Enrichment of hematopoietic stem/progenitor cells in the zebrafish kidney

Abstract

Hematopoietic stem cells (HScs) maintain the entire blood system throughout life and are utilized in therapeutic approaches for blood diseases. Prospective isolation of highly purified HSCs is crucial to understand the molecular mechanisms underlying regulation of HSCs. The zebrafish is an elegant genetic model for the study of hematopoiesis due to its many unique advantages. it has not yet been possible, however, to purify HSCs in adult zebrafish due to a lack of specific HSC markers. Here we show the enrichment of zebrafish HSCs by a combination of two HSC-related transgenes, gata2a:GFP and runx1:mCherry. the double-positive fraction of gata2a:GFP and runx1:mCherry (gata2a + runx1 + ) was detected at approximately 0.16% in the kidney, the main hematopoietic organ in teleosts. transcriptome analysis revealed that gata2a + runx1 + cells showed typical molecular signatures of HScs, including upregulation of gata2b, gfi1aa, runx1t1, pbx1b, and meis1b. transplantation assays demonstrated that long-term repopulating HScs were highly enriched within the gata2a + runx1 + fraction. in contrast, colony-forming assays showed that gata2a − runx1 + cells abundantly contain erythroid-and/or myeloid-primed progenitors. Thus, our purification method of HSCs in the zebrafish kidney is useful to identify molecular cues needed to regulate self-renewal and differentiation of HSCs.Hematopoietic stem cells (HSCs) are self-renewing multipotent cells that can generate all types of blood cells over the lifetime of an individual and can be used therapeutically to treat hematopoietic diseases 1 . In the adult, most HSCs present in bone marrow are quiescent and divide rarely under homeostatic conditions. HSCs produce a heterogeneous pool of hematopoietic progenitor cells (HPCs), which have limited or no self-renewal ability, but rapidly proliferate and differentiate to satisfy the requirements for new mature blood cells 2,3 . Although the frequency of HSCs is extremely rare in bone marrow, HSC potential can be evaluated by transplantation assays, whereby the relative hematopoietic reconstitution activity of co-transplanted donor and competitor cells are compared in a recipient 4 . Purification of HSCs from murine and human bone marrow has been facilitated via transplantation assays using combinations of multiple cell-surface markers [5][6][7][8][9] . Studies in mice revealed that a single CD150 + CD34 − c-kit + Sca-1 + Lineage-marker − cell in the bone marrow showed long-term and multilineage hematopoietic reconstitution following transplantation 10,11 . Prospective isolation of highly purified HSCs thus elucidated many aspects of HSC biology, including self-renewal, differentiation, and HSC niches.The zebrafish is an excellent model for the study of HSCs due to its many unique advantages. Many valuable tools and experimental methods have been established for the study of hematopoietic cells in zebrafish (e.g. transgenic/mutant animals, transplantation assays, cell culture assays, etc.) 12,13 . Moreover, genome-editing technology based on...

Definitive Hematopoietic Stem Cells Minimally Contribute to Embryonic Hematopoiesis

“…That HSCs have a delayed hematopoietic contribution during development indicates a disconnect between the timing of HSC emergence and their demonstrable long-term function. Additionally, our data contrasts the classically held interpretation that HSCs reside at the top of the hematopoietic hierarchy, maintaining lifelong erythroid, myeloid, and lymphoid hematopoiesis (Kobayashi, et al, 2019). Since nascent HSCs can engraft and reconstitute the blood system of a transplanted host, it was posited that they were the source of all blood cells from shortly after their emergence onward (Medvinsky and Dzierzak, 1996).…”

Uptake of osteoblast-derived extracellular vesicles promotes the differentiation of osteoclasts in the zebrafish scale

“…RNA-seq and qPCR. For sorted cells and EVs, whole-transcript amplification and double-strand cDNA synthesis was performed according to the Quartz-Seq method as previously described 53,54 . Cells were directly sorted in a lysis buffer containing 1 μg/ mL of polyinosinic-polycytidylic acid, and total RNA was extracted using RNeasy Mini Kit.…”

Single-cell ATAC-seq reveals GATA2-dependent priming defect in myeloid and a maturation bottleneck in lymphoid lineages

“…Gata2a has been implicated in lymphovascular development with some expression in HSPCs and eosinophils in adult hematopoiesis. [9][10][11][12] Gata2b, with 57% identity to gata2a and human GATA2, is restricted to hemogenic endothelium and emerging hematopoietic stem cells during development that contribute to adult hematopoiesis. 13 The tissue-restricted expression of the gata2 orthologs provides a potentially unique opportunity to investigate hematopoiesis-specific functions of GATA2.…”