Gene expression profiles of bone marrow cells from mice phenotype-selected for maximal or minimal acute inflammations: searching for genes in acute inflammation modifier loci

Abstract Abstract 2361 HOXB4 is a homeobox transcription factor that can induce hematopoietic stem cell (HSC) expansion both in vivo and in vitro. An interesting feature of HOXB4-induced HSC expansion is that HSC numbers do not exceed normal levels in vivo due to an unexplained physiological capping mechanism. To gain further insight into HOXB4 regulatory signals, we transplanted mice with bone marrow cells that had been transduced with a MSCV-HOXB4-ires-YFP vector and analyzed gene expression profiles in HSC-enriched populations 20 weeks after transplant, a time point at which HSC numbers have expanded to normal levels but no longer increasing beyond physiologic levels. We used Affymetrix arrays to analyze gene expression profiles in bone marrow cells sorted for a Lin−Sca-1+c-Kit+ (LSK), YFP+ phenotype. Using ANOVA, we identified1985 probe sets with >2 fold difference in expression (FDR<, 0.1) relative to a control vector-transduced LSK cells. A cohort of genes was identified that were known positive regulators of HSC self-renewal and proliferation. Hemgn, which we identified in a previous screen as a positive regulator of expansion and a direct transcriptional target of HOXB4, was 3.5 fold up-regulated in HOXB4 transduced LSKs. Other genes known to be important for HSCs survival, self-renewal and differentiation were upregulated to significant levels including N-myc, Meis1, Hoxa9, Hoxa10 and GATA2. Microarray data for selected genes was validated by quantitative real-time PCR on HOXB4 transduced CD34low LSK cells, a highly purified HSC population, obtained from another set of transplanted mice at the 20 week time point. In contrast, other gene expression changes were noted that would potentially limit or decrease stem cell numbers. PRDM16, a set domain transcription factor critical for HSC maintenance and associated with clonal hematopoietic expansions when inadvertently activated as a result of retroviral insertion, was dramatically down-regulated on the expression array and 7.6 fold decreased in the real time PCR assay of CD34low LSK cells. TFG-beta signaling is a well defined inhibitor HSC proliferation and utilize Smad proteins as downstream effectors. Expression of Smad1 and Smad7 were significantly upregulated on the LSK expression array and 8.1 and 3.5 fold up-regulated by qPCR in CD34low LSK cells. Another potential counter-regulatory signal was down regulation of Bcl3 mRNA, a potential anti-apoptotic effector in HSCs. We hypothesize that the HOXB4 expansion program involves activation of genes that lead to increased HSC numbers with later activation of counter-regulatory signals that limit expansion to physiologic numbers of HSCs in vivo. We are now examining how this program changes at various time points after transplantation and hypothesize the capping limits are set at relatively later time points during reconstitution. We also are studying the functional effects of these gene expression changes, and in particular, whether enforced expression of HOXB4 and PRMD16 will result in uncontrolled HSC proliferation and/or leukemia. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Xenografts of Pediatric Acute Lymphoblastic Leukemia Retain the Gene Expression Pattern From the Diagnostic Material They Originated From Over Serial Passages.

Blood ◽

10.1182/blood.v114.22.1629.1629 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1629-1629

Author(s):

Manon Queudeville ◽

Elena Vendramini ◽

Marco Giordan ◽

Sarah M. Eckhoff ◽

Giuseppe Basso ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Bone Marrow Cells ◽

Expression Profiles ◽

Lymphoblastic Leukemia ◽

Gene Expression Profiles ◽

Leukemia Cells ◽

Diagnostic Samples ◽

Xenotransplantation Model

Abstract Abstract 1629 Poster Board I-655 Primary childhood acute lymphoblastic leukemia (ALL) samples are very difficult to culture in vitro and the currently available cell lines only poorly reflect the heterogeneous nature of the primary disease. Many groups therefore use mouse xenotransplantation models not only for in vivo testing but also as a means to amplify the number of leukemia cells to be used for various analysis. It remains unclear as to what extent the xenografted samples recapitulate their respective primary leukemia. It has been suggested for example that transplantation may result in the selection of a specific clone present only to a small amount in the primary diagnostic sample. We used a NOD/SCID xenotransplantation model and injected leukemia cells isolated from fresh primary diagnostic material of 4 pediatric ALL patients [2 pre-B-ALL, 1 pro-B-ALL (MLL/AF4}, 1 cortical T-ALL] intravenously into the lateral tail vein of unconditioned mice. As soon as the mice presented clinical signs of leukemia, leukemia cells were isolated from bone marrow and spleen. Isolated leukemia cells were retransplanted into secondary and tertiary recipients. RNA was isolated from diagnostic material and serial xenograft passages and gene expression profiles were obtained using a human whole genome array (Affymetrix U133 2.0). Simultaneously, immunophenotypic analysis via multicolor surface and cytoplasmatic staining by flow cytometry was performed for the diagnostic samples and respective serial xenograft passages. In an unsupervised clustering analysis the diagnostic sample of each patient clustered together with the 3 derived xenograft samples, although the 3 xenograft samples clustered stronger to each other than to their respective diagnostic sample. Comparison of the 4 diagnostic samples vs. all xenograft samples resulted in a gene list of 270 genes upregulated at diagnosis and 8 genes upregulated in the xenograft passages (Wilcoxon, p< .05). The high number of genes upregulated at diagnosis is most likely due to contamination of primary patient samples with normal peripheral blood and/or bone marrow cells as 15% of genes are attributed to myeloid cells, 7% to erythroid cells, 7% to lymphoid cells, 32% to bone marrow in general as well as to innate immunity, chemokines, immunoglobulins. The remaining genes can not be attributed to a specific hematopoetic cell lineage and are not known to be related to leukemia or cancer in general. Accordingly, there are no statistically significant differences between the primary, secondary and tertiary xenograft passages. The immunophenotype analysis are also in accordance with these findings, as the diagnostic blast population retains its immunophenotypic appearance during serial transplantation, whereas the contaminating CD45-positive non- leukemic cells disappear after the first xenograft passage. The few genes upregulated in xenograft samples compared to diagnosis are mainly involved in cell cycle regulation, protein translation and apoptosis resistance. Some of the identified genes have already been described in connection with cancer subtypes, their upregulation therefore being indicative of a high proliferative state in general and could argue towards a more aggressive potential of the engrafted leukemia cells but alternatively could also simply be due to the fact that the xenograft samples are pure leukemic blasts and are not contaminated with up to 15% of non-cycling healthy bone marrow cells as in the diagnostic samples. We conclude that the gene expression profiles generated from xenografted leukemias are very similar to those of their respective primary leukemia and moreover remain stable over serial retransplantation passages as we observed no statistically significant differences between the primary, secondary and tertiary xenografts. The differentially expressed genes between diagnosis and primary xenotransplant are most likely to be due to contaminating healthy cells in the diagnostic samples. Hence, the NOD/SCID-xenotransplantation model recapitulates the primary human leukemia in the mouse and is therefore an appropriate tool for in vivo and ex vivo studies of pediatric acute leukemia. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Early and late stage MPN patients show distinct gene expression profiles in CD34+ cells

Annals of Hematology ◽

10.1007/s00277-021-04615-8 ◽

2021 ◽

Author(s):

Julian Baumeister ◽

Tiago Maié ◽

Nicolas Chatain ◽

Lin Gan ◽

Barbora Weinbergerova ◽

...

Keyword(s):

Gene Expression ◽

Late Stage ◽

Rna Splicing ◽

Bone Marrow Cells ◽

Myeloproliferative Neoplasms ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Primary Myelofibrosis ◽

Estrogen Signaling ◽

Therapeutic Modalities

AbstractMyeloproliferative neoplasms (MPN), comprising essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF), are hematological disorders of the myeloid lineage characterized by hyperproliferation of mature blood cells. The prediction of the clinical course and progression remains difficult and new therapeutic modalities are required. We conducted a CD34+ gene expression study to identify signatures and potential biomarkers in the different MPN subtypes with the aim to improve treatment and prevent the transformation from the rather benign chronic state to a more malignant aggressive state. We report here on a systematic gene expression analysis (GEA) of CD34+ peripheral blood or bone marrow cells derived from 30 patients with MPN including all subtypes (ET (n = 6), PV (n = 11), PMF (n = 9), secondary MF (SMF; post-ET-/post-PV-MF; n = 4)) and six healthy donors. GEA revealed a variety of differentially regulated genes in the different MPN subtypes vs. controls, with a higher number in PMF/SMF (200/272 genes) than in ET/PV (132/121). PROGENγ analysis revealed significant induction of TNFα/NF-κB signaling (particularly in SMF) and reduction of estrogen signaling (PMF and SMF). Consistently, inflammatory GO terms were enriched in PMF/SMF, whereas RNA splicing–associated biological processes were downregulated in PMF. Differentially regulated genes that might be utilized as diagnostic/prognostic markers were identified, such as AREG, CYBB, DNTT, TIMD4, VCAM1, and S100 family members (S100A4/8/9/10/12). Additionally, 98 genes (including CLEC1B, CMTM5, CXCL8, DACH1, and RADX) were deregulated solely in SMF and may be used to predict progression from early to late stage MPN. Graphical abstract

Download Full-text

730. Myocardial Infarction-Induced Acute Inflammation Impairs Therapeutic Efficacy of Bone Marrow Cells

Molecular Therapy ◽

10.1016/s1525-0016(16)37303-8 ◽

2011 ◽

Vol 19 ◽

pp. S279

Keyword(s):

Myocardial Infarction ◽

Bone Marrow ◽

Therapeutic Efficacy ◽

Acute Inflammation ◽

Bone Marrow Cells ◽

Marrow Cells

Download Full-text

Multiple Myeloma Cells Alter Adipogenesis, Increase Senescence-Related and Inflammatory Gene Transcript Expression, and Alter Metabolism in Preadipocytes

Frontiers in Oncology ◽

10.3389/fonc.2020.584683 ◽

2021 ◽

Vol 10 ◽

Author(s):

Heather Fairfield ◽

Samantha Costa ◽

Carolyne Falank ◽

Mariah Farrell ◽

Connor S. Murphy ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Bone Marrow ◽

Lipid Accumulation ◽

Expression Profiles ◽

Adipogenic Differentiation ◽

Gene Expression Profiles ◽

Mouse Bone Marrow ◽

Gene Transcript ◽

Bone Marrow Mscs

Within the bone marrow microenvironment, mesenchymal stromal cells (MSCs) are an essential precursor to bone marrow adipocytes and osteoblasts. The balance between this progenitor pool and mature cells (adipocytes and osteoblasts) is often skewed by disease and aging. In multiple myeloma (MM), a cancer of the plasma cell that predominantly grows within the bone marrow, as well as other cancers, MSCs, preadipocytes, and adipocytes have been shown to directly support tumor cell survival and proliferation. Increasing evidence supports the idea that MM-associated MSCs are distinct from healthy MSCs, and their gene expression profiles may be predictive of myeloma patient outcomes. Here we directly investigate how MM cells affect the differentiation capacity and gene expression profiles of preadipocytes and bone marrow MSCs. Our studies reveal that MM.1S cells cause a marked decrease in lipid accumulation in differentiating 3T3-L1 cells. Also, MM.1S cells or MM.1S-conditioned media altered gene expression profiles of both 3T3-L1 and mouse bone marrow MSCs. 3T3-L1 cells exposed to MM.1S cells before adipogenic differentiation displayed gene expression changes leading to significantly altered pathways involved in steroid biosynthesis, the cell cycle, and metabolism (oxidative phosphorylation and glycolysis) after adipogenesis. MM.1S cells induced a marked increase in 3T3-L1 expression of MM-supportive genes including Il-6 and Cxcl12 (SDF1), which was confirmed in mouse MSCs by qRT-PCR, suggesting a forward-feedback mechanism. In vitro experiments revealed that indirect MM exposure prior to differentiation drives a senescent-like phenotype in differentiating MSCs, and this trend was confirmed in MM-associated MSCs compared to MSCs from normal donors. In direct co-culture, human mesenchymal stem cells (hMSCs) exposed to MM.1S, RPMI-8226, and OPM-2 prior to and during differentiation, exhibited different levels of lipid accumulation as well as secreted cytokines. Combined, our results suggest that MM cells can inhibit adipogenic differentiation while stimulating expression of the senescence associated secretory phenotype (SASP) and other pro-myeloma molecules. This study provides insight into a novel way in which MM cells manipulate their microenvironment by altering the expression of supportive cytokines and skewing the cellular diversity of the marrow.

Download Full-text