Improved Method of Producing Human Neural Progenitor Cells of High Purity and in Large Quantities from Pluripotent Stem Cells for Transplantation Studies

Abstract

Transplantation of human neural progenitor cells (hNPCs) is a promising therapeutic approach for various diseases of the central nervous system (CNS). Reliable testing of hNPC transplantation in animal models of neurological diseases requires that these cells can be produced in sufficient amounts, show consistent homogeneity as a neural cell population, and be reliably labeled for in vivo tracking. In addition, the cells should be characterized as being at the optimal state of differentiation favoring successful engraftment. Here, we show that high numbers of purified hNPCs can be produced from human embryonic stem cells (hESCs) by manually selecting specifically sized and shaped spheres followed by fluorescence-activated cell sorting based on the relative cell size. In addition, we report that labeling of hNPCs with ultra-small superparamagnetic iron oxide (USPIO) particles does not affect the cellular morphology or growth. More importantly, we show that the transduction with lentiviral vector encoding green fluorescent protein (GFP) decreases the neurality of the cell population. We conclude that our cost-effective protocol of generating hNPCs is widely applicable for preclinical studies on CNS disorders. This improved method of producing large quantities of high-purity hNPCs maybe useful also when generating hNPCs from human induced pluripotent stem (hiPS) cell lines. However, caution should be used when lenti-GFP transduction is applied for hNPC labeling.

Evaluating reporter genes of different luciferases for optimized in vivo bioluminescence imaging of transplanted neural stem cells in the brain

“…However, potential side effects of imaging reporter overexpression cannot be excluded, as Puttonen et al . reported recently . The vulnerability of NSCs towards high reporter gene expression limited the imaging reporter expression levels.…”

Human neural progenitor cells retain viability, phenotype, proliferation, and lineage differentiation when labeled with a novel iron oxide nanoparticle, Molday ION Rhodamine B

“…Recombinant fluorescent proteins (eg, green fluorescent protein) have been used to unequivocally distinguish transplanted human neural progenitor cells from host brain cells. 9 Unfortunately, this approach is less suitable for human studies because of the added risk posed by genetic alteration of the cellular therapy. Antibodies against human cellular antigens, while useful for histological identification of transplanted neuroprogenitor cells in animal studies, are clearly not useful for human brain transplantation.…”

Induced Pluripotent Stem Cells Derived from a CLN5 Patient Manifest Phenotypic Characteristics of Neuronal Ceroid Lipofuscinoses

“…Neural differentiation of iPSCs was performed according to previously described protocols [50,51]. Briefly, iPSC colonies grown on feeders were first monitored by light microscopy followed by scraping the feeders from the plate using a pipette tip or a needle.…”