Immunocytochemistry, RT-PCR and Q-PCR analyses of neonatal ‘RSCs’ during <i>in vitro</i> cultivation.

Abstract

<p>Following expansion in the presence of growth factors ‘RSCs’ show immunoreactivity for <i>nestin</i>, <i>Sox2</i> and <i>Pax6</i> at the protein (A) and mRNA levels (B, C). Further gene expression examination using semi-quantitative RT-PCR revealed that throughout all passages analyzed ‘RSCs’ express components of the notch signaling pathway (<i>Notch1</i> receptor, <i>Hes1</i> and <i>Hes5</i>). Levels of transcription factors crucial for eye and retina development (primary retina, PN1) were highly variable: <i>Six3</i> expression was stable up to P20, <i>Lhx2</i> and <i>Six6</i> levels decreased with increasing passage number, and <i>Rax</i> and <i>Chx10</i> were no longer detectable beginning with passage 3 (B). In comparison, NSCs isolated from E14.5 spinal cord or striatum and cultured for 10 or 5 passages, respectively, showed expression of <i>nestin</i>, <i>Sox2</i>, <i>Pax6</i>, and notch pathway components, but were negative for <i>Rax</i>, <i>Chx10</i>, <i>Six3</i>, and <i>Six6</i>. Adult and PN1 primary retina served as a positive or negative control (B). Q-PCR analysis performed on peripheral ‘RSCs’ from P3 and on their primary counterparts (peripheral retinal cells from PN0) confirmed the above RT-PCR results: expanded cells from low passages expressed <i>Nes</i>, <i>Sox2</i>, <i>Pax6</i>, <i>Notch1</i>, <i>Hes1</i>, <i>Hes5</i>, <i>Six3</i> and <i>Six6</i> (C). Although <i>Lhx2</i> level in P3 ‘RSCs’ was as high as in primary retinal cells, <i>Rax</i> and <i>Chx10</i> genes were undetectable (C). Gene expression levels are related to the mean expression levels of housekeeping genes. Scale bar: 50 µm. Abbreviations: E, embryonic day; exp, expanded; NSC, neural stem cells; P, passage; PN, postnatal day; ‘RSCs’, retinal stem cells; spcord, spinal cord.</p