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T. Asami, Y.-H. Chen, W. Dailey, L. Y. Ho, M. Cheng, K. A. Drenser; Hematopoietic Stem Cell Integration and Differentiation Within the Murine Retina. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2639.
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Several studies have shown that hematopoietic stem cells (HSC) have the ability to differentiate into non-hematopoietic cell types. We describe our initial experience in isolating a specific collection of HSCs called side population (SP) cells, exposing them to various factors found in the Wnt-signaling pathway, to see if they integrate and differentiate into cell types normally found within the murine retina.
Flow cytometry was used to isolate the SP cells from the bone marrow of transgenic mice that express enhanced green fluorescent protein (EGFP) derived from the C57BL/6 strain. These "green" SP cells were injected into the vitreous of non-transgenic C57BL/6 mice alone as well as with various adjuncts including norrin or wingless-type MMTV integration site family member 3a (Wnt-3a) protein in the presence or absence of plasmin enzyme. The mice were then euthanized at various time points, the eyes were enucleated, fixed, embedded in OCT, frozen sections are made for immunofluorescent stains and evaluated by using fluorescent microscope.
Fluorescent microscopy shows that SP cell integration into the ganglion cell layer as well as inner nuclear layer of the murine retina is both feasible and reproducible. Immunohistochemistry techniques show that the SP cells which incorporate into the murine retina have neural progenitor cell markers. Mice receiving injections with SP cells and (Norrin or Wnt3a) have a higher SP cell number in the retina compared to those injected with SP cells alone. It also appears that Norrin may play an important role in delaying SP cell degradation. Plasmin, when used as an adjuvant to intravitreal injections, appears to allow for greater penetration of the SP cells into the retina.
Our preliminary results show that it is possible to have SP cells integrate and differentiate within the murine retina after intravitreal injection. In an attempt to enhance this process, we plan future investigations in which we will make additional injections of growth factors at periodic time points after SP cell injection. Further research is necessary before stem cells can be used in the treatment for diseases that cause severe retinal degeneration or vision loss.
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