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BMC Biotechnology (1)
Journal of developmental biology and tissue engineering (1)
Köllmer, Melanie (3)
Gemeinhart, Richard A. (2)
Buhrman, Jason S (1)
Buhrman, Jason S. (1)
Collins, John M. (1)
Gemeinhart, Richard A (1)
Hauk, Thomas G. (1)
Keskar, Vandana (1)
Rayahin, Jamie E (1)
Russell, Brenda (1)
Zhang, Yu (1)
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Markers Are Shared Between Adipogenic and Osteogenic Differentiated Mesenchymal Stem Cells
Buhrman, Jason S.
Gemeinhart, Richard A.
Journal of developmental biology and tissue engineering
The stem cell differentiation paradigm is based on the progression of cells through generations of daughter cells that eventually become restricted and committed to one lineage resulting in fully differentiated cells. Herein, we report on the differentiation of adult human mesenchymal stem cells (hMSCs) towards adipogenic and osteogenic lineages using established protocols. Lineage specific geneswere evaluated by quantitative real-time PCR relative to two reference genes. The expression of osteoblast-associated genes (alkaline phosphatase, osteopontin, and osteocalcin)was detected in hMSCs that underwent adipogenesis. When normalized, the expression of adipocyte marker genes (adiponectin, fatty acid binding protein P4, and leptin) increasedin a time-dependent manner during adipogenic induction. Adiponectin and leptin were also detected in osteoblast-induced cells. Lipid vacuoles that represent the adipocyte phenotype were only present in the adipogenic induction group. Conforming to the heterogeneous nature of hMSCs and the known plasticity between osteogenic and adipogenic lineages, these data indicatea marker overlap between MSC-derived adipocytes and osteoblasts. Weproposea careful consideration of experimental conditions such as investigated timepoints, selected housekeeping genesand the evidence indicating lack of differentiation into other lineageswhen evaluating hMSC differentiation.
mesenchymal stem cell; differentiation markers; cell plasticity; differentiation
Stem Cell Derived Extracellular Matrix Enables Survival and Multi Lineage Differentiation within Superporous Hydrogels
Hauk, Thomas G.
Collins, John M.
Gemeinhart, Richard A.
Hydrophilic poly(ethylene glycol) diacrylate (PEGDA) hydrogel surfaces resist protein adsorption and are generally thought to be unsuitable for anchorage dependent cells to adhere. Intriguingly, our previous findings revealed that PEGDA superporous hydrogel scaffolds (SPHs) allow anchorage of bone marrow derived human mesenchymal stem cells (hMSCs) and support their long term survival. Therefore, we hypothesized that the physicochemical characteristics of the scaffold impart properties that could foster cellular responses. We examined if hMSCs alter their microenvironment to allow cell attachment by synthesizing their own extracellular matrix (ECM) proteins. Immunofluorescence staining revealed extensive expression of collagen type I, collagen type IV, laminin and fibronectin within hMSC-seeded SPHs by the end of the third week. Whether cultured in serum-free or serum-supplemented medium, hMSC ECM protein gene expression patterns exhibited no substantial changes. The presence of serum proteins is required for initial anchorage of hMSCs within the SPHs but not for the hMSC survival after 24 hours. In contrast to 2D expansion on tissue culture plastic (TCP), hMSCs cultured within SPHs proliferate similarly in the presence or absence of serum. To test whether hMSCs retain their undifferentiated state within the SPHs, cell-seeded constructs were cultured for 3 weeks in stem cell maintenance medium and the expression of hMSC-specific cell surface markers were evaluated by flow cytometry. CD105, CD90, CD73 and CD44 were present to a similar extent in the SPH and in 2D monolayer culture. We further demonstrated multi lineage potential of hMSCs grown in the PEGDA SPHs whereby differentiation into osteoblasts, chondrocytes and adipocytes could be induced. The present study demonstrates the potential of hMSCs to alter the “blank” PEGDA environment to a milieu conducive to cell growth and multi-lineage differentiation by secreting adhesive ECM proteins within the porous network of the SPH scaffolds.
Porous; hydrogels; scaffolds; poly(ethylene glycol); mesenchymal stem cells; extracellular matrix; cell proliferation; cell differentiation
In-house preparation of hydrogels for batch affinity purification of glutathione S-transferase tagged recombinant proteins
Buhrman, Jason S
Rayahin, Jamie E
Gemeinhart, Richard A
Many branches of biomedical research find use for pure recombinant proteins for direct application or to study other molecules and pathways. Glutathione affinity purification is commonly used to isolate and purify glutathione S-transferase (GST)-tagged fusion proteins from total cellular proteins in lysates. Although GST affinity materials are commercially available as glutathione immobilized on beaded agarose resins, few simple options for in-house production of those systems exist. Herein, we describe a novel method for the purification of GST-tagged recombinant proteins.
Glutathione was conjugated to low molecular weight poly(ethylene glycol) diacrylate (PEGDA) via thiol-ene “click” chemistry. With our in-house prepared PEGDA:glutathione (PEGDA:GSH) homogenates, we were able to purify a glutathione S-transferase (GST) green fluorescent protein (GFP) fusion protein (GST-GFP) from the soluble fraction of E. coli lysate. Further, microspheres were formed from the PEGDA:GSH hydrogels and improved protein binding to a level comparable to purchased GSH-agarose beads.
GSH containing polymers might find use as in-house methods of protein purification. They exhibited similar ability to purify GST tagged proteins as purchased GSH agarose beads.
Glutathione; PEGDA; Glutathione S-transferase; Batch purification; Recombinant protein
Results 1-3 (3)
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