Abstract
Purpose :
Lacrimal gland (LG) is an exocrine gland that produces the aqueous layer of the tear film. Dry eye disease due to LG dysfunction leads to discomfort, visual problems, inflammation, infections and eventually loss of vision. Treatment options such as artificial tears focus on relieving the symptoms however fail to solve the underlying problem. Human induced pluripotent stem cells (iPSCs) have the capacity to differentiate into functional specialized cell types, and can be employed to construct 3D functional LG tissues in vitro. The use of microfluidics in tissue engineering enhances the recapitulation of native microenvironment by allowing specific control of physical and biochemical parameters. This study aims to develop a 3D LG construct from iPSCs in a microfluidic organ-on-a-chip system.
Methods :
Here we focus on mimicking the native development of the gland by differentiating iPSCs separately into periocular mesenchyme (POM) and conjuctival epithelial (CE) cells. Multi zonal ocular cells (MZOCs) derived from 2D matrigel cultures of iPSCs consist of four zones of ocular cells and can give rise to CE cells. These CE cells are isolated from the MZOCs with fluorescence activated cell sorting (FACS). Emergence of POM and CE cells is confirmed with immunostaining and gene expression analysis of the marker proteins. The bioengineered platform to differentiate and culture iPS cells has been designed and fabricated. Cells were cultured in photopolymerizable and biocompatible gelatin based hydrogel within custom made microfluidic system.
Results :
We have derived the pericolar mesenchymal cells through inhibition of TGFB and WNT pathways. Differentiation was confrmed by following the expression of periocular mesenchymal cell markers FOXC1 and PITX2 with immunofluorescence staining and qPCR analysis.
Differentiated conjunctival epithelial cells were found among the multi zone ocular cell colonies. Their presence were confirmed via conjunctival epithelial marker CK13 immunofluorescence staining. MZOCs were subjected to FACS during different time periods of differentiation and the cells were sorted using SSEA4 and ITGB4. SSEA4 negative ITGB4 positive cells are presumptive conjunctival cells. These cells were collected and cultured for further encapsulation studies.
Conclusions :
We have successfully bioengineered in vitro lacrimal gland tissue from induced pluripotent stem cells.
This is a 2020 ARVO Annual Meeting abstract.