Cultured cell‐derived extracellular matrices to enhance the osteogenic differentiation and angiogenic properties of human mesenchymal stem/stromal cells

Cell‐derived extracellular matrix (ECM) consists of a complex assembly of fibrillary proteins, matrix macromolecules, and associated growth factors that mimic the composition and organization of native ECM micro‐environment. Therefore, cultured cell‐derived ECM has been used as a scaffold for tissue engineering settings to create a biomimetic micro‐environment, providing physical, chemical, and mechanical cues to cells, and support cell adhesion, proliferation, migration, and differentiation. Here, we present a new strategy to produce different combinations of decellularized cultured cell‐derived ECM (dECM) obtained from different cultured cell types, namely, mesenchymal stem/stromal cells (MSCs) and human umbilical vein endothelial cells (HUVECs), as well as the coculture of MSC:HUVEC and investigate the effects of its various compositions on cell metabolic activity, osteogenic differentiation, and angiogenic properties of human bone marrow (BM)‐derived MSCs, vital features for adult bone tissue regeneration and repair. Our findings demonstrate that dECM presented higher cell metabolic activity compared with tissue culture polystyrene. More importantly, we show that MSC:HUVEC ECM enhanced the osteogenic and angiogenic potential of BM MSCs, as assessed by in vitro assays. Interestingly, MSC:HUVEC (1:3) ECM demonstrated the best angiogenic response of MSCs in the conditions tested. To the best of our knowledge, this is the first study that demonstrates that dECM derived from a coculture of MSC:HUVEC impacts the osteogenic and angiogenic capabilities of BM MSCs, suggesting the potential use of MSC:HUVEC ECM as a therapeutic product to improve clinical outcomes in bone regeneration.     

Osteogenic capacity of human BM‐MSCs,AT‐MSCs and their co‐cultures using HUVECs in FBS and PL supplemented media

Human bone marrow‐derived mesenchymal stem cells (BM‐MSCs) and human adipose tissue‐derived mesenchymal stem cells (AT‐MSCs) are the most frequently used stem cells in tissue engineering. Due to major clinical demands, it is necessary to find an optimally safe and efficient way for large‐scale expansion of these cells. Considering the nutritional source in the culture medium and method, this study aimed to analyze the effects of FBS‐ and PL‐supplemented media on osteogenesis in stem cell mono‐ and co‐cultures with human umbilical vein endothelial cells (HUVECs). Results showed that cell metabolic activity and proliferation increased in PL‐ compared to FBS‐supplemented media in mono‐ and co‐cultures for both BM‐MSCs and AT‐MSCs. In addition, calcium deposition was cell type dependent and decreased for BM‐MSCs but increased for AT‐MSCs in PL‐supplemented medium in both mono‐ and co‐cultures. Based on the effects of co‐cultures, BM‐MSCs/HUVECs enhanced osteogenesis compared to BM‐MSCs monocultures in both FBS‐ and PL‐supplemented media whereas AT‐MSCs/HUVECs showed similar results compared to AT‐MSCs monocultures. Copyright © 2013 John Wiley & Sons, Ltd.     

Electrical stimulation of adipose‐derived mesenchymal stem cells and endothelial cells co‐cultured in a conductive scaffold for potential orthopaedic applications

Electrical stimulation (ES) has emerged as a useful tool to regulate cell behaviour, but the effect of ES on mesenchymal stem cell (MSC)/vasculogenic cell co‐culture has not been investigated. Herein, human adipose‐derived MSCs (AD‐MSCs) and umbilical vein endothelial cells (HUVECs) were co‐cultured in an electrically conductive polypyrrole/chitosan scaffold. Compared with AD‐MSC monoculture, calcium deposition in the co‐culture without and with ES (200 μA for 4 h/day) was 139% and 346% higher, respectively, after 7 days. As the application of ES to AD‐MSC monoculture only increased calcium deposition by 56% compared with that without ES after 7 days, these results indicate that ES and co‐culture with HUVECs have synergistic effects on AD‐MSCs' osteogenic differentiation. ES application also significantly enhanced CD31 expression of HUVECs. In HUVEC monoculture, application of ES increased CD31 expression by 224%, whereas the corresponding increase in AD‐MSC/HUVEC co‐culture with ES application was 62%. The gene expression results indicate that ES enhanced the cellular functions in AD‐MSC and HUVEC monoculture via autocrine bone morphogenetic protein‐2 (BMP‐2) and vascular endothelial growth factor (VEGF), respectively. In co‐culture, crosstalk between AD‐MSCs and HUVECs due to paracrine BMP‐2 and VEGF enhanced the cellular functions compared with the respective monoculture. With application of ES to the AD‐MSC/HUVEC co‐culture, autocrine signalling was enhanced, resulting in further promotion of cellular functions. These findings illustrate that co‐culturing AD‐MSC/HUVEC in a conductive scaffold with ES offers potential benefits for bone defect therapy.     

Whatever their differentiation status,human progenitor derived – or mature – endothelial cells induce osteoblastic differentiation of bone marrow stromal cells

Association of the bone‐forming osteoblasts (OBs) and vascular endothelial cells (ECs) into a biomaterial composite provides a live bone graft substitute that can repair the bone defect when implanted. An intimate functional relationship exists between these cell types. This communication is crucial to the coordinated cell behaviour necessary for bone development and remodelling. Previous studies have shown that direct co‐culture of primary human osteoprogenitors (HOPs) with primary human umbilical vein endothelial cells (HUVECs) stimulates HOPs differentiation and induces tubular‐like networks. The present work aims to test the use of human bone marrow stromal cells (HBMSCs) co‐cultured with human endothelial progenitor cells in order to assess whether progenitor‐derived ECs (PDECs) could support osteoblastic differentiation as mature ECs do. Indeed, data generated from the literature by different laboratories considering these co‐culture systems appear difficult to compare. Monocultures of HUVECs, HOPs, HBMSCs (in a non‐orientated lineage), PDECs (from cord blood) were used as controls and four combinations of co‐cultures were undertaken: HBMSCs–PDECs, HBMSCs–HUVECs, HOPs–PDECs, HOPs–HUVECs with ECs (mature or progenitor) for 6 h to 7 days. At the end of the chosen co‐culture time, intracellular alkaline phosphatase (ALP) activity was detected in HOPs and HBMSCs and quantified in cell extracts. Quantitative real‐time polymerase chain reaction (qPCR) of ALP was performed over time and vascular endothelial growth factor (VEGF) was measured. After 21 days, calcium deposition was observed, comparing mono‐ and co‐cultures. We confirm that ECs induce osteoblastic differentiation of mesenchymal stem cells in vitro. Moreover, HUVECs can be replaced by PDECs, the latter being of great interest in tissue engineering. Copyright © 2012 John Wiley & Sons, Ltd.     

Effect of donor variation on osteogenesis and vasculogenesis in hydrogel cocultures

To introduce a functional vascular network into tissue‐engineered bone equivalents, human endothelial colony forming cells (ECFCs) and multipotent mesenchymal stromal cells (MSCs) can be cocultured. Here, we studied the impact of donor variation of human bone marrow‐derived MSCs and cord blood‐derived ECFCs on vasculogenesis and osteogenesis using a 3D in vitro coculture model. Further, to make the step towards cocultures consisting of cells derived from a single donor, we tested how induced pluripotent stem cell (iPSC)‐derived human endothelial cells (iECs) performed in coculture models. Cocultures with varying combinations of human donors of MSCs, ECFCs, or iECs were prepared in Matrigel. The constructs were cultured in an osteogenic differentiation medium. Following a 10‐day culture period, the length of the prevascular structures and osteogenic differentiation were evaluated for up to 21 days of culture. The particular combination of MSC and ECFC donors influenced the vasculogenic properties significantly and induced variation in osteogenic potential. In addition, the use of iECs in the cocultures resulted in prevascular structure formation in osteogenically differentiated constructs. Together, these results showed that close attention to the source of primary cells, such as ECFCs and MSCs, is critical to address variability in vasculogenic and osteogenic potential. The 3D coculture model appeared to successfully generate prevascularized constructs and were sufficient in exceeding the ~200 μm diffusion limit. In addition, iPSC‐derived cell lineages may decrease variability by providing a larger and potentially more uniform source of cells for future preclinical and clinical applications.     

Traumatized muscle‐derived multipotent progenitor cells recruit endothelial cells through vascular endothelial growth factor‐A action

Traumatized muscle, such as that debrided from blast injury sites, is considered a promising and convenient tissue source for multipotent progenitor cells (MPCs), a population of adult mesenchymal stem cell (MSC)‐like cells. The present study aimed to assess the regenerative therapeutic potential of human traumatized muscle‐derived MPCs, e.g., for injury repair in the blast‐traumatized extremity, by comparing their pro‐angiogenic potential in vitro and capillary recruitment activity in vivo to those of MSCs isolated from human bone marrow, a widely‐used tissue source. MPCs were tested for their direct and indirect effects on human microvascular endothelial cells (ECs) in vitro. The findings reported here showed that MPC‐conditioned culture medium (MPC‐CM), like MSC‐CM, promoted EC‐cord network branching. Silent (si)RNA‐mediated silencing of vascular endothelial growth factor‐A (VEGF‐A) expression in MPCs attenuated this effect. In a chick embryonic chorioallantoic membrane in vivo angiogenesis assay, MPCs encapsulated in photocrosslinked gelatin scaffold recruited blood vessels more efficiently than either MSCs or human foreskin fibroblasts. Together, these findings support the potential application of traumatized muscle‐derived MPCs in cell‐based regenerative medicine therapies as a result of their influence on EC organization. Copyright © 2017 John Wiley & Sons, Ltd.     

Human progenitor‐derived endothelial cells vs. venous endothelial cells for vascular tissue engineering: an in vitro study

The isolation of endothelial progenitor cells from human peripheral blood generates a great hope in vascular tissue engineering because of particular benefit when compared with mature endothelial cells. We explored the capability of progenitor‐derived endothelial cells (PDECs) to line fibrin and collagen scaffolds in comparison with human saphenous and umbilical cord vein endothelial cells (HSVECs and HUVECs): (a) in a static situation, allowing definition of the optimal cell culture conditions with different media and cell‐seeding densities to check cell behaviour; (b) under shear stress conditions (flow chambers or tubular vascular constructs), allowing investigation of cell response and mRNA expression on both substrates by oligonucleotide microarray analysis and quantitative real‐time PCR. Well characterized PDECs: (a) could not be expanded adequately with the usual mature ECs culture media; (b) were able to colonize and grow on fibrin glue; (c) exhibited higher resistance to oxidative stress than HSVECs and HUVECs; (d) withstood physiological shear stress when lining both substrates in flow chambers, and their gene expression was regulated; (e) colonized a collagen‐impregnated vascular prosthesis and were able to sense mechanical forces. Our results provide an improved qualification of PDECs for vascular tissue engineering. Copyright © 2010 John Wiley & Sons, Ltd.     

Point‐of‐care treatment of focal cartilage defects with selected chondrogenic mesenchymal stromal cells—An in vitro proof‐of‐concept study

Due to the poor self‐healing capacities of cartilage, innovative approaches are a major clinical need. The use of in vitro expanded mesenchymal stromal cells (MSCs) in a 2‐stage approach is accompanied by cost‐, time‐, and personnel‐intensive good manufacturing practice production. A 1‐stage intraoperative procedure could overcome these drawbacks. The aim was to prove the feasibility of a point‐of‐care concept for the treatment of cartilage lesions using defined MSC subpopulations in a collagen hydrogel without prior MSC monolayer expansion. We tested 4 single marker candidates (MSCA‐1, W4A5, CD146, CD271) for their effectiveness of separating colony‐forming units of ovine MSCs via magnetic cell separation. The most promising surface marker with regard to the highest enrichment of colony‐forming cells was subsequently used to isolate a MSC subpopulation for the direct generation of a cartilage graft composed of a collagen type I hydrogel without the propagation of MSCs in monolayer. We observed that separation with CD271 sustained the highest enrichment of colony‐forming units. We then demonstrated the feasibility of generating a cartilage graft with an unsorted bone marrow mononuclear cell fraction and with a characterized CD271 positive MSC subpopulation without the need for a prior cell expansion. A reduced volume of 6.25% of the CD271 positive MSCs was needed to achieve the same results regarding chondrogenesis compared with the unseparated bone marrow mononuclear cell fraction, drastically reducing the number of nonrelevant cells. This study provides a proof‐of‐concept and reflects the potential of an intraoperative procedure for direct seeding of cartilage grafts with selected CD271 positive cells from bone marrow.     

Ectopic bone formation by aggregated mesenchymal stem cells from bone marrow and adipose tissue: A comparative study

Tissue engineered constructs (TECs) based on spheroids of bone marrow mesenchymal stromal cells (BM‐MSCs) combined with calcium phosphate microparticles and enveloped in a platelet‐rich plasma hydrogel showed that aggregation of MSCs improves their ectopic bone formation potential. The stromal vascular fraction (SVF) and adipose‐derived MSCs (ASCs) have been recognized as an interesting MSC source for bone tissue engineering, but their ectopic bone formation is limited. We investigated whether aggregation of ASCs could similarly improve ectopic bone formation by ASCs and SVF cells. The formation of aggregates with BM‐MSCs, ASCs and SVF cells was carried out and gene expression was analysed for osteogenic, chondrogenic and vasculogenic genes in vitro. Ectopic bone formation was evaluated after implantation of TECs in immunodeficient mice with six conditions: TECs with ASCs, TECs with BM‐MSC, TECs with SVF cells (with and without rhBMP2), no cells and no cells with rhBMP2. BM‐MSCs showed consistent compact spheroid formation, ASCs to a lesser extent and SVF showed poor spheroid formation. Aggregation of ASCs induced a significant upregulation of the expression of osteogenic markers like alkaline phosphatase and collagen type I, as compared with un‐aggregated ASCs. In vivo, ASC and SVF cells both generated ectopic bone in the absence of added morphogenetic proteins. The highest incidence of bone formation was seen with BM‐MSCs (7/9) followed by SVF + rhBMP2 (4/9) and no cells + rhBMP2 (2/9). Aggregation can improve ectopic bone tissue formation by adipose‐derived cells, but is less efficient than rhBMP2. A combination of both factors should now be tested to investigate an additive effect.     

Angiogenic potential of human mesenchymal stromal cell and circulating mononuclear cell cocultures is reflected in the expression profiles of proangiogenic factors leading to endothelial cell and pericyte differentiation

Endothelial progenitors found among the peripheral blood (PB) mononuclear cells (MNCs) are interesting cells for their angiogenic properties. Mesenchymal stromal cells (MSCs) in turn can produce proangiogenic factors as well as differentiate into mural pericytes, making MSCs and MNCs an attractive coculture setup for regenerative medicine. In this study, human bone marrow‐derived MSCs and PB‐derived MNCs were cocultured in basal or osteoblastic medium without exogenously supplied growth factors to demonstrate endothelial cell, pericyte and osteoblastic differentiation. The expression levels of various proangiogenic factors, as well as endothelial cell, pericyte and osteoblast markers in cocultures were determined by quantitative polymerase chain reaction. Immunocytochemistry for vascular endothelial growth factor receptor‐1 and α‐smooth muscle actin as well as staining for alkaline phosphatase were performed after 10 and 14 days. Messenger ribonucleic acid expression of endothelial cell markers was highly upregulated in both basal and osteoblastic conditions after 5 days of coculture, indicating an endothelial cell differentiation, which was supported by immunocytochemistry for vascular endothelial growth factor receptor‐1. Stromal derived factor‐1 and vascular endothelial growth factor were highly expressed in MSC‐MNC coculture in basal medium but not in osteoblastic medium. On the contrary, the expression levels of bone morphogenetic protein‐2 and angiopoietin‐1 were significantly higher in osteoblastic medium. Pericyte markers were highly expressed in both cocultures after 5 days. In conclusion, it was demonstrated endothelial cell and pericyte differentiation in MSC‐MNC cocultures both in basal and osteoblastic medium indicating a potential for neovascularization for tissue engineering applications.     

人脐带和骨髓源间充质干细胞对脐血单个核细胞增殖能力的影响

目的：探讨人脐带间充质干细胞（UCMSCs）与骨髓间充质干细胞（BMMSCs）在体外对造血干细胞的支持作用。方法分别从人脐带和骨髓中分离、培养间充质干细胞,通过免疫细胞化学染色等方法对其进行表型鉴定;采用流式细胞仪测定脐血单个核细胞的周期分布,采用甲基纤维素法测定脐血单个核细胞混合集落形成单位（CFU-Mix）,比较 UCMSCs和BMMSCs对脐血单个核细胞细胞周期、CFU-Mix形成能力的影响。结果成功培养获得 UCMSCs和BMMSCs,鉴定结果符合预期;与非共培养组细胞相比,UCMSCs和 BMMSCs共培养均能促进脐血单个核细胞进入增殖周期,并增加其形成CFU-Mix的能力（P＜0．05）,但UCMSCs和BMMSCs共培养组之间比较差异无统计学意义（P＞0．05）。结论成功从人脐带和骨髓组织中培养获得间充质干细胞,两种来源的间充质干细胞均能提高脐血单个核细胞的体外增殖能力及 CFU-Mix形成能力,均具有造血支持作用。     

Production of human tissue‐engineered skin trilayer on a plasma‐based hypodermis

Full thickness wounds require a dermal component to achieve functional permanent skin restoration. Currently available tissue‐engineered skin substitutes lack a subcutaneous fat layer that would functionally contribute some of the mechanical and thermoregulatory properties of normal skin. To generate a trilayer engineered skin equivalent, we included bone marrow mesenchymal (BM‐MSC) or adipose tissue‐derived (ASC) stromal cells in a human plasma hydrogel exposed to adipogenic clues for three weeks. Approximately half of the cells differentiated under these conditions into mature adipocytes that survived for two years in culture with minimal medium change. In vitro generation of bona fide fully differentiated adipocytes was assessed by leptin secretion and ultrastructurally demonstrated through semithin to ultrathin sectioning and lipid staining with osmium tetroxide. Furthermore, presence of BM‐MSCs or ASCs within the subcutaneous layer contributed to the epidermal differentiation program, with more proliferating basal cells depositing basal membrane proteins and differentiating into mature keratinocytes that were able to generate a pluristratified epithelium. In conclusion, we engineered a fully differentiated human skin trilayer that could present multiple applications such as use for in vitro drug absorption tests and regenerative therapies. Copyright © 2012 John Wiley & Sons, Ltd.     

Comparison of in vitro‐cultivation of human mesenchymal stroma/stem cells derived from bone marrow and umbilical cord

Cell‐mediated therapy is currently considered as a novel approach for many human diseases. Potential uses range from topic applications with the regeneration of confined tissue areas to systemic applications. Stem cells including mesenchymal stroma/stem cells (MSCs) represent a highly attractive option. Their potential to cure or alleviate human diseases is investigated in a number of clinical trials. A wide variety of methods has been established in the past years for isolation, cultivation and characterization of human MSCs as expansion is presently deemed a prerequisite for clinical application with high numbers of cells carrying reproducible properties. MSCs have been retrieved from various tissues and used in a multitude of settings whereby numerous experimental protocols are available for expansion of MSCs in vitro. Accordingly, different isolation, culture and upscaling techniques contribute to the heterogeneity of MSC characteristics and the, sometimes, controversial results. Therefore, this review discusses and summarizes certain experimental conditions for MSC in vitro culture focusing on adult bone marrow‐derived and neonatal umbilical cord‐derived MSCs in order to enhance our understanding for MSC tissue sources and to stratify different procedures. Copyright © 2016 John Wiley & Sons, Ltd.     

人脐血单个核细胞和脐带间充质干细胞移植用于大鼠脊髓损伤的实验研究

目的:探讨人脐血单个核细胞和脐带间充质干细胞(MSCs)移植对脊髓损伤功能恢复的影响,寻找一种更适合治疗脊髓损伤的细胞源。方法:采集新鲜人脐带血和脐带,分离培养单个核细胞和MSCs。将脊髓损伤模型随机分成3组:单个核细胞移植组、MSCs移植组和低糖必需培养基(L-DMEM)培养组。采用免疫组化和免疫荧光检测细胞移植后1—4周细胞在脊髓内的存活情况和迁移情况,使用BBB行为学评分评估大鼠脊髓功能恢复情况。结果:L-DMEM培养液组在术后各时间点观察评分无明显差异,而细胞移植组脊髓功能处于逐渐恢复过程,与L-DMEM培养液比较,差异有显著性意义。单个核细胞移植组对损伤脊髓功能的修复作用较MSCs移植组显著,且差异有显著性意义。结论:与MSCs相比较,人脐血单个核细胞更适合作为治疗脊髓损伤的细胞源。     

Fucoidan‐induced osteogenic differentiation promotes angiogenesis by inducing vascular endothelial growth factor secretion and accelerates bone repair

Osteogenesis and angiogenesis, including cell–cell communication between blood vessel cells and bone cells, are essential for bone repair. Fucoidan is a chemical compound that has a variety of biological activities. It stimulates osteoblast differentiation in human mesenchymal stem cells (MSCs), which in turn induces angiogenesis. However, the mechanism by which this communication between osteoblasts and endothelial cells is mediated remains unclear. Thus, the aim of this study was to clarify the relationship between fucoidan‐induced osteoblastic differentiation in MSCs and angiogenesis in endothelial cells. First, the effect was confirmed of fucoidan on osteoblast differentiation in MSCs and obtained conditioned media from these cells (Fucoidan‐MSC‐CM). Next, the angiogenic activity of Fucoidan‐MSC‐CM was investigated and it was found that it stimulated angiogenesis, demonstrated by proliferation, tube formation, migration and sprout capillary formation in human umbilical vein endothelial cells. Messenger ribonucleic acid expression and protein secretion of vascular endothelial growth factor (VEGF) were dramatically increased during fucoidan‐induced osteoblast differentiation and that its angiogenic activities were reduced by a VEGF/VEGF receptor‐specific binding inhibitor. Furthermore, Fucoidan‐MSC‐CM increased the phosphorylation of mitogen‐activated protein kinase and PI3K/AKT/eNOS signalling pathway, and that its angiogenic effects were markedly suppressed by SB203580 and AKT 1/2 inhibitor. Finally, an in vivo study was conducted and it was found that fucoidan accelerated new blood vessel formation and partially promoted bone formation in a rabbit model of a calvarial bone defect. This is the first study to investigate the angiogenic effect of fucoidan‐induced osteoblastic differentiation through VEGF secretion, suggesting the therapeutic potential of fucoidan for enhancing bone repair.     

Dynamic cell–cell interactions between cord blood haematopoietic progenitors and the cellular niche are essential for the expansion of CD34+, CD34+CD38− and early lymphoid CD7+ cells

Most clinical applications of haematopoietic stem/progenitor cells (HSCs) would benefit from their ex vivo expansion to obtain a therapeutically significant amount of cells from the available donor samples. We studied the impact of cellular interactions between umbilical cord blood (UCB) haematopoietic cells and bone marrow (BM)‐derived mesenchymal stem cells (MSCs) on the ex vivo expansion and differentiative potential of UCB CD34⁺‐enriched cells. UCB cells were cultured: (a) directly in contact with BM MSC‐derived stromal layers (contact); (b) separated by a microporous membrane (non‐contact); or (c) without stroma (no stroma). Highly dynamic culture events occurred in HSC‐MSC co‐cultures, involving cell–cell interactions, which preceded HSC expansion. Throughout the time in culture [18 days], total cell expansion was significantly higher in contact (fold increase of 280 ± 37 at day 18) compared to non‐contact (85 ± 25). No significant cell expansion was observed in stroma‐free cultures. CD34⁺ cell expansion was also clearly favoured by direct contact with BM MSCs (35 ± 5‐ and 7 ± 3‐fold increases at day 18 for contact and non‐contact, respectively). Moreover, a higher percentage of CD34⁺CD38^? cells was consistently maintained during the time in culture under contact (8.1 ± 1.9% at day 18) compared to non‐contact (5.7 ± 1.6%). Importantly, direct cell interaction with BM MSCs significantly enhanced the expansion of early lymphoid CD7⁺ cells, yielding considerably higher (×3–10) progenitor numbers compared to non‐contact conditions. These results highlight the importance of dynamic cell–cell interactions between UCB HSCs and BM MSCs, towards the maximization of HSC expansion ex vivo to obtain clinically relevant cell numbers for multiple settings, such as BM transplantation or somatic cell gene therapy. Copyright © 2009 John Wiley & Sons, Ltd.     

Comparison of different culture conditions for human mesenchymal stromal cells for clinical stem cell therapy

Objective. Mesenchymal stromal cells (MSCs) from adult bone marrow (BM) are considered potential candidates for therapeutic neovascularization in cardiovascular disease. When implementing results from animal trials in clinical treatment, it is essential to isolate and expand the MSCs under conditions following good manufacturing practice (GMP). The aims of the study were first to establish culture conditions following GMP quality demands for human MSC expansion and differentiation for use in clinical trials, and second to compare these MSCs with MSCs derived from culture in four media commonly used for MSC cultivation in animal studies simulating clinical stem cell therapy. Material and methods. Human mononuclear cells (MNCs) were isolated from BM aspirates by density gradient centrifugation and cultivated in a GMP‐accepted medium (EMEA medium) or in one of four other media. Results. FACS analysis showed that the plastic‐adherent MSCs cultured in EMEA medium or in the other four media were identically negative for the haematopoietic surface markers CD45 and CD34 and positive for CD105, CD73, CD90, CD166 and CD13, which in combined expression is characteristic of MSCs. MSC stimulation with vascular endothelial growth factor (VEGF) increased expression of the characteristic endothelial genes KDR and von Willebrand factor; the von Willebrand factor and CD31 at protein level as well as the capacity to develop capillary‐like structures. Conclusions. We established culture conditions with a GMP compliant medium for MSC cultivation, expansion and differentiation. The expanded and differentiated MSCs can be used in autologous mesenchymal stromal cell therapy in patients with ischaemic heart disease.     

人脐带静脉灌注液间充质干细胞的分离和鉴定

本研究旨在探讨人脐带静脉灌注液中是否存在间充质干细胞及其分离和扩增方法。从正常产妇分娩后的脐带静脉灌注液分离间充质干细胞,观察细胞形态并用流式细胞仪分析其细胞表型和细胞周期,在体外分别诱导其成骨、成脂肪和成软骨细胞分化。结果表明:脐带静脉灌注液来源的间充质干细胞为成纤维细胞样,高表达CD29、HLA-ABC、CD166、CD105、CD73、CD44;不表达造血和内皮标志(CD34、CD45、CD144和CD14)。它们可向骨、脂肪和软骨细胞分化,符合间充质干细胞的基本功能特征。结论:人脐带静脉灌注液存在间充质干细胞,能够在体外培养和扩增,可作为今后细胞治疗和组织工程的种子细胞。     

Mesenchymal stem cells and ligand incorporation in biomimetic poly(ethylene glycol) hydrogels significantly improve insulin secretion from pancreatic islets

The main goal of this study was to investigate pancreatic islet function with mesenchymal stem cells (MSCs) in a ligand‐functionalized poly(ethylene glycol) (PEG) hydrogel for the treatment of type 1 diabetes (T1D). Rat bone marrow‐derived MSCs (rBM‐MSCs) were encapsulated within synthetic PEG hydrogel, and cell viability and apoptosis within this 3D environment was examined in detail. ATP content and caspase‐3 activity of encapsulated MSCs showed that fibronectin‐derived RGDS, laminin‐derived IKVAV and/or insulinotropic glucagon‐like peptide (GLP‐1) were required to maintain MSC survival. Incorporation of these peptides into the hydrogel environment also improved pancreatic islet viability, where combinations of peptides had altered effects on islet survival. GLP‐1 alone was the leading stimulator for insulin secretion. Cell adhesion peptides RGDS and IKVAV improved insulin secretion only when they were used in combination, but could not surpass the effect of GLP‐1. Further, when pancreatic islets were co‐encapsulated with MSCs within synthetic PEG hydrogel, a two‐fold increase in the stimulation index was measured. Synergistic effects of MSCs and peptides were observed, with a seven‐fold increase in the stimulation index. The results are promising and suggest that simultaneous incorporation of MSCs and ECM‐derived peptides and/or GLP‐1 can improve pancreatic islet function in response to altered glucose levels in the physiological environment. Copyright © 2014 John Wiley & Sons, Ltd.     

Control the fate of human umbilical cord mesenchymal stem cells with dual‐enzymatically cross‐linked gelatin hydrogels for potential applications in nerve regeneration

Stem‐cell‐based therapy is a promising strategy to treat challenging neurological diseases, while its application is hindered primarily by the low viability and uncontrolled differentiation of stem cell. Hydrogel can be properly engineered to share similar characteristics with the target tissue, thus promoting cell viability and directing cell differentiation. In this study, we proposed a new dual‐enzymatically cross‐linked and injectable gelatin hydrogel for regulating survival, proliferation, and differentiation of human umbilical cord mesenchymal stem cells (hUC‐MSCs) in a three‐dimensional matrix. This injectable gelatin hydrogel was formed by oxidative coupling of gelatin–hydroxyphenyl acid conjugates catalyzed by hydrogen horseradish peroxidase (HRP) and choline oxidase (ChOx). Modulus and H₂O₂ release can be well controlled by ChOx activity. Results from calcein‐AM/PI staining and Ki67 immunofluorescence tests demonstrated that the survival and proliferation behavior of hUC‐MSCs were highly enhanced in HRP_1UChOx_0.25U hydrogel with lower modulus and less H₂O₂ release compared with other groups. Attractively, the expression of neuron‐specific markers β‐III tubulin, neurofilament light chain (NFL), and synapsin‐1 was significantly increased in HRP_1UChOx_0.25U hydrogel as well. Additionally, in vitro hemolysis test and in vivo HE staining data highlighted the good biocompatibility. Undoubtedly, this injectable gelatin hydrogel's ability to control hUC‐MSCs' fate holds enormous potentials in nervous disorders' therapy and nerve regeneration.