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. 2021 Aug;23(3):294-302.
doi: 10.22074/cellj.2021.6836. Epub 2021 Jul 17.

Mir-106b Cluster Regulates Primordial Germ Cells Differentiation from Human Mesenchymal Stem Cells

Sadaf Mahboudi  1 , Kazem Parivar  2 , Zohreh Mazaheri  3 , S Hiva Irani  1
Affiliations

Mir-106b Cluster Regulates Primordial Germ Cells Differentiation from Human Mesenchymal Stem Cells

Sadaf Mahboudi et al. Cell J. 2021 Aug.

Abstract

Objective: Numerous evidence indicates that microRNAs (miRNAs) are critical regulators in the spermatogenesis process. The aim of this study was to investigate miR-106b cluster regulates primordial germ cells (PGCs) differentiation from human mesenchymal stem cells (MSCs).

Materials and methods: In this experimental study, samples containing male adipose (n: 9 samples- age: 25-40 years) were obtained from cosmetic surgeries performed for the liposuction in Imam Khomeini Hospital. The differentiation of MSCs into PGCs was accomplished by transfection of a lentivector expressing miR-106b. The transfection of miR-106b was also confirmed by the detection of a clear green fluorescent protein (GFP) signal in MSCs. MSCs were treated with bone morphogenic factor 4 (BMP4) protein, as a putative inducer of PGCs differentiation, to induce the differentiation of MSCs into PGCs (positive control). After 4 days of transfection, the expression of miR-106b, STELLA, and FRAGILIS genes was evaluated by real-time polymerase chain reaction (PCR). Also, the levels of thymocyte differentiation antigen 1 (Thy1) protein was assessed by the western blot analysis. The cell surface expression of CD90 was also determined by immunocytochemistry method. The cytotoxicity of miR-106b was examined in MSCs after 24, 48, and 72 hours using the MTT assay.

Results: MSCs treated with BMP4 or transfected by miR-106b were successfully differentiated into PGCs. The results of this study also showed that the expression of miR-106b was significantly increased after 48 hours from transfection. Also, we showed STELLA, FARGILIS, as well as the protein expression of Thy1, was significantly higher in MSCs transfected by lentivector expressing miR-106b in comparison with MSCs treated with BMP4 (P≤0.05). MTT assay showed miR-106b was no toxic during 72 hours in 1 μg/ml dose, that this amount could elevated germ cells marker significantly higher than other experimental groups (P≤0.05).

Conclusion: According to this findings, it appears that miR-106b plays an essential role in the differentiation of MSCs into PGCs.

Keywords: Mesenchymal Stem Cells; MiR-106b; MicroRNA.

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Conflict of interest statement

There is no conflict of interest in this study.

Figures

Fig.1
Fig.1
The cells isolated from ADSCs. A. Isolated stem cells 4 hours after incubation and B. ADSCs in the 4th passage (scale bar: 100 µm). ADSCs; Adipose-derived stem cells.
Fig.2
Fig.2
The in vitro osteogenesis and adipogenic differentiation. A. Adipose-derived stem cells (ADSCs) after incubation for 21 days in the adipogenic differentiation medium. The cells were visualized with Oil Red O staining, B. ADSCs after incubation for 21 days in the osteogenic differentiation medium. The cells were visualized with Alizarin Red S stain. The blue arrow indicates osteoblasts, and the red arrow shows adipocytes and the accumulated fat droplets (scale bar: 100 μm). C. Cell surface markers: CD90=79.1 ± 5.73, CD105=90.1 ± 3, CD73=75.8 ± 3.61, CD44=89.1 ± 6.49, CD34=5.98 ± 1.64, and CD45=7.15 ± 0.26. The number of positive cells for each marker was assayed by flow cytometry. The data was presented as mean ± SD.
Fig.3
Fig.3
Cytotoxicity of miR-106b expressing MSCs. The cytotoxicity level of miR-106b expressing MSCs was evaluated after 24, 48, 72 hours incubation at various concentrations of miR-106b. MSCs; Mesenchymal stem cells and h; Hour.
Fig.4
Fig.4
The differentiation of ADSCs into PGCs. A. The expression of the miR-106b measured by real-time PCR after the transfected by lentivector expressing miR-106b. B. Alkaline phosphatase-positive cells (scale bar: 50 µm). C, D. The expression of STELLA and FRAGILIS genes and E.Thy1 protein levels were evaluated as specific differentiation markers using real-time PCR and western blot analysis, respectively. ADSCs; Adipose-derived stem cells, PGCs; Primordial germ cells and PCR; Polymerase chain reaction. * demonstrates the significant changes in comparison to control (*; P≤0.05 and ***; P≤0.0001).
Fig.5
Fig.5
CD90 protein level measured as a marker of differentiation on germ cells (ADSCs) (scale bar: 50 µm).
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References

    1. Vahdati A, Fathi A, Hajihoseini M, Aliborzi G, Hosseini E. The regenerative effect of bone marrow-derived stem cells in spermatogenesis of infertile hamster. World J Plast Surg. 2017;6(1):18–25. - PMC - PubMed
    1. Huang P, Lin LM, Wu XY, Tang QL, Feng XY, Lin GY, et al. Differentiation of human umbilical cord Wharton’s jelly‐derived mesenchymal stem cells into germ‐like cells in vitro. J Cell Biochem. 2010;109(4):747–754. - PubMed
    1. Gnecchi M, Melo LG. Bone marrow-derived mesenchymal stem cells: isolation, expansion, characterization, viral transduction, and production of conditioned medium. Methods Mol Biol. 2009;482:281–294. - PubMed
    1. Gimble JM, Katz AJ, Bunnell BA. Adipose-derived stem cells for regenerative medicine. Circ Res. 2007;100(9):1249–1260. - PMC - PubMed
    1. Leatherman J. Stem cells supporting other stem cells. Front Genet. 2013;4:257–257. - PMC - PubMed

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