Maturation of Induced Pluripotent Stem Cell Derived Hepatocytes by 3D-Culture

Abstract

Induced pluripotent stem cell derived hepatocytes (IPSC-Heps) have the potential to reduce the demand for a dwindling number of primary cells used in applications ranging from therapeutic cell infusions to in vitro toxicology studies. However, current differentiation protocols and culture methods produce cells with reduced functionality and fetal-like properties compared to adult hepatocytes. We report a culture method for the maturation of IPSC-Heps using 3-Dimensional (3D) collagen matrices compatible with high throughput screening. This culture method significantly increases functional maturation of IPSC-Heps towards an adult phenotype when compared to conventional 2D systems. Additionally, this approach spontaneously results in the presence of polarized structures necessary for drug metabolism and improves functional longevity to over 75 days. Overall, this research reveals a method to shift the phenotype of existing IPSC-Heps towards primary adult hepatocytes allowing such cells to be a more relevant replacement for the current primary standard.

Requirements for human iPS cell-derived hepatocytes as an alternative to primary human hepatocytes for assessing absorption, distribution, metabolism, excretion and toxicity of pharmaceuticals

“…There are many reports on the improvements of hepatic functions in hES/iPSC-derived hepatocytes by threedimensional culture systems or spheroids (Gieseck et al, 2014;Nagamoto et al, 2012;Takayama et al, 2013;Sengupta et al, 2014). Further studies are needed to find the best combination of these culture methods and cell types and improve the functions of hiPSC-derived hepatocytes.…”

Induction of Hepatocyte Differentiation in Human Pluripotent Stem Cells

“…The most important ECM component was determined to be type 1 collagen. In a separate study, 3D culturing promoted the maturation of hepatocytes derived from hiPS cells [109] . For this culturing technique, cells are grown in hollow fibers similar to embryoid bodies.…”

Human hepatocytes derived from pluripotent stem cells: a promising cell model for drug hepatotoxicity screening

“…However, one of the remaining questions that arises when using HLCs derived from PSCs is the stability of their phenotype and function in long-term cultures, a problem commonly encountered with cultured adult hepatocytes (Schwartz et al 2014). Hence, distinct strategies to improve and stabilize the hepatic phenotype of derived HLCs, such as employing complex 3D culture systems (spheroidal aggregates, perfused bioreactors) (Sivertsson et al 2013;Gieseck et al 2014;Sengupta et al 2014;Subramanian et al 2014;Takayama et al 2014;Yao et al 2014;Kim et al 2015) or using different extracellular matrices (Hay et al 2011), have been promoted.…”

“…Despite the fact that distinct attempts to differentiate iPSCs into HLCs have been made, they still differ from adult hepatocytes (Baxter et al 2015). As with ESCs, their culture in 3D (Takayama et al 2013;Gieseck et al 2014), and other strategies such as their co-culture with Sjogren et al (2014) murine embryonic fibroblasts , has been described to improve their performance. Human iPSCs provide a unique opportunity to generate live cellular models of patient-specific diseases for personalized cell therapy and for screening candidate pharmacological molecules (Dianat et al 2013).…”