Human HSPCs Research Articles

Not just a marker: CD34 on human hematopoietic stem/progenitor cells dominates vascular selectin binding along with CD44.

CD34 is routinely used to identify and isolate human hematopoietic stem/progenitor cells (HSPCs) for use clinically in bone marrow transplantation, but its function on these cells remains elusive. Glycoprotein ligands on HSPCs help guide their migration to specialized microvascular beds in the bone marrow that express vascular selectins (E- and P-selectin). Here, we show that HSPC-enriched fractions from human hematopoietic tissue expressing CD34 (CD34pos) bound selectins, whereas those lacking CD34 (CD34neg) did not. An unbiased proteomics screen identified potential glycoprotein ligands on CD34pos cells revealing CD34 itself as a major vascular selectin ligand. Biochemical and CD34 knockdown analyses highlight a key role for CD34 in the first prerequisite step of cell migration, suggesting that it is not just a marker on these cells. Our results also entice future potential strategies to investigate the glycoforms of CD34 that discriminate normal HSPCs from leukemic cells and to manipulate CD34neg HSPC-enriched bone marrow or cord blood populations as a source of stem cells for clinical use.

Nemo-like Kinase Hyper-Activated in Erythroid Progenitors in Models of Diamond Blackfan Anemia

Diamond Blackfan Anemia (DBA) is associated with anemia, congenital abnormalities, and cancer. The disease typically presents within the first year of life. Approximately 70% of DBA patients possess a mutation in one of at least 12 ribosomal proteins, with RPS19 being the most prevalent, accounting for over 25% of cases. In mice, complete loss of RPS19 is not viable, yet mutations that result in haploinsufficiency result in failure of erythropoiesis due to a blockage during differentiation of early erythroblast progenitors. An increase in p53 stability has been attributed to the disease, but the downstream signaling molecules mediating aberrant erythropoiesis have not been completely characterized.Here, we report that Nemo-like Kinase (NLK) is chronically hyper-activated in RPS19 insufficient CD34+ HSPCs derived from fetal liver, cord blood and mobilized peripheral blood differentiating toward the erythroblast lineage, as well as DBA-patient derived EBV-transformed Human B lymphoblastic cell lines In in vitro kinase assays, significant increases in the extent of phosphorylation of three (3) NLK kinase substrates - NLK (3.86 fold), c-Myb (5.22 fold) and Raptor (9.82 fold) was observed in the presence of immunopurified NLK during RPS19 insufficiency. This activation is due to p53-mediated upregulation of TNFα that activates NLK in neighboring cells in a paracrine fashion. In differentiating HSPCs, RPS19 insufficiency results in increased phosphorylation of the critical erythroid transcription factor c-Myb (9.6 fold) and autophagy-regulating Raptor (5.3 fold), both of which are restored to basal levels upon expression of siRNA against NLK.Phosphorylation of c-Myb by NLK results in increased ubiquitination and proteasomal degradation with subsequent loss of c-Myb-induced genes critical for transcriptional regulation of erythropoiesis (including LMO2 and KLF1). Raptor phosphorylation by NLK inhibits mTOR activation, resulting in premature autophagy in erythroblast progenitors. Knockdown of NLK using siRNAs significantly increased c-Myb downstream gene expression (p<0.0001), reduced autophagy (p<0.001) and increased erythrocyte maturation (p<0.001) in RPS19 insufficient human HSPCs. NLK expression is high in HSPCs and committed erythroid progenitors but is significantly downregulated in lymphoid, myeloid and megakaryocyte committed progenitors. In contrast, TNFα receptors are expressed at low levels in stem cells but increases during differentiation. Collectively, we propose the synergy of high NLK and TNFα receptor expression in erythroid progenitors contributes to selective erythroid progenitor hyper-activation of NLK in the TNFα-rich bone marrow niche DBA patients that contributes to red blood cell aplasia.Current therapies for DBA have undesirable side effects, including iron overload from repeated transfusions or infections from immunosuppressive drugs and stem cell transplantation. Identification of aberrantly activated enzymes, such as NLK, offer therapeutic promise as potential druggable targets in the clinical management of DBA. DisclosuresGlader:Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

RNAi Screens for Maintenance of Hematopoietic Stem Cell Phenotype During Culture

Successful ex vivo expansion of hematopoietic stem cells (HSCs) could greatly enhance cell- and gene therapy applications in treatment of malignant and inherited hematological disorders. It has therefore 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. We have developed RNAi screening strategies to identify modifiers of self-renewal/proliferation in human hematopoietic stem- and progenitor cells (HSCPs). Employing pooled lentiviral shRNA libraries targeted to human cord blood derived CD34+ cells, we use the limited persistence of HSPCs under ex vivo culture conditions as a basis for functional selection of shRNAs conferring prolonged maintenance or expansion of undifferentiated cells. From the screens we have been able to identify directly targetable genes such as p38 MAPK, which can be pharmacologically inhibited to enhance the stem cell output of cultured cord blood cells. Additionally, we have found several epigenetic regulators among the top-scoring genes, including genes of the cohesin family, which modulate chromatin architecture, and JARID2, an important modifier of polycomb repressive complex 2 (PRC2). Depletion of either the cohesin genes or JARID2 by shRNA impairs differentiation and enhances both in vitro expansion and the in vivo reconstitution capacity of human HSPCs. However, whether these genes can be targeted pharmacologically to support HSC expansion remains to be explored. The screens have further identified a number of shRNAs that display a remarkable ability to expand HSPCs in culture, but whose gene targets have not been validated, indicating that they are affecting one or more genes in a non-specific manner. We performed gene expression profiling of cells transduced with these off-target shRNAs to gain insight about the molecular context under which HSPCs are propagated ex vivo. A common expression signature was the down-modulation of cellular stress response genes, such as p38 MAPK (previously identified in the screens) as well as genes involved in NF-□B signaling. Indeed, we found that pharmacological inhibition of NF-□B signaling leads to a significant improvement of stem cell function, from ex vivo cultured cord blood derived CD34+ cells, as assessed by transplantation to NSG mice. The effect of NF-□B inhibition was most critical early during the culture where it reduced the levels of several inflammatory and stress-related cytokines that are induced as an immediate response to culture initiation. Taken together, findings from our RNAi screens indicate that ex vivo HSC expansion is facilitated by targeting two distinct gene categories: cellular stress response genes activated by the culture conditions, as well as epigenetic regulators controlling the balance between renewal and differentiation. In order to define the most optimal expansion conditions we are currently assessing the combined targeting of several of these factors in conjunction with other compounds such as SR1 and UM171 using cellular barcoding. Here, cells are genetically marked by lentivirally delivered barcode sequences prior to expansion culture, which enables a precise detection and comparison of the stem cell output from multiple different expansion conditions in a directly competitive manner within a single xenografted animal. Disclosures No relevant conflicts of interest to declare.

The blood-borne sialyltransferase ST6Gal-1 is a negative systemic regulator of granulopoiesis.

Responding to systemic demands in producing and replenishing end-effector blood cells is predicated on the appropriate delivery and interpretation of extrinsic signals to the HSPCs. The data presented herein implicate the systemic, extracellular form of the glycosyltransferase ST6Gal-1 in the regulation of late-stage neutrophil development. ST6Gal-1 is typically a membrane-bound enzyme sequestered within the intracellular secretory apparatus, but an extracellular form is released into the blood from the liver. Both human and murine HSPCs, upon exposure to extracellular ST6Gal-1 ex vivo, exhibited decreased proliferation, diminished expression of the neutrophilic primary granule protein MPO, and decreased appearance of CD11b+ cells. HSPC suppression was preceded by decreased STAT-3 phosphorylation and diminished C/EBPα expression, without increased apoptosis, indicating attenuated G-CSF receptor signaling. A murine model to raise systemic ST6Gal-1 level was developed to examine the role of the circulatory enzyme in vivo. Our results show that systemic ST6Gal-1 modified the cell surface of the GMP subset of HSPCs and decreased marrow neutrophil reserves. Acute airway neutrophilic inflammation by LPS challenge was used to drive demand for new neutrophil production. Reduced neutrophil infiltration into the airway was observed in mice with elevated circulatory ST6Gal-1 levels. The blunted transition of GMPs into GPs in vitro is consistent with ST6Gal-1-attenuated granulopoiesis. The data confirm that circulatory ST6Gal-1 is a negative systemic regulator of granulopoiesis and moreover suggest a clinical potential to limit the number of inflammatory cells by manipulating blood ST6Gal-1 levels.

The Role of Cbx Proteins in Human Benign and Malignant Hematopoiesis

Introduction:Hematopoietic stem cells (HSCs) need to properly balance self-renewal and differentiation to prevent malignant transformation or HSC exhaustion. We recently showed that the presence of specific Cbx proteins in the Polycomb Repressive Complex 1 (PRC1) plays a crucial role in balancing self-renewal and differentiation of murine HSCs. Whereas Cbx7 induces self-renewal, Cbx4 and Cbx8 instead induce differentiation. In this project, we investigate the role of various CBX Polycomb proteins in human normal and malignant hematopoiesis. We aim to identify genes which are controlled by CBX7 in normal and malignant human hematopoiesis.Methods:We overexpressed CBX2, -4, -6, -7 and -8 in human CD34+HSPCs. We assessed functional consequences by measuring cobblestone area-forming cell (CAFC), colony-forming unit (CFU) frequencies, and performed stem cell xenotransplantation studies in NSG mice. To identify genes which are controlled by CBX7 or CBX8, we performed RNA- and Chip-Seq in CD34+ HSPCs. To explore the role of CBX7 in leukemic cells, we performed short hairpin RNA-mediated knockdown of protein expression in leukemic cell lines.Results:Overexpression of CBX7 and CBX8 in human CD34+ HSPCs led to a significant 5-10-fold increase of week 5 CAFC (fold increase: 9,62 for CBX7; 4,92 for CBX8) and 1,5-fold increase CFU-frequencies (fold increase: 1,32 for CBX7; 1,41 for CBX8) after 14 days. In contrast, overexpression of CBX4 and CBX2 led to equal or decreased CAFC- and CFU-frequencies. Transplantation of CBX7 overexpressing human CD34+ HSPCs in NSG mice led to higher long-term engraftment in comparison to the empty vector control (p<0,05 after 18 weeks). Even after one week of in vitro culture of CBX7-overexpressing CD34+cells significant engraftment levels were obtained. Conversely, downregulation of CBX7 in various leukemic cell lines resulted in markedly decreased proliferation and strongly induced differentiation. Expression analysis showed that genes that are upregulated upon CBX7 overexpression are preferentially expressed in primitive stem cells, and are repressed in more differentiated cell types.Conclusions:Our study indicates that CBX7 and CBX8 regulate self-renewal of human HSPCs. Furthermore, our data show that repression of CBX7 markedly inhibits proliferation of leukemic cells and can release the differentiation block of leukemic cells in vitro. Collectively our results indicate that targeting CBX7 may be a viable strategy to induce terminal differentiation of leukemic cells. DisclosuresNo relevant conflicts of interest to declare.

285. Foamy Viral Vector Expressing Human CD18 Results in High Levels of Transduction and Multilineage Engraftment with CD18+ LAD-1 Cells in NSG Mice

Compared to other classes of retroviral vectors used for HSPC gene therapy, foamy viral vectors (FVV) have distinct advantages, including their non-pathogenicity, a safer integration profile, and a high-efficiency transduction of quiescent cells. Proof-of principle of its therapeutic safety and efficacy in HSPCs was provided with the correction of canine Leukocyte Adhesion Deficiency (CLAD). Dogs with CLAD and individuals with LAD type 1 (LAD-1) suffer from recurrent and life-threatening infections caused by mutations in the β2 integrin CD18 subunit. In this study, G-CSF-mobilized CD34+ HSPCs were isolated from a 19 YO male with severe LAD-1 due to homozygous deletions in CD18. CD34+ cells were transduced for 16 hours at MOI of 0, 5, 10, and 20 with a FVV expressing a human codon optimized CD18 transgene. Flow cytometry of CD34+ cells cultured for 3 days after transduction demonstrated CD18+ cell surface expression in 40-45% of cells. Thirty NSG mice were transplanted with CD18-FVV-transduced human LAD-1 HSPCs (~1.2 × 105 cells/mouse), and human cell engraftment was measured in murine BM 5 months after transplantation using flow cytometry. Human CD45+ cells were detected in all mice (average ~1%). Mice transplanted with mock-transduced (MOI=0) LAD-1 CD34+ cells showed a 3.4-fold, 2-fold and 1.4-fold lower engraftment compared to mice injected with CD34+ cells transduced with FVV at MOI 5 (p 0.05), respectively, suggesting a selective homing/engraftment or survival/proliferative advantage of CD18+ cells. The inverse relationship between engraftment levels and MOI correlated with a gradual decrease in cell survival with increasing MOI, most likely due to toxicity from DMSO (required for FVV cryopreservation) during transduction; cell viabilities of 91%, 84%, 82% and 69% were obtained at MOI 0, 5, 10 and 20, respectively, indicating that further increase in MOI would lead to increasing toxicity. High-level, clinically relevant gene marking levels were obtained; the percentages of human cells expressing CD18 in the murine BM 5 months post-transplantation were 36.0 ± 3.9%, 33.9 ± 5.1%, and 44.5 ± 1.6% at MOI 5, 10 and 20, respectively. Quantitative PCR analysis of vector integrants within engrafted human cells indicated a single integration event occurred in the majority of long-term repopulating HSPCs at all MOI tested. Flow cytometry-based lineage analysis of bone marrow from mice transplanted with FVV-transduced LAD-1 CD34+ cells revealed human CD18+ cells in all lineages, with a predominance in the CD13+ myeloid compartment (88.3 ± 4.5%) compared to other lineages, including CD20+ lymphoid (9.1 ± 3.9%), CD235a+ erythroid (0.2 ± 0.1%) and CD41+ megakaryocytic (0.6 ± 0.2%) lineages. Interestingly, human myeloid engraftment was superior in recipient mice engrafted with human CD18+ cells (81.5 ± 4.3%) compared to animals transplanted with non-transduced (CD18-) LAD-1 cells (65.3 ± 11.3%). Integration site analysis of engrafted human cells is ongoing. Thus, FVV-mediated transduction of human LAD-1 CD34+ cells leads to clinically significant levels of CD18 expression, supporting the use of this CD18-expressing FVV in a human clinical trial.

Junctional Adhesion Molecule-A Is Highly Expressed on Human Hematopoietic Repopulating Cells and Associates with the Key Hematopoietic Chemokine Receptor CXCR4

Hematopoietic stem/progenitor cells (HSPCs) reside in specialized bone marrow microenvironmental niches, with vascular elements (endothelial/mesenchymal stromal cells) and CXCR4-CXCL12 interactions playing particularly important roles for HSPC entry, retention, and maintenance. The functional effects of CXCL12 are dependent on its local concentration and rely on complex HSPC-niche interactions. Two Junctional Adhesion Molecule family proteins, Junctional Adhesion Molecule-B (JAM)-B and JAM-C, are reported to mediate HSPC-stromal cell interactions, which in turn regulate CXCL12 production by mesenchymal stromal cells (MSCs). Here, we demonstrate that another JAM family member, JAM-A, is most highly expressed on human hematopoietic stem cells with in vivo repopulating activity (p < .01 for JAM-A(high) compared to JAM-A(Int or Low) cord blood CD34(+) cells). JAM-A blockade, silencing, and overexpression show that JAM-A contributes significantly (p < .05) to the adhesion of human HSPCs to IL-1β activated human bone marrow sinusoidal endothelium. Further studies highlight a novel association of JAM-A with CXCR4, with these molecules moving to the leading edge of the cell upon presentation with CXCL12 (p < .05 compared to no CXCL12). Therefore, we hypothesize that JAM family members differentially regulate CXCR4 function and CXCL12 secretion in the bone marrow niche. Stem Cells 2016;34:1664-1678.

Integrative Analysis of Copy Number and Gene Expression Data Suggests Novel Pathogenetic Mechanisms in Primary Myelofibrosis

Primary myelofibrosis (PMF) is a Myeloproliferative Neoplasm (MPN) characterized by megakaryocyte hyperplasia, progressive bone marrow fibrosis, extramedullary hematopoiesis and transformation to Acute Myeloid Leukemia (AML). A number of phenotypic driver (JAK2, CALR, MPL) and additional subclonal mutations have been described in PMF, pointing to a complex genomic landscape. To discover novel genomic lesions that can contribute to disease phenotype and/or development, we integrated gene expression and copy number (CN) signals and identified several genomic abnormalities leading to a concordant alteration in gene expression levels. In particular, copy number gain in the polyamine oxidase (PAOX) gene locus is accompanied by a coordinated transcriptional up-regulation in PMF patients. To assess the impact of PAOX deregulation on the survival of PMF and normal primary hematopoietic cells, PMF and normal donors cells were incubated with increasing doses of the PAOX inhibitor MDL-72,572. PAOX inhibition resulted in rapid cell death of PMF progenitor cells (5,7±0,9% of late apoptotic cells in NT sample vs 20,1±3,2% in 150μM treated-sample, p<0.05), while survival of normal CD34+ cells was not affected (0,97±0,09 of late apoptotic cells in NT sample vs 2,7±0,4 in 150μM treated-sample, p<0.05), as monitored by Annexin V/PI staining. These data suggest that PAOX inhibition could represent a therapeutic strategy to selectively target PMF cells without affecting normal hematopoietic cells' survival.Moreover, our integrative analysis of gene expression and CN data pointed out the concomitant copy number loss and transcriptional down-regulation in PMF patients of the chromatin modifier HMGXB4. Interestingly, silencing of HMGXB4 in CD34+ stem/progenitor cells induced megakaryocyte differentiation, as demonstrated by increased expression of CD41 (36±1,9% vs 26,1±2,9% at day 12, 52,5±4,9% vs 33±1% at day 14 of serum free liquid culture, p<0.05) and increased number of CFU-MK colonies (27,7±2,5 vs 18,1±1,3% in small CFU-MK colonies, p<0.05). On the other hand, HMGXB4 silencing repressed the erythroid differentiation, as indicated by a decrease in the percentage of cells positive for the erythroid marker GPA (10.8%±5.6 vs 24.8%±5, p<0.05) and significant decrease in Burst Forming Unit-Erythroid (BFU-E) and Colony-Forming Unit-Erythroid (CFU-E) (47,9±6 vs 60,5±7,6, p<0.05). Taken together, these data suggest that HMGXB4 silencing in human HSPCs favors megakaryocyte differentiation while restraining the erythroid lineage. These results highlight a previously un-reported, yet potentially interesting role of HMGXB4 in the hematopoietic system and suggest that genomic and transcriptional imbalances of HMGXB4 could contribute to the aberrant expansion of the megakaryocytic lineage that characterize PMF patients.In conclusion, our work sheds light on the influence of genomic abnormalities on gene expression regulation in PMF CD34+ cells and on their impact to features typical of PMF, such as a hyperplastic megakaryopoiesis and resistance to apoptosis, and therefore potentially contributing to the development of the myeloproliferative disease. DisclosuresVannucchi:Novartis: Other: Research Funding paid to institution (University of Florence), Research Funding; Shire: Speakers Bureau; Baxalta: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Multipotent Hematopoietic Progenitors Divide Asymmetrically to Create Progenitors of the Lymphomyeloid and Erythromyeloid Lineages

SummaryHematopoietic stem and progenitor cells (HSPCs) can self-renew and create committed progenitors, a process supposed to involve asymmetric cell divisions (ACDs). Previously, we had linked the kinetics of CD133 expression with ACDs but failed to detect asymmetric segregation of classical CD133 epitopes on fixed, mitotic HSPCs. Now, by using a novel anti-CD133 antibody (HC7), we confirmed the occurrence of asymmetric CD133 segregation on paraformaldehyde-fixed and living HSPCs. After showing that HC7 binding does not recognizably affect biological features of human HSPCs, we studied ACDs in different HSPC subtypes and determined the developmental potential of arising daughter cells at the single-cell level. Approximately 70% of the HSPCs of the multipotent progenitor (MPP) fraction studied performed ACDs, and about 25% generated lymphoid-primed multipotent progenitor (LMPP) as wells as erythromyeloid progenitor (EMP) daughter cells. Since MPPs hardly created daughter cells maintaining MPP characteristics, our data suggest that under conventional culture conditions, ACDs are lineage instructive rather than self-renewing.

Effect of endothelial cell-targeted soluble Notch ligand hD1R protein on expansion and engraftment of cord blood hematopoietic stem/progenitor cells

To evaluate the effects of endothelial cell- targeted soluble Notch ligand hD1R protein on expansion and engraftment of cord blood hematopoietic stem/progenitor cell (CB HSPCs). Recombinant hD1R protein was first induced and purified. Human cord blood CD34⁺ cells were co-cultured on human umbilical vein endothelial cells (HUVECs) supplemented with a cocktail containing 5 types of human cytokines including TPO, SCF, FL, IL-6, IL-3 (5GF) and soluble hD1R. The expansion of CD34⁺ cells was tested under different culture conditions including PBS group (PBS replaces HUVEC), hD1R group, sup group (HUVEC supernatant replaces HUVEC), fix group (fixed HUVEC replaces HUVEC), Day 0 group (Control). Cell cycle and apoptosis of cultured cells were also analyzed. Their progeny expanded in PBS or hD1R group were transplanted into sublethally irradiated NOD/SCID mice. The percentages of human CD45⁺ (hCD45⁺) cells in the marrow of recipient mice were determined by FACS 12 weeks later. hD1R induced more expansion in the total number of CD34⁺ cells cocultured with HUVECs plus 5GF, which was 87.50-fold increase compared to the Day 0 group, and 7.98-fold increase than that of PBS group. FACS analysis also showed that the percentage of CD34⁺ cells was 77.0% in G0/G1 phase in the hD1R group, which indicated that hD1R enhanced HSPCs expansion and inhibited apoptosis. Moreover, hD1R significantly promoted human HSPC engraftment after BM transplantation in irradiated mice. The Notch-mediated ex vivo expansion system has been established and hD1R promoted expansion and engraftment of human CB HSPCs, which provided the evidence for further clinical application.

Oncogenic fusion proteins expressed in immature hematopoietic cells fail to recapitulate the transcriptional changes observed in human AML.

Reciprocal chromosomal translocations are observed in one-third of acute myeloid leukemia (AML) cases. Targeting and understanding the effects of the resulting aberrant oncogenic fusion proteins may help developing drugs against specific leukemic subtypes, as demonstrated earlier by the use of ATRA in acute promyelocytic leukemia. Hematopoietic stem/progenitor (HSPCs) cells transduced with oncogenic fusion genes are regarded as promising in vitromodels of their corresponding AML subtypes. Here, we critically assessed the potential of such in vitro models using an integrative bioinformatics approach. Surprisingly, we found that the gene-expression profiles of CD34+ human HSPCs transformed with the potent oncogenic fusion proteins AML-ETO or MLL-AF9, only weakly resembled those derived from primary AML samples. Hence, our work raises concerns as to the relevance of the use of in vitro transduced cells to study the impact of transcriptional deregulation in human AML.

Lncrna Hematlas Defines Blood Lineage-Specific RNA Expression Signatures and Novel Lincrna Biomarkers

Long non-coding RNAs (lncRNAs) recently emerged as central regulators of chromatin and gene expression. We created a comprehensive lncRNA HemAtlas in human and murine blood cells. We sampled RNA from differentiated granulocytes, monocytes, erythroid precursors, in vitro maturated megakaryocytes, CD4-T and CD8-T cells, NK cells, B cells and stem cells (human CD34+ cord blood hematopoietic stem and progenitor cells [CB-HSPCs]) and subjected them to microarray analysis of mRNA and lncRNA expression. Moreover, the human LncRNA HemAtlas was complemented with human hematopoietic stem cells (HSCs; CD34+/CD38-), megakaryocytic/erythroid progenitors (MEPs; CD34+/CD38+/CD45RA-/CD123-), common myeloid progenitors (CMPs; CD34+/CD38+/CD45RA-/CD123+) and granulocytic/monocytic progenitors (GMPs; CD34+/CD38+/CD45RA+/CD123+) from fetal liver (FL), CB and peripheral blood (PB) HSPCs.The complete microarray profiling of the differentiated cells yielded a total of 1588 (on Arraystar® platform) and 1439 lncRNAs (on NCode® platform), which were more than 20-fold differentially expressed between the blood lineages. Thus, a core fraction of lncRNAs is modulated during differentiation. LncRNA subtype comparison for each lineage, schematics of mRNA:lncRNA lineage coexpression and genomic loci correlation revealed a complex genetic interplay regulating hematopoiesis.Integrated bioinformatic analyses determined the top 50 lineage-specific lncRNAs for each blood cell lineage in both species, while gene set enrichment analysis (GSEA) confirmed lineage identity. The megakaryocytic/erythroid expression program was already evident in MEPs, while monocytoc/granulocytic signatures were found in GMPs.Amongst all significantly associated genes, 46% were lncRNAs, while 5% belonged to the subgroup of long intervening non-coding RNAs (lincRNA).For human megakaryocytes, erythroid cells, monocytes, granulocytes and HSPCs we validated four lincRNA candidates, respectively, to be specifically expressed by qRT-PCR. RNAi knock-down studies using two shRNA constructs per candidate demonstrated an impact on proliferation, survival or lineage specification for at least one specific lincRNA per lineage. We detected a 3 to 4.5-fold increased colony-forming capacity upon knockdown of the HSPC-specific PTMAP6 lincRNA in methylcellulose colony-forming unit (CFU) assays. Inversely, knockdown of monocyte-specific DB519945 resulted in 3.5 to 5.5-fold reduction of the total number of CFUs. Likewise, the total CFU counts was 4.3-fold reduced upon knockdown of megakaryocyte-specific AK093872. Kockdown of the granulocyte-specific LINC00173 perturbed granulocytic in vitro differentiation as assessed by the percentage of CD66b+/CD13+ granulocytes (2-fold reduction) and nuclear lobulation (MGG-stained cytospins). The erythroid-specific transcript AY034471 showed 25 to 50% reduction in burst-forming units in collagen-based assays.Thus, our study provides a global human hematopoietic lncRNA expression resource and defines blood-lineage specific lncRNA marker and regulator genes. Disclosures:No relevant conflicts of interest to declare.

Perivascular support of human hematopoietic stem/progenitor cells

AbstractHematopoietic stem and progenitor cells (HSPCs) emerge and develop adjacent to blood vessel walls in the yolk sac, aorta-gonad-mesonephros region, embryonic liver, and fetal bone marrow. In adult mouse bone marrow, perivascular cells shape a “niche” for HSPCs. Mesenchymal stem/stromal cells (MSCs), which support hematopoiesis in culture, are themselves derived in part from perivascular cells. In order to define their direct role in hematopoiesis, we tested the ability of purified human CD146+ perivascular cells, as compared with unfractionated MSCs and CD146− cells, to sustain human HSPCs in coculture. CD146+ perivascular cells support the long-term persistence, through cell-to-cell contact and at least partly via Notch activation, of human myelolymphoid HSPCs able to engraft primary and secondary immunodeficient mice. Conversely, unfractionated MSCs and CD146− cells induce differentiation and compromise ex vivo maintenance of HSPCs. Moreover, CD146+ perivascular cells express, natively and in culture, molecular markers of the vascular hematopoietic niche. Unexpectedly, this dramatic, previously undocumented ability to support hematopoietic stem cells is present in CD146+ perivascular cells extracted from the nonhematopoietic adipose tissue.

A Novel Perspective On Hematopoietic Stem/Progenitor Cell Migration and homing—evidence That Cationic Antimicrobial Peptides (C3a, LL-37, and β2-defensin) Prime (enhance) the Responsiveness of These Cells to a Low SDF-1 Gradient by Promoting Secretion of ATP, Indicating the Involvement of an Autocrine Purinergic Loop in Their Migration.

Abstract 2989 Background.It has been reported that proteolytic enzymes are upregulated in BM after conditioning for transplantation and, by removal of three N-terminal amino acids from stromal derived factor-1 (SDF-1), abrogate its chemotactic activity, even if the SDF-1 peptide remains detectable by ELISA (Cancer Res. 2010;70:3402). We also reported that cationic antimicrobial peptides (CAMPs), such as complement cascade C3 protein cleavage fragment C3a, b2-defensin, and cathelicidin (LL-37) (Leukemia 2012, 26, 736), potently enhance the migration and homing responses of HSPCs to a low SDF-1 gradient, which is critical for retention of SDF-1 chemotactic activity in the highly proteolytic microenvironment of BM induced by conditioning for transplantation. This priming effect depends on incorporation of the CXCR4 receptor into membrane lipid rafts, a phenomenon that allows for more efficient interaction of the CXCR4 receptor with downstream signaling proteins (Blood 2005;101:3784). However, we were aware that inhibition of lipid raft formation by b-methylcyclodextrin inhibits only ∼50% of the CAMP priming effect, which strongly indicates the involvement of other mechanisms. Interestingly, it was reported recently that LL-37, by involving pannexin channels, enhances the release of ATP from cells (J Biol. Chem. 2008;263:30471) that had been described to be an autocrine chemottractant secreted at the leading edge of migrating macrophages and neutrophils (Science 2006;314:1792). Exogenous ATP has also been reported in Tranwell assays to chemoattract HSPCs (Blood 2004;104:1662). Hypothesis.We hypothesized that HSPCs, like monocytes and neutrophils, release at their leading edge ATP, and respond to this autocrine chemoattractant. Thus, this autocrine ATP migration-enhancing phenomenon could provide not only new insight into mechanisms regulating the migration of HSPCs but also better explain the priming effect of CAMPs to a low or decreasing SDF-1 gradient. Experimental approach.First, we tested the responsiveness of murine and human BM-, mobilized peripheral blood (mPB)-, and human umbilical cord blood (UCB)-derived HSPCs to different extracellular nucleotides including ATP by performing i) Transwell migration assays, ii) MAPKp42/44 and AKT phosphorylation studies, and iii) RQ-PCR expression of different purinergic receptors on normal HSPCs. The priming effect of CAMPs on autocrine secretion of ATP by HSPCS was studied in functional chemotaxis assays by employing the broad-spectrum ATP receptor antagonist suramin and apyrase, an enzyme that degrades extracellular ATP. Results.We noticed that for all the nucleotides tested (ATP, UTP, GTP, TTP, and CTP), ATP has the strongest chemotactic activity against murine and human BM-derived HSPCs, which correlated with the phosphorylation of MAPKp42/44 and AKT. In contrast to BM-derived HSPCs, the chemotactic effect of ATP and its induction of signaling were significantly attenuated for mPB- and UCB-derived HSPCs, which suggests a desensitization of purinergic surface receptors by free extracellular nucleotides circulating in blood plasma. Most importantly, we found that exposure of HSPCs to suramin or apyrase significantly diminished (by ∼50%) the priming effect of CAMPs in enhancing the responsiveness of these cells to a low SDF-1 gradient. Finally, we found that both ATP and CAMPs are secreted from irradiated BM stromal cells, which provides a heretofore underappreciated homing signal for HSPCs. Conclusions.We show that ATP secreted both as a paracrine factor by BM stromal cells during conditioning for transplantation and by migrating HSPCs at their leading edge enhances migration and homing of HSPCs to BM niches. Most importantly, our data demonstrates, for first time, the involvement of an autocrine ATP–purinergic receptor migration regulatory loop, which better explains the pro-migratory priming effect seen after exposure of HSPCs to CAMPs. Since CAMPs, which enhance migration of HSPCs to a low SDF-1 gradient are upregulated like ATP in irradiated BM, all these factors together orchestrate the responsiveness of HSPCs to a decreasing SDF-1 gradient in the proteolytic microenvironment of BM conditioned for transplantation. These mechanisms should be further explored to improve the homing of HSPCs after transplantation, and we are currently testing this possibility in appropriate animal models. Disclosures:No relevant conflicts of interest to declare.

A Novel Perspective on Stem Cell Homing – Emerging Interplay Between Bioactive Lipids As Potent Chemoattractants and Cationic Peptides As Underappreciated Modulators of Responsiveness to SDF-1 Gradients

Abstract 2957 Background.Hematopoietic stem progenitor cells (HSPCs) are retained in bone marrow (BM) niches due to the stromal-derived growth factor-1 (SDF-1)–CXCR4 receptor axis and interactions between Very Late Antigen-4 (VLA-4 or a4b1 integrin) and its ligand, Vascular Adhesion Molecule-1 (VCAM-1 or CD106). While HSPCs express CXCR4 and VLA-4, their corresponding ligands, SDF-1 and VCAM-1, are expressed by cells in the BM microenvironment (e.g., osteoblasts and fibroblasts). While a role for the SDF-1–CXCR4 axis in the retention of HSPCs in BM under steady state conditions is undisputed, its role in stem cell homing needs further clarification. For many years, it has been proposed that concentration differences between the chemotactic SDF-1 gradient across the BM–peripheral blood (PB) barrier determines whether cells will home from PB into the BM microenvironment. However, this simple explanation has been challenged by several observations supporting the existence of SDF-1–CXCR4-independent homing mechanisms. For example, i) CXCR4−/– fetal liver HSPCs can home to BM in an SDF-1-independent manner, ii) homing of murine HSPCs made refractory to SDF-1 by preincubation and co-injection with a CXCR4 receptor antagonist (AMD3100) is normal or only mildly reduced, iii) HSPCs in which CXCR4 expression is reduced by a SDF-1 intrakine strategy remain able to engraft in lethally irradiated recipients, and as we recently reported, iv) myeloablative conditioning for transplantation induces a highly proteolytic microenvironment in BM that leads to proteolytic degradation of SDF-1 (Leukemia 2011, doi: 10.1038/leu.2011.185). Aim of the study. Based on these observations that strongly suggest the involvement of other factors and/or supportive mechanisms in the SDF-1-mediated homing of HSPCs we become interested in a role of bioactive lipids (sphingosine-1 phosphate; S1P and ceramide-1 phosphate; C1P) as well as priming phenomenon of SDF-1-CXCR4 axis by cationic antimicrobial peptides (CAMPs) in HSPCs homing. Results. We present evidence that gradients of bioactive lipids, such as S1P and C1P, which are products of membrane lipid metabolism, are involved in stem cell lodgment into BM. In addition, we present another important mechanism that enhances stem cell response to an low SDF-1 concentration gradient, namely, the “priming effect”. This mechanism is based on a phenomenon in which CAMPs such as complement cascade cleavage fragment C3a and stroma fibroblast- and granulocytes-derived antimicrobial peptide LL-37 significantly increase (up to 25 times) the chemotactic responsiveness of HSPCs to very low “tissue concentration-relevant” SDF-1 gradients (1–3 ng/ml). To support this further murine HSPCs primed ex vivo with CAMPs engrafted by 3–5 days faster in lethally irradiated littermates. At the molecular level, this sensitization of the responsiveness to SDF-1 after exposure to CAMPs depends on the incorporation of the CXCR4 receptor into membrane lipid rafts. This phenomenon is specific for SDF-CXCR4 axis as we did not observe that this phenomenon influences S1P- and C1P-mediated in vitro chemotaxis. Overall, these changes of SDF-1, S1P, and C1P chemotactic gradients and the appearance of priming factors in the BM microenvironment and PB plasma are triggered during conditioning for transplantation by radio-chemotherapy by the induction of a proteolytic microenvironment in BM and the activation of the complement cascade (CC). Conclusions. We provide in vivo evidence that C3a and LL-37, which have primarily antimicrobial functions and are harmless to mammalian cells, could be clinically applied to accelerate engraftment as ex vivo priming agent for transplanted human HSPCs. This novel approach based on a short ex vivo incubation of HSPCs in the graft before transplantation by CAMPs (C3a and LL-37) is of particular important in umbilical cord blood transplantations, where the number of available HSPCs in single unit for transplant is limited. This strategy is currently tested in the clinic. Disclosures:No relevant conflicts of interest to declare.

A Novel Paradigm In Stem Cell Trafficking: The Ratio of Peripheral Blood Sphingosine-1 Phosphate (S1P) to Bone Marrow Ceramide-1 Phosphate (C1P) Regulates Mobilization and Homing of Hematopoietic Stem Cells

AbstractAbstract 554The stromal derived factor-1 (SDF-1)–CXCR4 axis plays an unquestioned role in developmental migration of hematopoietic stem cells (HSPCs) and their retention in the bone marrow (BM). However, changes in the SDF-1 gradient between BM and peripheral blood (PB) do not always support its having a crucial role as chemoattractant for mobilization or homing of HSPCs. As demonstrated by others (e.g., Bone Marrow Transplantation 2003; 31:651–654, and Transfus Apher Sci 2009;40:159) and us (Leukemia 2010;24:976–985) the plasma SDF-1 level does not correlate with mobilization of HSPCs. On the other hand, there is increasing doubt about an exclusive role for SDF-1 in homing of HSPCs in BM. This is based on evidence that i) CXCR4−/− fetal liver HSPCs may home to BM in an SDF-1–independent manner (Immunity 1999;10:463-471), ii) homing of murine HSPCs made refractory to SDF-1 by incubation and co-injection with a CXCR4 receptor antagonist is normal or only mildly reduced (Science 2004;305:1000), and finally iii) HSPCs in which CXCR4 has been knocked down by means of an SDF-1 intrakine strategy also engraft in lethally irradiated recipients (Blood 2000;96:2074–,2080). All this strongly suggests the existence of other factors involved in the mobilization and homing of HSPCs. Moreover, while SDF-1 is a potent chemoattractant for HSPCs when employed at supraphysiological concentrations in vitro, as a peptide it is highly susceptible to degradation by proteases that are elevated, for example, in PB during stem cell mobilization or in the BM microenvironment after myeloablative conditioning for transplantation. Employing ELISA for detection in the present study, we observed insignificant changes in SDF-1 level both in PB during mobilization and in BM after myeloablative conditioning. We also found that mobilized PB (mPB) plasma as well as conditioned media (CM) from lethally irradiated mice chemoattract HSPCs in an SDF-1–independent manner as demonstrated by i) normal chemotaxis of AMD3100 pre-treated cells and ii) preservation of chemotactic activity of plasma and BM-derived CM following heat inactivation. However, the chemotactic activity of mPB plasma and BM CM was inhibited after stripping by activated charcoal. This suggested the involvement of small molecule bioactive lipids. It is known that sphingolipids, which are important components of cell membranes, give rise to two bioactive derivatives, sphingosine-1 phosphate (S1P) and ceramide-1 phosphate (C1P), with S1P already identified as a chemoattractant for HSPCs (Ann N Y Acad Sci. 200;1044:84–89). To our surprise, we found that C1P is also a strong chemoattractant for human and murine HSPCs. In addition, we observed that at physiological concentrations both these bioactive lipids i) activate phosphorylation of MAPKp42/44 and AKT in HSPCs, ii) induce expression of matrix metalloproteinases (MMPs), and iii) modulate adhesion to stroma and endothelium. Interestingly, by employing ELISA and/or mass spectophotometry we found that, while the S1P level increases in PB during mobilization, the C1P level increases in BM after myeloablative conditioning for transplantation. Based on these findings, we propose a new paradigm in which the S1P:C1P ratio plays a role in mobilization and homing of HSPCs. While S1P is a major chemoattractant that directs egress of HSPCs from BM into PB, C1P released from damaged cells in BM after myeloablative conditioning creates a homing gradient for circulating HSPCs. We also postulate that the S1P:C1P ratio plays a more universal role and is involved in regulating migration of other types of stem cells, such as circulating mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs), and very small embryonic-like (VSEL) stem cells. Accordingly, while S1P plays a role in egress of stem cells into PB, C1P released from damaged cells (e.g., in infarcted myocardium or brain tissue after stroke) chemoattracts circulating stem cells for potential repair. Disclosures:No relevant conflicts of interest to declare.

Differential gene expression of human stem progenitor cells derived from early stages of in utero human hematopoiesis

AbstractHematopoietic stem progenitor cells (HSPCs) are highly enriched in a rare subset of Lin-CD34+CD38-cells. Independent of stage of human development, HSPC function segregates to the subset of Lin-CD34+CD38-cells. However, fetal-derived HSPCs demonstrate distinct self-renewal and differentiation capacities compared with their adult counterparts. Here, to characterize the molecular nature of fetal HSPCs, suppressive subtractive hybridization was used to compare gene expression of HSPCs isolated from fetal blood (FB-HSPCs) versus adult mobilized peripheral blood (MPB-HSPCs). We identified 97 differentially expressed genes that could be annotated into distinct groups that include transcription factors, cell cycle regulators, and genes involved in signal transduction. Candidate regulators, such as Lim only domain-2 (LMO2), nuclear factor–kappa B (NF-κB), tripartite motif 28 (Trim28), and N-myc protooncogene (MYCN), and a novel homeobox gene product were among transcripts that were found to be differentially expressed and could be associated with specific proliferation and differentiation properties unique to FB-HSPCs. Interestingly, the majority of genes associated with signal transduction belong toRaspathway, highlighting the significance of Ras signaling in FB-HSPCs. Genes differentially expressed in FB-HSPCs versus adult MPB-HSPCs were verified using quantitative real-time polymerase chain reaction (Q-PCR). This approach also resulted in the identification of a transcript that is highly expressed in FB-HSPCs but not detectable in more differentiated Lin-CD34+CD38+FB progenitors. Our investigation represents the first study to compare phenotypically similar, but functionally distinct, HSPC populations and to provide a gene profile of unique human HSPCs with higher proliferative capacity derived from early in utero human blood development.