Protocol to generate immunocompetent human heart-macrophage assembloids for the study of immune-cardiac interactions

Figures

• 

  Figure 1. Simplified schematic illustrating the protocol timeline, including key experimental steps and associated supporting literature for each stage

• 

  Figure 2. Phase-contrast imaging of monocyte differentiation and suspended-cell output (A) Representative phase-contrast images of EB-initiated monocyte differentiation culminating in a monocyte-producing structure. n ≥ 4 monocyte differentiations. Scale bar = 200 μm. (B) A photograph depicting optimal distribution of EBs in a single well of a 6-well plate for a monocyte differentiation. (C) Representative phase-contrast images of monocyte-producing structure borders, depicting the progression and relative quantity of suspended cells at days 18, 22, and 26. n ≥ 4 monocyte differentiations. Scale bars = 200 μm. (D) Day-by-day percent yield of CD14+ cells purified by MACS from the whole cell product generated from the differentiation. n = 7 monocyte differentiations. Value = mean ± s.d. (E) Day-by-day quantification of CD14+ cells collected from a 6-well plate that was seeded with 5 EBs (red) vs 10 EBs (blue). n ≥ 3 monocyte differentiations. Value = mean ± s.d.

• 

  Figure 3. Assessment of monocyte addition and handling conditions in hHOs (A–C) Phase-contrast microscopy images depicting hHOs/hHMAs at day 27, 29, 31, and 34 plus representative immunofluorescent microscopy images of hHO/hHMAs at day 44 with incremental additions of monocytes (10,000, 20,000, and 30,000) on day 22, 26, and 30. Immunofluorescence stainings: red = TNNT2, green = CD45, and blue = DAPI. After each monocyte addition, the hHOs/hHMAs and monocytes were either subjected to two full pipette resuspensions (A), continuous orbital shaking at 75 rpm on a plate shaker (B), or centrifuged at 50 x g for 2 min followed by continuous orbital shaking at 75 rpm (C). n = 8 hHOs/hHMAs. Scale bars = 200 μm. (D) Quantification of hHOs visibly beating under live phase-contrast microscopy at day 26, day 30, and day 34 that were subject to pipette resuspension (PR), shaking (S), and centrifuging plus shaking (CS). n = 8 hHOs/hHMAs. Value = mean ± 95% c.i.

• 

  Figure 4. Batch-level quantification of beat rate and irregular rhythms in atrial cardiomyocytes of CTRL and PFM hHMAs (A) Quantification of beat rate by individual CTRL and PFM hHMAs derived from the same hPSC source (cell line and passage number) by batch. n ≥ 14 hHMAs. Value = mean ± s.d. Student’s t test. (B) Percentage of CTRL and PFM hHMAs with spontaneous irregular rhythms and waveforms identified in atrial cardiomyocytes derived from the same hPSC source (cell line and passage number). n = 3 CTRL/PFM hHMA preparations. Value = mean ± s.d. Student’s t test.

• 

  Figure 5. Representative images of successful (left) and unsuccessful (right) monocyte differentiations over time n ≥ 10 monocyte differentiations. Scale bars = 200 μm.

• 

  Figure 6. Representative images of successful (left) and unsuccessful (right) hHO differentiations over time hHO differentiation success depends greatly on hPSC quality used to generate EBs (day 14). n ≥ 25 hHO differentiations. Scale bars = 200 μm.