Department of Clinical Biochemistry, Faculty of Medical Science, Tarbiat Modares University, Tehran, IR Iran.
Cell Mol Biol Lett. 2012 Mar;17(1):89-106. doi: 10.2478/s11658-011-0040-x. Epub 2011 Dec 28.
The emerging fields of tissue engineering and biomaterials have begun to provide potential treatment options for liver failure. The goal of the present study is to investigate the ability of a poly L-lactic acid (PLLA) nanofiber scaffold to support and enhance hepatic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs). A scaffold composed of poly L-lactic acid and collagen was fabricated by the electrospinning technique. After characterizing isolated hMSCs, they were seeded onto PLLA nanofiber scaffolds and induced to differentiate into a hepatocyte lineage. The mRNA levels and protein expression of several important hepatic genes were determined using RT-PCR, immunocytochemistry and ELISA. Flow cytometry revealed that the isolated bone marrow-derived stem cells were positive for hMSC-specific markers CD73, CD44, CD105 and CD166 and negative for hematopoietic markers CD34 and CD45. The differentiation of these stem cells into adipocytes and osteoblasts demonstrated their multipotency. Scanning electron microscopy showed adherence of cells in the nanofiber scaffold during differentiation towards hepatocytes. Our results showed that expression levels of liver-specific markers such as albumin, α-fetoprotein, and cytokeratins 8 and 18 were higher in differentiated cells on the nanofibers than when cultured on plates. Importantly, liver functioning serum proteins, albumin and α-1 antitrypsin were secreted into the culture medium at higher levels by the differentiated cells on the nanofibers than on the plates, demonstrating that our nanofibrous scaffolds promoted and enhanced hepatic differentiation under our culture conditions. Our results show that the engineered PLLA nanofibrous scaffold is a conducive matrix for the differentiation of MSCs into functional hepatocyte-like cells. This represents the first step for the use of this nanofibrous scaffold for culture and differentiation of stem cells that may be employed for tissue engineering and cell-based therapy applications.
组织工程和生物材料这两个新兴领域已经开始为肝衰竭提供潜在的治疗选择。本研究的目的是研究聚 L-乳酸（PLLA）纳米纤维支架支持和增强人骨髓间充质干细胞（hMSC）肝向分化的能力。通过静电纺丝技术制备了由聚 L-乳酸和胶原组成的支架。分离出 hMSC 后，将其接种到 PLLA 纳米纤维支架上，并诱导其向肝谱系分化。使用 RT-PCR、免疫细胞化学和 ELISA 测定了几种重要肝基因的 mRNA 水平和蛋白表达。流式细胞术显示，分离的骨髓源性干细胞对 hMSC 特异性标志物 CD73、CD44、CD105 和 CD166 呈阳性，对造血标志物 CD34 和 CD45 呈阴性。这些干细胞向脂肪细胞和成骨细胞的分化证明了它们的多能性。扫描电子显微镜显示，在向肝细胞分化过程中，细胞在纳米纤维支架上附着。我们的结果表明，在纳米纤维上分化的细胞中，肝特异性标志物如白蛋白、α-胎蛋白、细胞角蛋白 8 和 18 的表达水平高于在平板上培养的细胞。重要的是，在纳米纤维上分化的细胞比在平板上培养的细胞分泌更多的肝脏功能血清蛋白白蛋白和α-1 抗胰蛋白酶进入培养基，这表明我们的纳米纤维支架在我们的培养条件下促进和增强了肝向分化。我们的结果表明，工程 PLLA 纳米纤维支架是一种有利于 MSC 分化为功能性肝细胞样细胞的基质。这是该纳米纤维支架用于培养和分化可能用于组织工程和基于细胞的治疗应用的干细胞的第一步。
