Journal: Communications Biology

Article Title: Cells sorted off hiPSC-derived kidney organoids coupled with immortalized cells reliably model the proximal tubule

doi: 10.1038/s42003-023-04862-7

Figure Lengend Snippet: a The simplified protocol of hiPSC differentiation into intermediate mesoderm and formation of 3D kidney organoids, followed by dissociation, sorting, and seeding the cells as sorted. Dissociated organoid, LTL + and LTL–, refer to dissociated organoid cells, positive fraction of MACS, and negative fraction of MACS, respectively. b Select brightfield and confocal fluorescent images (z-intensity projected) of kidney organoids on day 22. Immunochemistry is for EpCAM and proximal tubule markers, LTL and megalin. c Effect of LTL concentration (dilution factors of 1/50 or 1/100) on the specificity of MACS, N = 4 experiments and the error bars represent standard deviation of data, * p < 0.05. d Relative gene expression levels obtained from cells as sorted and cultured for 7 days in culture dishes and PToC: Organoid, dissociated organoid cells on day 22; LTL+/–, positive/negative fraction of MACS products. White area corresponds to samples obtained from as-dissociated (organoid) and as-sorted cells, whereas the grayed area indicates samples from cells cultured for 7 days. Error bars represent the standard deviation with N = 3 biologically independent experiments. N.S., not significant for p > 0.05; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001. e Evolution of LTL+ cells cultured on the membrane into aggregates, Scale bar, 200 μm. The yellow frame shows the boundary between the cell aggregate and the monolayer. Scale bar, 200 μm. f Immunochemistry on day 7 for EpCAM, LTL, and megalin, markers of proximal tubules, in sorted cells extracted from kidney organoids and seeded on the PToC. For the LTL+ tissue fluorescent scans were conducted on select parts at the proximity of aggregates as framed in ( e ). Scale bar, 50 μm.

Article Snippet: Stock solutions of 2-NBD-Glucose, fluorescent glucose uptake probe (2-NBDG, Abcam, ab1462002) were prepared at 30 mM and stored at –30 °C for later use.

Techniques: Concentration Assay, Standard Deviation, Gene Expression, Cell Culture, Membrane

Journal: Communications Biology

Article Title: Cells sorted off hiPSC-derived kidney organoids coupled with immortalized cells reliably model the proximal tubule

doi: 10.1038/s42003-023-04862-7

Figure Lengend Snippet: a Timeline (in days) demonstrating the coculture assay. hiPSCs are plated 23 days prior to the coculture as outlined earlier, followed by RPTECs which are subcultured on day –5. Kidney organoids are harvested upon maturation, dissociated, and mixed in equal portions with RPTECs. The cell mixture is then introduced into the chip. b Brightfield images showing the evolution of LTL+ and cocultured tissues with time. The positive fraction-only cells begin to aggregate from ∼D4 and form separable spheroids by D7 (as indicated by red arrowheads), rendering the device impractical for filtration assessments. Whereas no detachment is observed in the coculture with RPTECs. Scale bar, 200 μm. c Z-intensity projected fluorescent images taken from immunostained samples on D7. DAPI, F-actin and EpCAM are represented in blue, yellow, and green, respectively. Once cocultured with RPTECs at a 50/50 ratio, LTL+ cells blend well and make a confluent tissue layer. Coculturing the LTL– fraction with RPTECs however, does not yield a confluent tissue layer. The tissue partially detaches as indicated by white arrowheads. EpCAM is faintly expressed throughout this tissue and mostly in the RPTECs. Gray dashed lines show the whereabouts of the PToC membrane. Scale bar, 200 μm. d Fluorescent, brightfield and merged images of the cocultured tissue on D14. Top rows show that the channel is covered by RPTECs and LTL+ cells equally in its entirety. High magnification pictures at the bottom row elucidate that the dissociated/sorted cells in the mixture not only form small aggregates but are also distributed almost evenly throughout the coculture tissue. Scale bars are in 1 mm and 100 μm on top and bottom rows, respectively. Dashed lines are to guide the eye.

Article Snippet: Stock solutions of 2-NBD-Glucose, fluorescent glucose uptake probe (2-NBDG, Abcam, ab1462002) were prepared at 30 mM and stored at –30 °C for later use.

Techniques: Co-culture Assay, Filtration, Membrane

Journal: Communications Biology

Article Title: Cells sorted off hiPSC-derived kidney organoids coupled with immortalized cells reliably model the proximal tubule

doi: 10.1038/s42003-023-04862-7

Figure Lengend Snippet: a The timetable of generic cell seeding and maintenance processes. HUVECs are not included for the single-layer devices. b Time course of reported resistances of the RPTEC-only (blue circles), bilayer (red squares), and HUVEC-only (green triangles) tissue layers. For the bilayer devices once HUVECs are introduced on D10, the overall resistance jumps upon formation of a confluent endothelial layer and then starts to decline until reaching steady state on D14/d4. Dashed lines indicate the average resistances obtained over the last 4 days of culture (colored bars) for each case. All the measured values are normalized to that of the RPTEC TEER at steady state, i.e. 60 Ω cm 2 (blue dashed line). Green and red two-headed arrows indicate, respectively, the reported resistance of the HUVEC-only layer and the resistance increment measured upon addition of HUVECs to the RPTEC-only tissue. A stable bilayer is formed on D14/d4. RPTECs were cultured on N = 6 devices, out of which N = 3 were rendered bilayer upon addition of HUVECs on D10. N = 3 devices were dedicated for the HUVEC-only tissue. c Immunohistochemistry for ZO-1, EpCAM, and megalin in RPTEC tissue of the bilayer system along with fluorescent images of corresponding RFP-tagged HUVECs, showing the evolution of tight junctions, degree of re-epithelialization, and distribution of the albumin transporter, respectively. Tissue activity and integrity is peaked between D14/d4 and D17/d7. Scale bar, 50 μm. d Effect of HUVECs on the intensity of ZO-1 expression; In the presence of HUVECs tight junctions appeared from D14/d4 (green arrowhead) onward and became clearly visible on D17/d7, while in the absence of HUVECs they appeared faintly, even at a considerably higher confocal laser transmissivity. No Triton X was used in this experiment. Scale bars are 100 μm. All fluorescent images are confocal z-intensity projected. Scale bar, 100 μm.

Article Snippet: Stock solutions of 2-NBD-Glucose, fluorescent glucose uptake probe (2-NBDG, Abcam, ab1462002) were prepared at 30 mM and stored at –30 °C for later use.

Techniques: Cell Culture, Immunohistochemistry, Activity Assay, Expressing

Journal: Communications Biology

Article Title: Cells sorted off hiPSC-derived kidney organoids coupled with immortalized cells reliably model the proximal tubule

doi: 10.1038/s42003-023-04862-7

Figure Lengend Snippet: a Select cross sectional fluorescent confocal image of the RPTEC/HUVEC bilayer system, immunostained for megalin, illustrating the definition of the relative distance of the protein of interest (e.g. megalin) measured from the center of nuclei. Scale bars are 10 μm. b Representative z-intensity profiles of fluorescent signals obtained by averaging emission intensities of various markers throughout a laser scan area of 0.1 cm 2 with a Δ z (pitch) of 200 nm. Average proximal tubule-specific marker-to-nuclei distance in RPTECs (bilayer) obtained under static and perfused culture conditions for ( c ) megalin, ( d ) LTL, and ( e ) SGLT2; N = 2 independent chips were used to make n = 3 random measurements from each; Error bars indicate standard deviation. Statistically significant differences between data pairs are indicated by asterisks, *, **, ***, ****, for p ≤ 0.05, 0.01, 0.001, and 0.0001, respectively. Select cross-sectional TEM images highlight the appearance of the apical membrane of RPTECs in various culture conditions on D14/d4: ( f ) single layer under static, ( g ) bilayer under static, and ( h ) single layer under perfusion culture conditions. The close-up TEM of ( i ) reveals a tight junction (yellow arrowhead) formed between two adjacent RPTECs under perfusion culture. j A HUVEC at the opposite side of the membrane. Scale bars are 2 μm. All the micrographs are from the cells fixated on D14/d4. M, mitochondria; N, nucleus; V, vacuole; mem, PET membrane, arrows point to tight junctions, and the dashed lines show cell-cell boundaries. k Quantification of the microvilli length/density and the RPTEC height. We define the villi density as the count of protrusions divided by the length of the cell membrane cross-section periphery, measured from individual snapshots. RPTECs/HUVECs are on D14/d4. Clearly RPTECs in the proximity of HUVECs developed denser apical microvilli, resulting a higher surface area. In addition, the microvilli density distribution is narrower in the bilayer configuration. No significant difference in villi lengths was observed between the single layer and bilayer cases in static culture condition. Flow induced shear stress manifested not only longer but also a larger density of microvilli. For quantification purposes, from a total of N = 3 independent TEM observations per condition, n = 9, 5, and 5 RPTEC micrographs were randomly selected from single layer (static), bilayer (static), and single layer (flow) culture conditions, respectively, to measure villi lengths and densities. To measure cell heights, n = 8 and n = 9 micrographs were examined for static and flow conditions, respectively. Error bars represent the standard deviation of data. l Average distances of basement and apical proteins from the nucleus illustrated for RPTEC-only and coculture tissues developed under flow culture condition. Data obtained by z-intensity profiling of fluorescent images. Statistics are derived in a similar fashion to ( c – e ).

Article Snippet: Stock solutions of 2-NBD-Glucose, fluorescent glucose uptake probe (2-NBDG, Abcam, ab1462002) were prepared at 30 mM and stored at –30 °C for later use.

Techniques: Marker, Standard Deviation, Membrane, Shear, Derivative Assay

Journal: Communications Biology

Article Title: Cells sorted off hiPSC-derived kidney organoids coupled with immortalized cells reliably model the proximal tubule

doi: 10.1038/s42003-023-04862-7

Figure Lengend Snippet: a The generic protocol for quantification of renal reabsorption and excretion using the PToC. Measurements commence on day 14 irrespective of the assay in hand and the tissue layer configuration. The media in both epithelial and vasculature microchannels are circulated to improve diffusion of large-molecule substrates (e.g. BSA-AF488). Transfer rates (reabsorption of albumin/glucose or apparent permeability to Rh123) were estimated by conducting linear regression on time course data. b Relative expression levels (vs. ACTB ) of structural genes, CDH6 (K-Cadherin) and EpCAM , and functional genes, namely, ABCB1 ( MDR1 ), AQP1 , SLC22A2 (OCT2), SLC5A2 (SGLT2), vs. the level of ACTB gene in the RPTEC-only and RPTEC/HUVEC systems. Four groups are presented, namely, (i), RPTECs on D14 in the culture dishes; (ii), RPTECs of the bilayer under static culture condition; (iii), RPTECs of the bilayer under perfused culture conditions; (iv) Single layer RPTECs under perfused media. Proximity with HUVECs under media perfusion has augmented the expression levels of AQP1 , EpCAM , MDR1 , OCT2 , and SGLT2 (compare groups iii and iv). Independently, perfusion has increased the expression levels of the same genes (with the possible exception of SGLT2 ) in the co-culture (compare groups ii and iii). n = 5 PCR replicates were analyzed for each gene/sample obtained from one set of experiment. c Transfer rates of the glucose probe, 2NBDG measured in static and perfused culture conditions. Both reabsorption (a → b) and reverse transfer rates (b → a) were quantified. The inhibitory effect of phlorizin (P.) on glucose transport was also examined; n = 3 independent chips. d Apparent permeabilities to Rh123 in both excretion (b → a) and reverse (a → b) directions. Verapamil (V.), a contender for Rh123 extrusion, was applied to attenuate Rh123 efflux and confirm the function of Pgp; N = 3 independent chips. e Fluorescent confocal z-stacked images of the RPTEC tissue layer in bilayer and single layer configurations. A considerably higher amount of BSA was precipitated in the basolateral milieu of RPTECs in the bilayer system indicating a higher intake of the substrate. Some BSA is precipitated in the single layer RPTEC on D17 (white arrowhead). Scale bar is 50 μm. f Transport rates of AF488-conjugated bovine serum albumin (BSA-AF488) in both directions. In this study, we examined the effect of reducing the incubation temperature (to 4 °C) on selective transport of BSA-AF488; minimum N = 6 independent chips. In ( b – d , f ) two-sample t-test was run between pair of data sets as indicated. Error bars represent standard deviation; N.D., not detected; N/A, not available; N.S., not significant; *, **, ***, ****, for p ≤ 0.05, 0.01, 0.001, and 0.0001, respectively. a → b, apical to basal; b → a, basal to apical.

Article Snippet: Stock solutions of 2-NBD-Glucose, fluorescent glucose uptake probe (2-NBDG, Abcam, ab1462002) were prepared at 30 mM and stored at –30 °C for later use.

Techniques: Diffusion-based Assay, Permeability, Expressing, Functional Assay, Co-Culture Assay, Incubation, Standard Deviation