Hematopoietic Defects In ACD/TPP1-Deficient Mice Reveal An Essential Role for the Shelterin Complex In Blood-Forming Stem Cell Homeostasis

Abstract Abstract 882 Bone marrow failure syndromes can be caused by loss-of-function mutations in telomerase components. Besides the enzymatic activity of telomerase, telomere maintenance requires the protective function of the shelterin protein complex. This complex consists of six proteins that bind to the telomere and prevent an inappropriate DNA damage response from being elicited by exposed telomeric DNA. Within this complex, TPP1, encoded by the gene Acd, plays a central role in complex organization as it links elements that bind the double-stranded portion of the telomere to those that bind the single-stranded overhang. We report an essential function for the shelterin complex and specifically for TPP1 in the maintenance of hematopoietic stem cells (HSCs). Utilizing adrenocortical dysplasia (acd), a spontaneous autosomal recessive mouse mutation causing profoundly hypomorphic Acd expression, we identified phenotypic and functional abnormalities in fetal hematopoietic progenitors. acd mutant Fetal liver HSCs were larger, more granular, and expressed higher levels of the cell surface protein Sca-1, as compared to wild-type counterparts. BrdU incorporation analysis showed an accumulation of Acd-deficient progenitors in the G2M phase of the cell cycle, consistent with G2M arrest. Absolute quantification of fetal liver HSCs revealed a significant reduction in stem cell numbers in acd mutants as compared to control littermates. p53 deficiency rescued the HSC depletion phenotype, but induced a paradoxical exacerbation of the increased progenitor cell size and Sca-1 expression. These findings suggest that p53-dependent and p53-independent elements contribute to the acd HSC phenotype. In competitive repopulation assays, acd HSCs failed to provide tri-lineage hematopoietic reconstitution in lethally irradiated recipients, indicating a profound functional defect. To further study the effects of Acd deficiency in adult HSCs, we generated an Acd allele in which exons 3–8 are flanked by loxP sites, allowing conditional inactivation following Mx1-Cre induction via poly(I:C) administration. Acd inactivation resulted in rapid HSC depletion within 2 weeks after initiation of poly(I:C) injections. To assess whether Acd exerted exclusively cell-autonomous effects in HSCs, we transplanted Acdf/–Mx1-Cre+ HSCs into wild-type recipients. Poly(I:C)-mediated Acd inactivation in these bone marrow chimeric mice resulted in rapid HSC loss and failure of tri-lineage hematopoiesis in animals reconstituted with Acd-deficient bone marrow. These data indicate an exquisite cell-autonomous requirement for Acd/TPP1 in adult mouse HSCs. In view of the extremely rapid HSC loss after Acd inactivation, these observations cannot be explained by telomere shortening. Together, our findings represent the first report of an essential role for Acd/TPP1 and the shelterin complex in HSC maintenance and hematopoietic homeostasis, independently of telomerase function. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

The Rho Family GTPase Cdc42 Regulates Multiple Hematopoietic Stem/Progenitor Cell Functions and Erythropoiesis.

Blood ◽

10.1182/blood.v104.11.32.32 ◽

2004 ◽

Vol 104 (11) ◽

pp. 32-32

Author(s):

Lei Wang ◽

Linda Yang ◽

Marie–Dominique Filippi ◽

David A. Williams ◽

Yi Zheng

Keyword(s):

Bone Marrow ◽

Fetal Liver ◽

Brdu Incorporation ◽

Low Density ◽

Actin Assembly ◽

Wild Type ◽

Hematopoietic Stem ◽

Rho Family

Abstract The Rho family GTPase Cdc42 has emerged as a key signal transducer in cell regulation. To investigate its physiologic function in hematopoiesis, we have generated mice carrying a gene targeted null allele of cdc42gap, a major negative regulatory gene of Cdc42 and mice with conditional targeted cdc42 allele (cdc42flox/flox). Deletion of the respective gene products in mice was confirmed by PCR genotyping and Western blotting. Low-density fetal liver or bone marrow cells from Cdc42GAP−/− mice displayed ~3 fold elevated Cdc42 activity and normal RhoA, Rac1 or Rac2 activity, indicating that cdc42gap deletion has a specific effect on Cdc42 activity. The Cdc42GAP-deficient hematopoietic stem/progenitor cells (HSC/Ps, Lin−c-Kit+) generated from Cdc42GAP−/− E14.5 fetal liver and the Cdc42−/− HSC/Ps derived by in vitro expression of Cre via a retrovirus vector from Cdc42flox/flox low density bone marrow showed a growth defect in liquid culture that was associated with increased apoptosis but normal cell cycle progression. Cdc42GAP-deficient HSC/Ps displayed impaired cortical F-actin assembly with extended actin protrusions upon exposure to SDF–1 in vitro and a punctuated actin structure after SCF stimulation while Cdc42−/− but not wild type HSC/Ps responded to SDF-1 in inducing membrane protrusions. Both Cdc42−/− and Cdc42GAP−/− HSC/Ps were markedly decreased in adhesion to fibronectin. Moreover, both Cdc42−/− and Cdc42GAP−/− HSC/Ps showed impaired migration in response to SDF-1. These results demonstrate that Cdc42 regulation is essential for multiple HSC/P functions. To understand the in vivo hematopoietic function of Cdc42, we have characterized the Cdc42GAP−/− mice further. The embryos and newborns of homozygous showed a ~30% reduction in hematopoietic organ (i.e. liver, bone marrow, thymus and spleen) cellularity, consistent with the reduced sizes of the animals. This was attributed to the increased spontaneous apoptosis associated with elevated Cdc42/JNK/Bid activities but not to a proliferative defect as revealed by in vivo TUNEL and BrdU incorporation assays. ~80% of Cdc42GAP−/− mice died one week after birth, and the surviving pups attained adulthood but were anemic. Whereas Cdc42GAP−/− mice contained small reduction in the frequency of HSC markers and normal CFU-G, CFU-M, and CFU-GM activities, the frequency of BFU-E and CFU-E were significantly reduced. These results suggest an important role of Cdc42 in erythropoiesis in vivo. Taken together, we propose that Cdc42 is essential for multiple HSC/P functions including survival, actin cytoskeleton regulation, adhesion and migration, and that deregulation of its activity can have a significant impact on erythropoiesis. Cdc42 regulates HSC/P functions and erythropoiesis Genotype/phenotype Apoptosis increase Adhesion decrease Migration decrease F-actin assembly HSC frequency decrease BFU-E, CFU-E decrease The numbers were indicated as fold difference compared with wild type. ND:not determined yet. Cdc42GAP−/− 2.43, p<0.005 0.97, p<0.01 1.01, p<0.01 protrusion (SDF-1); punctruated (SCF) 0.34, p<0.05 0.92, p<0.01; 0.38, p<0 Cdc42−/− 3.68, p<0.005 0.98, p<0.001 3.85, p<0.005 protrusion (SDF-1) ND ND

Download Full-text

Hematopoietic Stem Cell Engraftment in Bone Marrow Is Dependent upon Gsα.

Blood ◽

10.1182/blood.v108.11.857.857 ◽

2006 ◽

Vol 108 (11) ◽

pp. 857-857

Author(s):

Gregor B. Adams ◽

Ian R. Alley ◽

Karissa T. Chabner ◽

Ung-il Chung ◽

Emily S. Marsters ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Bone Marrow Cells ◽

Fetal Liver ◽

Conditional Knockout ◽

Wild Type ◽

Hematopoietic Stem ◽

Cell Engraftment

Abstract During development, hematopoietic stem cells (HSCs) translocate from the fetal liver to the bone marrow, which remains the site of hematopoiesis throughout adulthood. In the bone marrow the HSCs are located at the endosteal surface, where the osteoblasts are a key component of the stem cell niche. The exogenous signals that specifically direct HSCs to the bone marrow have been thought to include stimulation of the chemokine receptor CXCR4 by its cognate ligand stromal derived factor-1α (SDF-1α or CXCL12). However, experiments in which CXCR4−/− fetal liver hematopoietic cells were transplanted into wild-type hosts demonstrated efficient engraftment of the HSCs in the bone marrow. In addition, treatment of HSCs with inhibitors of Gαi-coupled signaling, which blocks transmigration towards SDF-1αin vitro, does not affect bone marrow homing and engraftment in vivo. Therefore, we examined whether Gsα-coupled mechanisms play a key role in the engraftment of the HSCs in the bone marrow environment. Utilizing an inducible-conditional knockout of Gsα, we found that deletion of the gene in hematopoietic bone marrow cells did not affect their ability to perform in the in vitro primitive CFU-C or LTC-IC assay systems. However, Gsα−/− cells were unable to establish effective hematopoiesis in the bone marrow microenvironment in vivo in a competitive repopulation assay (41.1% contribution from wild-type cells versus 1.4% from knockout cells). These effects were not due to an inability of the cells to function in the bone marrow in vivo as deletion of Gsα following establishment of hematopoiesis had no effects on the HSCs. Examining the ability of the HSCs to home to the bone marrow, though, demonstrated that deletion of Gsα resulted in a marked impairment of the ability of the stem cells to localize to the marrow space (approximately 9-fold reduction in the level of primitive cell homing). Furthermore, treatment of BM MNCs with an activator of Gsα augmented the cells homing and thus engraftment potential. These studies demonstrate that Gsα is critical to the localization of HSCs to the bone marrow. Which receptors utilize this pathway in this context remains unknown. However, Gsα represents a previously unrecognized signaling pathway for homing and engraftment of HSCs to bone marrow. Pharmacologic activation of Gsα in HSC ex vivo prior to transplantation offers a potential method for enhancing stem cell engraftment efficiency.

Download Full-text

Reduced lymphomyeloid repopulating activity from adult bone marrow and fetal liver of mice lacking expression of STAT5

Blood ◽

10.1182/blood.v99.2.479 ◽

2002 ◽

Vol 99 (2) ◽

pp. 479-487 ◽

Cited By ~ 103

Author(s):

Kevin D. Bunting ◽

Heath L. Bradley ◽

Teresa S. Hawley ◽

Richard Moriggl ◽

Brian P. Sorrentino ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Cell Function ◽

Fetal Liver ◽

Side Population ◽

Significant Proportion ◽

Cell Phenotype ◽

Wild Type ◽

Erythroid Progenitor ◽

Hematopoietic Stem

Abstract Signal transducers and activators of transcription (STATs) are intracellular mediators of cytokine receptor signals. Because many early-acting growth factors have been implicated in STAT5 activation, this study sought to investigate whether STAT5 may be a transcriptional regulator of hematopoietic stem cell (HSC) long-term repopulating activity. To test this possibility, bone marrow (BM) and fetal liver (FL) cells from mice containing homozygous deletions of both STAT5a and STAT5b genes (STAT5ab−/−) were characterized for hematopoietic repopulating activities. BM and FL grafts were capable of repopulating lymphoid and myeloid lineages of lethally irradiated primary and secondary hosts, with defects observed primarily in T-lymphocyte engraftment. Because only a fraction of normal HSC function is required to reconstitute hematopoiesis, competitive repopulation assays of adult BM or FL cells were used against wild type adult BM or FL cells to quantitate stem cell function. In these analyses, average 25-, 28-, 45-, and 68-fold decreases in normal repopulating activity were evident in granulocyte (Gr-1+), macrophage (Mac-1+), erythroid progenitor (Ter119+), and B-lymphocyte (B220+) populations, respectively, with T lymphocytes (CD4+) always undetectable from the STAT5ab−/− graft. Consistent with previous reports of divergence between stem cell phenotype and function in cases of perturbed hematopoiesis, the absolute number of cells within Sca-1+c-kit+lin− or lin− Hoechst 33342 side population fractions was not significantly different between wild type and STAT5ab−/−BM or FL cells. These results demonstrate that a significant proportion of the growth factor signals required for multilineage reconstitution potential of HSCs is STAT5 dependent.

Download Full-text

Necdin Regulates Hematopoietic Stem Cell Quiescence and Sensitivity to Genotoxic Stress.

Blood ◽

10.1182/blood.v114.22.379.379 ◽

2009 ◽

Vol 114 (22) ◽

pp. 379-379 ◽

Cited By ~ 1

Author(s):

Takashi Asai ◽

Yan Liu ◽

Silvana Di Giandomenico ◽

Anthony Deblasio ◽

Silvia Menendez ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Target Genes ◽

Fetal Liver ◽

Genotoxic Stress ◽

Wild Type ◽

Hematopoietic Stem ◽

Stem Cell Quiescence ◽

Cell Quiescence

Abstract Abstract 379 Necdin, a member of MAGE (melanoma antigen) family proteins, is a growth suppressing protein that was first identified in post mitotic neurons. The gene encoding necdin is one of several deleted in individuals with Prader-Willi syndrome, a neurobehavioural disorder associated with an increased risk of myeloid leukemia. It is reported that necdin interacts with p53 and represses p53-mediated apoptosis in neurons, but its role in hematopoiesis is largely unknown. Recently, we defined a critical role of p53 in regulating hematopoietic stem cell quiescence, and identified necdin as a target gene of p53, that is highly expressed in LT-HSCs (Liu Y et al., Cell Stem Cell, 2009). To define the role of necdin in hematopoiesis, we have analyzed the hematopoietic compartment of necdin-null mice. As necdin-null mice die perinatally, we first investigated fetal hematopoiesis and found no alteration in the frequency of fetal liver HSCs, defined as Lin-Sca1+Mac1+CD48-CD150+ within the fetal liver cells. Although necdin-null fetal liver HSCs increase serial replating capability in methylcellulose and maintain stemness in long-term stromal based cultures better than wild type HSCs, necdin-null fetal liver HSCs repopulate lethally irradiated recipient mice similar to wild type HSCs, in primary, secondary, and tertiary serial bone marrow transplantation assays. In addition, necdin-null HSCs show almost comparable repopulating ability as wild type HSCs, after secondary competitive bone marrow transplantation assays. These imply that necdin is dispensable for HSC self renewal. On the other hand, BM-derived necdin-null HSCs show decreased quiescence 4 months after transplantation, and increased proliferation as indicated by in vivo BrdU incorporation assays. Furthermore, recipient mice repopulated with necdin-null HSCs show enhanced sensitivity both to weekly 5-FU administration and to total body irradiation, as manifested by increased mortality. This suggests that the decreased quiescence of necdin-null HSCs leads to their depletion under conditions of genotoxic stress. Gene expression profiling studies have identified several deregulated signaling pathways in the necdin-null HSCs. Expression of several p53 target genes is altered in irradiated necdin-null HSCs, which may account for their enhanced radiosensitivity. We are now investigating these necdin target genes to clarify how necdin functions to critically regulate HSC quiescence. We are also determining whether targeting necdin could be a therapeutic approach to eliminate quiescent leukemia stem cells, using a murine CML model. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Essential roles for Pot1b in HSC self-renewal and survival

Blood ◽

10.1182/blood-2011-06-361527 ◽

2011 ◽

Vol 118 (23) ◽

pp. 6068-6077 ◽

Cited By ~ 22

Author(s):

Yang Wang ◽

Mei-Feng Shen ◽

Sandy Chang

Keyword(s):

Progenitor Cell ◽

Essential Role ◽

Dyskeratosis Congenita ◽

Telomere Maintenance ◽

Wild Type ◽

Telomere Shortening ◽

Damage Response ◽

First Time

Abstract Maintenance of mammalian telomeres requires both the enzyme telomerase and shelterin, which protect telomeres from inappropriately activating DNA damage response checkpoints. Dyskeratosis congenita is an inherited BM failure syndrome disorder because of defects in telomere maintenance. We have previously shown that deletion of the shelterin component Pot1b in the setting of telomerase haploinsufficiency results in rapid telomere shortening and fatal BM failure in mice, eliciting phenotypes that strongly resemble human syskeratosis congenita. However, it was unclear why BM failure occurred in the setting of Pot1b deletion. In this study, we show that Pot1b plays an essential role in HSC survival. Deletion of Pot1b results in increased apoptosis, leading to severe depletion of the HSC reserve. BM from Pot1bΔ/Δ mice cannot compete with BM from wild-type mice to provide multilineage reconstitution, indicating that there is an intrinsic requirement for Pot1b the maintenance of HSC function in vivo. Elimination of the p53-dependent apoptotic function increased HSC survival and significantly extended the lifespan of Pot1b-null mice deficient in telomerase function. Our results document for the first time the essential role of a component of the shelterin complex in the maintenance of HSC and progenitor cell survival.

Download Full-text

Protein Arginine Methyltransferase 1 (PRMT1) Dampens Self-Renewal and Promotes Differentiation of Hematopoietic Stem Cells in Mouse Models

Blood ◽

10.1182/blood-2019-127226 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2460-2460 ◽

Cited By ~ 1

Author(s):

Hairui Su ◽

Szu-Mam Liu ◽

Chiao-Wang Sun ◽

Mark T. Bedford ◽

Xinyang Zhao

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Bone Marrow Transplantation ◽

Arginine Methylation ◽

Poly I:C ◽

Differentiation Potential ◽

Self Renewal ◽

Arginine Methyltransferase ◽

Hematopoietic Stem

Protein arginine methylation is a common type of post-translational modification. PRMT1, the major type I protein arginine methyltransferase, catalyzes the formation of asymmetric dimethyl-arginine and is implicated in various cellular processes, including hematopoiesis and tumorigenesis. We have shown that PRMT1 expression is naturally low in hematopoietic stem cells (HSCs). However, the functions of PRMT1 in hematopoietic stem cell self-renewal and differentiation are yet to be revealed. We have found a cyanine-based fluorescent probe (E84) that can specifically label PRMT1 protein. E84 staining dynamically captures intracellular PRMT1 level and was used to separate live HSC populations with differential PRMT1 expression by flow cytometry. Subsequent bone marrow transplantation of E84high or E84low Lin−Sca1+cKit+ (LSK) cells showed that E84low LSK cells were much more advantageous in reconstituting each blood cell lineages, compared to the E84high counterparts, meaning that the stem-ness of HSCs is negatively correlated with endogenous PRMT1. Therefore, inhibition of PRMT1 was expected to enhance the number and differentiation potential of functional HSCs. The treatment of a PRMT1-specific inhibitor (MS023) to mice resulted in an enlarged LT-HSC population in bone marrow and decreased frequency of granulocyte progenitor cells. In vitro colony formation assays further demonstrated that PRMT1 is required for GMP differentiation. Then we asked whether copious expression of PRMT1 promotes the differentiation of HSC. In this line, we made a LoxP-STOP-LoxP-PRMT1 transgenic mouse model, which induces PRMT1 overexpression upon the expression of Cre recombinase from tissue-specific promoters. We established Mx1-Cre-PRMT1 (Mx1-Tg) mice. Mx1-Tg mice were injected with poly(I:C) for PRMT1 induction and analyzed at four weeks after the last dose. We found that, as predicted, LT-HSC population was reduced and the Pre-GM population was raised. Accordingly, more CFU-Gs but less GEMMs were grown on CFU assays. We further utilized this animal model to compare the blood reconstitution capabilities of bone marrow cells from Mx1-Tg vs. WT mice in the same repopulating conditions. We performed competitive bone marrow transplantation by injecting Mx1-Tg/WT (CD45.2) bone marrow plus supporting cells (CD45.1) to irradiated mice, followed by 5 doses of poly(I:C) induction. Recipient mice were analyzed during a course of approximately 16 weeks. Mx1-Tg cells were outcompeted by WT cells in reconstituting every blood lineages. Taken together, we conclude that PRMT1 promotes HSC differentiation and accelerates HSC exhaustion during the stress caused by bone marrow irradiation. To understand the mechanism on PRMT1-mediated stress hematopoiesis, we also made Pf4-Cre PRMT1 transgenic mice. When PRMT1 is specifically expressed in MK cells, the number of LT-HSCs was also reduced, implying that PRMT1 affects the self-renewal of LT-HSCs via communication between MK cells and HSCs. Mechanistically, two PRMT1 substrates - RBM15 and DUSP4 - are critical for stem cell self-renewal. We further characterized how PRMT1 activates p38 kinase pathway via directly methylating DUSP4 thus induces ubiquitylation and degradation of DUSP4. The arginine methylation site on DUSP4 has been identified. Moreover, introducing methyl-R mutated DUSP4 back to PRMT1-overexpressing cells partially rescued the loss of HSC differentiation potential. This data adds a new link between arginine methylation and protein phosphorylation mediated by MAP kinases/phosphatases. In addition, we discovered that RBM15 controls alternative RNA splicing and RNA processing in a PRMT1-dosage dependent manner. In this report, we will further address how RBM15 target genes, such as enzymes involved in fatty acid metabolic pathways, affect HSC differentiation. In summary, we report that arginine methylation is a novel regulator for the HSC differentiation via controlling p38-regulated stress pathway and metabolic reprogramming. Disclosures No relevant conflicts of interest to declare.

Download Full-text

The Interferon-Inducible GTPase LRG-47 Regulates Hematopoietic Stem Cell Proliferation and Is Required for Maintenance of Normal Hematopoiesis during Microbial Infection.

Blood ◽

10.1182/blood.v106.11.2266.2266 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2266-2266

Author(s):

David Weksberg ◽

Carl G. Feng ◽

Alan Sher ◽

Margaret A. Goodell

Keyword(s):

Bone Marrow ◽

Immune Response ◽

Stem Cell ◽

Knockout Mice ◽

Progenitor Cell ◽

Side Population ◽

Constant Number ◽

Wild Type ◽

Hematopoietic Stem ◽

Error Bars

Abstract Hematopoietic stem cells (HSCs) have a remarkable capacity to respond to proliferative stimuli, as they are able to reconstitute the blood following catastrophic injuries such as chemotherapy and lethal irradiation. Most work aimed at elucidating the genetic and molecular controls on this program of activation has focused on HSCs responding to these artificial stimuli, however there is a surprising paucity of information reflecting the response of HSCs to the types of stimuli encountered in a non-laboratory setting. Here we report that LRG-47, an interferon-inducible GTPase, is required for HSCs to respond to a variety of proliferative stimuli, including mycobaterial challenge. Previously studied solely in the context of the immune response to intracellular pathogens, LRG-47 is upregulated in HSCs during 5-fluorouracil-(5FU) induced proliferation, and we now show that LRG-47 −/− HSCs exhibit profound defects. LRG-47 −/− HSCs achieve only 4–8% of wild-type engraftment activity in competitive repopulation assays (Figure 1) and, strikingly, even transplantation in 25-fold excess over wild-type competitor fails to rescue this defect. We also demonstrate that LRG-47 −/− HSCs are impaired in colony-forming ability, and that LRG-47 −/− mice exhibit both a relative and absolute failure to expand the stem cell/progenitor compartments in response to 5FU (Figure 2). Intriguingly, we also show that infectious challenge with Mycobacterium avium stimulates an expansion of the progenitor cell (LSK) compartment in wild-type mice - and that LRG-47-deficient mice are unable to mount this response. These findings implicate LRG-47 as being required for effective proliferation of HSCs in response to various stimuli. Furthermore, these results imply that expansion at the progenitor cell level is a downstream effector mechanism of the cytokine-mediated immune response to infection. Ultimately, understanding the mechanisms by which HSCs sense and respond to proliferative stimuli has far-ranging applications, and our work establishes an important connection with the immune system as a regulator of this process. Infectious processes can now arguably join ex vivo HSC manipulation, mechanisms of hematologic malignancy, and transplantation medicine as areas of importance informed by an understanding of the controls on HSC activation, proliferation and quiescence. Figure 1. Competitive transplant of LRG-/- bone marrow. Whole bone marrow from wild type and LRG-47 -/- mice (CD45.2) admixed with a constant number of CD45.1 competitor cells (250,000) and transplanted into lethally irradiated recipients (CD45.1). Perecent chimerism was assessed every four weeks post-transplant (error bars = SEM). Figure 1. Competitive transplant of LRG-/- bone marrow. Whole bone marrow from wild type and LRG-47 -/- mice (CD45.2) admixed with a constant number of CD45.1 competitor cells (250,000) and transplanted into lethally irradiated recipients (CD45.1). Perecent chimerism was assessed every four weeks post-transplant (error bars = SEM). Figure 2. LRG-47 -/- fail to expand HSC compartment in response to SFU. Wild type and LRG-47 -/- mice were injected with SFU 6-days prior to side population (SP) analysis of HSC compartment. While wild-type mice showed the expected expansion of the HSC population (upper panels - gated), this response is impaired in the knockout mice (lower panels). Figure 2. LRG-47 -/- fail to expand HSC compartment in response to SFU. Wild type and LRG-47 -/- mice were injected with SFU 6-days prior to side population (SP) analysis of HSC compartment. While wild-type mice showed the expected expansion of the HSC population (upper panels - gated), this response is impaired in the knockout mice (lower panels).

Download Full-text

Integrin alpha 6 Is Essential for Fetal Hematopoietic Progenitor, but Not Fetal Stem Cell Homing to Bone Marrow.

Blood ◽

10.1182/blood.v106.11.1387.1387 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1387-1387

Author(s):

Hong Qian ◽

Sten Eirik W. Jacobsen ◽

Marja Ekblom

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Progenitor Cell ◽

Fetal Liver ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Integrin Alpha 6

Abstract Homing of transplanted hematopoietic stem cells (HSC) in the bone marrow (BM) is a prerequisite for establishment of hematopoiesis following transplantation. However, although multiple adhesive interactions of HSCs with BM microenviroment are thought to critically influence their homing and subsequently their engraftment, the molecular pathways that control the homing of transplanted HSCs, in particular, of fetal HSCs are still not well understood. In experimental mouse stem cell transplantation models, several integrins have been shown to be involved in the homing and engraftment of both adult and fetal stem and progenitor cells in BM. We have previously found that integrin a6 mediates human hematopoietic stem and progenitor cell adhesion to and migration on its specific ligands, laminin-8 and laminin-10/11 in vitro (Gu et al, Blood, 2003; 101:877). Furthermore, integrin a6 is required for adult mouse HSC homing to BM in vivo (Qian et al., Abstract American Society of Hematology, Blood 2004 ). We have now found that the integrin a6 chain like in adult HSC is ubiquitously (>99%) expressed also in fetal liver hematopoietic stem and progenitor cells (lin−Sca-1+c-Kit+, LSK ). In vitro, fetal liver LSK cells adhere to laminin-10/11 and laminin-8 in an integrin a6b1 receptor-dependent manner, as shown by function blocking monoclonal antibodies. We have now used a function blocking monoclonal antibody (GoH3) against integrin a6 to analyse the role of the integrin a6 receptor for the in vivo homing of fetal liver hematopoietic stem and progenitor cells to BM. The integrin a6 antibody inhibited homing of fetal liver progenitors (CFU-C) into BM of lethally irradiated recipients. The number of homed CFU-C in BM was reduced by about 40% as compared to the cells incubated with an isotype matched control antibody. To study homing of long-term repopulating stem cells, BM cells were first incubated with anti-integrin alpha 6 or anti-integrin alpha 4 or control antibody, and then injected intravenously into lethally irradiated primary recipients. After three hours, BM cells of the primary recipients were analysed by competitive repopulation assay in secondary recipients. Blood analysis up to 16 weeks after transplantation showed that no reduction of stem cell reconstitution from integrin a6 antibody treated cells as compared to cells treated with control antibody. In accordance with this, fetal liver HSC from integrin a6 gene deleted embryos did not show any impairment of homing and engraftment in BM as compared to normal littermates. These results suggest that integrin a6 plays an important developmentally regulated role for homing of distinct hematopoietic stem and progenitor cell populations in vivo.

Download Full-text

Bone Marrow-Derived Cells Regenerate Kit+ Cardiac Stem Cells (CSCs) in Damaged Heart.

Blood ◽

10.1182/blood.v108.11.1681.1681 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1681-1681

Author(s):

Francesco Cerisoli ◽

Lucio Barile ◽

Roberto Gaetani ◽

Letizia Cassinelli ◽

Giacomo Frati ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Troponin I ◽

Regulatory Elements ◽

Cardiac Cells ◽

Cardiac Differentiation ◽

Pcr Analysis ◽

Cardiac Stem Cells ◽

Wild Type

Abstract A growing amount of data indicates that the heart harbours stem cells (CSCs) with regenerative potential, however the origin(s) of adult CSCs is still unknown. The expression of Kit a marker of several stem cell types, including hematopoietic and cardiac stem cells, suggests that Kit positive-CSCs may derive, at least in part, from extracardiac sources. In addition, it has been suggested that bone marrow (BM) cells may be mobilized, home into the heart and trans-differentiate into cardiomyocytes, following myocardial infarction. To investigate whether BM cells can contribute to repopulate the cardiac Kit+ stem cell pool, we transplanted BM cells from a mouse line expressing transgenic Green Fluorescent Protein (GFP) under the control of Kit regulatory elements, into wild type irradiated recipients. After hematological reconstution (4–5 months) and following cardiac infarction, cardiac cells were grown in vitro into typical “cardiospheres” (Messina et al., Circ. Res. 95,911;2004). The cardiospheres obtained, although not numerous, were all GFP fluorescent; this result was confirmed by PCR analysis of genomic DNA of individual CSs. At confocal microscopy, cells at the periphery of CSs showed coexistence of low GFP with cardiac markers, such as Troponin I and the transcription factor NKx2.5, consistent with the expected kit downregulation during cardiac differentiation. Our results show that cells of bone marrow origin can give rise, after homing into the heart, to cells with properties of Kit+ CSC. In contrast, CSCs isolated from kit/GFP transgenic mice are not able, upon transplantation, to repopulate the bone marrow of wild-type irradiated recipients. Thus, at least in pathological conditions, part of the Kit-positive CSCs population may be generated by BM-derived cells, capable of adopting in the heart the same function and features of cardiac stem cells.

Download Full-text

Multifunctional Role of Caspase-3 in Regulating Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v108.11.861.861 ◽

2006 ◽

Vol 108 (11) ◽

pp. 861-861 ◽

Cited By ~ 1

Author(s):

Viktor Janzen ◽

Heather E. Fleming ◽

Michael T. Waring ◽

Craig D. Milne ◽

David T. Scadden

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Peripheral Blood ◽

Caspase 3 ◽

Brdu Incorporation ◽

Hematopoietic Stem ◽

Regulatory Pathways

Abstract The processes of cell cycle control, differentiation and apoptosis are closely intertwined in controlling cell fate during development and in adult homeostasis. Molecular pathways connecting these events in stem cells are poorly defined and we were particularly interested in the cysteine-aspartic acid protease, Caspase-3, an ‘executioner’ caspase also implicated in the regulation of the cyclin dependent kinase inhibitors, p21Cip1 and p27Kip1. These latter proteins are known to participate in primitive hematopoietic cell cycling and self-renewal. We demonstrated high levels of Caspase-3 mRNA and protein in immunophenotypically defined mouse hematopoietic stem cells (HSC). Using mice engineered to be deficient in Caspase-3, we observed a consistent reduction of lymphocytes in peripheral blood counts and a slight reduction in bone marrow cellularity. Notably, knockout animals had an increase in the stem cell enriched Lin−cKit+Sca1+Flk2low (LKSFlk2lo) cell fraction. The apoptotic rates of LKS cells under homeostatic conditions as assayed by the Annexin V assay were not significantly different from controls. However, in-vitro analysis of sorted LKS cells revealed a reduced sensitivity to apoptotic cell death in absence of Caspase-3 under conditions of stress (cytokine withdrawal or gamma irradiation). Primitive hematopoietic cells displayed a higher proliferation rate as demonstrated by BrdU incorporation and a significant reduction in the percentage of cells in the quiescent stage of the cell cycle assessed by the Pyronin-Y/Hoechst staining. Upon transplantation, Caspase-3−/− stem cells demonstrated marked differentiation abnormalities with significantly reduced ability to differentiate into multiple hematopoietic lineages while maintaining an increased number of primitive cells. In a competitive bone marrow transplant using congenic mouse stains Capase-3 deficient HSC out-competed WT cells at the stem cell level, while giving rise to comparable number of peripheral blood cells as the WT controls. Transplant of WT BM cells into Caspase-3 deficient mice revealed no difference in reconstitution ability, suggesting negligible effect of the Caspase-3−/− niche microenvironment to stem cell function. These data indicate that Caspase-3 is involved in the regulation of differentiation and proliferation of HSC as a cell autonomous process. The molecular bases for these effects remain to be determined, but the multi-faceted nature of the changes seen suggest that Caspase-3 is central to multiple regulatory pathways in the stem cell compartment.

Download Full-text