Cell culture system, its use, and cells derived therefrom

Abstract

The present disclosure relates to a cell culture system for generating cell aggregates and its use. The cell aggregates described herein can be used for studying embryonic development and hematopoiesis and for treating diseases.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 345,233, filed May 24, 2022, which is hereby incorporated by reference in its entirety.

[0002] Statement Regarding Federally Sponsored Research or Development This invention was made with government support under grant numbers HL141805, DK120531, and EB001026 awarded by the National Institutes of Health, and grant number 2134999 awarded by the National Science Foundation. The United States government has certain rights in this invention.

[0003] The present disclosure relates to in vitro and ex vivo culture systems for generating cell masses comprising one or more germ cell structures and one or more extra - embryonic cell structures.

Background Art

[0004] The decoding mechanism of human embryonic development has a major biomedical impact, from the treatment of congenital diseases and infertility to the manipulation of functional human organs. Immediately after implantation, the embryo and co-developing extraembryonic tissues are deeply remodeled, initiating morphological changes central to pregnancy success, including the formation of the amniotic cavity or the emergence of yolk sac hematopoiesis (Muller, F. 2004, Palis, J. & Yoder, M. C. 2001). However, due to its concealment within uterine tissue and limited access to this stage for both technical and ethical reasons, the critical steps of early post-implantation development in humans remain largely unachieved (Pera, M. F. 2017). In vitro models of human embryonic development have emerged as a means platform for investigating human-specific developmental mechanisms (Beccari, L. et al. 2018, Harrison, S. E., et al. 2017, Warmflash, A, et al. 2014, Zheng, Y. et al. 2019, Yu, L. et al. 2021, Sozen, B. et al. 2019, Rivron, N. C. et al. 2018, Liu, X. et al. 2021, Simunovic, M. et al. 2019, Li, R. et al. 2019, Veenvliet, J. V. et al. 2020, Karzbrun, E. et al. 2021, Kagawa, H. et al. 2021). However, they often suffer from limitations such as low efficiency and throughput, limited scalability, and high technical complexity. Traditionally, these studies use cocktails of growth factors at supra-physiological levels and encounter challenges in finding a common medium to support diverse cell fates. Additionally, human post-implantation embryo models with co-developing germ and extraembryonic layers are still lacking, which limits the ability to study the integrated morphogenetic events of the post-implantation human embryo. What is needed are in vitro and ex vivo cell culture systems for the study of embryonic development. The systems and methods disclosed herein address these and other needs. Summary of the Invention

[0005] Provided herein are in vitro or ex vivo cell culture systems for generating cell aggregates and their use. The generated cell aggregates may comprise one or more embryonic cell structures, one or more extraembryonic cell structures, and / or one or more hematopoietic stem cells.

[0006] Accordingly, in some aspects, disclosed herein is a method for generating ex vivo or in vitro a cell aggregate comprising one or more embryonic cell structures and one or more extraembryonic cell structures, a) obtaining a first stem cell population comprising an inducible transgene encoding a GATA binding protein 6 (GATA6) polypeptide; b) contacting the first stem cell population with an inducer that induces the expression of the transgene; c) mixing the first stem cell population of step b) with a second stem cell population; d) culturing the cell mixture of step c) on a surface for at least 7 days to thereby produce a cell aggregate.

[0007] In some embodiments, the second stem cell population does not have an inducible transgene encoding a GATA6 polypeptide. In some embodiments, the second stem cell population comprises an inducible transgene encoding an ETS variant transcription factor 2 (ETV2) polypeptide. In some embodiments, step c) further comprises contacting the first stem cell population of step b) with a second stem cell population and a third cell population, the third cell population comprising an inducible transgene encoding an ETS variant transcription factor 2 (ETV2) polypeptide.

[0008] In some embodiments, the first stem cell population comprises one or more nucleic acid sequences that are at least about 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 3-10. In some embodiments, the first stem cell population comprises one or more nucleic acid sequences that are at least about 80% identical to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the second stem cell population comprises one or more nucleic acid sequences that are at least about 80% identical to a sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 14. In some embodiments, the second stem cell population and / or the third stem cell population further comprises a nucleic acid sequence that is at least about 80% identical to SEQ ID NO: 15.

[0009] Cell aggregates generated by the methods disclosed herein can be used to evaluate, test, and / or screen for agents (e.g., therapeutic agents) that regulate embryonic development or hematopoietic differentiation, comprising generating a cell aggregate comprising one or more embryonic cell structures and one or more extraembryonic cell structures by a method of any preceding aspect, and contacting the cell aggregate with an agent. In some embodiments, the method further comprises determining biomarkers associated with embryonic development or hematopoietic differentiation.

[0010] Embryonic cell structures of the cell aggregate can include, but are not limited to, increased levels of octamer-binding transcription factor 4 (OCT4) polypeptide and decreased levels of GATA4 polypeptide. Extraembryonic cell structures can include, but are not limited to, increased levels of GATA6 polypeptide.

[0011] In some embodiments, the cell mass of any preceding aspect comprises one or more of a bilayer disk-like structure, an amnion-like domain, a primitive streak-like domain, and a yolk sac domain. In some embodiments, the amnion-like domain comprises one or more of increased levels of bone morphogenetic protein 4 (BMP4), bone morphogenetic protein receptor 1A (BMPR1A), distal-less homeobox 5 (DLX5), follistatin-like 1 (FSTL1), inhibitor of DNA binding 1 (ID1), lymphocyte enhancer-binding factor 1 (LEF1), msh homeobox (MSX)1, MSX2, SMAD family member 1 (SMAD1), and SMAD-specific E3 ubiquitin protein ligase 2 (SMURF2). In some embodiments, the cell mass comprises one or more neural characteristics (e.g., comprising one or more of increased levels of cerberus 1 (CER1) polypeptide and left-right determination factor 1 (LEFTY1) polypeptide).

[0012] In some embodiments, hematopoietic stem cells isolated from the cell mass of any preceding aspect have one or more of increased levels of CD45, CD11b, and CD34.

[0013] Also disclosed herein is an in vitro or ex vivo culture system for generating a cell mass comprising one or more embryonic cell structures and one or more extraembryonic cell structures, the system comprising a first stem cell population comprising an inducible transgene encoding a GATA binding protein 6 (GATA6) polypeptide, and a second stem cell population.

[0014] Also disclosed herein is an in vitro or ex vivo culture system for generating a cell mass comprising one or more hematopoietic stem cells, the system comprising a first stem cell population comprising an inducible transgene encoding a GATA binding protein 6 (GATA6) polypeptide, and a second stem cell population.

[0015] In some embodiments, the second stem cell population does not have an inducible transgene encoding a GATA6 polypeptide. In some embodiments, the second stem cell population comprises an inducible transgene encoding an ETS variant transcription factor 2 (ETV2) polypeptide. In some embodiments, step c) further comprises contacting the first stem cell population of step b) with a second stem cell population and a third cell population, the third cell population comprising an inducible transgene encoding an ETS variant transcription factor 2 (ETV2) polypeptide.

[0016] Also disclosed herein is a method of generating an extracellular matrix, a) obtaining one or more first stem cell populations comprising an inducible transgene encoding a GATA binding protein 6 (GATA6) polypeptide; b) contacting the one or more first stem cell populations with an inducer that induces expression of the transgene encoding the GATA6 polypeptide; c) mixing the one or more first stem cell populations of step b) with a second stem cell population; d) culturing the cell mixture of step c) on a surface for at least 3 days to thereby produce cell aggregates; e) isolating the extracellular matrix from the cell aggregates of step d).

[0017] In some embodiments, the second stem cell population does not have an inducible transgene encoding a GATA6 polypeptide. In some embodiments, the second stem cell population comprises an inducible transgene encoding an ETS variant transcription factor 2 (ETV2) polypeptide. In some embodiments, step c) further comprises contacting the first stem cell population of step b) with a second stem cell population and a third cell population, the third cell population comprising an inducible transgene encoding an ETS variant transcription factor 2 (ETV2) polypeptide. In some embodiments, the method of any preceding aspect further comprises contacting one or more of the first stem cell population, the second stem cell population, and / or the third stem cell population with an agent (e.g., an agent that improves the signature of trophoblast cells differentiated from the embryonic compartment of the system). In some embodiments, the agent is SB431542.

[0018] Also disclosed herein is an in vitro culture system comprising an extracellular matrix produced by the method of any preceding aspect. BRIEF DESCRIPTION OF THE DRAWINGS

[0019]

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Modes for Carrying Out the Invention

[0020] Disclosed herein are in vitro cell culture systems for generating cell aggregates and their use, the systems comprising one or more first stem cell populations and a second stem cell population comprising an inducible transgene encoding a GATA binding protein 6 (GATA6) polypeptide. This method has been shown to be surprisingly effective in generating cell aggregates comprising one or more embryonic cell structures and one or more extra-embryonic cell structures from stem cells. In some embodiments, the generated cell aggregates comprise one or more of a bilayer disk-like structure, an amnion-like domain, a primitive streak-like domain, and a yolk sac domain.

[0021] The term As used herein, the terms "can", "optionally", and "optionally can" are used interchangeably and mean including not only the case where the state occurs, but also the case where the state does not occur. Thus, for example, the statement that a formulation "can contain an excipient" means including not only the case where the formulation contains an excipient, but also the case where the formulation does not contain an excipient.

[0022] As used in this specification and the claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells and mixtures thereof.

[0023] As used herein, the term "about", when referring to a measurable value such as an amount, percentage, etc., means including a variation of ±20%, ±10%, ±5%, or ±1% from the measurable value.

[0024] "Administration" or "administering" to a subject includes any route by which a drug is introduced or delivered to the subject. Administration can be carried out by any suitable route, including oral, intravenous, intraperitoneal, etc. Administration includes self-administration and administration by others.

[0025] As used herein, the term "comprising" and variations thereof are used synonymously with the term "including" and variations thereof, and are open-ended and non-limiting terms. The terms "comprising" and "including" have been used herein to describe various embodiments, but the terms "consisting essentially of" and "consisting of" can be used in place of "comprising" and "including" to provide more specific embodiments and are also disclosed.

[0026] Contact: Direct physical association, e.g., placement in solid, liquid, or gaseous form. Contact includes, e.g., direct physical association of fully and partially solvated molecules.

[0027] A "control" is an alternative object or sample used in an experiment for comparison purposes. A control may be "positive" or "negative". "Decrease" can refer to any change that results in a lesser amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product containing that substance is less compared to the output of the gene product not containing that substance. Also, for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than those previously observed. A decrease can be any individual, median, or average decrease in a statistically significant amount of a state, symptom, activity, composition. Thus, a decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease as long as the decrease is statistically significant.

[0028] The "effective amount" of a drug refers to an amount of the drug sufficient to provide the desired effect. The amount of a drug that is "effective" will vary from subject to subject depending on many factors such as the age and general condition of the subject, the particular drug(s), etc. Thus, it is not always possible to specify a quantified "effective amount". However, the appropriate "effective amount" for any given subject may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, unless otherwise specified, the "effective amount" of a drug can also refer to an amount that covers both therapeutically effective amounts and prophylactically effective amounts. The "effective amount" of a drug required to achieve a therapeutic effect can vary according to factors such as the age, sex, and weight of the subject. The dosing schedule can be adjusted to provide an optimal therapeutic response. For example, the dose may be administered in divided amounts several times a day, or the dose may be proportionally reduced as indicated by the urgency of the therapeutic situation.

[0029] "To encode" refers to the unique property of a specific sequence of nucleotides in a polynucleotide such as a gene, cDNA, or mRNA that serves as a template for the synthesis of other polymers and macromolecules in a biological process having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, in the case of transcription and translation of mRNA, the gene encodes a protein.

[0030] As used herein, the term "engineered" and its grammatical forms can refer to one or more changes in a nucleic acid such as a nucleic acid within the genome of an organism. The term "engineered" can refer to changes, additions, and / or deletions of a gene. An "engineered cell" can also refer to a cell that contains an added, deleted, and / or altered gene.

[0031] The term "expression vector" refers to a vector containing a recombinant polynucleotide that includes an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression, and other elements for expression can be supplied by the host cell or in an in vitro expression system. Examples of expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0032] A "fragment" may include insertions, deletions, substitutions, or other selected modifications of a specific region or specific amino acid residues, whether or not attached to other sequences, provided that the activity of the fragment is not significantly changed or impaired compared to the unmodified peptide or protein. These modifications can provide some additional properties, such as removing or adding amino acids capable of forming disulfide bonds, extending its biological lifespan, and changing its secretion characteristics. In any case, the fragment must have biological activity characteristics, such as regulating the transcription of the target gene.

[0033] The term "gene" or "gene sequence" refers to a coding sequence or a control sequence, or a fragment thereof. A gene can include any combination of a coding sequence and a control sequence, or fragments thereof. Thus, the "gene" referred to herein can be all or part of a natural gene. The polynucleotide sequences referred to herein can be used interchangeably with the term "gene" or can include any coding sequence, non-coding sequence, or control sequence, fragments thereof, and combinations thereof. The term "gene" or "gene sequence" includes, for example, a control sequence (e.g., ribosome binding site) upstream of the coding sequence.

[0034] The term "genetically engineered cell" as used herein refers to a cell modified by genetic engineering.

[0035] In the context of two or more nucleic acid or polypeptide sequences, the terms "identical" or percent "identity" are the same as, or the same as, when measured using the BLAST or BLAST 2.0 sequence comparison algorithms with the default parameters described below, or by manual alignment and visual inspection (i.e., when compared and aligned for maximum match in a comparison window or specified region, about 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity) Two or more sequences or subsequences having the specified percentage of amino acid residues or nucleotides are referred to (see, for example, the NCBI website). Such sequences are then said to be "substantially identical". This definition can also refer to or apply to the praise of the test sequence. The definition also includes sequences with deletions and / or additions, as well as sequences with substitutions. As described below, preferred algorithms can take into account gaps and the like. Preferably, identity is present over a region of at least about 10 amino acids or 20 nucleotides in length, or more preferably, a region of 10 to 50 amino acids or 20 to 50 nucleotides in length. As used herein, percent (%) nucleotide sequence identity is defined as the percentage of amino acids in a candidate sequence that are identical to the nucleotides of a reference sequence after aligning the sequences and introducing gaps as necessary to achieve maximum percent sequence identity. Alignments for the purpose of determining percent sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software.Appropriate parameters for measuring the alignment, including any algorithm necessary to achieve the maximum alignment over the entire length of the arrays being compared, can be determined by known methods.

[0036] For array comparison, typically one array acts as the reference array and the test array is compared to it. When using an array comparison algorithm, the test and reference arrays are input into a computer, sub-array coordinates are specified as necessary, and array algorithm program parameters are specified. Preferably, default program parameters can be used or alternative parameters can be specified. The array comparison algorithm then calculates the percent sequence identity of the test array to the reference array based on the program parameters.

[0037] One example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST and BLAST 2.0 algorithms, described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm involves identifying high-scoring sequence pairs (HSPs) by first identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with words of the same length in the database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. (1990) J. Mol. Biol. 215:403-410). These initial neighborhood word hits serve as seeds for initiating a search to find longer HSPs that contain them. Word hits are extended in both directions along each sequence as long as the cumulative alignment score can be increased. The cumulative score for nucleotide sequences is calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction stops when the cumulative alignment score decreases from its maximum achieved value by an amount X, when the cumulative score goes below zero due to the accumulation of one or more negative-scoring residue alignments, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses, by default, a word length (W) of 11, an expectation (E) of 10, M = 5, N = -4, and comparison of both strands.For amino acid sequences, the BLASTP program by default has a word length of 3, an expectation (E) of 10, and an alignment (B) of 50 with the BLOSUM 62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915), an expectation (E) of 10, M = 5, N = -4, and uses comparison of both strands.

[0038] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the minimum total probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences occurs by chance. For example, if the minimum total probability in the comparison of a test nucleic acid to a reference nucleic acid is less than about 0.2, more preferably less than about 0.01, the nucleic acid is considered to be similar to the reference sequence.

[0039] As used herein, the terms "increased" or "increase" generally mean a statistically significant amount of increase, and to avoid any doubt, "increased" means at least a 10% increase compared to a reference level as long as the increase is statistically significant, for example, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or 100% or less increase compared to the reference level, or any increase between 10 - 100% compared to the reference level, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold increase, or any increase between 2-fold and 10-fold or more.

[0040] As used herein, the term "isolating" refers to isolation from a biological sample, i.e., blood, plasma, tissue, exosomes, or cells. As used herein, the term "isolated", when used, for example, in the context of a cell, refers to a cell of interest that contains at least 60%, at least 75%, at least 90%, at least 95%, at least 98%, or even at least 99% less of other compounds, materials, matter, mass, and / or substance that the cell was associated with prior to purification.

[0041] As used herein, the term "modulating" means mediating a detectable increase or decrease in the level of expression or response in a cell or subject as compared to the level of expression or response in the cell or subject in the absence of treatment or a compound, and / or as compared to the level of expression or response in a cell or subject that is otherwise identical except for being untreated. This term encompasses perturbing and / or affecting a natural signal, natural expression level, or response, thereby mediating a beneficial therapeutic response in a subject, preferably a human.

[0042] As used herein, the term "neural feature" is used to refer to an increase in the expression of markers associated with the neural plate and surface ectoderm, such as cerberus 1 (CER1) and left-right determination factor 1 (LEFTY1).

[0043] As used herein, the term "nucleic acid" means a polymer composed of nucleotides, e.g., deoxyribonucleotides (DNA) or ribonucleotides (RNA). As used herein, the terms "ribonucleic acid" and "RNA" mean a polymer composed of ribonucleotides. As used herein, the terms "deoxyribonucleic acid" and "DNA" mean a polymer composed of deoxyribonucleotides.

[0044] The term "polynucleotide" refers to a single-stranded or double-stranded polymer consisting of nucleotide monomers.

[0045] The term "polypeptide" refers to a compound consisting of a single chain of D- or L-amino acids, or a mixture of D- and L-amino acids linked by peptide bonds.

[0046] The term "promoter" or "regulatory element" refers to a region or sequence determinant that is located upstream or downstream from the start of transcription and is involved in the recognition and binding of RNA polymerase and other proteins for initiating transcription. The promoter does not have to be of bacterial origin; for example, promoters derived from viruses or other organisms can be used in the compositions, systems, or methods described herein.

[0047] "Pharmaceutically acceptable carrier" (sometimes referred to as "carrier") means a carrier or excipient that is generally safe and non-toxic and is useful in the preparation of pharmaceutical or therapeutic compositions, including carriers that are acceptable for veterinary and / or human pharmaceutical or therapeutic use. The term "carrier" or "pharmaceutically acceptable carrier" can include, but is not limited to, phosphate buffered saline, water, emulsions (such as oil / water or water / oil emulsions), and / or various types of wetting agents.

[0048] As used herein, the term "carrier" encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations. The choice of carrier for use in a composition will depend on the intended route of administration of the composition. The preparation of pharmaceutical acceptable carriers and formulations containing these materials is described, for example, in Remington’s Pharmaceutical Sciences, 21st Edition, ed. University of the Sciences in Philadelphia, Lippincott, Williams & Wilkins, Philadelphia, PA, 2005. Examples of physiologically acceptable carriers include buffers such as saline, glycerol, DMSO, phosphate buffers, citrate buffers, and buffers containing other organic acids; antioxidants containing ascorbic acid; polypeptides of low molecular weight (less than about 10 residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN™ (ICI, Inc., Bridgewater, New Jersey), polyethylene glycol (PEG), and PLURONICS™ (BASF, Florham Park, NJ). To provide for administration of such doses for the desired therapeutic treatment, the compositions disclosed herein can advantageously contain from about 0.1 wt% to 99 wt% of the total weight of one or more of the subject compounds, based on the total weight of the composition containing the carrier or diluent.

[0049] The term "tissue" refers to a group or layer of similarly specialized cells that together perform a particular specialized function. The term "tissue" is intended to include blood products such as blood, plasma, and serum, bone, joints, muscle, smooth muscle, lung tissue, and organs.

[0050] The term "transgene" refers to a gene that is artificially introduced into another organism or cell. An "inducible transgene" is a transgene that is expressed in a desired amount only in the presence of an inducer. In one embodiment, the inducer is doxycycline.

[0051] "Transplantation" refers to a biocompatible lattice or donor tissue, organ or cell to be transplanted. Examples of transplantation can include, but are not limited to, skin cells or tissue, bone marrow, and solid organs such as the heart, pancreas, kidney, lung, and liver. Transplantation can also refer to any material administered to a host. For example, transplantation can refer to nucleic acids or proteins.

[0052] As used herein, "treat", "treating", "treatment", and their grammatical variations refer to partially or completely delaying, alleviating, reducing, or lessening the intensity of one or more attendant symptoms of a disorder or condition, and / or alleviating, reducing, or interfering with one or more causes of a disorder or condition. Treatment according to the present invention can be applied preventively, prophylactically, palliatively, or therapeutically. Preventive treatment is administered to a subject before onset, during early onset, or after the development of an established disease or disorder. Preventive administration can be carried out from several minutes to several months before the signs of a disease or disorder. In some instances, "treat", "treating", "treatment", and their grammatical variations include reducing a disease and / or related symptoms in a subject as compared to before treatment of the subject or as compared to the incidence of such symptoms in the general population or a study population.

[0053] Cell culture system Disclosed herein is an in vitro or ex vivo culture system for generating a cell mass comprising one or more embryonic cell structures and one or more extraembryonic cell structures or one or more hematopoietic stem cells, the system comprising One or more first stem cell populations comprising an inducible transgene encoding a GATA binding protein 6 (GATA6) polypeptide, and a second stem cell population.

[0054] The embryonic cell structure is composed of pluripotent stem cells and their derivatives, for example, germ cell-like cells (characterized by one or more of OCT4, NANOG, SOX2, and DPPA4), amnion-like domains (characterized by one or more of ISL1, BMP4, DLX5, ID1, BMPR1A, FSTL1, LEF1, and MSX1), and / or primitive streak-like domains (characterized by one or more of TBXT and MIXL1). The extraembryonic cell structure includes, for example, yolk sac endoderm (characterized by one or more of GATA6, GATA4, PDGFRA, SOX17, and FOXA2), yolk sac mesoderm (characterized by one or more of BST2, COL2A1, VIM, and DES), foregut endoderm (characterized by one or more of CER1, HHEX, LHX1, and GSC), yolk sac endothelium (characterized by one or more of CD34, PECAM1, CDH5, and ETV2), yolk sac hematopoietic endothelium (characterized by one or more of CD34, RUNX1, TAL1, ERG, and ETV2), and hematopoietic cells (characterized by one or more of CD34, CD43, CD45, RUNX1, CD71, CD33, CX3CR1, and CD41).

[0055] The first stem cell population comprising an inducible transgene encoding a GATA6 polypeptide can further comprise additional transcription factors expressed by stem cells other than GATA6, including, for example, GATA4, SOX17, SOX7, TBX5, PDX1, HNF4A, FOXA2, MYOD, LMO2, GATA3, GATA1, GATA2, KLF2, KLF1, SOX9, SOX4, and / or ERG.

[0056] One or more first stem cell populations containing an inducible transgene are understood to be generated by an inducible expression system and are contemplated herein. An "inducible expression system" means all the components necessary to carry out an inducible expression protocol, i.e., a protocol in which the expression of a coding sequence in a cell occurs in response to an applied stimulus, e.g., contact with an expression mediator compound (e.g., doxycycline), as compared to a constitutive expression protocol, i.e., a protocol in which the expression of a coding sequence (also referred to as a transcription unit) in a cell is continuous regardless of the presence or absence of a particular expression mediator component. The inducible expression system of the present invention includes a transcriptional modulator (e.g., rtTA) and a transcriptional modulator-responsive element (e.g., a tetracycline-responsive element or TRE), and the transcriptional modulator binds to the transcriptional modulator-responsive element (in some cases, in the presence of an expression mediator) and inducibly controls the expression of a coding sequence (i.e., a transcription unit) as needed.

[0057] In some embodiments, the one or more first stem cell populations contain a doxycycline-inducible transgene encoding a GATA6 polypeptide.

Chemical formula

[0058] In some embodiments, the stem cells disclosed herein include any inducible system known in the art, for example, GAL4 (promoter: UAS (CGG-N11-CCG, where N can be any base), synthetic zinc finger (promoter: synthetic promoter designed to match the zinc finger sequence), TMP (for activation of DD-Cre for recombination of "floxed" genes enabling expression from a constitutive promoter), tamoxifen (for activation of Cre recombinase for recombination of "floxed" genes enabling expression from a constitutive promoter), Cumate (promoter: Cumate operator [CuO]), 17β-estradiol or synthetic activator (SynX) (promoter: estrogen receptor alpha).

[0059] In some embodiments, each of the one or more first stem cell populations includes one or more inducible transgenes encoding one or more GATA6 polypeptides. In some embodiments, the GATA6 polypeptide is encoded by a sequence selected from the group of SEQ ID NOs: 3-10 or a sequence that is at least about 80% (at least about 80%, 85%, 90%, 95%, 98%, or 99%) identical to a fragment thereof. In some embodiments, the transgene includes a sequence that is at least about 80% (at least about 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 10 or a fragment thereof. In some embodiments, each of the one or more first stem cell populations includes one or more transcriptional modulator-responsive elements (e.g., tetracycline-responsive element or TRE). In some embodiments, the transcriptional modulator-responsive element includes one or more sequences that are at least about 80% (at least about 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 11 or a fragment thereof.

[0060] In some embodiments, one or more first stem cell populations, second stem cell populations, and / or third stem cell populations are contacted with an agent (e.g., an agent that improves the signature of trophoblast cells differentiated from the embryonic compartment of the system). In some embodiments, the agent is SB431542.

Chemical formula

[0061] In some embodiments, one or more first stem cell populations comprise a nucleic acid sequence that is at least about 80% (at least about 80%, about 85%, about 90%, about 95%, about 98%, or about 99%) identical to SEQ ID NO: 1 or a fragment thereof. In some embodiments, two or more first stem cell populations comprise a nucleic acid sequence that is at least about 80% (at least about 80%, about 85%, about 90%, about 95%, about 98%, or about 99%) identical to SEQ ID NO: 1 or a fragment thereof. In some embodiments, the second stem cell population comprises a nucleic acid sequence that is at least about 80% (at least about 80%, about 85%, about 90%, about 95%, about 98%, or about 99%) identical to SEQ ID NO: 1 or a fragment thereof.

[0062] In some embodiments, the second stem cell population does not have an inducible transgene encoding a GATA6 polypeptide.

[0063] In some embodiments, the second stem cell population further comprises an inducible transgene encoding an ETS variant transcription factor 2 (ETV2) polypeptide. Thus, in some aspects, disclosed herein is an in vitro or ex vivo culture system for generating a cell mass comprising one or more embryonic cell structures and one or more extraembryonic cell structures or one or more hematopoietic stem cells, the system comprising one or more first stem cell populations comprising an inducible transgene encoding a GATA binding protein 6 (GATA6) polypeptide, and a second stem cell population, the second stem cell population further comprising an inducible transgene encoding an ETS variant transcription factor 2 (ETV2) polypeptide.

[0064] In some embodiments, the in vitro or ex vivo culture system disclosed herein further comprises a third cell population, the third cell population comprising an inducible transgene encoding an ETS variant transcription factor 2 (ETV2) polypeptide. Thus, in some aspects, what is disclosed herein is an in vitro or ex vivo culture system for generating a cell mass comprising one or more embryonic cell structures and one or more extraembryonic cell structures or one or more hematopoietic stem cells, the system comprising one or more first stem cell populations comprising an inducible transgene encoding a GATA binding protein 6 (GATA6) polypeptide, a second stem cell population, and a third cell population, the third cell population comprising an inducible transgene encoding an ETS variant transcription factor 2 (ETV2) polypeptide.

[0065] Organoids generated from iGATA6 or iETV2 engineered strains derived from single cell clones can exhibit enhanced characteristics different from those observed in heterogeneous strains as a result of different gene circuit copy numbers. As a result, they can be used in a plug-and-play approach to generate defined target tissues with specific functional capabilities in a different manner than organoids derived from heterogeneous iGATA6 mixtures. Different clone strains can have different gene circuit copy numbers and different expression ranges (or expression noise), which can be utilized to engineer designer tissues with extended behaviors such as improving blood production, improving a subset of mesoderm, or improving a subset of blood vessels. Thus, in some embodiments, one or more first stem cell populations comprise different numbers of inducible transgenes encoding a GATA6 polypeptide. In some embodiments, one or more first stem cell populations comprise the same number of inducible transgenes encoding a GATA6 polypeptide. It is understood and contemplated herein that one or more first stem cell populations express different levels of the GATA6 polypeptide. Different expression levels of the GATA6 polypeptide can be achieved by delivering the transgene encoding the GATA6 polypeptide one or more times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times) to different first stem cell populations. The transgene can be delivered by any expression vector known in the art (e.g., a DNA vector, a viral vector, or a nanoparticle). In some embodiments, different expression levels of the GATA6 polypeptide can be achieved by delivering a transgene comprising one or more copies (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more copies) of the GATA6 polynucleotide to different first stem cell populations. In some embodiments, different expression levels of the GATA6 polypeptide can be achieved by delivering one or more RNA sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more RNA sequences) encoding the GATA6 polypeptide.In some embodiments, different expression levels of the GATA6 polypeptide can be achieved by delivering one or more GATA6 polypeptides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GATA6 polypeptides).

[0066] In some embodiments, one or more first cell populations and second cell populations are pluripotent stem cells. As used herein, the term "pluripotent stem cell" refers to a cell that is capable of continuous self-renewal and, under appropriate conditions, can differentiate into all the cells of the three germ layers. Examples of pluripotent stem cells (PSCs) include embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). As used herein, the term "embryonic stem cell" or "ESC" means a pluripotent cell or population of pluripotent cells derived from the inner cell mass of a blastocyst. See, for example, Thomson et al, Science 282:1145-1147 (1998). These cells express Oct-4, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. As used herein, the term "induced pluripotent stem cell" or "iPSC" refers to a pluripotent cell or population of pluripotent cells that can vary with respect to the origin of their differentiated somatic cells, can vary with respect to a particular set of potency determinants, and can vary with respect to the culture conditions used to isolate them, but nevertheless are substantially genetically identical to the origin of their respective differentiated somatic cells and exhibit characteristics similar to those of higher potency cells, such as ESCs, as described herein. See, for example, Yu et al., Science 318:1917-1920 (2007). In some embodiments, one or more first cell populations and second cell populations are induced pluripotent stem cells (iPSCs). In some embodiments, one or more first cell populations and second cell populations are human cells.

[0067] In some embodiments, the first stem cell population and the second stem cell population are mixed at a ratio of about 1:20 to about 1:2, preferably about 1:10 to about 1:3, or preferably about 1:10 to about 1:4.

[0068] An introduced gene, polynucleotide, or polypeptide (e.g., GATA6 and ETV2) can be delivered to a stem cell or a subject in need thereof via any known delivery route, including, for example, transfection-based delivery of mRNA of GATA6 or ETV2, or protein delivery of GATA6 or ETV2. A DNA plasmid vector can also be delivered to engineered cells having a stable integrated gene circuit introduced via a piggybac transposase. In some embodiments, the cells can be generated in vitro and then transplanted in vivo. After transplantation, the cells can be induced in vivo.

[0069] Method Also disclosed herein is a method of generating, ex vivo or in vitro, a cell mass comprising one or more embryonic cell structures and one or more extraembryonic cell structures, a) obtaining one or more first stem cell populations comprising an inducible introduced gene encoding a GATA-binding protein 6 (GATA6) polypeptide; b) contacting the one or more first stem cell populations with an inducer that induces expression of the introduced gene; c) mixing the one or more first stem cell populations of step b) with a second stem cell population; d) culturing the mixture of cells of step c) on a surface for at least 7 days, thereby producing a cell mass.

[0070] As used herein, the term "cell mass" refers to a mass of cells having distinct boundaries. The cell mass can be a two-dimensional or three-dimensional structure. In some embodiments, the cell mass comprises one or more embryonic cell structures and one or more extraembryonic cell structures. In some embodiments, the cell mass comprises one or more hematopoietic stem cells.

[0071] In some embodiments, step b) comprises contacting the one or more first stem cell populations with the agent for at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 20, 25, or 30 days, or about 2 - 7 days, about 4 - 9 days, about 6 - 12 days, about 8 - 14 days, about 10 - 16 days, about 12 - 18 days, about 14 - 20 days, about 16 - 22 days, about 18 - 24 days, about 20 - 26 days, about 16 - 30 days, about 2 - 4 days, about 3 - 5 days, about 4 - 6 days, about 5 - 7 days, about 6 - 9 days, or about 7 - 10 days.

[0072] In some embodiments, each of one or more first stem cell populations comprises one or more inducible transgenes encoding one or more GATA6 polypeptides. In some embodiments, the GATA6 polypeptide is encoded by a sequence selected from the group consisting of SEQ ID NOs: 3-10 or a fragment thereof and one or more sequences that are at least about 80% (at least about 80%, 85%, 90%, 95%, 98%, or 99%) identical thereto. In some embodiments, the GATA6 transgene comprises a sequence that is at least about 80% (at least about 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 10. In some embodiments, each of one or more first stem cell populations comprises one or more transcriptional modulator-responsive elements (e.g., tetracycline-responsive element or TRE). In some embodiments, the transcriptional modulator-responsive element comprises one or more sequences that are at least about 80% (at least about 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 11 or a fragment thereof. In some embodiments, one or more first stem cell populations each comprise a different number of inducible transgenes encoding the GATA6 polypeptide. In some embodiments, one or more first stem cell populations each comprise the same number of inducible transgenes encoding the GATA6 polypeptide. It is understood and contemplated herein that one or more first stem cell populations express different levels of the GATA6 polypeptide. In some embodiments, one or more first stem cell populations comprise a doxycycline-inducible transgene encoding the GATA6 polypeptide. Different expression levels of the GATA6 polypeptide can be achieved by delivering the transgene encoding the GATA6 polypeptide one or more times to different first stem cell populations. The transgene can be delivered by any expression vector known in the art (e.g., a DNA vector, a viral vector, or a nanoparticle). In some embodiments, different expression levels of the GATA6 polypeptide can be achieved by delivering one or more RNA sequences encoding the GATA6 polypeptide. In some embodiments, different expression levels of the GATA6 polypeptide can be achieved by delivering one or more GATA6 polypeptides.

[0073] A first stem cell population comprising an inducible transgene encoding a GATA6 polypeptide can further comprise additional transcription factors expressed by stem cells other than GATA6, including, for example, GATA4, SOX17, SOX7, TBX5, PDX1, HNF4A, FOXA2, MYOD, LMO2, GATA3, GATA1, GATA2, KLF2, KLF1, SOX9, SOX4, and / or ERG.

[0074] In some embodiments, one or more first stem cell populations comprise a doxycycline-inducible transgene encoding a GATA6 polypeptide. The concentration of doxycycline in step b can be from about 1 ng / ml to about 10,000 ng / ml, from 1 ng / ml to about 1000 ng / ml, from about 10 ng / ml to about 750 ng / ml, from about 100 ng / ml to about 500 ng / ml, from about 100 ng / ml to about 250 ng / ml, or from about 10 ng / ml to about 150 ng / ml.

Chemical formula

[0075] In some embodiments, the stem cells disclosed herein comprise any inducible system known in the art, including, for example, GAL4 (promoter: UAS (CGG-N11-CCG, where N can be any base)), synthetic zinc finger (promoter: synthetic promoter designed to match the zinc finger sequence), TMP (for activation of DD-Cre for recombination of "floxed" genes to enable expression from a constitutive promoter), tamoxifen (for activation of Cre recombinase for recombination of "floxed" genes to enable expression from a constitutive promoter), Cumate (promoter: Cumate operator [CuO]), 17β-estradiol or synthetic activator (SynX) (promoter: estrogen receptor alpha).

[0076] In some embodiments, one or more first stem cell populations comprise a nucleic acid sequence that is at least about 80% (at least about 80%, about 85%, about 90%, about 95%, about 98%, or about 99%) identical to SEQ ID NO: 1 or a fragment thereof. In some embodiments, one or more first stem cell populations comprise a nucleic acid sequence that is at least about 80% (at least about 80%, about 85%, about 90%, about 95%, about 98%, or about 99%) identical to SEQ ID NO: 2 or a fragment thereof. In some embodiments, the second stem cell population comprises a nucleic acid sequence that is at least about 80% (at least about 80%, about 85%, about 90%, about 95%, about 98%, or about 99%) identical to SEQ ID NO: 1 or a fragment thereof.

[0077] In some embodiments, the second stem cell population does not have an inducible transgene encoding a GATA6 polypeptide. Thus, in those embodiments, the expression level of the GATA6 polypeptide in the second stem cell population is lower than the expression level of the GATA6 polypeptide in the first stem cell population.

[0078] In some embodiments, step c) comprises mixing one or more first stem cell populations and the second stem cell population at a ratio of about 1:20 to about 1:2, preferably about 1:10 to about 1:3, or preferably about 1:10 to about 1:4.

[0079] In some embodiments, a mixture of the first stem cell population and the second stem cell population is placed on a surface (e.g., a flat surface or a non-flat surface). In some embodiments, the stem cells form a single cell layer on the surface. The cells are then cultured on the surface for at least 7 days (e.g., at least 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 18 days, 20 days, 25 days, or 30 days), or about 4 - 10 days, about 6 - 12 days, about 8 - 14 days, about 10 - 16 days, about 12 - 18 days, about 14 - 20 days, about 16 - 22 days, 18 - 24 days, about 20 - 28 days, 24 - 30 days, or 25 - 35 days, thereby producing a cell mass comprising one or more embryonic cell structures and one or more extraembryonic cell structures or one or more hematopoietic stem cells. The process of generating the cell mass is self-organizing and self-timing. The cell mass can be generated from an initially randomly mixed state, and the cells of step c) reproducibly form one or more embryonic cell structures or extraembryonic cell structures described herein, with or without additional reagents, patterning cues, growth factors, etc. added to the culture system. One or more embryonic cell structures or extraembryonic cell structures can be formed in a consistent and reproducible schedule after circuit induction without adding additional reagents, patterning cues, growth factors, etc. to the culture system. In some embodiments, step d) of the method of any preceding aspect does not require changing the cell culture medium or adding reagents during the process of producing the cell mass. In some embodiments, step d) of the method of any preceding aspect further includes changing the cell culture medium or adding reagents during the process of producing the cell mass. In some embodiments, the generated cell mass includes one or more of a bilayer disk-like structure, an amnion-like domain, a primitive streak-like domain, and a yolk sac domain. In some embodiments, the embryonic cell structure includes an increased level of the octamer-binding transcription factor 4 (OCT4) polypeptide and a decreased level of the GATA4 polypeptide compared to a reference control. In some embodiments, the extraembryonic cell structure includes an increased level of the GATA6 polypeptide compared to a reference control.

[0080] "OCT4" as used herein refers to the polypeptide encoded by the POU5F1 gene in humans. In some embodiments, the OCT4 polypeptide is identified in one or more publicly available databases as follows: HGNC:9221, NCBI Entrez Gene:5460, Ensembl:ENSG00000204531, OMIM®:164177, UniProtKB / Swiss-Prot:Q01860. In some embodiments, the OCT4 polypeptide comprises the sequence of SEQ ID NO: 12, or a polypeptide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98% homology to SEQ ID NO: 12, or a polypeptide comprising a portion of SEQ ID NO: 12. The OCT4 polypeptide of SEQ ID NO: 12 may represent an immature or pre-processed form of mature OCT4, and thus, the mature or processed portion of the OCT4 polypeptide of SEQ ID NO: 12 is included herein.

[0081] "GATA4" as used herein refers to the polypeptide encoded by the GATA4 gene in humans. In some embodiments, the GATA4 polypeptide is identified in one or more publicly available databases as follows: HGNC:4173, NCBI Entrez Gene:2626, Ensembl:ENSG00000136574, OMIM®:600576, UniProtKB / Swiss-Prot:P43694. In some embodiments, the GATA4 polypeptide comprises the sequence of SEQ ID NO: 13, or a polypeptide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98% homology to SEQ ID NO: 13, or a polypeptide comprising a portion of SEQ ID NO: 13. The GATA4 polypeptide of SEQ ID NO: 13 may represent an immature or pre-processed form of mature GATA4, and thus, the mature or processed portion of the GATA4 polypeptide of SEQ ID NO: 13 is included herein.

[0082] In some embodiments, the cell mass of any preceding aspect comprises one or more of a bilayer disk-like structure, an amnion-like domain, a primitive streak-like domain, and a yolk sac domain. In some embodiments, the amnion-like domain comprises increased levels of one or more of bone morphogenetic protein 4 (BMP4), bone morphogenetic protein receptor 1A (BMPR1A), distal-less homeobox 5 (DLX5), follistatin-like 1 (FSTL1), DNA binding inhibitor 1 (ID1), lymphocyte enhancer binding factor 1 (LEF1), msh homeobox (MSX)1, MSX2, SMAD family member 1 (SMAD1), SMAD specific e3 ubiquitin protein ligase 2 (SMURF2), and ISL LIM homeobox 1 (ISL1).

[0083] In some embodiments, the cell mass comprises increased levels of one or more neural features (e.g., including one or more of the cerberus 1 (CER1) polypeptide and the left-right determination factor 1 (LEFTY1) polypeptide).

[0084] In some embodiments, the method of any preceding aspect further comprises contacting one or more first stem cell populations, second stem cell populations, and / or third stem cell populations with an agent (e.g., an agent that improves the signature of trophoblast cells differentiated from the systemic germ compartment). In some embodiments, the agent is SB431542.

[0085] The cell mass generated by the methods disclosed herein can be used to evaluate, test, and / or screen for agents (e.g., therapeutic agents) that regulate embryonic development or hematopoietic differentiation, comprising generating a cell mass comprising one or more embryonic cell structures and one or more extraembryonic cell structures by the method of any preceding aspect and contacting the cell mass with an agent. Thus, in some aspects, disclosed herein is a method for evaluating an agent that regulates embryonic development or hematopoietic differentiation, the method comprising generating, by the method of any preceding aspect, a cell mass comprising one or more embryonic cell structures and one or more extraembryonic cell structures, Contacting the cell mass with an agent; determining the effect of the agent on the cell mass as compared to a control.

[0086] In some embodiments, the method further comprises determining a biomarker associated with embryonic development or hematopoietic differentiation. Biomarkers associated with embryonic development or hematopoietic differentiation are well known in the art.

[0087] Also provided herein is a method for evaluating an agent that modulates embryonic development or hematopoietic differentiation, the method comprising: a) obtaining one or more first stem cell populations comprising an inducible transgene encoding a GATA binding protein 6 (GATA6) polypeptide; b) contacting the one or more first stem cell populations with an inducer that induces expression of the transgene; c) mixing the one or more first stem cell populations of step b) with a second stem cell population; d) culturing the cell mixture of step c) on a surface with an agent that modulates embryonic development for at least 7 days to thereby produce a cell mass and analyzing the cell mass as compared to a control.

[0088] In some embodiments, the method further comprises determining a biomarker associated with embryonic development or hematopoietic differentiation. Biomarkers associated with embryonic development or hematopoietic differentiation are well known in the art. In some embodiments, the agent is a therapeutic agent. In some embodiments, the agent is a toxin.

[0089] Also provided herein is a cell mass produced by the method of any preceding embodiment, the cell mass comprising one or more embryonic cell structures and one or more extraembryonic cell structures.

[0090] Also provided herein is a method for generating human primordial germ cell-like cells, a) obtaining one or more first stem cell populations comprising an inducible transgene encoding a GATA binding protein 6 (GATA6) polypeptide; b) contacting one or more first stem cell populations with an inducer that induces expression of the transgene; c) mixing the one or more first stem cell populations of step b) with a second stem cell population; d) culturing the cell mixture of step c) on a surface for at least 3 days, thereby producing cell aggregates; e) isolating human primordial germ cell-like cells from the cell aggregates.

[0091] Human primordial germ cell-like cells can be isolated from the cell aggregates on the 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, or 10th day after culturing the mixture of stem cells on a surface. In some embodiments, the generated human primordial germ cell-like cells have increased expression of one or more of CD38, BLIMP1, and AP2γ compared to a control.

[0092] Also disclosed herein are human primordial germ cell-like cells produced by the method of any preceding aspect, wherein the human primordial germ cell-like cells have increased expression of one or more of CD38, BLIMP1, and AP2γ compared to a control.

[0093] Also disclosed herein is a method of treating infertility in a subject in need thereof, generating a cell aggregate comprising one or more embryonic cell structures and one or more extraembryonic cell structures by the method of any preceding aspect, and transplanting one or more embryonic cell structures and / or one or more extraembryonic cell structures of the cell aggregate into the subject.

[0094] In some embodiments, the stem cells used to generate the cell aggregate are derived from the subject. In some embodiments, the stem cells used to generate the cell aggregate are engineered stem cells.

[0095] In some embodiments, the generated yolk sac has hematopoietic properties including erythropoiesis and myelopoiesis. Thus, in some aspects, disclosed herein is a method of generating hematopoietic stem cells, generating a cell mass comprising one or more embryonic cell structures and one or more extraembryonic cell structures by the method of any preceding aspect; isolating hematopoietic stem cells from the cell mass.

[0096] The hematopoietic stem cells can be isolated from the cell mass on day 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, or at least 7 days (e.g., at least 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 18 days, 20 days, 25 days, or 30 days) or about 4-10 days, about 6-12 days, about 8-14 days, about 10-16 days, about 12-18 days, about 14-20 days, about 16-22 days, 18-24 days, about 20-28 days, 24-30 days, or 25-35 days after culturing a mixture of stem cells on the surface. In some embodiments, the generated hematopoietic stem cells have increased expression of one or more of CD3, CD19, CD33, CD56, CD45, CD11b, and CD34 compared to a control.

[0097] Also disclosed herein is a method of treating a hematopoietic disorder in a subject in need thereof, generating a cell mass comprising one or more embryonic cell structures and one or more extraembryonic cell structures by the method of any preceding aspect; isolating hematopoietic stem cells from the cell mass; transplanting the hematopoietic stem cells into the subject.

[0098] In some embodiments, the stem cells used to generate the hematopoietic stem cells are derived from the subject. In some embodiments, the stem cells used to generate the hematopoietic stem cells are engineered stem cells.

[0099] Examples of hematopoietic disorders include, but are not limited to, megaloblastic (pernicious) anemia, sickle cell disease, thalassemia, leukemia, and myelodysplastic syndromes.

[0100] Also disclosed herein is a method of generating an extracellular matrix, a) obtaining one or more first stem cell populations comprising an inducible transgene encoding a GATA binding protein 6 (GATA6) polypeptide; b) contacting the one or more first stem cell populations with an inducer that induces expression of the transgene; c) mixing the one or more first stem cell populations of step b) with a second stem cell population; d) culturing the cell mixture of step c) on a surface, thereby producing cell aggregates; e) isolating the extracellular matrix from the cell aggregates of step d).

[0101] In some embodiments, the extracellular matrix can be isolated from the cell aggregates on the 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, or 30th day after culturing the mixture of stem cells on the surface. In some embodiments, the generated extracellular matrix is substantially composed of one or more of LAMA1, LAMA2, COL4A1, and COL2A1 as compared to a control.

[0102] The extracellular matrix can be the physical scaffold and / or protein architecture created by cells to establish and maintain the physical form of cell aggregates and their structures. The extracellular matrix can be isolated by killing and washing away the cells of the cell aggregate while leaving the scaffold intact. The decellularized extracellular matrix can be made receptor-compatible for transplantation. The extracellular matrix can then be used to culture completely different cell types that can benefit from having an existing structure patterned by the original iPSCs (such as cavities, vascular regions, etc.). These cells can be any cell type of interest, colonize this extracellular matrix, and be cultured under different media conditions.

[0103] Accordingly, in some aspects, disclosed herein is an in vitro or ex vivo culture system comprising an extracellular matrix generated by the method of any preceding aspect.

[0104] It is understood and contemplated herein that a transgene, polynucleotide, or polypeptide (e.g., GATA6 and ETV2) can be delivered to a stem cell or a subject in need thereof via any known delivery pathway, including, for example, transfection-based delivery of the mRNA of GATA6 or ETV2, or protein delivery of GATA6 or ETV2. A DNA plasmid vector can also be delivered to engineered cells having a stable integrated gene circuit introduced via a piggybac transposase. In some examples, cells can be generated in vitro and then transplanted in vivo. After transplantation, the cells can be induced in vivo.

Examples

[0105] The following examples are described below to illustrate compositions, methods, and results in accordance with the subject matter of the present disclosure. These examples are not intended to include all aspects of the subject matter disclosed herein, but rather are intended to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention, which will be apparent to those skilled in the art.

[0106] Example 1. Method. Ethics regarding the development of iDiscoid. This study was approved and conducted under the supervision of the University of Pittsburgh Human Stem Cell Research Oversight Committee (hSCRO #20-004) and complies with the 2021 ISSCR guidelines regarding the ethical standards of stem cell embryo models. For this purpose, iDiscoid is generated from human iPSCs derived from human somatic cells such as fibroblasts and does not contain human embryonic stem cells. iDiscoid is attached to cell culture dishes and lacks the extraembryonic trophoblast tissue essential for the development of a fully integrated embryo and implantation into the uterine cavity. The yolk sac cavities of iDiscoid are not closed, and no tissue can be harvested for any implantation without substantially destroying their structure. TBXT + The posterior domain was observed during the study but not maintained during the development of our system. These features comprehensively limit the ability of this model to undergo complete integrated development of human embryos in vitro and / or upon implantation to support further in vivo development of the concept.

[0107] Cell culture. All cells and tissues were cultured in a humidified incubator at 37 °C and 5% CO2. PGP1 and PGP9 were used in this study. The hiPSC line was cultured under sterile conditions in daily-changing mTeSR-1 (Stem Cell Technologies, Vancouver). Tissue culture plates were coated with BD ES-qualified Matrigel (BD Biosciences) diluted in ice-cold DMEM / F-12 containing 15 mM HEPES medium (Thermo Scientific) according to the manufacturer's instructions for 1 hour at room temperature. Routine passage was performed by incubating hiPSC colonies in Accutase (Sigma) at 37 °C for 5 minutes, collecting the suspension, adding 5 mL of DMEM / F-12 medium containing 10 mM Y-27632, centrifuging at 300 g for 5 minutes, and resuspending in DMEM / F-12 supplemented with 10 mM Y-27632 for counting. Cells were seeded at a cell density of 25,000 cells per 1 cm 2 2.

[0108] Generation of cell lines engineered with GATA6. rtTA (Guye, P. et al. 2016) that is expressed in previously generated hiPSCs was transfected using Lipofectamine 3000 (Thermo Fisher Scientific) together with Super PiggyBac transposase (System Biosciences) and a PiggyBac transposon vector together with hGATA6-2A-EGFP under the control of a tetracycline-responsive element promoter. Transfected cells were selected by adding 0.5 mg / mL puromycin to the mTeSR1 maintenance medium.

[0109] Generation of iDiscoids. hiPSCs engineered with GATA6 were seeded at a ratio of 4:1 with rtTA-expressing hiPSCs containing or lacking the mKate reporter gene at a total density of 25,000 cells per cm 2 in mTeSR-1 supplemented with 10 mM Y-27632. The next day, the medium was changed to mTeSR-1 with 1 mg / mL doxycycline to induce the expression of the GATA6 transgene and was exchanged daily for up to 5 days.

[0110] Cryopreservation of iDiscoids. Non-induced iDiscoid cells were incubated with Dispase for 10 minutes at 70% confluence or until the edges of the colonies were visible and lifted. After washing the cells twice with DMEM / F-12, the colonies were manually scraped from the plate. The colonies were centrifuged at 300 g for 5 minutes and then resuspended in Cryostor 10. The cells were cooled at -80 °C for 24 hours before transferring to liquid nitrogen for long-term storage. The cells were stored in liquid nitrogen for at least 24 hours before thawing.

[0111] Signal transduction pathway inhibition. On the third day of iDiscoid culture, BMP4 signal transduction was inhibited by applying 3 μM dorsomorphin to the normal medium. Starting from the second day of iDiscoid culture, NODAL signal transduction was inhibited via the application of 10 μM SB431542. Starting from the zeroth day of iDiscoid culture, CXCR4 signal transduction was inhibited via the application of 1 μM AMD3465.

[0112] Staining on glass coverslips. Cells were grown on Matrigel-coated circular glass coverslips with a diameter of 8 mm or Mattek 35 mm coverslip-bottom dishes. The cultures were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) for 10 minutes at room temperature. For the toluidine blue staining in Figure 12B, the cells were fixed in 2.5% glutaraldehyde in 0.01 M PBS (pH 7.4) for 1 hour at room temperature. The coverslips were then washed three times with PBS, followed by permeabilization with 0.2% Triton X-100 in PBS for 15 minutes. Subsequently, the coverslips were washed three times for 5 minutes each in wash buffer (0.05% Tween-20 in PBS) and blocked in 200 μl wash buffer and 5% normal donkey serum (Jackson ImmunoResearch Laboratories) for 20 minutes. The primary antibody was diluted in 5% normal donkey serum in PBS, incubated with the tissue for 1 hour at room temperature, and then washed three times for 5 minutes each in wash buffer. The secondary antibody was diluted in 5% normal donkey serum in PBS, incubated with the tissue for 1 hour at room temperature, and then washed three times for 5 minutes each in wash buffer. Thereafter, the 8 mm coverslips were mounted on microscope glass slides using ProLong Glass Antifade (Life Technologies), cured overnight at room temperature, and then sealed with nail polish. The coverslips in Mattek dishes were stored at 4 °C in PBS for 3D imaging before mounting.

[0113] Image acquisition and processing. Images were acquired using an EVOS M700 automated scanning microscope, a Leica SP8 confocal microscope, or a Nikon A1 confocal microscope and processed using ImageJ software (NIH). Any contrast adjustments were made on an individual channel and applied evenly across the entire image of that channel. Contrast and color balance for color images were applied evenly across the entire image. 3D reconstructions were generated using a Nikon A1 confocal microscope, generating a z-stack extending approximately 100 um deep into the tissue and constructing a 3D volume from the stack using ImageJ or Imaris (Bitplane).

[0114] Time-lapse imaging. iGATA6 cells were seeded at 25,000 cells / cm on wndicatiigel-coated 2 Cellstar 24-well tissue culture-treated plates and then induced with 1,000 ng / ml -1 Dox. After induction, the cells were placed under an Incucyte S3 live cell imaging platform at 37 °C and 5% CO2. Images were taken every 30 minutes for 3 days.

[0115] Analysis of wild-type cluster regions and radial expression. Images of the entire tiled coverslip were trimmed to the central 9000 μm 2 image for analysis. Wild-type compartment analysis was performed using an in-house Matlab pipeline. Briefly, wild-type compartments were programmatically detected by thresholding the nuclear dye channel for regions of high cell density. The maximum individual compartment area and average cell size were defined manually. Compartments were filtered to remove those with regions close to the defined cell area via the following equation.

Number

[0116] For compartment counting and wild-type region analysis, the properties of each compartment were recorded using the Matlab regionprops function. Additionally, the distance to the nearest wild-type compartment was recorded.

[0117] For the radial analysis of marker expression outside each wild-type compartment, an additional filtering step was applied. Compartments were excluded from the analysis if they were close to neighboring compartments or the image boundary, where the maximum radial analysis distance might overlap with another compartment or the image boundary. Radial analysis was performed on the remaining compartments by drawing a perpendicular outward line from each pixel around the compartment to the defined maximum analysis distance, and recording the intensity value of each marker at each point within this range. Values were skipped if the line intersected the compartment (e.g., if drawn from the inner edge of a concave shape). The intensity values at each distance were averaged to create a final expression curve per compartment.

[0118] The analysis of marker expression inward from the compartment perimeter was performed in a similar manner, except that the lines were drawn towards the centroid of each compartment rather than perpendicular to the compartment edge. The coverage of the WT clusters was evaluated via wide-field images of the entire coverslip, and regions where EGFP expression was observed within WT clusters exceeding 10% of the cluster radius were counted as being covered. The presence or absence of lumens / vacuoles was manually evaluated and detected via the presence of visible rings of PODXL expression within wild-type compartments.

[0119] Analysis of wild-type cluster characteristics. iGATA6 coverage was defined using the radial average of marker expression intensity from the GFP image channel. Following radial analysis, compartments with complete coverage, partial coverage, and no coverage were manually identified, and the other compartments were classified on a set-by-image basis using the average radial intensity curves of those compartments. Both complete coverage and partial coverage were considered as positive coverage in Figure 3G.

[0120] Wild-type clusters were classified into "early", "intermediate", or "late" categories based on the state and size of the lumen within each cluster. The "early" clusters were characterized by no or very minimal inward polarization, as established by contact with GATA6-expressing cells, and were characterized by PODXL. The "intermediate" clusters were characterized by the expansion of small lumens and the presence of multiple lumens within the cluster. The "late" clusters were characterized by a single continuous lumen centered within the cluster.

[0121] Enzyme-linked immunosorbent assay (ELISA). Samples were assayed for AFP, APOA1, and BMP4 using a commercially available ELISA kit (abcam). Sample dilutions were optimized to achieve detection within the linear range of the standard curve for each individual assay.

[0122] Preparation of 10× Genomics samples for next-generation array determination. Samples were prepared as described in the 10× Genomics Single Cell 30 v2 Reagent Kit User Guide. Day 5 iDiscoids, initially mixed at a 10:1 iGATA6:WT-iPSC ratio, were obtained in single-cell suspension by incubating with trypsin at 37 °C for 10 minutes, followed by gentle pipetting using a serological pipette to remove and dissociate aggregates. Two washes were performed in PBS- / - + 0.04% BSA on cells resuspended at a final concentration of 1000 cells / mL in PBS- / - + 0.04% BSA. Trypan blue was used to perform a live cell count using a hemocytometer to identify dead cells. Following the count, cells and 10× Genomics reagents were targeted to 6000 single cells for analysis, taking into account predicted cell loss and doublets, and loaded into a single-cell cassette as described in the user guide for the Chromium Single-Cell 3’ Reagent Kit (10× Genomics). After GEM generation, cDNA libraries were prepared by ASU Genomics Core staff according to the appropriate steps determined by the 10× Genomics user guide. Libraries were sent to Novogene for sequencing on an Illumina HiSeq X with 150 bp paired-end reads at an intended read depth of 100,000 reads per cell. Our downstream analysis from the sequencing data assumes that the actual number of sequenced cells is between 5000 and 7000, resulting in >80k reads per cell. The 10× Genomics CellRanger pipeline was used to align reads to a reference genome (GRCh38.84) augmented with the transgene sequence, assign reads to individual cells, and estimate gene expression based on UMI counts (Zheng, G.X. et al. 2017).

[0123] Single-cell data were excluded based on either a high mitochondrial genomic transcript ratio and either high or low features or UMI counts. Genes with UMI counts in fewer than 5 cells were excluded from consideration. For scRNA-Seq data processing and clustering analysis using Seurat (Butler, A. et al., 2018, Satija, R. et al., 215), this study used the following general normalization pipeline for processing Cell Ranger output: normalization, feature selection, scaling (including cell cycle regression), principal component analysis (PCA), and clustering. Elbow plots and permutation p-values were used to help determine the optimal number of principal components (PCs) needed to summarize the dataset without losing a significant amount of variation. The quality of the clustering resolution value range was evaluated using the enrichment of cluster marker genes (genes differentially upregulated within a given cluster relative to all other clusters) using germ cell type-specific genes. As a quality check, the PC and resolution metrics were adjusted to yield fewer or additional clusters to confirm that the selected parameters resulted in the most biologically relevant clustering. Visualization was achieved by using tSNE plots to identify cells, clusters, and the expression of selected genes in each cell, as well as heatmaps and violin plots showing the expression levels of genes by cluster.

[0124] Quantification of the statistical similarity between lists of marker genes. The similarity between two lists of marker genes, A and B, is quantified by using a hypergeometric test for overrepresentation. This corresponds to a one-sided Fisher's exact probability test.

[0125] Integration with publicly available datasets and label projection. To directly compare cell type annotations between human cell line data and publicly available non-human data, we follow a data integration protocol similar to (Minn, K. T. et al. 219). First, we select a subset of human cell line genes that have orthologs in mouse and NHP data. Using these orthologous gene mappings, we use a subset of genes from both datasets that are included in the orthologous gene set. Specifically, if our human cell line data is included in the dimensions (n cells, h genes) of the count matrix H and the non-human data is included in the dimensions (k cells, m genes) of the count matrix M, we subset both datasets into their respective matrices H’(n,g) and M’(k,g) such that both contain the same number of genes. Further, these g genes are orthologous between H’ and M’ and are ordered such that they correspond to each other between datasets. Note that since the orthologous gene mapping can be many-to-one, genes are duplicated and all of these orthologs are retained.

[0126] After obtaining H’ and M’, we proceed by integrating the datasets using the FindIntegrationAnchors and IntegrateData functions of the Seurat package (version 3) (Stuart, T. et al. 2019). Following the guide for performing this analysis (satijalab.org / seurat / v3.1 / integration.html#standard-workflow), this guide only considers the 2000 most variable genes among the g genes. This results in a new integrated data matrix X of shape (n+m, 2000).

[0127] To project cell type labels from the E16 - 19 data onto our human cell line data, we begin by calculating the first 30 principal components of this X data matrix (obtaining the shape of matrix X’ (n + m, 30)). Then, using only the labeled data in this reduced dimension (matrix shape (m, 30)), we train a k - nearest neighbor classifier (using k = 5). Then, we apply this classifier to predict the cell type labels for the (n, 30) human cell line subset of the data. These predicted labels are the final "projected labels". In vitro colony - forming unit assay. Cells were dissociated into single - cell solution using Accutase (StemCell Technologies). CD34+ cells were purified from a portion of the cells using the CD34 MicroBead Kit UltraPure and LS column (Miltenyi Biotec) according to the manufacturer's instructions. The colony - forming unit assay was performed using methylcellulose - containing media (MethoCult H4434 Enriched and MethoCult SF H4636, StemCell Technologies) according to the manufacturer's instructions. After 14 days of culture, plates were visually scored for CFU multi - lineage colonies and then analyzed immunophenotypically by flow cytometry.

[0128] Flow cytometry analysis. Input and output cells for the colony - forming unit assay were analyzed immunophenotypically via flow cytometry. Cells were stained on ice for 30 minutes with human anti - CD235a, anti - CD71, anti - CD45, anti - CD11b, and anti - CD34 antibodies (Biolegend). As a dead stain, 7 - AAD (ThermoFisher Scientific) was used, and cells were analyzed using an LSR II flow cytometer (BD Biosciences). Data analysis was performed using FlowJo software. An example of the gating strategy can be found in Figure 18. To prepare samples, iDiscoid was developed in wells 4 - 6 of a 6 - well plate, and samples were pooled prior to CD34+ cell isolation using the MACS separation system (Stemcell Tech).

[0129] Example 2. Modeling of human post-implantation development via extra-embryonic niche manipulation. Human embryo implantation initiates an important developmental stage that includes profound morphogenetic changes, axis formation, and gastrulation events in both embryonic and extra-embryonic tissues. Mechanical knowledge of this period of human life remains limited because access to natural healthy samples is restricted for both technical and ethical reasons. Additionally, there is a lack of early post-implantation human stem cell models that involve both embryonic and extra-embryonic morphogenesis. Here, this study presents iDiscoids generated from human induced pluripotent stem cells via engineered synthetic gene circuits. iDiscoids exhibit the co-development of human embryonic tissues and the engineered extra-embryonic niche. They display unexpected self-organization and tissue boundary formation that recapitulate the specification of yolk sac-like tissue with hematopoietic properties, the formation of a bilayered disc-like embryonic morphology, the development of an amniotic-like cavity, and the acquisition of the posterior axis. iDiscoids provide an easy-to-use, high-throughput, reproducible, and scalable platform for investigating multifaceted aspects of early post-implantation human development. This system provides a tractable human model for drug testing, developmental toxicology, and disease modeling.

[0130] This study presents a post-implantation embryo model derived from human stem cells, called "iDiscoids," which overcomes some of these limitations. iDiscoid engineering utilizes a synthetic biology approach to pre-program a population of human induced pluripotent stem cells (hiPSCs) with artificial gene circuits. Activation of the gene circuits forms an extra-embryonic niche that instructs the self-organization of adjacent wild-type hiPSCs. This study using iDiscoids has demonstrated the development of a human bilayer disc-like structure, luminescence, the formation of amnion-like tissue, symmetry breaking leading to posterior axis specification, and yolk sac specification with hematopoietic properties.

[0131] Manipulation of co-development of embryonic and extra-embryonic tissues. After addition of the small molecule, doxycycline (Dox) (hereinafter referred to as iGATA6), hiPSC cell lines were manipulated using an inducible gene circuit that expresses the human GATA6 transcription factor (Figure 1A, Figure 5A). These iGATA6 cells were engineered to express different levels of GATA6 upon circuit induction and are distinguishable by the expression of the reporter enhanced green fluorescent protein (EGFP) (Figure 5B). iGATA6-hiPSCs were mixed with wild-type hiPSCs (WT), and the mixed cell population was seeded on a 2D standard culture plate (Figure 1A).

[0132] The initial state of the system reflects the salt-and-pepper distribution of hypoblast (GATA6 + ) and epiblast (NANOG + ) in the inner cell mass of the embryonic (E) day 6 embryo in vivo (Figure 1B). After treatment with Dox, iGATA6 cells upregulate EGFP and exhibit loss of the pluripotency marker NANOG (Figure 1A). During the first 72 hours, from the initial state of random distribution, the induced iGATA6 cells and WT cells sort from each other. After 72 hours, WT cells form disk-shaped clusters (WT disks) (Figure 1A, 1C, and Figure 5C). At the same time, iGATA6 cells self-organize, and iGATA6 cells with low-level exogenous GATA6 induction (lower EGFP) are sorted from the WT disks and express characteristic markers of primitive endoderm, including PDGFRα and GATA4 (Figure 6A - 6B). Cells expressing the highest level of exogenous GATA6 (high EGFP) surrounding the WT disks lack the expression of these markers and show a fate bifurcation away from the primitive endoderm (Figure 6A - 6C).

[0133] Single-cell RNA transcriptomics reveals embryonic and extra-embryonic fate trajectories. To investigate the cell identity of each iGATA6 population, this study performed single-cell RNA sequencing (scRNAseq) analysis on 4,135 cells derived from day 5 cultures. This analysis identified the emergence of five cell clusters and indicated distinct identities (Figs. 1D–1E). Performing an unsupervised comparison of the transcriptomes with E16–19 human embryos (Tyser, R. C. V. et al. 2020) revealed the acquisition of distinct post-implantation cell fates in the iDiscoid subpopulation (Fig. 1F). The three largest clusters (clusters 1, 2, and 3) expressed the lowest mean positive EGFP and GATA6 transcript counts, and these clusters showed significant transcriptomic similarity to the human post-implantation yolk sac endoderm and hypoblast (Fig. 1F). These clusters expressed putative lineage markers associated with the human hypoblast, including LAMA1, GATA4, and PDGFRα. Two of the three clusters additionally expressed the human hypoblast markers CUBN, AMN, and NODAL (Fig. 1E) (Cindrova-Davies, T. et al. 2017). The WT population (cluster 0) showed very significant similarity to human post-implantation epiblast and primitive streak cells (Fig. 1F). These cells expressed the pluripotency-related factors SOX2 and OCT4 (POU5F1) and lacked GATA6 or EGFP transcripts (Fig. 1E).

[0134] The cluster containing the second highest average EGFP transcript expression (cluster 4) shows differential upregulation of genes involved in the regulation of the anterior visceral endoderm; LHX1, a significant activator of the anterior endoderm program in mouse and anterior visceral endoderm markers in humans, HHEX, a marker of the human anterior visceral endoderm, and CXCR4, a marker of the E16-19 anterior endoderm (Figure 1E, Figure 7A) Tyser, R.C.V. et al. 2020, Costello, I. et al. 2015, Mole, M.A. et al. 2021. Comparison with available datasets showed similarity to both the visceral endoderm and definitive endoderm of the E16-19 human embryo and strong correlation with the E6.5-E8.25 mouse embryonic visceral endoderm (emVE) (Figure 1F, Figure 7B). Immunofluorescence revealed that this population occupies the region adjacent to the WT disc (Figure 7C).

[0135] Analysis of the clusters with the highest expression of GATA6 and EGFP reveals two distinct subpopulations, both of which have significant similarity to the human yolk sac mesoderm (Fig. 1F, Fig. 7D). One (labeled 5.1) shows higher average expression of ECM proteins (COL1A2, COL4A1, LAMA1, LAMB1, FN1), and the other subcluster shows markers of hematopoietic tissues within the mouse allantois (TBXT, MIXL1, IFITM3 (Fragilis), DPPA3 (Stella), OCT4) and the highest average transcript numbers for both GATA6 and EGFP (labeled 5.2) (Fig. 1E, Fig. 7E, 8A) (Tyser, R.C.V. et al. 2020, Ross, C. & Boroviak, T.E. 2020, Wolfe, A.D., Rodriguez, 2017, Downs, K.M., 2009, Downs, K.M. 2020). Cluster 5.2 uniquely contains TBXT- and MIXL1-coexpressing cells and expresses erythropoiesis markers GATA1, GATA2, ALAS2, KLF1, GFI1B, and SMIM1 (Fig. 1F, Fig. 7E, Fig. 8A) (Barbarani, G. 2019, Zhang, Y. et al. 2017, Cvejic, A. et al. 2013, Lancrin, C. et al. 2012, Bruveris, F.F. et al. 2020). Further analysis via label transfer between populations additionally confirms the distinct identity of these subpopulations, which includes substantial alignment to yolk sac-derived erythrocytes (47%) for cluster 5.2 (Fig. 8B–8D).

[0136] Identification of yolk sac mesoderm-like cells with hematopoietic potential. In this study, the observed features of the yolk sac and their associated functional characteristics were further investigated. The human yolk sac, composed of extraembryonic endoderm and mesoderm, is a major source of serum proteins and lipids and a niche for the emergence of early primitive hematopoiesis and pro-definitive hematopoiesis around E16-18 of human development (Ross, C. & Boroviak, T. E et al., 2020, Nakazawa, F. et al., 2011, Palis, J. & Yoder, M. C. 2001). The human yolk sac mesoderm is suggested to differentiate from the yolk sac endoderm and appears in the form of spindle-shaped cells (Ross, C. & Boroviak, T. E et al., 2020, Enders, A. C. & King, B. F. 1988). Since the human embryo is not suitable for research, the use of a model system provides a means to identify the molecular and cellular mechanisms of yolk sac tissue. However, importantly, yolk sac tissue morphogenesis and its hematopoietic function have not been captured in past stem cell-based models of human embryogenesis.

[0137] In this experiment, in the culture medium where iDiscoid had grown by day 5, high levels of secreted AFP, a major serum protein associated with primitive erythropoiesis (An, J. et al., 2019), and APOA1, an apolipoprotein that plays a role in monocyte proliferation (Murphy, A. J. et al. 2011), were first identified (Figure 2A). Immunofluorescence analysis of iGATA6 cells was performed for CD34 + / ERG +Reveal spindle-shaped clusters of endothelial cells and confirm morphological adaptation (Figure 2B, Figure 9A). Importantly, these cells were also positive for TAL1 (scl), a major regulator of blood endothelial differentiation and hematopoietic development, and RUNX1, a master transcription factor required for the emergence of hematopoietic stem cells from endothelial cells (Vagapova, E.R. et al., 2018; Yzaguirre, A.D. et al., 2017). These spindle-shaped cells were consistently localized beneath the iGATA6 layer (Figure 2B), and this morphology clearly resembles the in vivo localization of the human yolk sac mesoderm, which is positioned basal to the yolk sac endoderm in the region beneath the yolk sac endoderm (Knoth, M. & Larsen, J.F. 1972) (Figure 2E). CD34 + / TAL1 + cells were also EGFP + , indicating that they developed from the parental population derived from iGATA6 (Figure 2B). Flow cytometry analysis of the erythroid marker CD71 showed higher levels among cells with high EGFP expression (also CD34 - ) (Figure 2D). This supports the presence of the erythroid signature observed in cluster 5.2 (Figure 8D). Colony-forming unit (CFU) assays in iDiscoid enriched for intact iDiscoid and CD34-expressing cells generated CD45 + cells (Figure 9B, 9C). Enrichment of myeloid-like cells (CD11b + ) was observed in the CD34-enriched population, while erythroid-like cells (CD71 + , CD235a - (early) or CD235a + (late)) were more abundant in the whole tissue population (Figure 9B, 9C). Collectively, these results indicate the presence of two forms of hematopoiesis-promoting cells in iDiscoid: one with an erythroid differentiation bias that occurs within GFP-high cells without transition through the CD34 + endothelial cell state, and one with a higher myeloid differentiation potential that arises from CD34 + endothelial cells. These data establish the relevance of this model for probing early hematopoiesis in humans.

[0138] The interaction between the embryo and extraembryonic cell progeny results in the formation of an amniotic cavity with a bilayer disc. After embryo implantation around E7 in humans, a cavity forms within the epiblast of the embryonic disc, followed by the formation of a bilayer disc (Shahbazi, M. N. et al. 2016). Beginning on day 3 of iDiscoid development in vitro, a conversion from a monolayer disc of the WT disc structure to a three-dimensional cell mass was observed (WT cluster, Figure 3A). Over 5 days, iGATA6 cells migrated over the top surface of these WT clusters, resulting in a continuous membrane of extraembryonic endoderm-like cells (Figures 3A - 3D, Figure 10A). CXCR4 / SDF-1 signaling was found to be essential for this migration, and inhibition of this chemokine pathway by administration of the CXCR4 antagonist AMD3465 substantially inhibited upward migration and continuous iGATA6 membrane formation (Figures 10A - 10B). scRNAseq also revealed that CXCR4 was exclusively expressed by iGATA6 cells (cluster 5), and SDF-1 was expressed by WT cells (Figure 10C), supporting the functional observations. CXCR4 is expressed in the hypoblast of the chick embryo and is present in the anterior human endoderm (Tyser, R. C. V. et al. 2020, Yusuf, F. 2015). Thus, these results indicate that reciprocal CXCR4-SDF1 signaling may have a role in the maintenance and stability of the human bilayer disc.

[0139] The process of upward iGATA6 migration proceeds in parallel with the deposition of laminin membranes surrounding the WT clusters. In WT clusters with complete iGATA6 coverage, a complete laminin envelope surrounding the WT clusters is observed (Figs. 10D - 10F), mimicking laminin deposition by primitive endoderm cells after separation from the epiblast in mouse embryos at E4.5 (Artus, J. 2011). Laminin deposition by iGATA6 cells can then trigger intracellular cell polarization of the WT clusters that promotes the onset of lumen formation (Li, S. et al., 2003, Bedzhov, I. & Zernicka - Goetz, 2014). Immunofluorescence analysis was performed for PODXL, an apical - expressed anti - adhesion surface protein involved in human primitive amniotic lumen and mouse primitive amniotic cavity formation, and for ZO - 1, an apical tight - junction protein (Fig. 3B) (Simunovic, M. et al. 2019, Kim, Y. S. et al. 2021, Shahbazi, M. N. et al. 2017, Sozen, B. et al. 2021, Wang, S. et al. 2021, Phua, D. C. et al. 2014). Following the upward migration of iGATA6 from day 3 to day 5, cavitation appears to initiate with the formation of local micro - lumens at the center of rosette - like cell structures (Figs. 3A - 3C). In WT clusters not surrounded by the iGATA6 layer, PODXL + , ZO - 1 + appears to initiate with the formation of local micro - lumens at the center of rosette - like cell structures (Figs. 3A - 3C). In WT clusters not surrounded by the iGATA6 layer, PODXL +It was observed that lumen formation was not observed (Figure 3C). As the migration of iGATA6 cells progressed in most clusters, the number of clusters without coverage gradually decreased by day 5 (Figure 3D). Cleaved caspase 3 concentrated within the region near the lumen was also detected, which coincided with the region of iGATA6 coverage in the WT clusters (Figure 11A). Apoptosis was most prominent in the intermediate-stage lumen, with high levels of apoptosis within the inner membrane separating multiple lumens, suggesting that cell death plays a role in the formation and expansion of the iDiscoid lumen (Figure 11A). Finally, the two cell monolayers of embryonic and extraembryonic fates were positioned on either side of the laminin membrane and polarized in opposite directions (Figure 11B; late stage), forming a cell arrangement similar to the bilaminar disc in the post-implantation E12 human embryo (O’Rahilly, R. et al., 1987).

[0140] In summary, this study presents a morphogenetic event that results in the assembly of a bilaminar disc-like structure within the iDiscoid system, starting with ECM deposition by iGATA6 cells migrating horizontally over the WT clusters and proceeding through cell detachment from the apical surface of polarized cells and cell apoptosis outside the disc (Figure 11A).

[0141] Identification of the amniotic-like domain of iDiscoid. Primate amnion formation is structurally and temporally distinct from that of mice, highlighting the need for human model systems to provide insights into human-specific developmental dynamics (Yang, R. et al. 2021). After luminescence within the epiblast of the post-implantation embryo, cells of the epiblast undergo dorsal-ventral patterning, separating the columnar epiblast from the flat epithelial amniotic ectoderm (Zheng, Y. et al. 2019, Hertig, A. T. & Rock, J. 1949). Similarly, the spontaneous emergence of domains within the WT disc expressing the amnion co-markers ISL1 and AP-2α was observed, but NANOG expression was absent (Figs. 3E–3G, 12A). This tissue is spatially separated within the cavitated sac, and the amnion-like tissue is positioned away from the bilayer iGATA6 / WT disc relative to the tissue culture dish. WT cells within the bilayer iGATA6 / WT disc display NANOG expression and exhibit a columnar morphology (Figs. 3E–3G, 12B).

[0142] Recently, an active role for primate amnion tissue in promoting BMP4 signaling, acting downstream of ISL1, has been shown (Yang, R. et al. 2021). BMP4 has also been reported to be able to promote amnion tissue formation in human embryo models (Zheng, Y. et al. 2019). Investigation of BMP4 secretion into the culture medium showed a strong increase by day 5, which correlated with the detection of BMP4 targets by scRNAseq on day 5 (Figs. 13A–13B). Immunofluorescent staining of the BMP4 effectors pSMAD1, pSMAD5, and pSMAD8 revealed BMP4 signaling in a ring pattern concentrated at the ends of the iDiscoid (Fig. 3H). This pattern was not perfectly aligned with the layer of amnion gene expression, indicating that BMP4-mediated signaling may not be active in all parts of the amnion-like population within the iDiscoid system. +

[0143] ​Immunofluorescent staining of pSMAD2, an agent for active NODAL signaling, showed a higher NODAL response in cells located in the amnion-like layer (Figure 3H). Investigation of the scRNAseq dataset on day 5 revealed the expression of NODAL transcripts within the iGATA6 compartment and the NODAL coreceptor TDGF1 (Cripto-1) within the WT cluster (Figure 13A). Inhibition of BMP signaling using the small molecule inhibitor dorsomorphin showed effective suppression of SMAD1 / 5 / 8 phosphorylation (Figure 13C), but did not completely prevent the specification of amnion-like tissue expressing ISL1 (Figure 3I, Figure 13C). However, NODAL inhibition via SB431562 resulted in substantial elimination of ISL1-expressing cells within iDiscoid (Figure 3I, Figure 13C). In particular, the expression of NANOG within the WT cluster was also substantially reduced, but cavitation was maintained (Figure 13C, Hoechst vertical slice). These data indicate an essential role for yolk sac-derived NODAL signaling for both the maintenance of pluripotent cells and alternative or complementary drivers of human amnion specification. This finding is also consistent with recent studies on NODAL-mediated actions in PSCs (Chhabra, S. et al., 2019, Guo, G. et al. 2021).

[0144] The primitive streak-like domain occurs in iDiscoid. The primitive streak, characterized by TBXT (Brachyury), establishes the onset of gastrulation and is formed in the posterior region of the embryo (Mikawa, T. et al., 2004). This study reveals the emergence of distinct domains expressing TBXT and MIXL1 in approximately 65% of day 4 WT clusters in iDiscoid (Figs. 4A - 4C, Fig. 14A). These domains lack EGFP expression and exhibit an asymmetric pattern, unlike the TBXT-expressing cells derived from iGATA6 (high GATA6-expressing population 5.2) (Figs. 1D, 1E, Figs. 7D, 7E) (Fig. 4A, 4B, Fig. 14A). This observation indicates symmetry breaking and the emergence of a primitive streak-like region within the iDiscoid structure that mimics the onset of gastrulation in epiblast cells after transplantation in natural human embryos. In particular, the domains that were positive for TBXT / MIXL1 had unique polarized expression within the WT cluster, while clusters that were positive for only a single marker lacked polarized compartmentalization (Fig. 4B). These domains were transiently identified, and the proportion of the observed streak-like cell mass decreased by day 5 (Fig. 4C). This decrease was accompanied by an increase in the average cluster size between day 4 and day 5 (Fig. 4D).

[0145] The following experiments sought to understand important parameters for the development of the TBXT-positive pole in WT cells. In non-human primate embryos at E11, CER1 has been shown to be expressed throughout the visceral endoderm, and over the course of several hours, the expression of CER1 becomes restricted to the region of the visceral endoderm near the epiblast cells (Sasaki, K. et al. 2016). In mice, the anterior positioning of Cer1 defines, positions, and enables the proper onset of primitive streak formation (Rodriguez, T. A. et al., 2005). In iDiscoid, the domain of CER1 expression was identified in the extra-embryonic cells derived from iGATA6 that surround the WT cluster (Figs. 4E, 4F). A comparison of the CER1 and TBXT expression intensities within iDiscoid shows the average hydrodynamic diameter of CER1 expression and TBXT +Reveal a sharp positive correlation with domain specification (Figure 4G). Slightly lower levels of CER1 expression result in substantially lower levels of TBXT expression within the associated WT clusters (Figure 4G). Investigation of the induced GATA6 (EGFP) expression intensity reveals that CER1 is expressed at higher levels around WT clusters with higher average local GATA6 induction, and these clusters have a higher TBXT positive rate (Figure 4H). This strong correlation with stripe domain specification was independent of the WT disc region (Figure 4I). These data emphasize the central role of CER1 involvement in stripe domain specification rather than in the region. These cells appeared to cover the stripe poles expressing TBXT rather than facing the positions of these poles. Currently, data on CER1 positioning in human and primate embryos are limited (Mole, M. A. et al. 2021). However, mouse and human Cerberus orthologs show structural differences, and investigation of the binding affinity of human CER1 shows high affinity for NODAL but minimal affinity for BMP2 and BMP4 (Katoh, M. & Katoh, M. 2006, Aykul, S. et al., 2015). In iDiscoid, spatial inhibition of NODAL signaling mediated by CER1 can prime WT cells for activation of TBXT and MIXL1 by BMP4 and mediate downregulation of NANOG at the basal tip of the cavity (Figure 14B) (Zhang, P. et al. 2008). These data indicate that the self-organization of the iGATA6 cell population that positions high GATA6-expressing cells around WT clusters contributes to the subsequent specification of the stripe domain. + TBXT + / MIXL1 + indicating its contribution to the subsequent specification of the stripe domain.

[0146] The iDiscoids presented here develop both embryonic and extra-embryonic lineages with several features that have been observed together for the first time in an in vitro human development model. These features indicate proximity to human embryogenesis post-transplantation, but are uniquely observed in hiPSC-based systems (Figure 15). The iDiscoid platform highlights the power of niche manipulation and tissue co-differentiation to promote morphogenetic events in vitro. This demonstrates that a combination of gene circuit-based heterotypic tissue manipulation and the formation of tissue-tissue boundaries in 2D enables geometric confinement and subsequent self-organization in 3D (i.e., luminescence).

[0147] The extra-embryonic tissues of iDiscoids are genetically pre-programmed into undifferentiated hiPSCs using inducible gene switches and mixed with WT hiPSCs. The undifferentiated cell mixture can be expanded, cryopreserved, and shipped for on-demand use using only a conventional 2D culture plate and the addition of a single small molecule, Dox (Figure 16). The high-throughput format, efficient generation, scalability, compatibility with live imaging, and easily implementable protocol enable consistent establishment across different laboratories, facilitating research in this field. These properties and the potential to be generated from hiPSCs with diverse genetic backgrounds (Figure 17) enable studies to elucidate the principles governing human developmental fate and morphology, opening up potential new routes for high-throughput drug testing, developmental toxicology, and easy-to-handle disease modeling with fewer ethical concerns.

[0148] Clonal replenishment of tissues by design function: Specific pre-programmed organ or hematopoietic progenitor cell lineages can be clonally isolated prior to system introduction and replenished into the system at higher levels to increase the amount of those lineages that develop, enabling custom-designed tissues pre-programmed in the present invention (Figures 20A - 20B).

[0149] Characteristics of self - vascularizing yolk sac: Cells engineered to inducibly express ETV2 can be incorporated into a cell culture system to enhance angiogenesis and endothelial cell formation in tissues, for use in understanding the capabilities of the yolk sac, and for contributing to the formation of a self - vascularizing yolk sac model system for the development of personalized therapeutics (Figure 21). ETV2 is expressed in adjacent cells that come into contact with GATA6 - expressing cells. In this case, the ETV2 gene circuit is engineered in an independent cell type and is controlled by doxycycline. In some cases, two cell lines are mixed to form the final organoids.

[0150] Engineered formation of advanced blood islands and hematopoietic lineages from human yolk sac: Cells engineered to inducibly express ETV2 can be combined with cells engineered to inducibly express GATA6, and together they express enhanced yolk sac characteristics, including cell identity equivalent to that of the in - vivo human yolk sac, enhanced yolk sac - like angiogenesis, and formation of yolk sac - derived hematopoietic cells in a physiologically relevant organized blood island structure (Figures 22A - 22C).

[0151] Anteriorizing activity from the anterior visceral endoderm - like tissue leading to nervous system development: This system can result in ectodermal growth with neural characteristics, including the expression of markers associated with the neural plate and surface ectoderm, due to anteriorizing signaling produced by GATA6 cells containing the proteins CER1 and LEFTY1. These neural characteristics develop into organized structures that can be harvested for analysis or independently cultured to enhance their characteristics (Figures 24A - 24C).

[0152] Maturation of the hematopoietic lineage over an extended maintenance period within the system: This system can be maintained in culture over an extended period and developed over time to show the development of a hematopoietic progenitor cell lineage, including hematopoietic stem cells, erythroid progenitor cells, and myeloid progenitor cells. By day 23, these lineages can have a gene regulatory network that is significantly similar to the in - vivo human embryonic hematopoietic lineage (Figures 27A - 27D).

[0153] In vivo functional reconstitution of the blood system: This technique can re-colonize and reconstruct the bone marrow and spleen in vivo with hematopoietic cells including B cells, T cells, NK cells, bone marrow cells, and erythrocyte cells, enabling their use in autologous genetic transplantation of healthy tissues after injury or irradiation (Figs. 28A - 28B).

[0154] Use of small molecules and proteins in the medium to affect the system state: The present invention can be altered by introducing chemical substances and exogenous proteins into cell culture media, changing the fate acquired by cells within the system. The NODAL inhibitor SB431542 can be added to the reprogramming system to acquire and maintain a state similar to early human and primate amnion before acquiring late amnion characteristics. This can also be used to induce the system to acquire markers of human trophoblasts, enabling the use of the system in the development of model placental tissue (Figs. 29A - 29E).

[0155] Re - establishment of embryonic identity in non - manipulated cell types exposed to the system: A specific combination of factors secreted by the system affects the gene regulatory network of cells exposed to the culture medium and can reprogram them to a younger, more embryo - like state. This alleviates age - related characteristics from cells associated with disease and tissue degeneration, enabling therapeutic implications in the context of age reversal and tissue regeneration (Figs. 30A - 30C).

[0156] Example 3. This study describes a new in vitro - induced pluripotent cell - based tool that can be used for research after the inner cell mass stage of human embryos. The design principles governing human embryogenesis remain enigmatic because access to post - implantation human embryos is limited for both technical and ethical reasons. Here, the study presents an in vitro stem - cell - based platform called iDiscoid. iDiscoid is generated from human induced pluripotent stem cells and exhibits, with self - timing and organization, the co - development of embryonic and extra - embryonic tissues, the separation of the epiblast and hypoblast, the formation of a human bilayer disc - like structure, lumenogenesis, the development of the amniotic cavity, the development of the posterior axis with primitive streak characteristics, and the development of a yolk sac with hematopoietic properties including erythropoiesis and myelopoiesis, as well as the reprogramming of induced pluripotent stem cells from a primed pluripotent state to a naive pluripotent state, making it a unique model for human embryo research. These events are achieved using a homogeneous genetic cell population containing populations of wild - type and genetically engineered cells, seeded and induced in 2D cultures to express different levels of the heterologous human transcription factor GATA6. Upon seeding and activation of the engineered gene circuit, this study shows the formation of the extra - embryonic endoderm lineage, self - organization, cell migration, and step - wise boundary establishment, which collectively recapitulate late - blastocyst - gastrula - like morphogenetic events in wild - type hiPSCs.

[0157] Current human embryo models focus on the development of a limited number of embryonic features and exclude the primitive endoderm and yolk sac from the research. Existing models develop subsets of embryonic - related features such as the amnion, somitogenesis, and posterior axis individually, while iDiscoid is unique in that it develops all the features listed together in a single self - organizing self - timing system. Additionally, no model has demonstrated the emergence of hematopoietic tissue within an organized embryoid system, nor has the novel reprogramming of iPSCs towards a naive state been shown. The self - organization and self - specification of these embryoid events are novel, and only a part of the overall process demonstrated in iDiscoid can be developed in other models.

[0158] A robust, scalable, and user-friendly model of human embryogenesis enables real-time interrogation of development across different laboratories. Subsequently, this model is used to study yolk sac hematopoiesis in hiPSCs, as well as the signaling events that govern cavitation and amnion fate acquisition. Collectively, iDiscoid provides an easy-to-use, high-throughput, scalable platform to investigate multifaceted aspects of post-implantation development in a human-based system using genetically pre-programmed niches. This system can be used to understand human developmental steps beyond implantation, for legal and ethical reasons where the actual embryo is hidden within the uterine wall and not observable. This model can be used for precision medicine, exploration of potential embryonic defects involving the yolk sac, early hematopoiesis, and amnion, as well as exploration and development of individualized-based therapies. This model can also be used to improve the effectiveness of in vitro fertilization through investigation of the properties of the yolk sac and epiblast, which lead to the most successful emergence of important embryonic features.

[0159] List any novel or unusual features of the invention.

[0160] · This system develops hematopoietic cells and tissues important for the blood system population, i.e., this technology can serve as a source of donor cells for patients in need of blood system regeneration after disease. · This system robustly develops tissues similar to the primitive endoderm, an important tissue for the success of in vitro fertilization pregnancies. Complementing low-quality embryos with tissues developed through this technology may significantly increase the number of high-quality embryos available to women seeking in vitro fertilization. · This system exhibits aspects of naive reprogramming of pluripotent tissues with applications for regenerative therapies and treatment of age-related diseases. · This system can function as a model of embryogenesis to test drugs and therapies, including pregnancy and prenatal tissues. · This system can be cryopreserved for long periods and thawed for use as needed.

[0161] This system depends only on the induction of gene circuits and can be cultured, stored, shipped, and activated as needed. This system is cultured in 2D on a cell culture dish and remains bound as it acquires 3D features in a bilayer disk region, resulting in a morphology similar to that of an "unrolled" embryo. This system develops many bilayer disk structures in a single cell culture well, enabling high-throughput analysis of hundreds of human disk features in a single experiment. With the development of these bilayer intervertebral disc structures, what is observed includes the development of cells with an identity similar to the amnion, primitive and pre-embryonic hematopoietic systems, and the primitive streak including the development of rare early embryonic cell types. Additionally, this system demonstrates the reprogramming of induced pluripotent stem cells to a naive state, which can be utilized in human developmental research and regenerative medicine.

[0162] This invention encompasses the use of genetic manipulation within a specific cell type and its combination with a second cell type to enable unique self-organization and the development of targeted secondary cell types.

[0163] The application of this technology is threefold, with a synergistic effect on the reproducibility and ease of use of the method, enabling commercialization and use in various laboratory environments with limited technical challenges.

[0164] · The first is as a diagnostic tool for evaluating the effects of drugs and toxins on the early stages of embryonic development, as a step that can be taken in drug development prior to mammalian in vitro testing. The early Phase 1 or Phase 1 clinical trials of 748 drugs or biologics were posted on ClinicalTrials.gov in 2021, and each clinical trial represents a company that may benefit from the use of this product. · Additionally, this product may be useful for improving the effectiveness of IVF. The quality of the primitive endoderm is one of the most important factors controlling embryo success during IVF, and self-genetic replenishment of the primitive endoderm in low-quality embryos may substantially improve IVF success. According to the CDC, in 2019, 330,773 women in the United States sought reproductive assistance therapy (ART), and 29% of these women sought ART due to decreased ovarian reserve, a condition in which the number of high-quality embryos produced is significantly reduced. This represents an annual U.S. market of approximately 96,000 people. · Thirdly, this product has the potential to be used for autologous or allogeneic generation of hematopoietic cells for hematopoietic stem cell transplantation. This product can take the form of a bioreactor-style platform that uses a patient's own cells for the expansion of hematopoietic stem cells for blood system regeneration. According to the HRSA, 23,000 bone marrow or cord blood transplants were performed in the United States in 2019. Additionally, approximately 18,000 people are diagnosed each year with life-threatening diseases for which a bone marrow or cord blood transplant is the best treatment option and for which our product could be an effective treatment.

[0165] Sequence SEQ ID NO:1 (pENTR_L1_rtTA3-2A-Hygro_L2(6)) CDS of rtTA3: 694 - 1398 of SEQ ID NO: 1 CDS of 2A: 1405 - 1458 of SEQ ID NO: 1 CDS of hygromycin: 1459 - 2499 of SEQ ID NO: 1 CDS of kanamycin resistance: 2787 - 3596 of SEQ ID NO: 1 SEQ ID NO: 2 (JVLR3_PB-TAG-ERP2_with_GATA6-2A-EGFP gateway (4)) Accession No. 3 (GATA6 CDS Sequence 1) Accession No. 4 (GATA6 CDS Sequence 2) Ccttcgagaccccggtgctgcacagcctgcagagccgcgccggagccccgctcccggtgccccggggtcccagt Accession No. 5 (GATA6 CDS Sequence 3) Gacctgctggaggacctgtccgagagccgcgagtgcgtgaactgcggctccatccagacgccgctgtggcggcgggacggcaccggccactacctgtgcaacgcctgcgggctctacagcaagatgaacggcctcagccggcccctcatcaagccgcagaagcg Accession No. 6 (GATA6 CDS Sequence 4) Ccttcatcacggcggcttggattgtcctgtgccaactgtcacaccacaactaccaccttatggcgcagaaacgccgagggtgaacccgtgtgcaatgcttgtggactctacatgaaactcca Accession No. 7 (GATA6 CDS Sequence 5) Gtgcccagaccacttgctatgaaaaaagagggaattcaaaccaggaaacgaaaacctaagaacataaataaatcaaagacttgc Accession No. 8 (GATA6 CDS Sequence 6) Gtaatagcaataattccattcccatgactccaacttccacctcttctaactcagatgattgcagcaaaaatacttcccccacaacacaacctacagcct Accession No. 9 (GATA6 CDS Sequence 7) gcgggtgccccggtgatgactggtgcgggagagagcaccaatcccgagaacagcgagctcaagtattcgggtcaagatgggctctacataggcgtcagtctcgcctcgccggccgaagtcacgtcctccgtgcgaccggattcctggtgcgccctggccctggcc Accession No. 10 (including the GATA6 CDS sequence) Array number 11 (tet operator) tccctatcagtgatagaga

[0166] Array number 12 (OCT4 polypeptide sequence) MAGHLASDFAFSPPPGGGGDGPGGPEPGWVDPRTWLSFQGPPGGPGIGPGVGPGSEVWGIPPCPPPYEFCGGMAYCGPQVGVGLVPQGGLETSQPEGEAGVGVESNSDGASPEPCTVTPGAVKLEKEKLEQNPEESQDIKALQKELEQFAKLLKQKRITLGYTQADVGLTLGVLFGKVFSQTTICRFEALQLSFKNMCKLRPLLQKWVEEADNNENLQEICKAETLVQARKRKRTSIENRVRGNLENLFLQCPKPTLQQISHIAQQLGLEKDVVRVWFCNRRQKGKRSSSDYAQREDFEAAGSPFSGGPVSFPLAPGPHFGTPGYGSPHFTALYSSVPFPEGEAFPPVSVTTLGSPMHSN Array number 13 (GATA4 polypeptide sequence) MYQSLAMAANHGPPPGAYEAGGPGAFMHGAGAASSPVYVPTPRVPSSVLGLSYLQGGGAGSASGGASGGSSGGAASGAGPGTQQGSPGWSQAGADGAAYTPPPVSPRFSFPGTTGSLAAAAAAAAAREAAAYSSGGGAAGAGLAGREQYGRAGFAGSYSSPYPAYMADVGASWAAAAAASAGPFDSPVLHSLPGRANPAARHPNLDMFDDFSEGRECVNCGAMSTPLWRRDGTGHYLCNACGLYHKMNGINRPLIKPQRRLSASRRVGLSCANCQTTTTTLWRRNAEGEPVCNACGLYMKLHGVPRPLAMRKEGIQTRKRKPKNLNKSKTPAAPSGSESLPPASGASSNSSNATTSSSEEMRPIKTEPGLSSHYGHSSSVSQTFSVSAMSGHGPSIHPVLSALKLSPQGYASPVSQSPQTSSKQDSWNSLVLADSHGDIITA Array number 14 (FMorg30 PB-TAG-ERP2 #80479-pENTR_L1_ETV2_L2) Accession No. 15 (ETV2 CDS)

[0167] Accession No. 16 (ETV2 polypeptide sequence) MDLWNWDEASPQEVPPGNKLAGLEGAKLGFCFPDLALQGDTPTATAETCWKGTSSSLASFPQLDWGSALLHPEVPWGAEPDSQALPWSGDWTDMACTAWDSWSGASQTLGPAPLGPGPIPAAGSEGAAGQNCVPVAGEATSWSRAQAAGSNTSWDCSVGPDGDTYWGSGLGGEPRTDCTISWGGPAGPDCTTSWNPGLHAGGTTSLKRYQSSALTVCSEPSPQSDRASLARCPKTNHRGPIQLWQFLLELLHDGARSSCIRWTGNSREFQLCDPKEVARLWGERKRKPGMNYEKLSRGLRYYYRRDIVRKSGGRKYTYRFGGRVPSLAYPDCAGGGRGAETQ

Claims

**Claim 1** A method for generating in ex vivo or in vitro a cell mass comprising one or more embryonic cell structures and one or more extraembryonic cell structures, comprising: a) obtaining one or more first stem cell populations comprising a first inducible transgene encoding a GATA binding protein 6 (GATA6) polypeptide; b) contacting the one or more first stem cell populations with an inducer that induces the expression of the transgene encoding the GATA6 polypeptide; c) mixing the one or more first stem cell populations of step b) with a second stem cell population; d) culturing the mixture of cells of step c) on a surface for at least 7 days to thereby produce the cell mass. The method as described above. **Claim 2** The method according to claim 1, wherein the second stem cell population does not have the inducible transgene encoding the GATA6 polypeptide. **Claim 3** The method according to claim 1 or 2, wherein the second stem cell population comprises an inducible transgene encoding an ETS variant transcription factor 2 (ETV2) polypeptide, and further comprising contacting the second stem cell population with an inducer that induces the expression of the transgene encoding the ETV2 polypeptide. **Claim 4** The method according to claim 1 or 2, wherein step c) further comprises contacting the one or more first stem cell populations of step b) with the second stem cell population and a third cell population, the third cell population comprises an inducible transgene encoding an ETV2 polypeptide, and further comprising contacting the third stem cell population with an inducer that induces the expression of the transgene encoding the ETV2 polypeptide. **Claim 5** The method according to any one of claims 1 to 4, wherein step b) comprises contacting the one or more first stem cell populations with the agent for at least about 4 days. **Claim 6** The method according to any one of claims 1 to 5, wherein the one or more first cell populations and the second stem cell population are induced pluripotent stem cells (iPSCs). **Claim 7** The method according to any one of claims 1 to 6, wherein the one or more first stem cell populations and the second stem cell population are human cells. **Claim 8** The method according to any one of claims 1 to 7, wherein the inducer is doxycycline. **Claim 9** The method according to any one of claims 1 to 8, wherein the introduced gene encoding the GATA6 polypeptide is at least about 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 3 to 10 or a fragment thereof.

10. The method according to any one of claims 1 to 8, wherein the one or more first stem cell populations comprise one or more nucleic acid sequences that are at least about 80% identical to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or a fragment thereof.

11. The method according to any one of claims 1 to 10, wherein the second stem cell population comprises one or more nucleic acid sequences that are at least about 80% identical to a sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 14 or a fragment thereof.

12. The method according to any one of claims 1 to 10, wherein the second stem cell population further comprises a nucleic acid sequence that is at least about 80% identical to SEQ ID NO: 15 or a fragment thereof.

13. The method according to any one of claims 4 to 12, wherein the third stem cell population comprises a nucleic acid sequence that is at least about 80% identical to SEQ ID NO: 15 or a fragment thereof.

14. The method according to any one of claims 1 to 13, wherein step c) comprises mixing the one or more first stem cell populations and the second stem cell population at a ratio of about 1:10 to about 1:

4.

15. The method according to any one of claims 1 to 14, wherein the cell mass comprises one or more of a bilayer disk-like structure, an amnion-like domain, a primitive streak-like domain, and a yolk sac domain.

16. The method according to any one of claims 1 to 15, wherein the cell mass comprises one or more neural features.

17. The method according to claim 16, wherein the one or more neural features comprise one or more of an increased level of the cerberus 1 (CER1) polypeptide and the left-right determinant 1 (LEFTY1) polypeptide.

18. The method according to any one of claims 1 to 17, wherein the embryonic cell structure comprises an increased level of the octamer-binding transcription factor 4 (OCT4) polypeptide and a decreased level of the GATA4 polypeptide.

19. The method according to any one of claims 1 to 18, wherein the extraembryonic cell structure comprises an increased level of the GATA6 polypeptide.

20. The method according to claim 15, wherein the amniotic-like domain comprises one or more of increased levels of bone morphogenetic protein 4 (BMP4), bone morphogenetic protein receptor 1A (BMPR1A), distal-less homeobox 5 (DLX5), follistatin-like 1 (FSTL1), DNA binding inhibitor 1 (ID1), lymphocyte enhancer-binding factor 1 (LEF1), msh homeobox (MSX) 1, MSX2, SMAD family member 1 (SMAD1), and SMAD-specific E3 ubiquitin protein ligase 2 (SMURF2).

21. A method for evaluating an agent for regulating embryonic development, comprising: generating a cell mass comprising one or more embryonic cell structures and one or more extra-embryonic cell structures by the method according to any one of claims 1 to 20; contacting the cell mass with the agent; The method, comprising:

22. A cell mass produced by the method according to any one of claims 1 to 20, the cell mass comprising one or more embryonic cell structures and one or more extra-embryonic cell structures.

23. The cell mass according to claim 22, comprising one or more of a bilayer disc-like structure, an amniotic-like domain, a primitive streak-like domain, and a yolk sac domain.

24. The cell mass according to claim 22 or 23, comprising one or more neural characteristics.

25. The cell mass according to claim 24, wherein the one or more neural characteristics comprise one or more of increased levels of cerberus 1 (CER1) polypeptide and lefty 1 (LEFTY1) polypeptide.

26. A method for generating hematopoietic stem cells, comprising: generating a cell mass comprising one or more embryonic cell structures and one or more extra-embryonic cell structures by the method according to any one of claims 1 to 20; isolating the hematopoietic stem cells from the cell mass; The method, comprising:

27. The method according to claim 26, wherein the hematopoietic stem cells have increased expression of one or more of CD45, CD11b, and CD34.

28. The method according to claim 26, further comprising administering the hematopoietic stem cells to a subject.

29. An in vitro or ex vivo culture system for generating a cell mass comprising one or more embryonic cell structures and one or more extra-embryonic cell structures, comprising: one or more first stem cell populations comprising an inducible transgene encoding a GATA binding protein 6 (GATA6) polypeptide; a second stem cell population; The in vitro or ex vivo culture system comprising the same.

30. The in vitro or ex vivo culture system according to claim 29, wherein the second stem cell group does not have the inducible transgene encoding the GATA6 polypeptide.

31. The in vitro or ex vivo culture system according to claim 29 or 30, wherein the stem cells of the second group comprise an inducible transgene encoding an ETS variant transcription factor 2 (ETV2) polypeptide.

32. The in vitro or ex vivo culture system according to any one of claims 29 to 31, further comprising a third cell group, wherein the third cell group comprises an inducible transgene encoding an ETS variant transcription factor 2 (ETV2) polypeptide.

33. The in vitro or ex vivo culture system according to claim 29, wherein the first cell group and the second cell group are induced pluripotent stem cells (iPSCs).

34. The in vitro or ex vivo culture system according to any one of claims 29 to 33, wherein the one or more first cell groups and second cell groups are human cells.

35. The in vitro or ex vivo culture system according to any one of claims 29 to 34, wherein the inducible transgene is a doxycycline-inducible transgene.

36. The in vitro or ex vivo culture system according to any one of claims 29 to 35, wherein the transgene encoding the GATA6 polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs: 3 to 10 or a sequence at least about 80% identical to a fragment thereof.

37. The in vitro or ex vivo culture system according to any one of claims 29 to 35, wherein the one or more first stem cell groups comprise a nucleic acid sequence at least about 80% identical to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or a fragment thereof.

38. The in vitro or ex vivo culture system according to any one of claims 29 to 37, wherein the second stem cell group comprises one or more nucleic acid sequences at least about 80% identical to a sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 14 or a fragment thereof.

39. The in vitro or ex vivo culture system according to any one of claims 29 to 37, wherein the second stem cell group further comprises a nucleic acid sequence at least about 80% identical to SEQ ID NO: 15 or a fragment thereof.

40. The in vitro or ex vivo culture system according to any one of claims 32 to 37, wherein the third stem cell group comprises a nucleic acid sequence that is at least about 80% identical to SEQ ID NO: 15 or a fragment thereof.

41. The in vitro or ex vivo culture system according to any one of claims 29 to 40, wherein the one or more first stem cell groups and the second stem cell group are mixed in a ratio of about 1:10 to about 1:

4.

42. A method for generating an extracellular matrix, comprising: a) obtaining one or more first stem cell groups comprising an inducible transgene encoding a GATA-binding protein 6 (GATA6) polypeptide; b) contacting the one or more first stem cell groups with an inducer that induces expression of the transgene; c) mixing the one or more first stem cell groups of step b) with a second stem cell group; d) culturing the cell mixture of step c) on a surface for at least 3 days to produce cell aggregates; e) isolating the extracellular matrix from the cell aggregates of step d). The method as described above.

43. An in vitro culture system comprising the extracellular matrix generated by the method according to claim 42.

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