ABSTRACT Platelet transfusions play life-saving roles for cancer therapy, bone marrow transplantation, bone marrow failure, sepsis, genetic platelet disorders, and other diseases. However, the limited shelf life of donor platelets causes frequent shortages. In addition, multiply transfused patients often become allosensitized making them refractory to treatment. The development of induced pluripotent stem cell (hIPSC) technology has raised the intriguing possibility of producing platelets in vitro for clinical use. This would provide a relatively limitless supply of on- demand platelets, including personalized and HLA matched/engineered products. However, current in vitro differentiation protocols fail to generate platelets at high enough efficiency for practical use. The major bottleneck involves the final stages of platelet production from their precursor cell, the megakaryocyte (Mk). This is currently at least 10 to 100-fold less efficient in vitro compared to in vivo. Overcoming this obstacle would therefore represent a major step forward in developing in vitro methods for clinical scale platelet production. We hypothesize that key spatially regulated signaling events occur when Mks engage the bone marrow vascular sinusoidal niche, where they normally produce platelets in vivo. We also hypothesize that current in vitro platelet production systems fail to adequately recapitulate these events. Further understanding these physiologic signaling events is therefore key to advancing this field. Given the complex microenvironment in which these events occur, it is critical to examine this problem in situ. The objective of this Stimulating Hematology Investigation: New Endeavors II (SHINE-II) proposal is to adapt new spatial transcriptomic technology to study gene expression events that occur when Mks interact with vascular sinusoids and begin producing platelets. It will utilize Multiplexed Error-robust Fluorescence In Situ Hybridization (MERFISH), a technique that provides sensitive and quantitative measurements of RNA expression of tens of thousands of genes in single cells while at the same time providing spatial information regarding their expression in tissue slices. This will involve developing MERFISH for the bone marrow vascular sinusoid using murine systems and pilot gene panels. This will then be extended to whole-transcriptome scale and applied to human bone marrow samples. The functional importance of select validated pathways will be explored using human CD34+ and hIPSCs in vitro differentiation methods. Successful completion of the project will have a significant positive impact on the field by expanding our knowledge of the physiologic signaling events that trigger Mks to mature and produce platelets in their natural microenvironment. As the vascular sinusoid is also a key hematopoietic stem cell (HSC) niche and MERFISH captures whole transcriptome gene expression measurements on all cells within the field, this project will also prov...