- Email: [email protected]
Transplantation of Keratinocyte Spore-Like Stem Cells
Transplantation of Keratinocyte Spore-Like Stem Cells W.L. Olszewskia,b,*, A. Domaszewska-Szosteka, M. Moscicka-Wesołowskaa, and M. Zaleskaa a
Department of Surgical Research Transplantation and Epigenetics, Medical Research Center, Polish Academy of Science, Warsaw, Poland; and bDepartment of Transplantation Surgery, Central Clinical Hospital, Ministry of Internal Affairs, Warsaw, Poland
ABSTRACT Background. Wound granulation tissue should be covered by epidermal cells migrating from the basal layer of the epidermis or hair “bulge” of the wound edge. However, new epidermal islands are frequently formed on the granulation tissue remote from the wound edge. Thus, current theory of “bulge”-originating stem cells does not necessarily correspond to the histological pictures of the healing wound. We took imprints of a leg ulcer surface and found single dispersed, large nucleated cells, some of them in mitosis. These cells resembled those from epidermal spinosum layer. The question arouse as to whether these cells might be the “spore-like” stem cells creating epidermal island. We found similarly shaped cells among the keratinocyte preserved in pulverized sodium chloride as the only surviving population in culture and revealing enzymatic activity. The aim of this work was to study whether the population of human keratinocytes surviving sodium chloride preservation and transplanted to SCID mice may form epidermis. Methods. The 12-month sodium chlorideepreserved and cultured keratinocytes (KC) were transplanted to the wound on the dorsum of SCID mice for 14 and 21 days. Results. Ninety-five percent of cultured KC were enzymatically active “large” cells; they did not express p63 and CD29 claimed as specific for stem cells, and they did not proliferate. Transplanted to the center of the wound, they formed small KC islands and became confluent after 14 days. Conclusions. The “large” epidermal keratinocytes survived the 12-month preservation in anhydrous sodium chloride. Transplanted to the wound, they formed epidermal islands of human phenotype. These cells may be the so-called “spore-like” stem cells.
D
ELAYED-HEALING and non-healing wounds are a world-wide problem. Wound granulation tissue should be covered by epidermal cells migrating from the basal layer of the epidermis or hair “bulge” of the wound edge. However, new epidermal islands are frequently formed on the granulation tissue remote from the wound edge. Thus, the current theory of “bulge” originating stem cells does not necessarily correspond to the histological images of the healing wound. The open question remains as to whether the keratinocyte islands on the granulation tissue are formed by the stem cells desquamated and sown from the wound edges. We took imprints of a leg ulcer surface and found single dispersed, large-nucleated cells, some of them in mitosis; these cells resembled those from epidermal spinosum layer (Fig 1). Previously, we found that human skin preserved in anhydrous pulverized sodium chloride for months can be successfully transplanted, and aggressive
proliferation of keratinocytes is seen on immunohistochemical sections stained with BrdU [1]. Moreover, we found that among the keratinocyte population preserved in pulverized sodium chloride, only “large”-nucleated cells survived in culture and revealed enzymatic activity. The question arose as to whether they may be “spore-like” stem cells, which, sown on the granulation tissue, form epidermal islands. The aim of this work was to study whether the population of human keratinocytes surviving sodium chloride preservation and transplanted to SCID mice may form epidermis. *Address correspondence to Waldemar L. Olszewski, Department of Surgical Research Transplantation and Epigenetics, Medical skiego Str., Research Centre, Polish Academy of Science, 5 Pawin 02-106 Warsaw, Poland. E-mail: [email protected]
ª 2014 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/14 http://dx.doi.org/10.1016/j.transproceed.2014.09.101
Transplantation Proceedings, 46, 2885e2886 (2014)
2885
2886
OLSZEWSKI, DOMASZEWSKA-SZOSTEK, MOSCICKA-WESOŁOWSKA ET AL
Fig 1. Human single metabolically active “large” keratinocytes from the epidermis preserved for 12 months in sodium chloride powder. Giesma stain; magnification 200.
METHODS Fragments of normal human leg skin harvested during elective vascular surgery were preserved in anhydrous pulverized sodium chloride. After 12 months, they were rehydrated, the epidermis was separated, and KC were isolated; they were cultured for 7 days. Viability was tested in the Live/Dead Viability/Cytoxicity test. The cultured KC were then transplanted to the wound on the dorsum of SCID mice (107 cells) for 14 and 21 days. Specimens of wound tissue were stained for confocal microscopy (Fig 2).
Fig 2. Epidermis formed from keratinocyte suspension preserved for 12 months in sodium chloride powder, desalinated, cultured for 7 days, and transplanted to SCID mice 3 weeks after grafting. Confocal microscopy, HLA I moAb stain; magnification 200.
The imprints of wound surface in lower legs show presence of “large” nucleated KC not only at the wound edge but also dispersed all over the wound. Unexpectedly, cells of the same shape were found, surviving dehydration of the epidermis by pulverized anhydrous sodium chloride. Transplanted to the wound, the cells formed epidermal islands of human phenotype. These cells may be the so called “spore-like” stem cells [2].
RESULTS
Ninety-five percent of cultured KC were enzymatically active “large” cells; they did not express p63 and CD29 claimed as specific for stem cells and they did not proliferate. Transplanted to the center of the wound, they formed small KC islands, becoming confluent after 14 days. CONCLUSIONS
The origin of epidermal islands on the surface of delayed healing wounds remains unknown. Remnants of hair “bulges” or basic epidermal layer might be the source; however, deep necrotic wounds preclude the presence of any such elements.
ACKNOWLEDGMENTS This work was supported by the Ministry of Science and Higher Education, Warsaw, Poland, grant No. NN 404/4942/38.
REFERENCES [1] Olszewski WL, Moscicka M, Zolich D. Human skin preserved long-term in anhydric pulverized sodium chloride retains cell molecular structure and resumes function after transplantation. Transplantation 2006;81:1583e8. [2] Vacanti MP, Roy A, Cortiella J, et al. Identification and initial characterization of spore-like cells in adult mammals. J Cell Biochem 2001;80:455.
Department of Surgical Research Transplantation and Epigenetics, Medical Research Center, Polish Academy of Science, Warsaw, Poland; and bDepartment of Transplantation Surgery, Central Clinical Hospital, Ministry of Internal Affairs, Warsaw, Poland
ABSTRACT Background. Wound granulation tissue should be covered by epidermal cells migrating from the basal layer of the epidermis or hair “bulge” of the wound edge. However, new epidermal islands are frequently formed on the granulation tissue remote from the wound edge. Thus, current theory of “bulge”-originating stem cells does not necessarily correspond to the histological pictures of the healing wound. We took imprints of a leg ulcer surface and found single dispersed, large nucleated cells, some of them in mitosis. These cells resembled those from epidermal spinosum layer. The question arouse as to whether these cells might be the “spore-like” stem cells creating epidermal island. We found similarly shaped cells among the keratinocyte preserved in pulverized sodium chloride as the only surviving population in culture and revealing enzymatic activity. The aim of this work was to study whether the population of human keratinocytes surviving sodium chloride preservation and transplanted to SCID mice may form epidermis. Methods. The 12-month sodium chlorideepreserved and cultured keratinocytes (KC) were transplanted to the wound on the dorsum of SCID mice for 14 and 21 days. Results. Ninety-five percent of cultured KC were enzymatically active “large” cells; they did not express p63 and CD29 claimed as specific for stem cells, and they did not proliferate. Transplanted to the center of the wound, they formed small KC islands and became confluent after 14 days. Conclusions. The “large” epidermal keratinocytes survived the 12-month preservation in anhydrous sodium chloride. Transplanted to the wound, they formed epidermal islands of human phenotype. These cells may be the so-called “spore-like” stem cells.
D
ELAYED-HEALING and non-healing wounds are a world-wide problem. Wound granulation tissue should be covered by epidermal cells migrating from the basal layer of the epidermis or hair “bulge” of the wound edge. However, new epidermal islands are frequently formed on the granulation tissue remote from the wound edge. Thus, the current theory of “bulge” originating stem cells does not necessarily correspond to the histological images of the healing wound. The open question remains as to whether the keratinocyte islands on the granulation tissue are formed by the stem cells desquamated and sown from the wound edges. We took imprints of a leg ulcer surface and found single dispersed, large-nucleated cells, some of them in mitosis; these cells resembled those from epidermal spinosum layer (Fig 1). Previously, we found that human skin preserved in anhydrous pulverized sodium chloride for months can be successfully transplanted, and aggressive
proliferation of keratinocytes is seen on immunohistochemical sections stained with BrdU [1]. Moreover, we found that among the keratinocyte population preserved in pulverized sodium chloride, only “large”-nucleated cells survived in culture and revealed enzymatic activity. The question arose as to whether they may be “spore-like” stem cells, which, sown on the granulation tissue, form epidermal islands. The aim of this work was to study whether the population of human keratinocytes surviving sodium chloride preservation and transplanted to SCID mice may form epidermis. *Address correspondence to Waldemar L. Olszewski, Department of Surgical Research Transplantation and Epigenetics, Medical skiego Str., Research Centre, Polish Academy of Science, 5 Pawin 02-106 Warsaw, Poland. E-mail: [email protected]
ª 2014 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/14 http://dx.doi.org/10.1016/j.transproceed.2014.09.101
Transplantation Proceedings, 46, 2885e2886 (2014)
2885
2886
OLSZEWSKI, DOMASZEWSKA-SZOSTEK, MOSCICKA-WESOŁOWSKA ET AL
Fig 1. Human single metabolically active “large” keratinocytes from the epidermis preserved for 12 months in sodium chloride powder. Giesma stain; magnification 200.
METHODS Fragments of normal human leg skin harvested during elective vascular surgery were preserved in anhydrous pulverized sodium chloride. After 12 months, they were rehydrated, the epidermis was separated, and KC were isolated; they were cultured for 7 days. Viability was tested in the Live/Dead Viability/Cytoxicity test. The cultured KC were then transplanted to the wound on the dorsum of SCID mice (107 cells) for 14 and 21 days. Specimens of wound tissue were stained for confocal microscopy (Fig 2).
Fig 2. Epidermis formed from keratinocyte suspension preserved for 12 months in sodium chloride powder, desalinated, cultured for 7 days, and transplanted to SCID mice 3 weeks after grafting. Confocal microscopy, HLA I moAb stain; magnification 200.
The imprints of wound surface in lower legs show presence of “large” nucleated KC not only at the wound edge but also dispersed all over the wound. Unexpectedly, cells of the same shape were found, surviving dehydration of the epidermis by pulverized anhydrous sodium chloride. Transplanted to the wound, the cells formed epidermal islands of human phenotype. These cells may be the so called “spore-like” stem cells [2].
RESULTS
Ninety-five percent of cultured KC were enzymatically active “large” cells; they did not express p63 and CD29 claimed as specific for stem cells and they did not proliferate. Transplanted to the center of the wound, they formed small KC islands, becoming confluent after 14 days. CONCLUSIONS
The origin of epidermal islands on the surface of delayed healing wounds remains unknown. Remnants of hair “bulges” or basic epidermal layer might be the source; however, deep necrotic wounds preclude the presence of any such elements.
ACKNOWLEDGMENTS This work was supported by the Ministry of Science and Higher Education, Warsaw, Poland, grant No. NN 404/4942/38.
REFERENCES [1] Olszewski WL, Moscicka M, Zolich D. Human skin preserved long-term in anhydric pulverized sodium chloride retains cell molecular structure and resumes function after transplantation. Transplantation 2006;81:1583e8. [2] Vacanti MP, Roy A, Cortiella J, et al. Identification and initial characterization of spore-like cells in adult mammals. J Cell Biochem 2001;80:455.