4 RESULTS
4.4 Recellularization of perfusion-decellularized kidneys
4.4.1 Reendothelialization with hiPSC-derived endothelial cells
The EC differentiation protocol was adapted from Patsch el al.109. In five days the hiPSCs differentiated into a confluent layer of 50,68% CD144 positive endothelial cells. The CD144 positive cells were magnetically sorted and expanded for six more days. During this time the cells matured to CD31/CD144 double positive ECs (Figure 40).
Figure 40: Differentiation scheme of hiPSC-derived endothelial cells. Endothelial cells were differentiated from hiPSCs in a 5-day protocol. At day 5, 50,68% of the cells were CD144+, as shown by flow cytometry.
The CD144+ cells were magnetically sorted and reseeded into expansion medium. After 6 days of expansion, on day 11, 98,54% of the cells matured into CD31/CD144 double positive endothelial cells. Brightfield images show the morphological changes during the differentiation process. Scale bar: 200 μm.
Special focus was laid on the expansion phase of the ECs, since millions of cells are needed to recellularize a whole rat kidney. The criteria for a successful expansion are cell proliferation, measured in population doublings (PDL), and EC marker stability, measured in the proportion of CD144/CD31 double positive cells. Furthermore, the cost of the expansion medium was considered. Patsch et al. proposed the expansion in StemPro-34 medium on fibronectin coating. StemPro-34 medium costs about 500€/l. Although the EC maturation and phenotype stability were excellent, this condition was not suitable for the expansion, since the cells did not proliferate. Additionally, gelatin coating was included as a comparison, since it is much less costly than fibronectin. The marker stability and PDL are comparable to fibronectin coating. EC-SFM medium142 also stabilized the phenotype over the whole tested period of 36 days and supported 11 PDL on fibronectin and costs only about
88 300 €/l. Notably, the PDL curve flattened over time, indicating a decreasing proliferation rate over time. EGM-FCS-SB medium141 stabilized the phenotype for 26 days. Thereafter, the percentage of double positive ECs dropped from 93% to 77%. However, the cell proliferation is higher than with any other tested medium; 16 PDL on fibronectin and 12 PDL on gelatin over the course of 36 days. Therefore, 26 days on gelatin will be sufficient to produce the number of cells needed for reendothelialization. Moreover, EGM-FCS-SB medium is with about 200 €/l by far the cheapest medium and was therefore chosen for the expansion of the hiPSC-derived ECs for the human kidney model.
0 10 20 30 40
0 20 40 60 80 100
Day
% CD144+ CD31+
0 10 20 30 40
0 20 40 60 80 100
Day
% CD144+ CD31+
0 10 20 30 40
0 20 40 60 80 100
Day
% CD144+ CD31+
10 20 30 40
-5 0 5 10 15 20
Day
PDL
10 20 30 40
-5 0 5 10 15 20
Day
PDL
10 20 30 40
-5 0 5 10 15 20
Day
PDL
Fibronectin Gelatin
StemPro-34 EC-SFM EGM-FCS-SB
Figure 41: Optimization of hiPSC-ECs expansion conditions. At day 5 of hiPSC-EC differentiation, the ECs were sorted for CD144 and seeded into three different expansion media with fibronectin or gelatin coating.
The expansion rate, given as population doubling (PDL), and the endothelial phenotype stability, given as the percentage of CD144 and CD31 double positive cells, were analyzed to identify the optimal expansion medium.
The phenotype was stable for 26 days in all conditions. The expansion was highest in EGM-FCS-SB medium on fibronectin coating.
To reendothelialize the rat kidney scaffold, 5x107 hiPSC-derived ECs were injected into the renal artery, analogous to the reendothelialization with HUVECs. The perfusion culture was commenced after an overnight attachment phase and continued for six days.
As already observed in the HUVEC reendothelialization, LDH release was highest shortly after seeding and decreased thereafter. Hence, cells died during seeding but survived thereafter. Glucose and lactate measurement were fluctuating around 0 mmol/d. These measurements are impaired by the high medium volume to cell ratio. The perfusion with resazurin detected a high cell viability in the scaffold, especially in the interlobar and arcuate
89 arteries. These data were corroborated by the histological analysis. The vessels were densely populated with ECs lining the vessel walls (Figure 42).
In comparison to the HUVEC experiment, however, slightly less cell nuclei and metabolic activity were detected. Therefore, it appears that hiPSC-derived ECs are not as robust as HUVECs. Nevertheless, a good reendothelialization result was achieved with hiPSC-derived ECs.
Figure 42: Arterial seeding of hiPSC-ECs. (A) hiPSC-ECs were injected through the IN port of the perfusion bioreactor into the renal artery of the decellularized rat kidney. Perfusion culture started after O/N static culture.
(B) DAPI stained cross-section after 6 days of perfusion culture. ECs were spread over the whole tissue, lining specifically the bigger blood vessels (white arrows), as shown in more detail in (C). (D) The resazurin-assay detected viable cells in the vascular tree. (E) LDH release was highest shortly after perfusion culture start.
Glucose, lactate metabolism was hardly detectable, due to the high medium volume.
CD31 staining on sections of a native human kidney, a HUVEC recellularized kidney and an hiPSC-EC recellularized kidney revealed the exact localization and morphology of the endothelial cells. In the native human kidney, CD31 positive ECs were detected lining the segmental, interlobar, arcuate and interlobular arteries and veins, the afferent and efferent arterioles and the delicate glomerular and peritubular capillaries. The recellularized rat kidneys showed similar staining patterns, which suggests an effective reendothelialization.
The cells did not migrate out of the vascular compartment. They lined the vessels walls, only some cell plugs were found inside the vessels. Merely the microstructure in the glomerular capillaries was not as well formed as in the native kidney.
90
Figure 43: CD31+ endothelial cells lined the blood vessels and the glomerular capillaries in the recellularized kidneys. The delicate structure of the human kidney glomerulus was not achieved in the recellularized kidneys. No differences between HUVEC and hiPSC-EC recellularizations were prominent.
Scale bar: 100 μm.
4.4.2 Recellularization of the kidney parenchyma with hiPSC-derived renal