Small-Molecule Induction of Canine Embryonic Stem Cells Toward Naïve Pluripotency

Abstract

Naïve and primed pluripotent stem cells (PSCs) reflect discrete pluripotent states that approximate the inner cell mass or the progressively lineage-restricted perigastrulation epiblast, respectively. Cells that occupy primed pluripotency have distinct epigenetic landscapes, transcriptional circuitry, and trophic requirements compared with their naïve counterparts. The existence of multiple pluripotent states has not been explored in dogs, which show promise as outbred biomedical models with more than 300 inherited diseases that also afflict humans. However, our understanding of canine embryogenesis and embryo-derived stem cells is limited. Herein, we converted leukemia inhibitory factor (LIF)-dependent and fibroblast growth factor 2 (FGF2)-dependent canine embryonic stem cells (cESCs) resembling primed PSCs toward a naïve pluripotent state using LIF and inhibitors of glycogen synthase kinase 3β and mitogen-activated protein kinase kinase 1/2 [called 2i and LIF (2iL)]. cESCs propagated in 2iL exhibited significant induction of genes associated with the naïve pluripotent state (eg, REX1, TBX3) and downregulation of primed pluripotency markers (eg, OTX2, FGF5) (P < 0.05). Differential phosphorylation of signal transducer and activator of transcription 3 (STAT3) and cell fate decisions on exposure to bone morphogenetic protein 4 (BMP4) suggested that a novel pluripotent identity has been established with 2iL. Accordingly, cESCs cultured with 2iL formed colonies at a greater efficiency than LIF-FGF2 cESCs following single-cell dissociation. Total genomic DNA methylation and histone H3 lysine 27 trimethylation signals were reduced in 2iL-treated cESCs. Our data suggest that 2iL culture conditions promote the conversion of cESCs toward an epigenetically distinct pluripotent state resembling naïve PSCs.

Stirred Suspension Bioreactor Culture of Porcine Induced Pluripotent Stem Cells

“…For chromosome preparations, cells were treated for 30 min with Colcemid (10 mg/mL), inducing metaphase arrest and consequently harvested, treated with a hypotonic solution (75 mM potassium chloride) and fixed with Carnoy’s solution (3:1 Methanol: Acetic Acid). Chromosome preparations were subjected to conventional GTG-banding protocol and standard karyotypes were obtained and analyzed as previous described (19). .…”

Characterization of companion animal pluripotent stem cells

“…However, since then other groups have derived canine ESCs which can be maintained for longer in vitro . Some of these cells have been taken forward to further work to determine their ability to undergo directed differentiation and to better determine their culture requirements . Success rates in deriving ESCs from canine blastocysts have also been reported to be in the region of 20% .…”

“…Cat ESCs have not been used in these assays and putative horse ESCs have not successfully generated teratomas . One report on dog ESCs generated teratomas , but another group have not been able to generate teratomas with their canine ESCs . However, given the limitations of teratoma assays described above it is difficult to interpret if a negative result is due to experimental variations (e.g., the starting number of cells not being sufficient) or due to an inability of the cells to form a teratoma.…”

“…This variability can also be seen across companion animal species, however there is also variability noted within species, with conflicting data on the exact SSEA and TRA markers present. For example horse and dog ESCs have been reported as being both positive , and negative for SSEA4 in different studies. However, SSEA‐3 and SSEA‐4 are epitopes of the same glycolipid and both have been found to be present in the inner cell mass of equine blastocysts .…”

“…However, very few of the antibodies which have been used in companion animal studies have been raised using equine, canine or feline proteins. Therefore, in order to appropriately gauge their biological activity it is important that they are tested by western blot to confirm their specificity, something that has only been conducted in one publication for stem cell markers .…”