Identification and Characterization of Hematopoietic Stem and Progenitor Populations from Cord Blood-Derived CD34+ Cells 2306499

Abstract Introduction Hematopoietic stem and progenitor cells (HSPCs) are essential for blood cell development and immune system function. Accurate identification and phenotypic characterization of these populations are critical for understanding hematopoiesis and optimizing stem cell-based therapies. CD34+ cells derived from cord blood represent a rich source of HSPCs. Here, we used standardized protocols for thawing, staining, and short-term culture of CD34+ cells, followed by multiparametric flow cytometric analysis using CytoFLEX mosaic 88 Spectral Detection Module to delineate stem and progenitor subsets. Methods Multiparametric flow cytometric analysis was performed using the CytoFLEX mosaic 88 Spectral Detection Module to identify hematopoietic stem cells (HSCs) and progenitor subsets, including multipotent progenitors (MPP), common myeloid progenitors (CMP), granulocyte-monocyte progenitors (GMP), and megakaryocyte-erythroid progenitors (MEP). Subsets were defined based on established immunophenotypic marker combinations. Comparative analysis was conducted across three platforms: CytoFLEX SRT, CytoFLEX LX, and CytoFLEX mosaic 88 Spectral Detection Module. Results The gating strategy enabled clear discrimination of HSCs (CD34+CD38⁻CD90+CD45RA⁻) and downstream progenitor populations across all CytoFLEX platforms. Resolution of these populations, assessed by staining index (SI), demonstrated the highest SI values with the CytoFLEX mosaic 88 Spectral Detection Module, indicating superior performance in resolving rare subsets. Conclusion This protocol provides a robust approach for immunophenotyping cord blood-derived CD34+ cells, enabling precise identification of HSPC subsets by flow cytometry. The ability to analyze and sort these populations supports downstream applications in stem cell research, transplantation biology, and regenerative medicine. Funding Source n/a Topic Categories Hematopoiesis and Immune System Development (HEM)