Mammalian Organ Regeneration Research Articles

Mammalian organ regeneration in spiny mice.

Fibrosis-driven solid organ failure is a major world-wide health burden with few therapeutic options. Spiny mice (genus: Acomys) are terrestrial mammals that regenerate severe skin wounds without fibrotic scars to evade predators. Recent studies have shown that spiny mice also regenerate acute ischemic and traumatic injuries to kidney, heart, spinal cord, and skeletal muscle. A common feature of this evolved wound healing response is a lack of formation of fibrotic scar tissue that degrades organ function, inhibits regeneration, and leads to organ failure. Complex tissue regeneration is an extremely rare property among mammalian species. In this article, we discuss the evidence that Acomys represents an emerging model organism that offers a unique opportunity for the biomedical community to investigate and clinically translate molecular mechanisms of scarless wound healing and regeneration of organ function in a mammalian species.

Progress on deer genome research.

Deer family is one of the most abundant mammalian families in the world. Deer species are distributed in wide geographic ranges including the North Pole, tropical regions and high-altitude mountains. Of these deer species, China accounts for more than 40% of them and is the main site for deer evolution. Besides the common phenotypical attributes for ruminants, deer family is evolved to possess the unique head gears with periodic regeneration, i.e. antlers. It is currently well accepted that deer is a very valuable model for the studies of ecology, behavior, evolution and biology, especially for the study of mammalian organ regeneration. Reference deer genome is the basis for systematically illustrating deer evolution, deciphering unique biological attributes of deer species, and is significant in protection and utilization of deer genetic resources. In this review, we summarize the recent progress in the field of deer genome research, including data of deer genetic variation, molecular basis of adaptive evolution, and key genes and functional genomics involved in deer antler origin and evolution. The overall aim of the paper is to provide the reference neccessary for in depth investigation of deer species.

The protein phosphatase PPM1A dephosphorylates and activates YAP to govern mammalian intestinal and liver regeneration.

The Hippo-YAP pathway responds to diverse environmental cues to manage tissue homeostasis, organ regeneration, tumorigenesis, and immunity. However, how phosphatase(s) directly target Yes-associated protein (YAP) and determine its physiological activity are still inconclusive. Here, we utilized an unbiased phosphatome screening and identified protein phosphatase magnesium-dependent 1A (PPM1A/PP2Cα) as the bona fide and physiological YAP phosphatase. We found that PPM1A was associated with YAP/TAZ in both the cytoplasm and the nucleus to directly eliminate phospho-S127 on YAP, which conferring YAP the nuclear distribution and transcription potency. Accordingly, genetic ablation or depletion of PPM1A in cells, organoids, and mice elicited an enhanced YAP/TAZ cytoplasmic retention and resulted in the diminished cell proliferation, severe gut regeneration defects in colitis, and impeded liver regeneration upon injury. These regeneration defects in murine model were largely rescued via a genetic large tumor suppressor kinase 1 (LATS1) deficiency or the pharmacological inhibition of Hippo-YAP signaling. Therefore, we identify a physiological phosphatase of YAP/TAZ, describe its critical effects in YAP/TAZ cellular distribution, and demonstrate its physiological roles in mammalian organ regeneration.

Techniques for Research in Deer Antlers, the Sole Mammalian Organ which undergoes Epimorphic Regeneration

The annual renewal of deer antlers offers the only opportunity to learn how nature has solved the problem of mammalian organ regeneration. Promotion of the antler model to the field of regenerative biology and medicine, however, requires understanding the mechanism underlying the generation and regeneration of this unique organ. During the course of nearly four decades of antler research, we developed a number of techniques specifically for carrying out the investigation of deer antler biology. In this paper, I summarized six of them including 1) Mechanical disintegration of antler stem cell (AnSC) tissue; 2) In vivo investigation of the interactions between antlerogenic tissue and overlying skin; 3) In vivo identification of skin tissue components required for establishing interactions with AP; 4) Alternative transplantation technique to reduce AP quantity required for antler induction; 5) In vivo evaluation of the role of interposing tissue layers in antler generation; 6) In vitro identification of the interactive molecules between AnSCs and niche cell populations. I believe if these techniques are adopted in the antler research field, it would greatly facilitate the progress for revealing the mechanisms of antler development and ultimately benefit regenerative medicine in general.

Proteomic Profiling of Stem Cell Tissues during Regeneration of Deer Antler: A Model of Mammalian Organ Regeneration.

As the only known mammalian organ that can fully and annually regenerate, deer antler has significant advantages over lower-order animal models when investigating the control of stem-cell-based organ regeneration. Antler regeneration is known to be initiated and maintained by neural-crest-derived stem cells in different states of activation. Antler stem cells can therefore be used as a model to study proteins and pathways involved in the maintenance of a stem cell niche and their activation and differentiation during organ formation. In this study, the MSC markers CD73, CD90, and CD105 were examined within the antler tip. Label-free quantification was performed to investigate the protein profiles of antler stem cells under different stages of activation and included dormant pedicle periosteum (DPP), antler growth center (GC), post-active stem cells from mid-beam antler periosteum (MAP), and deer facial periosteum (FP) as a control (n = 3 per group). PEAKS and IPA software were used to analyze the proteomic data. Our research confirmed the central role of stem cell activation in the development of this mammalian organ by localizing the MSC markers within the antler growth center. Label-free quantification revealed that the greatest number of unique proteins (87) was found in the growth center. There were only 12 proteins found with expression levels that significantly differed between DPP and FP. Protein profiles of these two groups indicated that antler stem cells may use similar mechanisms to maintain dormancy within a stem cell niche. The number of significantly regulated proteins among DPP, MAP, and GC was 153. Among them, the majority were upregulated in the growth center. Activation of antler stem cells was associated with many biological processes and signaling pathways, such as Hippo and canonical Wnt signaling. This work identifies the key pathways, molecular/cellular functions, and upstream regulators involved in mammal organ regeneration. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the iProX partner repository with the dataset identifier PXD016824.

Effects of macrophage-conditioned medium on sika deer (Cervus nippon) antler stem cells

Context Immune system has been claimed as the ‘main switch’ of tissue or organ regeneration. Among immune cells, macrophages stand out as important modulators in mutiple regeneration models, such as planarian, axolotl, mammalian hair and liver. As a unique model for mammals, deer antler is considered to ideal for studying complete mammalian organ regeneration. Studies have found that antler regeneration is a stem cell-based process and antler stem cells locate in the pedicle periosteum (PP). Although the regulatory roles of the immune system in other regeneration models have been extensively studied, they remain unstudied in antler regeneration. Aims To explore the possible role of macrophages in the PP cells (PPCs). Methods We treated PPCs with a macrophage-conditioned medium (MCM) and detected effects of MCM on proliferation, migration and apoptosis of the PPCs, and identified differentially expressed genes by using the RNA-seq technique. Key results We found that MCM enhanced proliferation rate and migration rate significantly and stimulated apoptosis of the PPCs. Using the RNA-seq technique, we identified 112 differentially expressed genes in the PPCs (38 downregulated and 74 upregulated) after the MCM treatment. Furthermore, gene-ontology annotation analyses showed that the upregulated genes were mainly involved in cell adhesion, chemotaxis, wound healing, growth factor-stimulated responses, and bone formation, and the downregulated genes were involved in regulation of biosynthesis. Conclusions MCM had a great influence on the antler stem cells, and macrophages might regulate antler regeneration through altering the microenvironment and gene-expression profiles of the PPCs. Implications We believe that the results of the present study would facilitate the discovery of the roles of immune system in antler stem cells and, thus, mammalian organ regeneration in general.

Identification of interactive molecules between antler stem cells and dermal papilla cells using an in vitro co-culture system.

Deer antlers are the only mammalian organs capable of complete renewal. Antler renewal is a stem cell-based [antler stem cells (ASCs)] process. Maintenance and activation of the ASCs require them to be located in a specialized microenvironment (niche), and to interact with the cells resident in the niche. Based on previous experiments we found that niche of the ASCs is provided by the closely associated enveloping skin, which currently was known includes dermal papilla cells (DPCs) and epidermal cells. Antler generation/regeneration are triggered by the interactions between ASCs and the niche. In the present study, we established an in vitro co-culture system in which ASCs and DPCs, were cultured together to mimic the in vivo state. A MLEFF strategy was adopted to identify the interactive molecules from the co-culture system. In total, 128 molecules were identified and over 60% belonged to exosomes. Important biological processes that were activated by these molecules included osteoblast differentiation, angiogenesis, and the PI3K-AKT signaling pathway. In so doing, we have significantly simplified the process for identifying interactive molecules, which may be the key signals for triggering antler formation/renewal. Further study of these molecules will help us to gain insights into the mechanism of mammalian organ regeneration.

Genetic basis of ruminant headgear and rapid antler regeneration.

Ruminants are the only extant mammalian group possessing bony (osseous) headgear. We obtained 221 transcriptomes from bovids and cervids and sequenced three genomes representing the only two pecoran lineages that convergently lack headgear. Comparative analyses reveal that bovid horns and cervid antlers share similar gene expression profiles and a common cellular basis developed from neural crest stem cells. The rapid regenerative properties of antler tissue involve exploitation of oncogenetic pathways, and at the same time some tumor suppressor genes are under strong selection in deer. These results provide insights into the evolutionary origin of ruminant headgear as well as mammalian organ regeneration and oncogenesis.

Single-cell transcriptome provides novel insights into antler stem cells, a cell type capable of mammalian organ regeneration.

Antler regeneration, a stem cell-based epimorphic process, has a potential as a valuable model for regenerative medicine. A pool of antler stem cells (ASCs) for antler development is located in the antlerogenic periosteum (AP). However, whether this ASC pool is homogenous or heterogeneous has not been fully evaluated. In this study, we produced a comprehensive transcriptome dataset at the single-cell level for the ASCs based on the 10× Genomics platform (scRNA-seq). A total of 4565 ASCs were sequenced and classified into a large cell cluster, indicating that the ASC resident in the AP are likely to be a homogeneous population. The scRNA-seq data revealed that tumor-related genes were highly expressed in these homogeneous ASCs, i.e., TIMP1, TMSB10, LGALS1, FTH1, VIM, LOC110126017, and S100A4. Results of screening for stem cell markers suggest that the ASCs may be considered as a special type of stem cell between embryonic (CD9) and adult (CD29, CD90, NPM1, and VIM) stem cells. Our results provide the first comprehensive transcriptome analysis at the single-cell level for the ASCs and identified only one major cell type resident in the AP and some key stem cell genes, which may hold the key to why antlers, the unique mammalian organ, can fully regenerate once lost.

Quantitative proteomic analysis of deer antler stem cells as a model of mammalian organ regeneration

The ability to activate and regulate stem cells during wound healing and tissue/organ regeneration is a promising field which could bring innovative approaches to regenerative medicine. The regenerative capacity of invertebrates has been well documented, however in mammals, stem cells that drive organ regeneration are rare. Deer antler is unique in providing a mammalian model of complete organ regeneration based on stem cells. The present study investigated the differentially regulated proteins (DRPs) between different antler stem cell populations (n = 3) using 2D-DIGE. Western blotting was used to validate the proteomics results. Comparative proteomics resulted in protein profiles which were similar for the biological replicates but different between the cells derived from two different stem cell niches involved in antler growth/regeneration and cells derived from facial periosteum. Ninety-two up- and down-regulated proteins were identified by MALDI-TOF MS. The work indicates that the epithelial-mesenchymal transition process may participate in the initiation of antler regeneration including the first stage of scar-less wound healing. Cell mobility is also highly regulated during antler regeneration. Energy and nucleotide metabolism may however be less active in antler regeneration as compared to that in antler generation phase. These results provide new insights into the underlying mechanisms of stem cell-based regeneration of mammalian organs.

MicroRNA profiling of antler stem cells in potentiated and dormant states and their potential roles in antler regeneration.

MicroRNAs (miRNAs) can effectively regulate gene expression at the post-transcriptional level and play a critical role in tissue growth, development and regeneration. Our previous studies showed that antler regeneration is a stem cell-based process and antler stem cells reside in the periosteum of a pedicle, the permanent bony protuberance, from which antler regeneration takes place. Antlers are the only mammalian organ that can fully regenerate and hence provide a unique opportunity to identify miRNAs that are involved in organ regeneration. In the present study, we used next generation sequencing technology sequenced miRNAs of the stem cells derived from either the potentiated or the dormant pedicle periosteum. A population of both conserved and 20 deer-specific miRNAs was identified. These conserved miRNAs were derived from 453 homologous hairpin precursors across 88 animal species, and were further grouped into 167 miRNA families. Among them, the miR-296 is embryonic stem cell-specific. The potentiation process resulted in the significant regulation (>±2 Fold, q value <0.05) of conserved miRNAs; 8 miRNA transcripts were down- and 6 up-regulated. Several GO biology processes and the Wnt, MAPK and TGF-beta signaling pathways were found to be up-regulated as part of antlerogenic stem cell potentiation process. This research has identified miRNAs that are associated either with the dormant or the potentiated antler stem cells and identified some target miRNAs for further research into their role played in mammalian organ regeneration.

MP8-02 CHARACTERIZATION OF THE MURINE BLADDER RESPONSE TO SUBTOTAL CYSTECTOMY: A MODEL OF MAMMALIAN ORGAN REGENERATION

You have accessJournal of UrologyUrodynamics/Incontinence/Female Urology: Basic Research I1 Apr 2015MP8-02 CHARACTERIZATION OF THE MURINE BLADDER RESPONSE TO SUBTOTAL CYSTECTOMY: A MODEL OF MAMMALIAN ORGAN REGENERATION Grace Delos Santos, Andrew Flum, Natalie Kukulka, Robert Dettman, and Edward Gong Grace Delos SantosGrace Delos Santos More articles by this author , Andrew FlumAndrew Flum More articles by this author , Natalie KukulkaNatalie Kukulka More articles by this author , Robert DettmanRobert Dettman More articles by this author , and Edward GongEdward Gong More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.269AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Bladder dysfunction is a significant source of urinary incontinence. Regeneration of adult bladders has been proposed to restore function. However the molecular mechanisms that mediate bladder regeneration are unknown. Regeneration of the rat bladder was reported in the literature, but rats, as a model, do not allow us to use the sophisticated genetic tools that are available in mice. We therefore investigated how mouse bladders regenerated after subtotal cystectomy (STC). METHODS STC (removal of 50% of the bladder) was performed on adult female CD1 mice. Mice that underwent low-midline laparotomy served as controls. Voiding stains on paper (VSOP) and urodynamics were used to demonstrate bladder function. Bladders were harvested at 1, 2, 4, and 8 weeks. Masson's trichrome stain identified collagen, as a proxy for bladder fibrosis. Immunohistochemistry expression of cytokeratin-5, smooth muscle myosin, and smooth muscle actin identified differentiated cells. Phospho-Histone H3 identified mitotically active cells. The movement of cells that divided during days 3–7 days post-STC was tracked using ethynyl deoxyuridine (EdU). RESULTS The STC group did not reach the same mean voided volumes as controls (Figure 1), with the majority of volume restoration occurring within the first two weeks. On cystometry, the STC bladders had higher filling pressure and higher maximum voiding pressures. Regenerating bladders had thinner walls with less mean muscle thickness, and they showed increased collagen deposition at the incision as well as throughout the bladder wall. Cell populations differed in their response to injury. Urothelial regeneration was observed by day 7. Urothelial cells incorporated EdU, indicating that they proliferated soon after STC. Smooth muscle cells represented only a small percentage of EdU+ cells indicating that smooth muscle growth was limited in the first week despite bladder volume increase. CONCLUSIONS Regeneration occurs in the adult mouse bladder, but is mostly limited to the urothelium. Though bladder volume increases after STC this appears to be as a result of tissue remodeling and not muscle regeneration. This tissue remodeling closely resembles bladder fibrosis. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e73-e74 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Grace Delos Santos More articles by this author Andrew Flum More articles by this author Natalie Kukulka More articles by this author Robert Dettman More articles by this author Edward Gong More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Histogenetic aspects of deer antler development

Deer antlers are the only mammalian organs that, once lost, can fully grow back; therefore, they offer a unique opportunity for investigating the mechanism underlying mammalian organ regeneration. This review summarizes the current knowledge of antler histogenesis. The axis of a pedicle (antecedent of antler) and a first antler consists of an internal component and an external component. Formation of the internal component commences from the proliferation of antlerogenic periosteal cells and undergoes 4 ossification stages: Formation of the external component goes through 3 distinguishable stages. Antler velvet transformation is mainly associated with alteration in the skin appendages. Subsequent antler regeneration is divided into 5 stages. The present account, together with the companion paper on antler morphogenesis in this special issue (E4, 1836-1842), provides a foundation for further mechanistic study of this fascinating model for mammalian organ regeneration.

Characterization of the early proliferative response of the rodent bladder to subtotal cystectomy: a unique model of mammalian organ regeneration.

Subtotal cystectomy (STC; surgical removal of ∼75% of the rat urinary bladder) elicits a robust proliferative response resulting in complete structural and functional bladder regeneration within 8-weeks. The goal of these studies was to characterize the early cellular response that mediates this regenerative phenomenon, which is unique among mammalian organ systems. STC was performed on eighteen 12-week-old female Fischer F344 rats. At 1, 3, 5 and 7-days post-STC, the bladder was harvested 2-hours after intraperitoneal injection of bromodeoxyuridine (BrdU). Fluorescent BrdU labeling was quantified in cells within the urothelium, lamina propria (LP), muscularis propria (MP) and serosa. Cell location was confirmed with fluorescently co-labeled cytokeratin, vimentin or smooth muscle actin (SMA), to identify urothelial, interstitial and smooth muscle cells, respectively. Expression of sonic hedgehog (Shh), Gli-1 and bone morphogenic factor-4 (BMP-4) were evaluated with immunochemistry. Three non-operated rats injected with BrdU served as controls. Less than 1% of cells in the bladder wall were labeled with BrdU in control bladders, but this percentage significantly increased by 5-8-fold at all time points post-STC. The spatiotemporal characteristics of the proliferative response were defined by a significantly higher percentage of BrdU-labeled cells within the urothelium at 1-day than in the MP and LP. A time-dependent shift at 3 and 5-days post-STC revealed significantly fewer BrdU-labeled cells in the MP than LP or urothelium. By 7-days the percentage of BrdU-labeled cells was similar among urothelium, LP and MP. STC also caused an increase in immunostaining for Shh, Gli-1 and BMP-4. In summary, the early stages of functional bladder regeneration are characterized by time-dependent changes in the location of the proliferating cell population, and expression of several evolutionarily conserved developmental signaling proteins. This report extends previous observations and further establishes the rodent bladder as an excellent model for studying novel aspects of mammalian organ regeneration.

Age‐dependent Impairments in Rat Bladder Regeneration

Models of de novo organ regeneration in mammals may provide guidance for harnessing natural regenerative capacities. We previously reported that removal of ~70% of the bladder (subtotal cystectomy; STC) in 12 week old female rats induces regeneration leading to functional recovery. In this study, 12 month (“older”) and 12 week (“younger”) old female rats underwent STC, and were followed up to 26 wks post‐STC with in vivo urodynamics, and pharmacological and histological analysis ex vivo. STC in older animals led to a higher mortality/morbidity rate (26%) than younger animals (11%), with histologic evidence of kidney damage. Bladder capacity 26 weeks post‐STC was 56% of age‐matched controls (AMC) in older rats, and 96% of AMCs in younger rats. There was no significant residual volume found in any group. Responses of bladder strips to several physiologically relevant stimuli were lower in younger STC animals than those from control bladders, but higher than older STC animals. Histology revealed age‐related differences post‐STC with respect to inflammation, angiogenesis, fibrosis, and proliferation. The impaired regeneration observed in older animals stands in stark contrast to the rather complete regeneration observed in younger animals. As such, this rodent model may aid in elucidating the basic biology of mammalian organ regeneration and identifying targetable pathways for optimizing regenerative capacity.

Initial Characterization of the Cellular Basis for Bladder Regeneration in a Rodent Model

PurposeA better understanding of intrinsic mammalian organ regeneration would benefit the field regenerative medicine. Our prior work shows functional bladder recovery in rats after removal of ~70% of the bladder (subtotal cystectomy/STC). Our objective is to begin identifying the spatiotemporal characteristics of cellular response to STC.MethodsTrigone‐sparing STC was performed on 12‐week‐old female Fischer F344 rats. At 1, 3, 5, and 7 days post‐STC, bladders were harvested 2 hours after IP 50mg/kg 5‐Bromo‐2’‐deoxyuridine (BrdU). Fluorescent BrdU labeling was used to quantify and localize S‐phase cells within the bladder wall. Immunohistochemistry for Shh, Gli‐1 and BMP‐4 was also performed. Normal rat bladders served as controls.ResultsSparse BrdU labeling was observed in native bladders (Fig. 1). The total number of BrdU‐positive cells/400x HPF was significantly higher post‐STC at all time points. A time‐depended location change in the percentage of BrdU‐positive cells was seen. Increased positive staining for Shh, Gli‐1 and BMP‐4 was also noted post‐STC.ConclusionsSTC induced a robust proliferative response with quantifiable spatiotemporal characteristics and up‐regulation of key targets in the Shh developmental pathway. Future studies will identify the cell source(s) and details of the molecular pathways that modulate functional bladder regeneration.

New concepts in liver regeneration

The unique ability of the liver to regenerate itself has fascinated biologists for years and has made it the prototype for mammalian organ regeneration. Harnessing this process has great potential benefit in the treatment of liver failure and has been the focus of intense research over the past 50 years. Not only will detailed understanding of cell proliferation in response to injury be applicable to other dysfunction of organs, it may also shed light on how cancer develops in a cirrhotic liver, in which there is intense pressure on cells to regenerate. Advances in molecular techniques over the past few decades have led to the identification of many regulatory intermediates, and pushed us onto the verge of an explosive era in regenerative medicine. To date, more than 10 clinical trials have been reported in which augmented regeneration using progenitor cell therapy has been attempted in human patients. This review traces the path that has been taken over the last few decades in the study of liver regeneration, highlights new concepts in the field, and discusses the challenges that still stand between us and clinical therapy.

Adult Stem Cells and Mammalian Epimorphic Regeneration-Insights from Studying Annual Renewal of Deer Antlers

Mammalian organ regeneration is the "Holy Grail" of modern regenerative biology and medicine. The most dramatic organ replacement is known as epimorphic regeneration. To date our knowledge of epimorphic regeneration has come from studies of amphibians. Notably, these animals have the ability to reprogram phenotypically committed cells at the amputation plane toward an embryonic-like cell phenotype (dedifferentiation). The capability of mammals to initiate analogous regeneration, and whether similar mechanisms would be involved if it were to occur, remain unclear. Deer antlers are the only mammalian appendages capable of full renewal, and therefore offer a unique opportunity to explore how nature has solved the problem of mammalian epimorphic regeneration. Following casting of old hard antlers, new antlers regenerate from permanent bony protuberances, known as pedicles. Studies through morphological and histological examinations, tissue deletion and transplantation, and cellular and molecular techniques have demonstrated that antler renewal is markedly different from that of amphibian limb regeneration (dedifferentiation-based), being a stem cell-based epimorphic process. Antler stem cells reside in the pedicle periosteum. We envisage that epimorphic regeneration of mammalian appendages, other than antler, could be made possible by recreating comparable milieu to that which supports the elaboration of that structure from the pedicle periosteum.