Efficient Gene Transfer to Human Epidermal Keratinocytes on Fibronectin: In Vitro Evidence for Transduction of Epidermal Stem Cells  Bharat G. Bajaj, Pedro Lei, Stelios T. Andreadis  Molecular Therapy  Volume 11, Issue 6, Pages 969-979 (June 2005) DOI: 10.1016/j.ymthe.2004.10.023 Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 1 rFN enhances gene transfer to human keratinocytes. (A) Structures of the two retroviral vectors used in this study. (B) Schematic shows various steps involved in each of the four transduction protocols. (C) Two retroviruses, LXCGN and BMN-I-GFP, were used to compare four transduction protocols. The fraction of transduced cells was measured using flow cytometry when cells reached 90–95% confluence (5–6 days postseeding). All values are the means ± SD of triplicate samples in a representative experiment. Asterisks (*) and daggers (†) indicate significant difference (P < 0.05) from TTCP control for each virus. Pounds (#) indicate significant difference (P < 0.05) between transduction efficiencies of the two viral preparations for each protocol. Molecular Therapy 2005 11, 969-979DOI: (10.1016/j.ymthe.2004.10.023) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 2 TFIR/TFN depends strongly on cell density. Human keratinocytes at the indicated densities were transduced with TFIR/TFN using either LXCGN or BMN-I-GFP retrovirus. The fraction of transduced cells was measured using flow cytometry when cells reached 90–95% confluence (5–14 days postseeding). All values are the means ± SD of triplicate samples in a representative experiment. *Significant difference (P < 0.05) from the lowest cell density for each virus. Molecular Therapy 2005 11, 969-979DOI: (10.1016/j.ymthe.2004.10.023) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 3 Effects of virus concentration on transduction kinetics. Human keratinocytes (12,500 cells/cm2) were transduced with TFN for the indicated times using retrovirus supernatant diluted in keratinocyte SFM as indicated. (A) LXCGN and (B) BMN-I-GFP. The fraction of transduced cells was measured using flow cytometry when cells reached 90–95% confluence (5–6 days postseeding). All values are the means ± SD of triplicate samples in a representative experiment. *Significant difference (P < 0.05) from the full-strength virus for each time point. Molecular Therapy 2005 11, 969-979DOI: (10.1016/j.ymthe.2004.10.023) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 4 rFN promotes gene transfer to the least differentiated keratinocytes. Human keratinocytes were separated into three fractions based on the rate of adherence to rFN preloaded with either BMN-I-GFP or LXCGN retrovirus: cells that attached in less than 5 min (LT5), cells that attached in greater than 5 min but in less than 15 min (GT5–LT15), and cells that failed to attach within 15 min but attached after overnight incubation (Overnight). In parallel, cells (12,500 cells/cm2) were seeded on rFN preloaded with either BMN-I-GFP or LXCGN retrovirus and allowed to adhere overnight (Unsorted). Approximately 24 h after cell seeding, the respective viral supernatant was reintroduced for a period of 2 h (TFIR/TFN). The fraction of transduced cells was measured using flow cytometry when cells reached 90–95% confluence (5–6 days postseeding). All values are the means ± SD of triplicate samples in a representative experiment. All samples were significantly different (P < 0.05) from the overnight samples for each virus. Molecular Therapy 2005 11, 969-979DOI: (10.1016/j.ymthe.2004.10.023) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 5 TFIR/TFN promotes gene transfer to stem cells but TTCP does not. (A) Two samples of human keratinocytes (culture 1 and culture 2) are genetically modified with the same overall efficiency. Each culture contains three cell compartments: a small population of stem cells (SC), some transit amplifying cells (TA), and terminally differentiated cells (TD). While the fraction of genetically modified cells in both samples is 33%, stem cells are modified only in culture 2. After several cell divisions, differentiated cells senesce and are replaced by progeny of transit amplifying cells, which continue to proliferate. Eventually transit amplifying cells also lose their proliferative potential and the whole culture is populated by the progeny of the original stem cells, which possess unlimited growth potential. Consequently, after a long time in culture (>30 cell generations [24]), the fraction of cells expressing the transgene should equal the fraction of transduced stem cells at the time of gene transfer. (B, C, and D) Human keratinocytes (12,500 cells/cm2) were transduced with TFIR/TFN or TTCP using the BMN-I-GFP retrovirus. (B and C) On reaching 80–90% confluence, the cells were trypsinized and ∼250,000 cells were replated on mitomycin-C-treated 3T3-J2 feeder layers in T-25 flasks. Cells were subcultured when they reached ∼90% confluence and the fraction of GFP+ cells was measured using flow cytometry. Cells were passaged until they exhausted their proliferative potential. Arrows indicate the time of transduction. (D) On reaching 80–90% confluence, the cells were trypsinized and either 800 or 1600 cells were plated in 100-mm dishes containing mitomycin-C-treated 3T3-J2 feeder layers. Keratinocytes were maintained for 14 days before the plates were washed and fixed. Clones that formed colonies greater than 5 mm in diameter were visually identified, numbered, and examined for GFP expression. Representative plates with colonies of TFIR/TFN or TTCP transduced cells are shown. Molecular Therapy 2005 11, 969-979DOI: (10.1016/j.ymthe.2004.10.023) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 6 Keratinocyte stratification increases the levels of transgene expression. Human keratinocytes (12,500 cells/cm2) were transduced with TFIR/TFN using the BMN-I-GFP retrovirus. After 4–5 days the cells were transferred onto mitomycin-C-treated 3T3-J2 cells for further expansion. Skin equivalents were generated by seeding keratinocytes (500,000 cells/cm2) onto the papillary side of decellularized dermis and raising them to the air/liquid interface for 7 days to allow complete stratification. (A) Paraffin sections were stained with hematoxylin and eosin (original magnification 25×). (B) Frozen sections were used to evaluate GFP expression (green). The nuclei were stained with a dsDNA binding dye Hoechst33258 (blue) and the basement membrane was stained with a polyclonal anti-human laminin antibody (red) (original magnification 25×). Molecular Therapy 2005 11, 969-979DOI: (10.1016/j.ymthe.2004.10.023) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions