We thank Suwei Gao for help in figure preparation. This work was supported by grants from the National Natural Science Foundation of China (81530004, 31425016) and the Ministry of Science and Technology of China (2016YFA0100500). J.L. performed the experiments and drafted the manuscript; F.L. edited the manuscript. Both authors read and approved the final manuscript.

All the procedures including animal subjects have been approved by the Ethical Review Committee in the Institute of Zoology, Chinese Academy of Sciences, Beijing, China.
1. Material Preparation
<ol>
	<li>Sterilize the 0.65 &#181;m filters with ultraviolet rays produced under the ultraviolet ray lamp on the clean bench for 4 h and turn the filters over after the 2 h mark. 
	NOTE: When working with UV light, wear appropriate protection.</li>
	<li>Sterilize the stainless steel meshes with the autoclave sterilizer at 121 &#176;C for 30 mins. 
	NOTE: A certain height of stainless steel mesh is needed to support the filters at the air-liquid interface and this can be realized with the wire on the steel mesh (Figure 1A).</li>
	<li>Dilute Fluoxetine to a final concentration of 10 &#181;M into the M5300 long-term culture medium and mix evenly. 
	NOTE: For a six-well plate, 2 mL M5300 long-term culture medium is appropriate for each well. Hydrocortisone at 10-6 M can be selectively diluted into the medium.</li>
	<li>Transfer the medium supplemented with Fluoxetine at 10 &#181;M into six-well plates or dishes and put the sterilized stainless steel meshes into the medium.</li>
	<li>Wash the 0.65 &#181;m filters with boiling water two times for 3 mins each time in the glass cell culture dish for sterilization. After each washing, put the filters into phosphate buffered saline (PBS) for 2 mins and place the wet filters onto the stainless steel meshes standing at the air-liquid interface as shown in Figure 1A [schematic in the center]. 
	NOTE: Boiling water can be generated with the autoclave sterilizer to achieve the asepsis and equipment should be taken out of autoclave sterilizer in a timely fashion to avoid a decrease in temperature.</li>
</ol>
2. AGM Explant Culture
<ol>
	<li>Carefully separate the AGM regions from the embryos at E10.5 or E11 with forceps.
	<ol>
		<li>Strip the uteruses from pregnant mice with the dissecting scissors and separate the embryos from the uteruses.</li>
		<li>Wipe off the yolk sac, umbilical cord, and viscera from the embryo body.</li>
		<li>Carefully remove the dorsal nerve tissues and surrounding muscular tissues.</li>
		<li>Cut off the tissues with the forceps at the site of anterior and hind limbs and separate the AGM region from the embryo body. Figures 1B-C show the schematic representation and morphology of dissected AGM.</li>
	</ol>
	</li>
	<li>Explant the dissected AGMs separately onto the filters at the air-liquid interface (Figure 1A) and culture in 5% CO2 at 37 &#176;C for 2 - 3 days. 
	NOTE: Do not place the dissected AGM onto the wire of the steel mesh and make sure the filters are at the air-liquid interface during culture. Besides the AGM, yolk sac and fetal liver can also be cultured with this system7.</li>
</ol>
3. Expression Analyses
<ol>
	<li>mRNA level
	<ol>
		<li>Collect the cultured AGMs into 1 mL PBS and centrifuge at 4 &#176;C, 310 x g for 6 min.</li>
		<li>After removing the supernatant, total RNA is extracted from the collected AGMs with a monophasic solution of phenol, according to the manufacturer&#39;s instructions.</li>
		<li>Total RNA (2 &#181;g) is then reverse transcribed using M-MLV reverse transcriptase to obtain cDNA as the template. The quantitative real-time PCR assays are performed with SYBR Green and Gapdh is used as the internal control.</li>
	</ol>
	</li>
	<li>Protein level
	<ol>
		<li>Collect the cultured AGM as described above in step 3.1.1.</li>
		<li>Prepare the protein from the cultured AGM with the cell lysis buffer (10 mM Tris-HCl, 10 mM NaCl, and 0.5% NP-40) containing protease inhibitor and use for western blotting to detect the protein level as previously described13.</li>
	</ol>
	</li>
</ol>
4. Colony-Forming Units in the Culture (CFU-C) Assay
<ol>
	<li>After culturing for 2 - 3 days, collect the AGM regions into 1 mL phosphate buffered saline (PBS) and centrifuge at 4 &#176;C, 310 x g for 6 mins and dissociate with 200 &#181;L collagenase (0.1% in PBS) at 37 &#176;C. 
	NOTE: The time needed for collagenase to generate single cell-suspensions is about 20 mins. Shaking the tube containing AGM every 5 mins can accelerate the digestion process. 200 &#181;L 0.1% collagenase is used for each AGM and 200 &#181;L 10% serum is used to stop the reaction.</li>
	<li>Culture single cell-suspensions in M3434 medium in ultra-low attachment 24-well plates. 
	NOTE: To avoid contamination, Penicillin-Streptomycin solution can be selectively added into the medium. Because the M3434 medium is viscous, a 1.0 cc syringe without a needle is used to mix the cells and medium.</li>
	<li>After 7 - 10 days culturing at 37 &#176;C in 5% CO2, the number for each type of colonies including Burst forming unit--erythroid (BFU-E), CFU-granulo-monocyte (CFU-GM) and CFU-granulocyte, erythrocyte, macrophage, megakaryocyte (CFU-GEMM) are distinguished based on the morphology and scored with an inverted microscope (Figure 2C).</li>
</ol>
5. In Vivo Transplantation Assay
<ol>
	<li>Colony-forming units-spleen (CFU-S) assay
	<ol>
		<li>After 2 - 3 days of explant culturing, collect the AGMs and prepare single cell-suspensions by incubating with 200 &#181;L collagenase (0.1% in PBS) for 20 min.</li>
		<li>Perform lethal irradiation (9 Gy of X-rays) of 8- to 10-week old male C57BL/6 mice 4 - 5 h before cell injection.</li>
		<li>Put the irradiated mouse into a restrainer to restrict its activity.</li>
		<li>Wipe the tail using a cotton swab steeped in 75% alcohol to promote vasodilatation of the vein.</li>
		<li>Inject 0.5 embryo equivalent (ee) or 1 ee single cell-suspensions of AGM intravenously into the tail vein of irradiated mouse with a 1.0 cc syringe. 
		NOTE: Cells are suspended with PBS and 0.5 mL PBS per recipient is a suitable volume. A needle of 25 gauge or 26 gauge size is used for injection with a 1.0 cc syringe. No air can be introduced into the syringe. Press the injection site with an antiseptic swab to stop bleeding.</li>
		<li>Eleven days post transplantation, collect and fix the spleens of the recipient mice in Bouin&#39;s solution (15 mL 1.22% Picric acid saturated aqueous solution, 2 mL 40% methanol and 1 mL acetic acid) for 1 - 2 days and wash with 80% alcohol for another 1 - 2 days. Count the visible colonies in the spleen macroscopically and determine the number of colonies per ee. 
		&#8203;NOTE: Bouin&#39;s fixative, wear appropriate protection.</li>
	</ol>
	</li>
	<li>Long-term transplantation assay
	<ol>
		<li>After 2 - 3&#160;days explant culturing, collect the AGMs and prepare single cell-suspensions by incubating with 200 &#181;L collagenase (0.1% in PBS) for 20 min.</li>
		<li>Perform lethal irradiation (9 Gy of X-ray) of 8- to 10-week old male C57BL/6 mice 4 - 5 hrs before cell injection.</li>
		<li>Put the irradiated mouse into a restrainer to restrict its activity.</li>
		<li>Wipe the tail using a cotton swab steeped in 75% alcohol to promote vasodilatation of the vein.</li>
		<li>Inject 0.5 embryo equivalent (ee) or 1 ee single cell-suspensions of AGM intravenously into the tail vein of an irradiated mouse with a 1.0 cc syringe. AGM are injected at a dose of 1 ee per recipient with 2&#215;105 bone marrow cells (CD45.1 background). 
		NOTE: No air can be introduced into the syringe. Press the injection site with an antiseptic swab to stop bleeding.</li>
		<li>Four months post-transplantation, collect the bone marrow of the recipient and use it for the reconstitution assay by FACS. Only the recipients with &#8805;10% donor-derived chimerism are considered to exhibit successful reconstitution.</li>
	</ol>
	</li>
</ol>

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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+tracing+reveals+the+endothelial+origin+of+hematopoietic+stem+cells.">Fate tracing reveals the endothelial origin of hematopoietic stem cells.</a> Cell Stem Cell. 3, 625-636 (2008).</li><li>Kumaravelu, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+developmental+anatomy+of+definitive+haematopoietic+stem+cells+long-term+repopulating+units+(HSC/RUs):+role+of+the+aorta-gonad-mesonephros+(AGM)+region+and+the+yolk+sac+in+colonisation+of+the+mouse+embryonic+liver.">Quantitative developmental anatomy of definitive haematopoietic stem cells long-term repopulating units (HSC/RUs): role of the aorta-gonad-mesonephros (AGM) region and the yolk sac in colonisation of the mouse embryonic liver.</a> Development. 129, 4891-4899 (2002).</li><li>Orkin, S. 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The authors have no conflicting financial interests.

It is well known that mature HSCs in the bone marrow can repopulate the blood system of irradiated recipients. Unlike these functional HSCs, the emerging HSCs in the AGM of mouse embryos are immature. Direct transplantation results showed that type I (VE-cad+ CD45- CD41+) and type II (VE-cad+ CD45+) pre-HSCs possess no reconstitution ability20. Taking advantage of the AGM explant culture system, the nascent HSCs in the AGM region can reconstit...

Hematopoietic stem cells (HSCs) are tissue-specific adult stem cells that possess multilineage potential including erythroid, myeloid, and lymphoid cells as well as the ability to self-renew. Recent studies have shown that the earliest HSCs arose from a specialized endothelial population, known as hemogenic endothelium (HE), through the endothelial to hematopoietic transition (EHT) at the ventral wall of dorsal aorta1,2,3,4. Once formed in the aorta-gonad-mesonephros (AGM) region from embryonic (E) 10.5 to E12.5 in the mouse embryo, HSCs will migrate into the fetal liver for expansion and finally colonize the bone marrow to maintain adult hematopoiesis throughout an individual&#39;s&#160;life5,6. Although this has been studied for many years, the underlying mechanisms of HSC emergence and development remain incompletely understood.
Unlike in vitro fertilization and development of zebrafish embryos, intrauterine development of mouse embryos makes the study of definitive hematopoiesis during embryogenesis much more inconvenient. Although genetic experiments using knockout (KO) mice are commonly utilized, the lack of certain KO mice also limits their use in the hematopoietic research field. In addition, in vivo rescue experiments are not easily performed in KO mice. Since 1996, the AGM explant culture has been developed for hematopoietic studies by the pioneers in the field7. With the help of this culture system, ventral tissues of the AGM region have been identified to promote HSC activity, while dorsal tissues exert an opposite effect8,9. The AGM explant culture system has also been applied to determine the roles of Serotonin, Mpl, SCF, BMP, and Hedgehog signaling in HSC development10,11,12,13,14. Importantly, it is also a popular method used to rescue hematopoietic defects in mutant embryos13,15.

<table><tbody><tr><td>durapore 0.65 &amp;micro;m filters</td><td>Millipore</td><td>R5BA63787</td><td>AGM explant culture</td></tr><tr><td>M5300 long-term culture medium</td><td>Stem Cell Technologies</td><td>5300</td><td>AGM explant culture</td></tr><tr><td>Trizol&amp;nbsp;</td><td>Tiangen</td><td>5829</td><td>RNA extration</td></tr><tr><td>M-MLV reverse transcriptase</td><td>Promega</td><td>90694</td><td>reverse transcription</td></tr><tr><td>SYBR Green</td><td>Qiagen</td><td>A6002</td><td>quantatitive real-time PCR</td></tr><tr><td>protease inhibitor</td><td>Roche</td><td>55622</td><td>Protein extration</td></tr><tr><td>collagenase</td><td>Sigma</td><td>C2674</td><td>digestion of AGM tissues</td></tr><tr><td>MethoCult GF M3434 medium</td><td>Stem Cell Technologies</td><td>3434</td><td>CFU-C assay</td></tr><tr><td>ultra-low attachment 24-well plates</td><td>Costar</td><td>3473</td><td>CFU-C assay</td></tr><tr><td>C57BL/6 CD45.2 mice</td><td>Beijing HFK Bioscience Co. Ltd</td><td></td><td>CFU-S assay</td></tr><tr><td>C57BL/6 CD45.1 mice</td><td>Beijing HFK Bioscience Co. Ltd</td><td></td><td>Long-term transplantation</td></tr><tr><td>phosphate buffered saline</td><td>Life Technologines</td><td>8115284</td><td>AGM Collection</td></tr><tr><td>Penicillin-Streptomycin solution</td><td>HyClone</td><td>SV30010</td><td>AGM explant culture</td></tr><tr><td>anti-CD45.2-PE-CY7</td><td>eBioscience</td><td>25-0454-80</td><td>Long-term transplantation</td></tr><tr><td>anti-CD45-FITC</td><td>eBioscience</td><td>11-0451-81</td><td>Long-term transplantation</td></tr><tr><td>UV lamp</td><td>Beijing jiangmorning yuan bio-technology co., LTD</td><td>039-14402</td><td>power: 30W</td></tr><tr><td>stainless steel mesh</td><td>AS ONE SHANGHAI Corporation</td><td>2-9817-10&amp;nbsp;</td><td>aperture diameter: 2.5 mm</td></tr><tr><td>hydrocortisone</td><td>Sigma</td><td>H0396</td><td>selectively diluted into M5300 medium</td></tr></tbody></table>

application of aorta-gonad-mesonephros explant culture system in developmental hematopoiesis

The limitation of using mouse embryos for hematopoiesis studies is the added inconvenience in operations, which is largely due to the intrauterine development of the embryo. Although genetic data from knockout (KO) mice are convincing, it is not realistic to generate KO mice for all genes as needed. In addition, performing in vivo rescue experiments to consolidate the data obtained from KO mice is not convenient. To overcome these limitations, the Aorta-Gonad-Mesonephros (AGM) explant culture was developed as an appropriate system to study hematopoietic stem cell (HSC) development. Especially for rescue experiments, it can be used to recover the impaired hematopoiesis in KO mice. By adding the appropriate chemicals into the medium, the impaired signaling can be reactivated or up-regulated pathways can be inhibited. With the use of this method, many experiments can be performed to identify the critical regulators of HSC development, including HSC related gene expression at mRNA and protein levels, colony formation ability, and reconstitution capacity. This series of experiments would be helpful in defining the underlying mechanisms essential for HSC development in mammals.

A recent publication reported endothelial cell-derived serotonin promotes the survival of HSCs by inhibiting the pro-apoptotic pathway in the AGM13. To confirm the promoting effect of serotonin on HSC development, Fluoxetine was included. As a selective serotonin re-uptake inhibitor (SSRI), Fluoxetine has been demonstrated to inhibit serotonin re-absorption in peripheral tissues16,17,<sup class="...

Watch this Scientific Journal Video about Application of Aorta-gonad-mesonephros Explant Culture System in Developmental Hematopoiesis at JoVE.com

Application of Aorta-gonad-mesonephros Explant Culture System in Developmental Hematopoiesis

This protocol describes using cultured Aorta-Gonad-Mesonephros for expression analyses, colony-forming units in the culture and spleen, and long-term reconstitution to determine the effect of regulatory factors and signaling pathways on hematopoietic stem cell development. This has been demonstrated as an effective system for studying hematopoietic stem cell biology and function.

The limitation of using mouse embryos for hematopoiesis studies is the added inconvenience in operations, which is largely due to the intrauterine ...

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8.4K Views.  Chinese Academy of Sciences. Dit protocol beschrijving van het gebruik van gekweekte Aorta-gonaden-Mesonephros voor expressie analyses, kolonie-vormende eenheden in de cultuur en milt, en op lange termijn reconstitutie om te bepalen van het effect van regelgevende factoren en signaalroutes op hematopoietische stamcellen cel ontwikkeling. Dit is aangetoond als een doeltreffend systeem voor het bestuderen van hematopoietische stamcelbiologie en functie.

Video: Application of Aorta-gonad-mesonephros Explant Culture System in Developmental Hematopoiesis

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop within the uterus. A newly developed ex vivo upright droplet culture method provides an attractive alternative to studies performed in utero. The ex vivo droplet culture provides the ability to examine and manipulate cellular interactions and diverse signaling pathways through use of various blocking and activating compounds; additionally, the effects of various pharmacological reagents on the development of specific organs can be studied without unwanted side effects of systemic drug delivery in utero. As compared to other in vitro systems, the droplet culture not only allows for the ability to study three-dimensional morphogenesis and cell-cell interactions, which cannot be reproduced in mammalian cell lines, but also requires significantly less reagents than other ex vivo and in vitro protocols. This paper demonstrates proper mouse fetal organ dissection and upright droplet culture techniques, followed by whole organ immunofluorescence to demonstrate the effectiveness of the method. The ex vivo droplet culture method allows the formation of organ architecture comparable to what is observed in vivo and can be utilized to study otherwise difficult-to-study processes due to embryonic lethality in in vivo models. As a model application system, a small-molecule inhibitor will be utilized to probe the role of vascularization in testicular morphogenesis. This ex vivo droplet culture method is expandable to other fetal organ systems, such as lung and potentially others, although each organ must be extensively studied to determine any organ-specific modifications to the protocol. This organ culture system provides flexibility in experimentation with fetal organs, and results obtained using this technique will help researchers gain insights into fetal development.

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

The&#160;ex vivo&#160;upright droplet culture is an alternative to current&#160;in vitro&#160;and&#160;in vivo&#160;experimental techniques. This protocol is easy to perform and requires smaller amounts of reagent, while permitting the ability to manipulate and study fetal vascularization, morphogenesis, and organogenesis.

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop ...

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

Hematopoietic stem cells require a unique microenvironment in order to sustain blood cell formation1; the bone marrow (BM) is a complex three-dimensional (3D) tissue wherein hematopoiesis is regulated by spatially organized cellular microenvironments termed niches2-4. The organization of the BM niches is critical for the function or dysfunction of normal or malignant BM5. Therefore a better understanding of the in vivo microenvironment using an ex vivo mimicry would help us elucidate the molecular, cellular and microenvironmental determinants of leukemogenesis6.
Currently, hematopoietic cells are cultured in vitro in two-dimensional (2D) tissue culture flasks/well-plates7 requiring either co-culture with allogenic or xenogenic stromal cells or addition of exogenous cytokines8. These conditions are artificial and differ from the in vivo microenvironment in that they lack the 3D cellular niches and expose the cells to abnormally high cytokine concentrations which can result in differentiation and loss of pluripotency9,10.
Herein, we present a novel 3D bone marrow culture system that simulates the in vivo 3D growth environment and supports multilineage hematopoiesis in the absence of exogenous growth factors. The highly porous scaffold used in this system made of polyurethane (PU), facilitates high-density cell growth across a higher specific surface area than the conventional monolayer culture in 2D11. Our work has indicated that this model supported the growth of human cord blood (CB) mononuclear cells (MNC)12 and primary leukemic cells in the absence of exogenous cytokines. This novel 3D mimicry provides a viable platform for the development of a human experimental model to study hematopoiesis and to explore novel treatments for leukemia.

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

A 3D culture system for hematopoiesis is described using human cord blood and leukemic bone marrow cells. The method is based on the use of a porous synthetic polyurethane scaffold coated with extracellular matrix proteins. This scaffold is adaptable to accommodate a wide range of cells.

Hematopoietic stem cells require a unique microenvironment in order to sustain blood cell formation1; the bone marrow (BM) is a complex three-dimensional ...

Bioengineering

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

Dissection and Culture of Mouse Embryonic Kidney

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney development, the two progenitor tissues, the ureteric bud and the metanephric mesenchyme, communicate and reciprocally induce cellular mechanisms to eventually form the collecting system and the nephrons of the kidney. As mammalian embryos grow intrauterine and therefore are inaccessible to the observer, an organ culture has been developed. With this method, it is possible to study epithelial-mesenchymal interactions and cellular behavior during kidney organogenesis. Furthermore, the origin of congenital kidney and urogenital tract malformations can be investigated. After careful dissection, the metanephric rudiments are transferred onto a filter that floats on culture medium and can be kept in a cell culture incubator for several days. However, one must be aware that the conditions are artificial and could influence the metabolism in the tissue. Also, the penetration of test substances could be limited due to the extracellular matrix and basal membrane present in the explant.
One main advantage of organ culture is that the experimenter can gain direct access to the organ. This technology is cheap, simple, and allows a large number of modifications, such as the addition of biologically active substances, the study of genetic variants, and the application of advanced imaging techniques.

This protocol describes a method for isolating and culturing metanephric rudiments from mouse embryos.

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic lamellae and smooth muscle cells (SMCs), and an outer layer of fibroblasts and extracellular matrix. In contrast to the widespread study of pathological models (e.g., atherosclerosis) in the adult aorta, much less is known about the embryonic and perinatal aorta. Here, we focus on SMCs and provide protocols for the analysis of the morphogenesis and pathogenesis of embryonic and perinatal aortic SMCs in normal development and disease. Specifically, the four protocols included are: i) in vivo embryonic fate mapping and clonal analysis; ii) explant embryonic aorta culture; iii) SMC isolation from the perinatal aorta; and iv) subcutaneous osmotic mini-pump placement in pregnant (or non-pregnant) mice. Thus, these approaches facilitate the investigation of the origin(s), fate, and clonal architecture of SMCs in the aorta in vivo. They allow for modulating embryonic aorta morphogenesis in utero by continuous exposure to pharmacological agents. In addition, isolated aortic tissue explants or aortic SMCs can be used to gain insights into the role of specific gene targets during fundamental processes such as muscularization, proliferation, and migration. These hypothesis-generating experiments on isolated SMCs and the explanted aorta can then be assessed in the in vivo context through pharmacological and genetic approaches.

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

Protocols for studying the embryonic and perinatal murine aorta using in vivo clonal analysis and fate mapping, aortic explants, and isolated smooth muscle cells are detailed here. These diverse approaches facilitate the investigation of the morphogenesis of the embryonic and perinatal aorta in normal development and the pathogenesis in disease.

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic ...

Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early embryo and the lack of assays that functionally identify the long-term multilineage engraftment potential of individual putative HSC precursors. Here, we describe methodology that enables the isolation and characterization of functionally validated HSC precursors at the single cell level. First, we utilize index sorting to catalog the precise phenotypic parameter of each individually sorted cell, using a combination of phenotypic markers to enrich for HSC precursors with additional markers for experimental analysis. Second, each index-sorted cell is co-cultured with vascular niche stroma from the aorta-gonad-mesonephros (AGM) region, which supports the maturation of non-engrafting HSC precursors to functional HSC with multilineage, long-term engraftment potential in transplantation assays. This methodology enables correlation of phenotypic properties of clonal hemogenic precursors with their functional engraftment potential or other properties such as transcriptional profile, providing a means for the detailed analysis of HSC precursor development at the single cell level.

Here we present methodology for the clonal analysis of hematopoietic stem cell precursors during murine embryonic development. We combine index sorting of single cells from the embryonic aorta-gonad-mesonephros region with endothelial cell co-culture and transplantation to characterize the phenotypic properties and engraftment potential of single hematopoietic precursors.

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early ...

Optimization of Renal Organoid and Organotypic Culture for Vascularization, Extended Development, and Improved Microscopy Imaging

Embryonic kidney organotypic cultures, and especially pluripotent stem cell-derived kidney organoids, are excellent tools for following developmental processes and modelling kidney disease. However, the models are limited by a lack of vascularization and functionality. To address this, an improved protocol for the method of xenografting cells and tissues to the chorioallantoic membrane (CAM) of an avian embryo to gain vascularization and restoration of blood flow was developed. The grafts are overlaid with custom-made minireservoirs that fix the samples to the CAM and supply them with culture medium that protects the grafts from drying. The improved culture method allows xenografts to grow for up to 9 days. The manuscript also describes how to provide optimal conditions for long-term confocal imaging of renal organoids and organotypic cultures using the previously published Fixed Z-Direction (FiZD) method. This method gently compresses an embryonic organ or organoid between a glass coverslip and membrane in a large amount of medium and provides excellent conditions for imaging for up to 12 days. Together, these methods allow vascularization and blood flow to renal organoids and organotypic kidney cultures with improved confocal imaging. The methods described here are highly beneficial for studying fundamental and applied functions of kidneys ex vivo. Both methods are applicable to various types of tissues and organoids.

This work describes two methods for studying organ development, an improved xenotransplantation setup on chorioallantoic membrane (CAM) from avian embryos that allows for vascularization of cultured embryonic organs and organoids and a novel fixed z-direction organ culture method with modified experimental conditions that allows for high-resolution time-lapse confocal imaging.

Embryonic kidney organotypic cultures, and especially pluripotent stem cell-derived kidney organoids, are excellent tools for following developmental ...

Modified ES / OP9 Co-Culture Protocol Provides Enhanced Characterization of Hematopoietic Progeny

The in vitro differentiation of ES cells towards a hematopoietic cell fate is useful when studying cell populations that are difficult to access in vivo and for characterizing the earliest genes involved in hematopoiesis, without having to deal with embryonic lethalities. The ES/OP9 co-culture system was originally designed to produce hematopoietic progeny, without the over production of macrophages, as the OP9 stromal cell line is derived from the calvaria of osteopetrosis mutant mice that lack functional M-CSF. The in vitro ES/OP9 co-culture system can be used in order to recapitulate early hematopoietic development. When cultured on OP9 stromal cells, ES cells differentiate into Flk-1+ hemangioblasts, hematopoietic progenitors, and finally mature, terminally differentiated lineages. The standard ES/OP9 co-culture protocol entails the placement of ES cells onto a confluent layer of OP9 cells; as well as, periodic replating steps in order to remove old, contaminating OP9 cells. Furthermore, current protocols involve evaluating only the hematopoietic cells found in suspension and are not optimized for evaluation of ES-derived progeny at each day of differentiation. However, with replating steps and the harvesting of only suspension cells one potentially misses a large portion of ES-derived progeny and developing hematopoietic cells. This issue becomes important to address when trying to characterize hematopoietic defects associated with knockout ES lines. Here we describe a modified ES/mStrawberry OP9 co-culture, which allows for the elimination of contaminating OP9 cells from downstream assays. This method allows for the complete evaluation of all ES-derived progeny at all days of co-culture, resulting in a hematopoietic differentiation pattern, which more directly corresponds to the hematopoietic differentiation pattern observed within the embryo.

mStrawberry OP9 cells allow for complete evaluation of all ES-derived progeny from co-culture.

The in vitro differentiation of ES cells towards a hematopoietic cell fate is useful when studying cell populations that are difficult to access in vivo ...

Biology

Osteo-Organoid-Derived Cells for Effective HSCT in Irradiated Mice

In Vivo Osteo-organoid Approach for Harvesting Therapeutic Hematopoietic Stem/Progenitor Cells

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an adequate source of HSPCs, we developed an in vivo osteo-organoid by implanting scaffolds loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) into an internal muscle pouch near the femur in mice. After 12 weeks of implantation, we retrieved the in vivo osteo-organoids and conducted flow cytometry analysis on HPSCs, revealing a significant presence of HSPC subsets within the in vivo osteo-organoids.
We then established a sublethal model of hematopoietic/immune system injury in mice through radiation and performed hematopoietic stem cell transplantation (HSCT) by injecting the extracted osteo-organoid-derived cells into the peripheral blood of radiated mice. The effect of hematopoietic recovery was evaluated through hematological, peripheral blood chimerism, and solid organ chimerism analyses. The results confirmed that in vivo osteo-organoid-derived cells can rapidly and efficiently reconstruct damaged peripheral and solid immune organs in irradiated mice. This approach holds potential as an alternative source of HSPCs for HSCT, offering benefits to a larger number of patients.

Here, we established in vivo osteo-organoids triggered by bone morphogenetic protein-2-loaded gelatin scaffolds to harvest therapeutic hematopoietic stem/progenitor cells for the reconstruction of a damaged hematopoietic and immune system. Overall, this approach can provide a promising cell source for cell therapies.

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

Application of Aorta-gonad-mesonephros Explant Culture System in Developmental Hematopoiesis

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