1. 정책이력 일반 현황 Telomerase reverse transcriptase (TERT) related with telomerase activity regulates tumorigenic potential of mouse embryonic stem cells Seung Tae Chung, Ph.D. Advanced Therapy Products Research Division

2. Introduction Embryonic stem cells  Development of biomarkers for the tumorigenic evaluation of embryonic stem cells to secure the safety of embryonic stem cell based therapies Embryonic stem cells (ES cells) are pluripotent stem cells derived from the inner cell mass of the blastocyst, an early-stage embryopluripotent stem cellsinner cell massblastocystembryo Because of their plasticity and potentially unlimited capacity for self-renewal, ES cell therapies have been proposed for regenerative medicine and tissue replacement after injury or disease. But the problems with the tumorigenicity of undifferentiated ESC must be solved 1

3. Telomere -A region of repetitive DNA (TTAGGG) at the end of chromosomes, which protects the end of the chromosome from destruction. -Some of telomere is lost during cell division. When the telomere becomes short, the chromosome can no longer replicate. -Cells are normally destroyed when telomeres are consumed. -An enzyme, telomerase, is elongates the telomere by adding DNA sequences. If telomerase is activated a cell, the cell will continue grow and divide. Back Ground and Purpose 2

4. Telomerase An enzyme that adds specific DNA sequence repeats ("TTAGGG" in all vertebrates) to the 3' ("three prime") end of DNA strands in the telomere regions. -Telomerase consists of two molecules, TERC as a RNA template and TERT is a reverse transcriptase. -A reverse transcriptase that carries its own RNA molecule, which is used as a template when it elongates telomeres, which are shortened after each replication cycle -Telomerase activity (TA) is not expressed in normal somatic cells, but highly expressed in stem cells or cancer cells, so reported that TA may be useful tools for early diagnosis of the cancer. Back Ground and Purpose 3

5. Telomerae activity was up-regulated in cancer cells (Hsu et al., 2005) and reported as an early detectable marker for cancer (Argyle & Nasir, 2003, Chen, 2003) TERT over-expression induced immortalization in somatic cells (Price & Figg, 2004) In our study, we attempt to find the possibility of telomerase activity as a biomarker for the prediction of tumor formation in ESC transplantation Back Ground and Purpose 4

6. General Scheme of the Study TERT-knockdown Stage 1 Stage 2 Stage 3 Stage 4 Stage 5 5-step method Tumorigenicity TEST Negative Control Evaluation of tumor incidence mass volume Histopathology Immunohistochemistry Evaluation of correlation between telomerase activity and tumorigenicity in vivo TERT-regulated mutant cell - TERT- transfection - TERT- specific shRNA TERT-related gene and protein expression TRAP assay  TRAP : telomeric repeat amplification protocol 5

7. Identification of Neuronal differentiation of mouse ESC Typical pictures of R1 cells at each stage (a:stage1, b:stage2, c:stage3, d:stage4 and e:stage5), f: Tuj1 (neuronal cell marker) and TH (dopaminergic marker) positive cells were observed by immunocytochemistry. 6

8. Identification of Neuronal differentiation of mouse ESC Analysis of gene expression by RT-PCR. Oct4 (undifferentiated cell marker) and PCNA (markers for cell proliferation) were decreased on process of differentiation, but genes related with neural development were increased as period. * Sox1, Nestin ; markers for neural precursor cells * Ntrk2, TH : neural marker 7

9. 8 Identification of neuronal differentiation of mouse ESC Identification of differentiation of ESC by immunocytochemistry. (A-C): Changes of Nestin and Ki-67 positive cells. Changes of SSEA1 and Oct3/4 positive cells (D-F): (G): Percentage of immunoreactive cells relative to the total number of cells. S3 vs. S4 or S5 (p<0.001). [SSEA-1, Oct3/4 : undifferentiated ESC marker, Ki-67 : cell proliferation marker, Nestin : neuronal precursor cell marker, Tuj1 : neuronal cell marker] G

10. 9 Proto-oncogene expressions during differentiation Analysis of proto-oncogene expression by RT-PCR. All gene’s expression levels were lower or the same compared with those of the control. * Control ; normal brain tissue of mice

11. Changes of telomerase activity during ESC differentiation Telomerase activity were decreased by the neuronal differentiation processes. Telomerase activity was measured at each stage using semi-quantitative TRAP (telomeric repeat amplification protocols) ELISA assay. The TERT gene expressions during mESCs differentiation was decreased and were correlated to the changes of telomerase activity 10

12. 11 pLKO.1-puro shRNA Vector Map  mouse TERT (NM_009354) MISSION shRNA Lentiviral Transduction Particle (Sigma)  antibiotics concentration for selection of ESC transduced with lentiviral particle : 7.5 ㎍ /ml puromycin Control TERT shRNA Formation of puromycin-resistant colony (white arrow) treated with TERT shRNA at stage1. Puromycin titration and selection Puromycin titration of lentiviral vector in mouse ESC Establishment of TERT-knock down ESCs

13. Identification of TERT-knock down mouse ESCs MOI 1 and 5 lentiviral vector concentration was administrated for transduction of TERT-shRNA. MOI 1 shows the formation of colony. TERT- knock down was identified by RT-PCR (A) and western blot(B) using TERT and 2C4 antibodies 12

14. Group1wk2wks3wks4wks N.C.0(0/8) ¤ 0(0/8) S10(0/8)100(8/8) S20(0/8) 75(6/8)100(8/8) S30(0/8) 87.5(7/8)100(8/8) S40(0/8) 50(4/8)75(6/8) S50(0/8) 25(2/8) TERT-K.D.0(0/8) 87.5(7/8)100(8/8) Scheme for tumorigenicity test of mouse ESC Tumorigenicity test of ESC in vivo Tumor incidence in tumorigenicity test of mESCs 13

15. 14 Changes of Body Weight Changes of Tumor Volume In vivo tumorigenicity of mESCs at each differentiation stage and TERT knockdown ES cells Body weights (A, B) and tumor volume (C, D) were evaluated during 4 weeks after ESCs injection to mice. TERT-KD: TERT knockdown ESC. A D C B

16. 15 Keratinized stratified squamous epi. (skin) Simple colummar epi. (digestive system) Transitional epi (urinary system) Nerve bundle Bone marrow tissueHyaline cartilageFibrocyteMixed gland Histopathological findings in the teratoma tissues

17. Conclusions Product Planning Of Utilizing  Establishment of the technique for neural differentiation using the mouse ES Cells  Establishment of TERT-knock down mouse ES cells  Analysis and Evaluation of telomerase activity effects on tumorigenesis in ES cells  mTert might be potentially beneficial as a biomaker for the safety assessment of ESCs tumorigenicity. 16

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