Initiation of puberty in mice following decellularized ovary transplant

Abstract

Clinical interventions to preserve fertility and restore hormone levels in female patients with therapy-induced ovarian failure are insufficient, particularly for pediatric cancer patients. Laparoscopic isolation of cortical ovarian tissue followed by cryopreservation with subsequent autotransplantation has temporarily restored fertility in at least 27 women who survived cancer, and aided in pubertal transition for one pediatric patient. However, reintroducing cancer cells through ovarian transplantation has been a major concern. Decellularization is a process of removing cellular material, while maintaining the organ skeleton of extracellular matrices (ECM). The ECM that remains could be stripped of cancer cells and reseeded with healthy ovarian cells. We tested whether a decellularized ovarian scaffold could be created, recellularized and transplanted to initiate puberty in mice. Bovine and human ovaries were decellularized, and the ovarian skeleton microstructures were characterized. Primary ovarian cells seeded onto decellularized scaffolds produced estradiol in vitro. Moreover, the recellularized grafts initiated puberty in mice that had been ovariectomized, providing data that could be used to drive future human transplants and have broader implications on the bioengineering of other organs with endocrine function.

Keywords: Bioactivity; Decellularization; ECM (extracellular matrix); Endocrine function; SEM (scanning electron microscopy); Xenotransplantation.

Conflict of interest statement

Competing Financial Interests: The authors declare no competing financial interests.

Copyright © 2015 Elsevier Ltd. All rights reserved.

Figures

Fig. 1

Human ovarian tissue information and SALL4 staining. Table of age, diagnosis and treatment information for participants in this study (a). ALL: acute lymphoblastic leukemia. Representative images of native ovarian cortical tissue stained with an oncogene expressed in ALL patients. Participant D was diagnosed with pancreatic cancer and the patient’s ovarian cortex contained no SALL4-positive cells (b, DNA, blue), while participant E was diagnosed with ALL and contained SALL4-positive cells (c, SALL4, green, arrows). Scale bar: 50 μm.

Fig. 2

Decellularization of bovine and human tissue. (a–h) Ovarian cortical tissues from four participants with ALL were selected. Primordial and primary follicles are visible within hematoxylin and eosin (H&E) stained native tissue sections (arrows in a,c,e,g) and nuclear material is stained blue. Tissue from these same participants that were decellularized with 0.1% SDS for 24 hours do not contain nuclear material, but only contain pink-staining extracellular matrix (ECM; b,d,f,h). This technique works for different ages and whether or not the patient has undergone chemotherapy. (a–b: A = 6yo, ALL, chemo, radiation; c-d: B = 14yo, ALL, chemo; e-f: C = 17yo; ALL; unknown treatment; g-h: E = 34yo, ALL, unknown treatment) Scale bar: 50 μm. (i–m) Tissue slices from cortex and medulla regions of bovine ovary were obtained. Follicles are visible within H&E stained native tissue sections (arrows in i,k) and nuclear material are stained blue. Tissue from these same animals that were decellularized with 0.1% SDS for 24 hours do not contain nuclear material (j,l). Scale bar: 50 μm. A whole bovine ovary was decellularized in 0.1% SDS for 28 days (m). Scale bar, 5 mm. Visible pores where a large recruitable follicle (3: 2.3mm), maturing and mature follicles (1: 4.1mm, 2: 3.6mm, 4: 4.3mm) are maintained within the ECM.

Fig. 3

Microstructures of the ovarian cortex and medulla architecture. Decellularized bovine ovarian cortical tissue (a–c,g,h) and medullary tissue (d–g,i,j), were imaged with a scanning electron microscope. The ovarian surface epithelium is visible in the cortical region (a, green arrow). Organized collagen fibers are visible within pore walls of cortical ECM (h, black arrows). (d–g) Tissue strips from medulla regions contain larger follicle pores and more vascular pores (d, red arrows). Some organized collagen fibers are visible within medulla pore walls (J, black arrow). These walls also contain flexible fibronectin fibers (j, white arrow). Organized collagen bundles are visible within the decellularized human ovary (k,l, white arrow). Follicle pore diameters were measured to be 1: 33.6 μm, 2: 119.2 μm, 3: 169.4 μm, 4: 24.8 μm, 5: 162.3 μm. Scale bar in a,d: 100 μm. Scale bar in b-d,e-f: 20 μm. Scale bar in g-l: 1 μm.

Fig. 4

ECM components in the decellularized human and bovine ovarian tissue. Decellularized bovine ovarian cortical tissue stained with collagen IV (a), laminin (c) and fibronectin (inset, d). Decellularized bovine ovarian medullary tissue stained with collagen IV (b), laminin (inset, c) and fibronectin (d). Decellularized human ovarian cortical tissue stained with collagen I (e), collagen IV (f–g) and fibronectin (h). Scale bar: 50 μm.

Fig. 5

Primary ovarian cells used to recellularize scaffold are FOXL2 or CYP17-positive, and create pores and blebs. Decellularized bovine ovary scaffold seeded with primary mouse ovarian cells and cultured for 2 days (a–e). Many primary ovarian stroma cells (blue, DNA, a-b) are positive for FOXL2 (red, a) or CYP17 (green, b). Several somatic cells have penetrated the decellularized bovine medulla scaffold (white dotted line, a-b). Scale bar in a-c: 50 μm. SEM images of primary cells seeded onto bovine scaffold (d–f). Sheets of cells are visible on top of bovine ECM (c,d, ECM is dark purple). False-colored granulosa cells form a pore (yellow, green and blue, d) and secrete blebs (d, light purple) characteristic of mural granulosa cells. Scale bar in c-e: 2 μm.

Fig. 6

Primary ovarian cells cultured on decellularized ovary scaffold produce estradiol and support follicle growth in vivo. (a–b) Renal grafts of decellularized bovine scaffolds with primary murine ovarian cells in ovariectomized mice produce comparable levels of estradiol (mean ± SEM: 9.17 ± 1.30 pg/ml, N=7) and inhibin A (4.03 ± 1.18 pg/ml, N=6) to age-matched cycling females (estradiol: 8.39 ± 0.57 pg/ml, N=8; inhibin A: 3.55 ± 1.84 pg/ml, N=8), while ovariectomized mice with sham grafts do not (“*” estradiol: 5.90 ± 0.24 pg/ml, N=6, ANOVA significant at p