In this study, the researchers isolated 12 blood lineage cells from the bone marrow of adult mice, totaling 1428 cells, and mapped the single-cell metabolome of the entire hematopoietic lineage using the hi-scMet technology platform. In addition, they introduced the TetO-H2B-GFP; R26rtTA mouse model, and classified HSCs into four subpopulations with different self-renewal capacities.

The researchers found that the metabolic levels of the hematopoietic stem cell subpopulations showed trend changes, and the more self-renewal capable HSCs were more inclined to be maintained in a state of low metabolic activity, in which 33 metabolites showed significant trend changes. Among these, a number of substances were involved in glucose metabolism, such as glucose, lactate, malate & 6-phosphogluconate (6PG).

To determine whether the pentose phosphate pathway, in which 6PG resides, can regulate the function of hematopoietic stem cells in vivo, the researchers designed and introduced 6PG synthase knockout mice, Pgls+/-, and the pentose phosphate pathway inhibitor, 6-AN, which showed that Pgls knockout HSCs exhibited weaker hematologic reconstitution during long-term graft reconstruction, and that 6-AN injections significantly inhibit the reconstruction process of hematopoietic stem and progenitor cells. This demonstrated that knockdown or pharmacological inhibition of the pentose phosphate pathway results in impaired self-renewal of HSCs.

This work established a single-cell metabolome analysis platform based on flow sorting, which remedied the past problems of metabolome analysis in terms of sensitivity, throughput, cellular integrity and targeting. It mapped the whole-cell metabolome of hematopoietic lineage, proved that HSCs are in lower metabolic activity, and discovered that the pentose phosphate pathway regulates the self-renewal ability of hematopoietic stem cells.