Xiaoyan Ding

Ex vivo expansion of hematopoietic stem cells (HSCs) is limited by culture-induced oxidative stress, yet the upstream transcriptional drivers remain poorly defined. Here, we show that the transcription factor Bcl11a is rapidly and persistently downregulated during HSC culture and that this decline drives functional deterioration. Bcl11a was both required and sufficient to maintain quiescence, clonogenic activity, and serially transplantable HSC function. Mechanistically, Bcl11a directly represses a Nox2 NADPH oxidase gene module, including Ncf1, encoding the p47phox organizer subunit; Ncf1 deletion reduced ROS and partially rescued the defects of Bcl11a-deficient HSCs. Rapamycin—but not the ROS scavenger N-acetylcysteine—restored Bcl11a expression, suppressed Ncf1 and ROS, and improved HSC function, identifying Bcl11a as a candidate effector linking mechanistic target of rapamycin complex 1 inhibition to redox control. This axis was conserved in human hematopoietic stem and progenitor cells, revealing a druggable transcriptional vulnerability of cultured HSCs and a framework for improving ex vivo–expanded grafts.

Retinal pigment epithelial (RPE) cells derived from human embryonic stem cells (hESCs) are superior cell sources for cell replacement therapy in individuals with retinal degenerative diseases; however, studies on the stable and secure banking of these therapeutic cells are scarce. Highly variable cell viability and functional recovery of RPE cells after cryopreservation are the most commonly encountered issues. In the present protocol, we aimed to achieve the best cell recovery rate after thawing by selecting the optimal cell phase for freezing based on the original experimental conditions. Cells were frozen in the exponential phase determined by using the 5-ethynyl-2′-deoxyuridine labeling assay, which improved cell viability and recovery rate after thawing. Stable and functional cells were obtained shortly after thawing, independent of a long differentiation process. The methods described here allow the simple, efficient, and inexpensive preservation and thawing of hESC-derived RPE cells. Although this protocol focuses on RPE cells, this freezing strategy may be applied to many other types of differentiated cells.

Sijia Ji, Wanzhi Tu, Chenwen Huang, Ziyang Chen, Xinyue Ren, Bingqing He, Xiaoyan Ding, Yuelei Chen, and Xin Xie. Mol. Cells 2022;45:923-34. https://doi.org/10.14348/molcells.2022.0077

Dear Editor,

Epithelial-mesenchymal transition (EMT) is a physiological process that has been recognized to occur during the progression of an increasingly large number of human diseases, including liver fibrosis, cirrhosis, and hepatocellular carcinoma. The activation of transforming growth factor β (TGF-β) signaling is considered a critical event during EMT, and efforts have been made to screen small molecules that interfere with the TGF-β signaling pathway during EMT. Here we report the identification of sorafenib, a clinical agent that inhibits TGF-β signaling. When applied to AML12 cells and primary hepatocytes, sorafenib strikingly suppressed TGF-β1-induced EMT and apoptosis. Additionally, sorafenib inhibited TGF-β1-induced signal transducer and activator of transcription 3 phosphorylation. We further present in vitro evidence that sorafenib ameliorates the proapoptotic and profibrotic effects of TGF-β1 in mouse primary hepatocytes, suggesting that this drug exerts a protective effect on hepatocytes and has therapeutic potential for the treatment of liver fibrosis. (Hepatology 2011;)