Methods for generation of pluripotent and multipotent cells
Inventors
Roberts, David D. • Kaur, Sukhbir • Isenberg, Jeffrey S.
Assignees
US Department of Health and Human Services
Publication Number
US-10407665-B2
Publication Date
2019-09-10
Expiration Date
2033-04-09
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Abstract
This disclosure relates to methods of producing induced pluripotent (iPS), multipotent, and/or lineage-committed stem cells from differentiated cells, maintaining iPS, multipotent, and/or lineage-committed cells in culture, and re-differentiating the iPS and multipotent stem cells into any desired lineage-committed cell type.
Core Innovation
This invention relates to methods for producing induced pluripotent (iPS), multipotent, and/or lineage-committed stem cells from differentiated cells, maintaining these cells in culture, and re-differentiating iPS and multipotent stem cells into any desired lineage-committed cell type. The methods are based on the blockade of cellular receptor CD47 signaling, which results in increased cellular lifespan and expansion of lineage-committed or differentiated cells in culture, and production of multipotent or iPS cells when cultured in appropriate media such as serum-free medium.
The problem solved by this invention stems from the challenges in regenerative medicine related to obtaining abundant and safe sources of pluripotent or multipotent stem cells. Embryonic stem (ES) cells are limited by possible immune rejection upon transplantation. Induced pluripotent stem (iPS) cells, generated by viral or plasmid expression of four stem cell transcription factors (c-Myc, Sox2, Klf4, Oct4), carry risks such as malignant transformation due to sustained c-Myc expression and insertional mutagenesis from viral integration. Attempts to replace gene expression approaches with small molecules have failed to identify any single agent capable of inducing iPS cells, revealing a need for agents that can produce stem cells without viral or plasmid-mediated gene expression.
The disclosed methods provide a non-genetic approach to generate and expand pluripotent, multipotent, and lineage-committed stem cells by blocking CD47 signaling, including blockade of its ligand thrombospondin-1 (TSP1). CD47 blockade can be achieved by contacting the cells with TSP1-derived peptides, anti-CD47 or anti-TSP1 antibodies, antisense oligonucleotides or morpholinos, or small molecules that inhibit CD47 expression or signaling. The blockade increases expression of stem cell transcription factors c-Myc, Sox2, Klf4, and Oct4, which supports self-renewal and de-differentiation. Subsequently, multipotent or iPS cells can be differentiated into desired lineages by culturing in differentiation media, while maintenance of these states can be controlled by continued, transient, or intermittent CD47 blockade.
Claims Coverage
This patent contains several independent claims directed to methods of inducing multipotent or lineage-committed stem cells from primary endothelial cells, generating differentiated cells from such stem cells, and methods specifying the composition of CD47 signaling blockers.
Induction of multipotent or lineage-committed stem cells by CD47 blockade
Obtaining primary endothelial cells from a mammal, transferring them into serum-free media, and contacting these cells with an agent that blocks CD47 signaling so as to induce multipotent or lineage-committed stem cells.
Identification and isolation of stem cells expressing key transcription factors
Identifying and isolating a subset of induced multipotent or lineage-committed stem cells that express stem cell markers including c-Myc, Sox2, Klf4, and Oct4.
Use of specific agents to block CD47 signaling for cell induction and differentiation
Agents capable of blocking CD47 signaling include anti-CD47 antibodies or fragments retaining specific binding activity, CD47-binding peptides, CD47 antisense oligonucleotides, CD47 morpholinos, anti-TSP1 antibodies or fragments thereof, TSP1-binding peptides, TSP1 antisense oligonucleotides, or TSP1 morpholinos.
Generation of differentiated cells from endothelial-derived stem cells
Obtaining primary endothelial cells, culturing and contacting them with CD47 signaling blockers to induce multipotent or lineage-committed stem cells, isolating these cells expressing specific stem cell markers, and culturing them in differentiation media to produce differentiated cells of various lineages.
Induction of embryoid-like multipotent cells without tumorigenicity
Inducing endothelial cells with serum-free media and contacting with CD47 signaling blockers to generate embryoid-like multipotent cells expressing c-Myc, Sox2, Klf4, Oct4, and lineage markers including neuronal, hepatocyte, and adipocyte markers, but which do not form teratomas.
The claims cover methods of inducing multipotent or lineage-committed stem cells and differentiated cells from primary endothelial cells via CD47 signaling blockade using specified molecular agents, with steps for identifying stem cell markers and differentiating cells into various lineages. These methods enable generation of stem cells exhibiting key transcription factor expression and embryoid body formation without malignant transformation.
Stated Advantages
The disclosed methods avoid the risk of malignant transformation associated with viral or plasmid-mediated expression of individual stem cell-inducing genes such as c-Myc.
Use of a single defined agent to generate and maintain pluripotent or multipotent stem cells greatly expands potential applications of autologous stem cell therapy.
The methods enable continuous expansion of stem cells or differentiated cells ex vivo without transformation or modification by viral vectors, suitable for clinical use.
CD47 blockade allows increased cellular lifespan, proliferation, and reprogramming capabilities in primary cells to generate stem cells.
CD47 signaling blockade facilitates repopulation and restoration of decellularized tissue matrices and enhances tissue regeneration including nephrogenesis and tracheal reconstruction.
Documented Applications
Ex vivo generation and expansion of induced pluripotent or multipotent stem cells or lineage-committed differentiated cells for cell-based therapies and tissue engineering.
Populating decellularized natural or synthetic tissue matrices or scaffolds with stem cells or differentiated cells for bioengineered organs or tissues suitable for transplantation.
Cell transplantation therapies including expansion of pancreatic islet cells, hematopoietic stem cells for marrow transplantation, cytotoxic T cells for adoptive immunotherapy, and other lineage-specific cells.
Direct administration of CD47 signaling inhibitors to enhance tissue regeneration in wounds, burns, bone fractures, and delayed healing conditions.
Treatment of diseases involving vision loss, such as retinal degeneration, macular degeneration, and glaucoma by administration of stem cells or CD47 inhibitors to the eye.
Therapeutic expansion or generation of stem cells in vivo by administering CD47 signaling inhibitors to subjects with tissue damage, genetic defects, diabetes, or hair loss like alopecia.
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