QualiStem® IPS Cell Hepatocyte Differentiation Kit

Differentiation of iPSCs to HLCs Driven by Small Molecules (SMs)

HLCs were differentiated from iPSCs using a protocol that only involves SMs. The protocol comprises of three phases (Fig. 1a). In the first phase, the cells were treated for 24 h with 3 μM CHIR99021, a potent and highly selective GSK-3β inhibitor, followed by 24 h of non-directed differentiation. In the second phase, the cells were treated with 1% DMSO for 5 days to specify a hepatic fate. In the final phase, cells were further treated for 10 days with dihexa, a potent and stable HGF receptor agonist, in combination with dexamethasone, a glucocorticoid mimetic, for hepatocyte maturation.

Fig. 1. a Schematic diagram showing the three-phase differentiation process. b Representative images showing sequential morphological changes at each stage of the differentiation. c Representative immunocytochemistry images demonstrating the expression of key markers at each stage of the differentiation (Gao, X. et al., 2020). ME, mesendoderm; DE, definitive endoderm; HP, hepatic progenitor; HLC, hepatocyte-like cell.

Hepatic Functions of HLCs Derived by SMs

One of the key functions of hepatocytes is the production of serum proteins. ELISA results showed that HLC_SM secreted both ALB and fibronectin into the medium at levels comparable to HLC_GF, with ALB higher for HLC_GF but fibronectin higher for HLC_SM (Fig. 2a). Ureogenic capacity is another indicator of functional liver cell. HLC_SM showed increased synthesis and release of urea into the culture medium upon incubation with NH4Cl, with levels significantly higher than HLC_GF (Fig. 2b). Another important function of hepatocytes is glycogen storage. PAS staining of HLC_SM displayed extensive cytoplasmic staining (pink to purple) indicative of glycogen storage, at levels similar to those in HLC_GF (Fig. 2c). Hepatocytes are capable of clearing xenobiotics via metabolism through cytochrome P450 (CYP) isoenzymes. Therefore, CYP activity is the most critical attribute of hepatocytes for toxicological applications. We used LC/MS to assess the basal activity of major CYP isoenzymes in HLC_SM. The result showed that HLC_SM had activity levels on par with HLC_GF for CYP1A, CYP2B6, CYP2C9, CYP2D6, and CYP3A (Fig. 2d).

Fig. 2. Functional analysis of hepatocyte-like cells derived using the small molecule-based protocol (HLC_SM) in comparison to those derived using the growth factor-based protocol (HLC_GF) (Gao, X. et al., 2020).