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 ...

Research

JoVE Journal

Developmental Biology

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, ...

Whole Retinal Explants from Chicken Embryos for Electroporation and Chemical Reagent Treatments

The retina is a good model for the developing central nervous system. The large size of the eye and most importantly the accessibility for experimental ...

Technique to Target Microinjection to the Developing Xenopus Kidney

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

An Efficient Method to Obtain Dedifferentiated Fat Cells

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used ...

In Ovo Electroporation in the Chicken Auditory Brainstem

In Ovo Electroporation in the Chicken Auditory Brainstem

Electroporation is a method that introduces genes of interest into biologically relevant organisms like the chicken embryo. It is long established that ...

Tracing Gene Expression Through Detection of &#946;-galactosidase Activity in Whole Mouse Embryos

Tracing Gene Expression Through Detection of ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²-galactosidase Activity in Whole Mouse Embryos

The Escherichia coli LacZ gene, encoding &#946;-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Fast and Efficient Expression of Multiple Proteins in Avian Embryos Using mRNA Electroporation

We report that mRNA electroporation permits fluorescent proteins to label cells in living quail embryos more quickly and broadly than DNA electroporation. ...

A Simplified Method for Generating Kidney Organoids from Human Pluripotent Stem Cells

Kidney organoids generated from hPSCs have provided an unlimited source of renal tissue. Human kidney organoids are an invaluable tool for studying kidney ...

A Simple and Effective Transplantation Device for Zebrafish Embryos

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex ...