Plasmacytoid dendritic cells (pDCs) constitute a rare type of immune cell with multifaceted functions, but their potential use as a cell-based immunotherapy is challenged by the scarce cell numbers that can be extracted from blood. Here, we systematically investigate culture parameters for generating pDCs from hematopoietic stem and progenitor cells (HSPCs). Using optimized conditions combined with implementation of HSPC pre-expansion, we generate an average of 465 million HSPC-derived pDCs (HSPC-pDCs) starting from 100,000 cord blood-derived HSPCs. Furthermore, we demonstrate that such protocol allows HSPC-pDC generation from whole blood HSPCs, and these cells display a pDC phenotype and function. Using GMP compliant medium, we observe a remarkable loss of TLR7/9 responses, which is rescued by ascorbic acid supplementation. Ascorbic acid induces transcriptional signatures associated with pDC-specific innate immune pathways suggesting an undescribed role of ascorbic acid for pDC functionality. This constitutes the first protocol for generating pDCs from whole blood, and lay the foundation for investigating HSPC-pDCs for cell-based immunotherapy.

HSPC-pDCs were stored in RNAprotect Cell Reagent at -80 degrees until total RNA was extracted using the RNeasy Plus Micro Kit (Qiagen), which efficiently eliminates genomic DNA without the need for DNase treatment. The total RNA was sent to BGI Europe for RNA-seq. Here, a non-stranded & polyA-selected mRNA library was prepared from the total RNA and subjected to PE100 sequencing using the BGISEQ platform. The samples generated on average about 4.84 Gb bases per sample on DNBSEQ. Low quality reads were filtered and the remaining reads were mapped to the genome with an average mapping ratio with the reference genome at 92.74%, the average gene mapping ratio at 79.90%.