Changes In Mesenchymal Stem Cells Research Articles

Insights into Mesenchymal Stem Cell Aging: Involvement of Antioxidant Defense and Actin Cytoskeleton

Progenitor cells such as mesenchymal stem cells (MSCs) have elicited great hopes for therapeutic augmentation of physiological regeneration processes, e.g., for bone fracture healing. However, regeneration potential decreases with age, which raises questions about the efficiency of autologous approaches in elderly patients. To elucidate the mechanisms and cellular consequences of aging, the functional and proteomic changes in MSCs derived from young and old Sprague-Dawley rats were studied concurrently. We demonstrate not only that MSC concentration in bone marrow declines with age but also that their function is altered, especially their migratory capacity and susceptibility toward senescence. High-resolution two-dimensional electrophoresis of the MSC proteome, under conditions of in vitro self-renewal as well as osteogenic stimulation, identified several age-dependent proteins, including members of the calponin protein family as well as galectin-3. Functional annotation clustering revealed that age-affected molecular functions are associated with cytoskeleton organization and antioxidant defense. These proteome screening results are supported by lower actin turnover and diminished antioxidant power in aged MSCs, respectively. Thus, we postulate two main reasons for the compromised cellular function of aged MSCs: (a) declined responsiveness to biological and mechanical signals due to a less dynamic actin cytoskeleton and (b) increased oxidative stress exposure favoring macromolecular damage and senescence. These results, along with the observed similar differentiation potentials, imply that MSC-based therapeutic approaches for the elderly should focus on attracting the cells to the site of injury and oxidative stress protection, rather than merely stimulating differentiation.

Mesenchymal Stem Cells Can Participate in Ischemic Neovascularization

Cells from the bone marrow contribute to ischemic neovascularization, but the identity of these cells remains unclear. The authors identify mesenchymal stem cells as a bone marrow-derived progenitor population that is able to engraft into peripheral tissue in response to ischemia. A murine model of skin ischemia was used. Bone marrow, blood, and skin were harvested at different time points and subjected to flow cytometric analysis for mesenchymal and hematopoietic markers (n = 3 to 7 per time point). Using a parabiotic model pairing donor green fluorescent protein (GFP)-positive with recipient wild-type mice, progenitor cell engraftment was examined in ischemic tissue by fluorescence microscopy, and engrafted cells were analyzed by flow cytometry for endothelial and mesenchymal markers. In vitro, the ability of both bone marrow- and adipose-derived mesenchymal stem cells to adopt endothelial characteristics was examined by analyzing (1) the ability of mesenchymal stem cells to take up DiI-acetylated low-density lipoprotein and Alexa Fluor lectin, and (2) phenotypic changes of mesenchymal stem cells co-cultured with GFP-labeled endothelial cells or under hypoxic/vascular endothelial growth factor stimulation. In vivo, the bone marrow mesenchymal stem cell population decreased significantly immediately after surgery, with subsequent engraftment of these cells in ischemic tissue. Engrafted cells lacked the panhematopoietic antigen CD45, consistent with a mesenchymal origin. In vitro, bone marrow- and adipose-derived mesenchymal stem cells took up DiI-acetylated low-density lipoprotein and Alexa Fluor lectin, and expressed endothelial markers under hypoxic conditions. The authors' data suggest that mesenchymal precursor cells can give rise to endothelial progenitors. Consequently, cell-based therapies augmenting the mesenchymal stem cell population could represent powerful alternatives to current therapies for ischemic vascular disease.

HRMAS1H-NMR measured changes of the metabolite profile as mesenchymal stem cells differentiate to targeted fat cellsin vitro: implications for non-invasive monitoring of stem cell differentiationin vivo

Mesenchymal stem cells (MSCs) have shown a great potential for clinical applications in regenerative medicine. However, it remains challenging to follow the transplanted cell grafts in vivo. Nuclear magnetic resonance spectroscopy (NMR or MRS) is capable of determining and quantifying the cellular metabolites in tissue and organs non-invasively, therefore it is an attractive method for monitoring and evaluating the differentiation and functions of transplanted stem cells in vivo. In this study, metabolic changes of MSCs undergoing adipogenic differentiation to targeted fat cells were investigated in vitro, using solid-state high-resolution magic angle spinning (1)H nuclear magnetic resonance spectroscopy. Quantification of metabolite concentrations before and after differentiation of MSCs showed decreased levels of intracellular metabolites, including choline, creatine, glutamate and myo-inositol, and a substantially increased level of fatty acids, when mesenchymal stem cells were differentiated preferentially to fat cells. Intracellular creatine, myo-inositol and choline reduced from 10.4 +/- 0.72, 16.2 +/- 1.2 and 8.22 +/- 0.51 mM to 3.27 +/- 0.34, 6.1 +/- 0.46 and 3.11 +/- 0.32 mM, respectively, while fatty acids increased from 32.6 +/- 1.5 to 91.2 +/- 3.2 mM after undergoing 3 weeks of differentiation. The increase of the fatty acid concentration measured by NMR is confirmed by the observation of 80% fat cells in differentiated cells by cell counting assay, suggesting resonances from fatty acids may be used as metabolite markers for monitoring MSC differentiation to fat cells in vivo, using the magnetic resonance spectroscopic technique readily available on MRI scanners.

Cytoskeletal changes of mesenchymal stem cells during differentiation.

Mesenchymal stem cells (MSCs) are progenitors for tissues such as bone and cartilage. In this report, the actin cytoskeleton and nanomechanobiology of human mesenchymal stem cells (hMSCs) were studied using fluorescence microscopy and atomic force microscopy (AFM). Human MSCs were differentiated into chondrocytes and osteoblasts as per previous approaches. Cytochalasin D (CytD) was used to temporarily disrupt cytoskeleton in hMSCs, hMSC-chondrocytes (hMSC-Cys) and hMSC-osteoblasts (hMSC-Obs). Fluorescence microscopy revealed a dose-dependent response to CytD. Removal of CytD from the media of cytoskeleton-disrupted cells led to the recovery of the cytoskeletal structures, as confirmed by both fluorescence microscopy and AFM. Force-volume imaging by AFM evaluated the nanomechanics of all three cell types before, during, and after CytD treatment. Cytochalasin D disruption of cytoskeleton had marked effects on hMSCs and hMSC-Cys, in comparison with limited cytoskeleton disruption in hMSC-Obs, as confirmed qualitatively by fluorescence microscopy and quantitatively by AFM. Treatment with CytD resulted in morphology changes of all cell types, with significant decreases in the observed Young's Moduli of hMSCs and hMSC-Cys. These data suggest human mesenchymal stem cells alter their cytoskeletal components during differentiation. Additional studies will address the mechanisms of cytoskeletal changes using biochemical and biophysical methods.

Metabolic Changes in Mesenchymal Stem Cells in Osteogenic Medium Measured by Autofluorescence Spectroscopy

The purpose of this study was to measure metabolic changes in mesenchymal stem cells (MSCs) placed in osteogenic medium by autofluorescence spectroscopy. MSCs were plated in stem cell-supporting or osteogenic medium and imaged. Shift from the basic growth environment to the inductive osteogenic environment was confirmed by reverse transcription-polymerase chain reaction. Reduced pyridine nucleotides were detected by exciting near 366 nm and measuring fluorescence at 450 nm, and oxidized flavoproteins were detected by exciting at 460 nm and measuring fluorescence at 540 nm. The ratio of these fluorescence measurements, reduction-oxidation (redox) fluorometry, is a noninvasive measure of the cellular metabolic state. The detected pyridine nucleotide to flavoprotein ratio decreased upon transitioning from the stem cell to the differentiated state, as well as with increasing cell density and cell-cell contact. MSC metabolism increased upon placement in differentiating medium and with increasing cell density and contact. Redox fluorometry is a feasible, noninvasive technique for distinguishing MSCs from further differentiated cells.

Toll-Like Receptor Agonists Induce Secretion of Immunomodulatory Soluble Factors from Human Mesenchymal Stem Cells.

Human mesenchymal stem cells (MSCs) suppress T-cell activation and proliferation by inducible expression and secretion of soluble inhibitory factors. We have previously shown that MSCs require activation by antigen-presenting cells (APCs) to secrete these factors. Toll-like receptors (TLRs) and their associated agonists have critical roles in APC activation, maturation and function. Therefore, we investigated whether TLR agonists induce cytokine and chemokine production from MSCs and if such soluble factors mediate inhibition of T-cell alloreactivity. Human MSCs (CD45-CD105+CD90+CD80-CD73+HLA−I+) were expanded from normal volunteer bone marrow aspirate specimens. MSCs were characterized morphologically by light microscopy, phenotypically by flow cytometry and functionally by ex vivo cell culture stimulation and inhibition of T-cell IFN-γ production. Cytokine and chemokine induction of third and fourth-passage MSCs co-cultured in triplicate with established TLR agonists were measured and compared to soluble factor induction from human IL-1β stimulated MSCs. TLR agonists included formalin-fixed Staphylococcus aureus Cowan A strain (SAC, TLR-2), Pam3CysSerLys4 (Pam3Cys, TLR-2), polyinosine:polycytidylic acid (poly I:C, TLR-3), Salmonella enteriditis lipopolysaccharide (LPS, TLR-4), R848 (TLR7/8) and unmethylated CpG oligodeoxynucleotides 1826 and 2216 (TLR-9). 24h MSC-culture supernatants were assessed for levels of soluble factors using standard and multiplex ELISA and for inhibition of T-cell alloreactivity using an established mixed lymphocyte reaction (MLR) IFN-γ ELISPOT. MLR was also performed in the presence of TLR agonists alone and agonist-stimulated MSCs. In general, TLR-MSC supernatants contained 2 to 5-fold lower levels of inducible IL-6, IL-8, IL-10 and RANTES than IL-1β-MSC supernatants. LPS- and poly I:C-MSC supernatants had comparable levels of inducible factors as IL-1β-MSC supernatant. Neither stimulation with IL-1β nor TLR agonists induced phenotypic changes in MSCs, as measured by surface expression of MSC markers as well as activation markers (HLA-DR, CD40, CD40L, CD80, HLA-I and HLA-II). However, supernatant from TLR-MSC cultures (CpG 1826 and poly I:C) and from IL-1β-MSC cultures did inhibit T-cell IFN-γ production. For example, percent IFN-γ inhibition using supernatant from MSC-CpG 1826 culture versus supernatant from IL-1β-MSC culture was 46.5% ± 22.8 and 66.8% ± 13.5, respectively (mean ± SEM, n=3 separate experiments involving different donor MSCs). Likewise, TLR- and IL-1β-stimulated MSCs themselves inhibited T-cell IFN-γ. Together, these results demonstrate that ex vivo culture with TLR agonists activates human MSCs to inhibit ex vivo T-cell alloreactivity likely via inducible soluble factors. Optimization of cell culture conditions is needed to define the soluble factors mediating this inhibitory effect. Our results suggest the presence of a potentially novel immunomodulatory interface at which TLR agonists are uniquely positioned to influence immune effector cell and mesenchymal stem cell responses.