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Figure 1. 3D cellular microarray platform for stem cell research. Stem cells dispersed in alginate or matrigel are encapsulated on a functionalized glass slide by spotting the cells on a PSMA-coated slide using a microarrayer. Cells can then be analyzed for growth and viability (shown above), protein expression, morphology, and toxicity in a high-throughput, high-content manner.

 

Deciphering and controlling the wealth of transcription factors and chemical signals involved in the self-renewal and differentiation of stem cells remains a challenge in cell biology that is hampering the use of stem cell technology in screening and clinical applications. The development of technologies that allow the high throughput cell-based screening of agents and culture conditions based on the analysis of multiple cellular features is expected to aid in elucidating complex interactions among the multiple signals involved in developmental pathways.

Following previous work with non-human stem cells and somatic cells (Fernandes et al., Anal. Biochem, 2008), we are printing human stem cells in a microarray format. The cells can be grown, induced to differentiate, and analyzed in three-dimensional (3D) high-throughput cellular microcultures. This is done by embedding the cells in a hydrogel, such as alginate or matrigel, printing the gels in spots as small as 20 nl on hydrophobic slides, and allowing gelation of hemispherical and discrete spots. The 3D cultures afford cells a microenvironment that can more accurately mimic the geometrical, mechanical, and biochemical aspects of different tissues. After growth on the slides, these whole-cell microarrays may be analyzed for protein expression using in-cell, on-chip immunofluorescence, as well as cell growth, viability, morphology, and toxicity in a high-throughput, high-content manner.

We have begun to assess the influence of matrix and ECM components and other compounds on self-renewal and differentiation of human embryonic stem cells (hESCs) and human neural stem cells (hNSCs) using in-cell, on-chip immunofluorescence analysis of key marker proteins. Cultures can be addressed either in groups of a few spots, or individually by means of complementary chips and/or add-on chambers that allow their incubation with components and marker antibodies of interest. Of particular interest is the evaluation of broader signaling pathways involved in self-renewal and differentiation. In collaboration with the Linhardt lab, we are also investigating the role of glycans in the hESC environment in early development using immunofluorescent analysis of protein expression coupled with glycan analysis. Finally, the differential responses of NSCs and hESCs to drug targets and toxins as compared to adult, terminally differentiated, primary cells and transformed cell lines are being studied using on-chip viability and toxicity screens.