Investigating the potential of the secretome of mesenchymal stem cells derived from sickle cell disease patients

Autoři: Tiago O. Ribeiro aff001;  Brysa M. Silveira aff001;  Mercia C. Meira aff001;  Ana C. O. Carreira aff002;  Mari Cleide Sogayar aff002;  Roberto Meyer aff001;  Vitor Fortuna aff001
Působiště autorů: Health Science Institute, Federal University of Bahia, Salvador, BA, Brazil aff001;  Cell and Molecular Therapy Center NUCEL-NETCEM, School of Medicine, Internal Medicine Department, University of São Paulo, São Paulo, SP, Brazil aff002;  Chemistry Institute, Biochemistry Department, University of São Paulo, São Paulo, SP, Brazil aff003
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article


Sickle cell disease (SCD) is a monogenic red cell disorder associated with multiple vascular complications, microvessel injury and wound-healing deficiency. Although stem cell transplantation with bone marrow-derived mesenchymal stem cells (BMSC) can promote wound healing and tissue repair in SCD patients, therapeutic efficacy is largely dependent on the paracrine activity of the implanted BM stromal cells. Since in vitro expansion and culture conditions are known to modulate the innate characteristics of BMSCs, the present study investigated the effects of normoxic and hypoxic cell-culture preconditioning on the BMSC secretome, in addition to the expression of paracrine molecules that induce angiogenesis and skin regeneration. BMSCs derived from SCD patients were submitted to culturing under normoxic (norCM) and hypoxic (hypoCM) conditions. We found that hypoxically conditioned cells presented increased expression and secretion of several well-characterized trophic growth factors (VEGF, IL8, MCP-1, ANG) directly linked to angiogenesis and tissue repair. The hypoCM secretome presented stronger angiogenic potential than norCM, both in vitro and in vivo, as evidenced by HUVEC proliferation, survival, migration, sprouting formation and in vivo angiogenesis. After local application in a murine wound-healing model, HypoCM showed significantly improved wound closure, as well as enhanced neovascularization in comparison to untreated controls. In sum, the secretome of hypoxia-preconditioned BMSC has increased expression of trophic factors involved in angiogenesis and skin regeneration. Considering that these preconditioned media are easily obtainable, this strategy represents an alternative to stem cell transplantation and could form the basis of novel therapies for vascular regeneration and wound healing in individuals with sickle cell disease.

Klíčová slova:

Angiogenesis – Endothelial cells – Hypoxia – Medical hypoxia – Tissue repair – Wound healing – Neurobiology of disease and regeneration – Sickle cell disease


1. Kato GJ. Sickle cell vasculopathy: Vascular phenotype on fire!. J Physiol. 2019 Feb; 597(4):993–994. doi: 10.1113/JP276705 30007009

2. Nguyen VT, Nassar D, Batteux F, Raymond K, Tharaux PL, Aractingi S. Delayed Healing of Sickle Cell Ulcers Is due to Impaired Angiogenesis and CXCL12 Secretion in Skin Wounds. J Invest Dermatol. 2016 Feb. 136(2):497–506. doi: 10.1016/j.jid.2015.11.005 26967481

3. Okwan-Duodu D, Hansen L, Joseph G, Lyle AN, Weiss D, Archer DR, et al. Impaired Collateral Vessel Formation in Sickle Cell Disease. Arterioscler Thromb Vasc Biol. 2018 May;38(5):1125–1133. doi: 10.1161/ATVBAHA.118.310771 29545241

4. Minniti CP, Delaney KM, Gorbach AM, Xu D, Lee CC, Malik N, et al. Vasculopathy, inflammation, and blood flow in leg ulcers of patients with sickle cell anemia. Am J Hematol. 2014 Jan;89(1):1–6. doi: 10.1002/ajh.23571 23963836

5. Potoka KP, Gladwin MT. Vasculopathy and pulmonary hypertension in sickle cell disease. Am J Physiol Lung Cell Mol Physiol. 2015 Feb 15;308(4):L314–24. doi: 10.1152/ajplung.00252.2014 25398989

6. Azar S, Wong TE. Sickle cell disease: a brief update. Med Clin North Am. 2017 Mar;101(2):375–393. doi: 10.1016/j.mcna.2016.09.009 28189177

7. Bou-Maroun LM, Meta F, Hanba CJ, Campbell AD, Yanik GA. An analysis of inpatient pediatric sickle cell disease: Incidence, costs, and outcomes. Pediatr Blood Cancer. 2018 Jan;65(1). doi: 10.1002/pbc.26758 28801954

8. Bronckaers A, Hilkens P, Martens W, Gervois P, Ratajczak J, Struys T, et al. Mesenchymal stem cells and vascular regeneration. Pharmacol Ther. 2014 Aug; 143(2):181–96. doi: 10.1016/j.pharmthera.2014.02.013 24594234

9. Gu W, Hong X, Potter C, Qu A, Xu Q. Mesenchymal stem/stromal cells as a pharmacological and therapeutic approach to accelerate angiogenesis. Microcirculation. 2017 Jan;24(1). doi: 10.1111/micc.12324 27681821

10. Kim HK, Lee SG, Lee SW, Oh BJ, Kim JH, Kim JA, et al. A Subset of Paracrine Factors as Efficient Biomarkers for Predicting Vascular Regenerative Efficacy of Mesenchymal Stromal/Stem Cells. Stem Cells. 2019 Jan;37(1):77–88. doi: 10.1002/stem.2920 30281870

11. Jiang RH, Wu CJ, Xu XQ, Lu SS, Zu QQ, Zhao LB, et al. Hypoxic conditioned medium derived from bone marrow mesenchymal stromal cells protects against ischemic stroke in rats. J Cell Physiol. 2019 Feb;234(2):1354–1368. doi: 10.1002/jcp.26931 30076722

12. Hoch AI, Leach JK. Concise review: optimizing expansion of bone marrow mesenchymal stem/stromal cells for clinical applications. Stem Cells Transl Med. 2014 May;3(5):643–52. doi: 10.5966/sctm.2013-0196 24682286

13. Zhu W, Chen J, Cong X, Hu S, Chen X. Hypoxia and serum deprivation-induced apoptosis in mesenchymal stem cells. Stem Cells. 2006 Feb;24(2):416–25. doi: 10.1634/stemcells.2005-0121 16253984

14. Dionigi B, Ahmed A, Pennington EC, Zurakowski D, Fauza DO. A comparative analysis of human mesenchymal stem cell response to hypoxia in vitro. J Pediatr Surg. 2014 Jun;49(6):915–8. doi: 10.1016/j.jpedsurg.2014.01.023 24888834

15. Buravkova LB, Andreeva ER, Gogvadze V, Zhivotovsky B. Mesenchymal stem cells and hypoxia: where are we? Mitochondrion. 2014 Nov;19 Pt A:105–12. doi: 10.1016/j.mito.2014.07.005 25034305

16. Bader AM, Klose K, Bieback K, Korinth D, Schneider M, Seifert M, et al. Hypoxic Preconditioning Increases Survival and Pro-Angiogenic Capacity of Human Cord Blood Mesenchymal Stromal Cells In Vitro. PLoS One. 2015 Sep 18;10(9):e0138477. doi: 10.1371/journal.pone.0138477 26380983

17. Antebi B, Rodriguez LA 2nd, Walker KP 3rd, Asher AM, Kamucheka RM, Alvarado L, et al. Short-term physiological hypoxia potentiates the therapeutic function of mesenchymal stem cells. Stem Cell Res Ther. 2018 Oct 11;9(1):265. doi: 10.1186/s13287-018-1007-x 30305185

18. Ferreira JR, Teixeira GQ, Santos SG, Barbosa MA, Almeida-Porada G, Gonçalves RM. Mesenchymal Stromal Cell Secretome: Influencing Therapeutic Potential by Cellular Pre-conditioning. Front Immunol. 2018 Dec 4;9:2837. doi: 10.3389/fimmu.2018.02837 eCollection 2018. 30564236

19. Elabd C, Ichim TE, Miller K, Anneling A, Grinstein V, Vargas V, et al. Comparing atmospheric and hypoxic cultured mesenchymal stem cell transcriptome: implication for stem cell therapies targeting intervertebral discs. J Transl Med. 2018 Aug 10;16(1):222. doi: 10.1186/s12967-018-1601-9 30097061

20. Konala VB, Mamidi MK, Bhonde R, Das AK, Pochampally R, Pal R. The current landscape of the mesenchymal stromal cell secretome: A new paradigm for cell-free regeneration. Cytotherapy. 2016 Jan;18(1):13–24. doi: 10.1016/j.jcyt.2015.10.008 26631828

21. Kusuma GD, Carthew J, Lim R, Frith JE. Effect of the Microenvironment on Mesenchymal Stem Cell Paracrine Signaling: Opportunities to Engineer the Therapeutic Effect. Stem Cells Dev. 2017 May 1;26(9):617–631. doi: 10.1089/scd.2016.0349 28186467

22. Salomon C, Ryan J, Sobrevia L, Kobayashi M, Ashman K, Mitchell M, et al. Exosomal Signaling during Hypoxia Mediates Microvascular Endothelial Cell Migration and Vasculogenesis. PLoS One. 2013 Jul 8;8(7):e68451. doi: 10.1371/journal.pone.0068451 23861904

23. Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest. 1973 Nov;52(11):2745–56. doi: 10.1172/JCI107470 4355998

24. Daltro GC, Fortuna V, de Souza ES, Salles MM, Carreira AC, Meyer R, et al. Efficacy of autologous stem cell-based therapy for osteonecrosis of the femoral head in sickle cell disease: a five-year follow-up study. Stem Cell Res Ther. 2015 May 29;6:110. doi: 10.1186/s13287-015-0105-2 26021713

25. Overath JM, Gauer S, Obermüller N, Schubert R, Schäfer R, Geiger H, et al. Short-term preconditioning enhances the therapeutic potential of adipose-derived stromal/stem cell-conditioned medium in cisplatin-induced acute kidney injury. Exp Cell Res. 2016 Mar 15;342(2):175–83. doi: 10.1016/j.yexcr.2016.03.002 26992633

26. Edwards SS, Zavala G, Prieto CP, Elliott M, Martínez S, Egaña JT, et al. Functional analysis reveals angiogenic potential of human mesenchymal stem cells from Wharton's jelly in dermal regeneration. Angiogenesis. 2014;17(4):851–66. doi: 10.1007/s10456-014-9432-7 24728929

27. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001;29:e45. doi: 10.1093/nar/29.9.e45 11328886

28. Nakatsu MN, Hughes CC. An optimized three-dimensional in vitro model for the analysis of angiogenesis. Meth Enzymol. 2008;443:65–82. doi: 10.1016/S0076-6879(08)02004-1 18772011

29. Newman AC, Chou W, Welch-Reardon KM, Fong AH, Popson SA, Phan DT, et al. Analysis of stromal cell secretomes reveals a critical role for stromal cell-derived hepatocyte growth factor and fibronectin in angiogenesis. Arterioscler Thromb Vasc Biol. 2013 Mar;33(3):513–22. doi: 10.1161/ATVBAHA.112.300782 23288153

30. Guedez L, Rivera AM, Salloum R, Miller ML, Diegmueller JJ, Bungay PM, et al. Quantitative assessment of angiogenic responses by the directed in vivo angiogenesis assay. Am J Pathol 2003;162:1431–1439, doi: 10.1016/S0002-9440(10)64276-9 12707026

31. Salz L, Driskell RR. The Sox2: GFP+/- knock-in mouse model does not faithfully recapitulate Sox2 expression in skin. Exp Dermatol. 2017 Nov;26(11):1146–1148. doi: 10.1111/exd.13396 28636810

32. Geyh S, Oz S, Cadeddu RP, Fröbel J, Brückner B, Kündgen A, et al. Insufficient stromal support in MDS results from molecular and functional deficits of mesenchymal stromal cells. Leukemia. 2013 Sep;27(9):1841–51. doi: 10.1038/leu.2013.193 23797473

33. Hamzic E, Whiting K, Gordon Smith E, Pettengell R. Characterization of bone marrow mesenchymal stromal cells in aplastic anaemia. Br J Haematol. 2015 Jun;169(6):804–13. doi: 10.1111/bjh.13364 25819548

34. Ribot J, Caliaperoumal G, Paquet J, Boisson-Vidal C, Petite H, Anagnostou F. Type 2 diabetes alters mesenchymal stem cell secretome composition and angiogenic properties. J Cell Mol Med. 2017 Feb;21(2):349–363. doi: 10.1111/jcmm.12969 27641937

35. Hu C, Li L. Preconditioning influences mesenchymal stem cell properties in vitro and in vivo. J Cell Mol Med. 2018 Mar;22(3):1428–1442. doi: 10.1111/jcmm.13492 29392844

36. Tsai CC, Yew TL, Yang DC, Huang WH, Hung SC. Benefits of hypoxic culture on bone marrow multipotent stromal cells. Am J Blood Res. 2012;2(3):148–59. 23119226

37. Moya A, Paquet J, Deschepper M, Larochette N, Oudina K, Denoeud C, et al. Human Mesenchymal Stem Cell Failure to Adapt to Glucose Shortage and Rapidly Use Intracellular Energy Reserves Through Glycolysis Explains Poor Cell Survival After Implantation. Stem Cells. 2018 Mar;36(3):363–376. doi: 10.1002/stem.2763 29266629

38. Nie Y, Han BM, Liu XB, Yang JJ, Wang F, Cong XF, et al. Identification of MicroRNAs involved in hypoxia- and serum deprivation-induced apoptosis in mesenchymal stem cells. Int J Biol Sci. 2011;7(6):762–8. doi: 10.7150/ijbs.7.762 21698002

39. Pezzi A, Amorin B, Laureano Á, Valim V, Dahmer A, Zambonato B, et al. Effects Of Hypoxia in Long-Term In Vitro Expansion of Human Bone Marrow Derived Mesenchymal Stem Cells. J Cell Biochem. 2017 Oct;118(10):3072–3079. doi: 10.1002/jcb.25953 28240374

40. Ferreira JR, Teixeira GQ, Santos SG, Barbosa MA, Almeida-Porada G, Gonçalves RM. Mesenchymal Stromal Cell Secretome: Influencing Therapeutic Potential by Cellular Pre-conditioning. Front Immunol. 2018 Dec 4; 9:2837. doi: 10.3389/fimmu.2018.02837 30564236

41. Vizoso FJ, Eiro N, Cid S, Schneider J, Perez-Fernandez R. Mesenchymal Stem Cell Secretome: Toward Cell-Free Therapeutic Strategies in Regenerative Medicine. Int J Mol Sci. 2017 Aug 25;18(9). pii:E1852. doi: 10.3390/ijms18091852 28841158

42. L PK, Kandoi S, Misra R, S V, K R, Verma RS. The mesenchymal stem cell secretome: A new paradigm towards cell-free therapeutic mode in regenerative medicine. Cytokine Growth Factor Rev. 2019 Apr;49:1–9. doi: 10.1016/j.cytogfr.2019.04.002 30954374

43. Todorova D, Simoncini S, Lacroix R, Sabatier F, Dignat-George F. Extracellular Vesicles in Angiogenesis. Circ Res. 2017 May 12;120(10):16581673. doi: 10.1161/CIRCRESAHA.117.309681 28495996

44. Bian S, Zhang L, Duan L, Wang X, Min Y, Yu H. Extracellular vesicles derived from human bone marrow mesenchymal stem cells promote angiogenesis in a rat myocardial infarction model. J Mol Med. 2014 Apr;92(4):387–97. doi: 10.1007/s00109-013-1110-5 24337504

45. Park H, Park H, Mun D, Kang J, Kim H, Kim M, et al. Extracellular vesicles derived from hypoxic human mesenchymal stem cells attenuate GSK3beta expression via mirna-26a in an ischemia-reperfusion injury model. Yonsei Med J. 2018 Aug; 59(6):736–45. doi: 10.3349/ymj.2018.59.6.736 29978610

46. Wysoczynski M, Pathan A, Moore JB 4th, Farid T, Kim J, Nasr M, et al. Pro-Angiogenic Actions of CMC-Derived Extracellular Vesicles Rely on Selective Packaging of Angiopoietin 1 and 2, but Not FGF-2 and VEGF. Stem Cell Rev. 2019 Aug;15(4):530–542. doi: 10.1007/s12015-019-09891-6 31102187

47. Marcus H, Attar-Schneider O, Dabbah M, Zismanov V, Tartakover-Matalon S, Lishner M, et al. Mesenchymal stem cells secretomes' affect multiple myeloma translation initiation. Cell Signal. 2016 Jun;28(6):620–30. doi: 10.1016/j.cellsig.2016.03.003 26976208

48. Konoshenko MY, Lekchnov EA, Vlassov AV, Laktionov PP. Isolation of Extracellular Vesicles: General Methodologies and Latest Trends. Biomed Res Int. 2018 Jan 30;2018:8545347. doi: 10.1155/2018/8545347 eCollection 2018. 29662902

49. Chang PY, Zhang BY, Cui S, Qu C, Shao LH, Xu TK, et al. MSC-derived cytokines repair radiation-induced intra-villi microvascular injury. Oncotarget. 2017 Sep 23;8(50):87821–87836. doi: 10.18632/oncotarget.21236 29152123

50. Oskowitz A, McFerrin H, Gutschow M, Carter ML, Pochampally R. Serum-deprived human multipotent mesenchymal stromal cells (MSCs) are highly angiogenic. Stem Cell Res. 2011 May;6(3):215–25. doi: 10.1016/j.scr.2011.01.004 21421339

51. Kim YS, Noh MY, Cho KA, Kim H, Kwon MS, Kim KS, et al. Hypoxia/reoxygenation-preconditioned human bone marrow-derived mesenchymal stromal cells rescue ischemic rat cortical neurons by enhancing trophic factor release Mol Neurobiol. 2015 Aug;52(1):792–803. doi: 10.1007/s12035-014-8912-5 25288154

52. Anderson JD, Johansson HJ, Graham CS, Vesterlund M, Pham MT, Bramlett CS, et al. Comprehensive proteomic analysis of mesenchymal stem cell exosomes reveals modulation of angiogenesis via nuclear factor-kappab signaling. Stem Cells 2016 Mar;34(3):601–13. doi: 10.1002/stem.2298 26782178

53. Chen L, Xu Y, Zhao J, Zhang Z, Yang R, Xie J, et al. Conditioned Medium from Hypoxic Bone Marrow-Derived Mesenchymal Stem Cells Enhances Wound Healing in Mice. PLoS One. 2014 Apr 29;9(4):e96161. doi: 10.1371/journal.pone.0096161 24781370

54. Carolina E, Kato T, Khanh VC, Moriguchi K, Yamashita T, Takeuchi K, et al. Glucocorticoid Impaired the Wound Healing Ability of Endothelial Progenitor Cells by Reducing the Expression of CXCR4 in the PGE2 Pathway. Front Med (Lausanne). 2018 Sep 28;5:276. doi: 10.3389/fmed.2018.00276 30324106

55. Assoni A, Coatti G, Valadares MC, Beccari M, Gomes J, Pelatti M, et al. Different Donors Mesenchymal Stromal Cells Secretomes Reveal Heterogeneous Profile of Relevance for Therapeutic Use. Stem Cells Dev. 2017 Feb 1;26(3):206–214. doi: 10.1089/scd.2016.0218 27762666

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2019 Číslo 10
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