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As a global market leader with numerous subsidiaries and distributors, Miltenyi Biotec is committed to providing our customers around the world with the highest quality products. In addition to direct selling in more than 20 countries in North America, Europe and Asia/Pacific, Miltenyi Biotec also provides support for our customers through an extensive distributor network covering dozens of additional countries.
In this application protocol, we describe the differentiation of human pluripotent stem cells (hPSCs) into cardiomyocytes, the isolation of the resulting cardiomyocytes, and their analysis using flow cytometry or immunofluorescence microscopy.
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With this protocol, cardiomyocytes can be generated from hPSCs with efficacies of up to 80%, giving rise to contracting cells in 9–12 days.
▲ Note: Testing of various cell numbers and CHIR99021 concentrations is recommended to optimize differentiation for each individual hPSC line. The diagram below provides a graphical overview of the procedure.
▲Note: The effectiveness of differentiation depends on the number of seeded cells. Therefore, it is crucial to determine the cell number that leads to optimal differentiation. Different cell numbers might be required for different hPSC lines.
Replace medium in each well with 1 mL StemMACS iPS-Brew XF.
Replace medium in each well with 2 mL of RPMI 1640 + L-glutamine + B-27 Supplement, minus insulin.
Replace medium in each well with 2 mL of RPMI 1640 + L-glutamine + B-27 Supplement, minus insulin.
Replace medium in each well with 2 mL of RPMI 1640 + L-glutamine + B-27 Supplement w/insulin, and continue to do so every 2–3 days.
The development of highly efficient cardiac-directed differentiation methods makes it possible to generate large numbers of cardiomyocytes.1,4 However, due to varying differentiation efficiencies, cardiomyocyte populations must be further enriched for downstream applications.
To achieve highest possible purity and recovery during cell separation, first dissociate the monolayer cultures into a single-cell suspension using the Multi Tissue Dissociation Kit 3.
▲ Note: Perform all steps under sterile conditions.
▲ Note: The dissociation protocol is optimized for use with 12-well or 6-well plates.
Separate the cardiomyocytes using the PSC-Derived Cardiomyocyte Isolation Kit, human. Follow the protocol of the kit data sheet.
The isolation is performed either as a single-step or two-step protocol, depending on the initial differentiation efficiency (see isolation strategy below). As a general guideline, we recommend performing the second (positive selection) step if the culture contains <50 % of cardiomyocytes.
The standard protocol enables enrichment of cardiomyocytes from up to 5×10⁶ total cells, and upscaling is possible for up to 1×10⁷ total cells.
Download kit data sheet
In recent years, various protocols to differentiate PSCs into cardiomyocytes have been published. However, the resulting populations are not homogeneous, but rather composed of a variety of cardiomyocyte subtypes or subpopulations, as well as non-cardiomyocyte cell types.
Different cardiomyocyte subpopulations can be identified and analyzed using techniques such as patch clamp, immunocytochemistry or flow cytometry. Compared to other techniques, flow cytometry is a fast and efficient method to analyze and quantify different cell types within a cell population, without difficult techniques or genetic engineering of the starting material.
Besides analysis by flow cytometry, cardiomyocytes are often examined by microscopy using immunofluorescence staining. Recombinantly engineered REAfinity™ Antibodies enable unambiguous analysis of cardiomyocytes.
The following is a list of relevant resources that might be of interest:
Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use.
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Column | Max. number of labeled cells | Max. number of total cells | Separator |
---|---|---|---|
Depletion or positive selection | |||
LS | 5×10⁶ | 5×10⁶ | MidiMACS™, QuadroMACS™, VarioMACS, SuperMACS II |
autoMACS | 1×107 | 1×107 | autoMACS Pro |
▲ Note: Column adapters are required to insert certain columns into the VarioMACS™ or SuperMACS™ II Separators. For details refer to the respective MACS Separator data sheet.
Copyright © 2019 Miltenyi Biotec and/or its affiliates. All rights reserved.