Skin wounds are characterized by injury to the skin due to trauma,tearing,cuts,or contusions.As such injuries are common to all human groups,they may at times represent a serious socioeconomic burden.Currently,increas...Skin wounds are characterized by injury to the skin due to trauma,tearing,cuts,or contusions.As such injuries are common to all human groups,they may at times represent a serious socioeconomic burden.Currently,increasing numbers of studies have focused on the role of mesenchymal stem cell(MSC)-derived extracellular vesicles(EVs)in skin wound repair.As a cell-free therapy,MSC-derived EVs have shown significant application potential in the field of wound repair as a more stable and safer option than conventional cell therapy.Treatment based on MSC-derived EVs can significantly promote the repair of damaged substructures,including the regeneration of vessels,nerves,and hair follicles.In addition,MSC-derived EVs can inhibit scar formation by affecting angiogenesis-related and antifibrotic pathways in promoting macrophage polarization,wound angiogenesis,cell proliferation,and cell migration,and by inhibiting excessive extracellular matrix production.Additionally,these structures can serve as a scaffold for components used in wound repair,and they can be developed into bioengineered EVs to support trauma repair.Through the formulation of standardized culture,isolation,purification,and drug delivery strategies,exploration of the detailed mechanism of EVs will allow them to be used as clinical treatments for wound repair.In conclusion,MSCderived EV-based therapies have important application prospects in wound repair.Here we provide a comprehensive overview of their current status,application potential,and associated drawbacks.展开更多
Mesenchymal stem cells(MSCs)have been widely used in regenerative medicine and clinical therapy due to their capabilities of proliferation,differentiation,and immune regulation.However,during in vitro expansion,MSCs a...Mesenchymal stem cells(MSCs)have been widely used in regenerative medicine and clinical therapy due to their capabilities of proliferation,differentiation,and immune regulation.However,during in vitro expansion,MSCs are prone to aging,which largely limits their application.Prostaglandin E-2(PGE-2)is a key effector secreted by MSCs to exert immunomodulatory effects.By screening the compound library for PGE-2 secretion,the antioxidant trolox was verified as a stimulator of MSCs to secrete PGE-2.The effect of antioxidant trolox on biological characteristics of MSCS,including aging,proliferation,and gene expression,was examined.The results demonstrated that trolox can resist aging,promote proliferation,and enhance PGE-2 secretion of MSCs without affecting their surface marker expression.Furthermore,trolox treatment up-regulates miR-17-92 clusters in MSCs and may contribute to its anti-aging effects.Thus,trolox addition might be beneficial for MSCs expansion and their application.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
In the present study, the morphological and ultrastructural characteristics of mesenchymal stem cells (MSCs) induced towards osteogenic, adipogenic, and chondrogenic lineages were investigated. The main objective of t...In the present study, the morphological and ultrastructural characteristics of mesenchymal stem cells (MSCs) induced towards osteogenic, adipogenic, and chondrogenic lineages were investigated. The main objective of this paper was to focus on the differentiation capacity of ovine [oMSCs] and equine MSCs [eMSCs]. Bone marrow [BM] MSCs were isolated from ovine and equine patients, expanded in monolayer culture and induced into osteogenic, adipogenic and chondrogenic differentiation. For chondrogenic differentiation, cells were cultured in micromass culture. Also, their ultrastructural phenotypes were studied by transmission electron microscopy [TEM]. This study was aimed to provide an indepth morphological description of BM-derived ovine and equine MSCs induced towards three lineages, it demonstrated that TEM analysis is useful in elucidating detailed structural information.展开更多
Effective bone regeneration through tissue engineering requires a combination of osteogenic progenitors,osteoinductive biofactors and biocompatible scaffold materials.Mesenchymal stem cells(MSCs)represent the most pro...Effective bone regeneration through tissue engineering requires a combination of osteogenic progenitors,osteoinductive biofactors and biocompatible scaffold materials.Mesenchymal stem cells(MSCs)represent the most promising seed cells for bone tissue engineering.As multipotent stem cells that can self-renew and differentiate into multiple lineages including bone and fat,MSCs can be isolated from numerous tissues and exhibit varied differentiation potential.To identify an optimal progenitor cell source for bone tissue engineering,we analyzed the proliferative activity and osteogenic potential of four commonly-used mouse MSC sources,including immortalized mouse embryonic fibroblasts(iMEF),immortalized mouse bone marrow stromal stem cells(imBMSC),immortalized mouse calvarial mesenchymal progenitors(iCAL),and immortalized mouse adipose-derived mesenchymal stem cells(iMAD).We found that iMAD exhibited highest osteogenic and adipogenic capabilities upon BMP9 stimulation in vitro,whereas iMAD and iCAL exhibited highest osteogenic capability in BMP9-induced ectopic osteogenesis and critical-sized calvarial defect repair.Transcriptomic analysis revealed that,while each MSC line regulated a distinct set of target genes upon BMP9 stimulation,all MSC lines underwent osteogenic differentiation by regulating osteogenesis-related signaling including Wnt,TGF-β,PI3K/AKT,MAPK,Hippo and JAK-STAT pathways.Collectively,our results demonstrate that adipose-derived MSCs represent optimal progenitor sources for cell-based bone tissue engineering.展开更多
BACKGROUND Mesenchymal stromal/stem cells (MSCs) constitute a promising tool in regenerative medicine and can be isolated from different human tissues. However, their biological properties are still not fully characte...BACKGROUND Mesenchymal stromal/stem cells (MSCs) constitute a promising tool in regenerative medicine and can be isolated from different human tissues. However, their biological properties are still not fully characterized. Whereas MSCs from different tissue exhibit many common characteristics, their biological activity and some markers are different and depend on their tissue of origin. Understanding the factors that underlie MSC biology should constitute important points for consideration for researchers interested in clinical MSC application. AIM To characterize the biological activity of MSCs during longterm culture isolated from: bone marrow (BM-MSCs), adipose tissue (AT-MSCs), skeletal muscles (SMMSCs), and skin (SK-MSCs). METHODS MSCs were isolated from the tissues, cultured for 10 passages, and assessed for: phenotype with immunofluorescence and flow cytometry, multipotency with differentiation capacity for osteo-, chondro-, and adipogenesis, stemness markers with qPCR for mRNA for Sox2 and Oct4, and genetic stability for p53 and c-Myc;27 bioactive factors were screened using the multiplex ELISA array, and spontaneous fusion involving a co-culture of SM-MSCs with BM-MSCs or AT-MSCs stained with PKH26 (red) or PKH67 (green) was performed. RESULTS All MSCs showed the basic MSC phenotype;however, their expression decreased during the follow-up period, as confirmed by fluorescence intensity. The examined MSCs express CD146 marker associated with proangiogenic properties;however their expression decreased in AT-MSCs and SM-MSCs, but was maintained in BM-MSCs. In contrast, in SK-MSCs CD146 expression increased in late passages. All MSCs, except BM-MSCs, expressed PW1, a marker associated with differentiation capacity and apoptosis. BM-MSCs and AT-MSCs expressed stemness markers Sox2 and Oct4 in long-term culture. All MSCs showed a stable p53 and c-Myc expression. BM-MSCs and AT-MSCs maintained their differentiation capacity during the follow-up period. In contrast, SK-MSCs and SM-MSCs had a limited ability to differentiate into adipocytes. BM-MSCs and AT-MSCs revealed similarities in phenotype maintenance, capacity for multilineage differentiation, and secretion of bioactive factors. Because AT-MSCs fused with SM-MSCs as effectively as BM-MSCs, AT-MSCs may constitute an alternative source for BM-MSCs. CONCLUSION Long-term culture affects the biological activity of MSCs obtained from various tissues. The source of MSCs and number of passages are important considerations in regenerative medicine.展开更多
To explore the way to induce mesenchymal stem cells (MSCs) to differentiate into dopaminergic neurons in vitro. Methods MSCs were obtained from rat bone marrow, cultured and passaged. MSCs used in this experi...To explore the way to induce mesenchymal stem cells (MSCs) to differentiate into dopaminergic neurons in vitro. Methods MSCs were obtained from rat bone marrow, cultured and passaged. MSCs used in this experiment had multipotency, which was indirectly proved by being induced to differentiate into chondrocytes and adipocytes. MSCs were cultured in medium containing 0.5 mmol/L IBMX for 2 days. Then the medium was replaced with induction medium, which contained GDNF, IL-1β, mesencephalic glial-cell-conditioned medium and flash-frozen mesencephalic membrane fragments. The surface markers of the differentiated neurons, such as NSE, nestin, MAP-2a, b and TH were detected by immunocytochemistry and Western blot after MSCs were cultured in induction medium for 7 days and 15 days. Results MSCs differentiated into neural progenitors and expressed nestin after MSCs were incubated with medium containing IBMX for 2 d. After the medium was replaced with induction medium containing many inducing agents, MSCs differentiated into neuron-like cells and dopaminergic neuron-like cells and expressed NSE, MAP-2a, b and TH. The percentage of NSE-positive cells, MAP-2a, b-positive cells and TH-positive cells was 30.032±2.489%, 41.580±5.101% and 34.958±5.534%, respectively after MSCs were induced in medium containing GDNF, IL-1β, mesencephalic glial-cell-conditioned medium and flash-frozen mesencephalic membrane fragments for 15 days. Conclusion MSCs can differentiate into dopaminergic neuron-like cells and are a new cell source for the treatment of neurodegeneration diseases and have a great potential for wide application展开更多
Background:Bone marrow mesenchymal stem cell (MSC) transplantation is a promising strategy in the treatment of myocardial infarction (MI). However, the time for transplanting cells remains controversial. The aim of th...Background:Bone marrow mesenchymal stem cell (MSC) transplantation is a promising strategy in the treatment of myocardial infarction (MI). However, the time for transplanting cells remains controversial. The aim of this study was to find an optimal time point for cell transplantation. Methods: MSCs were isolated and cultured from Sprague-Dawley (SD) rats. MI model was set up in SD rats by permanent ligation of left anterior descending coronary artery. MSCs were directly injected into the infarct border zone at 1 h, 1 week and 2 weeks after MI, respectively. Sham-operated and MI control groups received equal volume of phosphate buffered saline (PBS). At 4 weeks after MI, cardiac function was assessed by echocardiography; vessel density was analyzed on hematoxylin-eosin stained slides by light microscopy; the apoptosis of cardiomyocytes was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay; the expressions of proteins were analyzed by Western blot. Results: MSC transplantation improved cardiac function, reduced the apoptosis of cardiomyocytes and increased vessel density. These benefits were more obvious in 1-week group than in 1-h and 2-week groups. There are more obvious in-creases in the ratio of bcl-2/bax and the expression of vascular endothelial growth factor (VEGF) and more obvious decreases in the expression of cleaved-caspase-3 in 1-week group than those in other two groups. Conclusion: MSC transplantation was beneficial for the recovery of cardiac function. MSC transplantation at 1 week post-MI exerted the best effects on increases of cardiac function, anti-apoptosis and angiogenesis.展开更多
Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily and have diverse functions during development and organogenesis. BMPs play a major role in skeletal development and bone formation, and disrupti...Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily and have diverse functions during development and organogenesis. BMPs play a major role in skeletal development and bone formation, and disruptions in BMP signaling cause a variety of skeletal and extraskeletal anomalies. Several knockout models have provided insight into the mechanisms responsible for these phenotypes. Proper bone formation requires the differentiation of osteoblasts from mesenchymal stem cell (MSC) precursors, a process mediated in part by BMP signaling. Multiple BMPs, including BMP2, BMP6, BMP7 and BMP9, promote osteoblastic differentiation of MSCs both in vitro and in vivo. BMP9 is one of the most osteogenic BMPs, yet it is a poorly characterized member of the BMP family. Several studies demonstrate that the mechanisms controlling BMP9-mediated osteogenesis differ from other osteogenic BMPs, but little is known about these specific mechanisms. Several pathways critical to BMP9-mediated osteogenesis are also important in the differentiation of other cell lineages, including adipocytes and chondrocytes. BMP9 has also demonstrated translational promise in spinal fusion and bone fracture repair. This review will summarize our current knowledge of BMP-mediated osteogenesis, with a focus on BMP9, by presenting recently completed work which may help us to further elucidate these pathways.展开更多
Mesenchymal stem cells (MSCs) of nonembryonic origins possess the proliferation and multi-lineage differentiation potentials. It has been established that epigenetic mechanisms could be critical for determining the ...Mesenchymal stem cells (MSCs) of nonembryonic origins possess the proliferation and multi-lineage differentiation potentials. It has been established that epigenetic mechanisms could be critical for determining the fate of stem cells, and MSCs derived from different origins exhibited different expression profiles individually to a certain extent. In this study, ChiP-on-chip was used to generate genome-wide histone H3-Lys9 acetylation and dimethylation profiles at gene promoters in human bone marrow MSCs. We showed that modifications of histone H3-Lys9 at gene promoters correlated well with mRNA expression in human bone marrow MSCs. Functional analysis revealed that many key cellular pathways in human bone marrow MSC self-renewal, such as the canonical signaling pathways, cell cycle pathways and cytokine related pathways may be regulated by H3-Lys9 modifications. These data suggest that gene activation and silencing affected by H3-Lys9 acetylation and dimethylation, respectively, may be essential to the maintenance of human bone marrow MSC self-renewal and multi-potency.展开更多
Age related defect of the osteogenic differentiation of mesenchymal stem cells(MSCs) plays a key role in osteoporosis. Mechanical loading is one of the most important physical stimuli for osteoblast differentiation....Age related defect of the osteogenic differentiation of mesenchymal stem cells(MSCs) plays a key role in osteoporosis. Mechanical loading is one of the most important physical stimuli for osteoblast differentiation.Here, we compared the osteogenic potential of MSCs from young and adult rats under three rounds of 2 h of cyclic stretch of 2.5% elongation at 1 Hz on 3 consecutive days. Cyclic stretch induced a significant osteogenic differentiation of MSCs from young rats, while a compromised osteogenesis in MSCs from the adult rats.Accordingly, there were much more reactive oxygen species(ROS) production in adult MSCs under cyclic stretch compared to young MSCs. Moreover, ROS scavenger N-acetylcysteine rescued the osteogenic differentiation of adult MSCs under cyclic stretch. Gene expression analysis revealed that superoxide dismutase 1(SOD1) was significantly downregulated in those MSCs from adult rats. In summary, our data suggest that reduced SOD1 may result in excessive ROS production in adult MSCs under cyclic stretch, and thus manipulation of the MSCs from the adult donors with antioxidant would improve their osteogenic ability.展开更多
This study aimed to investigate the neural differentiation of human umbilical cord mesenchymal stem cells (hUCMSCs) under the induction of injured neural cells. After in vitro isolation and culture, passage 5 hUCMSC...This study aimed to investigate the neural differentiation of human umbilical cord mesenchymal stem cells (hUCMSCs) under the induction of injured neural cells. After in vitro isolation and culture, passage 5 hUCMSCs were used for experimentation, hUCMSCs were co-cultured with normal or AI31.4o-injured PC12 cells, PC12 cell supernatant or PC12 cell lysate in a Transwell co-culture system. Western blot analysis and flow cytometry results showed that choline acetyltransferase and microtubule-associated protein 2, a specific marker for neural cells, were expressed in hUCMSCs under various culture conditions, and highest expression was observed in the hUCMSCs co-cultured with injured PC12 cells. Choline acetyltransferase and microtubule-associated protein 2 were not expressed in hUCMSCs cultured alone (no treatment). Cell Counting Kit-8 assay results showed that hUCMSCs under co-culture conditions promoted the proliferation of injured PC12 cells. These findings suggest that the microenvironment during neural tissue injury can effectively induce neural cell differentiation of hUCMSCs. These differentiated hUCMSCs likely accelerate the repair of injured neural ceils.展开更多
To establish an in vitro system for isolating and culturing the mesenchymal stem cells (MSC) of Rhesus monkeys, and to provide research data for its further application, the bone marrow of Rhesus monkeys was collect...To establish an in vitro system for isolating and culturing the mesenchymal stem cells (MSC) of Rhesus monkeys, and to provide research data for its further application, the bone marrow of Rhesus monkeys was collected and separated by gradient centrifugation to discard most of the blood cells. The MSC contained in the monocyte centrifuging layer was obtained and cultured in Dulbecco's modified media (low glucose, L-DMEM) supplemented with 10% Fetal bovine serum (FBS) and 1 ng/ml basic fibroblast growth factor (bFGF). The non-MSC was screened out by continuously renewing the medium. A passage culture was undertaken while the MSC monolayer formed. The spindle-shaped MSC formed a monolayer after 18 days of primary culturing, and the cells appeared in an oriented array with a swirling and irradiating growth trend. In the anaphase of passage culture, the cell proliferation rate was decreased and the morphology changed into triangular, polygon and flat appearance. These results suggested that mesenchymal stem cells (MSC) of the Rhesus monkey can be passaged in vitro with the established optimized culture system.展开更多
Mesenchymal stem cells (MSCS) are pluripotent stem cells isolated from various tissues, but mostly from bone marrow, adipose tissue, and umbilical cord blood. Well known for their mesenchymal lineages differentiati...Mesenchymal stem cells (MSCS) are pluripotent stem cells isolated from various tissues, but mostly from bone marrow, adipose tissue, and umbilical cord blood. Well known for their mesenchymal lineages differentiation (e.g., bone, cartilage and fat tissues), it was suggested that MSCs possess plasticity prop- erties enabling them to differentiate into non-mesenchymal lineages. Indeed, several protocols claimed for differentiating MSCs to neurons in vitro, but concern was raised for the ef- fectiveness and in vivo relevance of such differentiation. Thus, though their neurogenic differentiation properties are still in debate, they were nevertheless, suggested as candidates for treat- ing neurodegenerative disorders such as Parkinson's diseases, multiple sclerosis and Alzheimer's disease (AD).展开更多
Our preliminary studies confirmed that an active principle region of Buyang Huanwu decoction, comprising alkaloid, polysaccharide, aglycon, glucoside and volatile oil, can induce bone marrow mesenchymal stem cell diff...Our preliminary studies confirmed that an active principle region of Buyang Huanwu decoction, comprising alkaloid, polysaccharide, aglycon, glucoside and volatile oil, can induce bone marrow mesenchymal stem cell differentiation into neurons. Mitogen-activated protein kinase signaling was identified as one of the key pathways underlying this differentiation process. The present study shows phosphorylated extracellular signal-regulated protein kinase and phosphorylated p38 protein expression was increased after differentiation. Cellular signaling pathway blocking agents, PD98059 and SB203580, inhibited extracellular signal-regulated protein kinase and p38 in mitogen-activated protein kinase signaling pathways respectively, mRNA and protein expression of the neuronal marker, neuron specific enolase, and neural stem cell marker, nestin, were decreased in bone marrow mesenchymal stem cells after treatment with the active principle region of Buyang Huanwu decoction. Experimental findings indicate that, extracellular signal-regulated protein kinase and p38 in mitogen-activated protein kinase signaling pathways participate in bone marrow mesenchymal stem cell differentiation into neuron-like cells, induced by the active principle region of Buyang Huanwu decoction.展开更多
Mesenchymal stromal cells (MSCs) can be obtained from several sources and the significant differences in their properties make it crucial to investigate the differentiation potential of MSCs from different sources to ...Mesenchymal stromal cells (MSCs) can be obtained from several sources and the significant differences in their properties make it crucial to investigate the differentiation potential of MSCs from different sources to determine the optimal source of MSCs. We investigated if this biological heterogeneity in MSCs from different sources results in different mechanisms for their differentiation. In this study, we compared the gene expression patterns of phenotypically defined MSCs derived from three ontogenically different sources: Embryonic stem cells (hES-MSCs), Fetal limb (Flb-MSCs) and Bone Marrow (BM-MSCs). Differentially expressed genes between differentiated cells and undifferentiated controls were compared across the three MSC sources. We found minimal overlap (5% - 16%) in differentially expressed gene sets among the three sources. Flb-MSCs were similar to BM-MSCs based on differential gene expression patterns. Pathway analysis of the differentially expressed genes using Ingenuity Pathway Analysis (IPA) revealed a large variation in the canonical pathways leading to MSC differentiation. The similar canonical pathways among the three sources were lineage specific. The Flb-MSCs showed maximum overlap of canonical pathways with the BM-MSCs, indicating that the Flb-MSCs are an intermediate source between the less specialised hES-MSC source and the more specialised BM-MSC source. The source specific pathways prove that MSCs from the three ontogenically different sources use different biological pathways to obtain similar differentiation outcomes. Thus our study advocates the understanding of biological pathways to obtain optimal sources of MSCs for various clinical applications.展开更多
基金supported by the National Key Research and Development Project Intergovernmental Cooperation in Science and Technology of China(2018YFE0126900)the Key R&D Program of Lishui City(2021ZDYF12)the National Natural Science Foundation of China(82271629)。
文摘Skin wounds are characterized by injury to the skin due to trauma,tearing,cuts,or contusions.As such injuries are common to all human groups,they may at times represent a serious socioeconomic burden.Currently,increasing numbers of studies have focused on the role of mesenchymal stem cell(MSC)-derived extracellular vesicles(EVs)in skin wound repair.As a cell-free therapy,MSC-derived EVs have shown significant application potential in the field of wound repair as a more stable and safer option than conventional cell therapy.Treatment based on MSC-derived EVs can significantly promote the repair of damaged substructures,including the regeneration of vessels,nerves,and hair follicles.In addition,MSC-derived EVs can inhibit scar formation by affecting angiogenesis-related and antifibrotic pathways in promoting macrophage polarization,wound angiogenesis,cell proliferation,and cell migration,and by inhibiting excessive extracellular matrix production.Additionally,these structures can serve as a scaffold for components used in wound repair,and they can be developed into bioengineered EVs to support trauma repair.Through the formulation of standardized culture,isolation,purification,and drug delivery strategies,exploration of the detailed mechanism of EVs will allow them to be used as clinical treatments for wound repair.In conclusion,MSCderived EV-based therapies have important application prospects in wound repair.Here we provide a comprehensive overview of their current status,application potential,and associated drawbacks.
基金supported by the Natural Science Foundation of Shandong Province(ZR2020MH327)Hebei Key Research and Development Program(19272405D)Jilin Scientific and Technological Development Program(Grant No.20190304041YY).
文摘Mesenchymal stem cells(MSCs)have been widely used in regenerative medicine and clinical therapy due to their capabilities of proliferation,differentiation,and immune regulation.However,during in vitro expansion,MSCs are prone to aging,which largely limits their application.Prostaglandin E-2(PGE-2)is a key effector secreted by MSCs to exert immunomodulatory effects.By screening the compound library for PGE-2 secretion,the antioxidant trolox was verified as a stimulator of MSCs to secrete PGE-2.The effect of antioxidant trolox on biological characteristics of MSCS,including aging,proliferation,and gene expression,was examined.The results demonstrated that trolox can resist aging,promote proliferation,and enhance PGE-2 secretion of MSCs without affecting their surface marker expression.Furthermore,trolox treatment up-regulates miR-17-92 clusters in MSCs and may contribute to its anti-aging effects.Thus,trolox addition might be beneficial for MSCs expansion and their application.
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In the present study, the morphological and ultrastructural characteristics of mesenchymal stem cells (MSCs) induced towards osteogenic, adipogenic, and chondrogenic lineages were investigated. The main objective of this paper was to focus on the differentiation capacity of ovine [oMSCs] and equine MSCs [eMSCs]. Bone marrow [BM] MSCs were isolated from ovine and equine patients, expanded in monolayer culture and induced into osteogenic, adipogenic and chondrogenic differentiation. For chondrogenic differentiation, cells were cultured in micromass culture. Also, their ultrastructural phenotypes were studied by transmission electron microscopy [TEM]. This study was aimed to provide an indepth morphological description of BM-derived ovine and equine MSCs induced towards three lineages, it demonstrated that TEM analysis is useful in elucidating detailed structural information.
基金by research grants from the Natural Science Foundation of China(82102696 to JF)the Chongqing Bayu Young Scholar Award(JF),the 2019 Chongqing Support Program for Entrepreneurship and Innovation(No.cx2019113 to JF)+4 种基金the 2019 Funding for Postdoctoral Research(Chongqing Human Resources and Social Security Bureau No.298 to JF),the National Institutes of Health(CA226303 to TCH,and DE030480 to RRR)supported by the Medical Scientist Training Program of the National Institutes of Health(T32 GM007281)supported in part by The University of Chicago Cancer Center Support Grant(P30CA014599)the National Center for Advancing Translational Sciences of the National Institutes of Health through Grant Number UL1TR002389-07.
文摘Effective bone regeneration through tissue engineering requires a combination of osteogenic progenitors,osteoinductive biofactors and biocompatible scaffold materials.Mesenchymal stem cells(MSCs)represent the most promising seed cells for bone tissue engineering.As multipotent stem cells that can self-renew and differentiate into multiple lineages including bone and fat,MSCs can be isolated from numerous tissues and exhibit varied differentiation potential.To identify an optimal progenitor cell source for bone tissue engineering,we analyzed the proliferative activity and osteogenic potential of four commonly-used mouse MSC sources,including immortalized mouse embryonic fibroblasts(iMEF),immortalized mouse bone marrow stromal stem cells(imBMSC),immortalized mouse calvarial mesenchymal progenitors(iCAL),and immortalized mouse adipose-derived mesenchymal stem cells(iMAD).We found that iMAD exhibited highest osteogenic and adipogenic capabilities upon BMP9 stimulation in vitro,whereas iMAD and iCAL exhibited highest osteogenic capability in BMP9-induced ectopic osteogenesis and critical-sized calvarial defect repair.Transcriptomic analysis revealed that,while each MSC line regulated a distinct set of target genes upon BMP9 stimulation,all MSC lines underwent osteogenic differentiation by regulating osteogenesis-related signaling including Wnt,TGF-β,PI3K/AKT,MAPK,Hippo and JAK-STAT pathways.Collectively,our results demonstrate that adipose-derived MSCs represent optimal progenitor sources for cell-based bone tissue engineering.
基金the National Science Center,No.N407121940the Wroclaw Centre of Biotechnology,the Leading National Research Centre(KNOW)program for the years 2014-2018
文摘BACKGROUND Mesenchymal stromal/stem cells (MSCs) constitute a promising tool in regenerative medicine and can be isolated from different human tissues. However, their biological properties are still not fully characterized. Whereas MSCs from different tissue exhibit many common characteristics, their biological activity and some markers are different and depend on their tissue of origin. Understanding the factors that underlie MSC biology should constitute important points for consideration for researchers interested in clinical MSC application. AIM To characterize the biological activity of MSCs during longterm culture isolated from: bone marrow (BM-MSCs), adipose tissue (AT-MSCs), skeletal muscles (SMMSCs), and skin (SK-MSCs). METHODS MSCs were isolated from the tissues, cultured for 10 passages, and assessed for: phenotype with immunofluorescence and flow cytometry, multipotency with differentiation capacity for osteo-, chondro-, and adipogenesis, stemness markers with qPCR for mRNA for Sox2 and Oct4, and genetic stability for p53 and c-Myc;27 bioactive factors were screened using the multiplex ELISA array, and spontaneous fusion involving a co-culture of SM-MSCs with BM-MSCs or AT-MSCs stained with PKH26 (red) or PKH67 (green) was performed. RESULTS All MSCs showed the basic MSC phenotype;however, their expression decreased during the follow-up period, as confirmed by fluorescence intensity. The examined MSCs express CD146 marker associated with proangiogenic properties;however their expression decreased in AT-MSCs and SM-MSCs, but was maintained in BM-MSCs. In contrast, in SK-MSCs CD146 expression increased in late passages. All MSCs, except BM-MSCs, expressed PW1, a marker associated with differentiation capacity and apoptosis. BM-MSCs and AT-MSCs expressed stemness markers Sox2 and Oct4 in long-term culture. All MSCs showed a stable p53 and c-Myc expression. BM-MSCs and AT-MSCs maintained their differentiation capacity during the follow-up period. In contrast, SK-MSCs and SM-MSCs had a limited ability to differentiate into adipocytes. BM-MSCs and AT-MSCs revealed similarities in phenotype maintenance, capacity for multilineage differentiation, and secretion of bioactive factors. Because AT-MSCs fused with SM-MSCs as effectively as BM-MSCs, AT-MSCs may constitute an alternative source for BM-MSCs. CONCLUSION Long-term culture affects the biological activity of MSCs obtained from various tissues. The source of MSCs and number of passages are important considerations in regenerative medicine.
基金This work was supported by a grant from the National Natural Science Foundation of China (No.39970741) a grant from the the Scienceand Technology Foundation of Jilin Health Administration (No. 200131) and a grant from the Youth Teacher Foundation o
文摘To explore the way to induce mesenchymal stem cells (MSCs) to differentiate into dopaminergic neurons in vitro. Methods MSCs were obtained from rat bone marrow, cultured and passaged. MSCs used in this experiment had multipotency, which was indirectly proved by being induced to differentiate into chondrocytes and adipocytes. MSCs were cultured in medium containing 0.5 mmol/L IBMX for 2 days. Then the medium was replaced with induction medium, which contained GDNF, IL-1β, mesencephalic glial-cell-conditioned medium and flash-frozen mesencephalic membrane fragments. The surface markers of the differentiated neurons, such as NSE, nestin, MAP-2a, b and TH were detected by immunocytochemistry and Western blot after MSCs were cultured in induction medium for 7 days and 15 days. Results MSCs differentiated into neural progenitors and expressed nestin after MSCs were incubated with medium containing IBMX for 2 d. After the medium was replaced with induction medium containing many inducing agents, MSCs differentiated into neuron-like cells and dopaminergic neuron-like cells and expressed NSE, MAP-2a, b and TH. The percentage of NSE-positive cells, MAP-2a, b-positive cells and TH-positive cells was 30.032±2.489%, 41.580±5.101% and 34.958±5.534%, respectively after MSCs were induced in medium containing GDNF, IL-1β, mesencephalic glial-cell-conditioned medium and flash-frozen mesencephalic membrane fragments for 15 days. Conclusion MSCs can differentiate into dopaminergic neuron-like cells and are a new cell source for the treatment of neurodegeneration diseases and have a great potential for wide application
基金Project (No. 2004QN018) supported by the Health Bureau of Zhejiang Province, China
文摘Background:Bone marrow mesenchymal stem cell (MSC) transplantation is a promising strategy in the treatment of myocardial infarction (MI). However, the time for transplanting cells remains controversial. The aim of this study was to find an optimal time point for cell transplantation. Methods: MSCs were isolated and cultured from Sprague-Dawley (SD) rats. MI model was set up in SD rats by permanent ligation of left anterior descending coronary artery. MSCs were directly injected into the infarct border zone at 1 h, 1 week and 2 weeks after MI, respectively. Sham-operated and MI control groups received equal volume of phosphate buffered saline (PBS). At 4 weeks after MI, cardiac function was assessed by echocardiography; vessel density was analyzed on hematoxylin-eosin stained slides by light microscopy; the apoptosis of cardiomyocytes was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay; the expressions of proteins were analyzed by Western blot. Results: MSC transplantation improved cardiac function, reduced the apoptosis of cardiomyocytes and increased vessel density. These benefits were more obvious in 1-week group than in 1-h and 2-week groups. There are more obvious in-creases in the ratio of bcl-2/bax and the expression of vascular endothelial growth factor (VEGF) and more obvious decreases in the expression of cleaved-caspase-3 in 1-week group than those in other two groups. Conclusion: MSC transplantation was beneficial for the recovery of cardiac function. MSC transplantation at 1 week post-MI exerted the best effects on increases of cardiac function, anti-apoptosis and angiogenesis.
文摘Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily and have diverse functions during development and organogenesis. BMPs play a major role in skeletal development and bone formation, and disruptions in BMP signaling cause a variety of skeletal and extraskeletal anomalies. Several knockout models have provided insight into the mechanisms responsible for these phenotypes. Proper bone formation requires the differentiation of osteoblasts from mesenchymal stem cell (MSC) precursors, a process mediated in part by BMP signaling. Multiple BMPs, including BMP2, BMP6, BMP7 and BMP9, promote osteoblastic differentiation of MSCs both in vitro and in vivo. BMP9 is one of the most osteogenic BMPs, yet it is a poorly characterized member of the BMP family. Several studies demonstrate that the mechanisms controlling BMP9-mediated osteogenesis differ from other osteogenic BMPs, but little is known about these specific mechanisms. Several pathways critical to BMP9-mediated osteogenesis are also important in the differentiation of other cell lineages, including adipocytes and chondrocytes. BMP9 has also demonstrated translational promise in spinal fusion and bone fracture repair. This review will summarize our current knowledge of BMP-mediated osteogenesis, with a focus on BMP9, by presenting recently completed work which may help us to further elucidate these pathways.
基金the National Basic Research Program of China (No 2005CB522404 and 2006CB910506)the Program for Changjiang Scholars and Innovative Research Team (PCSIRT) in Universities (No IRT0519)the National Natural Science Founda-tion of China (No 30771232 and 30671184)
文摘Mesenchymal stem cells (MSCs) of nonembryonic origins possess the proliferation and multi-lineage differentiation potentials. It has been established that epigenetic mechanisms could be critical for determining the fate of stem cells, and MSCs derived from different origins exhibited different expression profiles individually to a certain extent. In this study, ChiP-on-chip was used to generate genome-wide histone H3-Lys9 acetylation and dimethylation profiles at gene promoters in human bone marrow MSCs. We showed that modifications of histone H3-Lys9 at gene promoters correlated well with mRNA expression in human bone marrow MSCs. Functional analysis revealed that many key cellular pathways in human bone marrow MSC self-renewal, such as the canonical signaling pathways, cell cycle pathways and cytokine related pathways may be regulated by H3-Lys9 modifications. These data suggest that gene activation and silencing affected by H3-Lys9 acetylation and dimethylation, respectively, may be essential to the maintenance of human bone marrow MSC self-renewal and multi-potency.
基金financially supported by National Natural Science Foundation of China (81100240)‘985’ project of Sun Yat-Sen University grant+2 种基金Sun Yat-Sen university young teachers training project (13YKPY42)Natural Science Foundation of Guangdong Province,China(S2012010009495)Science and Technology Planning Project of Guangdong Province,China(2012B031800185)
文摘Age related defect of the osteogenic differentiation of mesenchymal stem cells(MSCs) plays a key role in osteoporosis. Mechanical loading is one of the most important physical stimuli for osteoblast differentiation.Here, we compared the osteogenic potential of MSCs from young and adult rats under three rounds of 2 h of cyclic stretch of 2.5% elongation at 1 Hz on 3 consecutive days. Cyclic stretch induced a significant osteogenic differentiation of MSCs from young rats, while a compromised osteogenesis in MSCs from the adult rats.Accordingly, there were much more reactive oxygen species(ROS) production in adult MSCs under cyclic stretch compared to young MSCs. Moreover, ROS scavenger N-acetylcysteine rescued the osteogenic differentiation of adult MSCs under cyclic stretch. Gene expression analysis revealed that superoxide dismutase 1(SOD1) was significantly downregulated in those MSCs from adult rats. In summary, our data suggest that reduced SOD1 may result in excessive ROS production in adult MSCs under cyclic stretch, and thus manipulation of the MSCs from the adult donors with antioxidant would improve their osteogenic ability.
文摘This study aimed to investigate the neural differentiation of human umbilical cord mesenchymal stem cells (hUCMSCs) under the induction of injured neural cells. After in vitro isolation and culture, passage 5 hUCMSCs were used for experimentation, hUCMSCs were co-cultured with normal or AI31.4o-injured PC12 cells, PC12 cell supernatant or PC12 cell lysate in a Transwell co-culture system. Western blot analysis and flow cytometry results showed that choline acetyltransferase and microtubule-associated protein 2, a specific marker for neural cells, were expressed in hUCMSCs under various culture conditions, and highest expression was observed in the hUCMSCs co-cultured with injured PC12 cells. Choline acetyltransferase and microtubule-associated protein 2 were not expressed in hUCMSCs cultured alone (no treatment). Cell Counting Kit-8 assay results showed that hUCMSCs under co-culture conditions promoted the proliferation of injured PC12 cells. These findings suggest that the microenvironment during neural tissue injury can effectively induce neural cell differentiation of hUCMSCs. These differentiated hUCMSCs likely accelerate the repair of injured neural ceils.
文摘To establish an in vitro system for isolating and culturing the mesenchymal stem cells (MSC) of Rhesus monkeys, and to provide research data for its further application, the bone marrow of Rhesus monkeys was collected and separated by gradient centrifugation to discard most of the blood cells. The MSC contained in the monocyte centrifuging layer was obtained and cultured in Dulbecco's modified media (low glucose, L-DMEM) supplemented with 10% Fetal bovine serum (FBS) and 1 ng/ml basic fibroblast growth factor (bFGF). The non-MSC was screened out by continuously renewing the medium. A passage culture was undertaken while the MSC monolayer formed. The spindle-shaped MSC formed a monolayer after 18 days of primary culturing, and the cells appeared in an oriented array with a swirling and irradiating growth trend. In the anaphase of passage culture, the cell proliferation rate was decreased and the morphology changed into triangular, polygon and flat appearance. These results suggested that mesenchymal stem cells (MSC) of the Rhesus monkey can be passaged in vitro with the established optimized culture system.
文摘Mesenchymal stem cells (MSCS) are pluripotent stem cells isolated from various tissues, but mostly from bone marrow, adipose tissue, and umbilical cord blood. Well known for their mesenchymal lineages differentiation (e.g., bone, cartilage and fat tissues), it was suggested that MSCs possess plasticity prop- erties enabling them to differentiate into non-mesenchymal lineages. Indeed, several protocols claimed for differentiating MSCs to neurons in vitro, but concern was raised for the ef- fectiveness and in vivo relevance of such differentiation. Thus, though their neurogenic differentiation properties are still in debate, they were nevertheless, suggested as candidates for treat- ing neurodegenerative disorders such as Parkinson's diseases, multiple sclerosis and Alzheimer's disease (AD).
基金sponsored by the National Natural Science Foundation of China,No.81102595the Natural Science Foundation of Guangxi,No.2012GXNSFAA053113
文摘Our preliminary studies confirmed that an active principle region of Buyang Huanwu decoction, comprising alkaloid, polysaccharide, aglycon, glucoside and volatile oil, can induce bone marrow mesenchymal stem cell differentiation into neurons. Mitogen-activated protein kinase signaling was identified as one of the key pathways underlying this differentiation process. The present study shows phosphorylated extracellular signal-regulated protein kinase and phosphorylated p38 protein expression was increased after differentiation. Cellular signaling pathway blocking agents, PD98059 and SB203580, inhibited extracellular signal-regulated protein kinase and p38 in mitogen-activated protein kinase signaling pathways respectively, mRNA and protein expression of the neuronal marker, neuron specific enolase, and neural stem cell marker, nestin, were decreased in bone marrow mesenchymal stem cells after treatment with the active principle region of Buyang Huanwu decoction. Experimental findings indicate that, extracellular signal-regulated protein kinase and p38 in mitogen-activated protein kinase signaling pathways participate in bone marrow mesenchymal stem cell differentiation into neuron-like cells, induced by the active principle region of Buyang Huanwu decoction.
文摘Mesenchymal stromal cells (MSCs) can be obtained from several sources and the significant differences in their properties make it crucial to investigate the differentiation potential of MSCs from different sources to determine the optimal source of MSCs. We investigated if this biological heterogeneity in MSCs from different sources results in different mechanisms for their differentiation. In this study, we compared the gene expression patterns of phenotypically defined MSCs derived from three ontogenically different sources: Embryonic stem cells (hES-MSCs), Fetal limb (Flb-MSCs) and Bone Marrow (BM-MSCs). Differentially expressed genes between differentiated cells and undifferentiated controls were compared across the three MSC sources. We found minimal overlap (5% - 16%) in differentially expressed gene sets among the three sources. Flb-MSCs were similar to BM-MSCs based on differential gene expression patterns. Pathway analysis of the differentially expressed genes using Ingenuity Pathway Analysis (IPA) revealed a large variation in the canonical pathways leading to MSC differentiation. The similar canonical pathways among the three sources were lineage specific. The Flb-MSCs showed maximum overlap of canonical pathways with the BM-MSCs, indicating that the Flb-MSCs are an intermediate source between the less specialised hES-MSC source and the more specialised BM-MSC source. The source specific pathways prove that MSCs from the three ontogenically different sources use different biological pathways to obtain similar differentiation outcomes. Thus our study advocates the understanding of biological pathways to obtain optimal sources of MSCs for various clinical applications.