Tomasz Kolanowski

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.

Techniques for the induction of human pluripotent stem cell differentiation towards cardiomyocytes.

The derivation of pluripotent stem cells from human embryos and the generation of induced pluripotent stem cells (iPSCs) from somatic cells opened a new chapter in studies on the regeneration of the post‐infarction heart and regenerative medicine as a whole. Thus, protocols for obtaining iPSCs were enthusiastically adopted and widely used for further experiments on cardiac differentiation. iPSC‐mediated cardiomyocytes (iPSC‐CMs) under in vitro culture conditions are generated by simulating natural cardiomyogenesis and involve the wingless‐type mouse mammary tumour virus integration site family (WNT), transforming growth factor beta (TGF‐β) and fibroblast growth factor (FGF) signalling pathways. New strategies have been proposed to take advantage of small chemical molecules, organic compounds and even electric or mechanical stimulation. There are three main approaches to support cardiac commitment in vitro: embryoid bodis (EBs), monolayer in vitro cultures and inductive co‐cultures with visceral endoderm‐like (END2) cells. In EB technique initial uniform size of pluripotent stem cell (PSC) colonies has a pivotal significance. Hence, some methods were designed to support cells aggregation. Another well‐suited procedure is based on culturing cells in monolayer conditions in order to improve accessibility of growth factors and nutrients. Other distinct tactics are using visceral endoderm‐like cells to culture them with PSCs due to secretion of procardiac cytokines. Finally, the appropriate purification of the obtained cardiomyocytes is required prior to their administration to a patient under the prospective cellular therapy strategy. This goal can be achieved using non‐genetic methods, such as the application of surface markers and fluorescent dyes. Copyright

Can apoptosis and necrosis coexist in ejaculated human spermatozoa during in vitro semen bacterial infection

PurposeTo evaluate whether ejaculated human spermatozoa undergo complete apoptosis or necrosis during experimental semen bacterial infection in vitro.MethodsApoptotic markers, including mitochondrial transmembrane potential (ΔΨm), phosphatidylserine (PS) externalization, and DNA fragmentation, have been detected simultaneously in ejaculated human sperm after their incubation with a known pathogenic (Escherichia coli), as well as with conditionally pathogenic bacterial strains (Staphylococcus haemolyticus, Bacteroides ureolyticus) and/or leukocytes. The ΔΨm and translocation of PS was evaluated using the JC-1 and Annexin V binding tests, respectively. A modified TUNEL assay with additional staining for sperm viability was used to detect the DNA fragmentation level.ResultsThe exposure of ejaculated spermatozoa to bacterial strains was associated with a simultaneous decrease in the percentage of sperm with normal ΔΨm and an increase in the proportion of Annexin V-positive sperm. Additionally, in the presence of S. haemolyticus, B. ureolyticus and/or leukocytes, a significant increase in the percentage of live TUNEL-positive (apoptotic) as well as dead TUNEL-positive (necrotic) sperm cells was also observed.ConclusionsThe cellular death observed in spermatozoa in the presence of inflammatory mediators may be due to both apoptosis and necrosis. Here, we demonstrate for the first time that direct contact of conditionally pathogenic bacteria with ejaculated human sperm may play an even greater role in the promotion of apoptosis than in case of some pathogenic bacterial strains. These findings suggest that significant bacteriospermia and leukocytospermia may be direct causes of subfertility or additional negative factors worsening the prognosis of fertility in natural and assisted procreation.

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).

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.

SPIN1 is a proto-oncogene and SPIN3 is a tumor suppressor in human seminoma.

SPIN1 is necessary for normal meiotic progression in mammals. It is overexpressed in human ovarian cancers and some cancer cell lines. Here, we examined the functional significance and regulation of SPIN1 and SPIN3 in the TCam-2 human seminoma cell line. We found that while SPIN1 overexpression reduced apoptosis in these cells, SPIN3 overexpression induced it. Similarly, SPIN1 upregulated and SPIN3 downregulated CYCD1, which is a downstream target of the PI3K/AKT pathway and contributes to apoptosis resistance in cancer cell lines. It appears that SPIN1 is pro-oncogenic and SPIN3 acts as a tumor suppressor in TCam-2 cells. To our knowledge, this is the first report of SPIN3 tumor suppressor activity. However, both SPIN1 and SPIN3 stimulated cell cycle progression. In addition, using luciferase reporters carrying SPIN1 or SPIN3 mRNA 3′UTRs, we found that PUM1 and PUM2 targeted and repressed SPINs. We also found that PUM1 itself strongly stimulated apoptosis and moderately slowed cell cycle progression in TCam-2 cells, suggesting that PUM1, like SPIN3, is a tumor suppressor. Our findings suggest that acting, at least in part, through SPIN1 and SPIN3, PUM proteins contribute to a mechanism promoting normal human male germ cell apoptotic status and thus preventing cancer.

The impact of in vitro cell culture duration on the maturation of human cardiomyocytes derived from induced pluripotent stem cells of myogenic origin

Ischemic heart disease, also known as coronary artery disease (CAD), poses a challenge for regenerative medicine. iPSC technology might lead to a breakthrough due to the possibility of directed cell differentiation delivering a new powerful source of human autologous cardiomyocytes. One of the factors supporting proper cell maturation is in vitro culture duration. In this study, primary human skeletal muscle myoblasts were selected as a myogenic cell type reservoir for genetic iPSC reprogramming. Skeletal muscle myoblasts have similar ontogeny embryogenetic pathways (myoblasts vs. cardiomyocytes), and thus, a greater chance of myocardial development might be expected, with maintenance of acquired myogenic cardiac cell characteristics, from the differentiation process when iPSCs of myoblastoid origin are obtained. Analyses of cell morphological and structural changes, gene expression (cardiac markers), and functional tests (intracellular calcium transients) performed at two in vitro culture time points spanning the early stages of cardiac development (day 20 versus 40 of cell in vitro culture) confirmed the ability of the obtained myogenic cells to acquire adult features of differentiated cardiomyocytes. Prolonged 40-day iPSC-derived cardiomyocytes (iPSC-CMs) revealed progressive cellular hypertrophy; a better-developed contractile apparatus; expression of marker genes similar to human myocardial ventricular cells, including a statistically significant CX43 increase, an MHC isoform switch, and a troponin I isoform transition; more efficient intercellular calcium handling; and a stronger response to β-adrenergic stimulation.