Selective Elimination of Human Induced Pluripotent Stem Cells Using Medium with High Concentration of L-Alanine

Abstract

Human pluripotent stem cells, including human induced pluripotent stem cells (hiPSCs), serve as highly valuable sources for both cell-based therapies and basic research, owing to their abilities to self-renew and differentiate into any cell type of the human body. However, tumorigenic risks of residual undifferentiated stem cells limit the clinical application of hiPSCs, necessitating methods to eliminate undifferentiated hiPSCs from differentiated cells. Here, we found that undifferentiated hiPSCs were more sensitive to the treatment with a medium supplemented with high concentration of L-alanine than human fibroblasts (hFBs), human skeletal muscle cells (hSkMCs), hiPSC-derived cardiomyocytes (iCMs) or hiPSC-derived fibroblast-like cells (iFLCs), which were used as differentiated cells. Undifferentiated hiPSCs co-cultured with differentiated cells were selectively eliminated following treatment. In addition, we found that the medium supplemented with high concentration of D-alanine or β-alanine also induced cell death of hiPSCs and the treatment at 4 °C didn’t induce cell death of hiPSCs. The cell death induced would be associated partly with high osmotic pressure of the medium supplemented with L-alanine. As L-alanine is a component of proteins in human body and popular ingredient of cell culture media, treatment with high concentration of L-alanine may be useful for eliminating tumorigenic residual hiPSCs for stem cell-based therapies.

Evaluation of Residual Human-Induced Pluripotent Stem Cells in Human Chondrocytes by Cell Type-Specific Glycosphingolipid Glycome Analysis Based on the Aminolysis-SALSA Technique

“…Since Gropp et al reported that a teratoma can be established by only a few hundred iPSCs, it is a prerequisite that final products used in regenerative medicine do not contain residual iPSCs when using iPSC-derived cells [17]. To overcome this problem, several strategies have been reported that promote the selective removal of residual iPSCs from a population of differentiated cells, such as the introduction of suicide genes into iPSCs [18], alteration of cell culture conditions [6], and cell sorting using antibodies against cell surface antigens [8]. In these reports, detection procedures for undifferentiated cells included fluorescent labeling, flow cytometry, and methods for confirming teratoma formation in vivo.…”

Compartmentalized three-dimensional human neuromuscular tissue models fabricated on a well-plate-format microdevice

“…The fabricated MN chamber was bonded onto the chamber for SkM tissues (27) The completed microdevices were exposed to UV light for at least 2 h for sterilization before use for the coculture. (31). hiPSCs were maintained with Stemfit AK02N on an iMatrix-511 coating (0.5 µg/cm 2 ).…”

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].…”