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PARK CITY, UT, United States

Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 106.85K | Year: 2010

DESCRIPTION (provided by applicant): An unmet need for expansion of primary cells and progenitor cells in three dimensions (3-D) is an extracellular matrix (ECM) substitute with user-controllable composition and compliance to which ECM proteins or synthetic peptides can be covalently attached by the end user. Glycosan BioSystems currently markets HyStem, an in situ-crosslinkable, semi-synthetic ECM composed of poly(ethylene glycol) diacrylate (PEGDA) and crosslinked thiol-modified hyaluronan (Glycosil) for culture of a variety of pluripotent cell types. The elastic modulus of the matrix determines to a large extent cell fate, and can be varied by changing crosslink density and concentration. In Phase I we will test the feasibility of a simple system for attachment of proteins to the hydrogels that would permit any synthetic polypeptide or any native matricellular protein (MP) to be covalently attached to the thiol-modified HA component of HyStem. In contrast to adding native ECM proteins that are not bound to the gel, and thus cannot provide mechanical support, this approach directly couples the pull of the cells on the proteins to the elastic modulus of the gel. We will compare MP-attached gels with the gelatin-containing HyStem-C hydrogel to identify conditions that maintain quiescence of mesenchymal stem cell (MSC) and that improve MSC differentiation capacity into adipocytes. Following the optimization of protein attachment conditions, we will determine the feasibility of this approach for growing MSCs in or on HyStem- MP hydrogels as autologous, animal-free feeder layers for the culturing of CD34+ stem cells derived from bone marrow. PUBLIC HEALTH RELEVANCE: An unmet need for expansion of primary cells and progenitor cells in three dimensions (3-D) is an extracellular matrix (ECM) substitute with user-controllable composition and compliance to which ECM proteins or synthetic peptides can be covalently attached by the end user. Glycosan BioSystems currently markets HyStem , an in situ-crosslinkable, semi-synthetic ECM composed of poly(ethylene glycol) diacrylate (PEGDA) and crosslinked thiol-modified hyaluronan (Glycosil ) for culture of a variety of pluripotent cell types. The goal of this proposal is to test the feasibility of a simple system for covalent attachment of extracellular matrix proteins to the HyStem hydrogel and to tailor the protein composition and stiffness for modulating the quiescence or differentiation of human mesenchymal stem cells.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 131.37K | Year: 2009

DESCRIPTION (provided by applicant): The realization of the remarkable potential of human embryonic stem cells or induced pluripotent stem cells to treat human disease cannot occur unless new animal-free methods for the expansion of undifferentiated stem cells are developed. An optimal extracellular matrix (ECM) substitute for the expansion of human embryonic stem cells (hESCs) in three dimensions (3-D) would be a soft hydrogel that includes two key features. First, the hydrogel should be composed of animal-free components to which researcher-provided ECM proteins or synthetic peptides can be covalently attached. Second, after multiple hESC divisions in an undifferentiated state, the hydrogel should allow rapid recovery of the expanded cell numbers without altering stemness. Glycosan BioSystems currently markets HyStem , an in situ-crosslinkable, semi-synthetic ECM composed of poly(ethylene glycol) diacrylate (PEGDA) and crosslinked thiol-modified hyaluronan ( Glycosil ) for culture of a variety of pluripotent cell types. In Phase I we will test the feasibility of a simple system for attachment of proteins to the hydrogels that would covalently bind any selected human matricellular protein to the thiol-modified HA component of HyStem. This approach directly couples the pull of the cells on the proteins to the elastic modulus of the gel. In our first Aim, we would establish the optimal combination of covalently-linked human collagen, vitronectin, fibronectin, and laminin (CVFL) proteins, as well as the minimum amounts required, for effective self-renewal of H9 hESCs. Cells will be recovered and analyzed for stem cell markers by flow cytometry. Second, for each combination that gave suitable cell growth, we will employ a new reductively-cleavable linker to form soft gels that will allow enzyme-free recovery of undifferentiated hESCs from the gel surface. After cell recovery with 25 mM NAc-Cys, stemness will be assessed as before. Third, H9 hESCs will be encapsulated in 3D in selected HyStem-CVFL hydrogels, expanded in 3-D, and analyzed. Finally, we will use optimized conditions to expand stem cells for a minimum of 6 passages, and compare the karyotypes and stem cell markers with cells cultured on murine fibroblast feeders, on Matrigel, or on CVFL- coated plastic. PUBLIC HEALTH RELEVANCE: The realization of the remarkable potential of human embryonic stem cells or induced pluripotent stem cells to treat human disease cannot occur unless new animal-free methods for the expansion of undifferentiated stem cells are developed. Glycosan BioSystems currently markets HyStem , an in situ-crosslinkable, semi- synthetic ECM composed of poly(ethylene glycol) diacrylate (PEGDA) and crosslinked thiol-modified hyaluronan (Glycosil ) for culture of a variety of pluripotent cell types. The goal of this proposal is to tailor HyStem for human embryonic stem cell culture by determining the optimal combination of covalently-bound attachment factors for proliferation without differentiation and by including a fall-apart crosslinker for gentle cell recovery.


Trademark
Biotime, Inc., Orthocyte Corporation and Glycosan Biosystems, Inc. | Date: 2010-11-16

Cell culture media, cell scaffold hydrogel for medical research or scientific research use; kit primarily containing thiol-modified hyaluronan, thiol modified gelatin and polyethylene glycol diacrylate for medical research or scientific research use.


Trademark
Biotime, Inc., Orthocyte Corporation and Glycosan Biosystems, Inc. | Date: 2010-11-16

Thiol-modified hyaluronan for medical research or scientific research use; kit primarily containing thiol-modified hyaluronan for medical research or scientific research use.


Trademark
Biotime, Inc., Orthocyte Corporation and Glycosan Biosystems, Inc. | Date: 2010-11-16

Thiol-modified hyaluronan and thiol-modified heparin for medical research or scientific research use.

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