Metallothionein in human immunomagnetically selected CD34+haematopoietic progenitor cells

Abstract

Human umbilical CD34(+) immature haematopoietic cells were rapidly and efficiently obtained from light density MNC (mononuclear cells) by MACS (magnetic cell sorting). An ex vivo expanded population of CD34(+) was cultured in serum-free medium supplemented with cytokines FL (flt3 ligand), SCF (stem cell factor) and TPO (thrombopoietin) in order to obtain a sufficient number of CD34(+) cells. CD34(+) cells expanded from cord blood for 7 days were demonstrated to increase in the absolute number of CD34(+) cells by 5.12 ± 2.47-fold (mean ± S.D., n = 3). Flow cytometric analysis demonstrated that the percentage of CD34 antigen expression after expansion of the culture was 97.81 ± 1.07%, whereas it was 69.39 ± 10.37% in none-expanded CD34(+) cells (mean ± S.D., n = 3), thus defining a system that allowed extensive amplification accompanied by no maturation. MTs (metallothioneins), low molecular weight, cysteine-rich metal-binding proteins, exhibit various functions, including metal detoxification and homoeostasis. We here examined the expression pattern of functional members of the MT gene family in immature CD34(+) cells and compared it with more mature CD34(-) cells in order to strengthen the proposed function of MT in differentiation. Cells were cultured in RPMI 1640 medium, with or without different zinc supplements for 24 h. Relative quantitative expression of MT isogenes in the mature CD34(-) cells was higher than in the immature CD34(+) cells. IHC (immunohistochemical staining) revealed an increased MT protein biosynthesis in CD34(-) cells, greater than in CD34(+) cells. Therefore, the role of MT in differentiation of human haematopoietic progenitor cells from human cord blood is reported for the first time.