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1.
PLoS Biol ; 20(9): e3001563, 2022 09.
Artículo en Inglés | MEDLINE | ID: mdl-36067211

RESUMEN

The development of stable specialized cell types in multicellular organisms relies on mechanisms controlling inductive intercellular signals and the competence of cells to respond to such signals. In developing cerebral cortex, progenitors generate only glutamatergic excitatory neurons despite being exposed to signals with the potential to initiate the production of other neuronal types, suggesting that their competence is limited. Here, we tested the hypothesis that this limitation is due to their expression of transcription factor Pax6. We used bulk and single-cell RNAseq to show that conditional cortex-specific Pax6 deletion from the onset of cortical neurogenesis allowed some progenitors to generate abnormal lineages resembling those normally found outside the cortex. Analysis of selected gene expression showed that the changes occurred in specific spatiotemporal patterns. We then compared the responses of control and Pax6-deleted cortical cells to in vivo and in vitro manipulations of extracellular signals. We found that Pax6 loss increased cortical progenitors' competence to generate inappropriate lineages in response to extracellular factors normally present in developing cortex, including the morphogens Shh and Bmp4. Regional variation in the levels of these factors could explain spatiotemporal patterns of fate change following Pax6 deletion in vivo. We propose that Pax6's main role in developing cortical cells is to minimize the risk of their development being derailed by the potential side effects of morphogens engaged contemporaneously in other essential functions.


Asunto(s)
Proteínas de Homeodominio , Factores de Transcripción Paired Box , Corteza Cerebral/metabolismo , Proteínas del Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Factor de Transcripción PAX6/genética , Factor de Transcripción PAX6/metabolismo , Factores de Transcripción Paired Box/genética , Factores de Transcripción Paired Box/metabolismo , Proteínas Represoras/metabolismo
2.
Blood ; 136(25): 2893-2904, 2020 12 17.
Artículo en Inglés | MEDLINE | ID: mdl-32614947

RESUMEN

Hematopoietic stem and progenitor cells (HSPCs) develop in distinct waves at various anatomical sites during embryonic development. The in vitro differentiation of human pluripotent stem cells (hPSCs) recapitulates some of these processes; however, it has proven difficult to generate functional hematopoietic stem cells (HSCs). To define the dynamics and heterogeneity of HSPCs that can be generated in vitro from hPSCs, we explored single-cell RNA sequencing (scRNAseq) in combination with single-cell protein expression analysis. Bioinformatics analyses and functional validation defined the transcriptomes of naïve progenitors and erythroid-, megakaryocyte-, and leukocyte-committed progenitors, and we identified CD44, CD326, ICAM2/CD9, and CD18, respectively, as markers of these progenitors. Using an artificial neural network that we trained on scRNAseq derived from human fetal liver, we identified a wide range of hPSC-derived HSPCs phenotypes, including a small group classified as HSCs. This transient HSC-like population decreased as differentiation proceeded, and was completely missing in the data set that had been generated using cells selected on the basis of CD43 expression. By comparing the single-cell transcriptome of in vitro-generated HSC-like cells with those generated within the fetal liver, we identified transcription factors and molecular pathways that can be explored in the future to improve the in vitro production of HSCs.


Asunto(s)
Antígenos de Diferenciación , Células Madre Hematopoyéticas , Aprendizaje Automático , Células Madre Pluripotentes , RNA-Seq , Análisis de la Célula Individual , Antígenos de Diferenciación/biosíntesis , Antígenos de Diferenciación/genética , Feto/citología , Feto/metabolismo , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Hígado/citología , Hígado/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo
3.
Eur Heart J ; 41(9): 1024-1036, 2020 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-31242503

RESUMEN

AIMS: Pluripotent stem cell-derived endothelial cell products possess therapeutic potential in ischaemic vascular disease. However, the factors that drive endothelial differentiation from pluripotency and cellular specification are largely unknown. The aims of this study were to use single-cell RNA sequencing (scRNA-seq) to map the transcriptional landscape and cellular dynamics of directed differentiation of human embryonic stem cell-derived endothelial cells (hESC-EC) and to compare these cells to mature endothelial cells from diverse vascular beds. METHODS AND RESULTS: A highly efficient directed 8-day differentiation protocol was used to generate a hESC-derived endothelial cell product (hESC-ECP), in which 66% of cells co-expressed CD31 and CD144. We observed largely homogeneous hESC and mesodermal populations at Days 0 and 4, respectively, followed by a rapid emergence of distinct endothelial and mesenchymal populations. Pseudotime trajectory identified transcriptional signatures of endothelial commitment and maturation during the differentiation process. Concordance in transcriptional signatures was verified by scRNA-seq analysis using both a second hESC line RC11, and an alternative hESC-EC differentiation protocol. In total, 105 727 cells were subjected to scRNA-seq analysis. Global transcriptional comparison revealed a transcriptional architecture of hESC-EC that differs from freshly isolated and cultured human endothelial cells and from organ-specific endothelial cells. CONCLUSION: A transcriptional bifurcation into endothelial and mesenchymal lineages was identified, as well as novel transcriptional signatures underpinning commitment and maturation. The transcriptional architecture of hESC-ECP was distinct from mature and foetal human EC.


Asunto(s)
Células Endoteliales , Células Madre Pluripotentes , Diferenciación Celular , Células Madre Embrionarias , Humanos , Análisis de Secuencia de ARN
4.
Eur Heart J ; 40(30): 2507-2520, 2019 08 07.
Artículo en Inglés | MEDLINE | ID: mdl-31162546

RESUMEN

AIMS: A better understanding of the pathways that regulate regeneration of the coronary vasculature is of fundamental importance for the advancement of strategies to treat patients with heart disease. Here, we aimed to investigate the origin and clonal dynamics of endothelial cells (ECs) associated with neovascularization in the adult mouse heart following myocardial infarction (MI). Furthermore, we sought to define murine cardiac endothelial heterogeneity and to characterize the transcriptional profiles of pro-angiogenic resident ECs in the adult mouse heart, at single-cell resolution. METHODS AND RESULTS: An EC-specific multispectral lineage-tracing mouse (Pdgfb-iCreERT2-R26R-Brainbow2.1) was used to demonstrate that structural integrity of adult cardiac endothelium following MI was maintained through clonal proliferation by resident ECs in the infarct border region, without significant contributions from bone marrow cells or endothelial-to-mesenchymal transition. Ten transcriptionally discrete heterogeneous EC states, as well as the pathways through which each endothelial state is likely to enhance neovasculogenesis and tissue regeneration following ischaemic injury were defined. Plasmalemma vesicle-associated protein (Plvap) was selected for further study, which showed an endothelial-specific and increased expression in both the ischaemic mouse and human heart, and played a direct role in regulating human endothelial proliferation in vitro. CONCLUSION: We present a single-cell gene expression atlas of cardiac specific resident ECs, and the transcriptional hierarchy underpinning endogenous vascular repair following MI. These data provide a rich resource that could assist in the development of new therapeutic interventions to augment endogenous myocardial perfusion and enhance regeneration in the injured heart.


Asunto(s)
Perfilación de la Expresión Génica/métodos , Infarto del Miocardio/metabolismo , Neovascularización Fisiológica/genética , Análisis de la Célula Individual/métodos , Transcriptoma/genética , Animales , Proliferación Celular/genética , Células Endoteliales/citología , Células Endoteliales/metabolismo , Humanos , Ratones , Ratones Transgénicos , Infarto del Miocardio/patología , Miocardio/metabolismo , Miocardio/patología
5.
BMC Musculoskelet Disord ; 18(1): 253, 2017 Jun 12.
Artículo en Inglés | MEDLINE | ID: mdl-28606129

RESUMEN

BACKGROUND: Heightened local inflammation due to muscle trauma or disease is associated with impaired bone regeneration. METHODS: We hypothesized that FK506, an FDA approved immunomodulatory compound with neurotrophic and osteogenic effects, will rescue the early phase of fracture healing which is impaired by concomitant muscle trauma in male (~4 months old) Lewis rats. FK506 (1 mg/kg; i.p.) or saline was administered systemically for 14 days after an endogenously healing tibia osteotomy was created and fixed with an intermedullary pin, and the overlying tibialis anterior (TA) muscle was either left uninjured or incurred volumetric muscle loss injury (6 mm full thickness biopsy from middle third of the muscle). RESULTS: The salient observations of this study were that 1) concomitant TA muscle trauma impaired recovery of tibia mechanical properties 28 days post-injury, 2) FK506 administration rescued the recovery of tibia mechanical properties in the presence of concomitant TA muscle trauma but did not augment mechanical recovery of an isolated osteotomy (no muscle trauma), 3) T lymphocytes and macrophage presence within the traumatized musculature were heightened by trauma and attenuated by FK506 3 days post-injury, and 4) T lymphocyte but not macrophage presence within the fracture callus were attenuated by FK506 at 14 days post-injury. FK506 did not improve TA muscle isometric torque production CONCLUSION: Collectively, these findings support the administration of FK506 to ameliorate healing of fractures with severe muscle trauma comorbidity. The results suggest one potential mechanism of action is a reduction in local T lymphocytes within the injured musculoskeletal tissue, though other mechanisms to include direct osteogenic effects of FK506 require further investigation.


Asunto(s)
Regeneración Ósea/efectos de los fármacos , Curación de Fractura/efectos de los fármacos , Inmunosupresores/uso terapéutico , Músculo Esquelético/lesiones , Tacrolimus/uso terapéutico , Fracturas de la Tibia/tratamiento farmacológico , Inmunidad Adaptativa/efectos de los fármacos , Animales , Biopsia , Clavos Ortopédicos , Callo Óseo/efectos de los fármacos , Callo Óseo/inmunología , Callo Óseo/patología , Modelos Animales de Enfermedad , Fijación Intramedular de Fracturas/instrumentación , Fijación Intramedular de Fracturas/métodos , Humanos , Inmunidad Innata/efectos de los fármacos , Inmunosupresores/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Masculino , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Enfermedades Musculares/complicaciones , Enfermedades Musculares/tratamiento farmacológico , Enfermedades Musculares/inmunología , Enfermedades Musculares/patología , Osteotomía , Ratas , Ratas Endogámicas Lew , Traumatismos de los Tejidos Blandos/complicaciones , Traumatismos de los Tejidos Blandos/tratamiento farmacológico , Traumatismos de los Tejidos Blandos/inmunología , Traumatismos de los Tejidos Blandos/patología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Tacrolimus/farmacología , Fracturas de la Tibia/fisiopatología , Fracturas de la Tibia/cirugía , Torque
6.
G3 (Bethesda) ; 13(10)2023 09 30.
Artículo en Inglés | MEDLINE | ID: mdl-37548242

RESUMEN

It is estimated that animals pollinate 87.5% of flowering plants worldwide and that managed honey bees (Apis mellifera) account for 30-50% of this ecosystem service to agriculture. In addition to their important role as pollinators, honey bees are well-established insect models for studying learning and memory, behavior, caste differentiation, epigenetic mechanisms, olfactory biology, sex determination, and eusociality. Despite their importance to agriculture, knowledge of honey bee biology lags behind many other livestock species. In this study, we have used scRNA-Seq to map cell types to different developmental stages of the worker honey bee (prepupa at day 11 and pupa at day 15) and sought to determine their gene expression signatures. To identify cell-type populations, we examined the cell-to-cell network based on the similarity of the single-cells transcriptomic profiles. Grouping similar cells together we identified 63 different cell clusters of which 17 clusters were identifiable at both stages. To determine genes associated with specific cell populations or with a particular biological process involved in honey bee development, we used gene coexpression analysis. We combined this analysis with literature mining, the honey bee protein atlas, and gene ontology analysis to determine cell cluster identity. Of the cell clusters identified, 17 were related to the nervous system and sensory organs, 7 to the fat body, 19 to the cuticle, 5 to muscle, 4 to compound eye, 2 to midgut, 2 to hemocytes, and 1 to malpighian tubule/pericardial nephrocyte. To our knowledge, this is the first whole single-cell atlas of honey bees at any stage of development and demonstrates the potential for further work to investigate their biology at the cellular level.


Asunto(s)
Ecosistema , Transcriptoma , Abejas/genética , Animales , Pupa/genética
7.
J Invest Dermatol ; 142(1): 114-123.e8, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-34274346

RESUMEN

Dupuytren's disease (DD) is a common, progressive fibroproliferative disease affecting the palmar fascia of the hands, causing fingers to irreversibly flex toward the palm with significant loss of function. Surgical treatments are limited; therefore, effective new therapies for DD are urgently required. To identify the key cellular and molecular pathways driving DD, we employed single-cell RNA sequencing, profiling the transcriptomes of 35,250 human single cells from DD, nonpathogenic fascia, and healthy dermis. We identify a DD-specific population of pathogenic PDPN+/FAP+ mesenchymal cells displaying an elevated expression of fibrillar collagens and profibrogenic genes. In silico trajectory analysis reveals resident fibroblasts to be the source of this pathogenic population. To resolve the processes governing DD progression, genes differentially expressed during fibroblast differentiation were identified, including upregulated TNFRSF12A and transcription factor SCX. Knockdown of SCX and blockade of TNFRSF12A inhibited the proliferation and altered the profibrotic gene expression of cultured human FAP+ mesenchymal cells, demonstrating a functional role for these genes in DD. The power of single-cell RNA sequencing is utilized to identify the major pathogenic mesenchymal subpopulations driving DD and the key molecular pathways regulating the DD-specific myofibroblast phenotype. Using this precision medicine approach, inhibition of TNFRSF12A has shown potential clinical utility in the treatment of DD.


Asunto(s)
Dermis/fisiología , Contractura de Dupuytren/genética , Fibroblastos/fisiología , Células Madre Mesenquimatosas/fisiología , Miofibrillas/patología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Endopeptidasas/metabolismo , Fibrosis/genética , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Receptor de TWEAK/genética , Receptor de TWEAK/metabolismo
8.
Cardiovasc Res ; 118(11): 2519-2534, 2022 08 24.
Artículo en Inglés | MEDLINE | ID: mdl-34528097

RESUMEN

AIMS: Endothelial cell (EC) dysfunction drives the initiation and pathogenesis of pulmonary arterial hypertension (PAH). We aimed to characterize EC dynamics in PAH at single-cell resolution. METHODS AND RESULTS: We carried out single-cell RNA sequencing (scRNA-seq) of lung ECs isolated from an EC lineage-tracing mouse model in Control and SU5416/hypoxia-induced PAH conditions. EC populations corresponding to distinct lung vessel types, including two discrete capillary populations, were identified in both Control and PAH mice. Differential gene expression analysis revealed global PAH-induced EC changes that were confirmed by bulk RNA-seq. This included upregulation of the major histocompatibility complex class II pathway, supporting a role for ECs in the inflammatory response in PAH. We also identified a PAH response specific to the second capillary EC population including upregulation of genes involved in cell death, cell motility, and angiogenesis. Interestingly, four genes with genetic variants associated with PAH were dysregulated in mouse ECs in PAH. To compare relevance across PAH models and species, we performed a detailed analysis of EC heterogeneity and response to PAH in rats and humans through whole-lung PAH scRNA-seq datasets, revealing that 51% of up-regulated mouse genes were also up-regulated in rat or human PAH. We identified promising new candidates to target endothelial dysfunction including CD74, the knockdown of which regulates EC proliferation and barrier integrity in vitro. Finally, with an in silico cell ordering approach, we identified zonation-dependent changes across the arteriovenous axis in mouse PAH and showed upregulation of the Serine/threonine-protein kinase Sgk1 at the junction between the macro- and microvasculature. CONCLUSION: This study uncovers PAH-induced EC transcriptomic changes at a high resolution, revealing novel targets for potential therapeutic candidate development.


Asunto(s)
Hipertensión Pulmonar , Hipertensión Arterial Pulmonar , Animales , Células Endoteliales/metabolismo , Hipertensión Pulmonar Primaria Familiar/metabolismo , Humanos , Ratones , Hipertensión Arterial Pulmonar/genética , Arteria Pulmonar , Ratas , Análisis de Secuencia de ARN
9.
Dev Cell ; 56(11): 1617-1630.e6, 2021 06 07.
Artículo en Inglés | MEDLINE | ID: mdl-34033756

RESUMEN

Central nervous system injury re-initiates neurogenesis in anamniotes (amphibians and fishes), but not in mammals. Activation of the innate immune system promotes regenerative neurogenesis, but it is fundamentally unknown whether this is indirect through the activation of known developmental signaling pathways or whether immune cells directly signal to progenitor cells using mechanisms that are unique to regeneration. Using single-cell RNA-seq of progenitor cells and macrophages, as well as cell-type-specific manipulations, we provide evidence for a direct signaling axis from specific lesion-activated macrophages to spinal progenitor cells to promote regenerative neurogenesis in zebrafish. Mechanistically, TNFa from pro-regenerative macrophages induces Tnfrsf1a-mediated AP-1 activity in progenitors to increase regeneration-promoting expression of hdac1 and neurogenesis. This establishes the principle that macrophages directly communicate to spinal progenitor cells via non-developmental signals after injury, providing potential targets for future interventions in the regeneration-deficient spinal cord of mammals.


Asunto(s)
Histona Desacetilasa 1/genética , Neurogénesis/genética , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Regeneración/genética , Médula Espinal/crecimiento & desarrollo , Proteínas de Pez Cebra/genética , Animales , Linaje de la Célula/genética , Regulación del Desarrollo de la Expresión Génica/genética , Macrófagos/citología , Macrófagos/metabolismo , RNA-Seq , Transducción de Señal/genética , Análisis de la Célula Individual , Médula Espinal/metabolismo , Células Madre/citología , Células Madre/metabolismo , Factor de Transcripción AP-1/genética , Pez Cebra/genética
10.
Cell Rep ; 29(7): 1832-1847.e8, 2019 11 12.
Artículo en Inglés | MEDLINE | ID: mdl-31722201

RESUMEN

Iterative liver injury results in progressive fibrosis disrupting hepatic architecture, regeneration potential, and liver function. Hepatic stellate cells (HSCs) are a major source of pathological matrix during fibrosis and are thought to be a functionally homogeneous population. Here, we use single-cell RNA sequencing to deconvolve the hepatic mesenchyme in healthy and fibrotic mouse liver, revealing spatial zonation of HSCs across the hepatic lobule. Furthermore, we show that HSCs partition into topographically diametric lobule regions, designated portal vein-associated HSCs (PaHSCs) and central vein-associated HSCs (CaHSCs). Importantly we uncover functional zonation, identifying CaHSCs as the dominant pathogenic collagen-producing cells in a mouse model of centrilobular fibrosis. Finally, we identify LPAR1 as a therapeutic target on collagen-producing CaHSCs, demonstrating that blockade of LPAR1 inhibits liver fibrosis in a rodent NASH model. Taken together, our work illustrates the power of single-cell transcriptomics to resolve the key collagen-producing cells driving liver fibrosis with high precision.


Asunto(s)
Células Estrelladas Hepáticas/metabolismo , Cirrosis Hepática/metabolismo , Análisis de la Célula Individual , Transcriptoma , Animales , Modelos Animales de Enfermedad , Células Estrelladas Hepáticas/patología , Humanos , Cirrosis Hepática/genética , Cirrosis Hepática/patología , Ratones , Ratones Transgénicos , Ratas , Ratas Wistar , Receptores del Ácido Lisofosfatídico/genética , Receptores del Ácido Lisofosfatídico/metabolismo
11.
PLoS One ; 13(1): e0191245, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29329332

RESUMEN

Minced muscle autografting mediates de novo myofiber regeneration and promotes partial recovery of neuromuscular strength after volumetric muscle loss injury (VML). A major limitation of this approach is the availability of sufficient donor tissue for the treatment of relatively large VMLs without inducing donor site morbidity. This study evaluated a laminin-111 supplemented hyaluronic acid based hydrogel (HA+LMN) as a putative myoconductive scaffolding to be co-delivered with minced muscle grafts. In a rat tibialis anterior muscle VML model, delivery of a reduced dose of minced muscle graft (50% of VML defect) within HA+LMN resulted in a 42% improvement of peak tetanic torque production over unrepaired VML affected limbs. However, the improvement in strength was not improved compared to a 50% minced graft-only control group. Moreover, histological analysis revealed that the improvement in in vivo functional capacity mediated by minced grafts in HA+LMN was not accompanied by a particularly robust graft mediated regenerative response as determined through donor cell tracking of the GFP+ grafting material. Characterization of the spatial distribution and density of macrophage and satellite cell populations indicated that the combination therapy damps the heightened macrophage response while re-establishing satellite content 14 days after VML to a level consistent with an endogenously healing ischemia-reperfusion induced muscle injury. Moreover, regional analysis revealed that the combination therapy increased satellite cell density mostly in the remaining musculature, as opposed to the defect area. Based on the results, the following salient conclusions were drawn: 1) functional recovery mediated by the combination therapy is likely due to a superposition of de novo muscle fiber regeneration and augmented repair of muscle fibers within the remaining musculature, and 2) The capacity for VML therapies to augment regeneration and repair within the remaining musculature may have significant clinical impact and warrants further exploration.


Asunto(s)
Ácido Hialurónico/administración & dosificación , Laminina/administración & dosificación , Músculo Esquelético/lesiones , Músculo Esquelético/trasplante , Animales , Modelos Animales de Enfermedad , Hidrogeles , Masculino , Fuerza Muscular , Músculo Esquelético/fisiología , Isoformas de Proteínas/administración & dosificación , Ratas , Ratas Endogámicas Lew , Regeneración/efectos de los fármacos , Regeneración/fisiología , Daño por Reperfusión/terapia , Andamios del Tejido/química , Trasplante Autólogo
12.
Stem Cell Res Ther ; 8(1): 142, 2017 06 09.
Artículo en Inglés | MEDLINE | ID: mdl-28599679

RESUMEN

BACKGROUND: The delivery of alternative myogenic cell sources to enhance the efficacy of minced muscle grafts (MG) for the treatment of volumetric muscle loss (VML) injuries is a promising strategy to overcome the demand on muscle-derived donor tissue that currently limits the translation of this therapy. METHODS: Using a rat model of VML, bone marrow mononuclear cells (BMNCs) were evaluated for their ability to directly contribute to de novo muscle fiber regeneration by transplanting MG in a collagen carrier at a dose of 50% of the VML injury both with and without concomitant delivery of 5 million BMNCs derived via density gradient centrifugation from the bone marrow of a syngeneic green fluorescent protein (GFP)+ donor. RESULTS: Histological, molecular, and functional analyses revealed that BMNCs can engraft with co-delivered MG and contribute to nascent myofiber, but do so at a low magnitude without resulting in significant changes to transcription of key myogenic genes or gains in whole muscle force generation relative to MG alone. CONCLUSION: As such, co-delivery of BMNCs with MG is a promising treatment paradigm to VML that will require further investigation to identify the phenotype and therapeutic dosing of the bone marrow-derived cell populations which engraft most efficiently.


Asunto(s)
Trasplante de Médula Ósea , Músculo Esquelético/trasplante , Enfermedades Musculares/terapia , Regeneración/genética , Animales , Terapia Combinada , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Humanos , Masculino , Desarrollo de Músculos/genética , Músculo Esquelético/patología , Enfermedades Musculares/patología , Ratas
13.
Physiol Rep ; 5(7)2017 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-28400501

RESUMEN

Volumetric muscle injury (VML) causes an irrecoverable loss of muscle fibers, persistent strength deficits, and chronic disability. A crucial challenge to VML injury and possible regeneration is the removal of all of the in situ native elements necessary for skeletal muscle regeneration. Our first goal was to establish a reliable VML model in the mouse tibialis anterior (TA) muscle. In adult male wild-type and nude mice, a non-repaired ≈20% VML injury to the TA muscle resulted in an ≈59% loss in nerve evoked muscle strength, ≈33% loss in muscle mass, and ≈29% loss of muscle fibers at 28 day post-injury. Our second goal was to investigate if minced muscle grafts (≈1 mm3 tissue fragments) promote recovery of muscle fibers after VML injury and to understand if the graft-derived progenitor cells directly contribute to fiber regeneration. To assess donor cell contribution, donor muscle tissue was derived from UBC-GFP mice in a subset of experiments. Minced grafts restored ≈34% of the lost fibers 28 days post-injury. The number of GFP+ fibers and the estimated number of regenerated fibers were similar, regardless of host mouse strain. The muscle tissue regeneration promoted by minced grafts did not improve TA muscle strength at this time post-injury. These findings demonstrate the direct contribution of minced muscle graft-derived myogenic stem/progenitor cells to recovery of muscle fibers after VML injury and signify the utility of autologous myogenic stem cell therapies for this indication.


Asunto(s)
Fibras Musculares Esqueléticas/trasplante , Músculo Esquelético/lesiones , Enfermedades Musculares/fisiopatología , Células Madre , Animales , Modelos Animales de Enfermedad , Ratones , Ratones Desnudos , Fibras Musculares Esqueléticas/fisiología , Recuperación de la Función/fisiología , Regeneración/fisiología
14.
Physiol Rep ; 5(14)2017 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-28747511

RESUMEN

The deleterious impact of concomitant muscle injury on fracture healing and limb function is commonly considered part of the natural sequela of orthopedic trauma. Recent reports suggest that heightened inflammation in the surrounding traumatized musculature is a primary determinant of fracture healing. Relatedly, there are emerging potential therapeutic approaches for severe muscle trauma (e.g., volumetric muscle loss [VML] injury), such as autologous minced muscle grafts (1 mm3 pieces of muscle; GRAFT), that can partially prevent chronic functional deficits and appear to have an immunomodulatory effect within VML injured muscle. The primary goal of this study was to determine if repair of VML injury with GRAFT rescues impaired fracture healing and improves the strength of the traumatized muscle in a male Lewis rat model of tibia open fracture. The most salient findings of the study were: (1) tibialis anterior (TA) muscle repair with GRAFT improved endogenous healing of fractured tibia and improved the functional outcome of muscle regeneration; (2) GRAFT repair attenuated the monocyte/macrophage (CD45+CDllb+) and T lymphocyte (CD3+) response to VML injury; (3) TA muscle protein concentrations of MCP1, IL-10, and IGF-1 were augmented in a proregenerative manner by GRAFT repair; (4) VML injury concomitant with osteotomy induced a heightened systemic presence of alarmins (e.g., soluble RAGE) and leukocytes (e.g., monocytes), and depressed IGF-1 concentration, which GRAFT repair ameliorated. Collectively, these data indicate that repair of VML injury with a regenerative therapy can modulate the inflammatory and regenerative phenotype of the treated muscle and in association improve musculoskeletal healing.


Asunto(s)
Curación de Fractura , Fuerza Muscular , Músculo Esquelético/trasplante , Fracturas de la Tibia/terapia , Trasplante de Tejidos/métodos , Alarminas/genética , Alarminas/metabolismo , Animales , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Masculino , Músculo Esquelético/lesiones , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiología , Ratas , Ratas Endogámicas Lew , Trasplante Autólogo
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