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• W1998307300 abstract "O419 Aims: Isolation of side population (SP) cells, those with a verapamil-sensitive ability to efflux Hoechst 33342, is known as a novel universal technique to enrich tissue stem cells. We reported that SP cells isolated from liver and bone marrow were co-cultuered with primary cultured hepatocyte in collagen gel and were capable of transdifferentiation into hepatocytes in vitro. Here we show that splenic SP cell has a potential to become hepatocyte and is another possible source for a stem cell transplantation to liver. Methods: 8-12-week-old transgenic rats that ubiquitously express Green Fluorecent Protein (GFP) were used for a culture study of SP cells, and 8-week-old B6 mice were used for a characterization of SP cells. Splenocytes were obtained by collagenase digestion and forcing the tissue through sterile mesh. Then mononuclear cells were separated by Ficoll density gradient centrifugation. Half of them was frozen at -80°C until use. The freshly isolated splenocytes or those kept at -80°C were resuspended at 106 cells/ml in Hanks Balanced Salt Solution (HBSS) containing 2% fetal calf serum, 1mM Hepes (HBSS+), and Hoechst 33342 w or w/o verapamil and were incubated at 37°C for 90min. Cells were washed twice with HBSS+ and then resuspended in HBSS+ with 2μg/ml propidium iodide. Cells were analyzed and sorted on a dual-laser FACStar Plus flow cytometer. RNA was extracted from isolated SP cells and RT-PCR was performed. SP cells were cultured with male rat primary cultured hepatocytes in collagen gel. Cell morphology was monitored under a phase-contrast microscope and a fluorescence microscope. The expressions of albumin, alpha-fetoprotein (AFP), cytokeratin 18 (CK18), and cytokeratin 19 (CK19) were determined by immunocytochemistry. In order to avoid direct contact of both splenic-SP cells and primary cultured hepatocytes, we laid collagen gel between them and cultured for some samples. Results: The rate of splenic-SP cells was 0.2% of fresh splenocytes, while 1.3% of cryopreservated splenocytes. Surface markers, such as c-kit, sca-1, CD45, and CD34, were not significantly different between SP cells isolated from fresh and frozen splenocytes. Splenic-SP cells were round and small. These cells did not express liver specific markers, such as albumin, AFP, CK18, or CK19. During one-week culture with hepatocytes in collagen gel, we could find no change in GFP-positive SP cells. We should observe several colonies of GFP-positive SP cells in about 10 days. We found GFP-positive cells were no longer round nor small. They were rather flat and polygonal. We could see similar result from splenic SP cells after freezing but the number of colonies from these splenic-SP cells was a little less. After 2-week-culture, most of GFP-positive cells were positive for albumin, AFP and CK18, and negative for CK19. Splenic-SP cells could not survive and form colonies unless they were co-cultured with primary cultured hepatocyte. Splenic-SP cells co-cultued with hepatocytes without any direct contact with hepatocytes to eliminate the possibilities of cell fusion, expressing liver specific protein. Conclusions: We succeeded in transdifferentiation of splenic-SP cells into hepatocytes in vitro. Our results indicated that splenic-SP cells have plasticity and obtain hepatic function. Thus, tissue stem cells, especially SP cells, are useful for cell transplantation. Splenic-SP cell could be a candidate for source of cell transplantation for treatment of liver cirrhosis." @default.
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• W1998307300 date "2004-07-01" @default.
• W1998307300 modified "2023-09-27" @default.
• W1998307300 title "SPLENIC SIDE POPULATION CELL IS A POSSIBLE SOURCE FOR STEM CELL TRANSPLANTATION TO LIVER." @default.
• W1998307300 doi "https://doi.org/10.1097/00007890-200407271-00432" @default.
• W1998307300 hasPublicationYear "2004" @default.
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