AP128C Sigma-AldrichGoat Anti-Mouse IgM Antibody, µ chain, Cy3 conjugate

Fetal bovine serum (FBS) is an animal product used as a medium supplement. The animal origin of FBS is a concern if cultured stem cells are to be utilized for human cell therapy. Therefore, a substitute for FBS is desirable. In this study, an industrial, xeno-free, pharmaceutical-grade supplement for cell culture (SCC) under development at Grifols was tested for growth of human mesenchymal stem cells (hMSCs), cell characterization, and differentiation capacity.SCC is a freeze-dried product obtained through cold-ethanol fractionation of industrial human plasma pools from healthy donors. Bone marrow-derived hMSC cell lines were obtained from two commercial suppliers. Cell growth was evaluated by culturing hMSCs with commercial media or media supplemented with SCC or FBS. Cell viability and cell yield were assessed with an automated cell counter. Cell surface markers were studied by indirect immunofluorescence assay. Cells were cultured then differentiated into adipocytes, chondrocytes, osteoblasts, and neurons, as assessed by specific staining and microscopy observation.SCC supported the growth of commercial hMSCs. Starting from the same number of seeded cells in two consecutive passages of culture with medium supplemented with SCC, hMSC yield and cell population doubling time were equivalent to the values obtained with the commercial medium and was consistent among lots. The viability of hMSCs was higher than 90%, while maintaining the characteristic phenotype of undifferentiated hMSCs (positive for CD29, CD44, CD90, CD105, CD146, CD166 and Stro-1; negative for CD14 and CD19). Cultured hMSCs maintained the potential for differentiation into adipocytes, chondrocytes, osteoblasts, and neurons.The tested human plasma-derived SCC sustains the adequate growth of hMSCs, while preserving their differentiation capacity. SCC can be a potential candidate for cell culture supplement in advanced cell therapies.

Layers 5 and 6 of primate primary visual cortex (V1) harbor morphologically diverse cell groups that have corticocortical and corticosubcortical projections. Layer 6 middle temporal area (MT)-projecting neurons are particularly interesting, as they are the only deep-layer cortical neurons that provide both corticocortical feedforward inputs (to area MT) and corticosubcortical feedback projections (to superior colliculus [SC]) (Fries et al. [1985] Exp Brain Res 58:613-616). However, due to limitations in anatomical tracing techniques, little is known about the detailed morphologies of these cells. We therefore applied a genetically modified rabies virus as a retrograde tracer to fill the dendrites of projection neurons with green fluorescent protein (GFP) (Wickersham et al. [2007] Nat Methods 4:47-49). We injected virus into SC or area MT of macaque monkeys and examined labeled cells in V1. Two-thirds of labeled neurons following SC injections were found in layer 5, consisting of "tall-tufted" and "nontufted" cells; the remaining cells were layer 6 "nontufted." Area MT injections labeled neurons in layers 4B and 6, as previously described (Shipp and Zeki [1989] Eur J Neurosci 1:309-332). The layer 6 neurons projecting to MT were remarkably similar to the layer 6 SC-projecting neurons. In contrast to the dense and focused dendritic arbors of layer 5 "tall-tufted" pyramids, all "nontufted" cells had sparse, but unusually long basal dendrites. The nontufted cells closely resemble Meynert cells (le Gros Clark [1942] J Anat 76:369-376; Winfield et al. [1983] Proc R Soc Lond B Biol Sci 217:129-139), but the full in vivo reconstructions presented here show that their basal dendrites can extend much further (up to 1.2 mm) and are less asymmetric than inferred from Golgi reconstructions.