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Poster Presentations/ Experimental Hematology 41 (2013) S23–S75
P1152 - MULTIPLE MICROENVIRONMENTS IN THE FETAL LIVER WITH DISTINCT FUNCTIONS Nathalie Brouard1,2, Poojabahen Gandhi2, Nadine Matthias2, Francois Lanza1, and Christian Gachet1 1 UMR-S949 INSERM, Strasbourg, France; 2Institute of Molecular Medicine, UTHSC, Houston, Texas, USA Hematopoiesis takes place during development in successive anatomical sites, each corresponding to different phases of hematopoiesis development. Hematopoietic Stem Cells (HSC) expansion and maintenance is controlled by a specific microenvironment termed niche. The niche during time and through the successive anatomical locations differs not only in terms of cellular and molecular composition, but also in terms of its function. First, it supports the emergence of HSC, then their amplification and finally in the adult, their maintenance. The fetal liver is the site of a major expansion of the HSC pool. Therefore we hypothesized that the fetal liver niche was the source of specific determinants capable of supporting a large expansion of HSC while maintaining their self-renewing potential. The present study focuses on defining the cellular components of the HSC niche. We first determined, using a combination of cell surface markers, the cell populations present in the mouse fetal liver at day 13.5. We identified cell populations that can produce adherent layers and will be referred to as stromal cells. We then tested in serum free conditions, their capacity to support the maintenance and expansion of HSC during a 2 week co-culture. Maintenance of serially transplantable HSC was observed only in cocultures with stromal layers derived from CD45-TER119-CD31-CD51+VCAM-1-PDGFRa+ cells. We concluded that this stromal cell population was an essential component of the fetal HSC niche. In the cocultures with stromal layers derived from CD45-TER119-CD31CD51+VCAM-1+PDGFRa- cells, we observed an expansion of megakaryocytes, and erythroid progenitors and a loss of serially transplantable HSC. This suggests that this stromal cell population recapitulates a progenitor microenvironment. Further characterization of these stromal cells and the factors they produce will guide the development of new methods for ex-vivo expansion of HSC or to direct their differentiation toward specific lineages.
P1153 - VARIABLE EFFECTS OF TUMOR SECRETED FACTORS ON HUMAN MESENCHYMAL STEM CELL Abdulaziz AlMusa, Mashael Al-toub, Mohammed Almajed, May Al-Nbaheen, Abdullah Aldahmash, and Nehad Alajez King Saud University, Riyadh, Saudi Arabia Objective: Recent years has witnessed huge interest in studying the tumor microenvironment, given its apparent role in driving tumor progression and metastasis. Of particular interest, mesenchymal stem cells (MSCs) have been the focus of many research groups as the exact role of MSCs in driving cancer progression remains controversial. Herein, we investigated the effects of tumor secreted factors from a panel of human cancer cell lines (breast (MCF7 and MDA-MB-231); prostate (PC-3); lung (NCIH522); and head & neck (FaDu)) on MSCs. Methods: Morphological changes were assesses using fluorescent microscopy. Changes in gene expression were assessed using Agilent microarray and qRT-PCR. Cell migration was assessed using transwell migration system. Results: Morphologically, MSCs exposed to secreted factor from FaDu, MDA-MB-231, PC-3, and NCI-H522, but not from MCF7, exhibited a spindle-shaped morphology, and the cells were more elongated with bipolar processes, compared to control MSCs which were larger and more flattened with multiple processes. Integrated analysis of gene expression and bioinformatics revealed a proinflammatory response of MSCs when exposed to conditioned media (CM) from all cell lines, but not MCF7. Nonetheless, MSCs exhibited significant tropism toward secreted factors from the aforementioned tumor cell lines. Conclusions: Thus our data suggest that MSCs might drive tumorigenicity through induction of inflammation.
S63
P1154 - EXPOSURE TO IONIZING RADIATIONS AND STARVATION CULTURE DOES NOT MODIFY PHENOTYPE, FUNCTIONS AND GENETIC PROFILE OF MESENCHYMAL STROMAL CELLS ISOLATED FROM BONE MARROW OF HEALTHY DONORS Simone Biagini, Antonella Conforti, Nadia Starc, Giusi Li Pira, Alessandra Proia, Franco Locatelli, and Maria Bernardo Pediatric Hematology/Oncology Department, Bambino Gesu Children’s Hospital, Rome, Italy Objective: Mesenchymal stromal cells (MSCs) are multipotent cells located in different human tissues, including bone marrow (BM), where they sustain the creation of the hematopoietic stem cell niche by regulating its differentiation and functions. Many studies have demonstrated that BM exposure to ionizing radiations induces the rapid depletion of hematopoietic precursors, however radiation effects on MSCs have been poorly investigated. Moreover, little is known about MSCs behavior in starvation culture conditions. In this study, we examined morphology, proliferative capacity (in terms of population doublings), immunophenotype, differentiation potential, immunomodulatory properties (PHA-induced T cell proliferation assay) and genetic profile (array-CGH and caryotype) of MSCs isolated from BM of healthy donors and exposed to ionizing radiations and starvation culture. Methods: MSCs were isolated from 10 healthy donors (median age: 16 years; range: 5-32) and expanded in culture medium supplemented with 5% platelet lysate up to passage 2. Thereafter MSCs were exposed both to escalating doses of ionizing radiations (3000, 10000 and 20000 rad) and to starvation culture conditions (culture medium supplemented with 1% platelet lysate instead of 5%). Results: With escalating doses of ionizing radiations, MSCs lose their typical spindle-shaped morphology, their boundaries are less regular and their growth rate slows down (at 3000 rad) or even stops (at 10000 and 20000 rad). Nonetheless, in the presence of 1% platelet lysate, although showing a slower growth rate as compared with non irradiated MSCs, the effects on morphology are less evident, thus suggesting that the lack of growing factors can slow down the senescent process induced by ionizing radiations. Furthermore, irradiated and starved MSCs maintain the same immunophenotype, differentiation potential, immunomodulatory properties and genetic profile of normal MSCs. Conclusions: Our data indicate that irradiated and starved MSCs, although presenting altered morphology and growth rate, maintain the typical characteristics of normal MSCs. P1155 - SELECTIVE RADIORESISTANCE OF BONE MARROW HEMATOPOIETIC PROGENITOR CELLS COMPARED TO STROMAL CELLS FROM FANCONI ANEMIA (FANCD2-/-) MICE Hebist Berhane, Ronny Kalash, Michael Epperly, Julie Goff, Darcy Franicola, Xichen Zhang, Donna Shields, Shaonan Cao, Frank Houghton, and Joel Greenberger Radiation Oncology, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania, USA The DNA repair defect in FancD2 -/- mice causes sensitivity to total body irradiation. We determined whether cell phenotype specific differences in radiosensitivity, DNA repair, and irradiation induced gene expression were detectable with FancD2 -/- bone marrow stromal cells compared to IL3 dependent hematopoietic progenitor cells. Radiation survival curves were performed with cell lines derived from C57BL/6 FancD2 +/+, FancD2 +/- and FancD2 -/- mice. Cells were irradiated over a range of 0 to 8 Gy, and day 7 colonies of 50 cells counted. Repair of DNA strand breaks was determined by Comet Assay. Gene transcripts were measured by Real Time Polymerase Chain Reaction (RT-PCR) 24 hr after 5 Gy. FancD2-/- marrow stromal cells were radiosensitive (Do 5 1.43 + 0.06 Gy, ~n 5 4.98 6 0.65) compared to FancD2 +/+ cells ((Do 5 1.70 6 0.09 Gy and ~n 5 8.33 6 0.72), p 5 0.0395 and 0.0040, respectively) or FancD2 +/- cells (Do 5 1.67 6 0.08 Gy and ~n 5 3.63 6 0.44, p 5 0.0348 and 0.1365, respectively). In contrast, IL3 dependent FancD2 -/- hematopoietic progenitor cells were radioresistant (Do 5 1.65 6 0.09 Gy and ~n 5 2.47 6 0.37) compared to FancD2+/+ or FancD2+/- hematopoietic progenitor cells (Do 5 1.39 6 0.09 Gy (p 5 0.043) and ~n 5 2.316 0.85), and (Do 5 1.64 6 0.08 Gy and ~n 5 4.40 6 0.99 (p50.004)). Comet assay demonstrated that IL3 dependent FancD2-/- hematopoietic cells repaired DNA strand breaks faster than FancD2 -/- stromal cells. There was an increase in p53 gene transcripts in FancD2 -/- IL-3 dependent cells after 5, compared to all three stromal cell lines or FancD2 +/+ or FancD2 +/- hematopoietic cells. Genes involved in cell cycle regulation including p21 and CHK-1 showed no relative increase in FancD2 -/- hematopoietic cells while p21 was increased in FancD2 -/- stromal cells. Levels of NFkB, Nrf2 and MnSOD remained unchanged after irradiation of FancD2-/- hematopoietic cells while FancD2 -/- stromal cells as well as FancD2 +/- and +/+ hematopoietic and stromal cells all showed increases. Absence of the FancD2 gene confers radiosensitivity to bone marrow stromal cells; however, hematopoietic progenitor cells from the same mice are radioresistant.
P1152 - MULTIPLE MICROENVIRONMENTS IN THE FETAL LIVER WITH DISTINCT FUNCTIONS Nathalie Brouard1,2, Poojabahen Gandhi2, Nadine Matthias2, Francois Lanza1, and Christian Gachet1 1 UMR-S949 INSERM, Strasbourg, France; 2Institute of Molecular Medicine, UTHSC, Houston, Texas, USA Hematopoiesis takes place during development in successive anatomical sites, each corresponding to different phases of hematopoiesis development. Hematopoietic Stem Cells (HSC) expansion and maintenance is controlled by a specific microenvironment termed niche. The niche during time and through the successive anatomical locations differs not only in terms of cellular and molecular composition, but also in terms of its function. First, it supports the emergence of HSC, then their amplification and finally in the adult, their maintenance. The fetal liver is the site of a major expansion of the HSC pool. Therefore we hypothesized that the fetal liver niche was the source of specific determinants capable of supporting a large expansion of HSC while maintaining their self-renewing potential. The present study focuses on defining the cellular components of the HSC niche. We first determined, using a combination of cell surface markers, the cell populations present in the mouse fetal liver at day 13.5. We identified cell populations that can produce adherent layers and will be referred to as stromal cells. We then tested in serum free conditions, their capacity to support the maintenance and expansion of HSC during a 2 week co-culture. Maintenance of serially transplantable HSC was observed only in cocultures with stromal layers derived from CD45-TER119-CD31-CD51+VCAM-1-PDGFRa+ cells. We concluded that this stromal cell population was an essential component of the fetal HSC niche. In the cocultures with stromal layers derived from CD45-TER119-CD31CD51+VCAM-1+PDGFRa- cells, we observed an expansion of megakaryocytes, and erythroid progenitors and a loss of serially transplantable HSC. This suggests that this stromal cell population recapitulates a progenitor microenvironment. Further characterization of these stromal cells and the factors they produce will guide the development of new methods for ex-vivo expansion of HSC or to direct their differentiation toward specific lineages.
P1153 - VARIABLE EFFECTS OF TUMOR SECRETED FACTORS ON HUMAN MESENCHYMAL STEM CELL Abdulaziz AlMusa, Mashael Al-toub, Mohammed Almajed, May Al-Nbaheen, Abdullah Aldahmash, and Nehad Alajez King Saud University, Riyadh, Saudi Arabia Objective: Recent years has witnessed huge interest in studying the tumor microenvironment, given its apparent role in driving tumor progression and metastasis. Of particular interest, mesenchymal stem cells (MSCs) have been the focus of many research groups as the exact role of MSCs in driving cancer progression remains controversial. Herein, we investigated the effects of tumor secreted factors from a panel of human cancer cell lines (breast (MCF7 and MDA-MB-231); prostate (PC-3); lung (NCIH522); and head & neck (FaDu)) on MSCs. Methods: Morphological changes were assesses using fluorescent microscopy. Changes in gene expression were assessed using Agilent microarray and qRT-PCR. Cell migration was assessed using transwell migration system. Results: Morphologically, MSCs exposed to secreted factor from FaDu, MDA-MB-231, PC-3, and NCI-H522, but not from MCF7, exhibited a spindle-shaped morphology, and the cells were more elongated with bipolar processes, compared to control MSCs which were larger and more flattened with multiple processes. Integrated analysis of gene expression and bioinformatics revealed a proinflammatory response of MSCs when exposed to conditioned media (CM) from all cell lines, but not MCF7. Nonetheless, MSCs exhibited significant tropism toward secreted factors from the aforementioned tumor cell lines. Conclusions: Thus our data suggest that MSCs might drive tumorigenicity through induction of inflammation.
S63
P1154 - EXPOSURE TO IONIZING RADIATIONS AND STARVATION CULTURE DOES NOT MODIFY PHENOTYPE, FUNCTIONS AND GENETIC PROFILE OF MESENCHYMAL STROMAL CELLS ISOLATED FROM BONE MARROW OF HEALTHY DONORS Simone Biagini, Antonella Conforti, Nadia Starc, Giusi Li Pira, Alessandra Proia, Franco Locatelli, and Maria Bernardo Pediatric Hematology/Oncology Department, Bambino Gesu Children’s Hospital, Rome, Italy Objective: Mesenchymal stromal cells (MSCs) are multipotent cells located in different human tissues, including bone marrow (BM), where they sustain the creation of the hematopoietic stem cell niche by regulating its differentiation and functions. Many studies have demonstrated that BM exposure to ionizing radiations induces the rapid depletion of hematopoietic precursors, however radiation effects on MSCs have been poorly investigated. Moreover, little is known about MSCs behavior in starvation culture conditions. In this study, we examined morphology, proliferative capacity (in terms of population doublings), immunophenotype, differentiation potential, immunomodulatory properties (PHA-induced T cell proliferation assay) and genetic profile (array-CGH and caryotype) of MSCs isolated from BM of healthy donors and exposed to ionizing radiations and starvation culture. Methods: MSCs were isolated from 10 healthy donors (median age: 16 years; range: 5-32) and expanded in culture medium supplemented with 5% platelet lysate up to passage 2. Thereafter MSCs were exposed both to escalating doses of ionizing radiations (3000, 10000 and 20000 rad) and to starvation culture conditions (culture medium supplemented with 1% platelet lysate instead of 5%). Results: With escalating doses of ionizing radiations, MSCs lose their typical spindle-shaped morphology, their boundaries are less regular and their growth rate slows down (at 3000 rad) or even stops (at 10000 and 20000 rad). Nonetheless, in the presence of 1% platelet lysate, although showing a slower growth rate as compared with non irradiated MSCs, the effects on morphology are less evident, thus suggesting that the lack of growing factors can slow down the senescent process induced by ionizing radiations. Furthermore, irradiated and starved MSCs maintain the same immunophenotype, differentiation potential, immunomodulatory properties and genetic profile of normal MSCs. Conclusions: Our data indicate that irradiated and starved MSCs, although presenting altered morphology and growth rate, maintain the typical characteristics of normal MSCs. P1155 - SELECTIVE RADIORESISTANCE OF BONE MARROW HEMATOPOIETIC PROGENITOR CELLS COMPARED TO STROMAL CELLS FROM FANCONI ANEMIA (FANCD2-/-) MICE Hebist Berhane, Ronny Kalash, Michael Epperly, Julie Goff, Darcy Franicola, Xichen Zhang, Donna Shields, Shaonan Cao, Frank Houghton, and Joel Greenberger Radiation Oncology, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania, USA The DNA repair defect in FancD2 -/- mice causes sensitivity to total body irradiation. We determined whether cell phenotype specific differences in radiosensitivity, DNA repair, and irradiation induced gene expression were detectable with FancD2 -/- bone marrow stromal cells compared to IL3 dependent hematopoietic progenitor cells. Radiation survival curves were performed with cell lines derived from C57BL/6 FancD2 +/+, FancD2 +/- and FancD2 -/- mice. Cells were irradiated over a range of 0 to 8 Gy, and day 7 colonies of 50 cells counted. Repair of DNA strand breaks was determined by Comet Assay. Gene transcripts were measured by Real Time Polymerase Chain Reaction (RT-PCR) 24 hr after 5 Gy. FancD2-/- marrow stromal cells were radiosensitive (Do 5 1.43 + 0.06 Gy, ~n 5 4.98 6 0.65) compared to FancD2 +/+ cells ((Do 5 1.70 6 0.09 Gy and ~n 5 8.33 6 0.72), p 5 0.0395 and 0.0040, respectively) or FancD2 +/- cells (Do 5 1.67 6 0.08 Gy and ~n 5 3.63 6 0.44, p 5 0.0348 and 0.1365, respectively). In contrast, IL3 dependent FancD2 -/- hematopoietic progenitor cells were radioresistant (Do 5 1.65 6 0.09 Gy and ~n 5 2.47 6 0.37) compared to FancD2+/+ or FancD2+/- hematopoietic progenitor cells (Do 5 1.39 6 0.09 Gy (p 5 0.043) and ~n 5 2.316 0.85), and (Do 5 1.64 6 0.08 Gy and ~n 5 4.40 6 0.99 (p50.004)). Comet assay demonstrated that IL3 dependent FancD2-/- hematopoietic cells repaired DNA strand breaks faster than FancD2 -/- stromal cells. There was an increase in p53 gene transcripts in FancD2 -/- IL-3 dependent cells after 5, compared to all three stromal cell lines or FancD2 +/+ or FancD2 +/- hematopoietic cells. Genes involved in cell cycle regulation including p21 and CHK-1 showed no relative increase in FancD2 -/- hematopoietic cells while p21 was increased in FancD2 -/- stromal cells. Levels of NFkB, Nrf2 and MnSOD remained unchanged after irradiation of FancD2-/- hematopoietic cells while FancD2 -/- stromal cells as well as FancD2 +/- and +/+ hematopoietic and stromal cells all showed increases. Absence of the FancD2 gene confers radiosensitivity to bone marrow stromal cells; however, hematopoietic progenitor cells from the same mice are radioresistant.