Genetic manipulation of neural progenitors derived from human embryonic stem cells.
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 1alpha, 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.
Regenerative Bioscience Center, University of Georgia, Athens, Georgia 30602, USA.
Sujoy K
SK
Dhara
Brian A
BA
Gerwe
Anirban
A
Majumder
Mahesh C
MC
Dodla
Nolan L
NL
Boyd
David W
DW
Machacek
Kowser
K
Hasneen
Steven L
SL
Stice
D002454:Cell Differentiation / Q000235:genetics; D049109:Cell Proliferation; D002470:Cell Survival / Q000235:genetics; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000502:physiology*; D024861:Genetic Enhancement / Q000379:methods*; D006801:Humans; D009474:Neurons / Q000166:cytology* / Q000502:physiology*