Anti aging Effect of Transplanted Amniotic Membrane Mesenchymal Stem Cells in a Premature Aging Model of Bmi 1 Deficiency 1Scientific RepoRts | 5 13975 | DOi 10 1038/srep13975 www nature com/scientifi[.]
www.nature.com/scientificreports OPEN received: 05 January 2015 accepted: 12 August 2015 Published: 15 September 2015 Anti-aging Effect of Transplanted Amniotic Membrane Mesenchymal Stem Cells in a Premature Aging Model of Bmi-1 Deficiency Chunfeng Xie*, Jianliang Jin*, Xianhui Lv, Jianguo Tao, Rong Wang & Dengshun Miao To determine whether transplanted amniotic membrane mesenchymal stem cells (AMSCs) ameliorated the premature senescent phenotype of Bmi-1-deficient mice, postnatal 2-day-old Bmi1−/− mice were injected intraperitoneally with the second-passage AMSCs from amniotic membranes of β-galactosidase (β-gal) transgenic mice or wild-type (WT) mice labeled with DiI Three reinjections were given, once every seven days Phenotypes of 5-week-old β-gal+ AMSC-transplanted or 6-weekold DiI+ AMSC-transplanted Bmi-1−/− mice were compared with vehicle-transplanted Bmi-1−/− and WT mice Vehicle-transplanted Bmi-1−/− mice displayed growth retardation and premature aging with decreased cell proliferation and increased cell apoptosis; a decreased ratio and dysmaturity of lymphocytic series; premature osteoporosis with reduced osteogenesis and increased adipogenesis; redox imbalance and DNA damage in multiple organs Transplanted AMSCs carried Bmi-1 migrated into multiple organs, proliferated and differentiated into multiple tissue cells, promoted growth and delayed senescence in Bmi-1−/− transplant recipients The dysmaturity of lymphocytic series were ameliorated, premature osteoporosis were rescued by promoting osteogenesis and inhibiting adipogenesis, the oxidative stress and DNA damage in multiple organs were inhibited by the AMSC transplantation in Bmi-1−/− mice These findings indicate that AMSC transplantation ameliorated the premature senescent phenotype of Bmi-1-deficient mice and could be a novel therapy to delay aging and prevent aging-associated degenerative diseases Aging is an inevitable physiological change It is a multifactorial process characterized by a progressive loss of physiological integrity that leads to impaired function and increased vulnerability to death1 Hallmarks of senescent cells include cellular DNA damage, mitochondrial dysfunction leading to increased reactive oxygen species (ROS) and decreased ATP, the production of proinflammatory cytokines, telomere shortening, the trigger of stem cell depletion and cell senescense2 As a representative and primary theory about senescence, the oxygen free radical hypothesis proposes that ROS attack cellular components and initiate and promote aging-associated degenerative diseases3,4 Oxidative stress damages cells, tissues and organs by causing imbalance between ROS generation and antioxidant defense, contributing to the aging process5 Superoxide dismutase and catalase are key antioxidant enzymes that reduce O2.− to H2O2 and water, delaying aging6 B lymphoma Mo-MLV insertion region (Bmi-1), a member of the polycomb family of transcriptional repressors, is involved in cell cycle regulation, self-renewal of stem cells and cell senescence7,8 Bmi-1 inhibits the p16INK4a/Rb and p19AFR/p53 pathways8–10 and is also involved in maintaining mitochondrial function and redox balance Persistent accumulation of ROS results from impaired mitochondrial function and imbalanced redox are sufficient to induce senescence via DNA damage in Bmi-1-deficient mice, The State Key Laboratory of Reproductive Medicine, Department of Anatomy, Histology and Embryology, Nanjing Medical University, Nanjing, China *These authors contributed equally to this work Correspondence and requests for materials should be addressed to D.M (email: dsmiao@njmu.edu.cn) or J.J (email: jinjianliang@njmu.edu.cn) Scientific Reports | 5:13975 | DOI: 10.1038/srep13975 www.nature.com/scientificreports/ which are characterized by growth retardation, dysmaturity, decreased ATP, dysfunction of organs, the induction of proinflammatory cytokines, stem cell exhaustion and expression of senescence-associatedβ -galactosidase and p16INK4a 7,8,11–13 These phenotypic features are consistent with the typical histological and functional hallmarks of a senescent model2 Increasing evidence suggests that exhaustion of functional stem cells is critical in aging and aging-associated degenerative diseases Stem cell transplantation is generally considered as a highly promising candidate method for regenerative applications because stem cells possess a high proliferative capacity and have the potential to differentiate into other cell types Stem cells also secrete paracrine cytokines and improve microenvironments14 Amniotic membrane mesenchymal stem cells (AMSCs) are a better prospect for cell therapy and regenerative medicine compared to other adult mesenchymal stem cells because they are abundant and easily and inexpensively acquired They can be obtained with little donor damage, have multipotency for all three germ layers and low immunogenicity, and are ethically acceptable15,16 AMSCs are reported to have the potential to differentiate into cells of different organs for treating diseases and to have immunomodulatory properties17,18 However, it is unclear whether AMSCs delay aging and prevent aging-associated degenerative diseases by migrating into organs and maintaining redox balance To investigate the potential of AMSCs, Bmi-1-deficient (Bmi-1−/−) mice were injected intraperitoneally with second-passage AMSCs derived from normal pregnant β -galactosidase (β -gal) transgenic mice or wild-type (WT) mice AMSCs derived from WT mice were labeled with DiI before transplantation The phenotype of the mice was compared with vehicle-transplanted Bmi-1−/− and WT mice Results Characterizations of donor AMSCs. Second-passage AMSCs from β -gal transgenic mice had a typical spindle-shaped fibroblast phenotype (Fig. 1Aa) Transgenic AMSCs were positive for β -gal as demonstrated by LacZ cytochemical staining (Fig. 1Ab) AMSCs derived from WT mice labeled with DiI display as red fluorescence observed under fluorescence microscopy (Fig. 1Ac) AMSCs derived from β -gal transgenic mice or from WT mice labeled with DiI were used as donor cells for transplantation To identify the stem cell potential of donor cells, osteogenic differentiation potential and immunophenotype of AMSCs were analyzed At the end of an osteogenic induction period, AMSCs had differentiated into osteoblast like cells which expressed alkaline phosphatase (Fig. 1Ad) AMSCs were expressed representative adult mesenchymal stem cell markers including CD29, CD44, CD73, CD90, CD105 (Fig. 1Ba–e), with low expression of the embryonic stem cell marker SSEA-4 (Fig. 1Bf) Little expression of hematopoietic stem cell markers CD34 and CD45 was detected (Fig. 1Bg,h) Genomic DNA of AMSCs contained Bmi-1 and the mRNA of AMSCs showed expression of embryonic stem cell markers including OCT-4, CXCR4 and Nanog (Fig. 1C–E) These results suggested that second-passage AMSCs from β -gal transgenic mice had good stem cell potential Growth retardation and premature aging were ameliorated by AMSC transplantation into Bmi-1−/− mice. Bmi-1−/− mice had significantly decreased survival rates and body weight compared to WT mice (Fig. 2A,B) The overall sizes of the body, thymus, spleen and kidney were decreased in Bmi1−/− mice, compared with WT mice (Fig. 2C,D) AMSC transplantation prolonged the median survival from 39 days to 92 days, and increased body weight, and overall size of the body, thymus, spleen and kidney in Bmi-1−/− mice (Fig. 2A–D) These results demonstrated that AMSC transplantation ameliorated growth retardation and premature aging in Bmi-1−/− mice To determine whether the rescue of growth retardation and premature aging in AMSC-transplanted Bmi-1−/− mice was associated with cell proliferation and apoptosis, the thymus and kidney were examined by immunohistochemistry for Ki67 and caspase3 and by TUNEL staining The results showed a decrease in the percentage of Ki67-positive thymocytes and renal cells and a significant increase in the percentages of caspase3-positive and TUNEL-positive thymocytes and renal cells in Bmi-1−/− mice compared to WT mice Compared to vehicle-transplanted Bmi-1−/− mice, in AMSC-transplanted Bmi1−/− mice, the percentages of Ki67-positive thymocytes and renal cells were increased, however, the percentages of Caspase3-positive and TUNEL-positive thymocytes and renal cells were decreased significantly (Figure E–J) These results demonstrated that AMSC transplantation promoted cell proliferation and inhibited cell apoptosis in Bmi-1−/− mice Decreased ratio of lymphocytic series was ameliorated by AMSC transplantation into Bmi1−/− mice. To determine the proportion of peripheral blood cell series, blood cells were analyzed using a peripheral blood cell counter The numbers of white blood cells, platelets and the ratio of lymphocytes relative to total white blood cells were decreased in Bmi-1−/− mice compared to WT mice, whereas the ratio of neutrophils relative to total white blood cells was significantly increased Compared with vehicle-transplanted Bmi-1−/− mice, the ratio of lymphocytes relative to total white blood cells was increased in AMSC-transplanted Bmi-1−/− mice (see Supplementary Table S1 online) These results demonstrated that AMSC transplantation increased the ratio of lymphocytic series relative to total white blood cells in Bmi-1−/− mice Scientific Reports | 5:13975 | DOI: 10.1038/srep13975 www.nature.com/scientificreports/ A LacZ a ALP DiI b c d 50 µm CD29 CD44 SSEA4 Gapdh Count h Count Count Marker CD90 g CD105 C CD73 f Count e d Count c Count Count b Bmi-1 Oct4 Count B a CD34 CXCR4 Nanog 2000 bp 1000 bp 750 bp 500 bp CD45 D tail WT AMSCs Bmi-1 200 bp β-actin 100 bp 346 bp E -/- Bmi-1 687 bp CXCR4 a 188 bp 480 bp 106 bp DAPI b Overlap c 10 µm Figure 1. Preparation and characterization of donor AMSCs The second-passage AMSCs from β -galactosidase (β -gal) transgenic mice or wild-type (WT) labeled with DiI were used as donor cells (Aa) Representative micrographs of cultured the second-passage AMSCs and (Ab) stained cytochemically for β -gal activity (Ac) Micrographs of DiI positive AMSCs observed under confocal microscopy on 549 nm (Ad) AMSCs were incubated in osteogenic medium to differentiate into osteoblasts and analyzed by cytochemical staining with Alizarin red (B) Flow cytometry analysis for (a–e) standard adult stem cell markers CD29, CD44, CD73, CD90, CD105, (f) embryonic stem cell marker SSEA-4, and (g,h) hematopoietic stem cell markers CD34, CD45 on AMSCs (C) Bmi-1 gene by PCR in DNA from AMSCs OCT-4, CXCR4 and Nanog mRNA were detected in AMSCs by RT-PCR GAPDH was used as the control (D) Western blots of 5-week-old WT or Bmi-1−/− tail and donor AMSCs extracts for Bmi-1 β -actin was used the loading control (E) Representative micrographs of donor cells stained by immunofluoresence for CXCR4 (red, panel a) with DAPI for nuclei (blue, panel b) and overlap (panel c) Scientific Reports | 5:13975 | DOI: 10.1038/srep13975 www.nature.com/scientificreports/ A B WT 80 -/- Bmi-1 60 Body weight (g) Percent survival 100 -/- Bmi-1 +AMSCs 40 20 0 20 40 60 80 100 120 140 160 180 200 24 22 20 18 16 14 12 10 Age (days after birth) C -/- Bmi-1 -/- -/- *** ## *** * # ** ** *** *** *** *** Lifespan (weeks) D Bmi-1 +AMSCs ### -/- Bmi-1 +AMSCs WT -/- Bmi-1 -/- Bmi-1 +AMSCs kidney spleen thymus WT WT Bmi-1 WT -/- Bmi-1 +AMSCs H kidney Ki67 Ki67 positive cells (%) thymus E -/- Bmi-1 20 18 16 14 12 10 10 µm thymus Caspase3 positive cells (%) I kidney Caspase3 F 10 µm G 30 25 20 15 ### ** ### * *** *** thymus kidney *** ## *** *** ### ** 10 thymus kidney kidney TUNEL TUNEL positive cells (%) thymus J 10 µm 18 16 14 12 10 *** ### *** thymus *** ## *** kidney Figure 2. Growth retardation and premature aging ameliorated by AMSC transplantation into Bmi1−/− mice (A) Percent survival of vehicle-transplanted wild-type (WT) and Bmi-1−/− mice (Bmi-1−/−) and AMSC-transplanted Bmi-1−/− mice (Bmi-1−/− + AMSCs) (B) Growth curves of mice by group (C) Representative appearance and (D) overall thymus, spleen or kidney size in 5-week-old mice of WT, Bmi-1−/− and β -gal+ Bmi-1−/− + AMSC groups Representative micrographs of paraffin-embedded thymus and kidney sections stained immunohistochemically for (E) Ki67, (F) Caspase and (G) TUNEL, respectively The percentage of (H) Ki67-positive cells, (I) Caspase 3-positive cells and (J) TUNEL positive cells relative to total cells or areas Values are mean ± SEM from determinations per group *P