Efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with lentiviral vector
© Nanba et al.; licensee BioMed Central Ltd. 2013
Received: 17 May 2013
Accepted: 14 October 2013
Published: 18 October 2013
The development of an appropriate procedure for lentiviral gene transduction into keratinocyte stem cells is crucial for stem cell biology and regenerative medicine for genetic disorders of the skin. However, there is little information available on the efficiency of lentiviral transduction into human keratinocyte stem/progenitor cells and the effects of gene transduction procedures on growth potential of the stem cells by systematic assessment.
In this study, we explored the conditions for efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with a lentiviral vector, by using the culture of keratinocytes on a feeder layer of 3 T3 mouse fibroblasts. The gene transduction and expansion of keratinocytes carrying a transgene were analyzed by Western blotting, quantitative PCR, and flow cytometry.
Polybrene (hexadiamine bromide) markedly enhanced the efficiency of lentiviral gene transduction, but negatively affected the maintenance of the keratinocyte stem/progenitor cells at a concentration higher than 5 μg/ml. Rho-assiciated kinase (ROCK) inhibitor Y-27632, a small molecule which enhanced keratinocyte proliferation, significantly interfered with the lentiviral transduction into cultured human keratinocytes. However, a suitable combination of polybrene and Y-27632 effectively expanded keratinocytes carrying a transgene.
This study provides information for effective expansion of cultured human keratinocyte stem/progenitor cells carrying a transgene. This point is particularly significant for the application of genetically modified keratinocyte stem/progenitor stem cells in regenerative medicine.
Recombinant retroviruses enable a transgene to permanently integrate into the genome of a target cell, and are widely used as a vector in gene therapy [1, 2]. Efficient and safe gene transduction into stem cells with viral vectors is indispensable for successful regenerative medicine, including the generation of genetically modified tissue stem cells and induced pluripotent stem (iPS) cells derived from patients affected by genetic disorders [3, 4]. Transduction and silencing of genes with viral vectors are also essential for analyzing stem cell function during development, homeostasis and tumorigenesis. Thus, the development of highly efficient and less-cytotoxic viral gene transduction into stem cells is crucial for advances in stem cell-based regenerative medicine and stem cell biology.
Human skin contains keratinocyte stem cells that are clonogenic when cultivated on a feeder layer of mouse 3 T3 fibroblasts, and show significant proliferative capacity in culture . Ex vivo maintenance and expansion of human keratinocyte stem cells have achieved the autologous transplantation of confluent sheets of cultured keratinocytes onto patients with extensive burns . The culture of keratinocyte stem cells has also enabled a gene therapy for a genetic disorder of the skin. De Luca and his colleagues  isolated epidermal keratinocyte stem cells from a patient who carries a null allele and a single point mutation in the LAMB 3 gene encoding laminin beta 3 subunit, and thus is affected by junctional epidermolysis bullosa. They transduced the full-length LAMB3 cDNA into the keratinocyte stem cells with a retroviral vector, and prepared genetically corrected cultured epidermal grafts. The grafts were engrafted and remained stable for at least one year in the absence of blisters, infections, inflammations or immune response. Thus, gene transduction into cultured keratinocyte stem cells assures the healing or alleviation of inherent genetic disorders of the skin.
Polybrene (hexadimethrine bromide), a cationic polymer, has been widely used to increase the efficiency of retroviral transduction . Cationic polymers, including polybrene, enhance virus adsorption on the surface of the cell by neutralizing the negative electrostatic repulsion between the cell surface and the virus particles [9, 10]. It has been reported that polybrene is utilized for introduction of DNA into keratinocytes as a DNA carrier [11, 12], and that it also enhances gene transduction into keratinocytes with adenoviruses [13, 14] and retroviruses [15, 16]. Besides polybrene, van den Bogaard et al. have recently reported that Rho-associated kinase (ROCK) inhibitor Y-27632 enhances lentiviral transduction into human keratinocytes . However, there is little information available on how polybrene and Y-27632 impact the efficiency of lentiviral transduction into human keratinocytes, keratinocyte proliferation and maintenance of the stem/progenitor cells. Here, we provide information for an efficient and less-cytotoxic gene transduction with a lentiviral vector into human epidermal keratinocyte stem/progenitor cells cultured on a feeder layer of 3 T3 mouse fibroblasts by using polybrene and Y-27632.
The culture of normal human epidermal keratinocytes (strain no. 685389; KURABO, Osaka, Japan) isolated from the foreskin of newborns was previously described . Briefly, frozen keratinocytes were thawed and cultivated at clonal density on a feeder layer of mitomycin C-treated 3 T3-J2 cells at 37°C as described [19, 20]. The medium was changed every four days. Cells were used between passage 4 and 10. For determination of colony-forming efficiency, 1 × 104 keratinocytes were cultured as described above. Cultures were maintained for eight days, and subsequently fixed in 3.7% buffered formaldehyde, and stained with 1% rhodamine B (Sigma-Aldrich, St. Louis, MO, USA).
Preparation of lentiviral vector
A plasmid for lentiviral expression of enhanced green fluorescent protein (EGFP) was generated by subcloning the EGFP cDNA from pEGFP-N1 (Clontech, Palo Alto, CA, USA) into the HIV-based self-inactivating lentiviral expression vector plasmid pCSII-CMV-MCS  (RIKEN BioResource Center, Tsukuba, Japan). The EGFP expression cassette under the control of CMV promoter was introduced upstream of woodchuck hepatitis virus posttranscriptional regulatory element in the transfer plasmid pCSII-CMV-MCS. Lentiviral vector pseudotyped with vesicular stomatitis virus glycoprotein was generated by standard DNA transfection. HEK293T cells were transfected with transfer, envelope and packaging plasmids using lipofectamine LTX (Invitrogen, Carlsbad, CA, USA). Viral vector particles were ultracentrifuged at 100,000 × g for 1 hr, and resuspended in Dulbecco-modified Eagle’s medium. To measure the functional titer, HEK293T cells were seeded into a 24-well culture plate, transduced with proper concentrations of viral vectors, and the functional titer was analyzed by flow cytometry (FACS Calibur; Becton Dickinson, Franklin Lakes, NJ, USA). The multiplicity of infection (MOI) of 1 was defined as a number of functional lentiviral particles which was sufficient to introduce a transgene into HEK293 cells with 100% efficiency.
A total of 104 keratinocytes were seeded in a 12-well cell culture plate with mitomycin C-treated 3 T3-J2 cells, grown for seven days, and analyzed by Western blotting, as previously described . The luminescence signals were detected by using ImageQuant LAS4010 (GE Healthcare, Buckinghamshire, UK). Primary antibodies used were as follows: rabbit polyclonal antibody to GFP (#598; 1:1,000; Medical & Biological Laboratories, Nagoya, Japan), and mouse monoclonal antibody to α-tubulin (#T6199; 1:5,000; Sigma-Aldrich). Secondary antibodies were horseradish peroxidase (HRP)-conjugated goat anti-mouse and rabbit IgG (#115-055-174 and #211-032-171; 1:5,000; Jackson ImmunoResearch, West Grove, PA, USA). Relative density of EGFP bands was measured with Volocity (PerkinElmer, Branchburg, NJ, USA) and normalized with density of α-tubulin bands. The values (means ± SD) were determined based on results in three independent experiments.
Cell proliferation assay
A total of 104 keratinocytes were seeded in a 12-well cell culture plate with mitomycin C-treated 3 T3-J2 cells. After seven days of cultivation, keratinocytes were trypsinized and stained with 0.4% Trypan blue (Invitrogen) to identify dead cells. Then, the number of cells was counted with a Countess™ automated cell counter (Invitrogen), which could eliminate the dead cells and large mitomycin C-treated 3 T3-J2 fibroblasts with irregular shape. The values (means ± SD) were determined based on results from three independent experiments.
A total of 2 × 103 keratinocytes were seeded at clonal density in a 35-mm cell culture dish with mitomycin C-treated 3 T3-J2 cells, grown for six days, and analyzed by immunofluorescence microscopy, as previously described . Rat monoclonal antibody to α6 integrin (clone GoH3; 1:200; Becton Dickinson), and mouse monoclonal antibody to p63 (clone 4A4; 1:500; Millipore, Bedford, MA, USA) and involucrin (clone SY5; 1:1,000; Abcam, Cambridge, UK) were used as primary antibodies. Cy3-conjugated goat anti-rat IgG and anti-mouse IgG (#112-165-167 and #715-025-140; 1:500; Jackson ImmunoResearch) were used as secondary antibodies.
A total of 2 × 105 keratinocytes were seeded in a 60-mm cell culture dish with mitomycin C-treated 3 T3-J2 cells, transduced EGFP cDNA with lentivirus vector in presence or absence of Y-27632, and grown for seven days. Trypsinized and resuspended keratinocytes were incubated with mouse monoclonal antibody against α6 integrin (clone 17D11; 1:20; a gift from Dr. Hirako, Nagoya University, Nagoya, Japan) for 1 hr on ice. Keratinocytes were incubated with phycoerythrin (PE)-conjugated goat polyclonal antibody against mouse IgG (sc-3738; 1:250; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 hr on ice. After three washes with Hank’s Balanced salt solution containing calcium and magnesium (HBSS (+)), the cells were then resuspended in HBSS (+) and analyzed by using the FACSCan flow cytometer (Becton Dickinson).
Calculation of EGFP-positive keratinocytes and 3 T3 feeder cells
The percentages of EGFP-positive keratinocytes and 3 T3 feeder cells were calculated by the following equations: EGFP-positive keratinocytes (%) = (% of EGFP-positive and ITGA6-positive cells)/(% of EGFP-positive and ITGA6-positive cells +% of EGFP-negative and ITGA6-positive cells) × 100, and EGFP-positive 3 T3 feeder cells (%) = (% of EGFP-positive and ITGA6-negative cells)/(% of EGFP-positive and ITGA6-negative cells +% of EGFP-negative and ITGA6-negative cells) × 100. The values (means ± SD) were determined based on results from three independent experiments.
A total of 2 × 105 keratinocytes were seeded in a 60 mm cell culture dish with mitomycin C-treated 3 T3-J2 cells, transduced EGFP cDNA with lentivirus vector in the presence or absence of Y-27632, and grown for seven days. Genomic DNA was extracted from keratinocytes and 3 T3-J2 cells with lysis buffer consisting of 50 mM Tris–HCl (pH 8.0), 100 mM NaCl, 20 mM EDTA, 1% SDS, and 5 units/mL of Proteinase K (Sigma-Aldrich). The DNA (50 ng) was subjected to quantitative PCR using Power SYBR Green PCR Master Mix Reagents and a 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Relative copy number of the transgene was calculated based on the ∆∆Ct method by normalization using the Ct-value of the human or mouse phosphoglycerate kinase (PGK) gene. The detection primers used were as follows: 5′-GTGAACGGATCTACAAATGGCAG and 5′- GTCTGTTGCTATTATGTCTACTA for the recombinant lentiviral vector, 5′- TGATTATTGGTGGTGGAATGGCTT and 5′-TGGAGGTCAGCATCTATACTAAGA for the human PGK gene, and 5′-TGCTAGACAAAGTCAATGAGATGA and 5′-TGATATGCAACCACTGTGAAAGGGT for the mouse PGK gene. The values (means ± SD) were determined based on results from three independent experiments.
Polybrene enhances transduction efficiency but reduces growth potential of keratinocytes
Clonal expansion of keratinocyte stem/progenitor cells expressing a transgene
Lower transduction efficiency of 3 T3 feeder cells
ROCK inhibitor interferes with lentiviral transduction into keratinocytes
The addition of the ROCK inhibitor Y-27632 into the culture medium results in the increased proliferation of human keratinocytes [28–30]. The mechanism is still unknown; however, it could have investigational and medical applications. Therefore, we examined the effect of Y-27632 on lentiviral transduction into human keratinocytes. Lentiviruses encoding EGFP cDNA were infected with or without 10 μM Y-27632, and the efficiency of gene transduction was analyzed by flow cytometry after seven days of cultivation (Figure 5A-D). In a condition in which lentiviruses were infected into keratinocytes at MOI 1 in the presence of 2.5 μg/ml polybrene, 23.4 ± 2.62% (mean ± SD) of ITGA6-positive keratinocytes expressed EGFP (Figure 5A and See Methods). However, only 8.51 ± 1.68% (mean ± SD) of ITGA6-positive keratinocytes cultured in the presence of 10 μM Y-27632 expressed EGFP (Figure 5B and See Methods). We further examined the integration of the transgene gene into the genome of keratinocytes and 3 T3 fibroblasts, with or without Y-27632. Quantitative PCR revealed that the integration of the exogenous EGFP gene into keratinocytes was significantly interfered with the treatment of Y-27632 (Figure 5E). This analysis also confirmed that EGFP-expressing lentiviruses easily infected into keratinocytes, compared to 3 T3 fibroblasts (Figure 5A), as shown in Figure 3. Interestingly, Y-27632 inhibited lentiviral transduction into keratinocytes, but not into a feeder layer of 3 T3 fibroblasts (Figure 5B, E). We also counted the number of EGFP-positive cells in the keratinocyte cultures two days after lentiviral transduction with or without 10 μM Y-27632, and confirmed that Y-27632 inhibited lentiviral transduction (Figure 5F). This result indicated that the decreased ratio of EGFP-expressing keratinocytes with Y-27632 after seven days of cultivation was not mainly due to the increased expansion of untransduced keratinocytes with Y-27632.
A combination of polybrene and Y-27632 efficiently expands keratinocytes carrying a transgene
Gene transduction with lentiviral vectors has already been used for basic and clinical studies of human keratinocytes. In this study, we demonstrated the efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with lentiviral vector by using polybrene and Y-27632. This work has three major findings concerning lentiviral transduction into keratinocytes. First, polybrene markedly enhances the efficiency of lentiviral gene transduction, but reduces keratinocyte proliferation and negatively impacts the maintenance of keratinocyte stem/progenitor cells at a concentration higher than 5 μg/ml. Second, ROCK inhibitor Y-27632 significantly interferes with the lentiviral transduction into cultured human keratinocytes. Third, a suitable combination of polybrene and Y-27632 can effectively expand keratinocytes carrying a transgene.
Polybrene is widely used in viral gene transduction. Polybrene has no cytotoxic activity at low concentrations, but negatively affects the proliferation of corneal keratinocytes at concentrations greater than 10 μg/ml . It also negatively impacts the proliferation of human mesenchymal stem cells, even if used at 1 μg/ml . It has been reported that treatment with 2 to 6 μg/ml of polybrene results in the highest efficiency of gene transduction with adenoviruses in human epidermal keratinocytes . In our experiments, 20 μg/ml of polybrene was required to induce the highest expression of transgene in lentiviral transduction. However, polybrene significantly reduced the proliferation of keratinocytes, even if used at low concentrations. Furthermore, we demonstrated that polybrene negatively impacted the maintenance of the keratinocyte stem/progenitor cells at a concentration higher than 5 μg/ml. In serial culture, keratinocyte stem cells progressively lose their proliferative capacity to become transient amplifying cells with limited growth, a phenomenon termed clonal conversion . Clonal conversion is accelerated by stress, suboptimal culture conditions, serial cultivation and age of donor . It is also governed by the balance of Rac1 and Akt signaling activity and actin filament organization . As polybrene modulates the charge of the cell surface, it might affect these intracellular signaling pathways and actin network, and then increase the rate of clonal conversion. Retroviral gene transfer into keratinocytes is also enhanced by another polycationic polymer, protamine sulfate  and by their cultivation on a substrate of fibronectin . The effects of these reagents on transduction efficiency and clonal conversion should be further investigated.
The process of retroviral infection involves the absorption of viral particles on the host plasma membrane, the entry of the viral protein-RNA complexes into the cytoplasm, the reverse transcription of viral RNA into DNA and the intracellular trafficking and entry of viral protein-DNA complexes into the nucleus, which is followed by the integration of viral DNA into the host genome . We have clearly demonstrated here that the ROCK inhibitor Y-27632 interfered with the lentiviral transduction in human epidermal keratinocytes. ROCK regulates both actin polymerization and phosphorylation of myosin regulatory light chain . Recently, it has also been shown that ROCK regulates microtubule dynamics through phosphorylation of the tubulin polymerization promoting protein 1 (TPPPA/p25) . The cytoskeleton, including actin filaments and microtubules in the host cells, contributes to intracellular transport of retroviral genomes from the cytoplasm into the nucleus [36–38]. As further investigations should be required, ROCK activity could be involved in intracellular trafficking of the retroviral genome.
Interestingly, van den Bogaard et al. have recently reported that Y-27632 enhances lentiviral transduction into human keratinocytes . This report seems to be contrary to our results. However, van den Bogaard et al. have used high-passage adult keratinocytes for lentivirus transduction and the generation of human skin equivalents (HSEs), and then assessed the efficiency of lentiviral transduction into keratinocytes in HSEs, but not in cultured keratinocytes. Human adult keratinocytes significantly decrease their proliferative capacity by serial cultivation and stress in culture, including gene transduction . Therefore, the enhanced expansion of EGFP-expressing adult keratinocytes in HSEs by Y-27632 might be due to the fact that the expansion of Y-27632-treated adult keratinocytes expressing EGFP is more efficient than that of untreated EGFP-expressing adult keratinocytes during the formation of HSEs.
van den Bogaard et al. has also reported high levels of GFP expression in the differentiated layers of HSEs . We also observed that EGFP was strongly expressed in stratified and differentiating cells in the center of colonies. These results suggest that CMV promoter activity is enhanced in the stratified and differentiating keratinocytes. Y-27632 inhibits keratinocyte differentiation [28–30]. These observations alone suggest that Y-27632 inhibits EGFP expression under the control of the CMV promoter, but not lentiviral transduction itself. However, our quantitative PCR analysis has revealed that the integration of the exogenous EGFP gene into keratinocytes significantly interfered with the treatment of Y-27632. Consequently, reduced expression of EGFP after lentiviral transduction with Y-27632 might be, at least partially, due to the inhibition of keratinocyte differentiation and, therefore, CMV promoter activity by Y-27632, as well as lower transduction efficiency by the treatment with Y-27632.
We have demonstrated in this study that keratinocytes gave rise to colonies derived from their stem/progenitor cells carrying a transgene after lentiviral transduction with lower concentration of polybrene. We have also shown that the keratinocyte stem/progenitor cells carrying EGFP transgene could maintain both self-renewing and terminal differentiation abilities that are indispensable for making a functional epidermal sheet ex vivo, and permanent engraftment after transplantation [33, 39]. These results indicate that a homologous clone of a keratinocyte stem cell carrying a transgene can be isolated and expanded massively for cell therapy for genetic disorders of the skin [7, 40].
A suitable combination of polybrene and Y-27632 efficiently expands keratinocyte stem/progenitor cells carrying a transgene, although polybrene and Y-27632 negatively affect growth and lentiviral transduction of human keratinocyte stem/progenitor cells, respectively. This point is particularly significant for the application of genetically modified keratinocyte stem/progenitor stem cells in regenerative medicine.
Enhanced green fluorescent protein
- HBSS (+):
Hank’s balanced salt solution containing calcium and magnesium
Human skin equivalents
Integrin alpha 3
Laminin beta 3
Multiplicity of infection
We thank Dr. Y. Hirako (Nagoya University, Japan) for providing us anti-α6 integrin antibody (clone 17D11), Dr. A.W. Amici (Ecole Polytechnique Fédérale de Lausanne, Switzerland) for critical reading of the manuscript, and Mrs. S. Matsushita for lentivirus production (Ehime University, Japan). We are also grateful to Japan Tissue Engineering Co., Ltd. (Gamagori, Japan) for providing us 3 T3-J2 cells. This work was supported by Grant-in Aid for Scientific Research from Japan Society for the Promotion of Science (No. 24791159) to D.N.
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