Regulatory mechanisms of stem cell differentiation: Biotechnological applications for neurogenesis.

The world population's life expectancy is growing, and neurodegenerative disorders common in old age require more efficient therapies. In this context, neural stem cells (NSCs) are imperative for the development and maintenance of the functioning of the nervous system and have broad therapeutic applicability for neurodegenerative diseases. Therefore, knowing all the mechanisms that govern the self-renewal, differentiation, and cell signaling of NSC is necessary. This review will address some of these aspects, including the role of growth and transcription factors, epigenetic modulators, microRNAs, and extracellular matrix components. Furthermore, differentiation and transdifferentiation processes will be addressed as therapeutic strategies showing their significance for stem cell-based therapy.

Strategic use of organoids and organs-on-chip as biomimetic tools.

Organoid development and organ-on-a-chip are technologies based on differentiating stem cells, forming 3D multicellular structures resembling organs and tissues in vivo. Hence, both can be strategically used for disease modeling, drug screening, and host-pathogen studies. In this context, this review highlights the significant advancements in the area, providing technical approaches to organoids and organ-on-a-chip that best imitate in vivo physiology.

Drugs and Endogenous Factors as Protagonists in Neurogenic Stimulation.

Neurogenesis is a biological process characterized by new neurons formation from stem cells. For decades, it was believed that neurons only multiplied during development and in the postnatal period but the discovery of neural stem cells (NSCs) in mature brain promoted a revolution in neuroscience field. In mammals, neurogenesis consists of migration, differentiation, maturation, as well as functional integration of newborn cells into the pre-existing neuronal circuit. Actually, NSC density drops significantly after the first stages of development, however in specific places in the brain, called neurogenic niches, some of these cells retain their ability to generate new neurons and glial cells in adulthood. The subgranular (SGZ), and the subventricular zones (SVZ) are examples of regions where the neurogenesis process occurs in the mature brain. There, the potential of NSCs to produce new neurons has been explored by new advanced methodologies and in neuroscience for the treatment of brain damage and/or degeneration. Based on that, this review highlights endogenous factors and drugs capable of stimulating neurogenesis, as well as the perspectives for the use of NSCs for neurological and neurodegenerative diseases.

Mouse Neural Stem Cell Differentiation and Human Adipose Mesenchymal Stem Cell Transdifferentiation Into Neuron- and Oligodendrocyte-like Cells With Myelination Potential.

Stem cell therapy is an interesting approach for neural repair, once it can improve and increase processes, like angiogenesis, neurogenesis, and synaptic plasticity. In this regard, adult neural stem cells (NSC) are studied for their mechanisms of proliferation, differentiation and functionality in neural repair. Here, we describe novel neural differentiation methods. NSC from adult mouse brains and human adipose-derived stem cells (hADSC) were isolated and characterized regarding their neural differentiation potential based on neural marker expression profiles. For both cell types, their capabilities of differentiating into neuron-, astrocyte- and oligodendrocytes-like cells (NLC, ALC and OLC, respectively) were analyzed. Our methodologies were capable of producing NLC, ALC and OLC from adult murine and human transdifferentiated NSC. NSC showed augmented gene expression of NES, TUJ1, GFAP and PDGFRA/Cnp. Following differentiation induction into NLC, OLC or ALC, specific neural phenotypes were obtained expressing MAP2, GalC/O4 or GFAP with compatible morphologies, respectively. Accordingly, immunostaining for nestin+ in NSC, GFAP+ in astrocytes and GalC/O4+ in oligodendrocytes was detected. Co-cultured NLC and OLC showed excitability in 81.3% of cells and 23.5% of neuron/oligodendrocyte marker expression overlap indicating occurrence of in vitro myelination. We show here that hADSC can be transdifferentiated into NSC and distinct neural phenotypes with the occurrence of neuron myelination in vitro, providing novel strategies for CNS regeneration therapy. Superior Part: Schematic organization of obtaining and generating hNSC from hADSC and differentiation processes and phenotypic expression of neuron, astrocyte and oligodendrocyte markers (MAP2, GFAP and O4, respectively) and stem cell marker (NES) of differentiating hNSC 14 days after induction. The nuclear staining in blue corresponds to DAPI. bar = 100 µm. Inferior part: Neural phenotype fates in diverse differentiation media. NES: nestin; GFAP: Glial fibrillary acidic protein. MAP2: Microtubule-associated protein 2. TUJ1: ß-III tubulin. PDGFRA: PDGF receptor alpha. Two-way ANOVA with Bonferroni post-test with n = 3. * p < 0.05 and ** p < 0.01: (NSCiM1 NSC induction medium 1) vs differentiation media.

Decoding epigenetic cell signaling in neuronal differentiation.

Neurogenesis is the process by which new neurons are generated in the brain. Neural stem cells (NSCs) are differentiated into neurons, which are integrated into the neural network. Nowadays, pluripotent stem cells, multipotent stem cells, and induced pluripotent stem cells can be artificially differentiated into neurons utilizing several techniques. Specific transcriptional profiles from NSCs during differentiation are frequently used to approach and observe phenotype alteration and functional determination of neurons. In this context, the role of non-coding RNA, transcription factors and epigenetic changes in neuronal development and differentiation has gained importance. Epigenetic elucidation has become a field of intense research due to distinct patterns of normal conditions and different neurodegenerative disorders, which can be explored to develop new diagnostic methods or gene therapies. In this review, we discuss the complexity of transcription factors, non-coding RNAs, and extracellular vesicles that are responsible for guiding and coordinating neural development.

The role of neurogenesis in neurorepair after ischemic stroke.

Stroke consists of an abrupt reduction of cerebral blood flow resulting in hypoxia that triggers an excitotoxicity, oxidative stress, and neuroinflammation. After the ischemic process, neural precursor cells present in the subventricular zone of the lateral ventricle and subgranular zone of the dentate gyrus proliferate and migrate towards the lesion, contributing to the brain repair. The neurogenesis is induced by signal transduction pathways, growth factors, attractive factors for neuroblasts, transcription factors, pro and anti-inflammatory mediators and specific neurotransmissions. However, this endogenous neurogenesis occurs slowly and does not allow a complete restoration of brain function. Despite that, understanding the mechanisms of neurogenesis could improve the therapeutic strategies for brain repair. This review presents the current knowledge about brain repair process after stroke and the perspectives regarding the development of promising therapies that aim to improve neurogenesis and its potential to form new neural networks.

Neural stem cell differentiation into mature neurons: Mechanisms of regulation and biotechnological applications.

The abilities of stem cells to self-renew and form different mature cells expand the possibilities of applications in cell-based therapies such as tissue recomposition in regenerative medicine, drug screening, and treatment of neurodegenerative diseases. In addition to stem cells found in the embryo, various adult organs and tissues have niches of stem cells in an undifferentiated state. In the central nervous system of adult mammals, neurogenesis occurs in two regions: the subventricular zone and the dentate gyrus in the hippocampus. The generation of the different neural lines originates in adult neural stem cells that can self-renew or differentiate into astrocytes, oligodendrocytes, or neurons in response to specific stimuli. The regulation of the fate of neural stem cells is a finely controlled process relying on a complex regulatory network that extends from the epigenetic to the translational level and involves extracellular matrix components. Thus, a better understanding of the mechanisms underlying how the process of neurogenesis is induced, regulated, and maintained will provide elues for development of novel for strategies for neurodegenerative therapies. In this review, we focus on describing the mechanisms underlying the regulation of the neuronal differentiation process by transcription factors, microRNAs, and extracellular matrix components.

Expression System Based on an MTIIa Promoter to Produce hPSA in Mammalian Cell Cultures.

Because of the limitations of standard culture techniques, the development of new recombinant protein expression systems with biotechnological potential is a key challenge. Ideally, such systems should be able to effectively and accurately synthesize a protein of interest with intrinsic metabolic capacity. Here, we describe such a system that was designed based on a plasmid vector containing promoter elements derived from the metallothionein MTIIa promoter, as well as processing and purification elements. This promoter can be induced by heavy metals in a culture medium to induce the synthesis of human prostate-specific antigen (hPSA), which has been modified to insert elements for purification, proteolysis, and secretion. We optimized hPSA production in this system by comparing the effects and contributions of ZnCl2, CdCl2, and CuSO4 in HEK293FT, HeLa, BHK-21, and CHO-K1 cells. We also compared the effectiveness of three different transfection agents: multi-walled carbon nanotubes, Lipofectamine 2000, and X-tremeGENE HP Reagent. hPSA production was confirmed via the detection of enhanced green fluorescent protein fluorescence, and cell viability was determined. The expression of hPSA was compared with that of the native protein produced by LNCaP cells, using enzyme-linked immunosorbent assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis. X-tremeGENE reagent, the BHK-21 cell line, and CuSO4 showed the highest hPSA production rates. Furthermore, BHK-21 cells were more resistant to the oxidative stress caused by 100 µM CuSO4. These results suggest that the proposed optimized inducible expression system can effectively produce recombinant proteins with desired characteristics for a wide range of applications in molecular biology.

Exploring the cell signalling in hepatocyte differentiation.

The liver is the second largest organ in the human body and is responsible for several functions that directly contribute to homeostasis. Hepatocytes are the main parenchymal liver cells that regulate multiple biochemical and metabolic functions and the synthesis of substances important to the body. Mesenchymal stem cells (MSCs) are a group of stem cells derived from the mesoderm, which can be obtained from various tissues. Under certain conditions, MSCs can differentiate into several cell types, including hepatocytes. Post-transcriptional regulations of liver development signalling and hepatocyte differentiation have been demonstrated. At the post-transcriptional level, microRNAs have emerged as precursors for determining cell fate during differentiation. MicroRNAs (miRNAs) are small non-coding RNAs involved in the post-transcriptional regulation of gene expression. They can determine the stem cell fate by repressing the translation of target mRNAs. In this review, we outline signalling pathways involved in stem cell differentiation to hepatocytes and its interplay with liver development. Hepatic differentiation models in two-dimensional and three-dimensional cultures used to analyse signalling mechanisms will be described. We also highlight the possible miRNAs involved in this process and the transdifferentiation signalling mechanisms present in hepatocytes.

Human adult stem cells from diverse origins: an overview from multiparametric immunophenotyping to clinical applications.

Stem cells are known for their capacity to self-renew and differentiate into at least one specialized cell type. Mesenchymal stem cells (MSCs) were isolated initially from bone marrow but are now known to exist in all vascularized organ or tissue in adults. MSCs are particularly relevant for therapy due to their simplicity of isolation and cultivation. The International Society for Cellular Therapy (ISCT) has proposed a set of standards to define hMSCs for laboratory investigations and preclinical studies: adherence to plastic in standard culture conditions; in vitro differentiation into osteoblasts, adipocytes, and chondroblasts; specific surface antigen expression in which ≥95% of the cells express the antigens recognized by CD105, CD73, and CD90, with the same cells lacking (≤2% positive) the antigens CD45, CD34, CD14 or CD11b, CD79a or CD19, and HLA-DR. In this review we will take an historical overview of how umbilical cord blood, bone marrow, adipose-derived, placental and amniotic fluid, and menstrual blood stem cells, the major sources of human MSC, can be obtained, identified and how they are being used in clinical trials to cure and treat a very broad range of conditions, including heart, hepatic, and neurodegenerative diseases. An overview of protocols for differentiation into hepatocytes, cardiomyocytes, neuronal, adipose, chondrocytes, and osteoblast cells are highlighted. We also discuss a new source of stem cells, induced pluripotent stem cells (iPS cells) and some pathways, which are common to MSCs in maintaining their pluripotent state.