Heterogeneity of high-potency multilineage hematopoietic stem cells and identification of 'Super' transplantability

• Unique molecular features of ‘Super’-class HSCs were identified through analyses of single-clone serial transplants.

• Low expression of CD27 is a key characteristic of ‘Super’-class HSCs and CD27– HSCs display superior transplantability.

Hematopoietic stem cells (HSCs) are heterogeneous, and the quality of HSCs - that is, 'transplantability' - is a key determinant for posttransplant hematopoietic reconstitution. However, molecular modalities of high-potency HSCs with superior transplantability still remain poorly understood. Here, we conducted large-scale single-clone serial-transplant experiments and tracked descendant cells of 288 HSC clones to quantify their intrinsic capability for hematopoietic reconstitution. Using integrated single-cell transcriptional, immunophenotypical, and Bayesian dynamic analyses, we uncovered three classes of HSC clones - 'Super', 'Flash', and 'Trickle' - that had higher output in the 1st generation but exhibited markedly different behavior in later generations. The 'Super'-class HSC clones comprised 4% of the HSCs and manifested persistent superior transplantability and balanced myeloid/lymphoid lineage outputs across generations in serial transplants. The 'Super'-class HSCs had a unique molecular signature, including low expression of CD27, that was distinct from previously known 'Classical HSC' signatures. Validation experiments indicated that CD27- HSCs had superior transplantability compared to CD27+ HSCs. Our study asserted an operational definition for 'Super' transplantability of HSCs, defined its molecular program, and suggested new directions for enriching high-potency HSCs in grafts.