Search

Additional file 1: Supplementary Figure 1. Analysis of albumin (A) and urea levels (B) by Mouse Albumin ELISA kit and Urea assay kit, respectively. Color changes of cell culture supernatants before reading of the absorbance by a microplate reader. Black and red boxes indicate serially diluted albumin or urea standards, and samples used for analysis, respectively. Supplementary Figure 2. Analysis of major liver function and Cyp enzyme-related genes in individual male and female mice. A, major liver function and mature related genes; B, major Cyp genes related to xenobiotic decomposition; C, sex predominant Cyp genes. Mail tail tissue was used as negative control. Five male and five female mice were used for each specific mRNA analysis. Each gene was normalized with tail mRNA of single male mouse1 randomly selected. Supplementary Figure 3. Sex determination of MEFs and karyotyping of random selected MEF. A, after isolation of MEF from each fetus, sex was identified by sorting sex chromosome genes using PCR analysis. Electrophoresis revealed 1, 2, and 5 MEF as male, and 3, 4, 6, and 7 as female. For further analysis, MEF 1 from male and MEF 4 from female were chosen randomly. B, karyotyping results of randomly chosen male (a) and female (b) MEFs showed a normal karyotype. Supplementary Figure 4. Expression analysis of the major CYP enzymes in miHeps. (A) The expression of the major Cyp enzymes, (a) m Cyp1a1, (b) m Cyp1a2, (c) mCyp2a5, (d) m Cyp2d22, (e) mCyp3a11, and (f) m Cyp3a13 was analyzed by real-time quantitative PCR in male and female miHeps. MEF and liver were used as negative and positive controls for each gene, respectively. Letters above the bars represent a significant difference at p < 0.05. The data represents relative quantification (RQ) ± min and max RQ values. (B) Expression of Cyp3a11 (red) and Cyp1a2/1a1 (green) proteins by immunofluorescence staining. MEF was used as a negative control. Blue fluorescence indicates DAPI staining of the nucleus. Scale bar = 100 μm. Supplementary Figure 5. Functional analysis of miHeps. (A) Histochemical analysis of miHeps by (a) Oil Red O staining, (b) PAS staining, (c) Dil-Ac-LDL uptake and (d-1,2) ICG uptake and release assay. Scale bar = 100 μm. (B) Analysis of mLpl and mPparγ by real-time quantitative PCR analysis. Superscript (*) above the bars represent a significant difference at p < 0.05. The data represents relative quantification (RQ) ± min and max RQ values. Supplementary Figure 6. Glucose/glycogen, lipoprotein/cholesterol and fatty acid metabolism of miHep by whole transcriptome analysis. A, Glucose/glycogen metabolism; B, lipoprotein/cholesterol metabolism; C, and fatty acid metabolism. Male MEF, male miHep, female miHep, male liver and female liver normalized with female MEF. male miHep / female miHep indicates male miHep normalized with female miHep. Male liver / female liver indicates male liver normalized with female liver. Supplementary Figure 7. Generation of a mouse model of acute liver injury. CCl4 diluted with Con oil was injected into the peritoneal cavity of immunodeficient mice. Sera were recovered from mice day 1 (n = 5) and 4 days (n = 5) after CCl4 injection and subjected to serum biochemical analysis [aspartate aminotransferase (AST, A), alanine aminotransferase (ALT, B), alkaline phosphatase (ALP, C), albumin (ALB, D), total bilirubin (T-BIL, E) and total protein (TP, F)] using a Hitachi clinical analyzer 7180 (Hitachi. Ltd., Tokyo, Japan). The data represent the means ± SD. Superscript (*) above the bars represent a significant difference by LSD post-hoc test at p < 0.05.

Additional file 2: Supplementary Table 1. Primers used for realtime PCR. Supplementary Table 2. Primers used for real-time PCR analysis. Supplementary Table 3. List of antibodies used for immunofluorescence staining. Supplementary Table 4. Ploidy percentage of miHep.

Background The liver is one of the vital organs involved in detoxification and metabolism. The sex-based differences between the functionality of male and female liver have been previously reported, i.e., male’s liver are good in alcohol clearance and lipid metabolism, while female’s liver are better in cholesterol metabolism. To date, studies on novel drug toxicity have not considered the sex-specific dimorphic nature of the liver. However, the use of hepatocyte-like cells to treat liver diseases has increased recently. Methods Mouse embryos were isolated from a pregnant female C57BL/6J mouse where mouse embryonic fibroblasts (MEFs) were isolated from back skin tissue of each embryo. MEFs were transduced with human transcription factors hHnf1α, hHnf4α, and hFoxa3 using the lentiviral system. The transduced MEFs were further treated with hepatocyte-conditioned media followed by its analysis through RT-qPCR, immunofluorescence, functional assays, and finally whole-transcriptome RNA sequencing analysis. For in vivo investigation, the mouse hepatocyte-like cells (miHep) were transplanted into CCl4-induced acute liver mouse model. Results In this study, we evaluated the sex-specific effect of miHep induced from male- and female-specific mouse embryonic fibroblasts (MEFs). We observed miHeps with a polygonal cytoplasm and bipolar nucleus and found that male miHeps showed higher mHnf4a, albumin secretion, and polyploidization than female miHeps. Transcriptomes from miHeps were similar to those from the liver, especially for Hnf4a of male miHeps. Male Cyps were normalized to those from females, which revealed Cyp expression differences between liver and miHeps. In both liver and miHeps, Cyp 4a12a and Cyp 4b13a/2b9 predominated in males and females, respectively. After grafting of miHeps, AST/ALT decreased, regardless of mouse sex. Conclusion In conclusion, activation of endogenic Hnf4a is important for generation of successful sex-specific miHeps; furthermore, the male-derived miHep exhibits comparatively enhanced hepatic features than those of female miHep.

Previously, we reported the successful regeneration of injured peripheral nerves using human dental pulp stem cells (DPSCs) or differentiated neuronal cells from DPSCs (DF-DPSCs) in a rat model. Here, we attempted to evaluate oxidative stress and supraspinal neuro-inflammation in rat brain after sciatic nerve injury (SNI). We divided our experimental animals into three SNI groups based on time. The expression of a microglial (Iba1) marker and reactive oxygen species (ROS) was lower in DPSCs and higher in DF-DPSCs. In contrast, the expression of an astroglial (GFAP) marker was higher in DPSCs and lower in DF-DPSCs at 2 weeks. However, the expression of ROS, Iba1 and GFAP gradually decreased at 8 and 12 weeks in the SNI DPSCs and DF-DPSCs groups compared to the SNI control. Furthermore, anti-inflammatory cytokine (IL-4 and TGF-β) expression was lower at 2 weeks, while it gradually increased at 8 and 12 weeks after surgery in the SNI DPSCs and DF-DPSCs groups. Similarly, SNI DPSCs had a high expression of pAMPK, SIRT1 and NFkB at the onset of SNI. However, 12 weeks after surgery, pAMPK and SIRT1 expression levels were higher and NFkB was down-regulated in both DPSCs and DF-DPSCs compared to the control group. Finally, we concluded that DPSCs responded early and more efficiently than DF-DPSCs to counterbalance peripheral nerve injury (PNI)-induced oxidative stress and supraspinal neuro-inflammation in rat brain.

Primary hepatocytes (PHs) are considered the ‘gold standard’ in drug screening owing to their ability to express many drug-metabolizing enzymes and transporters. Culturing hepatocytes and maintaining their fate in vitro is a major issue since last decade. The main problem with in vitro hepatocytes culture is that they rapidly lose their hepatic morphology and liver-specific functions in culture. Herein, we isolated rat PHs, and cultured them in monolayers (2-D) and spheroids (3-D). The 2-D-cultured PHs exhibited elongated morphology, whereas the 3-D-cultured PHs exhibited spheroid morphology with gradual diameter decrease until 7 days. After 7 days of in vitro culture, PHs were analyzed for the expression of hepatic (Alb, Tf, and Afp) and apoptotic markers (Bax and Bcl2), and co-expression of CYP3A1 and Abumin after 2 and 7 days. Furthermore, in both cultures, PHs were induced with 3-methylcholanthrene (3-MC, Cyp1a-specific inducer) and dexamethasone (Cyp3a-specific inducer) for 48 and 72 h, respe...

There is a growing interest in the application of primary hepatocytes for treatment of liver diseases in humans and for drug development. Several studies have focused on long-term survival and di-differentiation blocking of primary hepatocytes in an in vitro culture system. Therefore, the present study also aimed to optimize an in vitro culture system using primary rat hepatocytes. Primary rat hepatocytes from 6-week-old male Crl:CD rats were isolated using a modified two-step collagenase perfusion. Healthy 3.5 × 106 primary rat hepatocytes were seeded into a 2 dimensional (2D) culture in a 25T culture flask coated with collagen type I or into a 3D culture in a 125-ml spinner flask for 7 days. Production of plasma protein (ALB and TF), apoptosis (BAX and BCL2), and CYP (CYP3A1) related genes were compared between the 2D and 3D culture systems. The 3D culture system had an advantage over the 2D system because of the relatively high expression of ALB and low expression of BAX in the 3D system. However, the level of CYP3A1 did not improve in the 3D culture with and without the presence of a dexamethasone inducer. Therefore, 3D culture has an advantage for albumin production and primary rat hepatocyte survivability, but a low expression of CYP3A1 indicated that primary rat hepatocytes require a high?density culture for stress reduction by continuous flow.