Multipotent Stem Cells from the Brain and Retina of Green Mice

THE RECENT ISOLATION AND AMPLIFICATION of multipotent stem cells in the laboratory (1,2) has enlivened the fields of mammalian development and transplantation. Yet the very plasticity that makes these cells so interesting biologically makes them difficult to track as they integrate into host tissues. Here we describe the isolation of stem cells from the brain and retina of mice expressing the enhanced green fluorescent protein (eGFP) transgene, and the transplantation of these cells to the brain and retina of nontransgenic recipients. The most successful method for labeling stem cells has been transfection with a heritable reporter gene such as beta-galactosidase (b-GAL) (3) or GFP (4). These techniques are performed following isolation and prior to grafting. They are slow, labor-intensive, and require transfection of each cell type isolated, as well as repeated clonal selection for high level expression. Loss of transgene expression following differentiation or transplantation is an additional problem with these techniques. Furthermore, each transfection alters the genome unpredictably, risking unintentional changes in stem cell phenotype. While direct isolation of stem cells from b-Gal1 mice has been described, this label has the disadvantage of requiring an exogenous substrate for visualization. By harvesting from donors transgenic for GFP, it should be possible to obtain stem cell lines exhibiting uniformly high levels of constitutive reporter gene expression. Transgenic animals expressing GFP throughout their body have been generated in a number of species, including mice (5–7). We harvested tissue from newborn GFP transgenic mice and isolated stem cells from the brain (BSCs) and neuroretina (RSCs) using established methods (20 ng/mL EGF and bFGF). In both cases, GFP1 neurospheres appeared within the first 3 days and have now been maintained in culture for more than 1 year. To assess the capacity for self-renewal, spheres were broken up and plated as single cells. Individual cells formed new spheres over a period of 2–5 days, with number and size proportional to time in culture. Spheres labeled positively for nestin, an intermediate filament expressed by neuronal and glial precursors, and for Ki-67, which recognizes actively mitotic cells (Fig. 1a–1c). Spheres cultured in the presence of 10% fetal bovine serum generated cells that maintained their green fluorescence and co-labeled with neural markers. BSCs differentiated into cells labeled with markers of each neural lineage; that is, neurons (MAP-2), astrocytes (GFAP) and oligodendrocytes (GalC) (Fig. 1d–1f). RSCs did not differentiate into oligodendrocytes, a cell type not normally generated in the retina, but did differentiate into cells expressing the photoreceptor-specific marker rhodopsin. Green mouse neurospheres were injected into sites within the brain and eye of mature mice. Transplanted BSCs survived, maintained GFP expression (as evidenced by co-labeling after pulsing with BrdU in vitro), migrated widely and showed morphological evidence of integration with the host central nervous system (Fig. 1g). RSCs grafted to the subretinal space of the mechanically injured mouse retina showed morpho