Chemically-Defined, Xeno-Free, Scalable Production of hPSC-Derived Definitive Endoderm Aggregates with Multi-Lineage Differentiation Potential

For the production and bio-banking of differentiated derivatives from human pluripotent stem cells (hPSCs) in large quantities for drug screening and cellular therapies, well-defined and robust procedures for differentiation and cryopreservation are required. Definitive endoderm (DE) gives rise to respiratory and digestive epithelium, as well as thyroid, thymus, liver, and pancreas. Here, we present a scalable, universal process for the generation of DE from human-induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs). Optimal control during the differentiation process was attained in chemically-defined and xeno-free suspension culture, and high flexibility of the workflow was achieved by the introduction of an efficient cryopreservation step at the end of DE differentiation. DE aggregates were capable of differentiating into hepatic-like, pancreatic, intestinal, and lung progenitor cells. Scale-up of the differentiation process using stirred-tank bioreactors enabled production of large quantities of DE aggregates. This process provides a useful advance for versatile applications of DE lineages, in particular for cell therapies and drug screening.

Total number of DE cells after differentiation compared to adherent 2D culture (n = 3-5). (g) Growth rate of CA and SD conditions from d0 to d3 of differentiation compared to adherent 2D culture (n = 3-5). Scale bars, 100 μm. Each value of gene expression was first normalized to the reference gene(s) and then to d0 undifferentiated cells. Values are represented as the mean ± SEM. *P<.05, **P<0.01, ****P<0.0001. All n values correspond to independent experiments.
IF staining for Hepatic Differentiation Cells were washed three times with phosphate-buffered saline (PBS), fixed in 4% formaldehyde for 20 min at 4 °C, washed three times with PBS and permeabilized with 0.2% Tween 20 (Roth #9127.1) plus 0.1% IGEPAL® CA-630 (Sigma #I8896) in Tris-buffered saline (TBS) for 20 min. The permeabilized cells were washed three times with TBS, blocked with 3% BSA in TBS for 1 h, and washed once with TBS. Then, the cells were incubated with primary antibodies (Supplemental Figure 1) in TBS containing 1% BSA over night at 4°C. The next day cells were washed three times with TBS and incubated with secondary antibodies (Supplemental Figure 2) diluted in TBS 1h at RT. Cells were washed three times with TBS and counterstained with DAPI (Invitrogen #D1306). Staining was analyzed using an Olympus IX71 microscope equipped with appropriate filters for fluorescence detection, and images were processed using the cellSens software (Olympus).

IF staining for Pancreatic Differentiation
Differentiated cells were fixed for 10-20 min with 4% paraformaldehyde. Cell culture slides were washed with PBS and subsequently blocked for 20 min in PBS plus 0.2% Triton X-100 and 1 mg/ml NaBH4 and 5% donkey serum (Dianova #017-000-121). Primary and secondary antibodies (Supplemental Figure 1 and 2) were diluted in PBS with 0.1% Triton X-100 plus 0.1% donkey serum. Primary antibodies were incubated overnight at 4°C. Secondary antibodies were incubated for 1 h at room temperature. Nuclei were stained with DAPI. Images of pancreatic differentiation were taken using the Olympus IX81 microscope. Image processing was performed with cellSens software (Olympus).

IF Staining for Intestinal and Lung Differentiation
Cells were fixed with 4% PFA for 20 minutes and washed 3 times with PBS. The cells were incubated in blocking-permeabilization solution (TBS, 5% donkey serum, 0.025% Triton-X 100) for 20 minutes and then washed 3 times with PBS. Primary antibody (Supplemental Figure 1) was added for 1 hour at RT. The cells were washed 3 times with PBS, and incubated with secondary antibody (Supplemental Figure 2) for 1 hour at RT. The cells were washed 3 times with PBS, incubated with DAPI for 4 minutes, and washed with PBS. Images were taken with microscopes Zeiss Observer A1 and Z1. Image processing was performed with AxioVision software.
Total RNA isolation, reverse transcription and qRT-PCR For DE, intestinal and lung gene analysis, cell samples were collected in 500 µL Trizol® reagent to maintain RNA integrity and stored at -80 °C before further processing. For RNA isolation, samples were thawed and 100 µL chloroform was added. The samples were centrifuged at 12,000× g at 4 °C, leading to phase separation. The upper aqueous phase was transferred into a new tube and total RNA was isolated by use of the NucleoSpin® RNA II Kit (Macherey-Nagel #NZ740955250). The RevertAid™ H Minus First Strand cDNA Synthesis Kit (Thermo Fisher #K1632) was used to generate cDNA from 500 ng of RNA. qPCR was performed using Absolute™ qPCR SYBR® Green Mix (Thermo Fisher #AB1159A). The cDNA was diluted 1:5 and 5uL of cDNA was added to each 25 µL reaction. Each sample was run in duplicate and Ct values were averaged for analysis. For qRT-PCR analysis FOXA2, and SOX17, CDX2, and NKX2.1, and p63 were measured in duplicates, normalized to the housekeeping genes EEF1A1 and RPL0 and run on the Mastercycler® Realplex 2 (Eppendorf). Relative gene expression was normalized to the reference genes and to undifferentiated pluripotent stem cells. Primer sequences are listed in Supplemental Table 3.
For hepatic gene analysis, RNA was isolated using the peqGOLD Total RNA Kit (Peqlab #12-6634-02) according to the manufacturer's protocol. For cDNA synthesis, the SuperScript™ First-Strand Synthesis System (Invitrogen #11904-018) was used according to the manufacturer's protocol and reverse transcribed. For qRT-PCR analysis of hepatic cells, TaqMan gene expression assays and TaqMan 2× Master Mix (Applied Biosystems #4304437) for AFP, ALB, HNF4, TTR, CK7, CK19, CYP3A4 were used. The reactions were run in triplicate on a Step One Plus Real-Time PCR System (Applied Biosystems). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the endogenous control gene. Taqman sequences are listed in Supplemental Table 3.
For pancreatic gene analysis, RNA was isolated using the NucleoSpin® RNA Plus Kit (Macherey-Nagel) according to the manufacturer´s protocol. Then RNA was reverse transcribed using the RevertAid™ H Minus First Strand cDNA Synthesis Kit (Thermo Fisher #K1632) and 1 ng/µL of cDNA was applied in each 10 µL qPCR reaction performed using GoTaq® qPCR Master Mix (Promega #A6001). For pancreatic cells PDX1, SOX9, HLXB9 and HNF1B were measured in triplicates on a ViiA7 Real-Time PCR system (Applied Biosystems). Normalization was performed against the geometric mean of the housekeeping genes G6PD, TBP and TUB1A1. Primer sequences and are listed in Supplemental Table 3.

Definitive Endoderm Differentiation (CA)
Geltrex™ (Gibco #A1413202)   4. After 24 hours, collect aggregates in a 50 ml conical tube and centrifuge at 300 g for 2 minutes, and add 20 ml d0 SD or CA media to the Erlenmeyer flask. After centrifugation, aspirate supernatant, collect aggregates with a serological pipette, and place in flask.
Wash serological pipette and conical tube with medium to collect any remaining aggregates.
5. On d1 and d2, feed aggregates with d1 and d2 SD or CA media following step 4. 6. On day 3 of differentiation, dissociate aggregates with Accutase for 3 minutes or until you start seeing the solution become cloudy in a shaking water bath at 37°C 7. Add an equivalent amount of basis media such as DMEM/F12 supplemented with 10 µM Y-27632. Resuspend aggregates to break them apart, and then spin down at 300 g for 3 minutes.