This paper describes a gradient-generating microfluidic platform for optimizing proliferation and differentiation of neural stem cells (NSCs) in culture. Microfluidic technology has great potential to improve stem cell (SC) cultures, whose promise in cell-based therapies is limited by the inability to precisely control their behavior in culture. Compared to traditional culture tools, microfluidic platforms should provide much greater control over cell microenvironment and rapid optimization of media composition using relatively small numbers of cells. Our platform exposes cells to a concentration gradient of growth factors under continuous flow, thus minimizing autocrine and paracrine signaling. Human NSCs (hNSCs) from the developing cerebral cortex were cultured for more than 1 week in the microfluidic device while constantly exposed to a continuous gradient of a growth factor (GF) mixture containing epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2) and platelet-derived growth factor (PDGF). Proliferation and differentiation of NSCs into astrocytes were monitored by time-lapse microscopy and immunocytochemistry. The NSCs remained healthy throughout the entire culture period, and importantly, proliferated and differentiated in a graded and proportional fashion that varied directly with GF concentration. These concentration-dependent cellular responses were quantitatively similar to those measured in control chambers built into the device and in parallel cultures using traditional 6-well plates. This gradient-generating microfluidic platform should be useful for a wide range of basic and applied studies on cultured cells, including SCs.
Recently, various approaches for controlling the embryonic stem (ES) cell microenvironment have been developed for regulating cellular fate decisions. It has been reported that the lineage specific differentiation could be affected by the size of ES cell colonies and embryoid bodies (EBs). However, much of the underlying biology has not been well elucidated. In this study, we used microengineered hydrogel microwells to direct ES cell differentiation and determined the role of WNT signaling pathway in directing the differentiation. This was accomplished by forming ES cell aggregates within microwells to form different size EBs. We determined that cardiogenesis was enhanced in larger EBs (450 m in diameter), and in contrast, endothelial cell differentiation was increased in smaller EBs (150 m in diameter). Furthermore, we demonstrated that the EB-size mediated differentiation was driven by differential expression of WNTs, particularly noncanonical WNT pathway, according to EB size. The higher expression of WNT5a in smaller EBs enhanced endothelial cell differentiation. In contrast, the increased expression of WNT11 enhanced cardiogenesis. This was further validated by WNT5a-siRNA transfection assay and the addition of recombinant WNT5a. Our data suggest that EB size could be an important parameter in ES cell fate specification via differential gene expression of members of the noncanonical WNT pathway. Given the size-dependent response of EBs to differentiate to endothelial and cardiac lineages, hydrogel microwell arrays could be useful for directing stem cell fates and studying ES cell differentiation in a controlled manner.hydrogel microwells ͉ stem cell differentiation ͉ WNT signal pathway T he developmental versatility of embryonic stem (ES) cells offers a powerful approach for directing cell fate and is a promising source of progenitors for cell replacement therapy and tissue regeneration (1). ES cells can differentiate into a wide spectrum of cell types, such as cardiomyocytes and endothelial cells, by forming embryoid bodies (EBs) (2). Those lineages arise from distinct mesoderm subpopulations that develop sequentially from premesoderm cells (3). Such lineage specification is highly coordinated with differential changes in gene expression (4-8).Despite the therapeutic potential of ES cells, one of the significant challenges to their widespread clinical use, is the inability to homogeneously direct ES cell differentiation into specific lineages. One reason for the heterogeneity in EB differentiation is caused from variations in EB size (9, 10). To address this challenge for controlling the differentiation of ES cells, various microscale technologies (i.e., surface patterning, hydrogel microwells, and microfluidic systems) have been developed for directing the stem cell fate (11-15). Micropatterning techniques have been used to evaluate the effect of EB size on ES cell differentiation. For instance, microfabricated adhesive stencils were used to pattern ES cells for controlling initial ES cell aggregate sizes...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.