Nutrient broth comprising multipotent cells for use in propagation of mesodermal, neuroectodermal, and endodermal cells; tissue engineering
Inventors
Schiller, Paul C. • D'Ippolito, Gianluca
Assignees
GOVERNMENT OF United States, VETERANS AFFAIRS THE, Secretary of, Department of • University of Miami • US Department of Veterans Affairs
Publication Number
US-7807458-B2
Publication Date
2010-10-05
Expiration Date
2024-01-29
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Abstract
Described herein are post natal, multilineage inducible cells (MIAMI cells). In some examples, the cells are isolated from non fractionated, adult (males and females 3 to 72 years old) human bone marrow under cell culture conditions, which are believed to resemble an in vivo niche microenvironment in which primitive multipotent cells exist. MIAMI cells have a unique profile of molecular markers, and can be maintained in vitro (for more than 50 population doublings) without detectable changes in their characteristic molecular profile. MIAMI cells can be differentiated into mesodermal, neuroectodermal, and endodermal cell lineages. Methods of isolating, differentiating and using MIAMI cells are also described.
Core Innovation
The invention discloses isolated, post-natal, multilineage-inducible cells called MIAMI cells, which can be isolated from adult human bone marrow and other biological samples. These cells have a unique molecular marker profile, including positive expression of CD29, CD81, CD90, CD122, CD164, c-Met, BMP-receptor 1B, NTRK3, Oct-4, and Rex-1, while lacking expression of markers such as CD13, CD34. MIAMI cells are small, highly proliferative, and can be maintained in vitro for over 50 population doublings without loss of their characteristic phenotype or differentiation potential. They can differentiate into mesodermal, neuroectodermal, and endodermal lineages, including osteoblasts, chondrocytes, adipocytes, neurons, and pancreatic β-like cells.
The problem solved addresses the need for multipotent post-natal human cells that maintain a broad and multilineage differentiation capacity similar to embryonic stem cells but without the associated ethical and legal concerns. Prior adult stem cell populations often lacked the plasticity to differentiate into multiple germ layer-derived cell types, and established isolation methods commonly included cell fractionation that reduced yields. The invention provides cell culture methods that mimic the in vivo niche conditions—including low oxygen tension, extracellular matrix substrates, and co-culture with non-adherent cells—that allow reproducible isolation and expansion of MIAMI cells with extensive differentiation capacities.
Methods for isolating, culturing, and inducing differentiation of MIAMI cells are detailed, including low oxygen (about 3%) culture conditions, use of fibronectin-coated substrates, and co-culture periods with non-adherent cells to enhance selection. Differentiation protocols into osteogenic, chondrogenic, adipogenic, neural, and endodermal lineages are provided, employing media supplemented with specific factors and sequential exposure steps to induce lineage-specific phenotypes. The invention includes compositions of these isolated cells, pharmaceutical formulations, and methods for their therapeutic application and screening of differentiation-inducing agents.
Claims Coverage
The claims cover compositions comprising isolated post-natal multilineage-inducible cells with specific marker profiles, methods of inducing their differentiation into multiple lineages, methods of isolating the cells under defined conditions, and pharmaceutical compositions and kits containing such cells. Eleven main inventive features are identified.
Composition of multilineage-inducible cells with defined marker expression
The composition comprises isolated, post-natal, multilineage-inducible cells expressing at least CD29, CD81, CD90, CD122, and CD164, and not expressing CD13 or CD34; optionally further expressing c-Met, BMP receptor 1B, or NTRK3.
Methods of inducing osteogenic differentiation
Methods comprise culturing multilineage-inducible cells in osteogenic medium containing ascorbic acid 2-phosphate, β-glycerophosphate, and dexamethasone, optionally adding dexamethasone after cells adhere; osteogenic differentiation is indicated by expression of Runx2, osteocalcin, collagen I α1, or bone sialoprotein.
Methods of inducing chondrogenic differentiation
Methods comprise culturing multilineage-inducible cells in a serum-free chondrogenic medium containing dexamethasone, TGF-β3, ascorbic acid 2-phosphate, sodium pyruvate, proline, insulin, transferrin, and selenous acid, with pelleting of cells and culture for at least 4 to 6 weeks; differentiation includes collagen expression.
Methods of inducing adipogenic differentiation
Methods comprise culturing multilineage-inducible cells in adipogenic medium comprising hydrocortisone, isobutylmethylxanthine, and indomethacine; adipogenic differentiation involves expression of lipoprotein lipase or peroxisome proliferator-activated receptor γ-2.
Methods of inducing neural differentiation
Methods comprise sequential steps: (a) contacting cells with bFGF-containing medium; (b) then with medium comprising β-mercaptoethanol and NT-3; and (c) then with medium comprising NT-3, NGF, and BDNF; optionally plating cells at low density on extracellular matrix substrate before step (a).
Methods of inducing endodermal differentiation
Methods comprise sequential steps: (a) contacting cells with bFGF-containing medium; (b) then with medium comprising DMSO, BHA, and exendin-4; (c) then with medium comprising bFGF, EGF, and exendin-4; and (d) then with medium comprising nicotinamide, HGF, exendin-4, and activin-A.
Methods of identifying differentiation-inducing agents
Methods comprise providing multilineage-inducible cells, contacting them with an agent, and observing cellular differentiation to osteogenic, chondrogenic, adipogenic, neural, endodermal, or combined lineages to identify differentiation-inducing agents.
Methods of isolating multilineage-inducible cells under low-oxygen conditions
Methods comprise culturing a cell population from biological samples (bone marrow, vertebral bodies, peripheral blood, umbilical cord blood, iliac crest aspirate, fat, cartilage, muscle, skin, bone, teeth, liver, brain or mixtures) under low oxygen (≤3%) to produce adherent and non-adherent cells, removing non-adherent cells, and expanding adherent cells expressing CD29, CD81, CD90, CD122, CD164 and lacking CD13 or CD34, optionally co-cultured at least 7 days on extracellular matrix substrate.
Purified multilineage-inducible cell populations
Isolated post-natal multilineage-inducible mammalian cells expressing CD29, CD81, CD90, CD122, and CD164 but not CD13 or CD34, optionally expressing SSEA4, Oct-4, and Rex-1, isolated from biological samples including bone marrow and human postmortem tissue.
Pharmaceutical compositions and kits containing multilineage-inducible cells
Pharmaceutical compositions comprising the multilineage-inducible cells in pharmaceutically acceptable carriers and kits comprising purified populations of multilineage-inducible cells, optionally including growth factors, culture media, and instructions for use.
Use of cells and differentiated derivatives for therapeutic methods
Methods of administering therapeutically effective amounts of multilineage-inducible cells or their differentiated progeny locally or systemically for treatment of diseases or conditions involving bone, cartilage, neural tissue, diabetes, or adipose tissue augmentation.
The claims collectively cover novel isolated post-natal multilineage-inducible cells with defined marker expressions, methods to isolate and expand them under low oxygen and extracellular matrix conditions, their differentiation into multiple lineage cells via specific culture media and sequential protocols, pharmaceutical compositions and kits containing these cells, and use of the cells and their derivatives for therapeutic purposes and agent screening.
Stated Advantages
MIAMI cells maintain extensive proliferative capacity in vitro (>50 population doublings) without loss of characteristic molecular profile or differentiation capacity.
MIAMI cells have broad multilineage differentiation potential resembling embryonic stem cells, including differentiation into mesodermal, neuroectodermal, and endodermal lineages.
MIAMI cells can be reproducibly isolated using culture conditions mimicking in vivo niche microenvironment, improving efficiency over traditional fractionation techniques.
Neural differentiation yields mature neurons with electrophysiological properties without requiring co-culture with other cell types.
The invention provides ethically favorable post-natal stem cells with high plasticity as alternatives to embryonic stem cells for research and therapy.
Documented Applications
Treatment of neurological disorders including Parkinson's disease, Huntington's disease, Alzheimer's disease, epilepsy, familial dysautonomia, schizophrenia, amyotrophic lateral sclerosis, Lewy body dementia, multiple sclerosis, cerebellar ataxia, progressive supranuclear palsy, anxiety disorder, obsessive compulsive disorder, attention deficit disorder, Tourette Syndrome, Tay Sachs, Nieman Pick, HIV encephalopathy, spinal cord injury, and stroke.
Treatment of bone disorders including arthritis, osteoporosis, osteosclerotic metaphyseal dysplasia, osteomyelitis, Paget's disease of bone, hypophosphatasia, osteopetrosis, osteomalacia, and bone fractures.
Treatment of cartilage disorders including arthritis, pseudoachondroplasia, articular cartilage degeneration, osteogenesis imperfecta, and cartilage tears.
Fat augmentation applications such as cosmetic surgery to augment lips, breasts, cheeks, brows, to fill wrinkles, or to correct scarring.
Treatment of diabetes by administration of MIAMI cells or differentiated pancreatic β-like cells to restore insulin-producing capacity.
Use in tissue engineering of bone and cartilage in vitro.
Screening agents for differentiation-inducing capacity on multilineage-inducible cells.
Interested in licensing this patent?