Nutritional modulation of mouse and human liver bud growth through a branched-amino acid metabolism

Liver bud progenitors experience a transient amplification during early organ growth phase, yet the responsible mechanism was not fully understood. Collective evidence highlighted the specific requirements in stem cell metabolism for expanding organ progenitors during organogenesis and regeneration. Here, transcriptome analyses showed progenitors of mouse and human liver bud growth stage specifically expressed branched chain aminotransferase1 gene, a known breakdown enzyme of branched-chain amino acid (BCAA) for energy generation. Global metabolome analysis confirmed the active consumption of BCAA in the growing liver bud, but not in the later fetal or adult liver. Consistently, maternal dietary restriction of BCAA during pregnancy significantly abrogated the conceptus liver bud growth capability through a striking defect in hepatic progenitor expansion. Under defined conditions, the supplementation of L-valine among different BCAAs specifically promoted the rigorous growth of the human liver bud organoid in culture by selectively amplifying self-renewing bi-potent hepatic progenitor cells. These results highlight a previously underappreciated role of branched-chain amino acid metabolism in regulating mouse and human liver bud growth that can be modulated by maternal nutrition in vivo or cultural supplement in vitro. D ev el o pm en t • A dv an ce a rt ic le INTRODUCTION Organ bud progenitor cells, which have the remarkable capacity for rapid cell growth and differentiation into multi-lineage cells, play important roles in organ development. The hepatic progenitor cells (HPCs), or liver bud progenitors, specify from foregut endoderm at the embryonic day (E)9.5 in mice, followed by massive HPC expansion with a 10-fold population doubling from E9.5 to E13.5 in mice (Koike et al., 2014; Takebe et al., 2013). This tremendous growth is regulated by signals secreted from neighboring mesenchyme, such as HGF (Matsumoto et al., 2001), BMPs (Rossi et al., 2001) and FGFs (Serls et al., 2005), and by transcriptional networks that act intrinsically in the HPCs, such as Tbx3 (Suzuki et al., 2008), Smad2/3 (Weinstein et al., 2001) and beta-catenin (Micsenyi et al., 2004). Yet, the mechanism regulating this intensive and transient amplification in developing liver bud is largely unknown. Emerging studies of stem cell metabolism have elucidated a role for cell-type specific metabolic pathways that are modulated during proliferation, differentiation or reprogramming processes (Ito and Suda, 2014; Shyh-Chang et al., 2013a). For instance, embryonic stem cells (ESC) generally utilize the glycolytic system as a self-renewing propagation process (Kim et al., 2006), whereas differentiating ESC shift their metabolism to oxidative phosphorylation (Folmes et al., 2012). Consistently, during the cellular reprograming process to induced pluripotent stem cells (iPSC), glycolytic metabolism transitions from dependence on oxidative phosphorylation in adult fibroblast (Bukowiecki et al., 2014). The pivotal role of unique amino acid D ev el o pm en t • A dv an ce a rt ic le metabolism is also reportedly shown to maintain the pluripotency. Indeed, starvation of Thr, Met, Cys or Gln abolished mouse or human PSC growth in culture (Shiraki et al., 2014; Shyh-Chang et al., 2013b; Tohyama et al., 2016). Thus, the mechanism of energy creation is diverse depending on each cell/tissue types and its differentiation state. However, little is known about the metabolic dynamics during the early growth phase of hepatogenesis. The aim of this study was to identify the metabolic demands of rapid hepatic progenitor growth phase during mouse liver bud development and study their modulatory effects on ex vivo expansion by defined culture medium. Furthermore, in order to address the human specific metabolic dynamics during early growth phase of hepatogenesis, we took advantage of our recently developed liver bud organoid model from human iPSC (hiPSC) (Takebe et al., 2013) so as to determine if the implied metabolic mechanism is conserved in