scholarly journals Identification of Potent Quiescent Human Hematopoietic Stem Cells Using Mitochondrial Profile

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5602-5602
Author(s):  
Jiajing Qiu ◽  
Jana Gjini ◽  
Miao Lin ◽  
Tasleem Arif ◽  
Saghi Ghaffari
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Live Cells ◽  
Mitochondrial Activity ◽  
Hematopoietic Stem ◽  
Human Hscs ◽  
Time Point

Quiescence is the main property of the most potent hematopoietic stem cells (HSCs). Recent evidence suggests that mitochondrial activity may be implicated in the maintenance of stem cell quiescence. However, the potential function of mitochondria in the regulation of human HSCs remains largely unknown. To address this, we measured mitochondrial membrane potential (MMP) in subpopulations of human HSCs, using Tetramethylrhodamine, ethyl ester (TMRE) a cationic fluorescent dye sequestered by active mitochondria. We found that MMP profiles progressively shift towards lower levels in subpopulations with phenotypes of higher hematopoietic hierarchy. Subpopulation of CD34+CD38-CD45- cells within the 10% lowest MMP enriched for highly primitive CD90+ HSCs from 17.5% to 49.0% as compared to those within the 10% highest MMP (p=0.0049, n=5). These results are consistent with the murine HSC finding in our laboratory. To analyze the functional activity of HSCs with various MMP levels, subpopulations of CD90+ HSCs (CD34+CD38-CD45-CD90+) with the lowest and the highest 25% MMP were FACS-sorted as MMP-low and MMP-high HSCs respectively and subjected to long-term culture initiating cell (LTC-IC) limiting dilution assay. The results after 5 weeks revealed that the frequency of LTC-IC is much greater in MMP-low HSCs (1 in 7.85 cells) than in MMP-high HSCs (1 in 59.3 cells) (n=2). These results suggest that MMP-low HSCs maintain higher functional potential than MMP-high HSCs. The in vivo analysis of the MMP-low and MMP-high HSCs transplanted into NSG mice is ongoing (will be performed at 5 months). FACS-sorted MMP-low and high HSCs were subjected to cell cycle analysis by Pyronin Y/ Hoechst-33342 staining. We found that above 90% of both MMP-low and MMP-high HSCs were in a quiescence state (MMP-low: 4.48 %, MMP-high: 9.38% in G1; 0% in S/G2/M). Additionally, the expression of CDK6 that is associated with HSC activation was not detectable by confocal microscopy in either MMP-low or MMP-high HSCs (low: n=31, high: n=19). To identify potential distinct kinetic of cell cycle entry, FACS-sorted single HSCs were cultured in serum free medium (SFM), STEM SPAN, supplemented with cytokines (SCF 100ng/ml, TPO 50ng/ml, Flt3 50ng/ml). The occurrence of cell division in each well was monitored under microscopy every 12 hours for 6 days. At each time point the percentage of divided HSCs among total initial seeding HSCs were plotted and curve fitted to calculate the kinetics of cumulative first division (low: n=58, R2=0.9981; high: n=58, R2=0.9973). These analyses showed that MMP-low HSCs were delayed by 1.9 hrs as compared to MMP-high HSCs for cumulative 50% cells to complete the first division. The percentage of newly divided cells of first division at each time point was also plotted. Two waves of first division were revealed in both MMP-low and MMP-high HSCs. The peak of the first wave was delayed by 7 hours in MMP-low HSCs, while the second wave was delayed by 14 hours as compared to MMP-high HSCs. These results indicate that even though both MMP-low and MMP-high CD90+ HSCs are mostly in a quiescence state, upon cytokine exposure in vitro, MMP-high HSCs exist G0 phase more rapidly than MMP-low HSCs. In agreement with this interpretation, MMP-high HSCs express significantly higher level of CDK6 when cultured for 34 hours in the presence of the same cytokines as described above (low: n=14, high n=35; p=0.006). We also investigated whether MMP-low and MMP-high CD90+ HSCs can be maintained in vitro in the absence of cytokines. Both populations were cultured in 96 well plates for 7 days in SFM without cytokines. The percentage of live cells was significantly higher in MMP-low HSCs cultured for 7 days (64.9%) as compared to MMP-high HSCs (44.2%) (p=0.03). Furthermore, morphological analysis by mitochondrial specific probe TOM20 showed that MMP-low contained more fragmented mitochondria as compared to MMP-high HSCs. Our findings suggest that mitochondrial activity may be implicated in the regulation of HSC quiescence. Altogether these results support the notion that human MMP-low CD90+ HSCs are molecularly distinct from MMP-high CD90+ HSCs and maintain quiescence in vitro to a higher degree than MMP-high CD90+ HSCs which are more primed for activation. Disclosures Ghaffari: Rubius Therapeutics: Consultancy.

Adipocyte-Derived Adiponectin Positively Regulates Exit from Quiescence of Hematopoietic Stem Cells By Potentiating mTORC1 Activation after Myelotoxic Injury

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 777-777 ◽  
Author(s):  
Yosuke Masamoto ◽  
Shunya Arai ◽  
Tomohiko Sato ◽  
Iseki Takamoto ◽  
Naoto Kubota ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Adipose Tissue ◽  
Steady State ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem ◽  
Hematopoietic Recovery ◽  
Mtorc1 Activation

Abstract Myelotoxic injury unlocks the vigorous power of hematopoietic stem cells (HSCs) to replenish the hematopoietic system, making quiescent HSCs enter the cell cycle. Microscopically, it is well known that adipose tissue replaces cellular components in bone marrow (BM) after myeloablation by chemotherapeutic agents or irradiation. In a steady-state hematopoiesis, both HSC-intrinsic and -extrinsic mechanisms enforce quiescence of HSCs, and the interaction between HSCs and BM microenvironment has been drawing much attention in maintenance of the quiescence. In this meaning, it is supposed that the drastic change in BM microenvironment by myeloablation might trigger and promote the cell cycle entry of HSCs. We have previously reported that adiponectin, adipocyte-derived anti-diabetic hormone, indirectly enhances proliferation of murine immature myeloid progenitors upon granulocyte-colony stimulating factor treatment (emergency granulopoiesis) in Socs3-Stat3 dependent fashion by suppressing TNF-α production from macrophages (ASH meeting 2013, Abstract 221), however, its direct effect against HSCs in vivo is needed to be elucidated. Additionally, we have shown that both genetic loss and high-fat diet-induced reduction of adiponectin have no impact on steady-state hematopoiesis. Considering BM adipose tissue is an endocrine organ and adipocytes are the major cellular component in ablated marrow, we hypothesized that adiponectin derived from adipocytes might be implicated in HSC activation and subsequent hematopoietic recovery. Adiponectin-null (adipo-/-) mice showed significantly delayed hematopoietic recovery after 5-fluorouracil (5-FU) administration. In 5-FU-treated BM, adipo-/- SLAM-HSCs (CD150+ CD48- Lin- Sca-1+) and CD34- SLAM-HSCs were more quiescent than adipo+/+ counterparts. Adipo-/- mice survived longer than adipo+/+ control mice after serial 5-FU treatment. Taken into account our previous data showing that impaired emergency granulopoiesis of adipo-/- mice is Socs3-Stat3 dependent and Socs3 haploinsufficiency ameliorated the defect, we further investigated whether activation of adipo-/- HSCs on 5-FU treatment was potentiated by genetic loss of Socs3. But Socs3 haploinsufficiency had no capacity to revert impaired activation of adipo-/- HSCs, suggesting some mechanisms other than that of impaired emergency granulopoiesis in adipo-/- mice. Strikingly, adipo-/- HSCs were shown to be defective in mTORC1 activation, phosphorylation of S6 and mitochondrial activity after 5-FU treatment. In vivo rapamycin treatment cancelled the effect of adiponectin upon HSC activation by 5-FU, suggesting that adiponectin enhances HSC activation through mTORC1-dependent mechanism. Physiological isoform of adiponectin (full-length adiponectin) enhanced not only 5-FU-induced mTORC1 activation in vivo but also cytokine-induced activation in vitro, shortened the time to first division, without affecting subsequent proliferation of HSCs, in contrast to the previous report using non-physiological isoform (globular adiponectin) in vitro. The concentration of adiponectin in BM had a 4-fold increase after 5-FU treatment while the level in plasma remained unchanged. In a steady state, adiponectin level in adipocyte-rich tibia is higher than femur, suggesting local production of adiponectin constitutes a significant portion of BM adiponectin. Every BM cell components examined expressed adiponectin mRNA, and adipocytes had the highest. As 5-FU treatment had little effect on adiponectin expression in adipocytes, it was suggested that increased adipocytes in BM contributed to increased adiponectin upon myelotoxic injury. Furthermore, reciprocal transplants with adipo+/+ and adipo-/- mice demonstrated that adiponectin from BM environment of recipient mice plays a major role in the activation of HSC after in vivo 5-FU treatment and in vitro cytokine stimulation. These data reveal that adiponectin, produced mainly from increased adipocytes after myelotoxic injury, positively regulates HSC activation and subsequent hematopoietic recovery. Our data also highlight adipocytes as an essential source of adiponectin to ensure the proliferative burst of hematopoietic cells in myeloablated marrow. Adiponectin treatment could be clinically applied to relieve myelosuppression by chemotherapy. Disclosures No relevant conflicts of interest to declare.

scholarly journals Anti-CD117 antibody depletes normal and myelodysplastic syndrome human hematopoietic stem cells in xenografted mice

Blood ◽  
2019 ◽  
Vol 133 (19) ◽  
pp. 2069-2078 ◽  
Author(s):  
Wendy W. Pang ◽  
Agnieszka Czechowicz ◽  
Aaron C. Logan ◽  
Rashmi Bhardwaj ◽  
Jessica Poyser ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Mouse Models ◽  
Hematopoietic Cell Transplantation ◽  
Normal Donor ◽  
Hematopoietic Stem ◽  
Increased Risk ◽  
Human Hscs

Abstract The myelodysplastic syndromes (MDS) represent a group of clonal disorders that result in ineffective hematopoiesis and are associated with an increased risk of transformation into acute leukemia. MDS arises from hematopoietic stem cells (HSCs); therefore, successful elimination of MDS HSCs is an important part of any curative therapy. However, current treatment options, including allogeneic hematopoietic cell transplantation (HCT), often fail to ablate disease-initiating MDS HSCs, and thus have low curative potential and high relapse rates. Here, we demonstrate that human HSCs can be targeted and eliminated by monoclonal antibodies (mAbs) that bind cell-surface CD117 (c-Kit). We show that an anti-human CD117 mAb, SR-1, inhibits normal cord blood and bone marrow HSCs in vitro. Furthermore, SR-1 and clinical-grade humanized anti-human CD117 mAb, AMG 191, deplete normal and MDS HSCs in vivo in xenograft mouse models. Anti-CD117 mAbs also facilitate the engraftment of normal donor human HSCs in MDS xenograft mouse models, restoring normal human hematopoiesis and eradicating aggressive pathologic MDS cells. This study is the first to demonstrate that anti-human CD117 mAbs have potential as novel therapeutics to eradicate MDS HSCs and augment the curative effect of allogeneic HCT for this disease. Moreover, we establish the foundation for use of these antibody agents not only in the treatment of MDS but also for the multitude of other HSC-driven blood and immune disorders for which transplant can be disease-altering.

scholarly journals Changes in the Proliferative Activity of Human Hematopoietic Stem Cells in NOD/SCID Mice and Enhancement of Their Transplantability after In Vivo Treatment with Cell Cycle Inhibitors

2002 ◽  
Vol 196 (9) ◽  
pp. 1141-1150 ◽  
Author(s):  
J. Cashman ◽  
B. Dykstra ◽  
I. Clark-Lewis ◽  
A. Eaves ◽  
C. Eaves
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Transforming Growth Factor ◽  
Severe Combined Immunodeficiency ◽  
High Specific Activity ◽  
Scid Mice ◽  
Hematopoietic Stem ◽  
Human Hscs

Human hematopoietic tissue contains rare stem cells with multilineage reconstituting ability demonstrable in receptive xenogeneic hosts. We now show that within 3 wk nonobese diabetic severe combined immunodeficiency (NOD/SCID) mice transplanted with human fetal liver cells regenerate near maximum levels of daughter human hematopoietic stem cells (HSCs) able to repopulate secondary NOD/SCID mice. At this time, most of the human HSCs (and other primitive progenitors) are actively proliferating as shown by their sensitivity to treatments that kill cycling cells selectively (e.g., exposure to high specific-activity [3H]thymidine in vitro or 5-fluorouracil in vivo). Interestingly, the proliferating human HSCs were rapidly forced into quiescence by in vivo administration of stromal-derived factor-1 (SDF-1) and this was accompanied by a marked increase in the numbers of human HSCs detectable. A similar result was obtained when transforming growth factor-β was injected, consistent with a reversible change in HSCs engrafting potential linked to changes in their cell cycle status. By 12 wk after transplant, most of the human HSCs had already entered Go and treatment with SDF-1 had no effect on their engrafting activity. These findings point to the existence of novel mechanisms by which inhibitors of HSC cycling can regulate the engrafting ability of human HSCs executing self-renewal divisions in vivo.

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 CCND1–CDK4–mediated cell cycle progression provides a competitive advantage for human hematopoietic stem cells in vivo

2015 ◽  
Vol 212 (8) ◽  
pp. 1171-1183 ◽  
Author(s):  
Nicole Mende ◽  
Erika E. Kuchen ◽  
Mathias Lesche ◽  
Tatyana Grinenko ◽  
Konstantinos D. Kokkaliaris ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Competitive Advantage ◽  
Cell Cycle Progression ◽  
Cycle Phase ◽  
Cycle Progression ◽  
Hematopoietic Stem ◽  
Human Hscs

Maintenance of stem cell properties is associated with reduced proliferation. However, in mouse hematopoietic stem cells (HSCs), loss of quiescence results in a wide range of phenotypes, ranging from functional failure to extensive self-renewal. It remains unknown whether the function of human HSCs is controlled by the kinetics of cell cycle progression. Using human HSCs and human progenitor cells (HSPCs), we report here that elevated levels of CCND1–CDK4 complexes promoted the transit from G0 to G1 and shortened the G1 cell cycle phase, resulting in protection from differentiation-inducing signals in vitro and increasing human leukocyte engraftment in vivo. Further, CCND1–CDK4 overexpression conferred a competitive advantage without impacting HSPC numbers. In contrast, accelerated cell cycle progression mediated by elevated levels of CCNE1–CDK2 led to the loss of functional HSPCs in vivo. Collectively, these data suggest that the transition kinetics through the early cell cycle phases are key regulators of human HSPC function and important for lifelong hematopoiesis.

scholarly journals CD66e Enrichment Enhances Repopulation of Human Long-Term Hematopoietic Stem Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2156-2156
Author(s):  
Kuiying Ma ◽  
Riguo Fang ◽  
Lingling Yu ◽  
Yongjian Zhang ◽  
Chao Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Surface Markers ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Differentiation Pattern ◽  
Human Hscs

Abstract Gene-modified hematopoietic stem cells (HSCs) therapy has demonstrated remarkable success for the treatment of inherited blood disorders. As the origin of hematologic hierarchy, HSCs play an essential role in sustaining life-long hematopoiesis. HSCs identification via reliable and robust bio-markers could facilitate the development of HSC gene therapy. Previous studies showed that long-term hematopoietic stem cells (LT-HSCs) were enriched in the Lin -CD34 +CD38 -CD45RA -CD90 +CD49f + population which could support long-term hematopoietic reconstitution. However, several of these surface markers proved to be unreliable when ex vivo culturing, such as CD38 and CD49f. Thus, HSCs characterization is still hindered by lacking bona-fide bio-markers, and consequently identification of long-term HSCs still needs time-consuming in vivo transplantation. To this end, we performed in vitro screening and comprehensive functional evaluation to identify a novel surface marker of human HSCs. During initial screening, a cell surface antigen screen panel (including 242 human cell surface markers) and human CD34 and CD90 antibodies were used to perform flow cytometry analysis on CD34 + HSPCs enriched from umbilical cord blood. Compared with CD34 + cell population, we found that CD66 (a,c,d,e), CD200 and CD48 positive cells were more enriched in CD34 +CD90 + subset. Previous studies indicated that HSCs cannot be maintained during in vitro culturing. By tracking these candidate surface markers based on this principle, CD66e was selected as the potential HSCs bio-marker. Next, we examined the in vivo hematologic repopulating potential of HSCs by limiting dilution assay (LDA) on immune-deficient mouse model. We sorted CD66e + and CD66e - subsets from CD34 +CD90 +CD45RA - subpopulation, and transplanted into irradiated NOD-scid Il2rg −/− (NPG) mice respectively. At week16 post-transplantation, in contrast to the CD66e - group, CD66e + cells exhibited significantly higher reconstitution in peripheral blood (PB), bone marrow (BM) and spleen. Engraftment dynamics revealed that the CD66e - group were only capable of reconstitution 4 weeks post transplantation, even at the highest initial cell dose. Moreover, the CD66e - group displayed impaired multi-lineage differentiation pattern, especially in PB and BM samples, while the CD66e + group presented a robust multi-lineage reconstitution. Notably, LDA results showed that the CD66e + cells within CD34 +CD90 +CD45RA - population contained 1 out of 529 SCID repopulating cells (SRC), almost 60-fold greater than the CD66e - fraction. To further investigate the long-term repopulating potential of the CD66e + cells, we performed the secondary transplantation collected from the BM cells of primary recipients. CD66e + cells presented significant higher repopulating activity than CD66e- subset in the secondary recipients. These findings reveal that the major cells with homing and long-term reconstitution capacity among CD34 +CD90 +CD45RA - cells were CD66e positive. In order to determine the transcriptional profile of CD66e + cells, we performed RNA-sequencing analysis using the population of CD34 + cells, CD34 +CD90 +CD45RA - cells, CD66e + and CD66e - cells within CD34 +CD90 +CD45RA - subset. Remarkably, compared with other groups, the CD66e + cells displayed a bias toward the signature of HSC and early progenitors such as LMPP and CLP. Moreover, gene set enrichment analysis showed that hematopoietic lineage and long-term potentiation-related genes were highly enriched in the CD66e + cells. Further qRT-PCR experiment confirmed that several HSC-related genes were significantly higher expressed in CD34 +CD90 +CD45RA -CD66e + cells, compared to CD66e - population or CD34 + HSPCs, suggesting that the gene expression profile of CD66e + cells is reminiscent of HSC signature. Altogether, we demonstrate that CD66e is a robust functional HSC bio-marker that CD66e-positive population among CD34 +CD90 +CD45RA - cells exhibit typical HSC signature, enhanced in vivo engraftment potential and robust multilineage differentiation pattern, which will provide an invaluable tool to investigate the origin of human HSCs, paving the way for the therapeutic application. Figure 1 Figure 1. Disclosures Fang: EdiGene, Inc.: Current Employment.

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.

Endothelial stroma programs hematopoietic stem cells to differentiate into regulatory dendritic cells through IL-10

Blood ◽  
2006 ◽  
Vol 108 (4) ◽  
pp. 1189-1197 ◽  
Author(s):  
Hua Tang ◽  
Zhenhong Guo ◽  
Minghui Zhang ◽  
Jianli Wang ◽  
Guoyou Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Hematopoietic Stem Cells ◽  
Critical Role ◽  
Regulatory Function ◽  
Hematopoietic Stem ◽  
Regulatory Dendritic Cells

Abstract Regulatory dendritic cells (DCs) have been reported recently, but their origin is poorly understood. Our previous study demonstrated that splenic stroma can drive mature DCs to proliferate and differentiate into regulatory DCs, and their natural counterpart with similar regulatory function in normal spleens has been identified. Considering that the spleen microenvironment supports hematopoiesis and that hematopoietic stem cells (HSCs) are found in spleens of adult mice, we wondered whether splenic microenvironment could differentiate HSCs into regulatory DCs. In this report, we demonstrate that endothelial splenic stroma induce HSCs to differentiate into a distinct regulatory DC subset with high expression of CD11b but low expression of Ia. CD11bhiIalo DCs secreting high levels of TGF-β, IL-10, and NO can suppress T-cell proliferation both in vitro and in vivo. Furthermore, CD11bhiIalo DCs have the ability to potently suppress allo-DTH in vivo, indicating their preventive or therapeutic perspectives for some immunologic disorders. The inhibitory function of CD11bhiIalo DCs is mediated through NO but not through induction of regulatory T (Treg) cells or T-cell anergy. IL-10, which is secreted by endothelial splenic stroma, plays a critical role in the differentiation of the regulatory CD11bhiIalo DCs from HSCs. These results suggest that splenic microenvironment may physiologically induce regulatory DC differentiation in situ.

scholarly journals Synergy of NUP98-HOXA10 Fusion Gene and NrasG12D Mutation Preserves the Stemness of Hematopoietic Stem Cells on Culture Condition

Cells ◽  
2019 ◽  
Vol 8 (9) ◽  
pp. 951 ◽  
Author(s):  
Yong Dong ◽  
Chengxiang Xia ◽  
Qitong Weng ◽  
Tongjie Wang ◽  
Fangxiao Hu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Pluripotent Stem Cells ◽  
Culture Condition ◽  
Fusion Gene ◽  
Nras Mutation ◽  
Hematopoietic Stem ◽  
Bona Fide

Natural hematopoietic stem cells (HSC) are susceptible and tend to lose stemness, differentiate, or die on culture condition in vitro, which adds technical challenge for maintaining bona fide HSC-like cells, if ever generated, in protocol screening from pluripotent stem cells. It remains largely unknown whether gene-editing of endogenous genes can genetically empower HSC to endure the culture stress and preserve stemness. In this study, we revealed that both NUP98-HOXA10HD fusion and endogenous Nras mutation modifications (NrasG12D) promoted the engraftment competitiveness of HSC. Furthermore, the synergy of these two genetic modifications endowed HSC with super competitiveness in vivo. Strikingly, single NAV-HSC successfully maintained its stemness and showed robust multi-lineage engraftments after undergoing the in vitro culture. Mechanistically, NUP98-HOXA10HD fusion and NrasG12D mutation distinctly altered multiple pathways involving the cell cycle, cell division, and DNA replication, and distinctly regulated stemness-related genes including Hoxa9, Prdm16, Hoxb4, Trim27, and Smarcc1 in the context of HSC. Thus, we develop a super-sensitive transgenic model reporting the existence of HSC at the single cell level on culture condition, which could be beneficial for protocol screening of bona fide HSC regeneration from pluripotent stem cells in vitro.

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