Gopu Sriram

Efficient derivation of lateral plate and paraxial mesoderm subtypes from human embryonic stem cells through GSKi-mediated differentiation.

The vertebrae mesoderm is a source of cells that forms a variety of tissues, including the heart, vasculature, and blood. Consequently, the derivation of various mesoderm-specific cell types from human embryonic stem cells (hESCs) has attracted the interest of many investigators owing to their therapeutic potential in clinical applications. However, the need for efficient and reliable methods of differentiation into mesoderm lineage cell types remains a significant challenge. Here, we demonstrated that inhibition of glycogen synthase kinase-3 (GSK-3) is an essential first step toward efficient generation of the mesoderm. Under chemically defined conditions without additional growth factors/cytokines, short-term GSK inhibitor (GSKi) treatment effectively drives differentiation of hESCs into the primitive streak (PS), which can potentially commit toward the mesoderm when further supplemented with bone morphogenetic protein 4. Further analysis confirmed that the PS-like cells derived from GSKi treatment are bipotential, being able to specify toward the endoderm as well. Our findings suggest that the bipotential, PS/mesendoderm-like cell population exists only at the initial stages of GSK-3 inhibition, whereas long-term inhibition results in an endodermal fate. Lastly, we demonstrated that our differentiation approach could efficiently generate lateral plate (CD34(+)KDR(+)) and paraxial (CD34(-)PDGFRα(+)) mesoderm subsets that can be further differentiated along the endothelial and smooth muscle lineages, respectively. In conclusion, our study presents a unique approach for generating early mesoderm progenitors in a chemically directed fashion through the use of small-molecule GSK-3 inhibitor, which may be useful for future applications in regenerative medicine.

Efficient differentiation of human embryonic stem cells to arterial and venous endothelial cells under feeder- and serum-free conditions

BackgroundHeterogeneity of endothelial cells (ECs) is a hallmark of the vascular system which may impact the development and management of vascular disorders. Despite the tremendous progress in differentiation of human embryonic stem cells (hESCs) towards endothelial lineage, differentiation into arterial and venous endothelial phenotypes remains elusive. Additionally, current differentiation strategies are hampered by inefficiency, lack of reproducibility, and use of animal-derived products.MethodsTo direct the differentiation of hESCs to endothelial subtypes, H1- and H9-hESCs were seeded on human plasma fibronectin and differentiated under chemically defined conditions by sequential modulation of glycogen synthase kinase-3 (GSK-3), basic fibroblast growth factor (bFGF), bone morphogenetic protein 4 (BMP4) and vascular endothelial growth factor (VEGF) signaling pathways for 5 days. Following the initial differentiation, the endothelial progenitor cells (CD34+CD31+ cells) were sorted and terminally differentiated under serum-free conditions to arterial and venous ECs. The transcriptome and secretome profiles of the two distinct populations of hESC-derived arterial and venous ECs were characterized. Furthermore, the safety and functionality of these cells upon in vivo transplantation were characterized.ResultsSequential modulation of hESCs with GSK-3 inhibitor, bFGF, BMP4 and VEGF resulted in stages reminiscent of primitive streak, early mesoderm/lateral plate mesoderm, and endothelial progenitors under feeder- and serum-free conditions. Furthermore, these endothelial progenitors demonstrated differentiation potential to almost pure populations of arterial and venous endothelial phenotypes under serum-free conditions. Specifically, the endothelial progenitors differentiated to venous ECs in the absence of VEGF, and to arterial phenotype under low concentrations of VEGF. Additionally, these hESC-derived arterial and venous ECs showed distinct molecular and functional profiles in vitro. Furthermore, these hESC-derived arterial and venous ECs were nontumorigenic and were functional in terms of forming perfused microvascular channels upon subcutaneous implantation in the mouse.ConclusionsWe report a simple, rapid, and efficient protocol for directed differentiation of hESCs into endothelial progenitor cells capable of differentiation to arterial and venous ECs under feeder-free and serum-free conditions. This could offer a human platform to study arterial–venous specification for various applications related to drug discovery, disease modeling and regenerative medicine in the future.

Immunohistochemical evaluation of mast cells and vascular endothelial proliferation in oral submucous fibrosis

INTRODUCTION Oral submucous fibrosis (OSMF) is a chronic, progressive, scarring disease that predominantly affects the people of south-east Asian origin. AIM The present study is aimed at comparing and correlating the mast cell density (MCD) and micro vascular density (MVD) in normal mucosa and different grades of OSMF and to analyze their role in the disease progression. MATERIALS AND METHODS MCD was assessed immunohistochemically using anti mast cell tryptase and MVD was assessed using anti-factor VIII related von Willebrand factor. RESULTS The one way comparison of MVD and MCD in normal mucosa and among different grades of OSMF showed a significant increase in MCD and MVD among OSMF cases. Correlation analysis using Pearson correlation coefficient, showed positive correlation between MCD and MVD i.e. as MCD increases there is an exponential increase in MVD. CONCLUSION The increase in MVD and MCD reveals their role in the pathogenesis of OSMF, a lesion characterized by progressive fibrosis in early stages and failure of degradation or remodeling in the advanced stages.

Rhinosporidiosis of parotid duct

Rhinosporidiosis is a benign chronic granulomatous infection caused by Rhinosporidium seeberi. Rhinosporidiosis is endemic in south Asia, notably in southern India and Sri Lanka. Majority of the cases have been reported to occur in upper respiratory sites, notably anterior nares, nasal cavity, nasopharynx, larynx and soft palate. Only two rare cases of involvement of parotid duct, have been reported in literature. Hence, this case will probably be the third to be reported.

Comparison of three different methods of tissue processing

Biopsied tissue is cut into thin slices and stained suitably for microscopical examination. Enabling the tissue for sectioning by paraffin embedding is known as tissue processing. The three most commonly employed means of tissue processing are routine manual method, rapid manual method and the microwave method. In this study, sections obtained from the same site of the same tissue were processed by these three methods and stained by hematoxylin and eosin. These sections were then microscopically evaluated by various parameters to compare the three methods. The results that were obtained, after subjecting to statistical analysis, showed no significant differences between the three different processes in terms of quality of staining, clarity of nucleo-cytoplasmic differentiation in various cells and the presence of artifacts. Tissue shrinkage was less in microwave-processed tissue as compared to the other methods. Microwave tissue processing was also found to be more cost-effective than other methods.

In Vitro Osteogenic Potential of Green Fluorescent Protein Labelled Human Embryonic Stem Cell-Derived Osteoprogenitors

Cellular therapy using stem cells in bone regeneration has gained increasing interest. Various studies suggest the clinical utility of osteoprogenitors-like mesenchymal stem cells in bone regeneration. However, limited availability of mesenchymal stem cells and conflicting evidence on their therapeutic efficacy limit their clinical application. Human embryonic stem cells (hESCs) are potentially an unlimited source of healthy and functional osteoprogenitors (OPs) that could be utilized for bone regenerative applications. However, limited ability to track hESC-derived progenies in vivo greatly hinders translational studies. Hence, in this study, we aimed to establish hESC-derived OPs (hESC-OPs) expressing green fluorescent protein (GFP) and to investigate their osteogenic differentiation potential in vitro. We fluorescently labelled H9-hESCs using a plasmid vector encoding GFP. The GFP-expressing hESCs were differentiated into hESC-OPs. The hESC-OPsGFP+ stably expressed high levels of GFP, CD73, CD90, and CD105. They possessed osteogenic differentiation potential in vitro as demonstrated by increased expression of COL1A1, RUNX2, OSTERIX, and OPG transcripts and mineralized nodules positive for Alizarin Red and immunocytochemical expression of osteocalcin, alkaline phosphatase, and collagen-I. In conclusion, we have demonstrated that fluorescently labelled hESC-OPs can maintain their GFP expression for the long term and their potential for osteogenic differentiation in vitro. In future, these fluorescently labelled hESC-OPs could be used for noninvasive assessment of bone regeneration, safety, and therapeutic efficacy.

Immunohistochemical evaluation of mast cells and vascular endothelial proliferation in oral precancerous lesion-leukoplakia

Background: Oral leukoplakia is the best-known precursor lesion. Although a morphologic feature of oral epithelial dysplasia is well described, less is known about the pathobiologic changes within the cells and over the cell surfaces for malignant transformation. Aims: The present study is aimed at comparing and correlating the mast cell density (MCD) and micro vascular density (MVD) in Normal Mucosa (NM) and different grades of dysplasia and to analyze their role in disease progression. Materials and Methods: MCD was assessed using anti mast cell tryptase and MVD was assessed immunohistochemically using anti-Factor VIII related von Willibrand factor. Results: The Results of the present study showed an exponential increase in microvessel density as mast cell density increased. Conclusion: The role of mast cells in angiogenesis as it progresses from normal mucosa to dysplasia is in concordance with the study. The number of mast cells and microvessel can be used as indictors of disease progression.

Comparative study of Candida by conventional and CHROMagar method in non-denture and denture wearers by oral rinse technique

INTRODUCTION Candidal species colonizes the oral cavities of healthy individuals without dentures and also of denture wearers. Soft liners and tissue conditioning materials have been found to support the growth of Candida albicans which may predispose to lesions. The most important and common candidal species are C. albicans, C. tropicalis, and C. glabrata. C albicans is usually isolated from both the fitting surface of the denture and the denture-bearing mucosa of the affected patients. The aim of this study was to isolate, quantify, and speciate candidal species in non-denture wearers (controls) and denture wearers (study group) by the oral rinse technique. Isolation was done using Sabouraud dextrose agar (SDA). Speciation was done using conventional methods like the germ tube test, carbohydrate fermentation test, urease test, as well as the CHROMagar method. AIMS AND OBJECTIVE 1) To assess the prevalence of Candida in non-denture wearers and in denture wearers by oral rinse technique, with isolation on SDA; 2) to speciate and quantify Candida in non-denture wearers and denture wearers by using conventional methods (germ tube test, carbohydrate fermentation test, urease test) and the CHROMagar method; 3) to assess the influence of smoking and diabetes on candidal species among the denture wearers; and 4) to assess the sensitivity and specificity of SDA and CHRO Magar. MATERIALS AND METHODS Salivary samples for Candida evaluation were collected from the subjects in sterile sample containers, using the oral rinse technique. RESULTS C glabrata was the most commonly found species among denture wearers and non-denture wearers both by conventional and CHROMagar methods. In males, C. albicans was the predominant species, whereas C. glabrata was the predominant species in females. Candidal colonization was higher in denture wearers compared to non-denture wearers, especially among females. The CHROMagar method was more rapid compared to conventional methods. In the present study, CHROMagar Candida showed 100% specificity and 100% sensitivity when compared to SDA and conventional methods.

Fabrication and evaluation of electrohydrodynamic jet 3D printed polycaprolactone/chitosan cell carriers using human embryonic stem cell-derived fibroblasts:

Biological function of adherent cells depends on the cell–cell and cell–matrix interactions in three-dimensional space. To understand the behavior of cells in 3D environment and their interactions with neighboring cells and matrix requires 3D culture systems. Here, we present a novel 3D cell carrier scaffold that provides an environment for routine 3D cell growth in vitro. We have developed thin, mechanically stable electrohydrodynamic jet (E-jet) 3D printed polycaprolactone and polycaprolactone/Chitosan macroporous scaffolds with precise fiber orientation for basic 3D cell culture application. We have evaluated the application of this technology by growing human embryonic stem cell-derived fibroblasts within these 3D scaffolds. Assessment of cell viability and proliferation of cells seeded on polycaprolactone and polycaprolactone/Chitosan 3D-scaffolds show that the human embryonic stem cell-derived fibroblasts could adhere and proliferate on the scaffolds over time. Further, using confocal microscopy we demonstrate the ability to use fluorescence-labelled cells that could be microscopically monitored in real-time. Hence, these 3D printed polycaprolactone and polycaprolactone/Chitosan scaffolds could be used as a cell carrier for in vitro 3D cell culture-, bioreactor- and tissue engineering-related applications in the future.

Innate Immune Response of Human Embryonic Stem Cell-Derived Fibroblasts and Mesenchymal Stem Cells to Periodontopathogens

Periodontitis involves complex interplay of bacteria and host immune response resulting in destruction of supporting tissues of the tooth. Toll-like receptors (TLRs) play a role in recognizing microbial pathogens and eliciting an innate immune response. Recently, the potential application of multipotent stem cells and pluripotent stem cells including human embryonic stem cells (hESCs) in periodontal regenerative therapy has been proposed. However, little is known about the impact of periodontopathogens on hESC-derived progenies. This study investigates the effects of heat-killed periodontopathogens, namely, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, on TLR and cytokine expression profile of hESC-derived progenies, namely, fibroblasts (hESC-Fib) and mesenchymal stem cells (hESC-MSCs). Additionally, the serotype-dependent effect of A. actinomycetemcomitans on hESC-derived progenies was explored. Both hESC-Fib and hESC-MSCs constitutively expressed TLR-2 and TLR-4. hESC-Fib upon exposure to periodontopathogens displayed upregulation of TLRs and release of cytokines (IL-1β, IL-6, and IL-8). In contrast, hESC-MSCs were largely nonresponsive to bacterial challenge, especially in terms of cytokine production. Further, exposure of hESC-Fib to A. actinomycetemcomitans serotype c was associated with higher IL-8 production than serotype b. In contrast, the hESC-MSCs displayed no serotype-dependent response. Differential response of the two hESC progenies implies a phenotype-dependent response to periodontopathogens and supports the concept of immunomodulatory properties of MSCs.