Effects Of Extracellular Matrix From Dermal Substructure Of Human Hair Follicle On Biological Characteristics Of Dermal Cup Cell

Background and objectiveBased on different molecular markers,morphologies and functions,the dermal compartment of human hair follicle can be divided into three substructures,including dermal papilla(DP),dermal cup(DC),and non-peribulbar dermal sheath(DS).DP and DC cells play key roles in hair growth and are keenly interconnected.Dermal substructure cells of human hair follicle secrete abundant extracellular matrix(ECM)which constitutes their own microenvironment,playing important role in cell function and hair follicle growth.In this study,by isolating different dermal substructure cells of human hair follicle bulb and obtaining their ECM,we aimed to investigate:1.differences in cell morphology,proliferation and biological markers;2.differences in structure and composition of ECM;3.effects of different ECM on the morphology,proliferation and transcriptional profile of DC cells.Methods1.Isolation of dermal substructure cells of human hair follicle and preparation of different ECMCells from different dermal substructures of human hair follicle bulb(DP and DC)were isolated using a modified method(enzymatic digestion combined with microdissection)according to their different microscopic anatomy.Cell migration,morphology and proliferation was compared under light microscope;and alkaline phosphatase expression was evaluated by immunofluorescence staining.DPs and DCs were separately cultured and cellular components were washed away to obtain their ECM.Light microscope,scanning electron,elcian blue staining and sirius red staining were used to investigate the differences in their structure and composition.2.Effects of ECM from different dermal substructure of human hair follicle on DC cellsDC cells were seeded on the prepared DP-ECM,DC-ECM,and on plastic flask as control group.The morphology and distribution of cells after expansion on different substrate were evaluated through light microscope at 8h,2d and 8d,respectively.CCK8 assay were conducted to compare proliferation rate of DC cells in the three groups.The transcriptome profiles of DC cells cultured on DC-ECM,DP-ECM,and control group were investigated by RNA-sequencing,and up-regulated genes and related pathways were analyzed by bioinformatic analysis.ResultsThe modified method using enzymatic digestion combined with micro-dissection could successfully extract DP and DC cells from different substructures of human hair follicle,while did not significantly affect the cell morphology,growth pattern and ALP expression.Meanwhile,the isolated DP and DC cells showed good cell-migration and activity.Anatomically close in location,DP and DC cells were also similar in cell morphology.Compared with DC-ECM,DP-ECM was thicker and denser under light and electron microscopy,with smaller pores and higher content of proteoglycan and collagen after staining.After seeding DC cells,both ECMs promoted similar levels of cell attachment,which was significantly higher than that on plastic flask.DC cells on the ECMs adopted an elongated spindle-shape with smaller cell area and random cell distributions opposed to the spreading-out morphologies and directional cell-distribution on the control group.After 8 days,cells on DP-ECM were irregular in size and distribution with more vesicles inside the cells compared with those in DC-ECM and control group.DC-ECM promoted proliferation of DC cells while the DP-ECM inhibited cell proliferation.RNA-sequencing and bioinformatic analysis showed Notch1 was upregulated in DC cells from DP-ECM group,while up-regulated genes were related to BMP signaling pathway in DC-ECM group.ConclusionsThe novel modified method can successfully extract DP and DC cells from the dermal substructure of human hair follicles while did not significantly affect cellular biological characteristics.DP-ECM and DC-ECM were different in structures and composition.DP-ECM was thicker and denser,with smaller pores and higher content of proteoglycan and collagen.Both ECMs promoted similar levels of attachment and morphogical change of seeded DC cells;DC-ECM promoted proliferation of DC cells while the DP-ECM inhibited cell proliferation.DP-ECM and DC-ECM showed different effects on transcriptome profiles of DC cells,which provide basis for further studies on the mechanisms underlying the regulation of cellular biological characteristics in human hair follicle by ECM.