Methods of generating human endometrial stromal fibroblasts and three-dimensional multi-layered human endometrial tissue compositions
Disclosed herein are methods for obtaining endometrial stromal fibroblast cells from pluripotent stem cells, such as induced pluripotent stem cells. The present disclosure also provides methods of obtaining a three-dimensional, multilayered endometrial tissue composition. Methods of using the cells and tissue compositions in drug screening and therapeutic applications are also provided.
1 . A method comprising:
(a) culturing a monolayer of pluripotent stem cells (PSCs) with a single differentiation factor consisting of a first Wnt/β-catenin agonist, CHIR99021, for about 4 days;
(b) culturing the cells of (a) with a single differentiation factor consisting of a second Wnt/β-catenin agonist for about 4 day, wherein the second Wnt/β-catenin agonist consists of WNT7A;
(c) culturing the cells of (b) with Bone Morphogenetic Protein 2 (BMP-2) and β-estradiol for about 4 days;
wherein the cultured cells of (c) comprise a monolayer of PSC endometrial stromal fibroblasts (PSC-ESF) cells;
(d) combining:
(i) dissociated cells from endometrial epithelial organoids (EEO), and
(ii) dissociated PSC-ESF of (c);
(e) culturing the combined cells of (d) in a porous substrate comprising extracellular matrix components for at least 3 days, and
(f) during the culturing step of (e), forming, by cell self-assembly a three-dimensional human endometrial tissue comprising an outer layer of PSC-ESF and an inner layer of epithelial cells;
wherein CHIR99021 is provided in a range of about 1-20 μM, WNT7A is provided in a range of about 10-200 ng/ml, BMP-4 is provided in a range of about 1-20 ng/ml, and β-estradiol is provided in a range of about 1-20 nM; and
wherein the three-dimensional human endometrial tissue is responsive to cyclic hormone treatment.
2 . The method of claim 1 , wherein the EEO cells express EpCAM, FOXA2, Ki67 and PR.
3 . The method of claim 1 , wherein the porous substrate comprises a semi-solid culture medium or three dimensional (3D) porous biomaterial.
4 . The method of claim 1 , wherein the outer layer of PSC-ESF cells interface with the inner layer of epithelial cells along the Laminin+basolateral surface of the epithelial cells.
5 . The method of claim 1 , wherein the cells from endometrial epithelial organoids (EEO), the PSC-ESFs or both comprise a genetic mutation known to cause a medical disease or condition when the genetic mutation is present in a human subject.
6 . The method of claim 1 , wherein CHIR99021 is provided at about 8 μM, WNT7A is provided at about 100 ng/ml, BMP-4 is provided at about 10 ng/ml, and β-estradiol is provided at about 10 nM.