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.

[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.

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.