E-cadherin/ß-catenin expression is conserved in human and rat erythropoiesis and marks stress erythropoiesis.

E-cadherin is a crucial regulator of epithelial cell-to-cell adhesion and an established tumor suppressor. Aside epithelia, E-cadherin expression marks the erythroid cell lineage during human but not mouse hematopoiesis. However, the role of E-cadherin in human erythropoiesis remains unknown. Because rat erythropoiesis was postulated to reflect human erythropoiesis more closely than mouse erythropoiesis, we investigated E-cadherin expression in rat erythroid progenitors. E-cadherin expression is conserved within the erythroid lineage between rat and human. In response to anemia, erythroblasts in rat bone marrow (BM) upregulate E-cadherin as well as its binding partner ß-catenin. CRISPR/Cas9-mediated knock out of E-cadherin revealed that E-cadherin expression is required to stabilize ß-catenin in human and rat erythroblasts. Suppression of ß-catenin degradation by glycogen synthase kinase 3ß (GSK3ß) inhibitor CHIR99021 also enhances ß-catenin stability in human erythroblasts but hampers erythroblast differentiation and survival. In contrast, direct activation of ß-catenin signaling, using an inducible, stable ß-catenin variant, does not perturb maturation or survival of human erythroblasts but rather enhances their differentiation. Although human erythroblasts do not respond to Wnt ligands and direct GSK3ß inhibition even reduces their survival, we postulate that ß-catenin stability and signaling is mostly controlled by E-cadherin in human and rat erythroblasts. In response to anemia, E-cadherin-driven upregulation and subsequent activation of ß-catenin signaling may stimulate erythroblast differentiation to support stress erythropoiesis in the BM. Overall, we uncover E-cadherin/ß-catenin expression to mark stress erythropoiesis in rat BM. This may provide further understanding of the underlying molecular regulation of stress erythropoiesis in the BM, which is currently poorly understood.

Optimized Guide RNA Selection Improves Streptococcus pyogenes Cas9 Gene Editing of Human Hematopoietic Stem and Progenitor Cells.

Ribonucleoproteins (RNPs) are frequently applied for therapeutic gene editing as well as fundamental research because the method is fast, viral free, and shows fewest off target effects. We evaluated various parameters to genetically engineer human hematopoietic stem and progenitor cells (HSPCs) using Streptococcus pyogenes Cas9 (spCas9) RNPs, and achieve gene editing efficiencies up to 80%. We find that guide RNA (gRNA) design is critical to achieve high gene editing efficiencies. However, finding effective gRNAs for HSPCs can be challenging, while the contribution of numerous in silico models is unclear. By screening more than 120 gRNAs, our data demonstrate that in silico gRNA prediction models are ineffective. In this study, we established a time- and cost-efficient in vitro transcribed gRNA screening model in K562 cells that predicts effective gRNAs for HSPCs. RNP based screening thus outperforms in silico modeling and we report that gene editing is equally efficient in distinct CD34+ HSPC subpopulations. Furthermore, no effects on cell proliferation, differentiation, or in vitro hematopoietic lineage commitment were observed. Finally, no upregulation of p21 expression was found, suggesting unperturbed HSPC homeostasis.