RESUMO
Stem cell regulation and hierarchical organization of human skeletal progenitors remain largely unexplored. Here, we report the isolation of a self-renewing and multipotent human skeletal stem cell (hSSC) that generates progenitors of bone, cartilage, and stroma, but not fat. Self-renewing and multipotent hSSCs are present in fetal and adult bones and can also be derived from BMP2-treated human adipose stroma (B-HAS) and induced pluripotent stem cells (iPSCs). Gene expression analysis of individual hSSCs reveals overall similarity between hSSCs obtained from different sources and partially explains skewed differentiation toward cartilage in fetal and iPSC-derived hSSCs. hSSCs undergo local expansion in response to acute skeletal injury. In addition, hSSC-derived stroma can maintain human hematopoietic stem cells (hHSCs) in serum-free culture conditions. Finally, we combine gene expression and epigenetic data of mouse skeletal stem cells (mSSCs) and hSSCs to identify evolutionarily conserved and divergent pathways driving SSC-mediated skeletogenesis. VIDEO ABSTRACT.
Assuntos
Desenvolvimento Ósseo/fisiologia , Osso e Ossos/citologia , Células-Tronco Hematopoéticas/citologia , Animais , Osso e Ossos/metabolismo , Cartilagem/citologia , Diferenciação Celular , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Análise de Célula Única/métodos , Células-Tronco/citologia , Células Estromais/citologia , Transcriptoma/genéticaRESUMO
How are skeletal tissues derived from skeletal stem cells? Here, we map bone, cartilage, and stromal development from a population of highly pure, postnatal skeletal stem cells (mouse skeletal stem cells, mSSCs) to their downstream progenitors of bone, cartilage, and stromal tissue. We then investigated the transcriptome of the stem/progenitor cells for unique gene-expression patterns that would indicate potential regulators of mSSC lineage commitment. We demonstrate that mSSC niche factors can be potent inducers of osteogenesis, and several specific combinations of recombinant mSSC niche factors can activate mSSC genetic programs in situ, even in nonskeletal tissues, resulting in de novo formation of cartilage or bone and bone marrow stroma. Inducing mSSC formation with soluble factors and subsequently regulating the mSSC niche to specify its differentiation toward bone, cartilage, or stromal cells could represent a paradigm shift in the therapeutic regeneration of skeletal tissues.
Assuntos
Osso e Ossos/citologia , Células-Tronco Mesenquimais/citologia , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Cartilagem/citologia , Linhagem da Célula , Cruzamentos Genéticos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transdução de SinaisRESUMO
In lactating mothers, the high calcium (Ca2+) demand for milk production triggers significant bone loss1. Although oestrogen normally counteracts excessive bone resorption by promoting bone formation, this sex steroid drops precipitously during this postpartum period. Here we report that brain-derived cellular communication network factor 3 (CCN3) secreted from KISS1 neurons of the arcuate nucleus (ARCKISS1) fills this void and functions as a potent osteoanabolic factor to build bone in lactating females. We began by showing that our previously reported female-specific, dense bone phenotype2 originates from a humoral factor that promotes bone mass and acts on skeletal stem cells to increase their frequency and osteochondrogenic potential. This circulatory factor was then identified as CCN3, a brain-derived hormone from ARCKISS1 neurons that is able to stimulate mouse and human skeletal stem cell activity, increase bone remodelling and accelerate fracture repair in young and old mice of both sexes. The role of CCN3 in normal female physiology was revealed after detecting a burst of CCN3 expression in ARCKISS1 neurons coincident with lactation. After reducing CCN3 in ARCKISS1 neurons, lactating mothers lost bone and failed to sustain their progeny when challenged with a low-calcium diet. Our findings establish CCN3 as a potentially new therapeutic osteoanabolic hormone for both sexes and define a new maternal brain hormone for ensuring species survival in mammals.
Assuntos
Densidade Óssea , Osso e Ossos , Encéfalo , Hormônios , Mães , Proteína Sobre-Expressa em Nefroblastoma , Osteogênese , Adolescente , Animais , Feminino , Humanos , Masculino , Camundongos , Envelhecimento , Núcleo Arqueado do Hipotálamo/citologia , Núcleo Arqueado do Hipotálamo/metabolismo , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Remodelação Óssea , Reabsorção Óssea/metabolismo , Encéfalo/citologia , Encéfalo/metabolismo , Cálcio/administração & dosagem , Cálcio/metabolismo , Lactação/metabolismo , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Células-Tronco/metabolismo , Células-Tronco/citologia , Proteína Sobre-Expressa em Nefroblastoma/metabolismo , Hormônios/metabolismoRESUMO
Loss of skeletal integrity during ageing and disease is associated with an imbalance in the opposing actions of osteoblasts and osteoclasts1. Here we show that intrinsic ageing of skeletal stem cells (SSCs)2 in mice alters signalling in the bone marrow niche and skews the differentiation of bone and blood lineages, leading to fragile bones that regenerate poorly. Functionally, aged SSCs have a decreased bone- and cartilage-forming potential but produce more stromal lineages that express high levels of pro-inflammatory and pro-resorptive cytokines. Single-cell RNA-sequencing studies link the functional loss to a diminished transcriptomic diversity of SSCs in aged mice, which thereby contributes to the transformation of the bone marrow niche. Exposure to a youthful circulation through heterochronic parabiosis or systemic reconstitution with young haematopoietic stem cells did not reverse the diminished osteochondrogenic activity of aged SSCs, or improve bone mass or skeletal healing parameters in aged mice. Conversely, the aged SSC lineage promoted osteoclastic activity and myeloid skewing by haematopoietic stem and progenitor cells, suggesting that the ageing of SSCs is a driver of haematopoietic ageing. Deficient bone regeneration in aged mice could only be returned to youthful levels by applying a combinatorial treatment of BMP2 and a CSF1 antagonist locally to fractures, which reactivated aged SSCs and simultaneously ablated the inflammatory, pro-osteoclastic milieu. Our findings provide mechanistic insights into the complex, multifactorial mechanisms that underlie skeletal ageing and offer prospects for rejuvenating the aged skeletal system.
Assuntos
Envelhecimento/patologia , Osso e Ossos/patologia , Senescência Celular , Inflamação/patologia , Nicho de Células-Tronco , Células-Tronco/patologia , Animais , Proteína Morfogenética Óssea 2/metabolismo , Regeneração Óssea , Linhagem da Célula , Feminino , Consolidação da Fratura , Hematopoese , Fator Estimulador de Colônias de Macrófagos/metabolismo , Masculino , Camundongos , Células Mieloides/citologia , Osteoclastos/citologia , RejuvenescimentoRESUMO
BACKGROUND: Peripheral vascular disease remains a leading cause of vascular morbidity and mortality worldwide despite advances in medical and surgical therapy. Besides traditional approaches, which can only restore blood flow to native arteries, an alternative approach is to enhance the growth of new vessels, thereby facilitating the physiological response to ischemia. METHODS: The ActinCreER/R26VT2/GK3 Rainbow reporter mouse was used for unbiased in vivo survey of injury-responsive vasculogenic clonal formation. Prospective isolation and transplantation were used to determine vessel-forming capacity of different populations. Single-cell RNA-sequencing was used to characterize distinct vessel-forming populations and their interactions. RESULTS: Two populations of distinct vascular stem/progenitor cells (VSPCs) were identified from adipose-derived mesenchymal stromal cells: VSPC1 is CD45-Ter119-Tie2+PDGFRa-CD31+CD105highSca1low, which gives rise to stunted vessels (incomplete tubular structures) in a transplant setting, and VSPC2 which is CD45-Ter119-Tie2+PDGFRa+CD31-CD105lowSca1high and forms stunted vessels and fat. Interestingly, cotransplantation of VSPC1 and VSPC2 is required to form functional vessels that improve perfusion in the mouse hindlimb ischemia model. Similarly, VSPC1 and VSPC2 populations isolated from human adipose tissue could rescue the ischemic condition in mice. CONCLUSIONS: These findings suggest that autologous cotransplantation of synergistic VSPCs from nonessential adipose tissue can promote neovascularization and represents a promising treatment for ischemic disease.
Assuntos
Células-Tronco Mesenquimais , Neovascularização Fisiológica , Camundongos , Humanos , Animais , Neovascularização Fisiológica/fisiologia , Tecido Adiposo , Neovascularização Patológica , Isquemia/terapia , Modelos Animais de Doenças , Membro Posterior/irrigação sanguíneaRESUMO
During both embryonic development and adult tissue regeneration, changes in chromatin structure driven by master transcription factors lead to stimulus-responsive transcriptional programs. A thorough understanding of how stem cells in the skeleton interpret mechanical stimuli and enact regeneration would shed light on how forces are transduced to the nucleus in regenerative processes. Here we develop a genetically dissectible mouse model of mandibular distraction osteogenesis-which is a process that is used in humans to correct an undersized lower jaw that involves surgically separating the jaw bone, which elicits new bone growth in the gap. We use this model to show that regions of newly formed bone are clonally derived from stem cells that reside in the skeleton. Using chromatin and transcriptional profiling, we show that these stem-cell populations gain activity within the focal adhesion kinase (FAK) signalling pathway, and that inhibiting FAK abolishes new bone formation. Mechanotransduction via FAK in skeletal stem cells during distraction activates a gene-regulatory program and retrotransposons that are normally active in primitive neural crest cells, from which skeletal stem cells arise during development. This reversion to a developmental state underlies the robust tissue growth that facilitates stem-cell-based regeneration of adult skeletal tissue.
Assuntos
Regeneração Óssea , Mandíbula/citologia , Mandíbula/fisiologia , Crista Neural/citologia , Osteogênese por Distração , Células-Tronco/citologia , Animais , Cromatina/genética , Cromatina/metabolismo , Modelos Animais de Doenças , Proteína-Tirosina Quinases de Adesão Focal/antagonistas & inibidores , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Regulação da Expressão Gênica , Masculino , Mandíbula/cirurgia , Camundongos , Camundongos Endogâmicos C57BL , Retroelementos/genética , Transdução de Sinais , Células-Tronco/metabolismo , Transcrição GênicaRESUMO
The skeletal system is a highly complex network of mesenchymal, hematopoietic, and vasculogenic stem cell lineages that coordinate the development and maintenance of defined microenvironments, so-called niches. Technological advancements in recent years have allowed for the dissection of crucial cell types as well as their autocrine and paracrine signals that regulate these niches during development, homeostasis, regeneration, and disease. Ingress of blood vessels and bone marrow hematopoiesis are initiated by skeletal stem cells (SSCs) and their more committed downstream lineage cell types that direct shape and form of skeletal elements. In this chapter, we focus on the role of SSCs as the developmental origin of niches for hematopoietic stem and progenitor cells. We discuss latest updates in the definition of SSCs, cellular processes establishing and maintaining niches, as well as alterations of stem cell microenvironments promoting malignancies. We conclude with an outlook on future studies that could take advantage of SSC-niche engineering as a basis for the development of new therapeutic tools to not only treat bone-related diseases but also maladies stemming from derailed niche dynamics altering hematopoietic output.
Assuntos
Células-Tronco Hematopoéticas , Nicho de Células-Tronco , Medula Óssea , Diferenciação Celular , HematopoeseRESUMO
The postnatal skeleton undergoes growth, remodeling, and repair. We hypothesized that skeletal progenitor cells active during these disparate phases are genetically and phenotypically distinct. We identified a highly potent regenerative cell type that we term the fracture-induced bone, cartilage, stromal progenitor (f-BCSP) in the fracture callus of adult mice. The f-BCSP possesses significantly enhanced skeletogenic potential compared with BCSPs harvested from uninjured bone. It also recapitulates many gene expression patterns involved in perinatal skeletogenesis. Our results indicate that the skeletal progenitor population is functionally stratified, containing distinct subsets responsible for growth, regeneration, and repair. Furthermore, our findings suggest that injury-induced changes to the skeletal stem and progenitor microenvironments could activate these cells and enhance their regenerative potential.
Assuntos
Osso e Ossos/patologia , Fraturas Ósseas/patologia , Células-Tronco/citologia , Animais , Animais Recém-Nascidos , Desenvolvimento Ósseo , Calo Ósseo/citologia , Cartilagem/patologia , Proliferação de Células , Separação Celular , Fêmur/patologia , Perfilação da Expressão Gênica , Membro Posterior/efeitos da radiação , Integrina alfa6/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Osteogênese , Fenótipo , Células Estromais/citologiaRESUMO
Craniofacial surgery, since its inauguration, has been the culmination of collaborative efforts to solve complex congenital, dysplastic, oncological, and traumatic cranial bone defects. Now, 50 years on from the first craniofacial meeting, the collaborative efforts between surgeons, scientists, and bioengineers are further advancing craniofacial surgery with new discoveries in tissue regeneration. Recent advances in regenerative medicine and stem cell biology have transformed the authors' understanding of bone healing, the role of stem cells governing bone healing, and the effects of the niche environment and extracellular matrix on stem cell fate. This review aims at summarizing the advances within each of these fields.
Assuntos
Regeneração Óssea/fisiologia , Regeneração Tecidual Guiada/métodos , Procedimentos de Cirurgia Plástica/métodos , Crânio/cirurgia , Transplante de Células-Tronco/métodos , Humanos , Crânio/fisiologia , Alicerces TeciduaisRESUMO
Organs are composites of tissue types with diverse developmental origins, and they rely on distinct stem and progenitor cells to meet physiological demands for cellular production and homeostasis. How diverse stem cell activity is coordinated within organs is not well understood. Here we describe a lineage-restricted, self-renewing common skeletal progenitor (bone, cartilage, stromal progenitor; BCSP) isolated from limb bones and bone marrow tissue of fetal, neonatal, and adult mice. The BCSP clonally produces chondrocytes (cartilage-forming) and osteogenic (bone-forming) cells and at least three subsets of stromal cells that exhibit differential expression of cell surface markers, including CD105 (or endoglin), Thy1 [or CD90 (cluster of differentiation 90)], and 6C3 [ENPEP glutamyl aminopeptidase (aminopeptidase A)]. These three stromal subsets exhibit differential capacities to support hematopoietic (blood-forming) stem and progenitor cells. Although the 6C3-expressing subset demonstrates functional stem cell niche activity by maintaining primitive hematopoietic stem cell (HSC) renewal in vitro, the other stromal populations promote HSC differentiation to more committed lines of hematopoiesis, such as the B-cell lineage. Gene expression analysis and microscopic studies further reveal a microenvironment in which CD105-, Thy1-, and 6C3-expressing marrow stroma collaborate to provide cytokine signaling to HSCs and more committed hematopoietic progenitors. As a result, within the context of bone as a blood-forming organ, the BCSP plays a critical role in supporting hematopoiesis through its generation of diverse osteogenic and hematopoietic-promoting stroma, including HSC supportive 6C3(+) niche cells.
Assuntos
Osso e Ossos/metabolismo , Cartilagem/metabolismo , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/metabolismo , Transdução de Sinais/fisiologia , Nicho de Células-Tronco/fisiologia , Animais , Antígenos de Diferenciação/biossíntese , Antígenos de Diferenciação/genética , Osso e Ossos/citologia , Cartilagem/citologia , Citocinas/genética , Citocinas/metabolismo , Regulação da Expressão Gênica/fisiologia , Células-Tronco Hematopoéticas/citologia , Camundongos , Camundongos Transgênicos , Células Estromais/citologia , Células Estromais/metabolismoRESUMO
Little is known about the formation of niches, local micro-environments required for stem-cell maintenance. Here we develop an in vivo assay for adult haematopoietic stem-cell (HSC) niche formation. With this assay, we identified a population of progenitor cells with surface markers CD45(-)Tie2(-)alpha(V)(+)CD105(+)Thy1.1(-) (CD105(+)Thy1(-)) that, when sorted from 15.5 days post-coitum fetal bones and transplanted under the adult mouse kidney capsule, could recruit host-derived blood vessels, produce donor-derived ectopic bones through a cartilage intermediate and generate a marrow cavity populated by host-derived long-term reconstituting HSC (LT-HSC). In contrast, CD45(-)Tie2(-)alpha(V)(+)CD105(+)Thy1(+) (CD105(+)Thy1(+)) fetal bone progenitors form bone that does not contain a marrow cavity. Suppressing expression of factors involved in endochondral ossification, such as osterix and vascular endothelial growth factor (VEGF), inhibited niche generation. CD105(+)Thy1(-) progenitor populations derived from regions of the fetal mandible or calvaria that do not undergo endochondral ossification formed only bone without marrow in our assay. Collectively, our data implicate endochondral ossification, bone formation that proceeds through a cartilage intermediate, as a requirement for adult HSC niche formation.
Assuntos
Cartilagem/citologia , Células-Tronco Hematopoéticas/citologia , Osteogênese/fisiologia , Nicho de Células-Tronco/citologia , Nicho de Células-Tronco/fisiologia , Animais , Antígenos CD/metabolismo , Cartilagem/embriologia , Coristoma , Feto/citologia , Células-Tronco Hematopoéticas/metabolismo , Mandíbula/citologia , Mandíbula/embriologia , Camundongos , Camundongos Endogâmicos C57BL , Crânio/citologia , Crânio/embriologia , Fator de Transcrição Sp7 , Antígenos Thy-1/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Pluripotent cells represent a powerful tool for tissue regeneration, but their clinical utility is limited by their propensity to form teratomas. Little is known about their interaction with the surrounding niche following implantation and how this may be applied to promote survival and functional engraftment. In this study, we evaluated the ability of an osteogenic microniche consisting of a hydroxyapatite-coated, bone morphogenetic protein-2-releasing poly-L-lactic acid scaffold placed within the context of a macroenvironmental skeletal defect to guide in vivo differentiation of both embryonic and induced pluripotent stem cells. In this setting, we found de novo bone formation and participation by implanted cells in skeletal regeneration without the formation of a teratoma. This finding suggests that local cues from both the implanted scaffold/cell micro- and surrounding macroniche may act in concert to promote cellular survival and the in vivo acquisition of a terminal cell fate, thereby allowing for functional engraftment of pluripotent cells into regenerating tissue.
Assuntos
Regeneração Óssea , Células-Tronco Pluripotentes/transplante , Animais , Regeneração Óssea/fisiologia , Diferenciação Celular/fisiologia , Sobrevivência Celular/fisiologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/transplante , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/transplante , Masculino , Camundongos , Camundongos Nus , Osteogênese/fisiologia , Células-Tronco Pluripotentes/citologia , Crânio/diagnóstico por imagem , Crânio/lesões , Crânio/patologia , Nicho de Células-Tronco/fisiologia , Teratoma/patologia , Alicerces Teciduais , Transplante Heterólogo , Microtomografia por Raio-XRESUMO
A common genetic mutation found in clear cell renal cell carcinoma (CC-RCC) is the loss of the von Hippel-Lindau (VHL) gene, which results in stabilization of hypoxia-inducible factors (HIFs), and contributes to cancer progression and metastasis. CUB-domain-containing protein 1 (CDCP1) was shown to promote metastasis in scirrhous and lung adenocarcinomas as well as in prostate cancer. In this study, we established a molecular mechanism linking VHL loss to induction of the CDCP1 gene through the HIF-1/2 pathway in renal cancer. Also, we report that Fyn, which forms a complex with CDCP1 and mediates its signaling to PKCδ, is a HIF-1 target gene. Mechanistically, we found that CDCP1 specifically regulates phosphorylation of PKCδ, but not of focal adhesion kinase or Crk-associated substrate. Signal transduction from CDCP1 to PKCδ leads to its activation, increasing migration of CC-RCC. Furthermore, patient survival can be stratified by CDCP1 expression at the cell surface of the tumor. Taken together, our data indicates that CDCP1 protein might serve as a therapeutic target for CC-RCC.
Assuntos
Antígenos CD/fisiologia , Carcinoma de Células Renais/genética , Moléculas de Adesão Celular/fisiologia , Neoplasias Renais/genética , Proteínas de Neoplasias/fisiologia , Proteína Quinase C-delta/metabolismo , Regulação para Cima , Proteína Supressora de Tumor Von Hippel-Lindau/fisiologia , Antígenos CD/genética , Antígenos de Neoplasias , Moléculas de Adesão Celular/genética , Humanos , Mutação , Proteínas de Neoplasias/genética , Fosforilação , Transdução de Sinais , Proteína Supressora de Tumor Von Hippel-Lindau/genéticaRESUMO
In vivo molecular imaging tools are crucially important for elucidating how cells move through complex biological systems; however, achieving single-cell sensitivity over the entire body remains challenging. Here, we report a highly sensitive and multiplexed approach for tracking upward of 20 single cells simultaneously in the same subject using positron emission tomography (PET). The method relies on a statistical tracking algorithm (PEPT-EM) to achieve a sensitivity of 4 becquerel per cell and a streamlined workflow to reliably label single cells with over 50 becquerel per cell of 18F-fluorodeoxyglucose (FDG). To demonstrate the potential of the method, we tracked the fate of more than 70 melanoma cells after intracardiac injection and found they primarily arrested in the small capillaries of the pulmonary, musculoskeletal, and digestive organ systems. This study bolsters the evolving potential of PET in offering unmatched insights into the earliest phases of cell trafficking in physiological and pathological processes and in cell-based therapies.
Assuntos
Rastreamento de Células , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Análise de Célula Única , Imagem Corporal Total , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Animais , Análise de Célula Única/métodos , Rastreamento de Células/métodos , Imagem Corporal Total/métodos , Camundongos , Humanos , Fluordesoxiglucose F18 , Linhagem Celular Tumoral , Algoritmos , Melanoma/diagnóstico por imagem , Melanoma/patologiaRESUMO
In vivo molecular imaging tools are crucially important for elucidating how cells move through complex biological systems, however, achieving single-cell sensitivity over the entire body remains challenging. Here, we report a highly sensitive and multiplexed approach for tracking upwards of 20 single cells simultaneously in the same subject using positron emission tomography (PET). The method relies on a new tracking algorithm (PEPT-EM) to push the cellular detection threshold to below 4 Bq/cell, and a streamlined workflow to reliably label single cells with over 50 Bq/cell of 18F-fluorodeoxyglucose (FDG). To demonstrate the potential of method, we tracked the fate of over 70 melanoma cells after intracardiac injection and found they primarily arrested in the small capillaries of the pulmonary, musculoskeletal, and digestive organ systems. This study bolsters the evolving potential of PET in offering unmatched insights into the earliest phases of cell trafficking in physiological and pathological processes and in cell-based therapies.
RESUMO
Bone- and cartilage-related diseases, such as osteoporosis and osteoarthritis, affect millions of people worldwide, impairing their quality of life and increasing mortality. Osteoporosis significantly increases the bone fracture risk of the spine, hip, and wrist. For successful fracture treatment and to facilitate proper healing in the most complicated cases, one of the most promising methods is to deliver a therapeutic protein to accelerate bone regeneration. Similarly, in the setting of osteoarthritis, where degraded cartilage does not regenerate, therapeutic proteins hold great promise to promote new cartilage formation. For both osteoporosis and osteoarthritis treatments, targeted delivery of therapeutic growth factors, with the aid of hydrogels, to bone and cartilage is a key to advance the field of regenerative medicine. In this review article, we propose five important aspects of therapeutic growth factor delivery for bone and cartilage regeneration: (1) protection of protein growth factors from physical and enzymatic degradation, (2) targeted growth factor delivery, (3) controlling GF release kinetics, (4) long-term stability of regenerated tissues, and (5) osteoimmunomodulatory effects of therapeutic growth factors and carriers/scaffolds.
RESUMO
Mandibular distraction osteogenesis (DO) is mediated by skeletal stem cells (SSCs) in mice, which enact bone regeneration via neural crest re-activation. As peripheral nerves are essential to progenitor function during development and in response to injury, we questioned if denervation impairs mandibular DO. C57Bl6 mice were divided into two groups: DO with a segmental defect in the inferior alveolar nerve (IAN) at the time of mandibular osteotomy ("DO Den") and DO with IAN intact ("DO Inn"). DO Den demonstrated significantly reduced histological and radiological osteogenesis relative to DO Inn. Denervation preceding DO results in reduced SSC amplification and osteogenic potential in mice. Single cell RNA sequencing analysis revealed that there was a predominance of innervated SSCs in clusters dominated by pathways related to bone formation. A rare human patient specimen was also analyzed and suggested that histological, radiological, and transcriptional alterations seen in mouse DO may be conserved in the setting of denervated human mandible distraction. Fibromodulin (FMOD) transcriptional and protein expression were reduced in denervated relative to innervated mouse and human mandible regenerate. Finally, when exogenous FMOD was added to DO-Den and DO-Inn SSCs undergoing in vitro osteogenic differentiation, the osteogenic potential of DO-Den SSCs was increased in comparison to control untreated DO-Den SSCs, modeling the superior osteogenic potential of DO-Inn SSCs.
Assuntos
Osteogênese por Distração , Osteogênese , Humanos , Animais , Camundongos , Osteogênese/genética , Camundongos Endogâmicos C57BL , Mandíbula/fisiologia , Regeneração Óssea , Denervação , FibromodulinaRESUMO
Failure to properly form bone or integrate surgical implants can lead to morbidity and additional surgical interventions in a significant proportion of orthopedic surgeries. While the role of skeletal stem cells (SSCs) in bone formation and repair is well-established, very little is known about the factors that regulate the downstream Bone, Cartilage, Stromal, Progenitors (BCSPs). BCSPs, as transit amplifying progenitor cells, undergo multiple mitotic divisions to expand the pool of lineage committed progenitors allowing stem cells to preserve their self-renewal and stemness. Del1 is a protein widely expressed in the skeletal system, but its deletion led to minimal phenotype changes in the uninjured mouse. In this paper, we demonstrate that Del1 is a key regulator of BCSP expansion following injury. In Del1 knockout mice, there is a significant reduction in the number of BCSPs which leads to a smaller callus and decreased bone formation compared with wildtype (WT) littermates. Del1 serves to promote BCSP proliferation and prevent apoptosis in vivo and in vitro. Moreover, exogenous Del1 promotes proliferation of aged human BCSPs. Our results highlight the potential of Del1 as a therapeutic target for improving bone formation and implant success. Del1 injections may improve the success of orthopedic surgeries and fracture healing by enhancing the proliferation and survival of BCSPs, which are crucial for generating new bone tissue during the process of bone formation and repair.
Assuntos
Osso e Ossos , Osteogênese , Humanos , Animais , Camundongos , Idoso , Consolidação da Fratura , Peptídeos e Proteínas de Sinalização Intercelular , Apoptose , Camundongos KnockoutRESUMO
In lactating mothers, the high calcium (Ca 2+ ) demand for milk production triggers significant bone resorption. While estrogen would normally counteract excessive bone loss and maintain sufficient bone formation during this postpartum period, this sex steroid drops precipitously after giving birth. Here, we report that brain-derived CCN3 (Cellular Communication Network factor 3) secreted from KISS1 neurons of the arcuate nucleus (ARC KISS1 ) fills this void and functions as a potent osteoanabolic factor to promote bone mass in lactating females. Using parabiosis and bone transplant methods, we first established that a humoral factor accounts for the female-specific, high bone mass previously observed by our group after deleting estrogen receptor alpha (ER α ) from ARC KISS1 neurons 1 . This exceptional bone phenotype in mutant females can be traced back to skeletal stem cells (SSCs), as reflected by their increased frequency and osteochondrogenic potential. Based on multiple assays, CCN3 emerged as the most promising secreted pro-osteogenic factor from ARC KISS1 neurons, acting on mouse and human SSCs at low subnanomolar concentrations independent of age or sex. That brain-derived CCN3 promotes bone formation was further confirmed by in vivo gain- and loss-of-function studies. Notably, a transient rise in CCN3 appears in ARC KISS1 neurons in estrogen-depleted lactating females coincident with increased bone remodeling and high calcium demand. Our findings establish CCN3 as a potentially new therapeutic osteoanabolic hormone that defines a novel female-specific brain-bone axis for ensuring mammalian species survival.
RESUMO
Human skeletal stem cells (hSSCs) hold tremendous therapeutic potential for developing new clinical strategies to effectively combat congenital and age-related musculoskeletal disorders. Unfortunately, refined methodologies for the proper isolation of bona fide hSSCs and the development of functional assays that accurately recapitulate their physiology within the skeleton have been lacking. Bone marrow-derived mesenchymal stromal cells (BMSCs), commonly used to describe the source of precursors for osteoblasts, chondrocytes, adipocytes and stroma, have held great promise as the basis of various approaches for cell therapy. However, the reproducibility and clinical efficacy of these attempts have been obscured by the heterogeneous nature of BMSCs due to their isolation by plastic adherence techniques. To address these limitations, our group has refined the purity of individual progenitor populations that are encompassed by BMSCs by identifying defined populations of bona fide hSSCs and their downstream progenitors that strictly give rise to skeletally restricted cell lineages. Here, we describe an advanced flow cytometric approach that utilizes an extensive panel of eight cell surface markers to define hSSCs; bone, cartilage and stromal progenitors; and more differentiated unipotent subtypes, including an osteogenic subset and three chondroprogenitors. We provide detailed instructions for the FACS-based isolation of hSSCs from various tissue sources, in vitro and in vivo skeletogenic functional assays, human xenograft mouse models and single-cell RNA sequencing analysis. This application of hSSC isolation can be performed by any researcher with basic skills in biology and flow cytometry within 1-2 days. The downstream functional assays can be performed within a range of 1-2 months.