Effect of intrahepatic transplantation of embryonic stem cells-derived hepatic stem cells on host hepatic function and its safety evaluation

ABSTRACT

BACKGROUND:

In vitro differentiation of embryonic stem cells into hepatocytes has been successfully reported to a certain degree; however, whether embryonic stem cells are able to effectively enter hepatic plate of host after intrahepatic transplantation, whether embryonic stem cells can further differentiate into hepatocytes and express hepatocyte function, and risk factors for neoplastic formation are still unclear at present.

To study the intrahepatic transplantation of embryonic stem cells-derived hepatic stem cells in therapeutic liver repopulation models, and to investigate the liver tissue replacement, growth and differentiation in vivo, and neoplastic formation.

Randomized controlled animal study.

Department of Pediatric Surgery, the Second Hospital affiliated to Sun Yat-sen University. MATERIALS Twenty-four BALB/c mice, 6-8 weeks old, weighing 20-35 g, irrespective of gender, were provided by Guangzhou Experimental Animal Center. Embryonic stem cells-derived hepatic stem cells were differentiated from embryonic stem cells. E14 was provided by Stem cell Center of our hospital.

This study was performed at the Stem Cell Center, the Second Hospital affiliated to Sun Yat-sen University from July 2006 to June 2007. Twenty-four mice were randomly divided into a liver repopulation model + stem cell transplantation group (group A) and a liver resection + stem cell transplantation group (group B), with 12 mice in each group. Mice in the group A were intraperitoneally injected with 50 mg/kg retrorsine once every two weeks for totally twice. Four weeks after the second injection, about 70% liver was resected. And then, the embryonic stem cells-derived hepatic stem cells, labeled by 1×105 carboxy fluoresce in diacetate succinimidyl ester (CFDA-SE), were transplanted into mouse liver through portal vein. On the other hand, 70% liver of mice in the group B was resected and embryonic stem cells-derived hepatic stem cells were transplanted into mouse liver. MAIN OUTCOME

The distribution, incorporation, and proliferation of transplanted cells were observed under fluorescent microscopy. Two weeks later, hepatic function was stained with albumin fluorescence immunoassay (double fluorescence staining) and assayed by level of serum albumin. Embryonic stem cells-derived hepatic stem cells were poured into liver of remedial liver regeneration mice, and undifferentiated embryonic stem cells were transplanted into subcutaneous tissue in axillary region as the controls to observe neoplastic formation in embryonic stem cells-derived hepatic stem cells.

① Growth of hepatic stem cells in recipient mice One week after transplantation of CFDA-SE-labeled embryonic stem cells-derived hepatic stem cells, some scattered region was green under fluorescent microscopy. The area of green region increased apparently in 2 weeks, and cord-like structure could be observed. ② Liver function Immunofluorescent staining of albumin (double fluorescence staining) demonstrated that labeled cells expressed positive albumin (yellow fluorescence) in liver tissue of recipient mice, but there was not significant difference in serum albumin level between group A and group B (P > 0.05). ③ Reliability of hepatic stem cell transplantation Teratoma did not form over 6 months; however, transplantation of undifferentiated embryonic stem cells in the axillary region could cause formation of teratoma after 6 weeks.

The transplantation of embryonic stem cells-derived hepatic stem cells in therapeutic liver repopulation model mice can effectively and further grow and differentiate, or even partially express hepatocyte function; in particular, the transplantation is safe.