Towards an Autologous Ipsc-Derived Patient-On-A- Chip; Ramme Et Al

Paper supplements: Towards an autologous iPSC-derived patient-on-a- chip; Ramme et al.

Supplementary material and methods Flow cytometry Single cell suspension surface staining was performed, according to the manufacturer’s protocols, with the following antibodies: TRA-1-60-PE, SSEA-1-PE-Vio 770, SSEA-5-VioBlue, CD90-FITC, CD105•PE, CD73-APC, CD45-VioBlue and CD34-PE (Miltenyi Biotec). Intracellular staining was carried out with a Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific), according to the manufacturer’s protocols, with the following antibodies: Oct3/4 Isoform A-APC, Sox2-FITC, Nanog Isoform A-APC, (all antibodies purchased from Miltenyi Biotec). Data was acquired on a MACSQuant® Analyzer 10 (Miltenyi Biotec) flow cytometer and analyzed with FlowLogic (Miltenyi Biotec) software. Gates were adjusted according to the respective isotype controls.

Barrier genes high-throughput multiplex qPCR A total of 250 ng RNA per sample were transcribed to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Cat. No. 4368814), according to the manufacturer’s protocol. Ninety-four targets were investigated: PPIA, 18SrRNA, b-Actin, GAPDH, B2M, Claudin-1, Claudin-2, Claudin-3, Claudin-4, Claudin-5, Claudin-6, Claudin-7, Claudin-8, Claudin-9, Claudin-10a, Claudin-10b, Claudin-11, Claudin-12 tv1, Claudin-12 tv2, Claudin-12 tv3, Claudin-14, Claudin-15, Claudin-16, Claudin-17, Claudin-18 tv1b, Claudin-18 tv2a, Claudin-19, Claudin-20, Claudin-22, Claudin-23, Claudin- 24, Claudin-25, ZO-1, ZO-2, ZO-3, Jam-1, Jam-2, Jam-3, Occludin, Tricellulin, ABCB1, ABCC1, ABCC2, ABCC3, ABCC4, ABCC5, ABCG2, CAT1, CAT3, ENT1, LAT1, MCT1, MCT8, SLC1A1, SLC2A1, Insulin receptor, Transferrin receptor, LRP1, LRP8, E-cadherin, CDH5, VEGF, b-catenin, Vimentin, Fibronectin, Filagrin, E-selectin, vWF, Cytokeratin-1, Cytokeratin-4, Cytokeratin-5, Cytokeratin-8, Cytokeratin-10, Cytokeratin-13 tv1, Cytokeratin-13 tv2, Cytokeratin-14, Cytokeratin-16, Cytokeratin-18, Cytokeratin- 19, Aquaporin-1, Aquaporin-3, Aquaporin-4, Aquaporin-7, Aquaporin-10, Aquaporin-11, Mucin-1A, Mucin-1B, Mucin-2, Mucin-3A, Mucin-4, Mucin-5AC, Mucin-13, Mucin-18 and Mucin-20. They were preamplified using tenfold concentrated primer pools mixing with Qiagen Mastermix applying the following program: 15 min at 95 °C for HotStar Plus Taq Polymerase (Qiagen, Cat. No. 203603), 18 cycles (with 40 s at 95 °C, 40 s at 60 °C, 80 s at 72 °C) and 7 min at 72 °C. High-throughput qPCR was accomplished with a BiomarkTM System containing an IFC Controller HX and 96.96 Dynamic ArraysTM IFC, according to the manufacturer’s instructions. In brief, 96 sample wells were loaded with DNA Mix containing Tagman GeneExpression Mastermix, DNA binding dye sample loading reagent, EvaGreen binding dye and 1:8 diluted preamplified cDNA, whereas 96 target wells were filled with the Assay mix containing Assay loading reagent and according primers. After qPCR and data allocation, the Ct values of the targets were normalized to endogenous control PPIA. The ΔCt values were used for the following statistical analysis (PCA, Heatmaps) applying the software Qlucore Omics Explorer 3.3.

Supplementary Data iPSCs The integration-free StemUse101 was reprogrammed by episomal vectors containing the factors Oct4, Sox2, Lin28, Klf4 and L-Myc. This line was characterized by flat colony growth with high cell-cell contact morphology and minimum spontaneous differentiation (Figure S 1). Immunostaining is positive for the nuclear markers Nanog and OCT3/4, and the surface markers Tra-1-60 and SSEA5; the early differentiation markers SOX17 and nestin are negative (Figure S 3). Additionally, flow cytometry

1 analysis shows 98 % TRA-1-60+/SSEA1- cells (Figure S 2 e). SSEA1 is an early differentiation marker and, therefore, used as a negative surface marker of human iPSCs. A total of 99 % of the cells are positive for TRA-1-60 and SSEA5 (Figure S 2 f). Furthermore, 92 % of the cells show double positive expression of the intracellular marker Oct3/4 and SOX2 (Figure S 2 g). A total of 94 % of the cells are Nanog+ (Figure S 2 h). In addition, qPCR showed a high expression for stem cell markers OCT3/4, NANOG and SOX2 and low expression for endoderm marker FOXA2 and mesoderm marker Brachyury (T) and Desmin (Figure S 1 b). Intestinal tissue Several groups have already shown the direct differentiation of iPSCs into human intestinal organoids and further maturation and expansion in a 3D environment in Matrigel 1–3. Firstly, iPSCs were differentiated into a DE stage proved by FOXA2+/CXCR4+ cell expression (Figure S 2), proved by eight different iPSC lines (data only shown for the StemUse101 iPSC line). Furthermore, the mid- and hindgut stage was induced by KGR and retinoic acid over seven days 2. A dense 3D multilayer of cells formed, which was dissected into small pieces and transferred to a 3D Matrigel microenvironment. In comparison to other protocols, we did not only take the detached spheroids during the first transfer into Matrigel, instead we used small pieces of the complete 3D multilayer of cells for transfer. Nevertheless, we were repeatedly successful in generating self-assembled 3D intestinal organoids (Figure S 5 B and C) with two different iPSC lines (data only shown for StemUse101). The presence of Wnt activator (R-Spondin 1), epidermal growth factor (EGF) and BMP signaling inhibitor (Noggin) in the culture medium are essential for the continuous culture of intestinal organoids 4, 5. The latter were expanded for at least ten passages, although they were subsequently frozen due to time limitations. The mesenchyme differentiates in parallel with the epithelium, as we obtained rapidly self-renewing epithelium stained by Na+/K+-ATPase and Cytokeratin 8/18 as well as mesenchyme positive cells laying around the organoids positive for vimentin (data not shown). Evaluation of intestinal organoids was performed by qPCR (Figure S 5 A). The endoderm marker CXCR4 (C-X-C chemokine receptor type 4) is expressed more highly in the organoids than in the adult small intestine positive control as expected. We saw an expression of the hindgut marker KLF5 and the intestinal stem cell marker SOX9 and LGR5. The presence of small intestinal epithelia cells was shown in CDH1 (E-cadherin), CDX2 and DPP4 (Dipeptidyl peptidase-4). Enteroendocrine cell (CHGA – Chromogranin A), paneth cell (LYZ), goblet cell (MUCs) and enterocyte (Villin) markers could be detected. In addition, we could also see the expression of different cell membrane transporters, such as glucose transporter 1 (GLU1/SLC2A1), glucose transporter 3 (GLUT3/SLC2A3), multidrug resistance protein 1 (MDR1/ABCB1/P-gp) or Na+/K+-ATPase (ATP1A1), which indicate molecular transport capacity. Furthermore, the intestinal organoids can be thawed after cryopreservation and still show comparable morphology and gene expression (data not shown). Stromal cells were characterized by flow cytometry positive for CD90+ (95 %), CD73+ (87 %) and CD105+ (38 %) and negative for TRA-1-60-, CD45- and CD34- (data not shown) before they were co- cultured with the intestinal organoids. The iPSC markers NANOG, SOX2 and OCT3/4 were highly downregulated in intestinal organoids observed by qPCR (data not shown). Liver equivalents The DE differentiation in the monolayer follows hepatoblast differentiation with 1 % DMSO treatment for iPSC-derived hepatocyte differentiation 6. The maturation of the hepatoblast was achieved by promoters of hepatocyte differentiation HGF, oncostatin M and FGF 4, as well as dexamethasone, which induces the expression of mature hepatic-specific genes 7–9. At day 16 of the monolayer differentiation, the cells have characteristic hepatocyte morphology with polygonal-shaped cells with tight cell–cell contact, central single and multi-nucleated cells, and canaliculi-like structures between the hepatocytes (Figure S 6 B) 10. Liver capability to perform phase I and phase III metabolism was shown by the gene expression of CYP2E1, CYP3A4 and MRP2, respectively. Additionally, liver gene 2 expression of the fetal form of serum albumin – AFP, albumin, BSEP and CPS1 (Carbamoyl phosphate synthetase-1) were present. Moreover, it was shown that the liver equivalent formed by hepatocytes and stroma cell co-culture would lead to further differentiation present by the upregulation of albumin and MRP2 (Figure S 6 A). Interestingly, the bile-salt export pump is only expressed in spheroids and cannot be detected in the hepatocytes in the monolayer. Renal organoids Renal organoids were produced from iPSCs by mesoderm induction by CHIR99021 and Activin A. The renal vesicles were induced by FGF9 and CHIR99021 pulse 11, 12. Organoid self-assembly was already seen on day 12 of differentiation (Figure 3 A), predominantly in the denser part of the cell culture dishes. Renal cells showed a higher expression of JAG1, an early nephron marker, on day 12 of differentiation in comparison to the adult kidney (Figure 5 O: day -6 chip = day 12 static culture). Aquaporin, CD13, claudin 10 and megalin expression was lower in the renal cells on day 12 of differentiation in comparison to the adult kidney, as expected due to the early differentiation time point. Many other cells differentiated in addition to the renal organoid structure. Neuronal spheroids Differentiation of 3D iPSC aggregates to cortical neurospheres in a bioreactor system was induced by the inhibition of SMAD signaling via the application of transforming growth factor β signaling inhibitor SB431542 and bone morphogenetic signaling inhibitor LDN193189 13. When neural induction was induced, the iPSC spheroids had an average diameter of 113 µm, which increased over 32 days of cultivation to an average diameter of 344 µm. Vitality of the spheroids at day 32 was shown by fluorescein-diacetate and propidium iodide staining (Figure S 7 B). Neural tube-like structures with a distinct ZO-1 positive lumen surrounded by TUBB3 positive regions (Figure 3 D) were found inside the spheroids. Furthermore, a distinct fraction of PAX6-positive cells was distributed equally throughout the spheroid, which is a key regulator of brain development (Figure S 7 C)

Gene expression analysis showed high expression of the neural stem cell markers SOX2, PAX6 and nestin, compared to adult human brain tissue, which is expected due to the immature status of the spheroids. Expression of iPSC markers Nanog and Oct-4 were still detectable, indicating the iPSC origin of the spheroids. Expression of neuronal markers, such as ENO-2, SYP, TUBB3 and MAP2, and cortical markers, such as CTIP2, OTX1, SATB2 and TBR1, were detectable but at a lower level, with the exception of TUBB3 and OTX1, compared to adult human brain tissue. OTX 1 was only found in the developing cerebral cortex, which is why it was not detected in the primary brain sample. The high expression of TUBB3 together with the absent expression of the astrocyte marker GFAP indicated the predominantly neuronal identity of the spheroids at this time point (Figure S 7 D). When neurospheres were plated on Matrigel-coated plates, subsequent formation of neural rosettes and neuronal outgrowths were observed (data not shown). Regarding the chip, cultured neurospheres were transferred into 1-µm pore size 96-well Transwell inserts (Corning) to mimic the natural separation of the brain from the blood system. While only being a technical membrane in this setup, this design will allow the establishment of an endothelial cell-based blood-brain barrier equivalent in future experiments.

Supplementary Figures a b

Figure S 1: Representative iPSC characterization of the StemUse101 line. a: Microscopic images of StemUse101 iPSC colony morphology. Scale 250 µm. b: Transcriptional levels of pluripotency genes (OCT3/4, NANOG and SOX2) and early differentiation genes (FOXA2, T and DES) analyzed by qPCR of StemUse101 iPSCs in passage 17. The geometric mean with geometric standard derivation is shown.

Figure S 2: Flow cytometry characterization of StemUse101 iPSCs in passage 17. a – d: Isotype controls for gate adjustment. e: iPSCs show high TRA-1-60 expression and no expression for the early differentiation marker SSEA1. f: all iPSCs are double positive for the pluripotency marker TRA-1-60 and SSEA5. g: high double co-expression of SOX2 and OCT3/4. h: high expression of Nanog. %P = % of live single-cell population.

a b c

OCT3/4 TRA-1-60 OCT3/4 TRA-1-60 DAPI

d e

SSEA5 SSEA5 DAPI

f g h

NANOG NANOG DAPI Neg. Neg. DAPI i j k

SOX17 Nestin SOX17 Nestin DAPI

Figure S 3: StemUse101 characterization by immunofluorescence staining. a. OCT3/4 (red); b: TRA-1-60 (green); d: merge of OCT3/4, TRA-1-60 and DAPI; d: SSEA5 (green); e: merge of SSEA5 and DAPI; f: NANOG (red); g: merge of NANOG and DAPI; h: negative control without primary antibodies; i: SOX17 (red); j: nestin (green); k: merge of SOX17, nestin and DAPI. Scale: 100 µm.

Figure S 4: Definitive endoderm characterization of StemUse101 iPSCs by flow cytometry. High expression of the DE marker CXCR4 (d) and FOAX2 (e). Gating was adjusted to isotype controls (a – c). %P = % of live single-cell population.

B C

Figure S 5: Gene expression of intestinal organoids in static culture in comparison to a primary adult small intestine sample (A). Intestinal organoids morphology in passage 6 in 3D Matrigel (B and C). Scale: 500 µm. The geometric mean with geometric standard derivation is shown.

B C Albumin DAPI

Figure S 6: Gene expression of iPSC hepatocytes in static monolayer culture in comparison to iPSC liver spheroids with iPSC- derived stromal cells (A). Hepatocyte morphology (B) and albumin expression (C) on day 16 of differentiation. ND = no signal detected. Statistics were performed with the multiple T test using the Original FDR method of Benjamini and Hochberg. Each row was analyzed individually, without assuming a consistent SD. The geometric mean with geometric standard derivation is shown.

A B PI FDA DAPI C TUBB3 PAX6 DAPI

Figure S 7: Neurosphere morphology at day 32 of bioreactor DasBox cultivation (A). Live/dead staining with FDA (green) and PI (red). (C) TUBB3 (red) and PAX6 (green) staining in neurospheres. Scale: 200 µm. Gene expression of iPSC-derived neuronal spheroids hepatocytes in 3D spheroid DasBox culture in comparison to a primary adult brain sample (D). ND = no signal detected. The geometric mean with geometric standard derivation is shown.

adult positive control A d0 d7 ADME-MOC d14 ADME-MOC

intestine renal

B d0 d7 ADME-MOC d14 ADME-MOC

Figure S 8: Heatmap analyses of the high-throughput multiplex barrier qPCR data of the intestinal and kidney model on day 0 (blue), day 7 (yellow) and day 14 (pink) of co-culture in the ADME-MOC. An intestinal and renal adult positive sample was measured as a comparison (white). Forty-nine of 94 significant different target genes are shown. A multigroup comparison at p = 0.05 was performed. Three replicates per time point (A). Principle component analysis of the high-throughput multiplex barrier qPCR data of the intestinal and kidney model cultivated for 14 days in the ADME-MOC (B).

Figure S 9: KeyGenes prediction with the human adult training set (A) and the human fetal training set (B) on RNA sequencing data samples from the ADME-MOC experiment. The identity scores range from zero (black – no match) to one (green – high match). The rows represent the 11 (A) or 22 (B) organs from the human training set and the columns depict the ADME-MOC co-cultured RNA samples. Light grey and green boxes show the predicted tissues for the given samples by KeyGenes.

Figure S 10: iPSC-liver pathway. Time points of the liver were used as replicated and compared to the five iPSC replicates for differential expression analysis. Red: Expression is higher in the liver model in comparison to the iPSCs. Green: Expression is lower in the liver model in comparison to the iPSCs.

Figure S 11: iPSC-liver pathway. Time points of the liver were used as replicated and compared to the 5 iPSC replicates for differential expression analysis. Red: Expression is higher in the liver model in comparison to the iPSCs. Green: Expression is lower in the liver model in comparison to the iPSCs.

Figure S 12: iPSC-neuronal axon guidance pathway. Time points of the neuronal model were used as replicated and compared to the 5 iPSC replicates for differential expression analysis. Red: Expression is higher in the neuronal model in comparison to the iPSCs. Green: Expression is lower in the neuronal model in comparison to the iPSCs.

Figure S 13: iPSC-intestine WNT signaling pathway. Time points of the intestinal model were used as replicated and compared to the 5 iPSC replicates for differential expression analysis. Red: Expression is higher in the intestinal model in comparison to the iPSCs. Green: Expression is lower in the intestinal model in comparison to the iPSCs.

Supplementary Table Table S 1: Real-time qPCR primers.

Gene Full gene name Primer sequence 5’ to 3’ up and down CTTCCAAACAAAGGCAGCAACAG AFP Alpha Fetoprotein TCCTGCAGACAATCCAGCAC TCAGCTCTGGAAGTCGATGAAAC ALB Albumin AGTTGCTCTTTTGTTGCCTTGG CACACACAACTTCAGCAACCAC AQP1 Aquaporin 1 TCGGCATCCAGGTCATACTC BSEP / AACCAGGACGAAAGACAAATATCG ATP-binding Cassette, Subfamily B Member 11 ABCB11 GATTTTCCTCTCCTTTGCTCTGC AACCTCAATGTGACGGGCTATTAC CD13 / AAP Alanine Aminopeptidase CAATCAGGAAGAGGGTGTTGTTCA GATGAAGAAGGAGGCGGAGAAG CDH1 Cadherin 1 GCAGGGCGGGGAAGATAC AACCAGGACGAAAGACAAATATCG CDX2 Caudal Type Homeobox 2 GATTTTCCTCTCCTTTGCTCTGC CGAGGAAGAAGGCCCCACTG CHGA Chromogranin A TGCTCCTGTTCTCCCTTCCCT GGGCGATGCCAGAATAGATGCC CTIP2 B cell CLL/lymphoma 11B CGCCACACTGCTTCCTTTTGTG GGGCTGTGCTCAATGACTGG CLDN10 Claudin 10 GCCCCGTTGTATGTGTATCTGG CCCAGCCTCTCTTCCATCAG CPS1 Carbamoyl-Phosphate Synthase 1 GCGAGATTTCTGCACAGCTTC Cytochrome P450 Family 2, Subfamily A GTACCCTATGCTGGGCTCTGTG CYP2A6 Member 6 CCTTAGGTGACTGGGAGGACTTG Cytochrome P450 Family 3, Subfamily A GGAAGTGGACCCAGAAACTGC CYP3A4 Member 4 TTACGGTGCCATCCCTTGAC Cytochrome P450 Family 2, Subfamily E CATCAAGGATAGGCAAGAGATGC CYP2E1 Member 1 TCCAGAGTTGGCACTACGACTG GCAGCAGGTAGCAAAGTGACG CXCR4 C-X-C Motif Chemokine Receptor 4 GTAACCCATGACCAGGATGACC CAAAGCCCTGGTCGATGTTG DPP4 Dipeptidyl Peptidase 4 TGGCATGGTATTTTGAGGTGCT CCTGAAGCTGAGCCTGCAAC DES Desmin ACAACCTGCTCGACGACCTG TCAGGGACTATCCTGTGGTCTCC ENO2 Enolase 2 TTCCACTGCCCGCTCAATACG CGACGACATGTTCATGGAGC FOXA2 Forkhead Box A2 AGGGCTACTCCTCCGTGAG GGAACAGCAAAACAAGGCGCT GFAP Glial Fibrillary Acidic Protein GTGGCTTCATCTGCTTCCTGTCT GLUT1 / TCATTGTGGGCATGTGCTTC Solute Carrier Family 2 Member 1 SLC2A1 CAGCTCCTCGGGTGTCTTGT GLUT3/ TGGTTTATTGTGGCCGAACTC Solute Carrier Family 2 Member 3 SLC2A3 GGTAATGAGGAAGCCGGTGA ATGGGAACCCGATCAAGGAA JAG1 Jagged 1 TCCGCAGGCACCAGTAGAAG TTTGGAGAAACGACGCATCC KLF5 Kruppel Like Factor 5 GTAGTGGCGGGTCAGCTCAT Leucine-Rich Repeat Containing G Protein- AGTGCTGTGCATTTGGAGTGTG LGR5 Coupled Receptor 5 GAAGGGCTTTCAGGTCTTCCTC Low-density Lipoprotein-related Protein 2 or CAGGTGACAAAATGGAATCTCTTC LRP2 Megaline TTGGAGTTGGGTCTCTTCTCG CCTAGCAAACTGGATGTGTTTGG LYZ Lysozyme TAACTGCTCCTGGGGTTTTGC GTGGCTCTCTGAAGAACATCCGC MAP2 Microtubule Associated Protein 2 TCTGCCTGGGGACTGTGTAATG

MDR1 / TGGATGTTTCCGGTTTGGAG ATP Binding Cassette Subfamily B Member 1 ABCB1 TGTGGGCTGCTGATATTTTGG GCATCCACAGACATCAGGTTCAC MRP2 / ABCC2 Multidrug Resistance-associated Protein 2 CTGCGGCTCTCATTCAGTCTTTC CCGACCTCCAGCACAGTTTT MUC-2 Mucin-2 GATCCTTGACCGACCTGCAC Na+/K+-ATPase Sodium/Potassium-transporting ATPase Subunit ACAGCCCAGAAATCCCAAAAC / ATP1A1 Alpha-1 CAGCGGTCATCCCAGTCC TCCACCAGTCCCAAAGGCAA NANOG SRY-Box 2 TCTTCACCTGTTTGTAGCTGAGGTT CCTCAGCTTTCAGGACCCCAAG NES Nestin GAAAGGCTGGCACAGGTGTCT OCT3/4 / CCGAAAGAGAAAGCGAACCAGTATC Octamer-binding Transcription Factor 4 POU5F1 GAGACCCAGCAGCCTCAAAATC AACCGAGCAAGACAAGCCACTC OTX1 Orthodenticle Homeobox 1 ATGCCGTATGGGGGTTGTTTGAG GGCAACCTACGCAAGATGGCT PAX6 Paired Box 6 GCTGCTAGTCTTTCTCGGGCA CCAACAGATTGCCGTTAGCCG SATB2 SATB Homeobox 2 AGACAACAATCCCTGTGTGCGG ATGTCCCAGCACTACCAGAG SOX2 SRY-Box 2 GCACCCCTCCCATTTCCC GCCAGGTGCTCAAAGGCTAC SOX9 SRY-Box 9 CGCTTCTCGCTCTCGTTCA CAAGGAGATGCCTGTCTGCCG SYP Synaptophysin TTCAGGAAGCCGAACACCACC CCCTATGCTCATCGGAACAA T Brachyury ACAGGCTGGGGTACTGACTG AGGGAAGGCGCATGTTTCCTTT TBR1 T-Box, Brain 1 TGCACATTGGTGTCCGCTTTG CAGCAAGGTGCGTGAGGAG TUBB3 B Tubulin III TGCGGAAGCAGATGTCGTAG GGCCTCTGCCCATCTTCC VIL1 Villin AGGTTTTGTTGCTTCCATCGAG Housekeeper GTTGATATGGTTCCTGGCAAGC EF1 Elongation Factor 1-alpha GCCAGCTCCAGCAGCCTTC CCTTGTGCTCACCCACCAAC TBP TATA-Box Binding Protein TCGTCTTCCTGAATCCCTTTAGAATAG Succinate Dehydrogenase Complex TCCAGGGGCAACAGAAGAAG SDHA Flavoprotein Subunit A TTGTCTCATCAGTAGGAGCGAATG

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