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-8084023-B2
Publication Date
2011-12-27
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 culture in the absence of feeder cells. This is achieved by culturing MSCs on a specialized extracellular matrix (ECM) derived from marrow stromal cells, which resembles the bone marrow ECM and promotes self-renewal, retention of multipotentiality, and controls differentiation in response to signals.
The problem addressed is the difficulty in expanding MSCs ex vivo due to their rarity in bone marrow and their propensity to lose stem cell properties under standard culture conditions, commonly on tissue culture plastic. Traditional culture systems lack critical features of the in vivo marrow environment, particularly the specialized microenvironment or niche necessary to maintain MSCs' self-renewal and multipotentiality.
The marrow stromal cell-derived ECM comprises collagen types I, III, V, syndecan-1, perlecan, fibronectin, laminin, biglycan and decorin, and functions as a three-dimensional scaffold that uniquely supports MSC attachment, proliferation, and retention of undifferentiated state by sequestering pro-differentiation factors such as BMP2. MSCs cultured on this ECM show enhanced symmetric division producing identical daughter stem cells, in contrast to growth on plastic where differentiation toward osteoblast lineage predominates. Moreover, MSCs expanded on the ECM retain the capability to form bone tissue and support hematopoiesis in vivo post-transplantation.
Claims Coverage
The patent contains six independent claims focusing on compositions, matrices, methods, and devices for maintaining mesenchymal stem cells in an undifferentiated, proliferative state using a stromal cell-derived three-dimensional extracellular matrix.
Cellular composition comprising proliferating MSCs embedded in specific stromal cell-derived 3D ECM
The cellular composition includes proliferating, undifferentiated mammalian mesenchymal stem cells embedded within a stromal cell-derived three-dimensional extracellular matrix comprising collagen I, collagen III, syndecan-1, perlecan, fibronectin, laminin, biglycan, and decorin.
MSC composition essentially free of feeder cells
The cellular composition is essentially free of feeder cells, enabling MSC proliferation and maintenance without supporting feeder cell layers.
MSC identity selection
The MSCs in the composition can be selected from human or murine sources.
Capability of MSCs to undergo symmetrical division
The mammalian mesenchymal stem cells are capable of symmetrical division, facilitating self-renewal and expansion.
Use of marrow stromal cell-derived 3D ECM
The stromal cell-derived three-dimensional extracellular matrix is specifically a marrow stromal cell-derived matrix that supports MSC maintenance.
Method of manufacturing marrow stromal cell-derived ECM
The marrow stromal cell-derived 3D ECM is manufactured by culturing marrow stromal cells, lysing these cells, and removing the lysed cells by washing to obtain the cell-free extracellular matrix.
The independent claims cover cellular compositions of proliferating MSCs embedded in a stromal cell-derived 3D ECM, compositions and methods for maintaining MSCs in an undifferentiated state essentially free of feeder cells, the specific use of a marrow stromal cell-derived ECM, and methods for producing such ECM. The inventive features center on the ECM composition, maintenance of MSC stemness and proliferation, and the production of the matrix.
Stated Advantages
Promotion of self-renewal and proliferation of mammalian mesenchymal stem cells in culture.
Maintenance of MSCs in an undifferentiated state without the need for feeder cells.
Preservation of multipotentiality allowing differentiation into osteoblasts and adipocytes upon appropriate signals.
Restraint of spontaneous differentiation during culture, enhancing MSC expansion quality.
ECM sequesters pro-differentiation factors like BMP2, contributing to retention of stemness.
Expanded MSCs on marrow stromal cell-derived ECM generate significantly more bone and hematopoietic marrow in vivo post-transplantation compared to cells expanded on plastic.
Documented Applications
Ex vivo expansion and maintenance of undifferentiated mammalian mesenchymal stem cells for research or therapeutic use.
Generation of bone forming compositions comprising MSCs cultured on a marrow stromal cell-derived ECM combined with transplantation vehicles such as hydroxyapatite/tricalcium phosphate.
Treatment of conditions requiring bone formation in patients, such as fractures, delayed unions, non-unions, distraction osteogenesis, osteotomy, osseointegration, and osteoarthritis.
Use in transplantation therapy to promote bone development, remodeling, and hematopoiesis.
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