ESCDL-1, a new cell line derived from chicken embryonic stem cells, supports efficient replication of Mardiviruses

Abstract

Marek’s disease virus is the etiological agent of a major lymphoproliferative disorder in poultry and the prototype of the Mardivirus genus. Primary avian somatic cells are currently used for virus replication and vaccine production, but they are largely refractory to any genetic modification compatible with the preservation of intact viral susceptibility. We explored the concept of induction of viral replication permissiveness in an established pluripotent chicken embryonic stem cell-line (cES) in order to derive a new fully susceptible cell-line. Chicken ES cells were not permissive for Mardivirus infection, but as soon as differentiation was triggered, replication of Marek’s disease virus was detected. From a panel of cyto-differentiating agents, hexamethylene bis (acetamide) (HMBA) was found to be the most efficient regarding the induction of permissiveness. These initial findings prompted us to analyse the effect of HMBA on gene expression, to derive a new mesenchymal cell line, the so-called ESCDL-1, and monitor its susceptibility for Mardivirus replication. All Mardiviruses tested so far replicated equally well on primary embryonic skin cells and on ESCDL-1, and the latter showed no variation related to its passage number in its permissiveness for virus infection. Viral morphogenesis studies confirmed efficient multiplication with, as in other in vitro models, no extra-cellular virus production. We could show that ESCDL-1 can be transfected to express a transgene and subsequently cloned without any loss in permissiveness. Consequently, ESCDL-1 was genetically modified to complement viral gene deletions thus yielding stable trans-complementing cell lines. We herein claim that derivation of stable differentiated cell-lines from cES cell lines might be an alternative solution to the cultivation of primary cells for virology studies.

Three-dimensional culture of chicken primordial germ cells (cPGCs) in defined media containing the functional polymer FP003

“…In general, the somatic cells show a limited culture period and are difficult to derive cell line except of the transgenesis by oncogenes for immortalization. EB66 and ESCDL-1 cell lines are derived from duck and chicken embryonic stem cells (ESCs), respectively, both cell lines show the contributions on therapeutic applications [ 14 , 24 , 25 ]. Similar to ESCs [ 34 ], cPGCs [ 29 , 35 ] are pluripotent and can divide indefinitely when cultured under suitable conditions.…”

“…For the industrial interesting application, EB66 and other avian cell lines, e.g. ESCDL-1, AGE1.CR and QOR2/2E11, can also be adapted to cell suspension culture, which allows a scalable production in a large-scale bioreactor [ 14 , 22 – 24 ]. Additionally, these avian cell lines could also progressively replace the primary cells to become cell substrates for virus replication for the vaccine production [ 24 – 26 ].…”

Pluripotency in avian species

“…Additionally, until now REX1 (ZPF42) and UTF1 have not been found in the avian genome. Therefore, genes expressed in undifferentiated, long-term proliferating cESCs include SOX3, POUV (POU5F3), DNMT3B, EPCAM, CLDN1, VGLL2, EOMES, CFC1B, GATA5, NANOG, ESRP2, OTX2, TRIM71, GATA4, FOXA2, LIN28A, ASTL, ENS-1, CDH1 and SALL4 (Jean et al, 2015;Vautherot et al, 2017;Acloque et al, 2012;unpublished data). These genes encode transcription factors, DNA demethylases, and cell adhesion molecules.…”

“…Nuclei were stained with Hoechst 33342 (blue). (B) cESCs were induced into mesenchymal cells as described (Vautherot et al, 2017). They displayed a mesenchymal cell morphology with numerous stress fibers (yellow).…”

“…Hexamethylene bis acetamide (HMBA) during SDIA induction was also used to derive the ESCDL-1 mesenchymal cell line (Fig. 6B) (Vautherot et al, 2017).…”