Differentiation of Human Adipose-derived Stem Cells along the Keratocyte LineageIn vitro.
OBJECTIVE
To evaluate differentiation of human adipose-derived stem cells (hASCs) to the keratocyte lineage by co-culture with primary keratocytesin vitro.


METHODS
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.


RESULTS
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).


CONCLUSIONS
The co-culture method is a promising approach to induce differentiation of stem cell populations prior toin vivoapplications. This study reveals an important potential for bioengineering of corneal tissue using autologous multi-potential stem cells.
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.
Shijia
S
Zhang
Ladan
L
Espandar
Kathleen M P
KM
Imhof
Bruce A
BA
Bunnell
Bioengineered cornea; Co-culture system; Human adipose-derived stem cells; Keratocyte