Phenotypic analysis of bioactive cells for application in regenerative medicine.

The following chapter outlines methodologies to phenotypically characterize primary cells for the use in tissue-engineered and regenerative medicine applications. Methods covered include analyzing cells using immunocytochemistry, fluorescence-activated cell sorting, and confocal microscopy of adherent and suspended cells, as well as combinations of formulated cell-biomaterial constructs.

Characterization of the human smooth muscle cell secretome for regenerative medicine.

Smooth muscle cells (SMC) play a central role in maintaining the structural and functional integrity of muscle tissue. Little is known about the early in vitro events that guide the assembly of 'bioartificial tissue' (constructs) and recapitulate the key aspects of smooth muscle differentiation and development before surgical implantation. Biomimetic approaches have been proposed that enable the identification of in vitro processes which allow standardized manufacturing, thus improving both product quality and the consistency of patient outcomes. One essential element of this approach is the description of the SMC secretome, that is, the soluble and deposited factors produced within the three-dimensional (3D) extracellular matrix (ECM) microenvironment. In this study, we utilized autologous SMC from multiple tissue types that were expanded ex vivo and generated with a rigorous focus on operational phenotype and genetic stability. The objective of this study was to characterize the spatiotemporal dynamics of the first week of organoid maturation using a well-defined in vitro-like, 3D-engineered scale model of our validated manufacturing process. Functional proteomics was used to identify the topological properties of the networks of interacting proteins that were derived from the SMC secretome, revealing overlapping central nodes related to SMC differentiation and proliferation, actin cytoskeleton regulation, and balanced ECM accumulation. The critical functions defined by the Ingenuity Pathway Analysis included cell signaling, cellular movement and proliferation, and cellular and organismal development. The results confirm the phenotypic and functional similarity of the SMC generated by our platform technology at the molecular level. Furthermore, these data validate the biomimetic approaches that have been established to maintain manufacturing consistency.

Increased urothelial cell detection in the primary bladder smooth muscle cell cultures with dual MACS/qRT-PCR approach.

Bladder tissue has been regenerated in humans with neurogenic bladder using an implant produced from autologous urothelial (UC) and smooth muscle cells (SMC) expanded from bladder biopsies seeded onto a biodegradable synthetic scaffold. As the majority of bladder cancers are urothelial carcinomas (aka, transitional cell carcinoma), this 2-cell type autologous sourcing strategy presents significant challenges to product development. Entire bladders have been regenerated in cystectomized animals using a single-cell-type sourcing strategy: implants were seeded with bladder-derived SMC-only. Applying the bladder SMC-only sourcing strategy to produce clinical implants for bladder replacement or urinary diversion in bladder cancer patients requires methods for screening SMC cultures for the presence of potentially cancerous UC cells to provide evidence of SMC culture purity before seeding the scaffold. In this report, we show a 10-fold to 100-fold improvement in the sensitivity of qualitative and quantitative reverse-transcription PCR (qRT-PCR)-based assays for detecting UC positive for Cytokeratin 5 (CK5) in mixed SMC/UC cultures when the cell population was first subjected to magnetic activated cell sorting to enrich for cells expressing the epithelial cell adhesion molecule (known as EPCAM or CD326), a marker known to be present in normal UC and upregulated in the cancerous UC.

Induction of GADD45 and GADD153 in neuroblastoma cells by dopamine-induced toxicity.

Dopamine (DA) metabolism and oxidation produce both reactive oxygen species (ROS) and reactive quinones. These chemical species are implicated in dopamine neurotoxicity and neurodegeneration. In the present studies, human neuroblastoma (SK-N-SH) cells were exposed to toxic concentrations of dopamine (333 microM) in order to investigate molecular pathways involved in dopamine toxicity. cDNA hybridization array (microarray) technology demonstrated that GADD45 and GADD153 (growth arrest and DNA-damage inducible) gene expression was increased in dopamine-treated cells (333 microM for 18 h). Subsequent Northern and Western blot analysis confirmed these changes in GADD45 and GADD153 gene expression. The antioxidant, ascorbic acid, significantly reduced the increase in GADD45 gene expression but did not significantly reduce GADD153 gene expression. Currently, the precise function of the GADD gene products is not known. It is known, however, that these genes are upregulated in response to stress to allow cells time to repair macromolecular damage. In the present case, GADD gene expression (manifested as increased mRNA and protein levels) preceded dopamine-induced cytotoxicity. It appears that dopamine, through the formation of reactive oxygen species and quinones, may damage cellular macromolecules to the point of inducing GADD gene expression. Other genes that displayed changes, but that have not been subjected to post-hoc confirmation, include clusterin (increased), ubiquitin (increased), CD27 ligand (increased), CD27BP (increased), and rac-PK-beta (decreased).