Human Adipose-Derived and Amniotic Fluid-Derived Stem Cells: A Preliminary In Vitro Study Comparing Myogenic Differentiation Capability.

BACKGROUND Around the world, disabilities due to musculoskeletal disorders have increased and are a major health problem worldwide. In recent years, stem cells have been considered to be powerful tools for musculoskeletal tissue engineering. Human adipose-derived stem cells (hADSCs) and amniotic fluid-derived stem cells (hAFSCs) undergo typical differentiation process into cells of mesodermal origin and can be used to treat muscular system diseases. The aim of the present study was to compare the biological characteristic of stem cells isolated from different human tissues (adipose tissue and amniotic fluid) with respect to myogenic capacity and skeletal and smooth muscle differentiation under the same conditions. MATERIAL AND METHODS hAFSCs and hADSCs were isolated during standard medical procedures and widely characterized by specific markers expression and differentiation potential. Both cell types were induced toward smooth and striated muscles differentiation, which was assessed with the use of molecular techniques. RESULTS For phenotypic characterization, both stem cell types were assessed for the expression of OCT-4, SOX2, CD34, CD44, CD45, and CD90. Muscle-specific markers appeared in both stem cell types, but the proportion of positive cells showed differences depending on the experimental conditions used and the source from which the stem cells were isolated. CONCLUSIONS In this study, we demonstrated that hADSCs and hAFSCs have different capability of differentiation toward both muscle types. However, hADSCs seem to be a better source for myogenic protocols and can promote skeletal and smooth muscle regeneration through either direct muscle differentiation or by paracrine mechanism.

Influence of Mesenchymal Stem Cells Conditioned Media on Proliferation of Urinary Tract Cancer Cell Lines and Their Sensitivity to Ciprofloxacin.

Mesenchymal stem cells (MSCs) are known to interact with cancer cells through direct cell-to-cell contact and secretion of paracrine factors, although their exact influence on tumor progression in vivo remains unclear. To better understand how fetal and adult stem cells affect tumors, we analyzed viability of human renal (786-0) and bladder (T24) carcinoma cell lines cultured in conditioned media harvested from amniotic fluid-derived stem cells (AFSCs) and adipose-derived stem cells (ASCs). Both media reduced metabolic activity of 786-0 cells, however, decreased viability of T24 cells was noted only after incubation with conditioned medium from ASCs. To test the hypothesis that MSCs-secreted factors might be involved in chemoresistance acquisition, we further analyzed influence of mesenchymal stem cell conditioned media (MSC-CM) on cancer cells sensitivity to ciprofloxacin, that is considered as potential candidate agent for urinary tract cancers treatment. Significantly increased resistance to tested drug indicates that MSCs may protect cancer cells from chemotherapy. J. Cell. Biochem. 118: 1361-1368, 2017. © 2016 Wiley Periodicals, Inc.

Gene Expression and Activity Profiling Reveal a Significant Contribution of Exo-Phosphotransferases to the Extracellular Nucleotides Metabolism in HUVEC Endothelial Cells.

Purinergic signaling maintains local tissue homeostasis in blood vessels via the regulation of vascular tone, blood platelet aggregation, cell proliferation, and differentiation as well as inflammatory responses. Extracellular purines are important signaling molecules in the vasculature, and both purine-hydrolysing as well as -phosphorylating enzymes are considered to selectively govern extracellular nucleotide/nucleoside metabolism. Recent studies have provided some evidence for the existence of these enzymes in a soluble form in human blood and their secretion into the extracellular space under physiological and pathological conditions. However, the comprehensive analysis of endothelium-derived enzymes involved in purine metabolic pathways has received no attention so far. In the presented study, in vitro cultured human umbilical vein endothelial cells (HUVEC) are shown to be an abundant source of exo-nucleotidases comprising 5'-nucleotidase (exo-5'-NT), and nucleoside triphosphate diphosphohydrolases (exo-NTPDase) as well as phosphotransferases, represented by nucleoside diphosphate kinase (exo-NDPK) and adenylate kinase (exo-AK). An attempt is also made to demonstrate that, in contrast to the metabolic pattern determined on the endothelial cell surface, exo-phosphorylating activities markedly predominate over exo-hydrolytic ones. We present for the first time the expression profiles of genes encoding isoenzymes belonging to distinct nucleotide kinase and nucleotidase families. The genes encoding NDPK1, NDPK2, AK1, and AK2 phosphotransferases have been shown to be expressed at the highest level in HUVEC cells. The data indicate the coexistence of secreted and cell-associated factors of endothelial origin mediating ATP-consuming and ATP-generating pathways with the predominance of exo-phosphotransferases activity. The described enzymes contribute to the regulation of purinergic signal duration and extent in the venous vasculature. J. Cell. Biochem. 118: 1341-1348, 2017. © 2016 Wiley Periodicals, Inc.

High Quality Independent From a Donor: Human Amniotic Fluid Derived Stem Cells-A Practical Analysis Based on 165 Clinical Cases.

The aim of the study was to extend the potential use of human stem cells isolated from amniotic fluid in medical applications by confirming their high homogeneity and quality. Amniotic fluid samples were collected during amniocentesis from 165 women during pregnancy. The proliferation rate, clonogenicity, karyotype, aging process, pluripotent cell markers, expression of surface markers, and the potential to differentiate into adipose, bone and cartilage cells of hAFSCs were analyzed. Obtained results revealed that mesenchymal stem cells could be derived successfully from amniotic fluid, which exhibit properties comparable with MSCs of other origins. It is the first study, in which such a large group of patients was involved. Comprehensive statistical and biological analysis were conducted some of which clearly being innovative in relation to human amniotic fluid-derived stem cells. J. Cell. Biochem. 118: 116-126, 2017. © 2016 Wiley Periodicals, Inc.

Canine Adipose-Derived Stem Cells: Purinergic Characterization and Neurogenic Potential for Therapeutic Applications.

The presented results evidence that canine adipose-derived stem cells (ADSCs) represent the premature population of stem cells with great biological potential and properties. ADCS are easy to obtain and culture, able to differentiate into the neurogenic lineage as well as it is easy to control their proliferation rate with nucleotides and nucleosides or analogues. We report that in vitro cultured canine ADSCs response to adenosine- and ATP-mediated stimulation. Differences in canine ADSCs and human mesenchymal stem cells in ecto-nucleotidase activity have been observed. The ecto-nucleotidase activity changes during ADSCs in vitro transdifferentiation into neurogenic lineage are fast and simple to analyze. Therefore, the simple analysis of ecto-enzymes activity allows for verification of the stem cells quality: their stemness or initiation of the differentiation process. The biological potential of the cells isolated from canine fat, as well as the good quality control of this cell culture, make them a promising tool for both experimental and therapeutic usage. J. Cell. Biochem. 118: 58-65, 2017. © 2016 Wiley Periodicals, Inc.

Neurogenic Differentiation of Mesenchymal Stem Cells Induces Alterations in Extracellular Nucleotides Metabolism.

The presented results show for the first time that the neurogenic transdifferentiation of hUC-MSCs considerably changes the elements of purinergic signaling profile. Although, it has been demonstrated in the literature that extracellular nucleotides and nucleosides determine the fate of mesenchymal and neural stem cells, there is lack of comprehensive studies on the activity of ecto-enzymes metabolizing nucleotides on the surface of neurogenically induced cells. Our study shows that human UC-MSCs sense the microenvironment and adjust their response to the environmental signals for example, adenine nucleotides and nucleosides. Nucleotides, and not adenosine, signaling alters the biological potential of MSCs-decreases their proliferation rate, increases the neurogenic transdifferentiation efficiency expressed as the number of positively labeled NCAM+ and A2B5+ cells and simultaneously increases the ecto-nucleotidases activity on neural- and glial-committed precursors. Purines implication in the proliferative and neurogenic potential of hUC-MSCs is of strong importance for the in vitro propagation of hUC-MSCs and for their successive therapeutic applications. J. Cell. Biochem. 118: 478-486, 2017. © 2016 Wiley Periodicals, Inc.

Cloning and biochemical characterization of indole-3-acetic acid-amino acid synthetase PsGH3 from pea.

Phytohormone conjugation is one of the mechanisms that maintains a proper hormonal homeostasis and that is necessary for the realization of physiological responses. Gretchen Hagen 3 (GH3) acyl acid amido synthetases convert indole-3-acetic acid (IAA) to IAA-amino acid conjugates by ATP-dependent reactions. IAA-aspartate (IAA-Asp) exists as a predominant amide conjugate of auxin in pea tissues and acts as an intermediate during IAA catabolism. Here we report a novel recombinant indole-3-acetic acid-amido synthetase in Pisum sativum. In silico analysis shows that amino acid sequence of PsGH3 has the highest homology to Medicago truncatula GH3.3. The recombinant His-tag-PsGH3 fusion protein has been obtained in E. coli cells and is a soluble monomeric polypeptide with molecular mass of 69.18 kDa. The PsGH3 was purified using Ni(2+)-affinity chromatography and native PAGE. Kinetic analysis indicates that the enzyme strongly prefers IAA and L-aspartate as substrates for conjugation revealing Km(ATP) = 0.49 mM, Km(L-Asp) = 2.2 mM, and Km(IAA) = 0.28 mM. Diadenosine pentaphosphate (Ap5A) competes with ATP for catalytic site and diminishes the PsGH3 affinity toward ATP approximately 1.11-fold indicating Ki = 8.5 µM. L-Tryptophan acts as an inhibitor of IAA-amido synthesizing activity by competition with L-aspartate. Inorganic pyrophosphatase (PPase) hydrolyzing pyrophosphate to two phosphate ions, potentiates IAA-Asp synthetase activity of PsGH3. Our results demonstrate that PsGH3 is a novel enzyme that is involved in auxin metabolism in pea seeds.

Neurotoxic phospholipase A2 from rattlesnake as a new ligand and new regulator of prokaryotic receptor GLIC (proton-gated ion channel from G. violaceus).

Neurotoxic phospholipases A2 (sPLA2) from snake venoms interact with various protein targets with high specificity and potency. They regulate function of multiple receptors or channels essential to life processes including neuronal or neuromuscular chemoelectric signal transduction. These toxic sPLA2 exhibit high pharmacological potential and determination of PLA2-receptor binding sites represents challenging part in the receptor-channel biochemistry and pharmacology. To investigate the mechanism of interaction of neurotoxic PLA2 with its neuronal receptor at the molecular level, we used as a model crotoxin, a heterodimeric sPLA2 from rattlesnake venom and proton-gated ion channel GLIC, a bacterial homolog of pentameric ligand-gated ion channels. The three-dimensional structures of both partners, crotoxin and GLIC have been solved by X-ray crystallography and production of full-length pentameric GLIC (with ECD and TM domains) is well established. In the present study, for the first time, we demonstrated physical and functional interaction of full-length purified and solubilized GLIC with CB, (PLA2 subunit of crotoxin). We identified GLIC as a new protein target of CB and CB as a new ligand of GLIC, and showed that this non covalent interaction (PLA2-GLIC) involves the extracellular domain of GLIC. We also determined a novel function of CB as an inhibitor of proton-gated ion channel activity. In agreement with conformational changes observed upon formation of the complex, CB appears to be negative allosteric modulator (NAM) of GLIC. Finally, we proposed a possible stoichiometric model for CB - GLIC interaction based on analytical ultracentrifugation.

Chondrogenic Differentiation of Human Mesenchymal Stem Cells Results in Substantial Changes of Ecto-Nucleotides Metabolism.

Mesenchymal stem cells (MSCs) are population of adult stem cells and attractive candidates for cartilage repair due to their chondrogenic potential. Purinergic compounds (purinergic receptors and ecto-enzymes metabolizing nucleotides), together with nucleotides/nucleosides present in the extracellular environment, are known to play a key role in controlling the stem cells biological potential to proliferate and differentiate. Despite the available literature pointing to the importance of purinergic signaling in controlling the fate of MSCs, the research results linking nucleotides and ecto-nucleotidases with MSCs chondrogenic differentiation are indigent. Therefore, the aim of presented study was the characterization of the ecto-nucleotides hydrolysis profile and ecto-enzymes expression in human umbilical cord-derived MSCs and chondrogenically induced MSCs. We described substantial changes of ecto-nucleotides metabolism and ecto-enzymes expression profiles resulting from chondrogenic differentiation of human umbilical cord-derived MSCs. The increased rate of ADP hydrolysis, measured by ecto-nucleotidases activity, plays a pivotal role in the regulation of cartilage formation and resorption. Despite the increased level of NTPDase1 and NTPDase3 mRNA expression in chondrogenically induced MSCs, their activity toward ATP remains quite low. Supported by the literature data, we hypothesize that structure-function relationships in chondrogenic lineage dictate the direction of nucleotides metabolism. In early neocartilage tissue, the beneficial role of ATP in improving biomechanical properties of cartilage does not necessitate the high rate of enzymatic ATP degradation.

Cell therapy in Duchenne muscular dystrophy treatment: clinical trials overview.

Duchenne muscular dystrophy (DMD), the most common and most severe form of all muscular dystrophies, leads to progressive muscle fiber necrosis, fibroblast proliferation, and growth of fibrous tissue and fat. The most common cause of death in DMD patients is cardiac and respiratory failure. Current pharmacological and other treatment methods do not lead to full recovery. For this reason, new alternatives for skeletal muscle regeneration are being investigated. Transplantation of myoblasts from healthy donors is one studied approach to muscle treatment in DMD patients. However, the results of intramuscular injection of in vitro cultured myoblasts are still not satisfactory. The use of autologous stem cells is also proposed. Despite many ongoing studies, this therapy is still in preliminary testing and requires more experiments.

The expression of p63 and Ck HMW in magnum and infundibulum of Gallus domesticus oviduct.

The potential for proliferation and differentiation has a critical meaning in terms of the long-term in vitro culture of oviductal target cells. Therefore, it is important to characterize the oviduct epithelial cells, using approved markers. There is scarce data describing the epithelial cells lining the avian oviduct, most of it referring only to the magnum section of the oviduct. This study presents a comprehensive analysis of both magnum and infundibulum tissues, as the most preferred sources of epithelial cells for research on production of recombinant proteins in oviducts of birds. The main objective was to evaluate the expression of p63 and high molecular weight cytokeratins (anti- p63 antibody and anti- High Molecular Weight Cytokeratins) in epithelial cells (EC) of 2 oviduct sections: magnum (proximal and middle) and infundibulum (distal). IHC analysis and western blotting were performed using the mouse monoclonal anti- p63 antibody and anti-Ck HMW. Immunoreactivity was quantified based on the Remmele - Stegner scoring system (0-12). The expression of p63 in nuclei of luminal cells was significantly higher in the infundibulum (P < 0.05), compared to the magnum section. Cytokeratins were also highly expressed in the infundibulum, but the difference was non-significant. These findings reveal new characteristics of the oviduct EC and designate the location of the source of cells in the oviduct tissue for in vitro culture.

Chaperones are necessary for the expression of catalytically active potato apyrases in prokaryotic cells.

NTPDases (nucleoside triphosphate diphosphohydrolases) (also called in plants apyrases) hydrolyze nucleoside 5'-tri- and/or diphosphate bonds producing nucleosides di or monophosphate and inorganic phosphate. For years, studies have been carried out to use both plant and animal enzymes for medicine. Therefore, there is a need to develop an efficient method for the quick production of large amounts of homogeneous proteins with high catalytic activity. Expression of proteins in prokaryotic cells is the most common way for the protein production. The aim of our study was to develop a method of expression of potato apyrase (StAPY4, 5, and 6) genes in bacterial cells under conditions that allowed the production of catalytically active form of these enzymes. Apyrase 4 and 6 were overexpressed in BL21-CodonPlus (DE3) bacteria strain but they were accumulated in inclusion bodies, regardless of the culture conditions and induction method. Co-expression of potato apyrases with molecular chaperones allowed the expression of catalytically active apyrase 5. However, its high nucleotidase activity could be toxic for bacteria and is therefore synthesized in small amounts in cells. Our studies show that each protein requires other conditions for maturation and even small differences in amino acid sequence can essentially affect protein folding regardless of presence of chaperones.

[Epidermal stem cells--biology and potential applications in regenerative medicine]. / Komórki macierzyste naskórka--biologia i potencjalne zastosowanie w medycynie regeneracyjnej.

Stem cells are involved in the renewal and regeneration of the epithelium of various organs. The largest reservoir of epithelial stem cells in the human body is the skin. This organ is a specialized interior barrier protecting the body from the influence of physical, chemical and biological factors, ensuring at the same time the reception of signals from the external environment. Skin is also involved in numerous physiological processes which determine the homeostasis of the body. Renewal and regeneration of the epidermis which is the outer layer of the skin, is possible by the presence of different populations of stem cells that reside in microenvironments (niches), that creates specific conditions to preserve the biological properties of these cells. Because divisions of cells in niches are quite rare, it became possible to distinguish them from other rapidly proliferating cells of the skin. On this basis, the stem cells in the interfollicular epidermis, bulge region of the hair follicles, and within the sebaceous glands were located. Moreover hair follicles are suggested to be a niche for melanocyte progenitor cells and other multipotent stem cells derived from the neural crest, as well as mesenchymal stem cells. The presence of stem cells that are characterized by high proliferative potential and the ability to self-renew allow maintaing homeostasis and regeneration of epidermis. Identification, isolation and characterization of epithelial stem cells is necessary to understand skin diseases background, develop effective methods for their treatment and for wider use of stem cells in regenerative medicine, gene therapy or cosmetology.

Isolation and bioinformatic analysis of seven genes encoding potato apyrase. Bacterial overexpresssion, refolding and initial kinetic studies on some recombinant potato apyrases.

Here we have isolated seven apyrase encoding cDNA sequences (StAPY4-StAPY10) from the potato variety Saturna tuber cDNA library by affecting necessary modifications in the screening protocol. The cDNA sequences were identified with a pair of primers complementary to the most conserved sequences identified in potato variety Desiree apyrase genes. Our data strongly suggest the multigenic nature of potato apyrase. All deduced amino acid sequences contain a putative signal sequence, one transmembrane region at the amino terminus and five apyrase conserved regions (ACRs) (except StAPY6). Phylogenetic analysis revealed that encoded proteins shared high level of DNA sequence identity among themselves, representing a family of proteins markedly distinct from other eukaryotic as well as prokaryotic apyrases. Two cDNA sequences (StAPY4 and StAPY6) were overexpressed in bacteria and recombinant proteins were found accumulated in inclusion bodies, even thought they were fused with thioredoxin-tag. Additionally, we present the first successful in vitro attempt at reactivation and purification of recombinant potato apyrase StAPY6. The ratio of ATPase/ADPase hydrolysis of recombinant StAPY6 was determined as 1.5:1. Unlike other apyrases the enzyme lacked ACR5 and was endowed with lower molecular weight, high specificity for purine nucleotides and very low specificity for pyrimidine, suggesting that StAPY6 is a potato apyrase, not described so far.

[Prokaryotic expression systems]. / Prokariotyczne systemy ekspresyjne.

For overproduction of recombinant proteins both eukaryotic and prokaryotic expression systems are used. Choosing the right system depends, among other things, on the growth rate and culture of host cells, level of the target gene expression and posttranslational processing of the synthesized protein. Regardless of the type of expression system, its basic elements are the vector and the expression host. The most widely used system for protein overproduction, both on a laboratory and industrial scale, is the prokaryotic system. This system is based primarily on the bacteria E. coli, although increasingly often Bacillus species are used. The prokaryotic system allows one to obtain large quantities of recombinant proteins in a short time. A simple and inexpensive bacterial cell culture and well-known mechanisms of transcription and translation facilitate the use of these microorganisms. The simplicity of genetic modifications and the availability of many bacterial mutants are additional advantages of the prokaryotic system. In this article we characterize the structural elements of prokaryotic expression vectors. Also strategies for preparation of the target protein gene that increase productivity, facilitate detection and purification of recombinant protein and provide its activity are discussed. Bacterial strains often used as host cells in expression systems as well as the potential location of heterologous proteins are characterized. Knowledge of the basic elements of the prokaryotic expression system allows for production of biologically active proteins in a short time and in satisfactory quantities. 

[In vitro renaturation of proteins from inclusion bodies]. / Renaturacja in vitro bialek zakumulowanych w cialach inkluzyjnych.

Recombinant proteins and enzymes are commonly used in many areas of our life, such as diagnostics, industry and medicine, due to heterologous synthesis in prokaryotic expression systems. However, a high expression level of foreign protein in bacteria cells results in formation of inactive and insoluble aggregates--inclusion bodies. Reactivation of aggregated proteins is a complex and time-consuming process. Every protein requires experimental optimization of the process conditions. The choice of the refolding method depends on the type of recombinant protein and its physical, chemical and biological properties. Recovery of the activity of proteins accumulated in inclusion bodies can be divided into 4 steps: 1) inclusion bodies isolation, 2) solubilization of aggregates, 3) renaturation, 4) purification of catalytically active molecules. Efficiency of the refolding process depends on many physical factors and chemical and biological agents. The above parameters determine the time of the folding and prevent protein aggregation. They also assist the folding and have an influence on the solubility and stability of native molecules. To date, dilution, dialysis and chromatography are the most often used methods for protein refolding.

[The role of ectonucleotides metabolizing enzymes in purinergic signaling]. / Rola enzymów metabolizujacych ektonukleotydy w sygnalizacji z udzialem puryn.

Purinergic signaling plays an important role in the regulation of many physiological processes. The concentration of nucleotides in extracellular space is controlled by at least two families of nucleotidases: NPPases and NTPDases. These families are examples of convergent evolution of proteins. Above ezymes are not phylogenetically related, but they catalyze the same type of reaction. They hydrolyzed tri- and diphosphonucleosides to monophosphonucleosides and orthophosphate or pyrophosphate. This degradation terminates the nucleotide signaling process and also produces other signaling molecules like ADP, and with 5'-nucleotidase, adenosine. Most of known animal NPPases and NTPDases were found as membranous ectoenzymes or soluble proteins localized in tissue fluids. The aim of this work is to provide information about localization, structure, properties and function of NPPases and NTPDases in the regulation of extracellular concentration of nucleotides and purinergic signaling.