Viability and angiogenic activity of mesenchymal stromal cells from adipose tissue and bone marrow under hypoxia and inflammation in vitro

Progenitor stromal cells derived from adipose tissue (ADSC) and bone marrow (BMDSC) hold great promise for use in the cell-based therapy of ischemic diseases. It was demonstrated that these cells secrete a number of angiogenic cytokines that stimulate vascularization. It was demonstrated that ADSC or BMDSC injected intramuscularly or intravenously into the animals with experimental hind-limb ischemia improve vascularization. However, low oxygen levels and inflammation may impair the viability and functional activity of transplanted cells. We have examined ADSC and BMDSC properties in vitro under hypoxic and inflammatory conditions. ADSC and BMDSC derived from Balb/c mice have been cultivated under hypoxia or in the presence of inflammatory cytokines. The viability of cells assessed by annexin V-PE binding and 7AAD storage, as well as by the quantitative TUNEL method, was not changed under hypoxic conditions Cell exposure to inflammatory cytokines induced apoptosis in 70% of cells. Inflammatory cytokines did not stimulate gene expression of angiogenic growth factors. Under hypoxia conditions up-regulation of genes for pro-angiogenic factors and down-regulation of anti-angiogenic genes were more apparent in ADSC. Using angiogenesis models in vitro and in vivo, we demonstrated that stromal cell maintenance under hypoxic conditions increased their ability to stimulate the growth of blood vessels.     

Proliferation and differentiation of bone marrow stromal cells under hypoxic conditions

Low oxygen tension is a potent differentiation inducer of numerous cell types and an effective stimulus of many gene expressions. Here, we described that under 8% O(2), bone marrow stromal cells (MSCs) exhibited proliferative and morphologic changes. The level of differentiated antigen H-2Dd and the number of G(2)/S/M phase cells increased evidently under 8% O(2) condition. Also, the proportion of wide, flattened, and epithelial-like cells (which were alkaline phosphatase staining positive) in MSCs increased significantly. When cultured in adipogenic medium, there was a 5- to 6-fold increase in the number of lipid droplets under hypoxic conditions compared with that in normoxic culture. We also demonstrated the existence of MSC differentiation under hypoxic conditions by electron microscopy. Expression of Oct4 was inhibited under 8% O(2) condition, but after adipocyte differentiation in normoxic culture and hypoxia-mimicking agents cobalt chloride (CoCl(2)) and deferoxamine mesylate (DFX) treatments, Oct4 was still expressed in MSCs. These results indicate hypoxia accelerates MSC differentiation and hypoxia and hypoxia-mimicking agents exert different effects on MSC differentiation.     

A simple method for identifying bone marrow mesenchymal stromal cells with a high immunosuppressive potential

Background aimsThe beneficial activity of mesenchymal stromal cells (MSC) in allogeneic hematopietic stem cell transplantation requires correct use in terms of cell dose and timing of infusion and the identification of biomarkers for selection. The immunosuppressive bone marrow (BM)-derived MSC (BM-MSC) functions have been associated with the production of soluble HLA-G molecules (sHLA-G) via interleukin (IL)-10. We have established a reliable method for evaluating BM-MSC HLA-G expression without the influence of peripheral blood mononuclear cells (PBMC).MethodsThirteen BM-MSC from donors were activated with recombinant IL-10 or co-cultured with 10 different phytohemagglutinin (PHA)-treated PBMC (PHA-PBMC). Membrane-bound and sHLA-G expression was evaluated by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively; lymphoproliferation was measured by (methyl-³H)thymidine.ResultsThe results demonstrated the ability of IL-10 to stimulate both membrane-bound and sHLA-G production by BM-MSC. The levels of HLA-G expression induced by IL-10 in BM-MSC were associated with the inhibition of PHA-PBMC proliferation (sHLA-G, P = 0.0008, r = 0.9308; membrane HLA-G, P = 0.0005, r = 0.9502).ConclusionsWe propose the evaluation of sHLA-G production in IL-10-treated BM-MSC cultures as a possible marker of immunoregulatory function.     

Self-assembled extracellular macromolecular matrices and their different osteogenic potential with preosteoblasts and rat bone marrow mesenchymal stromal cells

Extracellular environment is a physical support that is critical to cell adhesion, migration, and differentiation. In this work, cell-derived matrices (CDMs) were obtained by separately culturing fibroblasts, preosteoblasts, and chondrocytes. The cells were grown on a coverslip and subjected to decellularization using detergents and enzymes. The resulting matrices were named fibroblast-derived matrix (FDM), preosteoblast-derived matrix (PDM), and chondrocyte-derived matrix (CHDM). We hypothesize that the unique compositional and structural feature of each CDM provides cells with a distinct microenvironment capable of functioning as a different signaling cue in the regulation of preosteoblast and rat bone marrow mesenchymal stromal cell (BMSC) osteogenic differentiation. SEM images show that each cell type creates its unique surface texture in a fibrillar structure. Three major macromolecules, fibronectin, type I collagen, and laminin, were clearly identified using both immunofluorescence and Western blot, in which FDM exhibited a much stronger signal of each ECM component than that of PDM or CHDM. For early cell morphology, BMSCs on the CDMs were highly elongated in a spindle-like shape. Both preosteoblasts and BMSCs proliferated well on CDMs comparable to the control. Once preosteoblasts were cultured for 2 weeks, their osteogenic activity was significantly different depending on the type of CDM. Using Alizarin red and von Kossa staining, we found that the cells on the FDM were much more osteogenic than the other groups. Furthermore, FDM was the most effective in upregulating the osteogenic markers, such as alkaline phosphatase (ALP), osteopontin, osteocalcin, and type I collagen. In particular, we observed a 2.5-fold increase in ALP activity with FDM compared to that of control and CHDM. In stark contrast, CHDM was very poor in stimulating osteogenic differentiation of preosteoblasts. Interestingly, these results were reproducible with the use of BMSCs, which are much more heterogeneous in cell populations than preosteoblasts. CHDM was still very weak in triggering the osteogenesis of BMSCs, whereas both FDM and PDM were equally competitive. This study demonstrates that a combination of factors (surface texture and composition) shape a unique cellular microenvironment, which serves as a physical cue toward the osteogenic differentiation of preosteoblasts and BMSCs.     

Multilineage potential of adult human mesenchymal stromal cells derived from bone marrow of patients with polycytaemia vera

Human mesenchymal stromal cells were isolated from the bone marrow of patients with polycyteamia vera (the myeloproliferative disorder) with the aim to characterize the properties of the mesenchymal stromal cells originating from the pathologically affected bone marrow. Their in vitro growth and potential to differentiate were determined. Isolated mesenchymal stromal cells were able to differentiate into three mesenchymal lineages under appropriate cultivation conditions.     

Inactivated human platelet lysate with psoralen: a new perspective for mesenchymal stromal cell production in Good Manufacturing Practice conditions

Background aimsMesenchymal stromal cells (MSC) are ideal candidates for regenerative and immunomodulatory therapies. The use of xenogeneic protein–free Good Manufacturing Practice–compliant growth media is a prerequisite for clinical MSC isolation and expansion. Human platelet lysate (HPL) has been efficiently implemented into MSC clinical manufacturing as a substitute for fetal bovine serum (FBS). Because the use of human-derived blood materials alleviates immunologic risks but not the transmission of blood-borne viruses, the aim of our study was to test an even safer alternative than HPL to FBS: HPL subjected to pathogen inactivation by psoralen (iHPL).MethodsBone marrow samples were plated and expanded in α-minimum essential medium with 10% of three culture supplements: HPL, iHPL and FBS, at the same time. MSC morphology, growth and immunophenotype were analyzed at each passage. Karyotype, tumorigenicity and sterility were analyzed at the third passage. Statistical analyses were performed.ResultsThe MSCs cultivated in the three different culture conditions showed no significant differences in terms of fibroblast colony-forming unit number, immunophenotype or in their multipotent capacity. Conversely, the HPL/iHPL-MSCs were smaller, more numerous, had a higher proliferative potential and showed a higher Oct-3/4 and NANOG protein expression than did FBS-MSCs. Although HPL/iHPL-MSCs exhibit characteristics that may be attributable to a higher primitive stemness than FBS-MSCs, no tumorigenic mutations or karyotype modifications were observed.ConclusionsWe demonstrated that iHPL is safer than HPL and represents a good, Good Manufacturing Practice–compliant alternative to FBS for MSC clinical production that is even more advantageous in terms of cellular growth and stemness.     

Age-dependent decrease in the chondrogenic potential of human bone marrow mesenchymal stromal cells expanded with fibroblast growth factor-2

Background aimsHuman bone marrow mesenchymal stromal cells are useful in regenerative medicine for various diseases, but it remains unclear whether the aging of donors alters the multipotency of these cells. In this study, we examined age-related changes in the chondrogenic, osteogenic and adipogenic potential of mesenchymal stromal cells from 17 donors (25–81 years old), including patients with or without systemic vascular diseases.MethodsAll stem cell lines were expanded with fibroblast growth factor-2 and then exposed to differentiation induction media. The chondrogenic potential was determined from the glycosaminoglycan content and the SOX9, collagen type 2 alpha 1 (COL2A1) and aggrecan (AGG) messenger RNA levels. The osteogenic potential was determined by monitoring the alkaline phosphatase activity and calcium content, and the adipogenic potential was determined from the glycerol-3-phosphate dehydrogenase activity and oil red O staining.ResultsSystemic vascular diseases, including arteriosclerosis obliterans and Buerger disease, did not significantly affect the trilineage differentiation potential of the cells. Under these conditions, all chondrocyte markers examined, including the SOX9 messenger RNA level, showed age-related decline, whereas none of the osteoblast or adipocyte markers showed age-dependent changes.ConclusionsThe aging of donors from young adult to elderly selectively decreased the chondrogenic potential of mesenchymal stromal cells. This information will be useful in stromal cell–based therapy for cartilage-related diseases.     

Comparison of epithelial differentiation and immune regulatory properties of mesenchymal stromal cells derived from human lung and bone marrow

Mesenchymal stromal cells (MSCs) reside in many organs including lung, as shown by their isolation from fetal lung tissues, bronchial stromal compartment, bronchial-alveolar lavage and transplanted lung tissues. It is still controversial whether lung MSCs can undergo mesenchymal-to-epithelial-transition (MET) and possess immune regulatory properties. To this aim, we isolated, expanded and characterized MSCs from normal adult human lung (lung-hMSCs) and compared with human bone marrow-derived MSCs (BM-hMSCs). Our results show that lung-MSCs reside at the perivascular level and do not significantly differ from BM-hMSCs in terms of immunophenotype, stemness gene profile, mesodermal differentiation potential and modulation of T, B and NK cells. However, lung-hMSCs express higher basal level of the stemness-related marker nestin and show, following in vitro treatment with retinoic acid, higher epithelial cell polarization, which is anyway partial when compared to a control epithelial bronchial cell line. Although these results question the real capability of acquiring epithelial functions by MSCs and the feasibility of MSC-based therapeutic approaches to regenerate damaged lung tissues, the characterization of this lung-hMSC population may be useful to study the involvement of stromal cell compartment in lung diseases in which MET plays a role, such as in chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis.     

Aging bone marrow mesenchymal stromal cells have altered membrane glycerophospholipid composition and functionality

Human mesenchymal stem/stromal cells (hMSC) are increasingly used in advanced cellular therapies. The clinical use of hMSCs demands sequential cell expansions. As it is well established that membrane glycerophospholipids (GPL) provide precursors for signaling lipids that modulate cellular functions, we studied the effect of the donor''s age and cell doublings on the GPL profile of human bone marrow MSC (hBMSC). The hBMSCs, which were harvested from five young and five old adults, showed clear compositional changes during expansion seen at the level of lipid classes, lipid species, and acyl chains. The ratio of phosphatidylinositol to phosphatidylserine increased toward the late-passage samples. Furthermore, 20:4n-6-containing species of phosphatidylcholine and phosphatidylethanolamine accumulated while the species containing monounsaturated fatty acids (FA) decreased during passaging. Additionally, in the total FA pool of the cells, 20:4n-6 increased, which happened at the expense of n-3 polyunsaturated FAs, especially 22:6n-3. The GPL and FA correlated with the decreased immunosuppressive capacity of hBMSCs during expansion. Our observations were further supported by alterations in the gene expression levels of several enzymes involved in lipid metabolism and immunomodulation. The results show that extensive expansion of hBMSCs harmfully modulates membrane GPLs, affecting lipid signaling and eventually impairing functionality.     

Differential mesengenic potential and expression of stem cell-fate modulators in mesenchymal stromal cells from human-term placenta and bone marrow

Placenta has attracted increasing attention over the past decade as a stem cell source for regenerative medicine. In particular, the amniochorionic membrane has been shown to harbor populations of mesenchymal stromal cells (MSCs). In this study, we have characterized ex vivo expanded MSCs from the human amniotic (hAMSCs) and chorionic (hCMSCs) membranes of human full-term placentas and adult bone marrow (hBMSCs). Our results show that hAMSCs, hCMSCs, and hBMSCs express typical mesenchymal (CD73, CD90, CD105, CD44, CD146, CD166) and pluripotent (Oct-4, Sox2, Nanog, Lin28, and Klf4) markers but not hematopoietic markers (CD45, CD34). Ex vivo expanded hAMSCs were found to be of fetal origin, while hCMSCs cultures contained only maternal cells. Cell proliferation was significantly higher in hCMSCs, compared to hAMSCs and hBMSCs. Integrin profiling revealed marked differences in the expression of α subunits between the three cell sources. Cadherin receptors were consistently expressed on a subset of progenitors (ranging from 1% to 60%), while N-CAM (CD56) was only expressed in hAMSCs and hCMSCs but not in hBMSCs. When induced to differentiate, hAMSCs and hCMSCs displayed strong chondrogenic and osteogenic differentiation potential but very limited capacity for adipogenic conversion. In contrast, hBMSCs showed strong differentiation potential along the three lineages. These results illustrate how MSCs from different ontological sources display differential expression of cell-fate mediators and mesodermal differentiation capacity.     

Photoactivation of bone marrow mesenchymal stromal cells with diode laser: Effects and mechanisms of action

Mesenchymal stromal cells (MSCs) are a promising cell candidate in tissue engineering and regenerative medicine. Their proliferative potential can be increased by low‐level laser irradiation (LLLI), but the mechanisms involved remain to be clarified. With the aim of expanding the therapeutic application of LLLI to MSC therapy, in the present study we investigated the effects of 635 nm diode laser on mouse MSC proliferation and investigated the underlying cellular and molecular mechanisms, focusing the attention on the effects of laser irradiation on Notch‐1 signal activation and membrane ion channel modulation. It was found that MSC proliferation was significantly enhanced after laser irradiation, as judged by time lapse videomicroscopy and EdU incorporation. This phenomenon was associated with the up‐regulation and activation of Notch‐1 pathway, and with increased membrane conductance through voltage‐gated K⁺, BK and Kir, channels and T‐ and L‐type Ca²⁺ channels. We also showed that MSC proliferation was mainly dependent on Kir channel activity, on the basis that the cell growth and Notch‐1 up‐regulation were severely decreased by the pre‐treatment with the channel inhibitor Ba²⁺ (0.5 mM). Interestingly, the channel inhibition was also able to attenuate the stimulatory effects of diode laser on MSCs, thus providing novel evidence to expand our knowledge on the mechanisms of biostimulation after LLLI. In conclusions, our findings suggest that diode laser may be a valid approach for the preconditioning of MSCs in vitro prior cell transplantation. J. Cell. Physiol. 228: 172–181, 2013. © 2012 Wiley Periodicals, Inc.     

Osteogenic differentiation of amniotic fluid mesenchymal stromal cells and their bone regeneration potential

In orthopedics, tissue engineering approach using stem cells is a valid line of treatment for patients with bone defects. In this context, mesenchymal stromal cells of various origins have been extensively studied and continue to be a matter of debate. Although mesenchymal stromal cells from bone marrow are already clinically applied, recent evidence suggests that one may use mesenchymal stromal cells from extra-embryonic tissues, such as amniotic fluid, as an innovative and advantageous resource for bone regeneration. The use of cells from amniotic fluid does not raise ethical problems and provides a sufficient number of cells without invasive procedures. Furthermore, they do not develop into teratomas when transplanted, a consequence observed with pluripotent stem cells. In addition, their multipotent differentiation ability, low immunogenicity, and anti-inflammatory properties make them ideal candidates for bone regenerative medicine. We here present an overview of the features of amniotic fluid mesenchymal stromal cells and their potential in the osteogenic differentiation process. We have examined the papers actually available on this regard, with particular interest in the strategies applied to improve in vitro osteogenesis. Importantly, a detailed understanding of the behavior of amniotic fluid mesenchymal stromal cells and their osteogenic ability is desirable considering a feasible application in bone regenerative medicine.     

Collection and culture of alveolar bone marrow multipotent mesenchymal stromal cells from older individuals

In this work, we examined the culture condition of alveolar bone marrow multipotent mesenchymal stromal cells (ABMMSCs), aiming to apply regenerative therapy to older periodontitis patients. To better understand the character of cultured cells from alveolar bone marrow, the expression profiles of well‐known genes and their responses to the induction of osteogenic, chondrogenic, or adipogenic differentiation were examined. Using αMEM‐based culture, ABMMSCs could be obtained from older individuals than in previous reports. Interestingly, ABMMSCs expressing Klf4 were able to differentiate into osteoblasts. The prediction of differentiation potential by Klf4 could be a useful guide for further improvement of the culture conditions required to culture ABMMSCs derived from older individuals. J. Cell. Biochem. 107: 1198–1204, 2009. © 2009 Wiley‐Liss, Inc.     

The immunosuppressive capacity of human mesenchymal stromal cells derived from amnion and bone marrow

Mesenchymal stromal cells derived from amnion (AM-MSCs) can be easily obtained in large quantity by less invasive method in comparison to bone marrow-derived MSCs (BM-MSCs). However, the biological and immunosuppressive properties of AM-MSCs are still poorly characterized. Previous studies demonstrated that BM-MSCs expressed indoleamine 2,3-dioxygenase (IDO) to suppress T-cell responses. This study was designed to address whether IDO contributes to the immunosuppressive function of AM-MSCs. MSCs isolated from amnion were cultured in complete medium similar to BM-MSCs. After culture, AM-MSCs exhibited spindle shape morphology and expressed MSC markers similar to that of BM-MSCs. In addition, AM-MSCs were able to differentiate into adipocytes and osteoblasts. Fascinatingly, AM-MSCs and BM-MSCs exhibited comparable degree of immunosuppressive effect when they were co-cultured with activated T-cells. In addition, IDO secreted by AM-MSCs was responsible for induction of immunosuppressive activities in the same manner as BM-MSCs. Taken together; the results of the present study demonstrate that while AM-MSCs and BM-MSCs show similar immunosuppressive effect, AM-MSCs may have additional advantage over the BM-MSCs in terms of availability. Therefore, AM-MSCs might be considered a potential source for therapeutic applications especially for treatment of immune related diseases.