Regenerative Stem Cell Therapy Research Articles

Regenerative stem cell therapy for stroke in Europe (RESSTORE): a multicenter randomized controlled efficacy clinical trial

Regenerative stem cell therapy for stroke in Europe (RESSTORE): a multicenter randomized controlled efficacy clinical trial

Interleukin 1β receptor blocker (Anakinra) and regenerative stem cell therapy: two novel approaches effectively ameliorating diabetic cardiomyopathy

Diabetic cardiomyopathy (DCM) is a serious common complication of diabetes. Unfortunately, there is no satisfied treatment for those patients and more studies are in critical need to cure them. Therefore, we aimed to carry out our current research to explore the role of two novel therapeutic approaches: one a biological drug aimed to block inflammatory signaling of the IL 1beta (IL1β) axis, namely, anakinra; the other is provision of anti-inflammatory regenerative stem cells. Wistar male rats were allocated into four groups: control group: type 2 diabetes mellitus (DM) induced by 6-week high-fat diet (HFD) followed by a single-dose streptozotocin (STZ) 35 mg/kg i.p., then rats were allocated into: DM: untreated; DM BM-MSCs: received a single dose of BM-MSCs (1 × 106 cell/rat) into rat tail vein; DM-Anak received Anak 0.5 μg/kg/day i.p. for 2 weeks. Both therapeutic approaches improved cardiac performance, fibrosis, and hypertrophy. In addition, blood glucose and insulin resistance decreased, while the antioxidant parameter, nuclear factor erythroid 2–related factor 2 (Nrf2) and interleukin 10 (IL10), and anti-inflammatory agent increased. Furthermore, there is a significant reduction in tumor necrosis factor alpha (TNFα), IL1β, caspase1, macrophage marker CD 11b, inducible nitric oxide synthase (iNOS), and T-cell marker CD 8. Both Anak and BM-MSCs effectively ameliorated inflammatory markers and cardiac performance as compared to non-treated diabetics. Improvement is mostly due to anti-inflammatory, antioxidant, anti-apoptotic properties, and regulation of TNFα/IL1β/caspase1 and Nrf2/IL10 pathways.

Real-Time, Non-Invasive Monitoring of Neuronal Differentiation Using Intein-Enabled Fluorescence Signal Translocation in Genetically Encoded Stem Cell-Based Biosensors.

Real-time and non-invasive monitoring of neuronal differentiation will help increase our understanding of neuronal development and help develop regenerative stem cell therapies for neurodegenerative diseases. Traditionally, reverse transcription-polymerase chain reaction (RT-PCR), western blotting, and immunofluorescence (IF) staining have been widely used to investigate stem cell differentiation; however, their limitations include endpoint analysis, invasive nature of monitoring, and lack of single-cell-level resolution. Several limitations hamper current approaches to studying neural stem cell (NSC) differentiation. In particular, fixation and staining procedures can introduce artificial changes in cellular morphology, hindering our ability to accurately monitor the progression of the process and fully understand its functional aspects, particularly those related to cellular connectivity and neural network formation. Herein, we report a novel approach to monitor neuronal differentiation of NSCs non-invasively in real-time using cell-based biosensors (CBBs). Our research efforts focused on utilizing intein-mediated protein engineering to design and construct a highly sensitive biosensor capable of detecting a biomarker of neuronal differentiation, hippocalcin. Hippocalcin is a critical protein involved in neurogenesis, and the CBB functions by translocating a fluorescence signal to report the presence of hippocalcin externally. To construct the hippocalcin sensor proteins, hippocalcin bioreceptors, AP2 and glutamate ionotropic receptor AMPA-type subunit 2 (GRIA2), were fused to each split-intein carrying split-nuclear localization signal (NLS) peptides, respectively, and a fluorescent protein was introduced as a reporter. Protein splicing (PS) was triggered in the presence of hippocalcin to generate functional signal peptides, which promptly translocated the fluorescence signal to the nucleus. The stem cell-based biosensor showed fluorescence signal translocation only upon neuronal differentiation. Undifferentiated stem cells or cells that had differentiated into astrocytes or oligodendrocytes did not show fluorescence signal translocation. The number of differentiated neurons was consistent with that measured by conventional IF staining. Furthermore, this approach allowed for the monitoring of neuronal differentiation at an earlier stage than that detected using conventional approaches, and the translocation of fluorescence signal was monitored before the noticeable expression of class III β-tubulin (TuJ1), an early neuronal differentiation marker. We believe that these novel CBBs offer an alternative to current techniques by capturing the dynamics of differentiation progress at the single-cell level and by providing a tool to evaluate how NSCs efficiently differentiate into specific cell types, particularly neurons.

Potential Effect of Bovine Colostrum on Mesenchymal Stem Cells for Regenerative Therapy

Bovine colostrum is the first mammary secretion after parturition; it is rich in Igs and bioactive compounds and could play a role in the development of naturally based products with positive effects on human health. In this discussion, we critically examine the effect of bovine colostrum on the properties of mesenchymal stem cells. Multipotent mesenchymal stem cells (MSCs) are a heterogeneous subset of fibroblast-like cells that can be isolated from various biological tissues, such as bone marrow, umbilical cord, and adipose tissues. They are characterized by their ability to self-renew and differentiate into cells of the mesodermal lineage, including adipocytes, osteocytes, and chondrocytes. Additionally, MSCs display an immunomodulatory capacity due to their ability to interact with effector cells typical of both innate and adaptive immune responses. Considering these important properties, MSCs have gained increasing attention in the field of regenerative medicine in recent decades. To date, most experimental protocols are based on cell culture media supplemented with fetal bovine serum (FBS) to promote the ex vivo expansion of MSCs while preserving their differentiative and immunomodulatory capacities. Future trends could involve the application of bovine colostrum in regenerative medicine.

Telomere dynamics in human pluripotent stem cells

ABSTRACT Pluripotent stem cells (PSCs) are a promising source of stem cells for regenerative therapies. Stem cell function depends on telomere maintenance mechanisms that provide them with the proliferative capacity and genome stability necessary to multiply and regenerate tissues. We show here that established human embryonic stem cells (hESCs) have stable telomere length that is dependent on telomerase but not on alternative mechanisms based on homologous recombination pathways. Here, we show that human-induced pluripotent stem cells (hiPSCs) reprogrammed from somatic cells show progressive telomere lengthening until reaching a length similar to ESCs. hiPSCs also acquire telomeric chromatin marks of ESCs including decreased abundance of tri-methylated histone H3K9 and H4K20 and HP1 heterochromatic marks, as well as of the shelterin component TRF2. These chromatin features are accompanied with increased abundance of telomere transcripts or TERRAs. We also found that telomeres of both hESCs and hiPSCs are well protected from DNA damage during telomere elongation and once full telomere length is achieved, and exhibit stable genomes. Collectively, this study highlights that hiPSCs acquire ESC features during reprogramming and reveals the telomere biology in human pluripotent stem cells (hPSCs).

Enhancing osteogenic differentiation in adipose-derived mesenchymal stem cells with Near Infra-Red and Green Photobiomodulation

Worldwide, osteoporosis is the utmost predominant degenerative bone condition. Stem cell regenerative therapy using adipose-derived mesenchymal stem cells (ADMSCs) is a promising therapeutic route for osteoporosis. Photobiomodulation (PBM) has sparked considerable international appeal due to its’ ability to augment stem cell proliferation and differentiation properties. Furthermore, the differentiation of ADMSCs into osteoblast cells and cellular proliferation effects have been established using a combination of osteogenic differentiation inducers and PBM. This in vitro study applied dexamethasone, β-glycerophosphate disodium, and ascorbic acid as differentiation inducers for osteogenic induction differentiation media. In addition, PBM at a near-infrared (NIR) wavelength of 825 nm, a green (G) wavelength of 525 nm, and the novel combination of both these wavelengths using a single fluence of 5 J/cm2 had been applied to stimulate proliferation and differentiation effectivity of immortalised ADMSCs into early osteoblasts. Flow cytometry and ELISA were used to identify osteoblast antigens using early and late osteoblast protein markers. Alizarin red Stain was employed to identify calcium-rich deposits by cells within culture. The morphology of the cells was examined, and biochemical assays such as an EdU proliferation assay, MTT proliferation and viability assay, Mitochondrial Membrane Potential assay, and Reactive Oxygen Species assay were performed. The Central Scratch Test determined the cells' motility potential. The investigative outcomes revealed that a combination of PBM treatment and osteogenic differentiation inducers stimulated promising early osteogenic differentiation of immortalised ADMSCs. The NIR-Green PBM combination did appear to offer great potential for immortalised ADMSC differentiation into early osteoblasts amongst selected assays, however, further investigations will be required to establish the effectivity of this novel wavelength combination. This research contributes to the body of knowledge and assists in the establishment of a standard for osteogenic differentiation in vitro utilising PBM.

Assessment of knowledge and attitude regarding stem cell regenerative dentistry amongst dental students

Objectives: An emerging area of dentistry is regenerative and stem cell therapy. For these treatments to be successfully implemented in clinical practice, dentists’ perspectives and understanding of the principles are crucial, to assess the knowledge of dental students on the potential therapeutic application of stem cells, to assess the attitude of dental students for the acceptance of regenerative treatment modalities, and to determine whether further training in the area is necessary. Materials and Methods: A questionnaire on stem cell regenerative dentistry was distributed to 250 dental students of Private Dental College of Nagpur (Maharashtra), consisting of 17 questions on knowledge and attitudes toward regenerative therapy. The frequencies underwent a descriptive analysis. Results: The postgraduate students have adequate knowledge about stem cell regenerative therapy (95.6%) and they are enthusiastic for the acceptance of regenerative treatment. The majority of dentistry students had no prior training in stem cell therapy (75.60%), although many were eager to pursue more education in this area (67.60%). Conclusion: Most dental students are eager to pursue further training in regenerative dentistry and are supportive of the use of stem cells and regenerative dental therapies. Most participants thought that the subject should be covered in the undergraduate course.

The Current State of Osteoarthritis Treatment Options Using Stem Cells for Regenerative Therapy: A Review.

Articular cartilage has very low metabolic activity. While minor injuries may be spontaneously repaired within the joint by chondrocytes, there is very little chance of a severely impaired joint regenerating itself when damaged. Therefore, any significant joint injury has little chance of spontaneously healing without some type of therapy. This article is a review that will examine the causes of osteoarthritis, both acute and chronic, and how it may be treated using traditional methods as well as with the latest stem cell technology. The latest regenerative therapy is discussed, including the use and potential risks of mesenchymal stem cells for tissue regeneration and implantation. Applications are then discussed for the treatment of OA in humans after using canine animal models. Since the most successful research models of OA were dogs, the first applications for treatment were veterinary. However, the treatment options have now advanced to the point where patients suffering from osteoarthritis may be treated with this technology. A survey of the literature was performed in order to determine the current state of stem cell technology being used in the treatment of osteoarthritis. Then, the stem cell technology was compared with traditional treatment options.

A Review of Novel Strategies for Human Periodontal Ligament Stem Cell Ex Vivo Expansion: Are They an Evidence-Based Promise for Regenerative Periodontal Therapy?

Periodontitis is a gingiva disease sustained by microbially associated and host-mediated inflammation that results in the loss of the connective periodontal tissues, including periodontal ligament and alveolar bone. Symptoms include swollen gingiva, tooth loss and, ultimately, ineffective mastication. Clinicians utilize regenerative techniques to rebuild and recover damaged periodontal tissues, especially in advanced periodontitis. Human periodontal ligament stem cells (hPDLSCs) are considered an appealing source of stem cells for regenerative therapy in periodontium. hPDLSCs manifest the main properties of mesenchymal stem cells, including the ability to self-renew and to differentiate in mesodermal cells. Significant progress has been made for clinical application of hPDLSCs; nevertheless, some problems remain, including the small number of cells isolated from each sample. In recent decades, hPDLSC ex vivo expansion and differentiation have been improved by modifying cell culture conditions, especially with the supplementation of cytokines' or growth factors' mix, chemicals, and natural compounds, or by using the decellularized extracellular matrix. Here, we analyzed the changes in stemness properties and differentiation potential of hPDLSCs when culturing in alternative media. In addition, we focused on the possibility of replacing FBS with human emoderivates to minimize the risks of xenoimmunization or zoonotic transmission when cells are expanded for therapeutic purposes.

"Stem Cell Therapy for Regeneration of Radiated Salivary Glands"

"Stem Cell Therapy for Regeneration of Radiated Salivary Glands"

Ultrastructural Characterization of Human Gingival Fibroblasts in 3D Culture.

Cell spheroids are applied in various fields of research, such as the fabrication of three-dimensional artificial tissues in vitro, disease modeling, stem cell research, regenerative therapy, and biotechnology. A preclinical 3D culture model of primary human gingival fibroblasts free of external factors and/or chemical inducers is presented herein. The ultrastructure of the spheroids was characterized to establish a cellular model for the study of periodontal tissue regeneration. The liquid overlay technique was used with agarose to generate spheroids. Fibroblasts in 2D culture and cell spheroids were characterized by immunofluorescence, and cell spheroids were characterized by optical and scanning electron microscopy, energy-dispersive X-ray spectroscopy, backscattered electrons, and Fourier transform infrared spectroscopy. Ostegenic related genes were analyzed by RT-qPCR. Gingival fibroblasts formed spheroids spontaneously and showed amorphous calcium phosphate nanoparticle deposits on their surface. The results suggest that human gingival fibroblasts have an intrinsic potential to generate a mineralized niche in 3D culture.

Effect of Hypoxia Preconditioning on the Regenerative Capacity of Adipose Tissue Derived Mesenchymal Stem Cells in a Model of Renal Artery Stenosis.

Atherosclerotic renal artery stenosis (ARAS) is associated with irreversible parenchymal renal disease and regenerative stem cell therapies may improve renal outcomes. Hypoxia preconditioning (HPC) may improve the regenerative functions of adipose tissue-derived mesenchymal stem cells (AMSC) by affecting DNA 5-hydroxymethylcytosine (5hmC) marks in angiogenic genes. Here, we investigated using a porcine ARAS model, whether growth of ARAS AMSCs in hypoxia (Hx) versus normoxia (Nx) would enhance renal tissue repair, and comprehensively analyze how HPC modifies DNA hydroxymethylation compared to untreated ARAS and healthy/normal pigs (n=5 each). ARAS pigs exhibited elevated serum cholesterol, serum creatinine and renal artery stenosis, with a concomitant decrease in renal blood flow (RBF) and increased blood pressure (BP) compared to healthy pigs. Renal artery injection of either autologous Nx or Hx AMSCs improved diastolic BP, reduced kidney tissue fibrosis, and inflammation (CD3+ T-cells) in ARAS pigs. In addition, renal medullary hypoxia significantly lowered with Nx but not Hx AMSC treatment. Mechanistically, levels of epigenetic 5hmC marks (which reflect gene activation) estimated using DNA immunoprecipitation technique were elevated in profibrotic and inflammatory genes in ARAS compared with normal AMSCs. HPC significantly reduced 5hmC levels in cholesterol biosynthesis and oxidative stress response pathways in ARAS AMSCs. Thus, autologous AMSCs improve key renovascular parameters and inflammation in ARAS pigs, with HPC mitigating pathological molecular effects on inflammatory and profibrotic genes which may play a role in augmenting regenerative capacity of AMSCs.

S100A6 as a Constituent and Potential Marker of Adult and Cancer Stem Cells.

Adult or tissue stem cells are present in various tissues of the organism where they reside in a specific environment called the niche. Owing to their ability to generate a progeny that can proliferate and differentiate into specialized cell types, adult stem cells constitute a source of new cells necessary for tissue maintenance and/or regeneration. Under normal conditions they divide with a frequency matching the pace of tissue renewal but, following tissue damage, they can migrate to the site of injury and expand/divide intensively to facilitate tissue repair. For this reason much hope is being placed on the use of adult stem cells in regenerative therapies, including tissue engineering. Identification and characterization of tissue stem cells has been a laborious process due to their scarcity and lack of universal markers. Nonetheless, recent studies, employing various types of transcriptomic analyses, revealed some common trends in gene expression pattern among stem cells derived from different tissues, suggesting the importance of certain genes/proteins for the unique properties of these cells. S100A6, a small calcium binding protein, has been recognized as an important factor influencing cell proliferation and differentiation. Accumulating results show that S100A6 is a constituent of adult stem cells and, in some cases, may even be considered as their marker. Thus, in this review we summarize literature data concerning the presence of S100A6 in adult and cancer stem cells and speculate on its potential role and usefulness as a marker of these cells.

Scalable manufacturing of clinical-grade differentiated cardiomyocytes derived from human-induced pluripotent stem cells for regenerative therapy.

Basic research on human pluripotent stem cell (hPSC)‐derived cardiomyocytes (CMs) for cardiac regenerative therapy is one of the most active and complex fields to achieve this alternative to heart transplantation and requires the integration of medicine, science, and engineering. Mortality in patients with heart failure remains high worldwide. Although heart transplantation is the sole strategy for treating severe heart failure, the number of donors is limited. Therefore, hPSC‐derived CM (hPSC‐CM) transplantation is expected to replace heart transplantation. To achieve this goal, for basic research, various issues should be considered, including how to induce hPSC proliferation efficiently for cardiac differentiation, induce hPSC‐CMs, eliminate residual undifferentiated hPSCs and non‐CMs, and assess for the presence of residual undifferentiated hPSCs in vitro and in vivo. In this review, we discuss the current stage of resolving these issues and future directions for realizing hPSC‐based cardiac regenerative therapy.

Odontogenic MSC Heterogeneity: Challenges and Opportunities for Regenerative Medicine.

Cellular heterogeneity refers to the genetic and phenotypic differences among cells, which reflect their various fate choices, including viability, proliferation, self-renewal probability, and differentiation into different lineages. In recent years, research on the heterogeneity of mesenchymal stem cells has made some progress. Odontogenic mesenchymal stem cells share the characteristics of mesenchymal stem cells, namely, good accessibility, low immunogenicity and high stemness. In addition, they also exhibit the characteristics of vasculogenesis and neurogenesis, making them attractive for tissue engineering and regenerative medicine. However, the usage of mesenchymal stem cell subgroups differs in different diseases. Furthermore, because of the heterogeneity of odontogenic mesenchymal stem cells, their application in tissue regeneration and disease management is restricted. Findings related to the heterogeneity of odontogenic mesenchymal stem cells urgently need to be summarized, thus, we reviewed studies on odontogenic mesenchymal stem cells and their specific subpopulations, in order to provide indications for further research on the stem cell regenerative therapy.

MUSSEL ADHESIVE PROTEIN-BASED ADHESIVE TO RETAIN STEM CELLS FOR CARTILAGE REGENERATION

Purpose: Osteoarthritis (OA) is a debilitating joint disease characterized by cartilage degeneration and pathologic remodeling of other joint tissues. Cartilage has limited intrinsic healing capacity, motivating the application of stem cells for regenerative therapies. Therapies to treat osteochondral defects using stem cell therapy-based tissue engineering have been developed and used for more than 20 years; however, the low viability and high possibility of dispersion of injected cells to target defect sites have remained challenge.

A 3D mathematical model of coupled stem cell-nutrient dynamics in myocardial regeneration therapy

Stem cell therapy is a promising treatment for the regeneration of myocardial tissue injured by an ischemic event. Mathematical modeling of myocardial regeneration via stem cell therapy is a challenging task, since the mechanisms underlying the processes involved in the treatment are not yet fully understood. Many aspects must be accounted for, such as the spread of stem cells and nutrients, chemoattraction, cell proliferation, stages of cell maturation, differentiation, angiogenesis, stochastic effects, just to name a few. In this paper we propose a 3D mathematical model with a free boundary that aims to provide a qualitative description of some main aspects of the stem cell regenerative therapy in a simplified scenario. The paper mainly focuses on the description of the shrinking of the necrotic core during treatment. The stem cell and nutrients dynamics are described through coupled reaction-diffusion problems. Proliferation, chemoattraction, tissue regeneration and nutrient consumption are included in the model.

Generation of iMyoblasts from Human Induced Pluripotent Stem Cells.

Skeletal muscle stem cells differentiated from human-induced pluripotent stem cells (hiPSCs) serve as a uniquely promising model system for investigating human myogenesis and disease pathogenesis, and for the development of gene editing and regenerative stem cell therapies. Here, we present an effective and reproducible transgene-free protocol for derivation of human skeletal muscle stem cells, iMyoblasts, from hiPSCs. Our two-step protocol consists of 1) small molecule-based differentiation of hiPSCs into myocytes, and 2) stimulation of differentiated myocytes with growth factor-rich medium to activate the proliferation of undifferentiated reserve cells, for expansion and cell line establishment. iMyoblasts are PAX3 + /MyoD1 + myogenic stem cells with dual potential to undergo muscle differentiation and to self-renew as a regenerative cell population for muscle regeneration both ex vivo and in vivo . The simplicity and robustness of iMyoblast generation and expansion have enabled their application to model the molecular pathogenesis of Facioscapulohumeral Muscular Dystrophy and Limb-Girdle Muscular Dystrophies, to both ex vivo and in vivo muscle xenografts, and to respond efficiently to gene editing, enabling the co-development of gene correction and stem cell regenerative therapeutic technologies for the treatment of muscular dystrophies and muscle injury. Graphical abstract.

Репаративная регенерация сухожилия под влиянием секретома мультипотентных мезенхимальных стромальных клеток: экспериментальное исследование

Introduction. Improving treatments for tendon injuries remains a topical issue in biology and medicine. Tendon structure is predisposed to scar formation, with significant regions of dystrophy leading to chronic and recurrent tendinitis. Stem cell regenerative therapy including mesenchymal stromal cell secretome is among promising treatments to improve patient outcomes. Standardized cytokine concentrations can be incorporated into the secretome, which may include both pro- and anti-inflammatory cytokines. This article addresses the use of stromal cell secretome for tendon injury regeneration, which is poorly studied. Materials and methods. The study included 95 white outbred rats (males, weight 120 g), which were divided into three groups: Group I comparison (n=35), Group II experimental (n=35), and Group III control (n=25). Animals in Groups I and II were anesthetized with isoflurane and their calcaneal tendons were dissected and sutured. Animals in Group I were treated with saline solution, whereas Group II received stromal cell secretome. The wounds were subsequently sutured. One week after the manipulation, the animals received the second treatment around the tendon. The animals were withdrawn from the experiment on days 1, 7, 14, and 60. We analyzed the samples using light microscopy with morphometry and transmission electron microscopy. Results. Treating tendons with stromal cell secretome (Group II) is accompanied by less pronounced inflammation and shows no dystrophic changes. The collagen bundles in the regenerate are parallel to each other, with vasculature being normalized and the number of fibroblasts having a slight increase. The control samples, however, showed inflammation, dystrophy, and scarring with hypovascularization of the regenerate. Moreover, the total fibroblast count in the tissue of the control samples elevated significantly. The regenerate in Group II was slightly thicker than in Group III, while in Group I, the regenerate was thinner than in Group III. Conclusion. Mesenchymal stromal cell secretome greatly contributes to tendon healing. Microscopic tendon regenerate structure is similar to that of the intact sample, suggesting its functional recovery. Keywords: multipotent mesenchymal stromal cells, secretome, cytokines, tendon, trauma, reparative regeneration

Extracellular Matrix Scaffolds - A Tissue-Specific Bioactive Niche for Stem Cells in Regenerative Therapy

Aiming to restore the normal function of diseased or injured tissues, regenerative therapy approaches are generally based on the engineering of complex tissue-mimicking grafts, encompassing biomaterial scaffolds, stem cells, or their combinations [1-4]. Due to the major role of stem cells in physiological regenerative mechanisms, regenerative therapies normally rely on either stem cells transplantation or stem cell recruitment from the neighboring tissue into the implanted scaffold