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 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.
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.
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
2. Preparing chicken embryos for transplantation
3. Intracelomic transplantation on day 4 of incubation
4. Injection of fluorescently labeled lectin
5. Collecting transplanted organoids on day 12 of incubation
6. Whole-mount immunofluorescence staining
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. The actions on day 0, 3, 4, and 12 of incubation are illustrated by photographs below the timeline. Organoids are transplanted at day 7 + 12 of differentiation into day 4 (HH 23-24) chicken embryos. LCA is injected into the venous system of the embryo 8 days after transplantation to stain the perfused vasculature, before sacrificing the embryo and retrieving the organoid. The assembled injection system is shown in Figure 1B.
Chicken embryos are sacrificed on day 12 of incubation (Figure 1A). Upon careful dissection of the embryo, the transplanted organoid can usually be found attached to the liver. Looking through a dissection microscope, it appears vascularized (Figure 1A; day 12; step 5.2.3). Confocal imaging of transplanted organoids injected with LCA and stained for nephron structures and human ECs confirms vascularization by perfused blood vessels (Figure 2A), which also invade glomerular structures (Figure 2B). The vasculature is chimeric: perfused human ECs (CD31+, LCA+), unperfused human ECs (CD31+, LCA-), and perfused chicken-derived ECs (CD31-, LCA+) can be distinguished (Figure 2A, panels III, IV, V).
Figure 1: Intracelomic transplantation method. (A) Timeline of the differentiation of hiPSCs to kidney organoids, incubation of fertilized chicken eggs, and intracelomic transplantation of kidney organoids. Differentiation of hiPSCs to kidney organoids is initiated 15 days before incubation of the chicken eggs is started (differentiation day 0 = incubation day -15) to enable transplantation of the kidney organoids on day 7 + 12 of differentiation in chicken embryos on day 4 of incubation. Days of incubation: Day 0: Fertilized chicken eggs are positioned horizontally on holders (step 2.1.1), which are placed in an incubator at 38 ºC ± 1 °C (protocol step 2.1.2). Day 3: A window is created in each egg by making a small hole in the upward-facing side of the egg with the sharp end of a pair of curved dissecting scissors (step 2.2.3) and cutting a circular window starting from this hole (step 2.2.4). The window is sealed with transparent tape before placing the egg back in the incubator. Day 4: Intracelomic transplantation of kidney organoids on day 7 + 12 of differentiation is performed. The embryo is in HH 23-24 and lying on its left side, with its right side facing the viewer (step 3.1.3). Between the wing and leg bud, an opening is made in the vitelline membrane, chorion, and amnion to obtain access to the celomic cavity, and the organoid is inserted through these openings into the celom. After transplantation, the organoid is visible as a white structure located just behind the wing bud. The edges of the opened vitelline and amnion membranes are visible (steps 3.2.3 and 3.2.3-Zoom). In some cases, the embryos are rotated, lying on their right instead of left side (3.1.3 NOTE), and must be turned around prior to transplantation. Day 12: Fluorescently labeled lectin is injected intravenously. Veins are distinguished from arteries by their color; the blood in the veins is oxygen-rich, coming from the CAM, so they are a slightly brighter red than the arteries that are coming from the embryo (steps 4.2.2., 4.2.2-Zoom, and 4.2.3.). The embryo is sacrificed and the organoid retrieved. The organoid (circled) has become attached to the chicken liver and appears to be vascularized (step 5.2.3). Scale bar = 1 mm. The schematic image depicting the intracelomic transplantation image in the top panel was reprinted with permission from Koning et al.28. (B) Image of the assembled injection system, consisting of a mouthpiece, two pieces of 38 cm silicone tubing, a 0.2 µm filter, a connector, and a glass microinjection needle that was generated by pulling glass microcapillaries in a micropipette puller. Abbreviations: A = allantois; Am = amnion; C = celom; CC = cut chorion membrane; CHIR = CHIR99021; FGF9 = fibroblast growth factor 9; hiPSCs = human induced pluripotent stem cells; IM = intermediate mesoderm; Lb = leg bud; O = organoid; PS = primitive streak; U = umbilical ring; V = vitelline membrane; Wb = wing bud; Y = yolk stalk. Please click here to view a larger version of this figure.
Figure 2: Vascularized transplanted kidney organoids. (A) Immunofluorescent images of (I) an untransplanted kidney organoid and (II) a transplanted kidney organoid. In both conditions, glomerular (NPHS1+, cyan) and tubular (LTL+, yellow) structures are visible. In the untransplanted organoids, some human ECs (CD31+, green) are present. In the transplanted organoid, a perfused vascular network (CD31+, green; injected rhodamine-labeled LCA+, white) is visible throughout the organoid. In panels III, IV, and V, magnifications of the boxed areas in panel II are shown, to demonstrate the three types of ECs that can be distinguished in transplanted organoids. Panel III contains perfused human ECs (CD31+, LCA+), marked with arrowheads. Panel IV contains unperfused human ECs (CD31+, LCA-), marked with arrowheads. Panel V contains perfused chicken-derived ECs (CD31-, LCA+), marked with arrowheads. Scale bar = 200 µm. (B) In untransplanted organoids (I), ECs (CD31+, green) surround glomerular structures (NPHS1+, cyan) but do not invade them. In transplanted organoids (II), glomerular structures (NPHS1+, blue) are vascularized by perfused capillaries (LCA+, white; CD31+, green). Scale bar = 50 µm. Please click here to view a larger version of this figure.
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 structures28. The transplantation is very efficient, taking ~5 min per embryo, and the only maintenance that the embryos require is a regular refill of the water basin in the incubator. This method is therefore very suitable for the analysis of large numbers of organoids.
Vascularization and maturation of kidney organoids have been previously demonstrated through transplantation in mice18,20. In these labor-intensive mouse models, organoids were transplanted for 2 to up to 12 weeks. In the intracelomic transplantation experiments shown here, the duration of transplantation was limited to 8 days. This allows for the sacrifice of the embryos before day 13 of incubation, when they are thought to start experiencing pain33,34. Since chicken embryos hatch on day 21, the duration of transplantation could be extended to 15 days, sacrificing the embryos on day 19 to avoid hatching. This would, however, require the use of anesthetics. Despite the relatively short transplantation duration in this model, it induces extensive vascularization and significant maturation of organoid nephrons compared to in vitro organoids, including the formation of a GBM between organoid podocytes and the invading ECs28.
When performing intracelomic transplantation experiments, it is important to consider that not all chicken embryos develop normally and survive. Usually, ~65% of the embryos placed in the incubator at day 0 reach the end point of the experiment. This is due to a combination of acute bleeding caused by vessel damage during transplantation (5%-10% in our hands) and the stress that is induced by the windowing and transplantation procedures. When embryos are in an earlier or later stage than HH 23-24, transplantation becomes complicated due to limited space and more extensive vasculature, respectively. If embryos are not in the correct stage at the expected time, this could be due to the temperature of the incubation, as a higher temperature generally leads to faster development.
Moreover, keeping fertilized eggs at room temperature for a prolonged period before incubation induces more variability in development. To avoid this, the temperature of the incubator must be kept stable throughout and between experiments, and incubation should be started within 3 days after delivery of the fertilized eggs. Unexpectedly high percentages of embryo death between procedures can be caused by dehydration. To avoid this, it is essential to add two or three drops of DPBS+/+ to each egg after opening it and after transplantation and seal the egg very carefully with tape, smoothing out creases in the tape as much as possible. Combining the windowing and transplantation steps to reduce the number of times the eggs are opened is not recommended, as windowing on day 4 considerably increases embryo death. This is the result of damage to blood vessels that have frequently become attached to the eggshell at this stage.
In conclusion, this method provides an efficient and powerful tool to induce vascularization and enhance maturation in kidney organoids. It can be used to study these processes in large numbers of organoids and has potential for the modeling of renal diseases that require a higher level of maturation than can currently be acquired in vitro.
The authors have nothing to disclose.
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 & Embryology, LUMC, Leiden, the Netherlands), Conny van Munsteren (Department of Anatomy & Embryology, LUMC, Leiden, the Netherlands), Manon Zuurmond (LUMC, Leiden, the Netherlands), and Annemarie de Graaf (LUMC, Leiden, the Netherlands). M. Koning is supported by 'Nephrosearch Stichting tot steun van het wetenschappelijk onderzoek van de afdeling Nierziekten van het LUMC'. This work was in part supported by the Leiden University Fund "Prof. Jaap de Graeff-Lingling Wiyadhanrma Fund" GWF2019-02. This work is supported by the partners of Regenerative Medicine Crossing Borders (RegMedXB) and Health Holland, Top Sector Life Sciences & 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).
0.2 µm filter: Whatman Puradisc 30 syringe filter 0.2 µm | Whatman | 10462200 | |
35 mm glass bottom dishes | MatTek Corporation | P35G-1.5-14-C | |
Aspirator tube assemblies for calibrated microcapillary pipettes | Sigma-Aldrich | A5177-5EA | Contains silicone tubes, mouth piece and connector |
Confocal microscope: Leica White Light Laser Confocal Microscope | Leica | TCS SP8 | |
Dissecting forceps, simple type. Titanium, curved, with fine sharp tips | Hammacher Karl | HAMMHTC091-10 | |
Dissecting forceps, simple type. Titanium, straight, with fine sharp tips | Hammacher Karl | HAMMHTC090-11 | |
Dissecting microscope | Wild Heerbrugg | 355110 | |
Dissecting scissors, curved, OP-special, extra sharp/sharp | Hammacher Karl | HAMMHSB391-10 | |
Donkey serum | Sigma-Aldrich | D9663 | |
Donkey-α-mouse Alexa Fluor 488 | ThermoFisher Scientific | A-212-02 | dilution 1:500 |
Donkey-α-sheep Alexa Fluor 647 | ThermoFisher Scientific | A-21448 | dilution 1:500 |
Double edged stainless steel razor blades | Electron Microsopy Sciences | 72000 | |
DPBS, calcium, magnesium (DPBS-/-) | ThermoFisher Scientific | 14040133 | |
DPBS, no calcium, no magnesium (DPBS+/+) | ThermoFisher Scientific | 14190094 | |
Egg cartons or custom made egg holders | NA | NA | |
Fertilized white leghorn eggs (Gallus Gallus Domesticus) | Drost Loosdrecht B.V. | NA | |
Incubator | Elbanton BV | ET-3 combi | |
Lotus Tetragonolobus lectin (LTL) Biotinylated | Vector Laboratories | B-1325 | dilution 1:300 |
Micro scissors, straight, sharp/sharp, cutting length 10 mm | Hammacher Karl | HAMMHSB500-09 | |
Microcapillaries: Thin wall glass capillaries 1.5 mm, filament | World Precision Instruments | TW150F-3 | |
Micropipette puller | Sutter Instrument Company | Model P-97 | We use the following settings: Heat 533, Pull 60, Velocity 150, Time 200 |
Microscalpel holder: Castroviejo blade and pins holder, 12 cm, round handle, conical 10 mm jaws. | Euronexia | L-120 | |
Mounting medium: Prolong Gold Antifade Mountant | ThermoFisher Scientific | P36930 | |
Olivecrona dura dissector 18 cm | Reda | 41146-18 | |
Parafilm | Heathrow Scientific | HS234526B | |
Penicillin-streptomycin 5,000 U/mL | ThermoFisher Scientific | 15070063 | |
Perforated spoon | Euronexia | S-20-P | |
Petri dish 60 x 15 mm | CELLSTAR | 628160 | |
Plastic transfer pipettes | ThermoFisher Scientific | PP89SB | |
Purified mouse anti-human CD31 antibody | BD Biosciences | 555444 | dilution 1:100 |
Rhodamine labeled Lens Culinaris Agglutinin (LCA) | Vector Laboratories | RL-1042 | This product has recently been discontinued. Vectorlabs does still produce Dylight 649 labeled LCA (DL-1048-1) and fluorescein labeled LCA (FL-1041-5) |
Sheep anti-human NPHS1 antibody | R&D systems | AF4269 | dilution 1:100 |
Sterile hypodermic needles, 19 G | BD microlance | 301500 | |
Streptavidin Alexa Fluor 405 | ThermoFisher Scientific | S32351 | dilution 1:200 |
Syringe 5 mL | BD Emerald | 307731 | |
Transparent tape | Tesa | 4124 | Available at most hardware stores |
Triton X | Sigma-Aldrich | T9284 | |
Tungsten wire, 0.25 mm dia | ThermoFisher Scientific | 010404.H2 |