| Popis: |
Introduction: Exploring techniques for differentiating and culturing canine hepatocytes serves as a means to establish systems for liver transplantation and drug metabolism testing. However, establishing consistent methods for culturing stable hepatocytes remains a challenge. Recently, several investigations have shown the reprogramming of mature hepatocytes into hepatic progenitor cells by applying specific small molecule compounds, including Y-27632, (a ROCK inhibitor), A-83-01 (a TGFβ inhibitor), and CHIR99021 (a GSK3 inhibitor) (termed YAC) in rat, mouse, and humans, respectively. However, reports or evidence of successful reprogramming using these small-molecule compounds in dogs are absent. This study aimed to induce the differentiation of mature canine hepatocytes into progenitor cells. Methods: Cryopreserved canine hepatocytes (cHep) were cultured for 14 d in a YAC-supplemented hepatocyte growth medium. Subsequently, an assessment was conducted involving morphological observations, quantitative real-time polymerase chain reaction (qRT-PCR), and immunocytochemistry. Results: Notably, cryopreserved cHep cells emerged and exhibited ongoing proliferation and concurrently developed colonies within the YAC-enriched culture. These observations indicated that the mature hepatocytes reprogrammed into hepatic progenitor cells. Moreover, qRT-PCR analysis revealed a notable enhancement in gene expression levels. Specifically, the genes encoding α-fetoprotein (AFP), epithelial cell adhesion molecule (EpCAM), Cytokeratin 19 (CK19) and SRY-box9 (Sox9) displayed approximately 12-, 2.2-, 517- and 2.9- increases in hepatic progenitor cells, respectively, on day 14 as compared to their state before induction of differentiation. Hepatocyte-related protein expression of AFP, EPCAM, SOX9 and CK19 was confirmed via immunocytochemistry on day 21. In contrast, ALB and MRP2, which are highly expressed in mature hepatocytes, were decreased compared to those before YAC addition, which is consistent with the characteristics of undifferentiated hepatocytes. Conclusions: Herein, we effectively promoted the reprogramming of cryopreserved cHep cells into hepatic progenitor cells using three small-molecule compounds. The mRNA and protein expression analyses demonstrated increased levels of hepatic progenitor cells-specific markers, whereas markers related to mature hepatocytes decreased, suggesting that reprogramming cryopreserved cHep cells to hepatic progenitor cells was achieved using YAC. Therefore, cultivating liver progenitor cells holds the potential to offer valuable insights into the development of artificial livers for drug discovery research and transplantation therapy aimed at addressing liver diseases in dogs. |
