Background Exosomes are extracellular vesicles that assume a significant role in local and systemic intercellular communication. Exosomes secreted by adipose tissue-derived stem cells (ASCs) possess therapeutic potential for various clinical conditions. Dedifferentiated fat cells (DFATs) are mature adipocyte-derived multipotent cells that show phenotypical and functional similarity to ASCs. Although expression profiles of humoral factors in DFATs are very similar to those in ASCs, there is limited knowledge on exosomes derived from DFATs. In this study, we isolated exosomes from the culture supernatant of DFATs and compared the microRNA (miRNA) expression profiles of DFAT-derived exosomes (DFAT-Exo) with those of ASC-derived exosomes (ASC-Exo). Additionally, we assessed the effect of DFAT-Exo administration on proliferation and phenotypic changes of nucleus pulposus (NP) cells and in T lymphocytes in an in vitro setting. Methods Donor-matched DFATs and ASCs were prepared from adipose tissue of three osteoarthritis patients. Exosomes were isolated from the supernatant of both cell types. The global miRNA expression profiles of DFAT-Exo and ASC-Exo were analyzed using miRNA microarrays. Following administration of DFAT-Exo and ASC-Exo, the cell proliferation capability and expression of chondrogenic marker genes in NP cells were evaluated. Flow cytometric analysis was performed to examine the effects of DFAT-Exo and ASC-Exo administration on the anti-CD3/CD28-stimulated proliferation of T cells and on the differentiation of naive T cells into regulatory T cell and Th17 cell subsets. Results Exosomes were successfully isolated from both DFATs and ASCs. MiRNA microarray analysis revealed that the miRNA profiles of DFAT-Exo closely resemble those of ASC-Exo, when compared to fibroblast-derived exosomes. Notably, certain miRNAs specifically expressed in DFAT-Exo were identified. Administration of DFAT-Exo and ASC-Exo promoted cell proliferation and the expression of cartilage-related genes Sox9 and Versican in NP cells. Both DFAT-Exo and ASC-Exo equivalently suppressed anti-CD3/CD28-stimulated proliferation of T cells. Moreover, DFAT-Exo and ASC-Exo exhibited similar capacities to promote the differentiation of naive T cells into regulatory T cell subsets and hinder the differentiation into Th17 cell subsets. Conclusions DFAT-Exo showed comparable miRNA expression profiles and biological activities to those of ASC-Exo. DFAT-Exo may be an alternative attractive tool for exosome-based therapeutic approaches.

The liver is capable of regeneration, regaining mass through compensatory hypertrophy even after resecting 70% of its mass. However, the underlying regenerative mechanism remains poorly understood. Thus, in this study, microarray data were procured over time, from 0 to 14 days post-70% partial hepatectomy (PH) in mice. This dataset was correlated with the body weight and liver weight measured at the time of liver harvest, allowing us to determine the progress of compensatory hypertrophy in each sample. Furthermore, the impact of individual differences in regenerative capacity was analyzed by preparing three biological replicates at each time point. The microarray data were confirmed to be of high quality based on the RNA integrity number and QC stats of the dataset. The reliability of the dataset was further supported by the results of hierarchical clustering and the expression variation of cell cycle-related genes. This microarray data will contribute to the elucidation of the regenerative mechanism in the 70% PH model.

Bone graft materials frequently employed in dental implant placement procedures, including hydroxyapatite and β-tricalcium phosphate (β-TCP), are typically granular in form, which complicates their manipulation and contributes to extended treatment durations. A tissue engineering approach utilizing readily manageable biomaterials in conjunction with mesenchymal stem cells (MSCs) represents the most promising approach in dentistry. This study assessed the bone-augmenting capacity at bone defect sites in inbred rats by seeding dedifferentiated fat (DFAT) cells onto a cotton-like bone graft scaffold composed of β-TCP and poly(L-lactic-co- glycolide) (PLGA), which was subsequently wrapped around titanium. As a control, cotton-like bone graft material without cells was used and wrapped around and transplanted. Four weeks post-implantation, computed tomography (CT) images of the DFAT group revealed a 1.25-fold enhancement in hard tissue formation compared to the control group. Histological analysis revealed compact structure in a dark red color surrounding the cotton-like bone graft material was observed on the titanium surface of DFAT group. Histomorphometric analysis revealed that the amount of hard tissue generated in the DFAT group was approximately 2.5 times higher than that observed in the control group. Moreover, this mineralized tissue demonstrated properties analogous to those observed in cortical bone. Collectively, these findings indicate that the composite of DFAT cells and cotton-like bone graft material holds potential for bone augmentation applications and represents a promising approach for regenerative therapies within the orofacial region.

Avian adipocyte differentiation differs from mammalian adipocyte differentiation; however, no useful preadipocyte cell line has been established to study avian adipocyte differentiation. This study investigated whether dedifferentiated fat (DFAT) cells derived from chick mature adipocytes have the same characteristics as stromal vascular fraction (SVF) cells derived from adipose tissues and whether DFAT cells can be used as an avian preadipocyte cell line. The floating top layer, which contained mature adipocytes, was isolated from chick abdominal fat tissue by collagenase digestion and filtration. To induce spontaneous dedifferentiation, the isolated mature adipocytes were cultured using the ceiling culture method. After 14 days of culture, the proliferation and differentiation potential of the resulting DFAT cells was evaluated. The isolated mature adipocytes successfully dedifferentiated into DFAT cells and actively proliferated in ceiling culture. The proliferative capacity of DFAT cells was similar to that of the SVF cells, although the ability of DFAT cells to differentiate into mature adipocytes was significantly higher than that of SVF cells. Subcultured DFAT cells maintained normal proliferation and differentiation into adipocytes for at least 33 passages. This study demonstrated that chicken DFAT cells provide a readily established preadipocyte cell line derived from mature adipocytes, which can be used as a model for investigating adipogenic differentiation in chickens.

Mature adipocyte-derived dedifferentiated fat (DFAT) cells show proliferative capabilities and multipotency. Given that the buccal fat pad (BFP) serves as a readily available resource for DFAT cell isolation, BFP-derived DFAT (BFP-DFAT) cells are a promising candidate in orofacial tissue engineering. In this research, we assessed the regenerative capacity of the periodontium through autologous BFP-DFAT cell transplantation in adult swine (micro-minipigs; MMPs). The BFP-DFAT cells were transplanted into inflammation-inducing two-walled infrabony periodontal defects located on the mesial of the second mandibular premolar (n = 6). Twelve weeks post-transplantation, a remarkable attachment gain was noted in the DFAT group, based on probing depths and clinical attachment levels. Histological and immunohistochemical analyses indicated new continuous cellular cementum and alveolar bone formation within the created infrabony defect. Well-organized periodontal ligament-like fibers were embedded between newly formed cementum and the alveolar bone. Histometric analysis demonstrated that the DFAT group had a 2.2-fold increase in new alveolar bone length and a 2.2-fold enhancement in vascularization than those in the control group. Except for minor inflammation in the lungs, no teratomas were detected in the recipient MMPs. BFP-DFAT cells significantly enhanced periodontal tissue regeneration, thus representing an optimal source for tissue engineering applications in dentistry.

IntroductionMature adipocyte-derived dedifferentiated fat cells (DFATs) represent a subtype of multipotent cells that exhibit comparable phenotypic and functional characteristics to adipose-derived stem cells (ASCs). In this study, we assessed the chondroprotective properties of intra-articularly administrated DFATs in a rat model of osteoarthritis (OA). We also investigated in vitro the expression of anti-inflammatory and chondroprotective genes in DFATs prepared from the infrapatellar fat pad (IFP) and subcutaneous adipose-tissue (SC) of human origin.MethodsIn the cell transplantation experiment, rats were assigned to the DFAT and Control group (n = 10 in each group) and underwent anterior cruciate ligament transection (ACLT) accompanied by medial meniscus resection (MMx) to induce OA. One week later, they received intra-articular injections of 1 × 106 DFATs (DFAT group) or PBS (control group) four times, with a weekly administration frequency. Macroscopic and microscopic evaluations were conducted five weeks post-surgery. In the in vitro experiments. DFATs derived from the IFP (IFP-DFATs) and SC (SC-DFATs) were prepared from donor-matched tissue samples (n = 3). The gene expression of PTGS2, TNFAIP6, PRG4, BMP2, and BMP6 under TNF-α or IFN-γ stimulation in these cells was evaluated using RT-PCR. Furthermore, the effect of co-culturing synovial fibroblasts with DFATs on the gene expression of ADAMTS4 and IL-6 were evaluated.ResultsIntra-articular injections of DFATs significantly inhibited cartilage degeneration in the rat OA model induced by ACLT and MMx. RT-PCR analysis revealed that both IFP-DFATs and SC-DFATs upregulated the expression of genes involved in immune regulation, anti-inflammation, and cartilage protection such as PTGS2, TNFAIP6, and BMP2, under stimulation by inflammatory cytokines. Co-culture with DFATs suppressed the expression of ADAMTS4 and IL6 in synovial fibroblasts.ConclusionsThe intra-articular injection of DFATs resulted in chondroprotective effects in the rat OA model. Both SC-DFATs and IFP-DFATs induced the expression of anti-inflammatory and chondroprotective genes in vitro. These results indicate that DFATs appear to possess therapeutic potential in inhibiting cartilage degradation and could serve as a promising cellular resource for OA treatment.

Background Mesenchymal stem cells (MSCs) are known to have different differentiation potential depending on the tissue of origin. Dedifferentiated fat cells (DFATs) are MSC-like multipotent cells that can be prepared from mature adipocytes by ceiling culture method. It is still unknown whether DFATs derived from adipocytes in different tissue showed different phenotype and functional properties. In the present study, we prepared bone marrow (BM)-derived DFATs (BM-DFATs), BM-MSCs, subcutaneous (SC) adipose tissue-derived DFATs (SC-DFATs), and adipose tissue-derived stem cells (ASCs) from donor-matched tissue samples. Then, we compared their phenotypes and multilineage differentiation potential in vitro. We also evaluated in vivo bone regeneration ability of these cells using a mouse femoral fracture model. Methods BM-DFATs, SC-DFATs, BM-MSCs, and ASCs were prepared from tissue samples of knee osteoarthritis patients who received total knee arthroplasty. Cell surface antigens, gene expression profile, and in vitro differentiation capacity of these cells were determined. In vivo bone regenerative ability of these cells was evaluated by micro-computed tomography imaging at 28 days after local injection of the cells with peptide hydrogel (PHG) in the femoral fracture model in severe combined immunodeficiency mice. Results BM-DFATs were successfully generated at similar efficiency as SC-DFATs. Cell surface antigen and gene expression profiles of BM-DFATs were similar to those of BM-MSCs, whereas these profiles of SC-DFATs were similar to those of ASCs. In vitro differentiation analysis revealed that BM-DFATs and BM-MSCs had higher differentiation tendency toward osteoblasts and lower differentiation tendency toward adipocytes compared to SC-DFATs and ASCs. Transplantation of BM-DFATs and BM-MSCs with PHG enhanced bone mineral density at the injection sites compared to PHG alone in the mouse femoral fracture model. Conclusions We showed that phenotypic characteristics of BM-DFATs were similar to those of BM-MSCs. BM-DFATs exhibited higher osteogenic differentiation potential and bone regenerative ability compared to SC-DFATs and ASCs. These results suggest that BM-DFATs may be suitable sources of cell-based therapies for patients with nonunion bone fracture.

Introduction: Mature adipocyte-derived dedifferentiated fat cells (DFATs) are mesenchymal stem cell (MSC)-like cells with high proliferative ability and multilineage differentiation potential. In this study, we first examined whether DFATs can be prepared from infrapatellar fat pad (IFP) and then compared phenotypic and functional properties of IFP-derived DFATs (IFP-DFATs) with those of subcutaneous adipose tissue (SC)-derived DFATs (SC-DFATs). Methods: Mature adipocytes isolated from IFP and SC in osteoarthritis patients (n = 7) were cultured by ceiling culture method to generate DFATs. Obtained IFP-DFATs and SC-DFATs were subjected to flow cytometric and microarray analysis to compare their immunophenotypes and gene expression profiles. Cell proliferation assay and adipogenic, osteogenic, and chondrogenic differentiation assays were performed to evaluate their functional properties. Results: DFATs could be prepared from IFP and SC with similar efficiency. IFP-DFATs and SC-DFATs exhibited similar immunophenotypes (CD73(+), CD90(+), CD105(+), CD31(-), CD45(-), HLA-DR-) and tri-lineage (adipogenic, osteogenic, and chondrogenic) differentiation potential, consistent with the minimal criteria for defining MSCs. Microarray analysis revealed that the gene expression profiles in IFP-DFATs were very similar to those in SC-DFATs, although there were certain number of genes that showed different levels of expression. The proliferative activity in IFP-DFATs was significantly (p < 0.05) higher than that in the SC-DFATs. IFP-DFATs showed higher chondrogenic differentiation potential than SC-DFATs in regard to production of soluble galactosaminogalactan and gene expression of type II collagen. Conclusions: IFP-DFATs showed higher cellular proliferative potential and higher chondrogenic differentiation capacity than SC-DFATs. IFP-DFAT cells may be an attractive cell source for chondrogenic regeneration. (C) 2022, The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.

Introduction The implantation of dedifferentiated fat (DFAT) cells has been shown to exert immunosuppressive effects. To develop DFAT cell therapy for antineutrophil cytoplasmic antibody (ANCA) glomerulonephritis, the effects of the implantation of DFAT cells on ANCA glomerulonephritis were investigated in mice. Methods PKH26-labeled DFAT cells (10 5 ) were infused through the posterior orbital venous plexus to investigate delivery of DFAT cells in ICR mice. DFAT cells (10 5 ) were also implanted in SCG mice as a model for ANCA glomerulonephritis. Expression of tumor necrosis factor-stimulated gene-6 (TSG-6) mRNA and protein in kidney was evaluated, and the expression of microRNAs associated with TSG-6 in plasma, lung and kidney was analyzed. Expressions of CD44, prostaglandin (PG) E2, interleukin (IL)-10, IL-1 β , tumor necrosis factor (TNF)- α mRNAs, C–C motif chemokine ligand 17 (CCL-17) and monocyte chemoattractant protein (MCP)-1 proteins were measured in kidney from SCG mice implanted with DFAT cells. Results After their intravenous infusion, almost all DFAT cells were trapped in the lung and not delivered into the kidney. Implantation of DFAT cells in SCG mice suppressed glomerular crescent formation, decreased urinary protein excretions and increased expression of TSG-6 mRNA, protein and immunostaining in kidney from these mice. Increased expression of microRNA 23b-3p in plasma, kidney and lung; decreased expression of CD44 mRNA; and increased expression of PGE2 and IL-10 mRNAs were also observed in kidney from these mice. Implantation of DFAT cells also decreased the expression of TNF- α and MCP-1 proteins and increased that of CCL-17 protein in kidney from the SCG mice. Survival rates were higher in SCG mice implanted with DFAT cells than in SCG mice without implantation. Conclusion Mechanisms underlying the effects of improvement of ANCA glomerulonephritis are associated with immunosuppressive effects by TSG-6 and the transition of M1–M2 macrophages, suggesting that implantation of DFAT cells may become a cell therapy for ANCA glomerulonephritis.

Dedifferentiated fat (DFAT) cells are mature adipocyte-derived multipotent cells that can be applicable to cell-based therapy for stress urinary incontinence (SUI). This study developed a persistence SUI model that allows long-term evaluation using a combination of vaginal distention (VD) and bilateral ovariectomy (OVX) in rats. Then, the therapeutic effects of DFAT cell transplantation in the persistence SUI model was examined. In total, 48 Sprague–Dawley rats were divided into four groups and underwent VD (VD group), bilateral OVX (OVX group), VD and bilateral OVX (VD + OVX group), or sham operation (Control group). At 2, 4, and 6 weeks after injury, leak point pressure (LPP) and histological changes of the urethral sphincter were evaluated. Next, 14 rats undergoing VD and bilateral OVX were divided into two groups and administered urethral injection of DFAT cells (DFAT group) or fibroblasts (Fibroblast group). At 6 weeks after the injection, LPP and histology of the urethral sphincter were evaluated. The VD + OVX group retained a decrease in LPP with sphincter muscle atrophy at least until 6 weeks after injury. The LPP and urethral sphincter muscle atrophy in the DFAT group recovered better than those in the fibroblast group. The persistence SUI model was created by a combination of VD and bilateral OVX in rats. Urethral injection of DFAT cells inhibited sphincter muscle atrophy and improved LPP in the persistence SUI model. These findings suggest that the DFAT cells may be an attractive cell source for cell-based therapy to treat SUI.

Adipose tissue is composed mostly of adipocytes that are in contact with capillaries. By using a ceiling culture method based on buoyancy, lipid-free fibroblast-like cells, also known as dedifferentiated fat (DFAT) cells, can be separated from mature adipocytes with a large single lipid droplet. DFAT cells can re-establish their active proliferation ability and transdifferentiate into various cell types under appropriate culture conditions. Herein, we sought to compare the regenerative potential of collagen matrix alone (control) with autologous DFAT cell-loaded collagen matrix transplantation in adult miniature pigs (microminipigs; MMPs). We established and transplanted DFAT cells into inflammation-inducing periodontal class II furcation defects. At 12 weeks after cell transplantation, a marked attachment gain was observed based on the clinical parameters of probing depth (PD) and clinical attachment level (CAL). Additionally, micro computed tomography (CT) revealed hard tissue formation in furcation defects of the second premolar. The cemento-enamel junction and alveolar bone crest distance was significantly shorter following transplantation. Moreover, newly formed cellular cementum, well-oriented periodontal ligament-like fibers, and alveolar bone formation were observed via histological analysis. No teratomas were found in the internal organs of recipient MMPs. Taken together, these findings suggest that DFAT cells can safely enhance periodontal tissue regeneration.

Regulation of the interaction between fibronectin and integrin α5 contributes to the reorganization of the actin cytoskeleton associated with the completion of adipocyte differentiation.

Background We established an adipogenic progenitor cell line derived from mature adipocytes and named these cells dedifferentiated fat (DFAT) cells, which have been shown to have characteristics very similar to those of mesenchymal stem cells (MSCs). The potential application of DFAT cells to support cell-based therapies for regenerative and immunosuppressive therapies has been suggested. The present study was designed to address beneficial ways that DFAT implantation can be used clinically as immunosuppressive therapy to treat immunological glomerulonephritis. Methods We evaluated distribution of DFAT cells after intravenous injection through the tail vein in Wistar rats. We examined effects of allogenic implantation of DFAT cells on BrdU incorporation into kidney from rats with monoclonal antibody (mAb) 1-22-3-induced glomerulonephritis. We compared effects of allogenic and autogenic implantations of DFAT cells on excretion of urinary protein, renal function, and glomerular and nephrotubular injuries in these rats, and serum levels of tumor necrosis factor-stimulated gene-6 (TSG-6), and expression of TSG-6 mRNA in kidney. Results The allogenic implantations of DFAT cells trapped in lung improved excretion of urinary protein and renal function, and significantly suppressed glomerular and nephrotubular injuries in the rats with mAb1-22-3-induced glomerulonephritis compared with the autogenic implantations. The allogenic implantation of DFAT cells increased serum levels of TSG-6 especially in mAb 1-22-3-induced glomerulonephritis and significantly increased the expression of TSG-6 mRNA in kidney compared to the autogenic implantation. Conclusion These findings suggest that allogenic implantation of DFAT cells could be clinically useful immunosuppressive therapy for immunological glomerulonephritis.

Here, we established dedifferentiated fat (DFAT) cells from mature bovine adipocytes and then examined the effects of volatile fatty acids on the differentiation of these DFAT cells into adipocytes in vitro. When mature adipocytes were isolated from bovine adipose tissue and cultured using the ceiling culture method, they were dedifferentiated into fibroblast-like cells without lipid droplets. These fibroblast-like cells, termed bovine DFAT (b-DFAT) cells, actively proliferated. After adipogenic induction, increased expression of adipocyte-specific genes occurred in b-DFAT cells and they redifferentiated into adipocytes with an accumulation of lipid droplets in their cytoplasm. The effects of volatile fatty acids on adipocyte differentiation in b-DFAT cells were also examined. Specifically, acetate, butyrate, and propionate added to adipogenic induction medium significantly enhanced the adipogenesis of b-DFAT cells compared with that observed in control cells; the addition of 10-3 mol of acetate enhanced adipogenesis of b-DFAT cells to the greatest extent. These results suggest that b-DFAT cells derived from bovine mature adipocytes are appropriate for the study of bovine adipocyte differentiation and that the optimum concentration treatment of acetate, a major energy source for ruminants, promotes adipogenesis of b-DFAT cells in vitro.

and surgery. Recently, regenerative medicine using stem cells has been applied in the field of orthopedics. Dedifferentiated fat (DFAT) cells are mature adipocyte-derived multipotent cells that have characteristics similar to mesenchymal stem cells. In this study, we examined whether DFAT cell transplantation into the fracture site promotes bone formation in a mouse femoral fracture model. As a result, we found that DFAT cell transplantation promoted callus formation and increased bone volume around the fracture site. These results suggest that DFAT cells are an attractive cell source for the treatment of refractory fracture.

The specification of cell identity depends on the exposure of cells to sequences of bioactive ligands. All-trans retinoic acid (ATRA) affects neuronal development in the early stage, and it is involved in neuronal lineage reprogramming. We previously established a fibroblast-like dedifferentiated fat cells (DFATs) derived from highly homogeneous mature adipocytes, which are more suitable for the study of cellular reprogramming. Canine cognitive dysfunction is similar to human cognitive dysfunction, suggesting that dogs could be a pathological and pharmacological model for human neuronal diseases. However, the effect of ATRA on neuronal reprogramming in dogs has remained unclear. Therefore, in this study, we investigated the effect of ATRA on the neuronal reprogramming of canine DFATs. ATRA induced the expression of neuronal marker mRNA/protein. The neuron-like cells showed Ca2+ influx with depolarization (50 mM KCl; 84.75 ± 4.05%) and Na+ channel activation (50 μM veratridine; 96.02 ± 2.02%). Optical imaging of presynaptic terminal activity and detection of neurotransmitter release showed that the neuron-like cells exhibited the GABAergic neuronal property. Genome-wide RNA-sequencing analysis shows that the transcriptome profile of canine DFATs is effectively reprogrammed towards that of cortical interneuron lineage. Collectively, ATRA can produce functional GABAergic cortical interneuron-like cells from canine DFATs, exhibiting neuronal function with > 80% efficiency. We further demonstrated the contribution of JNK3 to ATRA-induced neuronal reprogramming in canine DFATs. In conclusion, the neuron-like cells from canine DFATs could be a powerful tool for translational research in cell transplantation therapy, in vitro disease modeling, and drug screening for neuronal diseases.

Adipocyte differentiation is accompanied by a pronounced change in the actin cytoskeleton characterized by the reorganization of filamentous (F)-actin stress fibers into cortical F-actin structures. We previously showed that depolymerization of F-actin stress fibers induced by inactivation of RhoA-ROCK (Rho-associated kinase) signaling acts as a trigger for adipocyte differentiation. The relevance and underlying mechanism of the formation of cortical F-actin structures from depolymerized actin during adipocyte differentiation have remained unclear, however. We have now examined the mechanistic relation between actin dynamics and adipogenic induction. Transient exposure to the actin-depolymerizing agent latrunculin A (LatA) supported the formation of adipocyte-associated cortical actin structures and the completion of terminal adipocyte differentiation in the presence of insulin, whereas long-term exposure to LatA prevented such actin reorganization as well as terminal adipogenesis. Moreover, these effects of insulin were prevented by inhibition of phosphatidylinositol 3-kinase (PI3K)-Rac1 signaling and the actin-related protein 2/3 (Arp2/3) complex which is a critical component of the cortical actin networks. Our findings thus suggest that the insulin-PI3K-Rac1 axis leads to the formation of adipocyte-associated cortical actin structures which is essential for the completion of adipocyte differentiation.

Mature adipocyte-derived dedifferentiated fat (DFAT) cells have been identified to possess similar multipotency to mesenchymal stem cells, but a method for converting DFAT cells into hepatocytes was previously unknown. Here, using comprehensive analysis of gene expression profiles, we have extracted three transcription factors, namely Foxa2, Hnf4a and Sall1 (FHS), that can convert DFAT cells into hepatocytes. Hepatogenic induction has converted FHS-infected DFAT cells into an epithelial-like morphological state and promoted the expression of hepatocyte-specific features. Furthermore, the DFAT-derived hepatocyte-like (D-Hep) cells catalyzed the detoxification of several compounds. These results indicate that the transduction of DFAT cells with three genes, which were extracted by comprehensive gene expression analysis, efficiently generated D-Hep cells with detoxification abilities similar to those of primary hepatocytes. Thus, D-Hep cells may be useful as a new cell source for surrogate hepatocytes and may be applied to drug discovery studies, such as hepatotoxicity screening and drug metabolism tests.