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Southbridge, MA, United States

Ambady S.,Worcester Polytechnic Institute | Kashpur O.,Worcester Polytechnic Institute | Grella A.,Worcester Polytechnic Institute | Malcuit C.,Worcester Polytechnic Institute | And 5 more authors.

The POU5F1 gene codes for the OCT4 transcription factor, which is one of the key regulators of pluripotency. Its transcription, alternative splicing, and alternative translation leading to the synthesis of the active, nuclear localized OCT4A has been described in detail. Much less, however, is known about actively transcribed OCT4 pseudogenes, several of which display high homology to OCT4A and can be expressed and translated into proteins. Using RT-PCR followed by pseudogene-specific restriction digestion, cloning, and sequencing we discriminate between OCT4A and transcripts for pseudogenes 1, 3 and 4. We show that expression of OCT4 and its pseudogenes follows a developmentally- regulated pattern in differentiating hESCs, indicating a tight regulatory relationship between them. We further demonstrate that differentiated human cells from a variety of tissues express exclusively pseudogenes. Expression of OCT4A can, however be triggered in adult differentiated cells by oxygen and FGF2-dependent mechanisms. © 2014 Jez et al. Source

Grasman J.M.,Worcester Polytechnic Institute | Grasman J.M.,Bioengineering Institute | Page R.L.,Worcester Polytechnic Institute | Page R.L.,Bioengineering Institute | And 6 more authors.
Acta Biomaterialia

A significant challenge in the design of biomimetic scaffolds is combining morphologic, mechanical, and biochemical cues into a single construct to promote tissue regeneration. In this study, we analyzed the effects of different crosslinking conditions on fibrin biopolymer microthreads to create morphologic scaffolds with tunable mechanical properties that are designed for directional cell guidance. Fibrin microthreads were crosslinked using carbodiimides in either acidic or neutral buffer, and the mechanical, structural, and biochemical responses of the microthreads were investigated. Crosslinking in the presence of acidic buffer (EDCa) created microthreads that had significantly higher tensile strengths and moduli than all other microthreads, and failed at lower strains than all other microthreads. Microthreads crosslinked in neutral buffer (EDCn) were also significantly stronger and stiffer than uncrosslinked threads and were comparable to contracting muscle in stiffness. Swelling ratios of crosslinked microthreads were significantly different from each other and uncrosslinked controls, suggesting a difference in the internal organization and compaction of the microthreads. Using an in vitro degradation assay, we observed that EDCn microthreads degraded within 24 h, six times slower than uncrosslinked control threads, but EDCa microthreads did not show any significant indication of degradation within the 7-day assay period. Microthreads with higher stiffnesses supported significantly increased attachment of C2C12 cells, as well as increases in cell proliferation without a decrease in cell viability. Taken together, these data demonstrate the ability to create microthreads with tunable mechanical and structural properties that differentially direct cellular functions. Ultimately, we anticipate that we can strategically exploit these properties to promote site-specific tissue regeneration. © 2012 Acta Materialia Inc. Source

Kole D.,Worcester Polytechnic Institute | Ambady S.,Worcester Polytechnic Institute | Page R.L.,Worcester Polytechnic Institute | Page R.L.,CellThera Inc. | And 2 more authors.
Cellular Reprogramming

Direct reprogramming of a differentiated somatic cell into a developmentally more plastic cell would offer an alternative to applications in regenerative medicine that currently depend on either embryonic stem cells (ESCs), adult stem cells, or induced pluripotent stem cells (iPSCs). Here we report the potential of select Xenopus laevis egg extract fractions, in combination with exogenous fibroblast growth factor-2 (FGF2), to affect life span, morphology, gene expression, protein translation, and cellular localization of OCT4 and NANOG transcription factors, and the developmental potential of human dermal fibroblasts in vitro. A gradual change in morphology is accompanied by translation of embryonic transcription factors and their nuclear localization and a life span exceeding 60 population doublings. Cells acquire the ability to follow adipogenic, neuronal, and osteogenic differentiation under appropriate induction conditions in vitro. Analysis of active extract fractions reveals that Xenopus egg protein and RNAs as well as exogenously supplemented FGF2 are required and sufficient for induction and maintenance of this phenotypic change. Factors so far identified in the active fractions include FGF2 itself, transforming growth factor-β, maskin, and nucleoplasmin. Identification of critical factors needed for reprogramming may allow for nonviral, chemically defined derivation of human-induced multipotent cells that can be maintained by exogenous FGF2. © Mary Ann Liebert, Inc. Source

Page R.L.,Worcester Polytechnic Institute | Page R.L.,CellThera Inc. | Malcuit C.,Worcester Polytechnic Institute | Malcuit C.,CellThera Inc. | And 9 more authors.
Tissue Engineering - Part A

Large-scale musculoskeletal wounds, such as those seen in trauma injuries, present poor functional healing prognoses. In severe trauma, when the native tissue architecture is destroyed or lost, the regenerative capacity of skeletal muscle is diminished by scar formation. Here we demonstrate that a scaffold system composed of fibrin microthreads can provide an efficient delivery system for cell-based therapies and improve regeneration of a large defect in the tibialis anterior of the mouse. Cell-loaded fibrin microthread bundles implanted into a skeletal muscle resection reduced the overall fibroplasia-associated deposition of collagen in the wound bed and promoted in-growth of new muscle tissue. When fibrin microthreads were seeded with adult human cells, implanted cells contributed to the nascent host tissue architecture by forming skeletal muscle fibers, connective tissue, and PAX7-positive cells. Stable engraftment was observed at 10 weeks postimplant and was accompanied by reduced levels of collagen deposition. Taken together, these data support the design and development of a platform for microthread-based delivery of autologous cells that, when coupled to an in vitro cellular reprogramming process, has the potential to improve healing outcomes in large skeletal muscle wounds. © Copyright 2011, Mary Ann Liebert, Inc. 2011. Source

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