Effects of encapsulated rabbit mesenchymal stem cells on ex vivo expansion of human umbilical cord blood hematopoietic stem/progenitor cells

The expansion of umbilical cord blood mononuclear cells (UCB MNCs) was investigated in a novel co-culture system by means of encapsulation of rabbit bone marrow (BM) mesenchymal stem cells (MSCs) in alginate beads (Alg beads). Three kinds of media were applied and the experiments lasted for 7 days. The total nucleated cell density was measured every 24 h. Flow cytometric assay for CD34⁺ cells and methylcellulose colony assays were carried out at 0, 72 and 168 h. It was found that the encapsulated MSCs illustrated remarkable effects on UCB MNCs expansion regardless of whether serum is present in culture media or not. At the end of 168 h co-culture, the total nucleated cell number was multiplied by 15 ± 2.9 times, and CD34⁺ cells 5.3 ± 0.3 times and colony-forming units in culture (CFU-Cs) 5.6 ± 1.2 times in the serum-free media supplemented with conventional dose of cytokines, which was very similar to the results in the containing 20% serum media. While in the control, i.e. MNC expansion without encapsulated MSCs, however, total nucleated cells density changed mildly, CD34⁺ cells and CFU-Cs showed little effective expansion. It is demonstrated that the encapsulated stromal cells can support the expansion of UCB MNCs effectively under the experimental condition.     

人骨髓间充质干细胞联合细胞因子的无血清培养体系体外扩增脐血干细胞研究

目的 探讨以人骨髓间充质干细胞(BM-MSC)为基质的无血清培养方法,体外扩增脐血造血干细胞(CBSC),研究BM-MSC支持造血的功能和体外扩增时间的选择。方法 用含血小板生成素(TPO)、干细胞因子(SCF)、flt3／flk2配体(FL)和粒细胞-集落刺激因子(G-CSF)的无血清培养液,以人BM-MSC为基质,比较体外扩增脐血CD34^ 细胞7d及14d后总细胞(TC)、CD34^ 细胞和集落形成单位(CFU)数增加倍数。结果在以人BM-MSC为基质及TPO、FL、SCF和G-CSF这四种细胞因子作用下,经体外扩增7d后TC、CD34^ 细胞、CFU-GM和CFU-C数较起始分别增加了87、16、15和26倍;14d后较起始分别增加了427、38、125和104倍,与7d扩增倍数有显著性差异。结论以人BM-MSC为基质的无血清体外培养体系可以有效扩增CBSC,有潜在的临床应用价值,扩增时间应以14d为宜。     

转HOXB4基因人骨髓MSC促进脐血CD34~+细胞体外扩增

目的 研究转HOXB4基因的人骨髓间充质干细胞(MSC)对脐血CD34~+细胞体外扩增的支持作用.方法 用病毒载体质粒转染包装细胞293T,收获病毒上清(VCM)感染MSC后,用潮霉素筛选获得转HOXB4的MSC(MSC-HOX).分别将MSC(对照组)与MSC-HOX细胞(实验组)作为CD34~+细胞体外扩增的饲养层细胞,结合细胞因子Fh3/Flk3配体(FL)、促血小板生成素(TPO)、干细胞因子(SCF)和粒细胞-集落生长因子(G-CSF)体外扩增脐血CD34~+细胞10 d,收集所有脐血细胞,检测细胞总数、CD34~+细胞总数,集落细胞形成单位(CFU)数.结果 生产重组逆转录病毒可有效将HOXB4基因转入靶细胞内并表达.脐血CD34~+细胞经体外扩增10 d后,细胞总数和CFU数两组间差异无统计学意义(P>0.05),脐血CD34~+细胞总数和比例高于对照组(P<0.05).结论 转HOXB4基因的人骨髓MSC在脐血CD34~+细胞体外扩增中,可保持CD34~+细胞未分化状态,有潜在的应用价值.     

人脐血源性间充质干细胞的体外培养及生物学鉴定

目的 建立体外培养、扩增人脐带血间充质干细胞(MSCs)的方法．初步鉴定其生物学特性。方法 无菌条件下取正常足月剖宫产的脐带血,用淋巴细胞分离液分离脐血的单个核细胞．以偏酸性的Mesencult^TM作为培养基进行培养和纯化获得贴壁细咆层,测定MSCs的生长曲线．用流式细胞技术分析细胞的表面抗原。结果 来源于脐血的单个核细胞存体外用合适的培养基培养,细胞贴壁后出现破骨样及间充质样的细胞。间充质细胞为成纤维样的细胞形态,并表达MSCs相关的抗原CD29、CD44、CD105。但不表达CD34、CD45、CD106和HLA-DR。这与源于骨髓的MSCs一致。结论 脐带血中含有的MSCs可在体外培养、扩增,能够为实验和临床的应用提供一种新的间充质干细胞来源。     

Comparison of immunomodulatory effects of placenta mesenchymal stem cells with bone marrow and adipose mesenchymal stem cells

Mesenchymal stem cells (MSCs) are powerful sources for cell therapy in regenerative medicine because they can be isolated from various tissues, expanded, and induced into multiple-lineages. Of note, their immunomodulatory effects maximize the therapeutic effects of stem cells engrafted on host, making them an especially attractive choice. Recently, several varieties of placenta-derived stem cells (PDSCs) including chorionic plate-derived MSCs (CP-MSCs) have been suggested as alternative sources of stem cells. However, comparative studies of immunomodulatory effects for CP-MSCs among various MSCs are largely lacking. We examined and compared immunomodulatory function of CP-MSCs with that of BM-MSCs and AD-MSCs using co-culture system with activated T-cells derived from human umbilical cord blood (UCB) exposed to anti-CD3 and anti-CD28 which are T-cell activating monoclonal antibodies. All MSCs expressed markers of stem cells and three germ layers by RT-PCR. These cells also exhibited comparable immunomodulatory effects when they were co-cultured with activated T-cells in dose-dependent manner. However, expression of HLA-ABC and HLA-G was highly positive in CP-MSCs compared to other MSCs, and higher levels of cytokines of IL-2, IL-4, IL-13, and GM-CSF were detected in dose-dependent manner in CP-MSCs. Taken together, the results of the present study suggest that while CP-MSCs, BM-MSCs, and AD-MSCs all have immunomodulatory effects, CP-MSCs may have additional advantage over the other MSCs in terms of immunomodulation. In conjunction with other previous studies, CP-MSCs are suggested to be a useful stem cell source in cell therapy.     

Bone-derived MSCs encapsulated in alginate hydrogel prevent collagen-induced arthritis in mice through the activation of adenosine A2A/2B receptors in tolerogenic dendritic cells

Tolerogenic dendritic cells (tolDCs) facilitate the suppression of autoimmune responses by differentiating regulatory T cells (Treg). The dysfunction of immunotolerance results in the development of autoimmune diseases, such as rheumatoid arthritis (RA). As multipotent progenitor cells, mesenchymal stem cells (MSCs), can regulate dendritic cells (DCs) to restore their immunosuppressive function and prevent disease development. However, the underlying mechanisms of MSCs in regulating DCs still need to be better defined. Simultaneously, the delivery system for MSCs also influences their function. Herein, MSCs are encapsulated in alginate hydrogel to improve cell survival and retention in situ, maximizing efficacy in vivo. The three-dimensional co-culture of encapsulated MSCs with DCs demonstrates that MSCs can inhibit the maturation of DCs and the secretion of pro-inflammatory cytokines. In the collagen-induced arthritis (CIA) mice model, alginate hydrogel encapsulated MSCs induce a significantly higher expression of CD39⁺CD73⁺ on MSCs. These enzymes hydrolyze ATP to adenosine and activate A_2A/2B receptors on immature DCs, further promoting the phenotypic transformation of DCs to tolDCs and regulating naïve T cells to Tregs. Therefore, encapsulated MSCs obviously alleviate the inflammatory response and prevent CIA progression. This finding clarifies the mechanism of MSCs-DCs crosstalk in eliciting the immunosuppression effect and provides insights into hydrogel-promoted stem cell therapy for autoimmune diseases.     

Nanofiber technology in the ex vivo expansion of cord blood-derived hematopoietic stem cells

Umbilical cord blood (CB) can be used as an alternative source of hematopoietic stem cells (HSCs) for transplantation in hematological and non-hematological disorders. Despite several recognized advantages the limited cell number in CB one unit still restricts its clinical use. The success of transplantation greatly depends on the levels of total nucleated cell and CD34+ cell counts. Thus, many ex vivo strategies have been developed within the last decade in order to solve this obstacle, with more or less success, mainly determined by the degree of difficulty related with maintaining HSCs self-renewal and stemness properties after long-term expansion. Different research groups have developed very promising and diverse CB-derived HSC expansion strategies using nanofiber scaffolds. Here we review the state-of-the-art of nanofiber technology-based CB-derived HSC expansion.     

多种中药成分诱导大鼠骨髓间质干细胞转变为神经元样细胞

目的　体外定向诱导大鼠骨髓间质干细胞 (MSCs)分化为神经元样细胞。方法　通过贴壁法分离大鼠MSCs,体外扩增培养 ,流式细胞仪检测其表面抗原表达 ,中药成分定向诱导MSCs分化为神经元样细胞。光镜下观察细胞形态 ,免疫细胞化学法检测神经细胞特异性抗原标志。结果　大鼠MSCs可通过贴壁法成功分离并可在体外大量扩增。流式细胞仪检测结果显示CD1 4、CD1 1α、CD34、CD38、CD45、CD80、CD86为阴性 ,CD2 9、CD44、CD90、CD1 0 5、CD1 66呈阳性。黄芪、天麻、人参、当归、脑新舒、人参蜂王浆等多种中药诱导 1～ 3h后大部分MSCs转变为神经元样细胞 ,出现胞体和突起 ,免疫细胞化学染色神经元特异性烯醇酶 (NSE)、巢蛋白 (nestin)呈阳性 ,胶质纤维酸性蛋白 (GFAP)阴性。结论　多种传统中药成分及中药制剂体外能诱导大鼠MSCs分化为神经元样细胞     

Optimizing donor selection in order to establish a cord blood banking facility: maternal and obstetric factors impact

In this study, we analyzed the obstetric factors affecting total nucleated cells (TNC) content of cord blood units to establish the criteria for umbilical cord blood (UCB) donor selection in our geographic area. UCB was collected from normal uncomplicated pregnancies. In every case, following data were recorded: (1) gestation length; (2) type of delivery (cesarean or vaginal); and (3) newborn characteristics: weight and sex. For each sample, TNC content, percentage and number of CD34+ cells, and viability were analyzed. The results showed that TNC content increases with cord blood volume, gestational length and newborn weight. The mean blood volume and the mean TNC per unit were 42.37 ± 13.5 ml and 55.49 ± 19.4 × 10⁷, respectively. Stepwise regression analysis revealed a positive and significant correlation (r= 0.89) between these two variables. Meanwhile the CD34+ cell content remains unchanged in deliveries at 32–40 weeks of gestation. The mean CD34+ percentage obtained was 0.37 ± 0.06, and the total number of CD34+ cells was 4.827 ± 0.8204 × 10⁴ / mL UCB. Concluding, the maternal and obstetric factors have a significant impact on UCB cell quantity and quality. The main criteria for UCB collection and storage resulted to be: a gestational age higher than 36–40 weeks and newborn weight > 3200g; gestation number ≤ 2 and placental weight > 700g can be added to the standard criteria to improve the bank efficiency. Our results have also become helpful in evaluating stored UCB units to establish the adequacy for clinical transplant utilization.     

基于转录组数据探讨不同组织来源的间充质干细胞的生物学异质性

目的 基于转录组数据探讨脂肪组织（AD）、胎盘绒毛膜（HC）、胎盘羊膜（HA）和脐带（UC）来源的间充质干细胞 （MSCs） 的生物学异质性。方法 从人 AD、HC、HA 和 UC 中分离 MSCs,流式细胞术检测细胞表面阳性标志物（CD73、CD90、CD105）和阴性标志物（CD14、CD34、CD45、CD79a、HLA-DR）的表达,改良版茜素红染色、油红O 染色、阿利辛蓝染色检测细胞的三系分化能力;Trizol 法提取细胞总 RNA,用于转录组测序,应用 GFOLD（1.1.4）进行差异基因表达分析;使用 DAVID 数据库对差异表达的基因进行基因本体（GO）功能富集分析。结果 P2 代的不同来源的 MSCs,CD73、CD90、CD105 均为阳性表达,CD14、CD34、CD45、CD79a、HLA-DR 均为阴性表达,培养的 MSCs 均具有三系分化能力。新生儿来源的 MSCs（HA、HC 和 UC）相关性大于成人来源的 MSCs（AD）,在功能富集分析中,与来源于 AD 的MSCs 相比,HA 和 UC 来源的 MSCs 表现出更优异的增殖能力。来源于 AD 的 MSCs 有更好的分化潜力以及促进血管生成能力,而来源于 UC 的 MSCs 支持神经元的发育并分泌可以调节免疫环境的趋化因子和抗炎因子。结论 不同来源的 MSCs 具有不同的生物学特征,提示不同来源的 MSCs 可能具有不同临床应用的最佳选择。     

骨髓间充质干细胞对与平滑肌细胞共培养体系中细胞增殖和自身分化的影响

目的探讨骨髓间充质干细胞（marrow mesenchymal stem cells,MSCs）对联合培养细胞增殖及自身表达平滑肌肌动蛋白（smooth muscle alpha—aetin,α-actin SM）的影响。方法建立MSCs与平滑肌细胞（smooth muscle cells,SMCs）直接共培养体系,对MSCs与SMCs直接共培养组和单纯SMCs组及MSCs组在不同时间点细胞计数。对共培养组和单纯MSCs组行4’6’－二乙酰基－2－苯基吲哚（DAPI）（5ng／ml）标记,利用抗α—actin SM荧光抗体,检测2组MSCs胞浆内-aetin SM表达。结果共培养组细胞计数较单独SMCs培养组明显减少,以第4天为著（P〈0．01）,与SMCs共培养5d,MSCs在共培养组较单纯MSCs组表达α-actin SM阳性率明显降低（P〈0．05）。结论在无干预因素情况下,MSCs与SMCs直接共培养明显抑制共培养体系中细胞增殖,且抑制MSCs表达仪-α ctin SM。     

Effects of human umbilical cord-derived mesenchymal stem cells on anterior chamber-associated immune deviation

Introduction of antigen into anterior chamber (AC) induces a deviant immune response termed anterior chamber-associated immune deviation (ACAID) that protects the eye from inflammatory destruction consequent to a systemic immune response. Mesenchymal stem cells (MSCs) can modulate a variety of immune responses. However, the effects of systemic administration of MSCs on ACAID have not been explored. In this study, C57BL/6 mice were inoculated with ovalbumin in the AC to induce ACAID, control group received AC injection of solvent alone. Immediately after the AC injection, the mice were injected through the tail vein with human Umbilical Cord-derived MSCs (hUC-MSC) or phosphate buffer saline. All animals were subcutaneously immunized with ovalbumin one week later. Delayed-type hypersensitivity assay was performed another week following immunization. The splenic monocytes were then isolated, cultured and stimulated with ovalbumin. Levels of IL-10, TGF-β, and IFN-γ in culture media were measured by ELISA. The frequencies of CD4⁺CD25⁺Foxp3⁺ and CD8⁺Foxp3⁺ regulatory T cells (Tregs) were determined by flow cytometry. The results showed that the AC inoculation of ovalbumin induced significantly less ear swelling than controls, confirming the establishment of ACAID. MSCs potentiated IL-10 and TGF-β production, further suppressed IFN-γ secretion from splenic monocytes in ACAID mice, and enhanced expansion of CD4⁺CD25⁺Foxp3⁺ and CD8⁺Foxp3⁺ Tregs isolated from the spleen of ACAID mice. Therefore, our study, for the first time, provides clear evidence that systemic administration of MSCs augments cytokine production and Treg expansion from ACAID spleens, which may contribute to promotion and maintenance of ACAID.     

骨髓造血干细胞分离 保存的研究

目的探讨骨髓造血干细胞分离及保存的方法。方法应用羟乙基淀粉(HES)或percoll液分离骨髓造血干细胞;联合应用二甲基亚砜(DMSO)和HES对造血干细胞进行液氮保存。应用血细胞计数法、锥虫蓝拒染实验、粒-巨噬细胞集落生成单位(CFU-GM)的体外培养等方法对造血干细胞冷冻前后的有核细胞(NC)数、存活率、体外分化能力进行检测;应用流式分析法计数CD34+细胞数。结果利用HES沉降法分离的单个核细胞数、CD34+细胞数、CFU-GM集落数均比percoll液离心法明显增多;骨髓造血干细胞冷冻保存1年后的有核细胞数、CD34+细胞数、锥虫蓝活率、CFU-GM集落计数与保存前差异无统计学意义。结论HES法分离骨髓造血干细胞方法安全、有效;通过程序降温,联合使用DMSO及HES的低温冻存方法对骨髓造血干细胞的长期保存是适合的。     

兔骨髓间充质干细胞的分离、培养、鉴定及Dil荧光标记

摘要 目的　探讨兔骨髓间充质干细胞(MSCs)体外分离培养、表型鉴定和标记的方法。方法　采用密度梯度离心法及贴壁分离筛选法分离培养MSCs;采用免疫细胞化学方法检测细胞表面标志抗原CD29,CD106的表达,进行表型鉴定;DiI标记第3代MSCs,观察标记效率。结果　体外培养的原代MSCs48h内可见少量贴壁细胞,7-8d达到90%汇合;免疫细胞化学方法检测细胞表面标志抗原CD29,CD106为阳性;DiI进行细胞标记后,荧光显微镜下见所有MSCs均被标记为红色荧光,提示DiI标记法敏感性好,标记效率高。结论　此培养方法能在短时间内获得大量MSCs,操作简单,成功率高,可以作为培养兔MSCs的常规方法,为构建组织工程尿道提供充足的种子细胞,并进一步用于治疗重度尿道下裂、尿道下裂残废和较长的后尿道狭窄等顽症。     

人骨髓间充质干细胞体外分离培养方法的比较研究

目的 比较Ficoll密度梯度离心法分离人骨髓间充质干细胞(mesenchymal stem cells,MSCs)与全骨髓培养法之间的差异,以期建立一种简便、实用的基于临床移植需要的MSCs分离方法.方法 分别应用上述两种方法分离MSCs,比较用两种方法的细胞形态、MSCs细胞得率及以及细胞表面标志的差异.结果 两种方法均能有效培养出骨髓MSCs,且获得的第二代MSCs细胞形态无明显差异,细胞形态均一,为长梭形或三角形.5 ml骨髓细胞分离培养后,Ficoll密度梯度离心法获得的MSCs细胞总数显著小于全骨髓培养法(t=.639,P＜0.01).Ficoll密度梯度离心法与全骨髓培养法获得的第二代MSC8 CD29、CD105、CD105、CD34阳性表达率的差异无统计学意义(t分别为0.809、0.659、0.277、0.191,P均＞0.05),但前者的HLA-DR阳性表达率明显低于后者,差异具有统计学意义(t=2.347,P＜0.05).结论 与Ficoll密度梯度离心法相比较,全骨髓培养法分离MSCs具有培养时间较短,细胞得率较多的优点,虽纯度相对较低,但仍是一种较好的MSCs分离方法.     

六种重组细胞因子对人骨髓造血细胞的体外扩增效应

目的 :观察造血生长因子对骨髓CD34 +细胞的扩增效应及扩增细胞的分化特性。方法 :采用重组人白细胞介素 (rhIL) 1 +rhIL 3+rhIL6 +重组人粒细胞集落刺激因子 +重组人粒 巨噬细胞集落刺激因子和重组人干细胞因子对人骨髓单个核细胞 (MNC)进行刺激 ,动态观察了经上述细胞因子作用后MNC的增殖效应及液态扩增培养后粒 巨噬集落形成率 (CFU GM ) ,利用流式细胞技术 (FACS)分析了扩增前后CD34 +细胞及其亚群的动态变化。结果 :经上述 6种细胞因子作用 2 4d ,骨髓MNC总数是原代MNC的 38.33倍 ,液态扩增 1 0d后 ,CFU GM的数量达高峰 ,是原代MNC的 1 9.0 4倍 ,2 4d时CFU GM总数降至 3.94倍 ,且集落明显小于扩增初期。扩增至 6～ 1 0d ,CD34 +细胞总数是原代MNC的 1 36 .40～ 90 .40倍 ,1 9d时降为 6 1 .40倍。结论 :外源性造血生长因子对CD34 +骨髓细胞具有较强的扩增效应 ,选择适宜的扩增时间是保证骨髓移植后尽快重建造血功能的重要环节。     

Development of a clinical grade procedure for generation of mRNA transfected dendritic cells from purified frozen CD34(+) blood progenitor cells

Enriched CD34(+) peripheral blood progenitor cells (PBPC) are frequently used as stem cell support in cancer patients following high dose therapy. Since precursor dendritic cells (DCs) originate from haematopoietic progenitor cells, purified CD34(+) cells might also serve as starting cells for ex- vivo production of DC. In the present study we developed a clinical grade procedure for ex- vivo production of DC derived from enriched CD34(+) cells. Different concentrations of CD34(+) cells were grown in gas-permeable Teflon bags with different serum-free and serum-containing media supplemented with GM-CSF, IL-4, TNF-alpha, SCF, Flt-3L and INF-alpha. Serum-free CellGroSCGM medium for 7 days followed by CellGroDC medium in 7 days gave the same results as serum-containing medium. After incubation the cultured cells containing immature DCs were concentrated and transfected with tumour mRNA from human prostate cancer cell lines employing a highly efficient electroporation procedure. Thawed transfected DCs were able to elicit primary T-cell responses in vitro against antigens encoded by the prostate cancer mRNA as shown by ELISPOT assay using mock-transfected DCs as control. Our results show that frozen enriched CD34(+) cells can be an alternative and efficient source for production of DCs for therapeutic purpose.