Journal of Hematology & Thromboembolic Diseases
Open Access
ISSN: 2329-8790
ISSN: 2329-8790
Mini Review - (2025)Volume 13, Issue 1
WNT proteins constitute a family of secreted signaling molecules pivotal in orchestrating cell fate and behavior. This study investigated the potential impact of WNTs on hematopoietic cell destiny. Utilizing both Hematopoietic Progenitor Cell (HPC)-enriched embryonic avian bone marrow cells and the quail mesodermal stem cell line QCE6, comparable behaviors were observed under optimized conditions. These cells demonstrated identical differentiation into diverse blood cell types, including Red Blood Cells (RBCs), monocytes, macrophages, granulocytes and thrombocytes. The extensive spectrum of blood cell phenotypes exhibited by QCE6 cells was contingent upon their active expression of WNT11. Conversely, inhibition of WNT11 production in QCE6 cells, achieved through the expression of a stably transfected WNT11 antisense transgene, resulted in cultures dominated by highly vacuolated macrophages. Notably, RBCs were absent and monocyte presence was significantly reduced in these cultures. However, exposure of WNT11 antisense cells to soluble WNT11 or WNT5a effectively reinstated the diverse range of blood cell phenotypes seen in parental QCE6 cells. Augmenting WNT protein expression in QCE6 cells through overexpression accentuated the prevalence of RBCs and monocytes while markedly diminishing macrophage abundance. Intriguingly, treating HPC-enriched bone marrow cultures with soluble WNT11 or WNT5a impeded macrophage formation. Instead, WNT-treated bone marrow cultures predominantly showcased monocytes and RBCs. These findings collectively suggest a pivotal role for WNTs in regulating hematopoietic cell fate
WNT proteins; Hematopoiesis; Red blood cell; QCE6; Cell fate
The blood is composed of various cell types believed to originate from a common multipotent stem cell with mesodermal origins. Blood cell formation during embryogenesis involves at least two distinct episodes. Initially, primitive Hematopoietic Progenitor Cells (HPCs) emerge from ventral mesodermal cells in blood islands during early somite stage embryos. These cells primarily give rise to erythrocytes, providing circulating blood cells for the developing embryo. Subsequently, definitive hematopoiesis occurs as intra-embryonic dorsal mesoderm contributes to a second wave of blood cell formation. These HPCs, originating within the embryo, develop into myeloid and lymphoid cell lineages, serving as the foundation for adult hematopoiesis. As embryogenesis progresses, hematopoietic cells migrate to various sites such as the spleen, thymus and liver, with the bone marrow becoming the principal site for HPC regeneration and maturation in adulthood. While avian hematopoiesis closely resembles mammalian hematopoiesis, there are notable differences. Avian Red Blood Cells (RBCs) are nucleated and certain granulocytic cell types, including heterophils, are exclusive to birds. Additionally, thrombocytes, equivalent to mammalian platelets, are the progeny of mononuclear thromboblasts in birds. The study of avian hematopoiesis faces challenges due to the limited availability of cell identification markers, necessitating the use of cytological examination and enzymatic activity detection to identify blood cell types [1].
Numerous extracellular signaling factors regulate hematopoietic cell differentiation, including granulocyte-macrophage colonystimulating factor, erythropoietin, interleukins, bFGF, transforming Growth Factor β, BMP4, Stem Cell Factor (SCF) and WNT proteins. WNTs, a group of highly conserved signaling proteins, play a crucial role in embryonic tissue patterning and have been observed in hematopoietic tissue in mice and human bone marrow [2]. The present study explores the influence of WNTs on hematopoietic cell differentiation using the QCE6 mesodermal stem cell line derived from Japanese quail embryos. The results indicate that WNTs significantly impact the diversity of hematopoietic cell phenotypes exhibited by both QCE6 and bone marrow-derived cells, inhibiting macrophage formation and increasing the prevalence of monocyte and erythrocyte cell phenotypes.
The study involved the collection of femurs from Embryonic Day 14 (ED14) quail embryos to explore primary cell extraction and blood cell differentiation processes. The extraction of primary cells was carried out by flushing bone marrow cavities with Dulbecco's Modified Eagle's Medium (DMEM) supplemented with penicillin–streptomycin. The subsequent dissociation of bone marrow cords and isolation of the mononuclear cell fraction were achieved through specific techniques, including passage through a 20 gauge needle and Ficoll-Paque gradient centrifugation [3]. For cell cultures, purified avian bone marrow or QCE6 cells were utilized and seeded in dishes containing semisolid medium. This medium composition included various components such as methylcellulose, fetal bovine serum, chick serum, WeHi-3–conditioned medium, N-acetyl-D-glucosamine, deionized bovine serum albumin, ConA-stimulated spleen cell– conditioned medium, iron-saturated transferrin, erythropoietin, Stem Cell Factor (SCF), insulin-like growth factor I, hydrocortisone and pen/strep. The cultures were incubated for up to 7 days at 37°C [4].
Stable transfectants of QCE6 cells with altered WNT11 expression levels were produced by introducing full-length WNT11 complementary DNA (cDNA) into QCE6 cells. The resulting clones, designated as WNT11ox and WNT11αs, were selected for neomycin resistance. The study specifically used a single WNT11 sense clone (WNT11ox/3) and two antisense subclones (WNT11αs/4 and WNT11αs/6) [5]. The relative levels of WNT11 protein produced by these clones were assessed through immunoblot analysis. Additionally, WNT5a-expressing QCE6 cells were generated by amplifying Xenopus WNT5a cDNA and inserting it into the expression vector pcDNA3.1/myc-6xHis A. The resulting WNT5a/QCE6 clonal subline was selected based on high-secreting sublines identified by immunoblotting.
The influence of ectopic WNT protein on blood cell diversification was investigated by culturing embryonic bone marrow cells, QCE6 cells or QCE6 stably transfectants in Methylcellulose (MeC) or Fibrin Gel (FB) cultures. Conditioned medium from WNT11ox/3 or WNT5a/QCE6 cells, or Ni-NTA column-purified WNT5a protein, was used in place of DMEM. Both MeC and FB cultures were monitored for cell growth and differentiation, with colonies scored based on number and cell phenotype. Various staining techniques, such as May-Grünwald- Giemsa, esterase and phosphatase stains, were employed to assess blood cell phenotypes within the cultures [6]. Naphthol AS-D CAE, ANAE, AP and TARP stains were performed using Sigma kits to further characterize the cells. The study provided comprehensive insights into the methodologies and experimental setups used to investigate the impact of WNT proteins on blood cell differentiation. The molecular regulation of Hematopoietic Progenitor Cell (HPC) diversification in avian bone marrow. The researchers optimized conditions for obtaining a range of blood cell phenotypes from embryonic avian bone marrow. HPCs were isolated using Ficoll-Paque gradients and subsequent cultures in specific conditions led to the formation of distinct colonies, including erythroid, granulocyte/macrophage, granulocyte/erythroid/macrophage and macrophage colonies.
After extensive experimentation, the researchers developed culture conditions supporting complete hematopoiesis in both suspension cultures and fibrin gels [7]. A mixture of chicken and bovine sera, along with cytokines, proved optimal for supporting cell growth and differentiation. The cultures exhibited morphologically distinct colonies representing various blood cell lineages, including erythroid, granulocyte/macrophage, granulocyte/erythroid/ macrophage, granulocyte, macrophage/erythroid and macrophage. Further analyses were conducted on bone marrow cells cultured in both suspension and fibrin gels, showing maximal colony growth and differentiation by day 6. Cytological examination revealed the presence of various blood cell types, including erythroblasts, macrophages, granulocytes, monocytes and thrombocytes. The distribution of specific enzyme activities associated with leukocytes confirmed the differentiation of various cell types [8].
The researchers then explored the hematopoietic potential of the mesodermal stem cell line QCE6 under similar conditions. QCE6 cells exhibited a broad range of blood cell phenotypes, including RBCs, granulocytes, monocytes, macrophages and thrombocytes, in both suspension and fibrin gel cultures. The QCE6 cells were compared to bone marrow-derived cells in terms of enzyme activity and similar patterns were observed. Influence of WNT11 expression on the hematopoietic diversification of QCE6 cells. Different QCE6 subclones with altered levels of WNT11 expression were generated. Overexpression of WNT11 led to a shift toward the erythrocyte lineage and a decrease in macrophage formation. Conversely, reduced WNT11 expression resulted in a phenotypic shift toward macrophages. The study further explored the effect of WNT5a expression on QCE6 cells, revealing similar outcomes to WNT11 overexpression.
This study explores the impact of WNT proteins on the phenotypic distribution of blood cells, utilizing QCE6 cells as a model of hematopoiesis. QCE6 cells, initially characterized for cardiac and endothelial cell potential, were found to exhibit a broad hematopoietic cell potential, generating various blood cell types under optimized culture conditions. The study compares the behavior of QCE6 cells to bone marrow Hematopoietic Progenitor Cells (HPCs) and investigates the influence of WNT expression on blood cell diversification.
QCE6 cells, derived from early gastrula stage quail embryos, were observed to differentiate into Red Blood Cells (RBCs), granulocytes, monocytes, macrophages and thrombocytes, mirroring the phenotypes generated from bone marrow HPCs. This unexpected multipotentiality of QCE6 cells, originating from a mesodermal area not conventionally associated with blood cell production, suggests their utility as a tool to study hematopoiesis regulatory mechanisms. Notably, the study focuses on WNT11 expression in QCE6 cells, a property shared with a subset of anterior mesodermal cells. Modulation of WNT11 expression significantly influenced the formation of specific blood cell phenotypes. Inhibition of WNT11 production in QCE6 cells led to cultures dominated by macrophages, lacking RBCs and producing few monocytes. Conversely, exposure to ectopic WNT protein restored the formation of RBCs and monocytes. Furthermore, overexpressing WNT protein in QCE6 cells increased RBC and monocyte prevalence while inhibiting macrophage differentiation [9].
Comparison with bone marrow HPCs exposed to WNT11 or WNT5a revealed similar effects, indicating that QCE6 cells provide valuable insights into the regulated diversification of bone marrow HPCs. The study emphasizes the shared functional activities of WNT11 and WNT5a in influencing blood cell phenotypes. The data suggest that WNT5a or a similar signal, such as WNT11, shifts cell fate from macrophages to RBCs, while also inhibiting the differentiation of monocytes into macrophages. Overall, the study provides a compelling case for the role of WNT proteins in influencing blood cell formation, highlighting the intricate relationship between WNT signaling and the differentiation of various blood cell lineages. The findings prompt further exploration into the potential linkage between regulatory events impacting macrophage lineage and the closer relationship between RBCs and monocytes than previously suspected.
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Citation: Parker S (2025) WNT Proteins in Hematopoiesis: Regulatory Mechanisms Using the QCE6 Mesodermal Stem Cell Line. J Hematol Thrombo Dis. 13:645.
Received: 08-Dec-2023, Manuscript No. JHTD-23-28417 ; Editor assigned: 12-Dec-2023, Pre QC No. JHTD-23-28417 (PQ); Reviewed: 26-Dec-2023, QC No. JHTD-23-28417 ; Revised: 15-Jan-2025, Manuscript No. JHTD-23-28417 (R); Published: 22-Jan-2025 , DOI: 10.35248/2329-8790.25.13.645
Copyright: © 2025 Parker S. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.