134. DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLS TO MULLERIAN TISSUE

The human uterus develops from the distal Mullerian Duct, a derivative of the mesoderm germ layer. Unlike other mammalian species (eg. mouse) the endometrium of the human uterus develops prenatally during gestation. Little is known about the developmental process involved. A better understanding of human endometrial development may shed light on the mechanisms involved in endometrial regeneration and pathogenesis of adult proliferative endometrial diseases. Mouse neonatal uterine mesenchyme (mNUM) is inductive and can maintain the phenotype of normal adult human endometrial epithelial cells [1]. Both adult human endometrial stroma and neonatal mouse endometrial mesenchyme secrete growth factors of the TGF-beta family including BMPs which have been shown to play an important role in differentiation of human embryonic stem cells (HESC) [2, 3]. Hypothesis: mNUM will direct differentiation of HESC to form Mullerian Duct-like epithelium. Aim: to investigate the role of mNUM in differentiating HESC in vitro and in vivo using A tissue recombination technique. Method: Embryoid bodies (EB) were formed from GFP labelled HESC (ENVY) and GFP-MIXL1 HESC reporter line [4, 5] and recombined with 2 × 0.5 mm pieces of day 1 epithelial cell-free mNUM. Recombinant tissues were either harvested for gene expression analysis or grafted under the kidney capsule of NOD/SCID mice. Results: We found by qRT-PCR that mNUM induces HESC to form mesendoderm/mesoderm progenitors in vitro, obligate intermediates of the developing Mullerian Duct. After further incubation in vivo under the guidance of mNUM, HESC differentiated to form duct-like structures comprising mesoepithelial cells that co-expressed several key developmental proteins of the Mullerian Duct including Emx2, Pax2, Hoxa10, CA125, and also intermediate filament markers such as CK8/18, Vimentin (n = 8). Conclusion: Our study demonstrated for the first time that mNUM can direct HESC to form a mesodermally derived epithelium that is Mullerian Duct-like, providing a novel model for studying human uterine development. (1) Kurita T, et al., The activation function-1 domain of estrogen receptor alpha in uterine stromal cells is required for mouse but not human uterine epithelial response to estrogen. Differentiation, 2005. 73(6): 313–22.(2) Hu J, Gray CA, Spencer TE, Gene expression profiling of neonatal mouse uterine development. Biol Reprod, 2004. 70(6): 1870–6.(3) Stoikos CJ, et al., A distinct cohort of the TGFbeta superfamily members expressed in human endometrium regulate decidualization. Hum Reprod, 2008. 23(6): 1447–56.(4) Davis R, et al., Targeting a GFP reporter gene to the MIXL1 locus of human embryonic stem cells identifies human primitive streak-like cells and enables isolation of primitive hematopoietic precursors. Blood, 2008. 111(4): 1876–84.(5) Costa M, et al., The hESC line Envy expresses high levels of GFP in all differentiated progeny. Nat Methods, 2005. 2(4): 259–60.