STEMdiff™ Cardiomyocyte Differentiation Kit

Media for differentiation of human PSCs to cardiomyocytes and long-term maintenance of human PSC-derived cardiomyocytes

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STEMdiff™ Cardiomyocyte Differentiation Kit

Media for differentiation of human PSCs to cardiomyocytes and long-term maintenance of human PSC-derived cardiomyocytes

1 Kit
Catalog #05010
582 USD

Required Products

Overview

STEMdiff™ Cardiomyocyte Differentiation Kit (Catalog #05010) includes a medium for differentiation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells (human pluripotent stem cells [hPSCs]) into cardiomyocytes (cardiac troponin T-positive [cTnT+]), as well as a medium for maintenance of hPSC-derived cardiomyocytes. This kit can be used to generate cardiomyocytes derived from a clump culture of hPSCs maintained in mTeSR™1 (Catalog #85850), TeSR™-E8™ (Catalog #05990), or mTeSR™ Plus (Catalog #05825). Greater than 80% of these cells will be cTnT+.
An average of 1 x 10^6 cells can be harvested from a single well of a 12-well plate.

STEMdiff™ Cardiomyocyte Maintenance Kit (Catalog #05020) can be used for long-term maintenance of hPSC-derived cardiomyocytes for one month or longer. These cardiomyocytes can be used in various downstream applications and analyses.
Advantages:
• Supports the entire hPSC-derived cardiomyocyte workflow
• Simple monolayer protocol produces cardiomyocytes in 15 days
• One kit generates over 50 million cardiomyocytes (cTnT+)
• Robust performance with minimal variability across multiple hPSC lines
Components:
  • STEMdiff™ Cardiomyocyte Differentiation Basal Medium, 380 mL
  • STEMdiff™ Cardiomyocyte Differentiation Supplement A (10X), 10 mL
  • STEMdiff™ Cardiomyocyte Differentiation Supplement B (10X), 10 mL
  • STEMdiff™ Cardiomyocyte Differentiation Supplement C (10X), 20 mL
  • STEMdiff™ Cardiomyocyte Maintenance Basal Medium, 490 mL
  • STEMdiff™ Cardiomyocyte Maintenance Supplement (50X), 10 mL
Subtype:
Specialized Media
Cell Type:
Cardiomyocytes, PSC-Derived
Species:
Human
Application:
Cell Culture; Differentiation; Maintenance
Brand:
STEMdiff
Area of Interest:
Disease Modeling; Drug Discovery and Toxicity Testing; Stem Cell Biology
Formulation:
Serum-Free

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.

Research Area Workflow Stages for
Workflow Stages

Data and Publications

Data

Figure 1. Cardiomyocyte Differentiation Protocol

Two days before the differentiation protocol, hPSC colonies are harvested and seeded as single cells at 350,000 cells/well in a 12-well format in TeSR™ medium. After one day (Day -1), the medium is replaced with fresh TeSR™ medium. The following day (Day 0), the TeSR™ medium is replaced with Medium A (STEMdiff™ Cardiomyocyte Differentiation Basal Medium containing Supplement A) to begin inducing the cells toward a cardiomyocyte fate. On day 2, a full medium change is performed with fresh Medium B (STEMdiff™ Cardiomyocyte Differentiation Basal Medium containing Supplement B). On days 4 and 6, full medium changes are performed with fresh Medium C (STEMdiff™ Cardiomyocyte Differentiation Basal Medium containing Supplement C). On day 8, medium is switched to STEMdiff™ Cardiomyocyte Maintenance Medium with full medium changes on days 10, 12 and 14, to promote further differentiation into cardiomyocyte cells. Small beating areas of cardiomyocytes can be seen as early as day 8, progressing to a full lawn of beating cardiomyocytes that can be harvested as early as day 15.

Figure 2. Morphology of hPSC-Derived Cardiomyocytes

Representative images of (A) hES (H9) cells and (B) hiPS (WLS-1C) cells on day 15 of differentiation to cardiomyocytes using the STEMdiff™ Cardiomyocyte Differentiation Kit. Differentiated cells exhibit typical cardiomyocyte morphology as an adherent, tightly packed web-like monolayer of beating cells. (C) Representative confocal microscopy image of a single hPSC-derived cardiomyocyte generated with the STEMdiff™ Cardiomyocyte Differentiation Kit and stained with cTnT (green) and DAPI (blue).

Figure 3. Efficient and Robust Generation of cTnT-Positive Cardiomyocytes

hES and hiPS cells were cultured for 15 days in single wells of 12-well plates using the STEMdiff™ Cardiomyocyte Differentiation Kit. At the end of the culture period, cells were harvested and analyzed by flow cytometry for expression of cardiac troponin T (cTnT). (A) Histogram analysis for cardiomyocyte cell marker cTnT for cultures of hES (H9) and hiPS (WLS-1C and STiPS-M001) cells. (Filled = sample; blank = secondary antibody only control) (B,C) Percentages and total numbers of cells expressing cTnT in cultures of hES or hiPS cells are shown. Data shown as mean ± SEM; n=3.

Figure 4. hPSC-Derived Cardiomyocytes Exhibit a Robust and Stable Excitability Profile

Microelectrode array (MEA) voltage recordings of cardiomyocytes (day 27) derived from human pluripotent stem cells generated and maintained with the STEMdiff™ Cardiomyocyte Differentiation and Maintenance Kits. The hPSC-derived cardiomyocytes have a characteristic electrical profile and stable beat rate. A large depolarization spike followed by a smaller repolarization deflection is observed.

Publications

(7)
Leukemia 2020 jan

BCMA peptide-engineered nanoparticles enhance induction and function of antigen-specific CD8+ cytotoxic T lymphocytes against multiple myeloma: clinical applications.

J. Bae et al.

Abstract

The purpose of these studies was to develop and characterize B-cell maturation antigen (BCMA)-specific peptide-encapsulated nanoparticle formulations to efficiently evoke BCMA-specific CD8+ cytotoxic T lymphocytes (CTL) with poly-functional immune activities against multiple myeloma (MM). Heteroclitic BCMA72-80 [YLMFLLRKI] peptide-encapsulated liposome or poly(lactic-co-glycolic acid) (PLGA) nanoparticles displayed uniform size distribution and increased peptide delivery to human dendritic cells, which enhanced induction of BCMA-specific CTL. Distinct from liposome-based nanoparticles, PLGA-based nanoparticles demonstrated a gradual increase in peptide uptake by antigen-presenting cells, and induced BCMA-specific CTL with higher anti-tumor activities (CD107a degranulation, CTL proliferation, and IFN-$\gamma$/IL-2/TNF-$\alpha$ production) against primary CD138+ tumor cells and MM cell lines. The improved functional activities were associated with increased Tetramer+/CD45RO+ memory CTL, CD28 upregulation on Tetramer+ CTL, and longer maintenance of central memory (CCR7+ CD45RO+) CTL, with the highest anti-MM activity and less differentiation into effector memory (CCR7- CD45RO+) CTL. These results provide the framework for therapeutic application of PLGA-based BCMA immunogenic peptide delivery system, rather than free peptide, to enhance the induction of BCMA-specific CTL with poly-functional Th1-specific anti-MM activities. These results demonstrate the potential clinical utility of PLGA nanotechnology-based cancer vaccine to enhance BCMA-targeted immunotherapy against myeloma.
Stem cell research 2019 may

Characterization of the first induced pluripotent stem cell line generated from a patient with autosomal dominant catecholaminergic polymorphic ventricular tachycardia due to a heterozygous mutation in cardiac calsequestrin-2.

S. Ross et al.

Abstract

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmia syndrome characterized by adrenaline induced ventricular tachycardia. The primary genetic aetiologies underlying CPVT are either autosomal dominant or autosomal recessive inheritance, resulting from heterozygous mutations in cardiac ryanodine receptor (RYR2) and homozygous mutations in cardiac calsequestrin-2 (CASQ2), respectively. Recently, a large family with autosomal dominant CPVT due to a heterozygous mutation in CASQ2, p.Lys180Arg, was reported. This resource is the first induced pluripotent stem cell line generated from a patient with autosomal dominant CPVT due to a heterozygous mutation in CASQ2. Induced pluripotent stem cells were generated from the whole blood of a 40-year-old woman with severe CPVT who is heterozygous for the p.Lys180Arg CASQ2 mutation. Induced pluripotent stem cell (iPSC) characterization confirmed expression of pluripotency makers, trilineage differentiation potential, and the absence of exogenous pluripotency vector expression.
Biochemical and biophysical research communications 2019 aug

Differentiation of lymphoblastoid-derived iPSCs into functional cardiomyocytes, neurons and myoblasts.

H. Poulin et al.

Abstract

Human induced pluripotent stem cells (hiPSCs) are a valuable tool for investigating complex cellular and molecular events that occur in several human diseases. Importantly, the ability to differentiate hiPSCs into any human cell type provides a unique way for investigating disease mechanisms such as complex mental health diseases. The in vitro transformation of human lymphocytes into lymphoblasts (LCLs) using the Epstein-Barr virus (EBV) has been the main method for generating immortalized human cell lines for half a century. However, the derivation of iPSCs from LCLs has emerged as an alternative source from which these cell lines can be generated. We show that iPSCs derived from LCLs using the Sendai virus procedure can be successfully differentiated into cardiomyocytes, neurons, and myotubes that express neuron- and myocyte-specific markers. We further show that these cardiac and neuronal cells are functional and generate action potentials that are required for cell excitability. We conclude that the ability to differentiate LCLs into neurons and myocytes will increase the use of LCLs in the future as a potential source of cells for modelling a number of diseases.
Stem cell research 2019

Permeability analyses and three dimensional imaging of interferon gamma-induced barrier disintegration in intestinal organoids.

M. Bardenbacher et al.

Abstract

The aberrant regulation of the epithelial barrier integrity is involved in many diseases of the digestive tract, including inflammatory bowel diseases and colorectal cancer. Intestinal epithelial cell organoid cultures provide new perspectives for analyses of the intestinal barrier in vitro. However, established methods of barrier function analyses from two dimensional cultures have to be adjusted to the analysis of three dimensional organoid structures. Here we describe the methodology for analysis of epithelial barrier function and molecular regulation in intestinal organoids. Barrier responses to interferon-$\gamma$ of intestinal organoids with and without epithelial cell-specific deletion of the interferon-$\gamma$-receptor 2 gene were used as a model system. The established method allowed monitoring of the kinetics of interferon-$\gamma$-induced permeability changes in living organoids. Proteolytic degradation and altered localization of the tight junction proteins claudin-2, -7, and - 15 was detected using confocal spinning disc microscopy with 3D reconstruction. Hessian analysis was used for quantification of re-localization of claudins. In summary, we provide a novel methodologic approach for quantitative analyses of intestinal epithelial barrier functions in the 3D organoid model.
Stem cells translational medicine 2018

Ex Vivo Expansion of CD34+ CD90+ CD49f+ Hematopoietic Stem and Progenitor Cells from Non-Enriched Umbilical Cord Blood with Azole Compounds.

S. Bari et al.

Abstract

Umbilical cord blood (UCB) transplants in adults have slower hematopoietic recovery compared to bone marrow (BM) or peripheral blood (PB) stem cells mainly due to low number of total nucleated cells and hematopoietic stem and progenitor cells (HSPC). As such in this study, we aimed to perform ex vivo expansion of UCB HSPC from non-enriched mononucleated cells (MNC) using novel azole-based small molecules. Freshly-thawed UCB-MNC were cultured in expansion medium supplemented with small molecules and basal cytokine cocktail. The effects of the expansion protocol were measured based on in vitro and in vivo assays. The proprietary library of {\textgreater}50 small molecules were developed using structure-activity-relationship studies of SB203580, a known p38-MAPK inhibitor. A particular analog, C7, resulted in 1,554.1 ± 27.8-fold increase of absolute viable CD45+ CD34+ CD38- CD45RA- progenitors which was at least 3.7-fold higher than control cultures (p {\textless} .001). In depth phenotypic analysis revealed {\textgreater}600-fold expansion of CD34+ /CD90+ /CD49f+ rare HSPCs coupled with significant (p {\textless} .01) increase of functional colonies from C7 treated cells. Transplantation of C7 expanded UCB grafts to immunodeficient mice resulted in significantly (p {\textless} .001) higher engraftment of human CD45+ and CD45+ CD34+ cells in the PB and BM by day 21 compared to non-expanded and cytokine expanded grafts. The C7 expanded grafts maintained long-term human multilineage chimerism in the BM of primary recipients with sustained human CD45 cell engraftment in secondary recipients. In conclusion, a small molecule, C7, could allow for clinical development of expanded UCB grafts without pre-culture stem cell enrichment that maintains in vitro and in vivo functionality. Stem Cells Translational Medicine 2018;7:376-393.
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.