Gene Therapy for Tolerance to Factor VIII Induced with B Cells and Bone Marrow Transduced with FVIII Domains.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3051-3051
Author(s):  
David W. Scott ◽  
Tie Chi Lei
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Bone Marrow ◽  
B Cells ◽  
Factor Viii ◽  
Tolerance Induction ◽  
Class Ii ◽  
Hematopoietic Stem

Abstract Tolerance induction for hemophilia inhibitor formation has recently been achieved by gene therapy using B cells transduced with a fusion protein of C2 and A2 domains of factor VIII with an IgG heavy chain. We have shown that inhibitor titers can be reduced over 90% by this approach in both naïve and highly immunized FVIII knockout mice (FVIII−/−). This process requires the presence of CD25+ regulatory cells for induction, and is stable for at least three months (Lei, T-C. and Scott, D.W. 2005. Induction of tolerance to fVIII inhibitors by gene therapy with immunodominant A2 and C2 domains presented by B-cells as Ig fusion proteins. Blood, 105: 4865–4870). Herein, we wished to establish whether retroviral transduction and integration of the fusion Ig was necessary for tolerance. In addition, we also tested whether transduction of bone marrow could lead to long-term tolerance. B cells (resting and LPS blasts) from FVIII sufficient C57Bl/6 and FVIII−/− mice were pulsed with FVIII and injected into naïve FVIII−/− recipients that were subsequently challenged with therapeutic doses of FVIII in an immunogenic protocol. We reasoned that C57Bl/6 mice were FVIII sufficient and could display FVIII processed epitopes within their MHC class II. Moreover, pulsing with FVIII should lead to presentation of processed epitopes transiently in class II MHC. We found that specific tolerance to FVIII was only induced with B cells transduced with a combination of A2-IgG and C2-IgG, indicating that transient expression of FVIII epitopes and presentation by B cells is not sufficient for tolerance induction. Resting B-cells pulsed with fVIII in vitro are not tolerogenic Resting B-cells pulsed with fVIII in vitro are not tolerogenic In further studies, we examined whether tolerance could be induced by bone marrow transplantation of C2-Ig and A2-Ig transduced hematopoietic stem cells under a non-myeloablative regimen with busulfan in immunized FVIII−/− mice. This protocol could lead to long-term tolerance if pluripotential stem cells were transduced and engrafted. Our results indicate that tolerance to FVIII domains could be achieved even though the level of expression of GFP positive cells in peripheral blood of busulfan-treated mice was less than 1% (compared to >50% positive cells in lethally irradiated recipients of similarly transduced bone marrow cells). These results have implications for long-term therapy of inhibitor formation in hemophilia A. (Supported by NIH grants HL061883, AI035622 and a Lab Grant from the National Hemophilia Foundation.)

scholarly journals From Bone Marrow to Mobilized Peripheral Blood Stem Cells: The Circuitous Path to Clinical Gene Therapy for Fanconi Anemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2208-2208
Author(s):  
Pamela S Becker ◽  
Jennifer Adair ◽  
Grace Choi ◽  
Anne Lee ◽  
Ann Woolfrey ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Bone Marrow ◽  
Fanconi Anemia ◽  
Research Funding ◽  
Hematopoietic Stem ◽  
Post Infusion ◽  
Clinical Gene Therapy

Abstract For decades, it has remained challenging to achieve long-term engraftment and correction of blood counts using gene-modified hematopoietic stem cells for Fanconi anemia. Toward this goal, our group conducted preclinical studies using a safety modified lentiviral vector encoding full-length cDNA for FANCA in normal and affected patient hematopoietic progenitor cells, and in a mutant mouse model that supported the IND for a gene therapy clinical trial for Fanconi anemia, complementation group A (NCT01331018). These studies led us to incorporate methods such as addition of N-acetylcysteine and hypoxic incubation during transduction. Because of the low stem cell numbers of Fanconi patients and initial difficulty with using plerixafor off-label for mobilization, we began our study with bone marrow as the source of stem cells. Due to concerns regarding secondary cancers, no conditioning was administered prior to infusion of gene-modified cells. The US Food and Drug Administration approved adult patients initially, but later permitted pediatric patient enrollment with a minimum age of 4 years. The primary objective of our phase I trial was safety. Secondary objectives included in vitro correction of mitomycin C (MMC) sensitivity, procurement of sufficient cell numbers, and ultimately, long-term correction of blood counts in recipients. Eligibility included absolute neutrophil count ≥0.5, hemoglobin ≥8, platelet count ≥20,000, lack of matched family donor, adequate organ function, and not meeting criteria for diagnosis of MDS. Our three enrolled patients were ages 22, 10, and 5 years. All demonstrated defects in the FANCA gene, with two patients sequenced and one patient diagnosed by complementation. Due to in-process learning and the later addition of plerixafor mobilization to the protocol, three different laboratory procedures were used to prepare the gene-modified product for each patient. Cell products were CD34+ selected bone marrow, bone marrow mononuclear cells depleted of red cells by hetastarch, and G-CSF and plerixafor mobilized cells depleted of red blood cells and cells bearing lineage markers, respectively. Transduction efficiencies were 17.7, 42.7 and 26.3% of colony forming cells (CFC) in 0 nM MMC, and 80, 100, and 100% of CFC in 10 nM MMC. Growth of hematopoietic colonies in MMC indicated functional correction of the FANCA defect. The 1st patient received 6.1×10e4, the 2nd 2.9×10e5, and the 3rd 4.3×10e6 CD34+ cells/kg. Serious adverse events included cytopenias in all patients, and hospital admission for fever due to viral upper respiratory infection in one patient. The patients remain alive at 46, 38, and 12 months after receipt of gene-modified cells. Due to worsening cytopenias, the third patient underwent hematopoietic cell transplant from an unrelated donor 10 months after infusion of gene-modified cells. To date, he has done well with transplant, and no indication that prior gene therapy impacted the outcome. The blood counts for the first 2 patients who have not undergone allogeneic transplant remain stable at 1,111 and 1,077 days post infusion compared to the first blood counts when they arrived at our center. For the 1st patient, vector was detectable in white blood cells (WBC) up to 21 days, in the 2nd up to 582 days, and the 3rd up to 81 days post infusion. Thus, in these patients, despite dramatic improvement in cell dose during the study, there was lack of persistence in detection of gene-modified WBCs beyond 1.5 years. A number of factors may have contributed, including lack of conditioning, in vitro cell manipulation including cytokine exposure, inability to transduce primitive hematopoietic stem cells, and paucity of long-term repopulating cells at the ages of the patients, suggesting earlier collection may be beneficial. This study is now closed to enrollment. Valuable information gained as a result of this study will contribute to future clinical gene therapy trials. Current work focuses on how to evaluate stem cell fitness prior to attempting gene therapy, minimizing manipulation required for gene correction and/or in vivo genetic correction and non-chemotherapy-based conditioning to facilitate engraftment. We would like to personally thank each patient and their families for participating in this study, as we could not have learned these lessons without their support. Disclosures Becker: GlycoMimetics: Research Funding; Abbvie: Research Funding; Amgen: Research Funding; BMS: Research Funding; CVS Caremark: Consultancy; Trovagene: Research Funding; Rocket Pharmaceuticals: Research Funding; Novartis: Research Funding; Pfizer: Consultancy; JW Pharmaceuticals: Research Funding. Adair:Miltenyi Biotec: Honoraria; RX Partners: Honoraria; Rocket Pharmaceuticals: Patents & Royalties: PCT/US2017/037967 and PCT/US2018/029983. Kiem:Rocket Pharmaceuticals: Consultancy; Homology Medicine: Consultancy; Magenta: Consultancy.

Hybrid AAV6/2-Mediated Human β-Globin Expression in β-Thalassemia Patient Hematopoietic Stem Cells Transplanted Into Irradiated BALB/c Nude Mouse

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4715-4715
Author(s):  
Mengqun Tan ◽  
Zhenqing Liu ◽  
Juan Zhang ◽  
Zhiyan Li ◽  
Liujiang Song ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Bone Marrow ◽  
Globin Gene ◽  
Blood Transfusions ◽  
Rt Pcr ◽  
Thalassemia Patient ◽  
Hematopoietic Stem ◽  
Human Hscs

Abstract Abstract 4715 β -Thalassemia is one of the most common worldwide monogenic human diseases,caused by molecular defects in the human β -globin gene cluster leading to decrease or absence of β-globin. Loss of β -globin chains causes ineffective production of oxygen-carrying hemoglobin and therefore results in severe anemia. The treatment for β -Thalassemia major usually includes lifelong blood transfusions but chronic blood transfusion often causes iron overload, and accumulated iron produces tissue damage in multiple organs, so that iron chelating treatment is also needed. Bone marrow transplantation is another effective therapy, which can eliminate a patient's dependence on blood transfusions, however, it is difficult to find a matching donor for most patients; therefore it is only available for a minority of patients. Gene therapy is one potential novel therapy for treatment of inherited monogenic disorders. The long–term therapeutic strategy for this disease is to replace the defective β-globin gene via introduction of a functional gene into hematopoietic stem cells (HSCs). Adeno-associated virus type 2 (AAV), a nonpathogenic human parvovirus, has gained attention as a potentially useful vector for human gene therapy. AAV can infect both dividing and non-dividing cells and wild AAV integrates preferentially at a specific site on human chromosome 19. In the absence of helper virus, recombinant AAV will stably integrate into the host cell genome, mediating long-term and stable expression of the transgene. In this study, we used a hybrid rAAV6/2 vector carrying the human β-globin gene to transduce HSCs from a β -Thalassemia patient, followed by transplantation into irradiated BALB/c nude mice. One month post-transplantation, Hb was prepared from peripheral blood and analyzed by Western Blot and HPLC respectively. RNA and DNA were isolated from bone marrow cells (BMCs) from recipient mice transplanted with mock-infected or hybrid rAAV–globin-infected cells and analyzed by RT-PCR and PCR respectively. The results showed: 1. Human β-actin and β-globin transcripts were detected by RT-PCR in BMCs from all recipient mice, indicating that human HSCs were successfully transplanted in these mice and that the human β-globin gene was transcriptionally active in the donor cells. 2. The level of human hemoglobin expressed in peripheral red blood cells of recipient mice as measured by HPLC (ratio of β/α) was increased to 0.3 from 0.05 of pre-transplantation levels. Expression of human β-globin was also confirmed in recipient mice by Western Blot; a 2–3-fold increase compared with that of controls. Our results indicate that human HSCs from a β-Thalassemia patient can be efficiently transduced by a hybrid rAAV6/2-β-globin vector followed by expression of normal human β-globin protein. This study provides a proof-of-concept that rAAV6/2-mediated gene transfer into human HSCs might be a potential approach for gene therapy of β-Thalassemia. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Presence of mixed colony-forming cells in long-term hamster bone marrow suspension cultures: response to pokeweed spleen conditioned medium

Blood ◽  
1982 ◽  
Vol 60 (2) ◽  
pp. 495-502
Author(s):  
CE Eastment ◽  
FW Ruscetti ◽  
E Denholm ◽  
I Katznelson ◽  
E Arnold ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Conditioned Medium ◽  
In Vitro Study ◽  
Suspension Cultures ◽  
Rapid Expansion ◽  
Pokeweed Mitogen ◽  
Hematopoietic Stem

In contrast to the murine system, long-term hamster bone marrow suspension cultures maintain proliferation of both pluripotent and committed stem cells in the absence of an adherent layer and without addition of exogenous factors, such as hydrocortisone. Addition of pokeweed-mitogen-stimulated hamster spleen conditioned medium (SCM) to these long-term suspension cultures produces an increase in the number of mixed colonies assayed in soft-agar, These mixed colonies, which contained four cell lineages--granulocytic, erythroid, megakaryocytic, and macrophage--could be generated from cells grown in suspension for over 6 mo. Addition of SCM also induces an initial rapid expansion of the myeloid compartment, and this expansion results in 70% of the cells being terminally differentiated granulocytes. In contrast, addition of SCM to hamster bone marrow cultures containing both adherent cells and hematopoietic stem cells produced no change in the number of mixed colonies generated in the culture. This system allows the in vitro study of the process of stem cell proliferation and differentiation and also provides a means to examine the relationship of adherent and supernatant bone marrow populations.

scholarly journals An in vitro limiting-dilution assay of long-term repopulating hematopoietic stem cells in the mouse

Blood ◽  
1989 ◽  
Vol 74 (8) ◽  
pp. 2755-2763 ◽  
Author(s):  
RE Ploemacher ◽  
JP van der Sluijs ◽  
JS Voerman ◽  
NH Brons
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem ◽  
Dilution Assay ◽  
Limiting Dilution Assay ◽  
Colony Forming Units ◽  
Limiting Dilution

We have developed a limiting-dilution assay of long-term repopulating hematopoietic stem cells in the mouse using a miniturized stroma- dependent bone marrow culture assay in vitro. The cells were overlaid on irradiated stromal layers in microtiter wells in a range of concentrations, and frequencies of cobblestone area-forming cells (CAFC) were calculated by employing Poisson statistics. The production of secondary granulocyte/macrophage colony-forming units (CFU-G/M) in the adherent layer of individual wells was correlated with the presence of such cobblestone areas. CAFC frequencies were determined in bone marrow cell suspensions that were either enriched for marrow repopulating ability (MRA) in vivo, while depleted for spleen colony- forming units (CFU-S), or vice versa. The separation of bone marrow cells (BMC) was either based on centrifugal elutriation, or monoclonal antibody-mediated magnetic depletion of cells carrying cell surface differentiation antigens, and subsequent sorting on the basis of light scatter and rhodamine-123 retention as a measure of mitochondrial activity. In addition, 5-fluorouracil-resistant BMC were studied. Our investigations show that a time-dependent cobblestone area formation exists that reflects the turnover time and primitiveness of CAFC. The frequency of precursors forming cobblestone areas on day 28 after overlay is proposed to be a measure for MRA, whereas the day-7 CAFC frequency closely corresponds with day-12 CFU-S numbers in the suspensions tested.

Stable Polyclonal Murine Long-Term Hematopoiesis without Clonal Exhaustion of MGMT P140K Expressing Murine Hematopoietic Stem Cells after Extended Reduced Intensity Selection.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3271-3271
Author(s):  
Claudia R. Ball ◽  
Manfred Schmidt ◽  
Ingo H. Pilz ◽  
Fessler Sylvia ◽  
David A. Williams ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Retroviral Vector ◽  
Hematopoietic Stem ◽  
Reduced Intensity ◽  
Selection Of

Abstract Gene therapy is a promising approach for the therapy of hereditary diseases, but after the occurrence of adverse side effects in a SCID-X1 gene therapy trial increased biological safety has become a major goal of gene therapy. A reduction of the number of transplanted cells could help achieve this goal by reducing the statistical likelihood of insertional mutagenesis simply by simply reducing the number of transplanted cells carrying potentially untoward insertion sites. As we have previously shown, incorporation of the selectable marker gene MGMT P140K into a retroviral vector allows a reduced intensity and toxicity in vivo selection of low numbers of genetically modified hematopoietic cells by chemotherapy with O6-benzylguanine (O6BG) and nitrosourea drugs such as 1,3-bis-2 chloroethyl-1-nitrosourea (BCNU). However, it is still not known whether extended selection over longer periods of time influences the long-term proliferation and differentiation capacity of murine haematopoietic stem cells. To address this question, serial transplantations of murine MGMT-P140K-expressing hematopoiesis combined with repeated administrations of O6-BG and BCNU were performed. After ex vivo gene transfer of a MGMT/IRES/eGFP-encoding retroviral vector, bone marrow cells were transplanted into syngeneic C57 BL/6J mice and serially transplanted. First, 2nd and 3rd generation recipient mice were subsequently treated every four weeks in order to amplify treatment effects on the long-term clonal behaviour of modified hematopoietic stem cells. Lineage contribution of transduced hematopoiesis was monitored by FACS over a total of 17 rounds of selection and clonality was monitored by LAM-PCR over a total of 16 rounds of selection. In primary mice, the percentage of transduced blood cells increased from 4.7 ± 0.8 % to 36.4 ± 9.8 % (n=12) and in secondary mice from 29.9 ± 7.2 % to 65.1 ± 8.7 % (n=18) after selection without inducing persistent peripheral blood cytopenia. Lineage analysis showed an unchanged multilineage differentiation potential in the transduced compared to control cells in 1st and 2nd generation animals. LAM PCR analysis of peripheral blood revealed stable oligo- to polyclonal hematopoiesis in 1st, 2nd and 3rd generation mice. Evidence of predominant clones or clonal exhaustion was not observed despite of up to 16 rounds of BCNU/O6-BG treatment. Interestingly, pairs of secondary transplanted mice which had received bone marrow cells from identical donors showed very similar clonal composition, engraftment kinetics under selection and lineage contribution of the transduced hematopoiesis. This is molecular proof that extensive self-renewal of transplantable stem cells had occurred in the primary mice resulting in a net symmetric refilling of the stem cell compartment. In summary, we demonstrate that even extended selection of MGMT-P140K-expressing hematopoietic stem cells by repetitive chemotherapy does not affect differentiation or proliferation potential and does not result in clonal exhaustion. Our results have important implications for the clinical use of MGMT selection strategies intending to employ amplification of a limited number of genetically modified clones in clinical gene therapy.

Targeted deletion of the tachykinin 4 gene (TAC4−/−) influences the early stages of B lymphocyte development

Blood ◽  
2010 ◽  
Vol 116 (19) ◽  
pp. 3792-3801 ◽  
Author(s):  
Alexandra Berger ◽  
Patricia Benveniste ◽  
Steven A. Corfe ◽  
Anne H. Tran ◽  
Mary Barbara ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
B Cells ◽  
B Lymphocyte ◽  
De Novo ◽  
Targeted Deletion ◽  
Inhibitory Role ◽  
Proliferation And Differentiation

AbstractHemokinin-1 (HK-1), encoded by the TAC4 gene, is a tachykinin peptide that is predominantly expressed in non-neuronal cells, such as immune cells. We have disrupted the mouse TAC4 gene to obtain a better understanding of the actions of HK-1 during hematopoiesis. We demonstrate here that TAC4−/− mice exhibit an increase of CD19+CD117+HSA+BP.1− “fraction B” pro-B cells in the bone marrow, whereas pre-B, immature, and mature B cells are within the normal range. We show that in vitro cultures derived from TAC4−/− bone marrow, sorted “fraction B” pro-B cells or purified long-term reconstituting stem cells, contain significantly higher numbers of pro-B cells compared with controls, suggesting an inhibitory role for HK-1 on developing B cells. Supporting this idea, we show that addition of HK-1 to cultures established from long-term reconstituting stem cells and the newly described intermediate-term reconstituting stem cells leads to a significant decrease of de novo generated pro-B cells. Based on our studies, we postulate that HK-1 plays an inhibitory role in hematopoiesis, and we hypothesize that it may be part of the bone marrow microenvironment that supports and regulates the proliferation and differentiation of hematopoietic cells.

A Novel Nr4a1GFP Reporter System Reveals That Nr4a1 Expression Identifies Long-Term Hematopoietic Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2339-2339
Author(s):  
Ruben Land ◽  
Trevor Barlowe ◽  
Shwetha Manjunath ◽  
Sophie Eiger ◽  
Matthew Gross ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Conflicts Of Interest ◽  
Family Members ◽  
Reporter System ◽  
Hematopoietic Stem ◽  
And Function

Abstract Abstract 2339 Recent studies have highlighted the importance of the NR4A nuclear receptor family (Nur77 (Nr4a1), Nurr1 (Nr4a3), Nor1 (Nr4a2)) in the regulation of hematopoiesis. In murine models, NR4A gene deficiencies lead to aberrant proliferation of hematopoietic stem cells, and can lead to acute myeloid leukemia (AML). NR4A gene deficiencies also appear to be a feature in human AML cells. In order to better understand the pattern of expression and function of NR4A family members during normal hematopoiesis, we have developed a novel reporter mouse where the Nr4a1 promoter drives GFP expression (Nr4a1GFP). Our analyses reveal a hierarchy in Nr4a1 expression among bone marrow hematopoietic stem cells: long-term (LT) HSC's (CD150+CD48-LSKs) express the highest levels of Nr4a1GFP, more mature HSC's and multilineage progenitor populations (CD150+CD48+ and CD150-CD48+ LSKs) express intermediate levels, and common myeloid progenitors (CMLs, defined as Lin-c-kit+sca-1-) express no Nr4a1GFP. Interestingly, circulating LSK's in the spleen express Nr4a1GFP at higher levels than their bone marrow counterparts. In support of data suggesting that Nr4a family members regulate quiescence, we find that 1) all hematopoietic stem cells that remain in the bone marrow after acute (36h) 5-FU treatment express Nr4a1GFP, 2) Nr4a1GFP expression decreases among circulating splenic LSKs 48 hours after treatment with PolyI:C, and 3) Nr4a1GFP expression increases markedly when stem cells are cultured in vitro under conditions that promote quiescence. We will use this novel system to more directly address the role of Nr4a1 expression in hematopoiesis by evaluating the cell cycle status and defining the reconstitution potential of HSC's on the basis of their Nr4a1GFP expression. Disclosures: No relevant conflicts of interest to declare.

The Bone Marrow "Mystery Population" of Stem Cells 20 Years Later - a Puzzle Solved?

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2392-2392
Author(s):  
Malwina Suszynska ◽  
Daniel Pedziwiatr ◽  
Magdalena J Kucia ◽  
Mariusz Z Ratajczak ◽  
Janina Ratajczak
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Receptor Expression ◽  
Stem Cell Population ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Low Levels

Abstract Background . Almost 20 years ago, a "mystery" population of small stem cells with many of the phenotypic characteristics attributed to resting hematopoietic stem cells was identified in murine bone marrow (BM) (Stem Cells 1998, 16, 38-48). These cells expressed high levels of Sca-1, H-2K, and CD38 and low levels of Thy-1.1; they expressed CD45 antigen but were lineage-negative (lin-) for other hematopoietic markers. These cells incorporated only low levels of Rh123 and were resistant to the cytotoxic effects of 5-fluorouracil. The only phenotypic characteristic that distinguishes these cells from Sca-1+, Lin-, CD45+ Thy-1.1low long-term-reconstituting hematopoietic stem cell population is the lack of c-kit expression. In sum, this "mystery" population of small Sca-1+, lin-, c-kit- but CD45+ stem cells do not respond to hematopoietic growth factors in vitro, form in vivo spleen colonies, or reconstitute lethally irradiated mice. With our discovery of Sca-1+ Lin- CD45- very small embryonic-like stem cells (VSELs) in murine bone marrow (BM) (Leukemia 2006, 20, 857-869), we became interested in this "mystery" population of stem cells. VSELs, like the "mystery" population, are c-kit - and, if freshly isolated from BM, do not show any hematopoietic activity in standard in vitro and in vivo assays. In order to become specified to hematopoiesis, they need to be expanded over an OP-9 stromal support (Exp Hematol 2011;39:225-237). Hypothesis. Since (1) very small CD45- VSELs can be specified in OP-9 co-cultures into long-term reconstituting CD45+ HSCs, (2) the size of the "mystery" population is intermediate between VSELs and HSCs, and (3) VSELs and HSCs differ in cell surface receptor expression, we hypothesized that the "mystery" population is a missing developmental intermediate between VSELs and HSCs. Materials and Methods . Multicolor FACS analysis was employed to compare size and expression of surface markers between murine BM HSCs, the unknown population of stem cells, and VSELs. Next, the populations of small Sca-1+ H2-K+ lin- c-kit+ CD38+/- CD45+ cells (HSCs), smaller Sca-1+ H-2K+ lin- c-kit- CD38+ CD45+ cells (the "mystery" population), and very small in size Sca-1+ H-2K+ lin- c-kit- CD38+/- CD45- cells (VSELs) were purified by FACS from BM (Figure 1) and tested for in vitro colony formation. All these cell populations were primed/expanded over OP-9 support and subsequently evaluated for their hematopoietic potential after passaging in consecutive methylocellulose cultures (passages 1-4). RQ-PCR analysis was employed for detection of pluripotency marker expression as well as hematopoietic gene expression. Results . We found that, in contrast to HSCs, neither freshly sorted stem cells from the "mystery" BM population nor, as expected, VSELs grew hematopoietic colonies in standard methylcellulose cultures. This was also an important step in excluding contamination of our sorted populations with clonogenic cells. We also found that, while VSELs highly expressed Oct-4, this transcription factor was expressed at very low levels in the "mystery" population and was not detectable in HSCs. The most important observation was that the "mystery" population of stem cells became specified in OP-9-supported cultures into clonogenic HSPCs, and this specification occurred faster than the delayed specification of VSELs. VSELs first became enriched for HSPCs after acquiring CD45 antigen expression. Conclusions . Based on the results presented, we propose that the "mystery" population in murine BM is a population of stem cells intermediate between the most primitive population of BM-residing stem cells (VSELs) and the population of stem cells already specified to lympho-hematopoietic development (HSCs). Disclosures No relevant conflicts of interest to declare.

scholarly journals Diabetes impairs the interactions between long-term hematopoietic stem cells and osteopontin-positive cells in the endosteal niche of mouse bone marrow

2013 ◽  
Vol 305 (7) ◽  
pp. C693-C703 ◽  
Author(s):  
Hironori Chiba ◽  
Koji Ataka ◽  
Kousuke Iba ◽  
Kanna Nagaishi ◽  
Toshihiko Yamashita ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Mouse Bone Marrow ◽  
Diabetic Mice ◽  
Hematopoietic Stem ◽  
Coculture System ◽  
Endosteal Niche

Hematopoietic stem cells (HSCs) are maintained, and their division/proliferation and quiescence are regulated in the microenvironments, niches, in the bone marrow. Although diabetes is known to induce abnormalities in HSC mobilization and proliferation through chemokine and chemokine receptors, little is known about the interaction between long-term HSCs (LT-HSCs) and osteopontin-positive (OPN) cells in endosteal niche. To examine this interaction, LT-HSCs and OPN cells were isolated from streptozotocin-induced diabetic and nondiabetic mice. In diabetic mice, we observed a reduction in the number of LT-HSCs and OPN cells and impaired expression of Tie2, β-catenin, and N-cadherin on LT-HSCs and β1-integrin, β-catenin, angiopoietin-1, and CXCL12 on OPN cells. In an in vitro coculture system, LT-HSCs isolated from nondiabetic mice exposed to diabetic OPN cells showed abnormal mRNA expression levels of Tie2 and N-cadherin. Conversely, in LT-HSCs derived from diabetic mice exposed to nondiabetic OPN cells, the decreased mRNA expressions of Tie2, β-catenin, and N-cadherin were restored to normal levels. The effects of diabetic or nondiabetic OPN cells on LT-HSCs shown in this coculture system were confirmed by the coinjection of LT-HSCs and OPN cells into bone marrow of irradiated nondiabetic mice. Our results provide new insight into the treatment of diabetes-induced LT-HSC abnormalities and suggest that the replacement of OPN cells may represent a novel treatment strategy.

Long-Term Clinical Follow-Up and Safety/Toxicity Analysis in 3 X-CGD Patients Treated by Gene Therapy and Non-Myeloablative Conditioning.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 199-199 ◽  
Author(s):  
Marion G. Ott ◽  
Manfred Schmidt ◽  
Stefan Stein ◽  
Kerstin Schwarzwaelder ◽  
Ulrich Siler ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Myeloid Cells ◽  
Adult Patients ◽  
Hematopoietic Stem

Abstract Gene transfer into hematopoietic stem cells has been successfully used to correct immunodeficiencies affecting the lymphoid compartment. However, similar results have not been reported for diseases affecting myeloid cells, mainly due to low engraftment levels of gene-modified cells observed in unconditioned patients. Here we report on two adult patients (P1 and P2, follow up >24 months) and one child (P3, 6 years, follow up 15 months) who received gene-transduced hematopoietic stem cells in combination with nonmyeloablative bone marrow conditioning for the treatment of X-linked Chronic Granulomatous Disease (X-CGD), a primary immunodeficiency caused by a defect in the oxidative antimicrobial activity of phagocytes. Therapeutically significant gene marking was detected in neutrophils of both adult patients (P1 and P2) leading to large numbers (up to 60%) of functionally corrected phagocytes 24 months after gene therapy. This high correction resulted from an unexpected but temporarily restricted expansion of gene transduced myeloid cells in vivo. In contrast gene marking and functionally reconstitution levels in P3 have been low (1–2%). Both adult patients suffered from active infections prior to gene therapy (P1 of bacterial liver abscesses and P2 of lung aspergillosis) and were free of severe bacterial and fungal infections until 24 months after transplantation. P3 suffered from an Aspergillus infection of the spinal cord with paraparesis before transplantation and recovered after gene therapy despite low numbers of functionally corrected cells in the peripheral blood. Large-scale mapping of retroviral integration site distribution revealed that activating insertions in the zinc finger transcription factor homologs MDS1/EVI1, PRDM16, or in SETBP1 have expanded gene-corrected long term myelopoiesis 3- to 4-fold in both adults, providing direct evidence in humans that these genes may influence regulation of normal long-term hematopoiesis. The hematopoietic repopulation in P1 was polyclonal until 18 months after therapy. P1 died of a severe bacterial sepsis after colon perforation 27 months after gene therapy. No evidence of malignant transformation was found in peripheral blood or bone marrow aspirates from this patient. Gene marking at death was still 60%; however the function of gene transduced cells, the number of corrected cell clones and the activity of a predominant clone was greatly decreased. P2 has been free of infections since transplantation (last monitoring: month 26). Hematopoietic repopulation was polyclonal in P2 until day 560. In conclusion, gene therapy in combination with bone marrow conditioning has provided a transitory therapeutic benefit for all 3 patients. Further improvements in vector design and conditioning regimes are under investigation to provide a stable and long term correction of the disease.

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