Design of scaffolds for human stem cell expansion and differentiation: Influence of 3D structure, topographical and biochemical cues.

摘要

An ideal scaffold for tissue engineering application should mimic the natural microenvironment for natural tissue, coaxing the cultured cells to organize into functional tissue, provide a three-dimensional (3D) structure for mass transport, and present the appropriate biochemical and topography cues in a spatially controlled manner for cell proliferation and differentiation. In this dissertation, we studied the culture of different types of stem cells on scaffolds and surfaces designed to address the issues of topography and encapsulated biological signals in the form of controlled release factors, adhesion molecules, and entrapped cells.; The proliferation of embryoid body derived (EBD) cells was studied on fibrous and porous foam scaffold. The expansion of the EBD cells in 3D environment was significantly higher than their two-dimensional controls after 21 days. No apparent differentiation of the EBD cells cultured in the 3D environment, as indicated by histology and gene expression profile analysis, was evident.; When nerve growth factor (NGF) was surface-immobilized on the fibrous scaffold via chemically modified Pluronic, the EBD cells cultured in this scaffold showed evidence of entering the neural pathway. An upregulation of tyrosine hydroxylase mRNA expression was observed when EBD cells were cultured in the NGF-immobilized fibrous scaffold. Biochemical cues could also be delivered in a novel polyelectrolyte complexation (PEC) fibrous scaffold. NGF encapsulated in the PEC fibers or immobilized onto the PEC fiber stimulated the differentiation of PC12 cells into neurons. Cells, including, human mesenchymal stem cells (hMSCs), could also be incorporated into the fibers. The fiber encapsulated hMSCs were able to proliferate, and remained viable and metabolically active during the 6 weeks of culture. The encapsulated cells were also able to respond to the biochemical cues in the medium, and showed evidence of chondrogenesis and osterogenesis when cultured in the corresponding differentiation media. The fiber technology was further exploited to fabricate an hMSC-encapsulated fibrous scaffold for human embryonic stem (hES) cell expansion. The undifferentiated propagation of the hES cells was maintained in the co-culture system for a test-period of 4 weeks. HES cells on the scaffold and harvested from the scaffold, by washing the scaffold with culture medium, expressed undifferentiated cell markers such as Oct4, Tert-1 and SSEA4.; Influence of topographical cues on cellular behavior was studied on nanopatterns fabricated by nano-imprinting. The cellular behavior on the nanotopography was first tested with bovine smooth muscle cells (SMCs), which showed significant alignment and elongation along the grating axis. The proliferation of the SMCs was significantly reduced on the nanopatterned surfaces. The MTOC polarization of the SMCs was also altered during cell migration on the nanopattern. The question of whether nanometer scale pattern is necessary to produce a significant effect on cell behavior compared to micropattern has also been investigated. (Abstract shortened by UMI.)