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Basma F Benabdallah,
Emilie Allard, Shuyuan Yao,
Geoffrey Friedman,
Philip D Gregory,
Nicoletta Eliopoulos,
Julie Fradette,
Jeffrey L Spees,
Elie Haddad,
Michael C Holmes,
Christian M Beauséjour
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ABSTRACT: Gene-modified mesenchymal stromal cells (MSC) provide a promising tool for cell and gene therapy-based applications by potentially acting as a cellular vehicle for protein-replacement therapy. However, to avoid the risk of insertional mutagenesis, targeted integration of a transgene into a 'safe harbor' locus is of great interest.
We sought to determine whether zinc finger nuclease (ZFN)-mediated targeted addition of the erythropoietin (Epo) gene into the chemokine [C-C motif] receptor 5 (CCR5) gene locus, a putative safe harbor locus, in MSC would result in stable transgene expression in vivo.
Whether derived from bone marrow (BM), umbilical cord blood (UCB) or adipose tissue (AT), 30-40% of human MSC underwent ZFN-driven targeted gene addition, as determined by a combination of fluorescence-activated cell sorting (FACS)- and polymerase chain reaction (PCR)-based analyzes. An enzyme-linked immunosorbent assay (ELISA)-based analysis of gene-targeted MSC expressing Epo from the CCR5 locus showed that these modified MSC were found to secrete a significant level of Epo (c. 2 IU/10(6)cells/24 h). NOD/SCID/gammaC mice injected with ZFN-modified MSC expressing Epo exhibited significantly higher hematocrit and Epo plasma levels for several weeks post-injection, compared with mice receiving control MSC.
These data demonstrate that MSC modified by ZFN-driven targeted gene addition may represent a cellular vehicle for delivery of plasma-soluble therapeutic factors.
Cytotherapy 03/2010; 12(3):394-9. · 3.63 Impact Factor
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ABSTRACT: In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.(1) The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.(2) Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.(3) iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells. Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.
Journal of Visualized Experiments 01/2009;
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ABSTRACT: Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.(1) However, LIF is expensive and activation of the LIF/JAK/STAT3 pathway is not absolutely required to maintain the self-renewal state.(2) The SC1 small molecule may be an economical alternative to LIF. SC1 functions through dual inhibition of Ras-GAP and ERK1.(3) Illustration of its mechanism of action makes it a useful tool to study the fundamental molecular mechanism of self-renewal. Here we demonstrate the procedure for culturing mouse ES cells in the presence of SC1 and show that they are able to maintain self-renewal in the absence of LIF. Cells cultured with SC1 showed similar morphology compared to cells maintained with LIF. Both exhibited typical mouse ES morphology after five passages. Expression of typical pluripotency markers (Oct4, Sox2, Nanog, and SSEA1) was observed after five passages in the presence of SC1. Furthermore, SC1 caused no overt toxicity on mouse ES cells.
Journal of Visualized Experiments 01/2009;
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ABSTRACT: A cell-based screen of chemical libraries was carried out to identify small molecules that control the self-renewal of ES cells. A previously uncharacterized heterocycle, SC1, was discovered that allows one to propagate murine ES cells in an undifferentiated, pluripotent state under chemically defined conditions in the absence of feeder cells, serum, and leukemia inhibitory factor. Long-term SC1-expanded murine ES cells can be differentiated into cells of the three primary germ layers in vitro and also can generate chimeric mice and contribute to the germ line in vivo. Biochemical and cellular experiments suggest that SC1 works through dual inhibition of RasGAP and ERK1. Molecules of this kind may not only facilitate practical applications of stem cells in research and therapy, but also provide previously undescribed insights into the complex biology of stem cells.
Proceedings of the National Academy of Sciences 12/2006; 103(46):17266-71. · 9.68 Impact Factor
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ABSTRACT: Chemically defined medium (CDM) conditions for controlling human embryonic stem cell (hESC) fate will not only facilitate the practical application of hESCs in research and therapy but also provide an excellent system for studying the molecular mechanisms underlying self-renewal and differentiation, without the multiple unknown and variable factors associated with feeder cells and serum. Here we report a simple CDM that supports efficient self-renewal of hESCs grown on a Matrigel-coated surface over multiple passages. Expanded hESCs under such conditions maintain expression of multiple hESC-specific markers, retain the characteristic hESC morphology, possess a normal karyotype in vitro, as well as develop teratomas in vivo. Additionally, several growth factors were found to selectively induce monolayer differentiation of hESC cultures toward neural, definitive endoderm/pancreatic and early cardiac muscle cells, respectively, in our CDM conditions. Therefore, this CDM condition provides a basic platform for further characterization of hESC self-renewal and directed differentiation, as well as the development of novel therapies.
Proceedings of the National Academy of Sciences 06/2006; 103(18):6907-12. · 9.68 Impact Factor
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ABSTRACT: An obstacle confronting gene therapy in stem cells is transcriptional silencing of the vector. Here, we discuss recent data indicating that oncoretrovirus and lentivirus vectors are silenced by multiple epigenetic pathways that result in DNA methylation and histone modifications. Both vector types can be variegated in stem cells and expression is often extinguished during differentiation. We propose a novel model of retrovirus silencing in which epigenetic pathways compete to recruit histone deacetylases, de novo methyltransferases, histone H1 and MeCP2 to the provirus. These chromatin modifications may act in concert with heterochromatin at or near the integration site to establish silencing or variegation respectively. Retrovirus vector designs for stem cells should delete virus silencer elements, incorporate strong positive regulatory elements and insulators, and avoid non-mammalian reporter genes. In addition, cancer stem cells that continually repopulate a growing tumour may share silencing pathways with normal stem cells. Ultimately, optimized vector designs may prove to be valuable tools for gene therapy of both normal and cancer stem cells.
Current Gene Therapy 09/2005; 5(4):367-73. · 3.39 Impact Factor
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ABSTRACT: beta-Globin transgenes regulated by the locus control region (LCR) are dominantly silenced by linked bacterial reporter genes in transgenic mice. Enhanced green fluorescent protein (eGFP) from jellyfish is an alternative reporter used in retrovirus vectors to transfer LCRbeta-globin genes into bone marrow. We show here that the eGFP coding sequence silences LCRbeta-globin in transgenic mice, but the PGK promoter did not provoke such silencing. As eGFP contains 60 CpG dinucleotides, which are targets of DNA methylation, we synthesized a novel CpG-free variant called dmGFP. Its utility was demonstrated in MSCV retrovirus vectors transcriptionally controlled by the viral 5'LTR or internal PGK or EF1alpha promoter. Specific fluorescence was detected from eGFP, and at lower levels from dmGFP, in transduced mouse CFU-S and embryonic stem cells. While eGFP was rarely silenced in CFU-S, dmGFP was not silenced in these progenitors. Moreover, the dmGFP coding sequence did not silence LCRbeta-globin in transgenic mice, showing that the eGFP silencing mechanism acts primarily via CpG dinucleotides. However, LCRbeta-globin expression remained suboptimal, indicating that other silencing pathways recognize dmGFP in the absence of CpG dinucleotides. We conclude that dmGFP ameliorates silencing, but optimal LCRbeta-globin expression is obtained in the absence of nonmammalian reporters.
Molecular Therapy 05/2005; 11(4):591-9. · 6.87 Impact Factor
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ABSTRACT: Retrovirus silencing in stem cells produces silent or variegated provirus. Additional memory and extinction mechanisms act during differentiation. Here we show that retrovirus is silent or variegated in mouse embryonic stem (ES) cells that are de novo methyltransferase (dnmt3a and dnmt3b) null. Memory is maintained during differentiation, and extinction occurs on variegated retrovirus, indicating that DNA methylation is dispensable for all forms of retrovirus silencing. Silent and variegated provirus are marked by hypoacetylated histone H3 and bound H1. In wild-type ES cells, silent and variegated proviruses are methylated and bound by hypoacetylated H3, MeCP2, and less H1. Silencing, variegation, and extinction are partially reactivated by 5-AzaC in this context. Lentivirus vectors are also silent or variegated, marked by silent chromatin, and exhibit memory and extinction. We conclude that the universal epigenetic mark of retrovirus silencing is silent chromatin established via the dynamic interplay of multiple epigenetic modifications that include but do not require DNA methylation. A molecular mechanism of competitive H1 and MeCP2 binding may account for this epigenetic interplay, and a model for variegation is discussed.
Molecular Therapy 08/2004; 10(1):27-36. · 6.87 Impact Factor
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ABSTRACT: Silencing of retrovirus vectors poses a significant obstacle to genetic manipulation of stem cells and their use in gene therapy. We describe a mammalian silencer blocking assay using insulator elements positioned between retrovirus silencer elements and an LCRbeta-globin reporter transgene. In transgenic mice, we show that retrovirus silencers are blocked by the cHS4 insulator. Silencer blocking is independent of the CTCF binding site and is most effective when flanking the internal reporter transgene. These data distinguish silencer blocking activity by cHS4 from its enhancer blocking activity. Retrovirus vectors can be created at high titer with one but not two internal dimer cHS4 cores. cHS4 in the LTRs has no effect on expression in transduced F9 cells, suggesting that position effect blocking is not sufficient to escape silencing. The Drosophila insulators gypsy and Scs fail to block silencing in transgenic mice, but gypsy stimulates vector expression 2-fold when located in the LTRs of an infectious retrovirus. The silencer blocking assay complements existing insulator assays in mammalian cells, provides new insight into mechanisms of insulation and is a valuable tool to identify additional silencer blocking insulators that cooperate with cHS4 to improve stem cell retrovirus vector design.
Nucleic Acids Research 10/2003; 31(18):5317-23. · 8.03 Impact Factor
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ABSTRACT: Retrovirus vectors are de novo methylated and transcriptionally silent in mammalian stem cells. Here, we identify epigenetic modifications that mark retrovirus-silenced transgenes. We show that murine stem cell virus (MSCV) and human immunodeficiency virus type 1 (HIV-1) vectors dominantly silence a linked locus control region (LCR) -globin reporter gene in transgenic mice. MSCV silencing blocks LCR hypersensitive site formation, and silent transgene chromatin is marked differentially by a histone code composed of abundant linker histone H1, deacetylated H3 and acetylated H4. Retrovirus-transduced embryonic stem (ES) cells are silenced predominantly 3 days post-infection, with a small subset expressing enhanced green fluorescent protein to low levels, and silencing is not relieved in de novo methylase-null [dnmt3a-/-;dnmt3b-/-] ES cells. MSCV and HIV-1 sequences also repress reporter transgene expression in Drosophila, demonstrating establishment of silencing in the absence of de novo and maintenance methylases. These findings provide mechanistic insight into a conserved gene silencing mechanism that is de novo methylase independent and that epigenetically marks retrovirus chromatin with a repressive histone code.
The EMBO Journal 10/2000; 19(21):5884-5894. · 9.20 Impact Factor
