Protocol to Isolate a Large Amount of Functional Oligodendrocyte Precursor Cells from the Cerebral Cortex of Adult Mice and Humans

Abstract

During development, oligodendrocytes are generated from oligodendrocyte precursor cells (OPCs), a cell type that is a significant proportion of the total cells (3-8%) in the adult central nervous system (CNS) of both rodents and humans. Adult OPCs are responsible for the spontaneous remyelination that occurs in demyelinating diseases like Multiple Sclerosis (MS) and they constitute an interesting source of cells for regenerative therapy in such conditions. However, there is little data regarding the neurobiology of adult OPCs isolated from mice since an efficient method to isolate them has yet to be established. We have designed a protocol to obtain viable adult OPCs from the cerebral cortex of different mouse strains and we have compared its efficiency with other well-known methods. In addition, we show that this protocol is also useful to isolate functional OPCs from human brain biopsies. Using this method we can isolate primary cortical OPCs in sufficient quantities so as to be able to study their survival, maturation and function, and to facilitate an evaluation of their utility in myelin repair.

Endocannabinoids drive the acquisition of an alternative phenotype in microglia

“…Adult human biopsies of non-tumor origin (epilepsy) were obtained from the Neurosurgery Service at the Hospital La Princesa (Madrid, Spain). Biopsies were rapidly transported to the Instituto Cajal (Madrid, Spain) in DMEM: F10 + HEPES + 1% Fetal Bovine Serum at 4°C, and processed as described (Durafourt et al, 2013;Medina-Rodríguez et al, 2013), with some modifications. Briefly, meninges and blood vessels were removed with sterilized scalpels and forceps; brain cortices were washed with PBS to remove contaminating blood, and minced with a sterile surgical scalpel into small fragments.…”

Differential expression of sema3A and sema7A in a murine model of multiple sclerosis: Implications for a therapeutic design

“…OPCs were obtained from P0 and P60 CD1 mice as described [15,42]. Briefly, mice were decapitated and brains removed and placed in cold HBSS with Ca 2+ and Mg 2+ (Gibco).…”

“…At the end of the purification process, OPCs were counted and seeded in laminin-coated coverslips (Sigma). For OPCs immunostainings, 20,000 OPCs were seeded in Bottestein Sato medium containing DMEM (Gibco) with 1% FBS (Lonza BioWhittaker™), 1% L-glutamine (Gibco), 0.03% BSA (Sigma), 1% antibiotic/antimycotic Solution (Sigma), 0.3 ng/ml 3,3′,5-triiodo-L-thyronine (T3, Sigma), 0.4 ng/ml thyroxine (Sigma), 16 μg/ml putrescine (Sigma), 40 ng/ml sodium selenite (Sigma), 9.3 μg/ml insulin (Sigma), 0.1 mg/ml holo-transferrin (Sigma) and 62 ng/ml progesterone (Sigma) for 24 h. For immunostaining of differentiated oligodendroglia, 20,000 OPCs were seeded in BME:F12 medium containing BME:F12 (1:1, Gibco) supplemented with 100 μg/ml of holo-transferrin (Sigma), 20 μg/ml of putrescine (Sigma), 12.8 ng/ml of progesterone (Sigma), 10.4 ng/ml of sodium selenite (Sigma), 25 μg/ml of insulin (Sigma), 0.8 μg/ml of thyroxine (Sigma), 0.6% D(+)-glucose (Normapur® VWR International, Leics, England), 6.6 mM L-glutamine (Gibco) and 1% antibiotic/antimycotic Solution (Sigma) for 5 days [42,43].…”

Finding degrees of separation: Experimental approaches for astroglial and oligodendroglial cell isolation and genetic targeting

“…These procedures primarily used embryonic or neonatal mouse cortices. Interestingly, a recent revival of the shake-off/selective adhesion method was described for mouse OPCs not only from neonatal but also adult cerebral cortex (Medina-Rodriguez et al, 2013) as well as human adult biopsies. This protocol uses papain to aid the removal of meninges and choroid plexus, and to facilitate mechanical dissociation.…”

“…The papain concentration, incubation time and cell plating density are increased with age of the animal (up to P180). Unlike the McCarthy and deVellis protocol, PDGF-AA is included in the growth medium for plating of mixed glia of postnatal and adult mice, and the time in culture before shake-off varies from a minimum of 15 days for P15 cortices to 25 days for P180 (Medina-Rodriguez et al, 2013) (Table 1). When compared directly, the yield of cells with this method was reportedly an order of magnitude higher than with FACS (Medina-Rodriguez et al, 2013).…”