The Microarray Signature of Rhesus Macaque CD34+CD123 (IL-3 Receptor)+ Hematopoietic Stem/Progenitor Cells Isolated Following Mobilization with Plerixafor Alone or in Combination with Granulocyte Colony-Stimulating Factor (G-CSF)

Abstract Abstract 1301 CD34+ cells are a heterogeneous population of cells which contain the hematopoietic stem cell (HSC). Previously we determined that the microarray signature of CD34+ cells mobilized by G-CSF differed from those mobilized with either plerixafor (AMD3100) alone or in combination with G-CSF (Donahue RE, et al. Blood 2009; 114:2530–2541). Recently we also determined that a novel subpopulation of CD34+CD123+ cells was mobilized with plerixafor and not G-CSF in rhesus macaques (Uchida N, et al. Exp Hematol 2011; 39:795–805). To explore differences between CD34+CD123+ and CD34+CD123- cells, we evaluated their gene expression signatures. Following mobilization with plerixafor alone or in combination with G-CSF, we collected mononuclear cells by leukapheresis, isolated rhesus CD34+ cells by immunoselection, and further isolated CD34+CD123+ and CD34+CD123- by cell sorting. CD34+CD123+ cells were found not to express CXCR4 on their cell surface. Total RNA was isolated from cells and amplified to cRNA. Control cRNA was labeled with Cy3 dye and experimental cRNA was labeled with Cy5 dye. Control and experimental labeled cRNA were co-hybridized to a custom-made 17.5K cDNA (UniGene cluster) microarray. Gene expression was analyzed by quantifying the fluorescence from the microarray. The raw data set was filtered according to a standard procedure to exclude spots with minimum intensity. The relationship between the cells was analyzed using an unsupervised Eisen's hierarchical clustering method. Statistical analysis was done using Array Class Comparison analysis. Pathway analysis was carried out using Ingenuity Pathway Analysis. Principal Component Analysis demonstrated that CD34+CD123+ and CD34+CD123- cells cluster separately and are not distinguishable on the basis of mobilization regimen. Using Supervised Hierarchical Cluster Analysis for genes which were significantly different (p<0.05) there was further confirmation of clustering of the CD34+CD123+ cells versus the CD34+CD123- cells. Microarray analysis revealed that pathways including retinoic acid receptor activation and TGF-β and MAPK signaling were up-regulated in CD34+CD123+ cells, whereas glucocorticoid receptor and TREM-1 signaling, and genes involved in dendritic cell maturation and production of nitric oxide and reactive oxygen by macrophages were down regulated. The up regulated pathways and the down regulated pathways for the CD34+CD123+ population suggest that CD34+CD123+ cells are lymphoid progenitors. Upon comparing pathways between those previously reported to be unique to CD34+ cells mobilized with plerixafor alone or in combination with G-CSF, the majority are those that have been identified also to be in the CD34+CD123+ subpopulation. It appears as if the CD34+CD123+ population accounts for many of the differences in CD34+ cells mobilized with plerixafor observed in the earlier study. In conclusion, the microarray signature of the CD34+CD123+ cells subpopulation suggests that these cells are lymphoid progenitors which can be effectively mobilized with plerixafor. This may account for the more rapid lymphoid recovery we observed for rhesus macaques transplanted with CD34+ cells mobilized with plerixafor and suggests a therapeutic approach through immunoselection to selectively target CD34+CD123+ progenitor populations. Disclosures: No relevant conflicts of interest to declare.

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Identification of Gene Expression–Based Prognostic Markers in the Hematopoietic Stem Cells of Patients With Myelodysplastic Syndromes

Journal of Clinical Oncology ◽

10.1200/jco.2012.45.5626 ◽

2013 ◽

Vol 31 (28) ◽

pp. 3557-3564 ◽

Cited By ~ 32

Author(s):

Andrea Pellagatti ◽

Axel Benner ◽

Ken I. Mills ◽

Mario Cazzola ◽

Aristoteles Giagounidis ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Myelodysplastic Syndromes ◽

Mononuclear Cells ◽

Bone Marrow Cells ◽

Cox Proportional Hazards ◽

Sample Collection ◽

Data Set ◽

Hematopoietic Stem ◽

Cd34 Cells

Purpose The diagnosis of patients with myelodysplastic syndromes (MDS) is largely dependent on morphologic examination of bone marrow aspirates. Several criteria that form the basis of the classifications and scoring systems most commonly used in clinical practice are affected by operator-dependent variation. To identify standardized molecular markers that would allow prediction of prognosis, we have used gene expression profiling (GEP) data on CD34+ cells from patients with MDS to determine the relationship between gene expression levels and prognosis. Patients and Methods GEP data on CD34+ cells from 125 patients with MDS with a minimum 12-month follow-up since date of bone marrow sample collection were included in this study. Supervised principal components and lasso penalized Cox proportional hazards regression (Coxnet) were used for the analysis. Results We identified several genes, the expression of which was significantly associated with survival of patients with MDS, including LEF1, CDH1, WT1, and MN1. The Coxnet predictor, based on expression data on 20 genes, outperformed other predictors, including one that additionally used clinical information. Our Coxnet gene signature based on CD34+ cells significantly identified a separation of patients with good or bad prognosis in an independent GEP data set based on unsorted bone marrow mononuclear cells, demonstrating that our signature is robust and may be applicable to bone marrow cells without the need to isolate CD34+ cells. Conclusion We present a new, valuable GEP-based signature for assessing prognosis in MDS. GEP-based signatures correlating with clinical outcome may significantly contribute to a refined risk classification of MDS.

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Global Gene Expression Signatures in CD34+ Hematopoietic Cells Following Exposure to 16,16-Dimethylprostaglandin E2.

Blood ◽

10.1182/blood.v114.22.2523.2523 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2523-2523

Author(s):

Robyn S. Allen ◽

Ping Jin ◽

Trista North ◽

Wolfram Goessling ◽

Yu Tian ◽

...

Keyword(s):

Pathway Analysis ◽

Molecular Mechanisms ◽

Fold Increase ◽

Global Gene Expression ◽

Unigene Cluster ◽

Hematopoietic Stem ◽

Human Cd34 ◽

Cd34 Cells ◽

Custom Made ◽

Protein Receptors

Abstract Abstract 2523 Poster Board II-500 Prostaglandin E2 (PGE2), a member of the eicosanoid family, is a local signaling molecule released from mammalian blood vessel walls and other hematopoietic niches. PGE2 produces tissue specific effects by interacting with G protein receptors in cell membranes. It has been found to be an important regulator of hematopoietic stem cell (HSC) number during embryogenesis and is required for the development of HSCs (North et al., Nature 447:1007–1011, 2007). Additionally, PGE2 enhances HSC engraftment in competitive transplantation assays (North et al., Nature 447:1007–1011, 2007) through alterations in the survival, proliferation and homing of transplanted cells (Hoggatt et al., Blood 113:5444–5455, 2009). Immunoselected CD34+ cells were isolated from the leukapheresis products of both human and non-human primates following cytokine mobilization with G-CSF. CD34+ cells were incubated in X-Vivo 10™ with either 50μM or no PGE2 for one hour at 37°C on RetroNectin™ coated plates. After one hour incubation, media was replaced with X-Vivo 10™ supplemented with 10ng/mL SCF and IL-6 and cells were kept at 37°C and collected either prior to or at 2, 6, 12 and 24 hours following PGE2 exposure. Total RNA was isolated from cells and amplified to cRNA. Control cRNA was labeled with Cy3 dye and experimental cRNA was labeled with Cy5 dye. Control and experimental labeled cRNA was co-hybridized to a custom-made 17.5K cDNA (UniGene cluster) microarray. The relationship between the cells was analyzed using an unsupervised Eisen's hierarchical clustering method and gene regulation was analyzed at each time point using an Array scatter plot comparison. Statistical analysis was done using Array Class Comparison analysis and pathway analysis was carried out using Ingenuity Pathway Analysis. Many of the significantly up regulated genes were associated with pathways activated by PGE2, including genes involved in the inflammatory response, signal transduction, and G proteins. Interestingly the human CD34+ cells showed at 2 hours a 21.8-fold increase of 3′,5′-cyclic AMP phosphodiesterase mRNA consistent with prior observations in zebrafish and murine cells (Goessling et al. Cell 136: 1136–1148). Pathways that increased at 2 hours include molecular mechanisms of cancer, and those that were down regulated include CCR5, CTLA4, and leukocyte extravasation signaling. At 6 hours the molecular mechanisms of cancer pathway predominated even more (diminishing by 12 hours) while oxidative phosphorylation was down regulated. Genes involved in cAMP and WNT signaling are observed to be up regulated at earlier time points but diminish with time. These results correlate with protein expression observations that have been made in the zebrafish and murine models following PGE2 treatment (Goessling et al. Cell 136: 1136–1148) and are consistent with the observation that PGE2 may influence the self renewal of HSCs. Disclosures: Goessling: Fate Therapeutics: Consultancy, Patents & Royalties.

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Differential gene expression analysis of ankylosing spondylitis shows deregulation of the HLA-DRB, HLA-DQB, ITM2A, and CTLA4 genes

Egyptian Journal of Medical Human Genetics ◽

10.1186/s43042-021-00161-0 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Rowan AlEjielat ◽

Anas Khaleel ◽

Amneh H. Tarkhan

Keyword(s):

Gene Expression ◽

Ankylosing Spondylitis ◽

Differentially Expressed Genes ◽

Gene Network ◽

Disease Diagnosis ◽

Receptor Activation ◽

Functional Enrichment ◽

Differentially Expressed ◽

Inflammatory Disorder ◽

Data Set

Abstract Background Ankylosing spondylitis (AS) is a rare inflammatory disorder affecting the spinal joints. Although we know some of the genetic factors that are associated with the disease, the molecular basis of this illness has not yet been fully elucidated, and the genes involved in AS pathogenesis have not been entirely identified. The current study aimed at constructing a gene network that may serve as an AS gene signature and biomarker, both of which will help in disease diagnosis and the identification of therapeutic targets. Previously published gene expression profiles of 16 AS patients and 16 gender- and age-matched controls that were profiled on the Illumina HumanHT-12 V3.0 Expression BeadChip platform were mined. Patients were Portuguese, 21 to 64 years old, were diagnosed based on the modified New York criteria, and had Bath Ankylosing Spondylitis Disease Activity Index scores > 4 and Bath Ankylosing Spondylitis Functional Index scores > 4. All patients were receiving only NSAIDs and/or sulphasalazine. Functional enrichment and pathway analysis were performed to create an interaction network of differentially expressed genes. Results ITM2A, ICOS, VSIG10L, CD59, TRAC, and CTLA-4 were among the significantly differentially expressed genes in AS, but the most significantly downregulated genes were the HLA-DRB6, HLA-DRB5, HLA-DRB4, HLA-DRB3, HLA-DRB1, HLA-DQB1, ITM2A, and CTLA-4 genes. The genes in this study were mostly associated with the regulation of the immune system processes, parts of cell membrane, and signaling related to T cell receptor and antigen receptor, in addition to some overlaps related to the IL2 STAT signaling, as well as the androgen response. The most significantly over-represented pathways in the data set were associated with the “RUNX1 and FOXP3 which control the development of regulatory T lymphocytes (Tregs)” and the “GABA receptor activation” pathways. Conclusions Comprehensive gene analysis of differentially expressed genes in AS reveals a significant gene network that is involved in a multitude of important immune and inflammatory pathways. These pathways and networks might serve as biomarkers for AS and can potentially help in diagnosing the disease and identifying future targets for treatment.

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LYL1, Class II Basic Helix-Loop-Helix Transcription Factor, Induces the Differentiation of Common Marmoset Embryonic Stem Cells to Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v118.21.2348.2348 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2348-2348

Author(s):

Hirotaka Kawano ◽

Tomotoshi Marumoto ◽

Michiyo Okada ◽

Tomoko Inoue ◽

Takenobu Nii ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Lymphoblastic Leukemia ◽

Embryonic Stem ◽

Common Marmoset ◽

Hematopoietic Stem ◽

Helix Loop Helix ◽

Cd34 Cells

Abstract Abstract 2348 Since the successful establishment of human embryonic stem cells (ESCs) in 1998, transplantation of functional cells differentiated from ESCs to the specific impaired organ has been expected to cure its defective function [Thomson JA et al., Science 282:1145–47, 1998]. For the establishment of the regenerative medicine using ESCs, the preclinical studies utilizing animal model systems including non-human primates are essential. We have demonstrated that non-human primate of common marmoset (CM) is a suitable experimental animal for the preclinical studies of hematopoietic stem cells (HSCs) therapy [Hibino H et al., Blood 93:2839–48, 1999]. Since then we have continuously investigated the in vitro and in vivo differentiation of CM ESCs to hematopoietic cells by the exogenous hematopoietic gene transfer. In earlier study, we showed that the induction of CD34+ cells having a blood colony forming capacity from CM ESCs is promoted by lentiviral transduction of TAL1 cDNA [Kurita R et al., Stem Cells 24:2014-22,2006]. However those CD34+ cells did not have a bone marrow reconstituting ability in irradiated NOG (NOD/Shi-scid/IL-2Rγnull) mice, suggesting that transduction of TAL1 gene is not enough to induce functional HSCs which have self-renewal capability and multipotency. Thus we tried to find other hematopoietic genes being able to promote hematopoietic differetiation more efficiently than TAL1. We selected 6 genes (LYL1, HOXB4, BMI1, GATA2, c-MYB and LMO2) as candidates for factors that induce the differentiation from ESCs to HSCs, based on the comparison of gene expression level between human ESCs and HSCs by Digital Differential Display from the Uni-Gene database at the NCBI web site (http://www.ncbi.nlm.nih.gov/UniGene/). Then, we transduced the respective candidate gene in CM ESCs (Cj11), and performed embryoid body (EB) formation assay to induce their differentiation to HSCs for 9 days. We found that lentiviral transduction of LYL1, a basic helix-loop-helix transcription factor, in EBs derived from Cj11, one of CM ESC lines, markedly increased the number of cells positive for CD34, a marker for hematopoietic stem/progenitors. The lymphoblastic leukemia 1 (LYL1) was originally identified as the factor of a chromosomal translocation, resulting in T cell acute lymphoblastic leukemia [Mellentin JD et al., Cell 58:77-83.1989]. These class II bHLH transcription factors regulate gene expression by binding to target gene sequences as heterodimers with E-proteins, in association with Gata1 and Gata2 [Goldfarb AN et al., Blood 85:465-71.1995][Hofmann T et al., Oncogene 13:617-24.1996][Hsu HL et al., Proc Natl Acad Sci USA 91:5947-51.1994]. The Lyl1-deficient mice display the reduction of B cells and impaired long-term hematopoietic reconstitution capacity [Capron C et al., Blood 107:4678-4686. 2006]. And, overexpression of Lyl1 in mouse bone marrow cells induced the increase of HSCs, HPCs and lymphocytes in vitro and in vivo [Lukov GL et al., Leuk Res 35:405-12. 2011]. These information indicate that LYL1 plays important roles in hematopoietic differentiation in primate animals including human and common marmoset. To examine whether overexpression of LYL1 in EBs can promote hematopoietic differentiation in vitro we performed colony-forming unit (CFU) assay, and found that LYL1-overexpressing EBs showed the formation of multi-lineage blood cells consisting of erythroid cells, granulocytes and macrophages. Next, we analyzed gene expression level by RT-PCR, and found that the transduction of LYL1 induced the expression of various hematopoietic genes. These results suggested that the overexpression of LYL1 can promote the differentiation of CM ESCs to HSCs in vitro. Furthermore we found that the combined overexpression of TAL1 and LYL1 could enhance the differentiation of CD34+ cells from CM ESCs than the respective overexrpession of TAL1 or LYL1. Collectively, our novel technology to differentiate hematopoietic cells from ESCs by the transduction of specific transcription factors is novel, and might be applicable to expand human hematopoietic stem/progenitor cells in vitro for future regenerative medicine to cure human hematopoietic cell dyscrasias. Disclosures: No relevant conflicts of interest to declare.

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A-To-I RNA Editing By ADAR1 Is Essential For Hematopoiesis

Blood ◽

10.1182/blood.v122.21.1199.1199 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1199-1199 ◽

Cited By ~ 1

Author(s):

Brian Liddicoat ◽

Robert Piskol ◽

Alistair Chalk ◽

Miyoko Higuchi ◽

Peter Seeburg ◽

...

Keyword(s):

Gene Expression ◽

Rna Editing ◽

Fetal Liver ◽

Expression Profiles ◽

In Utero ◽

Tamoxifen Treatment ◽

Rna Seq ◽

Data Set ◽

Hematopoietic Stem

Abstract The role of RNA and its regulation is becoming increasingly appreciated as a vital component of hematopoietic development. RNA editing by members of the Adenosine Deaminase Acting on RNA (ADAR) gene family is a form of post-transcriptional modification which converts genomically encoded adenosine to inosine (A-to-I) in double-stranded RNA. A-to-I editing by ADAR directly converts the sequence of the RNA substrate and can alter the structure, function, processing, and localization of the targeted RNA. ADAR1 is ubiquitously expressed and we have previously described essential roles in the development of hematopoietic and hepatic organs. Germline ablation of murine ADAR1 results in a significant upregulation of interferon (IFN) stimulated genes and embryonic death between E11.5 and E12.5 associated with fetal liver disintegration and failed hemopoiesis. To determine the biological importance of A-to-I editing by ADAR1, we generated an editing dead knock-in allele of ADAR1 (ADAR1E861A). Mice homozygous for the ADAR1E861A allele died in utero at ∼E13.5. The fetal liver (FL) was small and had significantly lower cellularity than in controls. Analysis of hemopoiesis demonstrated increased apoptosis and a loss of hematopoietic stem cells (HSC) and all mature lineages. Most notably erythropoiesis was severely impaired with ∼7-fold reduction across all erythrocyte progenitor populations compared to controls. These data are consistent with our previous findings that ADAR1 is essential for erythropoiesis (unpublished data) and suggest that the ADAR1E861A allele phenocopies the null allele in utero. To assess the requirement of A-to-I editing in adult hematopoiesis, we generated mice where we could somatically delete the wild-type ADAR1 allele and leave only ADAR1E861A expressed in HSCs (hScl-CreERAdar1fl/E861A). In comparison to hScl-CreERAdar1fl/+ controls, hScl-CreERAdar1fl/E861A mice were anemic and had severe leukopenia 20 days post tamoxifen treatment. Investigation of marrow hemopoiesis revealed a significant loss of all cells committed to the erythroid lineage in hScl-CreERAdar1fl/E861A mice, despite having elevated phenotypic HSCs. Upon withdrawal of tamoxifen diet, all blood parameters were restored to control levels within 12 weeks owing to strong selection against cells expressing only the ADAR1E861A allele. To understand the mechanism through which ADAR1 mediated A-to-I editing regulates hematopoiesis, RNA-seq was performed. Gene expression profiles showed that a loss of ADAR1 mediated A-to-I editing resulted in a significant upregulation of IFN signatures, consistent with the gene expression changes in ADAR1 null mice. To define substrates of ADAR1 we assessed A-to-I mismatches in the RNA-seq data sets. 3,560 previously known and 353 novel A-to-I editing sites were identified in our data set. However, no single editing substrate discovered could account for the IFN signature observed or the lethality of ADAR1E861A/E861A mice. These results demonstrate that ADAR1 mediated A-to-I editing is essential for the maintenance of both fetal and adult hemopoiesis in a cell-autonomous manner and a key suppressor of the IFN response in hematopoiesis. Furthermore the ADAR1E861A allele demonstrates the essential role of ADAR1 in vivo is A-to-I editing. Disclosures: Hartner: TaconicArtemis: Employment.

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Disease-Relevant Expression Of TRAF6 In Mouse HSC Results In An MDS-Like Disease

Blood ◽

10.1182/blood.v122.21.101.101 ◽

2013 ◽

Vol 122 (21) ◽

pp. 101-101

Author(s):

Jing Fang ◽

Xiaona Liu ◽

Brenden Barker ◽

Lyndsey Bolanos ◽

Yue Wei ◽

...

Keyword(s):

Gene Expression ◽

Conflicts Of Interest ◽

Early Stage ◽

Bone Marrow Failure ◽

Gene Signature ◽

Sublethal Dose ◽

Hematopoietic Tissue ◽

Hematopoietic Stem ◽

Disease Evolution ◽

Cd34 Cells

Abstract Overexpression of immune-related genes is widely reported in Myelodysplastic Syndrome (MDS), and chronic immune stimulation increases the risk for developing MDS. We find that TNF receptor associated factor 6 (TRAF6), an innate immune protein, is overexpressed approximately 2-fold in CD34+ cells from 40% of MDS patients, and may explain immune pathway activation in the MDS-initiating hematopoietic stem/progenitor cell (HSPC). In support of these observations and our hypothesis that TRAF6 is important in the pathophysiology of MDS, a gene expression analysis revealed that TRAF6 controls an MDS gene signature in human cells. We, and others, have previously shown that retroviral overexpression of TRAF6 in mouse HSPC results in MDS and Acute Myeloid Leukemia (AML). However, interpretations of these findings are hampered by supra-physiological levels of TRAF6 (>10-fold overexpression) and the stress associated with HSPC transduction/transplantation. To investigate the consequences of TRAF6 overexpression to MDS, we generated a transgenic mouse model overexpressing TRAF6 from a hematopoietic-specific Vav promoter. Expression of TRAF6 in HSPC was approximately 2-fold higher as compared to endogenous TRAF6 and in line with MDS patient CD34+ cells. By 15 months of age, half of Vav-TRAF6 mice succumbed to a hematologic disease resembling MDS associated with bone marrow failure (BMF). In contrast to the retroviral overexpression approach, Vav-TRAF6 mice did not develop AML. Examination of sick mice revealed stage-specific disease evolution. Initially, all Vav-TRAF6 mice exhibit an inversion of myeloid/lymphoid proportions. For Vav-TRAF6 mice that develop a fatal disease, they present with a hypocellular marrow, dysplasic myeloid cells, and neutropenia. A subset of mice also display anemia with nucleated red blood cells, poikilocytosis, and extramedullular erythropoiesis. In support of a BMF phenotype, HSPC from Vav-TRAF6 mice form fewer colonies in methylcellulose. To investigate the consequences of an acute exposure to pathogen, early-stage Vav-TRAF6 mice were treated with a single sublethal dose of lipopolysaccharide (LPS). Unlike wild-type (WT) mice, Vav-TRAF6 mice developed a rapid and reversible anemia, suggesting environmental factors can influence the severity of the disease. To gain insight into the mechanism contributing to BMF, gene expression profiling was performed in WT and Vav-TRAF6 HSPC. One of the enriched pathways consisted of AKT activation and FOXO downregulation. Consistent with the microarray analysis, AKT is constitutively phosphorylated at Thr308 in hematopoietic tissue from Vav-TRAF6 mice. SOD2, a transcriptional target of FoxO3a that is suppressed by activated AKT, is decreased in Vav-TRAF6 HSPC. Given that AKT/FOXO regulate reactive oxygen species (ROS) in cells, we investigated ROS levels in HPSC from Vav-TRAF6 and WT mice. Intracellular ROS is significantly elevated in BM cells from Vav-TRAF6 mice, and restored to normal levels when AKT was inhibited. In conclusion, we propose the potential role of TRAF6 in the development of MDS-associated BMF, partly due to constitutive activation of AKT and subsequent ROS elevation in HSPC cells. Disclosures: No relevant conflicts of interest to declare.

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Inhibition of Lysine-Specific Demethylase 1A (LSD1) Supports Human HSCs in Culture By Preserving Their Immature State

Blood ◽

10.1182/blood-2018-194 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 194-194

Author(s):

Agatheeswaran Subramaniam ◽

Mehrnaz Safaee Talkhoncheh ◽

Kristijonas Zemaitis ◽

Shubhranshu Debnath ◽

Jun Chen ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Stem Cell ◽

Cell Division ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Enrichment Score ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Human Hscs

Abstract The molecular mechanisms that govern hematopoietic stem cell (HSC) fate decisions remain incompletely defined. It has been a long-standing goal in the field to gain a better understanding of the genes and pathways that regulate the self-renewal ability of HSCs in order to develop optimal culture conditions in which HSCs can be expanded for clinical benefit. Lysine-specific histone demethylase 1A (LSD1), also known as lysine (K)-specific demethylase 1A (KDM1A), regulates gene expression by specifically eliminating di- and mono-methyl groups on H3 lysine K4 and K9 residues. Studies in mice have shown that, conditional knockdown of LSD1 results in an expansion of bone marrow hematopoietic stem and progenitor cells (HSPCs). However, a complete knockout of LSD1 results in pancytopenia and a dramatic reduction of HSPCs. In this study, we asked whether inhibition of LSD1 would improve the maintenance or expansion of cultured human HSCs derived from umbilical cord blood (UCB). To evaluate the effect of LSD1 inhibition we treated UCB CD34+ cells with three different LSD1 inhibitors (2-PCPA, GSK-LSD1 and RN1) at their respective IC50 values (20µM, 16nM and 70nM) and expanded the cultures for 6 days in serum free medium supplemented with stem cell factor (SCF), thrombopoietin (TPO) and FMS-like tyrosine kinase 3 ligand (FLT3L). Since we (Subramaniam et. al. Haematologica 2018) and others recently have shown that EPCR is a reliable cell surface marker to track UCB derived HSCs during in vitro culture, we quantified the numbers of CD34+EPCR+ cells using flow cytometry and compared to DMSO treated control cultures. Remarkably, treatment with either 2-PCPA or GSK-LSD1 resulted in a more than 10-fold increase of CD34+EPCR+ cells, compared to controls. Further, from dose response experiments we found that 2-PCPA at 1.25 µM expanded the total CD34+ cell population more efficiently than GSK-LSD1, and we therefore used 2-PCPA at this concentration for the subsequent experiments. Using carboxyfluorescein succinimidyl ester (CFSE) labeling to monitor cell division, we found that 2-PCPA did not significantly alter the cell division rate of the cultured CD34+ cells compared to DMSO controls, suggesting that the expansion of CD34+EPCR+ cells is not due to increased proliferation, and that LSD1 inhibition rather may prevent differentiation of the immature HSPCs. To further explore this, we mapped the early transcriptional changes triggered by 2-PCPA in HSCs using gene expression profiling of CD34+CD38-CD45RA-CD90+ cells following 24 hours of culture with or without 2-PCPA treatment. We found that gene sets corresponding to UCB and fetal liver HSCs were significantly enriched upon 2-PCPA treatment compared to DMSO control (Normalized Enrichment Score (NES)=1.49, q=0.05). This suggest that 2-PCPA indeed restricts differentiation and preserves the HSC state upon ex vivo culture. Strikingly, the gene signature induced by LSD1 inhibition was highly similar to that induced by the known HSC expanding compound UM171 (NES=1.43, q=0.11). UM171 is a molecule with unknown target and has also been shown to dramatically expand the EPCR+ population in culture. Finally, the frequency of functional HSCs in DMSO and 2-PCPA treated cultures were measured using limiting dilution analysis (LDA). LDA was performed by transplanting 4 doses (day 0 equivalents of 20000, 1000, 300 and 100 CD34+ cells) of DMSO and 2-PCPA treated cultures into sub lethally irradiated (300cGy) NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Human CD45+ cell engraftment in the bone marrow was analyzed 18 weeks' post transplantation. Cultures treated with 2-PCPA showed a 5-fold higher content of long-term repopulating cells per day 0 CD34+ cell equivalent compared to the DMSO control (1 in 615 vs 1 in 3041, p=0.03). Thus, the 2-PCPA treated cultures had significantly enhanced HSCs numbers. To determine the absolute expansion rate, we are currently performing LDA using uncultured cells as well. Altogether our data suggest that LSD1 inhibition supports both phenotypic and functional HSCs in culture by preserving the immature state. Currently we are exploring the possibilities of using LSD1 inhibitors in combination with other known modifiers of HSC expansion. Disclosures No relevant conflicts of interest to declare.

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Hematopoiesis in Patients with Acquired Aplastic Anemia Is Supported By a Limited Number of CXCR4(-) Cells at the Common Myeloid Progenitor Level

Blood ◽

10.1182/blood.v124.21.4392.4392 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4392-4392

Author(s):

Takamasa Katagiri ◽

Atsuo Kasada ◽

Kana Motomiya ◽

Yoshitaka Zaimoku ◽

An T. T. Dao ◽

...

Keyword(s):

Gene Expression ◽

Aplastic Anemia ◽

Expression Profiles ◽

Cxcr4 Expression ◽

Healthy Individuals ◽

Wild Type ◽

Hematopoietic Stem ◽

Expression Levels ◽

Cd34 Cells ◽

Different Types

Abstract Background: HLA-A allele-lacking leukocytes (HLA-LLs) derived from hematopoietic stem cells (HSCs) with copy-number neutral loss of heterozygosity (LOH) in chromosome 6p (6pLOH) are detected in 13% of patients with acquired aplastic anemia (AA). These defective leukocytes account for more than 90% of granulocytes in some AA patients in remission after immunosuppressive therapy (IST). However, it remains unclear how a few HSC clones with genetic abnormalities sustain hematopoiesis for a long time in AA patients. The fate of 6pLOH(+) HSCs can be easily monitored by revealing HLA-LLs using monoclonal antibodies (mAbs) specific for HLA-A alleles. Phenotypic analyses of hematopoietic progenitor cells (HPCs) in bone marrow (BM) and gene expression profiling of HLA-A allele-lacking HPCs may help to clarify the mechanisms underlying clonal hematopoiesis by 6pLOH(+) HSCs in patients with AA. Methods: The HLA-A allele expression of BM CD34+ cell subsets, including common myeloid progenitors (CMPs), megakaryocyte/erythroid progenitors (MEPs) and granulocytic/monocyte progenitors (GMPs) in three patients possessing HLA-LLs was determined using mAbs specific for CD34, CD38, CD45RA, CD123 and three different HLA-A alleles. The HLA-A allele-lacking and wild-type CMPs were sorted, and the gene expression levels were compared between the two subsets using an Agilent Whole Human Genome Oligo Microarray (4x44K). The CXCR4 expression by CMPs was compared among different types of BM failure patients. Results: The percentages of HLA-A allele-lacking cells in peripheral blood granulocytes, GMPs, MEPs and CMPs of the three patients were 54.1%/67.3%/63%/1.7%, 98.1%//97.2%/98.8%/4.5% and 97.2%/97.8%/96.9%/12.9%, indicating that the hematopoiesis of these patients was being supported by HLA-A-lacking cells at the CMP level, which accounted for only a small percentage of the total CMPs. To characterize the CMPs capable of supporting hematopoiesis, the gene expression profiles were compared between HLA-A-lacking CMPs that substantially contributed to hematopoiesis and HLA-A-retaining (wild type) CMPs that did not contribute to hematopoiesis. One of the striking differences in the gene expression profile between the two groups was a lower expression of CXCL12 in the HLA-lacking CMPs compared to the wild type CMPs (1 vs. 27.6). Because CD34+ cells are known to negatively regulate themselves by secreting CXCL12, the expression of its receptor, CXCR4, on CMPs was examined using flow cytometry. Surprisingly, all HLA-A-lacking cells were negative for CXCR4, while most of the wild-type cells expressed the CXCL12 receptor (Figure). Examination of the CD34+ cell subsets from 10 healthy individuals showed that the percentages of CXCR4-negative cells were 25.9±8.7% (mean±SD) in CMPs, 98.7±6.4 in MEPs, 98.7±2.1 in GMPs and 77.1±11.5 in CD34+ cells. When the CXCR4 expression levels of CMPs were compared among different types of BM failure, the mean fluorescence intensity (MFI) of CXCR4 in the CMPs from four patients with AA (550±76, mean±SD) was significantly lower than that in three patients with MDS (901±332) and 10 healthy individuals (1440±229). Conclusions: Hematopoiesis in patients with AA is supported by a limited number of CXCR4(-) cells at the CMP level. Decreased CXCR4 expression by CMPs of AA patients may represent immune pressure applied to HSCs that elicits the participation of dormant CMPs in hematopoiesis. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Identification of Defects in the Transcriptional Program during Differentiation of CD34+Cells Selected from Patients with Both, Low- and High-Risk Myelodysplastic Syndromes.

Blood ◽

10.1182/blood.v104.11.201.201 ◽

2004 ◽

Vol 104 (11) ◽

pp. 201-201 ◽

Cited By ~ 2

Author(s):

Martina Komor ◽

Saskia Gueller ◽

Sven de Vos ◽

Oliver G. Ottmann ◽

Dieter Hoelzer ◽

...

Keyword(s):

Gene Expression ◽

High Risk ◽

Stress Responses ◽

Low Risk ◽

Transcriptional Analysis ◽

Transcriptional Program ◽

Hematopoietic Stem ◽

Specific Differentiation ◽

Cd34 Cells ◽

Transcriptional Pattern

Abstract The development of MDS is suggested to follow a multistep pathogenesis and is characterized by accumulation of molecular defects of the hematopoietic stem/progenitor cells. To detect alterations within the transcriptional program in MDS derived CD34+ cells during lineage-specific differentiation, CD34+ bone marrow cells were selected from healthy individuals (n=3) and patients with low-risk (IPSS, n=3) or high-risk (n=4) MDS and stimulated in vitro with EPO, TPO or G/GM-CSF to induce lineage-specific differentiation. Lineage-determined cells were harvested and if necessary purified by immunomagnetic beads at days 4, 7 and 11 for gene expression profiling. Gene expression was analyzed by oligonucleotide microarrays (HG-U133A, Affymetrix, Santa Clara, CA). The experiments were done in triplicates for each of the time points and each of the conditions. First, we identified 260 genes with a significant expression pattern associated with normal lineage-specific differentiation. These continuously up- or down-regulated genes are considered to be part of a specific genetic program of normal hematopoietic cells during lineage-specific differentiation. In MDS, 57% of these genes showed a different expression from the normal transcriptional pattern. Thirteen of 24 genes up-regulated during normal erythropoiesis were opponently expressed in MDS containing putative new erythro-specific genes like two GTPase activator proteins, RAP1GA1 and ARHGAP8, which regulate small Rho GTPases. Fourteen of 22 continuously up-regulated genes during normal granulopoietic development displayed a significantly different expression in MDS containing the putative candidate desmocollin 2, a gene which is involved in intercellular cell-adhesion. Delta-like 1 (DLK1) is known to be overexpressed in stem cells from patients with myelodysplastic syndrome. The role of DLK1 in normal hematopoiesis is still not defined. We found DLK1 with increasing expression during normal megakaryopoiesis but reverse expression during megakaryopoiesis in MDS. Interestingly, in erythropoiesis from both, high- and low risk MDS we found overexpression of Bladder cancer overexpressed (BLOV1) and Apoptosis inhibitor 5 (API5, which acts as a cellular survival factor by inhibiting apoptosis after growth factor withdrawal). These genes are not expressed in normal erythropoiesis. Furthermore, we identified the gene for a novel v-maf-like protein F, MafF-like (v-maf: musculoaponeurotic fibrosarcoma oncogene homolog F) to be significantly downregulated exclusively in low-risk MDS. MafF belongs to a basic leucine-zipper(bZIP)-transcription factor family normally involved in multiple physiological processes including hematopoiesis and stress responses. Our data provide the first comprehensive transcriptional analysis of differentiating human CD34+ cells derived from normal individuals compared to MDS. It gives new insights to understand the alteration of differentiation and proliferation of MDS derived CD34+ cells. In particular, the study could identify the gene encoding for the MafF-like protein that acts as a transcriptional regulator of normal hematopoiesis to be significantly down-regulated in low-risk MDS.

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Gene-Chip Analysis of Cord Blood Derived CD34+ Cells Expanded on Bioartificial Materials.

Blood ◽

10.1182/blood.v104.11.4131.4131 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4131-4131

Author(s):

Joachim Oswald ◽

Christine Steudel ◽

Katrin Salchert ◽

Christian Thiede ◽

Gerhard Ehninger ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Hematopoietic Stem Cells ◽

Cord Blood ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Fibrillar Collagen ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Colony Forming Units

Abstract Expansion of hematopoietic stem cells from neonatal cord blood is an important issue for clinical uses since the number of CD34+ cells in individual cord blood samples is limited and often not sufficient for a successful engraftment in adult individuals. In vivo, hematopoietic stem cells reside in the bone marrow in close vicinity to stromal cells and extracellular matrix molecules. We have established a culture system for the ex vivo expansion of CD34+ cord blood cells utilizing fibrillar collagen 1 as a bioartificial matrix to enable cellular adhesion during cell culture. CD34+ hematopoietic stem cells were isolated via immunomagnetic separation from umbilical cord blood after informed consent and cultivated in presence of recombinant cytokines and reconstituted collagen 1 fibrils as matrix. After seven days of cultivation, expansion of cells, expression of surface molecules cells and expansion of colony forming units were assessed. Additionally gene expression profiling was performed with Affymetrix HG U133A chips interrogating 22,253 probe sets. As control, CD34+ cells were expanded in liquid culture without fibrillar collagen. The overall expansion of CD34+ cells was 4.2 fold + 1.7 compared to 11.1 fold + 2.9 for the control sample. The number of colony forming units (CFU) was increased in the collagen 1 containing samples was elevated (65.1 + 10.3 compared to 26.1 + 7.6 in the control). Gene expression analysis with chip technology showed up regulation of several cytokines (e.g. interleukin 8, interleukin 1a) and also of transcription factors with antiproliferative features like BTG2. The chip data have been verified with quantitative PCR using the Taqman technology. Our data support the idea that direct contact of CD34+ cells with fibrillar collagen 1 results in a delay in cell cycle progression which prevents a subsequent differentiation into more committed progenitors. Therefore fibrillar collagen 1 may serve as supportive matrix for the ex vivo expansion of cord blood derived CD34+ cells.

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