Migration Of Hematopoietic Stem Cells Research Articles

2-AG-loaded and bone marrow-targeted PCL nanoparticles as nanoplatforms for hematopoietic cell line mobilization

BackgroundThe use of mobilizing agents for hematopoietic stem cell (HSC) transplantation is insufficient for an increasing number of patients. We previously reported lipid made endocannabinoid (eCB) ligands act on the human bone marrow (hBM) HSC migration in vitro, lacking long term stability to be therapeutic candidate. In this study, we hypothesized if a novel 2-AG-loaded polycaprolactone (PCL)-based nanoparticle delivery system that actively targets BM via phosphatidylserine (Ps) can be generated and validated.MethodsPCL nanoparticles were prepared by using the emulsion evaporation method and characterized by Zetasizer and scanning electron microscopy (SEM). The encapsulation efficiency and release profile of 2-AG were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The presence of cannabinoid receptors (CBRs) in HSCs and monocytes was detected by flow cytometry. Cell morphology and viability were assessed using transmission electron microscopy (TEM), SEM, and the WST-1 viability assay. The migration efficacy of the 2-AG and 2-AG-loaded nanoparticle delivery system on HSCs and HPSCs (TF-1a and TF-1) and monocytes (THP-1) was evaluated using a transwell migration assay.ResultsThe 140–225 nm PCL nanoparticles exhibited an increasing polydispersity index (PDI) after the addition of Ps and 2-AG, with a surface charge ranging from − 25 to -50 mV. The nanoparticles released up to 36% of 2-AG within the first 8 h. The 2-AG-Ps-PCL did not affect cellular viability compared to control on days 5 and 10. The HSCs and monocytes expressed CB1R and CB2R and revealed increased migration to media containing 1 µM 2-AG-Ps-PCL compared to control. The migration rate of the HSCs toward monocytes incubated with 1 µM 2-AG-Ps-PCL was higher than that of the monocytes of control. The 2-AG-Ps-PCL formulation provided a real time mobilization efficacy at 1 µM dose and 8 h time window via a specific CBR agonism.ConclusionThe newly generated and validated 2-AG-loaded PCL nanoparticle delivery system can serve as a stable, long lasting, targeted mobilization agent for HSCs and as a candidate therapeutic to be included in HSC transplantation (HSCT) protocols following scale-up in vivo preclinical and subsequent clinical trials.Graphical

Mobilization dynamics of bone marrow hematopoietic stem cells during hematopoietic regeneration

Under stress hematopoiesis, previous studies have suggested the migration of hematopoietic stem cells (HSCs) from bone marrow (BM) to extramedullary sites such as the spleen. However, there is little direct evidence of HSC migration from the BM to the spleen. Here, we induced myeloablation via 5-fluorouracil (5-FU) and showed direct evidence of HSC migration from BM to spleen during hematopoietic regeneration via a photoconvertible fluorophore. Moreover, during steady state, HSCs preferentially migrated to BM rather than spleen, but during hematopoietic regeneration, HSCs preferred spleen as a migration site equivalently or greater. Furthermore, in the early phase, HSCs egressed from BM through the attenuated HSC retention. However, HSCs in the late phase gained significantly enhanced cell-autonomous motility, which was independent of chemotaxis. Collectively, HSC mobilization from BM, before the migration to the spleen, was dynamically changed from passive to active events during hematopoietic regeneration.

Use of Combination Premedication Prior to Motixafortide Administration Reduced Severity and Frequency of AEs in the Phase 3 Genesis Trial - a Single Center Analysis

Background: Motixafortide is a novel, high affinity CXCR4 antagonist with extended receptor occupancy that facilitates the migration of hematopoietic stem and progenitor cells from the bone marrow into the peripheral blood in preparation for autologous stem cell transplantation (ASCT). In prior pre-clinical and clinical studies, motixafortide was associated with transient hypersensitivity and injection site reactions. Strategies to mitigate such adverse events (AEs) commonly include agents with antihistamine activity. The Phase 3 GENESIS trial demonstrated that 88.8% of Multiple Myeloma patients mobilized ≥6x10 6 CD34+ cells/kg (local labs) in 1 leukapheresis procedure (LP) following the administration of 1 dose of motixafortide in combination with GCSF. Per initial protocol, premedication with an antihistamine H1 blocker prior to motixafortide administration was provided to all patients. However, during the course of the GENESIS study a protocol amendment was implemented to include premedication with an antihistamine H1 blocker, an H2 blocker, a leukotriene inhibitor and acetaminophen prior to motixafortide administration. Here we report the safety findings in a cohort of patients at who received single drug premedication with an H1 antihistamine prior to the protocol amendment and those who received the updated combination pre-medication regimen following protocol amendment. Methods:The GENESIS trial was a multicenter, randomized, double-blind, placebo-controlled study in 122 transplant-eligible multiple myeloma patients, as previously described (PMID: 37069359). Included in this analysis are 46 patients enrolled at the Washington University in St. Louis; 32 patients were randomized to the motixafortide treatment arm and 14 patients were randomized to the placebo arm. Among motixafortide-treated patients, 22 received a single-agent H1 blocker pre-medication and 10 received a combination pre-medication regimen comprising of an H1 blocker, an H2 blocker, a leukotriene inhibitor and acetaminophen. All pre-medications were administered within 1 hour prior to motixafortide administration. Hypersensitivity AEs included all events coded to the MedDRA terms of urticaria, pruritus, flushing, erythema, rash, hypersensitivity or anaphylaxis. Injection site reactions included all events coded to the MedDRA High Level Term of Injection Site Reaction. All AEs were graded using CTCAE criteria and were recorded up to 30 days from the last dose of motixafortide. Results: Overall, motixafortide was safe and well tolerated with no episodes of anaphylaxis, no Grade 4 AEs and no deaths. The most commonly observed AEs included transient hypersensitivity and injection site reactions. The frequency of motixafortide-associated hypersensitivity AEs of any grade was reduced from 90.9% with single-agent H1 blocker pre-medication to 20% with the combination pre-medication regimen, similar to the 20-25% rate of hypersensitivity AEs reported in the placebo + GCSF cohort (Table 1). In addition, all hypersensitivity AEs that occurred following combination pre-medication were graded mild in severity (Figure 1). Meanwhile the frequency of injection site AEs of any grade was reduced from 95.5% with single-agent H1 blocker pre-medication to 80.0% with the combination pre-medication regimen (Table 1). Furthermore, a notable improvement in severity of injection site AEs was observed, with moderate-severe injection site AEs reduced from 27% with single-agent H1 blocker pre-medication to 0% with the combination pre-medication regimen (Figure 1). Conclusions:Motixafortide was safe and well tolerated with no episodes of anaphylaxis, no Grade 4 AEs and no deaths. Following implementation of a protocol amendment using combination pre-medication with an antihistamine H1 blocker, an H2 blocker, a leukotriene inhibitor and acetaminophen, the frequency and severity of hypersensitivity and injection site AEs associated with motixafortide were markedly improved.

A mouse model with high clonal barcode diversity for joint lineage, transcriptomic, and epigenomic profiling in single cells

Cellular lineage histories and their molecular states encode fundamental principles of tissue development and homeostasis. Current lineage-recording mouse models have insufficient barcode diversity and single-cell lineage coverage for profiling tissues composed of millions of cells. Here, we developed DARLIN, an inducible Cas9 barcoding mouse line that utilizes terminal deoxynucleotidyl transferase (TdT) and 30 CRISPR target sites. DARLIN is inducible, generates massive lineage barcodes across tissues, and enables the detection of edited barcodes in ∼70% of profiled single cells. Using DARLIN, we examined fate bias within developing hematopoietic stem cells (HSCs) and revealed unique features of HSC migration. Additionally, we established a protocol for joint transcriptomic and epigenomic single-cell measurements with DARLIN and found that cellular clonal memory is associated with genome-wide DNA methylation rather than gene expression or chromatin accessibility. DARLIN will enable the high-resolution study of lineage relationships and their molecular signatures in diverse tissues and physiological contexts.

Degree of leukochimerism with calf age in Angus-crossbred twin sets.

Twinning in cattle is infrequent and usually undesired. It can result in an increased occurrence of abortion and dystocia, reduced calf survival and a high likelihood of freemartinism in mixed-sex twins. Twin gestations are also commonly associated with the formation of placental vascular anastomoses (PVA) between twins. Through PVA they share blood, hormones (leading to freemartinism in mixed sex twins) and hematopoietic stem cells, which are the progenitors of white blood cells. The sharing of stem cells between twins can result in leukochimeric twin sets. These are twins that have white blood cells derived from both self and the co-twin owing to the fetal migration of hematopoietic stem cells from the extraembryonic mesoderm of the yolk sac to final sites like bone marrow and thymus. This study examined the degree to which this leukochimerism changes with age. DNA was extracted from hair bulbs containing mesenchymal dermal papilla to determine the individual's true genotype and blood samples were obtained at six time points from 1 week to 8 months of age to assess leukochimerism. Samples were genotyped using a medium density SNP chip, and quantitative estimates of allele frequency were determined using SNPs for which members of a twin set had alternative homozygous genotypes. The results indicate statistically significant changes in the proportion of self and co-twin with age and suggest that by 2-4 months of age the genotypic mix in white blood cells represents the hematopoetic stem cell population resident in the individual (i.e. permanently found in thymus and bone marrow).

Inflammation-related mRNA expression in patients with multiple myeloma undergoing hematopoietic stem cell mobilization

Mobilization of CD34+ cells is a key element in the therapy of patients with multiple myeloma undergoing autologous stem cell transplantation. The use of chemotherapy and the granulocyte colony-stimulating factor can significantly affect the expression of inflammation-related proteins and the migration of hematopoietic stem cells. We assessed the mRNA expression of selected proteins involved in the inflammatory landscape in MM patients (n=71). The aim of the study was to evaluate C-C motif chemokine ligand 3, 4, 5 (CCL3, CCL4, CCL5), leukocyte cell-derived chemotaxin 2 (LECT2), tumor necrosis factor (TNF), and formyl peptide receptor 2 (FPR2) levels in the course of mobilization and their role in the CD34+ collection efficacy. mRNA expression from peripheral blood plasma was evaluated by RT-PCR. We observed a deep decline in CCL3, CCL4, LECT2, and TNF mRNA expression on the day of the first apheresis (day A) as compared to baseline. A negative correlation was observed between CCL3, FPR2, LECT2, TNF level, and the CD34+ cells count in peripheral blood on day A, and the number of CD34+ cells obtained at first apheresis . Our results indicate that the investigated mRNAs significantly alter and may regulate the migration of CD34+ cells during mobilization. Moreover, in case of FPR2 and LECT2, the results obtained in patients differ from the murine models.

Expression Changes of Hypoxia-Inducible Factor-1α in G-CSF Induced Hematopoietic Stem Cell Mobilization

To investigate the expression and its relative mechanism of hypoxia-inducible factor-1α(HIF-1α) in bone marrow(BM) of mice during G-CSF mobilization of hematopoietic stem cells (HSC) . Flow cytometry was used to detect the proportion of Lin-Sca-1+ c-kit+ (LSK) cells in peripheral blood of C57BL/6J mice before and after G-CSF mobilization. And the expression of HIF-1α and osteocalcin (OCN) mRNA and protein were detected by RQ-PCR and immunohistochemistry. The number of osteoblasts in bone marrow specimens of mice was counted under the microscope. The proportion of LSK cells in peripheral blood began to increase at day 4 of G-CSF mobilization, and reached the peak at day 5, which was significantly higher than that of control group (P<0.05). There was no distinct difference in the expression of HIF-1α mRNA between bone marrow nucleated cells and osteoblasts of steady-state mice (P=0.073), while OCN mRNA was mainly expressed in osteoblasts, which was higher than that in bone marrow nucleated cells (P=0.034). After mobilization, the expression level of HIF-1α increased, but OCN decreased, and the number of endosteum osteoblasts decreased. The change of HIF-1α expression was later than that of OCN and was consistent with the proportion of LSK cells in peripheral blood. The expression of HIF-1α in bone marrow was increased during the mobilization of HSC mediated by G-CSF, and one of the mechanisms may be related to the peripheral migration of HSC induced by osteoblasts inhibition.

Mobilization and Hematopoietic Stem Cell Collection in Poor Mobilizing Patients with Lymphoma: Final Results of the German OPTIMOB Study

Introduction: Successful mobilization and collection of peripheral hematopoietic stem cells (HSCs) are necessary for lymphoma patients eligible for myeloablative chemotherapy with subsequent autologous stem cell transplantation (ASCT). Albeit G-CSF alone or combined with chemotherapy is well-established methods for HSC mobilization, up to 40% of the patients fail to mobilize (poor mobilizer, PM). Plerixafor (PLX) is commonly used in PM patients resulting in increased migration of HSCs into peripheral blood and thus improves the collection outcome. Methods: The prospective, multicenter, open-label, non-interventional OPTIMOB study assessed mobilization and collection parameter of patients with lymphoma or multiple myeloma to get deep insights in the treatment of those patients in clinical routine focusing on PM patients. PM was defined as follows: (1) no achievement of ≥20 CD34+ progenitor cells/µL before first apheresis, (2) PLX administration at any time point during the observational period, (3) reduction of the initially planned CD34+ progenitor cell yield as necessity due to failed mobilization or HSC collection, and (4) no performance of apheresis due to low CD34+ progenitor level. Primary objective of the study was to assess mobilization success by the proportion of PM patients achieving >2 × 106 CD34+ progenitor cells/kg body weight on the first day of apheresis. Here, the data of the lymphoma cohort are presented. Results: Out of 238 patients with lymphoma documented in the study, 32% were classified as PM. 87% of them received PLX. Demographic data revealed no obvious differences between PM and good mobilizing (GM) patients. All patients were treated highly individualized prior to mobilization. Majority of all PM patients were able to undergo apheresis (95%) and reached their individual requested CD34+ progenitor cell target (72%). 57% of the PM patients achieved >2.0 × 106 CD34+ progenitor cells/kg body weight on day 1 of apheresis and nearby 70% of them underwent ASCT. Median time to engraftment was similar in PM and GM patients of the lymphoma cohort. Conclusions: Majority of PM patients with lymphoma were successfully mobilized and underwent ASCT. Most of them received PLX during the study.

The eighth International Forum on Stem Cells: virtual meeting, October 20-21, 2022.

The eighth International Forum on Stem Cells: virtual meeting, October 20-21, 2022.

Longitudinal single-cell transcriptomics reveals distinct patterns of recurrence in acute myeloid leukemia

BackgroundAcute myeloid leukemia (AML) is a heterogeneous and aggressive blood cancer that results from diverse genetic aberrations in the hematopoietic stem or progenitor cells (HSPCs) leading to the expansion of blasts in the hematopoietic system. The heterogeneity and evolution of cancer blasts can render therapeutic interventions ineffective in a yet poorly understood patient-specific manner. In this study, we investigated the clonal heterogeneity of diagnosis (Dx) and relapse (Re) pairs at genetic and transcriptional levels, and unveiled the underlying pathways and genes contributing to recurrence.MethodsWhole-exome sequencing was used to detect somatic mutations and large copy number variations (CNVs). Single cell RNA-seq was performed to investigate the clonal heterogeneity between Dx-Re pairs and amongst patients.ResultsscRNA-seq analysis revealed extensive expression differences between patients and Dx-Re pairs, even for those with the same -presumed- initiating events. Transcriptional differences between and within patients are associated with clonal composition and evolution, with the most striking differences in patients that gained large-scale copy number variations at relapse. These differences appear to have significant molecular implications, exemplified by a DNMT3A/FLT3-ITD patient where the leukemia switched from an AP-1 regulated clone at Dx to a mTOR signaling driven clone at Re. The two distinct AML1-ETO pairs share genes related to hematopoietic stem cell maintenance and cell migration suggesting that the Re leukemic stem cell-like (LSC-like) cells evolved from the Dx cells.ConclusionsIn summary, the single cell RNA data underpinned the tumor heterogeneity not only amongst patient blasts with similar initiating mutations but also between each Dx-Re pair. Our results suggest alternatively and currently unappreciated and unexplored mechanisms leading to therapeutic resistance and AML recurrence.

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.

Interferon-alpha2 treatment of patients with polycythemia vera and related neoplasms favorably impacts deregulation of oxidative stress genes and antioxidative defense mechanisms.

Chronic inflammation is considered a major driving force for clonal expansion and evolution in the Philadelphia-negative myeloproliferative neoplasms, which include essential thrombocythemia, polycythemia vera and primary myelofibrosis (MPNs). One of the key mutation drivers is the JAK2V617F mutation, which has been shown to induce the generation of reactive oxygen species (ROS). Using whole blood gene expression profiling, deregulation of several oxidative stress and anti-oxidative defense genes has been identified in MPNs, including significant downregulation of TP53, the NFE2L2 or NRF2 genes. These genes have a major role for maintaining genomic stability, regulation of the oxidative stress response and in modulating migration or retention of hematopoietic stem cells. Therefore, their deregulation might give rise to increasing genomic instability, increased chronic inflammation and disease progression with egress of hematopoietic stem cells from the bone marrow to seed in the spleen, liver and elsewhere. Interferon-alpha2 (rIFNα) is increasingly being recognized as the drug of choice for the treatment of patients with MPNs. Herein, we report the first gene expression profiling study on the impact of rIFNα upon oxidative stress and antioxidative defense genes in patients with MPNs (n = 33), showing that rIFNα downregulates several upregulated oxidative stress genes and upregulates downregulated antioxidative defense genes. Treatment with rIFNα induced upregulation of 19 genes in ET and 29 genes in PV including CXCR4 and TP53. In conclusion, this rIFNα- mediated dampening of genotoxic damage to hematopoietic cells may ultimately diminish the risk of additional mutations and accordingly clonal evolution and disease progression towards myelofibrotic and leukemic transformation.

P1418: EXPANDED COMMON MYELOID PROGENITORS ACT IN CONCERT WITH THE SPLEEN AND SUPPORT EARLY HEMATOPOIETIC RECOVERY

Background: Hematopoietic stem cells (HSC) represent the apex of the hematopoietic hierarchy and their engraftment post-transplantation is essential for long-term donor hematopoiesis. After intravenous injection, HSCs are thought to home directly toward the bone marrow niche, and their homing mechanisms and interactions with the microenvironment have been intensively studied. However, the time course of the transplanted HSCs to reconstitute the entire blood system and the spatial transition within the whole body have remained a mystery. Aims: We aim to elucidate the spatiotemporal dynamics of hematopoiesis in the same individual in a murine HSC transplantation model. Methods: Current methods for tracking transplanted HSCs in vivo are limited due to their low sensitivity and their restriction to observe only previously known niches. To address these issues, we used the recently published, highly sensitive AkaLuc bioluminescence imaging system to visualize HSC migration in vivo. We combined Akaluc 3D images with CT scans to spatially capture early hematopoiesis after transplantation on a whole-body scale, and continuously monitored bioluminescence signals to clarify the dynamics of cellular distributions over time. Results: Surprisingly, our data show that the spleen acts as a major site of hematopoiesis and shows unique hematopoietic patterns with dynamic changes. Splenic hematopoiesis was distinct from that occurring in the bone marrow and we found that common myeloid progenitors (CMPs) use the spleen as a site for hematopoiesis, producing many cells early after transplantation. Based on these findings, we developed expanded CMPs (exCMPs) that leverage the spleen environment to improve early hematopoietic recovery after transplantation. exCMPs, which can be produced in large quantities in polyvinyl-alcohol (PVA)-based cultures (Wilkinson et al., Nature 2019), had lower CXCR4 expression, different integrin expression patterns, better cytokine responsiveness and mitogenic potential, as well as higher expression of platelet-related genes compared to fresh CMP. Therefore, exCMPs are more likely to migrate to the spleen and proliferate more rapidly than fresh CMPs. Exploiting these properties, we could show that weekly administration of exCMP allowed for the long-term survival of lethally irradiated mice in a spleen-dependent manner. Image:Summary/Conclusion: Our studies of post-transplant hematopoiesis using a next-generation luminescence imaging system showed that the spleen functioned as a major hematopoietic tissue and that CMPs and the spleen cooperated to contribute to early hematopoiesis post-SCT, which led to the concept of utilizing the spleen as an alternative hematopoietic site. These findings refine our understanding of the dynamics of hematopoietic reconstitution, reveal the unique collaboration of the spleen and transplanted cells and establish the therapeutic potential of exCMPs for early hematopoietic recovery.

Preparative Production and Purification of Recombinant Human Cyclophilin A.

In this work, we developed the method of preparative production of recombinant human cyclophilin A (rhCypA) in Escherichia coli. The full-length cDNA encoding the gene of human CypA (CYPA) was amplified by RT-PCR from the total RNA of human T cell lymphoma Jurkat. The nucleotide sequence of CYPA was optimized to provide highly effective translation in E. coli. Recombinant CYPA DNA was cloned into the pET22b(+) vector, and the resulted expression plasmid was used to transform E. coli strain BL21(DE3)Gold. The recombinant producer strain of E. coli produced soluble rhCypA in the bacterial cytoplasm. The synthesis efficiency of rhCypA was up to 50% of the total cell protein allowing to produce rhCypA in the amount of 1 g per liter of the culture. We also developed the method for rhCypA purification, consisting of a single-step tandem anion exchange chromatography on DEAE- and Q-Sepharose columns. The protein purity was 95% according to electrophoresis (SDS-PAGE), and its contamination with endotoxin did not exceed 0.05 ng per 1 mg of the protein that met the requirements of European pharmacopoeia for injectable preparations. The produced recombinant protein exhibited functional features of native CypA, i.e., isomerase activity and chemokine activity as assessed by stimulation of migration of mouse bone marrow hematopoietic stem cells in vivo. The generated producer strain of E. coli is a super-producer and could be used for large-scale experimental studies of rhCypA and in its preclinical and clinical trials as a drug.

SIRT1-SIRT7 Expression in Patients with Lymphoproliferative Disorders Undergoing Hematopoietic Stem Cell Mobilization.

Simple SummaryOne of the treatment lines of patients with lymphoproliferative disorders is autologous transplantation, preceded by an effective mobilization of hematopoietic stem cells from the bone marrow niche into the peripheral blood. Sirtuins (SIRT1-SIRT7) are members of the NAD + -dependent class III histone deacetylase family, which affect the inflammatory response, hematopoiesis, angiogenesis, and the behavior of hematopoietic cells during mobilization and transplantation. In our work, we wanted to investigate how sirtuins expression changes on the day of the first apheresis as compared to the period before mobilization and how they can influence the efficacy of CD34+ cell collection. Our study revealed a significant increase in sirtuin expression on the day of the first apheresis as compared to the baseline. We demonstrated that the expression of SIRT5, SIRT6, and in particular SIRT7, on the day of first apheresis, may be associated with the efficacy of hematopoietic stem cell mobilization.Sirtuins are involved in the fate of hematopoietic stem cells (HSCs), including their metabolism, stress response, differentiation, migration, and apoptosis. The aim of this study was to explore SIRT1-7 expression during HSC mobilization. The study included 50 patients with lymphoproliferative disorders (39 multiple myeloma, 11 lymphoma). Samples were taken before mobilization (day 0) and on the day of first apheresis (day A). The sirtuin expression was evaluated by the Droplet Digital PCR (ddPCR) method. A significant increase of the SIRT1, SIRT2, SIRT3, SIRT5, SIRT6, and SIRT7 levels measured at day A as compared to baseline was observed. The study revealed a positive correlation between SIRT5, SIRT6, and SIRT7 expression and the CD34+ peak value in peripheral blood and the number of CD34+ cells collected on day A. Patients from the SIRT7 “high expressors” group collected more CD34+ cells on day A than “low expressors”. Upregulated expressions of SIRT3 and SIRT7 on the day of first apheresis were observed in patients in complete remission status (CR) as compared to the non-CR group. Our results suggest that the investigated sirtuins may influence the HSC migration and hematopoietic landscape during mobilization. SIRT5, SIRT6, and SIRT7 may be associated with the efficacy of HSC mobilization.

Ginsenoside Rg1 as a Potential Regulator of Hematopoietic Stem/Progenitor Cells.

Ginsenoside Rg1 (Rg1), a purified, active component of the root or stem of ginseng, exerts positive effects on mesenchymal stem cells (MSCs). Many recent studies have found that hematopoietic stem cells (HSCs), which can develop into hematopoietic progenitor cells (HPCs) and mature blood cells, are another class of heterogeneous adult stem cells that can be regulated by Rg1. Rg1 can affect HSC proliferation and migration, regulate HSC/HPC differentiation, and alleviate HSC aging, and these findings potentially provide new strategies to improve the HSC homing rate in HSC transplantation and for the treatment of graft-versus-host disease (GVHD) or other HSC/HPC dysplasia-induced diseases. In this review, we used bioinformatics methods, molecular docking verification, and a literature review to systematically explore the possible molecular pharmacological activities of Rg1 through which it regulates HSCs/HPCs.

Immunomodulatory Activity of Granulocyte Colony-Stimulating Factor and its Therapeutic Effect on Liver Failure

Abstract Liver failure is characterized by the rapid deterioration of liver function, often accompanied by ascites, coagulation dysfunction, hepatic encephalopathy, and other critical complications. Owing to the complex multifaceted pathogenesis and consequential clinical manifestations of the disease, liver failure displays poor prognosis and warrants comprehensive clinical treatment and management. Liver transplantation remains the only well-established treatment for liver failure. However, several factors including transplantation cost and low organ donation rates limit the rate of liver transplantation. The development of a suitable therapy for liver failure is a significant challenge and remains a cause of concern for the medical world. Granulocyte colony-stimulating factor (G-CSF), a member of the cytokine family of hematopoietic growth factors, is involved in the migration of hematopoietic stem cells into the damaged liver, and effectuates their dedifferentiation into hepatocytes. Liver regeneration involves a complex crosstalk of multiple cell types, including hepatocytes, endothelial cells, and inflammatory cells. Neutrophils and monocytes/macrophages that present different types of innate immune cells were found to play a crucial role in the progression of inflammation and restoration of the liver tissue. G-CSF, known as the most common used cytokine, may also affect these immune cells by combining G-CSF receptors on their surface. The immunomodulatory activity of G-CSF should be studied and described in order to ascertain its therapeutic effect on liver failure.

Editorial introductions.

Editorial introductions.

Age-Related Features of the Response of the Liver and Stem Cells during Modeling of Liver Cirrhosis.

We studied the age-related characteristics of the response of stem cells and liver in male Wistar rats to administration of carbon tetrachloride (CCl4) and ethanol. It was shown that modeling of liver cirrhosis caused inflammation, fibrosis, damage to sinusoidal capillaries, necrosis, and disturbances in the functional activity of hepatocytes in young rats. These processes were accompanied by mobilization of profibrotic mesenchymal stem cells (MSC), proinflammatory hematopoietic stem cells (HSC) and lymphocytes (CD45hiCD133+) from the bone marrow into the blood and migration to the liver. On the other hand, the number of hepatocyte precursors expressing Sox9 (cells of Hering's canal), immature cholangiocytes, Ito cells, oval cells, and endothelial cells of the liver sinusoids) sharply increased in the liver. In young rats, mobilization and migration of MSC, HSC, and hepatocyte precursors against the background of liver cirrhosis were more intensive than in old animals. The higher resistance of old rats to exposure is associated with age-related changes in the niches as well as in mobilization and migration of cells.

Bone Marrow Stromal Antigen 2 Is Critical for IFNy-Dependent Hematopoietic Stem Cell Activation

Bone marrow failure is a significant complication of many chronic infections, which affect a third of the world's population. The inflammatory cytokine interferon-gamma (IFNy) contributes to bone marrow failure syndromes by activating hematopoietic stem cells (HSCs) and impairing their self-renewal. IFNy upregulation during many chronic infections such as tuberculosis, HIV and hepatitis B directly depletes hematopoietic stem cells (HSCs). The mechanisms by which IFNy drives the loss of quiescence and ultimate exhaustion of HSCs remain poorly understood, but may be related to changes in the interaction between HSCs and the bone marrow (BM) microenvironment, or BM niche. Current evidence suggests that quiescent HSCs reside predominantly in the vascular niche, where the production of stem cell factor (SCF) from endothelial cells and CXCL12 from perivascular CXCL12 abundant reticular (CAR) stromal cells are critical for maintaining their quiescence. The goal of our work was to determine whether IFNy signaling alters HSC interactions within the niche. We performed transcriptomic analysis of IFNy-stimulated HSCs and focused on changes in cell-surface expressed genes that may influence HSC-niche interactions. Our analysis revealed Bone Marrow Stromal Antigen 2 (BST2) as the only surface protein upregulated on HSCs upon 24-hour IFNy stimulation. To study the effects of BST2 on HSC-niche interactions, we performed intravital imaging using two reporter mouse models: CXCL12-GFP reporter mice in which CAR cells are labeled with GFP, and CXCL12-GFP Krt18-Cre LSL-tdTomato dual reporter mice in which CAR cells are labelled with GFP and HSCs are labeled with tdTomato. After exogenously labeling and transferring HSCs into CXCL12-GFP mice or endogenously labeling HSCs in the CXCL12-GFP Krt18-Cre LSL-tdTomato mice, we observed that HSCs stimulated with IFNy were significantly distanced from CAR cells compared to pre-treated controls. These findings are consistent with other reports that chemotherapeutic and inflammatory stress disrupts HSC interactions with the niche and promotes HSC migration. Conversely, we observed no change in HSC distancing from CAR cells after IFNy stimulation of IFNy-receptor deficient HSCs, suggesting that the observed HSC displacement was due to a cell autonomous mechanism. These changes were not due to a loss of CXCL12 receptor (CXCR4) expression or disrupted capacity of HSCs to migrate towards CXCL12. Interestingly, Intravital imaging using BST2-deficient HSCs revealed that BST2 KO HSCs do not re-localize from CAR cells during IFNy stimulation. Increased BST2 expression has been linked to the migration, adhesion and metastasis of various cancer cells and we explored whether it could serve a similar role in protein binding in HSCs. Using in vitro plate binding assays, we found that IFNy-treatment promoted increased HSC binding to E-selectin via BST2, as well as increased HSC homing to the bone marrow, a property that is dependent on E-selectin binding. Finally, to determine whether BST2 affects IFNy-dependent HSC activation we performed cell cycle analysis of WT and BST2 KO HSCs. We discovered that the loss of BST2 protects against HSC activation during Mycobacterium avium infection. Furthermore, HSC depletion during chronic infection was mitigated in BST2 KO mice. Our data identifies BST2 as a key protein that influences niche relocalization and activation in response to inflammatory stimulation. This study expands our understanding of factors that contribute to HSC activation and loss of quiescence. These findings could shed light on novel therapeutic interventions for patients who develop pancytopenia or bone marrow failure due to chronic inflammation. Disclosures No relevant conflicts of interest to declare.