Polycistronic Lentiviral Vector for “Hit and Run” Reprogramming of Adult Skin Fibroblasts to Induced Pluripotent Stem Cells

Stem Cells - Tập 27 Số 5 - Trang 1042-1049 - 2009
Chia‐Wei Chang1, Yi-Shin Lai1, Kevin M. Pawlik1, Kaimao Liu1, Chengzhen Sun1, Chao Li1, Trenton R. Schoeb2, Tim M. Townes1
1Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama, USA
2Department of Genetics, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, Alabama, USA.

Tóm tắt

Abstract We report the derivation of induced pluripotent stem (iPS) cells from adult skin fibroblasts using a single, polycistronic lentiviral vector encoding the reprogramming factors Oct4, Sox2, and Klf4. Porcine teschovirus-1 2A sequences that trigger ribosome skipping were inserted between human cDNAs for these factors, and the polycistron was subcloned downstream of the elongation factor 1 alpha promoter in a self-inactivating (SIN) lentiviral vector containing a loxP site in the truncated 3′ long terminal repeat (LTR). Adult skin fibroblasts from a humanized mouse model of sickle cell disease were transduced with this single lentiviral vector, and iPS cell colonies were picked within 30 days. These cells expressed endogenous Oct4, Sox2, Nanog, alkaline phosphatase, stage-specific embryonic antigen-1, and other markers of pluripotency. The iPS cells produced teratomas containing tissue derived from all three germ layers after injection into immunocompromised mice and formed high-level chimeras after injection into murine blastocysts. iPS cell lines with as few as three lentiviral insertions were obtained. Expression of Cre recombinase in these iPS cells resulted in deletion of the lentiviral vector, and sequencing of insertion sites demonstrated that remnant 291-bp SIN LTRs containing a single loxP site did not interrupt coding sequences, promoters, or known regulatory elements. These results suggest that a single, polycistronic “hit and run” vector can safely and effectively reprogram adult dermal fibroblasts into iPS cells. Disclosure of potential conflicts of interest is found at the end of this article.

Từ khóa


Tài liệu tham khảo

Takahashi, 2006, Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors, Cell, 126, 663, 10.1016/j.cell.2006.07.024

Hanna, 2007, Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin, Science, 318, 1920, 10.1126/science.1152092

Maherali, 2007, Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution, Cell Stem Cell, 1, 55, 10.1016/j.stem.2007.05.014

Meissner, 2007, Direct reprogramming of genetically unmodified fibroblasts into pluripotent stem cells, Nature Biotechnol, 25, 1177, 10.1038/nbt1335

Okita, 2007, Generation of germline-competent induced pluripotent stem cells, Nature, 448, 313, 10.1038/nature05934

Takahashi, 2007, Induction of pluripotent stem cells from fibroblast cultures, Nat Protoc, 2, 3081, 10.1038/nprot.2007.418

Takahashi, 2007, Induction of pluripotent stem cells from adult human fibroblasts by defined factors, Cell, 131, 861, 10.1016/j.cell.2007.11.019

Aasen, 2008, Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes, Nat Biotechnol, 26, 1276, 10.1038/nbt.1503

Aoi, 2008, Generation of pluripotent stem cells from adult mouse liver and stomach cells, Science, 321, 699, 10.1126/science.1154884

Brambrink, 2008, Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells, Cell Stem Cell, 2, 151, 10.1016/j.stem.2008.01.004

Dimos, 2008, Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons, Science, 321, 1218, 10.1126/science.1158799

Eminli, 2008, Reprogramming of neural progenitor cells into induced pluripotent stem cells in the absence of exogenous Sox2 expression, Stem Cells, 26, 2467, 10.1634/stemcells.2008-0317

Hanna, 2008, Direct reprogramming of terminally differentiated mature B lymphocytes to pluripotency, Cell, 133, 250, 10.1016/j.cell.2008.03.028

Huangfu, 2008, Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2, Nat Biotechnol, 26, 1269, 10.1038/nbt.1502

Kim, 2008, Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors, Nature, 454, 646, 10.1038/nature07061

Lowry, 2008, Generation of human induced pluripotent stem cells from dermal fibroblasts, Proc Natl Acad Sci USA, 105, 2883, 10.1073/pnas.0711983105

Okita, 2008, Generation of mouse induced pluripotent stem cells without viral vectors, Science, 322, 949, 10.1126/science.1164270

Park, 2008, Disease-specific induced pluripotent stem cells, Cell, 134, 877, 10.1016/j.cell.2008.07.041

Park, 2008, Generation of human-induced pluripotent stem cells, Nat Protoc, 3, 1180, 10.1038/nprot.2008.92

Park, 2008, Reprogramming of human somatic cells to pluripotency with defined factors, Nature, 451, 141, 10.1038/nature06534

Silva, 2008, Promotion of reprogramming to ground state pluripotency by signal inhibition, PLoS Biol, 6, e253, 10.1371/journal.pbio.0060253

Stadtfeld, 2008, Defining molecular cornerstones during fibroblast to iPS cell reprogramming in mouse, Cell Stem Cell, 2, 230, 10.1016/j.stem.2008.02.001

Stadtfeld, 2008, Induced pluripotent stem cells generated without viral integration, Science, 322, 945, 10.1126/science.1162494

Varas, 2008, Fibroblast derived induced pluripotent stem cells show no common retroviral vector insertions, Stem Cells

Wernig, 2008, Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson's disease, Proc Natl Acad Sci USA, 105, 5856, 10.1073/pnas.0801677105

Wu, 2006, Correction of sickle cell disease by homologous recombination in embryonic stem cells, Blood, 108, 1183, 10.1182/blood-2006-02-004812

Holst, 2006, Generation of T-cell receptor retrogenic mice, Nat Protoc, 1, 406, 10.1038/nprot.2006.61

Levasseur, 2003, Correction of a mouse model of sickle cell disease: lentiviral/antisickling beta-globin gene transduction of unmobilized, purified hematopoietic stem cells, Blood, 102, 4312, 10.1182/blood-2003-04-1251

Donnelly, 2001, Analysis of the aphthovirus 2A/2B polyprotein ‘cleavage’ mechanism indicates not a proteolytic reaction, but a novel translational effect: A putative ribosomal ‘skip’, J Gen Virol, 82, 1013, 10.1099/0022-1317-82-5-1013

Chinnasamy, 2006, Multicistronic lentiviral vectors containing the FMDV 2A cleavage factor demonstrate robust expression of encoded genes at limiting MOI, Virol J, 3, 14, 10.1186/1743-422X-3-14

Zufferey, 1998, Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery, J Virol, 72, 9873, 10.1128/JVI.72.12.9873-9880.1998

Pawlik, 1995, End joining of genomic DNA and transgene DNA in fertilized mouse eggs, Gene, 165, 173, 10.1016/0378-1119(95)00519-C

Silver, 1989, Novel use of polymerase chain reaction to amplify cellular DNA adjacent to an integrated provirus, J Virol, 63, 1924, 10.1128/jvi.63.5.1924-1928.1989

Sommner, 2009, iPS cell generation using a single lentiviral stem cell cassette, Stem Cells, 10.1634/stemcells.2008-1075

Carey, 2009, Reprogramming of murine and human somatic cells using a single polycistronic vector, Proc Natl Acad Sci USA, 106, 157, 10.1073/pnas.0811426106

Townes, 2008, Gene replacement therapy for sickle cell disease and other blood disorders, Hematology, 2008, 193, 10.1182/asheducation-2008.1.193

Thông tin
Thông tin xuất bản

Stem Cells

Tập 27 Số 5

1042-1049

Thông tin tác giả