Regulation of Immune Checkpoint Antigen CD276 (B7-H3) on Human Placenta-Derived Mesenchymal Stromal Cells in GMP-Compliant Cell Culture Media.

Therapies utilizing autologous mesenchymal cell delivery are being investigated as anti-inflammatory and regenerative treatments for a broad spectrum of age-related diseases, as well as various chronic and acute pathological conditions. Easily available allogeneic full-term human placenta mesenchymal stromal cells (pMSCs) were used as a potential pro-regenerative, cell-based therapy in degenerative diseases, which could be applied also to elderly individuals. To explore the potential of allogeneic pMSCs transplantation for pro-regenerative applications, such cells were isolated from five different term-placentas, obtained from the dissected maternal, endometrial (mpMSCs), and fetal chorion tissues (fpMSCs), respectively. The proliferation rate of the cells in the culture, as well as their shape, in vitro differentiation potential, and the expression of mesenchymal lineage and stem cell markers, were investigated. Moreover, we studied the expression of immune checkpoint antigen CD276 as a possible modulation of the rejection of transplanted non-HLA-matched homologous or even xeno-transplanted pMSCs. The expression of the cell surface markers was also explored in parallel in the cryosections of the relevant intact placenta tissue samples. The expansion of pMSCs in a clinical-grade medium complemented with 5% human platelet lysate and 5% human serum induced a significant expression of CD276 when compared to mpMSCs expanded in a commercial medium. We suggest that the expansion of mpMSCs, especially in a medium containing platelet lysate, elevated the expression of the immune-regulatory cell surface marker CD276. This may contribute to the immune tolerance towards allogeneic pMSC transplantations in clinical situations and even in xenogenic animal models of human diseases. The endurance of the injected comparably young human-term pMSCs may promote prolonged effects in clinical applications employing non-HLA-matched allogeneic cell therapy for various degenerative disorders, especially in aged adults.

CD24: A Marker for an Extended Expansion Potential of Urothelial Cancer Cell Organoids In Vitro?

BACKGROUND: Bladder cancer is the most cost-intensive cancer due to high recurrence rates and long follow-up times. Bladder cancer organoids were considered interesting tools for investigating better methods for the detection and treatment of this cancer. METHODS: Organoids were generated from urothelial carcinoma tissue samples, then expanded and characterized; the expression of immune modulatory antigens and tumor stem cells markers CD24 and CD44 was explored in early (P ≤ 3) and later (P ≥ 5) passages (P) by immunofluorescence and by quantitative PCR of cDNA. The expression of these factors was investigated in the corresponding cancer tissue samples by immunohistochemistry. RESULTS: The expression of the PD-L1 was detected on some but not all organoids. CD276 and CD47 were observed on organoids in all passages investigated. Organoids growing beyond passage 8 expressed both CD24 and CD44 at elevated levels in early and late cultures. Organoids proliferating to the eighth passage initially expressed both CD24 and CD44, but lost CD24 expression over time, while CD44 remained. Organoids growing only up to the 6th passage failed to express CD24 but expressed CD44. CONCLUSIONS: The data indicate that the expression of CD24 in urothelial cancer cell organoids may serve as an indicator for the prolonged proliferation potential of the cells.

Urinary Tract Tumor Organoids Reveal Eminent Differences in Drug Sensitivities When Compared to 2-Dimensional Culture Systems.

Generation of organoids from urinary tract tumor samples was pioneered a few years ago. We generated organoids from two upper tract urothelial carcinomas and from one bladder cancer sample, and confirmed the expression of cytokeratins as urothelial antigens, vimentin as a mesenchymal marker, and fibroblast growth factor receptor 3 by immunohistochemistry. We investigated the dose response curves of two novel components, venetoclax versus S63845, in comparison to the clinical standard cisplatin in organoids in comparison to the corresponding two-dimensional cultures. Normal urothelial cells and tumor lines RT4 and HT1197 served as controls. We report that upper tract urothelial carcinoma cells and bladder cancer cells in two-dimensional cultures yielded clearly different sensitivities towards venetoclax, S63845, and cisplatin. Two-dimensional cultures were more sensitive at low drug concentrations, while organoids yielded higher drug efficacies at higher doses. In some two-dimensional cell viability experiments, colorimetric assays yielded different IC50 toxicity levels when compared to chemiluminescence assays. Organoids exhibited distinct sensitivities towards cisplatin and to a somewhat lesser extent towards venetoclax or S63845, respectively, and significantly different sensitivities towards the three drugs investigated when compared to the corresponding two-dimensional cultures. We conclude that organoids maintained inter-individual sensitivities towards venetoclax, S63845, and cisplatin. The preclinical models and test systems employed may bias the results of cytotoxicity studies.

Elevated Expression of the Immune Checkpoint Ligand CD276 (B7-H3) in Urothelial Carcinoma Cell Lines Correlates Negatively with the Cell Proliferation.

The cell surface molecule CD276 (B7-H3) is an immune checkpoint antigen. The elevated expression of CD276 on tumors contributes to the suppression of anti-tumor T-cell responses and correlates with poor prognosis. METHODS: The expression of CD276 was explored in vitro on eight urothelial carcinoma cell lines (UM-UC) in comparison to eight normal urothelial cells (NUCs) by RT-qPCR, Western blotting, and flow cytometry. Cell proliferation was enumerated over consecutive passages. The expression of cancer stem cell markers CD24 and CD44, cytokeratins, and vimentin was investigated by immunofluorescence. The expression of CD276 in bladder tumor samples and metastases was explored by immunohistochemistry. RESULTS: Expression of CD276 on cell surfaces was elevated on UM-UCs when compared to NUCs. In UM-UCs, CD276 transcripts correlated moderately positive with CD276 protein expression (ρ = 0.660) and strongly positive with CD276 surface-expression (ρ = 0.810). CD276 mRNA expression (ρ = -0.475) and CD276 protein expression (ρ = -0.417) had a significant negative correlation with proliferation, while a significant correlation between proliferation and cell surface expression was not observed in UM-UCs. CONCLUSION: The expression of CD276 on UM-UC bladder tumor cell surfaces is elevated. Slow proliferating UM-UC cells express more CD276 mRNA and protein than fast proliferating cells. In patients, slow proliferating CD276high tumor (stem) cells may evade immune surveillance. However, cancer therapy targeting CD276 may be effective in the treatment of slow proliferating tumor cells.

Data-Driven Identification of Biomarkers for In Situ Monitoring of Drug Treatment in Bladder Cancer Organoids.

Three-dimensional (3D) organoid culture recapitulating patient-specific histopathological and molecular diversity offers great promise for precision medicine in cancer. In this study, we established label-free imaging procedures, including Raman microspectroscopy (RMS) and fluorescence lifetime imaging microscopy (FLIM), for in situ cellular analysis and metabolic monitoring of drug treatment efficacy. Primary tumor and urine specimens were utilized to generate bladder cancer organoids, which were further treated with various concentrations of pharmaceutical agents relevant for the treatment of bladder cancer (i.e., cisplatin, venetoclax). Direct cellular response upon drug treatment was monitored by RMS. Raman spectra of treated and untreated bladder cancer organoids were compared using multivariate data analysis to monitor the impact of drugs on subcellular structures such as nuclei and mitochondria based on shifts and intensity changes of specific molecular vibrations. The effects of different drugs on cell metabolism were assessed by the local autofluorophore environment of NADH and FAD, determined by multiexponential fitting of lifetime decays. Data-driven neural network and data validation analyses (k-means clustering) were performed to retrieve additional and non-biased biomarkers for the classification of drug-specific responsiveness. Together, FLIM and RMS allowed for non-invasive and molecular-sensitive monitoring of tumor-drug interactions, providing the potential to determine and optimize patient-specific treatment efficacy.

Rapid and precise delivery of cells in the urethral sphincter complex by a novel needle-free waterjet technology.

OBJECTIVES: To investigate the therapy of stress urinary incontinence in a preclinical setting cells were injected into the urethrae of minipigs; however, cells injected by William's needle were frequently misplaced or lost; thus, we investigated if needle-free cell injections using a novel waterjet technology facilitates precise injections in the urethral sphincter complex. MATERIALS AND METHODS: Porcine adipose tissue-derived stromal cells (pADSCs) were isolated from boars, expanded, labelled, and injected in the sphincter of female pigs by waterjet employing two different protocols. After incubation for 15 min or 3 days, the urethrae of the pigs were examined. Injected cells were visualised by imaging and fluorescence microscopy of tissue sections. DNA of injected male cells was verified by polymerase chain reaction (PCR) of the sex-determining region (SRY) gene. Cell injections by William's needle served as controls. RESULTS: The new waterjet technology delivered pADSCs faster and with better on-site precision than the needle injections. Bleeding during or after waterjet injection or other adverse effects, such as swelling or urinary retention, were not observed. Morphologically intact pADSCs were detected in the urethrae of all pigs treated by waterjet. SRY-PCR of chromosomal DNA and detection of recombinant green fluorescent protein verified the injection of viable cells. In contrast, three of four pigs injected by William's needle displayed no or misplaced cells. CONCLUSION: Transurethral injection of viable pADSCs by waterjet is a simple, fast, precise, and yet gentle new technology. This is the first proof-of-principle concept study providing evidence that a waterjet injects intact cells exactly in the tissue targeted in a preclinical in vivo situation. To further explore the clinical potential of the waterjet technology longer follow-up, as well as incontinence models have to be studied.

Expression patterns of the immune checkpoint ligand CD276 in urothelial carcinoma.

BACKGROUND: CD276 is an immune checkpoint molecule. Elevated CD276 expression by urothelial carcinoma is associated with poor prognosis, but little is known about its expression across different tumor stages. We therefore investigated CD276 expression in bladder cancer (BC) cells and in tissue samples of BC stages from pT2 to pT4. METHODS: CD276 expression was explored in 4 urothelial cancer cell lines and 4 primary normal urothelial cell populations by quantitative RT-PCR, Western blot and flow cytometry. CD276 was investigated in bladder tumors from 98 patients by immunohistochemistry using a score (0-300) incorporating both, staining intensity and area of CD276 staining. Normal appearing urothelium in the bladder of the same patients served as controls. RESULTS: The urothelial carcinoma cell lines expressed significantly higher levels of CD276 on transcript (p < 0.006), total protein levels (p < 0.005), and on the cell surface (p < 0.02) when compared to normal urothelial cells. In pT2-T4 tumor tissue samples, CD276 was overexpressed (median score 185) when compared to corresponding healthy tissues from the same patients (median score 50; p < 0.001). No significant differences in CD276 expression were recorded in late, locally advanced ≥ pT3a tumors (median score 185) versus organ-confined < pT3a tumors (median score 190), but it was significantly lower in the normal urothelial tissue associated with ≥ pT3a tumors (median score 40) versus < pT3a tumors (median score 80; p < 0.05). CONCLUSION: CD276 expression is significantly elevated in urothelial carcinoma cells in all stages but varies between individuals considerably. Reduced CD276 expression in normal urothelial cells may imply that these cells would be protected from CD276-mediated immuno therapies.

Allogenic Use of Human Placenta-Derived Stromal Cells as a Highly Active Subtype of Mesenchymal Stromal Cells for Cell-Based Therapies.

The application of mesenchymal stromal cells (MSCs) from different sources, including bone marrow (BM, bmMSCs), adipose tissue (atMSCs), and human term placenta (hPSCs) has been proposed for various clinical purposes. Accumulated evidence suggests that the activity of the different MSCs is indirect and associated with paracrine release of pro-regenerative and anti-inflammatory factors. A major limitation of bmMSCs-based treatment for autologous application is the limited yield of cells harvested from BM and the invasiveness of the procedure. Similar effects of autologous and allogeneic MSCs isolated from various other tissues were reported. The easily available fresh human placenta seems to represent a preferred source for harvesting abundant numbers of human hPSCs for allogenic use. Cells derived from the neonate tissues of the placenta (f-hPSC) can undergo extended expansion with a low risk of senescence. The low expression of HLA class I and II on f-hPSCs reduces the risk of rejection in allogeneic or xenogeneic applications in normal immunocompetent hosts. The main advantage of hPSCs-based therapies seems to lie in the secretion of a wide range of pro-regenerative and anti-inflammatory factors. This renders hPSCs as a very competent cell for therapy in humans or animal models. This review summarizes the therapeutic potential of allogeneic applications of f-hPSCs, with reference to their indirect pro-regenerative and anti-inflammatory effects and discusses clinical feasibility studies.

Novel Techniques to Improve Precise Cell Injection.

We noted recently that the injection of cells with a needle through a cystoscope in the urethral sphincter muscle of pigs failed to deposit them nearby or at the intended target position in about 50% of all animals investigated (n > 100). Increasing the chance for precise cell injection by shotgun approaches employing several circumferential injections into the sphincter muscle bears the risk of tissue injury. In this study, we developed and tested a novel needle-free technique to precisely inject cells in the urethral sphincter tissue, or other tissues, using a water-jet system. This system was designed to fit in the working channels of endoscopes and cystoscopes, allowing a wide range of minimally invasive applications. We analyze key features, including the physical parameters of the injector design, pressure ranges applicable for tissue penetration and cell injections and biochemical parameters, such as different compositions of injection media. Our results present settings that enable the high viability of cells post-injection. Lastly, the method is suitable to inject cells in the superficial tissue layer and in deeper layers, required when the submucosa or the sphincter muscle of the urethra is targeted.

Large Animal Models for Investigating Cell Therapies of Stress Urinary Incontinence.

Stress urinary incontinence (SUI) is a significant health concern for patients affected, impacting their quality of life severely. To investigate mechanisms contributing to SUI different animal models were developed. Incontinence was induced under defined conditions to explore the pathomechanisms involved, spontaneous recovery, or efficacy of therapies over time. The animal models were coined to mimic known SUI risk factors such as childbirth or surgical injury. However, animal models neither reflect the human situation completely nor the multiple mechanisms that ultimately contribute to the pathogenesis of SUI. In the past, most SUI animal studies took advantage of rodents or rabbits. Recent models present for instance transgenic rats developing severe obesity, to investigate metabolic interrelations between the disorder and incontinence. Using recombinant gene technologies, such as transgenic, gene knock-out or CRISPR-Cas animals may narrow the gap between the model and the clinical situation of patients. However, to investigate surgical regimens or cell therapies to improve or even cure SUI, large animal models such as pig, goat, dog and others provide several advantages. Among them, standard surgical instruments can be employed for minimally invasive transurethral diagnoses and therapies. We, therefore, focus in this review on large animal models of SUI.

Injection of Porcine Adipose Tissue-Derived Stromal Cells by a Novel Waterjet Technology.

Previously, we developed a novel, needle-free waterjet (WJ) technology capable of injecting viable cells by visual guided cystoscopy in the urethral sphincter. In the present study, we aimed to investigate the effect of WJ technology on cell viability, surface markers, differentiation and attachment capabilities, and biomechanical features. Porcine adipose tissue-derived stromal cells (pADSCs) were isolated, expanded, and injected by WJ technology. Cell attachment assays were employed to investigate cell-matrix interactions. Cell surface molecules were analyzed by flow cytometry. Cells injected by Williams Needle (WN), normal cannula, or not injected cells served as controls. Biomechanical properties were assessed by atomic force microscopy (AFM). pADSCs injected by the WJ were viable (85.9%), proliferated well, and maintained their in vitro adipogenic and osteogenic differentiation capacities. The attachment of pADSCs was not affected by WJ injection and no major changes were noted for cell surface markers. AFM measurements yielded a significant reduction of cellular stiffness after WJ injections (p < 0.001). WJ cell delivery satisfies several key considerations required in a clinical context, including the fast, simple, and reproducible delivery of viable cells. However, the optimization of the WJ device may be necessary to further reduce the effects on the biomechanical properties of cells.

Treatment of Stress Urinary Incontinence with Muscle Stem Cells and Stem Cell Components: Chances, Challenges and Future Prospects.

Urinary incontinence (UI) is a major problem in health care and more than 400 million people worldwide suffer from involuntary loss of urine. With an increase in the aging population, UI is likely to become even more prominent over the next decades and the economic burden is substantial. Among the different subtypes of UI, stress urinary incontinence (SUI) is the most prevalent and focus of this review. The main underlying causes for SUI are pregnancy and childbirth, accidents with direct trauma to the pelvis or medical treatments that affect the pelvic floor, such as surgery or irradiation. Conservative approaches for the treatment of SUI are pelvic physiotherapy, behavioral and lifestyle changes, and the use of pessaries. Current surgical treatment options include slings, colposuspensions, bulking agents and artificial urinary sphincters. These treatments have limitations with effectiveness and bear the risk of long-term side effects. Furthermore, surgical options do not treat the underlying pathophysiological causes of SUI. Thus, there is an urgent need for alternative treatments, which are effective, minimally invasive and have only a limited risk for adverse effects. Regenerative medicine is an emerging field, focusing on the repair, replacement or regeneration of human tissues and organs using precursor cells and their components. This article critically reviews recent advances in the therapeutic strategies for the management of SUI and outlines future possibilities and challenges.

A novel waterjet technology for transurethral cystoscopic injection of viable cells in the urethral sphincter complex.

AIMS: In a recent preclinical study, we noticed that injection of cells in the urethral sphincter by needle through a cystoscope under visual control frequently yielded in misplacement or loss of cells. We, therefore, investigated if a needle-free waterjet device delivers viable cells under defined settings, including injection volume and pressure, fluid velocity and transportation media, precisely through the urothelium and connective tissue close to the sphincter muscle without full penetration of the sphincter apparatus. METHODS: Mesenchymal stromal cells (MSCs) were prepared for needle-free waterjet injections. Upon injections into liquids cell viability and yield were investigated by trypan blue dye exclusion. Upon injection into cadaveric urethral tissue samples, cells were isolated from the urethrae and expanded to prove that this novel method delivered viable cells into the tissue. MSC injections by William's needle served as controls. RESULTS: Waterjet injections of MSCs into isotonic cell culture medium resulted in equal or better yields of viable cells when compared with needle injections. Upon injection in urethral tissue samples, the waterjet technology facilitated fast and precise injections of viable cells through urothelial, mucosal and submucosal layers to reach the sphincter muscle. By controlling the injection pressure, loss of cells due to insufficient thrust or unintended full penetration was avoided. CONCLUSIONS: Needle-free waterjet injections deliver cells in the urethra faster and more precisely when compared with needle injections without compromising their viability. This is the first proof-of-concept study providing evidence that a waterjet transports viable cells precisely into the targeted tissue.

Risk assessment of neuromuscular stimulation by energy-based transurethral resection devices: an ex vivo test standard.

BACKGROUND: During transurethral resection of bladder tumours (TURB), radio-frequency (RF) currents can lead to adverse neuromuscular stimulation (NMS). Here we present a novel ex vivo method to determine the risk of RF generators and their bipolar TURB modes to cause NMS. We aimed to develop an experimental platform for safety evaluation of new RF generators and their modes with a newly established test standard, suitable for replacement or reduction of animal testing. METHODS: We tested four contemporary RF generators with their bipolar modes for TURB in saline. A two-stage ex vivo approach was pursued: First, we recorded voltages at possible positions of the obturator nerve behind a porcine bladder wall in a TURB model using 18 RF applications per generator. Second, these voltage records were used as stimuli to evoke nerve compound action potentials (CAPs) in isolated porcine axillary nerves. The NMS potential was defined as the ratio between the observed area under the CAPs and the theoretical CAP area at maximum response and a firing rate of 250 Hz, which would reliably induce tetanic muscle responses in most human subjects. The measurement protocol was tailored to optimise reproducibility of the obtained NMS potentials and longevity of the nerve specimens. RESULTS: As prerequisite for the clinical translation of our results, the robustness of our test method and reproducibility of the NMS potential are demonstrated with an excellent correlation (r = 0.93) between two sets of identical stimuli (n = 72 each) obtained from 16 nerve segments with similar diameters (4.2 ± 0.37 mm) in the nerve model. The RF generators differed significantly (p < 0.0001) regarding NMS potential (medians: 0-3%). CONCLUSIONS: Our test method is suitable for quantifying the NMS potential of different electrosurgical systems ex vivo with high selectivity at a reasonable degree of standardization and with justifiable effort. Our results suggest that the clinical incidence of NMS is considerably influenced by the type of RF generator. Future generations of RF generators take advantage from the proposed test standard through higher safety and less animal testing. Health professionals and treated patients will benefit most from improved RF surgery using generators with a low NMS risk.

A sensitive refining of in vitro and in vivo toxicological behavior of green synthesized ZnO nanoparticles from the shells of Jatropha curcas for multifunctional biomaterials development.

ZnO nanoparticles (NPs) possess a wide range of biological functions in pharmaceutical and cosmetic applications due to their excellent antimicrobial, optical and UV protective properties. This study first reports the toxicological assessment of ZnO NPs green synthesized from Jatropha curcas shells for multifunctional biomedical applications. The hot water extract of J.curcas shells is utilized as a chelating agent for the reduction of zinc acetate and then, the prepared ZnO NPs are broadly characterized using X-ray spectroscopic and electron microscopic observations. The prepared ZnO NPs acquire high purity (100%) wurtzite crystal with hexagonal structure with the average particle size of 53 nm. In vitro and in vivo toxicity evaluation against human tumor cell lines and zebrafish embryos have ascertained the purpose of ZnO NPs in clinical research. Toxic effects of ZnO NPs were observed by a dose-dependent reduction of bacterial growth at ≥1   µg ml-1, by teratogenicity and genotoxicity in zebrafish embryos (from 3 to 90 µg ml-1) and by a significant nanoparticle uptake (0.5 ng µl-1) by a fish serum. In contrast, ZnO NPs fail to reduce the proliferation of human bladder tumor cells (UC6) and cell viability of A549 cells in vitro up to 500 µg ml-1. All these observations limit the unobstructed application of ZnO NPs at higher concentrations. Thus, abundantly used metal oxide nanoparticles like ZnO NPs examined in our present study in different animal models under in vitro and in vivo conditions will be the significant screening strategy to determine the nanotoxicity.

Establishing and monitoring of urethral sphincter deficiency in a large animal model.

BACKGROUND: Different methods for induction and monitoring of urethral sphincter deficiency were explored in a large animal model. METHODS: Sphincter deficiency was established in female pigs by dilatation and cauterization, and amount and frequencies of voiding were monitored and explored by pad test. Sphincteric closure pressures were recorded prior to and immediately after treatment of each animal, and on day 21 by two techniques: standard urethral pressure profilometry (s-UPP) and high-definition urethral pressure profilometry (HD-UPP). Tissue samples of the urethrae were analyzed by histochemistry (AZAN- and Sirius Red staining) and by immunohistochemistry detecting desmin and fast-myosin to depict muscular tissues. RESULTS: After 3 weeks of observation animals treated by dilatation plus electrocautery presented with sphincter deficiency: measurements by both, s-UPP and HD-UPP demonstrated the maximal closure pressure reduced to baseline levels and a diminished area under the curve. Histological analyses documented, that dilatation yielded a pitted connective tissue and cauterization lead to muscle damage. Animals treated by either dilatation only or proximal injury only recovered within 3 weeks. By pad test no significant differences between untreated and treated animals or between the differently treated groups were recorded. CONCLUSION: Significant urethral sphincter deficiency can be induced in female pigs by a combination of urethral dilatation and distal electrocautery. Sphincter deficiency can be measured by standard and high-definition urethral pressure profilometry. It was maintained over 21 days after induction and correlated with visible changes in the tissue structure of the distal urethra.

Precise injection of human mesenchymal stromal cells in the urethral sphincter complex of Göttingen minipigs without unspecific bulking effects.

AIM: To investigate if injection of cells in the urethral sphincter complex causes unspecific bulking effects. METHODS: Human mesenchymal stromal cells were isolated, expanded, and characterized. For transurethral injection, cells were labeled with the fluorescent dye PKH26 and in magnetic resonance imaging associated experiments with superparamagnetic particles. Aliquots of cells in 250 µL solvent were injected under vision in the urethral sphincter of immuno-suppressed Göttingen minipigs. Sphincteric closure pressure was recorded by standard and high-definition urethral pressure profilometry prior to and after cell injection. The animals were sacrificed after surgery or after 3 weeks, 3, 6, or 12 months of follow-up. The localisation of the injected cells was explored by histochemistry. Sham-treated animals served as controls. RESULTS: PKH26-labeled cells survive injections in sphincter tissue samples by Williams cystoscopic injection needle well. In our animal study, the cellular depots were detected in the submucosa or in deeper zones of the sphincter, depending of the length of the injection needle (4-8 mm). Adverse effects associated with injection of cells or solvent such as a noteworthy bleeding, incontinence, or obstruction, were not recorded (n = 96 minipigs). However, a transient infiltration of macrophages was detected 3 weeks after cell injection. Changes in the urethral pressure profiles were not observed in cell-treated (n = 72) compared to sham-treated animals (n = 24). CONCLUSIONS: Injection of small aliquots of cells to investigate cell therapies in minipigs is a feasible and safe procedure, and it does not bias the intrinsic urethral wall pressure.

Choice of xenogenic-free expansion media significantly influences the myogenic differentiation potential of human bone marrow-derived mesenchymal stromal cells.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) have great potential for use in cell-based therapies for restoration of structure and function of many tissue types including smooth muscle. METHODS: We compared proliferation, immunophenotype, differentiation capability and gene expression of bone marrow-derived MSCs expanded in different media containing human serum, plasma and platelet lysate in combination with commonly used protocols for myogenic, osteogenic, chondrogenic and adipogenic differentiation. Moreover, we developed a xenogenic-free protocol for myogenic differentiation of MSCs. RESULTS: Expansion of MSCs in media complemented with serum, serum + platelet lysate or plasma + platelet lysate were multipotent because they differentiated toward four mesenchymal (myogenic, osteogenic, chondrogenic, adipogenic) lineages. Addition of platelet lysate to expansion media increased the proliferation of MSCs and their expression of CD146. Incubation of MSCs in medium containing human serum or plasma plus 5% human platelet lysate in combination with smooth muscle cell (SMC)-inducing growth factors TGFß1, PDGF and ascorbic acid induced high expression of ACTA2, TAGLN, CNN1 and/or MYH11 contractile SMC markers. Osteogenic, adipogenic and chondrogenic differentiations served as controls. DISCUSSION: Our study provides novel data on the myogenic differentiation potential of human MSCs toward the SMC lineage using different xenogenic-free cell culture expansion media in combination with distinct differentiation medium compositions. We show that the choice of expansion medium significantly influences the differentiation potential of human MSCs toward the smooth muscle cell, as well as osteogenic, adipogenic and chondrogenic lineages. These results can aid in designing studies using MSCs for tissue-specific therapeutic applications.

The spatial organisation of joint surface chondrocytes: review of its potential roles in tissue functioning, disease and early, preclinical diagnosis of osteoarthritis.

Chondrocytes display within the articular cartilage depth-dependent variations of their many properties that are comparable to the depth-dependent changes of the properties of the surrounding extracellular matrix. However, not much is known about the spatial organisation of the chondrocytes throughout the tissue. Recent studies revealed that human chondrocytes display distinct spatial patterns of organisation within the articular surface, and each joint surface is dominated in a typical way by one of four basic spatial patterns. The resulting complex spatial organisations correlate with the specific diarthrodial joint type, suggesting an association of the chondrocyte organisation within the joint surface with the occurring biomechanical forces. In response to focal osteoarthritis (OA), the superficial chondrocytes experience a destruction of their spatial organisation within the OA lesion, but they also undergo a defined remodelling process distant from the OA lesion in the remaining, intact cartilage surface. One of the biological insights that can be derived from this spatial remodelling process is that the chondrocytes are able to respond in a generalised and coordinated fashion to distant focal OA. The spatial characteristics of this process are tremendously different from the cellular aggregations typical for OA lesions, suggesting differences in the underlying mechanisms. Here we summarise the available information on the spatial organisation of chondrocytes and its potential roles in cartilage functioning. The spatial organisation could be used to diagnose early OA onset before manifest OA results in tissue destruction and clinical symptoms. With further development, this concept may become clinically suitable for the diagnosis of preclinical OA.

Cell Therapy by Mesenchymal Stromal Cells Versus Myoblasts in a Pig Model of Urinary Incontinence.

The leading cause of stress urinary incontinence (SUI) in women is the urethral sphincter muscle deficiency caused by mechanical stress during pregnancy and vaginal delivery. In men, prostate cancer surgery and injury of local nerves and muscles are associated with incontinence. Current treatment often fails to satisfy the patient's needs. Cell therapy may improve the situation. We therefore investigated the regeneration potential of cells in ameliorating sphincter muscle deficiency and UI in a large animal model. Urethral sphincter deficiency was induced surgically in gilts by electrocautery and balloon dilatation. Adipose tissue-derived stromal cells (ADSCs) and myoblasts from Musculus semitendinosus were isolated from male littermates, expanded, characterized in depth for expression of marker genes and in vitro differentiation, and labeled. The cells were injected into the deficient sphincter complex of the incontinent female littermates. Incontinent gilts receiving no cell therapy served as controls. Sphincter deficiency and functional regeneration were recorded by monitoring the urethral wall pressure during follow-up by two independent methods. Cells injected were detected in vivo during follow-up by transurethral fluorimetry, ex vivo by fluorescence imaging, and in cryosections of tissues targeted by immunofluorescence and by polymerase chain reaction of the sex-determining region Y (SRY) gene. Partial spontaneous regeneration of sphincter muscle function was recorded in control gilts, but the sphincter function remained significantly below levels measured before induction of incontinence (67.03% ± 14.00%, n = 6, p < 0.05). Injection of myoblasts yielded an improved sphincter regeneration within 5 weeks of follow-up but did not reach significance compared to control gilts (81.54% ± 25.40%, n = 5). A significant and full recovery of the urethral sphincter function was observed upon injection of ADSCs within 5 weeks of follow-up (100.4% ± 23.13%, n = 6, p < 0.05). Injection of stromal cells provoked slightly stronger infiltration of CD45pos leukocytes compared to myoblasts injections and controls. The data of this exploratory study indicate that ADSCs inherit a significant potential to regenerate the function of the urethral sphincter muscle.