Induced pluripotent stem cells (iPSCs) are of increasing interest for regenerative medicine and disease modeling owing to their ability to differentiate into any cell type and accessibility on a patient-specific basis. iPSCs are typically cultured in clusters either on a 2D cell feeder layer (MEFs) or a feeder-free layer (Matrigel), maintaining pluripotency and proliferation but also introducing potential for variability. Here, we investigate the culture of single-cell iPSCs in three-dimensional (3D) culture using well-defined hydrogel-based matrices to examine the effect of geometry and integrin binding on iPSC proliferation and pluripotency (oct4+) over time. Synthetic poly(ethylene) glycol (PEG) hydrogels enable independent control of matrix physical and biochemical properties allowing hypothesis testing to determine key factors relevant for stem cell culture. Photo-initiated step-growth polymerization of PEG-8-norbornene (PEG-8-Nb) and cell-degradable dithiol crosslinker rapidly form hydrogels and enable iPSC encapsulation. Cell viability in these matrices correlates with matrix density (controlled with PEG wt%), as well as integrin binding. Specifically, peptide motifs (RGDS, YIGSR) from relevant extracellular matrix proteins in the basement membrane (fibronectin, laminin) were examined, and cell response was monitored over time using a fluorescent reporter system and qRT-PCR. Lastly, within these culture environments, single cell seeding and soluble cues are being used as handles to control differentiation of iPSCs into neural progenitor cells (PAX6+). Taken together, these studies demonstrate the power of tunable hydrogels for the recapitulation of aspects of <i>in vivo </i>cell microenvironments to determine key properties for iPSC pluripotency and differentiation.

Engineered environments for modulating iPSC function and fate in three-dimensional culture

253rd National Meeting (2017)

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