Human polarized 3D cell aggregates

Abstract

Provided are human polarized three-dimensional cell aggregates produced in vitro from one or more human pluripotent stem cells. Also provided are methods for obtaining human polarized three-dimensional cell aggregates and cells obtained from the human polarized three-dimensional cell aggregates.

Description

Technical Field

[0001] The present invention relates to human polarised three-dimensional cellular aggregates produced in vitro from one or more human pluripotent stem cells, methods for obtaining human polarised three-dimensional cellular aggregates, and cells obtained from human polarised three-dimensional cellular aggregates. Background Art

[0002] During development, the body-plan of the mammalian embryo is revealed through a process called 'gastrulation', which transforms an isotropic mass of cells into an organized entity of all three germ layers (mesoderm, endoderm and neuroectoderm) arranged along the embryonic axis. Although model organisms have provided us with insights into the processes occurring during early embryonic development, we know relatively little about the analogous stages in humans due to obvious ethical and practical limitations. Therefore, in vitro models are essential to gain a deeper understanding of the principles of body planning in the developing human.

[0003] Recent advances in the culture of human embryos in vitro have raised the possibility of exploring the earliest stages of human gastrulation. However, these studies are limited by technical challenges associated with keeping embryos alive and healthy during in vitro culture, as well as ethical regulations that prevent culture beyond day 14.

[0004] The derivation of human pluripotent stem cells (PSCs) from human embryos has opened up alternative options for functional studies of early cell fate decisions, especially in adherent culture. Under these conditions, human PSCs differentiate in a heterogeneous and heterochronous manner, challenging attempts to understand the mechanisms underlying cell fate decisions and tissue and organ formation. Restricting the growth of human PSCs to micropatterned surfaces reduces these heterogeneities and leads to complex gene expression patterns that resemble the germ layer organization of early mammalian embryos, with separate domains of ectoderm, endoderm, and mesoderm (Warmflash et al., 2014). However, although the geometry of the micropatterns (two-dimensional dishes) resembles that of the human embryo, exposure to signals results in radially symmetric patterns of gene expression rather than the multi-axial organization characteristic of the embryo.

[0005] When mouse PSCs are aggregated in three dimensions under defined conditions, they generate ‘gastruloids’, an in vitro experimental system that exhibits embryonic-like spatiotemporal organization (Turner et al., 2014; Turner et al., 2017). 1; and van den Brink et al., 2014).

[0006] Three-dimensional growth of human ESCs within the confines of artificial matrices leads to the appearance of structures resembling the amniotic sac at a low frequency (<10%), in which cells undergo initial gastrulation-like movements, including Bra expression and epithelial-to-mesenchymal transition (EMT) before collapse (Shao et al., 2017). In addition, growth of human ESCs as a mixture of gel-embedded cells has generated a 3D model of the human epiblast (Simunovic et al., 2018). Although these approaches have proven to be helpful in understanding some of the principles of pre-gastrulation development in humans, they are clearly different from the three-dimensional, axially organized, and multilineage differentiation characteristics of the early embryo. Summary of the invention

[0007] The present invention provides polarized three-dimensional cell aggregates (or human gastrula-like embryos) produced in vitro from one or more human pluripotent stem cells, methods for obtaining polarized three-dimensional cell aggregates, and human cells (e.g., progenitor cells and derivatives thereof) obtained from polarized three-dimensional cell aggregates. When the human pluripotent stem cells in the present application are human embryonic stem cells, the human embryonic stem cells are isolated or obtained using human embryos within 14 days of fertilization that have not undergone in vivo development, or are selected from mature and commercialized human embryonic stem cell lines. Tissues and organs comprising human cells derived from polarized three-dimensional cell aggregates are also provided.

[0008] Polarized three-dimensional cell aggregates derived from hPSCs (i.e., human gastrula-like embryos) have a wide range of applications, including: basic biology (understanding developmental events: gastrulation, specification of tissue and organ primordium, lineage and gene regulatory network principles); cell type characterization (combinatorial gene expression of rare and early cell types), cell type generation (generation of any derivative of the 3 germ layers or primordial germ cells for research or therapy); mutant analysis and disease modeling (patient-derived, patient-specific, or by, for example, gene knockout); experimental systems for analyzing gene expression, epigenetic regulatory mechanisms, and molecular mechanisms behind human development. Analysis of gene regulatory regions; drug screening; toxicity assays (especially in early development, or for multi-organ specificity / sensitivity analysis); cell line validation (PSC validation assays replace mouse teratoma assays or directed differentiation as a measure of pluripotent potential); IVF blastomere assessment (non-genetic prenatal screening); and the generation of functional cell types, organs, and tissues for regenerative medicine.

[0009] Polarized three-dimensional cell aggregates are dynamic entities like embryos. These entities have emerging embryo-like characteristics because over time they exhibit a sequence of different combinations of markers, gene expression patterns, and morphological changes described herein.

[0010] The present invention provides polarized three-dimensional cell aggregates produced in vitro from one or more human pluripotent stem cells, wherein:

[0011] (a) Polarized 3D cell aggregates contain

[0012] i. a cell comprising one or more markers characteristic of endodermal cells or derivatives thereof,

[0013] ii. cells comprising one or more markers characteristic of mesodermal cells or derivatives thereof, and

[0014] iii. cells comprising one or more markers characteristic of ectodermal cells or derivatives thereof; and

[0015] (b) The polarized three-dimensional cell aggregate is polarized along an anterior-posterior axis, wherein the anterior-posterior axis is defined by at least an anterior cell region and a posterior cell region, and wherein cells in the anterior region express higher or lower levels of one or more genes than cells in the posterior region.

[0016] The present invention provides polarized three-dimensional cell aggregates produced in vitro from one or more human pluripotent stem cells, wherein:

[0017] (a) the polarized three-dimensional cell aggregates contain cells comprising one or more markers characteristic of primordial germ cells or derivatives thereof; and

[0018] (b) The polarized three-dimensional cell aggregate is polarized along an anterior-posterior axis, wherein the anterior-posterior axis is defined by at least an anterior cell region and a posterior cell region, and wherein cells in the anterior region express higher or lower levels of one or more genes than cells in the posterior region.

[0019] The present invention provides polarized three-dimensional cell aggregates produced in vitro from one or more human pluripotent stem cells, wherein:

[0020] (a) Polarized 3D cell aggregates contain

[0021] i. a cell comprising one or more markers characteristic of endoderm cells or their derivatives,

[0022] ii. a cell comprising one or more markers characteristic of mesoderm cells or their derivatives,

[0023] iii. cells comprising one or more markers characteristic of ectoderm cells or their derivatives, and

[0024] iv. cells comprising one or more markers characteristic of primordial germ cells or their derivatives; and

[0025] (b) The polarized three-dimensional cell aggregate is polarized along an anterior-posterior axis, wherein the anterior-posterior axis is defined by at least an anterior cell region and a posterior cell region, and wherein cells in the anterior region express higher or lower levels of one or more genes than cells in the posterior region.

[0026] The one or more markers may be gDNA, RNA, polypeptides or other molecules. Preferably, the one or more markers are genes whose expression is characteristic of a given cell type.

[0027] The polarized three-dimensional cell aggregates can be polarized along a dorsal-ventral axis, wherein the dorsal-ventral axis is defined by at least a dorsal cell region and a ventral cell region, wherein cells in the dorsal region express higher or lower levels of one or more genes than cells in the ventral region.

[0028] Polarized three-dimensional cell aggregates can be polarized along an inside-outside axis, wherein the inside-outside axis is defined by at least an inside cell region and two outside cell regions, wherein cells in the inside region express higher or lower levels of one or more genes than cells in the outside regions.

[0029] Polarized three-dimensional cell aggregates can be polarized along a left-right axis, wherein the left-right axis is defined by at least a left cell region and a right cell region, wherein cells in the left region express higher or lower levels of one or more genes than cells in the right region.

[0030] The cells in the anterior region may express lower levels of one or more genes than the cells in 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. Preferably, the cells in the anterior region express lower levels of BRA than the cells in the posterior region.

[0031] The cells in the anterior region may express higher levels of one or more genes than the cells in the posterior region, and wherein the one or more genes are selected from GATA6, HAND2, PRDM1, TBX1, BMP2, CDH3, LHX1, PAX8 and BMP4. Preferably, the cells in the anterior region express higher levels of GATA6 than the cells in the posterior region.

[0032] The cells in the anterior region may express lower levels of BRA than the cells in the posterior region, and wherein the cells in the anterior region express higher levels of GATA6 than the cells in the posterior region.

[0033] Cells in the anterior region may express lower levels of BRA, WNT3A, CDX2, BMP7, CHRD, CYP26A1, DAND5, NOTO1, FOXA2, CER1, DLL1, DLL3, LEFTY1, LEFTY2, SHH and / or PTCH1 than cells in the posterior region, and wherein cells in the anterior region express higher levels of GATA6 than cells in the posterior region.

[0034] Cells in the anterior region may express lower levels of BRA, WNT3A, CDX2, BMP7, CHRD, CYP26A1, DAND5, NOTO1, FOXA2, CER1, DLL1, DLL3, LEFTY1, LEFTY2, SHH and / or PTCH1 than cells in the posterior region, and wherein cells in the anterior region express higher levels of HAND2 than cells in the posterior region.

[0035] Cells in the anterior region may express lower levels of BRA, WNT3A, CDX2, BMP7, CHRD, CYP26A1, DAND5, NOTO1, FOXA2, CER1, DLL1, DLL3, LEFTY1, LEFTY2, SHH and / or PTCH1 than cells in the posterior region, and wherein cells in the anterior region express higher levels of PRDM1 than cells in the posterior region.

[0036] Cells in the anterior region may express lower levels of BRA, WNT3A, CDX2, BMP7, CHRD, CYP26A1, DAND5, NOTO1, FOXA2, CER1, DLL1, DLL3, LEFTY1, LEFTY2, SHH and / or PTCH1 than cells in the posterior region, and wherein cells in the anterior region express higher levels of TBX1 than cells in the posterior region.

[0037] Cells in the anterior region may express lower levels of BRA, WNT3A, CDX2, BMP7, CHRD, CYP26A1, DAND5, NOTO1, FOXA2, CER1, DLL1, DLL3, LEFTY1, LEFTY2, SHH and / or PTCH1 than cells in the posterior region, and wherein cells in the anterior region express higher levels of BMP2 than cells in the posterior region.

[0038] Cells of the posterior region may express BRA, and wherein the polarized three-dimensional cell aggregate comprises one or more SOX17-expressing cells, wherein optionally the SOX17-expressing cells are adjacent to the posterior region.

[0039] The cells in the posterior region may express BRA, and wherein the polarized three-dimensional cell aggregates include a SOX2 expression region, and wherein the SOX2 expression region includes SOX2 expressing cells. The SOX2 expression region may be in front of the posterior region. The SOX2 expression region may be adjacent to the posterior region. The SOX2 expression region may overlap with the posterior region.

[0040] The front region can be composed of at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or 50% of the polarized three-dimensional cell aggregates. Preferably, the front region is composed of at least 5% of the polarized three-dimensional cell aggregates.

[0041] The rear region can be composed of at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or 50% of the polarized three-dimensional cell aggregates. Preferably, the rear region is composed of at least 5% of the polarized three-dimensional cell aggregates.

[0042] Polarized three-dimensional cell aggregates may include two or more of the following:

[0043] a. a region of cells expressing at least BRA,

[0044] b. a region of cells expressing at least SOX2,

[0045] c. a region of cells expressing at least TBX6,

[0046] d. a region of cells expressing at least MEOX1,

[0047] e. a region of cells expressing at least MESP2,

[0048] f. A region of cells expressing at least TCF15;

[0049] g. a region of cells expressing at least GATA6; and

[0050] h. A region of cells expressing at least BMP2;

[0051] (a) to (h) are arranged from back to front in polarized three-dimensional cell aggregates.

[0052] Polarized three-dimensional cell aggregates may contain:

[0053] a. a region of cells expressing at least BRA,

[0054] b. a region of cells expressing at least SOX2,

[0055] c. a region of cells expressing at least TBX6,

[0056] d. a region of cells expressing at least MEOX1,

[0057] e. a region of cells expressing at least MESP2,

[0058] f. A region of cells expressing at least TCF15;

[0059] g. a region of cells expressing at least GATA6; and

[0060] h. A region of cells expressing at least BMP2;

[0061] (a) to (h) are arranged from back to front in polarized three-dimensional cell aggregates.

[0062] The polarized three-dimensional cell aggregate may comprise a tail-bud-like cell region in the posterior region, optionally wherein cells of the tail-bud-like cell region express one or more of BRA, CDX2, WNT3a, WNT5a, FGF8 and CYP26a1.

[0063] The polarized three-dimensional cell aggregates may include nodule-like structures. The nodule-like structures may include cells expressing one or more of NODAL, CER1, DAND5, BMP7, FOXA2, NOTO1, SHH, CDH1, and LEFTY1, LEFTY2.

[0064] The anterior-posterior axis is further defined by a central cell region (or also referred to herein as a mid-gastruloid region) between the anterior and posterior cell regions, wherein cells in the central region express higher or lower levels of one or more genes than cells in the anterior or posterior regions.

[0065] Cells in the central region express higher levels of one or more genes than cells in 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.

[0066] Cells in the central region express higher levels of one or more genes than cells in the anterior or posterior regions, and wherein the one or more genes are selected from ALDH1A2, DKK1, MEOX1, MESP1, MESP2, OSR1, PITX2, TCF15, PAX3, UNCX, RIPPLY1, RIPPLY2 and / or SIX1.

[0067] The central region can be composed of at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or 50% of the polarized three-dimensional cell aggregates. Preferably, the central region is composed of at least 5% of the polarized three-dimensional cell aggregates.

[0068] The front-back axis can be further defined by a somite-like cell region between the posterior cell region and the central cell region, wherein the cells in the somite-like region express one or more genes at higher or lower levels than the cells in the anterior, posterior or central regions. The cells in the somite-like region can express one or more genes at higher levels than the cells in the anterior, posterior or central regions, and wherein the one or more genes are selected from BRA, MSGN, DLL1, MESP1, TBX6, MEOX1, MESP1 and MESP2. The cells in the somite-like region can express one or more genes at higher levels than the cells in the anterior, posterior or central regions, and wherein the one or more genes are selected from BRA, MSGN, DLL1, TBX6, MEOX1, MESP1, MESP2, RIPPLY1, RIPPLY2, TCF15 and UNCX. The cells in the somite-like region can be selected from one or more somite cells or their precursors, optionally wherein the precursor is one or more presomitic mesoderm cells.

[0069] The somitogenesis-like region can be composed of at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or 50% of the polarized three-dimensional cell aggregates. Preferably, the somitogenesis-like region is composed of at least 5% of the polarized three-dimensional cell aggregates.

[0070] The front-back axis can be further limited by the nodular cell region between the rear cell region and the central cell region, wherein the cells in the nodular region express one or more genes at higher or lower levels than the cells in the front, rear or central region. The cells in the nodular region can express one or more genes at higher levels than the cells in the front, rear or central region, and wherein the one or more genes are selected from group 1 (i.e., BRA, MSGN, DLL1, MESP1, TBX6, MEOX1, MESP1 and MESP2). The cells in the nodular region can express one or more genes at higher levels than the cells in the front, rear or central region, and wherein the one or more genes are selected from group 2 (i.e., NODAL, CER1, DAND5, BMP7, FOXA2, NOTO1, SHH and LEFTY1, LEFTY2). The expression level of one or more genes in group 2 in the nodular region can be higher than the expression level of one or more genes in group 1 in the nodular region.

[0071] The nodular region can be composed of at least 1%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or 50% of the polarized three-dimensional cell aggregates. Preferably, the nodular region is composed of at least 1% of the polarized three-dimensional cell aggregates.

[0072] The front-rear axis can be further limited by the middle and rear cell regions between the central cell region and the rear cell region, wherein the cells in the middle and rear regions are higher or lower than the cells in the front, rear or central regions. The cells in the middle and rear regions can be higher than the cells in the front, rear or central regions. One or more genes of the level, and wherein the one or more genes are selected from NODAL, CER1, DAND5, BMP7, FOXA2, NOTO1, SHH and LEFTY1, LEFTY2.

[0073] The middle and rear regions can be composed of at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or 50% of polarized three-dimensional cell aggregates. Preferably, the middle and rear regions are composed of at least 5% of polarized three-dimensional cell aggregates.

[0074] Polarized three-dimensional cell aggregates may exhibit spatial collinearity of HOX gene expression along the anterior-posterior axis. Polarized three-dimensional cell aggregates may exhibit spatial and temporal collinearity of HOX gene expression along the anterior-posterior axis. The spatial collinearity of HOX gene expression along the anterior-posterior axis may include sequential and ordered expression of HOX 1 to 13 from each of the A, B, C, and D clusters along the axis. The spatial collinearity of HOX gene expression along the anterior-posterior axis may include temporal sequential and ordered expression of HOX 1 to 13 from each of the A, B, C, and D clusters along the axis.

[0075] The cells of the dorsal region may express lower levels of one or more genes than cells of the ventral region, and wherein the one or more genes are selected from the group consisting of SHH, NODAL, LEFTY1, LEFTY2, TBX6 and KDR.

[0076] The cells in the dorsal region may express higher levels of one or more genes than cells in the ventral region, and wherein the one or more genes are selected from SOX2, OTX2, IRX3, SOX1, POU3F1, POU3F2 and PAX6.

[0077] Cells in the dorsal region may express lower levels of SHH, NODAL, LEFTY1, LEFTY2, TBX6 and / or KDR than cells in the ventral region, and wherein cells in the dorsal region express higher levels of SOX1 than cells in the ventral region.

[0078] Cells in the dorsal region may express lower levels of SHH, NODAL, LEFTY1, LEFTY2, TBX6 and / or KDR than cells in the ventral region, and wherein cells in the dorsal region express higher levels of SOX2 than cells in the ventral region.

[0079] Cells in the dorsal region may express lower levels of SHH, NODAL, LEFTY1, LEFTY2, TBX6 and / or KDR than cells in the ventral region, and wherein cells in the dorsal region express higher levels of SOX1 and SOX2 than cells in the ventral region.

[0080] The dorsal region can be composed of at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or 50% of the polarized three-dimensional cell aggregates. Preferably, the dorsal region is composed of at least 5% of the polarized three-dimensional cell aggregates.

[0081] The ventral region can be composed of at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or 50% of the polarized three-dimensional cell aggregates. Preferably, the ventral region is composed of at least 5% of the polarized three-dimensional cell aggregates.

[0082] The cells of the medial region may express lower levels of one or more genes than the cells of the lateral region, and wherein the one or more genes are selected from OSR1, PECAM, MEOX1, TBX6, PAX2, LEFTY1 and PITX2. The cells of the medial region may express lower levels of OSR1, MEOX1 and PAX2 than the cells of the lateral region.

[0083] The cells of the medial region express higher levels of one or more genes than the cells of the lateral region, and wherein the one or more genes are selected from SOX1, SOX2, DAND5, CER1, LFNG, FOXA2 and NOTO1.

[0084] The cells of the inner region may express lower levels of MEOX1 and / or TBX6 than the cells of the outer region, and wherein the cells of the inner region express higher levels of SOX2 than the cells of the outer region.

[0085] The cells of the inner region may express lower levels of MEOX1 and / or TBX6 than the cells of the outer region, and wherein the cells of the inner region express higher levels of SOX1 than the cells of the outer region.

[0086] The inner region can be composed of at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or 50% of the polarized three-dimensional cell aggregates. Preferably, the inner region is composed of at least 5% of the polarized three-dimensional cell aggregates.

[0087] The outer region can be composed of at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or 50% of the polarized three-dimensional cell aggregates. Preferably, the outer region is composed of at least 5% of the polarized three-dimensional cell aggregates.

[0088] The cells in the right region may express lower or higher levels of one or more genes than the cells in the left region, and wherein the one or more genes are selected from NODAL, LEFTY1, LEFTY2 and PITX2.

[0089] The cells in the right region may express lower or higher levels of one or more genes than the cells in the left region, or express lower or higher levels of one or more genes than the cells in the right region, and wherein the one or more genes are selected from NODAL, LEFTY1, LEFTY2 and PITX2.

[0090] The polarized three-dimensional axial aggregates can have an axial organization. The axial organization can be as described herein with reference to the anterior-posterior, dorsal-ventral, and medial-lateral axes.

[0091] The one or more endoderm cells or their derivatives characteristic markers can be one or more genes whose expression is characteristic of endoderm cells or their derivatives. The one or more genes whose expression is characteristic of endoderm cells or their derivatives can be selected from GSC, CDX2, NEDD9, PYY, SHH, SORCS2, CER1, SOX17, FOXA2, TRH1 and FOXA1.

[0092] The one or more genes whose expression is characteristic of endoderm cells or their derivatives may be one or more genes whose expression is characteristic of mesendodermal cells or their derivatives. The one or more genes whose expression is characteristic of mesendodermal cells or their derivatives may be selected from BRA, MIXL1, LEFTY1, LEFTY2, AXIN2, TRH1, NODAL, WNT3a, WMT5a, Dll1 and CDX2.

[0093] The one or more markers characteristic of endoderm cell derivatives may be one or more genes whose expression is characteristic of intestinal cells, optionally wherein the intestinal cells are foregut cells, midgut and / or hindgut cells and / or derivatives thereof (e.g. esophageal, lung, tracheal, pancreatic, liver, stomach, intestine and / or colon cells).

[0094] The three-dimensional cell aggregate may include an endoderm-like cell field. The cells of the endoderm-like cell field may express one or more of GSC, CDX2, NEDD9, PYY, SHH, SORCS2, CER1, SOX17, FOXA2, TRH1, and FOXA1. The cells of the endoderm-like cell field may express SOX17, and optionally, the cells of the endoderm-like cell field express one or more of GSC, CDX2, NEDD9, PYY, SHH, SORCS2, CER1, SOX17, FOXA2, TRH1, and FOXA1. The endoderm-like cell field may be arranged in one or more tubular structures.

[0095] The one or more mesodermal cells or their derivatives characteristic markers may be one or more genes whose expression is characteristic of mesodermal cells or their derivatives. The one or more mesodermal cells or their derivatives characteristic markers may be selected from BRA, MEOX1, OSR1, PAX2, ALDH1A2, MESP1, MESP2, TBX6, TCF15, MEOX1, FLK1 / KDR, FOXA2, PITX2 and TBX1.

[0096] The one or more genes whose expression is characteristic of mesoderm cells or their derivatives may be one or more genes whose expression is characteristic of axial mesoderm or their derivatives. The one or more genes whose expression is characteristic of axial mesoderm or their derivatives may be selected from BRA, FOXA2, NOTO1, CER1, SHH and NOGGIN.

[0097] The polarized three-dimensional cell aggregates may comprise fields of axial mesoderm-like cells, optionally wherein cells of the fields of axial mesoderm-like cells express one or more of BRA, FOXA2, NOTO1, CER1, SHH and NOGGIN.

[0098] The one or more genes whose expression is characteristic of mesoderm cells or their derivatives may be one or more genes whose expression is characteristic of paraxial mesoderm or their derivatives. The one or more genes whose expression is characteristic of paraxial mesoderm or their derivatives may be selected from MEOX1, MSGN1, TBX6, TCF15, MESP1, MESP2 and ALDH1A2.

[0099] The polarized three-dimensional cell aggregates may comprise a field of paraxial mesoderm-like cells, optionally wherein cells of the field of paraxial mesoderm-like cells express one or more of MEOX1, MSGN1, TBX6, TCF15, MESP1, MESP2 and ALDH1A2.

[0100] The three-dimensional cell aggregates may comprise neuromesodermal progenitor cells (NMPs), optionally wherein the neuromesodermal progenitor cells co-express SOX2, BRA and NKX1.2.

[0101] The one or more genes whose expression is characteristic of mesodermal cells or their derivatives may be one or more genes whose expression is characteristic of somitic mesoderm, optionally wherein the one or more genes are selected from TCF15, MESP1, MESP2, RIPPLY1, RIPPLY2, MEOX1 and UNCX4.1.

[0102] The polarized three-dimensional cell aggregates may comprise somitic mesoderm-like cell fields, optionally wherein cells of the somitic mesoderm-like cell fields express one or more of TCF15, MESP1, MESP2, MEOX1, UNCX4.1.

[0103] The one or more genes whose expression is characteristic of mesodermal cells or derivatives thereof may be one or more genes whose expression is characteristic of mid-mesoderm or derivatives thereof, optionally wherein the one or more genes are selected from OSR1 and PAX2.

[0104] The polarized three-dimensional cell aggregates may comprise a mid-mesoderm-like cell field, optionally wherein cells of the mid-mesoderm-like cell field express one or more of OSR1 and PAX2.

[0105] The one or more genes whose expression is characteristic of mesoderm cells or derivatives thereof may be one or more genes whose expression is characteristic of notochord, optionally wherein the one or more genes are selected from BRA, NOGGIN, NOTO1 and FOXA2.

[0106] The polarized three-dimensional cell aggregates may comprise nodule-like cells, optionally wherein the nodule-like cells express one or more of CHORDIN, NODAL, NOGGIN, NOTO1, DAND5, BMP7 and FOXA2.

[0107] The polarized three-dimensional cell aggregates may comprise cell clusters, and wherein the cells of the cell clusters express NODAL.

[0108] The one or more genes whose expression is characteristic of mesodermal cells or derivatives thereof may be one or more genes whose expression is characteristic of lateral plate mesoderm, optionally wherein the one or more genes are selected from FLK1 / KDR, LEFTY1, LEFTY2 and PITX2.

[0109] The polarized three-dimensional cell aggregates may comprise a lateral plate mesoderm-like cell field, optionally wherein cells of the lateral plate mesoderm-like cell field express one or more of FLK1 / KDR, LEFTY1, LEFTY2 and PITX2.

[0110] The one or more markers characteristic of ectoderm cells or their derivatives may be one or more genes whose expression is characteristic of ectoderm cells or their derivatives. The one or more genes whose expression is characteristic of ectoderm cells or their derivatives may be selected from OTX2, GBX2, SIX1, SIX3, SOX2, SOX3, DLX5, EYA2 and BARX1.

[0111] The one or more ectoderm cells or their derivatives characteristic markers are one or more neural cell characteristic markers. The one or more neural cell characteristic markers can be one or more genes that are expressed as neural cell characteristic, optionally wherein the one or more genes are selected from SOX1, SOX2, SOX3, POU3F1, POU3F2, PAX6, NKX1.2 and ZEB2.

[0112] The one or more neural cell characteristic markers may be one or more neural precursor characteristic markers. The one or more neural cell characteristic markers may be one or more markers characteristic of differentiated neural precursor cells. The one or more neural cell characteristic markers may be one or more markers characteristic of neural derivatives. The neural derivatives may be neurons and / or glial cells. The one or more neural precursor characteristic markers may be one or more genes whose expression is characteristic of neural precursors, optionally wherein the gene is selected from SOX2, POU3F1, POU3F2, OLIG2, PAX6, PAX7, NKX2.1, NKX2.5.

[0113] The polarized three-dimensional cell aggregates may comprise neural crest-like cells, optionally wherein the neural crest-like cells express one or more of PAX3, SOX5, SOX9 and SOX10.

[0114] The polarized three-dimensional cell aggregates may comprise a neuroectoderm-like cell region, optionally wherein the cells of the neuroectoderm-like region express one or more of SOX2, SOX3, OTX2, DLX5, EYA2 and BARX1.

[0115] The polarized three-dimensional cell aggregates may comprise an epithelial track, optionally wherein cells of the epithelial track express PAX6, SOX1, SOX2, SOX3.

[0116] The polarized three-dimensional cell aggregates may comprise epithelial tubes, optionally wherein cells of the epithelial tubes express PAX6, SOX1, SOX2 and / or SOX3.

[0117] Neuroectodermal derivatives may comprise neural plate or neural tube cells.

[0118] The one or more primordial germ cell characteristic markers may be one or more genes whose expression is characteristic of primordial germ cells. The one or more primordial germ cell characteristic markers may be one or more genes whose expression is characteristic of primordial germ cells. The one or more genes whose expression is characteristic of primordial germ cells may be selected from SOX17, PRDM1, PRDM14, DAZL, TFAP2C and NANOS3. The one or more primordial germ cell characteristic markers may be one or more primordial germ cell derivative characteristic markers.

[0119] The polarized three-dimensional cell aggregates can be elongated along the anterior-posterior axis. The anterior-posterior axis can be at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% longer than the dorsal-ventral axis. Preferably, the anterior-posterior axis is at least 10% longer than the dorsal-ventral axis.

[0120] A diameter of the polarized three-dimensional cell aggregate at the anterior end may be greater than a diameter of the polarized three-dimensional cell aggregate at the posterior end.

[0121] The polarized three-dimensional cell aggregates may be elongated along an anterior-posterior axis, optionally wherein cells in the anterior region express lower levels of BRA than cells in the posterior region.

[0122] Polarized three-dimensional cell aggregates may undergo one or more morphological elongations, optionally wherein the morphological elongations are convergent-extension and proliferation.

[0123] The polarized three-dimensional aggregate may include an oval polarized structure having different adhesion forces between its cells, serving as a source of axial mesoderm, within the BRA expression region.

[0124] Polarized three-dimensional cell aggregates may contain one or more of cavities, tubular structures, cyst pores, lumens, folds, plates, nerve bundles, and segments.

[0125] The polarised three-dimensional cell aggregates may have undergone one or more morphological shape changes, optionally wherein the morphological shape change is one or more of elongation, cavitation, cyst formation and epithelialisation.

[0126] The polarized three-dimensional cell aggregates may comprise lumenized structures, optionally wherein cells of the lumenized structures express GATA6.

[0127] Polarized three-dimensional cell aggregates can undergo bilaterally symmetrical budding at defined positions along the anterior-posterior axis. The bilaterally symmetrical buds can be limb buds.

[0128] The polarized three-dimensional cell aggregate may contain an internal morphological structure at the posterior end. The morphological structure may be an epithelial rosette and / or a lumen-like structure.

[0129] Polarized three-dimensional cell aggregates may contain one or more cells that have undergone mesenchymal to epithelial transition.

[0130] Polarized three-dimensional cell aggregates may contain one or more cells undergoing epithelial to mesenchymal transition.

[0131] The polarized three-dimensional cell aggregates can release one or more cells from the posterior region.

[0132] The polarized three-dimensional cell aggregates may contain primordial germ cell-like cells (PGCs). The PGCs may express SOX17, PRDM1, PRDM14, DAZL, TFAP2C and / or NANOS3.

[0133] Polarized three-dimensional cell aggregates may contain clusters of cells expressing PRDM1 in the anterior region.

[0134] The polarized three-dimensional cell aggregates may contain one or more of axial mesoderm derivatives, paraxial mesoderm derivatives, mid-segment mesoderm derivatives, and lateral plate mesoderm derivatives.

[0135] Paraxial mesoderm derivatives may contain somitic cells.

[0136] The mid-mesoderm derivative may comprise kidney cells and / or gonadal cells.

[0137] The lateral plate mesoderm derivative may be selected from one or more of cardiac cells, hematopoietic cells and limb cells.

[0138] Polarized three-dimensional cell aggregates can contain at least 50 cells, at least 100 cells, at least 200 cells, at least 300 cells, at least 400 cells, at least 500 cells, at least 600 cells, at least 800 cells, at least 900 cells, at least 1000 cells, at least 1500 cells, at least 2000, at least 2500 cells, at least 5000 cells, at least 10,000 cells, at least 15,000 cells, at least 20,000 cells, at least 30,000 cells, at least 40,000 cells, or at least 50,000 cells. Preferably, polarized three-dimensional cell aggregates contain at least 20,000 cells. Polarized three-dimensional cell aggregates may contain 50 to 100,000 cells, 100 to 75,000 cells, 200 to 50,000 cells, 300 to 25,000 cells, 400 to 10,000 cells, 500 to 5,000 cells, 750 to 2,500 cells, or 1000 to 2,000 cells. Preferably, polarized three-dimensional cell aggregates may contain 20,000 to 75,000 cells.

[0139] The length of the polarized three-dimensional cell aggregate can be at least 0.05mm, at least 0.1mm, at least 0.2mm, 0.3mm, at least 0.4mm, at least 0.5mm, at least 0.6mm, at least 0.7mm, at least 0.8mm, at least 0.9mm, at least 1mm or at least 1.5mm. Preferably, the length of the polarized three-dimensional cell aggregate is at least 0.2mm. The length of the polarized three-dimensional cell aggregate can be 0.05 to 2mm, 0.1 to 2mm, 0.2 to 2mm, 0.3 to 1.9mm, 0.5 to 1.8mm, 0.6 to 1.7mm, 0.7 to 1.6mm, 0.8 to 1.5mm, 0.9 to 1.4mm, 1.0 to 1.3mm or 1.1 to 1.2mm. Preferably, the length of the polarized three-dimensional cell aggregate is 0.2 to 2mm.

[0140] The polarized three-dimensional cell aggregates may comprise one or more progenitor cells or derivatives thereof.As used herein, the term "progenitor or progenitor cell" refers to both stem cells and progenitor cells.

[0141] The one or more progenitor cells or derivatives thereof may be:

[0142] a. Hematopoietic progenitor cells and / or their derivatives;

[0143] b. cardiac progenitor cells and / or their derivatives;

[0144] c. Paraxial mesoderm and / or its derivatives;

[0145] d. somites and / or their derivatives (eg, dermatomes, myotomes and / or sclerotomes);

[0146] e. Neural crest and / or its derivatives;

[0147] f. neuroectoderm and / or its derivatives (e.g., neural plate / tube cells and / or neurons);

[0148] g. placode ectoderm and / or its derivatives (e.g., ear and / or nasal primordium);

[0149] h. midsegmental mesodermal progenitors and / or their derivatives (e.g., kidney and / or gonadal primordium);

[0150] i. Axial mesodermal progenitor cells;

[0151] j. neural mesodermal progenitor cells and / or their derivatives (e.g., spinal cord neural progenitor cells and / or their derivatives, and / or paraxial mesoderm and / or their derivatives);

[0152] k. Lateral plate mesoderm and / or its derivatives;

[0153] l. Primordial germ cells and / or their derivatives;

[0154] m. nodule cells and / or their derivatives; and / or

[0155] n. Endoderm and / or its derivatives (eg, primordium of the esophagus, stomach, intestine, lung, pancreas, liver, trachea, thymus and / or thyroid gland).

[0156] The polarized three-dimensional cell aggregates may contain hematopoietic progenitor cells and / or progenitor cells of the vascular system.

[0157] Hematopoietic progenitor cells may express one or more of FLK1, GATA2, and SCL1.

[0158] Progenitor cells of the vascular system may express one or more of FLK1, SCL, RUNX1, GATA2, CXCR4, CKIT, CD41, CD35, and VE-CDH.

[0159] Polarized three-dimensional cell aggregates can contain a vascularization system of cells.

[0160] The polarized three-dimensional cell aggregates may comprise endothelial cells, optionally wherein the endothelial cells express one or more of VE-CDH, FLK1 and SCL.

[0161] The polarized three-dimensional aggregates may comprise cysts comprising clusters of endothelial cells expressing one or more of VE-CDH, CD41, CD43, and CD45.

[0162] Hematopoietic progenitor cells can express HB, optionally wherein the HB gene is fetal HB or adult HB. Hematopoietic progenitor cells can express genes characteristic of hemoglobin, optionally wherein the hemoglobin is fetal hemoglobin (HBf) or adult hemoglobin (HBa). Hematopoietic progenitor cells derived from polarized three-dimensional cell aggregates may be able to produce differentiated blood cells in vitro (for example, as measured by colony forming cells (Colony Forming Cell, CFC) assay), optionally wherein the differentiated blood cells are myeloid cells and / or lymphoid cells. Myeloid cells can be selected from one or more of monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes and platelets. Lymphoid cells can be selected from one or more of T cells, B cells and natural killer cells.

[0163] The three-dimensional cell aggregate may include a cardiac structure. The cardiac structure may be located in the anterior region of the three-dimensional cell aggregate, optionally wherein the cardiac structure is asymmetrically located in the anterior region of the three-dimensional cell aggregate. The cardiac structure may include components of a vascular system, optionally wherein the cardiac structure includes one or more blood vessels. The cardiac structure may include one or more cavities. The cardiac structure may include one or more tubular structures. The cardiac structure may beat or contract spontaneously. The cardiac structure may beat or contract at 10 to 250 beats per minute, 20 to 200 beats per minute, 30 to 175 beats per minute, 40 to 150 beats per minute, 50 to 125 beats per minute, or 60 to 100 beats per minute. The cells of the cardiac structure may express one or more cardiac-specific genes at any time point of its development. The cells of the cardiac structure may express one or more cardiac-specific genes. One or more cardiac-specific genes may be selected from GATA4, GATA6, HAND1, CTNT, NKX2.5, and TBX1. The cardiac structure may be located in the anterior region of the polarized three-dimensional cell aggregate. The cardiac-like cell region may be asymmetrically located in the anterior region of the polarized three-dimensional cell aggregate.

[0164] Polarized three-dimensional cell aggregates can be generated in vitro from one or more human embryonic stem cells (ESCs). The human ESCs can be primary human ESCs ( human ESCs).

[0165] Polarized three-dimensional cell aggregates can be generated in vitro from one or more human induced pluripotent stem cells (iPSCs).

[0166] Polarized three-dimensional cell aggregates can be generated in vitro from a single pluripotent stem cell.Polarized three-dimensional cell aggregates can be generated in vitro from a single colony derived from a single pluripotent stem cell.

[0167] Polarized three-dimensional cell aggregates can be generated in vitro from one or more blastomeres derived from the preimplantation epiblast.

[0168] The present invention provides a method for obtaining polarized three-dimensional cell aggregates, the method comprising:

[0169] (a) generating a cell suspension from one or more human pluripotent stem cells, wherein the cell suspension comprises one or more dissociated human pluripotent stem cells;

[0170] (b) culturing the cell suspension under conditions that promote conversion of at least one dissociated human pluripotent stem cell into three-dimensional cell aggregates; and

[0171] (c) culturing the three-dimensional cell aggregates under conditions that promote conversion of the three-dimensional cell aggregates into polarized three-dimensional cell aggregates;

[0172] Wherein the polarized three-dimensional cell aggregate is a polarized three-dimensional cell aggregate as defined herein. The present invention provides a method for obtaining a polarized three-dimensional cell aggregate, the method comprising:

[0173] (a) pre-treating one or more human pluripotent stem cells, wherein the pre-treating step comprises culturing the human pluripotent stem cells in a medium comprising an activator of Wnt signaling;

[0174] (b) generating a cell suspension from the pretreated human pluripotent stem cells, wherein the cell suspension comprises one or more dissociated human pluripotent stem cells;

[0175] (c) culturing the cell suspension under conditions that promote conversion of at least one dissociated human pluripotent stem cell into three-dimensional cell aggregates; and

[0176] (d) Three-dimensional cell aggregates are cultured under conditions that promote the conversion of three-dimensional cell aggregates into polarized three-dimensional cell aggregates.

[0177] Step (c) may comprise sorting the cell suspension (eg by flow cytometry) until three-dimensional cell aggregates are formed.

[0178] The method may further comprise culturing the polarized three-dimensional cell aggregates under conditions that promote differentiation of one or more cells of the polarized three-dimensional cell aggregates into progenitor cells or derivatives thereof. The progenitor cells or derivatives thereof may be progenitor cells or derivatives of any tissue or organ described herein.

[0179] In the methods, polarized three-dimensional cell aggregates may be cultured in the absence of extraembryonic cells or tissues, including primitive endoderm, amniotic sac, and / or trophoblast.

[0180] In the method, polarized three-dimensional cell aggregates may be cultured in the presence of extraembryonic cells or tissues, including primitive endoderm, amniotic sac, and / or trophoblast.

[0181] As used herein, "cell suspension" refers to a suspension comprising a single dissociated pluripotent stem cell, i.e., a single cell suspension, and / or a suspension comprising dissociated colonies comprising pluripotent stem cells, i.e., a colony suspension, and / or a suspension comprising dissociated colonies comprising pluripotent stem cells, wherein the colonies are derived from a single pluripotent stem cell, i.e., a clonal suspension.

[0182] The present invention provides a method for obtaining one or more progenitor cells or derivatives thereof, the method comprising:

[0183] (a) generating a cell suspension from one or more human pluripotent stem cells, wherein the cell suspension comprises one or more dissociated human pluripotent stem cells;

[0184] (b) culturing the cell suspension under conditions that promote conversion of at least one dissociated pluripotent stem cell into a three-dimensional cell aggregate;

[0185] (c) culturing the three-dimensional cell aggregates under conditions that promote conversion of the three-dimensional cell aggregates into polarized three-dimensional cell aggregates, wherein the polarized three-dimensional cell aggregates are as defined herein; and

[0186] (d) culturing the polarized three-dimensional cell aggregates under conditions that promote differentiation of one or more cells of the polarized three-dimensional cell aggregates into progenitor cells or derivatives thereof.

[0187] The present invention provides a method for obtaining one or more progenitor cells or derivatives thereof, the method comprising:

[0188] (a) pre-treating one or more human pluripotent stem cells, wherein the pre-treating step comprises culturing the human pluripotent stem cells in a medium comprising an activator of Wnt signaling;

[0189] (b) generating a cell suspension from the pretreated human pluripotent stem cells, wherein the cell suspension comprises one or more dissociated human pluripotent stem cells;

[0190] (c) culturing the cell suspension under conditions that promote conversion of at least one dissociated pluripotent stem cell into a three-dimensional cell aggregate;

[0191] (d) culturing the three-dimensional cell aggregates under conditions that promote conversion of the three-dimensional cell aggregates into polarized three-dimensional cell aggregates; and

[0192] (e) culturing the polarized three-dimensional cell aggregates under conditions that promote differentiation of one or more cells of the polarized three-dimensional cell aggregates into progenitor cells or derivatives thereof.

[0193] The one or more progenitor cells or derivatives thereof may be:

[0194] a. Hematopoietic progenitor cells and / or their derivatives;

[0195] b. cardiac progenitor cells and / or their derivatives;

[0196] c. Paraxial mesoderm and / or its derivatives;

[0197] d. somites and / or their derivatives (e.g., dermatomes, myotomes and / or osteotomes);

[0198] e. Neural crest and / or its derivatives;

[0199] f. neuroectoderm and / or its derivatives (e.g., neural plate / tube cells and / or neurons);

[0200] g. placode ectoderm and / or its derivatives (e.g., ear and / or nasal primordium);

[0201] h. midsegmental mesodermal progenitors and / or their derivatives (e.g., kidney and / or gonadal primordium);

[0202] i. Axial mesodermal progenitor cells;

[0203] j. neural mesodermal progenitor cells and / or their derivatives (e.g., spinal cord neural progenitor cells and / or their derivatives, and / or paraxial mesoderm and / or their derivatives);

[0204] k. Lateral plate mesoderm and / or its derivatives;

[0205] l. Primordial germ cells and / or their derivatives;

[0206] m. nodule cells and / or their derivatives; and / or

[0207] n. Endoderm and / or its derivatives (eg, primordium of the esophagus, stomach, intestine, lung, pancreas, liver, trachea, thymus and / or thyroid gland).

[0208] The polarized three-dimensional cell aggregates may be polarized three-dimensional cell aggregates as defined herein.

[0209] Polarized three-dimensional cell aggregates can have axial organization.

[0210] The pretreatment step may include culturing the human pluripotent stem cells in a medium that promotes pluripotency.

[0211] The pretreatment step may comprise culturing the human pluripotent stem cells in a medium that promotes pluripotency, optionally comprising an activator of Wnt signaling.

[0212] The pretreatment step may include culturing human pluripotent stem cells on a solid matrix. The solid matrix may be a coated solid matrix. The solid matrix may be coated with vitronectin. The solid matrix may be coated with a basement membrane matrix (e.g., GeltrexTM). The basement membrane matrix may include one or more of laminin, collagen (e.g., collagen IV), heparan sulfate proteoglycans, and entactin. The gel may be formed by a basement membrane extract, which may be separated from a suitable basement membrane secreting cell type, such as Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. The basement membrane extract produced by EHS cells may be commercially available under the trade names Matrigel (BD Biosciences, Franklin Lakes, NJ, USA), Cultrex (Trevigen Inc., Gaithersburg, MD, USA), and Geltrex (Invitrogen). Its main component is laminin, followed by collagen IV, heparan sulfate proteoglycans, and entactin.

[0213] Solid matrix can be coated with gel and / or matrix. Gel or matrix can include at least one extracellular matrix protein or its analog. Extracellular matrix protein can be one or more of collagen (e.g. collagen IV), laminin, fibronectin, vitronectin and / or gelatin. Preferably, extracellular matrix protein is collagen (e.g. collagen IV) and / or laminin. Matrix can activate signal transduction by beta-integrin receptor. Gel can be a hydrogel. Gel can include basement membrane matrix or be substantially composed of basement membrane matrix. Basement membrane matrix can be as described herein. Alternatively, gel can be polyacrylamide gel, for example, a gel comprising a cross-linked polymer matrix formed by polymerization of acrylamide and bisacrylamide (e.g., N, N'-methylene bisacrylamide). Other suitable gel types can include alginate gel, gel based on polyethylene glycol (PEG) and agarose gel.

[0214] The step of culturing the human pluripotent stem cells may include feeder-dependent culture, such as feeder-dependent culture on mouse embryonic fibroblasts.

[0215] The pretreatment step may include growing the human pluripotent stem cells to 60% to 85% confluence.

[0216] The pre-treatment step may comprise culturing the human pluripotent stem cells in a medium comprising an activator of Wnt signaling and an inhibitor of TGFβ, Nodal and / or Activin signaling.

[0217] The pre-treatment step may comprise culturing the human pluripotent stem cells in a medium comprising an activator of Wnt signaling and TGFβ, Nodal, Activin and / or an activator of signaling.

[0218] The pretreatment step may include culturing the human pluripotent stem cells in a medium comprising an activator of Wnt signaling and an inhibitor of BMP signaling,

[0219] The pretreatment step may include culturing the human pluripotent stem cells in a culture medium comprising an activator of Wnt signaling and an activator of BMP signaling.

[0220] The pretreatment step may include culturing human pluripotent stem cells in a medium comprising an activator of Wnt signaling and an inhibitor of TGFβ, Nodal and / or activin signaling. The inhibitor of TGFβ, Nodal and / or activin signaling may be an ALK4, ALK5, ALK7 inhibitor. The ALK4, ALK5, ALK7 inhibitor may be SB-431542 or SB-5051214.

[0221] The pretreatment step may include culturing human pluripotent stem cells with one or more organogenesis promoting factors. The organogenesis promoting factors may be selected from:

[0222] a. One or more activators of Wnt signaling, e.g. to promote the production of derivatives of the three germ layers and PGCs;

[0223] b. one or more activators of Wnt signaling and inhibitors of TGFβ, Nodal and / or Activin signaling, e.g. to promote the generation of proneural ectodermal and neuromesodermal progenitor cells;

[0224] c. one or more activators of Wnt signaling and one or more activators of TGFβ, Nodal and / or activin, e.g., to promote the production of endoderm and its derivatives and mesoderm derivatives (e.g., paraxial and / or axial mesoderm); and

[0225] d. One or more activators of Wnt signaling and one or more activators of BMP signaling, for example to promote the production of lateral and midsegmental mesoderm and / or their derivatives.

[0226] The step of culturing the cell suspension comprises centrifuging the one or more dissociated human pluripotent stem cells, optionally wherein the centrifugation of the one or more dissociated pluripotent stem cells induces the formation of three-dimensional cell aggregates.

[0227] The step of culturing the cell suspension may include culturing the cell suspension in a basic differentiation medium. The step of culturing the cell suspension may include culturing the cell suspension in a medium containing an activator of Wnt signaling. The step of culturing the cell suspension may include culturing the cell suspension in a medium containing an activator of Wnt signaling and a Rock inhibitor. The step of culturing the cell suspension may include culturing on a low-adhesion plate.

[0228] The step of culturing three-dimensional cell aggregates may include culturing three-dimensional cell aggregates in a basic differentiation medium. The step of culturing three-dimensional cell aggregates may include culturing three-dimensional cell aggregates in a medium containing an activator of Wnt signaling. The step of culturing three-dimensional cell aggregates may include culturing three-dimensional cell aggregates in a medium containing an activator of Wnt signaling and a Rock inhibitor.

[0229] The step of culturing three-dimensional cell aggregates may include culturing the three-dimensional cell aggregates in a culture medium containing an inhibitor of TGFβ, Nodal and / or activin signaling. The step of culturing three-dimensional cell aggregates may include culturing the three-dimensional cell aggregates in a culture medium containing an inhibitor of TGFβ, Nodal and / or activin signaling and a Rock inhibitor.

[0230] The step of culturing three-dimensional cell aggregates may include culturing three-dimensional cell aggregates in a culture medium comprising an activator of TGFβ, Nodal and / or activin signaling. The step of culturing three-dimensional cell aggregates may include culturing three-dimensional cell aggregates in a culture medium comprising an inhibitor of TGFβ, Nodal and / or activin signaling, an activator of Wnt signaling, and a Rock inhibitor.

[0231] The activator of Wnt signaling can be any agent or molecule that activates the Wnt signaling pathway including the downstream signaling network. The activator of Wnt signaling can be an activator of Wnt / β-catenin signaling. The activator of Wnt signaling can be a soluble protein. The activator of Wnt signaling can be a GSK inhibitor, optionally wherein the GSK3 inhibitor is CHIR99021 (Chi or Chiron). The activator of Wnt signaling can be selected from one or more of WNT3, WNT3a, WNT5, WNT8 and WNT11.

[0232] The activator of Nodal signaling may be soluble Nodal. The activator of activin signaling may be soluble activin.

[0233] The inhibitor of TGFβ, Nodal and / or activin signal transduction can be an ALK4, ALK5, ALK7 inhibitor. The ALK4, ALK5, ALK7 inhibitor can be SB-431542 or SB-5051214.

[0234] The step of culturing the three-dimensional cell aggregates may include culturing on a low-adhesion plate.

[0235] The one or more human pluripotent stem cells are one or more human embryonic stem cells (ESC).

[0236] The one or more human pluripotent stem cells may be one or more human induced pluripotent stem cells (iPSC).

[0237] The one or more dissociated human pluripotent stem cells may be a single pluripotent stem cell.

[0238] The one or more dissociated human pluripotent stem cells may be a colony from a single human pluripotent stem cell.

[0239] The one or more dissociated human pluripotent stem cells can be one or more blastomeres from the pre-implantation epiblast.

[0240] One or more steps of the method may be performed with suspended human pluripotent stem cells, suspended three-dimensional cell aggregates and / or suspended polarized three-dimensional cell aggregates. One or more steps of the method may be performed with three-dimensional cell aggregates and / or polarized three-dimensional cell aggregates not embedded in a gel (e.g., a hydrogel). Preferably, steps (b) to (e) are not performed with three-dimensional cell aggregates and / or polarized three-dimensional cell aggregates embedded in a gel (e.g., a hydrogel).

[0241] The cell suspension can contain 1×10 3 Up to 1×10 5 cells / ml, 5×10 3 Up to 5×10 4 cells / ml or 7.5×10 3 Up to 2.5×10 4 cells / ml.

[0242] The pretreatment step may include culturing the human pluripotent stem cells in a medium comprising an activator of Wnt signaling for 1 to 48 hours, 6 to 42 hours, 12 to 36 hours, 18 to 30 hours, or 24 hours.

[0243] The step of culturing the cell suspension may be performed for at least 10 minutes, at least 15 minutes, at least 30 minutes, at least 1 hour, at least 6 hours, at least 12 hours, or at least 24 hours.

[0244] The step of culturing the cell suspension can be performed for 10 minutes to 48 hours, 15 minutes to 42 hours, 20 minutes to 36 hours, 25 minutes to 30 hours, 30 minutes to 24 hours, 35 minutes to 18 hours, 40 minutes to 12 hours, 45 minutes to 12 hours, 50 minutes to 6 hours, 55 minutes to 1 hour.

[0245] The steps of culturing three-dimensional cell aggregates and culturing polarized three-dimensional cell aggregates can be performed for a total of at least 72 hours, at least 96 hours, at least 120 hours, at least 130 hours, at least 140 hours, at least 150 hours, at least 160 hours, at least 170 hours, at least 180 hours, at least 190 hours, at least 200 hours, at least 210 hours, at least 220 hours, at least 230 hours, at least 240 hours or at least 250 hours.

[0246] The step of culturing the three-dimensional cell aggregates may include shaking the three-dimensional cell aggregates.

[0247] The step of culturing the polarized three-dimensional cell aggregates may include shaking the polarized three-dimensional cell aggregates.

[0248] The step of culturing the cell suspension may include transferring one or more dissociated human pluripotent stem cells to the wells of the plate. The wells may be round-bottomed wells. The number of dissociated human pluripotent stem cells transferred to the wells of the plate may be 50 to 1000 dissociated human pluripotent stem cells, 200 to 800 dissociated human pluripotent stem cells, 300 to 800 dissociated human pluripotent stem cells, or 400 to 600 dissociated human pluripotent stem cells.

[0249] The present invention provides polarized three-dimensional cell aggregates (ie polarized human three-dimensional cell aggregates) obtainable by any of the methods defined herein.

[0250] The present invention provides progenitor cells or derivatives thereof (i.e., human progenitor cells or derivatives thereof) obtainable by any of the methods defined herein. The present invention also provides organs and / or tissues comprising one or more progenitor cells or derivatives thereof. Progenitor cells or derivatives thereof may be any one or more of the progenitor cells or derivatives thereof described herein. The organ or tissue may be blood, vascular tissue, kidney, heart, lung, somite, dermatome, myotome, osteomeme, neural crest, neural tube, neuron, sensory placode, gonad, notochord, neural mesodermal progenitor cells, primordial germ cells, nodules, esophagus, stomach, intestine, pancreas, liver, trachea, thymus and / or thyroid.

[0251] Polarized three-dimensional cell aggregates may not contain extraembryonic cells or tissues (including primitive endoderm, amniotic sac and / or trophoblast). Polarized three-dimensional cell aggregates may not be associated with extraembryonic cells or tissues (including primitive endoderm, amniotic sac and / or trophoblast). Polarized three-dimensional cell aggregates may not be associated with extraembryonic cells or tissues (including primitive endoderm, amniotic sac and / or trophoblast). Polarized three-dimensional cell aggregates may not be able to form yolk sac or placenta. Polarized three-dimensional cell aggregates may not contain yolk sac or placenta. Polarized three-dimensional cell aggregates may lack any anterior neural derivatives. Polarized three-dimensional cell aggregates may not be able to form brain tissue. Polarized three-dimensional cell aggregates may not contain brain tissue. Polarized three-dimensional cell aggregates do not have the inherent ability to develop into humans. BRIEF DESCRIPTION OF THE DRAWINGS

[0252] The present disclosure will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0253] Figure 1 The structure and morphology of human gastrula are shown. (a) Schematic diagram of the scheme for producing human gastrula. (b) The temporal dynamics of human gastrula formation and development, from independent cells (0 hours) to spherical aggregates (<24 hours) and finally elongated gastrula (72 to 96 hours), followed by elongated contraction (96 hours). Two representative examples from RUES2-GLR cell lines are shown. (c) Scanning electron micrograph (SEM) of human gastrula at 72 hours. Two representative examples are shown. (d) 3D projection of immunofluorescence-labeled gastrula at 72 hours (left) and 96 hours (right), respectively showing the anterior-posterior positioning of GATA6 and CDX2 (above), and the colocalization domain of SOX2 and BRA and CDH2 (N-cadherin) at 72 hours, which is resolved into the spatially different domains of SOX2 and BRA at 96 hours (below). Inset shows individual planes of each gastrula (bounded by dotted lines). (e) Lumenised rosette structure at the posterior-most end of a gastrula showing CDH2 localisation at the membrane, with SOX2 and BRA expression domains.

[0254] Figure 2:Dynamic polarization of three germ layers in human gastrula.(a) Schematic diagram of RUES2-GLR cell line used to generate human gastrula as germ layer reporter.(b) Time dynamics of human gastrula formation and development, from independent cells (0 hours) to spherical aggregates (<24 hours), and finally elongated gastrula (72 hours). Two representative examples are shown.(c) Real-time imaging of dynamics of breaking symmetry between 5.5 hours and 19.5 hours after aggregation, showing initial polarization of SOX2-mCitrine, and SOX17-tdTomato positive cells appearing at the other end.(d) Quantification of dynamics of SOX2-mCitrine, SOX17-tdTomato and BRA-mCerulean along the anterior-posterior axis of elongated gastrula. Thin lines represent independent gastrula. (e) Final localization of the three germ layer markers 72 hours after aggregation, with BRA located at the posterior side of the human gastruloid and the SOX2 and SOX17 domains located more anteriorly. Three representative examples are shown. CDS, coding sequence; tdTom, tdTomato; mCer, mCerulean.

[0255] Figure 3 : The presence of the anterior-posterior axis in human gastrula. (a) Schematic diagram of gastrula analysis by TOMO sequencing, involving slicing of embedded gastrula in an anterior-posterior direction and RNA sequencing of each slice. (b) The number of transcripts (left) and mapped genes (right) during TOMO sequencing of each slice. The blue bar represents the background, while the orange bar represents gastrula sequencing. (c) Heat map of selected variable expression genes, showing the hierarchical clustering of genes found to be particularly located in the anterior side (red phylogenetic tree), the central region (orange phylogenetic tree), the mid-posterior peak (blue phylogenetic tree), or the posterior side (green phylogenetic tree). (d) Some selected gene examples, showing the anterior side positioning (pink line), central positioning (orange line), peak mid-posterior positioning (blue line) and posterior positioning (green).

[0256] Figure 4 : Similarity of human gastrula and mouse embryo elements. (a) Schematic diagram of somitogenesis in mouse embryo (left) and comparison of gene expression patterns in human gastrula (right). Similar comparisons can be made for elements including the anterior cardiopharyngeal mesoderm and foregut (b) and nodule (c).

[0257] Figure 5:Pretreatment with Chiron promotes human gastrulation. (a) RT-qPCR analysis shows that those pretreated with Chiron show similar expression of pluripotency factors compared to hESCs maintained in pluripotency medium (Nutristem), but the expression of mesendoderm markers is significantly increased. The average of two independent experiments performed in triplicate with two cell lines (RUES2-GLR and MasterShef7) in technology. The error bar shows the standard deviation between the two cell line means. (b) Immunofluorescence of two-dimensional adherent cells maintained in Nutristem (above) or pretreated with Chiron (below), which shows the increased level of BRA and the positioning of N-cadherin (CDH2) at the membrane.

[0258] Figure 6 : Reporter lines and live imaging of human gastruloids. (a) Live imaging analysis of the GATA6-GFP line starting 24 to 35 hours after aggregation after Chiron pretreatment, showing the anterior localization of GATA6-GFP. (b) Six representative examples of human gastruloids at 72 hours generated with H9TV expressing the BRA-Venus reporter. (c) Live imaging of the RUES2-GLR line showing cells extruded from the elongated tip (black arrow) of the gastruloid between 55 and 62 hours after aggregation (AA).

[0259] Figure 7 : Effect of SB431542 on 'dorsalisation' of human gastruloids. (a) Schematic diagram of the human gastruloid scheme in the presence of SB431542 pretreatment. (b) Time course of SB431542 pretreated human gastruloids generated from the RUES2-GLR line showing altered kinetics and expression patterns compared to human gastruloids pretreated with Chiron alone (for comparison, see Figure 2 (b) 3 representative examples are shown. (c) TOMO sequencing analysis showed that the expression of posterior markers (including BRA and CYP26A1) was comparable between human gastrulae pretreated with Chiron alone and Chiron+SB431542. (d) However, some genes expressed in the central region of human gastrulae pretreated with Chiron alone were more anteriorly moved with additional SB431542 pretreatment. (e) Increased expression of neuroectodermal genes (including SOX2 and SOX3) suggests that human gastrulae pretreated with SB431542 may be more 'dorsalized'.

[0260] Figure 8: Human gastrulae produced by induced pluripotent stem cells. (a) Using the HYS01030 human induced pluripotent stem cell (iPSC) line pretreated with 3.5uM Chiron 1 day before aggregation and aggregation under Chiron and ROCK inhibitors, spherical aggregates were formed at 24 hours, which gradually became oval at 48 hours and polarized in their morphology at 72 hours after aggregation (a). 6 representative examples are shown. (b) At 96 hours after aggregation, human iPSC-derived gastrulae showed that BRA and SOX2 were clearly located at the posterior tip of the elongated gastrulae (marked as 'P'; the anterior region was marked as 'A') in a manner showing an anterior-posterior gradient. There are also some signs of rosettes or luminal structures expressing SOX2 at the posterior tip (arrow). 3 representative examples are shown.

[0261] Fig. 9 : Illustrate the structure and morphology of human gastrula. a, Schematic diagram of the protocol for generating human gastrula. Chi, CHIR99021 or 'Chiron'; ROCKi, ROCK inhibitor; E6, Essential 6 medium. b, Temporal dynamics of human gastrula development from 48 hours (h) to 96 hours after aggregation. Two representative examples from the MasterShef7 (mShef7) cell line are shown. Scale bar, 200 μm. c, Elongation of RUES2-GLR gastrula elongation from 24 hours to 72 hours with and without Chiron (Chi) pretreatment, plotted as a jitter and boxplot (central line, median; box limit, upper and lower quartiles; whiskers, interquartile range). Data from 2 to 4 independent biological replicates (Rep) are shown. Significance assessed by Welch two-sample t-test, p<2.2e-16 ( ****; Two-sided test; 24 hours, t = -12.673, degrees of freedom (df) = 95.579; 48 hours, t = -32.156, df = 226.52; 72 hours, t = -23.797, df = 121.45). The text below each figure indicates the number of gastrulae evaluated under each condition. d, the proportion of elongated gastrulae at 72 hours produced by the RUES2-GLR line. 7 independent biological replicates (left) and the average proportion of gastrulae shapes observed at 72 hours are shown (right). The text above each stacked bar indicates the number of gastrulae in each experimental replicate. e, scanning electron micrograph (SEM) of human gastrulae produced by the RUES2-GLR line at 72 hours. Two representative examples are shown. Scale bar: 200μm. f, 3D projections of RUES2-GLR human gastruloids immunofluorescently labeled at 72 hours (left) and 96 hours (right). Insets show individual sections of each type of gastruloid (bounded by dashed lines). Scale bar: 100 μm.

[0262] Fig.10 : Transcriptomic anterior-posterior organization of human gastrulae. Wide-field imaging of two 72-hour RUES2-GLR-derived Chiron-pretreated human gastrulae for tomo sequencing. Scale bar: 100 μm. mCer, mCerulean; tdTom, tdTomato; mCit, mCitrine. b, Detection of mRNA of fluorescent reporter transgenes integrated into the RUES2-GLR line by normalized expression. c, Transcriptomic analysis of 1,023 significantly reproducible genes mapped along the anterior-posterior (AP) axis of two 72-hour RUES2-GLR-derived Chiron-pretreated human gastrulae. The color map (left) shows the hierarchical clustering of gene expression behavior of two gastrulae along the AP axis. Two replicates are shown. d, Some examples of AP gradients of selected clusters and some of their enriched Gene Ontology (GO) terms. The number of overlaps in each figure corresponds to the number of genes observed within the cluster. The shaded ribbon corresponds to the standard deviation of the cluster profile for all genes within the cluster. e, Scaled gene expression along the AP axis using markers characteristic of all three germ layers. Selected genes are highlighted. The color scale is the same as for c. f, Localization of HOX gene expression along the AP axis by scaled z-scores. White bars indicate the absence of the paralog in the genome, while dark blue indicates levels below the limit of detection.

[0263] Fig.11: Comparative elements of early embryogenesis using gastrula as an in vitro model. A, schematic diagram of mammalian embryonic morphology and gene expression of the tail bud. B, AP organization of tail bud-related genes in human gastrula. The heat map (top) represents the expression of signaling components and wide-domain transcription factors, while the line graph (bottom) represents the localization of other gene expression. The line graph is shown as a smooth gene expression pattern (loess method) with a 50% confidence interval represented by a gray band. Only one of the replicates of human gastrula is shown. C, schematic diagram of mammalian embryonic morphology and gene expression of the nodule region. D, AP organization of nodule region-related genes in human gastrula. The figure is organized as in Figure c. E, immunostaining of human gastrula from 72-hour Chiron pretreatment of the RUES2-GLR line, showing the posterior localization of FOXA2 expression within a defined domain. Three representative examples are shown. Scale bar: 100 μm. f, Close-up of the FOXA2 expression domain, showing co-expression of FOXA2 and NOTCH1 intercellular domain (NICD) (bottom). Two representative examples are shown. Scale bar: 10 μm. Dashed border line indicates NOTCH1NICD expressing cells. g, Schematic diagram of the cardiac mesoderm region of mammals. h, AP organization of genes associated with the cardiac mesoderm region in human gastrulae. The figure is organized as in figure c. i, In situ hybridization of HOX gene paralogs in 72-hour human gastrulae generated from RUES2-GLR cells. Representative examples of each gene are shown. Scale bar: 200 μm.

[0264] Fig.12 : Comparison with mammalian embryos. a, Patterned organization of somitogenesis-related genes in two human gastrulae, including signaling gradients (top) and downstream genes associated with tail bud and developing somite organization (bottom). b, Illustration of Carnegie Stage (CS) 8 and 9, showing gross anatomical features, including somite boundaries. Adapted from Ref32. In both figures, yellow bars correspond to somite-related features, magenta bars correspond to developing somite-related and pre-somite mesoderm features, and green bars correspond to primitive streak and tail bud mesoderm-related features. Black bars involve nodular features. c, Heat map showing AP expression patterns of 253 genes in mouse and human gastrulae (left, average human gastrulae, n=2; right, average mouse gastrulae, n=5), 20 μm tomo-seq data. Only genes with interspecies homology between mouse and human, with significant correlations between mouse gastrula samples and between human gastrula replicates (P<0.05) were included in the analysis. Genes were clustered based on their AP expression patterns; numbered bars indicate clusters. Red asterisks indicate clusters whose expression positions deviate between the two systems.

[0265] Fig.13 : Chiron pretreatment optimization and morphological variability. A, a schematic diagram of a scheme that was not pretreated with Chiron but aggregated in Chiron (Chi) and ROCK inhibitor (ROCKi) culture medium. B, gastrula-like embryos produced by RUES2-GLR system without Chiron pretreatment at 24, 48 and 72 hours after aggregation. 3 representative examples of each time point are shown, which have all three fluorescent reporters (SOX2-mCitrine, SOX17-tdTomato and BRA-mCerulean; left) and without SOX2-mCitrine (right). Scale bar: 100 μm. C, examples of reporter patterns in differential morphological categories, as evaluated by automatic segmentation of gastrula-like outline boundaries (yellow line indicates boundaries for quantification). Three representative gastrula-like embryos of each category are shown. Scale bar, 100 μm. D, cell line-dependent optimization of Chiron conditions. Shown are gastrulae (left) derived from people using MasterShef7 cells with different Chiron pre-treatments (pre-treatment, PT) and aggregation concentrations, showing morphological variability. For each condition, two representative examples are shown. Scale bar: 100 μm. In contrast, gastrulae (right) derived from people using RUES2-GLR after different Chiron pre-treatments (PT) and aggregation concentrations are shown, showing morphological variability. For each condition, three representative examples are shown. Scale bar: 100 μm. The red bounding box indicates the concentration at which gastrulae are considered to be optimally elongated, as well as the resulting conditions for subsequent gastrulae derivatization.

[0266] Fig.14: Effect of Chiron pretreatment on human embryonic stem cells. a, Gene expression in adherent RUES2-GLR cells after Chiron pretreatment for 24 hours compared to unpretreated cells in Nutristem alone, as assessed by RT-qPCR. Mean values ​​from 5 biological replicates are shown, with bars showing mean values ​​and dots showing technical mean values ​​for each experimental replicate. Significance (ns, p>0.05, *, p<0.05; **, p<0.01; ***, p<0.001) was assessed by Welch's two-sided t-test (NANOG, p=0.005904, t=-3.7774, df=7.6264; POU5F1, p=0.3309, t=1.1047, df=4.025; SOX2, p=0.07455, t=2.3451, df=4.2888; AXIN, p=0.02146, t=-3.4691, df=4.4642; CDX 1, p=0.00639, t=-5.2284, df=4.0001; EOMES, p=0.01171, t=-4.3383, df=4.0864; LEFTY1, p=0.03737, t=-2.9402, df=4.430 6; LEFTY2, p=0.02911, t=-3.2499, df=4.212; MIXL1, p=0.01783, t=-3.8804, df=4.0003; BRA, p=0.01735, t=3.9131, df=4). B, brachyury, E-cadherin CADHERIN and N-cadherin (CDH1 and CDH2, respectively) of adherent colonies of RUES2-GLR cells were immunostained in conventional Nutristem medium (upper) or Chiron (Chi) pretreatment for 24 hours (lower). Scale bar: 100 μm. The dotted area on the colony (upper figure) shows the position of the enlarged area (lower figure). C, a histogram of the expression of immunostaining from RUES2-GLR cells as shown in Figure b was quantified. The entire image is used to generate this data. D, a spectrum of membrane localization of E- and N-cadherin from immunostaining of RUES2-GLR cell clones as shown in Figure b. A target line area is drawn across the center of each colony, and the resulting fluorescence intensity is plotted. The nuclear region is inferred by the fluorescence intensity of Hoechst greater than an arbitrary threshold. The nuclear region is demarcated by a light blue bar, and the peak of cadherin expression tends to appear, and the internuclear region indicates membrane localization.

[0267] Fig.15: Establishment and disruption of axial patterning in human gastrulae. a, Progressive polarization of GATA6-GFP fluorescence and restriction to the anterior pole of human gastrulae generated by the S4-GATA6-GFP cell line. Scale bar: 100 μm. b, Immunofluorescence imaging of RUES2-GLR human gastrulae stained for BRA and SOX2 expression, showing polarized expression as early as 24 hours. Confocal sections (top) and average projections (bottom) of gastrulae are shown. Scale bar: 100 μm. c, Human gastrulae generated by the MasterShef7 cell line at 72 hours after aggregation, showing BRA, SOX2 and N-cadherin (CDH2) localization. 3 representative examples are shown. Scale bar: 100 μm. d, Pretreatment with Wnt3a instead of Chiron for 24 hours in RUES2-GLR cells is not sufficient to promote elongation or BRA expression. The dark green bounding box indicates the pretreatment condition in Nutristem, and the cyan box indicates the aggregation medium composition in E6 and ROCK inhibitor. 2 representative examples of each condition are shown. Scale bar: 100 μm. e, Pretreatment with BMP4 instead of Chiron for 24 hours in RUES2-GLR cells is not sufficient to promote elongation or BRA expression. 2 representative examples of each condition are shown. The dark green bounding box indicates the pretreatment condition in Nutristem, and the cyan box indicates the aggregation medium composition in E6 and ROCK inhibitor. Scale bar: 100 μm. f, Application of BMP inhibitor LDN193189 (LDN; left) or end anchor polymerase inhibitor XAV-939 (XAV; right) during 24-hour pretreatment of RUES2-GLR cells. 2 representative examples of each condition are shown. The dark green bounding box indicates the pretreatment condition in Nutristem, and the cyan box indicates the aggregation medium composition in E6 and ROCK inhibitor. Scale bar: 100 μm. g, Application of Nodal signaling inhibitor SB43 (SB43) during 24 h pretreatment of RUES2-GLR cells. 2 representative examples of each condition are shown. The dark green border box represents the pretreatment condition in Nutristem, and the cyan box represents the aggregation medium composition in E6 and ROCK inhibitor. Scale bar: 100 μm.

[0268] Fig.16:The spatial transcriptomics by tomo-seq identified the clusters of gene expression. a, Quantification of the number of genes (left) and the number of unique transcripts (right) detected in each slice of the anterior-posterior (AP) axis of the Chiron-pretreated human gastrulae produced by 72 hours of RUES2-GLR gastrulae. The slices above the threshold and used for downstream gastrulae tomo-seq analysis are marked with blue, while the slices below the threshold are colored with dark gray. Two repetitions are shown. b, hierarchical clustering of reproducible tomo-seq gene expression patterns along the length of the AP axis (normalized between the 0 at the most anterior side and the 1 at the most posterior side). The band represents the standard deviation of a group of genes in each cluster. c, the selection of gene traces along the AP axis of two gastrulae indicates the reproducibility of individual genes.

[0269] Fig.17: Transcriptional profiles and anterior-posterior (AP) positioning in human gastrula. a, Normalized expression of anterior neural genes in human gastrula, usually showing low expression levels. Those with high expression levels may be due to cell types associated with epidermal fate (KRT8 and KRT18) and tail bud region (GBX2, FOXB1). b, Total expression (log10 conversion) of each HOX gene in all sections of gastrula 1 (upper) and gastrula 2 (lower) for all 4 clusters (HOXA, HOXB, HOXC and HOXD) and many of the 13 paralogs. White boxes indicate that genes do not exist in the human genome. c, Expression of ligands of the BMP (upper) and WNT (lower) signaling pathways. Red boxes indicate genes with particularly strong AP positioning biases. d, Wide-field images of human gastrula at 72 hours produced by SMAD1-RFP; H2B-mCitrine cell lines. Three representative examples are shown. Scale bar: 100 μm. e, Confocal image of a real-time human gastrula at 72 hours produced by the SMAD1-RFP; H2B-mCitrine cell line. Sections (left) and maximum projections (Max Proj.; right) through the gastrula show that SMAD1-RFP cells can be observed throughout the gastrula. Three representative examples are shown by maximum projection. Scale bar: 100 μm. f, Immunostaining of LEF1 and BRA expression in 96-hour RUES2-GLR human gastrula. LEF1 is mainly localized in a gradient in the anterior part of the gastrula. Two representative examples are shown. Scale bar: 100 μm. g, Immunostaining of WNT3A and BRA expression in 72-hour RUES2-GLR human gastrula, showing a close-up of the posterior end. A representative example is shown. Scale bar: 50 μm. Max Proj: Maximum projection. h, Expression of Nodal signaling-related genes localized toward the posterior side of Chiron-pretreated human gastruloids by tomo sequencing.

[0270] Fig.18 : Perturbation of Nodal signaling in human gastrulae. a, Schematic diagram of the protocol used to generate Chiron (Chi) and SB431542 (SB43) pretreated human gastrulae (Chi+SB43). b, Representative examples of the dynamic development of Chi and SB43 (Chi+SB43) pretreated gastrulae produced by the RUES2-GLR cell line. These show the oval morphology and polarization of gene expression at 48 to 72 hours (h) after aggregation, and elongation at 96 to 120 hours. Colors indicate the dynamic development of Chi and SB43 (Chi+SB43) pretreated gastrulae. Figure 2Reporter fluorescence shown in a. No SOX17-tdTomato reporter expression was observed. Scale bar, 100 μm. c, in situ hybridization for BRA and SOX2 mRNA in 96-hour Chi+SB43 gastrula embryos. Four representative examples of each gene are shown. d, wide-field imaging of two 120-hour RUES2-GLR-derived Chi+SB43-pretreated human gastrula embryos for tomo-sequencing. Scale bar: 100 μm. mCer, mCerulean; tdTom, tdTomato; mCit, mCitrine. e, Venn diagram of the number of genes reproducibly located in Chiron-pretreated human gastrula embryos (Chi hGld; green) and Chiron and SB43-pretreated human gastrula embryos (Chi+SB43 hGld; yellow). The numbers represent gene counts, and the percentage values ​​in brackets represent the proportion relative to the complete graph. F, from those differentially expressed genes that can be reproduced in one or both of the gastrulae pretreated by Chi or Chi+SB43. G, between the gastrulae pretreated by average Chi and the gastrulae pretreated by average Chi+SB43, on its axle from front (A) to back (P), the transcriptomics comparison of gene expression localization. The gene shown is those that are obviously reproducible under no matter which condition. Greyscale band (Greyscale band) represents the hierarchical clustering of the gene expression behavior of four gastrulae, and the colored bands show whether specific genes are reproducible under one or both of these conditions (red, only Chi+SB43; blue, only Chi; gray, both). The crimson frame represents the cluster (cluster 4) of its expression loss after SB43 pretreatment. The white row represents the expression of the gene that can not be detected.

[0271] Fig.19: Transcriptional profiles of gastrulae exposed to Nodal inhibition before aggregation. a, Quantification of the number of genes (left) and the number of unique transcripts (right) detected in each slice of the anterior-posterior (AP) axis of the gastrulae of Chiron+SB43 pretreated people generated by 120 hours of RUES2-GLR gastrulae. The slices above the threshold and used for downstream gastrulae tomo-seq analysis are marked with blue, while the slices below the threshold are colored with dark gray. Two repetitions are shown. b, the significantly reproducible gene expression patterns (left) of each repetition of the gastrulae of Chiron+SB43 pretreated people, and the resulting average gastrulae along the AP axis (right). c, hierarchical clustering of reproducible tomo-seq gene expression patterns along the length of the AP axis (normalized between 0 at the most anterior side and 1 at the most posterior side). The band represents the standard deviation of a group of genes in each cluster. d, the expression detected for markers of all three germ layers. The white line indicates that the expression of the gene is not detected. e, Gene expression traces along the AP axis of four human gastrulae (grey line, Chiron pretreatment; blue line, Chi+SB43 pretreatment; solid line, repeat 1; dashed line, repeat 2). The traces show persistent posterior expression of tail bud-related genes (BRA, SOX2, CDX2), lack of expression of Nodal signaling-related genes (NODAL, FOXA2, SHH, and DAND5), and abnormal expression of multiple other genes (CLDN11, CHRD, TFF3, FN1). Example

[0272] The inventor has shown that human pluripotent stem cells (hPSC) can be used to produce polarized three-dimensional cell aggregates (or people's gastrula). These 3-dimensional aggregates experience morphological rearrangement, differentiate into all three germ layers, and show the gene expression pattern of organization similar to that observed in developing mammalian embryos. Using this system, the inventor has shown that the aggregate of hPSC can spontaneously break symmetry without the need for other extraembryonic tissues. It also polarizes the expression of genes including GATA6, BRA and SOX2, and experiences dynamic morphological changes including elongation. Using TOMO sequencing to map transcriptional profiles spatially along the front-back axis, the inventor has shown that people's gastrula summarizes many transcriptional features observed in early mammalian embryos, including tail buds, mesoderm derivatives, somitogenesis networks, cardiovascular systems, baseplate ectoderm, cardiopharyngeal mesoderm, spinal cord elements. As an experimentally tractable system, people's gastrula can be proven to be invaluable in revealing the people-specific regulatory processes that occur during early development and enhancing our understanding of human embryo development and disease.

[0273] Example 1 - Cultivation of human embryonic stem cells using conditions previously used to generate mouse gastruloids

[0274] Using two human embryonic stem cell (hESC) lines, H9(T / Bra:GFP) and MasterShef 7, and subjecting them to equivalent conditions for generating mouse gastruloids (Baillie--Johnson, P., et al., 2015), we showed that although cell aggregation was achieved in some cases, we never observed signs of growth, polarization of BRA expression, or tissue elongation.

[0275] Example 2 - Cultivation of human embryonic stem cells and human induced pluripotent stem cells using the method of the present invention

[0276] Prior to aggregation, we pretreated hESCs for 1 day in pluripotency medium supplemented with 3 to 3.5 μM of the Wnt signaling agonist CHIR99021 (hereafter referred to as Chiron); effectively moving the Chiron pulse to before aggregation, rather than 48 hours after aggregation, as is the case in mouse gastrulae (Baillie--Johnson, P., et al., 2015). It was found that these Chiron-pretreated hESCs still expressed pluripotency markers at levels comparable to cells maintained in pluripotency medium alone ( Figure 5 a). However, pretreated cells showed slightly reduced SOX2 levels and increased expression of mesendoderm marker genes including Bra (indicated as "T"), MIXL1, LEFTY1, LEFTY2, AXIN2, EOMES, CDX1, indicating that they were ready to move toward a primitive streak-like fate. This was accompanied by expression of CDH1 (E-cadherin) and a shift in membrane localization toward CDH2 (N-cadherin; Figure 5 b) conversion.

[0277] After Chiron pretreatment, human ESCs were dissociated and 300 to 500 cells were plated in each well of a round-bottom 96-well plate in basal differentiation medium supplemented with 0.5 μM Chiron and ROCKi to aid aggregation. After 24 hours under this condition, the medium was supplemented with fresh E6 and the medium was changed every day ( Figure 1 a). Within 24 hours of plating, individual cells formed compact spherical aggregates, which then gradually polarized in morphology and formed long elongated structures within 48 hours ( Figure 1 b) This elongation phase reaches its maximum at about 72 hours after aggregation, after which the elongated structure tends to shrink or curl itself up ( Figure 1 c).

[0278] These elongated structures bear remarkable similarities to mouse embryonic stem cell-derived gastruloids, which display not only elongated morphological structures but also differentiation gene expression patterned along defined axes. To test whether these elongated structures resemble mouse gastruloids in this respect, we stained the 72-hour elongated aggregates for differentiation markers and noted a clear anterior-posterior distinction between the two ends: at the BRA-expressing end, we observed an overlap in expression with CDX2, and at the other end, there was a domain expressing GATA6 ( Figure 1 d); The polarization of GATA6 to the anterior and lateral ends was also confirmed using a reporter system ( Figure 6 a). BRA / CDX2 expressing cells are located at the tips of elongating structures, as in the embryo. This combination identifies the caudal-most region of the embryo, which we believe is also true in gastrulae and has also been observed using fluorescent reporter lines (Allison et al., 2018) ( Figure 6 b). This polarized region of the BRA was also positive for CDH2 (N-cadherin) and overlapped with the SOX2-positive region at 72 hours but gradually resolved into distinct BRA and SOX2 expression domains ( Figure 1 d). These often appear to be arranged so that the BRA and SOX2 domains separate along an axis roughly orthogonal to the AP axis, suggesting the presence of at least 2 axes. In a certain proportion of aggregates, we also observed internal morphological structures that appeared to display epithelial rosette-like or lumen-like structures at the posterior-most end ( Figure 1 e), and the extrusion of a single cell from the posteriormost tip of the gastruloid ( Figure 6 c) Due to their elongated morphology, polarized BRA expression, and similarity to mouse gastruloids derived from human ESC populations, we refer to these structures as human gastruloids.

[0279] However, to determine whether these human gastruloids are indeed capable of generating organized derivatives of the three germ layers as occurs during gastrulation, we used a germ layer reporter line (RUES2-GLR; Martyn et al., 2018). This hESC line utilizes fluorescent reporters for the BRA (mesoderm), SOX17 (endoderm and primordial germ cells), and SOX2 (neurectoderm) marker genes, which allows for dynamic assessment of germ layer development and organization ( Figure 2 a) Human gastrulae generated using this cell line initially express SOX2 as they are in a pluripotent state prior to aggregation ( Figure 2 b) They then rapidly polarize SOX2 expression to a coherent domain at the end of the aggregate, with independent SOX17-expressing cells appearing throughout the rest of the aggregate ( Figure 2c) Between 24 and 48 hours after aggregation, aggregates undergo elongation as BRA localizes to SOX2-positive regions of gastruloids ( Figure 2 d). By 72 hours after aggregation, elongating gastruloids express BRA in the posteriormost part of the elongation (with adjacent SOX17 and SOX2 expression domains) ( Figure 2 e).

[0280] Taken together, these observations suggest that hESC-derived gastruloids develop structures similar to mouse gastruloids and display an axially organized profile. To further explore the complexity and organization of these gastruloids, we decided to analyze the transcriptional profile of human gastruloids using TOMO sequencing (TOMO-seq), a technique that allows for the sampling of the spatial organization of transcription at the genome-wide level (Junker et al., 2014 and Kruse et al., 2016). Gastruloids were fixed 72 hours after aggregation, embedded for sectioning along their AP axis, and subjected to RNAseq analysis ( Figure 3 a).

[0281] When evaluating the spatial localization of all genes along the AP axis, we found four major gene expression categories ( Figure 3 c). These correspond to: (1) the most anteriorly expressed genes, including GATA6, HAND2, PRDM1, TBX1, and BMP2, (2) the most posteriorly expressed genes, including BRA(T), WNT3A, CDX2, BMP7, CHRDN, CYP26A1, MIXL1, DAND5, NOTO, and FOXA2, (3) the mid-gastruloid expressed genes, including MEOX1, TCF15, PAX3, SIX1, HOXA2, and HOXB1, and (4) genes whose peaks are just anterior to the most posterior genes, indicating nodule-like or somite-like structures, including DLL1, MESP1, MESP2, and NODAL.

[0282] Interestingly, our results are consistent with our results in the mouse gastrula context (Turner et al., 2017 1 ; Turner et al., 2017 2 ; and van den Brink et al., 2014) in a similar manner to that seen in the anterior neural tissues (e.g., forebrain, midbrain, or hindbrain). In an attempt to uncover these alternative elements of this architecture, we attempted to use the Nodal inhibitor SB431542 (herein referred to as SB431542; Figure 7a) 'dorsalize' the starting cell population. SB431542 has been shown to promote expression of multipotent caudal neural progenitor cell markers in hESCs together with Chiron, and to reduce mesoderm specification and promote neural development with nodal repression in mouse and human PSCs. Indeed, our gastruloids from this condition showed significantly larger domains of SOX2 expression (which overlaps with Bra expression), and showed fewer, if any, SOX17 expressing cells ( Figure 7 b) However, they do continue to express BRA, CYP26A1, and WNT3A in the posterior domain in a manner comparable to Chiron-pretreated human gastruloids ( Figure 7 c). However, in contrast to Chiron-pretreated gastrulae, SB431542-pretreated gastrulae showed no markers of nodal-like cell populations, no detectable expression of NODAL, NOTO, PITX2, or DAND5, and had very little expression of FOXA2, as expected following inhibition of Nodal signaling. We also saw little or no expression of the asymmetry markers LEFTY1 and LEFTY2, or the more anterior cardiac mesoderm markers GATA4, GATA6, or HAND2. We also observed a shift in somitogenesis-like genes, including MEOX and TCF15, which are normally expressed in the central region in Chrion-pretreated gastrulae, becoming localized anteriorly in SB431542-pretreated gastrulae ( Figure 7 d). In addition, we observed an increase in domain size and expression levels of SOX2 and SOX3 ( Figure 7 e), indicating an increase in neuroectodermal-like tissue. These observations lead us to believe that human gastruloids pretreated with SB431542 may be biased toward a more 'dorsal' fate, expressing more neuroectodermal markers and fewer endoderm markers.

[0283] Thus, Chiron-pretreated gastrula structures show a particularly high degree of organization along the AP axis, ranging from the most posterior BRA expression (which is located in the primitive streak and tail bud regions in mouse embryos) to the anterior GATA6-positive region (which is found in the cardiac crescent in mice). This suggests to us that human gastrula may follow the principles of spatial transcriptional regulation of the developing embryo, and we sought to examine whether the spatial localization of transcriptional markers of specific cell types is equivalently localized in gastrula as in mammalian embryos. Indeed, we observed that certain elements of the developing embryo were reflected in the gastrula system, including the tail bud-like posterior domain (expressing BRA, CDX2, WNT3A, FGF8, and CYP26A1), as well as anteriorly localized elements of the cardiac primordium and cranial mesoderm (GATA4, GATA6, HAND1, and TBX1). Within the elongation region, we observed expression of neural progenitor genes (SOX2) as well as low but detectable expression of neural differentiating cells (PAX6, SOX1, ZEB2) and, importantly, signs of organized somitogenesis in a sequential order along the AP axis (from posterior to anterior: BRA, TBX6, MEOX1, MESP2, TCF15). Importantly, between the tail bud and the onset of TBX6 expression, we observed localized expression of CHRD, NOTO1, NODAL and LEFTY1, LEFTY2, suggesting the presence of nodule-like structures in the gastruloids. Furthermore, at the anterolateral end, we observed expression of FLK1 / KDR and GATA2, predicting the emergence of endothelial and hematopoietic system components. Axial organization was supported by spatially organized expression of members of the four HOX clusters along the AP axis. Altogether, these results indicate that the human gastruloids develop into a body plan with all the hallmarks of mammalian embryos.

[0284] Interestingly, we also found that similar polarized and partially elongated structures could be derived from induced pluripotent stem cells (iPSCs) treated in the same manner as hESCs, suggesting that the human gastrula system can be used to generate both pluripotent cell types ( Figure 8 ab).

[0285] Our results show that, after 72 hours of aggregation, people's gastrulae are in the stage consistent with the embryos that experience gastrulation. The following observations provide anchors for staging with reference to the embryos in Carnegie and Kyoto collections: the people's gastrulae of 72 hours AA seem to be experiencing somitogenesis. In human embryos, gastrulation begins at the 13th day (Carnegie staging 6, CS6), and lasts for about 5 days. By the 17th day (CS9), the embryo shows signs of somitogenesis, which do not exist in CS8 and nodule-like structures. We use the ratio of pre-stomach mesoderm and body wall mesoderm in gastrulae (such as the expression of Tbx6 and Uncx4.1) and embryo (the distance between somites and nodules to the first somites) as an approximate value to match both. Comparison shows that people's gastrulae of 72 hours AA are in a state approximately equal to CS9.

[0286] Our results demonstrate that hPSCs can be organized into gastrula-like embryos that exhibit axial organization reminiscent of mammalian body configurations during mid-gastrulation and provide a useful experimental system to explore early stages of human development in vitro from hESCs.

[0287] Example 3 - Further analysis of the culture of human embryonic stem cells and human induced pluripotent stem cells using the method of the present invention

[0288] When hESCs were treated with the Wnt agonist Chiron for one day and then a limited number of cells were plated in a low adhesion plate in the presence of 3 to 3.5 μM Wnt signaling agonist CHIR99021 (hereinafter referred to as Chiron), they formed compact spherical aggregates within a few hours, which gradually broke the symmetry and became ovoid by 24 hours (h) after aggregation, and formed elongated structures by 48 hours (h). Fig. 9 a, b). This elongation reaches a maximum at 72 to 96 hours and is strictly dependent on Chiron pretreatment ( Fig. 9 bc and Fig.13 ab). After 72 hours, only some structures remained elongated until 96 hours, when most structures tended to curl or shrink. We observed that this elongation was reproducible, as on average, about 66% of the aggregates from each experiment exhibited an elongated morphology at 72 hours ( Fig. 9 d and Fig.13 c) and have a smooth cell surface along the outside of the aggregate ( Fig. 9 e). Different cell lines require different concentrations of Wnt signaling stimulation, indicating lineage-specific requirements ( Fig.13d). Prior to aggregation, pre-treated hESCs were found to still express the pluripotency markers POU5F1 (also known as OCT4) and SOX2 at levels comparable to cells maintained in pluripotency medium alone (Welch two-sided t-test, p = 0.33 and 0.075, respectively; Fig.14 a), but NANOG (p=0.0059) and mesendoderm marker genes, including BRA, MIXL1, LEFTY1, LEFTY2, AXIN2, EOMES, CDX1 (p=0.017, 0.018, 0.037, 0.029, 0.021, 0.012, and 0.0064, respectively; Fig.14 It also increases the expression of membrane-localized CDH2 (N-cadherin) based on the expression of CDH1 (E-cadherin) ( Fig.14 bd). These results indicate that hES cells are partially primed toward a primitive streak-like state following Chiron pretreatment and are therefore consistent with the observation that Wnt signaling induces mesodermal differentiation in hESCs.

[0289] These elongated structures bear remarkable similarities to mouse embryonic stem cell-derived gastruloids, which display not only elongated morphological structures but also differentiation gene expression patterned along defined axes. To test whether these elongated structures resemble mouse gastruloids in this respect, we stained the 72-hour elongated aggregates for differentiation markers and noted a clear anterior-posterior distinction between the two ends: at the BRA-expressing end, we observed an overlap in expression with CDX2, and at the other end, there was a domain expressing GATA6 ( Fig. 9 f); The polarization of GATA6 to the anterior end was also confirmed using a reporter system ( Fig.15 a). BRA / CDX2 expressing cells are located at the tips of elongating structures, as in the embryo. This combination identifies the most caudal region of the embryo, which we believe is also true in gastrulae. This polarized region of BRA is also positive for CDH2 (N-cadherin) and overlaps with the SOX2 positive region at 72 hours, but gradually resolves into distinct BRA and SOX2 expression domains ( Fig. 9 f). These often appear to be arranged so that the BRA and SOX2 domains separate along an axis roughly orthogonal to the AP axis, suggesting the presence of at least 2 axes. In a certain proportion of aggregates, we also observed internal morphological structures that appeared to display epithelial rosette or lumen-like structures at the posterior-most end ( Figure 1 e) Due to their elongated morphology, polarized BRA expression, and similarity to mouse gastruloids derived from human ESC populations, we refer to these structures as human gastruloids.

[0290] However, to determine whether these human gastruloids are indeed capable of generating organized derivatives of the three germ layers as occurs during gastrulation, we used a germ layer reporter line (RUES2-GLR; Martyn et al., 2018). This hESC line utilizes fluorescent reporters for the BRA (mesoderm), SOX17 (endoderm and primordial germ cells), and SOX2 (neurectoderm) marker genes, which allows for dynamic assessment of germ layer development and organization ( Figure 2 a) Human gastrulae generated using this cell line initially express SOX2 as they are in a pluripotent state prior to aggregation ( Figure 2 b) They then rapidly polarize SOX2 expression to a coherent domain at the end of the aggregate, with independent SOX17-expressing cells appearing throughout the rest of the aggregate ( Figure 2 c) Between 24 and 48 hours after aggregation, aggregates undergo elongation as BRA localizes to SOX2-positive regions of gastruloids ( Figure 2 d). By 72 hours after aggregation, elongating gastruloids express BRA in the posteriormost part of the elongation (with adjacent SOX17 and SOX2 expression domains) ( Figure 2 e).

[0291] Taken together, these observations suggest that hESC-derived gastruloids develop structures similar to mouse gastruloids and display an axially organized profile. To further explore the complexity and organization of these gastruloids, we decided to analyze the transcriptional profile of human gastruloids using TOMO sequencing (TOMO-seq), a technique that allows for the sampling of the spatial organization of transcription at the genome-wide level (Junker et al., 2014 and Kruse et al., 2016). Gastruloids were fixed 72 hours after aggregation, embedded for sectioning along their AP axis, and subjected to RNAseq analysis ( Fig.10 ab).

[0292] When evaluating the spatial localization of all genes along the AP axis, we found 22 gene expression categories ( Fig.10 cd and Fig.16 ). Six clusters (clusters 0 to 5) located in the posteriormost region of the gastruloid contained genes that localize to the tail bud in the mouse embryo, including BRA, CDX2, and CYP26A1. At the opposite end, we observed nine clusters (clusters 6 to 14) containing genes including KDR, SOX4, MEIS1 / 2, PBX1, TWIST1, ISL1, IRX1 / 2 / 3, JARID2, and PRDM1 ( Fig.10 c, d). In addition, we observed 7 additional gene clusters (clusters 15 to 21) located in the middle of the AP axis ( Fig.10 c, d), which includes many genes expressed in the trunk region of mammalian embryos, such as PAX3, TCF15, DKK1, MEOX2 (Cluster 16), RALDH2 (ALDH1A2), ZIC2, MEOX1 (Cluster 18), WNT5A, DLL3, CITED1, MESP1, RIPPLY2, and PTCH1 (Cluster 21; Fig.10 c, d). Cluster 21 is highly enriched for genes involved in somitogenesis (GO:0001756, Benjamini-corrected p-value=0.0135) and Notch signaling pathway (GO:0007219, p-value=0.0375).

[0293] Throughout the AP axis, we observed expression of genes associated with mesoderm, endoderm, and ectoderm, indicating that cell types associated with all three germ layers are represented in human gastrulae ( Fig.10 e). We found no evidence for expression of genes associated with the development of anterior neural structures ( Fig.17 a), but expression of many paralogs from the four HOX clusters was observed ( Fig.17 b) These HOX genes display variable expression domains along the length of the gastrula AP axis, including a broader domain for paralogs 1 to 5, and a more posterior distribution for the latter paralogs, including groups 5 to 8 and 9 to 13 ( Fig.10 f) This pattern of HOX gene expression along the length of the AP axis indicates that active axial extension is ongoing.

[0294] We also observed many Wnt ligands expressed at the posterior end ( WNT5A , WNT3A , WNT5B , and WNT3 ) and some BMP ligands at the anterior end ( BMP2 , BMP4 , and BMP5 ; Fig.17 c). These transcriptional patterns were reminiscent of the organization of signaling in the mouse embryo and led us to examine whether related signaling pathways were also active. In gastrulae generated from the BMP reporter line RUES2:SMAD1-RFP;H2B-mCitrine, SMAD1-RFP was predominantly localized to the anterior domain, but there were a few positive cells scattered throughout its length, indicating activity of the BMP ligand ( Fig.17 At the posterior end, we observed expression of WNT3a in the pericellular space, suggesting ligand secretion within the adjacent domain, as well as expression of LEF1, a target of WNT / β-catenin signaling, suggesting Wnt signaling ( Fig.17 fg). In addition, we observed peaks of Nodal signaling components and targets (including NODAL, LEFTY1 / 2, and CER1) in the posteriormost region of gastruloids ( Fig.17 h) These results indicate that human gastruloids display inverted gradients of BMP and Wnt / Nodal signaling along their long axis.

[0295] The organization of the posterior domain of gene expression in human gastruloids resembles the pattern in the tail bud region ( Fig.11 Within this region, we observed a nodule-like transcriptional domain with overlapping expression of SHH, BMP7, CHRD, CELSR1, and DAND5, with adjacent peaks for DLL1, NOTCH1, and PTCH1 ( Fig.11 cd), and localized domains of NOTCH signaling around FOXA2-expressing cells ( Fig.11 At the opposite end, we observed a pattern associated with cardiac mesoderm development, reflected in the consistent expression of BMP2 with peaks for FGF8 and FGF10 (overlapped by expression of TBX1 / 5, HAND1 / 2, NKX2.5, MEF2C, and ISL1) ( Fig.11 gh). This is accompanied by anterior-posterior expression of HOX genes, including HOXB3, HOXC8, and HOXB9 ( Fig.11 i).

[0296] The tomo-seq data show, in addition to very clear and patterned mesodermal differentiation signals, a small representation of neural gene expression: a posterior-to-anterior signature of somitogenesis, with peaks in expression of tailbud genes (BRA and CDX2) at the posteriormost side (overlapping with LFNG, which also peaks posteriorly), a short domain of MESP1 and MESP2, followed by a more anterior, broader domain of MEOX1 and TCF15 expression. This sequence of gene expression is capped by a gradient of WNT3A posteriorly, followed by FGF8, and then RALDH2 (ALDH1A2) more anteriorly ( Fig.12 a). This overall pattern of gene expression reflects the organization of paraxial mesoderm specification and differentiation in mammalian embryos, as well as the temporal sequence of somitogenesis in hESC directed differentiation, and together with the expression of HOX genes, indicates an active axially polarized somitogenesis transcriptional program in human gastruloids. The results presented here indicate that human gastruloids display elements of the mammalian embryonic AP axis, including polarized and organized arrangements of gene expression (e.g., those involved in early somitogenesis).

[0297] The axial organization of the somitogenic program of gene expression suggests the possibility of approximate staging of the gastrula in development. Examination of images of extant collections of human embryos revealed a major transition in morphogenetic events associated with the onset of somitogenesis between Carnegie stages (CS) 8 and 9 (corresponding to 17 to 19 days and 19 to 21 days, respectively). Fig.12b). Images of CS9 embryos reveal the presence of 1 to 3 somite pairs that are absent in CS8, with a short domain between the last somite and the tubercle-like structure. In gastrulae, the pattern of gene expression indicates the presence of a somite domain preceding the presomitic domain of similar length, allowing us to propose that 72-hour human gastrulae may serve as a model for some features of late CS8 or early CS9 stages of human development.

[0298] Materials and Methods for Examples 1 to 3

[0299] Cell lines

[0300] We used a wild-type hESC line (mSHEF7; REF), as well as two transgenic lines, the H9TV BRACHYURY-Venus reporter (Mendjan et al., 2014) and RUES2-GLR, a triple reporter of germ layer markers with BRACHYURY-H2B-mCerulean, SOX17-H2B-tdTomato, and SOX2-mCitrine (Simunovic et al., 2018). We also used a human induced pluripotent stem cell line, called HYS01030 (unpublished).

[0301] Cell culture conditions

[0302] Human ESCs were cultured in vitronectin-coated flat-bottom 6-well plates (Costar, Corning Inc.) in NutriStem hPSC XF medium (Biological Industries). For coating of the plates, 1 ml of MgCl-free medium was used per well. 2 and CaCl 2, Dulbecco's phosphate buffered saline (PBS- / -, Sigma-Aldrich) supplemented with vitronectin (1:100). The plate was then incubated at room temperature for 1.5 to 4 hours or at 4°C for 1 to 7 days. Before use, the plate was washed with PBS- / -. hESC was passaged at 70% to 90% confluence (approximately every 3 to 5 days). After washing twice with 3ml PBS- / - per well, 2ml 0.5mM EDTA (0.5M storage solution at pH 8.0, Invitrogen, Lifetechnologies) in PBS- / - was applied to each well, and the cells were incubated for 5 minutes at 37°C, 5% CO2 for separation. The cell clumps were then collected in a centrifuge tube, and PBS- / - was added to dilute EDTA, and then centrifuged at 1000rpm for 3 minutes. The supernatant was then discarded, and the cells were gently resuspended in 1ml Nutristem. The cells were split at a ratio of 1:5 to 1:10. The medium was changed daily.

[0303] Pretreatment before generating human gastruloids

[0304] Unless otherwise stated, hESCs cultured in 6-well plates were typically pretreated with 3 to 3.5 μM CHIR99021 (Chiron) in Nutristem for 24 hours prior to aggregation. The exact concentration of Chiron pretreatment was observed to be cell line specific, and each new cell line should be titrated with 1.5 to 5 μM Chiron for optimal gastrulation.

[0305] For signal modulation experiments, RUES2-GLR cells were pretreated in Nutristem supplemented with 100ng / ml recombinant human Wnt3a (5036-WN-010) or 50ng / ml BMP4 (314-BP) for 1 day and aggregated in E6 and ROCK inhibitors with additional supplements shown. Subsequent culture medium replacement was performed with separate E6 every day. In order to test the effect of signal modulation on gastrulation, unless otherwise stated, RUES2-GLR cells were pretreated in Nutristem supplemented with 1μM LDN193189 (04-0074), 1μM XAV-939 (04-0046) or 10μM SB431542 (1614) and 3.25μM Chiron, and then aggregated in E6 with 0.5μM Chiron and ROCK inhibitor. Subsequent culture medium replacement was performed with separate E6 every day.

[0306] An alternative 'dorsalization' protocol used 3 μM Chiron supplemented with 10 μM SB431542 Nodal inhibitor (SB431542), also in Nutristem, for 24 hours.

[0307] Generation of hESC gastrula-like embryos

[0308] After pretreatment, hESCs at 60% to 85% confluence were washed twice with PBS- / -. For detachment, 2 ml of 0.5 mM EDTA in PBS- / - was applied to each well, and the cells were incubated at 37°C, 5% CO2 for 6.5 minutes. The plate was then carefully tapped against a solid object to facilitate detachment, and the suspension was repeatedly pipetted up and down (5-10x) with a P1000 to break up the remaining clumps into single cells.

[0309] The suspension is then transferred to a 15ml centrifuge tube with 8ml PBS- / - and centrifuged at 1000rpm for 3 to 5 minutes. The supernatant is removed, the cells are washed with PBS- / -, and spun down again at 1000rpm for 3 to 5 minutes. Subsequently, according to the size of the precipitate, the cells are resuspended in 500μl to 2ml of Essential 6 (E6) culture medium (Gibco, lifetechnologies) and counted using an automatic cell counter (MoxiZ, Orflo). The optimal cell density should be about 1×10^6 / ml. The optimized specific cell number of each cell line (400 / well for RUES2-TR and 500 / well for mSHEF7 and H9TV systems) is then added to the E6 supplemented with 5μM Rock inhibitor and 3μM Chiron. Using a multichannel pipette, cells in this suspension were plated in 40 μl per well of an ultra-low adhesion, round-bottom 96-well plate (Costar, Corning Inc.). 24 hours after aggregation, 150 μl of E6 was added to each well using a multichannel pipette. E6 was then continuously replaced daily by removing 150 μl per well and adding the same amount (see Chen et al., 2011).

[0310] Immunofluorescence imaging

[0311] For fixation, use P1000 pipette to extract aggregates from 96-well plates and merge them into 30mm Drosophila glass dissection wells. After washing 3 times with PBS- / -, 1ml 4% paraformaldehyde (paraformaldehyde PFA) in PBS- / - is applied, and the sample is incubated at 4 ℃ for 2 hours under gentle horizontal rotation. Then the gastrula is washed 3 times with PBS- / - and transferred to a small net, which is then placed in each well of a 24-well plate. Aggregates are washed three times with PBS- / - containing 10% fetal bovine serum (FBS) and 0.2% Triton X-100 (PBSFT) under gentle horizontal rotation, each time for 10 minutes. For blocking, the sample is incubated at 4 ℃ for 1 hour in PBSFT on an orbital shaker. The primary antibodies included: rabbit anti-Cdx2 (ThermoScientific, EPR2764Y), rabbit anti-Brachyury (abcam, EPR18113), goat anti-Sox2 (R&D Systems, AF2018), mouse anti-N-cadherin (BD Biosciences, BD610920), chicken anti-GFP (invitrogen, ThermoScientific) and were diluted in PBSFT and applied overnight at 4°C with gentle horizontal rotation.

[0312] The next day, the aggregates were washed 9 times with PBSFT: 2 times for 5 minutes each time, 3 times for 15 minutes each time, and 4 times for 1 hour each time. The sample was kept at 4 ° C on an orbital shaker. The secondary antibody and nuclear Hoechst (Hoechst33342, invitrogen) were applied overnight in PBSFT. After 9 washing steps with PBSFT as described above, the aggregates were washed 5 times at room temperature with PBS- / - containing 0.2% fetal bovine serum (FBS) and 0.2% Triton X-100 (PBT): 2 times for 5 minutes each time, and 3 times for 15 minutes each time. Subsequently, the sample was incubated in the dark with a 1: 1 glycerol / PBT solution for 30 minutes, then incubated with a 7: 3 glycerol / PBT solution for 30 minutes, and then the solution was replaced with a mounting medium (90% glycerol, 0.1MTris-Cl pH 8.0, 0.05g n-propyl gallate). Each aggregate was taken up into a 3 μl droplet and mounted on a glass slide with a spacer.

[0313] Immunostaining

[0314] Unless otherwise stated, the staining of gastruloids was performed according to the existing protocol. 15Human gastruloids were fixed and immunostained. The antibodies used were: 1:200 rabbit anti-CDX2 (ThermoScientific, EPR2764Y); 1:200 goat anti-GATA6 (R&D Systems, AF1700); 1:200 rabbit anti-BRACHYURY (AbCam, ab209665); 1:200 goat anti-SOX2 (R&D Systems, AF2018); 1:200 mouse anti-CDH2 (BD Biosciences, BD10920); 1:200 rat anti-CDH1 (Takara, M108), 1:100 rabbit anti-Wnt3a (ab219412), 1:200 rabbit anti-LEF1 (ab137872), 1:200 rabbit anti-FOXA2 (ab108422), 1:200 mouse anti-NOTCH1 (552466). All secondary antibodies were diluted 1:500 and included Alexa-Fluor-488, Alexa-Fluor-568, and Alexa-Fluor-647 conjugated antibodies (Invitrogen).

[0315] Adherent cells were stained with 1:200 mouse anti-CDH2 (BD Biosciences, BD10920), 1:200 rat anti-CDH1 (Takara, M108) and 1:200 rabbit anti-Brachyury (AbCam, ab209665) primary antibodies. Quantification was performed using Fiji software for the entire image (histogram) or using a line ROI through the colony (line graph).

[0316] In situ hybridization

[0317] Human gastruloid embryos were collected at 72 hours or 96 hours after aggregation. After a brief wash in PBS, they were fixed in 4% PFA at 4°C overnight or for 2 hours and stored in 100% methanol at -20°C until further use. 10, with small modifications, the whole sealed gastrula was subjected to in situ hybridization. The gastrula was rehydrated by incubating in a series of methanol (75%, 50%, 25% and 0% respectively) of gradually decreasing concentration in TBST (20mM Tris 137mM NaCl, 2.7mM KCl, 0.1% Tween, pH=7.4) for 3 to 5 minutes. After washing the gastrula in TBST, it was incubated in proteinase K (2.5 μg / ml) for 2 minutes to make it permeable to the probe, and post-fixed (post-fix) 20 minutes in 4% PFA at room temperature, and then washed again in TBST. In order to block nonspecific interactions, it was pre-hybridized at 68°C for 4 to 5 hours. Hybridization was carried out by incubating it overnight at 68°C in a specific digoxigenin (DIG) labeled RNA probe of 200ng / ml. The probe sequence used can be found in Supplementary Table 2. The next day, after washing the gastrula at 68°C, it was incubated in a blocking solution with a solution at room temperature for 1.5 hours. Then incubate overnight in an anti-DIG antibody coupled to alkaline phosphatase (Sigma) at 1:3,000 dilution in blocking buffer at 4°C. The next day, it was washed overnight at 4°C in MABT (100mM maleic acid, 150mM NaCl, 0.1% Tween, pH7.5). The gastrula was then washed 3 times with TBST and washed 3 times in alkaline phosphatase buffer (0.1MTris pH 9.5, 100mM NaCl, 0.1% Tween), and incubated at 4°C or room temperature in BM purple solution (purple solution) (Sigma) until the signal is fully developed. The gastrula was washed in TBST and post-fixed in 4% PFA for 20 minutes at room temperature. For imaging, the gastrula was suspended in CUBIC-R1A tissue clearing reagent.

[0318] RT-qPCR

[0319] Gene expression was analyzed from adherent cells using Trizol (Ambion Life Technologies) according to the manufacturer's instructions. Total RNA was quantified using NanoDrop 2000C (Thermo Scientific) and 5 μg was added to the reverse transcription reaction with Superscript III (Invitrogen) according to the manufacturer's instructions. The resulting cDNA was quantified by qPCR with SYBR Green (Merck) using a liquid handling robot (Qiagility, Qiagen) and analyzed on a RotorGeneQ thermal cycler (Qiagen). Primer sequences can be found in Supplementary Table 1. The concentration of cDNA was estimated using an in-house MAK2 analysis method.

[0320] Wide field of view imaging

[0321] Confocal imaging was performed using LSM700 (Zeiss) on a Zeiss Axiovert 200M using a 40EC Plan-NeoFluar 1.3 NA DIC oil immersion objective. Image capture was performed using Zen2010 v6 (Carl Zeiss Microscopy Ltd, Cambridge UK). All samples were fixed and immunostained before imaging, except for SMAD1-RFP; H2B-mCitrine gastrulae, which were imaged live. For gastrulae produced by the RUES2-GLR reporter line, we never observed fluorescent signals for the reporter proteins according to our fixation protocol, and therefore used the same antibody design and microscope settings as described.

[0322] The RT-PCR system was connected to a Zeiss AxioObserver Z1 (Carl Zeiss, UK) and supplied with 5% CO as described in Turner et al., 2017. 2 Widefield imaging was performed in a 37°C incubation chamber. All images were analyzed using Fiji software (Schindelin et al., 2012) and any adjustments were consistent throughout the group. The images presented have been rotated to align their AP axes horizontally when necessary, as indicated by the dark grey background.

[0323] Scanning electron microscopy

[0324] The gastrula of the people produced by RUES2-GLR system 72 hours after aggregation was washed twice with HEPES buffer, and fixed overnight at 4 ℃ in 3% glutaraldehyde, 0.05M sodium cacodylate buffer pH 7.4. The sample was washed several times in deionized water (DIW) at room temperature (RT) to remove fixative. The polyester film (Melinex) coverslip with a diameter of 12mm was covered with a large drop of poly-L-lysine solution (Sigma P4707), and incubated at room temperature for 15 minutes. Excessive solution was discharged, and the coverslip was air-dried at 37 ℃. The gastrula was transferred to the coverslip of poly-L-lysine in DIW drops, and allowed to adhere to about 30 minutes at room temperature, while ensuring that the gastrula remained covered by DIW. Excessive DIW was carefully discharged using tissue paper, and the sample was immediately frozen suddenly in the ethane cooled by liquid nitrogen. After freeze drying overnight in a liquid nitrogen cooled turbo freeze dryer (Quorum Emitech K775X), the samples were mounted on an aluminum SEM stub using a sticky carbon pad and sputter coated with 35 nm Au followed by 15 nm iridium. The samples were observed in secondary electron mode in a FEI Verios 460 scanning electron microscope using an Everhart-Thornley detector at 2 keV accelerating voltage and 25 pA probe current.

[0325] Image analysis

[0326] The dynamic reporter expression of the gastrula of people produced using RUES2-GLR reporter system has been specifically analyzed. An internal MATLAB script has been developed to evaluate the dynamic fluorescent marker expression along the AP axis of the gastrula of people. The wide field image of the gastrula is obtained at 24 hours, 48 ​​hours and 72 hours, and aligned with reference to the fluorescent reporter expression along its front and back axis. For each sample, a binary image is produced in the bright field channel, and it is used as a mask for all fluorescent channels, and the main (length) axis is identified. Therefore, for each pixel along the length axis, the sum of the intensity values ​​of each channel along the width of the aggregate is divided by the width of the gastrula at this specific point, and the normalized fluorescence intensity along the sample length is obtained. Then, this process is repeated for each fluorescent channel obtained. The gastrula used for this analysis is a full set from an experimental batch, and only when the front and back alignment or binarization are unsuccessful, the image is excluded from the analysis.

[0327] To quantify the extent of elongation in human gastruloids, brightfield channel widefield images were imported into Fiji (Schindelin et al., 2012). The length of the longest axis was measured using the straight line tool, followed by the length of the perpendicular axis at the midpoint of the longest axis. The ratio of these two values ​​was calculated and plotted by time point and condition using R. Significance was assessed using the Welch two-sample t-test.

[0328] The proportion of gastrula shapes (spherical, ovoid, elongated-short and elongated-long) was estimated for multiple independent biological replicates. This was done using an in-house method from a previously described method (Turner 2017, doi: 10.1242 / dev.150391). Briefly, images were converted to single-channel 8-bit TIFF files using FIJI (Schindelin et al., 2012). These were then processed using Python 3.6 (Python Software Foundation, https: / / www.python.org / ) and the Open-CV package (Bradski 2000) to apply a Gaussianblur to assign a mask around each gastrula shape before performing Otsu's thresholding and floodfilling with corrosion. Multiple quantitative features were then extracted from these contours and further processed using R. The categories for each shape descriptor were defined as follows: spherical, with a circularity less than or equal to 1.1 or an aspect ratio (AR) greater than or equal to 0.95; ovoid, with a circularity less than or equal to 1.2 or an AR greater than or equal to 0.9; elongated-short, with a circularity greater than 1.2 and less than or equal to 1.4 and an AR less than 0.9; and elongated-long, with a circularity less than 1.4 and an AR greater than 0.9. Image quality control was performed for empty wells or wells with debris whose shape descriptors were compromised using quantification of area or circularity outliers and manual confirmation by image inspection.

[0329] Tomo sequencing and mapping

[0330] Tomo sequencing and analysis were performed using an updated version of published methods (Junker et al., 2014; and Kruse et al., 2016). Briefly, gastrula embryos were sliced ​​along their AP axis, and the mRNA content of each slice was extracted using SORT-seq (Muraro et al., 2016). The resulting RNA-seq library was sequenced by paired end (75 bp) using the Illumina Next-Seq sequencing platform. Read 1 contains a cell or slice barcode and a unique molecular identifier (UMI). Read 2 contains biological information. Read 2 with a valid cell / slice barcode was selected and mapped to the human GRCh38 genome (ENSEMBL version 93) using STAR-2.5.3a (with default parameters), and only reads mapped to gene bodies (exons or introns) were used for downstream analysis. Reads that mapped to both exons and introns were assigned to exons. The mappability of the different samples ranged between 44% and 47%. For each cell or section, the number of transcripts was obtained as previously described (Bradski 2000). We called transcripts unique molecules based on UMI correction.

[0331] After mapping, spike-ins, ribosomal genes, and mitochondrial genes were removed from downstream analysis along with KCNQ1OT1, LARS2, and MALAT1, as these genes appeared to be associated with mapping errors and had been shown to be erroneous in earlier studies. Data were then normalized to the median number of unique transcripts per section in each gastrula, and z scores for each gene were extracted across sections.

[0332] Gene expression data analysis

[0333] For each gene, the reproducibility of the AP expression pattern between different gastrula repeats was scored using a random background model to calculate a Pearson correlation coefficient p value. The p-value threshold for selected reproducible genes was set to 0.001. These significantly reproducible genes were then clustered using the Self-Organizing Map (SOM) method, followed by hierarchical clustering, to identify general patterns of gene expression along the AP axis.

[0334] The average of the z-scores along the AP axis was used to generate the average gastruloid spectrum. When the number of slices between replicates differed, the values ​​were quadratically interpolated to fill the space using the interp1d function in the package scipy.interpolate (Python 3.6).

[0335] Differential gene expression was performed by normalizing the transcripts of each section of all gastruloids to 100,000; then merging all sections of each gastruloid together; and finally assessing significantly differentially expressed genes based on total expression using a binomial test.

[0336] For smooth gene expression line graphs, the distribution of gene expression along the slice was plotted using R and smoothed using the geom_smooth() function (method = loess, span = 0.3, level = 0.5) to minimize background variability. Therefore, for each gene expression distribution, confidence intervals (at 0.5 confidence intervals) are shown as grey bands.

[0337] Using the ENSEMBL IDs run with the DAVID Annotation tool 46 Gene Ontology (GO) term analysis was performed on each hierarchical cluster of Chiron-pretreated human gastruloids with the human genome as background model, focusing on biological process terms. Statistical correction for multiple comparisons was achieved using the Benjamini adjustment.

[0338] Gene ontology for each hierarchical clustering of human and mouse gastruloids was performed using the python package goatools (Klopfenstein et al., 2018) and the p-value was set to 0.05. Either the list of reproducible genes in each corresponding condition or the complete human transcriptome was set as the background model, focusing on biological process terms. Statistical correction for multiple comparisons was achieved using the Bonferroni adjustment.

[0339] Example 4 - Cultivation of human embryonic stem cells and human induced pluripotent stem cells using the methods of the present invention (including pretreatment with a signal modulator)

[0340] Materials and Methods - See Materials and Methods of Examples 1 to 3, with the variations described below.

[0341] An alternative 'dorsalization' protocol used 3.25 μM Chiron supplemented with 10 μM SB431542 Nodal inhibitor (SB431542), also in Nutristem, for 24 hours. They were then aggregated in E6 with 3 μM Chiron and ROCK inhibitor.

[0342] result

[0343] Aggregation of gastrula-generating Chiron and SB-431542 (SB43 in this article) pretreated cells allowed them to elongate and polarize their gene expression in a manner similar to the original method ( Fig.18ab). However, it also shows a larger, well-defined SOX2 domain at its posterior end, diffuse expression of BRA that approximates an anterior-to-posterior gradient, and no detectable SOX17 expression ( Fig.18 We analyzed two 120-h SB43-treated gastrulae using tomo-sequencing and compared their transcriptional profiles with those without Nodal signaling inhibition during pretreatment ( Fig.18 d, g, and Fig.19 ac). Although 301 genes were reproducibly localized in both cases, SB43 treatment resulted in the acquisition of 944 genes with new spatial localizations and the loss of reproducible localizations for 509 genes ( Fig.18 e). A gene cluster lost in the case of SB43 pretreatment (cluster 4, Fig.18 g) was initially observed in the posterior-most region of Chiron-prepared gastruloids and included genes known to be involved in Nodal signaling, such as DAND5, DACT2, and SHH, as well as those normally expressed in or around the nodule of mammalian embryos, including FOXA2, CHRD, PTCH2, LEFTY2, and MIXL1, consistent with a loss of Nodal activity.

[0344] Of the genes that were reproducible under either or both conditions, 564 genes (33%) were differentially expressed between the two treatments ( Fig.18 f), including downregulation of many Nodal signaling components and targets. In contrast, we observed upregulation of several HOX genes (HOXC9, HOXA9, HOXC6) and SOX2 ( Fig.18 f). When we specifically examined germ layer markers, we noted decreased expression of genes associated with the definitive endoderm (SHH, LHX1, CER1, FOXA3, SORCS2, FOXA2), consistent with a role for Nodal in specification of this germ layer, and loss of many genes associated with cardiac development (TBX5, GATA6, LBX1, NKX2.5), likely a consequence of loss of endoderm induction ( Fig.19 de).

[0345] References

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[0355] Mendjan S,Mascetti VL,Ortmann D,Ortiz M,Karjosukarso DW,Ng Y,MoreauT,Pedersen RA.(2014)NANOG and CDX2 pattern distinct subtypes of humanmesoderm during exit from pluripotency.Cell Stem Cell 4;15(3):310-325.

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Claims

1. A polarized three-dimensional cell aggregate produced in vitro from one or more human pluripotent stem cells; wherein: (a) The polarized three-dimensional cell aggregate contains: (A)： i. a cell comprising one or more markers characteristic of an endoderm cell or an endoderm cell derivative selected from one or more of the primordium of the esophagus, stomach, intestine, lung, pancreas, liver, trachea, thymus and / or thyroid gland, ii. a cell comprising one or more markers characteristic of a mesoderm cell or a mesoderm cell derivative, wherein the mesoderm cell derivative is selected from the group consisting of paraxial mesoderm or axial mesoderm, and iii. a cell comprising one or more markers characteristic of an ectodermal cell or an ectodermal cell derivative, wherein the ectodermal cell derivative is selected from one or more of a neural plate or a neural tube cell; or (B)： cells containing one or more markers characteristic of primordial germ cells; or (C)： i. a cell comprising one or more markers characteristic of an endoderm cell or an endoderm cell derivative selected from one or more of the primordium of the esophagus, stomach, intestine, lung, pancreas, liver, trachea, thymus and / or thyroid gland, ii. a cell comprising one or more markers characteristic of a mesoderm cell or a mesoderm cell derivative selected from the group consisting of paraxial mesoderm or axial mesoderm, iii. A cell comprising one or more markers characteristic of an ectodermal cell or an ectodermal cell derivative, wherein the ectodermal cell derivative is selected from one or more of a neural plate cell or a neural tube cell, and iv. cells containing one or more markers characteristic of primordial germ cells; and (b) The polarized three-dimensional cell aggregate is polarized along an anterior-posterior axis, wherein the anterior-posterior axis is defined by at least an anterior cell region and a posterior cell region, and wherein cells in the anterior region express higher or lower levels of one or more genes than cells in the posterior region.

2. The polarized three-dimensional cell aggregate of claim 1, wherein: (i) the polarized three-dimensional cell aggregate is polarized along a dorsal-ventral axis, wherein the dorsal-ventral axis is defined by at least a dorsal cell region and a ventral cell region, wherein cells in the dorsal region express higher or lower levels of one or more genes than cells in the ventral region; and / or (ii) the polarized three-dimensional cell aggregates are polarized along an inside-outside axis, wherein the inside-outside axis is defined by at least an inside cell region and two outside cell regions, wherein cells in the inside region express higher or lower levels of one or more genes than cells in the outside regions; and / or (iii) the polarized three-dimensional cell aggregate is polarized along a left-right axis, wherein the left-right axis is defined by at least a left cell region and a right cell region, wherein cells in the left region express higher or lower levels of one or more genes than cells in the right region.

3. The polarized three-dimensional cell aggregate of claim 1 or claim 2, wherein: (i) the cells of the anterior region express lower levels 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) cells in the anterior region express higher levels of one or more genes than cells in the posterior region, and wherein the one or more genes are selected from GATA6, HAND2, PRDM1, TBX1, BMP2, CDH3, LHX1, PAX8 and BMP4.

4. The polarized three-dimensional cell aggregate of claim 1 or 2, wherein the polarized three-dimensional cell aggregate comprises two or more of the following: a. a region of cells expressing at least BRA, b. a region of cells expressing at least SOX2, c. a region of cells expressing at least TBX6, d. a region of cells expressing at least MEOX1, e. a region of cells expressing at least MESP2, f. A region of cells expressing at least TCF15; g. A region of cells expressing at least GATA6; and h. A region of cells expressing at least BMP2; Two or more of (a) to (h) are arranged from back to front in the polarized three-dimensional cell aggregate.

5. The polarized three-dimensional cell aggregate of claim 1 or 2, wherein the anterior-posterior axis is further defined by a central cell region between the anterior cell region and the posterior cell region, wherein cells in the central region express higher or lower levels of one or more genes than cells in the anterior or posterior regions.

6. The polarized three-dimensional cell aggregate of claim 5, wherein the cells in the central region express higher levels of one or more genes than the cells in 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.

7. The polarized three-dimensional cell aggregate of claim 1 or 2, wherein the three-dimensional cell aggregate exhibits spatial colinearity of HOX gene expression along the anterior-posterior axis.

8. The polarized three-dimensional cell aggregate of claim 2, wherein: (i) cells in the dorsal region express lower levels of one or more genes than cells in the ventral region, and wherein the one or more genes are selected from the group consisting of SHH, NODAL, LEFTY1, LEFTY2, TBX6 and KDR; and / or (ii) cells in the dorsal region express higher levels of one or more genes than cells in the ventral region, and wherein the one or more genes are selected from SOX2, OTX2, IRX3, SOX1, POU3F1, POU3F2 and PAX6; and / or (iii) cells in the medial region express lower levels of one or more genes than cells in the lateral region, 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 inner region express higher levels of one or more genes than the cells of the outer region, and wherein the one or more genes are selected from SOX1, SOX2, DAND5, CER1, FOXA2 and NOTO1; and / or (v) the cells in the right region express lower or higher levels of one or more genes than the cells in the left region, and wherein the one or more genes are selected from NODAL, LEFTY1, LEFTY2 and PITX2.

9. The polarized three-dimensional cell aggregate of claim 1 or 2, wherein the one or more endoderm cell or endoderm cell derivative characteristic markers are one or more genes whose expression is characteristic of endoderm cells or endoderm cell derivatives, wherein: (i) the one or more genes whose expression is characteristic of endoderm cells or endoderm cell derivatives are selected from the group consisting of GSC, CDX2, NEDD9, PYY, SHH, SORCS2, CER1, SOX17, FOXA2, TRH1 and FOXA1; or (ii) the one or more genes whose expression is characteristic of endoderm cells or endoderm cell derivatives is one or more genes whose expression is characteristic of mesendoderm cells or mesendoderm cell derivatives, wherein the one or more genes whose expression is characteristic of mesendoderm cells or mesendoderm cell derivatives are selected from BRA, MIXL1, LEFTY1, LEFTY2, AXIN2, TRH1, NODAL, WNT3a, WMT5a, Dll1 and CDX2.

10. The polarized three-dimensional cell aggregate of claim 1 or 2, wherein the one or more markers characteristic of mesodermal cells or mesodermal cell derivatives are one or more genes whose expression is characteristic of mesodermal cells or mesodermal cell derivatives, wherein the one or more markers characteristic of mesodermal cells or mesodermal cell derivatives are selected from BRA, MEOX1, OSR1, PAX2, ALDH1A2, MESP1, MESP2, TBX6, TCF15, MEOX1, FLK1 / KDR, FOXA2 and TBX1.

11. The polarized three-dimensional cell aggregate of claim 10, wherein the one or more markers characteristic of mesoderm cells or mesoderm cell derivatives are one or more genes whose expression is characteristic of mesoderm cells or mesoderm cell derivatives, and wherein the one or more genes whose expression is characteristic of mesoderm cells or mesoderm cell derivatives are one or more genes whose expression is characteristic of axial mesoderm.

12. The polarized three-dimensional cell aggregate of claim 11, wherein: (i) the one or more genes whose expression is characteristic of axial mesoderm are selected from BRA, FOXA2, NOTO1, CER1, SHH and NOGGIN; or (ii) the one or more genes whose expression is characteristic of axial mesoderm cells is one or more genes whose expression is characteristic of paraxial mesoderm.

13. The polarized three-dimensional cell aggregate of claim 1 or 2, wherein: The one or more markers characteristic of ectoderm cells or ectoderm cell derivatives are one or more genes whose expression is characteristic of ectoderm cells or ectoderm cell derivatives; and wherein the one or more markers characteristic of ectoderm cells or ectoderm cell derivatives are one or more markers characteristic of neural cells; and wherein the one or more markers characteristic of neural cells are one or more genes whose expression is characteristic of neural cells, wherein the one or more genes are selected from SOX1, SOX2, SOX3, POU3F1, POU3F2, PAX6, NKX1.2 and ZEB2.

14. The polarized three-dimensional cell aggregate of claim 1 or 2, wherein the one or more markers characteristic of ectoderm cells or ectoderm cell derivatives are one or more genes whose expression is characteristic of ectoderm cells, wherein the one or more genes are selected from OTX2, GBX2, SIX1, SIX3, SOX2, SOX3, DLX5, EYA2 and BARX1.

15. The polarized three-dimensional cell aggregate of claim 1 or 2, wherein the polarized three-dimensional cell aggregate comprises one or more progenitor cells.

16. The polarized three-dimensional cell aggregate of claim 15, wherein the one or more progenitor cells are: a. Hematopoietic progenitor cells; b. Cardiac progenitor cells; c.Paraxial mesoderm; d. a somite and / or a somite derivative selected from one or more of a dermatome, a myotome and an osteomeme cell; e. neural crest; f. neuroectoderm and / or neuroectoderm derivatives selected from one or more of neural plate cells, neural tube cells and neurons; g. a placode ectoderm and / or a placode ectoderm derivative selected from one or more of an ear primordium and a nasal primordium; h. mid-mesoderm progenitor cells and / or mid-mesoderm progenitor cell derivatives selected from one or more of the kidney primordium and the gonad primordium; i. Axial mesodermal progenitor cells; j. neural mesodermal progenitor cells and / or neural mesodermal progenitor cell derivatives selected from one or more of spinal cord neural progenitor cells and paraxial mesoderm; k. lateral plate mesoderm and / or lateral plate mesoderm derivatives selected from one or more of cardiac cells, hematopoietic cells and limb cells; l. Primordial germ cells; m. Nodule cells; and / or n. Endoderm.

17. The polarized three-dimensional cell aggregate of claim 1 or 2, wherein: (i) the polarized three-dimensional cell aggregates are produced in vitro from one or more human embryonic stem cells (ESCs), or the polarized three-dimensional cell aggregates are produced in vitro from one or more human induced pluripotent stem cells (iPSCs); and / or (ii) The three-dimensional cell aggregates are generated in vitro from a single pluripotent stem cell.

18. A method for obtaining polarized three-dimensional cell aggregates, the method comprising: (a) pre-treating one or more human pluripotent stem cells, wherein the pre-treating step comprises culturing the human pluripotent stem cells in a medium comprising an activator of Wnt signaling, wherein the activator of Wnt signaling is CHIR99021; (b) generating a cell suspension from the pretreated one or more human pluripotent stem cells, wherein the cell suspension comprises one or more dissociated human pluripotent stem cells; (c) culturing the cell suspension in a culture medium comprising an activator of Wnt signaling and a Rock inhibitor that promotes conversion of at least one of the dissociated human pluripotent stem cells into three-dimensional cell aggregates; and (d) culturing the three-dimensional cell aggregates in a basic differentiation medium that promotes conversion of the three-dimensional cell aggregates into polarized three-dimensional cell aggregates; Wherein the polarized three-dimensional cell aggregate is a polarized three-dimensional cell aggregate as defined in any one of claims 1 to 17.

19. A method for obtaining one or more progenitor cells, the method comprising: (a) pre-treating one or more human pluripotent stem cells, wherein the pre-treating step comprises culturing the human pluripotent stem cells in a medium comprising an activator of Wnt signaling, wherein the activator of Wnt signaling is CHIR99021; (b) generating a cell suspension from the pretreated one or more human pluripotent stem cells, wherein the cell suspension comprises one or more dissociated human pluripotent stem cells; (c) culturing the cell suspension in a medium comprising an activator of Wnt signaling and a Rock inhibitor that promotes conversion of at least one of the dissociated pluripotent stem cells into three-dimensional cell aggregates; (d) culturing the three-dimensional cell aggregate in a basic differentiation medium that promotes conversion of the three-dimensional cell aggregate into a polarized three-dimensional cell aggregate, wherein the polarized three-dimensional cell aggregate is a polarized three-dimensional cell aggregate as defined in any one of claims 1 to 17; and (e) culturing the polarized three-dimensional cell aggregates under conditions that promote differentiation of one or more cells of the polarized three-dimensional cell aggregates into progenitor cells.

20. The method of claim 19, wherein the one or more progenitor cells are: a. Hematopoietic progenitor cells; b. Cardiac progenitor cells; c.Paraxial mesoderm; d. a somite and / or a somite derivative selected from one or more of a dermatome, a myotome and an osteomeme cell; e. neural crest; f. neuroectoderm and / or neuroectoderm derivatives selected from one or more of neural plate cells, neural tube cells and neurons; g. a placode ectoderm and / or a placode ectoderm derivative selected from one or more of an ear primordium and a nasal primordium; h. mid-mesoderm progenitor cells and / or mid-mesoderm progenitor cell derivatives selected from one or more of the kidney primordium and the gonad primordium; i. Axial mesodermal progenitor cells; j. neural mesodermal progenitor cells and / or neural mesodermal progenitor cell derivatives selected from one or more of spinal cord neural progenitor cells and paraxial mesoderm; k. lateral plate mesoderm and / or lateral plate mesoderm derivatives selected from one or more of cardiac cells, hematopoietic cells and limb cells; l. Primordial germ cells; m. Nodule cells; and / or n. Endoderm.

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