Differentiation potential of bone marrow mesenchymal stem cells into hepatocyte-like cells

doi:10.3969/j.issn.2095-4344.2014.28.025

Abstract

Abstract:

BACKGROUND: Studies have demonstrated that bone marrow mesenchymal stem cells can be induced to differentiate into hepatocyte-like cells under certain condition, which provides a new idea for the treatment of end-sate liver diseases, such as acute hepatic failure.
OBJECTIVE: To review the discovery, isolation and culture, induction and differentiation, and application prospects of bone marrow mesenchymal stem cells.
METHODS: A computer-based online research of CNKI and PubMed databases was performed to collect articles about bone marrow mesenchymal stem cells and their differentiation into hepatocyte-like cells published between 1999 and 2014. The key words were “hepatocyte-like cells, bone marrow mesenchymal stem cells, cell differentiation” in Chinese and English. Finally, 52 articles were included in result analysis.
RESULTS AND CONCLUSION: Currently, the most important issue in liver tissue engineering is to find a kind of seed cells with stable traits and liver-specific functions. Mature hepatocytes are difficult to be harvested, and immune rejection and difficulty in in vitro culture also severely limit the development of liver transplantation. Bone marrow mesenchymal stem cells characterized as multiple differentiation, rapid self-renewal, easy to be cultured and proliferated have been considered as the most promising source of cells. Increasing studies have shown that bone marrow mesenchymal stem cells can differentiate into liver cells in vitro and in vivo, and differentiated cells have the synthesis and secretion functions same as hepatocytes. But how to proliferate such a large number of cells, to maintain the good differentiation potential of cells, to optimize the culture conditions in vitro as well as mechanisms of induction and differentiation mechanisms and clinical security need further studies.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: bone marrow, mesenchymal stem cells, cell differentiation, hepatocytes

CLC Number:

R318

Fan Jing-jing. Differentiation potential of bone marrow mesenchymal stem cells into hepatocyte-like cells[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(28): 4578-4582.

Figures/Tables 1

2.1 骨髓间充质干细胞的发现 1987年，Friedenstein等发现在塑料培养皿中培养的贴壁的骨髓单个核细胞在一定条件下可分化为骨细胞、软骨细胞、脂肪细胞和肌肉细胞，而且这些细胞扩增20-30代后仍能保持其多向分化潜能，这类细胞被称骨髓间充质干细胞。此研究不仅证实了骨髓间充质干细胞的存在，而且为应用于临床提供了可靠的理论基础。 2.2 骨髓间充质干细胞的生物学特点骨髓间充质干细胞最主要的生物学特点是体外扩增容易和黏附贴壁性强[16]，快速的增殖能力是应用其进行细胞治疗的基础，而黏附贴壁性有利于细胞的纯化培养。 2.2.1 表面标志目前为止，尚未发现骨髓间充质干细胞特异的表面标志[17-18]。但可表达区别于造血干细胞的表面标志，包括CD13、SH2、SH3、CD29、CD71、CD90、CD100、CD166、CD120a、CD124、CD49b等，表达低水平的CD44、CDW90，不表达造血系特征标志CD14、CD34、CD45、CD50，也不表达与人类白细胞抗原(human leukocyte antigen，HLA)识别有关的共刺激分子B7-1、B7-2及主要组织相容性复合物(major histocompability complex，MHC)Ⅱ类分子报告，如HLA-DR抗原等。 2.2.2 分离培养目前常用的分离骨髓间充质干细胞的方法主要有4种：①全骨髓贴壁培养法[19]：此方法根据骨髓间充质干细胞具有贴壁生长而造血细胞悬浮生长的特性对其进行筛选。应用贴壁培养法通过多次传代换液，除去悬浮生长的造血细胞，细胞增殖较快，可短时间内获得大量细胞，缺点是所得细胞的纯度不足，细胞均一性差，但其分化诱导特性不受影响。②密度梯度离心法：该法根据骨髓间充质干细胞与其他细胞的密度不同而采用Percoll分离液，能有效地将红细胞、白细胞和骨髓间充质干细胞分离开来，分离获得的细胞较为均一，但多向分化及增殖力较差。③流式细胞仪分选法及免疫磁珠法：此两种方法虽具有高度专一性，但成本较高、步骤复杂、分选后的细胞不易贴壁，对细胞的活性影响较大。根据骨髓间充质干细胞有贴壁生长的特点，在实际使用中往往更多地使用密度梯度离心法和贴壁培养法联合分离骨髓间充质干细胞。Petersen等[20]采用全骨髓贴壁培养法分离纯化骨髓间充质干细胞，通过多次换液、传代除去造血系细胞等非贴壁细胞，得到纯度较高的骨髓间充质干细胞，利用流式细胞技术进行鉴定，所得细胞表达CD29、CD90，不表达CD34、CD45等造血干细胞表面特异标志。此法能较大限度的保持干细胞活性，而且操作简便，有利于临床干细胞的移植。 2.3 骨髓间充质干细胞的诱导分化潜能骨髓间充质干细胞可塑性已得到证实，它具有定向分化或是横向分化为一些成体细胞的潜能。Petersen等1999年首先报道，用2-AFF阻断大鼠肝细胞增殖，CCL4造成肝细胞损伤，进行性别交叉骨髓细胞移植，或者全肝移植，在肝脏中发现来源于骨髓的肝细胞。Theise等[21]又报道了女性患者接受男性供体的骨髓移植后，受体的一些肝细胞和胆管上皮细胞核内含有Y染色体。两者均证明了骨髓干细胞或造血干细胞能够在鼠肝内转化为肝卵圆细胞甚至成熟的肝细胞和胆管细胞。Terai等[22]发现荧光蛋白阳性标记的骨髓细胞定植在肝小叶门脉周围，4周后肝脏中有25%的细胞是骨髓细胞，并横向分化为能分泌白蛋白的成熟肝细胞。以上均表明，体内环境和肝脏本身微环境是促进骨髓间充质干细胞向肝样细胞转化的关键因素，分化的肝样细胞能够对损伤肝脏起到一定的修复作用。骨髓间充质干细胞在动物体内可以分化为肝样细胞得到了证实，但对其诱导的最佳环境还处于摸索阶段。对于诱导分化的条件，目前尚未发现比较理想的统一方法。在体外诱导剂的作用下，骨髓间充质干细胞也可以向肝实质细胞转化[23-27]。细胞因子和生长因子如肝细胞生长因子[28]、表皮生长因子、成纤维细胞生长因子及肿瘤坏死因子1等在肝脏的发育和再生过程中起重要的作用，单独和联合应用会在体外诱导骨髓干细胞增殖并向肝系细胞分化。肝细胞生长因子主要在肝脏Kupfer细胞与窦内皮细胞中产生，对肝细胞有促进有丝分裂作用，在肝脏的发育成熟和再生过程中发挥重要作用，因此添加肝细胞生长因子被广泛用在肝相关干细胞体外诱导分化的实验中。成纤维细胞生长因子4也是一种多功能细胞生长因子，能刺激来源于中胚层和神经外胚层细胞的生长。ITS等营养物质也在肝细胞发育和再生中起着不可代替的作用。白蛋白、甲胎蛋白、细胞角蛋白18及糖原均为肝系细胞的特异性标志。甲胎蛋白和白蛋白都是一种胞浆蛋白，甲胎蛋白由肝前体细胞分泌，随着细胞的成熟而消失，成熟肝细胞不表达。细胞角蛋白18是肝细胞相对特异性的一种角蛋白，在幼稚的肝前体细胞不表达，当肝细胞趋于成熟或已成熟时表达。糖原合成是成熟肝细胞特有的功能。有实验采用肝细胞生长因子(20 μg/L)和成纤维细胞生长因子4(10 μg/L)作为诱导剂，辅以ITS、Dex等因子体外诱导骨髓间充质干细胞向肝细胞样细胞定向分化[29]。经过多个检测结果验证，支持骨髓间充质干细胞向成熟肝细胞定向分化，且随着诱导时间的延长，具有分泌白蛋白、合成糖原功能的肝细胞样细胞逐渐增多，提示此类细胞具有成熟肝细胞的功能，为替代肝细胞移植打下了一定基础。Schwartz等[30]从人、大鼠及小鼠骨髓中分离出间充质干细胞，在培养体系中加入成纤维细胞生长因子4、肝细胞生长因子等诱导后，检测到CK18、甲胎蛋白等一些肝细胞特有的表面标志。Lange等[31]用GFP标记骨髓间充质干细胞，纤维连接蛋白为底物，加入胎肝细胞共同培养，流式细胞仪分离GFP(+)、GFP(-)细胞，RT-PCR发现2周后表达甲胎蛋白、细胞角蛋白18、白蛋白，提示骨髓间充质干细胞和胎肝细胞共同培养的微环境不但有利于骨髓间充质干细胞向肝细胞的分化扩增，也利于胎肝细胞的分化。Chen等[32]将CCl4注射的小鼠肝脏进行培养，收集肝损伤血清，用于诱导小鼠骨髓单个核细胞分化。诱导后细胞呈类圆形肝细胞样变化。RT-PCR示：第7天甲胎蛋白、细胞角蛋白19基因表达；第14天白蛋白、细胞角蛋白18、肝细胞核因子3β基因表达；第21天酪胺酸胺基转移酶表达。免疫荧光示：第21天白蛋白、细胞角蛋白18、细胞角蛋白19、甲胎蛋白基因表达。Chivu等[33]也相继证实骨髓间充质干细胞暴露于体外诱导因素，可向肝样细胞分化。但是有关干细胞诱导的肝样细胞多是进行表型鉴定，而没有在功能水平进行分析，而肝样细胞的功能是细胞治疗的前提。所以应该就进入肝中定植的骨髓间充质干细胞发挥的具体作用机制做进一步深入研究。 2.4 骨髓间充质干细胞的应用前景及存在的问题中国是肝病大国，慢性乙型肝炎患者占全国总人数的1/10，约30%的肝炎患者最终因肝功能衰竭而死亡，虽然肝移植手术在临床上已有了广泛的开展，因供肝缺乏，费用昂贵，尚存在免疫排斥问题，严重限制了肝移植的开展[34-43]，故其临床价值受限，受益者极少。目前主要采取维持和支持疗法治疗各种急慢性肝病，生物人工肝和肝细胞移植因肝细胞来源所限而不能在临床上发挥更大作用，故应用骨髓间充质干细胞作为新型的种子细胞治疗终末期肝病具有很多优点：其体外分离、纯化、扩增、定向诱导分化技术及相应的分子调控机制等方面取得一定进展；取材于患者本人，传代扩增并定向分化为靶细胞后再回输给患者本人，安全性高；没有异体排斥反应，并且避免了胚胎干细胞研究的伦理学争论，故临床应用前景非常突出[44]。同时，骨髓间充质干细胞也存在一些不足：①由于人骨髓中具有能分化为肝细胞的骨髓间充质干细胞数量很少，约占骨髓中有核细胞数量的1/100 000，5 mL骨髓中仅能得到300-1 000个，所以原代获得的骨髓间充质干细胞数量远不能满足肝组织工程的需要。②骨髓间充质干细胞体外扩增纯化速度较慢，尚无法在短时间内获得足够数量的肝干细胞，来满足急性肝衰竭患者的急迫需要。③种植后的干细胞是否能够持续数代的有效分裂和存活，并发挥相应的功能。④移植数量需达到临床需要的移植要求。⑤选择合适的移植部位以避免肺栓塞的发生，以及进入受体体内细胞数量的多少均有待于进一步探讨。⑥干细胞的归巢和定位及体内增殖情况：目前常用的细胞追踪定位方法多为GFP绿色荧光蛋白及菲力磁等，但此类方法只表明细胞进入定植部位，但对定植体内后细胞的分化增殖及功能活性状态尚缺乏进一步的了解。"

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