Isolation and use of pluripotent stem cell population from adult neural crest-derived tissues
Inventors
Cheung, Herman S. • Pelaez, Daniel • Huang, C-Y Charles
Assignees
University of Miami • US Department of Veterans Affairs
Publication Number
US-10106773-B2
Publication Date
2018-10-23
Expiration Date
2033-03-01
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Abstract
The present invention relates to methods of isolating a substantially homogeneous population of pluripotent stem cells from adult neural crest tissue (e.g., periodontal ligament) as well as pharmaceutical compositions comprising such isolated pluripotent stem cells. Methods of inducing the isolated pluripotent stem cells into specific cell lineages, such as neurogenic and retinogenic lineages, are also described. The isolated pluripotent stem cells find use in various regenerative medicine applications and the treatment of degenerative diseases.
Core Innovation
The invention provides methods of isolating a substantially homogeneous population of pluripotent stem cells from adult neural crest-derived tissues, such as adult periodontal ligament, based on the discovery that connexin 43 can be used as a selection marker for such pluripotent stem cells. The isolated pluripotent stem cells are capable of differentiating into cells of all three germ layers: ectoderm, mesoderm, and endoderm, and are characterized by expression of stem cell markers (e.g., Oct4, Nanog, Sox2, Klf4) and neural crest markers (e.g., p75 neurotrophin receptor, Nestin, Sox10, N-Cadherin, Slug). These cells can be used in regenerative medicine and treatment of degenerative diseases.
The problem addressed by the invention lies in the current limitations and drawbacks of pluripotent stem cell sources, including ethical dilemmas and immunological concerns with embryonic stem cells, and complications in induced pluripotent stem cell (iPSC) technology such as aberrant epigenetic changes. Although adult tissues were known to contain remnant embryonic-like cells, including from neural crest-derived tissues like periodontal ligament, there was a need for methods to isolate a homogeneous population of pluripotent stem cells and to condition these cells for differentiation into desired lineages for regenerative applications.
The invention further provides methods for differentiating the isolated pluripotent stem cells into specific lineages, including neural progenitor cells or neural cells by exposure to growth factors such as epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), and retinal progenitor cells or retinal cells by treatment with antagonists of BMP and Wnt/beta-catenin signaling pathways such as Noggin and Dkk-1, optionally with insulin-like growth factor-1 (IGF-1). These differentiation methods enhance the applicability of the isolated cells in targeted tissue repair and treatment of degenerative conditions.
Claims Coverage
The patent includes two independent claims covering methods of isolating pluripotent stem cells and methods of repairing damaged tissue using these cells. The inventive features relate to cell extraction, culturing, selection, and therapeutic applications.
Method of isolating pluripotent stem cells from adult neural crest-derived tissue
The method comprises extracting cells from adult neural crest-derived tissue, specifically adult periodontal ligament or dental pulp from exfoliated deciduous teeth, culturing said cells under adherent conditions, and isolating the cultured cells expressing Connexin-43 using a reagent with affinity for Connexin-43, thereby obtaining a population comprising at least 80% pluripotent stem cells.
Screening for marker expression prior to isolation
The method optionally includes screening extracted cells for expression of Connexin-43, at least one stem cell marker or at least one neural crest marker prior to isolation. The stem cell or neural crest markers include Oct4, Nanog, Sox2, Klf4, p75 neurotrophin receptor, Nestin, Sox10, N-Cadherin, Notch1, BMP2, Snail, Slug, or combinations thereof.
Capability of isolated stem cells to differentiate and form teratomas
The isolated stem cells are capable of differentiating into cells of ectoderm, endoderm, and mesoderm lineages and can generate teratomas when injected into immunodeficient mice.
Method of repairing damaged tissue using isolated pluripotent stem cells
The method involves administering to a subject in need pluripotent stem cells isolated by the described method. The stem cells can be autologous. The damaged tissue includes neural tissue, retinal tissue, skeletal muscle tissue, cardiac tissue, bone, or cartilage.
The claims comprehensively cover the isolation of pluripotent stem cells from specific adult neural crest-derived tissues using Connexin-43 selection, characterization of these cells’ pluripotency, and their therapeutic use in repairing various damaged tissues, highlighting the novelty in both cell isolation and application methods.
Stated Advantages
Provides a method to isolate a substantially homogeneous population of pluripotent stem cells from adult neural crest-derived tissues using Connexin-43 as a selection marker.
Enables generation of patient-specific pluripotent stem cell lines from easily accessible adult tissues, overcoming ethical and immunological issues associated with embryonic and induced pluripotent stem cells.
Facilitates differentiation of isolated pluripotent stem cells into specific lineages (neural and retinal), allowing tailored regenerative medicine applications.
The isolated stem cells exhibit pluripotency demonstrated by differentiation into all three germ layers and teratoma formation, confirming their potential for versatile tissue repair.
Documented Applications
Regenerative medicine applications including repair of damaged tissue due to injury, ischemic events, or degenerative diseases.
Treatment of degenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, osteoarthritis, and macular degeneration.
Neural tissue repair including treatment of neurodegenerative disorders, brain injuries such as stroke, and spinal cord injuries.
Retinal tissue repair and treatment of ocular diseases including retinitis pigmentosa, age-related macular degeneration, glaucoma, and diabetic retinopathy.
Repair and regeneration of skeletal muscle tissue, cardiac tissue, bone, and cartilage.
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