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US 12,709,734 B2Granted
Human polarised three-dimensional cellular aggregates
AssigneeCambridge Enterprise Limited
InventorsAlfonso Martinez Arias, Naomi Moris
Life of this patent
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Abstract
Human polarised three-dimensional cellular aggregates generated in vitro from one or more human pluripotent stem cells are provided. Methods for obtaining human polarised three-dimensional cellular aggregates and cells obtained from the human polarised three-dimensional cellular aggregates are also provided.
Claims17
A polarised three-dimensional cellular aggregate generated in vitro from one or more human pluripotent stem cells, wherein:
- (a) the polarised three-dimensional cellular aggregate comprises
- i. cells comprising one or more markers characteristic of endodermal cells or derivatives of endodermal cells, wherein the one or more markers comprise CDX2 mRNA or polypeptide,
- ii. cells comprising one or more markers characteristic of mesodermal cells or derivatives of mesodermal cells, wherein the one or more markers comprise BRA mRNA or polypeptide and
- iii. cells comprising one or more markers characteristic of ectodermal cells or derivatives of ectodermal cells, wherein the one or more markers comprise SOX2 mRNA or polypeptide;
- (b) the polarised three-dimensional cellular aggregate is polarised along an anterior-posterior axis defined by at least an anterior region of cells and a posterior region of cells, wherein the polarised three-dimensional cellular aggregate exhibits spatial collinearity of HOX gene expression along the anterior-posterior axis;
- wherein the cellular aggregate does not comprise extra-embryonic cells, amnion or yolk sac;
- wherein the cellular aggregate does not comprise forebrain, midbrain or hindbrain;
- and wherein all cells of the cellular aggregate are genetically descendants of the one or more human pluripotent stem cells;
- (c) the polarised three-dimensional cellular aggregate is present in a culture plate, wherein the culture plate comprises a culture cultured in a medium comprising an activator of Wnt signalling and a Rock inhibitor;
- and
- (d) the polarised three-dimensional cellular aggregate does not have an inherent capacity of developing into a human being.
8 more elements
A polarised three-dimensional cellular aggregate generated in vitro from one or more human pluripotent stem cells, wherein:
- (a) the polarised three-dimensional cellular aggregate comprises cells comprising one or more markers characteristic of primordial germ cells or derivatives of primordial germ cells, wherein the one or more markers comprise SOX17 mRNA or polypeptide;
- (b) the polarised three-dimensional cellular aggregate is polarised along an anterior-posterior axis, and wherein the polarised three-dimensional cellular aggregate exhibits spatial collinearity of HOX gene expression along the anterior-posterior axis, wherein the cellular aggregate does not comprise extra-embryonic cells, amnion or yolk sac;
- wherein the polarized three-dimensional cellular aggregate does not comprise forebrain, midbrain or hindbrain;
- and wherein all cells of the cellular aggregate are genetically descendants of the one or more human pluripotent stem cells;
- (c) the polarised three-dimensional cellular aggregate is present in a culture plate, wherein the culture plate comprises a culture cultured in a medium comprising an activator of Wnt signalling and a Rock inhibitor;
- and
- (d) the polarised three-dimensional cellular aggregate does not have an inherent capacity of developing into a human being.
4 more elements
A polarised three-dimensional cellular aggregate generated in vitro from one or more human pluripotent stem cells, wherein:
- (a) the polarised three-dimensional cellular aggregate comprises
- i. cells comprising one or more markers characteristic of endodermal cells or derivatives of endodermal cells, wherein the one or more markers comprise CDX2 mRNA or polypeptide,
- ii. cells comprising one or more markers characteristic of mesodermal cells or derivatives of mesodermal cells, wherein the one or more markers comprise BRA mRNA or polypeptide,
- iii. cells comprising one or more markers characteristic of ectodermal cells or derivatives of ectodermal cells, wherein the one or more markers comprise SOX2 mRNA or polypeptide;
- and
- iv. cells comprising one or more markers characteristic of primordial germ cells or derivatives of primordial germ cells, wherein the one or more markers comprise SOX17 mRNA or polypeptide;
- (b) the polarised three-dimensional cellular aggregate is polarised along an anterior-posterior axis, and wherein the polarised three-dimensional cellular aggregate exhibits spatial collinearity of HOX gene expression along an anterior-posterior axis;
- wherein the cellular aggregate does not comprise extra-embryonic cells, amnion or yolk sac;
- wherein the cellular aggregate does not comprise forebrain, midbrain or hindbrain, and wherein all cells of the cellular aggregate are genetically descendants of the one or more human pluripotent stem cells;
- (c) the polarised three-dimensional cellular aggregate is present in a culture plate, wherein the culture plate comprises a culture cultured in a medium comprising an activator of Wnt signalling and a Rock inhibitor;
- and
- (d) the polarised three-dimensional cellular aggregate does not have an inherent capacity of developing into a human being.
9 more elements
A method for obtaining a polarised three-dimensional cellular aggregate generated in vitro from one or more human pluripotent stem cells, the method comprising:
- (a) generating a cell suspension from one or more human pluripotent stem cells, wherein the cell suspension comprises one or more disassociated human pluripotent stem cells;
- (b) culturing the cell suspension under conditions that promote the transformation of at least one of the disassociated human pluripotent stem cells into a three-dimensional cellular aggregate;
- and
- (c) culturing the three-dimensional cellular aggregate under conditions comprising in a medium comprising an activator of Wnt signalling and a Rock inhibitor that promote the transformation of the three-dimensional cellular aggregate into a polarised three-dimensional cellular aggregate, wherein the activator of Wnt signalling is selected from one or more of a GSK3 inhibitor, CHI99021, WNT3, WNT3a, WNT5, WNT8, and WNT11;
- whereby the polarised three-dimensional cellular aggregate is generated which is polarised along an anterior-posterior axis having spatial collinearity of expression of HOX genes along the anterior-posterior axis;
- and
- wherein the polarised three-dimensional cellular aggregate comprises:
- i. cells comprising one or more markers characteristic of endodermal cells or derivatives of endodermal cells, wherein the one or more markers comprise CDX2 mRNA or polypeptide,
- ii. cells comprising one or more markers characteristic of mesodermal cells or derivatives of mesodermal cells, wherein the one or more markers comprise BRA mRNA or polypeptide, and
- iii. cells comprising one or more markers characteristic of ectodermal cells or derivatives of ectodermal cells, wherein the one or more markers comprise SOX2 mRNA or polypeptide.
7 more elements
A method for obtaining one or more progenitor cells or derivatives thereof, the method comprising
- (a) generating a cell suspension from one or more human pluripotent stem cells, wherein the cell suspension comprises one or more disassociated human pluripotent stem cells;
- (b) culturing the cell suspension under conditions that promote the transformation of at least one of the disassociated human pluripotent stem cells into a three-dimensional cellular aggregate;
- and
- (c) culturing the three-dimensional cellular aggregate under conditions comprising in a medium comprising an activator of Wnt signalling and a Rock inhibitor that promote the transformation of the three-dimensional cellular aggregate into a polarised three-dimensional cellular aggregate:
- whereby the polarised three-dimensional cellular aggregate is generated which is polarised along an anterior-posterior axis having spatial collinearity of expression of HOX genes along the anterior-posterior axis;
- and
- wherein the polarised three-dimensional cellular aggregate comprises:
- i. cells comprising one or more markers characteristic of endodermal cells or derivatives of endodermal cells, wherein the one or more markers comprise CDX2 mRNA or polypeptide,
- ii. cells comprising one or more markers characteristic of mesodermal cells or derivatives of mesodermal cells, wherein the one or more markers comprise BRA mRNA or polypeptide, and
- iii. cells comprising one or more markers characteristic of ectodermal cells or derivatives of ectodermal cells, wherein the one or more markers comprise SOX2 mRNA or polypeptide;
- and
- (d) culturing the polarised three-dimensional cellular aggregate under conditions that promote the differentiation of one or more cells of the polarised-three dimensional cellular aggregate into progenitor cells or derivatives thereof;
- whereby one or more progenitor cells or derivatives thereof are generated and wherein the one or more progenitor cells or derivatives thereof are:
- a. haematopoietic progenitor cells and/or derivatives thereof;
- b. cardiac progenitor cells and/or derivatives thereof;
- c. paraxial mesoderm and/or derivatives thereof;
- d. somites and/or derivatives thereof;
- e. neural crest and/or derivatives thereof;
- f. neural ectoderm and/or derivatives thereof;
- g. placodal ectoderm and/or derivatives thereof;
- h. intermediate mesoderm progenitor cells and/or derivatives thereof;
- i. axial mesoderm progenitor cells;
- j. neuromesodermal progenitor cells and/or derivatives thereof;
- k. lateral plate mesoderm and/or derivatives thereof;
- I. primordial germ cells and/or derivatives thereof;
- m. node cells and/or derivatives thereof;
- and/or
- n. endoderm and/or derivatives thereof.
25 more elements
Dependencies are inferred from the claim text.