Percentage Of Hematopoietic Stem Cells Research Articles

Abstract LB081: Novel targeted oligonucleotide-based telomere disruptor for immunotherapy of diffuse large B-cell lymphoma

Abstract Telomerase (TERT) is an enzyme critical for carcinogenesis expressed in about 90% of human cancers, including aggressive diffuse large B-cell lymphoma (DLBCL). Targeting TERT proven difficult due to on-target toxicities to non-malignant hematopoietic stem cells and potentially to activated immune effector cells. To overcome these challenges, we developed novel TLR9+ cell-selective CpG-oligodeoxynucleotides (ODNs) to deliver telomere-damaging nucleoside analogue, 6-thio-2’-deoxy-guanosine (6tdG), into target B cell lymphoma cells in vitro and in vivo. Human and mouse TLR9+/TERT+ lymphoma cells rapidly internalized CpG(6tdG)-oligonucleotides through active endocytosis. 6tdG molecules were released from the 3’ end of the oligonucleotide into cytosol and cell nucleus. TERT-mediated 6tdG incorporation into telomeres, led to DNA damage and lymphoma cell death. In vitro, CpG(6tdG)-oligonucleotides showed potency in targeting variety of human DLBCL cells in nanomolar range without detectable toxicity to non-malignant TERT— cells or human TERT+ hematopoietic stem cells and activated T cells. Importantly, the repeated systemic administration of CpG(6tdG)-oligonucleotides was well-tolerated by humanized hCD34/NOG mice, without detectable decrease in the percentage of hematopoietic stem cells, myeloid cells or T cells although the numbers of B cells were partly reduced as expected. Intravenously injected CpG(6tdG)-oligonucleotides, but not 6tdG nucleoside, had potent and dose-dependent antitumor effects against xenotransplanted models of human GC- or ABC-subtypes of DLBCL, such as OCI-Ly18 or OCI-Ly3, respectively. Furthermore, we assessed CpG(6tdG)-oligonucleotide activity against aggressive, syngeneic TERT+ Eμ-myc/15A lymphoma in immunocompetent mice. CpG(6tdG)-oligonucleotide, but not the CpG-oligonucleotide alone, 6tdG alone or co-injection of thereof, was effective in triggering regression of established 15A tumors with long-term survival of the majority of mice. The effect of CpG(6tdG)-oligonucleotide correlated with the activation of tumor antigen-specific T cell-mediated antitumor immune responses. In contrast, when tested in immunodeficient mice, CpG(6tdG)-oligonucleotide induced significant but transient regression of 15A lymphoma. These results suggest that the oligonucleotide elicits two-pronged activity triggering direct lymphoma cell cytotoxicity and also immune-mediated antitumor effects. Overall, this cell-selective strategy could provide an effective and safer strategy against aggressive TERT+ DLBCL cells with potential to engage and support T cell-based antitumor immunity. Citation Format: Chunsong Yu, Yong Liang, Piotr Swiderski, Larry Kwak, Marcin Kortylewski. Novel targeted oligonucleotide-based telomere disruptor for immunotherapy of diffuse large B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB081.

Bletilla striata polysaccharide improves toxic and side effects induced by 5-FU: an untargeted metabolomics study

This study aimed to analyze the effect of Bletilla striata polysaccharide(BSP) on endogenous metabolites in serum of tumor-bearing mice treated with 5-fluorouracil(5-FU) by untargeted metabolomics techniques and explore the mechanism of BSP in alleviating the toxic and side effects induced by 5-FU. Male BALB/C mice were randomly divided into a normal group, a model group, a 5-FU group, and a 5-FU + BSP group, with eight mice in each group. Mouse colon cancer cells(CT26) were transplanted into the mice except for those in the normal group to construct the tumor-bearing mouse model by subcutaneous injection, and 5-FU chemotherapy and BSP treatment were carried out from the second day of modeling. The changes in body weight, diarrhea, and white blood cell count in the peripheral blood were recorded. The mice were sacrificed and sampled when the tumor weight of mice in the model group reached approximately 1 g. TUNEL staining was used to detect the cell apoptosis in the small intestine of each group. The proportions of hematopoietic stem cells and myeloid progenitor cells in bone marrow were measured by flow cytometry. Five serum samples were selected randomly from each group for untargeted metabolomics analysis. The results showed that BSP was not effective in inhibiting colon cancer in mice, but diarrhea, leukopenia, and weight loss caused by 5-FU chemotherapy were significantly improved after BSP intervention. In addition, apoptotic cells decreased in the small intestinal tissues and the percentages of hematopoietic stem cells and myeloid progenitor cells in bone marrow were significantly higher after BSP treatment. Metabolomics results showed that the toxic and side effects of 5-FU resulted in significant decrease in 29 metabolites and significant increase in 22 metabolites in mouse serum. Among them, 19 disordered metabolites showed a return to normal levels in the 5-FU+BSP group. The results of pathway enrichment indicated that metabolic pathways mainly involved pyrimidine metabolism, arachidonic acid metabolism, and steroid hormone biosynthesis. Therefore, BSP may ameliorate the toxic and side effects of 5-FU in the intestinal tract and bone marrow presumably by regulating nucleotide synthesis, inflammatory damage, and hormone production.

Umbilical cord blood derived cellular therapy: advances in clinical development.

While cord blood (CB) is primarily utilized in allogeneic hematopoietic cell transplantation (HCT), the development of novel cell therapy products from CB is a growing and developing field. Compared to adult blood, CB is characterized by a higher percentage of hematopoietic stem cells (HSCs) and progenitor cells, less mature immune cells that retain a high capacity of proliferation, and stronger immune tolerance that requires less stringent HLA-matching when used in the allogenic setting. Given that CB is an FDA regulated product and along with its unique cellular composition, CB lends itself as a readily available and safe starting material for the development of off-the-shelf cell therapies. Moreover, non-hematologic cells such as mesenchymal stem cell (MSCs) residing in CB or CB tissue also have potential in regenerative medicine and inflammatory and autoimmune conditions. In this review, we will focus on recent clinical development on CB-derived cellular therapies in the field of oncology, including T-cell therapies such as chimeric antigen receptor (CAR) T-cells, regulatory T-cells, and virus-specific T-cells; NK-cell therapies, such as NK cell engagers and CAR NK-cells; CB-HCT and various modifications; as well as applications of MSCs in HCT.

Dose-dependent effect of GFI1 expression in the reconstitution and the differentiation capacity of HSCs.

GFI1 is a transcriptional repressor and plays a pivotal role in regulating the differentiation of hematopoietic stem cells (HSCs) towards myeloid and lymphoid cells. Serial transplantation of Gfi1 deficient HSCs repopulated whole hematopoietic system but in a competitive setting involving wild-type HSCs, they lose this ability. The underlying mechanisms to this end are poorly understood. To better understand this, we used different mouse strains that express either loss of both Gfi1 alleles (Gfi1-KO), with reduced expression of GFI1 (GFI1-KD) or wild-type Gfi1/GFI1 (Gfi1-/GFI1-WT; corresponding to the mouse and human alleles). We observed that loss of Gfi1 or reduced expression of GFI1 led to a two to four fold lower number of HSCs (defined as Lin-Sca1+c-Kit+CD150+CD48-) compared to GFI1-WT mice. To study the functional influence of different levels of GFI1 expression on HSCs function, HSCs from Gfi1-WT (expressing CD45.1 + surface antigens) and HSCs from GFI1-KD or -KO (expressing CD45.2 + surface antigens) mice were sorted and co-transplanted into lethally irradiated host mice. Every 4weeks, CD45.1+ and CD45.2 + on different lineage mature cells were analyzed by flow cytometry. At least 16weeks later, mice were sacrificed, and the percentage of HSCs and progenitors including GMPs, CMPs and MEPs in the total bone marrow cells was calculated as well as their CD45.1 and CD45.2 expression. In the case of co-transplantation of GFI1-KD with Gfi1-WT HSCs, the majority of HSCs (81% ± 6%) as well as the majority of mature cells (88% ± 10%) originated from CD45.2 + GFI1-KD HSCs. In the case of co-transplantation of Gfi1-KO HSCs with Gfi1-WT HSCs, the majority of HSCs originated from CD45.2+ and therefore from Gfi1-KO (61% ± 20%); however, only a small fraction of progenitors and mature cells originated from Gfi1-KO HSCs (<1%). We therefore in summary propose that GFI1 has a dose-dependent role in the self-renewal and differentiation of HSCs.

Evaluation of Transplanted Stem Cell Dynamic Variables in the Bone Marrow of Children with Malignancies

Objective: According to risk classifications, the transplant of healthy hematopoietic stem cells in the bone marrow is a priority treatment option for hematopoietic diseases. The effectiveness of hematopoietic stem cell transplantation after bone marrow transplantation is largely dependent on the capacity of the bone marrow microenvironment to accept transplanted cells. Detailed analysis of hematopoietic stem cell kinetics after transplantation is very important, as initial transplantation of hematopoietic stem cells affects prognosis. In this study, hematopoietic stem and progenitor cell percentage values were evaluated together with other transplantation-related parameters to determine the capacity of stem cell dynamic variables in the bone marrow. Methods: Dynamic variables in hematopoietic stem cell fate in bone marrow transplantation include many parameters; hematopoietic stem cell transplantation rates in the apheresis product of 13 patients and cell counts per kilogram and engraftment times, CD34+ cell rates, CD34+ cell counts per kilogram, chimerism rates, Graft-versus-Host Disease grades, white blood cell counts, and post-transplant relapse status. In the apheresis products collected from the patients before transplantation, hematopoietic stem and progenitor cells were identified by the immunophenotypic barcode "CD34+, CD38-, CD45RA-, CD90+, CD49f+", and data analysis was performed with the BD FACSDivaTM software by studying on the BD FACSCanto II flow cytometer device. Results: According to the results of the pearson correlation analysis (non-parametric correlations), a strong positive and significant relationship was found between the number of hematopoietic stem cell transplantation per kg and the percentage of hematopoietic stem cell transplantation of the patients (r=.733, p&lt;.05). According to the results of the analysis, there was a moderately significant and positive relationship (r=.631, p&lt;.05) between the percentage of hematopoietic stem cell transplantation and platelet engraftment time, while there was a strong inverse relationship between erythrocyte engraftment time (r=-.730, p&lt;.01). A strong positive and significant relationship was found between the number of Hematopoietic stem cell transplantation cells per kilogram infused into the patients and the platelet engraftment times (r=.780, p&lt;.05). Conclusion: This study confirms that a mean cell dose of hematopoietic stem and progenitor cells ≥ 2.57 x 106/kg infused provides rapid short- and long-term platelet engraftment in pediatric patients undergoing autologous and allogeneic transplantation. This study is important in terms of revealing the possible relationship between pulling forward the platelet engraftment time and the number and percentage of Hematopoietic stem and progenitor cells in our transplanted patients. It is also important that a statistically significant inverse relationship was determined between HSPC values and mean erythrocyte engraftment times.

A Pilot Study on Clinical Scores, Immune Cell Modulation, and Microbiota Composition in Allergic Patients with Rhinitis and Asthma Treated with a Probiotic Preparation

Background: Specific drugs and/or immunotherapies are widely used to treat allergies, but drug-induced adverse effects recently led to explore new additional strategies. We studied whether a probiotic preparation (iPROB®; Anallergo SpA, Florence, Italy) is effective in allergic patients and the mechanisms underlying clinical outcomes. Methods: Eligible patients (n = 28), all suffering from allergic rhinitis with/without bronchial asthma, were consecutively recruited at the Allergology Medical Unit (Novara, Italy) and treated with this probiotic. From each patient, we collected blood and stool samples at the baseline, after 60 days of probiotic supplementation, and after 60 days from probiotic discontinuation. In each blood sample, the percentage of hematopoietic stem cells, eosinophils, and basophils was measured by FACS. To analyze stool microbiota composition, genomic DNA was extracted, bacterial 16S DNA libraries sequenced by Illumina platform (Miseq), and raw sequences processed. Generated data were statistically analyzed. Results: Probiotic-treated patients showed a significant decrease in Average Rhinitis Total Symptom Score (d = −10.5714), and Visual Analog Scale (d = −2.00) clinical indices, as well as important improvements in quality of life. In whole blood, a significant reduction in the percentage of activated eosinophils and basophils was determined, and this effect persisted after specific cell stimulation. Consistently, the serum levels of IL-4 and IL-5 decreased after probiotic treatment, suggesting a reduction in the Th2 cytokine profile. In addition, microbiome genomic analysis (n = 6) showed an increase in microbiome biodiversity, which positively correlates with clinical and cellular data. Conclusion: Present study suggests that iPROB® preparation has clinical/biological properties to be a valid add-on supplementation in allergic patients with asthma and rhinitis.

Angiotensin II modulates the murine hematopoietic stem celland progenitors cocultured with stromal S17 cells.

Although the existence of the renin-angiotensin system (RAS) in the bone marrow is clear, the exact role of this system in hematopoiesis has not yet been fully characterized. Here the direct role of angiotensin II (AngII) in hematopoietic stem cells (HSCs), common myeloid progenitors (CMPs), granulocyte/monocyte progenitors (GMPs), and megakaryocytes/erythroid progenitors (MEPs), using a system of coculture with stromal S17 cells. Flow cytometry analysis showed that AngII increases the percentage of HSC and GMP, while reducing CMP with no effect on MEP. According to these data, AngII increased the total number of mature Gr-1+ /Mac-1+ cells without changes in Terr119+ cells. AngII does not induce cell death in the population of LSK cells. In these populations, treatment with AngII decreases the expression of Ki67+ protein with no changes in the Notch1 expression, suggesting a role for AngII on the quiescence of immature cells. In addition, exposure to AngII from murine bone marrow cells increased the number of CFU-GM and BFU-E in a clonogenic assay. In conclusion, our data showed that AngII is involved in the regulation of hematopoiesis with a special role in HSC, suggesting that AngII should be evaluated in coculture systems, especially in cases that require the expansion of these cells in vitro, still a significant challenge for therapeutic applications in humans.

The combination of Radix Astragali and Radix Angelicae Sinensis attenuates the IFN-\u03b3-induced immune destruction of hematopoiesis in bone marrow cells

BackgroundRadix Astragali and Radix Angelicae Sinensis are two herbs that compose Danggui Buxue Tang (an herbal formula for treatment of anemia diseases). In this study, we explored the molecular mechanism and effective targets to immune destruction of bone marrow (BM) cells treated with Radix Astragali, Radix Angelicae Sinensis or a combination of two agents. The potential synergic advantages of two herbs should also be explored.MethodsThe constituents of Radix Astragali and Radix Angelicae Sinensis were analyzed by high performance liquid chromatography-electrospray ionization/mass spectrometer system BM cells were separated from limbs of BALB/c mice, and immune destruction was induced with IFN-γ. The percentages of hematopoietic stem cells (HSCs) and CD3+ T cells were detected by flow cytometry. The distribution of T-bet and changes in the combination of SAP and SLAM in BM cells were observed by immunofluorescence. Western blotting was used to assay the expression of key molecules of the eIF2 signaling pathway in BM cells.ResultsSeven constituents of Radix Astragali and six constituents of Radix Angelicae Sinensis were identified. The percentages of HSCs increased significantly after treatment with Radix Angelicae Sinensis, especially at high concentrations. The percentages of CD3+ T cells were significantly decreased after Radix Astragali and Radix Angelicae Sinensis treatment. However, the synergistic function of two-herb combinations was superior to that of the individual herbs alone. The distribution of T-bet in BM cells was decreased significantly after Radix Angelicae Sinensis treatment. The number of SLAM/SAP double-stained cells was increased significantly after Radix Astragali treatment at low concentrations. The phosphorylation levels of eIF2α were also reduced after Radix Astragali and Radix Angelicae Sinensis treatment.ConclusionsRadix Astragali and Radix Angelicae Sinensis could intervene in the immunologic balance of T lymphocytes, inhibit the apoptosis of BM cells induced by immune attack, restore the balance of the T cell immune response network and recover the hematopoietic function of HSCs. The synergistic effects of Radix Astragali and Radix Angelicae Sinensis were superior to those of each herb alone.

Structural and Functional Characterization of Deceased Donor Stem Cells: A Viable Alternative to Living Donor Stem Cells.

Stem cells can be isolated from various human tissues including bone marrow (BM) and adipose tissue (AT). Our study outlines a process to isolate adult stem cells from deceased donors. We have shown that cell counts obtained from deceased donor BM were within established living donor parameters. Evaluation of demographic information exhibited a higher percentage of hematopoietic stem cells (HSC) in males versus females, as well as a higher percentage of HSC in the age bracket of 25 years and under. For the first time, we show that deceased donor femur BM grew cell colonies. Our introduction of new technology for nonenzymatic AT processing significantly increased cell recovery over the traditional enzymatic processing method. Cell counts from the deceased donor AT exceeded living donor parameters. Furthermore, our data illustrated that AT from female donors yielded a much higher number of total nucleated cells (TNC) than males. Together, our data demonstrates that our approach to isolate stem cells from deceased donors could be a routine practice to provide a viable alternative to living donor stem cells. This will offer increased accessibility for patients awaiting stem cell therapies.

Low Iron Promotes Megakaryocytic Commitment of Megakaryocytic-Erythroid Progenitors in Human and Mice

The mechanism underlying thrombocythemia in patients with iron deficiency anemia remains unknown. Iron metabolism is highly conserved in humans and mice. We used Tmprss6-/- knockout (KO) mice as a genetic model of chronic iron deficiency anemia to study the effects of iron deficiency on lineage commitment. Tmprss6 is expressed in the liver, but not in hematopoietic cells, and is required for iron uptake from the diet. We observed that Tmprss6-/- mice have elevated platelet counts compared to littermate controls (average x1000/uL ± SD in wild type (WT): 477.9 ± 53.68, Tmprss6-/-: 915.9 ± 57; p<0.001; males and females 6-10 weeks old). Based on immunophenotype, the percentage of hematopoietic stem cells (HSC), megakaryocytic (Mk)-biased HSC (CD41+), megakaryocytic-erythroid progenitor (MEP), (Mk)- or erythroid (E)-committed progenitors in bone marrow (BM) is comparable between the two genotypes. To address whether thrombocythemia in iron deficiency anemia is related to altered commitment of MEP, we assayed the effects of low iron conditions on the function, transcriptome and signaling in MEP.CFU assays of single MEPs showed a striking increase in CFU-Mk (unilineage committed Mk progenitors) within the MEP gate in Tmprss6-/- at the expense of the bipotent CFU-Mk/E compared to WT (p<0.001). Thus, MEP in KO mice are significantly biased toward the Mk lineage, suggesting that low iron content may reprogram cell commitment. Consistent with this reprogramming effect, MEPs from KO mice have a 50% decrease in the labile iron pool (p=0.03, measured by Calcein-AM), and a 30% decrease (p=0.04) in proliferation relative to WT as measured by CFSE.To test the effect of altered iron content in vivo, we transplanted WT or KO BM into sublethally irradiated WT or Tmprss6-/- recipients. Tmprss6-/- recipients (of WT or KO BM) exhibited microcytic anemia and hypoferremia when compared to WT recipients (of WT and KO BM). Interestingly, the Mk-bias observed in non-transplanted Tmprss6-/- MEPs was recapitulated in Tmprss6-/-recipients (regardless of transplanted BM genotype) 6 months post-transplant (p < 0.009, n= 3 per group). We show using index single cell sorting followed by CFU assays that in WT mice, surface expression of transferrin receptor 1 (CD71) is a predictor of cell fate; cells from the MEP gate that grow as BFU-E have a higher CD71 mean fluorescence intensity (MFI) (179.9 ± 38.9) than cells that grow bipotent CFU-Mk/E colonies (65.9 ± 13.9) or Mk-only (CFU-Mk) colonies (24.9 ± 7.06; p<0.006). However, this correlation is lost in KO recipients, where the CD71 expression is elevated in all cells compared to WT (MFI: 279.9 ± 49.81, p<0.0001).RNAseq of WT and KO MEPs showed that 137 genes are differentially expressed (fold change: 1.5; p<0.05) between the two genotypes; of these, 84 are up regulated (e.g. Tfrc and Mybl2) and 53 are downregulated in the KO compared to WT. Surprisingly, mRNA encoding transcription factors that are known to regulate E vs. Mk maturation such as Myb, Tal, Fli1, and Eklf were unchanged. Pathway analysis of the differentially expressed genes highlighted metabolic pathways (asparagine and cholesterol biosynthesis and b-alanine degradation, p<0.008).We then asked if the iron regulation of the MEP commitment observed in iron deficient mice was also true for human MEP. In order to disrupt the iron sensing pathways in primary human MEP, we performed shRNA-based knockdown of Transferrin Receptor 2 (TFR2) using two different sequence targets and found a similar decrease in proliferation (p=0.03) and skewing toward Mk commitment (p<0.04) as was observed in Tmprss6-/- MEPs. Signal transduction analyses revealed that both human and murine MEPs in iron deficient conditions have lower levels of phospho-ERK (Thr202/Tyr204) compared to WT or scrambled controls.Regulation of erythroid differentiation by iron is thought to prevent inappropriate iron utilization when body stores are limited. To function in a protective manner, this checkpoint must act at stages prior to erythropoietin-mediated expansion of erythroid progenitors. Here we propose a model where iron acts on the bipotent progenitor, one possible source of Mk and E cells, to bias lineage commitment. These data support that low iron in the environment affects MEP metabolism, leading to lower ERK phosphorylation, slower proliferation and increased Mk commitment. DisclosuresNo relevant conflicts of interest to declare.

Leukemia cells impair normal hematopoiesis and induce functionally loss of hematopoietic stem cells through immune cells and inflammation

Bone marrow (BM) failure is often seen in leukemia patients, indicating an abnormal hematopoietic process. However, hematopoiesis in leukemic milieus is largely unknown. In the present study, we utilized one of the most frequent leukemogenic translocations MLL-AF9 to induce leukemia and investigated the hematopoiesis and the activity of hematopoietic stem and progenitor cells (HSPCs) in a leukemic milieu. We found that the phenotypes of the non-leukemic population in leukemic BM were drastically different than normal BM, including blockage of differentiation and a drastically reduced Lin-/Sca+/c-kit+ (LSK) population that contains all HSPCs in leukemic BM. Further, transplantation assays demonstrated that stem cell function of HSPCs from leukemic BM was significantly compromised. Intriguingly, BM from a patient-derived xenograft leukemia model and from immunocompromised mice transplanted with murine MLL-AF9 cells, showed comparable percentage of hematopoietic stem cells (HSCs) to normal controls, indicating that an immunocompetent microenvironment is critical for leukemia-induced loss of HSPCs. Mechanistically, we found that the non-leukemic cells from leukemic BM possessed a more inflammatory profile than either leukemic cells or normal BM counterparts. Co-culturing or co-transplantation with non-leukemic cells from leukemic BM impaired the stem cell function of normal HSPCs in vitro and in vivo respectively, suggesting that the highly inflammatory non-leukemic population in leukemic BM not only is functionally abnormal but displayed a ‘leukemia-like’ characteristic. Finally, we tested the effect of the anti-inflammation drug diclofenac on leukemia mice. However, no phenotypic changes of HSPCs were observed upon diclofenac treatment due to only mild repression of inflammatory genes by diclofenac, further indicating that inflammation is a powerful negative regulator of HSPCs. Together, our results suggest that leukemia impairs normal hematopoiesis and inflammation as well as immune cells play a critical role in leukemia-induced BM failure.

Reduced hematopoietic stem cell frequency predicts outcome in acute myeloid leukemia.

In patients with acute myeloid leukemia and low percentages of aldehyde-dehydrogenase-positive cells, non-leukemic hematopoietic stem cells can be separated from leukemic cells. By relating hematopoietic stem cell frequencies to outcome we detected poor overall- and disease-free survival of patients with low hematopoietic stem cell frequencies. Serial analysis of matched diagnostic and follow-up samples further demonstrated that hematopoietic stem cells increased after chemotherapy in patients who achieved durable remissions. However, in patients who eventually relapsed, hematopoietic stem cell numbers decreased dramatically at the time of molecular relapse demonstrating that hematopoietic stem cell levels represent an indirect marker of minimal residual disease, which heralds leukemic relapse. Upon transplantation in immune-deficient mice cases with low percentages of hematopoietic stem cells of our cohort gave rise to leukemic or no engraftment, whereas cases with normal hematopoietic stem cell levels mostly resulted in multi-lineage engraftment. Based on our experimental data, we propose that leukemic stem cells have increased niche affinity in cases with low percentages of hematopoietic stem cells. To validate this hypothesis, we developed new mathematical models describing the dynamics of healthy and leukemic cells under different regulatory scenarios. These models suggest that the mechanism leading to decreases in hematopoietic stem cell frequencies before leukemic relapse must be based on expansion of leukemic stem cells with high niche affinity and the ability to dislodge hematopoietic stem cells. Thus, our data suggest that decreasing numbers of hematopoietic stem cells indicate leukemic stem cell persistence and the emergence of leukemic relapse.

Preservation of Gene Edited Hematopoietic Stem Cells By Transient Overexpression of BCL-2 mRNA

Introduction: Site-specific gene correction of the point mutation causing sickle cell disease (SCD) in hematopoietic stem cells (HSCs) constitutes a precise strategy to generate a life-long source of gene-corrected erythrocytes that do not sickle. However, low efficiency of homology-directed repair (HDR) in primitive reconstituting HSCs is currently a limit to the use of therapeutic genome editing for treatment of severe genetic blood disorders. To identify the mechanism(s) that underlie decreased HDR efficacy in primitive HSCs relative to that in more mature progenitor populations, we assessed: efficiency of gene delivery and expression after electroporation of in vitro transcribed mRNA; functional ZFN-mediated endonuclease activity; cell cycle status; gene expression of key HDR genes; and cytotoxic responses; in the following immunophenotypically-defined human cell populations: HSCs (CD34+/CD38-/CD90+CD45RA-); multipotent progenitors (MPPs) (CD34+/CD38-/CD45RA-/CD90-); and progenitor cells (CD34+/CD38+).Methods: CD34+ cells were enriched from human G-CSF-mobilized peripheral blood and cultured for 1-3 days prior to electroporation of in vitro transcribed mRNA encoding GFP or a pair of zinc finger nucleases (ZFN). The ZFNs, designed to target the sickle mutation in exon 1 of the human beta-globin gene, were co-delivered with one of the homologous donor templates containing the corrective base (A/T): an integrase-deficient lentiviral vector (IDLV) or a 101bp single-stranded oligodeoxynucleotide (oligo). Percentages of alleles containing insertions/deletions (indels) and/or HDR-mediated gene correction were analyzed by high throughput sequencing (HTS). Acute cytotoxicity was determined by flow cytometry, identifying viable cells as 7AAD/AnnexinV neg. cells. To assess HDR-mediated gene correction in vivo after three months, gene-edited cells were transplanted (>1E6 viable CD34+ cells/mouse, I.V.) one day after electroporation into irradiated (250cGy) NOD/SCID/IL2R gamma-/- (NSG) mice.Results: In HSCs, MPPs and progenitor populations, no differences were observed in delivery and expression from electroporated GFP mRNA [%GFP(+) and MFI]. To assess the activity of ZFN mRNA in the stem and progenitor populations, ZFNs were delivered to CD34+ cells through electroporation of in vitrotranscribed mRNA. The CD34+ cells were then FACS-sorted into the respective populations and HTS was used to determine the percentage of alleles containing indels; the frequencies of indels were equivalent among the populations indicating equivalent ZFN mRNA activity. To evaluate the efficacy of site-specific HDR in HSCs and progenitor cells, ZFN mRNA was co-delivered with either an IDLV or an oligodeoxynucleotide donor template to modify the single base-pair involved in SCD. We observed lower percentage of HDR-mediated gene modification in the HSC population compared to progenitors with all donor templates.Due to the cell cycle phase restriction of HDR, we pre-stimulated CD34+ cells for 1-3 days prior to electroporation of ZFN mRNA and the oligo donor, and analyzed the cell cycle phases at the time of electroporation, and the frequencies of HDR and NHEJ produced by HTS. Only a small percentage of the immunophenotypic HSCs were in S/G2 phase after 24 hours of pre-stimulation; no HDR modification was observed in these cells. After 2-3 days of pre-stimulation, the HDR levels increased as the percentage of HSCs in S/G2 phase reached 20%. Importantly, assessment of relative cytotoxicity of the genome editing procedure (electroporation of ZFN mRNA and oligo donor) revealed a heightened sensitivity of HSCs/MPPs compared to progenitors, resulting in ~80% cell death in HSC vs. ~30% in progenitors under the conditions we are using. Transient expression of BCL-2 mRNA, co-electroporated with the genome editing reagents, improved HSC survival and significantly increased the numbers of HDR gene-corrected HSCs both in vitro and in vivo.Conclusions: These data indicate an elevated sensitivity to cytotoxicity from the gene editing process for HSCs compared to the mature progenitor cells under our conditions, which may explain the lower levels of gene modification seen using in vivo compared to in vitro assays. Transient overexpression of BCL-2 mRNA preserves HSC survival after HDR-based gene editing, increasing the frequency of gene-corrected HSCs. DisclosuresBjurström:UCLA: Patents & Royalties: 2016-290. Holmes:Sangamo BioSciences Inc: Employment.

PIM1 Inhibition Enhances and Prolongs Plerixafor-Induced Mobilization of HSCs

IntroductionThe CXCL12/CXCR4 axis was shown to be a major regulator for the interaction of hematopoietic stem cells (HSCs) with the niche and interruption of this pathway mobilizes HSCs from the bone marrow (BM).Therefore CXCR4 antagonists like AMD3100 (Plerixafor) are used in the clinic for the collection of HSCs from patients who fail to mobilize HSCs in response to G-CSF.In our previous studies, we could show that the serine/threonine kinase PIM1 is regulating the surface expression of the CXCR4 receptor on HSCs. In addition, PIM1-deficient HSCs fail to home to a wild type (WT) BM niche. Based on these results, we aimed to improve HSC mobilization by combining CXCR4 and PIM inhibition.MethodsIn order to study the mobilization efficiency in the murine model, mice were treated with AMD3100 alone or in combination with LGB321, a novel pan-PIM inhibitor. The mice were sacrificed at various timepoints and peripheral blood (PB) was isolated. The percentage of HSCs was then determined by flow cytometry. The mechanism of HSC mobilization was studied in isolated HSC and stroma populations by analyzing mRNA levels and surface expression of CXCR4, its ligand CXCL-12 and other factors, which are crucial for the interaction of HSCs and stromal cells.ResultsWe found that CXCR4 inhibition using AMD3100 leads to a compensatory upregulation of CXCR4 surface expression on total BM cells as well as HSCs. This effect can be reverted by deficiency or inhibition of PIM1 (Figure 1A). As a consequence, HSC mobilization using AMD3100 is strongly enhanced and prolonged in Pim1-deficient mice compared to WT animals (Figure 1B). Likewise, treatment of WT animals with AMD3100 in combination with LGB321 leads to increased and prolonged HSC mobilization compared to animals treated only with AMD3100 (Figure 1C). Besides the downregulation of CXCR4 on HSCs, we found that the Cxcl-12 expression as well as CXCR4 surface expression in CXCL12-abundant reticular (CAR) cells is dramatically decreased in Pim1-deficient mice, which even further increases the mobilization capacity of Pim1-deficient mice.ConclusionOur findings indicate, that PIM1 inhibition counteracts the compensatory upregulation of the CXCR4 receptor on HSCs after Plerixafor treatment and decreases CXCL12 levels within the bone marrow niche. Therefore, targeting PIM kinases in combination with CXCR4 inhibition could improve the collection of stem cells in patients at risk for poor mobilization. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A synthetic fragment of leptin increase hematopoietic stem cell population and improve its engraftment ability.

Several studies have shown the important actions of cytokine leptin that regulates food intake and energy expenditure. Additionally, the ability to modulate hematopoiesis has also been demonstrated. Previous reports have shown that some synthetic sequences of leptin molecules can activate leptin receptor. Herein, decapeptides encompassing amino acids from positions 98 to 122 of the leptin molecule were constructed to evaluate their effects on hematopoiesis. Among them, the synthetic peptide Lep(110-119)-NH2 (LEP F) was the only peptide that possessed the ability to increase the percentage of hematopoietic stem cells (HSC). Moreover, LEP F also produced an increase of granulocyte/macrophage colony-forming units and activated leptin receptor. Furthermore, LEP F also improves the grafting of HSC in bone marrow, but did not accelerate the recovery of bone marrow after ablation with 5-fluorouracil. These results show that LEP F is a positive modulator of the in vivo expansion of HSC and could be useful in bone marrow transplantation.

Microrna Mediated Regulation of Hematopoietic Stem Cell Aging

Aging hematopoietic stem cells (HSCs) exhibit numerous functional alterations including reduced capacity for self-renewal, myeloid-biased differentiation, and reduced production of mature lymphocytes and red blood cells. Interventions such as calorie restriction (CR) and rapamycin (Rapa) treatment have been shown to increase lifespan and to delay the onset of age-related diseases, and some studies have demonstrated that they may improve HSC function through poorly defined mechanisms. We and others have shown that microRNAs (miRNAs) are potent cell-intrinsic regulators of HSC self-renewal and lineage specification and also contribute to age-related disorders such as acute myeloid leukemia (AML) and the myelodysplastic syndromes (MDS). We hypothesized that miRNAs may underlie the recovery of HSC function observed in anti-aging mouse models, and thus we characterized miRNA expression profiles from HSCs (Lin-c-Kit+Sca-1+CD34-CD150+) from young mice (12-16 weeks old), old mice (20-22 months), and old mice that had been treated with anti-aging interventions. Evaluation of HSCs from CR and Rapa treated old mice revealed numerous changes consistent with inhibition/reversal of age-related HSC changes including a 5-fold reduction in HSC frequency (p=0.04), 2-fold increase in erythroid progenitors (pro-erythroblasts, p=0.04), 2.5 fold increase in common lymphoid progenitors (CLP; Lin-c-Kit+Sca-1+CD127+FLK2+, p=0.05), as well as 3.5-fold increase in peripheral blood B cells (p=0.002), 2.2 fold decrease in platelets (p=0.01), and increased red blood cells (p=0.04). These changes were associated with statistically significant increases in the percentage of HSCs in S/M/G2 (p=0.045), and undergoing apoptosis (p=0.05). Using a TaqMan-based qPCR expression profiling method evaluating 750 miRNAs, we found that old HSCs exhibited altered expression of 91 miRNAs compared to young (FDR <0.1, P <0.05). Moreover, HSCs from both CR and Rapa treated old mice exhibited expression of 60 miRNAs at levels similar to young, normal HSCs. miR-125b, a miRNA we and others previously showed to positively regulate HSC self-renewal, was reduced 2.2-fold in old mice, and its expression was restored in CR and Rapa treated HSCs. Lentivirally mediated expression of miR-125b in old HSCs increased their long-term reconstitution capacity 8.1-fold compared to control old HSCs based on donor chimerism levels at 16 weeks post-transplantation, resulting in chimerism levels similar to mice transplanted with young HSCs expressing miR-125b. The improved HSC engraftment capacity of old HSCs transduced with miR-125b was accompanied by statistically significant increases in the frequencies of lymphoid biased HSCs (Lin-c-Kit+Sca-1+CD34-CD150neg-low), megakaryocyte-erythroid progenitors (MEPs), CLPs, and peripheral blood B- and T-cells, compared to old HSCs transduced with control lentivirus (p<0.05 for all indicated cell types). While enforced expression of high levels of miR-125b in mouse HSPCs has been reported to induce myeloid leukemias, there was no evidence of a hematologic malignancy in mice transplanted with miR-125b transduced old HSCs up to 6 months post-transplantation. Overall, these results demonstrate that functional HSC aging phenotypes can be that inhibited/reversed by anti-aging interventions, that miR-125b regulates HSC aging, and that anti-aging interventions may exert their positive effects on HSC function by regulating miR-125b expression. DisclosuresNo relevant conflicts of interest to declare.

Exogenous VEGF promotes hematopoietic stem cell mobilization

This study was aimed to investigate the effect of exogenous VEGF on hematopoietic stem cell mobilization and immune system. The C57BL/6J mice were randomly divided into the normal control group, VEGF short-term group (5 d) and VEGF long-term group (27 d). Mice in the experimental group were injected ip with VEGF (100 ng/d); mice in control group were injected ip with PBS. The white blood cell (WBC) count and the ratio of lymphocyte in the peripheral blood at different time point were assayed by hemacytometer. The percentage of hematopoietic stem cell (HSC), lymphocyte subgroup, regulatory T cell (Treg), myeloid-derived suppressor cells (MDSC) in the peripheral blood and spleen of different groups were detected by flow cytometry. The morphological changes of spleen and spleen index of mice in the control and long-term group were observed by microscopy. The results showed that the absolute number of WBC in the peripheral blood of mice significantly increased after injection of VEGF, and the peak value was at day 3. The percentage of Lin(-)Sca-1(+)CD117(+) cells in the peripheral blood and spleen of the long-term group were significantly higher than that in the normal control group (P < 0.05). The spleen of the mice in VEGF long-term group was larger than that of the control group, the spleen index also increased (P < 0.05), and remarkable extramedullary hematopoietic signs were found in the HE stained sections. There was no significant change in the total ratio of lymphocytes in the peripheral blood after injection, but the percentage of CD3(+) cells and the CD3(+)/B220(+) ratio in the long-term group deceased; the percentages of Treg and Gr-1(+)CD11b(+) MDSC in the experimental groups increased (P < 0.05), which more significantly increased in the long-term group than that in the short-term group (P < 0.05). It is concluded that the exogenous VEGF promotes hematopoietic stem cell mobilization, and at same time up-regulates the many kinds of suppressive immune cell levels which leads to changes of immuno-function.

PTU-110 Circulating Bone Marrow-Derived Stem Cells and Stem Cell Factor Serum Level in Chronic Hepatitis C: Relation to Hepatic Proliferation and Fibrosis

IntroductionBone marrow-derived stem cells (BMSCs) are pluripotent cells that can be mobilised into circulation and recruited to sites of inflammation where they promote local tissue repair. Therefore, the present work...

Abstract 365: Circulating bone marrow-derived VEGFR-2+ progenitor cells in benign and malignant soft tissue tumors.

Abstract Background : Circulating endothelial progenitor cells (cEPCs) as recruited to the angiogenic vascular system of malignant tumors have been evaluated as a biomarker of tumor remission or progression through therapy in lung and breast cancer. We were interested to analyze the presence of bone marrow derived (BMD) cEPCs in soft tissue malignancies and correlate the changes to treatment efficacy. Methods: 75 treatment-naïve patients have (41 sarcoma, 21 gastro-intestinal stromal tumors GIST, 12 desmoid) and 11 healthy controls have been enrolled to the study. 25 ml of blood samples were drawn prior to therapy and 4-6 weeks after treatment by 20G iv cannula, collected in tubes containing sodium citrate (0.105 M) and processed within 1 h. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll density gradient centrifugation and cEPCs were characterized by quadruple staining using antibodies against CD133+, CD34, CD45dim and vascular endothelial growth factor-receptor (VEGF-R)-2+. The expression of cell-surface antigens was determined by four-color immunofluorescence. The frequency of cEPCs in peripheral blood was determined by a 2-dimensional side-scatter/fluorescence dot-plot analysis after exclusion of annexin V-positive cells and appropriate to exclude apoptotic cells. EPC counts are expressed as a percentage of total PBMCs. Serum-concentrations of VEGF and Angiopoetin-2 were determined by ELISA in triplicate samples from 5 ml serum. Results:The percentage of hematopoietic stem cells (CD34+, CD133+) was significantly increased in sarcoma and desmoid patients (0.44 ± 0.1 %) compared to GIST and healthy controls (0.13 ± 0.04 %; p=0.001). The percentage of VEGFR-2+ cells correspond to cEPC in sarcoma of 0.179 ± 0.190 % vs. healthy controls of 0.025 ± 0.018 %. Values for desmoid patients were similar to sarcoma whereas GIST patients showed values comparable to healthy patients. We found significantly increased mean VEGF levels in both sarcoma and desmoids (359 ± 157 pg/ml) in comparison to healthy controls (27 ± 13 pg/ml; p = 0.001). Ang-2 levels before therapy were significantly lower in healthy controls and desmoids (1665 ± 445 pg/ml) compared to sarcoma and GIST (2929 ± 960 pg/ml, p = 0.033) patients. Patients without signs of tumor recurrence showed normal cEPC values at post-treatment controls, whereas patients with progressive disease had increasing cEPC levels. Conclusion: This study demonstrates an increased mobilization of cEPCs in sarcoma and (non-metatasizing) desmoids tumors compared to healthy individuals and patients with GIST. cEPCs recruited for vasculogenesis and paracrine effects play a different role in the various subtypes of human soft tissue tumors. Citation Format: Kai Nowak, Elena Joas, Grietje Beck, Neysan Rafat, Peter Hohenberger. Circulating bone marrow-derived VEGFR-2+ progenitor cells in benign and malignant soft tissue tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 365. doi:10.1158/1538-7445.AM2013-365

Improvement of ginsenoside Rg1 on hematopoietic function in cyclophosphamide-induced myelosuppression mice

This study was aimed to investigate the effects of ginsenoside Rg1 (Rg1) on hematopoietic function of bone marrow in cyclophosphamide-induced bone marrow depression mice. Mice were given cyclophosphamide (150mg/kg, i.p. for three days) to produce bone marrow depression. Rg1 was then administrated at 7.5 and 15mg/kg by i.p. for seven days. Bone marrow cells number was counted, and the percentage of hematopoietic stem cells (Lin−Sca-1+c-kit+) was quantified by flow cytometry. The histology of femoral bone was examined by H&E staining. The expression of calcium-sensing receptor mRNA was determined by the real time RT-PCR. Rg1 (7.5 and 15mg/kg) protected against cyclophosphamide-induced bone marrow depression, as evidenced by increased bone marrow cell numbers and improved femoral bone morphology. The percentage of Lin−Sca-1+c-kit+ cells and lymphoid lineage CD3+ cells were lower in cyclophosphamide group, but returned towards normal after Rg1 treatment in both bone marrow and peripheral blood cells. Expression of calcium-sensing receptor mRNA was increased in bone marrow cells on the 10th day after cyclophosphamide, but it was returned to normal level after Rg1 treatment. Rg1 alone did not produce these changes in normal mice. These results demonstrated that Rg1 improved hematopoietic function of bone marrow in cyclophosphamide-induced myelosuppression.