Delineating the hierarchical organization and lineage heterogeneity of the mouse hsc and mpp network

The Lin–Sca1+cKit+ (LSK) compartment of the mouse bone marrow contains the most primitive hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs), which harbour a gradient of self-renewal and multilineage potential to long-term regeneration of the entire hematopoietic system. Using additional surface markers including CD34, CD48, CD135 and CD150, HSCs and distinct MPP subsets (MPP1-4) have been identified and extensively characterized regarding their self-renewal capacity, reconstitution kinetics and lineage potential. Recently, we have described two newly defined MPP subsets within LSK termed MPP5 (CD34+CD135-CD48-CD150-) and MPP6 (CD34-CD135-CD48-CD150-), which are molecularly and functionally located between HSC and MPP2-4. We performed serial transplantation of purified HSC/MPP populations and demonstrated that MPP6s behaved highly similar to HSCs regarding their long-term multi-lineage engraftment. In contrast, MPP5s displayed little reconstitution activity in secondary transplantation. Intriguingly, despite similar functional output in vivo, MPP6s transcriptionally clustered distinctly from HSCs, MPP1s and MPP5s. Furthermore, in vitro division and ontogeny tracking revealed similar proliferation history between HSCs and MPP6s, but distinct differentiation dynamics. Therefore, the hierarchical organization of MPP6s with regard to HSCs and other MPP subsets, as well as their lineage heterogeneity, remain highly ambiguous. In this study, cellular barcoding will be used to comprehensively map the landscape of lineage differentiation of HSC/MPP subsets. Combined with clonal ontogeny analysis and single-cell RNA sequencing, the developmental relationship within the HSC/MPP network will be deciphered with unprecedented clonal and temporal resolution. Collectively, this study will provide a comprehensive clonal characterization of the LSK compartment in vivo. The Lin–Sca1+cKit+ (LSK) compartment of the mouse bone marrow contains the most primitive hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs), which harbour a gradient of self-renewal and multilineage potential to long-term regeneration of the entire hematopoietic system. Using additional surface markers including CD34, CD48, CD135 and CD150, HSCs and distinct MPP subsets (MPP1-4) have been identified and extensively characterized regarding their self-renewal capacity, reconstitution kinetics and lineage potential. Recently, we have described two newly defined MPP subsets within LSK termed MPP5 (CD34+CD135-CD48-CD150-) and MPP6 (CD34-CD135-CD48-CD150-), which are molecularly and functionally located between HSC and MPP2-4. We performed serial transplantation of purified HSC/MPP populations and demonstrated that MPP6s behaved highly similar to HSCs regarding their long-term multi-lineage engraftment. In contrast, MPP5s displayed little reconstitution activity in secondary transplantation. Intriguingly, despite similar functional output in vivo, MPP6s transcriptionally clustered distinctly from HSCs, MPP1s and MPP5s. Furthermore, in vitro division and ontogeny tracking revealed similar proliferation history between HSCs and MPP6s, but distinct differentiation dynamics. Therefore, the hierarchical organization of MPP6s with regard to HSCs and other MPP subsets, as well as their lineage heterogeneity, remain highly ambiguous. In this study, cellular barcoding will be used to comprehensively map the landscape of lineage differentiation of HSC/MPP subsets. Combined with clonal ontogeny analysis and single-cell RNA sequencing, the developmental relationship within the HSC/MPP network will be deciphered with unprecedented clonal and temporal resolution. Collectively, this study will provide a comprehensive clonal characterization of the LSK compartment in vivo.