Di Cui

Macrophages are pivotal effectors of host immunity and regulators of tissue homeostasis. Understanding of human macrophage biology has been hampered by the lack of reliable and scalable models for cellular and genetic studies. Human induced pluripotent stem cell (hiPSC)-derived monocytes and macrophages, as an unlimited source of subject genotype-specific cells, will undoubtedly play an important role in advancing our understanding of macrophage biology and implication in human diseases. In this study, we present a fully optimized differentiation protocol of hiPSC-derived monocytes and granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF). We present characterization of iPSC-derived myeloid lineage cells at phenotypic, functional, and transcriptomic levels, in comparison with corresponding subsets of peripheral blood-derived cells. We also highlight the application of hiPSC-derived monocytes and macrophages as a gene-editing platform for functional validation in research and drug screening, and the study also provides a reference for cell therapies.

Dnmt3a2, a de novo DNA methyltransferase, is induced by neuronal activity and participates in long-term memory formation with the increased expression of synaptic plasticity genes. We wanted to determine if Dnmt3a2 with its partner Dnmt3L may influence motor behavior via the dopaminergic system. To this end, we generated a mouse line, Dnmt3a2/3L^Dat/wt, with dopamine transporter (DAT) promotor driven Dnmt3a2/3L overexpression. The mice were studied with behavioral paradigms (e.g., cylinder test, open field, and treadmill), brain slice patch clamp recordings, ex vivo metabolite analysis, and in vivo positron emission tomography (PET) using the dopaminergic tracer 6-[¹⁸F]FMT. The results showed that spontaneous activity and exercise performance were enhanced in Dnmt3a2/3L^Dat/wt mice compared to Dnmt3a2/3L^wt/wt controls. Dopaminergic substantia nigra pars compacta neurons of Dnmt3a2/3L^Dat/wt animals displayed a higher fire frequency and excitability. However, dopamine concentration was not increased in the striatum, and dopamine metabolite concentration was even significantly decreased. Striatal 6-[¹⁸F]FMT uptake, reflecting aromatic L-amino acid decarboxylase activity, was the same in Dnmt3a2/3L^Dat/wt mice and controls. [¹⁸F]FDG PET showed that hypothalamic metabolic activity was tightly linked to motor behavior in Dnmt3a2/3L^Dat/wt mice. Furthermore, dopamine biosynthesis and motor-related metabolic activity were correlated in the hypothalamus. Our findings suggest that Dnmt3a2/3L, when overexpressed in dopaminergic neurons, modulates motor performance via activation of the nigrostriatal pathway. This does not involve increased dopamine synthesis.

Circular RNAs (circRNAs) are an emerging class of RNA molecules that have been linked to human diseases and important regulatory pathways. Their functional roles are still under investigation, often hampered by inefficient circRNA formation in and ex vivo. We generated an intron-mediated enhancement (IME) system that—in comparison to previously published methods—increases circRNA formation up to 5-fold. This strategy also revealed previously undetected translation of circRNA, e.g., circRtn4. Substantiated by Western blots and mass spectrometry we showed that in mammalian cells, translation of circRtn4 containing a potential “infinite” circular reading frame resulted in “monomers” and extended proteins, presumably “multimer” tandem repeats. In order to achieve high levels of circRNA formation and translation of other natural or recombinant circRNAs, we constructed a versatile circRNA expression vector—pCircRNA-DMo. We demonstrated the general applicability of this method by efficiently generating two additional circRNAs exhibiting high expression levels. The circRNA expression vector will be an important tool to investigate different aspects of circRNA biogenesis and to gain insights into mechanisms of circular RNA translation.

Ageing, a leading cause of the decline/deficits in human learning, memory, and cognitive abilities, is a major risk factor for age-associated neurodegenerative disorders such as Alzheimer’s disease. Emerging evidence suggests that epigenetics, an inheritable but reversible biochemical process, plays a crucial role in the pathogenesis of age-related neurological disorders. DNA methylation, the best-known epigenetic mark, has attracted most attention in this regard. DNA methyltransferases (DNMTs) are key enzymes in mediating the DNA methylation process, by which a methyl group is transferred, faithfully or anew, to genomic DNA sequences. Biologically, DNMTs are important for gene imprinting. Accumulating evidence suggests that DNMTs not only play critical roles, including gene imprinting and transcription regulation, in early development stages of the central nervous system (CNS), but also are indispensable in adult learning, memory, and cognition. Therefore, the impact of DNMTs and DNA methylation on age-associated cognitive functions and neurodegenerative diseases has emerged as a pivotal topic in the field. In this review, the effects of each DNMT on CNS development and healthy and pathological ageing are discussed.

Human embryonic stem cells (hESCs) are thought to be a promising resource for cell therapy, while it has to face the major problem of graft immunological rejection. Major histocompatibility complex (MHC) class I expressed on the cell surface is the major cause of graft rejection. Transporter associated with antigen presentation 1 (TAP1) and TAP-associated glycoprotein (TAPBP) play important roles in regulating MHC class I expression. In this study, we generated TAP1- and TAPBP-deficient hESC lines, respectively, using transcription activator-like effector nucleases technique. These cells showed deficient expression of MHC class I on the cell surface and reduced immunogenicity compared with wild types, but maintained normal pluripotency, karyotypes, and differentiation ability. Thus, our findings are instrumental in developing a universal cell resource with both pluripotency and hypo-immunogenicity for transplantation therapy in the future.

Theoretically human embryonic stem cells (hESCs) have the capacity to self-renew and differentiate into all human cell types. Therefore, the greatest promise of hESCs-based therapy is to replace the damaged tissues of patients suffering from traumatic or degenerative diseases by the exact same type of cells derived from hESCs. Allograft immune rejection is one of the obstacles for hESCs-based clinical applications. Human leukocyte antigen (HLA) II leads to CD4+ T cells-mediated allograft rejection. Hence, we focus on optimizing hESCs for clinic application through gene modification. Transcription activator-like effector nucleases (TALENs) were used to target MHC class II transactivator (CIITA) in hESCs efficiently. CIITA −/− hESCs did not show any difference in the differentiation potential and self-renewal capacity. Dendritic cells (DCs) derived from CIITA −/− hESCs expressed CD83 and CD86 but without the constitutive HLA II. Fibroblasts derived from CIITA −/− hESCs were powerless in IFN-γ inducible expression of HLA II. We generated HLA II defected hESCs via deleting CIITA, a master regulator of constitutive and IFN-γ inducible expression of HLA II genes. CIITA −/− hESCs can differentiate into tissue cells with non-HLA II expression. It’s promising that CIITA −/− hESCs-derived cells could be used in cell therapy (e.g., T cells and DCs) and escape the attack of receptors’ CD4+ T cells, which are the main effector cells of cellular immunity in allograft.