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

• Kim WT
• Lee HM
• Kim MK
• Choi HS
• Ryu CJ

• KMID: 2362520
• DOI: http://doi.org/10.15283/ijsc16052

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

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