Application of sugars and starch in the cryopreservatio of cells

Fibroblasts, keratinocytes and mesenchymal stem cells (MSC) are a useful tool in the field of regenerative medicines and tissue engineering due to their ability of being functional cell types of mesodermal lineage. Storing and banking of these cells can provide to every potential patient with a source of allogenic or autologous cells and tissues which are an important factor for cell replacement therapies and tissue production. The most effective way to preserve cells and tissues is by cryopreservation. It usually involves the use of compounds that can be cytotoxic (DMSO) or xenogeneous (serum). The perfect cryoprotectant agent is non-toxic, non-antigenic and provide with high viability rate. Given that, sugars and starchers which are often use as natural cryoprotectors can be used as a cryoprotectant agents minimizing the adverse effect due to the presence of DMSO and serum. Here I showed the development of a novel DMSO- and serum-free cryosolution for the cryopreservation of stem cells based on HES, sorbitol and dextran in Ringer Acetate. MSC diffentiation potential and viability were not affected after cryopreservation demonstrating that MSCs are still functional. Furthermore, several attemtps in the cryopreservation field to find the perfect cooling rate and method are still in process. Fully comparison of different freezing rates and methods were performed on this dissertation for MSC, fibroblasts and keratinocytes cryopreservation. The 3 cell types can be cryopreserved with slow cooling and vitrification. The use of controlled rate freezing machine which is expensive and time consuming is unnecessary. In addition, I clearly demonstrated that primary and cell line fibroblasts, being both the same cell type, differ in the cryopreservation efficiency in suspension and as monolyer, being this dissertation the first report to show that freezing protocols cannot be applicable for primary cells and cell lines

Cryopreservation of human umbilical cord-derived mesenchymal stem cells in complex sugar based cryoprotective solutions

Mesenchymal stem cells (MSCs) are able to differentiate in vivo and in vitro giving rise to different cell types including osteoblasts, adipocytes, chondrocytes and neuronal cells, providing a valuable source for treatment of degenerative and age-associated diseases. Im-provement of protocols and procedures for human MSCs cryopreservation will contribute significantly to the development of cell replacement therapies. We developed an alternative cryopreservation solutions for stem cell cryopreservation. Most cryoprotectants need to be removed from the cells by washing after thawing, a procedure that can lead to a loss of precious stem cells. Additionally, the procedure is time and cost-consuming. In our study we used a combination of transfusable and non-toxic substances such as hydroxyethylstarch, sorbitol and dextran replacing DMSO and FCS. We found that a cryosolution containing 5% HES, 0.3M sorbitol and 5% dextran provide successful protection for human umbilical cord derived mesenchymal stem cells. These MSC retain a high viability and show multilineage differentiation

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

Background: Preservation 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. Results: We 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. Conclusion: We 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

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

Background: Preservation 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. Results: We 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. Conclusion: We 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

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

Here we describe some of the crucial steps to generate induced pluripotent stemcells (iPSCs) usingmRNA transfection. Our approach uses a V. virus-derived capping enzyme instead of a cap-analog, ensuring 100% proper cap orientation for in vitro transcribedmRNA. V. virus\'' 2′-O-Methyltransferase enzymecreates 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 48 h, 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

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

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

BACKGROUND: Mesenchymal 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. METHODS: In 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. RESULTS: Four 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. CONCLUSIONS: We 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

Pathogenic LRRK2 regulates centrosome cohesion via Rab10/RILPL1-mediated CDK5RAP2 displacement

Mutations in LRRK2 increase its kinase activity and cause Parkinson's disease. LRRK2 phosphorylates a subset of Rab proteins which allows for their binding to RILPL1. The phospho-Rab/RILPL1 interaction causes deficits in ciliogenesis and interferes with the cohesion of duplicated centrosomes. We show here that centrosomal deficits mediated by pathogenic LRRK2 can also be observed in patient-derived iPS cells, and we have used transiently transfected cell lines to identify the underlying mechanism. The LRRK2-mediated centrosomal cohesion deficits are dependent on both the GTP conformation and phosphorylation status of the Rab proteins. Pathogenic LRRK2 does not displace proteinaceous linker proteins which hold duplicated centrosomes together, but causes the centrosomal displacement of CDK5RAP2, a protein critical for centrosome cohesion. The LRRK2-mediated centrosomal displacement of CDK5RAP2 requires RILPL1 and phospho-Rab proteins, which stably associate with centrosomes. These data provide fundamental information as to how pathogenic LRRK2 alters the normal physiology of a cell.We are grateful to Erich Nigg and Francis Barr for providing a variety of constructs and antibodies, and to Dario Alessi for providing various A549 cell lines and MEF cells. We thank LauraMontosa for excellent technical assistance with confocal microscopy. This work was supported by The Michael J. Fox Foundation for Parkinson's research (to S.H.), intramural funding from Rutgers University (to S.H.), the Spanish Ministry of Economy and Competitiveness (SAF2017-89402-R to S.H.), the BBVA Foundation (to S.H., S.A.C., and R.W. M.), the Spanish Ministry of Education, Culture and Sport (FPU12/04367 to J.M. P., FPU15/05233 to A.J. L.O.), and the Spanish Ministry of Science, Innovation and Universities (EST18/00412 to A.J.L. O.)

Effects of single-dose antipurinergic therapy on behavioral and molecular alterations in the valproic acid-induced animal model of autism

Autism spectrum disorder (ASD) is characterized by deficits in communication and social interaction, restricted interests, and stereotyped behavior. Environmental factors, such as prenatal exposure to valproic acid (VPA), may contribute to the increased risk of ASD. Since disturbed functioning of the purinergic system has been associated with the onset of ASD and used as a potential therapeutic target for ASD in both clinical and preclinical studies, we analyzed the effects of suramin, a non-selective purinergic antagonist, on behavioral, molecular and immunological in an animal model of autism induced by prenatal exposure to VPA. Treatment with suramin (20 mg/kg, intraperitoneal) restored sociability in the three-chamber apparatus and decreased anxiety measured by elevated plus maze apparatus, but had no impact on decreased reciprocal social interactions or higher nociceptive threshold in VPA rats. Suramin treatment had no impact on VPA-induced upregulation of P2X4 and P2Y2 in hippocampus, and P2X4 in medial prefrontal cortex, but normalized an increased level of interleukin 6 (IL-6). Our results suggest an important role of purinergic modulation in behavioral, molecular, and immunological aberrations described in VPA model, and suggest that purinergic system might be a potential target for pharmacotherapy in preclinical studies of ASD