Field Of Hematopoiesis Research Articles

Rapid Degradation of mDia2 Protein during Terminal Erythropoiesis Via an In Vivo Aid System: An Alternative Approach for Loss-of-Function Studies

Loss-of-function manipulations are crucial methods for studying gene functions. Despite the availability of many chemical-induced genetic manipulation techniques, there are limitations associated with these technologies. Tamoxifen-induced conditional gene expression, gene knockdown, or cell tracking represent some of the most common genetic manipulations employed in mice. However, tamoxifen-induced gene or Cre expression in bone marrow cell populations is relatively weak, affecting only a low proportion of cells. Similar observations have been made with the in vivo doxycycline-inducible system. Given that inflammation could affect the dispersion of bone marrow hematopoietic cells, an inducible system based on the MX1 promoter and interferon-alpha is not suitable for long-term protein knockdown. In addition, the above-mentioned methods are not able to achieve rapid loss-of-function because it is largely determined by the half-life of the transcriptional and translational products of the targeted gene. Furthermore, none of these technologies could achieve reversible depletion of the protein of interest. Therefore, there is a need for enhanced techniques for inducible and reversible loss-of-function gene manipulations in the field of hematopoiesis. The capacity for quick, targeted protein elimination is particularly necessary when researching the functions of proteins that play roles in swift biological processes. The Auxin-inducible degron (AID) system is a chemically inducible tool, widely employed for targeted protein degradation to study protein functions in cultured mammalian cells. It holds significant potential as a powerful tool for studying protein functions in vivo within the bone marrow. The AID system comprises three components: the auxin signal, AID tag, and auxin receptor F-box proteins (AFBs). When an AID tag is fused to a protein of interest, the AFB proteins recognize the tag and, in the presence of the auxin signal, induce the protein's degradation via the proteasome. Additionally, due to its small molecular weight, auxin can potentially penetrate the bone marrow barrier and achieve the working concentration in the bone marrow, facilitating protein degradation. In this study, we aim to investigate the role of mDia2 in terminal erythropoiesis using AID system. The mDia family proteins are formin proteins that catalyze the assembly of F-actin. The loss of function of mDia2 achieved by Cre-flox system leads to deficiency in enucleation and anemia. We hypothesize that the mDia2 loss of function could be compensated by upregulated mDia1 in the Cre-flox mediated knockout system, and a rapid loss of mDia2 protein could lead to more severe phenotypes in vivo and ex-vivo. We knocked in minilAA7-eGFP to the end of the last exon of mDia2 gene to create an endogenous tagged mDia2 protein in C57/B6 mice. We also created an AFB protein transgenic mouse by knocking in the atAFB2 protein coding gene at the ROSA26 locus driven by the Vav1 promoter. After crossing the mDia2-minilAA7-eGFP transgenic mice with Vav1-atAFB2 mice, we obtained mDia2-minilAA7-eGFP and Vav1-atAFB2 double homozygous mice. We administered auxin via intraperitoneal (IP) injection at a concentration of 300 mg/kg and collected total bone marrow cells four hours post-injection. Quantification of the GFP signal through flow cytometry revealed a significant reduction following auxin administration compared to the control group. This protein degradation was further confirmed with Western blotting assays. To understand how an immediate loss of mDia2 influences terminal erythropoiesis, we isolated lineage-negative cells from auxin-administered mice and cultured them in an EPO-containing medium with 10 µM auxin for 48 hours. Compared to lineage-negative cells from VavCre-mDia2f/f mice, auxin-induced mDia2 degradation resulted in a significantly lower enucleation rate. Notably, auxin administration had no observable effects on either WT mice or atAFB2 homozygous mice. This suggests that rapid mDia2 loss, induced by the AID system, permits a better understanding of the targeted protein's function by avoiding potential compensatory effects from mDia1 during terminal enucleation. In conclusion, our established in vivo AID system enables rapid targeted protein degradation and serves as a powerful tool for studying protein functions potentially obscured by compensatory effects during erythropoiesis.

BCR-ABL promotes hematopoietic stem and progenitor cell formation in embryonic stem cells

Generating hematopoietic stem cells (HSCs) from pluripotent stem cells (PSCs) has been a long-lasting quest in the field of hematopoiesis. Previous studies suggested that enforced expression of BCR-ABL, the unique oncogenic driver of chronic myelogeneous leukemia (CML), in embryonic stem cells (ESCs)-derived hematopoietic cells is sufficient to confer long-term in vivo repopulating potential. To precisely uncover the molecular events regulated by the tyrosine kinase activity of BCR-ABL1 (p210) during the course of hematopoietic differentiation, we engineered a Tet-ON inducible system to modulate its expression in murine ESCs (mESCs). We showed in unique site-directed knock-in ESC model that BCR-ABL expression tightly regulated by doxycycline (dox) controls the formation and the maintenance of immature hematopoietic progenitors. Interestingly, these progenitors can be expanded in vitro for several passages in the presence of dox. Our analysis of cell surface markers and transcriptome compared with wild-type fetal and adult HSCs unraveled a similar molecular signature. Long-term culture initiating cell (LTC-IC) assay confirmed their self-renewal capacities albeit with a differentiation bias toward erythroid and myeloid cells. Collectively, our novel Tet-ON system represents a unique in vitro model to shed lights on ESC-derived hematopoiesis, CML initiation, and maintenance.

Niches of Hematopoietic Stem Cells in Bone Marrow

Hematopoietic stem cells (HSCs) exist in a close contact with their specific microenvironment, called a niche, which supports the HSC function and significantly influences the HSC properties. The existence of the HSC niche, which was proposed as a purely theoretical concept in 1978, finds increasing experimental evidence and is now generally accepted by specialists in the field of hematopoiesis. The review briefly describes various cell components of the HSC niche in bone marrow, considers the metabolic states of the niche and HSCs, and discusses other aspects of niche biology. Increasing knowledge of the HSC niche will help to create in vitro cell models of the HSC niche, to modulate the HSC properties, and to achieve multifold HSC expansion in culture for further applications in therapeutic practice.

Drosophila hematopoiesis under normal conditions and in response to immune stress.

The emergence of hematopoietic progenitors and their differentiation into various highly specialized blood cell types constitute a finely tuned process. Unveiling the genetic cascades that control blood cell progenitor fate and understanding how they are modulated in response to environmental changes are two major challenges in the field of hematopoiesis. In the last 20 years, many studies have established important functional analogies between blood cell development in vertebrates and in the fruit fly, Drosophila melanogaster. Thereby, Drosophila has emerged as a powerful genetic model for studying mechanisms that control hematopoiesis during normal development or in pathological situations. Moreover, recent advances in Drosophila have highlighted how intricate cell communication networks and microenvironmental cues regulate blood cell homeostasis. They have also revealed the striking plasticity of Drosophila mature blood cells and the presence of different sites of hematopoiesis in the larva. This review provides an overview of Drosophila hematopoiesis during development and summarizes our current knowledge on the molecular processes controlling larval hematopoiesis, both under normal conditions and in response to an immune challenge, such as wasp parasitism.

HSC-Explorer: A Curated Database for Hematopoietic Stem Cells

HSC-Explorer (http://mips.helmholtz-muenchen.de/HSC/) is a publicly available, integrative database containing detailed information about the early steps of hematopoiesis. The resource aims at providing fast and easy access to relevant information, in particular to the complex network of interacting cell types and molecules, from the wealth of publications in the field through visualization interfaces. It provides structured information on more than 7000 experimentally validated interactions between molecules, bioprocesses and environmental factors. Information is manually derived by critical reading of the scientific literature from expert annotators. Hematopoiesis-relevant interactions are accompanied with context information such as model organisms and experimental methods for enabling assessment of reliability and relevance of experimental results. Usage of established vocabularies facilitates downstream bioinformatics applications and to convert the results into complex networks. Several predefined datasets (Selected topics) offer insights into stem cell behavior, the stem cell niche and signaling processes supporting hematopoietic stem cell maintenance. HSC-Explorer provides a versatile web-based resource for scientists entering the field of hematopoiesis enabling users to inspect the associated biological processes through interactive graphical presentation.

Hematopoietic Stem Cell Gene Therapy: Assessing the Relevance of Preclinical Models

The modern laboratory mouse has become a central tool of for biomedical research with a notable influence in the field of hematopoiesis. Application of retroviral-based gene transfer approaches to mouse hematopoietic stem cells (HSCs) has led to a sophisticated understanding of the hematopoietic hierarchy in this model. However, the assumption that gene transfer methodologies developed in the mouse could be similarly applied to human HSCs for the treatment of human diseases left the field of gene therapy in a decade-long quandary. It is not until more relevant humanized xenograft mouse models and phylogenetically related large animal species were used to optimize gene transfer methodologies that unequivocal clinical successes were achieved. However, the subsequent reporting of severe adverse events in these clinical trials cast doubts on the predictive value of conventional preclinical testing, and encouraged the development of new assays for assessing the relative genotoxicity of various vector designs.

Hematopoietic development in the zebrafish

The model organism Danio rerio, also known as the zebrafish, is an excellent system for studying the developmental process of hematopoiesis. It is an ideal model for in vivo imaging, and it is useful for large-scale genetic screens. These have led to the discovery of previously unknown players in hematopoiesis, as well as helped our understanding of hematopoietic development. In this review, we will summarize hematopoiesis in the zebrafish and discuss how genetic approaches using the zebrafish system have helped to build our current knowledge in the field of hematopoiesis.

Hematopoietic Stem and Progenitor Cells: Clinical and Preclinical Regeneration of the Hematolymphoid System

A vast literature exists on the biology of blood formation and regeneration under experimental and clinical conditions. The field of hematopoiesis was recently advanced by the capacity to purify to homogeneity primitive hematopoietic stem and progenitor cells. Isolation of cells at defined maturational stages has enhanced the understanding of the fundamental nature of stem cells, including how cell fate decisions are made, and this understanding is relevant to the development of other normal as well as malignant tissues. This review updates the basic biology of hematopoietic stem cells (HSC) and progenitors, the evolving use of purified HSC as grafts for clinical hematopoietic cell transplantation (HCT) including immune tolerance induction, and the application of HSC biology to other stem cell fields.

3 Ontogeny of hematopoiesis: Examining the emergence of hematopoietic cells in the vertebrate embryo

Hematopoietic stem cells (HSCs) are responsible for generating all the lineages of the blood. During vertebrate development, waves of hematopoietic activity can be found in distinct anatomical sites, and they contribute to both embryonic and adult hematopoiesis. The origin of the HSCs that ultimately give rise to all the adult blood lineages has been a controversial issue in the field of hematopoiesis. Studies of amniotes have linked HSC activity to the aorta-gonad-mesonephros (AGM) region, whereas others suggest that the yolk sac is the true source of HSCs. This review describes both primitive and definitive hematopoiesis in mice, humans, chicks, frogs, and zebrafish and examines the current debate over the embryonic origins of HSCs.

Immune regulation of hematopoiesis

Effective hematopoiesis is a multistep phenomenon. It consists in the presence of pluripotent hematopoietic stem cells (HSC), their proliferation and self-maintenance, their differentiation into various committed lineages of specific progenitors, their orderly maturation into functional cells that are released into the circulation in an orderly fashion in response to the body's demand. Increasing numbers of hematopoietic factors are being purified to homogeneity and/or cloned. The availability of sufficient quantities of these regulators promises a new area for research into the physiology and pathophysiology of the hematopoietic system. The purpose of this overview is to consider some newly-developed concepts in the field of hematopoiesis, with regard to regulatory control mechanisms and cellular interactions.