Bilic Grozdana, Zeisberger Steffen M, Mallik Ajit S, Zimmermann Roland, Zisch Andreas H
Department of Obstetrics, University Hospital Zurich, Zurich, Switzerland.
Cell Transplant. 2008;17(8):955-68. doi: 10.3727/096368908786576507.
Emerging evidence suggests human amnion tissue as a valuable source of two distinct types of pluripotent cells, amnion epithelial cells (hAECs) and mesenchymal stromal cells (hAMSCs), for applications in cell replacement therapy. For some approaches, it may be necessary to culture and differentiate these cells before they can be transplanted. No systematic attempt has been yet made to determine the quantity and quality of amnion cells after isolation and culture. We looked at amnion cell isolates from 27 term placentas. Following our optimized protocol, primary yields were 6.3 x 10(6) hAECs and 1.7 x 10(6) hAMSCs per gram amnion. All 27 cases gave vital cultures of hAMSCs, while one third of cases (9 of 27) failed to give adherent cultures of hAECs. Primary cultures contained significantly more proliferating than apoptotic cells (hAECs: 16.4% vs. 4.0%; hAMSCs: 9.5% vs. 2.4%). Neither hAECs nor hAMSCs were clonogenic. They showed slow proliferation that almost stopped beyond passage 5. Microscopic follow-up revealed that hAEC morphology gradually changed towards mesenchymal phenotype over several passages. Flow cytometric characterization of primary cultures showed expression of mesenchymal progenitor markers CD73, CD90, CD105, and CD166, as well as the embryonic stem cell markers SSEA-3 and -4 on both amnion cell types. These profiles were grossly maintained in secondary cultures. Reverse transcriptase-PCR analysis exhibited transcripts of Oct-3/4 and stem cell factor in primary and secondary cultures of all cases, but no telomerase reverse transcriptase. Immunocytochemistry confirmed translation into Oct-3/4 protein in part of hAEC cultures, but not in hAMSCs. Further, both amnion cell types stained for CD90 and SSEA-4. Osteogenic induction studies with amnion cells from four cases showed significantly stronger differentiation of hAECs than hAMSCs; this capacity to differentiate greatly varied between cases. In conclusion, hAECs and hAMSCs in culture exhibit and maintain a similar marker profile of mesenchymal progenitors. hAECs were found as a less reliable source than hAMSCs and altered morphology during subculture.
新出现的证据表明，人羊膜组织是两种不同类型多能细胞的宝贵来源，即羊膜上皮细胞（hAECs）和间充质基质细胞（hAMSCs），可用于细胞替代治疗。对于某些方法，可能需要在这些细胞能够移植之前进行培养和分化。尚未进行系统的尝试来确定分离和培养后羊膜细胞的数量和质量。我们研究了来自27个足月胎盘的羊膜细胞分离物。按照我们优化的方案，每克羊膜的原代产量为6.3×10⁶个hAECs和1.7×10⁶个hAMSCs。所有27例均获得了有活力的hAMSCs培养物，而三分之一的病例（27例中的9例）未能获得hAECs的贴壁培养物。原代培养物中增殖细胞明显多于凋亡细胞（hAECs：16.4%对4.0%；hAMSCs：9.5%对2.4%）。hAECs和hAMSCs均无克隆形成能力。它们增殖缓慢，传代5次后几乎停止增殖。显微镜随访显示，hAECs的形态在几次传代后逐渐向间充质表型转变。原代培养物的流式细胞术表征显示，两种羊膜细胞类型均表达间充质祖细胞标志物CD73、CD90、CD105和CD166，以及胚胎干细胞标志物SSEA - 3和 - 4。这些特征在传代培养中基本保持。逆转录 - PCR分析显示，所有病例的原代和传代培养物中均有Oct - 3/4和干细胞因子的转录本，但没有端粒酶逆转录酶。免疫细胞化学证实部分hAECs培养物中有Oct - 3/4蛋白的翻译，但hAMSCs中没有。此外，两种羊膜细胞类型均对CD90和SSEA - 4染色。对4例羊膜细胞进行成骨诱导研究表明，hAECs的分化明显强于hAMSCs；这种分化能力在不同病例之间差异很大。总之，培养中的hAECs和hAMSCs表现并维持了相似的间充质祖细胞标志物谱。发现hAECs作为一种来源不如hAMSCs可靠，并且在传代培养过程中形态发生了改变。
