[0001] This application claims priority to U.S. Provisional Patent Application No.

61/540,507, filed September 28, 2011 , the entirety of which is hereby incorporated by reference.

BACKGROUND

[0002] This invention generally relates to multipotent stem cell-based research tools.

More particularly, the present invention relates to culture systems and 3-dimensional tissue models that may be used for identifying agents useful for treating diseases and conditions and that are suitable for high-throughput screening applications. Additionally, the present invention is directed to identifying genes that are involved in the process of acquisition of multipotency. The present invention is also directed to identifying genes that are involved in the process of lineage-specific differentiation. The present invention is based, in part, on the discovery of methods for propagation of multipotent stem cells from human skin fibroblast samples as is disclosed in International Patent Publication No. WO 2009/151844, which is incorporated herein by reference in its entirety.

[0003] International Patent Publication No. WO 2009/151844 describes methods for propagating multipotent stem cells from human skin fibroblast samples. These multipotent stem cells were then subsequently differentiated into cells of any of the three germ layers, including adipose, hepatic, muscle, and nerve cells.

[0004] The invention described in International Patent Publication No. WO

2009/151844 further provides that the multipotent stem cells could be propagated and differentiated to be used for regeneration, recreation repopulation and/or reconstitution of desired tissues and organs. For example, International Patent Publication No. WO 2009/151844 provides for autologous therapies based on the propagated multipotent stem cells for regeneration of tissues, for use as grafts, tissue/organ replacement or

supplementation.

DESCRIPTION OF THE EMBODIMENTS

[0005] This invention generally relates to multipotent stem cell-based research tools.

More particularly, the present invention relates to culture systems and 3-dimensional tissue models that may be used for identifying agents useful for treating diseases and conditions and that are suitable for high-throughput screening applications. Additionally, the present invention is directed to identifying genes that are involved in the process of acquisition of multipotency. The present invention is also directed to identifying genes that are involved in the process of lineage-specific differentiation. The present invention is based, in part, on the discovery of methods for propagation of multipotent stem cells from human skin fibroblast samples as is disclosed in International Patent Publication No. WO 2009/151844, which is incorporated herein by reference in its entirety.

[0006] To date, much of the work on stem cells has entailed obtaining stem cells from embryonic sources. However this is often accompanied by moral and ethical issues, as this typically involves the destruction of an embryo. Accordingly, scientists have sought alternative and uncontroversial means of acquiring stem cells. Some examples include using adult stem cells, amniotic stem cells, or induced pluripotent stem cells. Adult stem cells, also known as somatic stem cells, are found in adult tissues throughout the body. Amniotic stem cells are of mesenchymal origin extracted from amniotic fluid. Induced pluripotent stem cells are artificially derived, typically by taking an adult somatic cell and inducing pluripotency by forcing expression of specific genes, for example, by recombinant gene or protein transfer. Although each of these means is uncontroversial, they are not without drawbacks. For example, it is often difficult to get large numbers of stem cells and acquiring such cells may often require selecting and isolating out rare stem cells against a backdrop of non-stem cells. Moreover, induced pluripotent stem cells may result in the unwanted induction of genes, which may be oncogeneic. Therefore, there was a need in the art for an uncontroversial method for obtaining a large number of multipotent stem cells without the need for isolation or transfer of recombinant gene or protein.

[0007] International Patent Publication No. WO 2009/1 1844 describes methods for propagating, without the need for an initial isolation or for gene or viral transduction, multipotent stem cells from human skin fibroblasts of both sexes, of all races, using selective culture conditions. Selective culture conditions may consist of an appropriate medium comprising amniotic growth fluid media (AFM) and other media and various growth factors (as described in DeCoppi et ai, comprising a-MEM (Invitrogen), 15% ES-FBS (Invitrogen), 1% L-Glutamine, and 1% Pen/Strep, supplemented with 18% CHANG MEDIUM® B (Irvine Scientific) and 2% CHANG MEDIUM® C (Irvine Scientific)). The AFM comprises a-MEM media plus supplements. These multipotent stem cells may then be subsequently differentiated into cells of any of the three germ layers, including adipose, hepatic, muscle, and nerve cells. That is, these methods provide a relatively simple tissue culturing procedure to take cells— (frozen or otherwise) obtained from individuals of all ages— and grown them to a point whereby large numbers of multipotent cells can be reproducibly obtained in culture at various scales and subsequently differentiated along cell lineages that resemble cells of any of the three germ layers, including nerve, adipose, hepatic, and muscle cells. The methods underlying the invention provide an advantageous alternative to methods of

attaining/obtaining embryonic stem cells, adult stem cells, amniotic stem cells, or induced pluripotent stem cells. Even skin fibroblast cells that were from passages 8-10 were able to propagate substantial numbers of multipotent stem cells. Using these methods, after 3 passages, large numbers of cells that were CD1 17 ⁺ and/or NANOG ⁺ were observed. Both CD1 17 and NANOG are stem cell markers well known in the art. There was, however, an observed inverse relationship with age of the patient and number of CD1 17 ⁺ cells.

[0008] Microarray studies were conducted to measure the differential expression of the genes that are either up- or down-regulated upon transfer of the skin fibroblast cells (in an Eagles-based MEM media) into media that promotes the acquisition of multipotency fa- MEM media plus supplements). These studies show that once the skin fibroblasts cells are transferred to the culture media that promotes propagation of multipotent stem cells, the cells undergo a complex change in the gene pattern of expression involving numerous genes.

[0009] Once the multipotent stem cells are propagated, these cells may then subject to differentiation into cells of the 3 germ layers. The setting and culture conditions that promote any given lineage-specific differentiation are well known in the art. For example, multipotent stem cells may be subject to differentiation in standard tissue culture conditions, growing in a monolayer. However, in addition, the setting and culture conditions may be such that the multipotent stem cells may be encouraged to grow and differentiate 3-dimensionally onto a scaffold or a matrix, such as a plate with laminin-coated beads. This 3-dimensional tissue model provides setting and conditions for differentiation that more closely proximate the tissues/cells in their actual in vivo environment.

[0010] In addition, it is possible to conduct studies to identify and examine the differential gene expression, i.e., which genes are up- or down-regulated, during the lineage- specific differentiation process. For example, microarray analyses may be conducted at various time points during lineage-specific differentiation to examine gene expression patterns during that lineage-specific differentiation. These studies allow for the observation of how gene expression changes from the initiation of differentiation to the generation of each of the lineage-specific tissues. Such analysis may identify genes that are important in the function and/or development of that cell lineage. It is possible that, in the case of a 3- dimensional tissue model, microarray data may represent an improvement over microarray data generated by measuring the differential expression of genes during the process of lineage-specific differentiation when done under conventional means under selective culture conditions. That is, the microarray data generated from a 3-dimensional engineering model might be more accurate, since it more closely proximates the tissues/cells in their actual in vivo environment.

[0011] Neither chromosome nor Comparative Genomic Hybridization studies showed any anomalies. That is, based on studies thus far, no obvious chromosome aberrations were observed in multipotent stem cells or cells derived from multipotent stem cells generated by the methods described.

[0012] There are a plurality of setting and culture conditions for any given lineage- specific differentiation. By way of example, provided below are setting and culture conditions for adipogenic, hepatic, myogenic, and neurogenic differentiation. These examples are meant to be illustrative only and not limiting.

[0013] Adipogenic Differentiation: Cells were seeded at a density of 3,000 cells/cm ² onto chamber slides (Nunc). They were cultured in DMEM low-glucose medium (Sigma- Aldrich) with 10% FBS (Invitrogen), 1 % Pen/Strep, and the following adipogenic supplements: 1 μΜ dexamethasone (Sigma-Aldrich), ImM 3-isobutyl-l-methylxanthine (Sigma-Aldrich), 10 μg/ml insulin (Sigma Aldrich), and 60 μΜ indomethacin (Sigma- Aldrich). Cells were maintained in adipogenic differentiation media for up to 20 days.

[0014] Hepatic Differentiation: Cells were seeded at a density of 5,000 cells/ cm ² onto chamber slides coated with Matrigel (Sigma-Aldrich). The cells were first expanded for 3 days in AFM then placed in hepatic differentiation media: DMEM low-glucose with 15% FBS, 300 μΜ monothioglycerol (Sigma-Aldrich), 20 ng/ml hepatocyte growth factor (Sigma- Aldrich), 10 ng/ml oncostatin M (Sigma-Aldrich), 10-7 M dexamethasone (Sigma-Aldrich), 100 ng/ml FGF4 (Peprotech), lxITS (Invitrogen) and 1% Pen/Strep. The cells were maintained in this differentiation medium for 17 days, with medium changes every third day.

[0015] Myogenic Differentiation: Cells were seeded at a density of 3,000 cells/ cm ² onto chamber slides coated with Matrigel and grown in DMEM low-glucose with 10% horse serum (Invitrogen), 0.5% chick embryo extract, and 1% Pen/Strep. Twelve hours after seeding, 3 μΜ 5-aza-2'-deoxycytodine (5-azaC; Sigma-Aldrich) was added to the culture medium for 24 hours. Incubation continued in complete medium lacking 5-azaC, with medium changes every 3 days. Cells were maintained in myogenic differentiation media for up to 20 days.

[0016] Neurogenic Differentiation: Cells were seeded at a concentration of 3,000 cells/ cm ² onto either chamber slides or Nunc 6 well Petri dishes for micro array studies. These cells were cultured in DMEM/F12 media (Invitrogen), supplemented with 200uM BHA (Sigma-Aldrich), N2 (Invitrogen), 25ng/ml NGF (Invitrogen), 10 ng/ml bFGF

(Invitrogen) 15% ESFBS, 1 % Pen/Strep and 1 % L-GIutamine. Every two days an additional 25 ng/ml of NGF and 10 ng/ml of bFGF were added to the cultures. After 6 or 7 days the cultures were examined and photographed for nerve morphology or harvested for microarray analysis. The medium used in this neurogenic differentiation media contains no DMSO. A second set of experiments was set up using the above media but lacking DMSO and BHA

[0017] In one aspect, the invention may be used as a research tool for drug discovery, e.g., in a high throughput drug screen. This high throughput drug screen would provide a means to test the efficacy of a plurality of compounds or substances on tissues/cells, including a patient's own tissues/cells. In a further aspect, a high throughput drug screen may entail administration of compound libraries to plates harboring differentiated fibroblast- derived lineage-specific cells. Lineage-specific, in this context, refers to cells of any type, including, but not limited to, cells of the integumentary system, nervous system, teeth, nervous system, eyes, digestive system (stomach, intestine, gallbladder, exocrine pancreas), endocrine, respiratory, liver, urogenital, cartilage/bone/muscle, urinary, reproductive system, blood system, immune system, circulatory system. In yet a further aspect, the high throughput drug screen is applied to lineage-specific cells from a patient suffering from a disease or condition affecting cells of that lineage in vivo. Furthermore, large amounts of differentiated fibroblast-derived lineage-specific cells may be generated for use in the high throughput drug screen by employing the methods described in International Patent

Publication No. WO 2009/151844, which methods, including everything else in International Patent Publication No. WO 2009/151844 are incorporated herein by reference. One major advantage of this aspect of the invention is the ability to determine if a patient's tissues/cells respond to a battery of potential drug compounds or biological substances, in some cases, without the need to invasively obtain those tissues from the patient suffering from a disease or condition. Some diseases or conditions that may be useful for the high throughput drug screen include, but are not limited to, Alzheimer's Disease, Amyotrophic Lateral Sclerosis, Parkinson's Disease, Liver Sclerosis, Heart Failure, Type 1 Diabetes, and Muscular

Dystrophy.

[0018] In one aspect, the invention comprises a stem-cell based culture system that may be used for generating a 3-dimensional tissue engineering model. This may be done by altering the culturing methods described in International Patent Publication No. WO

2009/151844 such that the cells propagate in a culture setting that will foster 3-dimensional tissue growth, such as with a scaffold or matrix. Such methods are well known to one of ordinary skill in the art. One such example of an appropriate scaffold or matrix is a plate coated with laminin-coated beads . For example, the multipotent stem cells may be encouraged to grow and differentiate 3-dimensionally onto a scaffold or a matrix, such as a plate with laminin-coated beads. Other examples of appropriate scaffolds or matrices are microfluidic chambers and nanofiber membrane scaffolds. This 3-dimensional tissue model provides setting and conditions for differentiation that more closely proximate the tissues/cells in their actual in vivo environment. Moreover, in this capacity, the 3- dimensional tissue model may be used for drug discovery— to determine the efficacy of a drug or agent on an individual's patient's tissues/cells in the context of a 3-dimensional tissue engineering model. Some diseases or conditions that may be useful for drug discovery using a 3-dimensional tissue engineering model include, but are not limited to, Alzheimer's Disease, Amyotrophic Lateral Sclerosis, Parkinson's Disease, Liver Sclerosis, Heart Failure, Type 1 Diabetes, and Muscular Dystrophy.

[0019] In another aspect, the invention comprises a research tool to identify the function of genes involved in the acquisition of multipotency. Microarray analysis of the fibroblast cells during the propagation stage may reveal the identity of genes whose expression levels change during the process of acquiring multipotency. Accordingly, such analysis may reveal one or more genes that are important for the process of generating multipotent stem cells. This information may be useful as a research tool for developing new and different strategies for obtaining and generating large amounts of multipotent stem cells derived from adult tissues/cells. The genes identified may be useful to treat diseases and conditions such as Alzheimer's Disease, Amyotrophic Lateral Sclerosis, Parkinson's Disease, Liver Sclerosis, Heart Failure, Type 1 Diabetes, and Muscular Dystrophy. [0020] In another aspect, the invention comprises a research tool to identify the genes involved in the process of lineage-specific differentiation. Microarray analysis of the cells during the differentiation process reveals the identity of genes whose expression levels change during and as a result of lineage-specific differentiation. Identification of such genes maybe useful in various applications. For example, identification of such genes may reveal genes important for development of lineage-specific cells. Additionally, genes important in the lineage-specific development of a particular cell type may also function as suitable targets for therapeutic intervention to treat diseases and conditions affecting that specific cell type. The genes identified may be useful to treat diseases and conditions such as Alzheimer's Disease, Amyotrophic Lateral Sclerosis, Parkinson's Disease, Liver Sclerosis, Heart Failure, Type 1 Diabetes, and Muscular Dystrophy. In a related aspect, the lineage-specific differentiation may be in a 3-dimensional tissue model. That is, the multipotent stem cells may be encouraged to grow and differentiate 3-dimensionally onto a scaffold or a matrix, such as a plate with laminin-coated beads. This 3-dimensional tissue model provides setting and conditions for differentiation that more closely proximate the tissues/cells in their actual in vivo environment. In this way, it is possible that the microarray data generated by measuring the differential expression of genes during the process of lineage-specific differentiation in a 3-dimensional tissue modeling may represent an improvement over microarray data generated by measuring the differential expression of genes during the process of lineage-specific differentiation when done under conventional means under selective culture conditions. That is, the microarray data generated from a 3-dimensional engineering model might be more accurate, since it more closely proximate the tissues/cells in their actual in vivo environment.

[0021] In another aspect, the invention comprises a diagnostic tool for identifying one or more genes that may be defective in an individual. Microarray analysis of the cells during the differentiation process reveals the identity of genes whose expression levels change during and as a result of lineage-specific differentiation. The differentiation process may be in a 3-dimensional tissue model. That is, the multipotent stem cells may be encouraged to grow and differentiate 3-dimensionally onto a scaffold or a matrix, such as a plate with laminin-coated beads. This 3-dimensional tissue model provides setting and conditions for differentiation that more closely proximate the tissues/cells in their actual in vivo

environment. If microarray analysis reveals that one or more genes are differentially expressed in fibrob last-derived lineage-specific cells taken from an individual suffering from a disease or condition as compared to a normal individual, this may indicate that the expression of those one or more genes is altered, mutated, or defective in that individual. In this way this culture system may function as a diagnostic tool to identify underlying genetic causes of diseases or conditions. The genes identified may be useful to identify diseases and conditions such as Alzheimer's Disease, Amyotrophic Lateral Sclerosis, Parkinson's Disease, Liver Sclerosis, Heart Failure, Type 1 Diabetes, and Muscular Dystrophy.

[0022] In another aspect, the invention provides for autologous therapies based on the propagated multipotent stem cells for regeneration of tissues, for use as grafts, tissue/organ replacement or supplementation, as described in International Patent Publication No. WO 2009/151844. One advantage of the methods described in International Patent Publication No. WO 2009/151844, is that fibroblast-derived multipotent stem cells are propagated without the need for recombinant gene or protein transfer, rendering the multipotent stem cells of the invention safer for use in autologous therapy as compared to other methods that employ recombinant gene or protein transfer. Moreover, karyotype and comparative genomic hybridization (CGH) studies described in Example 6 reveal that the fibroblast- derived multipotent stem cells described herein and in International Patent Publication No. WO 2009/151844 do not exhibit elevated levels of mutations, suggesting the safety of the multipotent stem cells for various applications, such as for autologous therapies. The autologous therapies may be useful to treat disease and conditions such as such as

Alzheimer's Disease, Amyotrophic Lateral Sclerosis, Parkinson's Disease, Liver Sclerosis, Heart Failure, Type 1 Diabetes, and Muscular Dystrophy.

[0023] In one aspect, the invention provides for a method of generating a 3- dimensional tissue engineering model comprising the steps of: (a) propagating multipotent stem cells from human skin fibroblast culture by growing the cells in a culture containing amniotic fluid growth medium (AFM) and allowing the cells to propagate for at least 3 passages; and (b) subjecting said multipotent stem cells to lineage-specific differentiation by culturing said multipotent stem cells in cells in a culture setting that will foster 3-dimensional tissue growth, such as a scaffold or matrix. In a related aspect, the culture further comprises Embryonic Cell Qualified Fetal Bovine Serum (ES-FBS). In another aspect, the cells are subject to at least 3, 4, 5, 6, 7, or 8 passages in culture. In a related aspect, the method comprises the step of determining the number of multipotent stem cells in the culture, and, in another aspect, the number of CD 1 17 ⁺ multipotent stem cells in the culture can be determined after each passage. In another aspect, the human skin fibroblast culture is prolonged by continued passages in the culture until a high number of CD1 17 ⁺ multipotent stem cells is attained. In a related aspect, the propagated CD117 ⁺ multipotent stem cells are subject to differentiation when the CD1 17+ cell count reaches at least about 85%. In another aspect, the propagated cells are cryopreserved after step (a) but before step (b). In another aspect, the propagated multipotent stem cells are capable of differentiating into any of the three germ layers. In a related aspect, the propagated multipotent stem cells are capable of

differentiation into adipose, hepatic, muscle, or nerve cells under suitable culture conditions. In yet another aspect, the suitable culture conditions are conditions will foster 3-dimensional tissue growth are culture plates containing laminin-coated beads. In a related aspect, the culture plates containing laminin-coated beads are created by: (a) dissolving laminin in cold phosphate buffer saline (PBS) placed in a tissue culture plate; (b) adding sterile spherical glass beads or a mix of spherical glass beads to the laminin; (c) placing the culture plate in an incubator at 37°C for at least 12 hours in order to induce polymerization of laminin; and (d) removing excess PBS and allowing the culture plate to completely air dry.

[0024] In another aspect, the invention provides a method of generating a 3- dimensional tissue engineering model comprising the steps of: (a) propagating multipotent stem cells from human skin fibroblast culture by growing the cells in a culture containing amniotic fluid growth medium (AFM) and allowing the cells to propagate for at least 3 passages; (b) culturing the multipotent stem cells in the laminin-coated bead plates in a tissue culture media that promotes differentiation into one of the three germ layers, wherein the laminin-coated bead plates were created by: (1) dissolving laminin in cold phosphate buffer saline (PBS) placed in a tissue culture plate; (2) adding sterile spherical glass beads or a mix of spherical glass beads to the laminin; (3) placing the culture plate in an incubator at 37°C for at least 12 hours in order to induce polymerization of laminin; (4) removing excess PBS and allowing the culture plate to completely air dry; (5) adding the multipotent stem cells to the laminin-coated bead plates; and (6) plating the multipotent stem cells in the laminin- coated bead plates with the multipotent stem cells in an incubator at 37°C; and (c) subjecting the multipotent stem cells to lineage-specific differentiation under suitable conditions into cells of any of three germ layers.

[0025] In a related aspect, the invention provides a method for identifying one or more genes involved in the process of lineage-specific differentiation, said method comprising the steps of: (a) propagating multipotent stem cells from human skin fibroblast culture by growing the cells in a culture containing amniotic fluid growth medium (AFM) and allowing the cells to propagate for at least 3 passages; (b) subjecting said multipotent stem cells to lineage-specific differentiation by culturing said multipotent stem cells under culture conditions suitable for lineage-specific differentiation until differentiated cells result; (c) subjecting said differentiated cells to gene expression profiling using microarray technology; and (d) determining which one or more genes is upregulated or downregulated during the process of lineage-specific differentiation. In a related aspect, the culture containing amniotic fluid growth medium (AFM) further comprises Embryonic Cell

Qualified Fetal Bovine Serum (ES-FBS). In another aspect, the cells are subject to at least 3, 4, 5, 6, 7, or 8 passages in culture. In another aspsect, the method further comprises the step of determining the number of multipotent stem cells in the culture. In another aspect, the number of CD1 17 ⁺ multipotent stem cells in the culture can be determined after each passage. In a related aspect, the human skin fibroblast culture is prolonged by continued passages in the culture until a high number of CD1 17 ⁺ multipotent stem cells is attained. In a related aspect, the propagated CD1 17 ⁺ multipotent stem cells are subject to differentiation when the CD1 17+ cell count reaches at least about 85%. In another aspect, the propagated cells are cryopreserved after step (a) but before step (b). In another aspect, the propagated multipotent stem cells are capable of differentiating into any of the three germ layers. In a related aspect, the propagated multipotent stem cells are capable of differentiation into adipose, hepatic, muscle, or nerve cells under suitable culture conditions. In another aspect, the suitable culture conditions will foster 3 -dimensional tissue growth, such as a scaffold or matrix. In a related aspect, the culture conditions that will foster 3-dimensional tissue growth are culture plates containing laminin-coated beads. In another aspect, the culture plates containing laminin-coated beads are created by: (a) dissolving laminin in cold phosphate buffer saline (PBS) placed in a tissue culture plate; (b) adding sterile spherical glass beads or a mix of spherical glass beads to the laminin; (c) placing the culture plate in an incubator at 37°C for at least 12 hours in order to induce polymerization of laminin; and (d) removing excess PBS and allowing the culture plate to completely air dry.

1. In one aspect, the invention relates to an isolated multipotent stem cell, or a collection of culture of isolated multipotent stem cells, obtained by a method of propagating multipotent stem cells from human skin fibroblast culture by growing the cells in a culture containing amniotic fluid growth medium (AFM) and allowing the cells to propagate for at least 3 passages. In a related aspect, the the culture further comprises Embryonic Cell Qualified Fetal Bovine Serum (ES-FBS). In another aspect, the multipotent stem cells are capable differentiating into any of the three germ layers.

[0026] In another aspect, the invention is directed to an isolated differentiated cell, or a collection of culture of isolated differentiated cells, obtained by: (a) propagating multipotent stem cells from human skin fibroblast culture by growing the cells in a culture containing amniotic fluid growth medium (AFM) and allowing the cells to propagate for at least 3 passages; and (b) subjecting said multipotent stem cells to lineage-specific differentiation by culturing said multipotent stem cells under culture conditions suitable for lineage-specific differentiation until differentiated cells result. In a related aspect, the differentiated cells are cells of any of the three germ layers. In another aspect, the cells of any of the three germ layers include adipose, hepatic, muscle, or nerve cells. In a related aspect, the culture conditions suitable for lineage-specific differentiation foster 3-dimensional tissue growth. In another aspect, the culture conditions suitable for lineage-specific differentiation foster 3-dimensional tissue growth are culture plates containing laminin- coated beads. In another aspect, the culture plates containing laminin-coated beads are created by: (a) dissolving laminin in cold phosphate buffer saline (PBS) placed in a tissue culture plate; (b) adding sterile spherical glass beads or a mix of spherical glass beads to the laminin; (c) placing the culture plate in an incubator at 37°C for at least 12 hours in order to induce polymerization of laminin; and (d) removing excess PBS and allowing the culture plate to completely air dry..

EXAMPLES

[0027] FIGURES

[0028] FIG. 1 is a table showing the number of genes having at least a two-fold difference in expression levels (increase or decrease) from fibroblasts taken from 3 different patients cultured in tissue culture medium containing amniotic fluid growth medium and other media and various growth factors, as described in International Patent Publication No. WO 2009/151844, in order to drive propagation of multipotent stem cells, after passages 1, 2, and 3.

[0029] FIG. 2 is a Principle Component Analysis (PCA) plot showing the distribution of genes expressed from fibroblast-derived multipotent stem cells for 3 patients as the cells progress through adipose tissue differentiation, taken at day 0, 1, 3, 7, 10, 15, and 21.

[0030] FIG. 3 is a PCA plot showing the distribution of genes expressed from fibroblast-derived multipotent stem cells for 3 patients as the cells progress through hepatic tissue differentiation, taken at day 0, 1, 3, 6, 10, 12, 17, and 25.

[0031] FIG. 4 is a heat map and clustering diagram of the entire genome, comparing the gene expression of the undifferentiated fibroblast-derived multipotent stem cells from 3 patients against the gene expression of adult obese adipose tissue samples.

[0032] FIG. 5 is a heat map and clustering diagram of the entire genome, comparing the gene expression of the undifferentiated fibroblast-derived multipotent stem cells from 3 patients against the gene expression of adult lean adipose tissue samples. [0033] FIG. 6 is a heat map and clustering diagram of the entire genome, comparing the gene expression of the undifferentiated fibroblast-derived multipotent stem cells from 3 patients against the gene expression of adult hepatic tissue samples.

[0034] FIG. 7 is a heat map and clustering diagram showing gene clusters that have at least a two-fold difference in expression levels from fibroblast-derived multipotent stem cells from 3 patients as the cells progress through adipose tissue differentiation, taken at day 7, 10, 15, and 21.

[0035] FIG. 8 is a heat map and clustering diagram showing gene clusters that have at least a two-fold difference in expression levels from fibroblast-derived multipotent stem cells from 3 patients as the cells progress through hepatic tissue differentiation, taken at day 6, 10, 12, 17, and 25.

[0036] FIG. 9 is a heat map and clustering diagram of an individual 37 year old patient (Sample 970) showing the genes that have at least a two-fold difference in expression levels from the patient's fibroblasts samples (Control) as the cells differentiate into adipose tissue, taken at 24 hours, 3 days, 7 days, 10 days, 15 days, and 21 days after culture in media that promotes differentiation into adipose tissue.

[0037] FIG. 10 is a heat map and clustering diagram of an individual 3 day old patient (Sample 1650) showing the genes that have at least a two-fold difference in expression levels from the patient's fibroblasts samples (Control) as the cells differentiate into adipose tissue, taken at 24 hours, 3 days, 7 days, 10 days, 15 days, and 21 days after culture in media that promotes differentiation into adipose tissue.

[0038] FIG. 11 is a heat map and clustering diagram of an individual 96 year old patient (Sample 731) showing the genes that have at least a two-fold difference in expression levels from the patient's fibroblasts samples (Control) as the cells differentiate into adipose tissue, taken at 24 hours, 3 days, 7 days, 10 days, 15 days, and 21 days after culture in media that promotes differentiation into adipose tissue

[0039] FIG. 12 is a summary of the data from FIGS. 9-11.

[0040] FIG. 13 is a heat map and clustering diagram of the entire genome, comparing the gene expression of the differentiated adipose tissue from the 3 patients (after 21 days in media that promotes differentiation into adipose tissue) against the gene expression of adult lean adipose tissue samples.

[0041] FIG. 14 is a heat map and clustering diagram of the entire genome, comparing the gene expression of the differentiated adipose tissue from the 3 patients (after 21 days in media that promotes differentiation into adipose tissue) against the gene expression of adult obese adipose tissue samples.

[0042] FIG. 15 is a heat map and clustering diagram of the entire genome, comparing the gene expression of adult obese adipose tissue samples against the gene expression of adult lean adipose tissue samples.

[0043] FIG. 16 is a heat map and clustering diagram of the entire genome, comparing the gene expression of the differentiated hepatic tissue from the 3 patients (after 25 days in media that promotes differentiation into hepatic tissue) against the gene expression of adult liver tissue samples.

[0044] FIG. 17 is a collection of data showing a heat map and clustering diagram of the entire genome, comparing the gene expression of fibroblast-derived multipotent stem cells, differentiated adipose tissue from the 3 patients (after 21 days in media that promotes differentiation into adipose tissue), and adult lean adipose tissue samples, and adult obese adipose tissue samples.

[0045] FIG. 18 is a collection of data showing a heat map and clustering diagram of the entire genome, comparing the gene expression of fibroblast-derived multipotent stem cells, differentiated hepatic tissue from the 3 patients (after 25 days in media that promotes differentiation into hepatic tissue), and adult hepatic tissue samples.

[0046] FIG. 19 is a PCA plot showing the distribution of genes expressed from fibroblast-derived multipotent stem cells for 3 patients as the cells progress through muscle tissue differentiation, taken at day 0, 1.5, 4.5, 7.5, 10.5, 13.5, 16.5, and 19.5.

[0047] FIG. 20 is a PCA plot showing the distribution of genes expressed from fibroblast-derived multipotent stem cells for 3 patients as the cells progress through nerve tissue differentiation, taken at day 0, 0.5, 1, 2, 4, 6, and 8.

[0048] FIG. 21 is a heat map and clustering diagram showing gene clusters that have at least a two-fold difference in expression levels from fibroblast-derived multipotent stem cells from 3 patients as the cells progress through muscle tissue differentiation, taken at day 1.5, 4.5, 7.5, 10.5, 13.5, 16.5, and 19.5.

[0049] FIG. 22 is a PCA plot showing the distribution of genes expressed from skin fibroblasts from 3 patients when cultured in: (a) conventional media (Eagles-based MEM); and (b) media that propagates multipotent stem cells (a-MEM media plus supplements).

[0050] FIG. 23. like FIG. 8. is a heat map and clustering diagram showing gene clusters that have at least a two-fold difference in expression levels from fibroblast-derived multipotent stem cells from 3 patients as the cells progress through hepatic tissue differentiation, however, taken at day 7, 10, 15, and 21. [0051] FIG. 24 is a heat map and clustering diagram of an individual 37 year old patient (Sample 970) showing the genes that have at least a two-fold difference in expression levels from the patient's fibroblasts samples (Control) as the cells differentiate into hepatic tissue, taken at 24 hours, 3 days, 6 days, 10 days, 12 days, 17 days, and 25 days after culture in media that promotes differentiation into hepatic tissue.

[0052] FIG. 25 is a heat map and clustering diagram of an individual 3 day old patient (Sample 1650) showing the genes that have at least a two-fold difference in expression levels from the patient's fibroblasts samples (Control) as the cells differentiate into hepatic tissue, taken at 24 hours, 3 days, 6 days, 10 days, 12 days, 17 days, and 25 days after culture in media that promotes differentiation into hepatic tissue.

[0053] FIG. 26 is a heat map and clustering diagram of an individual 96 year old patient (Sample 731) showing the genes that have at least a two-fold difference in expression levels from the patient's fibroblasts samples (Control) as the cells differentiate into hepatic tissue, taken at 24 hours, 3 days, 6 days, 10 days, 12 days, 17 days, and 25 days after culture in media that promotes differentiation into hepatic tissue.

[0054] FIG. 27 is a summary of the data from FIGS. 24-26.

[0055] FIG. 28. like FIG. 8 and FIG. 23. shows a heat map and clustering diagram showing gene clusters that have at least a two-fold difference in expression levels from fibrob last-derived multipotent stem cells from 3 patients as the cells progress through hepatic tissue differentiation, however, taken at day 1, 3, 67, 10, 15, and 21.

[0056] FIG. 29 is a PCA plot showing the distribution of genes expressed from fibroblast-derived multipotent stem cells for 3 patients as the cells progress through nerve tissue differentiation, taken at day 0, 0.5, 1, 2, 4, 6, and 8. [0057] FIG. 30, a summary of FIGS. 34-36, is a heat map and clustering diagram of

3 patients showing the genes that have at least a two-fold difference in expression levels from the patient's fibroblasts samples (Control) as the cells differentiate into nerve tissue, taken at 12 hours, 24 hours, 2 days, 4 days, 6 days, and 8 days after culture in media that promotes differentiation into nerve tissue.

[0058] FIG. 31 is a heat map and clustering diagram of an individual 96 year old patient (Sample 731) showing the genes that have at least a two-fold difference in expression levels from the patient's fibroblasts samples (Control) as the cells differentiate into muscle tissue, taken at 1.5 days, 4.5 days, 7.5 days, 10.5 days, 13.5 days, and 16.5 days after culture in media that promotes differentiation into muscle tissue.

[0059] FIG. 32 is a heat map and clustering diagram of an individual 37 year old patient (Sample 970) showing the genes that have at least a two-fold difference in expression levels from the patient's fibroblasts samples (Control) as the cells differentiate into muscle tissue, taken at 1.5 days, 4.5 days, 7.5 days, 10.5 days, 13.5 days, and 16.5 days after culture in media that promotes differentiation into muscle tissue.

[0060] FIG. 33 is a heat map and clustering diagram of an individual 3 day old patient (Sample 1650) showing the genes that have at least a two-fold difference in expression levels from the patient's fibroblasts samples (Control) as the cells differentiate into muscle tissue, taken at 1.5 days, 4.5 days, 7.5 days, 10.5 days, 13.5 days, and 16.5 days after culture in media that promotes differentiation into muscle tissue.

[0061] FIG. 34 is a heat map and clustering diagram of an individual 96 year old patient (Sample 731) showing the genes that have at least a two-fold difference in expression levels from the patient's fibroblasts samples (Control) as the cells differentiate into nerve tissue, taken at 0.5, 1, 2, 4, 6, and 8 days after culture in media that promotes differentiation into nerve tissue.

[0062] FIG. 35 is a heat map and clustering diagram of an individual 37 year old patient (Sample 970) showing the genes that have at least a two-fold difference in expression levels from the patient's fibroblasts samples (Control) as the cells differentiate into nerve tissue, taken at 0.5, 1, 2, 4, 6, and 8 days after culture in media that promotes differentiation into nerve tissue.

[0063] FIG. 36 is a heat map and clustering diagram of an individual 3 day old patient (Sample 1650) showing the genes that have at least a two-fold difference in expression levels from the patient's fibroblasts samples (Control) as the cells differentiate into nerve tissue, taken at 0.5, 1, 2, 4, 6, and 8 days after culture in media that promotes differentiation into nerve tissue.

[0064] Example 1

[0065] Fibroblast were obtained from 3 different patients ranging in age from 3 days old (Sample 1650); 37 years old (Sample 970); and 96 years old (Sample 731). The fibroblasts were cultured in medium comprising amniotic growth fluid media (AFM) (as described in DeCoppi et al, comprising a-MEM (Invitrogen), 15% ES-FBS (Invitrogen), 1% L-Glutamine, and 1% Pen/Strep, supplemented with 18% CHANG MEDIUM® B (Irvine Scientific) and 2% CHANG MEDIUM® C (Irvine Scientific)), so as to propagate multipotent stem cells, as is described in International Patent Publication No. WO 2009/151844. The AFM comprises a-MEM media plus supplements.

[0066] After passages 1, 2, and 3, cells were harvested and subject to gene expression profiling using the Affymetrix GENECHIP® (Affymetrix GENECHIP® microarray technology) Human Gene 1.0 ST Array, as described in International Patent Publication No. WO 2009/151844.

[0067] Figure 1 is a table that shows the number of genes that exhibit at least a twofold difference in gene expression (increase or decrease) of fibroblast cells cultured in tissue culture medium containing amniotic fluid growth medium and other media and various growth factors, as described in International Patent Publication No. WO 2009/151844, in order to drive propagation of multipotent stem cells, after passages 1 , 2, and 3, as compared against the gene expression of the resting fibroblasts in traditional MEM media as described in International Patent Publication No. WO 2009/151844. Additionally, the table shows the number of genes exhibit at least a two-fold difference in expression levels (increase or decrease) that are common across all 3 patients. These studies demonstrate that these fibroblast cells obtained from 3 very different individuals are upregulating and

downregulating a fair number of the same genes when subject to the same media conditions. This suggests that the process of obtaining multipotency may involve a genetic expression profile shared by all humans, regardless of age.

[0068] Example 2

[0069] The fibroblast-derived multipotent stem cells from the 3 patients in Example 1 were subject to differentiation media conditions suitable to promote lineage-specific differentiation as described in International Patent Publication No. WO 2009/151844.

[0070] Figure 2 depicts a Principle Component Analysis (PCA) plot showing the distribution of genes expressed from fibroblast-derived multipotent stem cells for the 3 patients as the cells progress through adipose tissue differentiation, taken at day 0, 1 , 3, 7, 10, 15, and 21 of culture. Each of the 3 patients is represented by a sphere. Similarly, Figure 3 depicts a PCA plot showing the distribution of genes expressed from fibroblast-derived multipotent stem cells for the 3 patients as the cells progress through hepatic tissue differentiation, taken at day 0, 1, 3, 6, 10, 12, 17, and 25. Figure 19 depicts a PCA plot showing the distribution of genes expressed from fibroblast-derived multipotent stem cells for the 3 patients as the cells progress through muscle tissue differentiation, taken at day 0, 1.5, 4.5., 7.5, 10.5, 13.5, 16.5, and 19.5. Figure 20 depicts a PCA plot showing the distribution of genes expressed from fibroblast-derived multipotent stem cells for the 3 patients as the cells progress through nerve tissue differentiation, taken at day 0, 0.5, 1, 2, 4, 6, and 8. The clustering of the patient data points, in Figures 2, 3, 19, and 20, at each time point indicates that the cells for patient are undergoing many of the same genetic expression changes when undergoing the same lineage-specific differentiation process.

[0071] Example 3

[0072] Undifferentiated fibroblast-derived multipotent stem cells from the 3 patient samples and adult obese adipose tissue samples, adult lean adipose tissue samples, and adult liver tissue samples were subject to microarray analysis as described in International Patent Publication No. WO 2009, which protocols, methods, and materials are incorporated herein by reference. The data from the adult obese adipose, adult lean adipose, and adult liver tissue samples, subject to the same microarray analysis, were obtained from the Gene Expression Omnibus at the National Center for Biotechnology Information, NIH. Figures 4-6 show heat maps and clustering diagrams of the whole genome showing gene expression profiles of the undifferentiated fibroblast-derived multipotent stem cells against obese adipose tissue (Figure 4), lean adipose tissue (Figure 5), and liver tissue (Figure 6). Differential expression is observed by intensity of color (black or white) for the expression level of that corresponding gene. The data from Figures 4-6 show similar gene expression profiles as between the 3 different patients and similar gene expression profiles as between the individual adult tissues. However, the gene expression profiles are dramatically different as between the

undifferentiated fibroblast-derived multipotent stem cells and any of the adult tissue samples.

[0073] Example 4

[0074] The fibroblast-derived multipotent stem cells from the 3 patients were subject to differentiation media conditions suitable to promote lineage-specific differentiation as described in International Patent Publication No. WO 2009/151844, which protocols, methods, and materials are incorporated herein by reference. At various time points during the differentiation process, cells were collected and were subject to a microarray analysis as described in International Patent Publication No. WO 2009/151844. For adipose cell differentiation, cells were harvested at day 7, 10, 15, and 21 ; for hepatic differentiation, cells were harvested at day 6, 10, 12, and 25; and for muscle differentiation, cells were harvested at day 1.5, 4.5, 7.5, 10.5, 13.5, 16.5, and 19.5. Figure 7 shows a heat map and clustering diagram showing the expression profiles of genes exhibiting at least a two-fold change in expression for the fibroblast-derived multipotent stem cells from the 3 patients undergoing adipose cell differentiation. Figures 8 and 23 shows a heat map and clustering diagram showing the expression profiles of clusters of genes exhibiting at least a two-fold change in expression (increase or decrease) for the fibroblast-derived multipotent stem cells from the 3 patients undergoing hepatic cell differentiation. Figure 21 shows a heat map and clustering diagram showing the expression profiles of clusters of genes exhibiting at least a two-fold change in expression (increase or decrease) for the fibroblast-derived multipotent stem cells from the 3 patients undergoing muscle cell differentiation. The data in Figures 7, 8, 21 , and 23 show that the patient samples exhibit increasingly similar gene expression profiles toward the end of the differentiation cycle. Indeed, the gene expression profiles toward the end of the differentiation cycle resemble the gene expression profiles of the adult tissues samples from Example 3 (Figures 4-6).

[0075] These data were also analyzed on an individual patient basis. Figures 9-11 contain heat maps and cluster diagrams showing the expression profiles of genes exhibiting at least a two-fold change in expression (increase or decrease) for the fibroblast-derived multipotent stem cells from the 3 patients undergoing adipose cell differentiation at 1, 3, 7, 10, 15, and 21 days for Sample 970 (37 year old) (Figure 9), Sample 1650 (3 day old) (Figure 10), and Sample 731 (96 year old) (Figure 1 1). Figure 12 is a summary of the data from Figures 9-1 1. Figure 12 supports the data of Figures 7, 8, 21 , and 23, showing that the patient samples exhibit increasingly similar gene expression profiles toward the end of the differentiation cycle.

[0076] Similarly, Figures 24-26 contain heat maps and cluster diagrams showing the expression profiles of genes exhibiting at least a two-fold change in expression (increase or decrease) for the fibroblast-derived multipotent stem cells from the 3 patients undergoing hepatic cell differentiation at 1, 3, 6, 10, 12, 17, and 25 days for Sample 970 (37 year old) (Figure 24), Sample 1650 (3 day old) (Figure 25), and Sample 731 (96 year old) (Figure 26). Figure 27 is a summary of the data from Figures 24-26. Figure 27 supports the data of showing that the patient samples exhibit increasingly similar gene expression profiles toward the end of the differentiation cycle.

[0077] Example 5

[0078] Studies were conducted to determine if the lineage-specific differentiated cells, which were derived from fibroblasts, show similar gene expression profiles as those actual adult cells of the same corresponding lineage. To that end, fibroblast-derived multipotent stem cells from the 3 patient samples were subject to differentiation under conditions of adipose cell differentiation (for 21 days) or hepatic cell differentiation (for 25 days). These differentiated fibroblast-derived adipose cells were subject to microarray analysis along with samples of adult lean adipose tissue (Figure 13) and adult obese adipose tissue (Figures 14). Similarly, these differentiated fibroblast-derived hepatic cells were subject to microarray analysis along with samples of adult liver tissue (Figure 16). The data from the adult obese adipose, adult lean adipose, and adult liver tissue samples, subject to the same microarray analysis, were obtained from the Gene Expression Omnibus at the National Center for Biotechnology Information, NIH. Figure 13 and Figure 14 show a heat map and cluster diagram showing the expression profiles of the whole genome for differentiated fibroblast-derived adipose cells against adult lean adipose tissue and adult obese adipose tissue, respectively. Figure 15 shows a heat map and cluster diagram showing the expression profiles of the whole genome for adult lean adipose tissue and adult obese adipose tissue. Figure 16 shows a heat map and cluster diagram showing the expression profiles of the whole genome for differentiated fibroblast-derived hepatic cells against adult liver tissue.

[0079] Lastly, Figure 17 is a collection of data showing a heat map and clustering diagram of the entire genome, comparing the gene expression of fibroblast-derived multipotent stem cells, differentiated fibroblast-derived adipose tissue from the 3 patients, and adult lean adipose tissue samples, and adult obese adipose tissue samples. Similarly, Figure 18 is a collection of data showing a heat map and clustering diagram of the entire genome, comparing the gene expression of fibroblast-derived multipotent stem cells, differentiated fibroblast-derived hepatic tissue from the 3 patients, and adult hepatic tissue samples.

[0080] Collectively, these data show that from a gene expression profile perspective, the differentiated lineage-specific tissue generated from fibroblasts closely resemble those of actual tissue from that lineage. This is true for adipose and hepatic tissues. Thus, the data presented in this Example suggests that multipotent stem cells produced by the disclosed methods are capable of differentiation into cells of any of the 3 germ layers and that in terms of gene expression, the fibroblast-derived differentiated multipotent stem cells approximate normal differentiated tissue.

[0081] Example 6

[0082] DNA from fibroblast-derived multipotent stem cells from the 3 patients at passage 3 was analyzed by karyotyping and by comparative genomic hybridization (CGH). Results from both analyses show that the fibroblast-derived multipotent stem cells do not exhibit an increased rate of mutations (data not shown). These results indicate that the methods described herein for propagation of multipotent stem cells does not increase the rate of mutations, suggesting the safety of the multipotent stem cells.

[0083] Example 7

[0084] Skin fibroblasts from 3 patients of varying ages were subject to PCA analysis at two different time points: (1) while culturing in standard conventional media (Eagles-based MEM media); and (2) while culturing for 3 passages in media that promotes propagation of multipotent stem cells (a-MEM media plus supplements). The PCA, shown in Figure 22, detects gene changes that occur during the transfer in media. These studies show that the 3 patient samples appear to cluster together as cells under the 3 passages in media that promotes propagation of multipotent stem cells.

[0085] Example 8 [0086] A 3 -dimensional tissue model was generated and used to identify differentially expressed genes during hepatic differentiation, using lamin bead plates as a 3-dimensional scaffold or matrix for cellular growth and differentiation.

[0087] Laminin-Coated Bead Plates

[0088] The 3-dimensional tissue model was based on the use of plates with laminin- coated beads, or laminin-coated bead plates. These laminin-coated bead plates were generated by first obtaining a stock substrate of laminin at a concentration of 1 mg/ml dissolved in cold Phosphate Buffered Saline (PBS). Roughly 1 ml of this stock substrate of laminin was added to each well of a 6-well Falcon Tissue Culture plate. Spherical glass beads (Sartorius, Inc.) were then obtained and allowed to sit under ultraviolet (UV) excitation for sterilization. In this experiment, a mixture of beads (20-25 micron diameter (66.7%) and 17-20 micron diameter (33.3%)) was employed. After the roughly 1 ml of laminin was added to the wells, beads were added to the laminin. The plate was then placed in an incubator at 37°C for 12 hours in order to induce polymerization of the laminin. After this incubation period, the plate, any excess PBS was drawn off using a sterile pipette, and the plate was allowed to completely air dry.

[0089] Fibroblast-Derived Multipotent Stem Cells.

[0090] Fibroblast-derived multipotent stem cells from 3 patients were generated as described in Example 1 (i.e., in media comprising amniotic medium comprising amniotic growth fluid media (AFM) (as described in DeCoppi et al, comprising a-MEM (Invitrogen), 15% ES-FBS (Invitrogen), 1% L-Glutamine, and 1% Pen/Strep, supplemented with 18% CHANG MEDIUM® B (Irvine Scientific) and 2% CHANG MEDIUM® C (Irvine

Scientific)), so as to propagate multipotent stem cells, as described in International Patent Publication No. WO 2009/151844). The AFM comprises a-MEM media plus supplements. [0091] 3-Dimensional Tissue Model

[0092] These cells were then plated onto the laminin-coated bead plates at a concentration of 5,000 cells/cm ² and then placed back into the 37°C incubator at an atmosphere of 5% C0 ₂ for three days. The media was then removed from wells containing the fibroblast-derived multipotent stem cells in laminin-coated bead plates, and was replaced with culture media conditioned to promote hepatic differentiation. A coverslip (about 8 cm ² ) was then placed atop the cells in the wells of the laminin-coated bead plate, which created a 3-dimensional space for the cells to then grown and differentiate. Culture media from control (undifferentiated) wells was replaced again with media comprising amniotic medium comprising amniotic growth fluid media (AFM). The AFM comprises a-MEM media plus supplements.

[0093] For cells undergoing hepatic differentiation, fresh hepatic differentiation culture media was replaced every three days by gently removing old culture media and adding fresh new culture media. By contrast, undifferentiated control multipotent stem cells were given fresh media comprising amniotic medium comprising amniotic growth fluid media (AFM). The AFM comprises a-MEM media plus supplements.

[0094] Cells grown in the hepatic differentiation media on laminin-coated bead plates and undifferentiated control multipotent stem cells were harvested at days 1 , 3, 6, 10, 12, 17, 25, 33, 38, and 45 for microarray analysis. The microarray analysis was conducted to assess the gene expression changes over this period of time in the 3-dimensional model of differentiating hepatic cells as compared to control undifferentiated cells. Microarray analysis was conducted as described in International Patent Publication No. WO

2009/151844 [0095] The microarray results are shown in tabular format for hepatic differentiation after day 1 (Table 1), day 3 (Table 2), day 6 (Table 3), day 10 (Table 4), day 12 (Table 5), day 17 (Table 6), day 25 (Table 7), day 33 (Table 8), day 38 (Table 9), and day 45 (Table 10) for each of 3 individuals. Each individual is designated by their identification numbers, 731, 970, and 1650. These data show that individual 731, 970, and 1650 each had 93, 88, and 82 genes, respectively, which were up- or down-regulated 15-fold for at least two time points. Taken together, these studies show that there are a number of common genes that are differentially regulated across individuals during hepatic differentiation. These common genes may provide insight into genes that are important in hepatic differentiation and hepatic biology and function.

Table 1. Gene expression profiles for samples from three individuals (identified by numbers 731, 970, and 1650) of hepatic differentiated (day 1) vs. undifferentiated cell types. All genes displaying at least 2-fold up- or down- regulation common in all three individuals are shown.

Fold- Fold- Fold-

Probe mRNA Change Change Change Set ID Gene Symbol Accession (731) (970) (1650)

7947199 LGR4 NM 018490 -2.36058 -2.16172 -5.77244

7982889 NUSAP1 NM 016359 -2.3873 -2.77451 -2.37489

7969243 CKAP2 NM 018204 -2.43552 -2.65892 -2.46081

7917255 SSX2IP NM 014021 -2.4554 -3.49591 -3.04079

8095585 SLC4A4 NM 001098484 -2.49169 -2.26387 -3.181 14

8151 101 YBL1 NM 001080416 -3.06675 -3.94656 -3.77018

7923086 ASPM NM 018136 -3.23469 -3.68736 -3.54465

7971 104 TRPC4 NM 016179 -3.55001 -4.13931 -5.15296

7976567 BDKRB1 NM 000710 -4.20844 -3.6416 -5.43657

8015349 KRT19 NM 002276 -4.51085 -3.52882 -4.24316

Table 2. Gene expression profiles for samples from three individuals (identified by numbers 731, 970, and 1650) of hepatic differentiated (day 3) vs. undifferentiated cell types. All genes displaying at least 3-fold up- or down- regulation common in all three individuals are shown.

Fold- Fold- Fold-

Probe mRNA Change Change Change Set ID Gene Symbol Accession (731) (970) (1650)

8092177 NCEH1 NM 001 146276 -5.01826 -6.071 16 -3.58301

8046380 ITGA6 N 000210 -16.2581 -9.85997 -11.7434

7984540 KIF23 NM 138555 -6.04761 -5.69183 -3.35572

8085138 OXTR NM 000916 -8.44601 -6.57678 -5.00445

7947199 LGR4 NM 018490 -3.22619 -3.18139 -6.25298

7917255 SSX2IP NM 014021 -6.28932 -8.04475 -5.49439

8095585 SLC4A4 NM 001098484 -7.06149 -6.99498 -7.35258

8151101 MYBL1 NM 001080416 -6.79619 -10.2036 -7.42154

7923086 ASPM NM 018136 -6.62611 -8.59572 -3.58084

7971104 TRPC4 NM 016179 -5.3725 -4.47693 -5.68616

7987315 ACTC1 NM 005159 -13.1087 -10.4742 -23.0797

7976567 BDKRB1 NM 000710 -11.3348 -1 1.1869 -8.66277

8015349 KRT19 NM 002276 -8.5028 -6.84666 -6.31428

Table 3. Gene expression profiles for samples from three individuals (identified by numbers 731, 970, and 1650) of hepatic differentiated (day 6) vs. undifferentiated cell types. All genes displaying at least 10-fold up- or down- regulation common in all three individuals are shown.

Fold- Fold- Fold-

Probe mRNA Change Change Change Set ID Gene Symbol Accession (731) (970) (1650)

7906930 NUF2 NM 145697 -24.1246 -20.6648 -20.6703

8120838 TTK NM 003318 -17.0286 -28.0153 -15.2684

7983969 CCNB2 NM 004701 -14.0894 -15.2591 -13.5907

8054580 BUB1 NM 004336 -13.4537 -17.7715 -14.1001

7929258 KIF11 NM 004523 -14.511 -15.265 -12.9951

7909708 CENPF NM 016343 -10.5236 -12.817 -11.5195

8102643 CCNA2 NM 001237 -12.2259 -14.9277 -10.6569

8014974 TOP2A NM 001067 -12.9532 -17.4024 -12.5757

8109712 HM R NM 001142556 -1 1.8044 -10.9385 -1 1.4646

7984540 KIF23 NM 138555 -14.7154 -17.1207 -15.1236

8132318 ANLN NM 018685 -21.2876 -20.1581 -20.0911

7900699 CDC20 NM 001255 -22.4067 -14.9763 -14.7787

8108301 KIF20A NM 005733 -20.83 -25.5036 -22.3207

8149955 PBK NM 018492 -16.1135 -19.6492 -13.5689

7982889 NUSAP1 NM 016359 -10.9847 -15.6213 -11.5089

7994109 PLK1 NM 005030 -14.5652 -12.1821 -13.2616

8151 101 YBL1 NM 001080416 -10.36 -13.739 -15.2515

7979307 DLGAP5 NM 014750 -27.0698 -28.2352 -15.785

7923086 ASPM NM 018136 -29.3922 -34.6268 -23.3511

7987315 ACTC1 NM 005159 -11.9448 -12.6572 -28.2205

7976567 BDKRB1 NM 000710 -13.9694 -14.1704 -10.7645

Table 4. Gene expression profiles for samples from three individuals (identified by numbers 731, 970, and 1650) of hepatic differentiated (day 10) vs. undifferentiated cell types. All genes displaying at least 10-fold up- or down- regulation common in all three individuals are shown.

Fold- Fold- Fold-

Probe mRNA Change Change Change Set ID Gene Symbol Accession (731) (970) (1650)

8046380 ITGA6 N 000210 -34.8909 -22.8495 -22.6072

7929334 CEP55 NM 018131 -17.743 -11.7215 -18.6339

8054702 CKAP2L NM 152515 -12.228 -10.3821 -10.4132

8085754 SGOL1 NM 001012410 -16.8883 -13.0281 -14.6698

7982757 CASC5 NM 170589 -27.7392 -14.0897 -18.8298

8061579 TPX2 NM 012112 -19.7956 -10.0017 -13.5713

7974404 CDKN3 NM 005192 -28.3035 -20.3289 -21.6472

7906930 NUF2 NM 145697 -16.7132 -11.1472 -18.7732

8120838 TTK NM 003318 -21.2318 -15.547 -18.2735

7983969 CCNB2 NM 004701 -20.6167 -12.0802 -12.6952

8054580 BUB1 NM 004336 -18.9961 -11.8212 -19.9417

7929258 KIF11 NM 004523 -19.6767 -11.8609 -14.6221

7984540 KIF23 NM 138555 -18.1939 -11.6047 -13.1659

8132318 ANLN NM 018685 -27.7326 -11.6962 -19.1003

7900699 CDC20 NM 001255 -26.875 -13.2593 -13.6862

8108301 KIF20A NM 005733 -27.1896 -17.5099 -21.2622

8149955 PBK NM 018492 -21.0555 -11.7959 -1 1.9119

7982889 NUSAP1 NM 016359 -16.63 -12.7869 -1 1.7961

7994109 PLK1 NM 005030 -15.7272 -11.0327 -11.6765

8095585 SLC4A4 NM 001098484 -17.6935 -10.2597 -10.8457

7979307 DLGAP5 NM 014750 -29.9358 -11.7283 -16.6535

7923086 ASP NM 018136 -37.1396 -17.3282 -21.189

7987315 ACTC1 NM 005159 -12.8655 -11.1982 -29.4309

7976567 BDKRB1 NM 000710 -19.098 -12.0674 -12.1562

Table 5. Gene expression profiles for samples from three individuals (identified by numbers 731, 970, and 1650) of hepatic differentiated (day 12) vs. undifferentiated cell types. All genes displaying at least 10-fold up- or down- regulation common in all three individuals are shown.

Fold- Fold- Fold-

Probe mRNA Change Change Change Set ID Gene Symbol Accession (731) (970) (1650)

8142981 PODXL N 001018111 -25.3486 -28.3843 -20.1403

8001133 SHCBP1 N 024745 -14.0914 -16.9758 -11.2108

8117594 HIST1 H2BM NM 003521 -18.6634 -13.8923 -12.9928

8145570 ESC02 NM 001017420 -22.0196 -16.3605 -20.4903

7927710 CDK1 NM 001786 -15.1084 -17.8366 -12.4258

8094278 NCAPG NM 022346 -14.3838 -14.0245 -10.1197

8046380 ITGA6 NM 000210 -32.6746 -30.2244 -21.0493

7929334 CEP55 NM 018131 -15.454 -10.885 -10.1603

8085754 SGOL1 NM 001012410 -12.3814 -10.1293 -12.3605

7982757 CASC5 NM 170589 -17.3203 -14.7567 -13.371

7974404 CDKN3 NM 005192 -18.6409 -15.725 -1 1.9337

8054580 BUB1 NM 004336 -15.4831 -11.5552 -10.9449

7929258 KIF1 1 NM 004523 -16.9821 -15.356 -10.986

8132318 ANLN NM 018685 -16.1389 -10.5164 -10.2032

8108301 KIF20A NM 005733 -16.0288 -14.2485 -13.5796

8095585 SLC4A4 NM 001098484 -13.7244 -12.8051 -10.045

7979307 DLGAP5 NM 014750 -22.4725 -14.2664 -10.3782

7923086 ASP NM 018136 -25.3499 -17.7753 -12.9735

7987315 ACTC1 NM 005159 -15.1603 -12.5897 -31.8732

7976567 BDKRB1 NM 000710 -11.9456 -16.9748 -13.8472

Table 6. Gene expression profiles for samples from three individuals (identified by numbers 731, 970, and 1650) of hepatic differentiated (day 17) vs. undifferentiated cell types. All genes displaying at least 12-fold up- or down- regulation common in all three individuals are shown.

Fold- Fold- Fold-

Probe mRNA Change Change Change Set ID Gene Symbol Accession (731) (970) (1650)

8145570 ESC02 NM 001017420 -34.0606 -26.3714 -27.8274

7927710 CDK1 NM 001786 -14.5077 -16.2072 -15.8429

8046380 ITGA6 NM 000210 -41.0972 -32.0706 -24.6844

7929334 CEP55 NM 018131 -16.5164 -16.1579 -13.0563

8085754 SGOL1 NM 001012410 -13.876 -15.8975 -15.7542

7982757 CASC5 NM 170589 -18.9653 -15.34 -15.1125

8061579 TPX2 NM 012112 -14.8453 -13.1334 -12.2978

7974404 CDKN3 NM 005192 -23.8829 -19.48 -20.0499

7929258 KIF11 NM 004523 -16.8152 -12.3758 -13.6087

8132318 ANLN NM 018685 -20.1714 -12.7609 -13.1742

8108301 KIF20A NM 005733 -18.7512 -16.2361 -17.0865

7923086 ASP NM 018136 -24.3799 -19.9013 -19.6574

7987315 ACTC1 NM 005159 -14.9801 -15.71 17 -29.031

Table 7. Gene expression profiles for samples from three individuals (identified by numbers 731, 970, and 1650) of hepatic differentiated (day 25) vs. undifferentiated cell types. All genes displaying at least 15-fold up- or down- regulation common in all three individuals are shown.

Fold- Fold- Fold-

Probe mRNA Change Change Change Set ID Gene Symbol Accession (731) (970) (1650)

8149955 PBK N 018492 -27.9869 -41.2947 -18.237

7979307 DLGAP5 NM 014750 -57.8301 -41.697 -30.7483

7923086 ASPM NM 018136 -48.194 -66.0815 -35.0628

Table 8. Gene expression profiles for samples from three individuals (identified by numbers 731, 970, and 1650) of hepatic differentiated (day 33) vs. undifferentiated cell types. All genes displaying at least 15-fold up- or down- regulation common in all three individuals are shown.

Fold- Fold- Fold-

Probe mRNA Change Change Change Set ID Gene Symbol Accession (731) (970) (1650)

7923086 ASPM NM 018136 -42.0298 -38.6657 -27.6311

Table 9. Gene expression profiles for samples from three individuals (identified by numbers 731, 970, and 1650) of hepatic differentiated (day 38) vs. undifferentiated cell types. All genes displaying at least 15-fold up- or down- regulation common in all three individuals are shown.

Fold- Fold- Fold-

Probe mRNA Change Change Change Set ID Gene Symbol Accession (731) (970) (1650)

8149955 PBK N 018492 -21.2251 -27.3684 -30.4638

7982889 NUSAP1 N 016359 -22.6107 -25.0294 -24.9593

7994109 PLK1 NM 005030 -17.9671 -19.9368 -15.4117

7979307 DLGAP5 NM 014750 -30.8046 -58.6434 -35.4778

7923086 ASPM NM 018136 -34.9205 -37.761 -44.1499

7976567 BDKRB1 NM 000710 -24.9668 -34.2236 -24.0507

Table 10. Gene expression profiles for samples from three individuals (identified by numbers 731, 970, and 1650) of hepatic differentiated (day 45) vs. undifferentiated cell types. All genes displaying at least 15-fold up- or down- regulation common in all three individuals are shown.

Fold- Fold- Fold-

Probe mRNA Change Change Change Set ID Gene Symbol Accession (731 ) (970) (1650)

7974404 CDKN3 NM 005192 -31.3203 -34.6756 -26.6261

7906930 NUF2 NM 145697 -23.724 -20.8188 -19.014

8120838 TTK NM 003318 -32.3815 -26.2418 -22.913

7983969 CCNB2 NM 004701 -28.6658 -38.6225 -27.782

8054580 BUB1 NM 004336 -30.4457 -28.3179 -23.9106

7929258 KIF1 1 NM 004523 -16.3365 -25.3002 -29.3944

7909708 CENPF NM 016343 -19.5955 -15.9155 -21.2705

8102643 CCNA2 NM 001237 -17.4267 -17.7815 -15.8523

8014974 TOP2A NM 001067 -23.8265 -27.5626 -26.1444

8132318 ANLN NM 018685 -50.3464 -36.3315 -64.557

7900699 CDC20 NM 001255 -41.9813 -24.4716 -18.2385

8108301 KIF20A NM 005733 -43.3796 -49.1219 -68.7341

8149955 PBK NM 018492 -25.7263 -30.8167 -18.0083

7982889 NUSAP1 NM 016359 -21.9692 -31.7755 -25.5242

7979307 DLGAP5 NM 014750 -40.3228 -39.0419 -32.3877

7923086 ASP NM 018136 -57.0446 -47.2426 -49.2828

7976567 BDKRB1 NM 000710 -20.3252 -30.9189 -22.9959

[0096] While the present invention has been disclosed with reference to certain aspects and embodiments, persons of ordinary skill in the art will appreciate that numerous modifications, alterations, and changes to the described aspects are possible without departing from the sphere and scope of the present invention. Accordingly, it is intended that the present inventions not be limited to the described aspects and embodiments described herein, but that the inventions be understood consistent with the full spirit and scope in which they are intended to be understood, including equivalents of the particular aspects and embodiments described herein.