Purpose

Glial cell line-derived neurotrophic factor (GDNF) is neuroprotective of retinal neurons, and transduced retinal progenitor cells (RPCs) can deliver this cytokine for the treatment of retinal diseases, yet the potential effects of GDNF on RPCs have received little attention.

Methods

Murine RPCs were assessed under multiple conditions in the presence or absence of epidermal growth factor (EGF, 20 ng/ml) and/or GDNF (10 ng/ml) using a variety of techniques, including live-cell imaging, caspase-3 activity assay, whole genome microarray, quantitative polymerase chain reaction (qPCR), and western blotting.

Results

Live monitoring revealed that formation of initial aggregates resulted largely from the collision and adherence of dissociated RPCs, as opposed to clonal proliferation. Spheres enlarged in size and number, with more reaching the threshold criteria for cross-sectional areas in the EGF+GDNF condition. Proliferation was measurably augmented in association with EGF+GDNF, and K_i-67 expression was modestly increased (1.07 fold), as were hairy and enhancer of split 5 (Hes5), mammalian achaete-scute homolog 1 (Mash1), and Vimentin. However, global gene expression did not reveal a notable treatment-related response, and the expression of the majority of progenitor and lineage markers examined remained stable. GDNF reduced RPC apoptosis, compared to complete growth-factor withdrawal, although it could not by itself sustain mitotic activity.

Conclusions

These data support the feasibility of developing GDNF-transduced RPCs as potential therapeutic agents for use in retinal diseases.

Purpose

Following transplantation, cultured retinal progenitor cells (RPCs) integrate into the diseased host retina and exhibit morphologies and markers indicative of local cellular phenotypes. In vitro analysis of cultured RPCs allows detailed examination of marker gene expression during the initial phase of differentiation and can provide insight into the variables influencing this process.

Methods

Using cultured murine RPCs, this study compares the effects of fetal bovine serum (FBS) with those of ciliary neurotrophic factor (CNTF), individually or in combination with epidermal growth factor (EGF). Differentiation was assessed by way of the relative expression of 17 genes using quantitative PCR (qPCR) at five time points over a seven-day period.

Results

Both CNTF and FBS rapidly altered the gene expression of RPCs, with very marked upregulation of glial fibrillary acidic protein (GFAP; FBS>CNTF) and marked down-regulation of the proliferation marker K_i-67, consistent with the induction of differentiation. The evidence supports a preponderantly pro-glial influence for both the FBS and CNTF, however, neuronal markers were also upregulated to a lesser extent. Immunocytochemistry confirmed subpopulations labeling with neuronal markers, including rhodopsin. In the presence of sustained EGF stimulation, the differentiating influences of both FBS and CNTF remained perceptible as transient peaks of relative gene expression, but were markedly diminished overall.

Conclusions

This study shows that it is possible to compare the relative efficacy of in vitro differentiation protocols using murine RPCs and qPCR. The differentiating influences of both serum and CNTF were confirmed, but shown to be powerfully moderated by EGF. This suggests that EGF withdrawal is the dominant feature of these differentiation protocols and that exposure to either serum or CNTF is insufficient to irreversibly commit a cultured RPC population to terminal differentiation unless accompanied by concomitant cessation of mitogenic stimulation.

Work has shown that stem cell transplantation can rescue or replace neurons in models of retinal degenerative disease. Neural progenitor cells (NPCs) modified to overexpress neurotrophic factors are one means of providing sustained delivery of therapeutic gene products in vivo. To develop a nonrodent animal model of this therapeutic strategy, we previously derived NPCs from the fetal cat brain (cNPCs). Here we use bicistronic feline lentiviral vectors to transduce cNPCs with glial cell-derived neurotrophic factor (GDNF) together with a GFP reporter gene. Transduction efficacy is assessed, together with transgene expression level and stability during induction of cellular differentiation, together with the influence of GDNF transduction on growth and gene expression profile. We show that GDNF overexpressing cNPCs expand in vitro, coexpress GFP, and secrete high levels of GDNF protein-before and after differentiation-all qualities advantageous for use as a cell-based approach in feline models of neural degenerative disease.

Neural progenitor cells (NPCs) of feline origin (cNPCs) have demonstrated utility in transplantation experiments, yet are difficult to grow in culture beyond the 1 month time frame. Here we use an enriched, serum-free base medium (Ultraculture) and report the successful long-term propagation of these cells. Primary cultures were derived from fetal brain tissue and passaged in DMEM/F12-based or Ultraculture-based proliferation media, both in the presence of EGF + bFGF. Cells in standard DMEM/F12-based medium ceased to proliferate by 1-month, whereas the cells in the Ultraculture-based medium continued to grow for at least 5 months (end of study) with no evidence of senescence. The Ultraculture-based cultures expressed lower levels of progenitor and lineage-associated markers under proliferation conditions but retained multipotency as evidenced by the ability to differentiate into neurons and glia following growth factor removal in the presence of FBS. Importantly, later passage cNPCs did not develop chromosomal aberrations.

The development of the mammalian retina is a complicated process involving the generation of distinct types of neurons from retinal progenitor cells (RPCs) in a spatiotemporal-specific manner. The progression of RPCs during retinogenesis includes RPC proliferation, cell-fate commitment, and specific neuronal differentiation. In this study, by performing single-cell RNA sequencing of cells isolated from human embryonic stem cell (hESC)-derived 3D retinal organoids, we successfully deconstructed the temporal progression of RPCs during early human retinogenesis. We identified two distinctive subtypes of RPCs with unique molecular profiles, namely multipotent RPCs and neurogenic RPCs. We found that genes related to the Notch and Wnt signaling pathways, as well as chromatin remodeling, were dynamically regulated during RPC commitment. Interestingly, our analysis identified that CCND1, a G₁-phase cell-cycle regulator, was coexpressed with ASCL1 in a cell-cycle-independent manner. Temporally controlled overexpression of CCND1 in retinal organoids demonstrated a role for CCND1 in promoting early retinal neurogenesis. Together, our results revealed critical pathways and novel genes in early retinogenesis of humans.