Name: 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
Uploaded: 2023-12-01T12:50:00
Description: Watch this Scientific Journal Video about Application of Live Mitochondria Staining in Cell-Sorting to Purify Hepatocytes Derived from Human Induced Pluripotent Stem Cells at JoVE.com

这项工作得到了文部科学省的资助号[23390072]。

本研究使用商业获得的人 iPS 细胞（253G1 菌株）（参见 材料表）。
1. 人iPS细胞的维持
<ol><li>在涂有 0.25 μg/cm 2 iMatrix-511 的培养皿上，在 AK02 培养基中将人 iPS 细胞保持在无饲养层条件下（参见 材料表）。</li>
</ol>2. 人 iPS 细胞在 2D 培养物中的肝分化
注：请参阅 ?...

通过线粒体-ALCAM 分选纯化人 iPSC 来源的肝细胞

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由于肝细胞在营养代谢和解毒方面的功能，与其他细胞类型相比，肝细胞拥有相对较多的线粒体15。ALCAM是免疫球蛋白超家族的成员，在细胞粘附和迁移中发挥作用。它在多种细胞类型中表达，包括肝细胞、上皮细胞、淋巴细胞、髓样细胞、成纤维细胞和神经元细胞16。通过结合使用基于线粒体的方法和ALCAM抗体，成功鉴定了人iPS细胞来源的肝细胞。
<p class="jo...

胚胎干细胞和诱导多能干细胞（分别为 ES 和 iPS）被认为是再生疗法的有前途的细胞来源。然而，即使使用相同的细胞系、方案和实验者 1,2,3,4，将这些细胞分化为特定靶细胞类型的效率也可能有所不同。这种变异性可能归因于人类 ES/iPS 细胞对其环境的高敏感性。因此，目前很难一致地获得纯的靶细胞。为了实现高度安全的再生医学，消除治疗细胞中的增殖细胞和未分化干细胞至关重要，而针对靶细胞的先进纯化技术是必不可少的5,6,7。
细胞分选仪是一种设备，可以立即分析单个细胞，并根据荧光信号强度对感兴趣的活细胞进行分选，提供了一种有前途的解决方案。这可以通过对细胞类型特异性表面标记物进行抗体染色或利用细胞类型特异性报告基因表达来实现。使用这种技术，有几篇关于纯化多能干细胞来源的心肌细胞 8,9,10 和肝细胞11,12 的方法的报道。Hattori 等人使用细胞分选仪开发了一种创新的线粒体纯化方法13。利用心肌细胞通过线粒体活性对能量有高需求这一事实，用活线粒体指示染料 TMRM（四甲基罗丹明甲酯）染色细胞可用于通过 FACS 标记和高度纯化来自含有各种细胞类型的人 ES 细胞衍生的胚状体的心肌细胞。通过对纯化的心肌细胞进行畸胎瘤形成测定，证实了没有致瘤性。此外，Yamashita 等人出乎意料地发现了一种通过分离具有高线粒体活性和 ALCAM 阳性表达的组分从人 ES 细胞衍生的胚状体中纯化肝细胞的方法14。这种方法的基本原理是，肝细胞也具有相对较多的线粒体，因为它们大量消耗 ATP 进行营养代谢和解毒15，并且肝细胞表达 ALCAM，它是免疫球蛋白超家族的成员，在细胞粘附和迁移中发挥作用16。
以往对多能干细胞来源的肝细胞的高纯化方法需要基因修饰，非基因纯化方法效率低17。线粒体非遗传方法在实现高纯度方面具有优点。当需要肝祖细胞时，可以选择基于CD133和CD13或Dlk1的方法18,19。尽管基因组编辑技术的准确性已经提高，但无法完全消除不可预见的基因组变化（例如致癌）的潜在风险。基于线粒体活性的方法，不涉及基因改造，可以避免这种风险。
通过检查新生大鼠心肌细胞中的各种线粒体指标，观察到 TMRM 在 24 小时内完全消失，而其他染料保留至少 5 天13。此外，需要注意的是，使用 3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴化物 （MTT） 测定法评估时，TMRM 和 JC-1 不影响细胞活力，而其他染料对细胞活力的影响各不相同13。TMRM表现出更高的安全性。
迄今为止，与三维 （3D） 胚状体形成相比，已经开发了几种二维 （2D） 分化方法，作为更有效的诱导分化方法。这是因为逐步分化诱导化合物或细胞因子可以在 2D 平面上均匀地给药到细胞中，而不是在 3D 空间中。在这项研究中，对先前报道的方法20 进行了修改，以诱导分化为人 iPS 细胞来源的肝细胞。本文详细介绍了对源自人 iPS 细胞的肝细胞进行最新 2D 分化和纯化的程序。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>253G1 human iPS cell line</td>
			<td>RIKEN BioResource Research Center</td>
			<td>HPS0002</td>
			<td></td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>163-20145</td>
			<td></td>
		</tr>
		<tr>
			<td>Activin A Solution, Human, Recombinant</td>
			<td>NACALAI TESQUE, INC.</td>
			<td>18585</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Nuclei Antibody, clone 235-1</td>
			<td>Chemicon</td>
			<td>MAB1281</td>
			<td>Antibody against human nuclear antigen</td>
		</tr>
		<tr>
			<td>B-27 Supplement (50x), serum free</td>
			<td>Thermo Fisher Scientific</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSAria III</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Cell sorter</td>
		</tr>
		<tr>
			<td>CHIR-99021</td>
			<td>MedChemExpress</td>
			<td>HY-10182&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Ciclosporin A</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>035-18961</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>034-22363</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix</td>
			<td>Corning</td>
			<td>354230</td>
			<td>Gel-like basement membrane matrix</td>
		</tr>
		<tr>
			<td>CultureSure Y-27632</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>036-24023</td>
			<td>ROCK inhibitor</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>047-18863</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>047-29353</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-11055</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 546</td>
			<td>Thermo Fisher Scientific</td>
			<td>A10036</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-21206</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap</td>
			<td>Corning</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Biowest</td>
			<td>51820-500</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050061</td>
			<td>Dipeptide L-Alanyl-L-Glutamine</td>
		</tr>
		<tr>
			<td>Human/Mouse/Rat/Canine ALCAM/CD166 Antibody</td>
			<td>R&#38;D Systems</td>
			<td>AF1172</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone 21-hemisuccinate sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>H2270</td>
			<td></td>
		</tr>
		<tr>
			<td>iMatrix-511 silk</td>
			<td>Nippi</td>
			<td>892021</td>
			<td></td>
		</tr>
		<tr>
			<td>ImmunoBlock</td>
			<td>KAC</td>
			<td>CTKN001</td>
			<td>Blocking solution</td>
		</tr>
		<tr>
			<td>ITS-G Supplement(&#215;100)</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>090-06741</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s L-15 Medium</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>128-06075</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Hexanoic-Try-Ile-(6)-amino Hexanoic amide (Dihexa)</td>
			<td>Toronto Research Chemicals</td>
			<td>H293745</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyclonal Rabbit Anti-Human Albumin</td>
			<td>Dako</td>
			<td>A0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyoxyethylene Sorbitan Monolaurate (Tween 20)</td>
			<td>NACALAI TESQUE, INC.</td>
			<td>28353-85</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>189-02025</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium L-Ascorbate</td>
			<td>NACALAI TESQUE, INC.</td>
			<td>03422-32</td>
			<td></td>
		</tr>
		<tr>
			<td>StemFit AK02N</td>
			<td>REPROCELL</td>
			<td>RCAK02N</td>
			<td></td>
		</tr>
		<tr>
			<td>TBS (10x)</td>
			<td>NACALAI TESQUE, INC.</td>
			<td>12748-31</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylrhodamine, methyl ester (TMRM)</td>
			<td>Thermo Fisher Scientific</td>
			<td>T668</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>NACALAI TESQUE, INC.</td>
			<td>28229-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution</td>
			<td>NACALAI TESQUE, INC.</td>
			<td>20577-34</td>
			<td></td>
		</tr>
		<tr>
			<td>TRYPSIN 250</td>
			<td>Difco</td>
			<td>215240</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptose phosphate broth solution</td>
			<td>Sigma-Aldrich</td>
			<td>T8159</td>
			<td></td>
		</tr>
	</tbody>
</table>

application of live mitochondria staining in cell-sorting to purify hepatocytes derived from human induced pluripotent stem cells

人胚胎干细胞（ES）和诱导多能干细胞（iPS）由于其无限增殖和多能特性，在基于细胞的再生医学中具有治疗严重疾病器官的潜在应用。然而，由于人 ES/iPS 细胞对环境的高度敏感性，将人 ES/iPS 细胞分化为 100% 纯的靶细胞类型具有挑战性。移植后的肿瘤发生是由被污染的、增殖的和未分化的细胞引起的，因此高纯化技术对于安全实现再生医学至关重要。为了降低肿瘤发生的风险，已经开发了一种针对人 iPS 细胞来源的肝细胞的高纯度技术。该方法采用FACS（荧光激活细胞分选），使用高线粒体含量和细胞表面标记物ALCAM（活化的白细胞粘附分子）的组合，无需基因修饰。使用该方法的纯化肝细胞中±97%0.38%（n = 5）表现出白蛋白表达。本文旨在提供该方法的详细程序，应用于最新的人iPS细胞到肝细胞的二维分化方法。

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.

图中显示了通过2D培养诱导人iPS细胞分化为肝细胞的过程时间表（图1A）和代表性细胞特征（图1B）。大约在分化第 12 天，细胞开始表现出多边形细胞形状和圆形细胞核，这是肝细胞的特征。一些肝细胞也表现出多核。
在分化第 27 天使用细胞进行 FACS 分析。为了鉴定ALCAM+群体，有和没有第一种抗ALCAM抗体的比较图分别显示?...

Watch this Scientific Journal Video about Application of Live Mitochondria Staining in Cell-Sorting to Purify Hepatocytes Derived from Human Induced Pluripotent Stem Cells at JoVE.com

来源于多能干细胞的肝细胞可以通过细胞分选进行纯化，使用线粒体和活化的白细胞粘附分子（ALCAM，也称为CD166）染色的组合。

Human embryonic stem (ES) and induced pluripotent stem (iPS) cells have potential applications in cell-based regenerative medicine for treating severely ...

我们的研究范围是纯化人诱导的多能干细胞来源的肝细胞，无需基因改造。我们的目标是解决与未分化细胞相关的致瘤风险问题，即从人类 iPS 细胞分化的细胞中。目前的实验挑战之一是由于分选的机械存储，细胞纯度低。我们正在开发一种新的纯化方法，利用肝细胞独特的代谢特性，进行大规模纯化。一种针对肝祖细胞的非细胞分选方法无法获得纯肝细胞。另一种用于成熟肝细胞的药物只能治疗一小部分肝细胞，因为标记蛋白的表达有限。相比之下，我们的方法可以获得不同成熟水平的肝细胞，并且具有更高的前程。人iPS细胞来源的肝细胞极不成熟，纯化后肝细胞宏表达减少较多。我们未来的目标是利用类器官技术增强肝细胞调节，并利用再生医学和药物发现的日常进展来增强肝细胞调节。

application-live-mitochondria-staining-cell-sorting-to-purify

Research

JoVE Journal

Biology

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.

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

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

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.