The bone marrow of mouse-rat chimeras contains progenitors of multiple pulmonary cell lineages.

Radiation-induced lung injury (RILI) is a common complication of anti-cancer treatments for thoracic and hematologic malignancies. Bone marrow (BM) transplantation restores hematopoietic cell lineages in cancer patients. However, it is ineffective in improving lung repair after RILI due to the paucity of respiratory progenitors in BM transplants. In the present study, we used blastocyst injection to create mouse-rat chimeras, these are artificial animals in which BM is enriched with mouse-derived progenitor cells. FACS-sorted mouse BM cells from mouse-rat chimeras were transplanted into lethally irradiated syngeneic mice, and the contribution of donor cells to the lung tissue was examined using immunostaining and flow cytometry. Donor BM cells provided long-term contributions to all lung-resident hematopoietic cells which includes alveolar macrophages and dendritic cells. Surprisingly, donor BM cells also contributed up to 8% in pulmonary endothelial cells and stromal cells after RILI. To identify respiratory progenitors in donor BM, we performed single-cell RNA sequencing (scRNAseq). Compared to normal mouse BM, increased numbers of hematopoietic progenitors were found in the BM of mouse-rat chimeras. We also identified unique populations of hemangioblast-like progenitor cells expressing Hes1, Dntt and Ebf1, along with mesenchymal stromal cells expressing Cpox, Blvrb and Ermap that were absent or ultra-rare in the normal mouse BM. In summary, by using rats as "bioreactors", we created a unique mouse BM cell transplant that contributes to multiple respiratory cell types after RILI. Interspecies chimeras have promise for future generations of BM transplants enriched in respiratory progenitor cells.

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

RATIONALE: 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. While epithelial cell lineages were efficiently generated from ESC, other cell types were mosaic. A complete contribution of donor ESC to lung tissue has never been achieved. The mouse lung has never been generated in a rat. OBJECTIVE: To generate the mouse lung in a rat. METHODS: CRISPR/Cas9 genome editing was used to disrupt the Nkx2-1 gene in rat 1-cell zygotes. Interspecies mouse-rat chimeras were produced by injection of wild-type mouse ESC into Nkx2-1-deficient rat embryos with lung agenesis. The contribution of mouse ESC 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 ESC restored pulmonary and thyroid structures in mouse-rat chimeras leading to a near 99% contribution of ESC 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 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".

In vivo generation of bone marrow from embryonic stem cells in interspecies chimeras.

Generation of bone marrow (BM) from embryonic stem cells (ESCs) promises to accelerate the development of future cell therapies for life-threatening disorders. However, such approach is limited by technical challenges to produce a mixture of functional BM progenitor cells able to replace all hematopoietic cell lineages. Herein, we used blastocyst complementation to simultaneously produce BM cell lineages from mouse ESCs in a rat. Based on fluorescence-activated cell sorting analysis and single-cell RNA sequencing, mouse ESCs differentiated into multiple hematopoietic and stromal cell types that were indistinguishable from normal mouse BM cells based on gene expression signatures and cell surface markers. Receptor-ligand interactions identified Cxcl12-Cxcr4, Lama2-Itga6, App-Itga6, Comp-Cd47, Col1a1-Cd44, and App-Il18rap as major signaling pathways between hematopoietic progenitors and stromal cells. Multiple hematopoietic progenitors, including hematopoietic stem cells (HSCs) in mouse-rat chimeras derived more efficiently from mouse ESCs, whereas chondrocytes predominantly derived from rat cells. In the dorsal aorta and fetal liver of mouse-rat chimeras, mouse HSCs emerged and expanded faster compared to endogenous rat cells. Sequential BM transplantation of ESC-derived cells from mouse-rat chimeras rescued lethally irradiated syngeneic mice and demonstrated long-term reconstitution potential of donor HSCs. Altogether, a fully functional BM was generated from mouse ESCs using rat embryos as 'bioreactors'.

Endothelial progenitor cells stimulate neonatal lung angiogenesis through FOXF1-mediated activation of BMP9/ACVRL1 signaling.

Pulmonary endothelial progenitor cells (EPCs) are critical for neonatal lung angiogenesis and represent a subset of general capillary cells (gCAPs). Molecular mechanisms through which EPCs stimulate lung angiogenesis are unknown. Herein, we used single-cell RNA sequencing to identify the BMP9/ACVRL1/SMAD1 pathway signature in pulmonary EPCs. BMP9 receptor, ACVRL1, and its downstream target genes were inhibited in EPCs from Foxf1WT/S52F mutant mice, a model of alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). Expression of ACVRL1 and its targets were reduced in lungs of ACDMPV subjects. Inhibition of FOXF1 transcription factor reduced BMP9/ACVRL1 signaling and decreased angiogenesis in vitro. FOXF1 synergized with ETS transcription factor FLI1 to activate ACVRL1 promoter. Nanoparticle-mediated silencing of ACVRL1 in newborn mice decreased neonatal lung angiogenesis and alveolarization. Treatment with BMP9 restored lung angiogenesis and alveolarization in ACVRL1-deficient and Foxf1WT/S52F mice. Altogether, EPCs promote neonatal lung angiogenesis and alveolarization through FOXF1-mediated activation of BMP9/ACVRL1 signaling.

Generation of Pulmonary Endothelial Progenitor Cells for Cell-based Therapy Using Interspecies Mouse-Rat Chimeras.

Rationale: Although pulmonary endothelial progenitor cells (EPCs) hold promise for cell-based therapies for neonatal pulmonary disorders, whether EPCs can be derived from pluripotent embryonic stem cells (ESCs) or induced pluripotent stem cells remains unknown.Objectives: To investigate the heterogeneity of pulmonary EPCs and derive functional EPCs from pluripotent ESCs.Methods: Single-cell RNA sequencing of neonatal human and mouse lung was used to identify the heterogeneity of pulmonary EPCs. CRISPR/Cas9 gene editing was used to genetically label and purify mouse pulmonary EPCs. Functional properties of the EPCs were assessed after cell transplantation into neonatal mice with S52F Foxf1 mutation, a mouse model of alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). Interspecies mouse-rat chimeras were produced through blastocyst complementation to generate EPCs from pluripotent ESCs for cell therapy in ACDMPV mice.Measurements and Main Results: We identified a unique population of EPCs, FOXF1+cKIT+ EPCs, as a subset of recently described general capillary cells (gCAPs) expressing SMAD7, ZBTB20, NFIA, and DLL4 but lacking mature arterial, venous, and lymphatic markers. FOXF1+cKIT+ gCAPs are reduced in ACDMPV, and their transcriptomic signature is conserved in mouse and human lungs. After cell transplantation into the neonatal circulation of ACDMPV mice, FOXF1+cKIT+ gCAPs engraft into the pulmonary vasculature, stimulate angiogenesis, improve oxygenation, and prevent alveolar simplification. FOXF1+cKIT+ gCAPs, produced from ESCs in interspecies chimeras, are fully competent to stimulate neonatal lung angiogenesis and alveolarization in ACDMPV mice.Conclusions: Cell-based therapy using donor or ESC/induced pluripotent stem cell-derived FOXF1+cKIT+ endothelial progenitors may be considered for treatment of human ACDMPV.

Blastocyst complementation reveals that NKX2-1 establishes the proximal-peripheral boundary of the airway epithelium.

BACKGROUND: Distinct boundaries between the proximal conducting airways and more peripheral-bronchial regions of the lung are established early in foregut embryogenesis, demarcated in part by the distribution of SOX family and NKX2-1 transcription factors along the cephalo-caudal axis of the lung. We used blastocyst complementation to identify the role of NKX2-1 in the formation of the proximal-peripheral boundary of the airways in mouse chimeric embryos. RESULTS: While Nkx2-1-/- mouse embryos form primordial tracheal cysts, peripheral pulmonary structures are entirely lacking in Nkx2-1-/- mice. Complementation of Nkx2-1-/- embryos with NKX2-1-sufficient embryonic stem cells (ESCs) enabled the formation of all tissue components of the peripheral lung but did not enhance ESC colonization of the most proximal regions of the airways. In chimeric mice, a precise boundary was formed between NKX2-1-deficient basal cells co-expressing SOX2 and SOX9 in large airways and ESC-derived NKX2-1+ SOX9+ epithelial cells of smaller airways. NKX2-1-sufficient ESCs were able to selectively complement peripheral, rather than most proximal regions of the airways. ESC complementation did not prevent ectopic expression of SOX9 but restored ß-catenin signaling in Nkx2-1-/- basal cells of large airways. CONCLUSIONS: NKX2-1 and ß-catenin function in an epithelial cell-autonomous manner to establish the proximal-peripheral boundary along developing airways.

In Vivo Generation of Lung and Thyroid Tissues from Embryonic Stem Cells Using Blastocyst Complementation.

Rationale: The regeneration and replacement of lung cells or tissues from induced pluripotent stem cell- or embryonic stem cell-derived cells represent future therapies for life-threatening pulmonary disorders but are limited by technical challenges to produce highly differentiated cells able to maintain lung function. Functional lung tissue-containing airways, alveoli, vasculature, and stroma have never been produced via directed differentiation of embryonic stem cells (ESCs) or induced pluripotent stem cells. We sought to produce all tissue components of the lung from bronchi to alveoli by embryo complementation.Objectives: To determine whether ESCs are capable of generating lung tissue in Nkx2-1-/- mouse embryos with lung agenesis.Methods: Blastocyst complementation was used to produce chimeras from normal mouse ESCs and Nkx2-1-/- embryos, which lack pulmonary tissues. Nkx2-1-/- chimeras were examined using immunostaining, transmission electronic microscopy, fluorescence-activated cell sorter analysis, and single-cell RNA sequencing.Measurements and Main Results: Although peripheral pulmonary and thyroid tissues are entirely lacking in Nkx2-1 gene-deleted embryos, pulmonary and thyroid structures in Nkx2-1-/- chimeras were restored after ESC complementation. Respiratory epithelial cell lineages in restored lungs of Nkx2-1-/- chimeras were derived almost entirely from ESCs, whereas endothelial, immune, and stromal cells were mosaic. ESC-derived cells from multiple respiratory cell lineages were highly differentiated and indistinguishable from endogenous cells based on morphology, ultrastructure, gene expression signatures, and cell surface proteins used to identify cell types by fluorescence-activated cell sorter.Conclusions: Lung and thyroid tissues were generated in vivo from ESCs by blastocyst complementation. Nkx2-1-/- chimeras can be used as "bioreactors" for in vivo differentiation and functional studies of ESC-derived progenitor cells.

Maternal regulation of inflammatory cues is required for induction of preterm birth.

Infection-driven inflammation in pregnancy is a major cause of spontaneous preterm birth (PTB). Both systemic infection and bacterial ascension through the vagina/cervix to the amniotic cavity are strongly associated with PTB. However, the contribution of maternal or fetal inflammatory responses in the context of systemic or localized models of infection-driven PTB is not well defined. Here, using intraperitoneal or intraamniotic LPS challenge, we examined the necessity and sufficiency of maternal and fetal Toll-like receptor (TLR) 4 signaling in induction of inflammatory vigor and PTB. Both systemic and local LPS challenge promoted induction of inflammatory pathways in uteroplacental tissues and induced PTB. Restriction of TLR4 expression to the maternal compartment was sufficient for induction of LPS-driven PTB in either systemic or intraamniotic challenge models. In contrast, restriction of TLR4 expression to the fetal compartment failed to induce LPS-driven PTB. Vav1-Cre-mediated genetic deletion of TLR4 suggested a critical role for maternal immune cells in inflammation-driven PTB. Further, passive transfer of WT in vitro-derived macrophages and dendritic cells to TLR4-null gravid females was sufficient to induce an inflammatory response and drive PTB. Cumulatively, these findings highlight the critical role for maternal regulation of inflammatory cues in induction of inflammation-driven parturition.

Mthfd2 Modulates Mitochondrial Function and DNA Repair to Maintain the Pluripotency of Mouse Stem Cells.

The pluripotency of stem cells determines their developmental potential. While the pluripotency states of pluripotent stem cells are variable and interconvertible, the mechanisms underlying the acquisition and maintenance of pluripotency remain largely elusive. Here, we identified that methylenetetrahydrofolate dehydrogenase (NAD+-dependent), methenyltetrahydrofolate cyclohydrolase (Mthfd2) plays an essential role in maintaining embryonic stem cell pluripotency and promoting complete reprogramming of induced pluripotent stem cells. Mechanistically, in mitochondria, Mthfd2 maintains the integrity of the mitochondrial respiratory chain and prevents mitochondrial dysfunction. In the nucleus, Mthfd2 stabilizes the phosphorylation of EXO1 to support DNA end resection and promote homologous recombination repair. Our results revealed that Mthfd2 is a dual-function factor in determining the pluripotency of pluripotent stem cells through both mitochondrial and nuclear pathways, ultimately ensuring safe application of pluripotent stem cells.

Long non-coding RNAs involved in the regulatory network during porcine pre-implantation embryonic development and iPSC induction.

Long non-coding RNAs (lncRNA) play a key role in the orchestration of transcriptional regulation during development and many other cellular processes. The importance of the regulatory co-expression network was highlighted in the identification of the mechanism of these processes in humans and mice. However, elucidation of the properties of porcine lncRNAs involved in the regulatory network during pre-implantation embryonic development and fibroblast reprogramming to induced pluripotent stem cell (iPSC) has been limited to date. Using a weighted gene co-expression network analysis, we constructed the regulatory network and determined that the novel lncRNAs were functionally involved in key events of embryonic development during the pre-implantation period; moreover, reprogramming could be delineated by a small number of potentially functional modules of co-expressed genes. These findings indicate that lncRNAs may be involved in the transcriptional regulation of zygotic genome activation, first lineage segregation and somatic reprogramming to pluripotency. Furthermore, we performed a conservation and synteny analysis with the significant lncRNAs involved in these vital events and validated the results via experimental assays. In summary, the current findings provide a valuable resource to dissect the protein coding gene and lncRNA regulatory networks that underlie the progressive development of embryos and somatic reprogramming.

Pluripotent stem cells secrete Activin A to improve their epiblast competency after injection into recipient embryos.

It is not fully clear why there is a higher contribution of pluripotent stem cells (PSCs) to the chimera produced by injection of PSCs into 4-cell or 8-cell stage embryos compared with blastocyst injection. Here, we show that not only embryonic stem cells (ESCs) but also induced pluripotent stem cells (iPSCs) can generate F0 nearly 100% donor cell-derived mice by 4-cell stage embryo injection, and the approach has a "dose effect". Through an analysis of the PSC-secreted proteins, Activin A was found to impede epiblast (EPI) lineage development while promoting trophectoderm (TE) differentiation, resulting in replacement of the EPI lineage of host embryos with PSCs. Interestingly, the injection of ESCs into blastocysts cultured with Activin A (cultured from 4-cell stage to early blastocyst at E3.5) could increase the contribution of ESCs to the chimera. The results indicated that PSCs secrete protein Activin A to improve their EPI competency after injection into recipient embryos through influencing the development of mouse early embryos. This result is useful for optimizing the chimera production system and for a deep understanding of PSCs effects on early embryo development.

Tetraploid embryonic stem cells can contribute to the development of chimeric fetuses and chimeric extraembryonic tissues.

Our study examined the in vivo chimeric and survival capacities of chimeras created by injecting tetraploid embryonic stem cells (ESCs) expressing green fluorescent protein (GFP) into diploid embryos. At 3.5 days post-coitum (dpc) and 4.5 dpc, the tetraploid ESCs were able to contribute to the inner cell mass (ICM) just as diploid ESCs tagged with GFP. At 6.5 dpc, 8.0 dpc and 10.5 dpc, the tetraploid ESCs manifested in the same location as the diploid ESCs. The GFP cells in the extraembryonic tissues and fetuses of tetraploid ESC chimeras were tetraploid as determined by fluorescence activated cell sorting (FACS). Furthermore, tetraploid ESCs contributed to the development of the placenta, embryolemma and umbilical cord at 13.5 dpc and 16.5 dpc; however, very less GFP cells were found in the fetuses of tetraploid ESC chimeras. We further found that the proliferation of tetraploid ESCs was slower than that of diploid ESCs. In addition, the relative mRNA expression in the three germ layers and the trophoblast was abnormal in the EBs of tetraploid ESCs compared with diploid ESCs. In short, slower proliferation and abnormal differentiation potential of tetraploid ESCs might be two of the reasons for their poor survival and chimeric capacities.

Embryo development after mitochondrial supplementation from induced pluripotent stem cells.

PURPOSE: The purpose of this study was to evaluate the effects of mitochondrial supplementation (MS) on early embryonic development and to assess the safety of MS treatments using induced pluripotent stem cells (iPSCs) as the mitochondrial donor. METHODS: In this study, we evaluated the effect of MS on early embryonic development using induced pluripotent stem cells (iPSCs) as the donor. Mouse zygotes were injected with either mitochondria from iPSCs or a vehicle solution. Several parameters were evaluated, including the rates of blastocyst formation and implantation, the weight of E13.5 embryos and placentas, the distribution of the donor mitochondrial DNA (mtDNA), and the pattern of methylation in the differentially methylated regions (DMRs) of the H19 and Snrpn genes. RESULTS: We found that neither the rates of blastocyst formation and implantation nor the weights of E13.5 embryos and placentas were significantly different between the MS and control groups. Additionally, the mtDNA from the iPSC donors could be detected in the muscle tissue of four fetuses and all placentas in the MS group. Finally, the methylation patterns of H19 and Snrpn DMRs remained unchanged by MS. CONCLUSIONS: iPSC-derived mtDNA was directly involved in the process of embryonic development after MS. No adverse effects were seen when using iPSCs as a mitochondrial donor, but it remains to be seen whether this method can improve embryonic development, especially in older mice.

Improvement in Mouse iPSC Induction by Rab32 Reveals the Importance of Lipid Metabolism during Reprogramming.

Induced pluripotent stem cells (iPSCs) have variable expression levels of a series of genes that affect their pluripotent potential, but the regulatory mechanisms controlling reprogramming remain unclear. By testing the efficiency of iPSC generation using Oct4, Sox2, Klf4 (termed OSK) plus one additional gene, we found that Rab32 improved reprogramming efficiency. We established a system for detecting the number and the size of lipid droplets and autophagosomes per cell for tracking their morphological changes during reprogramming. Our results showed that Rab32 increased lipid storage during the early and middle stages, and also increased autophagy during the middle stage of reprogramming. These findings were further confirmed by the up-regulation of lipid biosynthesis and autophagosome formation related genes, of which their expression could improve iPSC induction. The inhibition of lipid biosynthesis and autophagosome formation significantly reduced reprogramming efficiency, and the inhibition of lipid synthesis phenotype could be rescued by the overexpression of Rab32. In addition, the expression of pluripotency genes such as Klf2, Nr5a2 and Tbx3, was up-regulated by Rab32. These results demonstrated that Rab32 could improve the induction of iPSCs through the enhancement of lipid biosynthesis, highlighting the importance of lipid metabolism during reprogramming.

Pluripotent and Metabolic Features of Two Types of Porcine iPSCs Derived from Defined Mouse and Human ES Cell Culture Conditions.

The domestic pig is an excellent animal model for stem cell research and clinical medicine. There is still no suitable culture condition to generate authentic porcine embryonic stem cells (pESCs) and high quality porcine induced pluripotent stem cells (piPSCs). In this study, we found that culture conditions affected pluripotent and metabolic features of piPSCs. Using defined human embryonic stem cell (hESC) and mouse ESC (mESC) culture conditions, we generated two types of piPSCs, one of which was morphologically similar to hESCs (here called hpiPSCs), the other resembled mESCs (here called mpiPSCs). Transcriptome analysis and signaling pathway inhibition results suggested that mpiPSCs shared more of mESC signaling pathways, such as the BMP pathway and JAK/STAT pathway and hpiPSCs shared more hESC signaling pathways, such as the FGF pathway. Importantly, the mpiPSCs performed embryonic chimera incorporation more efficiently than the hpiPSCs did. In addition, the mpiPSCs showed mitochondrial features of naive ESCs and lipid droplets accumulation. These evidences may facilitate understanding of the gene regulation network and metabolism in piPSCs and promote derivation of bona fide pESCs for translational medicine.

The histone deacetylase inhibitor Scriptaid improves in vitro developmental competence of ovine somatic cell nuclear transferred embryos.

Although the success rate of sheep cloning remains extremely low, using a histone deacetylase (HDAC) inhibitor to increase histone acetylation in SCNT embryos has significantly enhanced developmental competence in several species. The objective was to determine whether HDAC inhibitors trichostatin A (TSA) and the novel inhibitor Scriptaid enhance cloning efficiency in sheep cumulus cell (passage 2) reconstructed embryos. In this study, 0.2 µmol/L Scriptaid yielded a high blastocyst development rate, almost twice that of the untreated group (25/103 [24.3%] vs. 12/101 [11.9%]; P < 0.05). Furthermore, 0.2 µmol/L Scriptaid was more effective than 0.05 µmol/L TSA in terms of the blastocyst percentage for cloned ovine embryos in vitro (17/66 [25.7%] vs. 11/65 [16.8%]; P < 0.05). Furthermore, treatment with Scriptaid increased acetylation (compared with the Control, P < 0.05) at lysine residue 12 of histone H4 (acH4K12) and lysine residue 9 of histone H3 (acH3K9) in one-, two-, four-, and eight-cell stages, as well as blastocyst stages, in cloned embryos. In conclusion, Scriptaid was more effective than TSA to enhance in vitro developmental competence in ovine SCNT embryos; furthermore, Scriptaid improved epigenetic status.