Characterization and visualization of murine coagulation factor VIII-producing cells in vivo

Coagulation factors are produced from hepatocytes, whereas production of coagulation factor VIII (FVIII) from primary tissues and cell species is still controversial. Here, we tried to characterize primary FVIII-producing organ and cell species using genetically engineered mice, in which enhanced green fluorescent protein (EGFP) was expressed instead of theF8 gene. EGFP-positive FVIII-producing cells existed only in thin sinusoidal layer of the liver and characterized as CD31high, CD146high, and lymphatic vascular endothelial hyaluronan receptor 1 (Lyve1)+. EGFP-positive cells can be clearly distinguished from lymphatic endothelial cells in the expression profile of the podoplanin and C-type lectin-like receptor-2 (CLEC-2)+.

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Fig: F8 targeting strategy in F8 knock-in mice. (a) Schematic of F8 targeting construct. Diagrams show wild-type allele, targeting vector, targeting allele, floxed allele (F8flox knock-in), and deleted allele (F8Δ knock-in). The targeting vector was constructed by flanking the F8 exons 16–28 cDNA-SV40 polyA and PGK-Neo cassette with loxP sites, followed by linking 2A-EGFP cDNA-SV40 polyA. White boxes, F8 exon 16; dark gray boxes, F8 cDNA including exons 16–28 (stop codon); light gray boxes, 2A-EGFP cDNA; black boxes, SV40 poly A; white arrow, PGK-Neo cassette; black triangles, loxP sites; white triangles, FRT sites; black arrow, DTA. Restriction enzyme sites: Ap, ApaLI; S, SphI; A, ApaI. (b) Southern blotting of targeted embryonic stem cells. Genomic DNA was digested with ApaL I or Sph I, and probed with the 5′ or 3′ probe. Locations of probes are indicated in (a). (c) Confirmation of deletion of F8 cDNA (exons 16–28) in F8Δknock-in mice. PCR was performed using the indicated F and R primers in (a).

In embryogenesis, EGFP-positive cells began to emerge at E14.5 and subsequently increased according to liver maturation. Furthermore, plasma FVIII could be abolished by crossing F8 conditional deficient mice with Lyve1-Cre mice. In conclusion, in mice, FVIII is only produced from endothelial cells exhibiting CD31high, CD146high, Lyve1+, CLEC-2+, and podoplanin in liver sinusoidal endothelial cells.

Hayakawa, M., Sakata, A., Hayakawa, H. et al. Characterization and visualization of murine coagulation factor VIII-producing cells in vivo.Sci Rep 11, 14824 (2021). https://doi.org/10.1038/s41598-021-94307-0Hayakawa, M., Sakata, A., Hayakawa, H. et al. Characterization and visualization of murine coagulation factor VIII-producing cells in vivo.Sci Rep 11, 14824 (2021). https://doi.org/10.1038/s41598-021-94307-0

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