Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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The impact of early WNT pathway inhibition (day 1 of differentiation) during human pluripotent stem cell differentiation towards cardiomyocytes

ABSTRACT: Human pluripotent stem cells were aggregated in mTeSR medium for 4 days and subjected to an optimzed small molecule-based cardiac differentiation protocol (modified from Kempf et. al., Stem Cell Reports, 2014) in agitated Erlenmeyer flasks. Differentiation was induced using 7.5µM CHIR99021 (day 0). Cardiac mesoderm specification was carried out using 5µM IWP2 either directly after CHIR99021 exposure after 24h (day 1; optimized protocol) or after 72h hours (day 3; standard procedure). RNA samples were harvested at undifferentiated state (day 0), after differentiation induction (day 1) and after 3 days with and without IWP2 treatment.

ORGANISM(S): Homo sapiens

SUBMITTER: Henning Kempf

PROVIDER: E-MTAB-6828 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background: Evidence has recently accumulated suggesting that mature hepatocytes have the unique capacity to autonomously decide their replication fate. The molecular mechanism by which hepatocytes switch from an essentially quiescent state to rapidly proliferation after liver injury is not fully understood. To provide molecular insights into the self-renewal of mature hepatocytes, we have applied an ex vivo liver slice culture system. Results: After liver injury, activation of p38 and Erk1/2 began in mature hepatocytes within 5 min. Erk1/2 was activated at the edge of the cut as well as on the surface of liver slices. The number of hepatocytes that contain activated Erk1/2 increased within 1 h and then decreased. Concomitantly, immediate early genes (IEGs), such as Jun, early gene response 1 (Egr1) and Myc were induced and Ki67 positive hepatocytes appeared 24 h after liver injury in the slices. In agreement with in vivo studies, transcriptome analysis using 2 h slice culture reveals that 63% of upregulated genes were downstream of lipopolysaccharide (LPS) stimulation which induces interleukin 6 (IL 6) or tumor necrosis factor (TNF) alpha pathways. Furthermore, circadian clock regulator, such as members of the basic helix-loop-helix family, Bhlhe40 and Bhlhe41, were highly upregulated for 4 h after injury. Although upon injury Jun or Egr1 were induced in liver slices from proliferation-defective liver, no circadian clock regulator genes were upregulated. Moreover, using an in vitro cell culture system we show that Bhlhe40 is required for the G0-G1 transition. Conclusion: Our data suggest that the circadian clock regulator, Bhlhe40, is involved in the G0-G1 transition. An ex vivo system using normal and proliferation defective KO liver is a useful tool for identification of genes that trigger cell proliferation shortly after liver injury. This method may also be applied for measurement of the liver regeneration potential of individual livers at the priming phase. In one dual-color microarray hybridization, mRNA expression changes after 2h ex vivo incubation of liver slices were examined.

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The impact of early WNT pathway inhibition (day 1 of differentiation) during human pluripotent stem cell differentiation towards cardiomyocytes

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