Induction Of Epidermal Cells To Dedifferentiate With Non-external Gene Intervention And Clinical Research Of The Regeneration Of Sweat Glands

Objective:1.To establish the dedifferentiation-inducing models in vivo and in vitro and to determine the possible signaling pathway involved in the reversal process. 2.To explore the possibility of transdifferentiating bone marrow-derived mesenchymal stem cells(MSCs)into sweat gland cells(SGCs),and implanting the latter into fresh skin wound to generate functional sweat glands.Methods:1.In the in vivo study,epidermal sheets free of all epidermal stem cells(ESCs)in the basal layer were transplanted into full-thickness skin wounds in immunodeficient BALB/c nude.Five days later,immunohistochemial staining,flow cytometrical,Western bloting and RT-PCR analyses were used to observe the phenotypic change of the transplanted epidermal sheets.2.In the in vitro study, mature epidermal cells were exposed to heated water and basic fibroblast growth factor(bFGF)to induce dedifferentiation.Immunocytochemical staining,flow cytometrical and Western bloting analyses were used to determine the phenotypic change of epidermal cells;Giemsa staining and electron microscopic analyses were performed to observe the morphological change;the proliferation capacity was assessed by two-and feeder-layer culture systems and the redifferentiation potential by organotypic cultures;gene chip technology was utilized to analyze the differences of gene expression profile before and after induction.The phenotype and proliferation potential were also observed after the epidermal cells were pretreated withβ-catenin agonists;β-catenin-knockdown epidermal cells were cultured by use of small interfering RNA(siRNA)and again induced with heated water and bFGF to observe the changes in their phenotype;Smad4 knockout mice were used as a model of indirect activativation ofβ-catenin,the number and distribution of CK14- and Ki67-positive cells were observed in epidermis of this animal model.3.Human MSCs and SGCs were isolated,cultured,expanded and indentified in vitro respectively. MSCs were labeled with BrdU or GFP.The confluence SGCs(2～4×10⁵)were heat-shocked at 47℃for 40 min and cooled for 1～2 hours at 37℃,then 1～2×10⁵ BrdU-labeled or GFP-traced human MSCs were added and co-cultured with SGCs. The phenotype of MSCs was detected after 5 days of co-culture.The stem cells which subsequently exhibited the phenotype of sweat gland cells were implanted into scald injured paws of nude mice,and regeneration of functioning sweat glands was confirmed by perspiration test(iodine and starch)and histological examination.A male patient beating almost identical burn scars on the posterior aspect of both arms was enrolled for clinical trial.The scars were first proved to be anhydrotic with iodine and starch test.With patient's written consent,the clinical trial was carried out.Bone marrow-derived MSCs and SGCs were obtained from the patient.After being heat shocked,the SGCs were co-cultured with MSCs.Five days later,the scars of both arms were excised.MSCs(2.4×10⁵/ml,1.5ml)having acquired the phenotype of sweat gland cells after co-culture were evenly spread onto the excision wound on the right arm.They were covered with a piece of acellular allogeneic dermis,which was perforated with numerous micropores.On top of the latter,micrografls of autologous origin were transplanted,and the wound was finally covered with a piece of allogeneic skin graft.The wound on the left side was similarly covered,but without transdifferentiated MSCs.After complete healing of the wounds,perspiration test with iodine and starch was performed,and biopsy was taken from the MSC transplanted area.The components of the sweat collected from the implantation area were analyzed and compared with that from normal skin elsewhere on the body.Results:1.In the in vivo study,immunohistochemical analysis revealed no CK19 andβ1 integrin expression in sheets before transplantation but their expression at the wound-neighboring side of the skin grafts.Flow cytometry revealed increasing evidence of CK19- andβ1 integrin-positive cells,from their absence before transplantation,to a 7.54%and 5.24%proportion,respectively,5 days after transplantation.The proportion of CK10-positive cells in transplanted ultrathin epidermal sheets decreased,from 99.62%before transplantation to 86.56%after transplantation.The protein and mRNA levels of CK19 andβ1 integrin were significantly increased on days 3,5,and 7 after transplantation and peaked on day 5.2.In the in vitro study,after exposure to heated water and bFGF,some of the surviving cells reverted from a differentiated to a dedifferentiated state,as evidenced by the re-expression of CK19,β1 integrin andα6^briCD71^dim.They experienced morphological change to present smaller size with fewer cellular organelles and a relatively higher ratio of nucleus to cytoplasm.They also regained strong proliferation capacity and formed colonies with defined edges.And the skin equivalent constructed with these cells showed a well-organized structure,with CK14-,CK19- andβ1 integrin-positive cells in the basal layer.For the gene expression profile,genes controlling cell adhesion and mitotic cell cycle were upregulated during the reversal process,but those controlling epidermal cell development and differentiation,as well as keratinization,were downregulated.3.The activation of the Wnt/β-catenin signaling pathway in vitro by LiCl or GSK-3βinhibitor induced the dedifferentiation of cultured epidermal cells.In contrast,after successful specific knockdown ofβ-catenin by siRNA,these epidermal cells failed to revert to stem cell-like cells after induction of dedifferentiation.Furthermore,the study of Smad4 knockout mice showed that the indirect activation ofβ-catenin by disruption of smad4 increased the number of CK14- and Ki67-positive TA cells,which were distributed widely in both basal and suprabasal layers,as compared with their distribution in the basal layer of normal epidermis.4.Immunocytochemistry showed that MSCs expressed CD29, CD44,CD71,CD105 and CD166,but not the hematopoietic markers CD34 and CD45,as well as SGC markers CK19 and CEA.MSCs could be induced toward adipocytic lineage and osteogenic lineage differentiation.SGCs were strongly positive for CK19 and CEA.After SGCs were heat-shocked at 47℃for 40 min,the majority of them remained adherent but many cells lost cell-cell contact as their cytoplasms retracted.When MSCs were co-cultured with heat-shocked SGCs for 5 days,a significant number of BrdU-labeled MSCs and GFP-positive cells were also positive for CEA.In the animal experiment,there was regeneration of functional sweat glands in the burned paws of the nude mice,as indicated by perspiration test,histological and immunohistochemical analysis.In human patients,all wounds healed nicely.The areas where transdifferented MSCs were implanted showed positive iodine-starch perspiration test.Histological and immunohistochemical examination confirmed that the transformed MSCs bore CEA,the specific marker of sweat gland cells. Biochemical analysis of the excreted sweat contained similar components as that of sweat collected from normal skin.Conclusions:1.Change of microenvironment and some external factors could induce a phenotype reversion of epidermal cells from an adult-differentiated state to an immature-like dedifferentiated state.2.The Wnt signaling pathway,especiallyβ-catenin,plays a key role in regulating the phenotype reversion of epidermal cells to epidermal stem cells.3.MSCs can be transdifferentiated into SGCs in vitro,and they can be implanted into a flesh wound to form functional sweat glands.