Comparative Analysis of the Potential for Germ Cell (GC) Differentiation of Bovine Peripheral Blood Derived-Mesenchymal Stem Cells (PB-MSC) and Spermatogonial Stem Cells (SSC) in Co-Culture System with Sertoli Cells (SC)
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Ethics
2.2. Experimental Design
2.3. Isolation, Purification, and Characterization of Bull PB-MSC
2.4. Isolation, Purification, and Characterization of Bull SC
2.5. Isolation, Purification, and Characterization of Bull SSC
2.6. Co-Culture of PB-MSC or SSC with SC in 3D SACS
2.7. Q-PCR Analysis
2.8. Immunofluorescence Analysis
2.9. Statistical Analysis
3. Results
3.1. Morphology of Bull PB-MSC, SC and SSC
3.2. Morphology of PB-MSC or SSC Co-Cultured with SC in SACS 3D for 21 Days
3.3. Expression of MSC, SC and SSC Markers in Single Cultures
3.4. Expression of Pluripotency Markers in PB-MSC or SSC Co-Cultured with SC for 21 Days in 3D SACS
3.5. Expression of GC Markers in PB-MSC or SSC Co-Cultured with SC for 21 Days in 3D SACS
3.6. Gene Expression of MSC Markers in PB-MSC Co-Cultured with SC in 3D SACS for 21 Days
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Gene | Nucleotide Sequence (5′-3′) | Access Number | |
---|---|---|---|
Forward | Reverse | ||
Housekeeping genes | |||
β-ACTINA | CGCACCACTGGCATTGTCAT | TCCAAGGCGACGTAGCAGAG | NM_173979.3 |
GAPDH | CCTTCATTGACCTTCACTACATGGTCTA | TGGAAGATGGTGATGGCCTTTCCATTG | NM_001034034.2 |
Mesenchymal stem cell genes | |||
CD73 | TGGTCCAGGCCTATGCTTTTG | GGGATGCTGCTGTTGAGAAGAA | NM_174129.3 |
CD105 | CGGACAGTGACCGTGAAGTTG | TGTTGTGGTTGGCCTCGATTA | NM_00107639.1 |
Hematopoietic cell genes | |||
CD34 | CATGCCGTCTTAACCCATCT | CGGTCTACAGAGGTGGTGGT | NM_174009.1 |
CD45 | CCACGGGTATTCAGCAAGTT | CCCAGATCATCCTCCAGAAA | NM_001206523 |
Sertoli cell genes | |||
WT1 | CGTGCGTACCATGTAGGGAA | CTCGTGCTTGAAGGAGTGGT | XM_015474834.2 |
AR | CAGATGGCAGTCATTCAG | CTTGGTGAGCTGGTAGAAG | XM_001244127 |
Spermatogonial stem cell genes | |||
UCHL1 | AGAAGCAGCATCTCGGTTCC | CGTGGTTGAGGGTAAGTGCT | NM_001046172.2 |
CD90 | ACTCATACCGCTCCCGAACCA | CATGTGTATGTCCCCTCGTCCTT | NM_001034765 |
Germ cell genes | |||
DAZL | TCCAAGTTCACCAGTTCAGG | CGT CTG TAT GCT TCT GTC CAC | NM_001081725.1 |
STRA8 | TGTGCCCAGGTGTTCATCTC | GGGGACTGTCACCTCATTGG | XM_015463130 |
PIWIL2 | TCGTATTGATGATGTGGATTGG | GGGAGCAGCAGGATTTCAC | XM_617223.3 |
FRAGILIS | ATCTGCAGCGAGACCTCTGT | CCGATGGACATGATGATGAG | XM_002697323 |
STELLA | TGCAAGTTGCCACTCAACTC | TCTTACCCCTCTCCGCCTAT | NM_00111110 |
SCP3 | CTAGAATTGTTCAGAGCCAGAG | GTTCAAGTTCTTTCTTCAAAG | NM_001040588.2 |
VASA | TGCTACTCCTGGAAGACTGA | CGGTCTGCTGAACATCTCTA | NM_001007819.1 |
Pluripotency genes | |||
OCT4 | GAAAGAGAAAGCGGACGAG | GTGAAAGGAGACCCAGCAG | NM_174580.2 |
NANOG | TAAGCACAGGGGGCAAAAGT | ATGGCTAAAAGGGGTGGAGG | NM_001025344.1 |
SOX2 | CCCGTGGTTACCTCTTCTTCC | CGCTCTGCTAGTGCTGGGAC | NM_001105463.2 |
Gene | PB-MSC+SC | SSC+SC |
---|---|---|
CD73 | ↓ (Q-PCR) | - |
CD105 | ↓ (Q-PCR) | - |
DAZL | ↑ (Q-PCR) *(IF) | ↑(Q-PCR) *(IF) |
STRA8 | x (Q-PCR) | + (Q-PCR) *(IF) |
PIWIL2 | ↑ (Q-PCR) *(IF) | ↑ (Q-PCR) *(IF) |
FRAGILIS | x (Q-PCR) | < (Q-PCR) |
STELLA | x (Q-PCR) | < (Q-PCR) *(IF) |
OCT4 | x (Q-PCR) *(IF) | → (Q-PCR) *(IF) |
NANOG | x (Q-PCR) | → (Q-PCR) *(IF) |
SOX2 | x (Q-PCR) | + (Q-PCR) |
SCP3 | x (Q-PCR) | x (Q-PCR) |
VASA | x (Q-PCR) | x (Q-PCR) |
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Segunda, M.N.; Díaz, C.; Torres, C.G.; Parraguez, V.H.; De los Reyes, M.; Peralta, O.A. Comparative Analysis of the Potential for Germ Cell (GC) Differentiation of Bovine Peripheral Blood Derived-Mesenchymal Stem Cells (PB-MSC) and Spermatogonial Stem Cells (SSC) in Co-Culture System with Sertoli Cells (SC). Animals 2023, 13, 318. https://doi.org/10.3390/ani13020318
Segunda MN, Díaz C, Torres CG, Parraguez VH, De los Reyes M, Peralta OA. Comparative Analysis of the Potential for Germ Cell (GC) Differentiation of Bovine Peripheral Blood Derived-Mesenchymal Stem Cells (PB-MSC) and Spermatogonial Stem Cells (SSC) in Co-Culture System with Sertoli Cells (SC). Animals. 2023; 13(2):318. https://doi.org/10.3390/ani13020318
Chicago/Turabian StyleSegunda, Moisés N., Carlos Díaz, Cristian G. Torres, Víctor H. Parraguez, Mónica De los Reyes, and Oscar A. Peralta. 2023. "Comparative Analysis of the Potential for Germ Cell (GC) Differentiation of Bovine Peripheral Blood Derived-Mesenchymal Stem Cells (PB-MSC) and Spermatogonial Stem Cells (SSC) in Co-Culture System with Sertoli Cells (SC)" Animals 13, no. 2: 318. https://doi.org/10.3390/ani13020318
APA StyleSegunda, M. N., Díaz, C., Torres, C. G., Parraguez, V. H., De los Reyes, M., & Peralta, O. A. (2023). Comparative Analysis of the Potential for Germ Cell (GC) Differentiation of Bovine Peripheral Blood Derived-Mesenchymal Stem Cells (PB-MSC) and Spermatogonial Stem Cells (SSC) in Co-Culture System with Sertoli Cells (SC). Animals, 13(2), 318. https://doi.org/10.3390/ani13020318