Derivation of Rabbit Embryonic Stem Cells from Vitrified–Thawed Embryos

Abstract

The rabbit is a useful animal model for regenerative medicine. We previously developed pluripotent rabbit embryonic stem cell (rbESC) lines using fresh embryos. We also successfully cryopreserved rabbit embryos by vitrification. In the present work, we combined these two technologies to derive rbESCs using vitrified-thawed (V/T) embryos. We demonstrate that V/T blastocysts (BLs) can be used to derive pluripotent rbESCs with efficiencies comparable to those using fresh BLs. These ESCs are undistinguishable from the ones derived from fresh embryos. We tested the developmental capacity of rbESCs derived from V/T embryos by BL injection experiments and produced chimeric kits. Our work adds cryopreservation to the toolbox of rabbit stem cell research and applications and will greatly expand the available research materials for regenerative medicine in a clinically relevant animal model.

Embryonic stem‐like cells from rabbit blastocysts cultured with melatonin could differentiate into three germ layers in vitro and in vivo

“…The application of bFGF or LIF, however, is still controversial as the reliance on these growth factors appears to indicate cells in ESC‐primed and naïve states, respectively, which differ in cell characteristics and pluripotency for murine and human ESCs. bFGF and activin/nodal signaling, through Smad2/3 activation, appear to be indispensable for maintaining rabbit ESCs in an undifferentiated state (Catunda et al, ; Wang et al, ; Honda et al, ; Jiang et al, ; Du et al, ), while the irreplaceability of LIF (Xue et al, ) implies that rabbit ESCs exist in an intermediate state between naïve and primed. Thus, 10 ng/mL bFGF and 1,000 IU/mL LIF were both supplemented into our basic medium to ensure the maintenance of self‐renewal and pluripotency of our rES‐like cells, as conducted previously (Catunda et al, ; Honda et al, ).…”

“…Extensive knowledge has accumulated since the first reported derivation of rabbit ESCs (Graves and Moreadith, ), particularly with respect to the conditions need for their production. For example, (i) acceptable sources of the ICM include fresh or vitrified–thawed blastocysts (Du et al, ) and fertilized, parthenogenetic, or somatic cell nuclear transfer embryos (Fang et al, ). Superovulation does not decrease blastocyst quality (Honda et al, ).…”

“…(iii) Mouse embryonic fibroblasts, versus rabbit embryonic fibroblasts or STO cells, are the best feeder cell line to support the maintenance of rabbit ESCs (Intawicha et al, ). (iv) Basic fibroblast growth factor (bFGF) is needed to maintain self‐renewal and pluripotency of these cells (Fang et al, ; Catunda et al, ; Honda et al, ; Jiang et al, ; Du et al, ), although a few researchers insist that only adding leukemia inhibitory factor (LIF) can produce pluripotent lines (Intawicha et al, ; Xue et al, ). Finally, (v) small molecules such as Noggin (inhibits the bone morphogenetic protein [BMP] pathway) and Y‐27632 (inhibits apoptosis) can improve maintenance of the lines (Du et al, ).…”

“…(iv) Basic fibroblast growth factor (bFGF) is needed to maintain self‐renewal and pluripotency of these cells (Fang et al, ; Catunda et al, ; Honda et al, ; Jiang et al, ; Du et al, ), although a few researchers insist that only adding leukemia inhibitory factor (LIF) can produce pluripotent lines (Intawicha et al, ; Xue et al, ). Finally, (v) small molecules such as Noggin (inhibits the bone morphogenetic protein [BMP] pathway) and Y‐27632 (inhibits apoptosis) can improve maintenance of the lines (Du et al, ). Nevertheless, derivation and culture systems vary among laboratories, contributing to the inconsistent characteristics (i.e., morphology and pluripotency) of the reported rabbit ESC lines—which hampers research and utilization of rabbit ESCs.…”