BackgroundPancreatic progenitors (PPs) co-expressing the two transcription factors (TFs) PDX1 and NKX6.1 are recognized as the indispensable precursors of functional pancreatic β cells. Here, we aimed to establish an efficient protocol for maximizing generation of PDX1+/NKX6.1+ PPs from human pluripotent stem cells (hPSCs).MethodsIn order to enhance the PDX1+/NKX6.1+ population, we manipulated in vitro culture conditions during differentiation by dissociating densely formed endodermal cells and re-plating them at different densities. These dissociated cells were subjected to an augmented duration of retinoid and fibroblast growth factor (FGF)10 signaling to induce higher PDX1 and NKX6.1 expression.ResultsOur optimized protocol dramatically increased the expression of NKX6.1, leading to an increase in the proportion of PDX1+/NKX6.1+ progenitors (~90%) in monolayer, higher than the previously published protocols, as well as upregulated key TFs controlling pancreatic development. The improved efficiency of pancreatic differentiation was complemented by an inhibited hepatic specification and an increased proliferation of NKX6.1+ cells. Interestingly, we were able to enrich a novel PDX1–/NKX6.1+ population by manipulating the re-plating density; these oriented themselves in three-dimensional clusters. Further differentiation validated the ability of our PDX1+/NKX6.1+ progenitors to generate NGN3+ endocrine progenitors.ConclusionsWe provide a novel technique that facilitates appropriate cellular rearrangement in monolayer culture to yield a high proportion of PDX1+/NKX6.1+ PPs with an elevated self-replicating capacity, thereby aiding scalable production of functional β cells from hPSCs in vitro. Our innovative method also enriches a novel NKX6.1+/PDX1– population, with characteristics of proposed endocrine precursors, allowing further studies on deciphering routes to β-cell development.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-017-0759-z) contains supplementary material, which is available to authorized users.

The COVID-19 pandemic has negatively impacted the global public health and the international economy; therefore, there is an urgent need for an effective therapy to treat COVID-19 patients. Mesenchymal stem cells (MSCs) have been proposed as an emerging therapeutic option for the SARS-CoV-2 infection. Recently, numerous clinical trials have been registered to examine the safety and efficacy of different types of MSCs and their exosomes for treating COVID-19 patients, with less published data on the mechanism of action. Although there is no approved effective therapy for COVID-19 as of yet, MSC therapies showed an improvement in the treatment of some COVID-19 patients. MSC’s therapeutic effect is displayed in their ability to reduce the cytokine storm, enhance alveolar fluid clearance, and promote epithelial and endothelial recovery; however, the safest and most effective route of MSC delivery remains unclear. The use of poorly characterized MSC products remains one of the most significant drawbacks of MSC-based therapy, which could theoretically promote the risk for thromboembolism. Optimizing the clinical-grade production of MSCs and establishing a consensus on registered clinical trials based on cell-product characterization and mode of delivery would aid in laying the foundation for a safe and effective therapy in COVID-19. In this review, we shed light on the mechanistic view of MSC therapeutic role based on preclinical and clinical studies on acute lung injury and ARDS; therefore, offering a unique correlation and applicability in COVID-19 patients. We further highlight the challenges and opportunities in the use of MSC-based therapy.

The loss of functional β cells leads to development of diabetes. Several studies have shown that β cells are specified through several stages of progenitors during pancreas development, each stage defined by the expression of specific transcription factors (TFs). Understanding signalling pathways that control the differentiation and specification processes during embryogenesis will facilitate efforts to obtain functional β cells in vitro. Our current knowledge of the mechanisms involved in pancreatic β cell development and survival under normal or diabetic conditions has come largely from animal studies. However, there are marked differences in islet structure and physiological properties between humans and animals, and not all phenotypes of human diabetes can be recapitulated in animal models. Therefore, human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced PSCs (hiPSCs) offer a great opportunity for increasing our understanding of the pathways regulating human pancreatic β-cell development and survival. Furthermore, hPSCs provide a renewable source of functional pancreatic β cells for cell replacement therapy as well as disease modelling. Herein, we discuss the signalling pathways involved in the development of pancreatic β cells during embryogenesis. Additionally, we describe how these pathways are manipulated in vitro to differentiate hPSCs into functional β cells. Finally, we highlight the progress that has been made for the applications of those cells in treating and modelling diabetes.

Objective: Stroke is the main cause of adult disability in the world, leaving more than half of the patients dependent on daily assistance. Understanding the post-stroke biochemical and molecular changes are critical for patient survival and stroke management. The aim of this work was to investigate the photo-thrombotic ischemic stroke in male rats with particular focus on biochemical and elemental changes in the primary stroke lesion in the somatosensory cortex and surrounding areas, including the corpus callosum.Materials and Methods: FT-IR imaging spectroscopy and LA-ICPMS techniques examined stroke brain samples, which were compared with standard immunohistochemistry studies.Results: The FTIR results revealed that in the lesioned gray matter the relative distribution of lipid, lipid acyl and protein contents decreased significantly. Also at this locus, there was a significant increase in aggregated protein as detected by high-levels Aβ1-42. Areas close to the stroke focus experienced decrease in the lipid and lipid acyl contents associated with an increase in lipid ester, olefin, and methyl bio-contents with a novel finding of Aβ1-42 in the PL-GM and L-WM. Elemental analyses realized major changes in the different brain structures that may underscore functionality.Conclusion: In conclusion, FTIR bio-spectroscopy is a non-destructive, rapid, and a refined technique to characterize oxidative stress markers associated with lipid degradation and protein denaturation not characterized by routine approaches. This technique may expedite research into stroke and offer new approaches for neurodegenerative disorders. The results suggest that a good therapeutic strategy should include a mechanism that provides protective effect from brain swelling (edema) and neurotoxicity by scavenging the lipid peroxidation end products.

BackgroundEmbryonic stem (ES) cells have unlimited proliferation potential, and can differentiate into several cell types, which represent ideal sources for cell-based therapy. This high-level proliferative ability is attributed to an unusual type of cell cycle. The Signaling pathways that regulate the proliferation of ES cells are of great interest.Methodology/Principal FindingsIn this study, we show that murine ES cells specifically express brain natriuretic peptide (BNP), and its signaling is essential for ES cell proliferation. We found that BNP and its receptor (NPR-A, natriuretic peptide receptor-A) were highly expressed in self-renewing murine ES cells, whereas the levels were markedly reduced after ES cell differentiation by the withdrawal of LIF. Targeting of BNP with short interfering RNA (siRNA) resulted in the inhibition of ES cell proliferation, as indicated by a marked reduction in the cell number and colony size, a significant reduction in DNA synthesis, and decreased numbers of cells in S phase. BNP knockdown in ES cells led to the up-regulation of gamma-aminobutyric acid receptor A (GABAAR) genes, and activation of phosphorylated histone (γ-H2AX), which negatively affects ES cell proliferation. In addition, knockdown of BNP increased the rate of apoptosis and reduced the expression of the transcription factor Ets-1.Conclusions/SignificanceAppropriate BNP expression is essential for the maintenance of ES cell propagation. These findings establish BNP as a novel endogenous regulator of ES cell proliferation.

Diabetes mellitus (DM) is one of the most prevalent metabolic disorders. In order to replace the function of the destroyed pancreatic beta cells in diabetes, islet transplantation is the most widely practiced treatment. However, it has several limitations. As an alternative approach, human pluripotent stem cells (hPSCs) can provide an unlimited source of pancreatic cells that have the ability to secrete insulin in response to a high blood glucose level. However, the determination of the appropriate pancreatic lineage candidate for the purpose of cell therapy for the treatment of diabetes is still debated. While hPSC-derived beta cells are perceived as the ultimate candidate, their efficiency needs further improvement in order to obtain a sufficient number of glucose responsive beta cells for transplantation therapy. On the other hand, hPSC-derived pancreatic progenitors can be efficiently generated in vitro and can further mature into glucose responsive beta cells in vivo after transplantation. Herein, we discuss the advantages and predicted challenges associated with the use of each of the two pancreatic lineage products for diabetes cell therapy. Furthermore, we address the co-generation of functionally relevant islet cell subpopulations and structural properties contributing to the glucose responsiveness of beta cells, as well as the available encapsulation technology for these cells.

Diabetes mellitus is the most prevailing disease with progressive incidence worldwide. To date, the pathogenesis of diabetes is far to be understood, and there is no permanent treatment available for diabetes. One of the promising approaches to understand and cure diabetes is to use pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced PCSs (iPSCs). ESCs and iPSCs have a great potential to differentiate into all cell types, and they have a high ability to differentiate into insulin-secreting β cells. Obtaining PSCs genetically identical to the patient presenting with diabetes has been a longstanding dream for the in vitro modeling of disease and ultimately cell therapy. For several years, somatic cell nuclear transfer (SCNT) was the method of choice to generate patient-specific ESC lines. However, this technology faces ethical and practical concerns. Interestingly, the recently established iPSC technology overcomes the major problems of other stem cell types including the lack of ethical concern and no risk of immune rejection. Several iPSC lines have been recently generated from patients with different types of diabetes, and most of these cell lines are able to differentiate into insulin-secreting β cells. In this review, we summarize recent advances in the differentiation of pancreatic β cells from PSCs, and describe the challenges for their clinical use in diabetes cell therapy. Furthermore, we discuss the potential use of patient-specific PSCs as an in vitro model, providing new insights into the pathophysiology of diabetes.

Understanding the biology underlying the mechanisms and pathways regulating pancreatic β cell development is necessary to understand the pathology of diabetes mellitus (DM), which is characterized by the progressive reduction in insulin-producing β cell mass. Pluripotent stem cells (PSCs) can potentially offer an unlimited supply of functional β cells for cellular therapy and disease modeling of DM. Homeobox protein NKX6.1 is a transcription factor (TF) that plays a critical role in pancreatic β cell function and proliferation. In human pancreatic islet, NKX6.1 expression is exclusive to β cells and is undetectable in other islet cells. Several reports showed that activation of NKX6.1 in PSC-derived pancreatic progenitors (MPCs), expressing PDX1 (PDX1+/NKX6.1+), warrants their future commitment to monohormonal β cells. However, further differentiation of MPCs lacking NKX6.1 expression (PDX1+/NKX6.1−) results in an undesirable generation of non-functional polyhormonal β cells. The importance of NKX6.1 as a crucial regulator in MPC specification into functional β cells directs attentions to further investigating its mechanism and enhancing NKX6.1 expression as a means to increase β cell function and mass. Here, we shed light on the role of NKX6.1 during pancreatic β cell development and in directing the MPCs to functional monohormonal lineage. Furthermore, we address the transcriptional mechanisms and targets of NKX6.1 as well as its association with diabetes.