CD34 Hematopoietic Stem Cells Research Articles

Hyperglycemia epigenetically propels senescence-associated secretory phenotype of CD34 hematopoietic stem cells and their myeloid expansion into inflammatory monocyte subpopulations

Abstract Background The "leitmotif "underlying the cardiovascular disease initiation and progression in diabetic patients is the chronic inflammation in which monocytes/macrophages are the key players [1]. Diabetes mellitus (DM) showed to alter hematopoiesis, by promoting myelopoiesis and the generation of monocyte subsets with inflammatory and atherogenic phenotype [2-4]. Epigenetic modifications have been described in the inflammation and in control of monocyte-derived macrophage polarization [5,6]. However, there is no notion of when these epigenetic modifications take place and whether can be transferred to more differentiated cells. Purpose The aims of the study were 1) to assess in vitro if high glucose (HG) exposure promotes epigenetic changes and alteration in myeloid differentiation of hematopoietic stem/progenitor cells HSPCs; 2) if such epigenetic modifications are transferred to cell progeny and 3) if similar mechanisms are at work in bone marrow (BM)-derived HSPCs of DM patients. Methods HSPCs were isolated form the cord blood (CB) and from BM of DM patients. CB-HSPCs were grown in normal-glucose (NG;30 mM mannitol) or HG (30 mM) conditions and counted after 10, 20 and 30 days. The expression of p27, p21, IL6, TNFα, RELA/p65, KAT2B/PCAF, SetD7 genes and telomere length was assessed by qPCR; cytokines by ELISA. Western Blot was used to evaluate NFkB-p65 expression and acetylation at lysine-310. H3K9me3, H3K4me1 modifications and SetD7, RNA polymerase II at RELA/p65 gene promoter were assessed by ChIP-qPCR assay. Flow cytometry was used to assess ROS, CD14/CD16 monocyte subpopulations and NFkB-p65 nuclear translocation. Results HG-exposure of CB-HSPCs induced a senescent-associated secretory phenotype characterized by cell proliferation lowering, ROS production, telomere shortening, up-regulation of p21 and p27 genes, increased secretion of TNFα and IL6 cytokines, and upregulation of NFkB-p65 transcription factor (fig.1). NFkB-p65 gene promoter analysis showed H3K9me3 and H3K4me1 histone mark alterations associated with increased SetD7 and RNA polymerase II recruitment. Importantly, the upregulation of KAT2B gene linked with increased lysine-310 acetylation, nuclear translocation and binding activity of NFkB-p65 (fig.1). HG-HSPCs, once differentiated into myeloid lineage, generated higher level of intermediate (CD14++CD16+) proinflammatory monocyte subpopulations (Fig.2). Similarly, BM-HSPCs from DM patients displayed a senescent-associated secretory phenotype and abnormal intermediate (CD14++CD16+) monocyte output when differentiated into myeloid lineage. Importantly, epigenetic changes at the level of NFkB-p65 gene promoter persisted in both HG-HSPC- and DM-HSPC-derived monocytes populations (fig.2). Conclusions Overall, our data show that hyperglycemia elicits epigenetic changes in HSPCs that persist in monocyte progeny and are potentially involved in their inflammatory phenotype.fig.1fig.2

Comparison of the effect of Sumbawa honey and sucrose as extracellular cryoprotectants on viability, morphology, and phenotype stability of CD34+ hematopoietic stem cells from umbilical cord blood

Cryopreservation is a common practice for the long-term storage and maintenance of the quality of Hematopoietic Stem Cells (HSC) from Umbilical Cord Blood (UCB). Cryomedium containing 10% DMSO is the standard intracellular cryoprotectant agent (CPA) in HSC cryopreservation. However, DMSO is toxic to both cells and patients during transplantation. Therefore, the concentration of DMSO needs to be reduced by adding extracellular CPAs, such as sucrose or Sumbawa honey. The objective of this study was to compare the ability of Sumbawa honey and sucrose as extracellular CPAs to protect HSC CD34+ during cryopreservation. This in vitro study was designed using cryomedium consisting of 10% DMSO as a control, 5% DMSO + 5% Sumbawa honey, and 5% DMSO + 5% sucrose. The results showed that the cryoprotectant containing 5% DMSO + 5% Sumbawa honey had a positive effect and a significant difference (p<0.05) compared with 5% DMSO + 5% sucrose on the viability and morphology of HSC. However, the mean reduction in phenotype stability as indicated by the decrease in percentage CD34+ in the 10% DMSO (6.90 ± 8.60), 5% DMSO + 5% sucrose (10.60 ± 9.20), and 5% DMSO + 5% Sumbawa honey (8.60 ± 11.50) showed no significant difference (p>0.05). In conclusion, the combination of DMSO and Sumbawa honey was able to maintain the viability, morphology, and phenotype stability of HSC. Therefore, honey can be used as an alternative cryoprotectant for the cryopreservation of hematopoietic stem cells.

Characterize Biomarkers and Mechanisms of Resistance for MDM2 Inhibitors in AML

Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous disease with an aggressive phenotype and poor prognosis. Inactivation of the wild-type p53, through upregulation of the TP53 negative regulator MDM2, is observed in around 50% of AML patients, providing a rationale for targeting MDM2 for the treatment of AML. Small molecule inhibitors of MDM2, including Idasanutlin, have shown efficacy as monotherapy and in combinations. However, patients exhibit heterogeneous responses with frequent drug resistance. We integrated ex vivo drug sensitivity assay, clinical parameters, whole exome sequence (WES), RNAseq, cytokine stimulation, and CRISPR/Cas9 dropout screening data to identify biomarkers that predict idasanutlin response and to elucidate the underlying drug-resistant mechanisms. Surprisingly, besides TP53 itself, we did not find an essential role in losing any of the major TP53 downstream targets alone leading to Idasanutlin resistance from Idasanutlin treated resistant cell lines, CRISPR screening, or individually targeted knockout experiments. Besides TP53 mutations, other major AML mutations, including RAS pathway mutations do not drive Idasanutlin resistance indicated by the patient sample functional screening data and in vitro mutation overexpression models. We observed instead that AML with a high differentiation or mature myeloid signature: including low blast, a high percentage of monocytes/granulocytes/T cells, AML FAB M4/M5 morphology, and a high expression of a myeloid immune signature gene are relatively resistant to Idasanutlin. We further characterized that both healthy/leukemia-associated T cells, granulocytes, and monocytes are intrinsically resistant to idasnutlin due to low expression levels of MDM2 and/or TP53. To examine whether the presence of differentiated cells protects leukemia blast cells from Idasanutlin, we performed a co-culture drug treatment experiment. Strikingly, only leukemia-associated monocytes, but not healthy monocytes, healthy/leukemia T cells or granulocytes, or leukemia blast cells protected leukemia blast cells from apoptosis in the presence or absence of Idasanutlin. Similar results were obtained for the other two MDM2 inhibitors, AMG232 and DS-3032b, and a BCL2 inhibitor, venetoclax. To further uncover which cytokines secreted from the leukemia-associated monocytes confer MDM2 inhibitor resistance, we tested 11 common cytokines, including IL-1a, IL-1b, IL-3, IL-6, IL-8, M-CSF GM-CSF, FLT3, MCP-1, TNFa, and SCF. We observed that IL-1α, IL-1β, IL-3, and GM-CSF markedly induced idasanutlin treatment resistance for AML, but not ALL or CLL patient cells. Interestingly, IL-1α and IL-1β only protect leukemia blasts, but not healthy CD34 hematopoietic stem and progenitor cells from Idasanutlin-induced apoptosis. We further performed RNAseq and immunoblots on IL-1α and IL-1β treated AML, CLL, and ALL cells. We observed the major TP53 downstream pathways remain activated by idasanutlin in the presence of IL-1α and IL-1β. Interestingly, GATA2 is significantly upregulated in IL-1α and IL-1β treated AML, but not CLL or ALL cells in the presence or absence of Idasanutlin. Furthermore, overexpressing GATA2 conferred Idasanutlin resistance at high concentrations. Finally, we observed that combining a FLT3/IRAK dual inhibitor, pacritinib, or an IRAK1 /IL-1 receptor antagonist, anakinra, partially rescued IL-1α and IL-1β mediated drug resistance of MDM2 inhibitors. As such, we uncovered the role of leukemia-associated monocyte in driving intrinsic and extrinsic MDM2 inhibitor resistance and the potential underlying mechanisms.

Effect of Replacement of Wharton Acellular Jelly With FBS on the Expression of Megakaryocyte Linear Markers in Hematopoietic Stem Cells CD34.

Animal environments for the growth of stem cells cause the transmission of some diseases and immune problems for the recipient. Accordingly, replacing these environments with healthy environments, at least with human resources, is essential. One of the media that can be used as an alternative to animal serums is Wharton acellular jelly (AWJ). Therefore, in this study, we intend to replace FBS with Wharton jelly and investigate its effect on the expression of megakaryocyte-related genes and markers in stem cells. In this study, cord blood-derived CD34 positive HSCs were cultured and expanded in the presence of cytokines including SCF, TPO, and FLT3-L. Then, the culture of expanded CD34 positive HSCs was performed in two groups: 1) IMDM culture medium containing 10% FBS and 100 ng / ml thrombopoietin cytokine 2) IMDM culture medium containing 10% AWJ, 100 ng / ml thrombopoietin cytokine. Finally, CD41 expressing cells were analyzed with the flow cytometry method. The genes related to megakaryocyte lineage including FLI1 and GATA2 were also evaluated using the RT-PCR technique. Results: The expression of CD41, a specific marker of megakaryocyte lineage in culture medium containing Wharton acellular jelly was increased compared to the FBS group. Additionally, the expression of GATA2 and FLI1 genes was significantly increased related to the control group. This study provided evidence of differentiation of CD34 positive hematopoietic stem cells from umbilical cord blood to megakaryocytes in a culture medium containing AWJ.<br />.

Isolation and characterization of mesenchymal stem cells from umbilical cord of giant panda

Umbilical cord-derived mesenchymal stem cells (UC-MSCs) constitute a class of cells with significant self-renewal and multilineage differentiation properties and have great potential for therapeutic applications and the genetic conservation of endangered animals. In this study, we successfully isolated and cultured UC-MSCs from the blood vessels of giant panda umbilical cord (UC). The cells were arranged in a vortex or cluster pattern and exhibited a normal karyotype, showing the morphological characteristics of fibroblasts. In addition, we found that basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) promoted cell proliferation, whereas stem cell factor (SCF) did not promote cell proliferation. Cultured UC-MSCs were negative for CD34 (hematopoietic stem cell marker) and CD31 (endothelial cell marker), but positive for MSC markers (CD44, CD49f, CD105, and CD73) and stem cell markers (KLF4, SOX2, and THY1). Similar to other MSCs, giant panda UC-MSCs have multiple differentiation ability and can differentiate into adipocytes, osteoblasts and chondrocytes. Giant panda UC-MSCs are new resources for basic research as cell models following their differentiation into different cell types and for future clinical treatments of giant panda diseases.

The Effect of Different Doses of Melatonin on in Vitro Maturation of Human Follicular Fluid-Derived Oocyte-Like Cells.

ObjectiveHuman follicular fluid (FF) contains different cell populations including mesenchymal stem cells. Studies tried to improve their differentiation to oocyte and use them in infertility treatments. Using an antioxidant may improve the quality of these cells. The present study investigated the effects of different doses of melatonin on FF-derived cells grown to oocyte-like cells (OLC).MethodsCell viability (MTT assay), flow cytometry, and ICC staining were utilized to evaluate CD105 and CD34 expression; colony forming unit assay (CFU-F) capability, qRT-PCR were used to investigate ZP1, ZP2, ZP3, GDF9, and SCP3 expression. AMH, Estradiol and Progesterone levels in the supernatant were measured. Morphological characteristics of fibroblast-like cells changing to a round shape were seen specifically in the group treated with melatonin 10-7M after 2 weeks.ResultsThere was no difference between control and treatment groups for MTT and CFU assays. ICC staining was positive for CD105 marker and negative for CD34 hematopoietic stem cell marker. qRT-PCR results indicated that ZP1, ZP2, GDF9, and SCP3 expression increased in the group treated with melatonin 10-7M in Week 2, while ZP3 decreased in this group. Progesterone and AMH were detected in differentiation medium.ConclusionsMelatonin may improve in vitro formation of OLCs.

Comparative Somatic Mutational Profiling of CD34+ Hematopoietic Precursors (HSC) and Circulating Endothelial Cells (CEC) in Patients with Primary Myelofibrosis (PMF)

Comparative Somatic Mutational Profiling of CD34+ Hematopoietic Precursors (HSC) and Circulating Endothelial Cells (CEC) in Patients with Primary Myelofibrosis (PMF)

PB2385 EX VIVO EXPANSION OF HUMAN UMBILICAL CORD BLOOD HEMATOPOIETIC STEM CELLS WITH VALPROIC ACID OR NICOTINAMIDE IS ABLE TO ACCELERATE ENGRAFTMENT IN A NSG MOUSE TRANSPLANT MODEL

Background:The major obstacle against wider use of cord blood (CB) hematopoietic stem cells (HSC) is low level of HSC counts causing delay in both hematopoietic and immunological recovery. In recent years, HSC expansion strategies with small molecules ie Valproic acid (VPA) or nicotinamide (NAM) have yielded the most promising results. In Ankara University Cord Blood Bank, we have previously demonstrated increase in CD34 content and CFU‐GM in the presence of VPA and/or NAM.Aims:To ascertain the functionality of expanded HSCs, we have initiated a mice stem cell transplant model using non‐obese diabetic (NOD)Cg‐Prkdcscid IL2rgtmWjl stocks of Rag2null mice (NSG mice).Methods:Following consent CD34+ cells were isolated by positive selection using MACS CD34 MicroBead Kit‐UltraPure system (Miltenyi Biotec) from fresh CBUs collected at Ankara University or Hacettepe University, Departments of Obstetrics and Gynecology. Subsequently, 3.500 CD34+ cells per ml were plated in StemSpan medium in the presence of cytokine cocktail containing SCF, Flt3L, IL‐3 and IL‐6 (StemCell Technologies) w/wo VPA (1 mM) and/or NAM (5 mM). VPA, NAM and VPA + NAM added cultures were incubated at 37 °C and 5% CO2 for 8 days, 21 days and either 8 or 21 days, respectively. Following incubation CD34+ cells were washed and evaluated against their colony forming capacities using MethoCult Enriched wo EPO (StemCell Technologies) medium and cell viabilities were detected with 7‐AAD and HSC phenotyping were performed by flow cytometry (Beckman Coulter) using labeled CD34 and CD45 monoclonal antibodies. Expanded cells were transplanted to 8‐ to 12 weeks old NOD.Cg‐Prkdcscid IL2rgtmWjl/Sz (NSG) mice, purchased from The Jackson Laboratory and maintained under specific pathogen‐ free conditions at Laboratory of Animal Research Center in Ankara University. 24 hours prior to transplantation, mice were injected with busulfan through the tail vein (25 mg/kg, 50–100 g per dose). The next day, stem cell harvests (2,5 × 104 ‐ 5 × 105 total nucleated cells (TNC) suspended in 200 μl PBS) were transplanted by intravenous injection. Following transplantation complete blood count was performed on peripheral blood drawn from the tail vein in every 3‐7 days. As control unexpanded CD34 HSCs and cytokine only expanded HSCs were used.Results:Two CBUs were successfully expanded with or without VPA and/or NAM in the presence of cytokine cocktail. All expanded HSCs were found to be competent on CFU‐GM assays. Either freshly isolated or expanded HSCs were transplanted into 12 mice in total. One mouse was transplanted with non‐expanded HSCs; 2 mice with cytokine‐only‐expanded HSCs; 2 mice with VPA‐expanded HSCs and 2 mice with VPA + NAM expanded HSCs on the 8th day of the expansion. 2 mice with NAM‐expanded HSCs and 1 mouse with VPA + NAM expanded HSCs on the 21st day of expansion. WBC counts of mice in different time points are shown in Figure 1.Summary/Conclusion:Our preliminary findings show that expanded CB HSCs are capable of increasing the peripheral WBC counts. VPA and/or NAM mediated expanded HSC led to an increase in WBC counts (&gt;1 × 109/L) earlier than the non‐transplanted or unexpanded HSC transplanted mice. Since VPA mediated expansion can be achieved within 8 days, VPA‐expanded HSCs seem to be the most rapid way to achieve early engraftment in vivo. image

Arsenic Trioxide Enhances the NK Cell Cytotoxicity Against Acute Promyelocytic Leukemia While Simultaneously Inhibiting Its Bio-Genesis

Natural killer cells (NK) contribute significantly to eradication of cancer cells, and there is increased interest in strategies to enhance it’s efficacy. Therapeutic agents used in the treatment of cancer can impact the immune system in a quantitative and qualitative manner. In this study, we evaluated the impact of arsenic trioxide (ATO) used in the management of acute promyelocytic leukemia (APL) on NK cell reconstitution and function. In patients with APL treated with single agent ATO, there was a significant delay in the reconstitution of circulating NK cells to reach median normal levels from the time of diagnosis (655 days for NK cells vs 145 and 265 days for T cells and B cells, respectively). In vitro experiments demonstrated that ATO significantly reduced the CD34 hematopoietic stem cell (HSC) differentiation to NK cells. Additional experimental data demonstrate that CD34+ sorted cells when exposed to ATO lead to a significant decrease in the expression of IKZF2, ETS1, and TOX transcription factors involved in NK cell differentiation and maturation. In contrast, exposure of NK cells and leukemic cells to low doses of ATO modulates NK cell receptors and malignant cell ligand profile in a direction that enhances NK cell mediated cytolytic activity. We have demonstrated that NK cytolytic activity toward NB4 cell line when exposed to ATO was significantly higher when compared with controls. We also validated this beneficial effect in a mouse model of APL were the median survival with ATO alone and ATO + NK was 44 days (range: 33–46) vs 54 days (range: 52–75). In conclusion, ATO has a differential quantitative and qualitative effect on NK cell activity. This information can potentially be exploited in the management of leukemia.

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OBJECTIVES/SPECIFIC AIMS: To date, only 1 documented case of an individual cured of HIV has been reported. He received an allogeneic bone marrow transplant with cells harboring an HIV-resistant genotype. To mimic this result, we have initiated a Phase I to evaluate the safety of an autologous stem cell gene therapy bone marrow transplant in HIV-related lymphoma patients. METHODS/STUDY POPULATION: The first cohort of patients will receive a 1:1 ratio of unmanipulated CD34 hematopoietic stem cells (HSC) and lentivector modified CD34 HSC expressing a combination of HIV-resistant genes and a selectable marker for cell sorting prior to transplantation. Safety of the HIV-resistant stem cells will be assessed by evaluating engraftment, expression of the anti-HIV genes, and the stability and sequence of the vector. RESULTS/ANTICIPATED RESULTS: One patient has been enrolled and transplanted with the HIV-resistant stem cells. After 1 and 2 months post-transplant, patient blood samples were received, processed for genomic DNA, analyzed by quantitative PCR (qPCR), and displayed successful engraftment of 16 and 12 vector copies per 100 cells, respectively. Expression of all anti-HIV genes was confirmed by qPCR. PCR on genomic DNA confirmed the correct sizes and sequences of the integrated vector and confirmed the successful engraftment of our gene modified cells. Currently, we are enrolling more patients into the trial. DISCUSSION/SIGNIFICANCE OF IMPACT: If successful, this therapy has the potential to change HIV treatment.

TALEN-Mediated Knockout of CCR5 Confers Protection Against Infection of Human Immunodeficiency Virus.

Transcription activator-like effector nuclease (TALEN) represents a valuable tool for genomic engineering due to its single-nucleotide precision, high nuclease activity, and low cytotoxicity. We report here systematic design and characterization of 28 novel TALENs targeting multiple regions of CCR5 gene (CCR5-TALEN) which encodes the co-receptor critical for entry of human immunodeficiency virus type I (HIV-1). By systemic characterization of these CCR5-TALENs, we have identified one (CCR5-TALEN-515) with higher nuclease activity, specificity, and lower cytotoxicity compared with zinc-finger nuclease (CCR5-ZFN) currently undergoing clinical trials. Sequence analysis of target cell line GHOST-CCR5-CXCR4 and human primary CD4 T cells showed that the double-strand breaks at the TALEN targeted sites resulted in truncated or nonfunctional CCR5 proteins thereby conferring protection against HIV-1 infection in vitro. None of the CCR5-TALENs had detectable levels of off-target nuclease activity against the homologous region in CCR2 although substantial level was identified for CCR5-ZFN in the primary CD4 T cells. Our results suggest that the CCR5-TALENs identified here are highly functional nucleases that produce protective genetic alterations to human CCR5. Application of these TALENs directly to the primary CD4 T cells and CD34 hematopoietic stem cells (HSCs) of infected individuals could help to create an immune system resistant to HIV-1 infection, recapitulating the success of "Berlin patient" and serving as an essential first step towards a "functional" cure of AIDS.

Humanized mice efficiently engrafted with fetal hepatoblasts and syngeneic immune cells develop human monocytes and NK cells

Human liver chimeric mice are useful models of human hepatitis virus infection, including hepatitis B and C virus infections. Independently, immunodeficient mice reconstituted with CD34(+) hematopoietic stem cells (HSC) derived from fetal liver reliably develop human T and B lymphocytes. Combining these systems has long been hampered by inefficient liver reconstitution of human fetal hepatoblasts. Our study aimed to enhance hepatoblast engraftment in order to create a mouse model with syngeneic human liver and immune cells. The effects of human oncostatin-M administration on fetal hepatoblast engraftment into immunodeficient fah(-/-) mice was tested. Mice were then transplanted with syngeneic human hepatoblasts and HSC after which human leukocyte chimerism and functionality were analyzed by flow cytometry, and mice were challenged with HBV. Addition of human oncostatin-M enhanced human hepatoblast engraftment in immunodeficient fah(-/-) mice by 5-100 fold. In contrast to mice singly engrafted with HSC, which predominantly developed human T and B lymphocytes, mice co-transplanted with syngeneic hepatoblasts also contained physiological levels of human monocytes and natural killer cells. Upon infection with HBV, these mice displayed rapid and sustained viremia. Our study provides a new mouse model with improved human fetal hepatoblast engraftment and an expanded human immune cell repertoire. With further improvements, this model may become useful for studying human immunity against viral hepatitis. Important human pathogens such as hepatitis B virus, hepatitis C virus and human immunodeficiency virus only infect human cells which complicates the development of mouse models for the study of these pathogens. One way to make mice permissive for human pathogens is the transplantation of human cells into immune-compromised mice. For instance, the transplantation of human liver cells will allow the infection of these so-called "liver chimeric mice" with hepatitis B virus and hepatitis C virus. The co-transplantation of human immune cells into liver chimeric mice will further allow the study of human immune responses to hepatitis B virus or hepatitis C virus. However, for immunological studies it will be crucial that the transplanted human liver and immune cells are derived from the same human donor. In our study we describe the efficient engraftment of human fetal liver cells and immune cells derived from the same donor into mice. We show that liver co-engraftment resulted in an expanded human immune cell repertoire, including monocytes and natural killer cells in the liver. We further demonstrate that these mice could be infected with hepatitis B virus, which lead to an expansion of natural killer cells. In conclusion we have developed a new mouse model that could be useful to study human immune responses to human liver pathogens.

The frequency of multipotent CD133(+)CD45RA(-)CD34(+) hematopoietic stem cells is not increased in fetal liver compared with adult stem cell sources.

The cell surface marker CD133 has been used to describe a revised model of adult human hematopoiesis, with hematopoietic stem cells and multipotent progenitors (HSCs/MPPs: CD133(+)CD45RA(-)CD34(+)) giving rise to lymphomyeloid-primed progenitors (LMPPs: CD133(+)CD45RA(+)CD34(+)) and erythromyeloid progenitors (EMPs: CD133(low)CD45RA(-)CD34(+)). Because adult and fetal hematopoietic stem and progenitor cells (HSPCs) differ in their gene expression profile, differentiation capabilities, and cell surface marker expression, we were interested in whether the reported segregation of lineage potentials in adult human hematopoiesis would also apply to human fetal liver. CD133 expression was easily detected in human fetal liver cells, and the defined hematopoietic subpopulations were similar to those found for adult HSPCs. Fetal HSPCs were enriched for EMPs and HSCs/MPPs, which were primed toward erythromyeloid differentiation. However, the frequency of multipotent CD133(+)CD45RA(-)CD34(+) HSPCs was much lower than previously reported and comparable to that of umbilical cord blood. We noted that engraftment in NSG (NOD scid gamma [NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ])mice was driven mostly by LMPPs, confirming recent findings that repopulation in mice is not a unique feature of multipotent HSCs/MPPs. Thus, our data challenge the general assumption that human fetal liver contains a greater percentage of multipotent HSCs/MPPs than any adult HSC source, and the mouse model may have to be re-evaluated with respect to the type of readout it provides.

Possible biological and translational significance of mast cells density in colorectal cancer.

Mast cells (MCs), located ubiquitously near blood vessels, are descended from CD34(+) hematopoietic stem cells. Initially, although their role has been well defined in hypersensitivity reactions, the discovery of their sharing in both innate and adaptive immunity has allowed to redefine their crucial interplay on the regulatory function between inflammatory and tumor cells through the release of mediators granule-associated (mainly tryptase and vascular endothelial growth factor). In particular, in several animal and human malignancies it has been well demonstrated that activated c-Kit receptor (c-KitR) and tryptase (an agonist of the proteinase-activated receptor-2) take pivotal part in tumor angiogenesis after the MCs activation, contributing to tumor cells invasion and metastasis. In this review, we focused on crucial MCs density (MCD) role in colorectal cancer (CRC) development and progression angiogenesis-mediated; then, we will analyze the principal studies that have focused on MCD as possible prognostic factor. Finally, we will consider a possible role of MCD as novel therapeutic target mainly by c-KitR tyrosine kinase inhibitors (imatinib, masitinib) and tryptase inhibitors (gabexate and nafamostat mesylate) with the aim to prevent CRC progression.

High-affinity FRβ-specific CAR T cells eradicate AML and normal myeloid lineage without HSC toxicity.

Acute myeloid leukemia (AML) is an aggressive malignancy, and development of new treatments to prolong remissions is warranted. Chimeric antigen receptor (CAR) T-cell therapies appear promising but on-target, off-tumor recognition of antigen in healthy tissues remains a concern. Here, we isolated a high affinity (HA) folate receptor beta (FRβ)-specific scFv (2.48nM KD) for optimization of FRβ-redirected CAR T-cell therapy for AML. T-cells stably expressing the HA-FRβ CAR exhibited greatly enhanced antitumor activity against FRβ+ AML in vitro and in vivo compared to a low affinity (LA) FRβ CAR (54.3nM KD). Using the HA-FRβ IgG, FRβ expression was detectable in myeloid-lineage hematopoietic cells; however, expression in CD34+ hematopoietic stem cells (HSCs) was nearly undetectable. Accordingly, HA-FRβ CAR T-cells lysed mature CD14+ monocytes, while HSC colony formation was unaffected. Because of the potential for elimination of mature myeloid lineage, mRNA CAR electroporation for transient CAR expression was evaluated. mRNA-electroporated HA-FRβ CAR T-cells retained effective anti-tumor activity in vitro and in vivo. Together, our results highlight the importance of antibody affinity in target protein detection and CAR development and suggest that transient delivery of potent HA-FRβ CAR T-cells is highly effective against AML and reduces the risk for long-term myeloid toxicity.

Helicase-like transcription factor is a RUNX1 target whose downregulation promotes genomic instability and correlates with complex cytogenetic features in acute myeloid leukemia.

Helicase-like transcription factor is a SWI/SNF chromatin remodeling factor involved in various biological processes. However, little is known about its role in hematopoiesis. In this study, we measured helicase-like transcription factor mRNA expression in the bone marrow of 204 adult patients with de novo acute myeloid leukemia. Patients were dichotomized into low and high expression groups at the median level for clinicopathological correlations. Helicase-like transcription factor levels were dramatically reduced in the low expression patient group compared to those in the normal controls (n=40) (P<0.0001). Low helicase-like transcription factor expression correlated positively with French-American-British M4/M5 subtypes (P<0.0001) and complex cytogenetic abnormalities (P=0.02 for ≥3 abnormalities;P=0.004 for ≥5 abnormalities) but negatively with CEBPA double mutations (P=0.012). Also, low expression correlated with poorer overall (P=0.005) and event-free (P=0.006) survival in the intermediate-risk cytogenetic subgroup. Consistent with the more aggressive disease associated with low expression, helicase-like transcription factor knockdown in leukemic cells promoted proliferation and chromosomal instability that was accompanied by downregulation of mitotic regulators and impaired DNA damage response. The significance of helicase-like transcription factor in genome maintenance was further indicated by its markedly elevated expression in normal human CD34(+)hematopoietic stem cells. We further demonstrated that helicase-like transcription factor was a RUNX1 target and transcriptionally repressed by RUNX1-ETO and site-specific DNA methylation through a duplicated RUNX1 binding site in its promoter. Taken together, our findings provide new mechanistic insights on genomic instability linked to helicase-like transcription factor deregulation, and strongly suggest a tumor suppressor function of the SWI/SNF protein in acute myeloid leukemia.

Generation of Humanized Mice for Analysis of Human Dendritic Cells.

Transplantation of human CD34(+) hematopoietic stem and progenitor cells into severe immunocompromised newborn mice allows the development of a human hemato-lymphoid system (HHLS) including dendritic cells (DCs) in vivo. Therefore, it can be a powerful tool to study human DC subsets, residing in different lymphoid and nonlymphoid organs. We have recently generated novel mouse strains called human cytokine knock-in mice in which human versions of several cytokines are knocked into Rag2(-/-)γC(-/-) strains. In addition, human SIRPα, which is a critical factor to prevent donor cell to be eliminated by host macrophages, is expressed as transgene. These mice efficiently support human myeloid cell development and, indeed, allow the analysis of three major subsets of human DC lineages, plasmacytoid DCs and CD1c(+) and CD141(+) classical DCs. Moreover, these strains also support cytokine-mobilized peripheral blood CD34(+) cell engraftment and subsequent DC development. Here we describe our standard methods to characterize DCs developed in human cytokine knock-in mice.

Humanizing NOD/SCID/IL-2Rγnull (NSG) mice using busulfan and retro-orbital injection of umbilical cord blood-derived CD34(+) cells.

BackgroundHumanized mouse models are still under development, and various protocols exist to improve human cell engraftment and function.MethodsFourteen NOD/SCID/IL-2Rγnull (NSG) mice (4‒5 wk old) were conditioned with busulfan and injected with human umbilical cord blood (hUCB)-derived CD34+ hematopoietic stem cells (HSC) via retro-orbital sinuses. The bone marrow (BM), spleen, and peripheral blood (PB) were analyzed 8 and 12 weeks after HSC transplantation.ResultsMost of the NSG mice tolerated the regimen well. The percentage of hCD45+ and CD19+ cells rose significantly in a time-dependent manner. The median percentage of hCD45+cells in the BM was 55.5% at week 8, and 67.2% at week 12. The median percentage of hCD45+ cells in the spleen at weeks 8 and 12 was 42% and 51%, respectively. The median percentage of hCD19+ cells in BM at weeks 8 and 12 was 21.5% and 39%, respectively (P=0.04). Similarly, the median percentage of hCD19+ cells in the spleen at weeks 8 and 12 was 10% and 24%, respectively (P=0.04). The percentage of hCD19+ B cells in PB was 23% at week 12. At week 8, hCD3+ T cells were barely detectable, while hCD7+ was detected in the BM and spleen. The percentage of hCD3+ T cells was 2‒3% at week 12 in the BM, spleen, and PB of humanized NSG mice.ConclusionWe adopted a simplified protocol for establishing humanized NSG mice. We observed a higher engraftment rate of human CD45+ cells than earlier studies without any significant toxicity. And human CD45+ cell engraftment at week 8 was comparable to that of week 12.

Recent advances in haploidentical hematopoietic stem cell transplantation using ex vivo T cell-depleted graft in children and adolescents.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for children and adolescents with various malignant and non-malignant diseases. While human leukocyte antigen (HLA)-identical sibling donor is the preferred choice, matched unrelated volunteer donor is another realistic option for successful HSCT. Unfortunately, it is not always possible to find a HLA-matched donor for patients requiring HSCT, leading to a considerable number of deaths of patients without undergoing transplantation. Alternatively, allogeneic HSCT from haploidentical family members could provide donors for virtually all patients who need HSCT. Although the early attempts at allogeneic HSCT from haploidentical family donor (HFD) were disappointing, recent advances in the effective ex vivo depletion of T cells or unmanipulated in vivo regulation of T cells, better supportive care, and optimal conditioning regimens have significantly improved the outcomes of haploidentical HSCT. The ex vivo techniques used to remove T cells have evolved from the selection of CD34+ hematopoietic stem cell progenitors to the depletion of CD3+ cells, and more recently to the depletion of αβ+ T cells. The recent emerging evidence for ex vivo T cell-depleted haploidentical HSCT has provided additional therapeutic options for pediatric patients with diseases curable by HSCT but has not found a suitable related or unrelated donor. This review discusses recent advances in haploidentical HSCT, focusing on transplant using ex vivo T cell-depleted grafts. In addition, our experiences with this novel approach for the treatment of pediatric patients with malignant and non-malignant diseases are described.

Adipogenic Mesenchymal Stromal Cells from Bone Marrow and Their Hematopoietic Supportive Role: Towards Understanding the Permissive Marrow Microenvironment in Acute Myeloid Leukemia.

The role of bone marrow-derived mesenchymal stem/stromal cells (MSCs) in creating a permissive microenvironment that supports the emergence and progression of acute myeloid leukemia (AML) is not well established. We investigated the extent to which adipogenic differentiation in normal MSCs alters hematopoietic supportive capacity and we undertook an in-depth comparative study of human bone marrow MSCs derived from newly diagnosed AML patients and healthy donors, including an assessment of adipogenic differentiation capacity. MSCs from healthy controls with partial induction of adipogenic differentiation, in comparison to MSCs undergoing partial osteogenic differentiation, expressed increased levels of hematopoietic factors and induced greater proliferation, decreased quiescence and reduced in vitro hematopoietic colony forming capacity of CD34(+) hematopoietic stem and progenitor cells (HSPCs). Moreover, we observed that AML-derived MSCs had markedly increased adipogenic potential and delayed osteogenic differentiation, while maintaining normal morphology and viability. AML-derived MSCs, however, possessed reduced proliferative capacity and decreased frequency of subendothelial quiescent MSCs compared to controls. Our results support the notion of a bone marrow microenvironment characterized by increased propensity toward adipogenesis in AML, which may negatively impact normal hematopoiesis. Larger confirmatory studies are needed to understand the impact of various clinical factors. Novel leukemia treatments aimed at normalizing bone marrow niches may enhance the competitive advantage of normal hematopoietic progenitors over leukemia cells.