Battula Venkata Lokesh, Bareiss Petra M, Treml Sabrina, Conrad Sabine, Albert Ingrid, Hojak Sigrid, Abele Harald, Schewe Bernhard, Just Lothar, Skutella Thomas, Bühring Hans-Jörg
Department of Internal Medicine II, Division of Hematology, Immunology, Oncology and Rheumatology, University Clinic of Tübingen, Tübingen, Germany.
Differentiation. 2007 Apr;75(4):279-91. doi: 10.1111/j.1432-0436.2006.00139.x. Epub 2006 Dec 11.
Conventionally, mesenchymal stem cells (MSC) are generated by plating cells from bone marrow (BM) or other sources into culture flasks and selecting plastic-adherent cells with fibroblastoid morphology. These cells express CD9, CD10, CD13, CD73, CD105, CD166, and other markers but show only a weak or no expression of the embryonic markers stage-specific embryonic antigen-4 (SSEA-4), Oct-4 and nanog-3. Using a novel protocol we prepared MSC from BM and non-amniotic placenta (PL) by culture of Ficoll-selected cells in gelatin-coated flasks in the presence of a serum-free, basic fibroblast growth factor (b-FGF)-containing medium that was originally designed for the expansion of human embryonic stem cells (ESC). MSC generated in gelatin-coated flasks in the presence of ESC medium revealed a four-to fivefold higher proliferation rate than conventionally prepared MSC which were grown in uncoated flasks in serum-containing medium. In contrast, the colony forming unit fibroblast number was only 1.5- to twofold increased in PL-MSC and not affected in BM-MSC. PL-MSC grown in ESC medium showed an increased surface expression of SSEA-4 and frizzled-9 (FZD-9), an increased Oct-4 and nestin mRNA expression, and an induced expression of nanog-3. BM-MSC showed an induced expression of FZD-9, nanog-3, and Oct-4. In contrast to PL-MSC, only BM-MSC expressed the MSC-specific W8B2 antigen. When cultured under appropriate conditions, these MSC gave rise to functional adipocytes and osteoblast-like cells (mesoderm), glucagon and insulin expressing pancreatic-like cells (endoderm), as well as cells expressing the neuronal markers neuron-specific enolase, glutamic acid decarboxylase-67 (GAD), or class III beta-tubulin, and the astrocyte marker glial fibrillary acidic protein (ectoderm). In conclusion, using a novel protocol we demonstrate that adult BM-and neonatal PL-derived MSC can be induced to express high levels of FZD-9, Oct-4, nanog-3, and nestin and are able of multi-lineage differentiation.
传统上，间充质干细胞（MSC）是通过将来自骨髓（BM）或其他来源的细胞接种到培养瓶中，并选择具有成纤维细胞样形态的塑料贴壁细胞来生成的。这些细胞表达CD9、CD10、CD13、CD73、CD105、CD166和其他标志物，但仅微弱表达或不表达胚胎标志物阶段特异性胚胎抗原-4（SSEA-4）、Oct-4和nanog-3。我们采用一种新方法，通过在明胶包被的培养瓶中培养经Ficoll分离的细胞，在一种最初设计用于人胚胎干细胞（ESC）扩增的无血清、含碱性成纤维细胞生长因子（b-FGF）的培养基中，从BM和非羊膜胎盘（PL）制备MSC。在ESC培养基存在下于明胶包被的培养瓶中生成的MSC，其增殖率比在含血清培养基中于未包被的培养瓶中生长的传统制备的MSC高4至5倍。相比之下，PL-MSC中的集落形成单位成纤维细胞数量仅增加1.5至2倍，而BM-MSC则不受影响。在ESC培养基中生长的PL-MSC显示SSEA-4和卷曲蛋白-9（FZD-9）的表面表达增加，Oct-4和巢蛋白mRNA表达增加，以及nanog-3的诱导表达。BM-MSC显示FZD-9、nanog-3和Oct-4的诱导表达。与PL-MSC不同，只有BM-MSC表达MSC特异性W8B2抗原。在适当条件下培养时，这些MSC可分化为功能性脂肪细胞和成骨样细胞（中胚层）、表达胰高血糖素和胰岛素的胰腺样细胞（内胚层），以及表达神经元标志物神经元特异性烯醇化酶、谷氨酸脱羧酶-67（GAD）或III类β-微管蛋白的细胞，还有星形胶质细胞标志物胶质纤维酸性蛋白（外胚层）。总之，我们采用一种新方法证明，源自成人BM和新生儿PL的MSC可被诱导高表达FZD-9、Oct-4、nanog-3和巢蛋白，并具有多向分化能力。
