Prasanna Vidyasekar

Cardiogel: a nano-matrix scaffold with potential application in cardiac regeneration using mesenchymal stem cells.

3-Dimensional conditions for the culture of Bone Marrow-derived Stromal/Stem Cells (BMSCs) can be generated with scaffolds of biological origin. Cardiogel, a cardiac fibroblast-derived Extracellular Matrix (ECM) has been previously shown to promote cardiomyogenic differentiation of BMSCs and provide protection against oxidative stress. To determine the matrix composition and identify significant proteins in cardiogel, we investigated the differences in the composition of this nanomatrix and a BMSC-derived ECM scaffold, termed as ‘mesogel’. An optimized protocol was developed that resulted in efficient decellularization while providing the maximum yield of ECM. The proteins were sequentially solubilized using acetic acid, Sodium Dodecyl Sulfate (SDS) and Dithiothreitol (DTT). These proteins were then analyzed using surfactant-assisted in-solution digestion followed by nano-liquid chromatography and tandem mass spectrometry (nLC-MS/MS). The results of these analyses revealed significant differences in their respective compositions and 17 significant ECM/matricellular proteins were differentially identified between cardiogel and mesogel. We observed that cardiogel also promoted cell proliferation, adhesion and migration while enhancing cardiomyogenic differentiation and angiogenesis. In conclusion, we developed a reproducible method for efficient extraction and solubilization of in vitro cultured cell-derived extracellular matrix. We report several important proteins differentially identified between cardiogel and mesogel, which can explain the biological properties of cardiogel. We also demonstrated the cardiomyogenic differentiation and angiogenic potential of cardiogel even in the absence of any external growth factors. The transplantation of Bone Marrow derived Stromal/Stem Cells (BMSCs) cultured on such a nanomatrix has potential applications in regenerative therapy for Myocardial Infarction (MI).

Genome Wide Expression Profiling of Cancer Cell Lines Cultured in Microgravity Reveals Significant Dysregulation of Cell Cycle and MicroRNA Gene Networks

Zero gravity causes several changes in metabolic and functional aspects of the human body and experiments in space flight have demonstrated alterations in cancer growth and progression. This study reports the genome wide expression profiling of a colorectal cancer cell line-DLD-1, and a lymphoblast leukemic cell line-MOLT-4, under simulated microgravity in an effort to understand central processes and cellular functions that are dysregulated among both cell lines. Altered cell morphology, reduced cell viability and an aberrant cell cycle profile in comparison to their static controls were observed in both cell lines under microgravity. The process of cell cycle in DLD-1 cells was markedly affected with reduced viability, reduced colony forming ability, an apoptotic population and dysregulation of cell cycle genes, oncogenes, and cancer progression and prognostic markers. DNA microarray analysis revealed 1801 (upregulated) and 2542 (downregulated) genes (>2 fold) in DLD-1 cultures under microgravity while MOLT-4 cultures differentially expressed 349 (upregulated) and 444 (downregulated) genes (>2 fold) under microgravity. The loss in cell proliferative capacity was corroborated with the downregulation of the cell cycle process as demonstrated by functional clustering of DNA microarray data using gene ontology terms. The genome wide expression profile also showed significant dysregulation of post transcriptional gene silencing machinery and multiple microRNA host genes that are potential tumor suppressors and proto-oncogenes including MIR22HG, MIR17HG and MIR21HG. The MIR22HG, a tumor-suppressor gene was one of the highest upregulated genes in the microarray data showing a 4.4 log fold upregulation under microgravity. Real time PCR validated the dysregulation in the host gene by demonstrating a 4.18 log fold upregulation of the miR-22 microRNA. Microarray data also showed dysregulation of direct targets of miR-22, SP1, CDK6 and CCNA2.

Lowered expression levels of a tumor suppressor gene - caveolin-1 within dysregulated gene networks of Fanconi anemia.

Fanconi anemia (FA) is a genetic disorder characterized by progressive bone marrow failure and a predisposition to cancers like acute myeloid leukemia, lung and squamous cell carcinomas. DNA damage in a healthy cell activates the FA pathway where 15 individual FA proteins interact and function together to maintain genomic stability. The disruption of this pathway results in the characteristic cellular phenotype and clinical outcome of the disease. The diverse clinical symptoms of FA such as impaired immunity and predisposition to cancers may not be explained exclusively by a non-functional FA pathway. These symptoms could then be attributed to defects in other functions of the individual FA proteins. To identify the effects of a mutant FA protein, FANCC, a transcriptome analysis was carried out on a FANCC mutant cell line (EUFA 450) and its revertant isogenic control cell line (EUFA 450Rev). Microarray data revealed dysregulation of genes involved in regulation of cell death and immune response. This study reports for the first time, the lowered expression of a tumor suppressor gene - caveolin-1, in FANCC mutant cells. The downregulation of caveolin-1 can be significant as Fanconi anemia patients have an elevated predisposition to develop cancer.

Characterization of electrospun fibrous scaffold produced from Indian eri silk fibroin

Abstract A scaffold, synthesized from bio-degradable polymers and Bombyxmori silk fibroin in the form of films and fibrous assemblies, has been used as the bio-material for in-vivo applications. In the present work, the scaffold was prepared from the fibroin of Indian eri silk via the electrospinning method. The diameter of the fibre produced was in the range of 300 to 900 nm. The scaffold was subjected to ethanol treatment to improve its dimensional stability, as there was the problem of curling and shrinking when it was treated with solutions used for the cell culture. The scaffold was characterized for surface, thermal and tensile properties. The dimensional stability of the scaffold improved and the porosity reduced, due to the treatment of the scaffold with ethanol. The average failure stress of the raw and ethanol treated scaffold was 2.34 MPa and 4.91 MPa respectively and the mean strain was 13.63 % and 7.91 % respectively. Bone marrow stromal cells were isolated from the bone marrow of Swiss albino mice, and cultured on the ethanol treated electrospun fibrous scaffold. Scanning electron microscopy of the culture was carried out to evaluate the attachment and growth of cells on the scaffold at different incubation periods. Mouse bone marrow stromal cells adhered and grew on the electrospun fibrous scaffold prepared from eri silk fibroin, and the cell density increased with increasing incubation periods.

A simplified protocol for the isolation and culture of cardiomyocytes and progenitor cells from neonatal mouse ventricles

The neonatal heart is a very useful tool for the study of biochemical pathways and properties of cardiomyocytes and as it has the potential to regenerate for a brief period of time from birth; it is also useful to study cardiac regeneration. However, as the heart matures, this proficiency for regeneration is reduced. This regenerative potential may be influenced by the microenvironment of the heart in the early stages of postnatal development and therefore, cell cultures derived at this stage may contain functional cardiomyocytes and progenitor cells. The aim of this study was to identify key steps in the isolation and culture of such early stage-neonatal mouse hearts to allow maximum migration of cardiomyocytes from the explant and their maintenance as functional, long term cultures. Explant cultures of mouse ventricles preserved 3-dimensional structure and generated migrating layers of cardiomyocytes that expressed alpha sarcomeric actin which could be further sub-cultured by enzymatic dissociation. Western blotting demonstrated expression of c-KIT, GATA4, alpha sarcomeric actin and connexin43 proteins after 20 days of explant culture. ACTA1, GATA4, and CX43 continued to express in five weeks old explant cultures while the c-KIT protein was expressed up to two passages during sub-culture. Real time PCR and SQRT PCR also demonstrated gene expression of cardiomyocyte markers in long term cultures. Migrating cells from the explants assembled into contracting spheroids after subculture and expressed the c-KIT protein. Progenitor markers CD44, CD90, and extracellular proteins, periostin and vimentin demonstrated the preservation of cellular heterogeneity in such cultures. Supplementation with Hydrocortisone maintained a cardioprotective environment and reduced the non-myocyte population. This is an optimized and efficient method for the generation of neonatal heart cultures that is not labor intensive and does not require supplementation with cytokines.

Identification of novel target genes involved in Indian Fanconi anemia patients using microarray

Fanconi anemia (FA) is a genetic disorder characterized by progressive bone marrow failure and a predisposition to cancers. Mutations have been documented in 15 FA genes that participate in the FA-BRCA DNA repair pathway, a fundamental pathway in the development of the disease and the presentation of its characteristic symptoms. Certain symptoms such as oxygen sensitivity, hematological abnormalities and impaired immunity suggest that FA proteins could participate in or independently control other pathways as well. In this study, we identified 9 DNA repair genes that were down regulated in a genome wide analysis of 6 Indian Fanconi anemia patients. Functional clustering of a total of 233 dysregulated genes identified key biological processes that included regulation of transcription, DNA repair, cell cycle and chromosomal organization. Microarray data revealed the down regulation of ATXN3, ARID4A and ETS-1, which were validated by RTPCR in a subsequent sample set of 9 Indian FA patients. Here we report for the first time a gene expression profile of Fanconi anemia patients from the Indian population and a pool of genes that might aid in the acquisition and progression of the FA phenotype.

PTEN/FOXO3/AKT pathway regulates cell death and mediates morphogenetic differentiation of Colorectal Cancer Cells under Simulated Microgravity

Gravity is a major physical factor determining the stress and strain around cells. Both in space experiments and ground simulation, change in gravity impacts the viability and function of various types of cells as well as in vivo conditions. Cancer cells have been shown to die under microgravity. This can be exploited for better understanding of the biology and identification of novel avenues for therapeutic intervention. Here, we described the effect of microgravity simulated using Rotational Cell Culture System-High Aspect Ratio Vessel (RCCS-HARV) on the viability and morphological changes of colorectal cancer cells. We observed DLD1, HCT116 and SW620 cells die through apoptosis under simulated microgravity (SM). Gene expression analysis on DLD1 cells showed upregulation of tumor suppressors PTEN and FOXO3; leading to AKT downregulation and further induction of apoptosis, through upregulation of CDK inhibitors CDKN2B, CDKN2D. SM induced cell clumps had elevated hypoxia and mitochondrial membrane potential that led to adaptive responses like morphogenetic changes, migration and deregulated autophagy, when shifted to normal culture conditions. This can be exploited to understand the three-dimensional (3D) biology of cancer in the aspect of stress response. This study highlights the regulation of cell function and viability under microgravity through PTEN/FOXO3/AKT pathway.

Scaffold-free and scaffold-assisted 3D culture enhances differentiation of bone marrow stromal cells

Abstract3D cultures of stem cells can preserve differentiation potential or increase the efficiency of methods that induce differentiation. Mouse bone marrow-derived stromal cells (BMSCs) were cultured in 3D as scaffold-free spheroids or “mesoid bodies” (MBs) and as aggregates on poly(lactic) acid microspheres (MB/MS). 3D cultures demonstrated viable cells, interaction on multiple planes, altered cell morphology, and the formation of structures similar to epithelial cell bridges. Cell proliferation was limited in suspension cultures of MB and MB/MS; however, cells regained proliferative capacity when transferred to flat substrates of tissue culture plates (TCPs). Expanded as monolayer, cells retained expression of Sca-1 and CD44 stem cell markers. 3D cultures demonstrated enhanced potential for adipogenic and osteogenic differentiation showing higher triglyceride accumulation and robust mineralization in comparison with TCP cultures. Enhanced and efficient adipogenesis was also observed in 3D cultures generated in a rotating cell culture system. Preservation of multilineage potential of BMSC was demonstrated in 5-azacytidine treatment of 3D cultures and TCP by expression of cardiac markers GATA4 and ACTA1 although functioning cardiomyocytes were not derived.

Evaluation of platelet lysate as a substitute for FBS in explant and enzymatic isolation methods of human umbilical cord MSCs

Mesenchymal stem cells (MSCs) have immense potential for cell-based therapy of acute and chronic pathological conditions. MSC transplantation for cell-based therapy requires a substantial number of cells in the range of 0.5–2.5 × 106 cells/kg body weight of an individual. A prolific source of MSCs followed by in vitro propagation is therefore an absolute prerequisite for clinical applications. Umbilical cord tissue (UCT) is an abundantly available prolific source of MSC that are fetal in nature and have higher potential for ex-vivo expansion. However, the ex-vivo expansion of MSCs using a xenogeneic supplement such as fetal bovine serum (FBS) carries the risk of transmission of zoonotic infections and immunological reactions. We used platelet lysate (PL) as a xeno-free, allogeneic replacement for FBS and compared the biological and functional characteristics of MSC processed and expanded with PL and FBS by explant and enzymatic method. UCT-MSCs expanded using PL displayed typical immunophenotype, plasticity, immunomodulatory property and chromosomal stability. PL supplementation also showed 2-fold increase in MSC yield from explant culture with improved immunomodulatory activity as compared to enzymatically dissociated cultures. In conclusion, PL from expired platelets is a viable alternative to FBS for generating clinically relevant numbers of MSC from explant cultures over enzymatic method.

Enrichment of cardiomyocytes in primary cultures of murine neonatal hearts.

In vitro culture of neonatal murine cardiomyocytes is vital for understanding the functions of the heart. Cardiomyocyte cultures are difficult to maintain because they do not proliferate after birth. The maintenance of primary cultures of viable and functional cardiomyocytes is considerably affected by the yield from initial steps of isolation procedures. This protocol describes an efficient and rapid method for isolation and maintenance of long-term cultures of neonatal murine cardiomyocytes by effectively shortening the trypsin enzyme digestion period and the cardiomyocyte enrichment step.