| One of the important key points to realize adult stem cell therapy is to aquire sufficient amounts of stem cells and to effectively induce terminal differentiation of functional cells. Recently, several abnormal transformed cell populations derived from in vitro cultured bone marrow mesenchymal stem cells were found. Therefore, the safety of this traditional strategy of in vitro expansion of sufficient amounts of adult stem cells is becoming more and more concerned.In previous study, a transformed cell population derived from in vitro cultured rat bone marrow mesenchymal stem cells with characteristics of small round, clonogenic growth and capable of embryonic body formation was found. This cell population had been cultured and isolated by a combined method of Dextor's method, two-step trypsinization method and limiting dilution. These cells are able to maintain high proliferative potential (up to 220 passages) and cardiac differentiation after treated with 5-azacytidine (5-Aza, a DNA methyltransferase inhibitor). These cells are negative for mesenchymal stem cell (MSCs) marker:CD90; hematopoietic stem cell markers:CD34, CD45; endothelial progenitor cell markers:CD31, flk-1 and T cell marker:CD4. Phenotype, doubling time, cell cycle distribution and cardiac differentiation potential of these cells maintain stable after long-term culture. Replicative senescence of this cell population is not observed.In this doctoral dissertation research, we aimed to further investigate this isolated transformed cell population on (1)summary of formation of this cell population, (2)ultrastructure, (3)specific markers, (4)growth features, (5)time-lapse study, (6)nucleofection, labelling and in vivo tracking, (7)gene expression microarrays analysis, (8)the effect of Oct4 (a pluripotent transcription factor) shRNA knockdown on these cells, (9)telomerase activity detection, (10)karyotyping and (11)in vivo tumorgenesis. The results of this doctoral dissertation study are as follows:In vitro expansion of mouse (2-3 months age) bone marrow MSCs in DMEM medium did not show this transformed cell population. In vitro expansion of SD rat (3-6 months age) bone marrow MSCs in IMDM medium, abnormal transformed cell population was detected in culture at passage 0,0,3,4,5, and 5 respectively in 6 out of 10 rats. In vitro expansion of SD rats (>24 months age) bone marrow MSCs in IMDM medium, abnormal transformed cell population was detected in culture at passage 0,0,4 respectively in 3 out of 4 rats. Results of transmission electron microscopy and scanning electron microscopy showed that isolated representative cell population El single-cell clone are around 6-9μm in diameter, have high nuclear-plasmic ratio, and are particularly rich in mitochondria. A few lipid droplets and granules were also observed in cytoplasm. Results of Gene expression microarray and real-time RT-PCR further showed that these cells expressed some of mesenchymal stem cell marker: CD73, but negative for mesenchymal stem cell marker:CD105 and B cell marker: CD19. These cells expressed some particular markers:CD9, CD24, CD63, CD68, CD81, CD276 and CD302. These cells showed adherent growth, reduced contact inhibition, clonal growth in three dimensions, suspension growth, and adherent growth after suspension culture. Time-lapse microscopy and live cell imaging analysis proved these growth features and showed that time interval for cell division is about 6-7 hours. After co-culture with 2-day cultured neonatal cardiomyocytes for 3 days, fast-DiI labelled transformed cells were positive for cardiac specific marker-cardiac troponin I. After intravenous transplantation of fast-DiI labelled transformed cells into rat infarct model by ligating the LAD for 7 days, red-fluorescent cell mass expressed Cardiac troponin I. This suggested that these cells are capable of cardiac homing and cardiac differentiation. Differential gene expression of El single-cell clone stimulated by 5-Aza showed that 663 genes were dominantly affected by 5-Aza with fold change>2 in 26,419 genes, including 118 up-regulated>2-fold genes and 545 down-regulated>2-fold genes. The Gene Ontology (GO) tool MAS2.0 was utilized to categorize genes according to molecular function and biological process. On the basis of MAS2.0 analysis, there was a significant enrichment of downregulated genes in GO categories such as the following:rRNA binding, RNA binding and mRNA binding (categorized by molecular function); complement activation, classical pathwayn negative regulation of RNA splicing,hydrogen peroxide biosynthesis (categorized by biological process). Also, there was an enrichment of upregulated genes in categories such as calcium ion binding, L-lysine transporter activity and dimethyladenosine transferase activity (categorized by molecular function); cell adhesion, smoothened signaling pathway involved in spinal cord motor neuron cell fate specification and lysine transport (categorized by biological process). KEGG pathways that are significantly affected by 5-Aza are as follows:ECM-receptor interaction, Glycosphingolipid biosynthesis-ganglioseries, Chondroitin sulfate biosynthesis (top 3 in up-regulated genes list with p<0.05) and Complement and coagulation cascades, Oxidative phosphorylation, Alzheimer's disease (top 3 in down-regulated genes list with p<0.05). Results of TRAP-silver staining telomerase detection showed that telomerase activity were found in 70th passage and 212th passage E1 single-cell clone with levels of telomerase activity in 212th passage higher. These cells maintained their cell morphology, proliferation and differentiation potential after nucleofection. Transfection efficiency is independent of passage of these cells. GFP-nucleofected cells remained GFP positive after intracardiac transplantation for one week. Immunofluorescence staining of OCT4 on different passages of these cells was positive. After Oct4 downregulated by RNAi, these cells changed from small round into large flat shape and showed a dramatic reduction in growth rate and terminal differentiation. Giemsa-banding showed that karyotypes of D6, E1, G11 and H7 four single-cell clones of transformed cell population were abnormal; with 59.3% cells of D6 clone contain 39 chromosomes,70.5% cells of El clone contain 39 chromosomes,60.7% cells of G11 clone contain 39 chromosomes, 68.0% cells of H7 clone contain 39 chromosomes. These cells mainly lost No.19 and 20 (24.6% of D6 clone lost No.19 and 37.7% lost No.20; 30.5% of E1 clone lost No. 19 and 38.9% lost No.20; 23.7% of G11 clone lost No.19 and 40.6% lost No.20; 28.8% of H7 clone lost No.19 and 32.2% lost No.20). After intravenous/intracutaneous injection of this transformed cell population for 60 days showed absence of tumor formation. In summary, this study demonstrated that traditional strategy of in vitro expansion of rat bone marrow mesenchymal stem cells may induce their immortalization and non-tumorigenic transformation. This transformed cell population has not only stem-like characteristics of high proliferation, embryonic body formation and cardiac differentiation in vivo/in vitro, but also tumor cell-like characteristics of reduced contact inhibition, three dimensional growth and chromosomal abnormality. These cells may be in a transition state of mesenchymal stem cells and tumor cells and still need further investigation. It may not only offer better understanding of adult stem cells and tumor stem cells, but also promote the development of safe adult stem cell therapy strategy. This study also suggested that in vitro expansion of adult stem cells must be proved safety before clinical use. |
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