J Biomed. 2016 March; 1(1):e5347.

Published online 2016 March 21.

doi: 10.17795/jbm-5347.

Research Article

Optimization of Lentiviral Transduction Procedure for the Generationof Human Induced Pluripotent Stem Cells

Fatemeh Movahedi Motlagh,1,2 Bahareh Rajaei,1 Mohammad Massumi,1 Maryam Kabir Salmani,1 andHamid Reza Soleimanpour-lichaei1,*

1Department of Stem Cells and Regenerative Medicine, National Institute of Genetic Engineering and Biotechnology, Tehran, IR Iran2Sabzevar University of Medical Sciences, Sabzevar, IR Iran

*Corresponding author: Hamid Reza Soleimanpour-lichaei, Department of Stem Cells and Regenerative Medicine, National Institute of Genetic Engineering andBiotechnology, Tehran, IR Iran. Tel: +98-2144580324, Fax: +98-2189789727, E-mail: hrs@nigeb.ac.ir

Received 2016 January 10; Accepted 2016 January 26.

Abstract

Background: Human somatic cells are reprogrammed to induced pluripotent stem (iPS) cells when four transcription factors (Oct4,Sox2, Klf4, and Myc) are ectopically expressed in them. These iPS cells, which evade immunological rejection, are a valuable sourcefor patient-specific cell therapy. Lentiviral systems have been proved to be powerful tools for cellular reprogramming, an example ofwhich is the induction of iPS cells from somatic cells that requires a high transduction efficiency of lentiviruses harboring the fourreprogramming factors.Objectives: The purpose of this study was to define an optimized calcium phosphate transfection method to produce high-titer lentivi-ral vectors for the generation of human iPS cells.Materials andMethods: In this study, the calcium phosphate transfection method was used to generate lentiviruses. The virus super-natant was concentrated using Amicon Ultra-4 column.Results: This method resulted in 80% GFP-positive cells and viral preparations of 2.4 × 108 viral particles/mL.Conclusions: This method is both cost effective and simple to adopt.

Keywords: Lentiviral Transduction, Induced Pluripotent Stem Cells, Calcium Phosphate Transfection Method

1. Background

Lentiviruses such as the human immunodeficiencyvirus (HIV) are a subcategory of retroviruses used as aneffective gene delivery system. Their advantages for genetherapy include long-term expression owing to their inte-gration into the host genome, large capacity for delivery ofDNAs, ability to infect both growth-arrested and dividingcells, and biosafety (1, 2).

To abolish the pathogenicity of HIV, six of thepathogenic genes were removed from the HIV-derivedvector system without any alteration in its gene-transfercapability. Therefore, the parental virus cannot be recon-stituted.

Viral transduction of four transcription factors hasbeen used for generating induced pluripotent stem (iPS)cells in both humans and mice (3-8). Efficient lentivirusproduction is a prerequisite for transferring pluripotencygenes into the host cell. For a satisfactory gene expres-sion, a high number of virus particles per cell target,MOI, is needed (9). Owing to their high proliferativeyield and autologous nature, iPS cells provide numerouspromising applications in the fields of tissue engineering,

patient-specific cell therapy, personalized drug discovery,and modeling of several genetic and metabolic diseases.In addition to surpassing the immune system rejection,iPS cells are advantageous over embryonic stem cells be-cause their application obviates the need for the contro-versial destruction of early-stage embryo, thereby evadingthe subsequent ethical issues. Thus, iPS cells mimic manycanonical characteristics of ES cells, including the abilityto self-renew indefinitely and to differentiate into all threegerm layers.

2. Objectives

The purpose of this study was to define a simple andoptimized calcium phosphate transfection method to pro-duce high-titer lentiviral vectors by transient transfectionof 293T cells and concentrating the viral particles usingAmicon Ultra-4 column. This is a convenient methodthat maximizes the efficiency of lentiviral gene transfer,which is a prerequisite for efficient gene transfer. Calciumphosphate transfection is the only method that involvessmall ions that are the natural compounds in the culture

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Movahedi Motlagh F et al.

medium and have no impact on cell viability and productquality (10).

3. Materials andMethods

3.1. Cell Lines

HEK 293T cells and fibroblasts were cultured inhigh-glucose DMEM supplemented with 10% FBS (Gibco)(vol/vol), 2 mM GlutaMAX, 50 U/mL penicillin, and 50mg/mL streptomycin in a 37°C humidified incubator with5% CO2. The 293T cells were split in ratios of 1:4 to 1:6, threetimes per week. Frequent passages maintain the hightransfection ability of the 293T cells.

3.2. Isolation of Human Fibroblasts

Human fibroblasts were isolated according to themethod described by Raya et al. (2010) and the collagenaseI treatment method. Skin biopsies measuring 4 mm de-rived from T1DM patients were minced into smaller pieces(0.5 mm) using scalpels and then subjected to the above-mentioned two methods, as follows:

1) Tissue fragments were placed into a 60-mm tissueculture dish under a sterile coverslip, and the dishes wereincubated at 37°C in a humidified incubator (5% CO2) (11).

2) Tissue fragments were incubated with DMEM/F12containing 1 mg/mL collagenase I (Invitrogen) at 37°C. Col-lagenase was removed and washed with PBS. Cells werecultured in an appropriate volume of the medium (DMEMsupplemented with 10% FBS, glutamine, sodium pyruvate,nonessential amino acids, and penicillin/streptomycin).When the fibroblasts proliferated into sufficiently highnumbers, the cells were trypsinized and expanded.

3.3. Production of Lentivirus

293T cells were seeded at 6× 106 cells per 100-mm dishand incubated at 37°C under 5% CO2, 1 day before transfec-tion. The 293T cells were cotransfected with pMD2G (8 µg)encoding the vesicular stomatitis virus glycoprotein (VSV-G), psPax2 (15 µg) gag-pol plasmids, and eGFP plasmid (21µg) using CaPO4 precipitation method as described pre-viously (1). At about 16 hours post-transfection, the me-dia was replaced with fresh media, and 48 - 72 hours post-transfection, the media containing the viruses were col-lected, filtered through a 0.45-mm pore size filter (What-man), and then concentrated 100-fold using Amicon Ultra-4 column.

VSV-G pseudo-typed lentivectors have a half-life of 24hours at 37°C. The vector stocks aliquots were stored at 4°Cfor up to 24 hours, or frozen at -80°C for longer storage (12).

3.4. Titration

Viral titration was determined by transducing 3 × 105

cells in 2 mL of high-glucose DMEM in a six-well plate.The titer was determined using 1 and 0.5 µL of concen-trated fresh viruses with 8 µg/mL polybrene (Sigma). Thepercentage of labeled cells was determined 72 hours post-infection. Then, the biological titer (BT = TU/mL, transduc-ing units) was calculated according to the following for-mula:

(3× 105InfectedCells×%OfGFP− PositiveCells)

VirusVolume(uL)×100

(1)

To ensure the accuracy for titer calculations, the num-ber of GFP-positive cells was counted for two wells infectedwith two consecutive dilutions. The resulting counts cor-responded to the expected 1:2 ratio.

4. Results

Lentiviruses were generated by the cotransfection of293T cells with eGFP plasmid along with two plasmidsencoding the structural and envelope proteins accordingto the optimized calcium phosphate transfection method(Figure 1). The green fluorescent protein (GFP) was used asa suitable quantifiable marker to determine the biologicaltiter.

The virus supernatant was concentrated using AmiconUltra-4 column. To determine the quantity of infectiousparticles per milliliter of the supernatant, 293T cells wereinfected with lentiviruses for the titration of GFP viruses(Figure 2). We employed polybrene to enhance lentiviraltransduction (8 µg/mL polybrene). Next, the biologicaltiter was calculated as BT = TU/mL, in transducing units.

This method resulted in viral preparations of 2.4 × 108

viral particles/mL. We produced high-titer lentiviral vec-tors by the transient transfection of 293T cells and thenconcentrated the viral particles by centrifugation.

To confirm the efficiency of the generated viruses forthe transfection of human primary cells, we introducedGFP-harboring lentiviruses into human adult fibroblastsusing the abovementioned method. For this purpose, to-tal multiplicities of infection (MOIs) of 2 were used thatyielded a transduction efficiency of 80% (Figure 3).

5. Discussion

Transduction of exogenous genes via the lentiviralsystem into primary cells requires the preparation ofhigh-titer viral stocks, for which numerous considerationsshould be taken into account, including the type of cell line

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Figure 1. GFP Emission of Transfected 293T Cells With Lentiviral Vectors Harboring GFP Gene As a Traceable Marker (12 Hour Post-Transfection)

Figure 2. 293T Cells Transfected With GFP-Harboring Lentiviruses for 72 Hours to Calculate Biological Titer

used, the volume of medium added for the generation ofvector particles, the duration of infection, and the amountof polycations causing the binding of vector particles tothe surface of target cells (12).

The selection of a stable producer cell line, such as the293T cells, is very important for an efficient production oflentiviruses. During transfection, gene transfer would beenhanced due to DNA condensation and enhancement ofbinding to cells by calcium phosphate (13). Some parame-ters could impact the efficiency of transfection, includingthe pH of HEPES-buffered saline, the temperature, the qual-ity of FBS used, the cell density, the passage of cells used,and the amount and quality of DNA (1, 12).

For increasing the infectivity of the produced viruses,

consideration of some factors could be useful, such asharvesting of supernatants at an early time period afterthe start of virus production, collection in a serum-freemedium, and concentration of the produced viruses (2).

Logan et al. (2004) showed that the time of virus collec-tion between 48 and 72 hours does not change the ability ofinfection, but a longer incubation period decreases the in-fectivity of the produced viruses. They also demonstratedthat the presence of serum during virus production mayresult in a negative regulation of transduction (2).

Transduction efficiency can also be enhanced by copre-cipitation of viral vectors with calcium phosphate, whichis then boosted by VSV-G component of the lentiviral par-ticles, as this glycoprotein component of lentiviruses fa-

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Movahedi Motlagh F et al.

Figure 3. Isolated Human Primary Fibroblast Cells Transfected by GFP-Harboring Lentiviruses

cilitates their entry into the cells via interactions with thephospholipid component of the cell membrane (13). To in-crease the viral transduction efficiency, we concentratedthe virus supernatant using Amicon Ultra-4 column.

We chose the 293T cells for titration, because they arehighly amenable to transduction by lentiviral vectors andare easy to handle for cultivation (12). The titration of GFP-positive cells was determined by counting the green fluo-rescent cells visualized with a fluorescence microscope.

In conclusion, this study suggests that calcium phos-phate transfection is a simple, easy, and efficient methodto transfer lentivirus genes into 293T cells. Therefore, thismethod could be used as an efficient way for the transduc-tion of SOKM gene cassette, harboring a polycistron of thefour transcription factors (Oct4, Sox2, Klf4, and Myc), intohuman fibroblast cells to generate iPS cells.

References

1. Tiscornia G, Singer O, Verma IM. Production and purifica-tion of lentiviral vectors. Nat Protoc. 2006;1(1):241–5. doi:10.1038/nprot.2006.37. [PubMed: 17406239].

2. Logan AC, Nightingale SJ, Haas DL, Cho GJ, Pepper KA, Kohn DB. Factorsinfluencing the titer and infectivity of lentiviral vectors. Hum GeneTher. 2004;15(10):976–88. doi: 10.1089/hum.2004.15.976. [PubMed:15585113].

3. Maherali N, Sridharan R, Xie W, Utikal J, Eminli S, Arnold K, et al. Di-rectly reprogrammed fibroblasts show global epigenetic remodelingand widespread tissue contribution. Cell Stem Cell. 2007;1(1):55–70.doi: 10.1016/j.stem.2007.05.014. [PubMed: 18371336].

4. Okita K, Ichisaka T, Yamanaka S. Generation of germline-competentinduced pluripotent stem cells. Nature. 2007;448(7151):313–7. doi:10.1038/nature05934. [PubMed: 17554338].

5. Park IH, Lerou PH, Zhao R, Huo H, Daley GQ. Generation of human-induced pluripotent stem cells. Nat Protoc. 2008;3(7):1180–6. doi:10.1038/nprot.2008.92. [PubMed: 18600223].

6. Takahashi K, Yamanaka S. Induction of pluripotent stem cells frommouse embryonic and adult fibroblast cultures by defined factors.Cell. 2006;126(4):663–76. doi: 10.1016/j.cell.2006.07.024. [PubMed:16904174].

7. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al.Induction of pluripotent stem cells from adult human fibroblasts bydefined factors. Cell. 2007;131(5):861–72. doi: 10.1016/j.cell.2007.11.019.[PubMed: 18035408].

8. Wernig M, Meissner A, Foreman R, Brambrink T, Ku M, HochedlingerK, et al. In vitro reprogramming of fibroblasts into a pluripo-tent ES-cell-like state. Nature. 2007;448(7151):318–24. doi: 10.1038/na-ture05944. [PubMed: 17554336].

9. Zhang B, Xia HQ, Cleghorn G, Gobe G, West M, Wei MQ. A highlyefficient and consistent method for harvesting large volumesof high-titre lentiviral vectors. Gene Ther. 2001;8(22):1745–51. doi:10.1038/sj.gt.3301587. [PubMed: 11892843].

10. Jordan M, Wurm F. Transfection of adherent and suspendedcells by calcium phosphate. Methods. 2004;33(2):136–43. doi:10.1016/j.ymeth.2003.11.011. [PubMed: 15121168].

11. Raya A, Rodriguez-Piza I, Navarro S, Richaud-Patin Y, Guenechea G,Sanchez-Danes A, et al. A protocol describing the genetic correction ofsomatic human cells and subsequent generation of iPS cells. Nat Pro-toc. 2010;5(4):647–60. doi: 10.1038/nprot.2010.9. [PubMed: 20224565].

12. Zufferey R, Trono D. Production of High-Titer Lentiviral Vectors. Currprotoc hum genet. 2002 doi: 10.1002/0471142905.hg1210s26.

13. Sakoda T, Kasahara N, Kedes L, Ohyanagi M. Calcium phosphate co-precipitation greatly enhances transduction of cardiac myocytes andvascular smooth muscle cells by lentivirus vectors. Exp Clin Cardiol.2007;12(3):133–8. [PubMed: 18650994].

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