Regeneration of uterine horns in rats using collagen scaffolds loaded with human embryonic stem cell-derived endometrium-like cells

Abstract

A variety of diseases may lead to hysterectomies or uterine injuries, which may form a scar and lead to infertility. Due to the limitation of native materials, there are a few effective methods to treat such damages. Tissue engineering combines cell and molecular biology with materials and mechanical engineering to replace or repair damaged organs and tissues. The use of human embryonic stem cells (hESCs) as a donor cell source for the replacement therapy will require the development of simple and reliable cell differentiation protocols. This study aimed at efficiently generating endometrium-like cells from the hESCs and at using these cells with collagen scaffold to repair uterine damage. The hESCs were induced by co-culturing with endometrial stromal cells, and simultaneously added cytokines: epidermal growth factor (EGF), platelet-derived growth factor-b (PDGF-b), and E2. Expression of cell specific markers was analyzed by immunofluorescence and reverse trascription-polymerase chain reaction to monitor the progression toward an endometrium-like cell fate. After differentiation, the majority of cells (>80%) were positive for cytokeratin-7, and the expression of key transcription factors related to endometrial development, such as Wnt4, Wnt7a, Wnt5a, Hoxa11, and factors associated with endometrial epithelial cell function: Hoxa10, Intergrinβ3, LIF, ER, and PR were also detected. Then, we established the uterine full-thickness-injury rat models to test cell function in vivo. hESC-derived cells were dropped onto collagen scaffolds and transplanted into the animal model. Twelve weeks after transplantation, we discovered that the hESC-derived cells could survive and recover the structure and function of uterine horns in a rat model of severe uterine damage. The experimental system presented here provides a reliable protocol to produce endometrium-like cells from hESCs. Our results encourage the use of hESCs in cell-replacement therapy for severe uterine damage in future.

FIG. 1.

In vitro differentiation of human embryonic stem cells (hESCs) into endometrium-like cells. (A) Phase morphology of NJGLLhES1 hESCs. (B) Morphology of human endometrial stromal cells. The cells were isolated from normal endometrial tissues. (C) More than 95% of the cells were stromal cells, which were characterized by a spindle shape, positive vimentin staining, and a negative cytokeratin-7 immunohistochemical reaction. (D–F) The hESC-derived cells in the cytokines/stromal cells group at the end of differentiation. (D) Morphology of hESC-derived endometrium-like cells. The differentiated cells were uniform with the small and flat cell morphology of an endometrial cell. (E) A high percentage (more than 80%) of the cells was positive for cytokeratin-7. (F) Some cells (∼15%) were vimentin positive. (G, J) Representative images of the hESC-derived cells in the stromal cells group (G) and cells in the cytokine group (J). (H, K) CK7+ cells in the stromal cells group (H) and in the cytokine group (K). (I, L) Vimentin+ cells in the stromal cells group (I) and in the cytokine group (L). DAPI nuclear staining of the same field. Scale bars, 100 mm. DAPI, 4′, 6′-diamidino-2-phenylindole. Color images available online at www.liebertpub.com/tea

FIG. 2.

Expression profiles of hESC-derived cells after differentiation. Quantitative real-time–polymerase chain reaction was used to compare the mRNA expression pattern on stromal cell, stromal cell with cytokine, or cytokine treatment alone. Relative gene expression levels were calculated using the 2^−ΔΔCt method. When the stromal cells existed (the cytokines/stromal cells group and the stromal cells group), the transcription factors Wnt4, Wnt7a, Wnt5a, Hoxa11, Hoxa10, Intergrinβ3, LIF, ER, and PR were expressed at higher levels. Color images available online at www.liebertpub.com/tea

FIG. 3.

Transplantation experiment. (A) hESC-derived cells were isolated, resuspended, and dropped on to the collagen scaffold. (B) Collagen scaffolds with or without hESC-derived cells were grafted to the remaining uterine horns, which were ∼1.5 cm in length and 1/2–2/3 of the total circumference. (C) Gross view of intrauterine adhesions and distal hydrometra in natural regeneration group at 4 weeks after grafting. The arrowheads indicate hydrometra. (D, E) Gross view of reconstructed uterine horns in cells/collagen group at 4 weeks (D) and at 12 weeks (E) after grafting. The arrowheads indicate repair sites. (F) Immunostaining analyses on uterine slices of grafted rats killed at 12 weeks after transplantation in the cells/collagen group. Anti-human nucleus antigen (HN) was used to demonstrate the presence of human cells. HN staining is in red, and nuclei were stained with DAPI (blue). Scale bars, 100 mm. (G–I) Pregnancy in uterine horns at 12 weeks after surgery. Pregnancies in the natural regeneration (G) and collagen group (H) generally implanted in the normal tissue. In the cells/collagen group (I), embryos can be implanted in the grafted tissue. The arrowheads show the margins of the grafted tissue. Color images available online at www.liebertpub.com/tea

FIG. 4.

Histological structures of the regenerative uterine horns at 4 weeks (A1–D1) and 12 weeks (A2–D2) in the sham-operated (A1, A2), natural regeneration (B1, B2), collagen (C1, C2), and cells/collagen (D1, D2) groups. The arrowheads indicate repair sites in (A1-1 to D1-1) and (A2-1 to D2-1). Em, endometrium; Mm, myometrium; SCEp, simple columnar epithelium; UG, uterine gland. The scale bar indicates 500 μm. Color images available online at www.liebertpub.com/tea