Rodent malignancy bioassays indicate the aryl hydrocarbon receptor (AHR) agonist 2 3 7 8 of rHpScs consisting of a substratum of hyaluronans and Kubota’s medium (KM) a serum-free medium designed for endodermal stem/progenitor cells. image analysis. The AHR agonists improved proliferation of rHpSCs at concentrations producing a prolonged AHR activation as indicated by induction of and of hBTSCs hHpSCs hHBs and rHBs are summarized in many articles/evaluations.16 17 (See online product for further referrals.) Here we display that marker analyses for HpSCs versus HBs in humans and rodents are almost identical (Assisting Table S3). evidence for rules of progenitor functions by AHR is limited to studies using a transformed liver progenitor-like cell collection (i.e. WB-F344 cells) in which AHR activation correlates with proliferation through loss of contact-mediated growth inhibition.18 19 Increases in Jun D expression cyclin A/cyclin-dependent kinase 2 (CDK2) activity dysregulation of β-catenin signaling and changes in cell-cell adhesion N-(p-Coumaroyl) Serotonin proteins were observed.19-21 AHR activation offers been shown to modulate cell cycle progression in additional transformed cell lines.1 10 The effects are consistent with the part of TCDD like a tumor promoter and indicate that AHR plays a role in regulating cell proliferation. However the effects of AHR on HpSCs of any varieties have not been studied. Here we provide the 1st investigations of effects of AHR activation on rHpSCs versus rHBs using a combination of immunocytochemistry and high-content image analysis. Materials and Methods Most methods for ethnicities were as explained previously.15 Rat Hepatic Stem Cell Ethnicities Neonatal Sprague-Dawley rat livers were enzymatically dispersed and then cultured on substrata coated with 30 μg/cm2 hyaluronan and in Kubota’s medium (KM).22 Recombinant rat leukemia inhibitory element (LIF) was added at concentrations specified in experiments resulting in lineage restriction to hepatoblasts. Chemical Treatments AHR agonists were prepared in dimethyl sulfoxide (DMSO) at a 1 0 concentration and given at 1 μL/mL of medium. Assays Cultures were analyzed using immunocytochemistry (ICC) 15 quantitative reverse-transcription polymerase chain reaction (qRT-PCR) 23 and high content material image analyses.24 N-(p-Coumaroyl) Serotonin (See online product for details of the methods.) Results Hyaluronans: Essential Conditions for rHpSCs Neonatal rat liver cells were plated into KM and onto collagen types III IV or plastic. Mesenchymal cells rapidly overgrew ethnicities reaching confluence within a week; parenchymal cell growth was limited (Fig. S5). In contrast plating onto hyaluronans and in KM resulted in coordinated growth reactions of parenchymal and mesenchymal cells (Fig. 1A). By 10-12 days cells had created unique stem/progenitor colonies (Fig. 1A). Colony sizes improved indicating proliferation and contained both epithelial and mesenchymal cells. Hepatic lineage markers previously founded for either hHpSCs and hHBs or rHBs (Assisting Table S3) were used to characterize the ethnicities using immunocytochemistry. Both epithelial and mesenchymal cells were positive for CD44 the hyaluronan receptor (Fig. 1B). The epithelial Arf6 but not mesenchymal cells were positive for E-cadherin EpCAM and spread cells for alpha-fetoprotein (AFP) and/or albumin (ALB) (Fig. 1B). These phenotypic qualities are consistent with combined ethnicities of rHpSCs and of rHBs. Mesenchymal cells coexpressing desmin and CD44 were hepatic stellate precursors (Fig. 1B) as defined previously.15 Adult rat hepatocytes did not communicate EpCAM AFP or CD44 (Fig. S6). ALB and E-cadherin were indicated by hepatocytes but with a distinct manifestation pattern as compared to stem/progenitors. Occasional desmin+ mesenchymal cells were observed. Therefore hyaluronans plus KM supported survival and development of N-(p-Coumaroyl) Serotonin hepatic stem/progenitors and their mesenchymal partners. Expansion assorted from stable stable cell divisions for most colonies to some with limited divisions followed by degeneration due we presume to stem cells present in stable colonies versus committed progenitors in those that degenerated. Fig 1 Hyaluronan advertised selective development of rat hepatic stem/progenitor cells at LIF concentrations ≥0.5 ng/mL. A less pronounced effect was.