Directional Differentiation Of Mammary Progenitor Cells Into Sweat Gland Cells By 3D Bioprinted Microenvironment

Objective(1)To investigate the effect of mouse sweat gland extracellular matrix(ECM)protein on the differentiation of mouse mammary progenitor cells in vitro.(2)To explore the effect of three-dimensional(3D)bio-printing sweat gland microenvironment on the differentiation of MPCs in vitro,and to provide a reliable theoretical basis for the application of tissue engineering technology to construct a suitable cell growth microenvironment in vitro.(3)To explore the differentiation direction of MPCs in the sweat gland microenvironment constructed in vitro,and to provide a new research idea for the construction of an ideal microenvironment that can induce cell differentiation in vitro.(4)To explore the possible mechanism of MPCs differentiation induced by sweat gland microenvironment of 3D bioprinting,and to provide theoretical basis for clinical application of sweat gland microenvironment of 3D bioprinting.Material and methods(1)The foot homogenate of newborn mice was used as the source of sweat gland ECM protein.Mammary progenitor cells of newborn mice and sweat gland cells of adult mice were separately extracted.By consulting a large number of literatures and using immunofluorescence staining technology,the differential protein expressed in MPCs and sweat gland cells,namely sodium/potassium channel protein ATP1a1 during sweat gland development,was screened.MPCs were added with sweat gland ECM protein in culture under two-dimensional(2D)environmental conditions.After collecting cells,immunofluorescence and reverse transcription quantitative polymerase chain reaction(Real-time Quantitative polymerase chain reaction,RT-qPCR)technology to detect the expression of differential proteins to explore the role of sweat gland ECM protein on the differentiation of MPCs in a 2D culture environment.(2)Gelatin and sodium alginate were fully dissolved in phosphate buffered saline(PBS)and then proportioned into printable bioink and added to the sweat gland ECM protein.After wrapping the MPCs,they were printed into multi-layer mesh by 3D bioprinting technology to construct sweat gland microenvironment model in vitro.The control groups were set to have no sweat gland ECM protein group and unprinted group compared with the experimental group.The proliferation and development of cells in the microenvironment were observed under light microscope.Immunofluorescence analysis and RT-qPCR were used to detect the expression of differentially expressed proteins in MPCs and to explore the effect of sweat gland microenvironment on the differentiation of MPCs.(3)Isolation and extraction of MPCs cultured in 3D bioprinted sweat gland microenvironment for 7 and 14 days.Immunofluorescence staining and RT-qPCR were used to detect the expression of CK8 and CK14 in sweat glandular epithelial and myoepithelial cells in the induced cells at the protein and molecular levels respectively.Furthermore,the results of co-localization of two specific proteins in the induced cells with the differential protein ATP1a1 were observed by immunofluorescence double staining to further explore the differentiation direction of MPCs in the 3D bioprinted sweat gland microenvironment.(4)Previous experimental results and literature review demonstrated that the signal transduction pathways involved in mouse sweat gland development include EDA,NF-?b,and Shh signaling pathways.The cells induced in the sweat gland microenvironment for 7 and 14 days were collected,and the expression of the above pathway in the induced cells was analyzed by RT-qPCR.The signal pathway with obvious up-regulation was screened,and the inhibitor of the signal pathway was added during the induction process.RT-qPCR was used to further test the expression of this signal pathway in the induced cells and then explored the related mechanism of 3D bioprinting sweat gland microenvironment to induce differentiation of MPCs into sweat glands.The influence of microenvironment hardness on the differentiation direction of MPCs was estimated based on the measured hardness.Result(1)Under 2D culture conditions,the expression of the sweat gland cell specific protein ATP1a1 in MPCs was negative regardless of whether or not the sweat gland ECM protein was added in the culture environment.The results showed that the ECM protein of sweat gland could not specifically induce the differentiation of MPCs into sweat gland cells in 2D culture environment.(2)The immunofluorescence and RT-qPCR results of the cells cultured in the control group showed no significant specific protein ATP1a1 expression.The MPCs cultured in the 3D bioprinted sweat gland microenvironment expressed the differential protein ATP1a1,and the expression was significantly up-regulated on the 14 th day of induction.The results of immunofluorescence staining showed that the expression level of Estrogen Receptor-?(ER-?),which is specifically expressed in mammary gland cells,was significantly decreased in cells with up-regulated ATP1a1 expression.The results of spectrophotometer analysis showed that there were no residual cellular components in the ECM proteins of sweat glands,thus eliminating the interference of sweat gland cells on this induction process.This result demonstrated that the 3D bioprinted sweat gland microenvironment could induce the specific differentiation of MPCs into sweat gland cells and the application of 3D bioprinting in sweat gland regeneration is of great significance.(3)The results of immunofluorescence and RT-qPCR further showed that the expression of CK8 was up-regulated in cells after 14 days of 3D bioprinted sweat gland microenvironment.The results of immunofluorescence co-localization showed that ATP1a1 and CK8 were co-expressed in the induced cells and no co-expression with CK14.In summary,the 3D bioprinted sweat gland microenvironment constructed in this experiment induced the specific differentiation of MPCs into the sweat glandular epithelial cells,which provided a new idea for constructing the microenvironment of the in vitro cell and tissue development.(4)The results of RT-qPCR showed that the expression of Shh signaling pathway was significantly up-regulated during the differentiation of MPCs into sweat gland cells induced by 3D bioprinted sweat gland microenvironment.After microenvironment treatment with inhibitors of Shh signaling pathway,RT-qPCR results showed that the expression of ATP1a1 and CK8 in cells was significantly lower than that in cells without inhibitor treatment on the 14 th day of culture.This result demonstrated that the Shh signaling pathway played an important role in the differentiation of MPCs into sweat gland cells induced by 3D bioprinted sweat gland microenvironment,which lays a solid foundation for further study of sweat gland regeneration in vitro.The microenvironmental hardness test showed that the hardness of the control group was about twice that of the experimental group.According to the literature,it was suggested that the microenvironment of the experimental group with less hardness was more favorable for inducing the differentiation of MPCs into the epithelial cells of the sweat gland.ConclusionIn this experiment,the process of differentiation of MPCs into sweat gland cells induced by 3D bioprinted sweat gland microenvironment was studied and its related mechanism was explored.It was found that the sweat gland development-specific in vitro microenvironment constructed by sweat gland ECM proteins binding to 3D bioprinting can effectively induce the differentiation of MPCs into sweat glandular epithelial cells and express functional proteins compared with the 2D environment and the three-dimensional environment.The Shh signaling pathway played an important role in this induction process.The hardness of the microenvironment might also affect the differentiation direction of MPCs.Our study provided a good theoretical basis and new ideas for the future construction of the ideal microenvironment and regeneration of sweat glands in vitro using 3D bioprinting technology.