Progress and prospects: gene transfer into embryonic stem cells

Yates, F.; Daley, George Q.

doi:10.1038/sj.gt.3302854

Cited by 37 publications

(24 citation statements)

References 71 publications

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“…9 Transfection in hESC was beset with poor rate of stable integration. [10][11][12] Moreover, when successfully integrated, transgene expression, especially that of fluorescent proteins, was often not maintained for prolonged periods in transfected hESC.…”

Section: Introductionmentioning

confidence: 99%

Genetic Manipulation of Neural Progenitors Derived from Human Embryonic Stem Cells

Dhara

Gerwe

Majumder

et al. 2009

Tissue Engineering Part A

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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.

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Section: Introductionmentioning

confidence: 99%

Genetic Manipulation of Neural Progenitors Derived from Human Embryonic Stem Cells

Dhara

Gerwe

Majumder

et al. 2009

Tissue Engineering Part A

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“…To overcome concerns about irreversible genetic alterations, we used the PiggyBac transposon system which allows fully reversible gene delivery. In this regard, PiggyBac is not only superior to viral vectors which mediate irreversible gene integration but also to previously describe transposases including Sleeping Beauty based systems that can be excised but leave mutations in the host genome upon excision (Yates and Daley, 2006). To minimize the impact of ABO mismatch, we used H1 hESC since they have previously been shown to carry type O alleles (Chen et al, 2008).…”

Section: Discussionmentioning

confidence: 98%

Heterelogous expression of mutated HLA-G decreases immunogenicity of human embryonic stem cells and their epidermal derivatives

2014

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Human embryonic stem cells (hESCs) are capable of extensive self-renewal and expansion and can differentiate into any somatic tissue, making them useful for regenerative medicine applications. Allogeneic transplantation of hESC-derived tissues from results in immunological rejection absent adjunctive immunosuppression. The goal of our study was to generate a universal pluripotent stem cell source by nucleofecting a mutated human leukocyte antigen G (mHLA-G) gene into hESCs using the PiggyBac transposon. We successfully generated stable mHLA-G(EF1α)-hESC lines using chEF1α promoter system that stably expressed mHLA-G protein during prolonged undifferentiated proliferation andin differentiated embryoid bodies as well as teratomas. Morphology, karyotype, and telomerase activity of mHLA-G expressing hESC were normal. Immunofluorescence staining and flow cytometry analysis revealed persistent expression of pluripotent markers, OCT-3/4 and SSEA-4, in undifferentiated mHLA-G(EF1α)-hESC. Nucleofected hESC formed teratomas and when directed to differentiate into epidermal precursors, expressed high levels of mHLA-G and keratinocyte markers K14 and CD29. Natural killer cell cytotoxicity assays demonstrated a significant decrease in lysis of mHLA-G(EF1a)-hESC targets relative to control cells. Similar results were obtained with mHLA-G(EF1α)-hESC-derived epidermal progenitors (hEEP). One way mixed T lymphocyte reactions unveiled that mHLA-G(EF1a)-hESC and -hEEP restrained the proliferative activity of mixed T lymphocytes. We conclude that heterologous expression of mHLA-G decreases immunogenicity of hESCs and their epidermal differentiated derivatives.

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“…The ability to introduce viral DNA to virtually all cell types from any origin, including many different human cell lines [2,[95][96][97][98][99][100][101], have raised an interest in the use of baculoviruses for gene delivery. For many reasons, baculoviruses appear to be ideal gene transfer vectors for mammalian cells.…”

Section: Gene Delivery Into Mammalian Cells By Recombinant Baculovirusesmentioning

confidence: 99%

Application of Baculovirus-Insect Cell Expression System for Human Therapy

Rychłowska

Gromadzka²,

Bieńkowska-Szewczyk³

et al. 2011

CPB

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A major advantage of recombinant DNA technology is its flexibility allowing for "on demand" production of specific proteins with theurapeutic value in heterologous expression systems. Gene expression vectors based on baculovirus, insect virus attacking mostly lepidopteran species, are frequently used for relatively inexpensive and fast production of such proteins. This expression system is recognized as one of the most powerful technologies for commercial synthesis of glycoproteins originating from vertebrate themselves or from vertebrate viruses. Glycosylation pathways utilized by insects are not identical, though they are similar to vertebrate glycosylation pathways. In the review special attention is given to the development of new virus-like particles (VLPs) potential vaccines which represent a novel class of subunit vaccines that are able to stimulate efficiently cellular and humoral immune responses against viral agents. Apart from production of vertebrate proteins or VLPs "on demand " in insect cells, a new exciting field of using baculovirus as gene delivery system to vertebrate cells was recently open which has a great potential for future uses of baculovirus as effective gene therapy vector.

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Progress and prospects: gene transfer into embryonic stem cells

Cited by 37 publications

References 71 publications

Genetic Manipulation of Neural Progenitors Derived from Human Embryonic Stem Cells

Genetic Manipulation of Neural Progenitors Derived from Human Embryonic Stem Cells

Heterelogous expression of mutated HLA-G decreases immunogenicity of human embryonic stem cells and their epidermal derivatives

Application of Baculovirus-Insect Cell Expression System for Human Therapy

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