Comparison of the Efficiency of Viral Transduction and ... - Springer Link

DOI 10.1007/s10517-015-3112-5 Cell Technologies in Biology and Medicine, No. 3, November, 2015

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Comparison of the Efficiency of Viral Transduction and Episomal Transfection in Human Fibroblast Reprogramming A. S. Vdovin, A. Yu. Lupatov, I. V. Kholodenko*, and K. N. Yarygin Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 3, pp. 149-154, July, 2015 Original article submitted April 3, 2015 Induced pluripotent cells were derived from adult human skin fibroblast by using two methods of reprogramming. Episomal transfection with vectors containing oriP/EBNA-1 sequence for delivery of reprogramming genes Oct4, Sox2, Klf4, L-Myc, and Lin28 proved to be more effective than viral transduction with Sendai virus-based vector: ~200 and 8 colonies per 105 cells were found on day 21 of culturing, respectively. Colonies of induced pluripotent cells obtained by these two methods expressed pluripotency marker Tra1-60. Key Words: induced pluripotent stem cells; transfection; transduction; reprogramming; differentiation

Cell reprogramming cells is artificially induced dedifferentiation of somatic cells into a pluripotent state capable of differentiation into cells into cells representing the three major germ layers: endoderm, mesoderm or ectoderm. The methods of cell reprogramming are in high demand in the studies of fundamental mechanisms of cell differentiation and de-differentiation and creation of personalized cell cultures that can serve as the source of cell material for tissue engineering, cell therapy, and development of individual models of pathological processes. Pluripotent stem cells (iPS cells) were first successfully generated from somatic cells via introduction of Oct3/4, Sox2, Klf4 and cMyc genes delivered with a retroviral vector [6]. Now, the use of retroviral vectors, in particular lentiviral, is the most efficient method of reprogramming, as this system allows direct gene integration into the cell genome and ensures efficient and stable expression of the reprogramming factors. These systems are characterized by high reproducibility and are efficient even in somatic cells that are difficult to reprogram, for example, cells in the state of replicative senescence V. N. Orekhovich Research Institute of Biomedical Chemistry, Moscow, Russia. Address for correspondence: [email protected]. I. V. Kholodenko

and/or terminally differentiated cells. However, this approach has some limitations, first of all, for clinical application. Stable expression of transgenes possessing oncogenic properties and the possibility of insertional mutagenesis due to integration of the vector construction into the genome put in doubt the prospects of using this method in medicine. In later works, various non-integrating genetic elements with temporal expression, chemical agents, or proteins were used for somatic cell reprogramming were used to avoid the risk of malignant transformation [5,4,9]. Here we compare the effectiveness of two methods for reprogramming of skin fibroblasts: viral transduction and episomal transfection with vectors that do not integrate into the genome of somatic cells and carry genes responsible for the dedifferentiation.

MATERIALS AND METHODS Fibroblasts were isolated from forearm skin biopsy specimens taken from a male 23-year-old volunteer as described previously [1]. The cells were cultured in complete growth medium DMEM/F-12-GlutaMax supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin (all components were from Gibco) in 75-cm2 culture flasks (Greiner) in CO2-

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Cell Technologies in Biology and Medicine, No. 3, November, 2015

incubator (5% CO2, 37oC). After attaining 80-90% confluence, skin fibroblasts were subcultured. To this end, the cells were treated with trypsin/Versene (1:1; PanEco) for 3-5 min at 37oC, collected in tubes, and washed by centrifugation at 300g for 5 min. The pellet was resuspended in complete growth medium, cells were counted in a Goryaev chamber, and transferred into vials at a concentration of 5×104 cells/ml. For induction of differentiation, passage 3-5 skin fibroblasts were transferred in a 24-well plate (Greiner) at a concentration of 105 cells per well. After attaining 80-90% confluence, the growth medium was replaced with differentiation one. Osteogenic differentiation was induced in a medium based on DMEM/F-12-GlutaMax supplemented with 100 nM dexamethasone, 10 mM β-glycerophosphate, and 0.05 mM ascorbic acid (all reagents were from Sigma). Adipogenic differentiation was performed in the same medium DMEM/F-12-GlutaMax supplemented with 10 μg/ml insulin, 100 nM dexamethasone, 250 μM 3-isobutyl-1-methylxanthine (IBMX), and 200 μM indomethacin (all reagents were from Sigma). The medium was replaced every 3 days. Analysis of differentiation was performed in 3 weeks

by cytochemical detection of intracellular triglyceride (adipogenic differentiation) and calcium deposits (osteogenic differentiation) after staining with Oil Red O and alizarin red (Sigma), respectively. Bone marrow mesenchymal stromal cells (MSC) were used as the positive control of differentiation. The preparations of stained cells were photographed using an Axiovert 40 CFL inverted microscope (Carl Zeiss) and a Nikon D5000 digital camera. The procedure of skin fibroblast reprogramming was carried out using two vector systems: a mixture of 4 reprogramming Sendai virus-based vectors expressing a classic set of embryonic reprogramming genes Oct4, Sox2, Klf4, and Myc (CytoTune-iPS Sendai Reprogramming Kit, Invitrogen) and a system containing an optimized mixture of 3 oriP/EBNA-1(Epstein-Barr nuclear antigen-1)-based episomal vectors. The latter system uses Oct4, Sox2, Lin28, L-Myc, and Klf4 as the reprogramming genes and also contains vectors expressing mp53DD (dominant-negative mutation of p53 protein) and EBNA1 for improving reprogramming efficiency (The Epi5 Episomal iPSC Reprogramming Kit, Invitrogen).

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Fig. 1. Adipogenic (a, b) and osteogenic (b, d) differentiation of skin fibroblasts and bone marrow MSCs. Oil Red O staining, ×20 (a, c); alizarin red staining, ×10 (b), ×20 (d).

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Fig. 2. Efficiency of transfection of skin fibroblasts with plasmid vector pmCherry-N1. Abscissa: fluorescence intensity in relative units; ordinate: number of events (cells). Red peak: control nontransfected cells; blue peak: cells after transfection with plasmid vector pmCherry-N1.

Transduction with Sendai virus was performed in accordance with manufacturer’s instruction. Two days prior to transduction the cells were plated in a 6-well plate (Greiner) in a concentration of 105 cells per well. On the day of transduction, the growth medium was removed from wells with cultures (80-90% confluence) and 1 ml fresh medium with dissolved viral particles was added for 24 h. Then the medium was replaced daily over 6 days. In 7 days, the cells were harvested with trypsin/versene, centrifuged, counted in a Goryaev chamber, and seeded in wells of a 6-well plate on a feeder layer (3T3 fibroblasts pretreated with 10 μg/ml mitomycin C) in a concentration of 5×105 cells per well in complete growth medium. In 24 h, the cells were transferred to medium for iPS cell culturing containing DMEM/F-12, 100 μM MEM non-essential amino acids solution, GlutaMAXI Supplement (100×), penicillin/streptomycin (100×) (all components were from Gibco), β-mercaptoethanol (100 μM), and bFGF (4 ng/ml) (Sigma). The medium was replaced every day over 2 weeks and the formation of colonies was observed. The formed colonies were transferred into new wells. Transfection of cells with vectors based on DNA sequences of Epstein—Barr virus was performed by electroporation using Gene Pulser Xcell system (BioRad). Electroporation conditions were optimized using pmCherry-N1 vector plasmid (Clontech) carrying mCherry red fluorescent protein gene under cytomegalovirus promoter. Electroporation was carried out in Ca,Mg-free MEM medium. The cells were harvested

and transferred into electroporation medium at a concentration of 105 cells/ml. Cuvettes with a 0.2-cm gap between the plates were used; 100 μl cell suspension and 1 μg plasmid DNA were taken for one procedure. Transfection efficiency was evaluated in 2 days on a FACSAria flow cytofluorometer (BD Biosciences) by protein mCherry fluorescence. After electrotransfection with Epstein–Barr virus-based vector, the cells were cultured according to manufacturer’s protocol. Transfected cells were seeded into wells of a 6-well plate precoated with Geltrex matrix (Gibco) in complete growth medium containing DMEM/F-12 supplemented with 25 mM HEPES, N-2 Supplement (100×), B-27 Supplement (50×), 100 μM MEM non-essential amino acids solution, GlutaMAXI (100×) (all components were from Gibco), 100 μM β-mercaptoethanol, 100 ng/ml bFGF (Sigma). The medium was changed in 24 h and than every day over 14 days. In 2 weeks after transfection, the cells were transferred to medium for iPS cell culturing (Essential 8 Medium; Gibco). The medium was replaced every day and the formation of colonies was monitored. In 7 days, the formed colonies were selectively harvested with trypsin after their isolation from surrounding cells with a plastic cylinder. The cell suspension was transferred to Geltrex matrix-coated wells of a 6-well plate for further expansion. For both reprogramming approaches, the colonies formed during 21-day culturing were counted under a microscope. In 5 days of culturing, the cell colonies obtained by the two methods of reprogramming were stained with antibodies to Tra1-60, a surface markers of pluripotent cells. The initial fibroblast cultures were also stained with antibodies to this marker. The cells were incubated with anti-Tra1-60 antibodies (Santa Cruz) for 1 h, washed with PBS, and incubated with secondary antispecies FITC-labeled antibodies (BD Biosciences) for 40 min. After double washing with PBS, the staining was evaluated under an Axiovert 200 confocal microscope.

RESULTS We have previously demonstrated that adult human skin fibroblasts express some surface markers typical of MSK [1]. However, despite phenotypic similarity, adult donor skin fibroblasts are characterized by very limited differentiating potential in comparison with MSC. For instance, fibroblasts did not differentiate into osteogenic lineage cells in vitro, as was seen from the absence of calcium deposits (Fig. 1, b). Induction of adipogenic differentiation resulted in the formation of only minor quantities of small lipid droplets in cells (Fig. 1, a). Under these conditions, bone marrow MSC used as the positive control effectively differentiated


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Fig. 3. Culture of skin fibroblasts in 24 h (a) and 5 days (b) after episomal transfection. Phase-contrast microscopy, ×20.

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Fig. 4. iPS colonies in 21 days after transfection with episomal oriP/EBNA-1 vector (a) and viral transduction with Sendai virus-based vectors (b). Phase-contrast microscopy, ×40.

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Fig. 5. Colonies of iPS cells obtained by episomal transfection in 24 h (a) and 5 days (b) after seeding. Phase-contrast microscopy, ×20.

into adipogenic (Fig. 1, c) and osteogenic (Fig. 1, d) lineage cells. At the same time, skin fibroblast are more often than other cells used for cell reprogramming and generation of iPS [3], because they can be easily isolated from donors irrespective of their age and health status, are characterized by high proliferative potential (which is an important factor for efficient reprogramming), and enter replicative senescence in culture not earlier than after 50 division cycles, as was demonstrated for skin fibroblasts from elderly (above

70 years) individuals [2]. These facts make skin fibroblasts a convenient source of iPS cells. In our experiments, we used two different strategies for reprogramming skin fibroblasts into iPS cells without integration vectors into the cell genome. One of them is the use of Sendai virus-based vectors. Sendai virus is an RNA-containing virus that does not integrate into the genome and operates exclusively in the cytoplasm of cells without penetrating the nucleus and, consequently, does not change the cell genome. This

A. S. Vdovin, A. Yu. Lupatov, et al.

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Fig. 6. Expression of pluripotency marker Tra1-60 in colonies of iPS cells (a, b) and skin fibroblasts (c). a) Colonies obtained after episomal transfection, b) colonies obtained after viral transduction. Confocal microscopy, ×40 (a, c); ×20 (b).

method of reprogramming is not associated with risks typical of other systems based on viral vectors such as lentiviral or adenoviral vectors. Another approach implies reprogramming with oriP/EBNA-1-based vector that mediates import and retention of vector DNA in the cell nucleus without integration into the genome and enables efficient derivation of iPS cells after single transfection procedure [7]. Episomal transfection of vectors was carried out by electroporation. For choosing optimal conditions, plasmid vector pmCherry-N1 was used. The efficiency of transfection was evaluated

127 by mCherry fluorescence on a flow cytofluorometer. The efficiency of transfection under chosen electroporation conditions (exponential wave mode, voltage 150 V, capacitance 500 μF, distance between the electrodes 2 mm) was approximately 30% (Fig. 2). The dynamics of colony formation was similar for both reprogramming methods. On the next day after episomal transfection, multinucleated cells (result of electroporation) were detected in the culture (Fig. 3, a) colonies appeared starting from day 5 (Fig. 3, b). In 21 day after induction of reprogramming by the two methods, the formed colonies were counted (Fig. 4, a, b). It was found that the efficiency of colony formation was significantly higher in the episomal system employing oriP/EBNA-1 vectors. Transduction with Sendai virus-based vectors yielded 8 colonies per 105 cells, whereas episomal oriP/ EBNA-1 sequence-based vector system allows generation of not less than 200 colonies after transfection cycle. This difference can be due to the fact that the episomal system includes additional vectors carrying genes mp53DD and EBNA1. Reprogramming of somatic cells is associated with stress exposure of cells. It can be hypothesized that p53 plays a pivotal restrictive role in this process. During somatic cells reprogramming, activation of the p53 pathway induces cell apoptosis, aging, and/or cell-cycle arrest, which determines very low effi ciency of the reprogramming. Inhibition of tumor suppressor protein p53 is associated with the efficiency of iPS cell generation, elimination of p53 function substantially improves the efficiency of reprogramming of mouse and human somatic cells [8]. In our experiments, inactivation of endogenous p53 during mp53DD expression inhibits signaling pathways leading to cell cycle arrest and cell death, which improves transfection efficiency. Moreover, viral protein EBNA1 provides active expression of episomal vector genes. The obtained cultures of iPS cells after passaging and culturing for 5 days (Fig. 5, a, b) were analyzed for the expression of pluripotent cells marker Tra160 (Fig. 6, a, b). The expression of this marker in unmodified skin fibroblasts used as the source of iPS cells was evaluated and served as the control (Fig. 6, c). Skin fibroblasts do not express this marker, whereas virtually 100% iPS cells obtained by the two methods were positive by this marker. Thus, despite the fact that the efficiency of reprogramming and subsequent colony formation were differed significantly depending on the reprogramming technique, induced pluripotent stem cells expressing pluripotency markers Tra1-60 were obtained from adult skin fibroblasts in both cases. The study was supported by the Russian Research Foundation (grant No. 14-15-00648).

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