“…Scale bar, 25 μm. Adapted from Sinton et al 2021 , iScience, https://doi.org/10.1016/j.isci.2020.101931 Aspirate the supplemented mTeSR1™ and replace with 2 mL RPMI 1640 supplemented with 100 ng/mL Activin A and 50 ng/mL Wnt3A. Incubate the cells at 37°C/5% CO 2 and refresh the media every 24 h for 3 days.…”
Section: Step-by-step Methods Detailsmentioning
confidence: 99%
“… Figure 3 Differentiation process leads to loss of pluripotency and gain of hepatocyte maturation markers Temporal expression of pluripotency marker (NANOG) and hepatocyte markers (ALB and HNF4A) during differentiation of hPSCs to HLCs (data are expressed as mean± SD; n = 8). Adapted from Sinton et al 2021 , iScience, https://doi.org/10.1016/j.isci.2020.101931 …”
Section: Step-by-step Methods Detailsmentioning
confidence: 99%
“…Recently developed in silico tools enable mathematical modeling of oxygen gradients within liver tissue, providing insight into how these gradients will impact on cell phenotype ( Leedale et al., 2021 ). This platform can be scaled and provides an excellent tool to study liver biology and disease ( Lucendo-Villarin et al., 2020a , 2020b ; Sinton et al., 2021 ) . Figure 4 Maturation of HLCs is associated with functional activity typically associated with human hepatocytes Cytochrome P450 3A4 activity in HLCs following differentiation with or without the lactate, pyruvate, octanoic acid (LPO) cocktail (data are expressed as mean± SD; n = 8).…”
Section: Expected Outcomesmentioning
confidence: 99%
“…Furthermore, the renewable nature of our system, on a defined genetic background, permits in-depth mechanistic analysis, which may facilitate therapeutic target identification in the future. For complete details on the use and execution of this protocol, please refer to Sinton et al. (2021) .…”
mentioning
confidence: 99%
“…For complete details on the use and execution of this protocol, please refer to Sinton et al. (2021) .…”
Summary
This protocol describes the production of hepatocyte-like cells (HLCs) from human pluripotent stem cells and how to induce hepatic steatosis, a condition characterized by intracellular lipid accumulation. Following differentiation to an HLC phenotype, intracellular lipid accumulation is induced with a steatosis induction cocktail, allowing the user to examine the cellular processes that underpin hepatic steatosis. Furthermore, the renewable nature of our system, on a defined genetic background, permits in-depth mechanistic analysis, which may facilitate therapeutic target identification in the future.
For complete details on the use and execution of this protocol, please refer to
Sinton et al. (2021)
.
“…Scale bar, 25 μm. Adapted from Sinton et al 2021 , iScience, https://doi.org/10.1016/j.isci.2020.101931 Aspirate the supplemented mTeSR1™ and replace with 2 mL RPMI 1640 supplemented with 100 ng/mL Activin A and 50 ng/mL Wnt3A. Incubate the cells at 37°C/5% CO 2 and refresh the media every 24 h for 3 days.…”
Section: Step-by-step Methods Detailsmentioning
confidence: 99%
“… Figure 3 Differentiation process leads to loss of pluripotency and gain of hepatocyte maturation markers Temporal expression of pluripotency marker (NANOG) and hepatocyte markers (ALB and HNF4A) during differentiation of hPSCs to HLCs (data are expressed as mean± SD; n = 8). Adapted from Sinton et al 2021 , iScience, https://doi.org/10.1016/j.isci.2020.101931 …”
Section: Step-by-step Methods Detailsmentioning
confidence: 99%
“…Recently developed in silico tools enable mathematical modeling of oxygen gradients within liver tissue, providing insight into how these gradients will impact on cell phenotype ( Leedale et al., 2021 ). This platform can be scaled and provides an excellent tool to study liver biology and disease ( Lucendo-Villarin et al., 2020a , 2020b ; Sinton et al., 2021 ) . Figure 4 Maturation of HLCs is associated with functional activity typically associated with human hepatocytes Cytochrome P450 3A4 activity in HLCs following differentiation with or without the lactate, pyruvate, octanoic acid (LPO) cocktail (data are expressed as mean± SD; n = 8).…”
Section: Expected Outcomesmentioning
confidence: 99%
“…Furthermore, the renewable nature of our system, on a defined genetic background, permits in-depth mechanistic analysis, which may facilitate therapeutic target identification in the future. For complete details on the use and execution of this protocol, please refer to Sinton et al. (2021) .…”
mentioning
confidence: 99%
“…For complete details on the use and execution of this protocol, please refer to Sinton et al. (2021) .…”
Summary
This protocol describes the production of hepatocyte-like cells (HLCs) from human pluripotent stem cells and how to induce hepatic steatosis, a condition characterized by intracellular lipid accumulation. Following differentiation to an HLC phenotype, intracellular lipid accumulation is induced with a steatosis induction cocktail, allowing the user to examine the cellular processes that underpin hepatic steatosis. Furthermore, the renewable nature of our system, on a defined genetic background, permits in-depth mechanistic analysis, which may facilitate therapeutic target identification in the future.
For complete details on the use and execution of this protocol, please refer to
Sinton et al. (2021)
.
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