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The thymus is critical for the establishment of a functional and self-tolerant adaptive immune system, but it involutes with age, resulting in reduced naive T-cell output. Generation of a functional human thymus from human pluripotent stem cells (hPSCs) is an attractive regenerative medicine strategy. Direct differentiation of thymic epithelial progenitors (TEPs) from hPSCs has been demonstrated in vitro, but functional thymic epithelial cells (TECs) develop only after transplantation of TEPs in vivo. Functional human reaggregated thymic organoid cultures (RTOCs) and artificial thymic organoids (ATOs) cultured at the air–liquid interface support T-cell development in vitro and in vivo and permit the interrogation of human thymic function and T-cell development. However, these approaches require access to primary human tissues or murine bone marrow stromal cells, are allogeneic, and do not support negative selection. Recently, we reported the directed differentiation of induced PSCs (iPSCs) to functional thymic epithelial progenitors (TEPs) that support murine T-cell development after transplantation in nude mice. Here, we combined hPSC-derived TEPs, hematopoietic progenitor cells (HPCs), and mesenchymal cells, differentiated from the same hPSC line, and generated functional isogenic stem cell–derived thymic organoids (sTOs). Our revised protocol improves our TEP differentiation process and allows the generation of functional isogenic, patient-specific thymic organoids in vitro.

Heterozygous variants in the KAT6A gene encoding the histone lysine acetyltransferase KAT6A (MOZ, MYST3) cause Arboleda-Tham syndrome, a cognitive impairment syndrome. Histone acetylation is generally associated with active gene transcription. Genetic deletion of both alleles of the Kat6a gene in mice causes developmental defects including anterior homeotic transformation, cleft palate, interrupted aortic arch and cardiac septal defects. Loss of KAT6A impairs expression of HOX, DLX and TBX genes, which are essential for body segment identity specification, palate, heart and aortic arch development. However, the effects of loss of KAT6A on chromatin modifications and gene expression in neural cells, which are relevant to normal brain development and function, is still poorly understood. In this study, we used an automated high-throughput chromatin profiling method and RNA sequencing in mouse neural system and progenitor cells to assess the effects of loss of one or two alleles of Kat6a on gene expression, histone acetylation and methylation. We also assessed occupancy by a trithorax group protein and RNA polymerase II. Our data suggests two modes of action for KAT6A: (1) acetylation of histone H3 on lysine 23 at promoters and enhancers and (2) recruitment of the trithorax group protein MLL1 (KMT2A) to promote the expression of developmental genes, including SOX and homeodomain genes. Together, these two functions appear to be required for normal gene expression in neural progenitors and essential for proliferation and neuronal differentiation. Author summaryDuring embryonic development, specific families of transcription factors pattern the early embryo to lay down and define the body and organ structure. However, the mechanisms governing the onset of the expression of these developmental transcription factors in less well understood. KAT6A is thought to promote gene expression by acetylation histone proteins. Here we determine the effects of KAT6A on histone acetylation and gene expression in mouse neural stem and progenitor cells. Our data are relevant for the understanding of pathogenic genetic variants in one allele of the human KAT6A gene, which cause the Arboleda-Tham cognitive impairment syndrome.

Introduction:Human induced pluripotent stem cells (hiPSCs) can be rapidly converted into neurons via Neurogenin-2 (NGN2) overexpression, but many protocols require costly reagents during the initial induction phase that may limit adoption by labs without routine neuronal differentiation experience. We developed a simplified, low-cost protocol using a tetracycline-inducible (TET-on) NGN2 system in minimal media to generate cortical neurons in as few as 6 days.Methods:KOLF2.1J hiPSCs were stably transfected with a TET-on NGN2 cassette using the nonviral PiggyBac system and induced with doxycycline in Essential 6 media with or without the Notch inhibitor DAPT. Neurogenesis was evaluated with immunocytochemistry (ICC) and qRT-PCR, and cultures matured in defined conditions were characterized by multielectrode array (MEA) recordings to assess functional maturation.Results:DAPT markedly improved hiPSC-to-neuron conversion efficiency, and yielded glutamatergic neurons expressing cortical markers. MEA recordings showed spontaneous activity by day 14 and synchronous network firing by day 35. Secondary PB transfection enabled Td-Tomato labelling of KOLF2.1J:pB-TO-NGN2 hiPSCs, allowing 24-hour live imaging of neurite outgrowth.Conclusion:This streamlined, growth-factor-free workflow provides an accessible route for generating functional neurons from patient-derived hiPSCs, including in labs with limited hiPSC or neuronal culture experience.