Organs begin developing during the first few months of pregnancy, while the baby is still an embryo. These early stages of development are known as embryogenesis – a tightly organized process, during which the embryo forms different layers of stem cells. These cells can be activated to turn into a particular type of cell, such as a heart or a lung cell.

The heart and lungs develop from different layers within the embryo, which must communicate with each other for the organs to form correctly. For example, chemical signals can be released from and travel between layers of the embryo, activating processes inside cells located in the different areas.

In mouse models, chemical signals and cells travel between developing heart and lung, which helps both organs to form into the correct structure. But it is unclear how well the observations from mouse models translate to heart and lung development in humans.

To find out more, Ng et al. developed a human model of heart and lung co-development during embryogenesis using human pluripotent stem cells. The laboratory-grown stem cells were treated with chemical signals, causing them to form different layers that developed into early forms of heart and lung cells.

The cells were then transferred into a specific growing condition, where they arranged into three-dimensional structures termed microtissues. Ng et al. found that lung cells developed faster when grown in microtissues with accompanying developing heart cells compared to microtissues containing only developing lung cells. In addition, Ng et al. revealed that the co-developing heart and lung tissues automatically separate from each other during later stage, without the need for chemical signals.

This human cell-based model of early forms of co-developing heart and lung cells may help provide researchers with new strategies to probe the underlying mechanisms of human heart and lung interaction during embryogenesis.

Human embryogenesis is a highly orchestrated process that requires delicate coordination between organs that originate from different germ layers. As the two main organs within the chest cavity, the mesoderm-derived heart and endoderm-derived lung partake in have extensive mutual interaction that are essential for their proper morphogenesis (Peng et al., 2013; Hoffmann et al., 2009; Arora et al., 2012; Steimle, 2018). During mouse embryonic development, WNT derived from the second heart field induces specification of pulmonary endoderm, which in turn secretes SHH that signals back to the heart and regulates proper atrial septation (Steimle, 2018; Zhou, 2017; Hoffmann et al., 2009). This inter-lineage crosstalk is partly mediated by the multipotent mesodermal progenitors located between the developing heart and lung, which have the potential for lineage contribution to pulmonary endothelium, pulmonary smooth muscle and cardiomyocytes (Peng et al., 2013). However, the extent of translation of findings derived from rodent models to the understanding of developmental interplay between human cardio-pulmonary systems remains unclear. There is, therefore, a critical need for experimentally tractable systems for investigating human cardio-pulmonary co-development during organogenesis.

Much work has been done for directed differentiation of hiPSCs into either cardiomyocytes (Lian et al., 2012; Mummery et al., 2012; Burridge et al., 2014; Lian et al., 2015; Lee et al., 2017; Kattman et al., 2011), or pulmonary epithelium (Chen et al., 2017; Huang et al., 2013; Jacob et al., 2017; Dye et al., 2015; Gotoh et al., 2014; Wong et al., 2012), both of which often utilize stepwise differentiation strategies that recapitulate key developmental signaling events. To recapitulate cardiogenesis, hiPSCs were sequentially specified into mesoderm, cardiac mesoderm, and then NKX2.5⁺ cardiac progenitors (Lian et al., 2012; Mummery et al., 2012; Burridge et al., 2014; Lian et al., 2015; Lee et al., 2017; Kattman et al., 2011; Laflamme et al., 2007). For pulmonary induction, hiPSCs went through stages corresponding to definitive endoderm and anterior foregut endoderm, and then became NKX2.1⁺ lung epithelial progenitors (Chen et al., 2017; Huang et al., 2013; Jacob et al., 2017; Dye et al., 2015; Gotoh et al., 2014; Wong et al., 2012; D’Amour et al., 2005; Longmire et al., 2012). Despite the significant contributions of these models make to the mechanistic understanding of human heart and lung organogenesis, they generally focus on one organ parenchyma at a time. It remains challenging to model and investigate multi-organ co-development within a single differentiation of hiPSCs, especially when the organs of interest are derived from different germ layers, as is the case for the heart and lung.

Comparison of existing protocols for single-lineage cardiac and pulmonary differentiation from hiPSCs indicates shared regulators despite their distinct germ-layer origin. Firstly, both endodermal and mesodermal specification is facilitated by the inhibition of insulin and phosphoinositide 3-kinase signaling (Lian et al., 2012; Lian et al., 2013; Mou et al., 2012), and can be induced by a similar set of paracrine factors, including WNT, BMP, and TGF-β (Kattman et al., 2011; D’Amour et al., 2005; Loh, 2014). It is the quantitative combination of these signaling that determines endoderm versus mesoderm bifurcation (Kattman et al., 2011; Loh, 2014; Kim, 2015). This is consistent with the shared primitive streak origin of both germ layers during gastrulation (Levak-Svajger and Svajger, 1974; Lawson et al., 1991; Tam and Beddington, 1987). Secondly, WNT inhibition not only facilitates the transition from definitive endoderm to anterior foregut endoderm (Loh, 2014; Spence et al., 2011), but it also promotes cardiac mesoderm emergence (Lian et al., 2012; Willems et al., 2011; Wang et al., 2011; Ren et al., 2011; Tran, 2009). Lastly, retinoic acid (RA) signaling is required for the induction and maturation of both cardiac and pulmonary progenitors (Huang et al., 2013; Jacob et al., 2017; Mou et al., 2012; Chen, 2007; McCauley et al., 2017). These common paracrine regulation of paralleled cardiac and pulmonary specification is consistent with their close spatial coordinates within the embryonic body planning, as demonstrated by shared HOX genes expression and functional requirement (Lufkin et al., 1991; Makki and Capecchi, 2012; Chisaka and Capecchi, 1991).

In this study, we described a stepwise, growth-factor-free protocol for simultaneous induction of cardiac and pulmonary progenitors from a single culture of hiPSCs. This is accomplished by initial co-induction of mesoderm and definitive endoderm mixture, followed by their concurrent specification into cardiac (NKX2.5⁺) and lung (NKX2.1⁺) progenitors, respectively, using the same sets of small molecule cocktails modulating WNT, nodal and TGF-β signaling in a temporal specific manner. Using 3D suspension culture with continuing WNT activation, we engineered pulmonary-centered, cardio-pulmonary micro-Tissues (μTs), and demonstrated the accompanying cardiac lineage as an essential cellular niche that promoted effective alveolar maturation. Finally, following the withdrawal of WNT agonist, each concentric cardio-pulmonary μT reorganized and ultimately segregated into cardiac-only and pulmonary-only μTs. This work therefore offers an effective hiPSC-based model for investigating cardio-pulmonary co-development and tissue segregation during human embryogenesis.

Here, we described a novel strategy to model human cardio-pulmonary co-development using multi-lineage hiPSC differentiation. The current study primarily focused on co-induction of cardio-pulmonary progenitor cells, thus establishing the foundation for future investigations of how the crosstalk between these two organ lineages regulates their respective lineage maturation. We demonstrated that upon co-induction of mesoderm and endoderm, a series of shared signaling events were capable of driving simultaneous cardiac and pulmonary specification from their respective germ-layer progenitors. Upon transitioning the co-induced cardiac and pulmonary progenitors to 3D suspension culture, we observed expedited alveolar maturation within 3 days, which was supported by the accompanying cardiac lineage. In 3D suspension culture, each cardio-pulmonary μT effectively segregates into separate cardiac and pulmonary μTs, which was partially inhibited by WNT activation. This study therefore delivers an effective in vitro model for studying the mechanistic interplay between the developing heart and lung during human embryogenesis.

The extensive cardio-pulmonary mutual interaction during organogenesis has been well documented in the mouse model (Peng et al., 2013; Hoffmann et al., 2009; Steimle, 2018); however, the translatability of these findings to human embryogenesis remains elusive due to the lack of proper model systems. Human pluripotent stem cell differentiation has offered an effective means for recapitulating and investigating human organogenesis, and tremendous progress has been made toward directed cardiac or pulmonary specification (Lian et al., 2012; Mummery et al., 2012; Burridge et al., 2014; Lian et al., 2015; Lee et al., 2017; Kattman et al., 2011; Chen et al., 2017; Huang et al., 2013; Jacob et al., 2017; Dye et al., 2015; Gotoh et al., 2014; Wong et al., 2012; Laflamme et al., 2007; D’Amour et al., 2005; Longmire et al., 2012; Lian et al., 2013; Mou et al., 2012; Green et al., 2011). However, almost all existing models have been focusing on one parenchymal lineage at a time, and therefore lack the ability to support the investigation of inter-organ crosstalk. Here, building on the established understanding of signaling events necessary for cardiac and pulmonary induction (Lian et al., 2012; Mummery et al., 2012; Burridge et al., 2014; Lian et al., 2015; Lee et al., 2017; Kattman et al., 2011; Chen et al., 2017; Huang et al., 2013; Jacob et al., 2017; Dye et al., 2015; Gotoh et al., 2014; Wong et al., 2012; Laflamme et al., 2007; D’Amour et al., 2005; Longmire et al., 2012; Lian et al., 2013; Mou et al., 2012; Green et al., 2011), we have developed a robust protocol for the simultaneous co-differentiation of cardiac and pulmonary lineages from hiPSCs. Within our co-differentiation system, unrestricted interaction between cells of both lineages is enabled even before their lineage commitment.

Most current attempts on pulmonary induction from hiPSCs relies on initial nodal activation using growth factor (Activin A) supplementation, which is critical for definitive endoderm specification. Here, we showed that by fine tuning WNT signaling using a small-molecule inhibitor of GSK-3β (CHIR), robust induction of endoderm and subsequently lung progenitors can be achieved without any exogenous growth factors. This is consistent with the observation that CHIR was capable of inducing cardiac differentiation in replacement of combined effect of exogenous Activin A and BMP4 (Lian et al., 2012). In addition, Martyn et al., 2019 demonstrated that WNT is sufficient to induce primitive streak, which then creates a gradient of signals (BMP, WNT, Nodal) that could further specify fate towards mesoderm and endoderm lineages (Martyn et al., 2019). Nonetheless, Nodal and BMP signaling remains crucial in mesoderm and endoderm specification, as inhibition of these signals effectively terminated cardio-pulmonary co-induction.

Our study demonstrated the need for endogenous TGF-β signaling for effective cardio-pulmonary induction, as well as the critical role of endogenous BMP signaling in cardiogenesis. Furthermore, we found that temporal-specific action of the same set of small molecules regulating TGF-β and WNT signaling was capable of simultaneously driving mesoderm-to-cardiac and endoderm-to-pulmonary specification. Moreover, BMP4 has been shown to improve NKX2.1⁺ lung progenitor specification in both mouse and hiPSCs (Huang et al., 2013; Jacob et al., 2017; Serra et al., 2017). In our system, endogenous instead of exogenous BMP signaling was required during a developmental stage corresponding to foregut ventralization for effective co-emergence of cardiac and pulmonary progenitors. This is in line with the close spatial positioning of the developing heart and lung primordia within embryonic body patterning, which implies their exposure to a similar paracrine microenvironment (Steimle, 2018; Herriges and Morrisey, 2014).

To achieve alveolarization, NKX2.1⁺ lung progenitors are usually embedded in extracellular matrices, such as Matrigel and collagen (Jacob et al., 2017; Dye et al., 2015; de Carvalho et al., 2019). Here, we established an effective approach that enabled AT2 cell maturation within 3 days in suspension culture of 3D cell aggregates spontaneously formed from Day-15 cardiac and pulmonary progenitors. We further demonstrated that the presence of accompanying cardiac lineage is critical for robust alveolar induction. This observation is consistent with the recently reported inter-dependence between cardiac and pulmonary lineages during embryogenesis (Steimle, 2018). In the absence of cardiac lineage, NKX2.1⁺ lung progenitors could not achieve as effective AT2 maturation as what is observed in cardio-pulmonary μTs. This is again emphasizing that chemical cues derived from heart field also plays a role in lung development (Peng et al., 2013; Steimle, 2018). However, it should be noted that pulmonary mesenchyme could also augment distal lung differentiation, which requires further investigation with the ability to differentiate the participation and contribution of each mesodermal cell lineages (Hawkins et al., 2017; McCulley et al., 2015; Leeman et al., 2019). In addition, the presence of mesoderm-derived stromal cells have also been shown to be essential for effective alveolarization in vivo (Herriges and Morrisey, 2014; Domyan et al., 2011; Rankin et al., 2016) and in vitro (Gotoh et al., 2014; Hawkins et al., 2017). Furthermore, cells of the mesodermal lineage are known to be robust producers of extracellular matrix, which may also contribute to the effective alveolar maturation in the absence of external extracellular matrix support. The ability to enable effective alveolar induction from hiPSC-derived lung progenitors with the convenient of suspension culture also opens the door to large-scale production of alveolar cells, a critical challenge in regenerative medicine applications.

Using dual-lineage cardio-pulmonary μTs formed from the co-induced progenitors, we observed a novel process of cardio-pulmonary tissue segregation. The human body cavities are highly crowded spaces, filled with different tissues and organs that are in close contact with each other. It remains enigmatic how inter-organ boundaries are maintained to prevent undesired cell migration or tissue merging. Our cardio-pulmonary tissue segregation model suggests an intrinsic mechanism that effectively establishes a boundary between two distinct parenchymal lineages even when they are initially mingled together. Although no model of collective migration has been described in the context of cardio-pulmonary development, studies in other model systems suggest cell-cell communication and paracrine signaling (e.g. WNT) to be crucial for directed cell migration during development (Ciruna and Rossant, 2001; De Calisto et al., 2005; Carmona-Fontaine et al., 2008). Here, we found that exogenous WNT activation via GSK-3β inhibition effectively slowed down the cardio-pulmonary segregation, while inhibition of endogenous WNT (canonical and non-canonical) did not obviously affect the process. In consistence with our observation, it has been shown that the inhibition of non-canonical WNT signaling does not stop collective cell migration but distorting migration direction (Li and Wang, 2018).

Despite the presence of AT1-like cells in cardio-pulmonary μTs based on HOPX staining (Wang et al., 2018; Frank et al., 2019; Liebler et al., 2016), the organization of these cells remained unlike the thin, flat squamous cells found in the native lung (Yang et al., 2016; Williams, 2003). Although HOPX expression has been observed in cardiomyocytes (Friedman et al., 2018), we did not observe HOPX expression following establishment of cardio-pulmonary progenitors, and the induction of HOPX coincided with alveolar maturation during Day-15 to Day-18. Further study is needed to explore the possibility of obtaining AT1-like cells with a more mature phenotype using multiple markers. Our work demonstrates early emergence of SFTPC-expressing cells as early as Day-18, with CHIR and 3D suspension culture being key driving forces that promote alveologenesis (Huang et al., 2013; Jacob et al., 2017), Removal of CHIR on Day-18 led to a decreased in SFTPC expression on Day-22 (Figure 4—figure supplement 1c), suggesting that the complete alveolar niche was not fully established yet. Future investigation is needed to further clarify components of the supporting alveolar niche as well as its timeline of establishment. In parallel, cardiac contractility of the μTs was initiated 7 days following removal of CHIR, consistent with previous studies showing that GSK-3β inhibition promotes cardiomyocyte proliferation but hinders contraction by affecting myofibrillar architecture (Buikema et al., 2020; Wang et al., 2016; Tseng et al., 2006).

Heart begins to form around gestation week 3, which is the earliest organ to be developed during embryogenesis (Buckingham et al., 2005; Tan and Lewandowski, 2020). Lung development initiates soon afterwards during weeks 4–7 (Schittny, 2017). In the present study, we showed that the progenitor cells of both heart and lung can be simultaneously induced following 15 days of co-differentiation from hiPSCs. More comprehensive time-series analysis will be necessary to further delineate the fine temporal relationship between heart and lung lineage specification, which is expected to provide fundamental insights regarding cardio-pulmonary crosstalk during their paralleled organogenesis.

In conclusion, our work focuses on specification of cardio-pulmonary progenitors that have the potential to further mature into their respective descendent lineages. Further morphological and functional maturation of both cardiac and pulmonary lineages, as well as their crosstalk during this process will require future in-depth investigation. In addition, our work offers a novel model for investigating the molecular and cellular mechanisms underlying human cardio-pulmonary co-development and tissue boundary formation. We also expect this work to be of potential use for studying congenital diseases affecting both cardiovascular and pulmonary systems, such as congenital diaphragmatic hernia.

All data supporting the findings of this study are available within the article and its supplementary files. Source data files have been provided for Figures 1 to 6.

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Author details

1. Wai Hoe Ng

   Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review and editing

   Competing interests

   This author is a co-inventor of a related provisional patent application (No. 63/124422) entitled 'Methods for simultaneous cardio-pulmonary differentiation and alveolar maturation from human pluripotent stem cells'

   "This ORCID iD identifies the author of this article:" 0000-0002-6876-6542

2. Elizabeth K Johnston

   Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, United States

   Contribution

   Investigation, Methodology, Writing – review and editing

   Competing interests

   This author is a co-inventor of a related provisional patent application (No. 63/124422) entitled 'Methods for simultaneous cardio-pulmonary differentiation and alveolar maturation from human pluripotent stem cells'

3. Jun Jie Tan

   Advanced Medical and Dental Institute, Universiti Sains Malaysia, Penang, Malaysia

   Contribution

   Supervision, Writing – review and editing

   Competing interests

   No competing interests declared

4. Jacqueline M Bliley

   1. Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, United States
   2. Department of Materials Science and Engineering, Carnegie Mellon University, Pittsburgh, United States

   Contribution

   Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

5. Adam W Feinberg

   1. Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, United States
   2. Department of Materials Science and Engineering, Carnegie Mellon University, Pittsburgh, United States

   Contribution

   Resources, Supervision, Writing – review and editing

   Competing interests

   No competing interests declared

6. Donna B Stolz

   Center for Biologic Imaging, University of Pittsburgh, Pittsburgh, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

7. Ming Sun

   Center for Biologic Imaging, University of Pittsburgh, Pittsburgh, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

8. Piyumi Wijesekara

   Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, United States

   Contribution

   Investigation, Visualization

   Competing interests

   No competing interests declared

9. Finn Hawkins

   Center for Regenerative Medicine of Boston University and Boston Medical Center, Boston, United States

   Contribution

   Resources, Supervision, Writing – review and editing

   Competing interests

   No competing interests declared

10. Darrell N Kotton

    Center for Regenerative Medicine of Boston University and Boston Medical Center, Boston, United States

    Contribution

    Resources, Supervision

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-9604-8476

11. Xi Ren

    Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, United States

    Contribution

    Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Project administration, Resources, Supervision, Writing – original draft, Writing – review and editing

    For correspondence

    xiren@cmu.edu

    Competing interests

    This author is a co-inventor of a related provisional patent application (No. 63/124422) entitled 'Methods for simultaneous cardio-pulmonary differentiation and alveolar maturation from human pluripotent stem cells'

    "This ORCID iD identifies the author of this article:" 0000-0003-3187-1311

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