Discarded tissues, like human amniotic membranes and adipose tissue, were investigated for the application of Decellularized Human Amniotic Membrane (DAM) as a viable scaffold for transplantation of Adipose-derived stromal cells (ASCs) in bone regeneration of non-healing calvarial defects in rats. Amniotic membrane was decellularized to provide a scaffold for male Wistar rats ASCs expansion and transplantation. ASCs osteoinduction in vitro promoted the deposition of a mineralized bone-like matrix by ASCs, as calcified globular accretions associated with the cells on the DAM surface and inside the collagenous matrix. Non-healing calvarial defects on male Wistar rats were randomly divided in control without treatment, treatment with four layers of DAM, or four layers of DAM associated with ASCs. After 12 weeks, tissue blocks were examined by micro-computed tomography and histology. DAM promoted osteoconduction by increasing the collagenous matrix on both DAM treatments. DAM with ASCs stimulated bone deposition, demonstrated by a higher percentage of bone volume and trabecular bone number, compared to control. Besides the osteogenic capacity in vitro, ASCs stimulated the healing of calvarial defects with significant DAM graft incorporation concomitant with higher host bone deposition. The enhanced in vivo bone regeneration by undifferentiated ASCs loaded onto DAM confirmed the potential of an easily collected autologous cell source associated with a broadly available collagenous matrix in tissue engineering.

Biological scaffolds have become an attractive approach for repairing the infarcted myocardium and have been shown to facilitate constructive remodeling in injured tissues. This study aimed to investigate the possible utilization of bacterial cellulose (BC) membrane patches containing cocultured cells to limit myocardial postinfarction pathology. Myocardial infarction (MI) was induced by ligating the left anterior descending coronary artery in 45 Wistar rats, and patches with or without cells were attached to the hearts. After one week, the animals underwent echocardiography to assess for ejection fraction and left ventricular end-diastolic and end-systolic volumes. Following patch formation, the cocultured cells retained viability of >90% over 14 days in culture. The patch was applied to the myocardial surface of the infarcted area after staying 14 days in culture. Interestingly, the BC membrane without cellular treatment showed higher preservation of cardiac dimensions; however, we did not observe improvement in the left ventricular ejection fraction of this group compared to coculture-treated membranes. Our results demonstrated an important role for BC in supporting cells known to produce cardioprotective soluble factors and may thus provide effective future therapeutic outcomes for patients suffering from ischemic heart disease.

Biologic scaffolds have become an attractive approach for repairing the infarcted myocardium and have been shown to facilitate constructive remodeling in injured tissues. This study aimed to investigate the possible utilization of bacterial cellulose membrane patch containing cocultured cells to limit the myocardium's post-infarction pathology. Myocardial infarction was induced by ligating the left anterior descending coronary artery in 45 Wistar rats, and patches with or without cells were attached to the hearts. After one week, the animals underwent echocardiography for assessing ejection fraction and left ventricular end-diastolic and end-systolic volumes. Following the patch formation, cocultured cells retained viability of >90% over 14 days in culture. The patch was applied to the myocardial surface of the infarcted area after staying 14 days in culture. Interestingly, the bacterial cellulose membrane without cellular treatment showed higher preservation of cardiac dimensions; however, we did not observe improvement in the left ventricular ejection fraction of this group compared to coculture treated membranes. Our results demonstrated an important role for bacterial cellulose in supporting cells known to produce cardioprotective soluble factors and may thus provide effective future therapeutic outcomes for patients suffering from ischemic heart disease.

Mesenchymal stem cells (MSCs) can be derived from several human and animal sources. According to this systematic review, the immortalization of these cells, by viral or gene transfer techniques (plasmid) and non-viral methods, are useful to ensure the reproducibility of the experiments and the prospect of using these cells in clinical studies. The resultant immortalized MSCs cells must undergo through different validation assays in order to prove their safety and phenotypic and genotypic stability; these assays include flow cytometry for specific MSC markers, trilineage differentiation, RT-PCR and qRT-PCR expression analysis for pluripotency genes, karyotype and telomere length and in vivo tumorigenicity assays.

Acellular amniotic membrane (AM) has been studied, with promising results on the reconstruction of lesioned tissues, and has become an attractive approach for tracheal repair. This study aimed to evaluate the repair of the trachea with human umbilical cord mesenchymal stem cells (hucMSCs) differentiated in chondrocytes, grown on an experimental model. Tracheal defects were induced by surgical tracheostomy in 30 New Zealand rabbits, and the acellular amniotic membrane, with or without cells, was covering the defect. The hucMSCs were isolated and cultivated with chondrogenic differentiation over the culture of 14 days, and then grown on the AM. In this study, the AM was biocompatible and hucMSCs differentiated into chondrocytes. Our results demonstrated an important role for AM with cultured cells in the promotion of immature collagen, known to produce tissue regeneration. In addition, cartilaginous tissue was found at the tracheal defects, demonstrated by immunohistology results. This study suggests that this biomaterial implantation can be an effective future therapeutic alternative for patients with tracheal injury.

This a preprint and has not been peer reviewed. Data may be preliminary.

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.