Recapitulating human cardio-pulmonary co-development using simultaneous multilineage differentiation of pluripotent stem cells

  1. Wai Hoe Ng
  2. Elizabeth K Johnston
  3. Jun Jie Tan
  4. Jacqueline M Bliley
  5. Adam W Feinberg
  6. Donna B Stolz
  7. Ming Sun
  8. Piyumi Wijesekara
  9. Finn Hawkins
  10. Darrell N Kotton
  11. Xi Ren

  • Curated by eLife

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    Evaluation Summary:

    In this manuscript, the authors present at interesting strategy for directing simultaneous induction of both mesoderm-derived cardiac and endoderm-derived lung epithelial lineages from human induced pluripotent stem cells (hiPSC). All reviewers found the work to be of interest, but concerns were raised regarding the efficiency of the differentiation process (including % of differentiated cells in the final cultures) . In addition, it is noted that experiments presented are based on analysis of a single hiPSC cell line, and only part of the differentiation was repeated in another cell line, and thus the broader applicability of the presented protocol remains to be established. However, the interesting data support the conclusions presented. It is likely that the presented methods will be very useful for researchers focusing on heart and lung development, and may inspire others to take similar approaches for studying development of other organs.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

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Abstract

The extensive crosstalk between the developing heart and lung is critical to their proper morphogenesis and maturation. However, there remains a lack of models that investigate the critical cardio-pulmonary mutual interaction during human embryogenesis. Here, we reported a novel stepwise strategy for directing the simultaneous induction of both mesoderm-derived cardiac and endoderm-derived lung epithelial lineages within a single differentiation of human-induced pluripotent stem cells (hiPSCs) via temporal specific tuning of WNT and nodal signaling in the absence of exogenous growth factors. Using 3D suspension culture, we established concentric cardio-pulmonary micro-Tissues (μTs), and expedited alveolar maturation in the presence of cardiac accompaniment. Upon withdrawal of WNT agonist, the cardiac and pulmonary components within each dual-lineage μT effectively segregated from each other with concurrent initiation of cardiac contraction. We expect that our multilineage differentiation model will offer an experimentally tractable system for investigating human cardio-pulmonary interaction and tissue boundary formation during embryogenesis.

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  1. Version published to 10.7554/elife.67872 on eLife
  2. eLife

    Author Response:

    Reviewer #2:

    In this manuscript, Ng et al., report on a system where cardiac mesoderm and pulmonary endoderm co-develop from pluripotent stem cells. This is of potential interest, as it could provide an integrated model for the study of human cardiopulmonary development.

    The main weakness lies in the lack of thorough characterization of the resulting cells and tissues. The characterization relies almost entirely on reporter gene expression and PCR for a limited set of markers. The only indication that ATII cells are generated is expression of a SPC-dTomato reporter and SFTPC mRNA. No evidence is given of function, of expression of other markers or direct staining for SPC, or of ultrastructure. No data are provided whether the lung component contains other lung cells. Another outstanding question for the lung component is whether any pulmonary mesenchyme was generated.

    Thank you for the suggestion. In the revised manuscript, we have included further cellular characterization of the 3D µTs. We included additional characterization for alveolar type 2 (AT2) cells, including a direct immunofluorescence staining of Pro-SPC and transmission electron microscopy imaging of the lamellar bodies (Fig. 6). Besides AT2 cells, we also identified the emergence of AT1-like cells via the expression of HOPX (Fig. 6b). To characterize cell types beyond the alveolar epithelium, we observed positive staining for S100A4, which is a marker for mesenchyme in the µTs (Figure 6-figure supplement 1a). In the meantime, we did not detect any proximal airway epithelial cell types, such as cilia cells (FOXJ1), secretory cell (MUC5AC), and basal cells (p63) (Figure 6-figure supplement 1b-d).

    The same is true for the cardiac component. Which types of cardiac cells are generated: ventricular, atrial, endocardium, epicardium, conducting tissue? No benchmarking was done compared to either human tissues or similar cells generated using more focused differentiation protocols, and functional studies are very limited.

    We agree with the reviewer’s perspective that the present study was primarily focused on progenitor specification. Nonetheless, in the revised manuscript, we have provided additional characterization of the induced cardiac tissues via immunofluorescence staining of Sarcomeric Alpha Actinin. Further, we have included new data on assessing the cardiac contractile function using a calcium channel blocker (Verapamil), showing reduced contractility in response to increasing concentrations of Verapamil (Fig. 6e).

    Another weakness is that there is no characterization of early intermediate developmental stages: primitive streak, mesendoderm, definitive endoderm, cardiac mesoderm, first or second heart field. This type of analysis would be required to validate this complex model as an approach to study human cardiopulmonary development.

    Thank you for pointing this out. In the revised manuscript, we have added a new figure (Figure 1-figure supplement 1) to include data on characterizing the presence of primitive streak by staining for T (Brachyury) after 2 days CHIR treatment. We also showed the presence of the mesendodermal marker (MIXL1), endodermal marker (SOX17) and mesodermal marker (NCAM1) during Stage-1 co-differentiation, as indicated by qPCR and immunostaining (Fig. 1).

    There is also no quantification of differentiation efficiency and yield, and neither are data shown to document absence or presence of other endodermal or mesodermal lineages. NKX2.1, for example is also expressed in the forebrain and in the thyroid.

    Thank you for the suggestion. In the revised manuscript, we have included FACS analysis of Day-15 differentiated cells to quantify the percentage of NKX2.1+ lung and NKX2.5+ cardiac progenitor cell populations. To assess the possibility of other related endodermal and neuronal cell populations, we have included new data on characterizing the Day-15 differentiated cells and showed no co-expression of NKX2.1 with TUJ1 (neuronal marker) or PAX8 (thyroid marker), thus, further supporting the observed NKX2.1+ cells representing the lung lineage (Figure 2-figure supplement 1).

    A final limitation is that multiple pluripotent line should be used.

    In the revised manuscript, we have provided a comprehensive characterization of applying the co-differentiation protocol to another hiPSC line (BU1), including germ layer induction, cardio-pulmonary progenitor induction, 3D organoid formation, and alveolar maturation (Figure 4-figure supplement 5). We have included the data for mesoderm and endoderm induction during Stage-1 (Figure 4-figure supplement 5b) and cardio-pulmonary µT formation from Day-15 progenitor cells. On Day-18, we showed that BU1-derived cardio-pulmonary µTs were stained positive for NKX2.1 and NKX2.5 as what we have observed in BU3. These µTs were also able to further mature into distal lung epithelial cells as indicated by positive staining of SFTPC and HOPX. Meanwhile, the NKX2.5+ cardiac lineages expressed cTnT and Sarcomeric Alpha Actinin ( Figure 4-figure supplement 5e).

    This type of model could be very useful, but it not clear that the goal of integrated cardiopulmonary development was achieved.

    We thank the reviewer for the comment. The following findings from this study suggests that an in vitro hiPSC-based integration of cardio-pulmonary development is possible. First, we showed that following establishment of a mixture of endoderm and mesoderm, the same set of signaling molecules were capable of inducing parallel induction of endoderm-to-pulmonary and mesoderm-to-cardiac specification, echoing their close spatial coordinates with embryonic body patterning and shared requirement of paracrine signaling. Second, we showed that in the presence of cardiac accompaniment, alveolar maturation was expedited, implying inter-lineage crosstalk between the co-developing cardio-pulmonary systems. In the meantime, we agree to the overall suggestion from reviewers that this study is primarily focusing more on cardio-pulmonary progenitor specification, and future investigations are needed to further clarify the mechanism and outcome of integrated cardio-pulmonary co-development. We have added this clarification in our revised manuscript.

    Reviewer #3:

    Ng and Johnston et al. reported the successful multilineage co-differentiation of mesoderm-derived cardiac and endoderm-derived lung progenitors from human pluripotent stem cells (hPSCs). The authors achieved their goals through a stepwise strategy built on the knowledge from published cardiac and lung differentiation protocols. The authors first employed WNT activation using GSK3 inhibitor CHIR, an established WNT signaling agonist, at relatively high dosage to induce primitive streak formation from hPSCs maintained in pluripotent medium (days 1-2). This is supported by knowledge from vertebrate development that both mesodermal and endodermal germ layers are patterned by primitive streak. This is also consistent with recent findings by Martyn et al. (PMID 29795348, https://doi.org/10.1038/s41586-018-0150-y) that activation of WNT signaling is sufficient to induce primitive streak from hPSCs. In the subsequent step (days 2-4), the newly formed primitive streak provides a gradient of endogenous WNT, BMP and Nodal/Activin signaling, which allows the co-induction of both mesoderm and definitive endoderm (DE) from the remaining hPSCs in culture in a serum and morphogen free differentiation medium. Consistently, high Nodal (by exogenous Activin A) favors endodermal induction at the expense of mesodermal specification, and medium-high exogenous BPM4 is detrimental to lung endodermal specification but enhances cardiac mesodermal differentiation. The authors then demonstrated that dual TGF and WNT inhibition is efficient to pattern the mesoderm and endoderm simultaneously for future cardiac and lung induction (days 4-8). This agrees with the existing knowledge that lungs derive from anterior foregut endoderm, and cardiac progenitors, the major substance of heart, derive from cranial lateral mesoderm. Mesoderm and DE patterning was followed by lung and heart specification through the activation of WNT and RA signaling exogenously, in the presence of endogenous BMP4 signaling (days 8-15).

    The differentiation strategy developed by the authors follows the lung and cardiac developmental paradigm overall, the protocol yields efficient lung and heart progenitor specification on the tested hiPSC line. The work provides a new insight into cardiac and lung directed differentiation, and offers a valuable platform to study human heart and lung development in vitro. For cardiac and pulmonary progenitor differentiation (days 4-15), the protocol described in this manuscript relies mainly on the exogenous application of common key developmental signal events shared by heart and lung specification from meso- and endo- derms, respectively. For progenitor maturation (post day 15), the data shows expedite alveolar maturation process in cardio-pulmonary co-differentiation culture, suggesting paracrine signal(s) from cardiac cells positively regulate alveolar maturation. The authors did not report any data on whether/how paracrine signal(s) from lung lineages may influence cardiac maturation. The authors achieved their goals, and the results support the conclusion of the paper overall.

    We agree to the reviewer’s point of view that the present study was primarily focused on progenitor cell induction and the maturation of the pulmonary lineage. In response to the reviewer’s comment, we have provided additional discussion suggesting how this model can be used to further investigate how paracrine signals from the lung lineage may influence cardiac maturation. Also, thank you for suggesting the reference (PMID19795348), we have added it to the manuscript.

    The weaknesses of manuscript are: 1) Lack of evidence/characterization of primitive streak formation at 48 hours of differentiation. 2) Lack of a thorough characterization of the composition of the entire differentiation culture at progenitor stage (day 15): it is very likely that there are pulmonary mesenchymal/mesodermal cells generated in the differentiation culture, besides cardiac mesoderm. The pulmonary mesenchyme may not be abundant in quantity but it plays critical roles in promoting alveolar maturation that the authors observed at day 18 of co-differentiation culture. Before drawing a conclusion, the authors must examine rigorously whether alveolar maturation was promoted by cardiac mesoderm or pulmonary mesoderm.

    We thank the reviewer for bringing this to our attention. In the revised manuscript, we have provided additional data on characterizing the primitive streak at 48 hours of differentiation (Figure 1-figure supplement 1), as well as on characterizing Day-15 differentiated cells using FACS analysis (Figure 2-figure supplement 1a-c). Further, as the reviewer pointed out, it is possible that supporting function maybe coming from additional mesodermal lineages aside from cardiac mesoderm. This has been demonstrated in the mouse model by Peng et al. that the heart served as a reservoir of cardiac and pulmonary mesenchymal cells that play a major role in lung development. In the revised manuscript, we have also added staining of S100A4, a marker for mesenchyme, in the 3D µT (Figure 6-figure supplement 1), as well as an additional discussion (line 556-562) on future studies needed to further assess the regulation of alveolar maturation by cardiac mesoderm or pulmonary mesoderm.

    1. The paper can benefit from providing mechanistic insights into whether/how alveolar maturation medium (CDCIK, days 15-18, and KDCI days 18-25) influenced the downstream cardiac lineage fate specification from the cardiac progenitors. Besides contracting/beating cardia cells, are there any other type(s) of cardiac lineages present in d25 culture? Do the cardiac progenitors generated by this protocol mainly represent cells from primary heart field? Is there any second heart field potential?

    We thank the reviewer for the comments. We agree to the overall comments from the editor and reviewers that the present study was primarily focused on the induction of cardiac and pulmonary progenitors. We also agree with the reviewer that further investigation and understanding of the cellular composition of cardiac-related lineages is needed. Related to this comment, we found that CHIR within the CKDCI was inhibitory for cardiac contraction, which would not initiate until the removal of CHIR, which is consistent with prior studies where they show that GSK-3β inhibition promotes expansion of cardiomyocytes but causes disorganized myofibrillar architecture. We have provided additional related discussion in the revised manuscript.

  3. eLife

    Evaluation Summary:

    In this manuscript, the authors present at interesting strategy for directing simultaneous induction of both mesoderm-derived cardiac and endoderm-derived lung epithelial lineages from human induced pluripotent stem cells (hiPSC). All reviewers found the work to be of interest, but concerns were raised regarding the efficiency of the differentiation process (including % of differentiated cells in the final cultures) . In addition, it is noted that experiments presented are based on analysis of a single hiPSC cell line, and only part of the differentiation was repeated in another cell line, and thus the broader applicability of the presented protocol remains to be established. However, the interesting data support the conclusions presented. It is likely that the presented methods will be very useful for researchers focusing on heart and lung development, and may inspire others to take similar approaches for studying development of other organs.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    In this manuscript, an interesting strategy is presented for directing simultaneous induction of both mesoderm-derived cardiac and endoderm-derived lung epithelial lineages from human induced pluripotent stem cells (hiPSC). This follows from published observations by others showing mutual beneficial cross-talk between the developing heart and lung during embryogenesis. The culture model is partly based on such observations in e.g. mice as well as on comparing protocols for cardiac and pulmonary differentiation from hiPSC, and may be of value for studying such interactions in the developing human heart and lung. The availability of such a human model is important in view of frequent failures in translating findings from rodents to humans. The authors characterized the obtained alveolar and cardiac cells based on a limited number of markers, electron microscopic analysis of lamellar bodies, and by showing contractility of the cardiac tissue obtained. A drawback is that the efficiency of the differentiation process (including % of differentiated cells in the final cultures) was not fully elucidated. Furthermore, since the experiments presented are based on analysis of a single hiPSC cell line, and only part of the differentiation was repeated in another cell line, the broader applicability of the presented protocol remains to be established. However, the interesting data support the conclusions presented. It is likely that the presented methods will be very useful for researchers focusing on heart and lung development, and may inspire others to take similar approaches for studying development of other organs.

  5. eLife

    Reviewer #2 (Public Review):

    In this manuscript, Ng et al., report on a system where cardiac mesoderm and pulmonary endoderm co-develop from pluripotent stem cells. This is of potential interest, as it could provide an integrated model for the study of human cardiopulmonary development.

    The main weakness lies in the lack of thorough characterization of the resulting cells and tissues. The characterization relies almost entirely on reporter gene expression and PCR for a limited set of markers. The only indication that ATII cells are generated is expression of a SPC-dTomato reporter and SFTPC mRNA. No evidence is given of function, of expression of other markers or direct staining for SPC, or of ultrastructure. No data are provided whether the lung component contains other lung cells. Another outstanding question for the lung component is whether any pulmonary mesenchyme was generated.

    The same is true for the cardiac component. Which types of cardiac cells are generated: ventricular, atrial, endocardium, epicardium, conducting tissue? No benchmarking was done compared to either human tissues or similar cells generated using more focused differentiation protocols, and functional studies are very limited.

    Another weakness is that there is no characterization of early intermediate developmental stages: primitive streak, mesendoderm, definitive endoderm, cardiac mesoderm, first or second heart field. This type of analysis would be required to validate this complex model as an approach to study human cardiopulmonary development.

    There is also no quantification of differentiation efficiency and yield, and neither are data shown to document absence or presence of other endodermal or mesodermal lineages. NKX2.1, for example is also expressed in the forebrain and in the thyroid.

    A final limitation is that multiple pluripotent line should be used.

    This type of model could be very useful, but it not clear that the goal of integrated cardiopulmonary development was achieved.

  6. eLife

    Reviewer #3 (Public Review):

    Ng and Johnston et al. reported the successful multilineage co-differentiation of mesoderm-derived cardiac and endoderm-derived lung progenitors from human pluripotent stem cells (hPSCs). The authors achieved their goals through a stepwise strategy built on the knowledge from published cardiac and lung differentiation protocols. The authors first employed WNT activation using GSK3 inhibitor CHIR, an established WNT signaling agonist, at relatively high dosage to induce primitive streak formation from hPSCs maintained in pluripotent medium (days 1-2). This is supported by knowledge from vertebrate development that both mesodermal and endodermal germ layers are patterned by primitive streak. This is also consistent with recent findings by Martyn et al. (PMID 29795348, https://doi.org/10.1038/s41586-018-0150-y) that activation of WNT signaling is sufficient to induce primitive streak from hPSCs. In the subsequent step (days 2-4), the newly formed primitive streak provides a gradient of endogenous WNT, BMP and Nodal/Activin signaling, which allows the co-induction of both mesoderm and definitive endoderm (DE) from the remaining hPSCs in culture in a serum and morphogen free differentiation medium. Consistently, high Nodal (by exogenous Activin A) favors endodermal induction at the expense of mesodermal specification, and medium-high exogenous BPM4 is detrimental to lung endodermal specification but enhances cardiac mesodermal differentiation. The authors then demonstrated that dual TGF and WNT inhibition is efficient to pattern the mesoderm and endoderm simultaneously for future cardiac and lung induction (days 4-8). This agrees with the existing knowledge that lungs derive from anterior foregut endoderm, and cardiac progenitors, the major substance of heart, derive from cranial lateral mesoderm. Mesoderm and DE patterning was followed by lung and heart specification through the activation of WNT and RA signaling exogenously, in the presence of endogenous BMP4 signaling (days 8-15).

    The differentiation strategy developed by the authors follows the lung and cardiac developmental paradigm overall, the protocol yields efficient lung and heart progenitor specification on the tested hiPSC line. The work provides a new insight into cardiac and lung directed differentiation, and offers a valuable platform to study human heart and lung development in vitro. For cardiac and pulmonary progenitor differentiation (days 4-15), the protocol described in this manuscript relies mainly on the exogenous application of common key developmental signal events shared by heart and lung specification from meso- and endo- derms, respectively. For progenitor maturation (post day 15), the data shows expedite alveolar maturation process in cardio-pulmonary co-differentiation culture, suggesting paracrine signal(s) from cardiac cells positively regulate alveolar maturation. The authors did not report any data on whether/how paracrine signal(s) from lung lineages may influence cardiac maturation. The authors achieved their goals, and the results support the conclusion of the paper overall.

    The weaknesses of manuscript are: 1) Lack of evidence/characterization of primitive streak formation at 48 hours of differentiation. 2) Lack of a thorough characterization of the composition of the entire differentiation culture at progenitor stage (day 15): it is very likely that there are pulmonary mesenchymal/mesodermal cells generated in the differentiation culture, besides cardiac mesoderm. The pulmonary mesenchyme may not be abundant in quantity but it plays critical roles in promoting alveolar maturation that the authors observed at day 18 of co-differentiation culture. Before drawing a conclusion, the authors must examine rigorously whether alveolar maturation was promoted by cardiac mesoderm or pulmonary mesoderm. 3) the paper can benefit from providing mechanistic insights into whether/how alveolar maturation medium (CDCIK, days 15-18, and KDCI days 18-25) influenced the downstream cardiac lineage fate specification from the cardiac progenitors. Besides contracting/beating cardia cells, are there any other type(s) of cardiac lineages present in d25 culture? Do the cardiac progenitors generated by this protocol mainly represent cells from primary heart field? Is there any second heart field potential?

  7. Version published to 10.1101/2021.03.03.433714v1 on bioRxiv