Title: Building blocks of the human heart

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Cover Page

The handle http://hdl.handle.net/1887/78948 holds various files of this Leiden University dissertation.

Author: Giacomelli, E.

Title: Building blocks of the human heart

Issue Date: 2019-10-01

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CHAPTER 4

Co-differentiation of human pluripotent stem cells-derived cardiomyocytes and endothelial cells from cardiac mesoderm provides a three-dimensional model of cardiac microtissue

Elisa Giacomelli¹, Milena Bellin^{1, *}, Valeria V Orlova^{1, *}, Christine L Mummery^{1, 2}

1 Department of Anatomy and Embryology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC, Leiden, The Netherlands

2 Department of Applied Stem Cell Technologies, University of Twente, Building Zuidhorst, 7500 AE, Enschede, The Netherlands

* These authors contributed equally to this work

Published in Current protocols in human genetics; 2017 Oct 18; 95:21.9.1-21.9.22. doi: 10.1002/

cphg.46.

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Abstract

The formation of cardiac mesodermal subtypes is highly regulated in time and space during heart development. In vitro models based on human pluripotent stem cells (hPSCs) provide opportunities to study mechanisms underlying fate choices governing lineage specification from common cardiovascular progenitors in human embryos. The generation of cardiac endothelial cells in particular allows the creation of complex models of cardiovascular disorders in which either cardiomyocytes or endothelial cells are affected. Here, we describe our protocol for co-differentiation of cardiomyocytes and endothelial cells from cardiac mesoderm using hPSCs. We provide precise details for the enrichment of each cell population from heterogeneous differentiated cultures, describe how to maintain and dissociate enriched cardiomyocytes, and expand and cryopreserve enriched endothelial cells. We then describe how to generate and culture three-dimensional cardiac microtissues from these cell populations and finally provide guidelines for the characterization of microtissues by immunofluorescent staining and re-plating for downstream applications.

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4 Introduction

This unit describes two basic protocols for (i) simultaneous differentiation towards cardiac and endothelial cell fates from cardiac mesoderm using human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), collectively called human pluripotent stem cells (hPSCs) (Basic Protocol 1), and (ii) the generation and culture of three-dimensional (3D) cardiac microtissues from either enriched cardiomyocytes cultured alone (MT-CM), or in combination with enriched endothelial cells (MT-CMEC), (Basic Protocol 2), as described previously (Giacomelli et al., 2017).

All methods described here have been robustly reproduced in both NKX2.5^eGFP/w hESCs and in wild-type hiPSCs described previously (Elliott et al., 2011; Zhang et al., 2014). Details are provided in the protocols but globally, the process is as follows:

Prior to cardiac mesoderm induction, hPSCs are seeded on human recombinant vitronectin (VTN-N) and grown in chemically defined E8™ medium. E8™

medium is refreshed every day and both hESC and hiPSC lines are passaged non-enzymatically using EDTA at the same cell seeding density of 20 × 10³/ cm²or 12 × 10³/cm²every 3 or 4 days, respectively, by which time they have become 65% to 85% confluent. In hiPSCs only, RevitaCell™ supplement is used for 24 hr after splitting the culture to improve cell survival. Isolation of the resultant cardiomyocyte and endothelial cell populations is described in two support protocols: Support Protocol 1 describes isolation, maintenance and dissociation of VCAM1⁺ cardiomyocytes, while Support Protocol 2 describes isolation, expansion and cryopreservation of CD34⁺ endothelial cells. Finally, Support Protocol 3 describes characterization of 3D cardiac microtissues by immunofluorescent staining and re-plating for downstream applications.

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Basic Protocol 1

Co-differentiation of cardiomyocytes and endothelial cells from cardiac mesoderm using human pluripotent stem cells

The protocol for co-differentiation of cardiomyocytes and endothelial cells from cardiac mesoderm, termed CMEC (Figure 1A), is designed to be fast, reproducible and cost effective, taking only 6 and 9-10 days respectively from induction of cardiac mesoderm through to appearance of endothelial cells and cardiomyocytes (Giacomelli et al., 2017).

In this protocol, NKX2.5^eGFP/w hESCs (Elliott et al., 2011) and hiPSCs (Zhang et al., 2014) are seeded on Matrigel™ the day before differentiation (day -1). At day 0, differentiation toward cardiac mesoderm is induced in monolayer as previously described (Elliott et al., 2011; van den Berg et al., 2016) by changing E8™ to BPEL medium (Ng et al., 2008) supplemented with a mixture of cytokines (20 ng/mL BMP4; 20 ng/mL ACTIVIN A; 1.5 μM GSK3 inhibitor CHIR99021) (Figure 1B ; Movie 1). After 3 days, simultaneous specification into cardiomyocyte and endothelial cell fates is induced by combination of the Wnt inhibitor XAV939 (5 μM) and VEGF (50 ng/ml) (Figure 1B). BPEL medium supplemented with VEGF is refreshed on day 6 (Figure 1B) and after that every 3 days. Cell morphology and contracting areas of differentiated cultures on day 14 are shown in Figure 2A-B and in Movies 2-3, respectively.

The major factors requiring optimization for successful differentiation are the seeding density (12.5 – 25 × 10³/cm²range) and the time in hours from plating of hPSCs through to induction of cardiac mesoderm (~24).

Notably, we have previously demonstrated that supplementation of VEGF and XAV939 does not affect either cardiomyocyte electrophysiological phenotype or endothelial cell differentiation (Giacomelli et al., 2017). Furthermore, supplementation of XAV939 results in a higher yield of CD34⁺endothelial cells compared to the condition in which XAV939 is not present, termed EC condition (Table 1) (Giacomelli et al., 2017).

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Figure 1. (A) Schematic showing the differentiation protocol towards cardiomyocyte and endothelial cell fates, termed CMEC. Cardiac mesoderm is induced in BPEL medium supplemented with BMP4, ACTIVIN A and CHIR99021 from day 0-3, followed by BPEL supplemented with XAV939 and VEGF on day 3. BPEL medium supplemented with VEGF is then refreshed every 3 days. (B) Cell morphology at the indicated time points (d=day) of differentiating hESC (top panel) and hiPSC (bottom panel) cultures upon CMEC. Objective 4x; Scale Bar 200 μm

Condition Starting n. of wells (6-well plate)

Percentage of CD34⁺ Number of CD34⁺ cells

hESC-EC 2 19 ~300,000

hESC-CMEC 1 33 ~600,000

hiPSC-EC 2 10 ~180,000

hiPSC-CMEC 1 34 ~400,000

Table 1. Percentages and numbers of CD34⁺ endothelial cells obtained after isolation from either EC (no XAV supplementation) or CMEC (XAV supplementation) protocol of differentiation, starting from

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Materials

• Human embryonic stem cells (NKX2.5^eGFP/w hESCs; (Elliott et al., 2011)

• Human induced pluripotent stem cells (hiPSCs; (Zhang et al., 2014)

• E8™ medium (Gibco; cat. no. A1517001; with penicillin-streptomycin; see recipe)

• RevitaCell™ Supplement (100X; Gibco; cat. no. A2644501)

• EDTA 0.5 mM (see recipe)

• BPEL medium (see recipe)

• BPEL medium for cardiac mesoderm induction (with BMP; ACTIVIN A;

CHIR99021; see recipe)

• BPEL medium for cardiomyocyte and endothelial cells specification (with XAV939 and VEGF; see recipe)

• BPEL medium for cardiomyocyte and endothelial cells maintenance (with VEGF; see recipe)

• VTN-N-coated (see recipe) and Matrigel-coated (see recipe) 6-well plates (Greiner Bio-One; cat. no. 657160)

• Polystyrene conical tubes 15 ml (Corning Falcon; cat. no. 352097) and 50 ml (Corning Falcon; cat. no.352098)

• Dulbecco’s phosphate-buffered saline without Ca²⁺ or Mg²⁺ (DPBS 1X; Gibco, cat. no. 14190-094)

• Liquid Nitrogen (Liquid N₂)

• Centrifuge (Eppendorf; cat. no. 5810R)

• CO₂ cell culture incubator (Sanyo)

Figure 2. Cell Morphology of day 14 hESC (A) and hiPSC (B) cultures differentiated upon CMEC.

Objective 4x; Scale Bar 200 μm

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• Sterile biosafety cabinet (CleanAir)

• Sterile plastic pipette (Greiner Bio-One; 5 ml, cat. no. 606180; 10 ml, cat. no.

607180; 25 ml, cat. no. 760180)

• Filter tips (Corning; 10 μl, cat. no. 4807; 200 μl, cat. no. 4810; 1,000 μl, cat. no.

4809)

• Pipetman starter kit P2, P10 and P100 (Gilson International; cat. no. F167500S)

• Pipetman starter kit P20, P200 and P1000 (Gilson International; cat. no.

F167300)

• Manual Cell Counter

• Freezer, MDF-U73V, -80°C (Sanyo Electric)

• Freezer, -20°C (Liebherr profi line)

• Fridge, 4°C (Liebherr profi line)

• Microscope (Nikon Eclipse T)

• Microscope (Leitz Diavert; 987072 80 M7-469)

• Water bath (Julabo TW20; MCO-18AIC Serial no. 08010017)

Thawing and initial plating of hESCs and hiPSCs

1. Fill a 10 ml plastic pipet with 5 ml of E8™ medium and transfer the 5 ml to a 15 ml conical tube.

2. Remove 1 vial (either hESC or hiPSC line) from liquid N₂ and place it in a 37°C water bath until only a sliver of ice remains. With a P1000 (1 ml) pipet tip, transfer the content of the vial dropwise to the 15 ml conical tube containing 5 ml of medium while gently tapping the tube, wash out vial with 1 ml of E8™ medium and transfer the remainder to the conical tube.

3. Centrifuge 3 min at 300 x g, room temperature. Aspirate supernatant.

Resuspend in 2 ml of E8™ and transfer to 1 well of a VTN-N-coated 6-well plate.

For hiPSCs only, the addition of RevitaCell™ (1:200) in E8™ medium improves cell survival after dissociation, which enhances the consistency of plating.

4. Change medium every 24 hr with E8™ (without RevitaCell™).

Passaging and expansion of hPSCs

Ideally, cells should have reached 65% to 85% confluence in 3 to 4 days (adjust cell seeding density at 20 × 10³/cm²or 12 × 10³/cm²for passaging every 3 or 4 days respectively).

5. Aspirate culture medium from the well.

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8. Add 1 ml of 0.5 mM EDTA.

9. Incubate hESCs for 3 min in the incubator at 37°C, whereas hiPSCs for 4 min in the incubator at 37°C followed by 1 min at room temperature.

10. Aspirate EDTA.

11. Using a P1000 (1 ml) pipet tip, add 1 ml of E8™ medium to the well and blast medium against cell surface to dissociate cells (cells should come off easily after pipetting in this way ~10 times). Transfer the content of the well to a 15 ml conical tube, wash out the well with 1 ml of E8™ medium and transfer the remainder to the conical tube.

12. Count cells.

13. Aspirate VTN-N from two wells of a new 6-well plate and add 2 ml of E8™ to each well (supplemented with RevitaCell™ 1:200 for hiPSCs).

14. Plate 200,000 or 120,000 cells per well, which will be ready to be passaged after 3 or 4 days respectively.

15. Place the plate in the incubator at 37°C and change medium every 24 hr with E8™ (without RevitaCell™).

NOTE: In this protocol, we aim to keep the hPSCs in the logarithmic growth phase, as previously described in (Burridge et al., 2015).

Day -1: Seeding hPSCs for differentiation

From initial thawing, hPSCs usually require ~one week to adapt to culture conditions and become ready for differentiation.

16. In addition to steps 13, 14 and 15, seed 125,000 cells per well into a Matrigel- coated 6-well plate into 2 ml of E8™ medium per well (supplemented with RevitaCell™ 1:200 for hiPSCs).

17. Place the plate in the incubator at 37°C.

Day 0: Cardiac mesoderm induction 18. Aspirate medium from wells.

19. Wash each well with 2 ml of DPBS.

20. Aspirate DPBS.

21. Wash each well with 1 ml of BPEL medium.

22. Add 3 ml of BPEL medium for cardiac mesoderm induction (with BMP4;

ACTIVIN A; CHIR99021).

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NOTE: To improve differentiation efficiency and reproducibility, we recommend inducing cardiac mesoderm 24 hr after seeding of pluripotent cells.

Day 3: Co-differentiation of cardiomyocyte and endothelial cells 23. Aspirate medium from wells.

24. Add 3 ml per well of BPEL medium for cardiomyocyte and endothelial cells specification (with XAV939 and VEGF).

NOTE: To improve differentiation efficiency and reproducibility, we recommend performing this step 72 hr after induction of cardiac mesoderm.

Days 6, 9, 12, and every next 3 days: Maintenance of cardiomyocyte and endothelial cells

25. Aspirate medium from wells.

26. Add 3 ml of BPEL medium for cardiomyocyte and endothelial cells maintenance (with VEGF).

Support Protocol 1

Isolation, culture and dissociation of VCAM1⁺cardiomyocytes

HPSC-derived cardiomyocytes can be isolated from heterogeneous differentiated cultures by immunomag netic selection with anti-VCAM1 antibody–coupled magnetic beads, as described previously (Uosaki et al., 2011; Wang et al., 2014). Following 14 to 17 days in culture, differentiated hPSC cultures are first labeled with an anti-VCAM1-PE-conjugated antibody and next targeted with tetrameric antibody complexes recognizing PE and dextran- coated magnetic particles. VCAM1⁺ cells remain in the positive fraction tube while VCAM1^- cells are poured off in the negative or waste fraction tubes. Follow the manufacturer’s instructions for use of the magnet and isolation procedure.

Expected results are approximately ~80% VCAM1⁺ cells after isolation (Giacomelli et al., 2017). Re-plated VCAM1⁺ cells re-form spontaneously contracting networks (Figure 3A-B; Movies 4-5) and display characteristic sarcomeric structures that stain positively for troponin I (TNNI) and a-ACTININ (Figure 3C-D).

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Figure 3. Cell Morphology of VCAM1⁺cardiomyocytes differentiated from hESC (A) and hiPSC (B) after isolation and re-plating. Objective 10x; Scale Bar 100 μm (C) Immunofluorescence images of cardiac sarcomeric proteins TNNI (green) and a-ACTININ (red) in VCAM1⁺cardiomyocytes generated from NKX2.5^eGFP/w hESCs and (D) hiPSCs. Nuclei are stained in blue with DAPI. Objective 63x;

Scale bar: 50 μm.

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Materials

• hPSC-derived cardiomyocytes (Basic Protocol 1)

• TrypLE Select 1X (Gibco; cat. no. 12563029)

• TrypLE 2X (see recipe)

• FACS buffer (see recipe)

• EasySep™ buffer (see recipe)

• Matrigel-coated (see recipe) 24-well plates (Greiner Bio-One; cat. no.

662160)

• Dulbecco’s phosphate-buffered saline without Ca²⁺ or Mg²⁺ (DPBS 1X;

Gibco; cat. no. 14190-094)

• Round-bottom 5 ml FACS tubes (BD Biosciences; cat. no. 352058)

• EasySep™ Purple Easy magnet (StemCell Technologies; cat. no. 18000)

• EasySep™ PE Positive Selection Kit (StemCell Technologies; cat. no. 18557)

• Anti-VCAM1-PE antibody (R&D; cat. no. FAB5649P)

• Centrifuge (Eppendorf; cat. no. 5810R)

• CO₂cell culture incubator (Sanyo)

607180; 25 ml, cat. no. 760180)

• Filter tips (Corning; 10 μl, cat. no. 4807; 200 μl, cat. no. 4810; 1,000 μl, cat.

no. 4809)

F167500S)

F167300)

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Day 14-17: Isolation of VCAM1⁺ cardiomyocytes from hPSCs

To obtain a high cardiomyocyte yield (up to ~4 million VCAM1⁺ cardiomyocytes), we recommend starting from 6 wells of a 6-well plate of differentiated cultures in the absence of VEGF (CM condition) (Giacomelli et al., 2017).

3. Aspirate DPBS.

4. Add 1.5 ml of TrypLE 2X per well, then incubate for 5 min at 37°C.

5. Gently pipet up and down using a P-1000 (1 ml) pipet tip ~5 times to break up aggregates. Avoid forming bubbles.

6. Transfer cells to 2 x 50 ml conical tubes (3 wells per tube).

7. Wash each 3 wells with 1 ml of TrypLE 2X and transfer cells into the same 50 ml tubes.

8. Wash each 3 wells with 1 ml of BPEL medium and transfer cells into the same 50 ml tubes.

9. Top up each tube with 10 ml of BPEL medium.

10. Centrifuge 3 min at 300 x g at room temperature.

11. If needed, repeat step 10.

12. Aspirate supernatant.

13. Resuspend pellets in 1 ml of FACS buffer and combine them in one round- bottom 5 ml FACS tube.

16. Resuspend in VCAM1-PE antibody dilution containing 20 ml of VCAM1-PE antibody in 500 ml of FACS buffer.

17. Stain 30 min in the fridge at 4°C.

18. After 30 min, wash with 1 ml of FACS buffer.

21. Resuspend in 1 ml of EasySep™ buffer.

22. Take a 50 ml aliquot (pre isolation fraction), resuspend in 1 ml of FACS buffer, centrifuge 3 min at 300 x g at room temperature, resuspend in 200 ml of FACS buffer to measure the expression of VCAM1 by flow cytometer (step 39).

23. Add 100 ml of EasySep™ PE selection cocktail. Mix well and incubate 15 min at room temperature.

24. Prepare a 15 ml tube (VCAM1 negative fraction) and a 50 ml tube (waste fraction).

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25. Mix EasySep™ Magnetic Nanoparticles by pipetting up and down ~5 times using a P-200 pipet tip.

26. Add 50 µL of EasySep™ Magnetic Nanoparticles. Mix well and incubate 10 min at room temperature.

27. Top up the 5 ml tube with 1.5 ml of EasySep™ buffer.

28. Gently pipet up and down with a P-1000 (1 ml) pipet tip 2 times. Avoid forming bubbles.

29. Place the 5 ml FACS tube (without cap) into the EasySep™ magnet and set aside for 5 min.

30. Pick up the magnet (with the FACS tube still inserted) and in one continuous motion invert the magnet and the FACS tube, pouring off the supernatant fraction into the VCAM1 negative fraction 15 ml tube. The magnetically labelled cells will remain inside the tube.

31. Remove the 5 ml FACS tube from the magnet.

32. Wash with 2.5 mL of EasySep™ buffer (1^st wash).

33. Gently pipet up and down twice using a P-1000 (1 ml) pipet tip. Avoid forming bubbles.

34. Place the 5 ml FACS tube back in the magnet and set aside for 5 min.

35. Repeat Steps 30, 31 and 32 pouring off the supernatant fraction into the waste fraction 50 ml tube and for a total of 3 washes.

36. Remove the 5 ml FACS tube from the magnet and resuspend in 4 ml of BPEL medium.

37. Take a 50 ml aliquot (post isolation fraction), resuspend in 1 ml of FACS buffer, centrifuge 3 min at 300 x g at room temperature, resuspend in 200 ml of FACS buffer to measure the expression of VCAM1 by flow cytometer (step 39).

38. Centrifuge the waste and VCAM1 negative fractions 3 min at 300 x g at room temperature, aspirate supernatant, resuspend in 1 ml of EasySep™

buffer and take a 200 ml aliquot to measure the expression of VCAM1 by flow cytometer (step 39).

39. Analyze aliquots from steps 22, 37 and 38 with flow cytometer such as MACSQuant™ VYB (Miltenyi Biotech) equipped with a violet (405 nm), blue (488 nm) and yellow (561 nm) laser following instrument manufacturer’s instructions.

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Seeding and culture of VCAM1⁺ cardiomyocytes 40. Count cells from step 36.

41. Dilute cells in BPEL medium to seed ~450,000 cells per well in a Matrigel- coated 24-well-plate.

42. Change medium the day after and every 3-4 days.

43. Cells should re-form contracting networks after 2-3 days.

Dissociating and re-plating of VCAM1⁺ cardiomyocytes

This procedure describes dissociation of VCAM1⁺ cardiomyocytes from 1 well of a 24-well plate at a split ratio of 1:6 to 1:8. In our experience, this is suitable for re-plating of cardiomyocytes for downstream applications such as Patch Clamp electrophysiology or immunofluorescent staining (Giacomelli et al., 2017).

44. Aspirate medium from the well.

45. Wash well with 1 ml of DPBS.

46. Aspirate DPBS.

47. Add 1 ml of TrypLE 1X and incubate for 4 min at 37°C.

48. Dilute TrypLE by adding 1.5 ml of BPEL medium to the well.

49. Using a P-1000 (1 ml) pipet tip, transfer cells to a 15 ml conical tube.

50. Top up the 15 ml tube with 5 ml of BPEL medium.

53. Resuspend in 1 ml of BPEL medium.

54. Dilute cells in BPEL medium to distribute them in 6-8 wells (1:6-1:8 ratio) of a Matrigel-coated 24-well plate, in 500 µL of BPEL per well.

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Support Protocol 2

Isolation, culture and cryopreservation of CD34⁺endothelial cells As for the cardiomyocyte population, hPSC-derived endothelial cells can be isolated from heterogeneous differentiated cultures by immunomagnetic selection with anti-CD34 antibody–coupled magnetic beads, as described previously (Lian et al., 2014). Following 6 days in culture, hPSC- derived CD34⁺ cells are isolated using the EasySep™ CD34 Human Cord Blood Isolation Kit II according to manufacturer’s custom protocol. Briefly, endothelial cells are targeted with tetrameric antibody complexes recognizing the endothelial cell surface marker CD34 and dextran-coated magnetic particles.

As with the cardiomyocyte purification strategy, labeled cells are separated using a magnet; CD34⁺ cells remain in the positive fraction tube while CD34^- cells are poured off in the negative and waste fraction tubes.

Expected results are > 95% CD34⁺ cells after isolation (Giacomelli et al., 2017).

Re-plated CD34⁺cells are highly proliferative, reaching confluence within 3 or 4 days, and re-display the typical endothelial morphology after freezing and thawing (Figure 4A-B). Notably, when compared to primary cardiac and non cardiac endothelial cells by RT-qPCR, our day 6 CD34⁺ endothelial cells cluster with primary Human Cardiac Microvascular Endothelial Cells (HCMEC), suggesting cardiac endothelium identity (Giacomelli et al., 2017).

Figure 4. Cell Morphology of CD34⁺ endothelial cells differentiated from hESC (A) and hiPSC (B) after freezing and thawing. Objective 10x; Scale Bar 100 μm

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Materials

• hPSC-derived endothelial cells (Basic Protocol 1)

• TrypLE Select 1X (Gibco; cat. no. 12563029)

• FACS buffer (see recipe)

• EasySep™ buffer (see recipe)

• BPEL for endothelial cell maintenance (with VEGF; see recipe)

• Fibronectin-coated (see recipe) 6-well plates (Greiner Bio-One; cat. no.

657160)

• Dulbecco’s phosphate-buffered saline without Ca²⁺ or Mg²⁺ (DPBS 1X; Gibco, cat. no. 14190-094)

• Round-bottom 5 ml FACS tubes (BD Biosciences; cat. no. 352058)

• EasySep™ Purple Easy magnet (StemCell Technologies; cat. no. 18000)

• EasySep™ CD34 Human Cord Blood Isolation Kit II (StemCell Technologies;

cat. no. 18309)

• Anti-CD34-APC antibody (Miltenyi Biotech; cat. no.130-090-954)

• Cryovials (Greiner Bio-One; cat. No. 123263)

• Nalgene Cryo 1 °C freezing container (Thermo Scientific; cat. No. 6100-0001)

• Liquid Nitrogen (Liquid N₂)

• Centrifuge (Eppendorf, cat. no. 5810R)

607180; 25 ml, cat. no. 760180)

• Filter tips (Corning; 10 μl, cat. no. 4807; 200 μl, cat. no. 4810; 1,000 μl, cat. no.

4809)

• Pipetman starter kit P2, P10 and P100 (Gilson International; cat. no. F167500S)

F167300)

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Day 6: Isolation of CD34⁺ endothelial cells from hPSCs

To obtain a high endothelial cell yield (up to 4 million CD34⁺ endothelial cells), we recommend starting from 6 wells of a 6-well plate of differentiated cultures upon CMEC.

3. Aspirate DPBS.

4. Add 1 ml of TrypLE 1X per well, then incubate for 5 min at 37°C.

5. Dilute TrypLE by adding 1 ml of BPEL medium per well.

6. Gently pipet up and down using a P-1000 (1 ml) pipet tip and transfer cells to a 50 ml conical tube. Avoid forming bubbles.

7. Wash each well with 1 ml of BPEL medium and transfer cells into the same 50 ml tube.

10. Resuspend in 1 ml of EasySep™ buffer and transfer to a 5 ml FACS tube.

11. Take a 50 ml aliquot (pre isolation fraction) and place it in the fridge at 4°C.

12. From step 10, add 50 ml of EasySep™ CD34 antibody cocktail. Mix well and incubate 3 min at room temperature.

13. Prepare a 15 ml tube (CD34 negative fraction) and a 50 ml tube (waste fraction).

14. Vortex EasySep™ Dextran RapidSpheres™ for 30 sec to ensure they are in a uniform suspension.

15. Add 50 µL of EasySep™ Dextran RapidSpheres™. Mix well and incubate 3 min at room temperature.

16. Top up the 5 ml tube with 1.5 ml of EasySep™ buffer.

17. Gently pipet up and down using a P-1000 (1 ml) pipet tip 2 times. Avoid forming bubbles.

18. Place the 5 ml FACS tube (without cap) into the EasySep™ magnet and set aside for 3 min.

19. Pick up the magnet (with the FACS tube still inserted) and in one continuous motion invert the magnet and the FACS tube, pouring off the supernatant fraction into the CD34 negative fraction 15 ml tube. The magnetically labelled cells will remain inside the tube.

20. Remove the 5 ml FACS tube from the magnet.

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forming bubbles.

23. Place the 5 ml FACS tube back in the magnet and set aside for 3 min.

24. Repeat Steps 19, 20 and 21 pouring off the supernatant fraction into the waste fraction 50 ml tube and for a total of 3 washes.

25. Remove the 5 ml FACS tube from the magnet and resuspend in 4 ml of BPEL medium.

26. Take a 50 ml aliquot (post isolation fraction) and place it in the fridge at 4°C.

27. Centrifuge the waste and CD34 negative fractions 3 min at 300 x g at room temperature, aspirate supernatant, resuspend in 50 ml of FACS buffer and stain them together with aliquots from steps 11 and 27 with anti-CD34- APC antibody, 30 min at room temperature; wash with 1 ml of FACS buffer, centrifuge 3 min at 300 x g at room temperature, aspirate supernatant and resuspend in 200 ml of FACS buffer to measure the expression of CD34 by flow cytometer (step 28).

28. Analyze aliquots from steps 11, 26 and 27 with flow cytometer such as MACSQuant™ VYB (Miltenyi Biotech) equipped with a violet (405 nm), blue (488 nm) and yellow (561 nm) laser following instrument manufacturer’s instructions.

Seeding and culture of CD34⁺ endothelial cells 29. Count cells from step 25.

30. Dilute cells in BPEL medium with VEGF (50 ng/ml) to seed ~120,000 cells per well in a fibronectin-coated 6-well-plate.

31. Change medium the day after.

32. Endothelial cells should be confluent after ~4 days.

Cryopreservation of CD34⁺ endothelial cells

We recommend cryopreserving CD34⁺ endothelial cells in CryoStor™ CS10 medium:

3 wells of a 6-well plate can be cryopreserved in 1 cryovial, in 500 µl of CryoStor™

CS10 medium.

33. Aspirate medium from the wells.

34. Wash with 2 ml of DPBS per well.

35. Aspirate DPBS.

36. Add 1 ml of TrypLE 1X, then incubate for 5 min at 37°C.

37. Dilute TrypLE by adding 1 ml of BPEL medium per well.

38. Using a P-1000 (1 ml) pipet tip, transfer cells to a 15 ml conical tube.

39. Wash each well with 1 ml of BPEL medium and transfer cells into the same

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15 ml tube.

42. Resuspend in 1 ml of DPBS.

56. Resuspend in 500 µl of CryoStor™ CS10 medium, transfer to a cryvial and place in a Biocision CoolCell™. Place CoolCell™ at -80°C overnight and then transfer the vial to N_2.

Thawing and re-plating CD34⁺ endothelial cells

57. Fill a 10 ml plastic pipette with 5 ml of BPEL medium and transfer the 5 ml to a 15 ml conical tube.

58. Remove vial from N₂and place in a 37°C water bath for approximately 1 min until there is just a sliver of ice left.

59. Using a P-1000 (1 ml) pipet tip, transfer vial contents to the 15 ml conical tube.

62. Resuspend pellet in 6 ml of BPEL medium supplemented with 6 ml of VEGF and distribute in 3 wells of a Fibronectin-coated 6-well plate (2 ml per well).

63. After 24 hr, replace medium with BPEL medium supplemented with fresh VEGF (50 ng/ml).

Expect > 80% survival after thawing. Re-plated cells should be confluent within 2 days. If used for cardiac microtissues, CD34⁺ endothelial cells must be thawed at least 1 day before microtissues are generated.

Basic protocol 2

Generation and culture of 3D cardiac microtissues from enriched cardiomyocytes and endothelial cells differentiated from human pluripotent stem cells

This protocol is designed for developing 3D cardiac microtissues from either cardiomyocytes cultured alone (MT-CM) or in combination with endothelial cells (MT-CMEC) for medium-high scale production (1.5 million hPSCs can be used to generate up to 480 microtissues), and without use of any extracellular matrix.

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are isolated from heterogeneous differentiated cultures and next mixed in a defined ratio, which allows us to precisely control the system. For both MT- CM and MT-CMEC, cardiomyocytes are isolated the exact day that microtissues are formed as described in Support Protocol 1, whereas for MT-CMEC a vial of cryopreserved endothelial cells is thawed 1 to 3 days before microtissue formation. The day on which microtissues are generated, endothelial cells are detached using TrypLE 1X and resuspended in BPEL medium. In MT-CM, cardiomyocytes are diluted to 5,000 cells per 50 μl of BPEL, whereas in MT-CMEC cell suspensions are combined together to 5,000 cells (4,250 VCAM1⁺and 750 CD34⁺ cells, which corresponds to 85% cardiomyocytes and 15% endothelial cells) per 50 μl of BPEL medium supplemented with VEGF. Both MT-CM and MT- CMEC microtissues are seeded on V-bottom 96 well microplates and incubated at 37^oC with medium refreshment every 3 days, either growth factor-free (MT- CM) or VEGF-supplemented (MT-CMEC). Following 7 to 20 days in culture, microtissues are characterized by immunofluorescent staining, qRT-PCR, MEAs and contraction analyses as described previously (Giacomelli et al., 2017).

The major factors requiring optimization for successful generation of microtissues are (i) the number of cells (5,000) in the medium used (50 μl) for each aggregate, (ii) the ratio in which cell populations are mixed to form the aggregate, and (iii) the scaffold-free format that we used, which allows cell aggregation and subsequent tissue formation.

Materials

• Enriched VCAM1⁺ cardiomyocytes (Support Protocol 1)

• Enriched CD34⁺ endothelial cells (Support Protocol 2)

• TrypLE Select 1X (Gibco, cat. no. 12563029)

• MT-CM medium (BPEL medium; see recipe)

• MT-CMEC medium (BPEL medium for cardiomyocyte and endothelial cell maintenance, with VEGF; see recipe)

• Polystyrene conical tubes 15 ml (Corning Falcon, cat. no. 352097) and 50 ml (Corning Falcon; cat. no.352098)

• V-bottom 96 well microplates (Greiner Bio-one; cat. no. 651161)

• Dulbecco’s phosphate-buffered saline without Ca²⁺ or Mg²⁺ (DPBS 1X;

Gibco, cat. no. 14190-094)

• Round-bottom 5 ml FACS tubes (BD Biosciences, cat. no. 352058)

• Centrifuge (Eppendorf, cat. no. 5810R)

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607180; 25 ml, cat. no. 760180)

no. 4809)

• Pipetman starter kit P2, P10 and P100 (Gilson International, cat. no.

F167500S)

F167300)

• Multi channel 5-50 μl pipette (FinnPipette, Thermo Scientific)

• Dispenser Multipette plus (Eppendorf, cat. no. 4981 000.019)

Preparing cardiomyocytes and endothelial cells

We recommend isolating cardiomyocytes on the same day in which microtissues are generated, but thawing CD34⁺ endothelial cells at least 1 day before. To generate up to 480 microtissues, we recommend starting from 6 wells of a 6-well plate of non-enriched cardiomyocytes and 1 well of a 6-well plate of thawed CD34⁺ endothelial cells.

1. Isolate cardiomyocytes from heterogeneous cultures between days 14-17 as described in Support Protocol 1.

2. To generate MT-CM microtissues, follow steps 1 to 36 of Support Protocol 1 and count the cells.

3. Alternatively, to generate MT-CMEC microtissues, follow steps 1 to 36 of Support Protocol 1 and prepare endothelial cells during step 17 (30 min of staining with anti-VCAM1-PE antibody) as follows: dissociate previously thawed CD34⁺ endothelial cells following steps 33-41 of Support Protocol 2. Resuspend pellet in 1 ml of BPEL medium and count the cells.

4. Store endothelial cells in the incubator at 37°C while cardiomyocytes are

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Generation and culture of 3D Cardiac Microtissues

5. For MT-CM microtissues, prepare a 15 ml conical tube and dilute VCAM1⁺ cells in MT-CM medium (BPEL medium), combining 5,000 cells per 50 µL of medium per well of a V-bottom 96 well microplate, while for MT-CMEC microtissues, prepare a 15 ml conical tube and dilute VCAM1⁺ and CD34⁺ cells in MT-CMEC medium (BPEL medium supplemented with VEGF 50 ng/

ml), combining 5,000 cells (4,250 VCAM1⁺and 750 CD34⁺ cells) per 50 µL of medium per well of a V-bottom 96 well microplate.

6. Using a 5 or 10 ml plastic pipette, transfer the content of each 15 ml tube to a reservoir (one reservoir per tube) and seed cell aggregates using a 50 ml multichannel pipette and 50 ml filter tips.

8. Place microtissues in the incubator at 37°C.

9. Change medium every 3 days by replacing 25 µL of old medium with 25 µL of fresh medium either growth factor-free (MT-CM) or VEGF-supplemented (MT-CMEC).

NOTE: The entire procedure (from cardiomyocyte isolation to plating of microtissues) takes approximately 4 hr.

NOTE: We do not seed microtissues in the outermost wells of the V-bottom 96 well microplate due to possible medium evaporation. Prior to microtissue seeding, we fill each external well with 50 µL of DPBS. Each V-bottom 96 well microplate contains maximum 60 microtissues.

Support protocol 3

Characterization of 3D cardiac microtissues by immunofluorescent staining and re-plating for downstream applications

Following 7 days in culture, morphology and cellular architecture of 3D cardiac microtissues can be determined by immunofluorescent staining for cardiomyocyte- (TNNI) and endothelial- (CD31) specific cell markers, as shown in Figure 5A-B.

Alternatively, following 4 to 7 days in culture, microtissues can be re-plated in Matrigel-coated 24-well plates on top of plastic coverslips for downstream applications such as contraction measurements (Movies 6-7) (Giacomelli et al., 2017).

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Figure 5. Immunofluorescent staining of sarcomeric cardiomyocyte TNNI (green) and endothelial cell surface marker CD31 (red) of day 7 MT-CM (left panels) and MT-CMEC (right panels) microtissues generated from hESCs (A) and hiPSCs (B). Nuclei are stained in blue with DAPI. Objective 25x; Scale bar: 100 μm.

Materials

• MT-CM and MT-CMEC microtissues (Basic Protocol 2)

• Matrigel-coated (see recipe) 24-well plates (Greiner Bio-One; cat. no.

662160) with tissue culture coverslips, 13 mm, plastic (Sarstedt; cat. no.

83.1840.002).

• Dulbecco’s phosphate-buffered saline with Ca²⁺ and Mg²⁺ (DPBS 1X; Gibco;

cat. no. 14040-091)

• 1.5 ml Eppendorf tubes (Eppendorf; cat. no. 0030 120.086)

• Rabbit anti-human TNNI primary antibody, polyclonal (Santa Cruz; cat. no.

Sc-15368)

• Mouse anti-human CD31 primary antibody, monoclonal (Dako; cat.no.

M0823)

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• Donkey anti-rabbit AF488 secondary antibody, polyclonal (Invitrogen; cat.

no. A21206)

• DAPI nucleic acid stain, dilactate (Molecular Probes; cat. no. D3571)

• Fixative solution (4% wt/vol PFA in 0.2 M Phosphate buffer; see recipe)

• Permeabilization solution (0.2% vol/vol Triton™ X-100 in DPBS; See recipe)

• Blocking solution (5% FBS and 5% normal goat serum in DPBS; see recipe)

• ProLong™ Gold antifade Mountant with DAPI (Life Technologies; cat. no.

P36931)

• Microscope slides (VWR Collection; cat. no. ECN 631-1553)

• 12 mm round glass coverslips (Menzel-Glaser; cat. no. CB00140RA1)

607180; 25 ml, cat. no. 760180)

no. 4809)

• Filter tips (Biosphere plus; 2 – 100 μl, cat. no. 70.760.212)

F167500S)

F167300)

• SP8WLL confocal laser-scanning microscope (Leica)

Preparing and fixing microtissues for immunofluorescent staining

1. Using a P-1000 (1 ml) pipet tip, transfer microtissues to 2 x 15 ml conical tubes (combining MT-CM in one tube and MT-CMEC in the second one) and wait ~ 3 min until they sediment.

2. Carefully remove supernatant using a P-1000 (1 ml) pipet tip.

3. Gently, top up each tube with 3 ml of DPBS. Avoid pipetting microtissues up and down. Wait until they sediment.

4. Carefully remove supernatant using a P-1000 (1 ml) pipet tip.

5. Fix microtissues by adding 1 ml of 4% PFA per tube and incubate for 1hr in the fridge at 4°C.

6. Wash 3 times with DPBS, each time for 5 min, waiting for microtissues to sediment and carefully removing supernatant using a P-1000 (1 ml) pipet tip.

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Day 1 of staining

We recommend starting the staining within 7 days from initial fixation of the samples.

7. Using a P-200 pipet tip, transfer microtissues to 2 x 1.5 ml Eppendorf tubes (combining MT-CM in one tube and MT-CMEC in the second one). Do it in two steps to be sure to collect all microtissues.

8. Permeabilize with 200 μl of 0.2% Triton™ X-100 in DPBS. Incubate for 30 min at room temperature.

7. Wash 3 times with 200 μl of DPBS, each time for 10 min, waiting for microtissues to sediment.

8. Block with 200 μl of 5% FBS + 5% Goat serum in DPBS for at least 2 hr at room temperature.

9. Stain with 100 μl of first antibodies TNNI and CD31 at 1:500 and 1:200 dilutions respectively in blocking solution overnight at 4°C.

Day 2 of staining

11. Stain with 100 μl of secondary antibodies Cy™3 and AF488 at 1:100 and 1:200 dilutions respectively in blocking solution overnight at 4°C in the dark.

Day 3 of staining

13. Stain with 100 μl of DAPI at 1:500 dilutions in DPBS for 30 min at room temperature in the dark.

14. Resuspend no more than 60 microtissues in 8 μl of ProLong™ Gold antifade Mountant with DAPI and mount microtissues on microscope slides on top of 12 mm round coverslips.

15. Store slides at room temperature in the dark, waiting at least 24 to 48 hr to dry before imaging.

16. Acquire images with a fluorescent microscope such as SP8WLL confocal laser-scanning microscope (Leica) equipped with a violet (405 nm), blue (488 nm) and orange (532 nm) laser, using either a 20x or 25x objective

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marker TNNI (detected with the blue laser), while MT-CMEC microtissues will stain positively for DAPI (violet laser) and the endothelial marker CD31 (orange laser).

Alternative to steps 1-16, following 4 to 7 days in culture, microtissues can be re- plated in Matrigel-coated 24-well plates on top of plastic coverslips for downstream applications as described below.

Re-plating microtissues for downstream applications

Due to care required regarding disaggregation of microtissues, we recommend waiting at least 4 days before re-plating. Make sure microtissues are beating and compact before transferring them. After re-plating, they need at least 2 days to attach before further measurements can be performed.

1. Aspirate medium from the 24-well plate with plastic coverslips.

2. Add 300 µl per well of BPEL medium, either growth factor-free (MT-CM) or VEGF-supplemented (MT-CMEC).

3. Using a P-1000 (1 ml) pipet tip, transfer 3 microtissues per time (either MT- CM or MT-CMEC) from V-bottom 96 well microplates into each well of the 24-well plate, on top of each plastic coverslip.

4. Check under the microscope if microtissues are placed correctly on top of the coverslips; if not, gently move them on top of the coverslips with the help of a P-200 pipet tip.

5. Gently place the plate in the incubator at 37°C and wait at least 2 days before moving the plate.

Reagents and solutions

All reagents and solutions coming into contact with living cells must be sterile, and aseptic technique should be used accordingly. We do not place any media in a 37°C water bath before use due to concerns regarding the temperature stability of growth factors in the media. In our experience, bringing the media to room temperature is sufficient.

BPEL medium (Ng et al., 2008)

For 100 ml of BPEL medium mix and filter using a 0.2 μm filter (Puradisc, 30 sterile syringe filter, 0.2 μm; Whatman; cat. no. 10462200):

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• IMDM, 43 ml (Gibco; cat. no. 21056-023)

• Phenol red, 1 mg (Brocades; cat. no. FE557)

• F12, 43 ml (Gibco; cat. no. 31765-027)

• PFHMII, 5 ml (Gibco; 12040-077)

• BSA 10%, 2.5 ml (BovoStar BSA Bovogen Biologicals; cat. no. BSAS 0.5)

• PVA 5%, 2.5 ml (Sigma-Aldrich; cat. no. P8136)

• CDLC, 1ml (Gibco; 11905-031)

• ITS-X, 0,1 ml (Gibco; cat. no. 51500-056)

• a-MTG 300 µl (Sigma-Aldrich; cat. no. M6145)

• AA2P, 1ml (5 mg/ml in tissue culture water; Sigma-Aldrich; cat. no A8960)

• GlutaMAX™-I supplement, 1 ml (Gibco; cat. no. 35050-038)

• Penicillin-streptomycin 5,000 U ml⁻¹, 0,5 ml (Pen-strep; Gibco; cat no. 15070-063)

BPEL medium is stable at 4°C for up to 3 weeks. If the volume of BPEL is 1000 ml, prepare it in 2 steps adding the phenol red all together only in step 1. To add the phenol red, we recommend transferring the tube inside the filter and cleaning completely the tube using part of the volume of F12.

BPEL medium for cardiac mesoderm induction To BPEL medium add:

• BMP4, 20 ng/mL (R&D Systems; cat. no 314-BP-01M)

• ACTIVIN A, 20 ng/mL (Miltenyi Biotec; cat. no cat. no. 130-095-547)

• CHIR99021, 1.5 μM (Axon Medchem; cat. no Axon 1386)

This medium needs to be prepared fresh every time and storage is not recommended.

Thaw BMP4 and ACTIVIN A on ice whereas CHIR99021 at room temperature (protect from light); spin them down before using; after using, store them at 4°C for 1-2 weeks maximum.

BPEL medium for cardiomyocyte and endothelial cells specification To BPEL medium add:

• XAV939, 5 μM (Tocris Bioscience; cat. no 284028-89-3)

• VEGF, 50 ng/ml (R&D Systems; cat. no. 293-VE)

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Thaw XAV939 at room temperature whereas VEGF on ice; spin them down before using; after using, store them at 4°C for 1-2 weeks maximum.

BPEL medium for cardiomyocyte and endothelial cells maintenance To BPEL medium add:

• VEGF, 50 ng/ml (R&D Systems; cat. no. 293-VE)

Thaw VEGF on ice and spin it down before using. After using it, store at 4°C for 1-2 weeks maximum.

E8™ medium

To 500 ml of E8™ medium (Gibco; cat. no. A1517001) add:

• One thawed 10 ml aliquot of E8™ supplement (provided with E8™ bottle)

• Penicillin-streptomycin 5,000 U ml⁻¹

,

2,5 ml (Pen-strep; Gibco; cat no.

15070-063)

There is no need to filter sterilize. E8™ medium is stable at 4°C for up to 3 weeks.

EDTA, 0.5 mM

To 50 ml of DPBS (Gibco; cat. no. 14190-094) add 50 μl of 0.5 M EDTA (Sigma- Aldrich, cat. no. E5134).

Store for up to 6 months at room temperature.

Matrigel-coated plates

1. Thaw a bottle of growth factor-reduced Matrigel™ (Corning; cat. no 356230) on ice overnight; make 50 and 100 μl aliquots (containing 0.5 mg and 1 mg of protein respectively) and store them at -20°C.

2. Add 50 μl of Matrigel™ to 6 ml of 4°C DMEM/F12 (Gibco; cat. no. 31331-028) (enough for one 6-well plate), or 100 μl of Matrigel™ to 12 ml of 4°C DMEM/

F12 (enough for two 6-well plates).

3. Plate at 1 ml per well of a 6-well plate, or equivalent amounts for other size plates, or 0.4 ml per well of a 24-well plate with 13 mm plastic coverslips previously added to each well.

4. Leave the plate at room temperature for at least 1 hr, then either use it or

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4

add 1 extra ml of DMEM/F12 per well (of a 6-well plate) so that the plate does not dry out during storage.

5. Store the plate at 4°C.

During the entire procedure, Matrigel™ and DMEM/F12 need to be kept on ice and medium needs to be added quickly to the wells to prevent gelling. Matrigel-coated plates can be kept at 4°C for maximum 2 weeks.

VTN-N-coated plates

1. Thaw VTN-N recombinant human protein (Gibco; cat. no A14700) on ice;

make 60 μl aliquots and store them at -80°C.

2. Add 60 μl of VTN-N to 6 ml of DPBS (Gibco; cat. no. 14190-094) (enough for one 6-well plate).

3. Plate at 1 ml per well of a 6-well plate, or equivalent amounts for other size plates.

4. Leave the plate at room temperature for at least 1 hr, then either use it or add 1 extra ml of DPBS per well (of a 6-well plate) so that the plate does not dry out during storage.

VTN-N-coated plates can be kept at 4°C for maximum 2 weeks.

1. Fibronectin-coated plates

2. Add 30 μl of fibronectin (Bovine; Sigma-Aldrich, cat. no. F1141) to 6 ml of DPBS (Gibco; cat. no. 14190-094) (enough for one 6-well plate).

3. Plate at 1 ml per well of a 6-well plate, or equivalent amounts for other size plates.

4. Leave the plate at room temperature for at least 1 hr, then either use it or add 1 extra ml of DPBS per well (of a 6-well plate) so that the plate does not dry out during storage.

Fibronectin-coated plates can be kept at 4°C for maximum 2 weeks. We recommend using Fibronectin at a concentration of ~2-5 μg/ml.

TrypLE 2X

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FACS Buffer

Dissolve 1.25 g of BSA (Sigma-Aldrich, cat. no. A3311) in 250 ml of DPBS (Gibco;

cat. no. 14190-094) and add 1 ml of 0.5 M EDTA (Sigma-Aldrich, cat. no. E5134).

Sterilize the medium by using a Stericup filter (0.22 mm filter).

Store it up to 4 weeks at 4°C.

EasySep™ Buffer

Add 5 ml of FBS (Gibco, cat no. 10270-106) and 0.5 ml of 0.5 M EDTA (Sigma- Aldrich, cat. no. E5134) to 250 ml of DPBS (Gibco; cat. no. 14190-094). Sterilize the medium by using a Stericup filter (0.22 mm filter).

Store up to 4 weeks at 4°C.

Fixative solution

Add 1 volume of PFA 8% wt/vol (PFA Merck; cat. no. 1.04005.1000) to 1 volume of 0.2 M of Phosphate buffer (pH 7.4).

Cover the mixture with aluminum foil and keep it at 4°C for up to 2 weeks.

CAUTION: Work in a fume hood.

Permeabilization solution

Add 100 μl of Triton™ X-100 (Sigma-Aldrich, cat. no. T8787) to 50 ml of DPBS (Gibco; cat. no. 14190-094) and mix.

Blocking solution

Add 2.5 ml of FBS (Gibco, cat no. 10270-106) and 2.5 ml of normal goat serum (Dako, cat. no. X0907) to 50 ml of DPBS (Gibco; cat. no. 14190-094) and mix.

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Commentary

Background Information

We and others have previously described protocols for differentiation of hPSCs towards either cardiomyocyte or endothelial cell fates (Burridge et al., 2015;

2014; Elliott et al., 2011; Giacomelli et al., 2017; Lian et al., 2014; 2012; Murry and Keller, 2008; Orlova et al., 2014a; 2014b; van den Berg et al., 2016; Willems et al., 2011).

To date, several 3D models of the heart tissue derived from hanging drop cultures, hydrogels, cell sheets or patches have been generated from hPSCs (Caspi et al., 2007; Huebsch et al., 2016; Mannhardt et al., 2016; Masumoto et al., 2016; Narmoneva et al., 2004; Ravenscroft et al., 2016; Stevens et al., 2009; Tulloch et al., 2011). However, the majority of these models contain cardiomyocytes alone, without taking into account that multiple cell types are required to build the cardiac tissue in vivo, and without considering that drug-induced cardiotoxicity might have a multicellular contribution (Cross et al., 2015). Furthermore, when other cell types, such as endothelial cells, are included in these models, they have in most cases been primary or non-cardiac specific cells, such as HUVECs (human umbilical vein endothelial cells).

Recently, Palpant and colleagues developed a protocol for differentiation of cardiac and endothelial cells from hPSCs (Palpant et al., 2016; 2015). We provide here an extended protocol using our strategy to co-differentiate hPSCs towards cardiomyocytes and endothelial cells from cardiac mesoderm and formation of 3D cardiac microtissue that can be applied to test relevant drugs and to develop models of cardiovascular disorders in which not only cardiomyocytes but also endothelial cells are affected. To our knowledge, this is the first protocol that describes an in vitro 3D cardiac tissue model in which cardiomyocytes and endothelial cells are derived simultaneously from common mesodermal progenitors.

Our system requires a substantially smaller number of cells compared to others cardiac 3D models, such as engineered heart tissues (EHTs) (Mannhardt et al., 2016), making it particularly suitable to high scale production (low cost per data point). Finally, since both cardiomyocytes and endothelial cells are enriched prior to microtissue formation, we can precisely control the ratio in which these

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Critical Parameters

• Culturing cells in E8™ medium provides a reproducible environment for pluripotent stem cell growth and efficient subsequent differentiation:

hPSCs are passaged as single cells instead of colonies and differentiated as monolayers. Moreover, the use of EDTA allows passaging the cells non-enzymatically, which improves cell survival, and avoids the step of centrifugation, which simplifies the splitting procedure. To improve cell survival and split ratio reproducibility of hiPSCs, we use RevitaCell™

supplement for 24 hr after splitting.

• Our protocol for co-differentiation of cardiomyocytes and endothelial cells from hPSCs was tested and optimized using hPSCs maintained in E8™

medium from Invitrogen. Endothelial cells can be efficiently differentiated from hPSCs maintained in E8™ medium from StemCell Technologies. For differentiation of cardiomyocytes from hPSCs maintained in E8™ medium from StemCell Technologies we recommend to test seeding densities and split ratios, as we have observed variability in hPSC proliferation in E8™

medium from different vendors.

• Notably, methods described here were robustly reproducible in both hESCs and in hiPSCs. However, the CMEC protocol does not always result in beating cardiomyocytes: we have observed that cell lines go through periods of very successful differentiation followed by periods in which they become refractory to differentiate (usually after passage 70). We have also observed that within the same differentiation, some wells can differentiate better than others.

• With regards to the supplementation of VEGF on day 3 and onwards, we would advise starting from VEGF concentration at 50 ng/ml, and we did not observe significant variation within the different lots and commercial providers, whereas XAV939 might vary from 1 to 5 mM.

• With regards to the enrichment of endothelial cells from differentiated hPSCs using the EasySep™ CD34 Human Cord Blood Isolation Kit II from StemCell Technologies (cat. no. 18309), we recommend using fluorochrome- conjugated antibody clones that are not blocked by the antibody clone used in the tetrameric antibody complexes. For purity assessment by flow cytometry, we recommend using clones suggested by the company, which are listed in the product information sheet.

• Due to the fact that hPSC-derived cardiomyocytes are extremely sensitive to dissociation as well as enrichment and re-plating, we have found that enriched cardiomyocytes do not always survive after these procedures.

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Therefore, we recommend performing dissociation very gently, and VCAM1 isolation as fast as possible. After isolation, we recommend seeding VCAM1⁺ cells on Matrigel™ at high seeding density (~ 450,000 cells per well of a 24-well-plate).

• During the development of our microtissue protocol, we observed that microtissues generated using non-enriched cardiomyocytes tend to be more stable and less prone to disaggregate compared to microtissues generated using enriched cardiomyocytes. This might be due to the fact that residual fibroblasts/mesenchymal cells present in the non-enriched cardiomyocyte cell fraction might contribute to microtissue stabilization via secreting extracellular matrix proteins. Therefore, non-enriched cardiomyocyte can be used as a control if any issues with the stability of the microtissues happened.

Troubleshooting

• Interline variability and passage number variability in differentiation efficiency:

Pluripotent stem cells must be undifferentiated and growing at a fast rate, ideally achieving 65% to 85% confluence within 3 to 4 days. We have found that lines over passage 70 have a lower differentiation success rate.

• No beating cardiomyocytes: If cardiomyocytes are not beating by day 14, discard the plate and repeat differentiation. Do not use them for generating microtissues.

• No beating microtissues: If microtissues are not beating by day 7, discard the plate and repeat generation of microtissues from a different cardiac differentiation.

• Non-compact microtissues: If microtissues are not compact, they tend to disaggregate during immunofluorescent staining or during re-plating. In this case, discard the plate and repeat generation of microtissues from a different cardiac differentiation. Do not use microtissues for further downstream applications.

Anticipated Results

With regard to the cardiomyocyte population, differentiation with no VEGF supplementation should produce ~0.5 to 1 million VCAM1⁺ cells per well for a 6-well plate. Cardiomyocyte purity after immunomagnetic selection will be

~80% based on VCAM1 flow cytometry. Isolated cardiomyocytes will stain positively for cardiac markers such as TNNI and ACTN2 (Giacomelli et al., 2017).

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With regard to the endothelial cell population, CMEC differentiation should produce ~0.3 to 1 million CD34⁺cells per well for a 6-well plate. Endothelial cell purity after immunomagnetic selection will be > 95% based on CD34 flow cytometry. Isolated endothelial cells will show expression of endothelial- and cardiac-specific markers by FACS analysis and RT-qPCR (Giacomelli et al., 2017). When compared to primary cardiac and non cardiac endothelial cells on gene expression level, CD34⁺ cells will cluster with HCMEC suggesting cardiac endothelium identity (Giacomelli et al., 2017). After freezing and thawing, cell survival will be ~80% and cells will be ready in 1 day for generating microtissues.

6 wells of non-enriched cardiomyocytes and 1 well of CD34⁺ endothelial cells of a 6-well plate can generate up to 480 microtissues. Cardiac microtissues will aggregate and start beating within 3-4 days from initial plating. They will stain positively for the cardiac marker TNNI (MT-CM and MT-CMEC) and the endothelial marker CD31 (MT-CMEC) after 7 days in culture. Following 20 days in culture, further evidence of maturity, specifically for MT-CMEC, will result in increased expression of cardiac genes encoding ion channels and calcium-handling proteins (Giacomelli et al., 2017). Microtissues will display human dose-response to b-adrenergic stimulation and negative inotropy after treatment with the Ca²⁺ channel blocker Verapamil (Giacomelli et al., 2017).

Time Considerations

The CMEC differentiation protocol takes 6 days from induction of cardiac mesoderm through to appearance and enrichment of endothelial cells, whereas beating cardiomyocytes will appear at approximately days 9-10 of differentiation and can be isolated between days 14-17. After isolation, cardiac microtissues are generated and cultured at least 7 days to reach a suitable level of aggregation for successful characterization. The entire procedure for generating cardiac microtissues (from cardiomyocyte isolation to microtissue plating) requires approximately 4 hr.

Medium needs to be changed daily for pluripotent stem cells, while every 3 days for cardiac/endothelial cell differentiation and microtissue culture.

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Acknowledgements

This project was funded by the following grants: European Research Council (ERCAdG 323182 STEMCARDIOVASC); European Community’s Seventh Framework Programme (FP7/2007-2013) under grant agreement No. 602423; European Union’s Horizon 2020 research and innovation Programme (TECHNOBEAT) under grant agreement No. 668724.

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