouse distal Digit. Genetic fate mapping and clonal analysis of individual cells revealed that these stem cells are lineage restricted, mimicking Digit growth during development. Transplantation of cyan-fluorescent-protein-expressing haematopoietic stem cells, and parabiosis between genetically marked mice, confirmed that the stem/progenitor cells are tissue resident, including the cells involved in angiogenesis. These results, combined with those from appendage regeneration in other vertebrate subphyla, collectively demonstrate that tissue stem cells rather than pluripotent blastema cells are an evolutionarily conserved cellular mode for limb regeneration after amputation. Newborn and adult mice are able to regrow forelimb and hindlimb Digit Tips after their amputation through the distal interphalangeal joint, but the cellular origin of the regenerated tissue in adult mice has been unclear. Using in vivo genetic fate-mapping techniques, Rinkevich et al. show that a range of tissue-specific stem cells contribute to the restoration of the mouse's distal phalanx. These findings, together with work in chick and salamander limb-regeneration models, suggest that lineage restriction of tissue stem cells — as opposed to dedifferentiation into a mulTipotent cell state — is evolutionarily conserved.

Gemma Johnson - One of the best experts on this subject based on the ideXlab platform.

  • cellular heterogeneity and lineage restriction during mouse Digit Tip regeneration at single cell resolution
    Developmental Cell, 2020
    Co-Authors: Gemma Johnson, Erick J Masias, Jessica A Lehoczky
    Abstract:

    Summary Innate regeneration following Digit Tip amputation is one of the few examples of epimorphic regeneration in mammals. Digit Tip regeneration is mediated by the blastema, the same structure invoked during limb regeneration in some lower vertebrates. By genetic lineage analyses, the Digit Tip blastema has been defined as a population of heterogeneous, lineage-restricted progenitor cells. These previous studies, however, do not comprehensively evaluate blastema heterogeneity or address lineage restriction of closely related cell types. In this report, we present single-cell RNA sequencing of over 38,000 cells from mouse Digit Tip blastemas and unamputated control Digit Tips and generate an atlas of the cell types participating in Digit Tip regeneration. We computationally define differentiation trajectories of vascular, monocytic, and fibroblastic lineages over regeneration, and while our data confirm broad lineage restriction of progenitors, our analysis reveals 67 genes enriched in blastema fibroblasts including a novel regeneration-specific gene, Mest.

  • cellular heterogeneity and lineage restriction during mouse Digit Tip regeneration at single cell resolution
    bioRxiv, 2019
    Co-Authors: Gemma Johnson, Erick J Masias, Jessica A Lehoczky
    Abstract:

    Innate regeneration following Digit Tip amputation is one of the few examples of epimorphic regeneration in mammals. Digit Tip regeneration is mediated by the blastema, the same structure invoked during limb regeneration in some lower vertebrates. By genetic lineage analyses in mice, the Digit Tip blastema has been defined as a population of heterogeneous, lineage restricted progenitor cells. These previous studies, however, do not comprehensively evaluate blastema heterogeneity or address lineage restriction of closely related cell types. In this report we present single cell RNA sequencing of over 38,000 cells from mouse Digit Tip blastemas and unamputated control Digit Tips and generate an atlas of the cell types participating in Digit Tip regeneration. We define the differentiation trajectories of vascular, monocytic, and fibroblastic lineages over regeneration, and while our data confirm broad lineage restriction of progenitors, our analysis reveals an early blastema fibroblast population expressing a novel regeneration-specific gene, Mest.

Manjong Han - One of the best experts on this subject based on the ideXlab platform.

  • connective tissue fibroblast properties are position dependent during mouse Digit Tip regeneration
    PLOS ONE, 2013
    Co-Authors: Karen Wang, Jennifer Simkin, Manjong Han, Adrine Karapetyan, Warnakulusuriya Akash Fernando, Elizabeth L Rugg, Ken Muneoka
    Abstract:

    A key factor that contributes to the regenerative ability of regeneration-competent animals such as the salamander is their use of innate positional cues that guide the regeneration process. The limbs of mammals has severe regenerative limitations, however the distal most portion of the terminal phalange is regeneration competent. This regenerative ability of the adult mouse Digit is level dependent: amputation through the distal half of the terminal phalanx (P3) leads to successful regeneration, whereas amputation through a more proximal location, e.g. the subterminal phalangeal element (P2), fails to regenerate. Do the connective tissue cells of the mammalian Digit play a role similar to that of the salamander limb in controlling the regenerative response? To begin to address this question, we isolated and cultured cells of the connective tissue surrounding the phalangeal bones of regeneration competent (P3) and incompetent (P2) levels. Despite their close proximity and localization, these cells show very distinctive profiles when characterized in vitro and in vivo. In vitro studies comparing their proliferation and position-specific interactions reveal that cells isolated from the P3 and P2 are both capable of organizing and differentiating epithelial progenitors, but with different outcomes. The difference in interactions are further characterized with three-dimension cultures, in which P3 regenerative cells are shown to lack a contractile response that is seen in other fibroblast cultures, including the P2 cultures. In in vivo engraftment studies, the difference between these two cell lines is made more apparent. While both P2 and P3 cells participated in the regeneration of the terminal phalanx, their survival and proliferative indices were distinct, thus suggesting a key difference in their ability to interact within a regeneration permissive environment. These studies are the first to demonstrate distinct positional characteristics of connective tissue cells that are associated with their regenerative capabilities.

  • P3 cells are regeneration competent after expansion in vitro.
    2013
    Co-Authors: Karen Wang, Jennifer Simkin, Manjong Han, Adrine Karapetyan, Warnakulusuriya Akash Fernando, Elizabeth L Rugg, Ken Muneoka
    Abstract:

    A) LacZ positive P3 cells were injected into the Digit Tip of SCID mice 1 day prior to amputation and collected at 10 DPA when the regenerate at the blastema stage. LacZ positive cells are present at the injection site in the dorsal connective tissue (*) and are scattered throughout the blastema. B) During the differentiation stage (16 DPA) LacZ positive P3 cells are primarily found in the regenerating connective tissue with small clusters of cells present within the trabeculae of the regenerating bone (arrows). C–E) GFP+ human breast cancer cells injected into P3 connective tissue prior to amputation remained aggregated in the regeneration stump and did not enter the blastema. GFP positive cells (C) shown aggregated in the stump of a 16 DPA regenerate indicating that they survive engraftment. Immunohistochemical co-staining for the endothelial marker vWF (D, E) indicates that these cells differentiate in situ without participating in the regenerative response. A and B, scale bar  = 200 µm; C–E, scale bar  = 100 µm.

  • wound healing and blastema formation in regenerating Digit Tips of adult mice
    Developmental Biology, 2011
    Co-Authors: Warnakulasuriya Akash Fernando, Jennifer Simkin, Manjong Han, Eric Leininger, Carrie A Malcom, Shyam Sathyamoorthi, Ken Muneoka
    Abstract:

    Amputation of the distal region of the terminal phalanx of mice causes an initial wound healing response followed by blastema formation and the regeneration of the Digit Tip. Thus far, most regeneration studies have focused in embryonic or neonatal models and few studies have examined adult Digit regeneration. Here we report on studies that include morphological, immunohistological, and volumetric analyses of adult Digit regeneration stages. The regenerated Digit is grossly similar to the original, but is not a perfect replacement. Re-differentiation of the Digit Tip occurs by intramembranous ossification forming a trabecular bone network that replaces the amputated cortical bone. The Digit blastema is comprised of proliferating cells that express vimentin, a general mesenchymal marker, and by comparison to mature tissues, contains fewer endothelial cells indicative of reduced vascularity. The majority of blastemal cells expressing the stem cell marker SCA-1, also co-express the endothelial marker CD31, suggesting the presence of endothelial progenitor cells. Epidermal closure during wound healing is very slow and is characterized by a failure of the wound epidermis to close across amputated bone. Instead, the wound healing phase is associated with an osteoclast response that degrades the stump bone allowing the wound epidermis to undercut the distal bone resulting in a novel re-amputation response. Thus, the regeneration process initiates from a level that is proximal to the original plane of amputation.

  • development and regeneration of the neonatal Digit Tip in mice
    Developmental Biology, 2008
    Co-Authors: Manjong Han, Xiaodong Yang, Christopher H Allan, Jangwoo Lee, Ken Muneoka
    Abstract:

    The Digit Tips of children and rodents are known to regenerate following amputation. The skeletal structure that regenerates is the distal region of the terminal phalangeal bone that is associated with the nail organ. The terminal phalanx forms late in gestation by endochondral ossification and continues to elongate until sexual maturity (8 weeks of age). Postnatal elongation at its distal end occurs by appositional ossification, i.e. direct ossification on the surface of the terminal phalanx, whereas proximal elongation results from an endochondral growth plate. Amputation through the middle of the terminal phalanx regenerates whereas regenerative failure is observed following amputation to remove the distal 2/3 of the bone. Regeneration is characterized by the formation of a blastema of proliferating cells that appear undifferentiated and express Bmp4. Using chondrogenic and osteogenic markers we show that redifferentiation does not occur by endochondral ossification but by the direct ossification of blastema cells that form the rudiment of the Digit Tip. Once formed the rudiment elongates by appositional ossification in parallel with unamputated control Digits. Regenerated Digits are consistently shorter than unamputated control Digits. Finally, we present a case study of a child who suffered an amputation injury at a proximal level of the terminal phalanx, but failed to regenerate despite conservative treatment and the presence of the nail organ. These clinical and experimental findings expand on previously published observations, and initiate a molecular assessment of a mammalian regeneration model.

  • limb regeneration in higher vertebrates developing a roadmap
    The Anatomical Record Part B: The New Anatomist, 2005
    Co-Authors: Manjong Han, Xiaodong Yang, Gail Taylor, Carol Burdsal, Rosalie Anderson, Ken Muneoka
    Abstract:

    We review what is known about amphibian limb regeneration from the prospective of developing strategies for the induction of regeneration in adult mammals. Prominent in urodele amphibian limb regeneration is the formation of a blastema of undifferentiated cells that goes on to reform the limb. The blastema shares many properties with the developing limb bud; thus, the outgrowth phase of regeneration can be thought of as cells going through development again, i.e., redevelopment. Getting to a redevelopment phase in mammals would be a major breakthrough given our extensive understanding of limb development. The formation of the blastema itself represents a transition phase in which limb cells respond to injury by dedifferentiating to become embryonic limb progenitor cells that can undergo redevelopment. During this phase, rapid wound closure is followed by the dedifferentiation of limb cells to form the blastema. Thus, the regeneration process can be divided into a wound-healing/dedifferentiation phase and a redevelopment phase, and we propose that the interface between the wound-healing response and gaining access to developmentally regulated programs (dedifferentiation) lies at the heart of the regeneration problem in mammals. In urodele amphibians, dedifferentiation can occur in all of the tissues of the limb; however, numerous studies lead us to focus on the epidermis, the dermis, and muscle as key regulators of regeneration. Among higher vertebrates, the Digit Tip in mammals, including humans, is regeneration-competent and offers a unique mammalian model for regeneration. Recent genetic studies in mice identify the Msx1 gene as playing a critical role in the injury response leading to Digit Tip regeneration. The results from regeneration studies ranging from amphibians to mammals can be integrated to develop a roadmap for mammalian regeneration that has as its focus understanding the phenomenon of dedifferentiation.