Transurethral injection of autologous muscle precursor cells for treatment of female stress urinary incontinence: a prospective phase I clinical trial.

INTRODUCTION AND HYPOTHESIS: The purpose was to investigate the safety and feasibility of transurethral injections of autologous muscle precursor cells (MPCs) into the external urinary sphincter (EUS) to treat stress urinary incontinence (SUI) in female patients. METHODS: Prospective and randomised phase I clinical trial. Standardised 1-h pad test, International Consultation on Incontinence Questionnaire-Urinary Incontinence Short Form (ICIQ-UI-SF), urodynamic study, and MRI of the pelvis were performed at baseline and 6 months after treatment. MPCs gained through open muscle biopsy were transported to a GMP facility for processing and cell expansion. The final product was injected into the EUS via a transurethral ultrasound-guided route. Primary outcomes were defined as any adverse events (AEs) during follow-up. Secondary outcomes were functional, questionnaire, and radiological results. RESULTS: Ten female patients with SUI grades I-II were included in the study and 9 received treatment. Out of 8 AEs, 3 (37.5%) were potentially related to treatment and treated conservatively: 1 urinary tract infection healed with antibiotics treatment, 1 dysuria and 1 discomfort at biopsy site. Functional urethral length under stress was 25 mm at baseline compared with 30 mm at 6 months' follow-up (p=0.009). ICIQ-UI-SF scores improved from 7 points at baseline to 4 points at follow-up (p=0.035). MRI of the pelvis revealed no evidence of tumour or necrosis, whereas the diameter of the EUS muscle increased from 1.8 mm at baseline to 1.9 mm at follow-up (p=0.009). CONCLUSION: Transurethral injections of autologous MPCs into the EUS for treatment of SUI in female patients can be regarded as safe and feasible. Only a minimal number of expected and easily treatable AEs were documented.

Adult stem cell sources for skeletal and smooth muscle tissue engineering.

INTRODUCTION: Tissue engineering is an innovative field with enormous developments in recent years. These advances are not only in the understanding of how stem cells can be isolated, cultured and manipulated but also in their potential for clinical applications. Thus, tissue engineering when applied to skeletal and smooth muscle cells is an area that bears high benefit for patients with muscular diseases or damage. Most of the recent research has been focused on use of adult stem cells. These cells have the ability to rejuvenate and repair damaged tissues and can be derived from different organs and tissue sources. Recently there are several different types of adult stem cells, which have the potential to function as a cell source for tissue engineering of skeletal and smooth muscles. However, to build neo-tissues there are several challenges which have to be addressed, such as the selection of the most suitable stem cell type, isolation techniques, gaining control over its differentiation and proliferation process. CONCLUSION: The usage of adult stem cells for muscle engineering applications is promising. Here, we summarize the status of research on the use of adult stem cells for cell transplantation in experimental animals and humans. In particular, the application of skeletal and smooth muscle engineering in pre-clinical and clinical trials will be discussed.

Feasibility, technique and accuracy of ultrasound-guided transurethral injections into the urinary sphincter of female cadavers: proof of concept.

BACKGROUND: The injection of muscle precursor cells (MPC) into the external urinary sphincter muscle (EUS) is a promising therapeutic option for regenerative treatment of stress urinary incontinence (SUI). The objective of the present project was to conduct a pre-clinical trial to investigate the feasibility and accuracy of ultrasound (US) guided, transurethral injections into the EUS of female cadavers. METHODS: This is a prospective, anatomical, interventional and radiological cadaveric laboratory investigation. Two urologists performed transurethral US-guided injections to deliver nano-iron particles into the EUS. The intervention was performed in three unfixed, fresh female cadavers. Each cadaver received MRI before and CT as well as MRI of the pelvis after the injections. RESULTS: The precision and accumulation of nano-iron particles in the EUS was compared using a rating scale to evaluate left versus right and anterior versus posterior distribution in axial and sagittal orientation with US, MRI and CT. The accuracy of our US-guided injections into the anterior target region yielded 4 points on the rating scale. Adequate precision and accumulation of particles in the left versus right EUS were also demonstrated (3 vs. 3.33 points, respectively). Signal intensity in MRI revealed a mean ratio of 0.33 before and after injection. CT scans showed no relevant artefacts impairing the assessment. CONCLUSION: US-guided, transurethral injection into the EUS is feasible and imaging reveals a precise accumulation in the target region. Our method provides an appropriate approach to deliver MPC in the EUS muscle for a regenerative treatment of SUI in the near future.

Overcoming Endocytosis Deficiency by Cubosome Nanocarriers.

The use of lipid-based nanoparticles for the delivery of biomacromolecules has attracted considerable attention due to the current interest in protein-based therapeutics. Cubosomes protect the incorporated therapeutics, which are susceptible to degradation by enzymes, thereby improving their bioavailability, and concomitantly enhance cellular uptake. The cubosome nanoparticles presented herein were loaded with bovine serum albumin (BSA) and characterized by small-angle X-ray scattering and dynamic light scattering techniques, while the BSA encapsulation and its release were evaluated in vitro. The ability of this formulation to increase the cellular uptake of albumin by 2-fold was tested on various types of renal tubular cells and confirmed by in vivo renal uptake experiments in mice. The obtained results show that cubosomes are able to deliver BSA inside the cell through distinct uptake and intracellular routing. These data were substantiated, with evidence of a high cubosome-mediated uptake of BSA in Clcn5 knockout mice characterized by defective receptor-mediated endocytosis. The use of cubosomes as a delivery system thus represents a promising approach to overcome the low endocytic uptake in diseased epithelial cells and to treat dysfunctions of the kidney proximal tubule.

Human proximal tubule cells form functional microtissues.

The epithelial cells lining the proximal tubules of the kidney mediate complex transport processes and are particularly vulnerable to drug toxicity. Drug toxicity studies are classically based on two-dimensional cultures of immortalized proximal tubular cells. Such immortalized cells are dedifferentiated, and lose transport properties (including saturable endocytic uptake) encountered in vivo. Generating differentiated, organotypic human microtissues would potentially alleviate these limitations and facilitate drug toxicity studies. Here, we describe the generation and characterization of kidney microtissues from immortalized (HK-2) and primary (HRPTEpiC) human renal proximal tubular epithelial cells under well-defined conditions. Microtissue cultures were done in hanging drop GravityPLUS™ culture plates and were characterized for morphology, proliferation and differentiation markers, and by monitoring the endocytic uptake of albumin. Kidney microtissues were successfully obtained by co-culturing HK-2 or HRPTEpiC cells with fibroblasts. The HK-2 microtissues formed highly proliferative, but dedifferentiated microtissues within 10 days of culture, while co-culture with fibroblasts yielded spherical structures already after 2 days. Low passage HRPTEpiC microtissues (mono- and co-culture) were less proliferative and expressed tissue-specific differentiation markers. Electron microscopy evidenced epithelial differentiation markers including microvilli, tight junctions, endosomes, and lysosomes in the co-cultured HRPTEpiC microtissues. The co-cultured HRPTEpiC microtissues showed specific uptake of albumin that could be inhibited by cadmium and gentamycin. In conclusion, we established a reliable hanging drop protocol to obtain functional kidney microtissues with proximal tubular epithelial cell lines. These microtissues could be used for high-throughput drug and toxicology screenings, with endocytosis as a functional readout.