Standardized generation of human pluripotent stem cell–derived cardiomyocytes

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

Introduction 3 Analysis of cardiac differentiation

Human pluripotent stem cell (PSC)–derived cardio- to obtain the optimal differentiation efficiency and A myocytes (CMs) are of high interest for use in drug cell yield. Moreover, lot-to-lot variations of media testing, disease modeling, and therapeutic ap- components also influence the outcome of differen- plications. Therefore, standardized protocols for the tiation. These protocol optimizations are costly and efficient generation of CMs are needed. Even though time consuming. In order to circumvent these limita- several protocols for cardiac differentiation have tions, we developed a cardiac differentiation medi- been published, the majority of them have to be ad- um, the StemMACS™ CardioDiff Kit, enabling robust justed for each stem cell clone, e.g., by titration of and standardized cardiac differentiation, without the small molecule and cytokine concentrations, in order need for media adjustments. B Results

cTnT NKX2.5 Kv1.5 cTnT–FITC cTnT PLN DRAQ5 cTnT MLC2a 1 Cardiac differentiation of PSCs Figure 3 Final characterization by flow cytometry of CMs gener- Immunofluorescence staining for Kv1.5 and phospho- A ated by the StemMACS CardioDiff Kit showed the ex- lamban (PLN) showed the presence of CMs with electri- pression of typical CM markers such as cTnT, alpha cal properties and Ca2+ handling machinery, which en-

Day 0 Day 2 actinin, and MHC. Moreover, costaining of depletion ables further characterization through functional stud- Day 1 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Mesoderm Cardiac fate and enrichment markers revealed compatibility with ies based on the patch-clamp technique or the analysis induction induction the PSC-derived Cardiomyocyte Isolation Kit, human, of Ca2+ transients (fig. 3B).

Medium 1 Medium 2 Medium 3 Medium 2 thus enabling further purification of CMs (fig. 3A).

Early Cardiac Cardiomyocytes mesoderm progenitors Differentiation of hPSCs into atrial- or ventricular-like CMs B 4

A

Day 0 Day 2 Day 1 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Mesoderm Cardiac fate induction induction

Figure 1 Medium 1 Medium 2 Medium 3 Medium 2

Addition of signaling pathway regulator Cardiac differentiation of PSCs was induced stepwise days 4 to 6, cardiac clusters started to form (fig. 1). First by using three different media (fig. 1). First, PSCs were contracting cells were observed on days 6–8 of differ- seeded as single cells in mesoderm induction medium entiation. This new workflow protocol allows for robust, B C on plates coated with Matrigel®. Cardiac fate was in- highly efficient, and scalable generation of CMs within 10.00 Standard Modified condition duced on day 2 of differentiation. Depending on the less than 10 days of differentiation, thereby solving sev- 8.00 time point of differentiation, morphological changes eral technical issues related to the generation of PSC- 6.00 could be observed. On day 1, cell clusters became visi- derived CMs. 4.00 ble, which disappeared on day 3 of differentiation. From 2.00 0.00

PITX2 HEY1 TBX3 HCN4 KCNA5 KCNJ3 MYH6 MYL7 SHOX2 TNNT2 KNCJ2 −2.00 HAND1 COUP-TFII −4.00 StemMACS™ CardioDiff Kit HEY2 MYL2 cTnT COUP-TFII DRAQ5 cTnT COUP-TFII DRAQ5

Fold change (log2) RA/STFold change (log2) −6.00 MYH7 enables standardized CM differentiation IRX4 2 −8.00 −10.00 A C 100 100 1.0×108 Figure 4 90 90 9.0×107 7 cells 8.0×10 80 80 + cells cells + + 70 70 7.0×107 Expression analysis of CM subtype markers, e.g., IRX4 tion of ventricular markers such as MYL2, IRX4, HEY2 60 60 6.0×107 and MYL2, revealed a ventricular-like CM subtype. In- (fig. 4B). This was in line with immunofluorescence data 50 50 5.0×107 40 40 4.0×107 terestingly, addition of a signaling pathway modulator showing high expression levels of COUP-TFII after atrial 30 30 3.0×107 to the StemMACS CardioDiff Kit at the stage of cardiac fate induction. In contrast, COUP-TFII was not detected 20 20 2.0×107

Percentage of cTnT of Percentage mesoderm specification enabled induction of an atri- in CMs generated using the standard protocol (fig. 4C). Percentage of cTnT of Percentage 7

10 10 cTnT of number Total 1.0×10 0 0 0.0×107 al-like cell fate (fig. 4A). expression analysis by In conclusion, the StemMACS Cardio Diff Kit generates PSC PSC PSC iPSC iPSC iPS Brew 12-well 6-well 10-cm 1-layer clone 1 clone 2 clone 3 medium 1 medium 2 XF plate plate dish cell stack qRT-PCR showed the up-regulation of known atrial CMs biased towards a ventricular-like fate, while the ad- B markers, such as COUP-TFII, PITX2 (transcription fac- dition of signaling pathway modulators can lead to an 100 90 4.5×105 tors), KCNJ3, KCNA5 (ion channels), and down-regula- atrial-like fate switch. 2 80 4.0×105 cells + 70 3.5×105 5

60 cells/cm 3.0×10 + 50 2.5×105 40 2.0×105 30 1.5×105 20 1.0×105 Conclusion Percentage of cTnT of Percentage 10 0.5×105 0 Number of cTnT 0.0×105 12-well 6-well 10-cm 12-well 6-well 10cm The StemMACS CardioDiff Kit perfectly integrates into • The StemMACS CardioDiff Kit enables short, robust, plate plate dish plate plate dish our complete workflow covering controlled cardiac dif- and scalable cardiac differentiation that can be Figure 2 ferentiation as well as CM harvesting, purification, stor- applied to different stem cell clones. age, and analysis. Transferring this complete workflow • Generated CMs show typical cardiac marker Application of the protocol to different PSC clones ized, automated generation and large-scale differenti- and the reagents to the automated cell processing plat- expression. yielded differentiation efficiencies of up to 90% as ation of PSC-derived CMs in the functionally closed form of the CliniMACS Prodigy will pave the way • The StemMACS CardioDiff Kit generates CMs shown by flow cytometry analysis of cardiac troponin T system of the CliniMACS Prodigy®. In a next step, our for standardized, large-scale manufacturing of PSC- biased towards a ventricular-like fate, while the (cTnT, fig. 2A). Moreover, the StemMACS™ CardioDiff Kit workflow protocol for CM generation will be transferred derived CMs. addition of signaling pathway modulators can lead supported cardiac differentiation of PSCs that were cul- to this GMP-compliant cell manufacturing platform, to an atrial-like fate switch. tured in StemMACS iPS-Brew XF, human or two other enabling the scale-up of CM manufacturing. First commercially available stem cell media (fig. 2A). Further data indicated that 9×10⁷ CMs could be gene- experiments have shown the scalability of the protocol rated in a single automated production run of the to different multiwell plate formats or 10-cm dishes, CliniMACS Prodigy connected to a one-layer cell stack 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. yielding similar differentiation efficiencies and num- system (fig. 2C). All graphs: n ≥ 3; means±SD; except 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 2 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 bers of CMs/cm (fig. 2B). This is the basis for standard- data for one-layer cell stack (fig. 2C): n = 1. 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- iCARE has received funding from the German Federal Ministry of Education and Research within the funding 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 program “innovations for individualized medicine” (Fkz 01EK1601D) owners and are used for identification purposes only. Copyright © 2019 Miltenyi Biotec GmbH and/or its affiliates. All rights reserved.