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2.
Chin J Dent Res ; 23(2): 143-150, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32548605

RESUMO

OBJECTIVE: To compare the biological characteristics of dental pulp stem cells (DPSCs) and inflamed dental pulp derived stem cells (I-DPSCs) in vitro and their regeneration potential in Beagle immature premolars. METHODS: Pulpitis was induced in the premolars of one beagle dog by opening the pulp chamber for 2 weeks, and inflammation was histologically confirmed. DPSCs and I-DPSCs were isolated from normal and inflamed dental pulp, and cell morphology, expression of mesenchymal stem cell markers, clone formation ability, cell proliferation and osteogenic/odontogenic differentiation potential were compared. The dental pulp of 20 roots from 10 immature premolars was extracted and divided into two groups. DPSCs or I-DPSCs with scaffolds were transplanted into the root canals. The roots were extracted after 3 months, and pulp regeneration was evaluated by histological analysis. The data were statistically analysed using one-way ANOVA and a Student t test. RESULTS: Histological analyses showed lymphocyte infiltration and elevated TNF-α expression, which confirmed the diagnosis of pulpitis. I-DPSCs showed similar morphology, marker gene expression and clone formation ability but greater proliferation ability and osteogenic/odontogenic differentiation potential. Pulp-like tissue formation and bone- and dentine-like tissue deposition were observed in both DPSC- and I-DPSC-transplanted roots. CONCLUSION: DPSCs derived from inflammatory dental pulp tissue have similar biological characteristics to those from normal dental pulp and could mediate pulp and dentine regeneration in immature premolars.


Assuntos
Polpa Dentária , Regeneração , Animais , Dente Pré-Molar , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Cães , Humanos , Células-Tronco
3.
Life Sci ; 254: 117812, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32428596

RESUMO

AIMS: Since the role of the major mitochondrial NAD+-dependent deacetylase, sirtuin 3 (Sirt3), is differential in cancer, opposite to the well-known tumor-suppressing effect of hyperoxia, this study aimed to investigate the role of Sirt3 in triple-negative breast cancer (TNBC) cell line MDA-MB-231 upon hyperoxic (95% O2) conditions. MAIN METHODS: MDA-MB-231 cells were stably transfected with Flag-tagged Sirt-3 or empty plasmid. Western blot and real-time PCR were used to monitor the expression of proteins or genes involved in mitochondrial biogenesis, metabolic regulation and antioxidant defense. Immunocytochemistry and confocal microscopy were used to confirm the cellular localization and abundance of proteins. Flow cytometry was used to analyze mitochondrial mass, potential and ROS production, and MTT test as a measure of metabolic activity. Mitotic index analysis, colony-forming unit assay, DNA damage and Annexin V-FITC analyses were used to assess the differences in the growth and apoptosis rate. KEY FINDINGS: Although Sirt3 seemed to improve mitochondrial properties by increasing mitochondrial mass and potential, metabolic activity (Warburg effect) and antioxidative defense (SOD2, Cat), it also increased mitochondrial ROS, induced DNA damage, timp-1 expression, formation of multinucleated cells and apoptosis, and finally markedly reduced the proliferation of MDA-MB-231 cells. All these effects were even more evident upon the hyperoxic treatment, thus pointing towards combined negative effect of Sirt3 and hyperoxia on MDA-MB-231 cells. SIGNIFICANCE: Both Sirt3 and hyperoxia, alone or in combination, have the potential to negatively affect the malignant properties of the MDA-MB-231 cells and should be further explored as a possible therapy for TNBC.


Assuntos
Sobrevivência Celular/fisiologia , Hiperóxia/fisiopatologia , Mitocôndrias/fisiologia , Sirtuína 3/fisiologia , Neoplasias de Mama Triplo Negativas/fisiopatologia , Anexinas/metabolismo , Apoptose/fisiologia , Carcinogênese , Linhagem Celular Tumoral , Dano ao DNA , Regulação Neoplásica da Expressão Gênica , Humanos , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/metabolismo , Índice Mitótico , Proteínas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sirtuína 3/genética , Células-Tronco , Transfecção , Neoplasias de Mama Triplo Negativas/metabolismo
4.
Gene ; 752: 144758, 2020 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-32422235

RESUMO

Drugs targeting chromatin-modifying enzymes have entered clinical trials for myeloid malignancies, including INCB059872, a selective irreversible inhibitor of Lysine-Specific Demethylase 1 (LSD1). While initial studies of LSD1 inhibitors suggested these compounds may be used to induce differentiation of acute myeloid leukemia (AML), the mechanisms underlying this effect and dose-limiting toxicities are not well understood. Here, we used precision nuclear run-on sequencing (PRO-seq) and ChIP-seq in AML cell lines to probe for the earliest regulatory events associated with INCB059872 treatment. The changes in nascent transcription could be traced back to a loss of CoREST activity and activation of GFI1-regulated genes. INCB059872 is in phase I clinical trials, and we evaluated a pre-treatment bone marrow sample of a patient who showed a clinical response to INCB059872 while being treated with azacitidine. We used single-cell RNA-sequencing (scRNA-seq) to show that INCB059872 caused a shift in gene expression that was again associated with GFI1/GFI1B regulation. Finally, we treated mice with INCB059872 and performed scRNA-seq of lineage-negative bone marrow cells, which showed that INCB059872 triggered accumulation of megakaryocyte early progenitor cells with gene expression hallmarks of stem cells. Accumulation of these stem/progenitor cells may contribute to the thrombocytopenia observed in patients treated with LSD1 inhibitors.


Assuntos
Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Histona Desmetilases/antagonistas & inibidores , Leucemia Mieloide Aguda/metabolismo , Animais , Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Camundongos , Camundongos Endogâmicos C57BL , RNA-Seq , Análise de Célula Única/métodos , Células-Tronco/metabolismo , Células THP-1 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Sequenciamento Completo do Exoma/métodos
6.
Cell Prolif ; 53(6): e12831, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32441391

RESUMO

OBJECTIVES: AF4/FMR2 family member 1 (AFF1), known as a central scaffolding protein of super elongation complex (SEC), regulates gene transcription. We previously reported that AFF1 inhibited osteogenic differentiation of human mesenchymal stromal/stem cells (hMSCs). However, its role in adipogenic differentiation has not been elucidated. MATERIALS AND METHODS: hMSCs and 3T3-L1 pre-adipocytes were cultured and induced for adipogenic differentiation. Small interfering RNAs (siRNAs) were applied to deplete AFF1 while lentiviruses expressing HA-Aff1 were used for overexpression. Oil Red O staining, triglyceride (TAG) quantification, quantitative real-time PCR (qPCR), Western blot analysis, immunofluorescence staining, RNA sequencing (RNA-seq) analysis and ChIP-qPCR were performed. To evaluate the adipogenesis in vivo, BALB/c nude mice were subcutaneously injected with Aff1-overexpressed 3T3-L1 pre-adipocytes. RESULTS: AFF1 depletion leads to an enhanced adipogenesis in both hMSCs and 3T3-L1 pre-adipocytes. Overexpression of Aff1 in 3T3-L1 cells results in the reduction of adipogenic differentiation and less adipose tissue formation in vivo. Mechanistically, AFF1 binds to the promoter region of Tgm2 gene and regulates its transcription. Overexpression of Tgm2 largely rescues adipogenic differentiation of Aff1-deficient cells. CONCLUSIONS: Our data indicate that AFF1 inhibits adipogenic differentiation by regulating the transcription of TGM2.


Assuntos
Adipogenia/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica , Proteínas Nucleares/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Transglutaminases/genética , Células 3T3-L1 , Animais , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação ao GTP/biossíntese , Proteínas de Ligação ao GTP/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Nucleares/genética , Células-Tronco/metabolismo , Transcrição Genética , Fatores de Elongação da Transcrição/genética , Transglutaminases/biossíntese , Transglutaminases/metabolismo
7.
Cell Prolif ; 53(6): e12834, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32468637

RESUMO

OBJECTIVES: Advanced glycation end products (AGEs) are considered a cause of diabetic osteoporosis. Although adipose-derived stem cells (ASCs) are widely used in the research of bone regeneration, the mechanisms of the osteogenic differentiation of ASCs from diabetic osteoporosis model remain unclear. This work aimed to investigate the influence and the molecular mechanisms of AGEs on the osteogenic potential of ASCs. MATERIALS AND METHODS: Enzyme-linked immunosorbent assay was used to measure the change of AGEs in diabetic osteoporotic and control C57BL/6 mice. ASCs were obtained from the inguinal fat of C57BL/6 mice. AGEs, 5-aza2'-deoxycytidine (5-aza-dC) and DKK-1 were used to treat ASCs. Real-time cell analysis and cell counting kit-8 were used to monitor the proliferation of ASCs within and without AGEs. Real-time PCR, Western blot and Immunofluorescence were used to analyse the genes and proteins expression of osteogenic factors, DNA methylation factors and Wnt/ß-catenin signalling pathway among the different groups. RESULTS: The AGEs and DNA methylation were increased in the adipose and bone tissue of the diabetic osteoporosis group. Untreated ASCs had higher cell proliferation activity than AGEs-treatment group. The expression levels of osteogenic genes, Opn and Runx2, were lower, and mineralized nodules were less in AGEs-treatment group. Meanwhile, DNA methylation was increased, and the Wnt signalling pathway markers, including ß-Catenin, Lef1 and P-GSK-3ß, were inhibited. After treatment with 5-aza-dC, the osteogenic differentiation capacity of ASCs in the AGEs environment was restored and the Wnt signalling pathway was activated during this process. CONCLUSIONS: Advanced glycation end products inhibit the osteogenic differentiation ability of ASCs by activating DNA methylation and inhibiting Wnt/ß-catenin pathway in vitro. Therefore, DNA methylation may be promising targets for the bone regeneration of ASCs with diabetic osteoporosis.


Assuntos
Tecido Adiposo/citologia , Metilação de DNA , Complicações do Diabetes/metabolismo , Produtos Finais de Glicação Avançada/farmacologia , Osteogênese/efeitos dos fármacos , Osteoporose/metabolismo , Células-Tronco/metabolismo , Tecido Adiposo/metabolismo , Animais , Osso e Ossos/metabolismo , Proliferação de Células , Células Cultivadas , Decitabina/farmacologia , Complicações do Diabetes/patologia , Feminino , Produtos Finais de Glicação Avançada/metabolismo , Camundongos Endogâmicos C57BL , Osteoporose/patologia , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Via de Sinalização Wnt
8.
Basic Res Cardiol ; 115(4): 36, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32399655

RESUMO

There are no definitive therapies for patients with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Therefore, new therapeutic strategies are needed to improve clinical outcomes, particularly in patients with severe disease. This case series explores the safety and effectiveness of intravenous allogeneic cardiosphere-derived cells (CDCs), formulated as CAP-1002, in critically ill patients with confirmed coronavirus disease 2019 (COVID-19). Adverse reactions to CAP-1002, clinical status on the World Health Organization (WHO) ordinal scale, and changes in pro-inflammatory biomarkers and leukocyte counts were analyzed. All patients (n = 6; age range 19-75 years, 1 female) required ventilatory support (invasive mechanical ventilation, n = 5) with PaO2/FiO2 ranging from 69 to 198. No adverse events related to CAP-1002 administration were observed. Four patients (67%) were weaned from respiratory support and discharged from the hospital. One patient remains mechanically ventilated as of April 28th, 2020; all survive. A contemporaneous control group of critically ill COVID-19 patients (n = 34) at our institution showed 18% overall mortality at a similar stage of hospitalization. Ferritin was elevated in all patients at baseline (range of all patients 605.43-2991.52 ng/ml) and decreased in 5/6 patients (range of all patients 252.89-1029.90 ng/ml). Absolute lymphocyte counts were low in 5/6 patients at baseline (range 0.26-0.82 × 103/µl) but had increased in three of these five patients at last follow-up (range 0.23-1.02 × 103/µl). In this series of six critically ill COVID-19 patients, intravenous infusion of CAP-1002 was well tolerated and associated with resolution of critical illness in 4 patients. This series demonstrates the apparent safety of CAP-1002 in COVID-19. While this initial experience is promising, efficacy will need to be further assessed in a randomized controlled trial.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Ensaios de Uso Compassivo , Infecções por Coronavirus/terapia , Miocárdio/citologia , Pneumonia Viral/terapia , Células-Tronco/citologia , Idoso , Betacoronavirus , Biomarcadores/sangue , Estado Terminal/terapia , Feminino , Ferritinas/sangue , Humanos , Infusões Intravenosas , Los Angeles , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Pandemias , Adulto Jovem
10.
Autoimmun Rev ; 19(6): 102536, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32251718
11.
Zhonghua Shao Shang Za Zhi ; 36(3): 195-203, 2020 Mar 20.
Artigo em Chinês | MEDLINE | ID: mdl-32241045

RESUMO

Objective: To explore the effects and mechanism of rat epidermal stem cells (ESCs) that were treated with exogenous vascular endothelial growth factor (VEGF) on the healing of deep partial-thickness burn wounds in rats. Methods: ESCs were isolated and cultured from the trunk skin of a 3-month-old female Sprague-Dawley (SD) rat. The third passage of cultured cells in the logarithmic growth phase was used in experiments (1)-(3). (1) The cells were routinely cultured in keratinocytes-specified serum-free medium (K-SFM) (the same routine culture condition below). The morphology of cells cultured for 3 and 5 days was observed under the inverted optical microscope. (2) After 24 hours in routine culture, the expression of cell surface markers CD44, CD45, CD11b, and CD11c was detected by flow cytometer, with triplicate samples. (3) Four batches of cells were collected, and each batch was divided into VEGF group or blank control group according to the random number table. The cells in blank control group were routinely cultured, while the cells in VEGF group were cultured in K-SFM containing VEGF in the final mass concentration of 10 ng/mL. The protein expressions of cytokeratin 19 (CK19) and CK10 in cells cultured for 10 days were detected by Western blotting. The Nanog mRNA expression in cells cultured for 0 (immediately), 2, 4, 6, 8, and 10 day (s) was detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction. The absorbance value was detected with cell counting kit-8 in cells cultured for 2, 4, 6, 8, and 10 days. The clone number of more than 50 cells was observed and counted under the optical microscope in cells cultured for 10 days, and the cell colony formation rate was calculated. Three samples at each time point was analysed. (4) Thirty-six 3-month-old SD rats (either male or female) were used for the study, and two deep partial-thickness burn wounds (10 mm in diameter) were created in each rat by pressing a 100 ℃ electric iron plate on symmetric dorsal side. According to the random number table, the injured rats were divided into VEGF+ ESCs group, ESCs alone group, and blank control group, with 12 rats and 24 wounds in each group. From 0 (immediately) to 2 day (s) after injury, 20 µL phosphate buffer solution (PBS) was injected into each wound in the three groups in one time, once a day, with the solution in VEGF+ ESCs group containing 0.8×10(6) cells/mL of ESCs treated by 10 ng/mL VEGF for 10 days, the solution in ESCs alone group containing 0.8×10(6) cells/mL of ESCs without any treatment, and the solution in blank control group being PBS only. On post first injection day (PFID) 0 (immediately), 3, 7, and 14, three rats from each group were taken respectively according to the random number table for wound healing assessment, and the wound healing rates on PFID 3, 7, and 14 were calculated. The mice at each time point were sacrificed with wound tissue harvested for histology, and the skin structure was observed by hematoxylin-eosin staining. Data were statistically analyzed with independent sample t test, analysis of variance for factorial design, least significant difference test, and Bonferroni correction. Results: (1) By day 3 in culture, cells distributed in slowly-growing clusters. By day 5, the clusters were large and round, in which the cells mainly with large and round nuclei and little cytoplasm were observed. The above results were consistent with the morphological characteristics of ESCs. (2) The positive expression rate of CD44 was (94.3±1.2) %, and the expressions of CD45, CD11b, and CD11c were negative. The cells were confirmed as ESCs. (3) Compared with those of blank control group, the protein expression of CK19 in the cells of VEGF group was significantly increased after 10 days in culture (t=3.756, P<0.05), while the protein expression of CK10 was significantly decreased (t=3.149, P<0.05). Compared with those of blank control group, the Nanog mRNA expression in the cells cultured for 0 and 2 day (s) and absorbance values of the cells cultured for 2 and 4 day (s) were not significantly changed in VEGF group (t=0.58, 0.77, 0.53, 3.02, P>0.05), while the Nanog mRNA expression in the cells cultured for 4, 6, 8, and 10 days and absorbance values of the cells cultured for 6, 8, and 10 days were significantly increased in VEGF group (t=6.34, 5.00, 5.58, 4.61, 5.65, 10.78, 15.51, P<0.01). After 10 days in culture, the cell colony-forming rate in VEGF group was (56.4±1.3) %, significantly higher than (31.5±1.3) % of blank control group (t=13.96, P<0.01). (4) The burn wounds of rats in the three groups were confined to the superficial dermis of the skin on PFID 0. On PFID 3, normal skin tissue at wound margins slightly contracted in the rats of VEGF+ ESCs group, which was earlier than that in the other two groups. On PFID 7, the newly generated epidermis covered most parts of the rat wounds in VEGF+ ESCs group, and some of the epithelium crawled around the rat wounds in ESCs alone group, but no obvious epithelialization was observed in the rat wounds in blank control group. On PFID 14, the rat wounds in VEGF+ ESCs group were basically healed, while some parts of the rat wounds were unhealed in ESCs alone group, and most parts of the rat wounds were unhealed in blank control group. On PFID 3, the wound healing rates of rats in the three groups were similar (P>0.05). On PFID 7 and 14, the wound healing rates of rats in ESCs alone group, respectively (26.0±2.0) % and (64.4±4.7) %, were obviously higher than (12.4±1.1) % and (29.1±3.3) % of blank control group (P<0.01), all of which were obviously lower than (41.0±2.4) % and (91.3±3.5) % of VEGF+ ESCs group (P<0.01). On PFID 3, infiltration of a large number of inflammatory cells were observed in the rat wounds in VEGF+ ESCs group, which was earlier than those in the other two groups. On PFID 7, a large number of endothelial cells were observed in the rat wounds in VEGF+ ESCs group, while proliferation of a few endothelial cells were observed in the rat wounds in ESCs alone group, and a large number of inflammatory cells infiltrated the rat wounds in blank control group. On PFID 14, the newly generated epidermal cells covered nearly all the rat wounds in VEGF+ ESCs group and most parts of the rat wounds in ESCs alone group, while a large number of endothelial cells were observed and the newly generated epidermal cells covered some parts of the rat wounds in blank control group. Conclusions: ESCs of rats treated with exogenous VEGF can promote the healing of deep partial-thickness burn wounds in rats, which may be related to VEGF's roles in promoting the proliferation of ESCs and reducing its differentiation level, so as to maintain the potency of stem cells.


Assuntos
Queimaduras/terapia , Células-Tronco , Fator A de Crescimento do Endotélio Vascular , Animais , Células Endoteliais , Células Epidérmicas , Feminino , Masculino , Camundongos , Ratos , Ratos Sprague-Dawley , Cicatrização
12.
Cell Prolif ; 53(5): e12803, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32246537

RESUMO

OBJECTIVES: The aim of this study is to investigate the role of sensory nerve in tooth homeostasis and its effect on mesenchymal stromal/stem cells (MSCs) in dental pulp. MATERIALS AND METHODS: We established the rat denervated incisor models to identify the morphological and histological changes of tooth. The groups were as follows: IANx (inferior alveolar nerve section), SCGx (superior cervical ganglion removal), IANx + SCGx and Sham group. The biological behaviour of dental pulp stromal/stem cells (DPSCs) was evaluated. Finally, we applied activin B to DPSCs from sensory nerve-deficient microenvironment to analyse the changes of proliferation and apoptosis. RESULTS: Incisor of IANx and IANx + SCGx groups exhibited obvious disorganized tooth structure, while SCGx group only showed slight decrease of dentin thickness, implying sensory nerve, not sympathetic nerve, contributes to the tooth homeostasis. Moreover, we found sensory nerve injury led to disfunction of DPSCs via activin B/SMAD2/3 signalling in vitro. Supplementing activin B promoted proliferation and reduced apoptosis of DPSCs in sensory nerve-deficient microenvironment. CONCLUSIONS: This research first demonstrates that sensory nerve-deficient microenvironment impairs tooth haemostasis by inducing apoptosis of DPSCs via activin B/SMAD2/3 signalling. Our study provides the evidence for the crucial role of sensory nerve in tooth homeostasis.


Assuntos
Apoptose/fisiologia , Polpa Dentária/fisiologia , Homeostase/fisiologia , Células Receptoras Sensoriais/fisiologia , Células-Tronco/fisiologia , Dente/fisiologia , Animais , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Microambiente Celular/fisiologia , Técnicas de Cocultura/métodos , Polpa Dentária/metabolismo , Dentina/metabolismo , Dentina/fisiologia , Feminino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Ratos , Ratos Sprague-Dawley , Células Receptoras Sensoriais/metabolismo , Transdução de Sinais/fisiologia , Células-Tronco/metabolismo , Dente/metabolismo
13.
Arthroscopy ; 36(4): 981-982, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32247428

RESUMO

The use of stem cells in orthopaedics remains a controversial topic, stem cells remain experimental, and significant concerns exist. Studies evaluating diagnoses that may spontaneously resolve could be of low value absent a control group. Only same-day harvest of minimally manipulated stem cells is approved for use in the United States, and these minimally manipulated products may contain insufficient cells to affect outcomes. Extensively cultured cells do not qualify for use in the United States outside of an approved Investigational New Drug Application. Moreover, in other arenas, significant, serious adverse events have been reported after the use of manipulated stem cells. Both the US Food and Drug Administration and American Academy of Orthopaedic Surgeons have recognized the potential for abuse regarding this evolving technology. Published results using stems cells to treat rotator cuff disease are inconsistent, and the optimum source and preparation of the stem cells remains unknown.


Assuntos
Células-Tronco Mesenquimais , Lesões do Manguito Rotador , Artroscopia , Seguimentos , Humanos , Manguito Rotador , Células-Tronco , Estados Unidos
14.
Hua Xi Kou Qiang Yi Xue Za Zhi ; 38(2): 122-127, 2020 Apr 01.
Artigo em Chinês | MEDLINE | ID: mdl-32314882

RESUMO

OBJECTIVE: This study aimed to evaluate the effects of porcine acellular cartilaginous matrix (pACM) on the proliferation and differentiation of human adipose-derived stromal cells (hADSCs). METHODS: pACM was prepared from porcine articular cartilage through decellularization treatment. hADSCs were isolated from human adipose tissues and cultured with different pACM concentrations. No pACM was used as the control group. The effect of pACM on hADSCs proliferation was detected by CCK-8 method. Moreover, the effect of pACM on hADSCs chondrogenic differentiation was analyzed through fluorescence quantitative polymerase chain reaction and Western blot. RESULTS: hADSCs proliferation rate in 0.5, 1.0, and 2.0 mg·mL⁻¹ pACM groups was not significantly different from that in the control group, whereas that in 4.0 and 8.0 mg·mL⁻¹ pACM group was lower than that in the control group (P<0.05). The expression levels of pACM chondrogenic genes, including SOX-9, collagen type Ⅱ alpha 1 chain (COL2A1), and aggrecan (ACAN) and cell adhesion-related gene LAMININ in 0.5, 1.0, and 2.0 mg·mL⁻¹ pACM group were higher than those of the control group (P<0.05), but that of a stemness-related gene Notch-1 was lower than that of the control group (P<0.05). No statistical difference was found in the expression of a lipogenesis-related gene peroxisome proliferator-activated receptor-γ (PPAr-γ) (P>0.05). The expression levels of chondrogenic proteins (SOX-9, COL2A1, and ACAN) were higher than those of the control group (P<0.05). CONCLUSIONS: Appropriate pACM con-centrations do not affect hADSCs proliferation but can induce hADSCs chondrogenic differentiation.


Assuntos
Adipócitos , Células-Tronco , Tecido Adiposo , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Condrogênese , Humanos , Células Estromais , Suínos
15.
Hua Xi Kou Qiang Yi Xue Za Zhi ; 38(2): 128-132, 2020 Apr 01.
Artigo em Chinês | MEDLINE | ID: mdl-32314883

RESUMO

OBJECTIVE: This study aimed to investigate the distribution of Gli1+ cells in the periodontal ligament (PDL) and to evaluate their contribution in the development of periodontal tissue by using transgenic mouse lines. METHODS: Gli1lacZ/+ mice were harvested at different ages (3, 6, and 8 weeks), and the temporal and spatial distribution patterns of Gli1+ PDL cells were revealed by X-gal staining. Afterward, 3-week-old Gli1-CreERT2/+;R26RtdTomato/+ mice were administered with tamoxifen, and the fates of Gli1+ cells and their descendants were traced during periodontal development. RESULTS: A large number of Gli1+ cells were detected in the PDL of the 3-week-old mice; however, their number significantly decreased from 3 weeks to 8 weeks (P<0.05). Cell lineage tracing data showed that the descendants of Gli1+ cells dramatically increased from 3 weeks to 8 weeks (P<0.05) and gradually differentiated into fibroblasts, cementocytes, and osteocytes. CONCLUSIONS: The multi-differentiation potential of Gli1+ PDL cells was revealed, indicating that Gli1+ cells are an important cell source for periodontal development.


Assuntos
Ligamento Periodontal , Células-Tronco , Animais , Diferenciação Celular , Células Cultivadas , Camundongos , Camundongos Transgênicos , Proteína GLI1 em Dedos de Zinco
16.
Braz Oral Res ; 34: e033, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32267290

RESUMO

The aim of our study was to isolate populations of keratinocyte stem cells based on the expression of cell surface markers and to investigate whether the culture could affect their phenotype. keratinocytes from human oral mucosa were sorted based on the expression of the epithelial stem cell markers p75NTR and CD71. We also examined the co-expression of other epithelial stem markers such as integrins ß1 and α6 and their stem cell-like proprieties in in vitro assays. Three passages after being sorted by MACS, more than 93% of the p75NTR+ve cells lost the expression of p75NTR, while 5.46% of the p75NTR-ve gained it. Within the small population of the p75NTR+ve cells, 88% co-expressed other epithelial stem cell markers such as integrins ß1 and α6, while only 28% of p75NTR-ve cells co-expressed these markers. These results were confirmed by sorting cells by FACS. Additionally, when double staining was used for sorting cells, 99% of the p75NTR+veCD71-ve and 33% of the p75NTR-veCD71+ve cells expressed both integrins, but just one week after culture, only 1.74% of the p75NTR+veCD71-ve cells still expressed p75NTR and only 0.32% still expressed CD71. Similar results were obtained when co-culturing p75NTR+ve and p75NTR-ve populations before analysis. Our results suggest that phenotype changes may be part of an intrinsic cellular mechanism to conserve levels of protein expression as they may found in the human body. In addition, in vitro culture may not offer ideal conditions for epithelial stem cell maintenance due to phenotype changes under standard culture conditions.


Assuntos
Técnicas de Cultura de Células/métodos , Células Epiteliais/citologia , Queratinócitos/citologia , Mucosa Bucal/citologia , Fenótipo , Células-Tronco/citologia , Antígenos CD/análise , Biomarcadores/análise , Separação Celular/métodos , Citometria de Fluxo/métodos , Humanos , Proteínas do Tecido Nervoso/análise , Receptores de Fator de Crescimento Neural/análise , Receptores da Transferrina/análise , Reprodutibilidade dos Testes
17.
Braz Oral Res ; 34: e030, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32236319

RESUMO

The abnormal increase in proliferation rate of human periodontal ligament stem cells (PDLSCs) is considered to be involved in the pathogenesis of periodontitis, a disease in which the IL-10-mediated anti-inflammatory pathway plays a critical role. This study aimed to investigate the involvement of microRNA-466l in periodontitis and to explore the possible interaction between IL-10 and microRNA-466l. PDLSCs were obtained from periodontitis-affected teeth and healthy control teeth. The expression of microRNA-466l and IL-10 mRNA was measured in PDLSCs using RT-qPCR. The proliferation ability of PDLSCs was analyzed using CCK-8 assays. Overexpression of microRNA-466l in a PDLSC cell line was established using two different types of PDLSCs, and the effect of microRNA-466l overexpression on IL-10 expression and cell proliferation were detected by western blot and CCK-8 assays, respectively. We found that expression levels of microRNA-466l and IL-10 mRNA were significantly lower (P < 0.05) in PDLSCs derived from periodontitis-affected teeth compared to those derived from healthy teeth. However, the cell proliferation ability was significantly higher in the PDLSCs derived from periodontitis-affected teeth. Meanwhile microRNA-466l overexpression decreased cell proliferation rates of both types of PDLSCs and upregulated IL-10 expression. Together, these data suggest that microRNA-466l can upregulate IL-10 and reduce the proliferation rate of PDLSCs.


Assuntos
Proliferação de Células/fisiologia , Interleucina-10/metabolismo , Interleucina-10/uso terapêutico , MicroRNAs/metabolismo , Periodontite/terapia , Células-Tronco/metabolismo , Adulto , Western Blotting , Diferenciação Celular , Humanos , Periodontite/genética , Periodontite/metabolismo , Regulação para Cima
18.
PLoS One ; 15(4): e0229593, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32324791

RESUMO

Acute myeloid leukaemia (AML) is characterised by phenotypic heterogeneity, which we hypothesise is a consequence of deregulated differentiation with transcriptional reminiscence of the normal compartment or cell-of-origin. Here, we propose a classification system based on normal myeloid progenitor cell subset-associated gene signatures (MAGS) for individual assignments of AML subtypes. We generated a MAGS classifier including the progenitor compartments CD34+/CD38- for haematopoietic stem cells (HSCs), CD34+/CD38+/CD45RA- for megakaryocyte-erythroid progenitors (MEPs), and CD34+/CD38+/CD45RA+ for granulocytic-monocytic progenitors (GMPs) using regularised multinomial regression with three discrete outcomes and an elastic net penalty. The regularisation parameters were chosen by cross-validation, and MAGS assignment accuracy was validated in an independent data set (N = 38; accuracy = 0.79) of sorted normal myeloid subpopulations. The prognostic value of MAGS assignment was studied in two clinical cohorts (TCGA: N = 171; GSE6891: N = 520) and had a significant prognostic impact. Furthermore, multivariate Cox regression analysis using the MAGS subtype, FAB subtype, cytogenetics, molecular genetics, and age as explanatory variables showed independent prognostic value. Molecular characterisation of subtypes by differential gene expression analysis, gene set enrichment analysis, and mutation patterns indicated reduced proliferation and overrepresentation of RUNX1 and IDH2 mutations in the HSC subtype; increased proliferation and overrepresentation of CEBPA mutations in the MEP subtype; and innate immune activation and overrepresentation of WT1 mutations in the GMP subtype. We present a differentiation-dependent classification system for AML subtypes with distinct pathogenetic and prognostic importance that can help identify candidates poorly responding to combination chemotherapy and potentially guide alternative treatments.


Assuntos
Células-Tronco Hematopoéticas/metabolismo , Leucemia Mieloide Aguda/genética , Células Mieloides/metabolismo , Células-Tronco/metabolismo , ADP-Ribosil Ciclase 1/genética , Antígenos CD34/genética , Diferenciação Celular/genética , Linhagem da Célula/genética , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Células-Tronco Hematopoéticas/patologia , Humanos , Isocitrato Desidrogenase/genética , Leucemia Mieloide Aguda/patologia , Antígenos Comuns de Leucócito/genética , Masculino , Pessoa de Meia-Idade , Mutação/genética , Células Mieloides/patologia , Análise de Componente Principal , Análise de Regressão , Células-Tronco/patologia , Proteínas WT1/genética
19.
Stem Cell Rev Rep ; 16(3): 434-440, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32307653

RESUMO

The expressive number of deaths and confirmed cases of SARS-CoV-2 call for an urgent demand of effective and available drugs for COVID-19 treatment. CD147, a receptor on host cells, is a novel route for SARS-CoV-2 invasion. Thus, drugs that interfere in the spike protein/CD147 interaction or CD147 expression may inhibit viral invasion and dissemination among other cells, including in progenitor/stem cells. Studies suggest beneficial effects of azithromycin in reducing viral load of hospitalized patients, possibly interfering with ligand/CD147 receptor interactions; however, its possible effects on SARS-CoV-2 invasion has not yet been evaluated. In addition to the possible effect in invasion, azithromycin decreases the expression of some metalloproteinases (downstream to CD147), induces anti-viral responses in primary human bronchial epithelial infected with rhinovirus, decreasing viral replication and release. Moreover, resident lung progenitor/stem are extensively differentiated into myofibroblasts during pulmonary fibrosis, a complication observed in COVID-19 patients. This process, and the possible direct viral invasion of progenitor/stem cells via CD147 or ACE2, could result in the decline of these cellular stocks and failing lung repair. Clinical tests with allogeneic MSCs from healthy individuals are underway to enhance endogenous lung repair and suppress inflammation.


Assuntos
Antibacterianos/uso terapêutico , Azitromicina/uso terapêutico , Basigina/genética , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/terapia , Pandemias , Pneumonia Viral/terapia , Glicoproteína da Espícula de Coronavírus/genética , Transplante de Células-Tronco , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/virologia , Basigina/antagonistas & inibidores , Basigina/imunologia , Betacoronavirus/metabolismo , Betacoronavirus/patogenicidade , Ensaios Clínicos como Assunto , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Expressão Gênica , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/imunologia , Humanos , Pulmão/imunologia , Pulmão/virologia , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/imunologia , Pneumonia Viral/epidemiologia , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Ligação Proteica/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Glicoproteína da Espícula de Coronavírus/imunologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/imunologia , Células-Tronco/virologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/virologia , Carga Viral/efeitos dos fármacos
20.
Nature ; 580(7803): 386-390, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32296174

RESUMO

The aetiology of inflammatory bowel disease (IBD) is a multifactorial interplay between heredity and environment1,2. Here we report that deficiency in SETDB1, a histone methyltransferase that mediates the trimethylation of histone H3 at lysine 9, participates in the pathogenesis of IBD. We found that levels of SETDB1 are decreased in patients with IBD, and that mice with reduced SETDB1 in intestinal stem cells developed spontaneous terminal ileitis and colitis. SETDB1 safeguards genome stability3, and the loss of SETDB1 in intestinal stem cells released repression of endogenous retroviruses (retrovirus-like elements with long repeats that, in humans, comprise approximately 8% of the genome). Excessive viral mimicry generated by motivated endogenous retroviruses triggered Z-DNA-binding protein 1 (ZBP1)-dependent necroptosis, which irreversibly disrupted homeostasis of the epithelial barrier and promoted bowel inflammation. Genome instability, reactive endogenous retroviruses, upregulation of ZBP1 and necroptosis were all seen in patients with IBD. Pharmaceutical inhibition of RIP3 showed a curative effect in SETDB1-deficient mice, which suggests that targeting necroptosis of intestinal stem cells may represent an approach for the treatment of severe IBD.


Assuntos
Instabilidade Genômica , Histona-Lisina N-Metiltransferase/metabolismo , Doenças Inflamatórias Intestinais/metabolismo , Necroptose , Células-Tronco/metabolismo , Animais , Histona-Lisina N-Metiltransferase/genética , Humanos , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/patologia , Camundongos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Células-Tronco/citologia
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