Salaheddine Ali

MYCN amplification drives one in six cases of neuroblastoma. The supernumerary gene copies are commonly found on highly rearranged, extrachromosomal circular DNA (ecDNA). The exact amplicon structure has not been described thus far and the functional relevance of its rearrangements is unknown. Here, we analyze the MYCN amplicon structure using short-read and Nanopore sequencing and its chromatin landscape using ChIP-seq, ATAC-seq and Hi-C. This reveals two distinct classes of amplicons which explain the regulatory requirements for MYCN overexpression. The first class always co-amplifies a proximal enhancer driven by the noradrenergic core regulatory circuit (CRC). The second class of MYCN amplicons is characterized by high structural complexity, lacks key local enhancers, and instead contains distal chromosomal fragments harboring CRC-driven enhancers. Thus, ectopic enhancer hijacking can compensate for the loss of local gene regulatory elements and explains a large component of the structural diversity observed in MYCN amplification.

ABSTRACT Human induced pluripotent stem cells (hiPSCs) hold great potential for modeling human diseases and the development of innovative therapeutic approaches. Here, we report on a novel, simplified differentiation method for forming functional osteoclasts from hiPSCs. The three-step protocol starts with embryoid body formation, followed by hematopoietic specification, and finally osteoclast differentiation. We observed continuous production of monocyte-like cells over a period of up to 9 weeks, generating sufficient material for several osteoclast differentiations. The analysis of stage-specific gene and surface marker expression proved mesodermal priming, the presence of monocyte-like cells, and of terminally differentiated multinucleated osteoclasts, able to form resorption pits and trenches on bone and dentine in vitro. In comparison to peripheral blood mononuclear cell (PBMC)-derived osteoclasts hiPSC-derived osteoclasts were larger and contained a higher number of nuclei. Detailed functional studies on the resorption behavior of hiPSC-osteoclasts indicated a trend towards forming more trenches than pits and an increase in pseudoresorption. We used hiPSCs from an autosomal recessive osteopetrosis (ARO) patient (BIHi002-A, ARO hiPSCs) with compound heterozygous missense mutations p.(G292E) and p.(R403Q) in CLCN7, coding for the Cl−/H+-exchanger ClC-7, for functional investigations. The patient's leading clinical feature was a brain malformation due to defective neuronal migration. Mutant ClC-7 displayed residual expression and retained lysosomal co-localization with OSTM1, the gene coding for the osteopetrosis-associated transmembrane protein 1, but only ClC-7 harboring the mutation p.(R403Q) gave strongly reduced ion currents. An increased autophagic flux in spite of unchanged lysosomal pH was evident in undifferentiated ARO hiPSCs. ARO hiPSC-derived osteoclasts showed an increased size compared to hiPSCs of healthy donors. They were not able to resorb bone, underlining a loss-of-function effect of the mutations. In summary, we developed a highly reproducible, straightforward hiPSC-osteoclast differentiation protocol. We demonstrated that osteoclasts differentiated from ARO hiPSCs can be used as a disease model for ARO and potentially also other osteoclast-related diseases. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Multipotent stromal cells are considered attractive sources for cell therapy and tissue engineering. Despite numerous experimental and clinical studies, broad application of stromal cell therapeutics is not yet emerging. A major challenge is the functional diversity of available cell sources. Here, we investigated the regenerative potential of clinically relevant human stromal cells from bone marrow (BMSCs), white adipose tissue, and umbilical cord compared with mature chondrocytes and skin fibroblasts in vitro and in vivo. Although all stromal cell types could express transcription factors related to endochondral ossification, only BMSCs formed cartilage discs in vitro that fully regenerated critical-size femoral defects after transplantation into mice. We identified cell type-specific epigenetic landscapes as the underlying molecular mechanism controlling transcriptional stromal differentiation networks. Binding sites of commonly expressed transcription factors in the enhancer and promoter regions of ossification-related genes, including Runt and bZIP families, were accessible only in BMSCs but not in extraskeletal stromal cells. This suggests an epigenetically predetermined differentiation potential depending on cell origin that allows common transcription factors to trigger distinct organ-specific transcriptional programs, facilitating forward selection of regeneration-competent cell sources. Last, we demonstrate that viable human BMSCs initiated defect healing through the secretion of osteopontin and contributed to transient mineralized bone hard callus formation after transplantation into immunodeficient mice, which was eventually replaced by murine recipient bone during final tissue remodeling.