scholarly journals Glutamylation of deubiquitinase BAP1 controls self-renewal of hematopoietic stem cells and hematopoiesis

2019 ◽  
Vol 217 (2) ◽  
Author(s):  
Zhen Xiong ◽  
Pengyan Xia ◽  
Xiaoxiao Zhu ◽  
Jingjing Geng ◽  
Shuo Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Peripheral Blood Cells ◽  
Self Renewal ◽  
Hematopoietic Stem

All hematopoietic lineages are derived from a limited pool of hematopoietic stem cells (HSCs). Although the mechanisms underlying HSC self-renewal have been extensively studied, little is known about the role of protein glutamylation and deglutamylation in hematopoiesis. Here, we show that carboxypeptidase CCP3 is most highly expressed in BM cells among CCP members. CCP3 deficiency impairs HSC self-renewal and hematopoiesis. Deubiquitinase BAP1 is a substrate for CCP3 in HSCs. BAP1 is glutamylated at Glu651 by TTLL5 and TTLL7, and BAP1-E651A mutation abrogates BAP1 glutamylation. BAP1 glutamylation accelerates its ubiquitination to trigger its degradation. CCP3 can remove glutamylation of BAP1 to promote its stability, which enhances Hoxa1 expression, leading to HSC self-renewal. Bap1E651A mice produce higher numbers of LT-HSCs and peripheral blood cells. Moreover, TTLL5 and TTLL7 deficiencies sustain BAP1 stability to promote HSC self-renewal and hematopoiesis. Therefore, glutamylation and deglutamylation of BAP1 modulate HSC self-renewal and hematopoiesis.

scholarly journals Mitochondria in the maintenance of hematopoietic stem cells: new perspectives and opportunities

Blood ◽  
2019 ◽  
Vol 133 (18) ◽  
pp. 1943-1952 ◽  
Author(s):  
Marie-Dominique Filippi ◽  
Saghi Ghaffari
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Blood Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Cellular Energy ◽  
Cell Pool ◽  
Stem Cell Pool

Abstract The hematopoietic system produces new blood cells throughout life. Mature blood cells all derived from a pool of rare long-lived hematopoietic stem cells (HSCs) that are mostly quiescent but occasionally divide and self-renew to maintain the stem cell pool and to insure the continuous replenishment of blood cells. Mitochondria have recently emerged as critical not only for HSC differentiation and commitment but also for HSC homeostasis. Mitochondria are dynamic organelles that orchestrate a number of fundamental metabolic and signaling processes, producing most of the cellular energy via oxidative phosphorylation. HSCs have a relatively high amount of mitochondria that are mostly inactive. Here, we review recent advances in our understanding of the role of mitochondria in HSC homeostasis and discuss, among other topics, how mitochondrial dynamism and quality control might be implicated in HSC fate, self-renewal, and regenerative potential.

Loss of Krüppel-Like Factor 4 (KLF4) Leads to Increased Self-Renewal Under Stress Conditions and Improved Survival of Hematopoietic Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 861-861 ◽  
Author(s):  
Chun Shik Park ◽  
Takeshi Yamada ◽  
Koramit Suppipat ◽  
Maksim Mamonkin ◽  
H. Daniel Lacorazza
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Knockout Mice ◽  
Peripheral Blood ◽  
Fold Increase ◽  
Ki 67 ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Abstract 861 Hematopoiesis is a highly regulated process in which a small number of hematopoietic stem cells (HSC) generate all mature blood cells. In order to preserve homeostasis of the hematopoietic system throughout lifetime, this pool of HSC must be maintained by the processes of self-renewal and survival. Self-renewal requires a coordination of survival signals and control of proliferation uncoupled from differentiation. Even though extrinsic signals from the microenvironment and cell-intrinsic regulators are required for self-renewal of HSCs, the intricate transcriptional machinery that selectively regulates HSC self-renewal and survival is still poorly understood. Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor that regulates proliferation, differentiation, apoptosis, and self-renewal. The role of KLF4 in reprogramming adult somatic cells into pluripotent stem cells along with OCT3/4, c-Myc and SOX2 suggests that KLF4 is required for preservation of an undifferentiated state. To investigate the function of KLF4 in HSC maintenance, we used conditional Klf4 knockout mice (Klf4fl/flVav-iCre+) to specifically delete KLF4 gene in hematopoietic cells. We first analyzed the frequency of HSC and progenitor cells in the bone marrow (BM) of Klf4fl/flVav-iCre– (control) and Klf4fl/flVav-iCre+ (knockout) by flow cytometry. We found that KLF4 deficiency leads to a 2.4-fold increase in the number of long-term HSC (Lin–Sca-1+c-Kit+ CD150+ CD48–) and a 2.2-fold increase in short-term HSC compartements (Lin–Sca-1+c-Kit+ CD150+ CD48+) whereas no significant changes were found in myeloid and lymphoid progenitor cells. Consistent with this phenotypic analysis, KLF4 expression in HSC is higher than hematopoietic progenitor cells and mature lineages (n=3; P<0.05). Even though ablation of Klf4 gene does not affect multi-lineage potential of HSC upon transplantation, its deletion leads to a reduction of monocytes and T cell expansion. To assess the effect of Klf4 ablation in self-renewal, we performed serial competitive repopulation assays using a 1:1 mixture of BM cells from control (Klf4fl/flVav-iCre–; CD45.2+) or knockout (Klf4fl/flVav-iCre+; CD45.2+) with B6.SJL (CD45.1+) mice. In primary transplants, the contribution of knockout BM cells in peripheral blood was similar to controls. Interestingly, loss of KLF4 led to enhanced contribution to peripheral blood in secondary transplants (4.5-fold; P<0.005) and tertiary transplants (2.6-fold; P<0.005). Consistent with this result, we found a significant increased number of colony forming units only in the third replating on methylcellulose (P<0.0005). To further characterize the role of KLF4 in HSC proliferation, we determined expression of Ki-67 and DNA content in nuclei of LSK CD150+ cells. The fraction of G0 cells defined as Ki-67– within 2n DNA in Klf4-knockout LSK CD150+ cells was similar to control (control 74.3 ± 0.7% vs 73.2 ± 2.3%). However, Annexin V staining revealed a 2.4-fold increased survival of LSK CD150+ cells in Klf4-knockout mice compared to control mice but not in myeloid progenitor cells (Lin–c-Kit+Sca-1–) suggesting that KLF4 selectively regulates the survival of HSC. These studies indicate that KLF4 controls steady state HSC survival and self-renewal under stress conditions. Disclosures: No relevant conflicts of interest to declare.

MEIS1 Is Required for the Maintenance of Long Term Hematopoietic Stem Cells Wherein It Regulates Quiescence

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 551-551
Author(s):  
Zeenath Unnisa ◽  
Jason P Clark ◽  
Elizabeth Wojtowicz ◽  
Lino Tessarollo ◽  
Neal G. Copeland ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Bone Marrow Analysis ◽  
Marrow Cells

Abstract Abstract 551 Normal hematopoiesis is maintained by long-term hematopoietic stem cells (LT-HSCs) that are defined by their extensive self-renewal and multipotency. Self-renewal of LT-HSCs in turn is regulated by a complex network of intrinsic and extrinsic factors. The transcription factor MEIS1 is highly expressed in hematopoietic stem and progenitor cells and also in several leukemias, suggesting that MEIS1 might be important in regulating self-renewal. However, the role of MEIS1 in normal hematopoiesis has not been defined. To determine the role of MEIS1 in hematopoiesis, we studied conditional knockout mice. We generated transgenic mice bearing loxp sites flanking the homeodomain of MEIS1. The MEIS1-floxed mice were then bred to Rosa26-CreERT2 mice, the latter expressing cre-recombinase ubiquitously, that can be activated by estrogen or its analog Tamoxifen (Tam). Efficient, complete recombination was achieved in vivo by treating MEIS1-f/f-Cre (homozygous for MEIS1-flox) mice with Tam and in vitro by treating bone marrow cells with 4-hydroxy tamoxifen. Loss of MEIS1 expression was detected by QRT-PCR and western blotting. To determine the role of MEIS1 in the maintenance of adult hematopoiesis, MEIS1-f/f-Cre and control mice were treated with Tam and MEIS1 deletion confirmed by PCR. At three weeks post deletion, bone marrow analysis showed a significant reduction in the number of LT-HSCs defined as lin-/c-Kit+/Sca1+/CD48−/CD150+ in the MEIS1-depleted mice compared to controls (0.012% compared to 0.037%, N=6, p<0.05, t-test). However, the progenitor populations were unaffected by MEIS1 deletion. Over a period of 12 weeks of observation, the mice did not show any signs of distress and the peripheral blood counts of the experimental and control mice remained normal, indicating that short term hematopoiesis was not affected. Cell cycle analysis of LT-HSCs showed that MEIS1 deletion resulted in a significant shift of cells from G0 to G1 phase (G0 and G1 proportions respectively, 81.75±3.25% and 9.40±3% for control and 56.10±0.873% and 31.17±1.5% for MEIS1-deleted). To determine the effects of MEIS1 loss on intrinsic hematopoietic stem cell function, we performed competitive repopulation assays. Bone marrow cells harvested from MEIS1-f/f-Cre or MEIS1-f/+-Cre (control) mice were combined with equal numbers of bone marrow cells from BoyJ mice and transplanted via tail vein injection into lethally irradiated BoyJ mice. Four weeks after transplant, recipients were treated with Tam or vehicle for 5 days and deletion of MEIS1 confirmed by PCR on peripheral blood. Peripheral blood of recipient mice was analyzed at 1, 4, 8, 12 and 16 weeks after treatment and relative chimerism assessed by flow cytometry. At 1 and 4 weeks after treatment, the chimerism in the MEIS1 deleted group (Tam treated MEIS1-f/f-CreER) and the control groups (Tam treated MEIS1-f/+-CReER and vehicle treated MEIS1-f/f-CreER) was comparable (41%, 40.5% and 41.5% respectively, average, N=5 to 8). However, by 8 weeks after treatment, the MEIS1 deleted group showed a significant decline in chimerism compared to controls (18.2% compared to 43.1% and 35.1% respectively, p<0.02, t-test) and at 16 weeks the chimerism in the MEIS1-deleted group declined further (11.1% compared to 40.2% and 35.0% respectively, p<0.001). Subpopulation analysis showed loss of chimerism in granulocytes and in B and T lymphocytes. The latency and breadth of the effect of MEIS1 loss suggested an effect on the hematopoietic stem cell population. Indeed, bone marrow analysis of transplant recipients showed near complete loss of LT-HSC chimerism (3% compared to 70.25% and 75.6% respectively, p<0.001). Finally, we performed gene expression profiling on lineage negative bone marrow cells with and without MEIS1 deletion. Results showed that loss of MEIS1 was associated with decreased expression of hypoxia-responsive genes. Collectively, these results indicate that MEIS1 is required for the maintenance of the pool of LT-HSCs. Loss of MEIS1 promotes cycling and exhaustion of LT-HSCs. Further, we propose that activation of the hypoxia-response pathway may be one of the mechanisms by which MEIS1 exerts its effects on hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Rapid and sustained hematopoietic recovery in lethally irradiated mice transplanted with purified Thy-1.1lo Lin-Sca-1+ hematopoietic stem cells

Blood ◽  
1994 ◽  
Vol 83 (12) ◽  
pp. 3758-3779 ◽  
Author(s):  
N Uchida ◽  
HL Aguila ◽  
WH Fleming ◽  
L Jerabek ◽  
IL Weissman
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Blood Cells ◽  
Critical Role ◽  
White Blood Cells ◽  
Cell Types ◽  
Dose Dependence ◽  
Hematopoietic Stem ◽  
Hematopoietic Recovery

Abstract Hematopoietic stem cells (HSCs) are believed to play a critical role in the sustained repopulation of all blood cells after bone marrow transplantation (BMT). However, understanding the role of HSCs versus other hematopoietic cells in the quantitative reconstitution of various blood cell types has awaited methods to isolate HSCs. A candidate population of mouse HSCs, Thy-1.1lo Lin-Sca-1+ cells, was isolated several years ago and, recently, this population has been shown to be the only population of BM cells that contains HSCs in C57BL/Ka-Thy-1.1 mice. As few as 100 of these cells can radioprotect 95% to 100% of irradiated mice, resulting long-term multilineage reconstitution. In this study, we examined the reconstitution potential of irradiated mice transplanted with purified Thy-1.1lo Lin-Sca-1+ BM cells. Donor-derived peripheral blood (PB) white blood cells were detected as early as day 9 or 10 when 100 to 1,000 Thy-1.1lo Lin-Sca-1+ cells were used, with minor dose-dependent differences. The reappearance of platelets by day 14 and thereafter was also seen at all HSC doses (100 to 1,000 cells), with a slight dose-dependence. All studied HSC doses also allowed RBC levels to recover, although at the 100 cell dose a delay in hematocrit recovery was observed at day 14. When irradiated mice were transplanted with 500 Thy-1.1lo Lin-Sca-1+ cells compared with 1 x 10(6) BM cells (the equivalent amount of cells that contain 500 Thy-1.1lo Lin-Sca-1+ cells as well as progenitor and mature cells), very little difference in the kinetics of recovery of PB, white blood cells, platelets, and hematocrit was observed. Surprisingly, even when 200 Thy1.1lo Lin-Sca- 1+ cells were mixed with 4 x 10(5) Sca-1- BM cells in a competitive repopulation assay, most of the early (days 11 and 14) PB myeloid cells were derived from the HSC genotype, indicating the superiority of the Thy-1.1lo Lin-Sca-1+ cells over Sca-1- cells even in the early phases of myeloid reconstitution. Within the Thy-1.1lo Lin-Sca-1+ population, the Rhodamine 123 (Rh123)hi subset dominates in PB myeloid reconstitution at 10 to 14 days, only to be overtaken by the Rh123lo subset at 3 weeks and thereafter. These findings indicate that HSCs can account for the early phase of hematopoietic recovery, as well as sustained hematopoiesis, and raise questions about the role of non-HSC BM populations in the setting of BMT.

Cripto Selectively Expands a Distinct Population of Hematopoietic Stem Cells Expressing the Cell Surface Receptor GRP78 and Strongly Induces An Immature Phenotype In Vivo After Ex Vivo Culture

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 405-405
Author(s):  
Kenichi Miharada ◽  
Göran Karlsson ◽  
Jonas Larsson ◽  
Emma Larsson ◽  
Kavitha Siva ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Distinct Population ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Total Bone Marrow

Abstract Abstract 405 Cripto is a member of the EGF-CFC soluble protein family and has been identified as an important factor for the proliferation/self-renewal of ES and several types of tumor cells. The role for Cripto in the regulation of hematopoietic cells has been unknown. Here we show that Cripto is a potential new candidate factor to increase self-renewal and expand hematopoietic stem cells (HSCs) in vitro. The expression level of Cripto was analyzed by qRT-PCR in several purified murine hematopoietic cell populations. The findings demonstrated that purified CD34-KSL cells, known as highly concentrated HSC population, had higher expression levels than other hematopoietic progenitor populations including CD34+KSL cells. We asked how Cripto regulates HSCs by using recombinant mouse Cripto (rmCripto) for in vitro and in vivo experiments. First we tested the effects of rmCripto on purified hematopoietic stem cells (CD34-LSK) in vitro. After two weeks culture in serum free media supplemented with 100ng/ml of SCF, TPO and 500ng/ml of rmCripto, 30 of CD34-KSL cells formed over 1,300 of colonies, including over 60 of GEMM colonies, while control cultures without rmCripto generated few colonies and no GEMM colonies (p<0.001). Next, 20 of CD34-KSL cells were cultured with or without rmCripto for 2 weeks and transplanted to lethally irradiated mice in a competitive setting. Cripto treated donor cells showed a low level of reconstitution (4–12%) in the peripheral blood, while cells cultured without rmCripto failed to reconstitute. To define the target population and the mechanism of Cripto action, we analyzed two cell surface proteins, GRP78 and Glypican-1, as potential receptor candidates for Cripto regulation of HSC. Surprisingly, CD34-KSL cells were divided into two distinct populations where HSC expressing GRP78 exhibited robust expansion of CFU-GEMM progenitor mediated by rmCripto in CFU-assay whereas GRP78- HSC did not respond (1/3 of CD34-KSL cells were GRP78+). Furthermore, a neutralization antibody for GRP78 completely inhibited the effect of Cripto in both CFU-assay and transplantation assay. In contrast, all lineage negative cells were Glypican-1 positive. These results suggest that GRP78 must be the functional receptor for Cripto on HSC. We therefore sorted these two GRP78+CD34-KSL (GRP78+HSC) and GRP78-CD34-KSL (GRP78-HSC) populations and transplanted to lethally irradiated mice using freshly isolated cells and cells cultured with or without rmCripto for 2 weeks. Interestingly, fresh GRP78-HSCs showed higher reconstitution than GRP78+HSCs (58–82% and 8–40%, p=0.0038) and the reconstitution level in peripheral blood increased rapidly. In contrast, GRP78+HSC reconstituted the peripheral blood slowly, still at a lower level than GRP78-HSC 4 months after transplantation. However, rmCripto selectively expanded (or maintained) GRP78+HSCs but not GRP78-HSCs after culture and generated a similar level of reconstitution as freshly transplanted cells (12–35%). Finally, bone marrow cells of engrafted recipient mice were analyzed at 5 months after transplantation. Surprisingly, GRP78+HSC cultured with rmCripto showed higher reconstitution of the CD34-KSL population in the recipients' bone marrow (45–54%, p=0.0026), while the reconstitution in peripheral blood and in total bone marrow was almost the same. Additionally, most reconstituted CD34-KSL population was GRP78+. Interestingly freshly transplanted sorted GRP78+HSC and GRP78-HSC can produce the GRP78− and GRP78+ populations in the bone marrow and the ratio of GRP78+/− cells that were regenerated have the same proportion as the original donor mice. Compared to cultured cells, the level of reconstitution (peripheral blood, total bone marrow, HSC) in the recipient mice was almost similar. These results indicate that the GRP78 expression on HSC is reversible, but it seems to be “fixed” into an immature stage and differentiate with lower efficiency toward mature cells after long/strong exposure to Cripto signaling. Based on these findings, we propose that Cripto is a novel factor that maintains HSC in an immature state and may be a potent candidate for expansion of a distinct population of GRP78 expressing HSC. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Smad4 is critical for self-renewal of hematopoietic stem cells

2007 ◽  
Vol 204 (3) ◽  
pp. 467-474 ◽  
Author(s):  
Göran Karlsson ◽  
Ulrika Blank ◽  
Jennifer L. Moody ◽  
Mats Ehinger ◽  
Sofie Singbrant ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
In Vitro Proliferation ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Early Embryonic Lethality

Members of the transforming growth factor β (TGF-β) superfamily of growth factors have been shown to regulate the in vitro proliferation and maintenance of hematopoietic stem cells (HSCs). Working at a common level of convergence for all TGF-β superfamily signals, Smad4 is key in orchestrating these effects. The role of Smad4 in HSC function has remained elusive because of the early embryonic lethality of the conventional knockout. We clarify its role by using an inducible model of Smad4 deletion coupled with transplantation experiments. Remarkably, systemic induction of Smad4 deletion through activation of MxCre was incompatible with survival 4 wk after induction because of anemia and histopathological changes in the colonic mucosa. Isolation of Smad4 deletion to the hematopoietic system via several transplantation approaches demonstrated a role for Smad4 in the maintenance of HSC self-renewal and reconstituting capacity, leaving homing potential, viability, and differentiation intact. Furthermore, the observed down-regulation of notch1 and c-myc in Smad4−/− primitive cells places Smad4 within a network of genes involved in the regulation HSC renewal.

scholarly journals Hematopoietic stem cells fail to regenerate following inflammatory challenge

2020 ◽  
Author(s):  
Ruzhica Bogeska ◽  
Paul Kaschutnig ◽  
Malak Fawaz ◽  
Ana-Matea Mikecin ◽  
Marleen Büchler-Schäff ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Recovery Period ◽  
Cumulative Impact ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Inflammatory Stress ◽  
Kinetics Of

AbstractHematopoietic stem cells (HSCs) are canonically defined by their capacity to maintain the HSC pool via self-renewal divisions. However, accumulating evidence suggests that HSC function is instead preserved by sustaining long-term quiescence. Here, we study the kinetics of HSC recovery in mice, following an inflammatory challenge that induces HSCs to exit dormancy. Repeated inflammatory challenge resulted in a progressive depletion of functional HSCs, with no sign of later recovery. Underlying this observation, label retention experiments demonstrated that self-renewal divisions were absent or extremely rare during challenge, as well as during any subsequent recovery period. While depletion of functional HSCs held no immediate consequences, young mice exposed to inflammatory challenge developed blood and bone marrow hypocellularity in old age, similar to elderly humans. The progressive, irreversible attrition of HSC function demonstrates that discreet instances of inflammatory stress can have an irreversible and therefore cumulative impact on HSC function, even when separated by several months. These findings have important implications for our understanding of the role of inflammation as a mediator of dysfunctional tissue maintenance and regeneration during ageing.

scholarly journals Mitochondrial Role in Stemness and Differentiation of Hematopoietic Stem Cells

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Luena Papa ◽  
Mansour Djedaini ◽  
Ronald Hoffman
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Energy Production ◽  
Ex Vivo ◽  
Mitochondrial Activity ◽  
Mitochondrial Oxidative Phosphorylation ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Hypoxic Environment

Quiescent and self-renewing hematopoietic stem cells (HSCs) rely on glycolysis rather than on mitochondrial oxidative phosphorylation (OxPHOS) for energy production. HSC reliance on glycolysis is considered an adaptation to the hypoxic environment of the bone marrow (BM) and reflects the low energetic demands of HSCs. Metabolic rewiring from glycolysis to mitochondrial-based energy generation accompanies HSC differentiation and lineage commitment. Recent evidence, however, highlights that alterations in mitochondrial metabolism and activity are not simply passive consequences but active drivers of HSC fate decisions. Modulation of mitochondrial activity and metabolism is therefore critical for maintaining the self-renewal potential of primitive HSCs and might be beneficial for ex vivo expansion of transplantable HSCs. In this review, we emphasize recent advances in the emerging role of mitochondria in hematopoiesis, cellular reprograming, and HSC fate decisions.

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