Cyclic-RGD Peptides Increase the Adenoviral Transduction of Human Mesenchymal Stem Cells

Human mesenchymal stem cells (hMSCs) have been extensively explored for drug delivery applications due to their safety, immunomodulatory properties, and ability to differentiate into new tissues. The experiments presented in this study were designed to determine peptide-based mechanisms to increase the adenoviral transduction of hMSCs for the purpose of improving their capacity as drug delivery vehicles. Specifically, we demonstrated that cyclic- RGD peptides increased the internalization of adenoviruses into MSCs. MSCs treated with cyclic-RGD peptides had a transduction efficiency of 76.6%±4%, which was significantly greater than the 23.5%±12.2% transduction efficiency of untreated stem cells (P<0.05). Blocking endocytosis with inhibitors of dynamin or actin polymerization decreased the cyclic-RGD-mediated increase in transduction efficiency. MSCs treated with cyclic-RGD and adenoviruses carrying the gene for bone morphogenetic protein-2 produced significantly greater concentrations of this growth factor compared to stem cells treated with only adenoviruses or adenoviruses cocultured with cyclic-RAD peptides. Furthermore, this stem cell-produced bone morphogenetic protein induced alkaline phosphatase expression in C2C12 cells indicating growth factor bioactivity. Taken together, these studies suggest that cyclic-RGD peptides could be used to increase the adenoviral transduction of hMSCs and increase their therapeutic potential.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140200/1/scd.2012.0379.pd

Research and Discovery Science and the Future of Dental Education and Practice

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/153692/1/jddjde017040.pd

Ablation of Proliferating Marrow with 5‐Fluorouracil Allows Partial Purification of Mesenchymal Stem Cells

The ability to identify and maintain mesenchymal stem cells in vitro is a prerequisite for the ex vivo expansion of cells capable of effecting mesenchymal tissue regeneration. The aim of this investigation was to develop an assay to enrich and ultimately purify mesenchymal stem cells. To enrich the population of mesenchymal stem cell‐like cells, rats or mice were administered 5‐fluorouracil (5‐FU) in vivo. Limiting dilution analysis demonstrated that 5‐FU‐treated bone marrow had the potential to form colony‐forming units‐fibroblastic (CFU‐F) at a 10‐fold or sixfold enrichment compared to normal bone marrow in rats or mice, respectively. In vivo and in vitro differentiation assays supported the enrichment and purification effects. In vitro, bone marrow cultures from 5‐FU‐treated bone marrow demonstrated lineage‐specific gene expression in lineage‐specific medium conditions in contrast to the multilineage gene expression of control bone marrow cultures. In vivo implantation of 5‐FU‐treated cells that were not expanded in culture generated ossicles containing an intact bone cortex and mature hematopoietic components, whereas non‐5‐FU‐treated bone marrow only formed fibrous tissues. Our results demonstrate that enrichment of a quiescent cell population in the bone marrow by in vivo treatment of 5‐FU spares those undifferentiated mesenchymal stem cells and influences the differentiation of bone marrow stromal cells in vitro and in vivo. This prospective identification of a population of mesenchymal cells from the marrow that maintain their multilineage potential should lead to more focused studies on the characterization of a true mesenchymal stem cell.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90309/1/241573_ftp.pd

Mammalian EAK-7 activates alternative mTOR signaling to regulate cell proliferation and migration.

Nematode EAK-7 (enhancer-of-akt-1-7) regulates dauer formation and controls life span; however, the function of the human ortholog mammalian EAK-7 (mEAK-7) is unknown. We report that mEAK-7 activates an alternative mechanistic/mammalian target of rapamycin (mTOR) signaling pathway in human cells, in which mEAK-7 interacts with mTOR at the lysosome to facilitate S6K2 activation and 4E-BP1 repression. Despite interacting with mTOR and mammalian lethal with SEC13 protein 8 (mLST8), mEAK-7 does not interact with other mTOR complex 1 (mTORC1) or mTOR complex 2 (mTORC2) components; however, it is essential for mTOR signaling at the lysosome. This phenomenon is distinguished by S6 and 4E-BP1 activity in response to nutrient stimulation. Conventional S6K1 phosphorylation is uncoupled from S6 phosphorylation in response to mEAK-7 knockdown. mEAK-7 recruits mTOR to the lysosome, a crucial compartment for mTOR activation. Loss of mEAK-7 results in a marked decrease in lysosomal localization of mTOR, whereas overexpression of mEAK-7 results in enhanced lysosomal localization of mTOR. Deletion of the carboxyl terminus of mEAK-7 significantly decreases mTOR interaction. mEAK-7 knockdown decreases cell proliferation and migration, whereas overexpression of mEAK-7 enhances these cellular effects. Constitutively activated S6K rescues mTOR signaling in mEAK-7-knocked down cells. Thus, mEAK-7 activates an alternative mTOR signaling pathway through S6K2 and 4E-BP1 to regulate cell proliferation and migration

Failure to Attract and Retain Clinician/Scientist Faculty Puts Our Profession at Risk

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/67925/2/10.1177_00220345990780100101.pd

Substrate mineralization stimulates focal adhesion contact redistribution and cell motility of bone marrow stromal cells

Understanding the mechanisms of substrate based control of cell function is critical to the design of biomaterials. Cells interact with their extracellular matrix through cell adhesion contacts. We have previously described the self assembly of bone-like mineral onto an organic template and have shown that these biomimetic surfaces lead to an increased volume fraction of bone regenerated in vivo . In the present study, we compared the distribution of cell adhesion contacts, cell spreading, and cell motility of murine bone marrow stromal cells (BMSC) on mineralized vs. nonmineralized substrates. We developed a new approach for quantification of cell-material interactions and demonstrated that cell adhesion contacts on mineralized substrates were distributed throughout the cell surface contacting the substrate, whereas on nonmineralized substrates cell adhesion contacts were present near the cell periphery. We propose that mineralized substrates stimulate the predominant expression of fibrillar contacts, and nonmineralized substrates stimulate expression of focal adhesion contacts. Cell motility assays with colloidal gold demonstrated a statistically significant decrease in the average phagokinetic index of migrating cells on mineralized vs. nonmineralized substrates after 90 min of cell seeding. We propose that the physical–chemical properties of the substrate, altered by mineralization, cause expression of specific types of cell contacts and, as a result, modify molecular mechanisms responsible for cell spreading, motility, and possibly differentiation. © 2006 Wiley Periodicals, Inc. J Biomed Mater Res, 2006Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/55848/1/30786_ftp.pd

Role of Vascular Endothelial Growth Factor in Bone Marrow Stromal Cell Modulation of Endothelial Cells

One of the fundamental principles that underlies tissue-engineering strategies using cell transplantation is that a newly formed tissue must acquire and maintain sufficient vascularization in order to support its growth. Enhancing angiogenesis through delivery of growth factors is one approach to establishing a vascular network to these tissues. In this study, we tested the potential of bone marrow stromal cells (BMSCs) to modulate the growth and differentiation activities of blood vessel precursors, endothelial cells (ECs), by their secretion of soluble angiogenic factors. The growth and differentiation of cultured ECs were enhanced in response to exposure to BMSC conditioned medium (CM). Enzyme-linked immunosorbent assays demonstrated that both mouse and human BMSCs secreted significant quantities of vascular endothelial growth factor (VEGF) (2.4-3.1 ng/106 cells per day). Furthermore, eliminating the activity of BMSC-secreted VEGF with blocking antibodies completely blocked the CM effects on cultured ECs. These data demonstrate that human BMSCs secrete sufficient quantities of VEGF to enhance survival and differentiation of endothelial cells in vitro, and suggest they may be capable of directly orchestrating angiogenesis in vivo.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63256/1/107632703762687573.pd

Bone Marrow Stromal Cells: Characterization and Clinical Application

The bone marrow stroma consists of a heterogeneous population of cells that provide the structural and physiological support for hematopoietic cells. Additionally, the bone marrow stroma contains cells with a stem-cell-like character that allows them to differentiate into bone, cartilage, adipocytes, and hematopoietic supporting tissues. Several experimental approaches have been used to characterize the development and functional nature of these cells in vivo and their differentiating potential in vitro. In vivo, presumptive osteogenic precursors have been identified by morphologic and immunohistochemical methods. In culture, the stromal cells can be separated from hematopoietic cells by their differential adhesion to tissue culture plastic and their prolonged proliferative potential. In cultures generated from single-cell suspensions of marrow, bone marrow stromal cells grow in colonies, each derived from a single precursor cell termed the colony-forming unit-fibroblast. Culture methods have been developed to expand marrow stromal cells derived from human, mouse, and other species. Under appropriate conditions, these cells are capable of forming new bone after in vivo transplantation. Various methods of cultivation and transplantation conditions have been studied and found to have substantial influence on the transplantation outcome The finding that bone marrow stromal cells can be manipulated in vitro and subsequently form bone in vivo provides a powerful new model system for studying the basic biology of bone and for generating models for therapeutic strategies aimed at regenerating skeletal elements.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/68070/2/10.1177_10454411990100020401.pd

Tissue-Engineered Cartilage Constructs Using Composite Hyaluronic Acid/Collagen I Hydrogels and Designed Poly(Propylene Fumarate) Scaffolds

Our approach to cartilage tissue-engineering scaffolds combines image-based design and solid free-form (SFF) fabrication to create load-bearing constructs with user-defined parameters. In this study, 3-dimensional scaffolds with cubic and ellipsoidal pore architecture were fabricated using poly(propylene fumarate) (PPF). To increase seeding efficiency and cellular retention, hydrogels were used to deliver cells into the scaffolds. The first objective of this study was to evaluate the concentrations of composite hyaluronic acid (HyA) and collagen I hydrogels best able to stimulate proteoglycan synthesis in porcine chondrocytes in vitro and in vivo. The second objective was to evaluate the differences in extracellular matrix production due to pore geometry and scaffold design. For the in vitro assessment, chondrocytes were encapsulated in collagen I hydrogels with varying concentrations of HyA. Hydrogels were cultured for 1 and 2 weeks, and then the sulfated glycosaminoglycan (sGAG) content was quantified using a dimethyl-methylene blue assay. The concentration of HyA best able to increase ECM synthesis was 5% HyA/collagen I, or 0.23 mg/mL HyA. The results from the in vitro experiment were used as culture parameters for the in vivo analysis. Composite 5% HyA/collagen I or collagen I-only hydrogels were used to seed chondrocytes into SFF-fabricated scaffolds made of PPF with designed cubic or ellipsoidal pore geometry. The scaffolds were implanted subcutaneously in immunocompromised mice for 4 weeks. Histomorphometric analyses of sections stained with Safranin O were used to quantify the amount of ECM deposited by cells in the scaffolds. Scaffolds seeded with 5% HyA/collagen hydrogels had significantly greater areas of positive Safranin O staining (approximately 60%, compared with 30% for scaffolds with collagen I hydrogels only), indicating that greater numbers of chondrocytes retained their metabolic activity in the ectopic environment. These scaffolds also had greater stain intensities (corresponding to greater amounts of sGAG in the ECM) than their counterparts seeded with collagen I hydrogels alone. Significant differences in matrix production were not found between the scaffold pore designs. Overall, these results indicate that a combination of composite HyA hydrogels and designed SFF scaffolds could provide a functional tissue-engineered construct for cartilage repair with enhanced tissue regeneration in a load-bearing scaffold.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63183/1/ten.2006.0117.pd