Renal cancer secretome induces migration of mesenchymal stromal cells.

BACKGROUND: Advanced renal cell carcinoma (RCC) is therapeutically challenging. RCC progression is facilitated by mesenchymal stem/stromal cells (MSCs) that exert remarkable tumor tropism. The specific mechanisms mediating MSCs' migration to RCC remain unknown. Here, we aimed to comprehensively analyze RCC secretome to identify MSCs attractants. METHODS: Conditioned media (CM) were collected from five RCC-derived cell lines (Caki-1, 786-O, A498, KIJ265T and KIJ308T) and non-tumorous control cell line (RPTEC/TERT1) and analyzed using cytokine arrays targeting 274 cytokines in addition to global CM proteomics. MSCs were isolated from bone marrow of patients undergoing standard orthopedic surgeries. RCC CM and the selected recombinant cytokines were used to analyze their influence on MSCs migration and microarray-targeted gene expression. The expression of genes encoding cytokines was evaluated in 100 matched-paired control-RCC tumor samples. RESULTS: When compared with normal cells, CM from advanced RCC cell lines (Caki-1 and KIJ265T) were the strongest stimulators of MSCs migration. Targeted analysis of 274 cytokines and global proteomics of RCC CM revealed decreased DPP4 and EGF, as well as increased AREG, FN1 and MMP1, with consistently altered gene expression in RCC cell lines and tumors. AREG and FN1 stimulated, while DPP4 attenuated MSCs migration. RCC CM induced MSCs' transcriptional reprogramming, stimulating the expression of CD44, PTX3 and RAB27B. RCC cells secreted hyaluronic acid (HA), a CD44 ligand mediating MSCs' homing to the kidney. AREG emerged as an upregulator of MSCs' transcription. CONCLUSIONS: Advanced RCC cells secrete AREG, FN1 and HA to induce MSCs migration, while DPP4 loss prevents its inhibitory effect on MSCs homing. RCC secretome induces MSCs' transcriptional reprograming to facilitate their migration. The identified components of RCC secretome represent potential therapeutic targets.

The Role of COX-2 and PGE2 in the Regulation of Immunomodulation and Other Functions of Mesenchymal Stromal Cells.

The ability of MSCs to modulate the inflammatory environment is well recognized, but understanding the molecular mechanisms responsible for these properties is still far from complete. Prostaglandin E2 (PGE2), a product of the cyclooxygenase 2 (COX-2) pathway, is indicated as one of the key mediators in the immunomodulatory effect of MSCs. Due to the pleiotropic effect of this molecule, determining its role in particular intercellular interactions and aspects of cell functioning is very difficult. In this article, the authors attempt to summarize the previous observations regarding the role of PGE2 and COX-2 in the immunomodulatory properties and other vital functions of MSCs. So far, the most consistent results relate to the inhibitory effect of MSC-derived PGE2 on the early maturation of dendritic cells, suppressive effect on the proliferation of activated lymphocytes, and stimulatory effect on the differentiation of macrophages into M2 phenotype. Additionally, COX-2/PGE2 plays an important role in maintaining the basic life functions of MSCs, such as the ability to proliferate, migrate and differentiate, and it also positively affects the formation of niches that are conducive to both hematopoiesis and carcinogenesis.

The Intersection of Human and Veterinary Medicine-A Possible Direction towards the Improvement of Cell Therapy Protocols in the Treatment of Perianal Fistulas.

The effective treatment of perianal fistulizing Crohn's disease is still a challenge. Local administration of mesenchymal stromal cells (MSCs) is becoming a part of accepted treatment options. However, as a fledgling technique, it still can be optimized. A new trend in translational research, which is in line with "One Health" approach, bases on exploiting parallels between naturally occurring diseases affecting humans and companion animals. Canine anal furunculosis (AF) has been indicated as condition analogous to human perianal Crohn's disease (pCD). This narrative review provides the first comprehensive comparative analysis of these two diseases based on the published data. The paper also outlines the molecular mechanisms of action of MSCs which are likely to have a role in modulating the perianal fistula niche in humans, and refers them to the current knowledge on the immunomodulatory properties of canine MSCs. Generally, the pathogenesis of both diseases shares main determinants such as the presence of genetic predispositions, dysregulation of immune response and the relation to intestine microbiota. However, we also identified many aspects which should be further specified, such as determining the frequency of true fistulas formation in AF patients, elucidating the role of TNF and Th17 pathway in the pathogenesis of AF, or clarifying the role of epithelial-to-mesenchymal transition phenomenon in the formation of canine fistulae. Nevertheless, the available data support the hypothesis that the results from testing cell therapies in dogs with anal furunculosis have a significant translational value in optimizing MSC transplants procedures in pCD patients.

Ibuprofen in Therapeutic Concentrations Affects the Secretion of Human Bone Marrow Mesenchymal Stromal Cells, but Not Their Proliferative and Migratory Capacity.

Mesenchymal stromal cells (MSCs) are able to modulate the immune system activity and the regeneration processes mainly through the secretion of multiple soluble factors, including prostaglandin E2 (PGE2). PGE2 is produced as a result of cyclooxygenases (COX) activity. In the present study, we investigated how ibuprofen, a nonselective COX inhibitor, affects the proliferation, migration and secretion of human bone marrow MSCs (hBM-MSCs). For this purpose, six hBM-MSCs populations were treated with ibuprofen at doses which do not differ from maximum serum concentrations during standard pharmacotherapy. Ibuprofen treatment (25 or 50 µg/mL) substantially reduced the secretion of PGE2 in all tested populations. Following ibuprofen administration, MSCs were subjected to proliferation (BrdU), transwell migration, and scratch assays, while its effect on MSCs secretome was evaluated by Proteome Profiler and Luminex immunoassays. Ibuprofen did not cause statistically significant changes in the proliferation rate and migration ability of MSCs (p > 0.05). However, ibuprofen (25 µg/mL for 3 days) significantly decreased mean secretion of: CCL2 (by 44%), HGF (by 31%), IL-6 (by 22%), VEGF (by 20%) and IL-4 (by 8%) compared to secretion of control MSCs (p < 0.05). Our results indicate that ibuprofen at therapeutic concentrations may impair the pro-regenerative properties of hBM-MSCs.

PD-L1 CAR effector cells induce self-amplifying cytotoxic effects against target cells.

BACKGROUND: Immune checkpoint inhibitors and chimeric antigen receptor (CAR)-based therapies have transformed cancer treatment. Recently, combining these approaches into a strategy of PD-L1-targeted CAR has been proposed to target PD-L1high tumors. Our study provides new information on the efficacy of such an approach against PD-L1low targets. METHODS: New atezolizumab-based PD-L1-targeted CAR was generated and introduced into T, NK, or NK-92 cells. Breast cancer MDA-MB-231 and MCF-7 cell lines or non-malignant cells (HEK293T, HMEC, MCF-10A, or BM-MSC) were used as targets to assess the reactivity or cytotoxic activity of the PD-L1-CAR-bearing immune effector cells. Stimulation with IFNγ or with supernatants from activated CAR T cells were used to induce upregulation of PD-L1 molecule expression on the target cells. HER2-CAR T cells were used for combination with PD-L1-CAR T cells against MCF-7 cells. RESULTS: PD-L1-CAR effector cells responded vigorously with degranulation and cytokine production to PD-L1high MDA-MB-231 cells, but not to PD-L1low MCF-7 cells. However, in long-term killing assays, both MDA-MB-231 and MCF-7 cells were eliminated by the PD-L1-CAR cells, although with a delay in the case of PD-L1low MCF-7 cells. Notably, the coculture of MCF-7 cells with activated PD-L1-CAR cells led to bystander induction of PD-L1 expression on MCF-7 cells and to the unique self-amplifying effect of the PD-L1-CAR cells. Accordingly, PD-L1-CAR T cells were active not only against MDA-MD-231 and MCF-7-PD-L1 but also against MCF-7-pLVX cells in tumor xenograft models. Importantly, we have also observed potent cytotoxic effects of PD-L1-CAR cells against non-malignant MCF-10A, HMEC, and BM-MSC cells, but not against HEK293T cells that initially did not express PD-L1 and were unresponsive to the stimulation . Finally, we have observed that HER-2-CAR T cells stimulate PD-L1 expression on MCF-7 cells and therefore accelerate the functionality of PD-L1-CAR T cells when used in combination. CONCLUSIONS: In summary, our studies show that CAR-effector cells trigger the expression of PD-L1 on target cells, which in case of PD-L1-CAR results in the unique self-amplification phenomenon. This self-amplifying effect could be responsible for the enhanced cytotoxicity of PD-L1-CAR T cells against both malignant and non-malignant cells and implies extensive caution in introducing PD-L1-CAR strategy into clinical studies.

Interaction between Macrophages and Human Mesenchymal Stromal Cells Derived from Bone Marrow and Wharton's Jelly-A Comparative Study.

Despite intensive clinical research on the use of mesenchymal stromal cells (MSCs), further basic research in this field is still required. Herein, we compared human bone marrow MSCs (BM-MSCs, n = 6) and Wharton's jelly MSCs (WJ-MSCs, n = 6) in their ability to interact with human primary macrophages. Evaluation of secretory potential revealed that under pro-inflammatory stimulation, WJ-MSCs secreted significantly more IL-6 than BM-MSCs (2-fold). This difference did not translate into the effect of MSCs on macrophages: both types of MSCs significantly directed M1-like macrophages toward the M2 phenotype (based on CD206 expression) to a similar extent. This observation was consistent both in flow cytometry analysis and immunocytochemical assessment. The effect of MSCs on macrophages was sustained when IL-6 signaling was blocked with Tocilizumab. Macrophages, regardless of polarization status, enhanced chemotaxis of both BM-MSCs and WJ-MSCs (p < 0.01; trans-well assay), with WJ-MSCs being significantly more responsive to M1-derived chemotactic signals than BM-MSCs. Furthermore, WJ-MSCs increased their motility (scratch assay) when exposed to macrophage-conditioned medium while BM-MSCs did not. These results indicate that although both BM-MSCs and WJ-MSCs have the ability to reciprocally interact with macrophages, the source of MSCs could slightly but significantly modify the response under clinical settings.

Gene Expression Profile of Human Mesenchymal Stromal Cells Exposed to Hypoxic and Pseudohypoxic Preconditioning-An Analysis by RNA Sequencing.

Mesenchymal stromal cell (MSC) therapy is making its way into clinical practice, accompanied by research into strategies improving their therapeutic potential. Preconditioning MSCs with hypoxia-inducible factors-α (HIFα) stabilizers is an alternative to hypoxic priming, but there remains insufficient data evaluating its transcriptomic effect. Herein, we determined the gene expression profile of 6 human bone marrow-derived MSCs preconditioned for 6 h in 2% O2 (hypoxia) or with 40 µM Vadadustat, compared to control cells and each other. RNA-Sequencing was performed using the Illumina platform, quality control with FastQC and adapter-trimming with BBDUK2. Transcripts were mapped to the Homo_sapiens. GRCh37 genome and converted to relative expression using Salmon. Differentially expressed genes (DEGs) were generated using DESeq2 while functional enrichment was performed in GSEA and g:Profiler. Comparison of hypoxia versus control resulted in 250 DEGs, Vadadustat versus control 1071, and Vadadustat versus hypoxia 1770. The terms enriched in both phenotypes referred mainly to metabolism, in Vadadustat additionally to vesicular transport, chromatin modifications and interaction with extracellular matrix. Compared with hypoxia, Vadadustat upregulated autophagic, phospholipid metabolism, and TLR cascade genes, downregulated those of cytoskeleton and GG-NER pathway and regulated 74 secretory factor genes. Our results provide valuable insight into the transcriptomic effects of these two methods of MSCs preconditioning.

Stem cell therapies for autoimmune hepatitis.

Autoimmune hepatitis is a chronic inflammatory hepatic disorder which may cause liver fibrosis. Appropriate treatment of autoimmune hepatitis is therefore important. Adult stem cells have been investigated as therapies for a variety of disorders in latest years. Hematopoietic stem cells (HSCs) were the first known adult stem cells (ASCs) and can give rise to all of the cell types in the blood and immune system. Originally, HSC transplantation was served as a therapy for hematological malignancies, but more recently researchers have found the treatment to have positive effects in autoimmune diseases such as multiple sclerosis. Mesenchymal stem cells (MSCs) are ASCs which can be extracted from different tissues, such as bone marrow, adipose tissue, umbilical cord, and dental pulp. MSCs interact with several immune response pathways either by direct cell-to-cell interactions or by the secretion of soluble factors. These characteristics make MSCs potentially valuable as a therapy for autoimmune diseases. Both ASC and ASC-derived exosomes have been investigated as a therapy for autoimmune hepatitis. This review aims to summarize studies focused on the effects of ASCs and their products on autoimmune hepatitis.

The Effect of L-Ascorbic Acid and Serum Reduction on Tenogenic Differentiation of Human Mesenchymal Stromal Cells.

BACKGROUND AND OBJECTIVES: Despite significant improvement in the treatment of tendon injuries, the full tissue recovery is often not possible because of its limited ability to auto-repair. The transplantation of mesenchymal stromal cells (MSCs) is considered as a novel approach in the treatment of tendinopathies. The question about the optimal culture conditions remains open. In this study we aimed to investigate if serum reduction, L-ascorbic acid supplementation or a combination of both factors can induce tenogenic differentiation of human adipose-derived MSCs (ASCs). METHODS AND RESULTS: Human ASCs from 3 healthy donors were used in the study. The tested conditions were: 0.5 mM of ascorbic acid 2-phosphate (AA-2P), reduced serum content (2% FBS) or combination of these two factors. The combination of AA-2P and 2% FBS was the only experimental condition that caused a significant increase of the expression of all analyzed genes related to tenogenesis (SCLERAXIS, MOHAWK, COLLAGEN_1, COLLAGEN_3, DECORIN) in comparison to the untreated control (evaluated by RT-PCR, 5th day of experiment). Moreover, this treatment significantly increased the synthesis of SCLERAXIS, MOHAWK, COLLAGEN_1, COLLAGEN_3 proteins at the same time point (evaluated by Western blot method). Double immunocytochemical staining revealed that AA-2P significantly increased the extracellular deposition of both types of collagens. Semi-quantitative Electron Spin Resonance analysis of ascorbyl free radical revealed that AA-2P do not induce harmful transition metals-driven redox reactions in cell culture media. CONCLUSIONS: Obtained results justify the use of reduced content of serum with the addition of 0.5 mM of AA-2P in tenogenic inducing media.

Vadadustat, a HIF Prolyl Hydroxylase Inhibitor, Improves Immunomodulatory Properties of Human Mesenchymal Stromal Cells.

The therapeutic potential of mesenchymal stromal cells (MSCs) is largely attributed to their immunomodulatory properties, which can be further improved by hypoxia priming. In this study, we investigated the immunomodulatory properties of MSCs preconditioned with hypoxia-mimetic Vadadustat (AKB-6548, Akebia). Gene expression analysis of immunomodulatory factors was performed by real-time polymerase chain reaction (real-time PCR) on RNA isolated from six human bone-marrow derived MSCs populations preconditioned for 6 h with 40 µM Vadadustat compared to control MSCs. The effect of Vadadustat preconditioning on MSCs secretome was determined using Proteome Profiler and Luminex, while their immunomodulatory activity was assessed by mixed lymphocyte reaction (MLR) and Culturex transwell migration assays. Real-time PCR revealed that Vadadustat downregulated genes related to immune system: IL24, IL1B, CXCL8, PDCD1LG1, PDCD1LG2, HIF1A, CCL2 and IL6, and upregulated IL17RD, CCL28 and LEP. Vadadustat caused a marked decrease in the secretion of IL6 (by 51%), HGF (by 47%), CCL7 (MCP3) (by 42%) and CXCL8 (by 40%). Vadadustat potentiated the inhibitory effect of MSCs on the proliferation of alloactivated human peripheral blood mononuclear cells (PBMCs), and reduced monocytes-enriched PBMCs chemotaxis towards the MSCs secretome. Preconditioning with Vadadustat may constitute a valuable approach to improve the therapeutic properties of MSCs.

The influence of oxygen deprivation and donor age on the effect of statins on human mesenchymal stromal cells.

To date, no study evaluated the effect of oxygen deprivation together with statins pretreatment on human mesenchymal stromal cells (MSCs). The aim of our study was to establish the influence of atorvastatin and rosuvastatin on MSC proliferation and cytotoxicity in different oxygenic conditions. Human MSCs isolated from the bone marrow (n = 12) were incubated with statins. The proliferation rate and cytotoxic effect were evaluated in normoxic (21 %O2) and hypoxic (2%O2) conditions, also in relation to donor age. The treatment with atorvastatin was associated with significantly higher proliferation rate compared to control, both in hypoxic (19 % median increase) and normoxic conditions (20 %), p = 0.02 and p = 0.04, respectively. Atorvastatin had no significant cytotoxic effect on MSCs. Treatment with rosuvastatin in hypoxia resulted in significantly higher proliferation rate (15 %, p = 0.02) comparing to control with no significant cytotoxicity. In atmospheric oxygen concentration, rosuvastatin was associated with no significant change in proliferation and higher cytotoxicity compared to untreated control (p = 0.042 and p = 0.015, for 0.04 µM and 1 µM solutions respectively). There were no differences in the effect of statins on MSC from young donors vs. aged donors. These results suggest that statins could support MSC-based therapy of acute myocardial infarction.

Copper Does Not Induce Tenogenic Differentiation but Promotes Migration and Increases Lysyl Oxidase Activity in Adipose-Derived Mesenchymal Stromal Cells.

BACKGROUND: Copper belongs to the essential trace metals that play a key role in the course of cellular processes maintaining the whole body's homeostasis. As there is a growing interest in transplanting mesenchymal stromal cells (MSCs) into the site of injury to improve the regeneration of damaged tendons, the purpose of the study was to verify whether copper supplementation may have a positive effect on the properties of human adipose tissue-derived MSCs (hASCs) which potentially can contribute to improvement of tendon healing. RESULTS: Cellular respiration of hASCs decreased with increasing cupric sulfate concentrations after 5 days of incubation. The treatment with CuSO4 did not positively affect the expression of genes associated with tenogenesis (COL1α1, COL3α1, MKX, and SCX). However, the level of COL1α1 protein, whose transcript was decreased in comparison to a control, was elevated after a 5-day exposition to 25 µM CuSO4. The content of the MKX and SCX protein in hASCs exposed to cupric sulfate was reduced compared to that of untreated control cells, and the level of the COL3α1 protein, whose transcript was decreased in comparison to a control, was elevated after a 5-day exposition to 25 µM CuSO4. The content of the MKX and SCX protein in hASCs exposed to cupric sulfate was reduced compared to that of untreated control cells, and the level of the COL3. CONCLUSION: Copper sulfate supplementation can have a beneficial effect on tendon regeneration not by inducing tenogenic differentiation, but by improving the recruitment of MSCs to the site of injury, where they can secrete growth factors, cytokines and chemokines, and prevent the effects of oxidative stress at the site of inflammation, as well as improve the stabilization of collagen fibers, thereby accelerating the process of tendon healing.

The Influence of Cell Source and Donor Age on the Tenogenic Potential and Chemokine Secretion of Human Mesenchymal Stromal Cells.

BACKGROUND: Cellular therapy is proposed for tendinopathy treatment. Bone marrow- (BM-MSC) and adipose tissue- (ASC) derived mesenchymal stromal cells are candidate populations for such a therapy. The first aim of the study was to compare human BM-MSCs and ASCs for their basal expression of factors associated with tenogenesis as well as chemotaxis. The additional aim was to evaluate if the donor age influences these features. METHODS: Cells were isolated from 24 human donors, 8 for each group: hASC, hBM-MSC Y (age ≤ 45), and hBM-MSC A (age > 45). The microarray analysis was performed on RNA isolated from hASC and hBM-MSC A cells. Based on microarray results, 8 factors were chosen for further evaluation. Two genes were additionally included in the analysis: SCLERAXIS and PPARγ. All these 10 factors were tested for gene expression by the qRT-PCR method, and all except of RUNX2 were additionally evaluated for protein expression or secretion. RESULTS: Microarray analysis showed over 1,400 genes with a significantly different expression between hASC and hBM-MSC groups. Eight of these genes were selected for further analysis: CXCL6, CXCL12, CXCL16, TGF-ß2, SMAD3, COLLAGEN 14A1, MOHAWK, and RUNX2. In the subsequent qRT-PCR analysis, hBM-MSCs showed a significantly higher expression than did hASCs in following genes: CXCL12, CXCL16, TGF-ß2, SMAD3, COLLAGEN 14A1, and SCLERAXIS (p < 0.05, regardless of BM donor age). In the case of CXCL12, the difference between hASC and hBM-MSC was significant only for younger BM donors, whereas for COLLAGEN 14A1-only for elder BM donors. PPARγ displayed a higher expression in hASCs compared to hBM-MSCs. In regard to CXCL6, MOHAWK, and RUNX2 gene expression, no statistically significant differences between groups were observed. CONCLUSIONS: In the context of cell-based therapy for tendinopathies, bone marrow appears to be a more attractive source of MSCs than does adipose tissue. The age of cell donors seems to be less important than cell source, although cells from elder donors show slightly higher basal tenogenic potential than do cells from younger donors.

Targeting the thioredoxin system as a novel strategy against B-cell acute lymphoblastic leukemia.

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a genetically heterogeneous blood cancer characterized by abnormal expansion of immature B cells. Although intensive chemotherapy provides high cure rates in a majority of patients, subtypes harboring certain genetic lesions, such as MLL rearrangements or BCR-ABL1 fusion, remain clinically challenging, necessitating a search for other therapeutic approaches. Herein, we aimed to validate antioxidant enzymes of the thioredoxin system as potential therapeutic targets in BCP-ALL. We observed oxidative stress along with aberrant expression of the enzymes associated with the activity of thioredoxin antioxidant system in BCP-ALL cells. Moreover, we found that auranofin and adenanthin, inhibitors of the thioredoxin system antioxidant enzymes, effectively kill BCP-ALL cell lines and pediatric and adult BCP-ALL primary cells, including primary cells cocultured with bone marrow-derived stem cells. Furthermore, auranofin delayed the progression of leukemia in MLL-rearranged patient-derived xenograft model and prolonged the survival of leukemic NSG mice. Our results unveil the thioredoxin system as a novel target for BCP-ALL therapy, and indicate that further studies assessing the anticancer efficacy of combinations of thioredoxin system inhibitors with conventional anti-BCP-ALL drugs should be continued.

Intraurethral co-transplantation of bone marrow mesenchymal stem cells and muscle-derived cells improves the urethral closure.

BACKGROUND: Cell therapy constitutes an attractive alternative to treat stress urinary incontinence. Although promising results have been demonstrated in this field, the procedure requires further optimization. The most commonly proposed cell types for intraurethral injections are muscle derived cells (MDCs) and mesenchymal stem/stromal cell (MSCs). The aim of this study was to evaluate the effects of MDC-MSC co-transplantation into the urethra. METHODS: Autologous transplantation of labeled MDCs, bone marrow MSCs or co-transplantation of MDC-MSC were performed in aged multiparous female goats (n = 6 in each group). The mean number of cells injected per animal was 29.6 × 106(± 4.3 × 106). PBS-injected animals constituted the control group (n = 5). Each animal underwent urethral pressure profile (UPP) measurements before and after the injection procedure. The maximal urethral closure pressure (MUCP) and functional area (FA) of UPPs were calculated. The urethras were collected at the 28th or the 84th day after transplantation. The marker fluorochrome (DID) was visualized and quantified using in vivo imaging system in whole explants. Myogenic differentiation of the graft was immunohistochemically evaluated. RESULTS: The grafted cells were identified in all urethras collected at day 28 regardless of injected cell type. At this time point the strongest DID-derived signal (normalized to the number of injected cells) was noted in the co-transplanted group. There was a distinct decline in signal intensity between day 28 and day 84 in all types of transplantation. Both MSCs and MDCs contributed to striated muscle formation if transplanted directly to the external urethral sphincter. In the MSC group those events were rare. If cells were injected into the submucosal region they remained undifferentiated usually packed in clearly distinguishable depots. The mean increase in MUCP after transplantation in comparison to the pre-transplantation state in the MDC, MSC and MDC-MSC groups was 12.3% (± 11.2%, not significant (ns)), 8.2% (± 9.6%, ns) and 24.1% (± 3.1%, p = 0.02), respectively. The mean increase in FA after transplantation in the MDC, MSC and MDC-MSC groups amounted to 17.8% (± 15.4%, ns), 15.2% (± 12.9%, ns) and 17.8% (± 2.5%, p = 0.04), respectively. CONCLUSIONS: The results suggest that MDC-MSC co-transplantation provides a greater chance of improvement in urethral closure than transplantation of each population alone.

Limited accuracy of transurethral and periurethral intrasphincteric injections of cellular suspension.

AIMS: The efficacy of cell therapy in patients with stress urinary incontinence (SUI) is lower than expected. The aim of this study was to determine the injection accuracy rate both with transurethral and periurethral route. METHODS: Autologous intraurethral cell transplantation was performed in female goats. The cells were injected either periurethrally (PERI group, two depots/animal, n = 8) or transurethrally (TRANS group, eight depots/animal, n = 11). Transurethral injections were performed under endoscopic guidance. The number and distribution of cell depots in urethras were analyzed in the three-step protocol: 1) screening of whole explants by in vivo imaging system; 2) systematic microscopic analysis of raw 10 µm cross-sections; 3) immunohistochemistry. As control, four urethras collected 1 day after transurethral transplantation were used. Episodes of cell suspension leakages after needle withdrawal were noted. RESULTS: In all experimental animals depots were identified in the urethral wall 28 days after transplantation. The mean percentage of depots located in the urethral wall in relation to all performed injections amounted to 68.7% and 67.0% for PERI and TRANS groups, respectively. The mean proportions of depots which were identified in external urethral sphincter (EUS) amounted 18.8% and 17.1%, respectively. Suspension leakage was observed in 19% of transurethral injections. CONCLUSIONS: Although majority of cell depots were administrated accurately into the urethral wall, the precise delivery of cells into EUS is limited regardless of injection method. The insufficient accuracy of cell delivery into EUS and cell suspension leakage can contribute to the low efficacy of cell therapy in human patients with SUI.

In vivo imaging system for explants analysis-A new approach for assessment of cell transplantation effects in large animal models.

INTRODUCTION: Despite spectacular progress in cellular transplantology, there are still many concerns about the fate of transplanted cells. More preclinical studies are needed, especially on large animal models, to bridge the translational gap between basic research and the clinic. Herein, we propose a novel approach in analysis of cell transplantation effects in large animals explants using in vivo imaging system (IVIS®) or similar equipment. MATERIAL AND METHODS: In the in vitro experiment cells labeled with fluorescent membrane dyes: DID (far red) or PKH26 (orange) were visualized with IVIS®. The correlation between the fluorescence signal and cell number with or without addition of minced muscle tissue was calculated. In the ex vivo study urethras obtained from goats after intraurethral cells (n = 9) or PBS (n = 4) injections were divided into 0.5 cm cross-slices and analyzed by using IVIS®. Automatic algorithm followed or not by manual setup was used to separate specific dye signal from tissue autofluorescence. The results were verified by systematic microscopic analysis of standard 10 µm specimens prepared from slices before and after immunohistochemical staining. Comparison of obtained data was performed using diagnostic test function. RESULTS: Fluorescence signal strength in IVIS® was directly proportional to the number of cells regardless of the dye used and detectable for minimum 0.25x106 of cells. DID-derived signal was much less affected by the background signal in comparison to PKH26 in in vitro test. Using the IVIS® to scan explants in defined arrangement resulted in precise localization of DID but not PKH26 positive spots. Microscopic analysis of histological specimens confirmed the specificity (89%) and sensitivity (80%) of IVIS® assessment relative to DID dye. The procedure enabled successful immunohistochemical staining of specimens derived from analyzed slices. CONCLUSIONS: The IVIS® system under appropriate conditions of visualization and analysis can be used as a method for ex vivo evaluation of cell transplantation effects. Presented protocol allows for evaluation of cell delivery precision rate, enables semi-quantitative assessment of signal, preselects material for further analysis without interfering with the tissue properties. Far red dyes are appropriate fluorophores to cell labeling for this application.

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

AIM: 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). METHODS: UC-MSCs (n = 6) were obtained from tissue explants using an adherent method after two weeks of incubation. AM-MSCs (n = 6) were obtained by digestion with tripsin and collagenase. MSC phenotype was confirmed in vitro by performing flow cytometry, differentiation assays and vimentin staining. Supernatants were collected after 48 h culturing in serum-free conditions and the following concentrations were determined: epidermal growth factor (EGF), interleukin (IL)-6, IL-10, tumor necrosis factor-α, transforming growth factor-ß (TGF-ß), vascular endothelial growth factor-α (VEGF-α) and metalloproteinase (MMP) 1, 8 and 13, using multiplex supernatant cytokine assay. Data were compared with adipose tissue derived MSCs (AD-MSCs, n = 6). RESULTS: Both UC-MSC and AM-MSC populations were positively identified as MSCs by flow cytometry and differentiation potential into bone, cartilage and adipose tissue. Using a multiple cytokine detection assay, we proved that both UC-MSCs and AM-MSCs show high secretive capacity. However, the secretion profile differed between cells from various sources. UC-MSCs showed significantly higher production of TGF-ß and lower production of VEGF-α, compared to AD-MSCs (P = 0.004) and AM-MSCs (P = 0.039) and lower levels of EGF (P = 0005). AM-MSCs showed significantly lower levels of MMP-8 than UC-MSCs (P = 0.024); however, there was no difference in levels of released cytokines compared to AD-MSCs. CONCLUSION: AM-MSCs show similar IL production as AD-MSCs, while UC-MSCs have a significantly different profile, which suggests diverse biological potential of both cell types for immunomodulative and regenerative therapy.

Premature fetal tissues are possible source of valuable mesenchymal stem cells.

OBJECTIVES: Comparison of the ability to inhibit alloactivated lymphocytes proliferation of human Wharton Jelly (WJ) and amniotic membrane (AM) mesenchymal stem cells (MSCs) from preterm and term pregnancies. MATERIAL AND METHODS: Term-WJ-MSCs (n = 5) and Preterm-WJ-MSCs (n = 1) were obtained from tissue explants by adherent method. Term-AM-MSCs (n = 5) and Preterm-AM-MSCs (n = 1) were obtained by tripsin and collagenase digestion method. Term and Preterm MSCs phenotype was confirmed in vitro by flow cytometry. To evaluate the potential of fetal and adult MSCs to diminish immunological response mixed lymphocytes reaction (MLR) has been performed. RESULTS: Term and Preterm cells were positively identified as MSCs by the expression of CD73 and CD90 and CD105 with simultaneous absence of CD11b, CD14, CD19, CD34, CD45 and HLA-DR. The mean inhibition of allostimulated lymphocytes after addition of fetal derived MSCs amounted 64.8% for term AM-MSCs and 42.1% for term WJ-MSCs (for both populations the effect was statistically significant, p < 0.01). The addition of preterm-MSCs to MLR resulted in reduction of stimulated lymphocytes proliferation by 64.9% for AM-MSCs and 86.1% for WJ-MSCs. CONCLUSIONS: Presented results suggest that preterm fetal tissues contain MSCs which posses similar immunosuppressive capacity as those from term pregnancies. In the future MSCs from the umbilical cord and amnion can be potentially used to prevent immuno-dependent injuries in premature newborns.