Browse > Article

Effective Application of Multiplex RT-PCR for Characterization of Human Embryonic Stem Cells/ Induced Pluripotent Stem Cells  

Kim, Jung-Mo (Graduate School of Life Science, CHA Stem Cell Institute, CHA University, College of Medicine)
Cho, Youn-Jeong (Graduate School of Life Science, CHA Stem Cell Institute, CHA University, College of Medicine)
Son, On-Ju (Graduate School of Life Science, CHA Stem Cell Institute, CHA University, College of Medicine)
Hong, Ki-Sung (CHA Biotech & Diostech Co., Ltd.)
Chung, Hyung-Min (Graduate School of Life Science, CHA Stem Cell Institute, CHA University, College of Medicine)
Publication Information
Reproductive and Developmental Biology / v.35, no.1, 2011 , pp. 1-8 More about this Journal
Abstract
Techniques to evaluate gene expression profiling, such as sufficiently sensitive cDNA microarrays or real-time quantitative PCR, are efficient methods for monitoring human pluripotent stem cell (hESC/iPSC) cultures. However, most of these high-throughput tests have a limited use due to high cost, extended turn-around time, and the involvement of highly specialized technical expertise. Hence, there is an urgency of rapid, cost-effective, robust, yet sensitive method development for routine screening of hESCs/hiPSCs. A critical requirement in hESC/hiPSC cultures is to maintain a uniform undifferentiated state and to determine their differentiation capacity by showing the expression of gene markers representing all three germ layers, including ectoderm, mesoderm, and endoderm. To quantify the modulation of gene expression in hESCs/hiPSC during their propagation, expansion, and differentiation via embryoid body (EB) formation, we developed a simple, rapid, inexpensive, and definitive multimarker, semiquantitative multiplex RT-PCR platform technology. Among the 9 gene primers tested, 5 were pluripotent markers comprising set 1, and 3 lineage-specific markers were combined as set 2, respectively. We found that these 2 sets were not only effective in determining the relative differentiation in hESCs/hiPSCs, but were easily reproducible. In this study, we used the hES/hiPS cell lines to standardize the technique. This multiplex RT-PCR assay is flexible and, by selecting appropriate reporter genes, can be designed for characterization of different hESC/hiPSC lines during routine maintenance and directed differentiation.
Keywords
Human embryonic stem cells; Human induced pluripotent stem cells; Embryoid body; Multiplex RT-PCR; Pluripotency; Differentiation;
Citations & Related Records
Times Cited By KSCI : 1  (Citation Analysis)
연도 인용수 순위
1 Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM (1998): Embryonic stem cell lines derived from human blastocysts. Science 282:1145-1147.   DOI
2 Tropel P, Tournois J, Come J, Varela C, Moutou C, Fragner P, Cailleret M, Laabi Y, Peschanski M, Viville S (2010): High-efficiency derivation of human embryonic stem cell lines following pre-implantation genetic diagnosis. In Vitro Cell Dev BioI Anim 46:376-385.   DOI   ScienceOn
3 Tulpule A, Lensch MW, Miller JD, Austin K, D'Andrea A, Schlaeger TM, Shimamura A, Daley GQ (2010): Knockdown of Fanconi anemia genes in human embryonic stem cells reveals early developmental defects in the hematopoietic lineage. Blood 115:3453-3462.   DOI   ScienceOn
4 Mateizel I, Spits C, Verloes A, Mertzanidou A, Liebaers I, Sermon K (2009): Characterization of CD30 expression in human embryonic stem cell lines cultured in serum-free media and passaged mechanically. Hum Reprod 24:2477-2489.   DOI   ScienceOn
5 Moore JC, Atze K, Yeung PL, Toro-Ramos AJ, Camarillo C, Thompson K, Ricupero CL, Brenneman MA, Cohen RI, Hart RP (2010): Efficient, high-throughput transfection of human embryonic stem cells. Stem Cell Res Ther 1:23.   DOI
6 Olsen AL, Stachura DL, Weiss MJ (2006): Designer blood: creating hematopoietic lineages from embryonic stem cells. Blood 107:1265-1275.   DOI   ScienceOn
7 Park TS, Zambidis ET, Lucitti JL, Logar A, Keller BB, Peault B (2009): Human embryonic stem cell-derived hematoendothelial progenitors engraft chicken embryos. Exp Hematol 37:31-41.   DOI   ScienceOn
8 Schena M, Shalon D, Davis RW, Brown PO (1995): Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270:467-470.   DOI
9 Kim JB, Sebastiano V, Wu G, Arauzo-Bravo MJ, Sasse P, Gentile L, Ko K, Ruau D, Ehrich M, van den Boom D, Meyer J, Hubner K, Bernemann C, Ortmeier C, Zenke M, Fleischmann BK, Zaehres H, Scholer HR (2009): Oct4-induced pluripotency in adult neural stem cells. Cell 136:411-419.   DOI   ScienceOn
10 Klimanskaya I, Chung Y, Becker S, Lu SJ, Lanza R (2006): Human embryonic stem cell lines derived from single blastomeres. Nature 444:481-485.   DOI   ScienceOn
11 Mamidi MK, Pal R, Bhonde R, Zakaria Z, Totey S (2010): Application of multiplex PCR for characterization of human embryonic stem cells (hESCs) and its differentiated progenies. J Biomol Screen 15:630-643.   DOI   ScienceOn
12 Langmann T, Mauerer R, Schmitz G (2006): Human ATP-binding cassette transporter TaqMan low-density array: analysis of macrophage differentiation and foam cell formation. Clin Chern 52:310-313.
13 Lee MJ, Lee JH, Kim JM, Shin JM, Park SJ, Chung SH, Lee KI, Chae JI, Chung HM (2010): Differentiation of mesenchymal stem cell-like cell from feeder free cultured human embryonic stem cells using Direct Induction. Reprod Dev BioI 34:1-6.
14 Liao J, Cui C, Chen S, Ren J, Chen J, Gao Y, Li H, Jia N, Cheng L, Xiao H, Xiao L (2009): Generation of induced pluripotent stem cell lines from adult rat cells. Cell Stem Cell 4:11-15.   DOI   ScienceOn
15 Duggan DJ, Bittner M, Chen Y, Meltzer P, Trent JM (1999): Expression profiling using cDNA microarrays. Nat Genet 21:10-14.
16 Friedrich Ben-Nun I, Benvenisty N (2006): Human embryonic stem cells as a cellular model for human disorders. Mol Cell Endocrinol 252: 154-159.   DOI
17 Dvash T, Ben-Yosef D, Eiges R (2006): Human embryonic stem cells as a powerful tool for studying human embryogenesis. Pediatr Res 60:111-117.   DOI   ScienceOn
18 Edwards MC, Gibbs RA (1994): Multiplex PCR: advantages, development, and applications. PCR Methods Appl 3:S65-75.   DOI
19 Ek M, Soderdahl T, Kuppers-Munther B, Edsbagge J, Andersson TB, Bjorquist P, Cotgreave I, Jernstrom B, Ingelman-Sundberg M, Johansson I (2007): Expression of drug metabolizing enzymes in hepatocyte-like cells derived from human embryonic stem cells. Biochem Pharmacol 74:496-503.   DOI   ScienceOn
20 Horwitz EM (2003): Stem cell plasticity: the growing potential of cellular therapy. Arch Med Res 34:600-606.   DOI   ScienceOn
21 Draper JS, Andrews PW (2002): Embryonic stem cells: advances toward potential therapeutic use. Curr Opin Obstet Gynecol 14:309-315.   DOI   ScienceOn
22 Cho SW, Moon SH, Lee SH, Kang SW, Kim J, Lim JM, Kim HS, Kim BS, Chung HM (2007): Improvement of postnatal neovascularization by human embryonic stem cell derived endothelial-like cell transplantation in a mouse model of hindlimb ischemia. Circulation 116:2409-2419.   DOI   ScienceOn
23 Cowan CA, Klimanskaya I, McMahon J, Atienza J, Witmyer JP, Zucker S, Wang CC, Morton AP, McMahon J, Powers D, Melton DA (2004): Derivation of embryonic stem-cell lines from human blastocysts. N Engl J Med 350:1353-1356.   DOI   ScienceOn
24 Deb KD, Sarda K (2008): Human embryonic stem cells: preclinical perspectives. J Transl Med 6:7.   DOI
25 Takahashi K, Yamanaka S (2006): Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663-676.   DOI   ScienceOn
26 Brimble SN, Zeng X, Weiler DA, Luo Y, Liu Y, Lyons IG, Freed WJ, Robins AJ, Rao MS, Schulz TC (2004): Karyotypic stability, genotyping, differentiation, feeder-free maintenance, and gene expression sampling in three human embryonic stem cell lines derived prior to August 9, 2001. Stem Cells Dev 13: 585-597.   DOI   ScienceOn
27 Chen Y, Zhong JF (2008): Microfluidic devices for high-throughput gene expression profiling of single hESC-derived neural stem cells. Methods Mol BioI 438:293-303.
28 Chiao E, Kmet M, Behr B, Baker J (2008): Derivation of human embryonic stem cells in standard and chemically defined conditions. Methods Cell BioI 86:1-14.
29 Tzukerman M, Rosenberg T, Reiter I, Ben-Eliezer S, Denkberg G, Coleman R, Reiter Y, Skorecki K (2006): The influence of a human embryonic stem cell-derived microenvironment on targeting of human solid tumor xenografts. Cancer Res 66:3792-3801.   DOI   ScienceOn
30 Zeng X, Rao MS (2006): The therapeutic potential of embryonic stem cells: A focus on stem cell stability. Curr Opin Mol Ther 8:338-344.
31 Tettelin H, Radune D, Kasif S, Khouri H, Salzberg SL (1999): Optimized multiplex PCR: efficiently closing a whole-genome shotgun sequencing project. Genomics 62:500-507.   DOI   ScienceOn