Interleukin (IL)-6 is a pleiotropic cytokine involved in the regulation of hematological and immune responses. IL-6 is secreted chiefly by stromal cells, but little is known about its precise role in the homeostasis of human mesenchymal stromal cells (hMSCs) and the role it may play in hMSC-mediated immunoregulation. We studied the role of IL-6 in the biology of bone marrow derived hMSC in vitro by silencing its expression using short hairpin RNA targeting. Our results show that IL-6 is involved in immunosuppression triggered by hMSCs. Cells silenced for IL-6 showed a reduced capacity to suppress activated T-cell proliferation. Moreover, silencing of IL-6 significantly blocked the capacity of hMSCs to proliferate. Notably, increasing the intracellular level of IL-6 but not recovering the extracellular level could restore the proliferative impairment observed in IL-6-silenced hMSC. Our data indicate that IL-6 signals in hMSCs by a previously undescribed intracellular mechanism.

SummaryIn vertebrates, GATA2 is a master regulator of hematopoiesis and is expressed throughout embryo development and in adult life. Although the essential role of GATA2 in mouse hematopoiesis is well established, its involvement during early human hematopoietic development is not clear. By combining time-controlled overexpression of GATA2 with genetic knockout experiments, we found that GATA2, at the mesoderm specification stage, promotes the generation of hemogenic endothelial progenitors and their further differentiation to hematopoietic progenitor cells, and negatively regulates cardiac differentiation. Surprisingly, genome-wide transcriptional and chromatin immunoprecipitation analysis showed that GATA2 bound to regulatory regions, and repressed the expression of cardiac development-related genes. Moreover, genes important for hematopoietic differentiation were upregulated by GATA2 in a mostly indirect manner. Collectively, our data reveal a hitherto unrecognized role of GATA2 as a repressor of cardiac fates, and highlight the importance of coordinating the specification and repression of alternative cell fates.

SummaryNeurons obtained directly from human somatic cells hold great promise for disease modeling and drug screening. Available protocols rely on overexpression of transcription factors using integrative vectors and are often slow, complex, and inefficient. We report a fast and efficient approach for generating induced neural cells (iNCs) directly from human hematopoietic cells using Sendai virus. Upon SOX2 and c-MYC expression, CD133-positive cord blood cells rapidly adopt a neuroepithelial morphology and exhibit high expansion capacity. Under defined neurogenic culture conditions, they express mature neuronal markers and fire spontaneous action potentials that can be modulated with neurotransmitters. SOX2 and c-MYC are also sufficient to convert peripheral blood mononuclear cells into iNCs. However, the conversion process is less efficient and resulting iNCs have limited expansion capacity and electrophysiological activity upon differentiation. Our study demonstrates rapid and efficient generation of iNCs from hematopoietic cells while underscoring the impact of target cells on conversion efficiency.

Mesenchymal stem cells (MSCs), together with hematopoietic stem cells (HSCs), are the most frequently used cell type for cell-based therapeutics. As for other cell types intended for research and translational use, it is important to establish correctly typed cell lines from human tissue donations. Here, we describe methods for isolating, culturing, and identifying MSCs from various tissues obtained through human tissue donation. The methods have been used in the context of a biobank, prepared as standard operating procedures (SOPs), ensuring traceability and reproducibility of cell production.

The tumor suppressor p53 regulates the expression of genes involved in cell cycle progression, senescence and apoptosis. Here, we investigated the effect of single point mutations in the oligomerization domain (OD) on tetramerization, transcription, ubiquitylation and stability of p53. As predicted by docking and molecular dynamics simulations, p53 OD mutants show functional defects on transcription, Mdm2-dependent ubiquitylation and 26S proteasome-mediated degradation. However, mutants unable to form tetramers are well degraded by the 20S proteasome. Unexpectedly, despite the lower structural stability compared to WT p53, p53 OD mutants form heterotetramers with WT p53 when expressed transiently or stably in cells wild type or null for p53. In consequence, p53 OD mutants interfere with the capacity of WT p53 tetramers to be properly ubiquitylated and result in changes of p53-dependent protein expression patterns, including the pro-apoptotic proteins Bax and PUMA under basal and adriamycin-induced conditions. Importantly, the patient derived p53 OD mutant L330R (OD1) showed the more severe changes in p53-dependent gene expression. Thus, in addition to the well-known effects on p53 stability, ubiquitylation defects promote changes in p53-dependent gene expression with implications on some of its functions.