Nuclear magnetic resonance footprint of Wharton Jelly mesenchymal stem cells death mechanisms and distinctive in-cell biophysical properties in vitro.

The importance of the biophysical characterization of mesenchymal stem cells (MSCs) was recently pointed out for supporting the development of MSC-based therapies. Among others, tracking MSCs in vivo and a quantitative characterization of their regenerative impact by nuclear magnetic resonance (NMR) demands a full description of MSCs' MR properties. In the work, Wharton Jelly MSCs are characterized in a low magnetic field (LF) in vitro by using different approaches. They encompass various settings: MSCs cultured in a Petri dish and cell suspensions; experiments- 1D-T 1 , 1D-T 2 , 1D diffusion, 2D T 1 -T 2 and D-T 2 ; devices- with a bore aperture and single-sided one. Complex NMR analysis with the aid of random walk simulations allows the determination of MSCs T 1 and T 2 relaxation times, cells and nuclei sizes, self-diffusion coefficients of the nucleus and cytoplasm. In addition, the influence of a single layer of cells on the effective diffusion coefficient of water is detected with the application of a single-sided NMR device. It also enables the identification of apoptotic and necrotic cell death and changed diffusional properties of cells suspension caused by compressing forces induced by the subsequent cell layers. The study delivers MSCs-specific MR parameters that may help tracking MSCs in vivo.

Selective Cytotoxicity of Complexes with N,N,N-Donor Dipodal Ligand in Tumor Cells.

The present article demonstrates selective cytotoxicity against cancer cells of the complexes [Co(LD)2]I2∙CH3OH (1), [CoLD(NCS)2] (2) and [VOLD(NCS)2]∙C6H5CH3 (3) containing the dipodal tridentate ligand LD = N,N-bis(3,5-dimethylpyrazol-1-ylmethyl)amine), formed in situ. All tested complexes expressed greater anticancer activities and were less toxic towards noncancerous cells than cisplatin. Cobalt complexes (1 and 2) combined high cytotoxicity with selectivity towards cancer cells and caused massive tumour cell death. The vanadium complex (3) induced apoptosis specifically in cancer cells and targeted proteins, controlling their invasive and metastatic properties. The presented experimental data and computational prediction of drug ability of coordination compounds may be helpful for designing novel and less toxic metal-based anticancer species with high specificities towards tumour cells.

Highly Effective Protocol for Differentiation of Induced Pluripotent Stem Cells (iPS) into Melanin-Producing Cells.

Melanin is a black/brown pigment present in abundance in human skin. Its main function is photo-protection of underlying tissues from harmful UV light. Natural sources of isolated human melanin are limited; thus, in vitro cultures of human cells may be a promising source of human melanin. Here, we present an innovative in vitro differentiation protocol of induced pluripotent stem cells (iPS) into melanin-producing cells, delivering highly pigmented cells in quantity and quality incomparably higher than any other methods previously described. Pigmented cells constitute over 90% of a terminally differentiated population and exhibit features characteristic for melanocytes, i.e., expression of specific markers such as MITF-M (microphthalmia-associated transcription factor isoform M), TRP-1 (tyrosinase-related protein 1), and TYR (tyrosinase) and accumulation of black pigment in organelles closely resembling melanosomes. Black pigment is unambiguously identified as melanin with features corresponding to those of melanin produced by typical melanocytes. The advantage of our method is that it does not require any sophisticated procedures and can be conducted in standard laboratory conditions. Moreover, our protocol is highly reproducible and optimized to generate high-purity melanin-producing cells from iPS cells; thus, it can serve as an unlimited source of human melanin for modeling human skin diseases. We speculate that FGF-8 might play an important role during differentiation processes toward pigmented cells.

Carbon Fibers as a New Type of Scaffold for Midbrain Organoid Development.

The combination of induced pluripotent stem cell (iPSC) technology and 3D cell culture creates a unique possibility for the generation of organoids that mimic human organs in in vitro cultures. The use of iPS cells in organoid cultures enables the differentiation of cells into dopaminergic neurons, also found in the human midbrain. However, long-lasting organoid cultures often cause necrosis within organoids. In this work, we present carbon fibers (CFs) for medical use as a new type of scaffold for organoid culture, comparing them to a previously tested copolymer poly-(lactic-co-glycolic acid) (PLGA) scaffold. We verified the physicochemical properties of CF scaffolds compared to PLGA in improving the efficiency of iPSC differentiation within organoids. The physicochemical properties of carbon scaffolds such as porosity, microstructure, or stability in the cellular environment make them a convenient material for creating in vitro organoid models. Through screening several genes expressed during the differentiation of organoids at crucial brain stages of development, we found that there is a correlation between PITX3, one of the key regulators of terminal differentiation, and the survival of midbrain dopaminergic (mDA) neurons and tyrosine hydroxylase (TH) gene expression. This makes organoids formed on carbon scaffolds an improved model containing mDA neurons convenient for studying midbrain-associated neurodegenerative diseases such as Parkinson's disease.

Use of 3D Organoids as a Model to Study Idiopathic Form of Parkinson's Disease.

Organoids are becoming particularly popular in modeling diseases that are difficult to reproduce in animals, due to anatomical differences in the structure of a given organ. Thus, they are a bridge between the in vitro and in vivo models. Human midbrain is one of the structures that is currently being intensively reproduced in organoids for modeling Parkinson's disease (PD). Thanks to three-dimensional (3D) architecture and the use of induced pluripotent stem cells (iPSCs) differentiation into organoids, it has been possible to recapitulate a complicated network of dopaminergic neurons. In this work, we present the first organoid model for an idiopathic form of PD. iPSCs were generated from peripheral blood mononuclear cells of healthy volunteers and patients with the idiopathic form of PD by transduction with Sendai viral vector. iPSCs were differentiated into a large multicellular organoid-like structure. The mature organoids displayed expression of neuronal early and late markers. Interestingly, we observed statistical differences in the expression levels of LIM homeobox transcription factor alpha (early) and tyrosine hydroxylase (late) markers between organoids from PD patient and healthy volunteer. The obtained results show immense potential for the application of 3D human organoids in studying the neurodegenerative disease and modeling cellular interactions within the human brain.

Origin of the Induced Pluripotent Stem Cells Affects Their Differentiation into Dopaminergic Neurons.

Neuronal differentiation of human induced pluripotent stem (iPS) cells, both in 2D models and 3D systems in vitro, allows for the study of disease pathomechanisms and the development of novel therapies. To verify if the origin of donor cells used for reprogramming to iPS cells can influence the differentiation abilities of iPS cells, peripheral blood mononuclear cells (PBMC) and keratinocytes were reprogrammed to iPS cells using the Sendai viral vector and were subsequently checked for pluripotency markers and the ability to form teratomas in vivo. Then, iPS cells were differentiated into dopaminergic neurons in 2D and 3D cultures. Both PBMC and keratinocyte-derived iPS cells were similarly reprogrammed to iPS cells, but they displayed differences in gene expression profiles and in teratoma compositions in vivo. During 3D organoid formation, the origin of iPS cells affected the levels of FOXA2 and LMX1A only in the first stages of neural differentiation, whereas in the 2D model, differences were detected at the levels of both early and late neural markers FOXA2, LMX1A, NURR1, TUBB and TH. To conclude, the origin of iPS cells may significantly affect iPS differentiation abilities in teratomas, as well as exerting effects on 2D differentiation into dopaminergic neurons and the early stages of 3D midbrain organoid formation.

The Potential of Novel Chitosan-Based Scaffolds in Pelvic Organ Prolapse (POP) Treatment through Tissue Engineering.

The growing number of female reproductive system disorders creates a need for novel treatment methods. Tissue engineering brings hope for patients, which enables damaged tissue reconstruction. For this purpose, epithelial cells are cultured on three-dimensional scaffolds. One of the most promising materials is chitosan, which is known for its biocompatibility and biodegradability. The aim of the following study was to verify the potential of chitosan-based biomaterials for pelvic organ prolapse regeneration. The scaffolds were obtained under microwave-assisted conditions in crosslinking reactions, using dicarboxylic acids and aminoacid as crosslinkers, including l-glutamic acid, adipic acid, malonic acid, and levulinic acid. The products were characterized over their physicochemical and biological properties. FT-IR analysis confirmed formation of amide bonds. The scaffolds had a highly porous structure, which was confirmed by SEM analysis. Their porosity was above 90%. The biomaterials had excellent swelling abilities and very good antioxidant properties. The cytotoxicity study was performed on vaginal epithelial VK2/E6E7 and human colon cancer HCT116 cell lines. The results showed that after certain modifications, the proposed scaffolds could be used in pelvic organ prolapse (POP) treatment.

Regenerative Potential of the Product "CardioCell" Derived from the Wharton's Jelly Mesenchymal Stem Cells for Treating Hindlimb Ischemia.

In recent years, mesenchymal stem cells (MSCs) have emerged as a promising therapeutic modality in regenerative medicine. They hold great promise for treating civilization-wide diseases, including cardiovascular diseases, such as acute myocardial infarction and critical limb ischemia. MSCs isolated from Wharton's jelly (WJ-MSCs) may be utilized in both cell-based therapy and vascular graft engineering to restore vascular function, thereby providing therapeutic benefits for patients. The efficacy of WJ-MSCs lies in their multipotent differentiation ability toward vascular smooth muscle cells, endothelial cells and other cell types, as well as their capacity to secrete various trophic factors, which are potent in promoting angiogenesis, inhibiting apoptosis and modulating immunoreaction. Ischemic limb disease is caused by insufficient nutrient and oxygen supplies resulting from damaged peripheral arteries. The lack of nutrients and oxygen causes severe tissue damage in the limb, thereby resulting in severe morbidities and mortality. The therapeutic effects of the conventional treatments are still not sufficient. Cell transplantations in small animal model (mice) are vital for deciphering the mechanisms of MSCs' action in muscle regeneration. The stimulation of angiogenesis is a promising strategy for the treatment of ischemic limbs, restoring blood supply for the ischemic region. In the present study, we focus on the therapeutic properties of the human WJ-MSCs derived product, Cardio. We investigated the role of CardioCell in promoting angiogenesis and relieving hindlimb ischemia. Our results confirm the healing effect of CardioCell and strongly support the use of the WJ-MSCs in regenerative medicine.

Molecular and Functional Verification of Wharton's Jelly Mesenchymal Stem Cells (WJ-MSCs) Pluripotency.

The properties of mesenchymal stem cells (MSCs), especially their self-renewal and ability to differentiate into different cell lines, are widely discussed. Considering the fact that MSCs isolated from perinatal tissues reveal higher differentiation capacity than most adult MSCs, we examined mesenchymal stem cells isolated from Wharton's jelly of umbilical cord (WJ-MSCs) in terms of pluripotency markers expression. Our studies showed that WJ-MSCs express some pluripotency markers-such as NANOG, OCT-4, and SSEA-4-but in comparison to iPS cells expression level is significantly lower. The level of expression can be raised under hypoxic conditions. Despite their high proliferation potential and ability to differentiate into different cells type, WJ-MSCs do not form tumors in vivo, the major caveat of iPS cells. Owing to their biological properties, high plasticity, proliferation capacity, and ease of isolation and culture, WJ-MSCs are turning out to be a promising tool of modern regenerative medicine.

AFM-based Analysis of Wharton's Jelly Mesenchymal Stem Cells.

Wharton's jelly mesenchymal stem cells (WJ-MSCs) are multipotent stem cells that can be used in regenerative medicine. However, to reach the high therapeutic efficacy of WJ-MSCs, it is necessary to obtain a large amount of MSCs, which requires their extensive in vitro culturing. Numerous studies have shown that in vitro expansion of MSCs can lead to changes in cell behavior; cells lose their ability to proliferate, differentiate and migrate. One of the important measures of cells' migration potential is their elasticity, determined by atomic force microscopy (AFM) and quantified by Young's modulus. This work describes the elasticity of WJ-MSCs during in vitro cultivation. To identify the properties that enable transmigration, the deformability of WJ-MSCs that were able to migrate across the endothelial monolayer or Matrigel was analyzed by AFM. We showed that WJ-MSCs displayed differences in deformability during in vitro cultivation. This phenomenon seems to be strongly correlated with the organization of F-actin and reflects the changes characteristic for stem cell maturation. Furthermore, the results confirm the relationship between the deformability of WJ-MSCs and their migration potential and suggest the use of Young's modulus as one of the measures of competency of MSCs with respect to their possible use in therapy.

The Importance of HLA Assessment in "Off-the-Shelf" Allogeneic Mesenchymal Stem Cells Based-Therapies.

The need for more effective therapies of chronic and acute diseases has led to the attempts of developing more adequate and less invasive treatment methods. Regenerative medicine relies mainly on the therapeutic potential of stem cells. Mesenchymal stem cells (MSCs), due to their immunosuppressive properties and tissue repair abilities, seem to be an ideal tool for cell-based therapies. Taking into account all available sources of MSCs, perinatal tissues become an attractive source of allogeneic MSCs. The allogeneic MSCs provide "off-the-shelf" cellular therapy, however, their allogenicity may be viewed as a limitation for their use. Moreover, some evidence suggests that MSCs are not as immune-privileged as it was previously reported. Therefore, understanding their interactions with the recipient's immune system is crucial for their successful clinical application. In this review, we discuss both autologous and allogeneic application of MSCs, focusing on current approaches to allogeneic MSCs therapies, with a particular interest in the role of human leukocyte antigens (HLA) and HLA-matching in allogeneic MSCs transplantation. Importantly, the evidence from the currently completed and ongoing clinical trials demonstrates that allogeneic MSCs transplantation is safe and seems to cause no major side-effects to the patient. These findings strongly support the case for MSCs efficacy in treatment of a variety of diseases and their use as an "off-the-shelf" medical product.

Serum-resistant CpG-STAT3 decoy for targeting survival and immune checkpoint signaling in acute myeloid leukemia.

Targeting oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) in acute myeloid leukemia (AML) can reduce blast survival and tumor immune evasion. Decoy oligodeoxynucleotides (dODNs), which comprise STAT3-specific DNA sequences are competitive inhibition of STAT3 transcriptional activity. To deliver STAT3dODN specifically to myeloid cells, we linked STAT3dODN to the Toll-like receptor 9 (TLR9) ligand, cytosine guanine dinucleotide (CpG). The CpG-STAT3dODN conjugates are quickly internalized by human and mouse TLR9(+)immune cells (dendritic cells, B cells) and the majority of patients' derived AML blasts, including leukemia stem/progenitor cells. Following uptake, CpG-STAT3dODNs are released from endosomes, and bind and sequester cytoplasmic STAT3, thereby inhibiting downstream gene expression in target cells. STAT3 inhibition in patients' AML cells limits their immunosuppressive potential by reduced arginase expression, thereby partly restoring T-cell proliferation. Partly chemically modified CpG-STAT3dODNs have >60 hours serum half-life which allows for IV administration to leukemia-bearing mice (50% effective dose ∼ 2.5 mg/kg). Repeated administration of CpG-STAT3dODN resulted in regression of human MV4-11 AML in mice. The antitumor efficacy of this strategy is further enhanced in immunocompetent mice by combining direct leukemia-specific cytotoxicity with immunogenic effects of STAT3 blocking/TLR9 triggering. CpG-STAT3dODN effectively reducedCbfb/MYH11/MplAML burden in various organs and eliminated leukemia stem/progenitor cells, mainly through CD8/CD4 T-cell-mediated immune responses. In contrast, small-molecule Janus kinase 2/STAT3 inhibitor failed to reproduce therapeutic effects of cell-selective CpG-STAT3dODN strategy. These results demonstrate therapeutic potential of CpG-STAT3dODN inhibitors with broad implications for treatment of AML and potentially other hematologic malignancies.

A multiscale model for the simulation of cerebral and extracerebral blood flows and pressures in humans.

PURPOSE: Brain hemodynamics is fundamental for the functioning of the human being. Many biophysical factors affect brain circulation, so that a satisfactory understanding of its behavior is challenging. We developed a mathematical model to simulate cerebral and extracerebral flows and pressures in humans. METHODS: The model is composed of an anatomically informed 1-D arterial network, and two 0-D networks of the cerebral circulation and brain drainage, respectively. It takes into account the pulse-wave transmission properties of the 55 main arteries and the main hydraulic and autoregulation mechanisms ensuring blood supply and drainage to the brain. Proper pressure outputs from the arterial 1-D model are used as input to the 0-D models, together with the contribution to venous pressure due to breathing that simulates the drainage effect of the thoracic pump. RESULTS: The model we developed is able to link the arterial tree with the venous pathways devoted to the brain drainage, and to simulate important factors affecting cerebral circulation both for physiological and pathological conditions, such as breathing and hypo/hypercapnia. Finally, the average value of simulated flows and pressures is in agreement with the available experimental data. CONCLUSIONS: The model has the potential to predict important clinical parameters before and after physiological and/or pathological changes.

Caffeic Acid and Metformin Inhibit Invasive Phenotype Induced by TGF-ß1 in C-4I and HTB-35/SiHa Human Cervical Squamous Carcinoma Cells by Acting on Different Molecular Targets.

During the progression of epithelial cancer, the cells may lose epithelial markers and gain mesenchymal phenotype via Epithelial-Mesenchymal Transition (EMT). Such transformation of epithelial cancer cells to mesenchymal-like characteristic benefits plasticity and supports their ability to migrate. The aim of this study was to evaluate the influence of natural compound Caffeic Acid (CA) alone and in combination with antidiabetic drug Metformin (Met) on metastatic progression of two human cervical squamous cell cancer lines, C-4I and HTB-35/SiHa cells. EMT program was triggered by exposition of both epithelial cell lines to TGF-ß1. Gene expression patterns related to epithelial/mesenchymal phenotype were evaluated by Real-Time PCR analysis and the protein amount was detected by western blot. The treatment of human squamous cancer cells with CA and with Met, suppressed the motility of cells and the effect depended on a particular cell line. Both compounds regulated the EMT process in C4-I and HTB-35 cells by interfering with different molecular targets. In TGF-ß1-stimulated C4-I cells, CA suppressed the expression of mesenchymal transcription factor SNAI1 which resulted in enhanced expression of epithelial markers E-cadherin, Occludin and Claudin. Additionally, CA blocked MMP-9 and upregulated TIMP-1 expression, a specific inhibitor of MMP-9. In HTB-35 cells stimulated with TGF-ß1, Met decreased the expression of Vimentin. By suppressing hypoxia master regulator HIF-1α, Met caused downregulation of CAIX, an enzyme involved in metastasis of aggressive malignant cells. In this study we showed that CA and Met inhibited EMT process in cancer cells via different mechanisms. However, when applied together, compounds exerted the greater effect on EMT than each compound alone. This is the first report revealing that CA alone and co-treated with Met may reverse mesenchymal phenotype of TGF-ß1-treated cervical tumor cells and we believe that the use of the two small molecules may be considered as a potential therapeutic approach for metastatic cervical cancer.

Introduction of Exogenous HSV-TK Suicide Gene Increases Safety of Keratinocyte-Derived Induced Pluripotent Stem Cells by Providing Genetic "Emergency Exit" Switch.

Since their invention in 2006, induced Pluripotent Stem (iPS) cells remain a great promise for regenerative medicine circumventing the ethical issues linked to Embryonic Stem (ES) cell research. iPS cells can be generated in a patient-specific manner as an unlimited source of various cell types for in vitro drug screening, developmental biology studies and regenerative use. Having the capacity of differentiating into the cells of all three primary germ layers, iPS cells have high potential to form teratoma tumors. This remains their main disadvantage and hazard which, until resolved, prevents utilization of iPS cells in clinic. Here, we present an approach for increasing iPS cells safety by introducing genetic modification-exogenous suicide gene Herpes Simplex Virus Thymidine Kinase (HSV-TK). Its expression results in specific vulnerability of genetically modified cells to prodrug-ganciclovir (GCV). We show that HSV-TK expressing cells can be eradicated both in vitro and in vivo with high specificity and efficiency with low doses of GCV. Described strategy increases iPS cells safety for future clinical applications by generating "emergency exit" switch allowing eradication of transplanted cells in case of their malfunction.

Caffeic Acid Expands Anti-Tumor Effect of Metformin in Human Metastatic Cervical Carcinoma HTB-34 Cells: Implications of AMPK Activation and Impairment of Fatty Acids De Novo Biosynthesis.

The efficacy of cancer treatments is often limited and associated with substantial toxicity. Appropriate combination of drug targeting specific mechanisms may regulate metabolism of tumor cells to reduce cancer cell growth and to improve survival. Therefore, we investigated the effects of anti-diabetic drug Metformin (Met) and a natural compound caffeic acid (trans-3,4-dihydroxycinnamic acid, CA) alone and in combination to treat an aggressive metastatic human cervical HTB-34 (ATCC CRL-1550) cancer cell line. CA at concentration of 100 µM, unlike Met at 10 mM, activated 5'-adenosine monophosphate-activated protein kinase (AMPK). What is more, CA contributed to the fueling of mitochondrial tricarboxylic acids (TCA) cycle with pyruvate by increasing Pyruvate Dehydrogenase Complex (PDH) activity, while Met promoted glucose catabolism to lactate. Met downregulated expression of enzymes of fatty acid de novo synthesis, such as ATP Citrate Lyase (ACLY), Fatty Acid Synthase (FAS), Fatty Acyl-CoA Elongase 6 (ELOVL6), and Stearoyl-CoA Desaturase-1 (SCD1) in cancer cells. In conclusion, CA mediated reprogramming of glucose processing through TCA cycle via oxidative decarboxylation. The increased oxidative stress, as a result of CA treatment, sensitized cancer cells and, acting on cell biosynthesis and bioenergetics, made HTB-34 cells more susceptible to Met and successfully inhibited neoplastic cells. The combination of Metformin and caffeic acid to suppress cervical carcinoma cells by two independent mechanisms may provide a promising approach to cancer treatment.

Quality of life assessment in female patients 2 and 4 years after muscle-derived cell transplants for stress urinary incontinence treatment.

INTRODUCTION: Regenerative medicine for the treatment of urinary incontinence has become a popular area of focus in the search for therapies for this disease. The paper focused on women's quality of life assessment who were subjected to transplantation of MDSC (autologous muscle derived stem cells) to the urethral sphincter. METHODS: The procedure was conducted in 16 female patients who completed the observation stage. Assessment of quality of life before and after the treatment (two and four years post-operation) was conducted based on the validated I-QOL questionnaire (the Polish language version). RESULTS: The questionnaire study showed that autologous cell therapy significantly improves quality of life in female patients suffering from stress urinary incontinence (SUI). The total I-QOL score increased from 49 (SD ± 7.7) before therapy to 77 (SD ± 5.4) two years post-operation. Four years after the procedure, quality of life remained at a higher level than before therapy, although quality of life decreased by several points when compared with the results from the two-year follow-up - 63 (SD ± 7.2). Patients reported significantly less concern related to their ability to reach the toilet to avoid incontinence, improved sleep at night, a higher level of satisfaction with life, and more satisfaction with their sexual lives (p<0.05). CONCLUSION: The MDSC injection procedure for SUI treatment has significant improved quality of life in the majority of our patients in 2 and 4 year follow-up.

Regenerative medicine- techniques and methods of administering autologous derived stem cells in urinary incontinence.

The aim of the work is to present regenerative medicine achievement as an alternative SUI treatment and the variety of injected cells type as well as injection techniques itself with the analysis of their quality and possible the mechanism in which they reduce urinary incontinence symptoms. For over a decade numerous authors declare use of different type of autologous mesenchymal-derived stem cells (AMDC) in male and female SUI. The leakage improvement reached 80%, despite the number of injected cells as well as the injection technique. Important subject in the AMDC treatment is the precise cell material injection into the selected spot which might be possible with the use of the endoscopic assisting robot. The robotic supported system for cells procedure might bring the missing percentage in reaching the goal in SUI treatment.

Autologous muscle-derived cells for the treatment of female stress urinary incontinence: a 2-year follow-up of a Polish investigation.

AIMS: We evaluated the safety, feasibility and initial effects of therapy with muscle-derived cells (MDCs) for women with stress urinary incontinence (SUI). METHODS: MDCs were isolated from an upper-arm muscle biopsy from 16 women with SUI. Cells were isolated by enzymatic digestion and expanded in vitro for 8-10 weeks. A quantity of 0.6-25 × 10(6) of the obtained cells were injected transurethrally into the urethral rhabdosphincter of women under local anesthesia. The cells were placed circumferentially at the 9, 12, and 3 O'clock positions with endoscopic guidance. RESULTS: The initial results of the treatment of SUI with adult muscle-derived stem cells demonstrate the safety and feasibility of using these cells. The 2-year follow-up revealed a 75% success rate, with some patients achieving complete improvement (50%) and some patients achieving partial improvement (25%), suggesting that the prospects for this method are encouraging. CONCLUSIONS: Stem cell therapy promises to become a minimally invasive method for the regeneration of the urethral rhabdosphincter muscle. Injecting a small number of cells does not preclude obtaining the desired therapeutic result.

The Impact of Microorganisms on Canine Semen Quality.

Various microorganisms, including Mycoplasma spp., have been reported in canine ejaculate. The impact of these microorganisms on semen quality remains unclear. This study included 63 male intact healthy dogs aged 1-8 years. One dog exhibited azoospermia, indicating a relatively low incidence of this condition. Interestingly, 36.5% of the examined dogs tested negative for both aerobic bacteria and mycoplasmas, while 12.7% tested positive for bacterial presence. Additionally, 60.3% of the dogs tested positive for Mycoplasma spp. using PCR, with most carrying 1-2 Mycoplasma species. We found no significant difference in semen characteristics between Mycoplasma-positive and -negative dogs. The detection of Mycoplasma was not significantly linked to the presence of bacteria in semen. All the microorganisms identified were classified as saprophytic flora. Our findings indicate that Mycoplasma spp. is common in canine ejaculate. Semen quality parameters were not correlated with the presence of Mycoplasma spp. in semen. Mycoplasma HRC689 was the most common species. Some dogs exhibited no presence of aerobic bacteria or mycoplasmas in their semen. Our study highlights the common presence of Mycoplasma spp. in canine ejaculate. Semen quality shows no correlation with Mycoplasma presence. Some canine ejaculate is sterile. Our findings suggest the existence of undescribed species of canine mycoplasmas, necessitating advanced diagnostic techniques like NGS for their identification.