Hematopoietic Stem Cell Population Research Articles

In This Issue

B cell–depleting therapies impair antibody responses in 2-dose series of mRNA vaccination against SARS-CoV-2, urging research on additional protection against COVID-19 in patients with B-cell malignancies. Greenberger et al. characterize T-cell responses after 2 doses of mRNA vaccines, and antibody responses to a 3rd vaccine dose, in a real-world cohort of over 1,000 patients with hematologic cancers. The third dose boosted antibody titers of patients seropositive after 2 doses, with 97% reaching physiologically meaningful titers. Among the 40% of patients who were seronegative after two doses, the third dose elicited antibodies in quantities sufficient for virus neutralization in approximately one in five cases. T-cell responses to the 2nd vaccine dose in 514 patients correlated with humoral immunity but were less impacted by anticancer treatments.See article, p. 481.Although BCMA-directed CAR T cell therapy (BCMA-CART) leads to high rates of response in myeloma, many of these patients experience disease relapse. There is an urgent unmet need to identify determinants of durable responses. Dhodapkar, Cohen et al. combine several high-dimensional tools to analyze tumor and immune cells in the bone marrow of myeloma patients receiving BCMA-CART. They identify distinct features of tumor and immune cells both at baseline and following therapy that correlate with durability of clinical responses. These data illustrate dynamic cross-talk between CAR T cells, endogenous immunity, and residual tumors, and suggest novel strategies to improve durability of CAR T-cell therapy in myeloma.See article, p. 490.The functional precision medicine approach screens patient cell sensitivity to hundreds of drugs in a dish in order to identify a drug that kills cancer cells but not normal cells. The results are scored based on cancer markers by clinicians, limiting scalability. In a computational screen, Heinemann, Kornauth et al. identified a neural network distinguishing cancer cells from stroma by morphology features. When compared to marker-based scoring in a clinical trial, concordant recommendations by both approaches had best outcomes. In cases with discordant recommendations, progression-free survival was 144 days in deep morphology learning–concordant cases as compared to 72 days in marker-concordant therapy cases.See article, p. 502.In the past decade, numerous revolutionary therapeutic options were made available for adults with acute myeloid leukemia (AML) but direct extrapolation to children is particularly challenging due to the marked differences in leukemia genetics between young and old. Wang, Chan, Cheng, Ng et al. performed integrated drug and genomic profiling of patient biopsies and linked to clinical parameters to build the functional genomic landscape of childhood AML, representing a high-dimensional data set to characterize the biological uniqueness of the disease. Age-specific differences in drug response, new gene-drug interactions, and predictive biomarkers were identified. Feasibility of functional precision medicine-guided treatment of children with high-risk AML was successfully demonstrated in the clinic, marking a new approach to manage difficult cases.See article, p. 516.Myeloblast expansion is a clinical hallmark of myeloid neoplasms. Ferrall-Fairbanks, Dhawan et al. resolve myeloblasts and other hematopoietic stem and progenitor cell (HSPC) populations in chronic myelomonocytic leukemia (CMML) by high-dimensional single-cell characterization of sorted CD34+ HSPCs from 39 patients at diagnosis. They identify differentiation trajectories with three biases: normal, megakaryocyte-erythroid, and monocytic. The latter associates with adverse outcomes and is phenotypically characterized by stem cell marker depletion, CD120b+, and increased diversity and activation of cytokine receptor signaling. The monocytic bias is diminished in CMML patients following hypomethylating therapy, and is instigated by inflammation in preclinical mouse models.See article, p. 536.Mutations in the splicing factor SF3B1 are commonly found in patients with myelodysplastic syndrome with ringed sideroblasts (MDS-RS), but it is unknown how the dysregulated downstream iron metabolism pathways disrupt erythroid differentiation. Adema, Ma et al. leverage single-cell transcriptomics in SF3B1-mutant MDS-RS patient samples to identify buildup of late-stage erythroid progenitor cell types, which upregulate EIF2AK1-associated heme deficiency stress response and downstream autophagy genes. Knockout of EIK2AK1 in SF3B1-mutant erythroblasts and MDS-RS cells, as well as response to hypomethylating therapy in patients, was associated with rescue of erythroid progenitor differentiation, reduced autophagy, and increased heme metabolism gene expression, suggesting targeting of the EIF2AK1 pathway as a therapeutic strategy for MDS-RS.See article, p. 554.

Prenatal inflammation perturbs murine fetal hematopoietic development and causes persistent changes to postnatal immunity.

Adult hematopoietic stem and progenitor cells (HSPCs) respond directly to inflammation and infection, causing both acute and persistent changes to quiescence, mobilization, and differentiation. Here we show that murine fetal HSPCs respond to prenatal inflammation in utero and that the fetal response shapes postnatal hematopoiesis and immune cell function. Heterogeneous fetal HSPCs show divergent responses to maternal immune activation (MIA), including changes in quiescence, expansion, and lineage-biased output. Single-cell transcriptomic analysis of fetal HSPCs in response to MIA reveals specific upregulation of inflammatory gene profiles in discrete, transient hematopoietic stem cell (HSC) populations that propagate expansion of lymphoid-biased progenitors. Beyond fetal development, MIA causes the inappropriate expansion and persistence of fetal lymphoid-biased progenitors postnatally, concomitant with increased cellularity and hyperresponsiveness of fetal-derived innate-like lymphocytes. Our investigation demonstrates how inflammation in utero can direct the output and function of fetal-derived immune cells by reshaping fetal HSC establishment.

Role of reactive oxygen species in regulating 27-hydroxycholesterol-induced apoptosis of hematopoietic progenitor cells and myeloid cell lines

Oxysterols are oxygenated derivatives of cholesterol that contain an additional hydroxy, epoxide, or ketone group in the sterol nucleus and/or a hydroxyl group in the side chain of the cholesterol molecule. 27-Hydroxycholesterol (27HC) is a side-chain oxysterol that is oxygenated at the 27th carbon atom of cholesterol. The oxysterol (27HC) is produced via oxidation by sterol 27-hydroxylase (CYP27A1) and metabolized via oxysterol 7a-hydroxylase (CYP7B1) for bile acid synthesis in the liver. A previous study has demonstrated that treatment with the alternative Estrogen receptor alpha (ERα) ligand 27HC induces ERα-dependent hematopoietic stem cell (HSC) mobilization. In addition, Cyp27a1-deficient mice demonstrate significantly reduced 27HC levels and HSC mobilization. Here, we report that exogenous 27HC treatment leads to a substantial reduction in the hematopoietic stem and progenitor cell (HSPC) population owing to significantly increased reactive oxygen species (ROS) levels and apoptosis in the bone marrow (BM). However, 27HC does not influence the population of mature hematopoietic cells in the BM. Furthermore, exogenous 27HC treatment suppresses cell growth and promotes ROS production and apoptosis in leukemic cells. Moreover, acute myeloid leukemia (AML) patients with high CYP7B1 expression (expected to have inhibition of 27HC) had significantly shorter survival than those with low CYP7B1 expression (expected to have an elevation of 27HC). Single-cell RNA-sequencing (scRNA seq) analysis revealed that the expression of CYP7B1 was significantly increased in AML patients. Thus, our study suggests that 27HC may serve as a potent agent for regulating pools of HSPCs and may have an application as a novel therapeutic target for hematological malignancies. Collectively, pharmacological inhibition of CYP7B1 (expected to have an elevation of 27HC) would potentially have fewer long-term hematological side effects, particularly when used in combination with chemotherapy or radiation for the treatment of leukemia patients.

Criteria for Accepting or Rejecting Cord Blood Units

Rare hematopoietic stem cell populations are responsible for the transplantation engraftment process. Umbilical cord blood (UCB) is usually processed to the total nucleated cell (TNC), but not to the mononuclear cell (MNC) fraction. TNC counts are used to determine UCB unit storage, release for transplantation and correlation with time to engraftment [1]. It has been mentioned in the literature that there are several factors that affect harvesting of cord blood stem cells [2]. It was set to analyze the relationship between fetal weight and other factors that led to rejection of CBUs brought to DCRC from public and private donations. In this retrospective study, 375 donations of CBUs from January 2018 to October 2018 at DCRC were recorded. From literature, factors that are usually involved in rejection of CBUs were recorded and analyzed to mark the leading cause of rejection criteria. In this study, it showed that results did not find infants’ birth weight or mother’s age to have any value in rejection. Other factors’ results will be discussed.

Ubiquitin E3 ligase FBXO21 regulates cytokine-mediated signaling pathways, but is dispensable for steady-state hematopoiesis

Hematopoietic cell fate decisions such as self-renewal and differentiation are highly regulated through multiple molecular pathways. One pathway, the ubiquitin proteasome system (UPS), controls protein levels by tagging them with polyubiquitin chains and promoting their degradation through the proteasome. Ubiquitin E3 ligases serve as the substrate-recognition component of the UPS. By investigating the FBOX family of E3 ligases, we discovered that Fbxo21 was highly expressed in the hematopoietic stem and progenitor cell (HSPC) population, and exhibited low to no expression in mature myeloid populations. To determine the role of FBXO21 on HSPC maintenance, self-renewal, and differentiation, we generated shRNAs against FBXO21 and a hematopoiesis-specific Fbxo21 conditional knockout (cKO) mouse model. We found that silencing FBXO21 in HSPCs led to a loss in colony formation and an increase in cell differentiation in vitro. Additionally, stressing the HSPC populations in our Fbxo21 cKO mouse with 5-fluorouracil injections resulted in a decrease in survival, despite these populations exhibiting minimal alterations during steady-state hematopoiesis. Although FBXO21 has previously been proposed to regulate cytokine signaling via ASK and p38, our results indicate that depletion of FBXO21 led to altered ERK signaling in vitro. Together, these findings suggest ubiquitin E3 ligase FBXO21 regulates HSPCs through cytokine-mediated pathways.

Fate mapping of hematopoietic stem cells reveals two pathways of native thrombopoiesis

Hematopoietic stem cells (HSCs) produce highly diverse cell lineages. Here, we chart native lineage pathways emanating from HSCs and define their physiological regulation by computationally integrating experimental approaches for fate mapping, mitotic tracking, and single-cell RNA sequencing. We find that lineages begin to split when cells leave the tip HSC population, marked by high Sca-1 and CD201 expression. Downstream, HSCs either retain high Sca-1 expression and the ability to generate lymphocytes, or irreversibly reduce Sca-1 level and enter into erythro-myelopoiesis or thrombopoiesis. Thrombopoiesis is the sum of two pathways that make comparable contributions in steady state, a long route via multipotent progenitors and CD48hi megakaryocyte progenitors (MkPs), and a short route from HSCs to developmentally distinct CD48−/lo MkPs. Enhanced thrombopoietin signaling differentially accelerates the short pathway, enabling a rapid response to increasing demand. In sum, we provide a blueprint for mapping physiological differentiation fluxes from HSCs and decipher two functionally distinct pathways of native thrombopoiesis.

Bioengineered 3D microfibrous-matrix modulates osteopontin release from MSCs and facilitates the expansion of hematopoietic stem cells.

The osteopontin (OPN) released from mesenchymal stem cells (MSCs) undergoing lineage differentiation can negatively influence the expansion of hematopoietic stem cells (HSCs) in coculture systems developed for expanding HSCs. Therefore, minimizing the amount of OPN in the coculture system is important for the successful ex vivo expansion of HSCs. Towardthis goal, a bioengineered three dimensional (3D) microfibrous-matrix that can maintain MSCs in less OPN-releasing conditions has been developed, and its influence on the expansion of HSCs has been studied. The newly developed 3D matrix significantly decreased the release of OPN, depending on the MSC culture conditions used during the priming period before HSC seeding. The culture system with the lowest amount of OPN facilitated a more than 24-fold increase in HSC number in 1 week time period. Interestingly, the viability of expanded cells and the CD34+ pure population of HSCs were found to be the highest in the low OPN-containing system. Therefore, bioengineered microfibrous 3D matrices seeded with MSCs, primed under suitable culture conditions, can be an improved ex vivo expansion system for HSC culture.

Carbon dots for the treatment of cancer-related anemia.

Carbon dots for the treatment of cancer-related anemia.

Refining the migration and engraftment of short-term and long-term HSCs by enhancing homing-specific adhesion mechanisms

AbstractIn contrast to the short-term (ST) CD34+ stem cells, studies have suggested that long-term (LT) hematopoietic stem cells (HSCs) found in the CD34− stem cell pool have trouble migrating and engrafting when introduced through IV. To understand why these deficiencies exist, we set out to fully elucidate the adhesion mechanisms used by ST and LT-HSCs to migrate to the bone marrow(BM). Specifically focusing on murine ST-HSCs (Flk2−CD34+) and LT-HSCs (Flk2−CD34−), we observed a distinctive expression pattern of BM homing effectors necessary for the first step, namely sialyl Lewis-X (sLex) (ligand for E-selectin), and the second step, namely CXCR4 chemokine receptor (receptor for SDF-1). sLex expression was higher on Flk2−CD34+ ST-HSCs (>60%) compared with Flk2−CD34− LT-HSCs (<10%), which correlated to binding to E-selectin. Higher concentrations of CXCR4 were observed on Flk2−CD34+ ST-HSCs compared with Flk2−CD34− LT-HSCs. Interestingly, the expression of CD26, a peptidase known to deactivate chemokines (ie, SDF-1), was higher on Flk2−CD34− LT-HSCs. Given that both E-selectin–binding and CXCR4-mediated migration are compromised in Flk2−CD34− LT-HSCs, we aimed to enhance their ability to migrate using recombinant human fucosyltransferase 6 (rhFTVI) and the CD26 inhibitor, Dip A (diprotin A). To this end, we observed that although LT-HSCs expressed low concentrations of sLex, they were able to engraft when transplanted into recipient mice. Moreover, although both CD26 inhibition and fucosylation enhanced migration of both HSC populations in vitro, only pretreatment of LT-HSCs with Dip A enhanced engraftment in vivo after transplantation into recipient mice. Remarkably, fucosylation of Flk2−CD34+ ST-HSCs consistently led to their ability to transplant secondary recipients. These data suggest that using fucosylation and Dip A to overcome the molecular disparity in adhesion mechanisms among ST-HSCs and LT-HSCs differentially influences their abilities to migrate and engraft in vivo and promotes the ability of ST-HSCs to engraft secondary recipient mice, the gold standard for testing functionality of LT-HSCs.

P748: SINGLE CELL GENOTYPING OF MATCHED BONE MARROW AND PERIPHERAL BLOOD CELLS IN TREATMENT NAIVE AND AZA-TREATED MDS AND CMML

Background: Somatic mutations in hematopoietic stem cells (HSCs) are a central pathogenic event in myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) where they induce proliferative advantages and impaired differentiation with subsequent cytopenias in peripheral blood (PB). Patients with high-risk disease who are ineligible for allogenic stem cell transplantation are treated with hypomethylating agents, including 5-azacytidine (AZA). AZA treatment can improve PB counts and delay progression to Akute Myeloid Leukemia. However, AZA response does not require eradication of mutated HSCs and may be related to improved differentiation capacity of mutated hematopoietic stem and progenitor cells (HSPCs). However, the contribution of mutated HSPCs to steadystate hematopoiesis in MDS and CMML is unclear. Aims: In this project, we aimed to determine the distribution of individual subclones and even wild-type cells within the HSPC-compartment and the contribution of these subclones and wild-type cells to effective hematopoiesis in patients with MDS or CMML. Methods: We used a combination of index sorting and single cell genotyping to characterize the somatic mutations of individual stem- and progenitor cells and matched high-turnover circulating cells in three patients, one treatment naïve and two AZA-treated. Using this approach, myeloid driver mutations previously detected in a traditional approach were tracked in HSC/multipotent progenitors (MPP), MDS stem cells MDS-SC, progenitor cells (common myeloid progenitor (CMP), granulocyte monocyte progenitor (GMP), megakaryocyte erythroid progenitor (MEP)), as well as in monocytes, neutrophils and naïve B cells (nBC). Results: Patient H198302 and Patient H198303 both had CMML and had been treated with AZA for ̴10 years with complete response. Patient H198304 had MDS-EB1 and has never been treated with hypomethylating agents. For each patient, the figure shows the proportions of cells across the haematopoietic hierarchy carrying zero, one, two, three, or four mutations in the specified alleles. Only very small numbers of HSPCs carrying no mutation could be detected. In all three patients, most stem and progenitor cells carried a majority of tracked mutations, with no major differences between stem cells, progenitor cells and MDS-stem cells. In one patient, H198304, MDS-stem cells had a higher proportion of cells with 3 or 4 mutations detected. Overall, in all three patients, a similar frequency of mutations was observed in differentiated monocytes and neutrophils with a substantial proportion of the circulating cells derived from highly mutated progenitors. One notable exception were nBCs in Patient H198304, which were mostly wild-type, suggesting that the small wildtype HSC population is the predominant origin of nBCs in this individual. Analysis of additional lymphoid populations in PB from this patient revealed that naïve T, but not NK cells were also predominantly wild-type, suggesting specific impairment of B- and T-lineage maturation in the mutated cells. Image:Summary/Conclusion: In conclusion, attrition of highly mutated cells during myeloid maturation was not observed in any of the three patients, irrespective of HMA therapy. This suggests that in vivo, highly mutated stem and progenitor cells retain the capacity to differentiate to mature myeloid and in some patients mature lymphoid cells and contribute significantly to circulating blood cells in MDS and CMML, prior to and following AZA treatment.

Abstract 3159: CUX1 dosage-dependent regulation of hematopoietic stem and progenitor cells

Abstract The mechanism by which stem and progenitor cells commit to proliferate, self-renew, or differentiate towards different lineages remains unclear. Understanding this process is essential to decipher both normal tissue development and aberrant differentiation associated with malignant transformation. CUX1 is a non-clustered HOX-family transcription factor that is recurrently mutated in hematopoietic and solid tumours and regulates cell fate in multiple developmental systems. We reported that CUX1 is required for normal hematopoietic homeostasis of both murine and human hematopoietic stem and progenitor cells (HSPCs). To date, the transcriptional role of CUX1 in HSPCs has been unclear, partly because of the complexity of the Cux1 locus. Namely, Cux1 shares exons with a distinct gene, Casp, which encodes a highly expressed golgi-associated protein. As most transcriptomic studies combine Casp and Cux1 transcripts, the nuances of Cux1 expression are concealed. To identify the transcriptional mechanism by which CUX1 regulates HSPC functions, we compared scRNA-seq data of Kit+lin- HSPCs from transgenic mice expressing inducible shRNAs targeting Cux1, Cux1mid and Cux1low, to control counterparts expressing an shRNA targeting renilla luciferase. We demonstrate that separating Cux1 from Casp transcripts provides more power to detect the influence of Cux1 in scRNA-seq data analysis. While cell numbers within most HSPC sub-populations are largely unchanged after five days of Cux1 knockdown, the transcriptomes within multiple clusters are disrupted. The effect size is greatest in Cux1low cells, compared to Cux1mid, consistent with dosage dependent roles of CUX1 in HSPCs. Across sub-populations, Cux1 levels correlated with gene signatures of “stemness,” and Cux1 knockdown disrupted this gene signature in the core hematopoietic stem cell (HSC) population. Concomitantly, low levels of Cux1 led to upregulation of cellular metabolism and G2/M cell cycle transition pathways in core HSCs, indicating that CUX1 regulates stem cell quiescence at the hematopoietic apex. We are currently integrating this data with epigenetic datasets to determine the mechanism by which CUX1 regulates critical hematopoietic transcriptional programs. Overall, understanding the regulatory function of CUX1 will provide insight into how HSCs commit to different fates and may ultimately lead to novel cancer therapeutic interventions. Citation Format: Weihan Liu, Stephanie Konecki, Tanner Martinez, Megan McNerney. CUX1 dosage-dependent regulation of hematopoietic stem and progenitor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3159.

Bioinformatics analysis of molecular pathways and key candidate biomarkers associated with human bone marrow hematopoietic stem cells (HSCs) micro-array gene expression data

An identification of the molecular properties of glioma stem cells (GSCs) has led studies in clinical use, stem cell identification, and highly-effective usage. The GSE32719 contains a total expression of 54,676 genes of healthy human bone marrow HSCs in 14 young (20–31 years), 5 middle (42–61 years), and a lot of old (65–85 years) age groups. The researchers of this study described age-related changes in the human HSC population, using the gene expression profile of significance analysis to discover differentially expressed genes (DEGs) between each age group. The DEGs were subjected to significantly enriched biological processing that decoded the increase and functional decline in the HSC population. The GSE dataset analysis was conducted by the GEOquery package in Bioconductor. Using the Biobase and gplots packages, 453 DEGs were screened. DEGs analyses were conducted by gene ontology (GO) pathway enrichment and Kyoto Encylopedia of Genes and Genomes (KEGG) enrichment analysis. The Hippo signaling pathway was observed to be significant using the GO pathway enrichment analysis, which was previously reported as an effective pathway in cancers, including AML. A protein-protein interaction (PPI) network was constructed; then based on that, a subnetwork module analysis for the Hippo signaling pathway was made. Additionally, the GO pathway enrichment analysis revealed ‘cellular process’, ‘cellular metabolic process’, ‘metabolic process’, ‘biogenesis’, and ‘vasculogenesis biological processes’, which are involved in a wide of biological activities such as metabolic regulation, cell growth, and proliferation. Our findings offer silico evidence for candidate genes, such as the UBC, PTK2, and TCF7L2, that may be promising biomarkers for the translation approach associated with HSC population age-related diseases.

The Role of Ubiquitin E3 Ligase, FBOX21, in Regulating Healthy and Malignant Hematopoietic Stem and Progenitor Cells (HSPC)

Hematopoietic stem cells allow for the formation of all cell types in the blood and maintain these populations throughout a person’s life. Hematopoietic cell fate decisions such as self‐renewal and differentiation are highly regulated through multiple molecular pathways and dysregulation can lead to the initiation of malignant hematopoietic disorders like acute myeloid leukemia (AML), immune deficiencies, and anemia. One pathway that regulates hematopoietic cell fate decisions is the ubiquitin proteasome system (UPS). The UPS controls protein levels by tagging them with polyubiquitin chains and promoting their degradation through the proteasome. The substrate recognition component, the protein that decides which substrate will be chosen for degradation, of the UPS is the ubiquitin E3 ligase. Through investigating a specific family of ubiquitin E3 ligases, the Fbox family of proteins, we discovered that Fbxo21was highly expressed in the hematopoietic stem and progenitor (HSPC) population, and was more highly expressed in this population when compared to other Fbox genes suggesting a possible role in maintaining the HSPC population. Likewise, Western blot confirmed high levels of FBXO21 in HSPCs and revealed low to no protein in mature myeloid populations. To determine the role of FBXO21 on HSPC maintenance, self‐renewal, and differentiation, we generated shRNAs against both Fbxo21 and FBXO21. We found that silencing Fbxo21/FBXO21 in healthy HSPCs and in human AML cell lines lead to a loss in colony formation, a decrease in proliferation, and an increase in differentiation towards the mature myeloid lineage. While overexpression of FBXO21 lead to an increase in colony formation and proliferation. This leads us to hypothesize that the ubiquitin E3 ligase, FBXO21, maintains the hematopoietic stem cell population while loss of Fbxo21/FBXO21 leads to a promotion in myeloid differentiation.We then generated the first conditional Fbxo21 knockout (KO) mouse model to delete Fbxo21 in hematopoietic cells. Our Fbxo21 KO mice exhibited no significant difference in primary sites of hematopoietic development, but display changes in hematopoietic cell populations in both young and aged mice. To further identify the substrates and pathway of FBXO21, we performed TMT Mass Spectrometry to analyze changes in protein levels upon knockdown of Fbxo21/FBXO21 in healthy HSPC and human AML cell lines. We found that both populations displayed an increase in proteins involved in the inflammatory response, the immune response primarily maintained by the myeloid cells through cytokine signaling. Which among both our Fbxo21 KO mice and human AML cell lines with FBXO21 knocked down, we see alterations in the cell’s response to certain cytokines, including IL‐6 and G‐CSF, upon Fbxo21/FBXO21 being knocked down. Therefore, deciphering the role of FBXO21 could expand the current known molecular mechanisms that regulate hematopoietic lineage specification and stem cell maintenance.

Inflammation and accompanied disrupted hematopoiesis in adult mouse induced by rare earth element nanoparticles

Rare earth element nanoparticles (REE NPs) or agents have been used extensively in various fields. Human exposure to REE NPs is an increasing concern. To date, REE NP-mediated comprehensive immune responses after incorporation into the body remain unclear. In our study, using gadolinium oxide NPs (Gd2O3) as a typical REE NP, we systematically investigated immune responses in vivo. The liver and spleen were the main sites where Gd2O3 retained and accumulated, while Gd2O3 content per unit tissue mass in the spleen was 4.4 times higher than that in the liver. Gd2O3 increased the number of monocyte-derived macrophages and myeloid-derived dendritic cells (M-DCs) in the liver. In the spleen, Gd2O3 caused infiltration of neutrophils, M-DCs, and B cells. The accumulation of Gd2O3 in the liver or spleen also contributed to an increased concentration of cytokines in peripheral blood. In both the bone marrow and spleen, Gd2O3 led to increased populations of hematopoietic stem cells (HSCs), multipotent progenitors, and common lymphoid progenitors. Compared to the decreased monocytes in peripheral blood on day 2, a significant decrease of circulating lymphocytes on day 7 was still observed, suggesting the exposure duration led to variable effects. This might be explained by the sustained accumulation of Gd2O3 in the liver and spleen. Together, our study systemically depicted the alterations in mature immune alterations together with hematopoiesis in both myeloid and lymphoid lineages induced by Gd2O3 exposure. Our findings will facilitate a comprehensive understanding of the interactions of immune system with REE NPs in vivo.

Single cell analyses identify a highly regenerative and homogenous human CD34+\u2009hematopoietic stem cell population

The heterogeneous nature of human CD34+ hematopoietic stem cells (HSCs) has hampered our understanding of the cellular and molecular trajectories that HSCs navigate during lineage commitment. Using various platforms including single cell RNA-sequencing and extensive xenotransplantation, we have uncovered an uncharacterized human CD34+ HSC population. These CD34+EPCR+(CD38/CD45RA)− (simply as EPCR+) HSCs have a high repopulating and self-renewal abilities, reaching a stem cell frequency of ~1 in 3 cells, the highest described to date. Their unique transcriptomic wiring in which many gene modules associated with differentiated cell lineages confers their multilineage lineage output both in vivo and in vitro. At the single cell level, EPCR+ HSCs are the most transcriptomically and functionally homogenous human HSC population defined to date and can also be easily identified in post-natal tissues. Therefore, this EPCR+ population not only offers a high human HSC resolution but also a well-structured human hematopoietic hierarchical organization at the most primitive level.

Translocation of Helicobacter hepaticus synergizes with myeloid-derived suppressor cells and contributes to breast carcinogenesis

ABSTRACT Microbial dysbiosis plays an important role in the development of intestinal diseases. Recent studies suggest a link between intestinal bacteria and mammary cancer. Here, we report that female ApcMin/+ mice infected with Helicobacter hepaticus exhibited an increased mammary and small/large intestine tumor burden compared with uninfected littermates. H. hepaticus DNA was detected in small/large intestine, mammary tumors, and adjacent lymph nodes, suggesting a migration pathway. CD11b+Gr1+ myeloid-derived suppressor cells (MDSCs) infiltrated and expressed high levels of Wnts, likely enhancing tumorigenesis through activation of Wnt/β-catenin pathway. Our previous studies indicated that histidine decarboxylase (Hdc) marks a population of myeloid-biased hematopoietic stem cells and granulocytic MDSCs. Cytokines/chemokines secreted by IL-17-expressing mast cells and tumor tissues promoted Hdc+ MDSCs expansion and trafficking toward mammary tumors. Adoptive transfer of MDSCs isolated from H. hepaticus-infected mice increased MDSCs frequencies in peripheral blood, mesenteric lymph nodes, mammary gland, and lymph nodes in recipient ApcMin/+ mice. The adoptive transfer of H. hepaticus primed MDSCs also increased the size and number of mammary tumors. Our results demonstrate that H. hepaticus can translocate from the intestine to mammary tissues to promote mammary tumorigenesis with MDSCs. Targeting bacteria and MDSCs may be useful for the prevention and therapy of extraintestinal cancers. Abbreviations: Helicobacter hepaticus, Hh; myeloid-derived suppressor cell, MDSC; histidine decarboxylase, Hdc; Breast cancer, BC; T regulatory, TR; inflammatory bowel disease, IBD; fluorescence in situ hybridization, FISH; myeloid-biased hematopoietic stem cells, MB-HSCs; granulocytic MDSCs, PMN-MDSCs; Lipopolysaccharide, LPS; Toll-like receptors, TLRs; Mast cells, MCs; Granulocyte-macrophage colony-stimulating factor, GM-CSF; epithelial–mesenchymal transition, EMT; Intestinal epithelial cells, IECs.

The Reconstitution Dynamics of Cultivated Hematopoietic Stem Cells and Progenitors Is Independent of Age.

Hematopoietic stem cell transplantation (HSCT) represents the only curative treatment option for numerous hematologic malignancies. While the influence of donor age and the composition of the graft have already been examined in clinical and preclinical studies, little information is available on the extent to which different hematological subpopulations contribute to the dynamics of the reconstitution process and on whether and how these contributions are altered with age. In a murine model of HSCT, we therefore simultaneously tracked different cultivated and transduced hematopoietic stem and progenitor cell (HSPC) populations using a multicolor-coded barcode system (BC32). We studied a series of age-matched and age-mismatched transplantations and compared the influence of age on the reconstitution dynamics. We show that reconstitution from these cultured and assembled grafts was substantially driven by hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) independent of age. The reconstitution patterns were polyclonal and stable in all age groups independently of the variability between individual animals, with higher output rates from MPPs than from HSCs. Our experiments suggest that the dynamics of reconstitution and the contribution of cultured and individually transduced HSPC subpopulations are largely independent of age. Our findings support ongoing efforts to expand the application of HSCT in older individuals as a promising strategy to combat hematological diseases, including gene therapy applications.

The CCR5 Gene Edited CD34+CD90+ Hematopoietic Stem Cell Population Serves as an Optimal Graft Source for HIV Gene Therapy.

Transplantation of allogenic hematopoietic stem and progenitor cells (HSPCs) with C-C chemokine receptor type 5 (CCR5) Δ32 genotype generates HIV-1 resistant immune cells. CCR5 gene edited autologous HSPCs can be a potential alternative to hematopoietic stem cell transplantation (HSCT) from HLA-matched CCR5 null donor. However, the clinical application of gene edited autologous HSPCs is critically limited by the quality of the graft, as HIV also infects the HSPCs. In this study, by using mobilized HSPCs from healthy donors, we show that the CD34+CD90+ hematopoietic stem cells (HSCs) express 7-fold lower CD4/CCR5 HIV receptors, higher levels of SAMHD1 anti-viral restriction factor, and possess lower susceptibility to HIV infection than the CD34+CD90- hematopoietic progenitor cells. Further, the treatment with small molecule cocktail of Resveratrol, UM729 and SR1(RUS) improved the in vivo engraftment potential of CD34+CD90+ HSCs. To demonstrate that CD34+CD90+ HSC population as an ideal graft for HIV gene therapy, we sort purified CD34+CD90+ HSCs, treated with RUS and then gene edited the CCR5 with single sgRNA. On transplantation, 100,000 CD34+CD90+ HSCs were sufficient for long-term repopulation of the entire bone marrow of NBSGW mice. Importantly, the gene editing efficiency of ~90% in the infused product was maintained in vivo, facilitating the generation of CCR5 null immune cells, resistant to HIV infection. Altogether, CCR5 gene editing of CD34+CD90+ HSCs provide an ideal gene manipulation strategy for autologous HSCT based gene therapy for HIV infection.

Heterozygous Dnmt3a R878C induces expansion of quiescent hematopoietic stem cell pool

Somatic mutation of DNMT3A (DNA methyltransferase 3 alpha) is implicated in the development of a wide range of hematological disorders, including clonal hematopoiesis of indeterminate potential. To elucidate the functional roles of endogenous levels of a DNMT3A R882 mutant, we generated a novel Dnmt3a R878C conditional knock-in mouse model. In contrast to viable heterozygotes, mice homozygous for the Dnmt3a R878C mutation in the hematopoietic system were not viable (Dnmt3a R878C is homologous to human DNMT3A R882C). Hematopoietic cell-specific heterozygous expression of Dnmt3a R878C led to significant expansion of adult quiescent hematopoietic stem cells (HSCs); however, these mice had no hematological malignancies. The expanding HSC population in heterozygous Dnmt3a R878C knock-in mice had an accumulation of G0-phase cells. In contrast to aberrantly enhanced self-renewal capacity in vitro, heterozygous Dnmt3a R878C knock-in HSCs had no competitive repopulating advantage in vivo over wild-type HSCs. Considering the capacity of the heterozygous Dnmt3a R878C mutant for HSC pool expansion, our Dnmt3a R878C knock-in mouse line is a useful platform on which to dissect the pathophysiology of clonal hematopoiesis. This mouse line can also help to elucidate the biological and molecular actions of DNMT3A mutations in the malignant transformation of normal HSCs.

Deleterious effect of bone marrow-resident macrophages on hematopoietic stem cells in response to total body irradiation

AbstractBone marrow (BM) resident macrophages interact with a population of long-term hematopoietic stem cells (LT-HSCs) but their role on LT-HSC properties after stress is not well defined. Here, we show that a 2 Gy-total body irradiation (TBI)-mediated death of LT-HSCs is associated with increased percentages of LT-HSCs with reactive oxygen species (ROS) and of BM resident macrophages producing nitric oxide (NO), resulting in an increased percentage of LT-HSCs with endogenous cytotoxic peroxynitrites. Pharmacological or genetic depletion of BM resident macrophages impairs the radio-induced increases in the percentage of both ROS+ LT-HSCs and peroxynitrite+ LT-HSCs and results in a complete recovery of a functional pool of LT-HSCs. Finally, we show that after a 2 Gy-TBI, a specific decrease of NO production by BM resident macrophages improves the LT-HSC recovery, whereas an exogenous NO delivery decreases the LT-HSC compartment. Altogether, these results show that BM resident macrophages are involved in the response of LT-HSCs to a 2 Gy-TBI and suggest that regulation of NO production can be used to modulate some deleterious effects of a TBI on LT-HSCs.