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

Li, Enhong; Ustiyan, Vladimir; Wen, Bingqiang; Kalin, Gregory T.; Whitsett, Jeffrey A.; Kalin, Tanya V.; Kalinichenko, Vladimir V.

doi:10.1002/dvdy.298

Cited by 13 publications

(8 citation statements)

References 59 publications

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“…C57BL/6 mice were purchased from Jackson Lab. Interspecies mouse–rat chimeras were generated using blastocyst complementation as described ( Li et al, 2021 ; Wang et al, 2021 ). Briefly, blastocysts from SD rats were obtained at embryonic day 4.5 (E4.5), injected with 15 GFP-labeled mouse ESC cells (ESC-GFP, C57BL/6 background) ( Sun et al, 2021 ; Wen et al, 2021 ) and transferred into pseudopregnant SD rat females.…”

Section: Methodsmentioning

confidence: 99%

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

Wen

Wang

et al. 2022

eLife

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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 FACS 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 bone marrow was generated from mouse ESCs using rat embryos as 'bioreactors'.

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Section: Methodsmentioning

confidence: 99%

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

Wen

Wang

et al. 2022

eLife

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“…The expression level of α-Tub protein in the distal bronchus and terminal bronchus was decreased, which resulted in the failure of the formation of intact bronchial trees in the E2del pups. Besides the expression characteristics of the above genes, the Trp63 proteins of basal cells were abnormally expressed and lined most of the epithelial surfaces of the cystic structures in E2del similar to those in the E3stop lungs [ 15 ]. The specific gene of embryo mesenchyme HOXA4 was lowly expressed in the E2del lungs; this is different from its normal expression throughout lung development in the E3stop embryos [ 17 ].…”

Section: Discussionmentioning

confidence: 99%

“…Thyroid transcription factor 1 ( TTF-1 ) knockout mice have recently emerged as another potential SCOG host for lung regeneration [ 13 , 15 ]. TTF-1, also known as NKX2.1, is a tissue-specific transcription factor that regulates the embryonic development and differentiation of the thyroid and, more importantly, lungs [ 16 ].…”

Section: Introductionmentioning

confidence: 99%

Development of a Lung Vacancy Mouse Model through CRISPR/Cas9-Mediated Deletion of Thyroid Transcription Factor 1 Exon 2

Zhao

Yin

et al. 2022

Cells

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A developmental niche vacancy in host embryos is necessary for stem cell complementation-based organ regeneration (SCOG). Thyroid transcription factor 1 (TTF-1) is a tissue-specific transcription factor that regulates the embryonic development and differentiation of the thyroid and, more importantly, lungs; thus, it has been considered as a master gene to knockout in order to develop a lung vacancy host. TTF-1 knockout mice were originally produced by inserting a stop codon in Exon 3 of the gene (E3stop) through embryonic stem cell-based homologous recombination. The main problems of utilizing E3stop host embryos for lung SCOG are that these animals all have a tracheoesophageal fistula (TEF), which cannot be corrected by donor stem cells, and most of them have monolateral sac-like lungs. To improve the mouse model towards achieving SCOG-based lung generation, in this project, we used the CRISPR/Cas9 tool to remove Exon 2 of the gene by zygote microinjection and successfully produced TTF-1 knockout (E2del) mice. Similar to E3stop, E2del mice are birth-lethal due to retarded lung development with sac-like lungs and only a rudimentary bronchial tree, increased basal cells but without alveolar type II cells and blood vessels, and abnormal thyroid development. Unlike E3stop, 57% of the E2del embryos presented type I tracheal agenesis (TA, a kind of human congenital malformation) with a shortened trachea and clear separations of the trachea and esophagus, while the remaining 43% had TEF. Furthermore, all the E2del mice had bilateral sac-like lungs. Both TA and bilateral sac-like lungs are preferred in SCOG. Our work presents a new strategy for producing SCOG host embryos that may be useful for lung regeneration.

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“…C57BL/6 mice were purchased from Jackson Lab. Interspecies mouse-rat chimeras were generated using blastocyst complementation as described (35, 36). Briefly, blastocysts from SD rats were obtained at embryonic day 4.5 (E4.5), injected with fifteen GFP- labeled mouse ESC cells (ESC-GFP, C57BL/6 background) (32, 37) and transferred into pseudo pregnant SD rat females.…”

Section: Methodsmentioning

confidence: 99%

In Vivo Generation of Bone Marrow from Embryonic Stem Cells in Interspecies Chimeras

Wen

Wang

et al. 2021

Preprint

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Generation of bone marrow (BM) from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) 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 all BM hematopoietic cell lineages from mouse ESCs in a rat. Based on FACS analysis and single-cell RNA sequencing, mouse ESCs differentiated into hematopoietic progenitor cells and multiple hematopoietic cell types that were indistinguishable from normal mouse BM cells based on gene expression signatures and cell surface markers. Transplantation of ESC-derived BM cells from mouse-rat chimeras rescued lethally-irradiated syngeneic mice and resulted in long-term contribution of donor cells to hematopoietic cell lineages. Altogether, a fully functional bone marrow was generated from mouse ESCs using rat embryos as “bioreactors”.

show abstract

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

Cited by 13 publications

References 59 publications

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

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

Development of a Lung Vacancy Mouse Model through CRISPR/Cas9-Mediated Deletion of Thyroid Transcription Factor 1 Exon 2

In Vivo Generation of Bone Marrow from Embryonic Stem Cells in Interspecies Chimeras

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