Title: rDNA genes and development
The accumulated data suggest a link between the expression of rDNA clusters and differentiation. In early development, the formation of epigenetically silenced rDNA is associated with the formation of condensed chromatin structures outside of the nucleolus, as well as transcriptional activation of a set of differentiation genes. One possible underlying mechanism is the involvement of nucleoli in the global epigenetic regulation of thousands of developmental genes via the formation of direct inter-chromosomal contacts between rDNA clusters and different chromosomal regions. We used the 4C (circular chromosome conformation capture) approach to determine the whole-genome contacts of rDNA clusters (4C-rDNA) in HEK293T cells which were originally isolated from the human embryonic kidney. The numerous inter-chromosomal contacts were observed with different genomic regions that possessed in the close neighborhood (about 2.5 kb) hundreds of genes controlling nervous system development and cell morphogenesis. Among rDNA- contacting genes there were both active and silent genes. Among the latter’s been DUX4 genes residing at the tip of chr4. Numerous rDNA contacts were also detected at the chromosomal regions possessing very long (from 5 to 50 kb) stretches of the H3K27ac mark which is associated with super-enhancers. Another link between rDNA clusters and developmental genes we detected in the course of analysis of small RNAs originated almost exclusively from the transcribed regions of rDNA genes (small ribosomal RNAs, siRNAs) from specific regions of ribosomal RNAs shaping hairpins. We observed that diverged and rearranged fragments of rDNA units are scattered throughout the human genome and that srRNAs correspond to particular sites inside these fragments of rDNA that mostly reside in intergenic regions or the introns of about 1500 genes. The genes corresponding to GO items are associated with development and morphogenesis. A major part of these genes does not shape contacts with rDNA clusters. Amounts or srRNAs depend on activity of rDNA genes. We assume that both mechanisms either via direct contact of rDNA clusters with developmental genes or via rsRNAs could be behind the link between rDNA genes and differentiation.
Niсkolai A. Tchurikov, Doctor of Sciences (D. Sci.- molecular biology), now is a Head of the Department of Epigenetic Mechanisms of Gene Expression Regulation, Engelhardt Institute of Molecular Biology, Moscow, Russia. He got his Medicine Doctor's degree (M.D.) at Stavropol State Medical Institute in 1972. In 1977 he got his Molecular Biology Ph.D. at Engelhardt Institute of Molecular Biology, Moscow, USSR. Winner of the State Award of the USSR for the discovery on the molecular level of mobile elements in eukaryotes, 1983. 1977 – Ph.D.- “Discovery and properties of mobile element Dm225 in Drosophila genome”. 1989 – D.Sci. – “Molecular organization of the cut locus in Drosophila melanogaster”. In 1977-1980 discovered at the molecular level mobile genetic elements in Drosophila and clusters of different mobile elements. In 1981-1989 cloned the entire complex cut locus from Drosophila and detected multiple transposition events in the locus and several new mobile elements. In 1988-2000 detected the physical capacity of DNA and RNA molecules to form parallel duplexes. In 1998 sequenced completely a novel LTR element, burdock. In 1988-2015 detected large chromosomal domains corresponding to the units of coordinated gene expression, trans-RNAi. Current interests – epigenetic mechanisms in the regulation of gene expression, regulation of siRNA and piRNA pathways of silencing, the study of higher-order chromosomal structures by 4C, and development of RNAi approach for AIDS/HIV treatment.
Title: Role of Lyophilized Platelet-Rich Plasma in Lung Disorders
The concept of mesenchymal stem cells being proposed as a medicinal signaling agent is based on the fact that cells are not single molecular agents. MSCs, which more appropriately may be called ‘Living Drug’ molecules, are adaptive agents capable of complex functions involving many bioactive factors. These multipotent cells may be considered injury-specific drug stores that are home to the injury site and secrete multiple levels of immunomodulatory and trophic factors. MSCs reduce inflammation, modulate the immune system, as well as stimulate other cells to perform their functions effectively. In addition, MSCs can function as regulated delivery vehicles and target specific molecules that act against cancerous cells. This could be an interesting avenue of cancer immunotherapy research to enhance the outcomes of dendritic and natural killer cell therapies. Mesenchymal cell-based therapy is emerging as an extremely promising approach for tissue regeneration owing to the availability, ease of harvest, and transplant of cells. Capitalizing on the regenerative potential of the human body also ensures reduced side effects along with enhancing the overall health of individuals. Current research also includes the development of bioactive scaffolds that are capable of supporting the activation and differentiation of host stem cells at the required site. In the future, through these living drug molecules, it will be possible to use human native sites as micro-niche/micro-environment for potentiation of the human body's site-specific response. Research on cells and growth factors in the human body will expand the horizon of translational medicine and provide definitive therapeutic solutions to several acute, chronic, and lifestyle-mediated diseases. The ultimate aim is to REGENERATE and not REPAIR and change the scenario of medical therapy from the use of PILLS to CELLS.
Mahajan completed his master's in General Surgery from Marathwada University, Maharashtra, and pursued a Diploma in Urology at the University of Vienna, Austria. In a career-changing move, after three decades of being a successful general and uro-surgeon, he started his brainchild-StemRx Bioscience Solutions Pvt. Ltd. in the year 2011. This was to focus on in-depth research in Regenerative Medicine and Cell-Based Therapy which he believes is the solution to address the limitations of conventional therapeutic modalities. To this effect, he underwent and continues to undergo intensive training in the United States. He has devised personalized treatment protocols for more than 75 health conditions in cell-based therapy. Dr. Mahajan is a Life member and Joint Secretary of The Stem Cell Society of India as well as Vice President of the Anti-Aging Foundation, Delhi, and Scientific National Advisor, of the Indian Stem Cell Study Group. He has been appointed as Hon. Associate Professor of Regenerative Medicine and Cell-Based Therapy at Dr. R N Cooper Hospital, Mumbai, and Faculty for MD Transfusion Medicine at KEM Hospital, Mumbai. Examiner for various fellowship courses with D.Y. Patil Institute, Kolhapur.
Title: Tendon regeneration and functional enhancement for physical performance via Piezo1-Mkx axis
Tendons and ligaments are tough tissues that precisely connect muscle to bone and bone to bone, respectively. Once damaged; however, they are difficult to heal, and secondary osteoarthritis is known to occur in approximately 60% of cases. However, the development of treatment methods in current medicine has been challenging. One of the reasons for this is that the master transcription factors, the Operation System of our genome, that produce tendons have long been unknown. We created an expression catalog of all 1600 transcription factors and identified Mkx as the central transcription factor for tendons. First, we showed that Mkx could promote the differentiation into tenocytes (tendon cells) from human iPS cells. Using these tenocytes, we could produce tendon-like tissues and show the therapeutic effect of its transplantation in the tendon injury model. Next, we analyzed the potential function of Piezo1, mechano-sensor, and an Mkx upstream activator in tenocytes, by generating the mice in which an active type of Piezo1 was introduced into tenocytes. Surprisingly, in the tendon-specific gain of function Piezo1 mice, jumping ability and Max speed were significantly enhanced. Based on the results in mice, the role of Piezo1 in human athletic performance was tested. In collaboration with the Athrome Consortium, an international athlete genomics organization, we investigated the frequency of active PIEZO1 E756del in Olympic-level sprinters and the general population in Jamaica. Although the analysis is limited to a small number, the results showed that Jamaican sprinters had a significantly increased ratio of functional polymorphism compared to the general population. These discoveries in tendons have allowed us to understand the entire motor function system, contributing healthy society and medical care.
Hiroshi Asahara was trained as an Orthopedic Surgeon after graduating from Okayama University Medical School. His career as a researcher led him to be a postdoctoral fellow at Harvard Medical School and a staff scientist at Salk Institute, under Prof. Marc Montminy. Asahara now organizes his lab as a Professor of Molecular Medicine at The Scripps Research Institute, USA, and as Professor at Tokyo Medical and Dental University, Japan. Based on his unique Systems Biomedicine approaches combining a novel strategy and database, he and his lab are trying to uncover molecular mechanisms of musculoskeletal development and identify the critical pathway to regulate inflammatory diseases, including rheumatoid arthritis.
Title: Evaluation of Autologous Bone Marrow Derived Stromal Cell Therapy on Spinal Cord Injury in Companion Animals
Pet animals are vulnerable to spinal cord injuries, which are frequently accompanied by significant sensory and locomotor problems and can progress to irreversible paralysis. Through differentiation into neurons and related glial cells, stopping immunological attacks, preventing apoptosis and necrosis, and secreting neurotrophic substances that promote the regeneration process, stem cell therapies provide hope for healing spinal cord injuries. The study aimed to assess the effectiveness of a single intrathecal/intravenous injection of autologous bone marrow-derived stromal cells in a platelet-rich plasma carrier for a subset of clinical cases involving chronic spinal cord injury in dogs and cats. During clinical trials, stem cells were injected into Five dogs and three cats who had spinal cord injuries caused by disc protrusion and those who had not responded to traditional treatment. Three dogs suffered from degenerative Myelopathy for 2 years with no response to treatment, two dogs suffered from Hemiplegia following spinal surgery, and one tetraplegic dog. According to the results, physical therapy combined with the transplanted cells allowed for the restoration of weight-bearing locomotor function and spinal reflexes in less than 90 days. The magnetic resonance scans of the extruded discs indicated only slight modifications after the treatment. The combination intrathecal/intravenous injection of bone marrow stromal cells is a safe and effective method for treating chronic spinal cord injury in companion animals, according to the study's findings.
Ahmed Nour Eldine Abdallah is a Researcher at the Hormones department in the Stem cell Research Unit at the Medical research and clinical studies institute under National Research Center. he is affiliated with Animal Health Research Institute, Dokki, Egypt.
Title: Investigation of conditioned medium properties obtained from mesenchymal stem cells preconditioned with dimethyloxalylglycine
Background: Mesenchymal stem cells (MSCs) are multipotent progenitor cells with fibroblastic morphology, high proliferation capacities, and trilineage differentiation abilities1. They can be isolated from many tissues, such as bone marrow, umbilical cord, adipose tissue, and amniotic membrane, and reproduced on a large scale in vitro. Since the umbilical cord is a primitive tissue, cells isolated from it stand out for regenerative treatments with their high proliferation capacity, strong immunomodulation, and immunosuppression abilities. Recent research has shown that the therapeutic value of MSCs is related to secretomes rather than their ability to differentiate2,3. In this case, a better understanding and enhancement of the paracrine properties of MSCs will increase the therapeutic potential of these cells and the possibility of developing targeted therapy options. Regulation of the secretome profiles of MSCs involves cultivating cells following a specific topography, preconditioning them with hypoxia and various chemical agents4. Preconditioning with hypoxia improves the robustness and survival of MSCs and their therapeutic potential by increasing their angiogenic and immune modulatory capacity5.
Aim: This study aimed to compare the secretome profiles and exosome contents of two types of conditioned media (CM) obtained from human umbilical cord-derived MSCs preconditioned and non-preconditioned with dimethyloxalylglycine (DMOG), a hypoxia mimetic agent, and to evaluate the ultrastructural changes in both MSC groups.
Methods: First, MSCs were isolated from the human umbilical cord, and the cells were characterized by confirming their immunophenotypic and differentiation properties. The expression of Hif1a in MSCs was analyzed immunocytochemically to decide the appropriate dose and duration of use DMOG. Then, two different CMs were prepared by preconditioning MSCs with/without DMOG. CM contents were analyzed for total protein, IL-4, IL-10, IL-17, IFN-λ, VEGF, NGF, BDNF, and GDNF. Moreover, exosomes were isolated from both conditioned media using a commercial kit, and the isolated exosomes were shown by Western blot and transmission electron microscopy. To observe and compare the effects of conditioned media on proliferation and migration, an in vitro wound healing test was performed. Finally, MSCs in both groups were evaluated ultrastructurally.
Results: After isolation and characterization of MSCs, considering the Hif1a expression results, 1000μM DMOG was applied to MSCs for 24 hours to prepare the conditioned medium. VEGF, NGF, and IL-4 levels were found to be increased in CM from DMOG-preconditioned MSCs compared to CM from normal MSC. BDNF, GDNF, IL-10, IL-17, and IFN-γ levels were not detectable in both groups. Western blot and negative staining in TEM demonstrated the presence of exosomes isolated from CMs of both groups. According to the wound healing test results, both CM increased the migration and proliferation abilities of 3T3 cells. Finally, examination of MSCs by TEM showed that these cells were active, and it was observed that autophagosome, autolysosome, myelin figure, and microvesicular body structures increased in MSCs preconditioned with DMOG.
Conclusion: These findings suggested that preconditioning MSCs with DMOG could alter their secretion profile, modify their ultrastructural morphology accordingly, and make their conditioned medium a more potent therapeutic tool
Basak Isıldar completed her bachelor's degree in the fields of Molecular Biology and Genetics at Istanbul University University. She received her master's and doctorate degrees from Istanbul University-Cerrahpasa, Cerrahpasa Faculty of Medicine, Department of Histology and Embryology. In her master's thesis, she investigated the characteristics of mesenchymal stem cells isolated after freezing the umbilical cord in different cryopreservation solutions, and in her doctoral thesis, the effects of conditioned media obtained from MSCs cultured in 2D and 3D environments on experimental autoimmune type 1 diabetes (T1D). Her studies on extracellular vesicles and conditioned medium derived from MSCs and their effects on T1D are still ongoing. She also gives Histology and Embryology lectures at Balikesir University, Faculty of Medicine.
Title: Piezo Mechanosensory channels regulate centrosome integrity and mitotic entry
Mechanotransduction is a process by which mechanical stimuli are converted into biochemical signals in cells to elicit different physiological functions, including embryogenesis, hearing, touch, and muscle contractility. Piezo1 and 2 are evolutionarily conserved mechanosensory cation channels known to be responsible for sensing mechanical stimuli and transducing a mechanically activated ion current, on the cell membrane. Piezo channels are widely expressed, and play important roles in developmental and homeostatic processes. Piezo proteins have overlapping but distinct expression patterns in tissues and cells and therefore exhibit both common and unique biological functions. Both gain- and loss-of-function PIEZO1 and PIEZO2 mutations are associated with several severe human diseases, such as anemia, musculoskeletal disorders, and cancer. In addition, Piezo1 was shown to regulate the regenerative capacity of muscle stem cells. The majority of studies have focused on the Piezo mechanosensory function on the cell membrane, with transduction of currents to the cytoplasm in response to extracellular forces. Here, we show in myoblasts and multiple other cell types that Piezo proteins also exhibit concentrated intracellular localization at centrosomes. Both Piezo loss-of-function and Piezo1 activation by the small molecule Yoda1 produce supernumerary centrosomes due to premature centriole disengagement leading to multi-polar spindles, and mitotic delay. We further show that perturbations in Piezo modulate Ca2+ flux at centrosomes, indicated by a Ca2+ reporter and that photoactivation of a caged Piezo1 agonist at centrosomes leads to rapid centriole disengagement. Moreover, the inhibition of Polo-like kinase 1 eliminates Yoda1-induced centriole disengagement. Thus, we suggest that mechanotransduction by Piezo in the peri-centrosomal pool regulates centriole engagement by maintaining peri-centrosomal Ca2+ within a defined range, likely through sensing cell intrinsic forces from microtubules. Thus, Piezo proteins may represent an important new class of intracellular mechanotransducers.
David is a structural and cell biologist, currently an instructor at Harvard Medical School. She completed her graduate studies at the Technion, Israel Institute of Technology. For her Postdoctoral training, Liron joined the structural immunology lab of Prof. Hao Wu at the Harvard Medical School and was awarded the Cancer Research Institute postdoctoral fellowship. Liron obtained in-depth training in cryoelectron microscopy (cryo-EM) and cell biology including cellular imaging and other cutting-edge techniques and followed her passion to investigate large and challenging molecular assemblies and membrane proteins in the field of immunology, cancer research and mechanotransduction investigating piezo channels in myoblasts.
Title: Therapeutic potential of paracrine factors derived from mesenchymal stem cells
Mesenchymal stem cells (MSCs) are multipotent stromal cells that can be isolated from various types of tissues including bone marrow, adipose tissue, and umbilical cord, and can differentiate into mesodermal lineages including adipocytes, osteoblasts and chondrocytes 1–7 Mesenchymal stem cells (MSCs) have come up as a potential therapeutic for the treatment of various types of diseases 4,8. In the first place, the mechanism of the recuperative effect of MSCs was based on their potential of replacing damaged cells. However, entrapment of MSCs primarily in the lung microvasculature and other organs like the liver and spleen rather than target tissue following direct administration of MSCs via systemic route showed that the therapeutic potential of these cells could result from their secretome content. In addition, MSCs that are entrapped in the lung and not able to stay alive longer than 48 hours have also ameliorative effects 3. It was also shown that MSCs are providing tissue regeneration by inducing differentiation of resident progenitor cells rather than their direct differentiation 9. All these findings indicate that paracrine factors secreted by MSCs could be the main route of therapeutic action of MSCs. Furthermore, direct application of MSCs has its limitation including the risk of tumorigenicity, complications related to cryopreservation of the cells, and loss of stemness potential with increasing passages 10. Self-renewal and differentiation potential of MSCs tend to worsen with increased donor age, genetics, environmental stress, and increased number of in vitro passaging 5. These data imply the necessity of improving the therapeutic potential of MSCs, especially in the case of autologous isolation 11,12. At this point, preconditioning of MSCs with different strategies like treating them with certain chemical agents, incubating them in hypoxic conditions, or culturing them with scaffolds mimicking ideal microenvironment could increase or compensate for the disease-related loss of therapeutic potential 13,14. In this context, I will share our experience in the field of the therapeutic potential of CM derived from MSCs in the recent 5 years in correlation with new findings put forward in the literature.
Serbay Ozkan completed his bachelor’s degree in the fields of molecular biology and genetics and chemistry (double major program) at Bogaziçi University (which is one of the top universities in Turkey). He performed a comparative characterization of mesenchymal stem cells (MSCs) obtained from different regions of the umbilical cord and evaluated their ultrastructural morphologies during osteogenic and adipogenic differentiation in his master’s degree. He continued his studies in the field of MSCs during the doctorate program. In this respect, he evaluated the therapeutic potential of conditioned mediums obtained from MSCs which are preconditioned or not on diabetic nephropathy. His studies on the field of extracellular vesicles and conditioned medium derived from MSCs continue. He is also giving lectures as a histologist and embryologist at Izmir Katip Celebi University Medical Faculty.
Title: The superior regenerative potential of Mesangiogenic Progenitor Cells
Statement of the Problem: Even if an increased efficacy in cell isolation, expansion, and characterization leads to a consistent number of studies applying adult multipotent cells isolated from different tissues in various disease models, most of the clinical and pre-clinical investigations showed disappointing outcomes, related to the lack or inefficient vascularization of the new-formed tissue. Taking these considerations into account, MPC's power to couple melanogenesis and angiogenesis highlights their tissue regenerative potential and clinical value with particular reference to tissue engineering.
Methodology: We have isolated and compared MPCs from human bone marrow (hBM), a human stromal vascular fraction (hSVF) of adipose tissue, and human umbilical cord blood. We have also evaluated their differentiating potential. Flow cytometry and cell sorting have been performed to characterize the different populations.
Findings: MPCs are tissue-specific and can be isolated exclusively from hBM-mononuclear cells. MPCs can generate exponentially growing MSC cultures and in addition, MPCs retained angiogenic potential, suggesting the term "mesangiogenic". We also demonstrated a hierarchical multi-step model of mesenchymal differentiation with at least three different populations of multi-potent cells. Cell sorting experiments showed that a highly specific hBM subpopulation, described as Pop#8 and identified by the CD64brightCD31brightCD14negCD45dim phenotype, represents the only hBM subpopulation able to generate MPCs in culture under selective conditions.
Conclusion & Significance: We hypothesized that the suspected superior performances of hBM-MSCs in tissue regeneration could be explained by the presence of MPCs and/or their progenitors. It is reasonable to hypothesize that these cells could trigger new blood vessel formation in the early phases coupled with the essential chondrogenic and osteogenic differentiation capability. MPCs are found at frequencies from one to two logs higher than other MSC progenitors described in hBM. Consequently, millions of MPCs could be easily isolated, starting from 10-15 ml of fresh hBM in 4-6 days by applying a cheap and GMP-compliant culture method without the need for cell expansion. In summary, MPCs represent a valuable cell population for the proof of new concepts in tissue engineering, where neo-vascularization plays a crucial role in the establishment of successful therapies.
Serena Barachini has a degree in Biology and a Ph.D. in Experimental Medicine from the University of Rome where she worked for six years in the lab of Tumor Immunology and Cell Therapy and she had attended a Postgraduate School in Clinical Pathology at the Hospital of Pisa where she was active in on-hematology for five years. She is working with Prof. Iacopo Petrini in “Laboratory for Cell Therapy” at the University of Pisa, where she is coordinating a group of researchers. Her study focuses on the isolation of MSCs and MPCs in human bone marrow, cord blood, and adipose tissues. Her work also concerns the study of cancer stem cells isolated from hematological diseases, thymoma, and glioblastoma. In particular, she has expertise in 3D cultures, flows cytometry, and cell sorting combined with molecular and cell biology techniques, applied in research projects concerning human stem cells both in tissue repair and cancers.