scholarly journals Sphingolipid Perturbation Activates Proteostasis Programs to Govern Human Hematopoietic Stem Cell Self-Renewal

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 170-170
Author(s):  
Stephanie Zhi-Juan Xie ◽  
Laura Garcia Prat ◽  
Veronique Voisin ◽  
Alex Murison ◽  
Olga I. Gan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Ex Vivo ◽  
Fold Increase ◽  
Chromatin Accessibility ◽  
Self Renewal ◽  
Cellular Activation ◽  
Hematopoietic Stem ◽  
Risk Of Malignancy ◽  
Ex Vivo Culture

Abstract The hematopoietic stem cells (HSC) field has long been perplexed by how the blood system d (~10e12 cells produced daily) - yet hematologic malignancies remain relatively rare. The risk of malignancy is mitigated in part by a complex hierarchy in which the quiescent long-term hematopoietic stem cells (LT-HSC) with high self-renewal capacity undergo a restricted number of cell divisions. Nonetheless, such a high production demand over a lifetime raises an inherent risk of malignancy due to DNA replication errors, misfolded proteins and metabolic stress that cause cellular damage in HSC. Previously, HSC dormancy, largely thought to be controlled by transcription factor networks, was held responsible for preventing mutation acquisition. However, recent studies suggest that LT-HSC contain critical cellular networks centered around the coordination of distinct HSC metabolic programs with proteostasis, which serve as crucial decision nodes to balance persistence or culling of HSC for lifelong blood production. While HSC culling mechanisms are known, the linkage between cellular stress programs and the self-renewal properties that underlie human HSC persistence remains unknown. Here, we ask how this HSC fate choice is influenced by lipid biosynthesis - an underexplored area of HSC metabolism. We observed a distinct sphingolipid transcriptional signature in human HSC and examined the consequences of sphingolipid perturbation in human cord blood (CB) stem cells during ex vivo activation. DEGS1 (Delta 4-Desaturase, Sphingolipid 1) is the final enzyme in de novo sphingolipid synthesis, converting dihydroceramide (dhCer) to ceramide (Cer); ablation of DEGS1 either genetically or by treatment with the synthetic retinoid fenretinide/N-(4-hydroxyphenyl) retinamide (4HPR) is sufficient to activate autophagy in mouse cells and human cell lines. DEGS1 gene expression was higher in HSC than in progenitors and was significantly increased in LT-HSC following 6 hours of cytokine stimulation, suggesting that it plays a role in cellular activation. Sphingolipid composition was altered in CB cultured with 4HPR for 8 days with an increase in dhCer levels and decrease in Cer levels shown by lipidomics. Remarkably, 4HPR treatment significantly increased in vitro colony forming efficiency from LT-HSC (50% over control), but not from short-term HSC or granulocyte-macrophage progenitors. Ex vivo 4HPR treatment of CB followed by serial xenotransplantation resulted in a 2.5-fold increase in long-term repopulation cell (LTRC) frequency over control-treated cells, suggesting that 4HPR treatment affects HSC self-renewal. RNA-seq analysis showed that 4HPR activates a set of proteostatic quality control (QC) programs that coalesce around the unfolded protein response (UPR) and autophagy, the latter confirmed by immunofluorescence and flow cytometry in CB stem cells. Ex vivo culture perturbs these programs and results in loss of chromatin accessibility at sites associated with uncultured LT-HSC as determined by ATAC-Seq. Addition of 4HPR to the culture activates these proteostatic programs to sustain immunophenotypic and functional HSC. These results suggest that ceramide, the central component to all sphingolipids, may act as a "lipid biostat" for measuring cellular stress and activating stress responses. We further asked if 4HPR could synergize with known compounds to enhance HSC self-renewal. Treatment of CB with a combination of 4HPR plus CD34+ agonists UM171 and StemRegenin-1 during ex vivo culture maintains a chromatin state more similar to uncultured LT-HSC as demonstrated by ATAC-seq, and led to a 4-fold increase in serial repopulating ability in xenotransplant assays over treatment with UM171 and SR1 alone. These results suggest that sphingolipid perturbation not only activates proteostatic mechanisms that protect HSC organelles from damage incurred during cellular activation, but also regulates the landscape of chromatin accessibility in cultured HSC when combined with CD34+ agonists. This work identifies a new linkage between sphingolipid metabolism, proteostatic QC systems and HSC self-renewal, and identifies novel strategies by which to expand HSC numbers and improve HSC quality for clinical applications. Disclosures Takayama: Megakaryon co. Ltd.: Research Funding.

scholarly journals Engraftment and Retroviral Marking of CD34+ and CD34+CD38− Human Hematopoietic Progenitors Assessed in Immune-Deficient Mice

Blood ◽  
1998 ◽  
Vol 91 (4) ◽  
pp. 1243-1255 ◽  
Author(s):  
Mo A. Dao ◽  
Ami J. Shah ◽  
Gay M. Crooks ◽  
Jan A. Nolta
Keyword(s):  
Stem Cells ◽  
Ex Vivo ◽  
Human Stem Cells ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Low Levels ◽  
Ex Vivo Culture ◽  
Daunting Challenge

Abstract Retroviral-mediated transduction of human hematopoietic stem cells to provide a lifelong supply of corrected progeny remains the most daunting challenge to the success of human gene therapy. The paucity of assays to examine transduction of pluripotent human stem cells hampers progress toward this goal. By using the beige/nude/xid (bnx)/hu immune-deficient mouse xenograft system, we compared the transduction and engraftment of human CD34+progenitors with that of a more primitive and quiescent subpopulation, the CD34+CD38− cells. Comparable extents of human engraftment and lineage development were obtained from 5 × 105 CD34+ cells and 2,000 CD34+CD38− cells. Retroviral marking of long-lived progenitors from the CD34+ populations was readily accomplished, but CD34+CD38− cells capable of reconstituting bnx mice were resistant to transduction. Extending the duration of transduction from 3 to 7 days resulted in low levels of transduction of CD34+CD38− cells. Flt3 ligand was required during the 7-day ex vivo culture to maintain the ability of the cells to sustain long-term engraftment and hematopoiesis in the mice.

Use of Chromatin Modifying Agents for Ex Vivo Expansion of Human Umbilical Cord Blood Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4208-4208
Author(s):  
Hiroto Araki ◽  
Nadim Mahmud ◽  
Mohammed Milhem ◽  
Mingjiang Xu ◽  
Ronald Hoffman
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Functional Properties ◽  
Ex Vivo ◽  
Fold Increase ◽  
Scid Mice ◽  
Human Umbilical Cord ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract The fixed number of hematopoietic stem cells (HSCs) within a single cord blood (CB) unit has limited the use of CB grafts for allogeneic transplantation in adults. Efforts to promote self-renewal and expansion of HSCs have been met with limited success. Using presently available ex-vivo culture techniques HSCs lose their functional properties in proportion to the number of cellular divisions they have undergone. We hypothesized that chromatin modifying agents, 5-aza-2′-deoxycytidine (5azaD) and histone deacetylase inhibitor, trichostatin A (TSA) could reactivate pivotal genes required for retaining the functional properties of dividing HSC. We have demonstrated previously that the fate of human bone marrow CD34+ cells could be altered by the addition of 5azaD/TSA (Milhem et al. Blood.2004;103:4102). In our current studies we hypothesized that in vitro exposure of CB CD34+ cells to chromatin modifying agents might lead to optimal HSC expansion to permit transplantation of adults. A 12.5-fold expansion was observed in the 5azaD/TSA treated CD34+CD90+ cell cultures containing SCF, thrombopoietin and FLT3 ligand (cytokines) in comparison to the input cell number. Despite 9 days of culture, 35.4% ± 5.8% (n = 10) of the total cells in the cultures exposed to chromatin modifying agents were CD34+CD90+ as compared to 1.40 % ± 0.32% in the culture containing cytokines alone. The 12.5-fold expansion of CD34+CD90+ cells was associated with a 9.8-fold increase in the numbers of CFU-mix and 11.5-fold expansion of cobblestone area-forming cells (CAFC). The frequency of SCID repopulating cells (SRC) was 1 in 26,537 in primary CB CD34+CD90+ cells but was increased to 1 in 2,745 CD34+CD90+ cells following 9 days of culture in the presence of 5azaD/TSA resulting in a 9.6-fold expansion of the absolute number of SRC. In contrast, the cultures lacking 5azaD/TSA had a net loss of both CFC/CAFC as well as SRC. The expansion of cells maintaining CD34+CD90+ phenotype was not due to the retention of a quiescent population of cells since all of the CD34+CD90+ cells in the culture had undergone cellular division as demonstrated by labeling with a cytoplasmic dye. CD34+CD90+ cells that had undergone 5–10 cellular divisions in the presence of 5azaD/TSA but not in the absence still retained the ability to repopulate NOD/SCID mice. 5azaD/TSA treated CD34+CD90+ cells, but not CD34+CD90- cells were responsible for in vivo hematopoietic repopulation of NOD/SCID assay, suggesting a strong association between CD34+CD90+ phenotype and their ability to repopulate NOD/SCID mice. We next assessed the effect of 5azaD/TSA treatment on the expression of HOXB4, a transcription factor which has been implicated in HSC self-renewal. A significantly higher level of HOXB4 protein was detected by western blot analysis after 9 days of culture in the cells treated with 5azaD/TSA as compared to cells exposed to cytokines alone. The almost 10-fold increase in SRC achieved using the chromatin modifying agents should be sufficient to increase the numbers of engraftable HSC within a single human CB unit so as to permit these expanded grafts to be routinely used for transplanting adult recipients.

Enhanced Self-Renewal of Hematopoietic Stem Cells Mediated by the Polycomb Gene Product, Bmi-1.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1686-1686
Author(s):  
Hideyuki Oguro ◽  
Atsushi Iwama ◽  
Hiromitsu Nakauchi
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Target Genes ◽  
Ex Vivo ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Ex Vivo Culture ◽  
Deficient Mice

Abstract The Polycomb group (PcG) proteins form multiprotein complexes that play an important role in the maintenance of transcriptional repression of target genes. Loss-of-function analyses show abnormal hematopoiesis in mice deficient for PcG genes including Bmi-1, Mph-1/Rae28, M33, Mel-18, and Eed, suggesting involvement of PcG complexes in the regulation of hematopoiesis. Among them, Bmi-1 has been implicated in the maintenance of hematopoietic and leukemic stem cells. In this study, detailed RT-PCR analysis of mouse hematopoietic cells revealed that all PcG genes encoding components of the Bmi-1-containing complex, such as Bmi-1, Mph1/Rae28, M33, and Mel-18 were highly expressed in CD34−c-Kit+Sca-1+Lin− (CD34−KSL) hematopoietic stem cells (HSCs) and down-regulated during differentiation in the bone marrow. These expression profiles support the idea of positive regulation of HSC self-renewal by the Bmi-1-containing complex. To better understand the role of each component of the PcG complex in HSC and the impact of forced expression of PcG genes on HSC self-renewal, we performed retroviral transduction of Bmi1, Mph1/Rae28, or M33 in HSCs followed by ex vivo culture. After 14-day culture, Bmi-1-transduced but not Mph1/Rae28-transduced cells contained numerous high proliferative potential-colony forming cells (HPP-CFCs), and presented an 80-fold expansion of colony-forming unit-neutrophil/macrophage/Erythroblast/Megakaryocyte (CFU-nmEM) compared to freshly isolated CD34−KSL cells. This effect of Bmi-1 was comparable to that of HoxB4, a well-known HSC activator. In contrast, forced expression of M33 reduced proliferative activity and caused accelerated differentiation into macrophages, leaving no HPP-CFCs after 14 days of ex vivo culture. To determine the mechanism that leads to the drastic expansion of CFU-nmEM, we employed a paired daughter cell assay to see if overexpression of Bmi-1 promotes symmetric HSC division in vitro. Forced expression of Bmi-1 significantly promoted symmetrical cell division of daughter cells, suggesting that Bmi-1 contributes to CFU-nmEM expansion by promoting self-renewal of HSCs. Furthermore, we performed competitive repopulation assays using transduced HSCs cultured ex vivo for 10 days. After 3 months, Bmi-1-transduced HSCs manifested a 35-fold higher repopulation unit (RU) compared with GFP controls and retained full differentiation capacity along myeloid and lymphoid lineages. As expected from in vitro data, HSCs transduced with M33 did not contribute to repopulation at all. In ex vivo culture, expression of both p16INK4a and p19ARF were up-regulated. p16INK4aand p19ARF are known target genes negatively regulated by Bmi-1, and were completely repressed by transducing HSCs with Bmi-1. Therefore, we next examined the involvement of p19ARF in HSC regulation by Bmi-1 using p19ARF-deficient and Bmi-1 and p19ARF-doubly deficient mice. Although bone marrow repopulating activity of p19ARF-deficient HSCs was comparable to that of wild type HSCs, loss of p19ARF expression partially rescued the defective hematopoietic phenotypes of Bmi-1-deficient mice. In addition, transduction of Bmi-1 into p19ARF-deficient HSCs again enhanced repopulating capacity compared with p19ARF-deficient GFP control cells, indicating the existence of additional targets for Bmi-1 in HSCs. Our findings suggest that the level of Bmi-1 is a critical determinant for self-renewal of HSC and demonstrate that Bmi-1 is a novel target for therapeutic manipulation of HSCs.

Selective Activation of STAT5 Unveils Its Role in the Maintenance of Hematopoietic Stem Cells and the Development of Myeloproliferative Disorder.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3549-3549
Author(s):  
Yuko Kato ◽  
Atsushi Iwama ◽  
Hiromitsu Nakauchi
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Janus Kinase ◽  
Selective Activation ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Lineage Differentiation

Abstract Recent studies have implicated the janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway in the maintenance of stem cells, such as mouse embryonic stem cells and Drosophila germ cells. We have previously reported that thrombopoietin (TPO) can support in vitro self-renewal division of murine hematopoietic stem cells (HSCs) (CD34−/lowc-Kit+Sca-1+lineage marker-negative; CD34−KSL cells). Signal transducers and activators of transcription 5 (STAT5) is one of the major signaling molecules that mediate TPO signals. All these findings suggest that STAT5 could be an attractive candidate for therapeutic manipulation of HSCs. Cytokines activate JAK/STAT5 pathway along with other signaling pathways, causing difficulty to dissect STAT5-specific functions in hematopoietic stem cells (HSCs). Here we took advantage of constitutively active STAT5 mutants to selectively activate STAT5 signaling pathway in HSCs. The mutants used are STAT5A 1*6 that harbors two amino acid mutations S710F and H298R in the effecter domain, and STAT5A #2 that harbors a point mutation N642H in the SH2 domain. Retroviral transduction of either STAT5 1*6 or STAT5#2 mutant into purified CD34−KSL HSCs caused a drastic expansion of multipotential progenitors in vitro and promoted multi-lineage differentiation in vitro. During 7 days of culture supplemented with SCF and TPO, the number of high proliferative potential colonies (HPPC) increased ten-fold compared with the GFP control and half of them were derived from multipotential progenitor cells. Notably, even in the culture supplemented with SCF only, expression of STAT5 mutants in HSCs supported a similar mode of expansion of progenitors cells and multi-lineage differentiation, indicating that activation of STAT5 can substitute major biological effects of TPO in HSCs. In all in vitro experiments, STAT5 1*6 showed stronger effects than STAT5#2. To evaluate the effect of STAT5A mutants in the maintenance of long-term bone marrow repopulating HSC ex vivo, cultured transduced cells corresponding to 30 initial CD34−KSL HSCs were transplanted into lethally irradiated mice 7 to 10 days after transduction. Although rapid hamatopoietic repopulation was observed with HSCs expressing STAT5A 1*6, mice developed myeloproliferative disease (MPD) and succumbed to death within two months. In contrast, HSCs expressing STAT5A #2 presented significantly higher long-term repopulating capacity than the GFP control. These data indicate that selective activation of STAT5 maintains long-term repopulating ability of HSCs ex vivo. Oncostatin M, a well known STAT5 target gene, has been postulated to be involved in the development of MPD and was actually induced STAT5A 1*6-expressing cells. However, transplantation of OSM−/− HSCs expressing STAT5A 1*6 similarly caused a lethal MPD in wild-type mice, indicating that Oncostatin is not the main target for STAT5 in MPD development. Taken together, our findings establish a role for STAT5 in the self-renewal of HSCs and provide STAT5 as novel target for therapeutic manipulation of HSCs ex vivo.

A Possible Role for Wnt3a in Enhancing StemRegenin1-Mediated Expansion of Hematopoietic Stem Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4130-4130
Author(s):  
Lucia Duinhouwer ◽  
Elwin Rombouts ◽  
Nesrin Tüysüz ◽  
Jan Spanholtz ◽  
Derk ten Berge ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cell Biology ◽  
Ex Vivo ◽  
Synergistic Effects ◽  
Fold Increase ◽  
Differentiation Potential ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 4130 Insufficient engraftment after umbilical cord blood stem cell transplantation (UCB-SCT) may be improved by the administration of ex vivo expanded UCB-derived hematopoietic stem cells (HSC). Although culturing HSC with cytokines such as SCF, Flt3L and TPO results in robust proliferation, it is accompanied with extensive differentiation and loss of self renewal capacity. Inhibition of the Aryl hydrocarbon Receptor (AhR) leads to potent expansion of hematopoietic progenitor cells (HPC) (Boitano et al. Science 2010). Reportedly, Wnt3a inhibits differentiation in different types of stem cells including hematopoietic stem cells(Reya et al. Nature 2003, Ten Berge et al. Nature Cell Biology 2011). Here, we evaluated possible additive or synergistic effects of combining the AhR antagonist StemRegenin1 (SR1) with recombinant purified Wnt3a to expand hematopoietic stem cells ex vivo. UCB-derived CD34-selected cells were cultured in serum-free Glycostem Basal Growth Medium (GBGM) supplemented with the early-acting growth factors SCF, Flt3L and TPO (SFT medium) with or without the addition of SR1 and Wnt3a. Cell number, viability and subset composition within the CD34+ cells were measured using flowcytometry. The multilineage differentiation potential and self renewal capacity of expanded CD34+HPC were evaluated in stroma-supported long-term culture-initiating cell (LTC-IC) assays. Culturing CD34+ cells in SFT medium resulted in a mean 10.2-fold increase in CD34+ cells after 1 week of culture (n=3). Addition of SR1 to the SFT-medium resulted in a 16-fold increase of CD34+ input cells within 7 days, while on the other hand, addition of Wnt3a alone to the SFT-medium resulted in a 7-fold increase in CD34+ cells. However, combining SR1 and Wnt3a in the SFT medium resulted in a 20-fold expansion of CD34+ cells compared to input. The early additive effect of Wnt3a on SFT+SR1-induced expansion of CD34+ cells, however, disappeared upon prolonged culture up to 2–3 weeks. Approximately 3–10% of UCB-derived CD34+ cells could be characterized as phenotypic HSC, as was defined by CD34+CD38lowCD45RAlowCD90+ cells. After culture, we sorted different CD34+-populations to evaluate their functional capacity. Evaluation of LTC-IC frequencies yielded a frequency of 1/23 LTC-IC in the phenotypic HSC-subset (CD34+CD38lowCD45RAlowCD90+) after 28 days of culture in SFT medium with SR1. However, no LTC-IC appeared to be present in the multipotent progenitor subset (MPP, CD34+CD38lowCD45RAlowCD90low). These results indicate that phenotypic HSC maintain their functional LTC-IC capacity after expansion culture. Collectively, our results confirm that SR1 expands HSC with preservation of self-renewal capacity and ability to differentiate into various hematopoietic lineages. In addition, we show that Wnt3a initially enhances SFT+SR1-driven expansion of CD34+ HPC, but reduces the increase in number of CD34+ cells at later stages of culture. These data may suggest that the period of expansion needed for clinical application may be shortened by combining SR1 and Wnt3a. Disclosures: Spanholtz: Glycostem Therapeutics: Employment. Groenewegen:Glycostem Therapeutics: Equity Ownership.

Pyrimido-Indole Derivatives Are Novel Agonists of Human Cord Blood Hematopoietic Stem Cell Self-Renewal

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 650-650
Author(s):  
Iman Fares ◽  
Jalila Chagaroui ◽  
Yves Gareau ◽  
Stéphane Gingras ◽  
Nadine Mayotte ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Fed Batch ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract The widespread use of cord blood (CB) unit in transplantation is limited with low number of long-term hematopoietic stem cells (LT-HSCs) and progenitors. Several approaches have been developed to expand HSC ex vivo such as automated and continuous medium delivery (fed-batch), notch delta ligand and SR1 (antagonist of aryl hydrocarbon receptor (AhR)). Concurrent with these studies, we hypothesized that small molecule with potent LT-HSC stimulating activity might be identified and potentiated in fed-batch culture system. Accordingly, we tested a library of more than 5000 small molecules for their in vitro expansion of CD34+CD45RA- cells. Most of the identified hits, except one (UM729) synthesized in our institute, suppress AhR pathway. Structure activity relationship was performed on UM729 to generate a more potent analog named UM171. This optimized molecule was 10-20 times more potent with an effective concentration of 15-20 nM when tested for its ability to expand CD34+CD45RA- cells. When compared to SR1, UM171 delivered in a fed-batch system for 12 and 16 days showed a better expansion of HSC phenotypes and lower apoptotic cell number compared to SR1 or DMSO controls. Also, UM171-expaned cultures showed higher number in multipotent progenitors (CFU-GEMM) and long term initiating cells (LTC-IC) compared to DMSO controls. Further studies showed the UM171 did not affect division rate, and its effect in expanding HSC phenotype was reversible. When combined with SR1, UM171 showed a better suppression of differentiation and led to a higher CFU-GEMM expansion compared to the single treatment of the compounds or DMOS controls. These observations suggest that UM171+SR1 cooperate to enhance ex vivo expansion of progenitor cells and suppress differentiation. To determine the in vivo activity of the expanded CD34+ CB cells, we transplanted fresh (un-manipulated) and 12-day cultured cells in NSG mice and monitored the human hematopoietic reconstitution after 20 and 30 weeks post-transplantation. Frequencies of day0 equivalent LT-HSCs were 13-fold higher in UM171 expanded cultures compared to fresh or fed-batch cultures supplemented with DMSO or SR1. Secondary experiments indicated that UM171 ex vivo treatment did not appear to affect the capability of LT-HSC to expand in primary recipients and hence similarly reconstituted secondary animals for at least 18 more weeks. This suggests that UM171 expands LT-HSC ex vivo without losing their engraftment potential. To further investigate UM171 mechanism of action, RNA- Seq expression profiling was performed. Unlike SR1 or DMSO controls, UM171 treatment was accompanied by a marked suppression of transcripts associated with erythroid and megakaryocytic differentiation and up-regulation of membrane protein transcripts such as EPCR and TEMEM 183a. In summery, UM171 is the first molecule identified so far that enables a robust ex vivo expansion of human CD34+ CB cells that sustain their in vivo activity independent of AhR suppression. Conversely, AhR suppression was limited to expand cells with less durable self-renewal potential. This study could enhance the use of small yet well HLA-matched CB units to become a prioritized source for stem cells transplantation. 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.

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.

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