Genetically Modified Keratinocytes Transplanted to Wounds Reconstitute the Epidermis PETER M

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Proc. Nadl. Acad. Sci. USA Vol. 91, pp. 9307-9311, September 1994 Medical Sciences Genetically modified keratinocytes transplanted to wounds reconstitute the epidermis PETER M. VOGT*, SIMON THOMPSONt, CHRISTOPH ANDREE*, PAUL LIu*, KARL BREUING*, DIMITRiOs HATzIs*, HENRY BROWN*, RICHARD C. MULLIGANt, AND ELOF ERIKSSON** *Division of Plaftic Surgery, Brigham and Women's Hospital, Boston, MA 02115; and tWhitehead Institute for Biomedical Research, Cambridge, MA 02138 Communicated by Joseph E. Murray, May 23, 1994 (receivedfor review March 2, 1994) ABSTRACT Normal and reovaly tnfected kerati- study. Domestic female Yorkshire pigs, 3-4 months old and nocytegetentetatodsgeor weighing 40-45 kg, were used. Keratinocytes were cultured the human growth Moroe GH) gene were Xto frompartial-thickness skin cut0.012inch(0.03 cm) thickfrom chamber-endosed akin flil-Atikness wonds ofYorkshre pigs. the dorsal neck region of anesthetized pigs. To separate the Immg ofIseql bi es obtained for4 weeks epidermis from the dermis minced grafts were incubated in aftr arnshowed surivafte I phudd kera- serum-free medium containing 0.25% Dispase at 370C for 2-3 tinocytesbas well as eofrTeuialeo hr. Incubation ofthe tissue in 0.1% trypsin and 0.02% EDTA keratnocytes were first see in the ne l pi of the at 37C for 30 min with gentle mechanical disruption yielded wounds, then In the baail epidral layer, and suspensions of single keratinocytes. The suspension was finally In the ter lyd r ii cells of the sriam filtered through a 100-iAm mesh to rid debris and resuspended spinosum. When kai sfedwith the hGH gene in medium. Cells were then seeded into culture flasks at a were t pAted into sinia wounds, hGH was detected for 10 density of 0.12 x 106 cells per cm2 and grown in modified days In wound fluid. In contrIat, hGH was detected in vitro for Waymouth medium containing 20% fetal bovine serum. 47 days. Wounds t-ransted With either tafeted or normal At 60-801% confluence keratinocyte cultures were assigned keratinocytes restored the ephll barrier to gnifi- as controls or for gene transfer by infection with one or the canti faster than m n e conl (P < 0.05). The other of two recombinant retrovirus vectors: (i) an ampho- study confirms the _ tranlntatI of kern tropic helper-free murine leukemia virus vector, MFG-lacZ, ds -their reconstitution of the epidermis, and their containing the (-galactosidase gene, lacZ (R.C.M., unpub- acceleratdon of repair. Further, this apparently normal incor- lished), or (ii) a vector, a-SGC-hGH, containing the gene p n of ge ally en ed t nted keatin s sequence from hGH (L. Cohen and R.C.M., unpublished). ex eote r h~GH ----Athe _ Subconfluent keratinocyte cultures were incubated on 3 of itrodudg other genes e ssg therape p i into consecutive days with the retrovirus in Polybrene (8 pg/ml) wounds to favorably ffed healing. Woundfluid deeci ofthe and 10% fetal bovine serum containing Dulbecco's modified expreIned peptide provided early demonstration Of su fl Eagle's medium (DMEM; GIBCO). ranfer of the hGH gene. During transfection the modified Waymouth medium was removed and replaced daily by an equal amount of DMEM This work was originally started to test the hypothesis that containing the retrovirus at a concentration of 1-2 x 107 transplanted keratinocyte suspensions survive and regener- plaque-forming units/ml. After 6 hr the virus-containing ate the epidermis in a liquid wound chamber environment. medium was removed and replaced by Waymouth medium. The study was further designed to test the thesis that cells This procedure was repeated on 3 consecutive days. At transfected either with the ,-galactosidase gene or the human confluence, keratinocytes were released from the flasks with growth hormone (hGH) gene could live and express their 0.01% trypsin and resuspended in normal unbuffered saline encoded peptides in the same environment. The (-galacto- (0.9%O) containing 100 international units ofpenicillin and 100 sidase gene would then serve as a marker gene labeling the pg ofstreptomycin per ml, at a concentration of3 x 106 cells transplanted cells and its progeny. The hGH gene would be per ml. Keratinocytes resuspended in saline were found to be a model for transfer of a possibly therapeutic gene into the >90% viable, as determined by trypan blue staining. wound. The third hypothesis was that in this chamber envi- The procedure for making 170 reproducible standardized ronment wound fluid could be assayed for the expressed f-thickness skin wounds on the backs of 13 anesthetized peptide, simplifying verification of successful transduction. pigs for these experiments was as follows. Under aseptic The location of keratinocytes at the surface of the body conditions using a 2.25-cm2 square template the skin was conveniently places them for observation of their regenera- excised with a scalpel to the level ofthe panniculus carnosus tion as well as their ability to serve as recombinant gene muscle with careful hemostasis. Wound margins were tat- carriers. tooed with India ink, allowing for photoplanimetric evalua- The chamber system has been used in experimental portion of the surface area on sequential standardized photo- cine as well as clinical human wounds (1). It provides a graphs. The chambers, serving as an in vivo cell culture controlled wound environment and allows for the adminis- device, were applied to each wound (1, 4). The chamber tration ofmedia, additives, and cells. Conceptually it creates (P. A. Medical, Columbia, TN) consists of a flexible trans- an in vivo cell culture system. parent vinyl top bonded to an adhesive base. The base has a central opening fitting the wound margins. Chambers were MATERIAL AND METHODS filled with 1.2 ml of isotonic saline containing 100 pg of Porcine and human skin are similar in morphology and cell streptomycin and 100 international units penicillin per ml (1, turnover time (2, 3) and pigs were therefore chosen for this 4). Twenty-two wounds were treated with lacZ transfected The publication costs ofthis article were defrayed in part by page charge Abbreviations: hGH, human growth hormone; H&E, hematoxylin/ payment. This article must therefore be hereby marked "advertisement" eosin; X-Gal, 5-bromo-4-chloro-3-indolyl -D-galactoside. in accordance with 18 U.S.C. §1734 solely to indicate this fact. t~o whom reprint requests should be addressed. 9307 Downloaded by guest on September 30, 2021 9308 Medical Sciences: Vogt et al. Proc. Nad. Acad Sci. USA 91 (1994)

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FiG. 1. Macroscopic appearance of porcine full-thickness wounds on days 3, 6, 9, and 12 after wounding. (A-D) Transplanted wounds for those days. (E-H) Corresponding saline-treated controls. Keratinocyte-transplanted wounds began developing an epithelium on day 6 (B), the time saline-treated controls (E-H) still contained only a clot (F). The rate of wound contraction was not affected (D and H). (x3.) keratinocytes, and 10 other wounds were treated with hGH gradually thickening epithelium. Conversely, epithelializa- transfected keratinocytes. Seventy-one wounds treated with tion did not begin until after day 12 in saline controls (Fig. 1). nontransfected keratinocytes served as controls. As a further The reestablishment of the normal epithelialized barrier was control, 67 chambers were filled with only saline and antibi- also indicated by impermeability to protein (Fig. 2). This otics. In order not to disrupt transplanted cells growing on the impermeability was measured here by protein efflux as wound surface, chambers were undisturbed for 2 days. described previously (1). The rate of wound contraction, Beginning on that second day, the wound fluid was removed however, was similar in all three groups. and replaced with saline. Histologic examination of 105 wound biopsy specimens Fluid collected from individual wounds was centrifuged at showed that complete epithelialization occurred 12 days after 2000 x g for 10 min and then passed through a filter, with a keratinocyte transplantation, but not until 16 days in saline- pore size of 0.45 mm (Millipore). Concentrations of total treated wounds (P < 0.05). endogenous proteins, previously shown to be a reliable The transplanted keratinocytes, whether normal or trans- noninvasive marker of restoration of epithelial barrier func- fected, behaved in a characteristic fashion. On day 4 small tion, were measured with a turbidimetric assay (1). clusters of these cells were seen on the deep wound surface hGH concentrations in wound fluid and in vitro cell (Fig. 3 A andD). Two days later (Fig. 3 B andE) large clusters culture fluid were measured by a radioimmunoassay (Alle- of keratinocytes were seen in the upper neodermis. On day gro; Nichols Institute Diagnostics, San Juan Capistrano, 8 these keratinocytes were already incorporated into the new CA). Cross-sectional wound biopsies including margins of un- 1(JWu. wounded skin were excised from the wounds starting from day 4 and continuing until day 27. Half of the samples were fixed in 10%1 formalin, embedded in paraffin, and stained with w hematoxylin/eosin (H&E) or Masson's trichrome. The other half were flash-frozen in and la liquid nitrogen, sectioned, saw~~~-0NA TKEY(w.2 stained for the detection of ,-galactosidase activity with the 1003| substrate 5-bromo-4-chloro-3-indolyl (-D-galactoside (X-Gal chromogen), which forms a blue precipitate in transduced Is13 cells (5). a 100 Epithelial thickness was measured in histologic H&E cross .2 sections at the wound margins and four points equally spaced I across the wound. Means and standard errors for thickness 0 and protein concentration in the wound fluid were calculated a- for the groups. Statistical significance between groups was .0 analyzed with the nonparametric Mann-Whitney U test (6). 0 5 10 15 20 RESULTS Days After 4 days a fibrinous clot had developed in chamber- FIG. 2. Protein concentration in wound fluid. Disappearance of treated wounds of all groups. Beginning on day 6, wounds protein corresponds to complete epithelial regeneration. A compar- into which either unmodified or retrovirally transfected ke- ison is made of saline treatment and transplantation of keratinocyte ratinocytes had been transplanted were covered with a thin, suspensions (mean >95% confidence interval). Downloaded by guest on September 30, 2021 Medical Sciences: Vogt et aL Proc. Nati. Acad. Sci. USA 91 (1994) 9309

FIG. 3. Reconstitution of new epithelium of porcine full-thickness wounds in large part by transplanted keratinocytes. (A-C Low magnification (x25) of histologic cross section stained with Masson's trichrome to provide better contrast and to show collagen development. (F) Areas in squares at higher magnification (x260) with X-Gal staining to demonstrate lacZ expression. lacZ keratinocytes are first seen in clusters on the bottom of the wounds on day 4 (D), having migrated upward on day 6 (E), and are present in all layers of epithelium by day 8 (F). epidermis (Fig. 3 C and F). All wounds transplanted with increased to 0.35 ± 0.126 ng/ml at 6 days but then declined lacZ keratinocytes demonstrated the presence of this gene to become undetectable at day 10 (Fig. 4B). The histologic and the transfected keratinocytes had the location and be- pattern observed, nevertheless, was very similar after trans- havior which were similar to that of nontransfected kerati- plantation ofeither normal orhGH transfected keratinocytes. nocytes. Also, horizontal sections through the regenerated This finding might be due to the lack ofhGH receptors or the epidermis in wounds that had received transfected kerati- concentration used in this study had no effect on the regen- nocytes showed evidence of an even distribution of the erating porcine epithelium. No hGH was detected in control expressed reporter gene lacZ on day 12. wounds or in systemic blood samples. The average efficiency after MFG-lacZ transduction in An interesting observation of sections stained with H&E vitro was 1.2% ± 0.2%. After transplantation to 22 full- and with Masson's trichrome was that on day 6 these nests thickness wounds and with biopsies on day 4, every section were surrounded by collagen bundles (Fig. 5 A-C). By day 8 from each wound contained cells with the positive stain for lacZ-expressing keratinocytes were distributed in all cell (3-galactosidase (Fig. 3 D-F). After transduction with the layers of the newly regenerated epithelium including the hGH gene the production of the hGH in vitro was consistent stratum germinativum (Fig. 3F). Biopsies from wounds at 27 and increased for 45 days (Fig. 4A). days showed lacZ expression only in the upper portion ofthe All 10 wounds expressed hGH and the concentration as stratum granulosum and not in the stratum basale or the measured in wound fluid was 0.1 ± 0.0 ng/ml after 24 hr and stratum spinosum. At this time point no more nests of Downloaded by guest on September 30, 2021 9310 Medical Sciences: Vogt et al. Proc. Nad. Acad Sci. USA 91 (1994)

4 and normal keratinocytes (317.5 ± 59.5 gam vs. 297 ± 35.3 A U hin vio iAm; P = 0.788). 3- DIECUSSION For the purposes of study and treatment of wounds we have 2- developed a chamber system (1). Thi stanardies the wound environment and allows the addition of media, mediations, and cells. The chamber creates an in vivo culture system. Compared to in vitro systems, the in vivo arrangement allows I I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ for the generation of a three-dimensional tissue with intact angiogenesis, inflammatory, and immune responses. By 0 I. monitorng of the wound fluid significant aspects of healing 0 10 20 30 40 50 can be studied noninvasively. The disappearance of protein in wound fluid coincides with the reestablishment of an Al epithelial barrier (1). In 1975 and thereafter, Green et al. (7, 8) reported a series of experiments culturing keratinocytes. Further investiga- tions made it possible for O'Connor and others (9-12) to use cultured epidermal sheet grafts clinically. Fusenig and co- I workers (13) earlier, like our present work, r on the transplantation of keratinocyte suspensions. These experi- Iz ments, however, showed no data to prove survival of the transplanted keratinocytes. Our results not only show the feasibility ofthis method but also demonstrate the survival of these cells carrying the t-glactosidase gene. The presence of this gene also demonstrates the location of the transplanted cells, first deep in the wound and later in the epidermis. Days During the past several decades, the development ofmeth- FiG. 4. (A) Continuous production of hGH in vitro by primary ods for delivering genes to mammalian cells has given prom- porcine keratinocyte culture after retrovir gene transfer. Mean of ise to the possibility of treating human disease by gene two experiments. (8) hGH In vivo in porcine wound fluid in Ball- therapy (14). Several methods forgene transfer are described thickness wounds by transplaSntd suspensions ofretroviraily trans- in the literature (14). In this regard, Morgan et aL (15) first duced keratinocytes (mean ± SEM; see text). showed that human keratinocytes may be genetically engineered in vitro by the method of retrovirl gene transfer. transfected keratinocytes were seen in the subepidermal They further showed that these cells secrete hGH after tissue. The histologic appearance of the regenerated epithe- transplantation as sheets onto nude mice. In addition, plas- lium after transptation ofeither transfected or unmodified mids have been used for transfection of keratinocytes (16). keratinocytes was similar and was characteristic of a corni- The present study showed that cell suspensions could be fied multilayer epithelium. Epithelial thickness was not sig- transfected with the lacZ and hGH genes. These cells con- nificantly different between hGH-expressing keratinocytes tinue to express the gene after transplantation into filll-

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C FIG. 5. Representative transverse sections cut 6 daysfollowingfull-thickness skin wounding and seeding with lacZ-expressingkeratinocytes. (x130.) The clusters ofkeratinocytes seen with H&E stain (A) are shown to be surrounded by collagen bundles with Masson's trichrome stain (B) and to be P-galactosidase-positive with X-Gal stain (C). Downloaded by guest on September 30, 2021 Medical Sciences: Vogt et A Proc. Nadl. Acad. Sci. USA 91 (1994) 9311 thickness skin wounds in pigs, a finding which is strong 2. Weinstein, G. D. (1965) J. Invest. Dermatol. 44, 413-419. evidence that transplanted keratinocyte suspensions survive 3. Montagna, W. (1966) in Swine Biomedical Research, eds. in the wound. Bustad, L. K. & McClettan, R. 0. (Frayn, Seattle), pp. 285- On day 27, following keratinocyte transplantation, lacZ 2%. expression was observed only in the upper portion of the 4. Eriksson, E., Liu, P. Y., Zeckel, Y., Binder, T., Breuing, K. stratum granulosum, suggesting that these transfected kera- & Miller, D. R. (1991) Surg. Forum 41, 670-673. tinocytes may be undergoing early rapid terminal differenti- 5. Price, J., Cepko, C. & Turner, D. (1987) Proc. Nati. Acad. Sci. ation. We speculate that the high concentration ofcalcium in USA 84, 156-160. the wound fluid and perhaps other mediators as growth 6. Snedecor, G. W. & Cochran, W. G. (1989) in Statistical Meth- factors induce this rapid terminal keratinocyte differentiation ods (Iowa State Univ. Press, Ames, IA), 8th Ed., pp. 142-145. (17-21). It is also possible that the lacZ gene may be present 7. Rheinwald, J. G. & Green, H. (1975) Cell 6, 331-344. in basal cells with a loss of expression at transcriptional or 8. Green, H., Kehinde, 0. & Thomas, J. (1979) Proc. Natl. Acad. translational levels. Sci. USA 76, 5665-5668. The porcine keratinocytes could be permanently trans- 9. O'Connor, N. E., Mulliken, J. B., Banks-Schlegel, S., Ke- fected with the hGH gene in vitro. When these cells were hinde, 0. & Green, H. (1981) Lancet 1, 75-78. transplanted to the wound in an autologous fashion, hGH 10. Pittlekow, M. R. & Scott, R. E. (1986) Mayo Clin. Proc. 61, disappeared from wound fluid 10 This 771-777. on day (Fig. 4). 11. Kumagni, M., Nishina, H., Tanabe, H., Hosaka, T., Ishida, H. disappearance could depend on several factors. For example, & Ogino, Y. (1988) Plast. Reconstr. Surg. 82, 99-108. the regenerated epidermis could prevent diffusion of the 12. De Luca, M., Albanese, E., Bondanza, S., Megna, M., Ugoz- peptide into wound fluid. It could also be caused by terminal zoli, L., Molina, F., Cancedda, R., Santi, P. L., Bornioli, M., differentiation of the cells carrying the hGH gene. Other Stella, M. & Maglicani, G. (1989) Burns 15, 303-309. possibilities might be the loss of the transgene or a block of 13. Worst, P. K. M., Mackenzie, I. C. & Fusenig, N. E. (1982) transcription or of translation. The early detection of the Cell Tissue Res. 225, 65-77. hGH gene (on day 2) in wound fluid provides a noninvasive 14. Mulligan, R. C. (1993) Science 260, 926-932. way to demonstrate successful gene transfer. This experi- 15. Morgan, J. F., Barrandon, Y., Green, H. & Mulligan, R. C. mental approach not only demonstrates the feasibility ofgene (1987) Science 237, 1476-1479. transfer to keratinocytes but also may serve as a model for 16. Teumer, J., Lindahl, A. & Green, H. (1990) FASEB J. 4, short- or long-term gene therapy. 3245-3250. We believe that this approach might also be used success- 17. Marikowsky, M., Breuing, K., Liu, P. Y., Eriksson, E., Hi- fully to insert other genes with possible therapeutic value into gashiyama, S., Farber, P., Sasse, J. & Kagsbrun, M. (1992) keratinocytes. Since the expression of the transgenes might Proc. Natl. Acad. Sci. USA 90, 3889-3893. be transient in keratinocytes, this strategy may be useful for 18. Brem, H., Shing, Y., Watanabe, H., Tamvakopoulis, G., healing wounds in which only temporary production of Marikovsky, N. L., Vogt, P. M., Klagsbrun, M., Grosfeld, J., growth factors is required. Genetically engineered kerati- Eriksson, E. & Folkman, J. (1992) Surg. Forum 43, 664-667. nocytes may therefore be transplanted to the wound with the 19. Hennings, H., Holbrook, K. A. & Yuspa, S. H. (1983) J. dual purpose of providing temporary production of growth Invest. Dermatol. 81, 50s-SSs. factors as well as surface epithelium. 20. Lee, J. I. & Taichman, L. B. (1989) J. Invest. Dermatol. 92, 267-271. 1. Breuing, K., Eriksson,(1992)8.E., Liu, P. Y. & Miller, D. R. 21. Boyce, S. T. & Ham, R. G. (1983) J. Invest. Dermatol. 81, Surg. Res. 52, 50-58. 33-40. Downloaded by guest on September 30, 2021