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Endogenous growth factors secreted by mesenchymal stromal cells can be used to guide tissue regeneration
S48
Poster Presentations/ Experimental Hematology 42 (2014) S23–S68
P1100 - ENDOGENOUS GROWTH FACTORS SECRETED BY MESENCHYMAL STROMAL CELLS CAN BE USED TO GUIDE TISSUE REGENERATION Renaud Manuguerra1,2, Dave Vibhuti2, Mark Lesk1,2, and Denis-Claude Roy1,2 1 l’Universite de Montreal, Montreal, Quebec, Canada; 2H^opital MaisonneuveRosemont, Montreal, Quebec, Canada High intraocular pressure (IOP) is one of the hallmarks of open angle glaucoma. This is linked to a dysfunction of the trabecular meshwork (TM), the tissue regulating the outflow of aqueous humor. Studies have demonstrated that mesenchymal stem cells (MSC) exert regenerative properties through the production of soluble factors. We have previously demonstrated that MSC induce ocular regeneration and lower IOP in glaucoma models by producing factors leading to the reactivation of local progenitor cells in the ciliary body. This effect takes place even though these factors are only present in the eye for a short period of time. We hypothesized that these factors could alter the tissue environment near the TM and that such changes could result in the regenerative potential. Rats first underwent laser trabeculoplasty in order to increase IOP and generate a glaucoma model. Paracrine factors obtained from MSC cultured in either normoxic or hypoxic conditions were injected in the anterior chamber of the animal models. We measured the IOP and used immunofluorescent staining to assess the activation of progenitor cells and functional regeneration in the damaged eye. The MSC supernatant was analyzed by mass spectrometry and the production of endogenous growth factors in ocular tissue samples studied by quantitative PCR. We observed a significant drop in IOP on day 2 in the groups treated with hypoxic medium, along with a strong expression of the neuronal progenitor cell marker nestin (12% of cells) and proliferation marker ki67 (6% of cells). This was not observed in other control groups. Quantitative PCR measurements demonstrated significant upregulation of growth factors in the regenerating area 24 hours after injection of MSC medium. The injection of these purified growth factors in a glaucoma model has allowed us to partially recreate the regenerative effect of the hypoxic MSC media. These results indicate that MSC can induce the local production of growth factors and that these factors play an important role in the regenerative effect. These findings may have broad application to promote tissue regeneration in a large number of diseases.
P1101 - THE FANCONI ANEMIA C PROTEIN IS A TRANSCRIPTIONAL REGULATOR OF DICKKOPF-1 Delphine Masi1,2, Melody Mazon1,2, Caroline Huard1,2, and Madeleine Carreau1 1 CHUL, Quebec, Quebec, Canada; 2Universite Laval, Quebec, Quebec, Canada Children suffering from Fanconi anemia (FA) are at an increased risk of developing leukemia. FA is a devastating disease associated with a progressive decline in primitive hematopoietic cells due to accelerated cycling and hypersensitivity to various external cellular cues. These cellular defects favour the development of clonal proliferation and leads to leukemia. In an effort to understand the molecular basis of abnormal hematopoiesis in FA, we recently uncovered a novel function of proteins associated with FA that translates into elevated levels of the signaling molecule, Dickkopf-1 (DKK1). We found that the FA protein C forms a complex with b-catenin and the repressor protein CtBP1. We found that FANCC accumulates into the nucleus in response to b-catenin activation. This nuclear accumulation of FANCC requires a functional FA pathway as shown by lack of nuclear FANCC in FANCA-mutant cells in response to b-catenin activation. We also found that FANCC with CtBP1 act as transcriptional repressors of the DKK1 gene, while FANCC-mutant cells or CtBP1depleted cells show increased DKK1 expression. In addition, the FANCC diseasecausing mutation L554P impinge on FANCC ability to repress DKK1. Furthermore, we found increased levels of DKK1 in FA- and CtBP1-depleted cells as well as elevated levels of Dkk1 are found in sera from FancA and FancC knockout mice. Because elevated levels of DKK1 have been associated with altered hematopoiesis and malignancies similar to those found in children suffering from FA, our results suggest a possible mechanism explaining the progressive loss of bone marrow cells. Importantly, identification of FANCC as a transcriptional regulator of DKK1 is a crucial step for the development of novel strategies aimed at preventing bone marrow failure in FA patients.
P1102 - DETERMINATION OF 5-HYDROXYMETHYLCYTOSINE AT SINGLE BASE RESOLUTION USING NEXT GENERATION SEQUENCER Hirotaka Matsui, Akinori Kanai, and Toshiya Inaba Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Hiroshima, Japan TET2 and IDH1/2 genes, whose products play roles in converting 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC), frequently show mutations in myeloid diseases. However, the study of 5hmC has been hindered by the lack of methods that determine the proportion and the position of 5hmC precisely. Traditional bisulfite sequencing cannot distinguish 5hmC from 5mC as both of them are read as C. Meanwhile it is recently reported that Tet-assisted bisulfite sequencing (TAB-seq) successfully detected 5hmC at the whole genome level. Here we established a system that enables us to analyze the 5hmC level focusing on CpG rich gene promoters and CpG islands at relatively low cost by combining reduced representative bisulfite sequencing (RRBS) and TAB-seq methods. In our system, CpG-rich DNA fragments of 150-250bp are first selectively purified from genomic DNA. 5hmC sites in the fragments are then glucosylated to 5ghmC by b-glucosyltransferase, while 5mC are oxidized to 5-carboxycytosine (caC) by TET1 enzyme, followed by the bisulfite conversion. After these treatments, unmodified C and 5caC are sequenced as T, while only 5ghmC is read as C. In combination with traditional bisulfite sequencing, we are now able to estimate the proportions of C/5mC/5hmC in about a half million CpG sites. Taking advantage of this system, we analyzed 5hmC of erythroid progenitor cells. TET2 expression was downregulated through lentiviral transduction of shRNA that specifically targets TET2 and the difference of 5hmC between TET2 knocked-down cells and control cells were statistically evaluated using methylKit. In this experiment, we found that only 300 CpGs among evaluated 410,000 CpGs were hydroxy-methylated by TET2 in erythroid progenitor cells. In addition, we also noticed that these 300 CpGs were mostly outside of CpG islands or gene promoters. This unexpected small number of TET2-mediated hydroxyl-methylated CpGs might be partially explained by the structure of TET2 enzyme; unlike TET1 and TET3, TET2 lacks cxxc domain that preferentially binds CpG-rich DNA. It would be possible that TET2 is responsible for hydroxy-methylation of CpG-sparse region such as enhancers.
P1103 - DECLINED PRESENTATION CRITICAL ROLES FOR THE HOMEODOMAIN TRANSCRIPTION FACTOR HHEX IN LYMPHOID COMMITMENT AND SELF-RENEWAL OF HEMATPOIETIC AND LEUKAEMIC STEM CELLS Benjamin Shields1,3, Jacob Jackson1, Chayanica Nasa1, Don Metcalf1,3, Clifford Bogue2, Warren Alexander1,3, and Matthew McCormack1,3 1 Cancer and Hematology Division, Walter and Eliza Hall Institute, Parkville, Victoria, Australia; 2Yale University, New Haven, Connecticut, USA; 3University of Melbourne, Melbourne, Victoria, Australia Hhex is a homeodomain transcription factor that is highly expressed in hematopoietic stem cells (HSCs) and progenitors. Previous studies have implicated Hhex in the development of definitive HSCs and B cells during embryogenesis. However, as Hhex knockout mice are embryonic lethal, the role of Hhex in adult haematopoiesis has not been previously studied. To address this, we have used Hhex conditional knockout mice to delete Hhex throughout the hematopoietic system of adult mice. This revealed that whilst Hhex was dispensable for maintenance of myeloid lineages, it is essential for development of B-cell progenitors. Moreover, competitive transplantation experiments revealed that Hhex is essential for development of all lymphoid lineages beyond thr common lymphoid progenitor (CLP) including B-cells and T-cells. RNA sequencing revealed aberrant expression of a number of cell cycle regulators in the absence of Hhex. Overexpression of one of these, Cyclin D1, could restore lymphoid development in the absence of Hhex. Thus Hhex regulates lymphoid development acting at least in part via regulation of the cell cycle. Whilst Hhex was dispensable for HSC maintenance in the adult, serial transplantation and myeloablation recovery experiments revealed a severe defect in HSC self-renewal and stress haematopoesis in the absence of Hhex. In addition, Hhex-deleted blast colonies were incapable of secondary replating in vitro. In contrast, overexpression of Hhex blocked differentiation leading to immortalised promyelocyte cell lines. Thus Hhex is a dynamic regulator of HSC self-renewal. To assess the role of Hhex in self-renewal of leukaemic stem cells, we tested the ability of the fusion oncogene MLL-ENL to induce leukaemia in the absence of Hhex. Strikingly, this showed a critical role for Hhex in both initiation and maintenance of myeloid leukaemia in vivo. Thus, in addition to its role in lymphoid development, Hhex is essential for selfrenewal of hematopoietic and leukemic stem cells.
Poster Presentations/ Experimental Hematology 42 (2014) S23–S68
P1100 - ENDOGENOUS GROWTH FACTORS SECRETED BY MESENCHYMAL STROMAL CELLS CAN BE USED TO GUIDE TISSUE REGENERATION Renaud Manuguerra1,2, Dave Vibhuti2, Mark Lesk1,2, and Denis-Claude Roy1,2 1 l’Universite de Montreal, Montreal, Quebec, Canada; 2H^opital MaisonneuveRosemont, Montreal, Quebec, Canada High intraocular pressure (IOP) is one of the hallmarks of open angle glaucoma. This is linked to a dysfunction of the trabecular meshwork (TM), the tissue regulating the outflow of aqueous humor. Studies have demonstrated that mesenchymal stem cells (MSC) exert regenerative properties through the production of soluble factors. We have previously demonstrated that MSC induce ocular regeneration and lower IOP in glaucoma models by producing factors leading to the reactivation of local progenitor cells in the ciliary body. This effect takes place even though these factors are only present in the eye for a short period of time. We hypothesized that these factors could alter the tissue environment near the TM and that such changes could result in the regenerative potential. Rats first underwent laser trabeculoplasty in order to increase IOP and generate a glaucoma model. Paracrine factors obtained from MSC cultured in either normoxic or hypoxic conditions were injected in the anterior chamber of the animal models. We measured the IOP and used immunofluorescent staining to assess the activation of progenitor cells and functional regeneration in the damaged eye. The MSC supernatant was analyzed by mass spectrometry and the production of endogenous growth factors in ocular tissue samples studied by quantitative PCR. We observed a significant drop in IOP on day 2 in the groups treated with hypoxic medium, along with a strong expression of the neuronal progenitor cell marker nestin (12% of cells) and proliferation marker ki67 (6% of cells). This was not observed in other control groups. Quantitative PCR measurements demonstrated significant upregulation of growth factors in the regenerating area 24 hours after injection of MSC medium. The injection of these purified growth factors in a glaucoma model has allowed us to partially recreate the regenerative effect of the hypoxic MSC media. These results indicate that MSC can induce the local production of growth factors and that these factors play an important role in the regenerative effect. These findings may have broad application to promote tissue regeneration in a large number of diseases.
P1101 - THE FANCONI ANEMIA C PROTEIN IS A TRANSCRIPTIONAL REGULATOR OF DICKKOPF-1 Delphine Masi1,2, Melody Mazon1,2, Caroline Huard1,2, and Madeleine Carreau1 1 CHUL, Quebec, Quebec, Canada; 2Universite Laval, Quebec, Quebec, Canada Children suffering from Fanconi anemia (FA) are at an increased risk of developing leukemia. FA is a devastating disease associated with a progressive decline in primitive hematopoietic cells due to accelerated cycling and hypersensitivity to various external cellular cues. These cellular defects favour the development of clonal proliferation and leads to leukemia. In an effort to understand the molecular basis of abnormal hematopoiesis in FA, we recently uncovered a novel function of proteins associated with FA that translates into elevated levels of the signaling molecule, Dickkopf-1 (DKK1). We found that the FA protein C forms a complex with b-catenin and the repressor protein CtBP1. We found that FANCC accumulates into the nucleus in response to b-catenin activation. This nuclear accumulation of FANCC requires a functional FA pathway as shown by lack of nuclear FANCC in FANCA-mutant cells in response to b-catenin activation. We also found that FANCC with CtBP1 act as transcriptional repressors of the DKK1 gene, while FANCC-mutant cells or CtBP1depleted cells show increased DKK1 expression. In addition, the FANCC diseasecausing mutation L554P impinge on FANCC ability to repress DKK1. Furthermore, we found increased levels of DKK1 in FA- and CtBP1-depleted cells as well as elevated levels of Dkk1 are found in sera from FancA and FancC knockout mice. Because elevated levels of DKK1 have been associated with altered hematopoiesis and malignancies similar to those found in children suffering from FA, our results suggest a possible mechanism explaining the progressive loss of bone marrow cells. Importantly, identification of FANCC as a transcriptional regulator of DKK1 is a crucial step for the development of novel strategies aimed at preventing bone marrow failure in FA patients.
P1102 - DETERMINATION OF 5-HYDROXYMETHYLCYTOSINE AT SINGLE BASE RESOLUTION USING NEXT GENERATION SEQUENCER Hirotaka Matsui, Akinori Kanai, and Toshiya Inaba Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Hiroshima, Japan TET2 and IDH1/2 genes, whose products play roles in converting 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC), frequently show mutations in myeloid diseases. However, the study of 5hmC has been hindered by the lack of methods that determine the proportion and the position of 5hmC precisely. Traditional bisulfite sequencing cannot distinguish 5hmC from 5mC as both of them are read as C. Meanwhile it is recently reported that Tet-assisted bisulfite sequencing (TAB-seq) successfully detected 5hmC at the whole genome level. Here we established a system that enables us to analyze the 5hmC level focusing on CpG rich gene promoters and CpG islands at relatively low cost by combining reduced representative bisulfite sequencing (RRBS) and TAB-seq methods. In our system, CpG-rich DNA fragments of 150-250bp are first selectively purified from genomic DNA. 5hmC sites in the fragments are then glucosylated to 5ghmC by b-glucosyltransferase, while 5mC are oxidized to 5-carboxycytosine (caC) by TET1 enzyme, followed by the bisulfite conversion. After these treatments, unmodified C and 5caC are sequenced as T, while only 5ghmC is read as C. In combination with traditional bisulfite sequencing, we are now able to estimate the proportions of C/5mC/5hmC in about a half million CpG sites. Taking advantage of this system, we analyzed 5hmC of erythroid progenitor cells. TET2 expression was downregulated through lentiviral transduction of shRNA that specifically targets TET2 and the difference of 5hmC between TET2 knocked-down cells and control cells were statistically evaluated using methylKit. In this experiment, we found that only 300 CpGs among evaluated 410,000 CpGs were hydroxy-methylated by TET2 in erythroid progenitor cells. In addition, we also noticed that these 300 CpGs were mostly outside of CpG islands or gene promoters. This unexpected small number of TET2-mediated hydroxyl-methylated CpGs might be partially explained by the structure of TET2 enzyme; unlike TET1 and TET3, TET2 lacks cxxc domain that preferentially binds CpG-rich DNA. It would be possible that TET2 is responsible for hydroxy-methylation of CpG-sparse region such as enhancers.
P1103 - DECLINED PRESENTATION CRITICAL ROLES FOR THE HOMEODOMAIN TRANSCRIPTION FACTOR HHEX IN LYMPHOID COMMITMENT AND SELF-RENEWAL OF HEMATPOIETIC AND LEUKAEMIC STEM CELLS Benjamin Shields1,3, Jacob Jackson1, Chayanica Nasa1, Don Metcalf1,3, Clifford Bogue2, Warren Alexander1,3, and Matthew McCormack1,3 1 Cancer and Hematology Division, Walter and Eliza Hall Institute, Parkville, Victoria, Australia; 2Yale University, New Haven, Connecticut, USA; 3University of Melbourne, Melbourne, Victoria, Australia Hhex is a homeodomain transcription factor that is highly expressed in hematopoietic stem cells (HSCs) and progenitors. Previous studies have implicated Hhex in the development of definitive HSCs and B cells during embryogenesis. However, as Hhex knockout mice are embryonic lethal, the role of Hhex in adult haematopoiesis has not been previously studied. To address this, we have used Hhex conditional knockout mice to delete Hhex throughout the hematopoietic system of adult mice. This revealed that whilst Hhex was dispensable for maintenance of myeloid lineages, it is essential for development of B-cell progenitors. Moreover, competitive transplantation experiments revealed that Hhex is essential for development of all lymphoid lineages beyond thr common lymphoid progenitor (CLP) including B-cells and T-cells. RNA sequencing revealed aberrant expression of a number of cell cycle regulators in the absence of Hhex. Overexpression of one of these, Cyclin D1, could restore lymphoid development in the absence of Hhex. Thus Hhex regulates lymphoid development acting at least in part via regulation of the cell cycle. Whilst Hhex was dispensable for HSC maintenance in the adult, serial transplantation and myeloablation recovery experiments revealed a severe defect in HSC self-renewal and stress haematopoesis in the absence of Hhex. In addition, Hhex-deleted blast colonies were incapable of secondary replating in vitro. In contrast, overexpression of Hhex blocked differentiation leading to immortalised promyelocyte cell lines. Thus Hhex is a dynamic regulator of HSC self-renewal. To assess the role of Hhex in self-renewal of leukaemic stem cells, we tested the ability of the fusion oncogene MLL-ENL to induce leukaemia in the absence of Hhex. Strikingly, this showed a critical role for Hhex in both initiation and maintenance of myeloid leukaemia in vivo. Thus, in addition to its role in lymphoid development, Hhex is essential for selfrenewal of hematopoietic and leukemic stem cells.