258: Characterization and time lapse experiments of fetal neuroprogenitor cells development derived from cerebral spinal fluid

Abstract

Our objective was to capture and characterize the in vitro differentiation patterns of primary fetal neural progenitor cells (NPCs) isolated from fetal CSF obtained at the time of intrauterine myelomeningocele (MMC) repair. To optimize our differentiation of the MMC NPC cells, we developed a neonatal porcine model. We isolated NPCsfrom CSF as well as the sub ventricular zone (SVZ), which served as a positive control. Using fluorescent probes specific to progenitors (CDR3) and neurons (NeuO), we gated on porcine SVZ (“Brain”) and CSF populations during FACS. RT-qPCR and microscopy were employed in parallel as a means of molecular phenotyping aimed at confirmation of cellular phenotyping by FACS. We also performed a time lapse experiment over five days for observation of the differentiation pattern of these cells. Our fluorescence- activated cell sorting (FACS) results confirmed thatall of our neonatal piglet derivative cultures from SVZ differentiated into cells with NPC cellular phenotyping (CDR3+,NeuOlo/hi), but CSF from neonatal pigs retained a more heterogeneous population (Fig.1). Among human fetal MMC derived cells, we similarly derived NPCs with a minimum of 10% live cells which did not express markers of hematopoietic lineage cells (CDR3+/CD133+/CD45-). For both human and piglet derivatives, neurospheres were observed in culture on day 4, and persisted for at least 14 days (Fig.2) by time lapse microscopy. We have successfully demonstrated our capacity to isolate and differentiate NPCs from fetal human and neonatal piglet CSF. This is a key and novel in vitro model for future in vitro studies aimed at phenotyping neuronal single gene disorders and determining NPC susceptibility to congenital viral infections.View Large Image Figure ViewerDownload Hi-res image Download (PPT)