Microscale technologies for regulating human stem cell differentiation.

During development and regeneration, tissues emerge from coordinated sequences of stem cell renewal, specialization, and assembly that are orchestrated by cascades of regulatory factors. This complex in vivo milieu, while necessary to fully recapitulate biology and to properly engineer progenitor cells, is difficult to replicate in vitro. We are just starting to fully realize the importance of the entire context of cell microenvironment-the other cells, three-dimensional matrix, molecular and physical signals. Bioengineered environments that combine tissue-specific transport and signaling are critical to study cellular responses at biologically relevant scales and in settings predictive of human condition. We therefore developed microbioreactors that couple the application of fast dynamic changes in environmental signals with versatile, high-throughput operation and imaging capability. Our base device is a microfluidic platform with an array of microwells containing cells or tissue constructs that are exposed to stable concentration gradients. Mathematical modeling of flow and mass transport can predict the shape of these gradients and the kinetic changes in local concentrations. A single platform, the size of a microscope slide, contains up to 120 biological samples. As an example of application, we describe studies of cell fate specification and mesodermal lineage commitment in human embryonic stem cells and induced pluripotent stem cells. The embryoid bodies formed from these cells were subjected to single and multiple concentration gradients of Wnt3a, Activin A, bone morphogenic protein 4 (BMP4), and their inhibitors, and the gene expression profiles were correlated to the concentration gradients of morphogens to identify the exact conditions for mesodermal differentiation.