We have found a rare population of pNSCs
We have found a rare population of pNSCs in the adult mammalian brain (termed AdpNSCs). We isolated cells from the adult periventricular region that generate clonally derived, self-renewing, and multipotent colonies in vitro in the presence of LIF. These LIF colonies can be passaged to give rise to GFAP+, neurosphere-forming cells in the presence of EGF and FGF2. Most interestingly, the LIF colonies expressed Oct4 in vitro and contributed to the inner cell mass (ICM) of developing blastocysts after morula aggregation, which are characteristics attributed to pNSCs derived from embryonic stem cells (ESCs) (Hitoshi et al., 2004; Tropepe et al., 2001). We observed Oct4 expression in periventricular tissue by quantitative PCR (qPCR) of primary cells and in whole-mount sections from adult brains. Further, we asked whether these AdpNSCs could generate GFAP+, neurosphere-forming NSCs in vivo. We took advantage of a transgenic mouse that expresses herpes simplex virus thymidine kinase under control of the GFAP promoter (termed GFAP-TK mice), which enabled the selective ablation of proliferating GFAP+ cells in vitro and in vivo (Bush et al., 1998, 1999; Imura et al., 2003; Morshead et al., 2003) after exposure to the antiviral agent ganciclovir (GCV). We used multiple ablation paradigms and found that after an initial and complete loss, GFAP+ NSCs invariably recovered over time, thereby confirming the presence of a GFAP− cell upstream of the adult NSC in the lineage. Additionally, adult mice that are effectively AdpNSC null and do not generate LIF colonies are unable to repopulate the GFAP+ NSC population after ablation. Hence, these findings demonstrate the presence of a rare population of Oct4+ pNSCs in the adult forebrain whose progeny include GFAP+ type prostaglandin receptor that are indeed neurogenic in vivo (Doetsch et al., 1999a) and form neurospheres in vitro (Morshead et al., 2003).
Discussion Our results demonstrate the existence of a rare population of pNSCs in the adult brain that express Oct4 and have the ability to integrate into the ICM of blastocyst chimeras. This LIF-responsive population acts as a reserve pool capable of repopulating the neural lineage in the SE in vivo. Similar to its embryonic counterpart, the AdpNSC is a GFAP−, LIFR+ cell from the periventricular region of the brain. The inability to permanently ablate GFAP+ neurosphere-forming cells after a complete initial loss suggests that the AdpNSC is upstream of the GFAP+ adult NSC (Figure 7D). This lineage relationship is supported by both in vitro and in vivo findings. In vitro, the passaging of the LIF colonies into standard adult neurosphere conditions (EFH) revealed that LIF colonies gave rise to GFAP+, neurosphere-forming cells. In vivo, LIF-colony-derived cells devoid of GFAP+ cells at the time of transplantation were able to migrate along the RMS and contribute to neurogenesis. Further, we showed that Floxed Oct4-Sox1Cre mice that completely lacked AdpNSCs were unable to repopulate the GFAP+, neurosphere-forming NSCs after ablation in vivo. Hence, based on the studies described herein, we propose that the AdpNSC proliferates in response to injury and gives rise to GFAP+ adult NSCs that repopulate the SE after their ablation in vivo. Several pieces of evidence indicate that GFAP+ NSCs can be ablated completely in vivo after GCV treatment in GFAP-TK mice, and that they are subsequently reconstituted from a GFAP− cell in the NSC lineage. First, zero neurosphere-forming cells were observed immediately after GCV treatment for 21 days, or after AraC and GCV treatment in GFAP-TK mice. Second, GCV remains toxic to GFAP+ cells for periods of time exceeding the infusion times used here; however, GFAP+ NSC-derived clonal neurospheres inevitably returned in vitro and their proliferating progeny returned in vivo. Third, the neurospheres that returned at longer survival times after GCV infusion were lost when GCV was added in vitro in all instances, and therefore must have been derived from GFAP+ NSCs. Fourth, the observation that EGF alone did not support neurosphere formation immediately after GCV infusion indicates that the neurospheres were not derived from transit-amplifying cells. Finally, the finding that the kinetics of the return of GFAP+ NSCs was dramatically different in paradigms that did not completely eliminate neurosphere formation (i.e., AraC treatment alone) versus when there was a complete loss of GFAP+ NSCs (AraC and GCV treatment) suggests that different cell sources may be responsible for repopulation of the SE. Together, these data support the hypothesis that the GFAP− (resistant to GCV treatment) AdpNSC is able to repopulate the GFAP+ NSC population.