A patterned human heart tube organoid model generated by pluripotent stem cell self-assembly

Brett Volmert, Ashlin Riggs, Fei Wang, Aniwat Juhong, Artem Kiselev, Aleksandra Kostina, Colin O’Hern, Priyadharshni Muniyandi, Aaron Wasserman, Amanda Huang, Yonatan Lewis-Israeli, Sangbum Park, Zhen Qiu, Chao Zhou, Aitor Aguirre

Preprint posted on 20 December 2022

A patterned human heart tube organoid model generated by pluripotent stem cell self-assembly

Selected by Silvia Becca


Recent improvements in human organoid technology have gained substantial interest. “Organoids” represent a unique possibility to model human tissues and even organs starting from stem cells, particularly human induced pluripotent stem cells (hiPSCs). Some organoids recapitulate key physiological and metabolic functions. This makes them potentially suitable for a variety of applications ranging from developmental and disease modelling studies to drug screening and even cell therapy. Organoids also promise to overcome the ethical and biological limitations related to the use of animal models in many research areas.

Among other applications, organoids are an attractive model to study congenital heart defects. Due to the underlying complexity, this goal would ideally require reproducing the variety of cell types that are specified between the 4th to 6th week of human gestation, while also recapitulating various layers of organ architecture (Lewis-Israeli et al., 2022). Existing human heart organoid models have achieved the self-assembly of certain cell lineages derived from the first and second heart field (FHF and SHF) into chamber-like cave structures that can spontaneously contract (Drakhlis et al., 2021; Hofbauer et al., 2021b; Lewis-Israeli et al., 2022) . Despite the remarkable level of complexity achieved, these models do not yet replicate some key developmental milestones including heart tube polarization, looping, and vascularization (Hofbauer et al., 2021a).

The Aguirre laboratory previously published a cardiac organoid model comprising of several key cardiac cell types from both the FHF and SHF (approximately 59% cardiomyocytes, 16% epicardial cells, 14% endocardial cells, 12% cardiac fibroblasts, and 1.6% endothelial cells) (Lewis-Israeli et al., 2022). Nevertheless, they recognised that this model still lacked important cell types, like valve and conductance cells, and did not present right-left and anterior-posterior patterning. In this preprint the same group made important steps towards tackling these issues.


Major findings:

The goal of this study was to induce organoid maturation in order to more closely mimic the human heart at approximately gestational day 45 (GD45). To achieve this, Volmert and colleagues tested various culture conditions to better recapitulate the hormonal and metabolic changes that occur during in vivo heart development.

The authors gradually increased the complexity of the maturation medium, which was applied to cardiac organoids from day 20 to day 30 of their differentiation. Eventually, they identified a cocktail including fatty acids (FA) and L-carnitine provided in a low glucose medium that could recapitulate the physiological switch of the developing heart from glucose-based to FA-based metabolism. This was complemented by the temporally-controlled addition of two hormones with established roles in foetal heart growth and development: triiodothyronine (T3) and insulin growth factor 1 (IGF-1). All of these factors had been previously reported to improve ventricular cardiomyocyte maturation protocols (Karbassi et al., 2020), but both their application to cardiac organoids and the specific combination reported here is novel. The resulting protocol led to important effects on metabolic maturation, such as an increase in mitochondrial content and expression of genes involved in FA metabolism and oxidative phosphorylation (i.e. PPARA, UQCRB, PPARGC1, BNRF1).

Single cell RNA sequencing (sc-RNA-seq) experiments indicated the emergence of various cardiac cell types, particularly cardiomyocytes (CM) and stromal cells, but also proepicardial, valve, and conductance cells among other rarer cell types. Most notably, matured organoids showed the emergence of approximately 3 per 1000 epicardial cells, which while rare may have important functions, as discussed below. It is nevertheless worth noting that several key cardiac cell types seem to be still missing (i.e. cardiac neural crest cells, understandably given their ectodermal origin (George et al., 2020)) or greatly underrepresented (i.e. endothelial cells, which as noted by the authors are somehow reduced after maturation, which contrasts with their high prevalence and key developmental role in early cardiac development in vivo (Haack and Abdelilah-Seyfried, 2016)). Interestingly, the authors distinguished atrial and ventricular cardiomyocytes chiefly through the differential expression of sarcomeric protein isoforms (i.e. MYL7 vs MYL2/3 and MYH6 vs MYH7). Though, several such switches are most often regarded as indicators of developmental progression from immature to mature ventricular CMs (Karbassi et al., 2020), and alternative markers such as NR2F2 and IRX1/4 have been reported for atrial vs ventricular identity, respectively (Churko et al., 2018; Nelson et al., 2016; Schmidt et al., n.d.). Of note, the authors did not comment on whether the enhanced maturation protocol affects the specification of left versus right ventricular CMs, which arise from different embryonic progenitors in the FHF and SHF, respectively.

Having established their cardiac organoid model, the authors investigated its potential to replicate key changes in gene expression and electrophysiology during gestation. By comparing pseudo-bulk RNA seq with data from the Human Cell Atlas project (Asp et al., 2019), the authors proposed that their matured organoids are similar to the GD45 heart. For instance, there is comparable expression of certain epicardial cell and ventricular CM markers. It would be exciting to get a better understanding of the improvements driven by maturation by also comparing data at the single cell level. From the perspective of electrophysiology, the authors reported promising findings such as increased expression of mature cardiomyocytes ion channels and some T-tubule-like structures, even though their number is still low and it is unclear whether they are associated to RYR2 clustering (Parikh et al., 2017).

Remarkably, the authors reported having obtained, for the first time, some degree of anterior-posterior (A-P) patterning in the cardiac organoids. This is evident from immunofluorescence stainings that identified “atrial” and “ventricular” poles (Fig. 1). Moreover, they proposed that the atrial pole could be induced by an endogenous gradient of retinoic acid (RA). RA is known to be produced by (pro)epicardial cells in vivo, and it is a major morphogen involved in heart tube patterning (Duester, 2008). Accordingly, the atrial pole was found to contain a cluster of cells co-expressing the TBX18 (pro)epicardial marker and ALDH1A2, an enzyme responsible for converting retinol into retinoic acid. The presence of endogenous RA was further supported by Raman spectroscopy data. Matured organoids also exhibited a more elongated shape which reminisces the developing heart tube.

Fig 1. Developmental induction promotes the formation of well-developed atrial and ventricular chambers by self-organization. Representative surface and interior immunofluorescence images of individual day 30 organoids in control and EMM2/1 conditions. Three organoids are displayed for each condition (n=9-13 organoids per condition). DAPI (blue), MYL7 (red), MYL2 (green). Scale bars = 400 μm (Volmert et al., preprint).

Taken together with other results described in the preprint, the authors concluded having partially recapitulated the changes that occur during the first 4 to 6 weeks of development. They thus suggest that metabolically matured cardiac organoids could be a scalable model suitable for pharmacological testing applications.


What we liked about this preprint:

Heart tube organoids are an emerging and promising tool for several applications, but despite huge progress in the field we are still a long way for mimicking the more advanced morphogenetic changes involved in heart development: the A-P patterning reported in this preprint represents an important milestone towards this goal.

The RA gradient produced by the proepicardial pole, together with the WNT gradient, is a fundamental cue in in vivo heart development and contributes to the early specification of A-P patterning which drives the specification of atrial chambers versus ventricular ones. The most exciting outcome of this paper is that metabolic and hormonal maturation of organoids appears to be sufficient to support the differentiation and clustering of TBX18-ALDH1A2 at one pole of developing organoids, which may in turn drive an endogenous gradient able to break radial symmetry and drive A-P patterning of atrial and ventricular poles.


Questions to the authors:

  • Early IGF-1 treatment appears to have a major impact on later morphogenetic events: any ideas on the mechanism involved?
  • Do the atrial and ventricular cells derive from SHF and FHF progenitors, respectively, as during normal development? Alternatively, is the MYL7 to MYL2 gradient explained chiefly by selective maturation of FHF cells?
  • Could the causal role of RA gradient in A-P patterning be established, perhaps through genetic or pharmacological inhibition?
  • What is a key example of an application that would showcase the benefits of this model over earlier iterations?
  • How reproducible is the protocol across different batches of differentiations and, most importantly, different iPSC lines? What are, if any, the reagents that require batch testing/dosage optimization?


Asp, M., Giacomello, S., Larsson, L., Wu, C., Fürth, D., Qian, X., Wärdell, E., Custodio, J., Reimegård, J., Salmén, F., Österholm, C., Ståhl, P.L., Sundström, E., Åkesson, E., Bergmann, O., Bienko, M., Månsson-Broberg, A., Nilsson, M., Sylvén, C., Lundeberg, J., 2019. A Spatiotemporal Organ-Wide Gene Expression and Cell Atlas of the Developing Human Heart. Cell 179, 1647-1660.e19.

Churko, J.M., Garg, P., Treutlein, B., Venkatasubramanian, M., Wu, H., Lee, J., Wessells, Q.N., Chen, S.-Y., Chen, W.-Y., Chetal, K., Mantalas, G., Neff, N., Jabart, E., Sharma, A., Nolan, G.P., Salomonis, N., Wu, J.C., 2018. Defining human cardiac transcription factor hierarchies using integrated single-cell heterogeneity analysis. Nat. Commun. 9, 4906.

Drakhlis, L., Biswanath, S., Farr, C.-M., Lupanow, V., Teske, J., Ritzenhoff, K., Franke, A., Manstein, F., Bolesani, E., Kempf, H., Liebscher, S., Schenke-Layland, K., Hegermann, J., Nolte, L., Meyer, H., de la Roche, J., Thiemann, S., Wahl-Schott, C., Martin, U., Zweigerdt, R., 2021. Human heart-forming organoids recapitulate early heart and foregut development. Nat. Biotechnol. 39, 737–746.

Duester, G., 2008. Retinoic Acid Synthesis and Signaling during Early Organogenesis. Cell 134, 921–931.

George, R.M., Maldonado-Velez, G., Firulli, A.B., 2020. The heart of the neural crest: cardiac neural crest cells in development and regeneration. Development 147, dev188706.

Haack, T., Abdelilah-Seyfried, S., 2016. The force within: endocardial development, mechanotransduction and signalling during cardiac morphogenesis. Development 143, 373–386.

Hofbauer, P., Jahnel, S.M., Mendjan, S., 2021a. In vitro models of the human heart. Development 148, dev199672.

Hofbauer, P., Jahnel, S.M., Papai, N., Giesshammer, M., Deyett, A., Schmidt, C., Penc, M., Tavernini, K., Grdseloff, N., Meledeth, C., Ginistrelli, L.C., Ctortecka, C., Šalic, Š., Novatchkova, M., Mendjan, S., 2021b. Cardioids reveal self-organizing principles of human cardiogenesis. Cell 184, 3299-3317.e22.

Karbassi, E., Fenix, A., Marchiano, S., Muraoka, N., Nakamura, K., Yang, X., Murry, C.E., 2020. Cardiomyocyte maturation: advances in knowledge and implications for regenerative medicine. Nat. Rev. Cardiol. 17, 341–359.

Lewis-Israeli, Y.R., Volmert, B.D., Gabalski, M.A., Huang, A.R., Aguirre, A., 2022. Generating Self-assembling Human Heart Organoids Derived from Pluripotent Stem Cells.

Nelson, D.O., Lalit, P.A., Biermann, M., Markandeya, Y.S., Capes, D.L., Addesso, L., Patel, G., Han, T., John, M.C., Powers, P.A., Downs, K.M., Kamp, T.J., Lyons, G.E., 2016. Irx4 Marks a Multipotent, Ventricular-Specific Progenitor Cell. Stem Cells 34, 2875–2888.

Parikh, S.S., Blackwell, D.J., Gomez-Hurtado, N., Frisk, M., Wang, L., Kim, K., Dahl, C.P., Fiane, A., Tønnessen, T., Kryshtal, D.O., Louch, W.E., Knollmann, B.C., 2017. Thyroid and Glucocorticoid Hormones Promote Functional T-Tubule Development in Human-Induced Pluripotent Stem Cell–Derived Cardiomyocytes. Circ. Res. 121, 1323–1330.

Schmidt, C., Deyett, A., Ilmer, T., Caballero, A.T., Haendeler, S., Netzer, M.A., Ginistrelli, L.C., Cirigliano, M., Mancheno, E.J., Tavernini, K., Hering, S., Hofbauer, P., Mendjan, S., n.d. Multi-chamber cardioids unravel human heart development and cardiac defects.



Posted on: 13 February 2023 , updated on: 14 February 2023

doi: Pending

Read preprint (1 votes)

Author's response

The author team shared

  • Early IGF-1 treatment appears to have a major impact on later morphogenetic events: any ideas on the mechanism involved?

The authors’ response: That is a great question. IGF-1 is known to play a large role in the maturation of many organs during fetal development and postnatal growth (Li et al., 2011, Development; Liu et al., 1993, Cell; Ren et al., 1999, J Mol Cell Cardiol). For the heart, it is known to act through IGF1R (insulin-like growth factor 1 receptor) and INSR (insulin receptor) (Belfiore et al., 2017, Endocrine Reviews) – both receptor tyrosine kinases that signal through the mTOR and ERK pathways and ultimately regulate transcription. While IGF-2, another important mitogen involved in cardiac development and morphogenesis has been studied to an extent (Brisken et al., 2002, Developmental Cell; Li et al., 2011, Development), the exact role of IGF-1 is understudied. It is likely that IGF-1 acts via similar mechanisms at IGF-2 since they both act through IGF1R and INSR (Moral et al., 2021, Int J Mol Sci). Nonetheless, our patterned heart tube organoid model serves as a platform to better investigate and answer this question and more. 

  • Do the atrial and ventricular cells derive from SHF and FHF progenitors, respectively, as during normal development? Alternatively, is the MYL7 to MYL2 gradient explained chiefly by selective maturation of FHF cells?

The authors’ response: In our previous paper, we showed the emergence of the SHF and FHF through staining for HAND1, HAND2, and others. As shown in our scRNA-seq datasets, the atrial and ventricular cardiomyocyte populations possess key markers for their in vivo counterparts, as do many of the other cell types present in the organoid. With these recapitulations of normal development, among others, we believe that the atrial and ventricular cardiomyocytes do derive from the SHF and FHF, respectively. The beauty of our organoid system is that it is a completely self-organizing platform. Our developmental maturation strategies were inspired by facets of cardiac development, and through applying them to the organoids, we are biochemically manipulating the organoid, including every cell type within the organoid. In this way, it is not possible to selectively maturate just one cell type. If the organoid is a seed, we are simply trying to provide the right amount of water and sunlight for it to prosper.

  • Could the causal role of RA gradient in A-P patterning be established, perhaps through genetic or pharmacological inhibition?

The authors’response: This is an exciting question, and one that we are also considering. In short, yes, we are developing means to investigate the role of retinoic acid in A-P patterning. On one hand, we are exploring pharmacological inhibition of RA signalling, on the other, we are developing fluorescent-reporter systems to perform live tracking of the effects of RA in the organoids as they develop. For example, we designed a Retinoic Acid Response Element (RARE)-mCherry reporter system that functions through the nuclear effects of RA. RA acts through RAREs which are found in promoters of many genes and directly affect transcriptional activity.

  • What is a key example of an application that would showcase the benefits of this model over earlier iterations?

The authors’ response: A key example of how this model could be capitalized upon would be to study congenital heart diseases, which are the leading type of birth defects in humans, whether their source is genetic (such as a mutation) or environmental (an exposure, such as maternal diabetes). There is no access to first hand human tissues/organs at this stage, and animal models fail to recapitulate fine details specific to humans. Thus, the heart organoids provide a key tool in this field. The organoids can also be efficient tools for pharmacological and toxicological studies in the human heart, and can also be used to study normal heart development and provide insights independent of animal models (reducing our reliance on animal observations which might be inaccurate, and overall reducing animal use in general).

  • How reproducible is the protocol across different batches of differentiations and, most importantly, different iPSC lines? What are, if any, the reagents that require batch testing/dosage optimization?

The authors’ response: In our hands this protocol is reproducible across different passage numbers and human pluripotent stem cell lines, including both ESCs and iPSCs. The most important aspects are the medium (E8, our protocol does not work in other media), the quality of the PSCs, and the quality/testing of the reagents involved in the differentiation and maturation of the organoids: CHIR, BMP4, ActA, WntC59 and the fatty acids/growth factors used in the developmental maturation strategies.

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