STEMdiff™ Definitive Endoderm Kit

Defined animal component-free medium for the differentiation of human ES and iPS cells to definitive endoderm

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STEMdiff™ Definitive Endoderm Kit

Defined animal component-free medium for the differentiation of human ES and iPS cells to definitive endoderm

1 Kit
Catalog #05110
323 USD

Required Products

Overview

STEMdiff™ Definitive Endoderm Kit is a complete, serum- and animal component-free medium and supplement kit that supports highly efficient differentiation of human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells to definitive endoderm cells. Cells differentiated using STEMdiff™ Definitive Endoderm Kit express high levels of endoderm markers, including CD184 (CXCR4), SOX17, FOXA2, and c-KIT, and lack expression of ectoderm, mesoderm, and pluripotency markers. The definitive endoderm cells produced using this kit are multipotent and capable of further differentiation towards cells of the pancreatic, pulmonary, and hepatic lineages, thus providing a robust tool for developmental studies, disease modeling, and drug discovery.​

This kit is optimized for differentiation of cells maintained in mTeSR™1. For differentiation of cells maintained in TeSR™-E8™, please see the STEMDiff™ Definitive Endoderm Kit (TeSR™-E8™ Optimized).
Advantages:
• Defined, serum-free, animal component-free medium for the differentiation of human ES and iPS cells to definitive endoderm in a complete, ready-to-use format
• Efficient and reproducible differentiation of multiple ES cell and iPS cell lines
• Generates definitive endoderm cells capable of further differentiation to pancreatic, hepatic, intestinal and pulmonary cell lineages
• Optimized for use with cells maintained in TeSR™-E8™ cell culture medium
Components:
  • STEMdiff™ Definitive Endoderm Basal Medium, 100 mL
  • STEMdiff™ Definitive Endoderm Supplement A, 0.35 mL
  • STEMdiff™ Definitive Endoderm Supplement B, 1.1 mL
Subtype:
Specialized Media
Cell Type:
Airway Cells; Pluripotent Stem Cells; Intestinal Cells; Pancreatic Cells; Endoderm, PSC-Derived
Species:
Human
Application:
Differentiation; Cell Culture
Brand:
STEMdiff
Area of Interest:
Stem Cell Biology; Epithelial Cell Biology; Cancer Research
Formulation:
Serum-Free; Animal Component-Free; Defined

Scientific Resources

Product Applications

This product is designed for use in the following research area(s) as part of the highlighted workflow stage(s). Explore these workflows to learn more about the other products we offer to support each research area.

Data and Publications

Data

Definitive endoderm differentiation is efficient across multiple human ES and iPS cell lines

Figure 1. Definitive endoderm differentiation is efficient across multiple human ES and iPS cell lines

Quantitative analysis of definitive endoderm formulation on multiple human ES and iPS cell lines as measured by co-expression of CXCR4 and SOX17. Prior to differentiation using STEMdiff™ Definitive Endoderm, cells were maintained in their pluripotent state by culturing mTeSR™1 on Matrigel. Data are expressed as the mean percent of cells expressing both markers. Error bars indicate SEM, n = 4-18 per cell line.

Quantitative Analysis of Definitive Endoderm hES and iPS-Derived Using STEMdiff™ Definitive Endoderm

Figure 2. Quantitative Analysis of Definitive Endoderm hES and iPS-Derived Using STEMdiff™ Definitive Endoderm

Quantitative analysis of definitive endoderm in human ES and iPS cells previously maintained in TeSR™2 prior to differentiation on Matrigel using STEMdiff™ Definitive Endoderm. Data are expressed as the mean percent of cells expressing both markers. Error bars indicate SEM. n = 4-11 per cell line.

Efficient definitive endoderm differentiation in human ES and iPS cells

Figure 3. Efficient definitive endoderm differentiation in human ES and iPS cells

Representative Density plots showing CXCR4 and SOX17 expression in human ES cells (H1 and H9) and human iPS cells (WLS-4D1 and A13700) following 5 days of differentiation to definitive endoderm using STEMdiff™ Definitive Endoderm. Isotype controls were used to set quadrant gates.

STEMdiff™ Definitive Endoderm yields DE that retains potency for downstream lineage specification

Figure 4. STEMdiff™ Definitive Endoderm yields DE that retains potency for downstream lineage specification

Cultures differentiated using STEMdiff™ Definitive Endoderm maintain their ability to be directed towards pancreatic and hepatic lineages. A) Representative image of PDX-1 immunoreactivity in H9 cells following pancreatic specification. Scale bar 20 µm. B) Representative image of human serum albumin (HSA) immunoreactivity in H9 cells following hepatic specification. Scale bar, 100 µm.

Publications

(19)
Stem Cell Research 2016 MAR

Hepatic differentiation of human pluripotent stem cells in miniaturized format suitable for high-throughput screen

Carpentier A et al.

Abstract

The establishment of protocols to differentiate human pluripotent stem cells (hPSCs) including embryonic (ESC) and induced pluripotent (iPSC) stem cells into functional hepatocyte-like cells (HLCs) creates new opportunities to study liver metabolism, genetic diseases and infection of hepatotropic viruses (hepatitis B and C viruses) in the context of specific genetic background. While supporting efficient differentiation to HLCs, the published protocols are limited in terms of differentiation into fully mature hepatocytes and in a smaller-well format. This limitation handicaps the application of these cells to high-throughput assays. Here we describe a protocol allowing efficient and consistent hepatic differentiation of hPSCs in 384-well plates into functional hepatocyte-like cells, which remain differentiated for more than 3 weeks. This protocol affords the unique opportunity to miniaturize the hPSC-based differentiation technology and facilitates screening for molecules in modulating liver differentiation, metabolism, genetic network, and response to infection or other external stimuli.
Stem Cell Research 2016 MAR

The Forkhead box transcription factor FOXM1 is required for the maintenance of cell proliferation and protection against oxidative stress in human embryonic stem cells

Kwok CTD et al.

Abstract

Human embryonic stem cells (hESCs) exhibit unique cell cycle structure, self-renewal and pluripotency. The Forkhead box transcription factor M1 (FOXM1) is critically required for the maintenance of pluripotency in mouse embryonic stem cells and mouse embryonal carcinoma cells, but its role in hESCs remains unclear. Here, we show that FOXM1 expression was enriched in undifferentiated hESCs and was regulated in a cell cycle-dependent manner with peak levels detected at the G2/M phase. Expression of FOXM1 did not correlate with OCT4 and NANOG during in vitro differentiation of hESCs. Importantly, knockdown of FOXM1 expression led to aberrant cell cycle distribution with impairment in mitotic progression but showed no profound effect on the undifferentiated state. Interestingly, FOXM1 depletion sensitized hESCs to oxidative stress. Moreover, genome-wide analysis of FOXM1 targets by ChIP-seq identified genes important for M phase including CCNB1 and CDK1, which were subsequently confirmed by ChIP and RNA interference analyses. Further peak set comparison against a differentiating hESC line and a cancer cell line revealed a substantial difference in the genomic binding profile of FOXM1 in hESCs. Taken together, our findings provide the first evidence to support FOXM1 as an important regulator of cell cycle progression and defense against oxidative stress in hESCs.
Methods in molecular biology (Clifton, N.J.) 2016 JAN

Multisystemic Disease Modeling of Liver-Derived Protein Folding Disorders Using Induced Pluripotent Stem Cells (iPSCs).

Leung A and Murphy GJ

Abstract

Familial transthyretin amyloidosis (ATTR) is an autosomal dominant protein-folding disorder caused by over 100 distinct mutations in the transthyretin (TTR) gene. In ATTR, protein secreted from the liver aggregates and forms fibrils in target organs, chiefly the heart and peripheral nervous system, highlighting the need for a model capable of recapitulating the multisystem complexity of this clinically variable disease. Here, we describe detailed methodologies for the directed differentiation of protein folding disease-specific iPSCs into hepatocytes that produce mutant protein, and neural-lineage cells often targeted in disease. Methodologies are also described for the construction of multisystem models and drug screening using iPSCs.
Tissue engineering. Part C, Methods 2016 JAN

Recombinant Xeno-Free Vitronectin Supports Self-Renewal and Pluripotency in Protein-Induced Pluripotent Stem Cells.

Kaini RR et al.

Abstract

Patient safety is a major concern in the application of induced pluripotent stem cells (iPSCs) in cell-based therapy. Efforts are being made to reprogram, maintain, and differentiate iPSCs in defined conditions to provide a safe source of stem cells for regenerative medicine. Recently, human fibroblasts were successfully reprogrammed into pluripotent stem cells using four recombinant proteins (OCT4, c-Myc, KLF4, and SOX2) fused with a cell-penetrating peptide (9R). These protein-induced pluripotent stem cells (piPSCs) are maintained and propagated on a feeder layer of mouse embryonic fibroblasts. Use of animal-derived products in maintenance and differentiation of iPSCs poses risks of zoonotic disease transmission and immune rejection when transplanted into humans. To avoid potential incorporation of xenogenic products, we cultured piPSCs on recombinant human matrix proteins. We then tested whether recombinant human matrix proteins can support self-renewal and pluripotency of piPSCs. After long-term culture on recombinant human vitronectin in xeno-free conditions, piPSCs retained the expression of pluripotent markers. The pluripotency of these cells was further evaluated by differentiating toward ectoderm, mesoderm, and endoderm lineages in vitro. In conclusion, recombinant human vitronectin can support the long-term culture and maintain the stemness of piPSCs in defined nonxenogenic conditions.
Nature 2016 APR

Derivation and differentiation of haploid human embryonic stem cells.

Sagi I et al.

Abstract

Diploidy is a fundamental genetic feature in mammals, in which haploid cells normally arise only as post-meiotic germ cells that serve to ensure a diploid genome upon fertilization. Gamete manipulation has yielded haploid embryonic stem (ES) cells from several mammalian species, but haploid human ES cells have yet to be reported. Here we generated and analysed a collection of human parthenogenetic ES cell lines originating from haploid oocytes, leading to the successful isolation and maintenance of human ES cell lines with a normal haploid karyotype. Haploid human ES cells exhibited typical pluripotent stem cell characteristics, such as self-renewal capacity and a pluripotency-specific molecular signature. Moreover, we demonstrated the utility of these cells as a platform for loss-of-function genetic screening. Although haploid human ES cells resembled their diploid counterparts, they also displayed distinct properties including differential regulation of X chromosome inactivation and of genes involved in oxidative phosphorylation, alongside reduction in absolute gene expression levels and cell size. Surprisingly, we found that a haploid human genome is compatible not only with the undifferentiated pluripotent state, but also with differentiated somatic fates representing all three embryonic germ layers both in vitro and in vivo, despite a persistent dosage imbalance between the autosomes and X chromosome. We expect that haploid human ES cells will provide novel means for studying human functional genomics and development.
STEMCELL TECHNOLOGIES INC.’S QUALITY MANAGEMENT SYSTEM IS CERTIFIED TO ISO 13485. PRODUCTS ARE FOR RESEARCH USE ONLY AND NOT INTENDED FOR HUMAN OR ANIMAL DIAGNOSTIC OR THERAPEUTIC USES UNLESS OTHERWISE STATED.
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