scholarly journals Blocking TNF and IL-1 Sensitizes AML Stem Cells to NF-κB Inhibitor Treatment

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2302-2302
Author(s):  
Volk Andrew ◽  
Jing Li ◽  
Jun Zhang ◽  
Joseph Cannova ◽  
Caiqin Hao ◽  
...  
Keyword(s):  
Stem Cells ◽  
In Vitro Culture ◽  
Conflicts Of Interest ◽  
In Vivo Models ◽  
Hematopoietic Stem ◽  
Enhanced Sensitivity ◽  
Repressive Effect ◽  
Inhibitor Treatment

Abstract Background: NF-κB inhibition selectively eliminates leukemia stem cells (LSCs) but has less effect on healthy hematopoietic stem cells (HSCs), suggesting an advantageous target for leukemia therapy. However, NF-κB inhibition alone does not have a significant effect on in vivo models of leukemia. We have reported that many types of AML cells produce tumor necrosis factor-α (TNF) which appears to protect LSCs from NF-κB inhibition by stimulating JNK-AP1 survival/proliferative signaling parallel to NF-κB. Complete inactivation of TNF signaling cannot fully reproduce the effects of JNK-AP1 inhibition, suggesting that some other inflammatory cytokine(s) might also compensate for NF-κB inhibition through activation of JNK-AP1. We found that IL-1α and β (IL-1) is such a cytokine. Methods: TNF and IL-1 expression was examined in 430 AML samples by microarray and verified by qRT-PCR. IL-1α and β protein levels in the sera of AML patients and medium from cultured AML cells were examined by ELISA. MLL-AF9-transduced murine AML cells, human AML cell lines and primary patient samples were used in this study. TNF signal was blocked by genetic deletion of receptors Tnfr1 and 2, or treatment with a TNF-specific monoclonal antibody. IL-1 signal was blocked by shRNA knockdown of IL-1R or treatment with an IL-1R antagonist. The effects of IL-1 and TNF signal co-inactivation on growth and response to NF-κB inhibition in AML cells were evaluated by in vitro culture and colony-forming assay, and in vivo transplantation/leukemogenesis assays. Results: We found that most types of human AML cells both express and produce TNF and IL-1. Treatment with these cytokines stimulated the growth and colony-forming ability of AML cells but repressed the growth of normal HSPC in in vitro culture. Inactivation of either the TNF or IL-1 signaling axes suppressed the growth of and significantly sensitized AML stem cells to NF-κB inhibition both in vitro by targeting clonogenic AML progenitor cells, and in vivo through compromising leukemogenesis. Simultaneous inactivation of both TNF and IL-1 resulted in an enhanced repressive effect on AML cells compared to inactivation of either individual cytokine’s receptors alone. AML cells with inactivation of both TNF and IL-1 signals showed significantly lower clonogenic ability in vitro, further reduced leukemogenic capacity in vivo and enhanced sensitivity to NF-kB inhibitor treatment both in vitro and in vivo. Mechanistically, we found that both TNF and IL-1 stimulate JNK-AP1 and NF-κB, two parallel survival signaling pathways in AML cells, but induce JNK-mediated death signaling in normal HSPCs. Conclusion: Both TNF and IL-1 provide protection to LSCs from the effects of NF-κB inhibitor treatment in an autocrine manner and contribute to repression of normal hematopoiesis in a paracrine fashion. This protection is mediated by JNK-AP1. Our studies show inhibiting both TNF and IL-1 inflammatory signals can block this effect, and may be a better strategy to successfully treat AML when combined with NF-κB inhibitors. Disclosures No relevant conflicts of interest to declare.

Expansion of Murine Amniotic Fluid c-Kit High Lin- Cells Maintaining Their Hematopoietic Potential.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1475-1475
Author(s):  
Isabelle Andre-Schmutz ◽  
Andrea Ditadi ◽  
Amine Boudil ◽  
Sophie Ezine ◽  
Marina Cavazzana-Calvo
Keyword(s):  
Stem Cells ◽  
Amniotic Fluid ◽  
Conflicts Of Interest ◽  
Calf Serum ◽  
Embryonic Stem ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Serum Free

Abstract Abstract 1475 Poster Board I-498 We have recently described the hematopoietic potential of ckit+ Lin- cells from the murine and human amniotic fluid (1). These cells were able to generate all types of blood cells in vitro and as far as mice are concerned, to generate a complete hematopoietic system once transplanted to immunodeficient recipients. This strong hematopoietic potential was accompanied by a molecular signature measured by unicellular RT-PCR, characteristic of fetal hematopoietic progenitors. Indeed coexpression of Gata2, Lmo2 and Aml1 was found in 28% of ckit+Lin- AF cells. Intriguingly, murine ckit+Lin- AF cells can be subdivided into two fractions depending on the level of ckit expression (low or high). In in vitro assays, we demonstrated that hematopoietic potential was strictly restricted to the ckit high expressing fraction. The expansion of these cells would have great impact even in the clinical field as AF could be seen as a source of transplantable hematopoietic stem cells (HSCs). Many of the early studies that documented some expansion ability of HSCs included fetal calf serum in the protocol. Given the poorly defined combination of factors in serum and the variability between different serum lots, these protocols were often difficult to reproduce. Serum-free media supplied with specific inducers have been shown to bring several advances in driving direct differentiation of embryonic stem cells. Murine AF Lin-ckitlo and ckithi were cultured in serum- and feeder layer-free culture conditions. Lin-ckitlo AF cells died within a few days. Conversely, Lin-ckithi AF cells were maintained for up to 6 weeks, with a proliferation rate of more than 100 during the first three weeks. Their phenotype remained stable, ckithi CD45+ and Lin-. The hematopoietic potential tested in methylcellulose assays showed an increased frequency of mixed CFU-GEMM (from 24% to 84%). In vivo, CFU-S12 composed of erythroid, myeloid and Lin-ckit+Sca1+ progenitor cells were observed after the injection of AF ckithi in lethally irradiated recipients. Gene expression profile analyzed by single cell multiplex RT-PCR analysis correlated with the in vitro and in vivo results of differentiation. LMO2 was coexpressed with Gata2 and Aml1 in 66% of expanded cells, demonstrating the maintenance of an overall pattern of expression. Collectively, our results indicate that the hematopoietic potential of AF resides in the ckithi fraction and that these cells can be expanded in serum-free condition for prolonged periods of time without reduction of their hematopoietic potential. This strongly supports the idea that AF may be an excellent source of cells for therapeutic applications. 1. Ditadi 2009 Disclosures: No relevant conflicts of interest to declare.

TET Family Oncogene Fus Is Essential for the Maintenance of Self-Renewing Hematopoietic Stem Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2529-2529
Author(s):  
Takeaki Sugawara ◽  
Hideyuki Oguro ◽  
Atsushi Iwama
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Transcriptional Repression ◽  
Cell Activation ◽  
B Lymphocyte ◽  
Rna Binding ◽  
Conflicts Of Interest ◽  
Hematopoietic Stem

Abstract Abstract 2529 Poster Board II-506 A Proto-oncogene FUS (fusion derived from malignant liposarcoma), also known as TLS (translocated in liposarcoma), was originally identified in chromosomal translocation of human soft tissue sarcoma. FUS is also known to be fused with an ETS family transcription factor ERG in human myeloid leukemia with t(16;21) which is associated with poor prognosis. Based on its protein structure, DNA- and RNA-binding activity and involvement in many human cancers as the fusion with various transcription factors, FUS is now grouped with EWS and TAFII68 into TET (FET) oncogene family. Multiple functions have been postulated for FUS, including non-coding-RNA-mediated transcriptional repression, posttranscriptional RNA processing and the maintenance of genomic integrity. Fus-deficient (Fus−/−) mice showed a non-cell-autonomous defect in B lymphocyte development, defective B cell activation and increased sensitivity to radiation in previous studies. However, its physiological function in hematopoiesis remains unknown. In this study we performed detailed analyses of Fus−/− hematopoietic stem cells (HSCs). Fus−/− fetal livers at embryonic day 14.5 exhibited a mild reduction in numbers of hematopoietic stem and progenitor cells compared with the wild type. Disruption of Fus, however, did not grossly affect proliferation or differentiation of hematopoietic progenitors. Of note, Fus−/− HSCs had significantly reduced repopulating activity of hematopoiesis in competitive repopulation assays, and did not repopulate hematopoiesis at all in tertiary recipients. Moreover, Fus−/− HSCs were highly sensitive to radiation both in vitro and in vivo and showed a drastic reduction in numbers in recipient mice after sublethal irradiation. All these findings implicate Fus in the maintenance and radioprotection of HSCs. Studies of chromosome stability, telomere length, apoptosis and levels of reactive oxigen species (ROS) appeared not accountable for the apparent defect of Fus−/− HSCs. However, gene expression profiling identified changes in expression of several genes in Fus−/− HSCs, and dysregulated expression of some of these genes might be responsible for the defective function of Fus−/− HSCs. Disclosures: No relevant conflicts of interest to declare.

Stromal Cell Regulation of Murine Hematopoietic Stem Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1566-1566
Author(s):  
Stefan Wohrer ◽  
Keegan Rowe ◽  
Heidi Mader ◽  
Claudia Benz ◽  
Michael R Copley ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Stromal Cell ◽  
Cultured Cells ◽  
Conflicts Of Interest ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Daughter Cells

Abstract Abstract 1566 Recent advances in purifying murine hematopoietic stem cells (HSCs) to near homogeneity (>20%) have made it possible to analyze their in vivo clonal growth, self-renewal and differentiation properties over prolonged periods and the effects of various manipulations on these key functional parameters. However, conditions that allow genetically unaltered HSCs to maintain their original functional properties over equivalent periods of prolonged proliferation in vitro have not yet been identified. Since initial studies showed that the UG26 stromal cell could support murine HSC maintenance for limited periods, we first asked whether the addition of cytokines that also maintain HSCs for short periods might synergize with UG26 cells to enable HSC expansion to occur. Limiting dilution transplants that used a 6-month read-out of reconstituted blood elements (>1%) showed that the addition of 100 ng/ml Steel Factor (SF) and 20 ng/ml IL-11 to cultures containing UG26 cells and single purified (50%) HSCs (EPCR+CD150+CD48-, ESLAM cells) consistently stimulated a 3–5 fold HSC expansion after 7 days (3 expts). Furthermore, the effect of the UG26 cells could be replaced by UG26 conditioned medium (CM) and, in the presence of the CM+SF/IL-11 cocktail, the HSCs showed sustained longterm in vivo lympho-myeloid reconstituting activity in both primary and secondary recipients. Under these conditions, every ESLAM cell isolated proliferated several times within 7 days, but individual analysis of paired daughter cells showed that most first divisions (13/42) were, nevertheless, asymmetrical in terms of the numbers and types of different lineages produced by each of the 2 daughter cells for at least 4 months, although occasional evidence of symmetry was obtained (2/42 divisions). Interestingly, these first divisions showed a biphasic curve with 75% of the cells dividing before and 25% after 48 hours - the late dividers being more highly enriched for HSCs (95% vs 20%). We next asked whether TGF-β might be an important factor in UG26 CM, since UG26 cells exert a strong cell cycle inhibitory effect, and produce abundant TGF-beta. Accordingly, we next analyzed the effect of adding a neutralizing anti-TGF-β antibody or replacing the CM with TGF-β in the same type of single HSC cultures by tracking the survival and division kinetics of the cells as well as measuring the repopulating activity of their in vitro progeny present after 7 days. Strikingly, the addition of anti-TGF-β to the CM+SF/IL-11 supplemented HSC cultures eliminated the late wave of first cell divisions and caused an accompanying loss of myeloid reconstituting ability in recipients transplanted with the cultured cells. Conversely, replacement of the CM with TGF-β restored a biphasic division kinetics curve to cultures supplemented with SF/IL-11 but no CM. However, this did not protect against the early 50% loss of cells by apoptosis. These findings provide evidence of a new role of TGF-β in preserving the integrity of HSC functionality in vitro, but suggest a requirement for other types of factors released by certain stromal cells to achieve sustained symmetrical HSC self-renewal in vitro. Disclosures: No relevant conflicts of interest to declare.

A Subset of MicroRNAs and Genes Involved in AML Has a Pivotal Role in the in Vitro differentiation of Hematopoietic Stem Cell Precursors

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1290-1290
Author(s):  
Julian Pulecio ◽  
Leopoldo Laricchia-Robbio ◽  
Juan Carlos Izpisua ◽  
Montserrat Barragan ◽  
Marianna Vitaloni ◽  
...  
Keyword(s):  
Stem Cells ◽  
Conflicts Of Interest ◽  
Embryonic Stem ◽  
In Vitro Assays ◽  
Main Role ◽  
In Vitro Differentiation ◽  
Related Factors ◽  
Hematopoietic Stem

Abstract Abstract 1290 After the finding of a set of transcription factors capable of reprogram any somatic cell into an embryonic stem-like cell by Yamanaka's group a lot of effort has been put to differentiate and produce in-vitro engraftable cells that could replace and fix damaged tissues. One of the most attractive and promising fields is the differentiation towards blood, considering it is a tissue without a complex tridimensional structure and that the phenotypes of the different sublineages are already well characterized. Nonetheless, so far there are no reports of successful differentiation into blood progenitors which are able to completely recover functionally in vivo blood-depleted mice. We previously reported the differentiation from induced pluripotent stem cells (iPS) towards hematopoietic cells capable of distinguish into sub lineages in in vitro assays, while another group obtained blood precursors by transdifferentiation of fibroblasts; however a complete recovery of the hematopoietic lineages in vivo was not seen. Our hypothesis is that the gap missing in the current protocols to obtain repopulating blood stem cells can be filled by the microRNA profiling of Cord Blood (CB) progenitors, in order to find the key players in the maintenance of blood stemness. In particular, it has been shown that population with the highest capacity to be engrafted in mice is the CD34+/CD90+ from CB. Our preliminary results depict a set of miRNAs that are specifically overexpressed in the CD34+/CD90+ population from CB cells when compared against a less specific CD34+ population. These miRNAs are currently being tested as a tool to improve the efficiency of iPS differentiation and fibroblasts conversion towards blood progenitors by means of lentiviral infection of the miRNA precursors. Interestingly, we have found that these miRNAs have been previously reported to have a main role in the occurrence of Acute Myeloid Leukemia in humans and mice. These results led us to look for genes that are highly expressed in blood progenitors but also have been shown to be correlated with AML.As a safety study, we are currently evaluating the effect of overexpressing AML related factors (miRNAs and genes) when added to the established protocols to obtain blood progenitors from iPS and fibroblasts. Surprisingly, our initial results show that the overxpression of the above mentioned genes and miRNAs have an intrinsic potential to induce in vitro differentiation or conversion from iPS and fibroblasts towards blood progenitors. Disclosures: No relevant conflicts of interest to declare.

GPR84, a Proinflammatory Receptor, Sustains Wnt/β-Catenin Signaling In Leukemic Stem Cells For Maintenance Of MLL-AF9-Induced Leukemogenesis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3781-3781
Author(s):  
Philipp A Dietrich ◽  
Murray D Norris ◽  
Jenny Yingzi Wang
Keyword(s):  
Stem Cells ◽  
Conflicts Of Interest ◽  
Active Form ◽  
Leukemic Stem Cells ◽  
Poor Survival ◽  
Functional Link ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Inappropriate activation of Wnt/β-catenin signaling confers hematopoietic progenitors the property of self-renewal that promotes malignant transformation in MLL-rearranged acute myeloid leukemia (AML). However, it has been noted that activation of β-catenin is observed in tumors without clear mutations in the major components of the pathway or increase in Wnt signaling. This suggests that other developmental signaling pathways may be capable of inducing activation or downstream signaling of β-catenin. Recently, a number of G protein-coupled receptors (GPCRs) have been shown to activate β-catenin signaling to recruit the key downstream components of the canonical Wnt pathway in distinct cell types, including stem cells. GPCRs, the largest family of cell-surface molecules involved in signal transmission, have emerged as crucial players in tumor growth and metastasis, and represent one of the most important drug targets in pharmaceutical development. Given the close functional link with activation of β-catenin signaling, a GPCR signaling pathway may act as the upstream regulator of β-catenin signaling in the establishment of leukemic stem cells (LSC). In this study, our microarray analysis comparing genes differentially expressed between LSC and normal hematopoietic stem cells (HSC) identified GPR84, a proinflammatory GPCR, as a potential LSC-specific candidate target. An analysis of the comprehensive patient outcome database (Oncogenomics – maintained by the National Cancer Institute) showed that high levels of GPR84 were significantly associated with poor survival in patients with leukemia (P=0.0048), implying its potential clinical relevance in predicting disease prognosis. Western blot and flow cytometric analyses confirmed the microarray results and revealed a positive correlation between GPR84 and β-catenin expression. We previously demonstrated that β-catenin was highly expressed in HSC transformed by MLL-AF9 (HSC-MLLAF9) and had lower expression in HSC transduced with leukemic oncogenes Hoxa9/Meis1 (HSC-Hoxa9/Meis1), while increased β-catenin expression was correlated with a poor survival rate in vivo. Herein, our results showed that forced expression of GPR84 induced a robust upregulation of β-catenin in HSC-Hoxa9/Meis1. Conversely, shRNA-mediated ablation of GPR84 in HSC-MLLAF9 led to highly significant downregulation of both GPR84 (P=0.0003) and β-catenin (P=0.0008). Further in vitro functional studies showed that GPR84 knockdown significantly reduced HSC-MLL-AF9 colony forming units (P=0.0006), and induced a marked reduction of cells in S-phase (P=0.0017). This deficient phenotype could be rescued by expression of a constitutively active form of β-catenin. Importantly, subsequent in vivo survival studies using leukemia transplantation mouse models showed that GPR84 knockdown significantly reduced LSC frequency and severely impaired maintenance (P<0.0001; 11 mice per cohort) of HSC-MLL-AF9 induced leukemia, a highly aggressive and drug-resistant subtype of AML. The defect in disease phenotype resulted from inhibited expression of both GPR84 and β-catenin. Furthermore, forced overexpression of GPR84 alone was not sufficient for leukemic transformation of HSC but conferred a growth advantage in vivo to HSC-Hoxa9/Meis1 cells and significantly accelerated the onset of Hoxa9/Meis1-induced AML (P=0.0039), establishing a completely malignant phenotype similar to HSC-MLL-AF9 in vivo (P=0.9986). These data support an oncogenic role of GPR84 in MLL-AF9-induced leukemogenesis. In conclusion, our studies have identified a novel β-catenin regulator that contributes to leukemia maintenance by sustaining aberrant activation of a stem cell self-renewal pathway in LSC, and drugs targeting GPR84 may represent a novel and promising strategy for improving the therapy and outcome of AML patients. Disclosures: No relevant conflicts of interest to declare.

Remodeling Ca2+ Flux By ORP4L Is Essential for Leukemia Stem Cells (LSCs) Survival

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5257-5257
Author(s):  
Wenbin Zhong ◽  
Vesa Olkkonen ◽  
Xu Bing ◽  
Biying Zhu ◽  
Guoping Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Oxidative Phosphorylation ◽  
Mitochondrial Function ◽  
Conflicts Of Interest ◽  
Myelogenous Leukemia ◽  
Leukemia Stem Cells ◽  
Ip3 Receptor ◽  
Hematopoietic Stem

Abstract Acute myelogenous leukemia (AML) is one of the deadliest hematological malignancies and there is at present no efficient strategy to defeat it. New detailed insight into AML leukemia stem cells (LSCs) survival will facilitate the identification of targets for the development of new therapeutic approaches. Recent work has provided evidence that LSCs are defective in their ability to employ glycolysis, but are highly reliant on oxidative phosphorylation, and the maintenance of mitochondrial function is essential for LSCs survival. It is increasingly clear that Ca2+ released constitutively from endoplasmic reticulum (ER) is taken up by mitochondria to sustain optimal bioenergetics and cell survival. Here we report three striking findings: 1) oxysterol-binding protein (OSBP)-related protein 4 (ORP4L) is expressed in LSCs but not in normal hematopoietic stem cells (HSCs). 2) ORP4L is essential for LSC bioenergetics; It forms a complex with PLCβ3 and IP3 receptor 1 (ITPR1) to control Ca2+ release from the ER and subsequent cytosolic and mitochondrial parallel Ca2+ spike oscillations that sustain pyruvate dehydrogenase (PDH) activation and oxidative phosphorylation. 3) ORP4L inhibition eradicates LSCs in vitro and in vivo through impairment of Ca2+-dependent bioenergetics. These results suggest a novel role of ORP4L in governing Ca2+ release to sustain mitochondrial function and survival of LSCs and identify ORP4L as a putative new oncoprotein and therapeutic target for LSCs elimination. Disclosures No relevant conflicts of interest to declare.

scholarly journals β7 Integrin Regulates Intra-Marrow Trafficking of Hematopoietic Stem Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2899-2899
Author(s):  
Jodi Murakami ◽  
Baohui Xu ◽  
Christopher B. Franco ◽  
Xingbin Hu ◽  
Stephen J. Galli ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Cell Adhesion ◽  
Hematopoietic Stem Cells ◽  
Conflicts Of Interest ◽  
Hematopoietic Stem ◽  
Effector Cells ◽  
Cell Cycle Status

Abstract α4β7 integrin is a cell adhesion receptor that is crucial for the migration of hematopoietic progenitors and mature effector cells in the periphery, but its role in adult hematopoiesis remains controversial. To investigate this, we conducted studies using a mouse model in which β7 integrin is absent. Hematopoietic stem cells (HSCs) that lacked β7 integrin (β7KO) had significantly reduced engraftment potential. Intriguingly, the survival of β7KO mice was enhanced and their hematopoietic recovery after 5-fluorouracil-induced myeloablative stress was better compared to wild type (WT) mice, indicating that the decreased engraftment of β7KO HSCs was not caused by a defect in HSC hematopoietic activity. Next we examined the homing abilities of HSCs and we observed that β7KO HSCs had impaired migration abilities in vitro and BM homing capabilities in vivo. Lethal irradiation induced expression of the α4β7 integrin ligand - mucosal addressin cell adhesion molecule-1 (MAdCAM-1) on bone marrow (BM) endothelial cells. Moreover, blocking MAdCAM-1 reduced the homing of HSCs and impaired the survival of recipient mice. Altogether, these data indicate that β7 integrin, when expressed by HSCs, interacted with MAdCAM-1 in the BM microenvironment, thereby promoting HSC homing and engraftment. Interestingly, we also found that β7KO HSCs were retained in the BM, suggesting that β7 integrin may influence the localization of HSCs within different stem cell niches through interaction with MAdCAM-1. To examine the localization of HSCs within the BM, we used the hypoxyprobe pimonidazole to correlate oxygen status with niche localization. We observed that both β7KO and MAdCAM-1KO HSCs were more hypoxic compared to WT HSCs, demonstrating that the absence of either β7 integrin or MAdCAM-1 in mice causes HSCs to be localized in a more hypoxic region of the BM. To confirm these findings, we performed single-cell RT-PCR using Fluidigm Dynamic Array Chips and we discovered that β7KO HSCs differentially expressed genes associated with niche localization and cell cycle status compared to WT HSCs. Since hypoxia correlates with quiescence, we next assessed the cell cycle status of HSCs using Ki67 staining and in vivo BrdU assay and we found that β7KO HSCs may have reduced cell cycle activity. Collectively, these studies suggest that expression of β7 integrin on HSCs may promote exit from quiescence and influence HSC localization within the BM niche. Disclosures No relevant conflicts of interest to declare.

scholarly journals Development of Hematopoietic Stem Cells in the Human Embryo

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. SCI-13-SCI-13 ◽  
Author(s):  
Alexander Medvinsky
Keyword(s):  
Stem Cells ◽  
Human Development ◽  
Human Embryo ◽  
Conflicts Of Interest ◽  
Dorsal Aorta ◽  
Model Organisms ◽  
Hematopoietic Stem ◽  
Genetic Manipulations

Abstract Haematopoietic stem cells (HSCs) develop intra-embryonically in the aorta-gonad-mesonephros (AGM) region, more specifically in the ventral domain of the embryonic dorsal aorta. This process is highly conserved across vertebrate species. Key transcription factors and signalling pathways involved in HSC development have been identified using various model organisms. As a mammal, the mouse represents an excellent model for understanding HSC development in the human embryo. However, in utero development significantly reduces accessibility of the embryo for experimentation, which hampers analysis of dynamic developmental processes. We have overcome this hurdle by establishing AGM in vitro systems that can recapitulate in vivo HSC development. The generation of HSCs during embryonic development is a highly specific process. So far any significant expansion of HSCs from adult sources using solely external molecular cues have been largely unsuccessful. This is despite the fact that there is a pressing need to supply clinically relevant HSCs to some groups of patients. The generation of HSCs from pluripotent stem cells (ES/iPS cells) holds great hopes for regenerative medicine, however, this goal has not been achieved without genetic manipulations due to poor understanding of mechanisms acting in the embryo. Using a dissociation-reaggregation AGM culture system enabled developmental hierarchy of developing mouse HSCs to be reconstructed. On co-aggregation with AGM stroma or OP9 cells, embryonic precursors mature into adult definitive HSCs, which can provide multi-lineage long-term repopulation upon transplantation into adult irradiated recipients. We have established that during development, mouse HSCs mature through upregulation CD41, CD43 and CD45 haematopoietic markers. The analysis of the AGM niche revealed a highly heterogeneous signalling landscape that can regulate HSC development in a concerted manner. During human development, HSCs also emerge first in the ventral domain of the embryonic dorsal aorta and reside in a population, which express markers known from mouse HSC development. These first HSCs emerge in small numbers, possess an enormous regenerative/ self-renewing potential and can generate hundreds of fully functional daughter HSCs. However, it appears that establishing human AGM cultures, which recapitulate HSC development because of yet unclear reasons, is problematic. This makes it difficult to study the developing human HSC hierarchy and AGM stromal factors driving HSC development. In this talk, commonalities and differences in mouse and human development as well as routes to the solution of this hurdle will be discussed. Disclosures No relevant conflicts of interest to declare.

scholarly journals Tet2 Deficiency Rejuvenates Hematopoietic Stem and Progenitor Cells during Ageing

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 37-37
Author(s):  
Tingting Hong ◽  
Shengli Li ◽  
Shaohai Fang ◽  
Anna Guzman ◽  
Wei Han ◽  
...  
Keyword(s):  
Stem Cells ◽  
Conflicts Of Interest ◽  
Pool Size ◽  
Loss Of Function ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Increased Risk ◽  
Age Dependent

Ageing is accompanied by a significant reduction of hematopoietic competence driven by various causes including epigenetic alternations [1-4]. Ten-eleven translocation 2 (TET2) has been well delineated as a critical epigenetic regulator that affects hematopoietic progenitor and stem cells (HSPCs) function. Tet2 deficiency confers advantages in clonal expansion of HSPCs and skews myeloid lineage differentiation, giving rise to increased risk of hematological malignancy transformation [5-8]. TET2 loss-of-function mutations are frequently detected in aged HSPCs [10-11], thereby raising the question of how Tet2 deficiency affects HSPCs self-renewal and lineage specification during ageing. To address this question, we harvested HSPCs from wild-type (WT) or Tet2KO young and aged donor mice, followed by competitive bone marrow transplantations to monitor age-dependent functional alterations. Despite the enlargement of the HSC pool size (the number of cells with regenerative potential) in aged mice, the aged WT HSPCs exhibited lower self-renewal capability and displayed impaired hematopoietic differentiation when competed against young stem cells. However, we found that both aged and young Tet2-deficient HSPCs shared comparable peripheral blood reconstitution, indicating no engraftment defects were caused by age for Tet2-deficient HSPCs. In parallel, scRNA-seq analysis revealed that Tet2 deficiency and age promoted the expansion of HSC compartment in a synergistic manner, leading to the largely augmented pool size of Tet2-deficient aged HSCs. But unlike aged WT stem cells, these expanded aged Tet2-null stem cells retained high self-renewal potential and possessed a competitive advantage of lineage outputs both in vitro and in vivo. Overall, through conducting repopulation assays and single-cell transcriptomes analysis, we have demonstrated that Tet2 ablation alters age-dependent HSC functional decline, revealing a disparate ageing process in the Tet2-deficient haemopoietic system. Disclosures No relevant conflicts of interest to declare.

Strategies to Protect Hematopoietic Stem Cells from Culture-Induced Stress Conditions

2020 ◽  
Vol 15 ◽  
Author(s):  
Fatima Aerts-Kaya
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Stress Conditions ◽  
Stress Factors ◽  
Hematopoietic Stem ◽  
Induced Stress

: In contrast to their almost unlimited potential for expansion in vivo and despite years of dedicated research and optimization of expansion protocols, the expansion of Hematopoietic Stem Cells (HSCs) in vitro remains remarkably limited. Increased understanding of the mechanisms that are involved in maintenance, expansion and differentiation of HSCs will enable the development of better protocols for expansion of HSCs. This will allow procurement of HSCs with long-term engraftment potential and a better understanding of the effects of the external influences in and on the hematopoietic niche that may affect HSC function. During collection and culture of HSCs, the cells are exposed to suboptimal conditions that may induce different levels of stress and ultimately affect their self-renewal, differentiation and long-term engraftment potential. Some of these stress factors include normoxia, oxidative stress, extra-physiologic oxygen shock/stress (EPHOSS), endoplasmic reticulum (ER) stress, replicative stress, and stress related to DNA damage. Coping with these stress factors may help reduce the negative effects of cell culture on HSC potential, provide a better understanding of the true impact of certain treatments in the absence of confounding stress factors. This may facilitate the development of better ex vivo expansion protocols of HSCs with long-term engraftment potential without induction of stem cell exhaustion by cellular senescence or loss of cell viability. This review summarizes some of available strategies that may be used to protect HSCs from culture-induced stress conditions.

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