Jutta Krug

In Vitro Investigations of Tissue-Engineered Multilayered Urothelium Established from Bladder Washings

OBJECTIVEnHuman urothelial cells (HUCs) are commonly isolated from native urothelium requiring open or endoscopic surgery. The aim of this study was to raise primary monolayer cultures of HUCs from bladder washings, to generate multilayered urothelial sheets in vitro, to characterise the sheets immunologically, and to prove their viability.nnnMETHODSnIrrigation fluids were taken from 29 adult patients. Isolated cells were cultured in serum-free keratinocyte medium. Confluent monolayer cultures were stratified, and evolved cell sheets were harvested after 10-16 d. Pancytokeratins and cytokeratin 20 (CK20) in the stratified cultures and the detached sheets were immunologically detected. To exclude the presence of mesenchymal cells, antibodies against fibroblast surface antigen and smooth muscle alpha-actin were used. In addition, expression of p63 and uroplakin III was investigated. The viability of the detached cell sheets was proven by establishing explant cultures of small sheet sections.nnnRESULTSnConfluent primary HUC cultures were established in 55.2% of the collected bladder washings between days 15-20. Multilayered urothelium developed in 62.5% of the monolayers. Histology revealed stratified cell layers similar to native urothelium. Both stratified cultures and detached sheets stained 100% positive for pancytokeratins and partially for CK20, indicating differentiation into superficial cells. No positive staining was observed with the mesenchymal markers used. p63 was expressed partially. Uroplakin III expression was not observed. Cell sheet viability was confirmed by rapid cell outgrowth in explant cultures.nnnCONCLUSIONSnIsolation of HUCs from bladder washings is a minimally invasive approach to establish primary urothelial cultures for creating autologous multilayered urothelial sheets.

MYOGENIC DIFFERENTIATION OF HUMAN MESENCHYMAL STEM CELLS IN VITRO AND LONG-TERM SURVIVAL IN A NUDE RAT MODEL FOR URINARY INCONTINENCE

INTRODUCTION AND OBJECTIVES: Mesenchymal stem cells (MSCs) have the capacity to differentiate into smooth and skeletal muscle cells. Thus, autologous MSCs might be an option for a functional treatment of urinary incontinence (UI). The aim was to study myogenic differentiation of human MSCs in vitro and to examine their survival in bladder neck tissue of athymic rats in a long-term study. METHODS: To induce myogenic differentiation in vitro bone marrow-derived human MSCs in culture passage (P) 1 were exposed to 5-azacytidine (AZA). In vitro differentiation was examined up to P6 by FACS analysis with stem cell markers and intracellular F-actin, RT-PCR with primers against skeletal muscle-specific regulatory factor myoD1 (MyoD1), and myosin heavy chain (MHC) and by immuncytochemistry with monoclonal antibodies against smooth muscle -actin, / -actin, MyoD transcription factor, and skeletal slow muscle MHC. Native and AZA-exposed MSCs of P2, respectively, were directly injected into the bladder neck of two athymic rats in each case. For in vivo tracking MSCs were labeled with PKH26 red fluorescent cell linker. Integration of MSCs into host tissue was monitored histologically after 16 weeks of cell injection. RESULTS: FACS analysis of multiple MSC lines revealed that both native and AZA-exposed MSCs from P1-P6 were negative for the progenitor/endothelial antigen CD34, leukocytic CD45 and endothelial/ monocytic CD31. The MSC markers CD73, CD90, and CD105 increased over time in native, but not in AZA-exposed MSCs. Intracellular F-actin increased in AZA-exposed, but not in native MSCs until P3. Skeletal muscle mRNA expression decreased in native, but not in AZA-exposed MSCs from P1-P4. Native MSCs expressed / -actin and MyoD antigen only in P0 and P1, whereas -actin was expressed in P0-P6. AZAexposed MSCs expressed these muscle antigens homogeneously in P2-P6. There was no reactivity for skeletal slow muscle MHC in both groups of MSCs. In bladder neck tissues from all rats, well-defined MSC clusters with further continuous dissemination of transplanted MSCs were detected independent of AZA-exposure. CONCLUSIONS: Exposure of human MSCs to AZA in vitro is associated with increased myogenic differentiation and persistent expression of muscle antigens. Vital integration of MSCs in bladder neck tissue might lead to final differentiation into functional muscle cells in vivo. This model emphasizes the use of autologous adult MSCs for a functional treatment of UI.

CELL-BASED TREATMENT MODALITY OF URINARY INCONTINENCE WITH HUMAN MESENCHYMAL STEM CELLS IN A NUDE RAT MODEL

by Mini-MACS kits. The nude mice were subdivided into 5 groups (n= each group): normal group (N), saline injection group after cryo-injury (S), GIKVAV injection group after cryo-injury (G), human MDSC injection group after cryo-injury (M) and GIKVAV and human MDSC injection group after cryo-injury (GM). At 2 weeks after injection, we compared the contractility of bladder muscle strip of each group by organ bath and