Meta-analysis of the Mesenchymal Stem Cells Immortalization Protocols: A Guideline for Regenerative Medicine.

BACKGROUND: This systematic review describes the most common methodologies for immortalizing human and animal mesenchymal stem cells (MSCs). This study follows the rules of PRISMA and is registered in the Institutional Review Board of PROSPERO International of systematic reviews, numbered protocol code: CRD42020202465. METHOD: The data search systematization was based on the words "mesenchymal stem cell" AND "immortalization." The search period for publications was between 2000 and 2022, and the databases used were SCOPUS, PUBMED, and SCIENCE DIRECT. The search strategies identified 384 articles: 229 in the SCOPUS database, 84 in PUBMED, and 71 in SCIENCE DIRECT. After screening by titles and abstracts, 285 articles remained. This review included thirty-nine articles according to the inclusion and exclusion criteria. RESULT: In 28 articles, MSCs were immortalized from humans and 11 animals. The most used immortalization methodology was viral transfection. The most common immortalized cell type was the MSC from bone marrow, and the most used gene for immortalizing human and animal MSCs was hTERT (39.3%) and SV40T (54.5%), respectively. CONCLUSION: Also, it was observed that although less than half of the studies performed tumorigenicity assays to validate the immortalized MSCs, other assays, such as qRT-PCR, colony formation in soft agar, karyotype, FISH, and cell proliferation, were performed in most studies on distinct MSC cell passages.

State of the Art of microRNAs Signatures as Biomarkers and Therapeutic Targets in Parkinson's and Alzheimer's Diseases: A Systematic Review and Meta-Analysis.

Identifying target microRNAs (miRNAs) might serve as a basis for developing advanced therapies for Parkinson's disease (PD) and Alzheimer's disease. This review aims to identify the main therapeutic targets of miRNAs that can potentially act in Parkinson's and Alzheimer's diseases. The publication research was conducted from May 2021 to March 2022, selected from Scopus, PubMed, Embase, OVID, Science Direct, LILACS, and EBSCO. A total of 25 studies were selected from 1549 studies evaluated. The total number of miRNAs as therapeutic targets evidenced was 90 for AD and 54 for PD. An average detection accuracy of above 84% for the miRNAs was observed in the selected studies of AD and PD. The major signatures were miR-26b-5p, miR-615-3p, miR-4722-5p, miR23a-3p, and miR-27b-3p for AD and miR-374a-5p for PD. Six miRNAs of intersection were found between AD and PD. This article identified the main microRNAs as selective biomarkers for diagnosing PD and AD and therapeutic targets through a systematic review and meta-analysis. This article can act as a microRNA guideline for laboratory research and pharmaceutical industries for treating Alzheimer's and Parkinson's diseases and offers the opportunity to evaluate therapeutic interventions earlier in the disease process.

Three-Dimensional Bioprinting of an In Vitro Lung Model.

In December 2019, COVID-19 emerged in China, and in January 2020, the World Health Organization declared a state of international emergency. Within this context, there is a significant search for new drugs to fight the disease and a need for in vitro models for preclinical drug tests. This study aims to develop a 3D lung model. For the execution, Wharton's jelly mesenchymal stem cells (WJ-MSC) were isolated and characterized through flow cytometry and trilineage differentiation. For pulmonary differentiation, the cells were seeded in plates coated with natural functional biopolymer matrix as membrane until spheroid formation, and then the spheroids were cultured with differentiation inductors. The differentiated cells were characterized using immunocytochemistry and RT-PCR, confirming the presence of alveolar type I and II, ciliated, and goblet cells. Then, 3D bioprinting was performed with a sodium alginate and gelatin bioink in an extrusion-based 3D printer. The 3D structure was analyzed, confirming cell viability with a live/dead assay and the expression of lung markers with immunocytochemistry. The results showed that the differentiation of WJ-MSC into lung cells was successful, as well as the bioprinting of these cells in a 3D structure, a promising alternative for in vitro drug testing.

Laser Ablated Albumin Functionalized Spherical Gold Nanoparticles Indicated for Stem Cell Tracking.

Cell tracking in cell-based therapy applications helps distinguish cell participation among paracrine effect, neovascularization, and matrix deposition. This preliminary study examined the cellular uptake of gold nanoparticles (AuNPs), observing cytotoxicity and uptake of different sizes and AuNPs concentrations in Adipose-derived stromal cells (ASCs). ASCs were incubated for 24 h with Laser ablated Albumin functionalized spherical AuNPs (LA-AuNPs), with average sizes of 2 nm and 53 nm in diameter, in four concentrations, 127 µM, 84 µM, 42 µM, and 23 µM. Cytotoxicity was examined by Live/Dead assay, and erythrocyte hemolysis, and the effect on the cytoskeleton was investigated by immunocytochemistry for ß-actin. The LA-AuNPs were internalized by the ASCs in a size and concentration-dependent manner. Clusters were observed as dispersed small ones in the cytosol, and as a sizeable perinuclear cluster, without significant harmful effects on the cells for up to 2 weeks. The Live/Dead and hemolysis percentage results complemented the observations that the larger 53 nm LA-AuNPs in the highest concentrated solution significantly lowered cell viability. The demonstrated safety, cellular uptake, and labelling persistency with LA-AuNPs, synthesized without the combination of chemical solutions, support their use for cell tracking in tissue engineering applications.

The Neural Multilineage Differentiation Capacity of Human Neural Precursors from the Umbilical Cord-Ready to Bench for Clinical Trials.

Mesenchymal stem cells (MSC) are promising for regenerative medicine as they have a vast differentiation capacity, immunomodulatory properties and can be isolated from different tissues. Among them, the umbilical cord is considered a good source of MSC, as its collection poses no risk to donors and is unrelated to ethical issues. Furthermore, umbilical cord mesenchymal stem cells (UC-MSC) can differentiate into several cell lines, including neural lineages that, in the future, may become an alternative in the treatment of neurodegenerative diseases. This study used a natural functional biopolymer matrix (NFBX) as a membrane to differentiate UC-MSC into neurospheres and their Neural precursors without using neurogenic growth factors or gene transfection. Through the characterization of Neural precursors and differentiated cells, it was possible to demonstrate the broad potential for the differentiation of cells obtained through cultivation on this membrane. To demonstrate these Neural precursors' potential for future studies in neurodegenerative diseases, the Neural precursors from Wharton's jelly were differentiated into Schwann cells, oligodendrocytes, cholinergic-, dopaminergic- and GABAergic-like neurons.

Biodegradable Nanoparticles Loaded with Levodopa and Curcumin for Treatment of Parkinson's Disease.

Background: Parkinson's disease (PD) is the second most common age-related neurodegenerative disorder. Levodopa (L-DOPA) remains the gold-standard drug available for treating PD. Curcumin has many pharmacological activities, including antioxidant, anti-inflammatory, antimicrobial, anti-amyloid, and antitumor properties. Copolymers composed of Poly (ethylene oxide) (PEO) and biodegradable polyesters such as Poly (ε-caprolactone) (PCL) can self-assemble into nanoparticles (NPs). This study describes the development of NH2-PEO-PCL diblock copolymer positively charged and modified by adding glutathione (GSH) on the outer surface, resulting in a synergistic delivery of L-DOPA curcumin that would be able to pass the blood-brain barrier. Methods: The NH2-PEO-PCL NPs suspensions were prepared by using a nanoprecipitation and solvent displacement method and coated with GSH. NPs were submitted to characterization assays. In order to ensure the bioavailability, Vero and PC12 cells were treated with various concentrations of the loaded and unloaded NPs to observe cytotoxicity. Results: NPs have successfully loaded L-DOPA and curcumin and were stable after freeze-drying, indicating advancing into in vitro toxicity testing. Vero and PC12 cells that were treated up to 72 h with various concentrations of L-DOPA and curcumin-loaded NP maintained high viability percentage, indicating that the NPs are biocompatible. Conclusions: NPs consisting of NH2-PEO-PCL were characterized as potential formulations for brain delivery of L-DOPA and curcumin. The results also indicate that the developed biodegradable nanomicelles that were blood compatible presented low cytotoxicity.

Potential of Human Neural Precursor Cells in Diabetic Retinopathy Therapeutics - Preclinical Model.

PURPOSE: This study aimed to evaluate a cell therapy strategy with human neural precursor cells (hNPCs) to treat diabetic retinopathy (DR) in Wistar rats induced to diabetes by injecting streptozotocin. MATERIAL AND METHODS: The Wharton's jelly mesenchymal stem cells (WJ-MSCs) were isolated, expanded, and seeded onto a biopolymer substrate to develop neurospheres and obtain the hNPCs. The animals were divided into three groups: non-diabetic (ND) n = four, diabetic without treatment (DM) n = nine, and diabetic with cell therapy (DM + hNPCs) n = nine. After 8 weeks of diabetes induction and DR characteristics installed, intravitreal injection of hNPCs (1 × 106 cell/µL) was performed in the DM + hNPCs group. Optical Coherence Tomography (OCT) and Electroretinography (ERG) evaluations were conducted before and during diabetes and after cell therapy. Four weeks posttreatment, histopathological and immunohistochemistry analyses were performed. RESULTS: The repair of the retinal structures in the treated group (DM + hNPCs) was observed by increased thickness of neuroretinal layers, especially in the ganglion cell and photoreceptor layers, higher ERG oscillatory potentials (OPs) amplitudes, and transplanted hNPCs integration into the Retinal Pigment Epithelium. CONCLUSIONS: The results indicate that hNPCs reduced DR progression by a neuroprotective effect and promoted retinal repair, making them potential candidates for regenerating the neuroretinal tissue.

Natural Membrane Differentiates Human Adipose-Derived Mesenchymal Stem Cells to Neurospheres by Mechanotransduction Related to YAP and AMOT Proteins.

Adipose tissue-derived mesenchymal stem cells (ADMSCs) are promising candidates for regenerative medicine, as they have good cell yield and can differentiate into several cell lines. When induced to the neuronal differentiation, they form neurospheres composed of neural precursors (NPs) that can be an alternative in treating neurodegenerative diseases. This study aimed to characterize NPs from neurospheres obtained after seeding ADMSCs on a natural polyisoprene-based membrane. The ADMSCs were isolated from adipose tissue by enzymatic dissociation, were subjected to trilineage differentiation, and were characterized by flow cytometry for specific ADMSC surface markers. For neuronal differentiation, the cells were seeded on polystyrene flasks coated with the membrane and were characterized by immunocytochemistry and RT-PCR. The results demonstrated that the isolated cells showed characteristics of ADMSCs. At 15 to 25 days, ADMSCs seeded on the natural membrane developed neurospheres. Then, after dissociation, the cells demonstrated characteristic neuronal markers expressed on NPs: nestin, ß-III tubulin, GFAP, NeuN, and the YAP1/AMOT in the cytoplasm. In conclusion, it was demonstrated that this membrane differentiates the ADMSCs to NPs without any induction factors, and suggests that their differentiation mechanisms are related to mechanotransduction regulated by the YAP and AMOT proteins.

Human Mesenchymal Stem Cells Seeded on the Natural Membrane to Neurospheres for Cholinergic-like Neurons.

This study aimed to differentiate human mesenchymal stem cells (hMSCs) from the human umbilical cord in cholinergic-like neurons using a natural membrane. The isolation of hMSCs from Wharton's jelly (WJ) was carried out using "explant" and mononuclear cells by the density gradient from umbilical blood and characterized by flow cytometry. hMSCs were seeded in a natural functional biopolymer membrane to produce neurospheres. RT-PCR was performed on hMSCs and neurospheres derived from the umbilical cord. Neural precursor cells were subjected to a standard cholinergic-like neuron differentiation protocol. Dissociated neurospheres, neural precursor cells, and cholinergic-like neurons were characterized by immunocytochemistry. hMSCs were CD73+, CD90+, CD105+, CD34- and CD45- and demonstrated the trilineage differentiation. Neurospheres and their isolated cells were nestin-positive and expressed NESTIN, MAP2, ßIII-TUBULIN, GFAP genes. Neural precursor cells that were differentiated in cholinergic-like neurons expressed ßIII-TUBULIN protein and choline acetyltransferase enzyme. hMSCs seeded on the natural membrane can differentiate into neurospheres, obtaining neural precursor cells without growth factors or gene transfection before cholinergic phenotype differentiation.

Adipose-Derived Stromal Cells and Mineralized Extracellular Matrix Delivery by a Human Decellularized Amniotic Membrane in Periodontal Tissue Engineering.

Periodontitis is a prevalent disease characterized by the loss of periodontal supporting tissues, bone, periodontal ligament, and cementum. The application of a bone tissue engineering strategy with Decellularized Human Amniotic Membrane (DAM) with adipose-derived stromal cells (ASCs) has shown to be convenient and valuable. This study aims to investigate the treatments of a rat periodontal furcation defect model with DAM, ASCs, and a mineralized extracellular matrix (ECM). Rat ASCs were expanded, cultivated on DAM, and with a bone differentiation medium for four weeks, deposited ECM on DAM. Periodontal healing for four weeks was evaluated by micro-computed tomography and histological analysis after treatments with DAM, ASCs, and ECM and compared to untreated defects on five consecutive horizontal levels, from gingival to apical. The results demonstrate that DAM preserves its structure during cultivation and healing periods, supporting cell attachment, permeation, bone deposition on DAM, and periodontal regeneration. DAM and DAM+ASCs enhance bone healing compared to the control on the gingival level. In conclusion, DAM with ASC or without cells and the ECM ensures bone tissue healing. The membrane supported neovascularization and promoted osteoconduction.

Combined Biomaterials: Amniotic Membrane and Adipose Tissue to Restore Injured Bone as Promoter of Calcification in Bone Regeneration: Preclinical Model.

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.