Stem cells have been identified in many parts of the nervous system in embryonic life and in adulthood. However, there has been little evidence for the presence of stem cells in the cerebellum, and these cells have not been isolated or studied in detail36,37
. We have used flow cytometry to prospectively purify multipotent neural stem cells from the developing cerebellum. We show that these cells express stem cell markers, proliferate in response to bFGF and EGF, generate self-renewing neurospheres, and differentiate into neurons and glia in vitro
and after transplantation into neonatal mice. These findings have important implications for our understanding of normal cerebellar development and tumorigenesis.
Our identification of cerebellar stem cells originated from studies of bFGF responsiveness in GCPs. Although previous reports have suggested that bFGF is a weak mitogen for GCPs12
, we have observed that it is a potent inhibitor of Shh-induced GCP proliferation11
. To explain this discrepancy, we purified GCPs to homogeneity by FACS sorting GFP+
cells from Math1-GFP mice. Analysis of the resulting cells showed that highly purified GCPs did not proliferate to bFGF. On the other hand, we observed a significant proliferative response to bFGF in a small population of cells that co-fractionates with GCPs during the purification procedure. Thus, Math1-GFP mice represent a powerful tool for purifying different classes of neurons and glia from the cerebellum.
Among the GFP-
cells in the postnatal cerebellum, a subset expressed markers associated with neural stem cells, including prominin-1. Prominin is associated with plasma membrane protrusions in embryonic and adult epithelial cells, and is also a marker of hematopoietic stem cells22
. In the nervous system, prominin has been reported to colocalize with nestin in the ventricular zone25
. Moreover, antibodies to the human homolog of prominin, CD133, have been used to isolate multipotent stem cells from fetal brain and from brain tumor tissue26,38
. Thus, prominin is an important surface marker for neural stem cells. Expression of prominin has not been described previously in the postnatal cerebellum, but our studies suggest that it marks multipotent progenitors in this tissue as well.
We used antibodies to prominin to isolate progenitors from the postnatal cerebellum and tested their ability to form neurospheres. We found that prominin+
cells are highly enriched for neurosphere formation as compared to unfractionated cells from the postnatal cerebellum. Moreover, neurospheres derived from prominin+
cells can undergo self-renewal and differentiate into both neurons and glia. The fact that these cells can be prospectively isolated has also allowed us to examine their potential without subjecting them to prolonged culture in vitro
. This is important, because a number of recent studies have indicated that culturing cells under neurosphere-generating conditions can alter the types of cells generated39
. The fact that we can generate neurons, astrocytes and oligodendrocytes after transplantation of either single neurospheres or freshly isolated prominin+
cells suggests that such de-differentiation has not taken place in our studies. Rather, the tri-lineage potential of our cells seems to reflect an intrinsic characteristic of the cells we have isolated.
Our demonstration that purified cerebellar stem cells can generate neurons, astrocytes and oligodendrocytes in vitro
and after transplantation has important implications for cerebellar development. Although the postnatal cerebellum has long been known to contain precursors of basket and stellate neurons, astrocytes and oligodendrocytes2
, it has not been clear whether these cells arise from distinct precursors or from a single multipotent progenitor. Several studies have hinted at the latter possibility. For example, oncogene-immortalized cell lines derived from the postnatal cerebellum express neuronal and glial markers in vitro
and can generate neurons and glia after implantation in the cerebellum and other parts of the brain40,41
. Although it has been suggested that these cells represent GCPs whose differentiation potential has been altered by oncogenes40
, our studies suggest they may be stem cells that have been selectively immortalized in culture. Similarly, an elegant series of retroviral lineage-tracing studies8,42
has shown that the white matter contains progenitors that give rise to interneurons and glia. In these studies, a small proportion of retrovirally marked cells did not stain with lineage markers, and it was suggested that these might represent uncommitted stem cells42
. because we have detected expression of prominin primarily in white matter, the NSCs we have isolated may represent these uncommitted cells.
In addition to generating GABA and Pax2-expressing interneurons, prominin+
cells also generated glutamate and Zic-1-expressing granule cells. Although both granule and non-granule cells are generated in the postnatal cerebellum, several studies have suggested that they arise from distinct progenitors8,9,43
. One interpretation of our results is that cerebellar NSCs have the potential to generate granule cells in vitro
, but that in vivo
they generate exclusively non-granule neurons and glia. Consistent with this view, we have not observed any granule neurons generated after transplantation of cerebellar stem cells into the neonatal cerebellum. On the other hand, we cannot rule out the possibility that some of the granule cells generated during postnatal development come from multipotent progenitors rather than from restricted GCPs in the EGL. These cells, if they are rare, would be difficult to detect using conventional lineage tracing and transplantation studies. More saturating methods of fate mapping44
might shed light on this issue.
In addition to their importance for normal development, our studies also have significant implications for understanding medulloblastoma. The cell of origin for medulloblastoma has been a matter of longstanding debate. A subset of medulloblastomas express markers of GCPs and have mutations in the Shh pathway45,46
, and are therefore likely to arise from committed GCPs. However, the majority of medulloblastomas express distinct markers and show no evidence of Shh pathway activation46,47
. In light of recent studies demonstrating expression of CD133 and other NSC markers in human medulloblastoma38,48
, it is important to consider the possibility that some of these tumors arise from the stem cells we have described. If so, understanding the signaling pathways that control growth and differentiation of these cells may yield new approaches to the diagnosis or therapy of this devastating tumor.