Journal: Developmental cell

Article Title: Lineage-tracing hematopoietic stem cell origins in vivo to efficiently make human HLF+ HOXA+ hematopoietic progenitors from pluripotent stem cells.

doi: 10.1016/j.devcel.2024.03.003

Figure Lengend Snippet: Figure 1. Genetic lineage tracing reveals that artery endothelial cells generate HSCs in vivo (A) Experimental strategy. DA, dorsal aorta; FL, fetal liver; PB, peripheral blood; BM, bone marrow; E, embryonic day; P, postnatal day. (B) Mass spectrometry quantification of (Z)-4OHT levels in plasma of female adult Cx40-CreERT2 mice that intraperitoneally injected with (Z)-4OHT.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER H7 CAG-AkaLuc-PuroR hESCs Kyle Loh’s laboratory (Stanford University) This study WTC11 hiPSCs Coriell Institute for Medical Research Coriell Institute for Medical Research, GM25256, Kreitzer et al.148 OP9-DLL4-IRES-GFP feeder cells Juan Carlos Zúñiga-Pfl€ucker’s laboratory (University of Toronto) Mohtashami et al.149 MS5 feeder cells DSMZ ACC 441 Human cord blood CD34+ hematopoietic stem and progenitor cells (pooled from mixed donors) StemExpress CB3400.5C Experimental models: organisms/strains Mus musculus (mouse): Cx40-CreERT2 Lucile Miquerol’s laboratory (Aix-Marseille Université) Beyer et al.77 Mus musculus (mouse): Efnb2-CreERT2 Kyle Loh’s laboratory (Stanford University) This study (being deposited at The Jackson Laboratory [JAX], 038831) Mus musculus (mouse): Apj-CreERT2 Kristy Red-Horse’s laboratory (Stanford University) Chen et al.91 Mus musculus (mouse): Rosa26-CAG-LoxP-StopLoxP-ZsGreen (Ai6) The Jackson Laboratory JAX 007906 (Madisen et al.78) Mus musculus (mouse): FVB/NJ The Jackson Laboratory JAX 001800 Mus musculus (mouse): C57BL/6-CD45.2 The Jackson Laboratory JAX 000664 Mus musculus (mouse): C57BL/6-CD45.1 (Pepboy) The Jackson Laboratory JAX 002014 Mus musculus (mouse): NOD-SCID Il2rg-/- (NSG) The Jackson Laboratory JAX 005557 (Shultz et al.150) Oligonucleotides Primers See Table S4 N/A HCR3 probe for mouse Gja5 (Cx40), compatible with amplifier B3 Molecular Instruments Custom probe against Gja5 (sequence deposited at NCBI accession NM_001271628) HCR3 probe for CreERT2, compatible with amplifier B1 Molecular Instruments Custom probe against CreERT2 (sequence deposited at https:// www.addgene.org/14797/) HCR3 B3-Alexa Fluor 647 amplifier probe Molecular Instruments B3-Alexa Fluor 647 HCR3 B1-Alexa Fluor 488 amplifier probe Molecular Instruments B1-Alexa Fluor 488 Software and algorithms anndata Fabian Theis’s laboratory (Helmholtz Munich) https://github.com/ theislab/anndata AnnotationDbi Hervé Pagès, Marc Carlson, Seth Falcon, Nianhua Li (Bioconductor Team) https://github.com/ Bioconductor/ AnnotationDbi biomaRt Damian Smedley, Syed Haider, Benoit Ballester, Richard Holland, Darin London, Gudmundur Thorisson, Arek Kasprzyk (Bioconductor Team) https://github.com/ grimbough/biomaRt CellRanger 10x Genomics https://support.

Techniques: In Vivo, Mass Spectrometry, Clinical Proteomics, Injection

Journal: Developmental cell

Article Title: Lineage-tracing hematopoietic stem cell origins in vivo to efficiently make human HLF+ HOXA+ hematopoietic progenitors from pluripotent stem cells.

doi: 10.1016/j.devcel.2024.03.003

Figure Lengend Snippet: Figure 2. Artery-derived HSCs are functional in vivo (A–E) Arteries were lineage-traced in Cx40-CreERT2; Ai6 (ZsGreen reporter) embryos by administering 4OHT at either E8.0, E8.5, or E9.0. After embryos developed into adults, flow cytometry was performed to quantify ZsGreen+ cells in (B) and (C) peripheral blood and (C) and (D) bone marrow HSCs in 1- to 22-month-old adult mice. Line graphs depict the mean ± SEM. Related to Figure S3.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER H7 CAG-AkaLuc-PuroR hESCs Kyle Loh’s laboratory (Stanford University) This study WTC11 hiPSCs Coriell Institute for Medical Research Coriell Institute for Medical Research, GM25256, Kreitzer et al.148 OP9-DLL4-IRES-GFP feeder cells Juan Carlos Zúñiga-Pfl€ucker’s laboratory (University of Toronto) Mohtashami et al.149 MS5 feeder cells DSMZ ACC 441 Human cord blood CD34+ hematopoietic stem and progenitor cells (pooled from mixed donors) StemExpress CB3400.5C Experimental models: organisms/strains Mus musculus (mouse): Cx40-CreERT2 Lucile Miquerol’s laboratory (Aix-Marseille Université) Beyer et al.77 Mus musculus (mouse): Efnb2-CreERT2 Kyle Loh’s laboratory (Stanford University) This study (being deposited at The Jackson Laboratory [JAX], 038831) Mus musculus (mouse): Apj-CreERT2 Kristy Red-Horse’s laboratory (Stanford University) Chen et al.91 Mus musculus (mouse): Rosa26-CAG-LoxP-StopLoxP-ZsGreen (Ai6) The Jackson Laboratory JAX 007906 (Madisen et al.78) Mus musculus (mouse): FVB/NJ The Jackson Laboratory JAX 001800 Mus musculus (mouse): C57BL/6-CD45.2 The Jackson Laboratory JAX 000664 Mus musculus (mouse): C57BL/6-CD45.1 (Pepboy) The Jackson Laboratory JAX 002014 Mus musculus (mouse): NOD-SCID Il2rg-/- (NSG) The Jackson Laboratory JAX 005557 (Shultz et al.150) Oligonucleotides Primers See Table S4 N/A HCR3 probe for mouse Gja5 (Cx40), compatible with amplifier B3 Molecular Instruments Custom probe against Gja5 (sequence deposited at NCBI accession NM_001271628) HCR3 probe for CreERT2, compatible with amplifier B1 Molecular Instruments Custom probe against CreERT2 (sequence deposited at https:// www.addgene.org/14797/) HCR3 B3-Alexa Fluor 647 amplifier probe Molecular Instruments B3-Alexa Fluor 647 HCR3 B1-Alexa Fluor 488 amplifier probe Molecular Instruments B1-Alexa Fluor 488 Software and algorithms anndata Fabian Theis’s laboratory (Helmholtz Munich) https://github.com/ theislab/anndata AnnotationDbi Hervé Pagès, Marc Carlson, Seth Falcon, Nianhua Li (Bioconductor Team) https://github.com/ Bioconductor/ AnnotationDbi biomaRt Damian Smedley, Syed Haider, Benoit Ballester, Richard Holland, Darin London, Gudmundur Thorisson, Arek Kasprzyk (Bioconductor Team) https://github.com/ grimbough/biomaRt CellRanger 10x Genomics https://support.

Techniques: Derivative Assay, Functional Assay, In Vivo, Cytometry

Journal: Developmental Cell

Article Title: Lineage-tracing hematopoietic stem cell origins in vivo to efficiently make human HLF+ HOXA+ hematopoietic progenitors from pluripotent stem cells

doi: 10.1016/j.devcel.2024.03.003

Figure Lengend Snippet: Genetic lineage tracing reveals that artery endothelial cells generate HSCs in vivo (A) Experimental strategy. DA, dorsal aorta; FL, fetal liver; PB, peripheral blood; BM, bone marrow; E, embryonic day; P, postnatal day. (B) Mass spectrometry quantification of ( Z )-4OHT levels in plasma of female adult Cx40-CreERT2 mice that intraperitoneally injected with ( Z )-4OHT. (C) Cx40 and CreERT2 in situ staining of E8.5 Cx40-CreERT2 mouse embryos, using hybridization chain reaction v3.0 (HCR3). Arrows: paired dorsal aortae. Ant, anterior; post, posterior. (D) scRNA-seq of the entire E8.5 mouse embryo. (E–J) Arteries were lineage-traced in Cx40-CreERT2 ; Ai6 ( ZsGreen reporter) embryos by administering 4OHT at E8.5. The Cx40-CreERT2 allele also encodes RFP , which was used to visualize Cx40+ cells. (E, G, and I) Immunostaining and (F, H, and J) flow cytometry of E11.5 dorsal aorta, E11.5 yolk sac, and E16.5 fetal liver was performed. (K) Arteries were lineage-traced in Cx40-CreERT2 ; Ai6 ( ZsGreen reporter) embryos by administering a single 4OHT dose at the indicated times (E7.5–E12.5). Flow cytometry was performed to quantify artery-derived (i.e., ZsGreen+) HSCs in the E14.5–E18.5 fetal liver. Each dot: independent litter. For each time point, ≥8 independent embryos were analyzed. Inset: fetal liver HSCs labeled after E9.0 4OHT administration. (L) Arteries were lineage-traced in Efnb2-CreERT2 ; Ai6 ( ZsGreen reporter) embryos by administering 4OHT at E8.5. Flow cytometry was performed to quantify ZsGreen+ E14.5–E18.5 fetal liver HSCs. (M) Veins and capillaries were lineage-traced in Apj-CreERT2 ; Ai6 ( ZsGreen reporter) embryos by administering 4OHT at E9.5. Flow cytometry was performed to quantify ZsGreen+ E14.5–E18.5 fetal liver HSCs. Histograms depict the mean ± standard error of the mean (SEM). ∗ p < 0.05, ∗∗ p < 0.01. Scale bars, 50 μm. Related to Figures S1 and and Table S1 .

Article Snippet: HCR3 probe for CreERT2 , compatible with amplifier B1 , Molecular Instruments , Custom probe against CreERT2 (sequence deposited at https://www.addgene.org/14797/ ).

Techniques: In Vivo, Mass Spectrometry, Clinical Proteomics, Injection, In Situ, Staining, Hybridization, Immunostaining, Flow Cytometry, Derivative Assay, Labeling

Journal: Developmental Cell

Article Title: Lineage-tracing hematopoietic stem cell origins in vivo to efficiently make human HLF+ HOXA+ hematopoietic progenitors from pluripotent stem cells

doi: 10.1016/j.devcel.2024.03.003

Figure Lengend Snippet: Artery-derived HSCs are functional in vivo (A–E) Arteries were lineage-traced in Cx40-CreERT2 ; Ai6 ( ZsGreen reporter) embryos by administering 4OHT at either E8.0, E8.5, or E9.0. After embryos developed into adults, flow cytometry was performed to quantify ZsGreen+ cells in (B) and (C) peripheral blood and (C) and (D) bone marrow HSCs in 1- to 22-month-old adult mice. Line graphs depict the mean ± SEM. Related to Figure S3 .

Article Snippet: HCR3 probe for CreERT2 , compatible with amplifier B1 , Molecular Instruments , Custom probe against CreERT2 (sequence deposited at https://www.addgene.org/14797/ ).

Techniques: Derivative Assay, Functional Assay, In Vivo, Flow Cytometry

Journal: Developmental Cell

Article Title: Lineage-tracing hematopoietic stem cell origins in vivo to efficiently make human HLF+ HOXA+ hematopoietic progenitors from pluripotent stem cells

doi: 10.1016/j.devcel.2024.03.003

Figure Lengend Snippet: Artery-derived HSCs are functional in vivo upon transplantation (A and B) Arteries were lineage-traced by administering 4OHT to E8.5 Cx40-CreERT2 ; Ai6 ( ZsGreen reporter) embryos. B6, C57BL/6 mouse. (C and D) ZsGreen+ E16.5 fetal liver HSCs were (B) analyzed by flow cytometry and (C and D) transplanted into lethally irradiated primary recipient mice. 1–4 months post transplantation, flow cytometry was performed to quantify ZsGreen+ (C) peripheral blood cells and (D) bone marrow HSCs in primary recipients. (E and F) Bone marrow from primary recipient mice was transplanted into lethally irradiated secondary recipient mice. 1–4 months post transplantation, flow cytometry was performed to quantify ZsGreen+ (E) peripheral blood and (F) bone marrow HSCs in secondary recipients. Data depict the mean ± SEM. Each dot represents a single mouse. Related to Figure S4 .

Article Snippet: HCR3 probe for CreERT2 , compatible with amplifier B1 , Molecular Instruments , Custom probe against CreERT2 (sequence deposited at https://www.addgene.org/14797/ ).

Techniques: Derivative Assay, Functional Assay, In Vivo, Transplantation Assay, Flow Cytometry, Irradiation