Distinct roles of core autophagy-related genes (ATGs) in zebrafish definitive hematopoiesis

Despite the well-described discrepancy between some of the macroautophagy/autophagy-related genes (ATGs) in the regulation of hematopoiesis, the varying essentiality of core ATGs in vertebrate definitive hematopoiesis remains largely unclear. Here, we employed zebrafish (Danio rerio) to compare the function of six core atgs from the core autophagy machineries, which included atg13, beclin1 (becn1), atg9a, atg2a, atg5, and atg3, in vertebrate definitive hematopoiesis via CRISPR-Cas9 ribonucleoprotein targeting. Zebrafish embryos with various atg mutations showed autophagic deficiency throughout the body, including hematopoietic cells. The atgs mutations unsurprisingly caused distinctive hematopoietic abnormalities in zebrafish. Notably, becn1 or atg9a mutation resulted in hematopoietic stem cells (HSCs) expansion during the development of the embryo into a larva, which can be attributed to the proteomic changes in metabolism, HSCs regulators, and apoptosis. Besides, atg3 mutation lowered the leukocytes in developing zebrafish embryos. Intriguingly, a synergistic effect on HSCs expansion was identified in atg13+becn1 and atg9a+atg2a or atg3 double mutations, in which atg13 mutation and atg2a or atg3 mutation exacerbated and mitigated the HSCs expansion in becn1 and atg9a mutations, respectively. In addition, the myeloid cell type-specific effects of various atgs were also determined between neutrophils and macrophages. Of these, a skewed ratio of neutrophils versus macrophages was found in atg13 mutation, while both of them were reduced in atg3 mutation. These findings demonstrated the distinct roles of atgs and their interplays in zebrafish definitive hematopoiesis, thereby suggested that the vertebrate definitive hematopoiesis is regulated in an atgs-dependent manner.

Self-renewing tissue-resident endothelial-macrophage progenitor cells originate from yolk sac and are a local source of inflammation and neovascularization in postnatal aorta

Converging evidence indicates that extra-embryonic yolk sac is the source of both macrophages and endothelial cells in adult mouse tissues. Prevailing views are that these yolk sac-derived cells are maintained after birth by proliferative self-renewal in their differentiated states. Here we identify clonogenic, self-renewing endothelial-macrophage (EndoMac) progenitor cells in postnatal mouse aorta, heart and lung, that are independent of definitive hematopoiesis and derive from a CX3CR1+ and CSF1R+ yolk sac source. These bipotent progenitors are highly proliferative and vasculogenic, contributing to adventitial neovascularization in the aortic wall and forming perfused blood vessels after adoptive transfer into ischemic tissue. We establish a regulatory role for angiotensin II, which enhances their clonogenic, self-renewal and differentiation properties. Our findings demonstrate that tissue-resident EndoMac progenitors participate in local inflammatory and vasculogenic responses by contributing to the renewal and expansion of yolk sac-derived macrophages and endothelial cells postnatally.

The spliceosome factor sart3 regulates hematopoietic stem/progenitor cell development in zebrafish through the p53 pathway

Hematopoietic stem cells (HSCs) possess the potential for self-renew and the capacity, throughout life, to differentiate into all blood cell lineages. Yet, the mechanistic basis for HSC development remains largely unknown. In this study, we characterized a zebrafish smu471 mutant with hematopoietic stem/progenitor cell (HSPC) defects and found that sart3 was the causative gene. RNA expression profiling of the sart3smu471 mutant revealed spliceosome and p53 signaling pathway to be the most significantly enriched pathways in the sart3smu471 mutant. Knock down of p53 rescued HSPC development in the sart3smu471 mutant. Interestingly, the p53 inhibitor, mdm4, had undergone an alternative splicing event in the mutant. Restoration of mdm4 partially rescued HSPC deficiency. Thus, our data suggest that HSPC proliferation and maintenance require sart3 to ensure the correct splicing and expression of mdm4, so that the p53 pathway is properly inhibited to prevent definitive hematopoiesis failure. This study expands our knowledge of the regulatory mechanisms that impact HSPC development and sheds light on the mechanistic basis and potential therapeutic use of sart3 in spliceosome-mdm4-p53 related disorders.

Redundant mechanisms driven independently by RUNX1 and GATA2 for hematopoietic development

RUNX1 is essential for the generation of hematopoietic stem cells (HSCs). Runx1 null mouse embryos lack definitive hematopoiesis and die in mid-gestation. However, even though zebrafish embryos with a runx1 W84X mutation have defects in early definitive hematopoiesis, some runx1W84X/W84X embryos can develop to fertile adults with blood cells of multi-lineages, raising the possibility that HSCs can emerge without RUNX1. Here, using three new zebrafish runx1-/- lines we uncovered the compensatory mechanism for runx1-independent hematopoiesis. We show that, in the absence of a functional runx1, a cd41-GFP+ population of hematopoietic precursors still emerge from the hemogenic endothelium and can colonize the hematopoietic tissues of the mutant embryos. Single-cell RNA sequencing of the cd41-GFP+ cells identified a set of runx1-/--specific signature genes during hematopoiesis. Significantly, gata2b, which normally acts upstream of runx1 for the generation of HSCs, was increased in the cd41-GFP+ cells in runx1- /- embryos. Interestingly, genetic inactivation of both gata2b and its paralog, gata2a, did not affect hematopoiesis. However, knocking out runx1 and any three of the four alleles of gata2a and gata2b abolished definitive hematopoiesis. Gata2 expression was also upregulated in hematopoietic cells in Runx1-/- mice, suggesting the compensatory mechanism is conserved. Our findings indicate that RUNX1 and GATA2 serve redundant roles for HSC production, acting as each other's safeguard.

KIT Is Required for Fetal Liver Hematopoiesis

In the mouse embryo, endothelial cell (EC) progenitors almost concomitantly give rise to the first blood vessels in the yolk sac and the large vessels of the embryo proper. Although the first blood cells form in the yolk sac before blood vessels have assembled, consecutive waves of hematopoietic progenitors subsequently bud from hemogenic endothelium located within the wall of yolk sac and large intraembryonic vessels in a process termed endothelial-to-hematopoietic transition (endoHT). The receptor tyrosine kinase KIT is required for late embryonic erythropoiesis, but KIT is also expressed in hematopoietic progenitors that arise via endoHT from yolk sac hemogenic endothelium to generate early, transient hematopoietic waves. However, it remains unclear whether KIT has essential roles in early hematopoiesis. Here, we have combined single-cell expression studies with the analysis of knockout mice to show that KIT is dispensable for yolk sac endoHT but required for transient definitive hematopoiesis in the fetal liver.

Genetic and chemical inhibition of autophagy in zebrafish induced myeloproliferation

Autophagy is an evolutionary conserved and dynamic lysosomal degradation process for cellular homeostasis and remodelling, which is essential for the development and maintenance of different hematopoietic fates. However, the roles of autophagy in definitive hematopoiesis remain elusive. Here, we exploited zebrafish (Danio rerio) to investigate the effect of knocking-out unc-51 like autophagy activating kinase 1b and 2 (ulk1b and ulk2), homologous of human ULK1 and ULK2, respectively, on definitive hematopoiesis. Upon ulk1b or ulk2 mutation, autophagosome formation was blocked in zebrafish embryos. More importantly, pan-leukocytes (lcp1), common myeloid progenitors (spi1b), neutrophils (mpx), and macrophages (mpeg1.1) significantly elevated, while the hematopoietic stem and progenitor cell (HSPC) (myb), erythroid progenitors (gata1), and embryonic hemoglobin (hbae1.1) significantly reduced in the caudal hematopoietic tissue (CHT) of ulk1b or ulk2 mutant zebrafish embryos. On the other hand, chemically modulated autophagy induction by calpeptin, a downstream autophagy inducer for ulk complex, was insufficient to ameliorate dysregulated hematopoiesis in both ulk1b and ulk2 mutants. Conversely, autophagy inhibitor 3-Methyladenine functioned parallelly with the ulk mutants to maintain defective hematopoiesis. These observations raised a link between autophagy and definitive hematopoiesis and potentiates the fact that autophagy deficiency incorporates with myeloproliferation and anemia, which warrants the significance of autophagy in regulating definitive hematopoiesis.

A Benchmark Side-by-Side Comparison of Two Well-Established Protocols for in vitro Hematopoietic Differentiation From Human Pluripotent Stem Cells

The generation of transplantable hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) remains challenging. Current differentiation protocols from hPSCs generate mostly hematopoietic progenitors of the primitive HSC-independent program, and it remains unclear what is the best combination of cytokines and hematopoietic growth factors (HGFs) for obtaining functional hematopoietic cells in vitro. Here, we have used the AND1 and H9 hESC lines and the H9:dual-reporter RUNX1C-GFP-SOX17-Cherry to compare the hematopoietic differentiation in vitro based on the treatment of embryoid bodies (EBs) with the ventral mesoderm inducer BMP4 plus HGFs in the absence (protocol 1) or presence (protocol 2) of stage-specific activation of Wnt/β-catenin and inhibition of Activin/Nodal. Despite a slight trend in favor of protocol 1, no statistically significant differences were observed between protocols at any time point analyzed throughout EB development regarding the frequency of hemogenic endothelial (HE) precursors; CD43+ CD45−, CD45+, and CD45 + CD34 + hematopoietic derivatives; or the output of clonogenic progenitors. Similarly, the kinetics of emergence throughout EB development of both SOX17 + HE and RUNX1C + definitive hematopoiesis was very similar for both protocols. The expression of the early master mesendodermal transcription factors Brachyury, MIXL1, and KDR revealed similar gene expression kinetics prior to the emergence of RUNX1C + definitive hematopoiesis for both protocols. Collectively, the simpler protocol 1 is, at least, as efficient as protocol 2, suggesting that supplementation with additional morphogens/HGFs and modulation of Activin/Nodal and Wnt/β-catenin pathways seem dispensable for in vitro hematopoietic differentiation of hPSCs.

Mutation of Gemin5 Causes Defective Hematopoietic Stem/Progenitor Cells Proliferation in Zebrafish Embryonic Hematopoiesis

Fate determination and expansion of Hematopoietic Stem and Progenitor Cells (HSPCs) is tightly regulated on both transcriptional and post-transcriptional level. Although transcriptional regulation of HSPCs have achieved a lot of advances, its post-transcriptional regulation remains largely underexplored. The small size and high fecundity of zebrafish makes it extraordinarily suitable to explore novel genes playing key roles in definitive hematopoiesis by large-scale forward genetics screening. Here, we reported a novel zebrafish mutant line gemin5cas008 with a point mutation in gemin5 gene obtained by ENU mutagenesis and genetic screening, causing an earlier stop codon next to the fifth WD repeat. Gemin5 is an RNA-binding protein with multifunction in post-transcriptional regulation, such as regulating the biogenesis of snRNPs, alternative splicing, stress response, and translation control. The mutants displayed specific deficiency in definitive hematopoiesis without obvious defects during primitive hematopoiesis. Further analysis showed the impaired definitive hematopoiesis was due to defective proliferation of HSPCs. Overall, our results indicate that Gemin5 performs an essential role in regulating HSPCs proliferation.

Transfer to the clinic: refining forward programming of hPSCs to megakaryocytes for platelet production in bioreactors

The production of in vitro–derived platelets has great potential for transfusion medicine. Here, we build on our experience in the forward programming (FoP) of human pluripotent stem cells (hPSCs) to megakaryocytes (MKs) and address several aspects of the complex challenges to bring this technology to the bedside. We first identify clinical-grade hPSC lines that generate MKs efficiently. We design a bespoke media to maximize both production and maturity of MKs and improve platelet output. Crucially, we transition the lentiviral-based FoP of hPSCs to a nonviral inducible system. We also show how small molecules promote a definitive hematopoiesis phenotype during the differentiation process, thereby increasing the quality of the final product. Finally, we generate platelets using a bioreactor designed to reproduce the physical cues that promote platelet production in the bone marrow. We show that these platelets are able to contribute to both thrombus formation in vitro and have a hemostatic effect in thrombocytopenic mice in vivo.