Poster L2 - Impacts of Microenvironmental Modulation on Liver Cell Fate

Friday, October 25, 2024

10:00 AM – 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

KZ

Katie Zobus

Undergraduate Student
University of Illinois, Urbana-Champaign
Sherman, Illinois, United States

Co-Author(s)

BG

Brock Grenci

Graduate Research Assistant
University of Illinois Urbana-Champaign, United States

Primary Investigator(s)

GU

Gregory Underhill

Associate Professor of Bioengineering
University of Illinois Urbana Champaign, United States

Co-Author(s)

IJ

Ishita Jain, PhD

Postdoctoral Fellow
Stanford University, United States

Introduction: Induced pluripotent stem cells (iPSCs) have gained popularity for their potential to differentiate into various cell types, including hepatocytes, the primary cell type in the liver. The liver is the site of detoxification, making it an attractive model for drug screening, but primary liver tissue is in short supply. iPSCs are a promising solution to address the scarcity of these tissues and can come from a human directly, allowing for patient-specific studies or treatments. Organoids are three-dimensional tissues that can be derived from culturing stem cells in an attempt to better replicate differentiation and organ development in the body. Despite advances in organoid research, replicating differentiation accurately is difficult in vitro, barring organoids from being used as accurate models. To improve stem cell-derived liver cultures for disease modeling and drug screening, our research aims to develop well-defined, high-throughput 2D and 3D culture systems. This study has demonstrated the importance of extracellular matrix (ECM) modulation and how additional growth factors direct cell fate in the 2D microarray system. These results guide our research for the 3D microwell platform, allowing us to infer optimal microenvironmental conditions alongside seeding density and organoid size and geometry for the maturation and development of liver organoids. These approaches will facilitate the study of liver cell fate and provide more uniform platforms for drug screening and disease modeling. This research aims to create a robust and scalable platform for generating well-defined liver organoids that can be used for a wide range of applications in regenerative medicine.

Materials and

Methods: Cellular microarrays are fabricated by printing extracellular matrix (ECM) proteins onto polyacrylamide (PA) hydrogels with defined stiffnesses. ECM and growth factor combinations were used to study liver stem cell fate. hiHepatoblasts were seeded onto the microarrays and then differentiated into hepatocytes or cholangiocytes. The 3D microwell system was created using a polyethylene glycol (PEG) solution crosslinked with photoinitiator Irgacure, then added dropwise to a PDMS mold. hiHepatoblasts were then seeded into the system and further differentiated into hepatocytes or cholangiocytes, forming 3D tissues. Encapsulation of the organoid in the microwell was performed using a PEG-maleimide solution and a thiol crosslinker, allowing for ECM protein modulation. Immunohistochemistry was used to determine cell fate, patterning, and cell-cell interactions. Confocal microscopy was used to capture images, demonstrating the results of both platforms.

Results, Conclusions, and Discussions: Our study investigated the effects of different extracellular matrix (ECM) compositions and growth factor (GF) combinations on the differentiation of liver stem cells into cholangiocytes and hepatocytes, both key liver cell types. Figure 1 displays the impact on liver stem cell fate in response to different GF treatments and ECM combinations. In the 2D culture system, the combination of HGF and EGF significantly increased SOX9 expression, indicating an increase in cholangiocytic differentiation (Figure 1A). These results convey that these GFs synergistically promote cholangiocyte development, possibly due to the activation of signaling pathways that favor this differentiation. Conversely, hepatocytic differentiation, evidenced by HNF4α expression, was more influenced by ECM and less pronounced with the addition of growth factors (Figure 1B). Particularly, the ECM composition of collagen 3 (C3) exhibited the most substantial increase in HNF4α staining intensity.
For the 3D microwell system, CK19 (green) and HNF4α (red) were used as biomarkers for cholangiocytic and hepatocytic differentiation, respectively (Figure 2). Figure 2 demonstrates the effects of encapsulation and seeding times on liver cell differentiation. Figure 2C illustrates the importance of seeding timepoint optimization for organoid longevity, where cells seeded at an earlier stage (Figure 2B) express less HNF4α expression. Figure 2C also exhibits how mechanical forces can spatially dictate cell fate, with biliary cells lining the organoid and hepatocytic patterning in the organoid’s interior. Similar to the 2D system, the 3D platform was evaluated under various ECM combinations for its role in liver stem cell differentiation (Figure 3). Encapsulation with collagen I (C1) significantly increased the staining intensity of both CK19 and HNF4α when compared to fibronectin (FN) or no ECM modification (Figure 3). This finding suggests that C1 provides a supportive environment for both cholangiocyte and hepatocyte differentiation, likely strengthening cell-matrix interactions and mechanical signaling.
We deduced that while ECM components strongly influence hepatic fate, GFs may play a more pivotal role in biliary specification. Furthermore, the high throughput screening developed for the 2D platform will guide future 3D experimental designs. The preliminary results propose that optimizing ECM composition and GF combinations could aid in developing robust liver organoid models.

Acknowledgements (Optional): I would like to acknowledge and thank Brock Grenci, Dr. Ishita Jain, and Dr. Gregory Underhill for their guidance and support throughout this research project. I would also like to thank the Henry Luce Foundation through the Illinois Scholars Undergraduate Research Program for providing me with the opportunity and support to participate in this project.