Abstract
Cell Biology, Immunology and Biochemistry (basic and preclinical research)
Y M He, MD (she/her/hers)
Blood Center of Zhejiang Province
Hangzhou, Zhejiang, China (People's Republic)
Disclosure information not submitted.
The generation of MKs from stem cells in vitro to produce PLTs is appealing approach to providing an alternative source of PLTs. It is reported the intrinsic characteristics of MKs are distinct due to the different stem cell sources using single cell RNA sequencing(sc-RNAseq)technology. In order to fully understand the heterogeneity of MKs generated from HSC in vitro, we established a stable culture system about HSC derived from Cord blood(CB) inducing into MKs in vitro.
Study
Design/Methods:
CD34+ HSC were separated and enriched from CB, which could be induced into MKs in vitro through a culture process of 14-day in serum-free medium supplemented with different concentrations of cytokines (SCF, IL-3, IL-6 and TPO). The cell phenotype of different culture time was analyzed by Flow cytometry, the morphology, polyploid and subcellular structure of megakaryocytes were analyzed, the mRNA expression levels of megakaryocyte related genes (GATA1, GATA2, FOG1, NF-E2, FLI1, CD41 and CD61) at different time points were also detected by fluorescence quantitative PCR. Moreover, the activation levels of platelets in vitro and producing platelets in vivo were performed, respectively. Finally, the subpopulations of MKs were further analyzed by sc-RNAseq.
Results/Findings:
The 2D culture system of CD34+ HSC derived from CB induced into MKs (CB-MKs) in vitro was established. The CB-MKs possess typical characteristic of normal MKs, including morphology, polyploid and subcellular structure（FigA). The DNA polyploid was analyzed on day 12 by flow cytometry, approximately 44.45%±3.28% of CD41a+CD42b+ cells were 2N, 31.2±1.3% were 4 N, 15.4%±2.5% were 8N and 8.95%±1.27% >8N. The mRNA expression levels of all the detected genes except for GATA2 on day 10 and 14 was significantly higher than day 4, however, PF4 had obviously increased up to hundreds of times. Moreover, it was found that the expression of CD62P on the surface of CB-PLT has obviously increased with stimulation of Thrombin or TRAP6, which is similar to PLT derived from peripheral blood. Humanized platelets (0.24%±0.04) could be detected in peripheral blood of NSG mice after infusing CB-MKs within 30 minutes, platelets rapidly decreased after infusing CB-MKs for 6 hours until not be detected at 24h. According to the data of sc-RNAseq, the proportion of cell population with MK characteristics is 77.1% based on UMAP analysis, however, nine transcriptionally distinct clusters of the MKs labeled as C1-C9 were further identified in this MKs population. We have found only C9 population was related to immunity, and C1-C8 populations were shown with typical MK characteristics, which was the most common subtype.
Conclusions:
In conclusion, we have comprehensively and systematically described the biological characteristics and functions of MKs derived from cord blood, which will help to establish universal MKs through gene editing and promote the application of cell therapy.