Generation of Organotypic Multicellular Spheres by Magnetic Levitation: Model for the Study of Human Hematopoietic Stem Cells Microenvironment

BACKGROUND AND OBJECTIVE: The characteristics of human hematopoietic stem cells are conditioned by the microenvironment of the bone marrow, where they interact with other cell populations, such as mesenchymal stem cells and endothelial cells; however, the study of this microenvironment is complex. The objective of this work was to develop a 3D culture system by magnetic levitation that imitates the microenvironment of human HSC. METHODS AND RESULTS: Human bone marrow-mesenchymal stem cells, umbilical cord blood-hematopoietic stem cells and a non-tumoral endothelial cell line (CC2811, Lonza®) were used to develop organotypic multicellular spheres by the magnetic levitation method. We obtained viable structures with an average sphericity index greater than 0.6, an average volume of 0.5 mm3 and a percentage of aggregation greater than 70%. Histological studies of the organotypic multicellular spheres used hematoxylin and eosin stains, and an evaluation of vimentin expression by means of immunohistochemistry demonstrated an organized internal structure without picnotic cells and a high expression of vimentin. The functional capacity of human hematopoietic stem cells after organotypic multicellular spheres culture was evaluated by multipotency tests, and it was demonstrated that 3D structures without exogenous Flt3L are autonomous in the maintenance of multipotency of human hematopoietic stem cells. CONCLUSIONS: We developed organotypic multicellular spheres from normal human cells that mimic the microenvironment of the human hematopoietic stem cells. These structures are the prototype for the development of complex organoids that allow the further study of the biology of normal human stem cells and their potential in regenerative medicine.

Mesenchymal stem cells promote leukaemic cells aberrant phenotype from B-cell acute lymphoblastic leukaemia

The role of bone marrow-mesenchymal stem cells [BM-MSC] in leukaemic cell control is controversial. The purpose of this work was to evaluate BM-MSC role regarding the viability, proliferation and immunophenotype of normal B-cell precursors from control [Ct] patients and leukaemic cells from B-acute lymphoblastic leukaemia [B-ALL] patients. BM-MSC were isolated and characterised from voluntary donors. Mononuclear cells isolated from Ct and B-ALL bone marrow samples were cultured in the presence or absence of BM-MSC for 7 days. Cell viability was determined with LIVE/DEAD and proliferation index evaluated by CFSE labelling. Cell population immunophenotypes were characterised by estimating CD19, CD10, CD20 and CD45 antigens by flow cytometry. After co-culture, B-ALL cells exhibited higher viability [20-40%] as compared to just cells [3-10%]. Ct and B-ALL absolute cell counts were higher in the presence of BM-MSC [Ct: 25/mm[3] cf 8/mm[3], B-ALL: 15/mm[3] cf 3/mm[3]]. Normal B-cell subpopulations in co-culture had increased expression of CD19 and CD10 [Pre-pre B] and CD45 and CD20 antigens [Pre-B]. B-ALL cells co-cultured with BM-MSC showed an increase in CD19 and CD20, although the greatest increase was observed in the CD10 antigen. Lymphoid cell maintenance, at early stages of differentiation, was significantly promoted by BM-MSC in normal and leukaemic cells. Co-cultures also modulated the expression of antigens associated with the B-ALL asynchronous phenotype as CD10 co-expressed with CD19 and CD20. To our knowledge, this is the first time that CD10, CD19 and CD20 leukaemic antigens have been reported as being regulated by BM-MSC