Hepatocytes derived from pluripotent stem cells can be purified through cell sorting, using a combination of mitochondrial and activated leukocyte cell adhesion molecule (ALCAM, also known as CD166) staining.
Human embryonic stem (ES) and induced pluripotent stem (iPS) cells have potential applications in cell-based regenerative medicine for treating severely diseased organs due to their unlimited proliferation and pluripotent properties. However, differentiating human ES/iPS cells into 100% pure target cell types is challenging due to their high sensitivity to the environment. Tumorigenesis after transplantation is caused by contaminated, proliferating, and undifferentiated cells, making high-purification technology essential for the safe realization of regenerative medicine. To mitigate the risk of tumorigenesis, a high-purification technology has been developed for human iPS cell-derived hepatocytes. The method employs FACS (fluorescence-activated cell sorting) using a combination of high mitochondrial content and the cell-surface marker ALCAM (activated leukocyte cell adhesion molecule) without genetic modification. 97% ± 0.38% (n = 5) of the purified hepatocytes using this method exhibited albumin protein expression. This article aims to provide detailed procedures for this method, as applied to the most current two-dimensional differentiation method for human iPS cells into hepatocytes.
Embryonic and induced pluripotent stem cells (ES and iPS, respectively) are considered promising cell sources for regenerative therapies. However, the efficiency of differentiating these cells into specific target cell types can vary, even when using the same cell line, protocol, and experimenter1,2,3,4. This variability may be attributed to the high sensitivity of human ES/iPS cells to their environment. Therefore, it is currently difficult to consistently obtain pure target cells. To achieve highly safe regenerative medicine, it is crucial to eliminate proliferative cells and undifferentiated stem cells in therapeutic cells, and advanced purification technology for target cells is essential5,6,7.
A cell sorter is a device that instantaneously analyzes individual cells and sorts live cells of interest based on the fluorescent signal strengths, offering a promising solution. This can be accomplished through antibody staining of cell type-specific surface markers or by utilizing cell type-specific reporter gene expressions. Using this technique, there are several reports on methods for purifying pluripotent stem cell-derived cardiomyocytes8,9,10 and hepatocytes11,12. Hattori et al. developed an innovative mitochondrial purification method using a cell sorter13. Taking advantage of the fact that cardiomyocytes have high energy demands through mitochondrial activity, staining the cells with the live mitochondria-indicative dye TMRM (tetramethylrhodamine methyl ester) can be used to label and highly purify cardiomyocytes by FACS from human ES cell-derived embryoid bodies containing various cell types. The absence of tumorigenicity was confirmed by teratoma formation assays with the purified cardiomyocytes. Furthermore, Yamashita et al. unexpectedly discovered a method to purify hepatocytes from human ES cell-derived embryoid bodies by isolating fractions with high mitochondrial activity and ALCAM-positive expression14. The rationale for this method is that hepatocytes also have a relatively high number of mitochondria due to their high consumption of ATP for nutrient metabolism and detoxification15, and hepatocytes express ALCAM, a member of the immunoglobulin superfamily, which plays a role in cell adhesion and migration16.
Previous highly purifying methods for pluripotent stem cell-derived hepatocytes required genetic modifications, and non-genetic purification methods had low efficiency17. The mitochondrial non-genetic method holds merit for achieving high purity. When hepatic progenitor cells are needed, CD133 and CD13- or Dlk1-based methods18,19 can be chosen. Although the accuracy of genome editing technology has advanced, the potential risk of unforeseen genomic changes (e.g., carcinogenesis) cannot be entirely eliminated. Methods based on mitochondrial activity, without involving genetic modification, can be free from such risks.
As a result of examining various mitochondrial indicators in neonatal rat cardiomyocytes, TMRM was observed to disappear completely within 24 h, whereas other dyes remained for at least 5 days13. Moreover, it is important to note that TMRM and JC-1 did not impact cell viability when assessed with the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, while other dyes demonstrated varying effects on cell viability13. TMRM demonstrates higher safety.
To date, several two-dimensional (2D) differentiation methods have been developed as more efficient approaches for inducing differentiation compared to three-dimensional (3D) embryoid body formation. This is because step-wise differentiation-inducing compounds or cytokines can be uniformly administered to cells on a 2D plane, rather than in a 3D space. In this study, the previously reported method20 was modified to induce differentiation into human iPS cell-derived hepatocytes. Here, the details of the procedures for the up-to-date 2D differentiation and purification of hepatocytes derived from human iPS cells are described.
This study used commercially obtained human iPS cells (253G1 strain) (see Table of Materials).
1. Maintenance of human iPS cells
2. Hepatic differentiation of human iPS cells in 2D cultures
NOTE: Refer to Figure 1A for the daily schedule of hepatic differentiation. Throughout the process, use 6-well culture plates and add 2 mL of the designated wash or culture medium to each well. Maintain the cell culture condition at 100% humidity, 5% CO2, and 20% O2 at 37 °C.
3. Cell preparation for purification of human iPS cells-derived hepatocytes
4. FACS analyses and sorting of human iPS cells-derived hepatocytes
5. Immunocytochemistry for detection of human iPS cells-derived hepatocytes
The timeline of the processes inducing human iPS cells to differentiate into hepatocytes through 2D culture (Figure 1A) and representative cell features (Figure 1B) are shown. Approximately on differentiation day 12, cells began to exhibit polygonal cell shapes and round nuclei, characteristic of hepatocytes. Some hepatocytes also displayed multinucleation.
FACS analysis was performed using the cells on differentiation day 27. To identify the ALCAM+ population, comparative plots with and without the first anti-ALCAM antibody are shown in Figure 2D,E, respectively. Inducing hepatic differentiation through 2D culture, the population of hepatocytes (P2) was found to be 10.1% in this experiment, representing a significant increase from the 0.8% (data not shown) found using the 3D embryoid body formation method.
P1 and P2 cells that had been sorted were cultured on mouse embryonic fibroblasts treated with mitomycin C. P2 cells exhibited a compact, spherical colony formation, whereas P1 cells were dispersed and had a flatter morphology. Immunohistochemical staining for human nuclear antigen (hNA) and albumin confirmed the enrichment of hepatocyte-like cells in P2, as depicted in Figure 3. To assess the purity of hepatocytes in P1 and P2 cells, the ratio of albumin-positive cells to hNA-positive cells was calculated (n = 5). The results revealed that 0% of P1 cells and 97% ± 0.38% of P2 cells were derived from human iPSCs and displayed hepatocyte-like characteristics (Figure 3).
To confirm the successful sorting of hepatocytes during the FACS experiment, it is recommended to co-stain ASGR1 with its specific antibody conjugated with different fluorophores and confirm that P2 contains ASGR1-positive cells.
Figure 1: 2D Hepatic differentiation of human iPSCs. (A) The timeline for hepatic differentiation. This figure is adapted from Yamashita et al.14. (B) Morphological changes during hepatic differentiation. Scale bars = 200 µm. Please click here to view a larger version of this figure.
Figure 2: Gating strategy for sorting hepatocytes and non-hepatocytes in hepatic differentiated cells (day 27). (A) Gating the target cell population using the FSC-A vs. SSC-A plot. (B) Expanding the gated population from step (A) using the FSC-H vs. FSC-W plot and exclude doublet cells. (C) Further expanding the gated population from step (B) using the SSC-H vs. SSC-W plot and excluding doublet cells. (D) and (E) Expanding the gated population from step (C) using the FITC-A (ALCAM) vs. PE-A (TMRM) plot. (D) Stained cells with TMRM and the 2nd antibody. (E) Stained cells with TMRM and the 1st and 2nd antibodies. Please click here to view a larger version of this figure.
Figure 3: Immunohistochemistry results. Immunohistochemical detection of albumin and human nuclear antigen (hNA) in sorted P1 and P2 cells, and quantification of albumin (+) cell-fraction in hNA (+) cells. Scale bars = 100 µm. Data are represented as mean ± standard deviation (SD) (n = 5). Statistical analysis was conducted using the Student's t-test. *P < 0.05. This figure is adapted from Yamashita et al.14. Please click here to view a larger version of this figure.
Due to their functions in nutrient metabolism and detoxification, hepatocytes possess a relatively large number of mitochondria compared to other cell types15. ALCAM is a member of the immunoglobulin superfamily and plays a role in cell adhesion and migration. It is expressed in various cell types, including hepatic, epithelial, lymphocytic, myeloid, fibroblast, and neuronal cells16. By utilizing a combination of the mitochondria-based method and the ALCAM antibody, human iPS cell-derived hepatocytes were successfully identified.
While the assays to characterize the secretory function or enzyme activity of the purified hepatocytes were not performed, the mRNA expression of the CYP3A4 gene in human iPS cell-derived hepatocytes purified were verified. Furthermore, hepatocytes population with TMRM (high) and ALCAM (+) isolated from embryoid bodies derived from human ES cells, that were observed higher expressions of hepatocyte-associated gene sets (VTN, SERPINA1, CYP1A1, FGB, FGA, FGG, AFP, ALB, and APOB) compared to the ES cell-derived cardiomyocytes14.
A critical step within the protocol is to gently detach 2D hepatic differentiated cells from the culture dishes with minimal damage while ensuring the preservation of mitochondrial activity for TMRM staining. Depending on the state of differentiation, hepatocytes may exhibit resistance to detachment from the dish even with enzyme treatment, a challenge that can be effectively addressed by the addition of 0.25 mM EDTA.
Existing methods for the purification of hepatocytes via FACS involve the introduction of reporter genes downstream of promoters specific to hepatocyte-expressed genes. However, this approach carries the potential risk of side effects associated with genetic modifications. The mitochondrial method, on the other hand, eliminates such concerns. Several cell surface markers of adult or fetal liver stem/progenitor cells have been identified18, and hepatic progenitor cells derived from human pluripotent stem cells could be sorted using the CD13+CD133+ population19. On the other hand, among pluripotent stem cell-derived hepatocytes, a small fraction of the matured hepatocytes could be sorted using the ASGR1 (asialoglycoprotein receptor 1) antibody17, which is the only cell surface marker specific to hepatocytes. It was demonstrated that the population of cells exhibiting high mitochondrial and ALCAM positivity contained both populations marked with CD13+CD133+ cells and ASGR1+ cells. This suggests that high mitochondrial and ALCAM-positive populations may contain a wide range of hepatocytes with various maturities14. Consequently, this method may enable the purification of a hepatocyte population with little loss.
Analysis of mouse fetal whole viscera at embryonic day 14.5 using this method revealed the expression of ALCAM in both myocytes and hepatocytes14. Other previous studies have reported ALCAM expression in murine myocardial progenitor cells21. In the case of human pluripotent stem cell-derived cells, the cardiomyocytes were identified in ALCAM negative population. These data show that ALCAM expression in cardiac and hepatic lineages differs depending on both the species and the cell maturity stages. However, due to the higher mitochondrial fraction in cardiomyocytes compared to hepatocytes, it was actually succeeded that to separate the two cell types by FACS even from mixed cells derived from the mouse whole embryo. This suggests that the proposed method could be useful for purifying hepatocytes and cardiomyocytes from other animal species.
The hepatocyte purification technology described herein is anticipated to play a pivotal role in advancing the field of regenerative medicine by mitigating the potential risk of tumor formation associated with the transplantation of human iPS cell-derived hepatocytes. However, negation of a teratoma-forming ability in the hepatocyte population purified by this technique has not been done. Further studies for safety assurance have to be done.
Large and adherent cells such as hepatocytes are highly vulnerable to mechanical damage during FACS sorting, resulting in significant loss of viable cells. For clinical applications, it is essential to develop a new purification method that establishes a culture environment enabling the survival of hepatocytes exclusively such as lactate method for cardiomyocyte purification22.
The authors have nothing to disclose.
This work was supported by the Ministry of Education, Culture, Sports, Science and Technology grant number [23390072].
253G1 human iPS cell line | RIKEN BioResource Research Center | HPS0002 | |
4% paraformaldehyde | FUJIFILM Wako Pure Chemical Corporation | 163-20145 | |
Activin A Solution, Human, Recombinant | NACALAI TESQUE, INC. | 18585 | |
Anti-Nuclei Antibody, clone 235-1 | Chemicon | MAB1281 | Antibody against human nuclear antigen |
B-27 Supplement (50x), serum free | Thermo Fisher Scientific | 17504044 | |
BD FACSAria III | BD Biosciences | Cell sorter | |
CHIR-99021 | MedChemExpress | HY-10182 | |
Ciclosporin A | FUJIFILM Wako Pure Chemical Corporation | 035-18961 | |
Collagenase | FUJIFILM Wako Pure Chemical Corporation | 034-22363 | |
Corning Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix | Corning | 354230 | Gel-like basement membrane matrix |
CultureSure Y-27632 | FUJIFILM Wako Pure Chemical Corporation | 036-24023 | ROCK inhibitor |
Dexamethasone | FUJIFILM Wako Pure Chemical Corporation | 047-18863 | |
Dimethyl sulfoxide (DMSO) | FUJIFILM Wako Pure Chemical Corporation | 047-29353 | |
Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 | Thermo Fisher Scientific | A-11055 | |
Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 546 | Thermo Fisher Scientific | A10036 | |
Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 | Thermo Fisher Scientific | A-21206 | |
Falcon 5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap | Corning | 352235 | |
Fetal Bovine Serum | Biowest | 51820-500 | |
GlutaMAX Supplement | Thermo Fisher Scientific | 35050061 | Dipeptide L-Alanyl-L-Glutamine |
Human/Mouse/Rat/Canine ALCAM/CD166 Antibody | R&D Systems | AF1172 | |
Hydrocortisone 21-hemisuccinate sodium salt | Sigma-Aldrich | H2270 | |
iMatrix-511 silk | Nippi | 892021 | |
ImmunoBlock | KAC | CTKN001 | Blocking solution |
ITS-G Supplement(×100) | FUJIFILM Wako Pure Chemical Corporation | 090-06741 | |
Leibovitz's L-15 Medium | FUJIFILM Wako Pure Chemical Corporation | 128-06075 | |
N-Hexanoic-Try-Ile-(6)-amino Hexanoic amide (Dihexa) | Toronto Research Chemicals | H293745 | |
Polyclonal Rabbit Anti-Human Albumin | Dako | A0001 | |
Polyoxyethylene Sorbitan Monolaurate (Tween 20) | NACALAI TESQUE, INC. | 28353-85 | |
RPMI-1640 | FUJIFILM Wako Pure Chemical Corporation | 189-02025 | |
Sodium L-Ascorbate | NACALAI TESQUE, INC. | 03422-32 | |
StemFit AK02N | REPROCELL | RCAK02N | |
TBS (10x) | NACALAI TESQUE, INC. | 12748-31 | |
Tetramethylrhodamine, methyl ester (TMRM) | Thermo Fisher Scientific | T668 | |
Triton X-100 | NACALAI TESQUE, INC. | 28229-25 | |
Trypan Blue Solution | NACALAI TESQUE, INC. | 20577-34 | |
TRYPSIN 250 | Difco | 215240 | |
Tryptose phosphate broth solution | Sigma-Aldrich | T8159 |