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

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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. 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 …

  2. 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 …

  3. 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 …

  4. 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 …

  5. 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) …