Stem cells and cell therapy: on the eve of scientific approach

Cell therapy and the stem cells (SC) have become a popular topic during last time. The theme is cluttered with numerous publications of questionable reliability. Not all methods applied in praxis are founded on evidence-based research. In the abundant literature, there is a gap between the supposed SC's healing properties associated with their capability to migrate to and engraft in injured tissue, and lack of clear morphological evidence thereof. Accordingly, there is a gap between advertizing and the better part of professional literature: the former speaks about rejuvenation of tissues, and the latter explains sometimes questionable therapeutic effects by paracrine or immunomodulating mechanisms, secretion of cytokines and growth factors. However, a SC is an undifferentiated cell, and a specific and efficient paracrine function can hardly be awaited from it as compared to others, more differentiated cells. It should be noted in conclusion that the main problem with the SC and cell therapy is commercial influence. Probably, experience of some foreign countries should be studied, where moves have been made to stop the use of unproven treatments, including some stem cell therapy.     

Telocytes and stem cells in limbus and uvea of mouse eye

The potential of stem cell (SC) therapies for eye diseases is well‐recognized. However, the results remain only encouraging as little is known about the mechanisms responsible for eye renewal, regeneration and/or repair. Therefore, it is critical to gain knowledge about the specific tissue environment (niches) where the stem/progenitor cells reside in eye. A new type of interstitial cell–telocyte (TC) ( www.telocytes.com ) was recently identified by electron microscopy (EM). TCs have very long (tens of micrometres) and thin (below 200 nm) prolongations named telopodes (Tp) that form heterocellular networks in which SCs are embedded. We found TCs by EM and electron tomography in sclera, limbus and uvea of the mouse eye. Furthermore, EM showed that SCs were present in the anterior layer of the iris and limbus. Adhaerens and gap junctions were found to connect TCs within a network in uvea and sclera. Nanocontacts (electron‐dense structures) were observed between TCs and other cells: SCs, melanocytes, nerve endings and macrophages. These intercellular ‘feet’ bridged the intercellular clefts (about 10 nm wide). Moreover, exosomes (extracellular vesicles with a diameter up to 100 nm) were delivered by TCs to other cells of the iris stroma. The ultrastructural nanocontacts of TCs with SCs and the TCs paracrine influence via exosomes in the epithelial and stromal SC niches suggest an important participation of TCs in eye regeneration.     

The impact of physical,biochemical, and electrical signaling on Schwann cell plasticity

Peripheral nervous system (PNS) injuries are an ongoing health care concern. While autografts and allografts are regarded as the current clinical standard for traumatic injury, there are inherent limitations that suggest alternative remedies should be considered for therapeutic purposes. In recent years, nerve guidance conduits (NGCs) have become increasingly popular as surgical repair devices, with a multitude of various natural and synthetic biomaterials offering potential to enhance the design of conduits or supplant existing technologies entirely. From a cellular perspective, it has become increasingly evident that Schwann cells (SCs), the primary glia of the PNS, are a predominant factor mediating nerve regeneration. Thus, the development of severe nerve trauma therapies requires a deep understanding of how SCs interact with their environment, and how SC microenvironmental cues may be engineered to enhance regeneration. Here we review the most recent advancements in biomaterials development and cell stimulation strategies, with a specific focus on how the microenvironment influences the behavior of SCs and can potentially lead to functional repair. We focus on microenvironmental cues that modulate SC morphology, proliferation, migration, and differentiation to alternative phenotypes. Promotion of regenerative phenotypic responses in SCs and other non-neuronal cells that can augment the regenerative capacity of multiple biomaterials is considered along with innovations and technologies for traumatic injury.     

Immunomodulation of activated hepatic stellate cells by mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (MSCs) have been reported to prevent the development of liver fibrosis in a number of pre-clinical studies. Marked changes in liver histopathology and serological markers of liver function have been observed without a clear understanding of the therapeutic mechanism by which stem cells act. We sought to determine if MSCs could modulate the activity of resident liver cells, specifically hepatic stellate cells (SCs) by paracrine mechanisms using indirect cocultures. Indirect coculture of MSCs and activated SCs led to a significant decrease in collagen deposition and proliferation, while inducing apoptosis of activated SCs. The molecular mechanisms underlying the modulation of SC activity by MSCs were examined. IL-6 secretion from activated SCs induced IL-10 secretion from MSCs, suggesting a dynamic response of MSCs to the SCs in the microenvironment. Blockade of MSC-derived IL-10 and TNF-alpha abolished the inhibitory effects of MSCs on SC proliferation and collagen synthesis. In addition, release of HGF by MSCs was responsible for the marked induction of apoptosis in SCs as determined by antibody-neutralization studies. These findings demonstrate that MSCs can modulate the function of activated SCs via paracrine mechanisms provide a plausible explanation for the protective role of MSCs in liver inflammation and fibrosis, which may also be relevant to other models of tissue fibrosis.     

Applications of single cell RNA sequencing to research of stem cells

Stem cells(SCs) with their self-renewal and pluripotent differentiation potential,show great promise for therapeutic applications to some refractory diseases such as stroke, Parkinsonism, myocardial infarction, and diabetes. Furthermore, as seed cells in tissue engineering, SCs have been applied widely to tissue and organ regeneration. However, previous studies have shown that SCs are heterogeneous and consist of many cell subpopulations. Owing to this heterogeneity of cell states, gene expression is highly diverse between cells even within a single tissue,making precise identification and analysis of biological properties difficult, which hinders their further research and applications. Therefore, a defined understanding of the heterogeneity is a key to research of SCs. Traditional ensemble-based sequencing approaches, such as microarrays, reflect an average of expression levels across a large population, which overlook unique biological behaviors of individual cells, conceal cell-to-cell variations, and cannot understand the heterogeneity of SCs radically. The development of high throughput single cell RNA sequencing(scRNA-seq) has provided a new research tool in biology, ranging from identification of novel cell types and exploration of cell markers to the analysis of gene expression and predicating developmental trajectories. scRNA-seq has profoundly changed our understanding of a series of biological phenomena. Currently, it has been used in research of SCs in many fields, particularly for the research of heterogeneity and cell subpopulations in early embryonic development. In this review, we focus on the scRNA-seq technique and its applications to research of SCs.     

Roles for osteocalcin in proliferation and differentiation of spermatogonial cells cocultured with somatic cells

Spermatogonial cells (SCs) are key cells for spermatogenesis. These cells are affected by paracrine signals originated from nearby somatic cells, among them Leydig cells have receptors for osteocalcin, a hormone known for exerting positive roles in the promotion of spermatogenesis. The aim of this study was to evaluate roles for osteocalcin on SCs proliferative and differentiation features after coculture with Leydig cells. SCs and Leydig cells were isolated from neonate NMRI offspring mice and adult NMRI mice, respectively. SCs population were then enriched in a differential attachment technique and assessed for morphological features and identity. Then, SCs were cocultured with Leydig cells and incubated with osteocalcin for 4 weeks. Evaluation of proliferation and differentiation-related factors were surveyed using immunocytochemistry (ICC), Western blot, and quantitative real-time polymerase chain reaction (PCR). Finally, the rate of testosterone release to the culture media was measured at the end of 4th week. Morphological and flow cytometry results showed that the SCs were the population of cells able to form colonies and to express ID4, α6-, and β1-integrin markers, respectively. Leydig cells were also able to express Gprc6α as a specific marker for the cells. Incubation of SCs/Leydig coculture with osteocalcin has resulted in an increase in the rate of expressions for differentiation-related markers. Levels of testosterone in the culture media of SCs/Leydig was positively influenced by osteocalcin. It could be concluded that osteocalcin acts as a positive inducer of SCs in coculture with Leydig cells probably through stimulation of testosterone release from Leydig cells and associated signaling.     

Stem Cells: Properties and Prospective Medical Applications

The properties of some stem cells (SCs) that are most interesting in terms of their implications for medicine (embryonic, hematopoietic, and mesenchymal SCs) are considered. SCs are undifferentiated cells capable of both self-maintenance and differentiation into specialized cells. According to their origin, SCs are divided into embryonic and somatic ones. The former can be maintained in vitro for an infinitely long time and can differentiate into all cells of adult organisms. The latter have a limited capacity for differentiation and, probably, a limited proliferative potential. The plasticity of somatic SCs, i.e., their capacity for context-dependent differentiation into unrelated cell types, is of considerable therapeutic importance, although some researchers doubt this capacity. It is assumed that most types of SCs differentiate by the stepwise hierarchical maturation mechanism, one of the steps being rapidly proliferating progenitor cells. The use of SCs in medicine is currently at the stage of preclinical trials. Although embryonic SCs are promising for medicine, there are serious limitations of their use in therapy in the near future. However, the first clinical trials have demonstrated that the approaches involving autotransplantation of hematopoietic and mesenchymal SCs are effective for treating ischemia of extremities and the consequences of myocardial infarction. Obviously, the use of SCs in medicine promises dramatic progress in treating many severe diseases.     

Neural regeneration by regionally induced stem cells within poststroke brains: Novel therapy perspectives for stroke patients

Ischemic stroke is a critical disease which causes serious neurological functional loss such as paresis. Hope for novel therapies is based on the increasing evidence of the presence of stem cell populations in the central nervous system(CNS) and the development of stem-cell-based therapies for stroke patients. Although mesenchymal stem cells(MSCs) represented initially a promising cell source,only a few transplanted MSCs were present near the injured areas of the CNS.Thus, regional stem cells that are present and/or induced in the CNS may be ideal when considering a treatment following ischemic stroke. In this context, we have recently showed that injury/ischemia-induced neural stem/progenitor cells(i NSPCs) and injury/ischemia-induced multipotent stem cells(i SCs) are present within post-stroke human brains and post-stroke mouse brains. This indicates that i NSPCs/i SCs could be developed for clinical applications treating patients with stroke. The present study introduces the traits of mouse and human i NSPCs,with a focus on the future perspective for CNS regenerative therapies using novel i NSPCs/i SCs.     

Mitochondrial metabolism directs stemness and differentiation in P19 embryonal carcinoma stem cells

The relationship between mitochondrial metabolism and cell viability and differentiation in stem cells (SCs) remains poorly understood. In the present study, we compared mitochondrial physiology and metabolism between P19SCs before/after differentiation and present a unique fingerprint of the association between mitochondrial activity, cell differentiation and stemness. In comparison with their differentiated counterparts, pluripotency of P19SCs was correlated with a strong glycolytic profile and decreased mitochondrial biogenesis and complexity: round, low-polarized and inactive mitochondria with a closed permeability transition pore. This decreased mitochondrial capacity increased their resistance against dichloroacetate. Thus, stimulation of mitochondrial function by growing P19SCs in glutamine/pyruvate-containing medium reduced their glycolytic phenotype, induced loss of pluripotent potential, compromised differentiation and became P19SCs sensitive to dichloroacetate. Because of the central role of this type of SCs in teratocarcinoma development, our findings highlight the importance of mitochondrial metabolism in stemness, proliferation, differentiation and chemoresistance. In addition, the present work suggests the regulation of mitochondrial metabolism as a tool for inducing cell differentiation in stem line therapies.Embryonal carcinoma cells, including the P19 cell line, are pluripotent cancer stem cells (CSCs) derived from pluripotent germ cell tumors called teratocarcinomas. These have been described as the malignant counterparts of embryonic stem cells (ESCs) and are considered a good model to study stem cell (SC) differentiation. The P19 cell line can be maintained as undifferentiated cells (P19SCs) or differentiated (P19dCs) to any cell type of the three germ layers. Similar to ESCs, P19 cells differentiate with retinoic acid (RA) in a dose-dependent manner and depending on growth conditions.¹ Although differentiation generally yields a mixed population of differentiated cells, P19 cells grown in monolayer and treated with 1 μM RA primarily differentiate in endoderm or mesoderm, while retaining their immortality.^{2, 3}Although some therapeutic approaches for regenerative medicine and to targeting CSCs are based on differentiation⁴ and mitochondrial-targeted therapies,^{5, 6} very little is known about the role of mitochondrial metabolism in SC maintenance and differentiation.⁷ Several mitochondrial characteristics that distinguish transformed cells from healthy cells have been described,⁸ including increased mitochondrial transmembrane electric potential (Δψm), which may result from decreased mitochondrial ATP production under normoxia.⁹ Similarly, normal SCs primarily rely on glycolysis for energy supply, although the exact mechanism how this occurs in the presence of oxygen and the relationship between SC metabolism and cell fate control is not yet completely understood.¹⁰Given the mitochondrial involvement in stemness and differentiation,¹¹ one can ask whether manipulation of mitochondrial physiology results in an improvement of therapy efficacy. Therefore, characterizing the metabolic and mitochondrial profiles of both SCs and differentiated cells holds promise in order to explain the resistance of cancer cells expressing an embryonic signature to mitochondrial-targeted therapies. In the present work, we have two tandem hypotheses: (a) metabolic and mitochondrial remodeling accompanies P19SC differentiation and (b) P19SC differentiation results in a higher susceptibility to mitochondrial-directed therapies.     

Pleiotropic roles of autophagy in stem cell–based therapies

Stem cells (SCs) have been proven to possess regenerative and immunomodulatory properties and can be used to treat diseases that involve loss of cells due to tissue damage or inflammation. For this approach to succeed, SCs or their derivatives should be able to engraft in the target tissue at least for a short period of time. Unfortunately, once injected, therapeutic SCs will encounter a hostile environment, including hypoxia, lack of nutrients and stromal support, and cells may also be targeted and rejected by the immune system. Therefore, SC's stress-response mechanisms likely play a significant role in survival of injected cells and possibly contribute to their therapeutic efficacy. Autphagy, a stress-response pathway, is involved in many different cellular processes, such as survival during hypoxia and nutrient deprivation, cellular differentiation and de-differentiation, and it can also contribute to their immunovisibility by regulating antigen presentation and cytokine secretion. Autophagy machinery interacts with many proteins and signaling pathways that regulate SC properties, including PI3K/Akt, mammalian target of rapamycin (mTOR), Wnt, Hedgehog and Notch, and it is also involved in regulating intracellular reactive oxygen species (ROS) levels. In this review, we contend that autophagy is an important therapeutic target that can be used to improve the outcome of SC-based tissue repair and regeneration. Further research should reveal whether inhibition or stimulation of autophagy increases the therapeutic utility of SCs and it should also identify appropriate therapeutic regimens that can be applied in the clinic.     

Peripheral blood mononuclear cell secretome for tissue repair

For almost two decades, cell-based therapies have been tested in modern regenerative medicine to either replace or regenerate human cells, tissues, or organs and restore normal function. Secreted paracrine factors are increasingly accepted to exert beneficial biological effects that promote tissue regeneration. These factors are called the cell secretome and include a variety of proteins, lipids, microRNAs, and extracellular vesicles, such as exosomes and microparticles. The stem cell secretome has most commonly been investigated in pre-clinical settings. However, a growing body of evidence indicates that other cell types, such as peripheral blood mononuclear cells (PBMCs), are capable of releasing significant amounts of biologically active paracrine factors that exert beneficial regenerative effects. The apoptotic PBMC secretome has been successfully used pre-clinically for the treatment of acute myocardial infarction, chronic heart failure, spinal cord injury, stroke, and wound healing. In this review we describe the benefits of choosing PBMCs instead of stem cells in regenerative medicine and characterize the factors released from apoptotic PBMCs. We also discuss pre-clinical studies with apoptotic cell-based therapies and regulatory issues that have to be considered when conducting clinical trials using cell secretome-based products. This should allow the reader to envision PBMC secretome-based therapies as alternatives to all other forms of cell-based therapies.     

Differentiated human adipose-derived stromal cells exhibit the phenotypic and functional characteristics of mature Schwann cells through a modified approach

Background aimsTissue engineering technology is a promising therapeutic strategy in peripheral nerve injury. Schwann cells (SCs) are deemed to be a vital component of cell-based nerve regeneration therapies. Many methods for producing SC-like cells derived from adipose-derived stromal cells (ADSCs) have been explored, but their phenotypic and functional characteristics remain unsatisfactory.MethodsWe investigated whether human ADSCs can be induced to differentiate into mature and stable SC-like cells with the addition of insulin, progestero``ne and glucocorticoids. The phenotypic and functional characteristics of new differentiated ADSCs (modified SC-like cells) were evaluated by real-time quantitative polymerase chain reaction, enzyme-linked immunosorbent assay and immunocytochemistry in vitro. Cells loaded into collagen sponge biomaterials were implanted around transected sciatic nerves with a 10-mm gap in vivo. The axon regrowth and functional recovery of the regenerated nerves were assessed by immunohistochemistry and Walking footprint analysis.ResultsAfter differentiation induction, the modified SC-like cells showed significantly up-regulated levels of S100B and P0 and enhanced proliferative and migratory capacities. In addition, the modified SC-like cells showed increased secretion of neurotrophic factors, and their functional characteristics were maintained for more than 3 weeks after removing the induction reagents. The modified SC-like cells exhibited significantly enhanced axon regrowth, myelination and functional recovery after sciatic nerve injury.ConclusionsOverall, the results suggest that this modified induction method can induce human ADSCs to differentiate into cells with the molecular and functional properties of mature SCs and increase the promotion of peripheral nerve regeneration.     

Interactions between Sertoli cells and germ cells in the testis

The production of mature spermatozoa requires a complex interaction between Sertoli cells and germ cells. Sertoli cells regulate aspects of germ cell division and differentiation while germ cells provide signals that modulate Sertoli cell functions. Germ cells can undergo some differentiation independent of Sertoli cells but at certain crucial points the interaction with Sertoli cells is required. There are several means by which this interaction may occur: (1) direct contact of components of the plasma membrane may act as a signal; (2) secondary messengers could be exchanged via gap junctions; (3) the secretion of paracrine factors may facilitate intercellular communication.     

Interrelation between NO and H2O2 formation and their role in the regulation of ion homeostasis of cells

The review deals with some problems of formation, utilization and functioning two of the most important and the lest cytotoxic active oxidative metabolites--NO and H2O2. The interrelation of NO and H2O2 cellular metabolism is reviewed. There are literature data regarding NO and H2O2 significance in the cellular signalling transduction and tissues paracrine regulation. The peculiar attention is dedicated to NO and H2O2 significance in the smooth muscle cells ion homeostasis regulation particularly in the uterus. As well there are the own author's data evidencing about the composite and ambiguous concentration-dependent physiological action of these metabolites. Varying in level in the cells NO and H2O2 may have a cyclic character similar to the oscillations of some other signaling molecules concentrations, evidently playing an important role in the mechanisms of cellular signal transduction and in the paracrine system.     

Unconventional therapies for cancer: 1. Essiac

Physicians and patients have been frustrated by the lack of reliable information on unconventional therapies. To help fill this gap in the area of breast cancer therapy, the Canadian Breast Cancer Research Initiative formed a task force to advise it on how best to promote research into unconventional therapies. As part of the work of the task force, a review of the available literature was carried out for each of the following products: Essiac, green tea, Iscador, hydrazine sulfate, vitamins A, C and E, and 714-X. The first article in this series on unconventional therapies for cancer describes the methodology used to obtain and evaluate the information and provides a summary of the findings on Essiac. Subsequent articles will cover the other products. For most of the products reviewed, there has been some indication of possible benefit but no definitive evidence. Innovative and collaborative research needed to meet the information needs of growing numbers of patients and their physicians is now being sponsored by the Canadian Breast Cancer Research Initiative. Open communication between patients and physicians is also necessary for the maintenance of an appropriate therapeutic partnership and for the identification and control of side effects. The Ontario Division of the Canadian Cancer Society, a partner in the Canadian Breast Cancer Research Initiative, supported the preparation of a patient-information piece on unconventional therapies to accompany the series. This item will assist patients who are considering such therapies and will promote open communication between patients and their physicians.     

Animal Research Ethics in Africa: Is Tanzania Making Progress?

The significance of animals in research cannot be over‐emphasized. The use of animals for research and training in research centres, hospitals and schools is progressively increasing. Advances in biotechnology to improve animal productivity require animal research. Drugs being developed and new interventions or therapies being invented for cure and palliation of all sorts of animal diseases and conditions need to be tested in animals for their safety and efficacy at some stages of their development. Drugs and interventions for human use pass through a similar development process and must be tested pre‐clinically in laboratory animals before clinical trials in humans can be conducted. Therefore, animals are important players in research processes which directly and indirectly benefit animals and humans. However, questions remain as to whether these uses of animals consider the best interests of animals themselves. Various research and training institutions in Tanzania have established some guidelines on animal use, including establishing animal ethics committees. However, most institutions have not established oversight committees. In institutions where there may be guidelines and policies, there are no responsible committees or units to directly oversee if and how these guidelines and policies are enforced; thus, implementation becomes difficult or impossible. This paper endeavours to raise some issues associated with the responsible use of animals in research and training in Tanzania and highlights suggestions for improvement of deficiencies that exist in order to bridge the gap between what ought to be practised and what is practised.     

Considerations for the clinical use of stem cells in genitourinary regenerative medicine

The genitourinary tract can be affected by several pathologies which require repair or replacement to recover biological functions. Current therapeutic strategies are challenged by a growing shortage of adequate tissues. Therefore, new options must be considered for the treatment of patients, with the use of stem cells (SCs) being attractive. Two different strategies can be derived from stem cell use: Cell therapy and tissue therapy, mainly through tissue engineering. The recent advances using these approaches are described in this review, with a focus on stromal/mesenchymal cells found in adipose tissue. Indeed, the accessibility, high yield at harvest as well as anti-fibrotic, immunomodulatory and proangiogenic properties make adipose-derived stromal/SCs promising alternatives to the therapies currently offered to patients. Finally, an innovative technique allowing tissue reconstruction without exogenous material, the self-assembly approach, will be presented. Despite advances, more studies are needed to translate such approaches from the bench to clinics in urology. For the 21^st century, cell and tissue therapies based on SCs are certainly the future of genitourinary regenerative medicine.     

Co-localisation of Kir4.1 and AQP4 in rat and human cochleae reveals a gap in water channel expression at the transduction sites of endocochlear K+ recycling routes

Sensory transduction in the cochlea depends on perilymphatic-endolymphatic potassium (K⁺) recycling. It has been suggested that the epithelial supporting cells (SCs) of the cochlear duct may form the intracellular K⁺ recycling pathway. Thus, they must be endowed with molecular mechanisms that facilitate K⁺ uptake and release, along with concomitant osmotically driven water movements. As yet, no molecules have been described that would allow for volume-equilibrated transepithelial K⁺ fluxes across the SCs. This study describes the subcellular co-localisation of the K_ir4.1?K⁺ channel (K_ir4.1) and the aquaporin-4 water channel (AQP4) in SCs, on the basis of immunohistochemical double-labelling experiments in rat and human cochleae. The results of this study reveal the expression of K_ir4.1 in the basal or basolateral membranes of the SCs in the sensory domain of the organ of Corti that are adjacent to hair cells and in the non-sensory domains of the inner and outer sulci that abut large extracellular fluid spaces. The SCs of the inner sulcus (interdental cells, inner sulcus cells) and the outer sulcus (Hensen??s cells, outer sulcus cells) display the co-localisation of K_ir4.1 and AQP4 expression. However, the SCs in the sensory domain of the organ of Corti reveal a gap in the expression of AQP4. The outer pillar cell is devoid of both K_ir4.1 and AQP4. The subcellular co-localisation of K_ir4.1 and AQP4 in the SCs of the cochlea described in this study resembles that of the astroglia of the central nervous system and the glial Mueller cells in the retina.