Currently, clinical trials of regenerative medicine using human iPSC-derived retinal pigment epitheliums are about to start in ophthalmic field, and people worldwide are greatly expecting. In cardiac regenerative medicine using human
iPSCs, the hundreds of millions of cardiomyocytes will be needed. Thus, the risk of tumorigenesis due to contamination of residual undifferentiated stem cells cannot be neglected. Because of lethal complications, elimination of residual undifferentiated stem cells must be necessary.
Metabolomics is one of the most powerful tools for characterizing cell-specific metabolism. By understanding the global intracellular metabolism in cardiomyocytes and non-cardiac cells including undifferentiated stem cells, we developed a novel method for purifying the bulk of iPSC-derived cardiomyocytes using original culture media (glucosefree and lactate-rich environment). We believe that our inexpensive and unique technology will resolve the bottleneck and directly facilitate human cardiac regenerative therapies in the future. Recently, other groups have also reported the methods in elimination of undifferentiated stem cells, and I would like to introduce these methods and current situations in cardiac regenerative medicine.
Human induced pluripotent stem cells (hiPSCs) possess the capabilities of self-renewal and differentiation into multiple cell types, and they are free of the ethical problems associated with human embryonic stem cells. Currently, a lot of attempts are made to develop hiPSC-derived therapeutic products for regenerative medicine/cell therapy. There are significant obstacles, however, preventing the clinical use of hiPSC-derived products. One of the most obvious safety issues is the presence of residual undifferentiated cells that have tumorigenic potential. We recently characterized three in vitro assay methods for sensitive detection of tumorigenic/undifferentiated cells in hiPSC-derived products, i.e. soft agar colony formation assay, flow cytometry assay and quantitative real-time polymerase chain reaction assay
(qRT-PCR). The soft agar colony formation assay appeared to be unable to detect hiPSCs, presumably attributable to dissociation-induced apoptosis, a unique property of human pluripotent stem cells. The flow cytometry assay using anti-TRA-1-60 antibody detected 0.1% undifferentiated hiPSCs that were spiked in primary retinal pigment epithelial (RPE) cells. Moreover, qRT-PCR with a specific probe and primers was found to detect a trace amount of LIN28 mRNA, which is equivalent to that present in a mixture of a single hiPSC and 5.0×104 RPE cells. Our findings provide highly sensitive and quantitative in vitro assays essential for facilitating safety/quality profiling of hiPSC-derived RPE cells for their clinical use.
In the case of the regeneration treatment of human iPS cells (hiPSCs), the tumors may form in the transplanted tissue. It is thought that the tumors are caused by undifferentiated cells to remain in the transplanted cells and result in various problems. There are at least three methods before transplantation to overcome the tumor formation. 1. Transplanting the established cells with complete differentiation and no tumor formation, 2. Sorting and purified cells after differentiation induction, and 3. Removing unrequired cells after differentiation induction. We examined the residual undifferentiated cells in differentiation induction hiPSCs and investigated the effects of irradiation (IR), hyperthermia (HT) and drugs to the transplanted cells for preventing tumors. In differentiated induced cells treated with IR, HT and drugs, remaining undifferentiated cells were decreased. Fibroblasts and vascular endothelial cells were not affected and inhibition of cardiac muscle beating did not appear macroscopically after these treatments. Differentiated cells treated with these preventing methods were transplanted to SCID mice and the teratomatous formation was significantly decreased by IR. By flow cytometer, sorted cells after differentiation induction have some problems, which need rebuilding tissue from dissociated differentiated cells. Recently, it was reported that the hiPS lines have aberrant gene expressions and defective potential in neural differentiation, which need to be identified and eliminate before transplantation. When the high quality hiPSC lines are selected for differentiation and applied with IR, HT and drugs, residual undifferentiated cells are more eliminated. The combination with both the good hiPSCs and preventing methods would strongly inhibit post transplantation tumorigenesis.
Although c-kit expression on the murine hematopoieticstem cells (HSCs) is essential for the survival of HSCs and maintenance of hematopoiesis, the significance of the high level of expression of c-kit on HSCs has not been well determined. We show here that the intensity of c-kit expression correlates with the cycling status of the population. The HSCs expressinga lower level of c-kit on the cell surface also possess the lower level inside the cells, which cannot be explained by ligand receptor binding and internalization. And therefore it is assumed that the c-kit highly and modestly expressing HSCs are qualitatively different. In accordance with this, HSCs with low c-kit expression have higher p21
and p57 mRNA concentration and are slow to respond to cytokine stimulation. Despite the functional differences in the two populations, both HSCs are uniformly hypoxic and modestly accessible to blood perfusion.Therefore it is suggested that the two population reside in a similar niche environment.The cell fraction with very weak expression of c-kit obtains the ability to long-term reconstitute hematopoiesis, and to generate HSC population with high expression of c-kit, when transplanted in irradiated recipients.