Int J Stem Cells.  2016 Nov;9(2):264-270. 10.15283/ijsc16052.

In vivo Evaluation of Human Embryonic Stem Cells Isolated by 57-C11 Monoclonal Antibody

Affiliations
  • 1Institute of Anticancer Medicine Development, Department of Integrative Bioscience and Biotechnology, Sejong University, Seoul, Korea. semidivine@hanmail.net cjryu@sejong.ac.kr

Abstract

BACKGROUND
The normal cells derived from human embryonic stem cells (hESCs) are regarded as substitutes for damaged or dysfunctional adult cells. However, tumorigenicity of hESCs remains a major challenge in clinical application of hESC-derived cell transplantation. Previously, we generated monoclonal antibody (MAb) 57-C11 specific to the surface molecule on undifferentiated hESCs. The aim of this study is to prove whether 57-C11-positive hESCs are pluripotent and tumorigenic in immunodeficient mice.
METHODS
Undifferentiated hESCs were mixed with retinoic acid (RA)-differentiated hESCs at different ratios prior to 57-C11-mediated separation. To isolate 57-C11-positive hESCs from the mixture, biotinylated 57-C11 and streptavidin-coated magnetic beads were added to the mixture. Unbound 57-C11-negative hESCs were first isolated after applying magnet to the cell mixture, and 57-C11-bound hESCs were then released from the magnetic beads. In order to measure the efficiency of separation, 57-C11-positive or -negative hESCs were counted after isolation. To evaluate the efficiency of teratoma formation in vivo, 57-C11-positive or negative cells were further injected into left and right, respectively, testes of nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice.
RESULTS
Approximately 77~100% of undifferentiated hESCs were isolated after applying 57-C11-coated magnetic beads to the mixed cell populations. Importantly, teratomas were not observed in NOD/SCID mice after the injection of isolated 57-C11-negative hESCs, whereas teratomas were observed with 57-C11-positive hESCs.
CONCLUSION
57-C11-positive hESCs are pluripotent and tumorigenic. The combination of 57-C11 and magnetic beads will be useful to eliminate remaining undifferentiated hESCs for the safe cell transplantation.

Keyword

Human embryonic stem cells; Surface marker; 57-C11; Magnetic beads; Teratoma

MeSH Terms

Adult
Animals
Cell Transplantation
Human Embryonic Stem Cells*
Humans*
Mice
Teratoma
Testis
Transplants
Tretinoin
Tretinoin

Figure

  • Fig. 1 Characterization of biotinlyated 57-C11 antibody. (A) Biotinylation efficiency of 57-C11 was tested by flow cytometry analysis. H9 cells were reacted with 10 and 30 μg/ml of un-biotinylated or biotinylated 57-C11. The binding of antibody was measured with FITC-conjugated anti-mIgG or PE-conjugated streptavidin. (B) Flow cytometry analysis of H9 cells with 57-C11. After isolation with 20 μg/ml of biotinylated 57-C11 or CA142 from the mixed cells, the surface binding for 57-C11 was confirmed in 57-C11-positive/negative and CA142-positive/negative cells, respectively. (C) Recovery rate of undifferentiated H9 cells after isolation with 57-C11. Undifferentiated and RA-differentiated H9 cells were mixed at different ratios and undifferentiated H9 cells were recovered with 57-C11-mediated isolation procedure. The 57-C11-mediated isolation procedure nonspecifically recovered approximately 10% cells (1.08×104 cells) from 10×104 RA-differentiated H9 cells. Therefore, the number was subtracted from all the numbers of recovered cells in the calculation of the recovery rate.

  • Fig. 2 Strategy of isolation of undifferentiated hESCs with 57-C11. (A) The model of isolation process with biotinylated 57-C11 using Dynabeads. (B) The live cells were counted after the isolation with 57-C11 from the mixture of undifferentiated and RA-induced differentiated H9 cells. The percentage of live cells was estimated by flow cytometry analysis using PI-negative cell population.

  • Fig. 3 Teratoma formation of H9 cells. 0.1, 0.4, 1, and 2×106 of undifferentiated H9 or RA-induced differentiated H9 cells were injected into the right or left testis of NOD/SICD mice, respectively. The teratomas were recovered 8~18 weeks after injection.

  • Fig. 4 Teratoma formation of 57-C11-positive cells. (A) Table of teratoma formation of 57-C11-positive cells at 12 or 18 weeks after injection. (B) After the isolation of 57-C11 positive cells from the mixture of undifferentiated and RA-induced differentiated H9 cells, the 1×106 cells of 57-C11-positive or -negative cells were injected into the left or right testis of NOD/SCID mice. The teratomas were recovered 18 weeks postinjection and measured size and weight before fixation. The undifferentiated H9 (H9) and RA-induced differentiated H9 (RA) cells were used as positive and negative controls, respectively.


Reference

References

1. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Embryonic stem cell lines derived from human blastocysts. Science. 1998; 282:1145–1147. DOI: 10.1126/science.282.5391.1145. PMID: 9804556.
Article
2. Vogel G. Cell biology. Ready or not? Human ES cells head toward the clinic. Science. 2005; 308:1534–1538. DOI: 10.1126/science.308.5728.1534. PMID: 15947149.
3. Hentze H, Graichen R, Colman A. Cell therapy and the safety of embryonic stem cell-derived grafts. Trends Biotechnol. 2007; 25:24–32. DOI: 10.1016/j.tibtech.2006.10.010.
Article
4. Zhang SC, Wernig M, Duncan ID, Brüstle O, Thomson JA. In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat Biotechnol. 2001; 19:1129–1133. DOI: 10.1038/nbt1201-1129. PMID: 11731781.
Article
5. Laflamme MA, Gold J, Xu C, Hassanipour M, Rosler E, Police S, Muskheli V, Murry CE. Formation of human myocardium in the rat heart from human embryonic stem cells. Am J Pathol. 2005; 167:663–671. DOI: 10.1016/S0002-9440(10)62041-X. PMID: 16127147. PMCID: 1698736.
Article
6. Brederlau A, Correia AS, Anisimov SV, Elmi M, Paul G, Roybon L, Morizane A, Bergquist F, Riebe I, Nannmark U, Carta M, Hanse E, Takahashi J, Sasai Y, Funa K, Brundin P, Eriksson PS, Li JY. Transplantation of human embryonic stem cell-derived cells to a rat model of Parkinson’s disease: effect of in vitro differentiation on graft survival and teratoma formation. Stem Cells. 2006; 24:1433–1440. DOI: 10.1634/stemcells.2005-0393. PMID: 16556709.
Article
7. Roy NS, Cleren C, Singh SK, Yang L, Beal MF, Goldman SA. Functional engraftment of human ES cell-derived dopaminergic neurons enriched by coculture with telomerase-immortalized midbrain astrocytes. Nat Med. 2006; 12:1259–1268. DOI: 10.1038/nm1495. PMID: 17057709.
Article
8. Doi D, Morizane A, Kikuchi T, Onoe H, Hayashi T, Kawasaki T, Motono M, Sasai Y, Saiki H, Gomi M, Yoshikawa T, Hayashi H, Shinoyama M, Refaat MM, Suemori H, Miyamoto S, Takahashi J. Prolonged maturation culture favors a reduction in the tumorigenicity and the dopaminergic function of human ESC-derived neural cells in a primate model of Parkinson’s disease. Stem Cells. 2012; 30:935–945. DOI: 10.1002/stem.1060. PMID: 22328536.
Article
9. Seminatore C, Polentes J, Ellman D, Kozubenko N, Itier V, Tine S, Tritschler L, Brenot M, Guidou E, Blondeau J, Lhuillier M, Bugi A, Aubry L, Jendelova P, Sykova E, Perrier AL, Finsen B, Onteniente B. The postischemic environment differentially impacts teratoma or tumor formation after transplantation of human embryonic stem cell-derived neural progenitors. Stroke. 2010; 41:153–159. DOI: 10.1161/STROKEAHA.109.563015.
Article
10. Choi HS, Kim H, Won A, Kim JJ, Son CY, Kim KS, Ko JH, Lee MY, Kim CH, Ryu CJ. Development of a decoy immunization strategy to identify cell-surface molecules expressed on undifferentiated human embryonic stem cells. Cell Tissue Res. 2008; 333:197–206. DOI: 10.1007/s00441-008-0632-6. PMID: 18560898.
Article
11. Choi HS, Kim WT, Kim H, Kim JJ, Ko JY, Lee SW, Jang YJ, Kim SJ, Lee MJ, Jung HS, Kzhyshkowska J, Um SJ, Lee MY, Lee SH, Kim CH, Ryu CJ. Identification and characterization of adenovirus early region 1B-associated protein 5 as a surface marker on undifferentiated human embryonic stem cells. Stem Cells Dev. 2011; 20:609–620. DOI: 10.1089/scd.2010.0265.
Article
12. Gabler S, Schütt H, Groitl P, Wolf H, Shenk T, Dobner T. E1B 55-kilodalton-associated protein: a cellular protein with RNA-binding activity implicated in nucleocytoplasmic transport of adenovirus and cellular mRNAs. J Virol. 1998; 72:7960–7971. PMID: 9733834. PMCID: 110131.
Article
13. Kim WT, Seo Choi H, Min Lee H, Jang YJ, Ryu CJ. B-cell receptor-associated protein 31 regulates human embryonic stem cell adhesion, stemness, and survival via control of epithelial cell adhesion molecule. Stem Cells. 2014; 32:2626–2641. DOI: 10.1002/stem.1765. PMID: 24898727.
Article
14. Kim WT, Choi HS, Hwang HJ, Jung HS, Ryu CJ. Epitope mapping of antibodies suggests the novel membrane topology of B-cell receptor associated protein 31 on the cell surface of embryonic stem cells: the novel membrane topology of BAP31. PLoS One. 2015; 10:e0130670. DOI: 10.1371/journal.pone.0130670. PMID: 26102500. PMCID: 4478030.
Article
15. Choi HS, Lee HM, Jang YJ, Kim CH, Ryu CJ. Heterogeneous nuclear ribonucleoprotein A2/B1 regulates the self-renewal and pluripotency of human embryonic stem cells via the control of the G1/S transition. Stem Cells. 2013; 31:2647–2658. DOI: 10.1002/stem.1366. PMID: 23495120.
Article
16. Hentze H, Soong PL, Wang ST, Phillips BW, Putti TC, Dunn NR. Teratoma formation by human embryonic stem cells: evaluation of essential parameters for future safety studies. Stem Cell Res. 2009; 2:198–210. DOI: 10.1016/j.scr.2009.02.002. PMID: 19393593.
Article
17. Choi HS, Lee HM, Kim WT, Kim MK, Chang HJ, Lee HR, Joh JW, Kim DS, Ryu CJ. Detection of mycoplasma infection in circulating tumor cells in patients with hepatocellular carcinoma. Biochem Biophys Res Commun. 2014; 446:620–625. DOI: 10.1016/j.bbrc.2014.03.024. PMID: 24637212.
Article
18. Choo AB, Tan HL, Ang SN, Fong WJ, Chin A, Lo J, Zheng L, Hentze H, Philp RJ, Oh SK, Yap M. Selection against undifferentiated human embryonic stem cells by a cytotoxic antibody recognizing podocalyxin-like protein-1. Stem Cells. 2008; 26:1454–1463. DOI: 10.1634/stemcells.2007-0576. PMID: 18356574.
Article
19. Watanabe K, Ueno M, Kamiya D, Nishiyama A, Matsumura M, Wataya T, Takahashi JB, Nishikawa S, Muguruma K, Sasai Y. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat Biotechnol. 2007; 25:681–686. DOI: 10.1038/nbt1310. PMID: 17529971.
Article
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