Enrichment of xenograft-competent genetically modified pig cells using a targeted toxin, isolectin BS-I-B4 conjugate

Akasaka E, Watanabe S, Himaki T, Ohtsuka M, Yoshida M, Miyoshi K, Sato M. Enrichment of xenograft‐competent genetically modified pig cells using a targeted toxin, isolectin BS‐I‐B4 conjugate. Xenotransplantation 2010; 17: 81–89. © 2010 John Wiley & Sons A/S. : Background: The recent availability of α‐1,3‐galatosyltransferase knockout pigs has eliminated anti‐Gal antibodies to the galα1‐3gal (αgal epitope) as the major barrier to xenotransplantation. These αgal epitope‐negative animals can also be produced by somatic cell nuclear transfer of cells overexpressing endo‐β‐galactosidase (EndoGalC), an enzyme capable of digesting the αgal epitope. For this, selection of cells with highly reduced synthesis of αgal epitope is a prerequisite. In this study, we developed a novel method of selection using isolectin BS‐I‐B4‐conjugated saporin (IB4‐SAP), a targeted cytotoxin, that is specific for the terminal αgal epitope. Methods: A mixture of αgal epitope‐expressing and non‐expressing pig cells was obtained by transfection with an EndoGalC expression vector. These cells were incubated with a solution containing IB4‐SAP for 2 h at 37 °C, and subsequently cultivated for over 2 months under general conditions. Results: Almost all (98%) of surviving cells were completely negative for expression of αgal epitope, as confirmed by cytochemical staining using fluorescence‐labeled IB4. FACS analysis also confirmed that the IB4‐SAP‐treated cells exhibited a staining pattern similar to that of the IB4‐negative human cells. Extended cultivation (more than 6 months) of these IB4‐SAP‐treated cells did not alter the above staining pattern. RT‐PCR analysis revealed the presence of EndoGalC mRNA in these cells. Conclusions: This IB4‐SAP‐mediated method of selection of αgal epitope‐negative cells will provide an alternative to the present method of cytotoxicity‐based selection using specific antibody and complement.