Recent advances in the derivation of hepatocytes from pluripotent stem cells have raised hope for cell therapy to treat liver ailments. They have enhanced the prospects of developing reliable in vitro models for liver diseases and drug toxicity screening. We along with many other research laboratories have optimized a multi-stage protocol using a cocktail of growth factors to direct stem cells towards the hepatic fate and express key hepatic markers and functions. These efforts, while encouraging, still need to overcome major challenges of generating large quantities of cells for clinical applications and improving functional maturity as well as cell heterogeneity upon differentiation. We employed scalable cultivation methods for propagating undifferentiated stem cells and for their differentiation. Cell expansion was incorporated during differentiation to mitigate the reliance on large quantities of stem cells. Enrichment of cells was performed during the last stage of differentiation to obtain higher fraction of cells with the desired hepatic properties. Transcriptome data was generated through the time course of differentiation to identify key genes and pathways changing during the process of hepatic specification. To evaluate the state of art on hepatic differentiation, transcriptome data for differentiated hepatocyte like cells generated using several different protocols was collected from public data depositories. Interestingly, all the hepatocyte-like cells, including our cells clustered together irrespective of the methods of differentiation employed and the entire cohort of differentiated hepatocyte like cells were less mature compared to primary hepatocytes. This indicates that some inherent roadblocks need to be overcome for them to be comparable to their primary counterparts. To further understand the potential barriers for the in vitro differentiation to mature hepatocytes, data from embryonic liver development stage E9.5 to adult liver were compiled for comparative meta-analysis. The in vivo mouse liver development paths and the in vitro hepatic differentiation paths were aligned and all the human hepatocyte like cells were found to be more representative of the fetal stages of liver development. This analysis provided insights into the key regulators of liver development and enabled identification of potential roadblocks and targets for genetic intervention to increase the functional maturity of stem cell derived hepatocytes.

Cross species meta-analysis of transcriptome data to unveil the road block of stem cell differentiation to hepatocytes

249th National Meeting (2015)

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