ArticlesHuman embryonic stem-cell derivatives for full reconstruction of the pluristratified epidermis: a preclinical study
Introduction
For more than two decades, cell therapy has been essential in improving outcomes for patients with large burn injuries, by means of epidermal replacement following in-vitro expansion of the patient's own keratinocyte stem cells. The major drawback of this technique, however, is the 3-week delay needed to obtain the required amount of cells, which puts patients at risk of dehydration and infection.1 Decellularised cadaver skin has been used to cover wounds during the 3-week period, but the benefit of this temporary skin substitute is hampered by its limited availability. Furthermore, the tissue can be rapidly rejected by an immune reaction. To overcome the problem of accessibility, inert synthetic and biosynthetic matrices have been actively searched for and developed.2 Up to now, however, these substitutes have not replaced cadaver skin in large burns3 since they increase the risks of rapid graft rejection and disease transmission because they contain bovine collagen and adult allogenic skin cells.2
Human embryonic stem cells (hESCs) might offer another way to meet the challenge of temporary skin replacement because they can be expanded in unlimited amounts and differentiated into any human cell phenotype in vitro, provided the relevant differentiation protocol is developed.4 The potential of the derivatives of hESCs in cell therapy is currently being explored in applications as diverse as Parkinson's and Huntington's neurodegenerative diseases,5, 6 diabetes mellitus,7 and cardiac insufficiency following ischaemia.8 The rapid maturation of these studies has recently been exemplified by the authorisation given by the US Food and Drug Administration for a first clinical trial in patients with spinal trauma.9
So far, only one study has reported the formation of a skin equivalent by use of mouse embryonic stem cells.10 Subsequently, several studies tried to differentiate hESCs along the keratinocyte lineage. Cultured in the form of embryoid bodies or nodules produced in mice with severe combined immunodeficiency disorder, hESCs gave rise to epidermal cells, but they showed low proliferative potential.11 More recently, keratinocytes were derived from hESCs by use of short cytokine induction.10, 11, 12, 13, 14 However, production of a pluristratified epidermis has remained elusive in vitro and in vivo, suggesting that functional basal keratinocytes had not been obtained. We have met that functional challenge by designing a protocol that takes into account the long-term succession of biological steps that lead to epidermis formation during ontogenesis.
Section snippets
Cell culture
hESCs from two cell lines, SA01 (Cellartis, Götenborg, Sweden) and H9 (Wicell, Madison, WI, USA) were grown on STO mouse fibroblasts, inactivated with 10 mg/mL mitomycin C and seeded at 30 000 per cm2, and grown as previously described.6
For differentiation, hESC clumps were seeded onto mitomycin-C-treated 3T3 fibroblasts in FAD medium (3:1 mixture of Dulbecco's modified Eagle's medium [DMEM] and Ham's F12 media) and 10% fetal calf serum (FCII, Hyclone, Logan, UT, USA) supplemented with 5 μg/mL
Results
Figure 1 shows the experimental design. In differentiating hESCs from both H9 and SA01 cell lines, pluripotency gene markers OCT4 and NANOG decreased rapidly after 5 days, and were undetectable at 20 days (figure 2, webappendix p 2). FACS analysis showed a loss of the undifferentiated state marker SSEA3 at 40 days (figure 2, webappendix p 2).
The succession of epidermal markers during ontogenesis characterised by a strict temporal expression pattern of structural molecules17, 18, 19 was fully
Discussion
We have shown that keratinocytes can be derived from hESCs. K-hESCs can form a pluristratifed epithelium that resembles normal human epidermis in vitro and following grafting in vivo. To obtain these findings, we used a protocol that combined co-culture of hESCs with particular feeder cells and pharmacological treatment over 40 days. Growing human epidermis from hESCs could have clinical relevance as an unlimited resource for temporary skin replacement in patients with large burns awaiting
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Prof G Waksman died in Sept, 2007