Mobilization of bone marrow-derived mesenchymal stem cells into the injured tissues after intraarticular injection and their contribution to tissue regeneration

The purpose of present study was to evaluate active mobilization effect of mesenchymal stem cells (MSCs) into injured tissues after intraarticular injection of MSCs, and to evaluate their contribution to tissue regeneration. MSCs, which were obtained from green fluorescent protein (GFP) transgenic Sprague–Dawley (SD) rat and cultivated, were injected into normal SD rats in which multiple tissues had been injured including anterior cruciate ligament (ACL), medial meniscus, and articular cartilage of the femoral condyles. At 4 weeks after injection of MSCs, fluorescent microscopic observation, immunohistochemical or histological examinations were performed to evaluate mobilization of MSCs into injured tissue and their contribution to tissue regeneration. In the group of 1 × 10⁶ MSCs injection, GFP positive cells could mobilize into the injured ACL alone in all 8 knees. In the group of 1 × 10⁷ MSCs injection, GFP positive cells were observed in the injured site of ACL in all 8 knees and in the injured site of medial meniscus and cartilage of femoral condyles in 6 of 8 knees. More interestingly, extracellular matrix stained by toluidine blue was present around GFP positive cells in the injured femoral condyles cartilage and medial meniscus, indicating tissue regeneration. Intraarticularly injected MSCs could mobilize into the injured tissues, and probably contributed to tissue regeneration. This study demonstrated the possibility of intraarticular injection of MSCs for the treatment of intraarticular tissue injuries including ACL, meniscus, or cartilage. If this treatment option is established, it can be minimally invasive compared to conventional surgeries for these tissues.     

Anterior cruciate ligament regeneration using mesenchymal stem cells and collagen type I scaffold in a rabbit model

Purpose

The objective of this study was to determine whether using mesenchymal stem cells (MSC) seeded in a collagen type I scaffold would be sufficient to regenerate the torn anterior cruciate ligament (ACL).

Methods

Anterior cruciate ligament transection was performed on both knees in 10 New Zealand rabbits and then repaired with as follows: suture alone (suture-treated group, n = 6), suture associated with collagen type I scaffold (collagen type I scaffold-treated group, n = 8) or suture associated with autologous MSC seeded on collagen type I scaffold (MSC/collagen type I scaffold-treated group, n = 6). At 12-week post-intervention, the animals were killed and the ACLs were characterised macroscopically and histologically. Data of the 3 groups were against normal ACL (normal group, n = 10).

Results

Macroscopic observation found that in MSC/collagen type I scaffold group, 33 % of specimens showed a complete ACL regeneration, with a tissue similar to the normal ACL. Regeneration was not observed in the group treated with suture alone or associated with collagen type I scaffold without cells. In the latter, only a reparative attempt at the ends was observed. Histological analysis of the regenerated ACL showed a tissue with organised collagen and peripheric vessels.

Conclusions

These results provide evidence that the use of MSC seeded in a collagen type I scaffold in the treatment of ACL injuries is associated with an enhancement of ligament regeneration. This MSC-based technique is a potentially attractive tool for improving the treatment of ACL ruptures.     

间充质干细胞在组织修复与再生中的应用

由于间充质干细胞具有自我更新、多向分化潜能,且容易获取及体外扩增培养,成为近年来成体干细胞研究的热点。间充质干细胞既可以作为修复组织的细胞来源,还可能同时参与多种组织的更新。目前在多个领域显示出重要的应用前景,包括组织工程的种子细胞、基因转染的细胞载体等。     

BMP12诱导恒河猴骨髓间充质干细胞定向分化为腱细胞

目的研究骨髓间充质干细胞定向分化为腱细胞的可能性。方法通过电穿孔法将外源基因BMP12导入骨髓间充质干细胞中，诱导骨髓间充质干细胞定向分化为腱细胞；在形态学和分子生物学水平上对诱导后的细胞加以鉴定。结果光镜下，诱导后的细胞形态发生明显改变；RT-PCR结果表明，诱导后的细胞有BMP12和Collagen Ⅰ的mRNA表达，而没有Coilagen Ⅲ的mRNA表达；诱导后98.39％的细胞呈CD44^ 、HLA-DR^-。结论BMP12能够诱导骨髓间充质干细胞定向分化为腱细胞，间充质干细胞有可能成为肌腱组织工程种子细胞来源之一。     

应用间充质干细胞治疗急性辐射损伤的研究现状与进展

间充质干细胞是一类来源广泛的低免疫原性多能干细胞, 具有多向分化、造血支持、免疫调控等特点, 且易于分离、扩增和外源基因转染, 在临床治疗中有广泛的应用价值, 其用于急性放射损伤治疗的报道也较多。笔者主要介绍了间充质干细胞治疗急性放射损伤的研究现状和相关进展。     

EGFP标记的VEGF165重组质粒的构建及其在骨髓间充质干细胞中的表达

目的构建pIRES2-EGFP-VEGF165真核表达质粒,并在大鼠骨髓间充质干细胞内表达。方法应用DNA重组方法构建pIRES2-EGFP-VEGF165,并将其转染到大鼠骨髓间充质干细胞内,采用ELISA和MTT法检测转染后细胞培养上清液中VHGF165的含量及生物活性。结果成功构建了pIRES2-EGFP-VHGF165,将其转染骨髓间充质干细胞后,VEGF蛋白表达水平明显增高．转染后的细胞培养上清具有促使内皮细胞增殖的生物活性。结论成功构建了真核表达质粒pIRES2-EGFP-VEGFl65．将其转染骨髓间充质干细胞后可高水平地表达具有生物学活性的VEGF蛋白。     

Therapeutic gene expression in transduced mesenchymal stem cells can be monitored using a reporter gene

AimWe constructed a recombinant adenovirus construct Ad5-sr39tk-IRES-VEGF₁₆₅ (Ad5-SIV) that contained a mutant herpes viral thymidine kinase reporter gene (HSV1-sr39tk) and the human vascular endothelial growth factor 165 (VEGF₁₆₅) gene for noninvasive imaging of gene expression. The recombinant adenovirus Ad5-SIV was transfected into rat bone marrow-derived mesenchymal stem cells (MSCs), and we measured the expression of HSV1-sr39tk and VEGF₁₆₅ to evaluate the feasibility of monitoring VEGF₁₆₅ expression using reporter gene expression.MethodsThe MSCs were infected with Ad5-SIV at various levels of infection (MOI), ranging from 0 to 100 infectious units per cell (IU/cell). The mRNA and protein expression levels of the reporter and therapeutic genes were determined using real-time RT-PCR, Western blot, ELISA and immunofluorescence. The HSV1-sr39tk expression in the MSCs was also detected in vitro using a cellular uptake study of the reporter probe ¹³¹I-FIAU. Gene expression was also evaluated in vivo by micro-Positron Emission Tomography/Computed Tomography (micro-PET/CT) imaging 1 day after injecting Ad5-SIV-tranfected MSCs into the left foreleg of the rat. The right foreleg was injected with non-transfected MSCs and served as an internal control.ResultsThe real-time RT-PCR results demonstrated a good correlation between the expression levels of HSV1-sr39tk mRNA and VEGF₁₆₅ mRNA (R² = 0.93, P < 0.05). The cellular uptake of ¹³¹I-FIAU increased with increasing viral titers (R² = 0.89; P < 0.05), and in the group that received an MOI of 100, a peak value of 30.15% ± 1.11% was found at 3 hours of incubation. The uptake rates increased rapidly between 30 and 150 minutes and reached a plateau after 150 minutes. The uptake rates of ¹³¹I-FIAU by the Ad5-SIV-infected cells were significantly higher than by the Ad5-EGFP-infected cells for all time points (t = 18.43-54.83, P < 0.05). Moreover, the rate of VEGF₁₆₅ protein secretion was highly correlated with the uptake rate of ¹³¹I-FIAU (R² = 0.84, P < 0.05). The radioactivity on the micro-PET/CT images was significantly higher in the left foreleg (which received the transfected MSCs) compared with the control foreleg.ConclusionsThese results suggest that radionuclide reporter gene imaging may be used to monitor gene expression in vivo.     

人脐带组织间充质干细胞研究进展及应用前景

近年来对间充质干细胞（mesenchymal stem cells,MSCs）的研究越来越多,MSCs可以从骨髓、脂肪组织、肾和各种胎儿组织中提取,并能横向分化成成骨细胞、脂肪细胞和神经细胞等不同胚层的细胞。人脐带组织间充质干细胞是一类具有自我更新、增殖和多向分化潜能的干细胞,与其他来源的MSCs相比,具有来源广泛,易于采集、保存和运输,无异体排斥,避免伦理争议等诸多优点。本文就其生物学特征、优势以及临床应用前景等予以综述。     

In vivo tracking of 111In-oxine labeled mesenchymal stem cells following infusion in patients with advanced cirrhosis

Background

Several animal and few human studies suggest the beneficial role of bone marrow mesenchymal stem cells (MSCs) in liver cirrhosis. However, little is known about the fate of MSCs after infusion in cirrhotic patients. We evaluated stem cell biodistribution after peripheral infusion of MSCs in four cirrhotic patients.

Methods

After three passages of MSCs, the patients received a total of 250–400×10⁶ cells, of which only 50% of the cells were labeled. Specific activities of 0.21–0.67 MBq/10⁶ cells were maintained for the injected labeled MSCs. Planar whole-body acquisitions (anterior/posterior projections) were acquired immediately following infusion as well as at 2 h, 4 h, 6 h, 24 h, 48 h, 7th and 10th days after cell infusion.

Results

After intravenous infusion, the radioactivity was first observed to accumulate in the lungs. During the following hours to days, the radioactivity gradually increased in the liver and spleen, with spleen uptake exceeding that in the liver in all patients. Region-of-interest analysis showed that the percentage of cells homing to the liver (following decay and background corrections and geometric mean calculation) increased from 0.0%-2.8% at immediately post-infusion images to 13.0–17.4% in 10th-day post-infusion. Similarly, the residual activities in the spleen increased from 2.0%-10.2% at immediately post-infusion images to 30.1%-42.2% in 10th-day post-infusion. During the same period, the residual activities in the lungs decreased from 27.0–33.5% to 2.0–5.4%.

Conclusion

The infusion of MSCs labeled with ¹¹¹In-oxine through a peripheral vein is safe in cirrhosis. Cell labeling with ¹¹¹In-oxine is a suitable method for tracking MSC distribution after infusion.     

Labelling of human mesenchymal stem cells with indium-111 for SPECT imaging: effect on cell proliferation and differentiation

Purpose Stem cell therapy seems to be a new treatment option within cardiac diseases to improve myocardial perfusion and function. However, the delivery and traceability of the cells represent a problem. Radioactive labelling with ¹¹¹In could be a method for tracking mesenchymal stem cells (MSCs). However, ¹¹¹In could influence the viability and differentiation capacity of MSCs, which would limit its use. Therefore, the aim of this study was to evaluate the influence of ¹¹¹In labelling in doses relevant for SPECT imaging in humans on the viability and differentiation capacity of human MSCs.Methods and results Human MSCs isolated from bone marrow were incubated with ¹¹¹In-tropolone (15–800 Bq/cell). The labelling efficiency was approximately 25% with 30 Bq/cell ¹¹¹In. The MSC doubling time was 1.04±0.1 days and was not influenced by ¹¹¹In within the range 15–260 Bq/cell. Using 30 Bq ¹¹¹In/cell it was possible to label MSCs to a level relevant for clinical scintigraphic use. With this dose, ¹¹¹In had no effect on characteristic surface and intracellular markers of cultured MSCs analysed both by flow cytometry and by real-time polymerase chain reaction. Further, the labelled MSCs differentiated towards endothelial cells and formed vascular structures.Conclusion It is possible to label human MSCs with ¹¹¹In for scintigraphic tracking of stem cells delivered to the heart in clinical trials without affecting the viability and differentiation capacity of the MSCs. This creates an important tool for the control of stem cell delivery and dose response in clinical cardiovascular trials.     

兔骨髓间充质干细胞获取与特性的实验研究

目的：完善兔骨髓间充质干细胞（Mesenchymal stem cells，MSCs）体外扩增培养体系并观测其特性。方法：兔骨髓经密度梯度离心获得单核细胞后贴壁培养，光镜、透射电镜观察所获细胞形态，流式细胞术检测细胞周期，体外诱导MSCs向成骨细胞、成脂肪细胞分化并鉴定。结果：原代及传代MSCs为漩涡状贴壁生长的长梭形成纤维细胞样细胞，传代细胞具有类似的生长规律；透射电镜示所获细胞具有较强的自我更新能力；细胞周期分析显示87％以上细胞处于G0／G1期；MSCs经体外诱导可向成骨细胞、成脂肪细胞分化。结论：利用密度梯度离心联合贴壁培养法可分离、纯化MSCs，所获细胞具备成体干细胞特性，于体外可大量扩增，做为种子细胞和载体细胞满足实验需要。     

Survivin在骨髓间充质干细胞中的表达研究

目的：培养鉴定骨髓间充质干细胞（MSCs）,检测survivin的表达。方法：应用Ficoll法分离培养MSCs,流式细胞仪检测其特异性表面标志物,RT—PCR检测survivin mRNA表达。结果：培养MSCs的超微结构显示了干细胞特性,流式细胞仪检测证实为MSCs,survivin mRNA表达阳性。结论：Survivin在正常人骨髓间充质干细胞中有表达,提示它可能参与了其生物学行为并影响间充质干细胞存活。     

Transferrin receptor upregulation: in vitro labeling of rat mesenchymal stem cells with superparamagnetic iron oxide

PURPOSE: To prospectively evaluate the influence of superparamagnetic iron oxide (SPIO) or ultrasmall SPIO (USPIO) particles on the surface epitope pattern of adult mesenchymal stem cells (MSCs) by regulating the expression of transferrin receptor and to prospectively evaluate the influence of transfection agents (TAs) on the uptake of SPIO or USPIO particles in MSCs. MATERIALS AND METHODS: The study was approved by the institutional animal care committee of the University of Tübingen. MSCs were isolated from the bone marrow of four rats. To obtain highly homogeneous MSC populations, MSCs from one rat were single-cell cloned. One MSC clone was characterized and selected for the labeling experiments. The MSCs, which were characterized with flow cytometry and in vitro differentiation, were labeled with 200 microg/mL SPIO or USPIO or with 60 microg/mL SPIO or USPIO in combination with TAs. Aggregations of labeled cells were accommodated inside a defined volume in an agar gel matrix. Magnetic resonance (MR) imaging was performed to measure SPIO- or USPIO-induced signal voids. Quantification of cellular total iron load (TIL) (intracellular iron plus iron coating the cellular surface), determination of cellular viability, and electron microscopy were also performed. RESULTS: Labeling of MSCs with SPIO or USPIO was feasible without affecting cell viability (91.1%-94.7%) or differentiation potential. For MR imaging, SPIO plus a TA was most effective, depicting 5000 cells with an average TIL of 76.5 pg per cell. SPIO or USPIO particles in combination with TAs coated the cellular surface but were not incorporated into cells. In nontransfected cells, SPIO or USPIO was taken up. MSCs labeled with SPIO or USPIO but without a TA showed enhanced expression of transferrin receptor, in contrary to both MSCs labeled with SPIO or USPIO and a TA and control cells. CONCLUSION: SPIO or USPIO labeling without TAs has an influence on gene expression of MSCs upregulating transferrin receptor. Furthermore, SPIO labeling with a TA will coat the cellular surface.     

Ad-HGF修饰MSCs异体移植对烧伤创面愈合的作用

目的：探讨携带人肝细胞生长因子基因的重组腺病毒（Ad—HGF）修饰的骨髓间充质干细胞（MSCs）局部异体移植对烧伤创面愈合的作用。方法：密度梯度离心法体外分离培养雄性Wistar大鼠MSCs，并以感染复数（MOI=100）体外转染Ad—HGF。30只雌性Wistar大鼠随机分为：MSCs治疗组（A组）。Ad—HGF修饰MSCs治疗组（B组），Ad—GFP修饰MSCs治疗组（C组），Ad—HGF治疗组（D组）。空白对照组（E组）。复制大鼠深Ⅱ度烧伤模型，将细胞悬液、病毒悬液及生理盐水于创面下多点注射。伤后3、5、7、14、21d观察创面收缩率并采集创面标本，行HE染色观察创面愈合情况。结果：伤后7、14dB组与A组、C组、D组、E组比较，创口收缩率显著提高（P〈0．05）；HE染色可见B组表皮明显厚于其他各组。并可见钉脚下伸。结论：Ad—HGF修饰MSCs异体移植可显著提高烧伤创面愈合速度和质量，为烧伤创面修复提供了新的策略。     

MRI对自体骨髓间质干细胞移植于缺血心肌后动态监测价值的实验研究

目的 评价MRI对干细胞移植于缺血心肌后的动态监测价值.方法 中华小型猪6头,抽取髂骨骨髓并制备自体骨髓间质干细胞(mesenchymal stem cells,MSCs),以注射用超顺磁性氧化铁颗粒(SPIO)及4',6.二脒基-2.苯基吲哚(DAPI)标记细胞并进行标记率检测.开胸结扎冠状动脉左前降支制备小型猪心肌梗死模型.模型建立后14 d,MR延迟增强成像(DE-MRI)检测梗死面积,之后行第2次开胸,于每只动物心肌梗死周边区和正常心肌区直视下经心外膜各注射标记的MSCs 2个点,同时分别于各区注射培养基2个点作为对照点.细胞移植后24 h及21 d,快速梯度回波序列(F'GRE)T2*像检测十细胞移植点低信号区的面积及信号强度.T2*信号减低区范围的测量由FGRE面积法实现,其信号减低的程度以正常心肌区和该区的T2*值之差与正常心肌区T2*值的百分比表示.病理组织学检查心肌细胞形态、瘢痕形成、毛细血管密度及干细胞分布情况.不同时间点MSCs注射点低信号区面积及T2*信号强度的比较采用重复测量方差分析及配对t检验.干细胞注射点与对照点毛细血管密度的比较、梗死周边区及正常心肌区MSCs注射点信号衰减幅度和DAPI阳性细胞密度的比较采用成组资料t检验.结果 DAPI及SPIO对MSCs的标记率均达100%.心肌梗死模型建立后第14天,小型猪心肌梗死面积为(33.6±8.9)%.细胞移植后24 h,标记MSCs注射点于MRI显示为边界清晰的卵圆形T2*低信号区,梗死周边区与正常心肌区MSCs注射点T2*低信号区的信号强度[分别为(67.00±5.48)%、(61.92±7.76)%,t=1.65,P=0.1158)]及面积[分别为(0.56±0.24)、(0.52±0.25)cm2,t=0.39,P=0.7044.)]差异均无统计学意义.移植后3周,T2*低信号区与正常心肌区的对比程度均较前下降,梗死周边区减低至(40.12±5.93)%(t=9.53,P＜0.01);正常心肌区减低为(46.92±6.25)%(t=11.03,P＜0.01).梗死边缘区T2*减低的程度大于正常心肌区MSCs注射点,且2组间信号强度减弱幅度分别为(26.88±7.27)%和(15.00±4.51)%,差异有统计学意义(F:20.08,P=0.0003).正常心肌区MSCs注射点心肌组织中荧光标记的细胞核分布密度高于梗死边缘区.MSCs注射点[分别为(106±25)和(143±31)个/高倍镜(f=-2.47,P=0.0293)].梗死边缘区MSCs注射点组织中毛细血管分布密度高于该区对照点[分别为(13.4±4.0)和(9.4±3.1)个/高倍镜,f=2.49,P=0.0229].MSCs移植于心肌组织中3周后,普鲁十蓝染色阳性的铁颗粒仅位于部分移植MSCs细胞核周围.结论 MRI可在一定时间内实现对移植干细胞的示踪并反映移植MSCs在心肌局部的数量变化趋势,但是对于移植细胞的半定量监测存在局限性.移植干细胞所在的微环境是影响其牛存时间的重要因素之一.     

猴骨髓间充质干细胞的分离及原代培养特性的研究

目的：探讨猴骨髓间充质干细胞(MSCs)低分化干细胞方面的特性。方法：以Percoll(质量密度1073g/L)非连续密度梯度离心分离出骨髓悬液中的骨髓间充质干细胞，用含100mL/L胎牛血清的DMEM培养液培养，观察细脑的形态、生长状况及其超微结构。结果：原代培养的骨横间充质干细胞增殖缓侵，细胞增殖率和形态分化不均一。如有一些细脑在4wk的培养时间内末见明显增殖，这些增殖不明显的细胞有的未见明显的伸展，呈圆形，有的伸展充分，成片状；在培养2wk后也可见一些明显增殖的细胞，分别形成克隆团状或条状的增生细胞群；培养过程中可见有神经元样及多核细胞出现。原代培养的骨髓间充质干细胞的超微结构也显示了低分化的干细胞特点，如细胞表面具有微绒毛，脑浆内内质网丰富、激离核糖体数量多、线粒体小，核仁大而不规则、可见核裂，超微结构还显示了一些中胚层来源于细瘤的结构特点，如大量的微丝。结论：经Percoll细胞分离介质分离出的骨髓间充质干细胞，在非诱导条件下原代培养过程中，从形态分化、增殖率等方面显示了其多向分化潜能；从超微结构等方面显示了其中胚层来源低分化细胞的特点。这些特点可作为骨髓间充质干细胞鉴定的佐证之一，说明Percoll非连续密度梯度离心法对骨髓间充质干细胞的分离具有可靠性。     

骨髓间充质干细胞在肺损伤大鼠肺组织的分化

目的 探讨骨髓间充质干细胞(MSCs)分化为肺泡上皮细胞、支气管上皮细胞的可能性.方法 将SD大鼠MSCs用DAPI标记后注入受体大鼠体内.75只受体大鼠随机分为5组(n＝15):肺损伤组、MSCs治疗组、MSCs对照组、单个核细胞对照组和正常对照组.分别于MSCs、单个核细胞注入受体大鼠体内1、2、4周后观察肺组织结构变化,检测肺组织中植入细胞情况及广谱细胞角蛋白的表达水平.结果 MSCs治疗组大鼠肺组织在各时间点均有少量植入的细胞,并且部分植入的细胞表达广谱细胞角蛋白.MSCs植入后2周大鼠细支气管壁有少量植入的细胞同时表达广谱细胞角蛋白.其余各组未发现植入细胞的标记.结论 MSCs可在损伤的大鼠肺组织中分化为肺泡上皮细胞和支气管上皮细胞.     

MRI of the proximal femur predicts marrow cellularity and the number of mesenchymal stem cells

Purpose:

To determine whether the marrow conversion index (MCI) in MRI is related to the total number of mononuclear and mesenchymal stem cells (MSCs) in proximal femoral metaphysis of patients with hip osteoarthritis.

Materials and Methods:

Thirty‐two hips of 32 consecutive patients who underwent total hip arthroplasty (THA) for hip osteoarthritis were included in this study. MRI of the hip was performed preoperatively and MCI was subsequently calculated. Three‐milliliter bone marrow samples were obtained from the proximal femur during THA and the number of total mononuclear cells was determined using a hemocytometer. Colony forming unit‐fibroblasts (CFU‐Fs) assays of MSCs were performed by transferring a total of 2 × 10⁴ mononuclear cells to each of five 60‐mm plates. One week later, the numbers of colonies were counted.

Results:

The total number of mononuclear cells decreased with increasing MCI. Likewise, the prevalence and total number of CFU‐Fs increased with increasing number of total mononuclear cells, and decreased with increasing MCI.

Conclusion:

Our results suggest that measurement of MCI in MRI can be an objective and noninvasive method to predict marrow cellularity and the number of MSCs in patients with hip osteoarthritis. J. Magn. Reson. Imaging 2012;35:218‐222. © 2011 Wiley Periodicals, Inc.     

在体诱导骨髓间充质干细胞分化为表皮细胞的初步观察

目的：探讨骨髓间充质干细胞（MSCs)分化为表皮细胞的可行性，方法：抽取小型香猪的骨髓，经密度梯度离心分离、纯化间充质干细胞及培养扩增后，应用5－溴脱氧尿嘧啶（5－BardU)标记技术进行细胞标记。将已标记的细胞以注射方式回植到提供骨髓的香猪的皮内及皮下，分别于注射后1，2周取材，常规石蜡包埋，连续切片，行5－BrdU和角蛋白免疫组织化学染色，对比研究。结果：大多数5－BrdU阳性细胞聚集在真皮中的小血管周围。但有少数5－BrdU阳性标记细胞出现在表皮的棘层和颗粒层，并同时表达角蛋白，结论：在皮肤微环境下骨髓间充质干细胞具有分化为表歧细胞的潜能。