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., 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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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 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 ...

application-aorta-gonad-mesonephros-explant-culture-system

Research

JoVE Journal

Developmental Biology

8.3K Views.  Chinese Academy of Sciences. 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.

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

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations.

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human developmental processes. Stepwise directed differentiation that recapitulates developmental processes is one of the major ways to generate pancreatic cells including pancreas/duodenum homeobox protein 1+ (PDX1+) pancreatic progenitor cells. Conventional protocols initiate the differentiation with small colonies shortly after the passage. However, in the state of colonies or aggregates, cells are prone to heterogeneities, which might hamper the differentiation to PDX1+ cells. Here, we present a detailed protocol to differentiate hPSCs into PDX1+ cells. The protocol consists of four steps and initiates the differentiation by seeding dissociated single cells. The induction of SOX17+ definitive endoderm cells was followed by the expression of two primitive gut tube markers, HNF1&#946; and HNF4&#945;, and eventual differentiation into PDX1+ cells. The present protocol provides easy handling and may improve and stabilize the differentiation efficiency of some hPSC lines that were previously found to differentiate inefficiently into endodermal lineages or PDX1+ cells.

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Here, we present a detailed protocol to differentiate human pluripotent stem cells (hPSCs) into pancreas/duodenum homeobox protein 1+ (PDX1+) cells for the generation of pancreatic lineages based on the non-colony type monolayer growth of dissociated single cells. This method is suitable for producing homogenous hPSC-derived cells, genetic manipulation and screening.

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous transplants to treat hematological and malignant disorders. Hemogenic endothelium (HE) is an amenable platform to produce functional HSPCs by transcription factors or to induce lymphocytes in a robust manner. However, current methods to derive HE are either costly or variable in yield. In this protocol, we established a cost-effective and precise method to derive HE within 4 days in a feeder- and xeno-free defined condition. The resultant HE underwent an endothelial-to-hematopoietic transition and produced CD34+CD45+ clonogenic hematopoietic progenitors. The potential capacity of our platform for generating functional HSPCs will have broad applications in disease modeling and screening for therapeutic compounds.

Here, we present a protocol to precisely form hemogenic endothelium from human pluripotent stem cells in a feeder- and xeno-free condition. This method will have broad applications in disease modeling and screening for therapeutic compounds.

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous ...

In vitro Neuromuscular Junction Induced from Human Induced Pluripotent Stem Cells

The neuromuscular junction (NMJ) is a specialized synapse that transmits action potentials from the motor neuron to skeletal muscle for mechanical movement. The architecture of the NMJ structure influences the functions of the neuron, the muscle and the mutual interaction. Previous studies have reported many strategies by co-culturing the motor neurons and myotubes to generate NMJ in vitro with complex induction process and long culture period but have struggled to recapitulate mature NMJ morphology and function. Our in vitro NMJ induction system is constructed by differentiating human iPSC in a single culture dish. By switching the myogenic and neurogenic induction medium for induction, the resulting NMJ contained pre- and post- synaptic components, including motor neurons, skeletal muscle and Schwann cells in the one month culture. The functional assay of NMJ also showed that the myotubes contraction can be triggered by Ca++ then inhibited by curare, an acetylcholine receptor (AChR) inhibitor, in which the stimulating signal is transmitted through NMJ. This simple and robust approach successfully derived the complex structure of NMJ with functional connectivity. This in vitro human NMJ, with its integrated structures and function, has promising potential for studying pathological mechanisms and compound screening.

Here we provide a protocol to generate in vitro NMJs from human induced pluripotent stem cells (iPSCs). This method can induce NMJs with mature morphology and function in 1 month in a single well. The resulting NMJs could potentially be used to model related diseases, to study pathological mechanisms or to screen drug compounds for therapy.

The neuromuscular junction (NMJ) is a specialized synapse that transmits action potentials from the motor neuron to skeletal muscle for mechanical ...

A 3-D Tail Explant Culture to Study Vertebrate Segmentation in Zebrafish

Vertebrate embryos pattern their major body axis as repetitive somites, the precursors of vertebrae, muscle, and skin. Somites progressively segment from the presomitic mesoderm (PSM) as the tail end of the embryo elongates posteriorly. Somites form with regular periodicity and scale in size. Zebrafish is a popular model organism as it is genetically tractable and has transparent embryos that allow for live imaging. Nevertheless, during somitogenesis, fish embryos are wrapped around a large, rounding yolk. This geometry limits live imaging of PSM tissue in zebrafish embryos, particularly at higher resolutions that require a close objective working distance. Here, we present a flattened 3-D tissue culture method for live imaging of zebrafish tail explants. Tail explants mimic intact embryos by displaying a proportional slowdown of axis elongation and shortening of rostrocaudal somite lengths. We are further able to stall axis elongation speed through explant culture. This, for the first time, enables us to untangle the chemical input of signaling gradients from the mechanistic input of axial elongation. In future studies, this method can be combined with a microfluidic setup to allow time-controlled pharmaceutical perturbations or screening of vertebrate segmentation without any drug penetration concerns.

Here, we present the protocol for 3-D tissue culture of the zebrafish posterior body axis, enabling live study of vertebrate segmentation. This explant model provides control over axis elongation, alteration of morphogen sources, and subcellular resolution tissue-level live imaging.

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

Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human cancers. As a lining tissue, the skin epithelium is often subject to injury and has evolutionarily adapted by acquiring the cellular plasticity necessary to repair damaged tissue. Over the years, several efforts have been made to study epithelial plasticity using in&#160;vitro and ex vivo cell-based models. However, these efforts have been limited in their capacity to recapitulate the various phases of epithelial cell plasticity. We describe here a protocol for generating 3D epidermal spheroids and epidermal spheroid-derived cells from primary neonatal human keratinocytes. This protocol outlines the capacity of epidermal spheroid cultures to functionally model distinct stages of keratinocyte generative plasticity and demonstrates that epidermal spheroid re-plating can enrich heterogenous normal human keratinocytes (NHKc) cultures for integrin&#945;6hi/EGFRlo keratinocyte subpopulations with enhanced stem-like characteristics. Our report describes the development and maintenance of a high throughput system for the study of skin keratinocyte plasticity and epidermal regeneration.

Here we describe a protocol for the systematic cultivation of epidermal spheroids in 3D suspension culture. This protocol has wide-ranging applications for use in a variety of epithelial tissue types and for the modeling of several human diseases and conditions.

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human ...