Transduction of human embryonic stem cells by ecotropic retroviral vectors

Abstract

The steadily increasing availability of human embryonic stem (hES) cell lines has created strong interest in applying available tools for gene transfer in murine cells to human systems. Here we present a method for the transduction of hES cells with ecotropic retroviral vectors. hES cells were transiently transfected with a construct carrying the murine retrovirus receptor mCAT1. Subsequently, the cells were exposed to replication-deficient Moloney murine leukemia virus (MoMuLV) derivatives or pseudotyped lentiviral vectors. With oncoretroviral vectors, this procedure yields overall transduction efficiencies of up to 20% and permits selection of permanently transduced clones with high frequency. Selected clones maintained expression of pluripotency-associated markers and exhibited multi-germ layer differentiation both in vitro and in vivo. HES cell-derived somatic cells including neural progeny maintained high levels of transgene expression. Lentiviral vectors pseudotyped with the MoMuLV envelope could be introduced in the same manner with efficiencies of up to 33%. Transgene expression of lentivirally transduced hES cells remained permanent after differentiation even without selection pressure. Bypassing the regulatory issues associated with the use of amphotropic retroviral systems and exploiting the large pool of existing murine vectors, this method provides a safe and versatile tool for gene transfer and lineage analysis in hES cells and their progeny.

A rosette-type, self-renewing human ES cell-derived neural stem cell with potential for in vitro instruction and synaptic integration

“…S4). Some of the spotted cells had been engineered to express EGFP under control of the phosphoglycerate kinase (PGK) promoter element (10). In this coculture paradigm, 99 out of 1,071 deposited cells (9.2%) generated clones containing Ͼ20 cells within 2 weeks.…”

“…Human embryonic stem cells (hES cells; lines H9.2 passages 32-61, I3 passages 55-82, and I6 passages 42-55) were maintained according to standard protocols (1). Differentiation to neural rosettes was performed as described previously (4,5,10). Lt-hESNSCs were maintained as monolayer culture on polyornithine/laminin (both Sigma-Aldrich) coated plastic dishes in neural stem cell medium (NSCM) containing DMEM/F12, N2 supplement (1:100; both Invitrogen), 20 g/mL additional insulin (Sigma-Aldrich), 1.6 g/L glucose, 10 ng/mL FGF2 (R&D Systems), 10 ng/mL EGF (R&D Systems), and 1 L/mL B27 supplement (Invitrogen).…”

Electrophysiological evaluation of engrafted stem cell-derived neurons

“…The third technology additionally serves as a useful tool for studying the maturation and integration of donor cells in controlled, real-time scenarios. Crucial for studying vital donor cells in recipient tissues is the availability of genetically labeled donor cells that are readily identifiable in situ by expression of fluorescent indicator proteins (e.g., enhanced green fluorescent protein (EGFP)), under the control of either cell-type specific or constitutively active promoters [25][26][27][28][29][30] .…”

Genetic Manipulation of Neural Progenitors Derived from Human Embryonic Stem Cells

“…Previous reports have described genetic manipulation at the level of ES cells, with subsequent differentiation to neural cells, 6 or have used NPs isolated from fetal sources and modified them genetically. 7,8 For example, fetal rat NP cells were modified for spinal cord or brain transplantation utilizing a lentiviral delivery system and the cytomegalovirus (CMV) promoter driving expression of green fluorescent protein or neurotrophic factors (brain-derived neurotrophic factor, ciliary neurotrophic factor, glial cell derived neurotrophic factor, neurotrophin-3 and its mutated forms).…”