Name: application of aorta-gonad-mesonephros explant culture system in developmental hematopoiesis
Uploaded: 2017-11-03T13:00:00
Description: Watch this Scientific Journal Video about Application of Aorta-gonad-mesonephros Explant Culture System in Developmental Hematopoiesis at JoVE.com

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 ...

<ol>
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C., Robin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+founder+of+adult+hematopoiesis+in+the+mouse+embryo+aorta.">Imaging the founder of adult hematopoiesis in the mouse embryo aorta.</a> Cell cycle. 9, 2489-2490 (2010).</li><li>Kissa, K., Herbomel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+stem+cells+emerge+from+aortic+endothelium+by+a+novel+type+of+cell+transition.">Blood stem cells emerge from aortic endothelium by a novel type of cell transition.</a> Nature. 464, 112-115 (2010).</li><li>Zovein, A. 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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Q., Liu, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-hydroxytryptamine+synthesized+in+the+aorta-gonad-mesonephros+regulates+hematopoietic+stem+and+progenitor+cell+survival.">5-hydroxytryptamine synthesized in the aorta-gonad-mesonephros regulates hematopoietic stem and progenitor cell survival.</a> J Exp Med. 214, 529-545 (2017).</li><li>Souilhol, 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=Inductive+interactions+mediated+by+interplay+of+asymmetric+signalling+underlie+development+of+adult+haematopoietic+stem+cells.">Inductive interactions mediated by interplay of asymmetric signalling underlie development of adult haematopoietic stem cells.</a> Nature communications. 7, 10784 (2016).</li><li>Fitch, S. 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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 style="border-collapse:collapse;width:458pt" width="610">
	<colgroup>
		<col style="width:162pt" width="216" />
		<col style="width:81pt" width="108" />
		<col style="width:89pt" width="119" />
		<col style="width:125pt" width="167" />
	</colgroup>
	<tbody>
		<tr height="21" style="height:15.75pt">
			<td class="xl67" height="21" style="width:162pt;height:15.75pt" width="216">durapore 0.65 &#181;m filters</td>
			<td class="xl68" style="width:81pt" width="108">Millipore</td>
			<td class="xl69" style="width:89pt" width="119">R5BA63787</td>
			<td class="xl67" style="width:125pt" width="167">AGM explant culture</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl67" height="42" style="height:31.5pt">M5300 long-term culture medium</td>
			<td class="xl68" style="width:81pt" width="108">Stem Cell Technologies</td>
			<td class="xl69" style="width:89pt" width="119">5300</td>
			<td class="xl67">AGM explant culture</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl68" height="21" style="width:162pt;height:15.75pt" width="216">Trizol&#160;</td>
			<td class="xl68" style="width:81pt" width="108">Tiangen</td>
			<td class="xl69" style="width:89pt" width="119">5829</td>
			<td class="xl67">RNA extration</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl68" height="21" style="width:162pt;height:15.75pt" width="216">M-MLV reverse transcriptase</td>
			<td class="xl68" style="width:81pt" width="108">Promega</td>
			<td class="xl69" style="width:89pt" width="119">90694</td>
			<td class="xl67">reverse transcription</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl68" height="21" style="width:162pt;height:15.75pt" width="216">SYBR Green</td>
			<td class="xl68" style="width:81pt" width="108">Qiagen</td>
			<td class="xl69" style="width:89pt" width="119">A6002</td>
			<td class="xl67">quantatitive real-time PCR</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl68" height="21" style="width:162pt;height:15.75pt" width="216">protease inhibitor</td>
			<td class="xl68" style="width:81pt" width="108">Roche</td>
			<td class="xl69" style="width:89pt" width="119">55622</td>
			<td class="xl67">Protein extration</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl68" height="21" style="width:162pt;height:15.75pt" width="216">collagenase</td>
			<td class="xl68" style="width:81pt" width="108">Sigma</td>
			<td class="xl69" style="width:89pt" width="119">C2674</td>
			<td class="xl67">digestion of AGM tissues</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl68" height="42" style="width:162pt;height:31.5pt" width="216">MethoCult GF M3434 medium</td>
			<td class="xl68" style="width:81pt" width="108">Stem Cell Technologies</td>
			<td class="xl69" style="width:89pt" width="119">3434</td>
			<td class="xl67">CFU-C assay</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl68" height="42" style="width:162pt;height:31.5pt" width="216">ultra-low attachment 24-well plates</td>
			<td class="xl68" style="width:81pt" width="108">Costar</td>
			<td class="xl69" style="width:89pt" width="119">3473</td>
			<td class="xl67">CFU-C assay</td>
		</tr>
		<tr height="63" style="height:47.25pt">
			<td class="xl67" height="63" style="height:47.25pt">C57BL/6 CD45.2 mice</td>
			<td class="xl68" style="width:81pt" width="108">Beijing HFK Bioscience Co. Ltd</td>
			<td class="xl68" style="width:89pt" width="119"></td>
			<td class="xl67">CFU-S assay</td>
		</tr>
		<tr height="63" style="height:47.25pt">
			<td class="xl68" height="63" style="width:162pt;height:47.25pt" width="216">C57BL/6 CD45.1 mice</td>
			<td class="xl68" style="width:81pt" width="108">Beijing HFK Bioscience Co. Ltd</td>
			<td class="xl68" style="width:89pt" width="119"></td>
			<td class="xl67">Long-term transplantation</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl68" height="42" style="width:162pt;height:31.5pt" width="216">phosphate buffered saline</td>
			<td class="xl68" style="width:81pt" width="108">Life Technologines</td>
			<td class="xl69" style="width:89pt" width="119">8115284</td>
			<td class="xl68" style="width:125pt" width="167">AGM Collection</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl68" height="21" style="width:162pt;height:15.75pt" width="216">Penicillin-Streptomycin solution</td>
			<td class="xl68" style="width:81pt" width="108">HyClone</td>
			<td class="xl68" style="width:89pt" width="119">SV30010</td>
			<td class="xl68" style="width:125pt" width="167">AGM explant culture</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl68" height="42" style="width:162pt;height:31.5pt" width="216">anti-CD45.2-PE-CY7</td>
			<td class="xl68" style="width:81pt" width="108">eBioscience</td>
			<td class="xl68" style="width:89pt" width="119">25-0454-80</td>
			<td class="xl68" style="width:125pt" width="167">Long-term transplantation</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl68" height="42" style="width:162pt;height:31.5pt" width="216">anti-CD45-FITC</td>
			<td class="xl68" style="width:81pt" width="108">eBioscience</td>
			<td class="xl68" style="width:89pt" width="119">11-0451-81</td>
			<td class="xl68" style="width:125pt" width="167">Long-term transplantation</td>
		</tr>
		<tr height="105" style="height:78.75pt">
			<td class="xl68" height="105" style="width:162pt;height:78.75pt" width="216">UV lamp</td>
			<td class="xl68" style="width:81pt" width="108">Beijing jiangmorning yuan bio-technology co., LTD</td>
			<td class="xl68" style="width:89pt" width="119">039-14402</td>
			<td class="xl68" style="width:125pt" width="167">power: 30W</td>
		</tr>
		<tr height="63" style="height:47.25pt">
			<td class="xl68" height="63" style="width:162pt;height:47.25pt" width="216">stainless steel mesh</td>
			<td class="xl68" style="width:81pt" width="108">AS ONE SHANGHAI Corporation</td>
			<td class="xl68" style="width:89pt" width="119">2-9817-10&#160;</td>
			<td class="xl68" style="width:125pt" width="167">aperture diameter: 2.5 mm</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl68" height="42" style="width:162pt;height:31.5pt" width="216">hydrocortisone</td>
			<td class="xl68" style="width:81pt" width="108">Sigma</td>
			<td class="xl68" style="width:89pt" width="119">H0396</td>
			<td class="xl68" style="width:125pt" width="167">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 ...

The overall goal of this experiment is to use an aorta-gonad-mesonephros explant culture to investigate the critical regulators of hematopoietic stem cell development. This method can help answer key questions in HSC biology and function fields about how to identify the factors involved in the regulation of HSC development in mammals. The main advantage of this technique is that these regulators can be verified through DNA expression analysis and the colony formation ability and the reconstitution capacity of the HSC.Generally individuals new to this method will struggle because the precise separation of AGM from the surrounding tissues in the trunk region requires practice to master. Before beginning the procedure place stainless steel meshes into individual wells of a six-well plate containing two milliliters of freshly prepared fluoxetine-supplemented medium per well. Place one UV-sterilized 0.65 microliter filter per experimental well into a glass cell culture dish containing fresh boiling water for two three-minute washes, immersing the filters in a container of room temperature PBS for two minutes after each wash.Then place one filter onto each steel mesh at the air-liquid interface of each well. Next use the dissecting scissors to strip the uterus from an embryonic day-11 pregnant mouse and harvest the embryos from the uterus. Using dissecting scissors remove the yolk sac, umbilical cord and viscera from the embryo body.And carefully remove the dorsal nerve tissues and surrounding muscular tissues. Using forceps remove the tissues from the anterior and hind limbs. And separate the AGM region from the embryo body.Then place each AGM onto a single filter at the air-liquid interface of each well. Place the cultures in a 37 degree Celsius and 5%CO2 incubator. After two to three days, transfer the AGM samples into individual 1.5-milliliter microcentrifuge tubes containing 200 microliters of collagenase for their dissociation at 37 degrees Celsius for about 20 minutes with shaking every five minutes.At the end of the digestion stop the reactions with 200 microlilters of serum per explant. Transfer the entire contents of each tube into individual wells of a 24-well ultra-low attachment plate containing 500 to 600 microliters of M3434 medium per well. Mix the cells in each well with a one milliliter syringe without a needle.Place the plate in the cell culture incubator. The number of colony-forming units within each well can then be scored after seven to 10 days of culture. For in vivo AGM explant transplantation after two to three days of AGM culture as just demonstrated, lethally eradiate eight to 10-week-old male C57 black six mice with nine grays of radiation.Four to five hours later dissociate the harvested explants in 200 microliters of collagenase per samples as demonstrated. Load of 0.5 to one embryo equivalent of the resulting single-cell suspensions into one one-milliliter syringe per recipient animal. Place the first irradiated mouse into a restrainer and wipe the tail with a 75%ethanol-soaked cotton swab to promote tail vein vasodilation.Intravenously inject the entire contents of one syringe into a dilated tail vein. Use an antiseptic swab to stop the bleeding after removal of the needle. Then return the animal to its cage, and inject the next animal.11 days after the adoptive transfer the spleens can be fixed in Bouin's solution for macroscopic enumeration of the visible spleen colonies. Fluoxetine-supplemented AGM cultures demonstrate an increased expression of Runx1, a pivotal gene for HSC development at the mRNA and protein expression levels. Fluoxetine can also significantly increase the colony formation ability of HSC in the AGM including burst-forming unit erythroids, colony-forming unit granular monocytes and colony-forming unit granulocytes, erythrocytes, macrophages and megakaryocytes.Further in vivo transplantation of fluoxetine-treated HSC isolated from AGM explants results in increased numbers of colonies in the spleens of irradiated adult recipient animals. After watching this video, you should have a good understanding of how to isolate the AGM and to perform an AGM explant culture for the identification of the critical regulators of HSC development within the AGM.

Research

JoVE Journal

Developmental Biology

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Vertebrate embryos pattern their major body axis as repetitive somites, the precursors of vertebrae, muscle, and skin. Somites progressively segment from ...