The therapeutic potential of adipose tissue-derived mesenchymal stromal cells in the treatment of busulfan-induced azoospermic mice

Abstract

Purpose: The generation of germ cells from mesenchymal stromal cells (MSCs) provides a valuable in vitro platform for infertility modeling. The establishment of these cells is a new approach for assisted reproductive technology (ART) to help infertile patients who lack functional gametes.

Methods: Human adipose-derived MSCs were isolated and then characterized for multipotency by flow cytometry, differentiation capacity, and cytogenetic assays. These cells were used in a male germ cell differentiation study. The expression of male germ cell markers was evaluated at day 21 of differentiation using an immunofluorescence assay, flow cytometry, and RT-qPCR. Undifferentiated MSCs were used for transplantation in busulfan-induced azoospermic mice.

Results: In this study, MSCs were successfully isolated from human adipose tissues which were positive for cell markers such as CD90, CD105, CD73, and CD29 but negative for CD34 and CD45. The results of flow cytometry, immunocytochemistry, and RT-qPCR analysis at day 21 of differentiation showed that the undifferentiated adipose-derived MSCs are able to differentiate into male germ cells. Additionally, transplantation of undifferentiated MSCs in busulfan-induced azoospermic mice caused spermatogenesis recovery in the majority of seminiferous tubules.

Conclusion: In this study, we showed that differentiation of human adipose-derived MSCs into male germ cells is a useful tool for in vitro study of human germ cell development. Our results demonstrated that cell therapy with adipose-derived MSCs could help the repair of pathological changes in testicular seminiferous tubules. Therefore, it may have a clinical application for the treatment of azoospermia in infertile patients.

Keywords: Adipose tissue; Differentiation; Germ cell; Mesenchymal cells.

Fig. 1

Morphological and phenotypic characteristics of adipose tissue-derived mesenchymal stromal cells. These images are from the same cell line that show the morphology of isolated cells at 3 (A), 5 (B), 8 (C), and 12 days (D) after isolation. The cells were spindle-shaped and had fibroblast-like morphology. The images were captured by phase contrast microscope (Olympus CKX41, Tokyo, Japan). Scale bars, 100 µm (10 × magnification)

Fig. 2

A Metaphase spreads and karyotype of AD-MSCs, which indicate the absence of numerical abnormality in the cells. B Immunophenotyping of MSCs by flow cytometry. The cells were negative for hematopoietic markers (CD45 and CD34) and strongly expressed the mesenchymal markers (CD90, CD105, CD73, and CD29). All experiments were repeated at least three times. C, D The cells from the fourth passage were induced to differentiate by culturing in adipogenic and osteogenic medium for 21 days. C Adipogenic differentiation of cells demonstrated increased intracellular vesicles compared with growth media controls which stained positive for Oil Red O. D Osteogenic differentiation of adipose tissue-derived stem cells demonstrated increased alizarin red staining compared with MSC controls. Scale bars, 50 µm (40 × magnification)

Fig. 3

Human AD-MSCs were cultured on gelatin-coated plates and differentiated into male germ cell. The cells had the same fibroblast-like morphology in both day 1 of differentiation and day 21 of undifferentiation groups. In day 10 and 21 of differentiatied group, the cells tend to aggregate and form colony-like structures during the male germ cell differentiation. Scale bars, 100 µm (10 × magnification)

Fig. 4

A Protein expression of DAZL (green), VASA (red), and nuclei (blue) as specific male germ cell markers was detected in the differentiated cells at day 21 of differentiation and not detected in undifferentiated cells. Scale bars, 20 µm (40 × magnification). B The flow cytometric analysis of AD-MSC-differentiated cells into male germ cell revealed that the percentage of VASA and DAZL positive cells was significantly (p ≤ 0.001) increased in differentiated cells compared to undifferentiated cells

Fig. 5

A The RT-qPCR of differentiated cells into male germ cells revealed that the expression of VASA, DAZL, PLZF, NANOS3, SYCP3, and STELLA as male germ cell markers were significantly increased in differentiated cells compared to undifferentiated cells (*p ≤ 0.05, ***p ≤ 0.001, and ****p ≤ 0.0001)

Fig. 6

Azoospermia was induced in male mice by injection of two doses of busulfan 21 days apart. Thirty-five days after the last busulfan injection, male mice were anesthetized and AD-MSCs were transplanted into the right testes. A Sections of seminiferous tubule of control group showing normal morphology. B Sections of seminiferous tubule of the testes after busulfan treatment, without transplantation of AD-MSC, showing seminiferous tubules where the lumen is occupied by large vacuoles, indicative of no spermatogenic activity. C Sections of seminiferous tubules of busulfan-induced azoospermic mice treated with adipose-derived mesenchymal stromal cells; the majority of seminiferous tubules had normal morphology indicating recovery of spermatogenic activity. Scale bars, 100 μm (10 × magnification)