Expansion, in vivo-ex vivo cycling and genetic manipulation of primary human hepatocytes
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ABSTRACT:
PROVIDER: PRJNA534296 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR8946230.fastq.gz | Fastqsanger.gz | |||
| SRR8946231.fastq.gz | Fastqsanger.gz | |||
| SRR8946225.fastq.gz | Fastqsanger.gz | |||
| SRR8946226.fastq.gz | Fastqsanger.gz | |||
| SRR8946227.fastq.gz | Fastqsanger.gz |
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Project description:Primary human hepatocytes (PHH) are an essential tool for modeling drug metabolism and liver disease. However, variable plating efficiencies, short lifespan in culture and resistance to genetic manipulation have limited their use. Here we show that retrorsine improves human hepatocyte repopulation of chimeric mice to levels where poor donor PHH can be isolated for ex vivo cultures. Mouse-passaged (mp)PHH cultures overcome the marked donor-to-donor variability of cryopreserved PHH and remain functional for months, as demonstrated by metabolic assays and infection with hepatitis B virus and Plasmodium falciparum. mpPHH can be efficiently genetically modified in culture, mobilized and then re-cultured as spheroids or re-transplanted to create highly humanized mice carrying a genetically altered hepatocyte graft. Together, these advances provide flexible tools for studying human liver disease and evaluating hepatocyte-targeted gene therapy approaches
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Project description:Primary human hepatocytes (PHHs) are an essential tool for modeling drug metabolism and liver disease. However, variable plating efficiencies, short lifespan in culture, and resistance to genetic manipulation have limited their use. Here, we show that the pyrrolizidine alkaloid retrorsine improves PHH repopulation of chimeric mice on average 10-fold and rescues the ability of even poorly plateable donor hepatocytes to provide cells for subsequent ex vivo cultures. These mouse-passaged (mp) PHH cultures overcome the marked donor-to-donor variability of cryopreserved PHH and remain functional for months as demonstrated by metabolic assays and infection with hepatitis B virus and Plasmodium falciparum mpPHH can be efficiently genetically modified in culture, mobilized, and then recultured as spheroids or retransplanted to create highly humanized mice that carry a genetically altered hepatocyte graft. Together, these advances provide flexible tools for the study of human liver disease and evaluation of hepatocyte-targeted gene therapy approaches.
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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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Project description:Direct contact with mesenchymal stromal impacts on migratory behavior and gene expression profile of CD133+ hematopoietic stem cells during ex-vivo expansion Objective: To investigate the impact of direct contact between mesenchymal stromal cells (MSCs) and CD133+ hematopoietic stem cells (HSCs) in terms of expansion potential differentiation, migratory capacity and gene expression profile. Methods: CD133+ purified HSCs were cultured for 7 days on subconfluent MSCs supplemented with growth factor containing medium. After ex-vivo expansion, non-adherent and adherent cells were collected and analyzed separately. Results: The adherent cells were found to have a more immature phenotype compared to the non-adherent fraction. CXCR4 was up regulated in the adherent fraction which was associated with a higher migration capacity towards a SDF-1 gradient. CFU-GM and LTC-IC assays demonstrated a higher clonogenicity and repopulating capacity of the adherent fraction. Genes involved in adhesion, cell cycle control, motility, self-renewal and apoptosis were expressed at a higher level in the adherent fraction. Conclusion: Adhesion and direct cell-cell contact with a MSC feeder layer supports ex-vivo expansion, migratory potential and stemness of CD133+ HSCs. Keywords: co-culture hematopoietic stem cells (HSCs) on mesenchymal stromal cells (MSCs) Non-adherent and adherent fractions from three independent experiments were isolated and collected. Cells were stabilized in PreProtct™ buffer (Miltenyi Biotec, Germany) and stored at -80ºC. Samples were shipped to Miltenyi Biotec (Bergisch Gladbach, Germany) a Whole Human Genome expression analysis. Briefly RNA was extracted and overall quality of total RNA samples was checked via the Agilent 2100 Bioanalyzer platform (Agilent Technologies). RNA samples were amplified and labelled using the Agilent Low RNA Input Linear Amp Kit (Agilent Technologies). Non-adherent samples were pooled as “Non-adherent pool” and adherent samples were pooled as “adherent pool” and labelled with Cy3 and Cy5, respectively. Fluorescence signals of the hybridized Agilent Oligo Microarrays were detected using Agilent’s DNA microarray scanner and the microarray image files were processed with The Agilent Feature Extraction Software (FES).
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