Effect of systemic transplantation of bone marrow-derived mesenchymal stem cells on neuropathology markers in APP/PS1 Alzheimer mice.

AIMS: Mesenchymal stem cells (MSC) have recently attracted interest as a potential basis for a cell-based therapy of AD. We investigated the putative immune-modulatory effects in neuroinflammation of systemic transplantation of MSC into APP/PS1 transgenic mice. METHODS: 106 MSC were injected into APP/PS1 mice via the tail vein and histological analysis was performed for microglia and amyloid (pE3-Aß) plaque numbers, glial distribution and pE3-Aß plaque size. In addition, a biochemical analysis by qPCR for pro-inflammatory, chemoattractant and neurotrophic factors was performed. RESULTS: MSC are associated with pE3-Aß plaques. The effects of transplantation on microglia-associated pathology could be observed after 28 days. Animals showed a reduction in microglial numbers in the cortex and in microglia size. Gene expression was reduced for TNF-α, IL-6, MCP-1, and for NGF, in MSC recipients. Also, we investigated for the first time and found no changes in expression of IL-10, CCR5, BDNF, VEGF and IFNγ. PTGER2 expression levels were increased in the hippocampus but were reduced in the cortex of MSC recipients. While there were no transplant-related changes in pE3-Aß plaque numbers, a reduction in the size of pE3-Aß plaques was observed in the hippocampus of transplant recipients. CONCLUSION: This is the first study to show reduction in pE3-Aß plaque size. pE3-Aß plaques have gained attention as potential key participants in AD due to their increased aggregation propensity, the possibility for the initial seeding event, resistance against degradation and neurotoxicity. These findings support the hypothesis that MSC-transplants may affect AD pathology via an immune-modulatory function that includes an effect on microglial cells.

Generation of human induced pluripotent stem cells using non-synthetic mRNA.

Here we describe some of the crucial steps to generate induced pluripotent stem cells (iPSCs) using mRNA transfection. Our approach uses a V. virus-derived capping enzyme instead of a cap-analog, ensuring 100% proper cap orientation for in vitro transcribed mRNA. V. virus' 2'-O-Methyltransferase enzyme creates a cap1 structure found in higher eukaryotes and has higher translation efficiency compared to other methods. Use of the polymeric transfection reagent polyethylenimine proved superior to other transfection methods. The mRNA created via this method did not trigger an intracellular immune response via human IFN-gamma (hIFN-γ) or alpha (hIFN-α) release, thus circumventing the use of suppressors. Resulting mRNA and protein were expressed at high levels for over 48h, thus obviating daily transfections. Using this method, we demonstrated swift activation of pluripotency associated genes in human fibroblasts. Low oxygen conditions further facilitated colony formation. Differentiation into different germ layers was confirmed via teratoma assay. Reprogramming with non-synthetic mRNA holds great promise for safe generation of iPSCs of human origin. Using the protocols described herein we hope to make this method more accessible to other groups as a fast, inexpensive, and non-viral reprogramming approach.

Regulating aging in adult stem cells with microRNA.

Aging can be defined as the result of accumulated cellular damage and deregulation of the epigenome. These changes cause impaired cell maintenance systems, reduced tissue regeneration, weakening of the immune system and increased risk of malignancy. The higher mortality rate in older individuals is a result of these pathologies. The study of age-related changes in adult stem cells and their regenerative potential is crucial to our understanding of the physical deterioration of organs and tissues. The growing interest and knowledge in the field of microRNAs adds a further dimension to this field of research. MicroRNAs are important posttranscriptional regulators of gene expression. They co-regulate stem cell properties such as potency, differentiation, self-renewal and senescence. Various cell systems, e.g. defense mechanisms against reactive oxygen radicals (ROS), DNA repair and apoptosis are regulated by microRNAs. These properties and the assumption that microRNAs act as some kind of general switch make them highly relevant in aging research.

Hydroxyethylstarch in cryopreservation - mechanisms, benefits and problems.

As the progress of regenerative medicine places ever greater attention on cryopreservation of (stem) cells, tried and tested cryopreservation solutions deserve a second look. This article discusses the use of hydroxyethyl starch (HES) as a cryoprotectant. Charting carefully the recorded uses of HES as a cryoprotectant, in parallel to its further clinical use, indicates that some HES subtypes are a useful supplement to dimethysulfoxide (DMSO) in cryopreservation. However, we suggest that the most common admixture ratio of HES and DMSO in cryoprotectant solutions has been established by historical happenstance and requires further investigation and optimization.

Effect of age and diabetes on the response of mesenchymal progenitor cells to fibrin matrices.

Mesenchymal stem cells are showing increasing promise in applications such as tissue engineering and cell therapy. MSC are low in number in bone marrow, and therefore in vitro expansion is often necessary. In vivo, stem cells often reside within a niche acting to protect the cells. These niches are composed of niche cells, stem cells, and extracellular matrix. When blood vessels are damaged, a fibrin clot forms as part of the wound healing response. The clot constitutes a form of stem cell niche as it appears to maintain the stem cell phenotype while supporting MSC proliferation and differentiation during healing. This is particularly appropriate as fibrin is increasingly being suggested as a scaffold meaning that fibrin-based tissue engineering may to some extent recapitulate wound healing. Here, we describe how fibrin modulates the clonogenic capacity of MSC derived from young/old human donors and normal/diabetic rats. Fibrin was prepared using different concentrations to modulate the stiffness of the substrate. MSC were expanded on these scaffolds and analysed. MSC showed an increased self-renewal on soft surfaces. Old and diabetic cells lost the ability to react to these signals and can no longer adapt to the changed environment.

Age-related changes in human bone marrow-derived mesenchymal stem cells: consequences for cell therapies.

Human mesenchymal stem cells (hMSC) represent a promising cell-based therapy for a number of degenerative conditions. Understanding the effect of aging on hMSCs is crucial for autologous therapy development in older subject whom degenerative diseases typically afflict. Previous investigations into the effects of aging on hMSC have proved contradictory due to the relative narrow age ranges of subjects assessed and the exclusive reliance of in vitro assays. This study seeks to address this controversy by using a wider range of donor ages and by measuring indices of cellular aging as well as hMSC numbers ex vivo and proliferation rates. CFU-f analysis and flow cytometry analysis using a CD45(low)/D7fib(+ve)/LNGF(+ve) gating strategy were employed. In addition a variety of markers of cellular aging, oxidative damage and senescence measured. A reduction in CFU-f and CD45(low)/D7fib(+ve)/LNGF(+ve) cell numbers were noted in adulthood relative to childhood. Indices of aging including oxidative damage, ROS levels and p21 and p53 all increased suggesting a loss of MSC fitness with age. These data suggest that hMSC numbers obtained by marrow aspiration decline with age. Furthermore, there is an age-related decline in overall BM MSC "fitness" which might lead to problems when using autologous aged MSC for cell-based therapies.

The proteasome and its function in the ageing process.

The ageing process is characterized by a progressive loss of function and a decline in the functional capacities of the organism, leading to death. The nature of the processes involved in loss of functions is not well understood. A number of theories have been proposed, including a hypothesis that emphasizes the role of reactive oxygen species as a fundamental causal factor in the ageing process; among other things, oxidative damage to proteins through reactive oxygen species plays a key role in the ageing process. Oxidative modification of proteins generally causes them to become dysfunctional, and normally to undergo preferential degradation. Within the cell the main proteolytic machinery involved in the degradation of oxidized proteins is the proteasomal system, consisting of a multicatalytic protease complex--the proteasome--and numerous regulatory factors. The proteasome is a highly conserved structure that is distributed in the cytosol, nucleus and endoplasmatic reticulum of mammalian cells. As the proteasome itself is also exposed to oxidative stress during the ageing process several studies were carried out to investigate the role and the activity of the proteasomal system during ageing. This review will describe current knowledge of the activity of the protesomal system and its possible involvement in the ageing process.

Proteolysis, caloric restriction and aging.

The nature of the aging process has been the subject of considerable speculation. It is believed that free radical damage to cellular components is one of the main contributors to the aging process. Studies on proteins have shown age-related decline in enzyme activities, age-related accumulation of oxidized proteins and a decline of the proteolytic machinery of the cell. The proteasome, a highly regulated intracellular proteolytic system, is the major enzymatic system responsible for the degradation of damaged proteins. The current knowledge on regulation and of the properties of this unique proteolytic system with special emphasis to the aging process are discussed in this review. Since it is known that caloric restriction (CR) is the only method to delay the aging process and extend the maximal lifespan the effects of CR on the age-related decline in protein degradation is highlighted.