scholarly journals Long-Term, Clonal Tracking Comparing Autologous Transplantation of G-CSF/SCF-Primed Bone Marrow CD34+ Cells with G-CSF/SCF-Mobilized Peripheral Blood CD34+ Cells in Rhesus Macaques

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4524-4524
Author(s):  
Sanggu Kim ◽  
Robert E. Donahue ◽  
Aylin Bonifacino ◽  
Mark Metzger ◽  
Cynthia E. Dunbar ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Stem Cell ◽  
Peripheral Blood ◽  
Rhesus Macaques ◽  
Autologous Transplantation ◽  
Low Frequency ◽  
Time Points ◽  
Cd34 Cells

Abstract Introduction : Although autologous transplantation of peripheral blood stem cells (PBSC) - mobilized with a combination of granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) - has been well characterized, the efficacy of G-CSF/SCF-primed bone marrow stem cell (BMSC) transplantation, however, remains unclear and controversial. In our previous lentiviral vector-mediated PBSC and BMSC transplantation study, we reported efficient and long-term hematopoietic reconstitution by PBSC but not by BMSC - the later being associated with the gradual decline of vector markings in two BMSC-transplanted animals, with a loss of marking occurring in most lineages by 26 or 32 weeks after transplant. Follow-up analysis indicated that low-level yet consistent repopulation by BMSC continued in these animals for a longer period. Here we have compared peripheral blood (PB) markings and vector integration sites (VIS) in PBSC- and BMSC-transplanted animals for up to 12 years and 6 years, respectively. Methods : Young adult rhesus macaques were treated with G-CSF (10 mg/kg of body weight/day) and SCF (200 mg/kg/day) four days before the cell harvest for transplant. Mobilized PB leukapheresis cell products from five rhesus macaques (95E132, 2RC003, RQ5427, RQ3570, and 96E035) were harvested using a CS3000 Plus blood cell separator. Bone marrow (BM) cells from two animals (95E131 and 96E041) were surgically harvested from their femurs and iliac crests under anesthesia. After harvest, PBSC and BMSC were isolated by Ficoll-Hypaque density centrifugation followed by immunoselection of CD34+ cells, and transduced with HIV-based self-inactivating lentiviral vectors expressing EGFP. Vector-marked cells were then autologously transplanted into the host after total body irradiation (10 Gy). No further priming treatment was provided after transplant. PB from the 5 PBSC- and 2 BMSC-transplanted animals were serially collected over time and cryo-preserved for PCR, flow cytometry, and VIS analyses. Results: Both PBSC- and BMSC-transplanted animals showed long-term repopulation for lymphocytes, monocytes, granulocytes, platelets, and red blood cells. PBSC animals showed an average EGFP marking that ranged from 0.32 % to 10.24 %. From these animals, a total of 141 to 4,858 VIS were recovered. We found that the total number of VIS in each animal was proportional to the average EGFP marking in the same animals, and that both of these in turn were linearly correlated with the number of EGFP+ CD34+ cells initially transplanted (1.4 x106 - 28.8 x106 per animal). BMSC-transplanted animals did not show any such patterns. In two BMSC animals, the average EGFP marking levels remained at 0.05 % and 0.10 % until the end point (5 and 6 years) despite the fact that a comparatively large number of EGFP+ CD34+ cells had been transplanted (5.2 x106 and 17.7x106)and a large number of VIS recovered (793 and 680 VIS) in these animals. Temporal VIS analysis of PBSC animals showed that different groups of a large number of PBSC clones repopulated sequentially and reached a point of maximum repopulation at different time points, with some gradually declining after this. BMSC animals also showed a wave-like sequential repopulation similar to the patterns seen in PBSC animals. Unlike PBSC, however, nearly all BMSC clones were detected at a low frequency and at a single time point, except a few larger ones that were detected at multiple time points in a rising and falling pattern. There was no notable difference between the genomic features of VIS in PBSC- and BMSC-repopulating cells. Conclusions : Our data suggest that both the BMSC and PBSC consist of highly heterogeneous stem/progenitor cells that can provide long-term polyclonal repopulation through wave-like, sequential repopulation. Unlike PBSC, however, BMSC transplant was inefficient in PB repopulation resulting in only barely detectable markings in PB. The BMSC clonal profiles reflected the clonal patterns seen in PBSC animals, aside from BMSC animals having primarily low-frequency clones. We have previously shown significant differences in immunophenotype and cell cycle status between PBSC and BMSC, where BMSC were significantly lower in Thy-1 expression and had a higher percentage of cells in the S+G2/M phase of the cell cycle than PBSC. These differences may account for the inefficient differentiation and proliferation capabilities of BMSC compared to PBSC shown in this study. Disclosures Dunbar: National Institute of Health: Research Funding.

Mobilization as a Preparative Regimen for Hematopoietic Stem Cell (HSC) Transplantation in Rhesus Macaques.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1709-1709
Author(s):  
Andre Larochelle ◽  
Cynthia L. Perez ◽  
Allen Krouse ◽  
Mark Metzger ◽  
Simon Fricker ◽  
...  
Keyword(s):  
Stem Cell ◽  
Single Dose ◽  
Peripheral Blood ◽  
Rhesus Macaques ◽  
Myeloablative Conditioning ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Preparative Regimen ◽  
Hsc Transplantation

Abstract The myeloablative conditioning regimens currently used for hematopoietic stem cell (HSC) transplantation are associated with significant morbidity and mortality. Alternative strategies to promote engraftment of infused HSCs with increased safety warrant investigation. In a murine model, we previously demonstrated that, in absence of irradiation, mobilization with AMD3100 (a CXCR4 antagonist) before marrow transplantation vacated microenvironmental niches and resulted in higher levels of engraftment of transplanted HSCs compared to controls (no AMD3100 treatment before transplantation) (Abkowitz JL et al., Blood (ASH Annual Meeting Abstracts)104 (11): 1187, 2004). In this study, we hypothesized that AMD3100 mobilization before transplantation could also promote HSC engraftment in a large animal model, eliminating the need for toxic myeloablative conditioning. Peripheral blood cells from two rhesus macaques were collected by apheresis 3 hours after administration of a single dose of AMD3100 1mg/Kg. CD34+ cells were enriched and transduced for four days in the presence of cytokines and fibronectin with non-expression Moloney murine leukemia virus-derived retroviral vectors (G1PLI) that carry a bacterial neomycin phosphotransferase resistance gene (neoR). The neoR-marked CD34+ cells were reinfused in the non-myeloablated animals, immediately after AMD3100 mobilization and apheresis repeated on the day of transplantation. NeoR-marking levels of approximately 0.1% were detected in both peripheral blood MNC and granulocytes at two months (animal 2RC102) and four months (animal RQ4791) after transplantation. Previous transplantation studies performed without prior myeloablative conditioning or mobilization preparative regimen resulted in no long-term in vivo gene marking. We mathematically confirmed that this observed level of gene marking is what can be expected when AMD3100 mobilization is used as a conditioning regimen. Previous studies have estimated the number of long-term repopulating HSCs at 6 per 105 CD34+ cells (Abkowitz JL et al, Blood96: 3399, 2000). In animal RQ4791, approximately 4.5X107 CD34+ cells, and therefore 2700 HSCs, were mobilized after AMD3100 administration. The total number of HSCs per animal is thought to be conserved in mammals and has been estimated at 11,000 to 22,000 (Abkowitz JL et al, Blood100: 2665, 2002). Hence, 12–24% of HSCs were mobilized after a single dose of AMD3100, consequently opening 12–24% of microenvironmental niches for engraftment. If 1% of engrafted HSCs are marked, 0.12–0.24% long-term marking levels are expected, correlating well with the observed marking level of 0.1%. These results imply that the number of available niches in large animals, as in murine models, regulates the number of HSCs that engraft. As importantly, mobilization with AMD3100 could provide a non-toxic preparative approach in large mammals, including humans, to improve HSC engraftment in transplantation for genetic and other nonmalignant disorders.

scholarly journals Sustained long-term hematopoiesis after myeloablative therapy with peripheral blood progenitor cell support

Blood ◽  
1995 ◽  
Vol 85 (12) ◽  
pp. 3754-3761 ◽  
Author(s):  
R Haas ◽  
B Witt ◽  
R Mohle ◽  
H Goldschmidt ◽  
S Hohaus ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Progenitor Cell ◽  
Lymphoblastic Leukemia ◽  
Peripheral Blood Progenitor Cell ◽  
Myelogenous Leukemia ◽  
High Dose ◽  
Platelet Recovery ◽  
Cd34 Cells

A retrospective analysis of long-term hematopoiesis was performed in a group of 145 consecutive patients who had received high-dose therapy with peripheral blood progenitor cell (PBPC) support between May 1985 and December 1993. Twenty-two patients had acute myelogenous leukemia, nine had acute lymphoblastic leukemia, 43 had Hodgkin's disease, 57 had non- Hodgkin's lymphoma, and 14 patients had multiple myeloma. Eighty-four patients were male and 61 female, with a median age of 37 years (range, 16 to 58 years). In 46 patients, PBPC were collected after cytotoxic chemotherapy alone, while 99 patients received cytokines either during steady-state hematopoiesis or post-chemotherapy. Sixty patients were treated with dose-escalated polychemotherapy, and 85 patients had a conditioning therapy including hyperfractionated total body irradiation at a total dose of 14.4 Gy. The duration of severe pancytopenia posttransplantation was inversely related to the number of reinfused granulocyte-macrophage colony-forming units (CFU-GM) and CD34+ cells. Threshold quantities of 2.5 x 10(6) CD34+ cells per kilogram or 12.0 x 10(4) CFU-GM per kilogram became evident and were associated with rapid neutrophil and platelet recovery within less than 18 and 14 days, respectively. These numbers were also predictive for long-term reconstitution, indicating that normal blood counts are likely to be achieved within less than 10 months after transplantation. Conversely, 12 patients were autografted with a median of 1.75 x 10(4) CFU-GM per kilogram resulting in delayed recovery to platelet counts of greater than 150 x 10(9)/L between 1 and 6 years. Our study includes bone marrow examinations in 50 patients performed at a median follow-up time of 10 months (range, 1 to 85 months) posttransplantation. A comparison with normal volunteers showed a 3.2-fold smaller proportion of bone marrow CD34+ cells, which was paralleled by an even more pronounced reduction in the plating efficiency of CFU-GM and burst-forming unit-erythroid. No secondary graft failure was observed, even in patients autografted with relatively low numbers of progenitor cells. This suggests that either the pretransplant regimens were not myeloablative, allowing autochthonous recovery, or that a small number of cells capable of perpetual self-renewal were included in the autograft products.

AMD3100-Mobilized CD34+ Cells Are Phenotypically Different and Better Targets for Retroviral Transduction Than G-CSF-Mobilized CD34+ Cells in Rhesus Macaques.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2686-2686
Author(s):  
Andre Larochelle ◽  
Allen Krouse ◽  
Donald Orlic ◽  
Robert E. Donahue ◽  
Cynthia E. Dunbar ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Steady State ◽  
Genetic Manipulation ◽  
Rhesus Macaques ◽  
Post Transplantation ◽  
Alternative Source ◽  
Cd34 Cells

Abstract AMD3100 (AMD) has recently been shown to rapidly mobilize primitive hematopoietic cells in mice and humans, but little is known about the properties of cells mobilized with this agent. We initiated a study to determine retroviral (RV) in vivo gene marking efficiency in AMD-mobilized CD34+ cells in rhesus macaques. CD34+ cells collected 3 hours after administration of AMD to 2 animals were transduced using RV vectors containing the NeoR gene. Animals were irradiated and cells reinfused immediately after transduction. By molecular analysis, the levels of PB MNC and granulocyte NeoR gene marking at steady-state (up to 12 months post-transplantation) was 1–2% in animal RC909 and 30–40% in RQ2851. In two additional rhesus macaques, CD34+ cells were collected from steady-state BM and from the PB after mobilization with AMD or G-CSF (G). The two PB populations from each animal were transduced with one of two distinguishable NeoR vectors and simultaneously reinfused into irradiated animals. In animal RQ3590, 2% in vivo gene marking at steady-state (up to 4 months post-transplantation) was derived from AMD-mobilized cells compared to 0.05% from the G-mobilized fraction. Animal RQ3636 showed 10% in vivo marking from the AMD-mobilized fraction and no detectable marking from the G-mobilized cells. We also compared phenotypic and functional characteristics of CD34+ cells from BM, AMD-PB and G-PB. An average of 31% of the AMD-mobilized cells were in the Go phase of the cell cycle, compared to 79% of G-mobilized cells (p=0.02), and 45% for the BM fraction (p=0.24). In contrast, 64% AMD-mobilized cells were in G1 compared to 17% of G-mobilized cells (p=0.03) and 44% for the BM fraction (p=0.15). Flow cytometry showed CXCR4 expression on 59% AMD-mobilized cells, in comparison to 11% G-mobilized cells (p=0.02) and 22% BM cells (p=0.07). Similar results were obtained when comparing VLA-4 expression. The increased expression of CXCR4 on AMD-mobilized CD34+ cells correlated with their increased ability to migrate towards SDF-1α in vitro (45%) compared to G-mobilized cells (8%, p=0.01) and BM cells (17%, p=0.08). Our data indicate efficient long-term in vivo gene marking in the rhesus macaque model, validating the ability of AMD to induce mobilization of true long-term repopulating HSCs. AMD-mobilized PB HSCs represent an alternative source of HSCs amenable to genetic manipulation with integrating RV vectors, with potential applications in gene therapy approaches for patients with sickle cell anemia; documented complications have precluded mobilization using G or G/SCF in these patients. Also, cell cycle status and surface phenotype of AMD-mobilized CD34+ cells are more comparable to steady-state BM cells than G-mobilized PB HSCs. AMD-mobilized CD34+ cells are more actively cycling than G-mobilized CD34+ cells, correlating with the increased efficiency of replication-dependent retrovirus-mediated gene transduction. The increased expression of the adhesion receptors CXCR4 and VLA-4 on primitive AMD-mobilized cells compared to G-mobilized cells suggests fundamental differences in the mechanisms of AMD-mediated and cytokine-mediated stem cell mobilization.

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.

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

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 861-861 ◽  
Author(s):  
Viktor Janzen ◽  
Heather E. Fleming ◽  
Michael T. Waring ◽  
Craig D. Milne ◽  
David T. Scadden
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Caspase 3 ◽  
Brdu Incorporation ◽  
Hematopoietic Stem ◽  
Regulatory Pathways

Abstract The processes of cell cycle control, differentiation and apoptosis are closely intertwined in controlling cell fate during development and in adult homeostasis. Molecular pathways connecting these events in stem cells are poorly defined and we were particularly interested in the cysteine-aspartic acid protease, Caspase-3, an ‘executioner’ caspase also implicated in the regulation of the cyclin dependent kinase inhibitors, p21Cip1 and p27Kip1. These latter proteins are known to participate in primitive hematopoietic cell cycling and self-renewal. We demonstrated high levels of Caspase-3 mRNA and protein in immunophenotypically defined mouse hematopoietic stem cells (HSC). Using mice engineered to be deficient in Caspase-3, we observed a consistent reduction of lymphocytes in peripheral blood counts and a slight reduction in bone marrow cellularity. Notably, knockout animals had an increase in the stem cell enriched Lin−cKit+Sca1+Flk2low (LKSFlk2lo) cell fraction. The apoptotic rates of LKS cells under homeostatic conditions as assayed by the Annexin V assay were not significantly different from controls. However, in-vitro analysis of sorted LKS cells revealed a reduced sensitivity to apoptotic cell death in absence of Caspase-3 under conditions of stress (cytokine withdrawal or gamma irradiation). Primitive hematopoietic cells displayed a higher proliferation rate as demonstrated by BrdU incorporation and a significant reduction in the percentage of cells in the quiescent stage of the cell cycle assessed by the Pyronin-Y/Hoechst staining. Upon transplantation, Caspase-3−/− stem cells demonstrated marked differentiation abnormalities with significantly reduced ability to differentiate into multiple hematopoietic lineages while maintaining an increased number of primitive cells. In a competitive bone marrow transplant using congenic mouse stains Capase-3 deficient HSC out-competed WT cells at the stem cell level, while giving rise to comparable number of peripheral blood cells as the WT controls. Transplant of WT BM cells into Caspase-3 deficient mice revealed no difference in reconstitution ability, suggesting negligible effect of the Caspase-3−/− niche microenvironment to stem cell function. These data indicate that Caspase-3 is involved in the regulation of differentiation and proliferation of HSC as a cell autonomous process. The molecular bases for these effects remain to be determined, but the multi-faceted nature of the changes seen suggest that Caspase-3 is central to multiple regulatory pathways in the stem cell compartment.

The Polycomb Gene BMI1 Collaborates with BCR-ABL in Leukemic Transformation of Human Cord Blood CD34+ Cells.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1350-1350
Author(s):  
Aleksandra Rizo ◽  
Sandra Olthof ◽  
OS van Ronald ◽  
Bert HJ Dontje ◽  
Edo Vellenga ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
B Cells ◽  
Cord Blood ◽  
Bone Marrow Microenvironment ◽  
Leukemic Transformation ◽  
Human Cord Blood ◽  
Self Renewal ◽  
Cd34 Cells

Abstract Previously, we demonstrated that BMI1 acts as a stem cell maintenance factor for human stem/progenitor cells. Here, we report that BMI1 collaborates with BCR-ABL in inducing leukemogenic transformation of human cord blood (CB) CD34+ cells. BMI1 and BCR-ABL were co-expressed into CB CD34+ cells (further referred as B/B cells) using a retroviral approach and cells were transplanted into NOD-SCID mice. In two out of five mice we observed leukemia within 4 months after transplantation. Chimerism levels reached 80–90% in the bone marrow and peripheral blood and morphological analysis revealed the appearance of primitive blast-like human hematopoietic cells with features that recapitulate human lymphoid leukemia. The mice were lethargic, with splenomegaly and infiltration of leukemic cells in the spleen, liver and the bone marrow and immunophenotypical analyses revealed that the cells expressed CD34 and CD19. To further understand the mechanisms underlying the leukemic transformation we performed ex-vivo long-term cultures on bone marrow stroma. We observed that the double transduced B/B cells had a strong proliferative advantage and elevated self-renewal potential as compared to controls. Expanding cultures could be maintained for over 20 weeks and Cobblestone Area Forming Cells (CAFCs) could be harvested and replated to initiate new expanding cocultures. Stem cell frequencies were determined in Long-Term Culture-Initiating Cell (LTC-IC) assays and frequencies were enhanced over 100-fold as compared to controls. Depending on the MS5 co-culture conditions, both myeloid as well as lymphoid long-term cultures could be established, indicating that extrinsic factors might dictate the lineage fate of transformed cells. To determine the necessity of a bone marrow microenvironment, we performed stroma-free liquid cultures and observed that the B/B cells were capable of expanding over 23 weeks, BMI1 cells were able to grow for 16 weeks and, importantly, BCR-ABL cells were not able to propagate long-term in stromain-dependent cultures. Thus, these data suggest that BCR-ABL cells are still dependent on cues from the bone marrow microenvironment for long-term self-renewal, and that co-expression of the intrinsic stem cell regulator BMI1 might alleviate this necessity of BCR-ABL+ cells for a microenvironment. Experiments in which B/B-transduced cells were sorted into HSC, CMP, GMP and MEP populations indicated that long-term self-renewal and expansion could particularly be imposed on the HSC population, and much less efficiently on progenitor subpopulations. In order to study whether the B/B-leukemic stem cells could be targeted by Imatinib, we applied a short pulse of Imatinib to expanding MS5 cocultures for 7 days. While the vast majority of cells in all cultures did not survive, in the B/B-transduced group a population of immature cells remained that was capable of re-initiating proliferative cultures of self-renewing CAFCs with very high frequencies (1/96 as determined by LTC-IC assays). Finally, we asked whether retroviral introduction of BMI1 in BCR-ABL+ CD34+ cells isolated from CML patients in chronic phase that expressed low endogenous BMI1 levels would affect long-term growth and self-renewal. Upon overexpression of BMI1 we observed increased proliferation capacity of the BMI1 transduced CML cells, and cultures could be maintained for much longer periods than control-transduced cultures. In conclusion, our data indicate that BMI1 collaborates with BCR-ABL in leukemic transformation, and our human-based system should provide a useful model to study the pathology of leukemias and test new drug entities.

Increased Aldehyde Dehydrogenase Activity Indentifies a Subset of Human Leukemic Blasts with Stem Cell Characteristics and Correlates with Poor Prognosis.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1345-1345
Author(s):  
Dan Ran ◽  
Mario Schubert ◽  
Larissa Pietsch ◽  
Isabel Taubert ◽  
Christian Wallenwein ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Prognostic Factors ◽  
Aldehyde Dehydrogenase ◽  
Protein Level ◽  
Aldh Activity ◽  
Cd34 Cells ◽  
Dividing Cells

Abstract INTRODUCTION: Normal hematopoietic stem cells (HSC) are characterized by their ability to self-renew, to generate multiple cell-lineages, and show slow divisional kinetics. Leukemic stem cells (LSC) have been reported to show similar characteristics but their identification has been elusive. We have studied various methods and have identified aldehyde dehydrogenase (ALDH) staining as an optimal method for the enrichment of primary human LSC. MATERIAL&METHODS: Bone marrow samples were obtained from patients with newly diagnosed AML after informed consent. Mononuclear cells were stained with Aldefluor and sorted by flow cytometry according to their forward/side scatter characteristics and ALDH activity (ALDH+/ALDH−). Alternatively, primary AML samples were being enriched for CD34+ cells by magnetic column, then double-stained with CD34-antibodies and Aldefluor and sorted for the co-expression of CD34+ and ALDH+, respectively for CD34+ alone. Human Mesenchymal Stromal Cells (MSC), isolated from human bone marrow, were used as a surrogate model for the cellular microenvironment of the hematopoietic niche. Adhesion of various AML cell lines and subpopulations of primary leukemic cells (ALDH+, ALDH−, CD34+, CD34+/ALDH+, all blasts) to MSC was tested in the adhesion chamber assay. Semi-quantitative RT-PCR was used to analyze the gene expression of various adhesion molecules and Western- Blot analysis was performed to validate the PCR-results on protein level. The generation of secondary leukemic colonies was evaluated in a semi-solid methylcellulose medium, as well as in a long term co-culture system (LSC-IC assay; in analogy to the LTC-IC assay). RESULTS: The percentage of ALDH+ cells ranged from 0.01% to 13.2% with a median of 1.47% (n=55). Adhesion significantly differed in the ALDH+ and ALDH− subpopulations: 85±4% of ALDH+ cells but only 61±8% of ALDH− cells were adherent (n=11, p<0.001). Adhesion molecules, such as CXCR4 and CD44, were highly expressed on the ALDH+ subpopulation both on mRNA level and protein level, in contrast to the ALDH− subpopulation. Analysis of the initial divisional kinetics on single cell base showed that the ALDH+ subpopulation contained more slow dividing cells whereas the majority of the ALDH− subpopulation consisted of fast-dividing cells (n=3; p<0.01). The frequency of long term leukemic colony initiating cells (LSC-IC) was 3.82% in the ALDH+ but only 0.01% in the ALDH− (n=21; p<0.01). In the CD34+ the LSC-IC frequency was 1.96% versus 0.01% in the CD34− (n=5, p<0.01). The highest LSC-IC frequency could be monitored in ALDH+/CD34+ cells: 6.1% generated secondary leukemic colonies (n=5). These colonies, harvested after 7 weeks of cultivation, were examined for their immune phenotype and screened for cytogenetic aberrations by fluorescent in situ hybridization (FISH) and the chromosomal aberrations were consistent with the AML samples taken at diagnosis. Furthermore, the frequency of ALDH+ cells correlated significantly with adverse prognostic factors: patients with a high-risk karyotype had a mean of 2.9% ALDH+ cells (n=21); in contrast, patients with a normal karyotype had a mean of 0.4% ALDH+ cells in their bone marrow (n=34; p<0.001). The ability of ALDH+ versus ALDH− subsets to generate secondary leukemia in the animal model is concurrently examined. DISCUSSION: In summary, measurement of the ALDH activity provides a useful tool for the isolation of a distinct AML-blast subpopulation with stem-cell like features (LSC). The ALDH+ subsets showed higher affinity to the surrogate niche (MSC), elevated expression of CD44, Cadherin-2, and CXCR4 and were associated with an increased frequency of secondary leukemic colonies in vitro (LSC-IC). Above all, the frequency of ALDH+ blasts correlated with clinical prognostic factors, which substanciates LSC as a relevant therapeutic target.

A Novel View of Bone Marrow As a “stem Cell sensor” of Tissue/Organ Damage -Evidence That in Vivo Exposure to the Neurotoxin Kainic Acid (KA) Induces Proliferation and Neural Specification of Developmentally Early Stem Cells Directly in Bone Marrow Before They Are Mobilized Into Peripheral Blood

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1192-1192
Author(s):  
Katarzyna Grymula ◽  
Maciej Tarnowski ◽  
Malwina Suszynska ◽  
Katarzyna Piotrowska ◽  
Sylwia Borkowska ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Brain Damage ◽  
Peripheral Blood ◽  
Organ Damage ◽  
Organ Injury ◽  
Neural Lineage ◽  
Heart Infarct

Abstract Abstract 1192 Background. It is well known that various stem cells become mobilized into peripheral blood (PB) in response to tissue/organ injuries (e.g., heart infarct, stroke, or bleeding); however, the data on the immediate response of stem cells in BM during organ injuries are somewhat limited. We and others have demonstrated the presence of developmentally early stem cells in BM that we have named very small embryonic-like stem cells (VSELs). These Oct-4+SSEA-1+Sca-1+Lin–CD45– cells are kept quiescent in BM in the G0 phase of the cell cycle by erasure of the somatic imprint in the differentially methylated regions (DMRs) of some crucial paternally imprinted genes, (Igf2-H19, RasGRF1, and p57Kip2) that regulate proliferation of embryonic stem cells (Leukemia 2009;23:2042). These cells are mobilized into peripheral blood, for example, during heart infarct (J Am Coll Cardiol 2009;6:1–9.), stroke (Stroke 2009;40:1237–44.), or skin burns (Stem Cell Rev. 2012;8:184–94.). Hypothesis. We hypothesized that this population of BM-residing, small, quiescent, pluripotent cells should be able to respond to organ injury induced by a known neurotoxin, kainic acid (KA), in a brain damage model. We hypothesized that these quiescent cells would began to proliferate, expand, and become specified into the neural lineage. Experimental strategies. C57Bl6 mice were injected with increasing doses of KA and at various time intervals mice were sacrificed to harvest BM, PB samples, and brains for analysis. Brain damage was confirmed by histological analysis. The number of Sca-1+Lin–CD45– VSELs and Sca-1+Lin–CD45+ HSPCs was evaluated in BM and PB by FACS. The cell cycle status of VSELs and HSPCs was evaluated by FACS in cells isolated from mice that received bromodeoxyuridine (BrdU) after KA injection. By employing RQ-PCR, we also measured the expression of genes that regulate stem cell pluripotency (Oct-4, Nanog, Sox2, and Rex1) and regulate neuronal development (Nestin, βIII-tubulin, Olig1, Olig2, and GFAP). The expression of these genes was subsequently confirmed in sorted cells by immunohistochemical staining. The numbers of clonogenic CFU-GM and BFU-E progenitors residing in BM and circulating in PB were tested in methylcellulose cultures. Results. We found that 12 hrs after administration of KA (25 mg/kg bw) quiescent VSELs residing in BM enter the cell cycle: ∼2 ± 1% for control vs. 37 ± 6% for KA-treated cells. Interestingly, at the same time we did not observe significant changes in the proliferation rate of HSPCs (15±5% for control vs. 17±4% for KA-treated cells). The elevated number of VSELs in the cell cycle remained detectable for a few days and returned to control values (∼2%) after 1 week after KA administration. Furthermore, an increase in the number of cycling VSELs correlated with an increase in expression of pluripotent markers, according to RQ-PCR analysis. In parallel, 48 hrs after KA administration we observed the release from BM into PB of Sca-1+Lin–CD45–VSELs highly enriched for mRNAs characteristic of neural differentiation. Interestingly, while we observed a significant increase in VSEL number in BM and PB after KA-induced brain damage, no significant changes were observed for both BM-residing and circulating HSPCs. Conclusions. For the first time, we provide evidence that the compartment of developmentally early stem cells residing in BM responds robustly to brain damage induced by a neurotoxin. This effect seems to be specific for VSELs, as no significant changes were observed for HSPCs. The kinetics of changes in BM revealed that BM VSELs enter the cell cycle and, after they become specified into the neural lineage, egress from BM and enter the PB. Thus, our data provide novel evidence that developmentally early stem cells in BM “sense” the damage to brain tissue and respond to this type of organ injury. In parallel, we are studying the specificity of the response of BM-residing VSELs and HSPCs to other types of organ damage, such as heart infarct and acute limb ischemia. Disclosures: Ratajczak: Neostem Inc: Member of SAB Other.

Detection of a Stem Cell Population with Hemangioblastic Characteristics in Primary Myelofibrosis (PMF),

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3860-3860
Author(s):  
Ioanna N Trivai ◽  
Thomas Stuebig ◽  
Anita Badbaran ◽  
Ursula Gehling ◽  
Asterios Tsiftsoglou ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Peripheral Blood ◽  
Primary Myelofibrosis ◽  
Jak2v617f Mutation ◽  
Cell Subpopulation ◽  
Stem Cell Population ◽  
Allele Burden

Abstract Abstract 3860 Primary myelofibrosis (PMF) comprises a myeloproliferative neoplasia accompanied by imbalance of various tissues of the mesoderm, let alone the hematopoietic tissue. Involvement of multiple hematopoietic lineages during disease progression suggests the clonality of myelofibrosis that can be attributed to an initial stem cell defect at the very early stage of the stem cell hierarchy. Hematopoietic and endothelial phenotypes of circulating multipotent stem cells in patient peripheral blood, along with the increased microvascular density in the bone marrow, leads to the hypothesis that the critical event in PMF involves malignant transformation of a stem cell with hemangioblastic potential. Former studies have provided functional evidence that activated JAK2 signalling in primitive human hematopoietic cells is sufficient to drive key processes involved in the pathogenesis of the disease. In this study, the functionality and differentiation potential of circulating primitive JAK2V617F+ stem cells from primary myelofibrosis patients is assessed. Primitive stem cells were isolated from peripheral blood of 25 patients. All patients participating in the study were diagnosed with primary myelofibrosis, have been untreated, and were found positive for JAK2V617F mutation. Isolated stem cells were analysed for purity and assessed for the expression of markers characteristic for the hemangioblast phenotype (CD34, CD133, CD45, VEGFR2, VE-Cadherin, E-Cadherin, CD31) with flow cytometry. Genomic DNA was isolated from various stem cell populations to determine the mutational status by PCR. Our results indicate that long term repopulating stem cells circulating in peripheral blood bear the JAK2V617F mutation. Hemangioblast resembling populations within the isolated prime stem cells were also found positive for the mutation. Long term repopulating stem cells bearing different allele burden for JAK2V617F mutation from PMF patient peripheral blood were expanded for up to 4 months. Various colonies formed after seeding in semisolid media were characterised by morphological features (CFU-GEMM, CFU-GM, CFU-E, CFU-M, CFU-Endo) and expressing genes by quantitative PCR. Moreover, allele burden determination for various progenitors of both hematopoietic and endothelial lineages was performed. JAK2V617F allele burden varied within individual progeny phenotypes, indicating the acquisition of the mutation that boosts the outgrowth of the malignant clone within the hemangioblast compartment of the bone marrow. Endothelial and macrophage progenitors appear heterozygotic while all rest progenitors of various hematopoietic lineages can be either heterozygotic or homozygotic. This indicates high genomic instability of the JAK2V617F+ malignant clone as it is driven into hematopoietic differentiation. Our results indicate the existence of a malignant clone with hemangioblast phenotype in PMF which can differentiate into hematopoietic and/or endothelial progenitors in vitro. Our experiments shed light to the pathogenesis of PMF by characterising the potential of the defective stem cell subpopulation responsible for the disease. Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document