ORCID Profile
0000-0002-7926-7335
Current Organisation
CSIRO
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Research Topics
In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Top 5 Research Topics
ANZSRC Field of Research (FoR)
Pharmacology and Pharmaceutical Sciences | Haematology | Characterisation of Biological Macromolecules | Organic Chemistry | Natural Products Chemistry | Biological And Medical Chemistry | Biomolecular Modelling and Design | Organic Chemical Synthesis | Pharmaceutical Sciences | Medical Biochemistry and Metabolomics not elsewhere classified | Cardiorespiratory Medicine and Haematology | Medical Biochemistry and Metabolomics | Cellular Interactions (Incl. Adhesion, Matrix, Cell Wall) | Central Nervous System | Regenerative Medicine (incl. Stem Cells and Tissue Engineering) | Nanobiotechnology
ANZSRC Socio-Economic Objective (SEO)
Plant Production and Plant Primary Products not elsewhere classified | Nervous System and Disorders | Blood disorders | Cancer and related disorders | Other | Human Pharmaceutical Treatments (e.g. Antibiotics) | Cardiovascular System and Diseases | Expanding Knowledge in the Chemical Sciences | Blood Disorders | Health not elsewhere classified |
Related Links
Publications
The aging hematopoietic stem cell niche
Publisher: Elsevier
Date: 2020
Gene expression, synthesis and degradation of hyaluronan during differentiation of 3T3-L1 adipocytes
Publisher: Elsevier BV
Date: 08-2006
DOI: 10.1016/J.ABB.2006.05.010
Abstract: The high molecular weight glycosaminoglycan hyaluronan (HA) is an essential component of the extracellular matrix (ECM), however, the link between HA regulation and development of the adipocyte ECM, which is essential for differentiation, remains undefined. Hyaluronan synthase gene expression, HA synthetic rate and molecular weight during differentiation of 3T3-L1 pre-adipocytes were compared to undifferentiated 3T3-L1 pre-adipocytes and non-adipogenic NIH/3T3 fibroblasts. In the 3T3-L1 pre-adipocytes, the predominant genes associated with HA metabolism were found to be HA synthase-2 (Has-2) and hyaluronidase-2 (Hyal-2) demonstrating a co-regulation of expression which was stimulated by adipogenic induction consequently resulting in increased synthesis of high molecular weight HA (>10 MDa) and its simultaneous degradation. Accumulation of HA correlated positively with cell number, although synthetic rate was inversely related suggesting a regulatory feedback mechanism. Within 24h post-induction, pre-adipocytes responded with a higher HA synthetic rate and later, accumulated cytoplasmic lipid. In contrast, undifferentiated pre-adipocytes had a reduced HA synthetic rate during clonal expansion and did not accumulate lipid. HA was continuously and rapidly metabolised throughout 3T3-L1 adipogenesis, where terminal differentiation coincided with the increased generation of low molecular weight, angiogenic HA fragments, a likely prerequisite for concurrent neovascularisation of adipose tissue. This study has highlighted a relationship between HA metabolism and adipocyte differentiation, suggesting that the balance between the formation and regulation of the adipocyte extracellular matrix is finely coordinated in a growth phase-specific dependent manner.
FOXN1GFP/w reporter hESCs enable identification of integrin-a4, HLA-DR, and EpCAM as markers of human PSC-derived FOXN1 + thymic epithelial progenitors
Publisher: Elsevier BV
Date: 06-2014
Cells Capable of Bone Production Engraft from Whole Bone Marrow Transplants in Nonablated Mice
Publisher: Rockefeller University Press
Date: 15-02-1999
Abstract: Allogeneic and autologous marrow transplants are routinely used to correct a wide variety of diseases. In addition, autologous marrow transplants potentially provide opportune means of delivering genes in transfected, engrafting stem cells. However, relatively little is known about the mechanisms of engraftment in transplant recipients, especially in the nonablated setting and with regard to cells not of hemopoietic origin. In particular, this includes stromal cells and progenitors of the osteoblastic lineage. We have demonstrated for the first time that a whole bone marrow transplant contains cells that engraft and become competent osteoblasts capable of producing bone matrix. This was done at the in idual cell level in situ, with significant numbers of donor cells being detected by fluorescence in situ hybridization in whole femoral sections. Engrafted cells were functionally active as osteoblasts producing bone before being encapsulated within the bone lacunae and terminally differentiating into osteocytes. Transplanted cells were also detected as flattened bone lining cells on the periosteal bone surface.
Validating Eaton's Hypothesis: Cubane as a Benzene Bioisostere
Publisher: Wiley
Date: 05-02-2016
The identification and characterisation of transplanted hematopoietic stem cells in situ
Publisher: Oxford University Press (OUP)
Date: 08-2005
The development and establishment of hemopoiesis in fetal and newborn osteopetrotic (op/op) mice
Publisher: Elsevier BV
Date: 08-1994
Abstract: Previous studies of young CSF-1-less osteopetrotic (op/op) mice demonstrate a severe deficiency of both macrophages and osteoclasts, resulting in excessive bone formation, occlusion of the marrow cavity, and reduced hemopoietic activity. The accompanying splenomegaly and prolonged splenic hemopoiesis observed in these mice suggests that osteopetrosis may perturb the normal progression of fetal hemopoietic development and obstruct the seeding of hemopoietic precursors into the bone marrow. This study demonstrates that the absence of CSF-1 does not affect the progression of hemopoietic development in fetal op/op mice until after colonization of the bone marrow. Significant deficiencies in marrow cellularity and progenitor cell content in the long bones of op/op mice were not evident prior to Day 2 postnatal, suggesting that the altered hemopoietic state of young op/op mice is not a consequence of abnormal fetal hemopoietic development, but is primarily due to the lack of functional osteoclasts in op/op fetuses and hence, impaired remodeling of the marrow cavity after birth.
Stem cell engraftment and cell cycle phenotype
Publisher: Springer Science and Business Media LLC
Date: 04-1999
Hematopoietic Stem Cell Protocols
Publisher: Springer New York
Date: 2014
Reprogramming roadmap reveals route to human induced trophoblast stem cells
Publisher: Springer Science and Business Media LLC
Date: 16-09-2020
DOI: 10.1038/S41586-020-2734-6
Abstract: The reprogramming of human somatic cells to primed or naive induced pluripotent stem cells recapitulates the stages of early embryonic development
Primate-specific response of astrocytes to stroke limits peripheral macrophage infiltration
Publisher: Cold Spring Harbor Laboratory
Date: 10-05-2020
DOI: 10.1101/2020.05.08.083501
Abstract: Reactive astrocytes play critical roles after brain injuries but their precise function in stroke is not well defined. Here, we utilized single nuclei transcriptomics to characterize astrocytes after ischemic stroke in nonhuman primate (NHP) marmoset monkey primary visual cortex. We identified 19 putative subtypes of astrocytes from injured and uninjured brain hemispheres and observed nearly complete segregation between stroke and control astrocyte clusters. We then screened for genes that might be limiting stroke recovery and discovered that one neurite-outgrowth inhibitory protein, NogoA, previously associated with oligodendrocytes but not astrocytes, was expressed in numerous reactive astrocyte subtypes. NogoA upregulation on reactive astrocytes was confirmed in vivo for NHP and human, but not observed to the same extent in rodent. Further in vivo and in vitro studies determined that NogoA mediated an anti-inflammatory response which limits deeper infiltration of peripheral macrophages from the lesion during the subacute post-stroke period. Specifically, these findings are relevant to the development of NogoA-targeting therapies shortly after ischemic stroke. Our findings have uncovered the complexity and species specificity of astrocyte responses, which need to be considered more when investigating novel therapeutics for brain injury.
Osteopoietic stem cells: transplantable, but regeneratively limited
Publisher: American Society of Hematology
Date: 15-04-2008
Potent agonists of a hematopoietic stem cell cytokine receptor, c-Mpl
Publisher: Wiley
Date: 28-03-2013
Abstract: Several growth factors feature prominently in the control of hematopoiesis. Thrombopoietin, a class I hematopoietic cytokine, plays critical roles in regulating hematopoietic stem cell numbers and also stimulates the production and differentiation of megakaryocytes, the bone marrow cells that ultimately produce platelets. Thrombopoietin interacts with the c-Mpl cell-surface receptor. Recently, several peptide and small-molecule agonists and antagonists of c-Mpl have been reported. We conducted a bioinformatics and molecular modeling study aimed at understanding the agonist activities of peptides that bind to c-Mpl, and developed new potent peptide agonists with low nanomolar activity. These agonists also show very high activity in human CD34(+) primary cell cultures, and doubled the mean blood platelet counts when injected into mice.
Online Learners’ Reading Ability Detection Based on Eye-Tracking Sensors
Publisher: MDPI AG
Date: 10-09-2016
DOI: 10.3390/S16091457
The RNA-binding protein SRSF3 has an essential role in megakaryocyte maturation and platelet production
Publisher: American Society of Hematology
Date: 03-03-2022
Abstract: RNA processing is increasingly recognized as a critical control point in the regulation of different hematopoietic lineages including megakaryocytes responsible for the production of platelets. Platelets are anucleate cytoplasts that contain a rich repertoire of RNAs encoding proteins with essential platelet functions derived from the parent megakaryocyte. It is largely unknown how RNA binding proteins contribute to the development and functions of megakaryocytes and platelets. We show that serine-arginine–rich splicing factor 3 (SRSF3) is essential for megakaryocyte maturation and generation of functional platelets. Megakaryocyte-specific deletion of Srsf3 in mice led to macrothrombocytopenia characterized by megakaryocyte maturation arrest, dramatically reduced platelet counts, and abnormally large functionally compromised platelets. SRSF3 deficient megakaryocytes failed to reprogram their transcriptome during maturation and to load platelets with RNAs required for normal platelet function. SRSF3 depletion led to nuclear accumulation of megakaryocyte mRNAs, demonstrating that SRSF3 deploys similar RNA regulatory mechanisms in megakaryocytes as in other cell types. Our study further suggests that SRSF3 plays a role in sorting cytoplasmic megakaryocyte RNAs into platelets and demonstrates how SRSF3-mediated RNA processing forms a central part of megakaryocyte gene regulation. Understanding SRSF3 functions in megakaryocytes and platelets provides key insights into normal thrombopoiesis and platelet pathologies as SRSF3 RNA targets in megakaryocytes are associated with platelet diseases.
Dextran sulfate-amplified extracellular matrix deposition promotes osteogenic differentiation of mesenchymal stem cells
Publisher: Elsevier BV
Date: 03-2022
DOI: 10.1016/J.ACTBIO.2021.11.049
Abstract: The development of bone-like tissues in vitro that exhibit key features similar to those in vivo is needed to produce tissue models for drug screening and the study of bone physiology and disease pathogenesis. Extracellular matrix (ECM) is a predominant component of bone in vivo however, as ECM assembly is sub-optimal in vitro, current bone tissue engineering approaches are limited by an imbalance in ECM-to-cell ratio. We lified the deposition of osteoblastic ECM by supplementing dextran sulfate (DxS) into osteogenically induced cultures of human mesenchymal stem cells (MSCs). DxS, previously implicated to act as a macromolecular crowder, was recently demonstrated to aggregate and co-precipitate major ECM components, including collagen type I, thereby lifying its deposition. This effect was re-confirmed for MSC cultures undergoing osteogenic induction, where DxS supplementation augmented collagen type I deposition, accompanied by extracellular osteocalcin accumulation. The resulting differentiated osteoblasts exhibited a more mature osteogenic gene expression profile, indicated by a strong upregulation of the intermediate and late osteogenic markers ALP and OCN, respectively. The associated cellular microenvironment was also enriched in bone morphogenetic protein 2 (BMP-2). Interestingly, the resulting decellularized matrices exhibited the strongest osteo-inductive effects on re-seeded MSCs, promoted cell proliferation, osteogenic marker expression and ECM calcification. Taken together, these findings suggest that DxS-mediated enhancement of osteogenic differentiation by MSCs is mediated by the lified ECM, which is enriched in osteo-inductive factors. We have thus established a simple and reproducible approach to generate ECM-rich bone-like tissue in vitro with sequestration of osteo-inductive factors. STATEMENT OF SIGNIFICANCE: As extracellular matrix (ECM) assembly is significantly retarded in vitro, the imbalance in ECM-to-cell ratio h ers current in vitro bone tissue engineering approaches in their ability to faithfully resemble their in vivo counterpart. We addressed this limitation by leveraging a poly-electrolyte mediated co-assembly and lified deposition of ECM during osteogenic differentiation of human mesenchymal stem cells (MSCs). The resulting pericelluar space in culture was enriched in organic and inorganic bone ECM components, as well as osteo-inductive factors, which promoted the differentiation of MSCs towards a more mature osteoblastic phenotype. These findings thus demonstrated a simple and reproducible approach to generate ECM-rich bone-like tissue in vitro with a closer recapitulation of the in vivo tissue niche.
New agents in HSC mobilization
Publisher: Springer Science and Business Media LLC
Date: 30-11-2017
DOI: 10.1007/S12185-016-2156-2
Abstract: Mobilized peripheral blood (PB) is the most common source of hematopoietic stem cells (HSC) for autologous transplantation. Granulocyte colony stimulating factor (G-CSF) is the most commonly used mobilization agent, yet despite its widespread use, a considerable number of patients still fail to mobilize. Recently, a greater understanding of the interactions that regulate HSC homeostasis in the bone marrow (BM) microenvironment has enabled the development of new molecules that mobilize HSC through specific inhibition, modulation or perturbation of these interactions. AMD3100 (plerixafor), a small molecule that selectively inhibits the chemokine receptor CXCR4 is approved for mobilization in combination with G-CSF in patients with Non-Hodgkin's lymphoma and multiple myeloma. Nevertheless, identifying mobilization strategies that not only enhance HSC number, but are rapid and generate an optimal "mobilized product" for improved transplant outcomes remains an area of clinical importance. In recent times, new agents based on recombinant proteins, peptides and small molecules have been identified as potential candidates for therapeutic HSC mobilization. In this review, we describe the most recent developments in HSC mobilization agents and their potential impact in HSC transplantation.
Smad4 promotes diabetic nephropathy by modulating glycolysis and OXPHOS
Publisher: EMBO
Date: 09-01-2020
Frontispiece: Validating Eaton's Hypothesis: Cubane as a Benzene Bioisostere
Publisher: Wiley
Date: 29-02-2016
An innovative triple immunogold labeling method to investigate the hemopoietic stem cell niche in situ.
Publisher: Oxford University Press (OUP)
Date: 16-09-2009
DOI: 10.1017/S1431927609990924
Abstract: The ultrastructural study of rare cells within their niche in situ is very difficult. We have developed a method for locating in idual transplanted cells and simultaneously identifying and analyzing the molecules and cellular phenotypes surrounding them in situ using transmission electron microscopy. This innovative method involves triple immunogold labeling combined with serial ultrathin sectioning. We demonstrate the validity of this approach by examining the niche of in idual transplanted cells from a population highly enriched for hemopoietic stem cells and the ultrastructural expression of two key stem cell regulatory molecules, hyaluronic acid and osteopontin. In addition, we describe the phenotypes of the surrounding cells.
The over-expression of HAS2, Hyal-2 and CD44 is implicated in the invasiveness of breast cancer
Publisher: Elsevier BV
Date: 10-2005
DOI: 10.1016/J.YEXCR.2005.07.026
Abstract: Within tumors there appears to be an intricate balance between hyaluronan (HA) synthesis and degradation where the invading edges display increased HA metabolism. The metabolism of HA has not been characterized in breast cancer cell lines therefore, this study quantitatively identifies and characterizes the enzymes responsible for the synthesis and degradation of HA while correlating gene expression to cancer cell invasiveness and HA receptor status. In ten well-established breast cancer cell lines, the expression of the genes for each hyaluronan synthase (HAS) and hyaluronidase (Hyal) isoform was quantitated using real-time and reverse transcriptase polymerase chain reaction (PCR). The synthesis and degradation rates of hyaluronan were determined by ELISA, while quantitation of HA receptors, CD44 and RHAMM was performed by comparative Western blotting. The molecular weight of HA synthesized by each HAS isoform and the degradation products of each hyaluronidase were characterized by size exclusion chromatography. It was demonstrated that highly invasive cell lines preferentially expressed the HAS2 and Hyal-2 isoforms, while less invasive cells expressed HAS3 and Hyal-3. There was a correlation between elevated levels of HA synthesis, CD44 expression and cancer cell migration thereby highlighting the pivotal role that HA metabolism plays in the aggressive breast cancer phenotype.
The role of CD44 in fetal and adult hematopoietic stem cell regulation
Publisher: Ferrata Storti Foundation (Haematologica)
Date: 06-11-2016
Hemopoietic Stem Cells with Higher Hemopoietic Potential Reside at the Bone Marrow Endosteum
Publisher: Oxford University Press (OUP)
Date: 04-2007
DOI: 10.1634/STEMCELLS.2006-0528
Abstract: It is now evident that hemopoietic stem cells (HSC) are located in close proximity to bone lining cells within the endosteum. Accordingly, it is unlikely that the traditional method for harvesting bone marrow (BM) from mice by simply flushing long bones would result in optimal recovery of HSC. With this in mind, we have developed improved methodologies based on sequential grinding and enzymatic digestion of murine bone tissue to harvest higher numbers of BM cells and HSC from the endosteal and central marrow regions. This methodology resulted in up to a sixfold greater recovery of primitive hemopoietic cells (lineage−Sca+Kit+ [LSK] cells) and HSC as shown by transplant studies. HSC from different anatomical regions of the marrow exhibited important functional differences. Compared with their central marrow counterparts, HSC isolated from the endosteal region (a) had 1.8-fold greater proliferative potential, (b) exhibited almost twofold greater ability to home to the BM following tail vein injection and to lodge in the endosteal region, and (c) demonstrated significantly greater long-term hemopoietic reconstitution potential as shown using limiting dilution competitive transplant assays. Disclosure of potential conflicts of interest is found at the end of this article.
The role of Tenascin C in the lymphoid progenitor cell niche.
Publisher: Elsevier BV
Date: 12-2013
DOI: 10.1016/J.EXPHEM.2013.09.009
Abstract: Hemopoietic stem cells (HSCs) are extrinsically controlled by the bone marrow (BM) microenvironment. Mice devoid of the extracellular matrix molecule Tenascin-C (TNC) were reported to develop normally. The current study explores the relationship between TNC and hemopoiesis, from HSCs within their niche to maturing progenitors in alternate niches. Although the absence of TNC did not alter the size of the BM stem cell pool, we report decreased thymic T cell progenitors with redistribution to other lymphoid organs, suggesting an anchoring role for TNC. TNC did not play an essential role in stem and progenitor cell homing to BM, but significantly altered lymphoid primed progenitor cell homing. These cells express the TNC receptor, integrin α9β1, with the same reduced homing evident in the absence of this integrin. The absence of TNC also resulted in an increased proportion and number of mature circulating T cells. In addition, the absence of TNC significantly impaired hemopoietic reconstitution after transplant and increased stem and progenitor cell mobilization. In summary, our analysis revealed unidentified roles for TNC in hemopoiesis: in lineage commitment of thymic T cell progenitors, peripheral T cell migration, and hemopoietic reconstitution.
Periwound dopaminergic sprouting is dependent on numbers of wound macrophages
Publisher: Wiley
Date: 03-2002
DOI: 10.1046/J.1460-9568.2002.01914.X
Abstract: Injury to many regions of the central nervous system, including the striatum, results in a periwound or 'abortive' sprouting response. In order to directly evaluate whether macrophages play an important role in stimulating periwound sprouting, osteopetrotic (op/op) mice, which when young are deficient in a variety of macrophage subtypes, were given striatal wounds and the degree of dopaminergic sprouting subsequently assessed. Two weeks postinjury, significantly fewer wound macrophages were present in the striata of op/op mice compared with controls (144 +/- 30.1 in op/op mice vs. 416.6 +/- 82.3 in controls, P < 0.005, analysis performed on a section transecting the middle of the wound). Dopamine transporter immunohistochemistry revealed a marked decrease in the intensity of periwound sprouting in the op/op group of animals. Quantification of this effect using [H3]-mazindol autoradiography confirmed that periwound sprouting was reduced significantly in the op/op mice compared with controls (71.4 +/- 21.7 fmol/mg protein in op/op mice vs. 210.7 +/- 27.1 fmol/mg protein in controls, P < 0.0005). In the two groups of animals the magnitude of the sprouting response in in iduals was closely correlated with the number of wound macrophages (R = 0.83, R2 = 0.69). Our findings provide strong support for the crucial involvement of macrophages in inducing dopaminergic sprouting after striatal injury.
Vascular cell adhesion molecule-1 (CD106) is cleaved by neutrophil proteases in the bone marrow following hematopoietic progenitor cell mobilization by granulocyte colony-stimulating factor
Publisher: American Society of Hematology
Date: 09-2001
Abstract: Mobilized progenitor cells currently represent the most commonly used source of hematopoietic progenitor cells (HPCs) to effect hematopoietic reconstitution following myeloablative chemotherapies. Despite their widespread use, the molecular mechanisms responsible for the enforced egress of HPCs from the bone marrow (BM) into the circulation in response to mobilizing agents such as cytokines remain to be determined. Results of this study indicate that expression of vascular cell adhesion molecule-1 (VCAM-1) is strongly reduced in vivo in the BM during HPC mobilization by granulocyte colony-stimulating factor (G-CSF) and stem cell factor. Two serine proteases, namely, neutrophil elastase and cathepsin G, were identified, which cleave VCAM-1 and are released by neutrophils accumulating in the BM during the course of immobilization induced by G-CSF. The proposal is made that an essential step contributing to the mobilization of HPCs is the proteolytic cleavage of VCAM-1 expressed by BM stromal cells, an event triggered by the degranulation of neutrophils accumulating in the BM in response to the administration of G-CSF.
In memoriam: T. Ray Bradley (1923–2013)
Publisher: Elsevier BV
Date: 10-2013
Brief report: Factors released by megakaryocytes thrombin cleave osteopontin to negatively regulate hematopoietic stem cells
Publisher: Oxford University Press (OUP)
Date: 15-05-2015
DOI: 10.1002/STEM.2038
Abstract: Factor V (FV) and factor X (FX) activate and complex to form prothrombinase which subsequently cleaves prothrombin (PT), converting it to active thrombin. Thrombin cleaved osteopontin (tcOPN) contains a cryptic binding site for α4β1 and α9β1 integrins. We have previously shown that hematopoietic stem cells (HSC) bind to tcOPN via this site resulting in a decrease in their proliferation and differentiation. Therefore, tcOPN and the factors required for its generation are important components of the HSC niche. Herein we show mature megakaryocytes (MM, ≥8N) contain FV, FX, and PT mRNA and protein. Furthermore, we show 8N, 16N, 32N, and 64N MM all release the required factors to enable thrombin cleavage of OPN. Importantly, mice devoid of the myeloproliferative leukemia protein (Mpl), c-Mpl−/− mice, contain only approximately 10% of normal megakaryocyte numbers, showed significantly reduced FX and tcOPN protein levels in endosteal bone marrow (BM). In addition, WT hematopoietic progenitors and HSC showed reduced homing to the BM of c-Mpl−/− mice. This is the first report identifying MM as a key cellular component in the production of tcOPN in situ, allowing the BM microenvironment to self regulate HSC biology via tcOPN. Stem Cells 2015 :2351–2357
Combined Blockade of Smad3 and JNK Pathways Ameliorates Progressive Fibrosis in Folic Acid Nephropathy
Publisher: Frontiers Media SA
Date: 09-08-2019
Dental Abnormalities Associated with Failure of Tooth Eruption in src Knockout and op/op Mice
Publisher: Springer Science and Business Media LLC
Date: 07-1999
Abstract: c-src knockout and op/op mice develop osteopetrosis as a result of defective osteoclast function and osteoclast formation, respectively. The mutant mice can be distinguished readily from their wild-type littermates around 10-12 days after birth because their incisors do not erupt, but the morphology of their teeth and surrounding bone has not been reported previously in detail. Histologic examination of jaws of src-mutant mice reveals unerupted, abnormal incisors within their bony crypts. The tooth roots are distorted by foci of haphazard proliferation of odontogenic epithelium associated with primitive tooth structures that strongly resemble the tumor-like lesions in humans, known as odontomas. The crowns of the incisors are fused to the adjacent bone, and the developing periodontal ligament is disordered and hypocellular. Osteoclasts are present in the bone surrounding the distorted teeth, but as in other bones in these mice they lack ruffled borders and thus do not resorb effectively. Similar odontogenic proliferation is present around unerupted incisors in op/op mice which form very few osteoclasts, but the amount is significantly less than in src mutant mice. Molars fail to erupt in both types of mutant mice, but they are not accompanied by aberrant odontogenic proliferation. These findings and previous reports of similar abnormalities in jaws from op/op rats suggest that failure of incisor eruption and associated proliferation of odontogenic epithelium in osteopetrotic rodents are a direct result of defective osteoclastic bone resorption.
Factors that influence a mobilized HSC product
Publisher: Springer Science and Business Media LLC
Date: 24-12-2016
Osteoblast-specific gene expression after transplantation of marrow cells: Implications for skeletal gene therapy
Publisher: Proceedings of the National Academy of Sciences
Date: 22-06-1999
Abstract: Somatic gene therapies require targeted transfer of the therapeutic gene(s) into stem cells that proliferate and then differentiate and express the gene in a tissue-restricted manner. We have developed an approach for gene therapy using marrow cells that takes advantage of the osteoblast specificity of the osteocalcin promoter to confine expression of chimeric genes to bone. Adherent marrow cells, carrying a reporter gene [chlor henicol acetyltransferase (CAT)] under the control of a 1.7-kilobase rat osteocalcin gene promoter, were expanded ex vivo . After transplantation by intravenous infusion, engrafted donor cells in recipient mice were detected by the presence of the transgene in a broad spectrum of tissues. However, expression of the transgene was restricted to osteoblasts and osteocytes, as established by biochemical analysis of CAT activity and immunohistochemical analysis of CAT expression at the single cell level. Our data indicate that donor cells achieved long-term engraftment in various tissues of the recipients and that the CAT gene under control of the osteocalcin promoter is expressed specifically in bone. Thus, transplantation of multipotential marrow cells containing the osteocalcin promoter-controlled transgene provides an efficacious approach to deliver therapeutic gene expression to osteoblasts for treatment of bone disorders or tumor metastasis to the skeleton.
The role of hyaluronic acid in hemopoietic stem cell biology
Publisher: Future Medicine Ltd
Date: 07-2006
Abstract: The adult mammalian hemopoietic system maintains an extraordinarily large, yet well regulated supply of mature blood cells within the circulation throughout life. The system is capable of rapid recovery and compensation following injury, environmental stress or as a result of genetic disease such as the hemoglobinopathies. Despite the vast amount of research conducted there is still an incomplete understanding of hemopoietic regulation. Nevertheless, it is evident from transplantation studies that ongoing blood cell production is absolutely dependent upon hemopoietic stem cells (HSCs). These rare and potent cells have the capacity for extensive proliferation and the ability to differentiate into all blood cell types. An understanding of HSC regulation is fundamental to understanding hemopoiesis. There is now considerable evidence to demonstrate that in vivo, HSCs are located within defined anatomical sites or niches within the bone marrow. Regulation of HSC fate is mediated by both cell-autonomous mechanisms and extrinsic cues resulting from interactions between cells and extracellular components within the niche. This review focuses on the role of hyaluronic acid, a component of the HSC niche and moreover a HSC-associated glycosaminoglycan, in hemopoiesis and specifically HSC regulation. It is now evident that hyaluronic acid not only provides a physical scaffold or support within the marrow to facilitate localization and retention of HSCs to the stem cell niche but moreover, through ligation with its counter-receptors is able to directly affect the cellular functions of HSCs.
Homing of Purified Murine Lymphohematopoietic Stem Cells: A Cytokine-Induced Defect
Publisher: Mary Ann Liebert Inc
Date: 12-2002
DOI: 10.1089/152581602321080574
Abstract: This study was designed to establish a direct homing assay using purified lineage-negative Sca-1-positive (Lin(-) Sca(+)) murine bone marrow cells and to evaluate the effects of cytokines on homing. C57BL/6 Lin(-) Sca(+) marrow stem cells were labeled with 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) and then injected by tail vein into untreated C57BL/6 mice. Marrow was harvested at various times after cell infusion and analyzed on a high-speed MoFlo cell sorter for fluorescent positive events, using a large event analysis, with at least 16 million total events analyzed. We have shown that homing of Lin(-) Sca(+) cells plateaus by 1 h, and at 3 h post-infusion is linear between 50,000 and 1,000,000 infused cells. This forms a base for a homing assay in which 250,000 CFDA-SE labeled Lin(-) Sca(+) marrow cells are infused and then recovered from marrow 3 h later, followed by a large-event fluorescence-activated cell sorting (FACS) analysis. We found that 7.45-9.32% of infused cells homed and that homing of stem cells cultured for 48 h in interleukin-3 (IL-3), IL-6, IL-11, and steel factor cultured cells was defective when compared to noncultured cells. Exposure of marrow stem cells to IL-3, IL-6, IL-11, and steel factor induces a stem cell homing defect, which probably underlies the engraftment defect previously characterized under these conditions.
DELAYED HEMATOPOIETIC DEVELOPMENT IN OSTEOPETROTIC (OP/OP) MICE
Publisher: Rockefeller University Press
Date: 1993
Abstract: Changes in structure, cellularity, hematopoietic progenitor cell and macrophage content, and osteoclast activity were investigated in the hematopoietic organs of the colony-stimulating factor 1(CSF-1)-less osteopetrotic (op/op) mouse. The data indicated that op/op mice undergo an age-related hematopoietic recovery and resolution of osteopetrosis, suggesting that the hematopoietic system has the capacity to use alternative mechanisms to compensate for the absence of an important multifunctional growth factor, CSF-1. In young animals, op/op femurs were heavily infiltrated with bone, and marrow cellularity was significantly reduced. After 6 wk of age, there was an increase in the marrow space available for hematopoiesis. The femoral cavity of op/op mice progressively enlarged, and by 22 wk of age its appearance and marrow cellularity was comparable to that of controls. The percentage of op/op mononuclear phagocytes, defined by F4/80 antigen expression, progressively increased to normal levels by 35 wk of age. There was no difference in the incidence of both primitive and mononuclear phagocyte-committed, CSF-1-responsive progenitor cells in op/op marrow, but their femoral content was significantly reduced in young mice. During the period of reduced hematopoiesis in the marrow of young op/op mice, splenic hematopoietic activity was elevated. This mutant mouse represents a system for the study of the CSF-1-independent regulatory mechanisms involved in hematopoietic regulation.
Nicotinamide riboside attenuates age-associated metabolic and functional changes in hematopoietic stem cells.
Publisher: Springer Science and Business Media LLC
Date: 11-05-2021
DOI: 10.1038/S41467-021-22863-0
Abstract: With age, hematopoietic stem cells (HSC) undergo changes in function, including reduced regenerative potential and loss of quiescence, which is accompanied by a significant expansion of the stem cell pool that can lead to haematological disorders. Elevated metabolic activity has been implicated in driving the HSC ageing phenotype. Here we show that nicotinamide riboside (NR), a form of vitamin B3, restores youthful metabolic capacity by modifying mitochondrial function in multiple ways including reduced expression of nuclear encoded metabolic pathway genes, d ing of mitochondrial stress and a decrease in mitochondrial mass and network-size. Metabolic restoration is dependent on continuous NR supplementation and accompanied by a shift of the aged transcriptome towards the young HSC state, more youthful bone marrow cellular composition and an improved regenerative capacity in a transplant setting. Consequently, NR administration could support healthy ageing by re-establishing a more youthful hematopoietic system.
Immunofluorescence Characterization of Key Extracellular Matrix Proteins in Murine Bone Marrow In Situ
Publisher: SAGE Publications
Date: 03-1998
DOI: 10.1177/002215549804600311
Abstract: The mechanism of hemopoietic stem cell homing to the bone marrow involves molecular interactions that mediate the recognition and interaction of these cells with the marrow microenvironment, including the extracellular matrix. On selective binding, this environment, in combination with soluble cytokines, regulates stem cell proliferation and differentiation. Using immunofluorescence labeling, we analyzed the location of the prominent extracellular matrix proteins fibronectin, collagen Types I, III, and IV, and laminin in sections of murine femoral bone marrow. Collagen Types I, IV, and fibronectin were localized to the endosteum, the region of the femoral microenvironment for which homing stem cells have a high affinity. The results further demonstrated a strong spatial association of collagen Type IV and laminin with the bone marrow vessels, including arterioles, veins, and sinuses. Fibronectin was distributed throughout the central marrow region, and all the proteins analyzed except collagen Type III were present in the bone, although at different levels. Fibronectin, collagen Types III and IV, and laminin were also present in the periosteum. The distinct locations of particular extracellular matrix proteins support the notion that they may play an important mechanistic role in the homing of engrafting cells.
Thrombin-cleaved osteopontin regulates hemopoietic stem and progenitor cell functions through interactions with α9 β1 and α4 β1 integrins
Publisher: American Society of Hematology
Date: 02-07-2009
DOI: 10.1182/BLOOD-2009-01-197988
Abstract: Osteopontin (OPN), a multifunctional acidic glycoprotein, expressed by osteoblasts within the endosteal region of the bone marrow (BM) suppresses the proliferation of hemopoietic stem and progenitor cells and also regulates their lodgment within the BM after transplantation. Herein we demonstrate that OPN cleavage fragments are the most abundant forms of this protein within the BM. Studies aimed to determine how hemopoietic stem cells (HSCs) interact with OPN revealed for the first time that murine and human HSCs express α9β1 integrin. The N-terminal thrombin cleavage fragment of OPN through its binding to the α9β1 and α4β1 integrins plays a key role in the attraction, retention, regulation, and release of hemopoietic stem and progenitor cells to, in, and from their BM niche. Thrombin-cleaved OPN (trOPN) acts as a chemoattractant for stem and progenitor cells, mediating their migration in a manner that involves interaction with α9β1 and α4β1 integrins. In addition, in the absence of OPN, there is an increased number of white blood cells and, specifically, stem and progenitor cells in the peripheral circulation.
Endogenous fibroblastic progenitor cells in the adult mouse lung are highly enriched in the sca-1 positive cell fraction
Publisher: Oxford University Press (OUP)
Date: 03-2009
DOI: 10.1634/STEMCELLS.2008-0866
Abstract: Originally identified as a marker specifying murine hematopoietic stem cells, the Sca-1 antigen has since been shown to be differentially expressed by candidate stem cells in tissues including vascular endothelium, skeletal muscle, mammary gland, and prostate of adult mice. In the adult murine lung, Sca-1 has previously been identified as a selectable marker for the isolation of candidate nonhematopoietic (CD45−), nonendothelial (CD31−) bronchioalveolar stem cells (BASC) located at the bronchioalveolar duct junction that coexpress surfactant protein C and the Clara cell specific protein. Our systematic analysis of CD45−CD31−Sca-1+ cells in fetal, neonatal, and adult lung shows that very few of these cells are detectable prior to birth but expand exponentially postnatally coinciding with the transition from the saccular to the alveolar stage of lung development. Unlike candidate BASCs, the CD45−CD31−Sca-1+CD34+ cell fraction we describe coexpresses immunophenotypic markers (Thy-1 and platelet-derived growth factor receptor α) that define lung fibroblastic rather than epithelial cells. The mesenchymal “signature” of the CD45−CD31−Sca-1+CD34+ cell fraction is further confirmed by transcriptional profiling, by cell culture studies demonstrating enrichment for clonogenic lipofibroblastic and nonlipofibroblastic progenitors, and by immunohistochemical localization of Sca-1 in perivascular cells of the lung parenchyma. Although the CD45−CD31−Sca-1+CD34+ cell phenotype does define endogenous clonogenic progenitor cells in the adult murine lung, our data indicate that these progenitors are predominantly representative of mesenchymal cell lineages, and highlights the pressing need for the identification of alternative markers and robust functional assays for the identification and characterization of epithelial and fibroblastic stem and progenitor cell populations in the adult lung.
Osteopontin: a bridge between bone and blood
Publisher: Wiley
Date: 31-07-2006
DOI: 10.1111/J.1365-2141.2006.06218.X
Abstract: The production of mature blood cells within the bone marrow (BM) is attributed to a pool of haemopoietic stem cells (HSC). It is now evident that HSC reside preferentially at the endosteal region within the BM where bone-lining osteoblasts are a key cellular component of the HSC niche that directly regulates HSC fate. Osteoblasts synthesise proteins that stimulate and inhibit HSC proliferation. In addition to angiopoietin 1 (Ang-1), osteoblasts synthesise and express the highly acidic glycoprotein, osteopontin (Opn), which, like Ang-1, acts as a potent constraining factor on HSC proliferation. Overexpression of Opn is a feature of haemopoietic malignancies, such as multiple myeloma and chronic myeloid leukaemia, although its exact role in the aetiology and progression of these diseases remains unclear. Through osteoblasts and their cell surface and expressed proteins including Opn, bone is able to regulate the tissue that resides within it. In doing so, Opn can be considered a bridge between bone and blood.
Validating Eaton's Hypothesis: Cubane as a Benzene Bioisostere
Publisher: Wiley
Date: 05-02-2016
Abstract: Pharmaceutical and agrochemical discovery programs are under considerable pressure to meet increasing global demand and thus require constant innovation. Classical hydrocarbon scaffolds have long assisted in bringing new molecules to the market place, but an obvious omission is that of the Platonic solid cubane. Eaton, however, suggested that this molecule has the potential to act as a benzene bioisostere. Herein, we report the validation of Eaton's hypothesis with cubane derivatives of five molecules that are used clinically or as agrochemicals. Two cubane analogues showed increased bioactivity compared to their benzene counterparts whereas two further analogues displayed equal bioactivity, and the fifth one demonstrated only partial efficacy. Ramifications from this study are best realized by reflecting on the number of bioactive molecules that contain a benzene ring. Substitution with the cubane scaffold where possible could revitalize these systems, and thus expedite much needed lead candidate identification.
Isolation of Murine Bone Marrow Scavenging Sinusoidal Endothelial Cells
Publisher: Springer New York
Date: 29-10-2015
DOI: 10.1007/978-1-4939-1785-3_6
Abstract: The bone marrow (BM) is permeated with sinusoidal vessels lined with sinusoidal endothelial cells (SEC), which are crucial for BM physiology and hemopoietic stem cell (HSC) renewal. However, little is known about the characteristics or functional capacity of bone marrow sinusoidal endothelial cells (BMSEC). One significant barrier to the study of BMSEC is the lack of specific cell surface markers that can be used to isolate these cells. Nevertheless, BMSEC possess one exceptional property, namely, the ability to scavenge large amounts of soluble waste molecules such as advanced glycation end-products (AGE) and we have utilized this to label BMSEC for cell sorting and isolation. We describe the means to produce and fluorescently label AGE, its use as a functional in vivo marker of BMSEC and the isolation of these cells from murine BM using fluorescent activated cell separation (FACS).
The effect of bovine endosteum-derived particles on the proliferation of human mesenchymal stem cells
Publisher: Elsevier BV
Date: 07-2010
DOI: 10.1016/J.BIOMATERIALS.2010.03.054
Abstract: There is a large biomanufacturing and clinical need for cost-effective and simple techniques to expand mesenchymal stem cells whilst retaining their multipotency. Endosteum-derived particles were prepared, characterised and examined as a biomaterial to facilitate the in vitro expansion of human mesenchymal stem cells. Bovine endosteum-derived particles are composed of chondroitin sulphate glycosaminoglycans with 4- and 6-sulphation and N-sulphated heparan sulphate glycosaminoglycans. The particles were positive for perlecan, laminin and fibronectin by immunohistochemistry and alpha-mannose, alpha-glucose, terminal N-acetyl-alpha-D-glucosamine, N-acetyl-alpha-galactosamine and alpha-fucose, using lectin binding. Human mesenchymal stem cells showed greater than 96% attachment to the particles after one day in spinner culture. After 7 days, the stem cells on decalcified particles were viable and had a 5-fold higher growth than the stem cells grown on Cytodex-2 beads. Significantly more stem cells were recovered from decalcified particles compared with mineralised particles (P < 0.05). Differentiation to chondrogenic, osteogenic and adipogenic lineages was maintained after culturing stem cells on the demineralised particles. We conclude that bovine endosteum-derived particles can be extracted from bone marrow to retain sulphated proteoglycans and glycosylated proteins. These particles are a suitable biomaterial for supporting the growth and retaining the multipotency of human mesenchymal stem cells.
Comprehensive characterization of distinct states of human naive pluripotency generated by reprogramming
Publisher: Springer Science and Business Media LLC
Date: 25-09-2017
DOI: 10.1038/NMETH.4436
Abstract: Recent reports on the characteristics of naive human pluripotent stem cells (hPSCs) obtained using independent methods differ. Naive hPSCs have been mainly derived by conversion from primed hPSCs or by direct derivation from human embryos rather than by somatic cell reprogramming. To provide an unbiased molecular and functional reference, we derived genetically matched naive hPSCs by direct reprogramming of fibroblasts and by primed-to-naive conversion using different naive conditions (NHSM, RSeT, 5iLAF and t2iLGöY). Our results show that hPSCs obtained in these different conditions display a spectrum of naive characteristics. Furthermore, our characterization identifies KLF4 as sufficient for conversion of primed hPSCs into naive t2iLGöY hPSCs, underscoring the role that reprogramming factors can play for the derivation of bona fide naive hPSCs.
Hemopoietic stem cell engraftment
Publisher: Elsevier BV
Date: 02-2006
Stem Cell Regulation by the Haemopoietic Stem Cell Niche
Publisher: Informa UK Limited
Date: 23-09-2005
DOI: 10.4161/CC.4.10.2056
Abstract: Both cellular as well as extracellular matrix components of the stem cell microenvironment, or niche, are critical in stem cell regulation. Recent data highlight a central role for osteoblasts and their by-product osteopontin as a key part of the hematopoietic stem cell (HSC) niche. Herein we describe a model for the yin and yang of HSC regulation mediated by osteoblasts. In this respect, osteoblasts synthesise proteins with opposing effects on HSC proliferation and differentiation highlighting their pivotal role in adult hematopoiesis. Although osteoblasts play a central role in HSC regulation other stromal and microenvironmental cell types and their extracellular matrix proteins also contribute to this biology. For ex le, the glycosaminoglycan hyaluronic acid as well as the membrane bound form of stem cell factor are also key regulators of HSC. Osteopontin and these "niche" molecules are not only involved in regulation of HSC quiescence but also effect HSC homing, trans-marrow migration and lodgement. Accordingly this leads us to expand upon Schofield's niche hypothesis: we propose that the HSC niche is critical for attraction of primitive hematopoietic progenitors to the endosteal region and tightly tethering them within this location, and by doing so placing them into intimate contact with cells such as osteoblasts whose cellular products are able to exquisitely regulate their fate.
Spatial localization of transplanted hemopoietic stem cells: inferences for the localization of stem cell niches
Publisher: American Society of Hematology
Date: 15-04-2001
Abstract: The spatial distribution of subpopulations of hemopoietic progenitor cells following syngeneic transplantation was investigated at the single-cell level. The location of infused hemopoietic progenitor cells within the femoral bone marrow of nonablated recipients was determined by 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester labeling of cells and in situ fixation by perfusion. Analysis performed over 15 hours after infusion demonstrated that the spatial distribution of transplanted marrow cells is not a random process. Although the majority of cells enter the bone marrow from the central marrow vessels, the subsequent localization within the bone marrow varied according to their phenotype. Candidate “stem cells” demonstrated selective redistribution and were significantly enriched within the endosteal region, whereas mature terminally differentiated and lineage-committed cells selectively redistributed away from the endosteal region and were predominantly in the central marrow region. Together, these data strongly support historical evidence of the presence of endosteal hemopoietic stem cell niches.
Niche Extracellular Matrix Components and Their Influence on HSC
Publisher: Wiley
Date: 24-04-2017
DOI: 10.1002/JCB.25905
Abstract: Maintenance of hematopoietic stem cells (HSC) takes place in a highly specialized microenvironment within the bone marrow. Technological improvements, especially in the field of in vivo imaging, have helped unravel the complexity of the niche microenvironment and have completely changed the classical concept from what was previously believed to be a static supportive platform, to a dynamic microenvironment tightly regulating HSC homeostasis through the complex interplay between erse cell types, secreted factors, extracellular matrix molecules, and the expression of different transmembrane receptors. To add to the complexity, non-protein based metabolites have also been recognized as a component of the bone marrow niche. The objective of this review is to discuss the current understanding on how the different extracellular matrix components of the niche regulate HSC fate, both during embryonic development and in adulthood. Special attention will be provided to the description of non-protein metabolites, such as lipids and metal ions, which contribute to the regulation of HSC behavior. J. Cell. Biochem. 118: 1984-1993, 2017. © 2017 Wiley Periodicals, Inc.
The relationship between bone,,hemopoietic stem cells,and vasculature
Publisher: American Society of Hematology
Date: 11-08-2011
DOI: 10.1182/BLOOD-2010-08-303800
Abstract: A large body of evidence suggests hemopoietic stem cells (HSCs) exist in an endosteal niche close to bone, whereas others suggest that the HSC niche is intimately associated with vasculature. In this study, we show that transplanted hemopoietic stem and progenitor cells (HSPCs) home preferentially to the trabecular-rich metaphysis of the femurs in nonablated mice at all time points from 15 minutes to 15 hours after transplantation. Within this region, they exist in an endosteal niche in close association with blood vessels. The preferential homing of HSPCs to the metaphysis occurs rapidly after transplantation, suggesting that blood vessels within this region may express a unique repertoire of endothelial adhesive molecules. One candidate is hyaluronan (HA), which is highly expressed on the blood vessel endothelium in the metaphysis. Analysis of the early stages of homing and the spatial dis-tribution of transplanted HSPCs at the single-cell level in mice devoid of Has3-synthesized HA, provides evidence for a previously undescribed role for HA expressed on endothelial cells in directing the homing of HSPCs to the metaphysis.
Effector CD4+ T cells recognize intravascular antigen presented by patrolling monocytes
Publisher: Springer Science and Business Media LLC
Date: 21-02-2018
DOI: 10.1038/S41467-018-03181-4
Abstract: Although effector CD4 + T cells readily respond to antigen outside the vasculature, how they respond to intravascular antigens is unknown. Here we show the process of intravascular antigen recognition using intravital multiphoton microscopy of glomeruli. CD4 + T cells undergo intravascular migration within uninflamed glomeruli. Similarly, while MHCII is not expressed by intrinsic glomerular cells, intravascular MHCII-expressing immune cells patrol glomerular capillaries, interacting with CD4 + T cells. Following intravascular deposition of antigen in glomeruli, effector CD4 + T-cell responses, including NFAT1 nuclear translocation and decreased migration, are consistent with antigen recognition. Of the MHCII + immune cells adherent in glomerular capillaries, only monocytes are retained for prolonged durations. These cells can also induce T-cell proliferation in vitro. Moreover, monocyte depletion reduces CD4 + T-cell-dependent glomerular inflammation. These findings indicate that MHCII + monocytes patrolling the glomerular microvasculature can present intravascular antigen to CD4 + T cells within glomerular capillaries, leading to antigen-dependent inflammation.
The osteoblastic niche following TBI
Publisher: American Society of Hematology
Date: 10-09-2009
Methods to Analyze the Homing Efficiency and Spatial Distribution of Hematopoietic Stem and Progenitor Cells and Their Relationship to the Bone Marrow Endosteum and Vascular Endothelium
Publisher: Humana Press
Date: 2011
DOI: 10.1007/978-1-61779-145-1_14
Abstract: The tracking of immunofluorescent labeled hematopoietic stem and progenitor cells (HSC/HPC) within the bone marrow (BM) cavity allows the assessment of the regulatory processes involved in transendothelial migration, trans-marrow migration, and finally lodgement into the HSC niche. This is of interest as the extracellular and cellular components involved in the regulation of HSC quiescence and differentiation are still not completely understood. Homing of transplanted HSC is the first critical step in the interaction between HSC and the microenvironment of the BM. As a consequence, murine models allowing the evaluation of the structural relationship between migrating HSC, the endosteal bone surface, and the vascular components of the BM enhance our understanding of hematopoietic regulation.
The prospective isolation of viable, high ploidy megakaryocytes from adult murine bone marrow by fluorescence activated cell sorting
Publisher: Humana Press
Date: 2013
DOI: 10.1007/978-1-62703-508-8_10
Abstract: Mature megakaryocytes (MM) can be up to 65 μM in diameter and due to their size, viable and pure MM populations have been difficult to isolate in large numbers. Here in, we report a fluorescence activated cell sorting (FACS) method by which viable and pure populations of 8 N, 16 N, 32 N, and 64 N MM can be isolated from murine bone marrow (BM).
Detection and quantification of functionally defined hematopoietic progenitor cells and tissue specific mRNA within the peripheral blood of myeloma patients after administration of granulocyte colony‐
Publisher: Wiley
Date: 13-12-2007
DOI: 10.1111/J.1600-0609.2007.00983.X
Abstract: Hematopoietic progenitor cells (HPC) as well as tissue committed stem cells expressing mRNA specific to various somatic tissues are thought to be part of the CD34+ bone marrow compartment. In this study, we explore and quantify their mobilization in patients with multiple myeloma undergoing chemotherapy upon administration of granulocyte colony-stimulating factor (G-CSF) plus/minus erythropoietin (EPO). HPC were quantified by flow cytometry and functional assays within the blood of healthy donors and myeloma patients before and after chemotherapy followed by G-CSF or G-CSF + EPO given subcutaneously. The mRNA expression was studied by quantitative polymerase chain reaction (PCR). Cytokines and peripheral blood protease levels were measured by an enzyme-linked immunosorbent assay. EPO did not significantly alter the number of HPC mobilized by G-CSF alone, and mRNA specific for liver, brain, muscle and kidney was detected in both treatment groups. Quantitative PCR analysis revealed a 2.7-fold increased expression of glial fibrillary acidic protein after G-CSF + EPO administration compared to G-CSF alone (P = 0.003). The concentration of G-CSF rose from 62 +/- 22 pg/mL and 48 +/- 10 pg/mL to 28 +/- 9 ng/mL and 85 +/- 10 ng/mL after 10 d of treatment with G-CSF and G-CSF + EPO, respectively. The concentration of neutrophil elastase (NE) rose only in the G-CSF group by a factor 1.5. The alteration of G-CSF and NE levels as well as the expression of tissue committed RNA after the administration of EPO in addition to G-CSF indicate that different growth factors mobilize different stem cells that might potentially be used for the support of tissue repair in future treatment protocols.
Progress in bio-manufacture of platelets for transfusion
Publisher: Informa UK Limited
Date: 09-01-2017
DOI: 10.1080/09537104.2016.1257783
Abstract: Blood transfusion services face an ever-increasing demand for donor platelets to meet clinical needs. Whilst strategies for increasing platelet storage life and improving the efficiency of donor platelet collection are important, in the longer term, platelets generated by bio-manufacturing processes will be required to meet demands. Production of sufficient numbers of in vitro-derived platelets for transfusion represents a significant bioengineering challenge. In this review, we highlight recent progress in this area of research and outline the main technical and biological obstacles that need to be met before this becomes feasible and economic. A critical consideration is assurance of the functional properties of these cells as compared to their fresh, donor collected, counterparts. We contend that platelet-like particles and in vitro-derived platelets that phenotypically resemble fresh platelets must deliver the same functions as these cells upon transfusion. We also note recent progress with immortalized megakaryocyte progenitor cell lines, molecular strategies for reducing expression of HLA Class I to generate universal donor platelets and the move to early clinical studies with in vitro-derived platelets.
Megakaryocytes co-localise with hemopoietic stem cells and release cytokines that up-regulate stem cell proliferation
Publisher: Elsevier BV
Date: 09-2013
DOI: 10.1016/J.SCR.2013.05.007
Abstract: We report transplanted hemopoietic stem cells (HSC) preferentially lodge within two cells of mature megakaryocytes (MM). With both populations comprising ~0.2% of bone marrow cells, this strongly suggests a key functional interaction. HSC isolated from the endosteum (eLSKSLAM) showed significantly increased hemopoietic cell proliferation while in co-culture with MM. Furthermore, eLSKSLAM progeny retained HSC potential, maintaining long-term multi-lineage reconstitution capacity in lethally ablated recipients. Increased hemopoietic cell proliferation was not MM contact dependent and could be recapitulated with media supplemented with two factors identified in MM-conditioned media: insulin-like growth factor binding protein-3 (IGFBP-3) and insulin-like growth factor-1 (IGF-1). We demonstrate that HSC express the receptor for IGF-1 and that IGF-1/IGFBP-3 induced increased hemopoietic cell proliferation can be blocked by an anti-IGF-1 neutralising antibody. However, co-cultures of 8N, 16N or 32N MM with eLSKSLAM showed that MM of in idual ploidy did not significantly increase hemopoietic cell proliferation. Our data suggests that MM are an important component of the HSC niche and regulate hemopoietic cell proliferation through cytokine release.
Frontispiz: Validating Eaton's Hypothesis: Cubane as a Benzene Bioisostere
Publisher: Wiley
Date: 29-02-2016
Understanding the role of the microenvironment during definitive hemopoietic development
Publisher: Elsevier BV
Date: 09-2013
DOI: 10.1016/J.EXPHEM.2013.06.005
Abstract: Hemopoietic stem cells (HSCs) are sustained in a specific microenvironment known as the stem cell niche. Recent studies in adult bone marrow have identified osteoblasts and endothelial cells as two important supportive cell types within the niche and demonstrated that interactions between HSCs and cellular and extracellular components within the endosteal and perivascular regions are critical for HSC regulation. However, the understanding of the role of the microenvironment in definitive HSC establishment, expansion, and maintenance during embryonic development is extremely limited. This review focuses on what is known about the components of each HSC microenvironment at various developmental stages and their known functional roles.
Phenotypically identical hemopoietic stem cells isolated from different regions of bone marrow have different biologic potential
Publisher: American Society of Hematology
Date: 28-10-2010
DOI: 10.1182/BLOOD-2009-12-260703
Abstract: Hemopoietic stem cells (HSCs) reside within a specified area of the bone marrow (BM) cavity called a “niche” that modulates HSC quiescence, proliferation, differentiation, and migration. Our previous studies have identified the endosteal BM region as the site for the HSC niche and demonstrated that hemopoietic stem and progenitor populations (HSPCs, LSK) isolated from different BM regions exhibit significantly different hemopoietic potential. In this study, we have analyzed subpopulations of LSK cells isolated from different regions of the BM and showed that CD150+CD48−LSK HSCs within the endosteal BM region have superior proliferative capacity and homing efficiency compared with CD150+CD48−LSK HSCs isolated from the central BM. Furthermore, we show, for the first time, that a subset of CD150+CD48+LSK progenitor cells, previously defined as B-lymphoid primed hemopoietic cells, are capable of multilineage reconstitution, however, only when isolated from the endosteal region. In addition, we provide evidence for an unrecognized role of CD48 in HSC homing. Together, our data provide strong evidence that highly purified HSCs show functional differences depending on their origin within the BM and that the most primitive HSCs reside within the endosteal BM region.
Granulocyte colony stimulating factor expands hematopoietic stem cells within the central but not endosteal bone marrow region
Publisher: Elsevier BV
Date: 05-2012
DOI: 10.1016/J.CYTO.2012.01.014
Abstract: Granulocyte colony stimulating factor (G-CSF) is clinically well established for the mobilization of hematopoietic stem cells (HSC). Extensive data on the underlying mechanism of G-CSF induced mobilization is available however, little is known regarding the functional effect of G-CSF on HSC within the bone marrow (BM). In this study we analyzed the proportion and number of murine HSC in the endosteal and central bone marrow regions after 4 days of G-CSF administration. We demonstrate that the number of HSC, defined as CD150(+)CD48(-)LSK cells (LSKSLAM cells), increased within the central BM region in response to G-CSF, but not within the endosteal BM region. In addition the level of CD150 and CD48 expression also increased on cells isolated from both regions. We further showed that G-CSF mobilized proportionally fewer LSKSLAM compared to LSK cells, mobilized LSKSLAM had colony forming potential and the presence of these cells can be used as a measure for mobilization efficiency. Together we provide evidence that HSC in the BM respond differently to G-CSF and this is dependent on their location. These findings will be valuable in developing new agents which specifically mobilize HSC from the endosteal BM region, which we have previously demonstrated to have significantly greater hematopoietic potential compared to their phenotypically identical counterparts located in other regions of the BM.
i-bodies, Human Single Domain Antibodies That Antagonize Chemokine Receptor CXCR4
Publisher: Elsevier BV
Date: 06-2016
Recent Australian experience with hemopoietic stem and progenitor cell expansion
Publisher: Elsevier BV
Date: 2007
DOI: 10.1080/14653240701315296
Abstract: This review provides insight into two clinical trials conducted with ex vivo manipulated CD34+ cells. The first was an attempt to deliver a gene therapy for treatment of HIV and the second an attempt to improve rates of hemopoietic recovery with ex vivo generated myeloid cells.
Hematopoietic Progenitor Cell Mobilization Results in Hypoxia with Increased Hypoxia-Inducible Transcription Factor-1α and Vascular Endothelial Growth Factor A in Bone Marrow
Publisher: Oxford University Press (OUP)
Date: 03-05-2007
DOI: 10.1634/STEMCELLS.2006-0688
Abstract: Despite the fact that many hypoxia-inducible genes are important in hematopoiesis, the spatial distribution of oxygen in the bone marrow (BM) has not previously been explored in vivo. Using the hypoxia bioprobe pimonidazole, we showed by confocal laser scanning microscopy that the endosteum at the bone-BM interface is hypoxic, with constitutive expression of hypoxia-inducible transcription factor-1α (HIF-1α) protein in steady-state mice. Interestingly, at the peak of hematopoietic stem and progenitor cell (HSPC) mobilization induced by either granulocyte colony-stimulating factor or cyclophosphamide, hypoxic areas expand through the central BM. Furthermore, we found that HSPC mobilization leads to increased levels of HIF-1α protein and increased expression of vascular endothelial growth factor A (VEGF-A) mRNA throughout the BM, with an accumulation of VEGF-A protein in BM endothelial sinuses. VEGF-A is a cytokine known to induce stem cell mobilization, vasodilatation, and vascular permeability in vivo. We therefore propose that the expansion in myeloid progenitors that occurs during mobilization depletes the BM hematopoietic microenvironment of O2, leading to local hypoxia, stabilization of HIF-1α transcription factor in BM cells, increased transcription of VEGF-A, and accumulation of VEGF-A protein on BM sinuses that increases vascular permeability. Disclosure of potential conflicts of interest is found at the end of this article.
Role of CD44 variant exon 6 in acute lymphoblastic leukaemia: association with altered bone marrow localisation and increased tumour burden
Publisher: Springer Science and Business Media LLC
Date: 20-05-2004
NogoA-expressing astrocytes limit peripheral macrophage infiltration after ischemic brain injury in primates
Publisher: Springer Science and Business Media LLC
Date: 25-11-2021
DOI: 10.1038/S41467-021-27245-0
Abstract: Astrocytes play critical roles after brain injury, but their precise function is poorly defined. Utilizing single-nuclei transcriptomics to characterize astrocytes after ischemic stroke in the visual cortex of the marmoset monkey, we observed nearly complete segregation between stroke and control astrocyte clusters. Screening for the top 30 differentially expressed genes that might limit stroke recovery, we discovered that a majority of astrocytes expressed RTN4A/ NogoA, a neurite-outgrowth inhibitory protein previously only associated with oligodendrocytes. NogoA upregulation on reactive astrocytes post-stroke was significant in both the marmoset and human brain, whereas only a marginal change was observed in mice. We determined that NogoA mediated an anti-inflammatory response which likely contributes to limiting the infiltration of peripheral macrophages into the surviving parenchyma.
Haemopedia: An Expression Atlas of Murine Hematopoietic Cells
Publisher: Elsevier BV
Date: 09-2016
Analyzing hemopoietic stem cell homing, lodgment and engraftment to better understand the bone marrow niche
Publisher: Wiley
Date: 15-01-2014
DOI: 10.1111/NYAS.12329
Abstract: The existence of a bone marrow (BM) niche--the location in which hematopoietic stem cells (HSCs) reside--was proposed more than 30 years ago. Recent data suggest that the interaction of HSCs with cellular and extracellular components within the BM is critical for HSC regulation. The tracking of immunofluorescently labeled, prospectively isolated HSCs to and within the BM cavity allows the assessment of the regulatory processes involved in (1) homing, which involves transendothelial migration into the BM (2) lodgment, including transmarrow migration through the extravascular space and (3) BM reconstitution. Together, such analyses provide a better understanding of the cellular and extracellular components involved in the regulation of HSC quiescence and differentiation. Homing and lodgment of transplanted HSCs, the first critical steps in engraftment, involve multiple interactions between HSCs and the BM microenvironment. Herein, we describe a refined method of analyzing homing efficiency and spatial distribution of HSCs harvested from endosteal and/or central BM regions we also review alternate methods. Using these techniques, microenvironment modifications within the recipient or surface protein-expression modifications on donor HSCs in animal models provide insights into components influencing the homing, lodgment, and engraftment processes.
Design, synthesis and binding properties of a fluorescent α9β1/α4β1integrin antagonist and its application as anin vivoprobe for bone marrow haemopoi
Publisher: Royal Society of Chemistry (RSC)
Date: 2014
DOI: 10.1039/C3OB42332H
Abstract: A fluorescent α 9 β 1 integrin antagonist with nanomolar binding affinities has been demonstrated to bind bone marrow haemopoietic stem and progenitor cells in vivo .
Developmental Fate Determination and Marker Discovery in Hematopoietic Stem Cell Biology Using Proteomic Fingerprinting
Publisher: Elsevier BV
Date: 03-2008
Osteopontin, a key component of the hematopoietic stem cell niche and regulator of primitive hematopoietic progenitor cells
Publisher: American Society of Hematology
Date: 15-08-2005
DOI: 10.1182/BLOOD-2004-11-4422
Abstract: Although recent data suggests that osteoblasts play a key role within the hematopoietic stem cell (HSC) niche, the mechanisms underpinning this remain to be fully defined. The studies described herein examine the role in hematopoiesis of Osteopontin (Opn), a multidomain, phosphorylated glycoprotein, synthesized by osteoblasts, with well-described roles in cell adhesion, inflammatory responses, angiogenesis, and tumor metastasis. We demonstrate a previously unrecognized critical role for Opn in regulation of the physical location and proliferation of HSCs. Within marrow, Opn expression is restricted to the endosteal bone surface and contributes to HSC transmarrow migration toward the endosteal region, as demonstrated by the markedly aberrant distribution of HSCs in Opn–/– mice after transplantation. Primitive hematopoietic cells demonstrate specific adhesion to Opn in vitro via β1 integrin. Furthermore, exogenous Opn potently suppresses the proliferation of primitive HPCs in vitro, the physiologic relevance of which is demonstrated by the markedly enhanced cycling of HSC in Opn–/– mice. These data therefore provide strong evidence that Opn is an important component of the HSC niche which participates in HSC location and as a physiologic-negative regulator of HSC proliferation.
The location and cellular composition of the hemopoietic stem cell niche
Publisher: Elsevier BV
Date: 02-2012
Antisense-mediated suppression of hyaluronan synthase 2 inhibits the tumorigenesis and progression of breast cancer
Publisher: American Association for Cancer Research (AACR)
Date: 15-07-2005
DOI: 10.1158/0008-5472.CAN-04-1622
Abstract: The progression of several cancers is correlated with the increased synthesis of the glycosaminoglycan, hyaluronan. Hyaluronan is synthesized at the plasma membrane by various isoforms of hyaluronan synthases (HAS). The importance of HAS2 expression in highly invasive breast cancer was characterized by the antisense inhibition of HAS2 (ASHAS2). The effect of HAS2 inhibition on cell proliferation, migration, hyaluronan metabolism, and receptor status was characterized in vitro, whereas the effect on tumorigenicity and metastasis was established in vivo. HAS2 inhibition resulted in a 24-hour lag in proliferation that was concomitant to transient arrest of 79% of the cell population in G0-G1. Inhibition of HAS2 did not alter the expression of the other HAS isoforms, whereas hyaluronidase (HYAL2) and the hyaluronan receptor, CD44, were significantly down-regulated. ASHAS2 cells accumulated greater amounts of high molecular weight hyaluronan (& ,000 kDa) in the culture medium, whereas mock and parental cells liberated less hyaluronan of three distinct molecular weights (100, 400, and 3,000 kDa). The inhibition of HAS2 in the highly invasive MDA-MB-231 breast cancer cell line inhibited the initiation and progression of primary and secondary tumor formation following s.c. and intracardiac inoculation into nude mice, whereas controls readily established both primary and secondary tumors. The lack of primary and secondary tumor formation was manifested by increased survival times where ASHAS2 animals survived 172% longer than the control animals. Collectively, these unique results strongly implicate the central role of HAS2 in the initiation and progression of breast cancer, potentially highlighting the codependency between HAS2, CD44, and HYAL2 expression.
Potent In Vitro Peptide Antagonists of the Thrombopoietin Receptor as Potential Myelofibrosis Drugs
Publisher: Wiley
Date: 20-01-2021
Glycosylated Nanoparticles Derived from RAFT Polymerization for Effective Drug Delivery to Macrophages
Publisher: American Chemical Society (ACS)
Date: 28-07-2020
Abstract: The functional group tolerance and simplicity of reversible addition fragmentation chain transfer (RAFT) polymerization enable its use in the preparation of a wide range of functional polymer architectures for a variety of applications, including drug delivery. Given the role of tumor-associated macrophages (TAMs) in cancer and their dependence on the tyrosine kinase receptor FMS (CSF-1R), the key aim of this work was to achieve effective delivery of an FMS inhibitor to cells using a polymer delivery system. Such a system has the potential to exploit biological features specific to macrophages and therefore provide enhanced selectivity. Building on our prior work, we have prepared RAFT polymers based on a poly(butyl methacrylate-
Late fetal hematopoietic failure results from ZBTB11 deficiency despite abundant HSC specification
Publisher: American Society of Hematology
Date: 14-11-2023
DOI: 10.1182/BLOODADVANCES.2022009580
Abstract: Hematopoiesis produces all the erse blood cell lineages to meet basal needs and the sudden demands of injury or infection. Rapid response to such challenges requires expansion of specific lineages then prompt return to balanced steady-state levels, necessitating tightly coordinated regulation. We previously identified a requirement for the Zinc finger and BTB-domain containing 11 (ZBTB11) transcription factor in definitive hematopoiesis from a forward genetic screen for zebrafish myeloid mutants. To understand its relevance to mammalian systems, we extended these studies to mouse. When Zbtb11 was deleted in the hematopoietic compartment, embryos died at embryonic day (E) 18.5 with hematopoietic failure. Zbtb11 hematopoietic knockout (Zbtb11hKO) hematopoietic stem cells (HSCs) were overabundantly specified at E14.5 through E17.5 compared to controls. Overspecification was accompanied by loss of stemness, inability to differentiate into committed progenitors and mature lineages in fetal liver, failure to seed fetal bone marrow and total hematopoietic failure. Zbtb11hKO HSCs did not proliferate in vitro and were constrained in cell cycle progression, demonstrating a cell-intrinsic role for Zbtb11 in proliferation and cell cycle regulation in mammalian HSCs. scRNAseq analysis identified Zbtb11-deficient HSCs were underrepresented in an erythroid-primed subpopulation and showed downregulation of oxidative phosphorylation (OXPHOS) pathways and dysregulation of genes associated with the hematopoietic niche. We have identified a cell-intrinsic requirement for Zbtb11-mediated gene regulatory networks in sustaining a pool of maturation-capable hematopoietic stem and progenitor cells.
Hyaluronan is synthesized by primitive hemopoietic cells, participates in their lodgment at the endosteum following transplantation, and is involved in the regulation of their proliferation and differ
Publisher: American Society of Hematology
Date: 02-2003
DOI: 10.1182/BLOOD-2002-05-1344
Abstract: The localization of adult hemopoiesis to the marrow involves developmentally regulated interactions between hemopoietic stem cells and the stromal cell–mediated hemopoietic microenvironment. Although primitive hemopoietic cells exhibit a broad repertoire of adhesion molecules, little is known about the molecules influencing the site of cell lodgment within the marrow following transplantation. However, our recent studies indicate that hierarchically dependent patterns of migration of transplanted hemopoietic cells result in the retention of primitive cells within the endosteal and lineage-committed cells in the central marrow regions. Herein, we now demonstrate that these 2 subpopulations exhibit a striking difference in the expression of a cell surface adhesion molecule, with populations enriched for murine and human hemopoietic stem cells expressing the carbohydrate hyaluronic acid (HA). Furthermore, the presence of this glycosaminoglycan appears critical for the spatial distribution of transplanted stem cells in vivo. In addition, we also demonstrate that the binding of HA by a surrogate ligand results in marked inhibition of primitive hemopoietic cell proliferation and granulocyte differentiation. Collectively, these data describe an important yet previously unrecognized role for HA in the biology of primitive hemopoietic progenitor cells.
Differentiation of human embryonic stem cells to HOXA+ hemogenic vasculature that resembles the aorta-gonad-mesonephros
Publisher: Springer Science and Business Media LLC
Date: 17-10-2016
DOI: 10.1038/NBT.3702
Abstract: The ability to generate hematopoietic stem cells from human pluripotent cells would enable many biomedical applications. We find that hematopoietic CD34
Investigating the Interaction Between Hematopoietic Stem Cells and Their Niche During Embryonic Development: Optimizing the Isolation of Fetal and Newborn Stem Cells From Liver, Spleen, and Bone Marro
Publisher: Springer New York
Date: 2014
DOI: 10.1007/978-1-4939-1133-2_2
Abstract: Hematopoietic stem cells (HSCs) are maintained in a particular microenvironment termed a "niche." Within the niche, a number of critical molecules and supportive cell types have been identified to play key roles in modulating adult HSC quiescence, proliferation, differentiation, and reconstitution. However, unlike in the adult bone marrow (BM), the components of stem cell niches, as well as their interactions with fetal HSC during different stages of embryonic development, are poorly understood. During embryogenesis, hematopoietic development migrates through multiple organs, each with different cellular and molecular components and hence each with a potentially unique HSC niche. As a consequence, isolating fetal HSC from each organ at the time of hematopoietic colonization is fundamental for assessing and understanding both HSC function and their interactions with specific microenvironments. Herein, we describe methodologies for harvesting cells as well as the purification of stem and progenitors from fetal and newborn liver, spleen, and BM at various developmental stages following the expansion of hematopoiesis in the fetal liver at E14.5.
Membrane-bound stem cell factor is a key regulator in the initial lodgment of stem cells within the endosteal marrow region
Publisher: Elsevier BV
Date: 12-2003
DOI: 10.1016/J.EXPHEM.2003.08.015
Abstract: The transmembrane isoform of stem cell factor (tm-SCF) has been implicated in the adhesion of hemopoietic stem cells to the extracellular matrix within the bone marrow microenvironment in vitro. In addition, in vivo SCF has been shown to play a role in cell mobilization and migration. The aim of this study was to determine if SCF is an integral component of the hemopoietic "niche" of the bone marrow in situ. To analyze the role of tm-SCF in cell lodgment, purified populations of primitive progenitors and hemopoietic stem cells (HSC) were transplanted into a hemopoietic microenvironment devoid of tm-SCF, and the spatial distribution of engrafted cells was analyzed. In addition, populations of HSC were isolated using non-neutralizing and neutralizing antibodies to the SCF receptor c-kit, and their spatial distribution was analyzed post-transplant. The data demonstrated a significant impairment in the lodgment of transplanted cells within the endosteal marrow region in mice lacking tm-SCF, with a reduction of almost 30% by 15 hours post-transplant. The role of tm-SCF was confirmed by analyzing the spatial distribution of HSC isolated using a neutralizing antibody to c-kit. The data demonstrate that although tm-SCF does not appear to play a role in the homing of transplanted cells to the bone marrow, it is critical in the lodgment and detainment of HSC within their hemopoietic "niche."
A CX3CR1 Reporter hESC Line Facilitates Integrative Analysis of In-Vitro-Derived Microglia and Improved Microglia Identity upon Neuron-Glia Co-culture
Publisher: Elsevier BV
Date: 06-2020
Investigating the Interactions Between Haemopoietic Stem Cells and Their Niche: Methods for the Analysis of Stem Cell Homing and Distribution Within the Marrow Following Transplantation
Publisher: Humana Press
Date: 2009
DOI: 10.1007/978-1-59745-060-7_6
Abstract: Interactions between haemopoietic stem cells (HSC) and their microenvironment serve multiple functions including the attraction to and retention and regulation in the bone marrow HSC niche. However, the cell adhesion molecules involved, their HSC receptors and the mechanisms underpinning these processes remain poorly understood. An ability to thoroughly investigate the roles of specific molecules in this process relies on a variety of in vitro and in vivo assays including the assessment of a HSC ability to home to the bone marrow and analysis of its lodgement within the bone marrow.
Therapeutic targeting and rapid mobilization of endosteal HSC using a small molecule integrin antagonist
Publisher: Springer Science and Business Media LLC
Date: 15-03-2016
DOI: 10.1038/NCOMMS11007
Abstract: The inherent disadvantages of using granulocyte colony-stimulating factor (G-CSF) for hematopoietic stem cell (HSC) mobilization have driven efforts to identify alternate strategies based on single doses of small molecules. Here, we show targeting α 9 β 1 /α 4 β 1 integrins with a single dose of a small molecule antagonist (BOP ( N -(benzenesulfonyl)- L -prolyl- L - O -(1-pyrrolidinylcarbonyl)tyrosine)) rapidly mobilizes long-term multi-lineage reconstituting HSC. Synergistic engraftment augmentation is observed when BOP is co-administered with AMD3100. Impressively, HSC in equal volumes of peripheral blood (PB) mobilized with this combination effectively out-competes PB mobilized with G-CSF. The enhanced mobilization observed using BOP and AMD3100 is recapitulated in a humanized NODSCIDIL2Rγ −/− model, demonstrated by a significant increase in PB CD34 + cells. Using a related fluorescent analogue of BOP (R-BC154), we show that this class of antagonists preferentially bind human and mouse HSC and progenitors via endogenously primed/activated α 9 β 1 /α 4 β 1 within the endosteal niche. These results support using dual α 9 β 1 /α 4 β 1 inhibitors as effective, rapid and transient mobilization agents with promising clinical applications.
Effective macrophage delivery using RAFT copolymer derived nanoparticles
Publisher: Royal Society of Chemistry (RSC)
Date: 2018
DOI: 10.1039/C7PY01363A
Abstract: We use reversible addition fragmentation chain transfer (RAFT) polymerisation to prepare block copolymers that are subsequently assembled into nanoparticles. The prepared nanoparticles were extensively taken up by primary murine macrophages and are effective in the delivery of a cell impenetrable cargo.
Mobilisation strategies for normal and malignant cells
Publisher: Elsevier BV
Date: 10-2011
Stabilization and improved functionality of three-dimensional perfusable microvascular networks in microfluidic devices under macromolecular crowding
Publisher: Springer Science and Business Media LLC
Date: 19-04-2023
DOI: 10.1186/S40824-023-00375-W
Abstract: There is great interest to engineer in vitro models that allow the study of complex biological processes of the microvasculature with high spatiotemporal resolution. Microfluidic systems are currently used to engineer microvasculature in vitro , which consists of perfusable microvascular networks (MVNs). These are formed through spontaneous vasculogenesis and exhibit the closest resemblance to physiological microvasculature. Unfortunately, under standard culture conditions and in the absence of co-culture with auxiliary cells as well as protease inhibitors, pure MVNs suffer from a short-lived stability. Herein, we introduce a strategy for stabilization of MVNs through macromolecular crowding (MMC) based on a previously established mixture of Ficoll macromolecules. The biophysical principle of MMC is based on macromolecules occupying space, thus increasing the effective concentration of other components and thereby accelerating various biological processes, such as extracellular matrix deposition. We thus hypothesized that MMC will promote the accumulation of vascular ECM (basement membrane) components and lead to a stabilization of MVN with improved functionality. MMC promoted the enrichment of cellular junctions and basement membrane components, while reducing cellular contractility. The resulting advantageous balance of adhesive forces over cellular tension resulted in a significant stabilization of MVNs over time, as well as improved vascular barrier function, closely resembling that of in vivo microvasculature. Application of MMC to MVNs in microfluidic devices provides a reliable, flexible and versatile approach to stabilize engineered microvessels under simulated physiological conditions.
Expression profiling of a hemopoietic cell survival transcriptome implicates osteopontin as a functional prognostic factor in AML
Publisher: American Society of Hematology
Date: 26-11-2009
DOI: 10.1182/BLOOD-2009-02-204818
Abstract: Deregulated cell survival programs are a classic hallmark of cancer. We have previously identified a serine residue (Ser585) in the βc subunit of the granulocyte-macrophage colony-stimulating factor receptor that selectively and independently promotes cell survival. We now show that Ser585 phosphorylation is constitutive in 20 (87%) of 23 acute myeloid leukemia (AML) patient s les, indicating that this survival-only pathway is frequently deregulated in leukemia. We performed a global expression screen to identify gene targets of this survival pathway and report a 138-gene βc Ser585-regulated transcriptome. Pathway analysis defines a gene network enriched for PI3-kinase target genes and a cluster of genes involved in cancer and cell survival. We show that one such gene, osteopontin (OPN), is a functionally relevant target of the Ser585-survival pathway as shown by siRNA-mediated knockdown of OPN expression that induces cell death in both AML blasts and CD34+CD38−CD123+ leukemic progenitors. Increased expression of OPN at diagnosis is associated with poor prognosis with multivariate analysis indicating that it is an independent predictor of overall patient survival in normal karyotype AML (n = 60 HR = 2.2 P = .01). These results delineate a novel cytokine-regulated Ser585/PI3-kinase signaling network that is deregulated in AML and identify OPN as a potential prognostic and therapeutic target.
Bone marrow regulatory T cells are a unique population, supported by niche-specific cytokines and plasmacytoid dendritic cells, and required for chronic graft-vs-host disease control
Publisher: Frontiers Media SA
Date: 22-09-2021
DOI: 10.3389/FCELL.2021.737880
Abstract: Regulatory T cell (Treg) reconstitution is essential for reestablishing tolerance and maintaining homeostasis following stem-cell transplantation. We previously reported that bone marrow (BM) is highly enriched in autophagy-dependent Treg and autophagy disruption leads to a significant Treg loss, particularly BM-Treg. To correct the known Treg deficiency observed in chronic graft-versus-host disease (cGVHD) patients, low dose IL-2 infusion has been administered, substantially increasing peripheral Treg (pTreg) numbers. However, as clinical responses were only seen in ∼50% of patients, we postulated that pTreg augmentation was more robust than for BM-Treg. We show that BM-Treg and pTreg have distinct characteristics, indicated by differential transcriptome expression for chemokine receptors, transcription factors, cell cycle control of replication and genes linked to Treg function. Further, BM-Treg were more quiescent, expressed lower FoxP3, were highly enriched for co-inhibitory markers and more profoundly depleted than splenic Treg in cGVHD mice. In vivo our data are consistent with the BM and not splenic microenvironment is, at least in part, driving this BM-Treg signature, as adoptively transferred splenic Treg that entered the BM niche acquired a BM-Treg phenotype. Analyses identified upregulated expression of IL-9R, IL-33R, and IL-7R in BM-Treg. Administration of the T cell produced cytokine IL-2 was required by splenic Treg expansion but had no impact on BM-Treg, whereas the converse was true for IL-9 administration. Plasmacytoid dendritic cells (pDCs) within the BM also may contribute to BM-Treg maintenance. Using pDC-specific BDCA2-DTR mice in which diptheria toxin administration results in global pDC depletion, we demonstrate that pDC depletion h ers BM, but not splenic, Treg homeostasis. Together, these data provide evidence that BM-Treg and splenic Treg are phenotypically and functionally distinct and influenced by niche-specific mediators that selectively support their respective Treg populations. The unique properties of BM-Treg should be considered for new therapies to reconstitute Treg and reestablish tolerance following SCT.
The histo-morphometric relationship between bone, blood vessels and hemopoietic stem cells
Publisher: Oxford University Press (OUP)
Date: 08-2008
DOI: 10.1017/S1431927608083104
Abstract: Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008
Transplantable stem cells: home to specific niches
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 03-2004
DOI: 10.1097/01.MOH.0000133651.06863.9C
Abstract: Although the concept of engraftment and clinical reconstitution of the bone marrow was described several decades ago, the analysis of in idual steps within this process remains a major focus of much current research in stem cell biology. In particular, this extends to the identification and characterization of the specific stem cell niche first proposed by Schofield in 1978. It is appropriate, therefore, that on the 25th anniversary of this publication, that we review recent progress in our understanding of the location and composition of the bone marrow stem cell niche and of the mechanisms involved in the initial phases of hematopoietic stem cell engraftment. During the past 12 months there have been significant advancements in our understanding of the interplay of molecules involved in the homing of hematopoietic stem cells to the bone marrow. In addition, innovative methodologies have become available for the visualization of hematopoietic stem cells within the bone marrow in situ. In an important development in this area, studies our now focusing on events after transendothelial migration into the marrow cords, including mechanisms involved in hematopoietic stem cell migration to and lodgment within the hematopoietic stem cell niche. Furthermore, there have been numerous new reports analyzing the molecular regulation of hematopoietic stem cells within the bone marrow niche in situ. Overall, recent advancements in our understanding of hematopoietic stem cell biology and, in particular, the interaction of hematopoietic stem cells with the hematopoietic microenvironment paves the way for expanded use in regenerative medicine.
High ploidy large cytoplasmic megakaryocytes are hematopoietic stem cells regulators and essential for platelet production
Publisher: Springer Science and Business Media LLC
Date: 13-04-2023
DOI: 10.1038/S41467-023-37780-7
Abstract: Megakaryocytes (MK) generate platelets. Recently, we and others, have reported MK also regulate hematopoietic stem cells (HSC). Here we show high ploidy large cytoplasmic megakaryocytes (LCM) are critical negative regulators of HSC and critical for platelet formation. Using a mouse knockout model ( Pf4-Srsf3 Δ/Δ ) with normal MK numbers, but essentially devoid of LCM, we demonstrate a pronounced increase in BM HSC concurrent with endogenous mobilization and extramedullary hematopoiesis. Severe thrombocytopenia is observed in animals with diminished LCM, although there is no change in MK ploidy distribution, uncoupling endoreduplication and platelet production. When HSC isolated from a microenvironment essentially devoid of LCM reconstitute hematopoiesis in lethally irradiated mice, the absence of LCM increases HSC in BM, blood and spleen, and the recapitulation of thrombocytopenia. In contrast, following a competitive transplant using minimal numbers of WT HSC together with HSC from a microenvironment with diminished LCM, sufficient WT HSC-generated LCM regulates a normal HSC pool and prevents thrombocytopenia. Importantly, LCM are conserved in humans.
Intestinal stem cell aging signature reveals a reprogramming strategy to enhance regenerative potential
Publisher: Springer Science and Business Media LLC
Date: 16-06-2022
DOI: 10.1038/S41536-022-00226-7
Abstract: The impact of aging on intestinal stem cells (ISCs) has not been fully elucidated. In this study, we identified widespread epigenetic and transcriptional alterations in old ISCs. Using a reprogramming algorithm, we identified a set of key transcription factors ( Egr1, Irf1, FosB ) that drives molecular and functional differences between old and young states. Overall, by dissecting the molecular signature of aged ISCs, our study identified transcription factors that enhance the regenerative capacity of ISCs.
Expansion of Umbilical Cord Blood for Clinical Transplantation
Publisher: Bentham Science Publishers Ltd.
Date: 12-2007
DOI: 10.2174/157488807782793745
Abstract: Since the first successful cord blood transplant was performed in 1988 there has been a gradual increase in the use of cord blood for hemopoietic stem cell transplantation. Worldwide, over 8,000 unrelated cord blood transplants have been performed with the majority being for children with hemopoietic malignancies. Transplantation for adults has increased but is limited by the low number of nucleated cells and CD34(+) cells within a single cord blood collection. Cord blood hemopoietic stem cells are more primitive than their adult counterparts and have high proliferative potential. Cord blood ex vivo expansion is designed to improve transplant outcomes by increasing the number of hemopoietic stem cells with long term repopulating potential and their differentiated progeny. However, despite a large amount of research activity during the last decade, this aim has not been realized. Herein we discuss the rationale for this approach culture methods for ex vivo expansion, ways to assess the functional capacity of ex vivo generated hemopoietic stem cells and clinical outcomes following transplantation with ex vivo expanded cord blood.
Bone, microenvironment and hematopoiesis
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 07-2012
Adhesion receptor expression by hematopoietic cell lines and murine progenitors
Publisher: Elsevier BV
Date: 03-1999
DOI: 10.1016/S0301-472X(98)00037-X
Abstract: Hematopoietic progenitor cells are incubated with cytokine combinations for in vitro expansion of stem cells and to enhance retrovirus-mediated gene transfer. Optimization of the engraftment of these treated cells would be critical to the success of stem cell transplantation or gene therapy. Previous studies demonstrated that a 48-hour incubation of donor BALB/c bone marrow with a mixture of four cytokines (IL-3, IL-6, IL-11, and SCF), resulted in expansion of primitive progenitor/stem cells but a loss of long-term engraftment in nonmyeloablated or myeloablated recipients. We have established the expression pattern for a number of adhesion receptors by normal hematopoietic progenitors and cell lines and the modulation in expression induced by cytokines or cell cycle progression to ascertain the molecular basis for such defective engraftment. Northern blot analysis demonstrated that the cytokine combination of IL-3, IL-6, IL-11, and SCF dramatically down-regulated alpha 4 integrin receptor expression in HL-60 cells. Synchronized FDC-P1 cells exhibited modulation of alpha 4 expression through cell cycle progression, both by quantitative RT-PCR and flow cytometry. Normal murine bone marrow lineage-depleted, Sca+ cells expressed a number of adhesion receptors, including alpha L, alpha 1, alpha 3, alpha 4, alpha 5, alpha 6, beta 1, L-selectin, CD44, and PECAM as assessed by flow cytometry, immunofluorescence, and RT-PCR. There was modulation of the expression of several of these receptors after incubation in the four cytokines for 24 and/or 48 hours: the proportion of cells expressing alpha L, alpha 5, alpha 6, and PECAM increased, whereas the proportion of cells expressing alpha 4 and beta 1 decreased, after cytokine incubation. There was a demonstrable concomitant decline in adhesion of these cells to fibronectin after the cytokine incubation, a finding that correlates with the decrease in expression of alpha 4. These changes in adhesion receptor expression and function with cytokines and during cell cycle transit may be critical to stem cell homing and engraftment after transplantation, as multiple receptors could be involved in the process of rolling, attachment to endothelium, endothelial transmigration, and migration within the marrow space.
Osteopontin is An Important Regulative Component of the Fetal Bone Marrow Hematopoietic Stem Cell Niche
Publisher: MDPI AG
Date: 27-08-2019
DOI: 10.3390/CELLS8090985
Abstract: Osteopontin (OPN) is an important component in both bone and blood regulation, functioning as a bridge between the two. Previously, thrombin-cleaved osteopontin (trOPN), the dominant form of OPN in adult bone marrow (BM), was demonstrated to be a critical negative regulator of adult hematopoietic stem cells (HSC) via interactions with α4β1 and α9β1 integrins. We now demonstrate OPN is also required for fetal hematopoiesis in maintaining the HSC and progenitor pool in fetal BM. Specifically, we showed that trOPN is highly expressed in fetal BM and its receptors, α4β1 and α9β1 integrins, are both highly expressed and endogenously activated on fetal BM HSC and progenitors. Notably, the endogenous activation of integrins expressed by HSC was attributed to high concentrations of three alent metal cations, Ca2+, Mg2+ and Mn2+, which were highly prevalent in developing fetal BM. In contrast, minimal levels of OPN were detected in fetal liver, and α4β1 and α9β1 integrins expressed by fetal liver HSC were not in the activated state, thereby permitting the massive expansion of HSC and progenitors required during early fetal hematopoiesis. Consistent with these results, no differences in the number or composition of hematopoietic cells in the liver of fetal OPN-/- mice were detected, but significant increases in the hematopoietic progenitor pool in fetal BM as well as an increase in the BM HSC pool following birth and into adulthood were observed. Together, the data demonstrates OPN is a necessary negative regulator of fetal and neonatal BM progenitors and HSC, and it exhibits preserved regulatory roles during early development, adulthood and ageing.
Hematopoietic microenvironment and age
Publisher: Cambridge University Press
Date: 20-12-2007
In situ detection of individual transplanted bone marrow cells using FISH on sections of paraffin-embedded whole murine femurs.
Publisher: SAGE Publications
Date: 09-1996
DOI: 10.1177/44.9.8773573
Abstract: Studies of transplantation biology rely on the detection of donor hemopoietic cells in transplant recipients. Traditionally this has been achieved through ex vivo techniques, including flow cytometric analysis of cell surface markers to detect cells expressing specific epitopes, histochemical detection of cytoplasmic proteins, and the detection of Y chromosome-specific sequences by DNA hybridization. Studies using congenic models, such as the Ly5.1/5.2 mouse, or the utilization of fluorescent dyes, such as PKH-26, have allowed more in-depth analysis of transplantation, beginning to address key issues such as cell homing through cell tracking and elucidation of the "stem cell niche." However, these methods are limited by labeling sensitivity, specificity, crossreactivity and, in the case of PKH-26 labeling, the number of cell isions the transplanted cells can make before the signal disappears. We have developed a fluorescent in situ hybridization (FISH) technique that utilizes a murine Y chromosome-specific "painting" probe to identify in situ in idual transplanted male cells in paraffin-embedded sections of female whole bone marrow while maintaining good morphological integrity. This method is highly sensitive and specific, labeling more than 99% of male cells and no female cells, allowing each transplant to be assessed at the in idual cell level. The technique provides unique opportunities to follow the path taken by transplanted cells, both during homing into the marrow and through their maturation and differentiation into mature, functional hemopoietic cells.
Lymphohematopoietic Stem Cell Engraftmenta
Publisher: Wiley
Date: 04-1999
DOI: 10.1111/J.1749-6632.1999.TB08451.X
Abstract: Traditional dogma has stated that space needs to be opened by cytoxic myeloablative therapy in order for marrow stem cells to engraft. Recent work in murine transplant models, however, indicates that engraftment is determined by the ratio of donor to host stem cells, i.e., stem cell competition. One hundred centigray whole body irradiation is stem cell toxic and nonmyelotoxic, thus allowing for higher donor chimerism in a murine syngeneic transplant setting. This nontoxic stem cell transplantation can be applied to allogeneic transplant with the addition of a tolerizing step in this case presensitization with donor spleen cells and administration of CD40 ligand antibody to block costimulation. The stem cells that engraft in the nonmyeloablated are in G0, but are rapidly induced (by 12 hours) to enter the S phase after in vivo engraftment. Exposure of murine marrow to cytokines (IL-3, IL-6, IL-11 and steel factor) expands progenitor clones, induces stem cells into cell cycle, and causes a fluctuating engraftment phenotype tied to phase of cell cycle. These data indicate that the concepts of stem cell competition and fluctuation of stem cell phenotype with cell cycle transit should underlie any new stem cell engraftment strategy.
Haematopoietic Radioprotection by Cremophor EL: A Polyethoxylated Castor Oil
Publisher: Informa UK Limited
Date: 1995
DOI: 10.1080/09553009514550071
Abstract: The polyethoxylated castor oil, Cremophor EL (Cremophor) is approved for human use as a vehicle for oral and intravenous administration of water-insoluble compounds. Cremophor has also previously been shown to reverse the multidrug resistance phenotype at clinically acceptable doses. This study demonstrates that doses of Cremophor in the range of 25-50 microliters/kg intravenously (i.v.) administered 1 day prior to near-lethal irradiation protected the regenerative capacity of the marrow, resulting in haematopoietic radioprotection and long-term survival of near-lethally-irradiated mice. In normal mice, Cremophor administration (1) markedly reduced the level of serum haematopoietic inhibitory activity 4-8 h following injection (2) resulted in a transient decrease in femoral bone marrow cellularity and upregulated B220 (B cells), and 7/4 (neutrophils and activated macrophages), but not Thy-1 (T-cells) surface antigen expression in bone marrow cells within 24 h of injection and (3) transiently elevated the incidence of both primitive and committed haematopoietic progenitor cells detected in clonal agar culture within 48 h of injection. Bone marrow progenitor cell content, and peripheral blood white cell, platelet and reticulocyte counts were unaffected. This suggests that the haematopoietic radioprotection and recovery observed in irradiated mice pretreated with Cremophor may be the result of accessory cell activation and/or modulation of accessory factors regulating haematopoietic progenitor cells. Our data suggest a potential clinical use of Cremophor as an adjunct to, or as a substitute for, cytokines to minimize myelosuppression following cytotoxic therapy.
Related Organisations
Commonwealth Scientific And Industrial Research Organisation
Location: Australia
Related Funding Activities
ARC Future Fellowships - Grant ID: FT0991965
Start Date: 2010
End Date: 12-2013
Amount: $788,800.00
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View Funded ActivityIndustrial Transformation Training Centres - Grant ID: IC180100021
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End Date: 08-2025
Amount: $4,163,359.00
Funder: Australian Research Council
View Funded ActivityDiscovery Projects - Grant ID: DP130103858
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View Funded ActivitySpecial Research Initiatives - Grant ID: SR1101002
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