Cryopreservation Maintains Functionality of Human iPSC Dopamine Neurons and Rescues Parkinsonian Phenotypes In Vivo.

A major challenge for clinical application of pluripotent stem cell therapy for Parkinson's disease (PD) is large-scale manufacturing and cryopreservation of neurons that can be efficiently prepared with minimal manipulation. To address this obstacle, midbrain dopamine neurons were derived from human induced pluripotent stem cells (iPSC-mDA) and cryopreserved in large production lots for biochemical and transplantation studies. Cryopreserved, post-mitotic iPSC-mDA neurons retained high viability with gene, protein, and electrophysiological signatures consistent with midbrain floor-plate lineage. To test therapeutic efficacy, cryopreserved iPSC-mDA neurons were transplanted without subculturing into the 6-OHDA-lesioned rat and MPTP-lesioned non-human-primate models of PD. Grafted neurons retained midbrain lineage with extensive fiber innervation in both rodents and monkeys. Behavioral assessment in 6-OHDA-lesioned rats demonstrated significant reversal in functional deficits up to 6 months post transplantation with reinnervation of the host striatum and no aberrant growth, supporting the translational development of pluripotent cell-based therapies in PD.

Exploiting bacterial peptide display technology to engineer biomaterials for neural stem cell culture.

Stem cells are often cultured on substrates that present extracellular matrix (ECM) proteins; however, the heterogeneous and poorly defined nature of ECM proteins presents challenges both for basic biological investigation of cell-matrix investigations and translational applications of stem cells. Therefore, fully synthetic, defined materials conjugated with bioactive ligands, such as adhesive peptides, are preferable for stem cell biology and engineering. However, identifying novel ligands that engage cellular receptors can be challenging, and we have thus developed a high throughput approach to identify new adhesive ligands. We selected an unbiased bacterial peptide display library for the ability to bind adult neural stem cells (NSCs), and 44 bacterial clones expressing peptides were identified and found to bind to NSCs with high avidity. Of these clones, four contained RGD motifs commonly found in integrin binding domains, and three exhibited homology to ECM proteins. Three peptide clones were chosen for further analysis, and their synthetic analogs were adsorbed on tissue culture polystyrene (TCPS) or grafted onto an interpenetrating polymer network (IPN) for cell culture. These three peptides were found to support neural stem cell self-renewal in defined medium as well as multi-lineage differentiation. Therefore, bacterial peptide display offers unique advantages to isolate bioactive peptides from large, unbiased libraries for applications in biomaterials engineering.

Characterization of Matrigel interfaces during defined human embryonic stem cell culture.

Differences in attachment, proliferation, and differentiation were measured for human embryonic stem (hES) cells cultured on various substrata coated with Matrigel, a blend of extracellular matrix proteins derived from murine tumor cells. The authors observed that hES cells attach and grow poorly on Matrigel adsorbed onto polystyrene, while they proliferate when exposed to Matrigel adsorbed onto glass or oxygen plasma treated polystyrene (e.g., "tissue culture" treated polystyrene). Furthermore, hES cells grown on the Matrigel-coated tissue culture polystyrene are less likely to differentiate than those grown on the Matrigel-coated glass. To assess the mechanism for these observations, they replicated the cell culture interface in a quartz crystal microbalance with dissipation monitoring. In addition, they used ellipsometry and scanning electron microscopy to determine the thickness and topography of Matrigel on the varying surfaces. Matrigel formed a viscoelastic multilayer with similar thickness on all three surfaces; however, the network structure was different, where the adsorbed proteins formed a globular network on polystyrene, and fibrillar networks on the hydrophilic substrates. Matrigel networks on glass were denser than on oxygen plasma treated polystyrene, suggesting that the density and structure of the Matrigel network affects stem cell differentiation, where a denser network promoted uncontrolled hES cell differentiation and did not maintain the self-renewal phenotype.