Bone Marrow Mesenchymal Stromal Cells from Clinical Scale Culture: In Vitro Evaluation of Their Differentiation, Hematopoietic Support, and Immunosuppressive Capacities

Abstract

The differentiation capacity, hematopoietic support, and immunomodulatory properties of human bone marrow mesenchymal stromal cells (BM-MSCs) make them attractive therapeutic agents for a wide range of diseases. Clinical scale cultures (CSCs) have been used to expand BM-MSCs for their use in cell therapy protocols; however, little is known about the functionality of the expanded cells. The main goal of the present study was to evaluate the functional characteristics of BM-MSCs expanded from CSCs to determine the quality of the cells for cellular therapy protocols. To address this issue, we analyzed the morphology, immunophenotype, differentiation potential (adipogenic, osteogenic and chondrogenic), hematopoietic support, and immunosuppressive capacity of BM-MSCs from short scale cultures (SSCs) and CSCs in a comparative manner. After 12 days of culture in CSCs (HYPERFlask System), BM-MSCs reached cell numbers of 125.52 × 10(6) ± 25.6 × 10(6) MSCs, which corresponded to the number of cells required for transplantation (∼1.7 × 10(6) MSCs/kg for a 70-kg patient). After expansion, BM-MSCs expressed the characteristic markers CD73, CD90, and CD105; however, expansion decreased their differentiation capacity toward the adipogenic, osteogenic, and chondrogenic lineages and their ability to inhibit T-cell proliferation compared with SSCs-MSCs. Importantly, CSCs-MSCs maintained the ability to support the proliferation and expansion of hematopoietic progenitor cells and the capacity to express the molecules, cytokines, and extracellular matrix proteins involved in the regulation of hematopoiesis. Our study highlights the need to evaluate the functional properties of the expanded BM-MSCs for verification of their quality for cell therapy protocols.

Human Mesenchymal Stem/Stromal Cells from Umbilical Cord Blood and Placenta Exhibit Similar Capacities to Promote Expansion of Hematopoietic Progenitor Cells In Vitro

“…UCB and PL samples were collected according to the Declaration of Helsinki and the Local Ethics Committee of the Troncoso Hospital (IMSS, Mexico). MSCs from BM ( n = 6), UCB ( n = 6), and PL ( n = 6) were obtained as we previously reported [ 16 , 20 ]. Briefly, mononuclear cells (MNCs) were obtained from BM and UCB samples by density gradient centrifugation (specific gravity < 1.077 g/mL; GE Healthcare Bio-Sciences AB, Uppsala, Sweden).…”

“…Immunophenotypic analysis of MSCs was performed by flow cytometry [ 15 , 20 ]. Monoclonal antibodies against CD14, CD31, CD34, CD45, CD105, HLA-DR (Caltag Laboratories, USA), CD73, and CD90 (Becton Dickinson/PharMingen, USA) conjugated with FITC (fluorescein isothiocyanate), PE (phycoerythrin), or APC (allophycocyanin) were used.…”

“…Osteogenic and adipogenic differentiation was induced with Stem Cell Kits™ (STEMCELL Technologies Inc., Vancouver, BC, Canada), and chondrogenic differentiation was induced using chondrogenic differentiation medium (Cambrex Bio Science Walkersville Inc., Maryland, USA) supplemented with 10 ng/mL transforming growth factor beta (TGF β ; Cambrex). Differentiation capacities were determined using immunocytochemical stains, as we previously reported [ 16 , 20 ].…”

“…As we previously reported [ 20 ], MSC layers at 80% confluence were incubated with 0.3 μ g/mL mitomycin C to inhibit cell growth. Ten thousand cells enriched in CD34 + CD38 − Lin − cells were seeded on MSC layers in 6-well plates (Corning Inc., Costar, New York, NY, USA) in Stem Line medium (Sigma-Aldrich, St. Louis, MO, USA) with or without the early acting cytokines thrombopoietin (TPO), Flt-3 ligand (FL), stem cell factor (SCF), and interleukin-6 (IL-6) at a concentration of 10 ng/mL (Peprotech, USA).…”

The endometrium as a source of mesenchymal stem cells in domestic animals and possible applications in veterinary medicine

“…Similarly, most of the literature has described the application of MSCs from this source for cell therapy protocols. 66,71 Recent evidence indicates that the endometrium can be regenerated by BM-MSCs due to their capacity to promote neoangiogenesis and modify cellular fibrosis. Thus, in a rat model of severe uterine injury, BM-MSCs loaded onto degradable collagen membranes resulted in higher bFGF, IGF-1, TGFβ1 and VEGF levels in the wounded tissue adjacent to the collagen/BM-MSC constructs at four weeks post-transplantation compared to the corresponding tissues in rats who received the collagen construct alone or in the spontaneous regeneration group.…”