Investigations of Hematopoietic Stem Cells and Their Age-Associated Alterations

The hematopoietic stem cell (HSC) has and continues to be extensively investigated, and represents by far the most studied somatic stem cell. Development of various experimental technologies, including fluorescence activated cell sorting (FACS), have been crucial for research advancements within the hematopoietic field. However, utilization of flow cytometry techniques put high demands on study design, execution, and data analysis. In article I, we addressed a number of important aspects of flow cytometry-based experiments, particularly for experiments evaluating cells present at low frequencies, such as HSCs, and/or of limited sample amounts. Specifically, we highlighted the importance of different types of positive and negative controls. We also presented a 17-parameter analysis design for simultaneous investigation of numerous different mature and immature human hematopoietic cell populations, including HSCs, megakaryocyte/erythrocyte progenitors (MEPs), granulocyte/macrophage progenitors (GMPs), common lymphoid progenitors (CLPs), CD11b+ myeloid cells, CD19+ B cells, CD4+ T cells, and CD8+ T cells, in one bone marrow sample. Lastly, we discussed data visualization alternatives that allow for appropriate presentation of analyzed results.Serial transplantation is considered the gold standard approach for in vivo evaluation of long-term HSC capacity. Despite the extensive use of this methodology, such experiments are not conducted uniformly between laboratories. In article II, we therefore compared the two most common strategies for serial transplantation (serial transplantation of whole bone marrow [wBM] versus serial transplantation of purified HSCs). We revealed that donor-derived cells were not evenly distributed among separate bones within individual mice three months after transplantation – a time point that is frequently used for readout of bone marrow engraftment and/or isolation of cells for serial transplantations. We showed that such unequal distribution could impede isolation and serial transplantation of wBM with representative chimerism levels. Serial transplantation of purified HSCs would however correct for uneven chimerism levels. Furthermore, serial transplantation of wBM was associated with a relative lymphoid skewing, likely as a result of long-lived lymphoid-restricted non- HSCs. Again, serial transplantation of purified HSCs is preferential as it ensures evaluation of lineage output from the designated candidate HSC population. We also demonstrated that serial transplantation of purified HSCs enable distinction of long-term effects (occurring in secondary recipients) from short-term effects (presented in primary recipients), which may be hindered when wBM is transplanted. Therefore, we highly recommend utilization of purified HSCs for serial transplantation purposes. We also presented a proposed serial transplantation design that would maximize the chances to correctly evaluate long-term HSC competence.Lastly in this thesis, we investigated the effects of aging on human and murine hematopoietic stem and progenitor cells (HSPCs). Such investigations are important for understanding of the underlying mechanisms that cause age-associated alterations of the hematopoietic system, including immune impairments, and increased incidences of anemia and myeloid malignancies. Murine studies indicate that at least some age-associated hematologic changes are linked to alterations occurring in the most immature hematopoietic compartments. Such studies in humans are sparse, as well as conflicting, but necessary to establish species-conserved and species-specific agingpatterns, and to confirm mice as relevant model organisms for investigations of aging hematopoiesis. In article III, we revealed that humans and mice demonstrate several similar hematologic aging patterns, including increased HSC frequencies, and lymphoid differentiation impairments. Both species also exhibited prominent transcriptional lineage-skewing patterns of aged HSCs toward enrichments of megakaryocytic/erythroid signatures, while genes involved in lymphoid specification were markedly downregulated. In conclusion, these results confirmed several HSC aging similarities across the human-to-mouse species barrier, as well as demonstrated novel lineage-skewing patterns within aging HSPCs

Retinoic Acid Puts Hematopoietic Stem Cells Back To Sleep

Dormant hematopoietic stem cells (dHSCs) display superior serial reconstitution capacity compared to active HSCs, although their role in normal hematopoiesis has not been thoroughly investigated. Recently in Cell, Cabezas-Wallscheid et al. (2017) demonstrate involvement of retinoic acid signaling in murine dHSCs for preservation of the HSC pool

Probing hematopoietic stem cell function using serial transplantation: Seeding characteristics and the impact of stem cell purification.

Appropriate regulation of hematopoietic stem cell (HSC) numbers and function is a requisite for life-long blood cell replenishment. Knowledge of factors that regulate HSC activity is derived largely from murine model systems, with serial transplantation often considered a "gold standard" to assess longevity and self-renewal of HSCs. In the literature, we noted inconsistencies in how serial transplantations are conducted and decided to assess a set of parameters at play in such experiments. We found that HSCs distribute and expand unevenly among individual bones following transplantation, suggesting that isolation of a limited number of bone marrow cells for serial transplantation and/or analysis can influence experimental outcomes. Comparing donor cell output from transplanted unfractionated bone marrow cells, as opposed to fluorescence-activated cell-sorted HSCs, revealed distinct differences in the output of mature blood cells. Specifically, we found that long-lived progenitor and/or mature co-transplanted cells can severely affect the interpretation of ongoing HSC activity in secondary hosts. The implications of these data for the design and execution of serial transplantation experiments are discussed

Frequency determination of rare populations by flow cytometry: A hematopoietic stem cell perspective.

Flow cytometry allows for identification of cellular subsets based on cell intrinsic properties, most often by the use of fluorochrome-conjugated antibodies recognizing distinct cell-surface epitopes that define the cells of interest. Advances in technical instrumentation and the availability of an ever-increasing number of fluorophores, today enables identification of multicolor defined cellular populations to a previously unreachable resolution. However, these possibilities put an increasing demand on preparation, acquisition, and subsequent analysis of the investigated samples. Identification of very rare cellular subsets, such as the bone marrow-residing hematopoietic stem cells (HSCs), causes further complexity to such analysis. Here, we discuss considerations and aspects in multicolor flow cytometry as exemplified by analysis of human and mouse HSCs. We illustrate advantages and drawbacks of polychromatic flow cytometry and propose strategies, such as the use of internal reference populations, for sample analysis. © 2013 International Society for Advancement of Cytometry

Temporal dynamics of TNF-mediated changes in hematopoietic stem cell function and recovery

While tumor necrosis factor (TNF) is a critical mediator of appropriate immune response and tissue repair, its misregulation is linked to cancer, autoimmunity, bone marrow failure, and aging. Understanding the context-dependent roles of TNF is essential for elucidating normal and pathogenic conditions and to guide clinical therapy advancements. Prior studies suggested that TNF restricts the self-renewal capacity of hematopoietic stem cells (HSCs), but its long-term effect on HSCs remains unclear. Here, we demonstrate that in vivo TNF administration results in a transient exit of HSCs from quiescence, which coincides with a compromised repopulation capacity. These functional changes are; however, fully reversible even following prolonged/chronic transient exposure to TNF. Notably, antagonizing TNF signaling in transplantation recipients enhances donor HSC reconstitution. Our findings provide molecular and functional insight into HSC regulation, with implications for both acute and chronic inflammatory conditions

IRF1 regulates self-renewal and stress-responsiveness to support hematopoietic stem cell maintenance

Inflammatory mediators induce emergency myelopoiesis and cycling of adult hematopoietic stem cells (HSCs) through incompletely understood mechanisms. To suppress the unwanted effects of inflammation and preserve its beneficial outcomes, the mechanisms by which inflammation affects hematopoiesis need to be fully elucidated. Rather than focusing on specific inflammatory stimuli, we here investigated the role of transcription factor Interferon (IFN) regulatory factor 1 (IRF1), which receives input from several inflammatory signaling pathways. We identify IRF1 as a master HSC regulator. IRF1 loss impairs HSC self-renewal, increases stress-induced cell cycle activation, and confers apoptosis resistance. Transcriptomic analysis revealed an aged, inflammatory signature devoid of IFN signaling with reduced megakaryocytic/erythroid priming and antigen presentation in IRF1-deficient HSCs. Finally, we conducted IRF1-based AML patient stratification to identify groups with distinct proliferative, survival and differentiation features, overlapping with our murine HSC results. Our findings position IRF1 as a pivotal regulator of HSC preservation and stress-induced responses.Competing Interest StatementThe authors have declared no competing interest

IRF1 regulates self-renewal and stress responsiveness to support hematopoietic stem cell maintenance.

Hematopoietic stem cells (HSCs) are tightly controlled to maintain a balance between blood cell production and self-renewal. While inflammation-related signaling is a critical regulator of HSC activity, the underlying mechanisms and the precise functions of specific factors under steady-state and stress conditions remain incompletely understood. We investigated the role of interferon regulatory factor 1 (IRF1), a transcription factor that is affected by multiple inflammatory stimuli, in HSC regulation. Our findings demonstrate that the loss of IRF1 from mouse HSCs significantly impairs self-renewal, increases stress-induced proliferation, and confers resistance to apoptosis. In addition, given the frequent abnormal expression of IRF1 in leukemia, we explored the potential of IRF1 expression level as a stratification marker for human acute myeloid leukemia. We show that IRF1-based stratification identifies distinct cancer-related signatures in patient subgroups. These findings establish IRF1 as a pivotal HSC controller and provide previously unknown insights into HSC regulation, with potential implications to IRF1 functions in the context of leukemia

Human and murine hematopoietic stem cell aging is associated with functional impairments and intrinsic megakaryocytic/erythroid bias

Aging within the human hematopoietic system associates with various deficiencies and disease states, including anemia, myeloid neoplasms and reduced adaptive immune responses. Similar phenotypes are observed in mice and have been linked to alterations arising at the hematopoietic stem cell (HSC) level. Such an association is, however, less established in human hematopoiesis and prompted us here to detail characteristics of the most primitive human hematopoietic compartments throughout ontogeny. In addition, we also attempted to interrogate similarities between aging human and murine hematopoiesis. Coupled to the transition from human cord blood (CB) to young and aged bone marrow (BM), we observed a gradual increase in frequency of candidate HSCs. This was accompanied by functional impairments, including decreased lymphoid output and reduced proliferative potential. Downstream of human HSCs, we observed decreasing levels of common lymphoid progenitors (CLPs), and increasing frequencies of megakaryocyte/erythrocyte progenitors (MEPs) with age, which could be linked to changes in lineage-affiliated gene expression patterns in aged human HSCs. These findings were paralleled in mice. Therefore, our data support the notion that age-related changes also in human hematopoiesis involve the HSC pool, with a prominent skewing towards the megakaryocytic/erythroid lineages, and suggests conserved mechanisms underlying aging of the blood cell system

Genome-wide RNAi Screen Identifies Cohesin Genes as Modifiers of Renewal and Differentiation in Human HSCs

To gain insights into the regulatory mechanisms of hematopoietic stem cells (HSCs), we employed a genome-wide RNAi screen in human cord-blood derived cells and identified candidate genes whose knockdown maintained the HSC phenotype during culture. A striking finding was the identification of members of the cohesin complex (STAG2, RAD21, STAG1, and SMC3) among the top 20 genes from the screen. Upon individual validation of these cohesin genes, we found that their knockdown led to an immediate expansion of cells with an HSC phenotype in vitro. A similar expansion was observed in vivo following transplantation to immunodeficient mice. Transcriptome analysis of cohesin-deficient CD34+ cells showed an upregulation of HSC-specific genes, demonstrating an immediate shift toward a more stem-cell-like gene expression signature upon cohesin deficiency. Our findings implicate cohesin as a major regulator of HSCs and illustrate the power of global RNAi screens to identify modifiers of cell fate