Abstract TMP49: Translating Ribosome Affinity Purification for Qualitative Assessment of Human Neural Stem Cells Transcripts in the Ischemic Rat Brain
Introduction: Neural stem cell (NSC) transplantation is being explored as a potential therapy for ischemic stroke (IS). Although research has shown the efficacy of NSCs in IS, little is known about the gene expression profile of the NSCs after transplantation. To date this has been challenging due to the issue of trying to distinguish factors expressed by the transplanted cells from that of surrounding host cells. We have overcome this critical barrier by adapting the novel TRAP (Translating Ribosome Affinity Purification) approach to selectively purify mRNA from transplanted NSCs.
Hypothesis: Application of TRAP technology can enrich isolation of human NSC transcripts from IS rats and enabling identification of the in vivo gene expression profile of transplanted NSCs.
Methods: Nude rats were subjected to permanent distal cerebral artery occlusion and after 7 days fetal human NSCs expressing GFP under RPL10a promoter were transplanted into the motor cortex of IS and non-ischemic (NI) rats. Seven days post-transplantation, the graft site was dissected and processed using the TRAP methodology. Seven housekeeping genes (HKG) were analyzed by qPCR in TRAP and non-TRAP samples to determine the level of enrichment of human transcripts. The RNA isolated was converted to cDNA libraries and analyzed using RNAseq technology.
Results: The expression level of HKG was consistent between samples and groups when analyzed by the Pearson correlation (r ≥ 8.7), indicating that TRAP samples are compatible between them for sequencing analysis. Furthermore, the average enrichment of human HKG was 22.7-fold (range: 3.37-22.76) and 58.8-fold (range: 1.31-154.94) in IS and NI rats, respectively. Preliminary data from MiSeq analysis detected 317 genes differentially expressed between IS and NI rats. Gene ontology analysis of these genes showed enrichment of genes involved in the intracellular process, stem cell biology, extracellular region, and signaling and secreted proteins.
Conclusion: Our data demonstrate that the application of TRAP is feasible to enrich human NSC transcripts from rat cortex, making analysis of their expression profile in the stroke brain possible. This sets the stage to probe the molecular mechanism of action of transplanted stem cells.
Author Disclosures: R.L. Azevedo-Pereira: None. N.C. Manley: None. M.C. Winge: None. J. Berry: None. G. Sun: None. T. Bliss: None. G.K. Steinberg: Consultant/Advisory Board; Modest; Medtronic Neuroscience Strategic Advisory Board, Qool Therapeutics. Research Grant; Significant; NIH, NINDS, California Institute for Regenerative Medicine (CIRM).
- © 2016 by American Heart Association, Inc.