ALBERT

Description: 〈h3〉Abstract〈/h3〉 〈p〉For decades, megakaryocytopoiesis is believed to occur following a classical binary hierarchical developmental model. This model is based on an analysis of predefined flow-sorted cell populations by using cell surface markers. However, this classical model has been challenged by increasing evidences obtained with new techniques which integrating flow cytometric, transcriptomic and functional data at single-cell level and with lineage tracing technique. These recent advances in megakaryocytopoiesis proposed that commitment of haematopoietic stem cells (HSCs) towards megakaryocytic lineage occurs in much earlier stage than that postulated in the classical model. There may exist multipotent but megakaryocyte (MK)/platelet-biased HSCs within HSC compartment and even HSCs can directly differentiate into MKs in steady state or in response to stress. In this review, we focus on recent findings about differentiation from commitment of HSCs to MK and its regulation, and discuss future directions in this research field.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉The aim of this paper was to describe the outcome of therapeutic administration of mesenchymal stem cells (MSC) obtained from Wharton’s jelly (WJ-MSCs) in paediatric patients with spina bifida (SB) during a medical therapeutic experiment. We retrospectively analysed the records of twenty-eight patients aged 1–18 years (median age 4 years) recruited in daily clinical practice. Each patient received 0.9–5.0 × 10〈sup〉6〈/sup〉 WJ-MSCs/kg (median 2.6 × 10〈sup〉6〈/sup〉 WJ-MSCs/kg) administered in 1–5 injections as an experimental treatment for SB (allogenic administration). All the patients were examined by the same neurologist (study investigator, SI) on the day of each infusion. Based on the neurological examination, the SI used a six-point Likert scale to assess the quality of life and self-service of each patient. Twenty-six follow-up observations after MSC administration were analysed retrospectively. In addition, the assessments of the parents and other healthcare professionals were obtained for 5 patients and compared with the SI’s assessment. Twenty-one of 26 patients (81%) experienced some improvement in their health status. Twenty-one (81%) patients experienced increased quality of life (median 2.0) and 10 patients (38%) achieved a slight increase in their self-service level (median 1). Improvement was achieved in 12 out of 17 areas. Five were significant in low-power sign test: muscle tension, muscle strength, gross motor development, micturition/defecation control, and cognitive functions. Adverse events were mild and temporary. Age, body mass, single dose or poor response after the first administration were not significant predictors of later response to treatment in contrast to the total cell dose per one kg in the whole treatment course. WJ-MSC administration is a safe and effective procedure that improves motor functions, micturition/defecation control, and cognitive functions, and improves the quality of life in children with SB.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Hematopoietic stem cells (HSCs) are the best-characterized stem cells in adult tissues. Nevertheless, as of today, many open questions remain. First, what is the phenotype of the most primitive “pre-HSC” able to undergo asymmetric divisions during ex vivo expansion that gives rise to HSC for all hemato-lymphopoietic lineages. Next, most routine in vitro assays designed to study HSC specification into hematopoietic progenitor cells (HPCs) for major hematopoietic lineages are based on a limited number of peptide-based growth factors and cytokines, neglecting the involvement of several other regulators that are endowed with hematopoietic activity. Examples include many hormones, such as pituitary gonadotropins, gonadal sex hormones, IGF-1, and thyroid hormones, as well as bioactive phosphosphingolipids and extracellular nucleotides (EXNs). Moreover, in addition to regulation by stromal-derived factor 1 (SDF-1), trafficking of these cells during mobilization or homing after transplantation is also regulated by bioactive phosphosphingolipids, EXNs, and three ancient proteolytic cascades, the complement cascade (ComC), the coagulation cascade (CoA), and the fibrinolytic cascade (FibC). Finally, it has emerged that bone marrow responds by “sterile inflammation” to signals sent from damaged organs and tissues, systemic stress, strenuous exercise, gut microbiota, and the administration of certain drugs. This review will address the involvement of these unconventional regulators and present a broader picture of hematopoiesis.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Equine metabolic syndrome (EMS) is characterized by adiposity, insulin dysregulation and increased risk for laminitis. Increased levels of specific liver enzymes in the peripheral blood are typical findings in horses diagnosed with EMS. Current management of EMS is based on caloric restriction and increased physical activity. However, new potential treatment options are arising such as the transplantation of autologous adipose stem cells (ASC). However, cytophysiological properties of ASC derived from EMS horses are impaired which strongly limits their therapeutic potential. We hypothesized, that in vitro pharmacotherapy of those cells with 5-azacytidine (AZA) and resveratrol (RES) before their clinical application can reverse the aged phenotype of those cells and improve clinical outcome of autologous therapy. A 9 year old Dutch Warmblood Horse used for driving, was presented with severe obesity, insulin resistance. After EMS diagnosis, the animal received three intravenous injections of autologous, AZA/RES treated ASCs at weekly intervals. The therapeutic effect was assessed by the analysis of liver specific enzymes in the blood. ASC-transplantation reduced levels of glutamate dehydrogenase (GLDH), gamma-glutamyltransferase (GGT), lactate dehydrogenase (LDH) and aspartate transaminase (AST). This case report demonstrates the therapeutic potential of this intervention for EMS as well as apt utility of autologous, rejuvenated ASC injections.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Adipose stromal cells are promising tools for clinical applications in regeneration therapies, due to their ease of isolation from tissue and its high yield; however, their ability to transdifferentiate into neural phenotypes is still a matter of controversy. Here, we show that combined chemical and neurotrophin stimulation resulted in neuron-like morphology and regulated expression and activity of several genes involved in neurogenesis and neurotransmission as well as ion currents mediated by NMDA and GABA receptors. Among them, expression patterns of genes coding for kinin-B1 and B2, α7 nicotinic, M1, M3 and M4 muscarinic acetylcholine, glutamatergic (AMPA2 and mGlu2), purinergic P2Y1 and P2Y4 and GABAergic (GABA-A, β3-subunit) receptors and neuronal nitric oxide synthase were up-regulated compared to levels of undifferentiated cells. Simultaneously, expression levels of P2X1, P2X4, P2X7 and P2Y6 purinergic and M5 muscarinic acetylcholine receptors were down-regulated. Agonist-induced activity levels of the studied receptor classes also augmented during neuronal transdifferentiation. Transdifferentiated cells expressed high levels of neuronal β3-tubulin, NF-H, NeuN and MAP-2 proteins as well as increased ASCL1, MYT1 and POU3F2 gene expression known to drive neuronal fate determination. The presented work contributes to a better understanding of transdifferentiation induced by neurotrophins for a prospective broad spectrum of medical applications.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Origin of cancer stem cells (CSCs) and mechanisms by which oncogene 〈em〉PTTG1〈/em〉 contributes to tumor progression via CSCs is not known. Ovarian CSCs exhibit characteristics of self-renewal, tumor-initiation, growth, differentiation, drug resistance, and tumor relapse. A common location of putative origin, namely the ovarian surface epithelium, is shared between the normal stem and CSC compartments. Existence of ovarian stem cells and their co-expression with CSC signatures suggests a strong correlation between origin of epithelial cancer and CSCs. We hereby explored a putative oncogene 〈em〉PTTG1〈/em〉 (Securin), reported to be overexpressed in various tumors, including ovarian. We report a previously overlooked role of 〈em〉PTTG1〈/em〉 as a marker of CSCs thereby modulating CSC, germline, and stemness-related genes. We further characterized 〈em〉PTTG1〈/em〉’s ability to regulate (cancer) stem cell-associated self-renewal and epithelial-mesenchymal transition pathways. Collectively, the data sheds light on a potential target expressed during ovarian tumorigenesis and metastatically disseminated ascites CSCs in the peritoneal cavity. Present study highlights this unconventional, under-explored role of 〈em〉PTTG1〈/em〉 in regulation of stem and CSC compartments in ovary, ovarian cancer and ascites and highlights it as a potential candidate for developing CSC specific targeted therapeutics.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Neural progenitor cells (NPCs) are multipotent cells that have the potential to produce neurons and glial cells in the neural system. NPCs undergo identity maintenance or differentiation regulated by different kinds of transcription factors. Here we present evidence that ETV5, which is an ETS transcription factor, promotes the generation of glial cells and drives the neuronal subtype-specific genes in newly differentiated neurons from the human embryonic stem cells-derived NPCs. Next, we find a new role for ETV5 in the repression of NEUROG2 expression in NPCs. ETV5 represses NEUROG2 transcription via NEUROG2 promoter and requires the ETS domain. We identify ETV5 has the binding sites and is implicated in silent chromatin in NEUROG2 promoter by chromatin immunoprecipitation (ChIP) assays. Further, NEUROG2 transcription repression by ETV5 was shown to be dependent on a transcriptional corepressor (CoREST). During NPC differentiation toward neurons, ETV5 represses NEUROG2 expression and blocks the appearance of glutamatergic neurons. This finding suggests that ETV5 negatively regulates NEUROG2 expression and increases the number of GABAergic subtype neurons derived from NPCs. Thus, ETV5 represents a potent new candidate protein with benefits for the generation of GABAergic neurons.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉The promise of engineering specific cell types from stem cells and rebuilding damaged or diseased tissues has fascinated stem cell researchers and clinicians over last few decades. Mesenchymal Stem Cells (MSCs) have the potential to differentiate into non-mesodermal cells, particularly neural-lineage, consisting of neurons and glia. These multipotent adult stem cells can be used for implementing clinical trials in neural repair. Ongoing research identifies several molecular mechanisms involved in the speciation of neuroglia, which are tightly regulated and interconnected by various components of cell signalling machinery. Growing MSCs with multiple inducers in culture media will initiate changes on intricately interlinked cell signalling pathways and processes. Net result of these signal flow on cellular architecture is also dependent on the type of ligands and stem cells investigated in vitro. However, our understanding about this dynamic signalling machinery is limited and confounding, especially with spheroid structures, neurospheres and organoids. Therefore, the results for differentiating neurons and glia in vitro have been inconclusive, so far. Added to this complication, we have no convincing evidence about the electrical conductivity and functionality status generated in differentiating neurons and glia. This review has taken a step forward to tailor the information on differentiating neuroglia with the common methodologies, in practice.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉We have recently demonstrated that purinergic signaling in bone marrow (BM) microenvironment regulates mobilization of hematopoietic stem progenitor cells (HSPCs), mesenchymal stroma cells (MSCs), endothelial progenitor cells (EPCs), and very small embryonic like stem cells (VSELs) into the peripheral blood (PB). While extracellular adenosine triphosphate (ATP) promotes mobilization, its metabolite extracellular adenosine has an opposite effect. Since ATP is processed in extracellular space to adenosine by ectonucleotidases including cell surface expressed CD39 and CD73, we asked if inhibition of these enzymes by employing in vivo small molecular inhibitors ARL67156 and AMPCP of CD39 and CD73 respectively, alone or combined could enhance granulocyte stimulating factor (G-CSF)- and AMD3100-induced pharmacological mobilization of stem cells. Herein we report that pre-treatment of donor mice with CD39 and CD73 inhibitors facilitates the mobilization of HSPCs as well as other types of BM-residing stem cells. This data on one hand supports the role of purinergic signaling in stem cell trafficking, and on the other since both compounds are not toxic against human cells, they could be potentially employed in the clinic to enhance the mobilization of BM residing stem cells for clinical purposes.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Occurrence of stem cells (CSCs) in cancer is well established in last two decades. These rare cells share several properties including presence of common surface markers, stem cell markers, chemo- and radio- resistance and are highly metastatic in nature; thus, considered as valuable prognostic and therapeutic targets in cancer. However, the studies related to CSCs pave number of issues due to rare cell population and difficulties in their isolation ascribed to common stem cell marker. Various techniques including flow cytometry, laser micro-dissection, fluorescent nanodiamonds and microfluidics are used for the isolation of these rare cells. In this review, we have included the advance strategies adopted for the isolation of CSCs using above mentioned techniques. Furthermore, CSCs are primarily found in the core of the solid tumors and their microenvironment plays an important role in maintenance, self-renewal, division and tumor development. Therefore, 〈em〉in vivo〈/em〉 tracking and model development become obligatory for functional studies of CSCs. Fluorescence and bioluminescence tagging has been widely used for transplantation assay and lineage tracking experiments to improve our understanding towards CSCs behaviour in their niche. Techniques such as Magnetic resonance imaging (MRI) and Positron emission tomography (PET) have proved useful for tracking of endogenous CSCs which could be helpful in their identification in clinical settings.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉The current understanding and effective treatment of liver disease is far from satisfactory. Liver organoids and liver buds (LBs) transforming cell culture from two dimensions(2D) to three dimensions(3D) has provided infinite possibilities for stem cells to use in clinic. Recent technological advances in the 3D culture have shown the potentiality of liver organoids and LBs as the promising tool to model in vitro liver diseases. The induced LBs and liver organoids provide a platform for cell-based therapy, liver disease models, liver organogenesis and drugs screening. And its genetic heterogeneity supplies a way for the realization of precision medicine.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉T cell malignancies are aggressive diseases with no standard treatment available, often resulting in poor patient outcomes. Lately, the recent FDA approval of a CD19 CAR T cell therapy for B cell acute lymphoblastic leukemia has earned nationwide attention, leading to the possibility that success of CD19 CAR therapy can be extended to T cell malignancies. However, the impact of T cell depletion due to a shared antigen pool remains an issue to be resolved. Here, we describe a CD4CAR capable of eliminating CD4-positive T cell acute lymphoblastic leukemia in a systemic mouse model, with CAMPATH (alemtuzumab) as a natural safety switch to deplete the infused CD4CAR T cells to prevent toxicities associated with CD4 cell aplasia. Our data support the potential use of CD4CAR T cells for the treatment of CD4-postive T-cell acute lymphoblastic leukemia malignancies or refractory disease in clinical settings.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Retinal diseases were always difficult problem for clinical ophthalmology. Modern methods of their treatment only decrease risk of complications, however in Russia was created better technology for this purpose: peptide bioregulators, which were made by sequential adding of amino acids one to another, binding with the promoter region of genes, and promoting retinoprotective effect by regulation of their expression, improving the state of the retina.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Despite considerable advances made in understanding of lung cancer biology, there has been meek improvement in lung cancer treatment outcome with 4% to 5% increase in 5-year survival rates in the last four decades. Underlying problem of lung cancer recurrence and poor prognosis is attributed to the presence of cancer stem cells (CSCs) which possess the potential to differentiate, proliferate and trigger chemo-resistance, tumor progression and metastasis, despite initial elimination of the tumor. To address specific targeting of CSCs, we investigated the effects of a small molecule Verrucarin J (VJ) on lung cancer cell lines A549 and H1793. VJ significantly inhibited cell proliferation of both cell lines, with IC〈sub〉50〈/sub〉 values of approximately 10 nM for A549 and 20 nM for H1793 respectively after 48 h of treatment. A549 cell line when treated with VJ, induced cell apoptosis with concomitant down regulation of key CSC specific genes- ALDH1, LGR5, OCT4 and CD133 in a dose-dependent manner. To delineate the molecular mechanism by which VJ targets lung cancer cells and CSCs, we determined the effects of VJ on CSC self-renewal pathways Wnt1/β-catenin and Notch1. Treatment of A549 cell line with VJ inhibited significantly both the signalling pathways, suggesting inhibition of expression of CSC genes by VJ through the inhibition of CSC self-renewal signalling pathways. Taken together, our results suggest that VJ may serve as a potent anticancer drug to target cancer cells and CSCs.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Chemotherapy constitutes one of the key treatment modalities for solid and hematological malignancies. Albeit being an effective treatment, chemotherapy application is often limited by its damage to healthy tissues, and curative treatment options for chemotherapy-related side effects are largely missing. As mesenchymal stromal cells (MSCs) are known to exhibit regenerative capacity mainly by supporting a beneficial microenvironment for tissue repair, MSC-based therapies may attenuate chemotherapy-induced tissue injuries. An increasing number of animal studies shows favorable effects of MSC-based treatments; however, clinical trials for MSC therapies in the context of chemotherapy-related side effects are rare. In this concise review, we summarize the current knowledge of the effects of MSCs on chemotherapy-induced tissue toxicities. Both preclinical and early clinical trials investigating MSC-based treatments for chemotherapy-related side reactions are presented, and mechanistic explanations about the regenerative effects of MSCs in the context of chemotherapy-induced tissue damage are discussed. Furthermore, challenges of MSC-based treatments are outlined that need closer investigations before these multipotent cells can be safely applied to cancer patients. As any pro-tumorigenicity of MSCs needs to be ruled out prior to clinical utilization of these cells for cancer patients, the pro- and anti-tumorigenic activities of MSCs are discussed in detail.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Osteochondrosis (osteochondrosis dissecans; OCD) is a disease syndrome of growing cartilage related to different clinical entities such as epiphysitis, subchondral cysts and angular carpal deformities, which occurs in growing animals of all species, including horses. Nowadays, these disorders are affecting increasing numbers of young horses worldwide. As a complex multifactorial disease, OCD is initiated when failure in cartilage canals because of existing ischemia, chondrocyte biogenesis impairment as well as biochemical and genetic disruptions occur. Recently, particular attention have been accorded to the definition of possible relations between OCD and some metabolic disorders; in this way, implication of mitochondrial dysfunctions, endoplasmic reticulum disruptions, oxidative stress or endocrinological affections are among the most considered axes for future researches. As one of the most frequent cause of impaired orthopaedic potential, which may result in a sharp decrease in athletic performances of the affected animals, and lead to the occurrence of complications such as joint fragility and laminitis, OCD remains as one of the primary causes of considerable economic losses in all sections of the equine industry. It would therefore be important to provide more information on the exact pathophysiological mechanism(s) underlying early OC(D) lesions, in order to implement innovative strategies involving the use of progenitor stem cells, which are considered nowadays as a promising approach to regenerative medicine, with the potential to treat numerous orthopaedic disorders, including osteo-degenerative diseases, for prevention and reduction of incidence of the disease, not only in horses, but also in human medicine, as the equine model is already widely accepted by the scientific community and approved by the FDA, for the research and application of cellular therapies in the treatment of human conditions.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Stem cell aging underlies aging-associated disorders, such as steeply increased incidences of tumors and impaired regeneration capacity upon stress. However, whether and how the intestinal stem cells age remains largely unknown. Here we show that intestinal stem cells derived from 24-month-old mice hardly form typical organoids with crypt-villus structures, but rather mainly form big, rounded cysts devoid of differentiated cell types, which mimics the culturing of heterozygous APC-deficient cells from the 〈em〉APC〈/em〉〈sup〉〈em〉min〈/em〉〈/sup〉 mouse line. Further analysis showed that cultured crypts derived from aged mice exhibited reduced expression levels of differentiation genes and higher expression of Wnt target genes. Lowering the concentration of R-spondin-1 in the culture system significantly reduced formation of rounded cysts, accompanied by an increased formation of organoids from crypts derived from old mice. We are the first to uncover that intestinal stem cells derived from old mice harbor significant deficiency in differentiation that can be partially rescued through a reduction in R-spondin-1 exposure. This could be highly relevant to intestinal tumor development and the reduced regeneration potential observed in the aged population. Our study provides the first experimental evidence that an over-responsiveness to Wnt/beta-catenin signaling of aged intestinal stem cells mediates the aging-induced deficiency in differentiation, and could serve as a potential target to ameliorate aging-associated intestinal pathologies.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Primary stem cells, after several cell divisions, enter into a senescence state, that is characterized by alterations to spindle-shape typical morphology. This concern is one of the main problems in the use of human mesenchymal stem cells (hMSCs) in clinical applications which demand cells in large numbers. Short peptides had geroprotective properties and stimulated stem cell differentiation. The aim of the study is to demonstrate the role of AEDG and KED peptides in maintaining oral hMSCs morphology and functions over long-term expansion. 2 types of hMSCs were investigated: human periodontal ligament stem cells (hPLSCs) and human gingival mesenchymal stem cells (hGMSCs). Cells at the 25th passage were divided into 3 groups: 1 – control (without adding peptide), 2 – treated with AEDG peptide, 3 – treated with KED peptide. Cell cultures were analyzed by an immunofluorescence method and RT-PCR on the p16 and p21 senescence markers expression. AEDG peptide decreased p16 and p21 mRNA expression by 1.56–2.44 times in comparison with the control group. KED peptide decreased p16 and p21 mRNA expression by 1.82–3.23 times in comparison with the control group. These results were confirmed by immunofluorescent visualization. AEDG and KED peptides could be used as supplementary substances in a culture medium to delay the expression of senescence markers in long term stem cell cultivation in order to promote the large-scale in vitro expansion necessarily required for stem cell therapy clinical application. The data obtained confirm the geroprotective effect of AEDG and KED peptide, which was shown early in animal and cells models.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Duchenne Muscular Dystrophy (DMD) is a progressive lethal disease caused by X-linked mutations of the dystrophin gene. Dystrophin deficiency clinically manifests as skeletal and cardiac muscle weakness, leading to muscle wasting and premature death due to cardiac and respiratory failure. Currently, no cure exists. Since heart disease is becoming a leading cause of death in DMD patients, there is an urgent need to develop new more effective therapeutic strategies for protection and improvement of cardiac function. We previously reported functional improvements correlating with dystrophin restoration following transplantation of Dystrophin Expressing Chimeric Cells (DEC) of myoblast origin in the 〈em〉mdx〈/em〉 and 〈em〉mdx/scid〈/em〉 mouse models. Here, we confirm positive effect of DEC of myoblast (MB〈sup〉〈em〉wt〈/em〉〈/sup〉/MB〈sup〉〈em〉mdx〈/em〉〈/sup〉) and mesenchymal stem cells (MB〈sup〉〈em〉wt〈/em〉〈/sup〉/MSC〈sup〉〈em〉mdx〈/em〉〈/sup〉) origin on protection of cardiac function after systemic DEC transplant. Therapeutic effect of DEC transplant (0.5 × 10〈sup〉6〈/sup〉) was assessed by echocardiography at 30 and 90 days after systemic-intraosseous injection to the 〈em〉mdx〈/em〉 mice. At 90 days post-transplant, dystrophin expression in cardiac muscles of DEC injected mice significantly increased (15.73% ± 5.70 –MB〈sup〉〈em〉wt〈/em〉〈/sup〉/MB〈sup〉〈em〉mdx〈/em〉〈/sup〉 and 5.22% ± 1.10 – MB〈sup〉〈em〉wt〈/em〉〈/sup〉/MSC〈sup〉〈em〉mdx〈/em〉〈/sup〉 DEC) when compared to vehicle injected controls (2.01% ± 1.36) and, correlated with improved ejection fraction and fractional shortening on echocardiography. DEC lines of MB and MSC origin introduce a new promising approach based on the combined effects of normal myoblasts with dystrophin delivery capacities and MSC with immunomodulatory properties. Our study confirms feasibility and efficacy of DEC therapy on cardiac function and represents a novel therapeutic strategy for cardiac protection and muscle regeneration in DMD.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Pharmaceuticals and cell-based regenerative medicine for Parkinson’s disease (PD) offer palliative relief but do not arrest the disease progression. Cell therapy has emerged as an experimental treatment, but current cell sources such as human umbilical cord blood (hUCB) stem cells display only partial recapitulation of mature dopaminergic neuron phenotype and function. Nonetheless, stem cell grafts ameliorate PD-associated histological and behavioral deficits likely through stem cell graft-secreted therapeutic substances. We recently demonstrated the potential of hUCB-derived plasma in enhancing motor capabilities and gastrointestinal function, as well as preventing dopaminergic neuronal cell loss, in an 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP) rodent model of PD. Recognizing the translational need to test in another PD model, we now examined here the effects of an intravenously transplanted combination of hUCB and plasma into the 6-hydroxydopamine (6-OHDA) lesioned adult rats. Animals received three separate doses of 4 × 10〈sup〉6〈/sup〉 hUCB cells with plasma beginning at 7 days after stereotaxic 6-OHDA lesion, then behaviorally and immunohistochemically evaluated over 56 days post-lesion. Whereas vehicle-treated lesioned animals exhibited the typical 6-OHDA neurobehavioral symptoms, hUCB and plasma-treated lesioned animals showed significant attenuation of motor function, gut motility, and nigral dopaminergic neuronal survival, combined with diminished pro-inflammatory microbiomes not only in the nigra, but also in the gut. Altogether these data support a regenerative medicine approach for PD by sequestering inflammation and neurotoxicity through correction of gut dysbiosis.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉A potential therapeutic strategy for diabetes is the transplantation of induced-insulin secreting cells. Based on the common embryonic origin of liver and pancreas, we studied the potential of adult human liver stem-like cells (HLSC) to generate in vitro insulin-producing 3D spheroid structures (HLSC-ILS). HLSC-ILS were generated by a one-step protocol based on charge dependent aggregation of HLSC induced by protamine. 3D aggregation promoted the spontaneous differentiation into cells expressing insulin and several key markers of pancreatic β cells. HLSC-ILS showed endocrine granules similar to those seen in human β cells. In static and dynamic in vitro conditions, such structures produced C-peptide after stimulation with high glucose. HLSC-ILS significantly reduced hyperglycemia and restored a normo-glycemic profile when implanted in streptozotocin-diabetic SCID mice. Diabetic mice expressed human C-peptide and very low or undetectable levels of murine C-peptide. Hyperglycemia and a diabetic profile were restored after HLSC-ISL explant. The gene expression profile of in vitro generated HLSC-ILS showed a differentiation from HLSC profile and an endocrine commitment with the enhanced expression of several markers of β cell differentiation. The comparative analysis of gene expression profiles after 2 and 4 weeks of in vivo implantation showed a further β-cell differentiation, with a genetic profile still immature but closer to that of human islets. In conclusion, protamine-induced spheroid aggregation of HLSC triggers a spontaneous differentiation to an endocrine phenotype. Although the in vitro differentiated HLSC-ILS were immature, they responded to high glucose with insulin secretion and in vivo reversed hyperglycemia in diabetic SCID mice.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Mesenchymal stem cells (MSCs) represent a promising source of cell-based therapies for treatment of a wide variety of injuries and diseases. Their tropism and migration to the damaged sites, which are elicited by cytokines secreted from tissues around pathology, are the prerequisite for tissue repair and regeneration. Better understanding of the elicited-migration of MSCs and discovering conditions that elevate their migration ability, will help to increase their homing to pathologies and improve therapeutic efficacy. It is increasingly recognized that microRNAs are important regulators of cell migration. Here we summarize current understanding of the microRNA-regulated migration of MSCs.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉CD133 is a wildly used cancer stem cell marker. The purpose of this study was to explore the significance of 〈em〉CD133〈/em〉 mRNA in human cancers mainly based on The Cancer Genome Atlas (TCGA) database. Bioinformatic analyses were done by using public repositories, including BioGPS, SAGE Genie tools, Oncomine analysis, Regulome Explorer, COSMIC analysis, and Kaplan-Meier Plotter. The main findings in this study were: 1) High 〈em〉CD133〈/em〉 mRNA was correlated with a benign survival rate of gastric cancer and lung cancer; 2) Transmembrane protein 125 (TMEM125) in bladder urothelial carcinoma and intercellular adhesion molecule 2 (ICAM2) in ovarian serous cystadenocarcinoma were closely related to CD133 expression; 3) The location and the topological structure of CD133 protein were not determined by its transcript variant in cancer cells; 4) CD38 and CD200 may be used as novel surface markers for solid cancers. However, the mechanism of these findings is not completely clear, further studies have to be performed in the future.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Phenotype definition is driven by epigenetic mechanisms as well as directly influenced by the cell microenvironment and by biophysical signals deriving from the extracellular matrix. The possibility to interact with the epigenetic signature of an adult mature cell, reversing its differentiated state and inducing a short transient high plasticity window, was previously demonstrated. In parallel, in vitro studies have shown that 3D culture systems, mimicking cell native tissue, exert significant effects on cell behavior and functions. Here we report the production of “PTFE micro-bioreactors” for long-term culture of epigenetically derived high plasticity cells. The system promotes 3D cell rearrangement, global DNA demethylation and elevated transcription of pluripotency markers, that is dependent on WW domain containing transcription regulator 1 (TAZ) nuclear accumulation and SMAD family member 2 (SMAD2) co-shuttling. Our findings demonstrate that the use of 3D culture strategies greatly improves the induction and maintenance of a high plasticity state.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉The potential of human mesenchymal stromal/stem cells (MSCs) including oral stem cells (OSCs) as a cell source to derive functional neurons has been inconclusive. Here we tested a number of human OSCs for their neurogenic potential compared to non-OSCs and employed various neurogenic induction methods. OSCs including dental pulp stem cells (DPSCs), gingiva-derived mesenchymal stem cells (GMSCs), stem cells from apical papilla and non-OSCs including bone marrow MSCs (BMMSCs), foreskin fibroblasts and dermal fibroblasts using non-neurosphere-mediated or neurosphere-mediated methods to guide them toward neuronal lineages. Cells were subjected to RT-qPCR, immunocytofluorescence to detect the expression of neurogenic genes or electrophysiological analysis at final stage of maturation. We found that induced DPSCs and GMSCs overall appeared to be more neurogenic compared to other cells either morphologically or levels of neurogenic gene expression. Nonetheless, of all the neural induction methods employed, only one neurosphere-mediated method yielded electrophysiological properties of functional neurons. Under this method, cells expressed increased neural stem cell markers, nestin and SOX1, in the first phase of differentiation. Neuronal-like cells expressed 〈em〉βIII-tubulin, CNPase, GFAP, MAP-2, NFM, pan-Nav, GAD67, Nav1.6, NF1, NSE, PSD95〈/em〉, and 〈em〉synapsin〈/em〉 after the second phase of differentiation to maturity. Electrophysiological experiments revealed that 8.3% of DPSC-derived neuronal cells and 21.2% of GMSC-derived neuronal cells displayed action potential, although no spontaneous excitatory/inhibitory postsynaptic action potential was observed. We conclude that DPSCs and GMSCs have the potential to become neuronal cells in vitro, therefore, these cells may be used as a source for neural regeneration.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Bioactive phospholipids, including sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), lysophosphatidylcholine (LPC), and its derivative lysophosphatidic acid (LPA), have emerged as important mediators regulating the trafficking of normal and cancer cells. While the role of S1P in regulating migration of hematopoietic cells is well established, in this work we compared its biological effects to the effects of C1P, LPC, and LPA. We employed 10 human myeloid and lymphoid cell lines as well as blasts from AML patients. We observed that human leukemic cells express functional receptors for phospholipids and respond to stimulation by phosphorylation of p42/44 MAPK and AKT. We also found that bioactive phospholipids enhanced cell migration and adhesion of leukemic cells by downregulating expression of HO-1 and iNOS in a p38 MAPK-dependent manner but did not affect cell proliferation. By contrast, downregulation of p38 MAPK by SB203580 enhanced expression of HO-1 and iNOS and decreased migration of leukemic cells in vitro and their seeding efficiency to vital organs in vivo after injection into immunodeficient mice. Based on these findings, we demonstrate that, besides S1P, human leukemic cells also respond to C1P, LPC, and LPA. Since the prometastatic effects of bioactive phospholipids in vivo were mediated, at least in part, by downregulating HO-1 and iNOS expression in a p38 MAPK-dependent manner, we propose that inhibitors of p38 MAPK or stimulators of HO-1 activity will find application in inhibiting the spread of leukemic cells in response to bioactive phospholipids.〈/p〉

Description: 〈p〉Please note the following errors in the original version.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Our previous studies showed that nestin-expressing hair follicle-associated-pluripotent (HAP) stem cells, which reside in the bulge area of the hair follicle, could restore injured nerve and spinal cord and differentiate into cardiac muscle cells. Here we transplanted mouse green fluorescent protein (GFP)-expressing HAP stem-cell colonies enclosed on polyvinylidene fluoride membranes (PFM) into the severed thoracic spinal cord of nude mice. After seven weeks of implantation, we found the differentiation of HAP stem cells into neurons and glial cells. Our results also showed that PFM-captured GFP-expressing HAP stem-cell colonies assisted complete reattachment of the thoracic spinal cord. Furthermore, our quantitative motor function analysis with the Basso Mouse Scale for Locomotion (BMS) score demonstrated a significant improvement in the implanted mice compared to non-implanted mice with a severed spinal cord. Our study also showed that it is easy to obtain HAP stem cells, they do not develop teratomas, and do not loose differentiation ability when cryopreserved. Collectively our results suggest that HAP stem cells could be a better source compared to induced pluripotent stem cells (iPS) or embryonic stem (ES) cells for regenerative medicine, specifically for spinal cord repair.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Endothelial progenitors are a population of cells with the inherent capacity to differentiate into mature endothelial cells and proangiogenic paracrine action. These characteristics have led to extensive studies being performed and tested in the treatment of tissue ischemia. The natural course of diabetes mellitus (DM) results in multiple areas of vascular damage. Thus endothelial progenitor cells‘(EPCs) beneficial potential is particularly desirable in diabetic patients. In this review, we summarize contemporary knowledge of EPC biology in DM. It has been shown that EPC functions are considerably impaired by DM. The presence of peripheral arterial disease (PAD) seems to further exacerbate the deficiencies of EPCs. However, studies examining EPC counts in PAD and DM observed disparate results, which can be due to a lack of consensus on precise EPC immunotype used in the different studies. Nevertheless, the results of EPC-based autologous cell therapy (ACT) are promising. In addition, EPCs have been shown to bean independent predictor of cardiovascular risk and diabetic foot ulcer healing.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Multipotent mesenchymal stem/stromal cells (MSCs) have regenerative and immunomodulatory properties to restore and repair injured tissues, making them attractive candidates for cell-based therapies. Experimental and clinical evidence has demonstrated the effectiveness of MSC transplantation in managing diabetes mellitus (DM). Autologous MSCs are assumed to be favorable because patient-derived cells are readily available and do not entail sustained immunosuppressive therapy. DM is associated with hyperglycemia, oxidative stress and altered immune responses and inflammation. It may thus alter the biological characteristics and therapeutic qualities of human MSCs (hMSCs). Several studies have explored the effect of DM or the diabetic microenvironment on the engraftment and efficacy of transplanted MSCs, which are determined by proliferation, differentiation, senescence, angiogenesis supportive effect, migration, anti-oxidative capacity and immunomodulatory properties. This review aims to present the available data on how DM impacts MSC biology and functionality and identify future perspectives for autologous MSC-based therapy in diabetics.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Stroke is a major public health issue with limited treatment. The pharmacologically or mechanically removing of the clot is accessible to less than 10% of the patients. Stem cell therapy is a promising alternative strategy since it increases the therapeutic time window but many issues remain unsolved. To avoid a new dramatic failure when translating experimental data on the bedside, this review aims to highlight the indispensable checkpoints to make a successful clinical trial based on the current preclinical literature. The large panel of progenitors/ stem cells at the researcher’s disposal is to be used wisely, regarding the type of cells, the source of cells, the route of delivery, the time window, since it will directly affect the outcome. Mechanisms are still incompletely understood, although recent studies have focused on the inflammation modulation of most cells types.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Generation of proper controls is crucial in induced pluripotent stem cell (iPSC) studies. X-chromosomal disorders offer the potential to develop isogenic controls due to random X-chromosomal inactivation (XCI). However, the generation of such lines is currently hampered by skewed X-inactivation in fibroblast lines and X-chromosomal reactivation (XCR) after reprogramming. Here we describe a method to generate a pure iPSC population with respect to the specific inactivated X-chromosome (Xi). We used fibroblasts from Rett patients, who all have a causal mutation in the X-linked MeCP2 gene. Pre-sorting these fibroblasts followed by episomal reprogramming, allowed us to overcome skewness in fibroblast lines and to retain the X-chromosomal state, which was unpredictable with lentiviral reprogramming. This means that fibroblast pre-sorting followed by episomal reprogramming can be used to reliably generate iPSC lines with specified X-chromosomal phenotype such as Rett syndrome.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Long non-coding RNA (lncRNA) 〈em〉Xist〈/em〉 has emerged as a key modulator in dosage compensation by randomly inactivating one of the X chromosomes in mammals during embryonic development. Dysregulation of X chromosome inactivation (XCI) due to deletion of 〈em〉Xist〈/em〉 has been proven to induce hematologic cancer in mice. However, this phenomenon is not consistent in humans as growing evidence suggests 〈em〉Xist〈/em〉 can suppress or promote cancer growth in different organs of the human body. In this review, we discuss recent advances of XCI in human embryonic stem cells and provide an explanation for the seemingly contradictory roles of 〈em〉Xist〈/em〉 in development of human cancer.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉The pace of advances in the world of science have created new opportunities and insights that give us new and more understanding of our nature and environment. Among the different fields of science, new medical sciences have drawn a great deal of attention among medical science researchers and the society. The hope for finding treatments for incurable diseases and further improvement of man’s health is growing thanks to new medical technologies. Among the novel medical fields that have been extensively covered by medical and academic societies are cell therapy and gene therapy that are categorized under regenerative medicine. The present paper is an attempt to introduce the prospect of a curative cell-based therapy and new cellular and gene therapy drugs that have been recently approved by FDA (food and drug administration). Cellular and gene therapy are two very close fields of regenerative medicine and sciences which their targets and applications can be discussed together. What adds to the importance of this new field of science is the possibility to translate the hope for treatment of incurable diseases into actual treatments. What follows delves deeper into this new field of science and the drugs.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈span〉 〈h3〉Objectives〈/h3〉 〈p〉An immunotherapy was found to be effective in achieving long-term survival in some lung cancer patients. It has emerged to searching for new immune biomarkers for select the best candidates to this therapy. It is suggested that cancer stem cells (CSCs) are responsible for tumor initiation, maintenance and its metastatic potential. However, a role of CSCs in escape of cancer from immunosurveillance is unknown. The aim of the study was assess the phenotype of putative CSCs and to examine the expression of PD-L1 on CSCs in metastatic lymph nodes (LNs) in lung cancer patients.〈/p〉 〈/span〉 〈span〉 〈h3〉Material and Methods〈/h3〉 〈p〉Flow cytometry was used for CSCs evaluation in peripheral blood and EBUS/TBNA aspirates from N1,N2 lymph nodes in lung cancer patients.〈/p〉 〈/span〉 〈span〉 〈h3〉Results〈/h3〉 〈p〉Of 30 patients the LNs metastases were confirmed in 18 patients. We noticed presence of PD-L1 on putative lung CSCs- CD133 + EpCAM+ cells. A higher percentage of CD133 + EpCAM+PD-L1+ cells was observed in patients with metastatic in LNs- median value = 4.38% than in patients without LNs metastases– median value = 0,015% (〈em〉p〈/em〉 〈 0.05). The highest proportion of PD-L1+ CSCs was found in adenocarcinoma patients and in those with oncogene addiction what indicate an particular biology of this type of lung cancer.〈/p〉 〈/span〉 〈span〉 〈h3〉Conclusion〈/h3〉 〈p〉The presence of CSCs with expression of PD-L1 in the metastatic LNs might suggest their immunogenic potential. EBUS/TBNA is commonly used in diagnosis and staging of lung cancer, so the analysis of the cells in metastatic LNs may fit in “immunoscoring” before immunotherapy.〈/p〉 〈/span〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉POI is characterized by “absent not abnormal” menstruation with hormonal disorders in woman younger than 40 years of age, and etiological and pathophysiological mechanisms underlying the POI development have not been clearly defined. Recently, due to advantages such as abundant sources and non-invasive methods of harvest, MenSCs have been emerging as a promising treatment strategy for the recovery of female reproductive damage. Here, we demonstrated that MenSCs graft in POI mice after CTX treatment could restore ovarian function by regulating normal follicle development and estrous cycle, reducing apoptosis in ovaries to maintain homeostasis of microenvironment and modulating serum sex hormones to a relatively normal status. Moreover, MenSCs participated in the activation of ovarian transcriptional expression in ECM-dependent FAK/AKT signaling pathway and thus restored ovarian function to a certain extent. MenSCs transplantation was proved to be an effective way to repair ovarian function with low immunogenicity, suggesting its great potential for POI treatment.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉More than a decade ago, a pioneering study reported generation of induced Pluripotent Stem Cells (iPSCs) by ectopic expression of a cocktail of reprogramming factors in fibroblasts. This study has revolutionized stem cell research and has garnered immense interest from the scientific community globally. iPSCs hold tremendous potential for understanding human developmental biology, disease modeling, drug screening and discovery, and personalized cell-based therapeutic applications. The seminal study identified Oct4, Sox2, Klf4 and c-Myc as a potent combination of genes to induce reprogramming. Subsequently, various reprogramming factors were identified by numerous groups. Most of these studies have used integrating viral vectors to overexpress reprogramming factors in somatic cells to derive iPSCs. However, these techniques restrict the clinical applicability of these cells as they may alter the genome due to random viral integration resulting in insertional mutagenesis and tumorigenicity. To circumvent this issue, alternative integration-free reprogramming approaches are continuously developed that eliminate the risk of genomic modifications and improve the prospects of iPSCs from lab to clinic. These methods establish that integration of transgenes into the genome is not essential to induce pluripotency in somatic cells. This review provides a comprehensive overview of the most promising DNA-free reprogramming techniques that have the potential to derive integration-free iPSCs without genomic manipulation, such as sendai virus, recombinant proteins, microRNAs, synthetic messenger RNA and small molecules. The understanding of these approaches shall pave a way for the generation of clinical-grade iPSCs. Subsequently, these iPSCs can be differentiated into desired cell type(s) for various biomedical applications.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Stem cells at the origin of endothelial progenitor cells and in particular endothelial colony forming cells (ECFCs) subtype have been largely supposed to be positive for the CD133 antigen, even though no clear correlation has been established between its expression and function in ECFCs. We postulated that CD133 in ECFCs might be expressed intracellularly, and could participate to vasculogenic properties. ECFCs extracted from cord blood were used either fresh (〈em〉n〈/em〉 = 4) or frozen (n = 4), at culture days 〈30, to investigate the intracellular presence of CD133 by flow cytometry and confocal analysis. Comparison with HUVEC and HAEC mature endothelial cells was carried out. Then, CD133 was silenced in ECFCs using specific siRNA (siCD133-ECFCs) or scramble siRNA (siCtrl-ECFCs). siCD133-ECFCs (〈em〉n〈/em〉 = 12), siCtrl-ECFCs (n = 12) or PBS (n = 12) were injected in a hind-limb ischemia nude mouse model and vascularization was quantified at day 14 with H&E staining and immunohistochemistry for CD31. Results of flow cytometry and confocal microscopy evidenced the positivity of CD133 in ECFCs after permeabilization compared with not permeabilized ECFCs (〈em〉p〈/em〉 〈 0.001) and mature endothelial cells (〈em〉p〈/em〉 〈 0.03). In the model of mouse hind-limb ischemia, silencing of CD133 in ECFCs significantly abolished post-ischemic revascularization induced by siCtrl-ECFCs; indeed, a significant reduction in cutaneous blood flows (〈em〉p〈/em〉 = 0.03), capillary density (CD31) (〈em〉p〈/em〉 = 0.01) and myofiber regeneration (〈em〉p〈/em〉 = 0.04) was observed. Also, a significant necrosis (〈em〉p〈/em〉 = 0.02) was observed in mice receiving siCD133-ECFCs compared to those treated with siCtrl-ECFCs. In conclusion, our work describes for the first time the intracellular expression of the stemness marker CD133 in ECFCs. This feature could resume the discrepancies found in the literature concerning CD133 positivity and ontogeny in endothelial progenitors.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Stem cells (SCs) are discovered long back but the idea that SCs possess therapeutic potential came up just a few decades back. In a past decade stem cell therapy is highly emerged and displayed tremendous potential for the treatment of a wide range of diseases and disorders such as blindness and vision impairment, type I diabetes, infertility, HIV, etc. SCs are very susceptible to destruction after transplantation into the host because of the inability to sustain elevated stress conditions inside the damaged tissue/organ. Heat shock proteins (HSPs) are molecular chaperones/stress proteins expressed in response to stress (elevated temperature, harmful chemicals, ischemia, viruses, etc) inside a living cell. HSPs protect the cell from damage by assisting in the proper folding of cellular proteins. This review briefly summarises different types of HSPs, their classification, cellular functions as well as the role of HSPs in regulating SC self-renewal and survival in the transplanted host. Applications of HSP modulated SCs in regenerative medicine and for the treatment of ischemic heart disease, myocardial infarction (MI), osteoarthritis, ischemic stroke, spinocerebellar ataxia type 3 (SCA3), leukemia, hepatic ischemia-reperfusion injury, Graft-versus-host disease (GVHD) and Parkinson’s disease (PD) are discussed. In order to provide potential insights in understanding molecular mechanisms related to SCs in vertebrates, correlations between HSPs and SCs in cnidarians and planarians are also reviewed. There is a need to advance research in order to validate the use of HSPs for SC therapy and establish effective treatment strategies.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Mobilization of stem cells from bone marrow (BM) into peripheral blood (PB) in response to tissue or organ injury, infections, strenuous exercise, or mobilization-inducing drugs is as we postulated result of a “sterile inflammation” in the BM microenvironment that triggers activation of the Complement Cascade (ComC). Therefore, we became interested in the role of the Nlrp3 inflammasome in this process and show for the first time that its activation in ATP-dependent manner orchestrates BM egress of hematopoietic stem/progenitor cells (HSPCs) as well as other stem cells, including mesenchymal stroma cells (MSCs), endothelial progenitor cells (EPCs), and very small embryonic-like stem cells (VSELs). To explain this extracellular ATP is a potent activator of the Nrlp3 inflammasome, which leads to the release of interleukin 1β and interleukin 18, as well as several danger-associated molecular pattern molecules (DAMPs) that activate the mannan-binding lectin (MBL) pathway of the ComC, from cells of the innate immunity network. In support of this mechanism, we demonstrate that the Nlrp3 inflammasome become activated in innate immunity cells by granulocyte colony stimulating factor (G-CSF) and AMD3100 in an ATP-dependent manner. Moreover, administration of the Nlrp3 inflammasome activator nigericin induces mobilization in mice, and the opposite effect is obtained by administration of an Nlrp3 inhibitor (MCC950) to mice mobilized by G-CSF or AMD3100. In summary, our results further support the crucial role of innate immunity, BM sterile inflammation, and novel role of the ATP–Nlrp3–ComC axis in the egress of stem cells into PB.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Mesenchymal stem cells (MSCs) are currently being tested in several clinical trials. Mitochondria regulate many aspects of MSC function. Mitochondrial preproteins are rapidly translated and trafficked into the mitochondrion for assembly in their final destination, but whether coexisting cardiovascular risk factors modulate this process is unknown. We hypothesized that metabolic syndrome (MetS) modulates mitochondrial protein import in porcine MSCs. MSCs were isolated from porcine abdominal adipose tissue after 16 weeks of Lean or MetS diet (〈em〉n〈/em〉 = 5 each). RNA-sequencing was performed and differentially expressed mitochondrial mRNAs and microRNAs were identified and validated. Protein expression of transporters of mitochondrial proteins (presequences and precursors) and their respective substrates were measured. Mitochondrial homeostasis was assessed by Western blot and function by cytochrome-c oxidase-IV activity. Forty-five mitochondrial mRNAs were upregulated and 25 downregulated in MetS-MSCs compared to Lean-MSCs. mRNAs upregulated in MetS-MSCs encoded for precursor proteins, whereas those downregulated encoded for presequences. Micro-RNAs upregulated in MetS-MSCs primarily target mRNAs encoding for presequences. Transporters of precursor proteins and their substrates were also upregulated, associated with changes in mitochondrial homeostasis and dysfunction. MetS interferes with mitochondrial protein import, favoring upregulation of precursor proteins, which might be linked to post-transcriptional regulation of presequences. This in turn alters mitochondrial homeostasis and impairs energy production. Our observations highlight the importance of mitochondria in MSC function and provide a molecular framework for optimization of cell-based strategies as we move towards their clinical application.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉The lack of clear regulations for the use of veterinary stem cells has triggered the commercialization of unproven experimental therapies for companion animal diseases. Adult stem cells have complex biological characteristics that are directly related to the therapeutic application, but several questions remain to be answered. In order to regulate the use of these cells, well-conducted, controlled scientific studies that generate high-quality data should be performed, in order to assess the efficacy and safety of the intended treatment. This paper discusses the scientific challenges of mesenchymal stem cell therapy in veterinary regenerative medicine, and reviews published trials of adipose-tissue-derived stem cells in companion animal diseases that spontaneously occur.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Circular RNAs (circRNAs), which are produced by back-splicing events in genes, have emerged as factors in gene regulation and normal cellular homeostasis. They play an important role not only in normal development of tissues and organs, but also in disease pathogenesis and cell differentiation. However, the role of circRNAs in bone marrow stem cells (BMSCs) undergoing osteoblast differentiation remains largely unknown. We performed microarray analysis to determine the expression profiles of circRNAs during osteoblast differentiation. In total, 3938 circRNAs were upregulated and 1505 were downregulated in BMSCs at day 7 (D7) compared with day 0 (D0). About 95% of the differentially expressed circRNAs were derived from protein coding genes, and functional annotation analysis showed that the parental genes of differentially expressed circRNAs were enriched in osteogenesis-associated terms. We also analyzed the microRNA (miRNA) transcriptome since circRNAs have been suggested to interact with miRNAs. We then selected the circRNAs that were negatively correlated with miRNAs and possessed miRNA response elements to construct a circRNA-miRNA interaction network. Analysis of the hub nodes in the networks showed that the top five nodes were miRNAs. Some circRNAs were linked to miRNAs with osteogenic roles, indicating that these circRNAs potentially function in osteogenic differentiation of BMSCs. Moreover, we validated the expression of one hub miRNA, miR-199b-5p, and its linked circIGSF11. Silencing of circIGSF11 promoted osteoblast differentiation and increased the expression of miR-199b-5p. Our study suggests that circRNA-miRNA interaction actively contributes to the osteogenic differentiation of human BMSCs, suggesting potential avenues for further study.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉During the last two decades, mesenchymal stem cells (MSCs) gained a place of privilege in the field of regenerative medicine. Recently, extracellular vesicles (EVs) have been identified as major mediators of MSCs immunosuppressive as well as pro-regenerative activities in many disease models, including inflammatory/degenerative conditions as joint diseases and osteoarthritis. In order to shed light on EVs potential, a rigorous profiling of embedded proteins, lipids and nucleic acids (mRNA/miRNA) is mandatory. Nevertheless, reliable strategies to efficiently score miRNA cargo and modulation under diverse experimental conditions or treatments are missing. The aim of this work was to identify reliable reference genes (RGs) to analyze miRNA content in EVs secreted by adipose-derived MSCs (ASCs) and verify their consistency under inflammatory conditions that were proposed to enhance ASC-EVs immunomodulatory and regenerative potential. RefFinder algorithm, that integrates the currently available major computational programs (geNorm, NormFinder, BestKeeper, and Delta Ct method), allowed to identify miR-22-5p and miR-29a-5p as the most stable RGs. Notably, both miRNAs maintained the highest stability when EVs isolated from IFNg-treated ASCs were included in the analysis. In addition, considerable effects of suboptimal RGs choice on the reliable quantification of miRNAs involved at different levels (tissue homeostasis or macrophage polarization) in the osteoarthritis phenotype, and thus considered as promising therapeutic molecule, have clearly been demonstrated. In conclusion, a proper normalization method is not only needed for research purposes but also mandatory to characterize clinical products and predict their therapeutic potential, especially in the emerging field of MSCs derived-EVs as new tools for regenerative medicine.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Gelatin methacrylate (GelMA)-based hydrogels are gaining a great deal of attention as potentially implantable materials in tissue engineering applications because of their biofunctionality and mechanical tenability. Since different natural tissues respond differently to mechanical stresses, an ideal implanted material would closely match the mechanical properties of the target tissue. In this regard, applications employing GelMA hydrogels are currently limited by the low mechanical strength and biocompatibility of GelMA. Therefore, this review focuses on modifications made to GelMA hydrogels to make them more suitable for tissue engineering applications. A large number of reports detail rational synthetic processes for GelMA or describe the incorporation of various biomaterials into GelMA hydrogels to tune their various properties, e.g., physical strength, chemical properties, conductivity, and porosity, and to promote cell loading and accelerate tissue repair. A novel strategy for repairing tissue injuries, based on the transplantation of cell-loaded GelMA scaffolds, is examined and its advantages and challenges are summarized. GelMA-cell combinations play a critical and pioneering role in this process and could potentially accelerate the development of clinically relevant applications.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉There is a growing interest in the potential of adult stem cells for implementing regenerative medicine in the brain. We assessed the effect of intracerebroventricular (icv) administration of human umbilical cord perivascular cells (HUCPVCs) on spatial memory of senile (27 mo) female rats, using intact senile counterparts as controls. Approximately one third of the animals were injected in the lateral ventricles with a suspension containing 4.8 X 10〈sup〉5〈/sup〉 HUCPVC in 〈strong〉8〈/strong〉 μl per side. The other third received 4.8 X 10〈sup〉5〈/sup〉 transgenic HUCPVC overexpressing Insulin-like growth factor-1 (IGF-1) and the last third of the rats received no treatment. Spatial memory performance was evaluated using a modified version of the Barnes maze test. In order to evaluate learning ability as well as spatial memory retention, we assessed the time spent (permanence) by animals in goal sector 1 (GS〈sub〉1〈/sub〉) and 3 (GS〈sub〉3〈/sub〉) when the escape box was removed. Fluorescence microscopy revealed the prescence of Dil-labeled HUCPVC in coronal sections of treated brains. The HUCPVC were located in close contact with the ependymal cells with only a few labeled cells migrating into the brain parenchyma. After treatment with naïve or IGF-1 transgenic HUCPVC, permanence in GS〈sub〉1〈/sub〉 and GS〈sub〉3〈/sub〉 increased significantly whereas there were no changes in the intact animals. We conclude that HUCPVC injected icv are effective to improve some components of spatial memory in senile rats. The ready accessibility of HUCPVC constitutes a significant incentive to continue the exploration of their therapeutic potential on neurodegenerative diseases.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Adoptive cell therapy using CAR T cells has emerged as a novel treatment strategy with promising results against B cell malignancies; however, CAR T cells have not shown much success against solid malignancies. There are several obstacles which diminish the efficacy of CAR T cells, but the immunosuppressive tumor microenvironment (TME) of the tumor stands out as the most important factor. TME includes Tumor-Associated Stroma, Immunosuppressive cells and cytokines, tumor hypoxia and metabolism, and Immune Inhibitory Checkpoints which affect the CAR T cell efficacy and activity in solid tumors. A precise understanding of the TME could pave the way to engineer novel modifications of CAR T cells which can overcome the immunosuppressive TME. In this review, we will describe different sections of the TME and introduce novel approaches to improve the CAR T cells potential against solid tumors based on recent clinical and preclinical data. Also, we will provide new suggestions on how to modify CARs to augment of CAR T cells efficacy. Since there are also some challenges beyond the TME that are important for CAR function, we will also discuss and provide data about the improvement of CAR T cells trafficking and delivery to the tumor site and how to solve the problem of tumor antigen heterogeneity.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈span〉 〈h3〉Background〈/h3〉 〈p〉Acute myocardial infarction (AMI) and the ensuing ischemic heart disease are approaching an epidemic state. Limited stem cell retention following intracoronary administration has reduced the clinical efficacy of this novel therapy. Polymer based cell coating is biocompatible and has been shown to be safe. Here, we assessed the therapeutic utility of gelatin-based biodegradable cell coatings on bone marrow derived cell retention in ischemic heart.〈/p〉 〈/span〉 〈span〉 〈h3〉Methods〈/h3〉 〈p〉Gelatin based cell coatings were formed from the surface-mediated photopolymerization of 3% gelatin methacrylamide and 1% PEG diacrylate. Cell coating was confirmed using a multimodality approach including flow cytometry, imaging flow cytometry (ImageStream System) and immunohistochemistry. Biocompatibility of cell coating, metabolic activity of coated cells, and the effect of cell coating on the susceptibility of cells for engulfment were assessed using in vitro models. Following myocardial infarction and GFP+ BM-derived mesenchymal stem cell transplantation, flow cytometric and immunohistochemical assessment of retained cells was performed.〈/p〉 〈/span〉 〈span〉 〈h3〉Results〈/h3〉 〈p〉Coated cells are viable and metabolically active with coating degrading within 72 h in vitro. Importantly, cell coating does not predispose bone marrow cells to aggregation or increase their susceptibility to phagocytosis. In vitro and in vivo studies demonstrated no evidence of heightened immune response or increased phagocytosis of coated cells. Cell transplantation studies following myocardial infarction proved the improved retention of coated bone marrow cells compared to uncoated cells.〈/p〉 〈/span〉 〈span〉 〈h3〉Conclusion〈/h3〉 〈p〉Gelation based polymer cell coating is biologically safe and biodegradable. Therapies employing these strategies may represent an attractive target for improving outcomes of cardiac regenerative therapies in human studies.〈/p〉 〈/span〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Precise regulation of transcriptome modulates several vital aspects in an organism that includes gene expression, cellular activities and development, and its perturbation ensuing pathological conditions. Around 150 post-transcriptional modifications of RNA have been identified till date, which are evolutionarily conserved and likewise prevalent across RNA classes including messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), and detected less frequently in small nuclear RNA (snRNA) and microRNAs (miRNA). Among the RNA modifications documented, N6-methyladenosine (m〈sup〉6〈/sup〉A) is the best characterised till date. Also, N〈sup〉1〈/sup〉-methyladenosine (m〈sup〉1〈/sup〉A), 5-methylcytosine (m〈sup〉5〈/sup〉C) and pseudouridine (Ψ) are some of the other prominent modifications detected in coding and non-coding RNAs. “Epitranscriptome”, ensemble of these post-transcriptional RNA modifications, precisely coordinates gene expression and biological processes. Current literatures suggest the critical involvement of epitranscriptomics in several organisms during early development, contributing to cell fate specification and physiology. Indeed, epitranscriptomics similar to DNA epigenetics involves combinatorial dynamics provided by modified RNA molecules and associated protein complexes, which function as “writers”, “erasers” and “readers” of these modifications. A novel code orchestrating gene expression during cell fate determination is generated by the coordinated effects of different classes of modified RNAs and its regulator proteins. In this review, we summarize the current knowhow on N6-methyladenosine (m〈sup〉6〈/sup〉A), 5-methylcytosine (m〈sup〉5〈/sup〉C) and pseudouridine (ψ) modifications in RNA, the associated regulator proteins and enumerate how the epitranscriptomic regulations are involved in cell fate determination.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Mesenchymal stem cells (MSCs), also known as multipotent mesenchymal stromal stem cells, are found in the perivascular space of several tissues. These cells have been subject of intense research in the last decade due to their low teratogenicity, as well as their ability to differentiate into mature cells and to secrete immunomodulatory and trophic factors. However, they usually promote only a modest benefit when transplanted in experimental disease models, one of the limitations for their clinical application. The CRISPR-Cas system, in turn, is highlighted as a simple and effective tool for genetic engineering. This system was tested in clinical trials over a relatively short period of time after establishing its applicability to the edition of the mammalian cell genome. Similar to the research evolution in MSCs, the CRISPR-Cas system demonstrated inconsistencies that limited its clinical application. In this review, we outline the evolution of MSC research and its applicability, and the progress of the CRISPR-Cas system from its discovery to the most recent clinical trials. We also propose perspectives on how the CRISPR-Cas system may improve the therapeutic potential of MSCs, making it more beneficial and long lasting.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Gestational diabetes mellitus (GDM) has been associated with an increased risk of maternal and neonatal morbidity. The Wharton’s jelly (WJ) of the umbilical cord (UC) is a useful indicator of the deleterious effects of hyperglycemia on fetal tissues as it represents the fetus embryologically, physiologically and genetically. We studied WJ mesenchymal stem cells (hWJSCs) from UC from mothers without GDM (Normal; 〈em〉n〈/em〉 = 3); insulin-controlled GDM mothers (GDMi; n = 3) and diet-controlled GDM mothers (GDMd; n = 3)]. Cell proliferation, stemness markers, telomerase, osteogenic and chondrogenic differentiation, antioxidant enzymes and gene expression for mitochondrial function (〈em〉ND2〈/em〉, 〈em〉TFAM〈/em〉, 〈em〉PGC1α〈/em〉, and 〈em〉NDUFB9〈/em〉) were significantly lower in GDMi-hWJSCs and GDMd-hWJSCs compared to normal hWJSCs (〈em〉P〈/em〉 〈 0.05). On the other hand, cell cycle inhibitors (〈em〉p16〈/em〉, 〈em〉p21〈/em〉, 〈em〉p27〈/em〉) and 〈em〉p53〈/em〉 were remarkably up-regulated in GDMi-hWJSCs and GDMd-hWJSCs compared to normal hWJSCs. The results from this study confirmed that maternal hyperglycemia even though managed with insulin or diet, induced changes in the properties of the WJ and its cells. These changes may also be observed in fetal tissues and if true, prevention of the onset of gestational diabetes should be a priority over management. Generation of tissues that simulate those of the fetus such as pancreatic and cardiovascular cells from GDM-hWJSCs by direct differentiation or via induced pluripotent stem cell reprogramming provide possible platforms to evaluate the effects of glucose on specific fetal organ.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Fetal-maternal microchimerism describes the acquisition of fetal stem cells (FSC) by the mother during pregnancy and their long-term persistence after parturition. FSC may engraft in a variety of maternal tissues especially if there is organ/tissue injury, but their role and mechanism of persistence still remains elusive. Clinical applications due to their pluripotency, immunomodulatory effects and accessibility make them good candidates for ex-vivo manipulation and autologous therapies. The hair follicles contain a distinctive niche for pluripotent stem cells (PSC). To date, there is no published evidence of fetal microchimerism in the hair follicle. In our study, follicular unit extraction (FUE) technique allowed easy stem cell cultures to be obtained while simple hair follicle removal by pull-out technique failed to generate stem cells in culture. We identified microchimeric fetal stem cells within the primitive population of maternal stem cells isolated from the hair follicles with typical mesenchymal phenotype, expression of PSC genes and differentiation potential towards osteocytes, adypocites and chondrocytes. This is the first study to isolate fetal microchimeric stem cells in adult human hair long after parturition. We presume a sanctuary partition mechanism with PSC of the mother deposited during early embryogenesis could explain their long-term persistence.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Vascularization is a major hurdle in complex tissue and organ engineering. Tissues greater than 200 μm in diameter cannot rely on simple diffusion to obtain nutrients and remove waste. Therefore, an integrated vascular network is required for clinical translation of engineered tissues. Microvessels have been described as 〈150 μm in diameter, but clinically they are defined as 〈1 mm. With new advances in super microsurgery, vessels less than 1 mm can be anastomosed to the recipient circulation. However, this technical advancement still relies on the creation of a stable engineered microcirculation that is amenable to surgical manipulation and is readily perfusable. Microvascular engineering lays on the crossroads of microfabrication, microfluidics, and tissue engineering strategies that utilize various cellular constituents. Early research focused on vascularization by co-culture and cellular interactions, with the addition of angiogenic growth factors to promote vascular growth. Since then, multiple strategies have been utilized taking advantage of innovations in additive manufacturing, biomaterials, and cell biology. However, the anatomy and dynamics of native blood vessels has not been consistently replicated. Inconsistent results can be partially attributed to cell sourcing which remains an enigma for microvascular engineering. Variations of endothelial cells, endothelial progenitor cells, and stem cells have all been used for microvascular network fabrication along with various mural cells. As each source offers advantages and disadvantages, there continues to be a lack of consensus. Furthermore, discord may be attributed to incomplete understanding about cell isolation and characterization without considering the microvascular architecture of the desired tissue/organ.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Stroke remains a major unmet clinical need that warrants novel therapies. Following an ischemic insult, the cerebral vasculature secretes inflammatory molecules, creating the stroke vasculome profile. The present study evaluated the therapeutic effects of endothelial cells on the inflammation-associated stroke vasculome. qRT-PCR analysis revealed that specific inflammation-related vasculome genes BRM, IκB, Foxf1, and ITIH-5 significantly upregulated by oxygen glucose deprivation (OGD. Interestingly, co-culture of human endothelial cells (HEN6) with human endothelial cells (EPCs) during OGD significantly blocked the elevations of BRM, IκB, and Foxf1, but not ITIH-5. Next, employing the knockdown/antisense technology, silencing the inflammation-associated stroke vasculome gene, IκB, as opposed to scrambled knockdown, blocked the EPC-mediated protection of HEN6 against OGD. In vivo, stroke animals transplanted with intracerebral human EPCs (300,000 cells) into the striatum and cortex 4 h post ischemic stroke displayed significant behavioral recovery up to 30 days post-transplantation compared to vehicle-treated stroke animals. At 7 days post-transplantation, quantification of the fluorescent staining intensity in the cortex and striatum revealed significant upregulation of the endothelial marker RECA1 and a downregulation of the stroke-associated vasculome BRM, IKB, Foxf1, ITIH-5 and PMCA2 in the ipsilateral side of cortex and striatum of EPC-transplanted stroke animals relative to vehicle-treated stroke animals. Altogether, these results demonstrate that EPCs exert therapeutic effects in experimental stroke possibly by modulating the inflammation-plagued vasculome.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Bone marrow-derived endothelial progenitor cells (EPCs) play a key role in the maintenance of endothelial homeostasis and endothelial repair at areas of vascular damage. The quantification of EPCs in peripheral blood by flow cytometry is a strategy to assess this reparative capacity. The number of circulating EPCs is inversely correlated with the number of cardiovascular risk factors and to the occurrence of cardiovascular events. Therefore, monitoring EPCs levels may provide an accurate assessment of susceptibility to cardiovascular injury, greatly improving risk stratification of patients with high cardiovascular risk, such as those with an acute myocardial infarction. However, there are many issues in the field of EPC identification and quantification that remain unsolved. In fact, there have been conflicting protocols used to the phenotypic identification of EPCs and there is still no consensual immunophenotypical profile that corresponds exactly to EPCs. In this paper we aim to give an overview on EPCs-mediated vascular repair with special focus on acute coronary syndromes and to discuss the different phenotypic profiles that have been used to identify and quantify circulating EPCs in several clinical studies. Finally, we will synthesize evidence on the prognostic role of EPCs in patients with high cardiovascular risk.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉The global burden of diabetes has drastically increased over the past decades and in 2017 approximately 4 million deaths were caused by diabetes and cardiovascular complications. Diabetic cardiomyopathy is a common complication of diabetes with early manifestations of diastolic dysfunction and left ventricular hypertrophy with subsequent progression to systolic dysfunction and ultimately heart failure. An in vitro model accurately recapitulating key processes of diabetic cardiomyopathy would provide a useful tool for investigations of underlying disease mechanisms to further our understanding of the disease and thereby potentially advance treatment strategies for patients. With their proliferative capacity and differentiation potential, human induced pluripotent stem cells (iPSCs) represent an appealing cell source for such a model system and cardiomyocytes derived from induced pluripotent stem cells have been used to establish other cardiovascular related disease models. Here we review recently made advances and discuss challenges still to be overcome with regard to diabetic cardiomyopathy models, with a special focus on iPSC-based systems. Recent publications as well as preliminary data presented here demonstrate the feasibility of generating cardiomyocytes with a diabetic phenotype, displaying insulin resistance, impaired calcium handling and hypertrophy. However, capturing the full metabolic- and functional phenotype of the diabetic cardiomyocyte remains to be accomplished.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉The bioactive peptide bradykinin obtained from cleavage of precursor kininogens activates the kinin-B2 receptor functioning in induction of inflammation and vasodilatation. In addition, bradykinin participates in kidney and cardiovascular development and neuronal and muscle differentiation. Here we show that kinin-B2 receptors are expressed throughout differentiation of murine C2C12 myoblasts into myotubes. An autocrine loop between receptor activation and bradykinin secretion is suggested, since bradykinin secretion is significantly reduced in the presence of the kinin-B2 receptor antagonist HOE-140 during differentiation. Expression of skeletal muscle markers and regenerative capacity were decreased after pharmacological inhibition or genetic ablation of the B2 receptor, while its antagonism increased the number of myoblasts in culture. In summary, the present work reveals to date no functions described for the B2 receptor in muscle regeneration due to the control of proliferation and differentiation of muscle precursor cells.〈/p〉

Description: 〈h3〉Abstract〈/h3〉 〈p〉Aspirated follicular cells (AFCs) from the in vitro fertilization program can express various stem cell markers and are even able to differentiate into different types of cells in vitro. The female reproductive potential decreases with increasing age due to lowered ovarian reserve and oocyte quality, but data on the effect of female age on stem cell characteristics of AFCs are scarce. Therefore, the aim of this study was to elucidate whether female age affects the mesenchymal stem cell (MSC) characteristics of AFCs. Follicular aspirates were collected from 12 patients included in the in vitro fertilization programme with a normal ovarian reserve. Patients were divided into four age groups: Group A ≤ 30 years, Group B 31–35 years, Group C 36–39 years and Group D ≥ 40 years. After removal of the oocytes, AFCs were collected from follicular aspirates using hypo-osmotic technique and cultured in vitro, and their stemness was compared according to female age. The cultured AFCs were analysed for gene expression using the Human Mesenchymal Stem Cell RT〈sup〉2〈/sup〉 Profiler™ PCR Array, for their potential for differentiation into adipogenic and osteogenic lineage, and for their expression of MSC-related markers using immunocytochemistry. We found that female age can significantly influence their stemness: expression of pluripotency and MSC-related genes, and their differentiation potential. Despite the relatively high expression of MSC-related genes, the AFCs of the oldest patients had the lowest potential to differentiate into osteogenic and adipogenic lineages in vitro, which may be related to their age and the changed ovarian function.〈/p〉

Description: Hematopoietic stem cells (HSCs) are a valuable resource in transplantation medicine. Cytokines are often used to culture HSCs aiming at better clinical outcomes through enhancement of HSC reconstitution capability. Roles for each signal molecule downstream of receptors in HSCs, however, remain puzzling due to complexity of the cytokine-signaling network. Engineered receptors that are non-responsive to endogenous cytokines represent an attractive tool for dissection of signaling events. We here tested a previously developed chimeric receptor (CR) system in primary murine HSCs, target cells that are indispensable for analysis of stem cell activity. Each CR contains tyrosine motifs that enable selective activation of signal molecules located downstream of the c-Mpl receptor upon stimulation by an artificial ligand. Signaling through a control CR with a wild-type c-Mpl cytoplasmic tail sufficed to enhance HSC proliferation and colony formation in cooperation with stem cell factor (SCF). Among a series of CRs, only one compatible with selective Stat5 activation showed similar positive effects. The HSCs maintained ex vivo in these environments retained long-term reconstitution ability following transplantation. This ability was also demonstrated in secondary recipients, indicating effective transmission of stem cell-supportive signals into HSCs via these artificial CRs during culture. Selective activation of Stat5 through CR ex vivo favored preservation of lymphoid potential in long-term reconstituting HSCs, but not of myeloid potential, exemplifying possible dissection of signals downstream of c-Mpl. These CR systems therefore offer a useful tool to scrutinize complex signaling pathways in HSCs.

Description: The purpose of the present study was to evaluate the neural protein expression pattern of human multipotent mesenchymal stromal cells (hMSCs) treated with forskolin (free-form/FF). The study investigated forskolin’s capacity to enhance intracellular levels of cyclic adenosine monophosphate (cAMP) by activating adenylate cyclase and probably by inducing neuron-like cells in vitro. In addition, because nanotechnology is a growing field of tissue engineering, we also assessed the action of a new system called the nanostructured-forskolin (NF) to examine the improvement of drug delivery. Afterwards, the cells were submitted to low-level laser irradiation to evaluate possible photobiostimulatory effects. Investigations using the immunofluorescence by confocal microscopy and Western blot methods revealed the expression of the neuronal marker β-tubulin III. Fluorescence intensity quantification analysis using INCell Analyzer System for β-tubulin III was used to examine significant differences. The results showed that after low-level laser irradiation exposure, there was a tendency to increase the β-tubulin III expression in all groups, as expected in the photobiostimulation process. Notably, this process induced for irradiation was more pronounced in irradiated nanoforskolin cells (INF) compared to non-irradiated free-forskolin control cells (NFFC). However, there was also an increase in β-tubulin III protein expression in the groups: irradiated nanocontrol cells (INC) compared to non-irradiated free-forskolin control cells (NFF) and after treatment with non-irradiated free-forskolin (NFF) and non-irradiated nanoforskolin (NNFC). We concluded that the methods using low-level laser irradiation and/or nanoparticles showed an up-regulation of neural-protein expression in hMSCs that could be used to facilitate cellular therapy protocols in the near future.

Description: Adipogenesis is a complex process whereby the multipotent adipose-derived stem cell is converted to a preadipocyte before terminal differentiation into the mature adipocyte. Preadipocytes are present throughout adult life, exhibit adipose fat depot specificity, and differentiate and proliferate from distinct progenitor cells. The mechanisms that promote preadipocyte commitment and maturation involve numerous protein factor regulators, epigenetic factors, and miRNAs. Detailed characterization of this process is currently an area of intense research and understanding the roles of preadipocytes in tissue plasticity may provide insight into novel approaches for tissue engineering, regenerative medicine and treating a host of obesity-related conditions. In the current study, we analyzed the current literature and present a review of the characteristics of transitioning adipocytes and detail how local microenvironments influence their progression towards terminal differentiation and maturation. Specifically, we detail the characterization of preadipocyte via surface markers, examine the signaling cascades and regulation behind adipogenesis and cell maturation, and survey their role in tissue plasticity and health and disease.

Description: This study focuses on the interactions of human adipose tissue-derived stem cells (ADSCs) and malignant melanoma cells (MMCs) with regard to future cell-based skin therapies. The aim was to identify potential oncological risks as ADSCs could unintentionally be sited within the proximity of the tumor microenvironment of MMCs. An indirect co-culture model was used to analyze interactions between ADSCs and four different established melanoma cell lines (G-361, SK-Mel-5, MeWo and A2058) as well as two low-passage primary melanoma cell cultures (M1 and M2). Doubling time, migration and invasion, angiogenesis, quantitative real-time PCR of 229 tumor-associated genes and multiplex protein assays of 20 chemokines and growth factors and eight matrix metalloproteinases (MMPs) were evaluated. Co-culture with ADSCs significantly increased migration capacity of G-361, SK-Mel-5, A2058, MeWo and M1 and invasion capacity of G-361, SK-Mel-5 and A2058 melanoma cells. Furthermore, conditioned media from all ADSC-MMC-co-cultures induced tube formation in an angiogenesis assay in vitro. Gene expression analysis of ADSCs and MMCs, especially of low-passage melanoma cell cultures, revealed an increased expression of various genes with tumor-promoting activities, such as CXCL12, PTGS2, IL-6, and HGF upon ADSC-MMC-co-culture. In this context, a significant increase (up to 5,145-fold) in the expression of numerous tumor-associated proteins could be observed, e.g. several pro-angiogenic factors, such as VEGF, IL-8, and CCL2, as well as different matrix metalloproteinases, especially MMP-2. In conclusion, the current report clearly demonstrates that a bi-directional crosstalk between ADSCs and melanoma cells can enhance different malignant properties of melanoma cells in vitro.

Description: Stem cells are well-known to have prominent roles in tissue engineering applications. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can differentiate into every cell type in the body while adult stem cells such as mesenchymal stem cells (MSCs) can be isolated from various sources. Nevertheless, an utmost limitation in harnessing stem cells for tissue engineering is the supply of cells. The advances in biomaterial technology allows the establishment of ex vivo expansion systems to overcome this bottleneck. The progress of various scaffold fabrication could direct stem cell fate decisions including cell proliferation and differentiation into specific lineages in vitro. Stem cell biology and biomaterial technology promote synergistic effect on stem cell-based regenerative therapies. Therefore, understanding the interaction of stem cell and biomaterials would allow the designation of new biomaterials for future clinical therapeutic applications for tissue regeneration. This review focuses mainly on the advances of natural and synthetic biomaterials in regulating stem cell fate decisions. We have also briefly discussed how biological and biophysical properties of biomaterials including wettability, chemical functionality, biodegradability and stiffness play their roles.

Description: Thanks to their relative abundance and easier collection, adipose tissue (AT) is considered an alternative source for the isolation of mesenchymal stromal cells (MSCs). MSCs have great therapeutic values and are thus under investigations for several clinical indications such as regenerative medicine and immunomodulation. In this work, we aimed to identify, isolate and characterize AT-MSCs based on their aldehyde dehydrogenase (ALDH) activity known to be a classical feature of stem cells. FACS technology allowed to isolate two different populations of AT-MSCs according to their ALDH activity (referred as ALDH + and ALDH − ). Depending on their ALDH activity, the transcriptome analysis of both cell populations demonstrated a differential pattern of genes related to the main properties of MSCs (proliferation, response to hypoxia, angiogenesis, phenotype, stemness, multilineage, hematopoiesis, immunomodulation). Based on these profiling, both AT-MSC populations could differ in terms of biological responses and functionalities. Collectively, the use of ALDH for isolating and identifying sub-populations of MSCs with specific gene profile may represent an alternative method to provide solutions for targeted therapeutic applications.

Description: Over the past decade different stem cell (SC) based approaches were tested to treat Duchenne Muscular Dystrophy (DMD), a lethal X-linked disorder caused by mutations in dystrophin gene. Despite research efforts, there is no curative therapy for DMD. Allogeneic SC therapies aim to restore dystrophin in the affected muscles; however, they are challenged by rejection and limited engraftment. Thus, there is a need to develop new more efficacious SC therapies. Chimeric Cells (CC), created via ex vivo fusion of donor and recipient cells, represent a promising therapeutic option for tissue regeneration and Vascularized Composite Allotransplantation (VCA) due to tolerogenic properties that eliminate the need for lifelong immunosuppression. This proof of concept study tested feasibility of myoblast fusion for Dystrophin Expressing. Chimeric Cell (DEC) therapy through in vitro characterization and in vivo assessment of engraftment, survival, and efficacy in the mdx mouse model of DMD. Murine DEC were created via ex vivo fusion of normal ( snj) and dystrophin–deficient ( mdx) myoblasts using polyethylene glycol. Efficacy of myoblast fusion was confirmed by flow cytometry and dystrophin immunostaining, while proliferative and myogenic differentiation capacity of DEC were assessed in vitro. Therapeutic effect after DEC transplant (0.5 × 10 6 ) into the gastrocnemius muscle (GM) of mdx mice was assessed by muscle functional tests. At 30 days post-transplant dystrophin expression in GM of injected mdx mice increased to 37.27 ± 12.1% and correlated with improvement of muscle strength and function. Our study confirmed feasibility and efficacy of DEC therapy and represents a novel SC based approach for treatment of muscular dystrophies.

Description: The aim of this study was to compare effect of everolimus on growth of different renal cell carcinoma (RCC) populations and develop experimental design to measure the early response of everolimus in clear cell RCC (ccRCC) cell lines including renal cancer stem cells. Effect of everolimus on RCC cell lines which include primary (786-0) and metastatic (ACHN) RCC cell lines as well as heterogenous populations of tumor cells of different histological RCC subtypes (clear cell RCC and papillary RCC) was measured when treated with everolimus in the range of 1–9 µM. Gene expression profiling using microarray was performed to determine the early response to everolimus in ccRCC cell lines after optimizing concentration of drug. Gene Set Enrichment Analysis (GSEA) was done which mainly focused on basic genes related to mTOR, hormonal and metabolic pathways. Everolimus acts on RCC cells in a dose—dependent manner. In all examined cell lines IC50 dose was possible to calculate after the third day of treatment. In ccRCC lines (parental and stem cell) everolimus changes expression of mTOR complexes elements and elements of related pathways when treated with optimized doses of drug. Characteristic expression profile for ccRCC cells at an early exposure time to everolimus is to elucidate. Wevarie include some basic observations derived from data analysis in the context of mechanism of action of drug with a view to better understand biology of renal cancer cells.

Description: Identification of transcription factors that directly convert pluripotent stem cells (PSCs) into endothelial and blood cells and advances in the chemical modifications of messenger RNA (mRNA) offer alternative nucleic acid-based transgene-free approach for scalable production of these cells for drug screening and therapeutic purposes. Here we evaluated the effect of 5′ and 3′ RNA untranslated regions (UTRs) on translational efficiency of chemically-modified synthetic mRNA (modRNA) in human PSCs and showed that an addition of 5′UTR indeed enhanced protein expression. With the optimized modRNAs expressing ETV2 or ETV2 and GATA2 , we are able to produce VE-cadherin + endothelial cells and CD34 + CD43 + hematopoietic progenitors, respectively, from human PSCs as well as non-human primate (NHP) PSCs. Overall, our findings provide valuable information on the design of in vitro transcription templates being used in PSCs and its broad applicability for basic research, disease modeling, and regenerative medicine.

Description: Wharton’s jelly (WJ) is an important source of mesenchymal stem cells (MSCs) both in human and other animals. The aim of this study was to compare human and equine WJMSCs. Human and equine WJMSCs were isolated and cultured using the same protocols and culture media. Cells were characterized by analysing morphology, growth rate, migration and adhesion capability, immunophenotype, differentiation potential and ultrastructure. Results showed that human and equine WJMSCs have similar ultrastructural details connected with intense synthetic and metabolic activity, but differ in growth, migration, adhesion capability and differentiation potential. In fact, at the scratch assay and transwell migration assay, the migration ability of human WJMSCs was higher (P 〈 0.05) than that of equine cells, while the volume of spheroids obtained after 48 h of culture in hanging drop was larger than the volume of equine ones (P 〈 0.05), demonstrating a lower cell adhesion ability. This can also revealed in the lower doubling time of equine cells (3.5 ± 2.4 days) as compared to human (6.5 ± 4.3 days) (P 〈 0.05), and subsequently in the higher number of cell doubling after 44 days of culture observed for the equine (20.3 ± 1.7) as compared to human cells (8.7 ± 2.4) (P 〈 0.05), and to the higher (P 〈 0.05) ability to form fibroblast colonies at P3. Even if in both species tri-lineage differentiation was achieved, equine cells showed an higher chondrogenic and osteogenic differentiation ability (P 〈 0.05). Our findings indicate that, although the ultrastructure demonstrated a staminal phenotype in human and equine WJMSCs, they showed different properties reflecting the different sources of MSCs.