Biomedical subjects

Krishnendu Roy

Publications and source records attributed to Krishnendu Roy.

10 recordsLinked to original sources

PubMed2006

Biomaterial-based notch signaling for the differentiation of hematopoietic stem cells into T cells.

Thymocyte development takes place in a complex milieu of supportive cells and ECM that are responsible for the proliferation, adhesion, migration, and selection processes these cells undergo before reaching maturity. In recent years, the role of notch signaling in lymphocyte development, specifically T-cell development, has been extensively characterized. Although notch ligand mediated signals have been shown to be a necessary component of T-cell generation from stem cells, high-throughput, synthetic biomaterial-based systems for notch-directed stem-cell differentiation into lymphocytes are yet to be reported. Here, we present a microbead-based, artificial notch signaling system to study stem-cell differentiation into the T-cell lineage. Magnetic microbeads were functionalized with the notch ligand DLL4 using streptavidin-biotin binding and antibody-antigen coupling. Immunohistochemistry and flow cytometry analysis indicated approximately 65% conjugation efficiency. Efficient notch signaling through these functionalized microbeads was demonstrated through a myotube inhibition assay in C2C12 myoblasts. Thy1.2(+) early T cells were successfully generated from mouse bone marrow hematopoietic stem cells (BMHSCs) using DLL4 functionalized beads using both insert-based and mixed stromal cell (OP9) coculture conditions, indicating that stem cell-stromal cell physical contact is not necessary for DLL4 directed T-cell differentiation. Coculture studies with bead-to-cell ratios of 1:1 generated higher T-cell differentiation efficiencies, compared to bead-to-cell ratios of 5:1. These data demonstrate the promising potential of this biomaterial-based notch signaling system to generate T cells from stem cells and to elucidate the molecular interactions in T-cell development.

Animals↗

PubMed2006

Influence of scaffold physical properties and stromal cell coculture on hematopoietic differentiation of mouse embryonic stem cells.

Recent studies have suggested that three-dimensional (3D) biomaterial-based scaffolds and dynamic culture conditions could provide significant enhancement in the differentiation efficiency of embryonic stem cells (ESCs). Here we report that scaffold physical properties, like pore size, polymer concentration and compression modulus as well as specific culture conditions, e.g. cell seeding density and coculture with stromal cells can significantly influence hematopoietic differentiation of ESCs. PLLA scaffolds of various polymer concentrations (7.5%, 10% and 20% w/v) and pore size distributions (<150 microm, 150-425 microm, >425 microm) were fabricated using a standard solvent casting-salt leaching method. Mouse R1 ESCs were allowed to differentiate on these scaffolds either alone or in coculture with OP9 cells, a bone-marrow derived murine stromal cell line. Following one week of culture, cells were detached and analyzed using flow cytometry to evaluate the frequency of hematopoietic progenitor cells (HPC). Our results indicate that decreasing scaffold pore size increases hematopoietic differentiation of ESCs. In addition, increasing polymer concentration which resulted in increased scaffold compression modulus also provided significantly enhanced hematopoiesis. Furthermore, higher cell seeding densities as well as coculture with marrow-derived stromal cells increased HPC generation. Collectively, these results indicate that physical and mechanical properties of the 3D microenvironment as well as cell-cell and cell-stromal interactions might play a significant role in ESC differentiation and therefore warrants further investigation to elucidate the molecular mechanisms.

Animals↗

PubMed2006

Effect of 3D scaffold and dynamic culture condition on the global gene expression profile of mouse embryonic stem cells.

We have previously demonstrated that mouse embryonic stem (ES) cells differentiated on three-dimensional (3D), highly porous, tantalum-based scaffolds (Cytomatrixtrade mark) have significantly higher hematopoietic differentiation efficiency than those cultured under conventional two-dimensional (2D) tissue culture conditions. In addition, ES cell-seeded scaffolds cultured inside spinner bioreactors showed further enhancement in hematopoiesis compared to static conditions. In the present study, we evaluated how these various biomaterial-based culture conditions, e.g. 2D vs. 3D scaffolds and static vs. dynamic, influence the global gene expression profile of differentiated ES cells. We report that compared to 2D tissue culture plates, cells differentiated on porous, Cytomatrixtrade mark scaffolds possess significantly higher expression levels of extracellular matrix (ECM)-related genes, as well as genes that regulate cell growth, proliferation and differentiation. In addition, these differences in gene expression were more pronounced in 3D dynamic culture compared to 3D static culture. We report specific genes that are either uniquely expressed under each condition or are quantitatively regulated, i.e. over expressed or inhibited by a specific culture environment. We conclude that that biomaterial-based 3D cultures, especially under dynamic conditions, might favor efficient hematopoietic differentiation of ES cells by stimulating increased expression of specific ECM proteins, growth factors and cell adhesion related genes while significantly down-regulating genes that act to inhibit expression of these molecules.

Animals↗

PubMed2006

Prophylactic anti-tumor effects in a B cell lymphoma model with DNA vaccines delivered on polyethylenimine (PEI) functionalized PLGA microparticles.

Idiotypic sequences, specific to the hypervariable regions of immunoglobulins expressed by malignant B cells offer a therapeutic target in B cell lymphoma. Efficient approaches have been described to clone a single chain fragment of the tumor immunoglobulin (Ig) comprising of heavy and light Ig chains (sFv) fused with proinflammatory chemokines. Tumor associated, poorly immunogenic self antigens encoded by plasmid DNA (pDNA) have been rendered immunogenic by chemokine fusion, thereby targeting to antigen presenting cells (APCs) which differentially express chemokine receptors. Here we present an injectable (parenteral) approach using synthetic polymer based cationic microparticle formulations for enhancing the potency of such chemokine/self antigen expressing plasmid construct. Branched and linear polyethyleneimine (PEI) were conjugated on poly (D, L lactide-co-glycolide) (PLGA) microparticles using carbodiimide chemistry followed by efficient loading of plasmid DNA. In addition to imparting significant buffering ability to these cationic microparticles, flow cytometry studies indicate that these DNA loaded microparticles significantly up regulate CD80 and MHC class II markers in phagocytic RAW264.7 cells, indicating intrinsic adjuvant effects. Intradermal injections in Balb/c mice with these formulations induced significant protection upon tumor challenge with 2.5 times the minimal lethal dose. Long term survival rates were significant (p < 0.05) in comparison with saline injected controls or blank microparticles. Further studies indicated that intramuscular delivery might provide better protection compared to intradermal injections and perform similar to gene gun mediated administration. We conclude, based on these promising in vivo results, that such surface-functionalized microparticles offer an attractive strategy to improve the potency of self antigen-based cancer DNA vaccines.

Animals↗

PubMed2006

A digital micro-mirror device-based system for the microfabrication of complex, spatially patterned tissue engineering scaffolds.

Our ability to create precise, pre-designed, spatially patterned biochemical and physical microenvironments inside polymer scaffolds could provide a powerful tool in studying progenitor cell behavior and differentiation under biomimetic, three-dimensional (3D) culture conditions. We have developed a simple and fast, layer-by-layer microstereolithography system consisting of an ultra-violet light source, a digital micro-mirror masking device, and a conventional computer projector, that allows fabrication of complex internal features along with precise spatial distribution of biological factors inside a single scaffold. Photo-crosslinkable poly(ethylene glycol) diacrylates were used as the scaffold material, and murine bone marrow-derived cells were successfully encapsulated or seeded on fibronectin-functionalized scaffolds. Fluorescently-labeled polystyrene microparticles were used to show the capability of this system to create scaffolds with complex internal architectures and spatial patterns. We demonstrate that precisely controlled pore size and shapes can be easily fabricated using a simple, computer-aided process. Our results further indicate that multi-layered scaffolds with spatially distributed factors in the same layer or across different layers can be efficiently manufactured using this technique. These microfabricated scaffolds are conducive for osteogenic differentiation of marrow-derived stem cells, as indicated by efficient matrix mineralization.

Animals↗

PubMed2005

Laser-layered microfabrication of spatially patterned functionalized tissue-engineering scaffolds.

Understanding cell behavior inside complex, three-dimensional (3D) microenvironments with controlled spatiotemporal patterning of physical and biochemical factors would provide significant insights into the basic biology of organ development and tissue functions. One of the fundamental limitations in studying such behavior has been the inability to create patterned microenvironments within 3D scaffold structures. Here a simple, layer-by-layer stereolithography (SL) method that can precisely pattern ligands, extracellular-matrix (ECM) components, and growth factors, as well as controlled release particles inside a single scaffold, has been developed. The process also allows fabrication of predesigned internal architectures and porosities. Photocrosslinkable poly(ethylene glycol) dimethacrylate (PEGDMA) was used as the basic structural component of these microfabricated scaffolds. PEG acrylates, covalently modified with the cell adhesive peptide arginine-glycine-aspartic acid (RGD) or the ECM component heparan sulfate, was incorporated within the scaffolds to facilitate cell attachment and to allow spatial sequestration of heparan-binding growth factors. Fluorescently labeled polymer microparticles and basic fibroblast growth factor (FGF-2) were chosen to illustrate the capability of SL to spatiotemporally pattern scaffolds. The results demonstrate that a precise, predesigned distribution of single or multiple factors within a single 3D structure can be created, and specific internal architectures can be fabricated. Functionalization of these scaffolds with RGD is demonstrated, and heparan sulfate allows efficient cell attachment and spatial localization of growth factors. Such patterned scaffolds might provide effective systems to study cell behavior in complex microenvironments and could eventually lead to engineering of complex, hybrid tissue structures through predesigned, multilineage differentiation of a single stem-cell population.

Animals↗

PubMed2005

Covalent conjugation of polyethyleneimine on biodegradable microparticles for delivery of plasmid DNA vaccines.

Microparticle-based delivery of nucleic acids has gained particular attention in recent years in view of improving the potency of DNA vaccination. Such improvement has been reported by encapsulation of pDNA within biodegradable microparticles or through surface adsorption on cationic microparticles. However, the intrinsic intracellular barriers for gene delivery to antigen presenting cells (APCs) have not been adequately addressed in the rational design of delivery systems for DNA vaccines. Here we report synthesis and characterization of biodegradable microparticles that (a) can passively target phagocytic APCs, (b) have intrinsic buffering ability that might allow for enhanced phagosomal escape, (c) are not cytotoxic and (d) have improved APC transfection efficiency. Branched polyethyleneimine (b-PEI) was covalently conjugated using carbodiimide chemistry to the surface of poly(lactide-coglycolide) (PLGA) microparticles to create cationic microparticles capable of simultaneously delivering both DNA vaccines as well as other immunomodulatory agents (cytokines or nucleic acids) within a single injectable delivery vehicle. Our results indicate that covalent conjugation of b-PEI allows efficient surface loading of nucleic acids, introduces intrinsic buffering properties to PLGA particles and enhances transfection of phagocytic cells without affecting the cytocompatibility of PLGA carriers.

Absorbable Implants↗

PubMed2003

Cervical cancer chemoprevention, vaccines, and surrogate endpoint biomarkers.

At the Second International Conference on Cervical Cancer, held April 11-14, 2002, experts in cervical cancer prevention, detection, and treatment reviewed the need for more research in chemoprevention, including prophylactic and therapeutic vaccines, immunomodulators, peptides, and surrogate endpoint biomarkers. Investigators and clinicians noted the need for more rigorous Phase I randomized clinical trials, more attention to the risk factors that can affect study results in this patient population, and validation of optical technologies that will provide valuable quantitative information in real time regarding disease regression and progression. They discussed the role of the human papillomavirus (HPV) in cervical cancer development and the importance of developing strategies to suppress HPV persistence and progression. Results in Phase I randomized clinical trials have been disappointing because few have demonstrated statistically significant regression attributable to the agent tested. Researchers recommended using a transgenic mouse model to test and validate new compounds, initiating vaccine and immunomodulator trials, and developing immunologic surrogate endpoint biomarkers.

Biomarkers, Tumor↗

PubMed2003

Gene delivery with in-situ crosslinking polymer networks generates long-term systemic protein expression.

Two polyethylene oxide-based delivery systems comprised of reacting PEG polymers were designed for the delivery of DNA expression vectors. The polymers are formulated with the DNA and injected into the muscle, wherein a crosslinked matrix forms in-situ. The matrix resembles a viscous solution (formulation A) or a gel (formulation B). The reacting PEG polymers do not interact with, but entrap the DNA. The formation of the matrix does not affect the supercoiling of the incorporated DNA. The polymers are biocompatible and biodegradable due to the presence of hydrolytically labile bonds in one of the components. Measurement of degradation in vivo suggests that a significant amount of the polymer disappears from the injected muscle by 28 days post injection. Administration to mice of SEAP plasmid DNA formulated with the PEG polymers results in SEAP expression. Expression levels are similar to those of unformulated DNA, but the duration of gene expression is significantly longer in immunocompetent animals receiving the formulated DNA. Significantly lower anti-SEAP IgG titers are elicited by network-formulated DNA relative to unformulated DNA, even though expression levels are comparable. The data suggests that the matrix extends duration of expression by reducing the anti-SEAP immune response so that these delivery systems may be useful for prolonged gene expression following a single intramuscular injection.

Alkaline Phosphatase↗

PubMedDate not supplied

Biomimetic three-dimensional cultures significantly increase hematopoietic differentiation efficacy of embryonic stem cells.

Stem cell-based tissue engineering is a promising technology in the effort to create functional tissues of choice. To establish an efficient approach for generating hematopoietic cell lineages directly from embryonic stem (ES) cells and to study the effects of three-dimensional (3D) biomaterials on ES cell differentiation, we cultured mouse ES cells on 3D, highly porous, biomimetic scaffolds. Cell differentiation was evaluated by microscopy and flow cytometry analysis with a variety of hematopoiesis- specific markers. Our data indicate that ES cells differentiated on porous 3D scaffold structures developed embryoid bodies (EBs) similar to those in traditional two-dimensional (2D) cultures; however, unlike 2D differentiation, these EBs integrated with the scaffold and appeared embedded in a network of extracellular matrix. Most significantly, the efficiency of hematopoietic precursor cell (HPC) generation on 3D, as indicated by the expression of various HPC-specific surface markers (CD34, Sca-1, Flk-1, and c-Kit) and colony-forming cell (CFC) assays, was reproducibly increased (about 2-fold) over their 2D counterparts. Comparison of static and dynamic 3D cultures demonstrated that spinner flask technology also contributed to the higher hematopoietic differentiation efficiency of ES cells seeded on scaffolds. Continued differentiation of 3D-derived HPCs into the myeloid lineage demonstrated increased efficiency (2-fold) of generating myeloid compared with differentiation from 2D-derived HPCs.

Animals↗