Hematopoietic Stem Cell Repopulation Research Articles

Mitochondria-Mediated (MAVS) Innate Immune Signaling Drives Inflammation-Induced Bone Marrow Failure

Bone marrow failure syndromes (BMFS) are life threatening diseases, characterized by impaired fitness of hematopoietic stem cells (HSC) and ineffective hematopoiesis causing the absence of one or more hematopoietic lineages in the peripheral blood. Substantial clinical data indicate that hyperactivity of inflammatory cytokines, including TNFα, IL-6, and transforming growth factor-β (TGFβ), directly contribute to BMF. Inflammatory challenges result in irreversible HSC functional decline, including decreased repopulation potential and myeloid-bias differentiation. We recently showed that the combination of inflammatory challenges, including polyinosinc:polycytidilic acid (pIC - a double stranded RNA [dsRNA]) plus high TGFβ causes chronic pancytopenia, anemia, expanded hematopoietic stem and progenitor cell pools, and enhanced bone marrow dysplasia, mimicking a myelodysplastic syndrome (MDS) that developed 3-4 months after pIC challenge (Javier et.al. Haematologica 2022). However, the mechanisms behind this association and/or causality are still ill-defined. Here, we show that mitochondrial antiviral signaling (MAVS) protein can mediate the negative effect of pIC and TGFβ on HSC functions and BMF/ MDS development. Differential gene expression analyses in HSCs suggested that pIC plus TGFβ altered expression of nuclear-encoded mitochondrial genes. Mitochondria serve as platform for the activation of MAVS and downstream innate immune signaling in response to dsRNA. We thus examined the role of mitochondria and MAVS in pIC plus TGFβ-induced BMF/MDS. We show that overexpression TGFβ and pIC together cause alterations in mitochondrial proteins and membrane potential as well as enhanced caspase1 activity, but not the necroptotic signals p-RIP1 and p-RIP3, in HSCs. pIC exposure caused an increase in MAVS expression, and a redistribution into large aggregates that localize onto mitochondria in HSCs, indicative of MAVS activation (two days after pIC exposure in vivo). The MAVS aggregates resolved into smaller and dispersed clusters by 3 months after pIC exposure in control mice. In contrast, in HSCs from TGFβ overexpressing mice, MAVS aggregates persisted 3 months after pIC exposure. To determine the impact of augmented MAVS in dsRNA+TGFβ-driven BMF, we used MAVS deficient mice crossed with TGFβ overexpressing mice. We found that absence of MAVS protein restored normal white blood cell count, including neutrophils and lymphocytes, restored normal red blood cell but not platelet counts in TGFβ overexpressing mice after pIC challenge, compared to controls. Remarkably, MAVS deficiency partially rescued the dysplastic features of bone marrow myeloid cells in TGFβ overexpressing mice after pIC challenge. MAVS-deficiency also reduced the increased caspase1 activity and lowers mitochondria membrane potential in HSCs of these mice. In serial competitive transplant studies, MAVS-deficiency conferred HSC repopulation advantage. These findings suggest that pIC+TGFβ overexpression mediated BMF/MDS phenotype depends on chronic activation of MAVS. Because MAVS aggregation depends on mitochondrial fusion, we used genetic loss of the mitochondrial fission regulator Drp1. Drp1-deficiency caused severe mitochondrial aggregation in HSCs; this was associated with sustained MAVS aggregation. In response to pIC, Drp1-deficient mice exhibited increased caspase-1 activity, increased mitochondrial membrane potential and increase in HSC numbers 6 months after pIC challenges. Drp1 loss resulted in anemia and mild pancytopenia. Interestingly, Drp1-deficient bone marrow myeloid progenitors exhibited severe dysplastic features after pIC challenged. These findings suggest that chronic mitochondrial and MAVS aggregation are sufficient to drive BMF/MDS phenotype. Finally, we investigated the levels of epigenetic markers (acetyl H3K27 and methyl H3 Lys4) in HSCs after pIC stress. H4K4me3 levels were decreased in HSCs long-term after pIC challenges [6 months], this was further decreased in Drp1-deficient HSCs. In conclusion, these findings indicate that non-genetic factors contribute to the onset of BMF/MDS. Mitochondria and MAVS protein together mediate an innate immune mechanism that drives BMF/MDS. This study opens the possibility to target mitochondria-related proteins as a therapeutic strategy to restore ineffective hematopoiesis due to chronic inflammation.

Latexin regulates sex dimorphism in hematopoiesis via gender-specific differential expression of microRNA 98-3p and thrombospondin 1

Hematopoietic stem cells (HSCs) have the ability to self-renew and differentiate to all blood cell types. HSCs and their differentiated progeny show sex/gender differences. The fundamental mechanisms remain largely unexplored. We previously reported that latexin (Lxn) deletion increased HSC survival and repopulation capacity in female mice. Here, we find no differences in HSC function and hematopoiesis in Lxn knockout (Lxn-/-) male mice under physiologic and myelosuppressive conditions. We further find that Thbs1, a downstream target gene of Lxn in female HSCs, is repressed in male HSCs. Male-specific high expression of microRNA 98-3p (miR98-3p) contributes to Thbs1 suppression in male HSCs, thus abrogating the functional effect of Lxn in male HSCs and hematopoiesis. These findings uncover a regulatory mechanism involving a sex-chromosome-related microRNA and its differential control of Lxn-Thbs1 signaling in hematopoiesis and shed light on the process underlying sex dimorphism in both normal and malignant hematopoiesis.

BCLAF1 Regulates Expression of AP-1 Genes and Fetal Hematopoietic Stem Cell Repopulation Activity

BCLAF1 Regulates Expression of AP-1 Genes and Fetal Hematopoietic Stem Cell Repopulation Activity

EBF1 primes B-lymphoid enhancers and limits the myeloid bias in murine multipotent progenitors.

Hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) generate all cells of the blood system. Despite their multipotency, MPPs display poorly understood lineage bias. Here, we examine whether lineage-specifying transcription factors, such as the B-lineage determinant EBF1, regulate lineage preference in early progenitors. We detect low-level EBF1 expression in myeloid-biased MPP3 and lymphoid-biased MPP4 cells, coinciding with expression of the myeloid determinant C/EBPα. Hematopoietic deletion of Ebf1 results in enhanced myelopoiesis and reduced HSC repopulation capacity. Ebf1-deficient MPP3 and MPP4 cells exhibit an augmented myeloid differentiation potential and a transcriptome with an enriched C/EBPα signature. Correspondingly, EBF1 binds the Cebpa enhancer, and the deficiency and overexpression of Ebf1 in MPP3 and MPP4 cells lead to an up- and downregulation of Cebpa expression, respectively. In addition, EBF1 primes the chromatin of B-lymphoid enhancers specifically in MPP3 cells. Thus, our study implicates EBF1 in regulating myeloid/lymphoid fate bias in MPPs by constraining C/EBPα-driven myelopoiesis and priming the B-lymphoid fate.

Axin2/Conductin Is Required for Normal Haematopoiesis and T Lymphopoiesis.

The development of T lymphocytes in the thymus and their stem cell precursors in the bone marrow is controlled by Wnt signaling in strictly regulated, cell-type specific dosages. In this study, we investigated levels of canonical Wnt signaling during hematopoiesis and T cell development within the Axin2-mTurquoise2 reporter. We demonstrate active Wnt signaling in hematopoietic stem cells (HSCs) and early thymocytes, but also in more mature thymic subsets and peripheral T lymphocytes. Thymic epithelial cells displayed particularly high Wnt signaling, suggesting an interesting crosstalk between thymocytes and thymic epithelial cells (TECs). Additionally, reporter mice allowed us to investigate the loss of Axin2 function, demonstrating decreased HSC repopulation upon transplantation and the partial arrest of early thymocyte development in Axin2Tg/Tg full mutant mice. Mechanistically, loss of Axin2 leads to supraphysiological Wnt levels that disrupt HSC differentiation and thymocyte development.

Competitive inhibition of IL-2/IL-2R has a dual effect on HSC ex vivo expansion and IL-2R (CD25) content

Interleukin-2 (IL-2) and its receptor play a pivotal role in the regulation of immune response and possess both immune-regulatory and immune-stimulatory functions. As a cytokine of lymphoid cells, the role of IL-2 has been revealed in hematopoietic stem cell (HSC) maintenance and proper hematopoiesis. Here, we investigated that small molecule Ro 26-4550 trifluoroacetate (Ro) mediated competitive inhibition of IL-2 and its receptor alpha subunit (IL-2Rα) throughout ex vivo culture. Ro treatment induced murine and human ex vivo expansion of hematopoietic stem and progenitor cells (HSPCs). Ro treated HSPCs sustained self-renewal ability and low apoptotic activity. As a competitive inhibitor of IL-2/IL-2Rα interaction, Ro small molecule induced human HSPCs to entry into cell cycle. The proliferation of bone marrow mesenchymal stem cells (MSC) and fibroblasts were also highly increased post treatment. Besides, Ro treatment enhanced IL-2Rα (CD25) expression independent of IL-2 administration in human mPB-derived HSPCs and BM-derived HSPCs. Increased IL-2Rα (CD25) expression in BM-HSPCs was associated with the increase in the CD4+CD25+ T cell population. Xenotransplantation of immunodeficient mice with ex vivo expanded human CD34+ cells after Ro treatment revealed an efficient multi-lineage reconstitution in the recipient. These findings shed light on the role of IL-2/IL-2Rα interaction in HSC expansion, in vivo and in vitro HSC self-renewal ability and repopulation capacity as well as a possible mean for the induction of CD25 expressing cells in hematopoietic compartments.

Orchestrated delivery of PTH [1-34] followed by zoledronic acid prevents radiotherapy-induced bone loss but does not abrogate marrow damage.

Postradiotherapy bone fragility fractures are a frequent late-onset complication in cancer survivors. There is a critical need to develop preventative interventions, and the use of Food and Drug Administration-approved drugs remains an attractive option. Prior data from our lab and others have shown that parathyroid hormone [1-34] mitigates radiotherapy-induced bone loss, but only for the duration of drug delivery. Utilizing a murine hindlimb radiotherapy model, we investigated whether orchestrated delivery of single-dose zoledronic acid could extend these anabolic benefits after cessation of parathyroid hormone delivery. We then explored the potential use of parathyroid hormone as a bone marrow radioprotectant. While the addition of zoledronic acid to parathyroid hormone increased irradiated bone mass, there was no increase in femur bending strength. In this model, the parathyroid hormone was not effective as a marrow radioprotectant, although this could be due to the short course of parathyroid hormone treatment. Marrow repopulation kinetics differed from those in total body irradiation, with hematopoietic stem cell repopulation occurring relatively early at four weeks postirradiation. Furthermore, we found radiation induced a loss of marrow stromal cells and an increase in inflammatory monocytes. Statement of Clinical Significance: Staged delivery of parathyroid hormone and zoledronic acid shows promise as an off-the-shelf intervention to mitigate post-radiotherapy bone damage in cancer patients, but parathyroid hormone is unlikely to function as a broad-spectrum marrow radioprotectant.

Unique Roles for Grb2-Associated Binding (Gab) Adaptors in Hematopoietic Stem Cell Homeostasis and Repopulation

Homeostasis of hematopoietic stem cells (HSCs) is essential for the continuous replenishment of the hematopoietic system throughout the entire lifespan of an organism and is a tightly regulated process, controlled by intrinsic and extrinsic signals. Although a variety of molecules controlling HSC homeostasis are known, it remains unclear how signaling is regulated. The growth factor receptor-bound protein 2 (Grb2)-associated binding (Gab) proteins are a family of Pleckstrin homology domain containing adaptor proteins that dock with receptor tyrosine kinases (RTKs) and non-RTKs to control cell activation of ERK and PI3K pathways. The murine Gab family includes Gab1, Gab2, and Gab3. Gab1 and Gab2 are expressed throughout hematopoiesis including HSCs but Gab3 is limited mostly to macrophages, dendritic cells, and T-cells. Previously we reported that Gab2 is intrinsically required for hematopoietic cell responses to early-acting cytokines, with defective hematopoiesis in Gab2-/- mice (obtained from Toshio Hirano, Osaka, Japan; Nishida K et al, Blood, 99, 2002). Little is known about redundancy among Gabs in hematopoiesis and it is possible that roles for Gab1 and Gab3 have been obscured by the prominence of Gab2. Using Gab1flox/flox (Bard-Chapeau EA et al, Nat. Med. 11, 2005) and Gab3-/- mice (obtained from Pam Hankey-Giblin, Penn. State; Seiffert M et al, Mol. Cell. Biol. 23, 2003) we set out to make compound mutant mice. Gab3 knockout mice do not have reported hematopoietic phenotypes. First, we generated Gab2/3 double knockout mice. In the peripheral blood, we observed greater B-lymphocyte and erythroid deficiency compared to wild-type or Gab2-/- mice. In non-competitive bone marrow transplantation, Gab2/3-/- bone marrow was sufficient to reconstitute relatively normal myeloid hematology in irradiated recipients but resulted in significantly reduced donor derived B- and T-lymphocytes relative to wild type or Gab2-/- donor bone marrow cells, which were the same. In competitive bone marrow transplantation against Boy J (CD45.1) we observed sharp reductions in multilineage engraftment of Gab2/3-/- (13-15 fold) compared to Gab2-/- (4-6 fold) mice while Gab3-/- mice were normal. These data combined with the limited expression pattern for Gab3 suggest a novel critical extrinsic role in hematopoiesis. Despite severe multilineage engraftment defects, we surprisingly observed that Gab2/3-/- mice had significantly increased numbers of KLS cells (~2-fold) as well as LT-HSCs (KLSCD150+CD48-, ~3-fold) compared to the wild-type controls. Associated with the increased HSC numbers were cell cycle changes observed by staining with Pyronin Y/Hoechst 33342. During steady-state, Gab2/3-/- KLS cells were less quiescent with a lower percentage of cells in G0 (Gab2/3-/- vs WT: 40.5 ± 6.1 vs 57.4 ± 11.4, N=4, p=0.04) and more cells in G1 (Gab2/3-/- vs WT: 19.5 ± 0.7 vs 13.5 ± 3.5, N=4, p=0.01) phase of the cell cycle. Interestingly, KLS cells from Gab2/3-/- mice were less apoptotic than wild-type control (1.05 ± 0.49% in Gab2/3-/- (N=7) vs 5.97 ± 1.40% in WT KLS (N=10), p<0.001). Together these indicate that the HSCs were expanded but functionally defective. To further explore the mechanisms for Gab2/3-/- defects, we found that Gab1 expression levels were increased 2-fold. Since germline Gab1 deficiency results in embryonic lethality, Gab1 floxed alleles were deleted conditionally using Vav1-Cre to generate viable adult Gab1/2/3-/- mice. Real-time PCR confirmed the average of 94% Gab1 deletion efficiency (N=10) and Western analysis showed no detectable level of Gab1 in splenocytes. Interestingly, Gab1 deletion reversed the KLS expansion observed in the Gab2/3-/- mice indicating that Gab1 has effects that oppose Gab2/3 at the KLS level. However, Gab1/2/3-/- mice had severe defects in multilineage engraftment in competitive repopulation (39-52 fold). Altogether, our data indicate that Gab2/3 play positive roles while Gab1 plays a negative role in HSC homeostasis but all three Gabs are essential for multilineage repopulation. Further studies of Gab1 and Gab3 function in intrinsic and extrinsic regulation of HSC biology and repopulation are ongoing. DisclosuresNo relevant conflicts of interest to declare.

Osteoblastic Wntless deletion differentially regulates the fate and functions of bone marrow-derived stem cells in relation to age.

Although functional association between Wnt signaling and bone homeostasis has been well described through genetic ablation of Wntless (Wls), the mechanisms of how osteoblastic Wls regulates the fate of bone marrow stromal cells (BMSCs) and hematopoietic stem cells (HSCs) in relation to age are not yet understood. Here, we generated Col2.3-Cre;Wlsfl/fl mice that were free from premature lethality and investigated age-related impacts of osteoblastic Wls deficiency on hematopoiesis, BM microenvironment, and maintenance of BMSCs (also known as BM-derived mesenchymal stem/stromal cells) and HSCs. Ablation of osteoblastic Wls deteriorated BM microenvironment and bone mass accrual along with age-independent effects on functions of BMSCs. Osteoblastic Wls deletion impaired HSC repopulation and progeny with skewing toward myeloid lineage cells only at old stage. As proven by hallmarks of stem cell senescence, osteoblastic Wls ablation differentially induced senescence of BMSCs and HSCs in relation to age without alteration in their BM frequency. Our findings support that deletion of Wls in Col2.3-expressing cells induces senescence of BMSCs and impairs BM microenvironment in age-independent manner. Overall, long-term deterioration in BM microenvironment contributes to age-related HSC senescence with impaired progeny and hematopoiesis, which also suggests possible roles of osteoblastic Wls on the maintenance of BM HSCs.

Microphthalmia transcription factor expression contributes to bone marrow failure in Fanconi anemia.

Hematopoietic stem cell (HSC) attrition is considered the key event underlying progressive BM failure (BMF) in Fanconi anemia (FA), the most frequent inherited BMF disorder in humans. However, despite major advances, how the cellular, biochemical, and molecular alterations reported in FA lead to HSC exhaustion remains poorly understood. Here, we demonstrated in human and mouse cells that loss-of-function of FANCA or FANCC, products of 2 genes affecting more than 80% of FA patients worldwide, is associated with constitutive expression of the transcription factor microphthalmia (MiTF) through the cooperative, unscheduled activation of several stress-signaling pathways, including the SMAD2/3, p38 MAPK, NF-κB, and AKT cascades. We validated the unrestrained Mitf expression downstream of p38 in Fanca-/- mice, which display hallmarks of hematopoietic stress, including loss of HSC quiescence, DNA damage accumulation in HSCs, and reduced HSC repopulation capacity. Importantly, we demonstrated that shRNA-mediated downregulation of Mitf expression or inhibition of p38 signaling rescued HSC quiescence and prevented DNA damage accumulation. Our data support the hypothesis that HSC attrition in FA is the consequence of defects in the DNA-damage response combined with chronic activation of otherwise transiently activated signaling pathways, which jointly prevent the recovery of HSC quiescence.

Interferon Gamma, a Link between Hemophagocytic Lymphohistiocytosis and Dengue Fever Virus: A Literature Review of a Shared Inflammatory Pathway Resulting in Cytopenias and Potential Future Treatment Directions

Our patient was an elderly female, previously healthy, who presented with fever, anemia, thrombocytopenia, and enlarged spleen. Her complaints were progressive over the course of two months. She was treated with antibiotics with no improvement. Underlying lymphoproliferative disorder was suspected, and a bone marrow biopsy and aspiration was completed. While there was no evidence for lymphoproliferative disorder, there was hemophagocytosis. Further evaluation revealed ferritin 1,151 ng/mL, soluble IL2 receptor 32,186 u/mL, and natural killer activity was low. She was diagnosed with secondary hemophagocytic lymphohistiocytosis (HLH). During her evaluation, her condition acutely worsened; she became encephalopathic with hypotension requiring vasopressor support and respiratory failure. The patient was given etoposide with rapid improvement in overall clinical status though further cytopenias secondary to etoposide prevented further treatment. She was ultimately diagnosed with Dengue Fever Virus by IgM Antibody, 4.54, with positive result &gt;/= 2.84. A positive result is highly suggestive of acute disease, though cross reactivity is possible with other flavi viruses. The result was confirmed. The patient lived in a rural community in Eastern NC and had no travel history; the diagnosis was reported to the North Carolina public health department and the Center for Disease Control. Despite recognition and treatment of the HLH and Dengue fever, the patient died of complications. Both Dengue Fever and HLH cause significant morbidity and mortality. Their disease courses, while unique, do have some interesting parallels. They both can be associated with significant suppression of bone marrow function, frequently being associated with cytopenias. This is a major source of morbidity in both disease states. While Dengue Fever is a known, though rare, cause of HLH, it has not previously been reported in the continental United States. In this case report, we examine the known cytokine pathway activations in each disease state and their commonalities, focusing on bone marrow suppressive cytokines and subsequently suggesting mechanisms for targeted treatment of HLH. Even with our best treatments, in HLH, the associated mortality among all causes is around 42%. The mortality for untreated severe dengue is as high as 20% though with appropriate care, that is much improved at 1-2%. In HLH and Dengue, IL-1B, IFN-Ƴ, IL6, and TNF-α are increased. A common cytokine, IFN-Ƴ, directly affects bone marrow activity and can alter hematopoiesis. IFN-Ƴ is directly implicated in several bone marrow failure syndromes making it an attractive target to modulate. Based on results of an in vitro study of CD34+ hematopoietic stem cells (HSC) in the presence and absence of IFN-Ƴ in differing concentrations, IFN-Ƴ inhibited the ability of HSC to repopulate themselves though it did not affect their ability to differentiate. This was also shown in vivo in a mouse HSC transplant model with mirroring results. In our patient, it follows logical and chronological progression that her cytopenias worsened over time as her pool of CD34+ HSC was depleted. Additional studies of IFN-Ƴ and hematopoiesis suggest that IFN-Ƴ directly negatively affects the supportive bone marrow mesenchymal stoma cells which provide the cytokine environment that supports HSC repopulation. IFN-Ƴ is produced from the innate immune system via dendritic cells, natural killer (NK), and natural killer T (NKT) cell and in the adaptive immune system via Th1 T lymphocytes. IFN-Ƴ is a major activator of macrophages which in turn are responsible for secreting multiple other pro-inflammatory cytokines as previously mentioned. In Dengue, the innate immune system is activated via macrophage and dendritic cells. In HLH, IFN-Ƴ is primarily produced by T-cells. IFN-Ƴ is known to stimulate the janus kinase-STAT (JAK-STAT) signaling pathway in macrophages to increase inflammation. We suggest that modulation of the cytokines directly involved in the inflammatory syndrome might be considered. In summary, we report a case of Dengue fever with associated secondary HLH. This is the first report of Dengue fever in the continental United States in a person without travel history. In addition, this case report highlights the commonalities of the immune activation seen in both Dengue fever as well as secondary HLH and may indicate the potential for more targeted immunomodulation. Disclosures Liles: Imara: Other: PI on Clinical trial- Sickle cell ; Novartis: Other: PI on clinical trial Sickle cell ; Shire: Other: PI on clinical trial Sickle cell .

Chronic kidney failure mineral bone disorder leads to a permanent loss of hematopoietic stem cells through dysfunction of the stem cell niche

In chronic kidney disease (CKD), endothelial injury, is associated with disease progression and an increased risk for cardiovascular complications. Circulating cells with vascular reparative functions are hematopoietic and also reduced in CKD. To explore the mechanistic basis behind these observations, we have investigated hematopoietic stem cell (HSC) homeostasis in a mouse model for non-progressive CKD-mineral and bone disorder with experimentally induced chronic renal failure (CRF). In mice subjected to 12 weeks of CRF, bone marrow HSC frequencies were decreased and transplantation of bone marrow cells from CRF donors showed a decrease in long-term HSC repopulation compared to controls. This loss was directly associated with a CRF-induced defect in the HSC niche affecting the cell cycle status of HSC and could not be restored by the PTH-reducing agent cinacalcet. In CRF, frequencies of quiescent (G0) HSC were decreased coinciding with an increase in hematopoietic progenitor cells (HPC) in the S-and G2-phases of cell cycle. Moreover, in CRF mice, HSC-niche supporting macrophages were decreased compared to controls concomitant to impaired B lymphopoiesis. Our data point to a permanent loss of HSC and may provide insight into the root cause of the loss of homeostatic potential in CKD.

Latexin and hematopoiesis.

Hematopoietic stem cells (HSCs) produce mature blood cells throughout lifetime. Natural genetic diversity offers an important yet largely untapped reservoir for deciphering regulatory mechanisms of HSCs and hematopoiesis. In this review, we explore the role of latexin, identified by natural variation, in regulating homeostatic and stress hematopoiesis, unravel the underlying signaling pathways, and propose its therapeutic implication. Latexin acts endogenously in HSCs to negatively regulate their population size by enhancing apoptosis and by decreasing self-renewal. Deletion of latexin in vivo increases HSC repopulation capacity and survival, expands the entire hematopoietic system, and mitigates myelosuppression. Latexin inactivation downregulates thrombospondin 1 (Thbs1). It inhibits nuclear translocation of ribosomal protein subunit 3 (Rps3), a novel latexin-binding protein, and sensitizes hematopoietic cells to radiation-induced cell death. However, how latexin-Rps3 pathway regulates Thbs1 transcription is unclear. Latexin is downregulated in cancer cells because of promoter hypermethylation, but latexin-depleted mice do not inherently develop hematologic malignancies even with aging. The mechanism of action of latexin in tumorigenesis remains largely unknown. Understanding how latexin regulates HSC survival, self-renewal, and stress response will advance our knowledge of HSC biology. It will facilitate the development of a novel therapeutic strategy for hematopoietic regeneration and cancer treatment.

ICAM-1 Deficiency in the Bone Marrow Niche Impairs Quiescence andRepopulation of Hematopoietic Stem Cells.

SummaryThe bone marrow niche plays a critical role in controlling the fate of hematopoietic stem cells (HSCs) by integrating intrinsic and extrinsic signals. However, the molecular events in the HSC niche remain to be investigated. Here, we report that intercellular adhesion molecule-1 (ICAM-1) maintains HSC quiescence and repopulation capacity in the niche. ICAM-1-deficient mice (ICAM-1−/−) displayed significant expansion of phenotypic long-term HSCs with impaired quiescence, as well as favoring myeloid cell expansion. ICAM-1-deficient HSCs presented normal reconstitution capacity during serial transplantation; however, reciprocal transplantation experiments showed that ICAM-1 deficiency in the niche impaired HSC quiescence and repopulation capacity. In addition, ICAM-1 deletion caused failure to retain HSCs in the bone marrow and changed the expression profile of stroma cell-derived factors, possibly representing the mechanism for defective HSCs in ICAM-1−/− mice. Collectively, these observations identify ICAM-1 as a regulator in the bone marrow niche.

The transcription factor Zfp90 regulates the self-renewal and differentiation of hematopoietic stem cells

Hematopoietic stem cells (HSCs) can give rise to all blood cells that are essential to defend against pathogen invasion. The defective capability of HSC self-renewal is linked to many serious diseases, such as anemia. However, the potential mechanism regulating HSC self-renewal has not been thoroughly elucidated to date. In this study, we showed that Zfp90 was highly expressed in HSCs. Zfp90 deficiency in the hematopoietic system caused impaired HSPC pools and led to HSC dysfunction. We showed that Zfp90 deletion inhibited HSC proliferation, while HSC apoptosis was not affected. Regarding the mechanism of this effect on HSC proliferation, we found that Zfp90 interacted with Snf2l, a subunit of the NURF complex, to regulate Hoxa9 expression. Ectopic expression of Hoxa9 rescued the HSC repopulation capacity in Zfp90-deficient mice, which indicates that Hoxa9 is the downstream effector of Zfp90. In summary, our findings identify Zfp90 as a key transcription factor in determining the fate of HSCs.

Mesenchymal stem cells show functional defect and decreased anti-cancer effect after exposure to chemotherapeutic drugs

BackgroundMesenchymal stem cells (MSC) are used for several therapeutic applications to improve the functions of bone, cardiac, nervous tissue as well as to facilitate the repopulation of hematopoietic stem cells. MSC give rise to the non-hematopoietic stromal cells of the bone marrow and are important for the maintenance of normal hematopoiesis. Chemotherapeutic drugs used for treatment of leukemia extensively damage the stromal cells and alter their gene expression profiles.MethodsWe determined the changes in adipogenic, osteogenic differentiation, phenotypic and gene expression in MSC during treatment with chemotherapeutic drugs cytarabine, daunorubicin and vincristine. We also tested anti-cancer effects of drug treated MSC on leukemia cells.ResultsTreatment with the chemotherapeutic drugs resulted in functional defects in MSC, leading to reduced proliferation, osteogenic and adipogenic differentiation. The drug treated MSC also showed decreased expression of cell surface receptors, and the changes in proliferation, phenotype and differentiation defect was partially reversible after withdrawing the drugs from the cells. The drug treated MSC showed increased expression of cytokines, IL6, FGF2 and TNFA but reduced levels of differentiation markers SOX9 and ACTC1. Drug treated MSC also contributed to reduced anti-cancer effects in leukemia cells.ConclusionsChemotherapeutic drug treatment altered the phenotype, osteogenic and adipogenic differentiation potential of MSC and modified the gene expression profile of the cells to render them more chemoprotective of the leukemic cells. Thus, additional therapeutic efforts to target the stromal cell population will help in preventing chemoresistance, disease relapse in leukemia and to maintain a healthy bone marrow stroma.

Interferon Regulatory Factor-2 Regulates Hematopoietic Stem Cells

Objective: Interferon regulatory factor-2 (IRF-2) is highly expressed in hematopoietic stem cells (HSCs) in mouse bone marrow cells. The population of bone marrow Lin-c-Kit+Sca-1+(KSL) cells is increased in IRF-2-/- mice; however, there are less hematopoietic stem cells due to a reduction of the CD150+ population and type I interferon (IFN) signaling. We studied how HSCs lacking IRF-2 differentiate in bone marrow and peripheral blood. Methods: To study the function of IRF-2 in HSCs, bone marrow cells from IRF-2-/- mice were transplanted into wild-type mice. Results: Lineage cell population analysis showed that the B220 and CD19 populations from IRF-2-/- mouse bone marrow were enhanced in recipient mice. Conclusion: Bone marrow cells from IRF-2-/- mice can survive by lineage cell re-population. IRF-2 is important for the long-term repopulation of HSCs through not only the type I IFN response but also via the regulation of lineage-specific cell populations.

Mitochondrial Morphology Controls Hematopoietic Stem Cell Self-Renewal and Confers Them Divisional Memory

Hematopoietic stem cells (HSC) have extensive regenerative potential but limited self-renewal ability. The repopulation potential is gradually lost with HSC divisions and during stress hematopoiesis. Hence, we hypothesize that activation or inactivation of signaling pathways or other mechanisms, inherently occurring during HSC activation into cycle, drives their progressive functional decline. To address this, we used single cell RNA-sequencing and compared the transcriptome of HSCs [Lin-c-Kit+Sca-1+CD48-CD150+] that is expressed upon exit from quiescence and the one that is inherited by daughter cells upon HSC division. We compared HSCs with full self-renewal activity to HSCs that have lost self-renewal activity [i.e. before (NT-HSC) and after transplantation (T-HSC), respectively]. Analysis of the data was performed using unsupervised methods, namely iterative clustering and guide-gene selection (ICGS), [Olsson et al, Nature, 2016] and principal component (PCA) analysis. Each revealed that genes categorized in mitochondrial organization were dramatically upregulated in activated NT-HSC and remained highly expressed in their daughter cells. Remarkably, ICGS revealed at least 2 distinct regulatory states within T-HSC; one that clustered within NT-HSC, and another one that was clearly separated from NT-HSC. The latter was exclusively comprised of daughter T-HSCs, and quite unexpectedly was demarcated by a lack of expression of mitochondrial genes along with changes in expression of genes related to cytoskeleton organization. Using EGFP-mitochondria reporter mice; which labels mitochondria regardless of their membrane potential and activated state, we found that NT-HSC had 4-fold higher mitochondrial content than progenitors. High resolution confocal imaging indicated that mitochondria were punctuated and the network was dispersed in the entire cell volume in NT-HSC. However, in T-HSCs, mitochondria formed one or a few larger aggregates. Mitochondrial membrane potential was also severely reduced in T-HSC in vivo, indicating that HSC accumulate dysfunctional mitochondria after replicative stress during stress hematopoiesis. During cell division, in vitro, mitochondria were unequally distributed to daughter cells of single T-HSC division [one daughter cell had more aggregated mitochondria], whereas mitochondria were equally partitioned during division of NT-HSC, suggesting that HSC lose mitochondrial quality control after replicative stress. Further, mitochondria aligned along the microtubule network in NT-HSC but not in T-HSC. Mitochondrial morphology and distribution are maintained through regulated cycles of fission and fusion, which is essential for maintaining a functional organelle and for equal mitochondrial inheritance during division. We found that the mitochondrial fission protein Drp1, which normally accumulated and wrapped around mitochondria in NT-HSC during cell activation, failed to do so in T-HSCs, perhaps due to impaired mitochondrial-cytoskeleton association. Genetic loss of Drp1 caused severe mitochondrial aggregation in HSCs, and impaired HSC self-renewal and repopulation ability in competitive transplant studies, indicating that accumulation of aggregated and dysfunctional mitochondria causes HSC functional defects. To understand whether HSCs accumulated abnormal mitochondrial morphology with divisional history during homeostatic conditions, we used the GFP label-retaining H2B mouse model. Remarkably, mitochondria were well dispersed and homogeneously distributed in GFP labeled-retaining HSCs (i.e. HSC that had not divided), while HSCs with history of cell division (no GFP) had larger and more compact aggregates. Hence, once HSCs exit from quiescence, they irreversibly accumulate dysfunctional mitochondria over the course of divisions due to lack of mitochondrial quality control mechanisms, which drives gradual heterogeneity and loss of self-renewal ability. Thus, HSCs use mitochondrial architecture to remember their divisions, in turn affecting their self-renewal ability. Therefore, drugs or pharmacological inhibitors targeting mitochondrial organization or its quality can be new therapeutic approach to improve HSC function following transplantation. DisclosuresNo relevant conflicts of interest to declare.

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow.

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral gene therapy studies, IS assays, in combination with next-generation sequencing, have been used as a cell-tracking tool to characterize clonal stem cell populations sharing the same IS. For the accurate comparison of repopulating stem cell clones within and across different samples, the detection sensitivity, data reproducibility, and high-throughput capacity of the assay are among the most important assay qualities. This work provides a detailed protocol and data analysis workflow for bidirectional IS analysis. The bidirectional assay can simultaneously sequence both upstream and downstream vector-host junctions. Compared to conventional unidirectional IS sequencing approaches, the bidirectional approach significantly improves IS detection rates and the characterization of integration events at both ends of the target DNA. The data analysis pipeline described here accurately identifies and enumerates identical IS sequences through multiple steps of comparison that map IS sequences onto the reference genome and determine sequencing errors. Using an optimized assay procedure, we have recently published the detailed repopulation patterns of thousands of Hematopoietic Stem Cell (HSC) clones following transplant in rhesus macaques, demonstrating for the first time the precise time point of HSC repopulation and the functional heterogeneity of HSCs in the primate system. The following protocol describes the step-by-step experimental procedure and data analysis workflow that accurately identifies and quantifies identical IS sequences.

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral gene therapy studies, IS assays, in combination with next-generation sequencing, have been used as a cell-tracking tool to characterize clonal stem cell populations sharing the same IS. For the accurate comparison of repopulating stem cell clones within and across different samples, the detection sensitivity, data reproducibility, and high-throughput capacity of the assay are among the most important assay qualities. This work provides a detailed protocol and data analysis workflow for bidirectional IS analysis. The bidirectional assay can simultaneously sequence both upstream and downstream vector-host junctions. Compared to conventional unidirectional IS sequencing approaches, the bidirectional approach significantly improves IS detection rates and the characterization of integration events at both ends of the target DNA. The data analysis pipeline described here accurately identifies and enumerates identical IS sequences through multiple steps of comparison that map IS sequences onto the reference genome and determine sequencing errors. Using an optimized assay procedure, we have recently published the detailed repopulation patterns of thousands of Hematopoietic Stem Cell (HSC) clones following transplant in rhesus macaques, demonstrating for the first time the precise time point of HSC repopulation and the functional heterogeneity of HSCs in the primate system. The following protocol describes the step-by-step experimental procedure and data analysis workflow that accurately identifies and quantifies identical IS sequences.