1. Mohammad Hossein Nasr Esfahani 1
Transfection Efficiency Evaluation of Retinal Progenitor Cells Using Cationic
Lipid-based Reagents
Atefeh Atefi 1,3*,Pendar Shojaei Kojouri 2,Fereshteh Karamali 1,Kianoosh Dormiani 2,Shiva Irani 3,
Mohammad Hossein Nasr Esfahani 1
1. Department of Cellular Biotechnology ,Cell Science Research Center, Royan Institute for Biotechnology, ACECR ,Isfahan ,Iran
2. Department of Molecular Biotechnology , Cell Science Research Center, Royan Institute for Biotechnology, ACECR ,Isfanan ,Iran
3. Department of Biology, Science and Research branch ,Islamic Azad University ,Tehran ,Iran
Objectives
Results
Fig 3. The percentage of cells expressing green fluorescence as determined using flow cytometry and Real time PCR represents the transfection efficiency.
The retina is the significant neural tissue that localizes in the posterior layer of the eyeball(1). During development, retinal progenitor cells (RPCs) differentiate to different types of neural retinal cells(2). Although, recently, RPCs have been considered in many preclinical studies, its low transfection potential in genomic studies requires more investigations. For this purpose, In this study different lipid-based reagents (lipofectamine LTX, lipofectamin 2000(L2K) and lipofectamin 3000(L3K)) were applied as gene carrier to optimize the transfection efficiency.
Firstly, Three weeks after differentiation (Fig .1) RPCs characterized with immunostaining that expressed typical marker of RPCs (Fig .2A). hESC derived-RPCs expressed eye field markers RAX, PAX6, LHX2 and SIX3; Whereas the stemness markers such as NANOG and OCT4 were reduced in compared to hESCs (Fig .2B). The results of cationic lipid-mediated transfection of RPCs showed the highest transfection efficiency with lipofectamin LTX, whereas lipofectamin 2000 resulted in very low efficiency and also toxicity(Fig .3).
Methods
Fig 1. Microscopic view of differentiation RPCs from hESCs.
hESCs RPCs
Conclusions
Human embryonic stem cell line (hESC), RH6, was obtained from Royan institute. Under appropriate culture condition using combination of Noggin, IWR and IGF-1, RH6 cells were differentiated to RPCs(3). Next, gene expression profile of several eye field markers were characterized by immunocytochemistry (ICC) and quantitative real time PCR. The vector pEGFP-C1 (CLONTECH) containing the constitutive promoter of human CMV (driving the expression of EGFP gene) and SV40 polyadenylation signals; was used to allow visualization and analysis of transfected cells using lipofectamine LTX, 2000(L2K) and 3000(L3K). The transfection efficiency was analyzed with flow-cytometery and qRT-PCR.
Our data demonstrated that high yield of RPC could be obtained from hESCs. Moreover, RPCs showed the highest transfection potential with lipofectamin LTX in between different cationic reagents. These results could be used for genetic manipulation of RPC studies.
Fig 2A. Immunofluorescence stainings to characterize retinal progenitor cells.
DAPI RAX
DAPI NESTIN
DAPI LHX2
Key words
Retinal progenitor cells, Cationic lipids, Transfection, EGFP
References
Fig 2B. RT-PCR was performed with gene-specific primers for human retinal progenitor cells.
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