Yahaira Naaldijk

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.

Migrational changes of mesenchymal stem cells in response to cytokines, growth factors, hypoxia, and aging

Mesenchymal stem cells (MSCs) are non-immunogenic, multipotent cells with at least trilineage differentiation potential. They promote wound healing, improve regeneration of injured tissue, and mediate numerous other health effects. MSCs migrate to sites of injury and stimulate repair either through direct differentiation or indirectly through the stimulation of endogenous repair mechanisms. Using the in vitro scratch assay, we show that the inflammatory cytokines, chemokines, and growth factors TNF-α, SDF-1, PDGF, and bFGF enhance migration of rat MSCs under normoxic conditions, while TNF-α, IFN-γ, PDGF, and bFGF promote MSC migration under hypoxic conditions. This indicates that the oxygen concentration affects how MSCs will migrate in response to specific factors and, consistent with this, differential expression of cytokines was observed under hypoxic versus normoxic conditions. Using the transwell migration assay, we find that TNF-α, IFN-γ, bFGF, IGF-1, PDGF, and SDF-1 significantly increase transmigration of rat MSCs compared to unstimulated medium. MSCs derived from aged rats exhibited comparable migration to MSCs derived from young rats under hypoxic and normoxic conditions, even after application with specific factors. Similarly, migration in MSCs from aged, human donors did not statistically differ compared to migration in MSCs derived from human umbilical cord tissue or younger donors.

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

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.

Reprogramming of Human Huntington Fibroblasts Using mRNA

The derivation of induced pluripotent stem cells (iPS) from human cell sources using transduction based on viral vectors has been reported by several laboratories. Viral vector-induced integration is a potential cause of genetic modification. We have derived iPS cells from human foreskin, adult Huntington fibroblasts, and adult skin fibroblasts of healthy donors using a nonviral and nonintegrating procedure based on mRNA transfer. In vitro transcribed mRNA for 5 factors, oct-4, nanog, klf-4, c-myc, sox-2 as well as for one new factor, hTERT, was used to induce pluripotency. Reprogramming was analyzed by qPCR analysis of pluripotency gene expression, differentiation, gene expression array, and teratoma assays. iPS cells were shown to express pluripotency markers and were able to differentiate towards ecto-, endo-, and mesodermal lineages. This method may represent a safer technology for reprogramming and derivation of iPS cells. Cells produced by this method can more easily be transferred into the clinical setting.

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.ZusammenfassungAltern kann als die Summe aller zellulären Schäden und der Dysregulation des Epigenoms definiert werden. Gestörte Zellerhaltungsysteme, eine geringere Geweberegeneration, ein geschwächtes Immunsystems sowie ein erhöhtes Krebsrisiko resultieren aus diesen Veränderungen. Dies führt zu einem höheren Sterberisiko im Alter. Das Studium der altersinduzierten Veränderungen in den Stammzellen und deren regenerativen Potentials ist wichtig, um den funktionellen Abbau der Organ- und Gewebefunktion zu verstehen. Das wachsende Interesse und Wissen über die microRNA fügt eineweitere Dimension hinzu. MicroRNA sind wichtige posttranskriptionale Regulatoren der Genexpression und regulieren Stammzellfunktionen wie Wirksamkeit, Differenzierung, Selbsterneuerung und Seneszenz. Verschiedene Zellsysteme wie die Stressabwehrmechanismen gegen Sauerstoffradikale (ROS), DNA-Reparatur und Apoptose werden durch microRNA reguliert. Diese Funktionen und die Annahme, dass microRNA als ein genereller Schalter wirken, macht sie zu einem wichtigen Ziel der Altersforschung.

Mesenchymal stem cell-derived microvesicles modulate LPS-induced inflammatory responses to microglia cells

Microglia cells are the central nervous system immune cells and have been pointed out as the main mediators of the inflammation leading to neurodegenerative disorders. Mesenchymal stromal cells (MSCs) are a heterogeneous population of cells with very high self‐renewal properties and uncomplicated in vitro culture. Research has shown that MSCs have the capacity to induce tissue regeneration and reduce inflammation. Studies demonstrated that MSCs have complex paracrine machineries involving shedding of cell‐derived microvesicles (MVs), which entail part of the regulatory and regenerative activity of MSCs, as observed in animal models. We proposed MSC‐derived MVs as potent regulators of microglia activation and used an in vitro model of stimulation for BV‐2 cells, a microglia cell line, with lipopolysaccharides (LPS). Here we demonstrated that presence of MSCs‐derived MVs (MSC‐MVs) prevents Tumor necrosis factor‐α, Interleukin (IL)−1β and IL‐6 upregulation by BV‐2 cells and primary microglia cells toward LPS. Also, inducible isoform of nitric oxide synthases and Prostaglandin‐endoperoxide synthase 2 upregulation were hampered in presence of MSC‐MVs. Higher levels of the M2 microglia marker chemokine ligand‐22 were detectable in BV‐2 cells after coculture with MSC‐MVs in presence and absence of LPS. Moreover, upregulation of the activation markers CD45 and CD11b by BV‐2 cells was prevented when cocultured with MSC‐MVs. Furthermore, MSC‐MVs suppressed the phosphorylation of the extracellular signal kinases 1/2, c‐Jun N‐terminal kinases and the p38 MAP kinase (p38) molecules. Consequently, MSC‐MVs might represent a modulator of microglia activation with future therapeutic impact. Stem Cells 2017;35:812–823

Comparison of different cooling rates for fibroblast and keratinocyte cryopreservation

Easy, cost‐effective and reliable cryopreservation protocols are crucial for the successful and effective application of tissue engineering. Several different protocols are in use, but no comprehensive comparisons across different machine‐based and manual methods have been made. Here, we compare the effects of different cooling rates on the post‐thaw survival and proliferative capacity of two basic cell lines for skin tissue engineering fibroblasts and keratinocytes, cultured and frozen in suspension or as a monolayer. We demonstrate that effectiveness of cryopreservation cannot be reliably determined immediately after thawing: the results at this stage were not indicative of cell growth in culture 3 days post‐thaw. Cryopreservation of fibroblasts in an adherent state greatly diminishes their subsequent growth potential. This was not observed when freezing in suspension. In keratinocytes, however, adherent freezing is as effective as freezing in suspension, which could lead to significant cost and labour savings in a tissue‐engineering environment. The ‘optimal’ cryopreservation protocol depends on cell type and intended use. Where time, ease and cost are dominant factors, the direct freezing into a nitrogen tank (straight freeze) approach remains a viable method. The most effective solution across the board, as measured by viability 3 days post‐thaw, was the commonly used, freezing container method. Where machine‐controlled cryopreservation is deemed important for tissue‐engineering Good Manufacturing Practice, we present results using a portfolio of different cooling rates, identifying the ‘optimal’ protocol depending on cell type and culture method. Copyright

Distribution pattern following systemic mesenchymal stem cell injection depends on the age of the recipient and neuronal health

BackgroundMesenchymal stem cells (MSCs) show therapeutic efficacy in many different age-related degenerative diseases, including Alzheimer’s disease. Very little is currently known about whether or not aging impacts the transplantation efficiency of MSCs.MethodsIn this study, we investigated the distribution of intravenously transplanted syngeneic MSCs derived from young and aged mice into young, aged, and transgenic APP/PS1 Alzheimer’s disease mice. MSCs from male donors were transplanted into female mice and their distribution pattern was monitored by PCR using Y-chromosome specific probes. Biodistribution of transplanted MSCs in the brains of APP/PS1 mice was additionally confirmed by immunofluorescence and confocal microscopy.ResultsFour weeks after transplantation into young mice, young MSCs were found in the lung, axillary lymph nodes, blood, kidney, bone marrow, spleen, liver, heart, and brain cortex. In contrast, young MSCs that were transplanted into aged mice were only found in the brain cortex. In both young and aged mouse recipients, transplantation of aged MSCs showed biodistribution only in the blood and spleen. Although young transplanted MSCs only showed neuronal distribution in the brain cortex in young mice, they exhibited a wide neuronal distribution pattern in the brains of APP/PS1 mice and were found in the cortex, cerebellum, hippocampus, olfactory bulb, and brainstem. The immunofluorescent signal of both transplanted MSCs and resident microglia was robust in the brains of APP/PS1 mice. Monocyte chemoattractant-1 levels were lowest in the brain cortex of young mice and were significantly increased in APP/PS1 mice. Within the hippocampus, monocyte chemoattractant-1 levels were significantly higher in aged mice compared with younger and APP/PS1 mice.ConclusionsWe demonstrate in vivo that MSC biodistribution post transplantation is detrimentally affected by aging and neuronal health. Aging of both the recipient and the donor MSCs used attenuates transplantation efficiency. Clinically, our data would suggest that aged MSCs should not be used for transplantation and that transplantation of MSCs into aged patients will be less efficacious.

Protective effects of alpha phenyl-tert-butyl nitrone and ascorbic acid in human adipose derived mesenchymal stem cells from differently aged donors

Adipose-derived mesenchymal stem cells (ADSCs) are multipotent stem cells that promote therapeutic effects and are frequently used in autologous applications. Little is known about how ADSCs respond to genotoxic stress and whether or not donor age affects DNA damage and repair. In this study, we used the comet assay to assess DNA damage and repair in human ADSCs derived from young (20-40 years), middle-aged (41-60 years), and older (61+ years) donors following treatment with H2O2 or UV light. Tail lengths in H2O2-treated ADSCs were substantially higher than the tail lengths in UV-treated ADSCs. After 30 minutes of treatment with H2O2, ADSCs preconditioned with alpha phenyl-tert-butyl nitrone (PBN) or ascorbic acid (AA) showed a significant reduction in % tail DNA. The majority of ADSCs treated with PBN or AA displayed low olive tail movements at various timepoints. In general and indicative of DNA repair, % tail length and % tail DNA peaked at 30 minutes and then decreased to near-control levels at the 2 hour and 4 hour timepoints. Differently aged ADSCs displayed comparable levels of DNA damage in the majority of these experiments, suggesting that the age of the donor does not affect the DNA damage response in cultured ADSCs.

Cryopreservation of dermal fibroblasts and keratinocytes in hydroxyethyl starch–based cryoprotectants

BackgroundPreservation of human skin fibroblasts and keratinocytes is essential for the creation of skin tissue banks. For successful cryopreservation of cells, selection of an appropriate cryoprotectant agent (CPA) is imperative. The aim of this study was to identify CPAs that minimize toxic effects and allow for the preservation of human fibroblasts and keratinocytes in suspension and in monolayers.ResultsWe cryopreserved human fibroblasts and keratinocytes with different CPAs and compared them to fresh, unfrozen cells. Cells were frozen in the presence and absence of hydroxyethyl starch (HES) or dimethyl sulfoxide (DMSO), the latter of which is a commonly used CPA known to exert toxic effects on cells. Cell numbers were counted immediately post-thaw as well as three days after thawing. Cellular structures were analyzed and counted by labeling nuclei, mitochondria, and actin filaments. We found that successful cryopreservation of suspended or adherent keratinocytes can be accomplished with a 10% HES or a 5% HES, 5% DMSO solution. Cell viability of fibroblasts cryopreserved in suspension was maintained with 10% HES or 5% HES, 5% DMSO solutions. Adherent, cryopreserved fibroblasts were successfully maintained with a 5% HES, 5% DMSO solution.ConclusionWe conclude that skin tissue cells can be effectively cryopreserved by substituting all or a portion of DMSO with HES. Given that DMSO is the most commonly used CPA and is believed to be more toxic than HES, these findings are of clinical significance for tissue-based replacement therapies. Therapies that require the use of keratinocyte and fibroblast cells, such as those aimed at treating skin wounds or skin burns, may be optimized by substituting a portion or all of DMSO with HES during cryopreservation protocols.