Engineering of a Potent Recombinant Lectin-Toxin Fusion Protein to Eliminate Human Pluripotent Stem Cells

Abstract

The use of human pluripotent stem cells (hPSCs) such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) in regenerative medicine is hindered by their tumorigenic potential. Previously, we developed a recombinant lectin-toxin fusion protein of the hPSC-specific lectin rBC2LCN, which has a 23 kDa catalytic domain (domain III) of Pseudomonas aeruginosa exotoxin A (rBC2LCN-PE23). This fusion protein could selectively eliminate hPSCs following its addition to the cell culture medium. Here we conjugated rBC2LCN lectin with a 38 kDa domain of exotoxin A containing domains Ib and II in addition to domain III (PE38). The developed rBC2LCN-PE38 fusion protein could eliminate 50% of 201B7 hPSCs at a concentration of 0.003 μg/mL (24 h incubation), representing an approximately 556-fold higher activity than rBC2LCN-PE23. Little or no effect on human fibroblasts, human mesenchymal stem cells, and hiPSC-derived hepatocytes was observed at concentrations lower than 1 μg/mL. Finally, we demonstrate that rBC2LCN-PE38 selectively eliminates hiPSCs from a mixed culture of hiPSCs and hiPSC-derived hepatocytes. Since rBC2LCN-PE38 can be prepared from soluble fractions of E. coli culture at a yield of 9 mg/L, rBC2LCN-PE38 represents a practical reagent to remove human pluripotent stem cells residing in cultured cells destined for transplantation.

Lectin drug conjugate therapy for colorectal cancer

“…The IC 50 value for HT‐29 was 4888 pg/mL, supporting the low binding results we identified earlier. The SUIT‐2 cells, which we have previously shown to not bind rBC2LCN, 6 had an IC 50 with rBC2LCN‐PE38 of 93 161 pg/mL.…”

“…It was isolated from the gram‐negative bacterium Burkholderia cenocepacia 5 . Previously, we conjugated the rBC2LCN lectin with the 38‐kDa domain of pseudomonas exotoxin A containing domains Ib, II, and III (PE38) chemotherapeutic 6 . We showed that this rBC2LCN‐PE38 fusion protein was both safe and effective in treating pancreatic cancer in mouse models.…”

Simple derivation of skeletal muscle from human pluripotent stem cells using temperature‐sensitive Sendai virus vector

“…However, the differentiation efficiency remained around 20% due to the residual human ESCs, which had not to selectively remove undifferentiated pluripotent-state cells. 19,20 At first, we evaluated the positivity of MHC and rBC2LCN (Figure 4B)…”

Recent Updates on Induced Pluripotent Stem Cells in Hematological Disorders

“…Elimination of these unwanted pluripotent cells is crucial prior to use in the clinical setting. Approaches including positive selection of differentiated cells using specific surface markers [155], selective elimination of residual undifferentiated cells using compounds [156–159] or selective media [160, 161], and engineered safety switches such as inducible suicide genes in undifferentiated cells [162, 163], suicide-inducing virus-like particles [164], lectin-toxin fusion protein [165], or microRNA-302 switch [166] could be performed to minimize the risk of tumor formation. More detailed approaches have been extensively reviewed in Jeong et al [167].…”