BACKGROUND:Preliminary experiments have demonstrated that rat liver homogenate supernatants can induce the morphological changes of human umbilical cord mesenchymal stem cells. However, little is known about whether the induced cells have some phenotypic and functional features of hepatocytes.
OBJECTIVE:To investigate whether human umbilical cord mesenchymal stem cells have some phenotypic and functional characteristic of hepatocytes after being induced by liver homogenate supernatants.
METHODS:Passage 3 human umbilical cord mesenchymal stem cells were used and divided into control group (cells were cultured in basic culture medium) and liver homogenate supernatant group (cells were cultured in liver homogenate supernatants for 3, 5, 7 days). Meanwhile, positive control group (QSG-7701 human liver celllines) and negative control group (simple liver homogenate supernatants) were set up. The protein and mRNA level of hepatocyte markers, alpha-fetoprotein, cytokeratin 18 and tryptophan 2,3-dioxygenase enzyme, were detected at different time points.
RESULTS AND CONCLUSION:After inducement, the stem cells of fusiform shape began to lose their sharp edges and progressively shrunk, and then they changed into hepatocyte-like cells with the morphous of triangle, polygon and anomalism shape. Compared with the control group, the protein and mRNA level of alpha-fetoprotein, cytokeratin 18 and tryptophan 2,3-dioxygenase enzyme significantly increased time dependently after inducement with liver homogenate supernatants (P<0.01). This study demonstrated that human umbilical cord mesenchymal stem cells are able to differentiate into hepatocyte-like cells in vitro that possess some functions of liver cells.

BACKGROUND:Previous studies have demonstrated that normal rat liver homogenate supernatant can induce human umbilical cord mesenchymal stem cels to differentiate into hepatocyte-like cels with partial hepatocyte functions. However, whether fibrotic liver homogenate supernatant can work or how the inducing effect is remains unclear. OBJECTIVE:To investigate the differentiation potential of human umbilical cord mesenchymal stem cels into hepatocytes under the normal liver and fibrotic liver microenvironment in vitro. METHODS:Liver fibrosis was induced in the SD rats by repeated intraperitoneal injections of 3% thioacetamide at a dose of 200 mg/kg body mass, twice a week for 4 weeks, and then fibrotic liver tissues and normal liver tissues were used to prepare liver homogenate supernatants. Passage 3 human umbilical cord mesenchymal stem cels were used and divided into standard control group (cels were cultured in DMEM/F12 with 10% fetal bovine serum), fibrotic liver homogenate supernatants group (cels were cultured in DMEM/F12 with 10% fetal bovine serum and 50 g/L fibrotic liver homogenate supernatants), normal liver homogenate supernatants group (cels were cultured in DMEM/F12 with 10% fetal bovine serum and 100 g/L normal liver homogenate supernatants). The morphological changes of the cels in each group were recorded under inverted microscope; the protein levels of CK18, AFP, CYP3A4, CYP2E1, CYP2D6 and TPH2 were evaluated using western blot assay. Furthermore, the concentration of albumin in the cels was measured. RESULTS AND CONCLUSION:After a 7-day inducement, the stem cels in liver homogenate supernatants groups lost their fusiform shape and changed into hepatocyte-like cels with the morphous of round shape. Compared with the standard control group, the hepatocyte-like cels in the two liver homogenate supernatants groups exhibited human hepatocyte biomarkers, CK18 and AFP. The standard control group cels could express a little amount of CYP2E1, while cels in the two liver homogenate supernatants groups could express CYP3A4, CYP2E1, CYP2D6, TPH2. Compared with the standard control group, the expression level of CYP2E1 in the two liver homogenate supernatants groups increased significantly (P < 0.01), and however, the relative levels of CYP3A4, CYP2E1, CYP2D6, TPH2 in the two liver homogenate supernatants groups showed no statistical significance (P > 0.05). At the same time, compared with the standard control group, the concentration of albumin in the two liver homogenate supernatants groups markedly increased (P < 0.01), but there was no difference between the two liver homogenate supernatants groups (P > 0.05). Experimental findings demonstrated that both of normal liver tissue and fibrotic liver tissue microenvironments could induce human umbilical cord mesenchymal stem cels to differentiate into hepatocyte-like cels. To achieve the same effect, compared with normal liver tissue, fibrotic liver tissue required lower concentrations, suggesting that fibrotic liver tissue microenvironment may be more conducive to differentiation of umbilical cord mesenchymal stem cels into hepatocytes.

Objective To explore the effect of early application of pediauic amino acids on extrauterine growth and development of preterm and small for gestational age infants.Methods A total of 106preterm and small for gestational age infants was selected in our NICU from June 2011 to May 2013,and randomly divided into two groups:treatment group (group T) and control group (group C).Next,each group was divided into two subgroups according to gestational age and birth weight.Such as ≥34W group (group T1,group C1),＜ 34W group (group T2,group C2),＜ 1.5 kg group (group T3,group C3) and ≥ 1.5 kg group (group T4,group C4).They were observed the effect of extrauterine growth and correlative serum biochemical indicators with application of pediatric amino acid in postnatal 6 hours and 12 hours.Resuits After 2-week treatment,the head circumference and weight growth of group T were higher than that of group C (P ＜ 0.05,or P ＜ 0.01),and the time of birth weight recovery was significantly shortened(P ＜ 0.05,or P ＜ 0.01).The incidence rate of extrauterine growth retardation(EUGR) in the group T was lower than that in group C,there is significantly statistical differences(P ＜ 0.01).The concentration of blood urea nitrogen (BUN) in group T was obviously increased than that in the group C(P ＜0.01).The levels of ALT,aspartate aminotransferase (AST),total bilirubin (TBIL),cholesterol (CH) and triglycerides (TG) were not different between the group T and group C.The comparison of alanine aminotransferase (ALT) levels were statistically significant between group C4 and group T4.In the different gestational age groups,the concentration of BUN in group T was obviously higher than that in the group C after 2-week treatment(P ＜0.05),the levels of AST,TBIL,CH and TG were not different between the group T and group C (P ＞ 0.05).The incidence rate of EUGR in the 4th group C was increased significantly than that in the 4th group T(P ＜0.05).Conclusions The preterm and small for gestational age infants were safe after the pediatric amino acid was used at the 6 h after birth.Amino acid can promote growth of head circumference and weight,shorten the birth weight recovery time and reduce the incidence of EUGR.

OBJECTIVETo observe the in vivo migration of human umbilical cord mesenchymal stem cells (hUCMSCs) labeled with the PKH26 red fluorescent dye after transplantation into rats with liver cirrhosis.

METHODSFrozen hUCMSCs were resuscitated and labeled with PKH26. Labeling efficiency and fluorescent maintenance time of the PKH26-1abeled cells were measured. Morphology of the labeled and unlabeled (control) cells was observed by microscopy. The cell growth curve was determined using the MTT method. The PKH26-1abeled hUCMSCs were transplanted via tail vein injection into healthy (control) rats and rats with liver cirrhosis. Migration of the PKH26-1abeled hUCMSCs observed 48 h later in frozen liver sections under a fluorescence microscope.

RESULTSThe labeling ratio of PKH26 to hUCMSCs was 100%. Growth of the labeled cells was good. The cell morphology was not significantly different between the labeled and unlabeled cells; all cells were long and spindle-like. Cell proliferation was not impacted significantly by labeling.Fluorescence was maintained for at least 20 days, as detected by in vitro analysis. After transplantation into the rats, the PKH26-1abeled hUCMSCs were mainly distributed in the area surrounding the portal vein, the blood vessels, and the false lobule of the cirrhotic liver; a small amount ofhUCMSCs were present in the spleen and lung.

CONCLUSIONPKH26 is an ideal fluorescent dye to label hUCMSCs. The PKH26 labeling technique can be used to study the migration of hUCMSCs in cirrhotic liver.

Animals ; Cell Proliferation ; Fluorescent Dyes ; Humans ; Liver Cirrhosis ; Mesenchymal Stromal Cells ; Organic Chemicals ; Rats ; Umbilical Cord