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1.
J Crohns Colitis ; 14(10): 1424-1435, 2020 Oct 05.
Article in English | MEDLINE | ID: mdl-32318720

ABSTRACT

BACKGROUND AND AIMS: Mesenchymal stem cells [MSCs] are used in preclinical and clinical studies for treatment of immune-mediated disorders, thanks to their immunomodulatory properties. Cell therapy with MSCs induces multiple effects in the immune system which ultimately lead to increase in the number of immune cells with regulatory phenotype. In this study, we investigated whether the beneficial effects of MSC therapy are maintained in the long term in a clinically relevant mouse model of colitis. METHODS: A single dose of adipose-derived MSCs [aMSCs] was infused into dextran sulphate sodium [DSS]-induced colitic mice during the induction phase of the disease. Following a latency period of 12 weeks, mice were re-challenged with a second 7-day cycle of DSS. RESULTS: DSS-induced colitic mice treated with aMSCs showed significant reduction in their colitic disease activity index during the second DSS challenge when compared with non-aMSC treated DSS-induced colitic mice. Strikingly, the long-term protection induced by aMSC therapy was also observed in Rag-1-/- mice where no adaptive immune memory cell responses take place. Increased percentages of Ly6G+CD11b+ myeloid cells were observed 12 weeks after the first inflammatory challenge in the peritoneal cavity, spleen, and bone marrow of DSS-induced colitic mice that were infused with aMSCs. Interestingly, upon re-challenge with DSS, these animals showed a concomitant increase in the regulatory/inflammatory macrophage ratio in the colon lamina propria. CONCLUSIONS: Our findings demonstrate for the first time that MSC therapy can imprint an innate immune memory-like response in mice which confers sustained protection against acute inflammation in the long term.


Subject(s)
Colitis , Immunity, Innate/immunology , Immunologic Memory/immunology , Intestinal Mucosa , Mesenchymal Stem Cell Transplantation/methods , Myeloid Cells , Animals , Antigens, Ly/analysis , CD11b Antigen/analysis , Cell- and Tissue-Based Therapy/methods , Colitis/immunology , Colitis/therapy , Disease Models, Animal , Intestinal Mucosa/immunology , Intestinal Mucosa/pathology , Mice , Mice, Inbred C57BL , Myeloid Cells/immunology , Myeloid Cells/pathology , Time
2.
Stem Cell Res Ther ; 11(1): 164, 2020 04 28.
Article in English | MEDLINE | ID: mdl-32345365

ABSTRACT

BACKGROUND: CD18 is the common beta subunit of ß2 integrins, which are expressed on hematopoietic cells. ß2 integrins are essential for cell adhesion and leukocyte trafficking. METHODS: Here we have analyzed the expression of CD18 in different subsets of human hematopoietic stem and progenitor cells (HSPCs) from cord blood (CB), bone marrow (BM), and mobilized peripheral blood (mPB) samples. CD34+ cells were classified into CD18high and CD18low/neg, and each of these populations was analyzed for the expression of HSPC markers, as well as for their clonogenity, quiescence state, and repopulating ability in immunodeficient mice. RESULTS: A downregulated membrane expression of CD18 was associated with a primitive hematopoietic stem cells (HSC) phenotype, as well as with a higher content of quiescent cells and multipotent colony-forming cells (CFCs). Although no differences in the short-term repopulating potential of CD18low/neg CD34+ and CD18high CD34+ cells were observed, CD18low/neg CD34+ cells were characterized by an enhanced long-term repopulating ability in NSG mice. CONCLUSIONS: Overall, our results indicate that the downregulated membrane expression of CD18 characterizes a primitive population of human hematopoietic repopulating cells.


Subject(s)
Bone Marrow Cells , Hematopoietic Stem Cells , Animals , Antigens, CD34/genetics , Bone Marrow , Fetal Blood , Humans , Mice
3.
Sci Rep ; 10(1): 6997, 2020 04 24.
Article in English | MEDLINE | ID: mdl-32332829

ABSTRACT

The promising ability to genetically modify hematopoietic stem and progenitor cells by precise gene editing remains challenging due to their sensitivity to in vitro manipulations and poor efficiencies of homologous recombination. This study represents the first evidence of implementing a gene editing strategy in a murine safe harbor locus site that phenotypically corrects primary cells from a mouse model of Fanconi anemia A. By means of the co-delivery of transcription activator-like effector nucleases and a donor therapeutic FANCA template to the Mbs85 locus, we achieved efficient gene targeting (23%) in mFA-A fibroblasts. This resulted in the phenotypic correction of these cells, as revealed by the reduced sensitivity of these cells to mitomycin C. Moreover, robust evidence of targeted integration was observed in murine wild type and FA-A hematopoietic progenitor cells, reaching mean targeted integration values of 21% and 16% respectively, that were associated with the phenotypic correction of these cells. Overall, our results demonstrate the feasibility of implementing a therapeutic targeted integration strategy into the mMbs85 locus, ortholog to the well-validated hAAVS1, constituting the first study of gene editing in mHSC with TALEN, that sets the basis for the use of a new safe harbor locus in mice.


Subject(s)
Fanconi Anemia/genetics , Gene Editing/methods , Transcription Activator-Like Effector Nucleases/genetics , Animals , Blotting, Western , Chromosomal Instability/genetics , Female , Flow Cytometry , HEK293 Cells , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Humans , In Situ Hybridization, Fluorescence , Mice, Inbred C57BL , Pregnancy
4.
Cell Stem Cell ; 25(5): 607-621.e7, 2019 11 07.
Article in English | MEDLINE | ID: mdl-31543367

ABSTRACT

Non-homologous end-joining (NHEJ) is the preferred mechanism used by hematopoietic stem cells (HSCs) to repair double-stranded DNA breaks and is particularly increased in cells deficient in the Fanconi anemia (FA) pathway. Here, we show feasible correction of compromised functional phenotypes in hematopoietic cells from multiple FA complementation groups, including FA-A, FA-C, FA-D1, and FA-D2. NHEJ-mediated repair of targeted CRISPR-Cas9-induced DNA breaks generated compensatory insertions and deletions that restore the coding frame of the mutated gene. NHEJ-mediated editing efficacy was initially verified in FA lymphoblastic cell lines and then in primary FA patient-derived CD34+ cells, which showed marked proliferative advantage and phenotypic correction both in vitro and after transplantation. Importantly, and in contrast to homologous directed repair, NHEJ efficiently targeted primitive human HSCs, indicating that NHEJ editing approaches may constitute a sound alternative for editing self-renewing human HSCs and consequently for treatment of FA and other monogenic diseases affecting the hematopoietic system.


Subject(s)
CRISPR-Cas Systems/genetics , DNA End-Joining Repair/genetics , Fanconi Anemia Complementation Group A Protein/genetics , Fanconi Anemia/therapy , Gene Editing/methods , Genetic Therapy/methods , Hematopoietic Stem Cell Transplantation , Alleles , Animals , Antigens, CD34/metabolism , Cell Line , Cell Proliferation/genetics , DNA Breaks, Double-Stranded , Fanconi Anemia/genetics , Fanconi Anemia/metabolism , Fanconi Anemia Complementation Group A Protein/metabolism , Hematopoietic Stem Cell Transplantation/adverse effects , Hematopoietic Stem Cell Transplantation/methods , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , High-Throughput Nucleotide Sequencing , Humans , INDEL Mutation , Mice , Mice, Inbred NOD , Mice, Nude
5.
EMBO Mol Med ; 9(11): 1574-1588, 2017 11.
Article in English | MEDLINE | ID: mdl-28899930

ABSTRACT

Gene targeting constitutes a new step in the development of gene therapy for inherited diseases. Although previous studies have shown the feasibility of editing fibroblasts from Fanconi anemia (FA) patients, here we aimed at conducting therapeutic gene editing in clinically relevant cells, such as hematopoietic stem cells (HSCs). In our first experiments, we showed that zinc finger nuclease (ZFN)-mediated insertion of a non-therapeutic EGFP-reporter donor in the AAVS1 "safe harbor" locus of FA-A lymphoblastic cell lines (LCLs), indicating that FANCA is not essential for the editing of human cells. When the same approach was conducted with therapeutic FANCA donors, an efficient phenotypic correction of FA-A LCLs was obtained. Using primary cord blood CD34+ cells from healthy donors, gene targeting was confirmed not only in in vitro cultured cells, but also in hematopoietic precursors responsible for the repopulation of primary and secondary immunodeficient mice. Moreover, when similar experiments were conducted with mobilized peripheral blood CD34+ cells from FA-A patients, we could demonstrate for the first time that gene targeting in primary hematopoietic precursors from FA patients is feasible and compatible with the phenotypic correction of these clinically relevant cells.


Subject(s)
Antigens, CD34/metabolism , Fanconi Anemia Complementation Group A Protein/genetics , Fanconi Anemia/pathology , Gene Editing/methods , Hematopoietic Stem Cells/metabolism , Animals , Base Sequence , Cells, Cultured , Dependovirus/genetics , Fanconi Anemia/metabolism , Fanconi Anemia Complementation Group A Protein/metabolism , Fetal Blood/cytology , Genetic Vectors/genetics , Genetic Vectors/metabolism , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/cytology , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Mice, Transgenic , Reactive Oxygen Species/metabolism , Zinc Finger Nucleases/genetics , Zinc Finger Nucleases/metabolism
6.
Blood Adv ; 1(5): 319-329, 2017 Jan 24.
Article in English | MEDLINE | ID: mdl-29296947

ABSTRACT

Detectable clonal mosaicism for large chromosomal events has been associated with aging and an increased risk of hematological and some solid cancers. We hypothesized that genetic cancer predisposition disorders, such as Fanconi anemia (FA), could manifest a high rate of chromosomal mosaic events (CMEs) in peripheral blood, which could be used as early biomarkers of cancer risk. We studied the prevalence of CMEs by single-nucleotide polymorphism (SNP) array in 130 FA patients' blood DNA and their impact on cancer risk. We detected 51 CMEs (4.4-159 Mb in size) in 16 out of 130 patients (12.3%), of which 9 had multiple CMEs. The most frequent events were gains at 3q (n = 6) and 1q (n = 5), both previously associated with leukemia, as well as rearrangements with breakpoint clustering within the major histocompatibility complex locus (P = 7.3 × 10-9). Compared with 15 743 age-matched population controls, FA patients had a 126 to 140 times higher risk of detectable CMEs in blood (P < 2.2 × 10-16). Prevalent and incident hematologic and solid cancers were more common in CME carriers (odds ratio [OR] = 11.6, 95% confidence interval [CI] = 3.4-39.3, P = 2.8 × 10-5), leading to poorer prognosis. The age-adjusted hazard risk (HR) of having cancer was almost 5 times higher in FA individuals with CMEs than in those without CMEs. Regarding survival, the HR of dying was 4 times higher in FA individuals having CMEs (HR = 4.0, 95% CI = 2.0-7.9, P = 5.7 × 10-5). Therefore, our data suggest that molecular karyotyping with SNP arrays in easy-to-obtain blood samples could be used for better monitoring of bone marrow clonal events, cancer risk, and overall survival of FA patients.

7.
Cell Transplant ; 22(8): 1381-94, 2013.
Article in English | MEDLINE | ID: mdl-23044223

ABSTRACT

Mesenchymal stromal cell (MSC) immunosuppressive properties have been applied to treat graft-versus-host disease (GVHD) in allogeneic hematopoietic stem cell transplants (HSCTs). We have previously demonstrated that MSC infusions early after haplo-HSCT prevent GVHD in a haploidentical-HSCT mouse model. Now, we investigated the impact that MSCs' immunosuppressive properties have on the graft-versus-leukemia (GVL) effect. First, to mimic a chronic myeloid leukemia (CML) relapse after a haploidentical HSCT, lethally irradiated mice were coinfused with haploidentical donor bone marrow cells plus syngenic hematopoietic progenitors transduced with a retroviral vector encoding both the BCR/ABL oncogene and the ΔNGFR marker gene. As expected, a CML-like myeloproliferative syndrome developed in all the recipient animals. The addition of haploidentical splenocytes to the transplanted graft prevented CML development by a GVL effect, and all transplanted recipients died of GVHD. This GVL mouse model allowed us to investigate the impact of MSCs infused to prevent GVHD on days 0, 7, and 14 after HSCT, on the GVL effect, expecting an increase in leukemic relapse. Strikingly, a high mortality of the recipients was observed, caused by GVHD, and only few leukemic cells were detected in the recipient animals. In contrast, GVHD prevention by MSCs in the absence of BCR/ABL leukemic cells resulted in a significant survival of the recipients. In vitro data pointed to an inability of MSCs to control strong CTLs responses against BCR/ABL. Our results show that, although an evident increase in leukemic relapses induced by MSCs could not be detected, they showed a reduced efficacy in preventing GVHD that precluded us to draw clear conclusions on MSCs' impact over GVL effect.


Subject(s)
Bone Marrow Transplantation , Graft vs Host Disease/prevention & control , Haploidy , Leukemia/therapy , Mesenchymal Stem Cells/cytology , Adenoviridae/metabolism , Animals , Bone Marrow/pathology , Cell Proliferation , Disease Models, Animal , Female , Fusion Proteins, bcr-abl/metabolism , Hematopoietic Stem Cell Transplantation , Leukemia/blood , Mice , Mice, Inbred C57BL , Recurrence , Retroviridae/metabolism , Spleen/pathology , T-Lymphocytes/metabolism , Transduction, Genetic , Treatment Outcome
8.
Biochem J ; 422(1): 161-70, 2009 Jul 29.
Article in English | MEDLINE | ID: mdl-19473116

ABSTRACT

FA (Fanconi anaemia) is a hereditary disease characterized by congenital malformations, progressive bone marrow failure and an extraordinary elevated predisposition to develop cancer. In the present manuscript we describe an anomalous high level of the proinflammatory cytokine IL-1beta (interleukin-1beta) present in the serum of FA patients. The elevated levels of IL-1beta were completely reverted by transduction of a wild-type copy of the FancA cDNA into FA-A (FA group A) lymphocytes. Although the transcription factor NF-kappaB (nuclear factor-kappaB) is a well established regulator of IL-1beta expression, our experiments did not show any proof of elevated NF-kappaB activity in FA-A cells. However, we found that the overexpression of IL-1beta in FA-A cells is related to a constitutively activated PI3K (phosphoinositide 3-kinase)-Akt pathway in these cells. We provide evidence that the effect of Akt on IL-1beta activation is mediated by the inhibition of GSK3beta (glycogen synthase kinase 3beta). Finally, our data indicate that the levels of IL-1beta produced by FA-A lymphoblasts are enough to promote an activation of the cell cycle in primary glioblastoma progenitor cells. Together, these results demonstrate that the constitutive activation of the PI3K-Akt pathway in FA cells upregulates the expression of IL-1beta through an NF-kappaB-independent mechanism and that this overproduction activates the proliferation of tumour cells.


Subject(s)
Fanconi Anemia/enzymology , Fanconi Anemia/pathology , Interleukin-1beta/metabolism , Neoplasms/enzymology , Neoplasms/pathology , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Cell Proliferation/drug effects , Enzyme Activation/drug effects , Fanconi Anemia Complementation Group A Protein/deficiency , Glycogen Synthase Kinase 3/antagonists & inhibitors , Glycogen Synthase Kinase 3 beta , Humans , Lymphocytes/drug effects , Lymphocytes/metabolism , Mutation/genetics , NF-kappa B/metabolism , Phosphorylation/drug effects , Protein Kinase Inhibitors/pharmacology
9.
Exp Hematol ; 32(4): 360-4, 2004 Apr.
Article in English | MEDLINE | ID: mdl-15050746

ABSTRACT

OBJECTIVE: Previous studies have shown that the HS21/45 promoter of the vav protooncogene drives a predominant expression of exogenous transgenes in mouse hematopoietic cells, including clonogenic bone marrow (BM) progenitors. We investigated the activity of this promoter in the hematopoietic stem cell compartment of adult mice. MATERIALS AND METHODS: Inbred Ly5.1 transgenic mice expressing a nonfunctional human CD4 marker gene (hCD4) under the control of the HS21/45 promoter were generated. BM cells from these animals were sorted based on the intensity of hCD4 expression. Fractions characterized by high, intermediate, or low/negative expression of the transgene were then assessed for their competitive repopulation ability (CRA), using unfractionated BM cells from Ly5.2 mice as a reference competitor population. RESULTS: Data showed that BM cells having a low/negative or intermediate expression of hCD4 had a very poor hematopoietic CRA. In contrast, BM cells with high hCD4 expression were characterized by a high CRA. These observations were confirmed in the short- and long-term posttransplantation of primary and secondary recipients when analyzing the lymphoid and myeloid cells of recipient mice. CONCLUSIONS: Our results demonstrate for the first time that the regulatory HS21/45 sequence of the vav gene constitutes an efficient promoter for driving transgene expression in multipotent hematopoietic stem cells residing in the BM of adult mice. Thus, this promoter is proposed for the development of transgenic mice and gene therapy vectors that require restricted expression of exogenous transgenes in cells of the hematopoietic system, including primitive hematopoietic stem cells.


Subject(s)
Cell Cycle Proteins , Gene Expression Regulation/genetics , Hematopoietic Stem Cells/metabolism , Promoter Regions, Genetic/genetics , Proto-Oncogene Proteins/genetics , Recombinant Fusion Proteins/biosynthesis , Transgenes/genetics , Animals , Bone Marrow Cells/metabolism , CD4 Antigens/biosynthesis , CD4 Antigens/genetics , Clone Cells/metabolism , Humans , Mice , Mice, Inbred Strains , Mice, Transgenic , Multipotent Stem Cells/metabolism , Proto-Oncogene Proteins c-vav , Recombinant Fusion Proteins/genetics
10.
Blood ; 103(1): 128-32, 2004 Jan 01.
Article in English | MEDLINE | ID: mdl-12958075

ABSTRACT

Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome characterized by cellular sensitivity to genotoxic agents. In recent years, FA proteins have been associated with different molecules involved in signal transduction, which has raised the interest in FA-dependent signaling pathways. Here, we report that the c-Jun N-terminal kinase (JNK) fails to phosphorylate in response to UV radiation and treatment with mitomycin C in FA lymphoblast cells derived from type A patients (FA-A). Furthermore, defective kinase activity seems to be specific for JNK, because extracellular signal-regulated kinase (ERK) responded to the proper stimuli in FA-A cells. We also demonstrate that the early growth-response factor-1 (Egr-1), a JNK downstream target gene that is normally induced by genotoxic stress, is not upregulated in UV-treated FA-A cells. Moreover, FA-A cells are more sensitive to apoptosis than control lymphoblasts. Both JNK and Egr-1 may be part of a pathway triggered by FA proteins, because functional correction of FA-A cells by gene transfer restores, at least in part, JNK activation and Egr-1 expression after UV exposure. Together, our data suggest that activation of JNK and expression of Egr-1 gene in B lymphoblasts mediate a cellular response to genotoxic agents that may be induced by FA proteins.


Subject(s)
DNA-Binding Proteins/genetics , Fanconi Anemia/genetics , Fanconi Anemia/metabolism , Immediate-Early Proteins , Transcription Factors/genetics , B-Lymphocytes/metabolism , B-Lymphocytes/radiation effects , Base Sequence , Cell Line , DNA, Complementary/genetics , Down-Regulation , Early Growth Response Protein 1 , Enzyme Activation/radiation effects , Fanconi Anemia Complementation Group A Protein , Genetic Complementation Test , Humans , Proteins/genetics , Proteins/metabolism , Ultraviolet Rays
12.
Cancer Res ; 62(17): 5013-8, 2002 Sep 01.
Article in English | MEDLINE | ID: mdl-12208755

ABSTRACT

Tumor cell contamination of clinical grafts is a major concern in autologous hematopoietic stem cell transplantation because these contaminating cells can contribute to relapse. In the present work, we use a suicide gene therapy approach that successfully accomplishes the two main goals of any purging strategy: highly efficient elimination of contaminating tumor cells and preservation of the engraftment capability of the hematopoietic progenitor cells. Human CD34(+) cells spiked with breast cancer cells were infected with an adenoviral vector encoding the cytosine deaminase transgene (Ad-CMV-CD). In vitro, transduction with Ad-CMV-CD followed by exposure to 400 micro M 5-fluorocytosine resulted in complete elimination of clonogenic contaminating tumor cells without affecting the clonogenic potential of the human hematopoietic CD34(+) cells. Transplantation of nonobese diabetic/LtSz-scid/severe combined immunodeficient (NOD/SCID) mice with nonpurged contaminated grafts and purged contaminated grafts, allowed us to test the safety and efficacy of our procedure in two independent purging experiments. Hematopoietic engraftment kinetics as well as the quantity and quality of human engraftment were not affected by the purging therapy. Results showed a significant difference in survival between the nonpurged group (28%) and the purged group (100%; P = 0.012). Moreover, highly sensitive histological and molecular analyses confirmed the absence of tumor cells in the recipients of purged marrow. In contrast, metastatic tumors were detected in animals that received nonpurged grafts. We anticipate that this strategy will result in a safe and efficacious hematopoietic graft product for autologous transplantation for patients with multiple forms of epithelial cancers.


Subject(s)
Bone Marrow Purging/methods , Breast Neoplasms/pathology , Breast Neoplasms/therapy , Genetic Therapy/methods , Hematopoietic Stem Cell Transplantation/methods , Adenoviridae/genetics , Animals , Cytosine Deaminase , Female , Flucytosine/pharmacokinetics , Humans , Male , Mice , Mice, Inbred NOD , Mice, SCID , Nucleoside Deaminases/genetics , Nucleoside Deaminases/metabolism , Tumor Cells, Cultured
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