Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 59.569
Filtrar
1.
Stem Cell Res Ther ; 15(1): 199, 2024 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-38971781

RESUMO

BACKGROUND: Bone marrow-derived mesenchymal stem cell (BMMSC)-based therapy has become a major focus for treating liver fibrosis/cirrhosis. However, although these cell therapies promote the treatment of this disease, the heterogeneity of BMMSCs, which causes insufficient efficacy during clinical trials, has not been addressed. In this study, we describe a novel Percoll-Plate-Wait procedure (PPWP) for the isolation of an active cell subset from BMMSC cultures that was characterized by the expression of neuroglial antigen 2 (NG2/BMMSCs). METHODS: By using the key method of PPWP and other classical biological techniques we compared NG2/BMMSCs with parental BMMSCs in biological and functional characteristics within a well-defined diethylnitrosamine (DEN)-induced liver fibrosis/cirrhosis injury male C57BL/6 mouse model also in a culture system. Of note, the pathological alterations in the model is quite similar to humans'. RESULTS: The NG2/BMMSCs revealed more advantages compared to parentalBMMSCs. They exhibited greater proliferation potential than parental BMMSCs, as indicated by Ki-67 immunofluorescence (IF) staining. Moreover, higher expression of SSEA-3 (a marker specific for embryonic stem cells) was detected in NG2/BMMSCs than in parental BMMSCs, which suggested that the "stemness" of NG2/BMMSCs was greater than that of parental BMMSCs. In vivo studies revealed that an injection of NG2/BMMSCs into mice with ongoing DEN-induced liver fibrotic/cirrhotic injury enhanced repair and functional recovery to a greater extent than in mice treated with parental BMMSCs. These effects were associated with the ability of NG2/BMMSCs to differentiate into bile duct cells (BDCs). In particular, we discovered for the first time that NG2/BMMSCs exhibit unique characteristics that differ from those of parental BMMSCs in terms of producing liver sinusoidal endothelial cells (LSECs) to reconstruct injured blood vessels and sinusoidal structures in the diseased livers, which are important for initiating hepatocyte regeneration. This unique potential may also suggest that NG2/BMMSCs could be an novel off-liver progenitor of LSECs. Ex vivo studies revealed that the NG2/BMMSCs exhibited a similar trend to that of their in vivo in terms of functional differentiation responding to the DEN-diseased injured liver cues. Additionally, the obvious core role of NG2/BMMSCs in supporting the functions of BMMSCs in bile duct repair and BDC-mediated hepatocyte regeneration might also be a novel finding. CONCLUSIONS: Overall, the PPWP-isolated NG2/BMMSCs could be a novel effective cell subset with increased purity to serve as a new therapeutic tool for enhancing treatment efficacy of BMMSCs and special seed cell source (BDCs, LSECs) also for bioliver engineering.


Assuntos
Antígenos , Cirrose Hepática , Células-Tronco Mesenquimais , Camundongos Endogâmicos C57BL , Animais , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Cirrose Hepática/terapia , Cirrose Hepática/patologia , Cirrose Hepática/induzido quimicamente , Camundongos , Masculino , Antígenos/metabolismo , Transplante de Células-Tronco Mesenquimais/métodos , Proteoglicanas/metabolismo , Diferenciação Celular , Proliferação de Células , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Células Cultivadas
2.
Sci Rep ; 14(1): 15600, 2024 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-38971916

RESUMO

Binding of Staphylococcus aureus protein A (SPA) to osteoblasts induces apoptosis and inhibits bone formation. Bone marrow-derived mesenchymal stem cells (BMSCs) have the ability to differentiate into bone, fat and cartilage. Therefore, it was important to analyze the molecular mechanism of SPA on osteogenic differentiation. We introduced transcript sequence data to screen out differentially expressed genes (DEGs) related to SPA-interfered BMSC. Protein-protein interaction (PPI) network of DEGs was established to screen biomarkers associated with SPA-interfered BMSC. Receiver operating characteristic (ROC) curve was plotted to evaluate the ability of biomarkers to discriminate between two groups of samples. Finally, we performed GSEA and regulatory analysis based on biomarkers. We identified 321 DEGs. Subsequently, 6 biomarkers (Cenpf, Kntc1, Nek2, Asf1b, Troap and Kif14) were identified by hubba algorithm in PPI. ROC analysis showed that six biomarkers could clearly discriminate between normal differentiated and SPA-interfered BMSC. Moreover, we found that these biomarkers were mainly enriched in the pyrimidine metabolism pathway. We also constructed '71 circRNAs-14 miRNAs-5 mRNAs' and '10 lncRNAs-5 miRNAs-2 mRNAs' networks. Kntc1 and Asf1b genes were associated with rno-miR-3571. Nek2 and Asf1b genes were associated with rno-miR-497-5p. Finally, we found significantly lower expression of six biomarkers in the SPA-interfered group compared to the normal group by RT-qPCR. Overall, we obtained 6 biomarkers (Cenpf, Kntc1, Nek2, Asf1b, Troap, and Kif14) related to SPA-interfered BMSC, which provided a theoretical basis to explore the key factors of SPA affecting osteogenic differentiation.


Assuntos
Diferenciação Celular , Células-Tronco Mesenquimais , Osteogênese , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Osteogênese/genética , Diferenciação Celular/genética , Humanos , Biomarcadores/metabolismo , Quinases Relacionadas a NIMA/metabolismo , Quinases Relacionadas a NIMA/genética , Mapas de Interação de Proteínas/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Células da Medula Óssea/metabolismo , Células da Medula Óssea/citologia , Perfilação da Expressão Gênica , Redes Reguladoras de Genes
3.
Zhonghua Yi Xue Za Zhi ; 104(25): 2350-2358, 2024 Jul 02.
Artigo em Chinês | MEDLINE | ID: mdl-38951108

RESUMO

Objective: To investigate the role and underlying mechanisms of intercellular adhesion molecule-1 (ICAM-1) in the adhesion and migration of mesenchymal stem cells (MSCs) in patients with ankylosing spondylitis (AS). Methods: Bone marrow and ligament tissues were collected during surgery from patients with AS and thoracolumbar fractures (as controls, HC) treated from October 2021 to October 2022 at Nanjing University Medical School Affiliated Nanjing Drum Tower Hospital. MSCs were isolated and cultured from the bone marrow using the Ficoll separation method. Cell morphology was observed under high-resolution microscopy, and differences in the cytoskeletal features between AS-and HC-MSCs were analyzed through immunofluorescence staining. The expression of ICAM-1 was quantified in both groups using real-time quantitative polymerase chain reaction (RT-qPCR) and flow cytometry. Transwell migration assays and wound healing experiments were conducted to evaluate the differences in migration rates between the two groups of MSCs. Results: The interspinous ligament and bone marrow was acquired in AS (2 males and 1 female; 33, 37, 32 years old, respectively) and no-AS patients (2 males and 1 female; 35, 32, 38 years old, respectively). AS-MSCs exhibited broader cell morphology compared to HC-MSCs under bright field and fluorescence microscopy. Immunofluorescence staining of the interspinous ligament showed higher expression of ICAM-1 (68.38±3.42 vs 48.31±2.43) and CD105 (37.97±2.16 vs 23.36±2.06) in AS patients (both P<0.001). Western blot and RT-qPCR analysis revealed significantly stronger protein expression and transcription levels of ICAM-1 in AS-MSCs when compared to those in HC-MSCs (both P<0.001). Flow cytometry confirmed greater mean fluorescence intensity of ICAM-1 in AS-MSCs than in that in HC-MSCs (924.30±54.99 vs 636.47±40.03, P=0.002). Regarding cell adhesion efficiency, it showed no significant difference between AS-MSCs and HC-MSCs in the early stage of adhesion (0.5 h: 1 496±213 vs 1 205±163, P=0.133), but they were all significantly higher in AS-MSCs in the later stage (1 h: 2 894±172 vs 1 908±155, P=0.002; 2 h: 4 540±286 vs 3 334±188, P=0.004; 3 h: 5 212±281 vs 4 208±303, P=0.014). Finally, cell migration experiments demonstrated a stronger migration capability of AS-MSCs compared to HC-MSCs (5 449±172 vs 4 016±155, P<0.001), and the inhibition efficiency of A-205804 on the migration rate of AS-MSCs was stronger than that on HC-MSCs (2 145±239 vs 3 539±316, P=0.004). Conclusions: The aberrant expression of ICAM-1 markedly influences the adhesion and migration dynamics of MSCs. Elevated ICAM-1 levels in MSCs derives from patients with AS significantly enhance their migratory capabilities.


Assuntos
Adesão Celular , Movimento Celular , Molécula 1 de Adesão Intercelular , Células-Tronco Mesenquimais , Espondilite Anquilosante , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Espondilite Anquilosante/metabolismo , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Adulto , Feminino , Masculino , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Estudos Retrospectivos , Células Cultivadas
4.
Bone Res ; 12(1): 38, 2024 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-38961077

RESUMO

Bone marrow adipocytes (BMAds) affect bone homeostasis, but the mechanism remains unclear. Here, we showed that exercise inhibited PCNA clamp-associated factor (PCLAF) secretion from the bone marrow macrophages to inhibit BMAds senescence and thus alleviated skeletal aging. The genetic deletion of PCLAF in macrophages inhibited BMAds senescence and delayed skeletal aging. In contrast, the transplantation of PCLAF-mediated senescent BMAds into the bone marrow of healthy mice suppressed bone turnover. Mechanistically, PCLAF bound to the ADGRL2 receptor to inhibit AKT/mTOR signaling that triggered BMAds senescence and subsequently spread senescence among osteogenic and osteoclastic cells. Of note, we developed a PCLAF-neutralizing antibody and showed its therapeutic effects on skeletal health in old mice. Together, these findings identify PCLAF as an inducer of BMAds senescence and provide a promising way to treat age-related osteoporosis.


Assuntos
Adipócitos , Envelhecimento , Senescência Celular , Animais , Adipócitos/metabolismo , Senescência Celular/fisiologia , Camundongos , Envelhecimento/fisiologia , Camundongos Endogâmicos C57BL , Células da Medula Óssea/metabolismo , Osso e Ossos/metabolismo , Osso e Ossos/fisiologia , Masculino , Osteogênese/fisiologia , Transdução de Sinais , Macrófagos/metabolismo
5.
Stem Cell Res Ther ; 15(1): 168, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38886849

RESUMO

BACKGROUND: Mechanical stimulation (MS) significantly increases the release of adenine and uracil nucleotides from bone marrow-derived mesenchymal stem cells (BM-MSCs) undergoing osteogenic differentiation. Released nucleotides acting via ionotropic P2X7 and metabotropic P2Y6 purinoceptors sensitive to ATP and UDP, respectively, control the osteogenic commitment of BM-MSCs and, thus, bone growth and remodelling. Yet, this mechanism is impaired in post-menopausal (Pm)-derived BM-MSCs, mostly because NTPDase3 overexpression decreases the extracellular accumulation of nucleotides below the levels required to activate plasma membrane-bound P2 purinoceptors. This prompted us to investigate whether in vitro MS of BM-MSCs from Pm women could rehabilitate their osteogenic commitment and whether xenotransplantation of MS purinome-primed Pm cells promote repair of critical bone defects in an in vivo animal model. METHODS: BM-MSCs were harvested from the neck of femora of Pm women (70 ± 3 years old) undergoing total hip replacement. The cells grew, for 35 days, in an osteogenic-inducing medium either submitted (SS) or not (CTR) to MS (90 r.p.m. for 30 min) twice a week. Increases in alkaline phosphatase activity and in the amount of osteogenic transcription factors, osterix and osteopontin, denoted osteogenic cells differentiation, while bone nodules formation was ascertain by the alizarin red-staining assay. The luciferin-luciferase bioluminescence assay was used to quantify extracellular ATP. The kinetics of the extracellular ATP (100 µM) and UDP (100 µM) catabolism was assessed by HPLC. The density of P2Y6 and P2X7 purinoceptors in the cells was assessed by immunofluorescence confocal microscopy. MS-stimulated BM-MSCs from Pm women were xenotransplanted into critical bone defects drilled in the great trochanter of femora of one-year female Wistar rats; bone repair was assessed by histological analysis 10 days after xenotransplantation. RESULTS: MS-stimulated Pm BM-MSCs in culture (i) release 1.6-fold higher ATP amounts, (ii) overexpress P2X7 and P2Y6 purinoceptors, (iii) exhibit higher alkaline phosphatase activity and overexpress the osteogenic transcription factors, osterix and osteopontin, and (iv) form larger bone nodules, than CTR cells. Selective blockage of P2X7 and P2Y6 purinoceptors with A438079 (3 µM) and MRS 2578 (0.1 µM), respectively, prevented the osteogenic commitment of cultured Pm BM-MSCs. Xenotransplanted MS purinome-primed Pm BM-MSCs accelerated the repair of critical bone defects in the in vivo rat model. CONCLUSIONS: Data suggest that in vitro MS restores the purinergic cell-to-cell communication fostering the osteogenic differentiation and osteointegration of BM-MSCs from Pm women, a strategy that may be used in bone regeneration and repair tactics.


Assuntos
Diferenciação Celular , Células-Tronco Mesenquimais , Osteogênese , Pós-Menopausa , Feminino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Humanos , Osteogênese/efeitos dos fármacos , Animais , Idoso , Ratos , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Transplante de Células-Tronco Mesenquimais/métodos , Fator de Transcrição Sp7/metabolismo , Fator de Transcrição Sp7/genética , Células Cultivadas , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Ratos Wistar
6.
Cells ; 13(11)2024 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-38891064

RESUMO

Periodontal disease is characterized by inflammation and bone loss. Central to its pathogenesis is the dysregulated inflammatory response, complicating regenerative therapies. Mesenchymal stem cells (MSCs) hold significant promise in tissue repair and regeneration. This study investigated the effects of specialized pro-resolving mediators (SPMs), Resolvin E1 (RvE1) and Maresin 1 (MaR1), on the osteogenic differentiation of human bone marrow-derived MSCs under inflammatory conditions. The stem cells were treated with SPMs in the presence of lipopolysaccharide (LPS) to simulate an inflammatory environment. Osteogenic differentiation was assessed through alkaline phosphatase activity and alizarin red staining. Proteomic analysis was conducted to characterize the protein expression profile changes, focusing on proteins related to osteogenesis and osteoclastogenesis. Treatment with RvE1 and MaR1, both individually and in combination, significantly enhanced calcified deposit formation. Proteomic analysis revealed the differential expression of proteins associated with osteogenesis and osteoclastogenesis, highlighting the modulatory impact of SPMs on bone metabolism. RvE1 and MaR1 promote osteogenic differentiation of hBMMSCs in an inflammatory environment, with their combined application yielding synergistic effects. This study provides insights into the therapeutic potential of SPMs in enhancing bone regeneration, suggesting a promising avenue for developing regenerative therapies for periodontal disease and other conditions characterized by inflammation-induced bone loss.


Assuntos
Diferenciação Celular , Ácidos Docosa-Hexaenoicos , Ácido Eicosapentaenoico , Inflamação , Células-Tronco Mesenquimais , Osteogênese , Osteogênese/efeitos dos fármacos , Humanos , Ácido Eicosapentaenoico/farmacologia , Ácido Eicosapentaenoico/análogos & derivados , Ácidos Docosa-Hexaenoicos/farmacologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Inflamação/patologia , Proteômica , Células da Medula Óssea/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/citologia , Lipopolissacarídeos/farmacologia
7.
Cells ; 13(11)2024 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-38891119

RESUMO

Although discectomy is commonly performed for lumbar intervertebral disc (IVD) herniation, the capacity for tissue repair after surgery is limited, resulting in residual lower back pain, recurrence of IVD herniation, and progression of IVD degeneration. Cell-based therapies, as one-step procedures, are desirable for enhancing IVD repair. This study aimed to investigate the therapeutic efficacy of a combination of newly developed ultra-purified alginate (UPAL) gel and bone marrow aspirate concentrate (BMAC) implantation for IVD repair after discectomy. Prior to an in vivo study, the cell concentration abilities of three commercially available preparation kits for creating the BMAC were compared by measuring the number of bone marrow mesenchymal stem cells harvested from the bone marrow of rabbits. Subsequently, canine-derived BMAC was tested in a canine model using a kit which had the highest concentration rate. At 24 weeks after implantation, we evaluated the changes in the magnetic resonance imaging (MRI) signals as well as histological degeneration grade and immunohistochemical analysis results for type II and type I collagen-positive cells in the treated IVDs. In all quantitative evaluations, such as MRI and histological and immunohistochemical analyses of IVD degeneration, BMAC-UPAL implantation significantly suppressed the progression of IVD degeneration compared to discectomy and UPAL alone. This preclinical proof-of-concept study demonstrated the potential efficacy of BMAC-UPAL gel as a therapeutic strategy for implementation after discectomy, which was superior to UPAL and discectomy alone in terms of tissue repair and regenerative potential.


Assuntos
Alginatos , Modelos Animais de Doenças , Degeneração do Disco Intervertebral , Disco Intervertebral , Animais , Cães , Alginatos/química , Alginatos/farmacologia , Disco Intervertebral/cirurgia , Disco Intervertebral/patologia , Disco Intervertebral/efeitos dos fármacos , Coelhos , Degeneração do Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/cirurgia , Degeneração do Disco Intervertebral/terapia , Estudo de Prova de Conceito , Géis , Células da Medula Óssea/citologia , Células-Tronco Mesenquimais/citologia , Imageamento por Ressonância Magnética , Masculino , Transplante de Medula Óssea/métodos
8.
J Vis Exp ; (207)2024 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-38884476

RESUMO

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from appendicular bones, BMMSCs derived from the jaw have greater proliferative and osteogenic differentiation ability, gradually becoming important seed cells for jaw defect repair. However, the mandible has a complex bony structure and less cancellous content than appendicular bones. It is difficult to acquire a large number of high-quality jaw-derived marrow mesenchymal stem cells using traditional methods. This study presents a 'niche-based approach on stemness' for isolating and culturing rat jaw bone marrow mesenchymal stem cells (JBMMSCs). Primary rat JBMMSCs were isolated and cultured using the whole bone marrow adherent method combined with the bone slice digestion method. The isolated cells were identified as JBMMSCs through cell morphology observation, detection of cell surface markers, and multi-directional differentiation induction. The cells extracted by this method exhibit a 'fibroblast-like' spindle shape. The cells are long, spindle-shaped and fibroblast-like. The flow cytometry analysis shows these cells are positive for CD29, CD44, and CD90 but negative for CD11b/c, CD34, and CD45, which is congruent with BMMSCs characteristics. The cells show strong proliferation capacity and can undergo osteogenic, adipogenic, and chondrogenic differentiation. This study provides an effective and stable method for obtaining enough high-quality JBMMSCs with strong differentiation ability in a short time, which could facilitate further studies of the exploration of biological function, regenerative medicine, and related clinical applications.


Assuntos
Células da Medula Óssea , Células-Tronco Mesenquimais , Animais , Células-Tronco Mesenquimais/citologia , Ratos , Células da Medula Óssea/citologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Arcada Osseodentária/citologia , Técnicas Citológicas/métodos
9.
Cell Transplant ; 33: 9636897241260195, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38867486

RESUMO

Lymphedema is an intractable disease with few effective therapeutic options. Autologous mesenchymal stem cell (MSC) transplantation is a promising therapy for this disease. However, its use is limited by the cost and time for preparation. Recently, xenotransplantation of porcine MSCs has emerged as an alternative to autologous MSC transplantation. In this study, we aimed to clarify the usefulness of neonatal porcine bone marrow-derived MSC (NpBM-MSC) xenotransplantation for the treatment of lymphedema. One million NpBM-MSCs were xenotransplanted into the hind limbs of mice with severe lymphedema (MSC transplantation group). The therapeutic effects were assessed by measuring the femoral circumference, the volume of the hind limb, the number and diameter of lymphatic vessels in the hind limb, and lymphatic flow using a near-infrared fluorescence (NIRF) imaging system. We compared the effects using mice with lymphedema that did not undergo NpBM-MSC transplantation (negative control group). The condition of the transplanted NpBM-MSCs was also evaluated histologically. The femoral circumference and volume of the hind limb had been normalized by postoperative day (POD) 14 in the MSC transplantation group, but not in the negative control group (P = 0.041). NIRF imaging revealed that lymphatic flow had recovered in the MSC transplantation group by POD 14, as shown by an increase in luminance in the hind limb. Histological assessment also showed that the xenotransplantation of NpBM-MSC increased the proliferation of lymphatic vessels, but they had been rejected by POD 14. The xenotransplantation of NpBM-MSCs is an effective treatment for lymphedema, and this is mediated through the promotion of lymphangiogenesis.


Assuntos
Membro Posterior , Linfedema , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Transplante Heterólogo , Animais , Transplante de Células-Tronco Mesenquimais/métodos , Suínos , Camundongos , Linfedema/terapia , Transplante Heterólogo/métodos , Células-Tronco Mesenquimais/citologia , Vasos Linfáticos , Células da Medula Óssea/citologia , Animais Recém-Nascidos
10.
In Vivo ; 38(4): 1719-1730, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38936939

RESUMO

BACKGROUND/AIM: ClFdA is a second-generation antineoplastic agent that has demonstrated significant anticancer activity, particularly against acute lymphoblastic leukemia and has been shown to have radiosensitizing activity. The aim of the study was to explore the genotoxic, cytotoxic and radiosensitizing effects of clofarabine (ClFdA) on bone marrow cells (BMCs), normoblasts and leukocytes of mice in vivo. MATERIALS AND METHODS: Cytotoxicity was determined by the reduction in reticulocytes (RET), and genotoxicity was determined by the induction of micronucleated reticulocytes (MN-RET) in the peripheral blood and by DNA break induction in leukocytes determined by single-cell gel electrophoresis (SCGE). The radiosensitizing capacity of ClFdA was determined in leukocytes and BMCs by SCGE. RESULTS: Two mechanisms of MN-RET induction were identified according to the antecedents, that could be due to inhibition of DNA synthesis and demethylation of G-C regions, and subsequent chromosome fragility. ClFdA cytotoxicity causes two contiguous peaks, an early peak that seems to inhibit MN-RET induction and a second peak that seems to be caused by ribonucleotide reductase (RR) and/or DNA synthesis inhibitions. ClFdA induced early DNA damage in noncycling leukocytes, and also radiosensitizes leukocytes immediately after treatment. ClFdA-ionizing radiation (IR) causes two time-dependent episodes of DNA damage, the latest after 80 min triggers a major breakage of DNA. In terms of the number of damaged cells, leukocytes and BMCs are similarly sensitive to ionizing radiation; BMCs are slightly more sensitive than leukocytes to ClFdA, but BMCs are doubly sensitive to combined treatment. CONCLUSION: ClFdA causes early DNA damage and radiosensitivity in non-proliferating leukocytes, which rules out the most favored hypotheses of the participation of RR and DNA polymerase inhibition.


Assuntos
Clofarabina , Dano ao DNA , Leucócitos , Radiossensibilizantes , Animais , Clofarabina/farmacologia , Camundongos , Radiossensibilizantes/farmacologia , Leucócitos/efeitos dos fármacos , Leucócitos/efeitos da radiação , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Arabinonucleosídeos/farmacologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/efeitos da radiação , Células da Medula Óssea/metabolismo , Nucleotídeos de Adenina/farmacologia , Masculino , Reticulócitos/efeitos dos fármacos , Reticulócitos/efeitos da radiação , Antineoplásicos/farmacologia , Testes para Micronúcleos
11.
J Vis Exp ; (207)2024 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-38856198

RESUMO

Various techniques for isolating bone marrow from adult mice have been well established. However, isolating bone marrow from neonatal mice is challenging and time-consuming, yet for some models, it is translationally relevant and necessary. This protocol describes an efficient and straightforward method for preparing bone marrow cells from 7-9-day-old pups. These cells can then be further isolated or differentiated into specific cell types of interest. Macrophages are crucial immune cells that play a major role in inflammation and infection. During development, neonatal macrophages contribute significantly to tissue remodeling. Moreover, the phenotype and functions of neonatal macrophages differ from those of their adult counterparts. This protocol also outlines the differentiation of neonatal macrophages from the isolated bone marrow cells in the presence of L929-conditioned medium. Surface markers for differentiated neonatal macrophages were assessed using flow cytometric analysis. To demonstrate functionality, the phagocytic efficiency was also tested using pH-sensitive dye-conjugated Escherichia coli.


Assuntos
Animais Recém-Nascidos , Células da Medula Óssea , Macrófagos , Animais , Camundongos , Macrófagos/citologia , Células da Medula Óssea/citologia , Diferenciação Celular/fisiologia , Técnicas Citológicas/métodos , Citometria de Fluxo/métodos
12.
FASEB J ; 38(13): e23751, 2024 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-38923701

RESUMO

Mesenchymal stem cells (MSCs) reveal multifaceted immunoregulatory properties, which can be applied for diverse refractory and recurrent disease treatment including acute graft-versus-host disease (aGVHD). Distinguishing from MSCs with considerable challenges before clinical application, MSCs-derived exosomes (MSC-Exos) are cell-free microvesicles with therapeutic ingredients and serve as advantageous alternatives for ameliorating the outcomes of aGVHD. MSC-Exos were enriched and identified by western blotting analysis, NanoSight, and transmission electron microscopy (TEM). Bone marrow-derived MSCs (denoted as MSCs) and exosomes (denoted as MSC-Exos) were infused into the aGVHD SD-Wister rat model via tail vein, and variations in general growth and survival of rats were observed. The level of inflammatory factors in serum was quantized by enzyme-linked immunosorbent assay (ELISA). The pathological conditions of the liver and intestine of rats were observed by frozen sectioning. The ratios of CD4+/CD8+ and Treg cell proportions in peripheral blood, together with the autophagy in the spleen and thymus, were analyzed by flow cytometry. After treatment with MSC-Exos, the survival time of aGVHD rats was prolonged, the clinical manifestations of aGVHD in rats were improved, whereas the pathological damage of aGVHD in the liver and intestine was reduced. According to ELISA, we found that MSC-Exos revealed ameliorative effect upon aGVHD inflammation (e.g., TNF-α, IL-2, INF-γ, IL-4, and TGF-ß) compared to the MSC group. After MSC-Exo treatment, the ratio of Treg cells in peripheral blood was increased, whereas the ratio of CD4+/CD8+ in peripheral blood and the autophagy in the spleen and thymus was decreased. MSC-Exos effectively suppressed the activation of immune cells and the manifestation of the inflammatory response in the aGVHD rat model. Our data would supply new references for MSC-Exo-based "cell-free" biotherapy for aGVHD in future.


Assuntos
Exossomos , Doença Enxerto-Hospedeiro , Células-Tronco Mesenquimais , Animais , Exossomos/metabolismo , Doença Enxerto-Hospedeiro/terapia , Ratos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Ratos Wistar , Masculino , Ratos Sprague-Dawley , Transplante de Células-Tronco Mesenquimais/métodos , Linfócitos T Reguladores/imunologia , Células da Medula Óssea/citologia , Autofagia
13.
Cells ; 13(12)2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38920651

RESUMO

Bone formation is a complex process regulated by a variety of pathways that are not yet fully understood. One of the proteins involved in multiple osteogenic pathways is TID (DNAJA3). The aim of this work was to study the association of TID with osteogenesis. Therefore, the expression profiles of the TID splice variants (TID-L, TID-I) and their protein products were analyzed during the proliferation and differentiation of bone marrow mesenchymal stromal cells (B-MSCs) into osteoblasts. As the reference, the hFOB1.19 cell line was used. The phenotype of B-MSCs was confirmed by the presence of CD73, CD90, and CD105 surface antigens on ~97% of cells. The osteoblast phenotype was confirmed by increased alkaline phosphatase activity, calcium deposition, and expression of ALPL and SPP1. The effect of silencing the TID gene on the expression of ALPL and SPP1 was also investigated. The TID proteins and the expression of TID splice variants were detected. After differentiation, the expression of TID-L and TID-I increased 5-fold and 3.7-fold, respectively, while their silencing resulted in increased expression of SPP1. Three days after transfection, the expression of SPP1 increased 7.6-fold and 5.6-fold in B-MSCs and differentiating cells, respectively. Our preliminary study demonstrated that the expression of TID-L and TID-I changes under differentiation of B-MSCs into osteoblasts and may influence the expression of SPP1. However, for better understanding the functional association of these results with the relevant osteogenic pathways, further studies are needed.


Assuntos
Diferenciação Celular , Células-Tronco Mesenquimais , Osteoblastos , Osteogênese , Humanos , Osteoblastos/metabolismo , Osteoblastos/citologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Diferenciação Celular/genética , Osteogênese/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genética , Fosfatase Alcalina/metabolismo , Células da Medula Óssea/metabolismo , Células da Medula Óssea/citologia , Proliferação de Células
14.
Mol Biol Rep ; 51(1): 748, 2024 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-38874843

RESUMO

Background this study was conducted to assess the effects of vitamin D on differentiation of bone marrow- derived mesenchymal stem cells (BM-MSCs) into insulin producing cells (IPCs). Method BM-MSCs were isolated from femur and tibia of rats and incubated in low (LG) or high glucose (HG) (5mM or 25mM), or high glucose DMEM media supplemented with vitamin D (0.2nM) (HGD) for 14 days. Cells viability was analysis by MTT assay. Differentiation of SCs was confirmed using measuring genes expression level of pdx1 and insulin, and insulin secretion, glucose stimulated insulin secretion, and insulin content by ELISA method. Results Cell viability was significantly higher in HGD than LG (p < 0.05) in day 3, also, in HG and HGD than LG (p < 0.001), and HGD vs. HG (p < 0.001) in day 7. Pdx1 and insulin level was markedly higher in HGD than LG (p < 0.05 and p < 0.01). pdx1 expression was markedly higher in HGD (p < 0.05) than LG, also insulin expression the HG (p < 0.05), and HGD (p < 0.01) groups compared to the LG group. Insulin release at 5mM glucose was notably higher in the HGD group compared to LG (p < 0.05), and at 25mM glucose, both HG and HGD showed significant increases vs. LG (p < 0.05 and p < 0.01, respectively). Insulin content was significantly higher in both 5mM and 25mM glucose for HG and HGD vs. LG (p < 0.01 and p < 0.001, respectively). In conclusion, treatment BM-MSCs with vitamin D could increase their differentiation into IPCs and it can be considered as a potential supplementary agent in enhancing differentiation SCs into insulin generating cells.


Assuntos
Células da Medula Óssea , Diferenciação Celular , Células Secretoras de Insulina , Insulina , Células-Tronco Mesenquimais , Vitamina D , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/citologia , Animais , Diferenciação Celular/efeitos dos fármacos , Vitamina D/farmacologia , Vitamina D/metabolismo , Ratos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/citologia , Glucose/metabolismo , Glucose/farmacologia , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética , Células Cultivadas , Sobrevivência Celular/efeitos dos fármacos , Masculino , Transativadores/metabolismo , Transativadores/genética , Suplementos Nutricionais , Secreção de Insulina/efeitos dos fármacos
15.
Cells ; 13(11)2024 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-38891042

RESUMO

The bone marrow (BM) stromal cell microenvironment contains non-hematopoietic stromal cells called mesenchymal stromal cells (MSCs). MSCs are plastic adherent, form CFU-Fs, and give rise to osteogenic, adipogenic, chondrogenic progenitors, and most importantly provide HSC niche factor chemokine C-X-C motif ligand 12 (CXCL12) and stem cell factor (SCF). Different authors have defined different markers for mouse MSC identification like PDGFR+Sca-1+ subsets, Nestin+, or LepR+ cells. Of these, the LepR+ cells are the major source of SCF and CXCL12 in the BM microenvironment and play a major role in HSC maintenance and hematopoiesis. LepR+ cells give rise to most of the bones and BM adipocytes, further regulating the microenvironment. In adult BM, LepR+ cells are quiescent but after fracture or irradiation, they proliferate and differentiate into mesenchymal lineage osteogenic, adipogenic and/or chondrogenic cells. They also play a crucial role in the steady-state hematopoiesis process, as well as hematopoietic regeneration and the homing of hematopoietic stem cells (HSCs) after myeloablative injury and/or HSC transplantation. They line the sinusoidal cavities, maintain the trabeculae formation, and provide the space for HSC homing and retention. However, the LepR+ cell subset is heterogeneous; some subsets have higher adipogenic potential, while others express osteollineage-biased genes. Different transcription factors like Early B cell factor 3 (EBF3) or RunX2 help maintain this balance between the self-renewing and committed states, whether osteogenic or adipogenic. The study of LepR+ MSCs holds immense promise for advancing our understanding of HSC biology, tissue regeneration, metabolic disorders, and immune responses. In this review, we will discuss the origin of the BM resident LepR+ cells, different subtypes, and the role of LepR+ cells in maintaining hematopoiesis, osteogenesis, and BM adipogenesis following their multifaceted impact.


Assuntos
Células-Tronco Mesenquimais , Receptores para Leptina , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Animais , Humanos , Receptores para Leptina/metabolismo , Células da Medula Óssea/metabolismo , Células da Medula Óssea/citologia , Osso e Ossos/metabolismo , Hematopoese , Medula Óssea/metabolismo , Diferenciação Celular
16.
Adipocyte ; 13(1): 2365211, 2024 12.
Artigo em Inglês | MEDLINE | ID: mdl-38858810

RESUMO

microRNAs (miRNAs), a subclass of noncoding short RNAs, direct cells fate decisions that are important for cell proliferation and cell lineage decisions. Adipogenic differentiation contributes greatly to the development of white adipose tissue, involving of highly organized regulation by miRNAs. In the present study, we screened and identified 78 differently expressed miRNAs of porcine BMSCs during adipogenic differentiation. Of which, the role of miR-29c in regulating the proliferation and adipogenic differentiation was proved and detailed. Specifically, over-expression miR-29c inhibits the proliferation and adipogenic differentiation of BMSCs, which were reversed upon miR-29c inhibitor. Interference of IGF1 inhibits the proliferation and adipogenic differentiation of BMSCs. Mechanistically, miR-29c regulates the proliferation and adipogenic differentiation of BMSCs by targeting IGF1 and further regulating the MAPK pathway and the PI3K-AKT-mTOR pathway, respectively. In conclusion, we highlight the important role of miR-29c in regulating proliferation and adipogenic differentiation of BMSCs.


Assuntos
Adipogenia , Diferenciação Celular , Proliferação de Células , Células-Tronco Mesenquimais , MicroRNAs , Animais , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , MicroRNAs/genética , MicroRNAs/metabolismo , Suínos , Adipogenia/genética , Células Cultivadas , Transdução de Sinais , Adipócitos/citologia , Adipócitos/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo
17.
Adipocyte ; 13(1): 2350751, 2024 12.
Artigo em Inglês | MEDLINE | ID: mdl-38860452

RESUMO

INTRODUCTION AND PURPOSE: Mouse mesenchymal stem cells (MSCs) provide a resourceful tool to study physiological and pathological aspects of adipogenesis. Bone marrow-derived MSCs (BM-MSCs) and adipose tissue-derived MSCs (ASCs) are widely used for these studies. Since there is a wide spectrum of methods available, the purpose is to provide a focused hands-on procedural guide for isolation and characterization of murine BM-MSCs and ASCs and to effectively differentiate them into adipocytes. METHODS AND RESULTS: Optimized harvesting procedures for murine BM-MSCs and ASCs are described and graphically documented. Since macrophages reside in bone-marrow and fat tissues and regulate the biological behaviour of BM-MSCs and ASCs, we included a procedure to deplete macrophages from the MSC preparations. The identity and stemness of BM-MSCs and ASCs were confirmed by flow cytometry using established markers. Since the composition and concentrations of adipogenic differentiation cocktails differ widely, we present a standardized four-component adipogenic cocktail, consisting of insulin, dexamethasone, 3-isobutyl-1-methylxanthine, and indomethacin to efficiently differentiate freshly isolated or frozen/thawed BM-MSCs and ASCs into adipocytes. We further included visualization and quantification protocols of the differentiated adipocytes. CONCLUSION: This laboratory protocol was designed as a step-by-step procedure for harvesting murine BM-MSCs and ASCs and differentiating them into adipocytes.


Assuntos
Adipogenia , Tecido Adiposo , Células da Medula Óssea , Diferenciação Celular , Macrófagos , Células-Tronco Mesenquimais , Animais , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Separação Celular/métodos , Adipócitos/citologia , Adipócitos/metabolismo , Células Cultivadas
18.
Biosci Rep ; 44(6)2024 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-38836325

RESUMO

Natural and synthetic polymeric materials, particularly soft and hard tissue replacements, are paramount in medicine. We prepared calcium-incorporated sulfonated polyether-ether ketone (SPEEK) polymer membranes for bone applications. The bioactivity was higher after 21 days of immersion in simulated body fluid (SBF) due to calcium concentration in the membrane. We present a new biomaterial healing system composed of calcium and sulfonated polyether ether ketone (Ca-SPEEK) that can function as a successful biomaterial without causing inflammation when tested on bone marrow cells. The Ca-SPEEK exhibited 13 ± 0.5% clot with low fibrin mesh formation compared to 21 ± 0.5% in SPEEK. In addition, the Ca-SPEEK showed higher protein adsorption than SPEEK membranes. As an inflammatory response, IL-1 and TNF-α in the case of Ca-SPEEK were lower than those for SPEEK. We found an early regulation of IL-10 in the case of Ca-SPEEK at 6 h, which may be attributed to the down-regulation of the inflammatory markers IL-1 and TNF-α. These results evidence the innovative bioactivity of Ca-SPEEK with low inflammatory response, opening venues for bone applications.


Assuntos
Materiais Biocompatíveis , Células da Medula Óssea , Cálcio , Polímeros , Fator de Necrose Tumoral alfa , Animais , Camundongos , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Polímeros/química , Polímeros/farmacologia , Cálcio/metabolismo , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Benzofenonas/química , Benzofenonas/farmacologia , Inflamação/tratamento farmacológico , Polietilenoglicóis/química , Polietilenoglicóis/farmacologia , Cetonas/química , Cetonas/farmacologia , Teste de Materiais , Interleucina-1/metabolismo , Interleucina-10/metabolismo
19.
Int J Mol Sci ; 25(12)2024 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-38928452

RESUMO

Bone marrow mesenchymal stem cells (BMSCs) are key players in promoting ovarian cancer cell proliferation, orchestrated by the dynamic interplay between cytokines and their interactions with immune cells; however, the intricate crosstalk among BMSCs and cytokines has not yet been elucidated. Here, we aimed to investigate interactions between BMSCs and ovarian cancer cells. We established BMSCs with a characterized morphology, surface marker expression, and tri-lineage differentiation potential. Ovarian cancer cells (SKOV3) cultured with conditioned medium from BMSCs showed increased migration, invasion, and colony formation, indicating the role of the tumor microenvironment in influencing cancer cell behavior. BMSCs promoted SKOV3 tumorigenesis in nonobese diabetic/severe combined immunodeficiency mice, increasing tumor growth. The co-injection of BMSCs increased the phosphorylation of p38 MAPK and GSK-3ß in SKOV3 tumors. Co-culturing SKOV3 cells with BMSCs led to an increase in the expression of cytokines, especially MCP-1 and IL-6. These findings highlight the influence of BMSCs on ovarian cancer cell behavior and the potential involvement of specific cytokines in mediating these effects. Understanding these mechanisms will highlight potential therapeutic avenues that may halt ovarian cancer progression.


Assuntos
Proliferação de Células , Citocinas , Células-Tronco Mesenquimais , Neoplasias Ovarianas , Células-Tronco Mesenquimais/metabolismo , Feminino , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Humanos , Animais , Citocinas/metabolismo , Camundongos , Linhagem Celular Tumoral , Técnicas de Cocultura , Microambiente Tumoral , Movimento Celular , Meios de Cultivo Condicionados/farmacologia , Células da Medula Óssea/metabolismo , Camundongos SCID , Camundongos Endogâmicos NOD , Diferenciação Celular
20.
Nat Immunol ; 25(7): 1172-1182, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38871999

RESUMO

Natural killer (NK) cells traffic through the blood and mount cytolytic and interferon-γ (IFNγ)-focused responses to intracellular pathogens and tumors. Type 1 innate lymphoid cells (ILC1s) also produce type 1 cytokines but reside in tissues and are not cytotoxic. Whether these differences reflect discrete lineages or distinct states of a common cell type is not understood. Using single-cell RNA sequencing and flow cytometry, we focused on populations of TCF7+ cells that contained precursors for NK cells and ILC1s and identified a subset of bone marrow lineage-negative NK receptor-negative cells that expressed the transcription factor Eomes, termed EomeshiNKneg cells. Transfer of EomeshiNKneg cells into Rag2-/-Il2rg-/- recipients generated functional NK cells capable of preventing metastatic disease. By contrast, transfer of PLZF+ ILC precursors generated a mixture of ILC1s, ILC2s and ILC3s that lacked cytotoxic potential. These findings identified EomeshiNKneg cells as the bone marrow precursor to classical NK cells and demonstrated that the NK and ILC1 lineages diverged early during development.


Assuntos
Células Matadoras Naturais , Proteínas com Domínio T , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Animais , Proteínas com Domínio T/metabolismo , Proteínas com Domínio T/genética , Camundongos , Camundongos Knockout , Linhagem da Célula/imunologia , Camundongos Endogâmicos C57BL , Imunidade Inata , Diferenciação Celular/imunologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Análise de Célula Única
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...