Dorota Porowińska

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. J. Cell. Biochem. 116: 2915–2923, 2015.

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.

In vitro renaturation of proteins from inclusion bodies

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.

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.

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.

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.

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.

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.

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.