Purpose Loss of photoreceptors due to retinal degenerative diseases, such as Age-related macular degeneration (AMD) and retinitis pigmentosa, is the leading causes of incurable blindness. Recently, Very Small Embryonic-like Stem cells (VSELs) have been identified in the human bone marrow and in the adult peripheral blood. Human VSELs are populations of small (5 to 9 μm) Lin-, CD45- cells that have been observed to express CD133, CD34, CXCR4, as well as high levels of markers characteristic of embryonic stem cells such as Oct-4 and Nanog. To explore the regenerative potential of VSELs in the retina, we transplanted PKH26-labeled enriched human VSELs into the subretinal space or vitreous cavity of severe combined immunodeficiency (SCID) mouse eyes to assess the ability of human VSELs to engraft, survive and differentiate into retinal or neuroectodermal cells in the mouse retina.
Methods VSELs were provided by the NeoStem, Inc., and were prelabeled with PKH-26 (red dye) and quantified using trypan blue and Hemocytometer. 2 microliter of VSEls suspension were injected into the subretinal space of vitreous cavity of SCID mice. Mice were sacrificed by CO2 inhalation 2 to 4 weeks after transplantation. Immunohistochemistry study was performed with the following primary antibodies against human mitochondria, nestin, Ki67, PAX6, neuro-ectodermal cells such as MAP2 and beta-3-tubulin, rhodopsin, recoverin.
RESULTS At both 2 and 4 weeks after transplantation the injected human VSELs were able to engraft, survive and migrate within the retina. Furthermore, subsequent immunohistochemistry analysis revealed that the transplanted cells were able to differentiate by showing markers of retinal stem and developing progenitor cells such as Nestin and PAX6, neuro-ectodermal cells such as MAP2 and beta-3-tubulin, and even markers of retinal photoreceptors such as Rhodopsin and Recoverin.
Conclusions These studies indicate that human VSELs can engraft, migrate and differentiate into retinal cells, and support the further evaluation of these cells to assess their regenerative potential to treat retinal degenerative diseases. |
