CHARACTERIZATION OF Lin - STEM-PROGENITOR CELLS 125 Malays. Appl. Biol. (2014) 43(1): 125–132 * To whom correspondence should be addressed. MOLECULAR CHARACTERIZATION OF A LINEAGE NEGATIVE STEM-PROGENITOR CELL POPULATION FROM HUMAN PERIPHERAL BLOOD SITI NORHAIZA, H. 1 , INTAN ZARINA, Z.A. 1 , ROHAYA, M.A.W. 2 , SAHIDAN, S. 1 and SHAHRUL HISHAM, Z.A. 1* 1 School of Biosciences & Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi, 43600, Selangor, Malaysia 2 Department of Orthodontics, Faculty of Dentistry, Universiti Kebangsaan Malaysia, 50300, Kuala Lumpur, Malaysia * Email: shahroy8@gmail.com/hisham@ukm.edu.my ABSTRACT Current haematopoietic stem cell transplantation protocols rely heavily upon CD34 + cells to estimate haematopoietic stem and progenitor cell yield. However, several studies nowadays emerged with evidence that CD133 + cells represent a more primitive cell population than their CD34 + counterparts. The objective of the present study was to isolate CD133 + stem cells by a rapid negative isolation method which depletes human peripheral blood mononuclear cells (MNCs) from cells expressing haematopoietic lineage markers CD2, CD3, CD11b, CD14,CD15, CD16, CD19, CD56, CD123, and CD235a and isolates a discrete lineage negative (Lin - ) cell population (11±2% of MNC, n=6). Human peripheral blood MNCs were fractionated by density gradient centrifugation. After labelling with antibody against haematopoietic lineage markers, MNCs were subjected to magnetic assisted cell sorting (MACS). The expression of stem cells markers was examined by reverse transcriptase PCR (RT-PCR). We showed that Lin - stem-progenitor cell population encompassed the common markers used to characterize adult stem cells including SLAMF1, CD90 and CD133 and was also enriched for CD117, a surface receptor for stem cell factor. This cell population is more homogenous and primitive as compared to suspension MNCs which comprised of SLAMF1 and CD117 but the cells with haematopoietic lineage marker, CD14 and CD2 were also present. In conclusion, our approach was able to isolate a homogenous Lin – stem-progenitor cell population from human peripheral blood hence offers a promising alternative to current haematopoietic stem and progenitor selection methods. Key words: Lineage negative, mononuclear cells, peripheral blood, stem cells INTRODUCTION Adult stem cells are now becoming an attractive alternative to the use of embryonic stem cells in regenerative medicine and congenital disease because of their availability and the manipulation of this type of stem cell are not bounded by ethical concern (Si et al., 2011). Two examples of adult stem cells which are actively studied are hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) (Preston et al., 2003). Despite of increasing demands in adult stem cell research and their relevance in clinical field, it is agreed that a standard protocol for isolating homogeneous population is not yet established. The conventional isolation method of stem cells from various sources relies on the fractionation of mononuclear cells by gradient centrifugation and selection of fibroblast-like cells adhering to the culture plate surface by removing non-adherent floating cells. In order to obtain more homogenous stem cell populations, a cocktail antibody that depletes specific cell populations (Valenti et al. , 2008), fluorescence-activated cell sorting (FACS) isolation (Zohar et al. , 1997), and specific cell surface antibody selection (Battula et al. , 2009) have been employed, improving the purity of the final stem cell population isolated. However, although significant improvements have been made in purification techniques, these isolation methods have not yet been sufficient to isolate completely homogenous populations of stem cells. Therefore, this research is designed to isolate adult stem cell from human peripheral blood by adapting culture selection of peripheral blood mononuclear cells and enrichment of lineage negative stem-progenitor cells of human peripheral blood mononuclear cells.