hESCs (H7 line), supplied by Geron Corporation (Menlo Park, CA), were cultured on Matrigel-coated six-well plates in MEF-conditioned medium (MEF-CM), supplemented by 8 ng/mL recombinant human basic fibroblast growth factor (bFGF) and antibiotics (50 U/mL penicillin and 50 L/mL streptomycin). All procedures for producing MEF-CM in the presence of 4ng/mL bFGF, the subculturing of the H7 cells, and their differentiation via embryo bodies (EBs) were as described by Geron (http://www.geron.com/PDF/scprotocols.pdf), with the exception that mitomycin C, rather than irradiation, was used for mitotic β3-AR agonist 1 inactivation of the MEFs. and characterizing ESC cultures.1-3ESCs, derived from the inner cell mass of early stage of embryonic development, can self-renew and potentially differentiate into all cell types of the body. 4They can also serve as experimental models for the study of early embryonic development and differentiation, and may provide sources for cell therapy of various tissues and organs. However, despite this potential, their study so far usually involves cell fixation or destruction during sample preparation, and incorporate complex and laborious techniques. The culture of human embryonic stem cells (hESCs) is also still challenging and involves manual dissection and elimination of differentiated cells. Current methods are based on the observation of cultures under phase contrast microscope, and these can lead to errors in interpretation. Further, unless cells have been genetically modified to include a reporter gene driven by a pluripotency gene promoter (e.g., Oct-4-green fluorescent protein [GFP]), it is sometimes difficult to assess the cultures.5 Moreover, it is often necessary to derive differentiated cells from ESCs, and in most cases cell differentiation in β3-AR agonist 1 culture does not occur simultaneously, leaving a heterogeneous mixture of differentiated cell types.6The determination of the state of differentiation of ES-derived cells is thus also challenging. Although methods such as polymerase chain reaction (PCR) and proteomics are used with mixed cell populations,7there is a shortage of methods that can determine the phenotype and functional activity of individual cells within these populations, especially live cells as they grow and differentiate. More noninvasive methods β3-AR agonist 1 for monitoring ESC differentiation are required, preferably rapid ones and which IKK-alpha are applicable to live cells. We present here a method to monitor both undifferentiated and differentiated states of individual SCs in live cultures using the scanning ion conductance microscopy (SICM).8,9This is a unique imaging technique that uses similar principles to the atomic force microscope, but in contrast to physically tapping the preparation with a sharp tip, the SICM uses a noncontact nanopipette for the probe.9,10This allows high-resolution, non-invasive probing of the surface of unfixed cells. Fixed pipette-sample distance is maintained by reduction in ion flow as the pipette approaches the sample. SICM has been used to image living cell surfaces and their dynamics down to the level of individual protein complexes,11and determine the dynamics of microvilli of epithelial cells and how they assemble to produce more complex structures.12In this paper SICM is used to study individual SCs to monitor their status in time and identify the various cell types differentiated from SCs. == Materials and Methods == == Growth of hESCs == Two different hESC lines were used: hES-NCL1 and H7. hES-NCL1 cells were grown as described previously13on mouse embryonic fibroblasts (MEFs) until passages 43 and 47, respectively, and then transferred onto tissue culture plates (http://www.nuncbrand.com; Nunc, Roskilde, Denmark) precoated with human serum (HS) (Cat. No. H1388; Sigma-Aldrich, Dorset, UK). According to the manufacturer, HS had been derived from male clotted blood (all from the United States) tested and found negative for hepatitis B surface antigen, anti-hepatitis C virus, and anti-HIV/HIV-2 by U.S. Food and Drug Administration-approved tests. To coat plates with HS, their surfaces were overlaid with HS for 1 h at room temperature. Thereafter, HS was removed, and plates were dried for 1 h at room temperature. Colonies of hESCs were grown on HS in the presence of medium conditioned by the fibroblasts derived from differentiated hESCs (hES-dF), and medium was changed every 48 h. hESC colonies were disaggregated mechanically every 4-6 days and freshly re-plated.13 To study the distribution of specific cell-surface marker of undifferentiated hESCs on the cell surface, we performed live immunocytochemical staining of the hESCs cell-surface markers SSEA-4 (1:100) and TRA-1-60 (www.chemicon.com; Chemicon, Temecula, CA). First antibodies were added to hESCs for 20 min at 37C. The samples were gently washed three times with ES-medium before being incubated with the secondary antibodies (Sigma) conjugated to ALEXA Fluor-488 at 37C for 20 min. For Nanog, staining cells were fixed with 4% paraformaldehyde for 10 min and then permeabilized/blocked with 0.1% Triton X-100 and 5% fetal calf serum (FCS) in phosphate-buffered saline (PBS) at room temperature for 45 min. After blocking, the cells were incubated at room temperature for 1 h with Nanog (1:20; R&D Systems, Abingdon, United Kingdom) antibody. The cells were washed with 5% FCS and PBS before addition of the secondary antibody: rhodamine-conjugated anti-goat immunoglobulin G (1:100;http://www.jacksonimmuno.com; Jackson Immuno.
