Multiple regulatory mechanisms control osteoblast differentiation and function to ensure unperturbed skeletal formation and remodeling. In this study we identify histone lysine-specific demethylase 1(LSD1/KDM1 A) as a...Multiple regulatory mechanisms control osteoblast differentiation and function to ensure unperturbed skeletal formation and remodeling. In this study we identify histone lysine-specific demethylase 1(LSD1/KDM1 A) as a key epigenetic regulator of osteoblast differentiation. Knockdown of LSD1 promoted osteoblast differentiation of human mesenchymal stem cells(hMSCs)in vitro and mice lacking LSD1 in mesenchymal cells displayed increased bone mass secondary to accelerated osteoblast differentiation. Mechanistic in vitro studies revealed that LSD1 epigenetically regulates the expression of WNT7 B and BMP2. LSD1 deficiency resulted in increased BMP2 and WNT7 B expression in osteoblasts and enhanced bone formation, while downregulation of WNT7 B-and BMP2-related signaling using genetic mouse model or small-molecule inhibitors attenuated bone phenotype in vivo. Furthermore, the LSD1 inhibitor tranylcypromine(TCP) could increase bone mass in mice. These data identify LSD1 as a novel regulator of osteoblast activity and suggest LSD1 inhibition as a potential therapeutic target for treatment of osteoporosis.展开更多
目的建立人骨髓间充质干细胞体外分离、培养体系,并将其向成骨细胞定向诱导分化。方法采用梯度离心法从人骨髓血中提取骨髓间充质干细胞(m esenchym al stem cell,MSC),经体外培养扩增,观察其生物学特性。结果在体外培养条件下,骨髓MSC...目的建立人骨髓间充质干细胞体外分离、培养体系,并将其向成骨细胞定向诱导分化。方法采用梯度离心法从人骨髓血中提取骨髓间充质干细胞(m esenchym al stem cell,MSC),经体外培养扩增,观察其生物学特性。结果在体外培养条件下,骨髓MSC具有较强的增殖能力,并能被诱导向成骨细胞分化。结论建立了骨髓MSC体外分离、培养的体系,探讨了骨髓MSC的生物学特性,为进一步研究MSC作为骨组织工程的种子细胞打下了基础。展开更多
Arginine-glycine-aspartic acid （RGD） dendrimer-based nanopatterns on poly（L- lactic acid） were used as bioactive substrates to evaluate the impact of the RGD local surface density on the chondrogenic induction of ...Arginine-glycine-aspartic acid （RGD） dendrimer-based nanopatterns on poly（L- lactic acid） were used as bioactive substrates to evaluate the impact of the RGD local surface density on the chondrogenic induction of adult human mesenchymal stem cells. During chondrogenic commitment, active extracellular matrix （ECM） remodeling takes place, playing an instructive role in the differentiation process. Although three-dimensional environments such as pellet or micromass cultures are commonly used for in vitro chondrogenic differentiation, these cultures are rather limited with respect to their ability to interrogate cells in celI-ECM interactions. In the present study, the nanopatterns of the tunable RGD surface density were obtained as a function of the initial dendrimer concentration. The local RGD surface density was quantified through probability contour plots for the minimum interparticle distance, constructed from the corresponding atomic force microscopy images, and correlated with the cell adhesion and differentiation response. The results revealed that the local RGD surface density at the nanoscale acts as a regulator of chondrogenic commitment, and that intermediate adhesiveness of cells to the substrates favors mesenchymal cell condensation and early chondrogenic differentiation.展开更多
Nanotopographical cues endow biomaterials the ability to guide cell adhesion, proliferation, and differentiation. Cellular mechanical memory can maintain the cell status by retaining cellular information obtained from...Nanotopographical cues endow biomaterials the ability to guide cell adhesion, proliferation, and differentiation. Cellular mechanical memory can maintain the cell status by retaining cellular information obtained from past mechanical microenvironments. Here, we propose a new concept “morphology memory of small extracellular vesicles (sEV)” for bone regeneration. We performed nanotopography on titanium plates through alkali and heat (Ti8) treatment to promote human mesenchymal stem cell (hMSC) differentiation. Next, we extracted the sEVs from the hMSC, which were cultured on the nanotopographical Ti plates for 21 days (Ti8-21-sEV). We demonstrated that Ti8-21-sEV had superior pro-osteogenesis ability in vitro and in vivo. RNA sequencing further confirmed that Ti8-21-sEV promote bone regeneration through osteogenic-related pathways, including the PI3K-AKT signaling pathway, MAPK signaling pathway, focal adhesion, and extracellular matrix-receptor interaction. Finally, we decorated the Ti8-21-sEV on a 3D printed porous polyetheretherketone scaffold. The femoral condyle defect model of rabbits was used to demonstrate that Ti8-21-sEV had the best bone ingrowth. In summary, our study demonstrated that the Ti8-21-sEV have memory function by copying the pro-osteogenesis information from the nanotopography. We expect that our study will encourage the discovery of other sEV with morphology memory for tissue regeneration.展开更多
AIM： To establish a novel coculture system for ex vivo expansion of umbilical cord blood（UCB） hematopoietic progenitors using thrombopoietin （TPO）/FIt-3 ligand （FL）-transduced human marrow-derived mesenchymal s...AIM： To establish a novel coculture system for ex vivo expansion of umbilical cord blood（UCB） hematopoietic progenitors using thrombopoietin （TPO）/FIt-3 ligand （FL）-transduced human marrow-derived mesenchymal stem cells （tfhMSCs） as feeder. METHODS： UCB CD34^＋ cells were isolated and cultured using four culture systems in serum-containing or serumfree medium. Suitable aliquots of cultured cells were used to monitor cell production, clonogenic activity, and long-term culture-initiating culture （LTC-IC） output. Finally, the severe-combined immunodeficient （SCID） mouse-repopulating cell （SRC） assay was performed to confirm ability of the cultured cells to reconstitute longterm hematopoiesis. RESULTS： There were no significant differences in the number of total nudeated cells among different culture systems in serum-containing medium during 21-d culture. However, on d 14, the outputs of CD34^＋ cells, CFU-C and CFU-GEMM in tfhMSCs coculture system were significantly enhanced. LTC-IC assay demonstrated that the tfhMSCs coculture system had the most powerful activity. The severe-combined immunodeficient （SCID） mouse repopulating cell （SRC） assay confirmed extensive ability of the expanded cells to reconstitute long-term hematopoiesis. Furthermore, PCR analysis demonstrated the presence of human hematopoietic cells in the bone marrow and peripheral blood cells of NOD/SCID mice.CONCLUSION： The TPO/FL-transduced hMSCs, in combination with additive cytokines, can effectively expand hematopoietic progenitors from UCB in vitro and the tfhMSCs coculture system may be a suitable system for ex vivo manipulation of primitive progenitor cells under contact culture conditions.展开更多
基金supported in part by grants from 973 Program from the Chinese Ministry of Science and Technology (MOST) [2014CB964704, 2015CB964503]the National Natural Science Foundation of China (NSFC) [31371463]the "1000 Young Talents Program of China" and "the National Science Fund for Excellent Young Scholars" (NSFC) [81322027]
文摘Multiple regulatory mechanisms control osteoblast differentiation and function to ensure unperturbed skeletal formation and remodeling. In this study we identify histone lysine-specific demethylase 1(LSD1/KDM1 A) as a key epigenetic regulator of osteoblast differentiation. Knockdown of LSD1 promoted osteoblast differentiation of human mesenchymal stem cells(hMSCs)in vitro and mice lacking LSD1 in mesenchymal cells displayed increased bone mass secondary to accelerated osteoblast differentiation. Mechanistic in vitro studies revealed that LSD1 epigenetically regulates the expression of WNT7 B and BMP2. LSD1 deficiency resulted in increased BMP2 and WNT7 B expression in osteoblasts and enhanced bone formation, while downregulation of WNT7 B-and BMP2-related signaling using genetic mouse model or small-molecule inhibitors attenuated bone phenotype in vivo. Furthermore, the LSD1 inhibitor tranylcypromine(TCP) could increase bone mass in mice. These data identify LSD1 as a novel regulator of osteoblast activity and suggest LSD1 inhibition as a potential therapeutic target for treatment of osteoporosis.
文摘目的建立人骨髓间充质干细胞体外分离、培养体系,并将其向成骨细胞定向诱导分化。方法采用梯度离心法从人骨髓血中提取骨髓间充质干细胞(m esenchym al stem cell,MSC),经体外培养扩增,观察其生物学特性。结果在体外培养条件下,骨髓MSC具有较强的增殖能力,并能被诱导向成骨细胞分化。结论建立了骨髓MSC体外分离、培养的体系,探讨了骨髓MSC的生物学特性,为进一步研究MSC作为骨组织工程的种子细胞打下了基础。
文摘Arginine-glycine-aspartic acid （RGD） dendrimer-based nanopatterns on poly（L- lactic acid） were used as bioactive substrates to evaluate the impact of the RGD local surface density on the chondrogenic induction of adult human mesenchymal stem cells. During chondrogenic commitment, active extracellular matrix （ECM） remodeling takes place, playing an instructive role in the differentiation process. Although three-dimensional environments such as pellet or micromass cultures are commonly used for in vitro chondrogenic differentiation, these cultures are rather limited with respect to their ability to interrogate cells in celI-ECM interactions. In the present study, the nanopatterns of the tunable RGD surface density were obtained as a function of the initial dendrimer concentration. The local RGD surface density was quantified through probability contour plots for the minimum interparticle distance, constructed from the corresponding atomic force microscopy images, and correlated with the cell adhesion and differentiation response. The results revealed that the local RGD surface density at the nanoscale acts as a regulator of chondrogenic commitment, and that intermediate adhesiveness of cells to the substrates favors mesenchymal cell condensation and early chondrogenic differentiation.
基金This study was supported by the National Key R&D Program of China(2018YFB1105700)the National Natural Science Foundation of China(81902261,81772401)+2 种基金the Fundamental Research Funds for the Cen-tral Universities(2019kfyXMBZ063)the Application Foundation and Advanced Program of Wuhan Science and Technology Bureau(2019020701011457)We also thank the Medical Subcenter of HUST Analytical&Testing Center in data acquisition.
文摘Nanotopographical cues endow biomaterials the ability to guide cell adhesion, proliferation, and differentiation. Cellular mechanical memory can maintain the cell status by retaining cellular information obtained from past mechanical microenvironments. Here, we propose a new concept “morphology memory of small extracellular vesicles (sEV)” for bone regeneration. We performed nanotopography on titanium plates through alkali and heat (Ti8) treatment to promote human mesenchymal stem cell (hMSC) differentiation. Next, we extracted the sEVs from the hMSC, which were cultured on the nanotopographical Ti plates for 21 days (Ti8-21-sEV). We demonstrated that Ti8-21-sEV had superior pro-osteogenesis ability in vitro and in vivo. RNA sequencing further confirmed that Ti8-21-sEV promote bone regeneration through osteogenic-related pathways, including the PI3K-AKT signaling pathway, MAPK signaling pathway, focal adhesion, and extracellular matrix-receptor interaction. Finally, we decorated the Ti8-21-sEV on a 3D printed porous polyetheretherketone scaffold. The femoral condyle defect model of rabbits was used to demonstrate that Ti8-21-sEV had the best bone ingrowth. In summary, our study demonstrated that the Ti8-21-sEV have memory function by copying the pro-osteogenesis information from the nanotopography. We expect that our study will encourage the discovery of other sEV with morphology memory for tissue regeneration.
基金Supported by the grants of NIH-Heart,Lung & Blood,No.IR014L70593-01 and Zhejiang Scientific Foundation,No.2003C23015
文摘AIM： To establish a novel coculture system for ex vivo expansion of umbilical cord blood（UCB） hematopoietic progenitors using thrombopoietin （TPO）/FIt-3 ligand （FL）-transduced human marrow-derived mesenchymal stem cells （tfhMSCs） as feeder. METHODS： UCB CD34^＋ cells were isolated and cultured using four culture systems in serum-containing or serumfree medium. Suitable aliquots of cultured cells were used to monitor cell production, clonogenic activity, and long-term culture-initiating culture （LTC-IC） output. Finally, the severe-combined immunodeficient （SCID） mouse-repopulating cell （SRC） assay was performed to confirm ability of the cultured cells to reconstitute longterm hematopoiesis. RESULTS： There were no significant differences in the number of total nudeated cells among different culture systems in serum-containing medium during 21-d culture. However, on d 14, the outputs of CD34^＋ cells, CFU-C and CFU-GEMM in tfhMSCs coculture system were significantly enhanced. LTC-IC assay demonstrated that the tfhMSCs coculture system had the most powerful activity. The severe-combined immunodeficient （SCID） mouse repopulating cell （SRC） assay confirmed extensive ability of the expanded cells to reconstitute long-term hematopoiesis. Furthermore, PCR analysis demonstrated the presence of human hematopoietic cells in the bone marrow and peripheral blood cells of NOD/SCID mice.CONCLUSION： The TPO/FL-transduced hMSCs, in combination with additive cytokines, can effectively expand hematopoietic progenitors from UCB in vitro and the tfhMSCs coculture system may be a suitable system for ex vivo manipulation of primitive progenitor cells under contact culture conditions.
