Department of Pharmacology & Toxicology, Boonshoft School of Medicine, Wright State University, Dayton, Ohio, USA.
PLoS One. 2012;7(8):e44300. doi: 10.1371/journal.pone.0044300. Epub 2012 Aug 31.
Human endometrium is a high dynamic tissue that contains endometrial stromal stem cells (hESSCs). The hESSCs have been differentiated into a number of cell lineages. However, differentiation of hESSCs into megakaryocytes (MKs) has not yet been investigated. The aim of this study was to investigate the feasibility of MK generation from hESSCs and subsequent production of functional platelets (PLTs). In our study, hESSCs were cultured from endometrial stromal cells as confirmed by positive stromal cell specific markers (CD90 and CD29) and negative hematopoietic stem cell markers (CD45 and CD34) expression. Then, hESSCs were differentiated in a medium supplemented with thrombopoietin (TPO) for 18 days. The MK differentiation was analyzed by flow cytometry and confocal microscopy. The differentiation medium was collected for PLT production analysis by flow cytometry, transmission electron microscopy and functional measurements. Our results show: 1) MKs were successfully generated from hESSCs as identified by expression of specific markers (CD41a: 1 ± 0.09% and 39 ± 3.0%; CD42b: 1.2 ± 0.06% and 28 ± 2.0%, control vs. differentiation) accompanied with reduction of pluripotent transcription factors (Oct4 and Sox2) expression; 2) The level of PLTs in the differentiation medium was 16 ± 1 number/µl as determined by size (2-4 µm) and CD41a expression (CD41a: 1 ± 0.4% and 90±2.0%, control vs. differentiation); 3) Generated PLTs were functional as evidenced by the up-regulation of CD62p expression and fibrinogen binding following thrombin stimulation; 4) Released PLTs showed similar ultra-structure characteristics (alpha granules, vacuoles and dense tubular system) as PLTs from peripheral blood determined by electron microscopic analysis. Data demonstrate the feasibility of generating MKs from hESSCs, and that the generated MKs release functional PLTs. Therefore, hESSCs could be a potential new stem cell source for in vitro MK/PLT production.
人类子宫内膜是一种高动态组织，其中包含子宫内膜基质干细胞（hESSCs）。这些 hESSCs 已经分化为多种细胞谱系。然而，hESSCs 向巨核细胞（MKs）的分化尚未得到研究。本研究旨在探讨从 hESSCs 生成 MK 并随后产生功能血小板（PLTs）的可行性。在我们的研究中，通过阳性基质细胞特异性标志物（CD90 和 CD29）和阴性造血干细胞标志物（CD45 和 CD34）的表达，从子宫内膜基质细胞中培养 hESSCs。然后，在补充有血小板生成素（TPO）的培养基中培养 hESSCs 18 天。通过流式细胞术和共聚焦显微镜分析 MK 分化。收集分化培养基，通过流式细胞术、透射电子显微镜和功能测量分析 PLT 产生。我们的结果表明：1）成功地从 hESSCs 中生成了 MK，其特征是特异性标志物（CD41a：1 ± 0.09%和 39 ± 3.0%；CD42b：1.2 ± 0.06%和 28 ± 2.0%，对照与分化）的表达增加，多能转录因子（Oct4 和 Sox2）的表达减少；2）分化培养基中 PLTs 的水平为 16 ± 1 个/µl，通过大小（2-4 µm）和 CD41a 表达（CD41a：1 ± 0.4%和 90±2.0%，对照与分化）确定；3）生成的 PLTs 具有功能，如凝血酶刺激后 CD62p 表达和纤维蛋白原结合增加；4）释放的 PLTs 通过电子显微镜分析显示出与外周血 PLTs 相似的超微结构特征（α颗粒、空泡和致密管状系统）。数据表明，从 hESSCs 中生成 MK 是可行的，并且生成的 MK 释放功能 PLTs。因此，hESSCs 可能是体外 MK/PLT 生成的潜在新干细胞来源。
