Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

Cabral, Fatima; Miller, Colton M.; Kudrna, Katrina M.; Hass, Blake E.; Daubendiek, Jocelyn G.; Kellar, Brianna M.; Harris, Edward N.

doi:10.3791/56993

Cited by 57 publications

(44 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The isolated hepatoblasts were washed and maintained in culture on Matrigel-coated plates with William's medium E (Thermo Fisher Scientific). Primary hepatocytes were obtained from adult mice by liver enzymatic digestion by a previously described protocol [11].…”

Section: Methodsmentioning

confidence: 99%

Phenotype instability of hepatocyte-like cells produced by direct reprogramming of mesenchymal stromal cells

et al. 2020

View full text Add to dashboard Cite

Background: Hepatocyte-like cells (iHEPs) generated by transcription factor-mediated direct reprogramming of somatic cells have been studied as potential cell sources for the development of novel therapies targeting liver diseases. The mechanisms involved in direct reprogramming, stability after long-term in vitro expansion, and safety profile of reprogrammed cells in different experimental models, however, still require further investigation.Methods: iHEPs were generated by forced expression of Foxa2/Hnf4a in mouse mesenchymal stromal cells and characterized their phenotype stability by in vitro and in vivo analyses.Results: The iHEPs expressed mixed hepatocyte and liver progenitor cell markers, were highly proliferative, and presented metabolic activities in functional assays. A progressive loss of hepatic phenotype, however, was observed after several passages, leading to an increase in alpha-SMA + fibroblast-like cells, which could be distinguished and sorted from iHEPs by differential mitochondrial content. The resulting purified iHEPs proliferated, maintained liver progenitor cell markers, and, upon stimulation with lineage maturation media, increased expression of either biliary or hepatocyte markers. In vivo functionality was assessed in independent pre-clinical mouse models. Minimal engraftment was observed following transplantation in mice with acute acetaminophen-induced liver injury. In contrast, upon transplantation in a transgenic mouse model presenting host hepatocyte senescence, widespread engraftment and uncontrolled proliferation of iHEPs was observed, forming islands of epithelial-like cells, adipocytelike cells, or cells presenting both morphologies. Conclusion: The results have significant implications for cell reprogramming, suggesting that iHEPs generated by Foxa2/Hnf4a expression have an unstable phenotype and depend on transgene expression for maintenance of hepatocyte-like characteristics, showing a tendency to return to the mesenchymal phenotype of origin and a compromised safety profile.

show abstract

Section: Methodsmentioning

confidence: 99%

Phenotype instability of hepatocyte-like cells produced by direct reprogramming of mesenchymal stromal cells

et al. 2020

View full text Add to dashboard Cite

show abstract

“…This was especially evident in AOCs linked to gene activation. It is known that changes in chromatin accessibility precede changes in gene regulation [22], and the high temporal resolution of this dataset allowed us to identify a ~2h time shift between changes in AOCs and gene expression changes within some clusters.…”

Section: Discussionmentioning

confidence: 99%

“…Hepatocytes were isolated using a two-step perfusion protocol [22] Mice were euthanized by isoflurane followed by cervical dislocation. Liver Perfusion Medium and Liver Digest Medium (Gibco) were perfused for 10 minutes at 7 ml/minute flowrate.…”

Section: Hepatocyte Isolationmentioning

confidence: 99%

Rapid changes in chromatin structure during dedifferentiation of primary hepatocytes in vitro

Seirup

Sengupta

Swanson

et al. 2020

Preprint

View full text Add to dashboard Cite

Primary hepatocytes are widely used in the pharmaceutical industry to screen drug candidates for hepatotoxicity, but isolated hepatocytes quickly dedifferentiate and lose their mature metabolic function in culture. Attempts have been made to better recapitulate the in vivo liver environment in culture, but the full spectrum of signals required to maintain hepatocyte function in vitro remains elusive. Here we studied the dedifferentiation process in detail through RNA-sequencing of hepatocytes cultured over eight days. We identified three distinct phases of dedifferentiation. An early phase, where mature hepatocyte genes are rapidly downregulated in a matter of hours. A middle phase, where fetal genes are activated, leading to hepatocytes with a fetal phenotype. A late phase, where initially rare contaminating non-parenchymal cells over-grow the culture as the hepatocytes gradually die. Using genetically tagged hepatocytes, we demonstrate that the cells reactivating fetal marker alpha-fetoprotein arise from cells previously expressing the mature hepatocyte marker albumin, and not from albumin negative precursor cells, proving that hepatocytes undergo true dedifferentiation. To better understand the signaling events that result in the rapid down-regulation of mature hepatocyte genes, we examined changes in chromatin accessibility of hepatocytes during the first 24h of culture using ATAC-seq. We find that drastic and rapid changes in chromatin accessibility occurs immediately upon start of culture. Using binding motif analysis of the areas of open chromatin sharing similar temporal profiles, we identify several candidate transcription factors potentially involved in the dedifferentiation of primary hepatocytes in culture.

show abstract

“…Liver non-parenchymal cells (NPCs) were isolated from control mice and mice subjected to CCl 4 inhalation for 12 weeks as previously described with some modifications (67) . Briefly, liver cell suspensions were obtained by collagenase perfusion and were spun down at 100 xg for 5 min to remove hepatocytes.…”

Section: Cell Isolationmentioning

confidence: 99%

Single-cell transcriptomics reveals zone-specific alterations of liver sinusoidal endothelial cells in cirrhosis

Yang

Lai

et al. 2020

Preprint

View full text Add to dashboard Cite

Dysfunction of liver endothelial cells (ECs), particularly sinusoidal endothelial cells (LSECs), is permissive for the progression of liver fibrosis/cirrhosis and responsible for its clinical complications. Here, we have mapped the spatial distribution of heterogeneous liver ECs in normal versus cirrhotic mouse livers and identified zone-specific transcriptomic changes of LSECs associated with liver cirrhosis using single-cell RNA sequencing technology. We identified 6 clusters of liver EC populations including 3 clusters of LSECs, 2 clusters of vascular ECs and 1 cluster of lymphatic ECs. To add finer detail, we mapped the 3 clusters of LSECs to Zones 1 to 3. We found that heterogeneous liver EC identities are conserved even in liver cirrhosis and that Zone 3 LSECs are most susceptible to damage associated with liver cirrhosis, demonstrating increased capillarization and decreased ability to regulate endocytosis.Altogether, this study deepens our knowledge of the pathogenesis of liver cirrhosis at a spatial, cell-specific level, which is indispensable for the development of novel therapeutic strategies to target the most highly dysfunctional liver ECs. Words: 169

show abstract

Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

Cited by 57 publications

References 15 publications

Phenotype instability of hepatocyte-like cells produced by direct reprogramming of mesenchymal stromal cells

Phenotype instability of hepatocyte-like cells produced by direct reprogramming of mesenchymal stromal cells

Rapid changes in chromatin structure during dedifferentiation of primary hepatocytes in vitro

Single-cell transcriptomics reveals zone-specific alterations of liver sinusoidal endothelial cells in cirrhosis

Contact Info

Product

Resources

About