Human Epithelial Stem Cells Persist Within Tissue-Engineered Skin Produced by the Self-Assembly Approach

Abstract

To adequately and permanently restore organ function after grafting, human tissue-engineered skin substitutes (TESs) must ultimately contain and preserve functional epithelial stem cells (SCs). It is therefore essential that a maximum of SCs be preserved during each in vitro step leading to the production of TESs such as the culture process and the elaboration of a skin cell bank by cryopreservation. To investigate the presence and functionality of epithelial SCs within the human TESs made by the self-assembly approach, slow-cycling cells were identified using 5'-bromo-2'-deoxyuridine (BrdU) in the three-dimensional construct. A subset of basal epithelial cells retained the BrdU label and was positive for the SC-associated marker keratin 19 within TESs after a chase of 21 days in culture post-BrdU labeling. Moreover, keratinocytes harvested from TESs gave rise to SC-like colonies in secondary monolayer subcultures, indicating that SCs were preserved within TESs. To evaluate the effect of cryopreservation with dimethyl sulfoxide and storage in liquid nitrogen on SCs, human epithelial cells were extracted from skin samples, amplified in culture, and used to produce TESs, before cryopreservation as well as after thawing. We found that the proportion and the growth potential of epithelial SCs in monolayer culture and in TESs remained constant before and after cryopreservation. Further, the functionality of these substitutes was demonstrated by successfully grafting human TESs on athymic mice for 6 months. We conclude that human epithelial skin SCs are adequately preserved upon human tissue reconstruction. Thus, these TESs produced by the self-assembly approach are suitable for clinical applications.

Translating the combination of gene therapy and tissue engineering for treating recessive dystrophic epidermolysis bullosa

“…Next, the effect of the transduction enhancers and vectors on clonogenic behaviour of keratinocytes was evaluated. The percentage of colonies formed is indicated in Table 1 and it was in line with previous studies (Barrandon and Green, 1987;Lavoie et al, 2013;Rheinwald and Green, 1975). The transduction enhancers did not appear to affect the CFE.…”

“…Fibroblasts and keratinocytes were isolated by the two-step thermolysine and trypsin method, as previously described Lavoie et al, 2013). Briefly, skin biopsies were digested overnight at 4 °C in HEPES buffer [10 mM 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid (MP Biomedicals Inc., Montreal, QC, Canada), 6.7 mM KCl, 142 mM NaCl and 1 mM CaCl 2 ] containing 500 mg/mL thermolysine (SigmaAldrich).…”

“…Next, cryogenic vials were placed in a freezing container (Nalgene ® Labware) filled with 99 % ethanol and precooled at 4 °C and kept at −80 °C for 24 h to allow a freezing rate of −1 °C/min. Cells were stored in liquid nitrogen until use Lavoie et al, 2013).…”

“…For determination of colony-forming efficiency (CFE), 1 × 10 2 keratinocytes/cm 2 were plated in Petri dishes with iHFL (8 × 10 3 cells/cm 2 ) and maintained for 9 d in monolayer cultures (Barrandon and Green, 1987;Lavoie et al, 2013;Rheinwald and Green, 1975). Three Petri dishes per condition were seeded.…”

Improved Methods to Produce Tissue-Engineered Skin Substitutes Suitable for the Permanent Closure of Full-Thickness Skin Injuries

“…The resulting skin tissue shares many properties with native human skin and minimizes the host response after transplantation. The self-assembly method generates a highly functional and mechanically stable skin substitute 6 preserving epithelial stem cells, 7 which is suitable for autologous grafting in humans. 8 …”