This protocol outlines the derivation of Glial Restricted Precursors from fetal spinal cords and maintained in vitro either for transplantation or for the study of oligodendrocytic lineage.
This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors within the oligodendrocytic cell lineage. Recently, these cells have been studied as potential source for restorative therapies in white matter diseases. Periventricular leukomalacia (PVL) is the leading cause of non-genetic white matter disease in childhood and affects up to 50% of extremely premature infants. The data suggest a heightened susceptibility of the developing brain to hypoxia-ischemia, oxidative stress and excitotoxicity that selectively targets nascent white matter. Glial restricted precursors (GRP), oligodendrocyte progenitor cells (OPC) and immature oligodendrocytes (preOL) seem to be key players in the development of PVL and are the subject of continuing studies. Furthermore, previous studies have identified a subset of CNS tissue that has increased susceptibility to glutamate excitotoxicity as well as a developmental pattern to this susceptibility. Our laboratory is currently investigating the role of oligodendrocyte progenitors in PVL and use cells at the GRP stage of development. We utilize these derived GRP cells in several experimental paradigms to test their response to select stresses consistent with PVL. GRP cells can be manipulated in vitro into OPCs and preOL for transplantation experiments with mouse PVL models and in vitro models of PVL-like insults including hypoxia-ischemia. By using cultured cells and in vitro studies there would be reduced variability between experiments which facilitates interpretation of the data. Cultured cells also allows for enrichment of the GRP population while minimizing the impact of contaminating cells of non-GRP phenotype.
Introduction
In this protocol we show how to extract, select and plate glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors within the oligodendrocytic and astrocytic cell lineages and are defined by their expression of A2B5. In the GRP medium supplemented with FGF-2, the A2B5+ GRPs will begin expressing the early oligodendrocytic lineage markers PDGFαR and NG21,2. These oligodendrocyte lineage cells pass through a series of distinct phenotypic stages, each of them characterized by morphological changes, as well as expression of markers to specific developmental stages.
These precursor cells are currently being studied as a potential source for cell-based therapeutic approaches in disorders of the central nervous system white matter, including multiple sclerosis, leukodystrophies and periventricular leukomalacia (PVL)3,4,5,6. Studies using human brain derived oligodendrocyte lineage precursors have reported successful remyelination in brains of the shiverer mouse dysmyelination model as well as in demyelinated spinal cord rodent models6,7,8,9. These glial precursor cells have also been used in studies of other white matter disease models such as spinal cord injury and amyotrophic lateral sclerosis10,11,12,13.
Our laboratory has developed an early postnatal hypoxia-ischemia mouse model with which we are evaluating the efficacy of these spinal cord derived GRP cells as a restorative approach in PVL, the leading cause of cerebral palsy14,15,16. There is also a rodent brain derived OPC model that has been widely used for studying white matter injury since the GRP cells have a tendency to differentiate into the astrocytic pathway17. The OPC phenotype has already entered the oligodendrocyte lineage pathway, a phenomenon that seems irreversible. However, we are interested in assessing the response of a wider spectrum of oligodendrocyte progenitors including GRPs and possibly even the NEPs derived at E10.5. For this reason we have adopted the spinal cord derived GRP model for our work.
In addition to the use of GRPs for cell replacement, the derivation of these cells from wild type and transgenic rodent models of white matter diseases allows further study into glial differentiation under normal and disease conditions in an in vitro setting. Previous publications show evidence of selective vulnerability of oligodendrocytic lineage precursor cells to various external stressors like maturation dependent glutamate excitotoxicity, oxidative stress, and select factors implicated in neuroinflammation18,19. Our studies have focused on understanding the cellular and molecular mechanisms behind the development of these white matter injuries, evaluating the susceptibility of cells in the oligodendrocyte lineage spectrum to insult as well as analyzing putative therapeutic approaches.
Materials
All procedures involving animals conform to PHS policy and the JHU IACUC. All procedures should be performed in a laminar flow hood in order to maintain aseptic conditions. Commonly dissections are performed in a horizontal flow hood and in vitro work in a vertical flow hood. All media used cold during dissections. The mice used in our study are a wild type CD-1 strain and a transgenic GFP in a C57/BL6 background. One CD-1 mouse dam typically yields 10+ fetuses which are enough to seed 1-2 T25 flasks. Typically, two dams are sacrificed at a time and fetal spinal cords pooled and plated into 1 T25 flask per dam. Once cells have been derived they can be expanded and characterized in vitro for subsequent studies.
1. Preparation of Media and Flasks
2. Instruments and Other Material
3. Determining Timed Pregnancy
Pregnant mouse dams are either ordered from commercial sources specifying delivery on embryonic day E12 or E13 of fetal development or pregnancy determined in-house. Briefly, two females are placed together with one male in the late afternoon and left overnight together. The next day the females are observed for the presence of the vaginally located mucus plug. The mucus plug is only an indication that mating occurred so animals are subsequently weighed daily to monitor rate of weight increase. The day the mucus plug is observed is defined as embryonic day one (E1).
4. Tissue Derivation
5. Culture Establishment
6. Immunopanning the GRP Population
7. Freezing and Storage of Derived Cells
8. Representative Results
CO2 is not used for anesthetizing the animal because of the possible downstream effect on the viability of the fetuses and ultimately the derived cells. Furthermore, it will be very difficult to totally remove the meninges from the spinal cord during the derivation procedure but the combination of GRP medium and immunopanning will eliminate these fast growing cells. Similarly, the entire spinal cord is harvested despite a greater ventral concentration of the GRP population due to its originating in the ventral spinal cord and migrating dorsally20. However the GRP medium selects for the GRP phenotype and that population is maximized by A2B5 immunopanning. After plating spinal cord tissues, the in vitro cultures are incubated for two passages or about a week. After 2 days in culture, cells of different morphologies can be observed in the tissue culture flasks indicating the heterogeneity of the population but the GRP medium selects for the GRP phenotype. These cells are a combination of A2B5+ GRP, E-NCAM+ neuronal restricted precursors (NRP) and nestin+, A2B5- neuroepithelial cells and possibly fibronectin+ meningeal cells. In addition, more mature phenotypes may be present expressing markers including doublecortin and Tuj1 for neuronal lineage, GFAP for astrocytes and PDGFaR and GalC for oligo lineage In order to maximize a highly GRP enriched population from the starting heterogeneous pool (Figure 1A), cells can be double-immunopanned to select and eliminate the E-NCAM+ cells followed by selection for the A2B5+ GRP population once cell density has increased to about 85-90% confluency in T75 flasks. These GRP cells maintain their ability to become astrocytes despite being in a medium that selects for the oligodendrocyte phenotype so subsequent A2B5 immunopanning may be necessary to maintain a highly enriched GRP population. Immunopanning generally yields up to 95% A2B5+ cells but for even greater yield A2B5 conjugated magnetic beads (Miltenyi Biotec Inc.) can be used to provide a yield of up to 97%. Vigilance in the maintenance GRP cultures such as regular media changes will minimize the proliferation of non-GRP cell types. Even in the absence of BMP-4 astrocytes may still be found and depleted media in particular seems to induce differentiation to an astrocytic phenotype. Although the B27 supplement contains tri-iodothyronine hormone (T3), there has been no observed tendency to differentiate into oligodendrocytes in the GRP population, only when higher levels of T3 are added to the medium does this phenomenon occur. However, a T3 free B27 supplement can be substituted if there is a concern for contamination by mature oligodendrocytes (Figure 1B). These GRP cells can be readily identified by their morphology of small soma with 2 or 3 short processes but to screen for other cell types that may be present, immunocytochemistry can be performed for the previously named common developmental and cell type specific markers. There will commonly be a small persistent population of A2B5- cells that are neuroepithelial (NEP) and capable of becoming either GRP or NRP. Fortunately, the longer cells are maintained in the GRP medium the more likely that medium will select for the GRP phenotype.
Figure 1. a) Plated spinal cord cells of heterogeneous morphology are observed after 2 days in culture. b) Immunopanning maximizes a highly GRP-enriched population from the starting heterogeneous pool.
Disorders of central nervous system white matter include a large number of etiologies, including genetic, inflammatory, ischemic and toxic causes19,21,22,23,24. While multiple sclerosis and vasculopathies are the leading cause of white matter disease in adults, periventricular leukomalacia associated with prematurity is the most common cause of white matter injury in the childhood population. In order to further delineate disease mechanisms further study of cells ofoligodendrocytic lineage, which are greatly affected by the perinatal insult, is required. GRPs were isolated from the rodent embryonic spinal cord and shown to be tripotential in nature, giving rise to oligodendrocytes and two distinct astrocyte populations, but do not differentiate into neurons2,25,26. Likewise,oligodendrocyte precursor cells (OPC) derived from rat optic nerve and late embryonic or early postnatal rat brain have been demonstrated to mature into functioning oligodendrocytes in the appropriate media conditions27,28.Additionally, fetal or adult derived human OPC have been further manipulated into more mature oligodendrocyte phenotypes29,30.
GRP cells have been isolated from both the spinal cord of E13.5 rats and E12-13.5 in mice. In the rat differences have been reported between dorsal- and ventral-derived GRP cells in their response to conditions that promote generation of oligodendrocytes, or astrocytes with ventral-derived GRPs exhibiting a greater propensity to differentiate into the more mature phenotypes25,26,28. This is may be due to the ventral to dorsal migration of the nascent GRP cells, which could be less receptive to the differentiation cues. Immunopanning allows for the subpopulation that expresses A2B5 to be isolated and collected for maintenance in culture.
Our mouse GRP cultures are derived from total spinal cord tissue that yields a heterogeneous, pooled population of cells from which GRP cells are selected by chemical (medium) and antibody based techniques. While our studies have utilized the spinal cord derived mouse GRP cells, it has more recently been reported the derivation of brain derived mouse oligodendrocyte progenitor cells that are also capable of developing into mature oligodendrocytes31. Here the derived cells are initially cultured as oligospheres that using the appropriate culture medium are manipulated into an enriched PDGFαR+, OPC population31. Our studies have focused on the effect of our model of hypoxic-ischemic insult on cells in the oligodendrocyte lineage spectrum from A2B5+, PDGFαR- GRP precursors to the myelin basic protein expressing (MBP+) mature oligodendrocytes. Indeed our in vitro characterization has shown these immature GRP cells mature into MBP expressing cells either in monocultures or in co-cultures with cortical neurons. These cells can be manipulated in culture into specific differentiation pathways for studying the mechanisms involved in perinatal white matter injury. There are several mouse models of white matter diseases involving cells of oligodendrocyte lineage and the availability of this population allows for thorough investigation into the mechanisms that cause injury as well as to explore the usefulness of possible cell therapy options.
The authors have nothing to disclose.
We wish to thank Dr. Devin Gary for his insight and feedback on the use of GRP cells.
Name of the reagent | Company | Catalogue number |
---|---|---|
DMEM/ F12 1:1 | Invitrogen | 11320033 |
B27 | Invitrogen | 17504044 |
N2 | Invitrogen | 17502048 |
rH-FGF-basic | Invitrogen | PHG0026 |
Trypsin (0.05%) EDTA | Quality Biological, Inc. | 118-087-721 |
POLY-L-LYSINE HBr | Sigma | P1274-25MG |
Laminin | Sigma | L2020-1MG |
Dnase 1 | Sigma | DN25 |