Kidney Organoid Generation at the Air-Liquid Interface

Summary

This protocol describes asynchronous mixing of human embryonic stem cells derived kidney progenitors at the air-liquid interface to efficiently generate kidney organoids.

Abstract

The prevalence of kidney diseases continues to increase worldwide, driving the need to develop transplantable renal tissues. The kidney develops from four major renal progenitor populations: nephron epithelial, ureteric epithelial, interstitial and endothelial progenitors. Methods have been developed to generate kidney organoids but few or dispersed tubular clusters in the organoids hamper its use in regenerative applications. Here, we describe a detailed protocol of asynchronous mixing of kidney progenitors using organotypic culture conditions to generate kidney organoids tightly packed with tubular clusters and major renal structures including endothelial network and functional proximal tubules. This protocol provides guidance in the culture of human embryonic stem cells and their directed differentiation to kidney organoids. Our 18-day protocol provides a rapid method to generate kidney organoids that facilitate the study of different nephrological events including in-vitro tissue development, disease modeling and chemical screening. However, further studies are required to optimize the protocol to generate additional renal-specific cells types, interconnected nephron segments and physiologically functional renal tissues.

Introduction

Chronic kidney disease (CKD) is a worldwide healthcare problem with 13.4% global estimated prevalence¹. Approximately 10% of the adult population of the United States suffers from CKD². There is no curative treatment available for patients with CKD except renal transplantation. The lack of availability of transplantable organs warrants research into technologies to understand how new kidney tissues can be generated. In recent years, procedures have been reported to generate kidney organoids^3,4 from human embryonic stem cells (hESCs) and human induced pluripotent cells but differentiation into off-target cells^5,6 and lack of dense tubules within kidney organoids limit their use in modeling renal diseases, in-vitro chemical screening and transplantable renal tissue generation.

We followed a previously published protocol to generate kidney progenitors from hESCs³. The kidney functions as a 3D organ and an appropriate 3D environment allows kidney progenitors to self-organize to form a kidney organoid⁷. A 3D organotypic culture condition was selected to generate kidney organoids because it supports vigorous growth and differentiation of mammalian embryonic kidneys⁸. Using this approach, hESCs derived kidney progenitors were aggregated at the air-liquid interface, to provide a 3D organotypic culture environment for differentiation.

During kidney development, several stages of differentiating cells coexist within the embryonic kidney⁹, and their differentiation fate and spatial patterning along the nephron depends on the timing of their recruitment. To establish such a culture condition in-vitro, a method was developed for asynchronous mixing of kidney progenitors generated by directed differentiation³. Asynchronous mixing refers to the combination of two progenitor populations that are at different stages of differentiation. Directed differentiation cultures were staggered two days apart and newly differentiated cells were mixed with cells that have been cultured as aggregates in organotypic conditions for 2 days. Heterochronic mixing of two cell batches improve the fidelity of pluripotent stem cell-derived organoids¹⁰.

Here, we provide an efficient method of asynchronous mixing of the kidney progenitors at the air-liquid interface that potentiates nephrogenesis to produce tightly packed nephron epithelia with more tubular clusters in kidney organoids. These kidney organoids were filled with glomerular podocytes, proximal tubules, distal tubules, stromal cells, connecting tubule or collecting ducts. The protocol yields a complex and extensive network of endothelial cells. In addition, proximal tubules in kidney organoids were mature and functional, showing endocytic function confirmed by alexa flour 488 (AF488) labeled dextran uptake. In this protocol, a step by step methodology of asynchronous mixing of kidney progenitors to generate kidney organoids is presented which we recently published elsewhere¹⁰.

Protocol

Cell line WA09 (H9) was approved by the National Institutes of Health (registration number 0062) and was tested negative for mycoplasma infection.

1. Medium and plate preparation for hESCs culture

1. Dilute matrix (reduced growth factor basement membrane e.g., Geltrex) in DMEM/F12 (1:100) and add 1 mL/well in 2 wells of a 6 well plate (reagents utilized in the manuscript are summarized in the Table of Materials).
2. Incubate coated plate undisturbed at 37 °C for 1-2 h for effective coating.
3. Prepare 7 mL of culture medium by adding prewarmed basic culture medium (e.g., StemFit) supplemented with 100 ng/mL FGF2 and 10 µM Rock inhibitor Y-27632.

2. hESCs thawing and culture

1. Thaw a frozen vial containing 1 - 1.5 x 10⁶ H9 cells. Rapidly swirl the vial in a 37 °C water bath until just a small sliver of ice is left. This takes approximately 90-120 s.
2. Triturate once with 1 mL micropipette and transfer to a 15 mL conical tube. Slowly drip 2 mL of the medium prepared in step 1.3, dropwise into the thawed cell suspension.
3. Spin the cell suspension at 1000 x g for 3 min to pellet the cells.
4. Discard the supernatant and resuspend the cell pellet in 1 mL of fresh culture medium (as prepared in step 1.3) and triturate 2 times. Afterwards, add 3 mL of additional culture medium making a total volume of 4 mL.
5. Add 2 mL of the cell suspension into each of the matrix coated wells of the 6 well plate and culture at 37 °C, 5% CO₂ in the incubator.
6. Change the medium after 48 h with fresh basic culture medium supplemented with 50 ng/mL FGF2 only (now Y-27632 is not required).
7. Change the medium after 48 h with basic culture medium supplemented with 25 ng/mL FGF2.
8. Split cells once they reach 70% confluency.
9. Passage the cells at 1:10 ratio (i.e. distribute cells from 1 well into 10 wells of 6-well-plate) by following steps 3.5 to 3.8 for the cell culture maintenance.

3. Plating hESCs for directed differentiation

1. Dilute matrix in DMEM/F12 (1:100) and add 1 mL/well into 3 wells each of two 6 well plates.
2. Incubate the coated plate, undisturbed at 37 °C for 1-2 h for effective coating.
3. Prepare 18 mL of prewarmed basic culture medium supplemented with 100 ng/mL FGF2 and 10 µM Rock inhibitor Y-27632.
4. Remove the medium from 1 well of the 6 well hESCs culture plate and then wash the cells once with DPBS. After washing, add 1 mL prewarmed cell detachment solution (e.g., Accutase) to detach the cells from the plate. The 2^nd well can be used to freeze cells or to propagate cell culture.
5. Incubate in a 37 °C incubator for 10 min.
6. Triturate 3-4 times gently and then transfer the cells into a 15 mL conical tube.
7. Adjust the volume to 3 mL with medium. Take 10 µL cell suspension to count cells.
8. Spin the cell suspension at 1000 x g for 3 min to pellet the cells.
9. Discard the supernatant and resuspend the cell pellet in 1 mL of medium and triturate 2 times.
10. For the 1^st batch of asynchronous mixing, plate 1.7 x 10⁵ hESCs /well in 2 mL medium (Plate 1) and culture at 37 °C, 5% CO₂.
11. For the 2^nd batch of asynchronous mixing, plate 0.45 x 10⁵ hESCs/well in 2 mL medium in a 6 well plate (Plate 2) and culture at 37 °C, 5% CO₂.
12. Change the medium of both the plates after 48 h with fresh basic culture medium supplemented with 50 ng/mL FGF2 without Rock inhibitor Y-27632.
13. After 72 h, ensure that cells in Plate 1 is ~50% confluent. This is the correct time to start directed differentiation (proceed to section 4).
14. 96 h after seeding, change the medium in Plate 2 with fresh basic culture medium supplemented with 25 ng/mL FGF2.
15. 120 h after seeding, ensure that cells of the Plate 2 is ~50% confluent. This is the correct time to start directed differentiation (proceed to section 4).

4. Directed differentiation of hESCs into kidney progenitors (perform this procedure on both batches of cells staggered 2 days apart) (Figure 1A)

1. The hESCs should be ~50% confluent at the start of the differentiation. Remove medium and wash the cells once with DPBS.
2. Add 2 mL of advanced RPMI 1640 containing 1x L-glutamine supplement (e.g., GlutaMax) and 8 µM CHIR into each well of the 6 well plate and culture at 37 °C, 5% CO₂. This will be day 0 of differentiation for Plate 1.
3. On day 2, change the medium with fresh advanced RPMI 1640 containing 1x L-glutamine and 8 µM CHIR.
4. On day 4, change the medium with fresh advanced RPMI 1640 containing 1x L-glutamine and 10 ng/mL Activin A.
5. On day 6, change the medium with fresh advanced RPMI 1640 containing 1x L-glutamine and 10 ng/mL Activin A.
6. On day 7, change the medium with fresh advanced RPMI 1640 containing 1x L-glutamine and 10 ng/mL FGF9.
7. After 9 days of this treatment protocol (day 9), ensure that the cells are differentiated to kidney progenitors and adopt renal vesicle like morphology (Figure 2F). Now this 2D culture is ready for transition into 3D culture at the air-liquid interface to generate kidney organoids. For Plate 1 – proceed to section 5. For Plate 2 – proceed to section 6.

5. Making kidney progenitor cell aggregates at the air-liquid interface

1. Prepare a 24 well plate for culture of cells at the air-liquid interface.
   1. Prepare the medium containing APEL2, 1.5% PFHM II, 100 ng/mL BMP7, 100 ng/mL FGF9 and 1 µg/mL Heparin and add 1 mL/well into the 24 well plate.
   2. Float polycarbonate membrane on the medium in each well of the 24 well plate, using sterile forceps. Make sure not to flood the filters - wells with partially or entirely submerged filters should not be used. Keep plate aside in the hood for later use.
2. Wash the wells of the Plate 1 two times with DPBS and add 1 mL of cell dissociation enzyme (e.g., TrypLe express) into each well of the 6 well plate.
3. Incubate the plate for 5 min at 37 °C. Triturate 3-4 times to disperse cell clusters into single cell suspension.
4. Neutralize the cell dissociation enzyme by adding 8 mL/well advanced RPMI 1640 with 1 mL of FBS (Total 10% FBS) in a 50 mL conical tube.
5. Strain the cells through 40 µm strainer and use 10 µL of strained cell suspension to count the cells.
6. Centrifuge the cells at 300 x g for 5 min.
7. Resuspend the cells at 2.5 x 10⁵ cells/µL in APEL2 medium containing 1.5% PHFM II.
8. Spot 2 µL of cell suspension onto the polycarbonate membrane floating on the medium in the 24 well plate. Spot 6-8 cell aggregates/membrane.
9. Culture for 2 days at 37 °C, 5% CO₂ in the incubator.

6. Asynchronous mixing of kidney progenitors to generate kidney organoids

1. Prepare 24 well plate to spot cells following step 5.1.
2. Harvest and count the kidney progenitors in the Plate 2 following steps 5.2 - 5.7.
3. Remove membranes one at a time from the 24 well plate seeded in step 5.8 and break the cell aggregates into small fragments using 200 µL micropipette by triturating 7-10 times.
4. Mix these small fragments with fresh kidney progenitors from Plate 2 at a 1:1 ratio. (e.g., mix fragments from 1 cell aggregate of 5 x 10⁵ cells with 5 x 10⁵ cells of newly differentiated kidney progenitors from Plate 2, resuspend in 4 µL and make two cell aggregates on the membranes).
5. Spot mixed cells on the membranes floating at the surface of the medium in a 24 well plate. 6-8 cell aggregates/the membrane can be spotted.
6. Change medium on day 13 with fresh prewarmed APEL2 containing 1.5% PHFM II without any growth factors.
7. Change medium every 48 h with APEL2 containing 1.5% PHFM II.
8. On day 18, image the organoids under a stereo microscope and proceed to section 8 for marker expression (Table 1) analysis.

7. Evaluation of dextran uptake by proximal tubule cells in kidney organoids

1. On day 18, remove the membrane from the wells of 24 well plate and transfer kidney organoids such that 1 organoid is placed in each well of a ‘U’ bottom low attachment 96 well plate.
2. Replace the medium with 200 µL fresh prewarmed APEL2 containing 1.5% PHFM II and 10 µg/mL 10,000 MW dextran conjugated with Alexa flour 488.
3. Incubate the organoids at 37 °C, 5% CO₂ in the incubator for next 24 h on shaker.
4. Stain the organoids using ‘whole mount staining technique’ described in section 8 for proximal tubule specific markers to evaluate dextran uptake in proximal tubule cells.

8. Whole mount immunofluorescence staining on kidney organoids

1. On day 18 of the differentiation, remove the membrane from the wells and transfer each organoid to a ‘U’ bottom 96 well plate.
2. Fix the organoids with 150 µL 4% paraformaldehyde (PFA) for 15 min at room temperature.
3. Remove the PFA and wash the organoids 3 times with DPBS.
4. Add 150 µL of 1% Triton X-100 and incubate for 10 min at 4 °C on a shaker.
5. Remove the Triton X-100 and wash the organoids 3 times with DPBS.
6. Add 150 µL blocking buffer (5% serum from a species that matches the secondary antibody’s host species in DPBS) and incubate for 1 h at room temperature.
7. Incubate the organoids with primary antibodies diluted in blocking buffer at 4 ˚C overnight on shaker.
8. Remove the primary antibodies and wash the organoids with DPBS for next 8 h on shaker.
9. Incubate organoids with secondary antibodies diluted in blocking buffer at 4 ˚C overnight on shaker.
10. Remove the secondary antibodies and wash the organoids with DPBS for next 8 h on shaker.
11. Mount the organoids on glass slide and image under a fluorescent microscope.

Results

This protocol describes asynchronous mixing of kidney progenitor cells differentiated from hESCs (H9) at the air-liquid interface to generate kidney organoids with reproducible results and high success rates. We followed a previously published protocol to differentiate hESCs into kidney progenitors³ (Figure 1). The mix of cells that arises from the directed differentiation process is believed to represent the repertoire of developmental kidney progenitors that gives r...

Discussion

Asynchronous mixing of progenitors at the air-liquid interface (Figure 1) presents an efficient method to generate kidney organoids from hESCs. This work describes stepwise protocols for thaw and culture of hESCs, directed differentiation to kidney progenitors, making cell aggregates at the air-liquid interface, asynchronous mixing of progenitors to generate kidney organoids tightly packed with tubular clusters and major renal structures including endothelial network and functional proximal ...

Materials

Name	Company	Catalog Number	Comments
15 mL falcon tube	VWR	62406-200
24 well plate	VWR	29443-952
40 micron strainer	VWR	21008-949
50 mL falcon tube	VWR	21008-940
6 well plate	VWR	29442-042
96 well Clear Round Bottom Ultra-Low Attachment Microplate (U bottom)	Corning	7007
Accutase	STEMCELL Technologies	7920	Store at -20 °C
Activin A	R&D systems	338-AC-010	Aliquot and store at -20 °C
Advanced RPMI 1640	ThermoFisher Scientific	12633-012	Store at 4 °C
APEL2	STEMCELL Technologies	5270	Store at -20 °C
BMP7	R&D systems	354-BP-010	Aliquot and store at -20 °C
CHIR99021 IN SOLUTION	Reprocell	04-0004-10	Aliquot and store at -20 °C
Confocal microscope	Leica Microsystems	SP8
Dextran, AF 488, 10,000 MW	Thermo fisher Scientific	D22910	Store at -20 °C
DMEM/F12	Thermo fisher Scientific	11330-032	Store at 4 °C
DPBS	VWR	45000-434
FBS	Atlanta Biologicals	S11550	Store at -20 °C
FGF2	R&D systems	234-FSE-025	Aliquot and store at -20 °C
FGF9	R&D systems	273-F9-025	Aliquot and store at -20 °C
Forceps	Roboz	RS-5040
Geltrex	Thermo fisher Scientific	A1413301	Aliquot and Store at -20 °C
Glutamax	Thermo fisher Scientific	35050061
H9 cells	WiCell	WA09	Store in liquid Nitrogen
Heparin	Sigma	H3393-25KU
Isopore membrane	EMD Millipore	VCTP01300
Paraformaldehyde	P6148-500G	P6148-500G
PFHM II	Thermo fisher Scientific	12040077	Store at 4 °C
Rock inhibitor Y-27632	EMD Millipore	688002-1mg	Aliquot and store at -20 °C
StemFit Medium	amsbio	SFB-500	Store at -20 °C
Triton X-100	Sigma	X100-100ML
TrypLe express	Thermo fisher Scientific	12563029