3D Multicellular Spheroid for the Study of Human Hematopoietic Stem Cells: Synergistic Effect Between Oxygen Levels, Mesenchymal Stromal Cells and Endothelial Cells.

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.

TRPM8 Channel Promotes the Osteogenic Differentiation in Human Bone Marrow Mesenchymal Stem Cells.

Various families of ion channels have been characterized in mesenchymal stem cells (MSCs), including some members of transient receptor potential (TRP) channels family. TRP channels are involved in critical cellular processes as differentiation and cell proliferation. Here, we analyzed the expression of TRPM8 channel in human bone marrow MSCs (hBM-MSCs), and its relation with osteogenic differentiation. Patch-clamp recordings showed that hBM-MSCs expressed outwardly rectifying currents which were increased by exposure to 500 µM menthol and were partially inhibited by 10 µM of BCTC, a TRPM8 channels antagonist. Additionally, we have found the expression of TRPM8 by RT-PCR and western blot. We also explored the TRPM8 localization in hBM-MSCs by immunofluorescence using confocal microscopy. Remarkably, hBM-MSCs treatment with 100 µM of menthol or 10 µM of icilin, TRPM8 agonists, increases osteogenic differentiation. Conversely, 20 µM of BCTC, induced a decrease of osteogenic differentiation. These results suggest that TRPM8 channels are functionally active in hBM-MSCs and have a role in cell differentiation.

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.

Impact of donor characteristics on the quality of bone marrow as a source of mesenchymal stromal cells.

In recent years, the therapeutic use of mesenchymal stromal cells (MSC) has generated a valuable number of scientific studies that delve into their biological characteristics and their potential in regenerative medicine; however, the impact of the clinical characteristics of tissue donors, from which these cells are isolated, on their potential in applied clinical research is not yet clear. The objective of this study was to evaluate the impact of the clinical characteristics of bone marrow donors on the quality of this tissue as a source of MSC for therapeutic use. Human MSC were isolated, characterized and cultured (according to ISCT criteria) from bone marrow samples from volunteer donors (n = 70) attending the Department of Orthopedics and Traumatology of the Hospital Universitario San Ignacio (Bogota, Colombia) for surgery of prosthetic hip replacement that agreed to participate voluntarily in the study. Donor data such as age, gender, weight, smoker and type of anesthesia used during the surgical procedure were recorded, and the impact of these characteristics on the volume of tissue collection, mononuclear cell count and confluence time of cells with fibroblastoid morphology was evaluated. Correlation coefficients between quantitative variables were calculated with Spearman's correlation test, and the association between qualitative and quantitative variables was evaluated with biserial correlation coefficient. A significant correlation was observed between the age of the donors and the time necessary to obtain confluent cells in vitro (r = 0.2489, P = 0.0377); similarly, the correlation between the volume of bone marrow collected and the number of mononuclear cells obtained was significant (r = 0.7101, P = 0.0001). Although a negative correlation tendency was observed between the mononuclear cell count and the confluence time, this was not significant (r = -0.2041, P = 0.0950). No significant associations were observed between gender, smoking status or type of anesthesia and the expansion characteristics of human mesenchymal stromal cells. Bone marrow donor age and the tissue collection volume impact the time of obtaining MSC in vitro and the mononuclear cell count with which the culture starts. These conditions must be considered when the bone marrow is selected as the tissue for obtaining MSC.