This study aimed to differentiate human mesenchymal stem cells (hMSCs) from the human umbilical cord in cholinergic-like cells using a natural matrix. The isolation of hMSCs from Wharton's jelly (WJ) was carried out using "explant" and mononuclear cells by density gradient. hMSCs were plated in a natural functional biopolymer matrix for the production of neurospheres. Neural precursor cells were subjected to a standard cholinergic differentiation protocol. Dissociated neurospheres, neural precursor cells, and cholinergic-like cells were characterized by immunocytochemistry. The RT-PCR was performed. hMSCs were CD73+, CD90+, CD105+, CD34-and CD45-and demonstrated the trilineage differentiation. Neurospheres and their isolated cells were nestin-positive, and also expressed NESTIN, MAP2, ßIII-TUBULIN, GFAP genes. Neural precursor cells that were differentiated in cholinergic-like cells expressed ßIII-TUBULIN protein and choline acetyltransferase enzyme. hMSCs on the natural matrix were capable of differentiating hMSC into neurospheres, obtaining neural precursor cells without growth factors or gene transfection before cholinergic differentiation.
Hosted fileDIFFERENTIATION OF HUMAN MESENCHYMAL STEM CELLS THROUGH THE NATURAL MATRIX TO NEUROSPHERES FOR CHOLINERG available at https://authorea.com/users/339930/articles/473258-differentiation-of-humanmesenchymal-stem-cells-through-the-natural-matrix-to-neurospheres-for-cholinergic-likecells
Background
Considering the expressive number of individuals being diagnosed with diabetes worldwide, it is relevant to find medicines and treatments, in order to achieve diabetes complications, as diabetic retinopathy (DR) long-awaited regression and/or cure. The study aimed to evaluate cell therapy with human neural precursor cells (hNPCs) on induced diabetic retinopathy (DR) in Wistar rats.
Methods
Wharton's Jelly Mesenchymal stem cells (WJ-MSCs) were isolated, expanded, and seeded onto a biopolymer substrate without growth factors to develop neurospheres, then hNPCs were obtained and characterized by immunocytochemistry. The animals were divided into three groups; non-diabetic (ND) n = 4; diabetic without treatment (DM) n = 9; diabetic with cell therapy (DM + hNPCs) n = 9. Cells were transplanted by intravitreal injection (1 x 106 cel/µL) into each of Streptozotocin (STZ) induced diabetes rats. Evaluations by Optical Coherence Tomography (OCT) and Electroretinography (ERG) were done before and after diabetes induction and post cell therapy. Four weeks after treatment, eye enucleation allowed histopathological and immunohistochemistry (IHC) analysis.
Results
hNPCs increased the number of retina ganglion cells, ameliorated the photoreceptor layer, and decreased the microvessel points, evidenced by ERG, OCT, histopathological, and IHC findings. The most relevant differences in morphological analysis (treated vs untreated), exhibit the retinal improvement in many layers, notably in the retinal pigment epithelium and photoreceptors.
Conclusions
hNPCs reduced DR progression, as demonstrated by a neuroprotective effect and promotion of retinal regeneration.
Background
The immortalization of mesenchymal stem cells (MSCs) allows them to avoid senescence and be cultured through limitless cell passages. Thus, several experimental strategies, such as retrovirus-mediated gene transfer or viral oncogenesis, have been applied for the immortalization of MSCs. The aim was to identifier the most commonly used methodologies and their particularities for the immortalization of human and animal MSCs.
Methods
The search was conducted in June 2019 and developed in SCOPUS, PUBMED, and SCIENCE DIRECT. Statistical analysis was performed, obtaining the values of total n, mean and standard deviation, confidence interval (CI), and percentage (frequency) for all the predictors.
Results
The most used immortalization methodology was viral transfection, being the most common immortalized cell type was the bone marrow-derived MSC, and the most used gene for immortalizing both human and animal MSCs was hTERT (39.3%) and SV40T (54.5%). Among the articles analyzed in this review, only 39.3% and 36.4% of human and animal MSCs immortalization protocols, respectively, underwent the tumorigenicity test.
Conclusions
The virus-mediated gene transfection was observed as the most used and established technique. The insertion of the hTERT gene is still the most used gene for cell immortalization, suggesting that the maintenance of telomerase is efficient for maintaining cell proliferation and bypassing cell senescence. The review concluded that the tumorigenicity tests should become mandatory in order to safely use the immortalized MSCs for translation.
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