Non-viral gene transfer by nucleofection allows stable gene expression in human neural progenitor cells.

Human neural progenitor cells (hNPCs) are a promising source to treat various neurodegenerative diseases. Potential applications are to use such cells for reprogramming to induce pluripotent stem cells or for secretion of proteins into the brain. These applications usually involve expression of heterologously expressed genes which is difficult to achieve in hNPCs. We tested several protocols for non-viral gene transfer and different promoters. Nucleofection and the cytomegalovirus enhancer/chicken beta-actin promoter allowed expression of foreign genes in hNPCs for up to 6 months. Treatment with the antibiotic G418 enabled us to select stably transfected cells which were subcloned and continued to express the NPC marker nestin. Differentiation of stably nucleofected hNPCs revealed that multipotency was maintained following long-term expansion of subcloned hNPCs. After differentiation for 3 weeks in vitro or in vivo following striatal transplantations transfected hNPCs expressed voltage-gated sodium channels suggesting the development of functional properties during neuronal maturation. In conclusion, stably nucleofected hNPCs can be isolated, subcloned, and expanded for up to 6 months without loss of their differentiation potential. These data provide a basis for future studies using hNPCs to investigate the neuronal differentiation in vivo after transplantation, the feasibility as a vector for gene (protein) therapy, and the induction of pluripotent stem cells.

Uracil nucleotides stimulate human neural precursor cell proliferation and dopaminergic differentiation: involvement of MEK/ERK signalling.

Isolation and propagation of neural stem cells derived from human brain tissue uniquely enables the study of human neurogenesis in vitro. In addition, ex vivo-expanded human neural stem/precursor cells (NPCs) may offer novel therapeutic strategies. We investigated the effects of extracellular nucleotides on the proliferation and differentiation of human mesencephalic neural stem/precursor cells (hmNPCs). When combined with the mitogens epidermal growth factor and fibroblast growth factor 2, UTP (1 microm) boosted proliferation of hmNPCs as shown by increased expression of the proliferation marker proliferating cell nuclear antigen (330%). UTP-induced proliferation was abrogated by the preferential P2Y receptor blocker pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). UTP also stimulated dopaminergic differentiation. Treatment with UTP (100 microm) increased the number of tyrosine hydroxylase (TH)-positive cells and TH protein by 267 and 319% respectively. UTP-stimulated dopaminergic differentiation of hmNPCs was blocked by the P2 receptor antagonists suramin (10 microm) and PPADS (100 microm). In addition, UDP (1 microm) enhanced TH protein expression by 194%. During differentiation, treatment with UTP stimulated the extracellular signal-regulated kinase (ERK) pathway. Both ERK1/2 phosphorylation and dopaminergic differentiation were inhibited by U0126, a selective ERK kinase inhibitor, as well as by suramin. When other P2 receptor agonists (ATP, ADP and adenosine 5'-O-(2-thiophosphate) (ADPbetaS); all 100 microm) were applied, both proliferation and dopaminergic differentiation of NPCs were compromised. We conclude that uracil nucleotides exert specific P2 receptor-mediated effects on midbrain-derived human NPCs, and may be used to enhance both proliferation and dopaminergic differentiation.