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1.
Mater Sci Eng C Mater Biol Appl ; 128: 112313, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34474864

RESUMO

Tissue engineering strategies are widely used to model and study the bone marrow microenvironment in healthy and pathological conditions. Yet, while bone function highly depends on mechanical stimulation, the effects of biomechanical stimuli on the bone marrow niche, specifically on bone marrow adipose tissue (BMAT) is poorly understood due to a lack of representative in vitro loading models. Here, we engineered a BMAT analog made of a GelMA (gelatin methacryloyl) hydrogel/medical-grade polycaprolactone (mPCL) scaffold composite to structurally and biologically mimic key aspects of the bone marrow microenvironment, and exploited an innovative bioreactor to study the effects of mechanical loading. Highly reproducible BMAT analogs facilitated the successful adipogenesis of human mesenchymal bone marrow stem cells. Upon long-term intermittent stimulation (1 Hz, 2 h/day, 3 days/week, 3 weeks) in the novel bioreactor, cellular proliferation and lipid accumulation were similar to unloaded controls, yet there was a significant reduction in the secretion of adipokines including leptin and adiponectin, in line with clinical evidence of reduced adipokine expression following exercise/activity. Ultimately, this innovative loading platform combined with reproducibly engineered BMAT analogs provide opportunities to study marrow physiology in greater complexity as it accounts for the dynamic mechanical microenvironment context.


Assuntos
Tecido Adiposo , Medula Óssea , Células da Medula Óssea , Gelatina , Humanos , Engenharia Tecidual
2.
Acta Cytol ; 65(4): 354-357, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34350848

RESUMO

Morphological analysis of the bone marrow is an essential step in the diagnosis of hematological disease. The conventional analysis of bone marrow smears is performed under a manual microscope, which is labor-intensive and subject to interobserver variability. The morphological differential diagnosis of abnormal lymphocytes from normal lymphocytes is still challenging. The digital pathology methods integrated with advances in machine learning enable new diagnostic features/algorithms from digital bone marrow cell images in order to optimize classification, thus providing a robust and faster screening diagnostic tool. We have developed a machine learning system, Morphogo, based on algorithms to discriminate abnormal lymphocytes from normal lymphocytes using digital imaging analysis. We retrospectively reviewed 347 cases of bone marrow digital images. Among them, 53 cases had a clinical history and the diagnosis of marrow involvement with lymphoma was confirmed either by morphology or flow cytometry. We split the 53 cases into two groups for training and testing with 43 and 10 cases, respectively. The selected 15,353 cell images were reviewed by pathologists, based on morphological visual appearance, from 43 patients whose diagnosis was confirmed by complementary tests. To expand the range and the precision of recognizing the lymphoid cells in the marrow by automated digital microscopy systems, we developed an algorithm that incorporated color and texture in addition to geometrical cytological features of the variable lymphocyte images which were applied as the training data set. The selected images from the 10 patients were analyzed by the trained artificial intelligence-based recognition system and compared with the final diagnosis rendered by pathologists. The positive predictive value for the identification of the categories of reactive/normal lymphocytes and abnormal lymphoid cells was 99.04%. It seems likely that further training and improvement of the algorithms will facilitate further subclassification of specific lineage subset pathology, e.g., diffuse large B-cell lymphoma from chronic lymphocytic leukemia/small lymphocytic lymphoma, follicular lymphoma, mantle cell lymphoma or even hairy cell leukemia in cases of abnormal malignant lymphocyte classes in the future. This research demonstrated the feasibility of digital pathology and emerging machine learning approaches to automatically diagnose lymphoma cells in the bone marrow based on cytological-histological analyses.


Assuntos
Células da Medula Óssea/patologia , Exame de Medula Óssea , Diagnóstico por Computador , Interpretação de Imagem Assistida por Computador , Linfócitos/patologia , Linfoma/patologia , Aprendizado de Máquina , Microscopia , Humanos , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Estudos Retrospectivos
3.
Eur J Med Res ; 26(1): 94, 2021 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-34407888

RESUMO

BACKGROUND: The purpose of present study was to explore the mechanism of nuclear factor-kappa B (NF-κB), phosphatidylinositol 3-kinase (PI3K)/protein kinase B(PKB/Akt) and mitogen-activated protein kinase (MAPK) signaling pathways after intervention of advanced glycosylation end products (AGEs) on rat bone-marrow stromal cells (BMSCs). METHODS: Prepare and identify AGEs. BMSCs were isolated from 16 SD rats and cultured with different concentration of AGEs. Cell viability was detected by cell counting kit-8 (CCK-8). BMSCs were cultured with AGEs (0.25 mg/ml) for 30 min, 12 h, 24 h, 72 h and 120 h. In addition, BMSCs were cultured with AGEs, AGEs + JNK inhibitor and AGEs + P38 inhibitor for 24 h and 48 h, respectively. Western blotting and RT-PCR were used to determine the protein and mRNA expression levels, respectively. RESULTS: Cell viability of BMSCs was significantly correlated with concentration and effect time of AGEs (P < 0.05), and the most appropriate concentration was 0.25 mg/ml. AGEs stimulation significantly increased the protein expression levels of NF-κB p65, JNK, p38 (P < 0.05), decreased IκB (P < 0.05), but had no effect on the protein expression of Akt in BMSCs (P > 0.05). At the mRNA level, JNK and p38 inhibitors significantly reduced the levels of NF-κB p65, p38 and JNK, increased IκB (P > 0.05), but had no effect on Akt in BMSCs (P > 0.05). At the protein level, JNK and p38 inhibitors notably decreased the expression of NF-κB p65, p38, p-JNK, P-IκB and JNK (P < 0.001), and increased IκB (P < 0.05). CONCLUSION: Advanced glycosylation end products can inhibit the proliferation of bone-marrow stromal cells through activating MAPK pathway.


Assuntos
Células da Medula Óssea/metabolismo , Proliferação de Células , Produtos Finais de Glicação Avançada/metabolismo , Sistema de Sinalização das MAP Quinases , Células-Tronco Mesenquimais/metabolismo , Animais , Células da Medula Óssea/fisiologia , Células Cultivadas , MAP Quinase Quinase 4/metabolismo , Masculino , Células-Tronco Mesenquimais/fisiologia , NF-kappa B/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
4.
Arch Oral Biol ; 130: 105243, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34416564

RESUMO

OBJECTIVES: The aims of this study were to explore: (ⅰ) the effect of the polypeptide OP 3-4 on bone regeneration in vivo; (ⅱ) the effect of OP 3-4 on osteogenic differentiation of bone marrow mesenchymal stem cells in vitro; and (ⅲ) the potential mechanism of OP 3-4 in promoting osteogenic differentiation of bone marrow mesenchymal stem cells. DESIGNS: 30 Wistar rats (8-week, male) were randomly divided into Control group (n = 5), Hydrogel group (n = 5), and Hydrogel loaded OP 3-4 group (n = 5). Hematoxylin and eosin staining was used to evaluate the level of bone regeneration in mandibular defect. Immunohistochemistry staining was used to evaluate the expression of alkaline phosphatase, runt-related transcription factor 2, and type Ⅰ collagen. Flow cytometry was applied to identify the phenotype of bone marrow mesenchymal stem cells. Furthermore, LY294002, the inhibitor of protein kinase B, was applied to verify the role of OP 3-4 in promoting osteogenic differentiation via protein kinase B/glycogen synthase kinase 3ß/ß-catenin pathway through western blot. RESULTS: OP 3-4 promoted bone regeneration of rat mandibular defect. The expression of osteogenic differentiation related markers were increased after adding OP 3-4 to bone marrow mesenchymal stem cells. OP 3-4 promoted osteogenic differentiation of bone marrow mesenchymal stem cells via protein kinase B/glycogen synthase kinase 3ß/ß-catenin pathway. CONCLUSION: OP 3-4 could promote bone regeneration of mandibular defect and improve osteogenic differentiation through protein kinase B/glycogen synthase kinase 3ß/ß-catenin pathway.


Assuntos
Células-Tronco Mesenquimais , Osteogênese , Animais , Células da Medula Óssea/metabolismo , Regeneração Óssea , Cateninas , Diferenciação Celular , Células Cultivadas , Glicogênio Sintase Quinase 3 beta , Masculino , Células-Tronco Mesenquimais/metabolismo , Proteínas Proto-Oncogênicas c-akt , Ratos , Ratos Wistar , Via de Sinalização Wnt , beta Catenina/metabolismo
5.
Stem Cell Res Ther ; 12(1): 460, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34407863

RESUMO

BACKGROUND: Female sex hormone secretion and reproductive ability decrease with ageing. Bone marrow mesenchymal stem cells (BMMSCs) have been postulated to play a key role in treating ovarian ageing. METHODS: We used macaque ovarian ageing models to observe the structural and functional changes after juvenile BMMSC treatment. Moreover, RNA-seq was used to analyse the ovarian transcriptional expression profile and key pathways through which BMMSCs reverse ovarian ageing. RESULTS: In the elderly macaque models, the ovaries were atrophied, the regulation ability of sex hormones was reduced, the ovarian structure was destroyed, and only local atretic follicles were observed, in contrast with young rhesus monkeys. Intravenous infusion of BMMSCs in elderly macaques increased ovarian volume, strengthened the regulation ability of sex hormones, reduced the degree of pulmonary fibrosis, inhibited apoptosis, increased density of blood vessels, and promoted follicular regeneration. In addition, the ovarian expression characteristics of ageing-related genes of the elderly treatment group reverted to that of the young control group, 1258 genes that were differentially expressed, among which 415 genes upregulated with age were downregulated, 843 genes downregulated with age were upregulated after BMMSC treatment, and the top 20 differentially expressed genes (DEGs) in the protein-protein interaction (PPI) network were significantly enriched in oocyte meiosis and progesterone-mediated oocyte maturation pathways. CONCLUSION: The BMMSCs derived from juvenile macaques can reverse ovarian ageing in elderly macaques.


Assuntos
Células-Tronco Mesenquimais , Animais , Células da Medula Óssea , Senescência Celular , Feminino , Macaca mulatta , Folículo Ovariano , Ovário
6.
Stem Cell Res Ther ; 12(1): 448, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34372911

RESUMO

BACKGROUND: The skeletal muscle reconstruction occurs thanks to unipotent stem cells, i.e., satellite cells. The satellite cells remain quiescent and localized between myofiber sarcolemma and basal lamina. They are activated in response to muscle injury, proliferate, differentiate into myoblasts, and recreate myofibers. The stem and progenitor cells support skeletal muscle regeneration, which could be disturbed by extensive damage, sarcopenia, cachexia, or genetic diseases like dystrophy. Many lines of evidence showed that the level of oxygen regulates the course of cell proliferation and differentiation. METHODS: In the present study, we analyzed hypoxia impact on human and pig bone marrow-derived mesenchymal stromal cell (MSC) and mouse myoblast proliferation, differentiation, and fusion. Moreover, the influence of the transplantation of human bone marrow-derived MSCs cultured under hypoxic conditions on skeletal muscle regeneration was studied. RESULTS: We showed that bone marrow-derived MSCs increased VEGF expression and improved myogenesis under hypoxic conditions in vitro. Transplantation of hypoxia preconditioned bone marrow-derived MSCs into injured muscles resulted in the improved cell engraftment and formation of new vessels. CONCLUSIONS: We suggested that SDF-1 and VEGF secreted by hypoxia preconditioned bone marrow-derived MSCs played an essential role in cell engraftment and angiogenesis. Importantly, hypoxia preconditioned bone marrow-derived MSCs more efficiently engrafted injured muscles; however, they did not undergo myogenic differentiation.


Assuntos
Células-Tronco Mesenquimais , Animais , Medula Óssea , Células da Medula Óssea , Diferenciação Celular , Células Cultivadas , Hipóxia , Camundongos , Músculo Esquelético , Mioblastos , Células-Tronco , Suínos
8.
Nat Immunol ; 22(9): 1083-1092, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34429552

RESUMO

For decades, it was commonly accepted that the brain is secluded from peripheral immune activity and is self-sufficient for its maintenance and repair. This simplistic perception was based on the presence of resident immune cells, the microglia, and barrier systems within the brain, and the assumption that the central nervous system (CNS) lacks lymphatic drainage. This view was revised with the discoveries that higher functions of the CNS, homeostasis and repair are supported by peripheral innate and adaptive immune cells. The findings of bone marrow-derived immune cells in specialized niches, and the renewed observation that a lymphatic drainage system exists within the brain, further contributed to this revised model. In this Review, we describe the immune niches within the brain, the contribution of professional immune cells to brain functions, the bidirectional relationships between the CNS and the immune system and the relevance of immune components to brain aging and neurodegenerative diseases.


Assuntos
Encéfalo/imunologia , Imunidade/fisiologia , Microglia/imunologia , Doenças Neurodegenerativas/imunologia , Envelhecimento/imunologia , Barreira Hematoencefálica/imunologia , Células da Medula Óssea/imunologia , Líquido Cefalorraquidiano/citologia , Líquido Cefalorraquidiano/imunologia , Humanos , Subpopulações de Linfócitos/imunologia , Macrófagos/imunologia
9.
FASEB J ; 35(9): e21840, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34423881

RESUMO

With an aging world population, there is an increased risk of fracture and impaired healing. One contributing factor may be aging-associated decreases in vascular function; thus, enhancing angiogenesis could improve fracture healing. Both bone morphogenetic protein 2 (BMP-2) and thrombopoietin (TPO) have pro-angiogenic effects. The aim of this study was to investigate the effects of treatment with BMP-2 or TPO on the in vitro angiogenic and proliferative potential of endothelial cells (ECs) isolated from lungs (LECs) or bone marrow (BMECs) of young (3-4 months) and old (22-24 months), male and female, C57BL/6J mice. Cell proliferation, vessel-like structure formation, migration, and gene expression were used to evaluate angiogenic properties. In vitro characterization of ECs generally showed impaired vessel-like structure formation and proliferation in old ECs compared to young ECs, but improved migration characteristics in old BMECs. Differential sex-based angiogenic responses were observed, especially with respect to drug treatments and gene expression. Importantly, these studies suggest that NTN1, ROBO2, and SLIT3, along with angiogenic markers (CD31, FLT-1, ANGPT1, and ANGP2) differentially regulate EC proliferation and functional outcomes based on treatment, sex, and age. Furthermore, treatment of old ECs with TPO typically improved vessel-like structure parameters, but impaired migration. Thus, TPO may serve as an alternative treatment to BMP-2 for fracture healing in aging owing to improved angiogenesis and fracture healing, and the lack of side effects associated with BMP-2.


Assuntos
Envelhecimento , Células da Medula Óssea/citologia , Proteína Morfogenética Óssea 2/farmacologia , Células Endoteliais/efeitos dos fármacos , Pulmão/citologia , Neovascularização Fisiológica/efeitos dos fármacos , Caracteres Sexuais , Trombopoetina/farmacologia , Indutores da Angiogênese/metabolismo , Animais , Biomarcadores/metabolismo , Movimento Celular , Proliferação de Células , Células Endoteliais/citologia , Feminino , Consolidação da Fratura/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL
10.
Int J Mol Sci ; 22(15)2021 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-34361085

RESUMO

A novel aptamer-based competitive drug screening platform for osteoporosis was devised in which fluorescence-labeled, sclerostin-specific aptamers compete with compounds from selected chemical libraries for the binding of immobilized recombinant human sclerostin to achieve high-throughput screening for potential small-molecule sclerostin inhibitors and to facilitate drug repurposing and drug discovery. Of the 96 selected inhibitors and FDA-approved drugs, six were shown to result in a significant decrease in the fluorescence intensity of the aptamer, suggesting a higher affinity toward sclerostin compared with that of the aptamer. The targets of these potential sclerostin inhibitors were correlated to lipid or bone metabolism, and several of the compounds have already been shown to be potential osteogenic activators, indicating that the aptamer-based competitive drug screening assay offered a potentially reliable strategy for the discovery of target-specific new drugs. The six potential sclerostin inhibitors suppressed the level of both intracellular and/or extracellular sclerostin in mouse osteocyte IDG-SW3 and increased alkaline phosphatase activity in IDG-SW3 cells, human bone marrow-derived mesenchymal stem cells and human fetal osteoblasts hFOB1.19. Potential small-molecule drug candidates obtained in this study are expected to provide new therapeutics for osteoporosis as well as insights into the structure-activity relationship of sclerostin inhibitors for rational drug design.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Aptâmeros de Nucleotídeos/química , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteócitos/efeitos dos fármacos , Osteoporose/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Aptâmeros de Nucleotídeos/isolamento & purificação , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteócitos/citologia , Osteócitos/metabolismo , Osteoporose/metabolismo , Osteoporose/patologia
11.
Am J Vet Res ; 82(9): 770-776, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34432512

RESUMO

OBJECTIVE: To characterize the ultrastructure of mesenchymal stem cells (MSCs) that were harvested from the adipose tissue (AT-MSCs) and bone marrow (BM-MSCs) of horses and transfected with green fluorescent protein. SAMPLE: MSCs from adipose tissue and bone marrow of 6 adult female Hispano-Bretón horses. PROCEDURES: Harvested equine MSCs were cultivated and transfected with green fluorescent protein, and the immunophenotypes of the MSCs were characterized by use of anti-CD90 and anti-CD105 monoclonal antibodies. When stable transfection of MSCs was achieved, the morphological and ultrastructural characteristics of transfected and nontransfected AT-MSCs and BM-MSCs were compared with electron microscopy. RESULTS: The protocols for transfection and subsequent isolation of transfected cells with use of G418 were suitable for obtaining transfected MSCs. Transfection efficiency was 5% in AT-MSCs and 4% in BM-MSCs. Characterization of transfected and nontransfected MSCs revealed that they share immunocytochemical and morphological profiles. Expression of CD90 was significantly higher for transfected versus nontransfected AT-MSCs (97% vs 92%). Expression of CD105 was significantly lower for transfected versus nontransfected BM-MSCs (85% vs 94%). Transfected BM-MSCs had differences in organelles, compared with the other cell types, specifically including most commonly the rough endoplasmic reticulum with dilated cisternae and mitochondria. CONCLUSION AND CLINICAL RELEVANCE: These findings contribute to the knowledge base of the characteristics of equine AT-MSCs and BM-MSCs and of transfected versus nontransfected equine MSCs. The data provided a valuable starting point for researchers wishing to further study the morphological characteristics of equine MSCs.


Assuntos
Células-Tronco Mesenquimais , Tecido Adiposo , Animais , Medula Óssea , Células da Medula Óssea , Feminino , Proteínas de Fluorescência Verde/genética , Cavalos
12.
Blood Adv ; 5(16): 3120-3133, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34406376

RESUMO

How hematopoietic stem cells (HSCs) coordinate their divisional axis and whether this orientation is important for stem cell-driven hematopoiesis is poorly understood. Single-cell RNA sequencing data from patients with Shwachman-Diamond syndrome (SDS), an inherited bone marrow failure syndrome, show that ARHGEF2, a RhoA-specific guanine nucleotide exchange factor and determinant of mitotic spindle orientation, is specifically downregulated in SDS hematopoietic stem and progenitor cells (HSPCs). We demonstrate that transplanted Arhgef2-/- fetal liver and bone marrow cells yield impaired hematopoietic recovery and a production deficit from long-term HSCs, phenotypes that are not the result of differences in numbers of transplanted HSCs, their cell cycle status, level of apoptosis, progenitor output, or homing ability. Notably, these defects are functionally restored in vivo by overexpression of ARHGEF2 or its downstream activated RHOA GTPase. By using live imaging of dividing HSPCs, we show an increased frequency of misoriented divisions in the absence of Arhgef2. ARHGEF2 knockdown in human HSCs also impairs their ability to regenerate hematopoiesis, culminating in significantly smaller xenografts. Together, these data demonstrate a conserved role for Arhgef2 in orienting HSPC division and suggest that HSCs may divide in certain orientations to establish hematopoiesis, the loss of which could contribute to HSC dysfunction in bone marrow failure.


Assuntos
Hematopoese , Células-Tronco Hematopoéticas , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Apoptose , Células da Medula Óssea , Humanos , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fuso Acromático
13.
Gen Physiol Biophys ; 40(4): 329-339, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34350837

RESUMO

In this study, we aimed to identify the specific microRNAs (miRNAs) that are involved in the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) from ovariectomized (OVX) mice, and to further explore the mechanism by which these miRNAs regulate osteogenic differentiation. Based on the existing studies, the expression of seven miRNAs in BMSCs from OVX mice was evaluated using quantitative reverse transcription polymerase chain reaction (qRT-PCR). The expression of miR-133a-3p and osteogenesis-related genes (runt-related transcription factor 2 (Runx2), Osterix, alkaline phosphatase (ALP), and osteopontin) in BMSCs treated with miR-133a-3p mimics or inhibitors was detected by qRT-PCR or Western blotting. Osteogenesis efficiency was determined using ALP and alizarin red staining. The effector-target relationship between miR-133a-3p and ankyrin repeat domain 44 (ANKRD44) was confirmed by bioinformatics and a dual luciferase assay. Among the seven selected miRNAs, miR-133a-3p expression was significantly increased in BMSCs from OVX mice. Overexpression of miR-133a-3p dramatically inhibited the expression of osteogenesis-related genes in BMSCs and reduced ALP activity and mineralization. However, these processes were markedly ameliorated upon miR-133a-3p inhibition. Moreover, miR-133a-3p appeared to target ANKRD44, and the ANKRD44 expression was negatively regulated by miR-133a- 3p. Furthermore, ANKRD44 upregulation eliminated the anti-osteogenic differentiation effects of miR-133a-3p in BMSCs. Thus, our results indicated that miR-133a-3p inhibits the osteogenic differentiation of BMSCs by suppressing ANKRD44.


Assuntos
Células-Tronco Mesenquimais , MicroRNAs , Animais , Repetição de Anquirina , Células da Medula Óssea , Diferenciação Celular , Células Cultivadas , Camundongos , MicroRNAs/genética , Osteogênese/genética
14.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 29(4): 1224-1230, 2021 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-34362506

RESUMO

OBJECTIVE: To analyze the proliferation potential of bone marrow-derived mesenchymal stem cells (MSC) in patients with myelodysplastic syndrome (MDS). METHODS: The MSC derived from the 24 patients with newly diagnosed MDS (MDS-MSC group) and MSC derived from 15 patients with nutritional anemia (control group) in the Affiliated Hospital of Hebei University were used as the research objects. The proliferation potential of MSC was analyzed by colony-forming unit assay, doubling time, cumulative passaging, cell number after 10 days of culture with equal amount of MSC and MTT experiment. The mechanism of abnormal proliferation was analyzed by cell cycle experiment, apoptosis experiment and p21 gene expression assay. RESULTS: In the colony forming unit assay, the number of MDS-MSC colonies was 4.44±2.51, which was significantly lower than that of the control group (12.44±2.55)(P<0.01); the doubling time of MDS-MSC group was significantly longer than that of the control group (7.80±3.26 vs 3.63±0.85) (P<0.01); the number of MDS-MSC in 5×104 culture for 10 days was (39.40±14.18)×104, which was significantly lower than that of the control group ï¼»(85.30±9.49)×104 ï¼½(P<0.01); the number of cumulative passages in MDS-MSC group was 5.20±1.40, which was significantly lower than that in control group (11.60±1.96)(P<0.01); MTT results showed that the proliferation capability of MSC in MDS-MSC group was lower than that in the control group. The cell proportion of G0/G1 phase in MDS-MSC group was higher than that in the control group, while the cell proportion of S phase was lower (P<0.05). The percentage of early apoptotic cells in MDS-MSC group was higher than that in control group (P<0.05); the relative expression level of p21 mRNA in MDS-MSC group was significantly higher than that in control group(P<0.01). CONCLUSION: The proliferative capability of MDS-MSC is significantly reduced, which relates with the arrest of cell cycle in G0/G1 phase, the increase of early apoptotic cells and senescent cells of the MDS-MSC.


Assuntos
Células-Tronco Mesenquimais , Síndromes Mielodisplásicas , Apoptose , Células da Medula Óssea , Proliferação de Células , Humanos
15.
Nucleic Acids Res ; 49(15): 8505-8519, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34320202

RESUMO

The transcriptomic diversity of cell types in the human body can be analysed in unprecedented detail using single cell (SC) technologies. Unsupervised clustering of SC transcriptomes, which is the default technique for defining cell types, is prone to group cells by technical, rather than biological, variation. Compared to de-novo (unsupervised) clustering, we demonstrate using multiple benchmarks that supervised clustering, which uses reference transcriptomes as a guide, is robust to batch effects and data quality artifacts. Here, we present RCA2, the first algorithm to combine reference projection (batch effect robustness) with graph-based clustering (scalability). In addition, RCA2 provides a user-friendly framework incorporating multiple commonly used downstream analysis modules. RCA2 also provides new reference panels for human and mouse and supports generation of custom panels. Furthermore, RCA2 facilitates cell type-specific QC, which is essential for accurate clustering of data from heterogeneous tissues. We demonstrate the advantages of RCA2 on SC data from human bone marrow, healthy PBMCs and PBMCs from COVID-19 patients. Scalable supervised clustering methods such as RCA2 will facilitate unified analysis of cohort-scale SC datasets.


Assuntos
Algoritmos , Análise por Conglomerados , RNA Citoplasmático Pequeno/genética , RNA-Seq/métodos , Análise de Célula Única/métodos , Animais , Artrite Reumatoide/genética , Células da Medula Óssea/metabolismo , COVID-19/sangue , COVID-19/patologia , Estudos de Coortes , Conjuntos de Dados como Assunto , Humanos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Camundongos , Especificidade de Órgãos , Controle de Qualidade , RNA-Seq/normas , Análise de Célula Única/normas , Transcriptoma
16.
Stem Cell Res Ther ; 12(1): 382, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34233721

RESUMO

BACKGROUND: Tissue-engineered bone grafts (TEBGs) that undergo vascularization and neurotization evolve into functioning bone tissue. Previously, we verified that implanting sensory nerve tracts into TEBGs promoted osteogenesis. However, the precise mechanisms and interaction between seed cells were not explored. In this study, we hypothesized that neurotization may influence the osteogenesis of TEBGs through vascularization. METHODS: We cultured rat Schwann cells (SCs), aortic endothelial cells (AECs), and bone marrow-derived mesenchymal stem cells (BM-MSCs) and then obtained BM-MSC-derived induced endothelial cells (IECs) and induced osteoblasts (IOBs). IECs and AECs were cultured in an SC-conditioned medium (SC-CM) to assess proliferation, migration, capillary-like tube formation, and angiogenesis, and the vascular endothelial growth factor (VEGF) levels in the supernatants were detected. We established an indirect coculture model to detect the expression of nestin and VEGF receptors in IECs and tissue inhibitor of metalloproteinase (TIMP)-2 in SCs. Then, SCs, IECs, and IOBs were labeled and loaded into a ß-tricalcium phosphate scaffold to induce prevascularization, and the scaffold was implanted into a 6-mm-long defect of rat femurs. Three groups were set up according to the loaded cells: I, SCs, and IECs (coculture for 3 days) plus IOBs; II, IECs (culture for 3 days) plus IOBs; III, IOBs. Nestin and TIMP-2 expression and osteogenesis of TEBGs were evaluated at 12 weeks post-implantation through histological and radiological assessments. RESULTS: We found that SC-CM promoted IEC proliferation, migration, capillary-like tube formation, and angiogenesis, but no similar effects were observed for AECs. IECs expressed nestin extensively, while AECs barely expressed nestin, and SC-CM promoted the VEGF secretion of IECs. In the coculture model, SCs promoted nestin and VEGF receptor expression in IECs, and IECs inhibited TIMP-2 expression in SCs. The promotion of prevascularized TEBGs by SCs and IECs in group I augmented new bone formation at 6 and 12 weeks. Nestin expression was higher in group I than in the other groups, while TIMP-2 expression was lower at 12 weeks. CONCLUSIONS: This study demonstrated that SCs can promote TEBG osteogenesis via IECs and further revealed the related specific characteristics of IECs, providing preliminary cytological evidence for neurotization of TEBGs.


Assuntos
Células Endoteliais , Células-Tronco Mesenquimais , Osteogênese , Células de Schwann , Engenharia Tecidual , Animais , Células da Medula Óssea , Osso e Ossos , Neovascularização Fisiológica , Nestina , Ratos , Inibidor Tecidual de Metaloproteinase-2 , Fator A de Crescimento do Endotélio Vascular/genética
17.
Int J Mol Sci ; 22(13)2021 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-34206957

RESUMO

In recent decades, the conduct of uniform prospective clinical trials has led to improved remission rates and survival for patients with acute myeloid leukaemia and acute lymphoblastic leukaemia. However, high-risk patients continue to have inferior outcomes, where chemoresistance and relapse are common due to the survival mechanisms utilised by leukaemic cells. One such mechanism is through hijacking of the bone marrow microenvironment, where healthy haematopoietic machinery is transformed or remodelled into a hiding ground or "sanctuary" where leukaemic cells can escape chemotherapy-induced cytotoxicity. The bone marrow microenvironment, which consists of endosteal and vascular niches, can support leukaemogenesis through intercellular "crosstalk" with niche cells, including mesenchymal stem cells, endothelial cells, osteoblasts, and osteoclasts. Here, we summarise the regulatory mechanisms associated with leukaemia-bone marrow niche interaction and provide a comprehensive review of the key therapeutics that target CXCL12/CXCR4, Notch, Wnt/b-catenin, and hypoxia-related signalling pathways within the leukaemic niches and agents involved in remodelling of niche bone and vasculature. From a therapeutic perspective, targeting these cellular interactions is an exciting novel strategy for enhancing treatment efficacy, and further clinical application has significant potential to improve the outcome of patients with leukaemia.


Assuntos
Leucemia/tratamento farmacológico , Microambiente Tumoral/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Humanos , Leucemia/patologia , Transdução de Sinais
18.
Anticancer Res ; 41(8): 3891-3898, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34281851

RESUMO

BACKGROUND/AIM: Matrix metalloproteinases (MMPs) degrade extracellular matrix and process regulatory proteins. Recently, a membrane-bound 82kDa variant of proMMP-9 identified on myeloid blasts was shown to be associated with prognosis. PATIENTS AND METHODS: To investigate the role of 82kDa proMMP-9 with acute lymphoblastic leukemia (ALL) and chronic lymphoid leukemia (CLL), we performed flow-cytometry analysis of expression on ALL blasts (n=18) and CLL lymphocytes (n=21) from blood and correlated data with clinical parameters. RESULTS: In ALL, mature B-linear blasts expressed higher levels of 82kDa proMMP-9 compared to T-linear blasts. Elevated levels of 82kDa proMMP-9 were found in elderly patients and at patients with relapse. No correlation was observed on blood cells and extramedullary disease. In CLL, the 82kDa proMMP-9 expression did not correlate with any of the clinical parameters. CONCLUSION: Our findings suggest that higher levels of 82kDa proMMP-9 expression on blast cells may correlate with a more unfavorable ALL-subtype. Further studies are required to clarify the prognostic role of the 82kDa pro-MMP-9 expression.


Assuntos
Precursores Enzimáticos/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Linfócitos/imunologia , Metaloproteinase 9 da Matriz/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Adolescente , Adulto , Idoso , Células da Medula Óssea/citologia , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Adulto Jovem
19.
Aging (Albany NY) ; 13(13): 17190-17201, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34229300

RESUMO

Emerging evidence proves that exosomes contain specific microRNAs(miRNAs) contribute to osteogenic differentiation of bone marrow stem cells (BMSCs). However, the role and mechanism of bone marrow stem cells (BMSCs)-derived exosomes overexpressing miR-424-5p in osteoblasts remains unclear. Firstly, the BMSCs-derived exosomes were isolated, and identified by Western blot with the exosome surface markers CD9, CD81 and CD63. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to detect the level of miR-424-5p in exosomes, and western blot was implemented to verify the WIF1/Wnt/ß-catenin expression. The binding association between miR-424-5p and WIF1 was determined by the dual-luciferase reporter gene assay. Functional enhancement experiments were adopted to determine the role of exosome-carried miR-424-5p and WIF1/Wnt/ß-catenin in osteogenic differentiation. ALP staining was adopted, and levels of RUNX2, OCN, and OPN were monitored using qRT-PCR to determine osteogenic differentiation. As a result, In vivo experiments showed that RUNX2, OCN and OPN levels decreased and the ALP activity was dampened after miR-424-5p overexpression in exosomes. Besides, exosomes overexpressing miR-424-5p attenuated osteogenic development via WIF1/Wnt/ß-catenin. Our findings may bring evidence for miR-424-5p as a new biomarker for the treatment of osteoporosis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Exossomos/metabolismo , MicroRNAs/genética , Osteoblastos/metabolismo , Osteogênese/genética , Células-Tronco/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Humanos , Osteocalcina/genética
20.
Aging (Albany NY) ; 13(13): 16938-16956, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34292877

RESUMO

Macrophage accumulation and nitrosative stress are known mechanisms underlying age-related cardiovascular pathology and functional decline. The cardiac muscle microenvironment is known to change with age, yet the direct effects of these changes have yet to be studied in-depth. The present study sought to better elucidate the role that biochemical and biomechanical alterations in cardiac tissue have in the altered phenotype and functionality of cardiac resident macrophages observed with increasing age. To accomplish this, naïve bone marrow derived macrophages from young mice were seeded onto either functionalized poly-dimethyl-siloxane hydrogels ranging in stiffness from 2kPA to 64kPA or onto tissue culture plastic, both of which were coated with either young or aged solubilized mouse cardiac extracellular matrix (cECM). Both biomechanical and biochemical alterations were found to have a significant effect on macrophage polarization and function. Increased substrate stiffness was found to promote macrophage morphologies associated with pro-inflammatory macrophage activation, increased expression of pro-inflammatory inducible nitric oxide synthase protein with increased nitric oxide secretion, and attenuated arginase activity and protein expression. Additionally, exposure to aged cECM promoted attenuated responsivity to both canonical pro-inflammatory and anti-inflammatory cytokine signaling cues when compared to young cECM treated cells. These results suggest that both biomechanical and biochemical changes in the cardiovascular system play a role in promoting the age-related shift towards pro-inflammatory macrophage populations associated with cardiovascular disease development.


Assuntos
Microambiente Celular/fisiologia , Coração/fisiologia , Macrófagos/fisiologia , Macrófagos/ultraestrutura , Envelhecimento/patologia , Envelhecimento/fisiologia , Animais , Arginase/metabolismo , Fenômenos Biomecânicos , Células da Medula Óssea , Citocinas/metabolismo , DNA/biossíntese , Ativação de Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/patologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/biossíntese , Fenótipo , Transdução de Sinais , Técnicas de Cultura de Tecidos
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