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BackgroundThe rapid clinical translation of mesenchymal stem cells (MSC) has resulted in the development of cell-based strategies for multiple indications. Unfortunately one major barrier to widespread implementation of MSC-based therapies is the limited supply of fetal calf serum (FCS) used to expand cells to therapeutic numbers. Additionally, the xenogeneic element of fetal calf serum has been previously demonstrated to stimulate antibody mediated reactions and in some cases sensitization leading to anaphylaxis.MethodXcytePLUS™ media, a human platelet lysate based product, was used to supplement the culture medium at 5, 7.5 and 10% and compared to fetal calf serum at 10%, for human umbilical cord MSC expansion. Properties of the expanded cells were investigated.ResultsThis study demonstrated equivalent or superior effects of human platelet lysate compared to standard FCS supplemented media, based on doubling rate, without loss of identity or function, as demonstrated with flow cytometry characterization. Differentiation into osteocytes, adipocytes and chondrocytes was comparable from cells expanded in either media supplement.ConclusionsThese data support the implementation of human platelet lysate supplemented media as an alternative to xenogeneic containing preparations which may lead to safer MSC products with therapeutic uses.

We have previously reported that activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) results in potentiation of N-methyl-D-aspartate-induced currents (I NMDA ) of receptors contained in primary cultured cerebellar granule cells (CGCs). The purpose of this study was to identify which PKC isoform(s) was responsible for this effect by using the wholecell patch-clamp technique. Experiments were conducted on CGCs that expressed both the NR2A and NR2B NMDA receptor subunits as well as the PMA-sensitive PKC isoforms ␣, ␤⌱, ␤⌱⌱, ␦, , ␥, and . As observed previously, N-methyl-D-aspartate-induced peak currents (I Pk ) were enhanced by a 12.5-min, 100 nM PMA exposure at 37°C under normal recording conditions. Potentiation of receptor function was not observed when extracellular Ca 2ϩ was removed and 1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid was present inside the cell. PMA-induced potentiation of I Pk did not occur when PKC␣-specific antibody was introduced into the cell via the recording electrode. However, in similar experiments with antibodies specific for PKC␤⌱⌱, ␦, , ␥, and , PMA potentiation of I Pk was observed. Down-regulation of PMA-sensitive PKC isoforms by an overnight exposure of 100 nM PMA resulted in lack of potentiation by PMA that was rescued when catalytically active PKC␣ was introduced into the cell via the patch electrode. PMA potentiation of I Pk was not recovered when catalytically active PKC␤I, PKC␤II, or PKC␥ was introduced into the cell via the patch electrode. Collectively, our data provide strong evidence that PMA-enhanced function of native NMDA receptors expressed in primary cultured CGCs is mediated by activation of PKC␣.

Many intracellular proteins and signaling cascades contribute to the ethanol sensitivity of native N-methyl-d-aspartate receptors (NMDARs). One putative protein is the serine / threonine kinase, Protein kinase C (PKC). The purpose of this study was to assess if PKC modulates the ethanol sensitivity of native NMDARs expressed in primary cultured cerebellar granule cells (CGCs). With the whole-cell patch-clamp technique, we assessed if ethanol inhibition of NMDA-induced currents (INMDA) (100 μM NMDA plus 10 μM glycine) were altered in CGCs in which the novel and classical PKC isoforms were activated by phorbol-12-myristate-13-acetate (PMA). Percent inhibition by 10, 50 or 100 mM ethanol of NMDA-induced steady-state (ISS) or peak current amplitudes (IPk) of NMDARs expressed in CGCs in which PKC was activated by a 12.5 min, 100 nM PMA exposure at 37° C did not differ from currents obtained from receptors contained in control cells. However, PMA-mediated augmentation of IPk in the absence of ethanol was abolished after brief applications of 10 or 1 mM ethanol co-applied with agonists, and this suppression of enhanced receptor function was observed for up to eight minutes post-ethanol exposure. Because we had previously shown that PMA-mediated augmentation of INMDA of NMDARs expressed in these cells is by activation of PKCα, we assessed the effect of ethanol (1, 10, 50 and 100 mM) on PKCα activity. Ethanol decreased PKCα activity by 18% for 1 mM ethanol and activity decreased with increasing ethanol concentrations with a 50% inhibition observed with 100 mM ethanol. The data suggest that ethanol disruption of PMA-mediated augmentation of INMDA may be due to a decrease in PKCα activity by ethanol. However, given the incomplete blockade of PKCα activity and the low concentration of ethanol at which this phenomenon is observed, other ethanol-sensitive signaling cascades must also be involved.