STEMdiff™ 肝细胞试剂盒

用于将人多能干细胞分化为肝细胞样细胞的无血清分化试剂盒

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产品号 #100-0520_C

用于将人多能干细胞分化为肝细胞样细胞的无血清分化试剂盒

产品优势

可生成表达几种关键肝脏标志物（如白蛋白和 α1-抗胰蛋白酶）的肝细胞样细胞
在多个 hPSC 系中实现稳健、可重复的分化，具有多边形形态和双核特征
适用于 96 孔板中的高通量药物毒性和疾病建模应用
实验流程可与使用 HepatiCult™ 培养基生成肝脏类器官兼容
无血清培养基设计旨在最大限度地减少实验变量

产品组分包括

STEMdiff™ 内胚层基础培养基（肝脏），100 mL
STEMdiff™ 定型内胚层 100X 补充剂 MR，0.35 mL
STEMdiff™ 定型内胚层 100X 补充剂 CJ，1.1 mL
STEMdiff™ 肝脏前体细胞培养基，100 mL
STEMdiff™ 肝细胞培养基，150 mL

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总览

高效生成表达多种关键肝脏特异性标志物（包括白蛋白和α1-抗胰蛋白酶）的肝细胞样细胞 (HLC)。STEMdiff™ 肝细胞试剂盒可在多种人多能干细胞（hPSC）系中实现稳定重复的分化，为您的实验提供可靠的 HLC 来源。其无血清配方通过减少未定义成分，降低实验变量，并使您能够稳定地分化 HLC 培养物。使用 STEMdiff™ 肝细胞试剂盒生成的 HLC 适用于肝脏研究、疾病建模和肝毒性测试等多种应用。

了解更多关于补充方案的信息。

实验数据

Figure 1. Generation of Hepatocyte-Like Cells Within 21 Days Using the STEMdiff™ Hepatocyte Kit

hPSCs are seeded as single cells onto tissue culture-treated plates coated with Laminin-521 in *mTeSR™1, mTeSR™ Plus, or TeSR™-AOF supplemented with 10 µM ROCK inhibitor on day 0. On day 1, differentiation is initiated by replacing the medium with STEMdiff™ Definitive Endoderm (DE) Medium (Stage 1), then daily medium changes are performed. On day 5, cells are either harvested for downstream analyses or cultured for an additional 5 days, with full-medium changes using STEMdiff™ Hepatic Progenitor Medium on days 5, 6, 7, and 9. On day 10, hepatic progenitor cells (HPs) are either harvested for downstream analyses or cultured for an additional 11 days, with full-medium changes using STEMdiff™ Hepatocyte Medium every 2 days. On day 21, hepatocyte-like cells (HLCs) are harvested and used in downstream analyses and assays. hPSCs = Human pluripotent stem cells.

Figure 2. Cells Undergo Stage-Specific Changes in Expression of Hepatic Markers As Confirmed by Gene Expression Analysis

hPSCs, DE cells, HPs, and HLCs were collected on days 0, 5, 10, and 21, respectively, according to the STEMdiff™ Hepatocyte Kit protocol, and examined for gene expression through qPCR analysis. (A,B) Compared to hPSCs, expression of stem cell marker POU5F1 was down-regulated and expression of DE marker FOXA2 was up-regulated across all three stages of differentiation, demonstrating a successful endodermal lineage specification. (C-F) Up-regulation of hepatic lineage-specific markers AFP, CK19, ALB, and CYP3A4 demonstrates a successful differentiation of hPSCs to HPs and HLCs. Expression levels are normalized to housekeeping genes (ΔCT) and hPSCs for relative expression levels (**** represents an adjusted p-value <0.0001). hES = Human embryonic stem cells; hiPS = Human induced pluripotent stem cells; PHH = Primary human hepatocyte; hPSCs = Human pluripotent stem cells; DE = Definitive endoderm; HPs = Hepatic progenitors; HLCs = Hepatocyte-like cells; AFP = Alpha fetoprotein; CK19 = Cytokeratin 19; ALB = Albumin.

Figure 3. hPSC-derived Hepatic Progenitor Cells and Hepatocyte-Like Cells Express Hepatic Markers As Confirmed by Immunocytochemistry Analysis

Cells cultured to day 10 (HPs) and day 21 (HLCs) were fixed with 4% paraformaldehyde and permeabilized before being stained with primary and secondary antibodies. (A-C) HPs expressed the epithelial marker EPCAM, ductal marker CK19, fetal serum protein AFP, the hepatic transcription factors HNF6 and HNF4a, and the stage-specific transcription factor TBX3. (C) By day 10, some of the HPs also began to express the mature serum protein albumin. (D-F) Most HLCs expressed the mature hepatic markers ALB, CYP3A4, and A1AT by day 21. HPs = Hepatic progenitors; HLCs = Hepatocyte-like cells; CK19 = Cytokeratin 19; AFP = Alpha fetoprotein; ALB = Albumin.

Figure 4. STEMdiff™ Hepatocyte Kit Supports Efficient Differentiation of hES and hiPS Cells to Hepatocyte-Like Cells

hES (H9 and H1) cells and hiPS (WLS-1C and SCTi003- A) cells were cultured for 21 days using the STEMdiff™ Hepatocyte Kit. At the end of the culture period, cells were harvested and analyzed by flow cytometry for expression of hepatocyte markers ALB and A1AT. (A) Percentages and (B) total numbers of cells expressing ALB and A1AT following differentiation of hES or hiPS cells are shown. > 60% of HLCs are ALB+/A1AT+ at day 21 of the differentiation process. hES = Human embryonic stem cells; hiPS = Human induced pluripotent stem cells; ALB = Albumin; HLCs = Hepatocyte-like cells.

hPSC-Derived Hepatocyte-Like Cells Exhibit Key Liver Functionalities

Figure 5. hPSC-Derived HLCs Exhibit Key Liver Functionalities

Upon maturation of HPs to HLCs, the cells acquired the ability to (A) synthesize and secrete serum protein albumin (n=11), as detected by ELISA (Abcam Catalog #ab108788), (B) and exhibited CYP3A4 enzymatic activity (n=15), as assessed using the P450-Glo™ CYP3A4 Assay (Promega Catalog #V9002). (C) Day 21 HLCs were also capable of producing bile acids (n=2) (D) and synthesizing and secreting urea (n=2) at levels comparable to primary human hepatocytes (PHH; n=3), as detected by colorimetric assays (Abcam Catalog #ab239702, ab83362, respectively). Error bars = SD. Ordinary one-way ANOVA used for statistical testing (*** represents an adjusted p-value of 0.0007, ** represents an adjusted p-value of 0.0011, * represents an adjusted p-value of 0.0179, ns = not significant). HPs = Hepatic progenitors; HLCs = Hepatocyte-like cells; PHH = Primary human hepatocyte.

Figure 6. HLCs are More Sensitive Models of Drug-Induced Toxicity than Immortalized Cell Line HepG2

HLCs and HepG2 cells cultured in 96-well plates were treated with (A) ketoconazole, (B) rifampicin, a 0.1% DMSO vehicle control (DMSO Ctrl), or 1 μM staurosporine (Sts Ctrl) for 72 hours. Full-medium changes with fresh compound volumes were performed for the HLCs on day 18 and day 20, and analyses were performed on day 21, 24 hours after the final compound addition. Cell viability for both treated and untreated (No treatment) HLCs and HepG2 cells was assessed using the CellTiter-Glo® 3D Cell Viability Assay (Promega Catalog #G9681). HLCs exhibited ketoconazole-induced hepatotoxicity (IC50 for 1C-derived HLCs = 20.57 μM, IC50 for H9-derived HLCs = 23.79 μM) at a lower dose than HepG2 cells (IC50 = 37.74 μM). Similarly, HLCs were able to capture rifampicin-induced hepatotoxicity (IC50 for 1C-derived HLCs = 120.4 μM, IC50 for H1-derived HLCs = 197.4 μM) at a lower dose than HepG2 cells (IC50 = 332.3 μM). Error bars = SD. HLCs = Hepatocyte-like cells.

Figure 7. HP- and HLC-derived Liver Organoids Can Be Reliably Generated Using HepatiCult™ Organoid Kit

hPSC-derived liver organoids can be established from cells generated using the STEMdiff™ Hepatocyte Kit. HPs and HLCs were collected on days 10 and 21 of differentiation, respectively, seeded into Corning® Matrigel® domes, and grown using the HepatiCult™ Organoid Kit. Shown are representative images of HP- and HLC-derived organoids at passage 2, which were (A,B) established and expanded using HepatiCult™ Organoid Growth Medium (Human; hOGM) and then (C,D) further differentiated using HepatiCult™ Organoid Differentiation Medium (Human; hODM). hPSC = human pluripotent stem cells; HPs = Hepatic progenitors; HLCs = Hepatocyte-like cells.

Figure 8. HP- and HLC-derived Hepatic Organoids Exhibit Gene Expression Consistent with Hepatic Maturation When Differentiated Using hODM

hPSC-derived liver organoids were established, expanded for up to 10 passages, differentiated, and harvested for gene expression analysis through qPCR at each passage. Compared to two-dimensional cultures of HPs and HLCs collected on day 10 and day 21 of differentiation, respectively, three-dimensional organoid cultures established from these stages and further matured in hODM exhibited up-regulated expression of mature hepatic markers ALB, ASGR1, and CYP3A4 at levels comparable to PHH. Expression levels are normalized to housekeeping genes (**** represents an adjusted p-value <0.0001, ** represents an adjusted p-value of 0.0035, * represents an adjusted p-value of 0.0397). hPSC = human pluripotent stem cells; HPs = Hepatic progenitors; HLCs = Hepatocyte-like cells; ALB = Albumin; PHH = Primary human hepatocyte..

RNA Sequencing Analysis Confirms a More Mature Functional State of HLC-Derived Organoids upon Differentiation in HepatiCult™ Organoid Kit

Figure 9. RNA Sequencing Analysis Confirms a More Mature Functional State of HLC-Derived Organoids upon Differentiation in HepatiCult™ Organoid Kit

Relative to HLCs, HLC-derived organoids that have been further differentiated in hODM express increased levels of markers associated with mature hepatocyte identity and functionality, including serum protein synthesis (ALB), phase 1 and 2 drug metabolism (CYP2B6, CYP2C9, CYP3A4, UGT1A1, GSTA1), glucose metabolism (SLC2A2, GYS1, G6PC), lipid and cholesterol metabolism (FASN, PLIN2, NR1H3), bile acid synthesis (CYP27A1, BAAT), and urea synthesis (OTC, ARG1). Differentiated HLC-derived organoids also show reduced levels of the hepatic progenitor and fetal hepatocyte marker, AFP. HLC-derived organoids expanded in hOGM show gene expression that aligns with a proliferative state (PCNA, MKI67, POU5F1). Columns represent 2 biological replicates analyzed by bulk RNA-seq in the first passage after organoids are established from HLCs, on day 9 in hOGM and day 19 in hODM. The housekeeping gene, TBP, is included as a reference.

产品说明书及文档

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Product Information Sheet

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STEMdiff™ Hepatocyte Kit

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100-0520

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English

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Safety Data Sheet 1

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STEMdiff™ Hepatocyte Kit

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100-0520

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English

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Safety Data Sheet 2

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STEMdiff™ Hepatocyte Kit

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100-0520

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English

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Safety Data Sheet 3

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STEMdiff™ Hepatocyte Kit

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100-0520

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English

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Safety Data Sheet 4

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STEMdiff™ Hepatocyte Kit

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100-0520

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Safety Data Sheet 5

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STEMdiff™ Hepatocyte Kit

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100-0520

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应用领域

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文献 (2)

Single-nucleus multiomic analysis of Beckwith-Wiedemann syndrome liver reveals PPARA signaling enrichment and metabolic dysfunction S. Nirgude et al. Communications Biology 2025 Mar

Abstract

Beckwith-Wiedemann Syndrome (BWS) is an epigenetic overgrowth syndrome caused by methylation changes in the human 11p15 chromosomal locus. Patients with BWS may exhibit hepatomegaly,as well as an increased risk of hepatoblastoma. To understand the impact of these 11p15 changes in the liver,we performed a multiomic study [single nucleus RNA-sequencing (snRNA-seq) + single nucleus assay for transposable-accessible chromatin-sequencing (snATAC-seq)] of both BWS-liver and nonBWS-liver tumor-adjacent tissue. Our approach uncovers hepatocyte-specific enrichment of processes related to peroxisome proliferator—activated receptor alpha (PPARA). To confirm our findings,we differentiated a BWS induced pluripotent stem cell model into hepatocytes. Our data demonstrate the dysregulation of lipid metabolism in BWS-liver,which coincides with observed upregulation of PPARA during hepatocyte differentiation. BWS hepatocytes also exhibit decreased neutral lipids and increased fatty acid β-oxidation. We also observe increased reactive oxygen species byproducts in BWS hepatocytes,coinciding with increased oxidative DNA damage. This study proposes a putative mechanism for overgrowth and cancer predisposition in BWS liver due to perturbed metabolism. Subject terms: Paediatric research,Imprinting

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iRhom2 regulates HMGB1 secretion to modulate inflammation and hepatocyte senescence in an in vitro model of ischemia-reperfusion injury M. Calligaris et al. Cell Death & Disease 2026 Jan

Abstract

Ischemia-reperfusion injury (IRI) represents a major challenge in liver transplantation,driving acute dysfunction and contributing to long-term allograft rejection. This process triggers a robust inflammatory response,leading to hepatocyte damage,senescence,and impaired liver regeneration. While the underlying mechanisms remain incompletely understood,increasing evidence highlights macrophage-derived signaling as a pivotal driver of hepatocyte fate during IRI. Here,we identify iRhom2 as a key regulator of immune-mediated liver injury,orchestrating macrophage-driven inflammation and hepatocyte senescence. iRhom2 is known to modulate the secretion of multiple cytokines by macrophages,yet its specific contribution to IRI-driven hepatocyte senescence has not been fully elucidated. We reveal a significant upregulation of iRhom2 in IRI+ reperfused allografts,particularly in Kupffer cells and monocyte-derived macrophages. Functional characterization in iRhom2-deficient macrophages revealed reduced ER stress,preserved mitochondrial function,and attenuated apoptosis,indicating a protective role against IRI-induced cellular damage. Proteomic profiling further uncovers iRhom2-dependent secretion of inflammatory mediators,with HMGB1 emerging as a critical damage-associated molecular pattern (DAMP) molecule in this context. Notably,HMGB1 release occurs independently of TACE catalytic activity,suggesting an alternative unexplored regulatory mechanism. Furthermore,co-culture experiments confirm that macrophage-derived HMGB1 directly induces senescence of human induced pluripotent stem cell-derived hepatocytes (hiPSC-Heps) under in vitro IRI condition,driving the up-regulation of key senescence markers and disrupting cell cycle dynamics. Strikingly,HMGB1 neutralization enhances hepatocyte viability and mitigates senescence,underscoring its pathogenic role. Additionally,HMGB1 knockdown in macrophages protects hepatocytes,though p21 expression remains unaffected,hinting at additional senescence pathways. Our findings establish iRhom2 as a central orchestrator of macrophage-driven hepatocyte dysfunction in IRI and suggest that targeting the iRhom2-HMGB1 axis could represent a promising therapeutic strategy to improve post-transplant liver recovery and long-term graft survival.

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STEMdiff™ 肝细胞试剂盒

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