Exploring the Molecular Mechanisms for Enhancing MSC Homing and Lodgement within Sites of Liver Damage/Fibrosis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1690-1690
Author(s):  
Phillip E. Herrbrich ◽  
Todd E. Meyerrose ◽  
Jan A. Nolta
Keyword(s):  
Stem Cells ◽  
Growth Factor ◽  
Liver Injury ◽  
Hematopoietic Stem Cells ◽  
Liver Damage ◽  
Molecular Mechanisms ◽  
Cellular Protein ◽  
Urokinase Plasminogen Activator Receptor ◽  
Hematopoietic Stem ◽  
Pre Treatment

Abstract Exogenous stem cells such as human mesenchymal stem cells (MSC) and hematopoietic stem cells (HSC) have been reported to be applicable for tissue repair, as in the case of chronic liver injury. However, one of the limitations in achieving high enough level of stem cell engraftment at these damaged sites is the low level of recruitment of exogenous stem cells. Our goals in the current studies are to enhance recruitment of human MSC to sites of tissue damage and to equip the MSC with high enzymatic activity to digest through areas of fibrosis surrounding of the damaged tissue, in this case, an immune deficient mouse model of CCl4-mediated liver damage as we have previously reported (Wang et al, 2003). Pretreatment of HepG2 cells with hypoxia significantly increased the levels of the hepatocyte growth factor (HGF) receptor, c-met. Since HGF is both a growth factor and a chemotactic agent found at high levels within sites of hypoxic liver injury, we hypothesized that pre-treatment of MSC with hypoxia would increase the expression of c-met and thus, would enhance the sensitivity of MSC to the chemotactic effect of HGF. First, we demonstrated that in the absence of serum and any exogenous growth factor, a 16-hr hypoxic treatment of MSC increased the total cellular protein level of c-met, as well as the total cellular phosphotyrosine activity following HGF stimulation. 3T3 fibroblasts engineered to secrete human HGF were plated in the bottom well of the transmigration assay and human PKH26-labeled MSC pre-incubated in hypoxic vs. normoxic conditions were added to the upper transwell. Transmigrated cells were enumerated after 3 hours to measure the functional role of c-met induction. Preliminary results show enhanced directional transmigration of hypoxia pre- treated MSC toward an HGF gradient, when compared to normoxic treated MSC. Next, we addressed the issue of liver fibrosis as a possible physical barrier that could block the entry of exogenously recruited MSC into the sites of liver damage. We demonstrated that the combination of hypoxia and HGF modulated the degree of matrix metalloproteinase (mmp2 and mmp9) activity in MSC. In addition, recent studies identified the presence of urokinase plasminogen activator receptor (uPAR) on hematopoietic stem cells. We show here for the first time, that uPAR can also be found on MSC. Ongoing studies are assessing the changes in uPAR and uPA activity following hypoxic culture. We hypothesize that changes in MMP and uPA activity can affect the ability of MSC to engraft/lodge within the fibrotic tissue. In conclusion, we propose that the pre-treatment of MSC with hypoxia and HGF might not only enhance the migration of exogenous stem cells to the site of liver damage but also enhance their ability to degrade the accumulated fibrosis at sites of injury for entry into the regions of damaged tissue.

scholarly journals Basic fibroblast growth factor mediates its effects on committed myeloid progenitors by direct action and has no effect on hematopoietic stem cells

Blood ◽  
1995 ◽  
Vol 86 (6) ◽  
pp. 2123-2129 ◽  
Author(s):  
AC Berardi ◽  
A Wang ◽  
J Abraham ◽  
DT Scadden
Keyword(s):  
Stem Cells ◽  
Growth Factor ◽  
Hematopoietic Stem Cells ◽  
Fibroblast Growth Factor ◽  
Colony Formation ◽  
Fibroblast Growth ◽  
Colony Stimulating Factor ◽  
Hematopoietic Stem ◽  
Myeloid Progenitor ◽  
Stimulating Factor

Basic fibroblast growth factor or fibroblast growth factor-2 (FGF) has been shown to affect myeloid cell proliferation and hypothesized to stimulate primitive hematopoietic cells. We sought to evaluate the effect of FGF on hematopoietic stem cells and to determine if FGF mediated its effects on progenitor cells directly or through the induction of other cytokines. To address the direct effects of FGF, we investigated whether FGF induced production of interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha, IL-6, granulocyte colony- stimulating factor, or granulocyte-macrophage colony-stimulating factor by two types of accessory cells, bone marrow (BM) fibroblasts and macrophages. We further evaluated whether antibodies to FGF-induced cytokines affected colony formation. To determine if FGF was capable of stimulating multipotent progenitors, we assessed the output of different colony types after stimulation of BM mononuclear cells (BMMC) or CD34+ BMMC and compared the effects of FGF with the stem cell active cytokine, kit ligand (KL). In addition, a subset of CD34+ BMMC with characteristics of hematopoietic stem cells was isolated by functional selection and their response to FGF was evaluated using proliferation, colony-forming, and single-cell polymerase chain reaction (PCR) assays. We determined that FGF had a stimulatory effect on the production of a single cytokine, IL-6, but that the effects of FGF on colony formation were not attributable to that induction. FGF was more restricted in its in vitro effects on BM progenitors than KL was, having no effect on erythroid colony formation. FGF did not stimulate stem cells and FGF receptors were not detected on stem cells as evaluated by single-cell reverse transcription PCR. In contrast, FGF receptor gene expression was detected in myeloid progenitor populations. These data support a directly mediated effect for FGF that appears to be restricted to lineage-committed myeloid progenitor cells. FGF does not appear to modulate the human hematopoietic stem cell.

scholarly journals Inhibition of day-12 spleen colony-forming units by a monoclonal antibody to the murine macrophage/monocyte colony-stimulating factor receptor

Blood ◽  
1995 ◽  
Vol 85 (10) ◽  
pp. 2731-2734 ◽  
Author(s):  
GL Gilmore ◽  
RK Shadduck
Keyword(s):  
Stem Cells ◽  
Growth Factor ◽  
Hematopoietic Stem Cells ◽  
Growth Factor Receptor ◽  
Hematopoietic Growth Factor ◽  
Colony Stimulating Factor ◽  
Hematopoietic Stem ◽  
Colony Forming Units ◽  
Stimulating Factor ◽  
Specific Control

Primitive hematopoietic stem cells differentiate into committed progenitors that are thought to selectively express hematopoietic growth factor receptor(s), thereby acquiring hematopoietic growth factor responsiveness. To assess whether hematopoietic stem cells express hematopoietic growth factor receptors, the progenitor activity of bone marrow (BM) fractions, isolated by expression of receptors for macrophage/monocyte colony-stimulating factor (M-CSF), were examined. Recovery of day-12 spleen colony-forming units (CFU-S) is diminished in both M-CSF receptor-positive (M-CSFR+) and M-CSFR-fractions, indicating antibody inhibition of day-12 CFU-S. Incubation of BM cells with antibody without fractionation inhibits 50% to 60% of day-12 CFU-S. This inhibition is specific (control antibodies have no effect) and reversible by removal of bound antibody at low pH. Incubating BM cells with control or antireceptor antibody does not affect day-8 CFU-S, which are predominantly erythroid. Treating sublethally irradiated mice with antibody inhibits endogenous day-12 CFU-S. These results indicate that some early progenitors express M-CSFRs, and blocking M-CSFRs inhibits the ability of these progenitors to form colonies, possibly because of inactivation caused by prolonged receptor blockade.

Valproic Acid Results In Maintenance but Not Expansion of Transplantable Hematopoietic Stem Cells From Human Umbilical Cord Blood

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 827-827
Author(s):  
Hiroto Araki ◽  
Sudhakar Baluchamy ◽  
Benjamin Petro ◽  
Mirza Saqib Baig ◽  
Montha Suhangul ◽  
...  
Keyword(s):  
Stem Cells ◽  
Valproic Acid ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Molecular Mechanisms ◽  
Epigenetic Modifications ◽  
Human Umbilical Cord ◽  
Gene Transcript ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 827 Epigenetic modifications are considered to be important in determining the fate of hematopoietic stem cells (HSC). We previously demonstrated that the sequential addition of the chromatin-modifying agents (CMA) 5-aza-2′-deoxycytidine (5azaD) and trichostatin A (TSA) expands transplantable HSC (Araki et al. Blood 2007, Exp Hematol 2009). Others have shown that valproic acid (VPA), an HDAC inhibitor, also expands HSC (DeFelice et al. Cancer Res 2005). We thus compared the efficacy of 5azaD/TSA and VPA in promoting the ex vivo expansion of human cord blood (CB) HSC. Cells were incubated with cytokines alone (SCF, Flt3 ligand, TPO and IL-3) or with cytokines and either 5azaD/TSA or VPA, resulting in 2.2-fold, 10.7-fold or 65-fold expansion, respectively, of primitive CD34+CD90+ cells after 9 days (n=3, Cytokine alone vs. VPA p=0.004; Cytokine alone vs. 5azaD/TSA p=0.03; VPA vs. 5azaD/TSA p=0.003). Interestingly, the 10.7-fold expansion of CD34+CD90+ cells following 5azaD/TSA treatment correlated with a 10- and 10.5-fold expansion of short-term colony-forming cells (CFC) and long-term cobblestone area-forming cells (CAFC), respectively. However, the 65-fold expansion of CD34+CD90+ cells achieved with VPA treatment yielded only a 25.6- and 8.4-fold expansion of CFC and CAFC, respectively. These results suggest a marked discordance between the phenotype and function of CD34+CD90+ cells when they are expanded in VPA, but not in 5azaD/TSA. Thus, we examined the in vivo hematopoietic repopulation potential of CMA-expanded CB HSC by quantitating SCID mouse repopulating cells (SRC) using limiting dilution assays. The frequency of SRC was 1 in 22,000 in primary CB cells (n=29 mice), 1 in 123,315, in (cytokine) controls (n=16 mice), 1 in 21,720 with VPA-treatment (n=27 mice), and 1 in 3,147, in 5azaD/TSA-treated CD34+CD90+ cell cultures (n=22 mice). Unlike control, treatment with VPA prevents loss of SRC but only results in SRC maintenance, whereas 5azaD/TSA treatment leads to a 7-fold expansion of SRC. Furthermore, serial transplantation of bone marrow (BM) from primary recipients engrafted with unmanipulated CB cells resulted in engraftment in 2 of 5 secondary mice, while BM from mice engrafted with VPA-treated cells failed to display secondary engraftment (n=5 mice), whereas BM from mice engrafted with 5azaD/TSA-treated cells resulted in engraftment in 5 of 6 secondary mice. Hence, we conclude that treatment of CB CD34+ cells with 5azaD/TSA or VPA results in distinct SRC outcomes-expansion or maintenance, respectively. To dissect the molecular mechanisms that may mediate these distinct SRC fates, we examined genes implicated in HSC self-renewal including HoxB4, Bmi1, STAT3, Ezh2 and PU.1. These gene transcript levels were increased in CD34+ cells treated with either 5azaD/TSA or VPA when compared to control cultures as measured by real time quantitative PCR. In accordance with these studies, CHIP assays using antibody against acetylated histone H4 indicate increased acetylation of the promoters of HoxB4 and Bmi1 genes in both VPA- and 5azaD/TSA-treated cells. In addition, higher levels of HoxB4, Ezh2 and PU.1 proteins were observed in VPA- and 5azaD/TSA-expanded cells, compared to control cultures. Since VPA treatment does not result in SRC expansion, these observations raise questions as to the importance of the upregulation of these genes for HSC expansion. Since the pharmacologic activity of CMAs is short (hours) we hypothesize that temporal effects, including early epigenetic modifications, lead to changes in transcription factor expression, which directly or indirectly promote symmetric or asymmetric divisions ultimately resulting in expansion or maintenance of HSC. Importantly, our global microarray data (n=3) using a human genome affymetrix chip (U133 plus 2.0) revealed a set of differentially expressed genes present in 5azaD/TSA- but not in VPA-expanded CD34+ cells, thus uncovering a potential molecular signature for HSC expansion. Currently, we are examining the molecular interactions of these signature genes and the effects of silencing of these genes on HSC expansion or maintenance which should allow us to begin to unravel the molecular mechanisms involved. In summary our data indicate that treatment of HSCs with different CMAs results in distinct fates: expansion or maintenance of HSC, an observation of potential therapeutic importance. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The effect of recombinant mast cell growth factor on purified murine hematopoietic stem cells.

1991 ◽  
Vol 173 (5) ◽  
pp. 1205-1211 ◽  
Author(s):  
P de Vries ◽  
K A Brasel ◽  
J R Eisenman ◽  
A R Alpert ◽  
D E Williams
Keyword(s):  
Stem Cells ◽  
Mast Cell ◽  
Growth Factor ◽  
Cell Growth ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Fold Increase ◽  
Hematopoietic Stem ◽  
Mast Cell Growth Factor

Pluripotent hematopoietic stem cells (PHSC) are very rare cells whose functional capabilities can only be analyzed indirectly. For a better understanding and possible manipulation of mechanisms that regulate self-renewal and commitment to differentiation of PHSC, it is necessary to purify these cells and to develop assays for their growth in vitro. In the present study, a rapid and simple, widely applicable procedure to highly purify day 14 spleen colony-forming cells (day 14 CFU-S) is described. Low density bone marrow cells (rho less than or equal to 1.078 g/cm3) were enriched by two successive light-activated cell sorting procedures. In the first sort, cells within a predetermined light scatter (blast cell) window that are wheat germ agglutinin/Texas Red (WGA/TxR) positive and mAb 15-1.4.1/fluorescein isothiocyanate negative (granulocyte-monocyte marker) were selected. In the second sort, cells were selected on the basis of retention of the supravital dye rhodamine 123 (Rh123). Cells that take up little Rh123 (Rh123 dull cells) and those that take up more Rh123 (Rh123 bright cells) were 237-fold and 132-fold enriched, respectively, for day 14 CFU-S. Both Rh123 fractions were cultured for various time periods in vitro in the presence of mast cell growth factor (MGF), with or without interleukin 3 (IL-3) or IL-1 alpha. Both Rh123 fractions proliferated in response to MGF alone as determined by a [3H]TdR assay or by counting nucleated cells present in the cultures over time. MGF also acted synergistically with both IL-3 and IL-1 alpha to promote stem cell proliferation. Stimulation of both Rh123 fractions with MGF alone did not result in a net increase of day 14 CFU-S. Stimulation with MGF + IL-3 or MGF + IL-alpha resulted in a 4.4- or 2.6-fold increase of day 14 CFU-S in the Rh123 dull fraction, and an 11.6-fold or 2.6-fold increase of day 14 CFU-S in the Rh123 bright fraction, respectively. The data presented in this paper indicate that in vitro MGF acts on primitive hematopoietic stem cells by itself and also is a potent synergistic factor in combination with IL-3 or IL-1 alpha.

Differential gene expression profiling of adult murine hematopoietic stem cells

Blood ◽  
2002 ◽  
Vol 99 (2) ◽  
pp. 488-498 ◽  
Author(s):  
In-Kyung Park ◽  
Yaqin He ◽  
Fangming Lin ◽  
Ole D. Laerum ◽  
Qiang Tian ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Differential Gene Expression ◽  
Molecular Mechanisms ◽  
Expression Profiles ◽  
Cdna Clones ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Differential Gene

Abstract Hematopoietic stem cells (HSCs) have self-renewal capacity and multilineage developmental potentials. The molecular mechanisms that control the self-renewal of HSCs are still largely unknown. Here, a systematic approach using bioinformatics and array hybridization techniques to analyze gene expression profiles in HSCs is described. To enrich mRNAs predominantly expressed in uncommitted cell lineages, 54 000 cDNA clones generated from a highly enriched population of HSCs and a mixed population of stem and early multipotent progenitor (MPP) cells were arrayed on nylon membranes (macroarray or high-density array), and subtracted with cDNA probes derived from mature lineage cells including spleen, thymus, and bone marrow. Five thousand cDNA clones with very low hybridization signals were selected for sequencing and further analysis using microarrays on glass slides. Two populations of cells, HSCs and MPP cells, were compared for differential gene expression using microarray analysis. HSCs have the ability to self-renew, while MPP cells have lost the capacity for self-renewal. A large number of genes that were differentially expressed by enriched populations of HSCs and MPP cells were identified. These included transcription factors, signaling molecules, and previously unknown genes.

scholarly journals Vascular Endothelial Growth Factor and Angiopoietin-1 Stimulate Postnatal Hematopoiesis by Recruitment of Vasculogenic and Hematopoietic Stem Cells

2001 ◽  
Vol 193 (9) ◽  
pp. 1005-1014 ◽  
Author(s):  
Koichi Hattori ◽  
Sergio Dias ◽  
Beate Heissig ◽  
Neil R. Hackett ◽  
David Lyden ◽  
...  
Keyword(s):  
Stem Cells ◽  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Hematopoietic Stem Cells ◽  
Vegf Receptor ◽  
Vascular Endothelial ◽  
Hematopoietic Stem ◽  
Neutralizing Monoclonal Antibody ◽  
Endothelial Growth Factor ◽  
Angiopoietin 1

Tyrosine kinase receptors for angiogenic factors vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) are expressed not only by endothelial cells but also by subsets of hematopoietic stem cells (HSCs). To further define their role in the regulation of postnatal hematopoiesis and vasculogenesis, VEGF and Ang-1 plasma levels were elevated by injecting recombinant protein or adenoviral vectors expressing soluble VEGF165, matrix-bound VEGF189, or Ang-1 into mice. VEGF165, but not VEGF189, induced a rapid mobilization of HSCs and VEGF receptor (VEGFR)2+ circulating endothelial precursor cells (CEPs). In contrast, Ang-1 induced delayed mobilization of CEPs and HSCs. Combined sustained elevation of Ang-1 and VEGF165 was associated with an induction of hematopoiesis and increased marrow cellularity followed by proliferation of capillaries and expansion of sinusoidal space. Concomitant to this vascular remodeling, there was a transient depletion of hematopoietic activity in the marrow, which was compensated by an increase in mobilization and recruitment of HSCs and CEPs to the spleen resulting in splenomegaly. Neutralizing monoclonal antibody to VEGFR2 completely inhibited VEGF165, but not Ang-1–induced mobilization and splenomegaly. These data suggest that temporal and regional activation of VEGF/VEGFR2 and Ang-1/Tie-2 signaling pathways are critical for mobilization and recruitment of HSCs and CEPs and may play a role in the physiology of postnatal angiogenesis and hematopoiesis.

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