Efficient restricted gene expression in beta cells by lentivirus-mediated gene transfer into pancreatic stem/progenitor cells

Castaing, Muriel; Guerci, Aline; Mallet, Jacques; Czernichow, P; Ravassard, Philippe; Scharfmann, Raphaël

doi:10.1007/s00125-005-1694-6

Cited by 39 publications

(52 citation statements)

References 55 publications

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“…The pTrip RIP405 nlsCRE DeltaU3 (RIP-Cre) vector was generated by removing with BamHI and XhoI of the enhanced green fluorescent protein (eGFP) coding region from the pTrip RIP405 eGFP DeltaU3 vector (13), which contains a fragment of the rat insulin II gene from Ϫ405 to ϩ7 relative to the transcription start site. The resulting linearized plasmid was blunt-ended with DNA polymerase I Klenow fragment.…”

Section: Methodsmentioning

confidence: 99%

“…The reporter vector was constructed by amplifying the loxP-DsRed2-loxP cassette by PCR from a plasmid provided by Isabelle Houbracken (Free University, Brussles, Belgium) using the forward primer 5Ј-AATTCACTAGTGAACCTCTTC-3Ј and the reverse primer 5Ј-GATCCGATCATATTCAATAA-3Ј. The resulting PCR product was ligated into the blunt-ended BamHI site of the pTrip cytomegalovirus (CMV) eGFP DeltaU3 vector (13), resulting in the pTrip CMV-loxP-DsRed2-loxP-eGFP DeltaU3 lentiviral vector. Virus particles were produced in 293T cells after vector cotransfection with the pCMVdR8.91 and pMD2.G plasmids.…”

Section: Methodsmentioning

confidence: 99%

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In Vitro Proliferation of Cells Derived From Adult Human β-Cells Revealed By Cell-Lineage Tracing

et al. 2008

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OBJECTIVE-Expansion of insulin-producing ␤-cells from adult human islets could alleviate donor shortage for cellreplacement therapy of diabetes. A major obstacle to development of effective expansion protocols is the rapid loss of ␤-cell markers in the cultured cells. Here, we report a genetic celllineage tracing approach for following the fate of cultured ␤-cells.RESEARCH DESIGN AND METHODS-Cells dissociated from isolated human islets were infected with two lentiviruses, one expressing Cre recombinase under control of the insulin promoter and the other, a reporter cassette with the structure cytomegalovirus promoter-loxP-DsRed2-loxP-eGFP.RESULTS-␤-Cells were efficiently and specifically labeled by the dual virus system. Label ϩ , insulin Ϫ cells derived from ␤-cells were shown to proliferate for a maximum of 16 population doublings, with an approximate doubling time of 7 days. Isolated labeled cells could be expanded in the absence of other pancreas cell types if provided with medium conditioned by pancreatic non-␤-cells. Analysis of mouse islet cells by the same method revealed a much lower proliferation of labeled cells under similar culture conditions. CONCLUSIONS-Our findings provide direct evidence for survival and dedifferentiation of cultured adult human ␤-cells and demonstrate that the dedifferentiated cells significantly proliferate in vitro. The findings confirm the difference between mouse and human ␤-cell proliferation under our culture conditions. These findings demonstrate the feasibility of cell-specific labeling of cultured primary human cells using a genetic recombination approach that was previously restricted to transgenic animals. Diabetes 57:1575-1583, 2008 R estoration of an adequate ␤-cell mass is the central therapeutic goal in patients with type 1 diabetes. However, pancreatic islet transplantation is severely limited by the number of organ donors. Studies of adult islet renewal in vivo suggest that differentiated ␤-cells maintain a replication capacity (1). However, success in expansion of functional insulinproducing ␤-cells in vitro has been limited. Culture of adult human islets resulted in a small number of cell population doublings and loss of insulin expression (2-5). Insulin Ϫ cells derived from such cultures could be induced to re-express insulin; however, insulin levels were low and varied considerably among cells from different donors (6 -8). In the absence of stable markers for cultured ␤-cells, it was not possible to determine whether the loss of ␤-cell phenotype in the expanded cells reflected ␤-cell dedifferentiation or ␤-cell death accompanied by expansion of islet cells from a non-␤-cell origin. Efficient expansion of dedifferentiated human ␤-cells in vitro could be of therapeutic significance because these cells may retain enough of their specific open chromatin structure to facilitate their redifferentiation. Genetic cell-lineage tracing provided evidence for dedifferentiation of cultured mouse ␤-cells (9); however, it could not detect a significant proliferation of th...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

In Vitro Proliferation of Cells Derived From Adult Human β-Cells Revealed By Cell-Lineage Tracing

et al. 2008

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“…The lentiviral vector pTRIP ΔU3.RIP405-eGFP expresses eGFP under the control of the rat insulin II gene promoter (RIP) (41). A new lentiviral vector, pTRIP ΔU3.RIP405-hTERT, was constructed.…”

Section: Dna Constructs and Lentiviral Vector Productionsmentioning

confidence: 99%

“…RIP405-SV40LT, and the primary insulinomas were transduced with pTRIP ΔU3.RIP405-hTERT, as previously described (18,41). Briefly, tissues were transduced with a total amount of lentiviral vectors corresponding to 2 μg of p24 capside protein for 2 hours at 37°C in 200 μl of RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, HEPES (10 mM), l-glutamine (2 mM), nonessential amino acids (Invitrogen), penicillin-…”

Section: Dna Constructs and Lentiviral Vector Productionsmentioning

confidence: 99%

A genetically engineered human pancreatic β cell line exhibiting glucose-inducible insulin secretion

Ravassard¹,

Hazhouz²,

Pechberty³

et al. 2011

J. Clin. Invest.

498

521

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Despite intense efforts over the past 30 years, human pancreatic β cell lines have not been available. Here, we describe a robust technology for producing a functional human β cell line using targeted oncogenesis in human fetal tissue. Human fetal pancreatic buds were transduced with a lentiviral vector that expressed SV40LT under the control of the insulin promoter. The transduced buds were then grafted into SCID mice so that they could develop into mature pancreatic tissue. Upon differentiation, the newly formed SV40LT-expressing β cells proliferated and formed insulinomas. The resulting β cells were then transduced with human telomerase reverse transcriptase (hTERT), grafted into other SCID mice, and finally expanded in vitro to generate cell lines. One of these cell lines, EndoC-βH1, expressed many β cell-specific markers without any substantial expression of markers of other pancreatic cell types. The cells secreted insulin when stimulated by glucose or other insulin secretagogues, and cell transplantation reversed chemically induced diabetes in mice. These cells represent a unique tool for large-scale drug discovery and provide a preclinical model for cell replacement therapy in diabetes. This technology could be generalized to generate other human cell lines when the cell type-specific promoter is available.

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“…The 378-bp resulting PCR fragment was digested by KpnI and PacI restriction endonucleases and next inserted in the SV40 LT and hTERT lentiviral vectors to replace the ΔU3 3′ LTR by a loxP 3′ LTR. The pTRIP ΔU3 CMV-nlsCre and pTRIP ΔU3 CMV-eGFP vectors were described elsewhere (36,37).…”

Section: Dna Constructs and Lentiviral Vector Productionsmentioning

confidence: 99%

Development of a conditionally immortalized human pancreatic β cell line

Scharfmann¹,

Pechberty²,

Hazhouz³

et al. 2014

J. Clin. Invest.

167

193

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Diabetic patients exhibit a reduction in β cells, which secrete insulin to help regulate glucose homeostasis; however, little is known about the factors that regulate proliferation of these cells in human pancreas. Access to primary human β cells is limited and a challenge for both functional studies and drug discovery progress. We previously reported the generation of a human β cell line (EndoC-βH1) that was generated from human fetal pancreas by targeted oncogenesis followed by in vivo cell differentiation in mice. EndoC-βH1 cells display many functional properties of adult β cells, including expression of β cell markers and insulin secretion following glucose stimulation; however, unlike primary β cells, EndoC-βH1 cells continuously proliferate. Here, we devised a strategy to generate conditionally immortalized human β cell lines based on Cre-mediated excision of the immortalizing transgenes. The resulting cell line (EndoC-βH2) could be massively amplified in vitro. After expansion, transgenes were efficiently excised upon Cre expression, leading to an arrest of cell proliferation and pronounced enhancement of β cell-specific features such as insulin expression, content, and secretion. Our data indicate that excised EndoC-βH2 cells are highly representative of human β cells and should be a valuable tool for further analysis of human β cells.

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Efficient restricted gene expression in beta cells by lentivirus-mediated gene transfer into pancreatic stem/progenitor cells

Cited by 39 publications

References 55 publications

In Vitro Proliferation of Cells Derived From Adult Human β-Cells Revealed By Cell-Lineage Tracing

In Vitro Proliferation of Cells Derived From Adult Human β-Cells Revealed By Cell-Lineage Tracing

A genetically engineered human pancreatic β cell line exhibiting glucose-inducible insulin secretion

Development of a conditionally immortalized human pancreatic β cell line

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