Neuronal conditioning medium and nerve growth factor induce neuronal differentiation of collagen-adherent progenitors derived from human umbilical cord blood

Abstract

The aim of the study was to isolate and characterize a population of neuronal progenitors in the human umbilical cord blood (HUCB) mononuclear cell (MNC) fraction, for in vitro manipulation towards neuronal differentiation. Selection of the HUCB neuronal progenitors (HUCBNPs) was based on the neuronal prerequisite for adherence to collagen. Populations of collagen-adherent, nestin-positive (94.8+/-2.9%) progenitors expressing alpha1/2 integrin receptors, as revealed by Western blot and adhesion assay using selective antagonists, were isolated and survived for more than 14 days. In vitro differentiation of the HUCBNPs was achieved by treatment with 10% human SH-SY5Y neuroblastoma cell-conditioning media (CM) supplemented with 10 ng/ml nerve growth factor (NGF). Some 83+/-8.2% of the surviving progenitors acquired a neuronal-like morphology, expressed by cellular outgrowths of different lengths. About 35+/-6% of the HUCBNPs had long outgrowths with a length/cell diameter ratio greater than 2, typical of developing neurons. The majority of these progenitors, analyzed by immunocytochemistry and/or RT-PCR, expressed common neuronal markers such as microtubule-associated protein 2 (MAP-2; 98.5+/-2%), neurotrophin receptor (TrkA; 98.5+/-0.06%), neurofillament-160 (NF-160; 94.2+/-1%), beta-tubulin III (89.8+/-4.2%) and neuron specific enolase (NSE). Combined CM and NGF treatment induced constitutive activation of the mitogen-activated protein kinases ERK2 (36-fold vs control), p38alpha (nine-fold vs control) and p38beta (23-fold vs control), most likely related to survival and/or differentiation. The results point to operationally defined conditions for activating neuronal differentiation of HUCBNPs ex vivo and emphasize the crucial role of neuronal CM and NGF in this process.