背景:原代人肝细胞(PHHs)对于药物代谢评估非常有价值,肝脏疾病建模和肝细胞移植。然而,由于捐助来源有限,它们的供应受到很大限制,在体外培养时,它们的增殖能力受到限制,功能降低。为了应对这一挑战,我们的目的是开发一种新的方法,在体外有效扩增PHHs而不丧失功能。
方法:通过模拟体内肝脏再生途径,我们开发了一个两步策略,涉及去分化/扩增和随后成熟的PHHs,以在体外产生丰富的功能性肝细胞。最初,我们应用了SiPer,一种预测算法,鉴定能够激活肝脏再生转录因子的候选小分子,从而配制新的肝扩增培养基以将PHH去分化为增殖性人肝祖细胞样细胞(ProHPLC)。然后使用新的肝细胞成熟条件将这些ProHPLC再分化成功能成熟的肝细胞。此外,我们研究了在新条件下PHHs扩展的潜在机制。
结果:含有氢化可的松的新型肝扩增培养基促进PHHs去分化为ProHPLC,与在先前建立的扩增条件下培养的细胞相比,其表现出关键的肝祖细胞特征,并证明增殖能力显着增加。值得注意的是,这些随后成熟的肝细胞在转录组谱方面与PHH相当,药物代谢活性和体内植入能力。重要的是,我们的发现提示氢化可的松增强PHHs的扩增可能是通过PPARα信号通路和再生转录因子介导的。
结论:本研究提出了一个两步策略,该策略最初将PHH诱导为增殖状态(ProHPLC),以确保足够的细胞数量,随后ProHPLC成熟为功能齐全的肝细胞,以保证最佳的细胞质量。这种方法为基于肝细胞的应用提供了产生大量接种细胞的有希望的手段。
BACKGROUND: Primary human hepatocytes (PHHs) are highly valuable for drug-metabolism evaluation, liver disease modeling and hepatocyte transplantation. However, their availability is significantly restricted due to limited donor sources, alongside their constrained proliferation capabilities and reduced functionality when cultured in vitro. To address this challenge, we aimed to develop a novel method to efficiently expand PHHs in vitro without a loss of function.
METHODS: By mimicking the in vivo liver regeneration route, we developed a two-step strategy involving the de-differentiation/expansion and subsequent maturation of PHHs to generate abundant functional hepatocytes in vitro. Initially, we applied SiPer, a prediction algorithm, to identify candidate small molecules capable of activating liver regenerative transcription factors, thereby formulating a novel hepatic expansion medium to de-differentiate PHHs into proliferative human hepatic progenitor-like cells (ProHPLCs). These ProHPLCs were then re-differentiated into functionally mature hepatocytes using a new hepatocyte maturation condition. Additionally, we investigated the underlying mechanism of PHHs expansion under our new conditions.
RESULTS: The novel hepatic expansion medium containing hydrocortisone facilitated the de-differentiation of PHHs into ProHPLCs, which exhibited key hepatic progenitor characteristics and demonstrated a marked increase in proliferation capacity compared to cells cultivated in previously established expansion conditions. Remarkably, these subsequent matured hepatocytes rivaled PHHs in terms of transcriptome profiles, drug metabolizing activities and in vivo engraftment capabilities. Importantly, our findings suggest that the enhanced expansion of PHHs by hydrocortisone may be mediated through the PPARα signaling pathway and regenerative transcription factors.
CONCLUSIONS: This study presents a two-step strategy that initially induces PHHs into a proliferative state (ProHPLCs) to ensure sufficient cell quantity, followed by the maturation of ProHPLCs into fully functional hepatocytes to guarantee optimal cell quality. This approach offers a promising means of producing large numbers of seeding cells for hepatocyte-based applications.