ALBERT

Description: Abstract 4796 We have previously demonstrated the presence of very small (smaller than erythrocytes) Oct-4+SSEA-1+Sca-1+Lin-CD45- VSELs in bone marrow (BM) and in several murine adult organs (Leukemia 2006;20:857 and Cytometry 2009;73:1116). These small cells i) have large nuclei that contain primitive open chromatin, ii) express Oct-4 gene (as confirmed by our recent promoter methylation and chromatin structure analysis studies), and iii) posses bivalent domain-marked promoter regions of homeodomain-containing developmental master transcription factors, such as Dlx-, Irx-, Lhx-, Pou-, Pax-, and Six-family proteins. Furthermore, the epigenetic changes in selected somatic-imprinted genes (e.g., Igf2-H19 and RasGrf1) involved in insulin-factor signaling (Igf-1, Igf-2, and Insulin) govern their quiescent state, thus preventing them from unleashing proliferation and spontaneous growth of teratomas (Leukemia 2009;23:2042). O other hand it is well known that i) Igf-1 signaling negatively regulates lifespan in worms, flies, and mammals (Cell 2005;120:449) and that ii) Igf-1 and insulin levels in blood are positively regulated by caloric uptake. Indeed, we found that the pool size and pluripotentiality of VSELs decreases during aging. Accordingly, in our studies performed on young (4-week-old) and old (2-year-old) mice we found that i) the number of VSELs and their pluripotentiality decreases with age, ii) VSELs from old mice show lower expression of Oct-4, Nanog, Sox2, Klf4, and cMyc, iii) the Oct-4 promoter becomes hypermethylated with age and has a closed chromatin structure, iv) VSELs from old mice show somatic methylation in both Igf2-H19 and Rasgrf1 loci, and v), as a result, VSELs from these mice have increased sensitivity to insulin/insulin factors signaling. This suggests that chronic insulin receptor/Igf-1 receptor signaling in VSELs may contribute to age-related depletion of these cells. To explain better the role of insulin signaling in VSELs, we measured by FACS the number of VSELs in murine Laron dwarfs, which exhibit chronic Igf-1 deficiency and, as a result, live 30–40% longer than their normal littermates. We report here, for the first time, that the number of VSELs in the BM of Igf-1–deficient Laron dwarfs is 3–4 fold higher and is maintained at a higher level during aging compared to normal wild type (wt) littermates. Molecular analysis studies will confirm whether or not the molecular signature of VSELs in aging Laron dwarfs is somehow protected from age-related changes (e.g., by the methylation status of the Oct-4 promoter and/or genomic imprinted genes). Based on our data, we postulate novel linkages between Igf-1 level, aging, and the stem cell compartment. According to our hypothesis, early in development a population of VSELs would be deposited in developing organs as a backup for tissue-committed stem cells and play a role in rejuvenation of tissues and organ regeneration after damage. These cells would be protected from uncontrolled proliferation and age-related depletion by changes in imprinted genes that regulate insulin signaling. We further hypothesize that in the adult body the pool of VSEL cells is regulated by the circulating Igf-1 level. An increase in Igf-1 level (e.g., resulting from a chronically high calorie diet) would accelerate an age-dependent decrease in VSELs and their potential to rejuvenate tissues. By contrast, a low Igf-1 level (e.g., as seen in Laron dwarf mutants or due to caloric restriction) would have a protective effect on the overall pool of these cells. Thus, we present for the first time a hypothesis that reconciles aging, longevity, Igf-1 signaling, and caloric uptake and negative effect of Igf-1 and high calorie uptake on number/function of pluripotent VSELs deposited in adult tissues. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4818 Background. Caloric uptake elevates the plasma level of growth hormone (GH), which subsequently stimulates release of insulin-like growth factor-1 (IGF-1) from the liver. Evidence is accumulating that an increase in caloric intake, which leads to prolonged growth hormone (GH) and insulin/insulin-like growth factors signaling (IIS), accelerates aging. On the other hand, caloric restriction and a resulting decrease in IIS has the opposite effect and extends lifespan (Nature 2010;464:504). In support of this finding, mice with low circulating IGF-1 levels (Laron, Ames, and Snell dwarfs) live much longer than their normal littermates and, conversely, mice with high levels of circulating IGF-1 (e.g., transgenic mice that overexpress bovine growth hormone [bGH]) have significantly reduced life span. Aim of Hypothesis. To explain these phenomena, we hypothesized that prolonged IIS prematurely depletes adult tissues of very small embryonic-like stem cells (VSELs), which are the most developmentally primitive adult tissue-residing pluripotent stem cells (Leukemia 2006;20:857). We envision that VSELs play an important role in rejuvenation of the pool of tissue-committed stem cells and, as we observed previously, the number of these cells in murine BM decreases with age. We demonstrated that in bone marrow (BM), VSELs give rise to long-term repopulating hematopoietic stem cells (LT-HSCs) (Leukemia 2011; doi:10.1038/leu.2011.73, Exp. Hematology 2011;39:225–237). As previously reported, VSELs are kept quiescent in BM and protected from premature depletion by erasure of the somatic imprint in differentially methylated regions (DMRs) of some paternally imprinted genes involved in IIS (e.g., Igf2-H19 and RasGRF1). Results. In the current study, we provide direct evidence that the number of VSELs deposited in BM during ontogenesis is related to plasma IIS signaling, which is affected by the GH/IGF-1 level. In particular, mice with elevated IGF-I level in plasma due to expression of the bovine GH transgene and wild type mice injected for a sustained period with porcine GH both exhibit significant decreases in the number of VSELs and HSCs in BM compared to control animals. These decreases were paralleled by epigenetic changes in Igf2-H19 and RasGRF1 loci in which DMRs became hypermethylated over time. These changes in methylation lead to increases in IGF-2 and RasGRF1 expression and may explain why bovine GH transgenic mice have an increase in IIS and a significantly reduced life span. Conversely, mice with low circulating plasma IGF-1 levels (Laron and Ames dwarf mice ) have higher numbers of VSELs and HSCs in BM that, in contrast to aged-matched normal littermates, are maintained at high levels even into advanced age. The molecular signature of VSELs in these animals revealed prolonged retention of hypomethylation in the DMRs within the Igf2-H19 and RasGRF1 loci, which attenuated IIS signaling in these cells. The number of VSELs, however, decreased in these animals after prolonged treatment with porcine GH or human recombinant IGF-I. Conclusions. Our data shed new light on the relationships between senescence, high GH level, prolonged IIS, and depletion of VSELs and LT-HSCs. Accordingly, we propose a new paradigm in which chronic IIS (e.g., due to chronic high caloric intake and the resulting elevated GH and IGF-1 levels) prematurely depletes VSELs in BM, which leads to a decrease in the number of LT-HSCs. By contrast, caloric restriction and a decrease in IIS may delay the age-dependent elimination of VSELs from BM. This study also indicates that GH-based anti-aging therapies need careful re-evaluation of their potentially uncontrolled stimulation of VSELs in BM and downstream effects on hematopoiesis and the development of hematological malignancies. In support of this concern, elevated IIS may lead to hematological malignancies (uncontrolled proliferation of VSELs), while, by contrast, it is known that Laron dwarf mice and Laron dwarf patients, which have a GH-receptor deficiency and low plasma IGF-1 levels, do not develop leukemias. Disclosures: Ratajczak: Neostem Inc: Consultancy, Research Funding.

Description: Abstract 1551 Insulin-like growth factor-1 (Igf-1) or somatomedin is an important factor affecting proliferation of several types of cells, but its role in hematopoiesis remains controversial. Secretion of Igf-1 in the liver is stimulated directly by the growth hormone (GH)–GH receptor (GH-R) axis and indirectly by a high calorie diet. We have previously reported that Igf-1 does not directly stimulate proliferation of hematopoietic progenitors (J Clin Invest. 1994;94:320). However, our recent data indicate that Igf-1 stimulates proliferation of so-called very small embryonic-like stem cells (VSELs), that as we demonstrated, are the most developmentally primitive stem cells in adult bone marrow (BM) (Leukemia 2006;20:857) and may give rise to long-term repopulating hematopoietic stem cells (LT-HSCs) (Leukemia 2010; in press doi:10.1038/leu.2010.121). We envision that VSELs play a role in rejuvenation of the pool of tissue-committed stem cells in some tissues (e.g., HSCs and MSCs in BM) and we observed that the number of these cells in murine BM decreases with age. We also observed that erasure of the somatic imprint on some paternally imprinted genes (e.g., Igf2-H19 and RasGRF1) results in attenuation of insulin/insulin-like factors (e.g., Ins/Igf-1 signaling), keeps VSELs quiescent in BM, and protects them from uncontrolled proliferation. In the current work, to shed more light on the role of Igf-1 on hematopoiesis and stem cell compartment, we analyzed BM isolated from murine Laron dwarfs, which due to a genetic mutation in the GH-R, maintain very low levels of Igf-1 in peripheral blood (PB) and interestingly are long-living animals (Nature 2010;464:504). Analysis of PB cell counts, however, did not reveal any differences in the number of erythrocytes, platelets, and leucocytes between Laron dwarf mice and wild type controls. In striking contrast, however, we observed that Laron dwarf mice have in BM i) a ∼4–5-fold increase in the number of Sca-1+c-kit+lineage- (SKL) cells and ii) a 〉4-fold higher number of clonogenic CFU-Mix, CFU-GM, BFU-E, and CFU-Meg cells. Interestingly, Laron dwarfs also maintained ∼3-fold higher number of VSELs in BM tissue. Since the Igf-1 level is regulated by calorie uptake, these data shed new light on caloric restriction, senescence, and the hematopoietic stem cell compartment. Accordingly, we propose a new paradigm in which chronic Igf-1 deficiency somehow protects VSELs from age-related elimination from BM. This mimics a situation seen in chronic caloric restriction where the Igf-1 level is low and this results in longevity. Since the long-living Laron dwarf mice that maintain low levels of Igf-1 have higher numbers of VSELs and HSCs in BM, we postulate that chronically elevated levels of Igf-1, resulting e.g., from high calorie uptake, may lead to premature depletion of the stem cell pool, including VSELs and HSCs, and thus be responsible for premature aging. This hypothesis is currently being tested in animals that overexpress Igf-1, and interestingly, in contrast to IGF-1–deficient Laron dwarf mice, appear to have much shorter lifespans. Further studies are needed that will link the effect of chronic high Igf-1 signaling with the development of hematological malignancies. Of note, murine Laron dwarfs are significantly protected from developing cancer and human Laron dwarfs with chronic low Igf-1 level do not develop malignancies at all. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract LBA-5 Background: It is known that an increase in caloric intake leads to increase in plasma growth hormone (GH) level that subsequently induces secretion of insulin-like growth factor-1 (IGF-1) from liver, what leads to accelerated aging (Nature 2010;464:504). On the other hand, caloric restriction and a resulting decrease in plasma IGF-1 level has the opposite effect and extends lifespan. It also known that adult tissues contain a population of pluripotent very small embryonic like stem cells (VSELs) that play as postulated an important role in rejuvenation of long term hematopoietic stem cells (LT-HSCs) in bone marrow (BM) Leukemia 2011; doi:10.1038/leu.2011.73, Exp. Hematology 2011;39:225–237). As we observed previously, the number of these cells in murine BM decreases with age and VSELs are kept quiescent in BM and protected from premature depletion by erasure of the somatic imprint in differentially methylated regions (DMRs) of some paternally imprinted genes involved in insulin/insulin growth factors signaling (IIS) such as e.g., Igf2-H19 and RasGRF1 (Leukemia 2009;23:2042). Hypothesis: To explain and connect these phenomena together, we hypothesized that prolonged insulin/insulin growth factors signaling (IIS) prematurely depletes VSELs from the adult tissues and in BM may negatively impact on population of HSCs. Material and Methods: The number of VSELs and HSCs in long living murine strains with inborn low level of circulating IGF-1 (Laron- and Ames- dwarfs) as well as in short living mice with high levels of circulating IGF-1 (e.g., transgenic mice that overexpress bovine growth hormone; bGH) was evaluated by FACS. VSELs were isolated and epigenetic status of genes regulating pluripotency (e.g., Oct-4) as well as imprinted genes regulating IIS was evaluated by employing bisulfate modification of DNA followed by sequencing and by COBRE assay. We also challenged long living mice with low IGF-1 plasma level by daily injections of recombinant GH or IGF-1. Results: We found that the number of VSELs and HSCs residing in BM inversely correlates with plasma GH/IGF-1 level. To support this, mice with low circulating plasma IGF-1 levels (Laron- and Ames-dwarf mice) have higher numbers of VSELs and HSCs in BM that, in contrast to aged-matched normal littermates, are maintained at high levels even into advanced age. The analysis of molecular signature of VSELs in these animals revealed prolonged retention of hypomethylation in the DMRs within the Igf2-H19 and RasGRF1 loci, which attenuates IIS signaling in these cells. The number of VSELs, however, decreased in these animals after prolonged treatment with GH or recombinant IGF-I. Conversely, mice with elevated IGF-I level in plasma due to expression of the GH transgene or normal wild type mice injected for a sustained period with recombinant GH both exhibit significant decreases in the number of VSELs and HSCs in BM compared to control animals. These decreases were paralleled by epigenetic changes in Igf2-H19 and RasGRF1 loci in which DMRs became hypermethylated over time. These changes in methylation lead to increases in IGF-2 and RasGRF1 expression and may explain why GH transgenic mice have an increase in IIS that leads to shortening of life span in these animals. Conclusions: Our data shed new light on the relationships between senescence, GH/IGF-1 level, prolonged IIS, and number of VSELs and LT-HSCs. Accordingly, we propose a new paradigm in which a decrease in IIS (e.g., due to caloric restriction that lowers plasma IGF-1 level) may delay the age-dependent elimination of VSELs from adult tissues. By contrast, chronic IIS (e.g., due to chronic high caloric intake and the resulting elevated GH and IGF-1 levels) prematurely depletes VSELs residing in adult organs, which for example in BM leads to a decrease in the number of LT-HSCs. This study also indicates that GH-based anti-aging therapies need careful re-evaluation of their potentially uncontrolled stimulation of VSELs in BM that may lead to development of hematological malignancies. In support of this, elevated GH and IIS lead to hematological malignancies, while, by contrast, Laron dwarf mice and Laron dwarf patients, which have a low plasma IGF-1 levels, do not develop leukemias. Disclosures: Ratajczak: Neostem Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Description: Abstract 3422 Background: Growth hormone (GH) has been proposed as a rejuvenation factor that delays aging in older patients. The long-term results of such therapy, however, have recently become controversial. It is known that GH action is mediated by release of insulin-like growth factor-1 (IGF-1, also known as somatomedin C) from the liver. IGF-1 is an important factor affecting proliferation of several types of cells, including malignant hematopoietic blasts. A role for the GH-IGF-1 axis in normal hematopoiesis still remains controversial and some stimulatory effects to the clonogenicity of normal hematopoiteic stem/progenitor cells (HSPCs) have been described. We have previously reported that IGF-1 does not directly stimulate proliferation of normal clonogenic hematopoietic progenitors (J Clin Invest. 1994;94:320); however, it may promote erythroid differentiation and hemoglobinization of erythroblasts (Leukemia 1998;12:371). Aim of the study: Since long-term studies on the effect of GH-IGF-1 signaling on normal hematopoiesis in vivo have not been performed yet, we investigated the influence of these factors on the hematopoietic system in appropriate mouse models that express low or high levels of plasma GH and IGF-1. Experimental approach: We employed two mutant mouse strains that have low circulating plasma levels of IGF-1 due to a GH-receptor mutation (Laron dwarf mice) or to a defect in GH synthesis in the pituitary gland (Ames dwarf mice). We also employed transgenic mice that express the bovine GH gene (bGH), which results in a constitutively high level of circulating plasma IGF-1. In some experiments, normal wild type or Laron and Ames dwarf mice, with low circulating plasma IGF-1 levels, were injected for a prolonged period of time with recombinant GH or IGF-1. All these animals were analyzed by FACS for the presence of Sca-1+Lin–CD45+ HSPCs in bone marrow (BM) and the clonogenic growth of progenitors from all hematopoietic lineages monitored in standard methylcellulose (CFU-GM and BFU-E) and plasma clot (CFU-Meg) cultures. We also analyzed changes in peripheral blood count by Hemavet. The mice employed in our studies were at different ages (2 months, 6 months, and 2 years), and both female and male mice were analyzed. Results: We observed that Laron and Ames dwarf mice, with low circulating plasma levels of IGF-1, at the age of 1–2 years had ∼3–5 times more Sca-1+Lin–CD45+ in the BM than normal control littermates of the same age. Similarly, these mice also had ∼3–5 times more CFU-GM, BFU-E, and CFU-Meg in BM. The number of Sca-1+Lin–CD45+ cells and clonogenic progenitors from all lineages declined ∼5 fold in these animals (at 2 months and 8 months of age) after 4 weeks of treatment by daily injection of GH or IGF-1. However, interestingly, if this treatment was ceased the number of these cells showed over time a tendency to normalize. In another set of experiments, we evaluated the number of HSPCs in BM of 6-month- and 1-year-old mice that express the bovine GH transgene. We found that these mice have an ∼6-fold and ∼12-fold lower number in the BM of Sca-1+Lin–CD45+ and clonogenic progenitors, respectively, than control transgenic littermates expressing empty vector. Overall, a decrease in the number of HSPCs was accompanied by microcytic anemia. Conclusions: Our data, obtained from several in vivo animal models, show that prolonged exposure to GH-IGF-1 signaling depletes the number of HSPCs in the BM over time. This observation is highly relevant to GH rejuvenation therapies currently employed in humans. Our data indicate that this may lead to the premature depletion of HSPCs, and we also envision that prolonged stimulation of HSPCs by GH-IGF-1 may result in development of leukemias. In support of this notion, Laron dwarf mice and Laron dwarf patients do not develop hematological malignancies, while leukemias are often seen in bGH transgenic mice and in acromegalic patients. Disclosures: No relevant conflicts of interest to declare.

Description: LBA-5 Background: It is known that an increase in caloric intake leads to increase in plasma growth hormone (GH) level that subsequently induces secretion of insulin-like growth factor-1 (IGF-1) from liver, what leads to accelerated aging (Nature 2010;464:504). On the other hand, caloric restriction and a resulting decrease in plasma IGF-1 level has the opposite effect and extends lifespan. It also known that adult tissues contain a population of pluripotent very small embryonic like stem cells (VSELs) that play as postulated an important role in rejuvenation of long term hematopoietic stem cells (LT-HSCs) in bone marrow (BM) Leukemia 2011; doi:10.1038/leu.2011.73, Exp. Hematology 2011;39:225–237). As we observed previously, the number of these cells in murine BM decreases with age and VSELs are kept quiescent in BM and protected from premature depletion by erasure of the somatic imprint in differentially methylated regions (DMRs) of some paternally imprinted genes involved in insulin/insulin growth factors signaling (IIS) such as e.g., Igf2-H19 and RasGRF1 (Leukemia 2009;23:2042). Hypothesis: To explain and connect these phenomena together, we hypothesized that prolonged insulin/insulin growth factors signaling (IIS) prematurely depletes VSELs from the adult tissues and in BM may negatively impact on population of HSCs. Material and Methods: The number of VSELs and HSCs in long living murine strains with inborn low level of circulating IGF-1 (Laron- and Ames- dwarfs) as well as in short living mice with high levels of circulating IGF-1 (e.g., transgenic mice that overexpress bovine growth hormone; bGH) was evaluated by FACS. VSELs were isolated and epigenetic status of genes regulating pluripotency (e.g., Oct-4) as well as imprinted genes regulating IIS was evaluated by employing bisulfate modification of DNA followed by sequencing and by COBRE assay. We also challenged long living mice with low IGF-1 plasma level by daily injections of recombinant GH or IGF-1. Results: We found that the number of VSELs and HSCs residing in BM inversely correlates with plasma GH/IGF-1 level. To support this, mice with low circulating plasma IGF-1 levels (Laron- and Ames-dwarf mice) have higher numbers of VSELs and HSCs in BM that, in contrast to aged-matched normal littermates, are maintained at high levels even into advanced age. The analysis of molecular signature of VSELs in these animals revealed prolonged retention of hypomethylation in the DMRs within the Igf2-H19 and RasGRF1 loci, which attenuates IIS signaling in these cells. The number of VSELs, however, decreased in these animals after prolonged treatment with GH or recombinant IGF-I. Conversely, mice with elevated IGF-I level in plasma due to expression of the GH transgene or normal wild type mice injected for a sustained period with recombinant GH both exhibit significant decreases in the number of VSELs and HSCs in BM compared to control animals. These decreases were paralleled by epigenetic changes in Igf2-H19 and RasGRF1 loci in which DMRs became hypermethylated over time. These changes in methylation lead to increases in IGF-2 and RasGRF1 expression and may explain why GH transgenic mice have an increase in IIS that leads to shortening of life span in these animals. Conclusions: Our data shed new light on the relationships between senescence, GH/IGF-1 level, prolonged IIS, and number of VSELs and LT-HSCs. Accordingly, we propose a new paradigm in which a decrease in IIS (e.g., due to caloric restriction that lowers plasma IGF-1 level) may delay the age-dependent elimination of VSELs from adult tissues. By contrast, chronic IIS (e.g., due to chronic high caloric intake and the resulting elevated GH and IGF-1 levels) prematurely depletes VSELs residing in adult organs, which for example in BM leads to a decrease in the number of LT-HSCs. This study also indicates that GH-based anti-aging therapies need careful re-evaluation of their potentially uncontrolled stimulation of VSELs in BM that may lead to development of hematological malignancies. In support of this, elevated GH and IIS lead to hematological malignancies, while, by contrast, Laron dwarf mice and Laron dwarf patients, which have a low plasma IGF-1 levels, do not develop leukemias. Disclosures: Ratajczak: Neostem Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees.