Exploring the regulation of human neural precursor cell differentiation using arrays of signaling microenvironments

Abstract

Cells of a developing embryo integrate a complex array of local and long‐range signals that act in concert with cell‐intrinsic determinants to influence developmental decisions. To systematically investigate the effects of molecular microenvironments on cell fate decisions, we developed an experimental method based on parallel exposure of cells to diverse combinations of extracellular signals followed by quantitative, multi‐parameter analysis of cellular responses. Primary human neural precursor cells were captured and cultured on printed microenvironment arrays composed of mixtures of extracellular matrix components, morphogens, and other signaling proteins. Quantitative single cell analysis revealed striking effects of some of these signals on the extent and direction of differentiation. We found that Wnt and Notch co‐stimulation could maintain the cells in an undifferentiated‐like, proliferative state, whereas bone morphogenetic protein 4 induced an ‘indeterminate’ differentiation phenotype characterized by simultaneous expression of glial and neuronal markers. Multi‐parameter analysis of responses to conflicting signals revealed interactions more complex than previously envisaged including dominance relations that may reflect a cell‐intrinsic system for robust specification of responses in complex microenvironments. ### Synopsis The development of the nervous system depends on spatially and temporally programmed differentiation of precursors into specific neurons and glial cells. Understanding the regulation of precursor cell differentiation in complex microenvironments is a major challenge of developmental biology. However, despite remarkable progress that has been made in elucidating individual pathways and cell‐intrinsic factors, we are still a long way from an integrative understanding of how multiple pathways interact with one another and with cell‐intrinsic mechanisms to specify cellular fate and function. To systematically investigate mechanisms and logic by which extracellular signals specify the fate of human neural precursor cells, we developed and applied a signaling microenvironment microarray paradigm. This approach is based on parallel exposure of multi‐potent neural progenitor cells to a diverse array of defined signaling molecules (e.g. extracellular matrix components, morphogens, and other signaling proteins) presented individually and in combinations. Cells were cultured in the arrayed signaling microenvironments for varying periods, then analyzed by a scanning microscope to provide a high‐throughput quantitative analysis of multiple phenotypic outcomes at single cell resolution. Ensemble average responses to each microenvironment were determined by averaging over all the cells exposed to the same signaling combination, and higher level analyses were performed using novel computational tools for interpreting data with the scale and complexity produced by this experimental paradigm. The entire approach is very general. It can be applied to a wide range of cell types (different types of stem cells, tumor cells, immune cells, etc.) and can be used to study a broad spectrum of responses to diverse variations in a cell's signaling microenvironment. In this experiment, human neural precursors, capable of differentiating into neurons or glial cells, were captured on these printed signaling microarrays and cultured for several days under defined, differentiation conditions. Quantitative cell by cell analysis revealed consistent and striking effects of some of these signals on the differentiation of primary human neural stem cells ([Figure 2A][1]). We found that co‐stimulation with two factors, Wnt and Notch, could maintain the cells in an undifferentiated‐like, proliferative state, whereas a third factor, bone morphogenetic protein 4, induced an ‘indeterminate’ differentiation phenotype characterized by simultaneous expression of glial and neuronal markers. Multi‐parameter analysis of responses segregated all the various signaling combinations into four main groups based on their characteristic effects ([Figure 5][2]): (1) combinations that promoted neurogenesis, (2) combinations that promoted gliogenesis, (3) combinations that prevented both, and (4) combinations that elevated both neural and glial markers in the same cell (an indeterminate differentiation phenotype). To examine differentiation responses following simultaneous exposure to potentially conflicting signals (e.g. neurogenic and gliogenic signals), we compared the responses to individual signals with responses to the combined signals. We found that combinations of signals often promoted responses different from the effects of the individual signals. In some, however, the response to one of the signals appeared to dominate over the response to the others. Interestingly, some of the observed dominance relationships were complex—with the responses to different signals dominating with respect to different response parameters. Such dominance relations may reflect a cell‐intrinsic system for robust specification of responses in complex microenvironments. 1. Primary human neural precursor cells could be captured, cultured, and differentiated on a diverse array of printed signaling microenvironments. 2. Quantitative single cell analysis revealed striking effects of some of the signals on the extent and direction of differentiation. 3. Ensemble average phenotypic analysis implicated Wnt and Notch co‐stimulation in maintaining the cells in an undifferentiated, proliferative state, and revealed a surprising BMP‐induced indeterminate differentiation phenotype. 4. Multi‐parameter analysis of responses to conflicting signals revealed interactions more complex than previously envisaged, including reciprocal dominance relations. 5. New tools were introduced to analyze and interpret data...