Background: RT-qPCR analysis is a widely used method for the analysis of mRNA expression throughout the field of mesenchymal stromal cell (MSC) research. Comparison between MSC studies, both in vitro and in vivo, are challenging due to the varied methods of RT-qPCR data normalization and analysis. Therefore, this study focuses on putative housekeeping genes for the normalization of RT-qPCR data between heterogeneous commercially available human MSC, compared with more homogeneous populations of MSC such as MIAMI and RS-1 cells. Results: Eight genes including; ACTB, B2M, EF1α, GAPDH, RPL13a, YWHAZ, UBC and HPRT1 were tested as possible housekeeping genes based on their expression level and variability. EF1α and RPL13a were validated for RT-qPCR analysis of MIAMI cells during expansion in varied oxygen tensions, endothelial differentiation, neural precursor enrichment, and during the comparison with RS-1 cells and commercially available MSC. RPL13a and YWHAZ were validated as normalization genes for the cross-species analysis of MIAMI cells in an animal model of focal ischemia. GAPDH, which is one of the most common housekeeping genes used for the normalization of RT-qPCR data in the field of MSC research, was found to have the highest variability and deemed not suitable for normalization of RT-qPCR data. Conclusions: In order to make comparisons between heterogeneous MSC populations, as well as adult stem cell like MSC which are used in different laboratories throughout the world, it is important to have a standardized, reproducible set of housekeeping genes for RT-qPCR analysis. In this study we demonstrate that EF1α, RPL13a and YWHAZ are suitable genes for the RT-qPCR analysis and comparison of several sources of human MSC during in vitro characterization and differentiation as well as in an ex vivo animal model of global cerebral ischemia. This will allow for the comparative RT-qPCR analysis of multiple MSC populations with the goal of future use in animal models of disease as well as tissue repair.

The development of cytotoxic brain edema resulting in increased intracranial pressure is a major cause of death occurring in the early phase of traumatic brain injury (TBI). Such edema predominantly develops as a consequence of astrocyte swelling. We recently documented that fluid percussion injury (FPI) to cultured astrocytes causes cell swelling. Since aquaporin-4 (AQP4) has been strongly implicated in the development of brain edema/astrocyte swelling in various neurological conditions, this study examined the effect of in vitro trauma on AQP4 protein expression in cultured astrocytes. Exposure of astrocytes to FPI resulted in a significant upregulation of AQP4 protein in the plasma membrane due to neosynthesis, as cycloheximide blocked the trauma-induced AQP4 upregulation. Silencing the aqp4 gene by siRNA resulted in a significant reduction in trauma-induced astrocyte swelling, indicating a critical role of AQP4 in this process. We recently documented that oxidative/nitrative stress (ONS), the mitochondrial permeability transition (mPT), and activation of mitogen-activated protein kinases (MAPKs), contribute to trauma-induced astrocyte swelling in culture. We now show that inhibition of these factors reduces the upregulation of AQP4 following trauma. Since TBI has been shown to activate nuclear factor-kappa B (NF-κB), as well as the Na⁺,K⁺,Cl⁻ co-transporter (NKCC), both of which are implicated in brain edema/astrocyte swelling in other conditions, we also examined the effect of BAY 11-7082 and bumetanide, inhibitors of NF-κB and NKCC, respectively, and found that these agents also significantly inhibited the trauma-induced AQP4 upregulation. Our findings show that in vitro trauma upregulates AQP4, and that ONS, MAPKs, mPT, NF-κB, and NKCC are involved in its upregulation.

Chronic hepatic encephalopathy (CHE) is a major complication in patients with severe liver disease. Elevated blood and brain ammonia levels have been implicated in its pathogenesis, and astrocytes are the principal neural cells involved in this disorder. Since defective synthesis and release of astrocytic factors have been shown to impair synaptic integrity in other neurological conditions, we examined whether thrombospondin‐1 (TSP‐1), an astrocytic factor involved in the maintenance of synaptic integrity, is also altered in CHE. Cultured astrocytes were exposed to ammonia (NH₄Cl, 0.5–2.5 mM) for 1–10 days, and TSP‐1 content was measured in cell extracts and culture media. Astrocytes exposed to ammonia exhibited a reduction in intra‐ and extracellular TSP‐1 levels. Exposure of cultured neurons to conditioned media from ammonia‐treated astrocytes showed a decrease in synaptophysin, PSD95, and synaptotagmin levels. Conditioned media from TSP‐1 over‐expressing astrocytes that were treated with ammonia, when added to cultured neurons, reversed the decline in synaptic proteins. Recombinant TSP‐1 similarly reversed the decrease in synaptic proteins. Metformin, an agent known to increase TSP‐1 synthesis in other cell types, also reversed the ammonia‐induced TSP‐1 reduction. Likewise, we found a significant decline in TSP‐1 level in cortical astrocytes, as well as a reduction in synaptophysin content in vivo in a rat model of CHE. These findings suggest that TSP‐1 may represent an important therapeutic target for CHE. image Defective release of astrocytic factors may impair synaptic integrity in chronic hepatic encephalopathy. We found a reduction in the release of the astrocytic matricellular proteins thrombospondin‐1 (TSP‐1) in ammonia‐treated astrocytes; such reduction was associated with a decrease in synaptic proteins caused by conditioned media from ammonia‐treated astrocytes. Exposure of neurons to CM from ammonia‐treated astrocytes, in which TSP‐1 is over‐expressed, reversed (by approx 75%) the reduction in synaptic proteins. NF‐kB = nuclear factor kappa B; PSD95 = post‐synaptic density protein 95; ONS = oxidative/nitrative stress.

Astrocyte swelling and the subsequent increase in intracranial pressure and brain herniation are major clinical consequences in patients with acute hepatic encephalopathy. We recently reported that conditioned media from brain endothelial cells (ECs) exposed to ammonia, a mixture of cytokines (CKs) or lipopolysaccharide (LPS), when added to astrocytes caused cell swelling. In this study, we investigated the possibility that ammonia and inflammatory agents activate the toll‐like receptor 4 (TLR4) in ECs, resulting in the release of factors that ultimately cause astrocyte swelling. We found a significant increase in TLR4 protein expression when ECs were exposed to ammonia, CKs or LPS alone, while exposure of ECs to a combination of these agents potentiate such effects. In addition, astrocytes exposed to conditioned media from TLR4‐silenced ECs that were treated with ammonia, CKs or LPS, resulted in a significant reduction in astrocyte swelling. TLR4 protein up‐regulation was also detected in rat brain ECs after treatment with the liver toxin thioacetamide, and that thioacetamide‐treated TLR4 knock‐out mice exhibited a reduction in brain edema. These studies strongly suggest that ECs significantly contribute to the astrocyte swelling/brain edema in acute hepatic encephalopathy, likely as a consequence of increased TLR4 protein expression by blood‐borne noxious agents. image Ammonia and inflammatory agents, such as lipopolysaccharide (LPS) or cytokines (CKs), activate the toll‐like receptor 4 (TLR4) in endothelial cells (ECs) ultimately resulting in astrocyte swelling. TLR4 protein upregulation was detected in rat brain ECs in acute hepatic encephalopathy (AHE), whereas TLR4 knock‐out mice exhibited a reduction in brain edema after AHE. These studies suggest that ECs significantly contribute to the astrocyte swelling/brain edema in AHE.