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Enhancement of wound closure in diabetic mice by ex vivo expanded cord blood CD34+ cells

Abstract

Diabetes can impair wound closure, which can give rise to major clinical problems. Most treatments for wound repair in diabetes remain ineffective. This study aimed to investigate the influence on wound closure of treatments using expanded human cord blood CD34+ cells (CB-CD34+ cells), freshly isolated CB-CD34+ cells and a cytokine cocktail. The test subjects were mice with streptozotocin-induced diabetes. Wounds treated with fresh CB-CD34+ cells showed more rapid repair than mice given the PBS control. Injection of expanded CB-CD34+ cells improved wound closure significantly, whereas the injection of the cytokine cocktail alone did not improve wound repair. The results also demonstrated a significant decrease in epithelial gaps and advanced re-epithelialization over the wound bed area after treatment with either expanded CB-CD34+ cells or freshly isolated cells compared with the control. In addition, treatments with both CB-CD34+ cells and the cytokine cocktail were shown to promote recruitment of CD31+-endothelial cells in the wounds. Both the CB-CD34+ cell population and the cytokine treatments also enhanced the recruitment of CD68-positive cells in the early stages (day 3) of treatment compared with PBS control, although the degree of this enhancement was found to decline in the later stages (day 9). These results demonstrated that expanded CB-CD34+ cells or freshly isolated CB-CD34+ cells could accelerate wound repair by increasing the recruitment of macrophages and capillaries and the reepithelialization over the wound bed area. Our data suggest an effective role in wound closure for both ex vivo expanded CB-CD34+ cells and freshly isolated cells, and these may serve as therapeutic options for wound treatment for diabetic patients. Wound closure acceleration by expanded CB-CD34+ cells also breaks the insufficient quantity obstacle of stem cells per unit of cord blood and other stem cell sources, which indicates a broader potential for autologous transplantation.

Abbreviations

CB:

cord blood

DAPI:

4′,6-diamidino-2-phenylindole

Flt3-L:

Flt-3 ligand

HSC:

hematopoietic stem cell

IGF:

insulin-like growth factor

IL:

interleukin

MSC:

mesenchymal stem cell

PB:

peripheral blood

PDGF:

plateletderived growth factor

SCF:

stem cell factor

STZ:

streptozotocin

TGF-β:

transforming growth factor-β

TPO:

thrombopoietin

VEGF:

vascular endothelial growth factor

References

  1. Pradhan, L., Nabzdyk, C., Andersen, N.D., LoGerfo, F.W. and Veves, A. Inflammation and neuropeptides: the connection in diabetic wound healing. Expert Rev. Mol. Med. 11 (2009) e2.

    Article  PubMed  Google Scholar 

  2. Danaei, G., Finucane, M.M., Lu, Y., Singh, G.M., Cowan, M.J., Paciorek, C.J., Lin, J.K., Farzadfar, F., Khang, Y.H., Stevens, G.A., Rao, M., Ali, M.K., Riley, L.M., Robinson, C.A. and Ezzati, M. National, regional, and global trends in fasting plasma glucose and diabetes prevalence since 1980: systematic analysis of health examination surveys and epidemiological studies with 370 country-years and 2.7 million participants. Lancet 378 (2011) 31–40.

    Article  PubMed  CAS  Google Scholar 

  3. Junrungsee, S., Kosachunhanun, N., Wongthanee, A. and Rerkasem, K. History of foot ulcers increases mortality among patients with diabetes in Northern Thailand. Diabet. Med. 28 (2011) 608–611.

    Article  PubMed  CAS  Google Scholar 

  4. Iversen, M.M., Tell, G.S., Riise, T., Hanestad, B.R., Ostbye, T., Graue, M. and Midthjell, K. History of foot ulcer increases mortality among individuals with diabetes: ten-year follow-up of the Nord-Trondelag Health Study, Norway. Diabetes Care 32 (2009) 2193–2199.

    Article  PubMed  Google Scholar 

  5. Broughton, G., 2nd, Janis, J.E. and Attinger, C.E. The basic science of wound healing. Plast. Reconstr. Surg. 117 (2006) 12S–34S.

    Article  PubMed  CAS  Google Scholar 

  6. Blakytny, R. and Jude, E. The molecular biology of chronic wounds and delayed healing in diabetes. Diabet. Med. 23 (2006) 594–608.

    Article  PubMed  CAS  Google Scholar 

  7. Tsuboi, R., Shi, C.M., Sato, C., Cox, G.N. and Ogawa, H. Co-administration of insulin-like growth factor (IGF)-I and IGF-binding protein-1 stimulates wound healing in animal models. J. Invest. Dermatol. 104 (1995) 199–203.

    Article  PubMed  CAS  Google Scholar 

  8. Roberts, A.B. Transforming growth factor-beta: activity and efficacy in animal models of wound healing. Wound Repair Regen. 3 (1995) 408–418.

    Article  PubMed  CAS  Google Scholar 

  9. Heldin, C.H. and Westermark, B. Mechanism of action and in vivo role of platelet-derived growth factor. Physiol. Rev. 79 (1999) 1283–1316.

    PubMed  CAS  Google Scholar 

  10. Muangman, P., Muffley, L.A., Anthony, J.P., Spenny, M.L., Underwood, R.A., Olerud, J.E. and Gibran, N.S. Nerve growth factor accelerates wound healing in diabetic mice. Wound Repair Regen. 12 (2004) 44–52.

    Article  PubMed  Google Scholar 

  11. Behm, B., Babilas, P., Landthaler, M. and Schreml, S. Cytokines, chemokines and growth factors in wound healing. J. Eur. Acad. Dermatol. Venereol. 26 (2012) 812–820.

    Article  PubMed  CAS  Google Scholar 

  12. Barrientos, S., Stojadinovic, O., Golinko, M.S., Brem, H. and Tomic-Canic, M. Growth factors and cytokines in wound healing. Wound Repair Regen. 16 (2008) 585–601.

    Article  PubMed  Google Scholar 

  13. Gary Sibbald, R. and Woo, K.Y. The biology of chronic foot ulcers in persons with diabetes. Diabetes Metab. Res. Rev. 24Suppl 1 (2008) S25–30.

    Article  PubMed  Google Scholar 

  14. Lamers, M.L., Almeida, M.E., Vicente-Manzanares, M., Horwitz, A.F. and Santos, M.F. High glucose-mediated oxidative stress impairs cell migration. PLoS ONE 6 (2011) e22865–22873.

    Article  PubMed  CAS  Google Scholar 

  15. Smith, P.G. and Liu, M. Impaired cutaneous wound healing after sensory denervation in developing rats: effects on cell proliferation and apoptosis. Cell Tissue Res. 307 (2002) 281–291.

    Article  PubMed  CAS  Google Scholar 

  16. Kondo, M., Wagers, A.J., Manz, M.G., Prohaska, S.S., Scherer, D.C., Beilhack, G.F., Shizuru, J.A. and Weissman, I.L. Biology of hematopoietic stem cells and progenitors: implications for clinical application. Annu. Rev. Immunol. 21 (2003) 759–806.

    Article  PubMed  CAS  Google Scholar 

  17. Otrock, Z.K., Mahfouz, R.A., Makarem, J.A. and Shamseddine, A.I. Understanding the biology of angiogenesis: review of the most important molecular mechanisms. Blood Cells Mol. Dis. 39 (2007) 212–220.

    Article  PubMed  CAS  Google Scholar 

  18. Majka, M., Janowska-Wieczorek, A., Ratajczak, J., Ehrenman, K., Pietrzkowski, Z., Kowalska, M.A., Gewirtz, A.M., Emerson, S.G. and Ratajczak, M.Z. Numerous growth factors, cytokines, and chemokines are secreted by human CD34(+) cells, myeloblasts, erythroblasts, and megakaryoblasts and regulate normal hematopoiesis in an autocrine/ paracrine manner. Blood 97 (2001) 3075–3085.

    Article  PubMed  CAS  Google Scholar 

  19. Sivan-Loukianova, E., Awad, O.A., Stepanovic, V., Bickenbach, J. and Schatteman, G.C. CD34+ blood cells accelerate vascularization and healing of diabetic mouse skin wounds. J. Vasc. Res. 40 (2003) 368–377.

    Article  PubMed  CAS  Google Scholar 

  20. Caballero, S., Sengupta, N., Afzal, A., Chang, K.H., Li Calzi, S., Guberski, D.L., Kern, T.S. and Grant, M.B. Ischemic vascular damage can be repaired by healthy, but not diabetic, endothelial progenitor cells. Diabetes 56 (2007) 960–967.

    Article  PubMed  CAS  Google Scholar 

  21. Chan, R.K., Garfein, E., Gigante, P.R., Liu, P., Agha, R.A., Mulligan, R. and Orgill, D.P. Side population hematopoietic stem cells promote wound healing in diabetic mice. Plast. Reconstr. Surg. 120 (2007) 407–411; discussion 412–413.

    Article  PubMed  CAS  Google Scholar 

  22. Barcelos, L.S., Duplaa, C., Krankel, N., Graiani, G., Invernici, G., Katare, R., Siragusa, M., Meloni, M., Campesi, I., Monica, M., Simm, A., Campagnolo, P., Mangialardi, G., Stevanato, L., Alessandri, G., Emanueli, C. and Madeddu, P. Human CD133+ progenitor cells promote the healing of diabetic ischemic ulcers by paracrine stimulation of angiogenesis and activation of Wnt signaling. Circ. Res. 104 (2009) 1095–1102.

    Article  PubMed  CAS  Google Scholar 

  23. Pedroso, D.C., Tellechea, A., Moura, L., Fidalgo-Carvalho, I., Duarte, J., Carvalho, E. and Ferreira, L. Improved survival, vascular differentiation and wound healing potential of stem cells co-cultured with endothelial cells. PLoS ONE 6 (2011) e16114.

    Article  PubMed  CAS  Google Scholar 

  24. Elsharawy, M.A., Naim, M. and Greish, S. Human CD34+ stem cells promote healing of diabetic foot ulcers in rats. Interact. Cardiovasc. Thorac. Surg. 14 (2012) 288–293.

    Article  PubMed  Google Scholar 

  25. Motyl, K. and McCabe, L.R. Streptozotocin, type I diabetes severity and bone. Biol. Proced. Online 11 (2009) 296–315.

    Article  PubMed  CAS  Google Scholar 

  26. Nishio, Y., Koda, M., Kamada, T., Someya, Y., Yoshinaga, K., Okada, S., Harada, H., Okawa, A., Moriya, H. and Yamazaki, M. The use of hemopoietic stem cells derived from human umbilical cord blood to promote restoration of spinal cord tissue and recovery of hindlimb function in adult rats. J. Neurosurg. Spine 5 (2006) 424–433.

    Article  PubMed  Google Scholar 

  27. Templin, C., Grote, K., Schledzewski, K., Ghadri, J.R., Schnabel, S., Napp, L.C., Schieffer, B., Kurzen, H., Goerdt, S., Landmesser, U., Koenen, W. and Faulhaber, J. Ex vivo expanded haematopoietic progenitor cells improve dermal wound healing by paracrine mechanisms. Exp. Dermatol. 18 (2009) 445–453.

    Article  PubMed  CAS  Google Scholar 

  28. Badillo, A.T., Redden, R.A., Zhang, L., Doolin, E.J. and Liechty, K.W. Treatment of diabetic wounds with fetal murine mesenchymal stromal cells enhances wound closure. Cell Tissue Res. 329 (2007) 301–311.

    Article  PubMed  Google Scholar 

  29. Snarski, E., Milczarczyk, A., Torosian, T., Paluszewska, M., Urbanowska, E., Krol, M., Boguradzki, P., Jedynasty, K., Franek, E. and Wiktor-Jedrzejczak, W. Independence of exogenous insulin following immunoablation and stem cell reconstitution in newly diagnosed diabetes type I. Bone Marrow Transplant. 46 (2011) 562–566.

    Article  PubMed  CAS  Google Scholar 

  30. Couri, C.E., Oliveira, M.C., Stracieri, A.B., Moraes, D.A., Pieroni, F., Barros, G.M., Madeira, M.I., Malmegrim, K.C., Foss-Freitas, M.C., Simoes, B.P., Martinez, E.Z., Foss, M.C., Burt, R.K. and Voltarelli, J.C. C-peptide levels and insulin independence following autologous nonmyeloablative hematopoietic stem cell transplantation in newly diagnosed type 1 diabetes mellitus. JAMA 301 (2009) 1573–1579.

    Article  PubMed  CAS  Google Scholar 

  31. Feng, K., Xu, Y.W., Ye, F.G., Xiao, L., Ma, X.H., Gao, Y., Zhang, X., Yao, S.Z. and Shi, B.Y. [Autologous peripheral blood hematopoietic stem cell transplantation in the treatment of type 1 diabetic mellitus: a report of 16 cases]. Zhonghua Yi Xue Za Zhi 91 (2011) 1966–1969.

    PubMed  CAS  Google Scholar 

  32. Singer, A.J. and Clark, R.A. Cutaneous wound healing. N. Engl. J. Med. 341 (1999) 738–746.

    Article  PubMed  CAS  Google Scholar 

  33. Kim, J.Y., Song, S.H., Kim, K.L., Ko, J.J., Im, J.E., Yie, S.W., Ahn, Y.K., Kim, D.K. and Suh, W. Human cord blood-derived endothelial progenitor cells and their conditioned media exhibit therapeutic equivalence for diabetic wound healing. Cell Transplant. 19 (2010) 1635–1644.

    Article  PubMed  Google Scholar 

  34. Broughton, G.I., Janis, J.E. and Attinger, C.E. Wound Healing: An Overview. Plast. Reconstr. Surg. 117 (2006) 1e-S–32e-S.

    Google Scholar 

  35. Di Rocco, G., Gentile, A., Antonini, A., Ceradini, F., Wu, J.C., Capogrossi, M.C. and Toietta, G. Enhanced healing of diabetic wounds by topical administration of adipose tissue-derived stromal cells overexpressing stromal-derived factor-1: biodistribution and engraftment analysis by bioluminescent imaging. Stem Cells Int. 2011 (2010) 304562.

    PubMed  Google Scholar 

  36. Zhang, S., Anderson, D.F., Bradding, P., Coward, W.R., Baddeley, S.M., MacLeod, J.D., McGill, J.I., Church, M.K., Holgate, S.T. and Roche, W.R. Human mast cells express stem cell factor. J. Pathol. 186 (1998) 59–66.

    Article  PubMed  CAS  Google Scholar 

  37. Longley, B.J., Jr., Morganroth, G.S., Tyrrell, L., Ding, T.G., Anderson, D.M., Williams, D.E. and Halaban, R. Altered metabolism of mast-cell growth factor (c-kit ligand) in cutaneous mastocytosis. N. Engl. J. Med. 328 (1993) 1302–1307.

    Article  PubMed  Google Scholar 

  38. Weiss, R.R., Whitaker-Menezes, D., Longley, J., Bender, J. and Murphy, G.F. Human dermal endothelial cells express membrane-associated mast cell growth factor. J. Invest. Dermatol. 104 (1995) 101–106.

    Article  PubMed  CAS  Google Scholar 

  39. Huttunen, M., Naukkarinen, A., Horsmanheimo, M. and Harvima, I.T. Transient production of stem cell factor in dermal cells but increasing expression of Kit receptor in mast cells during normal wound healing. Arch. Dermatol. Res. 294 (2002) 324–330.

    PubMed  Google Scholar 

  40. Lam, C.R., Tan, M.J., Tan, S.H., Tang, M.B., Cheung, P.C. and Tan, N.S. TAK1 regulates SCF expression to modulate PKBalpha activity that protects keratinocytes from ROS-induced apoptosis. Cell Death Differ. 18 (2011) 1120–1129.

    Article  PubMed  CAS  Google Scholar 

  41. Morita, E., Tanaka, T., Shinoda, S., Kameyoshi, Y., Yamamoto, S., Lee, D.G. and Sugiyama, M. Expression of multiple forms of fetal liver kinase-2 (flk-2/flt-3) ligand in cultured human keratinocytes. Arch. Dermatol. Res. 289 (1997) 177–179.

    Article  PubMed  CAS  Google Scholar 

  42. Bohannon, J., Cui, W., Cox, R., Przkora, R., Sherwood, E. and Toliver-Kinsky, T. Prophylactic treatment with fms-like tyrosine kinase-3 ligand after burn injury enhances global immune responses to infection. J. Immunol. 180 (2008) 3038–3048.

    PubMed  CAS  Google Scholar 

  43. Bohannon, J., Cui, W., Sherwood, E. and Toliver-Kinsky, T. Dendritic cell modification of neutrophil responses to infection after burn injury. J. Immunol. 185 (2010) 2847–2853.

    Article  PubMed  CAS  Google Scholar 

  44. Toliver-Kinsky, T.E., Cui, W., Murphey, E.D., Lin, C. and Sherwood, E.R. Enhancement of dendritic cell production by fms-like tyrosine kinase-3 ligand increases the resistance of mice to a burn wound infection. J. Immunol. 174 (2005) 404–410.

    PubMed  CAS  Google Scholar 

  45. Ghazizadeh, M. Essential role of IL-6 signaling pathway in keloid pathogenesis. J. Nihon Med. Sch. 74 (2007) 11–22.

    Article  CAS  Google Scholar 

  46. Paquet, P. and Pierard, G.E. Interleukin-6 and the skin. Int. Arch. Allergy Immunol. 109 (1996) 308–317.

    Article  PubMed  CAS  Google Scholar 

  47. Sawamura, D., Meng, X., Ina, S., Sato, M., Tamai, K., Hanada, K. and Hashimoto, I. Induction of keratinocyte proliferation and lymphocytic infiltration by in vivo introduction of the IL-6 gene into keratinocytes and possibility of keratinocyte gene therapy for inflammatory skin diseases using IL-6 mutant genes. J. Immunol. 161 (1998) 5633–5639.

    PubMed  CAS  Google Scholar 

  48. Lee, M.J., Kim, J., Lee, K.I., Shin, J.M., Chae, J.I. and Chung, H.M. Enhancement of wound healing by secretory factors of endothelial precursor cells derived from human embryonic stem cells. Cytotherapy 13 (2011) 165–178.

    Article  PubMed  CAS  Google Scholar 

  49. Cheon, S.S., Cheah, A.Y., Turley, S., Nadesan, P., Poon, R., Clevers, H. and Alman, B.A. beta-Catenin stabilization dysregulates mesenchymal cell proliferation, motility, and invasiveness and causes aggressive fibromatosis and hyperplastic cutaneous wounds. Proc. Natl. Acad. Sci. USA 99 (2002) 6973–6978.

    Article  PubMed  CAS  Google Scholar 

  50. Cheon, S., Poon, R., Yu, C., Khoury, M., Shenker, R., Fish, J. and Alman, B.A. Prolonged beta-catenin stabilization and tcf-dependent transcriptional activation in hyperplastic cutaneous wounds. Lab. Invest. 85 (2005) 416–425.

    Article  PubMed  CAS  Google Scholar 

  51. Fathke, C., Wilson, L., Shah, K., Kim, B., Hocking, A., Moon, R. and Isik, F. Wnt signaling induces epithelial differentiation during cutaneous wound healing. BMC Cell Biol. 7 (2006) 4.

    Article  PubMed  Google Scholar 

  52. Slavik, M.A., Allen-Hoffmann, B.L., Liu, B.Y. and Alexander, C.M. Wnt signaling induces differentiation of progenitor cells in organotypic keratinocyte cultures. BMC Dev. Biol. 7 (2007) 9.

    Article  PubMed  Google Scholar 

  53. Kim, D.W., Lee, J.S., Yoon, E.S., Lee, B.I., Park, S.H. and Dhong, E.S. Influence of human adipose-derived stromal cells on Wnt signaling in organotypic skin culture. J. Craniofac. Surg. 22 (2011) 694–698.

    Article  PubMed  Google Scholar 

  54. Janowska-Wieczorek, A., Majka, M., Ratajczak, J. and Ratajczak, M.Z. Autocrine/paracrine mechanisms in human hematopoiesis. Stem Cells 19 (2001) 99–107.

    Article  PubMed  CAS  Google Scholar 

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Chotinantakul, K., Dechsukhum, C., Dejjuy, D. et al. Enhancement of wound closure in diabetic mice by ex vivo expanded cord blood CD34+ cells. Cell Mol Biol Lett 18, 263–283 (2013). https://doi.org/10.2478/s11658-013-0089-9

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