scholarly journals R-spondin 1 is required for specification of hematopoietic stem cells through Wnt16 and Vegfa signaling pathways

Development ◽  
2017 ◽  
Vol 144 (4) ◽  
pp. 590-600 ◽  
Author(s):  
Jamie R. Genthe ◽  
Wilson K. Clements
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Signaling Pathways ◽  
Hematopoietic Stem

scholarly journals Maintenance of Hematopoietic Stem Cells Through Regulation of Wnt and mTOR Pathways.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2309-2309
Author(s):  
Jian Huang ◽  
Peter S. Klein
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Signaling Pathways ◽  
Glycogen Synthase ◽  
Dependent Manner ◽  
Mtor Inhibition ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Abstract 2309 Hematopoietic stem cells (HSCs) maintain the ability to self-renew and to differentiate into all lineages of the blood. The signaling pathways regulating hematopoietic stem cell (HSCs) self-renewal and differentiation are not well understood. We are very interested in understanding the roles of glycogen synthase kinase-3 (Gsk3) and the signaling pathways regulated by Gsk3 in HSCs. In our previous study (Journal of Clinical Investigation, December 2009) using loss of function approaches (inhibitors, RNAi, and knockout) in mice, we found that Gsk3 plays a pivotal role in controlling the decision between self-renewal and differentiation of HSCs. Disruption of Gsk3 in bone marrow transiently expands HSCs in a b-catenin dependent manner, consistent with a role for Wnt signaling. However, in long-term repopulation assays, disruption of Gsk3 progressively depletes HSCs through activation of mTOR. This long-term HSC depletion is prevented by mTOR inhibition and exacerbated by b-catenin knockout. Thus GSK3 regulates both Wnt and mTOR signaling in HSCs, with opposing effects on HSC self-renewal such that inhibition of Gsk3 in the presence of rapamycin expands the HSC pool in vivo. In the current study, we found that suppression of the mammalian target of rapamycin (mTOR) pathway, an established nutrient sensor, combined with activation of canonical Wnt/ß-catenin signaling, allows the ex vivo maintenance of human and mouse long-term HSCs under cytokine-free conditions. We also show that combining two clinically approved medications that activate Wnt/ß-catenin signaling and inhibit mTOR increases the number of long-term HSCs in vivo. Disclosures: No relevant conflicts of interest to declare.

Critical Role for DOCK2 in CD8+/TCR− Graft Facilitating Cells Enhancing Engraftment of Hematopoietic Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 142-142
Author(s):  
Yujie Wen ◽  
Mary J Elliott ◽  
Yiming Huang ◽  
Deborah R Corbin ◽  
Yoshinori Fukui ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Signaling Pathways ◽  
Microarray Analysis ◽  
Control Analysis ◽  
Wild Type ◽  
Hematopoietic Stem

Abstract Abstract 142 CD8+/TCR− graft facilitating cells (FC) have a potent capability to facilitate relatively small numbers of highly purified hematopoietic stem cells (HSC) in both allogeneic and syngeneic recipients. The mechanisms by which FC promote HSC engraftment have not been fully elucidated. We previously found that FC from non-obese diabetic mice (NOD) were compromised in enhancing engraftment of syngeneic and allogeneic HSC compared with FC from diabetes free congenic NOR mice. We therefore compared gene expression profiling between NOD FC and control NOR FC by microarray analysis. Among 18 most significant differentially expressed genes revealed from 45101 genes by false discovery rate control analysis with a cut-off value at level 0.05, dedicator of cytokinesis 2 (DOCK2) was identified as the gene with the most significant difference (P = 1.07 × 10−7). DOCK2 is a hematopoietic cell-specific member of the Caenorhabditis elegans Ced-5, mammalian DOCK180 and Drosophila melanogaster myoblast city (CDM) family of guanine nucleotide exchange factors. DOCK2 activates the small GTPase Rac and is indispensable for migration of plasmacytoid dendritic cells (pDC). FC is a heterogeneous cell population, with a predominant subpopulation resembling plasmacytoid precursor DC (p-preDC FC). In vitro studies demonstrated that FC increased clonogenicity of HSC and improved the ability of the impaired stroma to support late cobblestone area formation by HSC, which suggests that FC homing to hematopoietic niche as a potential component might be a perquisite for FC to enhance HSC engraftment. Therefore, we hypothesized that signaling pathways controlling cell migration via DOCK2 are critical for FC to enhance HSC engraftment. To test our hypothesis, DOCK2 expression data from microarray analysis was further confirmed using relative quantitative real-time PCR and high content image analysis. The expression level of DOCK2 in FC was significantly lower in NOD mice compared with NOR mice (p < 0.01 vs. NOR FC). Functional phenotypes of DOCK2-deficient FC were determined by Transwell migration assay and colony-forming cell assay in vitro, and by co-transplantation of HSC with FC in vivo as well as enumeration of CellTrack Green labeled FC by flow cytometry in spleen, thymus, and bone marrow of femurs and tibias 18 hours post-transplantation. Deficiency of DOCK2 in FC did not affect the ability of FC in promoting HSC colony formation when the two were cultured together. However, DOCK2-deficient FC were compromised in migration to the α-cheemokine, stromal derived factor-1 (SDF-1) at dose 200 ng/ml (Fig. A, P < 0.01 vs. wild-type FC). Homing of FC to spleen and bone marrow of femurs and tibias was also significantly impaired in DOCK2-deficient FC. Moreover, deficiency of DOCK2 in FC abrogated enhancement of HSC engraftment by FC in the syngeneic and allogeneic in vivo assays (Fig. B, syngeneic model: 500 B6 HSC plus 30K B6 or DOCK2−/−FC into lethally irradiated B6 recipients; Fig. C, allogeneic model: 10K B6 HSC plus 30K B6 or DOCK2−/−FC into lethally irradiated B10.BR recipients, P < 0.05 vs. B6 HSC plus wild-type FC). Taken together, our results indicate that deficiency of DOCK2 in FC leads to the dysfunction in migration, and suggest that the signaling pathways involved in FC migration are crucial for FC to enhance HSC engraftment. Disclosures: Ildstad: Regenerex LLC: Equity Ownership.

scholarly journals Alterations in Secretome and Transcriptome of Bone Marrow Derived MSCs in Patients with Diffuse Large B-Cell Lymphoma without Bone Marrow Involvement

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1514-1514
Author(s):  
Nataliya A. Petinati ◽  
Natalia Sats ◽  
Nina J. Drize ◽  
Irina Malyants ◽  
Victoria Shender ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Complete Remission ◽  
Hematopoietic Stem Cells ◽  
Signaling Pathways ◽  
B Cell Lymphoma ◽  
Ion Source ◽  
Phenol Red ◽  
Hematopoietic Stem ◽  
Healthy Donors

Introduction Multipotent mesenchymal stromal cells (MSCs) differentiate into all mesenchymal lineages, regulate hematopoietic stem cells, and also take part in immunomodulation. MSCs are damaged in patients with leukemia. Most of the patients with DLBCL do not have bone marrow (BM) involvement. Despite the absence of proved BM damage in DLBCL patients, the properties of MSCs are changed. We aimed to analyze secretome and transcriptome of MSCs derived from BM of DLBCL patients without BM involvement. Methods The study included 16 DLBCL patients (7 males and 9 females), of which 6 were 42-60-year-old in the onset of the disease and a month after the end of treatment with NHL-BFM90; 10 were 48-78-year-old in complete remission for 6-14 years (5 received CHOP and 5 NHL-BFM90 treatment). Control group included 5 healthy donors (3 males, 2 females), median age 37. During diagnostic punctures BM was collected after informed consent. MSCs were cultured by standard method. Confluent MSCs layers after 1 passage were cultivated in serum-free RPMI1640 without phenol red for 24 hours; supernatants were studied for secretome and cells for transcriptome. The analysis of MSCs secretome was carried out using the LC-MS/MS analysis (TripleTOF 5600+ mass spectrometer with a NanoSpray III ion source coupled to a NanoLC Ultra 2D+ nano-HPLC System. Total RNA was isolated, applying standard procedures, from MSCs. Next-generation sequencing of complementary DNA libraries of polyA-enriched RNA was performed with Illumina HiSeq. Raw RNA-seq data were processed using STAR. Gene expression was compared using the limma R/Bioconductor package. Results The total cell production for 4 passages in primary patients' MSCs was higher than in donors (26.6 ± 2 versus 10.1 ± 4.4 x 106 per flask). It remained elevated regardless of the time passed after therapy. The patterns of secretome and transcriptome of patients' MSCs differed dramatically from the MSCs of healthy donors (Table). In MSCs of primary patients, the secretion and transcription of proteins involved in IL-17, TNF and Toll-like receptor signaling pathways, cytokine-cytokine receptor interaction, cytokine-mediated signaling pathway, cellular response to cytokine stimulus, regulation of signaling receptor activity, regulation of neutrophil chemotaxis, inflammatory and acute inflammatory response and its regulation, leukocyte activation involved in immune response, immune system process, extracellular matrix organization were elevated. Secretion and transcription of cytokines and chemokines (IL6, IL4, LIF, TNFa, CXCL1 and CXCL3), taking part in hematopoiesis regulation were increased in primary patients MSCs. One month after treatment, secretion of 332 proteins was decreased, only 2 of them (DKK1 and FKBP7) were previously overexpressed in primary patients. Many years after the end of both variants of treatment, the secretion and transcription of 32 proteins participating in the same pathways as before treatment remains elevated compared with healthy donors. In addition, the complement and coagulation cascades became upregulated. In MSCs of all patients, regardless of therapy and remission duration , expression/ secretion of following genes/proteins: ACAN, COL1A, MMP3, TGFb1, NDNF, CANX, LAP3, MGP, SERPINB2, STC1,TFPI,TMEM132A, BMP2, CFH, HILPDA, IDO1, IL1B, ITGA2, JUN, LMO2, MMP13, MMP3, TNFRSF1B,TNFSF4 was increased. Some of these proteins take part in bone and cartilage formation, hematopoietic stem cells regulation, blood coagulation and inflammation. These changes in secreted proteins reflect the response of MSCs at the organism level to the tumor presence. Moreover, NUCKS1 overexpression was observed in MSCs of all patients. This nuclear casein kinase plays a significant role in modulating chromatin structure and regulates replication, transcription, and chromatin condensation. Furthermore, this protein contributes to the susceptibility, occurrence, and development of several types of cancer and other diseases. NUCKS1 is considered to be a potent marker for such diseases. Conclusion The presence of a lymphoid tumor without BM involvement in the body leads to irreversible changes in the BM MSCs, thus affecting a lot of biological processes and signaling pathways, independent of the treatment and duration of complete remission. The work were supported by the Russian Foundation for Basic Research, Project No. 17-00-00170. Disclosures No relevant conflicts of interest to declare.

Cell staining for sorting of hematopoietic stem cells (HSC) and myeloid progenitors and isolating RNA from sorted cells

2006 ◽  
Author(s):  
Hideyo Hirai ◽  
Pu Zhang ◽  
Tajhal Dayaram ◽  
Christopher Hetherington ◽  
Shin-ichi Mizuno ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem ◽  
Cell Staining ◽  
Myeloid Progenitors
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