In this study, we loaded human umbilical cord mesenchymal stem cells onto human amniotic membrane with epithelial cells to prepare nerve conduits, i.e., a relatively closed nerve regeneration chamber. After neurolysis...In this study, we loaded human umbilical cord mesenchymal stem cells onto human amniotic membrane with epithelial cells to prepare nerve conduits, i.e., a relatively closed nerve regeneration chamber. After neurolysis, the injured radial nerve was enwrapped with the prepared nerve conduit, which was fixed to the epineurium by sutures, with the cell on the inner surface of the conduit. Simultaneously, a 1.0 mL aliquot of human umbilical cord mesenchymal stem cell suspension was injected into the distal and proximal ends of the injured radial nerve with 1.0 cm intervals. A total of 1.75 x 107 cells were seeded on the amniotic membrane. In the control group, patients received only neurolysis. At 12 weeks after cell transplantation, more than 80% of patients exhibited obvious improvements in muscular strength, and touch and pain sensations. In contrast, these improvements were observed only in 55-65% of control patients. At 8 and 12 weeks, muscular electrophysiological function in the region dominated by the injured radial nerve was significantly better in the transplantation group than the control group. After cell transplantation, no immunological rejections were observed. These findings suggest that human umbilical cord mesenchymal stem cell-loaded amniotic membrane can be used for the repair of radial nerve injury.展开更多
Objective To establish the method of isolation, purification, and identification of human amniotic mesenchymal stem cells (hAMSCs). Methods hAMSCs were isolated from human amniotic membrane by trypsin-collagenase dige...Objective To establish the method of isolation, purification, and identification of human amniotic mesenchymal stem cells (hAMSCs). Methods hAMSCs were isolated from human amniotic membrane by trypsin-collagenase digestion, and cultured in Dulbecco's modified Eagle's medinm/F12 medium supplemented with 10% fetal bovine serum. Phenotypic characteristics of these cells were analyzed by means of immunocytochemistry and flow cytometry. Results The cells successfully isolated from human amniotic membrane expressed representative mesenchymal cell surface markers CD44, CD90, and vimentin, but not CD45. Conclusions This study establishes a potential method for isolation of hAMSCs from human amnion, in vitro culture, and identification. The isolated cells show phenotypic characteristics of mesenchymal stem cells.展开更多
Amniotic membrane of human placenta is a source of abundant mesenchymal stem cell (hAMSC) which makes it a potential source of allogeneic multipotent cell for bone healing. However, much has to be explored about its i...Amniotic membrane of human placenta is a source of abundant mesenchymal stem cell (hAMSC) which makes it a potential source of allogeneic multipotent cell for bone healing. However, much has to be explored about its isolation procedure and the osteogenic differentiation potential. The aims of this study are to establish the procurement procedure of human amniotic membrane, the isolation and culture of hAMSC, the MSC phenotypic characterization, and the in vitro osteogenic differentiation of hAMSC. Results of the study are as follows. The quality of human amniotic membrane would be best if procured from Caesarean operation under highly aseptic condition to avoid fungal and bacterial contamination on the culture. Isolation procedure using modified Soncini protocol yielded large amount of MSC with high proliferative capacity in culture medium. Characterization of hAMSC showed that the majority of the target cells exhibited specific MSC markers (CD105 and CD90) with a small number of these cells expressing CD45, the marker of hematopoeitic cells. The in vitro osteogenic differentiation of hAMSC followed by Alizarin Red staining showed that osteoblastic differentiation was detected in a significantly high number of cells. This study concludes that hAMSCs isolated from human amniotic membrane have the capacity for in vitro osteogenesis which makes them be one of the potential allogeneic stem cells for application in maxillofacial bone reconstruction.展开更多
Experiments on maxillofacial bone tissue engineering showed the promising result;however, its healing mechanisms and effectiveness had not been fully understood. The aim of this study is to compare the bone healing me...Experiments on maxillofacial bone tissue engineering showed the promising result;however, its healing mechanisms and effectiveness had not been fully understood. The aim of this study is to compare the bone healing mechanism and osteogenic capacity between bovine bone mineral loaded with hAMSC and autogenous bone graft in the reconstruction of critical size mandibular bone defect. Critical size defects were made at the mandible of 45 New Zealand white rabbits reconstructed with BBM-hAMSC, BBM alone, and ABG, respectively. At the end of first, second, and twelfth weeks, five rabbits from each experimental week were sacrificed for histology and immunohistochemistry staining. Expressions of vascular endothelial growth factor (VEGF), bone mor-phogenic proteins-2 (BMP2), Runx2 and the amount of angiogenesis were analyzed in the first and second week groups, while expressions of Runx2, osteocalcin, collagen type-I fibres, trabecular area and bone incorporation were analyzed in the twelfth week groups. The result showed that expressions of VEGF, BMP2 and Runx2 as well as the amount of angiogenesis were higher in ABG compared with BBM-hAMSC group in the first and second weeks of healing. The result of twelfth week of healing showed that expressions of Runx2 and osteocalcin as well as the thickness of collagen type-I fibres were significantly higher in BBM-hAMSC compared to ABG group, while there was no statistically difference in trabecular area and bone incorporation between BBM-hAMSC and ABG group. This study concluded that early healing activities were higher in auto-genous bone graft than in BBM-hAMSC, while osteogenic activities in the late stage of healing were higher in BBM-hAMSC compared to autogenous bone graft. It was also concluded that the osteo-genic capacity of BBM-hAMSC was comparable to autogenous bone graft in the reconstruction of critical size defect in the mandible.展开更多
In orthopedics, tissue engineering approach using stem cells is a valid line of treatment for patients with bone defects. In this context, mesenchymal stromal cells of various origins have been extensively studied and...In orthopedics, tissue engineering approach using stem cells is a valid line of treatment for patients with bone defects. In this context, mesenchymal stromal cells of various origins have been extensively studied and continue to be a matter of debate. Although mesenchymal stromal cells from bone marrow are already clinically applied, recent evidence suggests that one may use mesenchymal stromal cells from extra-embryonic tissues, such as amniotic fluid, as an innovative andadvantageous resource for bone regeneration. The use of cells from amniotic fluid does not raise ethical problems and provides a sufficient number of cells without invasive procedures. Furthermore, they do not develop into teratomas when transplanted, a consequence observed with pluripotent stem cells. In addition, their multipotent differentiation ability, low immunogenicity, and anti-inflammatory properties make them ideal candidates for bone regenerative medicine. We here present an overview of the features of amniotic fluid mesenchymal stromal cells and their potential in the osteogenic differentiation process. We have examined the papers actually available on this regard, with particular interest in the strategies applied to improve in vitro osteogenesis. Importantly, a detailed understanding of the behavior of amniotic fluid mesenchymal stromal cells and their osteogenic ability is desirable considering a feasible application in bone regenerative medicine.展开更多
BACKGROUND To solve the problem of liver transplantation donor insufficiency,an alternative cell transplantation therapy was investigated.We focused on amniotic epithelial cells(AECs)as a cell source because,unlike in...BACKGROUND To solve the problem of liver transplantation donor insufficiency,an alternative cell transplantation therapy was investigated.We focused on amniotic epithelial cells(AECs)as a cell source because,unlike induced pluripotent stem cells,they are cost-effective and non-tumorigenic.The utilization of AECs in regenerative medicine,however,is in its infancy.A general profile for AECs has not been comprehensively analyzed.Moreover,no hepatic differentiation protocol for AECs has yet been established.To this end,we independently compiled human AEC libraries,purified amniotic stem cells(ASCs),and co-cultured them with mesenchymal stem cells(MSCs)and human umbilical vein endothelial cell(HUVECs)in a 3D system which induces functional hepatic organoids.AIM To characterize AECs and generate functional hepatic organoids from ASCs and other somatic stem cells METHODS AECs,MSCs,and HUVECs were isolated from the placentae and umbilical cords of cesarean section patients.Amnion and primary AEC stemness characteristics and heterogeneity were analyzed by immunocytochemistry,Alkaline phosphatase(AP)staining,and flow cytometry.An adherent AEC subpopulation was selected and evaluated for ASC purification quality by a colony formation assay.AEC transcriptomes were compared with those for other hepatocytes cell sources by bioinformatics.The 2D and 3D culture were compared by relative gene expression using several differentiation protocols.ASCs,MSCs,and HUVECs were combined in a 3D co-culture system to generate hepatic organoids whose structure was compared with a 3D AEC sphere and whose function was elucidated by immunofluorescence imaging,periodic acid Schiff,and an indocyanine green(ICG)test.RESULTS AECs have certain stemness markers such as EPCAM,SSEA4,and E-cadherin.One AEC subpopulation was also either positive for AP staining or expressed the TRA-1-60 and TRA-1-81 stemness markers.Moreover,it could form colonies and its frequency was enhanced ten-fold in the adherent subpopulation after selective primary passage.Bioinformatics analysis of ribose nucleic acid sequencing revealed that the total AEC gene expression was distant from those of pluripotent stem cells and hepatocytes but some gene expression overlapped among these cells.TJP1,associated with epidermal growth factor receptor,and MET,associated with hepatocyte growth factor receptor,were upregulated and may be important for hepatic differentiation.In conventional flat culture,the cells turned unviable and did not readily differentiate into hepatocytes.In 3D culture,however,hepatic gene expression of the AEC sphere was elevated even under a two-step differentiation protocol.Furthermore,the organoids derived from the MSC and HUVEC co-culture showed 3D structure with polarity,hepatic-like glycogen storage,and ICG absorption/elimination.CONCLUSION Human amniotic epithelial cells are heterogeneous and certain subpopulations have high stemness.Under a 3D co-culture system,functional hepatic organoids were generated in a multicellular microenvironment.展开更多
Inflammatory bowel diseases are inflammatory, chronic and progressive diseases of the intestinal tract for which no curative treatment is available. Research in other fields with stem cells of different sources and wi...Inflammatory bowel diseases are inflammatory, chronic and progressive diseases of the intestinal tract for which no curative treatment is available. Research in other fields with stem cells of different sources and with immunoregulatory cells(regulatory T-lymphocytes and dendritic T-cells) opens up new expectations for their use in these diseases. The goal for stem cell-based therapy is to provide a permanent cure. To achieve this, it will be necessary to obtain a cellular product, original or genetically modified, that has a high migration capacity and homes into the intestine, has high survival after transplantation, regulates the immune reaction while not being visible to the patient's immune system, and repairs the injured tissue.展开更多
In vivo,stem cells reside in a three-dimensional(3D)extracellular microenvironment in which complicated biophysical and biochemical factors regulate their behaviors.Biomimicking of the stem cellmatrix interactions is ...In vivo,stem cells reside in a three-dimensional(3D)extracellular microenvironment in which complicated biophysical and biochemical factors regulate their behaviors.Biomimicking of the stem cellmatrix interactions is an ideal approach for controlling the stem cell fate.This study investigates the effects of the incorporation of cell-adhesive ligands in 3D self-assembling peptide hydrogels to modulate stem cell survival,proliferation,maintenance of stemness,and osteogenic differentiation.The results show that the composite hydrogels were non-cytotoxic and effective for maintaining human amniotic mesenchymal stem cell(hAMSC)survival,proliferation and phenotypic characterization.The expression levels of pluripotent markers were also upregulated in the composite hydrogels.Under inductive media conditions,mineral deposition and mRNA expression levels of osteogenic genes of hAMSCs were enhanced.The increasing expression of integrin aand b-subunits for hAMSCs indicates that the ligandintegrin interactions may modulate the cell fate for hAMSCs in composite hydrogels.展开更多
Objective: To evaluate the use of hypothermically stored human amniotic membrane for cartilage repair in adult sheep. Studies show that human amniotic membrane contains pluripotent mesenchymal stem cells that can be i...Objective: To evaluate the use of hypothermically stored human amniotic membrane for cartilage repair in adult sheep. Studies show that human amniotic membrane contains pluripotent mesenchymal stem cells that can be influenced to produce chondrocytes. It is unknown if human amniotic cells can produce hyaline-like cartilage. This study evaluates the use of hypothermically stored amniotic membrane (HSAM) to fill chondral defects in a sheep model. We hypothesized HSAM would fill defects with hyaline-like cartilage with chondrocytes populating the matrix. One sheep was used as a control, and four sheep received amniotic membrane. Two of these sheep were used as a normal control comparison. A 1 cm2 defect was created on the trochlear grove in all specimens. Each membrane was sized and laid over with the stromal layer facing the subchondral bone and covered with Fibrin sealant. The knees were harvested at five months and underwent morphological, histological, and immunohistological evaluation based on the original validated scoring system by O’Driscoll. The control defect didn’t fill in with hyaline cartilage or fibrocartilage. The defects that successfully retained the graft had evidence of diffuse chondrocyte cell proliferation and showed a stromal matrix similar to hyaline cartilage. The graft samples showed a near 100% morphological fill in the HSAM defect contrasting to <10% fill in the control defect. The retained HSAM grafts scored 2.5 on a 0 - 3 cartilage appearance scale compared with 0.5 for the control defects. HSAM is a potential source of pluripotent cells that can influence chondrogenesis in a sheep model. The implications for application in a human model are promising and warrant further study.展开更多
目的:探究血管内皮生长因子(VEGF)基因修饰大鼠羊膜间充质干细胞对肾病综合征大鼠血液生化指标的影响。方法:选取60只雄性大鼠,随机分为4组,各15例,分别为对照组(健康)、模型组(肾病综合征)、治疗1组(羊膜间充质干细胞悬液)、治疗2组(V...目的:探究血管内皮生长因子(VEGF)基因修饰大鼠羊膜间充质干细胞对肾病综合征大鼠血液生化指标的影响。方法:选取60只雄性大鼠,随机分为4组,各15例,分别为对照组(健康)、模型组(肾病综合征)、治疗1组(羊膜间充质干细胞悬液)、治疗2组(VEGF基因修饰羊膜间充质干细胞悬液)。观察4组肾组织病理形态学及羊膜间充质干细胞存活、分布情况,并比较尿肌酐(SCr)、血尿素(BU)、总胆固醇(TC)、三酰甘油(TG)、低密度脂蛋白(LDL)、白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)、24 h尿蛋白定量(24 h UTP)、阻力指数(RI)、舒张期末期流速(EDV)、收缩期峰值流速(PSV)及肾组织VEGF相关表达。结果:对照组大鼠肾组织具有完整清晰结构,模型组大鼠肾组肾小球表现为肿胀,肾小球内存在较多细胞,系膜区明显增宽,系膜细胞及基质中到重度增生;相较于模型组,各治疗组病理改变均较轻,各治疗组病理改变程度均较模型组轻,其中治疗1组病理改变较治疗2组病理改变明显;对照组及模型组大鼠肾组织均未见荧光细胞;治疗1组、治疗2组大鼠肾组织均可见红色荧光的阳性细胞,且仅可见于肾间质及小管内,在肾小球中未见其表达。与治疗1组比较,治疗2组阳性细胞较多;与对照组比较,模型组SCr、BU、24 h UTP、TC、TG、LDL、IL-1β、TNF-α水平均明显升高(P<0.05),治疗1组、治疗2组SCr、BU、24 h UTP、TC、TG、LDL、IL-1β、TNF-α水平均较模型组明显降低,且治疗2组最低(P<0.05);与对照组比较,模型组RI明显升高,PSV、EDV明显下降(P<0.05),治疗1组、治疗2组PSV、EDV均较模型组显著升高,RI水平较模型组显著降低,且治疗2组RI最低,PSV、EDV最高(P<0.05);与对照组比较,模型组肾组织中VEGF蛋白及mRNA表达均明显下降(P<0.05),治疗1组、治疗2组肾组织中VEGF蛋白及mRNA表达均较模型组显著升高,且治疗2组最高(P<0.05)。结论:经VEGF基因修饰的羊膜间充质干细胞移植治疗,可显著改善肾病综合征大鼠血脂水平、微炎症状态、肾功能及肾动脉血流,效果更为显著,可为肾病综合征细胞移植治疗提供有力依据。展开更多
Bioengineered corneas are substitutes for human donor tissue that are designed to treat severe dis-ease affecting ocular surfaces. However,a shortage of candidate seed cells for bioengineering corneas is still a probl...Bioengineered corneas are substitutes for human donor tissue that are designed to treat severe dis-ease affecting ocular surfaces. However,a shortage of candidate seed cells for bioengineering corneas is still a problem. Bone-marrow mesenchymal stem cells (MSCs) are capable of multilineage differen-tiation. Therefore,we determined whether MSCs differentiate into corneal epithelial cells (ECs). We applied three exoteric-microenvironmental systems to induce MSCs to become ECs. Induced MSC were identified by means of morphologic examination,immunocytochemical analysis,and flow cytometry. MSCs grown in one microenvironment had characteristics similar to those of corneal epithelial pro-genitors. Induced MSCs expressed markers for EC,including integrin β1,Cx43,Pax6,and P63. MSCs were successfully induced to become corneal epithelial progenitors. Therefore,the use of MSCs may hold substantial promise for reconstructing the ocular surface after corneal injury.展开更多
Complete wound regeneration preserves skin structure and physiological functions,including sensation and perception of stimuli,whereas incomplete wound regeneration results in fibrosis and scarring.Amniotic fluid stem...Complete wound regeneration preserves skin structure and physiological functions,including sensation and perception of stimuli,whereas incomplete wound regeneration results in fibrosis and scarring.Amniotic fluid stem cells(AFSCs)would be a kind of cell population with self-renewing and non-immunogenic ability that have a considerable role in wound generation.They are easy to harvest,culture,and store;moreover,they are non-tumorigenic and not subject to ethical restrictions.They can differentiate into different kinds of cells that replenish the skin,subcutaneous tissues,and accessory organs.Additionally,AFSCs independently produce paracrine effectors and secrete them in exosomes,thereby modulating local immune cell activity.They demonstrate anti-inflammatory and immunomodulatory properties,regulate the physicochemical microenvironment of the wound,and promote full wound regeneration.Thus,AFSCs are potential resources in stem cell therapy,especially in scar-free wound healing.This review describes the biological characteristics and clinical applications of AFSCs in treating wounds and provide new ideas for the treatment of wound healing.展开更多
基金the Science and Technology Foundation of Shenyang in China,No.F10-217-1-00
文摘In this study, we loaded human umbilical cord mesenchymal stem cells onto human amniotic membrane with epithelial cells to prepare nerve conduits, i.e., a relatively closed nerve regeneration chamber. After neurolysis, the injured radial nerve was enwrapped with the prepared nerve conduit, which was fixed to the epineurium by sutures, with the cell on the inner surface of the conduit. Simultaneously, a 1.0 mL aliquot of human umbilical cord mesenchymal stem cell suspension was injected into the distal and proximal ends of the injured radial nerve with 1.0 cm intervals. A total of 1.75 x 107 cells were seeded on the amniotic membrane. In the control group, patients received only neurolysis. At 12 weeks after cell transplantation, more than 80% of patients exhibited obvious improvements in muscular strength, and touch and pain sensations. In contrast, these improvements were observed only in 55-65% of control patients. At 8 and 12 weeks, muscular electrophysiological function in the region dominated by the injured radial nerve was significantly better in the transplantation group than the control group. After cell transplantation, no immunological rejections were observed. These findings suggest that human umbilical cord mesenchymal stem cell-loaded amniotic membrane can be used for the repair of radial nerve injury.
基金Supported by Science and Technology Program of Shenyang (2009-090063, 2011-F10-222-4-00)
文摘Objective To establish the method of isolation, purification, and identification of human amniotic mesenchymal stem cells (hAMSCs). Methods hAMSCs were isolated from human amniotic membrane by trypsin-collagenase digestion, and cultured in Dulbecco's modified Eagle's medinm/F12 medium supplemented with 10% fetal bovine serum. Phenotypic characteristics of these cells were analyzed by means of immunocytochemistry and flow cytometry. Results The cells successfully isolated from human amniotic membrane expressed representative mesenchymal cell surface markers CD44, CD90, and vimentin, but not CD45. Conclusions This study establishes a potential method for isolation of hAMSCs from human amnion, in vitro culture, and identification. The isolated cells show phenotypic characteristics of mesenchymal stem cells.
文摘Amniotic membrane of human placenta is a source of abundant mesenchymal stem cell (hAMSC) which makes it a potential source of allogeneic multipotent cell for bone healing. However, much has to be explored about its isolation procedure and the osteogenic differentiation potential. The aims of this study are to establish the procurement procedure of human amniotic membrane, the isolation and culture of hAMSC, the MSC phenotypic characterization, and the in vitro osteogenic differentiation of hAMSC. Results of the study are as follows. The quality of human amniotic membrane would be best if procured from Caesarean operation under highly aseptic condition to avoid fungal and bacterial contamination on the culture. Isolation procedure using modified Soncini protocol yielded large amount of MSC with high proliferative capacity in culture medium. Characterization of hAMSC showed that the majority of the target cells exhibited specific MSC markers (CD105 and CD90) with a small number of these cells expressing CD45, the marker of hematopoeitic cells. The in vitro osteogenic differentiation of hAMSC followed by Alizarin Red staining showed that osteoblastic differentiation was detected in a significantly high number of cells. This study concludes that hAMSCs isolated from human amniotic membrane have the capacity for in vitro osteogenesis which makes them be one of the potential allogeneic stem cells for application in maxillofacial bone reconstruction.
文摘Experiments on maxillofacial bone tissue engineering showed the promising result;however, its healing mechanisms and effectiveness had not been fully understood. The aim of this study is to compare the bone healing mechanism and osteogenic capacity between bovine bone mineral loaded with hAMSC and autogenous bone graft in the reconstruction of critical size mandibular bone defect. Critical size defects were made at the mandible of 45 New Zealand white rabbits reconstructed with BBM-hAMSC, BBM alone, and ABG, respectively. At the end of first, second, and twelfth weeks, five rabbits from each experimental week were sacrificed for histology and immunohistochemistry staining. Expressions of vascular endothelial growth factor (VEGF), bone mor-phogenic proteins-2 (BMP2), Runx2 and the amount of angiogenesis were analyzed in the first and second week groups, while expressions of Runx2, osteocalcin, collagen type-I fibres, trabecular area and bone incorporation were analyzed in the twelfth week groups. The result showed that expressions of VEGF, BMP2 and Runx2 as well as the amount of angiogenesis were higher in ABG compared with BBM-hAMSC group in the first and second weeks of healing. The result of twelfth week of healing showed that expressions of Runx2 and osteocalcin as well as the thickness of collagen type-I fibres were significantly higher in BBM-hAMSC compared to ABG group, while there was no statistically difference in trabecular area and bone incorporation between BBM-hAMSC and ABG group. This study concluded that early healing activities were higher in auto-genous bone graft than in BBM-hAMSC, while osteogenic activities in the late stage of healing were higher in BBM-hAMSC compared to autogenous bone graft. It was also concluded that the osteo-genic capacity of BBM-hAMSC was comparable to autogenous bone graft in the reconstruction of critical size defect in the mandible.
文摘In orthopedics, tissue engineering approach using stem cells is a valid line of treatment for patients with bone defects. In this context, mesenchymal stromal cells of various origins have been extensively studied and continue to be a matter of debate. Although mesenchymal stromal cells from bone marrow are already clinically applied, recent evidence suggests that one may use mesenchymal stromal cells from extra-embryonic tissues, such as amniotic fluid, as an innovative andadvantageous resource for bone regeneration. The use of cells from amniotic fluid does not raise ethical problems and provides a sufficient number of cells without invasive procedures. Furthermore, they do not develop into teratomas when transplanted, a consequence observed with pluripotent stem cells. In addition, their multipotent differentiation ability, low immunogenicity, and anti-inflammatory properties make them ideal candidates for bone regenerative medicine. We here present an overview of the features of amniotic fluid mesenchymal stromal cells and their potential in the osteogenic differentiation process. We have examined the papers actually available on this regard, with particular interest in the strategies applied to improve in vitro osteogenesis. Importantly, a detailed understanding of the behavior of amniotic fluid mesenchymal stromal cells and their osteogenic ability is desirable considering a feasible application in bone regenerative medicine.
基金Supported by National Natural Science Foundation of China,No.81770621Ministry of Education,Culture,Sports,Science,and Technology of Japan,KAKENHI,No.16K15604,No.18H02866Japan Science and Technology Agency-Japan International Cooperation Agency’s(JST-JICA)Science and Technology Research Partnership for Sustainable Development(SATREPS)Project
文摘BACKGROUND To solve the problem of liver transplantation donor insufficiency,an alternative cell transplantation therapy was investigated.We focused on amniotic epithelial cells(AECs)as a cell source because,unlike induced pluripotent stem cells,they are cost-effective and non-tumorigenic.The utilization of AECs in regenerative medicine,however,is in its infancy.A general profile for AECs has not been comprehensively analyzed.Moreover,no hepatic differentiation protocol for AECs has yet been established.To this end,we independently compiled human AEC libraries,purified amniotic stem cells(ASCs),and co-cultured them with mesenchymal stem cells(MSCs)and human umbilical vein endothelial cell(HUVECs)in a 3D system which induces functional hepatic organoids.AIM To characterize AECs and generate functional hepatic organoids from ASCs and other somatic stem cells METHODS AECs,MSCs,and HUVECs were isolated from the placentae and umbilical cords of cesarean section patients.Amnion and primary AEC stemness characteristics and heterogeneity were analyzed by immunocytochemistry,Alkaline phosphatase(AP)staining,and flow cytometry.An adherent AEC subpopulation was selected and evaluated for ASC purification quality by a colony formation assay.AEC transcriptomes were compared with those for other hepatocytes cell sources by bioinformatics.The 2D and 3D culture were compared by relative gene expression using several differentiation protocols.ASCs,MSCs,and HUVECs were combined in a 3D co-culture system to generate hepatic organoids whose structure was compared with a 3D AEC sphere and whose function was elucidated by immunofluorescence imaging,periodic acid Schiff,and an indocyanine green(ICG)test.RESULTS AECs have certain stemness markers such as EPCAM,SSEA4,and E-cadherin.One AEC subpopulation was also either positive for AP staining or expressed the TRA-1-60 and TRA-1-81 stemness markers.Moreover,it could form colonies and its frequency was enhanced ten-fold in the adherent subpopulation after selective primary passage.Bioinformatics analysis of ribose nucleic acid sequencing revealed that the total AEC gene expression was distant from those of pluripotent stem cells and hepatocytes but some gene expression overlapped among these cells.TJP1,associated with epidermal growth factor receptor,and MET,associated with hepatocyte growth factor receptor,were upregulated and may be important for hepatic differentiation.In conventional flat culture,the cells turned unviable and did not readily differentiate into hepatocytes.In 3D culture,however,hepatic gene expression of the AEC sphere was elevated even under a two-step differentiation protocol.Furthermore,the organoids derived from the MSC and HUVEC co-culture showed 3D structure with polarity,hepatic-like glycogen storage,and ICG absorption/elimination.CONCLUSION Human amniotic epithelial cells are heterogeneous and certain subpopulations have high stemness.Under a 3D co-culture system,functional hepatic organoids were generated in a multicellular microenvironment.
文摘Inflammatory bowel diseases are inflammatory, chronic and progressive diseases of the intestinal tract for which no curative treatment is available. Research in other fields with stem cells of different sources and with immunoregulatory cells(regulatory T-lymphocytes and dendritic T-cells) opens up new expectations for their use in these diseases. The goal for stem cell-based therapy is to provide a permanent cure. To achieve this, it will be necessary to obtain a cellular product, original or genetically modified, that has a high migration capacity and homes into the intestine, has high survival after transplantation, regulates the immune reaction while not being visible to the patient's immune system, and repairs the injured tissue.
基金This work was supported by the National Natural Science Foundation of China(31860265)the Natural Science Research project of Education Department of Guizhou Province(Qian Jiao He KY Zi[2015]418)the National Natural Science Foundation of China(31360232).
文摘In vivo,stem cells reside in a three-dimensional(3D)extracellular microenvironment in which complicated biophysical and biochemical factors regulate their behaviors.Biomimicking of the stem cellmatrix interactions is an ideal approach for controlling the stem cell fate.This study investigates the effects of the incorporation of cell-adhesive ligands in 3D self-assembling peptide hydrogels to modulate stem cell survival,proliferation,maintenance of stemness,and osteogenic differentiation.The results show that the composite hydrogels were non-cytotoxic and effective for maintaining human amniotic mesenchymal stem cell(hAMSC)survival,proliferation and phenotypic characterization.The expression levels of pluripotent markers were also upregulated in the composite hydrogels.Under inductive media conditions,mineral deposition and mRNA expression levels of osteogenic genes of hAMSCs were enhanced.The increasing expression of integrin aand b-subunits for hAMSCs indicates that the ligandintegrin interactions may modulate the cell fate for hAMSCs in composite hydrogels.
文摘Objective: To evaluate the use of hypothermically stored human amniotic membrane for cartilage repair in adult sheep. Studies show that human amniotic membrane contains pluripotent mesenchymal stem cells that can be influenced to produce chondrocytes. It is unknown if human amniotic cells can produce hyaline-like cartilage. This study evaluates the use of hypothermically stored amniotic membrane (HSAM) to fill chondral defects in a sheep model. We hypothesized HSAM would fill defects with hyaline-like cartilage with chondrocytes populating the matrix. One sheep was used as a control, and four sheep received amniotic membrane. Two of these sheep were used as a normal control comparison. A 1 cm2 defect was created on the trochlear grove in all specimens. Each membrane was sized and laid over with the stromal layer facing the subchondral bone and covered with Fibrin sealant. The knees were harvested at five months and underwent morphological, histological, and immunohistological evaluation based on the original validated scoring system by O’Driscoll. The control defect didn’t fill in with hyaline cartilage or fibrocartilage. The defects that successfully retained the graft had evidence of diffuse chondrocyte cell proliferation and showed a stromal matrix similar to hyaline cartilage. The graft samples showed a near 100% morphological fill in the HSAM defect contrasting to <10% fill in the control defect. The retained HSAM grafts scored 2.5 on a 0 - 3 cartilage appearance scale compared with 0.5 for the control defects. HSAM is a potential source of pluripotent cells that can influence chondrogenesis in a sheep model. The implications for application in a human model are promising and warrant further study.
文摘目的:探究血管内皮生长因子(VEGF)基因修饰大鼠羊膜间充质干细胞对肾病综合征大鼠血液生化指标的影响。方法:选取60只雄性大鼠,随机分为4组,各15例,分别为对照组(健康)、模型组(肾病综合征)、治疗1组(羊膜间充质干细胞悬液)、治疗2组(VEGF基因修饰羊膜间充质干细胞悬液)。观察4组肾组织病理形态学及羊膜间充质干细胞存活、分布情况,并比较尿肌酐(SCr)、血尿素(BU)、总胆固醇(TC)、三酰甘油(TG)、低密度脂蛋白(LDL)、白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)、24 h尿蛋白定量(24 h UTP)、阻力指数(RI)、舒张期末期流速(EDV)、收缩期峰值流速(PSV)及肾组织VEGF相关表达。结果:对照组大鼠肾组织具有完整清晰结构,模型组大鼠肾组肾小球表现为肿胀,肾小球内存在较多细胞,系膜区明显增宽,系膜细胞及基质中到重度增生;相较于模型组,各治疗组病理改变均较轻,各治疗组病理改变程度均较模型组轻,其中治疗1组病理改变较治疗2组病理改变明显;对照组及模型组大鼠肾组织均未见荧光细胞;治疗1组、治疗2组大鼠肾组织均可见红色荧光的阳性细胞,且仅可见于肾间质及小管内,在肾小球中未见其表达。与治疗1组比较,治疗2组阳性细胞较多;与对照组比较,模型组SCr、BU、24 h UTP、TC、TG、LDL、IL-1β、TNF-α水平均明显升高(P<0.05),治疗1组、治疗2组SCr、BU、24 h UTP、TC、TG、LDL、IL-1β、TNF-α水平均较模型组明显降低,且治疗2组最低(P<0.05);与对照组比较,模型组RI明显升高,PSV、EDV明显下降(P<0.05),治疗1组、治疗2组PSV、EDV均较模型组显著升高,RI水平较模型组显著降低,且治疗2组RI最低,PSV、EDV最高(P<0.05);与对照组比较,模型组肾组织中VEGF蛋白及mRNA表达均明显下降(P<0.05),治疗1组、治疗2组肾组织中VEGF蛋白及mRNA表达均较模型组显著升高,且治疗2组最高(P<0.05)。结论:经VEGF基因修饰的羊膜间充质干细胞移植治疗,可显著改善肾病综合征大鼠血脂水平、微炎症状态、肾功能及肾动脉血流,效果更为显著,可为肾病综合征细胞移植治疗提供有力依据。
基金Supported by the Key Clinical Program of the Ministry of Health of China (Grant No. 2004468)the National Natural Science Foundation of China (Grant No. 30672275)+1 种基金Natural Science Foundation of Guangdong Province (Grant No. 06300679)the Post-doctor Foundation of China (Grant No. 2005037616)
文摘Bioengineered corneas are substitutes for human donor tissue that are designed to treat severe dis-ease affecting ocular surfaces. However,a shortage of candidate seed cells for bioengineering corneas is still a problem. Bone-marrow mesenchymal stem cells (MSCs) are capable of multilineage differen-tiation. Therefore,we determined whether MSCs differentiate into corneal epithelial cells (ECs). We applied three exoteric-microenvironmental systems to induce MSCs to become ECs. Induced MSC were identified by means of morphologic examination,immunocytochemical analysis,and flow cytometry. MSCs grown in one microenvironment had characteristics similar to those of corneal epithelial pro-genitors. Induced MSCs expressed markers for EC,including integrin β1,Cx43,Pax6,and P63. MSCs were successfully induced to become corneal epithelial progenitors. Therefore,the use of MSCs may hold substantial promise for reconstructing the ocular surface after corneal injury.
基金National Natural Science Foundation of China(Approval numbers 81871570 and 82072195)Science and Technology Plan of Guizhou Province(No.20204Y148 and No.20162910)Collaborative Innovation of 2011 for Tissue Injury Repair and Regenerative Medicine of Guizhou Province(No.201507)。
文摘Complete wound regeneration preserves skin structure and physiological functions,including sensation and perception of stimuli,whereas incomplete wound regeneration results in fibrosis and scarring.Amniotic fluid stem cells(AFSCs)would be a kind of cell population with self-renewing and non-immunogenic ability that have a considerable role in wound generation.They are easy to harvest,culture,and store;moreover,they are non-tumorigenic and not subject to ethical restrictions.They can differentiate into different kinds of cells that replenish the skin,subcutaneous tissues,and accessory organs.Additionally,AFSCs independently produce paracrine effectors and secrete them in exosomes,thereby modulating local immune cell activity.They demonstrate anti-inflammatory and immunomodulatory properties,regulate the physicochemical microenvironment of the wound,and promote full wound regeneration.Thus,AFSCs are potential resources in stem cell therapy,especially in scar-free wound healing.This review describes the biological characteristics and clinical applications of AFSCs in treating wounds and provide new ideas for the treatment of wound healing.