Capítulo 2 Revisión de la literatura sobre capacidad de absorción del conocimiento
2.3. La capacidad de absorción y sus categorías
2.3.1. Investigaciones del constructo como antecedentes o citación menor
The Taguchi 2014 protocol[2] differentiates the hiPSCs into metanephric mesenchyme. In 2017, Taguchi published an adapted protocol[3] based on his 2014 protocol, to grow metanephric nephron progenitors. We have executed both protocols and tried to extend the protocol of 2017 to induce epithelialised tubular structures.
3.2.1
Protocol of 2014
In the Taguchi protocol of 2014, the cells are differentiated towards the metanephric mesenchyme stage. BMP4 was added for 24h to induce em- bryonic bodies (EBs). In the next two days, the cells were driven towards the Epiblast stage by adding Activin and FGF2. To differentiate the cells into the posterior nascent mesoderm stage, BMP4 and CHIR were added for 6 days. At day 9, Activin, BMP4, CHIR and Retinoic Acid were added to get the cells in the posterior intermediate mesoderm stage. Two days later, CHIR and FGF9 induced the differentiation into metanephric mes- enchyme.
At day 14, the organoids were expected to be metanephric mesenchyme and were stained for PAX2 (metanephric mesenchyme), TBX6 (late prim- itive streak) and LHX1 (pretubular aggregates). The nuclei were counter- stained with DAPI. In most organoids (8) it was not possible to identify in- dividual cells and therefore it was not possible to make a statement on the expression of genes. Two organoids were probably less dense and showed specific staining (see figure 3.15). The top part of the organoid stained pos- itive for PAX2 and LHX1, indicating the differentiation towards nephron progenitors. The bottom part expressed TBX6, a marker of cells in the late primitive streak stage.
3.2 Taguchi protocols led to different expression patterns 39
Figure 3.15: Organoids grown in 3D at day 14 of differentiation in the Taguchi 2014 protocol show expression of both nephron progenitors and late primi- tive streak.Organoids were stained for PAX2 (metanephric Mesenchyme), LHX1 (pretubular aggregates), TBX6 (Late primitive streak). Scale bar: 200µm, magni-
fication: 20x. Z projection, maximum intensity. Most cells in the top part of the organoid seem to express PAX2. Small regions in the top part express LHX1. The bottom part expresses TBX6.
3.2.2
Protocol of 2017
In the Taguchi protocol of 2017, the cells were found to have differentiated into metanephric nephron progenitors within 11 days. In the first step, Ac- tivin was added for 24h to differentiate the cells towards the epiblast stage. CHIR, without additional BMP4, was added for 6 days to differentiate the cells towards posterior nascent mesoderm. Half of the medium was re- freshed every other day. To induce posterior intermediate mesoderm, the media was changed into CHIR, Activin, BMP4, Retinoic Acid. Two days later, at day 9, FGF9 and CHIR differentiated the cells in two days to the metanephric nephron progenitors.
At day 11, the organoids were expected to be metanephric nephron progenitors and were stained for PAX2 (Metanephric Mesenchyme), LHX1 (Pretubular aggregates) and TBX6 (late primitive streak). The organoids did not express TBX6, indicating that the organoids differentiated further than the late primitive streak stage. LHX1 was also not expressed, which
suggests that the cells did not start to form tubular structures. PAX2 was expressed in almost every cell (see figure 3.16) indicating that the cells were at the metanephric mesenchyme stage.
Figure 3.16: Organoids grown in 3D at day 11 of differentiation in the Taguchi 2017 protocol show expression metanephric mesenchyme. Organoids were stained for PAX2 (Metanephric mesenchyme) and TBX6 (late primitive streak). Scale bar: 50µm, 40x. Shown is probably a segment of a bigger organoid and not
a representative image. Other figures of bigger organoids were blurry and indi- vidual cells were not distinguishable. The TBX6 dot at the top right of the picture is not specific. In this fragment, all cells express PAX2.
3.2.3
Maturation of metanephric nephron progenitors
To further differentiate the metanephric nephron progenitors into epithe- lialized nephrons, Taguchi co-cultured the organoids with embryonic spinal cord or induced ureteric bud. Since other protocols like Morizane[1] can grow renal vesicles without embryonic spinal cord and ureteric bud, five experiments with growth factors were executed to mature the metanephric nephron progenitors. An overview of the experiments can be found in ta- ble 3.1. In the first experiment, spontaneous differentiation was promoted by refreshing basic differentiation medium on a regular basis. The sec- ond experiment was inspired by the protocol of Bonventre[27], CHIR was3.2 Taguchi protocols led to different expression patterns 41
added for 24h, which was then changed for FGF2 and Retinoic acid. Two days later the media was changed to FGF9 and Activin. After 3 days basic differentiation medium was added without additional growth factors. Ex- periments 3 and 4 are based on the idea that treatment with an activator of the Wnt signalling pathway for a short period would induce matura- tion of metanephric mesenchyme. In experiment 3 the non-canonical Wnt signalling pathway was activated with R-spondin 1 for 24 h. Experiment 4 activated the canonical Wnt pathway by adding CHIR for 24 h. The last experiment was inspired by the protocol of Morizane[1], where a pulse of CHIR was added next to FGF9 and ROCK inhibitor Y-27632 for 2 days. The media was then changed to only FGF9 and ROCK inhibitor Y-27632 for 3 days.
Day Exp 1 Exp 2 Exp 3 Exp 4 Exp 5
1 BDM CHIR Rspondin1 CHIR
CHIR FGF9 ROCK I 2 FGF2 RA BDM BDM 3 FGF9 ROCK I 4 BDM FGF9 Activin A BDM BDM 5 6 BDM BDM BDM BDM 7 BDM 8 BDM BDM BDM BDM
Table 3.1:Overview of the five experiments to differentiate metanephric nephron progenitors further into more mature nephron structures. The empty boxes in- dicate that the medium was not refreshed at that point. ROCK I: Rock inhibitor Y27632.BDM: basic differentiation medium.
After 6 days of treatment the organoids that could differentiate spon- taneously seem to grow bigger than the rest and showed dark and light regions. Which could indicate internal structures. The organoids of the second experiment showed small density differences within the organoid, their size was in between experiment 1 and the other experiments. The organoids of experiment 3, 4 and 5 were small and had no visible differ- ences in density. For the following days, all organoids became denser (see picture 3.17) and shrank every day. This indication made it very likely that
the organoids were not growing nor differentiating, on which the decision was based to abort the experiments.
Figure 3.17: Taguchi, 2017. Day 17. Representative pictures of organoids at day 17 after aggregation. The numbers correspond to the five experimental conditions (see table: 3.1). The organoids, kept shrinking and appeared to be in a non-vital state.
When executing the Morizane protocol, we expected to grow kidney organoids that would form epithelialised tubular structures. However, our organoids consisted only of podocytes. With the 2014 protocol of Taguchi, we aimed to grow metanephric mesenchyme. However, our organoids expressed, besides the metanephric mesenchyme marker PAX2, also TBX6 and LHX1 which are markers of the late primitive streak and pretubular aggregates respectively. The organoids that were grown ac- cording to the Taguchi protocol of 2017, did express the metanephric mes- enchyme marker PAX2. However, we were unable to let these organoids differentiate further into nephron-like structures.