Superficial angiomyxoma is a rare benign mesenchymal tumor that mainly occurs in the genital region. We report the case of a 51-year-old woman with a painless vulvar mass, well circumscribed on ultrasound. On gross fi...Superficial angiomyxoma is a rare benign mesenchymal tumor that mainly occurs in the genital region. We report the case of a 51-year-old woman with a painless vulvar mass, well circumscribed on ultrasound. On gross finding, it was a polypoid and bilobed mass, partially encapsulated. On histological examination, it was a proliferation of non-atypical spindle cells with an abundant, myxoid stroma and numerous medium-sized blood vessels. The diagnosis was superficial angiomyxoma. The clinical features do not often lead to the diagnosis of superficial vulvar angiomyxoma. It is based on histological examination and immunohistochemistry is helpful to differentiate it from other myxoid tumors.展开更多
Mesenchymal stromal/stem cells(MSCs)have shown significant therapeutic potential,and have therefore been extensively investigated in preclinical studies of regenerative medicine.However,while MSCs have been shown to b...Mesenchymal stromal/stem cells(MSCs)have shown significant therapeutic potential,and have therefore been extensively investigated in preclinical studies of regenerative medicine.However,while MSCs have been shown to be safe as a cellular treatment,they have usually been therapeutically ineffective in human diseases.In fact,in many clinical trials it has been shown that MSCs have moderate or poor efficacy.This inefficacy appears to be ascribable primarily to the heterogeneity of MSCs.Recently,specific priming strategies have been used to improve the therapeutic properties of MSCs.In this review,we explore the literature on the principal priming approaches used to enhance the preclinical inefficacy of MSCs.We found that different priming strategies have been used to direct the therapeutic effects of MSCs toward specific pathological processes.Particularly,while hypoxic priming can be used primarily for the treatment of acute diseases,inflammatory cytokines can be used mainly to prime MSCs in order to treat chronic immune-related disorders.The shift in approach from regeneration to inflammation implies,in MSCs,a shift in the production of functional factors that stimulate regenerative or anti-inflammatory pathways.The opportunity to fine-tune the therapeutic properties of MSCs through different priming strategies could conceivably pave the way for optimizing their therapeutic potential.展开更多
Objective： To investigate the methylation status of CpG island in E-cadherin（CDHl）, P16^INK4a（ P16）promoter region ,and to analyze their role in gastrointestinal stromal rumor （GISTs）. Methods： A total of 56 s...Objective： To investigate the methylation status of CpG island in E-cadherin（CDHl）, P16^INK4a（ P16）promoter region ,and to analyze their role in gastrointestinal stromal rumor （GISTs）. Methods： A total of 56 surgically resected GISTs were obtained from January 2003 to December 2005. The routine H＆E-stained sections and CD117, CD34-immunoreactions were reviewed to verify the morphologic diagnosis. Methylation status of the CDH1, P16^INK4a promoter region was analyzed by methylation specific polymerase chain reaction （MSP） from chemically modified DNA after Na-bisulfite treatment. Results： The frequency of CDHlgene methylation was 32% （18 of 56） in GISTs. The rate was 9% （1 of 11）, 21% （4 of 19）, 41.6% （5 of 12）, and 57% （8 of 14） for very low risk, low risk, intermediate risk, and high risk GISTs; P16^INK4a methylation was found in 19 of 56（34%） cases. The rate was 0% （0 of 11）, 16% （3 of 19）, 50% （6 of 12）, and 71% （10 of 14） for very low risk, low risk, intermediate risk, and high risk GISTs. Statistical analysis indicated that of the 56 cases, there was significant association of CDH^INK4a and/or P16^INK4a methylation status with tumor malignant behavior （methylation rate 23/56, 41%, P〈0.01） and site （P〈0.05）. Conclusion： E-cadherin （CDHI） and/or P16^INK4a promoter hypermethylation is strongly associated with risk grade, may be a useful biomarker for GISTs risk assessment, and may shed light on new therapeutic options to treat GISTs展开更多
AIM To investigate the prognostic value of preoperative fibrinogen concentration(FIB) and D-dimer-fibrinogen ratio(DFR) in gastrointestinal stromal tumors(GISTs).METHODS The purpose of this study was to retrospectivel...AIM To investigate the prognostic value of preoperative fibrinogen concentration(FIB) and D-dimer-fibrinogen ratio(DFR) in gastrointestinal stromal tumors(GISTs).METHODS The purpose of this study was to retrospectively analyze 170 patients with GISTs who were admitted to our hospital from January 2010 to December 2015. The optimal cutoff values of related parameters were estimated by receiver operating characteristic(ROC) curve analysis. The recurrence free survival(RFS) rate was evaluated using Kaplan-Meier curves. Univariate analysis and multivariate Cox regression models were used to analyze the prognostic factors of GISTs. The relationship between the FIB, D-dimer, DFR, platelet count(PLT), and the clinicopathological features of GISTs was described by the chi-square test or nonparametric rank sum test(MannWhitney test).RESULTS In ROC analysis, the optimal cutoff values of FIB, D-dimer, DFR, and PLT were 3.24 g/L, 1.24 mg/L, 0.354, and 197.5(× 109/L), respectively. Univariate analysis and the KaplanMeier survival curve showed that FIB, D-dimer, DFR, PLT,National Institutes of Health(NIH) risk category, tumor size, tumor location, and mitotic index were significantly relevant to the 3-year and 5-year survival rate of patients(P < 0.05). Cox multivariate regression analysis illustrated that FIB(RR : 0.108, 95%CI: 0.031-0.373), DFR(RR : 0.319, 95%CI: 0.131-0.777), and NIH risk category(RR : 0.166, 95%CI: 0.047-0.589) were independent prognostic factors of the RFS rate(P < 0. 05). Moreover, FIB, D-dimer, DFR, and PLT were correlated with the clinical features of GISTs.CONCLUSION FIB, D-dimer, DFR, and PLT are all related to the prognosis of GISTs. Moreover, FIB and DFR may be independent risk factors for predicting the prognosis of resectable GISTs.展开更多
The presence of endometrial tissue outside of the uterine cavity is named endometriosis and is the most common gynecologic disorder in women. Determining the inhibitory effect of a Deforolimus on angiogenesis in a thr...The presence of endometrial tissue outside of the uterine cavity is named endometriosis and is the most common gynecologic disorder in women. Determining the inhibitory effect of a Deforolimus on angiogenesis in a three-dimensional (3-D) culture of human endometrial stromal cells (hEnCs) in vitro. The important mechanism in the pathogenesis of endometriosis is angiogenesis, and deforolimus has been shown to have anti-angiogenic activity. This was an in vitro study of human endometrial stromal cells in 3-D culture of fibrin matrix. Endometrial stromal cells isolated and placed in a 3-D fibrin matrix culture system for angiogenesis with VEGF and inhibit angiogenesis by deforolimus. Finally these cells analyzed by CD31 antibodies. After 3 weeks, in cells treated with VEGF, endothelial cell branching was observed and rudimentary capillary-like structures formed. In the presence of 5μM of deforolimus, angiogenesis was reduced. The deforolimus were shown to be effective in inhibiting the mechanisms of angiogenesis.展开更多
Objective Gastrointestinal stromal tumors(GISTs)can rapidly proliferate through angiogenesis.Previous studies indicated the potential influence of microRNA on the progression of tumor immature angiogenesis.This study ...Objective Gastrointestinal stromal tumors(GISTs)can rapidly proliferate through angiogenesis.Previous studies indicated the potential influence of microRNA on the progression of tumor immature angiogenesis.This study aimed to explore the specific mechanism by which microRNA-409-5p(miR-409-5p)contributes to GIST.Methods To identify genes potentially involved in the development and progression of GIST,the differences of miR-409-5p between tumors and adjacent tissues were first analyzed.Following this analysis,target genes were predicted.To further investigate the function of miRNA in GIST cells,two GIST cell lines(GIST-T1 and GIST882)were transfected with lentiviruses that stably expressed miR-409-5p and scrambled miRNA(negative control).Later,the cells were subjected to Western blotting and ELSA to determine any differences in angiogenesis-related genes.Results In GISTs,there was a decrease in the expression levels of miR-409-5p compared to the adjacent tissues.It was observed that the upregulation of miR-409-5p in GIST cell lines effectively inhibited the proteins hypoxia-inducible transcription factor 1β(HIF1β)and vascular endothelial growth factor A(VEGF-A).Further investigations revealed that miR-409-5p acted as an inhibitor of angiogenesis by binding to the 3′-UTR of Lysine-specific demethylase 4D(KDM4D)mRNA.Moreover,the combination of miR-409-5p with imatinib enhanced its inhibitory effect on angiogenesis.Conclusion This study demonstrated that the miRNA-409-5p/KDM4D/HIF1β/VEGF-A signaling pathway could serve as a novel target for the development of therapeutic strategies for the treatment of imatinib-resistance in GIST patients.展开更多
Since 3D printed hard materials could match the shape of bone,cell survival and fate determination towards osteoblasts in such materials have become a popular research target.In this study,a scaffold of hardmaterial f...Since 3D printed hard materials could match the shape of bone,cell survival and fate determination towards osteoblasts in such materials have become a popular research target.In this study,a scaffold of hardmaterial for 3D fabrication was designed to regulate developmental signal(Notch)transduction guiding osteoblast differentiation.We established a polycaprolactone(PCL)and cell-integrated 3D printing system(PCI3D)to reciprocally print the beams of PCL and cell-laden hydrogel for a module.This PCI3D module holds good cell viability of over 87%,whereas cells show about sixfold proliferation in a 7-day culture.The osteocytic MLO-Y4 was engineered to overexpress Notch ligand Dll4,making up 25%after mixing with 75%stromal cells in the PCI3D module.Osteocytic Dll4,unlike other delta-like family members such as Dll1 or Dll3,promotes osteoblast differentiation and themineralization of primary mouse and a cell line of bone marrow stromal cells when cultured in a PCI3D module for up to 28 days.Mechanistically,osteocytic Dll4 could not promote osteogenic differentiation of the primary bone marrow stromal cells(BMSCs)after conditional deletion of the Notch transcription factor RBPjκby Cre recombinase.These data indicate that osteocytic Dll4 activates RBPjκ-dependent canonical Notch signaling in BMSCs for their oriented differentiation towards osteoblasts.Additionally,osteocytic Dll4 holds a great potential for angiogenesis in human umbilical vein endothelial cells within modules.Our study reveals that osteocytic Dll4 could be the osteogenic niche determining cell fate towards osteoblasts.This will open a new avenue to overcome the current limitation of poor cell viability and low bioactivity of traditional orthopedic implants.展开更多
Tumor growth is regulated by a diverse set of extraellular infuences,including paracrine crosstalk with stromal partners,and biophysical interactions with surrounding cells and tissues.Studies elucidating the role of ...Tumor growth is regulated by a diverse set of extraellular infuences,including paracrine crosstalk with stromal partners,and biophysical interactions with surrounding cells and tissues.Studies elucidating the role of physical force and the mechanical properties of the extracellular matrix(ECM)itself as regulators of tumor growth and invasion have been greatly catalyzed by the use of in ritro three-dimensional(3D)tumor models.These systems provide the ability to systematically isolate,manipulate,and evaluate impact of stromal components and extracellular mechanics in a platform that is both conducive to imaging and biologically relevant.However,recognizing that mechanoregulatory crosstalk is bi-directional and fully utilizing these models requires complementary methods for in situ measurements of the local mechanical environment.Here,in 3D tumor/fbroblast co-culture models of pancreatic canocer,a discase characterized by its prominent stromal involvement,we evaluate the use of particle-tracking microrhoology to probe dynamic mechanical changes.Using videos of fuorescently labeled polystyrene micro-spheres embedded in ollagen I ECM,we measure spatiotemporal changes in the Brownian motion of probes to report local ECM shear modulus and microheterogeneity.This approach reveals st ffening of collagen in fibroblast co-cultures relative to cultures with cancer cells only,which exhibit degraded ECM with heterogeneous microstructure.We further show that these effects are dependent on culture geometry with contrast ing behavior for embedded and overlay cultures.In addition to potential application to screening stroma targeted therapeutics,this work also provides insight into how the compoition and plating geometry impact the mechanical propertios of 3D cell cultures that are increasingly widey used in cancer biology.展开更多
Human mesenchymal stromal cells(hMSCs)are mechanically sensitive undergoing phenotypic alterations when subjected to shear stress,cell aggregation,and substrate changes encountered in 3D dynamic bioreactor cultures.Ho...Human mesenchymal stromal cells(hMSCs)are mechanically sensitive undergoing phenotypic alterations when subjected to shear stress,cell aggregation,and substrate changes encountered in 3D dynamic bioreactor cultures.However,little is known about how bioreactor microenvironment affects the secretion and cargo profiles of hMSC-derived extracellular vesicles(EVs)including the subset,“exosomes”,which contain therapeutic proteins,nucleic acids,and lipids from the parent cells.In this study,bone marrow-derived hMSCs were expanded on 3D Synthemax II microcarriers in the PBS mini 0.1L Vertical-Wheel bioreactor system under variable shear stress levels at 25,40,and 64 RPM(0.1-0.3 dyn/cm^(2)).The bioreactor system promotes EV secretion from hMSCs by 2.5-fold and upregulates the expression of EV biogenesis markers and glycolysis genes compared to the static 2D culture.The microRNA cargo was also altered in the EVs from bioreactor culture including the upregulation of miR-10,19a,19b,21,132,and 377.EV protein cargo was characterized by proteomics analysis,showing upregulation of metabolic,autophagy and ROS-related proteins comparing with 2D cultured EVs.In addition,the scalability of the Vertical-Wheel bioreactor system was demonstrated in a 0.5L bioreactor,showing similar or better hMSC-EV secretion and cargo content compared to the 0.1L bioreactor.This study advances our understanding of bio-manufacturing of stem cell-derived EVs for applications in cell-free therapy towards treating neurological disorders such as ischemic stroke,Alzheimer’s disease,and multiple sclerosis.展开更多
Background Human adipose tissue-derived stromal cells （hADSCs） can be induced to differentiate along an osteoblastic lineage under stimulation of dexamethasone （DEX）. Recent studies, however, have questioned the e...Background Human adipose tissue-derived stromal cells （hADSCs） can be induced to differentiate along an osteoblastic lineage under stimulation of dexamethasone （DEX）. Recent studies, however, have questioned the efficacy of glucocorticoids such as DEX in mediating the osteogenesis process of skeletal progenitor cells and processed lipoaspirate cells. Is it possible to find a substitute for DEX？ Therefore, this study was designed to investigate osteogenic capacity and regulating mechanisms for osteoblastic differentiation of hADSCs by comparing osteogenic media （OM） containing either 1, 25-dihydroxyvitamin D3 （VD） or DEX and determine if VD was an ideal substitute for DEX as an induction agent for the osteogenesis of hADSCs. Methods Osteogenic differentiation of hADSCs was induced by osteogenic medium （OM） containing either 10 nmol/L VD or 100 nmol/L DEX. Differentiation of hADSCs into osteoblastic lineage was identified by alkaline phosphatase （ALP） staining, von Kossa staining, and reverse transcription-polymerase chain reaction assays for mRNA expression of osteogenesis-related genes such as type Ⅰ collagen （COL Ⅰ）, bone sialoprotein （BSP）, osteocalcin （OC）, bone morphogenetic protein （BMP）-2, BMP-4, BMP-6, BMP-7, runt-related transcription factor 2/core binding factor α1 （Runx2/Cbfal）, osterix （Osx）, and LIM mineralization protein- 1 （LMP- 1）. Results von Kossa staining revealed that the differentiated cells induced by both VD and DEX were mineralized in vitro. They also expressed osteoblast-related markers, such as ALP, COL Ⅰ, BSP, and OC. Runx2/Cbfal, Osx, BMP-6, and LMP-1 were upregulated during VD and DEX-induced hADSC osteoblastic differentiation, but BMP-4, BMP-7 were not. BMP-2 was only expressed in VD-induced differentiated cells. Conclusions VD or DEX-induced hADSCs differentiate toward the osteoblastic lineage in vitro. Runx2/Cbfal, Osx, BMP-2, BMP-6, and LMP-1 are involved in regulating osteoblastic differentiation of hADSCs, but BMP-4, BMP-7 are not. VD, but not DEX, induces expression of BMP-2 during osteogenic induction of hADSCs. VD is an ideal substitute for DEX for osteogenic induction of hADSCs.展开更多
To the Editor:Gastrointestinal stromal tumors(GIST)is the most popular mesenchymal tumor in the gastrointestinal tract with approximately 80%of GIST harboring gain-of-function mutations at either the extracellular reg...To the Editor:Gastrointestinal stromal tumors(GIST)is the most popular mesenchymal tumor in the gastrointestinal tract with approximately 80%of GIST harboring gain-of-function mutations at either the extracellular region(exon 9)or the juxtamembrane domain(JMD,exon 11)of KIT,resulting in uncontrolled proliferation and resistance to apotosis1.展开更多
文摘Superficial angiomyxoma is a rare benign mesenchymal tumor that mainly occurs in the genital region. We report the case of a 51-year-old woman with a painless vulvar mass, well circumscribed on ultrasound. On gross finding, it was a polypoid and bilobed mass, partially encapsulated. On histological examination, it was a proliferation of non-atypical spindle cells with an abundant, myxoid stroma and numerous medium-sized blood vessels. The diagnosis was superficial angiomyxoma. The clinical features do not often lead to the diagnosis of superficial vulvar angiomyxoma. It is based on histological examination and immunohistochemistry is helpful to differentiate it from other myxoid tumors.
文摘Mesenchymal stromal/stem cells(MSCs)have shown significant therapeutic potential,and have therefore been extensively investigated in preclinical studies of regenerative medicine.However,while MSCs have been shown to be safe as a cellular treatment,they have usually been therapeutically ineffective in human diseases.In fact,in many clinical trials it has been shown that MSCs have moderate or poor efficacy.This inefficacy appears to be ascribable primarily to the heterogeneity of MSCs.Recently,specific priming strategies have been used to improve the therapeutic properties of MSCs.In this review,we explore the literature on the principal priming approaches used to enhance the preclinical inefficacy of MSCs.We found that different priming strategies have been used to direct the therapeutic effects of MSCs toward specific pathological processes.Particularly,while hypoxic priming can be used primarily for the treatment of acute diseases,inflammatory cytokines can be used mainly to prime MSCs in order to treat chronic immune-related disorders.The shift in approach from regeneration to inflammation implies,in MSCs,a shift in the production of functional factors that stimulate regenerative or anti-inflammatory pathways.The opportunity to fine-tune the therapeutic properties of MSCs through different priming strategies could conceivably pave the way for optimizing their therapeutic potential.
基金This work was supported by the NaturaScience Foundation of Shanxi Province (No. 2006011122)
文摘Objective： To investigate the methylation status of CpG island in E-cadherin（CDHl）, P16^INK4a（ P16）promoter region ,and to analyze their role in gastrointestinal stromal rumor （GISTs）. Methods： A total of 56 surgically resected GISTs were obtained from January 2003 to December 2005. The routine H＆E-stained sections and CD117, CD34-immunoreactions were reviewed to verify the morphologic diagnosis. Methylation status of the CDH1, P16^INK4a promoter region was analyzed by methylation specific polymerase chain reaction （MSP） from chemically modified DNA after Na-bisulfite treatment. Results： The frequency of CDHlgene methylation was 32% （18 of 56） in GISTs. The rate was 9% （1 of 11）, 21% （4 of 19）, 41.6% （5 of 12）, and 57% （8 of 14） for very low risk, low risk, intermediate risk, and high risk GISTs; P16^INK4a methylation was found in 19 of 56（34%） cases. The rate was 0% （0 of 11）, 16% （3 of 19）, 50% （6 of 12）, and 71% （10 of 14） for very low risk, low risk, intermediate risk, and high risk GISTs. Statistical analysis indicated that of the 56 cases, there was significant association of CDH^INK4a and/or P16^INK4a methylation status with tumor malignant behavior （methylation rate 23/56, 41%, P〈0.01） and site （P〈0.05）. Conclusion： E-cadherin （CDHI） and/or P16^INK4a promoter hypermethylation is strongly associated with risk grade, may be a useful biomarker for GISTs risk assessment, and may shed light on new therapeutic options to treat GISTs
文摘AIM To investigate the prognostic value of preoperative fibrinogen concentration(FIB) and D-dimer-fibrinogen ratio(DFR) in gastrointestinal stromal tumors(GISTs).METHODS The purpose of this study was to retrospectively analyze 170 patients with GISTs who were admitted to our hospital from January 2010 to December 2015. The optimal cutoff values of related parameters were estimated by receiver operating characteristic(ROC) curve analysis. The recurrence free survival(RFS) rate was evaluated using Kaplan-Meier curves. Univariate analysis and multivariate Cox regression models were used to analyze the prognostic factors of GISTs. The relationship between the FIB, D-dimer, DFR, platelet count(PLT), and the clinicopathological features of GISTs was described by the chi-square test or nonparametric rank sum test(MannWhitney test).RESULTS In ROC analysis, the optimal cutoff values of FIB, D-dimer, DFR, and PLT were 3.24 g/L, 1.24 mg/L, 0.354, and 197.5(× 109/L), respectively. Univariate analysis and the KaplanMeier survival curve showed that FIB, D-dimer, DFR, PLT,National Institutes of Health(NIH) risk category, tumor size, tumor location, and mitotic index were significantly relevant to the 3-year and 5-year survival rate of patients(P < 0.05). Cox multivariate regression analysis illustrated that FIB(RR : 0.108, 95%CI: 0.031-0.373), DFR(RR : 0.319, 95%CI: 0.131-0.777), and NIH risk category(RR : 0.166, 95%CI: 0.047-0.589) were independent prognostic factors of the RFS rate(P < 0. 05). Moreover, FIB, D-dimer, DFR, and PLT were correlated with the clinical features of GISTs.CONCLUSION FIB, D-dimer, DFR, and PLT are all related to the prognosis of GISTs. Moreover, FIB and DFR may be independent risk factors for predicting the prognosis of resectable GISTs.
文摘The presence of endometrial tissue outside of the uterine cavity is named endometriosis and is the most common gynecologic disorder in women. Determining the inhibitory effect of a Deforolimus on angiogenesis in a three-dimensional (3-D) culture of human endometrial stromal cells (hEnCs) in vitro. The important mechanism in the pathogenesis of endometriosis is angiogenesis, and deforolimus has been shown to have anti-angiogenic activity. This was an in vitro study of human endometrial stromal cells in 3-D culture of fibrin matrix. Endometrial stromal cells isolated and placed in a 3-D fibrin matrix culture system for angiogenesis with VEGF and inhibit angiogenesis by deforolimus. Finally these cells analyzed by CD31 antibodies. After 3 weeks, in cells treated with VEGF, endothelial cell branching was observed and rudimentary capillary-like structures formed. In the presence of 5μM of deforolimus, angiogenesis was reduced. The deforolimus were shown to be effective in inhibiting the mechanisms of angiogenesis.
基金supported by the National Natural Science Foundation of China(No.81372323 and No.81802426).
文摘Objective Gastrointestinal stromal tumors(GISTs)can rapidly proliferate through angiogenesis.Previous studies indicated the potential influence of microRNA on the progression of tumor immature angiogenesis.This study aimed to explore the specific mechanism by which microRNA-409-5p(miR-409-5p)contributes to GIST.Methods To identify genes potentially involved in the development and progression of GIST,the differences of miR-409-5p between tumors and adjacent tissues were first analyzed.Following this analysis,target genes were predicted.To further investigate the function of miRNA in GIST cells,two GIST cell lines(GIST-T1 and GIST882)were transfected with lentiviruses that stably expressed miR-409-5p and scrambled miRNA(negative control).Later,the cells were subjected to Western blotting and ELSA to determine any differences in angiogenesis-related genes.Results In GISTs,there was a decrease in the expression levels of miR-409-5p compared to the adjacent tissues.It was observed that the upregulation of miR-409-5p in GIST cell lines effectively inhibited the proteins hypoxia-inducible transcription factor 1β(HIF1β)and vascular endothelial growth factor A(VEGF-A).Further investigations revealed that miR-409-5p acted as an inhibitor of angiogenesis by binding to the 3′-UTR of Lysine-specific demethylase 4D(KDM4D)mRNA.Moreover,the combination of miR-409-5p with imatinib enhanced its inhibitory effect on angiogenesis.Conclusion This study demonstrated that the miRNA-409-5p/KDM4D/HIF1β/VEGF-A signaling pathway could serve as a novel target for the development of therapeutic strategies for the treatment of imatinib-resistance in GIST patients.
基金the National Natural Science Foundation of China(Nos.U1601220,82072450,and 81672118)Chongqing Science and Technology Commission-Basic Science and Frontier Technology Key Project(No.cstc2015jcyjBX0119)Chongqing Medical University Intelligent Medicine Research Project(No.ZHYX202115).
文摘Since 3D printed hard materials could match the shape of bone,cell survival and fate determination towards osteoblasts in such materials have become a popular research target.In this study,a scaffold of hardmaterial for 3D fabrication was designed to regulate developmental signal(Notch)transduction guiding osteoblast differentiation.We established a polycaprolactone(PCL)and cell-integrated 3D printing system(PCI3D)to reciprocally print the beams of PCL and cell-laden hydrogel for a module.This PCI3D module holds good cell viability of over 87%,whereas cells show about sixfold proliferation in a 7-day culture.The osteocytic MLO-Y4 was engineered to overexpress Notch ligand Dll4,making up 25%after mixing with 75%stromal cells in the PCI3D module.Osteocytic Dll4,unlike other delta-like family members such as Dll1 or Dll3,promotes osteoblast differentiation and themineralization of primary mouse and a cell line of bone marrow stromal cells when cultured in a PCI3D module for up to 28 days.Mechanistically,osteocytic Dll4 could not promote osteogenic differentiation of the primary bone marrow stromal cells(BMSCs)after conditional deletion of the Notch transcription factor RBPjκby Cre recombinase.These data indicate that osteocytic Dll4 activates RBPjκ-dependent canonical Notch signaling in BMSCs for their oriented differentiation towards osteoblasts.Additionally,osteocytic Dll4 holds a great potential for angiogenesis in human umbilical vein endothelial cells within modules.Our study reveals that osteocytic Dll4 could be the osteogenic niche determining cell fate towards osteoblasts.This will open a new avenue to overcome the current limitation of poor cell viability and low bioactivity of traditional orthopedic implants.
基金funding from the National Cancer Institute (R00 CA155045,PI Celli) which has supported this work.
文摘Tumor growth is regulated by a diverse set of extraellular infuences,including paracrine crosstalk with stromal partners,and biophysical interactions with surrounding cells and tissues.Studies elucidating the role of physical force and the mechanical properties of the extracellular matrix(ECM)itself as regulators of tumor growth and invasion have been greatly catalyzed by the use of in ritro three-dimensional(3D)tumor models.These systems provide the ability to systematically isolate,manipulate,and evaluate impact of stromal components and extracellular mechanics in a platform that is both conducive to imaging and biologically relevant.However,recognizing that mechanoregulatory crosstalk is bi-directional and fully utilizing these models requires complementary methods for in situ measurements of the local mechanical environment.Here,in 3D tumor/fbroblast co-culture models of pancreatic canocer,a discase characterized by its prominent stromal involvement,we evaluate the use of particle-tracking microrhoology to probe dynamic mechanical changes.Using videos of fuorescently labeled polystyrene micro-spheres embedded in ollagen I ECM,we measure spatiotemporal changes in the Brownian motion of probes to report local ECM shear modulus and microheterogeneity.This approach reveals st ffening of collagen in fibroblast co-cultures relative to cultures with cancer cells only,which exhibit degraded ECM with heterogeneous microstructure.We further show that these effects are dependent on culture geometry with contrast ing behavior for embedded and overlay cultures.In addition to potential application to screening stroma targeted therapeutics,this work also provides insight into how the compoition and plating geometry impact the mechanical propertios of 3D cell cultures that are increasingly widey used in cancer biology.
基金This work is supported by National Science Foundation(CBET-1743426 and CBET-1917618)Research reported in this publication was also partially supported by the National Institutes of Health(USA)under Award Number R01NS125016.
文摘Human mesenchymal stromal cells(hMSCs)are mechanically sensitive undergoing phenotypic alterations when subjected to shear stress,cell aggregation,and substrate changes encountered in 3D dynamic bioreactor cultures.However,little is known about how bioreactor microenvironment affects the secretion and cargo profiles of hMSC-derived extracellular vesicles(EVs)including the subset,“exosomes”,which contain therapeutic proteins,nucleic acids,and lipids from the parent cells.In this study,bone marrow-derived hMSCs were expanded on 3D Synthemax II microcarriers in the PBS mini 0.1L Vertical-Wheel bioreactor system under variable shear stress levels at 25,40,and 64 RPM(0.1-0.3 dyn/cm^(2)).The bioreactor system promotes EV secretion from hMSCs by 2.5-fold and upregulates the expression of EV biogenesis markers and glycolysis genes compared to the static 2D culture.The microRNA cargo was also altered in the EVs from bioreactor culture including the upregulation of miR-10,19a,19b,21,132,and 377.EV protein cargo was characterized by proteomics analysis,showing upregulation of metabolic,autophagy and ROS-related proteins comparing with 2D cultured EVs.In addition,the scalability of the Vertical-Wheel bioreactor system was demonstrated in a 0.5L bioreactor,showing similar or better hMSC-EV secretion and cargo content compared to the 0.1L bioreactor.This study advances our understanding of bio-manufacturing of stem cell-derived EVs for applications in cell-free therapy towards treating neurological disorders such as ischemic stroke,Alzheimer’s disease,and multiple sclerosis.
基金This study was supported by a grant from the National Natural Science Foundation of China (No. 30200319).
文摘Background Human adipose tissue-derived stromal cells （hADSCs） can be induced to differentiate along an osteoblastic lineage under stimulation of dexamethasone （DEX）. Recent studies, however, have questioned the efficacy of glucocorticoids such as DEX in mediating the osteogenesis process of skeletal progenitor cells and processed lipoaspirate cells. Is it possible to find a substitute for DEX？ Therefore, this study was designed to investigate osteogenic capacity and regulating mechanisms for osteoblastic differentiation of hADSCs by comparing osteogenic media （OM） containing either 1, 25-dihydroxyvitamin D3 （VD） or DEX and determine if VD was an ideal substitute for DEX as an induction agent for the osteogenesis of hADSCs. Methods Osteogenic differentiation of hADSCs was induced by osteogenic medium （OM） containing either 10 nmol/L VD or 100 nmol/L DEX. Differentiation of hADSCs into osteoblastic lineage was identified by alkaline phosphatase （ALP） staining, von Kossa staining, and reverse transcription-polymerase chain reaction assays for mRNA expression of osteogenesis-related genes such as type Ⅰ collagen （COL Ⅰ）, bone sialoprotein （BSP）, osteocalcin （OC）, bone morphogenetic protein （BMP）-2, BMP-4, BMP-6, BMP-7, runt-related transcription factor 2/core binding factor α1 （Runx2/Cbfal）, osterix （Osx）, and LIM mineralization protein- 1 （LMP- 1）. Results von Kossa staining revealed that the differentiated cells induced by both VD and DEX were mineralized in vitro. They also expressed osteoblast-related markers, such as ALP, COL Ⅰ, BSP, and OC. Runx2/Cbfal, Osx, BMP-6, and LMP-1 were upregulated during VD and DEX-induced hADSC osteoblastic differentiation, but BMP-4, BMP-7 were not. BMP-2 was only expressed in VD-induced differentiated cells. Conclusions VD or DEX-induced hADSCs differentiate toward the osteoblastic lineage in vitro. Runx2/Cbfal, Osx, BMP-2, BMP-6, and LMP-1 are involved in regulating osteoblastic differentiation of hADSCs, but BMP-4, BMP-7 are not. VD, but not DEX, induces expression of BMP-2 during osteogenic induction of hADSCs. VD is an ideal substitute for DEX for osteogenic induction of hADSCs.
基金supported by grants from Shanghai Municipal Science and Technology Major Project(TM202101H012,China)the Science and Technology Commission of Shanghai Municipality(No.18DZ2293200,China)the Yunnan Province Sciences and Technology plan(No.202102AA310026,China).
文摘To the Editor:Gastrointestinal stromal tumors(GIST)is the most popular mesenchymal tumor in the gastrointestinal tract with approximately 80%of GIST harboring gain-of-function mutations at either the extracellular region(exon 9)or the juxtamembrane domain(JMD,exon 11)of KIT,resulting in uncontrolled proliferation and resistance to apotosis1.
