Abstract
Purpose :
To characterize the gene expression changes and affected signaling pathways in the degenerating mouse retina in response to intravitreal injection of human CD34+ stem cells.
Methods :
C3H/HeJrd1/rd1 mice, systemically immunosuppressed, were injected intravitrealy with PBS (n=16) or human bone marrow CD34+ cells (n=16). Animals were euthanized at weeks 1 and 4 post-injection, eyes harvested, retina dissected and preserved in RNALater. Total RNA was isolated using Qiagen miRNeasy kit. Labeled probes were hybridized to the Affymetrix Mouse Transcriptome Assay 1.0 (MTA1) GeneChip microarrays. Raw microarray .cel files were analyzed using Affymetrix Expression Console followed by Transcriptome Analysis Console 3.0, to identify differentially expressed genes. Ingenuity Pathway Analysis and WikiPathways were used for biologically relevant changes of expression in signaling pathways.
Results :
Microarray analysis of the murine retina using the criteria of <-1.5 or >1.5 Fold change and ANOVA p-value <0.05 showed significant alteration in expression of 1114 transcript clusters at 1 week post- injection, including 98 coding and 386 non-coding RNAs (microRNAs, small nucleolar RNA, long-non-coding RNAs-lncRNA) and other novel transcripts. At 4 weeks post-injection, gene expression changes were observed in a total of 172 transcripts clusters. They included 19 coding and 20 non-coding genes. WikiPathways analysis identified expression changes in the Apoptosis pathway at both time points. Ingenuity Pathway Analysis (IPA) highlighted Phototransduction, Nervous System Development and Inherited Diseases affected at week 1, and Visual System Development &Function and Ophthalmic Diseases affected at week 4.
Conclusions :
Microarray analysis of the degenerating retina shows alteration in expression of both coding and noncoding genes in response to intravitreal administration of human CD34+ cells. The non-coding RNAs, including miRNAs, known to have a key role in gene expression regulation at the post-transcriptional level appear to be the prevalent retinal transcripts at week 1. Gene pathways affected are very relevant to pathogenesis of hereditary retinal degeneration, and alterations in expression of these genes support a potential trophic regenerative effect of human CD34+ stem cells on the degenerating mouse retina.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.