Flow cytometric characterization of perfused human bone marrow cultures: Identification of the major cell lineages and correlation with the CFU‐GM assay

Abstract

Background: Prolific cultures of human bone marrow mononuclear cells (BM MNCs) were recently developed that include a full spectrum of hematopoietic and accessory cells, with the presence of autofluorescent cells indicating adequate cell expansion. However, phenotypic and functional clonogenic characterizations of the autofluorescent cells and the various other subpopulations present in these cultures have not been carried out. Methods: Cells from a continuously perfused bioreactor inoculated with BM MNCs and cultured for 12 days in serum-containing medium with PIXY321, erythropoietin, and with or without FLT3-L were evaluated by using flow cytometry. Results: Two antibodies, CD71 and CD13, allowed the separation of the autofluorescent cells into two distinct populations. The CD71 ϩ CD13 ϩϩ autofluorescent population contained the colony-forming unit (CFU) fibroblast, and the CD71 ϩϩ CD13 ϩϩ autofluorescent population contained macrophage/dendritic like cells. The CFU-granulo-

Analysis of Immunophenotypic Changes during Ex Vivo Human Erythropoiesis and Its Application in the Study of Normal and Defective Erythropoiesis

“…This finding was similar to what had previously been reported [7]. Several studies state GPA as a late erythroid marker [7,24]; however, we observed that GPA expression is first observed on day 8 and was found to be maximal at day 18 and decreases further (Figure 1G). Moreover, benzidine staining revealed that hemoglobin accumulation appears on day eight and gradually increases during differentiation (Figure 1H(i,ii)).…”

Understanding Hematological Toxicities Using Mathematical Modeling

“…The first maturation stage of HSCs are multipotent progenitors (MPPs), which give rise to the lineage‐committed oligopotent progenitors: 1) megakaryocyte‐erythrocyte progenitors (MEPs), which can differentiate into red blood cells or platelets; 2) common myeloid progenitors (CMPs), which maturate in myeloid cells; and 3) common lymphoid progenitors (CLPs), which give birth to immune cells (mostly located in lymphoid organs). Cell types can be identified based on the expression of their surface antigen profiles, and the main cell surface markers for hematopoietic human cells are reported in the figure. Dotted boxes represent the last stages of cell maturation before their release into circulation.…”

“…In fact, the purified population of stem cells is always heterogeneous, containing a mixture of “true” stem cells and progenitors, which obstacles a clear separation of drug effects in these two key populations. Heterogeneity is also observed in the other cell compartments, due to the complexity of cell markers and differential analysis …”