Laboratory of Neurological Diseases and Brain Function, The Affiliated Hospital, Southwest Medical University, Luzhou, 646000, Sichuan, China.
Institute of Epigenetics and Brain Science, Southwest Medical University, Luzhou, 646000, Sichuan, China.
Stem Cell Res Ther. 2024 Sep 4;15(1):281. doi: 10.1186/s13287-024-03911-0.
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.
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.
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.
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.
原代人肝细胞（PHH）对于药物代谢评估、肝病建模和肝细胞移植非常有价值。然而，由于供体来源有限，以及在体外培养时增殖能力有限且功能降低，其可用性受到显著限制。为了解决这一挑战，我们旨在开发一种新的方法，在不丧失功能的情况下有效地在体外扩增 PHH。
通过模拟体内肝再生途径，我们开发了一种两步策略，包括 PHH 的去分化/扩增和随后的成熟，以在体外生成大量功能齐全的肝细胞。最初，我们应用 SiPer，一种预测算法，来鉴定能够激活肝再生转录因子的候选小分子，从而制定了一种新的肝扩展培养基，将 PHH 去分化为增殖性人肝祖细胞样细胞（ProHPLC）。然后，我们使用新的肝细胞成熟条件将这些 ProHPLC 重新分化为功能成熟的肝细胞。此外，我们研究了在我们的新条件下 PHH 扩增的潜在机制。
含有氢可体松的新型肝扩展培养基促进了 PHH 向 ProHPLC 的去分化，后者表现出关键的肝祖细胞特征，并与以前建立的扩展条件下培养的细胞相比，增殖能力显著提高。值得注意的是，这些随后成熟的肝细胞在转录组谱、药物代谢活性和体内植入能力方面与 PHH 相当。重要的是，我们的发现表明，氢可体松通过 PPARα 信号通路和再生转录因子增强 PHH 的扩增。
本研究提出了一种两步策略，首先诱导 PHH 进入增殖状态（ProHPLC）以确保足够的细胞数量，然后将 ProHPLC 成熟为完全功能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大量种子细胞的生产提供了一种有前途的手段。
