276 Side population cells derived from non-parenchymal liver cells and splenocytes are capable of differentiating into hepatocytes in vitro

HEPATOLOGY, Vol. 38, No. 4, Suppl. 1, 2003

AASLD ABSTRACTS

276 SIDE POPULATION CELLS DERIVED FROM NONPARENCHYMAL LIVER CELLS AND SPLENOCYTES ARE CAPABLE OF DIFFERENTIATING INTO HEPATOCYTES IN VITRO. Sayaka Inokuchi, Toshifumi Azuma, Takeshi Nishimura,

Kengo Tom#a, Naoto Kitamura, Go Wakabayashi, Masaki Kitajima, Hiroko Kouike, Yumi Matsuzaki, Hideyuki Okano, Hiromasa Ishii, Keio University School of Medicine, Tokyo, Japan Background: Bone marrow ceils and embryonic stem ceils are being used for cell transplantation. Although these ceils are known to be multipotent and capable to become hepatocyte, several problems have been pointed out, such as cell fusion and teratoma generation. Thus we paid attention to side population (SP) ceils, those with a verapamil-sensitive ability to efflux Hoechst 33342. SP ceils have been identified in several tissues. The bone marrow SP phenotype appears to be a common feature of stem ceils, but hepatic and splenic SP ceils have b e e n less well characterized. Aim: We tried to separate and culture SP ceils from non-parenchymal liver ceils (NPCs) and splenocytes, and examined whether they would obtain hepatic phenotype. Methods: 8-12-week-old female transgenic rats that ubiquitously express EGFP were used to isolate non-parencymal liver ceils by the standard collagenase two-step perfusion method, and splenocytes were isolated by forcing the tissue through sterile mesh, then mononuclear ceils were separated by Ficoll density gradient centrifugation. They were r e s u s p e n d e d at 106 cells/ml in Hanks Balanced Salt Solution (HBSS) containing 2% fetal calf serum, l m M Hepes (HBSS+), and 5ug/ml Hoechst 33342 (Hst) w or w/o Verapamil and were incubated at 37 degrees centigrade for 90rain. Cells were washed twice in HBSS+ and then r e s u s p e n d e d in HBSS+ with 2ug/ml propidium iodide. Cells were analyzed and sorted on a dual-laser FACStar Plus flow cytometer. RT-PCR was performed with RNA extracted from freshly isolated SP cells. Isolated SP cells were cultured with male rat primary cultured hepatocytes in collagen gel sandwich. Cell morphology was monitored under a phase-contrast microscope and a fluorescence microscope. The expression of albumin, cytokeratin 18, and cytokeratin 19 was analyzed by immunohistochemistry. Fluorescence in situ hybridization (FISH) for the sry gene was performed to exclude cell fusion. Results: The rate of NPC-SP was 0.05% of isolated NPCs. 89% of NPC-SP expressed CD45, while 55% of NPC main population (MP) did. Freshly isolated SP cells were smaller than mature hepatocytes. They looked like monocytes or lymphocytes. These cells did not express liver specific markers, such as albumin, alpha-fetoprotein, cytokeratin 18, or cytokeratin 19. During one-week culture with hepatocytes using collagen gel sandwich method, we could find no change in GFP-positive SP ceils, but after further culture, we found several colonies consisting of GFP-positive SP cells, whose shape became polygonal. After 2-week-culture, most of GFP-positive cells were stained positive for albumin. NPC-SP cells died within one week without hepatocyte co-culture. NPC-MP cells formed colonies after 2 weeks culture with hepatocytes, but unlike SP ceils, they looked like fibroblasts and were negative for albumin. Freshly isolated SP ceils from splenocytes were also small and monocyte-like, and expressed no liver specific markers. Spleen SP cells could culture for more than 2 weeks in the same way as NPC-SP cells, and looked like hepatocytes, but did not proliferate. After having b e e n cultured, most of GFP-positive ceils were stained positive for albumin and cytekeartin 18, and negative for cytokeartin 19; on the other hand splenic main population cells after 2 weeks' culture expressed those very little. Conclusion: We succeeded in culturing liver and spleen SP cells. SP cells which did not express mRNA of liver specific markers, such as albumin or cytokeratin 18, became to express those after the culture with hepatocytes. These results suggest that these SP cells have plasticity and obtain hepatic function. Thus, tissue stem cells, especially SP cells, are useful for ceil transplantation. Spleen SP cell might be a candidate for source of cell transplantation for treatment of liver cirrhosis. Disclosures: Toshifumi Azuma - No relationships to disclose Sayaka Inokuchi - No relationships to disclose Hiromasa Ishii - No relationships to disclose Masaki Kitajima - No relationships to disclose

289A

Naoto Kitamura - No relationships to disclose Hiroko Kouike - No relationships to disclose Yumi Matsuzaki - No relationships to disclose Takeshi Nishimura - No relationships to disclose Hideyuki Okano - No relationships to disclose Kengo Tomita - No relationships to disclose Go Wakabayashi - No relationships to disclose

277 EPIMORPHIN, A MESENCHYMAL CELL SURFACEASSOCIATED MOLECULE, IS INVOLVED IN DIFFERENTIATION OF HEPATIC STEM-LIKE CELLS THROUGH CELL CONTACT WITH HEPATIC STELLATE CELLS. Kouichi Miura, Takashi Goto, Ken-ichiro Mikami, Kunio

Nakane, Kazuo Yoneyama, Hirokazu Nagai, Kunihiko Terada, Toshihiro Sugiyama, Katsuyuki Imai, Haruki Senoo, Sumio Watanabe, Akita University, Akita, Japan Background: Epimorphin, a mesenchymal cell surface-associated molecule identified as an epithelial morphogen, is detected on stellate ceils in the liver. We have reported that a contact with hepatic stellate ceils (HSCs) promotes differentiation of hepatic stem-like ceils (HSLCs). Here, we show the involvement of epimorphin in that process.Materials and Methods: HSLCs and HSCs were isolated from healthy adult rats using two-step collagenase perfusion and Percoll gradient centrifugation, and maintained standard medium, Dulbecco's modified Eagle's medium with 10% fetal bovine serum. HSLCs were cultured in steilate ceil-conditioned medium, co-cultured with HSCs to maintain cellceil contact or in the presence of epimorphin. Phenotypical and morphologic changes were investigated by RT-PCR and with an electron microscopy, respectively. Results: HSLCs are polygonal in shape and assume a cobblestone appearance w h e n cultured in standard medium. Transmission electron microscopy showed that HSLCs , 20 to 25 micro-m in diameter, have a round to ovalshaped nucleus, a few vacuoles, scant organeiles and a high nucleus/cytoplasm ratio compared with normal hepatocytes. The phenotypic properties of HSLCs did not change as well as their size and shape during this experiment. HSLCs characterized by RT-PCR are follows: positive, c-kit, musashi-1 (a neural stem cell marker), alpha-fetoprotein, cytokeratin19, connexin43; negative, albumin, transferrin, tyrosine aminotransferase, gamma-glutamyl transpeptidase. HSLCs cultured in stellate cell-conditioned medium had no phenotypical and morphological changes. HSLCs co-cultured with HSCs expressed albumin, transferrin, and tyrosine aminotransferase, which were inhibited by an anti-epimorphin antibody. Furthermore, epimorphin induced the markers not only for hepatocytes including albumin, transferrin and tyrosine aminotransferase, but also for cholangiocytes including gammaglutamyl transpeptidase in addition to increased expression of connexin43 and cytokeratin19, with decreased expression of c-kit and musashi-1. In addition, C/EBP beta was enhanced, which has been reported to mediate through morphogenesis by epimorphin. HSLCs co-cultured with HSCs piled up and subsequent development of bile-canalicui-like structures, which was dramatically inhibited by an anti-epimoirphin antibody. HSLCs, close to epimorphin, stated piling up, changed their shape from flat to cuboidal, became rich in mitochondria and rough endoplasmic reticulum and formed bile-canalicui-like structures. Conclusions: HSLCs have a self-renewing capacity and multilineage differentiation potential. Epimorphin is involved in differentiation of HSLCs though a contact with HSCs. Disclosures: Takashi Goto - No relationships to disclose Katsuyuki Imai - No relationships to disclose Ken-ichiro Mikami - No relationships to disclose Kouichi Miura - No relationships to disclose Hirokazu Nagai - No relationships to disclose Kunio Nakane - No relationships to disclose Haruki Senoo - No relationships to disclose Toshihiro Sugiyama - No relationships to disclose Kunihiko Terada - No relationships to disclose Sumio Watanabe - No relationships to disclose Kazuo Yoneyama - No relationships to disclose