Hepatic Differentiation Research Articles

Sirt1/Nrf2/TNFα; TLR4/Myd88/NF-κB signaling pathways are involved in mediating hepatoprotective effect of bupropion in rat model of myocardial infarction

Background The aim of the current study is to identify the possible protective effect of bupropion (BUP) on liver injury in rat model of myocardial infarction (MI). BUP was administered in the presence and absence of MI. Materials and methods Thirty-two Wistar adult male rats were randomly arranged into four groups: control, BUP (30 mg/kg/day, intraperitoneal) for 28 days, isoproterenol (ISO) was injected subcutaneous (85 mg/kg) in the 26th and 27th days and BUP/ISO groups. Cardiac and hepatic enzymes were measured, also Hepatic oxidative stress indicators, as well as inflammatory and apoptotic biomarkers, were evaluated. Cardiac and hepatic histopathological examination and hepatic nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) immunohistochemical study were also detected. Results ISO significantly increased cardiac and hepatic enzymes, hepatic oxidative stress, inflammatory, apoptotic, with a histopathological picture of cardiac and hepatic damage and high hepatic NF-κB immunoexpression were detected. BUP significantly normalized the upraised oxidative, inflammatory, and apoptotic parameters, with an impressive improvement in the histopathological picture and a reduction in hepatic NF-κB immunoexpression. Conclusion BUP protects against liver injury on top of MI in rat model via modulation of Sirtuin type 1 (Sirt1)/Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/tumor necrosis factor α (TNFα); Toll-like receptor 4 (TLR4)/Hepatic myeloid differentiation primary response 88(Myd88)/NF-κB signaling pathways.

Primary Malignant Peripheral Nerve Sheath Tumor in the Liver with Glandular Differentiation

Abstract Introduction/Objective Malignant peripheral nerve sheath tumors (MPNST) most commonly affect the proximal extremities and paraspinal region. Primary hepatic MPNST is extremely rare. Here we report a primary hepatic MPNST with heterologous glandular differentiation in a patient with Neurofibromatosis type 1 (NF1). Methods/Case Report Review of the clinical features and histopathological findings of the resected specimen. A review of all the published cases of primary liver MPNST. Results (if a Case Study enter NA) A 69-year-old male patient with a history of NF1, Gastrointestinal Stromal Tumor and treated hepatitis C without pre-existed neurofibroma presented with jaundice. Imaging revealed an ill-defied hypodense mass in the left hepatic lobe. A segmentectomy of the liver revealed an infiltrating 10 cm heterogenous yellow-white-pink slightly firm tumor with 5% necrosis. The tumor showed biphasic histology composed of predominantly spindle cells and clusters of glandular components. The spindle cells showed fascicular growth pattern with vaguely marbled appearance. Focal osteoclast-like multinucleated cells were seen. The glands were well defined, mostly appeared to be low grade, and lined by cuboidal to columnar cells, with a bile-duct and intestinal-like appearance. However, focal malignant glands with hepatic differentiation were also noted. Immunohistochemistry showed patchy loss of H3K27me3 in the spindle cells. The glandular cells were positive for AE1/AE3, CK7 and CK20. The tumor cells showed rare reactivity to TLE1 and were negative for S-100, SOX10, CD34, CD117 and DOG1. These findings supported the diagnosis of MPNST with glandular differentiation. 13 cases of primary MPNST of liver have been reported, and only one case described epithelioid differentiation. Conclusion The differential diagnoses of a primary hepatic biphasic tumor include sarcomatoid carcinoma, carcinosarcoma, biphasic synovial sarcoma and MPNST. Our case illustrates a rare biphasic hepatic MPNST. The morphology and loss of H3K27me3 expression by immunohistochemistry in a patient with NF1 support this challenging diagnosis.

MRI radiomics model differentiates small hepatic metastases and abscesses in periampullary cancer patients

This multi-center, retrospective study focused on periampullary cancer patients undergoing MRI for hepatic metastasis and abscess differentiation. T1-weighted, T2-weighted, and arterial phase images were utilized to create radiomics models. In the training-set, 112 lesions in 54 patients (median age [IQR, interquartile range], 73 [63–80]; 38 men) were analyzed, and 123 lesions in 55 patients (72 [66–78]; 34 men) comprised the validation set. The T1-weighted + T2-weighted radiomics model showed the highest AUC (0.82, 95% CI 0.75–0.89) in the validation set. Notably, < 30% T1-T2 size discrepancy in MRI findings predicted metastasis (Ps ≤ 0.037), albeit with AUCs of 0.64–0.68 for hepatic metastasis. The radiomics model enhanced radiologists’ performance (AUCs, 0.85–0.87 vs. 0.80–0.84) and significantly increased diagnostic confidence (P < 0.001). Although the performance increase lacked statistical significance (P = 0.104–0.281), the radiomics model proved valuable in differentiating small hepatic lesions and enhancing diagnostic confidence. This study highlights the potential of MRI-based radiomics in improving accuracy and confidence in the diagnosis of periampullary cancer-related hepatic lesions.

Small Molecule-Mediated Stage-Specific Reprogramming of MSCs to Hepatocyte-Like Cells and Hepatic Tissue for Liver Injury Treatment.

Derivation of hepatocytes from stem cells has been established through various protocols involving growth factor (GF) and small molecule (SM) agents, among others. However, mesenchymal stem cell-based derivation of hepatocytes still remains expensive due to the use of a cocktail of growth factors, and a long duration of differentiation is needed, thus limiting its potential clinical application. In this study, we developed a chemically defined differentiation strategy that is exclusively based on SM and takes 14days, while the GF-based protocol requires 23-28days. We optimized a stage-specific differentiation protocol for the differentiation of rat bone marrow-derived mesenchymal stem cells (MSCs) into functional hepatocyte-like cells (dHeps) that involved four stages, i.e., definitive endoderm (DE), hepatic competence (HC), hepatic specification (HS) and hepatic differentiation and growth. We further generated hepatic tissue using human decellularized liver extracellular matrix and compared it with hepatic tissue derived from the growth factor-based protocol at the transcriptional level. dHep, upon transplantation in a rat model of acute liver injury (ALI), was capable of ameliorating liver injury in rats and improving liver function and tissue damage compared to those in the ALI model. In summary, this is the first study in which hepatocytes and hepatic tissue were derived from MSCs utilizing a stage-specific strategy by exclusively using SM as a differentiation factor.

Framework nucleic Acid-MicroRNA mediated hepatic differentiation and functional hepatic spheroid development for treating acute liver failure

The specific induction of hepatic differentiation presents a significant challenge in developing alternative liver cell sources and viable strategies for clinical therapy of acute liver failure (ALF). The past decade has witnessed the blossom of microRNAs in regenerative medicine. Herein, microRNA 122-functionalized tetrahedral framework nucleic acid (FNA-miR-122) has emerged as an unprecedented and potential platform for directing the hepatic differentiation of adipose-derived mesenchymal stem cells (ADMSCs), which offers a straightforward and cost-effective method for generating functional hepatocyte-like cells (FNA-miR-122-iHep). Additionally, we have successfully established a liver organoid synthesis strategy by optimizing the co-culture of FNA-miR-122-iHep with endothelial cells (HUVECs), resulting in functional Hep:HUE-liver spheroids. Transcriptome analysis not only uncovered the potential molecular mechanisms through which miR-122 influences hepatic differentiation in ADMSCs, but also clarified that Hep:HUE-liver spheroids could further facilitate hepatocyte maturation and improved tissue-specific functions, which may provide new hints to be used to develop a hepatic organoid platform. Notably, compared to transplanted ADMSCs and Hep-liver spheroid, respectively, both FNA-miR-122-iHep-based single cell therapy and Hep:HUE-liver spheroid-based therapy showed high efficacy in treating ALF in vivo. Collectively, this research establishes a robust system using microRNA to induce ADMSCs into functional hepatocyte-like cells and to generate hepatic organoids in vitro, promising a highly efficient therapeutic approach for ALF.

Development of a hepatic differentiation method in 2D culture from primary human hepatocyte-derived organoids for pharmaceutical research

Human liver organoids derived from primary human hepatocytes (PHHs) are expected to be a hepatocyte source for preclinical invitro studies of drug metabolism and disposition. Because hepatic functions of these organoids remain low, it is necessary to enhance the hepatic functions. Here, we develop a novel method for two dimensional (2D)-cultured hepatic differentiation from PHH-derived organoids by screening several compounds, cytokines, and growth factors. Hepatic gene expressions in the hepatocyte-like cells differentiated from PHH-derived organoids (Org-HEPs) were greatly increased, compared to those in PHH-derived organoids. The metabolic activities of cytochrome P450 (CYP) 1A2, CYP2C8, CYP2C19, CYP2E1, and CYP3A4 were at levels comparable to those in PHHs. The cell viability of Org-HEPs treated with hepatotoxic drugs was almost the same as that of PHHs. Thus, PHH-derived organoids could be differentiated into highly functional hepatocytes in 2D culture. Thus, Org-HEPs will be useful for pharmaceutical research, including hepatotoxicity tests.

Role of HNF4alpha-cMyc interaction in liver regeneration after partial hepatectomy.

Hepatocyte nuclear factor 4 alpha (HNF4α) is the master regulator of hepatic differentiation. Recent studies have also revealed the role of HNF4α in hepatocyte proliferation via negatively regulating the expression of proto-mitogenic genes, including cMyc. Here, we aimed to study the interaction between HNF4α-cMyc during liver regeneration after partial hepatectomy (PHX). Wild-type (WT), hepatocyte-specific knockout of HNF4α (HNF4α-KO), cMyc (cMyc-KO), and HNF4α-cMyc double knockout (DKO) mice were subjected to PHX to induce liver regeneration. Blood and liver tissue samples were collected at 0h, 24h, 48h, 7D, and 14D after PHX for further analysis. WT, HNF4α-KO, cMyc-KO and DKO mice regained liver weight by 14 days after PHX. The deletion of cMyc did not affect liver regeneration, which was similar to the WT mice. WT and cMyc-KO mice started regaining liver weight as early as 24 hours after PHX, with a peak proliferation response at 48 hours after PHX. HNF4α- KO and DKO showed a delayed response with liver weight increase by day 7 after PHX. The overall hepatocyte proliferation response by DKO mice following PHX was lower than that of other genotypes. Interestingly, the surviving HNF4α-KO and DKO mice showed re-expression of HNF4α at mRNA and protein levels on day 14 after PHX. This was accompanied by a significant increase in the expression of Krt19 and Epcam, hepatic progenitor cell markers, in the DKO mice on day 14 after PHX. These data indicate that, in the absence of HNF4α, cMyc contributes to hepatocyte-driven proliferation to compensate for the lost tissue mass. Furthermore, in the absence of both HNF4α and cMyc, HPC-driven proliferation occurs to support liver regeneration.

Fasudil and viscosity of gelatin promote hepatic differentiation by regulating organelles in human umbilical cord matrix-mesenchymal stem cells

BackgroundHuman mesenchymal stem cells originating from umbilical cord matrix are a promising therapeutic resource, and their differentiated cells are spotlighted as a tissue regeneration treatment. However, there are limitations to the medical use of differentiated cells from human umbilical cord matrix-mesenchymal stem cells (hUCM-MSCs), such as efficient differentiation methods.MethodsTo effectively differentiate hUCM-MSCs into hepatocyte-like cells (HLCs), we used the ROCK inhibitor, fasudil, which is known to induce endoderm formation, and gelatin, which provides extracellular matrix to the differentiated cells. To estimate a differentiation efficiency of early stage according to combination of gelatin and fasudil, transcription analysis was conducted. Moreover, to demonstrate that organelle states affect differentiation, we performed transcription, tomographic, and mitochondrial function analysis at each stage of hepatic differentiation. Finally, we evaluated hepatocyte function based on the expression of mRNA and protein, secretion of albumin, and activity of CYP3A4 in mature HLCs.ResultsFasudil induced endoderm-related genes (GATA4, SOX17, and FOXA2) in hUCM-MSCs, and it also induced lipid droplets (LDs) inside the differentiated cells. However, the excessive induction of LDs caused by fasudil inhibited mitochondrial function and prevented differentiation into hepatoblasts. To prevent the excessive LDs formation, we used gelatin as a coating material. When hUCM-MSCs were induced into hepatoblasts with fasudil on high-viscosity (1%) gelatin-coated dishes, hepatoblast-related genes (AFP and HNF4A) showed significant upregulation on high-viscosity gelatin-coated dishes compared to those treated with low-viscosity (0.1%) gelatin. Moreover, other germline cell fates, such as ectoderm and mesoderm, were repressed under these conditions. In addition, LDs abundance was also reduced, whereas mitochondrial function was increased. On the other hand, unlike early stage of the differentiation, low viscosity gelatin was more effective in generating mature HLCs. In this condition, the accumulation of LDs was inhibited in the cells, and mitochondria were activated. Consequently, HLCs originated from hUCM-MSCs were genetically and functionally more matured in low-viscosity gelatin.ConclusionsThis study demonstrated an effective method for differentiating hUCM-MSCs into hepatic cells using fasudil and gelatin of varying viscosities. Moreover, we suggest that efficient hepatic differentiation and the function of hepatic cells differentiated from hUCM-MSCs depend not only on genetic changes but also on the regulation of organelle states.

Role of Hepatocyte Nuclear Factor 4 Alpha in Liver Cancer.

Liver cancer is the sixth most common cancer and the fourth leading cause of cancer-related deaths worldwide. Hepatocellular carcinoma (HCC) is the most prevalent primary liver cancer and the incidence of HCC is on the rise. Liver cancers in general and HCC in particular do not respond to chemotherapy. Radiological ablation, surgical resection, and liver transplantation are the only medical therapies currently available. Hepatocyte nuclear factor 4 α (HNF4α) is an orphan nuclear receptor expressed only in hepatocytes in the liver. HNF4α is considered the master regulator of hepatic differentiation because it regulates a significant number of genes involved in various liver-specific functions. In addition to maintaining hepatic differentiation, HNF4α also acts as a tumor suppressor by inhibiting hepatocyte proliferation by suppressing the expression of promitogenic genes and inhibiting epithelial to mesenchymal transition in hepatocytes. Loss of HNF4α expression and function is associated with rapid progression of chronic liver diseases that ultimately lead to liver cirrhosis and HCC, including metabolism-associated steatohepatitis, alcohol-associated liver disease, and hepatitis virus infection. This review summarizes the role of HNF4α in liver cancer pathogenesis and highlights its potential as a potential therapeutic target for HCC.

CD157 selectively identifies hPSCs with enhanced hepatic differentiation capacity

CD157 selectively identifies hPSCs with enhanced hepatic differentiation capacity

Nanofibril guided spheroid formation for enhanced pluripotency and differentiation of human induced pluripotent stem cells

The spheroid formation derived from human-induced pluripotent stem cells (hiPSCs) generates a three-dimensional (3D) microenvironment that fosters cell–cell interactions, modulates epigenetic regulation, and imitates early embryonic development, all of which contribute to biomedical applications including drug discovery, disease modeling, and cell replacement therapy. In this study, we developed 3D hiPSC spheroids by incorporating surface-engineered nanofibrils to enhance their pluripotency and differentiation capability. Polymeric nanofibrils were chemically immobilized with cell adhesion peptides that mimic the properties of vitronectin and laminin. The incorporation of nanofibrils into hiPSC spheroids significantly improved their viability and undifferentiated state, which may be due to the facilitated mass transport across the spheroids, enabled by the loosely held structure of cells and nanofibrils. By harnessing the enhanced pluripotency of hiPSC spheroids with nanofibrils, we induced differentiation into the hepatic lineage and found a significant upregulation of mature hepatocyte-specific markers in spheroids with nanofibrils compared with those without nanofibrils. Therefore, the combination of 3D hiPSC spheroids with surface-engineered nanofibrils offers a promising approach to enhancing pluripotency and hepatic differentiation, with potential implications for regenerative medicine.

MicroRNA‐Modified DNA Hexahedron‐Induced Hepatocyte‐Like Cells Integrating 3D Printed Scaffold for Acute Liver Failure Therapy

AbstractAcute liver failure (ALF) involves extensive necrosis of liver cells and severe impairment of liver function. Hepatocyte transplantation holds promise for treating ALF by swiftly supporting liver functions and promoting liver regeneration. However, the scarcity of suitable cell sources requires strategies to obtain enough functional hepatocyte‐like cells (HLCs) and optimize their in vivo transplantation efficiency. A DNA hexahedral nanostructure (DHN) is developed loaded with microRNA‐122 to efficiently induce hepatic differentiation of human adipose‐derived mesenchymal stem cells into HLCs. These HLCs can serve as alternative hepatocyte sources, as confirmed by expression of liver‐specific genes and proteins, and the restoration of liver functions. To enhance in vivo survival efficiency of HLCs, a versatile scaffold is also created by 3D printing the calcium‐cross‐linked mixture bioink composed of sodium alginate, gelatin, and silk fibroin with excellent ROS scavenging capabilities. The scaffold is infused with chitosan‐DHN hydrogel containing HLCs for tissue engineering orthotopic transplantation in CCl4‐induced ALF mice. The transplanted composite scaffold‐HLCs successfully repair tissue necrosis in the liver injury area of mice and regulate expressions of genes and proteins associated with inflammation, oxidative stress, and hepatocyte function. Collectively, this study offers a novel approach and strategy for identifying alternative hepatocyte sources and treating ALF.

Cryopreserved cGMP-compliant human pluripotent stem cell-derived hepatic progenitors rescue mice from acute liver failure through rapid paracrine effects on liver cells

BackgroundLiver transplantation remains the only curative treatment for end-stage liver diseases. Unfortunately, there is a drastic organ donor shortage. Hepatocyte transplantation emerged as a viable alternative to liver transplantation. Considering their unique expansion capabilities and their potency to be driven toward a chosen cell fate, pluripotent stem cells are extensively studied as an unlimited cell source of hepatocytes for cell therapy. It has been previously shown that freshly prepared hepatocyte-like cells can cure mice from acute and chronic liver failure and restore liver function.MethodsHuman PSC-derived immature hepatic progenitors (GStemHep) were generated using a new protocol with current good manufacturing practice compliant conditions from PSC amplification and hepatic differentiation to cell cryopreservation. The therapeutic potential of these cryopreserved cells was assessed in two clinically relevant models of acute liver failure, and the mode of action was studied by several analytical methods, including unbiased proteomic analyses.ResultsGStemHep cells present an immature hepatic phenotype (alpha-fetoprotein positive, albumin negative), secrete hepatocyte growth factor and do not express major histocompatibility complex. A single dose of thawed GStemHep rescue mice from sudden death caused by acetaminophen and thioacetamide-induced acute liver failure, both in immunodeficient and immunocompetent animals in the absence of immunosuppression. Therapeutic biological effects were observed as soon as 3 h post-cell transplantation with a reduction in serum transaminases and in liver necrosis. The swiftness of the therapeutic effect suggests a paracrine mechanism of action of GStemHep leading to a rapid reduction of inflammation as well as a rapid cytoprotective effect with as a result a proteome reprograming of the host hepatocytes. The mode of action of GStemHep relie on the alleviation of inhibitory factors of liver regeneration, an increase in proliferation-promoting factors and a decrease in liver inflammation.ConclusionsWe generated cryopreserved and current good manufacturing practice-compliant human pluripotent stem cell-derived immature hepatic progenitors that were highly effective in treating acute liver failure through rapid paracrine effects reprogramming endogenous hepatocytes. This is also the first report highlighting that human allogeneic cells could be used as cryopreserved cells and in the absence of immunosuppression for human PSC-based regenerative medicine for acute liver failure.

3D Printing of a Vascularized Mini-Liver Based on the Size-Dependent Functional Enhancements of Cell Spheroids for Rescue of Liver Failure.

The emerging stem cell-derived hepatocyte-like cells (HLCs) are the alternative cell sources of hepatocytes for treatment of highly lethal acute liver failure (ALF). However, the hostile local environment and the immature cell differentiation may compromise their therapeutic efficacy. To this end, human adipose-derived mesenchymal stromal/stem cells (hASCs) are engineered into different-sized multicellular spheroids and co-cultured with 3D coaxially and hexagonally patterned human umbilical vein endothelial cells (HUVECs) in a liver lobule-like manner to enhance their hepatic differentiation efficiency. It is found that small-sized hASC spheroids, with a diameter of ≈50µm, show superior pro-angiogenic effects and hepatic differentiation compared to the other counterparts. The size-dependent functional enhancements are mediated by the Wnt signaling pathway. Meanwhile, co-culture of hASCs with HUVECs, at a HUVECs/hASCs seeding density ratio of 2:1, distinctly promotes hepatic differentiation and vascularization both in vitro and in vivo, especially when endothelial cells are patterned into hollow hexagons. After subcutaneous implantation, the mini-liver, consisting of HLC spheroids and 3D-printed interconnected vasculatures, can effectively improve liver regeneration in two ALF animal models through amelioration of local oxidative stress and inflammation, reduction of liver necrosis, as well as increase of cell proliferation, thereby showing great promise for clinical translation.

Human amniotic epithelial cells (HAECS); a potential cell type for the hepatic disorders.

To isolate a homogenous population of human amniotic epithelial cells (hAECs) from the amniotic membrane of the human placenta and differentiate them into hepatic-like cells with the help of small molecules. hAECs were isolated by using the enzymatic digestion method and characterized for the presence of specific stem cell markers. In-vitro, hepatic differentiation of hAECs was carried out by using a combination of small molecules. Differentiated cells were observed under a live cell imaging microscope for morphological changes followed by gene and protein expression analysis by qPCR and immunocytochemistry respectively. The isolated hAECs attained characteristic cuboid epithelial shape and express stem cells marker. The hepatic differentiation method was optimized based on soluble chemical compounds supplied in the culture medium. The differentiated hAECs phenotypically acquire hepatic-like cell features and expressed hepatic markers as well as hepatic protein albumin at immature levels. The isolated population of hAECs is highly proliferative. Moreover, hepatic markers expression in the isolated hAECs makes them an exclusive source for the treatment of chronic liver diseases.

In vitro characterization of 3D culture-based differentiation of human liver stem cells.

Introduction: The lack of functional hepatocytes poses a significant challenge for drug safety testing and therapeutic applications due to the inability of mature hepatocytes to expand and their tendency to lose functionality in vitro. Previous studies have demonstrated the potential of Human Liver Stem Cells (HLSCs) to differentiate into hepatocyte-like cells within an in vitro rotary cell culture system, guided by a combination of growth factors and molecules known to regulate hepatocyte maturation. In this study, we employed a matrix multi-assay approach to comprehensively characterize HLSC differentiation. Methods: We evaluated the expression of hepatic markers using qRT-PCR, immunofluorescence, and Western blot analysis. Additionally, we measured urea and FVIII secretion into the supernatant and developed an updated indocyanine green in vitro assay to assess hepatocyte functionality. Results: Molecular analyses of differentiated HLSC aggregates revealed significant upregulation of hepatic genes, including CYP450, urea cycle enzymes, and uptake transporters exclusively expressed on the sinusoidal side of mature hepatocytes, evident as early as 1day post-differentiation. Interestingly, HLSCs transiently upregulated stem cell markers during differentiation, followed by downregulation after 7days. Furthermore, differentiated aggregates demonstrated the ability to release urea and FVIII into the supernatant as early as the first 24h, with accumulation over time. Discussion: These findings suggest that a 3D rotation culture system may facilitate rapid hepatic differentiation of HLSCs. Despite the limitations of this rotary culture system, its unique advantages hold promise for characterizing HLSC GMP batches for clinical applications.

Schisandrin B promotes hepatic differentiation from human umbilical cord mesenchymal stem cells

Human umbilical cord mesenchymal stem cells (UC-MSCs)-derived hepatocyte-like cells (HLCs) have shown great promise in the treatment of liver diseases. However, most current induction protocols yield hepatocyte-like cells with limited function as compared with primary hepatocytes. Schisandrin B (Sch B) is one of the main components of Schisandra chinensis, which can prevent fibrosis progression and promote liver cell regeneration. Herein, we investigated the effects of Sch B on hepatic differentiation of UC-MSCs. We found that treatment with 10μM Sch B from the second stage of the differentiation process increased hepatic marker levels and hepatic function. Additionally, RNA-seq analysis revealed that Sch B promoted hepatic differentiation via activating the JAK2/STAT3 pathway. When transplanted HLCs into mice with CCL4-induced liver fibrosis, Sch B-treated HLCs exhibited significant therapeutic effects. This study provides an optimized hepatic differentiation protocol for UC-MSCs based on Sch B, yielding functioning cells for liver disease treatment.

Role of connexin 32 in the directional differentiation of induced pluripotent stem cells into hepatocytes.

This study aimed to explore the role of connexin 32 (Cx32) in the directional differentiation of induced pluripotent stem cells (iPSCs) into hepatocytes. Urine-derived epithelial cells were collected from the fresh urine of a healthy donor and transducted with reprogramming plasmid mixture to generate iPSCs. The iPSCs were then directionally differentiated into hepatocytes. During the differentiation, the upregulated and downregulated groups were treated with vitamin K2 (VK2) and 2-aminoethoxyboronate diphenylester (2-APB) to increase and inhibit Cx32 expression, respectively. The control group was not treated with the regulatory factor. Expression of Cx32 and hepatocyte-specific markers, including AFP, hepatocyte nuclear factor 4α (HNF-4α), albumin (ALB) and cytokeratin 18 (CK18) were detected. It indicated that Cx32 expression was not observed in iPSCs, but gradually increased during the process of hepatic differentiation from iPSCs. Upregulation of Cx32 expression by VK2 treatment promoted hepatocyte maturation and enhanced the expression of the aforementioned hepatic specific markers, whereas downregulation of Cx32 expression by 2-APB treatment had the opposite effects. In conclusion, urine-derived iPSCs could be directionally differentiated into hepatocytes. Up-regulation of Cx32 improves the efficiency and maturity of differentiation of iPSCs into hepatocytes, and Cx32 may be a promoting factor during the process of hepatic differentiation from iPSCs.

Microfluidic Fabrication of MicroRNA-Induced Hepatocyte-Like Cells/Human Umbilical Vein Endothelial Cells-Laden Microgels for Acute Liver Failure Treatment.

Acute liver failure (ALF) is a critical life-threatening disease that occurs due to a rapid loss in hepatocyte functions. Hepatocyte transplantation holds great potential for ALF treatment, as it rapidly supports liver biofunctions and enhances liver regeneration. However, hepatocyte transplantation is still limited by renewable and ongoing cell sources. In addition, intravenously injected hepatocytes are primarily trapped in the lungs and have limited efficacy because of the rapid clearance in vivo. Here, we designed a Y-shaped DNA nanostructure to deliver microRNA-122 (Y-miR122), which could induce the hepatic differentiation and maturation of human mesenchymal stem cells. mRNA sequencing analysis revealed that the Y-miR122 promoted important hepatic biofunctions of the induced hepatocyte-like cells including fat and lipid metabolism, drug metabolism, and liver development. To further improve hepatocyte transplantation efficiency and therapeutic effects in ALF treatment, we fabricated protective microgels for the delivery of Y-miR122-induced hepatocyte-like cells based on droplet microfluidic technology. When cocultured with human umbilical vein endothelial cells in microgels, the hepatocyte-like cells exhibited an increase in hepatocyte-associated functions, including albumin secretion and cytochrome P450 activity. Notably, upon transplantation into the ALF mouse model, the multiple cell-laden microgels effectively induced the restoration of liver function and enhanced liver regeneration. Overall, this study presents an efficient approach from the generation of hepatocyte-like cells to hepatocyte transplantation in ALF therapy.

Effects of green light-emitting diode irradiation on hepatic differentiation of hepatocyte-like cells generated from human adipose-derived mesenchymal cells

Light-emitting diode (LED) irradiation has been used in the differentiation of mesenchymal stem cells into a variety of cell types. This study investigated the effect of green LED (GLED) irradiation on the differentiation of adipocyte-derived mesenchymal cells into hepatocyte-like cells (HLCs) and the mechanism of its action. HLCs in the hepatocyte maturation phase were irradiated with GLED (520 nm, 21 W/m2, 5 min/day for 10 days). The cells were then assessed for expression of hepatocyte maturity genes and opsin 3 (OPN3), hepatocyte function, viability, apoptosis, and levels of reactive oxygen species (ROS), intracellular adenosine triphosphate (ATP) and calcium ions (Ca2+). GLED irradiation increased Alpha-1 antitrypsin and Ornithine transcarbamylase gene expression, promoted Cytochrome P450 3A4 activity and urea synthesis, and elevated intracellular ROS, ATP and Ca2+ levels. OPN3 expression was significantly more upregulated in GLED-irradiated HLCs than in the non-irradiated HLCs. No significant difference in cell viability or apoptosis was observed between GLED-irradiated and non-irradiated HLCs. GLED irradiation can promote hepatocyte maturation and functions through OPN3. GLED irradiation also stimulated mitochondrial function via Ca2+/ATP/ROS activation. GLED irradiation has potential to support cell-based transplantation in patients.