人脐带间充质干细胞诱导转化成神经干细胞及其移植治疗脑出血的研究
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摘要
背景：脑出血(intracerebral hemorrhage, ICH)是指原发性脑实质内血管破裂引起的出血,具有高发病率、高死亡率和高致残率的特点。目前临床上仍无有效的治疗方法用于改善幸存者的神经功能缺陷。实验证明神经干细胞移植能够促进脑出血后神经功能的恢复。目前,神经干细胞主要从胚胎干细胞诱导或是直接从发育中和成年哺乳动物的中枢神经系统中分离培养获得。但是伦理学、安全性问题以及细胞来源和数量的有限,在一定程度上都限制了神经干细胞的移植应用。因此,很有必要寻找其它能够获得神经干细胞的途径来克服这些限制。研究发现采用碱性成纤维细胞生长因子(basic fibroblast growth factor, bFGF)和表皮细胞生长因子(epidermal growth factor, EGF)可直接诱导骨髓间充质干细胞(bone marrow mesenchymal stem cell, BMSC)向神经干细胞(nerual stem cell, NSC)转化。最近,脐带(Umbilical cord, UC)被发现可以作为间充质干细胞的理想来源,因此脐带MSC (UC-MSC)是否可以诱导转化为神经干细胞以及获得的神经干细胞能否促进脑出血后神经功能的恢复,很值得我们去研究。
目的：探讨体外诱导人UC-MSC向神经干细胞转化的可行性；建立大鼠脑出血模型,探讨获得的神经干细胞移植后在大鼠脑内的存活、分布和分化情况以及对神经功能恢复的影响,为人UC-MSC在神经科学领域的临床应用提供理论依据和实验基础。方法：取足月妊娠剖宫产的新生儿脐带,利用酶消化法和贴壁法获得原代细胞,传4-6代后备用。在添加了bFGF、FGF8、SHH和LIF的DMEM／DF-12完全培养基中预诱导UC-MSC 6-8天,然后消化重新接种在添加了bFGF、FGF8、SHH和2%N2／B27的neurobasal media中,定向诱导大约20天后获得神经干细胞(NSC derived fromUC-MSC,UC-NSC)。一方面,通过real-time RT-PCR和免疫荧光染色分别检测mRNA和蛋白水平上Nestin、NeuroD1、Tubulin、GFAP、Galc以及Fibronectin的表达情况。另外,体外诱导UC-NSC向神经元和神经胶质细胞分化,进一步鉴定其是否具有神经干细胞的特点。另一方面,通过流式细胞学以及成骨和成脂能力来检测UC-NSC的细胞免疫表型以及向中胚层分化的能力,鉴定其是否丧失了UC-MSC的特性。此外,为了进一步研究UC-NSC的治疗潜能,我们将其移植至大鼠脑出血模型中,观察其对神经功能恢复的影响。建模后24小时,将CM-Dil标记的UC-MSC和UC-NSC移植至血肿周围。在移植后的7周每周都采用mNSS和MLPT两种方法进行一次神经功能评价。于移植后3天,通过“干湿重法”进行脑水容量分析。于移植后3天和7天,通过免疫组化染色观察IL-1β在大鼠脑内的表达情况；在移植后35天,制备脑冰冻切片,观察移植细胞在脑内的存活、分布和分化情况。此外,通过免疫荧光染色GFAP分析损伤区周围胶质细胞增殖情况,并测量了胶质瘢痕的厚度。同时,我们还进行结晶紫／速兰染色显示其损伤区域,检测了各组脑出血损伤体积的变化。
结果：人UC-MSC在体外可以诱导转化为UC-NSC,并且获得的UC-NSC不仅具有神经干细胞的特点,同时也丧失了UC-MSC的特性。UC-NSC移植至大鼠脑出血模型后,能够在宿主脑内存活、迁移和分化为神经元和星形胶质细胞。与PBS对照组相比,UC-NSC移植组的脑水肿和胶质瘢痕的发生明显减少,且损伤区周围IL-1β阳性细胞也少于对照组。此外,mNSS和MLPT评分也明显优于对照组。
结论：1.人UC-MSC在体外可以诱导转化为UC-NSC；2.UC-NSC可以有效地促进大鼠脑出血后神经功能的恢复。
背景：脊髓损伤(spinal cord injury, SCI)是造成截瘫的主要原因,同时也是人类致残率最高的疾病之一。目前,国内外治疗SCI的方法多局限于脊柱骨脱位的复位固定和药物治疗以达到解除脊髓压迫、减轻细胞水肿和继发性损伤以及改善微循环等对症治疗的目的,但疗效不佳。细胞移植治疗SCI是近年来的研究热点。研究表明移植的细胞可在损伤部位存活、迁移且能分化为神经细胞促进神经功能的恢复。胚胎干细胞、神经干细胞、间充质干细胞、脐血干细胞以及嗅鞘细胞等均己被作为移植细胞用于脊髓损伤的神经修复治疗。特别是间充质干细胞,相对于其它细胞具有多方面的优点,因此近年来倍受研究者的关注。实验证明移植骨髓间充质干细胞能够促进大鼠脊髓损伤后神经功能的恢复。但取材困难,供体有限,易并发病毒感染以及年龄增长造成的增殖能力和多向分化能力的下降等都使骨髓间充质干细胞的临床应用受到了一定的限制。最近,作为“废弃物”的脐带(Umbilical cord,UC)被发现可以作为间充质干细胞的理想来源,相对于骨髓,从脐带中分离的MSC具有组织来源丰富、细胞原始、增殖能力强和安全无病毒感染风险等优点,因此脐带间充质干细胞(UC-MSC)是否可以成为治疗脊髓损伤的理想种子细胞,值得进我们去研究。
目的：建立犬脊髓损伤模型,初步探讨人UC-MSC移植对犬脊髓损伤后神经功能恢复的影响,为细胞移植治疗寻找一种具有良好应用前景的种子细胞提供理论依据和实验基础。
方法：人UC-MSC来源于足月妊娠剖宫产的健康胎儿脐带,用酶消化法和贴壁法获得原代细胞,消化传代后,取P4-P6代的细胞备用。通过流式细胞学和成骨、成脂能力来检测细胞的免疫表型和多向分化能力从而进一步鉴定UC-MSC。采用闭合液压打击法制备犬脊髓损伤模型。将实验动物随机分为两组,即UC-MSC组和对照组(PBS组)。1)UC-MSC组：脊髓损伤后1周移植1×106个UC-MSC；2)对照组：脊髓损伤后1周移植同体积的PBS。分别于模型制备后1周和UC-MSC移植后1、2、4、6、8、16、24周,采用改良Tarlov评分对动物进行行为学评分。采用SIEMENS MagnetomVision超导MRI,分别于模型制备后1周、UC-MSC移植后1周及6周进行影像学检测,动态观察损伤后的脊髓。于移植24周后处死细胞移植组和对照组的实验动物,取出损伤的脊髓组织制备石蜡切片,Luxol fast blue/cresyl violet(结晶紫／速兰)染色观察组织病理改变情况。
结果：流式细胞学方法检测人UC-MSC的免疫表型,结果发现其高表达CD90、CD29、CD73和CD105；不表达造血干细胞标记CD34、CD45和内皮细胞特异性标记CD31。此外,UC-MSC表达中等水平的HLA-ABC而不表达HLA-DR,提示UC-MSC具有异体移植的可行性。在特定培养条件下,UC-MSC能够成骨和成脂,说明其具有多向分化能力。以上证实移植细胞为人UC-MSC。犬脊髓损伤后,UC-MSC移植组较对照组有明显的神经功能恢复,其改良Tarlov评分具有显著差异。MRI显示UC-MSC组细胞移植后,脊髓创伤区T2WI的高信号逐渐增多,而对照组则表现为不规则高信号环绕中心低的“环征信号”。Luxol fast blue/cresyl violet染色后发现UC-MSC移植组的脊髓填充坏死区的纤维组织较对照组明显减少,而且其周边可见散在的神经元分布,无核固缩,尼氏体染色较深。
结论：人UC-MSC能够促进犬脊髓损伤后的神经功能恢复。
Background:Intracerebral hemorrhage (ICH) refers to bleeding caused in vascular ruprure, namely primary parenchymal hemorrhage, with high morbidity、mortality and morbidity. There are still no effective treatment methods to improve the neural function of survivors. Experiments showed neural stem cell (NSC) transplantation could promote the neural function recovery after ICH. Currently, neural stem cells mainly derived from induction of the early embryonic stem cells or directly separation and culture from central nervous system of the developing and adult mammals. However, in a certain extent, transplantation of neural stem cells have been limited because of ethics, safety problems and the limited cell numbers as well as sources. Therefore, it is necessary to find other ways to obtain neural stem cells to overcome these limitations. Research demonstrated that bone marrow stromal cells could be directly induced into neural stem cell using basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). Umbilical cord (UC) were recently found as a good source of mesenchymal stem cells, so here we want to study whether Umbilical cord mesenchymal stem cells (UC-MSC) could be induced into the neural stem cells, which could promote the neural function recovery after incerebral hemorrhage.
Objective:We plan to explore the feasibility of generation of neural stem cells induced from UC-MSC, and then transplant these cells into the rat brain of intracerebral hemorrhage induced by collagenase and investigate the survival, distribution and differentiation of the grafted cells in order to provide the theory basis and the experimental data in neuroscience clinical application of UC-MSC.
Methods:We chose the umbilical cord from healthy neonatal with full-term pregnancy cesarean section using the method of enzymatic digestion and cell adherent to gain the primitive cells. Passage 4-6 were used in this study. UC-MSCs were pre-induced in DMEM/DF-12 media containing basic fibroblast growth factor (bFGF)、fibroblast growth factor 8 (FGF8)、sonic hedgehog (SHH) and leukemia inhibitory factor (LIF)within 6-8 days, and then replated in culture plastic dishes after trypsinization. After about 20 days of culture in 2%N2/B27containing neurobasal media with bFGF、FGF8 and SHH, we gained the floating nestin-positive aggregates with morphologies identical to typical neural stem cells(NSC). The characteristics of neural stem cells derived from UC-MSC were detected the expression of NeuroDl, Tubulin, Nestin, GFAP, Galc and Fibronectin in mRNA and protein level respectively by Real-time RT-PCR and immunofluorescence stain. The differentiation potentials to neurons and glia were also detected to further identify the the characteristics of neural stem cells derived from UC-MSC. In the other hand, the UC-MSC phenotypes of UC-NSC were confirmed by Flow cytometry analysis and the potentials to differentiate into osteoblasts and adipocytes by staining. In addition, to further determine the function of neurospheres derived from UC-MSCs (UC-NSC), we transplanted these cells into the collagenase-induced ICH rats. The CM-Dil labeled cells were transplanted in the boundary of hemotama 24 hours later. The neural function was estimated by two methods (mNSS and MLPT) every 7 days within 7 weeks. We also detected the brain water contents on day 3 after transplantation and the expression of IL-1βaround hemotama in the brain on day 3 and 7 after ICH. Otherwise, the glial thickness, hemorrhage volume and differentiation of grafted cells were investigated on day 35 after execution.
Results:The neural stem cells induced from UC-MSC grew in neurospheres-like structures, expressed high level of neural progenitor cell markers, but lost the characteristics of mesenchymal stem cells. In addition, they also could be differentiated into the neurons and astrocytes in vitro. Aftrer transplantation into ICH rat brains, we found they could decrease brain water content and glia thickness as well as the expression of IL-10 around hemotama in the brain, differentiate into neurons and astrocytes, and improve significant behavioral recovery. These results suggest that UC-NSC may be a new and useful therapeutic armament for ICH.
Conclusion:1. Nenral stem cells could be efficiently gained from human UC-MSC; 2. Nenral stem cells derived from human UC-MSC ameliorated neurological deficits in a rat model of incerebral hemorrhage.
Spinal cord injury (SCI) that is one of human diseases with highest morbidity mainly causes paraplegina. There is no effective treatment for patients with SCI at present. The methods all over the world are confined to fix and install the vertebral fracture and dislocation as well as drug treatment in order to relieve compression of spinal cord, reduce edema and secondary injury, and improve microcirculation. Cell transplantations for SCI have become a hot point of research in recent years. Studies showed that grafted cells could survive migration and differentiate into nerve cells and then promote neural function recovery. The transplanted cells were used for the treatment of spinal cord injury, including embryonic stem cells, neural stem cells, mesenchymal stem cells, umbilical cord blood stem cells, schwann cells and olfactory ensheathing cells. Among them, mesenchymal stem cells especially seem to be a more promising one regarding their advantages over other types of transplanted cells. It has been proven that mesenchymal stem cells derived from bone marrow (BMSC) could be beneficial to promote neural function recovery of SCI in rats. However, BMSC is restricted to clinical application because of difficult materials, limited donors and complication with virus infection as well as the declining proliferate capacity and multi-differentiation caused with age. Umbilical cord tissue, as a "waste", has been recently regarded as a rich and ideal source of mesenchymal stem cells. In comparison with BMSC, UC-MSCs derived from rich sources without virus infection risk are more primitive and powerful in proliferation ability, thus UC-MSCs are deserved to be.investigated in the treatment of SCI as seed cells.
Objective:This study was designed to preliminarily study if transplantation of UC-MSC could promote the recovery of neurological functions after canine spinal cord injury, in order to provide theoretical and experimental basis for seeking a promising seek cell to use in cell transplantation in SCI.
Methods:Human UC-MSCs are derived from the full-term pregnancy cesarean section umbilical cord from healthy children using the method of enzymatic digestion and cell adherent to gain the primitive cells. After digestion and passage, passage 4-6 were used to transplantation. The characteristics of UC-MSC were confirmed by Flow cytometry analysis to detect cellular markers and by the potential to differentiate into osteoblasts and adipocytes to detect the muti-differetiation ability. The canine spinal cord injury animal model was induced by closing hydraulic impact. The dogs were used and randomly divided into two groups:namely UC-MSC group and PBS group. 1) UC-MSC group:transplantation 1×106 UC-MSC into animals by lumbar puncture 1 weeks after spinal cord injury; 2) injection equal volume of PBS into animals by lumbar puncture 1 weeks after spinal cord injury. Functional outcome measurements were preformed using the modified Tarlov score at lday, 1、2、4、6、8、16、24 weeks after cells transplantation. In addition, Dynamic observations by MRI (SIEMENS MagnetomVision) were investigated to observe the injury spinal cord at 1 week after SCI and at 1,6 weeks after transplantation. The animals were sacrificed 24 weeks later transplantation and then removed injury spinal cord tissue to prepare paraffin sections. Luxol fast blue/cresyl violet staining was used to observe the pathological changes of spinal cord tissue.
Results:FCAS revealed that human UC-MSCs highly expressed cellular phenotypes of CD90, CD29, CD73 and CD105, but not hematopoietic markers CD34 and CD45, and endothelial cell markers CD31. In addition, they also expressed moderate level of HLA-ABC but not express HLA-DR, suggesting that UC-MSCs could be used to allograft. UC-MSCs were also able to differentiate into osteoblasts and adipocytes under special culture condition. After.canine SCI, significant improvements in neurological functional outcome were noted using Tarlov score in animals of human UC-MSCs transplantation group in comparison with PBS group. MRI showed that edema zone was observed around the damage in animals of human UC-MSCs transplantation group, however, hematoma around the damage in PBS group. Pathological changes were found by Luxol fast blue/cresyl violet staining. In contrast with PBS group, the fibrous tissue filling necrosis is significantly reduced in UC-MSC group. In addition, many neurons were found around the fibrous tissue without pyknotic and the Nissl bodies were dyeing deep.
Conclusion:Transplantation of human UC-MSCs following spinal cord injury may improve neurological outcome in dogs.
目的：探讨体外诱导人UC-MSC向神经干细胞转化的可行性；建立大鼠脑出血模型,探讨获得的神经干细胞移植后在大鼠脑内的存活、分布和分化情况以及对神经功能恢复的影响,为人UC-MSC在神经科学领域的临床应用提供理论依据和实验基础。方法：取足月妊娠剖宫产的新生儿脐带,利用酶消化法和贴壁法获得原代细胞,传4-6代后备用。在添加了bFGF、FGF8、SHH和LIF的DMEM／DF-12完全培养基中预诱导UC-MSC 6-8天,然后消化重新接种在添加了bFGF、FGF8、SHH和2%N2／B27的neurobasal media中,定向诱导大约20天后获得神经干细胞(NSC derived fromUC-MSC,UC-NSC)。一方面,通过real-time RT-PCR和免疫荧光染色分别检测mRNA和蛋白水平上Nestin、NeuroD1、Tubulin、GFAP、Galc以及Fibronectin的表达情况。另外,体外诱导UC-NSC向神经元和神经胶质细胞分化,进一步鉴定其是否具有神经干细胞的特点。另一方面,通过流式细胞学以及成骨和成脂能力来检测UC-NSC的细胞免疫表型以及向中胚层分化的能力,鉴定其是否丧失了UC-MSC的特性。此外,为了进一步研究UC-NSC的治疗潜能,我们将其移植至大鼠脑出血模型中,观察其对神经功能恢复的影响。建模后24小时,将CM-Dil标记的UC-MSC和UC-NSC移植至血肿周围。在移植后的7周每周都采用mNSS和MLPT两种方法进行一次神经功能评价。于移植后3天,通过“干湿重法”进行脑水容量分析。于移植后3天和7天,通过免疫组化染色观察IL-1β在大鼠脑内的表达情况；在移植后35天,制备脑冰冻切片,观察移植细胞在脑内的存活、分布和分化情况。此外,通过免疫荧光染色GFAP分析损伤区周围胶质细胞增殖情况,并测量了胶质瘢痕的厚度。同时,我们还进行结晶紫／速兰染色显示其损伤区域,检测了各组脑出血损伤体积的变化。
结果：人UC-MSC在体外可以诱导转化为UC-NSC,并且获得的UC-NSC不仅具有神经干细胞的特点,同时也丧失了UC-MSC的特性。UC-NSC移植至大鼠脑出血模型后,能够在宿主脑内存活、迁移和分化为神经元和星形胶质细胞。与PBS对照组相比,UC-NSC移植组的脑水肿和胶质瘢痕的发生明显减少,且损伤区周围IL-1β阳性细胞也少于对照组。此外,mNSS和MLPT评分也明显优于对照组。
结论：1.人UC-MSC在体外可以诱导转化为UC-NSC；2.UC-NSC可以有效地促进大鼠脑出血后神经功能的恢复。
背景：脊髓损伤(spinal cord injury, SCI)是造成截瘫的主要原因,同时也是人类致残率最高的疾病之一。目前,国内外治疗SCI的方法多局限于脊柱骨脱位的复位固定和药物治疗以达到解除脊髓压迫、减轻细胞水肿和继发性损伤以及改善微循环等对症治疗的目的,但疗效不佳。细胞移植治疗SCI是近年来的研究热点。研究表明移植的细胞可在损伤部位存活、迁移且能分化为神经细胞促进神经功能的恢复。胚胎干细胞、神经干细胞、间充质干细胞、脐血干细胞以及嗅鞘细胞等均己被作为移植细胞用于脊髓损伤的神经修复治疗。特别是间充质干细胞,相对于其它细胞具有多方面的优点,因此近年来倍受研究者的关注。实验证明移植骨髓间充质干细胞能够促进大鼠脊髓损伤后神经功能的恢复。但取材困难,供体有限,易并发病毒感染以及年龄增长造成的增殖能力和多向分化能力的下降等都使骨髓间充质干细胞的临床应用受到了一定的限制。最近,作为“废弃物”的脐带(Umbilical cord,UC)被发现可以作为间充质干细胞的理想来源,相对于骨髓,从脐带中分离的MSC具有组织来源丰富、细胞原始、增殖能力强和安全无病毒感染风险等优点,因此脐带间充质干细胞(UC-MSC)是否可以成为治疗脊髓损伤的理想种子细胞,值得进我们去研究。
目的：建立犬脊髓损伤模型,初步探讨人UC-MSC移植对犬脊髓损伤后神经功能恢复的影响,为细胞移植治疗寻找一种具有良好应用前景的种子细胞提供理论依据和实验基础。
方法：人UC-MSC来源于足月妊娠剖宫产的健康胎儿脐带,用酶消化法和贴壁法获得原代细胞,消化传代后,取P4-P6代的细胞备用。通过流式细胞学和成骨、成脂能力来检测细胞的免疫表型和多向分化能力从而进一步鉴定UC-MSC。采用闭合液压打击法制备犬脊髓损伤模型。将实验动物随机分为两组,即UC-MSC组和对照组(PBS组)。1)UC-MSC组：脊髓损伤后1周移植1×106个UC-MSC；2)对照组：脊髓损伤后1周移植同体积的PBS。分别于模型制备后1周和UC-MSC移植后1、2、4、6、8、16、24周,采用改良Tarlov评分对动物进行行为学评分。采用SIEMENS MagnetomVision超导MRI,分别于模型制备后1周、UC-MSC移植后1周及6周进行影像学检测,动态观察损伤后的脊髓。于移植24周后处死细胞移植组和对照组的实验动物,取出损伤的脊髓组织制备石蜡切片,Luxol fast blue/cresyl violet(结晶紫／速兰)染色观察组织病理改变情况。
结果：流式细胞学方法检测人UC-MSC的免疫表型,结果发现其高表达CD90、CD29、CD73和CD105；不表达造血干细胞标记CD34、CD45和内皮细胞特异性标记CD31。此外,UC-MSC表达中等水平的HLA-ABC而不表达HLA-DR,提示UC-MSC具有异体移植的可行性。在特定培养条件下,UC-MSC能够成骨和成脂,说明其具有多向分化能力。以上证实移植细胞为人UC-MSC。犬脊髓损伤后,UC-MSC移植组较对照组有明显的神经功能恢复,其改良Tarlov评分具有显著差异。MRI显示UC-MSC组细胞移植后,脊髓创伤区T2WI的高信号逐渐增多,而对照组则表现为不规则高信号环绕中心低的“环征信号”。Luxol fast blue/cresyl violet染色后发现UC-MSC移植组的脊髓填充坏死区的纤维组织较对照组明显减少,而且其周边可见散在的神经元分布,无核固缩,尼氏体染色较深。
结论：人UC-MSC能够促进犬脊髓损伤后的神经功能恢复。
Background:Intracerebral hemorrhage (ICH) refers to bleeding caused in vascular ruprure, namely primary parenchymal hemorrhage, with high morbidity、mortality and morbidity. There are still no effective treatment methods to improve the neural function of survivors. Experiments showed neural stem cell (NSC) transplantation could promote the neural function recovery after ICH. Currently, neural stem cells mainly derived from induction of the early embryonic stem cells or directly separation and culture from central nervous system of the developing and adult mammals. However, in a certain extent, transplantation of neural stem cells have been limited because of ethics, safety problems and the limited cell numbers as well as sources. Therefore, it is necessary to find other ways to obtain neural stem cells to overcome these limitations. Research demonstrated that bone marrow stromal cells could be directly induced into neural stem cell using basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). Umbilical cord (UC) were recently found as a good source of mesenchymal stem cells, so here we want to study whether Umbilical cord mesenchymal stem cells (UC-MSC) could be induced into the neural stem cells, which could promote the neural function recovery after incerebral hemorrhage.
Objective:We plan to explore the feasibility of generation of neural stem cells induced from UC-MSC, and then transplant these cells into the rat brain of intracerebral hemorrhage induced by collagenase and investigate the survival, distribution and differentiation of the grafted cells in order to provide the theory basis and the experimental data in neuroscience clinical application of UC-MSC.
Methods:We chose the umbilical cord from healthy neonatal with full-term pregnancy cesarean section using the method of enzymatic digestion and cell adherent to gain the primitive cells. Passage 4-6 were used in this study. UC-MSCs were pre-induced in DMEM/DF-12 media containing basic fibroblast growth factor (bFGF)、fibroblast growth factor 8 (FGF8)、sonic hedgehog (SHH) and leukemia inhibitory factor (LIF)within 6-8 days, and then replated in culture plastic dishes after trypsinization. After about 20 days of culture in 2%N2/B27containing neurobasal media with bFGF、FGF8 and SHH, we gained the floating nestin-positive aggregates with morphologies identical to typical neural stem cells(NSC). The characteristics of neural stem cells derived from UC-MSC were detected the expression of NeuroDl, Tubulin, Nestin, GFAP, Galc and Fibronectin in mRNA and protein level respectively by Real-time RT-PCR and immunofluorescence stain. The differentiation potentials to neurons and glia were also detected to further identify the the characteristics of neural stem cells derived from UC-MSC. In the other hand, the UC-MSC phenotypes of UC-NSC were confirmed by Flow cytometry analysis and the potentials to differentiate into osteoblasts and adipocytes by staining. In addition, to further determine the function of neurospheres derived from UC-MSCs (UC-NSC), we transplanted these cells into the collagenase-induced ICH rats. The CM-Dil labeled cells were transplanted in the boundary of hemotama 24 hours later. The neural function was estimated by two methods (mNSS and MLPT) every 7 days within 7 weeks. We also detected the brain water contents on day 3 after transplantation and the expression of IL-1βaround hemotama in the brain on day 3 and 7 after ICH. Otherwise, the glial thickness, hemorrhage volume and differentiation of grafted cells were investigated on day 35 after execution.
Results:The neural stem cells induced from UC-MSC grew in neurospheres-like structures, expressed high level of neural progenitor cell markers, but lost the characteristics of mesenchymal stem cells. In addition, they also could be differentiated into the neurons and astrocytes in vitro. Aftrer transplantation into ICH rat brains, we found they could decrease brain water content and glia thickness as well as the expression of IL-10 around hemotama in the brain, differentiate into neurons and astrocytes, and improve significant behavioral recovery. These results suggest that UC-NSC may be a new and useful therapeutic armament for ICH.
Conclusion:1. Nenral stem cells could be efficiently gained from human UC-MSC; 2. Nenral stem cells derived from human UC-MSC ameliorated neurological deficits in a rat model of incerebral hemorrhage.
Spinal cord injury (SCI) that is one of human diseases with highest morbidity mainly causes paraplegina. There is no effective treatment for patients with SCI at present. The methods all over the world are confined to fix and install the vertebral fracture and dislocation as well as drug treatment in order to relieve compression of spinal cord, reduce edema and secondary injury, and improve microcirculation. Cell transplantations for SCI have become a hot point of research in recent years. Studies showed that grafted cells could survive migration and differentiate into nerve cells and then promote neural function recovery. The transplanted cells were used for the treatment of spinal cord injury, including embryonic stem cells, neural stem cells, mesenchymal stem cells, umbilical cord blood stem cells, schwann cells and olfactory ensheathing cells. Among them, mesenchymal stem cells especially seem to be a more promising one regarding their advantages over other types of transplanted cells. It has been proven that mesenchymal stem cells derived from bone marrow (BMSC) could be beneficial to promote neural function recovery of SCI in rats. However, BMSC is restricted to clinical application because of difficult materials, limited donors and complication with virus infection as well as the declining proliferate capacity and multi-differentiation caused with age. Umbilical cord tissue, as a "waste", has been recently regarded as a rich and ideal source of mesenchymal stem cells. In comparison with BMSC, UC-MSCs derived from rich sources without virus infection risk are more primitive and powerful in proliferation ability, thus UC-MSCs are deserved to be.investigated in the treatment of SCI as seed cells.
Objective:This study was designed to preliminarily study if transplantation of UC-MSC could promote the recovery of neurological functions after canine spinal cord injury, in order to provide theoretical and experimental basis for seeking a promising seek cell to use in cell transplantation in SCI.
Methods:Human UC-MSCs are derived from the full-term pregnancy cesarean section umbilical cord from healthy children using the method of enzymatic digestion and cell adherent to gain the primitive cells. After digestion and passage, passage 4-6 were used to transplantation. The characteristics of UC-MSC were confirmed by Flow cytometry analysis to detect cellular markers and by the potential to differentiate into osteoblasts and adipocytes to detect the muti-differetiation ability. The canine spinal cord injury animal model was induced by closing hydraulic impact. The dogs were used and randomly divided into two groups:namely UC-MSC group and PBS group. 1) UC-MSC group:transplantation 1×106 UC-MSC into animals by lumbar puncture 1 weeks after spinal cord injury; 2) injection equal volume of PBS into animals by lumbar puncture 1 weeks after spinal cord injury. Functional outcome measurements were preformed using the modified Tarlov score at lday, 1、2、4、6、8、16、24 weeks after cells transplantation. In addition, Dynamic observations by MRI (SIEMENS MagnetomVision) were investigated to observe the injury spinal cord at 1 week after SCI and at 1,6 weeks after transplantation. The animals were sacrificed 24 weeks later transplantation and then removed injury spinal cord tissue to prepare paraffin sections. Luxol fast blue/cresyl violet staining was used to observe the pathological changes of spinal cord tissue.
Results:FCAS revealed that human UC-MSCs highly expressed cellular phenotypes of CD90, CD29, CD73 and CD105, but not hematopoietic markers CD34 and CD45, and endothelial cell markers CD31. In addition, they also expressed moderate level of HLA-ABC but not express HLA-DR, suggesting that UC-MSCs could be used to allograft. UC-MSCs were also able to differentiate into osteoblasts and adipocytes under special culture condition. After.canine SCI, significant improvements in neurological functional outcome were noted using Tarlov score in animals of human UC-MSCs transplantation group in comparison with PBS group. MRI showed that edema zone was observed around the damage in animals of human UC-MSCs transplantation group, however, hematoma around the damage in PBS group. Pathological changes were found by Luxol fast blue/cresyl violet staining. In contrast with PBS group, the fibrous tissue filling necrosis is significantly reduced in UC-MSC group. In addition, many neurons were found around the fibrous tissue without pyknotic and the Nissl bodies were dyeing deep.
Conclusion:Transplantation of human UC-MSCs following spinal cord injury may improve neurological outcome in dogs.
引文
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