Patent Application
20250223559
The present disclosure relates to a three-dimensional cellular aggregate termed axioloid generated in vitro from a pluripotent stem cell and to a method for producing the same.
The segmented body plan of vertebrates is established during somitogenesis, a well-studied process in model organisms, but remains largely elusive in humans due to ethical and technical limitations. Despite recent advances with pluripotent stem cell (PSC)-based approaches1-5, a system that robustly recapitulates the human embryo somitogenesis process in vitro in both space and time, including its characteristic morphogenetic features including the oscillatory expression of segmentation clock genes and the related sequential formation of epithelial somites along the anterior to posterior axis of the embryo, remains largely missing.
It is an object of the present disclosure to provide a new three-dimensional cellular aggregate.
In order to achieve the object above, the present disclosure provides a three-dimensional cellular aggregate generated in vitro from a pluripotent stem cell, comprising: a mesodermal cell, wherein the cellular aggregate has a polarity in an antero-posterior axis or a rostro-caudal axis and an apical-basolateral axis, and the cellular aggregate can reconstitute various aspects of somitogenesis and axial development, including axial elongation, segmentation, epithelial somite formation and patterning (formation of one or more somite like structures), and oscillation of the segmentation clock under a somitogenic culture condition.
The present disclosure can provide a new three-dimensional cellular aggregate.
The present disclosure relates to a three-dimensional cellular aggregate termed axioloid generated in vitro from a pluripotent stem cell and to a method for producing the same.
The present disclosure also relates to a three-dimensional cellular aggregate generated in vitro from a pluripotent stem cell, comprising: a mesodermal cell, wherein the cellular aggregate has a polarity in an antero-posterior axis or a rostro-caudal axis and an apical-basolateral axis, and the cellular aggregate can reconstitute various aspects of somitogenesis and axial development, including axial elongation, segmentation, epithelial somite formation and patterning (formation of one or more somite like structures), and oscillation of the segmentation clock under a somitogenic culture condition.
The present disclosure also relates to a three-dimensional cellular aggregate generated in vitro from a pluripotent stem cell, comprising: a mesodermal cell and/or progenitor cell wherein the cellular aggregate has a polarity in an antero-posterior axis or a rostro-caudal axis and an apical-basolateral axis, and the cellular aggregate can reconstitute various aspects of somitogenesis and axial development, including e.g. axial elongation, segmentation, epithelial somite formation and patterning (formation of one or more somite like structures), and oscillation of the segmentation clock under a somitogenic culture condition.
The present disclosure also relates to a three-dimensional cellular aggregate generated in vitro from a pluripotent stem cell, comprising: a mesodermal cell, wherein the cellular aggregate has a polarity in an antero-posterior axis or a rostro-caudal axis and an apical-basolateral axis, and a proportion of the mesodermal cell in the cellular aggregate is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, based on the number of cells.
The present disclosure also relates to a method for producing a three-dimensional cellular aggregate generated in vitro from a pluripotent stem cell, comprising the steps of: (a) culturing a pluripotent stem cell to induce a three-dimensional cellular aggregate comprising a mesodermal cell; and (b) culturing the cellular aggregate comprising the mesodermal cell to induce a three-dimensional cellular aggregate, wherein the three-dimensional cellular aggregate is the cellular aggregate.
The present disclosure also relates to a method for producing a progenitor cell or a differentiated cell, comprising the step of: culturing the cellular aggregate to induce the progenitor cell or the differentiated cell selected from the group consisting of the following (a) to (f): (a) neuro-mesodermal cell or a progenitor cell thereof; (b) a muscle cell or a progenitor cell thereof; (c) an osteocyte or a progenitor cell thereof; (d) a chondrocyte or a progenitor cell thereof; (e) a tenocyte or a progenitor cell thereof; and (f) an endothelial or hemogenic cell or a progenitor cell thereof.
The present disclosure also relates to a method for producing a progenitor cell or a differentiated cell, comprising the step of: culturing the cellular aggregate to induce the progenitor cell or the differentiated cell selected from the group consisting of the following (a) to (i): (a) neuro-mesodermal cell or a progenitor cell thereof; (b) a muscle cell or a progenitor cell thereof; (c) an osteocyte or a progenitor cell thereof; (d) a chondrocyte or a progenitor cell thereof; (e) a tenocyte or a progenitor cell thereof; and (f) an endotome, endothelial or hemogenic cell or a progenitor cell thereof, (g) an adipocyte cell including white, beige and brown cell or a progenitor cell thereof; (h) a dermis cell or a progenitor cell thereof; and (i) a neural tube cell or a progenitor cell thereof.
The present disclosure also relates to a method for evaluating a test substance, comprising the steps of: culturing a test substance in the presence of a three-dimensional cellular aggregate; and evaluating the three-dimensional cellular aggregate after the culture, wherein the three-dimensional cellular aggregate is the cellular aggregate.
The present disclosure also relates to a method for evaluating gene function or genome function, comprising the steps of: preparing a pluripotent stem cell in which a test gene or a test genome is modified; generating a three-dimensional cellular aggregate from the pluripotent stem cell; and evaluating a three-dimensional cellular aggregate after the culture, wherein the generation of the three-dimensional cellular aggregate is carried out by the method.
The present disclosure also relates to a method for evaluating gene function or genomic sequence function, comprising the steps of: preparing a pluripotent stem cell in which a test gene or a test genomic sequence is modified; generating a three-dimensional cellular aggregate from the pluripotent stem cell; and evaluating a three-dimensional cellular aggregate after the culture, wherein the generation of the three-dimensional cellular aggregate is carried out by the method.
In the present disclosure, the “lower” is used to intend a group having less number and/or amount of a subject compared with criterion, unless otherwise provided.
In the present disclosure, the “higher” is used to intend a group having more number and/or amount of a subject compared with criterion, unless otherwise provided.
In the present disclosure, suitable example of “one or more” may be the number of 1 to 6, in which the preferred one may be the number of 1 to 3.
In the present disclosure, the “marker” means a nucleic acid, a gene, a polypeptide, or a protein that is expressed to a different extent in a target cell. When the marker is a positive marker, the different extent means increased expression compared to the cells to be compared with. When the marker is a negative marker, the different extent means reduced expression compared to the cells to be compared with.
In the present disclosure, the “pluripotent cell” means a cell capable of differentiating into ectodermal, mesodermal, and endodermal cells. The pluripotent cell may also be referred to as a pluripotent stem cell when the pluripotent cell is capable of self-replication.
In the present disclosure, the “ectodermal cell” means a cell destined to be capable of differentiating into neural tissues such as nerves; epithelial tissues such as epidermis; and the like if there is a developmentally appropriate stimulation, and is a cell expressing an ectodermal cell marker to be described below.
In the present disclosure, the “mesodermal cell” means a cell destined to be capable of differentiating into connective tissue, such as bones, cartilages, blood vessels, lymphatic vessels and the like; muscle tissues; and the like if there is a developmentally appropriate stimulation, and is a cell expressing a mesodermal cell marker to be described below.
In the present disclosure, the “mesodermal cell” means a cell destined to be capable of differentiating into connective tissue, such as bones, cartilages, fat, blood vessels, lymphatic vessels and the like; muscle tissues; and the like if there is a developmentally appropriate stimulation, and is a cell expressing a mesodermal cell marker to be described below.
In the present disclosure, the “endodermal cell” means a cell destined to be capable of differentiating into thymus; digestive organs such as stomach, intestine, liver, and the like; respiratory organs such as trachea, bronchi, lungs, and the like; and urinary organs such as bladder, urethra, and the like; and the like if there is a developmentally appropriate stimulation, and is a cell expressing an endodermal cell marker to be described below.
In the present disclosure, the “three-dimensional cellular aggregate” means a structure in which cells are aggregated in three dimensions. The three-dimensional culture aggregate is different from, for example, a two-dimensional cellular aggregate (cell sheet) obtained in a planar culture, and forms a three-dimensional structure by, for example, accumulating cells in a thickness direction.
In the present disclosure, the sequence information of a protein or a nucleic acid encoding the same (e.g., DNA or RNA) described herein is available from Protein Data Bank, UniProt or Genbank.
The present disclosure refers to a cellular aggregate (hereinafter also referred to as “axioloid”) generated in vitro from one or more pluripotent stem cells, a method for producing such cellular aggregate, and the cell derived, obtained or obtainable from the cellular aggregate.
Certain aspect of the cellular aggregate (cell aggregate) described in the present disclosure includes being a three-dimensional cellular aggregate generated in vitro from pluripotent stem composed of mesodermal cells (including primitive streak and presomitic mesoderm cells), wherein the cellular aggregate has a polarity along its antero-posterior and apical-basolateral axis or can obtain, induce, or acquire a polarity along its antero-posterior and apical-basolateral axis, and the cellular aggregate can reconstitute various aspects of somitogenesis and axial development, including axial elongation, segmentation, epithelial somite formation and patterning (formation of one or more somite like structures), and oscillation of the segmentation clock under a somitogenic culture condition. The antero-posterior axis may also be referred to as a rostro-caudal axis.
Certain aspect of the cellular aggregate (cell aggregate) described in the present disclosure includes being a three-dimensional cellular aggregate generated in vitro from pluripotent stem cell-derived mesodermal cells (including primitive streak and presomitic mesoderm cells), wherein the cellular aggregate has a polarity along its antero-posterior and apical-basolateral axis or can establish, induce, or acquire a polarity along its antero-posterior and apical-basolateral axis, and the cellular aggregate can reconstitute various aspects of somitogenesis and axial development, including axial elongation, segmentation, epithelial somite formation and patterning (formation of one or more somite like structures), and oscillation of the segmentation clock under a somitogenic culture condition. The antero-posterior axis may also be referred to as a rostro-caudal axis.
In one another aspect, the cellular aggregate described in the present disclosure includes being a polarized three-dimensional cellular aggregate generated in vitro from pluripotent stem composed of mesodermal cell, wherein the cellular aggregate has a polarity along its antero-posterior and apical-basolateral axis or can obtain, induce, or acquire a polarity along its antero-posterior and apical-basolateral axis, and the cellular aggregate can form one or more somites or somite-like structures under a somitogenic culture condition.
In another aspect, the cellular aggregate described in the present disclosure includes being a polarized three-dimensional cellular aggregate generated in vitro from pluripotent stem cell-derived mesodermal cells and progenitor cells, wherein the cellular aggregate has a polarity along its antero-posterior and apical-basolateral axis or can obtain, induce, or acquire a polarity along its antero-posterior and apical-basolateral axis, and the cellular aggregate can form one or more somites or somite-like structures under a somitogenic culture condition.
In another aspect, a cellular aggregate of the present disclosure is a three-dimensional cellular aggregate generated in vitro from a pluripotent stem cell, composed of mesodermal cells, wherein the cellular aggregate has a polarity along its anteroposterior and apical-basolateral axis or can obtain, induce, or acquire a polarity along its antero-posterior and apical-basolateral axis, and a proportion of the mesodermal cells in the cellular aggregate is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, based on the number of cells. The proportion is preferably at least 50%, more preferably at least 90%.
In the present disclosure, the antero-posterior axis may be defined by an anterior (rostral) region and a posterior (caudal) region. An anterior region cell has, for example, a higher or lower expression of one or more markers as compared to a posterior region cell.
In the present disclosure, the anterior region cell may have a lower expression of one or more markers as compared to the posterior region cell. In this case, for example, the one or more markers are selected from the group consisting of TBXT, SOX2, CYP26 A1, FGF3, FGF4, FGF8, FGF17, WNT3a, WNT5a, WNT5b, TBX6, HES7, MSGN1, MEOX1, TCF15, HOXD13, HOXB, HOXA9, HOXA10 and CDX2, preferably TBXT, SOX2, TBX6, HES7, MEOX1 and TCF15, more preferably TBXT. One type or two or more types of them may be used as the marker.
In the present disclosure, the anterior region cell may have a higher or lower expression of one or more markers as compared to the posterior region cell. In this case, for example, the one or more markers are selected from the group consisting of LFNG, MEOX1, TCF15, UNCX, TBX18, ALDH1 A2 and RDH10. The anterior region may include somitic mesoderm (SM) and head mesoderm-like cells.
In the present disclosure, the posterior (caudal) region may include tailbud (TB) like-cells.
In the present disclosure, the apical-basolateral axis may be defined by an apical region and a basolateral region. In this case, for example, an apical region of a cell has a higher or lower expression of one or more markers comprising aPKC, CDH2, Ezrin and ZO1 as compared to a basolateral region of a cell.
In the present disclosure, the apical region of a cell may have a lower expression of one or more markers as compared to the basolateral region of a cell. In this case, for example, the one or more markers are selected from the group consisting of Fibronectin, Collagen and Laminin. One type or two or more types of them may be used as the marker.
In the present disclosure, the apical region of a cell may have a higher expression of one or more markers as compared to the basolateral region of a cell. In this case, for example, the one or more markers are selected from the group consisting of CDH2, aPKC, Ezrin, ZO1 and F-actin, and is preferably, CDH2 and/or aPKC. One type or two or more types of them may be used as the marker.
In the present disclosure, for example, a proportion of the mesodermal cell in the cellular aggregate is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, based on the number of cells. The proportion is preferably at least 50%, more preferably at least 90%.
The mesodermal cell expresses one or more markers (mesodermal cell marker) selected from the group consisting of BRA, MIXL1, NODAL, WNT3a, WNT5a, WNT5b, DLL1, CYP26 A1, TBX6, HES7, MSGN1, RIPPLY1, RIPPLY2, MESP1, MESP2, MEOX1, TCF15, TBX18, UNCX, ALDH1 A2, RDH10 and FLK1/KDR, for example. One type or two or more types of them may be used as the marker.
The mesodermal cell expresses one or more markers (mesodermal cell marker) selected from the group consisting of BRA, SOX2, MIXL1, NODAL, WNT3a, WNT5a, WNT5b, DLL1, CYP26 A1, TBX6, HES7, MSGN1, RIPPLY1, RIPPLY2, MESP1, MESP2, MEOX1, TCF15, TBX18, UNCX, ALDH1 A2, RDH10 and FLK1/KDR, for example. One type or two or more types of them may be used as the marker.
The cellular aggregate of the present disclosure can reconstitute various aspects of somitogenesis and axial development, including axial elongation, segmentation, epithelial somite formation and patterning (formation of one or more somite like structures), and oscillation of the segmentation clock under a somitogenic culture condition, for example. The somite and somite-like structure can be defined as the expression of one or more markers selected from the group consisting of MEOX1, TCF15, FOXC2, TBX18, UNCX, ALDH1 A2 and RDH10.
The cellular aggregate of the present disclosure can reconstitute various aspects of somitogenesis and axial development, including axial elongation, segmentation, epithelial somite formation and patterning (formation of one or more somite like structures), and oscillation of the segmentation clock under a somitogenic culture condition, for example. The somite and somite-like structure can be defined as the expression of one or more markers selected from the group consisting of MEOX1, TCF15, FOXC2, PAX3, TBX18, UNCX, ALDH1 A2 and RDH10.
The cellular aggregate of the present disclosure can form a somite or a somite-like structure under a somitogenic culture condition, for example. The somite and somite-like structure can be defined as the expression of one or more markers selected from the group consisting of MEOX1, TCF15, FOXC2, TBX18, UNCX, ALDH1 A2 and RDH10.
The cellular aggregate of the present disclosure can form a somite or a somite-like structure under a somitogenic culture condition, for example. The somite and somite-like structure can be defined as the expression of one or more markers selected from the group consisting of MEOX1, TCF15, FOXC2, PAX3, TBX18, UNCX, ALDH1 A2 and RDH10.
The somitogenic culture condition is, for example, a presence of a gel and/or a matrix and a retinoic acid, a retinoic acid precursor or its derivative and/or a retinoic acid receptor (RAR) agonist.
The somitogenic culture condition is, for example, a presence of a gel and/or a matrix and a retinoid, including retinoic acid, a retinoic acid precursor or its derivative and/or a retinoic acid receptor (RAR) agonist.
The RAR agonist includes, for example, Vitamin A, retinol, retinal, 9-cis retinoic acid, all-trans type retinoic acid (ATRA), TTNPB, AM580, AM80, LGD1550, E6060, AGN193312, AM555S, CD2314, AGN193174, LE540, CD437, CD666, CD2325, SR11254, SR11363, SR11364, AGN193078, TTNN(Ro19-0645), CD270, CD271, CD2665, SR3985, AGN193273, Ch55, 2AGN190521, CD2366, AGN193109 and/or Re80, preferably, retinal or retinol.
The retinoid includes, for example, Vitamin A, retinol, retinal, 9-cis retinoic acid, 13-cis-retinoic acid, all-trans type retinoic acid (ATRA), TTNPB, AM580, AM80, LGD1550, E6060, AM555S, CD2314, CD437, CD666, CD2325, SR11254, SR11364, TTNN(Ro19-0645), CD-270, CD271, SR3985, and/or Ch55 preferably, retinal or retinol.
The cellular aggregate is, for example, embedded in the gel or the matrix, or disposed inside the gel or the matrix.
The matrix includes, for example, an extracellular matrix. The extracellular matrix includes, for example, collagen, laminin, fibronectin, vitronectin, gelatin and/or entactin. The matrix can be used at various concentrations (e.g., 1%, or 5%, or 10% or 20%) per volume of utilized embedding media. The concentration is preferably 5%, more preferably 10%.
One type or two or more types of them used as the extracellular matrix.
The gel includes, for example, a hydrogel. The gel includes, for example, a basement membrane matrix. The basement membrane matrix includes, for example, one or more group comprising laminin, collagen, fibronectin, gelatin, vitronectin, heparan sulphate proteoglycan and/or entactin. One type or two or more types of them used as the basement membrane matrix. The gel includes, for example, an acrylamide gel, an arginine gel, an agarose gel and/or a polyethylene glycol hydrogel with various biomechanical properties.
In the present disclosure, the somite or the somite-like structure may include an anterior (rostral) portion and/or a posterior (caudal) portion in the antero-posterior axis. An anterior portion cell has, for example, a higher or lower expression of one or more markers as compared to a posterior portion cell.
The anterior (rostral) portion cell may have a higher expression of one or more markers than the posterior portion cell. In this case, for example, the one or more markers are selected from the group consisting of TBX18 and ALDH1 A2, and is preferably TBX18. One type or two or more types of them may be used as the marker.
The anterior portion cell may have a lower expression of one or more markers as compared to the posterior portion cell. In this case, for example, the one or more markers are selected from the group consisting of UNCX and LNFG, and is preferably UNCX. One type or two or more types of them may be used as the marker.
The cellular aggregate of the present disclosure may include anterior paraxial/presomitic mesoderm (aPSM). The aPSM means an area on the posterior (caudal) side of the axioloid located between PSM and SM. The aPSM can be defined by the expression of one or more makers selected from a group of markers consisting of MESP2, RIPPLY2, RIPPLY1 and PCDH8, preferably MESP2.
In the present disclosure, the somite is formed, for example, in a cycle of 3 to 7 hours, a cycle of 3.5 to 6.6 hours or a cycle of 4 to 6 hours. The somite is formed preferably in a cycle of 3.5 to 6.6 hours, more preferably in a cycle of 4 to 6 hours.
In the present disclosure, the length of the somite in the antero-posterior axis is, for example, 30 to 200 μm, 50 to 150 μm, 60 to 140 μm, 70 to 130 μm or 80 to 120 μm. The length of the somite is preferably 30 to 200 m, more preferably 80 to 120 m.
The cellular aggregate may include pluripotent stem cells.
In the present disclosure, the pluripotent stem cell expresses one or more markers (pluripotent stem cell marker) selected from the group consisting of OCT4, SOX2, NANOG, ABCG2, CRIPTO, FOXD3, Connexin43, Connexin45, hTERT, UTF1, ZFP42, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, LIN28 and REX1, for example. One type or two or more types of them (preferably OCT4, more preferably NANOG) may be used as the marker.
In the present disclosure, the pluripotent stem cell expresses one or more markers (pluripotent stem cell marker) selected from the group consisting of OCT4, SOX2, NANOG, ABCG2, CRIPTO, FOXD3, Connexin43, Connexin45, hTERT, UTF1, ZFP42, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, LIN28 and REX1. One type or two or more types of them (preferably OCT4, more preferably NANOG) may be used as the marker.
The proportion of the pluripotent stem cell in the cellular aggregate is, for example, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, based on the number of cells. The proportion is preferably 1%, more preferably less than 1%.
The proportion of the pluripotent stem cell defined by the expression of NANOG in the cellular aggregate is, for example, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, based on the number of cells. The proportion is preferably 1%, more preferably less than 1%.
The cellular aggregates do not substantially include, for example, an endodermal cell and/or an ectodermal cell.
The proportion of the endodermal cell in the cellular aggregate is 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, based on the number of cells. The proportion is preferably 1%, more preferably less than 1%.
The endodermal cell expresses one or more markers (endodermal cell marker) selected from the group consisting of GATA6, GSC, CDX2, NEDD9, PYY, SHH, SORCS2, CER1, SOX17, FOXA2, TRH1 and FOXA1, and preferably GATA6 and/or SHH, for example. One type or two or more types of them (preferably FOXA2, more preferably GATA6) may be used as the marker.
The proportion of the ectodermal cell in the cellular aggregate is, for example, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, based on the number of cells. The proportion is preferably 1%, more preferably less than 1%.
The ectodermal cell expresses one or more markers (ectodermal cell marker) selected from the group consisting of OTX2, GBX2, SIX1, SIX3, SOX1, SOX3, DLXS, EYA2 and BARX1, and preferably OTX2, for example. One type or two or more types of them (preferably SOX1, more preferably OTX2) may be used as the marker.
In the cellular aggregate, an expression of a segmentation clock gene may be subjected to gene oscillation. The gene oscillation means, for example, that the expression level of the target gene is periodically oscillating in space and time. The gene oscillation is also referred to as, for example, a gene expression oscillation.
The segmentation clock gene is a gene selected from the group consisting of LFNG, DKK1, DLL1, DLL3 and HES7, and is preferably HES7. One type or two or more types of them (preferably LFNG, more preferably HES7) may be used as the segmentation clock gene.
The cycle of the gene oscillation is, for example, a cycle of 3 to 7 hours, preferably a cycle of 3.5 to 6.6 hours or more preferably a cycle of 4 to 6 hours.
The pluripotent stem cell used for derivation of axioloids is, for example, a human pluripotent stem cell or a non-human animal pluripotent stem cell. Examples of the non-human animal include amniotes such as a mouse, a rat, a rabbit, a dog, a cat, a cow, a horse, a pig, a monkey, an ape, a dolphin, an elephant, a sea lion, a snake, a gecko, a chicken and the like. The pluripotent stem cell is, for example, an embryonic stem cell or an artificial pluripotent stem cell.
The pluripotent stem cell used for derivation of axioloids is, for example, a human pluripotent stem cell or a non-human animal pluripotent stem cell. Examples of the non-human animal include amniotes such as a mouse, a rat, a rabbit, a dog, a cat, a cow, a horse, a pig, a monkey, an ape, a dolphin, a whale, an armadillo, a tenrec, an elephant, a sea lion, a snake, a gecko, a chicken and the like. Non-human animal pluripotent stem cells also include monotreme species, such as a platypus or an echidna and marsupial species, such as an opossum, a kangaroo, a wombat or the like. The pluripotent stem cell is, for example, an embryonic stem cell or an artificially engineered pluripotent stem cell.
The pluripotent stem cell may be, for example, a pluripotent stem cell in which a gene or genome is modified. Examples of the modification include introduction of a mutation into a coding or non-coding gene or genome region, repair of a coding or non-coding (regulatory) gene or genome mutation, introduction of a foreign gene or non-coding (regulatory) genome region, and knockout of a gene or genome region. The modification of the gene or genome can be performed using, for example, genome editing techniques such as ZFN, TALEN, CRISPR/Cas systems and the like; gene recombination techniques; and the like.
The pluripotent stem cell may be, for example, a pluripotent stem cell in which a gene or genomic sequence is modified. Examples of the modification include introduction of a mutation into a coding or non-coding gene or genome region, repair of a coding or non-coding (regulatory) gene or genome mutation, introduction of a foreign gene or non-coding (regulatory) genome region, and knockout of a gene or genome region. The modification of the gene or genomic sequence can be performed using, for example, genome editing techniques such as ZFN, TALEN, CRISPR/Cas systems and the like; gene recombination techniques; and the like.
In the present disclosure, the cellular aggregate includes, for example, 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 cells, at least 2500 cells, at least 5000 cells, at least 10000 cells, at least 15000 cells, at least 20000 cells, at least 30000 cells, at least 40000 cells or at least 50000 cells. The number of the cellular aggregate is, for example, 100 to 100000 cells, 200 to 100000 cells, 300 to 100000 cells, 400 to 500000 cells, 600 to 100000 cells, 700 to 100000 cells, 800 to 100000 cells, 900 to 100000 cells, 1000 to 90000 cells, 1500 to 80000 cells, 2000 to 70000 cells, 2500 to 60000 cells, 5000 to 50000 cells, 10000 to 50000 cells, 15000 to 50000 cells, 20000 to 50000 cells, 30000 to 50000 cells or 40000 to 50000 cells. The number of the cellular aggregate is preferably at least 50 cells, more preferably 100 to 1000 cells.
In the present disclosure, the cellular aggregate has, for example, a length of at least 0.05 mm, at least 0.1 mm, at least 0.2 mm, at least 0.3 mm, at least 0.4 mm, at least 0.5 mm, at least 0.6 mm, at least 0.7 mm, at least 0.8 mm, at least 0.9 mm or at least 1 mm. The length of the cellular aggregate is, for example, 0.05 to 10 mm, 0.1 to 9 mm, 0.2 to 8 mm, 0.3 to 7 mm, 0.4 to 6 mm, 0.5 to 5 mm, 0.6 to 4 mm, 0.7 to 3 mm, 0.8 to 2 mm or 0.9 to 1 mm. The length of the cellular aggregate is preferably 0.05 mm to 10 mm, more preferably at least 1 mm.
In a further aspect, the method of the present disclosure is a method for producing a three-dimensional cellular aggregate generated in vitro from a pluripotent stem cell, including the steps of:
In a further aspect, the present disclosure is a method for producing a three-dimensional cellular aggregate generated in vitro from a pluripotent stem cell, including the steps of:
In the method of the present disclosure, the “culture” can be carried out, for example, using a medium optionally supplemented with a factor. In the method of the present disclosure, the medium may be replaced during the culture period.
The medium can be prepared using a medium used for culturing animal cells as a basal medium. Examples of the basal medium include IMDM, Medium199, Eagle's Minimum Essential Medium (EMEM), aMEM, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium, RPMI1640, Fischer's medium, a Neurobasal Medium (manufactured by Thermo Fisher Scientific), a stem cell culture medium (e.g., mTeSR-1 (manufactured by STEMCELL Technologies), TeSR-E8 (manufactured by STEMCELL Technologies), CDM-PVA, StemFit (registered trademark), AK02N (manufactured by ReproCELL Inc.), StemPRO (registered trademark) hESC SFM (manufactured by Life Technologies), E8 (manufactured by Life Technologies), Essential 6 (manufactured by Thermo Fisher scientific) and mixed media thereof. The medium may be supplemented with serum or may be serum-free. The medium may contain, for example, serum substitutes such as albumin, transferrin, Knockout Serum Replacement (KSR) (serum substitute for ES-cell culture), N2 supplement (manufactured by Invitrogen), B27 supplement (manufactured by Invitrogen), fatty acids, insulin, collagen progenitor, trace elements, 2-mercaptoethanol, 3′-thiol glycerol and the like. Further, the medium may contain additives such as lipids, amino acids, L-glutamine, Glutamax (manufactured by Invitrogen), non-essential amino acids (NEAA), vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts and the like. When a growth culture is performed using the structure, the medium is preferably a stem cell culture medium to which NEAA, glutamic acid and an antibiotic are added.
The culture conditions in the culture may adopt, for example, common conditions of cell culture. As a specific example, the culture temperature is, for example, 25° C. to 40° C., preferably 30° C. to 40° C. or more preferably about 37° C. The carbon dioxide concentration during the culture is 1 to 10%, preferably 3 to 7%, or more preferably about 5%. The culture is carried out, for example, in a wet environment.
The culture conditions implemented during the culture may adopt, for example, common conditions of cell culture. As a specific example, the culture temperature is, for example, 25° C. to 40° C., preferably 30° C. to 40° C. or more preferably about 37° C. The carbon dioxide concentration during the culture is 1 to 10%, preferably 3 to 7%, or more preferably about 5%. The culture is carried out, for example, in a wet environment.
The method of the present disclosure includes, for example, the steps of:
The method of the present disclosure includes, for example, the steps of:
The method of the present disclosure includes, for example, the steps of:
The method of the present disclosure includes, for example, the step of: culturing the three-dimensional cellular aggregate including the mesodermal cell, which is embedded in the gel or the matrix, or disposed inside the gel or the matrix, in a medium containing a retinoic acid, a retinoic acid precursor or its derivative and/or a retinoic acid receptor (RAR) agonist to induce the three-dimensional cellular aggregate.
The method of the present disclosure includes, for example, the step of: culturing the three-dimensional cellular aggregate including the mesodermal cell, which is embedded in the gel or the matrix, or disposed inside the gel or the matrix, in a medium containing a retinoid, retinoic acid, a retinoic acid precursor or its derivative and/or a retinoic acid receptor (RAR) agonist to induce the three-dimensional cellular aggregate.
In the steps (a1), (a2) and/or (b1), the FGF is, for example, bFGF (FGF2).
The concentration of bFGF in the medium is, for example, 1 to 1000 ng/ml or preferably 10 to 1000 ng/ml.
In the steps (a1), (a2) and/or (b1), the GSK3β inhibitor may be, for example, a substance that inhibits the kinase activity of GSK3β protein (e.g., phosphorylation ability to β catenin), specifically, an indirubin derivative such as BIO (GSK-3 P inhibitor IX: 6-bromoindirubin 3′-oxime) or the like; a maleimide derivative such as SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), SB415286 (3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrole-2,5-dione or the like; a phenyl a bromomethylketone compound such as SK-3β inhibitor VII (4-dibromoacetophenone) or the like; a cell membrane penetrating phosphorylated peptide such as L803-mts (GSK-3p peptide inhibitor; Myr-N-GKEAPPAPPQSpP-NH2) or the like; CHIR99021 (6-[2-[4-(2,4-Dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-ylamino]ethylamino]pyridine-3-carbonitrile); an expression suppressing the nucleic acid molecule of GSK3β protein (siRNA, shRNA, antisense or the like) and the like. The GSK3β inhibitor is preferably CHIR99021, because of its high selectivity to GSK3β. The GSK3β inhibitor is commercially available, for example, from Calbiochem, Biomol and the like. In the present disclosure, a WNT agonist including recombinant WNT proteins such as WNT3a as well as other canonical and non-canonical WNT agonists can be used instead of the GSK3β inhibitor.
The concentration of the GSK3β inhibitor in the medium is, for example, 1 nmol/l (also referred to as “M” hereinafter) to 1000 μmol/l, preferably 10 nmol/l to 100 μmol/l or more preferably 100 nmol/l to 100 μmol/l.
In the steps (a2) and/or (b1), the TGFβ inhibitor is a substance that inhibits SMAD mediated signaling caused by binding of TGFβ to a receptor. Examples of the TGFβ inhibitor include a substance that inhibits binding to the ALK family, which is a TGFβ acceptor, and a substance that inhibits phosphorylation of SMAD by the ALK family. Specific examples of the TGFβ inhibitor include Lefty-1 (NCBI Accession Number: NM-010094 (mouse), NM-020997 (human) SB431542 (4-(4-(benzo[d][1,3]dioxol-5-yl)-5-(pyridine-2-yl)-1H-imidazol-2-yl)benzamide), SB202190 (4-(4-Fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole), SB505124 (2-(5-Benzo1,3dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276 (Lilly Research Laboratories) and A-83-01 (WO2009/146408), and SB431542 is preferred.
The concentration of the TGFβ inhibitor in the medium is, for example, 1 nmol/l to 1000 μmol/l, preferably 10 nmol/l to 100 μmol/l or more preferably 100 nmol/l to 100 μmol/1.
In the steps (a2) and/or (b1), the ROCK inhibitor is a substance that can suppress the function of the Rho-kinase (ROCK). Examples of the ROCK inhibitor include Y27632 ((+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride), Fasudil/HA1077 (5-(1, 4-Diazepane-1-sulfonyl)isoquinoline), H-1152 ((S)-(+)-2-Methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]homopiperazine), Wf-536 ((+)-(R)-4-(1-Aminoethyl)-N-(4-pyridyl)benzamide) and an expression suppressing the nucleic acid molecule of ROCK protein (siRNA, shRNA, antisense, etc.), and Y27632 is preferred.
The concentration of the ROCK inhibitor in the medium is, for example, 1 nmol/l to 50 μmol/l, 10 nmol/l to 40 μmol/l, 50 nmol/l to 30 μmol/l, 100 nmol/l to 25 μmol/l, 500 nmol/l to 20 μmol/l or 750 nmol/l to 15 mol/l, and is preferably 1 nmol/l to 40 mol/l, more preferably 1 nmol/l to 15 mol/l.
In the step (b2), the RAR agonist is, for example, Vitamin A, retinol, retinal, 9-cis retinoic acid, all-trans type retinoic acid (ATRA), TTNPB, AM580, AM80, LGD1550, E6060, AGN193312, AM555S, CD2314, AGN193174, LE540, CD437, CD666, CD2325, SR11254, SR11363, SR11364, AGN193078, TTNN(Ro19-0645), CD270, CD271, CD2665, SR3985, AGN193273, Ch55, 2AGN190521, CD2366, AGN193109 and/or Re80, preferably, retinal or retinol.
In the step (b2), the retinoid includes, for example, Vitamin A, retinol, retinal, 9-cis retinoic acid, 13-cis-retinoic acid, all-trans type retinoic acid (ATRA), TTNPB, AM580, AM80, LGD1550, E6060, AM555S, CD2314, CD437, CD666, CD2325, SR11254, SR11364, TTNN(Ro19-0645), CD-270, CD271, SR3985, and/or Ch55 preferably, retinal or retinol.
The concentration of the retinoic acid, the retinoic acid precursor or derivative and/or the retinoic acid receptor (RAR) agonists in the medium is, for example, 1 nmol/l to 1000 μmol/l, preferably 10 nmol/l to 100 μmol/l or more preferably 100 nmol/l to 100 μmol/1.
The concentration of the retinoid, retinoic acid, the retinoic acid precursor or derivative and/or the retinoic acid receptor (RAR) agonists in the medium is, for example, 1 nmol/l to 1000 μmol/l, preferably 10 nmol/l to 100 μmol/l or more preferably 100 nmol/l to 100 μmol/l.
In the methods of the present disclosure, for example, the GSK3β inhibitor is CHIR99021, the FGF is bFGF, the TGFβ inhibitor is SB431542 and/or the ROCK inhibitor is Y-27632.
In the step (b2), for example, the matrix includes an extracellular matrix.
The matrix includes, for example, an extracellular matrix. The extracellular matrix includes, for example, collagen, laminin, fibronectin, vitronectin, gelatin and/or entactin. One type or two or more types of them may be used as the extracellular matrix.
In the step (b2), the gel includes, for example, a hydrogel. The gel includes, for example, a basement membrane matrix. The basement membrane matrix includes, for example, fibronectin, laminin, collagen, vitronectin, gelatin, heparan sulphate proteoglycan and/or entactin. One type or two or more types of them may be used as the basement membrane matrix. The gel includes an acrylamide gel, an arginine gel, an agarose gel and/or a polyethylene glycol hydrogel. The gel includes preferably an agarose gel, more preferably a polyethylene glycol hydrogel.
In the step (b2), the concentration of the gel and/or the matrix in the medium is, for example, a concentration capable of three-dimensional culture, and can be appropriately set according to the type of the gel and the matrix. As a specific example, the concentration of the gel and/or the matrix in the medium is, for example, 0.01 to 50% (v/v), 0.1 to 25% (v/v), 1 to 20% (v/v) or 5 to 10% (v/v). The concentration is preferably 0.01 to 50% (v/v), more preferably 5 to 10% (v/v).
In the step (b2), the concentration of the gel and/or the matrix in the medium is, for example, a concentration enabling three-dimensional culture, and can be appropriately adjusted according to the type of the gel and the matrix. As a specific example, the concentration of the gel and/or the matrix in the medium is, for example, 0.01 to 50% (v/v), 0.1 to 25% (v/v), 1 to 20% (v/v) or 5 to 10% (v/v). The concentration is preferably 0.01 to 50% (v/v), more preferably 5 to 10% (v/v).
In the method of the present disclosure, for example, a proportion of the mesodermal cell in the cellular aggregate including the mesodermal cell is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, based on the number of cells. The proportion is preferably at least 50%, more preferably at least 90%.
The mesodermal cell expresses one or more markers (mesodermal cell marker) selected from the group consisting of TBXT, SOX2, CYP26 A1, FGF3, FGF4, FGF8, FGF17, WNT3a, WNT5a, WNT5b, TBX6, HES7, MSGN1, MEOX1, TCF15, HOXD13, HOXB, HOXA9, HOXA10 and CDX2, for example. One type or two or more types of them may be used as the marker.
In the method of the present disclosure, for example, the cellular aggregate including the mesodermal cell substantially does not include an endodermal cell and/or an ectodermal cell.
The proportion of the endodermal cell in the cellular aggregate including the mesodermal cell is, for example, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, based on the number of cells. The proportion is preferably 10% or less, more preferably 1% or less.
The endodermal cell expresses one or more markers (endodermal cell marker) selected from the group consisting of GATA6, GSC, CDX2, NEDD9, PYY, SHH, SORCS2, CER1, SOX17, FOXA2, TRH1 and FOXA1, and preferably GATA6 and/or SHH, for example. One type or two or more types of them may be used as the marker.
The proportion of the ectodermal cell in the cellular aggregate including the mesodermal cell is, for example, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, based on the number of cells. The proportion is preferably 10% or less, more preferably 1% or less.
The ectodermal cell expresses one or more marker (ectodermal cell marker) selected from the group consisting of OTX2, GBX2, SIX1, SIX3, SOX1, SOX2, SOX3, DLXS, EYA2 and BARX1, and preferably OTX2, for example. One type or two or more types of them may be used as the marker.
The ectodermal cell expresses one or more marker (ectodermal cell marker) selected from the group consisting of OTX2, GBX2, SIX1, SIX3, SOX1, SOX3, DLXS, EYA2 and BARX1, and preferably OTX2, for example. One type or two or more types of them may be used as the marker.
The number of days of culture of the step (a) is, for example, 0.1 to 10 days, 0.25 to 6 days, 0.25 to 4 days, 0.5 to 3 days or 0.5 to 2 days. The number is preferably 0.1 to 10 days, more preferably 0.5 to 2 days.
The number of days of culture of the step (a1) is, for example, 0.1 to 5 days, preferably 0.25 to 3 days or more preferably 0.5 to 2 days.
The number of days of culture of the step (a2) is, for example, 0.1 to 5 days, preferably 0.25 to 3 days or more preferably 0.5 to 2 days.
The number of days of culture of the step (b) is, for example, 0.1 to 10 days, 1 to 9 days, 2 to 8 days or 3 to 7 days. The number is preferably 0.1 to 10 days, more preferably 2 to 8 days.
The number of days of culture of the step (b1) is, for example, 0.1 to 7 days preferably 0.25 to 5 days, or more preferably 0.5 to 4 days.
The number of days of culture of the step (b2) is, for example, 0.1 to 7 days preferably 0.25 to 5 days, or more preferably 0.5 to 4 days.
In the step (a1), for example, the pluripotent stem cell is one or more suitable dissociated pluripotent stem cell(s) or one or more suitable cell(s) suspension containing any pluripotent stem cell(s).
In the step (a1), the pluripotent stem cell is, for example, a human pluripotent stem cell or a non-human animal pluripotent stem cell. Examples of the non-human animal include amniotes such as a mouse, a rat, a rabbit, a dog, a cat, a cow, a horse, a pig, a monkey, an ape, a dolphin, an elephant, a sea lion, a snake, a gecko, a chicken, and the like. The pluripotent stem cell is, for example, an embryonic stem cell or an artificial pluripotent stem cell.
In the step (a1), the pluripotent stem cell is, for example, a human pluripotent stem cell or a non-human animal pluripotent stem cell. Examples of the non-human animal include amniotes such as a mouse, a rat, a rabbit, a dog, a cat, a cow, a horse, a pig, a monkey, an ape, a dolphin, a whale, an armadillo, a tenrec, an elephant, a sea lion, a snake, a gecko, a chicken and the like. Non-human animal pluripotent stem cells also include monotreme species, such as a platypus or an echidna and marsupial species, such as an opossum, a kangaroo, a wombat or the like. The pluripotent stem cell is, for example, an embryonic stem cell or an artificially engineered pluripotent stem cell.
The cellular aggregate including the mesodermal cell includes, for example, 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 cells, at least 2500 cells, at least 5000 cells, at least 10000 cells, at least 15000 cells, at least 20000 cells, at least 30000 cells, at least 40000 cells or at least 50000 cells. The cellular aggregate includes preferably at least 50 cells, more preferably at least 100 to 1000 cells.
The cellular aggregate including the mesodermal cell is made of, for example, 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 cells, at least 2500 cells, at least 5000 cells, at least 10000 cells, at least 15000 cells, at least 20000 cells, at least 30000 cells, at least 40000 cells or at least 50000 cells. The cellular aggregate includes preferably at least 50 cells, more preferably at least 100 to 1000 cells.
The cellular aggregate including the mesodermal cell has, for example, a length of at least 0.05 mm, at least 0.1 mm, at least 0.2 mm, at least 0.3 mm, at least 0.4 mm, at least 0.5 mm, at least 0.6 mm, at least 0.7 mm, at least 0.8 mm, at least 0.9 mm or at least 1 mm. The cellular aggregate has preferably at least 0.05 mm, more preferably at least 0.5 mm.
In another aspect, the present disclosure provides a cell obtained from the three-dimensional cellular aggregate of the present disclosure.
In yet another aspect, the present disclosure provides a method for producing a progenitor cell or a differentiated cell, including the step of:
In yet another aspect, the present disclosure provides a method for producing a progenitor cell or a differentiated cell, including the step of:
The method for inducing the differentiated cell and the progenitor cell thereof of (a) to (f) can be carried out in the same manner as, for example, a method for inducing each differentiated cell and a progenitor cell thereof from the mesodermal cell.
The method for inducing the differentiated cell and the progenitor cell thereof of (a) to (i) can be carried out in the same manner as, for example, a method for inducing each differentiated cell and a progenitor cell thereof from the mesodermal cell.
The production method of the present disclosure may include, for example, the step of inducing a three-dimensional cellular aggregate from a pluripotent stem cell prior to the inducing. In this case, for example, the inducing of the three-dimensional cellular aggregate may be performed by the method for producing the three-dimensional cellular aggregate of the present disclosure.
The production method of the present disclosure may include, for example, the step of forming a three-dimensional cellular aggregate from a pluripotent stem cell prior to the induction. In this case, for example, the formation of the three-dimensional cellular aggregate may be performed by the method for producing the three-dimensional cellular aggregate of the present disclosure.
In another aspect, the present disclosure provides a method for evaluating a test substance, including the steps of: culturing a test substance in the presence of a three-dimensional cellular aggregate; and evaluating the three-dimensional cellular aggregate through and/or after the culturing, wherein the three-dimensional cellular aggregate is the cellular aggregate of the present disclosure.
The type of the test substance is not particularly limited, and examples thereof include a protein, an antibody, a peptide, a nucleic acid molecule, a sugar chain, a lipid, an organic low molecular weight compound, an inorganic low molecular weight compound, a bacterial releasing substance, a fermentation product, a cell extract, a vacuole culture supernatant, a plant extract and an animal tissue extract. One type or two or more types of them (preferably an organic low molecular weight compound, more preferably a protein) may be used as the test substance.
In the evaluation, for example, a test substance that changes a polarity of the cellular aggregate, a shape of the cellular aggregate and/or a size of the cellular aggregate is selected as a candidate substance that modifies, promotes or suppresses the polarity of the cellular aggregate, the shape of the cellular aggregate and/or the size of the cellular aggregate.
The culture is, for example, a culture under a segmentation culture condition. The segmentation culture conditions can be referred to, for example, the above description. The culture conditions may adopt, for example, the common conditions of cell culture.
During the evaluation, for example, a test substance that changes somitogenesis of the cellular aggregate is selected as a candidate substance that modifies, promotes or suppresses the somitogenesis process of the cellular aggregate.
The culture is a culture inducing the progenitor cell or the differentiated cell selected from the group consisting of the following (a) to (f):
The culture is a culture inducing the progenitor cell or the differentiated cell selected from the group consisting of the following (a) to (i):
During the evaluation, for example, a test substance that promotes or suppresses induction of the progenitor cell or the differentiated cell selected from the group consisting of (a) to (f) is selected as a candidate substance that promotes or suppresses induction of the progenitor cell or the differentiated cell selected from the group consisting of (a) to (f).
During the evaluation, for example, a test substance that promotes or suppresses induction of the progenitor cell or the differentiated cell selected from the group consisting of (a) to (i) is selected as a candidate substance that promotes or suppresses induction of the progenitor cell or the differentiated cell selected from the group consisting of (a) to (i).
In the evaluation method, the evaluation is an evaluation using a control in which the test substance is not present as a reference.
During the evaluation, the method of evaluation uses a control in which the test substance is not present as a reference.
The culture is generation of a three-dimensional cellular aggregate from a pluripotent stem cell, and the generation of the three-dimensional cellular aggregate is carried out by the method for producing the three-dimensional cellular aggregate of the present disclosure.
The culture refers to the generation of a three-dimensional cellular aggregate from a pluripotent stem cell, where the generation of the three-dimensional cellular aggregate is carried out by the method for producing the three-dimensional cellular aggregate described in the present disclosure.
In another aspect, the present disclosure provides a method for evaluating gene function or genome function, including the steps of: preparing a pluripotent stem cell which a test gene or a test genome is modified; generating a three-dimensional cellular aggregate from the pluripotent stem cell; and evaluating a three-dimensional cellular aggregate through and/or after the culturing, wherein the generation of the three-dimensional cellular aggregate is carried out by the method for producing the three-dimensional cellular aggregate of the present disclosure.
In another aspect, the present disclosure provides a method for evaluating gene function or genomic sequence function, including the steps of: engineering a pluripotent stem cell in which a test gene or a test genomic sequence is modified; generating a three-dimensional cellular aggregate from the pluripotent stem cell; and evaluating a three-dimensional cellular aggregate through and/or after the culturing, wherein the generation of the three-dimensional cellular aggregate is carried out by the method for producing the three-dimensional cellular aggregate of the present disclosure.
During the evaluation, for example, a test gene or a test genome (coding and noncoding genome regions) that changes a polarity of the cellular aggregate, a shape of the cellular aggregate and/or a size of the cellular aggregate is evaluated as a candidate gene or a candidate genome (coding and non-coding genome regions) that modifies, promotes or suppresses the polarity of the cellular aggregate, the shape of the cellular aggregate and/or the size of the cellular aggregate.
During the evaluation, for example, a test gene or a test genomic sequence (coding and non-coding genome regions) that changes a polarity of the cellular aggregate, a shape of the cellular aggregate and/or a size of the cellular aggregate is evaluated as a candidate gene or a candidate genomic sequence (coding and non-coding genome regions) that modifies, promotes or suppresses the polarity of the cellular aggregate, the shape of the cellular aggregate and/or the size of the cellular aggregate.
The function evaluation method of the present disclosure includes, for example, culturing in the presence of a three-dimensional cellular aggregate under a somitogenic culture condition. In this case, during the evaluation, a test gene or a test genome (coding and non-coding genome regions) that changes somitogenesis, including axial elongation, segmentation, epithelialization or oscillation of the segmentation clock of the cellular aggregate is evaluated as a candidate gene or a candidate genome (coding or non-coding) that modifies, promotes or suppresses the somitogenesis, including axial elongation, segmentation, epithelialization or oscillation of the segmentation clock of the cellular aggregate.
The method to evaluate the function in the present disclosure includes, for example, culturing in the presence of a three-dimensional cellular aggregate under a somitogenic culture condition. In this case, during the evaluation, a test gene or a test genomic sequence (coding and non-coding genome regions) that changes somitogenesis, including axial elongation, segmentation, epithelialization or oscillation of the segmentation clock of the cellular aggregate is evaluated as a candidate gene or a candidate genomic sequence (coding or non-coding) that modifies, promotes or suppresses the somitogenesis, including axial elongation, segmentation, epithelialization or oscillation of the segmentation clock of the cellular aggregate.
In the present disclosure, the gene may be any gene and the example thereof is a gene related to human diseases (e.g., HES7). The test genes may be a gene having gene mutation related to human diseases (e.g., HES7R25W and spondylocostal dysostosis).
In the function evaluation method of the present disclosure, the genome is an exon region, an intron region, a promoter region, an enhancer region and/or a non-coding region of the genome.
In the function evaluation method of the present disclosure, the genomic sequence is an exon region, an intron region, a promoter region, an enhancer region and/or a noncoding region of the genome.
The following Preparations and Examples are given for the purpose of illustrating the present invention in more detail. However, the scope of the present invention is not limited to the following.
The present disclosure will be described specifically below with reference to examples. It is to be noted, however, that the present disclosure is by no means limited to embodiments described in the following examples.
Abstract The segmented body plan of vertebrates is established during somitogenesis, a well-studied process in model organisms, but remains largely elusive in humans due to ethical and technical limitations. Despite recent advances with pluripotent stem cell (PSC)-based approaches1-5, a system that robustly recapitulates human somitogenesis in both space and time remains missing. Here, a PSC-derived mesoderm-based 3D model of human segmentation and somitogenesis is introduced, which is termed Axioloids, that captures accurately the oscillatory dynamics of the segmentation clock as well as the morphological and molecular characteristics of segmentation and sequential somite formation in vitro. Axioloids show proper rostrocaudal patterning of forming segments and robust anterior-posterior FGF/WNT signaling gradients and Retinoic Acid (RA) signaling components. An unexpected critical role of RA signaling in the stabilization of forming segments is identified, indicating distinct, but also synergistic effects of RA and extracellular matrix (ECM) on the formation and epithelialization of somites. Importantly, comparative analysis demonstrates striking similarities of Axioloids to the human embryo, further validated by the presence of the HOX code in Axioloids. Lastly, the utility of the Axioloid system is demonstrated to study the pathogenesis of human congenital spine diseases, by using patient-like iPSC cells with mutations in HES7 and MESP2, which revealed disease-associated phenotypes including loss of epithelial somite formation and abnormal rostrocaudal patterning. These results suggest that Axioloids represent a promising novel platform to study axial development and disease in humans.
Previously, the human segmentation clock in vitro1,4,6 was able to be reconstructed. However, these systems lacked the ability to form proper axial segmental organization—a central feature of all vertebrates—limiting their utility to understand how higher-order tissue organization and more advanced stages of human embryonic development occur. As supplementation of extracellular matrix (ECM) molecules in vitro, have been shown to facilitate the formation of higher-order tissue structures in organoids as well as help mimic morphogenetic processes in mouse pluripotent stem cell-derived gas-truloids2 and trunk-like structures3, a single-germ layer, mesoderm-based 3D model of human axial development using human induced pluripotent stem cells (iPSCs) and ECM was set out to be established. iPSCs were exposed in a step-wise manner to signals promoting primitive streak (PS) and presomitic mesoderm (PSM) fates (
To determine the similarities of Axioloids to the vertebrate embryonic axis and tail, their morphological and molecular features were first assessed. In Axioloids, segments appeared with a periodicity of about 4-5 hours (
Further assessment of gene and protein expression patterns of MG embedded Axioloids revealed a striking similarity to the anatomically and functionally regionalized molecular features described for the growing tail of vertebrate embryos. A posterior-most TB region positive for TBXT and SOX2 could be clearly distinguished (
Besides the TB and PSM a narrow RIPPLY2 expressing anterior-PSM (aPSM) region (
A universal key feature of somitogenesis is the oscillatory activity of the segmentation clock, a molecular oscillator and gene regulatory network centered around Notch signalling active across the growing tail of the vertebrate embryo, and believed to control the pace and size of forming segments13-15. To determine dynamics of oscillatory activity within human Axioloids, a reporter system was utilized, which had previously been used to characterize the human segmentation clock in vitro1. Reproducible oscillatory activity of HES7, a well-studied segmentation clock gene1,4,16,17, with a periodicity of about 4-5 hours in Axioloids could be clearly observed regardless of the presence of MG (
To further understand the functional features of the model system and to characterize the prospective dynamic changes in the cellular composition and molecular complexity of human Axioloids, temporally-resolved single cell RNA-seq (scRNA-seq) analysis of various stages (48 h, 72 h, 96 h & 120 h) and conditions (+/−MG) of the system was performed. The analysis revealed the presence of multiple dynamically changing cell clusters, which could be matched to different mesodermal cell populations present in the developing axis and tail of vertebrate embryos, including tail bud (TB), presomitic mesoderm (PSM), anterior presomitic mesoderm (aPSM), somitic mesoderm (SM) and angioblast/endothelial-like (EC-like) cells (
As the embryonic tail of vertebrates is characterized by opposing gradients of FGF/WNT and Retinoic Acid (RA), believed to be involved in the establishment of the “wave front” during somitogenesis21-23, it was next asked whether similar gradients are also present within the human Axioloids. Pseudotime analysis of the scRNA-seq data, which matched well with the anterior-to-posterior organization and spatial distribution of the major cell populations found in Axioloids, was used to predict the expression patterns of multiple FGF, WNT and RA signaling associated transcripts within Axioloids (
Matrigel Only Potentiates In Vitro Segmentation but does not Stabilize them
The data indicated that while MG promotes axial elongation and the initial formation of segments, it is unlikely to be sufficient to maintain or stabilize these segments. To better understand the role of MG in the elongation and segmentation of human Axioloids, the scRNA-seq data of MG-containing and -lacking cultures of Axioloids were compared. The analysis revealed the MG-dependent emergence of an angioblast and endothelial cell-like (EC-like) population of cells at 96 h of Axioloid culture (
The scRNA-seq data, focusing on differentially expressed genes (DEGs) in PSM and SM cells (
Potential factors that may contribute to stabilizing the segmentation process and increase epithelialization of somites were next looked for. Although ALDH1 A2 and molecules associated with synthesizing RA, such as RDH10, are specifically expressed in the system, the precursors Retinol (ROL) and Retinal (RAL) were not present in the culture conditions. Axioloids are thus unable to generate RA de novo, raising the question as to the function of RA signaling in Axioloids. To address this question, either directly RA or its precursors ROL or RAL was added into the in vitro system, during the MG embedding phase between 72 h-120 h. Surprisingly, it was observed that presence of RA molecules, led to a dramatic improvement of the stability and epithelialization of forming segments within MG-embedded Axioloids at 96 h and 120 h. (
To further dissect how RA mediates its function and alters somite formation in Axioloids, Axioloid samples that were supplemented with RA and RAL at 96 h and 120 h using scRNA-seq analysis was analyzed. a clear segregation of the SM and TB clusters based on the presence or absence of RA and RAL was observed, while overall identities of the cell clusters remained stable and could still be matched with each other (
The findings, showing that RA has a critical role in somite formation and epithelialization, are surprising, as the loss of RA in embryos has generally been reported to cause smaller somites or asymmetric formation of bilateral somites21,27,34, rather than leading to a strong epithelialization-related phenotype. Furthermore, regarding symmetry and bilaterality in the system, in MG and RA treated Axioloids a single axis of sequentially forming epithelial somites with single central somitocoels was typically observed. Intriguingly, Axioloids also frequently displayed a superficial groove or midline-like structure, starting in the PSM and going through most of the forming segments (
Somitogenesis is a distinctive feature of vertebrate embryos and the number of somites allows an approximate assessment of the age of an embryo. Carnegie stage (CS) 10 human embryos are characterized by 4 to 12 pairs of somites, suggesting that 96 h and 120 h old Axioloids are, at least, in an equivalent stage. Comparison of the dimensions of the Axioloid somites with those of CS10 & 11 embryos suggests that they are similar in shape and size (
To further benchmark the morphogenetic and molecular features of Axioloids with that of actual human embryos, recently uploaded scRNA-seq data set of CS12 to CS16 human embryos was utilized 35. The CS12 embryo data for comparison was reanalyzed, and mutual nearest neighbors (MNN)-based integration analysis36 (
It was also found that a portion of Axioloid-derived angioblast/EC-like cells matched with cells marked as endothelium in the human CS12 data set (
Having established a robust in vitro system to model axial development, with the use of both MG and RA, next stage was to see whether there is a HOX Code, i.e. the spatiotemporally controlled expression of HOX genes, in human Axioloids. Combining the scRNA-seq data with HybISS-based spatial transcriptomics38, the spatial expression of major TB, PSM and SM markers was successfully recapitulated (
Based on the morphogenetic and molecular similarities between Axioloids and actual human embryos, it was then asked whether the latest iteration of the model system (MG+RAL) could be used to investigate the role of signaling pathways during human somitogenesis. Using HCR-based in situ hybridization, it was observed that Axioloids cultured in the presence of MG+RAL, still showed clear expression gradients of FGF8 and WNT3a in their TB and PSM region similar to Axioloids cultured in the presence of Matrigel only (
As RA signaling has a strong effect on somite formation and epithelialization, it was next asked whether it also influenced the oscillation and traveling wave-like activity of the segmentation clock within Axioloids. It was found that RA, RAL and ROL regardless of the presence or absence of MG in the system had largely no effect on the oscillatory activity including periodicity of the segmentation clock gene HES7, including robust presence of traveling wave-like expression in all treated Axioloids (
Then next stage was to modulate via small molecules also the FGF, WNT and Notch signaling pathways in human Axioloids. The observed alterations of the segmentation clock were similar to what had been previously reported in vitro1,4-40 and in vivo41. As expected, Notch inhibition with DAPT led to a quick damping and loss of oscillatory activity in Axioloids, while FGF and especially WNT inhibition had less severe effects on the segmentation clock (
Modeling Diseases of the Human Spine with Axioloids
Lastly, it was investigated whether Axioloids can be used to model genetically associated diseases of the human spine. Using patient-like iPSC-lines harboring loss-of-function mutations in the coding regions of genes known to be associated with segmentation defects of the vertebrae (SDV), focusing on HES744 or MESP245, Axioloids was generated and their morphological, molecular and functional features were assessed. Two different HES7 knock-out iPSC-lines were used, which showed a similar phenotype: a conspicuous loss of segments and epithelial somite formation despite evident axial elongation (
A similar range of phenotypes in Axioloids derived from patient-like iPSC lines containing a point mutation (rs113994160: c.73C>T) in HES7 was observed, resulting in a pathogenic missense mutation R25W in the helix-loop-helix domain of HES7 reported to cause segmentation defects of the vertebrae (SDV)44 (
Next, the effects of the loss of MESP2 were assessed, an aPSM associated transcription factor for which pathogenic mutations in patients with SDV have been previously reported45. Using MESP2 knock-out iPSC lines (MESP2 KO1 & MESP2 KO2) patient-like Axioloids was derived and various morphological, molecular and functional features were again assessed. MESP2 KO Axioloids elongated normally but were devoid of segments or epithelial somites (
In summary, a pluripotent stem cell-derived 3D mesodermal model of human axial development have been established and characterized in-depth, which could reconstitute various aspects of human somitogenesis and axial development in vitro. Axioloids recapitulated a range of complex developmental processes including axial elongation, segmentation, epithelialization to oscillation of the segmentation clock, while also sharing molecular and morphometric features with the tail and axis of the developing human embryo. The bottom-up approach revealed the remarkable self-organization potential of primitive and paraxial mesoderm, which can give rise to the metameric basic body plan of the human embryo even in the absence of other germ layers. A crucial role of RA signaling on the morphogenetic processes associated with segmentation and somite formation within Axioloids was also uncovered, suggesting that RA supplementation especially in combination with ECM components might also improve the morphogenetic features of other in vitro model systems of human and non-human early embryonic development.
The bottom-up experimental approach demonstrates that complex developmental events such as somitogenesis, can be deconstructed and dissected into discrete “building blocks” of developmental principles which are usually intricately connected and cannot be easily uncoupled in vivo. Axioloids, a self-organizing in vitro model of human axial development allowed us to individually assess and manipulate such building blocks at the molecular, cellular and morphogenetic level. Further iterations of this model system will likely incorporate still “missing” anatomical structures such as notochord or neutral tube, which will allow assessment of subsequent stages of somitic development and differentiation including compartmentalization of somites into sclerotome, dermomyotome and other functional derivatives. Axioloids, a surrogate model of the human embryonic tail and forming axis, are capable of recapitulating core features of human somitogenesis, and represent an exciting new platform to investigate axial development and disease in a human context.
Two human induced pluripotent stem cell (hiPSC) lines derived from healthy donors, 409B248 and 201B749, were used in this study, with the latter used mainly in the form of a HES7 reporter line described previously1. For disease modeling, patient-like iPSCs with pathogenic mutations in HES7 and MESP2 generated via CRISPR-Cas9-based genetic editing were used1. Human iPS cells were maintained in StemFit AK02N (Reprocell) medium supplemented with 50 U penicillin and 50 mg ml−1 streptomycin (Gibco) on iMatrix-511 silk (Nippi) coated dishes. StemFit AK02N (Reprocell) medium contains three components, A, B and C, all three of which were mixed and used for standard maintenance culture of hiPSCs in humidified incubators at 37° C. and 5% CO2. Utilized iPS cell lines were regularly tested and reported negative for mycoplasma contamination.
Human iPS cells were seeded on iMatrix-511 silk (Nippi) coated dishes at a density of 1.3×104 cells/well into 6 well plates 5 days prior to Axioloid induction and used at 60% confluency. All subsequent induction steps were performed in AK02N (Reprocell) medium without component C (AK02N-C). Initially, hiPSCs were pulsed with a strong mesoderm and primitive streak fate inducing combination of bFGF (20 ng ml-1) and WNT agonist CHIR99021 (5 μM) for 24 h. CHIR99021 concentration may need to be adjusted depending on the used iPSC line, but remains usually in the range of 3-5 μM. 24 h after the initial pulse, cells were dissociated with Accutase (Thermo Fisher Scientific) and cultured for 24 h in 96-well U-bottom low attachment plates (Sumilon) at 500 cells/well in 50 μl of AK02N—C based aggregation medium supplemented with CHIR99021 (5 μM), basic FGF (20 ng ml1), TGFβ inhibitor SB431542 (10 μM) and ROCK inhibitor Y27632 (10 μM). 24 h after aggregation, 150 μl of AK02N-C medium was added into each well and exchanged again after 24 h with 150 μl of fresh AK02N-C medium. 48 h after aggregation Axioloids were transferred one-by-one into BSA-treated 96-well flat-bottom low attachment plates (Watson) and embedded into 80 μl of AK02N-C medium containing 10% growth factor-reduced Matrigel (Corning) and cultured further at 37° C. and 5% CO2 for 24 to 48 h. Depending on the experimental setup Retinoic Acid signaling molecules, i.e. Retinoic Acid (RA) (100 nM), Retinol (ROL) (10 μM) or all trans-Retinal (RAL) (1 μM) were added to the MG containing medium throughout the embedding phase. Small molecule inhibitors of FGF (PD173074 (250 nM)), WNT (XAV939 (2 μM)), RA (BMS493 (2.5 μM)) and Notch (DAPT (25 μM)) signaling were also added during the embedding phase and their effects on Axioloids assessed accordingly. For details of the used recombinant human proteins, small molecule agonists and inhibitors, please see Supplementary Table 1.
Digital data of human embryos were obtained from the MRC/Wellcome-Trust funded Human Developmental Biology Resource (HDBR, www.www.hdbr.org) with appropriate maternal written informed consent and approval from the Newcastle and North Tyneside NHS Health Authority Joint Ethics Committee (REC reference 18/NE/0290 & 08/H0906/21+5). HDBR is regulated by the UK Human Tissue Authority (license #12534) and operates in accordance with the relevant HTA Codes of Practice. The embryos were staged as Carnegie Stage (CS) 10 (n=1) or CS11 (n=4) based on features that were visible externally in the unfixed sample (https://hdbratlas.org/staging-criteria/carnegie-staging.html). One CS11 embryo (N662) was imaged using Optical Projection Tomography (OPT)50. The remaining CS10 (13446) and CS11 (CS11-1021, 1177 and 1053) embryos were sectioned at a thickness of 4 μm (interval 20 im) for CS10 and 7 μm (interval 56 m for 1021 and 1053, and 35 μm for 1177) for CS11, stained with Hematoxylin and Eosin dye and imaged. The data related to the embryos utilized in this manuscript have been published51,52 and/or are publicly available on the HDBR Atlas website (https://hdbratlas.org/). OPT acquired image series of the CS11 embryo was 3D reconstructed and used for somite volume measurements, and scaled images of stained sections were used to measure the area of several visible and identifiable somite using ImageJ software.
Human Axioloid samples were washed twice with 0.1% BSA (Nacalai) in PBS (Takara), fixed with 4% paraformaldehyde (PFA) for 20 minutes at room temperature and then washed again twice with 0.1% BSA in PBS. Samples were permeabilized in 0.2% Triton X-100 (Nacalai) in PBS for 15 minutes at room temperature and blocked in 5% BSA in PBS for 1 hour at room temperature. Axioloid samples were stained with primary antibodies diluted in 0.5% BSA in PBST (1% Tween-20 (Nacalai) in PBS) for overnight (12-16 hours) at 4° C. Primary antibodies used in this study were: goat anti-TBXT (TBXT) (1:500, R&D Systems), mouse anti-Fibronectin (1:10, DSHB), mouse anti-Laminin (γ1) (1:10, DSHB), rabbit anti-MEOX1 (1:500, ATLAS), mouse anti-N-Cadherin (1:200, BD Biosciences), mouse anti-PKCζ (1:100, Santa Cruz), rabbit anti-SOX2 (1:400, Cell Signaling), and goat anti-TBX6 (1:500, R&D Systems). Samples were then washed twice with PBST and stained with secondary antibodies and Alexa Fluor 647 conjugated Phalloidin (Invitrogen) diluted in 0.5% BSA in PBST for 3 hours at room temperature, washed twice with PBST and counterstained with DAPI for 5 min at room temperature. Secondary antibodies used in this study (all diluted 1:500) were: Alexa Fluor 405 donkey anti-goat (Abcam), Alexa Fluor 405 donkey anti-mouse (Abcam), Alexa Fluor 488 donkey anti-goat (Invitrogen), Alexa Fluor 488 donkey anti-mouse (Invitrogen), and Alexa Fluor 555 donkey anti-rabbit (Invitrogen). Prior to imaging, stained Axioloid samples were submerged in and treated with Scale S4 clearing solution53 for overnight at 4° C. Images were taken using LSM980 (Carl Zeiss), Ti2 (Nikon) equipped with Dragonfly (Andor) or Nikon A1R MP (Multiphoton+N-STORM) fluorescence microscopes. For further details of used primary and secondary antibodies see Supplementary Table 2.
All probes, HCR amplifiers and buffers (hybridization, wash and amplification buffers) were purchased from Molecular Instruments and whole-mount in situ hybridization chain reaction (HCR) was performed as previously described54. Briefly, human Axioloids were collected in microtubes coated with 1% BSA in PBS, washed once with 1% BSA in PBS and then fixed with 4% PFA in PBS for 1 h at room temperature. Samples were then washed three times with PBST (0.1% Tween-20 in PBS) at room temperature 5 min each, post-fixed with 100% Methanol and stored at −30° C. until further use. Samples were rehydrated by washing with a series of graded 500 μL Methanol/PBST wash steps (75%, 50%, and 25%) for 5 min each at room temperature. Human Axioloid samples were then incubated for 5 min with hybridization buffer at room temperature, then for 30 min at 37° C. A mixture of probes (8 nM final for each) in hybridization buffer was incubated for 30 min at 37° C. prior to use. Samples were incubated for 12-16 h at 37° C. with probe-containing hybridization buffer. Samples were then washed with probe wash buffer 4 times 15 min each at 37° C. followed by washing with 5×SSCT 3 times at room temperature. Next, samples were pre-amplified by incubating at least 30 min in probe amplification buffer at room temperature. Amplifier hairpins were prepared by snap-cooling; heating each hi and h2 hairpins separately at 95° C. for 90 sec and then cool down at room temperature for 30 min in the dark. Hairpin mixtures were prepared at 6 nM each by adding h1 and h2 in 250 μL of amplification buffer. Axioloid samples were then incubated with the amplifier hairpin-containing solution for 12-16 h at room temperature in the dark. Finally, samples were washed with 5×SSCT and PBST, followed by counter staining with DAPI. During each wash and after addition of buffers, probes and hairpin mixtures into samples, tubes were inverted several times (5-20 times) to mix properly. HCR stained Axioloids were stored in 1% BSA in PBST at 4° C. not more than two weeks before imaging. HCR probe design and associated hairpins was as follows: ALDH1 A2 (Accession NM_003888.4, hairpin 514-B5); CYP26 A1 (Accession NM_000783.4, hairpin 594-B4); FGF8 (Accession NM_033163.5, hairpin 546-B2); HES7 (Accession NM_001165967.2, hairpin 594-B4); LFNG (Accession NM_001040167.2, hairpin 488-B1); MESP2 (Accession NM_001039958.2, hairpin 647-B3); MSGN1 (Accession NM_001105569.3, hairpin 514-B5); PCDH8 (Accession NM_002590.4, hairpin 647-B3); RIPPLY2 (Accession NM_001009994.3, hairpin 647-B3); TBX6 (Accession NM_004608.4, hairpin 647-B5); TBX18 (Accession NM_001080508.3, hairpin 546-B2); TCF15 (Accession NM_004609.3, hairpin 594-B4); UNCX (Accession NM_001080461.3, hairpin 488-B1); WNT3 A (Accession NM_033131.4, hairpin 488-B1).
Human Axioloids were washed with 0.1% BSA (Nacalai) in PBS twice and fixed with 0.4% PFA for 20 minutes at room temperature and then washed twice with 0.1% BSA in PBS. Axioloid samples were then transferred to a cryomold (Sakura Finetek), embedded in frozen section compound (Leica) and stored in −80° C. until sectioning. Tissues were cryosectioned at 8 m thickness and collected on a slide glass (MAS-01, MATSUNAMI). HybISS was performed as described previously38 with slight modifications. Briefly, slides were fixed with 3% formaldehyde for 5 minutes, washed with PBS twice, permeabilized with 0.1 M HCl for 5 minutes, and washed with PBS twice. After passing through 70% and 100% ethanol for dehydration, gaskets (SecureSeal Hybridization chambers, Grace Bio-Labs) were glued onto the sides enclosing the tissue. The mRNA was in situ reverse-transcribed to complementary DNA through following steps: Tissues were treated with random decamer (5 μM, obtained from IDT) for 5 minutes at 65° C. and cooled down on ice for 5 minutes. Then tissues were incubated with in-situ reverse transcription mix including superscript IV (20 U/μl, Thermo Fisher Scientific) and random decamer (5 μM) for 10 minutes RT and then overnight at 42° C. After reverse transcription, tissues were fixed again with 3% formaldehyde for 40 minutes at RT, followed by degradation of the mRNA with RNaseH (0.4 U/μl, NEB) for 30 minutes at 37° C., hybridization of padlock probes (PLPs) to the remaining cDNA (20 nM for each PLPs in 20% formamide), and ligation of padlock probes with Tth ligase (0.5 U/μl, BURT) for 90 minutes at 45° C. For the padlock probe design, 5 target sequences were selected per gene using Python padlock design software package (https://github.com/Moldia/multi_padlock_design) with the following parameters: arm length, 15 (if targets were not found, 20); Tm, low 65, high 75; space between targets, 15. Every set of padlock probes for a given gene carries a unique 20 nucleotide (nt) ID sequence as well as 20 nt sequences that is common among all of PLPs. After padlock probe hybridization and ligation, cDNA was digested with exonuclease I (0.5 U/μl, Thermo Fisher Scientific) for 3 hours at 37° C. Then rolling circle amplification (RCA) was performed with phi29 polymerase (1 U/μl, Monserate) overnight at 30° C. Resulting RCA products (RCPs) were subjected to sequencing by hybridization. Each round of sequencing contains the process of bridge probe hybridization (0.2 μM each) in 1× hybridization buffer (2×SSC, 20% formamide), detection probe hybridization (0.2 μM each) with Hoechst staining (1 μg/mL) in 1× hybridization buffer, imaging, and stripping with stripping solution (2×SSC, 65% formamide). The sequences of PLPs, bridge probes, and detection probes are shown in Supplementary Table 3.
Imaging was performed using a standard epifluorescence microscope (Nikon Ti2-E) connected to LED light source (Lumencor SPECTRA X light engine). Images were obtained with a CMOS camera (ORCA-Flash4.0V3, Hamamatsu) with CFI Plan Apochromat Lambda objective 40× (1.3 NA, oil). Filter cubes for wavelength separation were as follows: Chroma 89402X (Hoechst, Cy5), Chroma 89403X (AlexaFluor750), Semrock GFP-A-Basic (AlexaFluor488), Semrock Cy3-4040C (Cy3), and Semrock CFP-2432C (Atto425). For multiplex analyses, unique 4-digit code was assigned to each gene, in which each digit corresponds to one of four different fluorophore-conjugated detection probes (1: AlexaFluor750, 2: AlexaFluor488, 3: Cy3, 4: Cy5). Every digit code was reconstituted through 4 rounds of hybridization and imaging in situ. Several genes were imaged separately to avoid optical crowding: ACTB, HOXB9, HOXA3, HOXB3, and HOXD4 for tissue 1, 2, and 3 of 96 h and tissue 4 of 120 h. ACTB and HOXB9 for tissue 1 and 3 of 120 h. Multispectral image was obtained for multiple cycles. Each image consists of multiple tiles that together cover the tissue section (10% overlap), and each field of view consists of Z-stacks stepping 0.8 m through entire tissue thickness. Tiles were stitched and Z-stacks were merged to maximum-intensity projections (NIS-Elements). 8-bit TIF image of each channel from each round was exported for data analyses.
Data analyses of HybISS and subsequent quantification were performed with homemade Python code and Fiji. For decoding gene spot, firstly, images were roughly aligned to first round images using Hoechst staining. Then images were top-hat filtered and split into multiple smaller images, which will be referred as tiles hereafter. For each tile in each round, composite images of the four detection probe channels were created, aligned to the first-round composite images, and split into each channel again. Then each tile is stitched to create the whole image. Gene spots were detected using Laplacian of Gaussian Filter in the first-round image, and signal intensity of each channel at the spots was calculated in the rest of rounds. Spot intensity was normalized by dividing the intensity by the 99th percentile in the channel. Following spots were removed from the analysis due to the low quality: the maximum intensity in the set of channels is less than 0.15. The maximum intensity in the set of channels divided by the sum of the all of the channels is less than 0.5. Spots passed through the quality control were assigned to the gene according to their reconstituted 4-digit code. Density of gene spots along anterior-posterior axis was quantified as follows: firstly, anterior-posterior axis was manually drawn and tissues were divided so that each segment have the same area. Then density of gene spots, which was represented as number of spots/1,000 μm2, was calculated in each segment for each gene, and the center of mass of the segment whose density of MESP2 is highest was set as reference position. Distance between the center of mass of adjacent segments was calculated, and sum of distance from the reference position was shown in x-axis, in which posterior-to-anterior is minus-to-plus direction. To produce line graph, gene density was normalized so that maximum density was set to one. To produce heatmap, the value of gene density plus 1 was log-transformed (base e). All of the python code used in the analyses above is available via GitHub (xxxxx) or upon request from the corresponding author.
Human Axioloids were transferred into 1% BSA (Nacalai) treated 96 well flat-bottom low attachment plates (Watson) with one Axioloid per well in 80 μl of AKO2-N—C based embedding medium (+/−MG, +/−RA, RAL or ROL, +/−small molecule modulators of signaling pathways) and were cultured in a stage top incubator (Tokai Hit) which was set to 37° C. and 5% CO2. Brightfield live-cell imaging was performed with an inverted Ti2 microscope system (Nikon) using PlanApo λ 10× objective, with the microscope running in autofocus mode, focusing at 10 μm intervals over a total range of 190 μm. Images were taken every 3 minutes and processed with Fiji.
Bioluminescence live-cell imaging of HES7-reporter (201B7Luc) iPSC-derived human Axioloids (201B7Luc) was performed and signal was quantified as previously described55,56 Briefly, one day before imaging glass-bottom 96-well plates (Iwaki) were coated with 50 μl of 1.5% PVA (Nacalai), the PVA solution was hereby aspirated and the plates dried on top of a clean bench overnight at room temperature. Prior to imaging, Axioloids were set onto coated glass-bottom 96-well plates with 80 μl of AK02N-C medium containing 100 mM D-Luciferin and 10% Matrigel. Small molecule agonists and antagonists of signaling pathways were added according to the experimental setup. Bioluminescence signals of Axioloids induced from the HES7-reporter cell line were recorded on IX83 (Olympus) equipped with iXon EMCCD camera (Andor) cultured in a stage top incubator (Tokai Hit). Signal was acquired with 2×2 binning and 1 min exposure. Cosmic rays were removed from the raw images by applying spike noise filter, then smoothed by median filter. Temporal fluctuation of signal baseline was corrected by background subtraction. Kymograph was generated by averaging luminescence intensity values along the lateral axis of Axioloids and resulted values were aligned in temporal order.
For confocal imaging of stained Axioloids, samples were set onto glass-bottom 35 mm dishes or 8-well chamber covers (Matsunami) in BSA-containing PBS and imaged. Confocal microscopy was performed on LSM980 (Carl Zeiss Microscopy), Dragonfly (Andor) equipped Ti2 fluorescent microscope (Nikon) or Nikon A1R MP (Multiphoton+N-STORM) fluorescence microscopes. For HCR-stained samples, images were taken as tiled z-stacks with z-intervals of 10 μm. For immunostained samples, a center z-plane of each Axioloid was observed. For 3D reconstruction of immunostained Axioloids, samples were cleared and mounted in ScaleS4 clearing solution53 and tiled multi-stack images with z-intervals of 1.4-5 μm were acquired. 3D reconstruction Videos of immunostained samples were generated using NIS-Elements and the 3D Slicer software.
Measurement of somite volume was done using the 3D Slicer software (https://discourse.slicer.org/), based on z-stack images of immunostained Axioloids and CS11 human embryo data acquired previously using confocal microscope and OPT technic respectively. Using the segment editor, each somite was individually highlighted based on manual selections on several images of each stack, then global structure of each somite across each image was extrapolated automatically by the software using the fill between slices function. This updated selection was then manually cured before being used to recreate a 3D view of the highlighted structures. Voxel number and volume of each individual segment was then extracted automatically using the segment statistics function.
Periodicity of HES7 oscillation was quantified from time-series luminescence intensity data obtained from either live imaging or Kronos HT (Atto) measurements using HES7 reporter-derived Axioloids. For Kronos HT based oscillation measurements, Axioloids were transferred at 72 h (48 h after aggregation) into 24-well film-bottom plates (Eppendorf) covered in 400 μl of embedding medium (+/−MG) supplemented with D-Luciferin (100 μM) (bioWORLD). Axioloid containing culture plates were cultured at 37° C. and 5% CO2. Oscillations were measured for 48 hours and each well was measured for 10 seconds with 8 min intervals. Intensity values were processed using Matlab. Temporal trend was obtained by subtracting moving average (window size of 10 h), and the detrended signal was smoothed by Savitzky-Golay filter (window size of 3 h). Instantaneous oscillation phase was calculated by applying Hilbert transform, then peak detection was performed on cosine values of instantaneous oscillation phase. Peak-to-peak period was then quantified on each n-th oscillation.
Periodicity of segmentation was quantified from brightfield live-cell imaging data obtained with an inverted Ti2 microscope system (Nikon) using PlanApo λ 10× objective. All images were processed with Fiji and segments were assigned and corresponding time points of segment formation noted for the 24 h and 48 h periods following Axioloid embedding into MG.
Quantification of length or intensity based on imaging data was performed using Fiji57. Rostrocaudal length of formed segments was quantified from a center plane in z-stack images of Phalloidin-stained Axioloids. The longitudinal axes of Axioloids were measured on bright field images using Segmented Line tool. For quantifying fluorescent intensity of HCR or IHC staining, first, maximum intensity z projection images were created from multi-channel z-stack confocal images, then intensity of each channel was quantified along the longitudinal axis of Axioloids by Segmented Line tool, with a line width which corresponds to approximately 80% of the lateral length of Axioloids. Intensity values were further processed and plotted by using custom Python codes. Savitzky-Golay filter was applied on raw intensity values and then normalized. For HCR datasets including MESP2 staining, positional values were normalized using MESP2 peak before calculating average intensity of multiple samples.
pA-Tn5 Transposome Preparation
The recombinant Protein A-conjugated Tn5 transposase (pA-Tn5) was extracted and purified from bacterial cell lysates of T7 Express lysY/Iq Competent E. coli (NEB) harboring 3λFlag-pA-Tn5-Fl plasmid (a gift from Prof. Steven Henikoff, Addgene, #124601) using the columns filled with chitin slurry resin (NEB) after sonication-mediated solubilization as described before58. The transposase was assembled with a quarter of equimolar amount of the two types of oligo DNA adaptors (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3′ (SEQ ID NO: 1) and 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3′ (SEQ ID NO: 2)), each of which were pre-annealed with 5′-[PHO]CTGTCTCTTATACACATCT-3′ (SEQ ID NO: 3).
Axioloids were pooled and dissociated into single cell suspension in the same way as for the single-cell RNA-seq library preparation. Then, the cells were pelleted and snap-frozen in liquid nitrogen. Later on, the cells were processed according to previous literature58 with slight modifications. Briefly, 50,000 cells per experiment were first washed with wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 1× Protease Inhibitor (Roche)), and then resuspended again in the wash buffer. The cell suspension was then mixed with Concanavalin A-coated beads (Bangs Laboratories) resuspended in binding buffer (20 mM HEPES pH 7.9, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2). Next, cells were resuspended in 100 μL of ice-cold wash buffer, supplemented with 0.05% digitonin, 0.1 mM EDTA, and 0.1% BSA together with each 0.5 μl primary antibody. The antibodies used were mouse monoclonal IgG1 antibodies against H3K4me3 (MAB Institute, #MA304B) and H3K27me3 (MAB Institute, #MA323B). After incubation at room temperature for 2 hours, the buffer containing the primary antibody was replaced with 100 μl of ice-cold wash buffer, supplemented with 0.05% digitonin, together with 1 μl secondary rabbit anti-mouse IgG antibody (abcam, ab46540), and was incubated at 4° C. for overnight. After washing the beads with Dig-300 buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM spermidine, 0.01% digitonin, 1× Protease Inhibitor (Roche)) twice, 100 μl of the Dig-300 buffer containing 0.5 femtomol pA-Tn5 transposome assembled above was applied and incubated for 1 hour at room temperature. After washing the beads with Dig-300 buffer twice, the beads in 50 μl of Dig-300 buffer supplemented with 10 mM MgCl2 were resuspended and incubated at 37° C. for 1 hour. Then the supernatant was removed, and the beads were resuspended in 20 μl of 10 mM TAPS buffer (pH 8.5). Then thermolabile proteinase K (NEB) was added, and the mixture was incubated at 37° C. for 30 minutes, followed by incubation at 55° C. for 20 minutes to inactivate the proteinase. PCR-amplification of the library was directly performed from the tube after adding the reaction mixture (KAPA BIOSYSTEMS, #KK2502) using the primers listed in Supplementary Table 7.
The amplified libraries were purified with 1.3 volumes of AMPure XP beads. The libraries were sequenced with NovaSeq 6000 Sequencing System (Illumina) using NovaSeq 6000 SP Reagent Kit v1.5 (100 Cycles) (Illumina, #20028401) with the paired-end mode.
After the removal of the tagmentation adapter sequences with Cutadapt (version 3.4)59, paired-end reads were aligned to the human genome reference hg38 using Bowtie2 (version 2.2.4)60 with options—very-sensitive—X 2000. From mapped reads, properly paired (Samtools flag 0×2), MAPQ>=20, and non-mitochondrial chromosomal reads were extracted using Samtools (version 1.12)61. PCR duplicate reads were removed with Picard (version 2.25.2) (http://broadinstitute.github.io/picard/), and then reads mapped to blacklist (http://mitra.stanford.edu/kundaje/akundaje/release/blacklists/hg38-human/hg38.blacklist.bed.gz) were removed with bedtools (version 2.30)62. Bam files were converted to bigwig format with deepTools (version 3.5)63 and visualized with Integrative Genome Viewer (IGV) (version 2.12.2).
RNA Library Preparation for scRNA-Seq Analysis
All plastic materials used during the dissociation process of human Axioloids such as round bottom 96 well cell suspension plates, tubes, tips and cell strainers, were precoated with 0.1% BSA (SIGMA) in HBSS(−) (Wako). Axioloids were collected into wells of a 96-well plate and washed three times by transferring to another well filled with 0.1% BSA (SIGMA) in HBSS(−). Axioloids of the desired stage and number were then transferred to another well filled with 100 μl of enzyme P solution of the Neural Tissue Dissociation Kit (P) (Miltenyi Biotec) and incubated at 37° C. for 10 minutes. After adding 100 μl of 0.1% BSA in HBSS(−) Axioloids were dissociated by pipetting with an uncut P200 tip 20 times. The dissociated cells were filtered through Flowmi Cell Strainers with 40 m porosity (Merck) into a BSA-coated 2 ml tube (Eppendorf). Cells were washed twice in 1% BSA in HBSS(−) with centrifugation steps at 400×g for 6 minutes at room temperature. Cells were then resuspended in 1% BSA in HBSS(−) using a BSA-coated uncut-P200 tip and the concentration of the cell suspension was determined with hemocytometer. The cell suspension was subjected to library preparation using Chromium Next GEM Single Cell 3′ Kit v3.1 (10× Genomics), aiming for a target cell recovery of 5000-10000. Library preparation was preformed following the instructions provided by the manufacturer 10X Genomics (CG000315 Rev B).
scRNA-Seq Library Sequencing
The libraries were sequenced with NovaSeq 6000 Sequencing System (Illumina) using NovaSeq 6000 SP Reagent Kit v1.5 (100 Cycles) (Illumina, #20028401) and NovaSeq 6000 S1 Reagent Kit v1.5 (100 Cycles) (Illumina, #20028319) with the paired-end mode as described in the instructions by 10X Genomics (CG000315 Rev B).
scRNA-Seq Data Processing
scRNA-seq data were first mapped to the human reference genome (hg38) to make matrices of UMI count for each gene and each cell using Cell Ranger (10X Genomics, version 6.0.1). Putative doublet clusters were removed using Scrublet (version 0.2.3) with the expected_doublet_rate set as 0.0664. Then, the count data were imported into the Suerat package (version 4.0.3)65 for the downstream analysis. Cells with nfeature>1.500, nCount between 2,500 and 50,000, and proportions of mitochondrial gene counts between 2% and 12% were only considered for further analyses. The raw counts were normalized using the log-normalization method. The cell cycle scores and cell cycle phases were then determined as described before66.
Count matrices of different samples were first merged to be simultaneously projected on a UMAP plot and normalized variations due to differences of total UMI counts as well as the cell cycle phases with the SCTransform function employing the vars.to.regress option67. Principal component analysis (PCA) using the RunPCA function in the Seurat package was next performed. Then, the RunUMAP function was ran with default parameters except for dim=1:30 and the FindClusters function with a resolution of 0.5 for
the two replicates of single-cell transcriptome data of MG-embedded Axioloids at 96 h were integrated with the mutual nearest neighbor algorithm after reciprocal principal component analysis using the Seurat package to correct batch effects36. For this, the SCTransform normalization was first performed as described above and 3,000 integration features with SelectIntegrationFeatures function were selected. Next, the RunPCA function was ran with the selected features. Then, integration anchor sets using FindIntegrationAnchors was obtained with the normalization method and the reduction method set as “SCT” and “rpca”, respectively. The k.anchor parameter was set as 5. Then, the two replicates were integrated using the obtained anchor sets. After running PCA using the integration data, UMAP and clustering analysis were performed as described above with dim=1:20 and a resolution of 0.25 for
To call differentially expressed genes (DEGs) between different Axioloid culture conditions, the batch correction and cluster annotation were first performed as described above to define cell sets of the same cell types to be compared with each other condition. In each of the defined cell clusters, gene expression levels were compared based on the normalized count data using the FindMarkers function of Seurat. For the comparison between MG-plus and MG-minus, DEGs were first called with the log 2 fold changes threshold of 0.25 for each of the two replicate experiments. Then, genes that were commonly up- or down-regulated by MG in both replicates were listed. The expression changes with the two replicate data sets combined together using the FindMarkers function with a Wilcoxon rank sum test were also calculated to make the volcano plots in
To perform velocity analysis, the fastq sequence data were reanalyzed with Kallisto (version 0.46.0)69 and loompy (version 3.0.6) to obtain count matrices for both spliced and unspliced transcripts, followed by filtering out cells that were not subject to the UMAP and clustering analysis above. RNA velocity was then analyzed using scVelo (version 0.2.3) with the stochastic mode70. The parameters used were min_shared_counts=20 and n_top_genes=2000 for the scv.pp.filter_and_normalize function, and n_pcs=30 and n_neighbors=30 for the scv.pp.moments function. The velocities are projected onto the UMAP plot generated above. For the integrated data set of the two replicates of 96 h_MG, pseudotime analysis was subsequently performed based on the velocity graph, using the scv.tl.velocity_pseudotime function in the scVelo package. The cells except for those annotated as IM-like or EC-like cells according the to the rank of the pseudotime were ordered to have the heatmap plots in
Comparison with Human Embryo scRNA-Seq Data
To compare the Axioloids with human embryos, the single cell RNAseq data of a CS12 human embryo were used 35. From this dataset, Seurat object of the embryo was created using Seurat (version 4.0.6), and SCTransform was performed with options vars.to.regress=c(“S.Score”, “G2M.Score”), variable.features.n=5000. UMAP analysis and clustering were performed using the RunUMAP function with the option dims=1:60, and the FindClusters function with the resolution 0.65 for
To represent expression levels of each gene on the UMAP plots in
All single cell RNA sequencing data and CUT&Tag data used for this study have been deposited in the NCBI Gene Expression Omnibus under accession number GSE199576. To review the data please go to https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE199576 and enter reviewer token ktunygmklvclpwp into the box.
Computational codes and scripts used in this study are available at GitHub (https://github.com/Alev-Lab) and upon request from the corresponding author.
Live imaging of Axioloids derived from 409B2 (upper) and 201B7 Luc (lower) with +MG (right) or without −MG (left) embedding into Matrigel (MG) between 72 h and 120 h of culture. Data shown is representative of at least three independent experiments. Scale bar is 200 m.
Live imaging of the spatiotemporal morphogenetic expression of the HES7 gene in a 201B7 Luc-derived Axioloid embedded in MG from 72 h to 120 h of culture. BF video (left) and HES7:Luciferase signal (right). Data shown is representative of at least three independent experiments. Scale bar is 200 m.
Live imaging of Axioloids derived from 409B2 (Top panels) and 201B7 Luc (bottom panels) after embedding in MG only (left) or in MG supplemented with Retinal (RAL) (middle) or Retinoic Acid (RA) (right) from 72 h to 120 h of culture. Data shown is representative of at least three independent experiments. Scale bar is 200 m.
3D reconstruction of an Axioloid embedded in MG+RA at 120 h stained for F-actin (Phalloidin) in gray, TBXT (BRA) in blue, Fibronectin (FN) in green and MEOX1 in red.
Visualization of midline formation in a 409B2-derived Axioloid embedded in MG+RAL (Top) and of formation of a single bilateral somite in a 409B2-derived Axioloid embedded in MG+ROL (Bottom). Live imaging was performed between 72 and 120 h of culture. Scale bar is 200 m.
3D reconstruction of Axioloid segments derived from 409B2 (Right top) and 201B7 Luc (Right bottom) and reconstruction of the 8 posterior-most somites of a CS11 human embryo (right). Each somite-like structure is highlighted by a different color depending on its position along the antero-posterior axis.
Live imaging of the spatiotemporal morphogenetic expression of the HES7 gene in 201B7 Luc-derived Axioloids embedded in MG supplemented with either RAL (top left) or RA (top right) or RAL+DMSO (bottom left) or RAL+BMS493 (bottom right), from 72 h to 120 h of culture. BF video (left) and HES7:Luciferase signal (right) for each condition. Data shown is representative of at least three independent experiments. Scale bar is 200 m.
Live imaging of Axioloids derived from 201B7 Luc after embedding in MG+RAL supplemented with (from left to right) DMSO, BMS493, DAPT, PD173074 or XAV939 from 72 h to 120 h of culture. Data shown is representative of at least three independent experiments. Scale bar is 200 m.
Top panel, live imaging of Axioloids embedded in MG+RAL derived from 201B7 Luc (top left), HES7 KO1 (top middle) and HES7 KO2 (top right) cell lines. Data shown is representative of at least three independent experiments. Scale bar is 200 m. Bottom panel, 3D reconstruction of Axioloids embedded in MG+RAL derived from HES7 KO1 (middle) and HES7 KO2 (bottom) stained with F-actin (Phalloidin) in gray, TBXT (BRA) in blue, Fibronectin (FN) in green and MEOX1 in red.
Live imaging of the spatiotemporal expression of the HES7 gene in 201B7 Luc, (top), HES7 KO1 (middle) and HES7 KO2 (bottom)-derived Axioloids embedded in MG+RAL. BF video (left) and HES7:Luciferase signal (right) for each condition. Data shown is representative of at least three independent experiments. Scale bar is 200 m.
Top panel, live imaging of Axioloids embedded in MG+RAL derived from 201B7 Luc (left), HES7R25W MT1 (middle) and HES7R25W MT2 (right) cell lines. Data shown is representative of at least three independent experiments. Scale bar is 200 m. Bottom panel, 3D reconstruction of Axioloids embedded in MG+RAL derived from HES7R25W MT1 (top) and HES7R25W MT2 (bottom) stained with F-actin (Phalloidin) in gray, TBXT (BRA) in blue, Fibronectin (FN) in green and MEOX1 in red.
Live imaging of the spatiotemporal expression of the HES7 gene in 201B7 Luc, (top), HES7R25W MT1 (middle) and HES7R25W MT2 (bottom)-derived Axioloids. BF video (left) and HES7:Luciferase signal (right) for each condition. Data shown is representative of at least three independent experiments. Scale bar is 200 m.
Top panel, live imaging of Axioloids embedded in MG+RAL derived from 201B7 Luc (top left), MESP2 KO1 (top middle) and MESP2 KO2 (top right) cell lines. Data shown is representative of at least three independent experiments. Scale bar is 200 m. Bottom panel, 3D reconstruction of Axioloids embedded in MG+RAL derived from MESP2 KO1 (middle) and MESP2 KO2 (bottom) stained with F-actin (Phalloidin) in gray, TBXT (BRA) in blue, Fibronectin (FN) in green and MEOX1 in red.
Live imaging of the spatiotemporal expression of the HES7 gene in 201B7 Luc, (top), MESP2 KO1 (middle) and MESP2 KO2 (bottom)-derived Axioloids embedded in MG+RAL. BF video (left) and HES7:Luciferase signal (right) for each condition. Data shown is representative of at least three independent experiments. Scale bar is 200 m.
In MG and RA treated axioloids a single axis of sequentially forming epithelial somites with single central somitocoels was typically observed. Intriguingly, axioloids also frequently displayed a superficial groove or midlinelike structure, starting in the PSM and going through most of the forming segments (Supplementary
Supplementary Table 1 (Table 1-A to 1I): Genes expressed in identified clusters of scRNA-seq data.
Supplementary Table 2 (Table 2-A to 2-B): NMP and NE module score genes. List of neuromesodenrmal progenitor (NMP) and neuroectodenrm (NE) associated genes used to calculate the module scores for
Supplementary Table 3 (Table 3-A to 3-D) Recombinant proteins, small molecules, reagents & consumables/equipment used in this study. List of utilized recombinant proteins & small molecules (3.1), modulators of signaling pathways (3.2) reagents (3.3) and consumables/equipment (3.4).
Supplementary Table 4 (Table 4-A to 4-D): Antibodies used in this study. List of utilized primary antibodies for immunohistochemistry (4.1), secondary antibodies for immunohistochemistry (4.2), primary antibodies for CUT&Tag library preparation (4.3), secondary antibodies for CUT&Tag library preparation (4.4).
Supplementary Table 5 (Table 5): List of probes used for HCR. List of utilized probes for HCR-based whole-mount in situ hybridization analysis of human axioloid samples.
Supplementary Table 6 (Table 6-A to 6-R): List of probe sequences used for HybISS analysis. List of utilized probe sequences used for HybISS-based spatial transcriptomic analysis of human axioloid samples.
Supplementary Table 7 (Table 7): List of primers used for CUT&Tag experiments. List of utilized primers used for CUT&Tag-based analysis of human axioloids.
Live imaging of axioloids derived from 409B2 (upper) and 201B7 Luc (lower) between 24 h and 72 h of culture. Data shown is representative of at least three independent experiments. Scale bar is 200 m.
Live imaging of axioloids derived from 409B2 (upper) and 201B7 Luc (lower) with +MG (right) or without −MG (left) embedding into Matrigel (MG) between 72 h and 120 h of culture. Data shown is representative of at least three independent experiments. Scale bar is 200 m.
Live imaging of the spatiotemporal morphogenetic expression of the HES7 gene in a 201B7 Luc-derived axioloid embedded in MG from 72 h to 120 h of culture. BF video (left) and HES7:Luciferase signal (right). Data shown is representative of at least three independent experiments. Scale bar is 200 m.
Live imaging of axioloids derived from 409B2 (top panels) and 201B7 Luc (bottom panels) after embedding in MG only (left) or in MG supplemented with retinol (ROL), retinal (RAL) or retinoic acid (RA) (right) from 72 h to 120 h of culture. Data shown is representative of at least three independent experiments. Scale bar is 200 m.
3D reconstruction of 409B2-derived axioloids embedded in MG only (upper left) or in MG supplemented with retinol (ROL) (lower left), retinal (RAL) (lower right) or retinoic acid (RA) (upper right) at 120 h of culture stained for Factin (Phalloidin) in gray, TBXT (BRA) in blue, Fibronectin (FN1) in green and MEOX1 in red.
Live imaging of axioloids derived from 201B7 Luc after embedding in +MG+RAL supplemented with (from left to right) DMSO, BMS493, AGN193109 or ER50891 from 72 h to 120 h of culture. Data shown is representative of at least three independent experiments. Scale bar is 200 m.
Supplementary Video 7: 3D Visualization of Axioloids Treated with RA Pathway Inhibitors.
3D reconstruction of axioloids derived from 201B7 Luc embedded in +MG+RAL (upper left) or in +MG+RAL supplemented with three different RA inhibitors, including BMS493 (upper right), AGN193109 (lower left) or ER50891 (lower right) at 120 h of culture stained for F-actin (Phalloidin) in gray, TBXT (BRA) in blue, Fibronectin (FN1) in green and MEOX1 in red.
Visualization of midline formation in a 409B2-derived axioloid embedded in +MG +RAL (top) and of formation of a single bilateral somite in 409B2-derived axioloid embedded in +MG+ROL (bottom). Live imaging was performed between 72 and 120 h of culture. Scale bar is 200 m
3D reconstruction of somites formed in 409B2-derived MG embedded axioloids (top) treated retinol (ROL) (left), retinal (RAL) (middle) and retinoic acid (RA); and 3D reconstructions of somites in human embryos (bottom) found in CS9 (left), CS10 (middle) and CS11 (right) human embryos. Each somite-like structure is highlighted by a different color depending on its position along the antero-posterior axis.
Live imaging of the spatiotemporal morphogenetic expression of the HES7 gene in 201B7 Luc-derived axioloids embedded in MG only (upper left pair) or in MG supplemented with retinol (ROL) (lower left pair), retinal (RAL) (lower right pair) or retinoic acid (RA) (upper right pair) from 72 h to 120 h of culture. BF video (left) and HES7:Luciferase signal (right) for each condition. Data shown is representative of at least three independent experiments. Scale bar is 200 m.
Live imaging of the spatiotemporal morphogenetic expression of the HES7 gene in 201B7 Luc-derived axioloids embedded in MG supplemented with +RAL+DMSO (left pair) or +RAL+BMS493 (right pair), from 72 h to 120 h of culture. BF video (left) and HES7:Luciferase signal (right) for each condition. Data shown is representative of at least three independent experiments. Scale bar is 200 m.
Live imaging of axioloids derived from 201B7 Luc after embedding in +MG+RAL supplemented with DMSO (top left), DAPT (top right), PD173074 (middle left), PD0325901 (middle right), XAV939 (bottom left) or IWP2 (bottom right) from 72 h to 120 h of culture. Data shown is representative of at least three independent experiments. Scale bar is 200 m.
Top panel, live imaging of axioloids embedded in +MG+RAL derived from 201B7 Luc (top left), HES7 KO1 (top middle) and HES7 KO2 (top right) cell lines. Data shown is representative of at least three independent experiments. Scale bar is 200 m. Bottom panel, 3D reconstruction of axioloids embedded in +MG+RAL derived from HES7 KO1 (middle) and HES7 KO2 (bottom) stained with F-actin (Phalloidin) in gray, TBXT (BRA) in blue, Fibronectin (FN1) in green and MEOX1 in red.
Live imaging of the spatiotemporal expression of the HES7 gene in 201B7 Luc, (top pair), HES7 KO1 (middle pair) and HES7 KO2 (bottom pair)-derived axioloids embedded in +MG+RAL. BF video (left) and HES7:Luciferase signal (right) for each condition. Data shown is representative of at least three independent experiments. Scale bar is 200 m.
Top panel, live imaging of axioloids embedded in +MG+RAL derived from 201B7 Luc (top left), HES7R25W MT1 (top middle) and HES7R25W MT2 (top right) cell lines. Data shown is representative of at least three independent experiments. Scale bar is 200 m. Bottom panel, 3D reconstruction of axioloids embedded in +MG+RAL derived from HES7R25W MT1 (middle) and HES7R25W MT2 (bottom) stained with F-actin (Phalloidin) in gray, TBXT (BRA) in blue, Fibronectin (FN1) in green and MEOX1 in red.
Live imaging of the spatiotemporal expression of the HES7 gene in 201B7 Luc, (top pair), HES7R25W MT1 (middle pair) and HES7R25W MT2 (bottom pair)-derived axioloids. BF video (left) and HES7:Luciferase signal (right) for each condition. Data shown is representative of at least three independent experiments. Scale bar is 200 m.
Top panel, live imaging of axioloids embedded in +MG+RAL derived from 201B7 Luc (top left), MESP2 KO1 (top middle) and MESP2 KO2 (top right) cell lines. Data shown is representative of at least three independent experiments. Scale bar is 200 m. Bottom panel, 3D reconstruction of axioloids embedded in +MG+RAL derived from MESP2 KO1 (middle) and MESP2 KO2 (bottom) stained with F-actin (Phalloidin) in gray, TBXT (BRA) in blue, Fibronectin (FN1) in green and MEOX1 in red.
Live imaging of the spatiotemporal expression of the HES7 gene in 201B7 Luc (top pair), MESP2 KO1 (middle pair) and MESP2 KO2 (bottom pair)-derived axioloids embedded in +MG+RAL. BF video (left) and HES7:Luciferase signal (right) for each condition. Data shown is representative of at least three independent experiments. Scale bar is 200 m.
Somitogenesis is a core developmental event during which the metameric body plan is laid out in vertebrates. It is well studied in model organisms such as mouse, zebrafish or chick but remains poorly understood in human and other primates. Despite recent progress with pluripotent stem cell (PSC)-based in vitro models of organogenesis and embryonic development, an experimental model system that can robustly recapitulate core features of somitogenesis in vitro remained out of reach. Using in vitro-derived presomitic mesoderm (PSM), it has been previously succeeded to reconstitute and quantify oscillatory activity of the segmentation clock, a molecular oscillator believed to control somite formation. Extending on these earlier findings it was then asked whether not only the segmentation clock but also the actual process of segmentation and epithelial somite formation in vitro could be recapitulated.
To this end “axioloids” was generated, a self-organizing 3D in vitro model of human somitogenesis which shares morphological and molecular features of the emerging vertebrate embryonic tail and axis including presence of somitogenesis associated major cell populations and opposing morphogen gradients and signaling activities as well as periodic formation of properly patterned epithelial somites in synchrony with the segmentation clock. Using this model, an unknown function of retinoic acid (RA) signaling in the stabilization and epithelialization of the newly formed somite-like structures within axioloids was unveiled.
Here the effect on initial and final axioloid morphogenesis of variations in the concentration or nature of the molecules and reagents used during axioloid derivation was assessed and is reported, including effect of different CHIR mediated WNT activation levels, bFGF mediated FGF pathways activation levels, and different TGF-β inhibitor mediated TGF-β pathway activation levels. Moreover, the effect of various extracellular matrix compounds and a synthetic analog of the retinoic acid pathway was also evaluated.
Culture of Human Induced Pluripotent Stem Cells (iPSCs)
Human iPS cell lines derived from healthy donors, e.g. 409B2 were used in this study. Human iPS cells were maintained in StemFit AK02N (Reprocell) medium supplemented with 50 U penicillin and 50 g ml-1 streptomycin (Gibco) on iMatrix-511 silk-coated plates or dishes (Nippi). StemFit AK02N (Reprocell) medium contains three components, A, B and C, all three of which were mixed and used for standard maintenance culture of human iPS cells in humidified incubators at 37° C. and 5% CO2. Used iPS cells were regularly tested and reported negative for mycoplasma contamination.
Based on the axioloid protocol it was explored if alternative compounds, molecules or inhibitors could be used and would support the generation of axioloids from PSCs. It was started by testing if NDiff227 (Takara, Cat: Y40002) medium or RPMI 1640 (Nacalai, Cat: 30264-85) supplemented with B27 either with (Gibco, Cat: 17504-044) or without (in house) retinol could be used as an alternative to AK02N-C induction media. The effects of different concentrations of bFGF (from 5 to 250 ng ml-1), or CHIR99021 (from 2.5 to 10 μM), or SB431542 (from 1.25 to 15 μM) were then tested on axioloid morphology. It was also tested if another member of the FGF superfamily: FGF8b (PeproTech, Cat: 100-25) (from 20 to 250 ng ml−1) or another TGFβ inhibitor A83-01 (Selleck Chemicals, Cat: S7692) (from 1.25 to 15 μM) could have similar results as described for bFGF and SB431542. Finally, during the embedding phase it was tested if the alternative ECM containing compounds Geltrex (Gibco, Cat: A14132-0), Cultrex (R&D Systems, Cat: 3433-005-01), and ECMgel (Sigma-Aldrich, Cat: E6909) could be used instead of Matrigel and would support axioloid morphogenesis. It was also assessed whether a synthetic RA selective agonist, TTNPB (4-[(E)-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid) (Selleck Chemicals, Cat: S4627) could replace vitamin A derivatives such as retinol or retinal.
Basic Strategy for Axioloid Induction from Human Pluripotent Stem Cells (PSCs)
The generation of a mesoderm-based human induced pluripotent stem cell (iPSC)-derived 3D model of human axial development was recently reported, which is termed ‘axioloids’. This was achieved through an initial induction step in 2D, using both bFGF (20 ng/ml) and CHIR99421 (5 M) which efficiently and reproducibly promoted the formation of primitive streak-like cells from human PSCs. This was then followed by a 3D aggregation step in the presence of bFGF and CHIR (same concentrations as during 1st step) in addition to 10 μM of SB431542 (TGFβ inhibitor) and 10 μM of Y-27632 (ROCK inhibitor). This step aimed at promoting the commitment of the initial primitive streak cells to a presomitic or paraxial mesoderm (PSM) fate. Formed mesodermal aggregates spontaneously broke symmetry and started elongating and adopted an initial ‘bean-like’ morphology. At this point, embedding into 10% Matrigel (MG)-containing medium supplemented with retinoids led both to further elongation and the sequential emergence of somite-like epithelialized segments along the anterior-posterior axis of these structures, which is termed ‘axioloids’.
Different culture media in addition to the standard AK02N medium was tested. Several groups in the field have reported the successful generation of mouse and human PSC-derived gastruloids and similar structures using N2B27 media (PMID: 30283134). A quality controlled commercial version of the N2B27 medium called NDiff227 was initially tested, and it was shown that it can support axioloid formation (
Effective Range of Concentrations of bFGF, CHIR (WNT Agonist) & SB431542 (TGFβ Inhibitor)
It was shown in the corresponding publication (Yamanaka, Hamidi et al., Nature 2023) that for proper axioloid induction bFGF, CHIR99421 (a WNT agonist) and SB431542 (TGFβ-inhibitor) are necessary and sufficient. Here different concentrations of all three components were tested and their impact was assessed on axioloid induction and morphology. The results indicate that higher concentrations of bFGF do not largely affect overall axioloid morphology with regards to both elongation and segmentation observed at 96 h and 120 h, while structures at 120 h appear to get thinner at higher bFGF concentrations. Total absence of bFGF during axioloid induction results in loss of polarization, elongation and failure to generate axioloid structures from PSCs (
Different concentrations of the TGFβ pathway inhibitor SB431542 used for axioloid induction were furthermore assessed. The results indicate that SB431542 is active and effective over a large range of concentrations (
Basic FGF (bFGF) is a member of the fibroblast growth factor family that includes 23 heparin-binding peptides widely expressed during embryo development. A large number of recombinant FGFs were tested for a putative effect on axioloid induction and morphogenesis. An effect for recombinant FGF8b which behaved differently from bFGF was observed. At higher FGF8b concentrations (100 ng/ml) elongated structures were obtained but somites were not discernable within the forming structures (
Similarly, it was tested if A-83-01, an alternative TGFβ pathway inhibitor could be used to replace SB431542. The results indicate that axioloids generated in different concentrations of A-83-01 recapitulate hallmarks of proper axioloids morphogenesis, similar to what it has been described for SB431542, with best results obtained for smaller concentrations of A-83-01 e.g. 1.25 uM or 2.5 uM (
Matrigel (MG) is an extracellular matrix (ECM)-rich solubilized basement membrane secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells that can support the formation of complex tissue architectures and help mimic morphogenetic processes in vitro. In the experimental system addition of both 5% or 10% MG to the culture medium (during the embedding phase after initial symmetry breaking) is sufficient and leads to effective axioloid induction in the presence of retinoids. Here supplementation of the culture medium with alternative ECM-rich compounds (10%) such as Cultrex, Geltrex and ECMgel was assessed. All three tested ECM-rich compounds lead to variable morphological axioloid phenotypes, with axial elongation observed in all three compounds albeit to a lesser extent than Matrigel. It is believed that other ECM containing compounds can likely be used as well and may be able to replace MG. It is furthermore envisioned that distinct mixtures of defined recombinant matrix proteins and basement membrane components may be also able to replace MG or similar complex ECM-rich compounds in order to achieve reproducible and efficient axioloid induction and morphogenesis in the presence of active retinoid signaling which appears to be essential and working in synergy with MG.
In addition to assessing ECM-rich compounds it has been also investigated and confirmed that TTNPB, a synthetic analog of retinoic acid, selective for the retinoic acid receptor (RAR) subtype, could be used in replacement of retinoids. TTNPB showed overall similar effect on axioloid morphology and somite epithelialization than the other retinoids including retinol (ROL), retinal (RAL) and retinoic acid (RA).
While the present disclosure has been described above with reference to exemplary embodiments and example, the present disclosure is by no means limited thereto. Various changes and modifications that may become apparent to those skilled in the art may be made in the configuration and specifics of the present disclosure without departing from the scope of the present disclosure.
This application claims priority from U.S. Provisional Patent Application No. 63/326,611 filed on Apr. 1, 2022. The entire disclosure of this US provisional patent application is incorporated herein by reference.
Patents, patent applications, and references cited in the present specification are incorporated herein in their entirety by reference, as if fully and specifically set forth herein.
The whole or part of the exemplary embodiments and example disclosed above can be described as, but not limited to, the following Supplementary Notes.
A three-dimensional cellular aggregate generated in vitro from a pluripotent stem cell, comprising:
A three-dimensional cellular aggregate generated in vitro from a pluripotent stem cell, comprising:
The cellular aggregate according to Supplementary Note 1 or 2, wherein
The cellular aggregate according to Supplementary Note 3, wherein
The cellular aggregate according to Supplementary Note 3 or 4, wherein
The cellular aggregate according to Supplementary Note 5, wherein
The cellular aggregate according to any one of Supplementary Notes 1 to 6, wherein
The cellular aggregate according to Supplementary Note 3, wherein
The cellular aggregate according to Supplementary Note 3 or 4, wherein
The cellular aggregate according to any one of Supplementary Notes 1 and 3 to 9, wherein
The cellular aggregate according to any one of Supplementary Notes 1 to 10, wherein
The cellular aggregate according to any one of Supplementary Notes 2 to 11, wherein
The cellular aggregate according to Supplementary Note 1 or 12, wherein
The cellular aggregate according to Supplementary Note 13, wherein
The cellular aggregate according to Supplementary Note 13 or 14, wherein
The cellular aggregate according to any one of Supplementary Notes 13 to 15, wherein
The cellular aggregate according to any one of Supplementary Notes 13 to 16, wherein
The cellular aggregate according to any one of Supplementary Notes 13 to 17, wherein
The cellular aggregate according to any one of Supplementary Notes 13 to 18, wherein
The cellular aggregate according to Supplementary Note 19, wherein
The cellular aggregate according to any one of Supplementary Notes 13 to 17, wherein
The cellular aggregate according to any one of Supplementary Notes 1 and 12 to 21, wherein
The cellular aggregate according to Supplementary Note 22, wherein
The cellular aggregate according to Supplementary Note 22 or 23, wherein
The cellular aggregate according to any one of Supplementary Notes 1 and 12 to 24, comprising:
The cellular aggregate according to any one of Supplementary Notes 1 and 12 to 25, wherein
The cellular aggregate according to any one of Supplementary Notes 1 to 26, comprising:
The cellular aggregate according to Supplementary Note 27, wherein
The cellular aggregate according to any one of Supplementary Notes 1 to 28, substantially not comprising an endodermal cell and/or an ectodermal cell.
The cellular aggregate according to Supplementary Note 29, wherein
The cellular aggregate according to Supplementary Note 29 or 30, wherein
The cellular aggregate according to any one of Supplementary Notes 29 to 31, wherein
The cellular aggregate according to any one of Supplementary Notes 29 to 32, wherein
The cellular aggregate according to any one of Supplementary Notes 1 to 33, wherein
The cellular aggregate according to Supplementary Note 34, wherein
The cellular aggregate according to Supplementary Note 34 or 35, wherein
The cellular aggregate according to any one of Supplementary Notes 1 to 36, wherein
The cellular aggregate according to any one of Supplementary Notes 1 to 37, wherein
The cellular aggregate according to any one of Supplementary Notes 1 to 38, comprising:
The cellular aggregate according to any one of Supplementary Notes 1 to 39, which has a length of at least 0, 05 mm, at least 0, 1 mm, at least 0.2 mm, at least 0.3 mm, at least 0.4 mm, at least 0.5 mm, at least 0.6 mm, at least 0.7 mm, at least 0.8 mm, at least 0.9 mm, or at least 1 mm.
(Supplementary Note 41) A method for producing a three-dimensional cellular aggregate generated in vitro from a pluripotent stem cell, comprising the steps of:
The method according to Supplementary Note 41, comprising the steps of:
The method according to Supplementary Note 41 or 42, comprising the steps of:
The method according to Supplementary Note 42 or 43, comprising the step of:
The method according to Supplementary Note 43 or 44, wherein
The method according to any one of Supplementary Notes 42 to 45, wherein
The method according to any one of Supplementary Notes 42 to 46, wherein
The method according to any one of Supplementary Notes 42 to 47, wherein
The method according to any one of Supplementary Notes 42 to 48, wherein
The method according to any one of Supplementary Notes 42 to 49, wherein
The method according to Supplementary Note 50, wherein
The method according to any one of Supplementary Notes 42 to 51, wherein
The method according to any one of Supplementary Notes 41 to 52, wherein
The method according to any one of Supplementary Notes 41 to 53, wherein
The method according to any one of Supplementary Notes 41 to 54, wherein
The method according to Supplementary Note 55, wherein
The method according to Supplementary Note 55 or 56, wherein
The method according to any one of Supplementary Notes 55 to 57, wherein
The method according to any one of Supplementary Notes 55 to 58, wherein
The method according to any one of Supplementary Notes 41 to 59, wherein
The method according to any one of Supplementary Notes 41 to 60, wherein
The method according to any one of Supplementary Notes 41 to 61, wherein
The method according to any one of Supplementary Notes 41 to 62, wherein
The method according to any one of Supplementary Notes 41 to 63, wherein
A cell obtained from the cellular aggregate according to any one of Supplementary Notes 1 to 40.
A method for producing a progenitor cell or a differentiated cell, comprising the step of:
The method according to Supplementary Note 66, comprising the step of:
A method for evaluating a test substance, comprising the steps of:
The method according to Supplementary Note 68, wherein
The method according to Supplementary Note 68, wherein
The method according to Supplementary Note 70, wherein
The method according to Supplementary Note 68, wherein
The method according to Supplementary Note 72, wherein
The method according to any one of Supplementary Notes 68 to 73, wherein
The method according to Supplementary Note 68, wherein
A method for evaluating gene function or genome function, comprising the steps of:
The method according to Supplementary Note 75, wherein
The method according to Supplementary Note 75, comprising the step of:
The method according to any one of Supplementary Notes 75 to 78, wherein
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2023/013711 | 3/31/2023 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63326611 | Apr 2022 | US |
