Seamless and non-destructive monitoring of extracellular microRNAs during cardiac differentiation from human pluripotent stem cells.

Monitoring cardiac differentiation and maturation from human pluripotent stem cells (hPSCs) and detecting residual undifferentiated hPSCs are indispensable for the development of cardiac regenerative therapy. MicroRNA (miRNA) is secreted from cells into the extracellular space, and its role as a biomarker is attracting attention. Here, we performed an miRNA array analysis of supernatants during the process of cardiac differentiation and maturation from hPSCs. We demonstrated that the quantification of extracellular miR-489-3p and miR-1/133a-3p levels enabled the monitoring of mesoderm and cardiac differentiation, respectively, even in clinical-grade mass culture systems. Moreover, extracellular let-7c-5p levels showed the greatest increase with cardiac maturation during long-term culture. We also verified that residual undifferentiated hPSCs in hPSC-derived cardiomyocytes (hPSC-CMs) were detectable by measuring miR-302b-3p expression, with a detection sensitivity of 0.01%. Collectively, we demonstrate that our method of seamlessly monitoring specific miRNAs secreted into the supernatant is non-destructive and effective for the quality evaluation of hPSC-CMs.

Purification of cardiomyocytes and neurons derived from human pluripotent stem cells by inhibition of de novo fatty acid synthesis.

Here we describe a protocol to obtain highly pure cardiomyocytes and neurons from human induced pluripotent stem cells (hiPSCs) via metabolic selection processes. Compared to conventional purification protocols, this approach is easier to perform and scale up and more cost-efficient. The protocol can be applied to hiPSCs and human embryonic stem cells. For complete details on the use and execution of this protocol, please refer to Tohyama et al. (2016) and Tanosaki et al. (2020).

Efficient Large-Scale 2D Culture System for Human Induced Pluripotent Stem Cells and Differentiated Cardiomyocytes.

Cardiac regenerative therapies utilizing human induced pluripotent stem cells (hiPSCs) are hampered by ineffective large-scale culture. hiPSCs were cultured in multilayer culture plates (CPs) with active gas ventilation (AGV), resulting in stable proliferation and pluripotency. Seeding of 1 × 106 hiPSCs per layer yielded 7.2 × 108 hiPSCs in 4-layer CPs and 1.7 × 109 hiPSCs in 10-layer CPs with pluripotency. hiPSCs were sequentially differentiated into cardiomyocytes (CMs) in a two-dimensional (2D) differentiation protocol. The efficiency of cardiac differentiation using 10-layer CPs with AGV was 66%-87%. Approximately 6.2-7.0 × 108 cells (4-layer) and 1.5-2.8 × 109 cells (10-layer) were obtained with AGV. After metabolic purification with glucose- and glutamine-depleted and lactate-supplemented media, a massive amount of purified CMs was prepared. Here, we present a scalable 2D culture system using multilayer CPs with AGV for hiPSC-derived CMs, which will facilitate clinical applications for severe heart failure in the near future.

Differential influence of proteolysis by calpain 2 and Lp82 on in vitro precipitation of mouse lens crystallins.

The purpose of the present study was to compare the susceptibility of crystallins proteolyzed by ubiquitous calpain 2 and by lens-specific calpain Lp82 to insolubilization. To test this, transgenic (TG) mice expressing a calpain 2, in which the active site cysteine 105 was mutated to alanine, were produced. Expression of mutated calpain 2 was driven in lens by coupling the mutated gene to the betaB1-crystallin promoter. Light scattering was measured in solutions of lens proteins after activation of endogenous calpain 2 and/or Lp82. Mass spectrometric analysis was performed to determine the cleavage sites and the calpain responsible for insolubilization of crystallins. Lens proteins from TG mice incubated in vitro with calcium showed higher light scattering compared to proteins from wild type (WT) mice. alphaA-crystallin from TG mice was proteolyzed by Lp82. In contrast, alphaA-crystallin in lenses from WT mice were proteolyzed by both calpain 2 and Lp82. These results suggested that Lp82-induced proteolysis of crystallins caused increased susceptibility of truncated crystallins to in vitro precipitation. Since Lp82 is highest in young animals, Lp82-induced proteolysis and precipitation may be one of the factors responsible for the cataract formation in young rodents.