Request for Specialized Services
The ILCC also provides or assists in specialized services requiring a combination of the disease model, genetic model, FACS isolation, cell labeling and transplantation and/or newly developed procedures by the ILCC as described below.
- Genetic cell lineage and fate tracing: The ILCC now performs cell lineage and cell tracing with collaboration with the Animal Core by using Rosa26mTmGflox reporter mice crossed with Alb-Cre (hepatocyte and biliary), Ttr-Cre (hepatocyte), Col1a1-Cre (activated HSC), Mesp1-Cre (mesenchyme), Wt1-Cre (mesothelium), Lysozyme-Cre (myeloid) to label respective cells for tracing in mouse models of ALD and liver fibrosis. Labeled cells are isolated by the Core via FACS using GFP and a fluorophore-conjugated antibody against a marker for the cell type of interest for biochemical characterization, culture experiments, or cell fate analysis via transplantation.
- FACS isolation of activated HSC from Col1a1-GFP transgenic mice developing alcoholic liver fibrosis: In the past 20 years, we have learned that isolation of pure activated HSCs from experimental alcoholic liver fibrosis is very challenging if not impossible. This is primarily because HSC become severely depleted of vitamin A as compared to activated HSC from other models of liver fibrosis induced by CCl4 or bile duct ligation, making it impossible to isolate them based on their normal light density. Fatty HC and HM which phagocytized lipids also contaminate the 1.035-1.045 density interface from which HSC are usually recovered. To overcome these problems, the ILCC now applies a FACS-based isolation technique to Col1a1-GFP mice to separate HSC based on their UV fluorescence for vitamin A content and the activation marker of type I collagen promoter activity as detected by GFP. Using this method, a pure population of UV- or UV+ (mostly UV- but some UV+) and GFP+ cells is isolated as activated HSC as shown below in this figure.
- CD133+ liver progenitor isolation: As briefed above, the ILCC developed an isolation method of CD133+ liver progenitors. Livers from the mouse model of alcoholic hepatitis (1) are briefly perfused with 25mg/ml collagenase IV. CD45+ cells are depleted by MACS and CD45- cells are stained with CD133-APC, CD49f-PE, and CD45-eFlour 450 for FACS-based separation of CD133-CD49f+ cells. Note CD133+CD49f+ cell population is very low (<0.1%) in normal liver but can increase to >3% in AH liver. RNA-seq analysis of this population vs. CD133- cells, confirms CD133+ cells are indeed progenitors with higher expression of stem cell and progenitor markers. The presence of CD133+CD49f+ progenitor cells is only observed in this model of AH but not in chronic ASH, suggesting the former pathology uniquely induces progenitors.
- Assessment of infiltrating vs. resident HM and tracking on blood monocytes: Xu, now serving as one of two Associate Directors for the ILCC, has succeeded in analyzing the relative contributions of resident HM vs. monocyte-derived HM to M1 macrophage activation in mouse ASH model. After isolation of peripheral blood monocytes, the cells are labeled with PKH26 fluorescent dye and injected intravenously into recipient ASH mouse (0.5×106 cells/mouse). Then, HM are isolated and further labeled with anti-CD45-V450, F4/80-FITC, and CX3CR1-APC antibodies. Using FACS, infiltrating donor monocyte-derived HM are separated as CD45+PHK26+F4/80+CX3CR1high cells, and recipient infiltrating monocyte-derived HM and recipient resident HM are separated as CD45+PHK26-F4/80+CX3CR1high and CD45+PHK26-F4/80+CXCR1low cells, respectively. This method allows investigating how these 2 populations contribute to inflammation in ASH and the role of a particular gene of interest in monocyte transmigration into ASH livers by using donor monocytes with ablation of the gene as used to demonstrate the important role of Notch1 (2).
- Hypoxia system: As the liver is a relatively hypoxic organ and alcohol is known to exacerbate centrilobular hypoxia, the ability to perform cell culture studies under hypoxic conditions is important. The ILCC utilizes a complete hypoxia system from BioSpherix, NY which allows culture and manipulations of the cells under a constant hypoxic condition. This system has allowed a demonstration of cooperative actions of HIF-1 and NICD1 in Nos2 gene activation under hypoxia (2).
- Mesothelial cell (MC) isolation and culture: The first isolation and culture of mouse liver MC, have recently been accomplished by Asahina, the ILCC Associate Director (3). He has identified Glycoprotein M6A (GPM6A) as a specific marker for MC. Using antibodies against GPM6A, MC can be isolated from normal or injured mouse livers by MACS. In culture, MC form epithelial colonies. MC begin to lose the epithelial phenotype and differentiate into fibroblastic cells from Day 4 (click here to see figure). Quantitative RT-PCR shows expression of Gpm6a and Msln (Mesothelin) as markers for MCs (click here to see figure). In parallel to the morphologic changes of “mesothelial-mesenchyme transition”, cultured MC increase their expression of Acta2 mRNA, a marker for myofibroblasts (click here to see figure) .
If you are interested in the ILCC specialized services, please contact Stephanie Pan (email@example.com or 323-442-3850) or Dr. Kinji Asahina (firstname.lastname@example.org) in advance for the availability of the services, scheduling, and assessment of chargeback rate.