Endothelial protein C receptor CD201 is a better marker than stem cell antigen-1 to identify mouse long-term reconstituting hematopoietic stem cells following septic challenge.

Endothelial Protein C Receptor CD201 Enables Identification of Transplantable Mouse Hematopoietic Stem Cells in Inflammatory Conditions Unlike SCA1

Changes in the frequencies of human hematopoietic stem and progenitor cells with age and site

Transcriptome Analysis Identifies Regulators of Hematopoietic Stem and Progenitor Cells

Terminal differentiation induction as DNA damage response in hematopoietic stem cells by GADD45A.

Maintenance of the hematopoietic system is dependent on hematopoietic stem cells (HSCs). During homeoastasis HSCs are quiescent. However upon injury, HSCs can efficiently be activated, leading to repair of the system. Signals leading to the activation of quiescent HSCs are still largely unknown. Recently our group has shown that administration of IFNα leads to activation of mouse HSCs in vivo. This is mediated by activation of IFNAR/STAT1 signaling followed by the up-regulation of Sca-1, however the exact mechanism of cell cycle activation remains unclear. To get further insight into this process we performed microarray analysis on HSCs after treatment with IFNα. This screen identified several candidate genes, which are potentially involved in HSC activation, including cell cycle regulators like p57KIP2, Maged1 and Reprimo as well as cytokines like Ccl5 and Cxcl10, which are key regulators of inflammatory responses. Furthermore we identified interferon response genes like Ifitm1, Ifitm3, Iigp1, Iigp3 or Ddx58, which were previously linked to regulation of proliferation in different contexts. Along these studies we uncovered that Ifitm1 and Ifitm3 expression is highly enriched within hematopoietic stem and progenitor cells both on the RNA as well as on the protein level. Moreover expression is further induced by IFNα. However mice lacking the Ifitm family show normal hematopoiesis and normal HSC numbers and cycling behavior of HSCs in homeostatic conditions. Ifitm-deficient HSCs are capable to self-renew and differentiate similar to wild-type HSCs. This suggests that the Ifitm protein family is dispensable for HSCs during homeostasis. Notably Ifitm-deficient HSCs are also efficiently activated by IFNα, similar to wild type HSCs. Microarray analysis of HSCs from Ifitm deficient mice, both during homeostasis and after administration of IFNα, showed no differences in the expression profiles, indicating a role for the Ifitm family as terminal effectors rather than regulatory proteins within HSCs. During our study it was shown by others that the Ifitm family is a potent viral restriction factor in endothelial cells. We are currently investigating whether Ifitm proteins have a similar role in the immune defense of HSCs. Thus far it is still unclear whether human HSCs are similarly activated by IFNα as mouse HSCs. To elucidate this we established a xenotransplantation model with human cord blood cells, which allows testing of the effects of IFNα on human HSCs in vivo. Surprisingly, unlike mouse HSCs, human HSCs are not activated by IFNα in this model. Notably human HSCs in this model are already less quiescent during homeostasis compared to their mouse counterparts. In the mouse also the bacterial endotoxin LPS can induce cell cycle activation in HSCs. Surprisingly LPS similarly activates human HSCs in our model. Gene expression analysis showed a high overlap between the genes induced in mouse and human HSCs after LPS treatment, while IFNα only affected cell cycle regulatory genes in murine HSCs. One explanation for this phenotype could be an impaired interaction of murine stromal niche cells with human HSCs and we are currently investigating this in more detail. Finally we examined the effect of IFNα on quiescent leukemic stem cells (LSCs). While IFNα is known to activate normal mouse HSCs, it is unclear whether also LSCs are similarly affected. To address this we investigated the effects of IFNα on LSCs in a mouse model for chronic myeloid leukemia. Surprisingly LSC were less efficiently activated compared to normal HSCs. This can be explained by down-regulation of the IFNAR by BCR-ABL kinase activity, which was previously described in vitro. This also highlights the importance of exact timing of LSC activation and treatment in combination therapy approaches. Our group is currently using this model to further identify and optimize new possible combination therapies.

Frizzled-6 promotes hematopoietic stem/progenitor cell mobilization and survival during LPS-induced emergency myelopoiesis

A fundamental challenge in cancer biology is to understand why some people develop cancer and others do not despite similar genetic predispositions. Taking the blood system as an exemplar, we seek here to determine if the inflammatory milieu has distinct impact on the clonal fitness and selective advantage of mutant tissue stem cells at the expense of wildtype clones. Clonal hematopoiesis (CH) is an aging-associated phenomenon in which hematopoietic stem cells (HSC) acquire somatic mutations that result in clonal expansion of mutated blood cells and a 12-fold increased risk for myeloid neoplasms, cardiovascular disease, and other aging-associated diseases. Human lineage tracking studies have shown that pre-leukemic CH mutations often arising many decades before disease onset. Although CH has a prevalence as high as 1 in 3 in people over 60 years of age, only some individuals show a readily detectable clone size (≥2% and considered to have clonal hematopoiesis of indeterminate potential). The mechanisms regulating CH clone size are poorly understood, but inflammation is elevated in individuals with CH. Extensive data from murine models also shows that inflammation reprograms HSCs to impair their function. This likely contributes to clonal selection in murine models of CH as acute inflammation also activates murine HSCs, skews them towards myeloid differentiation, and impairs their self-renewal. Some of these inflammatory responses include activation of target genes downstream of TNFα via NFkB, which is known to regulate HSC survival in both murine and human settings. Repeated inflammatory challenges have also been associated with sustained epigenetic changes and accelerated aging of murine HSCs, as well as expansion of clones bearing CH-associated mutations in Dnmt3a and Tet2, the two most frequently mutated genes in CH cases. However, we have shown extensive molecular and functional heterogeneity in the human HSC compartment at steady-state, and it is unclear whether all human HSC respond homogeneously to inflammation. If HSC respond heterogeneously, it is also unclear whether this would impact clonal selection such as during CH. To address these two questions, we developed inflammation-recovery xenograft models to determine the long-term molecular and cellular sequelae of repeated inflammatory challenge on human HSCs in an empirical fashion (Zeng, et al, in revision). No in vivo studies of inflammatory stress on human HSCs have been previously reported. In parallel, we generated a TARGET-seq+ data set of bone marrow hematopoietic stem and progenitor cells from nine DNMT3A or TET2-mutated CH donors and four age-matched controls with no detectable CH mutations (Jakobsen, et al, Cell Stem Cell, 2024). TARGET-seq+ combines simultaneous single cell profiling of transcriptome, genotype, and cell surface immunophenotype, enabling comparisons of mutant (CHMUT) and wild-type (CHWT) HSC obtained from individuals with CH to HSC from age-matched controls without CH. First, we established that human cord blood (CB) HSC in xenotransplantation models recapitulate phenotypes previously observed in the murine system following acute inflammatory activation for 16 hours using human TNFα or lipopolysaccharide (LPS). However, when we mimicked repeated inflammatory challenge through acute treatment with TNFα or LPS at 2w and 10w post-transplantation followed by analysis at 20w, we found a reduced human graft at 20w compared to PBS controls. Thus, repeated challenge resulted in a long-lasting functional HSC impairment long after the treatment. Single cell multiome analysis of human HSCs isolated from this model showed two bona fide HSC subsets: HSC-I showed few molecular changes in response to prior inflammatory stress. However, the second HSC subset, termed HSC inflammatory memory (HSC-iM) showed extensive chromatin accessibility and transcriptional changes 2.5 months after recovery from TNFα or LPS treatment. These changes are primarily in the AP-1 and NF-kB gene regulatory networks and while distinct, also bear striking similarity to epigenetic changes seen in murine epithelial stem cells following wounding. We found HSC-iM share core molecular programs with human memory T cells. Importantly, a transcriptional program reflecting HSC-iM was enriched in HSCs from recovered COVID-19 patients and in HSC from aged individuals and in HSC from individuals with CH from our TARGET-seq+ data set. To gain insight into the impact of the HSC-I and HSC-iM states on selective advantage in CH, we turned to our TARGET-seq+ data set. First, we directly identified both the HSC-I and HSC-iM populations in human CH cases (named HSC1 and HSC2 respectively) validating the relevance of the inflammation-recovery xenograft model established with CB HSC from which they were first identified and confirming that this is not a xenograft artifact. Second, when CHMUT and CHWT within a BM of a subject with CH are compared to each other, gene expression changes occur predominantly in HSC-iM and not HSC-I. Moreover, the presence of the CH mutation results in decreased enrichment of specific inflammatory pathways. Third, we confirmed that CHWT within HSC2-dominant hierarchies were stalled in differentiation, with reduced production of downstream progenitor populations compared to CHWT cells within HSC1-dominant hierarchies. By contrast, mutations in DNMT3A and TET2 were associated with increased progenitor abundance within HSC-iM-dominant hierarchies. Thus, HSC2/HSC-iM is inherently growth restricted, but CH mutations relieve this impairment leading to an increased HSC2/HSC-iM contribution to hematopoiesis and an overall clonal growth advantage. This lends further support to our conclusion that accumulation of HSC-iM underlies age-related hematopoietic dysfunction. We posit that HSC-iM is a “protective adaptation” to inflammation, representing a reserve pool of stem cells that requires a subsequently stronger inflammatory trigger for activation. Additionally, a key hypothesis our work is the notion that selective advantage of CHMUT HSC arises due to a decrease in differentiation output of CHWT HSC-iM. We are currently testing this hypothesis with CRISPR-mediated DNMT3A- and TET2-mutant CB HSC in our xenograft-inflammatory recovery models. We show that HSC-iM progeny monocytes are pro-inflammatory compared to those from HSC-I in our xenograft models, in the COVID patient recovery data, and in steady-state human BM data sets. To gain insight into whether HSC-I or HSC-iM have differing functional capacity or biases to generate downstream progeny, raw RNA sequencing data from the CD34+CD38-CD45RA- (HSC/Progenitor) scMultiome was combined with scRNA-seq data from CD33+ mature myeloid cells isolated from those same xenografts. This allowed us to examine the differentiation output of clonally defined HSC-I and HSC-iM. HSC-iM progeny, including monocytes and dendritic cells, retain transcriptional features of HSC-iM including inflammatory signaling pathways, while HSC-I do not. From a biological viewpoint, this is a step forward to addressing a conundrum in the field: many of the mature myeloid cells that respond to inflammation are extremely short-lived raising the question of how long-term inflammation can be sustained and drive the deleterious phenotypes associated with CH. Our data suggests that HSC-iM continuously produces inflammatory primed myeloid cells nominating HSC-iM as a novel cellular player in exacerbating systemic inflammation. In sum, the discovery of HSC-iM implicates the importance of the inflammatory milieu and provides a framework to address whether HSC-iM emerges as a ‘cost’ of adaptation to inflammation that modifies wildtype HSC fitness to drive selective advantage of CH-bearing HSC clones. Citation Format: Stephanie Xie. Inflammatory memory and selective advantage in human clonal hematopoiesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr SY02-01.

Hematopoietic stem cell (HSC) function is tightly regulated by cytokine signaling. Although phospho-flow cytometry allows us to study signaling in defined populations of cells, there has been tremendous hurdle to carry out this study in rare HSCs due to unrecoverable critical HSC markers, low HSC number, and poor cell recovery rate. Here, we overcame these difficulties and developed a "HSC phospho-flow" method to analyze cytokine signaling in murine HSCs at the single-cell level and compare HSC signaling profile to that of multipotent progenitors (MPPs), a cell type immediately downstream of HSCs, and commonly used Lin(-) cKit(+) cells (LK cells, enriched for myeloid progenitors). We chose to study signaling evoked from three representative cytokines, stem cell factor (SCF) and thrombopoietin (TPO) that are essential for HSC function and granulocyte macrophage-colony-stimulating factor (GM-CSF) that is dispensable for HSCs. HSCs display a distinct TPO and GM-CSF signaling signature from MPPs and LK cells, which highly correlates with receptor surface expression. In contrast, although majority of LK cells express lower levels of cKit than HSCs and MPPs, SCF-evoked ERK1/2 activation in LK cells shows a significantly increased magnitude for a prolonged period. These results suggest that specific cellular context plays a more important role than receptor surface expression in SCF signaling. Our study of HSC signaling at the homeostasis stage paves the way to investigate signaling changes in HSCs under conditions of stress, aging, and hematopoietic diseases.

Activation of OCT4 Enhances Ex Vivo Expansion of Phenotypically Defined and Functionally Engraftable Human Cord Blood Hematopoietic Stem and Progenitor Cells By Regulating HOXB4 Expression

Ex Vivo Expanded Hematopoietic Stem Cells Overcome the MHC Barrier in Allogeneic Transplantation

Ex Vivo Expansion of Human Hematopoietic Stem Cells Via Lipid Nanoparticle-Mediated HOXB4 mRNA Delivery

The bone marrow (BM) contains the major reservoir of immature and maturing hematopoietic and immune cells throughout adult life while also harboring most hematopoietic stem and progenitor cells (HSPCs). BM-retained hematopoietic stem cells (HSCs) are mostly maintained in a quiescent, non-motile mode. A small fraction of BM-retained HSPC daily proliferate, differentiate, and migrate to the circulation, to replenish the blood with new immature and maturing blood and (all) immune (both myeloid and lymphoid) cells with a finite life span.1 The anti-coagulation and anti-inflammatory receptor EPCR is also functionally expressed by primitive BM-retained HSCs which are endowed with the highest competitive long-term repopulation potential (LT-HSC). Only BM-retained, quiescent EPCR-positive LT-HSCs are protected from DNA damaging insults including clinical chemotherapy and radiation treatments. Primitive EPCR-positive LT-HSC chemotherapy resistance requires the CXCL12-CXCR4 axis that also regulates HSC quiescence, cell cycle, and directional migration as well as the aPC/EPCR/PAR1 axis.2,3 The chemokine CXCL12 is highly expressed by many BM endothelial and stromal cells types including HSC niche supporting osteoprogenitor cells termed CXCL12 abundant reticular cells (CAR cells), while primitive EPCR-positive fetal liver and adult BM HSC functionally express its major receptor CXCR4.4 Most functional HSC studies involve mice experimental preclinical models as well as results obtained from clinical BM transplantation protocols. During fetal development, HSCs migrate from the fetal liver to the fetal BM and spleen for their lodgment and repopulation. Stem cell homing to the BM during development and in functional experimental transplantation assays including with human HSC in transplanted immune deficient mice is CXCL12/CXCR4-dependent.5,6 The precise timepoint of HSC colonization of the human fetal BM and spleen during pregnancy, as well as the identity of BM stromal niche cells and ligand–receptor interactions which regulate human BM HSC lodgment during fetal development are poorly understood. The group of Zheng et al7 have recently reported on the emergence of HSC in the human fetal BM and spleen and their landscape microenvironment during pregnancy by single-cell RNAseq analysis. This work revealed that functional, human fetal LT-HSC as assayed in transplanted immune deficient mice do not emerge before week 12 post conception. By careful examination of various types of fetal BM stromal and endothelial cells during weeks 10–14 post conception and their transcriptome, this study revealed that fetal BM CAR-like cells endowed with the high levels of CXCL12 production together with fetal endothelial cells which also highly express CXCL12 are the major LT-HSC niche supporting cells. Interestingly, the human fetal spleen was not repopulated with functional human LT-HSC before week 14 post conception, revealing that fetal BM is superior in providing critical signals for LT-HSC homing and lodgment over the fetal spleen. Importantly, self-renewing human CD146 positive osteoprogenitor CAR-like cells were detected in adult human BM and were found to provide a niche for adult human LT-HSC as well as in a miniature human bone implanted in immune deficient mice.8 In conclusion, this study provides evidence for the first functional fetal BM LT-HSC as well as their BM supporting microenvironment and niches.

Are transplantable stem cells required for adult hematopoiesis?

Antagonizing PPARγ Expands Human Hematopoietic Stem and Progenitor Cells By Switching on FBP1-Repressed Glycolysis and Preventing Differentiation

CD43 but Not CD41 Marks the First Hematopoietic Stem Cells in the Human Embryo