Two piglets and one juvenile pig were used to investigate closely what types of cells express green fluorescent protein (GFP) and if any, whether the GFP-tagged cells could be used for stem cell transplantation research as a middle-sized animal model in bone marrow cells of recloned GFP pigs. Bone marrow cells were recovered from the tibia, and further analyzed with various cell lineage markers to determine which cell lineage is concurrently expressing visible GFP in each individual animal. In the three animals, visible GFP were observed only in proportions of the plated cells immediately after collection, showing 41, 2 and 91% of bone marrow cells in clones #1, 2 and 3, respectively. The intensity of the visible GFP expression was variable even in an individual clone depending on cell sizes and types. The overall intensities of GFP expression were also different among the individual clones from very weak, weak to strong. Upon culture for 14 days in vitro (14DIV), some cell types showed intensive GFP expression throughout the cells; in particular, in cytoskeletons and the nucleus, on the other hand. Others are shown to be diffused GFP expression patterns only in the cytoplasm. Finally, characterization of stem cell lineage markers was carried out only in the clone #3 who showed intensive GFP expression. SSEA-1, SSEA-3, CD34, nestin and GFAP were expressed in proportions of the GFP expressing cells, but not all of them, suggesting that GFP expression occur in various cell lineages. These results indicate that targeted insertion of GFP gene should be pursued as in mouse approach to be useful for stem cell research. Furthermore, cell- or tissue-specific promoter should also be used if GFP pig is going to be meaningful for a model for stem cell transplantation.

Inflammatory bowel disease (IBD) is a group of chronic disorders of unknown etiology characterized by inflammation of the gastrointestinal tract. Recent data showed that the development of IBD is associated with the interplay of genetic, bacterial, and environmental factors and dysregulation of the intestinal immune system. We investigated how the gut cells were repaired after injury in Drosophila melanogaster. In this study we made D. melanogaster intestine damage model by oral feeding with variety IBD inducer such as pathogenic bacteria Serratia marcescens, Dextran Sulfate Sodium (DSS) and bleomycin, because its function is very similar with human, even though D. melanogaster has relatively simple organism. We repeated oral feeding with variety IBD inducer and got the survival rate and 50% lethal dose (LD50). After feeding with IBD inducer, we investigated the change of the intestinal stem cells, innate immune-related gene expression, and apoptosis in D. melanogaster gut. We examined the Delta, stem cell marker, staining image in the gut after feeding with DSS and S. marcescens with LD50 concentration. The Delta positive cells greatly increased in gut cells damaged by DSS or S. marcescens. This result supports the idea that intestinal gut stem cells are increased after gut cell damage and play very important role in damaged cell repair. Expression level of antimicrobial peptides was dramatically up-regulation after gut damage. As a result of TUNEL (terminal deoxynucleotidyl transferase mediated X-dUTP nick end labeling) assay, we confirmed that cell death by apoptosis was very increased in DSS feeding flies. Accordingly, we suggest that D. melanogaster is a proper IBD model organism to study how intestine damage can be repaired.

The HMG box containing protein (HBP) has a high mobility group domain and involved in the regulation of proliferation and differentiation of tissues. We screened HBP2 in glioblastoma using Suppression Subtractive Hybridization (SSH) and isolated human spermatogonial stem cell‐like cells (hSSC‐like cells) derived from patients of nonobstructive azoospermia (NOA). Expression of HBP2 was analyzed by RT‐PCR in undifferentiated stem cells (human Embryonic Stem Cells, hSSC‐like cells 2P) and spontaneous differentiated stem cells (hSSC‐like cells 4P). It was overexpressed in hESC and hSSC‐like cells 2P but not in hSSC‐like cells 4P. Also, the expression level of HBP2 was downregulated in colon tumor tissues compared to normal tissues. Specifically in synchronized WI‐38 cells, HBP2 was highly upregulated until the G1 phase of the cell cycle and gradually decreased during the S phase. Our results suggest that HBP2 was downregulated during the spontaneous differentiation of hSSC‐like cells. HBP2 was differently expressed in colon tissues and was related to G1‐progression in WI‐38 cells. It may play a role in the maintenance of an undifferentiated hSSC‐like cell state and transits from G1 to S in WI‐38 cells. This research was important that it identified a biomarker for an undifferentiated state of hSSC‐like cells and characterized its involvement to arrest during cell cycle in colon cancer.

Bone marrow (BM) cell harvesting is a crucial element in the isolation of mesenchymal stem cells (MSCs). A simple method for harvesting cat BM cells is described. The results show that a large number of BM cells can rapidly be harvested from the cat by this simple procedure. MSCs prepared by density-gradient method were spindle-shaped morphology with bipolar or polygonal cell bodies and strongly positive for CD9 and CD44 and negative for CD18 and CD45-like. They were capable of differentiation to adipocytic and osteocytic phenotypes when exposed to appropriate induction media. The advantages of this method are its rapidity, simplicity, low invasiveness, and low donor attrition and good outcome.