Tracking of human embryonic stem cell-derived mesenchymal stem cells in premature ovarian failure model mice

https://doi.org/10.1016/j.bbrc.2021.08.063Get rights and content

Highlights

  • More than 70% of ES-MSCs were successfully labeled with GFP for tracking in vivo.

  • Cyclophosphamide led to successful induction of premature ovarian failure (POF) in the mice.

  • Intravenously transplanted GFP-labeled ES-MSCs migrated into the various tissues of POF mice.

Abstract

Premature ovarian failure (POF) is defined by amenorrhea, hypoestrogenism, elevated gonadotropin levels, and infertility. Chemotherapeutic agents are the most gonadotoxic agents that lead to POF. Although some previous studies have presented that mesenchymal stem cells (MSCs) transplantation could rescue the ovary function of POF animal models through the paracrine pathway, these mechanisms require further investigation. However, mechanisms of embryonic stem cell-derived MSCs (ES-MSCs) therapeutic effects on POF animal models have not been fully investigated yet. This study aimed to evaluate the migration and distribution of ES-MSCs in a model of chemotherapy-induced POF.

Female mice received intraperitoneal injections of cyclophosphamide (Cy) to induce POF. Then, MSCs were labeled with green fluorescent protein (GFP) in vitro and injected intravenously into POF mice, and the distribution of MSCs was dynamically monitored at 1 week after transplantation. We harvested the lungs, liver, spleen, ovaries, heart, and kidneys 1 week after transplantation. The sections of these tissues were observed under the fluorescent microscope.

More than 70% MSCs were successfully labeled with GFP at 72 h after labeling. MSCs were uniformly distributed in multiple organs and tissues including lungs, liver, spleen, ovaries, heart, and kidneys of POF mice. In mice, at 1week after intravenous transplantation, GFP labeled ES-MSCs were observed in the lungs, liver, spleen, ovaries, heart, and kidneys of POF mice, and the number of GFP labeled ES-MSCs in lungs, ovaries, and heart were higher than that in the spleen, kidneys, and liver. Our results revealed intravenously implanted ES-MSCs could migrate into the various tissues in chemotherapy-induced damaged POF mice.

Introduction

Premature ovarian failure (POF) is a condition characterized by amenorrhea, hypoestrogenism, sex steroid deficiencies, high gonadotropins in women under 40 years old [1]. The etiology of POF is complex, which is caused by a variety of agents such as autoimmune disorders, genetic abnormalities, radiotherapy, and chemotherapy. POF is an adverse effect of chemotherapeutic, which leads to increased risk of infertility [2]. Chemotherapeutic agents that are frequently used in the treatment of various malignancies and autoimmune disorders are important factors for inducing POF. Cyclophosphamide (Cy), an alkylating agent, is one of the Chemotherapeutic agents that has irreversible cytotoxicity on major internal organs, especially on the ovary [3].

Recently, stem cell therapy has been introduced as a potential therapeutic procedure for repairing and restoring the proper function of affected tissues [4,5]. Previous studies have revealed that ES-MSCs have shown therapeutic potential for inflammatory bowel disease [6], fistulizing Crohn's disease [7], experimental autoimmune encephalitis [8], cardiac remodeling and dysfunction [9], pulmonary arterial hypertension [10], arthritis [11], lupus nephritis [12], cardiomyopathy [13], diabetes [14], acute hepatic failure [15], lethal fulminant hepatic failure models [16], and liver cirrhosis [17] in numerous animal models. It should be noted that the success of cell-based therapy is associated with the choice of a useful stem cell delivery tool. Some studies have already reported that straight injection of MSCs into the chemotherapy damaged ovaries can progress ovarian function and structure [18,19].

The intravenous injection of MSCs, in addition to being simple and quick, is also a less aggressive approach than local injection [20,21]. It is therefore more useful, particularly for chemotherapeutic toxicities that target different organs. In addition, intravenously implanted MSCs can migrate into and repair the injured tissues [22,23]. However, a few studies have reported the tracing of MSCs in different organs [24,25]. Green fluorescent protein (GFP) labeling is widely accepted as a tracking method for cell tracking in vivo [26]. Recently we have demonstrated that the implanted ES-MSCs can not only survive but also improve ovarian function and restore fertility in POF mice [27]. In this study, in order to evaluate the migration ability and distribution of ES-MSCs in the tissues injured by cyclophosphamide in POF mice, ES-MSCs were labeled with GFP in vitro before transplantation, then GFP-labeled ES-MSCs injected via the tail vein into chemotherapy-induced POF mice and their location in tissues were investigated.

Section snippets

Generation and characterization of ES-MSCs

To derive MSCs from ES cells, ES cells were cultured in Dulbecco's modified Eagle's medium/F12 (Life Technologies) supplemented with 20% knockout serum replacement (Life Technologies), 2 mM GlutaMAX, 0.1 mM non-essential amino acids (NEAAs), 1% insulin-transferrin-selenium (ITS) (all from Gibco), 0.1 mM β-mercaptoethanol (Sigma-Aldrich) without the addition of basic fibroblast growth factor (bFGF, Royan Biotech) to form embryoid body (EB). Then, EB were transferred onto gelatin-coated plates

Generation and characterization of ES-MSCs

MSCs were generated from ESC as it is shown in Fig. 1A. ES-MSCs were successfully differentiated into osteoblasts, adipocytes, and chondrocytes in vitro using osteogenic, adipogenic, and chondrogenic induction media (Fig. 1B). The flow cytometry results showed that ES-MSCs expressed CD44, CD73, CD90, CD105 but these cells were negative for CD11b, CD34, and CD45 (Fig. 1C). We transfected the ES-MSCs with lentiviral green fluorescent protein construct for identifying and tracking transplanted.

Discussion

In this study, we transplanted GFP- positive ES-MSCs into a mouse model of POF to investigate the migration and distribution of ES-MSCs in the tissues. In our study, ES-MSCs were identified to express CD44, CD73, CD90, and CD105, but not CD11b and CD34. Our results were consistent with previous reports that ES-MSCs expressed CD29, CD44, CD73.CD90, CD105, and negative expressions of hematopoietic cell-surface antigens, such as CD45, CD34, CD14 [30,31]. Green fluorescence protein (GFP) labeling

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by Royan Institute, the National Institute for Medical Research Development, No. 963255; and the Ministry of Health and Medical Education, No. 700/147.

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