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1.
BMC Bioinformatics ; 20(1): 369, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31262249

RESUMO

BACKGROUND: Single cell RNA sequencing (scRNA-seq) brings unprecedented opportunities for mapping the heterogeneity of complex cellular environments such as bone marrow, and provides insight into many cellular processes. Single cell RNA-seq has a far larger fraction of missing data reported as zeros (dropouts) than traditional bulk RNA-seq, and unsupervised clustering combined with Principal Component Analysis (PCA) can be used to overcome this limitation. After clustering, however, one has to interpret the average expression of markers on each cluster to identify the corresponding cell types, and this is normally done by hand by an expert curator. RESULTS: We present a computational tool for processing single cell RNA-seq data that uses a voting algorithm to automatically identify cells based on approval votes received by known molecular markers. Using a stochastic procedure that accounts for imbalances in the number of known molecular signatures for different cell types, the method computes the statistical significance of the final approval score and automatically assigns a cell type to clusters without an expert curator. We demonstrate the utility of the tool in the analysis of eight samples of bone marrow from the Human Cell Atlas. The tool provides a systematic identification of cell types in bone marrow based on a list of markers of immune cell types, and incorporates a suite of visualization tools that can be overlaid on a t-SNE representation. The software is freely available as a Python package at https://github.com/sdomanskyi/DigitalCellSorter . CONCLUSIONS: This methodology assures that extensive marker to cell type matching information is taken into account in a systematic way when assigning cell clusters to cell types. Moreover, the method allows for a high throughput processing of multiple scRNA-seq datasets, since it does not involve an expert curator, and it can be applied recursively to obtain cell sub-types. The software is designed to allow the user to substitute the marker to cell type matching information and apply the methodology to different cellular environments.


Assuntos
Células da Medula Óssea/citologia , Perfilação da Expressão Gênica/métodos , Análise de Sequência de RNA/métodos , Software , Algoritmos , Células da Medula Óssea/metabolismo , Análise por Conglomerados , Humanos , Análise de Componente Principal , Análise de Célula Única
3.
Gene ; 711: 143937, 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31228541

RESUMO

BACKGROUND & OBJECTIVES: Vaginal atrophy is characterized by thinning of vaginal epithelial layers and decreased local blood flow. We aimed to evaluate the regenerative effects of Adipose derived mesenchymal stem cells (ADMSC) and Bone marrow derived mesenchymal stem cells (BMDSC) on vaginal atrophy in rat menopause model. MATERIALS AND METHODS: Rats were randomly divided into 4 (four) groups: sham, control, ADMSC, BMDSC. Vaginal epithelial thickness, structure of the lamina propria, blood vessels in the lamina propria, collagen deposition, and muscle structure were evaluated. Anti ER α, VEGF, VEGFR 1, Bax and bcl-2 antibodies were analyzed. Beta actin gene was used as endogenous control. Genetical differences among the groups were compared by using Kruskal Wallis and Mann Whitney U test. p < 0.05 was regarded as statistically significant. RESULTS: Epithelial thickness of ADMSC group was higher than control group, but less than sham group Epithelial thickness of BMDSC group was less than sham group. Lamina propria and muscle tissue of ADMSC and BMDSC groups were found to be similar to sham group. VEGFR-1, VEGF, Bax and ER-α staining levels were higher in ADMSC and BMDSC groups than control group. ADMSC group stained stronger with VEGFR-1 and VEGF than BMDSC group. Bcl-2 staining level was increased in ADMSC applied group. No statistically significant difference was detected in Bax and Bcl-2 genes and Bax-/Bcl-2 ratio. CONCLUSIONS: Although genetic expression might have ended and could not be significantly demonstrated, histological and immunohistochemical results favor ADMSC application in vaginal atrophy rather than BMDSC.


Assuntos
Tecido Adiposo/citologia , Biomarcadores/metabolismo , Células da Medula Óssea/citologia , Menopausa/fisiologia , Transplante de Células-Tronco Mesenquimais/métodos , Vagina/patologia , Tecido Adiposo/metabolismo , Animais , Atrofia , Células da Medula Óssea/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Menopausa/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Ratos , Vagina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteína X Associada a bcl-2/metabolismo
4.
Nat Immunol ; 20(7): 852-864, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31213723

RESUMO

Dendritic cells (DC) are currently classified as conventional DCs (cDCs) and plasmacytoid DCs (pDCs). Through a combination of single-cell transcriptomic analysis, mass cytometry, in vivo fate mapping and in vitro clonal assays, here we show that, at the single-cell level, the priming of mouse hematopoietic progenitor cells toward the pDC lineage occurs at the common lymphoid progenitor stage, indicative of early divergence of the pDC and cDC lineages. We found the transcriptional signature of a pDC precursor stage, defined here, in the IL-7Rα+ common lymphoid progenitor population and identified Ly6D, IL-7Rα, CD81 and CD2 as key markers of pDC differentiation, which distinguish pDC precursors from cDC precursors. In conclusion, pDCs developed in the bone marrow from a Ly6DhiCD2hi lymphoid progenitor cell and differentiated independently of the myeloid cDC lineage.


Assuntos
Antígenos Ly/metabolismo , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Células Progenitoras Linfoides/citologia , Células Progenitoras Linfoides/metabolismo , Células Progenitoras Mieloides/citologia , Células Progenitoras Mieloides/metabolismo , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Biomarcadores , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Citometria de Fluxo , Proteínas Ligadas por GPI/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Camundongos , Transcriptoma
5.
Nat Commun ; 10(1): 2788, 2019 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-31243277

RESUMO

Many potentially therapeutic molecules have been identified for treating Duchenne muscular dystrophy. However, targeting those molecules only to sites of active pathology is an obstacle to their clinical use. Because dystrophic muscles become extensively inflamed, we tested whether expressing a therapeutic transgene in leukocyte progenitors that invade muscle would provide selective, timely delivery to diseased muscle. We designed a transgene in which leukemia inhibitory factor (LIF) is under control of a leukocyte-specific promoter and transplanted transgenic cells into dystrophic mice. Transplantation diminishes pathology, reduces Th2 cytokines in muscle and biases macrophages away from a CD163+/CD206+ phenotype that promotes fibrosis. Transgenic cells also abrogate TGFß signaling, reduce fibro/adipogenic progenitor cells and reduce fibrogenesis of muscle cells. These findings indicate that leukocytes expressing a LIF transgene reduce fibrosis by suppressing type 2 immunity and highlight a novel application by which immune cells can be genetically modified as potential therapeutics to treat muscle disease.


Assuntos
Terapia Genética , Fator Inibidor de Leucemia/metabolismo , Distrofia Muscular Animal/terapia , Animais , Células da Medula Óssea/metabolismo , Regulação da Expressão Gênica , Fator Inibidor de Leucemia/genética , Masculino , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético/patologia , Distribuição Aleatória , Organismos Livres de Patógenos Específicos , Transgenes
6.
Ecotoxicol Environ Saf ; 181: 89-95, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31176251

RESUMO

BACKGROUDS: Formaldehyde (FA) is an important chemicals that can induce sick house syndrome and may be an incentive of childhood leukemia, however the exact mechanism is unclear. Oxidative stress may be an underlying reason of cancer occurring, while diverse antioxidants can protect the bone marrow cells (BMCs) from damaged. PeroxiredoxinⅡ (PrxⅡ) is an important member of the peroxiredoxin family, can remove reactive oxygen species (ROS), and is closely related with the occurrence of tumor. The present study aimed to detect a possible relationship between PrxⅡ gene and FA-induced bone marrow toxicity. METHODS: The BMCs were taken out from BALB/c mice, then exposed to control and different doses of FA (50, 100, 200 µmol/L). The cell viability, ROS level and expressions of PrxⅡ gene were examined. Afterwards, we used a small interfering RNA (siRNA) to inhibit the expression of PrxⅡ gene, and chose 100 µmol/L FA for exposure dose, to examine the cell viability, ROS level, cell cycle, apoptotic rate, expressions of PrxⅡ gene in BMCs. RESULTS: After a 24 h exposure to different doses of FA, the cell viability, expressions of PrxⅡ gene were decreased with the increasing of FA concentration, while the ROS level was increased. Inhibiting PrxⅡ gene's expression could enhance above FA-induced events. Additionally, siRNA targeting of PrxⅡcould aggravate cell cycle arrest to inhibit cell's growth and development, as well as increase apoptotic rates induced by FA. CONCLUSION: These results demonstrated that PrxⅡ gene was involved in FA-induced bone marrow toxicity, and siRNA targeting of PrxⅡcould enhance this toxic process.


Assuntos
Células da Medula Óssea/efeitos dos fármacos , Formaldeído/toxicidade , Peroxirredoxinas/genética , Animais , Células da Medula Óssea/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Masculino , Camundongos Endogâmicos BALB C , Estresse Oxidativo , Peroxirredoxinas/antagonistas & inibidores , Peroxirredoxinas/metabolismo , RNA Interferente Pequeno , Espécies Reativas de Oxigênio/metabolismo
7.
Mol Cell Biochem ; 458(1-2): 11-26, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31165315

RESUMO

Dysregulation of microRNAs (miRNAs) has been found to disrupt the progression of oral cancer. However, which miRNAs are most effective against oral cancer and how these miRNAs should be delivered are major unanswered problems. We aimed at investigating if human bone marrow mesenchymal stem cells (hBMSCs)-derived exosomes affect oral cancer development, and the potential regulatory mechanism associated with COL10A1 and miR-101-3p. COL10A1 was upregulated, while miR-101-3p was downregulated in oral cancer, and miR-101-3p targeted COL10A1 as verified by dual-luciferase reporter gene assay. Meanwhile, exosomes derived from hBMSCs were isolated and then co-cultured with oral cancer cells to identify the role of exosomes, and the results suggested that hBMSCs-derived exosomes overexpressing miR-101-3p inhibited oral cancer progression. Furthermore, tumorigenicity assay in nude mice further confirmed the inhibitory effects of hBMSCs-derived exosomes, loaded with miR-101-3p, on oral cancer, which provides a new theoretical basis in the treatment of oral cancer.


Assuntos
Células da Medula Óssea/metabolismo , Movimento Celular , Proliferação de Células , Exossomos/transplante , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/biossíntese , Neoplasias Bucais/terapia , Idoso , Animais , Exossomos/metabolismo , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Invasividade Neoplásica , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Nat Cell Biol ; 21(6): 700-709, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31061465

RESUMO

Haematopoietic stem cells (HSCs) maintain balanced self-renewal and differentiation, but how these functions are precisely regulated is not fully understood. N6-methyladenosine (m6A) messenger RNA methylation has emerged as an important mode of epitranscriptional gene expression regulation affecting many biological processes. We show that deletion of the m6A methyltransferase Mettl3 from the adult haematopoietic system led to an accumulation of HSCs in the bone marrow and a marked reduction of reconstitution potential due to a blockage of HSC differentiation. Interestingly, deleting Mettl3 from myeloid cells using Lysm-cre did not impact myeloid cell number or function. RNA sequencing revealed 2,073 genes with significant m6A modifications in HSCs. Myc was identified as a direct target of m6A in HSCs. Mettl3-deficient HSCs failed to upregulate MYC expression following stimulation to differentiate and enforced expression of Myc rescued differentiation defects of Mettl3-deficient HSCs. Our results reveal a key role of m6A in governing HSC differentiation.


Assuntos
Adenosina/análogos & derivados , Diferenciação Celular/genética , Células-Tronco Hematopoéticas/citologia , Metiltransferases/genética , Proteínas Proto-Oncogênicas c-myc/genética , Adenosina/genética , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Células-Tronco Hematopoéticas/metabolismo , Metilação , Camundongos , RNA Mensageiro/genética , Análise de Sequência de RNA
10.
Epigenetics Chromatin ; 12(1): 25, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30992059

RESUMO

BACKGROUND: MMP-9-dependent proteolysis of histone H3 N-terminal tail (H3NT) is an important mechanism for activation of gene expression during osteoclast differentiation. Like other enzymes targeting their substrates within chromatin structure, MMP-9 enzymatic activity toward H3NT is tightly controlled by histone modifications such as H3K18 acetylation (H3K18ac) and H3K27 monomethylation (H3K27me1). Growing evidence indicates that DNA methylation is another epigenetic mechanism controlling osteoclastogenesis, but whether DNA methylation is also critical for regulating MMP-9-dependent H3NT proteolysis and gene expression remains unknown. RESULTS: We show here that treating RANKL-induced osteoclast progenitor (OCP) cells with the DNMT inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR) induces CpG island hypomethylation and facilitates MMP-9 transcription. This increase in MMP-9 expression results in a significant enhancement of H3NT proteolysis and OCP cell differentiation. On the other hand, despite an increase in levels of H3K18ac, treatment with the HDAC inhibitor trichostatin A (TSA) leads to impairment of osteoclastogenic gene expression. Mechanistically, TSA treatment of OCP-induced cells stimulates H3K27ac with accompanying reduction in H3K27me1, which is a key modification to facilitate stable interaction of MMP-9 with nucleosomes for H3NT proteolysis. Moreover, hypomethylated osteoclastogenic genes in 5-Aza-CdR-treated cells remain transcriptionally inactive after TSA treatment, because H3K27 is highly acetylated and cannot be modified by G9a. CONCLUSIONS: These findings clearly indicate that DNA methylation and histone modification are important mechanisms in regulating osteoclastogenic gene expression and that their inhibitors can be used as potential therapeutic tools for treating bone disorders.


Assuntos
Diferenciação Celular , Histonas/metabolismo , Osteoclastos/citologia , Osteogênese , Proteólise , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células Cultivadas , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Histonas/química , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Osteoclastos/metabolismo
11.
Biomed Pharmacother ; 114: 108806, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30928804

RESUMO

Agents that provide protection against irradiation-induced hematopoietic injury are urgently needed for radiotherapy. We examined the effects of the small molecule, 1,2-propanediol (PPD), on total body irradiation (TBI)-induced hematopoietic injury in C57BL/6 mice. PPD administration 1 h before TBI significantly increased hematopoietic parameters such as white blood cell, platelet, red blood cell, and lymphocyte counts in vivo and enhanced the survival of mice exposed to TBI (7.0 and 7.5 Gy). PPD administration 1 h before TBI improved bone marrow (BM) and spleen recovery after TBI, with increases in both BM cellularity and spleen index. The number of colony-forming-units in bone marrow mononuclear cells (BMNCs) in vitro also increased significantly. PPD pretreatment increased the numbers of hematopoietic stem cells and hematopoietic progenitor cells in BM. Importantly, PPD also maintained endogenous antioxidant status by decreasing levels of malondialdehyde and increasing the expression of reduced glutathione, superoxide dismutase and catalase in the serum of irradiated mice. PPD alleviated the levels of apoptosis in HSCs induced by TBI, thus increasing the proportion of dividing BMNCs. These results suggest that PPD protects against TBI-induced hematopoietic injury through the increased activities of antioxidant enzymes and the inhibition of apoptosis in HSCs. PPD increased the serum levels of granulocyte-colony stimulating factor and interleukin-6 irrespective of TBI. In conclusion, these data suggest that PPD acts as a radioprotector against radiation-induced hematopoietic injury.


Assuntos
Células-Tronco Hematopoéticas/efeitos dos fármacos , Propilenoglicol/farmacologia , Lesões Experimentais por Radiação/tratamento farmacológico , Animais , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Medula Óssea/efeitos dos fármacos , Medula Óssea/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Ensaio de Unidades Formadoras de Colônias/métodos , Fator Estimulador de Colônias de Granulócitos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Lesões Experimentais por Radiação/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Irradiação Corporal Total/métodos
12.
Nat Cell Biol ; 21(5): 560-567, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30988422

RESUMO

Haematopoietic stem cells (HSCs) are maintained by bone marrow niches in vivo1,2, but the ability of niche cells to maintain HSCs ex vivo is markedly diminished. Expression of niche factors by Nestin-GFP+ mesenchymal-derived stromal cells (MSCs) is downregulated upon culture, suggesting that transcriptional rewiring may contribute to this reduced HSC maintenance potential. Using an RNA sequencing screen, we identified five genes encoding transcription factors (Klf7, Ostf1, Xbp1, Irf3 and Irf7) that restored HSC niche function in cultured bone marrow-derived MSCs. These revitalized MSCs (rMSCs) exhibited enhanced synthesis of HSC niche factors while retaining their mesenchymal differentiation capacity. In contrast to HSCs co-cultured with control MSCs, HSCs expanded with rMSCs showed higher repopulation capacity and protected lethally irradiated recipient mice. Competitive reconstitution assays revealed an approximately sevenfold expansion of functional HSCs by rMSCs. rMSCs prevented the accumulation of DNA damage in cultured HSCs, a hallmark of ageing and replication stress. Analysis of the reprogramming mechanisms uncovered a role for myocyte enhancer factor 2c (Mef2c) in the revitalization of MSCs. These results provide insight into the transcriptional regulation of the niche with implications for stem cell-based therapies.


Assuntos
Diferenciação Celular/genética , Engenharia Celular/métodos , Células-Tronco Hematopoéticas/citologia , Nicho de Células-Tronco/genética , Animais , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Regulação da Expressão Gênica/genética , Células-Tronco Hematopoéticas/metabolismo , Humanos , Fator Regulador 3 de Interferon/genética , Fatores de Transcrição Kruppel-Like/genética , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Transgênicos , Nestina/genética , Peptídeos/genética , Análise de Sequência de RNA/métodos , Proteína 1 de Ligação a X-Box/genética
13.
Int J Mol Sci ; 20(7)2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30978983

RESUMO

The bone marrow (BM) microenvironment plays a crucial role in the maintenance and regeneration of hematopoietic stem (HSC) and progenitor cells (HSPC). In particular, the vascular niche is responsible for regulating HSC maintenance, differentiation, and migration of cells in and out of the BM. Damage to this niche upon exposure to ionizing radiation, whether accidental or as a result of therapy, can contribute to delays in HSC recovery and/or function. The ability of BM derived-endothelial cells (BMEC) to alter and/or protect HSPC after exposure to ionizing radiation was investigated. Our data show that exposure of BMEC to ionizing radiation resulted in alterations in Akt signaling, increased expression of PARP-1, IL6, and MCP-1, and decreased expression of MMP1 and MMP9. In addition, global analysis of gene expression of HSC and BMEC in response to mixed neutron/gamma field (MF) radiation identified 60 genes whose expression was altered after radiation in both cell types, suggesting that a subset of genes is commonly affected by this type of radiation. Focused gene analysis by RT-PCR revealed two categories of BMEC alterations: (a) a subset of genes whose expression was altered in response to radiation, with no additional effect observed during coculture with HSPC, and (b) a subset of genes upregulated in response to radiation, and altered when cocultured with HSPC. Coculture of BMEC with CD34+ HSPC induced HSPC proliferation, and improved BM function after MF radiation. Nonirradiated HSPC exhibited reduced CD34 expression over time, but when irradiated, they maintained higher CD34 expression. Nonirradiated HSPC cocultured with nonirradiated BMEC expressed lower levels of CD34 expression compared to nonirradiated alone. These data characterize the role of each cell type in response to MF radiation and demonstrate the interdependence of each cell's response to ionizing radiation. The identified genes modulated by radiation and coculture provide guidance for future experiments to test hypotheses concerning specific factors mediating the beneficial effects of BMEC on HSPC. This information will prove useful in the search for medical countermeasures to radiation-induced hematopoietic injury.


Assuntos
Células da Medula Óssea/efeitos da radiação , Técnicas de Cocultura , Células Endoteliais/efeitos da radiação , Células-Tronco Hematopoéticas/efeitos da radiação , Antígenos CD34/análise , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Diferenciação Celular/efeitos da radiação , Linhagem Celular , Proliferação de Células/efeitos da radiação , Técnicas de Cocultura/métodos , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Raios gama , Regulação da Expressão Gênica/efeitos da radiação , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Nêutrons , Fenótipo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Lesões por Radiação/prevenção & controle , Transdução de Sinais/efeitos da radiação
14.
ACS Appl Mater Interfaces ; 11(16): 15170-15180, 2019 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-30942571

RESUMO

The recruitment of endogenous mesenchymal stem cells (MSCs), as an alluring approach for in situ tissue regeneration, always accompanies with other types of cells. Therefore, it is of enormous value to bestow a substrate with the property of selective capture to MSCs. However, it was reported that when MSCs are cultured on a substrate with excessive affinity, their stemness diminished. Therefore, constructing a substrate with the balanced ability of selective capture and stemness maintenance becomes a big challenge. In this study, an Aptamer 19S (Apt19S)-modified substrate was fabricated by grafting Apt19S on a PEGylated glass substrate. The X-ray photoelectron spectroscopy results verified that the antifouling poly(ethylene glycol) (PEG) layer was created. Tracking by ellipsometry, the thicknesses of PEG layers were proved to increase with PEG concentration. The results of the quartz crystal microbalance also validated that the Apt19S densities could be modulated by the concentrations of the Apt19S solution. The results of the cell adhesion assay indicated that the modification of Apt19S caused a significant increase in the adhesion ratio and area of rBMSCs. Selective adhesion was confirmed by coculture of rBMSCs with macrophages and NIH3T3 cells, demonstrating that a higher proportion of rBMSCs adhered to the Apt19S-modified substrate. The results of specific capture were further confirmed by a flow model to simulate the body fluid flow. The comprehensive results of reverse transcription polymerase chain reaction, immunofluorescence staining, proliferation capacity, and differentiation assay showed that the stemness of rBMSCs was maintained better on a substrate with the appropriate Apt19S density. All of these results indicated that Apt19S modification is an effective strategy to endow a substrate with the specific capture ability of MSCs, and the balance between selective capture and stemness maintenance can be achieved by the precise regulation of the aptamer density.


Assuntos
Aptâmeros de Nucleotídeos , Células da Medula Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Polietilenoglicóis , Animais , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/farmacocinética , Células da Medula Óssea/citologia , Adesão Celular/efeitos dos fármacos , Células-Tronco Mesenquimais/citologia , Camundongos , Células NIH 3T3 , Polietilenoglicóis/química , Polietilenoglicóis/farmacologia , Ratos , Ratos Sprague-Dawley
15.
Methods Mol Biol ; 1971: 237-247, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30980307

RESUMO

Murine bone marrow-derived macrophages (BMMs) can be differentiated within 10 days from ex vivo bone marrow progenitor cells by supplementing the cell growth medium with colony stimulating factor-1 (CSF-1). Mature macrophages express specific myeloid markers which can be labeled and detected by flow cytometry (FACS).BMMs are a valuable tool to investigate the interactions between the Leishmania parasites and their host cell as well as to screen anti-Leishmania components. Options for the readout of in vitro infection experiments are diverse and may range from simple counting of intracellular parasites to the determination of metabolic changes of the intracellular parasite or the infected cell, thus providing the investigator with valuable results.


Assuntos
Células da Medula Óssea/parasitologia , Leishmania/crescimento & desenvolvimento , Leishmaniose/metabolismo , Macrófagos/parasitologia , Animais , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Diferenciação Celular , Células Cultivadas , Feminino , Leishmaniose/patologia , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos BALB C
16.
Mol Med Rep ; 19(6): 4779-4787, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30957181

RESUMO

Bone marrow stem cells (BMSCs) are a group cells that function as an underlying cell source for bone tissue regeneration. However, the molecular mechanisms of how BMSCs are induced into apoptosis remains unclear. In the present study, it was demonstrated that the molecular mechanisms of BMSCs were exerted via microRNA­15a­5p (miR­15a­5p) in femoral head necrosis (FHN). Briefly, miRNA­15a­5p expression was elevated in a rat model of FHN. Overexpression of miR­15a­5p promoted the apoptosis of BMSCs and reduced cell growth through the Wnt/ß­catenin/peroxisome proliferator­activated receptor γ (PPARγ) signaling pathway. Downregulation of miR­15a­5p reduced the apoptosis of BMSCs and promoted cell growth through the Wnt/ß­catenin/PPARγ signaling pathway. The activation of Wnt attenuated the effects of miR­15a­5p on the apoptosis of BMSCs via the ß­catenin/PPARγ signaling pathway. In conclusion, the present results indicated that miRNA­15a­5p was involved in the regulation of the apoptosis of BMSCs through regulating the Wnt/ß­catenin/PPARγ signaling pathway, which may serve an important role in the regulation of FHN.


Assuntos
Apoptose/efeitos dos fármacos , Células da Medula Óssea/efeitos dos fármacos , MicroRNAs/metabolismo , MicroRNAs/farmacologia , PPAR gama/metabolismo , Células-Tronco/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células , Regulação para Baixo , Necrose da Cabeça do Fêmur/patologia , Perfilação da Expressão Gênica , Masculino , Camundongos , MicroRNAs/genética , Modelos Animais , Ratos , Ratos Sprague-Dawley , beta Catenina/metabolismo
17.
Science ; 363(6430): 989-993, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30819964

RESUMO

To meet systemic metabolic needs, adipocytes release fatty acids and glycerol through the action of neutral lipases. Here, we describe a secondary pathway of lipid release from adipocytes that is independent of canonical lipolysis. We found that adipocytes release exosome-sized, lipid-filled vesicles (AdExos) that become a source of lipid for local macrophages. Adipose tissue from lean mice released ~1% of its lipid content per day via exosomes ex vivo, a rate that more than doubles in obese animals. AdExos and associated factors were sufficient to induce in vitro differentiation of bone marrow precursors into adipose tissue macrophage-like cells. Thus, AdExos are both an alternative pathway of local lipid release and a mechanism by which parenchymal cells can modulate tissue macrophage differentiation and function.


Assuntos
Adipócitos/metabolismo , Tecido Adiposo/imunologia , Exossomos/metabolismo , Metabolismo dos Lipídeos , Macrófagos/metabolismo , Tecido Adiposo/citologia , Animais , Células da Medula Óssea/metabolismo , Diferenciação Celular , Células Cultivadas , Lipase/metabolismo , Lipólise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/metabolismo
18.
Nat Biotechnol ; 37(4): 451-460, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30899105

RESUMO

Single-cell RNA sequencing studies of differentiating systems have raised fundamental questions regarding the discrete versus continuous nature of both differentiation and cell fate. Here we present Palantir, an algorithm that models trajectories of differentiating cells by treating cell fate as a probabilistic process and leverages entropy to measure cell plasticity along the trajectory. Palantir generates a high-resolution pseudo-time ordering of cells and, for each cell state, assigns a probability of differentiating into each terminal state. We apply our algorithm to human bone marrow single-cell RNA sequencing data and detect important landmarks of hematopoietic differentiation. Palantir's resolution enables the identification of key transcription factors that drive lineage fate choice and closely track when cells lose plasticity. We show that Palantir outperforms existing algorithms in identifying cell lineages and recapitulating gene expression trends during differentiation, is generalizable to diverse tissue types, and is well-suited to resolving less-studied differentiating systems.


Assuntos
Algoritmos , Diferenciação Celular/genética , Linhagem da Célula/genética , Análise de Sequência de RNA/estatística & dados numéricos , Análise de Célula Única/estatística & dados numéricos , Animais , Biotecnologia , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Eritropoese/genética , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese/genética , Humanos , Cadeias de Markov , Camundongos , Modelos Biológicos , Modelos Estatísticos
19.
Int J Mol Sci ; 20(6)2019 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-30917596

RESUMO

Cadmium is a common environmental pollutant that causes bone damage. However, the effects of cadmium on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs) and its mechanism of action in this process are unclear. Here, we determined the effects of cadmium chloride (CdCl2) on the osteogenic differentiation of BMMSCs and the potential mechanism involved in this process. As determined in the present investigation, CdCl2, in a concentration-dependent manner, affected the viability of BMMSCs and their cytoskeletons. Exposure to 0.1 or 0.2 µM CdCl2 inhibited osteogenic differentiation of BMMSCs, which was reflected in the down-regulation of osteoblast-related genes (ALP, OCN, Runx2, OSX, and OPN); in suppression of the protein expression of alkaline phosphatase (ALP) and runt-related transcription factor 2 (Runx2); and in decreased ALP activity and capacity for mineralization. Moreover, mRNA microarray was performed to determine the roles of these factors in BMMSCs treated with CdCl2 in comparison to control BMMSCs. As determined with the microarrays, the Wingless-type (Wnt), mothers against decapentaplegic and the C. elegans gene Sam (SMAD), and Janus kinase-Signal Transducers and Activators of Transcription (JAK-STAT) signaling pathways were involved in the effects caused by CdCl2. Moreover, during differentiation, the protein levels of Wnt3a, ß-catenin, lymphoid enhancer factor 1 (LEF1), and T-cell factor 1 (TCF1) were reduced by CdCl2. The current research shows that CdCl2 suppresses the osteogenesis of BMMSCs via inhibiting the Wnt/ß-catenin pathway. The results establish a previously unknown mechanism for bone injury induced by CdCl2.


Assuntos
Células da Medula Óssea/metabolismo , Cloreto de Cádmio/farmacologia , Diferenciação Celular , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Via de Sinalização Wnt , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteocalcina/genética , Osteocalcina/metabolismo , Osteopontina/genética , Osteopontina/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
20.
Res Vet Sci ; 124: 212-222, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30925336

RESUMO

Little information is currently available on therapeutic features of bovine mesenchymal stem cells (MSCs), despite the development of large animal experimental models including cattle may open alternative strategies for investigating MSC physiology and eventual applications for regenerative therapy. The aim of the present study was to compare in vitro immunomodulatory and immunogenic potentials of bovine fetal MSCs (bfMSCs) derived from bovine fetal bone marrow (BM-MSCs) and adipose tissue (AT-MSCs). Immunomodulatory analyses in bfMSCs were performed by determination of the effect of interferon-γ (IFNγ) on mRNA levels of indoleamine 2, 3-dioxygenase (IDO), transforming growth factor ß1 (TGFß1), prostaglandin E receptor 2 (PTGER2), interleukin-6 and -10 (IL-6 and IL-10), and IDO enzymatic activity. The effect of conditioned medium from IFNγ-stimulated bfMSCs on the proliferation of alloantigen-activated peripheral blood lymphocytes (PBLs) was assessed. Immunogenicity of bfMSCs was determined by quantification of mRNA levels of major histocompatibility complex I and II (MHC-I and -II), CD80 and CD86, and the proportion of cells positive for MHC-I and -II by flowcytometry (FACS) analyses. IFNγ treatment increased IL-6, PTGER2 and IDO gene expression and activity in bfMSCs but did not affect suppressive effect on proliferation of PBLs. Lower proportion of AT-MSCs expressed MHC-I and MHC-II in comparison to BM-MSCs. In conclusion, BM-MSCs and AT-MSCs upregulated expression of immunomodulatory genes in a similar way after IFNγ stimuli. BM-MSCs and AT-MSCs in basal condition and treated with IFNγ displayed similar in vitro immunomodulatory ability. Lower expression of MHC-I and MHC-II suggest that AT-MSCs might be less immunogenic compared to BM-MSCs.


Assuntos
Tecido Adiposo/metabolismo , Células da Medula Óssea/metabolismo , Imunomodulação , Células-Tronco Mesenquimais/imunologia , Animais , Medula Óssea/metabolismo , Bovinos , Feto
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