fluorescence-activated cell sorting of hepatocytes derived from human induced pluripotent stem cells

通过线粒体-ALCAM 分选纯化人 iPSC 来源的肝细胞

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.

作者聚焦：推进从人诱导多能干细胞中纯化肝细胞用于再生医学

活线粒体染色在细胞分选中纯化人诱导多能干细胞肝细胞的应用

The scope of our research is to purify human induced pluripotent stem cell-derived hepatocytes, using without genetic modification. We aim to address the issue of the tumorigenic risk associated with the undifferentiated cells, among those differentiated from human iPS cells. One of the current experimental challenges is a low cell purity, due to the mechanical storage of sorting.We are developing a new purification method, that utilizes the unique metabolism features of hepatocytes, for large-scale purification. One non-cell sorting method for hepatic progenitor cells cannot obtain pure hepatocytes. Another one for mature hepatocytes can only treat a small fraction of hepatocytes, because of the limited expression of the marker protein.In contrast, our approach can obtain hepatocytes at different maturation levels with a higher eve. Human iPS cell-derived hepatocytes are extremely immature, and show more reduced hepatocyte macro expression after purification. Our future goal is to enhance hepatocyte modulation using organoidal technology and day-to-day advancements to regenerative medicine and drug discovery.

荧光激活的肝细胞分选来自人诱导多能干细胞的肝细胞

首先，按照制造商的说明设置荧光激活细胞分选或 FACS 机器进行细胞分选。将样品管放在FACS机器上并装入。在 P1 区域对 TMRM-high 和 ALCAM 阴性群体进行门控，以对非肝细胞进行分选。然后在 P2 区域对 TMRM 高和 ALCAM 阳性群体进行门控，以对肝细胞进行分选。将分选的细胞收集在含有肝细胞成熟培养基的 15 毫升收集管中。分拣后，从FACS机器上取下收集管。将分选的细胞悬液以 200 G 在 18 摄氏度下离心 5 分钟。除去上清液，将沉淀重新悬浮在含有10微摩尔岩石抑制剂和20纳摩尔环孢菌素A的2毫升肝细胞成熟培养基中.将细胞接种在丝裂霉素C处理的小鼠胚胎成纤维细胞上用肝细胞成熟培养基在六孔板中。将细胞在 37 摄氏度的二氧化碳培养箱中培养 5 天，然后进行免疫染色进行肝细胞纯度测试。在肝分化第 27 天进行的 FACS 分析显示 TMRM 高和 ALCAM 阳性群体。显示了分选后在小鼠胚胎成纤维细胞上培养的 P1 和 P2 细胞的免疫组织化学染色。通过计数白蛋白阳性细胞进行的纯度测试显示 P1 为 0%，约 97% 的 P2 细胞为肝细胞样细胞。

fluorescence-activated-cell-sorting-hepatocytes-derived-from-human

Research

JoVE Journal

Biology

Fluorescence-Activated Cell Sorting of Hepatocytes Derived from Human Induced Pluripotent Stem Cells

22 次观看. 首先，按照制造商的说明设置荧光激活细胞分选或 FACS 机器进行细胞分选。将样品管放在FACS机器上并装入。在 P1 区域对 TMRM-high 和 ALCAM 阴性群体进行门控，以对非肝细胞进行分选。然后在 P2 区域对 TMRM 高和 ALCAM 阳性群体进行门控，以对肝细胞进行分选。将分选的细胞收集在含有肝细胞成熟培养基的 15 毫升收集管中。分拣后，从FACS机器上取下收集管。将分选的细...

视频: 荧光激活的肝细胞分选来自人诱导多能干细胞的肝细胞

Application of Live Mitochondria Staining in Cell-Sorting to Purify Hepatocytes Derived from Human Induced Pluripotent Stem Cells

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% &plusmn; 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.

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 ...

科研

生物学

Hepatic Differentiation of Human-Induced Pluripotent Stem Cells in 2D Cultures

To begin, coat each well of a six-well plate with one milliliter of basement membrane matrix and incubate at 37 degrees Celsius for one hour. Remove the coated matrix from the plate and add human-induced pluripotent stem cells prepared in AK02N medium supplemented with 10 micromolar ROCK inhibitor. Incubate the cells at 37 degrees Celsius and 5%carbon dioxide for three days.After three days, on day zero of the initiating hepatic differentiation, wash the cells once with RPMI 1640 medium. Next, to initiate endoderm differentiation, add RPMI B27 GlutaMAX supplemented with three to six micromolar CHIR 99021 and 100 nanograms per milliliter Activin A into the plate. Incubate the cells at 37 degrees Celsius and 5%carbon dioxide for 24 hours.On day one, replace the existing medium with RPMI B27 GlutaMAX supplemented with 50 nanograms per milliliter of Activin A.Incubate the cells at 37 degrees Celsius and 5%carbon dioxide for 24 hours. On day two, to induce hepatic progenitor differentiation, replace the existing medium with RPMI B27 GlutaMAX medium supplemented with 1%dimethyl sulfoxide, and continue incubation for up to five days. On day seven, to initiate hepatocyte maturation, switch to hepatocyte maturation medium and incubate for 20 days at 37 degrees Celsius and 5%carbon dioxide.On day 27, human-induced pluripotent stem cells displayed polygonal cell shapes and rounded nuclei, characteristics of hepatocytes.

人诱导多能干细胞在2D培养物中的肝分化

Cell Preparation for Purification of Human-Induced Pluripotent Stem Cells-Derived Hepatocytes

After 27 days of initiating hepatic differentiation of human induced pluripotent stem cells, wash the cells with two milliliters of PBS per well. Prepare the enzyme solution in ADS buffer using the given components. Add one milliliter of solution per well of a six well plate.Place the plate at 37 degrees Celsius on a rotating shaker for about two hours to detach the cells from the plate. Confirm the cell detachment under a microscope, then pipette the cell suspension to disperse into single cells, and collect them in a 50 milliliter tube containing the hepatocyte maturation medium. Centrifuge the cell suspension at 200G for five minutes at 18 degrees Celsius.And using an aspirator, remove the supernatant. To stain the mitochondria of cells, resuspend the pellet in five milliliters of hepatocyte maturation medium containing 100 nanomolar TMRM. Incubate the cells for 30 minutes at 37 degrees Celsius while protecting them from light.Again, centrifuge the cell suspension and resuspend the pellet in one to five milliliters of cold ADS buffer containing 2%FBS. After cell counting, aliquot 500, 000 cells to a 1.5 milliliter tube and label it as the no primary antibody control. Centrifuge the remaining cell suspension at 200G for five minutes at four degrees Celsius, and remove the supernatant.Add 100 microliters of diluted primary antibody per one million cells. Transfer the cell suspension to a 1.5 milliliter tube and incubate on ice for 50 minutes. After incubation, centrifuge the cell suspension and wash the cells twice with cold ADS buffer containing 2%FBS.Now add 100 microliters of diluted secondary antibody per one million cells to the primary antibody, stained or unstained cells, and incubate for 30 minutes on ice. Again, centrifuge the cell suspension and wash the cells twice with cold ADS buffer containing 2%FBS. After resuspending the cells in ADS buffer, filter them through a snap cap cell strainer and place the tube on ice.

用于纯化人诱导多能干细胞来源肝细胞的细胞制备

To begin, set up the fluorescence-activated cell sorting, or FACS machine, for cell sorting following the manufacturer's instructions. Place the sample tube on the FACS machine and load it. Gate TMRM-high and ALCAM negative population in the P1 region to sort non-hepatocytes.Then gate the TMRM high and ALCAM positive population in the P2 region to sort hepatocytes. Collect sorted cells in 15-milliliter collection tubes containing hepatocyte maturation medium. After sorting, remove the collection tube from the FACS machine.Centrifuge the sorted cell suspension at 200 G for five minutes at 18 degrees Celsius. Remove the supernatant, and re-suspend the pellet in two milliliters of hepatocyte maturation medium with 10 micromolar rock inhibitor and 20 nanomolar cyclosporine A.Seed the cells on mitomycin C-treated mouse embryonic fibroblasts with hepatocyte maturation medium in a six-well plate. Culture the cells in a 37-degree Celsius carbon dioxide incubator for five days before immuno staining for hepatocyte purity test.FACS analysis conducted on day 27 of hepatic differentiation revealed TMRM high and ALCAM positive population. The immunohistochemical staining of P1 and P2 cells cultured on mouse embryonic fibroblasts after sorting is shown. A purity test by counting albumin-positive cells showed 0%of P1, and approximately 97%of P2 cells were hepatocyte-like cells.