The purpose of this study was to evaluate the influence of bone marrow-mesenchymal stem cells (BM-MSC) and exogenously added cytokines on the proliferation, primitive cell subpopulation maintenance (including the c-kit+ marker) and clonogenic capacity of hematopoietic stem cells (HSC). BM-MSC were collected from volunteer donors, isolated and characterized. Umbilical cord blood (UCB) samples were collected from healthy full-term deliveries. UCB-CD34+ cells were cultured in the presence or absence of BM-MSC and/or cytokines for 3 and 7 days. CD34+ cell proliferation was evaluated using the CSFE method and cell phenotype was determined by CD34, c-kit, CD33, CD38, HLA-DR, cyCD22 and cyCD3 detection. Cell clonogenic ability was also assessed. Exogenously added SCF, TPO and FLT3L increasedCD34+ cell proliferation in the presence or absence of BM-MSC, but with concomitant cell differentiation. Without any added cytokines, BM-MSC are able to increase the percentage of primitive progenitors as evaluated by c-kit expression and CFU-GEMM increase. Interestingly, this latter effect was dependent on both cell-cell interactions and secreted factors. A 7-day co-culture period will be optimal for obtaining an increased primitive HSC level. Including c-kit as a marker for primitive phenotype evaluation has shown the relevance of BM-MSC and their secreted factors on UCB-HSC stemness function. This effect could be dissociated from that of the addition of exogenous cytokines, which induced cellular differentiation instead.

An understanding of the cell interactions occurring in the leukemic microenvironment and their functional consequences for the different cell players has therapeutic relevance. By co-culturing mesenchymal stem cells (MSC) with the REH acute lymphocytic leukemia (ALL) cell line, we have established an in vitro leukemic niche for the functional evaluation of hematopoietic stem/progenitor cells (HSPC, CD34+ cells). We showed that the normal homeostatic control exerted by the MSC over the HSPC is considerably lost in this leukemic microenvironment: HSPC increased their proliferation rate and adhesion to MSC. The adhesion molecules CD54 and CD44 were consequently upregulated in HSPC from the leukemic niche. Consequently, with this adhesive phenotype, HSPC showed less Stromal derived factor-1 (SDF-1)-directed migration. Interestingly, multipotency was severely affected with an important reduction in the absolute count and the percentage of primitive progenitor colonies. It was possible to simulate most of these HSPC alterations by incubation of MSC with a REH-conditioned medium, suggesting that REH soluble factors and their effect on MSC are important for the observed changes. Of note, these HSPC alterations were reproduced when primary leukemic cells from an ALL type B (ALL-B) patient were used to set up the leukemic niche. These results suggest that a general response is induced in the leukemic niche to the detriment of HSPC function and in favor of leukemic cell support. This in vitro leukemic niche could be a valuable tool for the understanding of the molecular events responsible for HSPC functional failure and a useful scenario for therapeutic evaluation.

Claudia Camila Mejía-Cruz, Emilia Barreto-Durán, María Alejandra Pardo-Pérez, María Camila Jimenez, Julieth Rincón, Karen Vanegas, Jorge Luis Rodríguez, Luis Fernando Jaramillo-Garcia, Juan Carlos Ulloa, Rodolfo Martínez Díaz, Efrain Leal-García, Rafael Pérez-Núñez, Alfonso Barreto, Viviana M Rodríguez-Pardo. IJSC 1999;0:. https://doi.org/10.15283/ijsc18061

Introduction: The human bone marrow microenvironment is composed of biological, chemical and physical factors that act in a synergistic way to modulate hematopoietic stem cell biology, such as mesenchymal stromal cells (MSCs), endothelial cells (ECs) and low oxygen levels; however, it is difficult to mimic this human microenvironment in vitro. Methods: In this work, we developed 3D multicellular spheroid (3D-MS) for the study of human hematopoietic stem cells (HSCs) with some components of perivascular niche. HSCs were isolated from umbilical cord blood, MSCs were isolated from human bone marrow and a microvasculature EC line (CC-2811, Lonza^®) was used. For the formation of a 3D structure, a magnetic levitation culture system was used. Cultures were maintained in 21%, 3% and 1% O₂ for 15 days. Culture volume, sphericity index and cell viability were determined. Also, human HSC proliferation, phenotype and production of reactive oxygen species were evaluated. Results: After 15 days, 3D-MS exhibited viability greater than 80%. Histology results showed structures without necrotic centers, and higher cellular proliferation with 3% O₂. An increase in the expression of the CD34 antigen and other hematopoietic antigens were observed to 1% O₂ with MSCs plus ECs and low ROS levels. Conclusion: These findings suggest that 3D-MS formed by MSCs, ECs and HSCs exposed to low concentrations of oxygen (1– 3% O₂) modulate human HSC behavior and mimics some features of the perivascular niche, which could reduce the use of animal models and deepen the relationship between the microenvironment of HSC and human hematological diseases development.

BACKGROUND AND OBJECTIVES: The role of bone marrow-mesenchymal stem cells (BM-MSC) in leukae-mic 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. PATIENTS AND METHODS: 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. RESULTS: 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/mm³ cf8/mm³, B-ALL: 15/ mm³ cf3/mm³). 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. CONCLUSIONS: 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.

The modulation of the tumor microenvironment by natural products may play a significant role in the response of tumor cells to chemotherapy. In this study, we evaluated the effect of extracts derived from P2Et (Caesalpinia spinosa) and Anamú-SC (Petiveria alliacea) plants, previously studied by our group, on the viability and ROS levels in the K562 cell line (Pgp− and Pgp+), endothelial cells (ECs, Eahy.926 cell line) and mesenchymal stem cells (MSC) cultured in 2D and 3D. The results show that: (a) the two botanical extracts are selective on tumor cells compared to doxorubicin (DX), (b) cytotoxicity is independent of the modulation of intracellular ROS for plant extracts, unlike DX, (c) the interaction with DX can be influenced by chemical complexity and the expression of Pgp, (d) the 3D culture shows a greater sensitivity of the tumor cells to chemotherapy, in co-treatment with the extracts. In conclusion, the effect of the extracts on the viability of leukemia cells was modified in multicellular spheroids with MSC and EC, suggesting that the in vitro evaluation of these interactions can contribute to the comprehension of the pharmacodynamics of the botanical drugs.