Conclusions
Our data show that combining Hh and mitogen signaling is sufficient to promote the expansion of RPCs in vitro, but it is insufficient to maintain competence of these cells for retinal differentiation.
Methods
Perinatal mouse retinal cells were treated with combinations of Hh agonist (Hh-Ag), epidermal growth factor (EGF)/fibroblast growth factor 2 (FGF2) and the cultures were assayed for long-term growth, gene expression, and dependence on Gli2. Differentiation was assessed in monolayer cultures, following in vivo transplantation and in cellular reaggregates.
Purpose
The in vitro expansion of multilineage competent primary neural progenitor cells is typically limited. Hedgehog (Hh) signaling is required in vivo for the maintenance of stem cell (SC) and progenitor populations in the central nervous system, including the retina. Here we investigated the impact of Hh signaling on in vitro expansion of perinatal mouse retinal progenitor cells (RPCs).
Results
Using a combination of Hh-Ag, EGF, and FGF2, we were able to establish long-term RPC cultures (termed Hh-RPCs). The ability of this combinatorial signaling approach to block quiescence of these was not associated with altered TP53/MDM2 levels or Hh-EGF cooperativity gene expression. Efficient Hh-RPC expansion and monolayer culture establishment requires Gli2, as Hh-RPCs derived from Gli2 knockout retinal tissue fail to generate cultures that can be passaged long-term in vitro. Hedgehog RPCs retain competence for neurogenic and gliogenic differentiation in vitro; however, they fail to engraft and differentiate into retinal cell types following in vivo transplantation to the eye or in vitro when mixed with acutely dissociated perinatal retinal cells. Conclusions: Our data show that combining Hh and mitogen signaling is sufficient to promote the expansion of RPCs in vitro, but it is insufficient to maintain competence of these cells for retinal differentiation.