Demonstrated here is an efficient and cost-effective method to purify, culture, and differentiate white matter stem cells from postnatal mouse cerebellum.
Most cerebellar neurons arise from two embryonic stem niches: a rhombic lip niche, which generates all the cerebellar excitatory glutamatergic neurons, and a ventricular zone niche, which generates the inhibitory GABAergic Purkinje cells, which are neurons that constitute the deep cerebellar nuclei and Bergman glia. Recently, a third stem cell niche has been described that arises as a secondary germinal zone from the ventricular zone niche. The cells of this niche are defined by the cell surface marker prominin-1 and are localized to the developing white matter of the postnatal cerebellum. This niche accounts for the late born molecular layer GABAergic interneurons along with postnatally generated cerebellar astrocytes. In addition to their developmental role, this niche is gaining translational importance in regards to its involvement in neurodegeneration and tumorigenesis. The biology of these cells has been difficult to decipher because of a lack of efficient techniques for their purification. Demonstrated here are efficient methods to purify, culture, and differentiate these postnatal cerebellar stem cells.
The cerebellum has been long recognized as a major neuronal circuit coordinating voluntary movement1. It receives input from the wide swathes of the neuroaxis, which includes proprioceptive information from the periphery, so as to fine tune motor output and coordinate motion. More recently, it has also been implicated in regulating cognition and emotions by potentially using similar information processing networks2,3,4.
The adult cerebellum is composed of an outer cerebellar cortex and inner white matter. Interspersed within these structures are deep intracerebellar nuclei. Similar to the rest of the nervous system, the development of the cerebellum is driven by the proliferation of multipotent progenitor cells (stem cells) that migrate and differentiate to yield this well-organized structure. In early development (E10.5–E13.5), a ventricular stem niche around the developing fourth ventricle generates GABAergic neurons (i.e., Purkinje cells, Lugaro cells, Golgi cells) along with Bergmann glia5,6,7,8.
Later in development (postnatal week one), a second stem cell niche in the rhombic lip generates MATH1- and Nestin-expressing progenitors that give rise to excitatory granule neurons9,10,11,12. Recently a third stem cell niche has been described13. These cells express prominin-1 (also known as CD133), a membrane-spanning glycoprotein that defines a subset of stem cells in the intestine and hematopoietic systems14,15,16. In vivo fate mapping shows that these stem cells generate key molecular layer interneurons (i.e., basket cells and stellate cells), along with astrocytes, during the first three postnatal weeks. In the past, it has been difficult to study these cells in vitro because prior methods have required costly and time-consuming techniques (i.e., fluorescence-activated cell sorting [FACS]) that are dependent on prominin-1 staining12,13,17. This protocol describes an immunomagnetic-based method for the isolation of these stem cells that can then be readily cultured and differentiated.
All animal experiments were performed in compliance with the NIH’s Guide for the Care and Use of Laboratory Animals (2011) and were approved by the Northwestern University IACUC (Protocol IS00011368).
1. Preparation of Solutions
2. Dissection of Cerebellum
3. Cell Suspension Preparation
4. Immunolabeling of Stem Cells
5. Magnetic Column Preparation, Cell Sorting, and Plating
NOTE: The magnetic separation from different genotype conditions (disease vs. control) must be performed at same time, since any delay may affect the neurosphere morphology.
6. Passaging of Neurospheres and Differentiation
Prominin-1-positive postnatal cerebellar stem cells formed neurospheres in neurosphere medium rich in growth factors (EGF and bFGF). These neurospheres were positive for prominin-1-staining, the marker used for isolation, and also as a stain for other stem cell markers such as Nestin and GFAP13 (Figure 1). The stem cell marker expression was maintained throughout culture and for up to at least eight passages20. Upon withdrawal of growth factors and in the presence of LIF and PDGF-AA (which are factors that support neuronal and glial differentiation21,22), the neurospheres differentiated into neuronal and glial lineages (Figure 2).
Figure 1: Isolation of prominin-1 stem cells from postnatal cerebellum. (A) Cerebellar stem cells were isolated using immunomagnetic prominin-1 beads. Top panel: Purified stem cells (column-bound) formed neurospheres with extensive proliferation and self-renewal properties. The cells unbound to the column (flowthrough) were unable to form neurospheres; instead, they became cerebellar neuronal/glial mixed cells (β-III tubulin/GFAP). Bottom panel: the neurospheres formed from prominin-1+ cells expressing stem cell-specific markers: Nestin, prominin-1, and GFAP. (B) Cells in the flowthrough stained negative for stem cell markers prominin-1 and nestin and positive for neuronal marker β-III tubulin. Please click here to view a larger version of this figure.
Figure 2: Differentiation of prominin-1 positive neurospheres. In the presence of differentiation factors (PDGF-AA or LIF), prominin-1-positive neurospheres differentiated into neurons (β-III tubulin), astrocytes (GFAP), and oligodendrocytes (O4). Please click here to view a larger version of this figure.
Prominin-1-expressing cerebellar stem cells reside in the prospective white matter during the first 3 weeks of postnatal life. Their proliferation is tightly controlled by the sonic hedgehog pathway supported by Purkinje cells17. These stem cells/progenitors contribute to later-born GABAergic interneurons called basket cells and stellate cells. These interneurons reside in the molecular layer, where they synapse onto Purkinje cells and sculpt PC topography and function via GABAergic inhibition13,17,23. Besides forming interneurons, this stem cell population also generates all postnatally derived cerebellar astrocytes17,24.
This protocol describes an easy and cost-effective method to purify prominin-1/CD133 stem cells from the postnatal mouse cerebellum. Stem cells must be cultured in ultra-low attachment plates. Culturing these stem cells in normal plates may cause the neurospheres to attach to the surface and lead to low stem cell proliferation and differentiation. Here, the yield of 200–300 neurospheres per 5,000 cells corresponds to a stem cell yield of around 1 x 107 cells from a single cerebellum. This is comparable to what has been described for an FACS-based strategy that is 10x more expensive. Moreover, FACS equipment requires an expensive set-up and highly trained personnel, and is not readily available.
These stem cells are also gaining increasing translational significance in research on cancer as well as neurodevelopmental and neurodegenerative disorders25,26,27. Uncontrolled proliferation of these stem cells in early life leads to medulloblastoma28, while research from our own lab suggests that their abnormal proliferation and differentiation can contribute to later cerebellar degeneration in the genetic disease spinocerebellar ataxia type 120. These new protocols will be valuable for studying these cells and provide novel insight into their roles in health and disease. These methods may also lead to advances in regenerative therapies after stroke or trauma and other insults to the brain that would warrant neuroregeneration. It is conceivable that these techniques can be generalized to extract prominin-1-expressing stem cells from other tissues, such as intestine and bone marrow, where they are also expressed15,29.
The authors have nothing to disclose.
We thank the Opal lab members for their suggestions. This work was supported by NIH grants 1RO1 NS062051 and 1RO1NS08251 (Opal P)
0.05%Trypsin | Thermo Fisher Scientific | 25300054 | 0.05% |
2% B27 | Gibco; Thermo Fisher Scientific | 17504001 | |
2mM EDTA solution | Corning | 46-034-CI | |
Anti- Prominin-1 microbeads | Miltenyi Biote | 130-092-333 | |
bovine serum albumin | Sigma | A9418 | |
Column MultiStand | Miltenyi Biotec | 130-042-303 | |
culture plates ultra – low attachment | Corning | 3473 | |
cysteine | Sigma | C7880 | |
DNase | Sigma | D4513-1VL | 250 U/ml |
Dulbecco’s Phosphate Buffer Saline | Thermo Fisher Scientific | 14040141 | |
Hank's balanced salt solution-HBSS | Gibco | 14025-092 | |
Human recombinant Basic Fibroblast Growth Factor | Promega | G507A | 20 ng/ml |
Human recombinant Epidermal Growth Factor | Promega | G502A | 20 ng/ml |
Leukemia Inhibitory Factor | Sigma | L5158 | |
l-glutamine | Gibco | 25030081 | |
Microscopy | Lieca TCS SP5 confocal microscopes | ||
MiniMACS separator | Miltenyi Biotec | 130-042-102 | |
mouse anti-Prominin-1 | Affymetrix eBioscience | 14-1331 | 1 in 100 |
Nestin | Abcam | ab27952 | 1 in 200 |
Neurobasal medium | Thermo Fisher | 25030081 | |
O4 | Millopore | MAB345 | |
Papain | Worthington | LS003126 | (100 U/ml) |
Platelet- Derived Growth Factor | Sigma | H8291 | 10 ng/ml |
Poly-D-Lysine | Sigma | P6407 | |
rabbit anti-tubulin, b-III | Sigma | T2200 | 1 in 500 |
Rabit anti-GFAP | Dako | Z0334 | 1 in 500 |
Separation columns-MS columns | Miltenyi Biotec | 130-042-201 | |
Sterile cell strainer | Fisher Scientific | 22363547 | 40um |