ERRγ enhances cardiac maturation with T-tubule formation in human iPSC-derived cardiomyocytes.

One of the earliest maturation steps in cardiomyocytes (CMs) is the sarcomere protein isoform switch between TNNI1 and TNNI3 (fetal and neonatal/adult troponin I). Here, we generate human induced pluripotent stem cells (hiPSCs) carrying a TNNI1EmGFP and TNNI3mCherry double reporter to monitor and isolate mature sub-populations during cardiac differentiation. Extensive drug screening identifies two compounds, an estrogen-related receptor gamma (ERRγ) agonist and an S-phase kinase-associated protein 2 inhibitor, that enhances cardiac maturation and a significant change to TNNI3 expression. Expression, morphological, functional, and molecular analyses indicate that hiPSC-CMs treated with the ERRγ agonist show a larger cell size, longer sarcomere length, the presence of transverse tubules, and enhanced metabolic function and contractile and electrical properties. Here, we show that ERRγ-treated hiPSC-CMs have a mature cellular property consistent with neonatal CMs and are useful for disease modeling and regenerative medicine.

Efficient, Selective Removal of Human Pluripotent Stem Cells via Ecto-Alkaline Phosphatase-Mediated Aggregation of Synthetic Peptides.

The incomplete differentiation of human induced pluripotent stem cells (iPSCs) poses a serious safety risk owing to their potential tumorigenicity, hindering their clinical application. Here, we explored the potential of phospho-D-peptides as novel iPSC-eliminating agents. Alkaline phosphatases overexpressed on iPSCs dephosphorylate phospho-D-peptides into hydrophobic peptides that aggregate and induce cell death. We isolated a peptide candidate, D-3, that selectively and rapidly induced toxicity in iPSCs within 1 hr but had little influence on various non-iPSCs, including primary hepatocytes and iPSC-derived cardiomyocytes. Two hours of D-3 treatment efficiently eliminated iPSCs from both single cultures and co-cultures spiked with increasing ratios of iPSCs. In addition, D-3 prevented residual iPSC-induced teratoma formation in a mouse tumorigenicity assay. These results suggest the enormous potential of D-3 as a low-cost and effective anti-iPSC agent for both laboratory use and for the safe clinical application of iPSC-derived cells in regenerative medicine.

Epigenetic Variation between Human Induced Pluripotent Stem Cell Lines Is an Indicator of Differentiation Capacity.

Variation in the differentiation capacity of induced pluripotent stem cells (iPSCs) to specific lineages is a significant concern for their use in clinical applications and disease modeling. To identify factors that affect differentiation capacity, we performed integration analyses between hematopoietic differentiation performance and molecular signatures such as gene expression, DNA methylation, and chromatin status, using 35 human iPSC lines and four ESC lines. Our analyses revealed that hematopoietic commitment of PSCs to hematopoietic precursors correlates with IGF2 expression level, which in turn depends on signaling-dependent chromatin accessibility at mesendodermal genes. Maturation capacity for conversion of PSC-derived hematopoietic precursors to mature blood associates with the amount and pattern of DNA methylation acquired during reprogramming. Our study therefore provides insight into the molecular features that determine the differential capacities seen among human iPSC lines and, through the predictive potential of this information, highlights a way to select optimal iPSCs for clinical applications.

Enhanced engraftment, proliferation, and therapeutic potential in heart using optimized human iPSC-derived cardiomyocytes.

Human pluripotent stem cell-derived cardiomyocytes (CMs) are a promising tool for cardiac cell therapy. Although transplantation of induced pluripotent stem cell (iPSC)-derived CMs have been reported in several animal models, the treatment effect was limited, probably due to poor optimization of the injected cells. To optimize graft cells for cardiac reconstruction, we compared the engraftment efficiency of intramyocardially-injected undifferentiated-iPSCs, day 4 mesodermal cells, and day 8, day 20, and day 30 purified iPSC-CMs after initial differentiation by tracing the engraftment ratio (ER) using in vivo bioluminescence imaging. This analysis revealed the ER of day 20 CMs was significantly higher compared to other cells. Transplantation of day 20 CMs into the infarcted hearts of immunodeficient mice showed good engraftment, and echocardiography showed significant functional improvement by cell therapy. Moreover, the imaging signal and ratio of Ki67-positive CMs at 3 months post injection indicated engrafted CMs proliferated in the host heart. Although this graft growth reached a plateau at 3 months, histological analysis confirmed progressive maturation from 3 to 6 months. These results suggested that day 20 CMs had very high engraftment, proliferation, and therapeutic potential in host mouse hearts. They also demonstrate this model can be used to track the fate of transplanted cells over a long time.

Efficient Detection and Purification of Cell Populations Using Synthetic MicroRNA Switches.

Isolation of specific cell types, including pluripotent stem cell (PSC)-derived populations, is frequently accomplished using cell surface antigens expressed by the cells of interest. However, specific antigens for many cell types have not been identified, making their isolation difficult. Here, we describe an efficient method for purifying cells based on endogenous miRNA activity. We designed synthetic mRNAs encoding a fluorescent protein tagged with sequences targeted by miRNAs expressed by the cells of interest. These miRNA switches control their translation levels by sensing miRNA activities. Several miRNA switches (miR-1-, miR-208a-, and miR-499a-5p-switches) efficiently purified cardiomyocytes differentiated from human PSCs, and switches encoding the apoptosis inducer Bim enriched for cardiomyocytes without cell sorting. This approach is generally applicable, as miR-126-, miR-122-5p-, and miR-375-switches purified endothelial cells, hepatocytes, and insulin-producing cells differentiated from hPSCs, respectively. Thus, miRNA switches can purify cell populations for which other isolation strategies are unavailable.