ABSTRACT
Cryopreservation is a procedure of a long-term storage of cells and/or tissues at a temperature that prevents cell divisions and metabolic processes. Due to ability to self-renewal and differentiation into more specialised cells, stem cells may be helpful in repairing of other damaged organs or tissues. Cryopreservation allows the frozen genetic material to maintain its biological properties for a long time. Therefore, there is a real chance for some samples to be used in the future therapy of the pathological conditions that at present remain incurable because of the current state of knowledge. The purpose of this review is to describe the modern methods of extraction, preservation, and storage of dental stem cells at low temperatures in particular procedure of collecting and transporting tissues intended for freezing, precise characteristics of stem cells of dentary origin and methods of their isolation using Enzymatic Digestion and Spontaneous Outgrowth. In the paper are also presented technical details of the protocols of rapid rate freezing, controlled rate milling and freezing in a magnetic field (magnetic freezing) which provides precise information about procedures of thawing cells and unfavourable effect of negative temperature on the biological properties of stem cells. Dental tissues may constitute a rich source of stem cells. The inexpensive, simple and quick procedure of their extraction is minimally invasive and does not pose a threat to the donor's organism. Transferring autologous cells within the same organism does not present a potential risk of transplant rejection and thereof does not raise ethical controversies. Laboratory procedures including cell preparation, its characteristics and genetic features, basics on the process of freezing, thawing, as well as quality control essentials have been also outlined.
Subject(s)
Cryopreservation , Stem Cells , Cold Temperature , Cell DifferentiationABSTRACT
PURPOSE: To identify clinically significant genomic copy number (CNV) and single nucleotide variants (SNV) in males with unexplained spermatogenic failure (SPGF). MATERIALS AND METHODS: Peripheral blood DNA from 97/102 study participants diagnosed with oligozoospermia, severe oligozoospermia, or non-obstructive azoospermia (NOA) was analyzed for CNVs via array comparative genomic hybridization (aCGH) and SNVs using whole-exome sequencing (WES). RESULTS: Of the 2544 CNVs identified in individuals with SPGF, > 90% were small, ranging from 0.6 to 75 kb. Thirty, clinically relevant genomic aberrations, were detected in 28 patients (~ 29%). These included likely diagnostic CNVs in 3/41 NOA patients (~ 7%): 1 hemizygous, intragenic TEX11 deletion, 1 hemizygous DDX53 full gene deletion, and 1 homozygous, intragenic STK11 deletion. High-level mosaicism for X chromosome disomy (~ 10% 46,XY and ~ 90% 47,XXY) was also identified in 3 of 41 NOA patients who previously tested normal with conventional karyotyping. The remaining 24 CNVs detected were heterozygous, autosomal recessive carrier variants. Follow-up WES analysis confirmed 8 of 27 (30%) CNVs (X chromosome disomy excluded). WES analysis additionally identified 13 significant SNVs and/or indels in 9 patients (~ 9%) including X-linked AR, KAL1, and NR0B1 variants. CONCLUSION: Using a combined genome-wide aCGH/WES approach, we identified pathogenic and likely pathogenic SNVs and CNVs in 15 patients (15%) with unexplained SPGF. This value equals the detection rate of conventional testing for aneuploidies and is considerably higher than the prevalence of Y chromosome microdeletions. Our results underscore the importance of comprehensive genomic analysis in emerging diagnostic testing of complex conditions like male infertility.
Subject(s)
DNA Copy Number Variations , Oligospermia , Azoospermia , Comparative Genomic Hybridization , DNA Copy Number Variations/genetics , Genetic Testing/methods , High-Throughput Nucleotide Sequencing , Humans , Male , Nucleotides , Oligospermia/diagnosis , Oligospermia/geneticsABSTRACT
Varicocele is a major entity defined within male infertility. In this report we have studied the influence of laparoscopic varicocelectomy on semen quality, biochemical parameters of seminal plasma and sperm DNA fragmentation. In this study, the semen samples from patients with left-side varicocele of grade II-III before and after laparoscopic varicocelectomy were compared to healthy individuals and separated into three groups. The volume of semen, sperm concentration (106/ml), motility (%), viability (%) and normal morphology (%) were assessed. Total antioxidant capacity (TAC), catalase (CAT), superoxide dismutase (SOD) and malondialdehyde (MDA) together with other biochemical substances in seminal plasma as alpha-glucosidase (α-Glu), fructose (Fr) and citric acid (CA) were determined by ELISA method. The spermatozoa activity including ion-transports through sodium, potassium ATPase (Na+, K+-ATPase) and calcium, magnesium ATPase (Ca2+, Mg2+-ATPase) were determined by using spectrophotometry. In addition, flow cytometry method for detection of sperm DNA fragmentation was used. The results showed, that three months after varicocelectomy such intervention led to significant postoperative improvement in volume of semen (p<0.001), total sperm count (p<0.001), sperm motility (p<0.001) and spermatozoa with normal morphology (p<0.001). We found decreased α-Glu levels due to varicocelectomy (p<0.05). There has been shown a high positive correlation between Na+, K+-ATPase and Ca2+, Mg2+-ATPase activity with total number of spermatozoa (p<0.05). The TAC levels and DNA fragmentation values after varicocelectomy can be considered as significant indicators of good prognosis after surgical intervention. It has to be emphasized that α-Glu levels and total sperm count expressed statistically significant both positive and negative predictive values for semen assessment. Varicocelectomy may lead to significant improvement of semen quality although the observations must be correlated with clinical pregnancies observed thereafter.
Subject(s)
Semen , Varicocele , Humans , Pregnancy , Female , Male , Semen Analysis , Varicocele/surgery , Sperm Motility , Spermatozoa/physiology , Sperm Count , DNA , Adenosine TriphosphatasesABSTRACT
Duchenne muscular dystrophy (DMD) is an X-linked lethal disorder caused by mutations in the dystrophin gene. Progression of this disease may lead to cardiomyopathy and respiratory failure, which are the main causes of death among DMD patients. Lack of dystrophin affects cellular myogenic function and related organelles. Dystrophin deficiency results in intracellular Ca2+ dysregulation, mitochondrial dysfunction and induces elevated production of reactive oxygen species (ROS). Due to current findings, mitochondria may be also a potential target for DMD therapy. In this review we attempted to provide an insight into the role of mitochondria in perpetuation of DMD disease.
Subject(s)
Muscular Dystrophy, Duchenne , Humans , Mitochondria , Muscular Dystrophy, Duchenne/genetics , Reactive Oxygen SpeciesABSTRACT
Stem cell therapy in combination with genetic modification (e.g., transfection with the coding sequence for the connexion 43 gene, GJA1) may solve the problems associated with the occurrence of additional (secondary) stimulation in the post-infarcted heart (arrhythmia). Human skeletal muscle-derived stem/progenitor cells (SkMDS/PCs) were transfected with the pCiNeo-GJA1 plasmid at an efficiency of approximately 96%. Gene overexpression was assessed using qPCR, and subsequent analysis revealed that GJA1 expression increased more than 40-fold in SkMDS/PCs transfected with the appropriate coding sequence (SkMDS/PCsCX43) compared to that of the 'native' SkMDS/PCs control (SkMDS/PCsWT). Enhanced (4-fold) protein expression of connexin-43 was also confirmed by Western immunoblotting. Furthermore, using the arrhythmic score, we demonstrated the positive effects of SkMDS/PCsCX43 cell intervention in reducing additional secondary stimulations in rat post-infarcted hearts compared with that of wild-type cell delivery. Selected gene responses (Kcnq1, Cacna1c, Ncx1, Serca2a, and Tgfb1) showed significantly altered expression profiles in the rat myocardium upon intervention with SkMDS/PCsCX43. The genetic modification of human skeletal muscle-derived stem/progenitor cells with connexin-43 prevented the pro-arrhythmic effects of myogenic implanted stem cells on the host myocardium and positively influenced myocardial gene expression profiles in respect to myocardium conductivity.
Subject(s)
Arrhythmias, Cardiac/prevention & control , Connexin 43/metabolism , Myocardial Infarction/therapy , Stem Cell Transplantation/methods , Animals , Arrhythmias, Cardiac/etiology , Connexin 43/genetics , Female , Gene Expression Regulation , Humans , Muscle, Skeletal/cytology , Myocardial Infarction/complications , Myocardium/metabolism , Rats , Rats, Wistar , Stem Cells/cytology , TransfectionABSTRACT
Reduced sperm motility, defined as asthenozoospermia, is a frequent cause of male infertility, and is mainly connected with the dysfunction of sperm mitochondria. The aim of this study was to identify the proteins, and thereby the metabolic pathways, responsible for asthenozoospermia, using 2-DE and MALDI-TOF MS, and correlate the results obtained with those of two mitochondrial tests: JC-1 and MitoSox Red. The JC-1 test was performed to test sperm mitochondrial activity, and the MitoSox Red test was performed to check whether the observed sperm poor motility is associated with mitochondrial reactive oxygen species (ROS) production. To identify proteins strictly connected with reduced sperm motility, men with isolated asthenozoospermia (n = 4 versus 10 normozoospermic controls) alone were included in the study. The proteomic analyses resulted in the identification of 25 sperm proteins that are differentially expressed in asthenozoospermic individuals. Most of the identified proteins were downregulated and were involved in energy production; however, we have also identified structural sperm proteins and proteins secreted by the epididymis. The latter, together with the results from MitoSox Red assay, may provide insights into the pathophysiological basis of asthenozoospermia.
Subject(s)
Asthenozoospermia/metabolism , Mitochondria/metabolism , Oxidative Stress , Spermatozoa/metabolism , Adult , Humans , Male , Proteomics , Young AdultABSTRACT
Stem cells constitute a non-specialised cell pool able for proliferation, self-renewal and differentiation into progeny constantly replacing used up tissue and/or organ fragments. They have been observed to be present in many tissue reservoirs, including stomatognathic system. Oral cavity seems to be a particularly attractive stem cells' source as these cells are richly present and easily accessible in dental and periodontal tissues and they can be used for therapeutic purposes. Their application is also morally and ethically non-controversial. Bone tissue structure restoration together with restoring its weight-bearing and nutritive function depend on the proper function of stem cells supported with other techniques including tissue engineering. Traditionally, bone regeneration means bone restoration supported by newly formed bones supplied by stem cells of tissue reservoir. It may be also indispensable to create a scaffold which may support the bone formation facilitating the transportation of cells and cell stimulating molecules involving both the matrix and bone-forming cells. The regenerative potential of stem cells present in oral cavity can be used, for instance, to restore maxillary and mandibular bones. The bones support natural teeth or prostheses and regress as soon as at least the one tooth is lost or extracted. Bone mass loss makes it difficult to conduct effective dental treatment and reduces the chances of obtaining positive, long-lasting therapeutic effects. It seems that modern and innovative therapies based on stem cells application may bring spectacular effects especially in patients in whom routine medical activities did not lead to satisfactory results.
Subject(s)
Bone Regeneration , Stem Cells , Animals , Dentistry , HumansABSTRACT
Cardiovascular diseases along with MI (myocardial infarction) lead to regional ischaemia and hypoxic conditions, which prevail after infarction. Diminished O2 saturation which is related to elevated level of hypoxia inducible factor 1 (HIF-1) transcription factor, may switch the expression of many genes. To maximize effect of therapies proposed by regenerative medicine, it is essential to verify (within different time points after MI) the expression of proangiogenic genes and their receptors that are regulated, along with the expression of HIF-1α. We demonstrated a connection between the expression of Hif-1α (in murine post infarcted heart model) and the proangiogenic genes Vegf-a; and Plgf and their receptors during myocardial hypoxia. The innovative part of the study required establishment of the most accurate in vitro O2 level corresponding to the hypoxia level prevailing in myocardium after MI. We determined the influence of hypoxia on the biology of human myoblasts in in vitro oxygen conditions (3%), corresponding to those prevailing in the heart after an infarction using a murine model. We also tested myoblasts that were genetically modified with VEGF-A/FGF-4 and PlGF under hypoxic conditions and compared their characteristics with cells cultured under normoxia and hyperoxia (standard in vitro conditions) with respect to myogenic gene expression, cell proliferation, fusion potential and proangiogenic function. The examination of genetically modified myoblasts under optimized in vitro hypoxia conditions led to the conclusion that hypoxia did not negatively influence the biological functions of the myoblasts, such as cell proliferation and/or proangiogenic characteristics. These results support the expected increased proregenerative effects of such genetically modified human myoblasts.
Subject(s)
Gene Expression/genetics , Hypoxia/genetics , Myoblasts/pathology , Myocardial Infarction/genetics , Neovascularization, Pathologic/genetics , Animals , Cell Line , Cell Proliferation/genetics , Disease Models, Animal , Fibroblast Growth Factor 4/genetics , Human Umbilical Vein Endothelial Cells , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Mice , Mice, SCID , Myocardium/pathology , Placenta Growth Factor/genetics , Vascular Endothelial Growth Factor A/geneticsABSTRACT
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.
Subject(s)
Cell Transplantation/methods , Gene Expression Profiling/methods , Gene Expression Regulation , Myoblasts/transplantation , Myocardial Infarction/surgery , RNA/genetics , Ventricular Remodeling/physiology , Animals , Anterior Cruciate Ligament/cytology , Cells, Cultured , Disease Models, Animal , Flow Cytometry , Humans , Immunohistochemistry , Mice , Mice, Inbred NOD , Mice, SCID , Myocardial Infarction/genetics , Myocardial Infarction/metabolism , Myocardium/metabolism , Myocardium/pathology , Real-Time Polymerase Chain Reaction , Ventricular Function, Left/physiologyABSTRACT
The aim of the study was to identify human sperm antigens reacting with polyclonal antisperm antibodies. Protein sperm extracts were subjected to electrofocusing, and next immune reactions (immunoblotting) were carried out with positive for antisperm antibodies and control (not containing antisperm antibodies) serum samples. Proteomic analysis of human sperm proteins resulted in identification of 80 sperm antigens that could be divided into three groups: antigens specific for patients with antisperm antibodies (32), antigens recognised by both infertile patients and control sera (35) and antigens detected by control serum samples only (13). Among antigens specific for infertile patients, there were 12 sperm entities known to be involved in fertilisation process. We have also characterised three protein entities identified only by sera of infertile women. Altogether, the proteomic analysis resulted in identification of 27 sperm entities not reported previously in human sperm proteome. Identified proteins are sperm antigens that could be potentially responsible for immunological infertility. The study also sheds new light on the sperm antigens in aspect of gender specificity. The investigation of human sperm proteome by the use of antisperm antibodies-containing sera of infertile individuals not only may indicate new proteins but also can draft their immunological nature.
Subject(s)
Autoantibodies/blood , Infertility, Male/immunology , Spermatozoa/immunology , Female , Humans , Infertility, Male/blood , Male , ProteomicsABSTRACT
BACKGROUND: The aim of this study is to present results of the implantation of autologous myoblasts into the external anal sphincter (EAS) in ten patients with fecal incontinence. METHODS: After anatomical and functional assessment of the patients' EAS, a vastus lateralis muscle open biopsy was performed. Stem cells were extracted from the biopsy specimens and cultured in vitro. Cell suspensions were then administered to the EAS. Patients were scheduled for follow-up visits in 6-week intervals. Total follow-up was 12 months. RESULTS: All biopsy and cell implantation procedures were performed without complications. Nine of the patients completed a full 12-month follow-up. There was subjective improvement in six patients (66.7 %). In manometric examinations 18 weeks after implantation, squeeze anal pressures and high-pressure zone length increased in all patients, with particularly significant sphincter function recovery in five patients (55.6 %). Electromyographic (EMG) examination showed an increase in signal amplitude in all patients, detecting elevated numbers of propagating action potentials. Twelve months after implantation two patients experienced deterioration of continence, which was also reflected in the deterioration of manometric and EMG parameters. The remaining four patients (44.4 %) still described their continence as better than before implantation and retained satisfactory functional examination parameters. CONCLUSIONS: Implantation of autologous myoblasts gives good short-term results not only in a subjective assessment, but also in objective functional tests. It seems that this promising technology can improve the quality of life of patients with fecal incontinence, but further study is required to achieve better and more persistent results.
Subject(s)
Anal Canal , Fecal Incontinence/surgery , Myoblasts/transplantation , Recovery of Function , Adult , Aged , Anal Canal/physiopathology , Anal Canal/surgery , Electromyography , Fecal Incontinence/physiopathology , Female , Follow-Up Studies , Humans , Male , Manometry , Middle Aged , Pilot Projects , Pressure , Prospective Studies , Quadriceps Muscle/cytology , Quadriceps Muscle/surgery , Transplantation, Autologous/methods , Treatment Outcome , Young AdultABSTRACT
Myocardial infarction results in cardiomyocyte loss and may eventually lead to cardiac failure. Skeletal myoblast transplantation into the scar area may compensate for this observed cell loss by strengthening the weakened myocardium and inducing myogenesis. Moreover, skeletal myoblasts may serve as potential transgene carriers for the myocardium (i.e., delivering pro-angiogenic factors, which may potentially improve blood perfusion in infarcted heart). We examined the influence of the simultaneous overexpression of two potent pro-angiogenic factors, fibroblast growth factor-4 (FGF-4) and vascular endothelial growth factor (VEGF), on human primary myoblast proliferation, cell cycle, resistance to hypoxic stress conditions and myogenic gene expression, as well as the induction of pro-angiogenic activities. We used a bicistronic plasmid vector encoding two factors introduced via an efficient myoblast electroporation method. The levels of overexpressed proteins were assessed, and their functionality at capillary formation was evaluated. This combined approach led to a high level of non-viral transient overexpression of both pro-angiogenic proteins, which proved to be potent regulators of blood vessel development assayed in capillary formation tests. We demonstrated in in vitro conditions that the transfection of human skeletal myoblasts with both FGF-4 and VEGF did not affect their basic biological properties such as the cell cycle, proliferation or expression of myogenic lineage-specific genes, and the modified cells adapted to oxidative stress conditions. Overall, the results obtained suggest that the applied combined approach with the use of two pro-angiogenic genes overexpressed in skeletal muscle stem cells may be an interesting alternative for the effective therapy of myocardial infarction in animal models and/or prospective clinical trials.
Subject(s)
Fibroblast Growth Factor 4/metabolism , Myoblasts, Skeletal/metabolism , Vascular Endothelial Growth Factor A/metabolism , Cell Cycle , Cell Proliferation , Electroporation , Gene Expression , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Myocardial Infarction/therapy , Neovascularization, Physiologic , TransfectionABSTRACT
BACKGROUND: Previously, connexin 43-modified skeletal myoblasts (MbCx) were shown to reduce the pro-arrhythmic effect during the regeneration of heart tissue in an animal model of infarction. To increase the relevance to clinical implementation, in this study, we introduced connexin 43 into human myoblasts using a highly safe non-viral vector and demonstrated that their transplantation had a positive effect on the function of the injured heart. METHODS AND RESULTS: Myoblasts were efficiently transfected with a pCiNeo-GJA1 plasmid (65.72%). qPCR analysis revealed over 32-fold higher expression of the connexin 43 gene in the MbCx cell population compared to 'native' controls. The susceptibility of the myoblasts to oxidative stress conditions (p<0.001) and the fusion index (p<0.01) were increased in the MbCx cells. Additionally, we observed changes in the MYOG and MYH2 gene expression levels in the GJA1-modified myoblasts. Finally, we observed a significant improvement in the post-infarction echocardiographic parameters after intervention using MbCx cells compared with non-transfected myoblasts (MbWt) and the control (0.9% NaCl), wherein a significant decrease in the left ventricular area change in the short axis (SAX AC%) was observed at the two-month follow-up (p<0.05 and p<0.01, respectively). CONCLUSIONS: We demonstrated the positive effect of connexin 43 overexpression on the biology and function of human skeletal myoblasts in the context of their potential clinical applications. Our preclinical studies using a mouse infarction model indicated the positive effect of MbCx implantation on the function of the injured heart.
Subject(s)
Connexin 43/biosynthesis , Heart Failure/metabolism , Heart Failure/therapy , Myoblasts, Skeletal/metabolism , Myoblasts, Skeletal/transplantation , Stem Cell Transplantation/methods , Animals , Cells, Cultured , Heart Failure/pathology , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Myocardial Infarction/metabolism , Myocardial Infarction/pathology , Myocardial Infarction/therapy , Prospective StudiesABSTRACT
The aim of the study was to examine an in vitro effect of the three bacterial strains (Escherichia coli, Staphylococcus haemolyticus and Bacteroides ureolyticus) on ejaculated spermatozoa with reference to sperm membrane integrity and mitochondrial activity. The study was carried out on swim-up-separated spermatozoa from 12 normozoospermic volunteers. Sperm plasma membrane stability was evaluated by the LIVE/DEAD Sperm Viability Kit and by the merocyanine 540 test. Mitochondrial activity was evaluated using the JC-1 test as well as the NADH-dependent NBT assay. The percentage of dead cells was significantly higher in spermatozoa treated with B. ureolyticus as compared to that of control spermatozoa (P < 0.01). All the bacterial strains applied affected sperm plasma membrane architecture measured by M540 test (P < 0.01). Moreover, the presence of E. coli or B. ureolyticus was connected with significant decrease in both the number of cells with high mitochondrial transmembrane potential (ΔΨm) and the cells with normal oxidoreductive function of mitochondria (P < 0.05 as compared to untreated cells). To conclude, the contact of bacteria with ejaculated spermatozoa can be a reason for severe injury of sperm membrane stability and mitochondrial activity with potential consequences for male fertility.
Subject(s)
Bacteroides Infections/physiopathology , Escherichia coli Infections/physiopathology , Mitochondria/physiology , Spermatozoa/microbiology , Staphylococcal Infections/physiopathology , Staphylococcus haemolyticus , Adult , Benzimidazoles , Carbocyanines , Cell Membrane/physiology , Cell Survival , Humans , Infertility, Male/microbiology , Male , Pyrimidinones , Spermatozoa/drug effects , Spermatozoa/physiologyABSTRACT
Cardiomyocyte loss in the ischaemic heart can be the reason of many complications, eventually being even the cause of patient's death. Despite many promises, cell therapy with the use of skeletal muscle stem cells (SMSC) still remains to be modified and improved. Combined cell and gene therapy seems to be a promising strategy to heal damaged myocardium. In the present study we have investigated the influence of a simultaneous overexpression of two potent pro-angiogenic genes encoding the fibroblast growth factor-4 (FGF-4) and the vascular endothelial growth factor-A (VEGF-A) on a myogenic murine C2C12 cell line. We have demonstrated in in vitro conditions that myoblasts which overexpressed these factors exhibited significant changes in the cell cycle and pro-angiogenic potential with only slight differences in the expression of the myogenic genes. There was not observed the influence of transient or stable overexpression of FGF-4 and VEGF on cell apoptosis/necrosis in standard or oxidative stress conditions comparing to non transfected controls. Overall, our results suggest that the possible transplantation of myoblasts overexpressing pro-angiogenic factors may potentially improve the functionality of the injured myocardium although the definite proof must originate from in situ conducted pre-clinical studies.
Subject(s)
Fibroblast Growth Factor 4/genetics , Myoblasts/physiology , Neovascularization, Physiologic/genetics , Transfection/methods , Vascular Endothelial Growth Factor A/genetics , Animals , Cell Line , Cell Proliferation , Cells, Cultured , Endothelial Cells/cytology , Endothelial Cells/physiology , Fibroblast Growth Factor 4/biosynthesis , Humans , Mice , Myoblasts/cytology , Neovascularization, Physiologic/physiology , Vascular Endothelial Growth Factor A/biosynthesisABSTRACT
Principal definitions of stem cell subdivisions as well as of the physiology of the renewal of their descendants have been elucidated in recent years. Regeneration mechanisms have been outlined using as an example the intestinal villus niche. Sources of stem cells for preclinical studies and the main conclusions from clinical trials have been developed in the vast majority in the 21st century. Meta-analyses and summaries have been focused so far on bone marrow stem cells and muscle-derived stem cells, which have been most often tried to date. Polish clinical trials on postinfarcted hearts have been consistent with the world literature regarding the major conclusions for myocardial regeneration. The controversies include possible side effects of stem cell applications. The necessity for genetic modification of the stem cells, which are mainly myoblasts, has been justified by the results of recently performed trials, initial examples including transfections of proangiogenic factors into human primary myoblast suspensions.
Subject(s)
Cell Transplantation , Heart/physiopathology , Myoblasts/cytology , Myocardial Infarction/therapy , Regeneration , Clinical Trials as Topic , Humans , Myocardial Infarction/pathologyABSTRACT
The transcription levels of stem cell factor (SCF) and c-kit were examined using real-time RT PCR in interstitial and intratubular cell fractions, as well as in tissue homogenates from normal, azoospermic and neoplasmic patients. Peripheral blood mononuclear cells (PBMC) were used as a systemic control. The observed level of c-kit expression in all investigated groups was generally higher than the expression of SCF. The highest (statistically significant) level of c-kit was noted in testicular tumours (the greater part of which were represented by seminomas) in contrast to SCF mRNA, which may indicate an association between c-kit overexpression and seminoma development. In Sertoli cell only syndrome, almost equal levels of SCF and c-kit transcripts were noted. These results may indicate Leydig cells as the alternative source of c-kit gene transcription. SCF transcript values were low and comparable among the analysed subgroups except that in maturation arrest at spermatocyte stage, the SCF gene expression was statistically higher than in testicular tumours. It appears from the study that c-kit has been a dynamic gene, changing its activity in a variety of testicular pathologies while being expressed in all testicular compartments but clearly overexpressed in testicular tumours of seminomatous origin.
Subject(s)
Azoospermia/metabolism , Proto-Oncogene Proteins c-kit/biosynthesis , Seminoma/metabolism , Stem Cell Factor/biosynthesis , Testicular Neoplasms/metabolism , Testis/metabolism , Adolescent , Adult , Aged , Aged, 80 and over , Gene Expression , Humans , Male , Sertoli Cell-Only Syndrome/metabolism , Spermatogenesis/geneticsABSTRACT
This aim of the study was to investigate whether human leukocyte antigen (HLA)-DQA1*0505 sharing or the maternal killer immunoglobulin-like receptor (KIR) repertoire is associated with recurrent spontaneous abortion (RSA) or repeated implantation failure (RIF). The study included 224 couples with RSA, 61 couples with RIF, 182 fertile couples, and 10 couples with successful in vitro fertilization and embryo transfer (IVF)/ET at first cycle. HLA-DQA1*0505 typing using polymerase chain reaction-sequence-specific oligonucleotide (PCR-SSO) was performed in 185 RSA (117 with alloimmune abnormalities and 68 of autoimmune etiology), 61 RIF and 182 control couples, and KIR genotyping using polymerase chain reaction-sequence-specific primer (PCR-SSP) in 167 RSA and 55 RIF cases as well as 46 RSA and 10 IVF controls. No differences in DQA1*0505 sharing were found between patients and controls. In RSA and RIF women, the ratio of inhibitory to activating KIRs was slightly lower (1.53 and 1.85 vs 2.03 in controls). The analysis of maternal inhKIR and fetal HLA-C molecule pairs showed that the 'less inhibiting' combination KIR2DL3-C1 was found in higher percentage in subfertile (mainly RIF) than in fertile couples. In contrast, the percentage of cases possessing the 'strong inhibiting' combination KIR2DL1-C2 was lower in the RSA and RIF groups in comparison with that in the control groups (17.36% vs 23.91 and 16.36% vs 40%, respectively). In women with >or= 6 implantation failures, the KIR2DL1-C2 combination was not found in any of them (P = 0.0014), and the KIR2DL3-C1 combination was not found in the control IVF group. The results oppose the suggestion that increased HLA-DQA1*0505 sharing predispose to RSA or RIF. The KIR2DL3-C1 combination (or lack of the KIR2DL1-C2 one) is associated with implantation failure.
Subject(s)
Abortion, Habitual/genetics , Abortion, Spontaneous/genetics , Autoimmune Diseases/genetics , Autoimmunity/genetics , HLA-DQ Antigens/genetics , Receptors, KIR/genetics , Abortion, Habitual/immunology , Abortion, Spontaneous/immunology , Adult , Embryo Implantation/genetics , Embryo Transfer , Female , Fertilization in Vitro , Genetic Predisposition to Disease , Genotype , HLA-DQ alpha-Chains , Homozygote , Humans , Male , Maternal-Fetal Relations , Young AdultABSTRACT
The chromosomal anomalies, microdeletions of AZF region of Y-chromosome and CFTR gene mutations have been studied among 80 infertile men with idiopathic spermatogenetic failure: 36 (45%) patients with aspermia, 19 (24%) patients with azoospermia and 25 (31%) patients with severe oligoasthenoteratozoospermia. In total 30% males with spermatogenetic failure genetic factor of infertility was observed. Karyotype anomalies were observed in 17.5% of infertile men, within 16.2% numerical and structural gonosomal anomalies and in 1.3%--Robertsonian translocation were revealed. In 11% males with spermatogenetic failure, Y-chromosome AZF region microdeletions were detected. The frequency of CFTR major mutation F508del among infertile men was 6.25%. 5T allele of polymorphic locus IVS8polyT was detected in 7.5% of examined men. The results obtained indicate the high complexity of cytogenetic and molecular-genetic studies of male infertility.