Contribution of HGF to Liver Regeneration by Strong Mobilization of Bone Marrow Stem Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1680-1680
Author(s):  
Fumihito Tajima ◽  
Hiroyuki Tsuchitya ◽  
Kenichi Nishikawa ◽  
Toshirou Okazaki ◽  
Goushi Shiota
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Liver Regeneration ◽  
Peripheral Blood ◽  
Wild Type Mouse ◽  
Bone Marrow Stem Cell ◽  
Bone Marrow Stem Cells ◽  
Wild Type ◽  
Cd34 Cells ◽  
Marrow Stem Cell

Abstract Cell plasticity of bone marrow stem cells to hepatocytes is known, however, the details are still unclear. hepatocyte growth factor (HGF) promotes an increase in liver stem cells in severely injured liver, but intervention to bone marrow stem cell is unclear. We examined a role of HGF in bone marrow stem cell-mediated liver regeneration in order to obtain effective liver regeneration. First, we found that the phenotype of stem cells, which can differentiate into hepatocytes, is Lin−c-kit+Sca-1+CD34− in bone marrow or Lin−c-kit+Sca−1+CD34+ in peripheral blood mobilized by G-CSF. We transplanted single Lin−c-kit+Sca-1+CD34− bone marrow cell harvested from male EGFP mouse to female wild type mouse, and then, using this GFP-chimera-mouse, we found that bone marrow origin GFP+ and Y chromosome+ hepatic cells were present in liver after acute liver damage. Next, single Lin−c-kit+Sca-1+CD34+ peripheral blood cell, which was mobilized in peripheral blood by administration of G-CSF, was transplanted into the portal vein of the wild type mouse which was given hepatic damage. We found that the GFP-positive cells also expressed albumin. Second, we investigated whether the HGF can mobilize stem cells from bone marrow to peripheral blood. In peripheral blood of HGF transgenic mice, 1.1% developed CD34+ cells and 20±3 colony forming cells of 1X106 peripheral blood mononuclear cells were shown. Colony forming cells were found in the mouse into which an HGF-expressing adenovirus was administered. After injection of rHGF to mice, a significant time-dependent increase of percentage of CD34+ cells in the PB was noted at the first 3 hours and CD34+ cells were increased in dose-dependent manner of rHGF and reached plateau level at 100 m/kg. The mice having transplantation with PB cells from 100 mg HGF-treated mice for 4days showed engraftment 2 months after transplantation. Upon activation of rHGF in mouse MS-5 stromal cells, phosphorylation c-Met and SCF were up-regulated, while VCAM -1, MMP-9, SDF -1 or CXCR4 were not changed. SCF level in conditioned media was also increased after the HGF stimulation. Finally, we examined whether the bone marrow stem cells in PB mobilized by HGF transdifferentiate into hepatocytes. Using GFP-chimera-mice given acute liver injury after administration of retorolusine and CCl4, the levels of GFP+ cells in liver of GFP-chimera-mice 2 months after treatment by PBS and HGF were 2.2±1.4% and 12.7±3.6%, respectively (p<0.01). In conclusion, HGF can mobilize stem cells with long-term engraftment capabilities from bone marrow to peripheral blood, resulting in contribution to liver regeneration.

Erythropoietin Signaling Inhibits the Short-Term Marrow Reconstitution.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3028-3028
Author(s):  
Ko-Tung Chang ◽  
Yves D. Pastore ◽  
Roberto H. Nussenzveig ◽  
Josef T. Prchal
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Peripheral Blood ◽  
Relative Proportion ◽  
Wild Type Mouse ◽  
Erythropoietin Receptor ◽  
Wild Type ◽  
Short Term ◽  
Gain Of Function ◽  
Hematopoietic Stem

Abstract Murine hematopoietic stem cells (HSCs) transfected with gain-of-function human erythropoietin receptor (EPOR) transgene were reported to have a competitive advantage over wild type mouse HSCs in a bone marrow transplantation (BMT) model (Kirby, Blood; 95(12):3710, 2000). However, EpoRs may not be normally expressed in early progenitor/stem cells, moreover, whether the Epo/EpoR signaling plays a role on homing and repopulating of hematopoietic progenitor/stem cells is also unknown. Our lab has previously created a mouse model harboring either a knocked-in human wild type (wthEPOR) hypomorphic gene or a mutant human gain-of-function EPOR (mthEPOR) gene into the mouse EPOR locus (Divoky, PNAS; 98(3):986, 2001). The wthEPOR mice are anemic, while mthEpoR mice are polycythemic. We tested the possible advantage of mthEPOR HSCs in the competitive bone marrow (BM) transplantation assay using C57/Bl6 congenic mice. BM from wthEPOR (CD45.1/45.2 heterozygous or CD45.1) and mthEPOR (CD45.2) mice were co-transplanted (1:1 ratio) into lethally irradiated (137Cs &gt;11Gy split) normal recipients (CD45.1 or CD45.1/45.2). Unexpectedly, in three independent groups the peripheral blood chimerism derived from wthEPOR-bearing cells significantly out-competed the mthEPOR-bearing cells two weeks after BMT (52% vs. 40%, 52% vs. 45% and 50% vs. 42%, n=11, 8 and 6, p&lt;0.01). Furthermore, administration of exogenous Epo (10U/day/mouse) further impaired peripheral blood engraftment (including Ter119+ erythroid lineages) of mthEPOR cells (45% vs. 39%, n=8, p&lt;0.05) though the relative proportion of erythroid cells within the engraftment was increased. Thus, an enhanced Epo/EpoR signaling interferes with the short-term repopulation of hematopoietic progenitors resulting in a decreased engraftment, possibly by interfering with homing. A possible long-term advantage of mthEPOR HSCs is being evaluated in ongoing studies; however, these short term data suggest that Epo administration to BM transplant patients may impede engraftment. Figure Figure

Cardiac chimerism in recipients of peripheral-blood and bone marrow stem cells

2004 ◽  
Vol 6 (4) ◽  
pp. 399-402 ◽  
Author(s):  
Antoni Bayes-Genis ◽  
Eduardo Muñiz-Diaz ◽  
Lluis Catasus ◽  
Marina Arilla ◽  
Carmen Rodriguez ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Peripheral Blood ◽  
Bone Marrow Stem Cells

scholarly journals Differential effects of recombinant thrombopoietin and bone marrow stromal-conditioned media on neonatal versus adult megakaryocytes

Blood ◽  
2006 ◽  
Vol 108 (10) ◽  
pp. 3360-3362 ◽  
Author(s):  
Karen M. Pastos ◽  
William B. Slayton ◽  
Lisa M. Rimsza ◽  
Linda Young ◽  
Martha C. Sola-Visner
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Peripheral Blood ◽  
Conditioned Media ◽  
Valuable Source ◽  
Cd34 Cells ◽  
Adult Bone

Abstract Umbilical cord blood (CB) is a valuable source of stem cells for transplantation, but CB transplantations are frequently complicated by delayed platelet engraftment. The reasons underlying this are unclear. We hypothesized that CB- and peripheral-blood (PB)–derived megakaryocytes (MKs) respond differently to the adult hematopoietic microenvironment and to thrombopoietin (Tpo). To test this, we cultured CB- and PB-CD34+ cells in adult bone marrow stromal conditioned media (CM) or unconditioned media (UCM) with increasing concentrations of recombinant Tpo and compared the effects of these conditions on CB-versus PB-MKs. PB-MKs reached highest ploidy in response to UCM + 100 ng/mL rTpo, and the addition of CM inhibited their maturation. In contrast, CB-MKs reached highest ploidy in CM without rTpo, and high rTpo concentrations (> 0.1 ng/mL) inhibited their maturation. This is the first evidence that human neonatal and adult MKs have substantially different biologic responses to Tpo and potentially to other cytokines.

Human Embryonic Stem Cell (hESC)-Derived Hematopoietic Elements Are Capable of Engrafting Primary as well as Secondary Fetal Sheep Recipients.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2685-2685
Author(s):  
A. Daisy Narayan ◽  
Jessica L. Chase ◽  
Adel Ersek ◽  
James A. Thomson ◽  
Rachel L. Lewis ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Peripheral Blood ◽  
Fetal Sheep ◽  
Hematopoietic Stem ◽  
Blood Samples ◽  
Human Dna ◽  
Cd34 Cells ◽  
Hematopoietic Activity

Abstract We used transplantation into 10 and 20 pre-immune fetal sheep recipients (55–65 days-old, term: 145 days) to evaluate the in vivo potential of hematopoietic elements derived from hESC. The in utero human/sheep xenograft model has proven valuable in assessing the in vivo hematopoietic activity of stem cells from a variety of fetal and post-natal human sources. Five transplant groups were established. Non-differentiated hESC were injected in one group. In the second and third group, embroid bodies differentiated for 8 days were injected whole or CD34+ cells were selected for injection. In the fourth and fifth group, hESC were differentiated on S17 mouse stroma layer and injected whole or CD34+ cells were selected for injection. The animals were allowed to complete gestation and be born. Bone marrow and peripheral blood samples were taken periodically up to over 12 months after injection, and PCR and flowcytometry was used to determine the presence of human DNA/blood cells in these samples. A total of 30 animals were analyzed. One primary recipient that was positive for human hematopoietic activity was sacrificed and whole bone marrow cells were transplanted into a secondary recipient. We analyzed the secondary recipient at 9 months post-injection by PCR and found it to be positive for human DNA in its peripheral blood and bone marrow. This animal was further challenged with human GM-CSF and human hematopoietic activity was noted by flowcytometry analyses of bone marrow and peripheral blood samples. Further, CD34+ cells enriched from its bone marrow were cultured in methylcellulose and human colonies were identified by PCR. We therefore conclude that hESC are capable of generating hematopoietic cells that engraft in 1° sheep recipients. These cells also fulfill the criteria for long-term engrafting hematopoietic stem cells as demonstrated by engraftment and differentiation in the 20 recipient.

Purified T Depleted Peripheral Blood and Bone Marrow Cd34 Transplantation from Haploidentical Mother to Child with Thalassemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3106-3106
Author(s):  
Pietro Sodani ◽  
Buket Erer ◽  
Javid Gaziev ◽  
Paola Polchi ◽  
Andrea Roveda ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
T Cell ◽  
Peripheral Blood ◽  
Therapeutic Option ◽  
B Cell Lymphoma ◽  
Marrow Transplant ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Approximately 60% of thalassemic patients can not apply to “gene therapy today” which the insertion of one allogenic HLA identical stem cell into the empty bone marrow as the vector of the normal gene for beta globin chain synthesis. We studied the use of the haploidentical mother as the donor of hematopoietic stem cells assuming that the immuno-tollerance established during the pregnancy will help to bypass the HLA disparity and allow the hemopoietic allogeneic reconstitution in the thalassemic recipient of the transplant. We have employed a new preparative regimen for the transplant in fourteen thalassemic children aged 3 to 12 years (median age 5 years) using T cell depleted peripheral blood stem cell (PBSCTs) plus bone marrow (BM) stem cells. All patients received hydroxyurea (OHU) 60 mg/kg and azathioprine 3 mg/kg from day -59 until day-11, fludarabine (FLU) 30 mg/m 2 from day -17 to day -11, busulphan (BU) 14 mg/kg starting on day -10, and cyclophosphamide(CY) 200mg/kg, Thiotepa 10 mg/kg and ATG Sangstat 2.5 mg/kg, followed by a CD34 + t cell depleted (CliniMacs system), granulocyte colony stimulating factor (G-csf) mobilized PBSC from their HLA haploidentical mother. The purity of CD34+ cells after MACS sorting was 98–99%, the average number of transplanted CD34+ cells was 15, 4 x 10 6/kg and the average number of infused T lymphocytes from BM was 1,8 x 10 5/Kg.The patients received cyclosporin after transplant for graft versus host disease(GVHD) prophylaxis during the first two months after the bone marrow transplantation. Results. Thirteen patients are alive. Four patients rejected the transplant and are alive with thalassemia One patients died six months after bone marrow transplant for central nervous system diffuse large B cell lymphoma EBV related. Nine patients are alive disease free with a median follow up of 30 months (range12–47). None of the seven patients showed AGVHD and CGVHD. This preliminary study suggest that the transplantation of megadose of haploidentical CD34+ cell from the mother is a realistic therapeutic option for those thalassemic patients without genotipically or phenotipically HLA identical donor.

Sparc Is Dispensable for Murine Hematopoiesis, Despite Its Suspected Role in 5q- Myelodysplastic Syndrome

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4822-4822
Author(s):  
Kavitha Siva ◽  
Pekka Jaako ◽  
Kenichi Miharada ◽  
Emma Rörby ◽  
Mats Ehinger ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Knockout Mice ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Lethal Dose ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Normal Peripheral Blood ◽  
Expression Levels

Abstract Abstract 4822 Hematopoiesis is a complex process where a limited number of stem cells give rise to all mature blood cells. It involves interplay of several factors, many of which are yet to be identified. In a search for novel regulators of hematopoiesis, we chose to study SPARC (Secreted Protein Acidic and Rich in Cysteine, also known as Osteonection and BM40) because it is downregulated upon hematopoietic differentiation (Bruno et al., Mol Cell Biol, 2004) and might therefore play a role in the regulation of hematopoietic stem cells (HSC). SPARC is a matricellular protein that forms a major component of bone and is ubiquitously expressed in a variety of tissues. It is the founding member of a family of SPARC-like proteins. Several publications have indicated an important role for SPARC in hematopoiesis. In particular – knockdown of SPARC in zebrafish embryos resulted in an altered number of circulating blood cells, and a knockout mouse model showed thrombocytopenia and reduced erythroid colony formation. We carried out an in depth phenotypic and functional analysis of the hematopoietic system of SPARC knockout mice; using it as a model to gain insight into the role of SPARC in hematopoiesis. These mice are viable and fertile but show severe osteopenia and age-onset cataract at about six months of age. They also show an altered response to tumour growth and wound healing. We used mice (129SVJ background) (Gilmour et al. EMBO, 1998) that were less than six months old. These mice had normal peripheral blood counts and the bone marrow and spleen showed no alterations in morphology or cellularity. A detailed phenotypic analysis of precursors within the bone marrow showed no significant differences in myelo-erythroid precursors as compared to wild types (n=6). Though in vitro, the precursors showed lower ability to form BFU-E (n=5, p=0.048). In transplantations of lethally irradiated recipient mice, SPARC knockout cells gave rise to multi-lineage long-term reconstitution. Also, when competed with wild type cells, they provided reconstitution as well as their wild type counterparts. When SPARC knockout mice (n=8) were transplanted with wild type cells, there was normal reconstitution, indicating that a SPARC deficient niche can fully support normal hematopoiesis. We also tested if SPARC deficient mice respond differently to hematopoietic stress. We subjected mice (n=7) to sub lethal dose of irradiation and to experimentally induced anemia (n=7) and followed recovery by analyzing peripheral blood counts. In both SPARC knockouts and wild type mice, the blood counts recovered in a similar fashion. In conclusion, we find that SPARC is dispensable for murine hematopoiesis. It is possible that there are compensatory mechanisms involving other members of the SPARC family that ultimately lead to normal hematopoiesis in the murine model. In humans, SPARC maps to the deleted region in 5q MDS and has been reported to be 71 % down regulated in patient samples (Lehmann et al. Leukemia, 2007). It is the most prominent gene that is up regulated in response to lenalidomide, a drug that inhibits the malignant clone (Pellagatti et al. PNAS, 2007). SPARC is thus increasingly speculated to be involved in the pathophysiology of this hematopoetic disease. We analysed the expression levels of SPARC mRNA in the hematopoietic stem/progenitor cell compartment and found high expression levels in the CD34+ fraction of human cord blood cells. In contrast, there is very low level of SPARC expression in all compartments of murine HSCs. Therefore SPARC function may play a more important role in human hematopoiesis than in murine blood cell regulation. Disclosures: No relevant conflicts of interest to declare.

Different kinases acting on stathmin (oncoprotein, Op18) in human healthy and malignant hematopoietic stem cells

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 17527-17527
Author(s):  
H. Lannert ◽  
T. Able ◽  
S. Leicht ◽  
R. Saffrich ◽  
V. Eckstein ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Expression Profiles ◽  
Proteome Analysis ◽  
Becton Dickinson ◽  
Hematopoietic Stem ◽  
Cd34 Cells

17527 Background: Stathmin/Op18 is a cytosolic phosphoprotein which regulates the dynamics of microtubules. This regulation is important in mitosis during cell division and in the migration of cells in modification of the cytoskeleton. The process of tumor proliferation and metastasis is characterized by high rates of mitosis and migration into distant tissues. Stathmin itself is regulated by kinases through phosphorylation of mainly 4 different serin sides. In this study, we investigated stathmin- and its kinases expression in native hematopoietic CD34+ stem cells (HSCs) from bone marrow (BM) in comparison to mobilized peripheral blood stem cells (mPBSCs) from G-CSF stimulated donors and leukemic CD34+ cells from patients with AML. Methods: Mononuclear cells were isolated by a standard Ficoll-Hypaque gradient separation method from the different blood sources. An Auto-MACS (Miltenyi) and FACS Vantage SE cell sorter (Becton Dickinson) was used to highly enrich (>99%) CD34+ cells fractions. In comparative proteome analysis, we detected the protein expression of stathmin in mPBSCs, AML CD34+ cells, and in native HSCs from BM. We performed microarray-based gene expression profiles of these cells and focused on kinases regulating stathmin’s activity. Furthermore, we monitored stathmin and its relevant kinases by FACS analyses of the enriched cell fractions and by fluorescence microscopy of bone marrow smears and cytospins. Results: In this study, we have shown in comparative proteome analysis (Q-TOF-MS/MS) that stathmin is expressed in G-CSF mobilized hematopoietic stem cells for the first time and in AML cells. In microarray analysis we indentified up- and down-regulated kinases: MAPK, PAK1, PKC beta/zeta, MEKK3 and CDKs. Accordingly, we demonstrated in FACS analyses and in immunofluorescence microscopy the high intracellular expression of PKCzeta in AML cells and MEKK3 as well PAK1 in mPBSCs. Conclusions: Our findings show that G-CSF stimulates Stathmin expression in mPBSCs and plays a key role in migration into peripheral blood. Furthermore, we show the different expression of kinases acting on stathmin in mPBSCs and AML cells. Consequently, stathmin and its relevant kinases promise to become a future target in therapies of malignant processes. No significant financial relationships to disclose.

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