Reversibly immortalized human umbilical cord-derived mesenchymal stem cells (UC-MSCs) are responsive to BMP9-induced osteogenic and adipogenic differentiation.

Human mesenchymal stem cells (MSCs) are a heterogeneous subset of nonhematopoietic multipotent stromal stem cells and can differentiate into mesodermal lineage, such as adipocytes, osteocytes, and chondrocytes, as well as ectodermal and endodermal lineages. Human umbilical cord (UC) is one of the most promising sources of MSCs. However, the molecular and cellular characteristics of UC-derived MSCs (UC-MSCs) require extensive investigations, which are hampered by the limited lifespan and the diminished potency over passages. Here, we used the piggyBac transposon-based simian virus 40 T antigen (SV40T) immortalization system and effectively immortalized UC-MSCs, yielding the iUC-MSCs. A vast majority of the immortalized lines are positive for MSC markers but not for hematopoietic markers. The immortalization phenotype of the iUC-MSCs can be effectively reversed by flippase recombinase-induced the removal of SV40T antigen. While possessing long-term proliferation capability, the iUC-MSCs are not tumorigenic in vivo. Upon bone morphogenetic protein 9 (BMP9) stimulation, the iUC-MSC cells effectively differentiate into osteogenic, chondrogenic, and adipogenic lineages both in vitro and in vivo, which is indistinguishable from that of primary UC-MSCs, indicating that the immortalized UC-MSCs possess the characteristics similar to that of their primary counterparts and retain trilineage differentiation potential upon BMP9 stimulation. Therefore, the engineered iUC-MSCs should be a valuable alternative cell source for studying UC-MSC biology and their potential utilities in immunotherapies and regenerative medicine.

3D computed tomography angiography as a novel post-processing approach in diagnosis of pediatric malignant bone tumors.

OBJECTIVE: This study aimed to investigate the application of 3D computed tomography (CT) angiography with a novel post-processing technique in diagnosis of malignant bone tumors in children. METHODS: Twenty-seven pediatric patients (15 males and 12 females; average age: 10±3.4 years old, with a range from 2 months to 14 years old) with suspected bone tumors were evaluated histopathologically using 3D CT angiography and a multislice scanner. CT angiography image data were analyzed with a novel post-processing technique that included separating, fusing opacifying false-coloring, and volume rendering. RESULTS: Among 27 cases, 20 (74%) osteosarcoma, 6 (22%) Ewing's sarcoma, and 1 (4%) non-Hodgkin lymohoma were diagnosed by histological examination of surgical specimens. The tumor features, including size, location, invasion into the adjacent tissue as well as distant metastases, were clearly visualized with the regular volume rendering method and rotational and stereoscopical videos. The post-processing technique provided the reconstructed structure images without any overlap or shelter independently and collectively. Special colors represented different tissue structures, aiding in identification of various anatomical structures and pre-surgical planning. CONCLUSIONS: Compared to traditional 3-D CT methods, 3-D CT angiography with rotational and stereoscopical videos provides more detailed information of bone tumor lesions. It offers a superior and effective imaging technique in pediatric patients with malignant bone tumors.

Therapeutic potential of urine-derived stem cells in renal regeneration following acute kidney injury: A comparative analysis with mesenchymal stem cells.

BACKGROUND: Acute kidney injury (AKI) is a common clinical syndrome with high morbidity and mortality rates. The use of pluripotent stem cells holds great promise for the treatment of AKI. Urine-derived stem cells (USCs) are a novel and versatile cell source in cell-based therapy and regenerative medicine that provide advantages of a noninvasive, simple, and low-cost approach and are induced with high multidifferentiation potential. Whether these cells could serve as a potential stem cell source for the treatment of AKI has not been determined. AIM: To investigate whether USCs can serve as a potential stem cell source to improve renal function and histological structure after experimental AKI. METHODS: Stem cell markers with multidifferentiation potential were isolated from human amniotic fluid. AKI severe combined immune deficiency (SCID) mice models were induced by means of an intramuscular injection with glycerol. USCs isolated from human-voided urine were administered via tail veins. The functional changes in the kidney were assessed by the levels of blood urea nitrogen and serum creatinine. The histologic changes were evaluated by hematoxylin and eosin staining and transferase dUTP nick-end labeling staining. Meanwhile, we compared the regenerative potential of USCs with bone marrow-derived mesenchymal stem cells (MSCs). RESULTS: Treatment with USCs significantly alleviated histological destruction and functional decline. The renal function was rapidly restored after intravenous injection of 5 × 105 human USCs into SCID mice with glycerol-induced AKI compared with injection of saline. Results from secretion assays conducted in vitro demonstrated that both stem cell varieties released a wide array of cytokines and growth factors. This suggests that a mixture of various mediators closely interacts with their biochemical functions. Two types of stem cells showed enhanced tubular cell proliferation and decreased tubular cell apoptosis, although USC treatment was not more effective than MSC treatment. We found that USC therapy significantly improved renal function and histological damage, inhibited inflammation and apoptosis processes in the kidney, and promoted tubular epithelial proliferation. CONCLUSION: Our study demonstrated the potential of USCs for the treatment of AKI, representing a new clinical therapeutic strategy.

Erratum: [Corrigendum] Hepa1­6­FLuc cell line with the stable expression of firefly luciferase retains its primary properties with promising bioluminescence imaging ability.

[This corrects the article DOI: 10.3892/ol.2018.8132.].

All-trans retinoic acid reverses malignant biological behavior of hepatocarcinoma cells by regulating miR-200 family members.

As a potential chemo-therapeutic agent, all-trans retinoic acid (ATRA) can significantly reverse epithelial-mesenchymal transition (EMT) of hepal-6 hepatocarcinoma cell line in vitro, but the mechanism is unclear. The expression profile of microRNA-200 (miR-200) families is different in hepatocellular carcinoma. In this study, we found that ATRA treatment could up-regulate the expression of miR-200a-3p, 200c-3p, and 141-3p, which were involved in ATRA regulated proliferation and apoptosis of hepal-6 cell, but not colony formation. Meanwhile, miR-200a-3p, 200c-3p, and 141-3p could recovery ATRA inhibited migration and invasion abilities of hepal-6 cells at various levels. miR-200a-3p and 200c-3p prevented ATRA from inducing the differentiation and hepatic functions of hepal-6 cells. Antagomir specific for miR-200a-3p and 200c-3p down-regulated the expression of CK18, but only miR-200a-3p antagomir played prominent role in regulating the expression of these mesenchymal markers, N-Cadherin, Snail and Twist. The transcriptional activities of 8 transcription factors were up-regulated and 35 transcription factors were down-regulated by ATRA. Compared with ATRA group, inhibition of miR-200a-3p, 200c-3p, and 141-3p significantly strengthened the expression of Fra1/Jun (AP1), Ets1/PEA3, Brn3, and Zeb1/AREB6 at varying degrees. Therefore, this result suggested that ATRA may suppress EMT through down-regulating miR-200a-3p, 200c-3p and 141-3p related transcription factors. miR-200 and their downstream genes might be the potentially specific targets for the treatment of hepatocarcinoma.

Identification and biological characteristics of clear cell renal cell carcinoma associated urine-derived stem cells.

Urine-derived stem cells (USC) are isolated from voided urine and have demonstrated potential for use in tissue engineering and regenerative medicine therapies. Clear cell renal cell carcinoma (ccRCC) is a common urological malignancy that originates in the kidney. Since USC also originate in the kidney, the objective of this study was to investigate any biological differences between USC isolated from healthy patients and those isolated from ccRCC patients (rc-USC). We found that USC can be isolated from the voided urine of ccRCC patients (rc-USC) and have a morphology and function similar to those isolated from healthy donors. However, the rc-USC showed greater proliferation and invasion capacity than USC, and possessed some features of cancer cells; but the rc-UC were not able to form xenografts when implanted in vivo. We further performed RNA sequencing of rc-USC and USC and found several differentially expressed lncRNAs and mRNAs; however subsequent GO and KEGG enrichment analysis showed few pathway differences between these cells. Bioinformatic analyses and RT-PCR showed the expression of several known ccRCC-related genes in rc-USC expressed, as compared to USC derived from healthy donors. This study demonstrates that rc-USC displayed several cellular and genetic features of ccRCC cells, which suggests that this population of cells could provide a non-invasive approach for for the diagnosis, predication, disease modeling and therapeutic strategies targeting ccRCC.

Erratum: Urine-derived stem cells accelerate the recovery of injured mouse hepatic tissue.

[This corrects the article on p. 5131 in vol. 12, PMID: 33042410.].

Isolation and Characterization of Human Synovial Fluid-Derived Mesenchymal Stromal Cells from Popliteal Cyst.

Mesenchymal stem cells (MSCs) are multipotent progenitor cells in adult tissues. The aim of this study is to isolate and identify synovial fluid-derived mesenchymal stromal cells (SF-MSCs) from the popliteal cyst fluid of pediatric patients. SF-MSCs were collected from the popliteal cyst fluid of pediatric patients during cystectomy surgery. After cyst fluid extraction and adherent culturing, in vitro morphology, growth curve, and cell cycle were observed. The expression of stem cell surface markers was analyzed by flow cytometry, and expression of cell marker protein was detected by immunofluorescence. SF-MSCs were cultured in osteogenic, adipogenic, and chondrogenic differentiation medium. The differentiation potential of SF-MSCs was analyzed by alkaline phosphatase (Alizarin Red), Oil Red O, and Alcian blue. Antibody detection of human angiogenesis-related proteins was performed compared with bone marrow mesenchymal stem cells (BM-MSCs). The results show that SF-MSCs from the popliteal cyst fluid of pediatric patients showed a shuttle appearance and logarithmic growth. Flow cytometry analysis revealed that SF-MSCs were negative for hematopoietic lineage markers (CD34, CD45) and positive for MSC markers (CD44, CD73, CD90, and CD105). Interstitial cell marker (vimentin) and myofibroblast-like cell marker alpha-smooth muscle actin (α-SMA) were positive. These cells could differentiate into osteogenic, adipogenic, and chondrogenic lineages, respectively. Several types of human angiogenesis-related proteins were detected in the cell secretory fluid. These results show that we successfully obtained SF-MSCs from the popliteal cyst fluid of pediatric patients, which have the potential to be a valuable source of MSCs.

All-trans-retinoic acid inhibits the malignant behaviors of hepatocarcinoma cells by regulating autophagy.

Hepatocellular carcinoma is the fourth leading cause of cancer-related deaths due to its high rate of recurrence and metastasis. All-trans-retinoic acid (ATRA) can inhibit the malignant behaviors of hepatocarcinoma cells. Autophagy is reportedly involved in the migration and metastasis of various cancer cells. This study aimed to investigate the effect of autophagy on the function of ATRA on hepatocarcinoma cells, and to explore its possible underlying mechanism. Hepatocarcinoma cell lines, Hepa1-6 and HepG2, were treated with ATRA and autophagy inhibitors, including 3-methyladenine (3-MA) and Bafilomycin (Baf). Transmission electron microscopy, laser scanning, western blot, and real-time PCR demonstrated that ATRA induces autophagy in hepatocarcinoma cells. Trypan blue staining, a wound healing assay, and a transwell assay showed that 3-MA and Baf reverses the inhibitory functions of ATRA on the proliferation, migration, and invasion of hepatocarcinoma cells. Flow cytometry, Hoechst staining, periodic acid-Schiff staining, and indocyanine green uptake validated that 3-MA and Baf reverses the function of ATRA on apoptosis and the differentiation of hepatocarcinoma cells. Real-time PCR, western blot, and an immunofluorescence assay demonstrated that the reversal of the epithelial-mesenchymal transition (EMT) process by ATRA is weakened when autophagy is inhibited. Additionally, we confirmed that Bcl-2 is associated with the induction of ATRA-induced autophagy instead of the PI3K/Akt/mTOR pathway. These findings suggest that ATRA induces autophagy and autophagic cell death through the Bcl-2/Beclin1 pathway. Furthermore, ATRA-induced autophagy is involved in the inhibitory effect of ATRA on the malignant behaviors of hepatocarcinoma cells by reversing the EMT process.

Urine-derived stem cells accelerate the recovery of injured mouse hepatic tissue.

Urine-derived stem cells (USCs) are autologous stem cells that exhibit self-renewal ability and multi-lineage differentiation potential. These characteristics make USCs an ideal cell source for hepatocellular transplantation. Here, we investigated the biological characteristics of USCs and their potential use for the treatment of chronic liver injury. We characterized the cell-surface marker profile of USCs by flow cytometry and determined the osteogenic, adipogenic, and hepatic differentiation capacities of USCs using histology. We established a chronic liver-injury model by intraperitoneally injecting carbon tetrachloride into nude mice. USCs were then transplanted via tail vein injection. To determine liver function and histopathology following chronic liver injury, we calculated the liver index, measured serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, and performed histological staining. USCs were small, adherent cells expressing mesenchymal but not hematopoietic stem-cell markers. Some induced USCs underwent osteogenic and adipogenic differentiation. When co-cultured with hepatic progenitor cells, about 10% of USCs underwent hepatic differentiation. The ALT and AST levels of the USC-transplanted group were lower than that of the chronic liver-injury model group, and there were no significant differences between the two USC-transplanted groups. However, hepatocyte degeneration and liver fibrosis substantially improved in the hypoxia-pretreated USC-transplanted group compared with the normoxia USC-transplanted group. Taken together, USCs display desirable proliferation and differentiation characteristics, and USC transplantation partially improves abnormal liver function and pathology associated with chronic liver injury. Furthermore, hypoxia pretreatment promotes cell proliferation, migration, and colony formation by inducing autophagy, leading to USC-elicited liver tissue recovery following injury in vivo.

ATRA induces the differentiation of hepatic progenitor cells by upregulating microRNA-200a.

Hepatic progenitor cells (HPCs) are potential seed cells for hepatocyte transplantation treatment of liver diseases. ATRA can induce the differentiation and mature function of hepatic progenitor cells, but the mechanism is still poorly understood. Here, by using microRNA array to analyze the expression profiles of microRNA (miR), we found that miR-200 family molecules in HPCs were upregulated after ATRA treatment, especially miR-200a-3p, 200c-3p, and 141-3p. ATRA induction could downregulate the expression of hepatic stem markers Oct4 and AFP, and improve the expression of hepatic markers ALB, CK18, and TAT, and the activity of ALB-GLuc, as well as indocyanine green uptake and glycogen storage function of HPCs. These above effects of ATRA on HPC differentiation were almost inhibited by blocking of miR-200a-3p, but not miR-200c-3p and 141-3p using antagomir. Cell autophagy is associated with ATRA regulation in HPCs, compared with control group, the expression of LC3 and Beclin1 increased in ATRA-treated HPCs, and orange and red fluorescent spot, which represents autophagy flow, also enhanced after ATRA treatment. However, ATRA-induced cell autophagy level was inhibited in antagomir-200a-3p+ATRA-treated cells. Therefore, the present study indicates that antagomir-200a-3p is related to ATRA-induced hepatic differentiation of HPCs through regulating cell autophagy, supporting the possible use of ATRA as a key inducer in HPC-based therapy of liver diseases.

[Changes in autophagy during maturation and differentiation of Hepa1-6 cells induced by all-trans retinoic acid].

OBJECTIVE: To investigate the effects of different concentrations of all-trans retinoic acid (ATRA) on the maturation, differentiation and autophagy of Hepa1-6 cells. MONTHOD: Hepa1-6 cells were treated with 0.1, 1, and 10 µmol/L ATRA, and the changes in the expressions of hepatic specific markers were detected using real-time PCR and Western blotting. Indocyanine green (ICG) and periodic acid-schiff (PAS) staining was used to assess the functional maturation of Hepa1-6 cells, and the cell-cell junction and autophagy were observed under transmission electron microscopy to determine the optimal concentration of ATRA for treatment. The expressions of autophagy-related markers in the cells were detected using Western blotting, and confocal microscopy was used to observe the autophagic flow in the cells transfected with ptfLC3 plasmid. RESULTS: Compared with the control cells, the hepatocytes treated with ATRA showed a concentration-dependent decrease in AFP expression and increase in the expressions of ALB, CK18, TAT and ApoB. ICG and PAS staining revealed significantly increased number of positive cells after ATRA treatment. Following ATRA treatment, the cells exhibited obviously increased tight junctions, cytoskeleton and number of autophagosomes under transmission electron microscopy. ATRA treatment resulted in significantly increased the expressions of autophagy-related markers LC3-II, Beclin-1, RAB7 and P62 and also an increased ratio of LC3-II/LC3-I(P<0.05). Confocal microscopy revealed obviously increased green and red spots in the cells after ATRA treatment. CONCLUSION: ATRA can induce the maturation and differentiation and enhance the level of autophagy in Hepa1-6 cells.

[Tumor necrosis factor-α and transforming growth factor-ß1 balance liver stem cell differentiation in cholestatic cirrhosis].

OBJECTIVE: To investigate the changes of tumor necrosis factor-α (TNF-α) and transforming growth factor-ß1 (TGF-ß1) in mice with cholestatic cirrhosis and their role in regulating the balance of liver stem cell differentiation. METHODS: Balb/c mice were subjected to bile duct ligation (BDL), and serum biochemical parameters were measured and hepatic histopathology was observed using HE staining to evaluate the modeling of cholestatic cirrhosis. Immunohistochemistry and Western blotting were used to detect the changes of TNF-α and TGF-ß1 in the mice after modeling. Mouse embryonic hepatic stem cells (HP14-19) were treated with different concentrations of TNF-α and TGF-ß1, and the cell differentiation was assessed using Western blotting, real-time PCR, and PAS staining. RESULTS: The mice receiving BDL showed significantly increased blood biochemical parameters (P<0.05), and HE staining revealed obviously increased collagen fibers in the liver with significantly increased expressions of TNF-α and TGF-ß1 (P<0.05). In HP14-19 cells, induction with TNF-α and TGF-ß1 for 3 days did not cause significant changes in cell differentiation, but induction for 5 days resulted in significantly increases intensity of PAS staining in the cells. The cells induced with 20, 40, and 80 ng/mL TNF-α for 5 days exhibited a significantly stronger expression of cytokeratin 18 than cytokeratin 19 (P<0.05), while induction with 20, 40, and 80 ng/mL TGF-ß1 produced opposite changes in cytokeratin 18 and cytokeratin 19 expressions. Further induction of the cells with TNF-α and TGF-ß1 for 10 days, did not alter the expression patterns of cytokeratin 18 and cytokeratin 19 observed on day 5, but their protein expression levels and PAS staining intensity of the cells were enhanced and their mRNA expressions became lowered. CONCLUSION: Common bile duct ligation can induce conditions simulating cholestatic cirrhosis in mice. TNF-α and TGF-ß1 are elevated in cholestatic cirrhosis and play opposite roles in regulating the differentiation balance of liver stem cells: the former promotes the differentiation of liver stem cells into hepatocytes, while the latter promotes the cell differentiation into colangiocytes.

Reversibly immortalized hepatic progenitor cell line containing double suicide genes.

A large number of functional hepatocytes is required for bioartificial liver therapy. Simian virus 40 T­antigen (SV40T) has been previously reported to improve the immortalized proliferation of primary hepatocytes to generate a sufficient number of cells; however, these long­term immortalized hepatocytes may induce further malignant transformation in vivo. In the present study, the SV40T immortalization gene and two suicide genes, herpes simplex virus thymidine kinase (HSV­tk) and cytosine deaminase (CD), were transducted into primary hepatocytes to construct a novel type of Cre/LoxP­mediated reversible immortalized hepatocyte line. Polymerase chain reaction analysis and western blotting confirmed that the SV40T, HSV­tk and CD genes were successfully inserted into hepatic progenitor cells and their expression was controlled by Cre/LoxP recombination. Total removal of SV40T could be achieved via the ganciclovir (GCV)/HSV­tk suicide system. Cells maintained their biosafety in vivo with CD gene expression and 5­fluorocytosine (5­FC) induced cell death. Following transplantation into the carbon tetrachloride (CCl4) model group, the majority of cells had survived after 14 days post­implantation and a number of the cells had transported into the liver parenchyma. When compared with the CCl4 model group, the transplanted cells repaired the liver biochemical index and pathological structure markedly. Thus, the present study reports a novel reversible immortalized hepatocyte with double suicide genes, which exhibited the cellular phenotype and recovery function of normal liver cells. This method maximally guaranteed the biological safety of immortalized hepatocytes for in vivo application, providing a reliable, safe and ideal cell material for the artificial liver technique.

Hepa1-6-FLuc cell line with the stable expression of firefly luciferase retains its primary properties with promising bioluminescence imaging ability.

Reliable animal models are required for the in vivo study of the molecular mechanisms and effects of chemotherapeutic drugs in hepatocarcinoma. In vivo tracing techniques based on firefly luciferase (FLuc) may optimize the non-invasive monitoring of experimental animals. The present study established a murine Hepa1-6-FLuc cell line that stably expressed a retrovirus-delivered FLuc protein gene. The cell morphology, proliferation, migration and invasion ability of Hepa1-6-FLuc cells were the same as that of the Hepa1-6 cells, and thus is suitable to replace Hepa1-6 cells in the construction of hepatocarcinoma animal models. No differences in subcutaneous tumor mass and its pathomorphology from implanted Hepa1-6-FLuc cells were observed compared with Hepa1-6 control tumors. Bioluminescence imaging indicated that the Luc signal of the Hepa1-6-FLuc cells was consistently strengthened with increases in tumor mass; however, the Luc signal of Hepa1-6-AdFLuc became weaker and eventually disappeared during tumor development. Therefore, compared with the transient expression by adenovirus, stable expression of the FLuc gene in Hepa1-6 cells may better reflect cell proliferation and survival in vivo, and provide a reliable source for the establishment of hepatocarcinoma models.

Periodic acid­Schiff staining method for function detection of liver cells is affected by 2% horse serum in induction medium.

Developing a thorough understanding of experimental methods of hepatic differentiation in hepatic progenitor cells (HPCs) should expand the knowledge of hepatocyte induction in vitro and may help to develop cell transplantation therapies for the clinical usage of HPCs in liver diseases. A previous induction method effectively induced differentiation and metabolic abilities in HPCs. Periodic acid­Schiff (PAS) staining is used to identify glycogen synthesis and hepatocyte function; however, this method failed to detect induced hepatocytes. The present study aimed to investigate the possible factors affecting the previous confusing results of PAS staining. Removal of single induction factors, including dexamethasone, hepatic growth factor and fibroblast growth factor 4 from the induction media did not restore PAS staining, whereas replacement of 2% horse serum (HS) with 10% fetal bovine serum (FBS) significantly increased the number of PAS positive cells. Following 12 days of basal induction, replacing the induction medium with media containing 10% FBS for 12­72 h significantly improved PAS staining, but did not influence indocyanine green uptake. Furthermore, incubation in induction medium with 10% FBS following 12 days of normal induction did not affect the expression of hepatic markers and mature function of HPCs. Therefore, the present study suggested that 2% HS in the induction medium did not affect the hepatic function of induced cells, but did affect glycogen storage, whereas replacement of medium with 10% FBS in advance of PAS staining may restore the failure of PAS staining in low serum concentrations of induced hepatocytes.

All-trans retinoic acid inhibits proliferation, migration, invasion and induces differentiation of hepa1-6 cells through reversing EMT in vitro.

Hepatocellular carcinoma (HCC) has the characristics of tumor invasiveness, frequent intrahepatic spread and extra hepatic metastases, which affects the therapy efficiency and prognosis. Epithelial-mesenchymal transition (EMT) is now recognized as a key process in tumor invasion, metastasis and the generation of cancer initiating cells. All-trans retinoic acid (ATRA) is currently used as a potential chemo-therapeutic or chemo-preventive agent because of its anti-proliferative, pro-apoptotic and antioxidant properties. This study investigated the effects of ATRA at different concentrations on the proliferation, migration, invasion, differentiation and functions of the mouse hepa1-6 hepatocarcinoma cell line and explored whether ATRA regulates EMT in the antitumor process. Trypan blue staining and colony formation assay were used to detect cell proliferation. Wound-healing assay and Transwell Matrigel assay were performed to examine migration. Invasion was assessed by using Transwell invasion assay. In the present study, ATRA significantly inhibited the cell growth, colony formation, migration, and invasion capability of hepa1-6 cells in a dose-dependent manner. Furthermore, ATRA at low concentration (0.1 µmol/l) could generate these influences. After treated in the ATRA medium, the expression of mature hepatic markers ALB (albumin), CK18 (cytokeratin 18), TAT (tyrosine aminotransferase), ApoB (apolipoprotein B) decreased and that of hepatocarcinoma marker AFP (α fetoprotein) increased. At day 7 after ATRA induction, hepa1-6 cells showed comparable indocyanine green (ICG) uptake and glycogen storage function to the blank control. The mRNA expression of mesenchymal markers N-cadherin, vimentin, snail and twist decreased, while expression of epithelial marker E-cadherin increased in hepa1-6 cells after treated with ATRA. Therefore, this study demonstrates that ATRA remarkably suppressed the proliferation, migration, invasion of hepa1-6 hepatocarcinoma cell line and effectively induced its differentiation and liver functions in vitro through the reversal of EMT. HCC may be more sensitive to ATRA than other cancers, suggesting the prospective usefulness of ATRA in the treatment of HCC.

[Construction of a luciferase reporter vector containing response element of activator protein 2α and its application in study of osteogenetic differentiation].

OBJECTIVE: To construct a luciferase reporter vector containing the response element of transcription protein AP2α for screening the effect of bone morphogenetic proteins (BMPs) on the transcriptional activity of AP2α. METHODS: Four tandem-linked response elements of AP2α were cloned to the pBGLuc luciferase reporter gene plasmid, which was digested with Bam HI and Mlu I to construct pBGLuc-AP2α-RE vector. The recombinant adenovirus Ad-AP2α and its dominant negative mutant Ad-dnAP2α were used to infect mouse mesenchymal stem cells C3H10; the changes in cellular AP2α mRNA and protein expressions were detected by real-time PCR and Western blotting, and electrophoretic mobility shift assay (EMSA) was carried out to assess the DNA-binding ability of AP2α. C3H10 cells were transfected with pBGLuc-AP2α-RE vector, and AP2α transcriptional activity was measured using luciferase reporter gene assay. In pBGLuc-AP2α-RE vector-transfected C3H10 cells infected with Ad-BMPs, luciferase reporter gene assay was performed to screen the effect of BMPs on AP2α transcriptional activity. RESULTS: The results of PCR, enzyme digestion and sequencing all confirmed correct cloning of AP2α-RE into pBGLuc-AP2α-RE luciferase reporter vector, and Ad-AP2α infection significantly increased AP2α expression and its DNA binding ability. The dominant negative mutants expressed the corresponding mutants, and EMSA results showed that Ad-dnAP2α-δbHLH significantly lowered while Ad-dnAP2α-δTAD enhanced the DNA-binding ability of AP2α. AP2α over-expression promoted AP2α transcriptional activity, which was suppressed by the two dominant negative mutants. AP2α transcriptional activity increased in the cells infected with the recombinant adenovirus BMPs, especially in cells with BMP9 infection. CONCLUSIONS: The luciferase reporter vector containing the response element of AP2α we constructed allows detection of AP2α transcriptional activity. BMP9 can significantly enhance AP2α transcriptional activity.

Comparison of proliferation and differentiation potential between mouse primary hepatocytes and embryonic hepatic progenitor cells in vitro.

Cell therapy may be a novel and effective treatment strategy for liver diseases, replacing liver transplantation. The potential of two alternative cell types (hepatic progenitor/stem cells and mature hepatocytes) has not yet been fully assessed; the issues of low amplification efficiency and recovery function remain to be resolved. In this study, we investigated the proliferation, differentiation and function of primary mouse mature hepatocytes and embryonic hepatic progenitor cells. Primary cells were obtained from the livers of mouse embryos at 14.5 days post coitus [hepatic progenitor 14.5d (HP14.5d) cells], as well as from the livers of 3-month-old mice [liver cells 3m (LC3m)]. Using trypan blue staining and crystal violet staining to detect cell viability, we found that compared with the limited growth capability of primary LC3m cells, primary HP14.5d cells exhibited an active cell proliferation; however, proliferative ability of passaged HP14.5d cells significantly decreased. After the HP14.5d cells were treated in hepatic induction medium, the expression of progenitor cell markers decreased and that of mature hepatic markers increased, to levels similar to those of LC3m cells. On day 12 of induction, the HP14.5d cells showed comparable indocyanine green (ICG) uptake and glycogen storage to that of the LC3m cells. Therefore, our study demonstrates that primary hepatic progenitor cells have a stronger proliferation capacity and differentiation potential, supporting their clinical application in liver cell transplantation.