I034 CD200-based cell sorting generates homogeneous subpopulations of transplantable striatal neuroblasts

<h3>Background</h3> Cell replacement therapy (CRT) holds promise for restoring atrophied tissue in Huntington’s Disease (HD) by replenishing degenerating striatal projection neurons (SPNs). However, heterogeneous cell compositions resulting from striatal differentiation protocols hamper clinical translation. Standardizing production is imperative for generating reproducible and safe cell products. <h3>Aim</h3> To devise a novel therapeutic strategy for brain tissue regeneration in HD, by ensuring the presence of specified SPN progenitors and the absence of overly proliferative cell types. <h3>Methods</h3> We developed a magnetic-associated cell sorting (MACS) pipeline to select post-mitotic neuroblasts (NBs) derived from human pluripotent stem cells (hPSCs). We characterized the identity of the selected cell subpopulations and evaluated their survival, safety, and fate following transplantation into the striatum of immunosuppressed adult mice. <h3>Results</h3> CD200 was identified as a marker of post-mitotic NBs, enabling consistent, high-yield enrichment of CD200^high NBs. Sorted cells exhibit hallmark characteristics of SPN-committed NBs, which undergo differentiation into the target SPN-like neurons <i>in vitro</i>. CD200^high NBs retain the expression of subpallial markers and differentiate for at least one month after transplantation. Markers of the striatal direct and indirect pathways are detected in the transplanted NBs, underscoring their regenerative potential for HD. Additionally, CD200-based selection significantly reduces the presence of proliferative cells in the intrastriatal grafts, minimizing the risk of graft overgrowth and tumor formation <i>in vivo</i>. <h3>Conclusion</h3> This work emphasizes the translational value of CD200-based cell sorting for addressing quality and safety requirements for an HD CRT. This study was supported by grants from the Ministerio de Ciencia e Innovación (PID2021-126961OB-I00, and PDC2022-133783-I00); Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and European Regional Development Fund (ERDF A way of making Europe) (Red de Terapias Avanzadas, RD21/0017/0020); European Union NextGenerationEU/PRTR; Generalitat de Catalunya (2021 SGR 01094); and ‘la Caixa’ Foundation under the grant agreement LCF/PR/HR21-00622, Spain. The project was also supported by European Union grants from: Marie Curie Programme, ASCTN Training Network, grant agreement N.813851; and European Innovation Council (EIC) grant agreement 101047099 – 4-DBR.