Reprogramming Of Fibroblasts To Uterine Glandular Epithelium By Chemical Cocktail And Its Functional Approach

Absolute uterine factor infertility(AUFI)affects 3–5% women and has multifactorial causes.Uterus transplantation(UTx)is the main treatment option for AUFI.However,due to the particularity of uterus,organs can be donated from dead bodies or living bodies.Limited organ sources and ethical issues limit the wide application of this technique.With the development of tissue engineering and regenerative medicine,artificial wombs could make by combining tissue materials and bioengineering.Alternatively,artificial uterus can be used as an incubator for embryos.A bioartificial uterus requires a complete structure which includes cell components,in order to achieve the reconstruction of the structure and physiological function of the artificial uterus,however,poor cell sources hamper the clinical applications of bioartificial uterus.In previous studies,somatic reprogramming and transdifferentiation techniques have been developed rapidly.Cells with pluripotency or specific functions can be obtained stably by means of virus-mediated transcription factors or small molecular compounds,which provides a possibility for regenerative medicine.Among them,the reprogramming method of virus and other mediated transcription factors has the risk in genetics,and the infinite amplification ability of pluripotent cells has also become the cause of its tumorigenic risk.In the method of transdifferentiation,clear objective function cells,avoiding the process of somatic cells into pluripotent cells,reducing the risk of tumorigenicity,which induced by chemical method gradually become important means of transdifferentiation experiment,because the structure and function of small molecule compounds with clear,operation simple is easy to control,the risk small and easy to standardize,genetics is useful in the clinical application of regenerative medicine.Currently,small molecular compounds that are often selected can be divided into epigenetic regulators,signaling regulators,metabolic regulators,nuclear receptor activators and inhibitors.Mammalian uterus glandular epithelium(GE)is necessary for blastocyst implantation and its deficiency can lead to infertility.GE responds to the ovarian hormones,estrogen(E2)and progesterone(P4),and induces the expression of leukemia inhibitory factor(Lif)for the embryo implantation.Direct reprogramming of the fibroblasts by specific transcription factors(TFs)or chemical molecules have been previously reported.However,obtaining uterine cells through fibroblasts reprogramming has rarely been reported.Uterine glands and their secretions are necessary to establish uterine receptivity and embryo implantation,early pregnancy failure is usually caused by the secretory phase defect.In this study,we reported the generation and characterization of full chemical-induced uterine glandular epithelium from fibroblasts with the ability of amplification in vitro and responding to ovarian hormones.In the culture system,proline hydroxylase inhibitors that can activate hypoxiainducible factor,Al K inhibitors that inhibit the fate of fibroblasts and GSK inhibitors that can activate WNT and promote cell proliferation were selected.Our study identified a new cell source that can be used for the reconstruction of the uterus or other organs and suggested a potential value for treating uterine infertility and the related reproductive medical disorders.We obtained ci GEs(chemical-induced glandular epithelial cells)with uterine glandular epithelial characteristics which respond to ovarian hormones,implying their application in endometrium replacement in clinical treatment.When the ci GEs and embryos were co-cultured,the ci GE cells showed a better cell connection mode than the MEF cells,so that the embryo showed better ductility and invasiveness.CiGEs were obtained by induction of fibroblasts using only a chemical cocktail.The chemical cocktail has several advantages including cell permeability,convenient handling,being nonimmunogenic,and ease of standardization.These advantages make it an attractive strategy for clinical application in treatment of uterine diseases like AUFI.Furthermore,we found the upregulation of functional related genes including estrogen and progesterone response genes in ci GEs.The molecular mechanism of the chemical cocktail-induced transdifferentiation needs to be subject to a single step in the future.Nevertheless,our research provides a clue to generate target cells from resident fibroblasts in situ in damaged or aging uterus through an induction by a chemical cocktail.At the same time,our results provide new insights into the treatment of uterine factor infertility,uterine regeneration and artificial uterine construction.