Optimization of Episomal Reprogramming for Generation of Human Induced Pluripotent Stem Cells from Fibroblasts

Overview

Journal Anim Cells Syst (Seoul)

Specialty Biology

Date 2018 Nov 22

PMID 30460090

Citations 14

Authors

Jin Seok Bang

Na Young Choi

Minseong Lee

Kisung Ko

Hye Jeong Lee

Yo Seph Park

Dahee Jeong

Hyung-Min Chung

Kinarm Ko

Affiliations

Soon will be listed here.

Abstract

Generation of induced pluripotent stem cells (iPSCs) by defined factors (, , , and ) from various human primary cells has been reported. Human fibroblasts have been widely used as a cellular source in reprogramming studies over recent decades. The original method of iPSC generation uses retro- or lentivirus vectors that require integration of viral DNA into the target cells. The integration of exogenous genes encoding transcription factors (, , , and ) can be detected in iPSCs, raising concern about the risk of mutagenesis and tumor formation. Therefore, stem cell therapy would ideally require generation of integration-free iPSCs using non-integration gene delivery system such as Sendai virus, recombinant proteins, synthetic mRNA, and episomal vectors. Several groups have reported that episomal vectors are capable of reprogramming human fibroblasts into iPSCs. Although vector concentration and cell density are important in the episomal vector reprogramming method, optimization of this method for human fibroblasts has not been reported. In this study, we determined optimal conditions for generating integration-free iPSCs from human fibroblasts through the use of different concentrations of episomal vectors (/, /, /) and different plating cell density. We found that optimized vector concentration and cell density accelerate reprogramming and improve iPSC generation. Our study provides a detailed stepwise protocol for improved generation of integration-free iPSCs from human fibroblasts by transfection with episomal vectors.

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References

Carlotti F, Bazuine M, Kekarainen T, Seppen J, Pognonec P, Maassen J . Lentiviral vectors efficiently transduce quiescent mature 3T3-L1 adipocytes. Mol Ther. 2004; 9(2):209-17. DOI: 10.1016/j.ymthe.2003.11.021. View

Okita K, Matsumura Y, Sato Y, Okada A, Morizane A, Okamoto S . A more efficient method to generate integration-free human iPS cells. Nat Methods. 2011; 8(5):409-12. DOI: 10.1038/nmeth.1591. View

Zhou Y, Zeng F . Integration-free methods for generating induced pluripotent stem cells. Genomics Proteomics Bioinformatics. 2013; 11(5):284-7. PMC: 4357834. DOI: 10.1016/j.gpb.2013.09.008. View

Wen W, Zhang J, Xu J, Su R, Neises A, Ji G . Enhanced Generation of Integration-free iPSCs from Human Adult Peripheral Blood Mononuclear Cells with an Optimal Combination of Episomal Vectors. Stem Cell Reports. 2016; 6(6):873-884. PMC: 4911493. DOI: 10.1016/j.stemcr.2016.04.005. View

Woltjen K, Michael I, Mohseni P, Desai R, Mileikovsky M, Hamalainen R . piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature. 2009; 458(7239):766-70. PMC: 3758996. DOI: 10.1038/nature07863. View

Jia F, Wilson K, Sun N, Gupta D, Huang M, Li Z . A nonviral minicircle vector for deriving human iPS cells. Nat Methods. 2010; 7(3):197-9. PMC: 2892897. DOI: 10.1038/nmeth.1426. View

Fusaki N, Ban H, Nishiyama A, Saeki K, Hasegawa M . Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc Jpn Acad Ser B Phys Biol Sci. 2009; 85(8):348-62. PMC: 3621571. DOI: 10.2183/pjab.85.348. View

Zhao H, Davies T, Ning J, Chang Y, Sachamitr P, Sattler S . A highly optimized protocol for reprogramming cancer cells to pluripotency using nonviral plasmid vectors. Cell Reprogram. 2014; 17(1):7-18. PMC: 4312798. DOI: 10.1089/cell.2014.0046. View

Palchetti S, Pozzi D, Marchini C, Amici A, Andreani C, Bartolacci C . Manipulation of lipoplex concentration at the cell surface boosts transfection efficiency in hard-to-transfect cells. Nanomedicine. 2016; 13(2):681-691. DOI: 10.1016/j.nano.2016.08.019. View

10.

Sia J, Sun R, Chu J, Li S . Dynamic culture improves cell reprogramming efficiency. Biomaterials. 2016; 92:36-45. PMC: 4983311. DOI: 10.1016/j.biomaterials.2016.03.033. View

11.

Zhou T, Benda C, Duzinger S, Huang Y, Li X, Li Y . Generation of induced pluripotent stem cells from urine. J Am Soc Nephrol. 2011; 22(7):1221-8. PMC: 3137570. DOI: 10.1681/ASN.2011010106. View

12.

Drozd A, Walczak M, Piaskowski S, Stoczynska-Fidelus E, Rieske P, Grzela D . Generation of human iPSCs from cells of fibroblastic and epithelial origin by means of the oriP/EBNA-1 episomal reprogramming system. Stem Cell Res Ther. 2015; 6:122. PMC: 4515927. DOI: 10.1186/s13287-015-0112-3. View

13.

Okita K, Hong H, Takahashi K, Yamanaka S . Generation of mouse-induced pluripotent stem cells with plasmid vectors. Nat Protoc. 2010; 5(3):418-28. DOI: 10.1038/nprot.2009.231. View

14.

Li D, Wang L, Hou J, Shen Q, Chen Q, Wang X . Optimized Approaches for Generation of Integration-free iPSCs from Human Urine-Derived Cells with Small Molecules and Autologous Feeder. Stem Cell Reports. 2016; 6(5):717-728. PMC: 4939659. DOI: 10.1016/j.stemcr.2016.04.001. View

15.

Yu J, Hu K, Smuga-Otto K, Tian S, Stewart R, Slukvin I . Human induced pluripotent stem cells free of vector and transgene sequences. Science. 2009; 324(5928):797-801. PMC: 2758053. DOI: 10.1126/science.1172482. View

16.

Kim D, Kim C, Moon J, Chung Y, Chang M, Han B . Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell. 2009; 4(6):472-6. PMC: 2705327. DOI: 10.1016/j.stem.2009.05.005. View

17.

Staerk J, Dawlaty M, Gao Q, Maetzel D, Hanna J, Sommer C . Reprogramming of human peripheral blood cells to induced pluripotent stem cells. Cell Stem Cell. 2010; 7(1):20-4. PMC: 2917234. DOI: 10.1016/j.stem.2010.06.002. View

18.

Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K . Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007; 131(5):861-72. DOI: 10.1016/j.cell.2007.11.019. View