Transmembrane channel activity in human hepatocytes and cholangiocytes derived from induced pluripotent stem cells

Abstract

The initial creation of human-induced pluripotent stem cells (iPSCs) set the foundation for the future of regenerative medicine. Human iPSCs can be differentiated into a variety of cell types in order to study normal and pathological molecular mechanisms. Currently, there are well-defined protocols for the differentiation, characterization, and establishment of functionality in human iPSC-derived hepatocytes (iHep) and iPSC-derived cholangiocytes (iCho). Electrophysiological study on chloride ion efflux channel activity in iHep and iCho cells has not been previously reported. We generated iHep and iCho cells and characterized them based on hepatocyte-specific and cholangiocyte-specific markers. The relevant transmembrane channels were selected: cystic fibrosis transmembrane conductance regulator, leucine rich repeat-containing 8 subunit A, and transmembrane member 16 subunit A. To measure the activity in these channels, we used whole-cell patch-clamp techniques with a standard intracellular and extracellular solution. Our iHep and iCho cells demonstrated definitive activity in the selected transmembrane channels, and this approach may become an important tool for investigating human liver biology of cholestatic diseases.

FIGURE 1

Generation and characterization of hepatocytes from human‐induced pluripotent stem cells (iPSCs). (A) Schematic depiction of the protocol used to differentiate human iPSCs to human‐induced iPSCs (iHep). (B) Immunofluorescence analysis exhibiting expression of a crucial endoderm marker using an antibody that recognized SRY‐box 17 (Sox17) at day 4. (C) Messenger RNA (mRNA) analyses of the expression of hepatocyte‐specific genes: adult isoform of hepatocyte nuclear factor 4α (HNF4α), liver X receptor (LXR), UDP glucuronosyltransferase family 1 member A1 (UGT1A1), bile salt export pump (BSEP), multidrug resistance‐associated protein 2 (MRP2), acetyl‐CoA carboxylase alpha (ACC), and fatty acid synthase (FASN); and cholangiocyte‐specific genes: Sox9, HNF1β, and inositol 1,4,5‐triphosphate receptor, type 3 (ITPR3). Values are determined relative to β‐actin and presented as fold change relative to the expression in human iPSCs at day 0, which is set as 1. Error bars represent mean ± SD of three independent experiments (*p < 0.05; **p < 0.01 and ***p < 0.001). (D) Immunofluorescence analyses exhibiting expression of key hepatocyte markers at day 18, while using antibodies that recognized the adult isoform of HNF4α, albumin, and alpha‐fetoprotein (AFP). Human adult hepatocytes (hAH) and human fetal hepatocytes (hFH) were used as controls. (E) Comparison of bile acid production between iHep and human adult hepatocytes. Values per million cells. Error bars represent mean ± SD of three independent experiments (***p < 0.001). PHC, primary hepatocellular carcinoma

FIGURE 2

Generation and characterization of iPSC‐derived cholangiocyte (iCho) cells from human iPSC. (A) Schematic depiction of the protocol used to differentiate human iPSCs to iCho cells. (B) Immunofluorescence analysis exhibiting expression of a crucial endoderm marker using an antibody that recognized Sox17 at day 4. (C) mRNA analyses of the expression of cholangiocyte‐specific genes: Sox9, HNF1β, ITPR3, secretin receptor (SCTR), Cl⁻/HCO₃ anion exchanger 2 (AE2), aquaporin 1 (AQP1), cholinergic receptor muscarinic 3 (CHRM3), P2Y purinoceptor 1 (P2Y1R), and hepatocyte‐specific genes: adult isoform of HNF4α, LXR, and UGT1A1. Values are determined relative to β‐actin and presented as fold change relative to the expression in human iPSCs at day 0, which is set as 1. Error bars represent mean ± SD of three independent experiments (*p < 0.05; **p < 0.01, and ***p < 0.001). (D) Immunofluorescence analyses exhibiting expression of key cholangiocyte markers at day 23, while using antibodies that recognized cytokeratin 7 (CK7), CK19, and Sox9. Primary human cholangiocyte was used as a control. (E) Measurement of intracellular cyclic adenosine monophosphate (cAMP) in response to secretin (10 µM) and forskolin (10 µM) stimuli. Error bars represent mean ± SD of three independent experiments (*p < 0.05 and ***p < 0.001). (F) Representative images demonstrating active export of the fluorescent bile acid cholyl‐lysyl‐fluorescein (CLF) from the lumen of human iCho organoids compared with controls loaded with fluorescein isothiocyanate (FITC). Results show the fluorescence intensity in the center of organoids was normalized to background; error bars represent mean ± SD of three independent experiments (**p < 0.01). (G) Acetyl‐α‐tubulin staining revealed that the iCho cells have a primary cilium as the primary human cholangiocyte. DAPI, 4′,6‐diamidino‐2‐phenylindole

FIGURE 3

Cystic fibrosis transmembrane conductance regulator (CFTR) characterization in human iHep and iCho. (A) mRNA analysis of the expression of CFTR in iCho and iHep. Values are determined relative to β‐actin and presented as fold change relative to the expression in human iPSCs at day 0, which is set as 1. Error bars represent mean ± SD of three independent experiments (***p < 0.001). (B) Representative image of the western blot analysis and quantification of CFTR in iCho (n = 3) at day 23 and iHep (n = 3) at day 14. For normalization, glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) was used. H69 cell (n = 3) and primary adult human hepatocytes (n = 3) were used as controls (*p < 0.05 and ***p < 0.001). (C) Representative whole cell currents of iCho measured during basal conditions and in response to forskolin (10 µM) and IBMX (100 µM). Currents measured at −100 mV (black circles) and at +100 mV (red circles) are shown. (D) Currents were measured in iCho during basal (control) conditions and in response to forskolin and IBMX using STEP protocol. (E) Current‐voltage (I‐V) plot generated by STEP protocol. (F) Representative whole cell currents of iHep measured during basal conditions and in response to forskolin (10 µM) and IBMX (100 µM). Currents measured at −100 mV (black circles) and at +100 mV (red circles) are shown. (G) Currents were measured in iHeps during basal (control) conditions and in response to forskolin and IBMX using STEP protocol. (H) I‐V plot generated by STEP protocol

FIGURE 4

Leucine rich repeat‐containing 8 subunit A (LRRC8A) characterization in human iHep and iCho. (A) mRNA analysis of the expression of LRRC8A in iCho and iHep. Values are determined relative to β‐actin and presented as fold change relative to the expression in human iPSCs at day 0, which is set as 1. Error bars represent mean ± SD of three independent experiments (*p < 0.05). (B) Representative image of the western blot analysis and quantification of LRRC8A in iCho (n = 3) at day 23 and iHep (n = 3) at day 14. For normalization, GAPDH was used. H69 cell (n = 3) and primary adult human hepatocytes (n = 3) were used as controls (**p < 0.01). (C) Representative whole cell currents in iCho in response to hypotonicity (190 mOsm). Currents measured at −100 mV (black circles) and at +100 mV (red circles) are shown. (D) Currents were measured in iCho during basal (control) conditions and in response to hypotonic exposure (Hypo) using STEP protocol. (E) I‐V plot generated by STEP protocol showing control (black circles) and hypotonic‐stimulated (red circles) currents. (F) Representative whole cell currents of iHeps measured during basal conditions and in response to hypotonicity. Currents measured at −100 mV (black circles) and at +100 mV (red circles) are shown. (G) Currents were measured in iHeps during basal (control) conditions and in response to hypotonicity using STEP protocol. (H) I‐V plot generated by STEP protocol showing control (black circles) and hypotonic‐stimulated (red circles) currents

FIGURE 5

Transmembrane member 16 subunit A (TMEM16A) characterization in human iHep and iCho. (A) mRNA analysis of the expression of TMEM16A in iCho and iHep. Values are determined relative to β‐actin and presented as fold change relative to the expression in human iPSCs at day 0, which is set as 1. Error bars represent mean ± SD of three independent experiments (*p < 0.05). (B) Representative image of the western blot analysis and quantification of TMEM16A in iCho (n = 3) at day 23 and iHep (n = 3) at day 14. For normalization, GAPDH was used. H69 cell (n = 3) and primary adult human hepatocytes (n = 3) were used as controls (**p < 0.01). (C) Representative whole cell currents in iCho in response to extracellular adenosine triphosphate (ATP) (100 µM). Currents measured at −100 mV (black circles) and at +100 mV (red circles) are shown. (D) Currents were measured in iCho during basal (control) conditions and in response to ATP using STEP protocol. (E) I‐V plot generated by STEP protocol showing control (black circles) and ATP‐stimulated (red circles) currents. (F) Representative whole cell currents of iHeps measured during basal conditions and in response to ATP. Currents measured at −100 mV (black circles) and at +100 mV (red circles) are shown. (G) Currents were measured in iHeps during basal (control) conditions and in response to ATP using STEP protocol. (H) I‐V plot generated by STEP protocol showing control (black circles) and ATP‐stimulated (red circles) currents