Intravital microscopy of intracameral adipose-derived mesenchymal stem cells in mice

Session Details

Session Title: Cornea Medical

Session Date/Time: Tuesday 16/09/2014 | 14:00-16:00

Paper Time: 15:12

Venue: Capital Hall A

First Author: : L.Espandar USA

Co Author(s): :    T. Blanco-Mezquita              

Abstract Details

Purpose:

There is lack of knowledge on stem cell dynamics under physiologic conditions. Previous studies used fixed ex vivo specimens at final time point to report on effectivity of stem cells despite the fact that artifacts resulting from sample fixation, slicing, and staining are inevitable. In this study, we used multi-photon microscopy for intravital observation of intracameral injection of adipose-derived mesenchymal stem cells (ASCs) during 4-week follow up in living animals

Setting:

Duke University Eye Center, Durham, NC, USA.

Methods:

GFP-ASCs were obtained from eGFP transgenic mice (C57BL/6-Tg (UBCGFP) 30Scha/J strain; Jackson Laboratory, Bar Harbor, ME). They were grown and expanded in complete culture medium and re-suspended in Hystem® hydrogel (Glycosan, San Francisco, CA). C57BL/6 mice were anaesthetized using an intraperitoneal (IP) injection of Ketamine/Xylazine, (90-100mg/kg, 5-10 mg/kg). Fully anesthetized mice (IP) were placed under a dissecting microscope. A 30G needle was used to make a scratch on posterior surface of cornea. Then 2-5 ul of GFP-mASCs in Hytem Hydrogel (10^4 cell/ul) was injected into anterior chamber. Then 1h, 12h, 1d, 1,2,3,4 weeks after injections mice were examined under multi-photon microscopy.

Results:

Intravital microscopies showed mASCs survived in anterior chamber, they transformed from round cell to elongated cell, they attached to bare descemet’s membrane and produced a barrier along the defect during four weeks follow up. They communicated with each other and even they constructed dendrite-like extensions to stroma.

Conclusions:

Intravital microscopy is an important part of understanding dynamics and behavior of MSCs on therapeutic application in corneal diseases. We showed for the first time that cells can survive in anterior chamber, they are non-toxic, non immunogenic and they will make a barrier to close the gap between cells and make a cellular plexus. We used ASCs re-suspended in HyStem, therefore cells maintained in anterior chamber and in contact with bare descemet’s membrane for a longer period of time. Further studies are essential to evaluate the function of the attached cells and possible differentiation to endothelial cells.

Financial Interest:

NONE