J Vet Sci.  2016 Jun;17(2):159-170. 10.4142/jvs.2016.17.2.159.

Differential regulation of senescence and in vitro differentiation by 17β-estradiol between mesenchymal stem cells derived from male and female mini-pigs

• Lee WJ
• Lee SC
• Lee JH
• Rho GJ
• Lee SL

• KMID: 2413165
• DOI: http://doi.org/10.4142/jvs.2016.17.2.159

Abstract

The characterization and potential of mesenchymal stem cells (MSCs) are gender dependent and estrogen influences these properties. This study demonstrated that supplementation with 17β-estradiol (E2) increases the proliferation of bone marrow-MSCs derived from male and female mini-pigs (Mp- and Fp-BMSCs) in a concentration-dependent manner, with 10(-12) M E2 suggested as the optimal dose of E2 that led to the greatest improvement in BMSCs proliferation. Supplementation of 10(-12) M E2 resulted in down-regulation of β-galactosidase activity and pro-apoptotic activity in both BMSCs, while anti-apoptotic activity was up-regulated in only Fp-BMSCs. Further, E2 increased the osteogenic ability of Fp-BMSCs. Based on these findings, optimal utilization of E2 can improve cellular senescence and apoptosis, as well as in vitro osteogenesis of BMSCs, and could therefore be useful in stem cell therapy, particularly in bone regeneration for adult females.

Keyword

17 beta-estradiol; gender differences; mesenchymal stem cells; mini-pig; senescence

MeSH Terms

Animals
*Apoptosis
*Cell Differentiation
Cell Proliferation
Estradiol/*pharmacology
Female
Male
Mesenchymal Stem Cell Transplantation/*veterinary
Mesenchymal Stromal Cells/*physiology
Sex Factors
Swine
Swine, Miniature/*physiology
Time Factors
Estradiol

Figure

• 

  Fig. 1 Identification of alkaline phosphatase (AP) activity and cell surface markers in the bone marrow-mesenchymal stem cells (MSCs) derived from male and female mini-pigs (Mp- and Fp-BMSCs). (A) Mp- and Fp-BMSCs showed AP activity based on BCIP/NBT staining. (B) Cell surface markers of Mp- and Fp-BMSCs at passage 3 were identified, positive for CD29 and CD44 and negative for CD45 by flow cytometry analysis. Scale bar = 100 µm.

• 

  Fig. 2 17β-estradiol (E2) concentration-dependent cell proliferation in Mp- and Fp-BMSCs by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were grown in steroid-free medium supplemented with different concentrations (0, 10-6, 10-8, 10-10, 10-12, and 10-14 M) of E2 for 14 days. The proliferation rates were quantified by measuring the absorbance at 540 nm. The black bars represent Mp-BMSCs, and the white bars represent Fp-BMSCs. The different superscript letters represent significant (p < 0.05) differences among the five replicates.

• 

  Fig. 3 Analysis of cellular senescence in Mp- and Fp-BMSCs following E2 supplementation using β-galactosidase (β-Gal) staining. (A) Detection of senescence-associated β-Gal staining of Mp- and Fp-BMSCs cultured in the control (0 M) and E2 (10-12 M). (B) Cell senescence was quantified in Mp- and Fp-BMSCs by measuring senescence-associated β-Gal staining at an absorbance of 405 nm. The white and black bars represent cells cultured in the control and E2, respectively. The different superscript letters represent significant (p < 0.05) differences. 200× (A).

• 

  Fig. 4 Detection of apoptosis in Mp- and Fp-BMSCs following E2 (10-12 M) supplementation using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Nuclei were counterstained with 4,6-diamidino 2-phenyindiol (DAPI; blue), and fragmented apoptotic bodies (arrow) were stained with TUNEL dye (red). Scale bars = 100 µm.

• 

  Fig. 5 Quantitative mRNA analysis of anti- and pro-apoptosis genes in Mp- and Fp-BMSCs following E2 supplementation. Cells were cultured in the control (0 M) and E2 (10-12 M) until they reached confluence. Expression of the anti-apoptosis related Bcl2 and Birc (A) and the pro-apoptosis related Bax, Bak and Hsp90 (B) was determined by qRT-PCR. Each experiment was performed in triplicate, and HMBS was used as the internal control gene. Different superscript letters represent significant (p < 0.05) differences among different cells.

• 

  Fig. 6 Cytochemical staining of Mp- and Fp-BMSCs in which differentiation was induced with various concentrations of E2. (A) Adipogenic induction in the E2-supplemented BMSCs was determined by Oil-red O staining of neutral lipid vacuoles, which showed intracellular accumulation. (B) Osteogenic induction in E2-supplemented BMSCs was determined by Alizarin-red S solution staining for identification of the mineralized matrix. (C) Chondrogenic induction in the E2-supplemented BMSCs was determined by Alcian blue 8GX solution staining for synthesis of glycosaminoglycans. The concentrations of E2 used were 0, 10-10, 10-12, and 10-14 M in the adipogenic, osteogenic, and chondrogenic induction medium. Scale bars = 100 µm.

• 

  Fig. 7 Relative quantification of adipogenic differentiation-related genes in Mp- and Fp-BMSCs following supplementation of E2 (10-12 M). Adipogenic differentiation was induced for 21 days and was demonstrated by the expression of adipocyte protein 2 (AP2), lipoprotein lipase (LPL), and fatty acid binding protein (FABP) by qRT-PCR. Five replicates were used, and HMBS was employed as an internal control gene. Error bars with asterisks indicate a significant (p < 0.05) differences between the undifferentiated control and differentiated cells.

• 

  Fig. 8 Relative quantification of osteogenic differentiation-related genes in Mp- and Fp-BMSCs following supplementation of E2 (10-12 M). Osteogenic differentiation was induced for 21 days and was demonstrated by the expression of osteonectin (ON), Runt-related transcription factor 2 (RUNX2), and biglycan (BG) by qRT-PCR. Five replicates were used, and HMBS was employed as an internal control gene. Error bars with asterisks indicate a significant (p < 0.05) difference between the undifferentiated control and differentiated cells. O, osteogenic differentiated cells.

• 

  Fig. 9 Relative quantification of chondrogenic differentiation-related genes in Mp- and Fp-BMSCs following supplementation with E2 (10-12 M). Chondrogenic differentiation was induced for 21 days and was evidenced by the expression of aggrecan (ACAN), collagen type2 (COL2), and collagen type10, alpha 1 (COL10A1) by qRT-PCR. Five replicates were used, and HMBS was employed as an internal control gene. Error bars with asterisks indicate a significant (p < 0.05) difference between the undifferentiated control and differentiated cells. C, chondrogenic differentiated cells.

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