Generation and single-cell characterization of functional megakaryocytes derived from umbilical cord blood

BACKGROUND: Generation of megakaryocytes (MKs) from stem cells in vitro to produce platelets (PLTs) is an appealing approach for providing an alternative source of PLTs. Understanding the transcriptomic characteristics of MKs in vitro is crucial for providing a theoretical foundation for producing functional MKs more efficiently in the future. METHODS: hematopoietic stem and progenitor cells (HSPCs) were separated from umbilical cord blood (UCB) and were induced to differentiate into MKs in vitro through a culture system. The phenotypes of these cells cultured for different durations were analyzed through flow cytometry. The mRNA expression levels of GATA1, GATA2, FOG1, NF-E2, FLI1, CD41, and CD61 at different time points were analyzed through quantitative PCR. The activation levels of PLTs in vitro and the PLTs produced in vivo were determined. The MK subpopulations were further analyzed through single-cell RNA sequencing (scRNA-seq). RESULTS: UCB-derived MKs exhibited typical characteristics of MKs in vivo, including morphology, polyploidy, and subcellular structure. The mRNA expression levels of GATA1, FOG1, NF-E2, FLI1, CD41, and CD61 on Days 10 and 14 were significantly greater than those on Day 4. CD62P expression on the surface of UCB-PLTs increased markedly in response to thrombin or TRAP6 stimulation. Humanized PLTs were also detected in the peripheral blood of NCG mice following the infusion of UCB-MKs. According to the results of the sc-RNAseq analysis, nine transcriptionally distinct clusters of UCB-MKs, labeled MK1-MK9, were identified, with only the MK9 population being related to immunity. The MK1-MK8 populations displayed typical MK characteristics and were the most prevalent subtypes. In addition, compared with hESCs, UCB-derived MKs exhibited a greater proportion of active-cycling MKs with strong differentiation potential. CONCLUSIONS: In conclusion, this study describes the biological functions and transcriptomic profile of MKs derived from UCB, which will aid in the further development of more efficient systems for generating MKs in vitro and promote their application in cellular therapy.