Biology subjects

Weinacht, K. G.

Publications and source records attributed to Weinacht, K. G..

3 recordsLinked to original sources

bioRxiv2022

Single cell transcriptomics of human prenatal anterior foregut-derived organs identifies distinct developmental signatures directing commitment and specialization of the thymic epithelial stroma

The thymus instructs T cell immunity and central tolerance, yet its therapeutic potential remains untapped. The quest to regenerate thymic function for clinical application is lagging while the signals that drive thymic epithelial cell differentiation remain incompletely understood. Here, we elucidate pathways instructing commitment and specialization of the human thymic epithelial stroma through complementary single cell transcriptomic approaches. First, we identify gene regulatory networks that define fetal thymic epithelium in the thus far unexplored context of other anterior foregut-derived organs; then, we characterize lineage trajectories within the thymic epithelial compartment across embryonic, fetal, and early postnatal stages. Activation of interferon response gene regulatory networks distinguished epithelial cells of the thymus from those of all other anterior foregut-derived organs. Interferon signals were processed differentially within thymic cortical and medullary lineages, reflected in distinct NF{kappa}B and IRF signatures, respectively. Our study reveals novel, translatable insights into the developmental programs underlying thymic epithelial cell differentiation that may advance the field of regenerative cell therapies. SUMMARYSingle cell transcriptomics of anterior foregut-derived organs identifies pathways governing thymic epithelial commitment and specialization.

developmental biology↗

bioRxiv2022

Multi-omics analysis reveals a crucial role for Retinoic Acid in promoting epigenetic and transcriptional competence of an in vitro model of human Pharyngeal Endoderm.

In vitro differentiation of human Pluripotent Stem Cells (hPSCs) into different cell types has enabled the study of developmental processes that are impossible to dissect in vivo. This innovation has allowed for the derivation of therapeutically relevant cell types that can be used for downstream applications and studies. The Pharyngeal Endoderm (PE) is considered an extremely relevant developmental tissue since it acts as a precursor to a plethora of organ systems such as Esophagus, Parathyroids, Thyroids, Lung, and Thymus. While several studies have highlighted the importance of these cells, an in vitro platform to generate human PE cells is still missing. Here we fill this knowledge gap, by providing a novel in vitro protocol for the derivation of bona fide PE cells from hPSCs. We demonstrated that our PE cells robustly express Pharyngeal Endoderm markers, they are transcriptionally similar to PE cells isolated from in vivo mouse development and represent a transcriptionally homogeneous population. Importantly, we elucidated the contribution of Retinoic Acid in promoting a transcriptional and epigenetic rewiring of PE cells. In addition, we defined the epigenetic landscape of PE cells by combining ATAC-Seq and ChIP-Seq of histone modifications. The integration of these data led to the identification of new putative regulatory regions and to the generation of a gene regulatory network orchestrating the development of PE cells. By combining hPSCs differentiation with computational genomics, our work reveals the epigenetic dynamics that occur during human PE differentiation, providing a solid resource and foundation for research focused on the development of PE derivatives and modeling of their developmental defects in genetic syndromes.

developmental biology↗

bioRxiv2021

Adenylate Kinase 2 deficiency causes NAD+ depletion and impaired purine metabolism during myelopoiesis

Reticular Dysgenesis is a particularly grave form of severe combined immunodeficiency that affects the adaptive and innate immune system. Patients suffer from congenital neutropenia, lymphopenia, and deafness. The disease is caused by biallelic loss of function in mitochondrial Adenylate Kinase 2 (AK2). AK2 mediates the phosphorylation of AMP to ADP, as substrate for ATP synthesis. Accordingly, declining oxidative phosphorylation has been postulated as the driver of disease pathology. The mechanistic basis, however, remains incompletely understood. Single cell RNA-sequencing of patient bone marrow cells implicated altered RNA catabolism and ribonucleoprotein synthesis in the pathogenesis of Reticular Dysgenesis. To investigate these findings, we developed a disease model based on CRISPR-mediated disruption of the AK2 gene in primary human hematopoietic stem cells. We found that AK2-deficient myeloid progenitor cells not only have compromised mitochondrial energy metabolism and increased AMP levels, but also NAD+ and aspartate depletion, metabolites that rely on TCA-cycle activity for regeneration and synthesis. Furthermore, AK2-deficient cells exhibited strikingly increased levels of the purine nucleotide precursor IMP, decreased cellular RNA content, ribosome subunit expression, protein synthesis and a profoundly hypo-proliferative phenotype. The rise in IMP levels stemmed from increased AMP deamination. Pharmacologic inhibition of AMP deaminase normalized IMP levels in AK2-deficient cells, but further aggravated the disease phenotype, pointing to AMP catabolism as a possible metabolic adaptation to mitigate AMP-mediated toxicity. Inducing an adenosine disequilibrium in control cells produced a similar myeloid maturation defect. This study shows that AK2 deficiency globally curtailed mitochondrial metabolism resulting in NAD+ and aspartate deficiency and disordered purine metabolism. AMP accumulation and its detrimental effects on ribonucleotide synthesis capacity may contribute to the failure of myelopoiesis in Reticular Dysgenesis.

cell biology↗