Fibroblast growth factor improves the motility of human mesenchymal stem cells expanded in a human plasma‐derived xeno‐free medium through αVβ3 integrin

Human mesenchymal stem cells (MSC) are being explored for cell therapies targeting varied human diseases. For that, cells are being expanded in vitro, many times with fetal bovine serum (FBS) as the main source of growth factors. However, animal‐derived components should not be used, to avoid immune rejection from the patient that receives the MSC. To solve this issue, different xeno‐free media are being developed, and an industrial‐grade human plasma fraction (SCC) is a promising candidate to substitute FBS. Indeed, we have previously shown that MSC expanded in SCC‐medium maintain their phenotype and genetic stability. However, a reduction on MSC motility was observed when comparing with MSC motility on FBS‐medium. Thus, in this present study, we have tested different factors to improve the motility of MSC in SCC‐medium. Time lapse assays and experiments with transwells revealed that supplementation of the xeno‐free medium with FGF or PDGF, but not TNF‐α or SDF‐1, increased MSC motility. Interestingly, FGF and PDGF supplementation also led to alterations on MSC morphology to a shape similar to the one observed when using FBS. The mechanism behind the effect of FGF on MSC motility involved the increased expression of αVβ3 integrin. Furthermore, assays with small molecule inhibitors revealed that the signalling molecule p38 MAPK is important for MSC motility and that MEK/ERK and PI3K/AKT also have a role on FGF‐supplemented expanded MSC. Thus, it was found that FGF supplementation can improve the motility of xeno‐free‐expanded MSC and that the cells motility is regulated by αVβ3 integrin.     

An in vitro expansion score for tissue‐engineering applications with human bone marrow‐derived mesenchymal stem cells

Human bone marrow‐derived mesenchymal stem cells (MSCs) have limited growth potential in vitro and cease to divide due to replicative senescence, which from a tissue‐engineering perspective has practical implications, such as defining the correct starting points for differentiation and transplantation. Time spent in culture before the loss of required differentiation potential is different and reflects patient variability, which is a problem for cell expansion. This study aimed to develop a score set which can be used to quantify the senescent state of MSCs and predict whether cells preserve their ability to differentiate to osteogenic, adipogenic and chondrogenic phenotypes, based on colony‐forming unit (CFU) assay, population doubling time (PDT), senescence‐associated β‐galactosidase (SA‐β‐Gal) activity, cell size, telomere length and gene expression of MSCs cultured in vitro over 11 passages. This set of morphological, physiological and genetic senescence markers was correlated to the ability of MSCs to differentiate. Differentiation efficiency was assessed by marker genes and protein expression. CFUs decreased with increasing passage number, whereas SA‐β‐Gal activity and PDT increased; however, the correlation with MSCs' differentiation potential was sometimes unexpected. The expression of genes related to senescence was higher in late‐passage cells than in early‐passage cells. Early‐passage cells underwent efficient osteogenic differentiation, with mid‐passage cells performing best in chondrogenic differentiation. Late‐passage cells preserve only adipogenic differentiation potential. Based on this marker set, we propose a senescence score in which combined markers give a reliable quality control of MSCs, not depending only on mechanistic passage number. Copyright © 2013 John Wiley & Sons, Ltd.     

Improved expansion of human bone marrow‐derived mesenchymal stem cells in microcarrier‐based suspension culture

Human bone marrow‐derived mesenchymal stem cells (hBM‐MSCs) have potential clinical utility in the treatment of a multitude of ailments and diseases, due to their relative ease of isolation from patients and their capacity to form many cell types. However, hBM‐MSCs are sparse, and can only be isolated in very small quantities, thereby hindering the development of clinical therapies. The use of microcarrier‐based stirred suspension bioreactors to expand stem cell populations offers an approach to overcome this problem. Starting with standard culture protocols commonly reported in the literature, we have successfully developed new protocols that allow for improved expansion of hBM‐MSCs in stirred suspension bioreactors using CultiSpher‐S microcarriers. Cell attachment was facilitated by using intermittent bioreactor agitation, removing fetal bovine serum, modifying the stirring speed and manipulating the medium pH. By manipulating these parameters, we enhanced the cell attachment efficiency in the first 8 h post‐inoculation from 18% (standard protocol) to 72% (improved protocol). Following microcarrier attachment, agitation rate was found to impact cell growth kinetics, whereas feeding had no significant effect. By serially subculturing hBM‐MSCs using the new suspension bioreactor protocols, we managed to obtain cell fold increases of 10³ within 30 days, which was superior to the 200‐fold increase obtained using the standard protocol. The cells were found to retain their defining characteristics after several passages in suspension. This new bioprocess represents a more efficient approach for generating large numbers of hBM‐MSCs in culture, which in turn should facilitate the development of new stem cell‐based therapies. Copyright © 2012 John Wiley & Sons, Ltd.     

Phenotypic and functional comparison of optimum culture conditions for upscaling of bone marrow‐derived mesenchymal stem cells

Human adult bone marrow‐derived mesenchymal stem cells (MSCs) are a promising tool in the newly emerging avenue of regenerative medicine. MSCs have already been translated from basic research to clinical transplantation research. However, there is still a lack of consensus on the ideal method of culturing MSCs. Here we have compared different culture conditions of human MSCs with an attempt to preserve their characteristics and multi‐lineage differentiation potential. We compare the different basal culture media DMEM‐F12, DMEM‐high glucose (DMEM‐HG), DMEM‐low glucose (DMEM‐LG), knock‐out DMEM (DMEM‐KO) and Mesencult^® on the proliferation rate, surface markers and differentiation potentials of MSCs. At every fifth passage until the 25th passage, the differentiation potential and the presence of a panel of surface markers was observed, using flow cytometry. We also compared the characteristics of human MSCs when cultured in reduced concentrations of fetal bovine serum (FBS), knockout serum replacement (KO‐SR) and human plasma. Data indicate that the presence of serum is essential to sustain and propagate MSCs cultures. The choice of basal medium is equally important so as to preserve their characteristics and multipotent properties even after prolonged culture in vitro. With MSCs emerging as a popular tool for regenerative therapies in incurable diseases, it is essential to be able to obtain a large number of MSCs that continue to preserve their characteristics following passaging. The data reveal the optimum basal medium for prolonged culture of MSCs while retaining their ability to differentiate and hence this may be used for up‐scaling to provide sufficient numbers for transplantation. Copyright © 2009 John Wiley & Sons, Ltd.     

Effective expansion of human adipose‐derived stromal cells and bone marrow‐derived mesenchymal stem cells cultured on a fragmin/protamine nanoparticles‐coated substratum with human platelet‐rich plasma

Fragmin/protamine nanoparticles (F/P NPs) can be stably coated onto plastic surfaces and used as a substratum for the absorption and controlled release of growth factors (GFs) secreted from human platelet‐rich plasma (PRP). In this study, we investigated the capability of F/P NP‐coated plates to act as a substratum for the proliferation of human adipose‐derived stromal cells (ASCs) and bone marrow‐derived mesenchymal stem cells (BMSCs) with GFs in PRP. Both cell types adhered well to the F/P NP‐coated plates and grew optimally, with a doubling time of 30 and 32 h in low‐concentration PRP (0.5%) medium supplemented with 5 ng/ml fibroblast growth factor‐2 (FGF‐2) on the F/P NP‐coated plates. These cells maintained their multilineage potential for differentiation into adipocytes or osteoblasts. Furthermore, ASCs and BMSCs grew well in medium without PRP and FGF‐2 on F/P NP‐coated plates pretreated with PRP and FGF‐2 in a concentration‐dependent manner. Thus, F/P NP‐coated plates are a useful substratum for the adherence and proliferation of ASCs and BMSCs in low‐concentration PRP medium supplemented with FGF‐2. No xenogeneic serum is required. Copyright © 2012 John Wiley & Sons, Ltd.     

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.     

间充质干细胞和内皮祖细胞对脐血造血干/祖细胞体外扩增效力的影响

目的比较脐带来源的间充质干细胞(Mesenchymal stem cells,MSCs)和内皮祖细胞(Endothelial progeni-tor cells,EPCs)对脐血造血干/祖细胞(Hematopoietic stem/progenitor cells,HSCs/HPCs)的体外扩增效力。方法正常人脐血中分离单个核细胞(Mononuclear cells,MNCs),流式细胞术检测其中HSCs/HPCs所占比例,将MNCs分别接种于处理后的MSCs或EPCs或仅接种于培养液中,比较不同培养条件对HSCs/HPCs扩增能力、表面抗原CD34的表达以及集落形成能力的影响。结果共培养过程中,MSC组和EPC组的MNCs扩增倍数均明显高于对照组,且EPC组更为显著。扩增7天后,对照组、MSC组和EPC组的HSCs/HPCs CD34的表达均较扩增前下降,其中EPC组下降的最为显著,MSC组最不显著。共培养4天后,MSC组的HSCs/HPCs集落形成总数为EPC组的2.47倍(**,P<0.01),共培养7天后,MSC组的HSCs/HPCs集落形成总数为EPC组的3.45倍(**,P<0.01);EPC组与对照组的HSCs/HPCs集落形成总数无显著性差异。结论 MSC和EPC均可为HSCs/HPCs的体外扩增提供适宜的微环境,MSC可抑制HSCs/HPCs的分化,有助于维持其表面抗原CD34的表达,并保持其造血重建潜能和归巢能力;而EPC则可有效促进HSCs/HPCs的分化。     

Large‐scale expansion of pre‐isolated bone marrow mesenchymal stromal cells in serum‐free conditions

The regenerative potential of mesenchymal stromal or stem cells (MSCs) has generated tremendous interest for treating various degenerative diseases. Regulatory preference is to use a culture medium that is devoid of bovine components for stem cell expansion intended for therapeutic applications. However, a clear choice an alternative to fetal bovine serum (FBS) has not yet emerged. We have screened five different commercially available serum‐free media (SFM) for their ability to support the growth and expansion of pre‐isolated undifferentiated bone marrow‐derived MSCs (BM‐MSCs) and compared the results with cells grown in standard FBS‐containing medium as control. In addition, based on initial screening results, BD Mosaic? Mesenchymal Stem Cell Serum‐free (BD‐SFM) medium was evaluated in large‐scale cultures for the performance and culture characteristics of BM‐MSCs. Of the five different serum‐free media, BD‐SFM enhanced BM‐MSCs growth and expansion in Cell STACK (CS), but the cell yield per CS‐10 was less when compared to the control medium. The characteristics of MSCs were measured in terms of population doubling time (PDT), cell yield and expression of MSC‐specific markers. Significant differences were observed between BD‐SFM and control medium in terms of population doublings (PDs), cell yield, CFU‐F and morphological features, whereas surface phenotype and differentiation potentials were comparable. The BD‐SFM‐cultured MSCs were also found to retain the differentiation potential, immune‐privileged status and immunosuppressive properties inherent to MSCs. Our results suggest that BD‐SFM supports large‐scale expansion of BM‐MSCs for therapeutic use. Copyright © 2014 John Wiley & Sons, Ltd.     

Long‐term in vitro expansion of human adipose‐derived stem cells showed low risk of tumourigenicity

In the field of cell‐based therapy and regenerative medicine, clinical application is the ultimate goal. However, one major concern is: does in vitro manipulation during culture expansion increases tumourigenicity risk on the prepared cells? Therefore, the aim of this study was to investigate the effect of long‐term in vitro expansion on human adipose‐derived stem cells (ASCs). The ASCs were harvested from lipo‐aspirate samples and cultured until passage 20 (P20), using standard culture procedures. ASCs at P5, P10, P15 and P20 were analysed for morphological changes, DNA damage (Comet assay), tumour suppressor gene expression level (quantitative PCR), p53 mutation, telomerase activity, telomere length determination and in vivo tumourigenicity test. Our data showed that ASCs lost their fibroblastic feature in long‐term culture. The population doubling time of ASCs increased with long‐term culture especially at P15 and P20. There was an increase in DNA damage at later passages (P15 and P20). No significant changes were observed in both p53 and p21 genes expression throughout the long‐term culture. There was also no p53 mutation detected and no significant changes were recorded in the relative telomerase activity (RTA) and mean telomere length (TRF) in ASCs at all passages. In vivo implantation of ASCs at P15 and P20 into the nude mice did not result in tumour formation after 4 months. The data showed that ASCs have low risk of tumourigenicity up to P20, with a total population doubling of 42 times. This indicates that adipose tissue should be a safe source of stem cells for cell‐based therapy. Copyright © 2012 John Wiley & Sons, Ltd.     

Integrated culture platform based on a human platelet lysate supplement for the isolation and scalable manufacturing of umbilical cord matrix‐derived mesenchymal stem/stromal cells

Umbilical cord matrix (UCM)‐derived mesenchymal stem/stromal cells (MSCs) are promising therapeutic candidates for regenerative medicine settings. UCM MSCs have advantages over adult cells as these can be obtained through a non‐invasive harvesting procedure and display a higher proliferative capacity. However, the high cell doses required in the clinical setting make large‐scale manufacturing of UCM MSCs mandatory. A commercially available human platelet lysate‐based culture supplement (UltraGRO^TM, AventaCell BioMedical) (5%(v/v)) was tested to effectively isolate UCM MSCs and to expand these cells under (1) static conditions, using planar culture systems and (2) stirred culture using plastic microcarriers in a spinner flask. The MSC‐like cells were isolated from UCM explant cultures after 11 ± 2 days. After five passages in static culture, UCM MSCs retained their immunophenotype and multilineage differentiation potential. The UCM MSCs cultured under static conditions using UltraGRO^TM‐supplemented medium expanded more rapidly compared with UCM MSCs expanded using a previously established protocol. Importantly, UCM MSCs were successfully expanded under dynamic conditions on plastic microcarriers using UltraGRO^TM‐supplemented medium in spinner flasks. Upon an initial 54% cell adhesion to the beads, UCM MSCs expanded by >13‐fold after 5–6 days, maintaining their immunophenotype and multilineage differentiation ability. The present paper reports the establishment of an easily scalable integrated culture platform based on a human platelet lysate supplement for the effective isolation and expansion of UCM MSCs in a xenogeneic‐free microcarrier‐based system. This platform represents an important advance in obtaining safer and clinically meaningful MSC numbers for clinical translation. Copyright © 2016 John Wiley & Sons, Ltd.     

人骨髓间充质干细胞体外培养方法的改良

目的改良人骨髓间充质干细胞(HBMSCs)体外分离培养的方法。方法采用含体积比为10%胎牛血清的α-MEM培养体系,通过密度梯度离心和贴壁筛选法从人骨髓中分离培养贴壁细胞,流式检测该细胞表面标志物,体外诱导成脂、成骨、成软骨分化并鉴定该细胞的多项分化能力,集落克隆形成和MTT试验检测该细胞的活力和增殖能力,并分别与经典的Dexter长期培养体系比较。结果采用改良方法从人骨髓中分离、培养出具有塑料底物粘附性的细胞,目的细胞不表达或低表达CD14、CD34、CD45,高表达CD73、CD90、CD105;体外成功诱导脂肪细胞、骨细胞、软骨细胞分化;Dexter-LTC体系培养的集落个数(为8.0±1.0)个、单个集落细胞数为(59.2±8.2)个,改良法培养的集落个数为(11.3±1.53)个、单个集落中细胞数为(66.5±14.4)个(P<0.05);在Dexter-LTC培养体系下,BMSCs于培养d10进入对数生长期、d19进入平台期,改良法培养BMSCs d4进入对数生长期、d15进入平台期。结论成功建立了改良的HBMSCs体外分离培养体系,且该方法培养的HBMSCs体外扩增速度、增殖能力、克隆形成能力明显改善。     

无血清培养体系原代培养脐带间充质干细胞

背景：以含血清培养基培养的脐带间充质干细胞,存在着进一步应用的障碍。目的：建立无血清培养体系原代培养脐带间充质干细胞。方法：取脐带华通氏胶(Wharton’s Jel y),采用机械法将组织胶切碎,1-3 mm3大小,分别培养到含胎牛血清完全培养液中以及无血清培养液中。培养第11,14,17天收获细胞并进行相关检测。在符合2006年制定的干细胞最低评估标准的基础上,以集落形成单元指标评估何种培养体系能获得较多高质量的原代细胞。结果与结论：无血清培养体系相对于经典的含血清的培养体系而言,细胞生长速度更快。另外,培养第11天,集落形成实验表明无血清培养体系下能获得更多的集落。因此,无血清培养体系可以保持间充质干细胞的特性,为替代含血清培养体系进行脐带间充质干细胞原代培养提供了可能。     

Three‐dimensional polymer scaffolds for enhanced differentiation of human mesenchymal stem cells to hepatocyte‐like cells: a comparative study

Stem cell‐based tissue engineering has emerged as a promising avenue for the treatment of liver diseases and as drug metabolism and toxicity models in drug discovery and development. The in vitro simulation of a micro‐environmental niche for hepatic differentiation remains elusive, due to lack of information about crucial factors for the stem cell niche. For generation of functional hepatocytes, an in vivo three‐dimensional (3D) micro‐environment and architecture should be reproduced. Towards this, we fabricated three scaffolds as dextran–gelatin (DG1), chitosan–hyaluronic acid (CH1) and gelatin–vinyl acetate (GEVAC). Hepatic differentiation of human umbilical cord‐derived mesenchymal stem cells (hUC‐MSCs) was induced by culturing hUC‐MSCs on these scaffolds. The scaffolds support hepatic differentiation by mimicking the native extracellular matrix (ECM) micro‐environment and architecture to facilitate 3D cell–cell and cell–matrix interactions. The expression of hepatic markers, glycogen storage, urea production, albumin secretion and cytochrome P450 (CYP450) activity indicated the hepatic differentiation of hUC‐MSCs. The differentiated hUC‐MSCs on the 3D scaffolds formed hepatospheroids (3D hepatocyte aggregates), as illustrated by scanning electron microscopy (SEM), confocal microscopy and cytoskeleton organization. It was observed that the 3D scaffolds supported improved cell morphology, expression of hepatic markers and metabolic activities, as compared to Matrigel‐coated plates. To the best of our knowledge, this is the first report demonstrating the use of a well‐characterized scaffold (GEVAC) for enhanced differentiation of hUC‐MSCs to hepatocyte‐like cells (HLCs). Copyright © 2016 John Wiley & Sons, Ltd.     

人血小板裂解液替代胎牛血清大规模扩增人脐带间充质干细胞

背景：目前国内外大多数实验仍采用经典的培养基和胎牛血清进行脐带间充质干细胞培养,血清培养潜在的不安全因素限制了未来临床应用的可行性。目的：以人血小板裂解液替代胎牛血清培养、鉴定人间充质干细胞,以期进一步应用于临床低密度扩增人间充质干细胞。方法：人血小板裂解液通过反复冻融、离心、过滤、浓缩等方法制备。以 IMDM 为基础培养基,添加5%浓缩血小板裂解液作为实验组培养基;以添加体积分数10%胎牛血清为对照组培养基。采用酶消化法分离培养人脐带间充质干细胞,以3000/cm2的浓度进行传代培养,待细胞扩增至P5代后,进行细胞形态与直径、免疫表型、成骨成脂分化、克隆形成率等细胞生物学特性检测,并比较两者之间的差别。结果与结论：人血小板裂解液扩增的脐带间充质干细胞形态细长,有更高的细胞累积群倍数;克隆形成检测结果显示两者形成的克隆效率差异无显著性意义,流式细胞术检测结果显示两者有相似的细胞表型,体外诱导分化显示两者都具有成骨、成脂分化能力,但人血小板裂解液扩增的间充质干细胞成骨分化能力更强。提示人血小板裂解液可以取代胎牛血清用于临床低密度扩增人间充质干细胞。     

体外诱导人骨髓间充质干细胞分化为肾小球系膜样细胞

目的:探讨血小板衍生生长因子(PDGF-BB)和全反式维甲酸(ATRA)在体外诱导人骨髓间充质干细胞分化为肾小球系膜样细胞的效果.方法:分离培养人骨髓间充质干细胞,在培养液中添加300 ng/mLPDGF-BB,2μmol/L ATRA进行诱导分化,对分化后的细胞进行鉴定.结果:经过7 d诱导分化后,细胞呈现典型的肾小球系膜细胞形态,波形蛋白基因,结蛋白基因与α-平滑肌肌动蛋白基因及相应的蛋白在分化组均有很强的阳性表达.结论:PDGF-BB和ATRA能够有效地在体外诱导人骨髓间充质干细胞分化为肾小球系膜样细胞.     

Allogenic human serum,a clinical grade serum supplement for promoting human periodontal ligament stem cell expansion

Exposing human periodontal ligament stem cells (hPDLSCs) to animal proteins during cell expansion would compromise quality and safety of the hPDLSCs for clinical applications. The current study aimed to evaluate the replacement of animal‐based serum by human serum for the expansion of hPDLSCs. hPDLSCs were cultured in culture media supplemented with four types of serums: Group A: fetal bovine serum (FBS); Group B: allogeneic human male AB serum (HS); Group C: in‐house autologous (Auto‐HS); and Group D: in‐house allogeneic human serums (Allo‐HS). Exhibitions of mesenchymal stem cell characteristics of hPDLSCs were examined. Then, growth and osteogenic (OS) differentiation potential of hPDLSCs in FBS and HS at passages 5 and 15 were compared to investigate the effects of serum supplements on growth and expansion stability of the expanded hPDLSCs. After that, growth and OS differentiation of hPDLSCs in Auto‐ and Allo‐HS were investigated. Flow cytometrical analyses, functional differentiations, cell growth kinetic, cytogenetic analysis, alkaline phosphatase and calcium content assays, and oil red O and von Kossa staining were performed. Results showed that at passage 5, HS promoted growth and OS differentiation of hPDLSCs and extensive cell expansion, decreased growth and differentiation potential of the expanded hPDLSCs, particularly in HS. Growth and OS differentiation of hPDLSCs in Auto‐HS and Allo‐HS were not different. In summary, allogeneic human serum could be a replacement to FBS for hPDLSC expansion. In vitro cell expansion of hPDLSCs should be minimal to ensure optimal cell quality. Copyright © 2016 John Wiley & Sons, Ltd.     

Human nail bed‐derived decellularized scaffold regulates mesenchymal stem cells for nail plate regeneration

Among hand trauma, nail bed is the most involved tissue in hospital emergency departments, resulting in the loss of nail plate, which leads to a disturbance of hand grasp function, long‐lasting digit tip pain, hyperpathia, and disesthesia. Treatment of nail bed defects is a significant clinical challenge due to the lack of uniform nail bed thickness and distinct regenerative ability. In this study, it is shown that the extracellular matrix of decellularized nail bed scaffolds can play an important role in inducing bone mesenchymal stem cells to differentiate into nail epithelial cells. Using decellularized nail bed scaffolds combined with bone mesenchymal stem cells, it is revealed that the engineered nail bed can promote nude mouse nail plate regeneration ectopically. The natural extracellular matrix of decellularized nail bed scaffolds can serve as a 3D structural template for bone mesenchymal stem cell differentiation into nail‐associated cells, initiating the nail plate regeneration. These results not only provide a proof‐of‐principle for the generation of transplantable nail grafts based on decellularized nail bed scaffolds derived from clinically wasted amputated fingers but also provide important considerations for clinical treatment for digit tip trauma.     

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.     

Co-culturing mesenchymal stem cells from bone marrow and periosteum enhances osteogenesis and neovascularization of tissue-engineered bone

Mesenchymal stem cells (MSCs) isolated from bone marrow and periosteum are often used as cellular sources for bone tissue engineering. This study showed that co‐cultured human bone marrow stem cells (hBMSCs) and periosteal‐derived stem cells (hPCs) resulted in a synergistic effect on osteogenic differentiation both in vitro and in vivo. Compared to hBMSCs and hPCs, co‐culturing MSCs showed abundant mineralization, robust calcium deposition, steadily increasing ALP activity, and upgraded mRNA expression of osteogenic specific genes (COL1A1, BMP‐2, osteopontin, osteocalcin) in vitro. Eight weeks after implantation of cellular β‐TCP scaffolds in immunodeficient mice, similar synergistic effects were confirmed during in vivo evaluation of total new bone formation, mature bone formation, and neovascularization. Based on these findings, the use of co‐cultured hBMSCs and hPCs can be recommended as a promising new approach for bone tissue engineering applications. Copyright © 2011 John Wiley & Sons, Ltd.