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A stem cell-derived ovarian regenerative patch restores ovarian function and rescues fertility in rats with primary ovarian insufficiency.

ABSTRACT: Rationale: Primary ovarian insufficiency (POI) normally occurs before age 40 and is associated with infertility. Hormone replacement therapy is often prescribed to treat vasomotor symptom, but it cannot restore ovarian function or fertility. Stem cell therapy has been studied for the treatment of POI. However, the application of live stem cells has suffered from drawbacks, such as low cell retention/engraftment rate, risks for tumorigenicity and immunogenicity, and lack of off-the-shelf feasibility. Methods: We developed a therapeutic ovarian regenerative patch (ORP) that composed of clinically relevant hydrolysable scaffolds and synthetic mesenchymal stem cells (synMSCs), which are microparticles encapsulating the secretome from MSCs. The therapeutic potency of ORP was tested in rats with cisplatin induced POI injury. Results: In vitro studies revealed that ORP stimulated proliferation of ovarian somatic cells (OSCs) and inhibited apoptosis under injury stress. In a rat model of POI, implantation of ORP rescued fertility by restoring sexual hormone secretion, estrus cycle duration, and follicle development. Conclusion: ORP represents a cell-free, off-the-shelf, and clinically feasible treatment for POI.

SUBMITTER: Zhang S

PROVIDER: S-EPMC8419036 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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A stem cell-derived ovarian regenerative patch restores ovarian function and rescues fertility in rats with primary ovarian insufficiency.

Zhang Sichen S Zhu Dashuai D Li Zhenhua Z Huang Ke K Hu Shiqi S Lutz Halle H Xie Mengjie M Mei Xuan X Li Junlang J Neal-Perry Genevieve G Wang Shaowei S Cheng Ke K

Theranostics 20210818 18

<b>Rationale:</b> Primary ovarian insufficiency (POI) normally occurs before age 40 and is associated with infertility. Hormone replacement therapy is often prescribed to treat vasomotor symptom, but it cannot restore ovarian function or fertility. Stem cell therapy has been studied for the treatment of POI. However, the application of live stem cells has suffered from drawbacks, such as low cell retention/engraftment rate, risks for tumorigenicity and immunogenicity, and lack of off-the-shelf f ...[more]

PMID: 34522217

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Project description:BackgroundEmerging drug-free in vitro activation (IVA) technique enables patients with premature ovarian insufficiency (POI) to restore ovarian function and conceive their own genetic offspring. However, various issues have greatly restricted its clinical application. Transplantation of adipose-derived stem cells (ADSCs) has promising roles in restoring ovarian function of rats with POI, but insufficient retention has greatly hampered their efficiency. Here, we designed a 3D-bioprinted engineering ovary composed of drug-free IVA and ADSCs, which may prolong the retention of ADSCs and construct an early vascular microenvironment, thus compensating for the disadvantages of drug-free IVA to some extent and ameliorating impaired ovarian function in the POI rats.MethodsAfter intraperitoneal injection of cyclophosphamide, the POI model rats were randomized into 5 groups: (1) POI group; (2) ovarian fragments group; (3) 3D scaffold combined with ovarian fragments group; (4) ovarian fragments combined with ADSCs group; (5) 3D scaffold with ADSCs combined with ovarian fragments as 3D-bioprinted engineering ovary group. Normal rats were identified as the control group. The localization of CM-Dil-labeled ADSCs and co-localization with CD31 were observed to examine the distribution and underlying mechanism of differentiation. Histomorphological and immunohistochemical analyses were performed to calculate follicle number and assess proliferation and apoptosis of granulosa cells (GCs). Immunofluorescence staining was used to evaluate angiogenesis. Hormone levels were measured to evaluate the restoration of endocrine axis. Western blot analysis and RT-PCR were conducted to explore the potential mechanism.ResultsCM-Dil-labeled ADSCs were distributed in the interstitium of ovaries and had significantly higher retention in the 3D-bioprinted engineering ovary group. Several regions of the co-staining for CM-Dil and CD31 were in the area of vascular endothelial cells. Meanwhile, the follicle counts, GCs proliferation, neoangiogenesis, and hormone levels were significantly improved in the 3D-bioprinted engineering ovary group, as compared with other groups. Furthermore, the ovarian function was ameliorated and angiogenesis was promoted through regulating the PI3K/AKT pathway.ConclusionOur results suggested that 3D-bioprinted engineering ovary had great potential for restoring impaired ovarian function of rats with POI, which could compensate for the disadvantages of drug-free IVA to some extent.

Project description:BackgroundPrimary ovarian insufficiency (POI) is an early decline in ovarian function that leads to ovarian failure. Conventional treatments for POI are inadequate, and treatments based on mesenchymal stem cells (MSCs) have emerged as an option. However, the lack of consideration of the estrogen niche in ovarian tissue significantly reduces the therapeutic efficacy, with an unclear mechanism in the MSCs in POI treatment. Furthermore, the disruption of circadian rhythm associated with POI has not been previously addressed.MethodsConditioned medium (CM) and estradiol-conditioned medium (E2-CM) were generated from estrogen receptor positive MSCs (ER+pcMSCs). Chemotherapy-induced POI models were established using C57BL/6 mice (in vivo) and KGN cells (in vitro) treated with cyclophosphamide (CTX) or 4-hydroperoxycyclophosphamide (4-OOH-CP). Gene/protein expressions were detected using RT-qPCR, Western blotting, and immunohistochemistry assays. Locomotor activity was monitored for behavioral circadian rhythmicity. Cytokine arrays and miRNA analysis were conducted to analyze potential factors within CM/E2-CM.ResultsThe secretome of ER+pcMSCs (CM and E2-CM) significantly reduced the CTX-induced defects in ovarian folliculogenesis and circadian rhythm. CM/E2-CM also reduced granulosa cell apoptosis and rescued angiogenesis in POI ovarian tissues. E2-CM had a more favorable effect than the CM. Notably, ER+pcMSC secretome restored CTX-induced circadian rhythm defects, including the gene expressions associated with the ovarian circadian clock (e.g., Rora, E4bp4, Rev-erbα, Per2 and Dbp) and locomotor activity. Additionally, the cytokine array analysis revealed a significant increase in cytokines and growth factors associated with immunomodulation and angiogenesis, including angiogenin. Neutralizing the angiogenin in CM/E2-CM significantly reduced its ability to promote HUVEC tube formation in vitro. Exosomal miRNA analysis revealed the miRNAs involved in targeting the genes associated with POI rescue (PTEN and PDCD4), apoptosis (caspase-3, BIM), estrogen synthesis (CYP19A1), ovarian clock regulation (E4BP4, REV-ERBα) and fibrosis (COL1A1).ConclusionThis study is the first to demonstrate that, in considering the estrogen niche in ovarian tissue, an estrogen-priming ER+pcMSC secretome achieved ovarian regeneration and restored the circadian rhythm in a CTX-induced POI mouse model. The potential factors involved include angiogenin and exosomal miRNAs in the ER+pcMSC secretome. These findings offer insights into potential stem cell therapies for chemotherapy-induced POI and circadian rhythm disruption.

Project description:Study questionCan host fertility be rescued by grafting of a fragment of a healthy ovary soon after chemotherapy?Summary answerWe found that grafting a green fluorescent protein (GFP)-positive fragment from a healthy isogenic ovary to the left ovary of a chemo-treated host rescued function and fertility of the grafted host ovary, and resulted in the production of host-derived offspring as late as the sixth litter after chemotherapy (CTx) treatment, whereas none of the ungrafted controls produced a second litter.What is known alreadyIn women and girls undergoing chemotherapy, infertility and premature ovarian failure are frequent outcomes. There are accumulating reports of improved endocrine function after autotransplantation of an ovarian fragment, raising the possibility that the transplant is beneficial to the endogenous ovary.Study design, size, durationWe first established a CTx treatment regimen that resulted in the permanent loss of fertility in 100% of female mice of the FVB inbred strain. We grafted an isogenic ovary fragment from a healthy female homozygous for a GFP transgene to the left ovary of 100 CTx-treated hosts, and compared fertility to 39 ungrafted controls in 6 months of continuous matings, using GFP to distinguish offspring derived from the graft, and those derived from the host.Participants/materials, setting, methodsImmunofluoresece and western blot analysis of 39 treated ovaries during and 15 days after CTx treatment revealed elevated apoptosis, rapid loss of granulosa cells and an increased recruitment of growing follicles. Using immunofluorescence and confocal imaging, we tracked the outcome of the grafted tissue over 4 months and its effect on the adjacent and contralateral ovary of the host.Main results and the role of chanceFifty-three percent of grafted females produced a second litter whereas none of the ungrafted females produced a second litter. The likelihood that this could occur by chance is very low (P < 0.0001).Limitations, reasons for cautionThese results are shown only in mice, and whether or how they might apply to chemotherapy patients subjected to different CTx regimens is not yet clear.Wider implications of the findingsOur experiments prove that rescue of a chemo-treated ovary is possible, and establish a system to investigate the mechanism of rescue and to identify the factors responsible with the long-term goal of developing therapies for preservation of ovarian endocrine function and fertility in women undergoing chemotherapy.Large scale dataNo large datasets were produced.Study funding/competing interestsDuke University Medical Center Chancellor's Discovery Grant to BC; ESJ was supported by an NRSA 5F31CA165545; SK was supported by NIH RO1 GM08033; RWT was supported by the Duke University School of Medicine Ovarian Cancer Research Fellowship; XBM was supported by CONICYT. The authors have no conflicts of interest to declare.

Project description:BackgroundHuman amnion-derived mesenchymal stem cells (hAD-MSCs) have the features of mesenchymal stem cells (MSCs). Low-intensity pulsed ultrasound (LIPUS) can promote the expression of various growth factors and anti-inflammatory molecules that are necessary to keep the follicle growing and to reduce granulosa cell (GC) apoptosis in the ovary. This study aims to explore the effects of LIPUS-pretreated hAD-MSC transplantation on chemotherapy-induced primary ovarian insufficiency (POI) in rats.MethodsThe animals were divided into control, POI, hAD-MSC treatment, and LIPUS-pretreated hAD-MSC treatment groups. POI rat models were established by intraperitoneal injection of cyclophosphamide (CTX). The hAD-MSCs isolated from the amnion were exposed to LIPUS or sham irradiation for 5 consecutive days and injected into the tail vein of POI rats. Expression and secretion of growth factors promoted by LIPUS in hAD-MSCs were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) in vitro. Estrous cycle, serum sex hormone levels, follicle counts, ovarian pathological changes, GC apoptosis, Bcl2 and Bax expression, and pro-inflammatory cytokine levels in ovaries were examined.ResultsPrimary hAD-MSCs were successfully isolated from the amnion. LIPUS promoted the expression and secretion of growth factors in hAD-MSCs in vitro. Both hAD-MSC and LIPUS-pretreated hAD-MSC transplantation increased the body and reproductive organ weights, improved ovarian function, and reduced reproductive organ injuries in POI rats. Transplantation of hAD-MSCs increased the Bcl-2/Bax ratio and reduced GC apoptosis and ovarian inflammation induced by chemotherapy in ovaries. These effects could be improved by pretreatment with LIPUS on hAD-MSCs.ConclusionBoth hAD-MSC transplantation and LIPUS-pretreated hAD-MSC transplantation can repair ovarian injury and improve ovarian function in rats with chemotherapy-induced POI. LIPUS-pretreated hAD-MSC transplantation is more advantageous for reducing inflammation, improving the local microenvironment, and inhibiting GC apoptosis induced by chemotherapy in ovarian tissue of POI rats.

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A stem cell-derived ovarian regenerative patch restores ovarian function and rescues fertility in rats with primary ovarian insufficiency.

Publications

A stem cell-derived ovarian regenerative patch restores ovarian function and rescues fertility in rats with primary ovarian insufficiency.

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