Standardized generation of human pluripotent stem cell–derived cardiomyocytes

Kristin Becker, Jens-Peter Derks, Jona Drushku, Andreas Bosio, Sebastian Knöbel, and Dominik EckardtMiltenyi Biotec GmbH, Bergisch Gladbach, Germany

IntroductionHuman pluripotent stem cell (PSC)–derived cardio-myocytes (CMs) are of high interest for use in drug testing, heart disease modeling, and therapeutic ap-plications. Therefore, standardized protocols for the efficient generation of CMs are needed. Even though several protocols for cardiac differentiation have been published, the majority of them have to be ad-justed for each stem cell clone, e.g., by titration of small molecule and cytokine concentrations, in order

to obtain the optimal differentiation efficiency and cell yield. Moreover, lot-to-lot variations of media components also influence the outcome of differen-tiation. These protocol optimizations are costly and time consuming. In order to circumvent these limita-tions, we developed a cardiac differentiation medi-um, the StemMACS™ CardioDiff Kit, enabling robust and standardized cardiac differentiation, without the need for media adjustments.

Analysis of cardiac differentiation 3

Differentiation of hPSCs into atrial- or ventricular-like CMs4

Results

Conclusion

Cardiac differentiation of PSCs1

Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use. The CliniMACS. System components, including Reagents, Tubing Sets, Instruments, and PBS/EDTA Buffer, are designed, manufactured and tested under a quality system certified to ISO 13485.In the EU, the CliniMACS System components are available as CE-marked medical devices for their respective intended use, unless otherwise stated. The CliniMACS Reagents and Biotin Conjugates are intended for in vitro use only and are not designated for therapeutic use or direct infusion into patients. The CliniMACS Reagents in combination with the CliniMACS System are intended to separate human cells. Miltenyi Biotec as the manufacturer of the CliniMACS System does not give any recommendations regarding the use of separated cells for therapeutic purposes and does not make any claims regarding a clinical benefit. For the manufacturing and use of target cells in humans the national legislation and regulations – e.g. for the EU the Directive 2004/23/EC (“human tissues and cells”), or the Directive 2002/98/EC (“human blood and blood components”) – must be followed. Thus, any clinical application of the target cells is exclusively within the responsibility of the user of a CliniMACS System.In the US, the CliniMACS CD34 Reagent System, including the CliniMACS Plus Instrument, CliniMACS CD34 Reagent, CliniMACS Tubing Sets TS and LS, and the CliniMACS PBS/EDTA Buffer, is FDA approved; all other products of the CliniMACS Product Line are available for use only under an approved Investigational New Drug (IND) application or Investigational Device Exemp-tion (IDE). CliniMACS MicroBeads are for research use only and not for human therapeutic or diagnostic use. CliniMACS, CliniMACS Prodigy, MACS, and the MACS logo are registered trade-marks or trademarks of Miltenyi Biotec GmbH and/ or its affiliates in various countries worldwide. All other trademarks mentioned in this document are the property of their respective owners and are used for identification purposes only. Copyright © 2019 Miltenyi Biotec GmbH and/or its affiliates. All rights reserved.

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Cardiac fateinduction

Early mesoderm

Cardiac progenitors Cardiomyocytes

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Mesoderminduction

Cardiac differentiation of PSCs was induced stepwise by using three different media (fig. 1). First, PSCs were seeded as single cells in mesoderm induction medium on plates coated with Matrigel®. Cardiac fate was in-duced on day 2 of differentiation. Depending on the time point of differentiation, morphological changes could be observed. On day 1, cell clusters became visi-ble, which disappeared on day 3 of differentiation. From

days 4 to 6, cardiac clusters started to form (fig. 1). First contracting cells were observed on days 6–8 of differ-entiation. This new workflow protocol allows for robust, highly efficient, and scalable generation of CMs within less than 10 days of differentiation, thereby solving sev-eral technical issues related to the generation of PSC- derived CMs.

StemMACS™ CardioDiff Kit enables standardized CM differentiation 2

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Application of the protocol to different PSC clones yielded differentiation efficiencies of up to 90% as shown by flow cytometry analysis of cardiac troponin T (cTnT, fig. 2A). Moreover, the StemMACS™ CardioDiff Kit supported cardiac differentiation of PSCs that were cul-tured in StemMACS iPS-Brew XF, human or two other commercially available stem cell media (fig. 2A). Further experiments have shown the scalability of the protocol to different multiwell plate formats or 10-cm dishes, yielding similar differentiation efficiencies and num-bers of CMs/cm2 (fig. 2B). This is the basis for standard-

ized, automated generation and large-scale differenti-ation of PSC-derived CMs in the functionally closed system of the CliniMACS Prodigy®. In a next step, our workflow protocol for CM generation will be transferred to this GMP-compliant cell manufacturing platform, enabling the scale-up of CM manufacturing. First data indicated that 9×10⁷ CMs could be gene- rated in a single automated production run of the CliniMACS Prodigy connected to a one-layer cell stack system (fig. 2C). All graphs: n ≥ 3; means±SD; except data for one-layer cell stack (fig. 2C): n = 1.

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Final characterization by flow cytometry of CMs gener-ated by the StemMACS CardioDiff Kit showed the ex-pression of typical CM markers such as cTnT, alpha actinin, and MHC. Moreover, costaining of depletion and enrichment markers revealed compatibility with the PSC-derived Cardiomyocyte Isolation Kit, human, thus enabling further purification of CMs (fig. 3A).

Immunofluorescence staining for Kv1.5 and phospho- lamban (PLN) showed the presence of CMs with electri-cal properties and Ca2+ handling machinery, which en-ables further characterization through functional stud-ies based on the patch-clamp technique or the analysis of Ca2+ transients (fig. 3B).

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B C

Expression analysis of CM subtype markers, e.g., IRX4 and MYL2, revealed a ventricular-like CM subtype. In-terestingly, addition of a signaling pathway modulator to the StemMACS CardioDiff Kit at the stage of cardiac mesoderm specification enabled induction of an atri-al-like cell fate (fig. 4A). Gene expression analysis by qRT-PCR showed the up-regulation of known atrial markers, such as COUP-TFII, PITX2 (transcription fac-tors), KCNJ3, KCNA5 (ion channels), and down-regula-

tion of ventricular markers such as MYL2, IRX4, HEY2 (fig. 4B). This was in line with immunofluorescence data showing high expression levels of COUP-TFII after atrial fate induction. In contrast, COUP-TFII was not detected in CMs generated using the standard protocol (fig. 4C). In conclusion, the StemMACS Cardio Diff Kit generates CMs biased towards a ventricular-like fate, while the ad-dition of signaling pathway modulators can lead to an atrial-like fate switch.

The StemMACS CardioDiff Kit perfectly integrates into our complete workflow covering controlled cardiac dif-ferentiation as well as CM harvesting, purification, stor-age, and analysis. Transferring this complete workflow and the reagents to the automated cell processing plat-form of the CliniMACS Prodigy will pave the way for standardized, large-scale manufacturing of PSC- derived CMs.

• The StemMACS CardioDiff Kit enables short, robust, and scalable cardiac differentiation that can be applied to different stem cell clones.• Generated CMs show typical cardiac marker expression.• The StemMACS CardioDiff Kit generates CMs biased towards a ventricular-like fate, while the addition of signaling pathway modulators can lead to an atrial-like fate switch.

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1-layercell stack

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Addition of signaling pathway regulator

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iCARE has received funding from the German Federal Ministry of Education and Research within the funding program “innovations for individualized medicine” (Fkz 01EK1601D)

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