(I-360) PolyloxExpress barcoding and tracking to examine adult mouse bone marrow endothelial cell differentiation potential

During embryonic development, the process of endothelial-to-hematopoietic transition (EHT) plays a crucial role in differentiating hemogenic endothelial cells into hematopoietic stem and progenitor cells (HSPCs). EHT is well-known to occur at different times in specific anatomical sites, including the yolk sac, placenta, and aorta-gonad-mesonephros (AGM). The HSPCs generated during development are believed to sustain adult hematopoiesis throughout life, producing all blood elements. Previous experiments in our laboratory revealed the presence of potential hemogenic endothelial cells within the adult murine bone marrow, as detected using fluorescence lineage tracking. To further substantiate this observation, we aim to employ lineage tracing through the inducible Cre-PolyloxExpress barcoding system. This investigation seeks to confirm that HSPCs are generated from bone marrow hemogenic endothelial cells, thus shedding light on the occurrence of EHT in adult mice within the bone marrow. Our goal is to expand current understanding on the potential role of adult bone marrow endothelial in hematopoiesis using this barcode system for lineage tracking. My project is aimed at developing an in vitro PolyloxExpress barcoding system for preliminary validation testing.

The PolyloxExpress barcode system, which allows specific recombination within individual single cells using the Cre recombinase enzyme, was utilized. The PolyloxExpress mouse strain was crossbred with ZsGreen and mTmG Cre reporter lines to identify bone marrow cell populations. Lentiviral constructs containing the Cre recombinase gene, along with the EGFP and puromycin resistance markers, were used to deliver Cre recombinase to pre-cultured (passage 1) murine primary bone marrow cells. Successful viral infection was confirmed by performing a western blot to detect GFP expression in infected HEK293T cells. The activity of Cre recombinase was induced using 4-Hydroxytamoxifen to generate specific barcodes within the lentivirus-infected bone marrow cells. Recombined PolyloxExpress barcode sequences were obtained through RNA extraction, RT-PCR, gel extraction, TA cloning, and Sanger sequencing.

Validation of the Cre-containing lentivirus infection of HEK293T cells was achieved through microscopy, western blotting, and flow cytometry. Fluorescence microscopy also demonstrated successful infection of murine primary bone marrow cells with the PolyloxExpress barcode. Upon treatment with 4-Hydroxytamoxifen, RNA was extracted from infected bone marrow cells, and RT-PCR confirmed the presence of recombined PolyloxExpress barcodes. Further analysis involved cloning the barcode sequences into a TA cloning vector and subsequent Sanger sequencing.

The potential existence of hemogenic endothelial cells within adult bone marrow, as suggested by fluorescence tracking, has significant implications for understanding EHT and its regulation. By genetically confirming the generation of HSPCs from bone marrow hemogenic endothelial cells using the PolyloxExpress barcoding system, this study challenges current knowledge and offers new insights into the EHT process. Additional studies using this in vitro barcoding system and the in vivo mouse lines promise to expand our knowledge on hemogenic endothelial cells and their contribution to adult hematopoiesis.