Methods for generating human amniotic membrane and uses thereof

Abstract

The technology described herein is directed to methods for producing an amnion composition or amnioid from at least one human stem cell or at least one human induced pluripotent stem cell. Further described herein are compositions produced by the methods described herein and cultures thereof, as well as screening methods.

Description

METHODS FOR GENERATING HUMAN AMNIOTIC MEMBRANE AND USESTHEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63 / 472,703, filed June 13, 2023, incorporated herein by reference in its entirety.GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant number AR075018, awarded by The National Institutes of Health. The government has certain rights in the invention.SEQUENCE LISTINGThe sequence listing submitted on June 13, 2024, as an .XML file entitled “11624-002W01 _ST26.xml” created on June 12, 2024, and having a file size of 15,429 bytes is, hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).TECHNICAL FIELD

[0003] The technology described herein relates to methods for producing human amniotic membrane compositions and their uses thereof.BACKGROUND

[0004] The amniotic sac protects the developing embryo from physical assaults and regulates fluid exchange between fetus and the mother. During embryogenesis, the amniotic membrane, or amnion, develops adjacent to the surface ectoderm and is closely associated with the extraembryonic mesoderm. The formation of amnion plays as essential role in normal versus abnormal pregnancy, yet the lack of an appropriate model hinders insights into this process in humans. In addition, the amnion are tissues rich in stem cells, growth factors, and structural proteins. As a result, amnion is used as grafts and dressings in surgeries to promote wound healing through epithelialization, reduction in pain, and minimization of inflammation. Methods for generating amnion would provide platforms to gain deeper insights into extraembryonic development and the surface ectoderm, to better understand pregnancy problems such as preclampsia and hydramnios, and to help in the reconstruction of injured tissues, e.g., cornea or tympanic membranes.SUMMARY

[0005] The technology described herein is an amnioid culture comprising: a. at least one human stem cell or at least one human induced pluripotent stem cell; and b. a Bone Morphogenetic Protein (BMP)-4 polypeptide, at least one inhibitor of Transforming Growth Factor Beta (TGFP), and / or a fibroblast growth factor (FGF) polypeptide.

[0006] Also disclosed herein, is an isolated three-dimensional amniotic membrane or amnioid comprising at least one of a unicellular / simple epithelium, at least one of a transparent membrane, at least one of a simple layer of mesenchymal cells, and a detectable level of at least one amnion marker.

[0007] Further disclosed herein is a method for producing a human amniotic membrane composition, the method comprising: a. culturing at least one human stem cell or at least one human induced pluripotent stem cell in a first cell culture medium comprising at least: a Bone Morphogenetic Protein (BMP)-4 polypeptide, at least one inhibitor of Transforming Growth Factor Beta (TGF0), and a fibroblast growth factor (FGF) polypeptide, for at least 1 day; b. contacting the cells resulting from step a) with a second cell culture medium comprising at least an antibiotic for 2 days; and c. contacting the cells resulting from step b) with Lateral Plate Mesoderm (LPM) for 6 days; and d. culturing the cells resulting from step c) in a third cell culture medium for at least 20 days, wherein the cell culture comprises a floating culture.

[0008] In some embodiments, the amnioid culture further comprises an antibiotic. In some embodiments, the culture media further comprises at least one Lateral Plate Mesoderm cell.

[0009] In one embodiment of any aspect described herein, the human stem cell or human induced pluripotent stem cell are provided for step a) at a concentration of less than 35,000 cells / mL. In one embodiment of any aspect described herein, the human stem cell or human induced pluripotent stem cell are provided for step a) at a concentration of no more than 20,000 cells / mL. In one embodiment of any aspect described herein, the human stem cell or human induced pluripotent stem cell are provided for step a) at a concentration of from 8,000 cells / mL to 20,000 cells / mL.

[0010] In one embodiment of any aspect described herein, the human stem cell or human induced pluripotent stem cell is first aggregated to 8,000 cells / mL - 20,000 cells / mL.

[0011] In one embodiment of any aspect described herein, the human stem cell or human induced pluripotent stem cell are provided for step a) as a cell aggregate at a concentration of less than 3,500 cells / aggregate. In one embodiment of any aspect described herein, the human stem cell or human induced pluripotent stem cell are provided for step a) as a cell aggregate at a concentration of no more than 2,000 cells / aggregate. In one embodiment of any aspect described herein, the human stem cell or human induced pluripotent stem cell are provided for step a) as a cell aggregate at a concentration of from 100 cell / aggregate to 2,000 cells / aggregate.

[0012] In one embodiment of any aspect described herein, the first cell culture medium further comprises at least one extracellular matrix material and / or at least one antibiotic.

[0013] In one embodiment of any aspect described herein, the first cell culture medium comprises a BMP-4 polypeptide, at least one inhibitor of TGFB, at least one extracellular matrix material, a FGF polypeptide, and at least one antibiotic.

[0014] In one embodiment of any aspect described herein, the cells are cultured in the first cell culture medium for 1-6 days. In one embodiment of any aspect described herein, the the cells are cultured in the first cell culture medium for 3 days.

[0015] In one embodiment of any aspect described herein, the first cell culture medium comprises the BMP4 polypeptide at 1-10 ng / mL. In one embodiment of any aspect described herein, the first cell culture medium comprises the BMP4 polypeptide at 5 ng / mL.

[0016] In one embodiment of any aspect described herein, the first cell culture medium comprises the at least one TGFP inhibitor at 1-20 uM. In one embodiment of any aspect described herein, the first cell culture medium comprises the at least one TGFP inhibitor at 10 M. In one embodiment of any aspect described herein, the at least one TGFP inhibitor is SB-431542.

[0017] In one embodiment of any aspect described herein, the first cell culture medium comprises the FGF polypeptide at 1-10 ng / mL, In one embodiment of any aspect described herein, the first cell culture medium comprises the FGF polypeptide at 4 ng / mL. In one embodiment of any aspect described herein, the FGF polypeptide is FGF2 (basic / bFGF).

[0018] In one embodiment of any aspect described herein, the second cell culture medium does not comprise a FGF polypeptide.

[0019] In one embodiment of any aspect described herein, the cells are cultured in the second cell culture medium for 1-6 days. In one embodiment of any aspect described herein, the cells are cultured in the second cell culture medium for 3 days.

[0020] In one embodiment of any aspect described herein, the second cell culture medium further comprises at least one BMP inhibitor.

[0021] In one embodiment of any aspect described herein, the cells are not contacted with a BMP inhibitor.

[0022] In one embodiment of any aspect described herein, the cells are contacted with LPM for 1-10 days. In one embodiment of any aspect described herein, the cells are contacted with LPM for 6 days.

[0023] In one embodiment of any aspect described herein, the third cell culture medium comprises a basal medium, a glutamine supplement, a neuronal culture supplement, a reducing agent, and an antibiotic. In one embodiment of any aspect described herein, the floating culture does not comprise basal medium.

[0024] In one embodiment of any aspect described herein, step d) is continued until the cells form a three-dimensional amniotic membrane composition comprising at least one of: a unicellular / simple epithelium, a transparent membrane, a simple layer of mesenchymal cells, and a detectable level of at least one amnion marker. In one embodiment of any aspect described herein, step d) is continued until the cells form a three-dimensional amniotic membrane composition comprising at least a unicellular / simple epithelium and a simple layer of mesenchymal cells. In one embodiment of any aspect described herein, step d) is continued until the cells form a three-dimensional amniotic membrane composition comprising: a unicellular / simple epithelium, a transparent membrane, a simple layer of mesenchymal cells and a detectable level of at least one amnion marker.

[0025] In one embodiment of any aspect described herein, the simple layer of mesenchymal cells expresses collagen markers selected from a group comprising but not limited to COL15A1, COL12A1, and / or COL3A1.

[0026] In one embodiment of any aspect described herein, the at least one amnion marker comprises KRT8, POSTN, KRT18, GABRP, IGFBP3, and / or VTCN1.

[0027] In one embodiment of any aspect described herein, the amniotic membrane composition comprises or is an amnioid.

[0028] In one embodiment of any aspect described herein, the amnioid has the capability of expanding in size to 0.1 cm- 10 cm. In one embodiment of any aspect described herein, theamnioid has the capability of expanding in size to at least 1 cm. In one embodiment of any aspect described herein, the amnioid has the capability of expanding in size to 4 cm.

[0029] In one embodiment of any aspect described herein, the amnioid is 0.1 cm- 10 cm in size. In one embodiment of any aspect described herein, the amnioid is at least 1 cm in size. In one embodiment of any aspect described herein, the amnioid is at least 4 cm in size.

[0030] In one embodiment of any aspect described herein is an amniotic membrane composition produced by the methods described herein.

[0031] In one embodiment of any aspect described herein is a composition comprising an amniotic membrane composition produced by the methods described herein.

[0032] Also disclosed herein is a method of screening one or more compounds to determine its / their effect upon amnion, amniotic fluid, or amniotic sac, the method comprising exposing the compound or compounds to the amnioid or amniotic membrane and determining the effect thereof.

[0033] In some embodiments, the compound(s) increase growth or expansion of the amnion, amniotic fluid, or amniotic sac and in some embodiments, the compound(s) decreases growth or expansion of the amnion, amniotic fluid, or amniotic sac. In some embodiments, the compound is compared to a control.

[0034] In some embodiments, more than one compound is used to determine their combined effect.

[0035] In some embodiments, the screening takes place in vitro.BRIEF DESCRIPTION OF THE DRAWINGS

[0036] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0037] FIG. l(A-C) shows schematics comparing in vivo and in vitro skin organogenesis. FIG. 1 A) A timeline of in vitro skin organogenesis in the skin organoid model. hPSCs form aggregates on day 0 of differentiation. These aggregates are treated with TGF-0 inhibitor (SB), BMP4 and a low concentration of bFGF, giving rise to surface ectoderm by day 3. The aggregates are then treated with BMP inhibitor (LDN) and a high concentration of bFGF. This day 3 treatment induces the development of non-epithelial cell populations consisting, in part, of cranial neural crest (CNC) cells, which further differentiate into diverse mesenchymal and neuro-glial cell populations contributing to the dermis layer of the organoid. Although rare, hair germs can be seen as early as day 56 of differentiation, with more mature hair pegsand hair follicles arising around day 70 through day 130 of differentiation. The fully mature skin organoid includes appendages, such as hair follicles, adipocytes, melanocytes, sensory neurons, sebaceous glands and sensory neurons. FIG. IB) A schematic of neurulation in vivo. Neurulation occurs around 3-4 weeks of development, corresponding to days 6-18 of organoid culture. The ectoderm folds and pinches inward, resulting in the formation of the neural tube and, in the cranial region, delaminating CNC cells. CNC cells give rise to diverse cell lineages, such as chondrocytes, fibroblasts, neurons and Schwann cells. FIG. 1C) A timeline of in vivo skin development. Following fertilization, the zygote undergoes many rounds of rapid division. By day 12, the epiblast (amniotic cavity) and hypoblast (yolk sac) compose the bilaminar disk. The epiblast gives rise to the definitive germ layers, the ectoderm, mesoderm and endoderm. In relation to the skin, the ectoderm gives rise to keratinocyte precursors (epidermis layer of skin), while the dermal fibroblasts (dermis layer) are derived primarily from the mesoderm in the body and CNCs in the face. By 6 weeks, melanocyte precursors, sensory neuron progenitors and other diverse cell progenitors appear. Hair germs, which develop into hair pegs and finally into hair follicles, start to form around 12 weeks of gestation. Fully stratified skin with erupted lanugo hairs is reached around week 18 of gestation. The skin further matures and develops diverse appendages, such as blood vessels, sweat glands, sebaceous glands and a network of sensory neurons (thermoreceptors, mechanoreceptors and nociceptors).

[0038] FIG. 2A-B shows the morphologies of developing skin organoids during differentiation. Representative differential interference contrast (DIC) (d(-2)-d90) images of skin organoids derived from DSP-GFP (WTC-11) hiPSCs. Representative images are taken at 50x (5x microscope objective x lOx eyepiece) and lOOx (1 Oxmicroscope objective x lOx eyepiece) magnifications as noted in each image. Scale bars, 500 pm. Day 55 images showing TFAP2A+KRT5+ and KRT17+KRT5+ basal layer, TFAP2A+ intermediate layer and KRT17+ periderm layer; the periderm layer is detectable only at initial stages of differentiation (A) prior to formation of the granular and cornified layers; the periderm is visible in organoids around days 25-60 of differentiation (B) .

[0039] FIG. 3A-C shows optimal timepoints to check differentiation and characterization of the skin organoid structure. Representative DIC images of skin organoids at d70-d90 when hair follicle formation begins (A, B). Day 75 high-magnification images representing PCAD+ hair placodes, PCAD+EDAR+LHX2-I- hair germs and PCAD+LHX2+ hair pegs; SOX2+ cells represent dermal condensates of hair germs and dermal papillae of hair pegs. Arepresentative IHC image of a day 140 skin organoid (C). The endogenous green fluorescence from the DSP-GFP cell line visualizes epithelium of the skin cyst in the center and the hair follicles protruding from the surface of the cyst. TUJ1+ neurons are wrapping around and innervating the epithelium and the hair follicles.

[0040] FIG. 4 shows a schematic of the challenges faced to with generating amnion and amnioids.

[0041] FIG. 5A-C shows the effect of day 3 LDN / bFGF treatment in determining the trajectory of differentiation (A). Without treatment of LDN / bFGF after day 3, the cells are fated towards unicellular epithelia instead of hair germs and hair follicles (B, C).

[0042] FIG. 6A-B shows the effect of concentration of BMP4 and the effect of the starting aggregate size on the growth and differentiation toward amnion (A, B).

[0043] FIG. 7 shows a schematic demonstrating the origins and development of various cell types.

[0044] FIG. 8A-B shows the differentiation path of cells when Lateral Plate Mesoderm (LPM) is added around day 3 (A). Cells begin to differentiate into migrating mesenchymal cells (B).

[0045] FIG. 9 shows single cell RNAseq (ScRNASeq) of day 6 lateral mesoderm organoids.

[0046] FIG. 10A-B shows a representative image of surface ectoderm cells being fused with lateral plate mesoderm (A, B is schematic).

[0047] FIG. 11A-C shows the size and growth of the lateral mesoderm organoids and amnioids after 27-96 days without confinement (A-C).

[0048] FIG. 12A-B shows the size and growth of the lateral mesoderm organoids and amnioids after 27-96 days of confinement (A, B is schematic).

[0049] FIG. 13 shows representative images of the stages of amnioid development.

[0050] FIG. 14A-B shows that amnioids express morphology similar to amniotic membrane and placenta (A, B).

[0051] FIG. 15 shows scRNAseq analysis revealing amnion markers.

[0052] FIG. 16 shows scRNAseq analysis revealing amnion markers of day 6 amnion or skin organoids developed under different conditions.

[0053] FIG. 17 shows scRNAseq analysis of day 6 and 28 amnion or skin developed under different conditions.

[0054] FIG. 18 shows scRNAseq analysis revealing amnion markers of day 6 and 28 amnion or skin developed under different conditions. Amnioids contain an upregulation of reported amnion markers.

[0055] FIG. 19 shows scRNAseq analysis of day 6 and 28 amnion or skin developed under different conditions. Amnioids have an absence of markers identifying periderm and basal epidermis.

[0056] FIG. 20 shows scRNAseq analysis of day 6 and 28 amnion or skin developed under different conditions revealing other markers specific for differentiation path.

[0057] FIG. 21 shows representative immunofluorescent images demonstrating amnion markers present in amnioids compared to skin and placenta.

[0058] FIG. 22 shows representative immunofluorescent images demonstrating amnion markers present in amnioids.

[0059] FIG. 23 shows representative immunofluorescent images demonstrating amnion markers present in amnioids compared to skin organoids.

[0060] FIG. 24 shows a representative image of a subset of KRT8+ cells in skin organoids.

[0061] FIG. 25 shows a schematic demonstrating the continuation of amnion epithelia and non-neural ectoderm during development.

[0062] FIG. 26A-C shows a schematic demonstrating proximity between embryonic and extraembryonic mesoderm (A-C, showing different views).

[0063] FIG. 27A-B shows a comparison between amnioids and skin organoids at different developmental stages in the presence or absence of LDN and LPM (A-B, B is schematic).

[0064] FIG. 28 shows a schematic demonstrating mechanical forces playing a role to direct fate commitment.

[0065] FIG. 29 shows the expression of mechanotransduction related, ion-channel related, immune cell related, and cell-cell adhesion genes in skin organoids and amnioids.

[0066] FIG. 30 shows scRNAseq analysis of day 6 and 28 amnion or skin developed under different conditions revealing YAP targets expression.

[0067] FIG. 31 shows a schematic demonstrating mechanical that may play a role in fate commitment.

[0068] FIG. 32 shows representative immunofluorescent images demonstrating beta-catenin, YAP-1, and KRT8 / 18 expression in day 93 skin-like and amnion-like fusion cells.

[0069] FIG. 33 shows that forskolin can rescue deflated amnioids.

[0070] FIG. 34 shows amnion-on- a-chip as a model to recapitulate epithelia-mesenchyme interaction.

[0071] FIG. 35 A-G shows amnioid development. FIG. 35A shows a schematic of Amnion and Epidermis development in embryos with relevant cell populations. FIG. 35B shows a schematic of Strategy to generate Organoids representing Amnioids. FIG. 35C shows bright- field representative of Amnioids morphology and size over time. FIG. 35D shows examples of Amnioids (Bright-field) cultured on 6-well plate. FIG. 35E shows examples of Amnioids growing on different floating platforms (6-well plate and 500ml Conical Flask). Amnioids can grow into gigantic sizes which cannot be seen in any other in vitro amniotic membrane generated in the lab. FIG. 35F shows quantification of size of Amnioid compared to Amnion epithelia (without LPM assembly on d6) and Skin Organoids (cSKO-3500) published in previous paper. FIG. 35G shows the quantification of Hair Follicle Production. Amnioid does not develop Hair Follicle.

[0072] FIG. 36A-C shows protocol optimization and comparison of organoid size at d29- d33. AdvDMEM is GlutaMAX + Norm + beta-mercaptoethanol, No B27 & N2 and No Neural basal. LPM basal medium is DMEM / F12 + 1% ITS + GlutaMAX + Normocin.

[0073] FIG. 37 shows expression of Amnion epithelia (KRT8 / 18) and Amnion fibroblast (COL15A1) in Amnioids as distinct markers.

[0074] FIG. 38A-E shows amnion morphology. FIG. 38A shows expression of Amnion epithelia markers (KRT8 / 18) in Amnioids in various timepoints. FIG. 38B shows expression of Amnion epithelia markers (KRT8 / 18) across various culture conditions. Prolonged KRT8 / 18 was not observed in cSKOs. FIG. 38C shows further validation of Amnion marker with IGFBP3 (RNA-Probes) and COL3A1 (Extraembryonic mesenchyme - Amnion mesenchyme marker). FIG. 38D shows comparison with Fetal samples. FIG. 38E shows the expression of GATA4 - extraembryonic mesoderm markers in the Amnioids.

[0075] FIG. 39 shows the epidermis stratification in the Amnioids compared to SKOs (lack of expression of keratinized layer).

[0076] FIG. 40 compares the top 10 Ion Channel genes for AE, AE-Epi, and Skin-Epi in Amnioids versus Skin Organoids.

[0077] FIG. 41 compares the top 10 Ion Channel genes for AE, AE-Epi, and Skin-Epi. FIG. 41 is a heatmap for scores that are standardized log-transformed average expression in each cell type for Ion Channel genes.

[0078] FIG. 42 shows the role of mesenchyme cells on amnion epithelia cell fate and variation of amnioid protocol to have different mesenchyme. Changing the time point (d38- d40) of combination of LPM does not change the phenotype but changes the efficiency of generating Amnioid. Changing the signaling to the LPM (adding CHIR, FGF8, not adding LPM at all - amnion epithelia group) changes the phenotype of the Amnioid.

[0079] FIG. 43 further shows how changing the signaling to the LPM (adding CHIR, FGF8, not adding LPM at all - amnion epithelia group) changes the phenotype of the Amnioid.DETAILED DESCRIPTION

[0080] The amniotic membrane is critical for healthy development of an embryo and has also been shown to act as a highly biocompatible natural scaffold and as a source of several types of stem cells and potent growth factors for uses in regenerative medicine. There is a currently a lack of an appropriate model of an amnion, and a lack of a method of producing an amnion ex-vivo from stem cells or from induced pluripotent stem cells. Described herein are methods for producing an amniotic membrane or amnion-like organoid from stem cells or induced pluripotent stem cells, e.g., for use in development basic research, regeneration materials, drug discovery, or gene therapy testing applications. In some embodiments, the amniotic membrane or amnion-like organoid is human and / or comprises human cells.

[0081] As used herein, “amniotic membrane” or “amnion” refers to a thin, cellular, extra- embryonic membrane that forms the inner membrane of a closed sac surrounding and protecting an embryo in reptiles, birds, and mammals. The sac contains the fetus and amniotic fluid, in which the embryo is immersed, nourished and protected. Typically, the amnion is a tough, transparent, nerve-free, and nonvascular membrane consisting of two layers of cells: an inner, single-cell-thick layer of ectodermal epithelium and an outer covering of mesodermal, connective, and specialized smooth muscular tissue. In some embodiments, an amniotic membrane composition as described herein comprises a single- cell-thick layer of ectodermal epithelium. In some embodiments, an amniotic membrane composition as described herein comprises mesodermal, connective, and / or specialized smooth muscular tissue. In some embodiments, an amniotic membrane composition as described herein comprises a single-cell-thick layer of ectodermal epithelium and a layer of mesodermal, connective, and / or specialized smooth muscular tissue. In some embodiments, an amniotic membrane composition as described herein comprises a single-cell-thick layer ofectodermal epithelium and a layer of mesodermal, connective, and specialized smooth muscular tissue.

[0082] An “amnioid” is an amnion organoid, that is 3-dimensional tissue which does not have the full size or dimensions of an amnion, but exhibits the key structural characteristics of an amnion, e.g., a single-cell-thick layer of ectodermal epithelium and a layer of mesodermal, connective, and / or specialized smooth muscular tissue.

[0083] As used herein, the term "stem cell" refers to an undifferentiated cell that can be induced to proliferate. The stem cell is capable of self-maintenance or self-renewal, meaning that with each cell division, one daughter cell will also be a stem cell. Stem cells can be obtained from embryonic, post-natal, juvenile, or adult tissue. Stem cells can be pluripotent or multipotent. The term "progenitor cell," as used herein, refers to an undifferentiated cell derived from a stem cell, and is not itself a stem cell. Some progenitor cells can produce progeny that are capable of differentiating into more than one cell type. Stem cells include pluripotent stem cells, which can form cells of any of the body's tissue lineages: mesoderm, endoderm and ectoderm. Therefore, for example, stem cells can be selected from a human embryonic stem (ES) cell; a human inner cell mass (lCM) / epiblast cell; a human primitive ectoderm cell, a human primitive endoderm cell; a human primitive mesoderm cell; and a human primordial germ (EG) cell. Stem cells also include multipotent stem cells, which can form multiple cell lineages that constitute an entire tissue or tissues, such as but not limited to hematopoietic stem cells or neural precursor cells. Stem cells also include totipotent stem cells, which can form an entire organism. In some embodiment, the stem cell is a mesenchymal stem cell. The term "mesenchymal stem cell" or "MSC" is used interchangeably for adult cells which are not terminally differentiated, which can divide to yield cells that are either stem cells, or which, irreversibly differentiate to give rise to cells of a mesenchymal cell lineage, e.g., adipose, osseous, cartilaginous, elastic and fibrous connective tissues, myoblasts) as well as to tissues other than those originating in the embryonic mesoderm (e.g., neural cells) depending upon various influences from bioactive factors such as cytokines. In some embodiments, the stem cell is a partially differentiated or differentiating cell.

[0084] In some embodiments, the stem cell is an induced pluripotent stem cell (iPSC), which has been reprogrammed or de-differentiated. A “induced pluripotent stem cell” refers to a somatic (e.g., mature or adult) cell that has been reprogrammed to an embryonic stem celllike state by being forced to express genes and factors important for maintaining the definingproperties of embryonic stem cells (see, e.g., Takahashi and Yamanaka, Cell 126 (4): 663-76, 2006, incorporated by reference herein). Induced pluripotent stem cell cells express stem cell markers and are capable of generating cells characteristic of all three germ layers (ectoderm, endoderm, mesoderm). Stem cells can be obtained from embryonic, fetal or adult tissues.

[0085] The term “reprogramming” as used herein refers to a process that alters or reverses the differentiation state of a differentiated cell (e.g., a somatic cell). Stated another way, reprogramming refers to a process of driving the differentiation of a cell backwards to a more undifferentiated or more primitive type of cell. The cell to be reprogrammed can be either partially or terminally differentiated prior to reprogramming. In some embodiments, reprogramming encompasses complete reversion of the differentiation state of a differentiated cell (e.g., a somatic cell) to a pluripotent state. In some embodiments, reprogramming also encompasses partial reversion of the differentiation state of a differentiated cell (e.g., a somatic cell) to a multipotent state. In some embodiments, reprogramming encompasses complete or partial reversion of the differentiation state of a differentiated cell (e.g., a somatic cell) to an undifferentiated cell. Reprogramming also encompasses partial reversion of the differentiation state of a somatic cell to a state that renders the cell more susceptible to complete reprogramming to a pluripotent state when subjected to additional manipulations.

[0086] Reprogramming involves alteration, e.g., reversal, of at least some of the heritable patterns of nucleic acid modification (e.g., methylation), chromatin condensation, epigenetic changes, genomic imprinting, etc., that occur during cellular differentiation as a zygote develops into an adult.

[0087] The term “derived from,” used in reference to a stem cell means the stem cell was generated by reprogramming of a differentiated cell to a stem cell phenotype. The term “derived from,” used in reference to a differentiated cell means the cell is the result of differentiation, e.g., in vitro differentiation, of a stem cell.

[0088] As used herein, a “cell culture” refers to an in vitro population of cells having a population of metabolically active cells. The number of these cells can be roughly stable over a period of at least 3 days or can grow. As used herein, “culturing” refers to continuing the viability of a cell or population of cells. In some embodiments of any of the aspects, the phenotype, morphology, number, or differentiation status of the cultured cells can change over time. Conditions suitable for cell culture for different cell types are well known in the art and cell culture media for various cell types is readily available. Exemplary media and conditions are provided elsewhere herein.

[0089] In some embodiments, the cell culture is a 3-D cell culture. As used herein, a “3-D cell culture” or “3-D culture” refers to a cell culture in which the cells extend into 3 dimensions, e.g., the cell culture is not a monolayer but has a depth of more than 1 cell.

[0090] As used herein, the term “organoid” refers to a 3D multicellular tissue culture construct comprising cells differentiated in vitro from stem cells, including, but not limited to iPS cells. Organoids can but need not necessarily include a plurality of different cell types.

[0091] In one aspect described herein, are methods for producing an amniotic membrane or amnion-like organoid from stem cells or induced pluripotent stem cells, e.g., for use in development basic research, regeneration materials, drug discovery, or gene therapy testing applications. In some embodiments, the methods described herein are for producing a mammalian amniotic membrane or a mammalian amnion-like organoid from stem cells or induced pluripotent stem cells. In some embodiments, the methods described herein are for producing a human amniotic membrane or a human amnion-like organoid from stem cells or induced pluripotent stem cells. In some embodiments, the methods described herein are for producing a human amniotic membrane or a human amnion-like organoid from human stem cells or human induced pluripotent stem cells.

[0092] In one aspect of any of the embodiments, the method for producing an amniotic membrane composition comprises culturing at least one stem cell or at least one induced pluripotent stem cell (hIPSC) in a first cell culture medium comprising at least: one Bone Morphogenetic Protein (BMP)-4 polypeptide, at least one inhibitor of Transforming Growth Factor Beta (TGFP), and a fibroblast growth factor (FGF) polypeptide then, then contacting the cells with a second cell culture medium comprising at least one antibiotic, then contacting the cells with Lateral Plate Mesoderm (LPM), and finally culturing the cells in a third cell culture comprising a floating culture.

[0093] In one aspect of any of the embodiments, described herein is a method for producing a amniotic membrane composition, the method comprising culturing or providing stem cells or induced pluripotent stem cells to a concentration of less than 35,000 cells / mL then contacting the cells with a first cell culture medium comprising at least a BMP-4 polypeptide, at least one inhibitor of TGF[>, and a FGF polypeptide. In some embodiments the cells are cultured to or provided at a concentration of less than 50,000 cells / mL, less than 45,000 cells / mL, less than 40,000 cells / mL, less than 35,000 cells / mL, less than 30,000 cells / mL, less than 25,000 cells / mL, less than 20,000 cells / mL, less than 15,000 cells / mL, less than 10,000 cells / mL, less than 5,000 cells / mL or less. In some embodiments, the cells arecultured to or provided at a concentration of 1,000 cells / mL-50,000 cells / mL, a concentration of 2,000 cells / mL-45,000 cells / mL, to a concentration of 3,000 cells / mL-40,000 cells / mL, a concentration of 4,000 cells / mL-35,000 cells / mL, a concentration of 5,000 cells / mL-30,000 cells / mL, a concentration of 6,000 cells / mL-25,000 cells / mL, a concentration of 7,000 cells / mL-20,000 cells / mL, a concentration of 8,000 cells / mL- 15,000 cells / mL, or a concentration of 9,000 cells / mL- 10,000 cells / mL.

[0094] In one aspect of any of the embodiments, described herein is a method for producing an amniotic membrane composition, the method comprising first culturing or providing stem cells or induced pluripotent stem cells to a concentration of 8,000 cells / mL-20,000 cells / mL to form a cell aggregate then contacting the aggregate with a first cell culture medium comprising at least a BMP-4 polypeptide, at least one inhibitor of TGFp, and a FGF polypeptide.

[0095] In one aspect of any of the embodiments, described herein is a method for producing a amniotic membrane composition, the method comprising culturing and providing stem cells or induced pluripotent stem cells that have been cultured to an aggregate at a concentration of less than 3,500 cells / aggregate then contacting the cells with a first cell culture medium comprising at least a BMP-4 polypeptide, at least one inhibitor of TGF|3, and a FGF polypeptide. In some embodiments the cells are cultured to and / or provided as an aggregate cultured to a concentration of less than 5,000 cells / aggregate, less than 4,500 cells / aggregate, less than 4,000 cells / aggregate, less than 3,500 cells / aggregate, less than 3,000 cells / aggregate, less than 2,500 cells / aggregate, less than 2,000 cells / aggregate, less than 1,500 cells / aggregate, less than 1,000 cells / aggregate, less than 500 cells / aggregate, less than 100 cells / aggregate, or less. In some embodiments, the cells are cultured to an aggregate and / or provided as an aggregate at a concentration of 100 cells / aggregate -5,000 cells / aggregate, a concentration of 200 cells / aggregate -4,500 cells / aggregate, to a concentration of 300 cells / aggregate -4,000 cells / aggregate, a concentration of 400 cells / aggregate -3,500 cells / aggregate, a concentration of 500 cells / aggregate -3,000 cells / aggregate, a concentration of 600 cells / aggregate-2,500 cells / aggregate, a concentration of 700 cells / aggregate -2,000 cells / aggregate, a concentration of 800 cells / aggregate -1,500 cells / aggregate, or a concentration of 900 cells / aggregate -1 ,000 cells / aggregate.

[0096] In one aspect of any of the embodiments, described herein is a method for producing an amniotic membrane composition, the method comprising first culturing stem cells or induced pluripotent stem cells to an aggregate a concentration of 100 cells / aggregate -2,000cells / aggregate then contacting the aggregate with a first cell culture medium comprising at least a BMP-4 polypeptide, at least one inhibitor of TGFp, and a FGF polypeptide.

[0097] In some embodiments, one or more BMP polypeptides or polypeptide fragments are used to promote a particular differentiation step of a pluripotent cell. In some embodiments, the BMP polypeptide is BMP4 or an active fragment of BMP4. BMP4 is a protein involved in a signal transduction pathway inducing the differentiation of pluripotent stem cells into mesodermal cells.

[0098] In some embodiments, the BMP4 polypeptide is present in the first cell culture medium at a final concentration of at least 5 ng / mL. In one embodiment, the BMP4 polypeptide is present in the first cell culture medium at a final concentration of at least 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, or more.

[0099] In some embodiments, the BMP4 polypeptide is a human BMP4 polypeptide. In some embodiments, the BMP4 polypeptide comprises, consists of, or consists essentially of a wildtype human BMP4 polypeptide sequence. BMP4 sequences are known in the art for a number of species, e.g., human BMP4 (NCBI Gene ID: 652) mRNA (e.g., isoform a: NCBI Ref Seq NM_001202.6, SEQ ID NO: 1) and polypeptide (e.g., isoform a: NCBI Ref Seq NP_001193.2, SEQ ID NO: 2). BMP4 can refer to human BMP4, including naturally occurring variants, molecules, and alleles thereof. BMP4 can refer to the mammalian BMP4, e.g., mouse, rat, rabbit, dog, cat, cow, horse, pig, and the like. The nucleic sequence of SEQ ID NO:1 comprises the nucleic sequence which encodes BMP4.

[0100] SEQ ID NO: 1 is a nucleic acid sequence that encodes isoform a of human BMP4.1 ggaagatgcg agaaggcaga ggaggaggga gggagggaag gagcgcggag cccggcccgg 61 aagctaggtg agtgtggcat ccgagctgag ggacgcgagc ctgagacgcc gctgctgctc 121 cggctgagta tctagcttgt ctccccgatg ggattcccgt ccaagctatc tcgagcctgc 181 agcgccacag tccccggccc tcgcccaggt tcactgcaac cgttcagagg tccccaggag 241 ctgctgctgg cgagcccgct actgcaggga cctatggagc cattccgtag tgccatcccg 301 agcaacgcac tgctgcagct tccctgagcc tttccagcaa gtttgttcaa gattggctgt 361 caagaatcat ggactgttat tatatgcctt gttttctgtc aagacaccat gattcctggt 421 aaccgaatgc tgatggtcgt tttattatgc caagtcctgc taggaggcgc gagccatgct 481 agtttgatac ctgagacggg gaagaaaaaa gtcgccgaga ttcagggcca cgcgggagga 541 cgccgctcag ggcagagcca tgagctcctg cgggacttcg aggcgacact tctgcagatg 601 tttgggctgc gccgccgccc gcagcctagc aagagtgccg tcattccgga ctacatgcgg 661 gatctttacc ggcttcagtc tggggaggag gaggaagagc agatccacag cactggtctt 721 gagtatcctg agcgcccggc cagccgggcc aacaccgtga ggagcttcca ccacgaagaa 781 catctggaga acatcccagg gaccagtgaa aactctgctt ttcgtttcct ctttaacctc 841 agcagcatcc ctgagaacga ggtgatctcc tctgcagagc ttcggctctt ccgggagcag901 gtggaccagg gccctgattg ggaaaggggc ttccaccgta taaacattta tgaggttatg 961 aagcccccag cagaagtggt gcctgggcac ctcatcacac gactactgga cacgagactg 1021 gtccaccaca atgtgacacg gtgggaaact tttgatgtga gccctgcggt ccttcgctgg 1081 acccgggaga agcagccaaa ctatgggcta gccattgagg tgactcacct ccatcagact 1141 cggacccacc agggccagca tgtcaggatt agccgatcgt tacctcaagg gagtgggaat 1201 tgggcccagc tccggcccct cctggtcacc tttggccatg atggccgggg ccatgccttg 1261 acccgacgcc ggagggccaa gcgtagccct aagcatcact cacagcgggc caggaagaag 1321 aataagaact gccggcgcca ctcgctctat gtggacttca gcgatgtggg ctggaatgac 1381 tggattgtgg ccccaccagg ctaccaggcc ttctactgcc atggggactg cccctttcca 1441 ctggctgacc acctcaactc aaccaaccat gccattgtgc agaccctggt caattctgtc 1501 aattccagta tccccaaagc ctgttgtgtg cccactgaac tgagtgccat ctccatgctg 1561 tacctggatg agtatgataa ggtggtactg aaaaattatc aggagatggt agtagaggga 1621 tgtgggtgcc gctgagatca ggcagtcctt gaggatagac agatatacac accacacaca 1681 cacaccacat acaccacaca cacacgttcc catccactca cccacacact acacagactg 1741 cttccttata gctggacttt tatttaaaaa aaaaaaaaaa aaaggaaaaa atccctaaac 1801 attcaccttg accttattta tgactttacg tgcaaatgtt ttgaccatat tgatcatata 1861 ttttgacaaa atatatttat aactacgtat taaaagaaaa aaataaaatg agtcattatt 1921 ttaaaggtaa a

[0101] SEQ ID NO: 2 is an amino acid sequence of Isoform a of human BMP4.1 mipgnrmlmv vllcqvllgg ashaslipet gkkkvaeiqg haggrrsgqs hellrdfeat61 llqmfglrrr pqpsksavip dymrdlyrlq sgeeeeeqih stgleyperp asrantvrsf121 hheehlenip gtsensafrf Ifnlssipen evissaelrl freqvdqgpd wergfhrini181 yevmkppaev vpghlitrll dtrlvhhnvt rwetfdvspa vlrwtrekqp nyglaievth241 Ihqtrthqgq hvrisrslpq gsgnwaqlrp llvtfghdgr ghaltrrrra krspkhhsqr301 arkknkncrr hslyvdfsdv gwndwivapp gyqafychgd cpfpladhln stnhaivqtl361 vnsvnssipk accvptelsa ismlyldeyd kvvlknyqem vvegcgcr

[0102] In some embodiments, the first cell culture medium comprises a TGF- b / Activin / Nodal signaling pathway inhibitor, e.g., SB-431542. Exemplary TGF- b / Activin / Nodal signaling pathway inhibitors include but are not limited to SB-431542, Galunisertib, A 83-01, RepSox, or LY364947. Additional TGF-b receptor inhibitors are known in the art.

[0103] In one embodiment, TGF-b / Activin / Nodal signaling pathway signaling inhibitor, e.g., SB-431542, is present in the first cell culture medium at a final concentration of at least 10 uM. In one embodiment, TGF-b / Activin / Nodal signaling pathway signaling inhibitor, e.g., SB-431542, is present in the first cell culture medium at a final concentration of at least 1 uM, 2 uM, 3 uM, 4 uM, 5 uM, 6 uM, 7 uM, 8 uM, 9 uM, 10 uM, 11 uM, 12 uM, 13 uM, 14 uM, 15 uM, 16 uM, 17 uM, 18 uM, 19 uM, 20 uM, or more.

[0104] In some embodiments, the first cell culture medium comprises a fibroblast growth factor (FGF) polypeptide, e.g., basic FGF (bFGF). Exemplary fibroblast growth factorsinclude but are not limited to bFGF or FGF2, acidic FGF (aFGF) or FGF1, FGF4, FGF5, FGF6, FGF3, FGF7, FGF10, FGF22, FGF8, FGF17, FGF18, FGF9, FGF16, FGF20, FGF19, FGF21, or FGF23. Additional fibroblast growth factors are known in the art.

[0105] In one embodiment, a fibroblast growth factor polypeptide, e.g., bFGF, is present in the cell culture medium at a final concentration of at least 4 ng / mL. In one embodiment, fibroblast growth factor polypeptide, e.g., bFGF, is present in the first cell culture medium at a final concentration of at least 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, or more.

[0106] In some embodiments, the FGF polypeptide is a human FGF polypeptide. In some embodiments, the FGF polypeptide comprises, consists of, or consists essentially of a wildtype human FGF polypeptide sequence. FGF sequences are known in the art for a number of species, e.g., human FGF2 (NCBI Gene ID: 2247) mRNA (e.g., isoform 34kDa: NCBI Ref Seq NM_002006.6, SEQ ID NO: 3) and polypeptide (e.g., isoform 34kDa: NCBI Ref Seq NP_001997.5, SEQ ID NO: 4). FGF can refer to human FGF, including naturally occurring variants, molecules, and alleles thereof. FGF can refer to the mammalian FGF, e.g., mouse, rat, rabbit, dog, cat, cow, horse, pig, and the like. The nucleic sequence of SEQ ID NO: 3 comprises the nucleic sequence which encodes FGF2.

[0107] SEQ ID NO: 3 is a nucleic acid sequence that encodes isoform 34 kDa of human FGF2.1 gtggccgaac cgccgaactc agaggccggc cccagaaaac ccgagcgagt agggggcggc 61 gcgcaggagg gaggagaact gggggcgcgg gaggctggtg ggtgtggggg gtggagatgt 121 agaagatgtg acgccgcggc ccggcgggtg ccagattagc ggacgcggtg cccgcggttg 181 caacgggatc ccgggcgctg cagcttggga ggcggctctc cccaggcggc gtccgcggag 241 acacccatcc gtgaacccca ggtcccgggc cgccggctcg ccgcgcacca ggggccggcg 301 gacagaagag cggccgagcg gctcgaggct gggggaccgc gggcgcggcc gcgcgctgcc 361 gggcgggagg ctggggggcc ggggccgggg ccgtgccccg gagcgggtcg gaggccgggg 421 ccggggccgg gggacggcgg ctccccgcgc ggctccagcg gctcggggat cccggccggg 481 ccccgcaggg accatggcag ccgggagcat caccacgctg cccgccttgc ccgaggatgg 541 cggcagcggc gccttcccgc ccggccactt caaggacccc aagcggctgt actgcaaaaa 601 cgggggcttc ttcctgcgca tccaccccga cggccgagtt gacggggtcc gggagaagag 661 cgaccctcac atcaagctac aacttcaagc agaagagaga ggagttgtgt ctatcaaagg 721 agtgtgtgct aaccgttacc tggctatgaa ggaagatgga agattactgg cttctaaatg 781 tgttacggat gagtgtttct tttttgaacg attggaatct aataactaca atacttaccg 841 gtcaaggaaa tacaccagtt ggtatgtggc actgaaacga actgggcagt ataaacttgg 901 atccaaaaca ggacctgggc agaaagctat actttttctt ccaatgtctg ctaagagctg 961 attttaatgg ccacatctaa tctcatttca catgaaagaa gaagtatatt ttagaaattt 1021 gttaatgaga gtaaaagaaa ataaatgtgt atagctcagt ttggataatt ggtcaaacaa 1081 ttttttatcc agtagtaaaa tatgtaacca ttgtcccagt aaagaaaaat aacaaaagtt1141 gtaaaatgta tattctccct tttatattgc atctgctgtt acccagtgaa gcttacctag1201 agcaatgatc tttttcacgc atttgcttta ttcgaaaaga ggcttttaaa atgtgcatgt1261 ttagaaacaa aatttcttca tggaaatcat atacattaga aaatcacagt cagatgttta1321 atcaatccaa aatgtccact atttcttatg tcattcgtta gtctacatgt ttctaaacat1381 ataaatgtga atttaatcaa ttcctttcat agttttataa ttctctggca gttccttatg1441 atagagttta taaaacagtc ctgtgtaaac tgctggaagt tcttccacag tcaggtcaat1501 tttgtcaaac ccttctctgt acccatacag cagcagccta gcaactctgc tggtgatggg1561 agttgtattt tcagtcttcg ccaggtcatt gagatccatc cactcacatc ttaagcattc1621 ttcctggcaa aaatttatgg tgaatgaata tggctttagg cggcagatga tatacatatc1681 tgacttccca aaagctccag gatttgtgtg ctgttgccga atactcagga cggacctgaa1741 ttctgatttt ataccagtct cttcaaaaac ttctcgaacc gctgtgtctc ctacgtaaaa1801 aaagagatgt acaaatcaat aataattaca cttttagaaa ctgtatcatc aaagattttc1861 agttaaagta gcattatgta aaggctcaaa acattaccct aacaaagtaa agttttcaat1921 acaaattctt tgccttgtgg atatcaagaa atcccaaaat attttcttac cactgtaaat1981 tcaagaagct tttgaaatgc tgaatatttc tttggctgct acttggaggc ttatctacct2041 gtacattttt ggggtcagct ctttttaact tcttgctgct ctttttccca aaaggtaaaa2101 atatagattg aaaagttaaa acattttgca tggctgcagt tcctttgttt cttgagataa2161 gattccaaag aacttagatt catttcttca acaccgaaat gctggaggtg tttgatcagt2221 tttcaagaaa cttggaatat aaataatttt ataattcaac aaaggttttc acattttata2281 aggttgattt ttcaattaaa tgcaaatttg tgtggcagga tttttattgc cattaacata2341 tttttgtggc tgctttttct acacatccag atggtccctc taactgggct ttctctaatt2401 ttgtgatgtt ctgtcattgt ctcccaaagt atttaggaga agccctttaa aaagctgcct2461 tcctctacca ctttgctgga aagcttcaca attgtcacag acaaagattt ttgttccaat2521 actcgttttg cctctatttt tcttgtttgt caaatagtaa atgatatttg cccttgcagt2581 aattctactg gtgaaaaaca tgcaaagaag aggaagtcac agaaacatgt ctcaattccc2641 atgtgctgtg actgtagact gtcttaccat agactgtctt acccatcccc tggatatgct2701 cttgtttttt ccctctaata gctatggaaa gatgcataga aagagtataa tgttttaaaa2761 cataaggcat ttgtctgcca tttttcaatt acatgctgac ttcccttaca attgagattt2821 gcccataggt taaacatggt tagaaacaac tgaaagcata aaagaaaaat ctaggccggg2881 tgcagtggct catgcctata ttccctgcac tttgggaggc caaagcagga ggatcgcttg2941 agcccaggag ttcaagacca acctggtgaa accccgtctc tacaaaaaaa cacaaaaaat3001 agccaggcat ggtggcgtgt acatgtggtc tcagatactt gggaggctga ggtgggaggg3061 ttgatcactt gaggctgaga ggtcaaggtt gcagtgagcc ataatcgtgc cactgcagtc3121 cagcctaggc aacagagtga gactttgtct caaaaaaaga gaaattttcc ttaataagaa3181 aagtaatttt tactctgatg tgcaatacat ttgttattaa atttattatt taagatggta3241 gcactagtct taaattgtat aaaatatccc ctaacatgtt taaatgtcca tttttattca3301 ttatgctttg aaaaataatt atggggaaat acatgtttgt tattaaattt attattaaag3361 atagtagcac tagtcttaaa tttgatataa catctcctaa cttgtttaaa tgtccatttt3421 tattctttat gtttgaaaat aaattatggg gatcctattt agctcttagt accactaatc3481 aaaagttcgg catgtagctc atgatctatg ctgtttctat gtcgtggaag cactggatgg3541 gggtagtgag caaatctgcc ctgctcagca gtcaccatag cagctgactg aaaatcagca3601 ctgcctgagt agttttgatc agtttaactt gaatcactaa ctgactgaaa attgaatggg3661 caaataagtg cttttgtctc cagagtatgc gggagaccct tccacctcaa gatggatatt3721 tcttccccaa ggatttcaag atgaattgaa atttttaatc aagatagtgt gctttattct3781 gttgtatttt ttattatttt aatatactgt aagccaaact gaaataacat ttgctgtttt3841 ataggtttga agaacatagg aaaaactaag aggttttgtt tttatttttg ctgatgaaga3901 gatatgttta aatatgttgt attgttttgt ttagttacag gacaataatg aaatggagtt 3961 tatatttgtt atttctattt tgttatattt aataatagaa ttagattgaa ataaaatata 4021 atgggaaata atctgcagaa tgtgggtttt cctggtgttt ccctctgact ctagtgcact4081 gatgatctct gataaggctc agctgcttta tagttctctg gctaatgcag cagatactct4141 tcctgccagt ggtaatacga ttttttaaga aggcagtttg tcaattttaa tcttgtggat4201 acctttatac tcttagggta ttattttata caaaagcctt gaggattgca ttctattttc4261 tatatgaccc tcttgatatt taaaaaacac tatggataac aattcttcat ttacctagta4321 ttatgaaaga atgaaggagt tcaaacaaat gtgtttccca gttaactagg gtttactgtt4381 tgagccaata taaatgttta actgtttgtg atggcagtat tcctaaagta cattgcatgt4441 tttcctaaat acagagttta aataatttca gtaattctta gatgattcag cttcatcatt4501 aagaatatct tttgttttat gttgagttag aaatgccttc atatagacat agtctttcag4561 acctctactg tcagttttca tttctagctg ctttcagggt tttatgaatt ttcaggcaaa4621 gctttaattt acactaagct taggaagtat ggctaatgcc aacggcagtt tttttcttct4681 taattccaca tgactgaggc atatatgatc tctgggtagg tgagttgttg tgacaaccac4741 aagcactttt ttttttttta aagaaaaaaa ggtagtgaat ttttaatcat ctggacttta4801 agaaggattc tggagtatac ttaggcctga aattatatat atttggcttg gaaatgtgtt4861 tttcttcaat tacatctaca agtaagtaca gctgaaattc agaggaccca taagagttca4921 catgaaaaaa atcaatttat ttgaaaaggc aagatgcagg agagaggaag ccttgcaaac4981 ctgcagactg ctttttgccc aatatagatt gggtaaggct gcaaaacata agcttaatta5041 gctcacatgc tctgctctca cgtggcacca gtggatagtg tgagagaatt aggctgtaga5101 acaaatggcc ttctctttca gcattcacac cactacaaaa tcatctttta tatcaacaga5161 agaataagca taaactaagc aaaaggtcaa taagtacctg aaaccaagat tggctagaga5221 tatatcttaa tgcaatccat tttctgatgg attgttacga gttggctata taatgtatgt5281 atggtatttt gatttgtgta aaagttttaa aaatcaagct ttaagtacat ggacattttt5341 aaataaaata tttaaagaca atttagaaaa ttgccttaat atcattgttg gctaaataga5401 ataggggaca tgcatattaa ggaaaaggtc atggagaaat aatattggta tcaaacaaat5461 acattgattt gtcatgatac acattgaatt tgatccaata gtttaaggaa taggtaggaa5521 aatttggttt ctatttttcg atttcctgta aatcagtgac ataaataatt cttagcttat5581 tttatatttc cttgtcttaa atactgagct cagtaagttg tgttagggga ttatttctca5641 gttgagactt tcttatatga cattttacta tgttttgact acctgactat taaaaataaa5701 tagtagatac aattttcata aagtgaagaa ttatataatc actgctttat aactgacttt5761 attatattta tttcaaagtt catttaaagg ctactattca tcctctgtga tggaatggtc5821 aggaatttgt tttctcatag tttaattcca acaacaatat tagtcgtatc caaaataacc5881 tttaatgcta aactttactg atgtatatcc aaagcttctc attttcagac agattaatcc5941 agaagcagtc ataaacagaa gaataggtgg tatgttccta atgatattat ttctactaat6001 ggaataaact gtaatattag aaattatgct gctaattata tcagctctga ggtaatttct6061 gaaatgttca gactcagtcg gaacaaattg gaaaatttaa atttttattc ttagctataa6121 agcaagaaag taaacacatt aatttcctca acatttttaa gccaattaaa aatataaaag6181 atacacacca atatcttctt caggctctga caggcctcct ggaaacttcc acatattttt6241 caactgcagt ataaagtcag aaaataaagt taacataact ttcactaaca cacacatatg6301 tagatttcac aaaatccacc tataattggt caaagtggtt gagaatatat tttttagtaa6361 ttgcatgcaa aatttttcta gcttccatcc tttctccctc gtttcttctt tttttggggg6421 agctggtaac tgatgaaatc ttttcccacc ttttctcttc aggaaatata agtggttttg6481 tttggttaac gtgatacatt ctgtatgaat gaaacattgg agggaaacat ctactgaatt6541 tctgtaattt aaaatatttt gctgctagtt aactatgaac agatagaaga atcttacaga6601 tgctgctata aataagtaga aaatataaat ttcatcacta aaatatgcta ttttaaaatc6661 tatttcctat attgtatttc taatcagatg tattactctt attatttcta ttgtatgtgt6721 taatgatttt atgtaaaaat gtaattgctt ttcatgagta gtatgaataa aattgattag6781 tttgtgtttt cttgtctccc a

[0108] SEQ ID NO: 4 is an amino acid sequence of isoform 34 kDa of human FGF2.1 mvgvgggdve dvtprpggcq isgrgargcn gipgaaawea alprrrprrh psvnprsraa 61 gsprtrgrrt eerpsgsrlg drgrgralpg grlggrgrgr apervggrgr grgtaapraa 121 paargsrpgp agtmaagsit tlpalpedgg sgafppghfk dpkrlyckng gfflrihpdg 181 rvdgvreksd phiklqlqae ergvvsikgv canrylamke dgrllaskcv tdecffferl 241 esnnyntyrs rkytswyval krtgqyklgs ktgpgqkail flpmsaks

[0109] In some embodiments, the first cell culture medium does not contain a fibroblast growth factor polypeptide. In some embodiments, the second cell culture medium does not contain a FGF polypeptide. In some embodiments of any of the aspects, the cells are not contacted with exogenous FGF polypeptide during the time the cells are contacted with the second cell culture medium. In some embodiments, the third cell culture medium does not contain a fibroblast growth factor polypeptide. In some embodiments of any of the aspects, the cells are not contacted with exogenous FGF polypeptide during the time the cells are contacted with the third cell culture medium.

[0110] In some embodiments, the stem cells or IPSCs are contacted in a first cell culture medium comprising at least one BMP4, at least one TGF|3 inhibitor, and at least one FGF polypeptide for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or more.

[0111] In some embodiments, the step of culturing in the first cell culture medium is at least 1 day. In one embodiment, the step of culturing in the first cell culture medium is at least 2 days. In one embodiment, the step of culturing in the first cell culture medium is at least 3 days.

[0112] In some embodiments, the first cell culture medium further comprises at least one extracellular matrix material. Examples of extracellular matrix materials include but are not limited to collagen, fibronectin, fibrinogen, poly-lysine, vitronectin, laminin, elastin, tenascin, and Matrigel®. Other extracellular matrix formulations and proteins are known in the art. In some embodiments of any of the aspects, the extracellular matrix material comprises a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. In some embodiments, the ECM composition comprises one or more of laminin, Collagen IV, heparan sulfate proteoglycans, entactin / nidogen, fibronectin, or any combination thereof. In some embodiments of any of the aspects, the gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells is MATRIGEL™. MATRIGEL™ is commercially available, for example, from CORNING® [MATRIGEL® Matrix (Catalog # 356231)]. In certain embodiments, the ECM may be or may include at least one of Matrigel®, poly L-lysine, Geltrex™, gelatin, nitrogen, fibronectin, collagen 1,collagen IV, fibrinogen, gelatin methacrylate, fibrin, silk, pegylated gels, collagen methacrylate, basement membrane proteins, or any other biomaterial, or a combination thereof.

[0113] In some embodiments, the first cell culture medium further comprises at least one antibiotic, e.g., normocin.

[0114] In some embodiments of any of the aspects, the first cell culture medium comprises, consists of, or consists essentially of Essential 6 medium (Gibco) with MATRIGEL™, growth factor reduced (Coming), human BMP4, at least one inhibitor of TGFp, e.g., SB-431542, bFGF, and one or more antibiotics, e.g., Normocin (InvivoGen). In some embodiments of any of the aspects, the first cell culture medium comprises, consists of, or consists essentially of Essential 6 medium (Gibco) with MATRIGEL™, growth factor reduced (Corning), human BMP4, SB-431542, bFGF, and normocin.

[0115] In some aspect of any of the embodiments, the stem cells or IPSCs are cultured in a first cell culture medium comprising at least one BMP4, at least one TGFp inhibitor, at least one FGF polypeptide, at least one extracellular matrix material, and at least one antibiotic for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or more. In some aspect of any of the embodiments, the stem cells or IPSCs are cultured in a first cell culture medium comprising at least one BMP4, at least one TGFP inhibitor, at least one FGF polypeptide, at least one extracellular matrix material, and at least one antibiotic for at least 3 days,

[0116] In some aspects of any of the embodiments, the stem cells or IPSCs, after being cultured in a first cell culture medium are contacted with or cultured in a second cell culture medium, the second cell culture medium comprising at least one antibiotic. In some embodiments, the second cell culture medium does not contain an FGF polypeptide. In some embodiments, the second cell culture medium does not contain a BMP4 polypeptide. In some embodiments, the second cell culture medium does not contain a TGFP inhibitor. In some embodiments, the second cell culture medium does not contain a BMP inhibitor.

[0117] In some aspects of any of the embodiments, the second cell culture medium further comprises at least one extracellular matrix material. In some aspects of any of the embodiments, the second cell culture medium further comprises at least one BMP4 polypeptide and at least one TGFP inhibitor.

[0118] In some embodiments of any of the aspects, the second cell culture medium comprises, consists of or consists essentially of Essential 6 medium (Gibco) and one or more antibiotics, e.g., normocin. In some embodiments of any of the aspects, the second cell culture medium comprises, consists of or consists essentially of Essential 6 medium (Gibco) and normocin.

[0119] In some embodiments of any of the aspects, the second cell culture medium comprises, consists of or consists essentially of Essential 6 medium (Gibco) with MATRIGEL™, growth factor reduced (Corning), human BMP4, at least one inhibitor of TGF0, e.g., SB-431542, and one or more antibiotics, e.g., Normocin. In some embodiments of any of the aspects, the second cell culture medium comprises, consists of or consists essentially of Essential 6 medium (Gibco) with MATRIGEL™, growth factor reduced (Corning), human BMP4, SB-431542, and normocin.

[0120] In some embodiments, the second cell culture medium comprises a BMP inhibitor. BMP inhibitors are known in the art. For example, BMP inhibitors can include LDN-193189 (LDN). Exemplary BMP inhibitors include DMH1, K02288, LDN-212854, LDN-214117, and ML347.

[0121] In some aspects of any of the embodiments, the stem cells or IPSCs are contacted with or cultured in a second cell culture medium comprising at least one antibiotic for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or more. In some aspects of any of the embodiments, the stem cells or IPSCs are contacted with or cultured in a second cell culture medium comprising at least one antibiotic for at least 3 days.

[0122] In some aspects of any of the embodiments, the stem cells or IPSCs after being contacted with or cultured in the first cell culture medium and then the second cell culture medium, are contacted with lateral plate mesoderm (LPM).

[0123] The term "lateral plate mesoderm" as used herein refers to a type of mesoderm that is found at the periphery of the embryo. LPM is a precursor tissue of vascular lineages that can give rise to the heart, blood vessels, and blood cells of the circulatory system as well as to the mesodermal component of the limbs. The lateral plate mesoderm is split into two layers, the somatic lateral plate mesoderm, which forms the future body wall, and the splanchnic lateral plate mesoderm, which forms the circulatory system.

[0124] The term "mesoderm" as used herein refers to one of the three primary germ layers in the early embryo. The other two layers are the ectoderm (outside layer) andendoderm (inside layer), with the mesoderm as the middle layer between them. It is formed through a process called gastrulation. There are three important components, the paraxial mesoderm, the intermediate mesoderm and the lateral plate mesoderm. The paraxial mesoderm forms the somitomeres, which give rise to mesenchyme of the head and organize into somites in occipital and caudal segments, forming sclerotome (cartilage and bone), and dermatome (subcutaneous tissue of the skin). Signals for somite differentiation are derived from surroundings structures, including the notochord, neural tube and epidermis. The intermediate mesoderm connects the paraxial mesoderm with the lateral plate, eventually it differentiates into urogenital structures consisting of the kidneys, gonads, their associated ducts, and the adrenal glands.

[0125] In some embodiments, the stem cells, IPSCs, or cultured aggregates are contacted with LPM after 6 cumulative days in contact with or cultured in the first cell culture medium and then the second cell culture medium. In some embodiments, the stem cells, IPSCs, or cultured aggregates are contacted with LPM after at least 2 days, 3 days, 4 days, 5 days, 6 days, 7 days 8 days, 9 days, 10 days, or more days cumulatively in contact with or cultured in the first cell culture medium and then the second cell culture medium.

[0126] In some embodiments, the stem cells, IPSCs, or cultured aggregates are contacted with or cultured in LPM for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or more. In some embodiments, the stem cells, IPSCs, or cultured aggregates are contacted with or cultured in LPM for at least 6 days.

[0127] In some embodiments of any of the aspects, after being contacted with or cultured with LPM the stem cell, IPSC, or cultured aggregate are cultured in a third cell culture medium in a floating culture.

[0128] As used herein, the term "floating culture" refers to culturing cells in a state of suspension in a liquid without fixing the cells to a substate or the like. In a floating culture, the cells are suspended in a liquid and the culture undergoes constant agitation or movement to prevent the cells from adhering to a surface. The floating culture can undergo constant movement or agitation using a stirrer such as a spinner or a rotational chamber such as an orbital shaker.

[0129] As used herein, the term “cell aggregate” refers to a cell having a three- dimensional structure formed by self-aggregation of cells cultured in an environment such as a floating culture that allows three-dimensional growth rather than a monolayer. The cell aggregate produced as a result of the three-dimensional culture provides an environmentsimilar to the in vivo tissue from which stem cells are derived and may be spherical or non- spherical depending on the size and number of self-assembled cells.

[0130] In some embodiments, the third cell culture medium comprises, consists of, or consists essentially of a basal medium, a glutamine supplement, a neuronal culture supplement, a reducing agent, and an antibiotic.

[0131] In some embodiments, the third cell culture medium comprises a basal medium, e.g., Neurobasal medium. Additional examples of basal medium are known in the art.

[0132] In some embodiments, the third cell culture medium comprises a glutamine supplement, e.g., GlutaMax. Additional examples of glutamine supplements are known in the art.

[0133] In some embodiments, the third cell culture medium comprises a neuronal culture supplement, e.g., N-2. Additional examples of neuronal culture supplements are known in the art.

[0134] In some embodiments, the third cell culture medium comprises a reducing agent, e.g., mercaptoethanol. Additional examples of reducing agents are known in the art.

[0135] In some embodiments, the third cell culture medium comprises a reducing agent, e.g., normocin. Additional examples of antibiotics are known in the art.

[0136] In some embodiments, the third cell culture medium comprises, consists of, or consists essentially of Advanced DMEM / F12 medium (Gibco) supplemented with GlutaMax supplement (Gibco), B27 supplement minus vitamin A, N2 supplement, 2-Mercaptoethanol, and an antibiotic, e.g., normocin.

[0137] In some embodiments, the third cell culture medium comprises, consists of, or consists essentially of Advanced DMEM / F12 medium (Gibco), Neurobasal medium (Gibco), GlutaMax supplement (Gibco), B27 supplement minus vitamin A, N2 supplement, 2- Mercaptoethanol, and an antibiotic, e.g., normocin.

[0138] In some embodiments of any of the aspects, the cells are cultured in a third cell culture medium in a floating culture for at least 10 days, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least21 , at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least85, at least 86, at least 87, at least 88, at least 89, at least 90, at least 91, at least 92, at least93, at least 94, at least 95, at least 96, at least 97, at least 98, at least 99, or more days.

[0139] In some embodiments, the floating culture provides conditions to promote self-assembly of cells into a three-dimensional amnion comprising at least one of: a unicellular / simple epithelium, a transparent membrane, a simple layer of mesenchymal cells, and amnion markers. In some embodiments, the floating culture provides conditions to promote self-assembly of cells into a three-dimensional amnion comprising unicellular / simple epithelium, transparent membrane, simple layer of mesenchymal cells, and amnion markers.

[0140] In some embodiments, the simple layer of mesenchymal cells expresses collagen markers. In some embodiments, the simple layer of mesenchymal cells expresses COL3A, COL15A. Other exemplary collagen markers include, but are not limited to, COL1A, COL2A, COL4A, COL5A, COL6A, COL7A, COL8A, COL9A, COL10A, COL11A, COL12A, COL13A, COLMA, COL16A, COLMA, COL18A, COL19A, COL20A, COL21A, COL22A, COL23A, COL24A, COL25A, COL27A, COL27A, and COL28A.

[0141] Amnion produced by the methods disclosed herein express detectable levels of amnion markers. Exemplary amnion expression markers that are that permit identification / selection include, but are not limited to, KRT8, KRT18, KRT19, GABRP, VTCN1, IGFBP3, and KCNMA1. Amnion can also be characterized by the down-regulation of markers characteristic of periderm and basal epidermis, including but not limited to KRT1, KRT4, CLIC3, and KRT5.

[0142] A “marker” as used herein refers to one or more characteristics that contribute to or are associated with the phenotype of a given cell or tissue. Markers can be used for selection of cells or compositions comprising characteristics of interest. Markers will vary with specific cells, and can be characteristics, whether morphological, functional or biochemical, that are particular to a cell or tissue type or phenotype and include both extracellular and intracellular molecules, e.g., proteins, RNAs, glycosylation patterns, etc. expressed or exhibited by the cell or tissue. In some embodiments, such markers are proteins, including,but not limited to proteins that possess an epitope for an antibody or other binding molecule available in the art. Other markers can include peptides, lipids, polysaccharides, nucleic acids and steroids, among others. Examples of morphological characteristics or traits include, but are not limited to, shape, size, and nuclear to cytoplasmic ratio. Markers can be detected by any appropriate method available to one of skill in the art.

[0143] Methods for detecting the expression of such markers can include, for example, RT- PCR and immunological methods that detect the presence of the encoded polypeptides, such as Western blots or flow cytometric analyses. In some embodiments, detection does not involve only RT-PCR, but also includes detection of protein markers. Intracellular markers may be best identified via RT-PCR, while cell surface markers are readily identified, e.g., by immunocytochemistry .

[0144] In some embodiments, described herein is a method for producing an amniotic membrane composition, the method comprising contacting the stem cells or induced pluripotent stem cells with Lateral Plate Mesoderm (LPM). In some embodiments, the stem cells or induced pluripotent stem cells are contacted with LPM for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or more. In some embodiments, the stem cells or induced pluripotent stem cells are contacted with LPM for 6 days.

[0145] In some embodiments, described herein is a method for producing a amniotic membrane composition, the method comprising culturing at least one stem cell or at least one induced pluripotent stem cell in a first culture medium comprising at least: a Bone Morphogenetic Protein (BMP)-4 polypeptide, at least one inhibitor of Transforming Growth Factor Beta (TGFP), and a fibroblast growth factor (FGF) polypeptide for at least 1 day then contacting the cells with a second cell culture medium comprising at least one antibiotic for 2 days; and then contacting the cells with LPM for 6 days; and then culturing the cells in a third cell culture medium comprising a floating culture for at least 20 days.

[0146] In some embodiments, described herein is a method for producing a amniotic membrane composition, the method comprising contacting stem cells or induced pluripotent stem cell cultured to a concentration of less than 35,000 cells / mL in a first culture medium comprising at least: a BMP-4 polypeptide, at least one inhibitor of TGFP, and a FGF polypeptide for at least 1 day then contacting the cells with a second cell culture medium comprising at least one antibiotic for 2 days; and then contacting the cells with LPM for 6 days; and then culturing the cells in a third cell culture medium comprising a floating culture for at least 20 days.

[0147] In some embodiments, described herein is a method for producing a amniotic membrane composition, the method comprising contacting stem cells or induced pluripotent stem cell cultured to an aggregate at a concentration of less than 3,500 cells / aggregate in a first culture medium comprising at least: a BMP-4 polypeptide, at least one inhibitor of TGFp, and a FGF polypeptide for at least 1 day then contacting the cells with a second cell culture medium comprising at least one antibiotic for 2 days; and then contacting the cells with LPM for 6 days; and then culturing the cells in a third cell culture medium comprising a floating culture for at least 20 days.

[0148] In some embodiments, described herein is a method for producing an amniotic membrane composition, the method comprising not contacting cells with a BMP inhibitor.

[0149] In some embodiments, described herein is a method for producing a amniotic membrane composition, the method comprising: culturing at least one stem cell or at least one induced pluripotent stem cell in a first culture medium comprising at least: a BMP-4 polypeptide, at least one inhibitor of TGFp, and a FGF polypeptide for at least 1 day; then contacting the cells with a second cell culture medium comprising at least one antibiotic for 2 days; and then contacting the cells with LPM for 6 days; and then culturing the cells in a third cell culture medium comprising a floating culture for at least 20 days, wherein the cells are not contacted with a BMP inhibitor.

[0150] In some embodiments, described herein is a method for producing a human amniotic membrane composition, comprising culturing cells in a floating culture that does not comprise basal medium.

[0151] In some embodiments, described herein is a method for producing a amniotic membrane composition, the method comprising: culturing at least one stem cell or at least one induced pluripotent stem cell in a first cell culture medium comprising at least: a BMP-4 polypeptide, at least one inhibitor of TGFp, and a FGF polypeptide for at least 1 day then contacting the cells with a second cell culture medium comprising at least one antibiotic for 2 days; and then contacting the cells with LPM for 6 days; and then culturing the cells in a third cell culture comprising a floating culture for at least 20 days, wherein the floating culture does not comprise basal medium.

[0152] In some embodiments, described herein is a method for producing a human amniotic membrane composition, the method comprising: culturing human stem cells or human induced pluripotent stem cells to a concentration of less than 35,000 cells / mL and contacting the cells with a first cell culture medium comprising at least: a BMP-4 polypeptide, at leastone inhibitor of TGF , and a FGF polypeptide for at least 1 day; and then contacting the cells with a second cell culture medium comprising at least one antibiotic for 2 days; and then contacting the cells with LPM for 6 days; and then culturing the cells in a third cell culture comprising a floating culture for at least 20 days, wherein the cells are not contacted with a BMP inhibitor, and wherein the floating culture does not comprise basal medium.

[0153] In some embodiments, described herein is a method for producing a human amniotic membrane composition, the method comprising: culturing human stem cells or human induced pluripotent stem cells to an aggregate at a concentration of less than 3,500 cells / aggregate and contacting the cells with a first cell culture medium comprising at least: a BMP-4 polypeptide, at least one inhibitor of TGFp, and a FGF polypeptide for at least 1 day; and then contacting the cells with a second cell culture medium comprising at least one antibiotic for 2 days; and then contacting the cells with LPM for 6 days; and then culturing the cells in a third cell culture comprising a floating culture for at least 20 days, wherein the cells are not contacted with a BMP inhibitor, and wherein the floating culture does not comprise basal medium.

[0154] In some embodiments, the amniotic membrane composition produced by the methods described herein comprises or is an amnioid.

[0155] In some embodiments, described herein is a method for producing an amnioid. In some embodiments, described herein is a method for producing a mammalian amnioid. In some embodiments, described herein is a method for producing a human amnioid. In some embodiments, is a method for producing a human amnioid, the method comprising: culturing human stem cells or human induced pluripotent stem cells to an aggregate at a concentration of less than 3,500 cells / aggregate and contacting the cells with a first cell culture medium comprising at least: a BMP-4 polypeptide, at least one inhibitor of TGFp, and a FGF polypeptide for at least 1 day; and then contacting the cells with a second cell culture medium comprising at least one antibiotic for 2 days; and then contacting the cells with LPM for 6 days; and then culturing the cells in a third cell culture comprising a floating culture for at least 20 days, wherein the cells are not contacted with a BMP inhibitor, and wherein the floating culture does not comprise basal medium.

[0156] In some embodiments, the amnioid produced by the methods described herein has the capability of expanding in size to 0.1 cm, to 0.2 cm, to 0.3 cm, to 0.4 cm, to 0.5 cm, to 0.6 cm, to 0.7 cm, to 0.8 cm, to 0.9 cm, to 1 cm, to 2 cm, to 3 cm, to 4 cm, to 5 cm, to 6 cm, to 7 cm, to 8 cm, to 9 cm, to 10 cm, or more. In some embodiments, the amnioid produced by themethods described herein has the capability of expanding in size to a range of 0.1 cm - 10 cm, to a range of 0.4 cm - 9 cm, to a range of 0.6 cm - 8 cm, to a range of 0.8 cm - 7 cm, to a range of 1 cm - 6 cm, to a range of 2 cm - 5 cm, to a range of 3 cm - 4 cm.

[0157] In one aspect of any of the embodiments, described herein is an amniotic membrane composition or amnioid produced by the methods described herein.

[0158] In one aspect of any of the embodiments, described herein is the use of an amnion composition or amnioid as described herein, or produced by the methods described herein, in modeling the molecular, cellular, and structural development of amnion. In one aspect of any of the embodiments, is the use of an amnion composition or amnioid as described herein, or produced by the methods described herein, to enhance understanding of the typical developmental processes and to problems that arise during development, e.g., preeclampsia and hydramnios.

[0159] In one aspect of any of the embodiments, described herein is the use of an amnion composition or amnioid as described herein, or produced by the methods described herein in drug screening or drug testing. In one aspect of any of the embodiments, described herein is a method of drug screening or drug testing, comprising generating an amnion composition or amnioid as described herein, and contacting the amnion composition or amnioid with a candidate drug. In some embodiments, a marker or activity of the amnion composition or amnion is measured after being contacted with the candidate drug.

[0160] In one aspect of any of the embodiments, described herein is the use of an amnion composition or amnioid as described herein, or produced by the methods described herein, as regeneration materials in a subject in need thereof. In some embodiments, described herein is the use of an amnion composition or amnioid as described herein, or produced by the methods described herein, for screening compounds..

[0161] As utilized herein, “and / or” means any one or more of the items in the list joined by “and / or.” As an example, “x and / or y” means any element of the three-element set { (x), (y), (x, y)}. As another example, “x, y, and / or z” means any element of the sevenelement set { (x), (y), (z), (x, y), (x, z), (y, z), (x, y, z) } . As utilized herein, the term “exemplary” means serving as a non-limiting example, instance, or illustration. As utilized herein, the terms “e.g.” and “for example” set off lists of one or more non-limiting examples, instances, or illustrations.

[0162] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodimentincludes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed the “less than or equal to 10” as well as “greater than or equal to 10” is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0163] The components, steps, features, objects, benefits and advantages which have been discussed are merely illustrative. None of them, nor the discussions relating to them, are intended to limit the scope of protection in any way. Numerous other embodiments are also contemplated. These include embodiments which have fewer, additional, and / or different components, steps, features, objects, benefits and advantages. These also include embodiments in which the components and / or steps are arranged and / or ordered differently.

[0164] Unless otherwise stated, all measurements, values, ratings, positions, magnitudes, sizes, and other specifications that are set forth in this specification, including in the claims that follow, are approximate, not exact. They are intended to have a reasonable range that is consistent with the functions to which they relate and with what is customary in the art to which they pertain.

[0165] The terms “decrease,” “reduced,” “reduction,” “decrease,” or “inhibit” as used herein generally refer to a decrease by a statistically significant amount. However, for avoidance of doubt, “reduced”, “reduction” or “decrease” or “inhibit” mean a decrease byat least 10% as compared to a reference level, for example a decrease by 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 up to and including a 100% decrease (e.g. absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level.

[0166] The terms “increased,” “increase,” or “enhance,” or “activate” as used herein generally refer to an increase by a statically significant amount. However, for the avoidance of doubt, the terms “increased”, “increase” or “enhance” or “activate” mean an increase of at least 10% as compared to a reference level, for example an increase of 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 up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4- fold, or at least about a 5 -fold or at least about a 10-fold increase or more, relative to a reference level.

[0167] The term “statistically significant" or “significantly" refers to statistical significance and generally means a two-standard deviation (2SD) or greater difference.

[0168] As used herein, the term “modulates” refers to an effect including increasing or decreasing a given parameter as those terms are defined herein.

[0169] As used herein the term "comprising" or "comprises" is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not.

[0170] As used herein the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0171] The term "consisting of" refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0172] As used in this specification and the appended claims, the singular forms “a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, references to "the method" includes one or more methods, and / or steps of thetype described herein and / or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0173] It is understood that the foregoing detailed description and the following examples are illustrative only and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments, which will be apparent to those of skill in the art, may be made without departing from the spirit and scope of the present invention. Further, all patents, patent applications, and publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the correctness of the dates or contents of these documents.

[0174] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0175] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biologyand Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081- 569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; laneway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), W. W. Norton & Company, 2016 (ISBN 0815345054, 978-0815345053); Lewin’s Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN- 1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties.

[0176] Disclosed herein is an amnioid culture, used to create the amnioid, comprising at least one human stem cell or at least one human induced pluripotent stem cell; and a Bone Morphogenetic Protein (BMP)-4 polypeptide, at least one inhibitor of Transforming Growth Factor Beta (TGFP), and / or a fibroblast growth factor (FGF) polypeptide. This culture can be used to create an amnioid, and the conditions upon which the aminiod can be created are described herein. The culture can further comprise an antibiotic, which can be added to the initial culture or at a subsequent time point, as described herein. The culture media can further comprise at least one Lateral Plate Mesoderm cell. This can also be added at a subsequent time, as described herein.

[0177] The human stem cell or human induced pluripotent stem cell can be provided to the culture at a concentration of less than 35,000 cells / mL, less than 20,000 cells / mL. or at a concentration of from about 8,000 cells / mL to about 20,000 cells / mL. The human stem cell or human induced pluripotent stem cell can first be aggregated to about 8,000 cells / mL - to about 20,000 cells / mL. The human stem cell or human induced pluripotent stem cell can be provided as a cell aggregate at a concentration of less than about 3,500 cells / aggregate. Thehuman stem cell or human induced pluripotent stem cell can be provided as a cell aggregate at a concentration of no more than about 2,000 cells / aggregate. The human stem cell or human induced pluripotent stem cell can be provided as a cell aggregate at a concentration of from 100 cell / aggregate to 2,000 cells / aggregate.

[0178] BMP4 polypeptide can be provided at a concentration of about 1-10 ng / mL, such as about 5 ng / mL.

[0179] TGFP inhibitor can be provided at a concentration of about 1-20 pM, such as about 10 pM. An example of a TGFP inhibitor is SB-431542.

[0180] The FGF polypeptide can be provided at a concentration of about 1-10 ng / mL. For example, the concentration can be about 4 ng / mL. The FGF polypeptide can be, for example, FGF2 (basic / bFGF).

[0181] Amnioid culture can further comprise a basal medium, a glutamine supplement, a neuronal culture supplement, a reducing agent, and an antibiotic.

[0182] The amnioid culture can be cryopreserved or can be cultured on a plate or a well. The growth of the amnioid described herein, as for any mammalian cell culture, depends in part upon the particular medium selected for growth. During culture, the amnioid described herein adhere to a substrate in culture, e.g. the surface of a tissue culture container (e.g., tissue culture dish plastic, fibronectin-coated plastic, and the like) and form a monolayer.

[0183] In another aspect, provided herein is a composition, e.g., a pharmaceutical composition, comprising any of the amnion derived adherent cells, or populations of cells comprising the amnion derived adherent cells, provided herein. In a specific embodiment, the composition is a matrix or scaffold, e.g., a natural tissue matrix or scaffold, for example, a permanent or degradable decellularized tissue matrix or scaffold; or synthetic matrix or scaffold. In a more specific embodiment, said matrix or scaffold is shaped in the form of a tube or other three-dimensional form of an organoid.

[0184] Also disclosed herein is the amnioid itself.. This is an isolated three- dimensional amniotic membrane (amnioid) comprising at least one of a unicellular / simple epithelium, at least one of a transparent membrane, at least one of a simple layer of mesenchymal cells, and a detectable level of at least one amnion marker. The simple layer of mesenchymal cells can express collagen markers selected from a group comprising but not limited to COL15A1, COL12A1, and / or COL3A1. The can also express at least one marker selected from a group comprising but not limited to KRT8, POSTN, KRT18, GABRP,IGFBP3, and / or VTCN1. The amnioid can have the capability of expanding in size to 0.1 cm- 10 cm. The amnioid can also have the capability of expanding in size to at least 1 cm. The amnioid can have the capability of expanding in size to about 4 cm.

[0185] In some embodiments, the amnioid is used for disease modeling and drug development. The quality of the amnioid described herein makes them ideally suited for such research uses, particularly high-throughput analysis. Agents are contacted with the cells to determine the effect of the agent. Cells derived from the amnioid may also be modified to include a marker and used either in vitro or in vivo as diagnostic compositions to assess properties of the cells in response to changes in the in vitro or in vivo environment.

[0186] In some embodiments, the amniods described herein can be used in drug testing or drug toxicity screening applications. For example, in some embodiments, drugs or biological or environmental agents are tested. Indications for drug testing include any compound or biological agent in the pharmaceutical discovery and development stages, or drugs approved by drug regulatory agencies, like the US Federal Drug Agency. All classes of drugs, over-the-counter and nutraceuticals for any medical indications are known or suspected environmental toxicant may be utilized. In some embodiments, drugs are screened using the amnioid or cells derived therefrom to identify drugs that are potentially toxic to embryos or fetuses or general toxicity, or which can be used to increase or reproduce amnion. In some embodiments, candidate infertility drugs are screened using the amnioid or cells derived from the aminioid to identify drugs to treat infertility (e.g., by promoting embryonic development, germ-line cell development, etc.).

[0187] Disclosed herein is a population of cells, e.g., human cells, referred to as the amnioid, wherein at least about 50%, 60%, 70%, 80%, 90%, 95% or 98% of cells in said isolated population of cells are amnion derived cells which express at least one of the markers disclosed herein.

[0188] In certain embodiments, provided herein is a cell that expresses, or a population of cells, wherein at least about 50%, 60%, 70%, 80%, 90%, 95% or 98% of cells in said isolated population of cells are amnion derived adherent cells that express one or more of, or all of, ACTA2 (actin, alpha 2, smooth muscle, aorta), ADAMTS1 (ADAM metallopeptidase with thrombospondin type 1 motif, 1), AMOT (angiomotin), ANG (angiogenin), ANGPT1 (angiopoietin 1), ANGPT2, ANGPTL1 (angiopoietin-like 1), ANGPTL2, ANGPTL4, BAI1 (brain-specific angiogenesis inhibitor 1), CD44, CD200, CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1), CHGA(chromogranin A), COL15A1 (collagen, type XV, alpha 1), COL18A1 (collagen, type XVIII, alpha 1), COL4A1 (collagen, type IV, alpha 1), COL4A2 (collagen, type IV, alpha 2), COL4A3 (collagen, type IV, alpha 3), CSF3 (colony stimulating factor 3 (granulocyte), CTGF (connective tissue growth factor), CXCL12 (chemokine (CXC motif) ligand 12 (stromal cell-derived factor 1)), CXCL2, DNMT3B (DNA (cytosine-5-)-methyltransferase 3 beta), ECGF1 (thymidine phosphorylase), EDG1 (endothelial cell differentiation gene 1), EDIL3 (EGF-like repeats and discoidin I-like domains 3), ENPP2 (ectonucleotide pyrophosphatase / phosphodiesterase 2), EPHB2 (EPH receptor B2), FBLN5 (FIBULIN 5), F2 (coagulation factor II (thrombin)), FGF1 (acidic fibroblast growth factor), FGF2 (basic fibroblast growth factor), FIGF (c-fos induced growth factor (vascular endothelial growth factor D)), FLT4 (fms-related tyrosine kinase 4), FN1 (fibronectin 1), FST (follistatin), F0XC2 (forkhead box C2 (MFH-1, mesenchyme forkhead 1)), GRN (granulin), HGF (hepatocyte growth factor), HEY1 (hairy / enhancer-of-split related with YRPW motif 1), HSPG2 (heparan sulfate proteoglycan 2), IFNB1 (interferon, beta 1, fibroblast), IL8 (interleukin 8), IL12A, ITGA4 (integrin, alpha 4; CD49d), ITGAV (integrin, alpha V), 1TGB3 (integrin, beta 3), MDK (midkine), MMP2 (matrix metalloprotease 2), MY0Z2 (myozenin 2), NRP1 (neuropilin 1), NRP2, PDGFB (platelet-derived growth factor |3), PDGFRA (platelet-derived growth factor receptor a), PDGFRB, PEC AMI (platelet / endothelial cell adhesion molecule), PF4 (platelet factor 4), PGK1 (phosphoglycerate kinase 1), PR0X1 (prospero homeobox 1), PTN (pleiotrophin), SEMA3F (semophorin 3F), SERPINB5 (serpin peptidase inhibitor, clade B (ovalbumin), member 5), SERPINC1, SERPINF1, TIMP2 (tissue inhibitor of metalloproteinases 2), TIMP3, TGFA (transforming growth factor, alpha), TGFB1, THBS1 (thrombospondin 1), THBS2, TIE1 (tyrosine kinase with immunoglobulin- like and EGF-like domains 1), TIE2 / TEK, TNF (tumor necrosis factor), TNNI1 (troponin I, type 1), TNFSF15 (tumor necrosis factor (ligand) superfamily, member 15), VASH1 (vasohibin 1), VEGF (vascular endothelial growth factor), VEGFB, VEGFC, VEGFR1 / FLT1 (vascular endothelial growth factor receptor 1), and / or VEGFR2 / KDR.

[0189] Other terms are defined herein within the description of the various aspects of the invention.

[0190] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describingand disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the correctness of the dates or contents of these documents.

[0191] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0192] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting.EXAMPLESExample 1:

[0193] The amnion is the extraembryonic structures deriving from the epiblast that co-develop with the embryo during mammalian development. The technology described herein relates to a protocol for deriving, from pluripotent stem cells, human amnion-like organoid for use in human development basic research, regeneration materials, drugdiscovery or gene therapy testing applications. The present technology covers novel techniques for generating amniotic membrane consisting of epithelia and mesenchyme subtypes that matched those in the human amnion. The organoid generated from this technology can be maintained in vitro for long-term, recapitulating morphological features, genetic markers and behaviors of the human amnion.

[0194] Amniotic membrane can be used for reconstructing injured cornea or tympanic membrane. For example, preserved amniotic membrane has been used to repair cornea and other ocular damages. Also, amniotic membrane can be applied to tympanic membrane repair.Example 2:Understanding extraembryonic development by building human amniotic membrane from induced pluripotent stem cells

[0195] The amniotic sac protects the developing embryo from physical assaults and regulates fluid exchange between the fetus and the mother. During embryogenesis, the amniotic membrane develops adjacent to the surface ectoderm and is closely associated with extraembryonic mesoderm. The formation of amnion plays an essential role in normal versus abnormal pregnancy, yet the lack of an appropriate model hinders insights into this process in humans. Described herein is the successful generation of an amnion sac derived from human induced pluripotent stem cells. The organoid maintained the unicellular epithelia, transparent membrane, and amnion marker expressions until day 90 while expanding into a gigantic structure - an amnioid.

[0196] A combination of single cell sequencing, immunostaining, live cell imaging, and pharmacological perturbation was used to compare amnioids with hair-bearing skin organoids. The data reveal potential biochemical and mechanotransduction signaling pathways that may determine cell fate commitment within the surface ectoderm or amniotic membrane. The amnioid cells maintain nuclear YAP localization, pronounced ZO-1 at cellcell junctions, a large cell area, and prolonged expression of YAP downstream targets and ion channels, suggesting an inter-regulatory role of mechanical pressure, cell proliferation, and cell fate commitment. The skin organoid and the amnioid were also used to uncover potential epithelia-mesenchyme interactions that regulate the mesoderm contribution to epidermis and amnion development. In conclusion, the human amnioid model can be used to gain deeper insights into the logic of surface ectoderm and extraembryonic development and provide anin vitro platform to understand pregnancy problems such as pre-eclampsia and hydramnios.Example 3:

[0197] Amnioid Generation Protocol* Every step in the biosafety cabinet* This protocol is standardized to preparation of culture in 2 plates of the 96-well plate* Protocol is paired with Lateral Plate Mesoderm (LPM) generation protocol on d6-d8A. Day (-2): Cell aggregationMaterials: E8 Flex medium (hereafter, E8), Normocin (stock concentration: 50 mg / ml), Y27632 (hereafter, Y, stock concentration: lOmM), IX DPBS, Accutase, 15 ml and 50 ml conical tubes, test tubes with cell strainer snap cap, Trypan blue, Automated cell counter (Invitrogen Countess II, recommended) or Hemocytometer, multi-channel pipette, low attachment 96-well U-bottom plates, 25 ml reservoirs.Cells that are at about 75% confluency are used. Prior to starting the experiment, 32 ml of E8 medium with 64 pl of Normocin (Final concentration of 100 pg / ml) is prepared and this E8 medium with Normocin is distributed into 2 conical tubes as 10 ml of medium to a 15 ml conical tube and 22 ml of medium to a 50 ml conical tube. Then, 10 pl of Y is added to the 10 ml medium, which brings the final concentration to 10 pM Y (hereafter, E8-10Y), and 44 pl of Y to the 22 ml medium, which brings the final concentration to 20 pM Y (hereafter, E8- 20Y).Cell dissociation:Spent medium is aspirated off the cells in one well of a 6-well culture plate. The cells are then washed with ~2.5 ml of IX DPBS twice, and the DPBS from the last wash is aspirated. 500 pl of Accutase is added and incubated for 3-4 mins in 37°C incubator with 5.0% CO2; after 3 mins of incubation, the plate is shaken horizontally and checked under a microscope to confirm that the cells are all detached from the surface of the plate. If clusters of adhered cells are still present, the cells are incubated for an extra 20 secs to 1 min (total 4 min), based on the percentage of adhered cell portion, until the cells are fully detached and partially singlecelled. Using a 5 ml serological pipette, 5 ml of E8-10Y are aliquoted to a sterile 15 ml tube. Using 3 ml from the step #4 E8-10Y with a 5 ml serological pipette, gently pipette up-and- down is used to break up the cell clusters into single cells in the well where cells weredetached by Accutase treatment. Then, all the cell suspension is collected back into the same 15 ml tube (final volume: ~5.5 ml). The tube is centrifuged for 5 min-30 sec at 230 ref to pellet cells. The supernatant is carefully and completely removed by aspiration (with caution not to disturb the cell pellet) - NOTE: The tube is tilted to flow the medium away from the pellet for aspiration. A pl 000 tip is used to resuspend cells in 1 ml of E8-10Y by gently pipetting up-and-down.Cell aggregation:To equilibrate a cell strainer, 1 ml of E8-10Y medium is forcefully pipetted through the strainer mesh. The resuspended cells in E8-10Y are pipetted onto the cell strainer in a dropwise manner. To thoroughly collect cells, the conical tube where the cells were resuspended are rinsed and transferred, with 1 ml of E8-10Y medium onto the cell strainer in a drop- wise manner. The cell strainer snap cap is carefully removed and discarded. 50 pl of Trypan blue is prepared in a 500 pl tube. Using plOOO tip, the cell suspension is pipetted up-and-down in the test tube to mix evenly. Immediately, using a p200 tip, 50 pl of cell suspension is collected from the center of the volume in the test tube, and transferred into the 50 pl of Trypan blue prepared ahead - this makes a 1 :1 dilution of the cells and Trypan blue. The cells are evenly mixed with trypan blue by carefully pipetting up-and-down. 11 pl of CelkTrypan blue mixture is added into both sides of the cell counting chamber slide for Invitrogen Countess II automated counter or on the hemocytometer (Disposable cell counting chamber slides for Invitrogen Countess II; Invitrogen, Cat. No. C10228). The number of cells is calculated, considering the 1: 1 trypan blue dilution - NOTE: The Invitrogen Countess II automated counter automatically calculates and displays the number while considering trypan blue dilution. The volume of cell suspension needed for cell culture is calculated.Note: The protocol was tested with a starting aggregate of 3000 cells / well, 1600 cells / well, 800 cells / well, 400 cells / well, and 100 cells / well. Reducing the aggregate starting size for dO increases the amount of cells that can form epithelia (amnion epithelia) and reduces the amount of mesenchymal and neural crest cells. All will result in amnion-like organoid later. For the best result (amount of epithelia is optimized balanced with the size of aggregate that can be transferred, use somewhere between 800 cells / well (8000 cells / mL to 2000 cells / well (20000 cells / ml)).After the calculation, the same volume of the cell suspension that needed for culture was removed from 22 ml of E8-20Y medium that was prepared earlier, and then, the adequatevolume of the cell suspension is added to that E8-20Y medium. For example, if the cell suspension concentration is 5 x 105cells per ml, 1.54 ml of the cell suspension is needed for culture in 22 ml of medium (easy to memorize equation: total number of cells needed for culture / number of cells in 1 ml of cell suspension). Therefore, 1.54 ml of E8-20Y is removed from the 22 ml and 1.54 ml of cell suspension is added into the 20.46 ml of E8-20Y medium, which brings the final cell concentration to 7.7 x 105cells in 22 ml of E8-20Y medium. To mix the cell suspension evenly the tube is inverted or swirled several times. The 22 ml cell suspension is poured into a 25 ml reservoir. Then, using a multi-channel pipette, 100 pl of cell suspension is aliquoted into each well of a 96-well U-bottom plates. The plates are centrifuged at 110 ref for 6 min at room temperature and incubated in a 37 °C incubator with 5.0% CO2 for 24 hrs.B. Day (-1): Dilution of Y solutionMaterials: E8 medium, Normocin, Multi-channel pipette, 25 ml reservoir.22 ml of fresh E8 medium containing 44 pl of Normocin ( WITHOUT Y) is prepared. 22 ml of fresh E8 medium is poured into a 25 ml reservoir, then, using a multi-channel pipette, 100 pl of E8 medium is added into each well, which brings total volume to 200 pl per well. The plates are then incubated in 37°C incubator with 5.0% CO2 for 24 hrs.C. Differentiation Day 0: Transition to differentiation in E6 medium (perform all procedures on ice)Materials: Ice in a bucket, E6 medium, Normocin, Matrigel, SB431542 (hereafter, SB, stock concentration: 10 mM), basic-FGF (hereafter, FGF, stock concentration: 200 pg / ml), BMP4 (stock concentration: 100 pg / ml), 2 ml round bottom tubes, Wide-orifice (W-O) p200 and pl 000 tips (cut the tips with a sterile blade or purchase from Rainin, Cat. No. 17014294 and 17014297, respectively), 100 mm petri-dishes, 96-well U-bottom plates.A day before, 600 pl of Matrigel is thawed on ice overnight at 4°C. E6 medium containing 2% Matrigel, 10 pM SB, 4 ng / ml FGF, and 2.5 ng / ml BMP4 is prepared - hereafter, E6SFB. 30 ml of E6 medium containing 60 pl of Normocin is prepared and kept on ice. 2% Matrigel (Final concentration, v / v) is made in 29.4 ml of E6 medium, by adding 600 pl of Matrigel. 30 pl of SB, 0.6 pl of FGF, and 0.75 pl of BMP4 are added. The tube is inverted several times to mix evenly and placed on ice.3 ml of E6 medium is prepared in a 15 ml falcon tube for washing step. All aggregates are collected from 96-well U-bottom plates to a 2 ml round-bottom tube. Using W-0 p200 tips and a multi-channel pipette - wherein the pipette is set to 170 pl (i.e., about 30 pl less than the total volume in each well) - all aggregates are collected on a 100 mm petri-dish. By gently swirling the petri-dish, all aggregates are concentrated into the center of the dish. Using a W- O plOOO tip, all aggregates are collected into a 2 ml round-bottom tube. Excessive E8 medium is carefully removed from the tube. The aggregates are washed with 1 ml of E6 medium three times to completely remove traces of E8 medium. 1 ml of E6SFB medium is then added to the tube containing aggregates. A new 100 mm petri-dish is placed on ice and ~15 ml of E6SFB medium prepared ahead is added. Using a W-0 plOOO tip, all aggregates are transferred, including 1 ml of E6SFB, to the petri-dish containing 15 ml of E6SFB on ice. An extra 1 ml of E6SFB medium is used to collect and transfer any remaining aggregates in the tube to the petri-dish. Using a W-0 p200 tip, individual aggregates are transferred in 100 pl of E6SFB medium into each well in a new 96-well U-bottom plate (more medium is poured in as it goes). The plate is then incubated in a 37°C incubator with 5.0% CO2 and morphological changes are observed every day.D. Differentiation Day 3: adding medium (to match the medium condition in SKO protocol)Materials: E6 medium, Normocin, Multi-channel pipette, 10 ml reservoir5 mL of E6 medium + 10 uL Normocin (E6+) is prepared and 5 ml of prepared E6+ is poured into a 10 ml reservoir. Using a multi-channel pipette, 25 pl of the E6+ medium is added per well into the aggregate culture in 96-well U-bottom plates. The plates are gently tapped to mix the medium and incubated in 37°C incubator with 5.0% CO2.Note: this step was tested with 0.5uL LDN or 6.25 uL FGF. Including LDN does not change the result, while including FGF makes organoid form SKO.E. Differentiation Day 6: Providing nutrition and combine with LPMNotes: Fresh E6 medium containing Normocin was added at 75 pl per well, making final volume of 200 pl per well.Materials: E6 medium, Normocin, 10 ml reservoir, Multi-channel pipette.11ml of E6 medium containing 22 l of Normocin (E6+) was prepared.To have the epithelia-only amnioid (called pure amnion hereon): 11 ml of prepared E6 medium is poured into a 10 ml reservoir. Using a multi-channel pipette, 75 pl is added per well into each well in 96-well U-bottom plates, making the final volume of 200 pl per well. The plates are gently tapped to mix the medium and incubated in 37°C incubator with 5.0% CO2.To have the full amnioid (called combined amnion hereon):Method 1: combine organoids: The Lateral Plate Mesoderm (LPM) plate is taken out and aggregate to combine is chosen. 100 uL medium is removed from the LPM aggregates. IOOUL E6+ is added into the wells containing the LPM. Using a W-0 pipette, 75uL of medium + LPM is transferred into the epithelial amnion.Method 2: dissociate LPM cells: The appropriate cell number is ascertained to dissociate the LPM so that it matches a 1-1 combination. (1 LPM + 1 amnion organoid). The LPM is dissociated into single cells by TriplE + CEPT (gentle dissociation). The single cells are spun down at 3 min, 450 ref and resuspended in appropriate volume of E6+. The cells are plated on top of the amnion to recombine. The 96-well containing amnion + dissociated LPM is centrifuged at 6 min 110 ref.F. Differentiation Day 8: Providing fresh medium by half-medium changeNotes: 100 pl of spent medium is removed, and fresh E6 medium containing Normocin is added at 100 pl per well, making final volume of 200 pl.Materials: E6 medium, Normocin, 25 ml reservoir, Multi-channel pipette, W-0 p200 tips.14 ml of E6 medium containing 28 pl of Normocin is prepared. Using W-0 p200 tips and a multi-channel pipette, at about 60° angle, 100 pl of spent medium is removed very carefully from each well of 96-well U-bottom plate, with 100 pl of medium remaining in each well. All fresh E6 medium is poured into a 25 ml reservoir. Using a multi-channel pipette, 100 pl of fresh E6 medium is added into each well, making final volume of 200 pl per well. The plates are gently tapped to mix the medium and then incubated in 37 °C incubator with 5.0% CO2.G. Differentiation Day 12: Transition to floating culture in organoid mediumMaterials: Two alternatives of basal media can be used. Under testing of the optimization. a. OM medium (OMM): 24.5 ml Advanced DMEM / F12, 24.5 ml Neurobasal medium, 500 pl GlutaMax (final concentration: I X), 500 pl B-27 supplement minus Vitamin A (final concentration 0.5X), 250 pl N-2 supplement (final concentration: 0.5X), 91 pl 2- Mercaptoethanol (final concentration: 0.1 mM), and 100 pl Normocin (final concentration: 100 pg / ml) b. AdvDMEM+: 49 ml Advanced DMEM / F12 500 pl GlutaMax (final concentration: IX), 500 pl B-27 supplement minus Vitamin A (final concentration 0.5X), 250 pl N-2 supplement (final concentration: 0.5X), 91 pl 2-Mercaptoethanol (final concentration: 0.1 mM), and 100 pl Normocin (final concentration: 100 pg / ml)A day before, 630 pl of Matrigel is thawed on ice overnight at 4°C. 100 mm petri-dishes are placed on ice. OM containing 1% Matrigel (OM1%M) (or AdvDMEM+l%M) is prepared. To make 1% Matrigel (final concentration, v / v) in 63 ml of OM (or AdvDMEM+), 630 pl (volume of Matrigel) of OM (or AdvDMEM+) is removed, and then, 630 pl of Matrigel is added. The tube is inverted several times to mix evenly and placed on ice. 3 ml of Advanced DMEM / F12 medium is prepared in a 15 ml tube for washing steps.Using a W-0 pl 000 tip, all aggregates are collected in a 2 ml round bottom tube. Excessive medium is carefully removed. The aggregates were washed with 1 ml of Advanced DMEM / F12 medium 3 times. After removing all excessive washing medium, the tube with aggregates is placed on ice. 1 ml of OM1%M is added into the tube containing aggregates. Media (first pour about 15 ml of OM1%M (or AdvDMEM+l%M) is poured and continuously added as it goes) into the cooled petri-dish placed on ice. Using a W-0 plOOO tip, all aggregates that are in 1 ml of OM1%M (or AdvDMEM+l%M) are collected, and transferred, with medium, into the OM1%M (or AdvDMEM+l%M) prepared in petri-dish on ice. Using a W-0 plOOO tip, each aggregate is transferred in 500 pl of OM1%M (or AdvDMEM+l%M) into each well in 24- well low-attachment plates (i.e., one aggregate per well in a total volume of 500 pl per well). The plates are gently swirled to make sure each well is completely covered by medium, and aggregates are not floating on the surface of the medium and incubated in a 37 °C incubator with 5.0% CO .H. Differentiation Day 15: Half medium change with OM containing 1% MatrigelNotes: Half-medium change is performed - half volume of spent medium is removed from each well and half volume of fresh medium was added into each well to provide nutrition.Materials: OM, Matrigel, Ice in a bucket.A day before, 320 pl of Matrigel is thawed on ice overnight at 4°C. OM containing 1% Matrigel (OM1%M) (or AdvDMEM+1 %M) is prepared. To make 1% Matrigel (final concentration, v / v) in 32 ml of OM (or AdvDMEM+), 320 pl (volume of Matrigel) of OM (or AdvDMEM+1) is removed, and then, 320 pl of Matrigel is added. The tube inverted several times to mix evenly and keep on ice. 250 pl of spent medium is removed from each well of the 24- well plates, leaving ~ 250 pl per well. 250 pl of freshly prepared OM1%M (or AdvDMEM+1 %M) is added into each well, making a final volume of ~ 500 pl per well. The plates are gently swirled to evenly mix the medium and incubated in 37°C incubator with 5.0% CO2.I. Differentiation Day 18: Half medium change for Matrigel dilutionNotes: Half-medium change to dilute out Matrigel and provide nutrition.Materials: OM (or AdvDMEM+).32 ml of fresh OM (or AdvDMEM+) WITHOUT Matrigel is prepared. 250 pl of spent medium is removed from each well of the 24-well low-attachment plates, leaving - 250 pl per well. 250 pl of freshly prepared OM (or AdvDMEM+) is added into each well, making the final volume of ~ 500 pl per well. The plates are gently swirled to mix the medium evenly and then incubated in 37°C incubator with 5.0% CO2.Note for long-term culture: From differentiation day 18, a full medium change is performed once every week by completely removing medium (500 pl) from each well and fresh medium (500 pl) is added back. Within that one-week period, for experiments up to differentiation day 45, half-medium changes are performed every three days (i.e., Full-medium change on Monday, half-medium changes on Thursday and Sunday, then full-medium change on Tuesday). For longer experiments after differentiation day 45, half medium changes are performed every other day. Also, based on the organoid sizes (i.e., larger) and medium consumption rate (i.e., color of medium changing to yellow), total volume is increased to 1 ml per well to provide proper nutrition. From d20 to d40, the organoid expands very fast.Once it reaches % of the size of a well in 24-well plate, moving to another container is necessary. Using an in-house 3D printed scooper the organoids are moved to both 6-well plates and 10cm dishes. The dishes are coated with anti-adhesion molecules that could be beneficial for preventing adhesion.Procedure to move the amnioid from plates to plates: The amnioid is submerged in excess medium. The 6-well plate is filled with 5ml of medium (the 10cm plate with 10-12 ml of medium). The plate is tilted gently, and the scooper is submerged to the side of the medium. It is important to not poke the amnioid. The amnoid is scooped up from the bottom. The amnioid is gently moved to the new plate, submerged in the medium and the scooper is removed from the bottom.

Claims

CLAIMS1. An amnioid culture comprising: a. at least one human stem cell or at least one human induced pluripotent stem cell; and b. a Bone Morphogenetic Protein (BMP)-4 polypeptide, at least one inhibitor of Transforming Growth Factor Beta (TGFP), and / or a fibroblast growth factor (FGF) polypeptide2. The amnioid culture of claim 1, further comprises an antibiotic.

3. The amnioid culture of claim 1 , wherein the culture media further comprises at least one Lateral Plate Mesoderm cell.

4. The amnioid culture of claim 1 , wherein the human stem cell or human induced pluripotent stem cell are provided for step a) at a concentration of less than 35,000 cells / mL.

5. The amnioid culture of claim 1 , wherein the human stem cell or human induced pluripotent stem cell are provided for step a) at a concentration of no more than 20,000 cells / mL.

6. The amnioid culture of claim 1 , wherein the human stem cell or human induced pluripotent stem cell are provided for step a) at a concentration of from 8,000 cells / mL to 20,000 cells / mL.

7. The amnioid culture of claim 1 , wherein the human stem cell or human induced pluripotent stem cell is first aggregated to 8,000 cells / mL - 20,000 cells / mL.

8. The amnioid culture of claim 1 , wherein the human stem cell or human induced pluripotent stem cell are provided as a cell aggregate at a concentration of less than 3,500 cells / aggregate.

9. The amnioid culture of claim 1, wherein the human stem cell or human induced pluripotent stem cell are provided for step a) as a cell aggregate at a concentration of no more than 2,000 cells / aggregate.

10. The amnioid culture of claim 1 , wherein the human stem cell or human induced pluripotent stem cell are provided for step a) as a cell aggregate at a concentration of from 100 cell / aggregate to 2,000 cells / aggregate.

11. The amnioid culture of any one of claims 1-10, wherein the BMP4 polypeptide is at a concentration of 1-10 ng / mL.

12. The amnioid culture of any one of claims 1-11, wherein the BMP4 polypeptide is at a concentration of 5 ng / mL.

13. The amnioid culture of any one of claims 1-12, wherein the at least one TGFP inhibitor is at a concentration of 1-20 pM.

14. The amnioid culture of any one of claims 1-13, wherein the at least one TGF0 inhibitor is at a concentration of 10 pM.

15. The amnioid culture of any one of claims 1-14, wherein the at least one TGFP inhibitor is SB-431542.

16. The amnioid culture of any one of claims 1-15, wherein the FGF polypeptide is at a concentration of 1-10 ng / mL.

17. The amnioid culture of any one of claims 1-16, wherein the wherein the FGF polypeptide is at a concentration of 4 ng / mL.

18. The amnioid culture of any one of claims 1-17, wherein the FGF polypeptide is FGF2 (basic / bFGF).

19. The amnioid culture of any one of claims 1-18, wherein amnioid culture further comprises a basal medium, a glutamine supplement, a neuronal culture supplement, a reducing agent, and an antibiotic.

20. An isolated three-dimensional amniotic membrane or amnioid comprising at least one of a unicellular / simple epithelium, at least one of a transparent membrane, at least one of a simple layer of mesenchymal cells, and a detectable level of at least one amnion marker.

21. The isolated three-dimensional amniotic membrane or amnioid of claim 20, wherein the simple layer of mesenchymal cells expresses collagen markers selected from a group comprising but not limited to COL15A1, COL12A1, and / or COL3A1.

22. The isolated three-dimensional amniotic membrane or amnioid of claim 20, wherein the at least one amnion marker is selected from a group comprising but not limited to KRT8, POSTN, KRT18, GABRP, IGFBP3, and / or VTCN1.

23. The isolated three-dimensional amniotic membrane or amnioid of any one of the claims 20-22, wherein the amnioid has the capability of expanding in size to 0.1 cm- 10 cm.

24. The isolated three-dimensional amniotic membrane or amnioid of any one of claims 20-23, wherein the amnioid has the capability of expanding in size to at least 1 cm.

25. The isolated three-dimensional amniotic membrane or amnioid of any one of claims 20-24, wherein the amnioid has the capability of expanding in size to 4 cm.

26. The isolated dimensional amniotic membrane or amnioid of any one of claims 20-25, wherein the amnioid is 0. 1 cm- 10 cm in size.

27. The isolated three-dimensional amniotic membrane or amnioid of any one of claims 20-26, wherein the amnioid is at least 1 cm in size.

28. The isolated three-dimensional amniotic membrane or amnioid of any one of claims 20-27, wherein the amnioid is at least 4 cm in size.

29. A method for producing a human amniotic membrane composition, the method comprising: a. culturing at least one human stem cell or at least one human induced pluripotent stem cell in a first cell culture medium comprising at least: a Bone Morphogenetic Protein (BMP)-4 polypeptide, at least one inhibitor of Transforming Growth Factor Beta (TGFP), and a fibroblast growth factor (FGF) polypeptide for at least 1 day; b. contacting the cells resulting from step a) with a second cell culture medium comprising at least an antibiotic for 2 days; and c. contacting the cells resulting from step b) with Lateral Plate Mesoderm (LPM) for 6 days; and d. culturing the cells resulting from step c) in a third cell culture medium for at least 20 days, wherein the cell culture comprises a floating culture.

30. The method of claim 29, wherein the human stem cell or human induced pluripotent stem cell are provided for step a) at a concentration of less than 35,000 cells / mL.

31. The method of claim 29, wherein the human stem cell or human induced pluripotent stem cell are provided for step a) at a concentration of no more than 20,000 cells / mL.

32. The method of claim 29, wherein the human stem cell or human induced pluripotent stem cell are provided for step a) at a concentration of from 8,000 cells / mL to 20,000 cells / mL.

33. The method of claim 29, wherein the human stem cell or human induced pluripotent stem cell is first aggregated to 8,000 cells / mL - 20,000 cells / mL.

34. The method of claim 29, wherein the human stem cell or human induced pluripotent stem cell are provided for step a) as a cell aggregate at a concentration of less than 3,500 cells / aggregate.

35. The method of claim 29, wherein the human stem cell or human induced pluripotent stem cell are provided for step a) as a cell aggregate at a concentration of no more than 2,000 cells / aggregate.

36. The method of claim 29, wherein the human stem cell or human induced pluripotent stem cell are provided for step a) as a cell aggregate at a concentration of from 100 cell / aggregate to 2,000 cells / aggregate.

37. The method of claim 29, wherein the first cell culture medium further comprises at least one extracellular matrix material and / or at least one antibiotic.

38. The method of claim 29, wherein the first cell culture medium comprises a BMP-4 polypeptide, at least one inhibitor of TGFB, at least one extracellular matrix material, a FGF polypeptide, and at least one antibiotic.

39. The method of claim 29, where the cells are cultured in the first cell culture medium for 1-6 days.

40. The method of claim 29, where the cells are cultured in the first cell culture medium for 3 days.

41. The method of claim 29, wherein the first cell culture medium comprises the BMP4 polypeptide at 1-10 ng / mL.

42. The method of claim 29, wherein the first cell culture medium comprises the BMP4 polypeptide at 5 ng / mL.

43. The method of claim 29, wherein the first cell culture medium comprises the at least one TGF0 inhibitor at 1-20 pM.

44. The method of claim 29, wherein the first cell culture medium comprises the at least one TGF|3 inhibitor at 10 pM.

45. The method of claim 29, wherein the at least one TGFp inhibitor is SB-431542.

46. The method of claim 29, wherein the first cell culture medium comprises the FGF polypeptide at 1-10 ng / mL.

47. The method of claim 29, wherein the first cell culture medium comprises the FGF polypeptide at 4 ng / mL.

48. The method of claim 29, wherein the FGF polypeptide is FGF2 (basic / bFGF).

49. The method of claim 29, wherein the second cell culture medium does not comprise a FGF polypeptide.

50. The method of claim 29, wherein the cells are cultured in the second cell culture medium for 1-6 days.

51. The method of claim 29, wherein the cells are cultured in the second cell culture medium for 3 days.

52. The method of claim 29, wherein the second cell culture medium further comprises at least one BMP inhibitor.

53. The method of claim 29, wherein the cells are not contacted with a BMP inhibitor.

54. The method of claim 29, wherein the cells are contacted with LPM for 1-10 days.

55. The method of claim 29, wherein the cells are contacted with LPM for 6 days.

56. The method of claim 29, wherein the third cell culture medium comprises a basal medium, a glutamine supplement, a neuronal culture supplement, a reducing agent, and an antibiotic.

57. The method of claim 29, wherein the floating culture does not comprise basal medium.

58. The method of claim 29, wherein step d) is continued until the cells form a three- dimensional amniotic membrane composition comprising at least one of: a unicellular / simple epithelium, a transparent membrane, a simple layer of mesenchymal cells, and a detectable level of at least one amnion marker.

59. The method of claim 29, wherein step d) is continued until the cells form a three- dimensional amniotic membrane composition comprising at least a unicellular / simple epithelium and a simple layer of mesenchymal cells.

60. The method of claim 29, wherein step d) is continued until the cells form a three- dimensional amniotic membrane composition comprising: a unicellular / simple epithelium, a transparent membrane, a simple layer of mesenchymal cells and a detectable level of at least one amnion marker.

61. The method of any one of claims 58-60, wherein the simple layer of mesenchymal cells expresses collagen markers selected from a group comprising but not limited to COL15 A 1 , COL12A 1 , and / or COL3 A 1.

62. The method of any one of claims 58-61, wherein the at least one amnion marker comprises KRT8, POSTN, KRT18, GABRP, IGFBP3, and / or VTCN1.

63. The method of claim 29, wherein the amniotic membrane composition comprises or is an amnioid.

64. The method of claim 29, wherein the amnioid has the capability of expanding in size to 0.1 cm- 10 cm.

65. The method of claim 29, wherein the amnioid has the capability of expanding in size to at least 1 cm.

66. The method of claim 29, wherein the amnioid has the capability of expanding in size to 4 cm.

67. The method of claim 29, wherein the amnioid is 0.1 cm-10 cm in size.

68. The method of claim 29, wherein the amnioid is at least 1 cm in size.

69. The method of claim 29, wherein the amnioid is at least 4 cm in size.

70. An amniotic membrane composition produced by the method of any one of claims 29- 69.

71. A composition comprising an amniotic membrane composition produced by the method of any one of claims 29-70.

72. A method of screening one or more compounds to determine its / their effect upon amnion, amniotic fluid, or amniotic sac, the method comprising exposing the compound or compounds to the amnioid or amniotic membrane of claims 20-28 and determining the effect thereof.

73. The method of claim 72, wherein the compound(s) increase growth or expansion of the amnion, amniotic fluid, or amniotic sac.

74. The method of claim 72, wherein the compound(s) decreases growth or expansion of the amnion, amniotic fluid, or amniotic sac.

75. The method of any one of claims 72-74, wherein the compound is compared to a control.

76. The method of any one of claims 72-75, wherein more than one compound is used to determine their combined effect.

77. The method of any one of claims 72-76, wherein the screening takes place in vitro.

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