Characterization Of Hematopoietic Cells Research Articles

The role of flow cytometry in the classification of myeloid disorders.

The World Health Organization classification (WHO-HAEM5) and the International Consensus Classification (ICC 2022) of myeloid neoplasms are based on the integration of clinical, morphologic, immunophenotypic, and genomic data. Flow cytometric immunophenotyping (FCIP) allows the identification, enumeration, and characterization of hematopoietic cells, and is therefore apowerful tool in the diagnosis, classification, and monitoring of hematological neoplasms. The vast majority of flow cytometry (FCM) studies in chronic myeloid neoplasms focus on its role in myelodysplastic neoplasms (MDS). FCM can also be helpful for the assessment of myeloproliferative neoplasms (MPN) and MDS/MPN, including the early detection of evolving myeloid or lymphoid blast crisis and the characterization of monocytic subsets. The classification of acute myeloid leukemia (AML) is primarily based on cytogenetic and molecular findings; however, FCIP is needed for subclassification of AML, not otherwise specified (NOS; ICC)/AML defined by differentiation (WHO-HAEM5). The main role of FCM in AML remains in making arapid diagnosis and as atool for measurable residual disease monitoring. Machine learning and artificial intelligence approaches can be used to analyze and classify FCM data. This article, based on an invited lecture at the 106th Annual Meeting of the German Society of Pathology in 2023, reviews the role of FCM in the classification of myeloid neoplasms, including recent publications on the application of artificial intelligence.

Characterization of hematopoietic cells generated from normal and leukemic human induced pluripotent stem cells (hiPSCs) in teratomas

Characterization of hematopoietic cells generated from normal and leukemic human induced pluripotent stem cells (hiPSCs) in teratomas

Localization and characterization of hematopoietic cells in the mouse embryonic head

The midgestation mouse head harbors hematopoietic stem cells (HSCs) at the same time HSCs are detected in the aorta, gonads, mesonephros region (AGM). Within the complex structure of the embryonic head, the localization and phenotype of HSCs and hematopoietic progenitor cells (HPCs) remains unknown. Since the Ly6a(Sca1)GFP transgene is a reporter for all AGM HSCs and some hemogenic endothelial cells (HEC), we examined these embryos for head HSC, HEC and HPC. Wholemount staining reveals some GFP+ cells in E10.5 head vasculature. By FACS analysis, 0.6% of E11.5 head cells are GFP+. To examine whether head HPC/HSC are enriched in the Ly6aGFP+ population, we sorted E11.5-E12.5 head cells and performed functional assays. Preliminary data show that head HSCs are GFP+. When we subdissected the head into forebrain (FB), midbrain (MB), and hindbrain with brachial arches (HBA) to localize HPCs, we found erythro-myeloid progenitor cells in GFP+ and GFP- fractions of all three brain regions at E10-11.5. Interestingly, in OP9 co-cutures, B cell potential was mostly observed in CD31+GFP+ of the three subregions. These results suggest that head HSCs (Ly6aGFP) are phenotypically and functionally similar to AGM HSCs. We are continuing to identify the precise head subregion(s) in which HSCs occur, examine the hemogenic potential of the head vasculature, and also the pathways affecting their development.

Identification of a population of cells with hematopoietic stem cell properties in mouse aorta–gonad–mesonephros cultures

The aorta–gonad–mesonephros (AGM) region is a primary source of definitive hematopoietic cells in the midgestation mouse embryo. In cultures of dispersed AGM regions, adherent cells containing endothelial cells are observed first, and then non-adherent hematopoietic cells are produced. Here we report on the characterization of hematopoietic cells that emerge in the AGM culture. Based on the expression profiles of CD45 and c-Kit, we defined three cell populations: CD45 low c-Kit + cells that had the ability to form hematopoietic cell colonies in methylcellulose media and in co-cultures with stromal cells; CD45 low c-Kit − cells that showed a granulocyte morphology; CD45 high c-Kit low/− that exhibited a macrophage morphology. In co-cultures of OP9 stromal cells and freshly prepared AGM cultures, CD45 low c-Kit + cells from the AGM culture had the abilities to reproduce CD45 low c-Kit + cells and differentiate into CD45 low c-Kit − and CD45 high c-Kit low/− cells, whereas CD45 low c-Kit − and CD45 high c-Kit low/− did not produce CD45 low c-Kit + cells. Furthermore, CD45 low c-Kit + cells displayed a long-term repopulating activity in adult hematopoietic tissue when transplanted into the liver of irradiated newborn mice. These results indicate that CD45 low c-Kit + cells from the AGM culture have the potential to reconstitute multi-lineage hematopoietic cells.

Characterization of Hematopoietic Stem/Progenitor Property of Cells in the Culture of the Mouse Aorta-Gonad-Mesonephros Region.

The aorta-gonad-mesonephros (AGM) region is a primary source of definitive hamotopoietic cells in the midgestation mouse embryo. In the culture of the dispersed AGM region with cytokines such as stem cell factor, basic fibroblast growth factor, and oncostatin M, adherent cells containing the endothelial cells were firstly observed, and then nonadherent hematopoietic cells were produced. In the present study, we report on the characterization of hematopoietic cells that emerge in the AGM culture. Based on the expression profile of CD45 and c-Kit examined by flow cytometry, we defined three cell populations: CD45low c-Kit+ (population 1), CD45low c-Kit− (population 2), and CD45high c-Kitlow/− (population 3) cells. Cells in the population 1 exhibited immature morphology with a large nucleus and cells in populations 2 and 3 displayed morphology characteristic to granulocytes and macrophages, respectively. Expression levels of the CD31, GATA-2, and AML-1 genes were higher in cells in population 1 than in cells in the other two populations. The number of colonies formed in the semi-solid culture of cells in the above-mentioned three populations was assessed. The semi-solid colony-forming activity was much higher in cells in the population 1 than those in the other two populations. It has been reported that hematopoietic stem/progenitor cells can be detected in cobble stone area forming cells in the coculture system with stromal cells. Only the population 1 cells had cobble stone area-forming activity when cultured with OP9 stromal cells. A part of the population 1 was positive for Sca-1. Among the population 1 cells, cobble stone area-forming activity of Sca-1+ cells (i.e., CD45low c-Kit+ Sca-1+ cells) was 6-fold higher than that of Sca-1− cells (i.e., CD45low c-Kit+ Sca-1− cells). When the population 1 cells were plated and cultured on a monolayer of OP9 stromal cells, three types of cells whose characters fitted all the above-mentioned population 1, 2, and 3, in terms of the CD45/c-Kit expression profile and microscopically observed morphology, emerged again. In contrast, cells in the populations 2 and 3 had no such features. Similarly, when GFP+ population 1 cells from the GFP retrovirus-infected AGM culture were cultured again with the freshly prepared dispersed AGM region, we found all populations 1, 2, and 3 with GFP expression. These results suggested that immature hematopoietic cells in the AGM culture were highly enriched in the population 1. We next performed intrahepatic injection of the population 1 cells into irradiated newborn mice. Four months after transplantation, myeloid reconstitution was found in the spleen, bone marrow, and peripheral blood of mice transplanted with population 1 cells. Lymphoid reconstitution was also observed in the spleen and thymus of the same recipients. These results indicate that the CD45low c-Kit+ cells produced in the AGM culture have the potential to reconstitute multi-lineage hematopoietic cells.

Immunophenotyping of acute leukemias and myelodysplastic syndromes.

Alberto Orfao,* Francisco Ortuno, Maria de Santiago, Antonio Lopez, and Jesus San Miguel Servicio General de Citometria, Universidad de Salamanca, Salamanca, Spain Centro de Investigacion del Cancer y Departamento de Medicina, Universidad de Salamanca, Salamanca, Spain Servicio de Hematologia y Oncologia Medica, Hospital General Universitario J.M. Morales Meseguer, Murcia, Spain Servicio de Hematologia, Hospital Universitario de Salamanca, Salamanca, Spain

Functional evaluation of DC-SIGN monoclonal antibodies reveals DC-SIGN interactions with ICAM-3 do not promote human immunodeficiency virus type 1 transmission.

DC-SIGN, a type II membrane-spanning C-type lectin that is expressed on the surface of dendritic cells (DC), captures and promotes human and simian immunodeficiency virus (HIV and SIV) infection of CD4(+) T cells in trans. To better understand the mechanism of DC-SIGN-mediated virus transmission, we generated and functionally evaluated a panel of seven monoclonal antibodies (MAbs) against DC-SIGN family molecules. Six of the MAbs reacted with myeloid-lineage DC, whereas one MAb preferentially bound DC-SIGNR/L-SIGN, a homolog of DC-SIGN. Characterization of hematopoietic cells also revealed that stimulation of monocytes with interleukin-4 (IL-4) or IL-13 was sufficient to induce expression of DC-SIGN. All DC-SIGN-reactive MAbs competed with intercellular adhesion molecule 3 (ICAM-3) for adhesion to DC-SIGN and blocked HIV-1 transmission to T cells that was mediated by THP-1 cells expressing DC-SIGN. Similar but less efficient MAb blocking of DC-mediated HIV-1 transmission was observed, indicating that HIV-1 transmission to target cells via DC may not be dependent solely on DC-SIGN. Attempts to neutralize DC-SIGN capture and transmission of HIV-1 with soluble ICAM-3 prophylaxis were limited in success, with a maximal inhibition of 60%. In addition, disrupting DC-SIGN/ICAM-3 interactions between cells with MAbs did not impair DC-SIGN-mediated HIV-1 transmission. Finally, forced expression of ICAM-3 on target cells did not increase their susceptibility to HIV-1 transmission mediated by DC-SIGN. While these findings do not discount the role of intercellular contact in facilitating HIV-1 transmission, our in vitro data indicate that DC-SIGN interactions with ICAM-3 do not promote DC-SIGN-mediated virus transmission.

Quantitative Long-Term Culture-Initiating Cell Assays Require Accessory Cell Depletion That Can Be Achieved by CD34-Enrichment or 5-Fluorouracil Exposure

Characterization of hematopoietic cells and measurement of their proliferative potential is critical in many research and clinical applications. Because in vivo assay of human cells is not possible and xenogeneic assays are not yet routine, in vitro assays such as the long-term culture-initiating cell (LTC-IC) assay have been widely adopted. This study investigated LTC-IC assay linearity and reproducibility and resulting implications with respect to quantitation of primitive cell expansion. Measurement of secondary colony-forming cells (2° CFCs) from 5-week cultures of bone marrow (BM) mononuclear cells (MNCs) showed that 2° CFC frequency varied with assay plating density in a nonlinear fashion. The measured 2° CFC frequency increased from 4.6 to 63.8 (per 105 MNCs) as assay plating density was decreased from 5 × 105 to 2 × 104 MNCs per well (P < 10−6, n = 37). In contrast, assay of CD34-enriched cells was linear within the range studied. Assays of cells obtained from expansion cultures initiated with either MNCs or CD34-enriched cells were also nonlinear. Consequently, calculated 2° CFC expansion ratios were ambiguous and dependent on the assay plating densities used. Limiting dilution analysis (LDA) results were also nonlinear, with LTC-IC frequency increasing from 8.2 to 22.4 per 105 MNCs (P < 10−4, n = 100) as assay plating densities were decreased. Despite the nonlinearity, 2° CFC and LTC-IC assay results were consistent and reproducible over time with different samples and techniques and gave a semiquantitative indication of relative primitive cell frequency. Although CD34-enriched cells gave linear assay output, purification of cells for every assay is impractical. Therefore, exposure of cells to 5-fluorouracil (5-FU) was explored for improving assay linearity. Incubation of MNCs in 250 μg/mL 5-FU for 1 to 2 hours depleted accessory cells and resulted in a cell population that gave linear 2° CFC readout. The 5-FU–resistant LTC-ICs accounted for 49% of the total LTC-IC population, adding the potential benefit of restricting assay measurement to more primitive noncycling LTC-ICs. Consequently, similar linear assay results can be obtained with either the bulk 2° CFC or LDA LTC-IC methods after 5-FU, but multiple plating densities are nevertheless still required in both methods due to the greater than 100-fold range in primitive cell frequency present in normal human donor BM.