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Figure 1. STEMdiff™ Hepatic Organoid Media Enables Establishment, Expansion, and Differentiation of hPSC-Derived Hepatic Organoids

(A) Mature hepatic organoids can be generated starting from hPSC-derived HLCs or HPs. For the generation of HLCs or HPs, see documentation for STEMdiff™ Hepatocyte Kit (Catalog #100-0520). HLCs (recommended) or HPs are mechanically dissociated and replated as fragments in Matrigel® domes and cultured in STEMdiff™ Hepatic Organoid Growth Medium (Catalog #100-1773) to generate confluent cultures within 7 to 14 days. These organoids can be expanded every 6 - 8 days via fragment passaging in STEMdiff™ Organoid Growth Medium, and can also be cryopreserved as fragments. (B) After one passage, cultures can be switched to STEMdiff™ Hepatic Organoid Differentiation Medium (Catalog #100-1774) to differentiate organoids toward more mature hepatic cell types. hPSCs = human pluripotent stem cells; HPs = hepatic progenitor cells; HLCs = hepatocyte-like cells.

Figure 2. HP- and HLC-Derived Liver Organoids Can Be Reliably Generated and Expanded Using STEMdiff™ Hepatic Organoid Media

hPSC-derived organoids can be established from HPs and HLCs generated using the STEMdiff™ Hepatocyte Kit, and then expanded for at least 10 passages. Shown are representative images of HP- and HLC-derived organoids cultured in STEMdiff™ Hepatic Organoid Growth Medium at (A, C) passage 1 and (B, D) passage 9. The HP- and HLC-derived organoids were further differentiated using STEMdiff™ Hepatic Organoid Differentiation Medium at (E, G) passage 1, and (F, H) passage 9. The HP- and HLC-derived organoids shown were derived from the same hPSC line. hPSC = human pluripotent stem cells; HPs = hepatic progenitor cells; HLCs = hepatocyte-like cells.

Figure 3. Proliferating Hepatic Organoids Display Characteristics of Hepatic Progenitors While Differentiated Hepatic Organoids Display Characteristics of Mature Hepatocytes

(A-C) Hepatic organoids cultured in STEMdiff™ Hepatic Organoid Growth Medium organoids express epithelial marker EPCAM, proliferation marker KI67, ductal markers CK19 and SOX9, and the fetal serum protein marker AFP. (D-F) Differentiated hepatic organoids cultured in STEMdiff™ Hepatic Organoid Differentiation Medium express mature hepatocyte markers ALB and A1AT as well as hepatic transcription factor HNF4A. Some organoid regions also retain expression of hepatic progenitor/ductal markers AFP and CK19. Nuclei are counterstained with DAPI.

Figure 4. STEMdiff™ Hepatic Organoid Growth Medium Enables Efficient Establishment and Expansion of hPSC-Derived Hepatic Organoids

(A) STEMdiff™ Hepatic Organoid Growth Medium supports more efficient organoid establishment from HLCs as compared to HPs, generating significantly more organoids from 1 well of HLCs in a 24-well plate than 1 well of HPs in a 24-well plate. (N = 3 cell lines) (B) Once established, HP- and HLC-derived organoids expanded in STEMdiff™ Hepatic Organoid Growth Medium exhibit similar average growth rates across 10 passages. (N = 3 cell lines). Error bars = SD. Welch’s t-test used for statistical testing (**** p-value <0.0001). HPs = Hepatic progenitor cells; HLCs = Hepatocyte-like cells.

Figure 5. HLC-Derived Organoids Maintained in STEMdiff™ Organoid Growth Medium Can Be Cryopreserved and Efficiently Recovered and Expanded Post-Thaw

(A) HLC-derived organoids can be cryopreserved as organoid fragments, and successfully thawed with high post-thaw recovery of fragments (N = 3 cell lines). (B) Recovered fragments retain their proliferative capacity and can be expanded in STEMdiff™ Organoid Growth Medium for several passages. Growth rates for organoids established from cryopreserved fragments are comparable to matched cultures that were never cryopreserved (N = 3 cell lines). Error bars = SD.

Figure 6. HP- and HLC-Derived Hepatic Organoids Exhibit Gene Expression Consistent with Hepatic Maturation When Differentiated Using STEMdiff™ Hepatic Organoid Differentiation Medium

hPSC-derived liver organoids were established, expanded for up to 10 passages, differentiated, and harvested for gene expression analysis through qPCR at each passage. Compared to two-dimensional cultures of HPs and HLCs collected on Day 10 and Day 21 of differentiation, respectively, three-dimensional organoid cultures established from these stages and further matured in STEMdiff™ Hepatic Organoid Differentiation Medium (hODM) exhibited up-regulated expression of mature hepatic markers ALB, ASGR1, and CYP3A4 at levels comparable to PHH. Expression levels are normalized to housekeeping genes (**** represents an adjusted p-value <0.0001, ** represents an adjusted p-value of 0.0035, * represents an adjusted p-value of 0.0397). hPSC = human pluripotent stem cells; HPs = hepatic progenitors; HLCs = hepatocyte-like cells; ALB = albumin; PHH = primary human hepatocyte, hOGM = STEMdiff™ Hepatic Organoid Growth Medium, hODM = STEMdiff™ Hepatic Organoid Differentiation Medium.

Figure 7. RNA Sequencing Analysis Confirms a More Mature Functional State of HLC-Derived Organoids Upon Differentiation Using STEMdiff™ Hepatic Organoid Media

HLC-derived organoids further differentiated in STEMdiff™ Hepatic Organoid Differentiation Medium express increased levels of markers associated with mature hepatocyte identity and functionality, including serum protein synthesis (ALB), phase 1 and 2 drug metabolism (CYP2B6, CYP2C9, CYP3A4, UGT1A1, GSTA1), glucose metabolism (SLC2A2, GYS1, G6PC), lipid and cholesterol metabolism (FASN, PLIN2, NR1H3), bile acid synthesis (CYP27A1, BAAT), and urea synthesis (OTC, ARG1). Differentiated HLC-derived organoids also show reduced levels of fetal hepatocyte marker AFP. Columns represent 2 biological replicates analyzed by bulk RNA-seq in the first passage after organoids are established from HLCs, on Day 9 in STEMdiff™ Hepatic Organoid Growth Medium, and on Day 19 in STEMdiff™ Hepatic Organoid Differentiation Medium. The housekeeping gene TBP is included as a reference. hPSC = human pluripotent stem cells; HPs = hepatic progenitors; HLCs = hepatocyte-like cells; ALB = albumin; PHH = primary human hepatocyte; AFP = Alpha-fetoprotein.