Fig. 2. Nestin-positive clones obtained from the selected human cord blood subpopulation. Phase-contrast images of CB-derived cells. (A) The CD34⁺-depleted cells that were characterised by FACS in Fig. 1B. (B) Cells from A after being re-seeded and expanded in DMEM/10% FCS and 10 ng/ml EGF to form a monolayer of homogenous, round, proliferating cells. An example of a typical, single, clonogenic cell from this culture is shown in the insert. (C) A clone growing for 14 days in the presence of EGF, after low-density (10 cells/cm²) suspension of cells from culture presented in B. (D) The same clone as in C after another 7 days in culture displays a 10-fold increase in the number of cells as quantified by cell counting. (E) Immunocytochemical staining with the anti-human nestin polyclonal antibody shows that the majority of cells are immunoreactive in growing the clone. The insert shows a higher magnification (40x) of a nestin-positive cell with a Hoechst 33258-stained nucleus, showing a typical filamentous pattern of immunostaining. (F) RT-PCR analysis of a nestin gene expression in cells growing in clones (lines 1 and 2) contrasted with an almost complete lack of signal in mRNA sample extracted from CB-derived (not EGF expanded) cells growing in a monolayer as shown in A. Expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in parallel samples served as a semi-quantitative control for RT-PCR products. Bars: A, 50 µm; B-D, 100 µm; E, 20 µm.