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Haematopoietic stem cells drive blood production, but their population size and lifetime dynamics have not been quantified directly in humans. Here we identified 129,582 spontaneous, genome-wide somatic mutations in 140 single-cell-derived haematopoietic stem and progenitor colonies from a healthy 59-year-old man and applied population-genetics approaches to reconstruct clonal dynamics. Cell divisions from early embryogenesis were evident in the phylogenetic tree; all blood cells were derived from a common ancestor that preceded gastrulation. The size of the stem cell population grew steadily in early life, reaching a stable plateau by adolescence. We estimate the numbers of haematopoietic stem cells that are actively making white blood cells at any one time to be in the range of 50,000–200,000. We observed adult haematopoietic stem cell clones that generate multilineage outputs, including granulocytes and B lymphocytes. Harnessing naturally occurring mutations to report the clonal architecture of an organ enables the high-resolution reconstruction of somatic cell dynamics in humans. Analysis of blood from a healthy human show that haematopoietic stem cells increase rapidly in numbers through early life, reaching a stable plateau in adulthood, and contribute to myeloid and B lymphocyte populations throughout life.

Haematopoietic stem cells (HSCs) are a rare cell type that reconstitute the entire blood and immune systems after transplantation and can be used as a curative cell therapy for a variety of haematological diseases 1 , 2 . However, the low number of HSCs in the body makes both biological analyses and clinical application difficult, and the limited extent to which human HSCs can be expanded ex vivo remains a substantial barrier to the wider and safer therapeutic use of HSC transplantation 3 . Although various reagents have been tested in attempts to stimulate the expansion of human HSCs, cytokines have long been thought to be essential for supporting HSCs ex vivo 4 . Here we report the establishment of a culture system that allows the long-term ex vivo expansion of human HSCs, achieved through the complete replacement of exogenous cytokines and albumin with chemical agonists and a caprolactam-based polymer. A phosphoinositide 3-kinase activator, in combination with a thrombopoietin-receptor agonist and the pyrimidoindole derivative UM171, were sufficient to stimulate the expansion of umbilical cord blood HSCs that are capable of serial engraftment in xenotransplantation assays. Ex vivo HSC expansion was further supported by split-clone transplantation assays and single-cell RNA-sequencing analysis. Our chemically defined expansion culture system will help to advance clinical HSC therapies. A culture system allows the long-term expansion of human haematopoietic stem cells (HSCs) in vivo without the use of recombinant cytokines or albumin, with potential applications for clinical therapies involving HSCs.

About 10% of myeloproliferative neoplasms carry mutations in both TET2 and JAK2 . Clinical presentation, risk of thrombosis, and rates of tumor progression are affected by which gene mutation is acquired first. Cancers evolve as a consequence of the stepwise accumulation of somatic lesions, with competition between subclones and sequential subclonal evolution. 1 , 2 Darwinian selection of variant subclones results in acquisition of biologic attributes required for tumor formation. 3 Genetic interaction is central to this process, but it is unclear how mutated genes interact to generate the phenotypic hallmarks of cancer, and the influence, if any, of the order in which mutations are acquired is unknown. 4 Cooperation between different genetic lesions has been observed in cell-line models of transformation 5 and in mouse models of several cancers. 6 , 7 Moreover, the consequences of an early . . .

Mouse haematopoietic stem cells (HSCs) undergo a postnatal transition in several properties, including a marked reduction in their self-renewal activity. We now show that the developmentally timed change in this key function of HSCs is associated with their decreased expression of Lin28b and an accompanying increase in their let-7 microRNA levels. Lentivirus-mediated overexpression of Lin28 in adult HSCs elevates their self-renewal activity in transplanted irradiated hosts, as does overexpression of Hmga2, a well-established let-7 target that is upregulated in fetal HSCs. Conversely, HSCs from fetal Hmga2 −/− mice do not exhibit the heightened self-renewal activity that is characteristic of wild-type fetal HSCs. Interestingly, overexpression of Hmga2 in adult HSCs does not mimic the ability of elevated Lin28 to activate a fetal lymphoid differentiation program. Thus, Lin28b may act as a master regulator of developmentally timed changes in HSC programs with Hmga2 serving as its specific downstream modulator of HSC self-renewal potential. Fetal haematopoietic stem cells (HSCs) display higher self-renewal potential than their adult counterparts. Eaves and colleagues show that adult HSCs express lower levels of Lin28b and higher levels of let-7 miRNA. They demonstrate that Lin28 overexpression, as well as that of Hmga2 (a target of let-7), induces fetal properties in adult HSCs. Conversely, HMGA2 loss in fetal HSCs results in premature induction of adult HSC self-renewal properties.

Adult blood contains a mixture of mature cell types, each with specialized functions. Single hematopoietic stem cells (HSCs) have been functionally shown to generate all mature cell types for the lifetime of the organism. Differentiation of HSCs toward alternative lineages must be balanced at the population level by the fate decisions made by individual cells. Transcription factors play a key role in regulating these decisions and operate within organized regulatory programs that can be modeled as transcriptional regulatory networks. As dysregulation of single HSC fate decisions is linked to fatal malignancies such as leukemia, it is important to understand how these decisions are controlled on a cell-by-cell basis. Here we developed and applied a network inference method, exploiting the ability to infer dynamic information from single-cell snapshot expression data based on expression profiles of 48 genes in 2,167 blood stem and progenitor cells. This approach allowed us to infer transcriptional regulatory network models that recapitulated differentiation of HSCs into progenitor cell types, focusing on trajectories toward megakaryocyte–erythrocyte progenitors and lymphoid-primed multipotent progenitors. By comparing these two models, we identified and subsequently experimentally validated a difference in the regulation of nuclear factor, erythroid 2 (Nfe2) and core-binding factor, runt domain, alpha subunit 2, translocated to, 3 homolog (Cbfa2t3h) by the transcription factor Gata2. Our approach confirms known aspects of hematopoiesis, provides hypotheses about regulation of HSC differentiation, and is widely applicable to other hierarchical biological systems to uncover regulatory relationships.

[...]the production and quality control of the aforementioned drug product is substantial and results in equally substantial cost implications which inevitably limit the future application of successful gene therapies. While it is still at the mouse model stage, this approach achieves the important benchmark of successful in vivo editing of functional (i.e., secondarily transplantable) HSCs—something that is essential for long term success of a gene-edited graft of cells in patient populations. If achievable, a safe in vivo gene editing protocol would have the enormous attractiveness of being much less expensive to produce and require much less specialized posttransplantation processing and monitoring.