Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (11): 2186-2201.doi: 10.3864/j.issn.0578-1752.2023.11.012

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles     Next Articles

Effects of Pladienolide B on Expression of Pluripotency Related Genes and Cell Viability of Bovine Embryonic Stem Cells

ZHAO Fang1(), DING Qiang1, XIA ShuWen1, GAO YunDong2, LAN GuoCheng3, LIN ZhiPing4, WANG HuiLi1(), ZHONG JiFeng1()   

  1. 1 Institute of Animal Science, Jiangsu Academy of Agricultural Sciences/Jiangsu Province Engineering Research Center for Precision Animal Breeding/Key Laboratory of Crop and Animal Integrated Farming, Ministry of Agriculture and Rural Affairs, Nanjing 210014
    2 Shandong OX Livestock Breeding Co., Ltd, Jinan 250100
    3 Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong 999077
    4 Jiangsu Youyuan Dairy Industry Research Institute Co. Ltd, Nanjing 211100
  • Received:2022-01-20 Accepted:2022-09-07 Online:2023-06-01 Published:2023-06-19

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Abstract:

【Background】 Due to high pluripotency of bovine embryonic stem cells (BESCs), they have important application values in cattle breed conservation, breed selection, and regulation mechanism study of livestock embryo development. However, the studies on the maintenance of pluripotency and differentiation of BESCs are limited, and the regulative mechanism remains unclear. 【Objective】The aim of this study was to investigate the effects of different concentrations of Pladienolide B (PlaB) on the expression of pluripotent markers, totipotent markers and embryonic cell-lineage genes as well as the cell viability of BESCs, so as to provide the reference and theoretical basis for improving the developmental potency of BESCs.【Method】Immunofluorescence was used to detect the expression of pluripotent markers of bovine BESCs, and real-time fluorescence quantitative PCR (RT-qPCR) was used to detect the effects of different concentrations of PlaB on the expression of spliceosome, totipotent markers and embryonic cell-lineage genes of BESCs. RT-qPCR and Western Blot were used to detect the effects of different concentrations of PlaB on both mRNA and protein expression of pluripotent markers of BESCs. CCK8 and EDU staining was used to detect the effects of different concentrations of PlaB on the proliferation of BESCs. 【Result】 RT-qPCR results showed that the mRNA expression levels of SF3B1 and SF3B2 in EPSCM-BESCs were significantly down-regulated by 0.5 nmol·L-1 to 1.5 nmol·L-1 PlaB; when the PlaB concentration was 1.5 nmol·L-1, the mRNA expressions of SF3B1 and SF3B2 in CTFR-BESCs were decreased; when the PlaB concentration ranged from 0.5 nmol·L-1 to 1.5 nmol·L-1, the mRNA expression levels of SF3B4 and SF3B5 in both CTFR-BESCs and EPSCM-BESCs were increased in a dose-dependent manner. Furthermore, PlaB significantly down-regulated mRNA expression of SF3B6 in the CTFR-BESCs. When PlaB concentration ranged from 0.5 nmol·L-1 to 1.5 nmol·L-1, the mRNA expression of spliceosome LSM4 both in EPSCM-BESCs and CTFR-BESCs were significantly down-regulated. The concentration from 0.5 nmol·L-1 to 1.5 nmol·L-1 PlaB significantly down-regulated the expression levels of EFTUD2 mRNA in CTFR-BESCs; the mRNA expression of EFTUD2 mRNA was significantly down-regulated in BEPSCM-BESCs with 1 nmol·L-1 and 1.5 nmol·L-1 PlaB while PlaB concentration from 0.5 to 1.5 nmol·L-1, both the mRNA expression and protein levels of the pluripotent markers OCT4, SOX2 and NANOG in CTFR-BESCs and EPSCM-BESCs were up-regulated in a dose-dependent manner. By the concentration range from 0.5 to 1.5 nmol·L-1, PlaB dose-dependently up-regulated the mRNA levels of totipotent markers such as MDM2, PID1 and BTG2 in CTFR-BESCs and EPSCM-BESCs, while the mRNA levels of DDIT4 and PDRG1 were down-regulated. The mRNA expression of embryonic cell lineage genes in the CTFR-BESCs were up-regulated while the PlaB was added. The addition of PlaB in EPSCM-BESCs significantly reduced the expression of GATA4, GATA6, SOX7 and other embryonic cell lineage genes, but had no significant effect on ZIC1. The cell viability of CTFR-BESCs and EPSCM-BESCs showed a downward trend with increasing of PlaB dose and treatment time, while CTFR-BESCs was more sensitive than EPSCM-BESCs. 【Conclusion】PlaB significantly up-regulated the expression of pluripotent markers and partial totipotent markers in CTFR-BESCs and EPSCM-BESCs, and the expression of gene lineages and cell viability in EPSCM-BESCs were decreased. The effective concentration and effects on gene expression of PlaB in the two types BESCs were not completely consistent. Due to the inhibiting effect of PlaB on cell viability of BESCs, the further studies were needed to optimize the culture system.

Key words: bovine embryonic stem cells, pladienolide B, spliceosome, pluripotency related genes, cell viability

Fig. 1

Establishment of bovine embryonic stem cell lines A. Attached blastocyst; B. Outgrowth of Day 1; C. Outgrowth of Day 2; D. Outgrowth of Day 4"

Table 1

Primer sequences used in RT-qPCR"

基因 Gene NCBI登录号 NCBI Genebank 引物对序列 Sequence of primers（5′→3′)
OCT4 NM_174580.3 F: GTGTTCAGCCAAACGACTATCT
R: TCTGCCTTGCATATCTCCTG
SOX2 NM_001105463.2 F: ATGTGAGGGCCGGACGGTGAACT
R: GCTGTTTCTTGCTGTCCTCCATTTCC
NANOG NM_001025344.1 F: AAAACAACTGGCCGAGGAATAGC
R: TGGTAGGAATAGAAGCCTGGGTA
SF3B1 NM_001192994.1 F: TGTGCTAAAGGTGGTAAA
R: CTATCCAAAGCCATTCTA
SF3B2 NM_001103271.2 F: GGAGCGATTGCAGACTTAC
R: GCTGTGCTGACATAGGAGG
SF3B3 NM_001077851.1 F: TCGCCCTGATCCTAACAC
R: CAGAATAACAATCCGACCT
SF3B4 NM_001205584.1 F: TATGGGAAGCCAATACGG
R: AACCTTTGGAGTTGCCTGT
SF3B5 NM_001025350.3 F: AAAAGCGGTGAGATGACG
R: GCAATGGCGAAGTAGTTGAG
SF3B6 NM_001046017.1 F: GTATCTGATGCCTTGTTG
R: GATTTACTTCTGGTGGAA
PHF5A NM_001083382.1 F: GGTTGCCCAAAGATTGTC
R: GGTGATGCTCTGTGCTCC
MDM2 NM_001099107.1 F: TAGCAACTAAACCATCCACCAC
R: TTGACTCCCACCCTCCCAC
PID1 NM_001079584.2 F: CCCTGTTCTTATTGTATTCG
R: GAGGGATGGTGTAGTGGA
DDIT4L NM_001081519.1 F: CTCTGGCAAGTTGTCTCC
R: TGGCAGTTTGAGCAGTAA
BTG2 XM_002693879.5 F: CGTAGTGAGGGACTTTGC
R: AAGGTAGCTTGGGATTTG
PDRG1 NM_001078115.1 F: TGAAGGTCAATCGCCTCT
R: AGTTCTGGGTCCTCCATG
EFTUD2 NM_001083396.1 F: TCAGGAGGAGGACACCCA
R: CTGCCAAGGAATCCATTT
PUF60 NM_001046133.3 F: GGGCAGACCCAGCAACAT
R: CCAGCGTGCAGGATTTGA
LSM4 NM_001035436.2 F: CCCACCCAGAGGCTGCTTT
R: AGTTGGCTGCGGCTTCAC
HAND1 NM_001075761.1 F: CCTACCTGATGGACGTGTTG
R: CTGTGCGCCCTTTAATCC
GATA4 NM_001192877.1 F: GGAAGCCCAAGAACCTTAACAA
R: GACCGAGAACGTCTGCGACA
GATA6 XM_024984608.1 F: GCTGAGTCGGTTCTTGAGGT
R: CTTCGGGTCAGTCGCACA
SOX7 XM_024996047 F: GGTCGTTGCTGAGGACGCTATTT
R: GATGCGAATGTTGCTGGGCTTTT
PAX6 NM_001040645.1 F: CAGTTTCAGCACCAGCGTCTA
R: GCGGCAGAGCACTGTATGTG
ZIC1 XM_024995953.1 F: GCGGACCGGAGCAGAGTAAC
R: TCAGAACGGGCGAAGACC
GAPDH NC_037332.1 F: ATGCTGGTGCTGAGTATGTG
R: CAATCTTGAGGGTGTTGTTAT

Fig. 2

Pluripotency identification of BESCs A. Immunofluorescence staining was used to identify the expression of pluripotent markers in BESCs. B. BESCs passage 26 were used to prepare teratoma, HE staining revealed that the teratoma tissue sections contained ectoderm (yellow arrow indicates myelinated nerve), mesoderm (yellow arrow indicates muscle cells), and endoderm (yellow arrow indicates columnar epithelial cells). C. BESCs retained a normal karyotype at passage 48"

Table 2

Composition comparison of two different culture mediums"

成分
Composition		 培养液 Culture medium
CTFR EPSCM
mTeSR1 + +
FGF2 + +
IWR1 + +
TGFβ - +
Activin A - +
CHIR-99021 - +
WH-4-023 - +
Vitamin C - +
β-mercaptoethanol - +

Fig. 3

Characteristics of bovine embryonic stem cells cultured by different culture systems A. EPSCM-BESCs under 4X microscope; B. EPSCM-BESCs under 10X microscope; C. Alkaline phosphatase staining of EPSCM-BESCs; D. CTFR -BESCs under 4X microscope; E. CTFR -BESCs under 10X microscope; F. Alkaline phosphatase staining of CTFR-BESCs; G. mRNA expression of pluripotent markers; H. mRNA expression of embryonic cell lineage genes"

Fig. 4

Effect of PlaB on mRNA expression of SF3Bs in BESCs The same as below"

Fig. 5

Effect of PlaB on mRNA expression of splicing factors in BESCs"

Fig. 6

Effect of PlaB on expression of pluripotent markers in BESCs A-C. Effects of different concentrations of PlaB on mRNA expression of pluripotent markers in BESCs; D. Effects of different concentrations of PlaB on protein expression of pluripotent markers in EPSCM-BESCs; E. Quantification results of protein expression of pluripotent markers"

Fig. 7

Effect of PlaB on mRNA expression of totipotent markers in BESCs"

Fig. 8

Effect of PlaB on mRNA expression of embryonic cell-lineage genes in BESCs"

Fig. 9

Effect of PlaB on cell viability of BESCs A-D. The proliferation of BESCs cells in different treatment groups was detected by CCK8. E. EDU detected the cell proliferation of CTFR-BESCs and EPSCM-BESCs treated by different concentration PlaB for 72 h"

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