Low-oxygen culture conditions extend the multipotent properties of human retinal progenitor cells

Abstract

Purpose: Development of an effective cell-based therapy is highly dependent upon having a reproducible cell source suitable for transplantation. One potential source, isolated from the developing fetal neural retina, is the human retinal progenitor cell (hRPC). One limiting factor for the use of hRPCs is their in vitro expansion limit. As such, the aim of this study was to determine whether culturing hRPCs under 3% O2 would support their proliferative capacity while maintaining multipotency.

Methods: To determine the effect of low oxygen on the ability of hRPCs to self-renew, rates of proliferation and apoptosis, telomerase activity, and expression of proliferative, stemness, and differentiation markers were assessed for hRPCs cultured in 3% and 20% oxygen conditions.

Results: Culture under 3% oxygen increases the proliferation rate and shifts the proliferation limit of hRPCs to greater 40 divisions. This increased capacity for proliferation is correlated with an upregulation of Ki67, CyclinD1, and telomerase activity and a decrease in p53 expression and apoptosis. Increased expression of cMyc, Klf4, Oct4, and Sox2 in 3% O₂ is correlated with stabilization of both HIF1α and HIF2α. The eye field development markers Pax6, Sox2, and Otx2 are present in hRPCs up to passage 16 in 3% O₂ . Following in vitro differentiation hRPCs expanded in the 3% O₂ were able to generate specialized retinal cells, including rods and cones.

Conclusions: Low-oxygen culture conditions act to maintain both multipotency and self-renewal properties of hRPCs in vitro. The extended expansion limits permit the development of a clinical-grade reagent for transplantation.

FIG. 1.

Morphology of human fetal retina. H&E and immunohistochemistry of human fetal retina. All scale bars are 100 μm. (a) Twenty-week gestational age: outer and inner nuclear layers are not completely separated and outer segments are not present, while the inner limiting membrane (black arrows) and ganglion cell layer have been formed. Total retina thickness is consistent at about 250 μm. (b–e) Eighteen-week gestational age. (b) Proliferative marker Ki67 is present in the middle of the inner and outer nuclear layers, but not in the layers expressing recoverin. (c) Neural progenitor marker Sox2 is expressed in all cells within the neural retina, with slightly decreased levels in the outer nuclear layer. (d) Recoverin (photoreceptor precursor marker) expression was limited to the outer nuclear layer and single cells in the ganglion cell layer (white arrows). (e) Pax6 is present in both the outer and inner nuclear layers. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Color images available online at www.liebertpub.com/tea

FIG. 2.

Primary culture of human retinal progenitor cells (hRPCs). Immunocytochemical expression of progenitor and eye field developmental markers in primary culture 3 days after isolation. While Otx2 (c) and Pax6 (b) were expressed only in some of the cells, Sox2 (f) was expressed in almost all of the cultured cells. Ki67 (a) expression in primary culture was low compared with higher passages, while some recoverin- (d) positive cells were observed. Tight junctions were present after isolation (e) After passage 1, cell clumps (g) were dissociated and cultured cells became more homogenous, obtaining higher levels of Ki67 expression, losing recoverin and increasing in size (h) Scale bars are 200 μm. Color images available online at www.liebertpub.com/tea

FIG. 3.

Growth kinetics, proliferation, and apoptosis of hRPCs. (a) Estimated number of hRPCs, expanded in 3% (red) and 20% (green) oxygen conditions, established for each of six different donations (A–F). Each point on the graph represents cell counts at the time of passaging. Expression of proliferative marker Ki67 (c) and cell cycling marker CyclinD1 (d) in 3% (red) and 20% (green) oxygen conditions for passages 1, 3, 5, 7, 10, and 16 (for the 3% oxygen condition only). Cycle checkpoint protein expression p53 (e) and phosphorylated Rb were higher in 20% oxygen conditions on all passages investigated (H1, H5, and H10—3% oxygen passages 1, 5, and 10; N1, N5, and N10—20% oxygen passages 1, 5, and 10). Percentage of apoptotic cell death (as determined by TUNEL labeling) in 3% (red) and 20% (green) oxygen conditions on passages 1–9 (b). There was no difference in the percentage of apoptotic cells in low-oxygen conditions on passages 1–7, and a significant increase on passages 8 and 9 (paired t-test with Bonferroni correction). For 20% oxygen conditions there was no difference between passages 1–4. However, apoptosis significantly increases at passage 5 in 20% oxygen, which correlates with pRb upregulation. Starting from passage 3 the ratio of TUNEL-positive cells was significantly higher in 20% oxygen compared with 3% oxygen. Color images available online at www.liebertpub.com/tea

FIG. 4.

Telomerase activity (TRAPeze method). Telomerase activity (b) in hRPCs, obtained for passages 1, 3, 5, 7, 10, and 16 in 3% and 20% oxygen conditions. Fifty-base-pair telomerase products are marked with white arrows (a) and are not present in negative controls (right of each sample). Telomerase activity in hRPCs at passage 1 in 20% oxygen is set as 100%, and other bars (normalized by internal loading control) show the relative telomerase activity.

FIG. 5.

Expression of stemness markers. Relative expression of immature markers in 3% (red) and 20% (green) oxygen conditions on passages 1, 5, and 10. Beta-actin was used as loading control. H1, H5, and H10—passages 1, 5, and 10 in 3% oxygen. N1, N5, and N10—passages 1, 5, and 10 in 20% oxygen, respectively. Oct4, cMyc, and Sox2 were upregulated at all passages in low-oxygen conditions and HIF transcription factors were stabilized in 3% oxygen.

FIG. 6.

Differentiation pattern of hRPCs. Percentage of cells expanded in 3% oxygen expressing specialized photoreceptor cell markers in maintenance conditions (red bars) and 2 days (orange bars), 5 days (green bars), and 9 days (blue bars) postdifferentiation on passages 1, 5, 10, and 16. Representative pictures of cells that were expanded up to passage 16 and differentiated for 9 days are shown in (a). All scale bars are 200 μm. The RT-PCR (b) and WB (c) analysis confirm the expression of photoreceptor markers after differentiation. Color images available online at www.liebertpub.com/tea