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-10676718-B2
Publication Date
2020-06-09
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 without the use of feeder cells. Specifically, it involves culturing MSCs on a cell-free three-dimensional extracellular matrix (ECM) derived from marrow stromal cells, which recreates the MSC niche that supports their self-renewal capability and multipotentiality while allowing differentiation upon appropriate signals.
The problem being solved is that MSCs are extremely rare in bone marrow, and conventional ex vivo expansion has proven difficult because MSCs tend to lose their stem cell properties under traditional cell culture conditions. This loss is attributed to the absence of the specialized marrow microenvironment or niche in culture systems. Previously, attempts to culture MSCs on plastic or two-dimensional ECM coatings failed to maintain their undifferentiated state and multipotentiality efficiently.
Claims Coverage
The patent contains one independent claim focusing on a method for generating bone in a patient using cultured mesenchymal stem cells on a specific extracellular matrix. The claim covers the composition of the matrix, sources of MSCs, methods of manufacture, and administration to patients.
Use of a three-dimensional marrow stromal cell derived extracellular matrix for MSC culture
The method involves culturing mammalian mesenchymal stem cells on a three-dimensional marrow stromal cell derived extracellular matrix that includes type I collagen, type III collagen, type V collagen, syndecan-1, fibronectin, decorin, biglycan, perlecan, and laminin to produce an undifferentiated MSC composition.
Production of bone forming composition combining cultured MSCs with a transplantation vehicle
After culturing MSCs on the extracellular matrix and isolating undifferentiated cells, these cells are combined with a transplantation vehicle to produce a bone forming composition.
Administration of bone forming composition to a patient
The method includes administering the bone forming composition locally or systemically to a patient in need of bone generation, including human and aging human patients.
Manufacture of extracellular matrix by lysing and removal of marrow stromal cells
The extracellular matrix is manufactured by culturing marrow stromal cells on a substrate, lysing these cells, and removing them by washing to leave a cell-free matrix suitable for MSC culture.
MSC selection from human or murine sources and autologous use
The MSCs used in the method are selected from human and murine sources and may be autologous to the patient receiving the bone forming composition.
The independent claim covers a method for generating bone that comprises culturing MSCs on a specific three-dimensional marrow stromal cell derived ECM to maintain their undifferentiated state, combining these cells with a transplantation vehicle, and administering the composition to a patient, highlighting features of the ECM composition, manufacture, MSC sources, and administration routes.
Stated Advantages
The marrow stromal cell derived extracellular matrix promotes self-renewal of MSCs and maintains them in an undifferentiated and multipotent state without feeder cells.
The ECM prevents spontaneous differentiation of MSCs during in vitro culture, preserving their ability to respond to differentiation signals.
MSCs expanded on the ECM form more bone and hematopoietic marrow in vivo after transplantation compared to those expanded on plastic or other matrices.
The described system enables expansion of functional MSCs ex vivo, facilitating practical applications including bone regeneration.
Documented Applications
Expansion and maintenance of mammalian mesenchymal stem cells in vitro in an undifferentiated state for research and therapeutic uses.
Production of bone forming compositions by culturing MSCs on marrow stromal cell derived ECM and combining them with transplantation vehicles such as hydroxyapatite/tricalcium phosphate ceramics.
Generation of bone in patients needing bone formation, including treatment of fractures, delayed unions, non-unions, distraction osteogenesis, osteotomy, osseointegration, and osteoarthritis.
Subcutaneous transplantation of MSCs expanded on marrow stromal cell derived ECM into immunocompromised mice to generate bone and hematopoietic marrow as an in vivo model.
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