Conditional blastocyst complementation of a defective Foxa2 lineage efficiently promotes the generation of the whole lung
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eLife assessment
This current study provides a new model of lung agenesis to explore the generation of the ability of blastocyst complementation to generate an entire organ. These studies will provide new avenues for organ bioengineering and additional insight into early contribution of mesoendoderm to lung development.
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Abstract
Millions suffer from incurable lung diseases, and the donor lung shortage hampers organ transplants. Generating the whole organ in conjunction with the thymus is a significant milestone for organ transplantation because the thymus is the central organ to educate immune cells. Using lineage-tracing mice and human pluripotent stem cell (PSC)-derived lung-directed differentiation, we revealed that gastrulating Foxa2 lineage contributed to both lung mesenchyme and epithelium formation. Interestingly, Foxa2 lineage-derived cells in the lung mesenchyme progressively increased and occupied more than half of the mesenchyme niche, including endothelial cells, during lung development. Foxa2 promoter-driven, conditional Fgfr2 gene depletion caused the lung and thymus agenesis phenotype in mice. Wild-type donor mouse PSCs injected into their blastocysts rescued this phenotype by complementing the Fgfr2-defective niche in the lung epithelium and mesenchyme and thymic epithelium. Donor cell is shown to replace the entire lung epithelial and robust mesenchymal niche during lung development, efficiently complementing the nearly entire lung niche. Importantly, those mice survived until adulthood with normal lung function. These results suggest that our Foxa2 lineage-based model is unique for the progressive mobilization of donor cells into both epithelial and mesenchymal lung niches and thymus generation, which can provide critical insights into studying lung transplantation post-transplantation shortly.
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Author Response
Reviewer #1 (Public Review):
In this manuscript, Drs. Miura, Mori, and colleagues, first present lineage tracing data using PDGFRa-CreERT2 and Foxa2-Cre drivers to show that PDGFRa+ cells, when lineage-labeled early in development go on to form the lung mesenchyme (but little to none of the epithelium), whereas FOXA2 expressing cells go on to contribute to both the lung epithelium and lung mesenchyme. However, it is already well known that FOXA2 is expressed in the mesendoderm around the time of gastrulation, and that this population generates both endoderm and mesodermal derivatives. As a result, it is not surprising that lineage labeling this population would contribute to both the lung epithelium and lung mesenchyme. The authors use the term bona fide lung (BFL) generative lineage. However, since the mesendoderm …
Author Response
Reviewer #1 (Public Review):
In this manuscript, Drs. Miura, Mori, and colleagues, first present lineage tracing data using PDGFRa-CreERT2 and Foxa2-Cre drivers to show that PDGFRa+ cells, when lineage-labeled early in development go on to form the lung mesenchyme (but little to none of the epithelium), whereas FOXA2 expressing cells go on to contribute to both the lung epithelium and lung mesenchyme. However, it is already well known that FOXA2 is expressed in the mesendoderm around the time of gastrulation, and that this population generates both endoderm and mesodermal derivatives. As a result, it is not surprising that lineage labeling this population would contribute to both the lung epithelium and lung mesenchyme. The authors use the term bona fide lung (BFL) generative lineage. However, since the mesendoderm contributes to both the endoderm and mesoderm, but is by no means specific to the lung, and as shown in this paper (Figure 2G) the FOXA2 population only generates 30-40% of the mesenchyme, the term BFL is both confusing and misleading.
We deleted the BFL concept and the sentences from the entire manuscript.
In the second portion of the manuscript, the authors conditionally delete Fgfr2 using a Foxa2-Cre driver. Although loss of Fgf10 or Fgfr2 is known to result in lung agenesis, deletion of Fgfr2 within the FOXA2+ expressing cells is novel. However, since FOXA2 is broadly expressed within the nascent lung epithelium and Fgfr2 is known to be expressed within the lung epithelium, it isn't entirely clear how much information this adds beyond what already known from other Fgfr2 knockout studies. Perhaps the most interesting aspect of the reported phenotype is that the other organs (e.g. intestine) in these knockout animals appears to be relatively spared. This should be better characterized by the authors, as currently only a few H&E images are shown.
As the reviewer described, Foxa2 is broadly expressed in the epithelium of several organs. We analyzed the other organs of Foxa2Cre/+; Fgfr2cnull mice shown in new Figures 4 - figure supplement 1C and 2A outlined in the manuscript, lines 267-275. We found that the intestine and other major organs were tdTomato-labelled but intact. Significantly, we discovered that thymus agenesis phenotype in Foxa2Cre/+; Fgfr2cnull mice because of the Fgfr2 requirement for their development (Dooley et al., 2007).
The authors then used conditional blastocyst complementation with nGFP+iPSCs from wild-type mice to rescue the phenotype of the Fgfr2 conditional knockout mice, showing that an embryonic lung is formed. However, blastocyst complementation has previously been performed with other knockout mouse models with severe lung hypoplasia/aplasia, including Dr. Mori's previous Nature Medicine paper. Although most of the previous mouse models target the endoderm/early epithelial cells (e.g. conditional deletion of Ctnnb1, Fgfr2, or global knockout of Nkx2.1; see Li E, et al. Dev Dyn 2021 Jul;250(7):1001-1020; Wen B, Am J Resp Crit Care Med. 2020; in addition to Mori M, Nature Medicine, 2019), Kitahara A, et al (Cell Rep. May 12 2020;31(6):107626) previously reported blastocyst complementation in in Fgf10 null mouse model, so it is not clear what the current study significantly adds contributes to this existing body of literature. The lungs of the mice undergoing blastocyst complementation are also incompletely characterized in the current version of this study. For example, it is unclear how functional these lungs are and whether they are capable of gas exchange after birth.
Our new Foxa2-lineage-based CBC model mice showed novel evidence of the co-generation of lung and thymus. We also added evidence that those rescued mice of the Foxa2-lineage-based CBC model survived until adulthood with normal lung function. These new findings were included in Figure 5, and described in the manuscript, lines 318-344.
Reviewer #2 (Public Review):
For most organs including lung produced by blastocyst complementation, certain cells including the blood vessels are still derived from host tissues, making them unfit for transplantation. To address this issue, Miura et al. explored the origin and the program of whole lung epithelium and mesenchyme, and identified the crucial Foxa2 lineage for lung organogenesis by using lineage tracing mice and human iPSC derived lung differentiation. They found that Foxa2 lineage cells contribute to both lung epithelium and mesenchyme formation, which suggest targeting Fox2 lineage cells could create an empty developmental niche for blastocyst complementation in mice. They further deplete Fgfr2 gene in Foxa2 lineage cells to induce the lung agenesis phenotype in mice, and donor mouse iPSCs injected into Fgfr2 mutant blastocysts occupied the empty niche and formed the missing lung.
Strengths:
To fill our knowledge gap of the origin of all lung cell types, especially pulmonary mesenchyme and endothelium, the authors investigated the lineage hierarchy of specified lung precursors in gastrulating mesendoderm. Using mouse lineage trancing and human iPSC derived lung differentiation, they clarified the msendoderm gene Expression pattern and progression, and compared the contributions of Pdgfra and Foxa2 lineage cells during lung development. They further demonstrate that the defective Foxa2 lineage in critically important for efficient lung complementation, which provide insight for next generation lung transplant therapies.
Weakness:
- Several lineage tracing experiment lack rigorous quantification, the authors using "partially labels" or "labels a part of" in the text to describe their finding and conclusion, which make the evidence less solid.
As described above, we quantified the lineage tracing mice and added results in new Figures 1C and 1G.
We quantified the lineage-tracing results by morphometric analyses described in Figures 1C and 1F. We provided the quantification of Foxa2 lineage tracing studies in early embryogenesis and removed the unqualified results from Figure 1, and the manuscript was corrected in lines 136-144 and 155-161.
Regarding Figure 1C, we have tried to have more numbers of embryos for these analyses using PdgfraCreERT2; Rosa tdTomato/+ mice. However, we often encountered embryo miscarriage due to the effect of Tamoxifen, even with the titration of tamoxifen or using the co-injection of progesterone (Nikita et al., 2019). Through more than twenty times experimental trials of Tm injection, we finally obtained a total of four embryos, three at E12.5 and one at E14.5. Those results were added in the new Figures 1A and B. This data was outlined in the manuscript, lines 134-141.
- The ideal lung for transplant should be functional for gas exchange, the lung complementation was only analyzed at E17.5 and E14.5, these two stages were too early to determine the function of the lungs generated via CBC.
We showed additional evidence of the rescued mice in adulthood. We confirmed that Foxa2Cre; Fgfr2cnull injected with donor PSCs survived until adulthood, and there are no differences in the respiratory function compared to Foxa2Cre; Fgfr2hetero injected with donor PSCs. We added this result in new Figure 5 and described it in the manuscript lines 318-344.
- Immune cells contribute large proportion in the lung, and are critical for lung transplant, the chimerism analysis of immune cells is missing in this study.
We analyzed the chimerism of hematopoietic cells in the E17.5 experiment, but there were no differences among all chimeric mice (see Table 1 and Figure 4 - figure supplement 3D). We thought this was because the origin of hematopoietic cells is the Liver and Yolk Sac (Yokomizo et al., 2022), which are off-target for our CBC model. However, we found that the thymus was also complemented in this model, as we described above. Since the thymus is a specialized primary lymphoid organ responsible for the education of T cells, essential for the maturation of T cells, this complementation may help for future successful transplantation, which can avoid post-transplantation graft versus host disease (GvHD). This data and discussion were added in Figure 4 - figure supplement 3D and Table 1, and the manuscript lines 293-295, and 417-427.
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eLife assessment
This current study provides a new model of lung agenesis to explore the generation of the ability of blastocyst complementation to generate an entire organ. These studies will provide new avenues for organ bioengineering and additional insight into early contribution of mesoendoderm to lung development.
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Reviewer #1 (Public Review):
In this manuscript, Drs. Miura, Mori, and colleagues, first present lineage tracing data using PDGFRa-CreERT2 and Foxa2-Cre drivers to show that PDGFRa+ cells, when lineage-labeled early in development go on to form the lung mesenchyme (but little to none of the epithelium), whereas FOXA2 expressing cells go on to contribute to both the lung epithelium and lung mesenchyme. However, it is already well known that FOXA2 is expressed in the mesendoderm around the time of gastrulation, and that this population generates both endoderm and mesodermal derivatives. As a result, it is not surprising that lineage labeling this population would contribute to both the lung epithelium and lung mesenchyme. The authors use the term bona fide lung (BFL) generative lineage. However, since the mesendoderm contributes to both …
Reviewer #1 (Public Review):
In this manuscript, Drs. Miura, Mori, and colleagues, first present lineage tracing data using PDGFRa-CreERT2 and Foxa2-Cre drivers to show that PDGFRa+ cells, when lineage-labeled early in development go on to form the lung mesenchyme (but little to none of the epithelium), whereas FOXA2 expressing cells go on to contribute to both the lung epithelium and lung mesenchyme. However, it is already well known that FOXA2 is expressed in the mesendoderm around the time of gastrulation, and that this population generates both endoderm and mesodermal derivatives. As a result, it is not surprising that lineage labeling this population would contribute to both the lung epithelium and lung mesenchyme. The authors use the term bona fide lung (BFL) generative lineage. However, since the mesendoderm contributes to both the endoderm and mesoderm, but is by no means specific to the lung, and as shown in this paper (Figure 2G) the FOXA2 population only generates 30-40% of the mesenchyme, the term BFL is both confusing and misleading.
In the second portion of the manuscript, the authors conditionally delete Fgfr2 using a Foxa2-Cre driver. Although loss of Fgf10 or Fgfr2 is known to result in lung agenesis, deletion of Fgfr2 within the FOXA2+ expressing cells is novel. However, since FOXA2 is broadly expressed within the nascent lung epithelium and Fgfr2 is known to be expressed within the lung epithelium, it isn't entirely clear how much information this adds beyond what already known from other Fgfr2 knockout studies. Perhaps the most interesting aspect of the reported phenotype is that the other organs (e.g. intestine) in these knockout animals appears to be relatively spared. This should be better characterized by the authors, as currently only a few H&E images are shown.
The authors then used conditional blastocyst complementation with nGFP+iPSCs from wild-type mice to rescue the phenotype of the Fgfr2 conditional knockout mice, showing that an embryonic lung is formed. However, blastocyst complementation has previously been performed with other knockout mouse models with severe lung hypoplasia/aplasia, including Dr. Mori's previous Nature Medicine paper. Although most of the previous mouse models target the endoderm/early epithelial cells (e.g. conditional deletion of Ctnnb1, Fgfr2, or global knockout of Nkx2.1; see Li E, et al. Dev Dyn 2021 Jul;250(7):1001-1020; Wen B, Am J Resp Crit Care Med. 2020; in addition to Mori M, Nature Medicine, 2019), Kitahara A, et al (Cell Rep. May 12 2020;31(6):107626) previously reported blastocyst complementation in in Fgf10 null mouse model, so it is not clear what the current study significantly adds contributes to this existing body of literature. The lungs of the mice undergoing blastocyst complementation are also incompletely characterized in the current version of this study. For example, it is unclear how functional these lungs are and whether they are capable of gas exchange after birth.
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Reviewer #2 (Public Review):
For most organs including lung produced by blastocyst complementation, certain cells including the blood vessels are still derived from host tissues, making them unfit for transplantation. To address this issue, Miura et al. explored the origin and the program of whole lung epithelium and mesenchyme, and identified the crucial Foxa2 lineage for lung organogenesis by using lineage tracing mice and human iPSC derived lung differentiation. They found that Foxa2 lineage cells contribute to both lung epithelium and mesenchyme formation, which suggest targeting Fox2 lineage cells could create an empty developmental niche for blastocyst complementation in mice. They further deplete Fgfr2 gene in Foxa2 lineage cells to induce the lung agenesis phenotype in mice, and donor mouse iPSCs injected into Fgfr2 mutant …
Reviewer #2 (Public Review):
For most organs including lung produced by blastocyst complementation, certain cells including the blood vessels are still derived from host tissues, making them unfit for transplantation. To address this issue, Miura et al. explored the origin and the program of whole lung epithelium and mesenchyme, and identified the crucial Foxa2 lineage for lung organogenesis by using lineage tracing mice and human iPSC derived lung differentiation. They found that Foxa2 lineage cells contribute to both lung epithelium and mesenchyme formation, which suggest targeting Fox2 lineage cells could create an empty developmental niche for blastocyst complementation in mice. They further deplete Fgfr2 gene in Foxa2 lineage cells to induce the lung agenesis phenotype in mice, and donor mouse iPSCs injected into Fgfr2 mutant blastocysts occupied the empty niche and formed the missing lung.
Strengths:
To fill our knowledge gap of the origin of all lung cell types, especially pulmonary mesenchyme and endothelium, the authors investigated the lineage hierarchy of specified lung precursors in gastrulating mesendoderm. Using mouse lineage trancing and human iPSC derived lung differentiation, they clarified the msendoderm gene Expression pattern and progression, and compared the contributions of Pdgfra and Foxa2 lineage cells during lung development. They further demonstrate that the defective Foxa2 lineage in critically important for efficient lung complementation, which provide insight for next generation lung transplant therapies.
Weakness:
1. Several lineage tracing experiment lack rigorous quantification, the authors using "partially labels" or "labels a part of" in the text to describe their finding and conclusion, which make the evidence less solid.
2. The ideal lung for transplant should be functional for gas exchange, the lung complementation was only analyzed at E17.5 and E14.5, these two stages were too early to determine the function of the lungs generated via CBC.
3. Immune cells contribute large proportion in the lung, and are critical for lung transplant, the chimerism analysis of immune cells is missing in this study.
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