Engineering patient-specific valves using stem cells generated from skin biopsy specimens.
Engineering patient-specific valves using stem cells generated from skin biopsy specimens.
复制标题
DOI：
10.1016/j.athoracsur.2014.04.075
复制
发表时间：
2014-09
期刊：
影响因子：
--
通讯作者：
D. Simpson;B. Wehman;Yekaterina Galat;Sudhish Sharma;Rachana Mishra;V. Galat;S. Kaushal
中科院分区：
文献类型：
--
作者：
D. Simpson;B. Wehman;Yekaterina Galat;Sudhish Sharma;Rachana Mishra;V. Galat;S. Kaushal
背景：由于瓣膜耐久性逐渐恶化，修复和生长潜力有限，需要瓣膜置换术的儿科患者可能需要再次手术。为了解决这些问题，我们试图使用患者特异性瓣膜细胞和去细胞化的人肺瓣膜来产生具有生物活性的肺瓣膜。方法对皮肤成纤维细胞进行重编程，制备诱导多能干细胞（iPSCs）。然后，我们使用有利于上皮细胞向间充质细胞转化的培养条件将iPSCs分化为间充质干细胞（iPSCs - mscs）。接下来，在静态和动态混合培养条件下，将去细胞化的人肺脏瓣膜植入iPCS-MSCs，培养30天。结果iPSCs-MSCs经4次传代后，CD105和CD90的表达量均超过90%，可分化为骨细胞、软骨细胞和脂肪细胞（n = 4）。与MSC表型一致，iPSCs-MSCs缺乏CD45和CD34的表达。与骨髓间充质干细胞相比，iPSCs-MSC更容易增殖两倍，但保持了与骨髓间充质干细胞相同的80%以上的基因表达谱。与去细胞化的肺瓣膜相比，免疫组化结果显示，再填充的肺瓣膜细胞数量增加，α-平滑肌肌动蛋白表达增加，细胞外基质成分（如蛋白聚糖和糖胺聚糖）的存在增加，表明细胞功能和成熟得以维持。结论本研究结果表明，利用可持续增殖的细胞源构建具有生物活性的人肺瓣膜是可行的。生物活性肺动脉瓣预计比现有的瓣膜替代物有优势，这需要进一步的验证。
BackgroundPediatric patients requiring valve replacement will likely require reoperations due to a progressive deterioration of valve durability and limited repair and growth potential. To address these concerns, we sought to generate a biologically active pulmonary valve using patient-specific valvular cells and decellularized human pulmonary valves.MethodsWe generated induced pluripotent stem cells (iPSCs) by reprogramming skin fibroblast cells. We then differentiated iPSCs to mesenchymal stem cells (iPCSs-MSCs) using culture conditions that favored an epithelial-to-mesenchymal transition. Next, decellularized human pulmonary heart valves were seeded with iPCS-MSCs using a combination of static and dynamic culture conditions and cultured up to 30 days.ResultsThe iPSCs-MSCs displayed cluster of differentiation CD105 and CD90 expression exceeding 90% after four passages and could differentiate into osteocytes, chondrocytes, and adipocytes (n = 4). Consistent with an MSC phenotype, iPSCs-MSCs lacked expression of CD45 and CD34. Compared with bone marrow MSCs, iPSCs-MSC proliferated more readily by twofold but maintained a gene expression profile exceeding 80% identical to bone marrow MSCs. In repopulated pulmonary valves compared with decellularized pulmonary valves, immunohistochemistry demonstrated increased cellularity, α-smooth muscle actin expression, and increased presence of extracellular matrix components, such as proteoglycans and glycosaminoglycans, suggesting sustained cell function and maturation.ConclusionsOur results demonstrate the feasibility of constructing a biologically active human pulmonary valve using a sustainable and proliferative cell source. The bioactive pulmonary valve is expected to have advantages over existing valvular replacements, which will require further validation.