Search

Hematopoietic Stem and Progenitor Cells are crucial in the maintenance of lifelong production of all blood cells. These Stem Cells are highly regulated to maintain homeostasis through a delicate balance between quiescence, self-renewal and differentiation. However, this balance is altered during the hematopoietic recovery after Hematopoietic Stem and Progenitor Cell Transplantation. Transplantation efficacy can be limited by inadequate Hematopoietic Stem Cells number, poor homing, low level of engraftment, or limited self-renewal. As recent evidences indicate that estrogens are involved in regulating the hematopoiesis, we sought to examine whether natural estrogens (estrone or E1, estradiol or E2, estriol or E3 and estetrol or E4) modulate human Hematopoietic Stem and Progenitor Cells. Our results show that human Hematopoietic Stem and Progenitor Cell subsets express estrogen receptors, and whose signaling is activated by E2 and E4 on these cells. Additionally, these natural estrogens cause different effects on human Progenitors in vitro. We found that both E2 and E4 expand human Hematopoietic Stem and Progenitor Cells. However, E4 was the best tolerated estrogen and promoted cell cycle of human Hematopoietic Progenitors. Furthermore, we identified that E2 and, more significantly, E4 doubled human hematopoietic engraftment in immunodeficient mice without altering other Hematopoietic Stem and Progenitor Cells properties. Finally, the impact of E4 on promoting human hematopoietic engraftment in immunodeficient mice might be mediated through the regulation of mesenchymal stromal cells in the bone marrow niche. Together, our data demonstrate that E4 is well tolerated and enhances human reconstitution in immunodeficient mice, directly by modulating human Hematopoietic Progenitor properties and indirectly by interacting with the bone marrow niche. This application might have particular relevance to ameliorate the hematopoietic recovery after myeloablative conditioning, especially when limiting numbers of Hematopoietic Stem and Progenitor Cells are available.

Pyruvate kinase deficiency (PKD) is a monogenic metabolic disease caused by mutations in the PKLR gene that leads to hemolytic anemia of variable symptomatology and that can be fatal during the neonatal period. PKD recessive inheritance trait and its curative treatment by allogeneic bone marrow transplantation provide an ideal scenario for developing gene therapy approaches. Here, we provide a preclinical gene therapy for PKD based on a lentiviral vector harboring the hPGK eukaryotic promoter that drives the expression of the PKLR cDNA. This therapeutic vector was used to transduce mouse PKD hematopoietic stem cells (HSCs) that were subsequently transplanted into myeloablated PKD mice. Ectopic RPK expression normalized the erythroid compartment correcting the hematological phenotype and reverting organ pathology. Metabolomic studies demonstrated functional correction of the glycolytic pathway in RBCs derived from genetically corrected PKD HSCs, with no metabolic disturbances in leukocytes. The analysis of the lentiviral insertion sites in the genome of transplanted hematopoietic cells demonstrated no evidence of genotoxicity in any of the transplanted animals. Overall, our results underscore the therapeutic potential of the hPGK-coRPK lentiviral vector and provide high expectations toward the gene therapy of PKD and other erythroid metabolic genetic disorders.

Hematopoietic Stem Cells (HSCs) is a rare cell population that sits atop a hierarchy of progenitors that become progressively restricted to several or a single blood lineage. HSCs are capable of self-renewal and multipotent differentiation to all blood cell lineages. HSCs are crucial in the maintenance of lifelong production of all blood cells. HSCs are highly regulated to maintain homeostasis through a delicate balance between quiescence, self-renewal and differentiation. However, this balance is altered during the hematopoietic recovery after Hematopoietic Stem Cell Transplantation (HSCT). HSCT is routinely used to reconstitute hematopoiesis after myeloablation, being the most commonly-used cell therapy. HSCT efficacy and multilineage reconstitution can be limited by inadequate HSC number, poor homing, engraftment, or limited self-renewal. Recent evidence indicates that estrogens are involved in regulating the hematopoietic system homeostasis. Estrogens are the primary female sex hormones and are responsible for controlling many cellular processes including growth, differentiation and function of the reproductive system. However, estrogens have also been proposed to regulate HSCs. b-Estradiol (E2) was shown to promote the cell cycle of HSCs and multipotent progenitors (MPPs) and increase erythroid differentiation in females (1). On the other hand, tamoxifen reduces the number of MPPs and short-term HSCs but activates proliferation of long-term HSCs (2). The potential clinical application of estrogens in HSCT mainly derives from the possibility that these drugs may enhance the engraftment of transplanted HSCs, thus reducing side effects associated to myeloablative conditioning. Here, we show that a short-term treatment of immunodeficient mice transplanted with hCD34+ cells with estrogens such as E2 and estetrol (E4) improves human hematopoietic engraftment. Fifty-thousand cord blood CD34+ cells (CB-CD34+) were transplanted into sublethally irradiated immunodeficient NSG mice. Three days after transplantation, mice were treated for four days with daily subcutaneous doses of E2, E4 or vehicle. Human hematopoietic engraftment was evaluated in the BM of transplanted mice at four months later. E2 and E4 estrogens increased the proportion of hCD45+ cells 1.8-fold and 2.4-fold as compared to values determined in control mice, without modifying the proportion of myeloid and lymphoid lineages. Significantly, animals treated with either estrogen had significantly higher levels of human hematopoietic progenitors (hCD45+CD34+). To study the engraftment of long-term engraftment HSCs in transplanted mice, human CD45+ cells from primary recipients were sorted and transplanted in secondary NSG recipients. Three months after transplants, the proportion of human hematopoietic cells in secondary recipients was also higher when primary recipients were treated with E2 or E4 than in vehicle-treated animals. Improved engraftments associated to the administration of E2 or E4 estrogens were confirmed when very low doses of CB-CD34+ cells were transplanted (5x103 hCD34+/mouse) in recipients of either sex. Collectively, our data support a new application of estrogens to improve the hematopoietic recovery after HSCT. This application may have particular relevance to enhance the hematopoietic recovery after myeloablative conditioning and when limiting numbers of HSCs are available. Disclosures Bueren: Rocket Pharmaceuticals Inc: Consultancy, Equity Ownership, Patents & Royalties, Research Funding. Segovia:Rocket Pharmaceuticals Inc: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

Hydroxytyrosol is a phenol found in olive oil. To verify the effect of hydroxytyrosol on the development of atherosclerosis, two groups of apo E deficient male mice on a standard chow diet were used: the control group receiving only water, and the second group an aqueous solution of hydroxytyrosol in order to provide a dose of 10 mg/kg/day to each mouse. This treatment was maintained for 10 weeks. At the moment of sacrifice, blood was drawn and heart removed. Plasma lipids, apolipoproteins and monocyte Mac-1 expression were assayed as well as aortic atherosclerotic areas in both groups. Data showed no significant changes in HDL cholesterol, paraoxonase, apolipoprotein B or triglyceride levels. However, hydroxytyrosol administration decreased apolipoprotein A-I and increased total cholesterol, atherosclerotic lesion areas and circulating monocytes expressing Mac-1. The latter was highly correlated with lesion areas (r = 0.65, P < 0.01). These results indicate that administration of hydroxytyrosol in low cholesterol diets increases atherosclerotic lesion associated with the degree of monocyte activation and remodelling of plasma lipoproteins. Our data supports the concept that phenolic-enriched products, out of the original matrix, could be not only non useful but also harmful. Our results suggest that the formulation of possible functional foods should approximate as much as possible the natural environment in which active molecules are found., This research was supported by grants FEGA-FEOGA (CAO99-0014), CICYT (SAF2004-08173-C03-02 and AGL2002-00495), Junta de Andalucía (CAO01-002), FISS 01/0202, Redes DGA (A-26) and FISS de investigación cooperativa C03-01 and G03-140 and by Fundación Española del Corazón., This research was supported by grants FEGA-FEOGA (CAO99-0014), CICYT (SAF2004-08173-C03-02 and AGL2002-00495), Junta de Andalucía (CAO01-002), FISS 01/0202, Redes DGA (A-26) and FISS de investigación cooperativa C03-01 and G03-140 and by Fundación Española del Corazón. R.C., S.A., M.A.N. and N.G. were recipient of DGA, FEGA-FEOGA and Fundación Cuenca Villoro fellowships.

Epidemiological studies have demonstrated the benefits of nut consumption on cardiovascular risk factors and CHD, attributed to their fatty acid profile, rich in unsaturated fatty acids, and also to other nutrients. The effect of nuts on atherosclerotic lesions was studied in female and male apoE-knockout mice fed a diet supplemented with 3 % (w/w) mixed nuts (mix: almonds, hazelnuts and walnuts in a proportion of 0·25:0·25:0·50, respectively), and compared with mice receiving an isoenergetic diet of similar fat content provided as palm oil. After 12 weeks, plasma lipid parameters and aortic lesions were measured. Males receiving nuts had lower plasma cholesterol than the palm oil group, and both sex groups had lower plasma non-HDL-cholesterol and lower content of reactive oxygen species in LDL than mice receiving the palm oil diet, the latter decrease being more pronounced in females than in males. Females consuming the nut diet showed a smaller aortic lesion area than those consuming palm oil, whereas no differences were observed in males. In females, hepatic paraoxonase 2 (Pon2) mRNA increased, and no change was observed in prenylcysteine oxidase 1 (Pcyox1) expression after the consumption of the nut-containing diet. In addition, aortic atherosclerotic lesions correlated directly with total plasma cholesterol and inversely with hepaticPon2expression. The results suggest that the beneficial effect of nut intake in female apoE-deficient mice may be attributed to reduced non-HDL-cholesterol levels and enhanced PON2 antioxidant activity.

AIM Research suggests that sex may condition atherosclerosis development against different genetic backgrounds. This study addresses the hypothesis that this effect would be exerted by changes in the different apolipoproteins present in high-density lipoproteins. METHODS ApoE-deficient mice of both sexes with Ola 129 and C57BL/6J genetic backgrounds were fed a chow diet for 14 weeks. At the end of the dietary intervention, the development of atherosclerotic lesions, apolipoproteins, lipid metabolism, inflammation and paraoxonase were assessed. RESULTS Differences between atherosclerotic lesions in Ola 129 and C57BL/6J strains of apoE-deficient mice were sex-dependent and were only statistically significant in females. Plasma levels of HDL cholesterol and apolipoproteins related to these lipoparticles, such as apoA-I, apoA-II, apoA-IV, apoA-V and apoJ, were significantly different between these two strains and there were sex-related differences in some of these apolipoproteins. Hepatic steatosis was also related to the strain and was independent of sex. In females, changes in HDL cholesterol and apolipoproteins A-I and A-II were important determinants of atherosclerosis, while this was not the case in males. CONCLUSIONS Our results demonstrate that atherosclerosis-related differences between Ola129 and C57BL/6J genetic backgrounds in apoE-deficient mice are sex-dependent and that this finding is explained by the differences in HDL cholesterol and its apolipoprotein components, mainly apoA-I and A-II. Overall, our findings highlight the importance of taking sex into account in the analysis of atherosclerosis and lipid metabolism in animal models.

BACKGROUND: Squalene is an intermediate of cholesterol biosynthesis which can be obtained from the diet where it is abundant, for example, in olive oil. The effect of this isoprenoid on the development of atherosclerosis was investigated on apoE-knockout mice. METHODS AND RESULTS: Two groups of animals, separated according to sex, were fed on standard chow diet: the control group receiving only vehicle and the second group an aqueous solution of squalene to provide a dose of 1g/kg/day in male and female mice. This treatment was maintained for 10 weeks. At the end of this period, plasma lipid parameters, oxidative stress markers and hepatic fat were measured as well as cross-sectional lesion area of aortic root in both groups. Data showed that in males squalene feeding reduced atherosclerotic lesion area independently of plasma lipids and activation of circulating monocytes. In contrast, squalene intake did not decrease lesion area in females, despite reducing plasma cholesterol and triglycerides, isoprostane and percentage of Mac-1 expressing white cells. In males, atherosclerotic lesion area was positively and significantly associated with hepatic fat content and the plasma triglycerides were also strongly associated with liver weight. CONCLUSIONS: These results indicate that administration of squalene modulates lesion development in a gender specific manner, and that accumulation of hepatic fat by liver is highly correlated with lesion progression in males. Hence, squalene administration could be used as a safe alternative to correct hepatic steatosis and atherosclerosis particularly in males.

Oils enriched in monounsaturated fatty acids do not seem to behave similarly in protecting against the development of atherosclerosis in animal models, which has been attributed to the presence of soluble phenolic compounds. To test the relevance of other components of oils in the prevention of atherosclerosis, two olive oils from the same cultivar devoid of soluble phenolic compounds were prepared using different procedures (pressure or centrifugation), characterized and fed to apolipoprotein E-deficient mice as 10% (w/w) of their diet. The 2 olive oils had similar levels of monounsaturated fatty acids and squalene, but they differed in their content of linoleic, phytosterols, tocopherols, triterpenes and waxes, which were particularly enriched in the test olive oil obtained by centrifugation. In mice that received a diet enriched in the olive oil derived through centrifugation, the progression of atherosclerosis was delayed compared to the mice that received standard olive oil. That effect was associated with decreases in plasma triglycerides, total and non-high-density lipoprotein cholesterol and isoprostane 8-iso-prostaglandin F2α. Our results clearly indicate that the preparation of olive oil is crucial in determining its antiatherosclerotic effect, which extends beyond the presence of phenolic compounds. The test olive oil exerted its antiatherosclerotic effects by modifying plasma lipids and oxidative stress, and it might be a good candidate to replace other fats in functional foods., This research was supported by grants FEGA-FEOGA (CAO99-014), CICYT (SAF2004-08173-C03-02 and AGL2002-00495), Junta de Andalucía (CAO01-002), FISS 01/0202, Redes DGA (A-26) and FISS de investigación cooperativa C03-01 and G03-140 and by Fundación Española del Corazón.

Pyruvate Kinase Deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene. This gene encodes the erythroid specific Pyruvate Kinase (RPK) enzyme, implying that this defective enzyme fails to produce normal levels of ATP and consequently, erythrocytes from PKD patients show an energetic imbalance. Hematopoietic stem cell gene therapy using gene editing will be the safest approach to treat PKD patients. However, its direct application in hematopoietic progenitor cells (HPCs) is challenging. Different gene editing approaches are being explored to correct PKD, either by Knock-In of an optimized cDNA sequence in the first introns of the gene or by site-specific correction using ssODN. While Knock-In strategy will allow the treatment of most PKD mutations, an ssODN-mediated gene editing will correct specific PKD patient-mutations. In our Knock-In system, a recombination matrix carrying an exon 3-11 partial codon optimized version of RPK cDNA and a puromycin selection cassette is inserted in the second intron of the PKLR gene assisted by TALEN® nucleases. We have previously shown that this approach was effective to correct PKD phenotype in PKDiPSC lines. Here, we have tested the feasibility of our approach in hematopoietic progenitors. Thus, the therapeutic matrix together with specific TALEN® for the second intron of PKLR was electroporated in purified cord blood CD34+ from healthy donors. Cells were then expanded and puromycin selected to enrich the population for gene edited ones. Up to 96% of the colony forming units that survived the puromycin selection showed the specific integration of the donor cassette. After transplantation into immunodeficient NSG mice, a low though detectable percentage of gene edited cells was . Additionally, CRISPR-Cas9 site-specific correction is being developed by combining guide RNAs directed against PKD patient's mutation and precise ssODN to restore RPK protein. Overall, our data show that gene editing in engraftable HPCs is feasible, although further improvements are needed to increase efficiency of this gene therapy appraoch prior to consider its therapeutic application in PKD patients. Disclosures Gouble: Cellectis: Employment. Galetto:Cellectis SA: Employment. Poirot:Cellectis: Employment.