Methods of generating human endometrial stromal fibroblasts and three-dimensional multi-layered human endometrial tissue compositions

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.