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

ABSTRACT: Loss of long-term hematopoietic stem cell (LT-HSC) function ex vivo hampers the success of clinical protocols that rely on culture. However, the kinetics and mechanisms through which this occurs remain incompletely characterized. In this study, through time-resolved single-cell RNA sequencing, matched in vivo functional analysis, and the use of a reversible in vitro system of early G1 arrest, we defined the sequence of transcriptional and functional events that occur during the first ex vivo division of human LT-HSCs. We demonstrated that the sharpest loss in 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, limit the global variability in gene expression, and transiently upregulate gene networks involved in signaling and stress responses. From 24 hours, LT-HSC progression past early G1 contributes to the establishment of differentiation programs in culture. However, contrary to the current assumptions, we demonstrated that the loss of HSC function ex vivo is independent of cell cycle progression. Finally, we showed that targeting LT-HSC adaptation to culture by inhibiting the early activation of JAK/STAT signaling improves HSC long-term repopulating function ex vivo. Collectively, our study demonstrated that controlling early LT-HSC adaptation to ex vivo culture, for example, via JAK inhibition, is critically important to improve HSC gene therapy and expansion protocols.

Single nuclei transcriptomics of the in situ human limbal stem cell niche.

The corneal epithelium acts as a barrier to pathogens entering the eye; corneal epithelial cells are continuously renewed by uni-potent, quiescent limbal stem cells (LSCs) located at the limbus, where the cornea transitions to conjunctiva. There has yet to be a consensus on LSC markers and their transcriptome profile is not fully understood, which may be due to using cadaveric tissue without an intact stem cell niche for transcriptomics. In this study, we addressed this problem by using single nuclei RNA sequencing (snRNAseq) on healthy human limbal tissue that was immediately snap-frozen after excision from patients undergoing cataract surgery. We identified the quiescent LSCs as a sub-population of corneal epithelial cells with a low level of total transcript counts. Moreover, TP63, KRT15, CXCL14, and ITGß4 were found to be highly expressed in LSCs and transiently amplifying cells (TACs), which constitute the corneal epithelial progenitor populations at the limbus. The surface markers SLC6A6 and ITGß4 could be used to enrich human corneal epithelial cell progenitors, which were also found to specifically express the putative limbal progenitor cell markers MMP10 and AC093496.1.

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

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.

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.

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.

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.