Cerebrospinal fluid promotes neuron-like differentiation of mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.1695

Abstract

Abstract:

BACKGROUND: Current research indicates that regulation of endogenous neural stem cells or transplantation of exogenous neural stem cells is the latest and most promising method for the treatment of neurological diseases such as dementia. The success of the differentiation of stem cells from other sources into neural stem cells has provided a new way for the source of neural stem cells. Recent studies have shown that cerebrospinal fluid cannot only nourish nerve cells, but also become an effective and suitable inducer to add new ways for the acquisition of neural stem cells.
OBJECTIVE: To review the effects of cerebrospinal fluid on stem cells at home and abroad, in order to provide assistance for the clinical application of neural stem cells in the future.
METHODS: “Cerebrospinal fluid, stem cells, induced differentiation, neural stem cells, stem cell transplantation therapy” were used as English keywords to search the PubMed database and the Embase database by computer. “Cerebrospinal fluid, stem cells, induced differentiation, proliferation, differentiation, neural stem cells, mesenchymal stem cells, embryonic stem cells, ginkgo biloba extract, stem cell transplantation therapy, neurodegenerative diseases” were used as Chinese keywords to search CNKI database, WanFang database, VIP database and other scientific journal databases. The search time was from December 10^th, 1998 to December 10^th, 2018. The effects of cerebrospinal fluid on mesenchymal stem cells, embryonic stem cells, and neural stem cells were summarized and analyzed, providing assistance for the clinical use of neural stem cells in the future.
RESULTS AND CONCLUSION: (1) Interaction of cerebrospinal fluid and stem cells is the hope of treating nervous system diseases. (2) Cerebrospinal fluid can promote the differentiation of mesenchymal stem cells into neuron-like cells. (3) Cerebrospinal fluid can induce human embryonic stem cells to differentiate into neuron-like cells, but the proportion of different types of cells obtained is different. (4) Cerebrospinal fluid can induce the proliferation and differentiation of endogenous neural stem cells.

Key words: cerebrospinal fluid, stem cells, induced differentiation, proliferation, differentiation, neural stem cells, mesenchymal stem cells, embryonic stem cells, ginkgo biloba extract, stem cell transplantation therapy, neurodegenerative diseases

CLC Number:

Ren Chao1, Ji Yongqiang2, Guan Lina3. Cerebrospinal fluid promotes neuron-like differentiation of mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(17): 2770-2775.

Figures/Tables 1

2.1 脑脊液对间充质干细胞的影响骨髓间充质干细胞具有多向分化和增殖能力，且免疫原性低，易于获得、分离和培养。颜连启等[18]使用自体脑脊液诱导骨髓间充质干细胞，培养四五天即可出现神经样细胞形态，分化为神经元、星形胶质细胞和少突胶质细胞，并表现相应的特征结构和生物学特性。有研究将骨髓间充质干细胞与神经细胞共培养，尝试建立更加类似人体的微环境从而诱导骨髓间充质干细胞的分化，培养1周后发现骨髓间充质干细胞形成突起，细胞逐渐变为星状，星状细胞数目逐渐增并互相形成连接，具有神经样形态结构，免疫荧光显示神经元特异性烯醇化酶阳性。而对照组骨髓间充质干细胞未形成神经样形态结构，免疫荧光显示神经元特异性烯醇化酶呈阴性。因此，研究者认为神经细胞生长过程中可分泌一些诱导骨髓间充质干细胞分化的物质，其提供的微环境对骨髓间充质干细胞分化为神经元具有促进作用。课题组的研究团队亦在既往研究中发现类似的结果[4，13，19-26]，且发现脐源间充质干细胞有类似骨髓间充质干细胞的结果[4，13，19-20，24]。 Farivar等[27]也证明脑脊液可以诱导脐源间充质干细胞分化为神经样细胞，不过使用分化诱导的脑脊液浓度不同、诱导分化获得神经细胞所需的天数略有不同。以上说明间充质干细胞可在脑脊液中生长，并可维持其分化为神经细胞的潜能。沈忆新等[28]认为间充质干细胞在脑脊液中和在一般培养液中具有相似的生长特性，和课题组认为的一样：间充质干细胞迅速生长的同时，悬浮的各种血细胞减少，提示造血干细胞的生长或分化受抑，表明脑脊液培养液环境有助于间充质干细胞的生长而不利于造血干细胞的生长，该培养环境有助于从骨髓/脐血(脐带)中分离、培养间充质干细胞，但要注意脑脊液培养、诱导方案的具体制定，这是因为由于间充质干细胞具有多向分化潜能，在不同的培养液中可分化为不同的组织细胞。因此不同的培养目的需要有不同的培养成分，但是具体机制目前探讨较少，理论上推导认为：间充质干细胞在脑内或脊髓内可诱导分化为神经细胞，脑内或脊髓内的微环境为其的定向诱导分化提供了必要的条件，具体条件仍不确定。而近期Zhu等[29]研究发现脑脊液是通过胰岛素样生长因子1调节干细胞增殖和迁移的；而Glage等[30]却发现胰高血糖素样肽1可能是其中的重要调控因子。也有学者认为是脑脊液中的脉络丛细胞与间充质干细胞两种细胞之间直接相互作用的结果[31-34]，亦或是两种细胞融合的结果，甚至有学者提出脉络丛微生态信号是调节干细胞分化、迁移的重要因素[35-36]，并有研究发现PRDM16可能是该信号通路上的重要调控因子[36]。另外，有学者发现肌萎缩侧索硬化患者脑脊液也能促进间充质干细胞分化成为神经元样细胞[37]。脑脊液对间充质干细胞影响的研究不仅局限于诱导实验，目前已经有将诱导后获得神经细胞用于治疗的相关研究[4，19，21，26，38]，且提示一定的疗效。当然，也有临床试验未取得预期效果，如有研究将间充质细胞注入9例阿尔茨海默症患者的海马和楔前叶，观察24个月，患者认知状态未取得任何改善[39]。 2.2 脑脊液对胚胎干细胞的影响卞林等[40]使用正常成人脑脊液能诱导人胚胎源干细胞分化为神经样细胞，如神经元、星形胶质细胞、少突胶质细胞，但所得不同类型细胞的比例不同，其中以胶质细胞比例较高，认为这可能由于脑脊液中含有更多能使干细胞偏向于胶质细胞方向分化的因子有关。陈新生等[41]研究中，血性脑脊液比正常脑脊液组神经干细胞分化为神经元的比例高，能获得更多比例的胶质细胞，这可能与脑损伤后脑脊液中促胶质细胞增生的成分增多有一定的关系。尹国才等[42]在研究“人胎脑神经干细胞在发育期脑脊液中的迁移和分化”时发现在胚胎期，处于不同发育阶段的胚胎干细胞分泌的信息物质差异大，且脑屏障发育尚未完成，处于开放状态，因而其对脑脊液的分泌有影响，而不同发育时期的脑脊液所承载的效应成分有差异，并进一步证实胶质细胞的出现可能与特定的脑脊液环境密切相关。和以上研究诱导获得胶质细胞为主不同，徐杰等[43]在其专利中指出“先采用一定浓度的抗坏血酸诱导分化，后采用正常成人脑脊液进一步使胚胎中脑来源的干细胞向多巴胺能神经细胞方向分化，可以获得最高比例的多巴胺能神经元”。 Zappaterra等[44]发现脑脊液的流体压力是促使胚胎干细胞分化、迁移的重要促发因素。而Martín等[45]提出成纤维细胞生长因子2是脑脊液诱导胚胎干细胞分化的重要因子。Parada等[46]通过基因分析证实了脑脊液中的因子对早期胚胎发育过程，尤其是神经细胞的分化、形成的有重要影响。Kiiski等[47]发现健康人脑脊液促进人类胚胎干细胞分化为神经细胞并能促进其形成保留自发活动的神经元网络，但Ma等[48]的研究却认为成人脑脊液不支持胚胎干细胞的神经发生，也有研究表明脑脊液抑制胚胎干细胞分化成神经元但却可促进其分化为胶质细胞[49]，这可能和他们使用的胚胎干细胞和脑脊液来源不同所致。 2.3 脑脊液对神经干细胞的影响李青[50]研究发现脊髓损伤后通过改变脑脊液的成分会对脊髓内源性神经干细胞增殖和分化产生影响。刘国平[51]通过对比研究发现：①神经干细胞在脑外伤血性脑脊液和脑积水清亮脑脊液中均能存活、增殖和分化；②神经干细胞在脑外伤血性脑脊液中贴壁分化的速度比在脑积水清亮脑脊液中要快，贴壁分化比例要高；③脑外伤血性脑脊液和脑积水清亮脑脊液对神经干细胞分化的种类上有差异。神经干细胞在脑外伤血性脑脊液中倾向于向胶质细胞方向分化，在脑积水清亮脑脊液中倾向于向神经元方向分化。滕弘等[52]发现脑缺血大鼠脑脊液可促进体外培养神经干细胞存活并且促进神经干细胞向神经元和星形胶质细胞分化。Nozaki等[53]研究蛛网膜下腔出血患者的脑脊液发现含血脑脊液是激活并促进内源性神经干细胞增殖、分化的有效刺激物。Haines等[54]发现多发性硬化患者的脑脊液可影响神经干细胞中的少突胶质细胞祖细胞的转录变化。以上说明病态下的脑脊液可能是患者启动自身内源性神经修复的一个重要因素[55-56]，但修复中脑脊液促进神经干细胞分化方向有一定差异[57]，多以胶质细胞为主[16]，少数可见神经元样细胞为主[58]。如何获得想要的目的细胞用于治疗，目前含药脑脊液[59]，尤其是中药脑脊液药理学的创立是目前的诱导内源性干细胞治疗神经系统疾病的研究热点，课题组团队正在开展此方面的探索(EGb761脑脊液药理学方法介导循环式干细胞移植治疗痴呆研究)，并提出了脑脊液介导的“治疗与获取诱导剂”同步的循环式干细胞移植模式，见图1，这是将来临床值得推广的、可以“一箭双雕”的个体化干细胞移植治疗优选方案之一。"

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