Co-transplantation of Human Ovarian Tissue with Engineered Endothelial Cells: A Cell-based Strategy Combining Accelerated Perfusion with Direct Paracrine Delivery

Summary

For some patients, the only option for fertility preservation is cryopreservation of ovarian tissue. Unfortunately, delayed revascularization undermines follicular viability. Here, we present a protocol to co-transplant human ovarian tissue with endothelial cells for utilization as a cell-based strategy combining accelerated perfusion with a direct paracrine delivery of bioactive molecules.

Abstract

Infertility is a frequent side effect of chemotherapy and/or radiotherapy and for some patients, cryopreservation of oocytes or embryos is not an option. As an alternative, an increasing number of these patients are choosing to cryopreserve ovarian tissue for autograft following recovery and remission. Despite improvements in outcomes among patients undergoing auto-transplantation of cryopreserved ovarian tissue, efficient revascularization of grafted tissue remains a major obstacle. To mitigate ischemia and thus improve outcomes in patients undergoing auto-transplantation, we developed a vascular cell-based strategy for accelerating perfusion of ovarian tissue. We describe a method for co-transplantation of exogenous endothelial cells (ExECs) with cryopreserved ovarian tissue in a mouse xenograft model. We extend this approach to employ ExECs that have been engineered to constitutively express Anti-Mullerian hormone (AMH), thus enabling sustained paracrine signaling input to ovarian grafts. Co-transplantation with ExECs increased follicular volume and improved antral follicle development, and AMH-expressing ExECs promoted retention of quiescent primordial follicles. This combined strategy may be a useful tool for mitigating ischemia and modulating follicular activation in the context of fertility preservation and/or infertility at large.

Introduction

Cancer remains among the leading causes of death in the developed world, yet decades of research have yielded significant progress for most types of cancer, and in some cases nearly doubled survival rates¹. Unfortunately, chemotherapeutic agents are often gonadotoxic, depleting the reserve of primordial follicles in ovaries and reducing fertility². This growing population can benefit from various methods of fertility preservation including oocyte and/or embryo cryopreservation, however, patients requiring prompt initiation of cancer therapy and pre-pubertal patients are ineligible for these options. As an alternative, so....

Protocol

All procedures involving animal subjects have been approved by the Institutional Animal Care and Use Committee (IACUC) at Weill Cornell Medical College. All xenotransplantation experiments using ovarian tissue were performed in accordance with relevant guidelines and regulations. Human ovarian tissue was collected from patients scheduled for chemotherapy or radiotherapy for cancer treatment or prior bone marrow transplantation. The institutional review board (IRB) Committee of Weill Cornell Medical College approved the collection of tissue for potential autologous use, and upon the patient's informed consent a donation of up to 10% of their ovarian tissue for rese....

Results

To determine whether co-transplantation of ExECs provides a benefit to patients' tissue, thawed ovarian cortical strips were divided into equal sized pieces and engrafted bilaterally into immuno-compromised, NOD scid gamma (NSG), mice. With one side embedded in a fibrin clot alone (no ECs) and the other containing ExECs (Figure 1a), each mouse served as its own control. ExECs were obtained via isolation of primary endothelium from human umbilical cords and su.......

Discussion

Here we demonstrate that co-transplantation of exECs provides a significant benefit to ovarian tissue viability and function following xenograft in mice. Standards for clinical application of ovarian tissue auto-transplantation for fertility preservation have not been set and the optimal parameters (size, transplantation site, duration of graft, etc.)^32,33,34 for enhanced recovery of the follicular pool remain undefined.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Leibovitz’s L-15 medium	Gibco	11415064
Antibiotic-Antimycotic	Gibco	15240062	Anti-Anti X100
Sucrose	Sigma	S 1888
Fibrinogen	Sigma	F 8630	from bovine plasma
Thrombin	Sigma	T 1063	from human plasma
DMSO	Sigma	D 2650
DMEM	Gibco	12491015
Enzyme Cell Detachment Medium	Invitrogen	00-4555-56	Accutase
Plastic paraffin film	Bemis NA		Parafilm M
Surgical paper tape 2.5 cm	3M	1530-1	Micropore
Surgical Paper tape 1.25 cm	3M	1530-0	Micropore
Perforated plastic Surgical tape 1.25 cm	3M	1527-0	Transpore
Monofilament Absorbable Suture	Covidien	UM-203	Biosyn
Braided Absorbable Suture	Covidien	GL-889	Polysorb
Povidone-iodine Solution USP 10%	Purdue Products	67618-153-01	Betadine Solution Swab Stick
Cryoviales	Nunc	377267	CryoTube
sterile ocular lubricant	Dechra	17033-211-38	Puralube
1.7 ml micro-centrifuge tube	Denville	C-2172	Eppendorf
Anasthesia system	VetEquip	V-1 table top system with scavenging
Endothelial cells	Angiocrine Biosciences, Inc., San Diego, CA, USA		Isolated, transfected with E4-ORF- 1 and labeled endothelial cells
Trichrome stain	Sigma	HT15-1kt	Trichrome Stain (Masson) Kit
Isolectin	Invitrogen	I32450	isolectin GS-IB4 From Griffonia simplicifolia, Alexa Fluor™ 647 Conjugate