Figure 1.
Characterisation of hESC-derived FRT epithelium in GF treated heterotypic (nMUM) recombinant 8 week xenograft.
β-tubulin expression in (A) ciliated simple columnar epithelia of human adult proliferative endometrium and (B) in hESC derived ciliated, simple columnar FRT epithelium (arrows indicates cilia). (C) immunofluorescence analysis of hESC derived FRT epithelium (green) where β-tubulin (yellow orange) is expressed on cell surface (inset shows a close up view of the cilia), the hESC origin of epithelial cells was assigned on the basis of GFP expression. Note that adjacent mouse uterine stromal cells are GFP−. hESC derived FRT epithelium co-localised (D) cytoplasmic CK18, (E) CA125 on epithelial surface (arrow), and (F) ERα (pink) with GFP+ hESC derived epithelial cells. Weak ERα expression was also evident in mouse uterine stromal cells (filled arrows). (G) ERα expression in human proliferative endometrial gland (full arrows), dotted line indicates epithelium. Ki67 expression in hESC derived FRT epithelium before (H) and after (I) estrogen injections showing estrogen-induced epithelial cell proliferation. The expression of GdA in hESC derived FRT epithelium without (J) and with (K) estrogen injections showing estrogen-induced cytoplasmic expression of GdA. H&E of hESC derived FRT epithelium lined by simple columnar cells without (L) and with (M) estrogen treatment, showing estrogen-induced increase in epithelial height and stromal oedema (arrows), classical hormonal responses of adult uterine epithelium (N). (O) Three hESC derived FRT epithelial structures surrounded by α-SMA positive cells. Inset in (K) is concentration matched mouse IgG1 negative control. Hoechst stained serial sections corresponding to (I) and (K) are found in Figure S6E–F. Abbreviations: α-SMA, α-smooth muscle actin; CA125, cancer antigen 125; E, epithelium; ERα, estrogen receptor alpha; E2, estrogen; G, gland; GdA, glycodelin A; S, stroma.
Figure 2.
Neonatal mouse uterine mesenchyme induces MIXL1 expression in MIXL1GFP/w EBs.
(A) nMUM induced GFP expression in MIXL1GFP/w EB recombinants after three days of co-culture (3rd panel), representative example of n>100. The duration of reporter activation is comparable to that observed in growth factor treated EBs (2nd panel, days 3,5), while little to no reporter activity was detected in the control (1st panel) (n>100) (×40 magnification, first 3 panels are fluorescent images, images in 4th panel represent phase contrast images of recombinants in 3rd panel). (B) Co-culture of two nMUM pieces (0.5 mm) with a larger EB (>4000 cells) activated reporter activity locally (dotted area is the EB) (×20 magnification). (C) No reporter activity was detected in embryoid bodies co-cultured with neonatal uterine epithelium (0.5 mm) (×40 magnification). Areas marked as mesenchyme in recombinants are based on morphological evidence and recombination experiments using ENVY hESC (refer to Figure S2A–C). Abbreviations: EB, embryoid body; E, neonatal mouse uterine epithelium; GF, growth factors; M, neonatal mouse uterine mesenchyme.
Figure 3.
Neonatal mouse uterine mesenchyme induces expression of primary germ layer markers in ENVY/nMUM recombinants.
Real time PCR analysis of RNA collected from EB and recombinants cultured in serum-free BPEL medium. Growth factors (BMP4, 50 ng.ml−1 and ACTIVIN A, 20 ng.ml−1) were added immediately after forced aggregation of hESCs. Neonatal mouse uterine mesenchyme was added 24 hours after EB formation into either growth factor treated or untreated EB culture. Expression of genes relative to GAPDH were analysed by quantitative RT-PCR after 3, 5, 7 and 9 days incubation. Expression of target genes in undifferentiated hESCs is indicated as day 0 of differentiation (Data is plotted as mean±s.e.m., n = 3 independent differentiation experiments). Abbreviations: GF, growth factor; nMUM, neonatal mouse uterine mesenchyme.
Figure 4.
Morphological and immunohistochemical characteristics of hESC derived FRT epithelial structures in grafted GF treated recombinants.
(A) H&E showing pseudostratification in the hESC derived FRT epithelium surrounded by co-transplanted mouse stroma in a 4 week graft. (B) no PAX2 or HOXA10 (C) expression in week 2 hESC derived FRT epithelium (B, C) were serial sections representative of two week 2 graft) (arrows point to HOXA10+ mouse stromal cells). (D–I) Representative sections from two different 4 week grafts, (E–J) serial sections of a single gland (sectioned at 3 µm), (D, E) Nuclear expression of HOXA10 and PAX2 in hESC derived FRT epithelium at 4 weeks. (F) representative serial sections of the same gland depicted from (E–J) showing GFP+ hESC derived epithelium surrounded by GFP− mouse uterine stromal cells. The gland co-localised cytoplasmic expression of (G) CK18 and (H) Vimentin in hESC derived FRT epithelium. (I) CA125 expressed on hESC derived FRT epithelium surface. (J) ERα expressed in transplanted mouse stromal cells but absent from hESC derived FRT epithelium. Insets in (B) is a positive control of PAX2 expression in adult human endometrial epithelium, inset in (C) concentration matched goat IgG negative control, inset in (E) concentration matched rabbit IgG negative control, inset in (I) mouse IgG1 negative control for (G–I). (K.1–2) 3D reconstruction of hESC derived FRT epithelium from serial sections depicted in (E–J) (arrow) shown on the Y-axis, (K.2) shown on the Z-axis view as a duct. Abbreviations: E, epithelium; hFRT, hESC derived FRT; HOXA10, Homeobox A10; PAX2, Pair box gene 2; S, stroma.
Figure 5.
Schematic of the proposed Human Müllerian Duct development model and its potential applications.
Stages of development of the hESCs/nMUM recombinant in vitro and in vivo. Key stages are numbered from 0–5 accompanied by some stage specific markers. (0): Undifferentiated hESC cells (white circles) were recombined with nMUM (black ovals), (1): primitive streak formation: nMUM (black ovals) induced some hESC to differentiate into MIXL1+ mesendodermal cells (green circles) whilst the rest remained either as undifferentiated (white circles) or belonging to the ectodermal lineage (blue circles), (2): germ layer induction: mesodermal cells (yellow circles) derived from MIXL1+ mesendodermal cells in the previous stage, other colored circles represent a variety of differentiated (purple, orange, blue) and undifferentiated cells (white circles), (3): in vivo differentiation, and formation of a pre-Müllerian epithelium (yellow) possibly derived from mesodermal cell types in the EB (the presence of tissues from other lineages is omitted from this figure for illustration purposes), (4): transition from the pre- Müllerian epithelium into MD epithelium (red), (5): maturation of MD epithelium into adult FRT epithelium (pink) containing residual MD epithelial cells (in red). Text boxes (below schematic) include potential applications of the model at different stages of development.
Table 1.
Primary antibody used in this study.