Differentiation Of Hepatic Stem Cells Research Articles

Guiding Hepatic Differentiation of Pluripotent Stem Cells Using 3D Microfluidic Co-Cultures with Human Hepatocytes.

Human pluripotent stem cells (hPSCs) are capable of unlimited proliferation and can undergo differentiation to give rise to cells and tissues of the three primary germ layers. While directing lineage selection of hPSCs has been an active area of research, improving the efficiency of differentiation remains an important objective. In this study, we describe a two-compartment microfluidic device for co-cultivation of adult human hepatocytes and stem cells. Both cell types were cultured in a 3D or spheroid format. Adult hepatocytes remained highly functional in the microfluidic device over the course of 4 weeks and served as a source of instructive paracrine cues to drive hepatic differentiation of stem cells cultured in the neighboring compartment. The differentiation of stem cells was more pronounced in microfluidic co-cultures compared to a standard hepatic differentiation protocol. In addition to improving stem cell differentiation outcomes, the microfluidic co-culture system described here may be used for parsing signals and mechanisms controlling hepatic cell fate.

SiNiSan alleviates liver injury by promoting hepatic stem cell differentiation via Wnt/β-catenin signaling pathway

BackgroundSiNiSan, a Traditional Chinese Medicine containing Radix Bupleuri, Radix Paeoniae Alba, Fructus Aurantii Immaturus, and Radix Glycyrrhizae, has been shown to be clinically effective in treating liver damage, its underlying molecular mechanisms however remains unclear. PurposeThe aim of the current study was to understand the molecular mechanisms of SiNiSan in the treatment of liver damage utilizing mice and cell culture models. MethodsHere, mice were gavaged with 0.2% CCl4 to obtain acute liver injury model and with alcohol to obtain chronic liver injury model. H&E staining was performed to detect liver histomorphology. HPLC-MS was performed to analyze the composition of SiNiSan decoction and SiNiSan-medicated serum (SMS). In addition, western blots were done to analyze the representative protein expression in Wnt/β-catenin signaling. Immunofluorescence staining was done to analyze the protein levels in WB-F344 cells. Finally, in an attempt to measure the influence of SiNiSan on liver regeneration in rats, we constructed a rats partial hepatectomy models. ResultsWe demonstrated that SiNiSan treatment mitigated liver damage in mice, as evidenced by the decrease in serum AST and ALT levels, as well as improved liver tissue morphology. HPLC-MS results showed that SMS contained a variety of components from the SiNiSan decoction. Next, our results showed that SMS reduced the expression of α-fetoprotein (AFP) and enhanced the expression of albumin (ALB) and cytokeratin 19 (CK19) in WB-F344 cells. Further, SMS treatment induced the accumulation of β-catenin. After 14 days of SMS treatment, β-catenin protein underwent nuclear translocation and bound to the LEF1 receptor in the nucleus, which regulated c-Myc and Cyclin D1 factors to activate Wnt/β-catenin signaling and promoted differentiation of WB-F344 cells. In addition, we demonstrated that SiNiSan increased liver regeneration in rat hepatectomy. ConclusionCollectively, the current study revealed that SiNiSan alleviated the acute liver injury induced by CCl4 as well as the chronic liver damage triggered by alcohol and sucrose in vitro. Concurrently, SMS treatment induced hepatic stem cell differentiation by activating Wnt/β-catenin signaling in vivo. Further study showed that SiNiSan promoted the regeneration of rats liver. The current study provides a theoretical basis for the clinical treatment of liver-related diseases with SiNiSan.

Co-growth of Stem Cells With Target Tissue Culture as an Easy and Effective Method of Directed Differentiation.

The long-term co-culture of mouse embryonic stem cells (mESC) with rat endothelial cells (EC) was tested for contact differentiation into the endothelial lineage. Serial passaging of rat ECs mixed with mESC in ratio 10:1 resulted in the emergence of a homogeneous cell population expressing mouse endothelial surface markers CD102, CD29, CD31. Rat endothelial surface marker RECA-1 completely disappeared from the co-cultured population after 2 months of weekly passaging. Co-incubation of mESC with rat ECs without cell-to-cell contact did not result in the conversion of mESC into ECs. After co-cultivation of adult mesenchymal stem cells from human endometrium (eMSC) with pre-hepatocyte-like cells of human hepatocarcinoma Huh7 the resulting co-culture expressed mature liver markers (oval cell antigen and cytokeratin 7), none of which were expressed by any of co-cultivated cultures, thus proving that even an immature (proliferating) pre-hepatocyte-like line can induce hepatic differentiation of stem cells. In conclusion, we have developed conditions where long-term co-proliferation of embryonic or adult SC with fully or partially differentiated cells results in stem cell progeny expressing markers of target tissue. In the case of endothelial differentiation, the template population quickly disappeared from the resulted culture and the pure endothelial population of stem cell progeny emerged. This approach demonstrates the expected fate of stem cells during various in vivo SC-therapies and also might be used as an effective in vitro differentiation method to develop the pure endothelium and, potentially, other tissue types of desirable genetic background.

Hepatic Differentiation of Stem Cells in 2D and 3D Biomaterial Systems.

A critical shortage of donor livers for treating end-stage liver failure signifies the urgent need for alternative treatment options. Hepatocyte-like cells (HLC) derived from various stem cells represent a promising cell source for hepatocyte transplantation, liver tissue engineering, and development of a bioartificial liver assist device. At present, the protocols of hepatic differentiation of stem cells are optimized based on soluble chemical signals introduced in the culture medium and the HLC produced typically retain an immature phenotype. To promote further hepatic differentiation and maturation, biomaterials can be designed to recapitulate cell–extracellular matrix (ECM) interactions in both 2D and 3D configurations. In this review, we will summarize and compare various 2D and 3D biomaterial systems that have been applied to hepatic differentiation, and highlight their roles in presenting biochemical and physical cues to different stem cell sources.

Biliary Obstruction Promotes Multilineage Differentiation of Hepatic Stem Cells.

Because of their high regenerative potential, stem cells are an ideal resource for development of therapies that replace injured tissue mass and restore function in patients with end‐stage liver diseases. Using a rat model of bile duct ligation (BDL) and biliary fibrosis, we investigated cell engraftment, liver repopulation, and ectopic tissue formation after intrasplenic transplantation of epithelial stem/progenitor cells. Fetal liver cells were infused into the spleens of Fisher 344 rats with progressing biliary fibrosis induced by common BDL or rats without BDL. Cell delivery was well tolerated. After migration to the liver, donor‐derived stem/progenitor cells engrafted, differentiated into hepatocytes and cholangiocytes, and formed large cell clusters at 2 months in BDL rats but not controls. Substantial numbers of donor cells were also detected at the splenic injection site where they generated hepatic and nonhepatic tissue. Transplanted cells differentiated into phenotypes other than hepato/cholangiocytic cells only in rats that underwent BDL. Quantitative reverse‐transcription polymerase chain reaction analyses demonstrated marked up‐regulation of tissue‐specific genes of nonhepatic endodermal lineages (e.g., caudal type homeobox 2 [Cdx2], pancreatic and duodenal homeobox 1 [Pdx1], keratin 13 [CK‐13]), confirmed by immunohistochemistry. Conclusion: BDL and its induced fibrosis promote liver repopulation by ectopically transplanted fetal liver‐derived cells. These cell fractions contain multipotent stem cells that colonize the spleen of BDL rats and differentiate into multiple gastrointestinal tissues, including liver, pancreas, intestine, and esophagus. The splenic microenvironment, therefore, represents an ideal niche to assess the differentiation of these stem cells, while BDL provides a stimulus that induces their differentiation.

Technological advancements for the development of stem cell-based models for hepatotoxicity testing.

Stem cells are characterized by their self-renewal capacity and their ability to differentiate into multiple cell types of the human body. Using directed differentiation strategies, stem cells can now be converted into hepatocyte-like cells (HLCs) and therefore, represent a unique cell source for toxicological applications in vitro. However, the acquired hepatic functionality of stem cell-derived HLCs is still significantly inferior to primary human hepatocytes. One of the main reasons for this is that most in vitro models use traditional two-dimensional (2D) setups where the flat substrata cannot properly mimic the physiology of the human liver. Therefore, 2D-setups are progressively being replaced by more advanced culture systems, which attempt to replicate the natural liver microenvironment, in which stem cells can better differentiate towards HLCs. This review highlights the most recent cell culture systems, including scaffold-free and scaffold-based three-dimensional (3D) technologies and microfluidics that can be employed for culture and hepatic differentiation of stem cells intended for hepatotoxicity testing. These methodologies have shown to improve in vitro liver cell functionality according to the in vivo liver physiology and allow to establish stem cell-based hepatic in vitro platforms for the accurate evaluation of xenobiotics.

Distinct roles of Dlk1 isoforms in bi-potential differentiation of hepatic stem cells

BackgroundFully understanding the developmental process of hepatic stem cells (HSCs) and the mechanisms of their committed differentiation is essential for optimizing the generation of functional hepatocytes for cell therapy in liver disease. Delta-like 1 homolog (Dlk1), primarily the membrane-bound form (Dlk1M), is generally used as a surface marker for fetal hepatic stem cell isolation, while its soluble form (Dlk1S) and the functional roles of different Dlk1 isoforms in HSC differentiation remain to be investigated.MethodsHepatic spheroid-derived cells (HSDCs) were isolated from E12.5 mouse livers to obtain Dlk1+ and Dlk1−subpopulations. Colony formation, BrdU staining, and CCK8 assays were used to evaluate the cell proliferation capacity, and hepatic/cholangiocytic differentiation and osteogenesis/adipogenesis were used to assess the multipotency of the two subpopulations. Transformation of Dlk1+ cells into Dlk1− cells was detected by FACS, and the expression of Dlk1 isoforms were measured by western blot. The distinct roles and regulatory mechanisms of Dlk1 isoforms in HSC differentiation were investigated by overexpressing Dlk1M.ResultsHSDCs were capable of differentiating into liver and mesenchymal lineages, comprising Dlk1+ and Dlk1− subpopulations. Dlk1+ cells expressed both Dlk1M and Dlk1S and lost expression of Dlk1M during passaging, thus transforming into Dlk1− cells, which still contained Dlk1S. Dlk1− cells maintained a self-renewal ability similar to that of Dlk1+ cells, but their capacity to differentiate into cholangiocytes was obviously enhanced. Forced expression of Dlk1M in Dlk1− cells restored their ability to differentiate into hepatocytes, with an attenuated ability to differentiate into cholangiocytes, suggesting a functional role of Dlk1 in regulating HSC differentiation in addition to acting as a biomarker. Further experiments illustrated that the regulation of committed HSC differentiation by Dlk1 was mediated by the AKT and MAPK signaling pathways. In addition, bFGF was found to serve as an important inducement for the loss of Dlk1M from Dlk1+ cells, and autophagy might be involved.ConclusionsOverall, our study uncovered the differential expression and regulatory roles of Dlk1 isoforms in the commitment of HSC differentiation and suggested that Dlk1 functions as a key regulator that instructs cell differentiation rather than only as a marker of HSCs. Thus, our findings expand the current understanding of the differential regulation of bi-potential HSC differentiation and provide a fine-tuning target for cell therapy in liver disease.

A practice-changing culture method relying on shaking substantially increases mitochondrial energy metabolism and functionality of human liver cell lines.

Practice-changing culturing techniques of hepatocytes are highly required to increase their differentiation. Previously, we found that human liver cell lines HepaRG and C3A acquire higher functionality and increased mitochondrial biogenesis when cultured in the AMC-Bioartificial liver (BAL). Dynamic medium flow (DMF) is one of the major contributors to this stimulatory effect. Recently, we found that DMF-culturing by shaking of HepaRG monolayers resulted in higher mitochondrial biogenesis. Here we further investigated the effect of DMF-culturing on energy metabolism and hepatic functionality of HepaRG and C3A monolayers. HepaRG and C3A DMF-monolayers were incubated with orbital shaking at 60 rpm during the differentiation phase, while control monolayers were maintained statically. Subsequently, energy metabolism and hepatic functionality were compared between static and DMF-cultures. DMF-culturing of HepaRG cells substantially increased hepatic differentiation; transcript levels of hepatic structural genes and hepatic transcription regulators were increased up to 15-fold (Cytochrome P450 3A4) and nuclear translocation of hepatic transcription factor CEBPα was stimulated. Accordingly, hepatic functions were positively affected, including ammonia elimination, urea production, bile acid production, and CYP3A4 activity. DMF-culturing shifted energy metabolism from aerobic glycolysis towards oxidative phosphorylation, as indicated by a decline in lactate production and glucose consumption, and an increase in oxygen consumption. Similarly, DMF-culturing increased mitochondrial energy metabolism and hepatic functionality of C3A cells. In conclusion, simple shaking of monolayer cultures substantially improves mitochondrial energy metabolism and hepatic differentiation of human liver cell lines. This practice-changing culture method may prove to prolong the in-vitro maintenance of primary hepatocytes and increase hepatic differentiation of stem cells.

The combination of miR-122 overexpression and Let-7f silencing induces hepatic differentiation of adipose tissue-derived stem cells.

Human adipose tissue-derived stem cells (hADSCs) have been considered as a promising source for cell therapy of liver diseases due to their accessibility, abundance, and expression of hepatocyte markers. Currently, the important role of certain microRNAs (miRNAs) has been reported during hepatic differentiation of stem cells. However, the combination effect of miRNAs on hepatic differentiation of these cells needs to be more investigated. The present study seeks to determine whether the combination of miRNAs can enhance hepatic differentiation of hADSCs in the absence of any other stimulation. First, lentiviral transduction was used to overexpress miR-122 and silence d let-7f in hADSCs for up to 21 days. Then, hepatic functionality was evaluated by analyzing specific hepatocyte genes and biochemical markers at different time points of differentiation induction. Stable miR-122 overexpression and let-7f silencing together in hADSCs resulted in increased expression of hepatocyte markers including ALB, AFP, CK18, CK19, and HNF4a. In addition, urea and albumin production, immunocytochemistry, and glycogen staining confirmed that the treated cells differentiated toward hepatocyte-like cells. Therefore, our findings demonstrate the possibility of using microRNAs to induce hADSCs into functional hepatocyte-like cells.

FXR-Gankyrin axis is involved in development of pediatric liver cancer.

The development of hepatoblastoma (HBL) is associated with failure of hepatic stem cells (HSC) to differentiate into hepatocytes. Despite intensive investigations, mechanisms of the failure of HSC to differentiate are not known. We found that oncogene Gankyrin (Gank) is involved in the inhibition of differentiation of HSC via triggering degradation of tumor suppressor proteins (TSPs) Rb, p53, C/EBPα and HNF4α. Our data show that the activation of a repressor of Gank, farnesoid X receptor, FXR, after initiation of liver cancer by Diethylnitrosamine (DEN) prevents the development of liver cancer by inhibiting Gank and rescuing tumor suppressor proteins. We next analyzed FXR-Gank-Tumor suppressor pathways in a large cohort of HBL patients which include 6 controls and 53 HBL samples. Systemic analysis of these samples and RNA-Seq approach revealed that the FXR-Gank axis is activated; markers of hepatic stem cells are dramatically elevated and hepatocyte markers are reduced in HBL samples. In the course of these studies, we found that RNA binding protein CUGBP1 is a new tumor suppressor protein which is reduced in all HBL samples. Therefore, we generated CUGBP1 KO mice and examined HBL signatures in the liver of these mice. Micro-array studies revealed that the HBL-specific molecular signature is developed in livers of CUGBP1 KO mice at very early ages. Thus, we conclude that FXR-Gank-TSPs-Stem cells pathway is a key determinant of liver cancer in animal models and in pediatric liver cancer. Our data provide a strong basis for development of FXR-Gank-based therapy for treatment of patients with hepatoblastoma.

Regulatory effect of faciogenital dysplasia 6 gene on hepatic stem cell differentiation

Objective: To investigate the regulatory effect of faciogenital dysplasia 6 (FGD6) gene on hepatic stem cell differentiation. Methods: FGD6 gene was selected for the co-intervention of target sequence, the AdEasy system was used for the construction of adenovirus vector and the packaging and multiplication of the recombinant adenovirus vector pSES-FGD6-siRNA, and the HP14.5 cells were infected. Immunofluorescence assay was used to measure the expression of FGD6 protein in HP14.5 cells, quantitative real-time PCR was used to measure the mRNA expression of FGD6, alpha-fetoprotein (AFP), and albumin (Alb), and Western blot was used to measure the protein expression of FGD6, AFP, and Alb. The empty pSES-Ad-RFP adenovirus vector was constructed as control in each group. All data were expressed as x±s, and a one-way analysis of variance was performed. Results: FGD6 protein was mainly expressed in the nucleus of HP14.5 cells. The pSES-FGD6-siRNA adenovirus vector was successfully constructed and it downregulated the expression of FGD6 gene and the mRNA and protein expression of AFP in HP14.5 cells and upregulated the mRNA and protein expression of Alb (P < 0.01). Conclusion: The inhibition of the expression of FGD6 gene in HP14.5 cells may differentiate HP14.5 cells into hepatocytes. Therefore, FGD6 gene plays an important role in the differentiation regulation of hepatic stem cells.

High‐throughput sequencing to identify microRNA signatures during hepatic differentiation of human umbilical cord Wharton's jelly‐derived mesenchymal stem cells

MicroRNAs (miRNAs) constitute a class of small non-coding RNAs involved in regulation of cognate mRNAs post-transcriptionally. MicroRNAs have been implicated in regulating the stem cell differentiation process. Limited regulatory miRNAs have been reported to date during hepatic differentiation of stem cells. The present study was designed to identify the signature miRNAs implicated in hepatic differentiation of stem cells using next-generation sequencing methods. We undertook sequencing of miRNAs isolated from three different time points during hepatic differentiation of human umbilical cord Wharton's jelly-derived mesenchymal stem cells (hUC-MSCs) from two biological replicates. Out of a total known 2588 miRNAs (according to miRBase version 21), 880 miRNAs were identified in our study. A total of 63 significantly expressed miRNAs during hepatic differentiation, with at least 2-fold change and a false discovery rate value <0.05, were considered for further analysis. The putative target genes of significantly downregulated miRNAs during hepatic differentiation appeared to be mostly associated with biological processes that are essential for hepatic differentiation and maintenance of mature hepatic phenotype-like liver development, stem cell differentiation, Wnt receptor signaling pathway, and drug and cholesterol metabolic processes. Putative target genes of significantly upregulated miRNAs are highly enriched in regulating processes that block hepatic differentiation of hUC-MSCs like epithelial-mesenchymal transition, transforming growth factor-β receptor signaling pathway, and stem cell maintenance. The study provides a new insight for investigation of miRNA-regulated pathways during the differentiation process.

In Vitro and In Vivo Hepatic Differentiation of Adult Somatic Stem Cells and Extraembryonic Stem Cells for Treating End Stage Liver Diseases.

The shortage of liver donors is a major handicap that prevents most patients from receiving liver transplantation and places them on a waiting list for donated liver tissue. Then, primary hepatocyte transplantation and bioartificial livers have emerged as two alternative treatments for these often fatal diseases. However, another problem has emerged. Functional hepatocytes for liver regeneration are in short supply, and they will dedifferentiate immediately in vitro after they are isolated from liver tissue. Alternative stem-cell-based therapeutic strategies, including hepatic stem cells (HSCs), embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and mesenchymal stem cells (MSCs), are more promising, and more attention has been devoted to these approaches because of the high potency and proliferation ability of the cells. This review will focus on the general characteristics and the progress in hepatic differentiation of adult somatic stem cells and extraembryonic stem cells in vitro and in vivo for the treatment of end stage liver diseases. The hepatic differentiation of stem cells would offer an ideal and promising source for cell therapy and tissue engineering for treating liver diseases.

Melatonin promotes hepatic differentiation of human dental pulp stem cells: clinical implications for the prevention of liver fibrosis.

Melatonin's effect on hepatic differentiation of stem cells remains unclear. The aim of this study was to investigate the action of melatonin on hepatic differentiation as well as its related signaling pathways of human dental pulp stem cells (hDPSCs) and to examine the therapeutic effects of a combination of melatonin and hDPSC transplantation on carbon tetrachloride (CCl4 )-induced liver fibrosis in mice. In vitro hepatic differentiation was assessed by periodic acid-Schiff (PAS) staining and mRNA expression for hepatocyte markers. Liver fibrosis model was established by injecting 0.5mL/kg CCl4 followed by treatment with melatonin (5mg/kg, twice a week) and hDPSCs. In vivo therapeutic effects were evaluated by histopathology and by means of liver function tests including measurement of alanine transaminase (ALT), aspartate transaminase (AST), and ammonia levels. Melatonin promoted hepatic differentiation based on mRNA expression of differentiation markers and PAS-stained glycogen-laden cells. In addition, melatonin increased bone morphogenic protein (BMP)-2 expression and Smad1/5/8 phosphorylation, which was blocked by the BMP antagonist noggin. Furthermore, melatonin activated p38, extracellular signal-regulated kinase (ERK), and nuclear factor-κB (NF-κB) in hDPSCs. Melatonin-induced hepatic differentiation was attenuated by inhibitors of BMP, p38, ERK, and NF-κB. Compared to treatment of CCl4 -injured mice with either melatonin or hDPSC transplantation alone, the combination of melatonin and hDPSC significantly suppressed liver fibrosis and restored ALT, AST, and ammonia levels. For the first time, this study demonstrates that melatonin promotes hepatic differentiation of hDPSCs by modulating the BMP, p38, ERK, and NF-κB pathway. Combined treatment of grafted hDPSCs and melatonin could be a viable approach for the treatment of liver cirrhosis.

Telocytes in liver regeneration: possible roles.

Telocytes (TCs) are a novel type of interstitial cells which are potentially involved in tissue regeneration and repair (http://www.telocytes.com). Previously, we documented the presence of TCs in liver. However, the possible roles of TCs in liver regeneration remain unknown. In this study, a murine model of partial hepatectomy (PH) was used to induce liver regeneration. The number of TCs detected by double labelling immunofluorescence methods (CD34/PDGFR-α, CD34/PDGFR-ß and CD34/Vimentin) was significantly increased when a high level of hepatic cell proliferation rate (almost doubled) as shown by 5-ethynyl-2′-deoxyuridine (EdU) immunostaining and Western Blot of Proliferating cell nuclear antigen (PCNA) was found at 48 and 72 hrs post-PH. Meanwhile, the number of CK-19 positive-hepatic stem cells peaked at 72 hrs post-PH, co-ordinating with the same time-point, when the number of TCs was most significantly increased. Taken together, the results indicate a close relationship between TCs and the cells essentially involved in liver regeneration: hepatocytes and stem cells. It remains to be determined how TCs affect hepatocytes proliferation and/or hepatic stem cell differentiation in liver regeneration. Besides intercellular junctions, we may speculate a paracrine effect via ectovesicles.

Human Embryonic and Hepatic Stem Cell Differentiation Visualized in Two and Three Dimensions Based on Serial Sections.

Pluripotent human embryonic stem cells (hESCs) are characterized by two defining properties, self-renewal and differentiation. Self-renewing hESCs express transcription factors OCT4, SOX2, and NANOG, and surface markers SSEA-4 and TRA-1-60 and TRA-1-81 and their ability to differentiate into derivatives of the three germ layers show the differentiating potential. Studies suggest a certain microheterogeneity of the hESC colonies, in which not all cells in one colony of apparently undifferentiated cells express all the expected markers. We describe a technique to paraffin embed an entire hESC colony, and prepare 3-5μm thick serial sections. Immunohistochemistry applied to individual sections produces a 2-dimensional survey of the developing hESC colony. Based on serial paraffin sections of the 2D-expression pattern, a new and useful 3D-visualization can be modeled. The actual 3D rendering of an entire colony is accomplished using 3D image processing software such as Mimics(®) or Amira(®). An extended version of this technique even allows for a high-magnification 3D-reconstruction of, e.g., hepatic stem cells in developing liver. These techniques combined allow for both a 2- and a 3-dimensional visualization of hESC colonies and stem cells in organs, which leads to new insights into and information about the interaction of stem cells with their surroundings.

Differential hepatic stem cell proliferation and differentiation after partial hepatectomy in rats

Stem cell‑derived hepatocyte precursor cells represent a promising model for clinical transplantation to diseased livers, as well as for establishment of in vitro systems for drug metabolism and toxicology studies. The present study aimed to establish a new method of induction of hepatocyte differentiation using various factors and evaluate the effect of different partial hepatectomies and the duration of collagenase perfusion on hepatic stem cell proliferation and differentiation. A rat model of hepatic oval cell proliferation was established by partial hepatectomy (PH). Following 73.1 and 83.4% PH, rats underwent perfusion with IV collagenase for 10, 20 and 30 min. Density gradient centrifugation was performed and cells in the supernatant were cultured in various combinations of factors to induce oval cells to differentiate into mature hepatocytes. Cells were characterized for hepatocyte marker expression by morphology, flow cytometry, immunofluorescence and western blot analysis. Hepatic oval cells isolated from rats at 7 and 14 days post‑PH exhibited properties of hepatic stem/progenitor cells. Following culturing in RPMI‑1640 medium with hepatocyte growth factor and fibroblast growth factor‑4, the cells resembled primary human hepatocytes with regard to morphology and expression of the hepatocyte markers, cytokeratin 18 (CK‑18) and α‑1‑fetoprotein (AFP). Optimal differentiation of hepatic stem cells to CK‑18‑ and AFP‑positive cells was observed when stem cells isolated from 83.4% PH rats (7 days following surgery) were perfused with IV collagenase for 20 min. The results of this study provide novel insights into characteristics of rat hepatic stem cells.

Transcriptional characterization of Wnt pathway during sequential hepatic differentiation of human embryonic stem cells and adipose tissue-derived stem cells

Human embryonic stem cells (hESs) and adipose-derived stem cells (hADSCs) are able to differentiate into hepatocytes. However, a role of Wnt signaling in hepatic differentiation of stem cells is unclear. This study characterized the transcriptional expression pattern of Wnt signaling genes during the sequential hepatocytes differentiation of hES and hADSC. The sequential hepatocytes differentiation of hES and hADSC was induced by three steps including induction, differentiation and maturation steps with the treatment of cytokines. Hepatocytes differentiation was more efficient in hES than hADSC in terms of the expression of hepatocyte-specific genes and the cellular uptake of ICG. The expression of WNT2B, WNT5A, and WISP1 increased at late hepatic differentiation of hES, but the expression of DKK1 and CCND1 decreased during early hepatic differentiation of hES. During hepatic differentiation of hADSC, the expression of WNT2B and WISP1 decreased, but the expression of WNT5B and DKK1 increased at late hepatic differentiation. These results showed that Wnt signaling appears to be activated in hepatic differentiation of hES, but repressed in hepatic differentiation of hADSC in a time-dependent manner, which suggests the differential regulation of Wnt signaling for hepatic differentiation of hES and hADSC.

Metabolic oxidation controls the hepatic stem cells (HpSCs) fate and the hepatic lineage organization in physiologic and pathologic conditions

The zonation of liver functions across the hepatic acinus from the periportal region (zone 1) to the pericentral region (zone 3) is marked by patterns of gene expression, enzyme activity, and redox state.1, 2 The hepatic lineage starts within the stem cell compartment, the canals of Hering located periportally, progresses through the midacinar region, and terminates with mature polyploid hepatocytes in the pericentral zone.3 Clues to the mechanisms governing the maturational process and the functional differentiation across the hepatic lobule are highlighted in the recent discovery of a metabolic pathway underlying the control of embryonic stem cell (ESC) fate by Yanes et al.4 These authors found that experimentally maintained high levels of unsaturated molecules perpetuatd ESC pluripotency, whereas a downstream increase of oxidized metabolites and pro-oxidative substrates promoted differentiation, highlighting the metabolome relevance on stem cell biology. The higher activity of redox enzymes found in the pericentral zone, and the metabolic adaptation to the pericentral lower oxygen concentration related to increased NADH/NAD and NADPH/NADP ratios,1, 2 could be in tight connection with a gradient of molecular saturation through the hepatic lobule, being the highly unsaturated molecules predominant in the periportal zone. The human hepatic stem cell differentiation and maturational lineage organization could be regulated and maintained by the gradient of saturation of small molecules (oxidized and polyunsaturated) along the zones of the acinus, depending primarily on an oxygen gradient metabolic adaptation. Dezso et al.5 have proposed a model of progenitor cell-driven liver regenerative growth. Applying the 2-acetylaminofluorene/partial hepatectomy experimental model in the rat, they discovered how the regeneration is initiated by proliferation of so-called “oval cells” in the periportal region, resulting in the formation of a seemingly random collection of small hepatocytes called foci. Interestingly, the polarized lobular vasculature architecture and the hepatic zonation were preserved in this model, and the foci can easily remodel to reconstitute a normal lobular structure. These results suggest that the preservation of the normal vasculature organization as well as the oxygen gradient along the acinus zones could drive the refolding of the foci and the entrance of the small hepatocytes in the lineage by sustaining the metabolites saturation gradient. In conclusion, the effect of metabolome on stem cell fate could be a pivotal phenomena regulating the hepatic lineage in physiologic conditions and during liver regeneration, and a candidate mechanism responsible for the progressive failure of cirrhotic liver. Vincenzo Cardinale M.D.*, Guido Carpino M.D. , Alfredo Cantafora M.D.*, Lola M. Reid M.D.§, Eugenio Gaudio M.D. , Domenico Alvaro M.D.* ¶, * Department of Medico-Surgical Sciences and Biotechnologies, Polo Pontino, Sapienza University of Rome, Rome, Italy, Department of Anatomical, Histological, Forensic Medicine and Orthopedics Sciences, Sapienza University of Rome, Rome, Italy, Department of Health Sciences, University of Rome “Foro Italico”, Rome, Italy, § Department of Cell and Molecular Physiology, Program in Molecular Biology and Biotechnology, UNC School of Medicine, Chapel Hill, NC, ¶ Eleonora Lorillard Spencer-Cenci Foundation, Rome, Italy.

Characteristics of hepatic stem/progenitor cells in the fetal and adult liver

The liver is an essential organ that maintains vital activity through its numerous important functions. It has a unique capability of fully regenerating after injury. Regulating a balance between self-renewal and differentiation of hepatic stem cells that are resources for functional mature liver cells is required for maintenance of tissue homeostasis. This review describes the characteristics of hepatic stem/progenitor cells and the regulatory mechanism of their self-renewal and differentiation capacity. In liver organogenesis, undifferentiated hepatic stem/progenitor cells expand their pool by repeated self-renewal in the early stage of liver development and then differentiate into two different types of cell lineage, namely hepatocytes and cholangiocytes. Liver development is regulated by expression of stem cell transcription factors in a complex multistep process. Recent studies suggest that stem cells are maintained by integrative regulation of gene expression patterns related to self-renewal and differentiation by epigenetic mechanisms such as histone modification and DNA methylation. Analysis of the proper regulatory mechanism of hepatic stem/progenitor cells is important for regenerative medicine that utilizes hepatic stem cells and for preventing liver cancer through clarification of the carcinogenetic mechanism involved in stem cell system failure.