Generation and Characterization of Leukemia Inhibitory Factor-Dependent Equine Induced Pluripotent Stem Cells from Adult Dermal Fibroblasts

Abstract

In this study we have reprogrammed dermal fibroblasts from an adult female horse into equine induced pluripotent stem cells (equiPSCs). These equiPSCs are dependent only on leukemia inhibitory factor (LIF), placing them in striking contrast to previously derived equiPSCs that have been shown to be co-dependent on both LIF and basic fibroblast growth factor (bFGF). These equiPSCs have a normal karyotype and have been maintained beyond 60 passages. They possess alkaline phosphatase activity and express eqNANOG, eqOCT4, and eqTERT mRNA. Immunocytochemistry confirmed that they produce NANOG, REX1, SSEA4, TRA1-60, and TRA1-81. While our equiPSCs are LIF dependent, bFGF co-stimulates their proliferation via the PI3K/AKT pathway. EquiPSCs lack expression of eqXIST and immunostaining for H3K27me3, suggesting that during reprogramming the inactive X chromosome has likely been reactivated to generate cells that have two active X chromosomes. EquiPSCs form embryoid bodies and in vitro teratomas that contain derivatives of all three germ layers. These LIF-dependent equiPSCs likely reflect a more naive state of pluripotency than equiPSCs that are co-dependent on both LIF and bFGF and so provide a novel resource for understanding pluripotency in the horse.

Derivation of Mesenchymal Stromal Cells from Canine Induced Pluripotent Stem Cells by Inhibition of the TGFβ/Activin Signaling Pathway

“…Of particular consideration, given both their derivation from iPSCs, and our eventual intent to transplant these cells in vivo, is the ability of the ciPSC-MSCs to form teratomalike tissues. Using an in vitro terminal differentiation assay, the ciPSCs gave rise to tissues that were derivatives of all three germ layers, although the mesodermal lineage was more extensively represented than the endoderm or ectoderm; we have observed a similar bias toward mesoderm derivatives in iPSCs from other domestic species assayed with this system [31]. However, of key importance was the observation that neither the ciPSC-MSCs nor the adult adipose-derived MSCs formed teratoma-like tissues.…”

“…While originally this protocol was applied to human ESCs and iPSCs, we have also used it successfully with horse [31] and platypus iPSCs (Whitworth, unpublished data). ciPSCs, ciPSC-MSCs and adult MSCs were passaged with TrypLE.…”

“…For this reason, both PEG and HA/PEG hydrogels were examined. As well as its widespread use in the veterinary field for applications in joint repair, there is further evidence for the utility of PPS for chondrogenic differentiation of MSCs [34] and so recently developed gels containing a form of PPS covalently bound to the HA backbone of the hydrogel matrix (HA-PPS) were also used [31]. ciPSC-MSCs encapsulated in each of these three hydrogel types (PEG, PEG/HA and PEG/ HA + HA-PPS) were cultured in basal medium, as well as medium supplemented with osteo-or chondrogenic factors.…”

Platypus Induced Pluripotent Stem Cells: the Unique Pluripotency Signature of a Monotreme

“…Because the core body temperature of the platypus is 32°C, and platypus cells do not survive at temperatures above this, the use of mice for the generation of conventional teratomas was not possible. However, we have devised and fully characterised a methodology for generating teratomas in vitro (Whitworth et al 2012;Whitworth et al, 2014a;Whitworth et al, 2014b).…”

Pluripotent stem cells and livestock genetic engineering

“…Interestingly, many of the studies summarized in Table 1 describing derivation of naïve state livestock iPSCs do not evaluate X-chromosome reactivation in female cells. Only three studies (Whitworth et al 2014a; Zhang et al 2014, 2015) evaluated H3K27me3 of X-chromosome by immunostaining showing promising results. X-chromosome reactivation is an important feature of naïve state pluripotency and its detection can add valuable information to the epigenetic profile of reprogrammed somatic cells.…”