Mika Pietilä

HIF-1α is upregulated in human mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) are multipotent cells that have aroused great expectations in regenerative medicine. They are assumed to originate from hypoxic stem cell niches, especially in the bone marrow. This suggests that O2 is of importance in their regulation. In order to characterize regulation of the oxygen sensing pathway in these cells, we studied hMSCs isolated from three origins, adult and pediatric bone marrow and umbilical cord blood (UCB). Surprisingly, pediatric bone marrow and UCB MSCs showed normoxic stabilization of hypoxia‐inducible factor‐1α (HIF‐1α) that is normally degraded completely by HIF prolyl 4‐hydroxylases in the presence of oxygen. This was due to a high expression level of HIF‐1α mRNA rather than inappropriate post‐translational degradation of HIF‐1α protein. HIF‐1α mRNA was also induced in normoxic adult bone marrow MSCs, but 40% less than in the pediatric cells, and this was apparently not enough to stabilize the protein. The high normoxic HIF expression in all the hMSCs studied was accompanied by increased expression of a large number of glycolytic HIF target genes and increased glycolysis. Osteogenic differentiation of bone marrow‐derived hMSCs reduced HIF‐1α mRNA and protein expression and the expression of glycolytic mRNAs, resulting in decreased glycolysis and induction of oxidative metabolism. Induced mitochondrial biogenesis, changes in mitochondrial morphology and size indicative of increased oxidative phosphorylation, and induction of extracellular matrix synthesis were observed following osteogenic differentiation. Altogether, these data suggest that HIF‐1α is a general regulator controlling the metabolic fate and multipotency of the hMSCs. Stem Cells 2013;31:1902‐1909

Cell Surface Structures Influence Lung Clearance Rate of Systemically Infused Mesenchymal Stromal Cells

The promising clinical effects of mesenchymal stromal/stem cells (MSCs) rely especially on paracrine and nonimmunogenic mechanisms. Delivery routes are essential for the efficacy of cell therapy and systemic delivery by infusion is the obvious goal for many forms of MSC therapy. Lung adhesion of MSCs might, however, be a major obstacle yet to overcome. Current knowledge does not allow us to make sound conclusions whether MSC lung entrapment is harmful or beneficial, and thus we wanted to explore MSC lung adhesion in greater detail. We found a striking difference in the lung clearance rate of systemically infused MSCs derived from two different clinical sources, namely bone marrow (BM‐MSCs) and umbilical cord blood (UCB‐MSCs). The BM‐MSCs and UCB‐MSCs used in this study differed in cell size, but our results also indicated other mechanisms behind the lung adherence. A detailed analysis of the cell surface profiles revealed differences in the expression of relevant adhesion molecules. The UCB‐MSCs had higher expression levels of α4 integrin (CD49d, VLA‐4), α6 integrin (CD49f, VLA‐6), and the hepatocyte growth factor receptor (c‐Met) and a higher general fucosylation level. Strikingly, the level of CD49d and CD49f expression could be functionally linked with the lung clearance rate. Additionally, we saw a possible link between MSC lung adherence and higher fibronectin expression and we show that the expression of fibronectin increases with MSC culture confluence. Future studies should aim at developing methods of transiently modifying the cell surface structures in order to improve the delivery of therapeutic cells. STEM CELLS2013;31:317–326

Mitochondrial function and energy metabolism in umbilical cord blood- and bone marrow-derived mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) are an attractive choice for a variety of cellular therapies. hMSCs can be isolated from many different tissues and possess unique mitochondrial properties that can be used to determine their differentiation potential. Mitochondrial properties may possibly be used as a quality measure of hMSC-based products. Accordingly, the present work focuses on the mitochondrial function of hMSCs from umbilical cord blood (UCBMSC) cells and bone marrow cells from donors younger than 18 years of age (BMMSC <18) and those more than 50 years of age (BMMSC >50). Changes of ultrastructure and energy metabolism during osteogenic differentiation in all hMSC types were studied in detail. Results show that despite similar surface antigen characteristics, the UCBMSCs had smaller cell surface area and possessed more abundant rough endoplasmic reticulum than BMMSC >50. BMMSC <18 were morphologically more UCBMSC-like. UCBMSC showed dramatically higher mitochondrial-to-cytoplasm area ratio and elevated superoxide and manganese superoxide dismutase (MnSOD) levels as compared with BMMSC >50 and BMMSC <18. All hMSCs types showed changes indicative of mitochondrial activation after 2 weeks of osteogenic differentiation, and the increase in mitochondrial-to-cytoplasm area ratio appears to be one of the first steps in the differentiation process. However, BMMSC >50 showed a lower level of mitochondrial maturation and differentiation capacity. UCBMSCs and BMMSCs also showed a different pattern of exocytosed proteins and glycoproteoglycansins. These results indicate that hMSCs with similar cell surface antigen expression have different mitochondrial and functional properties, suggesting different maturation levels and other significant biological variations of the hMSCs. Therefore, it appears that mitochondrial analysis presents useful characterization criteria for hMSCs intended for clinical use.

CD200 Positive Human Mesenchymal Stem Cells Suppress TNF-Alpha Secretion from CD200 Receptor Positive Macrophage-Like Cells

Human mesenchymal stem cells (hMSCs) display immunosuppressive properties in vitro and the potential has also been transferred successfully to clinical trials for treatment of autoimmune diseases. OX-2 (CD200), a member of the immunoglobulin superfamily, is widely expressed in several tissues and has recently been found from hMSCs. The CD200 receptor (CD200R) occurs only in myeloid-lineage cells. The CD200-CD200R is involved in down-regulation of several immune cells, especially macrophages. The present study on 20 hMSC lines shows that the CD200 expression pattern varied from high (CD200Hi) to medium (CD200Me) and low (CD200Lo) in bone marrow-derived mesenchymal stem cell (BMMSC) lines, whereas umbilical cord blood derived mesenchymal stem cells (UCBMSCs) were constantly negative for CD200. The role of the CD200-CD200R axis in BMMSCs mediated immunosuppression was studied using THP-1 human macrophages. Interestingly, hMSCs showed greater inhibition of TNF-α secretion in co-cultures with IFN-γ primed THP-1 macrophages when compared to LPS activated cells. The ability of CD200Hi BMMSCs to suppress TNF-α secretion from IFN-γ stimulated THP-1 macrophages was significantly greater when compared to CD200Lo whereas UCBMSCs did not significantly reduce TNF-α secretion. The interference of CD200 binding to the CD200R by anti-CD200 antibody weakened the capability of BMMSCs to inhibit TNF-α secretion from IFN-γ activated THP-1 macrophages. This study clearly demonstrated that the efficiency of BMMSCs to suppress TNF-α secretion of THP-1 macrophages was dependent on the type of stimulus. Moreover, the CD200-CD200r axis could have a previously unidentified role in the BMMSC mediated immunosuppression.

Sodium valproate induces mitochondrial respiration dysfunction in HepG2 in vitro cell model.

Sodium valproate (VPA) is a potentially hepatotoxic antiepileptic drug. Risk of VPA-induced hepatotoxicity is increased in patients with mitochondrial diseases and especially in patients with POLG1 gene mutations. We used a HepG2 cell in vitro model to investigate the effect of VPA on mitochondrial activity. Cells were incubated in glucose medium and mitochondrial respiration-inducing medium supplemented with galactose and pyruvate. VPA treatments were carried out at concentrations of 0-2.0mM for 24-72 h. In both media, VPA caused decrease in oxygen consumption rates and mitochondrial membrane potential. VPA exposure led to depleted ATP levels in HepG2 cells incubated in galactose medium suggesting dysfunction in mitochondrial ATP production. In addition, VPA exposure for 72 h increased levels of mitochondrial reactive oxygen species (ROS), but adversely decreased protein levels of mitochondrial superoxide dismutase SOD2, suggesting oxidative stress caused by impaired elimination of mitochondrial ROS and a novel pathomechanism related to VPA toxicity. Increased cell death and decrease in cell number was detected under both metabolic conditions. However, immunoblotting did not show any changes in the protein levels of the catalytic subunit A of mitochondrial DNA polymerase γ, the mitochondrial respiratory chain complexes I, II and IV, ATP synthase, E3 subunit dihydrolipoyl dehydrogenase of pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and glutathione peroxidase. Our results show that VPA inhibits mitochondrial respiration and leads to mitochondrial dysfunction, oxidative stress and increased cell death, thus suggesting an essential role of mitochondria in VPA-induced hepatotoxicity.

Transient Proteolytic Modification of Mesenchymal Stromal Cells Increases Lung Clearance Rate and Targeting to Injured Tissue

Systemic infusion of therapeutic cells would be the most practical and least invasive method of administration in many cellular therapies. One of the main obstacles especially in intravenous delivery of cells is a massive cell retention in the lungs, which impairs homing to the target tissue and may decrease the therapeutic outcome. In this study we showed that an alternative cell detachment of mesenchymal stromal/stem cells (MSCs) with pronase instead of trypsin significantly accelerated the lung clearance of the cells and, importantly, increased their targeting to an area of injury. Cell detachment with pronase transiently altered the MSC surface protein profile without compromising cell viability, multipotent cell characteristics, or immunomodulative and angiogenic potential. The transient modification of the cell surface protein profile was sufficient to produce effective changes in cell rolling behavior in vitro and, importantly, in the in vivo biodistribution of the cells in mouse, rat, and porcine models. In conclusion, pronase detachment could be used as a method to improve the MSC lung clearance and targeting in vivo. This may have a major impact on the bioavailability of MSCs in future therapeutic regimes.

Tumor necrosis factor alpha promotes the expression of immunosuppressive proteins and enhances the cell growth in a human bone marrow-derived stem cell culture

Mesenchymal stem cells (MSCs) are widely used in experimental treatments for various conditions that involve normal tissue regeneration via inflammatory repair. It is known that MSCs can secrete multiple soluble factors and suppress inflammation. Even though the effect of MSCs on inflammation has been extensively studied, the effect of inflammation on MSCs is poorly understood. One of the major cytokines released at the site of inflammation is tumor necrosis factor alpha (TNF-α) which is known to induce MSC invasion and proliferation. Therefore, we wanted to test the effects of TNF-α exposure on MSCs derived from human bone marrow. We found, as expected, that cell proliferation was significantly enhanced during TNF-α exposure. However, according to the cell surface marker analysis, the intensity of several antigens in the minimum criteria panel for MSCs proposed by International Society of Cellular Therapy (ISCT) was decreased dramatically, and in certain cases, the criteria for MSCs were not fulfilled. In addition, TNF-α exposure resulted in a significant but transient increase in human leukocyte antigen and CD54 expression. Additional proteomic analysis by two-dimensional difference gel electrophoresis and mass spectrometry revealed three proteins whose expression levels decreased and 8 proteins whose expression levels increased significantly during TNF-α exposure. The majority of these proteins could be linked to immunosuppressive and signalling pathways. These results strongly support reactive and immunosuppressive activation of MSCs during TNF-α exposure, which might influence MSC differentiation stage and capacity.

Mortalin antibody-conjugated quantum dot transfer from human mesenchymal stromal cells to breast cancer cells requires cell–cell interaction

The role of tumor stroma in regulation of breast cancer growth has been widely studied. However, the details on the type of heterocellular cross-talk between stromal and breast cancer cells (BCCs) are still poorly known. In the present study, in order to investigate the intercellular communication between human mesenchymal stromal cells (hMSCs) and breast cancer cells (BCCs, MDA-MB-231), we recruited cell-internalizing quantum dots (i-QD) generated by conjugation of cell-internalizing anti-mortalin antibody and quantum dots (QD). Co-culture of illuminated and color-coded hMSCs (QD655) and BCCs (QD585) revealed the intercellular transfer of QD655 signal from hMSCs to BCCs. The amount of QD double positive BCCs increased gradually within 48h of co-culture. We found prominent intercellular transfer of QD655 in hanging drop co-culture system and it was non-existent when hMSCs and BBCs cells were co-cultured in trans-well system lacking imminent cell-cell contact. Fluorescent and electron microscope analyses also supported that the direct cell-to-cell interactions may be required for the intercellular transfer of QD655 from hMSCs to BCCs. To the best of our knowledge, the study provides a first demonstration of transcellular crosstalk between stromal cells and BCCs that involve direct contact and may also include a transfer of mortalin, an anti-apoptotic and growth-promoting factor enriched in cancer cells.

Mesenchymal Stromal Cells from Female Donors Enhance Breast Cancer Cell Proliferation in vitro

The interplay between tumor stroma and breast cancer cells (BCCs) is thought to play a significant role in breast cancer. The current knowledge of human mesenchymal stromal cell (MSC) and BCC interaction is contradictory, and the donor sex issue is not addressed at all. We hypothesized that donor sex could have an effect on proliferation of MSCs or BCCs in co-culture in vitro. Three estrogen receptor-negative BCC lines, 19 primary human MSCs and breast tissue-derived fibroblasts from 4 donors were used. MSCs from female donors enhanced BCC proliferation (p = 0.005). The change in BCC proliferation was only partly due to soluble factors excreted by MSCs. The highly aggressive BCC line MDA-MB- 231 induced the proliferation of MSCs (p < 0.001) and fibroblasts (p = 0.037) in co-culture experiments. The magnitude in proliferation change was cell line dependent and partly sex dependent.

Monitoring mitochondrial inner membrane potential for detecting early changes in viability of bacterium-infected human bone marrow-derived mesenchymal stem cells

IntroductionOne of the most challenging safety issues in the manufacture of cell based medicinal products is the control of microbial risk as cell-based products cannot undergo terminal sterilization. Accordingly, sensitive and reliable methods for detection of microbial contamination are called for. As mitochondrial function has been shown to correlate with the viability and functionality of human mesenchymal stem cells (hMSCs) we have studied the use of a mitochondrial inner membrane potential sensitive dye for detecting changes in the function of mitochondria following infection by bacteria.MethodsThe effect of bacterial contamination on the viability of bone marrow-derived mesenchymal stem cells (BMMSCs) was studied. BMMSC lines were infected with three different bacterial species, namely two strains of Pseudomonas aeruginosa, three strains of Staphylococcus aureus, and three strains of Staphylococcus epidermidis. The changes in viability of the BMMSCs after bacterial infection were studied by staining with Trypan blue, by morphological analysis and by monitoring of the mitochondrial inner membrane potential.ResultsMicroscopy and viability assessment by Trypan blue staining showed that even the lowest bacterial inocula caused total dissipation of BMMSCs within 24 hours of infection, similar to the effects seen with bacterial loads which were several magnitudes higher. The first significant signs of damage induced by the pathogens became evident after 6 hours of infection. Early changes in mitochondrial inner membrane potential of BMMSCs were evident after 4 hours of infection even though no visible changes in viability of the BMMSCs could be seen.ConclusionsEven low levels of bacterial contamination can cause a significant change in the viability of BMMSCs. Moreover, monitoring the depolarization of the mitochondrial inner membrane potential may provide a rapid tool for early detection of cellular damage induced by microbial infection. Accordingly, mitochondrial analyses offer sensitive tools for quality control and monitoring of safety and efficacy of cellular therapy products.