Addition of synthetic polymer in the freezing solution of mesenchymal stem cells from equine adipose tissue as a future perspective for reducing of DMSO concentration.

The regenerative therapies with stem cells (SC) has been increased by the cryopreservation, permitting cell storage for extended periods. However, the permeating cryoprotectant agents (CPAs) such as dimethylsulfoxide (DMSO) can cause severe adverse effects. Therefore, this study evaluated equine mesenchymal stem cells derived from adipose tissue (eAT-MSCs) in fresh (Control) or after slow freezing (SF) in different freezing solutions (FS). The FS comprise DMSO and non-permeating CPAs [Trehalose (T) and the SuperCool X-1000 (X)] in association or not, totalizing seven different FS: (DMSO; T; X; DMSO+T; DMSO+X; T+X, and DMSO+T+X). Before and after cryopreservation were evaluated, viability, colony forming unit (CFU), and cellular differentiation capacity. After freezing-thawing, the viability of the eAT-MSCs reduced (P< 0.05) in all treatments compared to the control. However, the viability of frozen eAT-MSCs in DMSO (80.3 ± 0.6) was superior (P<0.05) to the other FS. Regarding CFU, no difference (P>0.05) was observed between fresh and frozen cells. After freezing-thawing, the eAT-MSCs showed osteogenic, chondrogenic, and adipogenic lineages differentiation potential. Nonetheless, despite the significative reduction in the osteogenic differentiation capacity between fresh and frozen cells, no differences (P > 0.05) were observed among FS. Furthermore, the number of chondrogenic differentiation cells frozen in DMSO+X solution reduced (P<0.05) comparing to the control, without differ (P>0.05) to the other FS. The adipogenic differentiation did not differ (P>0.05) among treatments. In conclusion, although these findings confirm the success of DMSO to cryopreserve eAT-MSCs, the Super Cool X-1000 could be a promise to reduce the DMSO concentration in a FS.

As terapias regenerativas com células-tronco (CT) têm sido incrementadas pela criopreservação, permitindo o armazenamento celular. No entanto, os agentes crioprotetores (ACPs) penetrantes, como DMSO, podem causar efeitos adversos graves. Portanto, este estudo avaliou células-tronco mesenquimais equinas derivadas de tecido adiposo (CTM-TAe) in natura (Controle) ou após congelamento lento (CL) em diferentes soluções de congelamento (SC). As SCs compreendem DMSO e ACPs não permeáveis [Trealose (T) e o SuperCool X-1000 (X)] associados ou não: (DMSO; T; X; DMSO+T; DMSO+X; T +X e DMSO+T+X). Antes e após a criopreservação foram avaliados, viabilidade, unidade formadora de colônia (UFC) e capacidade de diferenciação celular. Após o congelamento-descongelamento, a viabilidade das CTM-TAe reduziu (P< 0,05) em todos os tratamentos em relação ao controle. Entretanto, a viabilidade das CTM-TAe congeladas em DMSO (80,3 ± 0,6) foi superior (P<0,05) às demais SC. Em relação às UFC, não houve diferença (P>0,05) entre células frescas e congeladas. Após congelamento-descongelamento, as CTM-TAe apresentaram potencial de diferenciação de linhagens osteogênicas, condrogênicas e adipogênicas. No entanto, apesar da redução significativa na capacidade de diferenciação osteogênica entre células frescas e congeladas, não foram observadas diferenças (P > 0,05) entre SCs. Além disso, o número de células de diferenciação condrogênica congeladas em solução de DMSO+X reduziu (P<0,05) em relação ao controle, sem diferir (P>0,05) das demais SCs. A diferenciação adipogênica não diferiu (P>0,05) entre os tratamentos. Em conclusão, embora esses achados confirmem o sucesso do DMSO para criopreservar CTM-TAe, o Super Cool X-1000 pode ser uma promessa para reduzir a concentração de DMSO.

Polyol additives modulate the in vitro stability and activity of recombinant human phenylalanine hydroxylase.

Phenylketonuria (PKU; OMIM 261600), the most common disorder of amino acid metabolism, is caused by a deficient activity of human phenylalanine hydroxylase (hPAH). Although the dietetic treatment has proven to be effective in preventing the psycho-motor impairment, much effort has been made to develop new therapeutic approaches. Enzyme replacement therapy with hPAH could be regarded as a potential form of PKU treatment if the reported in vitro hPAH instability could be overcome. In this study, we investigated the effect of different polyol compounds, e.g. glycerol, mannitol and PEG-6000 on the in vitro stability of purified hPAH produced in a heterologous prokaryotic expression system. The recombinant human enzyme was stored in the presence of the studied stabilizing agents at different temperatures (4 and -20 degrees C) during a 1-month period. Protein content, degradation products, specific activity, oligomeric profile and conformational characteristics were assessed during storage. The obtained results showed that the use of 50% glycerol or 10% mannitol, at -20 degrees C, protected the enzyme from loss of its enzymatic activity. The determined DeltaG(0) and quenching parameters indicate the occurrence of conformational changes, which may be responsible for the observed increase in catalytic efficiency.

Modulation of the activity of newly synthesized human phenylalanine hydroxylase mutant proteins by low-molecular-weight compounds.

Phenylketonuria, the most frequent disorder of amino acid metabolism, is caused by a deficient activity of human phenylalanine hydroxylase (hPAH). Rescue of the enzyme activity of several recombinant hPAH mutant forms (I65T, R261Q, R270K and V388M) by low molecular weight compounds namely glycerol, trimethylamine N-oxide (TMAO) and sodium 4-phenylbutyrate (4-PB) was investigated using a prokaryotic expression model. The studied compounds were added to the culture medium, in a concentration dependent manner, simultaneously to induction of protein expression. Among the tested molecules glycerol and TMAO were able to increase the enzyme activity of the studied mutant proteins. Furthermore, a decrease in aggregates and a recovery of the active tetrameric and dimeric forms were detected. Since the addition of the studied compounds to the medium did not change the expression level of E. Coli molecular chaperones we postulate that glycerol and TMAO rescue results from a direct stabilizing effect of the newly synthesized mutant hPAH enzymes.

Co-expression of different subunits of human phenylalanine hydroxylase: evidence of negative interallelic complementation.

To study the interaction between two different subunits of the heteromeric human phenylalanine hydroxylase (hPAH), present in hyperphenylalaninemic (HPA) compound heterozygous patients, heteroallelic hPAH enzymes were produced. A dual vector expression system was used (PRO Bacterial Expression System) in which each mutant subunit was expressed from a separate compatible vector, with different epitope tags, in a single bacterial host. Experimental conditions were selected in order that each plasmid produced equivalent levels of mutant subunits. In this study, we demonstrated that both subunits were expressed and that the purified heteroallelic enzymes, were catalytically active. As expected, the produced proteins displayed enzymatic activities levels lower than the predicted catalytic activity, calculated by averaging in vitro PAH activities from both alleles, and were strongly dependent on the proteins subunit composition. The obtained data suggest that interactions between the studied hPAH subunits, namely the I65T, R261Q, R270K and V388M, and the wild-type protein occurred. As postulated, this phenomenon could be a source of phenotypic variation in genetic diseases involving multimeric proteins.