Bioactive factors secreted by differentiating embryonic stem cells.

摘要

Temporal patterns of phenotypic gene expression, including vasculogenesis, have been relatively well characterized during the course of differentiation; coincident patterns of endogenous extracellular matrix (ECM) and growth factor expression that accompany pluripotent stem cell differentiation remain poorly defined. To examine the expression of extracellular factors within embryoid bodies (EBs), gene expression patterns obtained from low-density PCR arrays in conjunction with a variety of analytical tools were utilized to gain an understanding of the importance of extracellular factors in embryonic stem cell (ESC) differentiation. Gene expression analysis of ECM and growth factors by ESCs differentiating as EBs for up to 14 days was assessed using PCR arrays (168 unique genes total), and the results were examined by a series of different clustering and systems pathway analyses. As expected, significant decreases in molecules regulating pluripotent stem cell fate preceded subsequent increases in morphogen expression associated with differentiation. Pathway analysis indicated strong correlations between the expression patterns of ECM and growth factor genes and morphogenic cell phenomena, such as cell growth, migration, and intercellular signaling required for primitive tissue and organ developmental events.;Stem cells are being investigated as catalysts of tissue regeneration to either directly replace or promote cellularity due to traumatic injuries and degenerative diseases. In many instances, despite low numbers of stably integrated cells, the transient presence of cells delivered or recruited to the site of tissue remodeling globally benefited functional recovery. Such reports have motivated subsequent studies to determine how paracrine factors secreted from transplanted cells may be capable of positively impacting endogenous repair processes. Thus, the effects of soluble factors secreted by ESCs during early stages of differentiation on exogenous cells (fibroblasts and endothelial cells) commonly involved in tissue remodeling events were investigated.;This soluble fraction of secreted factors contained within EB-conditioned media was compared to the matrix-associated factors produced by EBs, which led to the development of novel ESC-derived matrices via mechanical acellularization methods. Several acellularization protocols were applied to EBs, to develop a potential route to deliver ESC-derived molecules, independent of cells, to damaged tissues. Acellularization methods were developed to physically disrupt EBs via lyophilization or freeze-thaw cycling, and in combination with DNase treatment, while the efficacy of acellularization was based upon cell viability, DNA removal, and protein retention. Mechanical disruption and DNase treatment of EBs efficiently inhibited viability and removed DNA while retaining protein content to produce an acellular EB matrix.;The acellular embryonic stem cell-derived matrix was examined for its retention of bioactive factors that potentially could stimulate aspects of angiogenesis, including cell proliferation, chemotaxis, and structure formation as well as tissue morphogenesis. Protein extractions from EBM revealed that growth factors were harbored within the matrix in varying amounts depending on the day of EB differentiation. Following heat inactivation, BMP-4 and FGF-2 were no longer identified, contrary to IGF-2 and VEGF-A, which both showed increased amounts compared to non-heat inactivated EBM. The reaction of fibroblasts to EBM extractions was typically enhanced compared to basal level controls, in contrast to the inhibition of endothelial cells in response to EBM extractions. Endothelial cells were stimulated upon heat inactivation of EBM, especially during tube formation assays. The impact of EBMs on CAM angiogenesis was not as obvious of a response compared to EC migration and proliferation, yet altogether these bioactivity studies demonstrated the retention of bioactive factors within EBM.;In conclusion, this work has derived a novel embryonic stem cell matrix that is comprised of bioactive morphogens that further impact aspects of tissue remodeling, particularly angiogenesis. Moreover, these studies have elucidated the modulation of extracellular factors that reflect the progression of EB differentiation, while providing a means to deliver these ESC-secreted factors in a cell-free manner. Future work will continue to elucidate the unique milieu of factors secreted by ESCs that will further advance the innovation of novel therapeutics that harness the potential of ESCs for tissue regeneration applications. (Abstract shortened by UMI.).