Effect of testosterone and hypoxia on the expansion of umbilical cord blood CD34+ cells in vitro

Zhou,Liping; Zhang,Xiaowei; Zhou,Panpan; Li,Xue; Xu,Xuejing; Shi,Qing; Li,Dong; Ju,Xiuli

doi:10.3892/etm.2017.5026

Effect of testosterone and hypoxia on the expansion of umbilical cord blood CD34+ cells in vitro

Authors:
- Liping Zhou
- Xiaowei Zhang
- Panpan Zhou
- Xue Li
- Xuejing Xu
- Qing Shi
- Dong Li
- Xiuli Ju
View Affiliations

Affiliations: Department of Pediatrics, Qilu Hospital, Shandong University, Jinan, Shandong 250012, P.R. China, Department of Pediatrics, The Sixth People's Hospital of Jinan, Jinan, Shandong 250200, P.R. China
Published online on: August 24, 2017 https://doi.org/10.3892/etm.2017.5026
Pages: 4467-4475

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Successfully expanding hematopoietic stem cells (HSCs) is advantageous for clinical HSC transplantation. The present study investigated the influence of testosterone on the proliferation, antigen phenotype and expression of hematopoiesis‑related genes in umbilical cord blood‑derived cluster of differentiation (CD)34+ cells under normoxic or hypoxia conditions. Cord blood (CB) CD34+ cells were separated using magnetic activated cell sorting. A cytokine cocktail and feeder cells were used to stimulate the expansion of CD34+ cells under normoxic (20% O2) and hypoxic (1% O2) conditions for 7 days and testosterone was added accordingly. Cells were identified using flow cytometry and reconstruction capacity was determined using a colony‑forming unit (CFU) assay. The effects of oxygen concentration and testosterone on the expression of hematopoietic‑related genes, including homeobox (HOX)A9, HOXB2, HOXB4, HOXC4 and BMI‑1, were measured using reverse transcription‑quantitative polymerase chain reaction. The results indicated that the number of CFUs and total cells in the testosterone group increased under normoxic and hypoxic conditions compared with the corresponding control groups. Furthermore, the presence of testosterone increased the number of CFU‑erythroid colonies. In liquid culture, the growth of CD34+ cells was rapid under normoxic conditions compared with under hypoxic conditions, however CD34+ cells were maintained in an undifferentiated state under hypoxic conditions. The addition of testosterone under hypoxia promoted the differentiation of CD34+ cells into CD34+CD38+CD71+ erythroid progenitor cells. Furthermore, it was determined that the expression of hematopoietic‑related genes was significantly increased (P<0.05) in the hypoxia testosterone group compared with the other groups. Therefore, the results of the current study indicate that a combination of hypoxia and testosterone may be a promising cultivation condition for HSC/hemopoietic progenitor cell expansion ex vivo.

View Figures

View References

1	Walasek MA, van Os R and de Haan G: Hematopoietic stem cell expansion: Challenges and opportunities. Ann N Y Acad Sci. 1266:138–150. 2012. View Article : Google Scholar : PubMed/NCBI
2	Möbest D, Mertelsmann R and Henschler R: Serum-free ex vivo expansion of CD34(+) hematopoietic progenitor cells. Biotechnol Bioeng. 60:341–347. 1998. View Article : Google Scholar : PubMed/NCBI
3	Ballen KK, Gluckman E and Broxmeyer HE: Umbilical cord blood transplantation: The first 25 years and beyond. Blood. 122:491–498. 2013. View Article : Google Scholar : PubMed/NCBI
4	Rocha V, Wagner JE Jr, Sobocinski KA, Klein JP, Zhang MJ, Horowitz MM and Gluckman E: Graft-versus-host disease in children who have received a cord-blood or bone marrow transplant from an HLA-identical sibling. Eurocord and International Bone Marrow Transplant Registry Working Committee on Alternative Donor and Stem Cell Sources. N Engl J Med. 342:1846–1854. 2000. View Article : Google Scholar : PubMed/NCBI
5	Foeken LM, Green A, Hurley CK, Marry E, Wiegand T and Oudshoorn M: Monitoring the international use of unrelated donors for transplantation: The WMDA annual reports. Bone Marrow Transplant. 45:811–818. 2010. View Article : Google Scholar : PubMed/NCBI
6	Gao L, Chen X, Zhang X, Liu Y, Kong P, Peng X, Liu L, Liu H and Zeng D: Human umbilical cord blood-derived stromal cell, a new resource of feeder layer to expand human umbilical cord blood CD34⁺ cells in vitro. Blood Cells, Molecules and Diseases. 36:322–328. 2006. View Article : Google Scholar
7	Horwitz ME and Frassoni F: Improving the outcome of umbilical cord blood transplantation through ex vivo expansion or graft manipulation. Cytotherapy. 17:730–738. 2015. View Article : Google Scholar : PubMed/NCBI
8	Pawliuk R, Eaves C and Humphries RK: Evidence of both ontogeny and transplant dose-regulated expansion of hematopoietic stem cells in vivo. Blood. 88:2852–2858. 1996.PubMed/NCBI
9	Iscove NN and Nawa K: Hematopoietic stem cells expand during serial transplantation in vivo without apparent exhaustion. Curr Biol. 7:805–808. 1997. View Article : Google Scholar : PubMed/NCBI
10	Osawa M, Hanada K, Hamada H and Nakauchi H: Long term lymp hematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science. 273:242–245. 1996. View Article : Google Scholar : PubMed/NCBI
11	Sauvageau G, Iscove NN and Humphries RK: In vitro and in vivo expansion of hematopoietic stem cells. Oncogene. 23:7223–7232. 2004. View Article : Google Scholar : PubMed/NCBI
12	Schugar RC, Robbins PD and Deasy BM: Small molecules in stem cell self-renewal and differentiation. Gene Therapy. 15:126–135. 2008. View Article : Google Scholar : PubMed/NCBI
13	Hermitte F, de la Grange Brunet P, Belloc F, Praloran V and Ivanovic Z: Very low O2 concentration (0. 1%) favors G0 return of dividing CD34⁺ cells. Stem Cells. 24:65–73. 2006. View Article : Google Scholar : PubMed/NCBI
14	Ivanovic Z, Hermitte F, de la Grange Brunet P, Dazey B, Belloc F, Lacombe F, Vezon G and Praloran V: Simultaneous maintenance of human cord blood SCID-repopulating cells and expansion of committed progenitors at low O2 concentration (3%). Stem Cells. 22:716–724. 2004. View Article : Google Scholar : PubMed/NCBI
15	Ivanovic Z, Dello Sbarba P, Trimoreau F, Faucher JL and Praloran V: Primitive human HPCs are better maintained and expanded in vitro at 1 percent oxygen than at 20 percent. Transfusion. 12:1482–1488. 2000. View Article : Google Scholar
16	Eliasson P, Rehn M, Hammar P, Larsson P, Sirenko O, Flippin LA, Cammenga J and Jönsson JI: Hypoxia mediates low cell-cycle activity and increases the proportion of long-term reconstituting hematopoietic stem cells during in vitro culture. Experimental Hematology. 38:301–310. 2010. View Article : Google Scholar : PubMed/NCBI
17	Rehn M, Olsson A, Reckzeh K, Diffner E, Carmeliet P, Landberg G and Cammenga J: Hypoxic induction of vascular endothelial growth factor regulates murine hematopoietic stem cell function in the low-oxygenic niche. Blood. 118:1534–1543. 2011. View Article : Google Scholar : PubMed/NCBI
18	Penn A, Mohr AM, Shah SG, Sifri ZC, Kaiser VL, Rameshwar P and Livingston DH: Dose-response relationship between norepinephrine and erythropoiesis: Evidence for a critical threshold. J Surg Res. 163:85–90. 2010. View Article : Google Scholar
19	Kröpfl JM, Stelzer I, Mangge H, Pekovits K, Fuchs R and Allard N: Exercise-induced norepinephrine decreases circulating hematopoietic stem and progenitor cell colony-forming capacity. PLoS One. 9:106–120. 2014. View Article : Google Scholar
20	Kim SW, Hwang JH, Cheon JM, Park NS, Park SE, Park SJ, Yun HJ, Kim S and Jo DY: Direct and indirect effects of androgens on survival of hematopoietic progenitor cells in vitro. J Korean Med Sci. 20:409–416. 2005. View Article : Google Scholar : PubMed/NCBI
21	Beckman B and Fisher JW: Decreased erythroidcolony-forming cell response of XTfm/Y mice to testosterone and 5 betadihydrotestosterone. Endocrinology. 107:1587–1592. 1980. View Article : Google Scholar : PubMed/NCBI
22	Huang CK, Luo J, Lee SO and Chang C: Concise review: Androgen receptor differential roles in stem/progenitor cells including prostate, embryonic, stromal and hematopoietic lineages. Stem Cells. 32:2299–2308. 2014. View Article : Google Scholar : PubMed/NCBI
23	Chen C, Cao J, Song X, Zeng L, Li Z, Li Y and Xu K: Adrenaline administration promotes the efficiency of granulocyte colony stimulating factor-mediated hematopoietic stem and progenitor cell mobilization in mice. Int J Hematol. 97:50–57. 2013. View Article : Google Scholar : PubMed/NCBI
24	Duchez P, Chevaleyre J, de la Grange Brunet P, Vlaski M, Boiron JM, Wouters G and Ivanovic Z: Cryopreservation of hematopoietic stem and progenitor cells amplified ex vivo from cord blood CD34⁺ cells. Transfusion. 53:2012–2019. 2013. View Article : Google Scholar : PubMed/NCBI
25	Dumont N, Boyer L, Émond H, Celebi-Saltik B, Pasha R, Bazin R, Mantovani D, Roy DC and Pineault N: Medium conditioned with mesenchymal stromal cell-derived osteoblasts improves the expansion and engraftment properties of cord blood progenitors. Exp Hematol. 42:741–752. 2014. View Article : Google Scholar : PubMed/NCBI
26	Zhuang Y, Li D, Fu J, Shi Q, Lu Y and Ju X: Comparison of biological properties of umbilical cord-derived mesenchymal stem cells from early and late passages: Immunomodulatory ability is enhanced in aged cells. Mol Med Rep. 11:166–174. 2015. View Article : Google Scholar : PubMed/NCBI
27	Robinson S, Niu T, de Lima M, Ng J, Yang H, McMannis J, Karandish S, Sadeghi T, Fu P and del Angel M: Ex vivo expansion of umbilical cord blood. Cytotherapy. 3:243–250. 2005. View Article : Google Scholar
28	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2 (-Delta Delta C(T)) Method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
29	Bock TA, Ziegler BL, Bühring HJ, Scheding S, Brugger W and Kanz L: Characterization of purified and ex vivo manipulated human hematopoietic progenitor and stem cells in xenograft recipients. Ann N Y Acad Sci. 872:200–207. 1999. View Article : Google Scholar : PubMed/NCBI
30	Frascoli M, Proietti M and Grassi F: Phenotypic analysis and isolation of murine hematopoietic stem cells and lineage-committed progenitors. J Vis Exp. 65:pii: 3736. 2012.
31	Zhang QS, Benedetti E, Deater M, Schubert K, Major A, Pelz C, Impey S, Marquez-Loza L, Rathbun RK, Kato S, et al: Oxymetholone therapy of fanconi anemia suppresses osteopontin transcription and induces hematopoietic stem cell cycling. Stem Cell Reports. 4:90–102. 2015. View Article : Google Scholar : PubMed/NCBI
32	Gallien-Lartigue O: Differential effects of external agents on the G1-S transit rate of murine pluripotent hemopoietic stem cells (CFUs) after their release from G0. Stem Cells. 2:218–228. 1982.PubMed/NCBI
33	Freedman MH and Saunders EF: Factors affecting erythroid colony growth (CFU-E) from human marrow. Exp Hematol. 5:250–253. 1977.PubMed/NCBI
34	Reissmann KR, Udupa KB and Kawada K: Effects of erythropoietin and androgens on erythroid stem cells after their selective suppression by BCNU. Blood. 44:649–657. 1974.PubMed/NCBI
35	Katayama Y, Battista M, Kao WM, Hidalgo A, Peired AJ, Thomas SA and Frenette PS: Signals from the sympathetic nervous system regulate hematopoietic stem cell egress from bone marrow. Cell. 124:407–421. 2006. View Article : Google Scholar : PubMed/NCBI
36	Ivanović Z, Bartolozzi B, Bernabei PA, Cipolleschi MG, Rovida E, Milenković P, Praloran V and 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. 108:424–429. 2000. View Article : Google Scholar : PubMed/NCBI
37	Ivanović Z, Dello Sbarba P, Trimoreau F, Faucher JL and Praloran V: Primitive human HPCs are better maintained and expanded in vitro at 1 percent oxygen than at 20 percent. Transfusion. 40:1482–1488. 2000. View Article : Google Scholar : PubMed/NCBI
38	Ivanovic Z, Belloc F, Faucher JL, Cipolleschi MG, Praloran V and Dello Sbarba P: Hypoxia maintains and interleukin-3 reduces the pre-colony-forming cell potential of dividing CD34(+) murine bone marrow cells. Experimental Hematology. 30:67–73. 2002. View Article : Google Scholar : PubMed/NCBI
39	Ishikawa Y and Ito T: Kinetics of hemopoietic stem cells in a hypoxic culture. Eur J Haematol. 40:126–129. 1988. View Article : Google Scholar : PubMed/NCBI
40	Lu L and Broxmeyer HE: Comparative influences of phytohemagglutinin-stimulated leukocyte conditioned medium, hemin, prostaglandin E and low oxygen tension on colony formation by erythroid progenitor cells in normal human bone marrow. Exp Hematol. 13:989–993. 1985.PubMed/NCBI
41	Huang CK, Tsai MY, Luo J, Kang HY, Lee SO and Chang C: Suppression of androgen receptor enhances the self renewal of mesenchymal stem cells through helevated expression of EGFR. Biochim Biophys Acta. 1833:1222–1234. 2013. View Article : Google Scholar : PubMed/NCBI
42	Chang CY, Hsuuw YD, Huang FJ, Shyr CR, Chang SY, Huang CK, Kang HY and Huang KE: Androgenic and antiandrogenic effects and expression of androgen receptor in mouse embryonic stem cells. FertilSteril. 85:1195–1203. 2006.
43	Rogers HM, Yu X, Wen J, Smith R, Fibach E and Noguchi CT: Hypoxia alters progression of the erythroid program. Exp Hematol. 1:17–27. 2008. View Article : Google Scholar
44	Fried W and Morley C: Effects of androgenic steroids on erythropoiesis. Steroids. 46:799–826. 1985. View Article : Google Scholar : PubMed/NCBI
45	Huang CK, Luo J, Lee SO and Chang C: Concise review: Androgen receptor differential roles in stem/progenitor cells including prostate, embryonic, stromal and hematopoietic lineages. Stem Cells. 9:2299–2308. 2014. View Article : Google Scholar
46	Calado RT, Yewdell WT, Wilkerson KL, Regal JA, Kajigaya S, Stratakis CA and Young NS: Sex hormones, acting on the TERT gene, increase telomerase activity in human primary hematopoietic cells. Blood. 114:2236–2243. 2009. View Article : Google Scholar : PubMed/NCBI
47	Lane TA: Umbilical cord blood grafts for hematopoietic transplantation in adults: A cup half empty or half full? Transfusion. 45:1027–1034. 2005. View Article : Google Scholar : PubMed/NCBI