Expansion, in vivo-ex vivo cycling and genetic manipulation of primary human hepatocytes
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PROVIDER: PRJNA534296 | ENA |
REPOSITORIES: ENA
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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
2019-12-02 | GSE130219 | GEO
Project description: Not available
| S-EPMC6983380 | biostudies-literature
Project description:Successful ex vivo expansion of hematopoietic stem cells (HSCs) would greatly benefit the treatment of disease and the understanding of critical questions of stem cell biology. Microarray studies showed that the HSC supportive mouse fetal liver CD3+ cells specifically expressed angiopoietin-like 2 (Angptl2) and angiopoietin-like 3 (Angptl3). When highly enriched HSCs were cultured in the presence of Angptl2 or Angptl3 together with saturating levels of other growth factors for 10 days, a 24 or 30 fold net expansion of long-term HSCs was observed by reconstitution analysis. The coiled-coil domain of Angptl2 mediated the effects of Angptl2 on HSC expansion. Furthermore, angiopoietin-like 5, angiopoietin-like 7, and microfibril-associated glycoprotein 4 also supported expansion of HSCs in culture. Keywords: Cell type comparison
2006-03-01 | GSE3609 | GEO
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).
2008-11-12 | E-GEOD-13566 | biostudies-arrayexpress
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)
2008-11-13 | GSE13566 | GEO
Project description:Ex vivo activation and expansion of natural killer (NK) cells is a strategy to produce sufficient numbers of these effector cells for adoptive immunotherapy. Therefore we aimed at the development of an automated, clinical scale NK cell expansion process and compared NK cells after manual and automated expansion.We found only a small subset of 124 genes that significantly varied between both sample groups. These genes were associated with movement of leukocytes were identified including a group of genes for NK cell movement (CMKLR1, CX3CR1, S1PR5, GNLY and CXCR1) which were slightly higher expressed after automated compared to manual expansion. Nevertheless, the expansion profiles of NK cells after automated or manual expansion were rather similar in comparison to the remarkable difference between primary and expanded NK cells in general represented by the vast majority of regulated genes between the analyzed sample groups. Pathway analysis revealed that most of regulated genes upon expansion were associated with cell cycle regulation, DNA replication, DNA recombination and DNA repair, regulation of apoptosis and cell survival as well as cytokine signaling. Furthermore, the expression of many NK cell relevant markers was changed upon expansion with the strongest effects on up regulation of TRAIL and FASL, inhibitory TIGIT and chemokine receptors CCR2, CCR5 and CXCR6. In addition, granzyme M was down regulated but other important effector molecules like TNFa, perforin and granzymes A, B and K were up regulated. In summary we could show that manual and automatically expanded NK cells show a similar expansion profile while the differ significantly from primary NK cells. Up regulation of many NK cell relevant markers indicate for an enhanced NK cell functionaility after ex vivo activation and expansion. 24 samples were analyzed in total. 6 biological replicates represented by cells from different donors. 4 different samples per donor: freshly isolated NK cells (d0) and NK cells expanded for 14 days under 3 different conditions: in co-culture with EBV-LCL feeder cells and 500 U/mL IL2 either cultivated manually in T75 flasks (T) or automatically using the CliniMACS Prodigy system (P); NK cells cultured with 500 U/ml IL2 without feeder cells (I)
2015-04-23 | E-GEOD-62654 | biostudies-arrayexpress
Project description:Bone marrow mesenchymal stem cells (BMSCs) is a promising cell type for tissue engineering, however, the application of BMSCs are largely hampered for the limited number harvested from bone marrow cells. The methods or strategies that focused on promoting the capacity of BMSCs expansion ex vivo become more and more important. Tanshinone IIA (Tan IIA), the main active components of Danshen, has been found to promote BMSCs proliferation, but the underlying mechanism is still unclear. This present work employed biological techniques combined with quantitative proteome analysis to explore the effect and underlying mechanism of Tan IIA on the ex vivo expansion capacity of BMSCs from human. The results showed that a total of 84 differentially expressed proteins were annotated and among them 51 upregulated proteins and 33 downregulated proteins were identified. Besides, Tan IIA could promote hBMSCs proliferation by regulating the progression of S phase via increasing the release of fibroblast growth factor 2 (FGF2), FGF-mediated PI3K/AKT signaling pathways may play an important role in Tan IIA’s effect on hBMSCs expansion. The results of this study may provide further evidence about the potential of Tan IIA to be used for the research of hBMSCs therapies.
2022-11-28 | PXD036093 | Pride
Project description:Transplantation of genetically corrected hepatocytes is an attractive alternative to liver transplantation but is hampered by the low amplification potential of these cells in vitro. Here, we describe a method for generating proliferative hepatic progenitor cells (iHPC) from human hepatocytes as an expandable cell source for liver therapy. Dedifferentiation of primary hepatocytes to iHPC was achieved in less than 7 days by culturing the cells in medium with a cocktail of growth factors and small molecules. In culture, iHPC expressed a combination of endoderm hepatic progenitor and mesenchymal stem cell markers and proliferated vigorously, allowing for their expansion by at least 104 times. RNA sequencing of iHPC demonstrated that they displayed far more subtle changes in both transcriptome and transposcriptome, compared to hepatocyte-derived iPSC. Finally, transplantation of iHPC into the liver of immuno-deficient mice showed iHPC differentiation potential in vivo, without triggering detectable tumor development.
2019-09-25 | GSE87559 | GEO
Project description:Transplantation of genetically corrected hepatocytes is an attractive alternative to liver transplantation but is hampered by the low amplification potential of these cells in vitro. Here, we describe a method for generating proliferative hepatic progenitor cells (iHPC) from human hepatocytes as an expandable cell source for liver therapy. Dedifferentiation of primary hepatocytes to iHPC was achieved in less than 7 days by culturing the cells in medium with a cocktail of growth factors and small molecules. In culture, iHPC expressed a combination of endoderm hepatic progenitor and mesenchymal stem cell markers and proliferated vigorously, allowing for their expansion by at least 104 times. RNA sequencing of iHPC demonstrated that they displayed far more subtle changes in both transcriptome and transposcriptome, compared to hepatocyte-derived iPSC. Finally, transplantation of iHPC into the liver of immuno-deficient mice showed iHPC differentiation potential in vivo, without triggering detectable tumor development.
2019-09-25 | GSE87558 | GEO
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.
2015-12-31 | E-GEOD-65995 | biostudies-arrayexpress