Name: efficient vascularization of kidney organoids through intracelomic transplantation in chicken embryos
Uploaded: 2023-02-17T13:10:00
Description: Watch this Scientific Journal Video about Efficient Vascularization of Kidney Organoids through Intracelomic Transplantation in Chicken Embryos at JoVE.com

We thank George Galaris (LUMC, Leiden, the Netherlands) for his help with chicken embryo injection. We acknowledge the support of Saskia van der Wal-Maas (Department of Anatomy &#38; Embryology, LUMC, Leiden, the Netherlands), Conny van Munsteren (Department of Anatomy &#38; Embryology, LUMC, Leiden, the Netherlands), Manon Zuurmond (LUMC, Leiden, the Netherlands), and Annemarie de Graaf (LUMC, Leiden, the Netherlands). M. Koning is supported by &#39;Nephrosearch Stichting tot steun van het wetenschappelijk onderzoek van de afdeling Nierziekten van het LUMC&#39;. This work was in part supported by the Leiden University Fund &#34;Prof. Jaap de Graeff-Lingling Wiyadhanrma Fund&#34; GWF2019-02. This work is supported by the partners of Regenerative Medicine Crossing Borders (RegMedXB) and Health Holland, Top Sector Life Sciences &#38; Health. C.W. van den Berg and T.J. Rabelink are supported by The Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW), The Novo Nordisk Foundation Center for Stem Cell Medicine is supported by Novo Nordisk Foundation grants (NNF21CC0073729).

In accordance with Dutch law, approval by the animal welfare committee was not required for this research.
1. Preparing hiPSC-derived kidney organoids for transplantation
<ol>
	<li>Differentiate hiPSCs to kidney organoids using the protocol developed by Takasato et al.4,18,31. Culture the organoids following this protocol on polyester cell culture inserts with 0.4 &#181;m pores (cel...

<ol>
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In this manuscript, a protocol for intracelomic transplantation of hiPSC-derived kidney organoids in chicken embryos is demonstrated. Upon transplantation, organoids are vascularized by perfused blood vessels that consist of a combination of human organoid-derived and chicken-derived ECs. These are spread throughout the organoid and invade the glomerular structures, enabling interaction between the ECs and podocytes. It was previously shown that this leads to enhanced maturation of the organoid glomerular and tubular str...

Human induced pluripotent stem cell (hiPSC)-derived kidney organoids have been shown to have potential for developmental studies1,2,3,4, toxicity screening5,6, and disease modeling5,7,8,9,10,11,12,13. However, their applicability for these and eventual clinical transplantation purposes is limited by the lack of a vascular network. During embryonic kidney development, podocytes, mesangial cells, and vascular endothelial cells (ECs) interact to form the intricate structure of the glomerulus. Without this interaction, the glomerular filtration barrier, consisting of podocytes, the glomerular basement membrane (GBM), and ECs, cannot develop properly14,15,16. Although kidney organoids in vitro do contain some ECs, these fail to form a proper vascular network and diminish over time17. It is therefore not surprising that the organoids remain immature. Transplantation in mice induces vascularization and maturation of the kidney organoids18,19,20,21. Unfortunately, this is a labor-intensive process that is unsuitable for the analysis of large numbers of organoids.
Chicken embryos have been used to study vascularization and development for over a century22. They are easily accessible, require low maintenance, lack a fully functional immune system, and can develop normally after opening the eggshell23,24,25,26. The transplantation of organoids on their chorioallantoic membrane (CAM) has been shown to lead to vascularization27. However, the duration of transplantation on the CAM, as well as the level of maturation of the graft, are limited by CAM formation, which takes until embryonic day 7 to complete. Therefore, a method was recently developed to efficiently vascularize and mature kidney organoids through intracelomic transplantation in chicken embryos28. The celomic cavity of chicken embryos has been known since the 1930s to be a favorable environment for the differentiation of embryonic tissues29,30. It can be accessed early in embryonic development and allows for relatively unlimited expansion of the graft in all directions.
This paper outlines a protocol for the transplantation of hiPSC-derived kidney organoids in the celomic cavity of day 4 chicken embryos. This method induces vascularization and enhanced maturation of the organoids within 8 days. Injection of fluorescently labeled lens culinaris agglutinin (LCA) prior to sacrificing the embryos enables visualization of perfused blood vessels within the organoids through confocal microscopy.

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	<tbody>
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			<td>0.2 &#181;m filter: Whatman Puradisc 30 syringe filter 0.2 &#181;m</td>
			<td>Whatman</td>
			<td>10462200</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm glass bottom dishes&#160;</td>
			<td>MatTek Corporation</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Aspirator tube assemblies for calibrated microcapillary pipettes</td>
			<td>Sigma-Aldrich</td>
			<td>A5177-5EA</td>
			<td>Contains silicone tubes, mouth piece and connector</td>
		</tr>
		<tr>
			<td>Confocal microscope: Leica White Light Laser Confocal Microscope&#160;</td>
			<td>Leica</td>
			<td>TCS SP8</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting forceps, simple type. Titanium, curved, with fine sharp tips</td>
			<td>Hammacher Karl</td>
			<td>HAMMHTC091-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting forceps, simple type. Titanium, straight, with fine sharp tips</td>
			<td>Hammacher Karl</td>
			<td>HAMMHTC090-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope&#160;</td>
			<td>Wild Heerbrugg</td>
			<td>355110</td>
			<td></td>
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			<td>Dissecting scissors, curved, OP-special, extra sharp/sharp</td>
			<td>Hammacher Karl</td>
			<td>HAMMHSB391-10</td>
			<td></td>
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		<tr>
			<td>Donkey serum</td>
			<td>Sigma-Aldrich</td>
			<td>D9663</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey-&#945;-mouse Alexa Fluor 488</td>
			<td>ThermoFisher Scientific</td>
			<td>A-212-02</td>
			<td>dilution 1:500</td>
		</tr>
		<tr>
			<td>Donkey-&#945;-sheep Alexa Fluor 647</td>
			<td>ThermoFisher Scientific</td>
			<td>A-21448</td>
			<td>dilution 1:500</td>
		</tr>
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			<td>Double edged stainless steel razor blades</td>
			<td>Electron Microsopy Sciences</td>
			<td>72000</td>
			<td></td>
		</tr>
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			<td>DPBS, calcium, magnesium (DPBS-/-)</td>
			<td>ThermoFisher Scientific</td>
			<td>14040133</td>
			<td></td>
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		<tr>
			<td>DPBS, no calcium, no magnesium (DPBS+/+)</td>
			<td>ThermoFisher Scientific</td>
			<td>14190094</td>
			<td></td>
		</tr>
		<tr>
			<td>Egg cartons or custom made egg holders&#160;</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Fertilized white leghorn eggs (Gallus Gallus Domesticus)&#160;</td>
			<td>Drost Loosdrecht B.V.</td>
			<td>NA</td>
			<td></td>
		</tr>
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			<td>Incubator</td>
			<td>Elbanton BV</td>
			<td>ET-3 combi</td>
			<td></td>
		</tr>
		<tr>
			<td>Lotus Tetragonolobus lectin (LTL) Biotinylated</td>
			<td>Vector Laboratories</td>
			<td>B-1325</td>
			<td>dilution 1:300</td>
		</tr>
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			<td>Micro scissors, straight, sharp/sharp, cutting length 10 mm</td>
			<td>Hammacher Karl</td>
			<td>HAMMHSB500-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcapillaries: Thin wall glass capillaries 1.5 mm, filament</td>
			<td>World Precision Instruments</td>
			<td>TW150F-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instrument Company</td>
			<td>Model P-97</td>
			<td>We use the following settings: Heat 533, Pull 60, Velocity 150, Time 200</td>
		</tr>
		<tr>
			<td>Microscalpel holder: Castroviejo blade and pins holder, 12 cm, round handle, conical 10 mm jaws.</td>
			<td>Euronexia</td>
			<td>L-120</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting medium: Prolong Gold Antifade Mountant&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>Olivecrona dura dissector 18 cm&#160;</td>
			<td>Reda</td>
			<td>41146-18</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm&#160;</td>
			<td>Heathrow Scientific</td>
			<td>HS234526B</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin 5,000 U/mL</td>
			<td>ThermoFisher Scientific</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr>
			<td>Perforated spoon&#160;</td>
			<td>Euronexia</td>
			<td>S-20-P</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 60 x 15 mm&#160;</td>
			<td>CELLSTAR</td>
			<td>628160</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic transfer pipettes&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>PP89SB</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified mouse anti-human CD31 antibody</td>
			<td>BD Biosciences</td>
			<td>555444</td>
			<td>dilution 1:100</td>
		</tr>
		<tr>
			<td>Rhodamine labeled Lens Culinaris Agglutinin (LCA)</td>
			<td>Vector Laboratories</td>
			<td>RL-1042</td>
			<td>This product has recently been discontinued. Vectorlabs does still produce Dylight 649 labeled LCA (DL-1048-1) and fluorescein labeled LCA (FL-1041-5)</td>
		</tr>
		<tr>
			<td>Sheep anti-human NPHS1 antibody</td>
			<td>R&#38;D systems</td>
			<td>AF4269</td>
			<td>dilution 1:100</td>
		</tr>
		<tr>
			<td>Sterile hypodermic needles, 19 G</td>
			<td>BD microlance</td>
			<td>301500</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin Alexa Fluor 405</td>
			<td>ThermoFisher Scientific</td>
			<td>S32351</td>
			<td>dilution 1:200</td>
		</tr>
		<tr>
			<td>Syringe 5 mL</td>
			<td>BD Emerald</td>
			<td>307731</td>
			<td></td>
		</tr>
		<tr>
			<td>Transparent tape&#160;</td>
			<td>Tesa</td>
			<td>4124</td>
			<td>Available at most hardware stores</td>
		</tr>
		<tr>
			<td>Triton X</td>
			<td>Sigma-Aldrich</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten wire, 0.25 mm dia&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>010404.H2</td>
			<td></td>
		</tr>
	</tbody>
</table>

efficient vascularization of kidney organoids through intracelomic transplantation in chicken embryos

Kidney organoids derived from human induced pluripotent stem cells contain nephron-like structures that resemble those in the adult kidney to a certain degree. Unfortunately, their clinical applicability is hampered by the lack of a functional vasculature and consequently limited maturation in vitro. The transplantation of kidney organoids in the celomic cavity of chicken embryos induces vascularization by perfused blood vessels, including the formation of glomerular capillaries, and enhances their maturation. This technique is very efficient, allowing for the transplantation and analysis of large numbers of organoids. This paper describes a detailed protocol for the intracelomic transplantation of kidney organoids in chicken embryos, followed by the injection of fluorescently labeled lectin to stain the perfused vasculature, and the collection of transplanted organoids for imaging analysis. This method can be used to induce and study organoid vascularization and maturation to find clues for enhancing these processes in vitro and improve disease modeling.

The method and timeline for the differentiation of hiPSCs to kidney organoids, incubation of fertilized chicken eggs, transplantation of kidney organoids, injection of LCA, and collection of the organoids are summarized in Figure 1A. It is important to coordinate the timing of organoid differentiation and chicken egg incubation, starting differentiation 15 days before incubation.&#160;The actions on day 0, 3, 4, and 12 of incubation are illustrated by photographs below the timeline. Organoid...

Watch this Scientific Journal Video about Efficient Vascularization of Kidney Organoids through Intracelomic Transplantation in Chicken Embryos at JoVE.com

Efficient Vascularization of Kidney Organoids through Intracelomic Transplantation in Chicken Embryos

Here, we present a detailed protocol for the transplantation of kidney organoids in the celomic cavity of chicken embryos. This method induces vascularization and enhanced maturation of the organoids within 8 days and can be used to study these processes in an efficient manner.

Kidney organoids derived from human induced pluripotent stem cells contain nephron-like structures that resemble those in the adult kidney to a certain ...

Kidney organoids cultured in vitro remain immature, limiting their applicability. And this method of intracoelomic transplantation induces vascularization and enhances maturation of the organoids. The procedure is very efficient, allowing for the transplantation and analysis of large number of organoids in one experiment.After preparing HIPSC-derived kidney organoids and incubating the fertilized leghorn eggs create a window in the eggshell on day three of incubation. Place a small piece of transparent tape on the pointed tip of the egg. Make a small hole in the eggshell in the middle of the transparent tape by tapping it with the sharp end of a pair of dissecting scissors.Next, insert a 19-gauge needle on a five-milliliter syringe into the hole at a 45-degree angle. Aspirate two to three milliliters of albumin from the egg to lower the embryo inside the egg. Conceal the hole with a second piece of transparent tape, then place a large piece of transparent tape on the pencil marked upward facing side of the egg.Make a hole in the eggshell in the middle of the transparent tape by tapping it with the sharp end of a pair of dissecting scissors. Cut a small circular window in the eggshell from this hole using curved dissecting scissors. Look through this window to locate the embryo.Then, enlarge the window to optimize access to the embryo. Remove any large pieces of eggshell that may have fallen on top of the embryo using forceps. Remove smaller pieces by placing a few drops of DPBS with calcium and magnesium on the embryo with a plastic transfer pipette, then aspirating the DPBS with the eggshell into the pipette.Using a plastic transfer pipette, add three drops of DPBS, supplemented with 0.5%penicillin streptomycin to the egg. Carefully seal the window with a large piece of transparent tape before placing the egg back in the incubator until day four of incubation. Cut the tape from the window with curved dissecting scissors.Place the egg under a dissecting microscope on a rubber holder or egg carton. The chicken embryo is now in Hamburger-Hamilton Stage 23 to 24 and lying on its left side with its right side facing the viewer. Locate the right wing and leg bud of the embryo as the coelom will be assessed between these two limb buds.In the area between the right wing and leg bud, create an opening consecutively in the vitelline membrane, the chorion, and the amnion by holding them with two pairs of dissecting forceps and gently pulling them in opposite directions. Check whether there's unobstructed access to the coelomic cavity. Gently take hold of the edge of the body wall between the wing and limb bud with a pair of dissecting forceps and pull it slightly toward the viewer.The coelomic cavity must be clearly visible. Carefully insert a blunt but slim instrument into the coelomic cavity. Place half an organoid inside the egg on top of the allantois using a dura dissector.Using dissecting forceps, carefully take hold of the edge of the body wall and pull it slightly toward the viewer to make the opening to the coelum visible. Gently move the organoid toward and through the opening in the body wall into the coelum with a blunt tungsten wire and a micro scalpel holder. Push the organoid slightly cranially to lodge it inside the coelum.It is now visible just behind the wing bud. Add three drops of DPBS to the egg using a plastic transfer pipette. Carefully seal the window with a large piece of transparent tape before placing the egg back in the incubator until day 12 of incubation.Cut the tape from the window with curved dissecting scissors. Place the egg under a dissecting microscope in a rubber holder. Evaluate the vasculature and locate the veins, which can be distinguished from the arteries by their slightly brighter red color.To improve access to the vasculature, carefully enlarge the window by cutting it with curved dissecting scissors. After selecting the vein, insert the tip of the micro capillary needle into the vein at a zero to 20-degree angle. Ensure the needle is in the vein by gently moving the tip from side to side.Gently and steadily blow into the injection system to inject the lens culinaris agglutinin. And place the egg back in the incubator for 10 minutes to let it circulate. Place the egg in a rubber holder on the bench.Then, cut through the membranes surrounding the embryo with curved dissecting scissors. Scoop the embryo up from the egg with a perforated spoon and immediately decapitate the embryo using scissors. Under the dissection microscope, place the body of the embryo in a Petri dish.Place the embryo on its back in the Petri dish and spread its limbs. Carefully open the abdominal wall of the embryo along the longitudinal axis using forceps. Locate the organoid inside the embryo.The organoid most frequently becomes attached to the right liver lobe, either at the coddle tip or cranially, just below the rib cage. So, it's advisable to start by looking at these locations. Once the organoid is located, remove it from the embryo by cutting around it with micro scissors.Place the organoid in the chicken tissue that is inevitably attached to it in a Petri dish under the dissecting microscope. Remove as much chicken tissue as possible with a double-edged stainless steel razor blade. Immunofluorescent images of an non-transplanted kidney organoid and a transplanted kidney organoid both contain glomerular and tubular structures.In the untransplanted organoids, some human endothelial cells are present. In the transplanted organoid, a perfused vascular network is visible throughout the organoid. Magnified images of the boxed areas demonstrate the three types of endothelial cells that can be distinguished in transplanted organoids:perfused human endothelial cells, unperfused human endothelial cells, and perfused chicken-derived endothelial cells.In untransplanted organoids, endothelial cells surround glomerular structures but do not invade them. However, in transplanted organoids, glomerular structures are vascularized by perfused capillaries. Transplanted organoids can be analyzed using different techniques depending on the aim of the experiment.Direct comparison to untransplanted controls can provide insight into the process of vascularization and maturation.

Research

JoVE Journal

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