天然有機化合物討論会講演要旨集
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Chemical Approaches to Selective Elimination of Human Pluripotent Stem Cells
Di MaoYing QinTing-Fang Kuo川瀬 栄八郎佐藤 慎一平田 直中辻 憲夫上杉 志成
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Human induced pluripotent stem cells (hiPSCs) serve as highly valuable resource both for basic research and regeneration therapy. Despite the advantages, however, there are still a number of problems that limit the clinical application. One of them is a tumorigenic risk of undifferentiated cells during transplantation. For safer transplantation, it is necessary to eliminate the undifferentiated cells selectively.

We previously discovered Kyoto probe 1(KP-1), a fluorescent molecule that selectively labels hiPSCs. Mechanistic study showed that its selectivity results primarily from a distinct expression pattern of ABC transporters in human pluripotent stem cells and from the transporter selectivity of KP-1. Expression of ABCB1 (MDR1) and ABCG2 (BCRP), both of which cause the efflux of KP-1, is repressed in human pluripotent stem cells1 (Figure 1).

The different expression levels of ABCB1 and ABCG2 on hiPS cell and differentiated cell membrane allowed us to develop the strategies for selective elimination of hiPSCs. In one approach, we screened a chemical library of 333 anticancer drugs, cytotoxic natural products, and their derivatives and found an okadaic acid derivative (molecule 1) to be a substrate both for ABCB1 and ABCG2. This synthetic derivative of okadaic acid selectively eliminated hiPSCs from cell mixtures2 (Figure 2). For example, we compared the IC50 values of molecule 1 in hiPSCs, human somatic primary cells, and human cancer cells. Molecule 1 was highly toxic to hiPSCs, with an IC50 value of 0.78 μM, even lower than the IC50 value of astrocytes (2.1 μM), a neuronal cell lineage known to express low levels of ABC transporters. In contrast, cells primarily involved in secretory functions (adrenal gland), metabolic functions (liver), barrier functions (bronchia), and reproductive organs (prostate) tend to express higher levels of ABC transporters and exhibited greater resistance to molecule 1 (IC50 > 5 μM).

Another approach to developing molecules that eliminate hiPSCs is direct conjugation of KP-1 with cytotoxic anticancer drugs (Figure 3), hoping that the conjugation maintains the KP-1’s selectivity. To seek potential conjugation sites in KP-1, we carried out structure activity-relationship (SAR) studies. Fifteen KP-1 analogs were synthesized and estimated for their selectivity for ABCB1, ABCG2, and ABCC1 in cultured cells. The ABC transporter selectivity of KP-1 was tolerated with the alkylation at one of the exocyclic amines, which was subsequently selected as a conjugation site. Initially, combretastatin A-4 (CA4), a cytotoxic natural product that is not a substrate of any of the ABC transporters, was selected for conjugation with KP-1. Unfortunately the conjugate (conjugate 1) failed to maintain its selectivity for ABCG2, although the ABCB1 selectivity was intact.

To recover the selectivity for ABCG2, we next tested ABCG2 selective cytotoxic natural products: SN38 and Mitoxantrone. Their conjugates with KP-1 (conjugate 2 and 3) exhibited selectivity both for ABCB1 and ABCG2. The selectivity of these two conjugates was evaluated with partially differentiated human iPS cells. Just as KP-1 does, conjugate 2 and 3 fluorescently labeled undifferentiated cells significantly stronger than differentiated cells. Longer incubation led to selective elimination of hiPSCs from the cell mixtures. Alkaline Phosphatase (ALP), a marker of pluripotency, was utilized for detection of hiPSCs. Treatment with conjugate 2 or 3 eliminated ALP-positive hiPS cells from the cell mixtures, while the SNL feeder cells and differentiated cells had little effects.

The present study offers two chemical approaches to selective elimination of pluripotent stem cells for safer transplantation. The results

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