Applying single cell-based technologies to the characterisation of human haematopoietic stem cell heterogeneity

Haematopoiesis is essential for all vertebrate life. It involves the generation of terminally differentiated blood cells from a small pool of self-renewing multipotent haematopoietic stem cells (HSCs) through a series of lineage-restricted progenitors. HSCs form the basis of bone marrow (BM) transplantations and enable the treatment of patients with genetic blood disorders and leukaemia. It has long been assumed that individual HSCs have the capacity to produce all blood lineages in equal proportions. However, recently, single cell and serial transplantation studies in mice demonstrated a profound degree of heterogeneity within murine HSCs with a substantial proportion displaying a restricted pattern of lineage output. Consequently, these studies in mice have challenged the traditional view of a functionally homogeneous HSC population and instead suggested that individual HSCs differ in their properties. In contrast, human HSC heterogeneity has not yet been fully elucidated. The aim of this work was to characterise heterogeneity in human adult BM-derived HSCs, particularly focusing on the identification of human HSCs with distinct lineage outputs and their transcriptional differences. For this purpose, human HSCs were purified from adult BM, barcoded with unique DNA sequences encoded by lentiviruses, and transplanted into non irradiated, immunedeficient NBSGW mice. We performed single-cell RNA sequencing of the engrafted barcoded HSCs and bulk RNA sequencing of their cellular in vivo output. We identified several human HSC subtypes with biased and restricted lineage outputs, as well as associated transcriptional differences, revealing correlated functional and molecular heterogeneity of human adult BM HSCs. This work may lead to the identification of potential molecular and/or surface markers that can help to determine HSC subsets with specific haematopoietic fate restrictions. Ultimately, characterisation of the human HSC heterogeneity in terms of the molecular signatures of specific HSC subtypes with distinct lineage outputs may allow us to predict how transplanted HSCs behave in patients