scholarly journals Albumin-expressing hepatocyte-like cells develop in the livers of immune-deficient mice that received transplants of highly purified human hematopoietic stem cells

Blood ◽  
2003 ◽  
Vol 101 (10) ◽  
pp. 4201-4208 ◽  
Author(s):  
Xiuli Wang ◽  
Shundi Ge ◽  
George McNamara ◽  
Qian-Lin Hao ◽  
Gay M. Crooks ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Liver Damage ◽  
Human Albumin ◽  
Cell Populations ◽  
Human Stem Cells ◽  
Hematopoietic Stem ◽  
Stem Cell Populations

AbstractRodent bone marrow cells can contribute to liver. If these findings are applicable to humans, marrow stem cells could theoretically be harvested from a patient and used to repair his/her damaged liver. To explore this potential, CD34+ or highly purified CD34+CD38−CD7− human hematopoietic stem cells from umbilical cord blood and bone marrow were transplanted into immunodeficient mice. One month after transplantation, carbon tetrachloride (CCl4) was administered into the mice to induce liver damage and hepatocyte proliferation. Mice were analyzed in comparison with CCl4-injured mice that did not receive transplants and noninjured controls that received transplants with the same stem cell populations, one month after liver damage. Human-specific albumin mRNA and protein were expressed in the mouse liver and human albumin was detected in the serum of mice that had received CCl4 injury. Human alpha-fetoprotein was never expressed, but in some mice, human cytokeratin 19 was expressed, which may indicate bile duct development in addition to the albumin-secreting hepatocyte-like cells. Human albumin was not expressed in the starting stem cell populations in injured mice that did not receive transplants nor in noninjured mice that had received transplants of human stem cells. Human albumin expression was detected only in CCl4-treated mice that received transplants of human stem cells, and recovery was increased by administration of human hepatocyte growth factor 48 hours after the CCl4-mediated liver injury. Our studies provide evidence that human “hematopoietic” stem/progenitor cell populations have the capacity to respond to the injured liver microenvironment by inducing albumin expression.

Use of the NOD/SCID/γcnull Mouse Model To Assess the Hepatocyte-Producing Ability of Human Hematopoietic Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1695-1695
Author(s):  
Hisanori Fujino ◽  
Hidefumi Hiramatsu ◽  
Atsunori Tsuchiya ◽  
Haruyoshi Noma ◽  
Mitsutaka Shiota ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mouse Model ◽  
Hematopoietic Stem Cells ◽  
Liver Damage ◽  
Hematopoietic Cells ◽  
Human Albumin ◽  
Hematopoietic Stem ◽  
Hepatic Cells ◽  
Cd34 Cells

Abstract Hematopoietic cells have been shown to generate nonhematopoietic cells, although the true plasticity of stem cells has been questioned. Here we used the NOD/SCID/γcnull mouse model, which permits efficient engraftment of human hematopoietic stem cells and their multi-lineage differentiation including T cells, to investigate whether human hematopoietic stem cells can differentiate into human hepatocytes. Freshly collected cord blood was depleted of phagocytes with Silica® followed by CD34 positive selection using auto MACS®. These cells were intravenously transplanted into irradiated mice, after which the liver was either undamaged or damaged by chemicals. The livers of these mice contained hepatocyte-specific (albumin, CYP family, TAT, alpha1AT, CPSI, prealbumin, transferrin and RBP4), cholangiocyte-specific (CK19) and vascular endothelial cell-specific (eNOS) human mRNAs. Immunohistochemistry detected the human hepatocyte specific antigens, albumin and alpha-1-antitrypsin-positive hepatocytes, cholangiocytes and CD68+ Kupffer cells. We also found human albumin in the murine bloodstream. Human albumin levels in the peripheral blood of transplanted mice correlate with the degree of PB chimerism and increase with time after transplantation. Furthermore, after obtaining liver cells by collagenase perfusion, flow cytometry revealed the presence of human albumin-positive cells that bear both human and murine MHC molecules, suggesting cell fusion occurs. All of the above phenomena were found in both liver-damaged and undamaged mice. In addition, we found human CD34+ cells are recruited from the murine bone marrow to the liver only in the case of acute liver injury but do not acquire hepatic stem/progenitor characteristics. Our observation suggests there are two pathways that yield hepatic cells from hematopoietic stem cells. The first requires liver damage that recruits CD34+ cells from the bone marrow via the circulation while the second pathway does not involve liver damage and appears to represent a constitutive default pathway of hematopoietic to nonhematopoietic transition. Our model is thus a versatile tool for investigating the development of functional human hepatic cells from hematopoietic cells and the feasibility of using hematopoietic cells in clinical situations.

Embryonic and hematopoietic stem cells express a novel SH2–containing inositol 5′-phosphatase isoform that partners with the Grb2 adapter protein

Blood ◽  
2001 ◽  
Vol 98 (7) ◽  
pp. 2028-2038 ◽  
Author(s):  
Zheng Tu ◽  
John M. Ninos ◽  
Zhengyu Ma ◽  
Jia-Wang Wang ◽  
Maria P. Lemos ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Cells ◽  
Embryonic Stem ◽  
Cell Populations ◽  
Adapter Protein ◽  
Hematopoietic Stem ◽  
Stem Cell Populations

SH2–containing inositol 5′-phosphatase (SHIP) modulates the activation of immune cells after recruitment to the membrane by Shc and the cytoplasmic tails of receptors. A novel SHIP isoform of approximately 104 kd expressed in primitive stem cell populations (s-SHIP) is described. It was found that s-SHIP is expressed in totipotent embryonic stem cells to the exclusion of the 145-kd SHIP isoform expressed in differentiated hematopoietic cells. s-SHIP is also expressed in primitive hematopoietic stem cells, but not in lineage-committed hematopoietic cells. In embryonic stem cells, s-SHIP partners with the adapter protein Grb2 without tyrosine phosphorylation and is present constitutively at the cell membrane. It is postulated that s-SHIP modulates the activation threshold of primitive stem cell populations.

scholarly journals Non-Genotoxic Conditioning Efficiently Depletes Host Hematopoietic Stem Cells and Facilitates Robust Multi-Lineage Engraftment in a Mouse Model of Fanconi Anemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2041-2041
Author(s):  
Meera Srikanthan ◽  
Kevin G. Haworth ◽  
Christina M. Ironside ◽  
Jerry Chen ◽  
Adam J. Hartigan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Marrow Transplant ◽  
Cell Populations ◽  
Employment Equity ◽  
Hematopoietic Stem ◽  
Donor Cells ◽  
Equity Ownership ◽  
Stem Cell Populations

Abstract Conditioning chemotherapy is used to deplete hematopoietic stem cells in the recipient's marrow prior to a bone marrow transplant to facilitate engraftment of donor cells. While effective, some major issues with chemotherapy remain which includes off-target genotoxic effects increasing the risk of secondary malignancies. These complications are compounded in disease settings that arise from a deficit in DNA repair pathways where cytotoxic treatments can result in oncogenic transformation, such as Fanconi anemia (FA). FA is an inherited bone marrow failure disorder resulting from an intrinsic defect in DNA repair affecting approximately 130,000 children each year. Currently mutations in 22 genes have been implicated in the pathogenesis of FA. While novel gene-therapy based protocols are showing early promise for this patient populations, the standard treatment for the hematologic complications for FA is a bone marrow transplant. However, secondarily to an underlying sensitivity to DNA damage, these patients are unable to receive standard myeloablative conditioning, which can reduce the efficiency of reconstitution. Avoiding alkylating agents could improve outcomes and success rates in these patients. Furthermore, this methodology could be translated to the allogeneic bone marrow transplantation setting, decreasing the toxicity of this treatment modality. Our approach for characterizing an alternative conditioning regimen for FA patients utilizes immunotoxin conjugates specifically targeting hematopoietic stem cell populations. These non-genotoxic antibody-based drug conjugates utilize saporin (SAP), a ribosomal toxin, to eliminate cells in a targeted manner while leaving the remainder of the marrow compartment intact. Antibodies targeting either CD45 or CD117 were used in a mouse model of FA where expression of their FANCA gene, one of the most common mutations in humans, has been knocked out. Mice conditioned with either of the drugs received various doses of whole marrow from healthy heterozygous littermates. On the day of transplant, mice conditioned with either immunotoxin demonstrated significantly reduced LSK stem cell populations in the marrow similar to cyclophosphamide (Cy) controls (Figure 1A). Peripheral engraftment of donor cells was monitored for 6 months, after which mice were sacrificed for complete analysis of engraftment in the bone marrow compartment. No significant difference was observed in engraftment between Cy and immunotoxin conditioned mice (Figure 1B), and all treatment groups exhibited robust multilineage reconstitution in a cell dose dependent manner. Additionally, Cy treated mice demonstrated greater and sustained weight loss and lower gastrointestinal losses compared to immunotoxin treated mice. Ongoing clonal analysis studies of engrafted cells has demonstrated polyclonal reconstitution, indicating a large number of donor stem cells are actively contributing to hematopoiesis. In this study, we demonstrate that non-genotoxic conditioning approaches both facilitate multilineage engraftment of donor marrow and significantly deplete host hematopoietic stem cell populations. These are crucial since persistence of host hematopoiesis could eventually result in clonal evolution and leukemogenesis in post-transplant FA patients. Achieving both of these conditions through targeted elimination with immunotoxin conjugates represents a major advancement in bone marrow transplantation for FA. We now are initiating studies using immunotoxin-based conditioning for the transplantation of gene-modified syngeneic stem cells and allogeneic cells. We think these studies will inform future clinical trials and provide the groundwork for the next-generation of therapy for FA patients. Disclosures Hartigan: Magenta Therapeutics: Employment. Palchaudhuri:Harvard University: Patents & Royalties; Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Kiem:Homology Medicine: Consultancy; Magenta: Consultancy; Rocket Pharmaceuticals: Consultancy.

scholarly journals Cellular and molecular characterization of the role of the flk-2/flt-3 receptor tyrosine kinase in hematopoietic stem cells

Blood ◽  
1994 ◽  
Vol 84 (8) ◽  
pp. 2422-2430 ◽  
Author(s):  
FC Zeigler ◽  
BD Bennett ◽  
CT Jordan ◽  
SD Spencer ◽  
S Baumhueter ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Tyrosine Kinase ◽  
Hematopoietic Stem Cell ◽  
Receptor Tyrosine Kinase ◽  
Lymphoid Cells ◽  
Stem Cell Population ◽  
Cell Populations ◽  
Hematopoietic Stem ◽  
Stem Cell Populations

The flk-2/flt-3 receptor tyrosine kinase was cloned from a hematopoietic stem cell population and is considered to play a potential role in the developmental fate of the stem cell. Using antibodies derived against the extracellular domain of the receptor, we show that stem cells from both murine fetal liver and bone marrow can express flk-2/flt-3. However, in both these tissues, there are stem cell populations that do not express the receptor. Cell cycle analysis shows that stem cells that do not express the receptor have a greater percentage of the population in G0 when compared with the flk-2/flt-3- positive population. Development of agonist antibodies to the receptor shows a proliferative role for the receptor in stem cell populations. Stimulation with an agonist antibody gives rise to an expansion of both myeloid and lymphoid cells and this effect is enhanced by the addition of kit ligand. These studies serve to further illustrate the importance of the flk-2/flt-3 receptor in the regulation of the hematopoietic stem cell.

scholarly journals Osteoblast ablation reduces normal long-term hematopoietic stem cell self-renewal but accelerates leukemia development

Blood ◽  
2015 ◽  
Vol 125 (17) ◽  
pp. 2678-2688 ◽  
Author(s):  
Marisa Bowers ◽  
Bin Zhang ◽  
Yinwei Ho ◽  
Puneet Agarwal ◽  
Ching-Cheng Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem Cell ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Key Points ◽  
Leukemia Development

Key Points Bone marrow OB ablation leads to reduced quiescence, long-term engraftment, and self-renewal capacity of hematopoietic stem cells. Significantly accelerated leukemia development and reduced survival are seen in transgenic BCR-ABL mice following OB ablation.

Mac-1 and F4/80 Surface Markers Are Present on Murine Hematopoietic Stem Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1677-1677
Author(s):  
Toska J. Zomorodian ◽  
Debbie Greer ◽  
Kyle Wood ◽  
Bethany Foster ◽  
Delia Demers ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Muscle Fibers ◽  
Stem Cell Marker ◽  
Stem Cell Markers ◽  
Surface Markers ◽  
Hematopoietic Stem ◽  
Cell Markers

Abstract Transplanted bone marrow donor cells with tissue specific phenotypes have been found in the brain, liver, heart, skin, lung, kidney, and gut of transplanted humans and mice. Such observations have led to the controversial hypothesis that hematopoietic stem cells (HSC) might be intrinsically plastic, and through transdifferentiation or fusion lead to the repair of damaged tissues throughout the body. Alternately, it is suggested that fusion of macrophages to the recipient cells may explain this phenomenon. We have shown recently that purified HSC are the cells responsible for GFP positive donor-derived muscle fibers in the recipient mice post bone marrow transplantation. However, further studies sorting for macrophage markers Mac-1 and F4/80 also resulted in donor-derived muscle fibers in the host. To address this discrepancy, we investigated subpopulations of Mac-1 and F4/80 positive cells, in the presence or absence of stem cell markers (Sca-1 and C-kit). We demonstrate that only the subpopulations of Mac-1 and F4/80 positive cells harboring stem cell markers, Sca-1 or c-kit, were capable of contributing to the regenerating muscle post transplantation. Furthermore, these same subpopulations demonstrated single cell High Proliferative Potential (HPP) (6–26%) in a 7 factor cytokine cocktail, compared to the Mac-1 or F4/80 cells with no stem cell markers (0%). Additionally, they demonstrated long-term engraftment in all three lineages at 1-year (average chimerism of 55% versus 0% in stem cell marker negative groups). These subpopulations were also evaluated for morphology using Hematoxylin/Eosin (H/E), Wright-Giemsa, and Nonspecific Esterase staining. In the Mac-1 and F4/80 positive groups, those negative for stem cell markers resembled differentiated cells of the myeloid origin (macrophages, granulocytes), while those with positive stem cell markers demonstrated stem cell characteristics. We did not observe any engraftability, donor-derived muscle fibers, or HPP potential for CD14 or cfms positive cells coexpressing stem cell markers, indicating that these markers are more appropriate for identifying macrophages. In conclusion, our studies demonstrate that both Mac-1 and F4/80 surface markers are present on HSC and therefore caution must be taken in the interpretation of data using these macrophage markers. It is reasonable to believe that the use of Mac-1 and/or F4/80 surface markers in a lineage depletion process may result in the loss of a subpopulation of stem cells, and other markers such as CD14 or c-fms may be more appropriate for eliminating differentiated macrophages.

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

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 861-861 ◽  
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.

Antibody-Based Depletion of Hematopoietic Stem Cells Empties Niches for Efficient Transplantation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. LB2-LB2
Author(s):  
Agnieszka Czechowicz ◽  
Daniel L. Kraft ◽  
Deepta Bhattacharya ◽  
Irving L. Weissman
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Conditioning Regimen ◽  
Myeloablative Conditioning ◽  
Hematopoietic Stem ◽  
Exact Function ◽  
Potential Applications ◽  
Conditioning Regimens

Abstract Hematopoietic stem cells (HSCs) are used therapeutically in bone marrow/hematopoietic stem cell transplantation (BMT/HSCT) to correct hematolymphoid abnormalities. Upon intravenous transplantation, HSCs can home to specialized bone marrow niches, self-renew and differentiate and thus generate a new, complete hematolymphoid system. Unfortunately BMT has had limited applications, due to the risks associated with the toxic conditioning regimens, such as irradiation and chemotherapy, that are deemed necessary for HSC engraftment. Elimination of these toxic conditioning regimens could expand the potential applications of BMT to include many non-malignant hematologic disorders, a wide variety of autoimmune disorders such as diabetes and multiple sclerosis, as well as in the facilitation of organ transplantation. The exact function of these traditional myeloablative conditioning regimens is not clear. To elucidate the barriers of HSC engraftment, we transplanted 50–1000 purified HSCs (Ckit+Lin−Sca1+CD34+CD150−) into immunodeficient, Rag2−/− or Rag2−/−gc−/− recipient mice and show that HSC engraftment levels rarely exceed 0.5% following transplantation without toxic conditioning, indicating that the immune system is not the only barrier to engraftment. Additionally, we did not observe a significant increase in HSC engraftment when HSC doses of >250 cells were transplanted. Even when up to 18000 HSC were transplanted, we did not see a linear increase in HSC engraftment, indicating that the increased doses of HSCs transplant inefficiently. We believe this is due to the naturally low frequency of available HSC niches, which we postulate may result from the physiologic migration of HSCs into circulation. Conversely, separation of the graft into small fractions and the subsequent time-delayed transplantation of these doses did result in increased engraftment due to the natural physiologic creation of new available HSC niches. When 1800 HSC were transplanted daily for seven days, the engraftment was 6.1-fold higher than transplantation of 12800 HSC in a single bolus. Here, we provide evidence that, aside from immune barriers, donor HSC engraftment is restricted by occupancy of appropriate niches by host HSCs. Through elimination of host HSCs we are able to increase available HSC niches for engraftment. We have developed a novel system where HSCs can be eliminated by targeting C-kit, a cell surface antigen that is highly expressed on the surface of HSCs. Cultivation of HSCs with ACK2, a depleting antibody specific for c-kit, prevented stem-cell factor (SCF) dependent HSC proliferation in vitro and resulted in cell death. Administration of ACK2 to mice led to the rapid and transient removal of >98% of endogenous HSCs in vivo thus resulting in equal numbers of available niches for engraftment. Following ACK2 clearance from serum, transplantation of these animals with donor HSCs led to chimerism levels of up to 90%, representing a 180-fold increase as compared to unconditioned animals. This non-myeloablative conditioning regimen had few side effects, other than temporary loss of coat color. The HSCs in even untransplanted animals rapidly recovered and animals remained healthy and fertile. This work redefines the way we approach BMT/HSCT, and places great emphasis on the necessity to create available HSC niches prior to transplantation. Extrapolation of these methods to humans may enable efficient yet mild conditioning regimens for transplantation, thus expanding the potential applications of BMT/HSCT.

Metabolic Regulation of Hematopoietic Stem Cells During Stress

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. SCI-42-SCI-42
Author(s):  
Toshio Suda
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Metabolic Regulation ◽  
Conflicts Of Interest ◽  
Hypoxia Inducible Factor ◽  
Ros Generation ◽  
Hematopoietic Stem

Abstract Abstract SCI-42 Tissue homeostasis over the life of an organism relies on both self-renewal and multipotent differentiation of stem cells. Hematopoietic stem cells (HSCs) are sustained in a specific microenvironment known as the stem cell niche. Adult HSCs are kept quiescent during the cell cycle in the endosteal niche of the bone marrow. Normal HSCs maintain intracellular hypoxia, stabilize the hypoxia-inducible factor-1a (HIF-1a) protein, and generate ATP by anaerobic metabolism. In HIF-1a deficiency, HSCs became metabolically aerobic, lost cell cycle quiescence, and finally became exhausted. An increased dose of HIF-1a protein in VHL-mutated HSCs and their progenitors induced cell cycle quiescence and accumulation of HSCs in the bone marrow (BM), which were not transplantable. This metabolic balance promotes HSC maintenance by limiting the production of reactive oxygen species (ROS), but leaves HSCs susceptible to changes in redox status (1). We have performed the metabolomic analysis in HSCs. Upregulation of pyruvate dehydrogenase kinases enhanced the glycolytic pathway, cell cycle quiescence, and stem cell capacity. Thus, HSCs directly utilize the hypoxic microenvironment to maintain their slow cell cycle by HIF-1a-dependent metabolism. Downregulation of mitochondrial metabolism might be reasonable, since it reduces ROS generation. On the other hand, at the time of BM transplantation, HSCs activate oxidative phosphorylation to acquire more ATP for proliferation. Autophagy also energizes HSCs by providing amino acids during transplantation. ATG (autophagy-related) 7 is essential for transplantation and metabolic homeostasis. The relationship between mitochondrial heat shock protein, mortalin, and metabolism in HSCs will also be discussed. Disclosures: No relevant conflicts of interest to declare.

The Zinc Finger Transcription Factor Growth Factor Independence 1b (Gfi1b) Regulates The Wnt/Beta-Catenin Signaling Pathway In Hematopoietic Stem Cells Through Interaction With Inhibitory Proteins

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2417-2417
Author(s):  
Peiman Shooshtarizadeh ◽  
Ryan Chen ◽  
Tarik Moroy
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Amino Acid ◽  
Hematopoietic Stem Cells ◽  
Signaling Pathway ◽  
Beta Catenin ◽  
Hematopoietic Stem ◽  
Endosteal Niche ◽  
Canonical Wnt

Abstract Hematopoietic stem cells (HSCs) reside in the bone marrow in specific niches at the border between bone cells and the bone marrow (endosteal niche) or around blood vessels (perivascular niche). In the endosteal niche, HSCs are maintained at low oxygen levels in a quiescent (dormant) state by adhesion to niche cells. We have previously shown that Gfi1b restricts the expansion and proliferation of HSCs as well as their mobilization or re-localization into peripheral blood. We have proposed that Gfi1b exerts this function by regulating the expression of surface molecules such as integrins on HSCs that are required to maintain them in their bone marrow niche at a quiescent state. The objective of this study was to gain more insight into the precise molecular mechanisms by which Gfi1b regulates HSCs dormancy and mobilization and to obtain insights that may be exploited in the future to improve stem cell therapies or the expansion of human hematopoietic stem cells for clinical use. Immune precipitation and mass spectrometry identified a series of Gfi1b interacting proteins, most notably a group of regulators of the canonical Wnt/beta-catenin pathway. Independent protein IP validation of these findings suggested that Gfi1b can interact with several inhibitors of the canonical Wnt/beta catenin pathway namely with APC (Adenomatous polyposis coli) a tumor suppressor protein and important factor in the beta-catenin destruction complex, with the DNA helicase and chromatin remodeling factor CHD8, which silences beta catenin mediated transcription, with CtBP which antagonizes beta-catenin activity and is part of the LSD1/CoRest histone demethylase complex and with the direct beta-catenin inhibitors TLE1 and TLE3 (also called Groucho). Of particular interest was that the interactions between the Groucho proteins and Gfi1b were dependent on a previously unidentified Groucho binding domain (GBD) in Gfi1b. This is a well-conserved six-amino acid stretch that is found in the middle part of the Gfi1b protein. In addition, the binding of CtBP was dependent on the presence of the 20 amino acid N-terminal SNAG domain in Gfi1b that also mediates LSD1 binding. Using luciferase reporter gene assays (TOP/FOP reporter assay), we found that Gfi1b was able to significantly up-regulates TCF/beta-catenin-dependent transcription upon activation by LiCl or Wnt3A in HEK293 cells. This activity of Gfi1b was dependent on both the presence of the SNAG domain and the newly identified Groucho binding domain. Also, Gfi1b was able to reverse partially the inhibitory effect of CtBP and TLE3 on beta-catenin activity in the TOP/FOP reporter assays. To obtain further evidence that Gfi1b is indeed implicated in regulating the Wnt/beta catenin signaling pathway in hematopoietic stem cells, we FACS sorted Lin-Kit1+Sca+ hematopoietic progenitors (LSK cells) from wt and Gfi1b deficient mice and tested them for expression of Wnt effector genes using a Wnt signaling specific PCR array. We observed that the majority of Wnt target genes were significantly down regulated in Gfi1 deficient LSKs compared to wt LSKs. Among the genes affected the most were typical Wnt targets such as Axin2, Frz7, Tcf4, Klf5, Vegfa and Ccnd1. To show that Gfi1b is able to regulate Wnt pathway effectors in vivo in HSCs, we crossed Gfi1b flox/flox, Mx-Cre mice with animals that carry a NLS-lacZ reporter gene under the control of the endogenous Axin2 promoter/enhancer region. Treatment with pIpC, which deletes Gfi1b correlated with a significant decrease of Axin2 expression in HSCs and MPP1, which are high Gfi1b expressing cells. The Axin2 reporter was not affected by Gfi1b deletion in MPP2 or GMPs, which express low levels or no Gfi1b. The canonical Wnt/b-catenin signaling pathway is recognized as one of the elements that are critically important in the regulation of HSC function. Here we have identified Gfi1b as a potential new player in the Wnt-beta catenin signaling pathway. Our data suggest that Gfi1b acts on at least two inhibitory complexes of this pathway, on the TLE family of Groucho proteins and the CtBP/LSD1 complex and regulates effectors of the Wnt/beta-catenin signaling cascade. We propose therefore that Gfi1b may titer the level of activation of the Wnt/beta-catenin signaling pathway in HSCs, which offers an explanation of the hematopoietic stem cell phenotype seen in mice lacking Gfi1b. Disclosures: No relevant conflicts of interest to declare.

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