Elsevier

Cytotherapy

Volume 16, Issue 2, February 2014, Pages 203-212
Cytotherapy

Original paper
Mesenchymal stromal cells
Evaluation of gametogenic potential of vitrified human umbilical cord Wharton's jelly–derived mesenchymal cells

https://doi.org/10.1016/j.jcyt.2013.10.015Get rights and content

Abstract

Background aims

Vitrification as an advanced cryopreservation method is recommended for cell storage toward future applications. The purpose of this report was to appraise whether gametogenic potential of these cells is altered by vitrification.

Methods

A two steps method was applied for hUCM cells vitrification. An n-hUCM group of hUCM cells served as control. In order to differentiation of hUCM cells into male germ cells, the cells were induced by retinoic acid, testosterone and testicular-cell-conditioned medium. To evaluate induced hUCM cells toward germ cells, we used immunocytochemistry and karyotyping methods.

Results

v-hUCM cells similar to n-hUCM cells formed flat cells after gametogenic induction, and showed protein expression of germ-cell-specific markers DAZL, VASA (DDX4) and SCP3. Karyotyping pattern remained unchanged in the either groups.

Conclusions

The analysis of these results demonstrates that vitrification does not alter differentiation potential of hUCMs to male germ like cells. These results may set an in vitro pattern to study germ-cell formation from hUCM cells and also as a potential source of sperms for male infertility.

Introduction

Human umbilical cord matrix–derived mesenchymal (hUCM) cell culture has advanced in recent years. Proliferative capacity, multipotentiality and noninvasive procedure to access and isolate hUCM cells are among the advantages of these cells (1). Invasive procedures for harvesting of bone marrow stem cells (BMSCs) (2) and adipose tissue–derived MSCs (3), the low rate of successfully isolating umbilical cord blood mesenchymal stromal cells (MSCs) (4), reduction of the quantities, differentiation potential and frequency of BMSCs with aging 2, 5, 6 and ethical reasons regarding the use of of embryonic stem cells (ESCs) (7) create limitations in the use of these stem cells. In contrast, MSCs isolated from Wharton's jelly of the human umbilical cord is closely lacking of imperfections, as previously mentioned (8).

To bank cells for future applications in clinics and laboratories, cryopreservation is critical (9). Vitrification (glass-like solidification of a solution) is more desirable than is conventional slow freezing. In vitrification, the defects of slow freezing such as damage caused by cytoplasmic ice crystal formation (10), the requirement of an expensive programmable freezer and time-consuming procedures are eliminated (11). As a cryopreservation method, vitrification has been successfully applied for human cord blood hematopoietic progenitor cells (12), human embryonic stem cells (hESCs) 13, 14 and human amnion-derived MSCs (15). We have previously reported on the vitrification of hUCMs (16).

Trials for application of stem cells in reproductive medicine have been an important concern for many years. In the past decade, researchers have reported in vitro differentiation of ESCs 17, 18, 19, 20, 21, 22 and adult stem cells such as BMSCs (23), fetal porcine skin stem cells (24) and clonal pancreatic stem cells (25) into germ line cells. One study was carried out wherein hUCM cells were induced toward male germ cells (26). Human UCM cells are being considered as a non-immunogenic (27) source of stem cells capable of differentiating into various cell types suitable for experimental 28, 29 and clinical studies, especially regenerative medicine (30). Whether vitrification would alter differentiation capacity of hUCM cells has not been investigated. Regardless of existent results, there is long way to achievement of final goals. Therefore, in the present study, we evaluate the gametogenic potential of hUCM cells after vitrification.

Section snippets

Isolation and culture of hUCM cells

All materials were purchased from Sigma-Aldrich Co (Sigma-Aldrich, St. Louis, MO, USA) except for the cases mentioned in the text. Ethics approval was obtained from the ethics committee at Kerman University of Medical Sciences, Kerman, Iran. After informed consent was given by mothers in the Afzalipour hospital gynecology ward in Kerman University of Medical Sciences, fresh human umbilical cords (UCs) from normal, full-term newborn infants were obtained during cesarean section. UCs were

Features of hUCM cells

Ten days after the Wharton's jelly pieces were cultured, spindle-shaped, fibroblast-like cells were observed at the periphery of the explants. Flow cytometric analysis demonstrated that the cells expressed mesenchymal cell markers (CD44 and CD90) but did not express hematopoietic lineage markers (CD34, CD45) (Figure 1).

Osteogenic differentiation of hUCM cells was examined after 2–3 weeks with alizarin red S and alkaline phosphatase staining (Figure 2A,B). Calcium phosphate mineral accumulation

Discussion

Our research attempts to evaluate whether differentiation potential of hUCM cells toward male germ cells is affected by vitrification. To achieve this aim, a two-step vitrification protocol that was based on ethylene glycol as a cryoprotectant and rapid warming was used.

Our criterion for the evaluation of hUCM cells was provided by the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy guidelines (32). Adipogenic, osteogenic and chondrogenic

Acknowledgments

This work was supported by Physiology Research Centre of Kerman University of Medical Sciences. The authors thank the Yazd Genetic Laboratory of Research and Clinical Centre of infertility staff for their valuable assistance in the karyotyping study.

Disclosure of interests: The authors have no commercial, proprietary, or financial interest in the products or companies described in this article.

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