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1.
Hum Reprod Open ; 2024(2): hoae020, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38650655

RESUMO

STUDY QUESTION: Is the Tcte1 mutation causative for male infertility? SUMMARY ANSWER: Our collected data underline the complex and devastating effect of the single-gene mutation on the testicular molecular network, leading to male reproductive failure. WHAT IS KNOWN ALREADY: Recent data have revealed mutations in genes related to axonemal dynein arms as causative for morphology and motility abnormalities in spermatozoa of infertile males, including dysplasia of fibrous sheath (DFS) and multiple morphological abnormalities in the sperm flagella (MMAF). The nexin-dynein regulatory complex (N-DRC) coordinates the dynein arm activity and is built from the DRC1-DRC7 proteins. DRC5 (TCTE1), one of the N-DRC elements, has already been reported as a candidate for abnormal sperm flagella beating; however, only in a restricted manner with no clear explanation of respective observations. STUDY DESIGN SIZE DURATION: Using the CRISPR/Cas9 genome editing technique, a mouse Tcte1 gene knockout line was created on the basis of the C57Bl/6J strain. The mouse reproductive potential, semen characteristics, testicular gene expression levels, sperm ATP, and testis apoptosis level measurements were then assessed, followed by visualization of N-DRC proteins in sperm, and protein modeling in silico. Also, a pilot genomic sequencing study of samples from human infertile males (n = 248) was applied for screening of TCTE1 variants. PARTICIPANTS/MATERIALS SETTING METHODS: To check the reproductive potential of KO mice, adult animals were crossed for delivery of three litters per caged pair, but for no longer than for 6 months, in various combinations of zygosity. All experiments were performed for wild-type (WT, control group), heterozygous Tcte1+/- and homozygous Tcte1-/- male mice. Gross anatomy was performed on testis and epididymis samples, followed by semen analysis. Sequencing of RNA (RNAseq; Illumina) was done for mice testis tissues. STRING interactions were checked for protein-protein interactions, based on changed expression levels of corresponding genes identified in the mouse testis RNAseq experiments. Immunofluorescence in situ staining was performed to detect the N-DRC complex proteins: Tcte1 (Drc5), Drc7, Fbxl13 (Drc6), and Eps8l1 (Drc3) in mouse spermatozoa. To determine the amount of ATP in spermatozoa, the luminescence level was measured. In addition, immunofluorescence in situ staining was performed to check the level of apoptosis via caspase 3 visualization on mouse testis samples. DNA from whole blood samples of infertile males (n = 137 with non-obstructive azoospermia or cryptozoospermia, n = 111 samples with a spectrum of oligoasthenoteratozoospermia, including n = 47 with asthenozoospermia) was extracted to perform genomic sequencing (WGS, WES, or Sanger). Protein prediction modeling of human-identified variants and the exon 3 structure deleted in the mouse knockout was also performed. MAIN RESULTS AND THE ROLE OF CHANCE: No progeny at all was found for the homozygous males which were revealed to have oligoasthenoteratozoospermia, while heterozygous animals were fertile but manifested oligozoospermia, suggesting haploinsufficiency. RNA-sequencing of the testicular tissue showed the influence of Tcte1 mutations on the expression pattern of 21 genes responsible for mitochondrial ATP processing or linked with apoptosis or spermatogenesis. In Tcte1-/- males, the protein was revealed in only residual amounts in the sperm head nucleus and was not transported to the sperm flagella, as were other N-DRC components. Decreased ATP levels (2.4-fold lower) were found in the spermatozoa of homozygous mice, together with disturbed tail:midpiece ratios, leading to abnormal sperm tail beating. Casp3-positive signals (indicating apoptosis) were observed in spermatogonia only, at a similar level in all three mouse genotypes. Mutation screening of human infertile males revealed one novel and five ultra-rare heterogeneous variants (predicted as disease-causing) in 6.05% of the patients studied. Protein prediction modeling of identified variants revealed changes in the protein surface charge potential, leading to disruption in helix flexibility or its dynamics, thus suggesting disrupted interactions of TCTE1 with its binding partners located within the axoneme. LARGE SCALE DATA: All data generated or analyzed during this study are included in this published article and its supplementary information files. RNAseq data are available in the GEO database (https://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE207805. The results described in the publication are based on whole-genome or exome sequencing data which includes sensitive information in the form of patient-specific germline variants. Information regarding such variants must not be shared publicly following European Union legislation, therefore access to raw data that support the findings of this study are available from the corresponding author upon reasonable request. LIMITATIONS REASONS FOR CAUTION: In the study, the in vitro fertilization performance of sperm from homozygous male mice was not checked. WIDER IMPLICATIONS OF THE FINDINGS: This study contains novel and comprehensive data concerning the role of TCTE1 in male infertility. The TCTE1 gene is the next one that should be added to the 'male infertility list' because of its crucial role in spermatogenesis and proper sperm functioning. STUDY FUNDING/COMPETING INTERESTS: This work was supported by National Science Centre in Poland, grants no.: 2015/17/B/NZ2/01157 and 2020/37/B/NZ5/00549 (to M.K.), 2017/26/D/NZ5/00789 (to A.M.), and HD096723, GM127569-03, NIH SAP #4100085736 PA DoH (to A.N.Y.). The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

2.
Sci Rep ; 13(1): 16563, 2023 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-37783880

RESUMO

Infertility is a problem that affects approximately 15% of couples, and male infertility is responsible for 40-50% of these cases. The cause of male infertility is still poorly diagnosed and treated. One of the prominent causes of male infertility is disturbed spermatogenesis, which can lead to nonobstructive azoospermia (NOA). Whole-genome sequencing (WGS) allows us to identify novel rare variants in potentially NOA-associated genes, among others, in the ESX1 gene. The aim of this study was to activate the ESX1 gene using CRISPRa technology in human germ cells (testicular seminoma cells-TCam-2). Successful activation of the ESX1 gene in TCam-2 cells using the CRISPRa system was achieved, and the expression level of the ESX1 gene was significantly higher in modified TCam-2 cells than in WT cells or the negative control with nontargeted gRNA (p < 0.01). Using RNA-seq, a network of over 50 genes potentially regulated by the ESX1 gene was determined. Finally, 6 genes, NANOG, CXCR4, RPS6KA5, CCND1, PDE1C, and LINC00662, participating in cell proliferation and differentiation were verified in azoospermic patients with and without a mutation in the ESX1 gene as well as in men with normal spermatogenesis, where inverse correlations in the expression levels of the observed genes were noted.


Assuntos
Azoospermia , Infertilidade Masculina , Humanos , Masculino , Azoospermia/genética , Azoospermia/metabolismo , Infertilidade Masculina/genética , Espermatogênese/genética , Mutação , Testículo/metabolismo
3.
Sci Rep ; 12(1): 4016, 2022 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-35256767

RESUMO

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuronal disorder characterized by neuronal degeneration and currently no effective cure is available to stop or delay the disease from progression. Transplantation of murine glial-restricted precursors (mGRPs) is an attractive strategy to modulate ALS development and advancements such as the use of immune modulators could potentially extend graft survival and function. Using a well-established ALS transgenic mouse model (SOD1G93A), we tested mGRPs in combination with the immune modulators synthetic PreImplantation Factor (sPIF), Tacrolimus (Tac), and Costimulatory Blockade (CB). We report that transplantation of mGRPs into the cisterna magna did not result in increased mice survival. The addition of immunomodulatory regimes again did not increase mice lifespan but improved motor functions and sPIF was superior compared to other immune modulators. Immune modulators did not affect mGRPs engraftment significantly but reduced pro-inflammatory cytokine production. Finally, sPIF and CB reduced the number of microglial cells and prevented neuronal number loss. Given the safety profile and a neuroprotective potential of sPIF, we envision its clinical application in near future.


Assuntos
Esclerose Lateral Amiotrófica , Transtornos Motores , Neuroglia , Peptídeos , Transplante de Células-Tronco , Esclerose Lateral Amiotrófica/genética , Animais , Modelos Animais de Doenças , Inflamação , Camundongos , Camundongos Transgênicos , Transtornos Motores/tratamento farmacológico , Transtornos Motores/terapia , Neuroglia/citologia , Neuroglia/transplante , Peptídeos/farmacologia , Células-Tronco/citologia
4.
Cells ; 12(1)2022 12 26.
Artigo em Inglês | MEDLINE | ID: mdl-36611889

RESUMO

Stem cells seem to hold major promise for contemporary medicine, one which could almost be more significant than a discovery of DNA and ultimate its relevance for organismal integration in the past century [...].


Assuntos
Medicina Regenerativa , Transplante de Células-Tronco , Biologia
5.
Int J Mol Sci ; 22(22)2021 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-34830471

RESUMO

Cardiovascular diseases (CVD), with myocardial infarction (MI) being one of the crucial components, wreak havoc in developed countries. Advanced imaging technologies are required to obtain quick and widely available diagnostic data. This paper describes a multimodal approach to in vivo perfusion imaging using the novel SYN1 tracer based on the fluorine-18 isotope. The NOD-SCID mice were injected intravenously with SYN1 or [18F] fluorodeoxyglucose ([18F]-FDG) radiotracers after induction of the MI. In all studies, the positron emission tomography-computed tomography (PET/CT) technique was used. To obtain hemodynamic data, mice were subjected to magnetic resonance imaging (MRI). Finally, the biodistribution of the SYN1 compound was performed using Wistar rat model. SYN1 showed normal accumulation in mouse and rat hearts, and MI hearts correctly indicated impaired cardiac segments when compared to [18F]-FDG uptake. In vivo PET/CT and MRI studies showed statistical convergence in terms of the size of the necrotic zone and cardiac function. This was further supported with RNAseq molecular analyses to correlate the candidate function genes' expression, with Serpinb1c, Tnc and Nupr1, with Trem2 and Aldolase B functional correlations showing statistical significance in both SYN1 and [18F]-FDG. Our manuscript presents a new fluorine-18-based perfusion radiotracer for PET/CT imaging that may have importance in clinical applications. Future research should focus on confirmation of the data elucidated here to prepare SYN1 for first-in-human trials.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Coração/diagnóstico por imagem , Infarto do Miocárdio/genética , Proteínas de Neoplasias/genética , Serpinas/genética , Tenascina/genética , Animais , Meios de Contraste/farmacologia , Fluordesoxiglucose F18/farmacologia , Frutose-Bifosfato Aldolase/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Coração/efeitos dos fármacos , Humanos , Imageamento por Ressonância Magnética , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Infarto do Miocárdio/patologia , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Ratos , Receptores Imunológicos/genética , Distribuição Tecidual/efeitos dos fármacos
6.
Int J Mol Sci ; 22(19)2021 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-34639225

RESUMO

Current treatment protocols for myocardial infarction improve the outcome of disease to some extent but do not provide the clue for full regeneration of the heart tissues. An increasing body of evidence has shown that transplantation of cells may lead to some organ recovery. However, the optimal stem cell population has not been yet identified. We would like to propose a novel pro-regenerative treatment for post-infarction heart based on the combination of human skeletal myoblasts (huSkM) and mesenchymal stem cells (MSCs). huSkM native or overexpressing gene coding for Cx43 (huSKMCx43) alone or combined with MSCs were delivered in four cellular therapeutic variants into the healthy and post-infarction heart of mice while using molecular reporter probes. Single-Photon Emission Computed Tomography/Computed Tomography (SPECT/CT) performed right after cell delivery and 24 h later revealed a trend towards an increase in the isotopic uptake in the post-infarction group of animals treated by a combination of huSkMCx43 with MSC. Bioluminescent imaging (BLI) showed the highest increase in firefly luciferase (fluc) signal intensity in post-infarction heart treated with combination of huSkM and MSCs vs. huSkM alone (p < 0.0001). In healthy myocardium, however, nanoluciferase signal (nanoluc) intensity varied markedly between animals treated with stem cell populations either alone or in combinations with the tendency to be simply decreased. Therefore, our observations seem to show that MSCs supported viability, engraftment, and even proliferation of huSkM in the post-infarction heart.


Assuntos
Células-Tronco Mesenquimais/citologia , Imagem Molecular/métodos , Mioblastos Esqueléticos/citologia , Infarto do Miocárdio/patologia , Miocárdio/patologia , Animais , Modelos Animais de Doenças , Genes Reporter , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mioblastos Esqueléticos/metabolismo , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo
7.
Sci Rep ; 11(1): 19825, 2021 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-34615887

RESUMO

Preclinical and clinical studies have shown that stem cells can promote the regeneration of damaged tissues, but therapeutic protocols need better quality control to confirm the location and number of transplanted cells. This study describes in vivo imaging while assessing reporter gene expression by its binding to a radiolabelled molecule to the respective receptor expressed in target cells. Five mice underwent human skeletal muscle-derived stem/progenitor cell (huSkMDS/PC EF1-HSV-TK) intracardial transplantation after induction of myocardial infarction (MI). The metabolic parameters of control and post-infarction stem progenitor cell-implanted mice were monitored using 2-deoxy-18F-fluorodeoxyglucose ([18F]-FDG) before and after double promotor/reporter probe imaging with 9-(4-18F-fluoro-3-[hydroxymethyl]butyl)guanine ([18F]-FHBG) using positron emission tomography (PET) combined with computed tomography (CT). Standardized uptake values (SUVs) were then calculated based on set regions of interest (ROIs). Experimental animals were euthanized after magnetic resonance imaging (MRI). Molecular [18F]-FHBG imaging of myogenic stem/progenitor cells in control and post-infarction mice confirmed the survival and proliferation of transplanted cells, as shown by an increased or stable signal from the PET apparatus throughout the 5 weeks of monitoring. huSkMDS/PC EF1-HSV-TK transplantation improved cardiac metabolic ([18F]-FDG with PET) and haemodynamic (MRI) parameters. In vivo PET/CT and MRI revealed that the precise use of a promotor/reporter probe incorporated into stem/progenitor cells may improve non-invasive monitoring of targeted cellular therapy in the cardiovascular system.


Assuntos
Fluordesoxiglucose F18 , Imagem Molecular , Mioblastos Cardíacos/metabolismo , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Células-Tronco Adultas/metabolismo , Animais , Modelos Animais de Doenças , Ecocardiografia , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Imageamento por Ressonância Magnética , Masculino , Camundongos , Camundongos SCID , Imagem Molecular/métodos , Infarto do Miocárdio/diagnóstico por imagem , Infarto do Miocárdio/etiologia , Poliésteres
8.
Cells ; 10(7)2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34359973

RESUMO

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease, causing motor neuron and skeletal muscle loss and death. One of the promising therapeutic approaches is stem cell graft application into the brain; however, an immune reaction against it creates serious limitations. This study aimed to research the efficiency of glial restricted progenitors (GRPs) grafted into murine CNS (central nervous system) in healthy models and the SOD1G93A ALS disease model. The cellular grafts were administered in semiallogenic and allogeneic settings. To investigate the models of immune reaction against grafted GRPs, we applied three immunosuppressive/immunomodulatory regimens: preimplantation factor (PiF); Tacrolimus; and CTLA-4, MR1 co-stimulatory blockade. We tracked the cells with bioluminescence imaging (BLI) in vivo to study their survival. The immune response character was evaluated with brain tissue assays and multiplex ELISA in serum and cerebrospinal fluid (CSF). The application of immunosuppressive drugs is disputable when considering cellular transplants into the immune-privileged site/brain. However, our data revealed that semiallogenic GRP graft might survive inside murine CNS without the necessity to apply any immunomodulation or immunosuppression, whereas, in the situation of allogeneic mouse setting, the combination of CTLA-4, MR1 blockade can be considered as the best immunosuppressive option.


Assuntos
Sistema Nervoso Central/efeitos dos fármacos , Terapia de Imunossupressão , Imunossupressores/uso terapêutico , Doenças Neurodegenerativas/tratamento farmacológico , Células-Tronco , Animais , Sistema Nervoso Central/imunologia , Modelos Animais de Doenças , Tolerância Imunológica/efeitos dos fármacos , Terapia de Imunossupressão/métodos , Camundongos , Doenças Neurodegenerativas/imunologia , Transplante de Células-Tronco/métodos , Células-Tronco/imunologia
9.
Sci Rep ; 10(1): 14336, 2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32868771

RESUMO

Although less attention was paid to understanding physical localization changes in cell nuclei recently, depicting chromatin interaction maps is a topic of high interest. Here, we focused on defining extensive physical changes in chromatin organization in the process of skeletal myoblast differentiation. Based on RNA profiling data and 3D imaging of myogenic (NCAM1, DES, MYOG, ACTN3, MYF5, MYF6, ACTN2, and MYH2) and other selected genes (HPRT1, CDH15, DPP4 and VCAM1), we observed correlations between the following: (1) expression change and localization, (2) a gene and its genomic neighbourhood expression and (3) intra-chromosome and microscopical locus-centromere distances. In particular, we demonstrated the negative regulation of DPP4 mRNA (p < 0.001) and protein (p < 0.05) in differentiated myotubes, which coincided with a localization change of the DPP4 locus towards the nuclear lamina (p < 0.001) and chromosome 2 centromere (p < 0.001). Furthermore, we discuss the possible role of DPP4 in myoblasts (supported by an inhibition assay). We also provide positive regulation examples (VCAM1 and MYH2). Overall, we describe for the first time existing mechanisms of spatial gene expression regulation in myoblasts that might explain the issue of heterogenic responses observed during muscle regenerative therapies.


Assuntos
Diferenciação Celular , Cromatina/metabolismo , Dipeptidil Peptidase 4/metabolismo , Expressão Gênica , Mioblastos Esqueléticos/metabolismo , Humanos , Mioblastos Esqueléticos/citologia
10.
Cells ; 9(5)2020 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-32365922

RESUMO

Duchenne muscular dystrophy (DMD) is a genetic disorder associated with a progressive deficiency of dystrophin that leads to skeletal muscle degeneration. In this study, we tested the hypothesis that a co-transplantation of two stem/progenitor cell populations, namely bone marrow-derived mesenchymal stem cells (BM-MSCs) and skeletal muscle-derived stem/progenitor cells (SM-SPCs), directly into the dystrophic muscle can improve the skeletal muscle function of DMD patients. Three patients diagnosed with DMD, confirmed by the dystrophin gene mutation, were enrolled into a study approved by the local Bioethics Committee (no. 79/2015). Stem/progenitor cells collected from bone marrow and skeletal muscles of related healthy donors, based on HLA matched antigens, were expanded in a closed MC3 cell culture system. A simultaneous co­transplantation of BM-MSCs and SM-SPCs was performed directly into the biceps brachii (two patients) and gastrocnemius (one patient). During a six­month follow­up, the patients were examined with electromyography (EMG) and monitored for blood kinase creatine level. Muscle biopsies were examined with histology and assessed for dystrophin at the mRNA and protein level. A panel of 27 cytokines was analysed with multiplex ELISA. We did not observe any adverse effects after the intramuscular administration of cells. The efficacy of BM­MSC and SM­SPC application was confirmed through an EMG assessment by an increase in motor unit parameters, especially in terms of duration, amplitude range, area, and size index. The beneficial effect of cellular therapy was confirmed by a decrease in creatine kinase levels and a normalised profile of pro-inflammatory cytokines. BM-MSCs may support the pro-regenerative potential of SM-SPCs thanks to their trophic, paracrine, and immunomodulatory activity. Both applied cell populations may fuse with degenerating skeletal muscle fibres in situ, facilitating skeletal muscle recovery. However, further studies are required to optimise the dose and timing of stem/progenitor cell delivery.


Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Desenvolvimento Muscular , Músculo Esquelético/fisiopatologia , Distrofia Muscular de Duchenne/fisiopatologia , Doadores de Tecidos , Adolescente , Adulto , Biópsia , Fusão Celular , Criança , Citocinas/sangue , Distrofina/genética , Distrofina/metabolismo , Eletromiografia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/sangue , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Neurônios Motores/patologia , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/sangue , Distrofia Muscular de Duchenne/patologia , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Resultado do Tratamento , Adulto Jovem
11.
Cell Transplant ; 28(9-10): 1140-1154, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31124369

RESUMO

Neurodegeneration can be defined as a process in which neuronal structures and functions undergo changes leading to reduced neuronal survival and increased cell death in the central nervous system (CNS). Neuronal degeneration in specific regions of the CNS is a hallmark of many neurodegenerative disorders, and there is reliable proof that neural stem cells bring therapeutic benefits in treatment of neurological lesions. However, effective therapy with neural stem cells is associated with their biological properties. The assessment of immunological properties and comprehensive studies on the biology of glial restricted progenitors (GRP) are necessary prior to the application of these cells in humans. This study provides an in vitro characterization of the QSV40 glial human cell line, as well as murine and canine primary culture suspensions of GRPs and their mature, astrocytic forms using flow cytometry and immunohistochemical staining. Cytokines and chemokines released by GRPs were assessed by Multiplex ELISA. Some immunological differences observed among species suggest the necessity of reconsidering the pre-clinical model, and that careful testing of immunomodulatory strategies is required before cell transplantation into the CNS can be undertaken.


Assuntos
Doenças Neurodegenerativas , Neuroglia , Transplante de Células-Tronco , Células-Tronco , Animais , Técnicas de Cultura de Células , Linhagem Celular Transformada , Citocinas/metabolismo , Modelos Animais de Doenças , Cães , Humanos , Camundongos , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/terapia , Neuroglia/metabolismo , Neuroglia/patologia , Células-Tronco/metabolismo , Células-Tronco/patologia
12.
Oncotarget ; 9(65): 32466-32477, 2018 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-30197756

RESUMO

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.

13.
Cell Transplant ; 27(7): 1047-1067, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29947252

RESUMO

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 ß-adrenergic stimulation.


Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Adulto , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Cariótipo , Masculino , Desenvolvimento Muscular , Mioblastos Esqueléticos/citologia , Mioblastos Esqueléticos/metabolismo , Miócitos Cardíacos/metabolismo , Fatores de Tempo , Adulto Jovem
14.
Sci Rep ; 8(1): 3682, 2018 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-29487326

RESUMO

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.


Assuntos
Diagnóstico por Imagem/métodos , Nanopartículas de Magnetita/química , Mioblastos/metabolismo , Apoptose , Meios de Contraste/química , Compostos Férricos/química , Humanos , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Nanopartículas de Magnetita/ultraestrutura , Espectrometria de Massas , Microscopia Eletrônica de Transmissão , Espécies Reativas de Oxigênio/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier
15.
Arch Immunol Ther Exp (Warsz) ; 66(5): 341-354, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29536116

RESUMO

Muscular dystrophies represent a group of diseases which may develop in several forms, and severity of the disease is usually associated with gene mutations. In skeletal muscle regeneration and in muscular dystrophies, both innate and adaptive immune responses are involved. The regenerative potential of mesenchymal stem/stromal cells (MSCs) of bone marrow origin was confirmed by the ability to differentiate into diverse tissues and by their immunomodulatory and anti-inflammatory properties by secretion of a variety of growth factors and anti-inflammatory cytokines. Skeletal muscle comprises different types of stem/progenitor cells such as satellite cells and non-satellite stem cells including MSCs, interstitial stem cells positive for stress mediator PW1 expression and negative for PAX7 called PICs (PW1+/PAX7- interstitial cells), fibro/adipogenic progenitors/mesenchymal stem cells, muscle side population cells and muscle resident pericytes, and all of them actively participate in the muscle regeneration process. In this review, we present biological properties of MSCs of bone marrow origin and a heterogeneous population of muscle-resident stem/progenitor cells, their interaction with the inflammatory environment of dystrophic muscle and potential implications for cellular therapies for muscle regeneration. Subsequently, we propose-based on current research results, conclusions, and our own experience-hypothetical mechanisms for modulation of the complete muscle regeneration process to treat muscular dystrophies.


Assuntos
Células da Medula Óssea/fisiologia , Inflamação/imunologia , Células-Tronco Mesenquimais/fisiologia , Desenvolvimento Muscular , Músculo Esquelético/fisiologia , Distrofias Musculares/imunologia , Células-Tronco/fisiologia , Animais , Regeneração Tecidual Guiada , Humanos , Imunidade , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Fator de Transcrição PAX7/metabolismo
16.
Arch Immunol Ther Exp (Warsz) ; 66(2): 145-159, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28951939

RESUMO

Cardiovascular diseases are a growing problem in developing countries; therefore, there is an ongoing intensive search for new approaches to treat these disorders. Currently, cellular therapies are focused on healing the damaged heart by implanting stem cells modified with pro-angiogenic factors. This approach ensures that the introduced cells are capable of fulfilling the complex requirements of the environment, including the replacement of the post-infarction scar with cells that are able to contract and promote the formation of new blood vessels that can supply the ischaemic region with nutrients and oxygen. This study focused on the genetic modification of human skeletal muscle cells (SkMCs). We chose myoblast cells due to their close biological resemblance to cardiomyocytes and the placental growth factor (PlGF) gene due to its pro-angiogenic potential. In our in vitro studies, we transfected SkMCs with the PlGF gene using electroporation, which has previously been proven to be efficient and generate robust overexpression of the PlGF gene and elevate PlGF protein secretion. Moreover, the functionality of the secreted pro-angiogenic proteins was confirmed using an in vitro capillary development assay. We have also examined the influence of PlGF overexpression on VEGF-A and VEGF-B, which are well-known factors described in the literature as the most potent activators of blood vessel formation. We were able to confirm the overexpression of VEGF-A in myoblasts transfected with the PlGF gene. The results obtained in this study were further verified in an animal model. These data were able to confirm the potential therapeutic effects of the applied treatments.


Assuntos
Proteínas de Membrana/metabolismo , Músculo Esquelético/citologia , Mioblastos/fisiologia , Infarto do Miocárdio/terapia , Miócitos Cardíacos/fisiologia , Transplante de Células-Tronco , Animais , Diferenciação Celular , Modelos Animais de Doenças , Engenharia Genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos SCID , Mioblastos/transplante , Neovascularização Fisiológica/genética , Transgenes/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator B de Crescimento do Endotélio Vascular/metabolismo
17.
Postepy Hig Med Dosw (Online) ; 71(0): 975-987, 2017 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-29225198

RESUMO

There is a variety of mechanisms(s) factor(s) that may influence stem cell therapies for heart regeneration. Among the best candidates for stem cell source are: mesenchymal stem cells (also those isolated from adipose tissue), cardiac cell progenitors (CPC) and descendants of iPSC cells. iPSC/s can be potentially beneficial although their pluripotent induction has been still in question due to: low propagation efficacy, danger of genomic integration/instability, biological risk of current vector system teratoma formation etc. which have been discussed in this review. Optimization protocols are required in order to enhance stem cells resistance to pathological conditions that they may encounter in pathological organ and to increase their retention. Combination between gene transfer and stem cell therapy is now more often used in pre-clinical studies with the prospect of subsequent clinical trials. Complementary substances have been contemplated to support stem cell viability (mainly anti-inflammatory and anti- apoptotic agents), which have been tested in animal models with promising results. Integration of nanotechnology both for efficient stem cell imaging as well as with the aim to provide cell supporting scaffolds seem to be inevitable for further development of cellular therapies. The whole organ (heart) reconstruction as well as biodegradable scaffolds and scaffold-free cell sheets have been also outlined.


Assuntos
Doenças Cardiovasculares/terapia , Coração/fisiologia , Transplante de Células-Tronco Mesenquimais/métodos , Regeneração , Transplante de Células-Tronco/métodos , Engenharia Tecidual/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Diferenciação Celular/fisiologia , Sobrevivência Celular/fisiologia , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Masculino , Células-Tronco Mesenquimais/fisiologia , Pessoa de Meia-Idade
18.
Postepy Hig Med Dosw (Online) ; 71(0): 881-894, 2017 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-29151064

RESUMO

The purpose of this review is to present the current knowledge regarding the hierarchy of stem cells originating from the oral cavity, which could have a potential value when applied to regenerative stomatology. It must be particularly emphasized that the heterogenous nature of its biology and function within oral compartment may predispose them to different types of applications. Stem cells can be perceived as immature, primitive and unspecialized types of cells with the ability to proliferate, self-renew and differentiate into specialized progeny according to the compartmental signaling. Their presence in tissue reservoirs was already discovered in many organs and tissues as well as in the stomatognathic system. The oral cavity appears to be an exceptionally attractive site to acquire stem cells. The common presence and easy access to these cells in dental and peridental tissues provides a real chance to apply them for therapeutic purposes. Such an opportunity would also be neutral to bioethical and moral issues, assuming autologous stem cells employment. Many authors suspect that stem cells have epigenetic memory, so some of their features can be inherited through generations. They are not connected, however, with DNA sequence modifications. It is, therefore, justified to apply the cells, which have the oral cavity as their natural reservoir, in interventions associated with tissue engineering within the stomatognathic system. An increasing number of clinical trials, among which the number of randomized studies with large group of patients is progressively carried out, allows for a prediction that shortly therapeutic methods based on stem cells of dental origin may be implemented to the routine repertoire of clinical practice.


Assuntos
Células-Tronco Mesenquimais , Células-Tronco , Humanos , Boca , Regeneração , Engenharia Tecidual/métodos
19.
Vaccine ; 35(42): 5714-5721, 2017 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-28917537

RESUMO

Hepatitis B core Antigen (HBcAg) assembled into Capsid-Like Particles (CLPs) is investigated as a therapeutic vaccine in treatment of chronic hepatitis B (CHB) and in diagnostic tests or as a carrier for various epitopes. While the expression of HBcAg has been thoroughly clarified in E. coli and yeast, it has also been investigated in other expression systems. Stably transformed tobacco expressed HBcAg at a level of 110-250µg/g fresh weight, therefore in view of its large leaf biomass it offers a production platform comparable with transient expression systems regarding the final yield of HBcAg. Several extraction and purification methods were tested and finally the antigen was purified up to 43% using sucrose density gradient centrifugation. The purified HBcAg retained its antigenicity, as confirmed by ELISA and western blot, while maintaining its CLP-structure as observed in TEM. In mice HBcAg intramuscularly delivered at 2×10µg triggered a significant response (serum anti-HBc titre around 150,000), being statistically equivalent to that induced by the reference antigen. Among anti-HBc IgG isotypes, IgG2a and then IgG1 were increasing during immune response. However IgG2b and IgG3 were also induced, especially in mice immunised with the plant-derived antigen. Analysis of the isotype profile indicates mainly Th1 polarisation, but completed with Th2 response. Obtained results indicate a considerable potential of plant-derived HBcAg as a therapeutic vaccine, since a mixed immune response with a stronger Th1 component is particularly required for treatment of CHB.


Assuntos
Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Nicotiana/genética , Plantas Geneticamente Modificadas/genética , Células Th1/imunologia , Células Th2/imunologia , Vacinas/imunologia , Animais , Epitopos/imunologia , Escherichia coli/genética , Feminino , Anticorpos Anti-Hepatite B/imunologia , Antígenos do Núcleo do Vírus da Hepatite B/genética , Vírus da Hepatite B/genética , Imunização/métodos , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Vacinas/genética
20.
Eur J Heart Fail ; 19(1): 148-157, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-28052545

RESUMO

AIMS: 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). METHODS AND RESULTS: Thirteen subjects with advanced HF, New York Heart Association (NYHA) class II-III were enrolled and treated with targeted injection of Cx-43-MDS/PCs and then monitored for at least 6 months. Overexpression of Cx43 (Cx43+) was significantly higher in all but one subject (Cx43-). Injection of MDS/PCs was associated with significant improvement of exercise capacity: NYHA (3 ± 0 vs. 1.8 ± 0.7, P = 0.003), exercise duration (388.69 ± 141.83 s vs. 462.08 ± 176.69 s, P = 0.025), peak oxygen consumption (14.38 ± 3.97 vs. 15.83 ± 3.74 ml/kg.min, P = 0.022) and oxygen pulse (10.58 ± 2.89 vs. 18.88 ± 22.63 mLO2 /heart rate, P = 0.012). Levels of BNP, left ventricular (LV) ejection fraction and LV end-diastolic volumes tended to improve. There was a significant improvement of the mean unipolar voltage amplitudes measured for the injected segments and the entire left ventricle (9.62 ± 2.64 vs. 11.62 ± 3.50 mV, P = 0.014 and 8.83 ± 2.80 vs. 10.22 ± 3.41 mV, P = 0.041, respectively). No deaths were documented, Cx43+ (n = 12) subjects presented no significant ventricular arrhythmia; one Cx43- subject suffered from ventricular tachycardia (successfully treated with amiodarone). CONCLUSIONS: Injection of Cx-43-MDS/PCs in patients with severe HF led to significant improvement in exercise capacity and myocardial viability of the injected segments while inducing no significant ventricular arrhythmia. This may arise from improved electrical coupling of the injected cells and injured myocardium and thus better in-situ mechanical cooperation of both cell types. Therefore, further clinical studies with Cx43+ MDS/PCs are warranted.


Assuntos
Conexina 43/genética , Terapia Genética/métodos , Insuficiência Cardíaca/terapia , Músculo Esquelético/citologia , Mioblastos/transplante , Transplante de Células-Tronco/métodos , Idoso , Técnicas de Cultura de Células , Doença Crônica , Estudos de Viabilidade , Feminino , Insuficiência Cardíaca/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , Miocárdio , Projetos Piloto , Estudos Prospectivos , Regeneração , Índice de Gravidade de Doença , Transfecção , Transplante Autólogo , Resultado do Tratamento
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