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Development of a method of passaging and freezing human iPS cell-derived hepatocytes to improve their functions [1]

['Jumpei Inui', 'Laboratory Of Biochemistry', 'Molecular Biology', 'Graduate School Of Pharmaceutical Sciences', 'Osaka University', 'Osaka', 'Yukiko Ueyama-Toba', 'Laboratory Of Functional Organoid For Drug Discovery', 'National Institute Of Biomedical Innovation', 'Health']

Date: 2023-06

Human induced pluripotent stem (iPS) cell-derived hepatocyte-like cells (HLCs) are expected to replace primary human hepatocytes as a new source of functional hepatocytes in various medical applications. However, the hepatic functions of HLCs are still low and it takes a long time to differentiate them from human iPS cells. Furthermore, HLCs have very low proliferative capacity and are difficult to be passaged due to loss of hepatic functions after reseeding. To overcome these problems, we attempted to develop a technology to dissociate, cryopreserve, and reseed HLCs in this study. By adding epithelial-mesenchymal transition inhibitors and optimizing the cell dissociation time, we have developed a method for passaging HLCs without loss of their functions. After passage, HLCs showed a hepatocyte-like polygonal cell morphology and expressed major hepatocyte marker proteins such as albumin and cytochrome P450 3A4 (CYP3A4). In addition, the HLCs had low-density lipoprotein uptake and glycogen storage capacity. The HLCs also showed higher CYP3A4 activity and increased gene expression levels of major hepatocyte markers after passage compared to before passage. Finally, they maintained their functions even after their cryopreservation and re-culture. By applying this technology, it will be possible to provide ready-to-use availability of cryopreserved HLCs for drug discovery research.

Although HLCs are expected to be utilized for pharmaceutical applications, several issues remain. The first is that HLCs have low drug-metabolizing enzyme activities [ 10 ]. They are reported to be similar to fetal hepatocytes rather than adult hepatocytes [ 11 ]. Several groups have tried to sort high-functioning HLCs from their low-functioning counterparts using hepatocyte-specific cell surface markers such as asialoglycoprotein receptor 1 (ASGR1) [ 12 ] and sodium taurocholate cotransporting polypeptide (NTCP) [ 13 ]. However, the utility of the sorted HLCs is limited, because they tend to lose their functions after reseeding. We previously succeeded in expressing a neomycin resistance gene downstream of the CYP3A4 gene in HLCs, which allowed purification of only high-functioning HLCs expressing CYP3A4 by neomycin selection. However, this approach necessitated the use of genome editing to introduce the neomycin resistance gene to human iPS cells [ 14 ]. In addition, the span of about a month is required to differentiate HLCs from human iPS cells. Many researchers have developed alternative hepatocyte differentiation protocols from human iPS cells [ 15 – 18 ], but most of them have not been able to shorten the culture period. Finally, HLCs have a very low proliferative capacity and are difficult to be passaged due to their loss of hepatic functions after reseeding. Due to these problems, HLCs have not replaced PHHs in pharmaceutical research. Development of a method to overcome these barriers would facilitate the wider use of HLCs.

Primary (cryopreserved) human hepatocytes (PHHs) are the main cell source used for preclinical in vitro studies of drug metabolism and disposition. However, PHHs have some issues, such as lot-to-lot variability, limited supply, and loss of hepatic functions in culture. Human induced pluripotent stem (iPS) cell-derived hepatocyte-like cells (HLCs) are expected to replace PHHs as a new stable source of functional hepatocytes. Many studies have been conducted to generate HLCs for drug discovery research. For example, disease models in vitro have been established using HLCs differentiated from patient-derived iPS cells [ 1 – 3 ]. Human iPS cells derived from individuals with single nucleotide polymorphisms (SNPs) in cytochrome P450 (CYP) 2D6 recapitulated the poor metabolizer phenotype by differentiation into HLCs [ 4 ]. In addition, genome editing technology successfully recapitulated the phenotypes of poor metabolizers in HLCs and revealed the contribution of specific CYP enzymes in pharmacokinetics [ 5 , 6 ]. Moreover, hepatocyte transplantation technologies using HLCs sheets [ 7 ], spheroids [ 8 ], and organoids [ 9 ] have been developed as an alternative to living donor liver transplantation.

Results

Effects of passage on the functions of HLCs First, we examined whether it was indeed impossible to passage and re-culture HLCs. For this purpose, we prepared HLCs according to our previously reported method [4], and then passage-culture of HLCs as shown in Fig 1A. Cell morphology of HLCs was observed by phase-contrast microscopy before passage and again 7 days after passage. The gene expression levels of hepatocyte markers (albumin, ALB; hepatocyte nuclear factor 4 alpha, HNF4α; cytochrome P450 3A4, CYP3A4) were analyzed by real-time RT- PCR. After passage, HLCs lost their polygonal morphology and showed a fibroblast-like morphology (Fig 1B). The gene expression levels of hepatocyte markers were significantly lower in HLCs after passage than in those before passage. The ALB expression level in HLCs after passage decreased by about 50-fold compared to that before passage (Fig 1C). The gene expression level of CYP3A4 increased in HLCs after passage, while the expression levels of CYP2C9 and CYP2C19 did not change significantly (Figs 1C and S1). These results suggested that HLCs lost their functions after passage. PPT PowerPoint slide

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TIFF original image Download: Fig 1. Effect of passage on HLCs. Human iPS cells (Tic) were differentiated into hepatocyte-like cells (HLCs) as described in the Materials and Methods section. (A) The schematic overview shows the protocol for hepatic differentiation and passage of HLCs. (B) Phase-contrast micrographs of HLCs before passage and HLCs cultured for 7 days after passage are shown. (C, D) The gene expression levels of hepatocyte (C) (ALB, HNF4α, CYP3A4), mesenchymal (D) (SNAI1 and Fibronectin) or epithelial (E) (E-CAD) markers were examined in HLCs before passage and HLCs cultured for 7 days after passage by real-time RT-PCR. The gene expression levels in HLCs before passage were taken as 1.0. Data represent the means ± SD (n = 3). Statistical significance was evaluated by unpaired two-tail Student t test (*p < 0.05, **p < 0.01, ***p < 0.005: compared with “before passage”). https://doi.org/10.1371/journal.pone.0285783.g001 To identify the cause of the loss of hepatic functions in HLCs following the passage operation, we analyzed the differences in gene expression in HLCs between before and after passage in more detail. The gene expression levels of mesenchymal cell markers (snail family transcriptional repressor 1, SNAI1; Fibronectin) increased, while those of epithelial cell marker (E-cadherin, E-CAD) decreased in HLCs after passage (Fig 1D). Thus, the passage and re-culture might cause epithelial-mesenchymal transition (EMT) in HLCs and thereby eradicate their functions as hepatocytes.

Effects of EMT inhibitors on the functions of HLCs after passage We next attempted to improve the hepatic functions in HLCs after passage by inhibiting EMT. We examined the effects of a MEK inhibitor PD0325901 (P), a TGFβ inhibitor SB431542 (S), and a ROCK inhibitor Y-27632 (Y), which have previously been reported to inhibit EMT [19, 20]. Following the protocol shown in Fig 2A, the functions in HLCs after passage were examined when these compounds were administered alone or in combination. The results showed that the combined treatment of P, S and Y (PSY group) improved the cell morphology of HLCs after passage (Fig 2B) and increased hepatocyte marker gene expression (Fig 2C). In addition, the gene expression levels of mesenchymal cell markers (SNAI1, Fibronectin) decreased, while those of the epithelial cell marker (E-CAD) increased in the PSY group compared to the control group (S2A Fig). The PSY treatment itself did not greatly increase the gene expression levels of hepatocyte markers in the HLCs without passage (S2B Fig). These results suggested that PSY treatment could improve hepatic functions in HLCs after passage by inhibiting EMT. On the other hand, the improvement of cell morphology by PSY treatment was not sufficient, and the gene expression levels of the hepatocyte markers (ALB, HNF4α) were still low (Fig 2C), suggesting that further improvement of the passage manipulation was needed. PPT PowerPoint slide

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TIFF original image Download: Fig 2. Effects of small molecule compounds on HLCs after passage. Human iPS cells (Tic) were differentiated into hepatocyte-like cells (HLCs) as described in the Materials and Methods section. (A) The schematic overview shows the protocol for passage of HLCs. HLCs were passaged and cultured for 7 days with vehicle control (DMSO), with a single EMT inhibitor (P, 0.5 μM PD0325901; S, 2 μM SB43154; Y, 10 μM Y-27632), and a combination of EMT inhibitors (PS, PY, SY, PSY). (B) Phase-contrast micrographs of HLCs before passage and HLCs after passage cultured with each condition are shown. (C) The gene expression levels of hepatocyte markers (ALB, HNF4α, CYP3A4) were examined in HLCs before passage and HLCs after passage cultured with each condition for 7 days by real-time RT-PCR. The gene expression levels in HLCs before passage were taken as 1.0. Data represent the means ± SD (n = 3). Statistical significance was evaluated by one-way ANOVA followed by Tukey’s post-hoc tests to compare all groups. Groups that do not share the same letter are significantly different from each other (p<.05). https://doi.org/10.1371/journal.pone.0285783.g002

Optimum cell-dissociation time for passage of HLCs Passage of HLCs required 1 hr of cell dissociation time to recover as many cells as possible. This was a longer dissociation time than typically required for ordinary cell lines. Because there was a risk that the HLCs would be injured by the cell dissociation enzyme, resulting in decreased hepatic functions after passage, we investigated the optimum cell dissociation time for passage. Following the protocol shown in Fig 3A, individual HLCs were recovered after four different cell-dissociation times. Then the number of recovered cells and cell viability under each condition were evaluated. As the cell dissociation time shortened, the recovery of HLCs and cell viability decreased, although there was no significant difference in the cell viability (Fig 3B and 3C). Next, we passaged the HLCs under each dissociation time and cultured them to evaluate the cell morphology and the gene expression levels of hepatocyte markers. Cell morphology was worse in the 60 min of dissociation (60-min dissociation group), but greatly improved after 15 min of dissociation (15-min dis sociation group). The morphology in the 15-min dissociation group was similar to that before passage (Fig 3D). With the shortening of cell dissociation time, the gene expression levels of hepatocyte markers in HLCs after passage increased (Figs 3E and S3). This trend was also observed in another human iPS cell line, the DOO line (S4 Fig). Note that the experiment could not be performed in the 5-min dissociation group because the number of cells recovered was very small. These results suggested that shortening the cell dissociation time suppressed the decline of hepatic gene expressions, while long duration of the cell dissociation might damage HLCs and decreased hepatic functions. We further examined which cell dissociation enzyme was the most suitable for passage of HLCs. When the TrypLE Select was used, the cells showed the highest gene expression levels of hepatocyte markers and the highest cell recovery (S5 Fig). Therefore, we decided to use 15-min dissociation with TrypLE Select for HLCs passage. PPT PowerPoint slide

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TIFF original image Download: Fig 3. Optimum cell-dissociation time for passage of HLCs. Human iPS cells (Tic) were differentiated into hepatocyte-like cells (HLCs) as described in the Materials and Methods section. (A) The schematic overview shows the protocol for passage of HLCs. HLCs were passaged under four different cell dissociation times. (B) The number of dissociated cells per cell culture area was counted at different dissociation times. (C) Cell viability was calculated under different dissociation times. (D) Phase-contrast micrographs of HLCs before passage and HLCs cultured for 7 days after passage under different dissociation times. (E) The gene expression levels of hepatocyte markers (ALB, HNF4α, CYP3A4) were examined in HLCs before passage and HLCs cultured for 7 days after passage under different dissociation times by real-time RT-PCR. The gene expression levels in HLCs before passage were taken as 1.0. Data represent the means ± SD (n = 3). Statistical significance was evaluated by one-way ANOVA followed by Tukey’s post-hoc tests to compare all groups. Groups that do not share the same letter are significantly different from each other (p<.05). https://doi.org/10.1371/journal.pone.0285783.g003

Evaluation of HLCs passaged under the optimized passage conditions Based on the results shown in Figs 1–3, we have developed a passage method for HLCs (Fig 4A). In this protocol, the cell dissociation time was shortened, and PSY were added as EMT inhibitors. Various analyses were performed on HLCs cultured for 7 days after passage under this condition. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Characteristics of HLCs passaged under optimized passage conditions. Human iPS cells (Tic) were differentiated into hepatocyte-like cells (HLCs) as described in the Materials and Methods section. (A) The schematic overview shows the protocol for passage of HLCs. (B) The CYP3A4 activity was examined in HLCs before passage, HLCs cultured for 7 days after passage and PHHs cultured for 48 hours (PHH 48hr). Statistical significance was evaluated by one-way ANOVA followed by Tukey’s post-hoc tests to compare all groups. Groups that do not share the same letter are significantly different from each other (p<.05). (C) The expression of the hepatocyte markers (ALB, HNF4α, CYP3A4, αAT) in HLCs before and after passage was examined by immunohistochemistry. (D) LDL uptake was examined in HLCs before and after passage by Alexa-488-labeled LDL. (E) Cytoplasmic accumulation of glycogen was determined in HLCs before and after passage by PAS staining. (F) Comparative analysis of the gene expression levels of major hepatocyte markers was performed between HLCs before passage, HLCs after passage, PHHs cultured for 48 hours (PHH 48hr) and HepG2 cells. The gene expression levels of hepatocyte markers were examined by real-time RT-PCR. Heatmap was generated using Morpheus (https://software.broadinstitute.org/morpheus) by comparing and normalizing the gene expressions (ΔΔCt) between HLCs before passage, HLCs after passage, PHHs cultured for 48 hours (PHH 48hr) and HepG2 cells. https://doi.org/10.1371/journal.pone.0285783.g004 The activity of CYP3A4, a major drug-metabolizing enzyme expressed in human hepatocytes, was evaluated in HLCs before and after passage. The results showed that CYP3A4 activity in HLCs after passage was higher than that in PHHs cultured for 48 hr (PHH 48hr) or in HLCs before passage (Fig 4B). Therefore, it was suggested that the drug-metabolizing enzyme activity in HLCs was enhanced by use of the appropriate passage method. Immunofluorescence staining confirmed that hepatocyte marker proteins (ALB, HNF4α CYP3A4, and αAT) were expressed in HLCs after passage as well as before passage (Fig 4C). FACS analysis was performed to further investigate the levels of ALB and CYP3A4 protein expression in HLCs after passage. The results showed that the percentage of ALB- or CYP3A4-positive cells increased in the HLCs after passage compared to that before passage, suggesting that the HLCs matured into more highly functional hepatocytes by passage (S6 Fig). The capacity of low-density lipoprotein (LDL) uptake in HLCs was evaluated using fluorescently labeled LDL. Intracellular uptake of LDL was observed in HLCs after passage, indicating that they maintained LDL uptake capacity after passage (Fig 4D). PAS (periodic acid-Schiff) staining was performed to evaluate the glycogen storage capacity of HLC. As before passage, HLCs after passage were stained by PAS, suggesting that they had glycogen storage capacity (Fig 4E). We further compared the gene expression levels of major hepatocyte markers in HLCs after passage with those in PHHs, HLCs before passage and those in HepG2 cells (Fig 4F). The gene expression levels of most markers in HLCs after passage were comparable or higher than those in HLCs before passage. In particular, the gene expression levels of the ALB and CYP genes (2B6, 2C9, 2C19, 3A4) in HLCs after passage were higher than those in PHH 48hr. We confirmed that expression levels of many hepatocyte marker genes in HLCs were more increased by passage comparing to extended culture without passage (S7 Fig). These results indicated that the passage method developed in the present study not only maintained hepatic functions, but also upregulated hepatic gene and protein expression and CYP3A4 activity in HLCs.

Extended culture of HLCs after passage We also examined whether the hepatic functions in HLCs after passage could be maintained for a longer period. Following the protocol shown in Fig 5A, we examined the cell morphology and the gene expression levels of hepatocyte markers in HLCs after passage. The results showed that the HLCs after passage maintained their polygonal morphology for 15 days (Fig 5B). The expression levels of ALB, HNF4α and CYP3A4 peaked at 3 days after passage (Fig 5C). Thereafter, their expression levels showed a decreasing trend, but remained at the same or higher levels as compared with those before passage until 15 days after passage (Fig 5C). These results suggested that HLCs maintained higher hepatic functions for at least 2 weeks after passage. PPT PowerPoint slide

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TIFF original image Download: Fig 5. Extended culture of HLCs after passage. Human iPS cells (Tic) were differentiated into hepatocyte-like cells (HLCs) as described in the Materials and Methods section. (A) The schematic overview shows the protocol for passage of HLCs. (B) Phase-contrast micrographs of HLCs before passage and HLCs cultured for 3 to 15 days after passage. (C) The gene expression levels of hepatocyte markers (ALB, HNF4α, CYP3A4) were examined in HLCs before passage and HLCs cultured for 3 to 15 days after passage by real-time RT-PCR. The gene expression levels in HLCs before passage were taken as 1.0. Data represent the means ± SD (n = 3). Statistical significance was evaluated by one-way ANOVA followed by Tukey’s post-hoc tests to compare all groups. Groups that do not share the same letter are significantly different from each other (p<.05). https://doi.org/10.1371/journal.pone.0285783.g005

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