Publications by authors named "Yuval Rinkevich"

36 Publications

Fibroblasts as confederates of the immune system.

Immunol Rev 2021 May 25. Epub 2021 May 25.

Institute of Regenerative Biology and Medicine, Helmholtz Zentrum München, Munich, Germany.

Fibroblastic stromal cells are as diverse, in origin and function, as the niches they fashion in the mammalian body. This cellular variety impacts the spectrum of responses elicited by the immune system. Fibroblast influence on the immune system keeps evolving our perspective on fibroblast roles and functions beyond just a passive structural part of organs. This review discusses the foundations of fibroblastic stromal-immune crosstalk, under the scope of stromal heterogeneity as a basis for tissue-specific tutoring of the immune system. Focusing on the skin as a relevant immunological organ, we detail the complex interactions between distinct fibroblast populations and immune cells that occur during homeostasis, injury repair, scarring, and disease. We further review the relevance of fibroblastic stromal cell heterogeneity and how this heterogeneity is central to regulate the immune system from its inception during embryonic development into adulthood.
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http://dx.doi.org/10.1111/imr.12972DOI Listing
May 2021

Distinct fibroblasts in scars and regeneration.

Curr Opin Genet Dev 2021 May 19;70:7-14. Epub 2021 May 19.

Institute of Lung Biology and Disease, Helmholtz Zentrum München, Munich, Germany; Institute of Regenerative Biology and Medicine, Helmholtz Zentrum München, Munich, Germany. Electronic address:

The skin is home to a collection of fibroblastic cell types from varying embryonic origins. These varying fibroblastic lineages display unique genetic programs and in vivo functions. Studying the diversity of fibroblastic cells is emerging as an important area for cutaneous biology, wound repair and regenerative medicine. In this mini-review we discuss the distinct embryonic origins, microenvironments, and transcriptomic profiles of fibroblastic lineages, and how these varying lineages shape the skin's wound response across injury depths, anatomic locations, and developmental time to promote either scarring or regeneration. We outline how the development of single cell sequencing has led to our improved understanding of fibroblastic lineages at the molecular level and discuss existing challenges and future outlook on developing regenerative therapies that are based on this emerging field of eclectic fibroblasts.
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http://dx.doi.org/10.1016/j.gde.2021.04.005DOI Listing
May 2021

Connexin43 gap junction drives fascia mobilization and repair of deep skin wounds.

Matrix Biol 2021 03 27;97:58-71. Epub 2021 Jan 27.

Helmholtz Zentrum München, Institute of Lung Biology and Disease, Comprehensive Pneumology Center, Munich, Germany; Helmholtz Zentrum München, Institute of Regenerative Biology and Medicine, Munich, Germany. Electronic address:

Deep and voluminous skin wounds are repaired with scars, by mobilization of fibroblasts and extracellular matrix from fascia, deep below the skin. The molecular trigger of this novel repair mechanism is incompletely understood. Here we reveal that the gap junction alpha-1 protein (Connexin43, Cx43) is the key to patch repair of deep wounds. By combining full-thickness wound models with fibroblast lineage specific transgenic lines, we show Cx43 expression is substantially upregulated in specialized fibroblasts of the fascia deep beneath the skin that are responsible for scar formation. Using live imaging of fascia fibroblasts and fate tracing of the fascia extracellular matrix we show that Cx43 inhibition disrupts calcium oscillations in cultured fibroblasts and that this inhibits collective migration of fascia EPFs necessary to mobilize fascia matrix into open wounds. Cell-cell communication through Cx43 thus mediates matrix movement and scar formation, and is necessary for patch repair of voluminous wounds. These mechanistic findings have broad clinical implications toward treating fibrosis, aggravated scarring and impaired wound healing.
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http://dx.doi.org/10.1016/j.matbio.2021.01.005DOI Listing
March 2021

Injury triggers fascia fibroblast collective cell migration to drive scar formation through N-cadherin.

Nat Commun 2020 11 6;11(1):5653. Epub 2020 Nov 6.

Helmholtz Zentrum München, Institute of Lung Biology and Disease, Group Regenerative Biology and Medicine, Munich, Germany.

Scars are more severe when the subcutaneous fascia beneath the dermis is injured upon surgical or traumatic wounding. Here, we present a detailed analysis of fascia cell mobilisation by using deep tissue intravital live imaging of acute surgical wounds, fibroblast lineage-specific transgenic mice, and skin-fascia explants (scar-like tissue in a dish - SCAD). We observe that injury triggers a swarming-like collective cell migration of fascia fibroblasts that progressively contracts the skin and form scars. Swarming is exclusive to fascia fibroblasts, and requires the upregulation of N-cadherin. Both swarming and N-cadherin expression are absent from fibroblasts in the upper skin layers and the oral mucosa, tissues that repair wounds with minimal scar. Impeding N-cadherin binding inhibits swarming and skin contraction, and leads to reduced scarring in SCADs and in animals. Fibroblast swarming and N-cadherin thus provide therapeutic avenues to curtail fascia mobilisation and pathological fibrotic responses across a range of medical settings.
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http://dx.doi.org/10.1038/s41467-020-19425-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7648088PMC
November 2020

Post-surgical adhesions are triggered by calcium-dependent membrane bridges between mesothelial surfaces.

Nat Commun 2020 06 17;11(1):3068. Epub 2020 Jun 17.

Helmholtz Zentrum München, Institute of Lung Biology and Disease, Regenerative Biology and Medicine, Member of the German Center for Lung Research (DZL), Munich, Germany.

Surgical adhesions are bands of scar tissues that abnormally conjoin organ surfaces. Adhesions are a major cause of post-operative and dialysis-related complications, yet their patho-mechanism remains elusive, and prevention agents in clinical trials have thus far failed to achieve efficacy. Here, we uncover the adhesion initiation mechanism by coating beads with human mesothelial cells that normally line organ surfaces, and viewing them under adhesion stimuli. We document expansive membrane protrusions from mesothelia that tether beads with massive accompanying adherence forces. Membrane protrusions precede matrix deposition, and can transmit adhesion stimuli to healthy surfaces. We identify cytoskeletal effectors and calcium signaling as molecular triggers that initiate surgical adhesions. A single, localized dose targeting these early germinal events completely prevented adhesions in a preclinical mouse model, and in human assays. Our findings classifies the adhesion pathology as originating from mesothelial membrane bridges and offer a radically new therapeutic approach to treat adhesions.
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http://dx.doi.org/10.1038/s41467-020-16893-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7299976PMC
June 2020

Scars or Regeneration?-Dermal Fibroblasts as Drivers of Diverse Skin Wound Responses.

Int J Mol Sci 2020 Jan 17;21(2). Epub 2020 Jan 17.

Comprehensive Pneumology Center, Institute of Lung Biology and Disease, Helmholtz Zentrum München, Max-Lebsche-Platz 31, 81377 Munich, Germany.

Scarring and regeneration are two physiologically opposite endpoints to skin injuries, with mammals, including humans, typically healing wounds with fibrotic scars. We aim to provide an updated review on fibroblast heterogeneity as determinants of the scarring-regeneration continuum. We discuss fibroblast-centric mechanisms that dictate scarring-regeneration continua with a focus on intercellular and cell-matrix adhesion. Improved understanding of fibroblast lineage-specific mechanisms and how they determine scar severity will ultimately allow for the development of antiscarring therapies and the promotion of tissue regeneration.
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http://dx.doi.org/10.3390/ijms21020617DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7014275PMC
January 2020

Patch repair of deep wounds by mobilized fascia.

Nature 2019 12 27;576(7786):287-292. Epub 2019 Nov 27.

Group Regenerative Biology and Medicine, Institute of Lung Biology and Disease, Helmholtz Zentrum München, Munich, Germany.

Mammals form scars to quickly seal wounds and ensure survival by an incompletely understood mechanism. Here we show that skin scars originate from prefabricated matrix in the subcutaneous fascia. Fate mapping and live imaging revealed that fascia fibroblasts rise to the skin surface after wounding, dragging their surrounding extracellular jelly-like matrix, including embedded blood vessels, macrophages and peripheral nerves, to form the provisional matrix. Genetic ablation of fascia fibroblasts prevented matrix from homing into wounds and resulted in defective scars, whereas placing an impermeable film beneath the skin-preventing fascia fibroblasts from migrating upwards-led to chronic open wounds. Thus, fascia contains a specialized prefabricated kit of sentry fibroblasts, embedded within a movable sealant, that preassemble together diverse cell types and matrix components needed to heal wounds. Our findings suggest that chronic and excessive skin wounds may be attributed to the mobility of the fascia matrix.
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http://dx.doi.org/10.1038/s41586-019-1794-yDOI Listing
December 2019

Local and transient inhibition of p21 expression ameliorates age-related delayed wound healing.

Wound Repair Regen 2020 01 21;28(1):49-60. Epub 2019 Oct 21.

Department of Dermatology and Allergic Diseases, University of Ulm, Ulm, Germany.

Nonhealing chronic wounds in the constantly growing elderly population represent a major public health problem with high socioeconomic burden. Yet, the underlying mechanism of age-related impairment of wound healing remains elusive. Here, we show that the number of dermal cells expressing cyclin-dependent kinase inhibitor p21 was elevated upon skin injury, particularly in aged population, in both man and mouse. The nuclear expression of p21 in activated wound fibroblasts delayed the onset of the proliferation phase of wound healing in a p53-independent manner. Further, the local and transient inhibition of p21 expression by in vivo delivered p21-targeting siRNA ameliorated the delayed wound healing in aged mice. Our results suggest that the increased number of p21 wound fibroblasts enforces the age-related compromised healing, and targeting p21 creates potential clinical avenues to promote wound healing in aged population.
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http://dx.doi.org/10.1111/wrr.12763DOI Listing
January 2020

Neutrophil and monocyte kinetics play critical roles in mouse peritoneal adhesion formation.

Blood Adv 2019 09;3(18):2713-2721

Institute for Stem Cell Biology and Regenerative Medicine and.

Peritoneal adhesions are pathological fibroses that ensnare organs after abdominal surgery. This dense connective tissue can cause small bowel obstruction, female infertility, and chronic abdominal pain. The pathogenesis of adhesions is a fibrotic response to tissue damage coordinated between mesothelial cells, fibroblasts, and immune cells. We have previously demonstrated that peritoneal adhesions are a consequence of mechanical injury to the mesothelial layer sustained during surgery. Neutrophils are among the first leukocytes involved in the early response to tissue damage. Here, we show that when subjected to mechanical stress, activated mesothelial cells directly recruit neutrophils and monocytes through upregulation of chemokines such as CXCL1 and monocyte chemoattractant protein 1 (MCP-1). We find that neutrophils within the adhesion sites undergo cell death and form neutrophil extracellular traps (NETosis) that contribute to pathogenesis. Conversely, tissue-resident macrophages were profoundly depleted throughout the disease time course. We show that this is distinct from traditional inflammatory kinetics such as after sham surgery or chemically induced peritonitis, and suggest that adhesions result from a primary difference in inflammatory kinetics. We find that transient depletion of circulating neutrophils significantly decreases adhesion burden, and further recruitment of monocytes with thioglycolate or MCP-1 also improves outcomes. Our findings suggest that the combination of neutrophil depletion and monocyte recruitment is sufficient to prevent adhesion formation, thus providing insight for potential clinical interventions.
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http://dx.doi.org/10.1182/bloodadvances.2018024026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6759736PMC
September 2019

Surgical adhesions in mice are derived from mesothelial cells and can be targeted by antibodies against mesothelial markers.

Sci Transl Med 2018 11;10(469)

Comprehensive Pneumology Center, Institute of Lung Biology and Disease,Helmholtz Zentrum München, Member of the German Center for Lung Research (DZL), Munich, Germany.

Peritoneal adhesions are fibrous tissues that tether organs to one another or to the peritoneal wall and are a major cause of postsurgical and infectious morbidity. The primary molecular chain of events leading to the initiation of adhesions has been elusive, chiefly due to the lack of an identifiable cell of origin. Using clonal analysis and lineage tracing, we have identified injured surface mesothelium expressing podoplanin (PDPN) and mesothelin (MSLN) as a primary instigator of peritoneal adhesions after surgery in mice. We demonstrate that an anti-MSLN antibody diminished adhesion formation in a mouse model where adhesions were induced by surgical ligation to form ischemic buttons and subsequent surgical abrasion of the peritoneum. RNA sequencing and bioinformatics analyses of mouse mesothelial cells from injured mesothelium revealed aspects of the pathological mechanism of adhesion development and yielded several potential regulators of this process. Specifically, we show that PDPNMSLN mesothelium responded to hypoxia by early up-regulation of hypoxia-inducible factor 1 alpha (HIF1α) that preceded adhesion development. Inhibition of HIF1α with small molecules ameliorated the injury program in damaged mesothelium and was sufficient to diminish adhesion severity in a mouse model. Analyses of human adhesion tissue suggested that similar surface markers and signaling pathways may contribute to surgical adhesions in human patients.
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http://dx.doi.org/10.1126/scitranslmed.aan6735DOI Listing
November 2018

Defining Skin Fibroblastic Cell Types Beyond CD90.

Front Cell Dev Biol 2018 22;6:133. Epub 2018 Oct 22.

Comprehensive Pneumology Center, Institute of Lung Biology and Disease, Helmholtz Zentrum München, Munich, Germany.

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http://dx.doi.org/10.3389/fcell.2018.00133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6204438PMC
October 2018

Mesothelial to mesenchyme transition as a major developmental and pathological player in trunk organs and their cavities.

Commun Biol 2018 16;1:170. Epub 2018 Oct 16.

Comprehensive Pneumology Center, Institute of Lung Biology and Disease, Helmholtz Zentrum München, Munich, 81377, Germany.

The internal organs embedded in the cavities are lined by an epithelial monolayer termed the mesothelium. The mesothelium is increasingly implicated in driving various internal organ pathologies, as many of the normal embryonic developmental pathways acting in mesothelial cells, such as those regulating epithelial-to-mesenchymal transition, also drive disease progression in adult life. Here, we summarize observations from different animal models and organ systems that collectively point toward a central role of epithelial-to-mesenchymal transition in driving tissue fibrosis, acute scarring, and cancer metastasis. Thus, drugs targeting pathways of mesothelium's transition may have broad therapeutic benefits in patients suffering from these diseases.
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http://dx.doi.org/10.1038/s42003-018-0180-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6191446PMC
October 2018

Partial Lobular Hepatectomy: A Surgical Model for Morphologic Liver Regeneration.

J Vis Exp 2018 05 31(135). Epub 2018 May 31.

Comprehensive Pneumology Center, Institute of Lung Biology and Disease, Helmholtz Zentrum München; German Center for Lung Research (DZL);

Morphological organ regeneration following acute tissue loss is common among lower vertebrates, but is rarely observed in mammalian postnatal life. Adult liver regeneration after 70% partial hepatectomy results in hepatocyte hypertrophy with some replication in remaining lobes with restoration of metabolic activity, but with permanent loss of the injured lobe's morphology and architecture. Here, we detail a new surgical method in the neonate that leaves a physiologic environment conducive to regeneration. This model involves amputation of the left lobe apex and a subsequent conservative management regimen, and lacks the necessity for ligation of major liver vessels or chemical injury, leaving a physiologic environment where regeneration may occur. We extend this protocol to amputations on juvenile (P7-14) mice, during which the injured liver transitions from organ regeneration to compensatory growth by hypertrophy. The presented, brief 30 min protocol provides a framework to study the mechanisms of regeneration, its age-associated decline in mammals, and the characterization of putative hepatic stem or progenitors.
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http://dx.doi.org/10.3791/57302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6101439PMC
May 2018

Two succeeding fibroblastic lineages drive dermal development and the transition from regeneration to scarring.

Nat Cell Biol 2018 04 28;20(4):422-431. Epub 2018 Mar 28.

Comprehensive Pneumology Centre/Institute of Lung Biology and Disease, Helmholtz Zentrum München, Munich, Germany.

During fetal development, mammalian back-skin undergoes a natural transition in response to injury, from scarless regeneration to skin scarring. Here, we characterize dermal morphogenesis and follow two distinct embryonic fibroblast lineages, based on their history of expression of the engrailed 1 gene. We use single-cell fate-mapping, live three dimensional confocal imaging and in silico analysis coupled with immunolabelling to reveal unanticipated structural and regional complexity and dynamics within the dermis. We show that dermal development and regeneration are driven by engrailed 1-history-naive fibroblasts, whose numbers subsequently decline. Conversely, engrailed 1-history-positive fibroblasts possess scarring abilities at this early stage and their expansion later on drives scar emergence. The transition can be reversed, locally, by transplanting engrailed 1-naive cells. Thus, fibroblastic lineage replacement couples the decline of regeneration with the emergence of scarring and creates potential clinical avenues to reduce scarring.
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http://dx.doi.org/10.1038/s41556-018-0073-8DOI Listing
April 2018

Localized hepatic lobular regeneration by central-vein-associated lineage-restricted progenitors.

Proc Natl Acad Sci U S A 2017 04 22;114(14):3654-3659. Epub 2017 Mar 22.

Comprehensive Pneumology Center, Institute of Lung Biology and Disease, Helmholtz Zentrum München, 81377 Munich, Germany;

The regeneration of organ morphology and function following tissue loss is critical to restore normal physiology, yet few cases are documented in mammalian postnatal life. Partial hepatectomy of the adult mammalian liver activates compensatory hepatocyte hypertrophy and cell division across remaining lobes, resulting in restitution of organ mass but with permanent alteration of architecture. Here, we identify a time window in early postnatal life wherein partial amputation culminates in a localized regeneration instead of global hypertrophy and proliferation. Quantifications of liver mass, enzymatic activity, and immunohistochemistry demonstrate that damaged lobes underwent multilineage regeneration, reforming a lobe often indistinguishable from undamaged ones. Clonal analysis during regeneration reveals local clonal expansions of hepatocyte stem/progenitors at injured sites that are lineage but not fate restricted. Tetrachimeric mice show clonal selection occurs during development with further selections following injury. Surviving progenitors associate mainly with central veins, in a pattern of selection different from that of normal development. These results illuminate a previously unknown program of liver regeneration after acute injury and allow for exploration of latent regenerative programs with potential applications to adult liver regeneration.
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http://dx.doi.org/10.1073/pnas.1621361114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389277PMC
April 2017

Dynamic Patterns of Clonal Evolution in Tumor Vasculature Underlie Alterations in Lymphocyte-Endothelial Recognition to Foster Tumor Immune Escape.

Cancer Res 2016 Mar 30;76(6):1348-53. Epub 2015 Dec 30.

Stanford Ludwig Center for Cancer Stem Cell Research and Medicine, Stanford University School of Medicine, Stanford, California. Departments of Pathology and Developmental Biology, Stanford University School of Medicine, Stanford, California.

Although tumor blood vessels have been a major therapeutic target for cancer chemotherapy, little is known regarding the stepwise development of the tumor microenvironment. Here, we use a multicolor Cre-dependent marker system to trace clonality within the tumor microenvironment to show that tumor blood vessels follow a pattern of dynamic clonal evolution. In an advanced melanoma tumor microenvironment, the vast majority of tumor vasculature clones are derived from a common precursor. Quantitative lineage analysis reveals founder clones diminish in frequency and are replaced by subclones as tumors evolve. These tumor-specific blood vessels are characterized by a developmental switch to a more invasive and immunologically silent phenotype. Gene expression profiling and pathway analysis reveals selection for traits promoting upregulation of alternative angiogenic programs such as unregulated HGF-MET signaling and enhanced autocrine signaling through VEGF and PDGF. Furthermore, we show a developmental switch in the expression of functionally significant primary lymphocyte adhesion molecules on tumor endothelium, such as the loss in expression of the mucosal addressin MAdCAM-1, whose counter receptor a4β7 on lymphocytes controls lymphocyte homing. Changes in adhesive properties on tumor endothelial subclones are accompanied by decreases in expression of lymphocyte chemokines CXCL16, CXCL13, CXCL12, CXCL9, CXCL10, and CCL19. These evolutionary patterns in the expressed genetic program within tumor endothelium will have both a quantitative and functional impact on lymphocyte distribution that may well influence tumor immune function and underlie escape mechanisms from current antiangiogenic pharmacotherapies.
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http://dx.doi.org/10.1158/0008-5472.CAN-15-1150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4794394PMC
March 2016

fibroblasts and melanoma.

Melanoma Manag 2015 Aug 10;2(3):191-192. Epub 2015 Aug 10.

Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Plastic & Reconstructive Surgery, Stanford University School of Medicine, Stanford, CA, USA.

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http://dx.doi.org/10.2217/mmt.15.23DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6094637PMC
August 2015

The use of lineage tracing to study kidney injury and regeneration.

Nat Rev Nephrol 2015 Jul 12;11(7):420-31. Epub 2015 May 12.

Paediatric Stem Cell Research Institute &Division of Paediatric Nephrology, Edmond and Lily Safra Children's Hospital, Sheba Medical Centre, Tel Aviv University, Tel Aviv 52621, Israel.

Lineage tracing is a powerful tool to track cells in vivo and provides enhanced spatial, temporal, and kinetic resolution of the mechanisms that underlie tissue renewal and repair. The data obtained from novel mouse models engineered for lineage tracing has started to transform our understanding of the changes in cell fate that underlie renal pathophysiology, the role of stem and/or progenitor cells in kidney development, and the mechanisms of kidney regeneration. The complexity of the genetic systems that are engineered for lineage tracing requires careful analysis and interpretation. In this Review we emphasize that close attention in lineage tracing studies must be paid to the specificity of the promoter, the use of drug-controlled activation of Cre recombinase as a genetic switch, and the type of reporter that should be engineered into lineage tracing genetic constructs. We evaluate the optimal experimental conditions required to achieve the pre-specified aims of the study and discuss the novel genetic techniques that are becoming available to study putative renal progenitor cells and the mechanisms of kidney regeneration.
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http://dx.doi.org/10.1038/nrneph.2015.67DOI Listing
July 2015

Skin fibrosis. Identification and isolation of a dermal lineage with intrinsic fibrogenic potential.

Science 2015 Apr;348(6232):aaa2151

Institute for Stem Cell Biology and Regenerative Medicine, Departments of Pathology and Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.

Dermal fibroblasts represent a heterogeneous population of cells with diverse features that remain largely undefined. We reveal the presence of at least two fibroblast lineages in murine dorsal skin. Lineage tracing and transplantation assays demonstrate that a single fibroblast lineage is responsible for the bulk of connective tissue deposition during embryonic development, cutaneous wound healing, radiation fibrosis, and cancer stroma formation. Lineage-specific cell ablation leads to diminished connective tissue deposition in wounds and reduces melanoma growth. Using flow cytometry, we identify CD26/DPP4 as a surface marker that allows isolation of this lineage. Small molecule-based inhibition of CD26/DPP4 enzymatic activity during wound healing results in diminished cutaneous scarring. Identification and isolation of these lineages hold promise for translational medicine aimed at in vivo modulation of fibrogenic behavior.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5088503PMC
http://dx.doi.org/10.1126/science.aaa2151DOI Listing
April 2015

Injuries to appendage extremities and digit tips: A clinical and cellular update.

Dev Dyn 2015 May;244(5):641-50

Institute for Stem Cell Biology and Regenerative Medicine, Departments of Pathology and Developmental Biology, Stanford University School of Medicine, Stanford, California.

Background: The regrowth of amputated appendage extremities and the distal tips of digits represent models of tissue regeneration in multiple vertebrate taxa. In humans, digit tip injuries, including traumatic amputation and crush injuries, are among the most common type of injury to the human hand. Despite clinical reports demonstrating natural regeneration of appendages in lower vertebrates and human digits, current treatment options are suboptimal, and are complicated by the anatomical complexities and functions of the different tissues within the digits.

Results: In light of these challenges, we focus on recent advancements in understanding appendage regeneration from model organisms. We pay special attention to the cellular programs underlying appendage regeneration, where cumulative data from salamanders, fish, frogs, and mice indicate that regeneration occurs by the actions of lineage-restricted precursors. We focus on pathologic states and the interdependency that exists, in both humans and animal models, between the nail organ and the peripheral nerves for successful regeneration.

Conclusions: The increased understanding of regeneration in animal models may open new opportunities for basic and translational research aimed at understanding the mechanisms that support limb regeneration, as well as amelioration of limb abnormalities and pathologies.
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http://dx.doi.org/10.1002/dvdy.24265DOI Listing
May 2015

Live fibroblast harvest reveals surface marker shift in vitro.

Tissue Eng Part C Methods 2015 Mar 17;21(3):314-21. Epub 2014 Dec 17.

1 Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Plastic and Reconstructive Surgery, Stanford University School of Medicine , Stanford, California.

Current methods for the isolation of fibroblasts require extended ex vivo manipulation in cell culture. As a consequence, prior studies investigating fibroblast biology may fail to adequately represent cellular phenotypes in vivo. To overcome this problem, we describe a detailed protocol for the isolation of fibroblasts from the dorsal dermis of adult mice that bypasses the need for cell culture, thereby preserving the physiological, transcriptional, and proteomic profiles of each cell. Using the described protocol we characterized the transcriptional programs and the surface expression of 176 CD markers in cultured versus uncultured fibroblasts. The differential expression patterns we observed highlight the importance of a live harvest for investigations of fibroblast biology.
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http://dx.doi.org/10.1089/ten.TEC.2014.0118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4346232PMC
March 2015

Clonal analysis reveals nerve-dependent and independent roles on mammalian hind limb tissue maintenance and regeneration.

Proc Natl Acad Sci U S A 2014 Jul 23;111(27):9846-51. Epub 2014 Jun 23.

Institute for Stem Cell Biology and Regenerative Medicine, Department of Pathology, and Department of Developmental Biology,Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Plastic and Reconstructive Surgery, and

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.
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http://dx.doi.org/10.1073/pnas.1410097111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4103362PMC
July 2014

In vivo clonal analysis reveals lineage-restricted progenitor characteristics in mammalian kidney development, maintenance, and regeneration.

Cell Rep 2014 May 15;7(4):1270-83. Epub 2014 May 15.

Pediatric Stem Cell Research Institute, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Tel Hashomer, Sackler School of Medicine, Tel Aviv University, Tel Aviv 52621, Israel. Electronic address:

The mechanism and magnitude by which the mammalian kidney generates and maintains its proximal tubules, distal tubules, and collecting ducts remain controversial. Here, we use long-term in vivo genetic lineage tracing and clonal analysis of individual cells from kidneys undergoing development, maintenance, and regeneration. We show that the adult mammalian kidney undergoes continuous tubulogenesis via expansions of fate-restricted clones. Kidneys recovering from damage undergo tubulogenesis through expansions of clones with segment-specific borders, and renal spheres developing in vitro from individual cells maintain distinct, segment-specific fates. Analysis of mice derived by transfer of color-marked embryonic stem cells (ESCs) into uncolored blastocysts demonstrates that nephrons are polyclonal, developing from expansions of singly fated clones. Finally, we show that adult renal clones are derived from Wnt-responsive precursors, and their tracing in vivo generates tubules that are segment specific. Collectively, these analyses demonstrate that fate-restricted precursors functioning as unipotent progenitors continuously maintain and self-preserve the mouse kidney throughout life.
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http://dx.doi.org/10.1016/j.celrep.2014.04.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4425291PMC
May 2014

Repeated, long-term cycling of putative stem cells between niches in a basal chordate.

Dev Cell 2013 Jan 20;24(1):76-88. Epub 2012 Dec 20.

Institute of Stem Cell Biology and Regenerative Medicine, Departments of Pathology and Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.

The mechanisms that sustain stem cells are fundamental to tissue maintenance. Here, we identify "cell islands" (CIs) as a niche for putative germ and somatic stem cells in Botryllus schlosseri, a colonial chordate that undergoes weekly cycles of death and regeneration. Cells within CIs express markers associated with germ and somatic stem cells and gene products that implicate CIs as signaling centers for stem cells. Transplantation of CIs induced long-term germline and somatic chimerism, demonstrating self-renewal and pluripotency of CI cells. Cell labeling and in vivo time-lapse imaging of CI cells reveal waves of migrations from degrading CIs into developing buds, contributing to soma and germline development. Knockdown of cadherin, which is highly expressed within CIs, elicited the migration of CI cells to circulation. Piwi knockdown resulted in regeneration arrest. We suggest that repeated trafficking of stem cells allows them to escape constraints imposed by the niche, enabling self-preservation throughout life.
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http://dx.doi.org/10.1016/j.devcel.2012.11.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3810298PMC
January 2013

Identification and prospective isolation of a mesothelial precursor lineage giving rise to smooth muscle cells and fibroblasts for mammalian internal organs, and their vasculature.

Nat Cell Biol 2012 Dec 11;14(12):1251-60. Epub 2012 Nov 11.

Institute for Stem Cell Biology and Regenerative Medicine, Department of Pathology, Stanford University School of Medicine, Stanford, California 94305, USA.

Fibroblasts and smooth muscle cells (FSMCs) are principal cell types of connective and adventitial tissues that participate in the development, physiology and pathology of internal organs, with incompletely defined cellular origins. Here, we identify and prospectively isolate from the mesothelium a mouse cell lineage that is committed to FSMCs. The mesothelium is an epithelial monolayer covering the vertebrate thoracic and abdominal cavities and internal organs. Time-lapse imaging and transplantation experiments reveal robust generation of FSMCs from the mesothelium. By targeting mesothelin (MSLN), a surface marker expressed on mesothelial cells, we identify and isolate precursors capable of clonally generating FSMCs. Using a genetic lineage tracing approach, we show that embryonic and adult mesothelium represents a common lineage to trunk FSMCs, and trunk vasculature, with minimal contributions from neural crest, or circulating cells. The isolation of FSMC precursors enables the examination of multiple aspects of smooth muscle and fibroblast biology as well as the prospective isolation of these precursors for potential regenerative medicine purposes.
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http://dx.doi.org/10.1038/ncb2610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3685475PMC
December 2012

Isolation of primitive endoderm, mesoderm, vascular endothelial and trophoblast progenitors from human pluripotent stem cells.

Nat Biotechnol 2012 May 27;30(6):531-42. Epub 2012 May 27.

Institute of Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California, USA.

To identify early populations of committed progenitors derived from human embryonic stem cells (hESCs), we screened self-renewing, BMP4-treated and retinoic acid-treated cultures with >400 antibodies recognizing cell-surface antigens. Sorting of >30 subpopulations followed by transcriptional analysis of developmental genes identified four distinct candidate progenitor groups. Subsets detected in self-renewing cultures, including CXCR4(+) cells, expressed primitive endoderm genes. Expression of Cxcr4 in primitive endoderm was confirmed in visceral endoderm of mouse embryos. BMP4-induced progenitors exhibited gene signatures of mesoderm, trophoblast and vascular endothelium, suggesting correspondence to gastrulation-stage primitive streak, chorion and allantois precursors, respectively. Functional studies in vitro and in vivo confirmed that ROR2(+) cells produce mesoderm progeny, APA(+) cells generate syncytiotrophoblasts and CD87(+) cells give rise to vasculature. The same progenitor classes emerged during the differentiation of human induced pluripotent stem cells (hiPSCs). These markers and progenitors provide tools for purifying human tissue-regenerating progenitors and for studying the commitment of pluripotent stem cells to lineage progenitors.
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http://dx.doi.org/10.1038/nbt.2239DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3672406PMC
May 2012

The "Stars and Stripes" Metaphor for Animal Regeneration-Elucidating Two Fundamental Strategies along a Continuum.

Cells 2012 Dec 27;2(1):1-18. Epub 2012 Dec 27.

Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford 94305-5323, CA, USA.

A number of challenges have hindered the development of a unified theory for metazoan regeneration. To describe the full range of complex regeneration phenomena in Animalia, we suggest that metazoans that regenerate missing body parts exhibit biological attributes that are tailored along a morpho-spatial regeneration continuum, illustrated in its polar scenarios by the USA "stars and stripes" flag. Type 1 organisms ("T1, 'stars'") are typical colonial organisms (but contain unitary taxa) that are able to regenerate "whole new stars", namely, whole bodies and colonial modules, through systemic induction and sometimes multiple regeneration foci (hollow regeneration spheres, resembling the blastula) that compete for dominance. They regenerate soma and germ constituents with pluripotent adult stem cells and exhibit somatic-embryogenesis mode of ontogeny. Type 2 organisms ("T2, 'stripes'") are capable of limited regeneration of somatic constituents via fate-restricted stem cells, and regenerate through centralized inductions that lead to a single regeneration front. T2 organisms are unitary and use preformistic mode of ontogeny. T1 and T2 organisms also differ in interpretation of what constitutes positional information. T2 organisms also execute alternative, less effective, regeneration designs (i.e., scar formation). We assigned 15 characteristics that distinguish between T1/T2 strategies: those involving specific regeneration features and those operating on biological features at the whole-organism level. Two model organisms are discussed, representing the two strategies of T1/T2 along the regeneration continuum, the Botrylloides whole body regeneration (T1) and the mouse digit-tip regeneration (T2) phenomena. The above working hypothesis also postulates that regeneration is a primeval attribute of metazoans. As specified, the "stars and stripes" paradigm allows various combinations of the biological features assigned to T1 and T2 regeneration strategies. It does not consider any concentration gradient or thresholds and does not refer to the "epimorphosis" and "morphallaxis" terms, regeneration types across phyla or across body plans. The "stars and stripes" paradigm also ignores, at this stage of analysis, cases of regeneration loss that may obscure biological trajectories. The main advantage of the "stars and stripes" paradigm is that it allows us to compare T1/T2 regeneration, as well as other modes of regeneration, through critical determining characteristics.
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http://dx.doi.org/10.3390/cells2010001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3972663PMC
December 2012

Germ-layer and lineage-restricted stem/progenitors regenerate the mouse digit tip.

Nature 2011 Aug 24;476(7361):409-13. Epub 2011 Aug 24.

Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California 94305-5323, USA.

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.
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http://dx.doi.org/10.1038/nature10346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3812235PMC
August 2011

Piwi positive cells that line the vasculature epithelium, underlie whole body regeneration in a basal chordate.

Dev Biol 2010 Sep 27;345(1):94-104. Epub 2010 May 27.

Israel Oceanographic and Limnological Research, National Institute of Oceanography, PO Box 8030, Tel Shikmona, Haifa 31080, Israel.

The colonial tunicate Botrylloides leachi can regenerate functional adults from minute vasculature fragments, in a poorly understood phenomenon termed Whole Body Regeneration (WBR). Using Piwi expression (Bl-Piwi), blood cell labeling and electron microscopy, we show that WBR develops through activation, mobilization and expansion of 'dormant' cells which normally line the internal vasculature epithelium of blood vessels. Following a mechanical insult, these cells express Bl-Piwi de novo, change morphology and invade niches of the vasculature lumen, where they proliferate and differentiate, regenerating a functional organism. Mitomycin C treatments and siRNA knockdown of Bl-Piwi result in deficient cells incapable of expanding or differentiating and to subsequent regeneration arrest. Last, we find similar transient mobilization of Piwi(+) cells recurring every week, as part of normal colony development, and also during acute environmental stress. This recurrent activation of Piwi(+) cells in response to developmental, physiological and environmental insults may have enabled the adaptation of colonial tunicates to the imposed varied conditions in the marine, shallow water environment.
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http://dx.doi.org/10.1016/j.ydbio.2010.05.500DOI Listing
September 2010