Isolation, characterization and preclinical development of human glial-restricted progenitor cells for treatment of neurological disorders.

AIM: Glial-restricted progenitor cells (GRPs), a neural cell population that gives rise to astrocytes and oligodendrocytes both in vitro and in vivo, hold great promise as a cellular therapeutic for the treatment of demyelinating and neurodegenerative diseases of the CNS. The manufacturing and characterization protocols of human-derived GRPs (hGRPs; trade name Q-Cells) for use in a clinical setting that adhere to rigorous standards for their isolation, propagation, characterization and storage are presented. MATERIALS & METHODS: hGRPs, defined by their immunoreactivity with A2B5 antibodies, were isolated from fetal cadaver forebrain tissue of mice 17-24 weeks gestational age using Miltenyi paramagnetic bead cell separation technology. GRPs were grown in a defined xenobiotic-free medium for 6 days. At harvest, hGRPs were characterized using immunocytochemical techniques. Long-term cryopreservation and storage conditions, and viability upon freeze-thaw were determined. The phenotypic differentiation potential of hGRPs was determined by implantation experiments into the CNS of shiverer mice. RESULTS: hGRPs were isolated from over 50 neural tissues of either sex during gestational ages of 17-24 weeks. Cells expanded out to 6 days in vitro in a xenobiotic-free medium demonstrated very consistent immunocytochemical profiles. No residual antibody used in the purification process was detected after 6 days of growth in vitro. GRPs could be frozen at up to 24 million cells/ml and were over 70% viable upon freeze-thaw. Thawed hGRPs transplanted into the brain of the dysmyelinated shiverer mouse model were observed to differentiate into both glial fibrillary acidic protein-positive astrocytes and myelin basic protein-positive oligodendrocytes; no human-derived NeuN-positive neuronal cells were observed and no abnormal cell proliferation was observed. CONCLUSION: We demonstrate that hGRPs can be consistently obtained, propagated, cryopreserved and characterized using protocols that can be transferred to a good laboratory practice/good manufacturing practice setting for the manufacture of clinical-grade hGRP cellular therapeutics. Functional data demonstrate that cells manufactured under these conditions are able to differentiate into appropriate cellular phenotypes in an animal model of dysmyelination.

Modulation of group III metabotropic glutamate receptors by hydrogen ions.

Protons act as neuromodulators and produce significant effects on synaptic transmission. The molecular basis of neuromodulation by extracellular protons is partially explained by their effects on certain neurotransmitter receptors and ion channels. The metabotropic glutamate receptors (mGluRs) are a family of eight receptor subtypes that are widely expressed throughout the mammalian CNS. In this study, the effects of physiologically relevant changes in extracellular pH were examined in mammalian cells expressing the mGluR subtypes: human mGluR1a, mGluR4a, mGluR5d or mGluR8b. The signal transduction coupling properties of mGluR4a and mGluR8b were switched from the adenylate cyclase (G(i)) pathway to the phospholipase C (G(q)) pathway by coexpression of a promiscuous G protein. Fluorometric imaging plate reader was used to measure changes in cytoplasmic calcium concentrations in response to agonist. Extracellular acidification from pH 8.0 to pH 6.5 progressively diminished mGluR4 responsiveness to the agonists L-glutamate and (2S,1'S,2'R)-2-(carboxycyclopropyl)glycine (L-CCG-I), and this inhibition was characterized by insurmountable antagonism. By comparison, extracellular acidification did not significantly alter mGluR8 responses to agonists. Furthermore, agonist activation of mGluR1a and mGluR5d was virtually unaffected by changes in pH. Because mGluR4 is expressed presynaptically and its activation inhibits the release of neurotransmitters such as glutamate and GABA, we propose that the net effect of proton inhibition of mGluR4 would be to reverse or prevent that suppression of neurotransmitter release. As such, local decreases in pH could have significant effects on the regulation of transmitter release and synaptic tone via modulation of mGluR4.