Human polarised three-dimensional cellular aggregates

1 . 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.

2 . 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.

3 . 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.

4 . The polarised three-dimensional cellular aggregate of claim 1 , wherein:

(i) the polarised three-dimensional cellular aggregate is polarised along the dorso-ventral axis, wherein the dorsal-ventral axis is defined by at least a dorsal region of cells and a ventral region of cells, wherein the cells of the dorsal region express a higher or lower level of one or more genes than the cells of the ventral region; and/or

(ii) the polarised three-dimensional cellular aggregate is polarised along the medio-lateral axis, wherein the medio-lateral axis is defined by at least a medial region of cells and two lateral regions of cells, wherein the cells of the medial region express a higher or lower level of one or more genes than the cells of the lateral regions; and/or

(iii) the polarised three-dimensional cellular aggregate is polarised along the left-right axis, wherein the left-right axis is defined by at least a left region of cells and a right region of cells, wherein the cells of the left region express a higher or lower level of one or more genes than the cells of the right region.

5 . The polarised three-dimensional cellular aggregate of claim 1 , wherein:

(i) the cells of the anterior region express a lower level of one or more genes than the cells of the posterior region, and wherein the one or more genes are selected from BRA, WNT3a, CDX2, CDH2 (N-cadherin), BMP7, CHRD, CYP26A, DAND5, NOTO1, FOXA2, CER1, DLL1, DLL3, LEFTY1, LEFTY2, SHH and PTCH1; and/or

(ii) the cells of the anterior region express a higher level of one or more genes than the cells of the posterior region, and wherein the one or more genes are selected from GATA6, HAND2, PRDM1, TBX1, BMP2, CDH3, LHX1, PAX8 and BMP4.

6 . The polarised three-dimensional cellular aggregate of claim 1 , wherein the polarised three-dimensional cellular aggregate comprises two or more of:

a. a region of cells expressing at least BRA RNA or polypeptide,

b. a region of cells expressing at least SOX2 RNA or polypeptide,

c. a region of cells expressing at least TBX6 RNA or polypeptide,

d. a region of cells expressing at least MEOX1 RNA or polypeptide,

e. a region of cells expressing at least MESP2 RNA or polypeptide,

f. a region of cells expressing at least TCF15 RNA or polypeptide;

g. a region of cells expressing at least GATA6 RNA or polypeptide; and

h. a region of cells expressing at least BMP2 RNA or polypeptide;

wherein two or more of (a)-(h) are in any order along the anterior-posterior axis in the polarised three-dimensional cellular aggregate.

7 . The polarised three-dimensional cellular aggregate of claim 1 , wherein the anterior-posterior axis has is further defined by a central region of cells between the anterior region of cells and the posterior region of cells, wherein the cells of the central region express a higher or lower level of one or more genes than the cells of the anterior or posterior regions, optionally wherein the cells of the central region express a higher level of one or more genes than the cells of the anterior or posterior regions, and wherein the one or more genes are selected from ALDH1A2, DKK1, MEOX1, MESP1, MESP2, OSR1, PITX2, TCF15, PAX3 and/or SIX1.

8 . The polarised three-dimensional cellular aggregate of claim 4 , wherein:

(i) the cells of the dorsal region express a lower level of one or more genes than the cells of the ventral region, and wherein the one or more genes are selected from SHH, NODAL, LEFTY1, 2, TBX6 and FLK1 (KDR); and/or

(ii) the cells of the dorsal region express a higher level of one or more genes than the cells of the ventral region, and wherein the one or more genes are selected from SOX2, OTX2, IRX3, SOX1, POU3F1, POU3F2 AND PAX6; and/or

(iii) the cells of the medial region express a lower level of one or more genes than the cells of the lateral regions, and wherein the one or more genes are selected from OSR1, PECAM, MEOX1, TBX6, PAX2, PAX2, LEFTY1 and PITX2; and/or

(iv) the cells of the medial region express a higher level of one or more genes than the cells of the lateral regions, and wherein the one or more genes are selected from SOX1, SOX2, DAND5, CER1, FOXA2, and NOTO1; and/or

(v) the cells of the right region express a lower or higher level of one or more genes than the cells of the left region, and wherein the one or more genes are selected from NODAL, LEFTY1, LEFTY2 and PITX2.

9 . The polarised three-dimensional cellular aggregate of claim 1 ,

(i) wherein the one or more markers characteristic of endodermal cells or derivatives of endodermal cells are one or more genes the expression of which is characteristic of endodermal cells or derivatives of endodermal cells (i) are selected from GSC, NEDD9, PYY, SHH, SORCS2, CER1, SOX17, FOXA2, TRH1 and FOXA1; or

(ii) the one or more genes the expression of which is characteristic of endodermal cells or derivatives of endodermal cells are one or more genes the expression of which is characteristic of mesendodermal cells or derivatives of mesendodermal cells are selected from MIXL1, LEFTY1, LEFTY2, AXIN2, TRH1, NODAL, WNT3a, WMT5a, Dll1 and CDX2.

10 . The polarised three-dimensional cellular aggregate of claim 1 , wherein the one or more markers characteristic of mesodermal cells or derivatives of mesodermal cells are one or more genes the expression of which is characteristic of mesodermal cells or derivatives of mesodermal cells are selected from MEOX1, OSR1, PAX2, ALDH1A2, MESP1, MESP2, TBX6, TCF15, FLK1 (KDR), and TBX1.

11 . The polarised three-dimensional cellular aggregate of claim 1 , wherein the one or more markers characteristic of mesodermal cells are one or more genes the expression of which is characteristic of mesodermal cells, and wherein the one or more genes the expression of which is characteristic of mesodermal cells are one or more genes the expression of which is characteristic of axial mesoderm cells, wherein:

(i) the one or more genes the expression of which is characteristic of axial mesoderm cells are selected from BRA, NOTO1 and NOGGIN; or

(ii) the one or more genes the expression of which is characteristic of axial mesodermal cells are one or more genes the expression of which is characteristic of paraxial mesoderm cells, wherein the one or more genes the expression of which is characteristic of paraxial mesoderm cells are selected from MEOX1, MSGN1, TBX6, TCF15, MESP1, MESP2, and ALDH1A2.

12 . The polarised three-dimensional cellular aggregate of claim 1 , wherein either:

(i) the one or more markers characteristic of ectodermal cells or derivatives of ectodermal cells are one or more genes the expression of which is characteristic of ectodermal cells or derivatives of ectodermal cells are selected from OTX2, GBX2, SIX1, SIX3, SOX3, DLX5, EYA2and BARX1.

13 . The polarised three-dimensional cellular aggregate of claim 1 , wherein:

the one or more markers characteristic of ectodermal cells or derivatives of ectodermal cells are one or more genes the expression of which is characteristic of ectodermal cells or derivatives of ectodermal cells, and wherein the one or more markers characteristic of ectodermal cells or derivatives of ectodermal cells are one or more genes characteristic of neural cells, selected from SOX1, SOX2, SOX3, POU3F1, POU3F2, PAX6, NKX1.2 and ZEB2; and/or the polarised three-dimensional cellular aggregate is elongate along the anterior-posterior axis.

14 . The polarised three-dimensional cellular aggregate of claim 1 , wherein the polarised three-dimensional cellular aggregate comprises one or more progenitor cells or derivatives of progenitor cells, and wherein the one or more progenitor cells or derivatives of progenitor cells are:

a. haematopoietic progenitor cells and/or derivatives of haematopoietic progenitor cells;

b. cardiac progenitor cells and/or derivatives of cardiac progenitor cells;

c. paraxial mesoderm and/or derivatives of paraxial mesoderm;

d. somites and/or derivatives of somites;

e. neural crest and/or derivatives of neural crest;

f. neural ectoderm and/or derivatives of neural ectoderm;

g. placodal ectoderm and/or derivatives of placodal ectoderm;

h. intermediate mesoderm progenitor cells and/or derivatives of intermediate mesoderm progenitor cells;

i. axial mesoderm progenitor cells;

j. neuromesodermal progenitor cells and/or derivatives of neuromesodermal progenitor cells;

k. lateral plate mesoderm and/or derivatives of lateral plate mesoderm;

l. primordial germ cells and/or derivatives of primordial germ cells;

m. node cells and/or derivatives of node cells; and/or

n. endoderm and/or derivatives of endoderm.

15 . The polarised three-dimensional cellular aggregate of claim 1 , wherein either:

(i) the polarised three-dimensional cellular aggregate is generated in vitro from one or more human embryonic stem cells (ESCs) or the polarised three-dimensional cellular aggregate is generated in vitro from one or more human induced pluripotent stem cells (iPSCs); and/or

(ii) the three-dimensional cellular aggregate is generated in vitro from a single pluripotent stem cell.

16 . 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.

17 . 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.