Andrographolide induced heme oxygenase-1 expression in MSC-like cells isolated from rat bone marrow exposed to environmental stress.

BACKGROUND AND OBJECTIVES: Mesenchymal stem cells (MSC-like cells) are the most important stem cells that are used in transplantation clinically in various applications. The survival rate of MSC-like cells is strongly reduced due to adverse conditions in the microenvironment of transplantation, including environmental stress. Heme oxygenase-1 (HO-1) is a member of the heat shock protein, as well as a stress-induced enzyme, present throughout the body. The present study was conducted to investigate the effect of andrographolide, an active derivative from andrographolide paniculate, on HO-1 expression in mesenchymal stem cells derived from rat bone marrow. MATERIALS AND METHODS: The rat bone marrow-derived mesenchymal stem cells (BMSC-like cells) were extracted and proliferated in several passages. The identity of MSC-like cells was confirmed by morphological observations and differential tests. The flow cytometry method was used to verify the MSC-specific markers. Isolated MSC-like cells were treated with different concentrations of andrographolide and then exposed to environmental stress. Cell viability was assessed using the MTT colorimetric assay. A real-time PCR technique was employed to evaluate the expression level of HO-1 in the treated MSC-like cells. RESULTS: Isolated MSC-like cells demonstrated fibroblast-like morphology. These cells in different culture mediums differentiated into osteocytes and adipocytes and were identified using alizarin red and oil red staining, respectively. As well, MSC-like cells were verified by the detection of CD105 surface antigen and the absence of CD14 and CD45 antigens. The results of the MTT assay showed that the pre-treatment of MSC-like cells with andrographolide concentration independently increased the viability and resistance of these cells to environmental stress caused by hydrogen peroxide and serum deprivation (SD). Real-time PCR findings indicated a significant increase in HO-1 gene expression in the andrographolide-receiving groups (p < 0.01). CONCLUSION: Our results suggest that andrographolide creates a promising strategy for enhancing the quality of cell therapy by increasing the resistance of MSC-like cells to environmental stress and inducing the expression of HO-1.

Cryopreservation of swine colostrum-derived cells.

Mesenchymal stromal cells (MSCs) have been demonstrated to possess anti-inflammatory and antimicrobial properties and are of interest in biotechnologies that will require cryopreservation. Recently, MSC-like cells were isolated from colostrum and milk. We used an interrupted slow freezing procedure to examine cryoinjury incurred during slow cooling and rapid cooling of MSC-like cells from swine colostrum. Cells were loaded with either dimethyl sulfoxide (Me2SO) or glycerol, cooled to a nucleation temperature, ice-nucleated, and further cooled at 1 °C/min. At several temperatures along the cooling path, cells were either thawed directly, or plunged into liquid nitrogen for storage and later thawed. The pattern of direct-thaw and plunge-thaw responses was used to guide optimization of cryopreservation protocol parameters. We found that both 5% Me2SO (0.65 M, loaded for 15 min on ice) or 5% glycerol (0.55 M, loaded for 1 h at room temperature) yielded cells with high post-thaw membrane integrity when cells were cooled to at least -30 °C before being plunged into, and stored in, liquid nitrogen. Cells cultured post-thaw exhibited osteogenic differentiation similar to fresh unfrozen control. Fresh and cryopreserved MSC-like cells demonstrated antimicrobial activity against S. aureus. Also, the antimicrobial activity of cell-conditioned media was higher when both fresh and cryopreserved MSC-like cells were pre-exposed to S. aureus. Thus, we were able to demonstrate cryopreservation of colostrum-derived MSC-like cells using Me2SO or glycerol, and show that both cryoprotectants yield highly viable cells with osteogenic potential, but that cells cryopreserved with glycerol retain higher antimicrobial activity post-thaw.

Mesenchymal stem cells in peripheral blood of severely injured patients.

PURPOSE: Mesenchymal stem cells (MSCs) are primarily stromal cells present in bone marrow and other tissues that are crucial for tissue regeneration and can be mobilized into peripheral blood after different types of organ damage. However, little is known about MSC appearance in blood in the setting of polytrauma. METHODS: We conducted a monocentered and longitudinal observational clinical study in 11 polytraumatized patients with an injury severity score (ISS) ≥ 24 to determine the numbers of MSCs in peripheral blood. Blood was collected from healthy volunteers and patients after polytrauma in the emergency room and 4, 12, 24, 48 h, 5 and 10 day later, and cells carrying MSC-surface markers (negative for CD45, positive for CD29, CD73, CD90, CD105, and CD166 in different combinations also employing the more stringent markers STRO1 and MSCA1) were detected and characterized using flow cytometry. Relative numbers of MSC-like cells were correlated with clinical parameters to evaluate if specific injury patterns had an influence on their presence in the blood cell pool. RESULTS: We were able to detect MSC marker-positive cells in both cohorts; however, the percentage of those cells present in the blood of patients during the first 10 day after injury was mostly similar to healthy volunteers, and significantly lowers starting at 4 h post trauma for one marker combination when compared to controls. Furthermore, the presence of a pelvis fracture was partly correlated with reduced relative numbers of MSC-like cells detectable in blood. CONCLUSIONS: Polytrauma in humans was associated with partly reduced relative numbers of MSC-like cells detected in peripheral blood in the time course after injury. Further studies need to define if this reduction was due to lower mobilization from the bone marrow or to active migration to the sites of injury.

Transcriptomic Analysis of Adult Renal Derived Mesenchymal Stem-Like Cells.

Mesenchymal stem cells (MSC) from bone marrow or adult tissues are widely studied to evaluate their potential for tissue repair. Differences in tissue of origin, donor variation, or in vitro handling exist and it is still unclear how they affect cell function and regenerative potential. Large-scale gene expression analysis of these cells not only allows researchers to compare and contrast the differences between each MSC subset but also allows for the development of better analytical tools for their characterization and utilization. Here, we describe a protocol for transcriptomics analysis of MSC-like cells derived from adult kidneys.