TITLE: Generating Blood-Generating Heart-Forming Organoids from human pluripotent stem cells
AUTHORS: Miriana Dardano, Lika Drakhlis, Robert Zweigerdt

[DESCRIPTION]
Our established human pluripotent stem cell (hPSC)-derived heart-forming organoids (HFOs) recapitulate aspects of heart, vasculature, and foregut co-development. Modulating HFO differentiation, we here report the protocol for generating blood-generating (BG)-HFOs; while maintaining a functional ventricular-like heart anlagen, BG-HFOs comprise a mesenchyme embedded hemogenic endothelial layer encompassing multiple hematopoietic derivatives and hematopoietic progenitor cells with erythro-myeloid and lymphoid potential, reflecting aspects of primitive and definitive hematopoiesis. The model enables the morphologically structured co-development of cardiac, endothelial, and multipotent-hematopoietic tissues equivalent to the intra-embryonic hematopoietic region in vivo, promoting research on hematopoiesis in vitro.

[GUIDELINES]
Our research complies with all relevant ethical regulations; experiments using hESCs lines were performed under allowance ‘108 Genehmigung nach dem Stammzellgesetz’; granted by the Robert Koch Institute.

[STEPS]
SECTION: Pre-culture
1. Grow the human pluripotent stem cell (hPSC) lines of interest (in our case hES3 NKX2.5-eGFP1, 2 and HSC_ADCF_SeV-iPS23) on irradiated embryonic mouse fibroblasts in the incubator at 37 °C, 5%CO2. 

At 80% colony confluence, passage the cells and seed either onto fresh irradiated fibroblasts or, to start organoid differentiation, transfer to Geltrex-coated T25 flasks in Essential 8 (E8) medium supplemented with 10 micromolar (µM) Y-27632.

SECTION: Pre-culture
2. Passage the cells every 3-4 days and change the medium daily except the day immediately after the passaging.

SECTION: Generation of BG-HFOs
3. 
 

SECTION: Generation of BG-HFOs
4. On the starting day (day minus 4; d-4), detach hPSCs from Geltrex-coated flasks using Accutase. 

SECTION: Generation of BG-HFOs
9. 2 days afterwards, on d3, add RB− supplemented with 5 micromolar (µM) IWP2, 50 ng ml-1 VEGF, 10 ng ml-1 bFGF, 100 ng ml-1 SCF, 17 ng ml-1 EPO, 10 ng ml-1 IL-6, 10 ng ml- 1 IL-11 and 25 ng ml-1 IGF-1,  for 2880 min.

SECTION: Generation of BG-HFOs
5. After 52 h (d−2), only the hPSC aggregates looking round and with a sharp edge will be further differentiated. 

Embedd the selected aggregates in a 20 µL Matrigel droplet. Embedding is performed seeding Matrigel droplets using a p200 pipet set to 20 µl, and in each droplet seed one single aggregate using a p20 pipet set to 3 µl. 
In order to maintain the integrity of the aggregates, the edge of the 20 µl plastic tip is enlarged by cutting it with a pair of sterilized scissors; for specifics please refer to the HFO protocol4.

 

SECTION: Generation of BG-HFOs
8. After 24 h, on d1, exchange the medium by RB− supplemented with 50 ng ml-1 VEGF and 10 ng ml-1 bFGF.

SECTION: Generation of BG-HFOs
6. After embedding, place the plate at 37 °C in the incubator for 50 min to let the Matrigel solidify. 

Afterwards,  supplement E8 medium with 10 ng ml-1 BMP4 is added on top of the Matrigel-embedded aggregates. 
Add 100 µL for each well. Further details are described in our previous publication4.

SECTION: Generation of BG-HFOs
7. After 2 days, on d0, replace E8 with 200 µL per well of RB− supplemented with 7.5 μM CHIR 10 ng ml-1 BMP4, and bFGF 5 ng ml-1. 

Remove the medium by using first a P1000 pipet to remove the majority of the medium and then a p200 pipet to remove the remaining medium. 
It is important to do not disturb the Matrigel droplet containing the aggregate during the media change, but gently moving it with the edge of the tip. 
Perform all the subsequent media changes following this criteria.

SECTION: Generation of BG-HFOs
10. After 2 days, on d5, exchange the medium by RB- supplemented with the same molecules of d3 plus 30 ng ml-1 TPO, 10 ng ml-1 FLT3, 30 ng ml-1 IL-3, 10 ng ml-1 BMP4, and 20 ng ml-1 SHH.

SECTION: Generation of BG-HFOs
11. 2 days after, from d7 onwards, cultivate BG-HFOs in RB+ supplemented with the same cytokines added on d5.

SECTION: Generation of BG-HFOs
12. After 3 days, on d10, complete BG-HFO differentiation; keep the organoids in culture for downstream analysis until d14.

 

SECTION: Generation of BG-HFOs
4.1. Count the cells using a hemocytometer and trypan blue solution; seed 5000 cells per well in a U-shaped ultralow-attachment 96-well plate in E8 medium supplemented with 10 μM Y-27632. 

SECTION: Generation of BG-HFOs
4.2. Centrifuge the plate is at 300 x g, 6 min, 4 °C and placed in the incubator to let one hPSC aggregate per well form  .