Investigaciones utilizadas para las hipótesis o proposiciones del modelo de absorción

In the first experiment, the protocol of Morizane was followed without any adaptions. The cells that were cultured in a 12 well plate were kept in 2D until day 28. The cells that were cultured in a 24 well plate were transferred to a 96 well plate at day 9.

One 12-well plate in which coverslips were placed and a 24 well plate were prepared with Geltrex, as stated in section 5.1.1. The iPSCs cells were prepared as described in section 5.1.5.

The cells were re-suspended in mTesR1 + 0.5× 100 U/ml penicillin/ streptomycin and 10µM ROCK inhibitor (Y27632). Cells were plated with a density of 1.2×104 cells/cm2 in 1 ml of media or 500 µl of media per well of a 12-well or 24-well plate, respectively. Due to the dissociation, the cells lose contact with the matrix and to other cells, which results in a ten- dency of the cells to die (apoptosis). ROCK inhibitor is added to prevent apoptosis[37]. The media, mTesR + 0.5×100 U/ml penicillin/streptomycin, was refreshed everyday.

2D

The differentiation process was started 72 hours after preparation. At 50% confluence, the cells were washed once with PBS. The cells were cultured for 4 days in Basic Differentiation Medium (BDM), 1 ml per well, consist- ing of Advanced RPMI 1640 with 1×Glutamax and 0.5×100 U/ml peni- cillin/streptomycin. Glutamax is a stabilized form of L-glutamine which is an energy source for the cell. The penicillin and streptomycin are antibi- otics and prevent bacterial growth. The BDM was supplemented with 8 µM CHIR99021 and 5 ng/ml of Noggin to induce the growth of late prim- itive streak cells. The culture medium was refreshed after two days. The cells were then cultured in BDM supplemented with 10 ng/ml ActivinA

for 3 days to induce posterior intermediate mesoderm. To start the induc- tion of nephron progenitor cells, the cells were cultured in BDM supple- mented with 10 ng/ml FGF9 for 2 days. At day 9, the media was changed to BDM with 10 ng/ml FGF9 and 3 µM CHIR99021. After 2 days, the medium was changed to BDM supplemented with only 10 ng/ml FGF9. Three days later the cells were cultured in BDM without additional fac- tors. The next 14 days the media was changed on Monday, Wednesday and Friday.

At different time points, coverslips were fixed as described in section 5.4. Immunostaining was done according to section 5.5. Before imaging, the coverslips were mounted as written in section 5.5.3. When the cover- slips were fixed and for which genes they where stained is summed up below.

At day 8, a coverslip was stained for rabbit anti-PAX2 (1:100) and mouse anti-LHX1 (1:50). Using the secondary antibodies: Alexa Fluor 647 donkey anti-rabbit (1:200), Alexa Fluor 555 Donkey anti-mouse (1:200) and 4‘,6- diamidino-2-phenylindole (DAPI) (1µg/ml) to counterstain the nuclei of the cells.

At day 14, a coverslip was stained for rabbit anti-PAX2 (1:100), mouse anti-LHX1 (1:25) and goat anti-TBX6 (1:200). Another coverslip was stained for rabbit anti-SIX2 (1:100) and mouse anti-CITED1 (1:500). Using the sec- ondary antibodies: Alexa Fluor 647 donkey anti-rabbit (1:200), Alexa Fluor 555 Donkey anti-mouse (1:200), Alexa Fluor 488 Donkey anti-goat. (1:200) and DAPI (1µg/ml) to counterstain the nuclei of the cells.

Coverslips of day 14 showed unclear staining, presumably due to the multiple cell layers that have grown over the 14 days. To improve imaging, coverslips of day 21 and day 28 were treated according to the clearing protocol (see section 5.5.2). The fructose-glycerol clearing solution (300 µl) was only added to the coverslip of day 21, after which the coverslip was washed 3 times with 1 ml PBS + 0.05% Tween. Both coverslips were mounted as described in section 5.5.3.

The coverslips of day 21 and day 28 were both stained for LTL (1:200), SYNPO (1:100) and TMEM100 (1:100). Using the secondary antibodies Alexa Fluor 647 donkey anti-rabbit (1:200) and Alexa Fluor 555 Donkey anti-mouse (1:200) and DAPI (1 µg/ml) to counterstain the nuclei of the cells.

From 2D to 3D

The cells in the 24 well plate were treated the same from day 0 to day 9 of differentiation as described above. Only 500 µl of the medium was

5.2 Morizane Protocol 2015 51

used per well. At day 9, when FGF9 and CHIR were added to the media, the cells were transferred to a 3D culture environment. Therefore 250 µl trypLEselect was added to each 24 well, for 5 min at 37°C, to dissociate the cells. The cells were re-suspended in BDM supplemented with 3 mM CHIR99021 and 10 ng/ml FGF9 and replated at 100 000 cells per well in a 96-well plate with an ultra-low-attachment and round bottom. The plates were centrifuged at 200×g for 1 min. After 2 days, 80 µl of the culturing medium was changed for 100µl BDM supplemented with only 10 ng/ml FGF9. Three days hereafter the cells were cultured in BDM without ad- ditional factors. The next 14 days the media was refreshed on Monday, Wednesday and Friday by replacing 80µl of the media in a well with 100 µl of fresh media.

At different time points, organoids were fixed as described in section 5.4. Immunostaining was done according to the clearing protocol de- scribed in section 5.5.2. For which genes the organoids were stained at which day is stated below.

At day 14, organoids were stained for rabbit anti-PAX2 (1:100), mouse anti-LHX1 (1:25) and goat anti-TBX6 (1:200). Other organoids were stained for rabbit anti-SIX2 (1:100) and mouse anti-CITED1 (1:500). Using the sec- ondary antibodies Alexa Fluor 647 donkey anti-rabbit (1:200), Alexa Fluor 555 Donkey anti-mouse (1:200), Alexa Fluor 488 Donkey anti-goat. (1:200) and DAPI (1µg/ml) to counterstain the nuclei of the cells.

Organoids from day 21 and day 28 of differentiation were both stained for LTL (1:200), SYNPO (1:100) and TMEM100 (1:100). The secondary an- tibodies, Alexa Fluor 647 donkey anti-rabbit (1:200) and Alexa Fluor 555 Donkey anti-mouse (1:200), were added along with DAPI (1 µg/ml) to counterstain the nuclei of the cells.

Organoids of day 28 were prepared for sectioning (see section 5.6). The sections of 20 µm thickness were stained for LTL (1:100), SYNPO (1:100) and CDH1 (1:200) (see section 5.6). The secondary antibodies Alexa Fluor 555 Donkey anti-mouse (1:200) and Alexa Fluor 647 donkey anti-rabbit (1:200), were added along with DAPI (1µg/ml) to counterstain the nuclei of the cells.

5.2.2

Experiment II

Upon repeating the 3D protocol of Morizane, both minor and major mod- ifications were applied. In this section, the slightly adapted protocol will be discussed. The protocol with major changes - pretreatment of cells with DMSO and starting in 3D from the start - is discussed in section 5.2.2 and

section 5.2.2 respectively.

A 24 well plate was prepared with Geltrex (see section 5.1.1) and the cells were prepared (see section 5.1.5) for differentiation. The cells were resuspended in mTesR1 + 0.5× 100 U/ml penicillin/streptomycin, sup- plemented with 10µM of ROCK inhibitor (Y27632) and plated with a den- sity of 25 000 cells/well in 500µl of media per well. The media, mTesR + 0.5× 100 U/ml penicillin/streptomycin, was refreshed 2 days later. The differentiation was started 24h after the media was refreshed. At 50% confluence, the cells were washed once with PBS. For the next four days, the cells were cultured in Basic Differentiation Medium (BDM), 500µl per well, consisting of Advanced RPMI 1640 with 1×Glutamax and 0.5×100 U/ml penicillin/streptomycin. The BDM was supplemented with 8 µM CHIR99021 and 5 ng/ml Noggin. The culture medium was refreshed af- ter two days. The cells were then cultured in BDM supplemented with 10 ng/ml ActivinA for 3 days. After 2 days the BDM was supplemented with 10 ng/ml FGF9. At day 9 the cells were moved to a 3D culture. The cells were washed once with PBS, 300µl trypLEselect was added to each well and put at 37°C for 7 min, to dissociate the cells. The cells were re- suspended in BDM supplemented with 3 mM CHIR99021 and 10 ng/ml FGF9 and plated at 100 000 cells per well in a 96-well plate with an ultra- low-attachment and V-bottom. The plate was not centrifuged. After 2 days the aggregates were transferred to a 96-well plate with an ultra-low- attachment and round bottom. The media was changed to BDM supple- mented with 10 ng/ml FGF9. Three days hereafter the cells were cul- tured in BDM without additional factors. The next 14 days the media was changed on Monday, Wednesday and Friday. At every medium change in the 3D culture, 80µl of media was replaced for 80µl of fresh media.

DMSO

The cells in the DMSO experiment were treated exactly as described above, but 24 h before differentiation they were pretreated with 2% Dimethyl sul- foxide (DMSO).

3D from the start

Instead of starting the experiment in a 2D culture environment, it was started in a 3D environment from the start. Therefore the cells were plated at a density of 4000 cells per well in a 96-well plate with an ultra-low- attachment and round bottom and centrifuged at 100×g for a few sec- onds. Two days later when the media, mTesR + 0.5× 100 U/ml peni-