In vitro expanded stem cells from the developing retina fail to generate photoreceptors but differentiate into myelinating oligodendrocytes.

Cell transplantation to treat retinal degenerative diseases represents an option for the replacement of lost photoreceptor cells. In vitro expandable cells isolated from the developing mammalian retina have been suggested as a potential source for the generation of high numbers of donor photoreceptors. In this study we used standardized culture conditions based on the presence of the mitogens FGF-2 and EGF to generate high numbers of cells in vitro from the developing mouse retina. These presumptive 'retinal stem cells' ('RSCs') can be propagated as monolayer cultures over multiple passages, express markers of undifferentiated neural cells, and generate neuronal and glial cell types upon withdrawal of mitogens in vitro or following transplantation into the adult mouse retina. The proportion of neuronal differentiation can be significantly increased by stepwise removal of mitogens and inhibition of the notch signaling pathway. However, 'RSCs', by contrast to their primary counterparts in vivo, i.e. retinal progenitor cells, loose the expression of retina-specific progenitor markers like Rax and Chx10 after passaging and fail to differentiate into photoreceptors both in vitro or after intraretinal transplantation. Notably, 'RSCs' can be induced to differentiate into myelinating oligodendrocytes, a cell type not generated by primary retinal progenitor cells. Based on these findings we conclude that 'RSCs' expanded in high concentrations of FGF-2 and EGF loose their retinal identity and acquire features of in vitro expandable neural stem-like cells making them an inappropriate cell source for strategies aimed at replacing photoreceptor cells in the degenerated retina

<i>In vivo</i> myelin-formation by pre-differentiated ‘RSCs’ derived from peripheral regions of the developing retina.

<p>Analysis of wild-type retinas four weeks after transplantation of oligo-primed <i>actin-EGFP</i>-‘RSCs’ (green) from P3 into adult mice. Many GFP-expressing donor cells form MBP-positive elongated structures on the vitreal side of the retina (red; some are labeled by arrows in B; B is an enlarged view of the boxed area in A) showing MBP-positive fibers radiating towards the optic disc (white star). Scale bars: 100 µm (A), 50 µm (B).</p

Oligodendrocyte differentiation of ‘RSCs’ <i>in vitro</i>.

<p>Whereas primary cells isolated at E14.5 from different regions of the CNS, i.e. spinal cord, striatum, and cortex, showed strong MBP expression (B) when subjected to the oligodendroglial differentiation protocol detailed in (A), primary retinal cells remained MBP-negative (B). However, after <i>in vitro</i> expansion central and peripheral ‘RSC’ (P3) cultures generated by appropriate dissection procedures (C) responded to the oligodendrocyte differentiation protocol with expression of MBP as shown by immunocytochemistry (D) and RT-PCR performed on RNA isolated from undifferentiated, oligo-primed and oligo-differentiated ‘RSCs’ and NSCs from P3 (E). Expanded peripheral ‘RSCs’ (P3) as well as their primary counterparts (peripheral retinal cells from PN0) were subjected to the oligo-differentiation protocol <i>in vitro</i> and their gene expression profile was investigated in detail using Q-PCR array for oligodendrocyte-related gene expression (F). Primary cells expressed moderate levels of <i>Olig1/2</i> genes, whereas expanded ‘RSCs’ contained very high levels of these transcripts. While primary cells subjected to oligo-differentiation conditions did not respond to the treatment with an increase in the expression of oligodendrocyte-related genes, expanded ‘RSCs’ exhibited a significant increase in <i>MBP</i> transcript levels along with other oligodendrocyte-related genes that were analyzed (<i>Vcan</i>, <i>Nkx6.2</i>, <i>Sox10</i>). Generation of oligodendrocytes from expanded ‘RSCs’ was Shh-independent since we could not detect endogenous <i>Shh</i> nor its downstream target <i>Gli1</i> (F). Scale bars: 100 µm (B) and 50 µm (D). Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; E, embryonic day; MBP, myelin basic protein; Oligo1/1+2, oligodendroglial differentiation step 1/1+2; PN, postnatal day; T3, 3,3,5-triodothyronine; Rho, rhodopsin; Rxrg, retinoid X receptor gamma; Rcvrn - recoverin.</p

Differentiation of ‘RSCs’ <i>in vitro</i>.

<p>Following differentiation in 1% newborn calf serum expanded ‘RSCs’ showed immunoreactivity for the pan-neuronal markers β-III-tubulin (A, red) and MAP2 (B, red) or the glial marker GFAP (A, green). A subfraction of cells expressed the interneuron markers calretinin (B, green) or calbindin (B, red) or, after prolonged maintenance in differentiation conditions, the mature neuron marker NeuN (B, red). In contrast to primary neonatal retinal cells (D) subjected to the same differentiation conditions, expanded ‘RSC’ cultures are devoid of recoverin (red) or rhodopsin (green) expressing photoreceptors (C). Scale bars: 50 µm. Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein; MAP2, microtubule-associated protein 2; NeuN, neuron-specific nuclear antigen.</p

Table of used primers.

<p>Table of used primers.</p

Transplantation of ‘RSCs’ into adult mouse retina.

<p>Immunohistochemical analysis on adult wild-type retinas transplanted with GFP-positive ‘RSCs’ (green in A, B, C, and D) revealed that the majority of integrated donor cells expressed GFAP, indicative for extensive glial differentiation (A, arrows). While some transplanted ‘RSCs’ differentiated along the neuronal lineage and expressed β-III-tubulin (B, arrows), grafted cells did not show recoverin (C) or rhodopsin (D) immunoreactivity. Also following transplantation into the degenerative retina of rho−/− mice (E) donor ‘RSCs’ (green) did not show immunoreactivity for recoverin (red). Scale bars: 50 µm. Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; GCL, ganglion cell layer; GFAP, glial fibrillary acidic protein; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer.</p

Increased neuronal differentiation of ‘RSCs’ by priming and notch inhibition.

<p>Generation efficiency of neuronal cell types by expanded ’RSCs’ is dependent on differentiation conditions. ’RSC’ cultures subjected to different differentiation conditions detailed in (A) and immunolabeled with antibodies directed against GFAP (C, green) and β-III-tubulin (C, red) contain different percentages of neurons in dependence of the applied protocol (B, C). The percentage of β-III-tubulin -positive neurons is significantly increased by ’neuronal-priming’ from <17% to <32% when compared to differentiation conditions where mitogens are replaced by NCS and can be further increased to <76% by inhibition of notch-signaling using DAPT (B). DMSO represents the DAPT control experiment with neuron numbers (<37%) corresponding to ’priming’ (B, C). Scale bars: 50 µm. Abbreviations: d, days; DAPI, 4,6-diamidino-2-phenylindole; DAPT, [N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butylester; DMSO, dimethyl sulfoxide; GFAP, glial fibrillary acidic protein. **p<0.01, ***p<0.001.</p