Cell and Tissue Research

, Volume 358, Issue 1, pp 135–147 | Cite as

Deletion of Alox5 gene decreases osteogenic differentiation but increases adipogenic differentiation of mouse induced pluripotent stem cells

  • Yanru Wu
  • Hualing Sun
  • Fangfang Song
  • Cui Huang
  • Jiawei WangEmail author
Regular Article


Induced pluripotent stem cells (iPSCs) have great potential in bone tissue engineering to repair large bone defects. Before their clinical application, investigations are needed to discover the genes and osteoconductive scaffolds that influence their differentiation toward an osteogenic lineage. Alox5 plays controversial and complex roles in the regulation of bone and fat metabolism. To detect the effect of Alox5 on osteogenic and adipogenic differentiation of iPSCs, both Alox5 knockout mouse iPSCs (Alox5-KO-iPSCs) and wild-type mouse iPSCs (Wild-iPSCs) were developed. The mRNA levels of many osteogenic markers in Alox5-KO-iPSCs were significantly reduced, while many adipogenic markers were enhanced. Furthermore, when implanted in rat cranial critical-sized defects with collagen/chitosan/hydroxyapatite scaffolds (CCHS), Alox5-KO-iPSCs produced significantly less new bone than Wild-iPSCs and both cell-scaffold groups had no tumor formation. There was a significant difference in the expression of Cox2 during the osteogenic and adipogenic differentiation between the two kinds of iPSCs in vitro. In conclusion, firstly, Alox5 knockout reduced the osteogenic but increased the adipogenic differentiation potential of mouse iPSCs. These disorders might be related to the change of Cox2 expression. Secondly, combined with iPSCs, CCHS can serve as a potential substrate to repair critical-sized bony defects. However, more studies are required to confirm the mechanisms through which Alox5 affects the osteogenic and adipogenic abilities of iPSCs in vivo and the effect of Cox2 inhibition in this system.


Alox5 iPSCs Collagen/chitosan/hydroxyapatite scaffolds Bone regeneration Calvarial defects 



This work was financially supported by The Fundamental Research Fund for the Central Universities of China (2012304020207) and a grant from The Office of Science and Technology of Hubei Province (2011CDB470).


  1. Akino K, Mineda T, Mori N, Hirano A, Imaizumi T, Akita S (2006) Attenuation of cysteinyl leukotrienes induces human mesenchymal stem cell differentiation. Wound Repair Regen 14:343–349PubMedCrossRefGoogle Scholar
  2. Avis I, Hong SH, Martinez A, Moody T, Choi YH, Trepel J, Das R, Jett M, Mulshine JL (2001) Five-lipoxygenase inhibitors can mediate apoptosis in human breast cancer cell lines through complex eicosanoid interactions. FASEB J 15:2007–2009PubMedGoogle Scholar
  3. Baudry A, Bitard J, Mouillet-Richard S, Locker M, Poliard A, Launay JM, Kellermann O (2010) Serotonergic 5-HT (2B) receptor controls tissue-nonspecific alkaline phosphatase activity in osteoblasts via eicosanoids and phosphatidylinositol-specific phospholipase C. J Biol Chem 285:26066–26073PubMedCrossRefPubMedCentralGoogle Scholar
  4. Bitencourt CS, Pereira PA, Ramos SG, Sampaio SV, Arantes EC, Aronoff DM, Faccioli LH (2011) Hyaluronidase recruits mesenchymal-like cells to the lung and ameliorates fibrosis. Fibrogenesis Tissue Repair 4:3PubMedCrossRefPubMedCentralGoogle Scholar
  5. Chen XS, Sheller JR, Johnson EN, Funk CD (1994) Role of leukotrienes revealed by targeted disruption of the 5-lipoxygenase gene. Nature 372:179–182PubMedCrossRefGoogle Scholar
  6. Collet C, Schiltz C, Geoffroy V, Maroteaux L, Launay JM, de Vernejoul MC (2008) The serotonin 5-HT2B receptor controls bone mass via osteoblast recruitment and proliferation. FASEB J 22:418–427PubMedCrossRefGoogle Scholar
  7. Darios F, Davletov B (2006) Omega-3 and omega-6 fatty acids stimulate cell membrane expansion by acting on syntaxin 3. Nature 440:813–817PubMedCrossRefGoogle Scholar
  8. Gallwitz WE, Mundy GR, Lee CH, Qiao M, Roodman GD, Raftery M, Gaskell SJ, Bonewald LF (1993) 5-Lipoxygenase metabolites of arachidonic acid stimulate isolated osteoclasts to resorb calcified matrices. J Biol Chem 268:10087–10094PubMedGoogle Scholar
  9. Kawai M, Rosen CJ (2010) PPARgamma: a circadian transcription factor in adipogenesis and osteogenesis. Nat Rev Endocrinol 6:629–636PubMedCrossRefPubMedCentralGoogle Scholar
  10. Kawai M, Sousa KM, MacDougald OA, Rosen CJ (2010) The many facets of PPARgamma: novel insights for the skeleton. Am J Physiol Endocrinol Metab 299:E3–9PubMedCrossRefPubMedCentralGoogle Scholar
  11. Kwak HJ, Park KM, Choi HE, Lim HJ, Park JH, Park HY (2010) The cardioprotective effects of zileuton, a 5-lipoxygenase inhibitor, are mediated by COX-2 via activation of PKC delta. Cell Signal 22:80–87PubMedCrossRefGoogle Scholar
  12. Le Blanc K, Pittenger M (2005) Mesenchymal stem cells: progress toward promise. Cytotherapy 7:36–45PubMedCrossRefGoogle Scholar
  13. Le P, Kawai M, Bornstein S, Demambro VE, Horowitz MC, Rosen CJ (2011) A high-fat diet induces bone loss in mice lacking the alox5 gene. Endocrinology 153:6–16PubMedCrossRefPubMedCentralGoogle Scholar
  14. Lee HW, Kim SY, Kim AY, Lee EJ, Choi JY, Kim JB (2009) Adiponectin stimulates osteoblast differentiation through induction of COX2 in mesenchymal progenitor cells. Stem Cells 27:2254–2262PubMedCrossRefGoogle Scholar
  15. Levi B, Hyun JS, Nelson ER, Li S, Montoro DT, Wan DC, Jia FJ, Glotzbach JC, James AW, Lee M, Huang M, Quarto N, Gurtner GC, Wu JC, Longaker MT (2011) Nonintegrating knockdown and customized scaffold design enhances human adipose-derived stem cells in skeletal repair. Stem Cells 29:2018–2029PubMedCrossRefGoogle Scholar
  16. Liao J, Cui C, Chen S, Ren J, Chen J, Gao Y, Li H, Jia N, Cheng L, Xiao H, Xiao L (2009) Generation of induced pluripotent stem cell lines from adult rat cells. Cell Stem Cell 4:11–15PubMedCrossRefGoogle Scholar
  17. Liu H, Zhu F, Yong J, Zhang P, Hou P, Li H, Jiang W, Cai J, Liu M, Cui K, Qu X, Xiang T, Lu D, Chi X, Gao G, Ji W, Ding M, Deng H (2008) Generation of induced pluripotent stem cells from adult rhesus monkey fibroblasts. Cell Stem Cell 3:587–590PubMedCrossRefGoogle Scholar
  18. Liu Q, Spusta SC, Mi R, Lassiter RN, Stark MR, Hoke A, Rao MS, Zeng X (2012) Human neural crest stem cells derived from human ESCs and induced pluripotent stem cells: induction, maintenance, and differentiation into functional schwann cells. Stem Cells Transl Med 1:266–278PubMedCrossRefPubMedCentralGoogle Scholar
  19. Ma L, Gao C, Mao Z, Zhou J, Shen J, Hu X, Han C (2003) Collagen/chitosan porous scaffolds with improved biostability for skin tissue engineering. Biomaterials 24:4833–4841PubMedCrossRefGoogle Scholar
  20. Mehrabian M, Allayee H, Stockton J, Lum PY, Drake TA, Castellani LW, Suh M, Armour C, Edwards S, Lamb J, Lusis AJ, Schadt EE (2005) Integrating genotypic and expression data in a segregating mouse population to identify 5-lipoxygenase as a susceptibility gene for obesity and bone traits. Nat Genet 37:1224–1233PubMedCrossRefGoogle Scholar
  21. Mimeault M, Batra SK (2009) Recent insights into the molecular mechanisms involved in aging and the malignant transformation of adult stem/progenitor cells and their therapeutic implications. Ageing Res Rev 8:94–112PubMedCrossRefGoogle Scholar
  22. Mostoslavsky G, Fabian AJ, Rooney S, Alt FW, Mulligan RC (2006) Complete correction of murine Artemis immunodeficiency by lentiviral vector-mediated gene transfer. Proc Natl Acad Sci USA 103:16406–16411PubMedCrossRefPubMedCentralGoogle Scholar
  23. Nakagawa M, Koyanagi M, Tanabe K, Takahashi K, Ichisaka T, Aoi T, Okita K, Mochiduki Y, Takizawa N, Yamanaka S (2008) Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol 26:101–106PubMedCrossRefGoogle Scholar
  24. Okita K, Ichisaka T, Yamanaka S (2007) Generation of germline-competent induced pluripotent stem cells. Nature 448:313–317PubMedCrossRefGoogle Scholar
  25. Park JK, Kim HS, Uh KJ, Choi KH, Kim HM, Lee T, Yang BC, Kim HJ, Ka HH, Kim H, Lee CK (2013) Primed pluripotent cell lines derived from various embryonic origins and somatic cells in pig. PLoS ONE 8:e52481PubMedCrossRefPubMedCentralGoogle Scholar
  26. Rajan N, Habermehl J, Cote MF, Doillon CJ, Mantovani D (2006) Preparation of ready-to-use, storable and reconstituted type I collagen from rat tail tendon for tissue engineering applications. Nat Protoc 1:2753–2758PubMedCrossRefGoogle Scholar
  27. Sawada R, Kono K, Isama K, Haishima Y, Matsuoka A (2013) Calcium-incorporated titanium surfaces influence the osteogenic differentiation of human mesenchymal stem cells. J Biomed Mater Res A 101:2573–2585PubMedCrossRefGoogle Scholar
  28. Seong JM, Kim BC, Park JH, Kwon IK, Mantalaris A, Hwang YS (2010) Stem cells in bone tissue engineering. Biomed Mater 5:062001PubMedCrossRefGoogle Scholar
  29. Sommer CA, Stadtfeld M, Murphy GJ, Hochedlinger K, Kotton DN, Mostoslavsky G (2009) Induced pluripotent stem cell generation using a single lentiviral stem cell cassette. Stem Cells 27:543–549PubMedCrossRefGoogle Scholar
  30. Sommer CA, Sommer AG, Longmire TA, Christodoulou C, Thomas DD, Gostissa M, Alt FW, Murphy GJ, Kotton DN, Mostoslavsky G (2010) Excision of reprogramming transgenes improves the differentiation potential of iPS cells generated with a single excisable vector. Stem Cells 28:64–74PubMedGoogle Scholar
  31. Sun H, Wu Y, Fu D, Liu Y, Huang C (2014a) SIRT6 regulates osteogenic differentiation of rat bone marrow mesenchymal stem cells partially via suppressing the nuclear factor-kappa B signaling pathway. Stem Cells. doi: 10.1002/stem.1671 Google Scholar
  32. Sun HL, Wu YR, Huang C, Wang JW, Fu DJ, Liu YC (2014b) The Effect of SIRT6 on the Odontoblastic Potential of Human Dental Pulp Cells. J Endod 40:393–398PubMedCrossRefGoogle Scholar
  33. Taiani JT, Krawetz RJ, Yamashita A, Pauchard Y, Buie HR, Ponjevic D, Boyd SK, Rancourt DE, Matyas JR (2013) Embryonic stem cells incorporate into newly formed bone and do not form tumors in an immunocompetent mouse fracture model. Cell Transplant 22:1453–1462PubMedCrossRefGoogle Scholar
  34. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663–676PubMedCrossRefGoogle Scholar
  35. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861–872PubMedCrossRefGoogle Scholar
  36. Tashiro K, Kawabata K, Sakurai H, Kurachi S, Sakurai F, Yamanishi K, Mizuguchi H (2008) Efficient adenovirus vector-mediated PPAR gamma gene transfer into mouse embryoid bodies promotes adipocyte differentiation. J Gene Med 10:498–507PubMedCrossRefGoogle Scholar
  37. Tashiro K, Inamura M, Kawabata K, Sakurai F, Yamanishi K, Hayakawa T, Mizuguchi H (2009) Efficient adipocyte and osteoblast differentiation from mouse induced pluripotent stem cells by adenoviral transduction. Stem Cells 27:1802–1811PubMedCrossRefGoogle Scholar
  38. Traianedes K, Dallas MR, Garrett IR, Mundy GR, Bonewald LF (1998) 5-Lipoxygenase metabolites inhibit bone formation in vitro. Endocrinology 139:3178–3184PubMedCrossRefGoogle Scholar
  39. Wagner W, Bork S, Horn P, Krunic D, Walenda T, Diehlmann A, Benes V, Blake J, Huber FX, Eckstein V, Boukamp P, Ho AD (2009) Aging and replicative senescence have related effects on human stem and progenitor cells. PLoS ONE 4:e5846PubMedCrossRefPubMedCentralGoogle Scholar
  40. Wang X, Tan Y, Zhang B, Gu Z, Li X (2009) Synthesis and evaluation of collagen-chitosan-hydroxyapatite nanocomposites for bone grafting. J Biomed Mater Res A 89:1079–1087PubMedCrossRefGoogle Scholar
  41. Wernig M, Meissner A, Cassady JP, Jaenisch R (2008) c-Myc is dispensable for direct reprogramming of mouse fibroblasts. Cell Stem Cell 2:10–12PubMedCrossRefGoogle Scholar
  42. Ye L, Zhang S, Greder L, Dutton J, Keirstead SA, Lepley M, Zhang L, Kaufman D, Zhang J (2013) Effective cardiac myocyte differentiation of human induced pluripotent stem cells requires VEGF. PLoS ONE 8:e53764PubMedCrossRefPubMedCentralGoogle Scholar
  43. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science 318:1917–1920PubMedCrossRefGoogle Scholar
  44. Zhang X, Schwarz EM, Young DA, Puzas JE, Rosier RN, O’Keefe RJ (2002) Cyclooxygenase-2 regulates mesenchymal cell differentiation into the osteoblast lineage and is critically involved in bone repair. J Clin Invest 109:1405–1415PubMedCrossRefPubMedCentralGoogle Scholar
  45. Zhang X, You X, Wang Q, Zhang T, Du Y, Lv N, Zhang Z, Zhang S, Shan C, Ye L (2012) Hepatitis B virus X protein drives multiple cross-talk cascade loops involving NF-kappaB, 5-LOX, OPN and Capn4 to promote cell migration. PLoS ONE 7:e31458PubMedCrossRefPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Yanru Wu
    • 1
  • Hualing Sun
    • 1
  • Fangfang Song
    • 1
  • Cui Huang
    • 1
  • Jiawei Wang
    • 1
    Email author
  1. 1.Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory of Oral Biomedicine Ministry of Education School & Hospital of StomatologyWuhan UniversityWuhanPeople’s Republic of China

Personalised recommendations