UNS: Stem Cell Differentiation and Teratoma-forming Potential in hiPSC-derived Neural Cultures

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PI: Kirby, Brian J. Proposal Number: 1511914The investigators will develop techniques that allow human cells from patients to be reprogrammed into neural cells that can be used to repair nerve damage or neurodegenerative disease. Although techniques exist to reprogram cells, these reprogramming techniques at present pose risks to the patient because some reprogrammed cells can form tumors instead of serving a therapeutic purpose. This work will process populations of reprogrammed cells to remove those with the potential to form tumors. This work is broadly significant because, by eliminating the potential for tumor formation, it will enable tissue regeneration to be performed with therapeutic benefit and minimal risk of harming the patient.The long-term goal of this research is to enable efficient, teratoma-free engineering of neuronal cells. The objective of the proposed work is to delineate the relationships between adhesive mechanobiology, teratoma-related markers and differentiation in neurally reprogrammed human induced pluripotent stem cell (hiPSC) populations. The central hypotheses are that (a) neural rosette cells will have an adhesive signature (i.e., integrins, focal adhesions, and adhesive strength), distinct from contaminating cells, that will lead to their selective removal using the micro stem cell high-efficiency adhesion-based recovery (uSHEAR) platform, and (b) teratoma-forming cells will have a unique expression pattern of integrins and glycans, including stage-specific embryonic antigen (SSEA)-5, which will enable microfluidic-based selective capture of teratoma-forming cells. This hypothesis has been formulated based on the previous discovery of differential adhesiveness of hiPSCs and neural cells and label-free isolation of hiPSCs for partially reprogrammed cultures, as well as selective rare cell isolation with geometrically enhanced differential immunocapture (GEDI) devices. The rationale of the proposed work is that the relationships between adhesive mechanobiology, teratoma-related markers, and differentiation are most directly applicable to contaminant-extraction approaches that improve the time, yield, and purity of neural differentiation and facilitate its direct application in regenerative medicine. This work will determine relationships between adhesion signature, surface markers, and teratoma formation through two specific aims, which determine the relationships between teratoma-related markers, adhesive signature, and directed differentiation of hiPSCs along the neural lineage, and determine the relationship between SSEA5-based negative selection in differentiating cells and removal of teratoma-forming risk in progenitor/neuron cultures. The proposed work will determine relationships between surface expression and adhesion during the differentiation process, and evaluate the potential for differentiation and teratoma formation in immune-compromised mice. By exploiting the differences in adhesive signature and molecular fingerprints of cells at different stages of differentiation from hiPSC to neuron, immunocapture of SSEA5+ cells using geometrically enhanced differential immunocapture will be used to eliminate teratoma-forming cells and reduce teratoma formation in vivo. Flow cytometry, immunostaining, and in vivo teratoma formation studies will be used to evaluate the potential for negative selection to limit teratoma-forming risk and link teratoma outcomes to surface markers in flowthrough and captured subpopulations, and these results will allow (a) early purification of radially structured, multipotent neural rosettes for accelerated and enhanced yield and purity of neural differentiation, (b) purification of terminally differentiated neural cell populations, and (c) enhanced characterization of teratoma-specific surface markers. Taken together, the expected outcome of the specific aims is identification of the links between adhesive signature of neural cells in continuously differentiating hiPSC cultures, their surface expression of integrins and SSEA5, and teratoma formation in SCID mice, quantified by measurement of reduction of teratoma formation upon SSEA5-based negative cell selection in a novel microfluidic device. The broad impact of this work is that the knowledge gained will inform devices that use adhesion strength for hiPSC isolation and immunocapture for isolation of rare populations of teratoma-forming cells. The Investigators plan to broaden STEM participation through a series of summer activities designed to use GEDI devices to explain cellular transport to high school women as part of a Cornell leadership academy.