From the Bone Marrow to the Thymic Niche

Abstract To generate T cells throughout adult life, the thymus must import hematopoietic progenitor cells from the bone marrow via the blood. The cellular and molecular mechanisms governing the circulation of thymus-seeding progenitor cells are well characterized in mice but not in humans. The aim of the present study was to characterize the molecular mechanisms and cellular components involved in thymus colonization by lymphoid progenitors (CD34+/CD10+/CD7-/CD24-) and the early steps of thymopoiesis under physiological conditions in humans. Our results demonstrate that circulating lymphoid progenitor cells express CCR9 and CXCR4 chemokine receptors, VLA-4, VLA-5 and VLA-6 integrins and PSGL-1 and CD44 adhesion molecules. We used in vitro migration and adhesion assays to validate the functional status of these markers. As in the mouse, human circulating progenitor cells enter the thymus at the corticomedullary junction (CMJ). Once in the thymus, crosstalk with thymic epithelial cells causes the circulating progenitors to commit to the T-cell differentiation pathway. In order to characterize thymic niches and interactions between circulating progenitors and the thymic stroma, we undertook a chemokine/chemokine-receptor-focused gene expression analysis of sorted lymphoid progenitor cells and CMJ epithelial cells (based on the expression of EpCAM and Delta-like-4). We observed an unexpected gene expression profile for chemokines and chemokine regulators in thymus-seeding CD34+/CD10+/CD7-/CD24- cells and epithelial cells at the CMJ. The present results should help us to highlight candidate genes involved in the early steps of human thymopoiesis. Disclosures No relevant conflicts of interest to declare.

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Human thymic epithelial cells maintain long-term survival of clonogenic myeloid and erythroid progenitor cells in vitro

British Journal of Haematology ◽

10.1046/j.1365-2141.2000.02337.x ◽

2000 ◽

Vol 111 (1) ◽

pp. 363-370 ◽

Cited By ~ 1

Author(s):

Katsuto Takenaka ◽

Mine Harada ◽

Tomoaki Fujisaki ◽

Koji Nagafuji ◽

Shinichi Mizuno ◽

...

Keyword(s):

Epithelial Cells ◽

Progenitor Cells ◽

Thymic Epithelial Cells ◽

Erythroid Progenitor ◽

Term Survival ◽

Long Term Survival ◽

Erythroid Progenitor Cells

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PU.1 and p53 Double Mutant Mice Develop Aggressive AML with Dysplastic Features

Blood ◽

10.1182/blood.v120.21.769.769 ◽

2012 ◽

Vol 120 (21) ◽

pp. 769-769

Author(s):

Petra Vlckova ◽

Libor Stanek ◽

Pavel Burda ◽

Karin Vargova ◽

Filipp Savvulidi ◽

...

Keyword(s):

Bone Marrow ◽

Transcription Factor ◽

Progenitor Cells ◽

Double Mutant ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Regulatory Element ◽

Tumour Suppressor ◽

Mutant Mice ◽

Stem And Progenitor Cells

Abstract Abstract 769 Introduction: Downregulation of tumour suppressor transcription factor PU.1 in haematopoietic stem and progenitor cells represents primary underlying mechanism for the development of acute myeloid leukaemia (AML) in mice with homozygous deletion of the upstream regulatory element (URE) of PU.1 gene. Human AML often display differences in aggressiveness that are associated with mutations of a well known tumour suppressor p53. We produced murine model carrying mutations of p53 and URE that develops highly aggressive AML and focused on molecular mechanisms that are responsible for AML aggressiveness. Mouse models: PU.1ure/ure (Rosenbauer F, et al. 2004) and p53−/− (Jacks T, et al. 1994) mice were used. Conditional deletion of the URE leads to downregulation of PU.1 and is marked by clonal accumulation of myeloid c-Kit+Mac-1low Gr-1low blast cells within bone marrow, spleen, and peripheral blood mirrored by lower numbers of lymphoid and erythroid cells. AML development in PU.1ure/ure mice involves a preleukaemic phase (at 2–3 months) marked by proliferation of myeloid c-Kit+Gr-1+ cells and splenomegaly. Interestingly, p53−/−mice do not develop AML, instead loss of p53 predisposes mice to solid tumours, mostly lymphomas, by 6 months of age. Results: Deletion of TP53 in the PU.1ure/ure mice (PU.1ure/ure p53−/−) results in more aggressive AML with significantly shortened overall survival, prominent hepatosplenomegaly and cachexia (wasting syndrome). Mild differences in cell surface phenotype of bone marrow derived cells were observed between PU.1ure/ure and PU.1ure/ure p53−/− mice by flow cytometry (these included: blasts expansion and lymphopenia). Next, the PU.1 expression was determined in all genotypes at progenitor and stem cell levels. PU.1 mRNA level in more aggressive PU.1ure/ure p53−/− murine AML is decreased in the entire c-Kit+tumour cell population compared to AML in PU.1ure/ure mice including haematopoietic stem and progenitor cells (HSPCs). Correspondingly to RNA level, in the PU.1ure/ure progenitors the PU.1 protein was decreased compared to p53−/− progenitors and is yet further reduced in the PU.1ure/ure p53−/− c-Kit+ Mac1+progenitors. p53−/− progenitors express similar level of PU.1 as wild type progenitors indicating that despite p53 can bind DNA as a transcription factor, it does not regulate PU.1 level directly. In addition to URE deletion we searched for other mechanisms that control PU.1 levels and found that PU.1-inhibiting microRNA miR-155 gene display altered chromatin structure and expression of both pri-miR-155 as well as its spliced mature form in the AML of PU.1ure/ure and (to higher extent in) PU.1ure/ure p53−/− murine progenitors. Upregulation of miR-155 coincides with upregulation of the Mir155hg activators: Myc and Myb. Finally, upon inhibition of either Myb or miR-155 in vitro the AML progenitors restore PU.1 levels and lose leukaemic cell growth. Conclusion: In summary, PU.1 and p53 double mutant mice develop aggressive AML with dysplastic features. Defective control of PU.1 levels in PU.1ure/ure and PU.1ure/ure p53−/−AML involves miR-155. Lastly, restored PU.1 level and cell differentiation capacity are achieved by inhibiting either Myb or miR-155 in the PU.1ure/ure p53−/− progenitors. (Grant support: P305/12/1033, UNCE 204021, PRVOUK-P24/LF1/3, SVV-2012-264507, P301/12/P380. MK was sponsored by GAUK 251070 45410, 251135 82210) 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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Hedgehog Promotes Neovascularization through the Regulation of Bone-Marrow Derived Progenitors.

Blood ◽

10.1182/blood.v114.22.3048.3048 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3048-3048

Author(s):

Yusuke Mizukami ◽

Junpei Sasajima ◽

Kazumasa Nakamura ◽

Kazuya Sato ◽

Yoshiaki Sugiyama ◽

...

Keyword(s):

Bone Marrow ◽

Cancer Cells ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Tumor Vasculature ◽

Ductal Adenocarcinoma ◽

Mrna Levels ◽

Development Organization ◽

Hh Signaling

Abstract Abstract 3048 Poster Board II-1024 The hedgehog (Hh) pathway has been implicated in the development of embryonic blood vessels and pathogenesis of cancer. Smoothened (Smo), one of the receptors in Hh signaling, is a promising molecular target for the treatment of malignancies. Pancreatic ductal adenocarcinoma (PDAC) is one of the tumors in which sonic hedgehog (Shh) is misexpressed. Although there are cell-autonomous effects of Hh on the proliferation of tumor cells, recent studies have demonstrated an oncogenic function of Hh in stromal cells. Cyclopamine antagonizes Smo and can attenuate PDAC growth in mice, resulting in regression of the tumor vasculature with reduced pericyte coverage. However, the inhibitory effect of cyclopamine on proliferation of KP-1N cells, a human PDAC line highly expressing Shh, was modest, indicating additional effects of Hh signaling on tumor progression. Here, we have identified novel molecular mechanisms by which Hh regulates tumor angiogenesis. Expression of Gli2 protein in the stroma, but not in cancer cells, was attenuated markedly by cyclopamine administration, consistent with the general absence of autocrine Hh signaling in PDAC cells. Cyclopamine significantly attenuated the homing of bone marrow (BM)-derived cells into KP-1N xenografts and their interaction with the tumor vasculature, suggesting that Hh signaling may play a role during migration and differentiation of BM-derived progenitors to participate in neovascularization. Host derived Ang-1 and IGF-1 mRNA levels in xenografts were strongly downregulated by cyclopamine, which may contribute to the maintenance and maturation of tumor vasculature. In vitro co-culture experiments demonstrated that KP-1N cells induced Ang-1/IGF-1 production in BM-progenitors (c-Kit+ fraction of BM mononuclear cell), and this induction was significantly attenuated either by cyclopamine or lentiviral shRNA targeting Smo. In addition, in vitro tube formation assay with the mouse endothelial line MS-1 and a matrigel plug assay supports the role of Shh secreted from PDAC cells to induce migration and capillary formation of BM-derived progenitors. IGF-1 is a crucial target of Hh signaling in BM-derived cells during neovascularization, since anti-IGF-1 neutralizing antibody blocked the induction of the capillary morphogenesis by BM-progenitors. Finally, this “paracrine” effect of Hh seems to be a late event during pancreatic tumorigenesis, as stromal Patch1/Gli2 expression was detected within PDAC lesions in Pdx1-Cre;LSL-KrasG12D;p53lox/+ mice, but not in PanIN lesion, a potential precursor of PDAC, in Pdx1-Cre;LSL-KrasG12D mice. We also observed upregulation of VE-cadherin and Ptch1 mRNA in lineage–/c-Kit+ fraction of BM mononuclear cells (primitive BM-derived progenitors) from PDAC mice as compared to wild-type/PanIN mice, suggesting that pro-angiogenic conditions are prepared at the level of the BM in cancer-bearing hosts. The primitive progenitors derived from ‘activated BM’ are imported to the tumor microenvironment where they become fully activated. Hh-ligand from cancer cells can therefore have a profound effect on neovascularization through the regulation of the progenitors during late stages of tumorigenesis. This work was supported by New Energy and Industrial Technology Development Organization (NEDO) of Japan. Disclosures No relevant conflicts of interest to declare.

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Primary Bone Marrow-Derived MSC Subsets Have Distinct Wnt-Signatures Compared to Conventionally Cultured MSC and Differ in Their Capacity to Support Hematopoiesis in Vitro,

Blood ◽

10.1182/blood.v118.21.3414.3414 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3414-3414 ◽

Cited By ~ 1

Author(s):

Marijke W Maijenburg ◽

Marion Kleijer ◽

Kim Vermeul ◽

Erik P.J. Mul ◽

Floris P.J. van Alphen ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Wnt Signaling ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Cell Types ◽

Hematopoietic Stem ◽

Wnt Proteins

Abstract Abstract 3414 Mesenchymal stromal cells (MSC) are of promising therapeutic use to suppress immunogenic responses following transplantation, and to support expansion of hematopoietic stem- and progenitors cells (HSPC) from small transplants derived for instance from cord blood. Culture-expanded MSC produce a wide variety and quantity of Wnt-proteins and the crucial role of Wnt-signaling in the hematopoietic stem cell niche is well established. However, studies addressing Wnt-signaling in MSC have (i) only focused on culture-expanded MSC and (ii) did not discriminate between phenotypically distinct subpopulations which are present in bulk cultures of expanded MSC. Recently we identified three new subpopulations of MSC in human bone marrow (BM) based on expression of CD271 and CD146: CD271brightCD146−, CD271brightCD146+, CD271−CD146+. These fractions co-express the “classical” MSC markers CD90 and CD105 and lack expression of CD45 and CD34 (Maijenburg et al, Blood 2010, 116, 2590). We and others demonstrated that the adult BM-derived CD271brightCD146− and CD271brightCD146+ cells contain all colony forming units-fibroblasts (Maijenburg et al, Blood 2010, 116, 2590; Tormin et al, Blood 2010, 116, 2594). To investigate how these primary subsets functionally compare to conventional, culture-expanded MSC, we investigated their Wnt-signature and hematopoietic support capacity. To this end, we sorted CD271brightCD146− and CD271brightCD146+ cells from human adult BM (n=3) and compared their Wnt-signatures obtained by Wnt-PCR array to the profiles from cultured MSC from the same donors. Fifteen genes were consistently differentially expressed in the two sorted uncultured subsets compared to their conventionally cultured counterparts. Expression of CCND1, WISP1 and WNT5B was strongly increased, and WNT5A was only detected in the conventionally cultured MSC. In contrast, WNT3A was exclusively expressed by sorted primary CD271brightCD146− and CD271brightCD146+ cells, that also expressed higher levels of JUN, LEF1 and WIF1. The differences in Wnt (target)-gene expression between CD271brightCD146− and CD271brightCD146+ cells were more subtle. The Wnt-receptors LRP6 and FZD7 were significantly higher expressed in CD271brightCD146+ cells, and a trend towards increased expression in the same subset was observed for CTNNB1, WNT11 and MYC. When the sorted subsets were cultured for 14 days (one passage), the differences in Wnt-related gene expression between the subsets was lost and the expanded sorted cells acquired an almost similar Wnt-signature as the MSC cultured from BM mononuclear cells from the same donors. The cultured subsets lost the expression of Wnt3a and gained the expression of Wnt5a, similar to the unsorted MSC cultured from the same donors in parallel. Despite the loss of a distinct Wnt-signature, co-culture experiments combining the sorted MSC subsets with human HSPC revealed that CD271brightCD146+ cells have a significantly increased capacity to support HSPC in short-term co-cultures (2 weeks) compared to CD271brightCD146− cells (p<0.021, n=3), which was analyzed in hematopoietic colony assays following co-culture. In contrast, a trend towards better long-term hematopoietic support (co-culture for 6 weeks) was observed on CD271brightCD146− cells. In conclusion, we demonstrate for the first time that primary sorted uncultured MSC subsets have a distinct Wnt-signature compared to cultured unsorted MSC and display differences in hematopoietic support. As it was recently shown that CD271brightCD146− and CD271brightCD146+ MSC localize to separate niches in vivo (Tormin et al, Blood 2011), our data indicate that the two MSC subsets are not necessarily distinct cell types and that the different Wnt-signature may be a reflection of these distinct microenvironments. Cell culturing for only one passage dramatically changed the Wnt-signature of the sorted MSC subsets, indicating that Wnt-signaling in in vitro expanded MSC does not resemble the Wnt-signature in their tissue resident counterparts in vivo. Disclosures: No relevant conflicts of interest to declare.

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The Bleeding Defect Exhibited by Aryl Hydrocarbon Receptor-Null Mice Is Due to Defective Collagen-Dependent Outside-in Signaling

Blood ◽

10.1182/blood.v120.21.3294.3294 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3294-3294

Author(s):

Stephan C. Lindsey ◽

Jinlin Jiang ◽

Donna Woulfe ◽

Eleftherios T. Papoutsakis

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Surface Area ◽

Platelet Function ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Bone Marrow Niche ◽

Inside Out

Abstract Abstract 3294 We previously identified AHR as a novel regulator of megakaryocytic (Mk) polyploidization and differentiation (Lindsey et al. Brit J of Haem, 2011). Best known as a mediator of toxicological signals, we propose that AHR influences multiple aspects of normal hematopoietic differentiation (Lindsey et al. Stem Cell Rev, 2012), including unpublished data suggesting a role for AHR in mediating platelet function. AHR-null mice had 9% fewer platelets and 10.4% fewer reticulated, young RNA-containing platelets than WT mice. Abnormal Mk maturation played a role in this phenotype, as AhR-null mice had ca. 25% fewer high ploidy (= 32n) Mks residing within the murine bone marrow niche compared to WT mice. While investigating if AHR influenced platelet function, we found that AHR-null mice bleed 5.3 times longer (8 minutes for AHR-null mice compared to 1.5 minutes for WT mice) and lose 3 times as much blood as WT mice during bleeding time assays. Although significant, we felt that the decreased Mk polyploidization and resulting reduced platelet counts were not enough to explain the drastic bleeding phenotype in AHR-null mice. In agreement with our hypothesis that AHR impacts platelet function, others have suggested AHR is critical for blood clotting during Oryzias latipes embryogenesis (Kawamura et al. Zoolog Sci, 2002). We previously showed that treatment of bone marrow-derived progenitor cells with AHR ligands such as TCDD (dioxin, a prototypic AHR ligand and activator) during ex vivo expansion could produce polyploid CD41-expressing cells in the absence of any cytokines. Here, we show that ex vivo expansion of murine progenitor cells with 10 mM of the AHR inhibitor 6',2',4'-trimethoxyflavone (TMF) resulted in 37% fewer highly polyploid (≥32n) Mks by day 7 (n=3, p=0.017), effectively blocking the effects of TPO on Mk differentiation and suggesting that AHR activation is downstream of TPO signaling. To examine the dramatic bleeding phenotype present in AHR-null mice, we next turned our attention toward platelet function, mediated by both outside-in and inside-out signaling. Defects in either or both of these signaling cascades could result in the bleeding defect present in AHR-null mice. In our initial experiments, we found that platelets from AHR-null mice bind fibrinogen equivalently to WT platelets (n=3), suggesting that AHR is not involved in inside-out platelet signaling. As we investigated other measures of platelet activation, we found that although platelets from AHR-null mice efficiently aggregated in response to ADP and the PAR-4 agonist AYPGKF (n=3 p=0.897 and 0.914, respectively), only 20 percent of AHR-null murine platelets (compared to 60 percent for WT platelets) aggregated in response to collagen (n=3, p=0.013). Spreading assays further demonstrated defective collagen-dependent outside-in signaling in AHR-null mice. We found that 5 times as many AHR-null platelets remained round (lacking filipodia or lamellipodia) as WT platelets after resting on collagen-coated slides (100 ug/mL) for 5 minutes. Over 60 percent of WT platelets were fully spread after a 20 minute incubation on collagen, compared to only 35 percent of AHR-null platelets. Additionally, roughly 20 percent of AHR-null platelets failed to respond and maintained a round morphology, representing 8 times as many unresponsive platelets as WT mice. The extent of spreading also appeared altered in AHR-null platelets, as the surface area of AHR-null platelets spreading on collagen was reduced by 42% after 5 minutes and 39% after 20 minutes compared to WT (n=3; p<0.001 for both experiments). Similar responses were seen when AHR-null platelets were allowed to spread on fibrinogen (100 ug/mL) after activation by 1 ug/mL collagen, with a 39% and 28% reduction in the surface area of AHR-null platelets after 5 and 20 minutes, respectively (n=3; p<0.001 for both experiments). Based on these findings, we are now investigating the molecular mechanisms of the collagen signaling defects present in AHR-null platelets, beginning with known interactions between AHR and vav genes, critical mediators of collagen-dependant platelet outside-in signaling. Our work is significant in that it builds upon our previously reported data and provides evidence that AHR is a critical component of the physiologic response platelets undergo in response to collagen. This information may provide novel treatment options for patients with bleeding disorders. Disclosures: No relevant conflicts of interest to declare.

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Molecular Basis of Proliferation/Survival and Migration of CLL in Peripheral Blood, Bone Marrow and Lymph Nodes

Blood ◽

10.1182/blood.v112.11.546.546 ◽

2008 ◽

Vol 112 (11) ◽

pp. 546-546

Author(s):

Amit K Mittal ◽

Javeed Iqbal ◽

Tara Marie Nordgren ◽

Margaret Moragues ◽

R. Gregory Bociek ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

B Cell ◽

Chemokine Receptors ◽

Expression Patterns ◽

Gene Expression Patterns ◽

Bcr Signaling ◽

And Migration

Abstract B-cell chronic lymphocytic leukemia (CLL) is a heterogenous and incurable B-cell malignancy. CLL cells migrate and accumulate in different sites including the peripheral blood (PB), bone marrow (BM) and lymph nodes (LN) in vivo, but undergo apoptosis in vitro. Therefore, we hypothesized that CLL cells at these sites are different and receive different microenvironmental signals that regulate their proliferation/survival and migration. Most reports on the microenvironmental influence on CLL cells have used in vitro models consisting of stromal and CLL cells. However, in this study, to better understand the influence of site-specific microenvironments in vivo, gene expression patterns of CLL cells obtained from PB, BM and LN were investigated. CLL cells were isolated from patients’ PB (PB-CLL, n= 20), BM (BM-CLL, n=14) and LN (LN-CLL, n=15) and used to determine the gene expression patterns by microarray analysis. In addition, we also included PB-CLL cases from our previous study (n=40) to further validate the findings of this study. Significant Analyses of Microarray (SAM) revealed differential expression of more than 500 genes among these three sites. To understand the potential roles of these differentially-expressed genes and their association with relevant functional pathways in CLL, Gene Set Enrichment Analysis (GSEA) was performed. The validation of pathway specific genes was further confirmed by quantitative real time PCR. Among the pathways identified, the most active pathways associated with the migration and proliferation/survival of CLL cells, namely chemokine-signaling, BCR signaling, BAFF/APRIL-signaling, and NFκB-signaling pathways, were selected for further analyses. We hypothesized that chemokines and their receptors mediate the migration of CLL cells between PB and LN or BM, and that molecules of the BCR, BAFF/APRIL and NFκB pathways regulate proliferation/survival. To determine the role of chemokines and their receptors in CLL cell migration, we studied the expression of 52 chemokine/chemokine receptors and found that PB-CLL cells significantly (p<0.005) overexpressed CXCR4 and CCR7 compared to BM-CLL and LN-CLL cells. The ligands CCL21 and CXCL13 were significantly overexpressed (p<0.005 and p<0.01 respectively) in LN-CLL. These results indicate that PB-CLL cells express distinct chemokine receptors which may lead them to home to BM or LN and receive stimuli to form proliferation centers. Based on GSEA analysis, the stimuli for proliferation/survival for CLL cells in the LN and BM are provided by Syk and Btk (BCR signaling), BAFF and TRAF2 (BAFF/APRIL signaling), and several targets of the NFκB pathways. Syk and Btk were significantly overexpressed in LN-CLL (p<0.05) and PB-CLL (p<0.005) compared to BM-CLL, with the highest expression in LN-CLL, suggesting chronic activation of CLL cells in lymph node. Similarly, BAFF and TRAF2 were significantly overexpressed (p<0.03) in LN-CLL compared to PB-CLL and BM-CLL. Furthermore, the NFκB pathway, which is important for the proliferation and survival, also showed distinct association in different CLL-cell compartments. The RELA, NFκB1, NFκB2, TNFAIP3 and NFκB regulators such as NFκBIA, NFκBIE were also significantly (p<0.01) overexpressed in PB-CLL and BM-CLL compared to LN-CLL with highest expression in BM-CLL. Whereas few NFκB associated genes such as NFκB1L1 and RelB were significantly (p<0.02) expressed in LN-CLL cells. Thus, differentially-expressed NFkB genes among PB-CLL, BM-CLL and LN-CLL cells indicate that these different CLL cells utilize different NFκB molecules for proliferation/survival. Together, our results show that CLL cells from different in vivo microenvironments such as PB, BM and LN exhibit differential gene expression patterns, and many of the genes are involved in regulation of migration and proliferation/survival. Furthermore, LN-CLL cells expressing chemokine ligands, BCR, BAFF and NFκB signaling molecules attract other cells including more CLL cells to form an optimal microenvironment which provide prosurvival and proliferative signals to CLL cells.

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Diamond-Blackfan Anemia: Erythropoiesis Lost in Ribosome Biosynthesis

Blood ◽

10.1182/blood.v118.21.sci-2.sci-2 ◽

2011 ◽

Vol 118 (21) ◽

pp. SCI-2-SCI-2

Author(s):

Stefan Karlsson ◽

Johan Flygare ◽

Pekka Jaako ◽

David Bryder

Keyword(s):

Bone Marrow ◽

Platelet Count ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Macrocytic Anemia ◽

Hematopoietic Progenitors ◽

Hematopoietic Stem ◽

Diamond Blackfan Anemia

Abstract Abstract SCI-2 Diamond-Blackfan anemia (DBA) is a rare congenital erythroid hypoplasia that presents early in infancy. The classic hematologic profile of DBA consists of macrocytic anemia with selective absence of erythroid precursors in a normocellular bone marrow, normal or slightly decreased neutrophil, and variable platelet count. During the course of the disease some patients show decreased bone marrow cellularity that often correlates with neutropenia and thrombocytopenia. DBA is a developmental disease since almost 50% of the patients show a broad spectrum of physical abnormalities. All known DBA disease genes encode for ribosomal proteins that collectively explain the genetic basis for approximately 55% of DBA cases. Twenty-five percent of the patients have mutations in a gene encoding for ribosomal protein S19 (RPS19). All patients are heterozygous with respect to RPS19 mutations suggesting a functional haploinsufficiency of RPS19 as basis for disease pathology. Despite the recent advances in DBA genetics, the pathophysiology of the disease remains elusive. Cellular studies on patients together with successful marrow transplantation have demonstrated the intrinsic nature of the hematopoietic defect. DBA patients have a variable deficit in burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) progenitors. The frequency of immature hematopoietic progenitors in DBA patients is normal but their proliferation is impaired in vitro. Generation of animal models for RPS19-deficient DBA is pivotal to understand the disease mechanisms and to evaluate novel therapies. Several DBA models have been generated in mice or zebrafish. Although these models have provided important insights on DBA, they are limited in a sense that the hematopoietic phenotype and molecular mechanisms are likely to be influenced by the level of RPS19 downregulation. We have generated mouse models for RPS19-deficient DBA by taking advantage of transgenic RNAi. These models are engineered to contain a doxycycline-regulatable RPS19-targeting shRNA, allowing a reversible and dose-dependent downregulation of RPS19 expression. We demonstrate that the RPS19-deficient mice develop a macrocytic anemia together with leukocytopenia and variable platelet count and the severity of the phenotype depends on the level of RPS19 downregulation. We show further that a chronic RPS19 deficiency leads to irreversible exhaustion of hematopoietic stem cells and subsequent bone marrow failure. Overexpression of RPS19 following gene transfer rescues the proliferative and apoptotic phenotype of RPS19-deficient hematopoietic progenitors in vitro, demonstrating that the phenotype is specifically caused by the RPS19 deficiency. Expression analysis of RPS19-deficient hematopoietic progenitors reveals an activation of the p53 pathway. By intercrossing the DBA mice with p53 null mice we demonstrate that inactivation of p53 in vivo results in a variable rescue of the hematopoietic phenotype depending on the level of RPS19 downregulation. Therefore, we conclude that increased activity of p53 plays a major role in causing the DBA phenotype but that other hitherto unidentified pathways also play a role, specifically in patients that have low levels of functional RPS19. Disclosures: No relevant conflicts of interest to declare.

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Molecular Signatures of Self-Renewal, Differentiation, and Lineage Choice in Multipotential Hemopoietic Progenitor Cells In Vitro

Molecular and Cellular Biology ◽

10.1128/mcb.24.2.741-756.2004 ◽

2004 ◽

Vol 24 (2) ◽

pp. 741-756 ◽

Cited By ~ 68

Author(s):

Ludovica Bruno ◽

Reinhard Hoffmann ◽

Fraser McBlane ◽

John Brown ◽

Rajeev Gupta ◽

...

Keyword(s):

Gene Expression ◽

Progenitor Cells ◽

Cell Fate ◽

Molecular Mechanisms ◽

Morphological Changes ◽

Self Renewal ◽

Cell Fate Decisions ◽

Hemopoietic Progenitor ◽

Hemopoietic Progenitor Cells

ABSTRACT The molecular mechanisms governing self-renewal, differentiation, and lineage specification remain unknown. Transcriptional profiling is likely to provide insight into these processes but, as yet, has been confined to “static” molecular profiles of stem and progenitors cells. We now provide a comprehensive, statistically robust, and “dynamic” analysis of multipotent hemopoietic progenitor cells undergoing self-renewal in response to interleukin-3 (IL-3) and multilineage differentiation in response to lineage-affiliated cytokines. Cells undergoing IL-3-dependent proliferative self-renewal displayed striking complexity, including expression of genes associated with different lineage programs, suggesting a highly responsive compartment poised to rapidly execute intrinsically or extrinsically initiated cell fate decisions. A remarkable general feature of early differentiation was a resolution of complexity through the downregulation of gene expression. Although effector genes characteristic of mature cells were upregulated late, coincident with morphological changes, lineage-specific changes in gene expression were observed prior to this, identifying genes which may provide early harbingers of unilineage commitment. Of particular interest were genes that displayed differential behavior irrespective of the lineage elaborated, many of which were rapidly downregulated within 4 to 8 h after exposure to a differentiation cue. These are likely to include genes important in self-renewal, the maintenance of multipotentiality, or the negative regulation of differentiation per se.

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Clonogenic MM Progenitor Cells Reside In CD19-CD38++ Plasma Cells

Blood ◽

10.1182/blood.v116.21.1897.1897 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1897-1897

Author(s):

Naoki Hosen ◽

Haruo Sugiyama

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

B Cells ◽

Progenitor Cells ◽

Plasma Cells ◽

Conflicts Of Interest ◽

The Other ◽

Xenograft Models ◽

Nog Mice

Abstract Abstract 1897 For effective treatment of multiple myeloma (MM), it is important to identify clonogenic MM progenitor cells and target them. However, it is still controversial where MM progenitor cells reside. It was reported that CD19+ B cells from MM patients generated MM upon transplantation to NOD/SCID mice, while CD19-CD38++ plasma cells generated MM disease in SCID-hu or rab model. In this study, we performed both of those two xenograft models with a series of MM patient samples. To increase engraftment efficiencies, we used highly immuno-deficient NOD/Scid, IL-2Rγnull (NOG) mice as recipients, and cells were injected directly into bone marrow (BM) or intravenously to new born pups. CD19+ cells from 10 MM samples were transplanted, but none of them engrafted in NOG mice. Thus, the significance of CD19+ B cells was unclear in the MM patients examined in this study. On the other hand, in SCID-rab model, CD19-CD38++ plasma cells, but not CD19+ B cells, generate MM disease in 5 out of 13 MM patient samples. In addition, in vitro MM colony-forming cells were highly enriched in CD19-CD38++ plasma cells. Taken together, our results indicate that clonogenic MM progenitors reside in CD19-CD38++ cells and are essential targets to eradicate MM clones. Disclosures: No relevant conflicts of interest to declare.

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