CRISPR-Cas9 Genome Editing Allows Generation of the Mouse Lung in a Rat

Abstract

Rationale

Recent efforts in bioengineering and embryonic stem cell (ESC) technology allowed the generation of ESC-derived mouse lung tissues in transgenic mice that were missing critical morphogenetic genes. Epithelial cell lineages were efficiently generated from ESC, but other cell types were mosaic. A complete contribution of donor ESCs to lung tissue has never been achieved. The mouse lung has never been generated in a rat.

Objective

We sought to generate the mouse lung in a rat.

Methods

Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 genome editing was used to disrupt the Nkx2-1 gene in rat one-cell zygotes. Interspecies mouse–rat chimeras were produced by injection of wild-type mouse ESCs into Nkx2-1–deficient rat embryos with lung agenesis. The contribution of mouse ESCs to the lung tissue was examined by immunostaining, flow cytometry, and single-cell RNA sequencing.

Measurements and Main Results

Peripheral pulmonary and thyroid tissues were absent in rat embryos after CRISPR-Cas9–mediated disruption of the Nkx2-1 gene. Complementation of rat Nkx2-1^−/− blastocysts with mouse ESCs restored pulmonary and thyroid structures in mouse–rat chimeras, leading to a near-99% contribution of ESCs to all respiratory cell lineages. Epithelial, endothelial, hematopoietic, and stromal cells in ESC-derived lungs were highly differentiated and exhibited lineage-specific gene signatures similar to those of respiratory cells from the normal mouse lung. Analysis of receptor–ligand interactions revealed normal signaling networks between mouse ESC-derived respiratory cells differentiated in a rat.

Conclusions

A combination of CRISPR-Cas9 genome editing and blastocyst complementation was used to produce mouse lungs in rats, making an important step toward future generations of human lungs using large animals as “bioreactors.”

At a Glance Commentary

Scientific Knowledge on the Subject

Recent efforts in bioengineering and embryonic stem cell (ESC) technology allowed the generation of ESC-derived mouse lung tissues in transgenic mice missing critical morphogenetic genes. However, a complete contribution of donor ESC to lung tissue has never been achieved.

What This Study Adds to the Field

Using a combination of CRISPR-Cas9–mediated disruption of the Nkx2-1 gene and blastocyst complementation, we produced mouse fetal lung tissues in a rat with nearly 99% ESC contribution to all respiratory cell lineages. The present study represents an important step toward future generations of human transplantable lung tissues using large animals as “bioreactors.”

Recent single-cell omics identified over 40 resident cell types in the mammalian lung (1), which is a complex organ that plays a vital role in supplying the entire body with the oxygen required for terrestrial life. The process of lung development across mammalian species is dependent on a series of specific signaling events mediated through receptor–ligand interactions between respiratory cell types derived from endodermal, mesodermal, and ectodermal progenitors. In mice, the trachea and lung buds start to form around Embryonic Day 9.5 postconception (E9.5), whereas the lung development in a rat is delayed by approximately 1.5 days (2–4). In humans, lung specification takes place between 3 and 6 weeks of gestation (1). The initiation of lung morphogenesis requires the expression of homeobox transcription factor NKX2-1, also known as thyroid transcription factor 1, or TTF1. NKX2-1 is a critical regulator of specification and differentiation of all respiratory epithelial cell lineages (1, 5, 6). Primitive respiratory epithelial progenitors of early primordial lung bud express NKX2-1, which is the earliest identified marker of lung and thyroid epithelial lineages (7). Primordial lung cells give rise to all epithelial cell lineages (7), and NKX2-1-expressing progenitors differentiate into multiple respiratory epithelial cell types (8). Alveoli begin to form before and after birth, when NKX2-1 progenitors differentiate into alveolar type 1 (AT1) and type 2 (AT2) epithelial cells. Endothelial progenitor cells give rise to general capillary cells (CAP1, gCAP) and alveolar capillary cells (CAP2, aCAP) (9–11), forming efficient gas exchange units by interacting with epithelial cells and mesenchyme. Mice lacking the Nkx2-1 gene exhibit lung and thyroid agenesis and die at birth after respiratory failure (12). Mutations in the human NKX2-1 gene are linked to congenital lung defects (13), highlighting the crucial role of NKX2-1 in lung embryonic development.

There is a pressing need for the generation of transplantable lung tissues for severe pediatric lung diseases associated with respiratory failure, such as alveolar capillary dysplasia, acinar dysplasia, bronchopulmonary dysplasia, and surfactant deficiencies in newborns (1, 14–18). Recent studies have focused on producing respiratory epithelial cell lineages through the directed differentiation of pluripotent embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). In vitro differentiation protocols have been used to generate AT1 cells, AT2 cells, basal cells, and club cells from mouse and human ESCs and iPSCs (19–22). Lung organoids increase the complexity of ESC- and iPSC-derived structures; however, they do not recapitulate the remarkable diversity of natural lung tissue, which consists of multiple epithelial, stromal, hematopoietic, and endothelial cell types. On implantation into experimental animals, ESC/iPSC-derived cells are capable of undergoing further morphogenesis and tissue remodeling, leading to the formation of complex 3D structures that typically consist of ESC/iPSC-derived epithelial cells and host-derived stromal and endothelial cells that are recruited to the site of implantation (23). Although organoids display similarities in histological structures that often resemble developing respiratory tubules, gene expression studies reveal significant differences between ESC/iPSC-derived cells and endogenous lung cells that have undergone normal morphogenesis in the embryo (23–26).

Blastocyst complementation has been recently utilized to facilitate in vivo differentiation of ESC. In this method, donor ESCs are injected into the blastocysts of recipients that lack critical morphogenetic genes, leading to the formation of chimeras. Pioneering work from the Nakauchi lab led to the generation of the pancreas from mouse, rat, and pig ESCs/iPSCs in intraspecies (within species) and interspecies (between species) chimeras that lack the Pdx1 gene in host embryos (27, 28). The entire pancreas, consisting of donor exocrine and endocrine cells derived from ESCs/iPSCs, was generated in apancreatic Pdx1^−/− embryos, whereas stromal and vascular cells were mosaic (27, 28). Recently, lung tissues were generated from mouse ESCs in mouse transgenic embryos deficient in Fgfr2, β-catenin, Fgf10, and Nkx2-1 (29–31). Rat ESCs contributed to lung tissues in mouse embryos with global deletion of Fgfr2b in a germline (32). All of these published studies required stable knockouts of host embryos and did not result in the generation of donor lungs with a complete contribution from ESCs to all respiratory lineages.

In the present study, we used a combination of clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 genome editing and blastocyst complementation to produce the mouse ESC-derived lung in a rat. Our studies represent an important next step toward the ultimate goal to generate transplantable lungs from patient-derived iPSCs using large animals as “bioreactors.”

Methods

Generation of Rat Nkx2-1^−/− Embryos and Blastocyst Complementation

Sprague Dawley (SD) rat 1-cell embryos were obtained from rat oviducts at E0.5 and injected with Nkx2-1 single-guide (sgRNA) (10 ng/μl) and recombinant Cas9 protein (20 ng/μl; Thermo Fisher) into the cytoplasm to disrupt the rat Nkx2-1 gene (see Figure E1A in the online supplement). The resulting embryos were transferred into oviducts of pseudo-pregnant rat SD females. Blastocysts were harvested at E4.5 and injected with GFP-labeled mouse ESCs (C57Bl/6 background) as described elsewhere (33, 34). All procedures were approved by institutional animal care and use committees.

Fluorescence-activated Cell Sorter (FACS) Analysis, Histology, and Immunostaining

FACS analysis of lung cell suspensions was performed as described elsewhere (17, 30). Tissue sections were stained with hematoxylin and eosin (HandE) or used for immunostaining as previously described (35–39). Information about primary antibodies is provided elsewhere (40–43) (see Table E3). Secondary antibodies conjugated with Alexa Fluor 488, 594, or 647 were used for colocalization experiments as described elsewhere (44, 45). Images were obtained using a Zeiss Axioplan 2 microscope with AxioCam MRc5 and AxioCam MRm digital cameras and AxioVision 4.8 software (Carl Zeiss).

Single-Cell RNA Sequencing (RNAseq)

Read alignment, quality controls, false discovery rate, identification of cell clusters, and quantification of cluster-specific gene expression were conducted according to previously published methods (30, 46–48). Details of tissue processing and single-cell RNAseq analysis of transcriptome cell signatures and receptor–ligand interactions are provided elsewhere (see the online supplement). To evaluate the transcriptomic similarity of respiratory cell types from wild-type (wt) and Nkx2-1^−/− mouse–rat chimeras, we compared the transcriptomic profiles of both single-cell RNAseq datasets using batch correction methods.

Statistical Analysis

The statistical significance of cell counts was determined using one-way ANOVA and nonparametric Kruskal-Wallis test. P values ⩽ 0.05 were considered statistically significant. Data were presented as mean ± SEM. For the single-cell RNAseq analysis, the log-transformation was applied for skewed data to confirm normality. We used the R package, which uses the Wilcoxon rank sum test implemented through the FindAllMarkers function in Seurat (Version 4.3.0) to analyze single-cell RNAseq datasets.

Results

Efficient Generation of Rats with Lung and Thyroid Agenesis Using CRISPR-Cas9

To create Nkx2-1^−/− rat embryos, two sgRNAs were designed to target exon 2 of the rat Nkx2-1 gene (Figure E1A). The sgRNAs, along with the recombinant Cas9 protein, were injected into rat zygotes, which were then transferred into surrogate rat females. E20.5 rat embryos were subsequently collected (Figure 1A). Whereas two sgRNAs efficiently deleted DNA fragments located between the targeted Nkx2-1 regions, a single sgRNA was also capable of deleting multiple nucleotides, disrupting the frame of the rat Nkx2-1 gene (Figures E1B–E1D). Approximately 25% of embryos were homozygotes (Nkx2-1^−/−), whereas the remaining embryos were wt (Nkx2-1^+/+), heterozygotes (Nkx2-1^+/−), or mosaic (see Table E1). The lungs of Nkx2-1^−/− rats were absent at E20.5 (Figures 1B, 1C, and E1C). NKX2-1 protein was detected in the tracheal, bronchial, and alveolar epithelia of wt embryos (Figures 1C–1E); however, it was absent in Nkx2-1^−/− embryos (Figures 1C–1E). Immunostaining for pro–surfactant protein C (pro-SPC), endomucin, Ki-67, and activated caspase 3 was not detected in the lung region within the thoracic cavity of Nkx2-1^−/− embryos (Figures E2A–E2D), which is consistent with the lack of peripheral lung tissues in E20.5 Nkx2-1^−/− rat embryos. In the thyroid domain, Nkx2-1^−/− embryos lacked NKX2-1–expressing cells (Figure E3). In addition, the esophagus and trachea were fused, and the thyroid was absent in Nkx2-1^−/− rat embryos as shown by HandE and immunostaining for NKX2-1 and PAX8 (Figures 1D–1G and E4). Thus, CRISPR-Cas9–mediated Nkx2-1 gene editing disrupted the development of the lung, thyroid, and trachea in rats.

Figure 1.

Efficient generation of Nkx2-1–deficient rat embryos using clustered regularly interspaced short palindromic repeat-Cas9. (A) Schematic representation of generation of rat Nkx2-1^−/− embryos. Nkx2-1 sgRNA and Cas9 protein were injected into rat zygotes. Embryos were implanted into surrogate rat females and harvested at Embryonic Day 20.5 (E20.5). (B) Hematoxylin and eosin (HandE) staining shows the absence of lung lobes in the thoracic cavity of Nkx2-1^−/− rats at E20.5. Scale bars, 500 μm. (C) Immunostaining for NKX2-1 shows the presence of lung tissue in wild-type (wt) embryos and the absence of lung tissue in Nkx2-1^−/− embryos. Inserts show high magnification images. Scale bars: 500 μm; insert, 50 μm. (D) HandE staining of transversal sections shows tracheoesophageal fusion (Tr/Es) near thymus in E20.5 Nkx2-1^−/− rat embryos. The trachea and esophagus are separated in wt embryos. Scale bars, 200 μm. (E) NKX2-1 staining shows tracheoesophageal fusion in rat E20.5 Nkx2-1^−/− embryos and the absence of NKX2-1 expression in the trachea. Scale bars: 500 μm; insert, 50 μm. (F) HandE staining of transversal sections shows Tr/Es fusion and the absence of thyroid in E20.5 Nkx2-1^−/− embryos. Thyroids are present in E20.5 wt embryos. Scale bars, 200 μm. (G) Immunostaining of frozen sections for PAX8 shows the absence of thyroid tissue in Nkx2-1^−/− rat embryos. Scale bars: 500 μm. DAPI (blue) was used as a counterstain. Es = esophagus; La = left atrium; Lu = lung; Lv = left ventricle; Ra = right atrium; Rv = right ventricle; sgRNA = single-guide RNA; Th = thyroid; Thy = thymus; Tr = trachea; Ve = vertebra.

Blastocyst Complementation with Mouse ESCs Induces Lung and Thyroid Morphogenesis in Nkx2-1^−/− Rats

To investigate whether mouse ESCs could rescue lung morphogenesis in rat embryos with disrupted Nkx2-1 gene, we performed blastocyst complementation by injecting GFP-labeled mouse wt ESCs into rat Nkx2-1^−/− blastocysts (Figure 2A). The interspecies mouse–rat blastocysts were then transferred into surrogate rat females, and E20.5 chimeric embryos were collected. Histological evaluation of E20.5 Nkx2-1^−/− mouse–rat chimeras showed the presence of lung lobes in the thoracic cavity (Figures 2B and E5). However, the body and lung sizes in Nkx2-1^−/− mouse–rat chimeras were smaller compared with wt rat embryos of the same age (Figures E6A and E6B). GFP fluorescence was abundant in the lung lobes of mouse–rat chimeras (Figure 2C), consistent with the presence of mouse ESC-derived cells. Mouse epithelial cells marked with GFP were detected in the lung of mouse–rat chimeras and expressed NKX2-1 (Figure 2C). Peripheral lung lobes in mouse–rat chimeras contained bronchioles, alveolar saccules, and blood vessels containing erythrocytes within the lumen (Figures 2B and E5). On the basis of ESC contribution, mouse–rat chimeras exhibited four phenotypes 1): GFP contribution to both epithelium and mesenchyme (ubiquitous GFP); 2) GFP contribution to epithelium (epithelial GFP); 3) no GFP contribution (cystic lung); and 4) regional GFP contribution (regional GFP) (Figures E7 and E8 and Table E2). The cystic lung phenotype in mouse–rat chimeras was rare and accounted for only 7% complemented embryos (Figure E8). This phenotype resembled cystic lungs in Nkx2-1^−/− mice (6, 30) and showed the integration of GFP-negative endogenous (rat) cells into the cystic structures (Figures E7 and E8). The regional GFP phenotype was defined as the presence of GFP-expressing areas in some, but not all, lung regions (Figure E7), possibly because of incomplete disruption of Nkx2-1 in all cells by CRISPR-Cas9. Because our primary goal was to generate a complete mouse lung in a rat, we next selected Nkx2-1^−/− chimeric embryos with the ubiquitous phenotype (Figures 2C and 2D) for further characterization by immunostaining, flow cytometry, and single-cell RNAseq. Thus, complementation with mouse ESCs promotes and enables lung morphogenesis in Nkx2-1^−/− rats.

Figure 2.

Mouse embryonic stem cells (mESCs) generate lung tissue in rat Nkx2-1^−/− embryos after blastocyst complementation. (A) Schematic diagram shows blastocyst complementation in Nkx2-1^−/− embryos. GFP-labeled mESCs were injected into rat Nkx2-1^−/− blastocysts, which were generated using the injection of wild-type (wt) zygotes with Nkx2-1–specific sgRNA and Cas9 protein. Chimeric embryos were implanted into surrogate rat females and harvested at Embryonic Day 20.5 (E20.5). (B) Hematoxylin and eosin staining shows the presence of peripheral lung lobes in the thoracic cavity of E20.5 Nkx2-1^−/− mouse–rat chimeras. High-magnification images (yellow box) show the lung, Ra, and Rv in Nkx2-1^−/− mouse–rat chimeras. Scale bars: top images, 500 μm; bottom images, 100 μm. (C) Immunostaining shows expression of NKX2-1 in the lungs of Nkx2-1^−/− mouse–rat chimeras. After mESC complementation, NKX2-1–positive epithelial cells (red) are present in peripheral lung regions of Nkx2-1^−/− mouse–rat chimeras. mESC-derived cells were detected with GFP (green). Nuclei were counterstained with DAPI (blue). High-magnification images are shown in inserts. Scale bars: left panels, 200 μm; middle and right panels, 50 μm; and insert, 5 μm. (D) Percentages of GFP+ and GFP− cells were counted using high-magnification images (n = 15 sections for each group: 3 wt rat embryos, 6 wt + mESC embryos, and 4 Nkx2-1^−/− + mESC embryos). **P < 0.01. Es = esophagus; La = left atrium; Lu = lung; Lv = left ventricle; Rv = right ventricle; Ra = right atrium; sgRNA = single-guide RNA; Tr = trachea; Ve = vertebra.

Mouse ESCs Differentiate into Multiple Respiratory Epithelial Cell Types in Nkx2-1^−/− Mouse–Rat Chimeras

In Nkx2-1^−/− mouse–rat chimeras with the ubiquitous GFP phenotype, mouse AT1 cells were abundant and expressed T1α, a marker of AT1 cells (Figure 3A). The T1α antibody was mouse specific and did not recognize rat AT1 cells. Mouse AT2 cells in Nkx2-1^−/− mouse–rat chimeras were also abundant and expressed pro-SPC and mature surfactant protein B (SPB) (Figures 3A and 3B). Pro-SPC and mature SPB antibodies recognized both mouse and rat AT2 cells (Figures 3A and 3B). There was no difference in the number of AT2 cells between experimental groups (Figures E9A and E9B). Notably, AT1 and AT2 cells in Nkx2-1^−/− mouse–rat chimeras expressed GFP (Figures 3A–3D), indicating their origin from mouse ESCs. In airway epithelium, club cells were derived from mouse ESCs, as evidenced by colocalization of club cell secretory protein (CCSP) and GFP (Figures E10A and E10B). Moreover, the apical surfaces of ciliated cells stained positive for β-tubulin, and all ciliated cells in conducting airways of Nkx2-1^−/− mouse–rat chimeras expressed GFP (Figures E10C and E10D). Thus, both alveolar and airway epithelial cells in chimeric lungs originated from mouse cells.

Figure 3.

Mouse embryonic stem cells (mESCs) differentiate into epithelial alveolar type 1 and 2 (AT1 and AT2) cells in Nkx2-1^−/− mouse–rat chimeras. (A) Immunostaining shows the expression of T1α and pro–surfactant protein C (Pro-SPC) in alveolar epithelial cells of E20.5 Nkx2-1^−/− mouse–rat chimeras. After mESC complementation, Pro-SPC–positive AT2 cells (white) and T1α-positive AT1 cells (red) are present in peripheral lung regions of Nkx2-1^−/− mouse–rat chimeras. mESC-derived cells were detected with GFP (green). High-magnification images of AT2 and AT1 cells are shown in the inserts. T1α antibodies are mouse specific and do not recognize rat T1α. Sections were counterstained with DAPI (blue). Scale bars; 50 μm; insert, 5 μm. wt = wild-type. (B) Expression of mature surfactant protein B (mSPB) in lungs of Nkx2-1^−/− mouse–rat chimeras. Immunostaining of frozen lung sections for mSPB shows the presence of fully differentiated AT2 cells in the lungs of E20.5 Nkx2-1^−/− mouse–rat chimeras. Scale bars: 50 μm; insert, 5 μm. (C) Percentages of Pro-SPC+/GFP+ and Pro-SPC+/GFP− AT2 cells were counted using high-magnification images (n = 15 sections for each group: 3 wt rat embryos, 6 wt + mESC embryos, and 4 Nkx2-1^−/− + mESC embryos). **P < 0.01. (D) Percentages of mSPB+GFP+ and mSPB+GFP− AT2 cells were counted using high-magnification images (n = 15 sections for each group: 3 wt rat embryos, 6 wt + mESC embryos, and 4 Nkx2-1^−/− + mESC embryos). **P < 0.01.

Mouse ESCs Differentiate into Respiratory Stromal and Vascular Cells during Lung Morphogenesis in Nkx2-1^−/− Mouse–Rat Chimeras

The contribution of mouse ESCs to various mesenchymal and vascular cell types was evaluated by examining the expression of cell-specific proteins in the lungs of Nkx2-1^−/− and wt (control) mouse–rat chimeras. Immunostaining revealed that mouse ESCs contributed to endothelial cells marked by endomucin, and nearly all endothelial cells in Nkx2-1^−/− mouse–rat chimeras were positive for GFP (Figures 4A and 4B). Both gCAP (CAP1) and aCAP (CAP2) capillary endothelial cells identified by GPIHBP1 and CAR4, respectively, had high contribution from mouse ESCs (Figures E11A and E11B). Smooth muscle cells (SMCs) marked by αSMA (see Figure E12A) were predominantly derived from mouse ESCs in the Nkx2-1^−/− mouse–rat chimeras (Figure 4C). Furthermore, immune cells expressing PTPRC (CD45) (Figure E12B), pericytes marked by NG2 (Figures 4D and E13A), and fibroblasts expressing PDGFRA (Figure E13B) had high contributions from mouse ESCs in Nkx2-1^−/− mouse–rat chimeras. ESC complementation did not influence cellular proliferation and apoptosis in respiratory cell types at E20.5, as demonstrated by immunostaining for Ki-67 and activated (cleaved) caspase 3 (Figures E14A and E14B). Thus, multiple stromal and vascular cell types in newly formed lungs of Nkx2-1^−/− mouse–rat chimeras are derived from mouse ESCs.

Figure 4.

Mouse embryonic stem cells (mESCs) differentiate into endothelial and stromal cells during lung morphogenesis in Nkx2-1^−/− mouse–rat chimeras. (A) Immunostaining of frozen lung sections for endomucin shows extensive microvascular networks in the lungs of Nkx2-1^−/− mouse–rat chimeras at Embryonic Day 20.5. mESC-derived cells are detected with GFP. Nkx2-1^−/− and wild-type (wt) blastocysts were injected with GFP-labeled mESCs to generate mouse–rat chimeras. Sections were counterstained with DAPI. Scale bars: low-magnification images in left merge/DAPI column, 200 μm; high-magnification images (yellow box) in right three columns, 50 μm; and insert, 5 μm. (B) Percentages of endothelial GFP+ and GFP− cells were counted using high-magnification images of endomucin-stained sections (n = 15 sections for each group: 3 wt rat embryos, 6 wt + mESC embryos, and 4 Nkx2-1^−/− + mESC embryos). (C) Percentages of smooth muscle GFP+ and GFP− cells were counted using high-magnification images of αSMA-stained sections (n = 15 sections for each group: 3 wt rat embryos, 6 wt + mESC embryos, and 4 Nkx2-1^−/− + mESC embryos). (D) Percentages of GFP+ and GFP− pericytes were counted using high-magnification images of NG2-stained sections (n = 15 sections for each group: 3 wt rat embryos, 6 wt + mESC embryos, and 4 Nkx2-1^−/− + mESC embryos). **P < 0.01.

Mouse ESC Complementation Induces Thyroid Morphogenesis but Does Not Correct the Tracheoesophageal Fusion in Nkx2-1^−/− Mouse–Rat Chimeras

In Nkx2-1^−/− mouse–rat chimeras, fusion of the trachea and esophagus was observed above the tracheal bifurcation (Figure E5), which is consistent with observations in Nkx2-1^−/− mouse–mouse chimeras (30). Within the area of tracheoesophageal fusion, mouse ESC-derived cells were primarily found in the tracheal epithelium, as indicated by immunostaining for CCSP and β-tubulin (see Figures E15A and E15B). Additionally, an examination of E20.5 Nkx2-1^−/− mouse–rat chimeras revealed the presence of thyroid tissue (Figure E5), which was absent in Nkx2-1^−/− rat embryos that did not receive mouse ESC complementation (Figure E4). Mouse ESCs efficiently contributed to the development of thyrocyte progenitor cells expressing NKX2-1 and PAX8 (Figures 5A–5D). The contribution of mouse ESCs to the chimeric forebrain was also high (Figure E16). Altogether, mouse ESC complementation of Nkx2-1^−/− rat embryos induced thyroid morphogenesis but was unable to correct the tracheoesophageal fusion in Nkx2-1^−/− mouse–rat chimeras.

Figure 5.

Mouse embryonic stem cell (mESC) complementation induces thyroid (Th) morphogenesis but does not correct the Tr/Es in Nkx2-1^−/− mouse–rat chimeras. (A) Immunostaining of frozen sections for NKX2-1 shows Th tissue in E20.5 Nkx2-1^−/− mouse–rat chimeras. The Th is absent in Nkx2-1^−/− embryos without ESC complementation. NKX2-1–positive thyrocytes in Nkx2-1^−/− mouse–rat chimeras express GFP, indicating mESC origin. Nuclei were counterstained with DAPI (blue). Scale bars: low-magnification images in left merge/DAPI column, 500 μm; high-magnification images (yellow box) in right three columns, 200 μm. (B) Percentages of NKX2-1–positive mouse and rat cells were counted using high-magnification images (n = 6–12 sections for each group: 3 wild-type [wt] rat embryos, 6 wt + mESC embryos and 4 Nkx2-1^−/− + mESC embryos). (C) Immunostaining for PAX8 shows PAX8-positive thyrocytes in Nkx2-1^−/− mouse–rat chimeras. Scale bars: low magnification images in left merge/DAPI column, 500 μm; and high magnification images (yellow box) in right three columns, 200 μm. (D) Percentages of PAX8-positive mouse and rat thyrocytes were counted using high-magnification images (n = 6–12 sections for each group: 3 wt rat embryos, 6 wt + mESC embryos, and 4 Nkx2-1^−/− + mESC embryos). Es = esophagus; Tr = trachea; Tr/Es = tracheoesophageal fusion.

Single-Cell RNAseq Identifies Gene Expression Signatures of Mouse ESC-Derived Respiratory Cell Types in nkx2-1^−/− Mouse–Rat Chimeras

Single-cell RNAseq was conducted using the 10× platform to compare gene signatures in the lungs of Nkx2-1^−/− and wt mouse–rat chimeras with high contribution from mouse ESCs. High GFP expression in chimeric lungs was confirmed by flow cytometry (see Figures E17A–E17C). Both mouse and rat cells were sequenced. Using known gene expression signatures (1, 46–48), 13 major mouse cell subtypes were identified in lungs from wt and Nkx2-1^−/− E20.5 mouse–rat chimeras, including airway epithelial (ciliated and club) cells, AT1 cells, AT2 cells, AT1/AT2 cells, hematopoietic cells, endothelial cells, proliferative mesenchymal progenitors, SMCs, fibroblasts, matrix fibroblasts, pericytes, and mesothelial cells (Figures 6A, E18A, E18B, E19A, and E19B). Seven major rat cell subtypes were identified in lungs from wt and Nkx2-1^−/− mouse–rat chimeras, including macrophages, endothelial and epithelial cells, SMCs, fibroblasts, matrix fibroblasts, and pericytes (Figures 6B, E18C, E18D, E20A, and E20B). The analysis revealed that 98.5% of cells in newly formed lungs of Nkx2-1^−/− mouse–rat chimeras were mouse cells (Figures 6C and 6D). Nearly all epithelial, endothelial, and fibroblast cells were derived from mouse ESCs in Nkx2-1^−/− mouse–rat chimeras (Figure 6D). To confirm that the Nkx2-1^−/− chimeras contained homozygous Nkx2-1 disruption in endogenous rat cells, we examined mouse and rat Nkx2-1 mRNAs. Epithelial cells expressed mouse Nkx2-1 mRNA (Figure E21), whereas rat Nkx2-1 mRNA was absent in Nkx2-1^−/− mouse–rat chimeras (Figures E22A and E22B). Furthermore, HandE staining showed a fusion of the esophagus and trachea in Nkx2-1^−/− chimeric embryos, the lung of which was sequenced (Figure E22C), confirming the Nkx2-1^−/− phenotype in host rat embryos. Thus, mouse ESCs successfully formed the mouse lung in Nkx2-1^−/− mouse–rat chimeras.

Figure 6.

Single-cell RNA sequencing identifies pulmonary cell types in Nkx2-1^−/− mouse–rat chimeras. (A) Uniform Manifold Approximation and Projection (UMAP) plot of mouse single cells. Left: mouse cells from E20.5 wild-type (wt) mouse–rat chimeric lung complemented with mouse embryonic stem cells (mESCs) (n = 6,631 cells). Right: mouse cells form Embryonic Day 20.5 Nkx2-1^−/− mouse–rat chimeric lung complemented with mESCs (n = 8,018 cells). Cells are colored on the basis of cell subtypes. (B) UMAP plot of rat single cells. Left: rat cells from wt chimera (n = 1,173 cells). Right: rat cells from Nkx2-1^−/− chimera (n = 114 cells). (C and D) Diagrams and bar graphs show proportions of mouse and rat cells in (C) wt and (D) Nkx2-1^−/− mouse–rat chimeric lungs as well as proportions of mouse and rat cells in Endo, Hematop, Epi, Fib, and Peri. AT1/2 = alveolar type ½; AT1 = alveolar type 1; AT2 = alveolar type 2; Ciliated = ciliated cells; Club = club cells; Endo = endothelial cells; Epi = epithelial cells; Fib = fibroblasts; Hematop = hematopoietic cells; Matrixfib = matrix fibroblasts; Meso = mesothelial cells; Peri = pericytes; PMP = proliferative mesenchymal progenitors; SMC = smooth muscle cells.

Single-Cell RNAseq Identified Normal Gene Expression Profiles and Signaling Receptor–Ligand Interaction between Mouse Lung Cell Types in Nkx2-1^−/− Mouse–Rat Chimeras

Gene expression analysis revealed normal gene signatures in all ESC-derived cell types (Figures 7A and E23). ESC-derived AT2 cells that were isolated from Nkx2-1^−/− mouse–rat chimeras expressed AT2-specific genes, including Sftpc, Lyzl, and Lamp3 (Figure 7A). ESC-derived AT1 cells expressed Ager, Hopx, and Akap5, whereas Clic3, Cxcl15, and Ctsh were restricted to AT1/2 cells (Figure 7A). Gene expression signatures in ESC-derived gCAPs, aCAPs, arterial cells, venous cells, lymphatic cells, ciliated cells, club cells, hematopoietic cells, fibroblasts, and other cell types in Nkx2-1^−/− mouse–rat chimeras were similar to the control (Figures 7A and E23). Thus, the cellular composition of the lung tissue and gene expression signatures of all respiratory cell types were normal in Nkx2-1^−/− mouse–rat chimeras.

Figure 7.

Single-cell RNA sequencing (RNAseq) analysis shows similarities in gene expression and major signaling networks between respiratory cell types in wild-type (wt) and Nkx2-1^−/− mouse–rat chimeras. (A) Gene expression signatures in mouse embryonic stem cell (mESC)-derived pulmonary cells are similar in wt and Nkx2-1^−/− mouse–rat chimeras. Mouse cell-type signature genes were obtained from the Lung Gene Expression Analysis website. The heatmap shows mouse gene expression signatures between wt chimeric lung (yellow) and Nkx2-1^−/− chimeric lung (orange). (B) Single-cell RNAseq shows remarkable similarities in major receptor–ligand interactions between mouse alveolar type 1 (AT1) receptors and other mouse cell types in wt and Nkx2-1^−/− mouse–rat chimeras. Single cells were obtained from Embryonic Day 20.5 lungs using enzymatic digestion. Single-cell RNAseq was performed to identify cell types on the basis of gene expression signatures. The R package Cellchat was used to analyze the expression of ligands and receptors and to identify intercellular communication patterns between AT1 and other ESC-derived cell types. aCAP = alveolar capillary cells; AT1/2 = alveolar type 1/2; AT2 = alveolar type 2; Endo = endothelial cells; Fib = fibroblasts; gCAP = general capillary cells; Hematop = hematopoietic cells; Matrixfib = matrix fibroblasts; Peri = pericytes; PMP = proliferative mesenchymal progenitors.

To examine cell signaling pathways between different ESC-derived mouse cell types of Nkx2-1^−/− mouse–rat chimeras, maps of potential ligand–receptor interactions were generated using single-cell RNAseq datasets. There were remarkable similarities in major receptor–ligand interactions between mouse AT1 and other lung cell types (Figure 7B). These included FGF, WNT, and midkine signaling pathways. However, there were differences in expression of Egfr and Fzd5 in mouse AT1 cells of wt and Nkx2-1^−/− mouse–rat chimeras (Figure 7B). ESC-derived AT2 cells interacted with other mouse cell types in Nkx2-1^−/− mouse–rat chimeras through similar receptor–ligand networks compared with those in control lungs (see Figure E24A). Furthermore, signaling networks between ESC-derived fibroblasts (Figure E24B), gCAPs (see Figure E25A), aCAPs (Figure E25B), and other mouse cell types in Nkx2-1^−/− mouse–rat chimeras were relatively similar but not fully identical to controls. It is interesting that receptor–ligand interactions in mouse epithelial–mouse stromal cells were not conserved when compared with mouse epithelial–rat stromal cells (see Figure E26). Altogether, our results demonstrate that mouse ESC-derived respiratory cell types in Nkx2-1^−/− mouse–rat chimeras use common signaling pathways to form the mouse lung in a rat.

Discussion

The inactivation of the Nxk2–1 gene in the mouse causes severe defects in the development of the lung, thyroid, and structures derived from the forebrain (12). Published studies have also demonstrated that Nkx2-1 plays a crucial role in the development of respiratory epithelial cell lineages (1, 2), and mutations in human NKX2-1 cause the brain–lung–thyroid syndrome (49). Because of key requirements for Nkx2-1 in lung morphogenesis, we recently used Nkx2-1^−/− mouse embryos to generate lung tissue from mouse ESCs. ESC-derived donor lungs in Nkx2-1^−/− mouse–mouse chimeric embryos contained a near-complete contribution of ESCs in epithelial cell lineages (30). However, nonepithelial cell types, such as endothelial, stromal, and hematopoietic cells, were derived from both endogenous and donor ESCs (30). Similar results were observed after epithelial-specific inactivation of Fgfr2 or β-catenin (29) and after global deletion of Fgf10 in recipient embryos (31). An important contribution of the present study is that we achieved a near-complete engraftment of donor ESCs into the lung tissue using a combination of CRISPR-Cas9 genome editing and blastocyst complementation. Whereas half of the chimeric embryos exhibited mostly epithelial contributions to the lung tissue, as was expected by disrupting the Nkx2-1 gene, approximately 30% of Nkx2-1^−/− mouse–rat chimeras contained almost complete contribution of mouse ESCs to all respiratory cell lineages, including epithelial, endothelial, stromal, and hematopoietic cells. These results are surprising and suggest that, during lung development, mouse ESCs had a competing advantage over resident rat cells to form the lung tissue. It is interesting that rat embryos develop more slowly than mouse embryos by approximately 1.5 days (3, 4, 30). Therefore, it is possible that mouse cells migrate faster into the developing lung bud to initiate lung morphogenesis in Nkx2-1^−/− mouse–rat chimeras. This hypothesis is consistent with our recent studies demonstrating that hemogenic endothelium in dorsal aortas of mouse–rat chimeras develops primarily from mouse ESCs (33). Furthermore, the migration of mouse ESC-derived hematopoietic stem cells from the fetal liver into bone marrow of mouse–rat chimeras occurs faster compared with rat hematopoietic stem cells, resulting in early colonization and expansion of bone marrow by mouse progenitor cells in a rat (33). The mechanism of this observation is currently unknown.

The present study showed that, despite the presence of peripheral lung and thyroid tissues in Nkx2-1^−/− mouse–rat chimeras, the chimeras exhibited a lack of proper tracheoesophageal separation and mispatterning of the proximal trachea, which is consistent with the phenotype in Nkx2-1^−/− mice (30). Therefore, the complementation of rat Nkx2-1^−/− embryos with mouse ESCs was inadequate in correcting the tracheoesophageal fusion. It is possible that CRISPR-Cas9–mediated inhibition of genes required for tracheoesophageal patterning in rat embryos can correct this developmental defect and lead to the generation of the complete respiratory system from donor ESCs. One of the interesting differences between Nkx2-1^−/− mouse–rat and mouse–mouse chimeras generated previously (30) is that the formation of endogenous thoracic cysts was rarely observed in the rat but was common in mouse embryos (6, 30). It is possible that the lack of competition between ESC-derived mouse cells and endogenous basal rat cells can prevent the cyst formation and contribute to efficient generation of peripheral lung tissue from donor ESCs in Nkx2-1^−/− mouse–rat chimeras.

The combination of CRISPR-Cas9 technology and blastocyst complementation overcomes interspecies barriers and can provide an opportunity to use large animals as “biological reactors” for the generation of patient-specific lungs using human iPSCs. However, this technology is not ready for human use, because ESCs and iPSCs have the capacity to integrate into various embryonic tissues, such as the brain and sensory and reproductive organs (34, 50). The lack of precise ESC/iPSC integration into lung tissue will raise significant ethical concerns regarding the utilization of human–animal chimeras in the field of lung regenerative medicine. To advance the technology, it may be necessary to introduce additional genetic modifications to improve the selectivity of ESC/iPSC integration, including those necessary to correct the tracheoesophageal fistula and prevent immunological rejection. The creation of Nkx2-1^−/− interspecies chimeras in large animals, such as pigs or sheep, may be possible for generating human lungs from patient-specific iPSCs, potentially leading to lung transplantation without the need for immunosuppression.

In summary, a combination of CRISPR-Cas9 genome editing and blastocyst complementation was used to generate mouse thyroid and lung tissues in a rat. The newly generated lung exhibited a near-complete contribution from mouse ESCs into epithelial, endothelial, stromal, and hematopoietic cell lineages. Our study represents an important step toward the generation of transplantable lung tissue from patient-specific iPSC using large animals as “bioreactors.”