Efficient Chemical Reprogramming of Human T Cells into Functional Megakaryocytes and Platelets

The generation of megakaryocytes (MKs) from human somatic cells through chemical reprogramming represents a promising strategy for developing alternative platelet sources. Building on our prior chemical reprogramming protocol for converting erythroblasts to MKs, we established a robust method that successfully generated induced MKs (iMKs) from human cord blood-derived CD3? T cells, which is a more abundant source. This method utilized a five- small-molecule (5M) cocktail containing a reprogramming booster, AZD4205, to promote erasure of T cell identity and facilitate fate transition towards MKs. T cell-derived iMKs exhibited characteristic MK cellular and molecular signatures, demonstrating the capacity to produce proplatelets and release functional platelets in vitro and in vivo. ScRNA-sequencing further revealed that iMKs were heterogeneous with distinct functional profiles, including cycling, immune, and thrombopoiesis-biased MKs. Our findings highlight an optimized chemical reprogramming pathway that enables efficient conversion of T cells to MKs, providing a practical and convenient approach to generate clinically relevant MKs and platelets. Overall design: We performed Bulk RNA-seq and single cell RNA-Seq to analyse the molecular mechanism of chemical reprogramming system.Bulk RNA-seq:CD3? T cells cultured under medium containing 4M (Bix01294, RG108, PD0325901, and VPA) and/or AZD4205 conditions for 7 days were collected for bulk RNA-seq. CD3? T cells induced with 5M (Bix01294, RG108, PD0325901, VPA, and AZD4205)were collected for bulk RNA-seq. Single-cell RNA-seq: Isolated CD3+ T cells (day 0) and viable cells from the iMK induction culture on day 7 and day 14 were collected and analyzed using the Chromium system (10X Genomics) according to the manufacturer's instructions for an expected capture rate of approximately 10,000 single cells per sample.