Genetic Manipulation of Neural Progenitors Derived from Human Embryonic Stem Cells

Abstract

Human embryonic stem cell-derived neural progenitors (NP) present an important tool for understanding human development and disease. Optimal utilization of NP cells, however, requires an enhanced ability to monitor these cells in vitro and in vivo. Here we report production of the first genetically modified self-renewing human embryonic stem cell-derived NP cells that express fluorescent proteins under constitutive as well as lineage-specific promoters, enabling tracking and monitoring of cell fate. Nucleofection, transfection, and lentiviral transduction were compared for optimal gene delivery to NP cells. Transduction was most efficient in terms of transgene expression (37%), cell viability (39%), and long-term reporter expression (>3 months). Further, the constitutive gene promoters, cytomegalovirus, elongation factor 1a, and ubiquitin-C, exhibited comparable silencing (20-30%) in NP cells over a 2-month period, suggesting their suitability for long-term reporter expression studies. Transduced NP cells maintained their progenitor state and differentiation potential, as demonstrated by expression of endogenous NP markers and neuronal markers after differentiation. We also detected reporter expression in astrocytes generated from NP cells transduced with an astrocyte-specific gene promoter, glial fibrillary acidic protein, demonstrating the usefulness of this approach. The genetically manipulated NP cells described here offer great potential for live cell-tracking experiments, and a similar approach can as well be used for expression of proteins other than reporters.

Nuclear and cytoplasmic localization of neural stem cell microRNAs

“…The cells are commercially available with rigorous and welldescribed quality control procedures (Dhara et al 2009). Cells were harvested while in linear growth, and lysates were quickly partitioned into nuclear and cytoplasmic fractions with a Norgen Biotek system.…”

“…Cells used in our experiments were neural stem cells derived from human embryonic WA09 cell lines (Aruna Biomedical) as described by Dhara and colleagues (Dhara et al 2009). According to the goals of the provider are optimal differentiation of human embryonic stem cells (hESCs) to NP cells and their maintenance as a self-renewing population, available for further generation of pure populations of specified cell types in culture.…”

Neurotrophic Effects of Leukemia Inhibitory Factor on Neural Cells Derived from Human Embryonic Stem Cells

“…We have previously derived a self-renewing but fate-restricted neural progenitor (NP) cell line from hESC that can generate highly enriched neuronal populations under specific conditions [2,3], thus serving as a potentially unlimited source of human neurons for both basic [4] and applied research [5]. This progenitor line has previously been used to study neurotoxicity, differentiation to specific neuronal phenotypes, drug screening, and transplantation experiments in rodent models of stroke [4,5].…”

“…We have previously derived a self-renewing but fate-restricted neural progenitor (NP) cell line from hESC that can generate highly enriched neuronal populations under specific conditions [2,3], thus serving as a potentially unlimited source of human neurons for both basic [4] and applied research [5]. This progenitor line has previously been used to study neurotoxicity, differentiation to specific neuronal phenotypes, drug screening, and transplantation experiments in rodent models of stroke [4,5]. However, the ultimate success of NP cell-based therapy in transplantation experiments may depend on identifying factors that enhance the capacity of these cells to expand in vitro and resisting suboptimal conditions such as the generation of reactive oxygen species (ROS) [6][7][8] and to survive and function in a diseased or injured niche.…”

Gene Transduction Approaches in Human Embryonic Stem Cells

“…Additionally, these drug-selected cardiomyocytes exhibited a gene expression profile similar to that of adult human cardiomyocytes and generated force and action potentials consistent with normal fetal cardiomyocytes. Lentiviral approaches have also been used to engineer hESCs with glial fibrillary acidic protein (GFAP) (Dhara et al, 2009) and alpha-fetoprotein (AFP) (Chiao et al, 2008) promoters driving expression of genes encoding fluorescent proteins to enrich for hESCs differentiating towards the neural and hepatic lineages, respectively.…”