Cell Fate Decisions in a Human Retinal Precursor Cell Line: Basic Fibroblast Growth Factor- and Transforming Growth Factor-α-Mediated Differentiation

Abstract

The purpose of this study was to determine if immortalized human retinal precursor cells could serve as a model to investigate cues that modulate cell fate and differentiation. We investigated the effects of a variety of growth factors broadly but specifically tested the effects of basic fibroblast growth factor (bFGF) and transforming growth factor (TGF)a in retinal cell differentiation and commitment. To determine the role of exogenously added growth factors in a human retinal precursor cell line (KGLDMSM), established from a first-trimester retina, cells were adapted to grow in a defined medium and exposed to a variety of trophic factors (epidermal growth factor [EGF], neuron growth factor [NGF], TGFalpha, TGFbeta, acidic FGF, and bFGF). Dose-response curves were developed to arrive at optimal concentrations. The neurotrophic potential of growth factors was determined by 3H-thymidine incorporation and bromodeoxyuridine (BrdU) labeling. The identity of the emerging neuronal phenotypes were determined by phase-contrast microscopy, immunolabeling for the neuron-specific antigens neurofilament protein (NF) and neuron-specific enolases (NSE), and photoreceptor-specific antigens (Rho1D4, 7G6) using immunocytochemistry and Western blot analysis. To identify some of the early response genes (c-fos, c-myc) expressed in response to growth factors, Northern blot analysis was performed. Almost all of the factors tested increased the total number of cells with a neuronal phenotype. Potency of growth factors to generate neurons was TGFalpha > bFGF > EGF > NGF. Both TGFalpha and bFGF, alone or in combination, increased the total number of neurons. Most of the neurons generated were photoreceptors, as depicted by the polarized phenotype, expression of photoreceptor-specific antigens, and processes resembling rudimentary outer segments. The increase in photoreceptor-like neurons is possibly attributable to an increase in numbers rather than greater survival. Additionally, the majority of the photoreceptors generated labeled with BrdU and for photoreceptor-specific antigens, suggesting that an inductive effect of bFGF and TGFalpha could occur in the cell cycle or shortly thereafter. Both bFGF and TGFalpha induced the expression of the early response gene c-fos while not altering the expression of c-actin or c-myc. The emergence of a photoreceptor phenotype was confirmed by both immunocytochemistry and Western blot analysis. The immortalized retinal precursor cell line could prove valuable in determining the role of exogenously added growth factors in retinal development and differentiation. Both bFGF and TGFalpha enhance the photoreceptor phenotype in medium-density cultures under conditions of defined medium. The same was confirmed by phase-contrast microscopy, immunocytochemistry, and Western blot analysis. Furthermore, cell fate determination in cultured precursor cells could occur during the late part of the cell cycle or shortly after completion of cell division. The effects of TGFalpha and bFGF seem to be slightly additiv...

RPE-secreted factors: influence differentiation in human retinal cell line in dose- and density-dependent manner

“…Our studies confirm their previously published findings that bFGF enhances photoreceptor differentiation. We think that the neurotrophin most likely responsible for the increase in the number of photoreceptors in low-density cultures exposed to 50% conditioned medium is bFGF [10,38,60].…”

“…This density revealed the highest degree of neuronal differentiation, and the RPE-conditioned medium was tested at 50%. Cells were plated on polylysine-coated coverslips and exposed to conditioned medium for 36-48 h and processed for immunolabeling by our previously published protocols [23,38]. Cells plated at low density in serum-free culture were exposed to 50% conditioned medium and assessed for the expression of various antigens by western blot analysis to confirm immunophenotyping data at the same time.…”

“…The cell line is from first trimester spontaneously aborted fetus [23,38,43,44]. The cell line grows in a contact inhibited manner and is SV40T antigen negative (data not included).…”

“…In Xenopus, pigment epithelium treated with bFGF transdifferentiates, generating all retinal cell types including both neural retina and glia [37]. We have reported that bFGF and transforming growth factor alpha (TGFα) induce photoreceptor differentiation in this retinal progenitor cell line in a density-dependent manner [23,38].…”

“…In the present study, we have explored the role of RPE-secreted factors in monolayer cultures (conditioned media on a nontransformed human retinal cell line) [43,44] characterized in monolayer culture [23,38] and 3-D culture [10,11,42].…”