Oct 07, 2024
Generating Blood-Generating Heart-Forming Organoids from human pluripotent stem cells
- Miriana Dardano1,
- Lika Drakhlis1,
- Robert Zweigerdt1
- 1Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery (HTTG), REBIRTH–Research Center for Translational Regenerative Medicine, Hannover Medical School, Hannover, Germany.
Protocol Citation: Miriana Dardano, Lika Drakhlis, Robert Zweigerdt 2024. Generating Blood-Generating Heart-Forming Organoids from human pluripotent stem cells. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw1nkdlmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 28, 2024
Last Modified: October 07, 2024
Protocol Integer ID: 106662
Keywords: Organoids, Cardiogenesis, Embryo development, Hematopoiesis, Hematopoietic progenitor cells, Human pluripotent stem cells
Abstract
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.
Materials
Materials:
*synthesized by the Institute for Organic Chemistry (Leibniz University Hannover)
Accutase Gibco - Thermo FischerCatalog # A1110501
L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #A8960
B-27™ Supplement, minus insulinGibco - Thermo FisherCatalog #A1895601
B-27 SupplementGibco - Thermo FischerCatalog #17504044
Animal-Free Recombinant Human FGF-basicpeprotechCatalog #AF-100-18B
Recombinant Human BMP-4peprotechCatalog #120-05
DMEM/F-12, HEPESThermo FisherCatalog #11330057
Recombinant Human EPOpeprotechCatalog #100-64
Recombinant Human Flt3-LigandpeprotechCatalog #300-19
Geltrex™ LDEV-Free Reduced Growth Factor Basement Membrane MatrixThermo FisherCatalog #A1413202
Insulin solution humanMerck MilliporeSigma (Sigma-Aldrich)Catalog #I9278
Recombinant Human IGF-IpeprotechCatalog #100-11
Recombinant Human IL-3peprotechCatalog #200-03
Recombinant Human IL-6peprotechCatalog #200-06
Recombinant Human IL-11peprotechCatalog #200-11
IWP 2TocrisCatalog #3533
Corning® Matrigel® Basement Membrane Matrix, LDEV-free, 10 mLCorningCatalog #354234
PBS (10X), pH 7.4Thermo Fisher ScientificCatalog #70011051
Penicillin-StreptomycinMerck MilliporeSigma (Sigma-Aldrich)Catalog #P0781
Y-27632 dihydrochlorideTocrisCatalog #1254
RPMI 1640 MediumThermo FisherCatalog #21875091
Recombinant Human Sonic HedgehogpeprotechCatalog #100-45
Sodium bicarbonateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S5761
Sodium seleniteMerck MilliporeSigma (Sigma-Aldrich)Catalog #S5261
Recombinant Human SCFpeprotechCatalog #300-07
STEMdiff™ Cardiomyocyte Dissociation KitSTEMCELL Technologies Inc.Catalog #05025
SDS Tissue-Tek® Sakura FinetekCatalog #4583
Recombinant Human TGF-β1 (CHO derived)peprotechCatalog #100-21C
Recombinant Human TPOpeprotechCatalog #300-18
Transferrin humanMerck MilliporeSigma (Sigma-Aldrich)Catalog #T3705
Recombinant Human VEGFpeprotechCatalog #100-20
Trypan Blue solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #T8154
Culture Media:
Equipment:
Conical tube, 15 mlgreiner bio-oneCatalog #188271
Tissue Culture FlasksTechno Plastic Products (tpp)Catalog #90026
Nunclon™ Sphera™ Microplates, 96U-Well PlateThermo FisherCatalog #174925
BRAND® counting chamber BLAUBRAND® Neubauer improved New without clips, double ruledMerck MilliporeSigma (Sigma-Aldrich)Catalog #BR717805
Pre-culture
Pre-culture
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.
Passage the cells every 3-4 days and change the medium daily except the day immediately after the passaging.
Generation of BG-HFOs
Generation of BG-HFOs
2d 0h 56m
2d 0h 56m
Note
- The protocol for generation of BG-HFOs relies on the published protocol for generating Heart-Forming Organoids (HFOs)4, 5; to have specifics regarding the key protocol passages please refer to the cited manuscript4.
- The HFO protocol adaptation in order to generate BG-HFOs consists of the addition of growth factors and cytokines at specific time-points during the differentiation, described as follows:
On the starting day (day minus 4; d-4), detach hPSCs from Geltrex-coated flasks using Accutase.
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.
Centrifuge the plate is at 300 x g, 4°C, 00:06:00 and placed in the incubator to let one hPSC aggregate per well form Overnight .
6m
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.
hES3 NKX2.5-eGFP-derived aggregate at d-2 of the protocol with round and sharp edge; such organoids are selected for subsequent Matrigel embedding.
After embedding, place the plate at 37 °C in the incubator for 00:50:00 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.
50m
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.
After 24 h, on d1, exchange the medium by RB− supplemented with 50 ng ml-1 VEGF and 10 ng ml-1 bFGF.
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 48:00:00 .
2d
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.
2 days after, from d7 onwards, cultivate BG-HFOs in RB+ supplemented with the same cytokines added on d5.
After 3 days, on d10, complete BG-HFO differentiation; keep the organoids in culture for downstream analysis until d14.
hES3 NKX2.5-eGFP-derived BG-HFO at d10 of the differentiation. NKX2.5-eGFP, expressed in early cardiomyocytes, allows for the visualization of the organoid´s structure; an inner core (IC) NKX2.5-eGFPneg, a myocardial layer (ML) NKX2.5-eGFPpos, and an outer layer (OL) only partially positive for NKX2.5eGFP. Such organoids are used for downstream analyses.
Protocol references
1. Elliott, D. A. et al. NKX2-5 eGFP/w hESCs for isolation of human cardiac progenitors and cardiomyocytes. Nat. Methods 8, 1037–1043 (2011).
2. Den Hartogh, S. C. et al. Dual reporter MESP1mCherry/w-NKX2-5eGFP/w hESCs enable studying early human cardiac differentiation. Stem Cells 33, 56–67 (2015).
3. Haase, A., Göhring, G. & Martin, U. Generation of non-transgenic iPS cells from human cord blood CD34 + cells under animal component-free conditions. Stem Cell Res. 21, 71–73 (2017).
4. Drakhlis, L., Devadas, S. B. & Zweigerdt, R. Generation of heart-forming organoids from human pluripotent stem cells. Nature Protocols vol. 16 5652–5672 at
5. Drakhlis, L. et al. Human heart-forming organoids recapitulate early heart and foregut development. Nat. Biotechnol. 39, 737–746 (2021).