BMP4 preserves the developmental potential of mESCs through Ube2s- and Chmp4b-mediated chromosomal stability safeguarding

Chemically defined medium is widely used for culturing mouse embryonic stem cells (mESCs), in which N2B27 works as a substitution for serum, and GSK3β and MEK inhibitors (2i) help to promote ground-state pluripotency. However, recent studies suggested that MEKi might cause irreversible defects that compromise the developmental potential of mESCs. Here, we demonstrated the deficient bone morphogenetic protein (BMP) signal in the chemically defined condition is one of the main causes for the impaired pluripotency. Mechanistically, activating the BMP signal pathway by BMP4 could safeguard the chromosomal integrity and proliferation capacity of mESCs through regulating downstream targets Ube2s and Chmp4b. More importantly, BMP4 promotes a distinct in vivo developmental potential and a long-term pluripotency preservation. Besides, the pluripotent improvements driven by BMP4 are superior to those by attenuating MEK suppression. Taken together, our study shows appropriate activation of BMP signal is essential for regulating functional pluripotency and reveals that BMP4 should be applied in the serum-free culture system. Supplementary Information The online version contains supplementary material available at 10.1007/s13238-021-00896-x.


Supplementary Figure legends
(I) Confirmation of the indicated all-ESC mice by simple sequence length polymorphism (SSLP) assay. Genomic DNA of C57 and 129 mice were used as positive controls and genomic DNA of DBA/2 mice was used as a negative control.
Data are represented as the mean ± SEM in (A), (G) and (H), the mean ± SD in (C).
(D) ChIP-qPCR analysis of indicated histone modifications occupancy at promoter regions of Ube2s and Chmp4b in N/2i-and N/2i+BMP4-mESCs. Two and one potential regulator regions of Ube2s and Chmp4b were detected, respectively.
Relative enrichment was normalized to IgG ChIP signals at the same regions. A male mESC line #S2 was tested with 2 independent experiments. Data are represented as the mean ± SEM. Statistical analysis was performed using a two-tailed unpaired Welch's t-test. *p < 0.05. (H) Induced knockdown of Ube2s or Chmp4b in mESC line #S2 for 5 passages caused an increased resorption rate in chimera assay. The control-, ishUbe2s-and ishChmp4b-mESCs were cultured under N/2i+BMP4 condition with Dox for 5 passages before microinjection.
(I) Validation of overexpression of Ube2s (left) and Chmp4b (right) in N/2i-mESCs. mESC line #S2 was used in this test. Hprt was set as an endogenous control. n = 3 replicates.
(I) Karyotyping validation of S-, N/2i-and N/2i-S-mESCs. N/2i-S means that N/2i-mESCs were switched back to S condition for 15 days' culturing. Note that N/2i-S mESCs showed a high proportion of aneuploidy similar to that of N/2i-mESCs.
The mESC line #S2 was used in this test. More than 40 mitoses phases were counted for each group. n=3 biological replicates.
Data are represented as the mean ± SEM in (H) and (I). Statistical analysis was performed using a two-tailed unpaired Welch's t-test. *p < 0.05; *** p < 0.001, n.s., not significant.