Maintenance and propagation of mesenchymal stem cells
Inventors
Chen, Xiao-Dong • Jilka, Robert L.
Assignees
US Department of Veterans Affairs • University of Arkansas at Little Rock
Publication Number
US-8388947-B2
Publication Date
2013-03-05
Expiration Date
2027-01-22
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Abstract
Various embodiments of the present invention include compositions, materials and methods for maintaining and propagating mammalian mesenchymal stem cells in an undifferentiated state in the absence of feeder cells and applications of the same.
Core Innovation
The invention provides compositions, materials, and methods for maintaining and propagating mammalian mesenchymal stem cells (MSCs) in an undifferentiated state in vitro without the need for feeder cells. A key aspect is the use of an extracellular matrix (ECM) produced by marrow stromal cells, which reconstitutes the MSC niche that supports self-renewal and retention of multipotentiality of MSCs. This ECM comprises components such as type I, III, and V collagens, syndecan-1, fibronectin, decorin, biglycan, perlecan, and laminin, forming a three-dimensional structure that promotes MSC proliferation and prevents spontaneous differentiation.
The problem addressed by the invention is the difficulty in expanding MSCs ex vivo due to their rarity in bone marrow and their tendency to lose stem cell properties under traditional culture conditions, such as plastic substrates. Standard culture systems lack the specialized microenvironment or niche cues present in vivo that regulate MSC self-renewal and multipotentiality. The invention demonstrates that culturing MSCs on a cell-free, stromal cell-derived ECM preserves stemness, restrains osteoblastic differentiation, and enables effective expansion of functional MSCs for therapeutic and research applications.
The invention further shows that the stromal cell-derived ECM can sequester endogenous pro-osteoblastic factors, such as BMP2, thereby restricting spontaneous MSC differentiation. MSCs cultured on this ECM maintain responsiveness to exogenous differentiation signals, allowing controlled lineage specification. Moreover, MSCs expanded on this ECM exhibit enhanced bone and hematopoietic marrow formation in vivo compared to MSCs cultured on plastic, demonstrating improved functional capacity for bone regeneration and marrow tissue formation.
Claims Coverage
The patent contains one independent claim defining a composition for maintaining mammalian mesenchymal stem cells in an undifferentiated state, focusing on the extracellular matrix composition and its functional properties.
Composition comprising an in vitro extracellular matrix with specific components
The composition includes an extracellular matrix comprising type I collagen, type III collagen, type V collagen, syndecan-1, fibronectin, decorin, biglycan, perlecan, and laminin that maintains mammalian mesenchymal stem cells in culture in an undifferentiated state.
Extracellular matrix restraining mesenchymal stem cell differentiation
The extracellular matrix functions to restrain differentiation of mammalian mesenchymal stem cells, thereby preserving their stemness during in vitro culture.
Extracellular matrix is essentially free of feeder cells
The composition is essentially free of feeder cells, allowing MSC maintenance without support from other cell types.
Extracellular matrix derived from marrow stromal cells
The extracellular matrix is a marrow stromal cell derived extracellular matrix prepared by culturing marrow stromal cells, lysing them, and removing cell remnants through washing.
Three-dimensional structure of the extracellular matrix
The extracellular matrix presents a three-dimensional configuration that supports MSC propagation and maintenance.
Extracellular matrix comprising a BMP2 activity inhibitor
The extracellular matrix includes components that inhibit BMP2 activity, contributing to the maintenance of MSCs in an undifferentiated state.
The independent claim covers a composition containing a three-dimensional, marrow stromal cell-derived extracellular matrix with specific ECM proteins that restrain differentiation of mesenchymal stem cells, enabling their maintenance in culture without feeder cells and including inhibition of BMP2 activity as a functional feature.
Stated Advantages
Maintenance and propagation of mammalian mesenchymal stem cells in an undifferentiated state without feeder cells.
Promotion of MSC self-renewal and retention of multipotentiality during culture.
Inhibition of spontaneous osteoblastic differentiation of MSCs via sequestration of BMP2 by the ECM.
Enhanced proliferation and colony-forming capacity of MSCs compared to traditional plastic or 2D ECM substrates.
Improved in vivo bone and hematopoietic marrow formation by MSCs expanded on the stromal cell-derived ECM.
Provision of a defined, cell-free culture substrate that mimics the MSC niche for reliable ex vivo expansion.
Documented Applications
Ex vivo expansion and maintenance of functional mammalian mesenchymal stem cells for therapeutic use.
Preparation of bone forming compositions combining MSCs cultured on the ECM with transplantation vehicles such as hydroxyapatite/tricalcium phosphate for bone regeneration.
Treatment of bone-related conditions in patients, including fracture repair, delayed unions, non-unions, distraction osteogenesis, osteotomy, osseointegration, and osteoarthritis.
Use in research to identify ECM components affecting MSC behavior and studying the effects of aging or hormonal changes on MSC function.
Generation of bone in patients by obtaining MSCs, expanding them on the ECM in vitro, and administering the bone forming composition.
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