Combined mild hypoxia and bone marrow mesenchymal stem cells improve expansion and HOXB4 gene expression of human cord blood CD34+ stem cells


  • Fatemeh Mohammadali Department of Hematology and Blood Banking, Faculty of Medical Sciences, Tarbiat Modares University, Tehran
  • Saeid Abroun Department of Hematology and Blood Banking, Faculty of Medical Sciences, Tarbiat Modares University, Tehran
  • Amir Atashi 1. Department of Hematology and Blood Banking, Faculty of Medical Sciences, Tarbiat Modares University, Tehran; 2. Stem Cell and Tissue Engineering Research Center, Shahroud University of Medical Sciences, Shahroud


cord blood, hematopoietic stem cells, CD34 cells, mesenchymal stem cells, hypoxia, HOXB4


Paper description:

  • Expansion modalities of hematopoietic stem cells have been performed with the ultimate goal of improving engraftment of cord blood
  • Expansion in culture remains a challenge because of our limited knowledge of factors that drive self-renewal beside expansion.
  • Combining bone marrow mesenchymal stem cells and mild hypoxia (5% O2 tension) in serum-free culture not only improves expansion but can also significantly enhance self-renewal of hematopoietic stem cells.
  • Our findings contribute to the development of more efficient culture systems for the ex vivo expansion of cord blood hematopoietic stem cells for cellular therapy.

Abstract: Cord blood (CB) is a rich source of hematopoietic stem cells (HSC). It has been used successfully to treat a variety of hematological and non-hematological disorders. Beside the advantages of CB, its main disadvantages are the limited number of stem cells available for transplantation and delayed engraftment. Identifying strategies to enhance expansion and self-renewal of HSCs can improve transplantation efficiency. The aim of this study was to examine different culture conditions on ex vivo expansion and homeobox protein Hox-B4 gene (HOXB4) expression in cord blood CD34+ stem cells. Human cord blood CD34+ HSC were cultured in serum-free medium supplemented with cytokines with and without a feeder layer in normoxia (21% O2) and mild hypoxia (5% O2).At the end of 7 days of culture, the highest number of total nucleated cells (TNC), CD34+ cells, colony forming units (CFUs) and HOXB4  mRNA were observed in a co-culture of  HSC with a bone marrow mesenchymal stem cell(MSC) feeder layer at 5% O2.We concluded that the combination of  bone marrow (BM)-MSC and mild hypoxia (5% O2) not only improved HSC expansion but also enhanced HOXB4 gene expression as a self-renewal marker of HSC, and better mimicked the niche microenvironment conditions.

Received: October 28, 2017; Revised: December 21, 2017; Accepted: January 14, 2018; Published online: January 26, 2018

How to cite this article: Mohammadali F, Abroun S, Atashi A. Combined mild hypoxia and bone marrow mesenchymal stem cells improve expansion and HoxB4 gene expression of human cord blood CD34+ stem cells. Arch Biol Sci. 2018;70(3):…


Download data is not yet available.


Wang LD, Wagers AJ. Dynamic niches in the origination and differentiation of hematopoietic stem cells. Nat Rev Mol Cell Biol. 2011;12:643-55.

Mendelson A, Frenette PS. Hematopoietic stem cell niche maintenance during homeostasis and regeneration. Nat Med. 2014;20:833-46.

Lilly AJ, Johnson WE, Bunce CM. The hematopoietic stem cell niche: new insights into the mechanisms regulating hematopoietic stem cell behavior. Stem Cells Int. 2011;2011:274.

Shizuru JA, Negrin RS, Weissman IL. Hematopoietic stem and progenitor cells: clinical and preclinical regeneration of the hemato-lymphoid system. Annu Rev Med. 2005;56:509-38.

Hordyjewska A, Popiołek Ł, Horecka A. Characteristics of hematopoietic stem cells of umbilical cord blood. Cytotechnology. 2015;67(3):387-96.

Jacobson CA, Turki AT, McDonough SM, Stevenson KE, Kim HT, Kao G, Herrera MI, Reynolds CG, Alyea EP, Ho VT, Koreth J, Armand P, Chen YB, Ballen K, Soiffer RJ, Antin JH, Cutler CS, Ritz J. Immune reconstitution after double umbilical cord blood stem cell transplantation: comparison with unrelated peripheral blood stem cell transplantation. Biol Blood Marrow Transplant. 2012;18:565-74.

Majhail NS, Brunstein CG, Tomblyn M, Thomas AJ, Miller JS, Arora M, Kaufman DS, Burns LJ, Slungaard A, McGlave PB, Wagner JE, Weisdorf DJ. Reduced-intensity allogeneic transplant in patients older than 55 years: unrelated umbilical cord blood is safe and effective for patients without a matched related donor. Biol Blood Marrow Transplant. 2008;14:282-9.

Brunstein CG, Gutman JA, Weisdorf DJ, Woolfrey AE, Defor TE, Gooley TA, Verneris MR, Appelbaum FR, Wagner JE, Delaney C. Allogeneic hematopoietic cell transplantation for hematologic malignancy: relative risks and benefits of double umbilical cord blood. Blood. 2010;116:4693-9.

Mehta RS, Rezvani K, Olson A, Oran B, Hosing C, Shah N, Parmar S, Armitage S, Shpall E..Novel Techniques for Ex Vivo Expansion of Cord Blood: Clinical Trials. Front Med. 2015;2:89.

Tanavde VM, Malehorn MT, Lumkul R , Gao Z, Wingard J, Garrett ES, Civin CI. Human stem-progenitor cells from neonatal cord blood have greater hematopoietic expansion capacity than those from mobilized adult blood. Exp Hematol. 2002;30:816-23.

Mendez-Ferrer S. Mesenchymal and hematopoietic stem cells form a unique bone marrow niche. Nature. 2010;466(7308):829-34.

Jing D, Fonseca A-V, Alakel N, Fierro FA, Muller K, Bornhauser M, Ehninger G, Corbeil D, Ordemann R. Hematopoietic stem cells in co-culture with mesenchymal stromal cells - modeling the niche compartments in vitro. Haematologica. 2010;95(4):542-50.

Majumdar MK, Thiede MA, Mosca JD, Moorman M, Gerson SL.Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells (MSCs) and stromal cells. J Cell Physiol. 1998;176:57-66.

Dellatore SM, Garcia AS, Miller WM. Mimicking stem cell niches to increase stem cell expansion. Curr Opin Biotechnol. 2008;19(5):534-40

Hermitte F, Brunet de la Grange P, Belloc F, Praloran V, Ivanovic Z. Very low O2 concentration (0.1%) favors G0 return of dividing CD34+ cells. Stem Cells. 2006;24:65.

Cipolleschi MG, Dello Sbarba P, Olivotto M. The role of hypoxia in the maintenance of hematopoietic stem cells. Blood. 1993;82(7):2031-7.

Cipolleschi MG, Rovida E, Ivanovic Z, Praloran V, Olivotto M, Dello Sbarba P. The expansion of murine bone marrow cells preincubated in hypoxia as an in vitro indicator of their marrow-repopulating ability. Leukemia. 2000;14:735-9.

Ivanović Z, Bartolozzi B, Bernabei PA, Cipoleschi MG, Rovida E, Milenkovic P, Praloran V, DeloSbarba P. Incubation of murine bone marrow cells in hypoxia ensures the maintenance of marrow-repopulating activity together with the expansion of committed progenitors. Br J Haema. 2000;108:424.

Jiang BH, Semenza GL, Bauer C, Marti HH. Hypoxia-inducible factor 1 levels vary exponentially over a physiologically relevant range of O2 tension. Am J Physiol. 1996;271:C1172-80.

Möbest D, Mertelsmann R, Henschler R. Ex vivo expansion of CD34+ blood progenitor cells in serum-free medium. Biotechnol Bioeng. 1998;60:341-7.

Lebkowski JS, Schain LR, Okarma TB. Serum-free culture of hematopoietic stem cells: a review. Stem Cells. 1995;13:607-12.

Boitano AE, Wang J, Romeo R, Bouchez LC, Parker AE, Sutton SE, Walker JR, Flaveny CA, Perdew GH, Denison MS, Schultz PG, Cooke MP. Aryl hydrocarbon receptor antagonists promote the expansion of human hematopoietic stem cells. Science. 2010;329:1345-8.

Wilson A, Oser GM, Jaworski M, Blanco-Bose WE, Laurenti E, Adolphe C, Essers MA, Macdonald HR, Trumpp A. Dormant and Self Renewing Hematopoietic Stem Cells and Their Niches. Ann NY Acad Sci. 2007;1106(1):64-75.

Goncalves R, Lobato da Silva C, Cabral JM, Zanjani ED, Almeida-Porada G. A Stro-1(+) human universal stromal feeder layer to expand/maintain human bone marrow hematopoietic stem/progenitor cells in a serum-free culture system. Exp Hematol. 2006;34:1353-9.

Zhang Y, Chai C, Jiang XS, Teoh SH, Leong KW. Co-culture of umbilical cord blood CD34 + cells with human mesenchymal stem cells. Tissue Eng. 2006;12:2161-70.

Walenda T, Bork S, Horn P, Wein F, Saffrich R, Diehlmann A, Eckstein V, Ho AD, Wagner W. Coculture with mesenchymal stromal cells increases proliferation and maintenance of hematopoietic progenitor cells. J Cell Mol Med. 2010;14(1‐2):337-50.

Breems DA, Blokland EA, Siebel KE, Mayen AE, Engels LJ, Ploemacher RE. Stroma-contact prevents loss of hematopoietic stem cell quality during ex vivo expansion of CD34+ mobilized peripheral blood stem cells. Blood. 1998;91(1):111-7.

Chute JP, Saini AA, Chute DJ, Wells MR, Clark WB, Harlan DM, Park J, Stull MK, Civin C, Davis TA. Ex vivo culture with human brain endothelial cells increases the SCID-repopulating capacity of adult human bone marrow. Blood. 2002;100:4433-9.

Amirizadeh N, Oodi A, Nikougoftar M, Soltanpour MS. Expression and promoter methylation changes of the P15INK4b during ex vivo cord blood CD34+ cell expansion following co-culture with mesenchymal stromal cells. Hematology. 2013;18.5:260-8.

Jang YY, Sharkis SJ. A low level of reactive oxygen species selects for primitive hematopoietic stem cells that may reside in the low-oxygenic niche. Blood. 2007;110:3056-63.

Guitart AV, Hammoud M, Dello Sbarba P, Ivanovic Z, Praloran V. Slow-cycling/quiescence balance of hematopoietic stem cells is related to physiological gradient of oxygen. Exp Hematol. 2010;38(10):847-51.

Danet GH, Pan Y, Luongo JL, Bonnet DA, Simon MC. Expansion of human SCIDrepopulating cells under hypoxic conditions. J Clin Invest. 2003;112:126-35.

Shima H, Takubo K, Iwasaki H, Yoshihara H, Gomeia Y, Hosokawa K, Arai F, Takahash T Suda T. Reconstitution activity of hypoxic cultured human cord blood CD34-positive cells in NOG mice. Biochem Biophys Res Commun. 2009;378:467-72.

Zhang Y, Gao Y. Novel chemical attempts at ex vivo hematopoietic stem cell expansion. Int J Hematol. 2016;103:519-29.

Sauvageau G, Lansdorp PM, Eaves CJ, Hogge DE, Dragowska WH, Reid DS, Largman C, Lawrence HJ, Humphries RK. Differential expression of homeobox genes in functionally distinct CD34+ subpopulations of human bone marrow cells. Proc Natl Acad Sci USA. 1994;91:12223-7.

Pineault N, Helgason CD, Lawrence HJ, Humphries RK. Differential expression of Hox, Meis1, and Pbx1 genes in primitive cells throughout murine hematopoietic ontogeny. Exp Hematol. 2002;30:49-57.

Antonchuk J, Sauvageau G, Humphries RK. HOXB4 overexpression mediates very rapid stem cell regeneration and competitive hematopoietic repopulation. Exp Hematol. 2001;29:1125-34.

Thorsteinsdottir U, Sauvageau G, Humphries RK. Enhanced in vivo regenerative potential of HOXB4-transduced hematopoietic stem cells with regulation of their pool size. Blood. 1999;94(8):2605-12.

Antonchuk J, Sauvageau G, Humphries RK. HOXB4-induced expansion of adult hematopoietic stem cells ex vivo. Cell. 2002;109:39-45.

Kumar S, Geiger H. HSC Niche Biology and HSC Expansion Ex Vivo. Trends Mol Med. 2017;23(9):799-819.

IvanovićZ, Bartolozzi B, Bernabei PA, Cipolleschi MG, Rovida E, Milenković P, Praloran V, Dello Sbarba P. Incubation of murine bone marrow cells in hypoxia ensures the maintenance of marrow repopulating ability together with the expansion of committed progenitors. Br J Haematol. 2000;108: 424-9.

Ivanović Z, DelloSbarba P, Trimoreau F ,Faucher JL, Praloran V. Primitive human HPCs are better maintained and expanded in vitro at 1 percent oxygen than at 20 percent. Transfusion. 2000;40:1482-8.




How to Cite

Mohammadali F, Abroun S, Atashi A. Combined mild hypoxia and bone marrow mesenchymal stem cells improve expansion and HOXB4 gene expression of human cord blood CD34+ stem cells. Arch Biol Sci [Internet]. 2018Aug.20 [cited 2022May29];70(3):433-41. Available from: