Use of hybrid chitosan membranes and human mesenchymal stem cells from the Wharton jelly of umbilical cord for promoting nerve regeneration in an axonotmesis rat model

Many studies have been dedicated to the development of scaffolds for improving post-traumatic nerve regeneration. The goal of this study was to assess the effect on nerve regeneration, associating a hybrid chitosan membrane with non-differentiated human mesenchymal stem cells isolated from Wharton＇s jelly of umbilical cord, in peripheral nerve reconstruction after crush injury. Chromosome analysis on human mesenchymal stem cell line from Wharton＇s jelly was carried out and no structural alterations were found in metaphase. Chitosan membranes were previously tested in vitro, to assess their ability in supporting human mesenchymal stem cell survival, expansion, and differentiation. For the in vivo testing, Sasco Sprague adult rats were divided in 4 groups of 6 or 7 animals each： Group 1, sciatic axonotmesis injury without any other intervention （Group 1-Crush）; Group 2, the axonotmesis lesion of 3 mm was infiltrated with a suspension of 1 250 -1 500 human mesenchymal stem cells （total volume of 50 pL） （Group 2-CrushCell）; Group 3, axonotmesis lesion of 3 mm was enwrapped with a chitosan type Ill membrane covered with a monolayer of non-differentiated human mesenchymal stem cells （Group 3-CrushChitlllCell） and Group 4, axonotmesis lesion of 3 mm was enwrapped with a chitosan type III membrane （Group 4-CrushChiUll）. Motor and sensory functional recovery was evaluated throughout a healing period of 12 weeks using sciatic functional index, static sciatic index, extensor postural thrust, and withdrawal reflex latency. Stereological analysis was carded out on regenerated nerve fibers. Results showed that infiltration of human mesenchymal stem cells, or the combination of chitosan membrane enwrapment and human mesenchymal stem cell enrichment after nerve crush injury provide a slight advantage to post-traumatic nerve regeneration. Results obtained with chitosan type III membrane alone confirmed that they significantly improve post-traumatic axonal regrowth and may represent a very promising clinical tool in peripheral nerve reconstructive surgery. Yet, umbilical cord human mesenchymal stem cells, that can be expanded in culture and induced to form several different types of cells, may prove, in future experiments, to be a new source of cells for cell therapy, including targets such as peripheral nerve and muscle.

WJSC 6^(th) Anniversary Special Issues(2):Mesenchymal stem cells Umbilical cord-derived mesenchymal stem cells:Their advantages and potential clinical utility

Human umbilical cord(UC)is a promising source of mesenchymal stem cells(MSCs).Apart from their prominent advantages,such as a painless collection procedure and faster self-renewal,UC-MSCs have shown the ability to differentiate into three germ layers,to accumulate in damaged tissue or inflamed regions,to promote tissue repair,and to modulate immune response.There are diverse protocols and culture methods for the isolation of MSCs from the various compartments of UC,such as Wharton’s jelly,vein,arteries,UC lining and subamnion and perivascular regions.In this review,we give a brief introduction to various compartments of UC as a source of MSCs and emphasize the potential clinical utility of UC-MSCs for regenerative medicine and immunotherapy.

Human umbilical cord Wharton's jelly-derived oligodendrocyte precursor-like cells for axon and myelin sheath regeneration

Human umbilical mesenchymal stem cells from Wharton＇s jelly of the umbilical cord were induced to differentiate into oligodendrocyte precursor-like cells in vitro. Oligodendrocyte precursor cells were transplanted into contused rat spinal cords. Immunofluorescence double staining indicated that transplanted cells survived in injured spinal cord, and differentiated into mature and immature oligodendrocyte precursor cells. Biotinylated dextran amine tracing results showed that cell transplantation promoted a higher density of the corticospinal tract in the central and caudal parts of the injured spinal cord. Luxol fast blue and toluidine blue staining showed that the volume of residual myelin was significantly increased at 1 and 2 mm rostral and caudal to the lesion epicenter after cell transplantation. Furthermore, immunofluorescence staining verified that the newly regenerated myelin sheath was derived from the central nervous system. Basso, Beattie and Bresnahan testing showed an evident behavioral recovery. These results suggest that human umbilical mesenchymal stem cell-derived oligodendrocyte precursor cells promote the regeneration of spinal axons and myelin sheaths.

Effect of Human Umbilical Cord Mesenchymal Stem Cells on GRP78/ATF4 Pathway in Alzheimer s Disease Model Mice

[Objectives]To study the effect of human umbilical cord mesenchymal stem cells(hUC-MSCs)on GRP78/ATF4 pathway in APP/PS1 mice.[Methods]Twelve 6-month-old female APP/PS1 mice were randomly divided into model group(MOD,n=6)and human umbilical cord mesenchymal stem cell treatment group(MSC,n=6);six 6-month-old C57BL/6N mice were used as control group(CON,n=6).The mice in each group were treated with the fourth generation of human umbilical cord mesenchymal stem cells through tail vein.Four weeks later,the mice in each group were killed.The expression of GFP78 and ATF4 in the cortex of mice in each group was detected by Western blotting and real-time fluorescence quantitative PCR.[Results]The results of immunoblotting and real-time fluorescence quantitative PCR showed that the expression of GRP78 in MOD group was lower than that in CON group and the expression of ATF4 increased.The expression of GRP78 protein in MSC group was higher than that in MOD group,but the expression of ATF4 protein was lower.The results of real-time fluorescence quantitative PCR showed that the mRNA level of GRP78 decreased and the mRNA level of ATF4 increased in MOD group compared with CON group.The mRNA level of GRP78 in MSC group was higher than that in MOD group,while the mRNA level of ATF4 in MSC group was lower than that in MOD group.[Conclusions]Human umbilical cord mesenchymal stem cells can regulate the expression of GRP78/ATF4 pathway in APP/PSI mice,which may be related to the stress level of endoplasmic reticulum in the brain of APP/PS1 mice mediated by human umbilical cord mesenchymal stem cells.

Umbilical cord:an unlimited source of cells differentiable towards dopaminergic neurons

Cell replacement therapy utilizing mesenchymal stem cells as its main resource holds great promise for ultimate treatment of human neurological disorders.Parkinson＇s disease（PD）is a common,chronic neurodegenerative disorder hallmarked by localized degeneration of a specific set of dopaminergic neurons within a midbrain sub-region.The specific cell type and confined location of degenerating neurons make cell replacement therapy ideal for PD treatment since it mainly requires replenishment of lost dopaminergic neurons with fresh and functional ones.Endogenous as well as exogenous cell sources have been identified as candidate targets for cell replacement therapy in PD.In this review,umbilical cord mesenchymal stem cells（UCMSCs）are discussed as they provide an inexpensive unlimited reservoir differentiable towards functional dopaminergic neurons that potentially lead to long-lasting behavioral recovery in PD patients.We also present mi RNAs-mediated neuronal differentiation of UCMSCs.The UCMSCs bear a number of outstanding characteristics including their non-tumorigenic,low-immunogenic properties that make them ideal for cell replacement therapy purposes.Nevertheless,more investigations as well as controlled clinical trials are required to thoroughly confirm the efficacy of UCMSCs for therapeutic medical-grade applications in PD.

Editor's Choice——Umbilical cord blood mesenchymal stem cell differentiation and transplantation in neural regeneration

Previous in vivo experiments have shown that human umbilical cord blood mesenchymal stem cells can promote the proliferation and differentiation of damaged celts, and help to repair damaged sites, Recent studies have reported that umbilical cord blood-derived mesenchymal stem cells can differentiate into neurons and glial cells. Recent studies have reported that the repair mechanisms underlying cord blood stern cells involve the replacement of damaged cells and mediation of the local micro-environment.

Editor's Choice--Umbilical cord mesenchymal stem cell transplantation for the treatment of peripheral nerve injury

Schwann cells are the predominant seed cells for cell transplantation in the treatment of peripheral nerve injury. However, the source of Schwann cells is limited and amplification remains difficult. Studies have shown that mesenchymal stem cells, an alternative cell type, can be used for transplantation treatment of peripheral nerve defects. Umbilical cord mesenchymal stem cells are pluripotent stem cells derived from newborn umbilical cord tissues.

Mesenchymal stromal cell therapy for damaged retinal ganglion cells, is gold all that glitters?

Mesenchymal stromal cells are an excellent source of stem cells because they are isolated from adult tissues or perinatal derivatives, avoiding the ethical concerns that encumber embryonic stem cells. In preclinical models, it has been shown that mesenchymal stromal cells have neuroprotective and immunomodulatory properties, both of which are ideal for central nervous system treatment and repair. Here we will review the current literature on mesenchymal stromal cells, focusing on bone marrow mesenchymal stromal cells, adipose-derived mesenchymal stromal cells and mesenchymal stromal cells from the umbilical cord stroma, i.e.,Wharton’s jelly mesenchymal stromal cells. Finally, we will discuss the use of these cells to alleviate retinal ganglion cell degeneration following axonal trauma.

Methods of isolation, expansion, differentiating induction and preservation of human umbilical cord mesenchymal stem cells

Objective This literature review aims to summarize the methods of isolation, expansion, preservation of human umbilical cord mesenchymal stem cells （hUCMSCs）, for comprehensive practical use in preclinical research and clinical trials. differentiation and understanding and Data sources All the literature reviewed was published over the last 10 years and is listed in PubMed and Chinese National Knowledge Infrastructure （CNKI）. Studies were retrieved using the key word ＂human umbilical cord mesenchymal stem cells＂. Results Explants culture and enzymatic digestion are two methods to isolate hUCMSCs from WJ and there are modifications to improve these methods. Culture conditions may affect the expansion and differentiating orientations of hUCMSCs. In addition, hUCMSCs can maintain their multi-potential effects after being properly frozen and thawed. Conclusion Considering their multi-potential, convenient and non-invasive accessibility, low immunogenicity and the reported therapeutic effects in several different preclinical animal models, hUCMSCs have immense scope in regeneration medicine as a substitute for MSCs derived from bone marrow or umbilical cord blood.

人脐带Wharton’s jelly间充质干细胞生物学特性的研究

目的探讨人脐带Wharton’s jelly间充质干细胞的培养方法及生物学特性。方法将脐带Wharton’s jelly剪成约0.5 mm3种植到培养瓶,3 h后添加培养液。细胞达90%融合时用胰酶消化传代。用免疫组化检测波形蛋白的表达;用流式检测细胞膜表型;用诱导液检测其分化为脂肪样细胞和神经样细胞的潜能。结果培养6 d左右,细胞从组织块游出,为长梭形的成纤维样细胞,传代后细胞增殖加快,呈放射状分布。间充质干细胞标记物CD90等为阳性,而CD34等为阴性;波形蛋白呈强阳性。成脂诱导后,细胞质出现脂滴,油红O染色为阳性。经黄芪诱导24 h,细胞表达Nestin和NF,而NSE和GFAP为阴性。结论人脐带Wharton’s jelly中有间充质干细胞,且有多向分化潜能。

人脐带Wharton’s Jelly来源间充质干细胞与人牙周膜干细胞成骨分化能力的对比研究

目的:比较人脐带Wharton’s Jelly来源间充质干细胞(human umbilical cord Wharton’s Jelly-derived mesechymal stem cells,h UCWJMSCs)与人牙周膜干细胞(periodontal mesenchymal stem cells,h PDLSCs)成骨分化能力。方法:体外培养h UCWJMSCs和h PDLSCs。MTT法检测细胞增殖情况;成骨诱导后测定细胞的ALP活性,茜素红染色检测细胞矿化能力,Real-time PCR分析OPN和Runx2基因的表达。结果:hUCWJMSCs增殖能力高于hPDLSCs;经矿化诱导后hPDLSCsALP表达、矿化结节形成高于hUCWJMSCs(P<0.05);Runx2在hPDLSCs中表达高于hUCWJMSCs(P<0.05);而hUCWJMSCs中OPN表达高于hPDLSCs(P<0.05)。结论:h UCWJMSCs、hPDLSCs均具有成骨分化能力,hPDLSCs成骨分化能力较强。

人脐带Wharton's Jelly来源间充质干细胞和人牙周膜干细胞复合PHBHHx、PHBV支架成骨分化能力的体外研究

目的:探讨人脐带Wharton's Jelly来源间充质干细胞(h UCWJMSCs)和人牙周膜干细胞(h PDLSCs)复合聚(3-羟基丁酸酯-3-羟基己酸酯)(PHBHHx)、聚羟基丁酸-羟基戊酸酯(PHBV)支架成骨分化的能力。方法:将2种干细胞分别接种在2种支架中,MTT法及粘附率分析2种干细胞和PHBHHx、PHBV的生物相容性;成骨诱导后ALP活性检测、茜素红染色和扫描电镜观察研究两种干细胞在两种支架上的成骨分化情况。结果:2种干细胞与PHBV具有更好的黏附效果及增殖效率(P <0. 05),且h UCWJMSCs组优于h PDLSCs组; h UCWJMSCs和h PDLSCs与PHBV组ALP活性检测、茜素红染色阳性表达明显高于与PHBHHx组; h UCWJMSCs与2种支架组的表达高于h PDLSCs组。扫描电镜显示2种干细胞与PHBV组可见大量矿化基质形成。结论:h UCWJMSCs与PHBV生物相容性和成骨能力优于h PDLSCs组。

Effect of hUC-MSCs on the NLRP3/Caspase-1 Pathway in APP/PS1 Mice

[Objectives] To investigate the effect of human umbilical cord mesenchymal stem cells (hUC-MSCs) on the NOD-like receptor protein 3 (NLRP3)/cysteinyl aspartate specific proteinase (Caspase-1) pathway within the cerebral cortex of a mouse model of Alzheimer s disease (AD).[Methods] Twelve 6-month-old female APP/PS1 mice were randomly assigned to two groups: the model group (MOD, n =6) and the hUC-MSCs treatment group (MSC, n =6). Six 6-month-old C57BL/6N mice were utilized as a control group (CON, n =6). All mice underwent caudal vein injections of hUC-MSCs. Following a 4-week treatment, the mice from each group were euthanized. The expression levels of NLRP3, Caspase-1 protein, and mRNA in the cerebral cortex of each group were assessed using Western blotting and real-time fluorescence quantitative PCR assays.[Results] The results of immunoblotting showed that the expression levels of NLRP3 and Caspase-1 proteins in the MOD group were significantly higher than those observed in the CON group. Furthermore, the expression levels of NLRP3 and Caspase-1 proteins in the MSC group were found to be lower than those in the MOD group. Additionally, the findings from real-time fluorescence quantitative PCR assay demonstrated that the mRNA levels of NLRP3 and Caspase-1 in the MOD group were elevated compared to the CON group. Conversely, the mRNA levels of NLRP3 and Caspase-1 in the MSC group were reduced in comparison to the MOD group.[Conclusions] hUC-MSCs have the capacity to modulate the expression of the NLRP3/Caspase-1 pathway within the cerebral cortex of APP/PS1 mice. This modulation may be associated with the neuroinflammatory processes mediated by hUC-MSCs in the brains of APP/PS1 mice.

人WJ-MHCs移植对心肌梗死后心力衰竭大鼠TNF-α及NT-proBNP的影响

目的观察人华通胶间充质干细胞(WJ-MHCs)对心肌梗死后心力衰竭大鼠肿瘤坏死因子α(TNF-α)及N末端B型脑利钠肽前体(NT-proBNP)的影响。方法采用80只雄性SD大鼠通过异丙肾上腺素多点皮下注射,剂量为200mg/kg,隔24h注射1次共2次。待大鼠存活1周建立模型后各取12只随机均衡分入WJ-MHCs移植组、普通对照组、空白对照组各12只健康大鼠,3组再分为移植前和移植后4周两个亚组。WJ-MHCs移植组于心肌梗死后1周移植以DAPI标记的WJ-MHCs,空白对照组及普通对照组不做处理正常饲养。分别于移植前和移植后4周检测大鼠心脏组织中TNF-α水平,移植后4周WJ-MHCs细胞在大鼠心脏中的情况,大鼠TNF-α和NT-proBNP的水平,左室射血分数(LVEF)。结果移植后WJ-MHCs移植组较移植前LVEF明显提高(P<0.05),较移植前及普通对照组血清TNF-α及NT-proBNP明显降低(P<0.05);移植后WJ-MHCs移植组较普通对照组心脏组织中TNF-α表达明显减少;WJ-MHCs移植组病死率(16.67%)较普通对照组病死率(33.33%)明显降低;免疫荧光显示移植后4周仍可检测到移植的WJ-MHCs。结论移植人WJ-MHCs可明显降低心梗后心衰大鼠心脏组织及循环中的TNF-α,降低循环中的NT-proBNP,提高心功能。

脓毒症大鼠来源的外泌体对WJ-MSC的免疫调控能力的影响

目的探究脓毒症大鼠外周血来源外泌体对人脐带华通胶来源间充质干细胞(WJ-MSC)免疫调控能力的影响。方法采用盲肠穿孔结扎(CLP)方法建立脓毒症大鼠模型,分别提取对照组(假手术组)和实验组(CLP模型组)大鼠外周血的外泌体,鉴定其表面标志物及形态特征,并分别处理WJ-MSC,CCK8检测WJ-MSC的细胞活性,Annexin-VFITC/PI检测凋亡细胞比例,q PCR检测细胞因子(IL-10、TNF-α)的mRNA水平;分别将两组外泌体处理后的WJ-MSC与LPS刺激后的人单核巨噬细胞(THP-1)共培养,q PCR检测THP-1细胞中细胞因子(IL-6、IL-10、TNF-α、IL-1β)的mRNA水平变化。结果两组外泌体在表面标志物和形态特征上无明显差异;实验组外泌体不影响WJ-MSC的增殖活性,但可抑制其凋亡,同时能够上调IL-10的mRNA表达,下调TNF-α的mRNA表达;经过两组外泌体处理后的WJ-MSC,均能够降低LPS刺激下THP-1细胞中促炎性细胞因子(IL-1β,TNF-α)的表达,两组无明显差异。结论脓毒症大鼠外周血来源的外泌体能够上调WJ-MSC中IL-10的表达,下调TNF-α的表达,可增强其免疫调控能力。

具有抗氧化能力的人脐带间充质干细胞制备及评价策略研究

目的:从人脐带中分离制备一种具有抗氧化能力的间充质干细胞(antioxidant mesenchymal stem cell,AO-MSC)并评价其细胞生物学特性。方法:采用无血清培养体系,结合胶原酶消化培养法以及胶原酶消化组织块培养法,从人脐带华通氏胶组织分离培养MSC。使用0.2%Ⅱ型胶原酶消化,未完全消化的组织块贴壁培养收获AO-MSC。以常规消化培养法为对照。成纤维细胞集落形成实验检测MSC集落形成能力;CCK-8检测MSC增殖能力;流式细胞术及免疫荧光染色检测MSC表面标志物;体外诱导分化评价MSC成脂成骨能力,实时荧光定量PCR(RT-qPCR)检测成骨和成脂关键转录因子的表达差异;RT-qPCR检测MSC抗氧化还原物质SOD-1、GSH、GAT、NQO1的表达情况。结果:本策略分离的AO-MSC在18 d汇合率为80%-90%,细胞呈漩涡状生长。流式细胞术及免疫荧光染色检测结果显示,AO-MSC高表达CD73、CD29、CD105、CD90,低表达CD31、CD45、HLA-DR;胶原酶消化组织块培养法收获的AO-MSC自我更新及分化能力强于对照组MSC;对照组MSC体外成脂成骨能力强于胶原酶消化组织块培养法所得AO-MSC;RT-qPCR检测结果显示,胶原酶消化组织块培养法收获的AO-MSC其表达的抗氧化还原物质水平高于对照组。结论:成功制备了基于无血清体系的具备抗氧化能力的人脐带MSC。

Multipotent mesenchymal stromal cells are fully permissive for human cytomegalovirus infection

Congenital human cytomegalovirus(HCMV) infection is a leading infectious cause of birth defects.Previous studies have reported birth defects with multiple organ maldevelopment in congenital HCMV-infected neonates. Multipotent mesenchymal stromal cells(MSCs) are a group of stem/progenitor cells that are multi-potent and can self-renew, and they play a vital role in multiorgan formation. Whether MSCs are susceptible to HCMV infection is unclear. In this study, MSCs were isolated from Wharton's jelly of the human umbilical cord and identified by their plastic adherence, surface marker pattern, and differentiation capacity. Then, the MSCs were infected with the HCMV Towne strain, and infection status was assessed via determination of viral entry,replication initiation, viral protein expression, and infectious virion release using western blotting,immunofluorescence assays, and plaque forming assays. The results indicate that the isolated MSCs were fully permissive for HCMV infection and provide a preliminary basis for understanding the pathogenesis of HCMV infection in non-nervous system diseases, including multi-organ malformation during fetal development.

脐带华通胶间充质干细胞的分离培养及鉴定

目的探讨人脐带华通胶间充质干细胞体外分离、培养方法,并通过成脂成骨诱导及表面标记物的检测进行鉴定。方法剔除脐带动脉、静脉和外膜,取遗留的华通胶组织,将其剪成细小的碎块,用浓度为0.2%的Ⅱ型胶原酶消化,提取脐带华通胶间充质干细胞,流式细胞仪检测细胞表面抗原CD44、CD90、CD105、CD73、HLA-ABC、CD34、CD45及HLA-DR,成骨细胞、成脂细胞分化诱导。结果脐带华通胶经胶原酶消化后,可提取大量间充质干细胞,流式细胞仪检测脐带华通胶干细胞不表达造血干细胞的表面特征,而表达间充质干细胞的标志,例如CD44、CD90、CD105及CD73,并且干细胞HLA-ABC阳性表达,CD34、CD45及HLA-DR呈阴性。组织化学染色显示茜素红、碱性磷酸酶及油红染色强阳性。结论该方法可较好地分离脐带华通胶间充质干细胞,将为实验研究和临床应用提供一种新的干细胞来源。

脐带Wharton胶来源MSCs生物学特性及其优越性的研究进展

目的总结脐带Wharton胶来源MSCs(Wharton’s jelly-MSCs,WJ-MSCs)生物学特性及其优越性的研究进展。方法查阅近年来关于WJ-MSCs的生物学特性及其与脐带血MSCs(umbilical cord blood MSCs,UBMSCs)和BMSCs相比较的文献,进行综述分析。结果从脐带Wharton胶中可分离获得大量具有自我复制、自我更新、高度增殖和多向分化潜能的MSCs。与UBMSCs和BMSCs相比较,WJ-MSCs在分离时间、分离成功率、倍增时间、传代数量和扩增潜能等方面均具有优越性。结论 WJ-MSCs具有取材简便、来源丰富、相对纯净、无伦理问题等优点,是细胞移植治疗、基因治疗和组织工程器官构建的理想种子细胞,为组织再生修复与原位重建提供了新思路。

人脐带华通胶间充质干细胞的体外分离培养及鉴定

目的研究由脐带华通胶组织(Wharton’s jelly)在体外分离、培养、扩增获得脐带间充质干细胞(umbilical cord derived mesenchymal stem cells,以下简称UC-MSCs)的方法,并进行UC-MSCs的各项鉴定。方法脐带华通胶组织采用胶原酶以及胰酶序贯消化法分离得到UC-MSCs,用流式细胞仪分析其表型特征,向成骨、成脂以及成神经元细胞诱导分化并鉴定。结果由人脐带华通胶可以有效获得间充质干细胞。原代细胞培养24～48h内细胞开始贴壁生长,5～7d左右可以传代培养,在2～3周时间内可以迅速扩增至107到108数量级。各项分化鉴定试验证实UC-MSCs具有多向分化潜能,能分化成骨、成脂细胞以及神经元细胞。UC-MSCs在体外培养传至20～30代仍保持稳定的细胞表面标记。结论由人脐带华通胶可以有效地获得间充质干细胞,这种UC-MSCs能在体外长期传代培养,生物学特性稳定,具有多向分化的潜能,是今后细胞治疗很有前景的种子细胞。