scholarly journals PEDF Can Rescue the Inhibition of Injured Endothelial Cells on Hematopoietic Stem Cell Expansion Ex Vivo

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5008-5008
Author(s):  
Lingyu Zeng ◽  
Wenyi Lu ◽  
Lan Ding ◽  
Wen Ju ◽  
Jianlin Qiao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem Cell ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Mesenchymal Transition ◽  
Endothelial To Mesenchymal Transition

Introduction: Endothelial cells (ECs) provide a fertile niche for hematopoietic stem cell (HSC) maintenance, differentiation, and migration.Several studies have indicated that bone marrow (BM) vascular niche was impaired after HSC transplantation and severely inhibited hematopoietic reconstruction. Pigment epithelium-derived factor (PEDF) is an important potential cytoprotection and therapeutic agent for injured cells. The direct role of the injured endothelial cells on hematopoietic stem cells and whether PEDF has protective effect in this system remain unknown. This study aims to observe the influence of enjured ECs on HSCs and to explore the role of PEDF in endothelial-HSC coculture system. Methods: Injury of Endothelial cells by two important preparative regimenconditioning radiation and Busulfan respectively was evaluated with CCK8 assay. The expression of endothelial tight junctions(TJs),adherent junctions related molecules and endothelial to Mesenchymal Transition molecules such as ZO-1, Occludin,VE-cadherin, ICAM, α-SMA, CD31 and VCAM were detected by RT-qPCR, flow cytometry, immunofluorescence and western blot. The effects of injured endothelial cells on HSC self-renewal, differentiation, cell cycle and apoptosis were evaluated by flow cytometry, photography, viable cell count and clone formation assay. Hematopoiesis regulation factors SCF, IL-6, TGF-β and TNF-α were detected by ELISA. The protective effect of PEDF was also explored. Results: Both radiation and Busulfan could decrease cell viability of endothelial cells. The expression level of ZO-1, Occludin, VE-cadherin, ICAM, CD31 and VCAM were decreased and α-SMA was increased when EC exposed to radiation or Busulfan suggesting endothelial activation, impaired EC permeability and endothelial to Mesenchymal Transition after EC injured. Compared with normal endothelial cells and hematopoietic stem cell co-culture group, the HSC% of injured endothelial cells and hematopoietic stem cells co-cultured group were significantly decreased, the cell colony formation ability was decreased, the proportion of mature cells increased, and the damage of endothelial cells could not maintain the characteristics of HSC, weakened the self-renewal and multidirectional differentiation potential of HSC and promoted the maturation of HSC. After the administration of PEDF, endothelial to Mesenchymal Transition of EC was suppressed and the EC permeability was improved. Most importantly, the proportion of HSC was significantly increased, and the proportion of mature cells decreased in the coculture system. Conclusion: Injured endothelial cells can inhibit proliferation of hematopoietic stem cells, self-renewal and promote HSC differentiation. PEDF could ameliorate endothelial injury and promote HSC expansion by suppressing endothelial-mesenchymal transition and protecting TJs and AJs. Disclosures No relevant conflicts of interest to declare.

scholarly journals Clonal-level lineage commitment pathways of hematopoietic stem cells in vivo

2019 ◽  
Vol 116 (4) ◽  
pp. 1447-1456 ◽  
Author(s):  
Rong Lu ◽  
Agnieszka Czechowicz ◽  
Jun Seita ◽  
Du Jiang ◽  
Irving L. Weissman
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem Cell ◽  
Lineage Commitment ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Different Levels

While the aggregate differentiation of the hematopoietic stem cell (HSC) population has been extensively studied, little is known about the lineage commitment process of individual HSC clones. Here, we provide lineage commitment maps of HSC clones under homeostasis and after perturbations of the endogenous hematopoietic system. Under homeostasis, all donor-derived HSC clones regenerate blood homogeneously throughout all measured stages and lineages of hematopoiesis. In contrast, after the hematopoietic system has been perturbed by irradiation or by an antagonistic anti-ckit antibody, only a small fraction of donor-derived HSC clones differentiate. Some of these clones dominantly expand and exhibit lineage bias. We identified the cellular origins of clonal dominance and lineage bias and uncovered the lineage commitment pathways that lead HSC clones to different levels of self-renewal and blood production under various transplantation conditions. This study reveals surprising alterations in HSC fate decisions directed by conditioning and identifies the key hematopoiesis stages that may be manipulated to control blood production and balance.

scholarly journals Osteoblast ablation reduces normal long-term hematopoietic stem cell self-renewal but accelerates leukemia development

Blood ◽  
2015 ◽  
Vol 125 (17) ◽  
pp. 2678-2688 ◽  
Author(s):  
Marisa Bowers ◽  
Bin Zhang ◽  
Yinwei Ho ◽  
Puneet Agarwal ◽  
Ching-Cheng Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem Cell ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Key Points ◽  
Leukemia Development

Key Points Bone marrow OB ablation leads to reduced quiescence, long-term engraftment, and self-renewal capacity of hematopoietic stem cells. Significantly accelerated leukemia development and reduced survival are seen in transgenic BCR-ABL mice following OB ablation.

Hematopoietic Stem Cell Expansion by HOXB4 Is Greatly Enhanced in p21 Deficient Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1688-1688 ◽  
Author(s):  
Noriko Miyake ◽  
Ann C.M. Brun ◽  
Mattias Magnusson ◽  
David T. Scadden ◽  
Stefan Karlsson
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem Cell ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Hox transcription factors have emerged as important regulators of hematopoiesis. In particular, enforced expression of HOXB4 is a potent stimulus for murine hematopoietic stem cell (HSC) self-renewal. Murine HSCs engineered to overexpress HoxB4 expand significantly more than control cells in vivo and ex vivo while maintaining a normal differentiation program. HSCs are regulated by the cell proliferation machinery that is intrinsically controlled by cyclin-dependent kinase inhibitors such as p21Cip1/Waf1(p21) and p27Kip1 (p27). The p21 protein restricts cell cycling of the hematopoietic stem cell pool and maintains hematopoietic stem cell quiescence. In order to ask whether enhanced proliferation due to HOXB4 overexpression is mediated through suppression of p21 we overexpressed HOXB4 in hematopoietic cells from p21−/− mice. First, we investigated whether human HOXB4 enhances in vitro expansion of BM cells from p21−/− mice compared to p21+/+ mice. 5FU treated BM cells from p21−/− or p21+/+ mice were pre-stimulated with SCF, IL-6, IL-3 for 2 days followed by transduction using retroviral vector expressing HOXB4 together with GFP (MIGB4) or the control GFP vector (MIG). The cells were maintained in suspension cultures for 13 days and analyzed for GFP positive cells by flow-cytometry. Compared to MIG transduced BM cells from p21+/+ mice (MIG/p21+), the numbers of GFP positive cells were increased 1.1-fold in MIG/p21−, 3.2-fold in MIGB4/p21+ and 10.0-fold in MIGB4/p21− respectively (n=5). CFU assays were performed after 13 days of culture. The numbers of CFU were increased 4.8-fold in MIG/p21−, 19.5-fold in MIG/p21+ and 33.9 -fold in MIGB4/p21− respectively. Next, we evaluated level of HSCs expansion by bone marrow repopulation assays. After 12-days of culture, 1.5 x 105 MIGB4 or MIG-transduced cells (Ly5.2) were transplanted into lethally irradiated mice in combination with 8 x 105 fresh Ly5.1 bone marrow cells. Sixteen weeks after transplantation, no Ly5.2 cells could be detected in MIG/p21+ or MIG/p21− transplanted mice (n=6). In contrast, Ly5.2 positive cells were detected in both MIGB4/p21+/+ and MIGB4/p21−/− cells. The % of Ly5.2 positive cells in MIGB4/p21− transplanted mice was 9.9-fold higher than MIGB4/p21+ transplanted mice. (38.4 % vs 3.9 %, P<0.02, n=5). These Ly5.2 positive cells differentiated into all lineages, as determined by proportions of Mac-1, B-220, CD3 and Ter119 positive populations. Currently, we are enumerating the expansion of HOXB4 transduced HSCs in p21 deficient BM cells using the CRU assay. Our findings suggest that HOXB4 increases the self-renewal of hematopoietic stem cells by a mechanism that is independent of p21. In addition, the findings demonstrate that deficiency of p21 in combination with enforced expression of HOXB4 can be used to rapidly and effectively expand hematopoietic stem cells.

Genetic Uncoupling of the Regulation of Hematopoietic Stem Cell Quiescence and Self-Renewal.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1347-1347
Author(s):  
Yan Liu ◽  
Yasuhiko Miyata ◽  
Goro Sashida ◽  
Anthony Debalsio ◽  
Yuhui Liu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem Cell ◽  
Quiescent State ◽  
Double Knockout ◽  
Ki 67 ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract It is usually stated that HSCs must choose to either self-renew or to differentiate and lose some of their multi potentiality. Recently, we demonstrated that MEF, an ETS family of transcription factor, played an important role in regulating HSC quiescence, illustrating a third choice for the HSC, namely to make an “active” choice and remain quiescent, without undergoing either self-renewal, or differentiation. MEF null HSCs are more quiescent than normal HSCs. In addition, MEF null mice exhibit greater numbers of hematopoietic stem cells and show resistance to chemotherapy and radiation. Little is known about the regulation of self-renewal vs. quiescence of HSCs, however the cdk inhibitor p21 has been implicated in regulating both HSC quiescence and proliferation. In the absence of p21, hematopoietic stem cell numbers are reported to be increased, but so is proliferation, leading to stem cell exhaustion. This implies that while p21 may maintain HSCs in their quiescent state, MEF functions to facilitate the entry of quiescent HSCs into the cycle, To investigate the potential opposing roles of MEF and p21 in HSC quiescence and self-renewal and to test whether the quiescent state of MEF null HSCs is dependent on the presence of p21, we have generated MEF / p21 double-knockout (DKO) mice. These mice are viable and born at normal mendelian frequency. MEF / p21 DKO mice have a higher than normal proportion of HSCs in the G0 phase, based on Pyronin Y/Hoechst staining and staining for the proliferation antigen Ki-67. Thus, the increased quiescence is not dependent on the presence of p21. However, by measuring LSK cells, we have observed a normal number of HSCs in the bone marrow of MEF / p21 DKO mice, in contrast to the increased number of HSCs in the bone marrow of MEF null mice. This suggests that the increased number of hematopoietic stem cells in MEF null mice is dependent on p21. Ongoing studies will further address the unique mechanisms that control HSC vs. stem cell expansion.

Foxo3 Modulation of ATM and Oxidative Stress Mediates Distinct Functions in the Regulation of Hematopoietic Stem and Progenitor Cell Fate.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1272-1272 ◽  
Author(s):  
Safak Yalcin ◽  
Julia P. Luciano ◽  
Xin Zhang ◽  
Cecile Vercherat ◽  
Reshma Taneja ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Cell Fate ◽  
Hematopoietic Stem Cell ◽  
Progenitor Cell ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Atm Gene

Abstract FOXO transcription factors are required for hematopoietic stem cell self renewal. In this study, we demonstrate that Foxo3 plays a specific and essential function in the regulation of both hematopoietic stem and progenitor cell fate. Foxo3 null mice display a myeloproliferative syndrome characterized by splenomegaly, a major expansion of the myeloid compartment in the blood, bone marrow and spleen, cytokine hypersensitivity of progenitors in hematopoietic organs and associated with the repression of the B lymphoid compartment. In addition, loss of Foxo3 leads to significant defects in hematopoietic stem cell numbers and activity. In particular, the numbers of long-term culture initiating cells (LTC-IC) was significantly reduced and the ability to repopulate lethally irradiated mice was severely compromised in Foxo3-defcient mice. This effect was mediated at least partially by enhanced accumulation of reactive oxygen species (ROS) in Foxo3-deficient hematopoietic stem cells as demonstrated by reduced QRT-PCR expression of several anti-oxidant enzymes leading to accumulation of ROS, (as measured by chloromethyl,dichlorodihydrofluorescein diacetate assay) in Foxo3 null hematopoietic stem cells, and in vitro and in vivo rescue of the phenotype using ROS scavengers. Furthermore, we demonstrate that while ROS accumulation results in suppression of Foxo3 null hematopoietic stem cell compartment, it enhances the activity of multipotential cells. By measuring RNA versus DNA content, and BrdU uptake, we determined that Foxo3-deficient hematopoietic stem cells exit quiescence (G0) and are impaired in their cycling at the G2/M phase. In particular, we identified ROS activation of p19ARF/p53 pathway and ROS-independent modulation of ataxia telangiectasia mutated (ATM) gene and p16INK4a, as major contributors to the interference with Foxo3-deficient hematopoietic stem cell self renewal and cycling. Loss of ATM has been shown to lead to hematopoietic stem cell deficiency. Importantly, we show that ATM gene expression is significantly suppressed in Foxo3-deficient hematopoietic stem cells suggesting that ATM lies downstream of Foxo3. Retroviral expression of a constitutively active form of Foxo3 in Foxo3-deficient bone marrow mononuclear cells enhances significantly the ATM expression suggesting that Foxo3 regulate expression of ATM gene. These combined findings suggest that Foxo3 functions in a tumor suppressor network to protect hematopoietic stem cells against deleterious effects of oxidative damage, to maintain hematopoietic lineage fate determination and to restrict the activity of long term repopulating hematopoietic stem cells. These findings provide insights into the mechanisms underlying hematopoietic stem cell fate.

ETO2 Controls Hematopoietic Stem Cell Expansion.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 396-396
Author(s):  
Stephane Barakat ◽  
Julie Lambert ◽  
Guy Sauvageau ◽  
Trang Hoang
Keyword(s):  
Stem Cells ◽  
Transcription Factor ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Short Term ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Abstract 396 Hematopoietic stem cells that provide short term reconstitution (ST-HSCs) as well as hematopoietic progenitors expand from a small population of long term hematopoietic stem cells (LT-HSCs) that are mostly dormant cells. The mechanisms underlying this expansion remain to be clarified. SCL (stem cell leukemia), is a bHLH transcription factor that controls HSC quiescence and long term competence. Using a proteomics approach to identify components of the SCL complex in erythroid cells, we and others recently showed that the ETO2 co-repressor limits the activity of the SCL complex via direct interaction with the E2A transcription factor. ETO2/CBF2T3 is highly homologous to ETO/CBFA2T1 and both are translocation partners for AML1. We took several approaches to identify ETO2 function in HSCs. We initially found by Q-PCR that ETO2 is highly expressed in populations of cells enriched in short-term HSC (CD34+Flt3-Kit+Sca+Lin-) and lympho-myeloid progenitors (CD34+Flt3+Kit+Sca+Lin-) and at lower levels in LT-HSCs (CD34-Kit+Sca+Lin- or CD150+CD48-Kit+Sca+Lin-). Next, the role of ETO2 was studied by overexpression or downregulation combined with transplantation in mice. Ectopic ETO2 expression induces a 100 fold expansion of LT-HSCs in vivo in transplanted mice associated with differentiation blockade in all lineages, suggesting that ETO2 overexpression overcomes the mechanisms that limit HSC expansion in vivo. We are currently testing the role of the NHR1 domain of ETO2 in this expansion. Conversely, shRNAs directed against ETO2 knock down ET02 levels in Kit+Sca+Lin- cells, causing a ten-fold decrease in this population after transplantation, associated with reduced short-term reconstitution in mice. Finally, proliferation assays using Hoechst and CFSE indicate that ETO2 downregulation affects cell division (CFSE) and leads to an accumulation of Kit+Sca+Lin-cells in G0/G1 state (Hoescht). In conclusion, we show that ETO2 is highly expressed in ST-HSCs and lymphoid progenitors, and controls their expansion by regulating cell cycle entry at the G1-S checkpoint. In addition, ETO2 overexpression converts the self-renewal of maintenance into self-renewal of expansion in LT-HSCs. Disclosures: No relevant conflicts of interest to declare.

Identification of a Stroma-Mediated Wnt Signal Promoting Self-Renewal of Hematopoietic Stem Cells in the Stem Cell Niche.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2208-2208
Author(s):  
Young-Ju Kang ◽  
Eek-hoon Jho ◽  
Hanjun Kim ◽  
Gyeongsin Park ◽  
Jae-Seung Shim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Stem Cell Niche ◽  
Stable Form ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Stromal Component ◽  
Cell Niche

Abstract With contrasting results recently reported on the effects of b-catenin on hematopoietic stem cells (HSCs), the precise role of Wnt on HSC regulation remains in question. Here, we show that Wnt-b-catenin signaling triggers distinct biological effects on HSCs depending on the target of activation within the hematopoietic microenvironment. Retroviral transduction of a stable form of b-catenin into HSCs caused a loss of competitive repopulating units (CRUs) in a limiting-dilution assay, whereas stabilized b-catenin in stromal cells CRU frequencies of co-cultured HSCs with higher preservation of undifferentiated state and caused enhanced levels of reconstitution in a manner dependent on direct contact between HSC and stroma. The enhancing effect of b-catenin stabilized stroma on HSC was also observed for human HSCs exhibiting higher frequencies of lympho-myeloid repopulating cells after transplantation into NOD/SCID mice. Interestingly, gene expression patterns of Wnt signaling molecules revealed compartmentalization in a manner that canonical Wnt ligands were preferentially expressed in the hematopoietic cells while molecules for reception of the signal such as Frizzled receptors or their co-receptors are preferentially expressed in stromal component, suggesting the role of stromal component as a target of Wnt signals in the niche. Furthermore, b-catenin accumulated selectively in the endosteal stroma of the trabecule region in “stressed” marrows, but not in “steady-state” marrows. Taken together, these results suggest stroma-mediated Wnt signals may function as microenvironmental cues for HSC self-renewal in the stem cell niche.

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