Agnieszka Malcher

Potential biomarkers of nonobstructive azoospermia identified in microarray gene expression analysis

OBJECTIVE To identify potential biomarkers of azoospermia to determine a particular stage of spermatogenetic differentiation. DESIGN GeneChip Human Gene 1.0 ST microarray with validation at mRNA and protein levels. SETTING Basic research laboratory. PATIENT(S) Men with various types of nonobstructive azoospermia (n = 18) and with normal spermatogenesis (n = 4). INTERVENTION(S) Obtaining 31 testicular biopsy samples. MAIN OUTCOME MEASURE(S) Gene expression analysis using the Affymetrix Human Gene 1.0 ST microarrays on 14 selected genes according to the highest fold change, verified with quantitative polymerase chain reaction and on independent set of microarray samples. Western blot and immunohistochemistry were additionally performed. RESULT(S) The comparative analysis of gene expression profiles in the infertile and control groups resulted in the selection of 4,946 differentially expressed genes. AKAP4, UBQLN3, CAPN11, GGN, SPACA4, SPATA3, and FAM71F1 were the most significantly down-regulated genes in infertile patients. Global analysis also led to identification of up-regulated genes-WBSCR28, ADCY10, TMEM225, SPATS1, FSCN3, GTSF1L, and GSG1-in men with late maturation arrest. Moreover, the results from quantitative polymerase chain reaction and Western blot largely confirmed the microarray data. CONCLUSION(S) The set of selected genes can be used to create a molecular diagnostic tool to determine the degree of spermatogenic impairment for men with idiopathic nonobstructive azoospermia.

Characterisation of Nuclear Architectural Alterations during In Vitro Differentiation of Human Stem Cells of Myogenic Origin

Cell differentiation is based on a synchronised orchestra of complex pathways of intrinsic and extrinsic signals that manifest in the induced expression of specific transcription factors and pivotal genes within the nucleus. One cannot ignore the epigenetic status of differentiating cells, comprising not only histones and DNA modifications but also the spatial and temporal intranuclear chromatin organisation, which is an important regulator of nuclear processes. In the present study, we investigated the nuclear architecture of human primary myoblasts and myocytes in an in vitro culture, with reference to global changes in genomic expression. Repositioning of the chromosomal centromeres, along with alterations in the nuclear shape and volume, was observed as a consequence of myotube formation. Moreover, the microarray data showed that during in vitro myogenesis cells tend to silence rather than induce gene expression. The creation of a chromosome map marked with gene expression changes that were at least 2-fold confirmed the observation. Additionally, almost all of the chromosomal centromeres in the differentiated cells preferentially localised near the nuclear periphery when compared to the undifferentiated cells. The exceptions were chromosomes 7 and 11, in which we were unable to confirm the centromere repositioning. In our opinion, this is the first reported observation of the movement of chromosomal centromeres along differentiating myogenic cells. Based on these data we can conclude that the myogenic differentiation with global gene expression changes is accompanied by the spatial repositioning of chromosomes and chromatin remodelling, which are important processes that regulate cell differentiation.

Successful implantation of autologous muscle-derived stem cells in treatment of faecal incontinence due to external sphincter rupture

Dear Editor:The most common pathological mechanism of faecalincontinence is the insufficiency of the external anal sphinc-ter (EAS) caused by neurological or myogenic dysfunction.The myogenic mechanism of EAS insufficiency is usuallydue to direct mechanical damage during childbirth, traumaor surgery in anorectal region, whereas neurologicalaetiology involves either spinal or peripheral nerves disrup-tion—in most cases the pudendal nerve. Unfortunately, co-incidence of sphincter rupture with damage to pudendalnerves is quite common.Each skeletal muscle, including EAS, has the ability toregenerate to some degree and repair sustained damage. Inresponse to injury and/or muscle damage, so-called satellitecells are activated and become myoblasts—capable of in-tense proliferation. Myoblasts then differentiate and fusetogether to form new muscle fibres and connect withexisting ones, adding new portions of contractile tissue toexisting motoric units [1].Attempts of autotransplantation of myoblasts into dam-aged skeletal muscle were already made in animal models ofmuscular dystrophy, post-infarction myocardial dysfunctionand urethral sphincter insufficiency [2]. The results showedthat the transplanted myoblasts differentiate into musclefibres, connect with host motoric units, increase the amountof contractile elements in the muscle and improve its con-tractile activity. In 2001, Menasche et al. first transplantedautologous myoblasts into the post-infarction myocardialscar in human patients with cardiac failure, with significantimprovements in contractile function and clinical condition[3]. In Poland, the method of treating post-infarction heartfailure was performed for the first time a year later, withsimilar results [4].Basedonthose encouraging results,a pioneerexperimen-tal study was designed in attempt to enhance the function ofexternal anal sphincter using injections of autologousmuscle-derived stem cells. The study is designed as a pro-spective experimental study. It is being conducted by twocooperating research centres—the 3rd Department of Gen-eral Surgery, Jagiellonian University in Cracow and theDepartment of Reproductive Biology and Stem Cells, Insti-tute of Human Genetics, Polish Academy of Sciences inPoznan. We would like to present a case of the representa-tive patient enrolled to our study.A 20-year old male withfaecal incontinenceduetoanoldexternal anal sphincter rupture in a road accident was en-rolled to the study. Sphincter rupture had been repairedsurgically right after the accident (with an end-to-endsphincteroplasty). The patient underwent 6 months of bio-feedback training after the wounds were healed. At the timeof enrolment, he still complained of gas and loose stoolincontinence, daily soiling, with necessity to wear pads.Endoanal ultrasound showed a 8–10-mm scar on the leftcircumference of internal and external sphincter muscle,where anal canal was ruptured during the accident, andsurgically repaired afterwards. Anorectal manometryshowed decreased both mean resting and maximum squeezepressure, with short high pressure zone length. Endoanal

Safety, feasibility and effectiveness of first in‐human administration of muscle‐derived stem/progenitor cells modified with connexin‐43 gene for treatment of advanced chronic heart failure

To assess the safety and efficacy of transendocardial delivery of muscle‐derived stem/progenitor cells with connexin‐43 overexpression (Cx‐43‐MDS/PC) in advanced heart failure (HF).

The Gene Expression Analysis of Paracrine/Autocrine Factors in Patients with Spermatogenetic Failure Compared with Normal Spermatogenesis

The aim of this study was to examine the expression levels of IL‐1 family members, IL‐6, IL‐10, TNF family, SCF, and c‐kit in infertile patients with idiopathic non‐obstructive azoospermia (NOA) compared with men with normal spermatogenesis.

Expression of genes coding for proangiogenic factors and their receptors in human placenta complicated by preeclampsia and intrauterine growth restriction.

The aim of the study was to investigate the expression of genes coding for vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF) as well as their receptors, fms-like tyrosine kinase receptor 1 (VEGFR-1/Flt-1) and VEGF receptor 2 (VEGFR-2/KDR) in the placentae of patients with pregnancies complicated by preeclampsia (PE) and intrauterine growth restriction (IUGR). Tissue samples were collected from placentae of women with PE (n=31) and IUGR syndrome (n=25) as well as of healthy control women (n=31). Total RNA was extracted and purified, mRNA reversely transcribed, and amplified using real-time PCR. Expression of the examined genes was normalized to β-actin. Higher levels of PlGF (p<0.001) and Flt-1 (p<0.05) transcription were found in PE placentae compared to normal ones. A positive correlation between PlGF and Flt-1 expression was revealed in the PE patients. In conclusion, the presented data indicate the upregulation of both PlGF and Flt-1 in placentae of women with PE, which could be induced by a pathological process possibly due to endothelial dysfunction.

Genetically modified human myoblasts with eNOS may improve regenerative ability of myogenic stem cells to infarcted heart

BACKGROUND Modern therapies of post infarcted heart failure are focused on perfusion improvement of the injured myocardium. This effect can be achieved by, among other means, implanting stem cells which could be genetically modified with factors inducing the formation of new blood vessels in the post infarction scar area. Combined stem cell and gene therapy seems to be a promising strategy to heal an impaired myocardium. The creation of new blood vessels can be indirectly stimulated via factors inducing vascular endothelial growth factor synthesis, for example endothelial nitric oxide synthase (eNOS). The product of this enzyme, nitric oxide, is a molecule that can influence numerous physiological activities; it can contribute to vasodilation, stimulation of endothelial cell growth, prevention of platelet aggregation and leukocyte adhesion to the endothelium. AIM To verify the pro-angiogenic and regenerative potential of human primary myoblasts and murine myoblast cell line C2C12 transiently transfected with eNOS gene. METHODS Stem cells (either human or murine) were maintained in standard in vitro conditions. Next, both types of myoblasts were modified using electroporation and lipofection (human and murine cells), respectively. The efficacy of the transfection method was evaluated using flow cytometry. The concentration of eNOS protein was measured by ELISA immunoassay. The biological properties of modified cells were assessed using an MTT proliferation test and DAPI cell cycle analysis. To verify the influence of oxidative stress on myoblasts, cytometric tests using Annexin V and propidium iodide were applied. To check possible alterations in myogenic gene expression of stem cells transduced by genetic modification, the myogenic regulatory factors were evaluated by real-time PCR. The function of genetic modification was confirmed by a HUVEC capillary sprouting test using myoblasts supernatants. RESULTS Electroporation turned out to be an efficient transfection method. High amounts of secreted protein were obtained (in the range 2,000 pg/mL) in both cell types studied. Moreover, the functionality of gene overexpression product was confirmed in capillary development assay. Human myoblasts did not exhibit any changes in cell cycle; however, eNOS transfected murine myoblasts revealed a statistically significant reduction in cell cycle ratio compared to controls (p < 0.001). In the case of myogenic gene expression, a decrease in Myogenin level was only detected in the human transfected myoblast population (p < 0.05). CONCLUSIONS The results of our study may suggest that transplantation of myoblasts overexpressing eNOS could be promising for cell therapy in regenerating the post infarction heart.

Human myoblast transplantation in mice infarcted heart alters the expression profile of cardiac genes associated with left ventricle remodeling

BACKGROUND Myocardial infarction (MI) and left ventricle remodeling (LVR) are two of the most challenging disease entities in developed societies. Since conventional treatment cannot fully restore heart function new approaches were attempted to develop new strategies and technologies that could be used for myocardial regeneration. One of these strategies pursued was a cell therapy--particularly applying skeletal muscle stem cells (SkMCs). METHODS AND RESULTS Using NOD-SCID murine model of MI and human skeletal myoblast transplantation we were able to show that SkMC administration significantly affected gene expression profile (p<0.05) (NPPB, CTGF, GATA4, SERCA2a, PLB) of the heart ventricular tissue and this change was beneficial for the heart function. We have also shown, that the level of heart biomarker, NT-proBNP, decreased in animals receiving implanted cells and that the NT-proBNP level negatively correlated with left ventricle area fraction change (LVFAC) index which makes NT-proBNP an attractive tool in assessing the efficacy of cell therapy both in the animal model and prospectively in clinical trials. CONCLUSIONS The results obtained suggest that transplanted SkMCs exerted beneficial effect on heart regeneration and were able to inhibit LVR which was confirmed on the molecular level, giving hope for new ways of monitoring novel cellular therapies for MI.

Potential use of superparamagnetic iron oxide nanoparticles for in vitro and in vivo bioimaging of human myoblasts

Myocardial infarction (MI) is one of the most frequent causes of death in industrialized countries. Stem cells therapy seems to be very promising for regenerative medicine. Skeletal myoblasts transplantation into postinfarction scar has been shown to be effective in the failing heart but shows limitations such, e.g. cell retention and survival. We synthesized and investigated superparamagnetic iron oxide nanoparticles (SPIONs) as an agent for direct cell labeling, which can be used for stem cells imaging. High quality, monodisperse and biocompatible DMSA-coated SPIONs were obtained with thermal decomposition and subsequent ligand exchange reaction. SPIONs’ presence within myoblasts was confirmed by Prussian Blue staining and inductively coupled plasma mass spectrometry (ICP-MS). SPIONs’ influence on tested cells was studied by their proliferation, ageing, differentiation potential and ROS production. Cytotoxicity of obtained nanoparticles and myoblast associated apoptosis were also tested, as well as iron-related and coating-related genes expression. We examined SPIONs’ impact on overexpression of two pro-angiogenic factors introduced via myoblast electroporation method. Proposed SPION-labeling was sufficient to visualize firefly luciferase-modified and SPION-labeled cells with magnetic resonance imaging (MRI) combined with bioluminescence imaging (BLI) in vivo. The obtained results demonstrated a limited SPIONs’ influence on treated skeletal myoblasts, not interfering with basic cell functions.

Human sperm proteins identified by 2-dimensional electrophoresis and mass spectrometry and their relevance to a transcriptomic analysis

The aim of this study was to identify and analyse human sperm proteins from normozoospermic men using 2-dimensional electrophoresis (2-DE) and mass spectrometry (MS). We identified 73 different sperm proteins, including two less characterized human sperm proteins, Annexin A7 (ANXA7) and c14orf105. Bioinformatic analysis of detected sperm proteins revealed new carbohydrate and lipid metabolic pathways, which supply energy to motile sperm. A comparison of our data with available mRNA microarray data from the human testis allows for validation of identified sperm proteins and aids in the recognition of their physiological pathways.