Sweat Gland Organoid-like Spheroids Originating From Reprogrammed Epidermal Keratinocytes Recapitulated Damaged Skin Functionally

Objective（1）To explore the effect ofβ₂-AR signaling pathway on epidermal keratinocyte cell stemness.（2）Reprogramming the human epidermal keratinocyte into a sweat gland-like cell（3）To explore the ways of construction and maintenance sweat gland organoids in vitro.（4）Established the burn model in nude mouse paw and perform in vivo transplantation experiments to verify the role of sweat gland organodis in promoting the regeneration of sweat glands and accelerating the healing of wounds.Materials and methods（1）The HEK and Ha Ca T cells were used for cell experiments in vitro.Isoproterenol was used to activateβ2-AR signaling pathway,and screen out the up-regulated genes related to stemness with down-regulated genes related to cell differentiation for analysis by RNA sequence.further,We performed RT-PCR and western blot analysis in HEK and Ha Ca T to detect the expression of stemness gene after treatment of isoproterenol with or without ICI-118,551,a selectiveβ2-AR antagonist.（2）Constructed plasmids of directly overexpressed EDA genes,lentivirus transfection to obtained HEK overexpression EDA genes,and named HEK-E.Designed the EDA gene promoter sequences and used CRISPR/d Cas9 to get Ha Ca T-E cell,which of overexpressed EDA genes.Identification sweat gland markers by RT-PCR,western blot and immunofluorescence staining.Observation of cell ultrastructure by TEM.（3）HEK-E and Ha Ca T-E were implanted in Matrigel with Sw G-specific medium for3D cultured,immunofluorescence staining was performed when cells developed into cluster to detect sweat gland marker expression.Further,the cluster was transplanted into microfluidics chip and cultured in a Sw G-specific medium with a small concentration gradient.Finally,IF staining detection was performed.（4）Established BALB/c Nude paw burn model,Respectively grafted m Sw G-M,i Sw G and i Sw GO to calculate the healing area at different times and draw a graph.Three weeks later to detected perspiration function,HE staining and IF staining were used to observe the presence of new sweat glands.Results（1）Analysis of RNA-seq results showed that the stemness genes of HEK cells were significantly up-regulated after treatment with isoproterenol.Among them,the expressions of SOX9,OCT4,LGR5 and LGR6 were significantly up-regulated compared with the control group after verification by RT-PCR and Western blot.And,isoproterenol was inhibited by ICI-118,551,a selective inhibitor ofβ2-AR.The above results indicate that,isoproterenol has the effect of improving the stemness potential of hacat cells.（2）Lentivirus system was transfected into HEK cells after overexpression EDA plasmid was packaged to obtain HEK-E expressing GFP.Packaged plasmids sg RNA and d Cas9,respectively,transfected in sequence for Ha Ca T to obtain Ha Ca T-E cells expressing GFP and G418 resistance.The two types of cells were induced by Sw G-specific medium and expressed the marker of sweat gland cells.Transmission electron microscopy also observed a rich microvilli structure.These results indicate that HEK and Ha Ca T was successfully reprogrammed into sweat gland like cells.（3）HEK-E and Ha Ca T-E developed into organoid after culture in Matrigel containing Sw G-specific medium.IF staining showed sweat gland makers of CK18、CK19、α-SMA、APQ5、NA-K-ATPase was positive.Put organoid into microfluidic chip to further cluture,the morphology of the organoid developed from a circular cystic structure to a luminal structure with a long axis,and IF staining showed positive expression of CK19 andα-SMA.The above results indicated that after three dimensional culture,the cells had formed functional sweat gland organoid,which were then inducted into a sweat glandular structure closer to the original sweat glands by microfluidic medium with a small molecular gradient.（4）The results of burn model show that compared with m Sw G-M and i Sw G,i Sw GO can accelerate the healing of scald wounds on the paw.Three weeks after transplantation,the sweat test showed positive results in the i Sw GO group.HE staining of the three groups of paw revealed the presence of sweat glan structures in the i Sw GO and i Sw G.However,The structure of i Sw GO is closer to the original state than i Sw G.IF staining showed positive GFP expression in new sweat gland and expression of CK18 andα-SMA.ConclusionThis study preliminarily explored the effect of isoproterenol on the stemness potential of Ha Ca T cells,and found that could improve stemness.On this basis,it reduces cross-lineage difficulty and provides intrinsic motivation for direct reprogramming of epidermal cells into sweat gland cells.In addition,we used reprogrammed sweat gland-like cells for in vitro sweat gland-like organ culture and directed induction with microfluidic chip to achieve sweat gland-like tissue regeneration.It provides a new idea for the research and treatment of sweat gland regeneration in the future.In addition,we cultured in vitro sweat glandular organs by using reprogrammed sweat gland-like cells and induced the regeneration of sweat glands by microfluidic chip.It provides new ideas for future research and treatment in this field.