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1.
Nat Protoc ; 18(7): 2256-2282, 2023 07.
Artículo en Inglés | MEDLINE | ID: mdl-37316563

RESUMEN

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.


Asunto(s)
Células Madre Mesenquimatosas , Humanos , Ratones , Animales , Linaje de la Célula , Reproducibilidad de los Resultados , Diferenciación Celular/fisiología , Huesos , Células de la Médula Ósea , Células Cultivadas
2.
Arterioscler Thromb Vasc Biol ; 43(7): 1262-1277, 2023 07.
Artículo en Inglés | MEDLINE | ID: mdl-37051932

RESUMEN

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.


Asunto(s)
Células Madre Mesenquimatosas , Neovascularización Fisiológica , Ratones , Humanos , Animales , Neovascularización Fisiológica/fisiología , Tejido Adiposo , Neovascularización Patológica , Isquemia/terapia , Modelos Animales de Enfermedad , Miembro Posterior/irrigación sanguínea
3.
Elife ; 102021 07 19.
Artículo en Inglés | MEDLINE | ID: mdl-34280086

RESUMEN

Skeletal stem and progenitor cell populations are crucial for bone physiology. Characterization of these cell types remains restricted to heterogenous bulk populations with limited information on whether they are unique or overlap with previously characterized cell types. Here we show, through comprehensive functional and single-cell transcriptomic analyses, that postnatal long bones of mice contain at least two types of bone progenitors with bona fide skeletal stem cell (SSC) characteristics. An early osteochondral SSC (ocSSC) facilitates long bone growth and repair, while a second type, a perivascular SSC (pvSSC), co-emerges with long bone marrow and contributes to shape the hematopoietic stem cell niche and regenerative demand. We establish that pvSSCs, but not ocSSCs, are the origin of bone marrow adipose tissue. Lastly, we also provide insight into residual SSC heterogeneity as well as potential crosstalk between the two spatially distinct cell populations. These findings comprehensively address previously unappreciated shortcomings of SSC research.


Asunto(s)
Desarrollo Óseo , Huesos/metabolismo , Células del Estroma/metabolismo , Tejido Adiposo , Animales , Médula Ósea , Células de la Médula Ósea , Regulación del Desarrollo de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Pericitos , Nicho de Células Madre , Transcriptoma
4.
Nat Med ; 26(10): 1583-1592, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32807933

RESUMEN

Osteoarthritis (OA) is a degenerative disease resulting in irreversible, progressive destruction of articular cartilage1. The etiology of OA is complex and involves a variety of factors, including genetic predisposition, acute injury and chronic inflammation2-4. Here we investigate the ability of resident skeletal stem-cell (SSC) populations to regenerate cartilage in relation to age, a possible contributor to the development of osteoarthritis5-7. We demonstrate that aging is associated with progressive loss of SSCs and diminished chondrogenesis in the joints of both mice and humans. However, a local expansion of SSCs could still be triggered in the chondral surface of adult limb joints in mice by stimulating a regenerative response using microfracture (MF) surgery. Although MF-activated SSCs tended to form fibrous tissues, localized co-delivery of BMP2 and soluble VEGFR1 (sVEGFR1), a VEGF receptor antagonist, in a hydrogel skewed differentiation of MF-activated SSCs toward articular cartilage. These data indicate that following MF, a resident stem-cell population can be induced to generate cartilage for treatment of localized chondral disease in OA.


Asunto(s)
Cartílago Articular/fisiología , Regeneración/fisiología , Células Madre/fisiología , Adulto , Animales , Cartílago Articular/citología , Diferenciación Celular , Células Cultivadas , Condrocitos/citología , Condrocitos/fisiología , Condrogénesis/fisiología , Trasplante de Tejido Fetal , Feto/citología , Xenoinjertos , Humanos , Masculino , Células Madre Mesenquimatosas/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Madre/citología , Ingeniería de Tejidos/métodos
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