Inducing Pluripotency in the Domestic Cat (Felis catus)

Abstract

Domestic cats suffer from a range of inherited genetic diseases, many of which display similarities with equivalent human conditions. Developing cellular models for these inherited diseases would enable drug discovery, benefiting feline health and welfare as well as enhancing the potential of cats as relevant animal models for translation to human medicine. Advances in our understanding of these diseases at the cellular level have come from the use of induced pluripotent stem cells (iPSCs). iPSCs are capable of differentiating into derivatives of all three germ layers, therefore overcoming the limitations of primary differentiated cells and the ethical concerns of using embryonic stem cells. No studies however report the generation of iPSCs from domestic cats (fiPSCs). Feline adipose derived fibroblasts were infected with amphotropic retrovirus containing the coding sequences for human Oct4, Sox2, Klf4, cMyc and Nanog. Isolated iPSC clones were expanded on mouse inactivated embryonic fibroblasts in the presence of feline leukaemia inhibitory factor (LIF). Retroviral delivery of human pluripotent genes gave rise to putative fiPSC colonies within 5-7 days. These iPS-like cells required foetal bovine serum and feline LIF for maintenance. Colonies were domed with refractile edges, similar to mouse iPSCs. Immunocytochemistry demonstrated positive staining for stem cell markers: alkaline phosphatase, Oct4, Sox2, Nanog and SSEA1. Cells were negative for SSEA4. Expression of endogenous feline Nanog was confirmed by qPCR. The cells were able to differentiate in vitro into cells representative of the three germ layers. These results confirm the generation of the first induced pluripotent cells from domestic cats. These cells will provide valuable models to study genetic diseases and explore novel therapeutic strategies.

Viability, Yield and Expansion Capability of Feline MSCs Obtained from Subcutaneous and Reproductive Organ Adipose Depots

“…In comparison with canine cells, feline amniotic MSCs showed a much lower expression of Sox2 and less immunostaining of the protein compared to canine amniotic stem cells [39]. In addition, a number of labs have shown that induced pluripotent stem cells derived from feline fibroblasts express Sox2 [40,41]. However, the presence of Sox2 in feline adipose-derived MSCs is controversial.…”

Large Animal Models in Regenerative Medicine and Tissue Engineering: To Do or Not to Do

“…Feline iPSCs have recently been reported for the first time by our group, the development of which represents a significant step in the generation of iPSC derived cardiomyocytes from a veterinary species (Dutton et al, 2019). It paves the way for generating further cell lines from feline patients carrying the HCM causing MYBPC3/R820W mutation to test novel therapeutics for modifying the disease.…”

Anatomical templates for tissue (re)generation and beyond

“…It is worth noting that only very recently, in 2019, the cat ( Felis silvestris catus ; Linnaeus, 1758) contributed to expand the list of companion animals used as source of iPSCs (fiPSCs), although already in 2012 Verma et al (2012, 2013) generated these cells from wild felines. In 2019, Dutton et al (2019) isolated feline fibroblasts from adipose tissue and transfected them via Moloney murine leukemia virus containing human KOSM along with Nanog used as ESC‐like marker. By testing different derivation conditions, they set‐up a protocol for feline iPSCs induction observing that these cells require fetal bovine serum and feline leukemia inhibitory factor (LIF) to be maintained in culture for more than 3–5 days (Dutton et al, 2019).…”

“…In 2019, Dutton et al (2019) isolated feline fibroblasts from adipose tissue and transfected them via Moloney murine leukemia virus containing human KOSM along with Nanog used as ESC‐like marker. By testing different derivation conditions, they set‐up a protocol for feline iPSCs induction observing that these cells require fetal bovine serum and feline leukemia inhibitory factor (LIF) to be maintained in culture for more than 3–5 days (Dutton et al, 2019).…”

“…The majority of studies on pluripotency, differentiation, self‐renewal, and iPSCs have been mainly based on murine models aimed to obtain new data for human regenerative medicine (Attanasio & Netti, 2017; Attanasio, Latancia, Otterbein, & Netti, 2016). However, from 2008 onwards several research groups reported encouraging data about the derivation of iPSC lines from animals other than rodents (Chan, Cheng, Neumann, & Yang, 2010; Dutton, Dudhia, Guest, & Connolly, 2019; Esteban et al, 2009; Nagy et al, 2011; Pillai et al, 2019; Ren et al, 2011; Shimada et al, 2010; Verma, Holland, Temple‐Smith, & Verma, 2012; Figure 1) due to several factors. First, numerous companion and farm animal injuries resemble those of humans favoring the replacement of mouse models, which are increasingly contested as they often fall short in mimicking human diseases (Nagy et al, 2011; Weil et al, 2012).…”