3.1 Morizane protocol led to podocyte-like cells The Morizane protocol was executed twice In experiment I, the protocol
3.1.2 Experiment
The Morizane protocol was executed again in the original form (Control) and in an adapted form (DMSO pre-treatment and 3D from the start). Dur- ing the control experiment, a small modification was made when the cells were transferred from a 2D cell culture to a 3D environment. Instead of ag- gregating the cells in a U-bottom plate and centrifuging the plate, the cells were replated in a 96-well V-bottom plate for 2 days without centrifuga- tion. At day 11 the cells were transferred to a U-bottom plate. This small modification of the protocol resulted in more spherical like structures (see figure 3.11, control) compared to the flat structures in experiment I.
3.1 Morizane protocol led to podocyte-like cells 31
Control experiment
The Morizane protocol to grow kidney organoids in 3D was executed sim- ilarly as described in section 3.1.1. At day 14, the organoids were expected to have formed renal vesicles and were stained for PAX2 (metanephric mesenchyme), LHX1 (renal vesicles) and TBX6 (late primitive streak). The nuclei were counterstained with DAPI. The staining did not give a clear indication if certain genes were expressed, figure A.4 can be found in the Supplementary.
Figure 3.8: Cells grown in 3D at day 28 of differentiation in the Morizane protocol control experiment formed a curved structure of podocytes. Sections were stained with DAPI, PTH1R (S-shaped body) and SYNPO (podocytes). Top pictures, scale bar: 200 µm, magnification: 20x. Other pictures, scale bar: 20 µm, magnification: 100x, maximum intensity z-projection. Several cells, with a
big nucleus (see white arrowhead), which is a characteristic of podocytes, clus- ter together and express SYNPO. The clustered cells seemed to have formed a glomerulus-like structure (see yellow arrowheads). The cells were negative for PTH1R.
Organoids harvested at day 28 were sectioned (see methods 5.6) and stained for SYNPO (podocytes), LTL (proximal tubules), PTH1R (s-shaped
body), CDH1 (s-shaped body), MAFB (podocytes precursors) and CLDN7 (renal collecting duct system). The nuclei were counterstained with DAPI. The stainings for LTL, CDH1 and CLDN7 were negative (data not shown), therefore, the organoids did not form epithelialised tubular structures. However, SYNPO was clearly expressed, see figure 3.8, indicating that the organoids have differentiated into podocyte-like cells.
Figure 3.9: Cells grown in 3D at day 28 of differentiation in the Morizane pro- tocol control experiment did not show clear staining for podocyte precursors: MAFB and PTH1RSections were stained with DAPI, PTH1R (podocyte precur- sors) and MAFB (podocytes precursors). Top picture, scale bar: 200µm, magni-
fication: 20x. Other pictures, scale bar: 20 µm, magnification: 100x, maximum
intensity z-projection. Deconvoluted, Land Weber, 5 iterations. MAFB was ex- pressed at its expected localisation, that did not apply to PTH1R staining which seemed to overlap MAFB staining.
To indicate the developmental stage of the podocytes, sections were stained for MAFB. This is a marker for podocyte progenitor cells. MAFB is expected to be expressed in the nucleus and the cytosol[40]. According to the staining in figure 3.9, MAFB seems to be expressed by the whole cell. The staining might be specific, but the signal of MAFB is very low. The staining of PTH1R, another marker of podocytes, seems to be real staining
3.1 Morizane protocol led to podocyte-like cells 33
because the high signal is twice as high as the background signal outside the nucleus (5000:2000). The staining for PTH1R also seems to overlap with MAFB, but PTH1R should be expressed on the plasma membrane and in the nucleus. Based on their signal intensity and localisation, both PTH1R and MAFB are not stained specifically.
DMSO pre-treatment
To increase the efficiency of differentiation of the organoids into mature nephron cells, in the DMSO pre-treatment experiment, the cells were treated with 2% DMSO (Dimethylsulfoxide) for 24 h before differentiation, based on the protocol of Sambo[43]. DMSO is expected to extend the G1 phase of the cell cycle, the phase in which the cell decides to proliferate or differenti- ate[44]. The hiPSCs are characterised by a short stay in the G1 phase, due to which pluripotent stem cells are more willing to multiply[43]. By in- creasing the G1 phase, the DMSO prepares the cells for differentiation which increases the chance that cells follow the differentiation path to ma- ture nephron cells.
Figure 3.10: Cells grown in 2D from day 2 to 9 of differentiation in the Morizane protocol pre-treated with DMSO showed a different density pattern compared to cells of the control experiment. Bright-field images at different time points of one well that was pre-treated with DMSO (bottom) and one with- out DMSO pre-treatment (top). Scalebar day 2: 1 mm, scalebar day 4,7 and 9: 2 mm. From day 4 the control is more confluent than the DMSO well. Gaps of low density also appear in the control well, as can be clearly seen at day 7, but are more present in the DMSO well. On the contrary, the density of the concentrated cells in the DMSO well are much higher compared to the control well. Day 2, 1 tile. Day 4, tiled 3x3, Day 7, tiled 5x5, day 9, tiled 6x6.
Although the cells were only cultured in 2% DMSO for 24h, the DMSO had a great influence on their way of growing in a 2D culture dish, which became visible at day 4 of differentiation (see figure 3.10). The cells formed more dense areas, leaving a less confluent well. At day 9 the cells were dissociated into single cells and transferred to a 3D environment. At this time point, the viability, as well as the number of cells, was the same for both wells.
Figure 3.11: Organoids grown in 3D from day 14 to 18 of differentiation in the Morizane protocol pre-treated with DMSO decreased in size. Rows represent the same organoid at different time points. Scale bar: 500 µm. At day 10 the
organoids are cultured in a V-bottom 96-well plate and transferred the next day to a U-bottom 96-well plate. From day 14 the organoids were cultured in basic dif- ferentiation medium without growth factors. After which all organoids decreased in size. However, the DMSO pre-treated organoids shrunk more compared to the organoids in the control experiment.
The cells pretreated with DMSO seemed to be prone to form high- density clusters, which was expected to be beneficial when the cells were transferred to a 3D cell culture. When the organoids were cultured in the V-bottom well at day 9 and 10, the DMSO organoids resembled those of the control experiment (see figure 3.11). From day 14, the organoids were cultured in basic differentiation medium without additional growth factors. At this point the DMSO pretreated organoids looked different from the control, there seemed to be more cell death around the DMSO organoids. Between day 14 and day 18 all organoids reduced in size, but
3.1 Morizane protocol led to podocyte-like cells 35
the DMSO organoids shrunk more. The control organoids were consistent in size from day 18 on and stayed condense until day 28. This condense structure seemed to disappear in the DMSO pretreated organoids from day 25 when an increase of cell death was observed.
Figure 3.12: Cells grown in 3D at day 14 of differentiation in the Morizane pro- tocol pre-treated with DMSO show PAX2 staining in small clusters.Organoids were stained for PAX2 (metanephric mesenchyme). Scale bar: 200µm, magnifi-
cation: 20x. The PAX2 staining seemed to be spread in small clusters around the organoid.
At day 14, the organoids were expected to have formed renal vesi- cles. The results of the organoids in the control experiment are discussed above in section 3.1.2. The organoids pretreated with DMSO were also stained for PAX2 (metanephric mesenchyme), LHX1 (renal vesicles) and TBX6 (late primitive streak). The nuclei were counterstained with DAPI. The DMSO organoids at day 14 were less dense than the control, making it possible to recognise individual cells (see figure 3.12). LHX1 and TBX6 were not expressed (data not shown). However, PAX2 had a sporadic ex- pression throughout the organoid. Compared to the organoids at day 14 in experiment I (see figure 3.5) where PAX2 seems to be expressed in larger regions, the DMSO treatment resulted in a different expression pattern.
3D from the start
The development of the kidney occurs in a 3D manner within the em- bryo, therefore, to mimic the development of the kidney we started the differentiation in a 3D culture instead of a 2D culture. But having a 3D culture is also a challenge, the aggregates are not attached and can easily
be destroyed when the media is changed. It is therefore not possible to completely refresh the media in contrast to a 2D culture. The Morizane protocol starts in 2D and moves to 3D after 9 days, the moment when the nephron progenitors start to form 3D tubular structures. But to go from 2D to 3D the cells need to be dissociated, a process that might negatively influence the cells. Single-cell dissociation of hiPSCs increase cell death by apoptosis. To circumvent the step from 2D to 3D, we cultured the cells in a 96-well U-bottom plate from the start at a density of 4000 cells per well. The plate was centrifuged until it reached 100×g, to promote aggregation of the single cells. The organoids were treated with growth-factors based on the Morizane protocol.
The organoids kept growing until day 14, from then the media did not contain any additional growth factors. Comparable to the control and DMSO organoids (see figure 3.11) these organoids become smaller after day 14. The amount of cell death increased and at day 28, most of the organoids do not appear vital anymore (see figure 3.13).
Figure 3.13: Cell death increased from day 18 to day 28 in organoids that were grown in 3D from the start in the Morizane protocol. Scalebar: 500µm. Rows
represent the same organoid at different time points. The organoids grow in size until day 14. At that time point, the organoids were left to differentiate without additional growth factors. During this process, the organoids reduced in size, which was stable from day 18 until day 21. The amount of cell death increased from day 18 and the compact structure seemed to fall apart at day 28.
At day 14 the organoids were expected to have formed renal vesicles. The organoids were stained for LHX1 (renal vesicles), TBX6 (late primitive streak) and PAX2 (metanephric mesenchyme). The nuclei were counter- stained with DAPI. LHX1 and TBX6 were not expressed (data not shown).
3.1 Morizane protocol led to podocyte-like cells 37
In figure 3.14 PAX2 expression seemed to be concentrated around a small area, indicating that a tiny region is metanephric mesenchyme. Probably the organoids are too condense to get a clear image on the individual cells, making it difficult to determine if the staining is specific.
Figure 3.14: Cells grown in 3D at day 14 of differentiation in the Morizane pro- tocol cultered in 3D from the start expressed PAX2 in a small region.Organoids were stained for PAX2. Pictures on the right, scale bar: 50µm, 20x. Other pictures,
scale bar: 200µm, 20x. In a small region cells seem to express PAX2.
To conclude the results of the Morizane protocol, at day 14 the hiP- SCs were expected to have formed renal vesicles, but in all experiments, the marker LHX1 for epithelialised tubular structures was not expressed. The organoids only express the metanephric mesenchyme marker PAX2 in a small region. This expression pattern was different in the DMSO pre- treated organoids. At day 28, all organoids expressed the podocyte marker SYNPO. In the control experiment, when the organoids were more spheri- cal than in experiment I, podocyte-like cells clustered to form a glomerulus- like structure.