Generation and characterization of bat-induced pluripotent stem cells

Abstract

Induced pluripotent stem cells (iPSCs) were first generated from mouse embryonic fibroblasts in the year 2006. These cells resemble the typical morphology of embryonic stem cells, express pluripotency markers, and are able to transmit through germlines. To date, iPSCs of many species have been generated, whereas generation of bat iPSCs (biPSCs) has not been reported. To facilitate in-depth study of bats at the molecular and cellular levels, we describe the successful derivation of biPSCs with a piggyBac (PB) vector that contains eight reprogramming factors Oct4, Sox2, Klf4, Nanog, cMyc, Lin28, Nr5a2, and miR302/367. These biPSCs were cultured in media containing leukemia inhibitory factor and three small molecule inhibitors (CHIR99021, PD0325901, and A8301). They retained normal karyotype, displayed alkaline phosphatase activity, and expressed pluripotency markers Oct4, Sox2, Nanog, TBX3, and TRA-1-60. They could differentiate in vitro to form embryoid bodies and in vivo to form teratomas that contained tissue cells of all three germ layers. Generation of biPSCs will facilitate future studies on the mechanisms of antiviral immunity and longevity of bats at the cellular level.

Generation and characterization of induced pluripotent stem cells from guinea pig fetal fibroblasts

“…Despite their tendency to randomly and stably integrate into chromosomes, which may increase the risk of tumorigenesis (26), they have a high reprogramming efficiency when compared to other methods (14). …”

Genetic signatures of evolution of the pluripotency gene regulating network across mammals

“…The prevalence of evolutionary conserved genes across the PGRN indicates overall comparability of the regulating mechanisms for pluripotency and self-renewability of mammalian PSCs. In context of the reprogramming somatic cells into induced pluripotent stem cells, although human sequences are effective (Ben-Nun et al 2011), the efficient combination of reprogramming factors may differ among species (Tomioka et al 2010;Verma et al 2013;Mo et al 2014;Weeratunga et al 2018). Among the commonly used reprogramming factor, OSKM, we found genetic conservation of SOX2, KLF4, and MYC supporting the efficiency of these reprogramming factors in variety of species (Ezashi et al 2016).…”

“…Recent advances in somatic cell reprogramming into induced PSCs (iPSCs) have broadened the opportunity to obtain PSCs from variety of mammals, including endangered species (Ben-Nun et al 2011). The iPSC technology has been applied successfully to a wide range of taxonomic groups, including Carnivora (Shimada et al 2009;Verma et al 2012Verma et al , 2013Menzorov et al 2015), Cetartiodactyla (Ezashi et al 2009;Han et al 2011;Liu et al 2012), Chiroptera (Mo et al 2014), Lagomorpha (Osteil et al 2013), Metatheria (Weeratunga et al 2018), Monotremata (Whitworth et al 2019), Perissodactyla (Ben-Nun et al 2011;Breton et al 2013), Rodentia (Takahashi & Yamanaka 2006;Liao et al 2009;Miyawaki et al 2016; Lee et al 2017), and Primates (Takahashi et al 2007;Tomioka et al 2010;Marchetto et al 2013;Wunderlich et al 2014;Ramaswamy et al 2015). However, there continues to be discussion about species variation in the properties of the PSCs, such as pluripotent state, reprogramming efficiency, and optimal culture condition.…”

Induced pluripotent stem cells: Mechanisms, achievements and perspectives in farm animals

“…The integrated transposon can be removed seamlessly by supplying the transposase in trans [98] , which makes the system more attractive and relevant in producing the safe and clean iPS cells. Up to six reprogramming factors have been connected by selfcleaving peptide sequences allowing for coexpression from a single cassette [91,[99][100][101][102][103] . Individual proteins are then produced by the self-cleaving peptide [104][105][106] .…”