Molecular characterisation of human peripheral blood stem cells

  • Ruzanna Ab Kadir School of Biosciences and Biotechnology, Faculty of Science and Technology, National University of Malaysia, Bangi, Selangor
  • Shahrul Hisham Zainal Ariffin School of Biosciences and Biotechnology, Faculty of Science and Technology, National University of Malaysia, Bangi, Selangor
  • Rohaya Megat Abdul Wahab Department of Orthodontics, Faculty of Dentistry, National University of Malaysia, Bangi, Selangor
  • Sahidan Senafi School of Biosciences and Biotechnology, Faculty of Science and Technology, National University of Malaysia, Bangi, Selangor
Keywords: adherent cells, haematopoietic stem cell, mesenchymal stem cells, peripheral blood, suspension cells


Peripheral blood mononucleated cells consist of haematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). To date there is no well-defined isolation or characterisation protocol of stem cells from human adult peripheral blood mononucleated cells. Our aim in this study was to isolate and characterise mononucleated cells from human peripheral blood. Peripheral blood mononucleated cells were isolated using the Ficoll-Paque density gradient separation method and cultured in complete medium. After 4 days of culture, adherent and suspension mononucleated cells were separated and cultured for 14 days in an in-vitro culture selection. Stem cells in the isolated mononucleated cells were characterised using a multidisciplinary approach which was based on the expression of stem cell markers, morphology and the capacity to self-renew or proliferate and differentiate into specialised cells. Reverse transcription polymerase chain reaction was used to identify the expression of an HSC marker (signalling lymphocytic activation molecule family member 1, SLAMF1) and a MSC marker (CD105). Results revealed that adherent mononucleated cells were positive for MSC markers, whereas mononucleated cells in suspension were positive for HSC markers. The isolated adherent and suspension mononucleated cells were able to maintain their stem cell properties during in-vitro culture by retaining their capacity to proliferate and differentiate into osteoclast and osteoblast cells, respectively, when exposed to the appropriate induction medium. The isolated mononucleated cells consisted of suspension HSCs and adherent MSCs, both of which have the capability to proliferate and differentiate into mature cells. We have shown that suspension HSCs and adherent MSCs can be obtained from an in-vitro culture of peripheral blood mononucleated cells.


1. Weissman I, Anderson D, Gage F. Stem and progenitor cells: Origins, phenotypes, lineage commitments, and transdifferentiations. Annu Rev Cell Dev Biol. 2001;17:387–403., PMid:11687494

2. Shahrul Hisham ZA, Intan Zarina ZA, Sahidan S, Nor Muhammad M, Rohaya MAW, Zaidah ZA. Stem cells in blood development. Sains Malaysiana. 2005;34(1):21–26.

3. Shahrul Hisham ZA, Rohaya MAW, Ismanizan I, Nor Muhammad M, Zaidah ZA. Stem cells, cytokines and their receptors. As Pac J Mol Biol Biotechnol. 2005;13(1):1–13.

4. Hentze H, Graichen R, Colman A. Cell therapy and the safety of embryonic stem cell-derived grafts. Trends Biotechnol. 2007;25(1):24–32., PMid:17084475

.5 Togel F, Westenfelder C. Adult bone marrow-derived stem cells for organ regeneration and repair. Dev Dyn. 2007;236(12):3321–3331., PMid:17685479

6. Alhadlaq A, Mao JJ. Mesenchymal stem cells: Isolation and therapeutics. Stem Cells Dev. 2004;13(4):436–448., PMid:15345137

7. Wognum AW, Eaves AC, Thomas TE. Identification and isolation of hematopoietic stem cells. Arch Med Res. 2003;34(6):461–475., PMid:14734086

8. Arslan O, Moog R. Mobilization of peripheral blood stem cells. Transfus Apher Sci. 2007;37(2):179–185., PMid:17980665

9. Ariffin SH, Abidin IZ, Yazid MD, Wahab RM. Differentiation analyses of adult suspension mononucleated peripheral blood cells of Mus musculus. Cell Commun Signal. 2010;8:29., PMid:20969794

10. Bobis S, Jarocha D, Majka M. Mesenchymal stem cells: Characteristics and clinical applications. Folia Histochem Cytobiol. 2006;44(4):215–230. PMid:17219716

11. Barber RD, Harmer DW, Coleman RA, Clark BJ. GAPDH as a housekeeping gene: Analysis of GAPDH mRNA expression in a panel of 72 human tissues. Physiol Genomics. 2005;21(3):389–395.​1152/​physiolgenomics.​00025.​2005, PMid:15769908

12. Bhatia M, Bonnet D, Murdoch B, Gan OI, Dick JE. A newly discovered class of human hematopoietic cells with SCID-repopulating activity. Nat Med. 1998;4(9):1038–1045., PMid:9734397

13. Guo Y, Lübbert M, Engelhardt M. CD34 hematopoietic stem cells: Current concepts and controversies. Stem Cells. 2003;21(1):15–20., PMid:12529547

14. Larochelle A, Vormoor J, Hanenberg H, et al. Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: Implications for gene therapy. Nat Med. 1996;2(12):1329–1337., PMid:8946831

15. Peichev M, Naiyer AJ, Pereira D, et al. Expression of VEGFR-2 and AC133 by circulating human CD34(+) cells identifies a population of functional endothelial precursors. Blood. 2000;95(3):952–958. PMid:10648408

16. Kent DG, Copley MR, Benz C, et al. Prospective isolation and molecular characterization of hematopoietic stem cells with durable self-renewal potential. Blood. 2009;113(25):6342–6350., PMid:19377048

17. Engel P, Eck MJ, Terhorst C. The SAP and SLAM families in immune responses and X-linked lymphoproliferative disease. Nat Rev Immunol. 2003;3(10):813–821., PMid:14523387

18. Sidorenko SP, Clark EA. The dual-function CD150 receptor subfamily: The viral attraction. Nat Immunol. 2003;4(1):19–24., PMid:12496974

19. Kiel MJ, Yilmaz OH, Iwashita T, Yilmaz OH, Terhorst C, Morrison SJ. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell. 2005;121(7):1109–1121., PMid:15989959

20. Howie D, Okamoto S, Rietdijk S, et al. The role of SAP in murine CD150 (SLAM)-mediated T-cell proliferation and interferon gamma production. Blood. 2002;100(8):2899–2907., PMid:12351401

21. Wang N, Satoskar A, Faubion W, et al. The cell surface receptor SLAM controls T cell and macrophage functions. J Exp Med. 2004;199(9):1255–1264., PMid:15123745

22. Gaebel R, Furlani D, Sorg H, et al. Cell origin of human mesenchymal stem cells determines a different healing performance in cardiac regeneration. PLoS One. 2011;6(2):e15652., PMid:21347366

23. Duff SE, Li C, Garland JM, Kumar S. CD105 is important for angiogenesis: Evidence and potential applications. FASEB J. 2003;17(9):984–992., PMid:12773481

24. Shi Z, Silveira A, Patel P, Feng X. YY1 is involved in RANKL-induced transcription of the tartrate-resistant acid phosphatase gene in osteoclast differentiation. Gene. 2004;343(1):117–126., PMid:15563837

25. Feng X, Novack DV, Faccio R, et al. A Glanzmann’s mutation in beta 3 integrin specifically impairs osteoclast function. J Clin Inves. 2001;107(9):1137–1144., PMid:11342577

26. Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation. Nature. 2003;423(6937):337–342., PMid:12748652

27. Yazid MD, Ariffin SH, Senafi S, Razak MA, Wahab RM. Determination of the differentiation capacities of murines’ primary mononucleated cells and MC3T3-E1 cells. Cancer Cell Int. 2010;10:42., PMid:20979664

28. Matsuo K, Irie N. Osteoclast–osteoblast communication. Arch Biochem Biophys. 2008;473(2):201–209. 2008.03.027, PMid:18406338

29. Kartsogiannis V, Ng KW. Cell lines and primary cells cultures in the study of bone cell biology. Mol Cell Endocrinol. 2004;228(1–2):79–102., PMid:15541574

30. Valenti MT, Dalle Carbonare L, Donatelli L, Bertoldo F, Zanatta M, Lo Cascio V. Gene expression analysis in osteoblastic differentiation from peripheral blood mesenchymal stem cells. Bone. 2008;43(6):1084–1092., PMid:18761114

31. Li Z, Zhou Z, Saunders MM, Donahue HJ. Modulation of connexin43 alters expression of osteoblastic differentiation markers. J Cell Physiol. 2006;290:1248–1255.​1152/​ajpcell.​00428.​2005, PMid:16319124, PMid:15345137