Image cytometry differentiates CD34 ⁺ and CD133 ⁺ profiles of very small embryonic-like stem cells

Very small embryonic-like stem cells (VSELs) have been identified as potential precursors of the endothelial lineage in humans. Traditionally characterized by CD34 and CD133 markers, these populations remain poorly delineated, particularly regarding their morphological traits. Leveraging recent advances in imaging flow cytometry (iFC), this study sought to refine the characterization of VSELs by analyzing CD34⁺ and CD133⁺ Lin^⁻ CD45^⁻ cells from various sources, including bone marrow (BM), mobilized blood (MB), peripheral blood (PB), and cord blood (CB). Using high-resolution iFC, we assessed cell circularity and size, identifying significant morphological differences within subpopulations smaller than 7 µm. CD133⁺ cells predominantly exhibited irregular shapes, whereas CD34⁺ cells, regardless of CD133 co-expression, were mostly circular. Quantification across multiple sources revealed an enrichment of small circular CD34⁺ cells, especially in BM, MB, and CB. A comparative analysis with conventional flow cytometry (cFC) showed similar counts for CD34⁺ cells between methods (<i>P</i> = 0.06) but a significantly higher number of CD133⁺ cells with cFC (<i>P</i> < 0.0001), reflecting its inability to assess morphology. These findings highlight iFC as a powerful tool for rare stem cell analysis and propose CD34, rather than CD133, as a more reliable marker for identifying circular VSELs under 7 µm, refining future stem cell-isolation strategies.