Characterisation of human Haematopoietic Stem Cell differentiation and exit from quiescence at single cell resolution

Blood formation is coordinated by a set of functionally heterogeneous haematopoietic stem cell (HSC) compartments both in mouse and in human. However, how this diversity is regulated at the cellular and molecular level and when HSC multipotency is lost is still not known, in particular in humans. Quiescence is an important property of HSCs and fine regulation of the balance between quiescence and cell cycle entry is of vital importance for maintaining a healthy HSC pool and avoid haematological malignancies. Very little is known about how molecular networks change during exit from quiescence and no studies have formally examined if cell fate decisions occur during this process or later on during the cell cycle. Here, I combine index sorting, in-vitro single cell functional assays and single cell RNA-sequencing with xenotransplantation assays to profile single human HSC properties in the purest HSC compartment reported to date (CD49+ HSCs) and to understand when the first steps of lineage restriction occur during HSC differentiation. Moreover, I use an in-vitro model system to comprehensively study HSC activation and to investigate whether HSC self-renewal is lost during quiescence exit or during cell cycle progression. First, I unveil an unexpected degree of intrinsic functional and molecular heterogeneity within the human CD49f+ HSC pool. I demonstrate that the first restriction step towards the lymphoid lineage occurs already within the CD49f+ HSC compartment and generates erythroid-deficient myeloid-lymphoid committed cells. Within this compartment, transcriptional programmes and lineage potential progressively change along a gradient of opposing cell surface expression of CLEC9A and CD34. Two functionally distinct populations can be identified and purified. CLEC9Ahi CD34lo cells contain long-term repopulating multipotent HSCs with slow quiescence exit kinetics, whereas CLEC9Alo CD34hi cells are restricted to myelo-lymphoid differentiation and display infrequent but durable repopulation capacity. Second, I demonstrate that a drastic transcriptional remodelling reflective of the fast metabolic activation seen in ex-vivo cultured HSCs occurs during quiescence exit, and independently of cell cycle progression. In-vivo data show that the reduction in repopulation capacity that accompanies HSC culture also occurs independently of cell cycle progression. Recent work highlighted the active role of mitochondria and metabolism in HSC fate decision and self-renewal. My data is consistent with a model in which the metabolic remodelling seen during exit from quiescence represents the first step towards loss of self-renewal and differentiation. This has important implications for improving ex-vivo protocols for HSC culture and HSC transplants

Beyond "to divide or not to divide": Kinetics matters in hematopoietic stem cells.

Lifelong blood production is ensured by a population of rare and largely quiescent, long-lived hematopoietic stem cells (HSCs). The advent of single-cell technologies has recently highlighted underlying molecular and functional heterogeneity within the HSC pool. Despite heterogenous HSC behaviors, quiescence remains as the most uncontroversial and unifying property of HSCs. Nonetheless, a multifaceted and complex continuum of states has recently been identified within what was previously described as just "quiescent." Here we review such evidence and discuss how it challenges preconceived ideas on the contribution of cell cycle kinetics to HSC function. Specifically, we detail how both the frequency and kinetics of HSC division, largely determined by a network of molecular regulators linked to early G1, influence long-term HSC functionin vivo. In addition, we present data that indicate lengthening the duration of G1 by inhibiting CDK6 decreases lymphoid differentiation of a subset of lymphoid-primed human HSCs, thus linking cell cycle kinetics to cell fate decisions in HSCs. Finally, we reflect on how these new insights can be helpful to fully harness HSC potential in clinical applications that require ex vivo culture.We would like to thank the Cambridge Blood and Stem Cell Biobank, specifically Joanna Baxter and the team of nurses consenting and collecting cord bloos samples; the Cambridge NIHR BRC Cell Phenotyping Hub for their flow cytometry services. E.L. is supported by a Sir Henry Dale fellowship from Wellcome/Royal Society (107630/Z/15/Z). Research in E.L.’s laboratory is supported by Wellcome, BBSRC, EHA, BIRAX, Royal Society and by core support grants by Wellcome and MRC to the Wellcome-MRC Cambridge Stem Cell Institute (203151/Z/16/Z). C.J. is supported by an MRC iCASE PhD studentship and S.B. by a CRUK Cambridge Cancer Centre PhD fellowship

Adaptation to ex vivo culture reduces human hematopoietic stem cell activity independently of cell cycle

Loss of long-term hematopoietic stem cell (LT-HSC) function ex vivo hampers the success of clinical protocols reliant on culture. However, the kinetics and mechanisms by which this occurs remain incompletely characterized. Here, through time-resolved scRNA-Seq, matched in vivo functional analysis and the use of a reversible in vitro system of early G1 arrest, we define the sequence of transcriptional and functional events occurring during the first ex vivo division of human LT-HSCs. We demonstrate that the sharpest loss of LT-HSC repopulation capacity happens early on, between 6 and 24 hours of culture, before LT-HSCs commit to cell cycle progression. During this time window, LT-HSCs adapt to the culture environment, limiting global variability in gene expression and transiently upregulating gene networks involved in signaling and stress responses. From 24 hours, LT-HSC progression past early G1 contributes to the establishment of differentiation programmes in culture. However, contrary to current assumptions, we demonstrate that loss of HSC function ex vivo is independent of cell cycle progression. Finally, we show that targeting LT-HSC adaptation to culture by inhibiting early activation of JAK/STAT signaling improves HSC long-term repopulating function ex vivo. Collectively, our study demonstrates that controlling early LT-HSC adaptation to ex vivo culture, for example via JAK inhibition, is of critical importance to improve HSC gene therapy and expansion protocols

Myelo-lymphoid lineage restriction occurs in the human haematopoietic stem cell compartment before lymphoid-primed multipotent progenitors.

Capturing where and how multipotency is lost is crucial to understand how blood formation is controlled. Blood lineage specification is currently thought to occur downstream of multipotent haematopoietic stem cells (HSC). Here we show that, in human, the first lineage restriction events occur within the CD19-CD34+CD38-CD45RA-CD49f+CD90+ (49f+) HSC compartment to generate myelo-lymphoid committed cells with no erythroid differentiation capacity. At single-cell resolution, we observe a continuous but polarised organisation of the 49f+ compartment, where transcriptional programmes and lineage potential progressively change along a gradient of opposing cell surface expression of CLEC9A and CD34. CLEC9AhiCD34lo cells contain long-term repopulating multipotent HSCs with slow quiescence exit kinetics, whereas CLEC9AloCD34hi cells are restricted to myelo-lymphoid differentiation and display infrequent but durable repopulation capacity. We thus propose that human HSCs gradually transition to a discrete lymphoid-primed state, distinct from lymphoid-primed multipotent progenitors, representing the earliest entry point into lymphoid commitment.We thank the Cambridge NIHR BRC Cell Phenotyping Hub, particularly Anna Petrunkina-Harrison and Esther Perez for their flow cytometry advice; the Cambridge Blood and Stem Cell Biobank, specifically Joanna Baxter and the team of nurses consenting and collecting cord blood samples; David Kent for critical reading of the manuscript. E.L. is supported by a Sir Henry Dale fellowship from the Wellcome Trust (WT)/Royal Society. S.B. is supported by a CRUK Cambridge Cancer Center PhD fellowship. Research in the E.L. and B.G. laboratories is supported by the WT, EHA, CRUK, Bloodwise, MRC, BBSRC, NIH-NIDDK, and core support grants by the WT and MRC to the WT-MRC Cambridge Stem Cell Institute

Hematopoietic stem cells retain functional potential and molecular identity in hibernation cultures.

Advances in the isolation and gene expression profiling of single hematopoietic stem cells (HSCs) have permitted in-depth resolution of their molecular program. However, long-term HSCs can only be isolated to near purity from adult mouse bone marrow, thereby precluding studies of their molecular program in different physiological states. Here, we describe a powerful 7-day HSC hibernation culture system that maintains HSCs as single cells in the absence of a physical niche. Single hibernating HSCs retain full functional potential compared with freshly isolated HSCs with respect to colony-forming capacity and transplantation into primary and secondary recipients. Comparison of hibernating HSC molecular profiles to their freshly isolated counterparts showed a striking degree of molecular similarity, further resolving the core molecular machinery of HSC self-renewal while also identifying key factors that are potentially dispensable for HSC function, including members of the AP1 complex (Jun, Fos, and Ncor2), Sult1a1 and Cish. Finally, we provide evidence that hibernating mouse HSCs can be transduced without compromising their self-renewal activity and demonstrate the applicability of hibernation cultures to human HSCs

The transcription factor HIF2α partakes in the differentiation block of acute myeloid leukemia

Abstract One of the defining features of acute myeloid leukemia (AML) is an arrest of myeloid differentiation whose molecular determinants are still poorly defined. Pharmacological removal of the differentiation block contributes to the cure of acute promyelocytic leukemia (APL) in the absence of cytotoxic chemotherapy, but this approach has not yet been translated to non‐APL AMLs. Here, by investigating the function of hypoxia‐inducible transcription factors HIF1α and HIF2α, we found that both genes exert oncogenic functions in AML and that HIF2α is a novel regulator of the AML differentiation block. Mechanistically, we found that HIF2α promotes the expression of transcriptional repressors that have been implicated in suppressing AML myeloid differentiation programs. Importantly, we positioned HIF2α under direct transcriptional control by the prodifferentiation agent all‐trans retinoic acid (ATRA) and demonstrated that HIF2α blockade cooperates with ATRA to trigger AML cell differentiation. In conclusion, we propose that HIF2α inhibition may open new therapeutic avenues for AML treatment by licensing blasts maturation and leukemia debulking

Adaptation to<i>ex vivo</i>culture drives human haematopoietic stem cell loss of repopulation capacity in a cell cycle independent manner

Loss of long-term haematopoietic stem cell function (LT-HSC) hampers the success of ex vivo HSC gene therapy and expansion procedures, but the kinetics and the mechanisms by which this occurs remain incompletely characterized. Here through time-resolved scRNA-Seq, matched in vivo functional analysis and the use of a reversible in vitro system of early G 1 arrest, we define the sequence of transcriptional and functional events occurring during the first ex vivo division of human LT-HSCs. We demonstrate that contrary to current assumptions, loss of long-term repopulation capacity during culture is independent of cell cycle progression. Instead it is a rapid event that follows an early period of adaptation to culture, characterised by transient gene expression dynamics and constrained global variability in gene expression. Cell cycle progression however contributes to the establishment of differentiation programmes in culture. Our data have important implications for improving HSC gene therapy and expansion protocols