Primary human hepatocytes (PHHs) represent a scarce resource to build human liver models due to shortages of donor livers and thus these cells are not suitable for high-throughput drug screening. Induced pluripotent stem cell-derived human hepatocyte-like cells (iPSC-hHs) can address the aforementioned limitations of PHHs, allowing for the creation of genetically diverse donor panels to help elucidate inter-individual variations in drug response and disease progression. However, we and others have shown that iPSC-hHs need to be further differentiated towards the adult PHH phenotype. In vivo, a complex extracellular matrix (ECM) and a complex stromal compartment with multiple cell types modulate the hepatic phenotype. However, systematic evaluation of these cues on iPSC-hH functional maturation has not been evaluated to any considerable de- gree. It is well known that 3D microenvironments mimic human physiology better than 2D culture formats for many cell types (i.e. cancer, liver). Our main hypothesis is that a 3D microenvironment, which contains com- plex liver-inspired ECM coupled with key liver stromal cell types, will significantly differentiate iPSC-hHs to- wards the adult PHH phenotype. The challenge to testing this hypothesis using bulk hydrogels is that they are too labor/time intensive and costly to produce for high throughput exploration of optimal culture conditions. Thus, here we will utilize a high-throughput microtissue technology (>45,000 protein-based uniform microtis- sues per hour) to explore the aforementioned cues, which will allow us to have a large numbers of microtissues in each condition (>75) to obtain high statistical power in the results.
In aim 1, we will investigate differentiated functions of iPSC-hHs in 3D microtissues of varying ECM compositions, while in aim 2, we will investigate dif- ferentiated functions of iPSC-hHs in co-culture with a complex liver-like stromal compartment in 3D microtis- sues with optimal ECM. Thus, our studies will create the first high-throughput 3D iPSC-hH / stromal co-culture platform with tunable ECM microenvironment, which can be used to investigate the chronic impacts of various stimuli (i.e. differentiation cues, chemicals, viruses, implantation sites for therapies) on liver functions.

Public Health Relevance

Induced pluripotent stem cell-derived human hepatocyte-like cells (iPSC-hHs) can address the sourcing limita- tions of primary human hepatocytes for testing drug toxicity in a high-throughput setting as well as allow the creation of genetically diverse donor panels to help elucidate inter-individual variations in drug response and disease progression; however, it is well known that iPSC-hHs need to be further differentiated further towards the adult hepatic phenotype. Here, we will use a high-throughput 3D microtissue technology to explore with statistical power the effects of complex extracellular matrix and a complex liver-like stromal compartment on functional maturation of iPSC-hHs over several weeks. Our high-throughput 3D iPSC-hH / stromal co-culture platform with tunable ECM microenvironment could be used to investigate the chronic impacts of various stimuli (i.e. differentiation cues, chemicals, viruses, implantation sites for therapies) on liver functions.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21ES027622-01
Application #
9226831
Study Section
Hepatobiliary Pathophysiology Study Section (HBPP)
Program Officer
Reinlib, Leslie J
Project Start
2017-04-01
Project End
2019-03-31
Budget Start
2017-04-01
Budget End
2018-03-31
Support Year
1
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of Illinois at Chicago
Department
Biomedical Engineering
Type
Schools of Medicine
DUNS #
098987217
City
Chicago
State
IL
Country
United States
Zip Code
60612
Underhill, Gregory H; Khetani, Salman R (2018) Bioengineered Liver Models for Drug Testing and Cell Differentiation Studies. Cell Mol Gastroenterol Hepatol 5:426-439.e1
Ware, Brenton R; Durham, Mitchell J; Monckton, Chase P et al. (2018) A Cell Culture Platform to Maintain Long-term Phenotype of Primary Human Hepatocytes and Endothelial Cells. Cell Mol Gastroenterol Hepatol 5:187-207
Meseguer-Ripolles, Jose; Khetani, Salman R; Blanco, Javier G et al. (2017) Pluripotent Stem Cell-Derived Human Tissue: Platforms to Evaluate Drug Metabolism and Safety. AAPS J 20:20