Stem cells based three-dimensional embryo model
AssigneeCambridge Enterprise Limited
InventorsAzim SURANI, Jitesh NEUPANE
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Abstract
The present invention relates to an in vitro human embryo organoid at a post-gastrulation stage of development. Methods of forming the organoid and derivatives therefrom are described as well as their uses. The in vitro human embryo organoid at a post-gastrulation stage of development demonstrates early human development that recapitulates the formation of three germ layers, and hPGCLCs without exogenous BMP signalling.
Claims81
An in vitro human embryo organoid at a post-gastrulation stage of development comprising:
- cells expressing markers characteristic of endodermal cells,
- cells expressing markers characteristic of mesodermal cells,
- cells expressing markers characteristic of ectodermal cells, and
- primordial germ cell-like cells (hPGCLCs).
The in vitro human embryo organoid at a post-gastrulation stage of development according to any one of the preceding claims, wherein human embryo organoid comprises neural precursors, wherein the neural precursors are one or more genes selected from a list comprising SOX2, ZIC5, POU3F2, neural progenitors, wherein the neural progenitors are one or more genes selected from a list comprising ISI1, ZEB2, FOXC1, neural crest markers, wherein the neural crest are one or more genes selected from a list comprising FOXD3, S100B, HOXB9, SOX2, SOX10, CRABP1, TFAP2A, TFAP2B, neuron markers, wherein the neuron markers are one or more genes selected from a list comprising STMN2, ASCL1, ELAVL3, NEUROG1, and NEUROD4, neural tube markers, wherein the neural tube markers are one or more genes selected from a list comprising SOX3, PAX6, PAX7, and HES5, spinal cord markers, wherein the spinal cord markers are one or more genes selected from a list comprising OLIG3, SOX1, ZIC1, PAX7, and FGF8, and neural ectoderm markers, wherein the neural ectoderm markers are one or more genes selected from a list comprising SOX1 and PAX6.
The in vitro human embryo organoid at a post-gastrulation stage of development according to any one of the preceding claims, wherein the in vitro human embryo organoid comprises Hematopoietic Stem and Progenitor Cells (HSPC), wherein the HSPCs comprise one or more genes selected from a list comprising RUNX1, SOX7 and CD34.
The in vitro human embryo organoid at a post-gastrulation stage of development according to any one of the preceding claims, wherein the in vitro human embryo organoid comprises one or more selected from a list comprising hematopoietic cells CD45+RUNX1+, RUNX1+CD45−, CD45+RUNX1−, CD31+CD45−, CD31+CD45+, CD31−CD45+, CD31+CD34+CD45+, CD34+CD45− and CD34−CD45+.
The in vitro human embryo organoid at a post-gastrulation stage of development according to any one of the preceding claims, wherein the in vitro human embryo organoid expresses one or more markers indicative of primordial germ cell-like cells selected from a group comprising NANOS3, POU5F1, PDPN, TFAP2C, PRDM1, PRDM14, CD38, and NANOG.
The in vitro human embryo organoid at a post-gastrulation stage of development according to any one of the preceding claims, wherein the in vitro human embryo organoid comprises contractile cardiomyocytes, wherein the markers for cardiomyocytes are one or more genes selected from a list comprising NKX2-5 and MYH2, TNNI1, ACTC1, TNNT2, MYH6, MYL7 and IRX3.
The in vitro human embryo organoid at a post-gastrulation stage of development according to any one of the preceding claims, wherein the in vitro human embryo organoid comprises amnion cells, wherein the markers for amnion cells are one or more genes selected from a list comprising GABRP, VTCN1, WNT6 and TFAP2A,
The in vitro human embryo organoid at a post-gastrulation stage of development according to any one of the preceding claims, wherein the in vitro human embryo organoid comprises mesenchyme cells, wherein the markers for mesenchyme cells are one or more genes selected from a list comprising PCOLCE, COL1A2, COL6A3 and HAND1.
The in vitro human embryo organoid at a post-gastrulation stage of development according to any one of the preceding claims, wherein the in vitro human embryo organoid comprises hepatocytes, wherein the markers for hepatocytes are one or more genes selected from a list comprising AFP, ALB and CEBPA.
The in vitro human embryo organoid at a post-gastrulation stage of development according to any one of the preceding claims, wherein the in vitro human embryo organoid comprises blood progenitors, wherein the markers for blood progenitors are one or more genes selected from a list comprising PECAM1/CD31, CD34, CDH5, RUNX1, PTPRC/CD45 and CD44.
The in vitro human embryo organoid at a post-gastrulation stage of development according to any one of the preceding claims, wherein the in vitro human embryo organoid comprises splanchnic mesoderm, wherein the markers for splanchnic mesoderm are one or more genes selected from a list comprising WT1, LHX2 and GATA4.
The in vitro human embryo organoid at a post-gastrulation stage of development according to any one of the preceding claims, wherein the in vitro human embryo organoid comprises paraxial mesoderm, wherein the markers for paraxial mesoderm are one or more genes selected from a list comprising MEOX1, SIX4 and FOXC2.
The in vitro human embryo organoid at a post-gastrulation stage of development according to any one of the preceding claims, wherein the in vitro human embryo organoid comprises erythroid cells, wherein the markers for erythroid cells are one or more genes selected from a list comprising GATA1, KLF1, HBA1, HBA2, HBE1, HBE2, HBG1 and HBG2,
The in vitro human embryo organoid at a post-gastrulation stage of development according to any one of the preceding claims, wherein the in vitro human embryo organoid comprises endothelial cells, wherein the markers for endothelial cells are one or more genes selected from a list comprising CD34, CD31, CDH5, KDR, SOX7, THY1, ITGB1, CXCR4, NRARP, GJA4, IGFBP4, VEGF1, FLT1, TMEM88, TPM1, PRTG, HOXB7, MEIS2, RNF19A, CAV1, REN, BNIP3, STC1 and FGF23.
The in vitro human embryo organoid at a post-gastrulation stage of development according to any one of the preceding claims, wherein the in vitro human embryo organoid comprises hepatocytes (foetal liver progenitors), wherein the markers for hepatocytes are one or more genes selected from a list comprising AFP, APOA1, APOB, ALB and TF.
The in vitro human embryo organoid at a post-gastrulation stage of development according to any one of the preceding claims, wherein the in vitro human embryo organoid comprises musculoskeletal precursors, wherein the musculoskeletal precursors are one or more genes selected from a list comprising NEFM, SIX1 and TNNT1.
The in vitro human embryo organoid at a post-gastrulation stage of development according to any one of the preceding claims, wherein the in vitro human embryo organoid comprises hindgut, wherein the markers for hindgut are one or more genes selected from a list comprising CDX2, SOX2, SOX17, SHH and EPCAM.
The in vitro human embryo organoid at a post-gastrulation stage of development according to any one of the preceding claims, wherein the in vitro human embryo organoid is derived in vitro from one or more human embryonic stem cells (ESCs).
The in vitro human embryo organoid at a post-gastrulation stage of development according to any one of the preceding claims, wherein the in vitro human embryo organoid is derived in vitro from one or more human induced pluripotent stem cells (iPSCs).
A method of producing an in vitro human embryo organoid at a post-gastrulation stage of development comprising the steps of:
- a) treating human embryonic stem cells or human induced pluripotent stem cells to disassociate them into single cells,
- b) formation of aggregates,
- c) advancement of aggregates to human embryo organoid at the peri-gastrulation stage of development;
- and
- d) advancement of aggregates to human embryo organoid at the post-gastrulation stage of development, wherein the aggregates are incubated in the presence of a GSK3β inhibitor, preferably wherein the GSK3β inhibitor is Chiron, and a TGF-β inhibitor, preferably wherein the TGF-β inhibitor is SB43.
The method of producing an in vitro human embryo organoid at a post-gastrulation stage of development according to any one of claims 35 to 40, wherein one or more of the method steps are performed under conditions of about 1% to about 10% oxygen and 1% to about 6% carbon dioxide, preferably about 5% oxygen and about 5% carbon dioxide, optionally, wherein all of the method steps are performed under conditions of about 1% to about 10% oxygen and 1% to about 6% carbon dioxide, preferably about 5% oxygen and about 5% carbon dioxide.
A method of advancing an in vitro human embryo organoid at the peri-gastrulation stage of development to a post-gastrulation stage of development comprising incubating the human embryo organoid at the peri-gastrulation stage of development in the presence of a GSK3β inhibitor, preferably wherein the GSK3β inhibitor is Chiron, and a TGF-β inhibitor, preferably wherein the TGF-β inhibitor is SB43.
Dependencies are inferred from the claim text.