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Rao, Rajesh Raghavendran

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Rao

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Rajesh Raghavendran

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Rao, Rajesh Raghavendran
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    Analysis of Embryoid Bodies Derived from Human Induced Pluripotent Stem Cells as a Means to Assess Pluripotency
    (Hindawi Publishing Corporation, 2012) Sheridan, Steven D.; Surampudi, Vasudha; Rao, Rajesh Raghavendran
    Human induced pluripotent stem cells (hiPSCs) have core properties of unlimited self-renewal and differentiation potential and have emerged as exciting cell sources for applications in regenerative medicine, drug discovery, understanding of development, and disease etiology. Key among numerous criteria to assess pluripotency includes the in vivo teratoma assay that has been widely proposed as a standard functional assay to demonstrate the pluripotency of hiPSCs. Yet, the lack of reliability across methodologies, lack of definitive clinical significance, and associated expenses bring into question use of the teratoma assay as the “gold standard” for determining pluripotency. We propose use of the in vitro embryoid body (EB) assay as an important alternative to the teratoma assay. This paper summarizes the methodologies for creating EBs from hiPSCs and the subsequent analyses to assess pluripotency and proposes its use as a cost-effective, controlled, and reproducible approach that can easily be adopted to determine pluripotency of generated hiPSCs.
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    Effect of substrate stiffness on early human embryonic stem cell differentiation
    (BioMed Central, 2013) Eroshenko, Nikolai Alexandrovich; Ramachandran, Rukmani; Yadavalli, Vamsi K; Rao, Rajesh Raghavendran
    Background: The pluripotency and self renewing properties of human embryonic stem cells (hESC) make them a valuable tool in the fields of developmental biology, pharmacology and regenerative medicine. Therefore, there exists immense interest in devising strategies for hESC propagation and differentiation. Methods involving simulation of the native stem cell microenvironment, both chemical and physical, have received a lot of attention in recent years. Equally important is evidence that cells can also sense the mechanical properties of their microenvironment. In this study, we test the hypothesis that hESCs accept mechanical cues for differentiation from the substrate by culturing them on flexible polydimethylsiloxane (PDMS) of varying stiffness. Results: PDMS substrates were prepared using available commercial formulations and characterized for stiffness, surface properties and efficiency of cell attachment and proliferation. Across different substrate stiffness, cell numbers, cell attachment and cell surface area were found to be similar. Expression of pluripotency markers decreased with increased time in culture across all PDMS substrates of varying stiffness. Analysis of gene expression of differentiation markers indicates that the differentiation process becomes less stochastic with longer culture times. Conclusions: We evaluated the utility of PDMS substrates for stem cell propagation and substrate mediated differentiation. The stiffness affected gene expression of pluripotent and differentiation markers with results indicating that these substrate systems could potentially be used to direct hESC fate towards early mesodermal lineages. This study suggests that coupled with soluble factors, PDMS substrates could potentially be useful in generating defined populations of differentiated cells.