Summary Cytokine-activated STAT proteins dimerize and bind to high-affinity motifs, and N-terminal domain-mediated oligomerization of dimers allows tetramer formation and binding to low-affinity tandem motifs, but the functions of dimers versus tetramers are unknown. We generated Stat5a-Stat5b double knock-in (DKI) N-domain mutant mice that form dimers but not tetramers, identified cytokine-regulated genes whose expression required STAT5 tetramers, and defined dimer versus tetramer consensus motifs. Whereas Stat5- deficient mice exhibited perinatal lethality, DKI mice were viable; thus, STAT5 dimers were sufficient for survival. Nevertheless, STAT5 DKI mice had fewer CD4+CD25+ T cells, NK cells, and CD8+ T cells, with impaired cytokine-induced and homeostatic proliferation of CD8+ T cells. DKI CD8+ T cell proliferation following viral infection was diminished and DKI Treg cells did not efficiently control colitis. Thus, tetramerization of STAT5 is dispensable for survival but is critical for cytokine responses and normal immune function, establishing a critical role for tetramerization in vivo.

There has been considerable progress in engineering cardiac scaffolds for the treatment of myocardial infarction (MI). However, it is still challenging to replicate the structural specificity and variability of cardiac tissues using traditional bioengineering approaches. In this study, a four-dimensional (4D) cardiac patch with physiological adaptability has been printed by beam-scanning stereolithography. By combining a unique 4D self-morphing capacity with expandable microstructure, the specific design has been shown to improve both the biomechanical properties of the patches themselves and the dynamic integration of the patch with the beating heart. Our results demonstrate improved vascularization and cardiomyocyte maturation in vitro under physiologically relevant mechanical stimulation, as well as increased cell engraftment and vascular supply in a murine chronic MI model. This work not only potentially provides an effective treatment method for MI but also contributes a cutting-edge methodology to enhance the structural design of complex tissues for organ regeneration.

Targeted genome engineering to robustly express transgenes is an essential methodology for stem cell-based research and therapy. Although designer nucleases have been used to drastically enhance gene editing efficiency, targeted addition and stable expression of transgenes to date is limited at single gene/locus and mostly PPP1R12C/AAVS1 in human stem cells. Here we constructed transcription activator-like effector nucleases (TALENs) targeting the safe-harbor like gene CLYBL to mediate reporter gene integration at 38%–58% efficiency, and used both AAVS1-TALENs and CLYBL-TALENs to simultaneously knock-in multiple reporter genes at dual safe-harbor loci in human induced pluripotent stem cells (iPSCs) and neural stem cells (NSCs). The CLYBL-TALEN engineered cell lines maintained robust reporter expression during self-renewal and differentiation, and revealed that CLYBL targeting resulted in stronger transgene expression and less perturbation on local gene expression than PPP1R12C/AAVS1. TALEN-mediated CLYBL engineering provides improved transgene expression and options for multiple genetic modification in human stem cells.