On January 2, 2024, Cell Metabolism published online the collaborative results of Prof. ZHOU Bo's group from the Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences, Prof. WAN Jingjing's group from East China Normal University, and Prof. QIAN Kun's group from Shanghai Jiao Tong University. This work has built from scratch a high-throughput single-cell metabolomics (hi-scMet) platform combining antibody staining, pre-fixation, flow sorting, automated sample processing, and matrix-assisted laser desorption ionization mass spectrometry (MS), which is capable of detecting more than 100 metabolites from single cells. Based on this technology, this work successfully mapped the whole-cell metabolome of hematopoietic lineage, revealed the metabolic shifts during hematopoietic stem cell expansion, and also demonstrated the role of pentose phosphate pathway in hematopoietic stem cell activation.
Hematopoietic stem cells (HSCs) are a rare class of stem cells that can establish a complete and mature blood system through self-renewal and multi-lineage differentiation. The metabolic state of HSCs changes dramatically at different stages of development. In order to meet the demand for hematopoiesis, HSCs proliferate and divide rapidly in the embryonic period, and then after the birth of mice, when the demand for hematopoiesis gradually decreases, the HSCs pause their division and enter into a quiescent state. Thanks to the development of mass spectrometry technology, the metabolism of HSCs has become more and more abundant, and a number of researchers have demonstrated that several metabolites and their pathways are directly involved in the regulation of HSC function using liquid chromatography-mass spectrometry. However, since HSCs are not a homogeneous whole, traditional liquid chromatography-mass spectrometry is difficult to be used to analyze cellular heterogeneity, and there are obvious limitations in detection throughput, so how to analyze the metabolome of hematopoietic stem cells at the single-cell level in a high-throughput manner will be the next challenge.
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, the team introduced the TetO-H2B-GFP; R26rtTA mouse model, and classified HSCs into four subpopulations with different self-renewal capacities. The results demonstrated 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 are 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 also significantly inhibit the reconstruction process of hematopoietic stem and progenitor cells. This demonstrates that knockdown or pharmacological inhibition of the pentose phosphate pathway results in impaired self-renewal of HSCs.
In summary, this work successfully 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, and successfully 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.