A Systems Approach to Studying the Differentiation of Stem Cells Towards Hepatocytes

Abstract

The recent advancements in stem cell biology have allowed for new and exciting opportunities to use stem cells in clinical and industrial applications. Stem cells have the unique ability to self-renew and differentiate into any specialized cell type found in the body. Using certain mechanical and biochemical cues, stem cells can be directed to become any specific cell type, such as hepatocytes. A robust and efficient process for expansion and differentiation to generate large quantities of functional hepatocytes from stem cells will be essential to establishing a stem cell bioprocess in the future for therapeutic and industrial applications of hepatocytes. In this study, a differentiation protocol with soluble growth factors and cytokines was used to mimic the key signaling cues during embryonic development. However, most directed differentiation processes have run into issues with limited scalability and lack of functionality in the differentiated cells. In an effort to bring stem cell therapy closer to reality, our strategy was to use a systems-based approach to enhance the quality and yield of stem cell-derived hepatocytes. To achieve higher cell yield, we modified an existing differentiation protocol to incorporate a cell expansion stage to facilitate simultaneous differentiation and cell growth. Using transcriptome analysis and mass cytometry, we showed how the population of cells changed over time on both the transcript and protein level. Both analyses revealed that with the new expansion stage, we obtained a higher quantity of hepatocytes within the same time frame compared to the conventional method of differentiation. We then showed the capability to scale up our differentiation for larger scale cultures by adapting the expansion stage onto Cytodex 3 microcarriers. Using the same culture volume as a tissue plate culture, we demonstrated the ability to achieve up to a 5-fold increase in cell number with a final cell density in the range of 4-5x106 cells/ml. These strategies show that the demand for large quantities of hepatocytes can be met by translating the conventional method of differentiation to suspension microcarrier differentiation. Encouraged by our ability to yield higher cell density using microcarrier culture, we explored assessing the functional maturity of our stem cell-derived hepatocytes using transcriptome analysis. We showed that stem cell-derived hepatocytes are still clearly different when compared to primary hepatocytes at the transcriptome level. In addition to evaluating cells using transcriptome analysis, we wanted to be able to compare the current in-vitro processes to embryonic liver development to understand the genetic roadblocks. The transcriptome data from hESCs hepatocyte differentiation was integrated with mouse liver development using principal component analysis and batch corrections. This allowed us to create a unified developmental scale to compare samples from different species and in-vitro to in-vivo platforms. The meta-analysis revealed that stem cell-derived hepatocytes are equivalent to the functional maturity of developing cells at E15 in mouse development. From the transcriptome analysis, we observed many different genes in energy metabolism with dynamic behavior over the course of differentiation. We sought to understand the effect of changes in different metabolic genes and the impacts on metabolic transition during differentiation. We characterized the energy metabolism of hESCs and assessed the metabolic demand of cells at different stages of differentiation. hESCs and early differentiated cells exhibited a high glycolytic flux. transitioning towards an oxidative metabolism as the differentiation progressed. Furthermore, using confocal microscopy, we also characterized the activity and morphology of the mitochondria in the cells at different stages of differentiation. Using the consumption rates of different nutrients as an input to our metabolic flux model along with our transcriptome findings, we were able to gain a deeper understanding of the metabolic behavior of cells during differentiation. Our analysis revealed that cells consume lower amounts of glucose over the course of the differentiation but become more efficient at transporting pyruvate into the mitochondria leading to increased oxidative phosphorylation. However, our metabolic and transcriptome data revealed that our stem cell-derived hepatocytes are not capable of mature metabolic functions such as gluconeogenesis, supporting the immature phenotype that has been described in literature. Together, these studies reveal that stem cells can provide a renewable and scalable source of hepatocytes for therapeutic applications. These cells demonstrate some phenotypic and functional properties of primary hepatocytes but have some contrasting elements compared to their in-vivo counterparts that will need further genetic intervention to enhance their maturation before cellular therapy can become a reality. However, this work is invaluable as it contributes to the current status of the field and facilitates the translation of laboratory practices of stem cell culture into a scalable technology.