Elucidating an Essential Role of Promoting Endothelial-to-Hematopoietic Transition By a Key EMT-Regulator ZEB2 Using a Human iPSC-Based Model

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ZEB2 (Zinc finger E-box binding homeobox 2; also known as SMAD interacting protein-1 or SIP1) is a DNA-binding transcriptional regulator associated with epithelial-to-mesenchymal-transition (EMT). It has been reported that it is important in many other developmental processes, including mesoderm formation during gastrulation and neural crest formation. More recently, a study reported that inactivation of ZEB2 in mice results in differentiation defects in multiple hematopoietic cell lineages (1). In order to examine the roles of ZEB2 in human developmental process and hematopoiesis, we decided to apply human induced pluripotent stem cells (iPSCs) as a model with a pair of isogenic iPSCs with or without functional ZEB2 expression. We used a validated iPSC-based hematopoietic differentiation under a feeder- and xeno-free culture condition was used to study the role of ZEB2 in hematopoietic cell formation and to avoid other EMT-inducing factors in serum. We found that ZEB2 expression is abundant in either cord blood cells or human iPSCs-derived hematopoietic stem/progenitor cells (HSPCs), indicating that ZEB2 deficiency may hinder human hematopoietic differentiation. To knock out the ZEB2 gene and generate ZEB2 deficient human iPSCs, we used two different CRISPR guide RNAs as well as SpCas9 in a human iPSC line BC1. An engineered isogenic BC1 iPSC line that has both alleles 155-bp deletion in ZEB2 exon 1 (ZEB2 -/-) was successfully isolated and expanded, resulting in the frameshift and the consequent disappearance of ZEB2 protein expression. The complete ZEB2 knockout had little effects on human iPSCs expansion. However, we observed that the differentiation of ZEB-/- BC1 into CD34+CD45+ HSPCs was decreased. The number of ZEB-/- BC1-derived HSPCs was 5-fold lower than that from wide type BC1 control. Moreover, ZEB2-deleted HSPCs were defected in hematopoietic colony formation. To figure out at which hematopoietic differentiating stage the defects occurred, we applied inducible iPSC's endothelium-hematopoietic transformation (EHT) system to mimic hemogenic endothelial cells (ECs) giving rise to HSPCs. By using this established EHT system, ZEB2 deficient iPSCs were differentiated in order into CD34+CD31+CD144+ ECs, and then to HSPCs (2). We found that ZEB2 depletion did not adversely affect the formation of ECs from human iPSCs in this EHT experimental system but did impact on HSPCs generation from ECs. This study is unique in several ways to elucidate the roles of ZEB2 in human hematopoietic development. This prospective genetic model allowed us to pinpoint ZEB2 is critical at the stage of EHT during human hematopoietic cell formation. Our results indicate that contribution of ZEB2 deficiency in blood disorders might result from the inhibition of HSPC formation. The human iPSC-based hematopoietic differentiation system coupled with precisely edited isogenic iPSC lines also provide a more controlled genetic model for studying functions of others gene in their regulation of human hematopoiesis.