Ki Dae Park

Biological characterization of long-term cultured human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) have generated a great deal of interest in clinical applications. The reason is that they may have the plasticity needed to differentiate into multiple lineages and the ability to expand ex vivo. For the therapeutic applications of hMSCs to be of practical use, it is crucial to assess the efficacy and safety of hMSCs in long-term ex vivo expansion. In this study, we cultured hMSCs by population doubling (PD) 60, and investigated their growth, osteogenic and adipogenic differential abilities, change of surface markers, telomerase activity, telomere length, and gene expression related to tumorigenesis. An in vivo tumorigenesis assay was also carried out. In long-term expanded hMSCs, the cells became aged above PD 30 and their adipogenic and osteogenic differentiation potential decreased. Telomerase activity unchanged whereas telomere length decreased and karyotypes were not changed. Gene expressions related to tumorigenesis decreased in proportion as the PD of hMSCs increased. In vivo transplantation of long-term cultured hMSCs to nude mice did not result in tumor formation. These findings suggest that diverse tests for cellular therapy should be considered during the ex vivo culture of hMSCs, particularly when a prolonged and extended propagation period is required.

Telomerase Reverse Transcriptase Related with Telomerase Activity Regulates Tumorigenic Potential of Mouse Embryonic Stem Cells

Embryonic stem cell (ESC) research gave rise to the possibility that stem cell therapy could be used in the treatment of incurable diseases such as neurodegenerative disorders. However, problems related to the tumorigenicity of undifferentiated ESCs must be resolved before such cells can be used in the application of cell replacement therapies. In the present study, we attempted to determine biomarkers that predicted tumor formation of undifferentiated ESCs in vivo. We differentiated mouse ESCs (R1 cell line) into neural lineage using a 5-step method, and evaluated the expression of oncogenes (p53, Bax, c-myc, Bcl2, K-ras), telomerase-related genes (TERT, TRF), and telomerase activity and telomere length during differentiation of ESCs. The expression of oncogenes did not show a significant change during differentiation steps, but the expression of telomerase reverse transcriptase (TERT) and telomerase activity correlated with mouse ESCs differentiation. To investigate the possibility of mouse TERT (mTERT) as a biomarker of tumorigenicity of undifferentiated ESCs, we established mTERT knockdown ESCs using the shRNA lentivirus vector and evaluated its tumorigenicity in vivo using nude mice. Tumor volumes significantly decreased, and appearances of tumor formation in mice were delayed in the TERT-knockdown ESC treated group compared with the undifferentiated ESC treated group. Altogether, these results suggested that mTERT might be potentially beneficial as a biomarker, rather than oncogenes of somatic cells, for the assessment of ESCs tumorigenicity.

Biodistribution and in vivo efficacy of genetically modified human mesenchymal stem cells systemically transplanted into a mouse bone fracture model

Human mesenchymal stem cells (hMSCs) have generated a great deal of interest in clinical application due to their ability to undergo multi-lineage differentiation. Recently, ex vivo genetic modification of hMSCs was attempted to increase their differentiation potential. The present study was conducted to evaluate the biodistribution and in vivo efficacy of genetically modified hMSCs. To accomplish this, Runx2, which is a key transcription factor associated with osteoblast differentiation, was transduced into hMSCs using lentiviral vectors expressing green fluorescent protein (GFP) or luciferase. Here, we developed an experimental fracture in mice femur to investigate the effects of Runx2-transduced hMSCs on bone healing and migration into injury site. We conducted bio-luminescence imaging (BLI) using luciferase-tagged vector and quantitative real-time PCR using GFP probe to investigate the biodistribution of Runx2-transduced hMSCs in the fracture model. The biodistribution of hMSC cells in the fractured femur was observed at 14xa0days post-transplantation upon both BLI imaging and real-time PCR. Moreover, the fractured mice transplanted with Runx2-transduced hMSCs showed superior bone healing when compared to mock-transduced hMSC and MRC5 fibroblasts which were used as control. These data suggested that transplanted genetically modified hMSCs systemically migrate to the fractured femur, where they contribute to bone formation in vivo.

Embryotoxicity of Lead (II) Acetate and Aroclor 1254 Using a New End Point of the Embryonic Stem Cell Test

We developed a new end point of the mouse stem cell test (EST) for developmental neurotoxicity. We tested 2 developmental neurotoxicants, namely, lead (II) acetate and Aroclor 1254, using this EST. Our results showed that lead (II) acetate is nonembryotoxic, and Aroclor 1254 is weakly embryotoxic. To identify a new end point for developmental neurotoxicity, we used the default method of neuronal differentiation for D3 mouse embryonic stem cells with basic fibroblast growth factor (bFGF) and ascorbic acid. Flow cytometry and real-time polymerase chain reaction were used to quantify the inhibition of neuronal differentiation. Our results showed that both lead (II) acetate and Aroclor 1254 reduced the percentage of microtubule-associated protein 2 (MAP-2)-positive cells and the messenger RNA (mRNA) expression level of MAP-2 in a dose-dependent manner. These results suggested that these methods can be used to develop an additional end point of the EST for developmental neurotoxicity using default differentiation of mouse embryonic stem cells.

Transferability of a modified embryonic stem cell test using a new endpoint for developmental neurotoxicity

We developed and analyzed a new surrogate endpoint of the mouse embryonic stem cell test (EST) for developmental neurotoxicity. To determine the sensitivity, specificity, and transferability of the new endpoint, a pre-validation team from three independent laboratories optimized and standardized the protocol for neuronal differentiation of mouse embryonic stem cells (mESCs) by measuring the neuronal differentiation rates of mESCs under different culture conditions, such as the presence or absence of basic fibroblast growth factor (bFGF) in the growth media and varying lengths of culture. In addition, a component ratio of neuronal cells was measured by using flow cytometry analysis of β-III tubulin (Tuj1)-positive cells and real-time polymerase chain reaction analysis of microtubule-associated protein 2 (MAP2) mRNA. Our results showed that the best growth was achieved by culturing mESCs for 12 d in N2B27 medium without bFGF or ascorbic acid. Lead (II) acetate and aroclor 1254 were used to test the usefulness of the new endpoint. When we used the known ID50 values for lead (II) acetate in the EST model, it was classified as non-embryotoxic; however, when we used the new ID50 values that we determined in this study, it was classified as weakly embryotoxic. Aroclor 1254 and penicillin G were also classified as weakly embryotoxic and non-embryotoxic compounds, respectively, when cardiac and neuronal differentiation ID50 values were used. Therefore, our new surrogate endpoint for developmental neurotoxicity is not only sensitive and specific but also transferable among laboratories.

Decreased Diethylnitrosamine-induced Liver Preneoplastic Lesions by Estradiol-3-benzoate Treatment

To clarify whether inhibitory effect of estrogen on liver tumor is associated with cell proliferation, we investigated its role in diethylnitrosamine (DEN)-induced rat preneoplastic lesions, with time sequenced manners. F344 male rats (n = 90) were divided into three groups at 5 weeks of age. The mini-osmotic pumps providing a continuous infusion of DEN was implanted into the abdominal cavity of each animal in group 1, 2 and 3 at 6 weeks of age. To see the effect of estrogen, pellet containing 1 or 10 μg of estradiol- 3-benzoate (EB) was implanted subcutaneously in the animals of groups 2 or 3, respectively, one week prior to DEN treatment. Ten animals of each group were euthanized at 10, 14 and 18 weeks after DEN treatment. Liver tissues at each time point were fixed in 10% phosphate-buffered formalin and were processed and embedded in paraffin and 5 μm sections mounted on a silanized slide. Glutathione S-transferase placental form (GST-P) positive foci and 5-bromo-2-deoxyuridine (BrdU) labeling cells were detected at each time point. Area of GST-P positive foci in DEN+EB 1 or 10 μg group was significantly decreased compared to DEN alone at 14 weeks (p < 0.01 or p < 0.05, respectively) an at 18 weeks (p < 0.05 or p < 0.01, respectively). BrdU index in DEN+EB 1 or 10 μg groups was significantly decreased compared to DEN alone at 14 weeks and at 18 weeks (p < 0.01). Taken together, we conclude that EB treatment decrease the DEN-induced liver preneoplastic lesions and this may be associated with decrease of cellular proliferation.