Wharton’s jelly mesenchymal stem cells: Future regenerative medicine for clinical applications in mitigation of radiation injury

Wharton’s jelly mesenchymal stem cells(WJ-MSCs)are gaining significant attention in regenerative medicine for their potential to treat degenerative diseases and mitigate radiation injuries.WJ-MSCs are more naïve and have a better safety profile,making them suitable for both autologous and allogeneic transplantations.This review highlights the regenerative potential of WJ-MSCs and their clinical applications in mitigating various types of radiation injuries.In this review,we will also describe why WJ-MSCs will become one of the most probable stem cells for future regenerative medicine along with a balanced view on their strengths and weaknesses.Finally,the most updated literature related to both preclinical and clinical usage of WJ-MSCs for their potential application in the regeneration of tissues and organs will also be compiled.

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.

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.

Allogeneic mesenchymal stem cells may be a viable treatment modality in cerebral palsy

BACKGROUND Cerebral palsy(CP)describes a group of disorders affecting movement,balance,and posture.Disturbances in motor functions constitute the main body of CP symptoms.These symptoms surface in early childhood and patients are affected for the rest of their lives.Currently,treatment involves various pharmacotherapies for different types of CP,including antiepileptics for epilepsy and Botox A for focal spasticity.However,none of these methods can provide full symptom relief.This has prompted researchers to look for new treatment modalities,one of which is mesenchymal stem cell therapy(MSCT).Despite being a promising tool and offering a wide array of possibilities,mesenchymal stem cells(MSCs)still need to be investigated for their efficacy and safety.AIM To analyze the efficacy and safety of MSCT in CP patients.METHODS Our sample consists of four CP patients who cannot stand or walk without external support.All of these cases received allogeneic MSCT six times as 1×106/kg intrathecally,intravenously,and intramuscularly using umbilical cord-derived MSCs(UC-MSC).We monitored and assessed the patients pre-and post-treatment using the Wee Functional Independence Measure(WeeFIM),Gross Motor Function Classification System(GMFCS),and Manual Ability Classification Scale(MACS)instruments.We utilized the Modified Ashworth Scale(MAS)to measure spasticity.RESULTS We found significant improvements in MAS scores after the intervention on both sides.Two months:Rightχ^(2)=4000,P=0.046,leftχ^(2)=4000,P=0.046;four months:Rightχ^(2)=4000,P=0.046,leftχ^(2)=4000,P=0.046;12 months:Rightχ^(2)=4000,P=0.046,leftχ^(2)=4000,P=0.046.However,there was no significant difference in motor functions based on WeeFIM results(P>0.05).GMFCS and MACS scores differed significantly at 12 months after the intervention(P=0.046,P=0.046).Finally,there was no significant change in cognitive functions(P>0.05).CONCLUSION In light of our findings,we believe that UC-MSC therapy has a positive effect on spasticity,and it partially improves motor functions.

Neural differentiation of human Wharton＇s jelly-derived mesenchymal stem cells improves the recovery of neurological function after transplantation in ischemic stroke rats

Human Wharton＇s jelly-derived mesenchymal stem cells（h WJ-MSCs）have excellent proliferative ability,differentiation ability,low immunogenicity,and can be easily obtained.However,there are few studies on their application in the treatment of ischemic stroke,therefore their therapeutic effect requires further verification.In this study,h WJ-MSCs were transplanted into an ischemic stroke rat model via the tail vein 48 hours after transient middle cerebral artery occlusion.After 4 weeks,neurological functions of the rats implanted with h WJ-MSCs were significantly recovered.Furthermore,many h WJ-MSCs homed to the ischemic frontal cortex whereby they differentiated into neuron-like cells at this region.These results confirm that h WJ-MSCs transplanted into the ischemic stroke rat can differentiate into neuron-like cells to improve rat neurological function and behavior.

Human Wharton’s jelly mesenchymal stem cells inhibit cytokine storm in acute respiratory distress syndrome in a rat model

Objective:To evaluate the potential effect of human Wharton’s jelly mesenchymal stem cells(hWJMSCs)on acute respiratory distress syndrome in lipopolysaccharide(LPS)-induced rats.Methods:The hWJMSCs(5×10^(4)/mL,5×10^(5)/mL,5×10^(6)/mL)were administered to rats on day 1 and day 8 after being induced by LPS(5 mg/kg body weight).TNF-αlevels in the lung and IL-18 and IL-1βlevels in the serum were measured using ELISA.In addition,caspase-1 expression in lung tissues was quantified using qRT-PCR,and NF-κB and IL-6 expressions were assessed using immunohistochemistry.Results:The hWJMSCs decreased TNF-αlevels in the lung and plasma IL-18 and IL-1βlevels.Moreover,the hWJMSCs downregulated the expressions of caspase-1,IL-6,and NF-κB in lung tissues.Conclusions:The hWJMSCs can decrease inflammatory markers of acute respiratory distress syndrome in a rat model and may be further investigated for the treatment of acute respiratory distress syndrome.

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.

Wharton's jelly mesenchymal stem cells differentiate into retinal progenitor cells

Human Wharton＇s jelly mesenchymal stem cells were isolated from fetal umbilical cord. Cells were cultured in serumfree neural stem cellconditioned medium or neural stem cellconditioned medium supplemented with Dkk1, a Wnt/13 catenin pathway antagonist, and LeftyA, a Nodal signaling pathway antagonist to induce differentiation into retinal progenitor cells. Inverted microscopy showed that after induction, the spindleshaped or fibroblastlike Wharton＇s jelly mesenchymal stem cells changed into bulbous cells with numerous processes. Immunofluorescent cytochemical stain ing and reversetranscription PCR showed positive expression of retinal progenitor cell markers, Pax6 and Rx, as well as weakly downregulated nestin expression. These results demonstrate that Wharton＇s jelly mesenchymal stem cells are capable of differentiating into retinal progenitor cells in vitro.

Comparison between the therapeutic effects of differentiated and undifferentiated Wharton’s jelly mesenchymal stem cells in rats with streptozotocin-induced diabetes

BACKGROUND Despite the availability of current therapies,including oral antidiabetic drugs and insulin,for controlling the symptoms caused by high blood glucose,it is difficult to cure diabetes mellitus,especially type 1 diabetes mellitus.AIM Cell therapies using mesenchymal stem cells(MSCs)may be a promising option.However,the therapeutic mechanisms by which MSCs exert their effects,such as whether they can differentiate into insulin-producing cells (IPCs) beforetransplantation, are uncertain.METHODSIn this study, we used three types of differentiation media over 10 d to generateIPCs from human Wharton’s jelly MSCs (hWJ-MSCs). We further transplantedthe undifferentiated hWJ-MSCs and differentiated IPCs derived from them intothe portal vein of rats with streptozotocin-induced diabetes, and recorded thephysiological and pathological changes.RESULTSUsing fluorescent staining and C-peptide enzyme-linked immunoassay, we wereable to successfully induce the differentiation of hWJ-MSCs into IPCs.Transplantation of both IPCs derived from hWJ-MSCs and undifferentiated hWJMSCshad the therapeutic effect of ameliorating blood glucose levels andimproving intraperitoneal glucose tolerance tests. The transplanted IPCs homedto the pancreas and functionally survived for at least 8 wk after transplantation,whereas the undifferentiated hWJ-MSCs were able to improve the insulitis andameliorate the serum inflammatory cytokine in streptozotocin-induced diabeticrats.CONCLUSIONDifferentiated IPCs can significantly improve blood glucose levels in diabetic ratsdue to the continuous secretion of insulin by transplanted cells that survive in theislets of diabetic rats. Transplantation of undifferentiated hWJ-MSCs cansignificantly improve insulitis and re-balance the inflammatory condition indiabetic rats with only a slight improvement in blood glucose levels.

Differentiation of Wharton's jelly mesenchymal stem cells into neurons in alginate scaffold

Alginate scaffold has been considered as an appropriate biomaterial for promoting the differentiation of embryonic stem cells toward neuronal cell lineage. We hypothesized that alginate scaffold is suitable for culturing Wharton’s jelly mesenchymal stem cells（WJMSCs） and can promote the differentiation of WJMSCs into neuron-like cells. In this study, we cultured WJMSCs in a three-dimensional scaffold fabricated by 0.25% alginate and 50 m M Ca Cl2 in the presence of neurogenic medium containing 10 μM retinoic acid and 20 ng/m L basic fibroblast growth factor. These cells were also cultured in conventional two-dimensional culture condition in the presence of neurogenic medium as controls. After 10 days, immunofluorescence staining was performed for detecting β-tubulin（marker for WJMSCs-differentiated neuron） and CD271（motor neuron marker）. β-Tubulin and CD271 expression levels were significantly greater in the WJMSCs cultured in the three-dimensional alginate scaffold than in the conventional two-dimensional culture condition. These findings suggest that three-dimensional alginate scaffold cell culture system can induce neuronal differentiation of WJMSCs effectively.

Human umbilical cord Wharton's Jelly-derived mesenchymal stem cells differentiation into nerve-like cells

Background The two most basic properties of mesenchymal stem cells （MSCs） are the capacities to selfrenew indefinitely and differentiate into multiple cells and tissue types. The cells from human umbilical cord Wharton＇ s Jelly have properties of MSCs and represent a rich source of primitive cells. This study was conducted to explore the possibility of inducing human umbilical cord Wharton＇ s Jelly-derived MSCs to differentiate into nerve-like cells.Methods MSCs were cultured from the Wharton＇ s Jelly taken from human umbilical cord of babies delivered after full-term normal labor. Salvia miltiorrhiza and [3-mercaptoethanol were used to induce the human umbilical cord-derived MSCs to differentiate The expression of neural protein markers was shown by immunocytochemistry. The induction process was monitored by phase contrast microscopy, electron microscopy （EM）, and laser scanning confocal microscopy （LSCM） . The pleiotrophin and nestin genes were measured by reverse transcription-polymerase chain reaction （RT-PCR）.Results MSCs in the Wharton＇ s Jelly were easily attainable and could be maintained and expanded in culture. They were positive for markers of MSCs, but negative for markers of hematopoietic cells and graft-versus-host disease （GVHD）-related cells. Treatment with Salvia mihiorrhiza caused Wharton＇ s Jelly cells to undergo profound morphological changes. The induced MSCs developed rounded cell bodies with multiple neurite-like extensions. Eventually they developed processes that formed networks reminiscent of primary cultures of neurons. Salvia mihiorrhiza and β-mercaptoethanol also induced MSCs to express nestin, β-tubulin Ⅲ, neurofilament （NF） and glial fibrillary acidic protein （GFAP）. It was confirmed by RT-PCR that MSCs could express pleiotrophin both before and after induction by Salvia miltiorrhiza. The expression was markedly enhanced after induction and the nestin gene was also expressed.Conclusions MSCs could be isolated from human umbilical cord Wharton＇ s Jelly. They were capable ofdifferentiating into nerve-like cells using Salvia miltiorrhiza or 15-mercaptoethanol. The induced MSCs not only underwent morphologic changes, but also expressed the neuron-related genes and neuronal cell markers. They may represent an alternative source of stem cells for central nervous system cell transplantation.

“生物药”——Wharton's jelly源间充质干细胞

干细胞治疗代表生物冶疗进入到了一个崭新的时代。间充质干细胞是存在于胚胎或成体组织中来源于中胚层具有多向分化潜能的干细胞。由于成体间充质干细胞的质量与数量自身缺陷,使之应用受到了很大限制。Wharton's jelly组织,是起始于胚胎发育第13天的胚外中胚层组织。使用基因微阵列分析及功能分析,首次发现Wharton's jelly源间充质干细胞(Wharton's jelly derived mesenchymal stem cells,WJMSCs)高表达胚胎早期干性基因及心肌细胞分化早期特异转录因子,可分化心肌细胞等多种细胞。进而,应用临床级WJMSCs经冠状动脉移植治疗ST抬高型急性心肌梗死患者的随机双盲临床试验,首次证明WJMSCs可明显改善心肌活力及心脏功能。因此,WJMSCs具有极其重要益处;无伦理涉及,有强的分化潜能,无致瘤性;加之,WJMSCs可作为产品,在任何时候病情需要时立即应用。为此,WJMSCs作为真正意义上的干细胞族,将最有希望成为具有应用前景的于细胞生物药。

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。

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。

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.

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。

人脐带非酶解法培养MSCs的方法建立

目的建立从人脐带Wharton's jelly组织中直接培养间充质干细胞(MSCs)的方法。方法将人脐带切成5 cm左右的片段,纵向剖开,剔除血管,把富含Wharton's jelly组织的脐带面置于10 cm2塑料培养皿的培养面上,加入含20%FBS的LG-DMEM培养基连续培养10 d;移去脐带组织后,培养皿中的细胞继续培养至80%-90%融合。传代和扩增3代(次)后,以倒置光学显微镜观察细胞形态,流式细胞仪检测细胞免疫表型及细胞周期,用含有成骨细胞诱导剂、脂肪细胞诱导剂对传代培养至第3代的细胞分别定向诱导分化为成骨细胞、脂肪细胞,并对诱导后细胞用碱性磷酸酶检测试剂、Von-Kossa染色检测试剂和茜苏红染色试剂作细胞生物学检测。结果脐带组织在贴壁培养5 d,组织周围可见有少量细胞贴壁生长,主要呈"成纤维样",形状不规则,移去脐带组织后继续培养至14-15 d时,细胞达到80%-90%汇合;细胞表面表达CD73、CD105、CD90、CD44、CD29、CD71及CD13,不表达CD34、CD14、CD133、CD45、HLA-DR;培养至第4代时约72.724%的细胞处在G1期,S期细胞占18.069%,第6代时,G1期细胞约为83.875%、S期细胞仅为9.606%左右。细胞经成骨细胞诱导分化后,碱性磷酸酶染色显示呈强阳性,茜苏红染色和Von-Kossa染色显示细胞有钙盐沉积并形成钙结节;细胞经成脂细胞诱导分化后,油红O染色呈红色,显示胞浆内有大量甘油三酯的聚集。结论采用非酶解法从人脐带Wharton's jelly中分离及培养扩增的细胞具备MSCs的基本特性,具有分化为成骨细胞和脂肪细胞的能力。