Higher number of stem cells in bone marrow of circulating Igf-1 level low Laron dwarf mice - novel view on Igf-1, stem cells and aging

Aging-associated changes in the nuclear architecture, chromatin structure, altered expression and activity of chromatin remodeling factors, and the change in pattern of epigenetic marks (DNA methylation and histone modification) affect all cells in the adult body, including the population of stem cells (SCs) that is responsible for proper tissue rejuvenation.¹ The elucidation of these precise mechanisms will help to develop more efficient anti-aging strategies as well as facilitate a better understanding of age-related risks for cancer genesis.

Insulin-like growth factor-1 (Igf1), Insulin-like growth factor-2 (Igf2) and Insulin (Ins) are described as stimulators of proliferation of normal and malignant hematopoietic stem progenitor cells (HSPCs).² While Igf1 and Igf2 signal through tyrosine kinase receptor Igf1R, Ins together with Igf2 may activate classical insulin receptor (InsR) and all factors together may activate a hybrid Igf1R/InsR. In contrast Igf2R serves as a “molecular bin” that prevents Igf2 from binding to Igf1R or InsR. Activation of Igf1R and InsR depending on cell type affects cell metabolism and/or proliferation. Addition of these factors in clonogenic assays may enhance survival of human CD34⁺ HSPCs and increase size of hematopoietic colonies, in particular in erythroid lineage.² However insulin factors as reported may stimulate proliferation of leukemic blasts.

On other hand it is well known that changes in Ins/Igf signaling have important implications for aging³. Accordingly, i) Igf1 signaling negatively regulates lifespan in worms, flies, and mammals and ii) Igf1 and insulin level in blood is regulated positively by caloric uptake. While Ins is secreted after calorie uptake from pancreatic islands, Igf1 (known also as somatomedin-C) is secreted from liver into peripheral blood in growth hormone (GH) dependent manner.

Interestingly expression of Igf2, Igf2R and Rasgrf1 (a small GTP exchange factor for Ras protein that is involved in Ins/Igf signaling) are regulated by changes in somatic imprinting. The imprinted genes play a crucial role in embryogenesis, fetal growth, the totipotential state of the zygote, and the pluripotency of developmentally early stem cells.⁴ The expression of imprinted genes is regulated by DNA methylation within differential methylated regions, which are CpG-rich cis-elements within their gene loci.

Recently, our group demonstrated the presence of pluripotent Oct4⁺ SSEA-1⁺Sca-1⁺Lin⁻CD45⁻ very small embryonic-like stem cells (VSELs) in adult murine bone marrow,⁵ and postulated that these pluripotent stem cells (PSCs) are deposited during early embryogenesis as a backup for long term repopulating hematopoietic stem cells (LT-HSC).⁶ In fact as recently demonstrated BM-derived VSELs could be specified into hematopoietic lineage in co-cultures over OP-9 stroma cell support.⁶ Furthermore, molecular analysis of VSELs revealed that their quiescence in adult BM and potentially premature depletion is controlled by epigenetic changes of some imprinted genes that regulate signaling of Ins/Igf (e.g., Igf2-H19 locus, Igf2R and Rasgrf1).⁷ Accordingly, we observed that murine BM-sorted VSELs erase the paternally methylated imprints (e.g., at Igf2-H19 and Rasgrf1 loci); however, they hypermethylate the maternally methylated imprints (e.g., Igf2R).⁷ This epigenetic reprogramming of genomic imprinting negatively affects Ins/Igf signaling and maintains the quiescence of VSELs and protects them from premature aging and tumor formation.

Based on this we became interested in a role of Ins/Igf signaling in homeostasis of pool of BM VSELs and VSELs-derived LT-HSC. First, our studies performed on normal young (4-week-old) and old (2-year-old) mice revealed that the number of VSELs and their pluripotentiality decrease during ageing.⁸ Accordingly, VSELs from old mice show lower expression of the pluripotentiality master-regulators such as Oct4, Nanog, Sox2, Klf4, and cMyc (Figure 1A) and, at the molecular level, the Oct4 promoter in VSELs becomes hypermethylated with age and shows a closed chromatin structure (Figure 1B). Furthermore, VSELs from old mice show the somatic type of methylation at both Igf2-H19 and Rasgrf1 loci, which suggests that VSELs from old mice have increased sensitivity to insulin factors signaling (Figure 1C and Supplementary Figure 1). This suggests that chronic Ins/Igf signaling in VSELs may contribute to age-related depletion of these cells.

Figure 1. Change of pluriportentiality genes and genomic imprinting in VSELs from young vs. old wild type mice.

(a) RQ-PCR analysis of pluripotency (Oct4, Nanog, Sox2, Klf4 and cMyc) in VSELs isolated by FACS from young (4 weeks, 4wk) and old (18 months, 18M) C57BL/6 mice. The relative expression level is represented as the ratio of 18M value to 4wk one and shown as the mean ± S.D.; n=4. The DNA methylation level of Oct4 promoter (b) and DMRs for imprinted genes (c). Bisulfite sequencing results of DNA methylation of Oct4 promoter in VSELs isolated from the indicated aged mice. Methylated and unmethylated CpG sites are shown in filled and open circles, respectively. Mean values for percentage of methylated CpG sites in Oct4 promoter (b, n=4) and DMRs (c, n=3) are shown as the mean ± S.D.; Student t test was used for statistic analysis. *p<0.05, **p<0.01 compared to 4wk. KvDMR: DMR for Kcnq1 locus.

Thus, to test the hypothesis that chronic Ins/Igf signaling may accelerate depletion of the pool of VSELs in adult BM, we became interested in Laron dwarf mice. Due to a deficiency of GH receptor (GH-R), these animals display a severe reduction in the Igf1 plasma level and do not display increase in GH-mediated Igf-1 plasma level in response to caloric uptake.⁹ Interestingly, these animals live 30–40% longer than their normal littermates.¹⁰ Several possible explanations of this effect have been proposed, but we became interested in whether a reduced level of Igf1 in the plasma of these animals may directly impact/protect the survival of VSELs in adult murine BM.

To address this issue, we measured the number of VSELs in BM of 8 month and 20 month old murine Laron dwarfs (GHR^−/−) and corresponding normal heterozygous littermates (GHR^+/−) by FACS. Interestingly, we noticed that the number of VSELs in the BM of plasma Igf1-deficient Laron dwarfs is maintained at a 3–4-fold higher level than normal GHR^+/− littermates during aging (Figure 2A). Our molecular analysis studies have additionally demonstrated that the Oct4 promoter in these animals shows also a higher level of demethylation (Figure 2B). Furthermore, while analysis of peripheral blood cell counts did not reveal any differences in the number of erythrocytes, platelets, and leucocytes between Laron dwarf mice and wild type controls and all these mice have normal histology of bone marrow tissue (data not shown), we observed that Laron dwarf mice have in BM i) a ~3-fold increase in the number of Sca-1⁺c-kit⁺lineage⁻ (SKL) hematopoietic stem cells (8 month olds) and ii) up to 4-fold higher number of clonogenic CFU-GM, BFU-E, and CFU-Meg cells (8 month and 20 month old) (Figure 2C).

Figure 2. Increased number of VSELs and HSPCs in Laron dwarf mice.

(a) The analysis of body weight, BM cellularity, VSELs and HSCs ratio between the indicated aged wild-type and Laron dwarf mice. The ratio for Sca-1⁺Lin⁻CD45⁻ (VSELs) and Sca-1⁺Lin⁻CD45⁺ (HSCs) was evaluated as the number of events per 1×10⁶ BMMNC by employing LSR II FACS analyzer. Statistic analysis is done using one way Analysis of Variance (ANOVA) with Bonferroni’s Multiple Comparison Test. *p<0.05, **p<0.01 as compared to wild-type counterparts. (b) Bisulfite-sequencing results of DNA methylation of the Oct4 promoter in VSELs isolated from 20 month old normal heterozygotes (GHR^+/−) and Laron dwarf mice (GHR^−/−). The numbers indicated below bisulfite-sequencing profiles presents the percentage of methylated CpG sites. (c) The number of clonogenic BFU-E, CFU-GM and CFU-Meg from BM MNC isolated from 8 or 20 months old normal heterozygotes GHR^+/− and Laron dwarf GHR^−/− mice. Statistic analysis is done using two way ANOVA with Bonferroni post-tests. *p<0.05, **p<0.01 as compared to wild-type counterparts.

Since the plasma Igf1 level is regulated in mice by caloric uptake and in humans by caloric uptake providing that protein uptake is also low,¹¹ these data shed new light on caloric restriction, senescence, and the size of hematopoietic stem cell compartment. Based on this, we propose a new paradigm in which chronic Igf1 deficiency somehow protects VSELs from age-related elimination from BM. This mimics a situation seen in chronic caloric restriction in mice, where the Igf1 level is low and longevity is extended Since the long-living Laron dwarf mice that have extremely low levels of circulating Igf1 have higher numbers of VSELs and HSPCs in BM, we postulate that chronically elevated levels of Igf1, resulting, for example, from high caloric uptake, may lead to premature depletion of the stem cell pool, including VSELs and HSPCs, and thus be responsible for premature aging in mice. This hypothesis is currently being tested in mice that overexpress GH and thus have high plasma Igf1 level, and, interestingly, in contrast to the long-lived Laron dwarf mice (GHR^−/−), show a markedly reduced life span.¹²

Further studies are needed to link the effect of chronic high Igf1 signaling in VSELs with the development of cancer. Of note, both murine Laron dwarfs and human Laron dwarfs with chronically low Igf1 levels are significantly protected from developing solid cancer and leukemia.¹³ Since many human malignancies are activated by Igf1 and Igf2 signaling (e.g., due to loss of heterozygosity or loss of imprinting at the Igf2-H19 locus),¹⁴ we hypothesize that excessive activation of VSELs in an Ins/Igf-dependent manner could promote malignant transformation of these cells. However, the hypothetical reverse effect of low Ins/Igf signaling on a pool of VSELs that could become cancer/leukemia initiating cells requires further experimental study.

Based on our data, we postulate novel linkages between the Igf1 level, aging, and the stem cell compartment (Figure 3). 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 that plays a role in rejuvenation of tissues and organ regeneration after damage.⁵ These cells seem to be protected from uncontrolled proliferation and age-related depletion by changes in imprinted genes that regulate insulin signaling.⁷ We hypothesize that the pool of VSELs residing in adult tissues including BM is regulated by the circulating Igf1 level. An increase in Igf1 level (e.g., resulting from a chronically high caloric uptake) would accelerate in an Ins/Igf-dependent manner an age-dependent depletion of the pool of VSELs and their potential to rejuvenate tissues (e.g., in BM to supply LT-HSCs). By contrast, a low Igf1 level (e.g., as seen in Laron dwarf mutants or as a result of caloric restriction) would have an opposite and protective effect on these cells.

Figure 3. Hypothesis of developmental deposition of Oct4⁺ epiblast-derived VSELs in adult tissues and their depletion by chronic Insulin/Igf signaling.

Epiblast-derived VSELs are deposited in developing tissues as a backup population of SCs for production of tissue committed stem cells (TCSCs). Thus, we postulate that VSELs play a role in rejuvenation of tissues and organs as a source of stem cells committed to these various tissues. We envision that in bone marrow VSELs are a backup population for long term repopulating hematopoietic stem cells (LT-HSCs) that give rise to hematopoietic stem/priogenitor cells (HSPCs) (middle part of the Figure). Prolonged signaling by Insulin, Igf-1, and Igf-2 (e.g., due to high caloric uptake) may lead to premature depletion of these cells. By contrast, a decrease in Insulin/Igf signaling (e.g., by caloric restriction) may ameliorate the age-related decrease both in their number and pluripotency (e.g., as indicated by the Oct4 promoter state) of these cells. Thus, we present for the first time a hypothesis that reconciles aging, longevity, insulin factor signaling, and high caloric uptake with the abundance and function of pluripotent VSELs deposited in adult tissues. A decrease in the number of VSELs in bone marrow will directly affect a pool of HSPCs.

Thus, we present for the first time a hypothesis that relates aging, longevity, insulin factor signaling, and high caloric uptake to the abundance and function of pluripotent VSELs deposited in adult tissues. A decrease in the number of these cells will affect pools of TCSCs and have an impact on tissue rejuvenation and life span.

Figure 1. Bisulfite-sequencing results of DNA methylation of DMRs of paternally methylated (Igf2-H19 and Rasgrf1) and maternally methylated (Igf2R and Kcnq1) loci in VSELs and HSCs isolated from young (4 weeks, 4wk) and old (18 months, 18M) C57BL/6 mice. The percentage of methylated CpG sites is shown under the bisulfite-sequencing results. KvDMR: DMR for Kcnq1 locus.