LCDM medium supports the derivation of bovine extended pluripotent stem cells with embryonic and extraembryonic potency in bovine–mouse chimeras from iPSCs and bovine fetal fibroblasts

Abstract

Cattle have emerged as one of the most important domestic animals widely used for meat, milk, and fur. Derivation of bovine pluripotent stem cells (PSCs) can be applied in drug selecting and human disease modeling and facilitated agriculture‐related applications such as production of genetically excellent cattle by gene editing. Extended PSCs (EPSCs), capable of differentiating into embryonic and extraembryonic parts, have been generated in mouse, human, and pig. Whether bovine EPSCs could be generated, and their chimeric competency remains unclear. This study focused on derivation of bovine EPSCs using LCDM medium and exploring the characteristics of EPSCs among different species, including bovine, mouse, and human EPSCs. Here, using LCDM medium (consisting of hLIF, CHIR99021, (S)‐(+)‐dimethindene maleate, and minocycline hydrochloride) enables the derivation of bovine EPSCs from induced PSCs (iPSCs) and bovine fetal fibroblasts (BFF) with stable morphology, pluripotent marker expression, and in vitro differentiation ability. Notably, bovine EPSCs exhibited interspecies chimeric contribution to embryonic and extraembryonic tissues in pre‐implantation blastocysts and postimplantation bovine–mouse chimeras. Transcriptome analysis revealed the unique molecular characteristics of bovine EPSCs compared with iPSCs. The similarities and differences in molecular features across bovine, human, and mouse EPSCs were also described by transcriptome analysis. Taken together, the LCDM culture system containing chemical cocktails can be used for the establishment and long‐term passaging of bovine EPSCs with embryonic and extraembryonic potency in bovine–mouse chimeras. Our findings lay the foundation of generating PSCs in domestic animals and open avenues for basic and applied research in biology, medicine, and agriculture. Database Gene expression data of bovine EPSCs and bovine iPSCs are available in the GEO databases under the accession number PRJNA693452.

Simplification of culture conditions and feeder-free expansion of bovine embryonic stem cells

“…On the other hand, pig ESCs strongly expressed SSEA1, SSEA4, TRA-1-60 and TRA-1-81 11 , with pig EPSCs also expressing SSEA1 and SSEA4 10 . Contrary, bovine EPSCs showed only minimal signal after immunostaining against SSEA1 and SSEA4 8 . With large variation in cell surface marker expression between species and cell lines, it is difficult to ascertain the value of characterizing these markers in new, conversely, transcription factors have a clear functional role in maintaining pluripotency and are therefore highly valuable for characterizing pluripotent stem cells.…”

“…Therefore, it is not surprising that N2B27 base medium, which is also based on DMEM/F12 30 , supported long-term culture of bESCs after BSA supplementation. N2B27 has also been demonstrated to support the culture of PSCs in several other species, including cattle 8 , 16 , 24 , 30 . Interestingly, even though Essential 6 is also based on DMEM/F12, it did not show the same potential for replacing custom mTeSR1, even after BSA supplementation.…”

Strategy to Establish Embryo-Derived Pluripotent Stem Cells in Cattle

“…Why is it difficult to establish ESCs? Recent reports [75][76][77] have proposed several suggestions to explain this (Figure 1). One possible explanation is that the optimal time when pluripotency is sufficiently expressed in the developmental stage has yet to be determined.…”

“…Moreover, bovine EPSCs (bEPSCs) were captured under the same conditions [ 75 ]. Like mEPSCs and human EPSCs (hEPSCs), a single bEPSC could differentiate into both cell fates, embryonic and extraembryonic lineages, when injected into the mouse embryo.…”

“…Although these culture conditions are not perfect for establishing bESCs, new possibilities are available for capturing the pluripotency of bovine (stem) cells. In addition, one study [ 75 ] provided new insights into the similarities and differences in establishing pluripotency between species. In particular, the mechanism of bovine pluripotency would be different from other species, and it may be closer to those of EPSCs contributing to both lineages than those of conventional PSCs.…”