The Sooner the Better: Rapid Transduction Enhances the Engraftment/Selection of Gene Corrected Fanconi Anemia HSC.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 194-194 ◽  
Author(s):  
Lars U.W. Muller ◽  
Michael Milsom ◽  
Chad E. Harris ◽  
Jeff Bailey ◽  
David A. Williams
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Bone Marrow ◽  
Stem Cell ◽  
Reporter Gene ◽  
Fanconi Anemia ◽  
Peripheral Blood ◽  
Bone Marrow Cells

Abstract Fanconi anemia (FA) is amenable to genetic correction of hematopoietic stem cells (HSCs). However, as demonstrated in previous clinical gene therapy trials, successful extension of murine studies into human therapies is limited by low numbers of target HSC and poor engraftment of transduced FA HSC (Kelly et al., Mol Ther, 2007). To examine the potential biological consequences/benefits of shortened transduction we used a FA mouse model in which HSC are deficient and prone to excessive loss during in vitro manipulation. We applied a rapid transduction protocol (Mostoslavsky et al., Mol Ther, 2005) utilizing lentiviral vectors and demonstrate that this shortened transduction preserves engraftment of FA HSC to the level of C57BL/6 wt cells. Lin− Sca-1+ c-Kit+ bone marrow cells were isolated from Fanca−/− CD45.2 mice and underwent 4-hr rapid (RT) vs. 96-hr conventional (CT) transduction. An equivalent number of transduced cells were transplanted into lethally irradiated CD45.1 BoyJ mice. Analysis of engraftment chimerism three months post transplantation revealed a significantly higher level of engraftment in animals receiving RT vs. CT cells (90% +/− 14% vs. 26% +/− 31%, respectively, p=<0.01). Rapid transduction also resulted in a significant reduction of engraftment failure (0/36 animals RT vs. 20/36 animals CT). Importantly--emphasizing the FA disease-specific stem cell phenotype, RT vs. CT of C57BL/6 wt cells was associated with no significant difference in engraftment of these cells (93% +/− 1.2% RT vs. 84 +/− 19% CT, p=0.33). Analysis of peripheral blood cells expressing the proviral enhanced green fluorescent protein (eGFP) reporter gene revealed a normal distribution of B-lymphocytes (B220), T-lymphocytes (CD3 epsilon), and granulocytes (MAC-1), indicating multi-lineage engraftment of gene modified cells. In spite of this engraftment advantage, transduction efficiency was low (<30%) using RT. The 6-benzylguanine (6-BG) resistant P140K mutant of O6-methylguanine DNA methyltransferase (MGMTP140K) confers a selective advantage to tranduced HSC treated with alkylating drugs. Following RT with a MGMTP140K/ eGFP expressing lentivirus, 5/6 mice treated with 6-BG and the alkylating drug temozolomide showed a significant rise in the percentage of GFP reporter gene expression in peripheral blood. We extended this approach to the FA model by generating a tri-cistronic lentiviral vector expressing the FANCA cDNA, MGMTP140K, and eGFP. Despite modest in vivo gene marking with this vector, up to 37-fold selection (85% GFP-positive cells) was achieved following exposure of bone marrow of transplant recipients to 6-BG and the alkylating drug temozolomide in vitro. Concurrently, phenotypic correction of mitomycin C hypersensitivity of transduced Fanca−/− bone marrow cells was observed. These data suggest that RT improves stem cell engrafting capacity of FA stem cells in a relevant animal model of stem cell gene therapy. The combination of RT and in vivo selection may allow more successful reconstitution of the lympho-hematopoietic system in gene therapy applications.

Novel In Vivo Evidence That Not Only Androgens But Also Pituitary Gonadotropins and Prolactin Directly Stimulate Murine Bone Marrow Stem Cells – Implications For Potential Treatment Strategies In Aplastic Anemias

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2476-2476
Author(s):  
Kasia Mierzejewska ◽  
Ewa Suszynska ◽  
Sylwia Borkowska ◽  
Malwina Suszynska ◽  
Maja Maj ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Sex Hormones ◽  
Peripheral Blood ◽  
Hematopoietic Stem ◽  
Clonogenic Potential

Abstract Background Hematopoietic stem/progenitor cells (HSPCs) are exposed in vivo to several growth factors, cytokines, chemokines, and bioactive lipids in bone marrow (BM) in addition to various sex hormones circulating in peripheral blood (PB). It is known that androgen hormones (e.g., danazol) is employed in the clinic to treat aplastic anemia patients. However, the exact mechanism of action of sex hormones secreted by the pituitary gland or gonads is not well understood. Therefore, we performed a complex series of experiments to address the influence of pregnant mare serum gonadotropin (PMSG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), androgen (danazol) and prolactin (PRL) on murine hematopoiesis. In particular, from a mechanistic view we were interested in whether this effect depends on stimulation of BM-residing stem cells or is mediated through the BM microenvironment. Materials and Methods To address this issue, normal 2-month-old C57Bl6 mice were exposed or not to daily injections of PMSG (10 IU/mice/10 days), LH (5 IU/mice/10 days), FSH (5 IU/mice/10 days), danazol (4 mg/kg/10 days) and PRL (1 mg/day/5days). Subsequently, we evaluated changes in the BM number of Sca-1+Lin–CD45– that are precursors of long term repopulating hematopoietic stem cells (LT-HSCs) (Leukemia 2011;25:1278–1285) and bone forming mesenchymal stem cells (Stem Cell & Dev. 2013;22:622-30) and Sca-1+Lin–CD45+ hematopoietic stem/progenitor cells (HSPC) cells by FACS, the number of clonogenic progenitors from all hematopoietic lineages, and changes in peripheral blood (PB) counts. In some of the experiments, mice were exposed to bromodeoxyuridine (BrdU) to evaluate whether sex hormones affect stem cell cycling. By employing RT-PCR, we also evaluated the expression of cell-surface and intracellular receptors for hormones in purified populations of murine BM stem cells. In parallel, we studied whether stimulation by sex hormones activates major signaling pathways (MAPKp42/44 and AKT) in HSPCs and evaluated the effect of sex hormones on the clonogenic potential of murine CFU-Mix, BFU-E, CFU-GM, and CFU-Meg in vitro. We also sublethally irradiated mice and studied whether administration of sex hormones accelerates recovery of peripheral blood parameters. Finally, we determined the influence of sex hormones on the motility of stem cells in direct chemotaxis assays as well as in direct in vivo stem cell mobilization studies. Results We found that 10-day administration of each of the sex hormones evaluated in this study directly stimulated expansion of HSPCs in BM, as measured by an increase in the number of these cells in BM (∼2–3x), and enhanced BrdU incorporation (the percentage of quiescent BrdU+Sca-1+Lin–CD45– cells increased from ∼2% to ∼15–35% and the percentage of BrdU+Sca-1+Lin–CD45+ cells increased from 24% to 43–58%, Figure 1). These increases paralleled an increase in the number of clonogenic progenitors in BM (∼2–3x). We also observed that murine Sca-1+Lin–CD45– and Sca-1+Lin–CD45+ cells express sex hormone receptors and respond by phosphorylation of MAPKp42/44 and AKT in response to exposure to PSMG, LH, FSH, danazol and PRL. We also observed that administration of sex hormones accelerated the recovery of PB cell counts in sublethally irradiated mice and slightly mobilized HSPCs into PB. Finally, in direct in vitro clonogenic experiments on purified murine SKL cells, we observed a stimulatory effect of sex hormones on clonogenic potential in the order: CFU-Mix > BFU-E > CFU-Meg > CFU-GM. Conclusions Our data indicate for the first time that not only danazol but also several pituitary-secreted sex hormones directly stimulate the expansion of stem cells in BM. This effect seems to be direct, as precursors of LT-HSCs and HSPCs express all the receptors for these hormones and respond to stimulation by phosphorylation of intracellular pathways involved in cell proliferation. These hormones also directly stimulated in vitro proliferation of purified HSPCs. In conclusion, our studies support the possibility that not only danazol but also several other upstream pituitary sex hormones could be employed to treat aplastic disorders and irradiation syndromes. Further dose- and time-optimizing mouse studies and studies with human cells are in progress in our laboratories. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Stem cell factor increases colony-forming unit-spleen number in vitro in synergy with interleukin-6, and in vivo in Sl/Sld mice as a single factor

Blood ◽  
1992 ◽  
Vol 79 (4) ◽  
pp. 913-919 ◽  
Author(s):  
DM Bodine ◽  
D Orlic ◽  
NC Birkett ◽  
NE Seidel ◽  
KM Zsebo
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Growth Factors ◽  
Stem Cell Factor ◽  
Bone Marrow Cells ◽  
Cells Cultured

Abstract Hematopoiesis is thought to be modulated by interactions of progenitor cells with hematopoietic growth factors. We have shown that colony- forming units-spleen (CFU-S) and repopulating stem cells require interleukin-3 (IL-3) to survive in vitro, and that CFU-S number and long-term repopulating ability can be increased by culture in the combination of IL-3 and IL-6. In this report, we describe the effects of stem cell factor (SCF) on CFU-S and repopulating stem cells. Injection of SCF into anemic Sl/Sld mice caused a twofold and 20-fold increase in CFU-S number in the bone marrow and spleen of treated animals, respectively. After 6 days in suspension culture, CFU-S number increased threefold in cultures supplemented with SCF and IL-6, or SCF, IL-3, and IL-6 relative to the number at day 0. The long-term repopulating ability of cells cultured in SCF, IL-3, and IL-6 was approximately sevenfold better than that of cells cultured in IL-3 or SCF. Similar experiments were performed on populations of bone marrow cells enriched for, or depleted of, CFU-S by elutriation and lineage subtraction. The combination of SCF and IL-6 increased CFU-S number approximately fourfold to eightfold in the CFU-S-enriched fraction, but had no effect on the CFU-S-depleted cells. These results show that SCF alone can increase CFU-S number in vivo, and in combination with other growth factors increases CFU-S numbers in vitro.

Hematopoietc Stem Cell Targeted Neonatal Gene Therapy Cures oc/oc Mice from Osteopetrosis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 456-456
Author(s):  
Johan Richter ◽  
Maria Johansson ◽  
Teun J. de Vries ◽  
Mats Ehinger ◽  
Vince Everts ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Bovine Bone ◽  
Wild Type ◽  
Hematopoietic Stem

Abstract Infantile malignant osteopeterosis (IMO) is a progressive, rare autosomal recessive disorder affecting osteoclast function. 50% of the affected children have a mutation in the Tcirg1 gene coding for one subunit of an osteoclast specific proton pump, OC116. The non-resorbed dense, sclerotic bones cause symptoms including pancytopenia and progressive visual loss and ultimately death. So far, the only curative treatment is hematopoietic stem cell (HSC) transplantation. The oc/oc mouse has a mutation in the gene homologous to Tcirg1 giving rise to similar symptoms as in patients leading to death of the mice at the age of 3–4 weeks. We have previously shown that the oc/oc mouse can be successfully treated with neonatal transplantation of normal HSC leading to prolonged survival and reversal of osteopetrosis (M. Johansson et al., Exp. Hematology34;242, 2006). In the current study we set out to develop HSC directed gene therapy for osteopetrosis in the oc/oc mouse model. As the bone marrow compartment is severely reduced in the oc/oc mouse fetal liver (FL) cells depleted of Ter119+ erythroid cells were used as a source of hematopoietic stem cells. We first established that wild type Ter119 depleted FL cells marked with a GFP vector and transplanted to newborn oc/oc mice i.p. could correct the osteopetrotic phenotype just as was shown for fresh bone marrow cells previously. Subsequently, Ter119 depleted FL cells from oc/oc mice were transduced with a retroviral vector expressing OC116 and GFP. In vitro transduction efficiency was 60–85%. One-day-old oc/oc mice were irradiated (400cGy) and transplanted i.p. with the transduced FL cells (1–3.5x106). 7 out of 14 mice survived past the expected lifespan and had 8–53% GFP+ cells in the peripheral blood at 3, 6 and 12 weeks. Analysis of bone structure with X-ray and histopathology showed an improvement at 8 weeks and an almost normal structure at 18 weeks, indicating induction of osteoclast activity. In vitro culture of osteoclasts from bone marrow from transplanted animals on bovine bone slices showed GFP marked osteoclasts and bone resorption, albeit at lower levels than for wild type cells. In the oc/oc mouse there is a block in B-lymphopoiesis leading to a reduced number of B-lymphocytes in the peripheral blood. In treated mice a reversal of this deficiency was observed. In summary we have demonstrated that the osteoclast defect seen in oc/oc mice can be successfully corrected by neonatal transplantation of gene modified hematopoietic stem cells and that this can lead to long-term survival of treated mice. This represents a significant step towards the development of gene therapy for osteopetrosis.

scholarly journals Stem cell factor increases colony-forming unit-spleen number in vitro in synergy with interleukin-6, and in vivo in Sl/Sld mice as a single factor

Blood ◽  
1992 ◽  
Vol 79 (4) ◽  
pp. 913-919 ◽  
Author(s):  
DM Bodine ◽  
D Orlic ◽  
NC Birkett ◽  
NE Seidel ◽  
KM Zsebo
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Growth Factors ◽  
Stem Cell Factor ◽  
Bone Marrow Cells ◽  
Cells Cultured

Hematopoiesis is thought to be modulated by interactions of progenitor cells with hematopoietic growth factors. We have shown that colony- forming units-spleen (CFU-S) and repopulating stem cells require interleukin-3 (IL-3) to survive in vitro, and that CFU-S number and long-term repopulating ability can be increased by culture in the combination of IL-3 and IL-6. In this report, we describe the effects of stem cell factor (SCF) on CFU-S and repopulating stem cells. Injection of SCF into anemic Sl/Sld mice caused a twofold and 20-fold increase in CFU-S number in the bone marrow and spleen of treated animals, respectively. After 6 days in suspension culture, CFU-S number increased threefold in cultures supplemented with SCF and IL-6, or SCF, IL-3, and IL-6 relative to the number at day 0. The long-term repopulating ability of cells cultured in SCF, IL-3, and IL-6 was approximately sevenfold better than that of cells cultured in IL-3 or SCF. Similar experiments were performed on populations of bone marrow cells enriched for, or depleted of, CFU-S by elutriation and lineage subtraction. The combination of SCF and IL-6 increased CFU-S number approximately fourfold to eightfold in the CFU-S-enriched fraction, but had no effect on the CFU-S-depleted cells. These results show that SCF alone can increase CFU-S number in vivo, and in combination with other growth factors increases CFU-S numbers in vitro.

scholarly journals THE EFFECT OF HETEROLOGOUS ANTI-LYMPHOCYTE SERUM ON MOUSE HEMOPOIETIC STEM CELLS

1968 ◽  
Vol 127 (4) ◽  
pp. 731-748 ◽  
Author(s):  
Tom R. DeMeester ◽  
Norman D. Anderson ◽  
Charles F. Shaffer
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Cell Function ◽  
Bone Marrow Cells ◽  
Hemopoietic Stem Cell ◽  
Hemopoietic Stem Cells ◽  
Marrow Cells

The present study has demonstrated that rabbit anti-mouse lymphocyte serum (RAMLS) has the capability of destroying bone marrow cells and suppressing hemopoietic stem cell function. The in vitro incubation of bone marrow suspensions with RAMLS caused extensive cell lysis with an apparent preferential destruction of lymphoid, erythroid, and blastoid elements. Using the spleen colony assay, the number of functional hemopoietic stem cells was found to be markedly reduced in bone marrow populations exposed to RAMLS in vitro. Further, this loss of stem cell function could be produced by exposing marrow suspensions to small concentrations of antiserum which did not produce detectable cytotoxic effects on the general marrow population. A similar effect of RAMLS upon hemopoietic stem cells was found in vivo. The intravenous injection of RAMLS into lethally irradiated mice immediately after the infusion of isogeneic marrow cells reduced the number of spleen colonies formed, indicating that the antiserum could exhibit a deleterious effect upon stem cells in the bloodstream of the intact animal. Normal animals treated with daily subcutaneous injections of RAMLS for 3 wk had a significantly reduced marrow content of functional hemopoietic stem cells, suggesting that RAMLS can affect stem cells located in situ in the bone marrow. The experiments indicate that RAMLS possesses potential marrow toxicity.

Bone marrow-derived mesenchymal stem cells inhibit T follicular helper cell in lupus-prone mice

Lupus ◽  
2017 ◽  
Vol 27 (1) ◽  
pp. 49-59 ◽  
Author(s):  
X Yang ◽  
J Yang ◽  
X Li ◽  
W Ma ◽  
H Zou
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Lupus Nephritis ◽  
Bone Marrow Cells ◽  
Tfh Cells ◽  
Follicular Helper

Background The objective of this paper is to analyze the role of bone marrow-derived mesenchymal stem cells (BM-MSCs) on the differentiation of T follicular helper (Tfh) cells in lupus-prone mice. Methods Bone marrow cells were isolated from C57BL/6 (B6) mice and cultured in vitro, and surface markers were identified by flow cytometry. Naïve CD4+ T cells, splenocytes and Tfh cells were isolated from B6 mice spleens and co-cultured with BM-MSCs. The proliferation and the differentiation of CD4+ T cells and Tfh cells were analyzed by flow cytometry. Lupus-prone MRL/Mp-lpr/lpr (MRL/lpr) mice were treated via intravenous injection with expanded BM-MSCs, the differentiation of Tfh cells was detected, and the relief of lupus nephritis was analyzed. Results MSCs could be successfully induced from bone marrow cells, and cultured BM-MSCs could inhibit T cell proliferation dose-dependently. BM-MSCs could prevent Tfh cell development from naïve CD4+ T cells and splenocytes. BM-MSCs could inhibit IL-21 gene expression and cytokine production and inhibit isolated Tfh cells and STAT3 phosphorylation. In vivo study proved that BM-MSCs intravenous injection could effectively inhibit Tfh cell expansion and IL-21 production, alleviate lupus nephritis, and prolong the survival rate of lupus-prone mice. Conclusions BM-MSCs could effectively inhibit the differentiation of Tfh cells both in vitro and in vivo. BM-MSC treatment could relieve lupus nephritis, which indicates that BM-MSCs might be a promising therapeutic method for the treatment of SLE.

Involvement of the Integrin Alpha 6 Receptor in the Homing and Engraftment of Hematopoietic Stem and Progenitor Cells to Bone Marrow.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1293-1293
Author(s):  
Hong Qian ◽  
Sten Eirik W. Jacobsen ◽  
Marja Ekblom
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Bone Marrow Cells ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Functional Blocking

Abstract Within the bone marrow environment, adhesive interactions between stromal cells and extracellular matrix molecules are required for stem and progenitor cell survival, proliferation and differentiation as well as their transmigration between bone marrow (BM) and the circulation. This regulation is mediated by cell surface adhesion receptors. In experimental mouse stem cell transplantation models, several classes of cell adhesion receptors have been shown to be involved in the homing and engraftment of stem and progenitor cells in BM. We have previously found that integrin a6 mediates human hematopoietic stem and progenitor cell adhesion to and migration on its specific ligands, laminin-8 and laminin-10/11 in vitro (Gu et al, Blood, 2003; 101:877). Using FACS analysis, the integrin a6 chain was now found to be ubiquitously (>95%) expressed in mouse hematopoietic stem and progenitor cells (lin−Sca-1+c-Kit+, lin−Sca-1+c-Kit+CD34+) both in adult bone marrow and in fetal liver. In vitro, about 70% of mouse BM lin−Sca-1+c-Kit+ cells adhered to laminin-10/11 and 40% adhered to laminin-8. This adhesion was mediated by integrin a6b1 receptor, as shown by functional blocking monoclonal antibodies. We also used a functional blocking monoclonal antibody (GoH3) against integrin a6 to analyse the role of the integrin a6 receptor for the in vivo homing of hematopoietic stem and progenitor cells. We found that the integrin a6 antibody inhibited the homing of bone marrow progenitors (CFU-C) into BM of lethally irradiated recipients. The number of homed CFU-C was reduced by about 40% as compared to cells incubated with an isotype matched control antibody. To study homing of long-term repopulating stem cells (LTR), antibody treated bone marrow cells were first injected intravenously into lethally irradiated primary recipients. After three hours, bone marrow cells of the primary recipients were analysed by competitive repopulation assay in secondary recipients. Blood analysis 16 weeks after transplantation revealed an 80% reduction of stem cell activity of integrin a6 antibody treated cells as compared to cells treated with control antibody. These results suggest that integrin a6 plays an important role for hematopoietic stem and progenitor cell homing in vivo.

Short-Term Foamyviral Transduction with Virions Encoding Recombinant Corrects the Mitomycin C Hypersensitivity of Fancc −/− Progenitor Cells In Vitro and Restores Long Term Repopulating Activity of Fancc −/− Stem Cells In Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3171-3171
Author(s):  
Yue Si ◽  
Cordula Leurs ◽  
Edward Srour ◽  
Samantha Ciccone ◽  
Helmut Hanenberg ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Mitomycin C ◽  
Bone Marrow Cells ◽  
Genetic Disorder ◽  
Foamy Virus ◽  
Alkylating Agents

Abstract Fanconi anemia (FA) is a complex autosomal recessive genetic disorder characterized within the hematological system by progressive bone marrow aplasia, a high propensity to develop acute myeloid leukemia, and hypersensitivity to alkylating agents including mitomycin c. The identification of individual FA genes raises the potential of using gene transfer technology to express/introduce the functional cDNA in/into deficient autologous stem cells. We have previously shown that in the absence of genetic correction with a retroviral mediated Fancc transgene, ex vivo culture of Fancc−/− stem/progenitor cells (HSPC) predisposes uncorrected Fancc−/− HSPC cells to clonal hematopoiesis (Haneline, Blood 2003). Therefore we examined the potential of a helper-free human foamy virus (HFV) derived construct that encodes both the human FANCC and EGFP transgenes to transduce murine Fancc−/− HSC in the absence of prestimulation. In initial experiments, we determined that 40–80% of progenitors were transduced following a single overnight HFV infection using a 20:1 moiety of infection. Subsequent studies demonstrated that HFV efficiently transduced primitive hematopoietic progenitors in G0 and G1 phases of the cell cycle as evidenced both by using multicolor fluorescence activated cell sorting and subsequent culture of sorted cell populations in high proliferating potential (HPP-CFC) and low proliferating potential colony forming assays. Aliquots of HFV transduced cells that were transduced with the construct encoding both Fancc and EGFP, or the reporter transgene only were transplanted into irradiated recipient mice. Four months following transplantation, bone marrow cells were isolated from the reconstituted recipients and clonogenic assays were established in a range of mitomycin c (MMC) concentrations. In these experiments, the MMC hypersensitivity of Fancc−/− progenitors was corrected to wild-type levels. To assess quantitatively the potential of HFV expressed FANCC to correct stem cell repopulating ability, we next utilized the competitive repopulating assay. In two replicate experiments, we determined that the repopulating activity of HFV-transduced Fancc−/− stem cells was comparable to wildtype controls six months following transplantation in primary and secondary recipients. Collectively, these data provide in vivo evidence that the HFV vector is an efficient vehicle for introducing a functional hFANCC transgene into quiescent Fancc−/− HSC in the absence of prestimulation and for complementing the cellular FA defect in vitro and in vivo.

Donor Origin of Multipotent Adult Progenitor Cells in Radiation Chimeras.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1395-1395
Author(s):  
Morayma Reyes ◽  
Jeffrey S. Chamberlain
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Y Chromosome ◽  
Stromal Cells ◽  
Mononuclear Cells ◽  
Multipotent Adult Progenitor Cells

Abstract Multipotent Adult Progenitor Cells (MAPC) are bone marrow derived stem cells that can be extensively expanded in vitro and can differentiate in vivo and in vitro into cells of all three germinal layers: ectoderm, mesoderm, endoderm. The origin of MAPC within bone marrow (BM) is unknown. MAPC are believed to be derived from the BM stroma compartment as they are isolated within the adherent cell component. Numerous studies of bone marrow chimeras in human and mouse point to a host origin of bone marrow stromal cells, including mesenchymal stem cells. We report here that following syngeneic bone marrow transplants into lethally irradiated C57Bl/6 mice, MAPC are of donor origin. When MAPC were isolated from BM chimeras (n=12, 4–12 weeks post-syngeneic BM transplant from a transgenic mouse ubiquitously expressing GFP), a mixture of large and small GFP-positive and GFP-negative cells were seen early in culture. While the large cells stained positive for stroma cell markers (smooth muscle actin), mesenchymal stem cell makers (CD73, CD105, CD44) or macrophages (CD45, CD14), the small cells were negative for all these markers and after 30 cell doublings, these cells displayed the classical phenotype of MAPC (CD45−,CD105−, CD44−, CD73−, FLK-1+(vascular endothelial growth factor receptor 2, VEGFR2), Sca-1+,CD13+). In a second experiment, BM obtained one month post BM transplant (n=3) was harvested and mononuclear cells were sorted as GFP-positive and GFP-negative cells and were cultured in MAPC expansion medium. MAPC grew from the GFP-positive fraction. These GFP positive cells displayed the typical MAPC-like immunophenotypes, displayed a normal diploid karyotype and were expanded for more than 50 cell doublings and differentiated into endothelial cells, hepatocytes and neurons. To rule out the possibility that MAPC are the product of cell fusion between a host and a donor cell either in vivo or in our in vitro culture conditions, we performed sex mismatched transplants of female GFP donor BM cells into a male host. BM from 5 chimeras were harvested 4 weeks after transplant and MAPC cultures were established. MAPC colonies were then sorted as GFP-positive and GFP- negative and analyzed for the presence of Y-chromosome by FISH analysis. As expected all GFP-negative (host cells) contained the Y-chromosome whereas all GFP-positive cells (donor cells) were negative for the Y-chromosome by FISH. This proves that MAPC are not derived from an in vitro or in vivo fusion event. In a third study, BM mononuclear cells from mice that had been previously BM-transplanted with syngeneic GFP-positive donors (n=3) were transplanted into a second set of syngeneic recipients (n=9). Two months after the second transplant, BM was harvested and mononuclear cells were cultured in MAPC medium. The secondary recipients also contained GFP-positive MAPC. This is the first demonstration that BM transplantation leads to the transfer of cells that upon isolation in vitro generate MAPCs and, whatever the identity of this cell may be, is eliminated by irradiation. We believe this is an important observation as MAPC hold great clinical potential for stem cell and/or gene therapy and, thus, BM transplant may serve as a way to deliver and reconstitute the MAPC population. In addition, this study provides insight into the nature of MAPC. The capacity to be transplantable within unfractionated BM transplant renders a functional and physiological distinction between MAPC and BM stromal cells. This study validates the use of unfractionated BM transplants to study the nature and possible in vivo role of MAPC in the BM.

Differential Role for VLA-5 in Mobilization of Heamotopoieic Stem Cells Toward Peripheral Blood and Homing to Bone Marrow and Spleen.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2190-2190 ◽  
Author(s):  
Pieter K. Wierenga ◽  
Ellen Weersing ◽  
Bert Dontje ◽  
Gerald de Haan ◽  
Ronald P. van Os
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Hematopoietic Stem ◽  
Late Antigen ◽  
Cell Mobilization

Abstract Adhesion molecules have been implicated in the interactions of hematopoietic stem and progenitor cells with the bone marrow extracellular matrix and stromal cells. In this study we examined the role of very late antigen-5 (VLA-5) in the process of stem cell mobilization and homing after stem cell transplantation. In normal bone marrow (BM) from CBA/H mice 79±3 % of the cells in the lineage negative fraction express VLA-5. After mobilization with cyclophosphamide/G-CSF, the number of VLA-5 expressing cells in mobilized peripheral blood cells (MPB) decreases to 36±4%. The lineage negative fraction of MPB cells migrating in vitro towards SDF-1α (M-MPB) demonstrated a further decrease to 3±1% of VLA-5 expressing cells. These data are suggestive for a downregulation of VLA-5 on hematopoietic cells during mobilization. Next, MPB cells were labelled with PKH67-GL and transplanted in lethally irradiated recipients. Three hours after transplantation an increase in VLA-5 expressing cells was observed which remained stable until 24 hours post-transplant. When MPB cells were used the percentage PKH-67GL+ Lin− VLA-5+ cells increased from 36% to 88±4%. In the case of M-MPB cells the number increased from 3% to 33±5%. Although the increase might implicate an upregulation of VLA-5, we could not exclude selective homing of VLA-5+ cells as a possible explanation. Moreover, we determined the percentage of VLA-5 expressing cells immediately after transplantation in the peripheral blood of the recipients and were not able to observe any increase in VLA-5+ cells in the first three hours post-tranpslant. Finally, we separated the MPB cells in VLA-5+ and VLA-5− cells and plated these cells out in clonogenic assays for progenitor (CFU-GM) and stem cells (CAFC-day35). It could be demonstared that 98.8±0.5% of the progenitor cells and 99.4±0.7% of the stem cells were present in the VLA-5+ fraction. Hence, VLA-5 is not downregulated during the process of mobilization and the observed increase in VLA-5 expressing cells after transplantation is indeed caused by selective homing of VLA-5+ cells. To shed more light on the role of VLA-5 in the process of homing, BM and MPB cells were treated with an antibody to VLA-5. After VLA-5 blocking of MPB cells an inhibition of 59±7% in the homing of progenitor cells in bone marrow could be found, whereas homing of these subsets in the spleen of the recipients was only inhibited by 11±4%. For BM cells an inhibition of 60±12% in the bone marrow was observed. Homing of BM cells in the spleen was not affected at all after VLA-5 blocking. Based on these data we conclude that mobilization of hematopoietic progenitor/stem cells does not coincide with a downregulation of VLA-5. The observed increase in VLA-5 expressing cells after transplantation is caused by preferential homing of VLA-5+ cells. Homing of progenitor/stem cells to the bone marrow after transplantation apparantly requires adhesion interactions that can be inhibited by blocking VLA-5 expression. Homing to the spleen seems to be independent of VLA-5 expression. These data are indicative for different adhesive pathways in the process of homing to bone marrow or spleen.

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