Retina–committed Stem Cells Reside in the Bone Marrow and Migrate in vitro to Signals From Damaged Retina

Abstract

Abstract: : Purpose: To investigate whether retina–committed stem cells exist in the bone marrow and migrate in vitro to chemoattractants secreted from damaged retina. Methods: Patchy RPE loss was created in C57/BL6 mice by a single i.v. injection of NaIO₃ (50 mg/kg). RPE/choroidal tissue from untreated and NaIO₃ – treated animals (on days 3, 5, and 7) was cultured for 24 hours under serum free conditions. The collected supernatants were used in an in vitro assay to assess chemotaxis of Sca–1⁺ bone marrow mononuclear cells (MNC). The migrated cells were counted by FACS (Forward Scatter vs. Sideward Scatter) and the expression of RPE markers (MITF, Pax–6, Six–3 and OTX) was analyzed by real time RT–PCR after 4 hr incubation. In addition, RT–PCR was used to identify mRNA and ELISA/Western–blot/immunohistochemistry was used to identify proteins of chemoattractants involved in the migration process. Furthermore, to block the migration, T–140 – a non–peptide antagonist of SDF–1 – was used in the chemotaxis assay. Results: The expression of SDF–1, C3 and HGF was significantly increased in RPE/choroidal tissue after NaIO₃ injection with the maximum increase reached at day 3. Additionally, in the chemotaxis assay, a higher number of Sca–1⁺ bone marrow MNC migrated to supernatants from damaged retina. The expression of RPE markers in the migrated cells was also significantly increased in NaIO₃–related samples. The migration of these cells was blocked with the SDF–1 antagonist T–140. Conclusions: Retina–committed stem cells characterized by the expression of retina–specific markers reside in the bone marrow. Damaged retina secretes factors such as SDF–1, C3 and HGF which can attract stem cells to migrate along their gradient. These tissue–committed stem cells may serve as a new source for tissue regeneration after RPE damage.

Keywords: retinal degenerations: cell biology • retinal pigment epithelium