Zhang Shijia, Espandar Ladan, Imhof Kathleen M P, Bunnell Bruce A
Center for Stem Cell Research and Regenerative Medicine, School of Medicine, Tulane University, New Orleans, LA, USA ; Department of Pharmacology, School of Medicine, Tulane University, New Orleans, LA, USA.
J Clin Exp Ophthalmol. 2013 Feb 27;4(270). doi: 10.4172/2155-9570.1000270.
To evaluate differentiation of human adipose-derived stem cells (hASCs) to the keratocyte lineage by co-culture with primary keratocytes .
A co-culture system using transwell inserts to grow hASCs on bottom and keratocytes on top in keratocyte differentiating medium (KDM) was developed. hASCs that were cultured in complete culture medium (CCM) and KDM were used as control. After 16 days, hASCs were examined for morphologic changes and proliferation by cell count. qRT-PCR and flow cytometry were used to detect the expression of aldehyde dehydrogenase 3 family, member A1 (ALDH3A1) and keratocan.
hASCs became more dendritic and elongated in co-culture system relative to CCM and KDM. The doubling time of the cells was longer as differentiation progressed. qRT-PCR showed a definite trend towards increased expression of both ALDH3A1 and keratocan in co-culture system despite statistically non-significant p-values. Flow cytometry showed significantly increased protein levels of ALDH3A1 and keratocan in co-culture system relative to CCM group (p < 0.001) and even relative to KDM group (p < 0.001 for ALDH3A1 and p < 0.01 for keratocan).
The co-culture method is a promising approach to induce differentiation of stem cell populations prior to applications. This study reveals an important potential for bioengineering of corneal tissue using autologous multi-potential stem cells.
通过与原代角膜细胞共培养，评估人脂肪来源干细胞（hASCs）向角膜细胞谱系的分化情况。
开发了一种共培养系统，使用Transwell小室，将hASCs接种于底部，角膜细胞接种于顶部，置于角膜细胞分化培养基（KDM）中培养。将在完全培养基（CCM）和KDM中培养的hASCs作为对照。16天后，通过细胞计数检查hASCs的形态变化和增殖情况。采用qRT-PCR和流式细胞术检测醛脱氢酶3家族成员A1（ALDH3A1）和角蛋白聚糖的表达。
与CCM和KDM相比，hASCs在共培养系统中变得更具树突状且更长。随着分化进展，细胞的倍增时间延长。qRT-PCR显示，尽管p值在统计学上无显著意义，但在共培养系统中ALDH3A1和角蛋白聚糖的表达均有明确的增加趋势。流式细胞术显示，与CCM组相比，共培养系统中ALDH3A1和角蛋白聚糖的蛋白水平显著升高（p < 0.001），甚至与KDM组相比也显著升高（ALDH3A1，p < 0.001；角蛋白聚糖，p < 0.01）。
共培养方法是一种在应用前诱导干细胞群体分化的有前景的方法。本研究揭示了使用自体多能干细胞进行角膜组织生物工程的重要潜力。
