Application of Live Mitochondria Staining in Cell-Sorting to Purify Hepatocytes Derived from Human Induced Pluripotent Stem Cells
Summary
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.
Abstract
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.
Introduction
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.
Protocol
Representative Results
Discussion
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 i…
Declarações
The authors have nothing to disclose.
Acknowledgements
This work was supported by the Ministry of Education, Culture, Sports, Science and Technology grant number [23390072].
Materials
| 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 |
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