Improvement of transfection with reprogramming factors in urinederived cells

Human-induced pluripotent stem cells (iPSCs) are an accessible source of adult-derived, patient-specific pluripotent stem cells for use in basic research, drug discovery, disease modeling, and stem cell therapy. Improving the accessibility of methods to obtain iPSCs regardless of the cell source can enhance their clinical application. Therefore, our purpose is to report a simple protocol to obtain iPS-like cells from urine-derived renal epithelial cells (RECs) using different extracellular matrices and transfection reagents. In this study, we began by culturing urine-derived cells from healthy donors to establish a primary culture of renal epithelial cells, followed by their characterization. Subsequently, we generated iPS-like cells by transfecting renal epithelial cells (RECs) with vectors expressing Oct4, Sox2, L-Myc, Lin-28, and Klf4, and we compared the efficacy of different extracellular matrices and transfection reagents. The resultant iPS-like cells showed a human embryonic stem cell-like morphology and expressed the specific pluripotency markers Oct3/4, Nanog, Lin28, and Klf4. We concluded that Lipofectamine Stem Cell transfection reagent is more effective than FuGENE in obtaining iPS-like cells under the conditions tested. Moreover, the three matrices are similar in their efficiency of obtaining iPS-like cells. This report provides an experimental protocol for obtaining and generating iPS-like cells from urine samples for further cell therapy research on different human diseases.     

Determination of chloride efflux by X-ray microanalysis versus MQAE-fluorescence

The importance of chloride channels for the cell is demonstrated by a number of serious human diseases that are due to mutations in chloride channels. The most well-known of these diseases is cystic fibrosis. Investigations into the mechanisms of the disease and possible treatments require the study of chloride fluxes at the level of individual cells. The present study compares two methods for studies of chloride transport: X-ray microanalysis and MQAE fluorescence with image analysis. As an experimental system, the cAMP-activated chloride channel in cultured respiratory epithelial cells was chosen. Both methods showed that stimulation with the cAMP-elevating agents forskolin and IBMX decreased the chloride content of the cells by about 20-27%. Inducing a driving force for chloride by replacing extracellular chloride by nitrate resulted in a chloride efflux that was significantly increased in the presence of forskolin and IBMX. This study shows that X-ray microanalysis and MQAE fluorescence are adequate and comparable methods for measuring cAMP-dependent chloride transport in individual cells.     

From theory to therapy: implications from an in vitro model of ramified microglia

Microglia are the principal immune cells in the central nervous system (CNS), characterized by a highly specific morphology and unusual antigenic phenotype. An increasing number of studies have focused on the role of microglia in the pathogenesis of neurodegenerative diseases. To elucidate the function of microglial cells under several neuropathological conditions, we have studied and established a cell culture model that allows us to cultivate microglial cells in their inactive, resting (ramified) phenotype. In the first part of this work, we describe the interaction of microglia cells with their epithelial (astrocytic) microenvironment. The second part reviews experiments with microglia cell cultures to elucidate underlying signalling pathways and summarizes recent advances of our knowledge in microglial molecular pathways that may ultimately lead to neurodegeneration.     

分子影像技术活体追踪移植干细胞的研究进展

干细胞具有分化、再生能力,通过体外扩增和体内移植,可以治疗各种组织坏损和退化性疾病(如心脑血管疾病、脑脊髓外伤和糖尿病等),具有极大的应用前景,是目前国际、国内的研究热点。利用核素显像、磁共振成像和光成像等分子影像技术,通过体外直接标记、报告基因或功能显示等追踪策略,可以显示干细胞在活体内的分布和变化,明确其最终归宿和产生的功能。合理选择这些分子影像技术和追踪策略,或通过互补结合,将有助于阐明干细胞在活体内的作用机制和相关的影响因素,指导临床干细胞治疗抉择和疗效评估。     

Comparative characterization of human fetal neural stem cells and induced neural stem cells from peripheral blood mononuclear cells

Human-induced neural stem cells (iNSCs) transplantation is a potential treatment of neurodegeneration diseases. However, whether the reprogrammed cells have the same characterizations as human fetal neural stem cells needs further exploration. Here we isolated human fetal neural stem cells from aborted 12-week fetal brains and compared with iNSCs reprogrammed from human peripheral blood mononuclear cells in gene expression, proliferation ability, differentiation capacity, and the responses to tumor necrosis factor-α. We found that iNSCs and NSCs both expressed neural stem cell markers Nestin, SOX1, and SOX2. However, only iNSCs can be patterned into dopaminergic neurons and motor neurons. Furthermore, both iNSCs and NSCs can differentiate into oligodendrocyte progenitor cells. In addition, a low dose of tumor necrosis factor-α did not inhibit the proliferation and differentiation of iNSCs and NSCs. In conclusion, iNSCs have properties similar to, and even better than, fetal neural stem cells and may be suitable for disease modeling and transplantation.     

Phospholipids as cancer biomarkers: Mass spectrometry‐based analysis

Lipids, particularly phospholipids (PLs), are key components of cellular membrane. PLs play important and diverse roles in cells such as chemical‐energy storage, cellular signaling, cell membranes, and cell–cell interactions in tissues. All these cellular processes are pertinent to cells that undergo transformation, cancer progression, and metastasis. Thus, there is a strong possibility that some classes of PLs are expected to present in cancer cells and tissues in cellular physiology. The mass spectrometric soft‐ionization techniques, electrospray ionization (ESI), and matrix‐assisted laser desorption/ionization (MALDI) are well‐established in the proteomics field, have been used for lipidomic analysis in cancer research. This review focused on the applications of mass spectrometry (MS) mainly on ESI‐MS and MALDI‐MS in the structural characterization, molecular composition and key roles of various PLs present in cancer cells, tissues, blood, and urine, and on their importance for cancer‐related problems as well as challenges for development of novel PL‐based biomarkers. The profiling of PLs helps to rationalize their functions in biological systems, and will also provide diagnostic information to elucidate mechanisms behind the control of cancer, diabetes, and neurodegenerative diseases. The investigation of cellular PLs with MS methods suggests new insights on various cancer diseases and clinical applications in the drug discovery and development of biomarkers for various PL‐related different cancer diseases. PL profiling in tissues, cells and body fluids also reflect the general condition of the whole organism and can indicate the existence of cancer and other diseases. PL profiling with MS opens new prospects to assess alterations of PLs in cancer, screening specific biomarkers and provide a basis for the development of novel therapeutic strategies. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 37:107‐138, 2018     

MSCs derived extracellular vesicles as a therapeutic paragon for neurodegenerative disorders: A viewpoint

Neurodegenerative disorders are a vicious woe to the public health and wellness. Uncertainty in their underlying causes, lack of effective biomarkers for their early detection, existence of only supportive therapy, and their ever rising incidence creates an unmatched need for targeted therapies. Mesenchymal Stem Cells (MSCs) have found to be promising candidates for regenerative and remedial therapy in neurodegenerative disorders, however several biological risks and practical issues impede in their translational utility. Deriving from MSCs are certain Extracellular Vesicles (EVs), which aid in the paracrine action of MSCs and have lately gained the scientific interest for their implacability in diverse set ups. Their cargo is of utmost importance and is being explored in various different diseases like heart diseases, neuronal diseases, respiratory diseases and hepatic diseases. They thereby hold the position of a likely prospective remedial candidate for therapy against neurodegenerative disorders.     

Role of GM3 ganglioside in the pathology of some progressive human diseases and prognostic importance of serum anti-GM3 antibodies

Glycosphingolipids (gangliosides) have been characterized as important biological molecules with a key role as regulators in many physiological processes on cellular, tissue, organ, and organism levels. The deviations in their normal amounts, production, and metabolism are very often related to the development of many multi-factor socially important diseases. GM3 ganglioside, as a small molecule, plays important roles in the cascade regulatory pathways in the pathology of many disorders like neurodegenerative diseases, autoimmune diseases, inflammation, diabetes, malignant transformation, and others. Ganglioside GM3 and its derivatives are membrane-bound glycosphingolipids composed of an oligosaccharide head structure containing one sialic acid residue. These molecules transduce signals involved in cell surface events, including the phosphorylation of transmembrane receptors. This ganglioside is the most widely distributed among tissues, and it serves as a precursor for most of the more complex ganglioside species. GM3 inhibits the function of fibroblast growth factor receptor, and cell growth is regulated by GM3-enriched microdomain. GM3 is thought to inhibit immunologic functions, such as the proliferation and production of cytokines by T cells. On the other hand, the anti-ganglioside antibodies (AGAs) are important in many acquired demyelinating immunemediated neuropathies, like Multiple sclerosis (MS), Guillain–Barré syndrome (GBS) and its variation, Miller–Fisher syndrome (MFS) and could be suggested as important diagnostic and prognostic markers about the describe diseases and their etiology. We show that the complexes of anti-ganglioside antibodies to GM3 (detected by ELISA) may be useful diagnostic and prognostic tool markers for autoimmune diseases, neurodegenerative disorders, malignancy, diabetes, and inflammation. Our pilot studies suggest increased serum IgG anti-GM3 antibodies titers in patients with secondary progressive MS (SPMS), throat cancer, elder people with diabetes (89–96 years), old Lewis rats (30–33 months), and in the serum of subjected on lead intoxication BALB/c mice treated by salinomycin. We observed no changes in the titers in healthy elder people (89–96 years), in 70-year-old woman on dialysis, in relapsing-remitting MS (RRMS) patients on long-term treatment with Glatiramer acetate, Laquinimod, and Interferons, as well as in 18– 22 months old Wistar rats and subjected on lead intoxication BALB/c mice treated by monensin and dimercaptosuccinic acid (DMSA). Considerable decrease of serum GM3 in early MS correlate with early damage and severe destruction of the blood–brain barrier, which provides impetus to initiate early therapy.     

Mesenchymal stem cell-derived exosomes as new remedy for the treatment of inflammatory eye diseases

Detrimental immune response has a crucially important role in the development and progression of inflammatory eye diseases. Inflammatory mediators and proteolytic enzymes released by activated immune cells induce serious injury of corneal epithelial cells and retinal ganglion cell which may result in the vision loss. Mesenchymal stem cells (MSCs) are regulatory cells which produce various immunosuppressive factors that modulate phenotype and function of inflammatory immune cells. However, several safety issues, including undesired differentiation and emboli formation, limit clinical use of MSCs. MSC-derived exosomes (MSC-Exos) are nano-sized extracellular vesicles which contain all MSC-derived immunoregulatory factors. Intraocular administration of MSC-Exos efficiently attenuated eye inflammation and significantly improved visual acuity in experimental animals without causing any severe side effects. As cell-free product, MSC-Exos addressed all safety issues related to the transplantation of MSCs. Therefore, MSC-Exos could be considered as potentially new remedy for the treatment of inflammatory eye diseases which efficacy should be explored in up-coming clinical trials.     

Dental pulp stem cells and banking of teeth as a lifesaving therapeutic vista

Exfoliated deciduous or an extracted healthy adult tooth can be used to harvest, process, and cryogenically preserve dental pulp stem cells. Future stem cell-based regenerative medicine methods could benefit significantly from these mesenchymal stem cells. Teeth serve as a substantial source of mesenchymal stem cells, otherwise disposed of as medical waste. Care should be taken to store this treasure trove of stem cells. Collective responsibility of patients, dentists, and physicians is necessary to ensure that this valuable resource is not wasted and that every possible dental pulp stem cell is available for use in the future. The dental pulp stem cells (DPSC) inside teeth represent a significant future source of stem cells for regenerative medicine procedures. This review describes the ontogeny, the laboratory processing and collection, and isolation methods of DPSC. This review also discusses currently available stem cell banking facilities and their potential use in regenerative medicine procedures in dental and general medical applications in the future.     

S100B in brain damage and neurodegeneration

S100B is a calcium-binding peptide produced mainly by astrocytes that exert paracrine and autocrine effects on neurons and glia. Some knowledge has been acquired from in vitro and in vivo animal experiments to understand S100B's roles in cellular energy metabolism, cytoskeleton modification, cell proliferation, and differentiation. Also, insights have been gained regarding the interaction between S100B and the cerebral immune system, and the regulation of S100B activity through serotonergic transmission. Secreted glial S100B exerts trophic or toxic effects depending on its concentration. At nanomolar concentrations, S100B stimulates neurite outgrowth and enhances survival of neurons during development. In contrast, micromolar levels of extracellular S100B in vitro stimulate the expression of proinflammatory cytokines and induce apoptosis. In animal studies, changes in the cerebral concentration of S100B cause behavioral disturbances and cognitive deficits. In humans, increased S100B has been detected with various clinical conditions. Brain trauma and ischemia is associated with increased S100B concentrations, probably due to the destruction of astrocytes. In neurodegenerative, inflammatory and psychiatric diseases, increased S100B levels may be caused by secreted S100B or release from damaged astrocytes. This review summarizes published findings on S100B regarding human brain damage and neurodegeneration. Findings from in vitro and in vivo animal experiments relevant for human neurodegenerative diseases and brain damage are reviewed together with the results of studies on traumatic, ischemic, and inflammatory brain damage as well as neurodegenerative and psychiatric disorders. Methodological problems are discussed and perspectives for future research are outlined.     

Stem cells in intervertebral disc regeneration–more talk than action?

Pain and lifestyle changes are common consequences of intervertebral disc degeneration (IVDD) and affect a large part of the aging population. The stemness of cells is exploited in the field of regenerative medicine as key to treat degenerative diseases. Transplanted cells however often face delivery and survival challenges, especially in tissues with a naturally harsh microniche environment such as the intervertebral disc. Recent interest in the secretome of stem cells, especially cargo protected from microniche-related decay as frequently present in degenerating tissues, provides new means of rejuvenating ailing cells and tissues. Exosomes, a type of extracellular vesicles with purposeful cargo gained particular interest in conveying stem cell related attributes of rejuvenation, which will be discussed here in the context of IVDD.     

Proteomics in neurosciences

This review provides an outline of the most important proteomic applications in the study of neurodegenerative disorders including Alzheimer's (AD), Parkinson's (PD), Huntington's (HD), and prion diseases, and also discusses advances in cancer and addiction. One of the scopes is to illustrate the potential of proteomics in the biomarkers discovery of these diseases. Finally, this article comments the advantages and drawbacks of the most commonly used techniques and methods for samples preparation.     

Optimization of culture conditions for an efficient xeno‐feeder free limbal cell culture system towards ocular surface regeneration

Ex vivo expansion of limbal stem cells from a small biopsy and its subsequent transplantation is the golden choice of treatment for limbal stem cell deficiency. Use of murine 3T3 feeder layer is a prerequisite for this ex vivo expansion. There is an ever‐increasing demand for feeder free cultures to avoid xenotoxicity and transmission of xeno‐diseases to human system. This study was aimed to establish an efficient xeno‐feeder free limbal culture system towards ocular surface regeneration. To study the effect of initial dispase treatment and culture system used, migratory distance of cells from explants was analyzed from phase contrast images using “interactive measurements” of Qwin software (Leica). Expression of p63 in different culture systems was studied by immunofluorescent staining, followed by quantitative confocal microscopy (Carl Zeiss). Results showed dispase treatment was not necessary for establishing limbal explant culture. A combination of Iscove's modified Dulbecco's medium and Panserin 801 resulted in formation of autofeeder layer with maintenance of progenitor characteristics, thus mimicking natural tissue architecture. Further analysis of this culture system showed that cells could be cultured till confluency. Immunofluorescent staining of ABCG2 revealed presence of stem cell marker in the confluent cell layer. Scanning Electron Micrographs demonstrated homogenous population of tightly packed cells in this culture system. Replacement of bovine serum with autologous serum did not affect morphology or growth of cells in this culture system. This study will be a major step in the development of xeno‐feeder free epithelial equivalents towards ocular surface reconstruction. Microsc. Res. Tech. 73:1045–1052, 2010. © 2010 Wiley‐Liss, Inc.     

Mesenchymal stem cells derived secretome as an innovative cell-free therapeutic approach

The paracrine and immunomodulatory cytokines secreted by mesenchymal stem cells (MSCs), generally referred to as the MSCs derived secretome, has substantial potential for the treatment of many chronic and degenerative diseases. MSCs secretome contains both common and disease specific cytokines and modulators that can be beneficial against a wide range of chronic diseases. Herein, we discuss the MSCs secretome composition profile and its translational applicability and the challenges surrounding its use in clinical settings.     

Glycoproteomics in neurodegenerative diseases

Protein glycosylation regulates protein function and cellular distribution. Additionally, aberrant protein glycosylations have been recognized to play major roles in human disorders, including neurodegenerative diseases. Glycoproteomics, a branch of proteomics that catalogs and quantifies glycoproteins, provides a powerful means to systematically profile the glycopeptides or glycoproteins of a complex mixture that are highly enriched in body fluids, and therefore, carry great potential to be diagnostic and/or prognostic markers. Application of this mass spectrometry‐based technology to the study of neurodegenerative disorders (e.g., Alzheimer's disease and Parkinson's disease) is relatively new, and is expected to provide insight into the biochemical pathogenesis of neurodegeneration, as well as biomarker discovery. In this review, we have summarized the current understanding of glycoproteins in biology and neurodegenerative disease, and have discussed existing proteomic technologies that are utilized to characterize glycoproteins. Some of the ongoing studies, where glycoproteins isolated from cerebrospinal fluid and human brain are being characterized in Parkinson's disease at different stages versus controls, are presented, along with future applications of targeted validation of brain specific glycoproteins in body fluids. © 2009 Wiley Periodicals, Inc., Mass Spec Rev 29:79–125, 2010     

Precise tissue bioengineering and niches of mesenchymal stem cells: Their size and hierarchy matter

Stem cell microterritories (niches), as a specialized part of the extracellular matrix (ECM), are considered an important target and tool for the development of new materials, medical implants, and devices. However, tissue bioengineering products that have stem cell niches of known size on the surface or in the bulk structure of artificial materials are practically unknown. This brief review attempts to draw attention to the problematic aspects of niches as specific parts of the ECM, such as their hierarchy and size for mesenchymal stromal/stem cells (MSCs). These parameters arise directly from numerous definitions of stem cell niches as specialized morphological microterritories found in various tissues. The authors of this review analyze the known information on the hierarchy of MSC microterritories by analogy with that of hematopoietic stem cells. Occasional reports on the size of artificial MSC niches compared to natural niche candidates are summarized. A consensus on a hierarchy and optimal range of niche sizes for MSCs and other stem cells is needed to accelerate the development of prototyping technologies and additive manufacturing in applications to precise tissue bioengineering and regenerative medicine.     

Neural stem cell-conditioned medium upregulated the PCMT1 expression and inhibited the phosphorylation of MST1 in SH-SY5Y cells induced by Aβ_25-35

A progressive neurodegenerative disease, Alzheimer’s disease (AD). Studies suggest that highly expressed protein isoaspartate methyltransferase 1 (PCMT1) in brain tissue. In the current study, we explored the effects of neural stem cell-conditioned medium (NSC-CDM) on the PCMT1/MST1 pathway to alleviate Aβ_25-35-induced damage in SH-SY5Y cells. Our data suggested that Aβ25-35 markedly inhibited cell viability. NSC-CDM or Neural stem cell-complete medium (NSC-CPM) had a suppression effect on toxicity when treatment with Aβ_25-35, with a greater effect observed with NSC-CDM. Aβ_25-35 + NSC-CDM group exhibited an increase in PCMT1 expression. sh-PCMT1 markedly decreased cell proliferation and suppressed the protective role of NSC-CDM through the induction of apoptosis and improved p-MST1 expression. Overexpression of PCMT1 reversed the Aβ_25-35-induced decrease in cell proliferation and apoptosis. In summary, our findings suggest that NSC-CDM corrects the Aβ_25-35- induced damage to cells by improving PCMT1 expressions, which in turn reduces phosphorylation of MST1.     

Ultrastructural localization of prion proteins: physiological and pathological implications

The transmissible spongiform encephalopathies (TSE) or prion diseases are fatal neurodegenerative disorders in which the central event is the conversion of a normal host-encoded protein (PrP(c)) into an abnormal isoform (PrP(sc)) which accumulates as amyloid in TSE brain. The two PrP(c) and PrP(sc) prion protein isoforms are membrane sialoglycoproteins synthesized in the central nervous system and various peripheral organ tissues. In this review, we describe the ultrastructural localization of prion proteins in human and animal cerebral and non-cerebral tissues whether or not infected by TSE agents. In addition to the plasma membrane of several cells, PrP(c) was found in association with cytoplasmic organelles of central and nerve-muscle synapses, and secretory granules of epithelial cells. Fibrils of amyloid plaques, synaptic structures, and lysosome-like organelles constitute the subcellular sites harboring PrP(sc). These findings have led to discussions on the physiological role of PrP(c) and the pathological mechanisms underlying prion spongiform encephalopathies.