Anna Burdzinska

Characterization of bone-marrow-derived rat mesenchymal stem cells depending on donor age

It is generally accepted that autologous transfers, as non‐immunogenic, constitute the safest approach in cellular transplantations. However, this attitude is often associated with the need for isolation and extracorporeal propagation of cells derived from aged patients. Thus the knowledge about relationship between aging and the properties of MSCs (mesenchymal stem cells) is crucial in developing new clinical strategies. The aim of this study was to perform complex comparison of MSC derived from young and aged individuals, which included phenotype, proliferating rate, osteogenic and adipogenic potential and secretory activity. Evaluated populations were isolated from bone marrow of 3‐month‐old and 24‐month‐old rats. There was no significant difference in membrane antigen expression and PDT (population doubling time). Additionally, the adipogenic and osteogenic potential did not vary between studied populations. The reaction of MSCs to either mitogen [bFGF (basic fibroblast growth factor)] or oxidative stress (H2O2) in vitro displayed a very similar pattern in both analysed populations. There was no difference in TGFβ1 (transforming growth factor β1) and VEGF (vascular endothelial growth factor) secretion measured by ELISA test and gene expression evaluated by real‐time PCR. However, the expression of the gene for IL‐1α (interleukin‐1α) was 8‐fold lower in oMSC (MSC isolated from old rats). These results indicate that aging individuals can be considered as candidates for autologous transplantation of bone‐marrow‐derived MSCs.

Uremic toxins impair human bone marrow-derived mesenchymal stem cells functionality in vitro

Mesenchymal stem cells (MSCs) are becoming therapeutic agents of interest in many areas of medicine, including renal diseases and kidney transplantations. However, the effect of uremia on cell properties is still unclear. Therefore, we examined the in vitro influence of uremic toxins, p-cresol (PC) and indoxyl sulfate (IS), on human bone marrow-derived MSC functionality. Cultured MSCs were treated with PC and IS at concentrations corresponding to subsequent stages of chronic kidney disease. Cell viability was characterized by metabolic activity (MTT assay) and proliferation rate (BrdU assay). Apoptosis (Annexin V test) and cell membrane damage (LDH assay) were also tested. MSC secretory properties were determined by measuring cytokine/growth factor levels in media from toxin-treated cells (ELISA). Uremic concentrations of PC and IS resulted in significant inhibition of MSC metabolic activity and proliferation. Toxins did not induce apoptosis, but damaged cell membranes. MSC paracrine activity was also altered - a decrease of VEGF and TGF-β1 levels and an increase in IGF-1 and IL-8 secretion was detected. Presented data indicate a negative influence of uremic toxins on functional characteristics of human bone marrow-derived MSCs. Therefore, their use as autologous therapeutic agents for kidney disease may be questionable and requires further investigations. The observed phenomenon may be attributable to many other MSC therapies, because of the high prevalence of chronic kidney disease in adult population.

Urethral distension as a novel method to simulate sphincter insufficiency in the porcine animal model

Objectives:  To describe a novel animal model of intrinsic sphincter deficiency.

Preincubation with bFGF but not sodium ascorbate improves efficiency of autologous transplantation of muscle-derived cells into urethral wall.

OBJECTIVES Myogenic cell therapy is considered a new method to treat urinary incontinence. One of the crucial limitations in cellular transplantation is poor cell survival after cell transfer. Preconditioning of cells before transfer is a new approach in improving the efficacy of graft. METHODS In this study, autologous cell transplantation was performed on a rat model. Muscle-derived cells were isolated, identified with immunofluorescence and immunoblotting, preincubated or not with 10 ng/mL of basic fibroblast growth factor (bFGF) or 0.1 mM of sodium ascorbate for 24 hours, and transfected with the beta-galactosidase encoding gene using lipofectamine. Cell suspension was injected into the urethral wall. The tissue was collected 2 weeks after injection. RESULTS The presence of transplanted cells was confirmed by X-Gal staining. Moreover, the effects of preincubation with either sodium ascorbate or bFGF 24 hours before transfer were evaluated. A semiqualitative analysis using chlorophenol-red-beta-d-galactopyranoside substrate demonstrated that preincubation with ascorbate had no effect on the number of transplanted cells but that bFGF significantly increased the amount of cells in the engrafted tissue by 46% (P < .05). CONCLUSIONS Preconditioning of myogenic cells with bFGF should be seriously considered as a new tool for improving cell viability after cell transfer in the treatment of urinary incontinence.

The Effect of Endoscopic Administration of Autologous Porcine Muscle-derived Cells Into the Urethral Sphincter

OBJECTIVE To verify the fate of autologous porcine myogenic cells after endoscopic administration into the urethral sphincter. METHODS This study was performed on pig animal models. The muscle-derived cells (MDCs) were isolated and identified. After the third passage, the 6 × 10(7) of PKH26 labeled cells were injected into the urethral sphincter using a urethrocystoscope. The urethras were collected after 28 days. To analyze the fate of injected cells, the PKH26 presence, the desmin expression, and the distribution of acetylcholine receptors were evaluated in the tissue sections. Moreover, the maximal urethral closure pressure (MUCP) was assessed in experimental and control groups at day 1 and day 28. RESULTS The isolated porcine MDCs expressed desmin and were able to differentiate into myotubes in vitro. At day 28 after the transplantation, the depots of PKH26-positive cells were observed in the muscular layer, but also in the submucosa. The staining for desmin revealed that cells located in the muscle layer were integrated with muscle fibers that possessed acetylcholine receptors. However, cells administered into nonmuscle tissue did not express desmin. Urethral pressure profilometry demonstrated no significant differences between MUCP in the transplanted group in comparison to the control group at day 28. CONCLUSION The present study demonstrates the successful endoscopic transplantation of myogenic cells into the urethral sphincter. The experiments indicated the key importance of precise cell administration in terms of their fate after the injection.

Dynamics of Acute Local Inflammatory Response after Autologous Transplantation of Muscle-Derived Cells into the Skeletal Muscle

The vast majority of myoblasts transplanted into the skeletal muscle die within the first week after injection. Inflammatory response to the intramuscular cell transfer was studied in allogeneic but not in autologous model. The aim of this study was to evaluate immune reaction to autotransplantation of myogenic cells and to assess its dynamics within the first week after injection. Muscle-derived cells or medium alone was injected into the intact skeletal muscles in autologous model. Tissue samples were collected 1, 3, and 7 days after the procedure. Our analysis revealed the peak increase of the gene expression of all evaluated cytokines (Il-1α, Il-1 β, Il-6, Tgf-β, and Tnf-α) at day 1. The mRNA level of analyzed cytokines normalized in subsequent time points. The increase of Il- β gene expression was further confirmed at the protein level. Analysis of the tissue sections revealed rapid infiltration of injected cell clusters with neutrophils and macrophages. The inflammatory infiltration was almost completely resolved at day 7. The survived cells were able to participate in the muscle regeneration process. Presented results demonstrate that autotransplanted muscle-derived cells induce classical early immune reaction in the site of injection which may contribute to cellular graft elimination.

Protective effect of sodium ascorbate on efficacy of intramuscular transplantation of autologous muscle-derived cells

The possible reason for elimination of myogenic cells after transplantation is inflammation at the injection site associated with oxidative stress. The aim of this study was to determine whether preconditioning of muscle‐derived cells with an antioxidant, sodium ascorbate, can influence the fate of transplanted cells.

Comparison of the paracrine activity of mesenchymal stem cells derived from human umbilical cord, amniotic membrane and adipose tissue

The study was conducted to investigate secretory activity and define the paracrine potential of mesenchymal stem cells from human umbilical cord and amniotic membrane (UC‐MSCs and AM‐MSCs, respectively).

The Mutual Interactions between Mesenchymal Stem Cells and Myoblasts in an Autologous Co-Culture Model

Both myoblasts and mesenchymal stem cells (MSC) take part in the muscle tissue regeneration and have been used as experimental cellular therapy in muscular disorders treatment. It is possible that co-transplantation approach could improve the efficacy of this treatment. However, the relations between those two cell types are not clearly defined. The aim of this study was to determine the reciprocal interactions between myoblasts and MSC in vitro in terms of the features important for the muscle regeneration process. Primary caprine muscle-derived cells (MDC) and bone marrow-derived MSC were analysed in autologous settings. We found that MSC contribute to myotubes formation by fusion with MDC when co-cultured directly, but do not acquire myogenic phenotype if exposed to MDC-derived soluble factors only. Experiments with exposure to hydrogen peroxide showed that MSC are significantly more resistant to oxidative stress than MDC, but a direct co-culture with MSC does not diminish the cytotoxic effect of H2O2 on MDC. Cell migration assay demonstrated that MSC possess significantly greater migration ability than MDC which is further enhanced by MDC-derived soluble factors, whereas the opposite effect was not found. MSC-derived soluble factors significantly enhanced the proliferation of MDC, whereas MDC inhibited the division rate of MSC. To conclude, presented results suggest that myogenic precursors and MSC support each other during muscle regeneration and therefore myoblasts-MSC co-transplantation could be an attractive approach in the treatment of muscular disorders.

Transplantation of mesenchymal stem cells into the skeletal muscle induces cytokine generation

Mesenchymal stem cells due to the high proliferative potential, capacity of multilineage differentiation became a hope of regenerative medicine. However, the organisms response to the transplantation of MSCs is not fully elucidated. The aim of the present study was to evaluate the acute local tissue response to syngeneic MSCs administration into the muscle. Rat syngeneic MSCs were transplanted into the skeletal muscle and the tissue surrounding the injection site was collected after 24h. Analogous samples from untreated and PBS treated muscles served as controls. The analysis of genes expression using real-time PCR revealed significant up-regulation of proinflammatory cytokines: IL-1α, IL-1β, IL-6 in MSCs treated muscles in comparison to the PBS group. The evaluation of protein concentration (ELISA) in collected samples showed that injection of MSCs caused significant elevation of IL-1β. Immunofluorescent assessment of the tissue revealed infiltration of leukocytes and macrophages. Quantitative analysis of the samples immunostained for CD68 and CD43 antigens revealed that the number of phagocytes was significantly higher in MSC treated muscle when compared to the samples from PBS group. To conclude, the administration of mesenchymal stem cells into the muscle in syngeneic model induces the features of acute inflammation that affects cell engraftment.