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1.
Wound Repair Regen ; 31(1): 111-119, 2023 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-36053799

RESUMEN

Myeloid angiogenic cells (MACs) and pericyte-like cells, derived from peripheral blood mononuclear cells (MNCs) by in vitro culturing, are suggested as relevant cell types for angiogenesis and tissue repair. However, the in vivo existence and relevance of these cells has so far remained unknown. Our aim was thus to study, if MACs and pericyte-like cells exist in circulation during the wound healing of skin graft patients, and to evaluate the cellular features of wound repair. MNCs were isolated from blood samples of healthy controls (n = 4) and patients with a traumatic full thickness skin defect (n = 4) before skin grafting and on postoperative days 1 and 6. The numbers of circulating CD14+ CD45+ CD31+ CD34- MACs and CD14+ CD45+ NG2+ pericyte-like cells were assessed by flow cytometry, and gene expression of various pro-angiogenic factors was analysed by qPCR. Wound bed biopsies were taken on postoperative days 6 and 14, and MAC (CD31, CD14 and CD45) and pericyte-related markers (NG2 and PDGFRß) were histologically studied. MACs and pericyte-like cells were detected in both healthy controls and in patients. Before reconstruction, on average 18% of all circulating MNCs represented MACs and 2% pericyte-like cells in wound patients. Number of MACs significantly increased 1.1-1.7-fold in all patients 1 day after skin grafting (p < 0.01). In addition, histological analysis demonstrated effective vascularization of skin grafts, as well as presence of pericytes, and CD14 and CD45 expressing myeloid cells during wound healing. In conclusion, our data shows, for the first time, the presence and mobilisation of MACs and pericyte-like cells in human circulation.


Asunto(s)
Pericitos , Trasplante de Piel , Humanos , Pericitos/metabolismo , Leucocitos Mononucleares , Cicatrización de Heridas , Células Mieloides
2.
Stem Cells Dev ; 30(6): 309-324, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33499756

RESUMEN

Mesenchymal stromal cells (MSCs) are known to stimulate the survival and growth of endothelial cells (ECs) by producing paracrine signals, as well as to differentiate into pericytes and thereby support blood vessel formation and stability. On the other hand, cells with an EC-like phenotype have been found within the CD14+ and CD34+ cell populations of peripheral blood (PB) mononuclear cells (MNCs). The aim of this study was to investigate the proangiogenic differentiation potential of human MSC-MNC co-cultures. Bone marrow-derived MSCs (2,500 cells/cm2) were co-cultured with MNCs (50,000 cells/cm2), which were isolated from the PB of healthy donors. MSCs and MNCs cultured alone at same cell densities were used as controls. Cells in MNC fraction and in co-cultures were isolated for CD14, CD34, and CD31 surface markers with magnetic-activated cell sorting. Co-cultures were analyzed for cell proliferation and morphology, as well as for the expression of various hematopoietic, endothelial, and pericyte markers by immunocytochemistry, quantitative PCR (qPCR), and flow cytometry. Vascular endothelial growth factor (VEGF) expression and secretion was measured with qPCR and enzyme-linked immunosorbent assay, respectively. Our results show that in co-cultures with MSCs, CD14+CD45+ MNCs differentiated into spindle-shaped, nonproliferative, EC-like, myeloid angiogenic cells (MACs) expressing CD31, but also into pericyte-like cells expressing neural/glial antigen 2 (NG2) and CD146. Functionality of the isolated MACs was demonstrated in co-cultures with human umbilical vein endothelial cells, where they supported the formation of tube-like structures. NG2+ cells of MNC-origin were found among both CD34-CD14+ and CD34-CD14- cell populations, indicating the existence of different subtypes of pericyte-like cells. In addition, VEGF was shown to be secreted in MSC-MNC co-cultures, mainly by MSCs. In conclusion, MSCs were shown to possess proangiogenic capacity in MSC-MNC co-cultures as they supported the differentiation of functional MACs, as well as the differentiation of pericyte-like cells of MNC origin. This phenomenon was mediated at least partially via secreted VEGF.


Asunto(s)
Diferenciación Celular/fisiología , Células Endoteliales/citología , Leucocitos Mononucleares/citología , Células Madre Mesenquimatosas/citología , Pericitos/citología , Adulto , Antígenos CD34/metabolismo , Diferenciación Celular/genética , Células Cultivadas , Técnicas de Cocultivo/métodos , Células Endoteliales/metabolismo , Femenino , Expresión Génica , Humanos , Leucocitos Mononucleares/metabolismo , Receptores de Lipopolisacáridos/metabolismo , Células Madre Mesenquimatosas/metabolismo , Neovascularización Fisiológica/genética , Neovascularización Fisiológica/fisiología , Pericitos/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Adulto Joven
3.
J Tissue Eng Regen Med ; 12(3): 775-783, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-28593699

RESUMEN

Endothelial progenitors found among the peripheral blood (PB) mononuclear cells (MNCs) are interesting cells for their angiogenic properties. Mesenchymal stromal cells (MSCs) in turn can produce proangiogenic factors as well as differentiate into mural pericytes, making MSCs and MNCs an attractive coculture setup for regenerative medicine. In this study, human bone marrow-derived MSCs and PB-derived MNCs were cocultured in basal or osteoblastic medium without exogenously supplied growth factors to demonstrate endothelial cell, pericyte and osteoblastic differentiation. The expression levels of various proangiogenic factors, as well as endothelial cell, pericyte and osteoblast markers in cocultures were determined by quantitative polymerase chain reaction. Immunocytochemistry for vascular endothelial growth factor receptor-1 and α-smooth muscle actin as well as staining for alkaline phosphatase were performed after 10 and 14 days. Messenger ribonucleic acid expression of endothelial cell markers was highly upregulated in both basal and osteoblastic conditions after 5 days of coculture, indicating an endothelial cell differentiation, which was supported by immunocytochemistry for vascular endothelial growth factor receptor-1. Stromal derived factor-1 and vascular endothelial growth factor were highly expressed in MSC-MNC coculture in basal medium but not in osteoblastic medium. On the contrary, the expression levels of bone morphogenetic protein-2 and angiopoietin-1 were significantly higher in osteoblastic medium. Pericyte markers were highly expressed in both cocultures after 5 days. In conclusion, it was demonstrated endothelial cell and pericyte differentiation in MSC-MNC cocultures both in basal and osteoblastic medium indicating a potential for neovascularization for tissue engineering applications.


Asunto(s)
Inductores de la Angiogénesis/metabolismo , Diferenciación Celular , Células Endoteliales/citología , Leucocitos Mononucleares/metabolismo , Células Madre Mesenquimatosas/metabolismo , Neovascularización Fisiológica , Pericitos/citología , Biomarcadores/metabolismo , Diferenciación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Medios de Cultivo , Células Endoteliales/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Células Madre Mesenquimatosas/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Pericitos/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Factor A de Crecimiento Endotelial Vascular/farmacología , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Factor de von Willebrand/genética , Factor de von Willebrand/metabolismo
4.
Sci Rep ; 8(1): 859, 2018 01 16.
Artículo en Inglés | MEDLINE | ID: mdl-29339787

RESUMEN

Sperm flagellar protein 2 (SPEF2) is essential for motile cilia, and lack of SPEF2 function causes male infertility and primary ciliary dyskinesia. Cilia are pointing out from the cell surface and are involved in signal transduction from extracellular matrix, fluid flow and motility. It has been shown that cilia and cilia-related genes play essential role in commitment and differentiation of chondrocytes and osteoblasts during bone formation. Here we show that SPEF2 is expressed in bone and cartilage. The analysis of a Spef2 knockout (KO) mouse model revealed hydrocephalus, growth retardation and death prior to five weeks of age. To further elucidate the causes of growth retardation we analyzed the bone structure and possible effects of SPEF2 depletion on bone formation. In Spef2 KO mice, long bones (tibia and femur) were shorter compared to wild type, and X-ray analysis revealed reduced bone mineral content. Furthermore, we showed that the in vitro differentiation of osteoblasts isolated from Spef2 KO animals was compromised. In conclusion, this study reveals a novel function for SPEF2 in bone formation through regulation of osteoblast differentiation and bone growth.


Asunto(s)
Diferenciación Celular , Proteínas/genética , Animales , Densidad Ósea , Cartílago/metabolismo , Cartílago/patología , Células Cultivadas , Condrocitos/citología , Condrocitos/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Fémur/diagnóstico por imagen , Fémur/patología , Fémur/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteoblastos/citología , Osteoblastos/metabolismo , Osteogénesis , Proteínas/metabolismo , Tibia/diagnóstico por imagen , Tibia/patología , Tibia/fisiología , Microtomografía por Rayos X
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