评估牙髓间充质干细胞聚集物表达三种生殖细胞谱系标志物的能力。

Evaluation of the Capability of the Wharton's Jelly Mesenchymal Stem Cell Aggregates to Express the Markers of Three Germ Cell Lineages.

机构信息

Laboratory for Stem Cell Research, Department of Anatomical Sciences, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.

出版信息

Arch Iran Med. 2019 Feb 1;22(2):85-90.

PMID:30980644

Abstract

BACKGROUND

The ability of stem cells to differentiate into different cell types makes them a key component of healing damage in regenerative medicine. As human umbilical cord Wharton's jelly (HUCWJ) is available non-invasively, HUCWJ does not raise any ethical issues with higher differentiation potential compared to adult stem cells. With the ability to express embryonic stem cell markers, HUCWJ can be considered as a good candidate in regenerative medicine applications. The objective of this study was to find if these cells form cell aggregates with the same features as that formed by embryonic stem cells (embryoid body) and could form three germ layers.

METHODS

Eighteen umbilical cords were of healthy infants with parent permission. The umbilical cords were cut into small pieces and the explants were cultured. At the third passage, 1000, 5000 and 10000 cells/ 20 µL were cultured in hanging drops for 3 days. Then, they were incubated for additional 3 days in non-adhesive dishes. As the center of cell aggregates formed from 5000 and 10000 cells were darker than those formed from 1000 cells, this study focused on the aggregates formed by 1000 cells for further assessments. The immunocytochemistry and flowcytometry were performed using 3 color antibodies to detect the markers for three germ cell lineages.

RESULTS

The immunohistochemistry data showed that the embryoid-body-like aggregates expressed a low amount of ectodermal and endodermal markers and most of the cells expressed mesodermal markers. The flowcytometry percentage of the cells in each aggregate that expressed ectodermal marker Otx2 was17.1% and endodermal marker, Sox 17 was 5.49%. The frequency of cells expressing mesodermal marker Brachyury was high (75.0%). Flowcytometry also showed the percentages by mathematical evaluation and we did this three times for our result accuracy.

CONCLUSION

These aggregates mainly kept their mesenchymal state and showed a poor differentiation potential toward ectoderm and endoderm identity.

摘要

背景

干细胞能够分化为不同的细胞类型，这使它们成为再生医学中修复损伤的关键组成部分。由于人脐带华通氏胶（HUCWJ）可以非侵入性地获得，并且与成体干细胞相比，它具有更高的分化潜力，因此不会引起任何伦理问题。由于能够表达胚胎干细胞标志物，HUCWJ 可以被认为是再生医学应用中的一个良好候选物。本研究的目的是确定这些细胞是否能形成具有与胚胎干细胞（胚状体）相同特征的细胞聚集体，并能形成三个胚层。

方法

18 条脐带来自有父母同意的健康婴儿。将脐带切成小块进行培养。在第 3 代时，将 1000、5000 和 10000 个细胞/20µL 在悬滴中培养 3 天。然后，将它们在非粘附培养皿中再孵育 3 天。由于 5000 和 10000 个细胞形成的细胞聚集体的中心比 1000 个细胞形成的细胞聚集体的中心暗，因此本研究重点关注由 1000 个细胞形成的聚集体以进行进一步评估。使用 3 种颜色的抗体进行免疫细胞化学和流式细胞术，以检测三个胚层谱系的标志物。

结果

免疫组化数据显示，胚状体样聚集体表达少量外胚层和内胚层标志物，大多数细胞表达中胚层标志物。流式细胞术显示，每个聚集体中表达外胚层标志物 Otx2 的细胞百分比为 17.1%，表达内胚层标志物 Sox 17 的细胞百分比为 5.49%。表达中胚层标志物 Brachyury 的细胞频率较高（75.0%）。流式细胞术还显示了通过数学评估的细胞百分比，我们为此进行了三次以确保结果的准确性。