Methods for generation of pluripotent and multipotent cells
Inventors
Roberts, David D. • Kaur, Sukhbir • Isenberg, Jeffrey S.
Assignees
Usa Represented By Secretary Department Of Health And Human Services AS • US Department of Health and Human Services
Publication Number
US-11692175-B2
Publication Date
2023-07-04
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 disclosure 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 involve blocking CD47 signaling on cells, which significantly increases cellular lifespan and expansion in culture and induces production of multipotent or iPS cells when grown in appropriate media such as serum-free medium.
The background problem addressed is the challenge in regenerative medicine of obtaining abundant and safe sources of multipotent or pluripotent stem cells. While embryonic stem cell lines have potential, their use faces issues such as immune rejection. Induced pluripotent stem cells (iPS) offer advantages but are typically produced by viral or plasmid-mediated over-expression of transcription factors, which carries risks of malignant transformation due to sustained c-Myc expression or genome integration of vectors. Current efforts to produce iPS cells using small molecule mixtures have not identified a single agent capable of inducing iPS cells.
The disclosure reveals that blockade of CD47 signaling increases expression of c-Myc and stem cell-inducing transcription factors Sox2, Klf4, and Oct4, enabling generation and expansion of iPS and multipotent stem cells from primary or differentiated cells without the need for genetic manipulation. CD47 blockade can be achieved via various agents like TSP1-derived peptides, anti-CD47 or anti-TSP1 antibodies, antisense oligonucleotides, morpholinos, or small molecules that inhibit CD47 expression or function. The methods further encompass maintaining stem cells or differentiated cells in culture by exposure to CD47 blocking agents and differentiating the generated iPS or multipotent stem cells into desired cell types.
Claims Coverage
The patent discloses multiple inventive features across independent claims focused on methods for inducing multipotent or lineage-committed stem cells, generating differentiated cells from such stem cells, and the use of agents that block CD47 signaling.
Induction of multipotent or lineage-committed stem cells by CD47 signaling blockade
A method comprising obtaining primary cells from an animal, culturing these cells, and contacting them with an agent that blocks CD47 signaling to induce multipotent or lineage-committed stem cells.
Isolation of stem cells expressing key transcription factors
Identifying and isolating a subset of multipotent or lineage-committed stem cells expressing at least one of c-Myc, Sox2, Klf4, and Oct4 from the contacted cells.
Culture of primary cells in serum-free media to promote stem cell formation
Culturing primary cells in serum-free medium to facilitate the production of multipotent or lineage-committed stem cells that form embryoid bodies.
Use of various agents to block CD47 signaling
Employing agents that block CD47 signaling including anti-CD47 antibodies or fragments, CD47-binding peptides, CD47 antisense oligonucleotides or morpholinos, anti-TSP1 antibodies or fragments, TSP1-binding peptides, TSP1 antisense oligonucleotides or morpholinos, or small molecules binding CD47 or TSP1.
Induction of differentiated cells from stem cells generated by CD47 blockade
A method including producing differentiated cells by obtaining primary cells, culturing and contacting them with an agent blocking CD47 signaling, isolating multipotent or lineage-committed stem cells that express c-Myc, Sox2, Klf4, and/or Oct4, and culturing these stem cells in differentiation media to produce differentiated cells.
Differentiation into specific germ layer-derived lineages
Differentiating stem cells into ectoderm-derived lineages (neuronal cells, astrocytes), mesoderm-derived lineages (smooth muscle cells, endothelial cells, hematopoietic cells, myeloid cells), or endoderm-derived lineages (hepatocytes, adipocytes) by culturing in appropriate differentiation media.
Use of primary cells from various tissues
Methods apply to primary cells comprising endothelial cells, fibroblasts, hematopoietic cells, adipose cells, mucosal tissue cells, umbilical cord cells, or placenta cells, including human umbilical vein endothelial cells.
The claims cover methods of inducing multipotent or lineage-committed stem cells and generating differentiated cells by blocking CD47 signaling using diverse agents, from various primary cell types, with subsequent culture in specific media including serum-free and differentiation media. The features encompass identifying stem cell markers, forming embryoid bodies, and differentiation into all three germ layers.
Stated Advantages
The disclosed methods enable generation and expansion of iPS and multipotent stem cells without the need for plasmid- or retroviral-mediated gene expression, minimizing risks of malignant transformation.
Use of a single defined agent that blocks CD47 signaling simplifies iPS cell production and allows expansion of stem cells and lineage-committed cells in culture.
The methods increase cellular lifespan and proliferation capacity in culture, overcoming senescence issues observed in primary cells.
CD47 blockade permits maintenance of stem cells in a self-renewing de-differentiated state and supports efficient differentiation into desired cell types.
The approach enables generation of autologous stem cell therapies with potential for ex vivo genetic correction before re-administration.
Documented Applications
Ex vivo generation and expansion of iPS or multipotent stem cells from primary cells for cell-based therapies and tissue engineering.
Populating or repopulating decellularized organ or tissue matrices versus synthetic tissue scaffolds to bioengineer organs or tissues including liver, kidney, heart, lung, bladder, trachea, skin grafts, heart valves, bone grafts, nerves, retina, and cartilage.
Generation and expansion of differentiated cells such as pancreatic islet cells or hematopoietic cells for transplantation.
Use of expanded stem cells or differentiated cells for transplantation via local (e.g., injection or graft) or systemic administration to treat tissue damage, genetic defects, or diseases.
Administration of CD47 blocking agents or cells generated thereby to promote tissue regeneration in wounds, burns, non-healing injuries, bone fractures, hair loss, or vision loss conditions such as retinal degeneration, macular degeneration, or glaucoma.
Use of CD47 blockade to enhance hematopoietic stem cell function for bone marrow transplantation or cancer immunotherapy.
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