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1.
Int J Mol Sci ; 22(13)2021 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-34206740

RESUMO

N-Glycosylations are an important post-translational modification of proteins that can significantly impact cell function. Terminal sialic acid in hybrid or complex N-glycans has been shown to be relevant in various types of cancer, but its role in non-malignant cells remains poorly understood. We have previously shown that the motility of human bone marrow derived mesenchymal stromal cells (MSCs) can be modified by altering N-glycoforms. The goal of this study was to determine the role of sialylated N-glycans in MSCs. Here, we show that IFN-gamma or exposure to culture media low in fetal bovine serum (FBS) increases sialylated N-glycans, while PDGF-BB reduces them. These stimuli alter mRNA levels of sialyltransferases such as ST3Gal1, ST6Gal1, or ST3Gal4, suggesting that sialylation of N-glycans is regulated by transcriptional control of sialyltransferases. We next show that 2,4,7,8,9-pentaacetyl-3Fax-Neu5Ac-CO2Me (3F-Neu5Ac) effectively inhibits sialylations in MSCs. Supplementation with 3F-Neu5Ac increases adhesion and migration of MSCs, as assessed by both videomicroscopy and wound/scratch assays. Interestingly, pre-treatment with 3F-Neu5Ac also increases the survival of MSCs in an in vitro ischemia model. We also show that pre-treatment or continuous treatment with 3F-Neu5Ac inhibits both osteogenic and adipogenic differentiation of MSCs. Finally, secretion of key trophic factors by MSCs is variably affected upon exposure to 3F-Neu5Ac. Altogether, our experiments suggest that sialylation of N-glycans is tightly regulated in response to environmental cues and that glycoengineering MSCs to reduce sialylated N-glycans could be beneficial to increase both cell migration and survival, which may positively impact the therapeutic potential of the cells.


Assuntos
Movimento Celular , Células-Tronco Mesenquimais/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Polissacarídeos/metabolismo , Sialiltransferases/metabolismo , Adipócitos/metabolismo , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Humanos , Interferon gama/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/fisiologia , Osteoblastos/citologia , Sialiltransferases/antagonistas & inibidores
2.
OMICS ; 25(6): 342-357, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34115524

RESUMO

Early cell biology reports demonstrated the presence of cells with stem-like properties in bone marrow, with both hematopoietic and mesenchymal lineages. Over the years, various investigations have purified and characterized mesenchymal stromal/stem cells (MSCs) from different human tissues as cells with multilineage differentiation potential under the appropriate conditions. Due to their appealing characteristics and versatile potentials, MSCs are leveraged in many applications in medicine such as oncology, bioprinting, and as recent as therapeutics discovery and innovation for COVID-19. To date, studies indicate that MSCs have varied differentiation capabilities into different cell types, and demonstrate immunomodulating and anti-inflammatory properties. Different microenvironments or niche for MSCs and their resulting heterogeneity may influence attendant cellular behavior and differentiation capacity. The potential clinical applications of MSCs and exosomes derived from these cells have led to an avalanche of research reports on their properties and hundreds of clinical trials being undertaken. There is ample reason to think, as discussed in this expert review that the future looks bright and promising for MSC research, with many clinical trials under way to ascertain their clinical utility. This review provides a synthesis of the latest advances and trends in MSC research to allow for broad and critically informed use of MSCs. Early observations of the presence of these cells in the bone marrow and their remarkable differentiation capabilities and immunomodulation are also presented.


Assuntos
Diferenciação Celular , Imunomodulação , Células-Tronco Mesenquimais/imunologia , Humanos , Células-Tronco Mesenquimais/fisiologia , Medicina Regenerativa , Nicho de Células-Tronco , Engenharia Tecidual
3.
Int J Mol Sci ; 22(10)2021 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-34067978

RESUMO

Galectin-3 (Gal-3) is a ß-galactoside-binding protein that influences various cell functions, including cell adhesion. We focused on the role of Gal-3 as an extracellular ligand mediating cell-matrix adhesion. We used human adipose tissue-derived stem cells and human umbilical vein endothelial cells that are promising for vascular tissue engineering. We found that these cells naturally contained Gal-3 on their surface and inside the cells. Moreover, they were able to associate with exogenous Gal-3 added to the culture medium. This association was reduced with a ß-galactoside LacdiNAc (GalNAcß1,4GlcNAc), a selective ligand of Gal-3, which binds to the carbohydrate recognition domain (CRD) in the Gal-3 molecule. This ligand was also able to detach Gal-3 newly associated with cells but not Gal-3 naturally present on cells. In addition, Gal-3 preadsorbed on plastic surfaces acted as an adhesion ligand for both cell types, and the cell adhesion was resistant to blocking with LacdiNAc. This result suggests that the adhesion was mediated by a binding site different from the CRD. The blocking of integrin adhesion receptors on cells with specific antibodies revealed that the cell adhesion to the preadsorbed Gal-3 was mediated, at least partially, by ß1 and αV integrins-namely α5ß1, αVß3, and αVß1 integrins.


Assuntos
Proteínas Sanguíneas/metabolismo , Adesão Celular , Junções Célula-Matriz/metabolismo , Galectinas/metabolismo , Células Endoteliais da Veia Umbilical Humana/fisiologia , Integrinas/metabolismo , Células-Tronco Mesenquimais/fisiologia , Sítios de Ligação , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Células-Tronco Mesenquimais/citologia , Ligação Proteica
4.
Int J Mol Sci ; 22(10)2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-34064719

RESUMO

Inflammation is a major cause of several chronic diseases and is reported to be recovered by the immuno-modulation of mesenchymal stem cells (MSCs). While most studies have focussed on the anti-inflammatory roles of MSCs in stem cell therapy, the impaired features of MSCs, such as the loss of homeostasis by systemic aging or pathologic conditions, remain incompletely understood. In this study, we investigated whether the altered phenotypes of human placenta-derived MSCs (hPD-MSCs) exposed to inflammatory cytokines, including TNF-α and IFN-γ, could be protected by MIT-001, a small anti-inflammatory and anti-necrotic molecule. MIT-001 promoted the spindle-like shape and cytoskeletal organization extending across the long cell axis, whereas hPD-MSCs exposed to TNF-α/IFN-γ exhibited increased morphological heterogeneity with an abnormal cell shape and cytoskeletal disorganization. Importantly, MIT-001 improved mitochondrial distribution across the cytoplasm. MIT-001 significantly reduced basal respiration, ATP production, and cellular ROS levels and augmented the spare respiratory capacity compared to TNF-α/IFN-γ-exposed hPD-MSCs, indicating enhanced mitochondrial quiescence and homeostasis. In conclusion, while TNF-α/IFN-γ-exposed MSCs lost homeostasis and mitochondrial quiescence by becoming over-activated in response to inflammatory cytokines, MIT-001 was able to rescue mitochondrial features and cellular phenotypes. Therefore, MIT-001 has therapeutic potential for clinical applications to treat mitochondrion-related inflammatory diseases.


Assuntos
Citoesqueleto/fisiologia , Células-Tronco Mesenquimais/fisiologia , Mitocôndrias/fisiologia , Compostos Orgânicos/farmacologia , Placenta/citologia , Citoesqueleto/efeitos dos fármacos , Feminino , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Consumo de Oxigênio , Placenta/efeitos dos fármacos , Placenta/metabolismo , Gravidez , Espécies Reativas de Oxigênio/metabolismo
5.
Int J Mol Sci ; 22(10)2021 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-34065598

RESUMO

Bone transplantation is regarded as the preferred therapy to treat a variety of bone defects. Autologous bone tissue is often lacking at the source, and the mesenchymal stem cells (MSCs) responsible for bone repair mechanisms are extracted by invasive procedures. This study explores the potential of autologous mesenchymal stem cells derived from the hair follicle outer root sheath (MSCORS). We demonstrated that MSCORS have a remarkable capacity to differentiate in vitro towards the osteogenic lineage. Indeed, when combined with a novel gelatin-based hydrogel called Osteogel, they provided additional osteoinductive cues in vitro that may pave the way for future application in bone regeneration. MSCORS were also compared to MSCs from adipose tissue (ADMSC) and bone marrow (BMMSC) in a 3D Osteogel model. We analyzed gel plasticity, cell phenotype, cell viability, and differentiation capacity towards the osteogenic lineage by measuring alkaline phosphatase (ALP) activity, calcium deposition, and specific gene expression. The novel injectable hydrogel filled an irregularly shaped lesion in a porcine wound model displaying high plasticity. MSCORS in Osteogel showed a higher osteo-commitment in terms of calcium deposition and expression dynamics of OCN, BMP2, and PPARG when compared to ADMSC and BMMSC, whilst displaying comparable cell viability and ALP activity. In conclusion, autologous MSCORS combined with our novel gelatin-based hydrogel displayed a high capacity for differentiation towards the osteogenic lineage and are acquired by non-invasive procedures, therefore qualifying as a suitable and expandable novel approach in the field of bone regeneration therapy.


Assuntos
Tecido Adiposo/fisiologia , Medula Óssea/fisiologia , Gelatina/química , Folículo Piloso/fisiologia , Hidrogéis/química , Células-Tronco Mesenquimais/fisiologia , Osteogênese/fisiologia , Tecido Adiposo/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Medula Óssea/metabolismo , Células da Medula Óssea/metabolismo , Células da Medula Óssea/fisiologia , Regeneração Óssea/fisiologia , Cálcio/metabolismo , Diferenciação Celular/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Expressão Gênica/fisiologia , Folículo Piloso/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , Modelos Animais , Suínos , Tecidos Suporte/química
6.
Int J Mol Sci ; 22(11)2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34063955

RESUMO

The extracellular matrix (ECM) is the principal structure of bone tissue. Long-term spaceflights lead to osteopenia, which may be a result of the changes in composition as well as remodeling of the ECM by osteogenic cells. To elucidate the cellular effects of microgravity, human mesenchymal stromal cells (MSCs) and their osteocommitted progeny were exposed to simulated microgravity (SMG) for 10 days using random positioning machine (RPM). After RPM exposure, an imbalance of MSC collagen/non-collagen ratio at the expense of a decreased level of collagenous proteins was detected. At the same time, the secretion of proteases (cathepsin A, cathepsin D, MMP3) was increased. No significant effects of SMG on the expression of stromal markers and cell adhesion molecules on the MSC surface were noted. Upregulation of COL11A1, CTNND1, TIMP3, and TNC and downregulation of HAS1, ITGA3, ITGB1, LAMA3, MMP1, and MMP11 were detected in RPM exposed MSCs. ECM-associated transcriptomic changes were more pronounced in osteocommitted progeny. Thus, 10 days of SMG provokes a decrease in the collagenous components of ECM, probably due to the decrease in collagen synthesis and activation of proteases. The presented data demonstrate that ECM-associated molecules of both native and osteocommitted MSCs may be involved in bone matrix reorganization during spaceflight.


Assuntos
Osso e Ossos/metabolismo , Osso e Ossos/fisiologia , Matriz Extracelular/metabolismo , Matriz Extracelular/fisiologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Osteogênese/fisiologia , Doenças Ósseas Metabólicas/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Colágeno/metabolismo , Regulação para Baixo/fisiologia , Proteínas da Matriz Extracelular/metabolismo , Humanos , Peptídeo Hidrolases/metabolismo , Transcriptoma/fisiologia , Regulação para Cima/fisiologia , Ausência de Peso , Simulação de Ausência de Peso/métodos
7.
Nat Commun ; 12(1): 2860, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001878

RESUMO

Bone regenerates by activation of tissue resident stem/progenitor cells, formation of a fibrous callus followed by deposition of cartilage and bone matrices. Here, we show that mesenchymal progenitors residing in skeletal muscle adjacent to bone mediate the initial fibrotic response to bone injury and also participate in cartilage and bone formation. Combined lineage and single-cell RNA sequencing analyses reveal that skeletal muscle mesenchymal progenitors adopt a fibrogenic fate before they engage in chondrogenesis after fracture. In polytrauma, where bone and skeletal muscle are injured, skeletal muscle mesenchymal progenitors exhibit altered fibrogenesis and chondrogenesis. This leads to impaired bone healing, which is due to accumulation of fibrotic tissue originating from skeletal muscle and can be corrected by the anti-fibrotic agent Imatinib. These results elucidate the central role of skeletal muscle in bone regeneration and provide evidence that skeletal muscle can be targeted to prevent persistent callus fibrosis and improve bone healing after musculoskeletal trauma.


Assuntos
Regeneração Óssea/fisiologia , Calo Ósseo/fisiologia , Consolidação da Fratura/fisiologia , Fraturas Ósseas/fisiopatologia , Células-Tronco Mesenquimais/fisiologia , Músculo Esquelético/citologia , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência/métodos , Osteogênese/fisiologia
8.
Aging (Albany NY) ; 13(8): 12031-12045, 2021 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-33888646

RESUMO

Dysfunction of bone marrow mesenchymal stem cells (BMSCs), osteoblasts and osteocytes may be one of the main causes of bone loss in the elderly. In the present study, we found osteogenic cells from aged rats all exhibited senescence changes, with the most pronounced senescence changes in osteocytes. Meanwhile, the proliferative capacity and functional activity of osteogenic cells from aged rats were suppressed. Osteogenic differentiation capacity of BMSCs from aged rats decreased while adipogenic capacity increased. The mineralization capacity, ALP activity and osteogenic proteins expression of osteoblasts from aged rats decreased. Additionally, osteocytes from aged rats up-expressed sclerosteosis protein, a negative regulator of bone formation. To inhibit osteogenic cell senescence, we use low magnitude vibration (LMV) to eliminate the senescent osteogenic cells. After LMV treatment, the number of osteogenic cells staining positively for senescence-associated-ß-galactosidase (SA-ß-Gal) decreased significantly. Besides, the expression of anti-aging protein SIRT1 was upregulated significantly, while p53 and p21 were downregulated significantly after LMV treatment. Thus, the LMV can inhibit the senescence of osteogenic cells partly through the Sirt1/p53/p21 axis. Furthermore, LMV was found to promote bone formation of aged rats. These results suggest that the inhibition of osteogenic cell senescence by LMV is a valuable treatment to prevent or delay osteoporosis.


Assuntos
Osteogênese/fisiologia , Osteoporose/terapia , Vibração/uso terapêutico , Animais , Células Cultivadas , Senescência Celular/fisiologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Modelos Animais de Doenças , Regulação para Baixo/fisiologia , Feminino , Humanos , Masculino , Células-Tronco Mesenquimais/fisiologia , Osteoblastos/fisiologia , Osteoporose/fisiopatologia , Cultura Primária de Células , Ratos , Sirtuína 1/genética , Proteína Supressora de Tumor p53/genética , Regulação para Cima/fisiologia
9.
J Biosci Bioeng ; 131(6): 671-678, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33849774

RESUMO

Inflammation is one of major contributors of diabetic osteoporosis. Adipose derived mesenchymal stem cells (AD-MSCs) show great potential to inhibit inflammation. We investigated the anti-osteoporosis role of AD-MSCs-derived exosomes in diabetic osteoporosis and the underlying molecular mechanism. Cellular and animal diabetic osteoporosis models were created through high glucose exposure and streptozotocin injection. AD-MSCs-derived exosomes were isolated and characterized. Pro-inflammatory cytokines and osteoclast markers were determined by ELISA. Bone mineral content and density were detected to evaluate bone loss. qRT-PCR and Western blots were performed to detect the expression of target genes. AD-MSCs-derived exosomes inhibited the secretion of IL-1ß and IL-18 in HG treated osteoclasts and restored the bone loss in streptozotocin-induced diabetic osteoporosis rats. Mechanistically, AD-MSCs-derived exosomes suppress NLRP3 inflammasome activation in osteoclasts, and then reduce bone resorption and recover bone loss. AD-MSCs-derived exosomes alleviate diabetic osteoporosis through suppressing NLRP3 inflammasome activation in osteoclasts, which might be a potential cell-free therapeutic approach for diabetes-induced bone loss treatment.


Assuntos
Diabetes Mellitus Experimental/complicações , Exossomos/fisiologia , Células-Tronco Mesenquimais/citologia , Osteoporose/prevenção & controle , Animais , Células Cultivadas , Citocinas/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Exossomos/metabolismo , Inflamassomos/metabolismo , Masculino , Células-Tronco Mesenquimais/fisiologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Osteoclastos/metabolismo , Osteoclastos/patologia , Osteoclastos/fisiologia , Osteoporose/etiologia , Osteoporose/metabolismo , Osteoporose/patologia , Ratos , Ratos Sprague-Dawley , Estreptozocina
10.
Life Sci ; 277: 119520, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-33887345

RESUMO

AIMS: Mouse bone marrow mesenchymal stem cells (BMSCs) are pluripotent cells with self-renewal and differentiation abilities. Since the effects of senescent BMSCs on C2C12 cells are not fully clear, the present study aimed to elucidate these effects. MAIN METHODS: Senescence-associated ß-galactosidase staining and western blotting were performed to confirm the senescence of BMSCs. Immunofluorescence and western blotting were used to assess myoblast differentiation in each group. The role of the AKT/P70 signaling pathway and forkhead box O3 (FOXO3) nuclear translocation was explored by western blotting. BMSC-derived exosomes were injected into the tibialis anterior of mice, and RT-qPCR was used to assess the role of exosomes in promoting muscle differentiation. KEY FINDINGS: Conditioned medium (CM) from early-senescent BMSCs promoted myogenic differentiation in vitro, which was detected as enhanced expression of myosin heavy chain (MHC), myogenin (MYOG), and myogenic differentiation 1 (MyoD). The AKT signaling pathway was found to be regulated by CM, which inhibited FOXO3 nuclear translocation. RT-qPCR analysis results showed that MHC, MyoD, and MYOG mRNA expression increased in the tibialis anterior of mice after exosome injection. SIGNIFICANCE: The present study demonstrated that early-senescent BMSCs accelerated C2C12 cell myogenic differentiation, and the transcription factor, FOXO3, was the target of senescent cells. Collectively, our results suggest that the AKT/P70 signaling pathway mediates the effect of BMSCs on neighboring cells.


Assuntos
Diferenciação Celular/fisiologia , Proteína Forkhead Box O3/metabolismo , Células-Tronco Mesenquimais/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Células da Medula Óssea/citologia , Linhagem Celular , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Células-Tronco Mesenquimais/fisiologia , Camundongos , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Miogenina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo
11.
Int J Mol Sci ; 22(6)2021 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-33799588

RESUMO

We have been studying mesenchymal stem cells (MSCs) in synovial fluid and the intra-articular injection of synovial MSCs in osteoarthritis (OA) knees. Here, mainly based on our own findings, we overview the characteristics of endogenous MSCs in the synovial fluid of OA knees and their mode of action when injected exogenously into OA knees. Many MSCs similar to synovial MSCs were detected in the synovial fluid of human OA knees, and their number correlated with the radiological OA grade. Our suspended synovium culture model demonstrated the release of MSCs from the synovium through a medium into a non-contacting culture dish. In OA knees, endogenous MSCs possibly mobilize in a similar manner from the synovium through the synovial fluid and act protectively. However, the number of mobilized MSCs is limited; therefore, OA progresses in its natural course. Synovial MSC injections inhibited the progression of cartilage degeneration in a rat OA model. Injected synovial MSCs migrated into the synovium, maintained their MSC properties, and increased the gene expressions of TSG-6, PRG-4, and BMP-2. Exogenous synovial MSCs can promote anti-inflammation, lubrication, and cartilage matrix synthesis in OA knees. Based on our findings, we have initiated a human clinical study of synovial MSC injections in OA knees.


Assuntos
Condrogênese/genética , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/fisiologia , Osteoartrite do Joelho/terapia , Líquido Sinovial/fisiologia , Animais , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Movimento Celular , Proliferação de Células , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Injeções Intra-Articulares , Células-Tronco Mesenquimais/citologia , Osteoartrite do Joelho/genética , Osteoartrite do Joelho/metabolismo , Osteoartrite do Joelho/patologia , Proteoglicanas/genética , Proteoglicanas/metabolismo , Ratos , Líquido Sinovial/citologia , Transplante Heterólogo , Resultado do Tratamento
12.
Int J Mol Sci ; 22(7)2021 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-33805311

RESUMO

It is evident that depletion of interstitial cells and dysfunction of nitric oxide (NO) pathways are key players in development of several gastrointestinal (GI) motility disorders such as diabetic gastroparesis (DGP). One of the main limitations of DGP research is the lack of isolation methods that are specific to interstitial cells, and therefore conducting functional studies is not feasible. The present study aims (i) to differentiate telomerase transformed mesenchymal stromal cells (iMSCs) into platelet-derived growth factor receptor-α-positive cells (PDGFRα-positive cells) using connective tissue growth factor (CTGF) and L-ascorbic acids; (ii) to investigate the effects of NO donor and inhibitor on the survival rate of differentiated PDGFRα-positive cells; and (iii) to evaluate the impact of increased glucose concentrations, mimicking diabetic hyperglycemia, on the gene expression of neuronal nitric oxide synthase (nNOS). A fibroblastic differentiation-induction medium supplemented with connective tissue growth factor was used to differentiate iMSCs into PDGFRα-positive cells. The medium was changed every day for 21 days to maintain the biological activity of the growth factors. Gene and protein expression, scanning electron and confocal microscopy, and flow cytometry analysis of several markers were conducted to confirm the differentiation process. Methyl tetrazolium cell viability, nitrite measurement assays, and immunostaining were used to investigate the effects of NO on PDGFRα-positive cells. The present study, for the first time, demonstrated the differentiation of iMSCs into PDGFRα-positive cells. The outcomes of the functional studies showed that SNAP (NO donor) increased the survival rate of differentiated PDGFRα-positive cells whereas LNNA (NO inhibitor) attenuated these effects. Further experimentations revealed that hyperglycemia produced a significant increase in expression of nNOS in PDGFRα-positive cells. Differentiation of iMSCs into PDGFRα-positive cells is a novel model to conduct functional studies and to investigate the involvement of NO pathways. This will help in identifying new therapeutic targets for treatment of DGP.


Assuntos
Diferenciação Celular , Células Intersticiais de Cajal/enzimologia , Células-Tronco Mesenquimais/fisiologia , Modelos Biológicos , Óxido Nítrico Sintase Tipo I , Receptor alfa de Fator de Crescimento Derivado de Plaquetas , Animais , Medula Óssea , Células Cultivadas , Complicações do Diabetes , Gastroparesia , Humanos , Células Intersticiais de Cajal/metabolismo
13.
Knee ; 29: 418-425, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33721626

RESUMO

BACKGROUND: Cell based therapy in cartilage repair predominantly involves the use of chondrocytes and mesenchymal stromal cells (MSC). Co-culture systems, due to their probable synergistic effect on enhancement of functional chondrogenesis and reduction in terminal differentiation have also been attempted. Chondroprogenitors, derived from articular cartilage and regarded as MSCs, have recently garnered interest for consideration in cartilage regeneration to overcome limitations associated with use of conventional cell types. The aim of this study was to assess whetherco-culturing bone marrow (BM)-MSCs and chondroprogenitors at different ratios would yield superior results in terms of surface marker expression, gene expression and chondrogenic potential. METHODS: Human BM-MSCs and chondroprogenitors obtained from three osteoarthritic knee joints and subjected to monolayer expansion and pellet cultures (10,000 cells/cm2) as five test groups containing either monocultures or co-cultures (MSC: chondroprogenitors) at three different ratios (75:25, 50:50 and 25:75) were utilized. RESULTS: Data analysis revealed that all groups exhibited a high expression of CD166, CD29 and CD49e. With regard to gene expression, high expression of SOX9, Aggrecan and Collagen type I; a moderate expression of Collagen type X and RUNX2; with a low expression of Collagen type II was seen. Analysis of pellet culture revealed that chondroprogenitor monoculture and chondroprogenitor dominant coculture, exhibited a subjectively larger pellet size with higher deposition of Collagen type II and glycosaminoglycan. CONCLUSION: In conclusion, this study is suggestive of chondroprogenitor monoculture superiority over MSCs, either in isolation or in a coculture system and proposes further analysis of chondroprogenitors for cartilage repair.


Assuntos
Cartilagem Articular/citologia , Técnicas de Cultura de Células/métodos , Células-Tronco Mesenquimais/citologia , Osteoartrite do Joelho/patologia , Agrecanas/genética , Agrecanas/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Cartilagem Articular/fisiologia , Diferenciação Celular , Condrogênese/genética , Técnicas de Cocultura , Colágeno Tipo I/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Feminino , Expressão Gênica , Humanos , Articulação do Joelho/citologia , Masculino , Células-Tronco Mesenquimais/fisiologia , Pessoa de Meia-Idade
14.
Biomed Pharmacother ; 138: 111425, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33756154

RESUMO

Cardiovascular disease is one of the leading causes of mortality worldwide. Cardiac tissue engineering strategies focusing on biomaterial scaffolds incorporating cells and growth factors are emerging as highly promising for cardiac repair and regeneration. The use of stem cells within cardiac microengineered tissue constructs present an inherent ability to differentiate into cell types of the human heart. Stem cells derived from various tissues including bone marrow, dental pulp, adipose tissue and umbilical cord can be used for this purpose. Approaches ranging from stem cell injections, stem cell spheroids, cell encapsulation in a suitable hydrogel, use of prefabricated scaffold and bioprinting technology are at the forefront in the field of cardiac tissue engineering. The stem cell microenvironment plays a key role in the maintenance of stemness and/or differentiation into cardiac specific lineages. This review provides a detailed overview of the recent advances in microengineering of autologous stem cell-based tissue engineering platforms for the repair of damaged cardiac tissue. A particular emphasis is given to the roles played by the extracellular matrix (ECM) in regulating the physiological response of stem cells within cardiac tissue engineering platforms.


Assuntos
Microambiente Celular/fisiologia , Cardiopatias/terapia , Transplante de Células-Tronco Mesenquimais/métodos , Miócitos Cardíacos/transplante , Engenharia Tecidual/métodos , Transplante Autólogo/métodos , Animais , Cardiopatias/fisiopatologia , Humanos , Transplante de Células-Tronco Mesenquimais/tendências , Células-Tronco Mesenquimais/fisiologia , Miócitos Cardíacos/fisiologia , Engenharia Tecidual/tendências , Transplante Autólogo/tendências
15.
FASEB J ; 35(4): e21285, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33710643

RESUMO

The endometrium is a dynamic tissue that exhibits remarkable resilience to repeated episodes of differentiation, breakdown, regeneration, and remodeling. Endometrial physiology relies on a complex interplay between the stromal and epithelial compartments with the former containing a mixture of fibroblasts, vascular, and immune cells. There is evidence for rare populations of putative mesenchymal progenitor cells located in the perivascular niche of human endometrium, but the existence of an equivalent cell population in mouse is unclear. We used the Pdgfrb-BAC-eGFP transgenic reporter mouse in combination with bulk and single-cell RNA sequencing to redefine the endometrial mesenchyme. In contrast to previous reports we show that CD146 is expressed in both PDGFRß + perivascular cells and CD31 + endothelial cells. Bulk RNAseq revealed cells in the perivascular niche which express the high levels of Pdgfrb as well as genes previously identified in pericytes and/or vascular smooth muscle cells (Acta2, Myh11, Olfr78, Cspg4, Rgs4, Rgs5, Kcnj8, and Abcc9). scRNA-seq identified five subpopulations of cells including closely related pericytes/vascular smooth muscle cells and three subpopulations of fibroblasts. All three fibroblast populations were PDGFRα+/CD34 + but were distinct in their expression of Ngfr/Spon2/Angptl7 (F1), Cxcl14/Smoc2/Rgs2 (F2), and Clec3b/Col14a1/Mmp3 (F3), with potential functions in the regulation of immune responses, response to wounding, and organization of extracellular matrix, respectively. Immunohistochemistry was used to investigate the spatial distribution of these populations revealing F1/NGFR + cells in most abundance beside epithelial cells. We provide the first definitive analysis of mesenchymal cells in the adult mouse endometrium identifying five subpopulations providing a platform for comparisons between mesenchymal cells in endometrium and other adult tissues which are prone to fibrosis.


Assuntos
Endométrio/citologia , Células-Tronco Mesenquimais/fisiologia , Animais , Biomarcadores , Feminino , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde , Homeostase , Camundongos , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Análise de Sequência de RNA , Análise de Célula Única , Transcriptoma
16.
Mol Med Rep ; 23(6)2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33786622

RESUMO

The loosening and displacement of prostheses after dental implantation and arthroplasty is a substantial medical burden due to the complex correction surgery. Three­dimensional (3D)­printed porous titanium (pTi) alloy scaffolds are characterized by low stiffness, are beneficial to bone ingrowth, and may be used in orthopedic applications. However, for the bio­inert nature between host bone and implants, titanium alloy remains poorly compatible with osseointegration, especially in disease conditions, such as osteoporosis. In the present study, 3D­printed pTi scaffolds with ideal pore size and porosity matching the bone tissue, were combined with pulse electromagnetic fields (PEMF), an exogenous osteogenic induction stimulation, to evaluate osseointegration in osteoporosis. In vitro, external PEMF significantly improved osteoporosis­derived bone marrow mesenchymal stem cell proliferation and osteogenic differentiation on the surface of pTi scaffolds by enhancing the expression of alkaline phosphatase, runt­related transcription factor­2, osteocalcin, and bone morphogenetic protein­2. In vivo, Microcomputed tomography analysis and histological evaluation indicated the external PEMF markedly enhanced bone regeneration and osseointegration. This novel therapeutic strategy has potential to promote osseointegration of dental implants or artificial prostheses for patients with osteoporosis.


Assuntos
Ligas/química , Campos Eletromagnéticos , Osseointegração , Osteoporose/cirurgia , Impressão Tridimensional , Engenharia Tecidual/métodos , Tecidos Suporte/química , Titânio/química , Fosfatase Alcalina/metabolismo , Animais , Células da Medula Óssea/metabolismo , Células da Medula Óssea/fisiologia , Células da Medula Óssea/efeitos da radiação , Proteína Morfogenética Óssea 2/metabolismo , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Feminino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Células-Tronco Mesenquimais/efeitos da radiação , Osteocalcina/metabolismo , Coelhos
17.
Am J Physiol Endocrinol Metab ; 320(4): E760-E771, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33645251

RESUMO

Apigenin (API), a natural plant flavone, is abundantly found in common fruits and vegetables. As a bioactive flavonoid, API exhibits several activities including antiproliferation and anti-inflammation. A recent study showed that API could retard osteoporosis progress, indicating its role in the skeletal system. However, the detailed function and mechanism remain obscure. In the present study, API was found to promote osteogenic differentiation of mesenchymal stem cells (MSCs). And further investigation showed that API could enhance the expression of the critical transcription factor ß-catenin and several downstream target genes of Wnt signaling, thus activated Wnt/ß-catenin signaling. Using a rat femoral fracture model, API was found to improve new bone formation and accelerate fracture healing in vivo. In conclusion, our data demonstrated that API could promote osteogenesis in vitro and facilitate the fracture healing in vivo via activating Wnt/ß-catenin signaling, indicating that API may be a promising therapeutic candidate for bone fracture repair.NEW & NOTEWORTHY 1) API promoted osteogenic differentiation of human MSCs in vitro; 2) API facilitated bone formation and accelerated fracture healing in vivo; 3) API stimulated Wnt/ß-catenin signaling during osteogenesis of human MSCs.


Assuntos
Apigenina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Consolidação da Fratura/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Adulto , Animais , Apigenina/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Fraturas Ósseas/tratamento farmacológico , Fraturas Ósseas/fisiopatologia , Humanos , Masculino , Células-Tronco Mesenquimais/fisiologia , Ratos , Ratos Sprague-Dawley , Via de Sinalização Wnt/efeitos dos fármacos
18.
Nucleic Acids Res ; 49(8): 4203-4219, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33706382

RESUMO

Sirtuin 3 (SIRT3) is an NAD+-dependent deacetylase linked to a broad range of physiological and pathological processes, including aging and aging-related diseases. However, the role of SIRT3 in regulating human stem cell homeostasis remains unclear. Here we found that SIRT3 expression was downregulated in senescent human mesenchymal stem cells (hMSCs). CRISPR/Cas9-mediated depletion of SIRT3 led to compromised nuclear integrity, loss of heterochromatin and accelerated senescence in hMSCs. Further analysis indicated that SIRT3 interacted with nuclear envelope proteins and heterochromatin-associated proteins. SIRT3 deficiency resulted in the detachment of genomic lamina-associated domains (LADs) from the nuclear lamina, increased chromatin accessibility and aberrant repetitive sequence transcription. The re-introduction of SIRT3 rescued the disorganized heterochromatin and the senescence phenotypes. Taken together, our study reveals a novel role for SIRT3 in stabilizing heterochromatin and counteracting hMSC senescence, providing new potential therapeutic targets to ameliorate aging-related diseases.


Assuntos
Envelhecimento/metabolismo , Heterocromatina/metabolismo , Sirtuína 3/fisiologia , Envelhecimento/genética , Animais , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Células Cultivadas , Senescência Celular/genética , Senescência Celular/fisiologia , Técnicas de Inativação de Genes , Células HEK293 , Heterocromatina/genética , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Camundongos , Camundongos Nus , Camundongos SCID , Membrana Nuclear/metabolismo , Domínios Proteicos , Sirtuína 3/química , Sirtuína 3/genética
19.
Int J Mol Sci ; 22(5)2021 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-33673409

RESUMO

Mesenchymal stem cells (MSCs) have been identified in many adult tissues and they have been closely studied in recent years, especially in view of their potential use for treating diseases and damaged tissues and organs. MSCs are capable of self-replication and differentiation into osteoblasts and are considered an important source of cells in tissue engineering for bone regeneration. Several epigenetic factors are believed to play a role in the osteogenic differentiation of MSCs, including microRNAs (miRNAs). MiRNAs are small, single-stranded, non-coding RNAs of approximately 22 nucleotides that are able to regulate cell proliferation, differentiation and apoptosis by binding the 3' untranslated region (3'-UTR) of target mRNAs, which can be subsequently degraded or translationally silenced. MiRNAs control gene expression in osteogenic differentiation by regulating two crucial signaling cascades in osteogenesis: the transforming growth factor-beta (TGF-ß)/bone morphogenic protein (BMP) and the Wingless/Int-1(Wnt)/ß-catenin signaling pathways. This review provides an overview of the miRNAs involved in osteogenic differentiation and how these miRNAs could regulate the expression of target genes.


Assuntos
Células-Tronco Mesenquimais/metabolismo , MicroRNAs/metabolismo , Osteogênese , Transdução de Sinais , Animais , Osso e Ossos , Humanos , Células-Tronco Mesenquimais/fisiologia
20.
Int J Mol Sci ; 22(5)2021 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-33673509

RESUMO

Fusion cages composed of titanium and its alloys are emerging as valuable alternative to standard polyetheretherketone (PEEK) ones routinely used in cervical and lumbar spine surgery. Aim of this study was to evaluate osteo-inductive and osteo-conductive ability of an innovative trabecular titanium (T-Ti) scaffold on human mesenchymal stem cells (hMSCs), in both absence and presence of biochemical osteogenic stimuli. Same abilities were assessed on PEEK and standard 2D plastic surface, the latter meant as gold-standard for in vitro differentiation studies. hMSCs adhered and colonized both T-Ti and PEEK scaffolds. In absence of osteogenic factors, T-Ti triggered osteogenic induction of MSCs, as demonstrated by alkaline phosphatase activity and calcium deposition increments, while PEEK and standard 2D did not. Addition of osteogenic stimuli reinforced osteogenic differentiation of hMSCs cultured on T-Ti in a significantly higher manner with respect to standard 2D plastic culture surfaces, whereas PEEK almost completely abolished the process. T-Ti driven differentiation towards osteoblasts was confirmed by gene and marker expression analyses, even in absence of osteogenic stimuli. These results clearly indicate superior in vitro osteo-inductive and osteo-conductive capacity of T-Ti compared to PEEK, and make ground for further studies supporting the use of T-Ti cages to improve bone fusion.


Assuntos
Cetonas , Células-Tronco Mesenquimais/fisiologia , Osteogênese , Polietilenoglicóis , Tecidos Suporte/química , Titânio , Adulto , Diferenciação Celular , Feminino , Regulação da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Próteses e Implantes
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