Characteristics Of Hepatocyte Polarity During The Hepatocyte Differentiation By Adult Bone Marrow Mesenchymal Stem Cells In Vitro

Background and ObjectiveThe cell polarity is the asymmetry of the membrane, organelle andcellular skeleton according to the biological effect. The differentmembrane domains face the different extra cellular environment andperform different biological functions. The polarization is widely foundin the various organisms, ranged from the prokaryote, such as bacteria,to the mammalian, furthermore to the human being.Hepatocytes are highly polarized cells in the human body. It playsan important role in the development, formation and function of theorgans. Hepatocyte polarization is essencial for maintainingphysiological function of the hepatocyte. The liver plate separates thebile and the blood by the connected hepatocytes. The mechanism ofdevelopment and maintenance of hepatocyte polarity is unknown. Studyinghepatocyte polarizationis of significance both to the liver biology andto the clinic treatment of various liver diseases.Bone marrow stem cells(BMSCs) can be differentiated into liverprecursor cells and liver cells, and they can be easily acquired, cultured,generated and extended in vitro, and have strong ability to proliferate.The main purpose of this research is to further characterize the phenotypeof the bone marrow derived hepatocytes, and to understand the cellpolarization, the gerneration and maintenance factors impacting on thepolarization. This study may provide fundamental basement forunderstanding clinical diseases due to the disturbance of hepatocytepolarity. Methods1,Hepatocytes differentiated from the adult bone marrow cells wereinvestigated through the morphology and expression of HNF-4 alph,ALB mRNAby Reverse Transcription Polymerase Chain Reaction (RT-PCR). The cellswere stained immunohistochemically by the hepatocytes specificantibodies. Moreover, the differentiated hepatocytes were cryo-preserved and recovered for survival analysis.2,The polarization molecules, such as SRBI, MRP, DPPIV and LDLr, wereinvestigated by semi-quantitative RT-PCR. Moreover, the cells werestained immunohistochemically by polarization molecule antibodies invivo and in vitro.3,Through the spheroidal culture and the modification of extracellular matrix(ECM), such as Matrigel from Engelbreth-Holm-Swarm(EHS),the cells were stained immunohistochemically by polarization moleculeantibodies and examined under a confocal microscope. Furthermore, therelation between the uptake of DiI AcLDL and the distribution of actinwas studied. Under the inducing ofursodeoxycholic acid (UDCA), the uptakeand transport of the fluorescein diacetate (FD) was oberved.Results1,Adult bone marrow cells were differentiated into functionalhepatocytes in vitro. We found that the expression of several hepatocytespecific markers, for example, ALB and HNF-4 alpha mRNA, was increased.At the protein level, we found clear positive staining of ALB in thecytoplasm. And BMSCs-derived liver cells gained a high survival rate forthe further reasch use by the modified and improved cryopreservationmethods.2,With hepatocyte differentiation, among the polarization molecules,DPPIV, MRP, LDLr and SR-BI mRNA were expressed increasingly. Thepolarization molecules were characteristically expressed in vivo and in vitro when observed using immunohistochemical stainingand analyzed byRT-PCR.3,Using the spheroidal culture and matrigel coating, the celladhesion was shown by actin staining. The distribution of SR-BI, MRP,DPPIV and LDLr was in the patterns of typical hepatocyte polarity. Theuptake of DiI AcLDL is related to the construction of actin. The FD wasfound in bile canacules when induced by UDCA.Conclusions1,Adult bone marrow cells can differentiated into functionalhepatocytes in vitro. The hepatocytes stored by cryopreservation can beused for the further research.2,Human bone marrow-derived hepatocytes have potential topolarized. The molecules related to hepatocyte distributedcharacteristically in the differentiated hepatocytes.3,Through the spheroidal culture and modification of extra cellularmatrix, the hepatocytes drived from bone marrow stem cells expressedtypical hepatocyte polarity. This model can be used in studying themechanism of hepatocyte polarization under physilogical conditions.