WO2016187451A1

WO2016187451A1 - Multi-pathway induction of stem cell differentiation with rna

Info

Publication number: WO2016187451A1
Application number: PCT/US2016/033320
Authority: WO
Inventors: Jiwu Wang; Yuhui NI
Original assignee: Allele Biotechnology And Pharmaceuticals, Inc.
Priority date: 2015-05-19
Filing date: 2016-05-19
Publication date: 2016-11-24

Abstract

Method of changing the pluripotent state or progenitor state of stem cells or progenitor cells towards a specific lineage or tissue cell type, comprising at least one of: generate cells expressing critical cell fate genes (collectively referred as stem cells), including key cell fate factors and fusions between conventional transcription factors (TFs) with transactivation domains, optimized for directing stem cells towards different types of cells; introducing these factors as synthetic messenger RNA (mRNA) into cultured pluripotent stem cells at the preferred density by methods that result in appropriate levels of transgene expression; maintaining cell under optimized conditions to result in high efficiency of specific differentiation.

Description

MULTI-PATHWAY INDUCTION OF STEM CELL DIFFERENTIATION WITH RNA

RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. provisional application 62/163,648, filed on May 19, 2015.

FIELD OF THE INVENTION

[0002] The present disclosure relates to directing induction of pluripotent stem cells through kinetically controlled cell growth processes utilizing specific combinations and ranges of cell density, reagent concentrations, and specific combinations of mRNAs.

BACKGROUND

[0003] In order to realize the great therapeutic potential of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), the scientific community as a whole has put in great efforts to develop differentiation methods that will derive cells of various tissue types from pluripotent state. Although significant advancements have been made in the past years to guide cell fate towards different pathways, the current state of the overall efficiency of cell differentiation is far from being efficient, particularly if judged by the lack of readiness to mass production for clinical use of stem cell-derived tissue cells.

[0004] Most of the current protocols for differentiation are based on knowledge gained from embryology and developmental biology in respect to how intercellular signals and cellular response pathways affect the expression of batteries of genes that are ultimately responsible for cell fate determination. Accordingly, most of the protocols require the use of combinations of growth factors, hormones, cytokines, signal peptides and other intercellular signal molecules

(collectively referred to growth factors for simplicity in this text) at each step along a differentiation cascade. Unfortunately, the growth factors are in most cases expensive when supplied as purified polypeptides, unstable, and inconsistent from batch-to-batch, making them difficult to use. Additionally, because growth factors function in a highly specific yet combinatorial manner, each stage of differentiation is dictated by a different set of growth factors, which is difficult to figure out and expensive to create. Given that differentiation from pluripotent to terminally differentiated state often takes multiple steps, requiring a time frame of several weeks to even months, the growth factor-based, stepwise strategy is intrinsically inefficient and tedious. One of the goals of the current invention was to fundamentally remove the need of most, if not all, growth factors in guiding tissue cell generation.

[0005] To alleviate the burden of cost and inconsistency, those of skill in the art often resort to finding small molecules that can influence signal pathways as an agonist or antagonist of growth factor receptors, thereby in a way substituting growth factors. Small molecules are typically much cheaper than growth factors. However, one major disadvantage of small molecules is the non-specific effects they may exert on unintended targets, such as cell membrane-bound receptors, intracellular organelles, or genomic components, etc. The present disclosure also provide novel methods of achieving cell fate determination with no or minimum use of small molecules.

[0006] Another key component of a typical differentiation protocol is the media for culturing cells, which may be composed of nutrients (lipids, amino acids, carbohydrates, vitamins, etc.), proper concentrations of salts, pH buffering agents, critical elements, and common protein factors such as insulin or serum albumin. Different types of cells have different requirements of nutrients and media components, further complicated by cell type specific growth factors and small molecules for signaling, therefore, a special "differentiation medium" is often

painstakingly tested by removing or adding one component at a time. A major benefit of the current invention is the simplicity of establishing differentiation medium through use of properly supplied mRNAs of differentiation-directing genes. Optimal combination of mRNAs and appropriate medium, however, can still benefit the process and is often an integral part of the current invention.

[0007] In clinical applications of stem cell derived tissue cells, most components of the established differentiation media require individual certification under the current good manufacturing practice (cGMP) regulations; for example, growth factors need to be produced by special procedures, some in bacteria, some in insect cells, and some in mammalian systems, each requiring individual certification. Likewise, some small molecules added to specific differentiation media are produced through special chemical synthesis processes that vary in purity, stability, and toxicity. In the field of stem cell cultures, some previously published protocols also rely on animal products such as serum or matrigel. One incentive behind the current invention is to create a new method that is primarily based on a single type of molecule suitable to uniform certification and quality control processes.

[0008] In the current disclosure, the inventors describe the novel, enabling processes that involve how cell density and rate of division should be managed to achieve desired

differentiation results. Further disclosure teaches the optimization of timing, order of addition, RNA doses and ratios among different RNAs during transfection of RNAs, and their duration or number of repeats. The invention further relates to the choices of surface of culture vessels and environmental conditions such as oxygen concentration. The invention further includes processes and methods of selection of desired cells or enhancement of their percentage in the overall population, methods of cryopreserve and re-culture differentiated cells, and managing physical stress during handling throughout the differentiation process.

SUMMARY OF THE INVENTION [0009] Accordingly, the present invention provides methods for inducing stem cells and differentiation by modulating cell growth kinetics and associated parameters whereby specific combination of cells density, reagent concentrations, and combinations of mRNAs are used to control the direction of the differentiation/induction.

[0010] In certain embodiments, the exemplary stem cell induction protocol can include exemplars as represented by the regimen and steps as described and set forth in examples 1-13.

[0011] The present disclosure provides differentiation methods that utilize highly efficient and well-controlled expression of master control genes in tissue specific differentiation. More specifically, master control genes are introduced into pluripotent stem cells in the form of properly modified and purified mRNA molecules demonstrated through provided exemplars.

[0012] In one aspect, the present disclosure provides a method of inducing cell differentiation comprising: generating stem cells expressing critical cell fate genes (collectively referred as stem cells), including key cell fate factors and fusions between conventional transcription factors (TFs) with transactivation domains, optimized for directing stem cells towards different types of cells; introducing these factors as synthetic messenger RNA (mRNA) into cultured pluripotent stem cells at the preferred density by methods that result in appropriate levels of transgene expression; maintaining cell under optimized conditions to result in high efficiency of specific differentiation whereby the pluripotent state or progenitor state of stem cells or progenitor cells towards a specific lineage or tissue cell type, is induced.

[0013] In another aspect, the disclosure provides methods for changing the pluripotent state or progenitor state of stem cells or progenitor cells towards a specific lineage or tissue cell type, comprising at least one of: generating cell expressing critical cell fate genes (collectively referred as stem cells), including key cell fate factors and fusions between conventional transcription factors (TFs) with transactivation domains, optimized for directing stem cells towards different types of cells; introducing these factors as synthetic messenger RNA (mRNA) into cultured pluripotent stem cells at the preferred density by methods that result in appropriate levels of transgene expression; maintaining cell under optimized conditions to result in high efficiency of specific differentiation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Aspects of the present invention will now be described in relation to the drawings as described below:

[0015] FIG. 1 Exemplary embodiment of endoderm induction: stage 1 ; 2.5 uM CHIR99021

[0016] FIG. 2 Exemplary embodiment of endoderm induction: stage 1 ; 5 uM CHIR99021

[0017] FIG. 3 Exemplary embodiment of endoderm induction: stage 1 ; 7.5 uM CHIR99021

[0018] FIG. 4 Exemplary embodiment of endoderm induction: stage 1 ; 10 uM CHIR99021

[0019] FIG. 5 Exemplary embodiment of endoderm induction: stage 2; seeding density: 1χ10^Λ5 + Soxl7(10 ng/well ), 3 transfection + Endoderm

[0020] FIG. 6 Exemplary embodiment of endoderm induction: stage 2; seeding density: 3χ10^Λ5; Soxl7(100ng/well ), 4transfection endoderm;

[0021] FIG. 7 Exemplary embodiment of endoderm induction: stage 2; seeding density: 1χ10^Λ5; Soxl7(50 ng/well ) , 3 transfection endoderm

[0022] FIG. 8 Exemplary embodiment of endoderm induction: stage 2; seeding density: 4χ10^Λ5; Soxl7(100 ng/well ) , 6 transfection endoderm;

[0023] FIG. 9 Exemplary embodiment of hepatic cell induction: stage 1; Seeding density: lxl 0^Λ6 progenitor cells; [0024] FIG. 10 Exemplary embodiment of hepatic induction: stage 1; Seeding density: 4χ10^Λ5 progenitor;

[0025] FIG. 11 Exemplary embodiment of hepatic induction: stage 1; Seeding density: 1χ10^Λ5 progenitor;

[0026] FIG. 12 Exemplary embodiment of hepatic induction: stage 2; Hepatocyte induction

[0027] FIG. 13 Exemplary embodiment of lung cell/tissue induction: stage 1; condition 1 progenitor : optimized condition except RA luM;

[0028] FIG. 14 Exemplary embodiment of lung cell/tissue induction: stage 1; condition 2 progenitor : optimized condition except RA 0.7uM

[0029] FIG. 15 Exemplary embodiment of lung cell/tissue induction: stage 2; condition 1 lung epithelial cells;

[0030] FIG. 16 Exemplary embodiment of lung cell/tissue induction: stage 2: condition 2 lung epithelial cells;

[0031] FIG. 17 Exemplary embodiment of pancreatic cell/tissue induction: stage 1 : Seeding Density: lxl 0^Λ5 cells per ml; 50ng/ml KGF pre-treatment; PDX 50ng/well, 3 transfection Pancreatic Progenitor Like

[0032] FIG. 18 Exemplary embodiment of pancreatic cell/tissue induction: stage 1 : Seeding density: 1χ10^Λ5 cells per ml; PDX1 20ng/well , 4 transfection ; Pancreatic Progenitor Like

[0033] FIG. 19 Exemplary embodiment of pancreatic cell/tissue induction: stage 1 : Seeding density: 2χ10^Λ5 cells per ml medium ; PDX1 50ng/well, 3 transfection Pancreatic Progenitor Like

[0034] FIG. 20 Exemplary embodiment of pancreatic cell/tissue induction: stage 1 : Seeding density: 1χ10^Λ6 cells per ml medium; PDXl lOOng/well, 6 transfection; Pancreatic Progenitor Like

[0035] FIG. 21 Exemplary embodiment of pancreatic cell/tissue induction: stage 1 : Seeding density: 4χ10^Λ5 cells per ml medium ; PDXl lOOng/well, 3 transfection; Pancreatic

Progenitor Like

[0036] FIG. 22 Exemplary embodiment of pancreatic cell/tissue induction: stage 2:

PDXl(10ng/well)+NKX6.1 (50ng/well), 3 transfection; endocrine progenitor like

[0037] FIG. 23 Exemplary embodiment of pancreatic cell/tissue induction: stage 2:

PDXl(10ng/well)+NKX6. l(50ng/well), 3 transfection; Ngn3(50ng/well), single tansfection on first day; endocrine progenitor like

[0038] FIG. 24 Exemplary embodiment of pancreatic cell/tissue induction: stage 2: Ngn3(100 ng/well), single transfection on first day; NKX6.1(100ng/well) , 3 transfection ; endocrine progenitor like

[0039] FIG. 25 Exemplary embodiment of pancreatic cell/tissue induction: stage 3:

NKX6.1(50ng/well)+mafa(50ng/well), 3 transfection; Beta cells like

[0040] FIG. 26 Exemplary embodiment of pancreatic cell/tissue induction: stage 3:

NKX6.1(20ng/well)+mafa(30ng/well) , 4 transfection ; Beta cell like

[0041] FIG. 27 Exemplary embodiment of pancreatic cell/tissue induction: stage 3:

NKX6.1(25ng/well)+mafa(25ng/well) , 5 transfection ; Beta cell like

[0042] FIG. 28 NKX6.1(5 Ong/ well)+maf a(5 Ong/ well) , 6 transfection ; Beta cell like

[0043] FIG. 29 Exemplary induction protocol from Example 3

[0044] FIG. 30 Exemplary induction protocol from Example 4

[0045] FIG. 31 Exemplary induction protocol from example 6 [0046] FIG. 32 Exemplary induction protocol from Example 2

[0047] FIG. 33 Exemplary protocol using Adipocytes

[0048] FIG. 34 Representative features and/or embodiments of the present disclosure

[0049] FIG. 35 Representative features and/or embodiments of the present disclosure

[0050] FIG. 36 Representative features and/or embodiments of the present disclosure

[0051] FIG. 37 Representative features and/or embodiments of the present disclosure

[0052] FIG. 38 Representative features and/or embodiments of the present disclosure

[0053] FIG. 39 Representative features and/or embodiments of the present disclosure

[0054] FIG. 40 Representative features and/or embodiments of the present disclosure

[0055] FIG. 41 Representative features and/or embodiments of the present disclosure

[0056] FIG. 42 Representative features and/or embodiments of the present disclosure

[0057] FIG. 43 Exemplary embodiment of cardiac cell/tissue induction.

DETAILED DESCRIPTION

[0058] When describing the present invention, all terms not defined herein have their common meanings recognized in the art. To the extent that the following description is of a specific embodiment or a particular use of the invention, it is intended to be illustrative only, and not limiting of the claimed invention. The following description is intended to cover all alternatives, modifications and equivalents that are included in the spirit and scope of the invention.

[0059] The concept of "master control" gene, i.e. one key gene (typically a transcription factor gene, sometimes a small number of genes working together) can decide the fate of cells and tissues and eventually the formation of an entire organ during development, has been generally accepted based on studies in muscle (MyoD), eye (Pax6), and other fields of developmental biology. Shinya Yamanaka's discovery that differentiated cells can be reverted to a pluripotent state by the expression of a select group of transcription factors expressed in stem cells demonstrated the power of a small number of key transcription factors in driving cells through a lengthy, multi-stage fate change. Work by other groups on iPSC generation expanded the choices of reprogramming factors and showed that some variations can be tolerated in transcription factor choices for the purpose of reprogramming. In Yamanaka's original work, expression of the reprogramming factors was achieved through the application of viral vectors which integrate into the genome because prolonged expression of these factors is required to effect cell transformation. The attendant modification of the genome represents an important hurdle to therapeutic application of iPSCs, while the possibility of reactivated expression from integrated viral cassettes is a concern even for in vitro studies. The application of mRNA transfection to reprogramming as most recently disclosed by the current inventor group is particularly appealing as this system allows the expression of reprogramming cocktails and even individual component factors to be modulated in short time frames simply by changing which transcripts are added to the cell culture media. Once transfection of a particular factor is terminated, ectopic expression within the target cells ceases quickly due to the rapid decay of mRNA in the cytoplasm. Even though mRNA does not persist in the target cell, its ability to be directly translated in the cytoplasm, without the need of rate-limiting nuclear translocation as in the case of transfected DNA and integrating viral vectors, more than compensate for mRNA's short half-life to result in highly efficient expression but well within a small time window, which is critical for cell fate determination.

[0060] Long-lasting DNA vectors, such as episomal plasmids, when used for cell fate alteration, require weaning to reduce any risk of random genomic integration. RNA viruses or virus- derivatives, such as the Sendai virus or Venezuelan equine encephalitis (VEE) virus, even after being stripped to be a modified noninfectious RNA replicon, still carries viral elements, prone to recombination with viral elements hidden in the host genome. It is always difficult to be completely sure that the cells are rid of the viral vectors without tedious finding of proof in the form of negative data. The current invention discloses multiple inventive steps aimed at applying the advantages of mRNA-based cell fate determination to directed differentiation and transdifferentiation. In summary, the current disclosure teaches a single or multiple rounds of ectopic transcription factor expression in a streamlined method to direct cell differentiation.

[0061] Nonetheless, there are technical barriers to mRNA-based stem cell differentiation. Not all stem cell types and culture media are equally conducive to efficient mRNA delivery, and this is currently an impediment to mRNA-based differentiation. It has also been commonly known that stem cells, particularly most human stem cell lines, are rather difficult to culture without forming transfecti on-resistant patches. It is part of the current invention's teaching that pluripotent stem cells can be grown under conditions that most of the cells can be transfected with modified mRNAs. In another embodiment, the dose of RNA and transfecti on reagent (both of which have associated toxicities) are to be provided to the cells at levels capable of exerting master control gene effects while supporting the viability of the target cells in the face of the pro-apoptotic and cytostatic forces engendered by the cell fate changing process.

[0062] Accordingly, in view of the problems associated with the previously known stem cell differentiation procedures, the novel methods, materials, and protocols described herein produce different cell types from iPSCs or ESCs with improved efficiency of the process and quality of the resultant cells. The current invention achieved significant improvements through potentiation of the RF mRNA delivered to the target stem cells. The current invention also provides novel protocols which support the production of footprint-free tissue cells from human stem cells without the use of feeder cells or any other potentially xeno-contaminated reagents. The new protocols extend the benefits of the modified mRNA and help clear remaining roadblocks to the therapeutic application of stem cell derivation technology.

[0063] Given that differentiation from pluripotent to terminally differentiated state often takes multiple steps, requiring a time frame of several weeks to even months, the growth factor-based, stepwise strategy is intrinsically inefficient and tedious. Accordingly, embodiments of the present invention fundamentally remove the need of most, if not all, growth factors in guiding tissue cell generation.

[0064] More specifically, this invention relates to changing the pluripotent state or progenitor state of stem cells or progenitor cells towards a specific lineage or tissue cell type by expressing critical cell fate genes (collectively referred as stem cells), including key cell fate factors and fusions between conventional transcription factors (TFs) with transactivation domains, optimized for directing stem cells towards different types of cells; introducing these factors as synthetic messenger RNA (mRNA) into cultured pluripotent stem cells at the preferred density by methods that result in appropriate levels of transgene expression; maintaining cell under optimized conditions to result in previously unattainable efficiency of specific differentiation. Factors expressed through introduction of mRNA can also include growth factors, cytokines, hormones, signal peptides and other cell fate influencing secreted factors or modifying enzymes. Using similar procedure, microRNAs (miRNAs) or other non-proti en-coding RNAs can be introduced into cells under cell state transition in order to direct differentiation. Compared to other methods that are known in the art, the current invention dramatically reduces the time, cost and effort involved in stem cell differentiation. The invented materials and procedures can be used for creating essentially any cell type from stem cells.

[0065] Method of changing the pluripotent state or progenitor state of stem cells or progenitor cells towards a specific lineage or tissue cell type, comprising at least one of: expressing critical cell fate genes (collectively referred as stem cells), including key cell fate factors and fusions between conventional transcription factors (TFs) with transactivation domains, optimized for directing stem cells towards different types of cells; introducing these factors as synthetic messenger RNA (mRNA) into cultured pluripotent stem cells at the preferred density by methods that result in appropriate levels of transgene expression; maintaining cell under optimized conditions to result in high efficiency of specific differentiation.

[0066] In certain embodiments, the methods disclosed herein can enable rapidly generation of Human iPSCs (e.g. under 10 days).

[0067] In certain embodiments, the cells ready in 4-5 weeks from start of reprogramming

[0068] In certain embodiments, the fully stabilized, expanded hiPSCs are provided.

[0069] In certain embodiments, there is no need to clear episomes or RNA virus (e.g., Sendai), which can take 10+ passages of hiPSCs post-isolation.

[0070] In certain embodiments, the process is Feeder-free: elimination of key reprogramming variable

[0071] Xeno-free: all synthetic or human reagents.

[0072] In certain embodiments, the process is footprint-free: no random integration of DNA into genome (as often happens with episomal).

[0073] In certain embodiments, the process yields a fully-customized genetic background via patient-specific starting tissue and/or genome-editing.

[0074] DEFINITIONS

[0075] The term "cardiomyocyte-like cell" is intended to mean a cell sharing features with a cardiomyocyte. Cardiomyocyte-like cells are further defined by morphological characteristics as well as by specific marker characteristics. As induced pluripotent stem cell-derived

cardiomyocyte-like cells share similar characteristics (including marker and electrophysiological characteristics) with cardiomyocytes, induced pluripotent derived cardiomyocyte-like cells may be used interchangeably with induced pluripotent stem cell-derived cardiomyocytes.

[0076] Cardiovascular agent refers to agents that have potential to ameliorate, control, eliminate, prevent, reduce and/or treat a wide variety of disorders related to the heart and/or circulation.

[0077] An "embryoid body" refers to an aggregate of cells derived from pluripotent cells, where cell aggregation can be initiated by any method that prevents the cells from adhering to a surface to form typical colony growth.

[0078] As used herein, the term "induced pluripotent stem cells" refers to a pluripotent stem cell derived from a somatic cell (e.g. an adult somatic cell). Induced pluripotent stem cells are similar to embryonic stem cells in their differentiation abilities to form any adult cell types, but are not derived from an embryo.

[0079] As used herein, the term "pluripotent" refers to the potential of a stem cell to make any differentiated cell type of an organism. Pluripotent stem cells can give rise to any fetal or adult cell type. However, alone they cannot develop into a fetal or adult organism because they lack the potential to contribute to extraembryonic tissue, such as the placenta.

[0080] "Risk of and/or "predisposition to" cardiac arrhythmia in a subject, as used in this specification, refers to the probability that the subject has, or will develop, cardiac arrhythmia in the future or near future. For example, a subject may consider various options for treatment or take up various preventative measures if a subject predicts risk of cardiac arrhythmia as far in advance as possible. Risk of cardiac arrhythmia in a subject may be due to genetic and non- genetic factors, for example a family history of cardiac arrhythmia, scarring of heart tissue (such as from a heart attack), electrolyte imbalances in blood, coronary artery disease, or other risk factors. Accordingly, severity of this risk in a subject is determined in comparison to the probability of cardiac arrhythmia in control groups, for example a group of normal individuals (i.e. individuals without cardiac arrhythmia risk factors) and/or a group of individuals with cardiac arrhythmia or having risk factors for cardiac arrhythmia.

[0081] As used herein, "cell," "cell line," and "cell culture" include progeny. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological property, as screened for in the originally transformed cell, are included.

[0082] As used herein, "composition" refers to a combination of active agent and at least one other compound or molecule, inert (for example, a detectable agent or label) or active, such as an adjuvant.

[0083] As used herein, "culturing" refers to maintaining cells under conditions in which they can proliferate and avoid senescence as a group of cells. "Culturing" can also include conditions in which the cells also or alternatively differentiate.

[0084] As used herein, "differentially expressed," refers to the differential production of RNA, including but not limited to mRNA, tRNA, miRNA, siRNA, snRNA, and piRNA transcribed from a gene or regulatory region of a genome or the protein product encoded by a gene as compared to the level of production of RNA by the same gene or regulator region in a normal or a control cell. In another context, "differentially expressed," also refers to nucleotide sequences or proteins in a cell or tissue which have different temporal and/or spatial expression profiles as compared to a normal or control cell.

[0085] As used herein, "overexpressed" or "overexpression" refers to an increased expression level of an RNA or protein product encoded by a gene as compared to the level of expression of the RNA or protein product in a normal or control cell. [0086] As used herein, "underexpressed" or "underexpression" refers to decreased expression level of an RNA or protein product encoded by a gene as compared to the level of expression of the RNA or protein product in a normal or control cell.

[0087] As used herein, "differentiate" or "differentiation," refers to the process by which precursor or progenitor cells (i.e., chondrogenic progenitor cells) differentiate into specific cell types, e.g., chondrogenic cells.

[0088] As used herein, "effective amount" is an amount sufficient to effect beneficial or desired biological, emotional, medical, or clinical response of a cell, tissue, system, animal, or human. An effective amount can be administered in one or more administrations, applications, or dosages. The term also includes, within its scope, amounts effective to enhance normal physiological function.

[0089] As used herein, "expansion" or "expandeds" in the context of cells, refers to an increase in the number of a characteristic cell type, or cell types, from an initial population of cells, which may or may not be identical. The initial cells used for expansion need not be the same as the cells generated from expansion. For instance, the expanded cells may be produced by ex vivo or in vitro growth and differentiation of the initial population of cells.

[0090] As used herein, "expression" refers to the process by which polynucleotides are transcribed into RNA transcripts. In the context of mRNA and other translated RNA species, "expression" also refers to the process or processes by which the transcribed RNA is subsequently translated into peptides, polypeptides, or proteins.

[0091] As used herein, "induced pluripotent stem cell" or "iPS cell" refers to a cell capable of differentiating into multiple cell types that is artificially derived (not naturally derived) from a non-pluripotent cell. [0092] As used herein, "integration free iPS cell" refers to an iPS cell that does not contain an exogenous transgene integrated into the genome of the non-pluripotent cell.

[0093] As used herein, "isolated" means separated from constituents, cellular and otherwise, in which the polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, are normally associated with in nature. A non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, do not require "isolation" to distinguish it from its naturally occurring counterpart.

[0094] As used herein, "concentrated" refers to a molecule, including but not limited to a polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, that is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume is greater than that of its naturally occurring counterpart.

[0095] As used herein, "diluted" refers to a molecule, including but not limited to a polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, that is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume is less than that of its naturally occurring counterpart.

[0096] As used herein, "separated" refers to the state of being physically divided from the original source or population such that the separated compound, agent, particle, or molecule can no longer be considered part of the original source or population.

[0097] As used herein, "embryoid body" refers to a three-dimensional spheroid aggregate of pluripotent stem cells, including but not limited to embryonic stem cells derived from the blastocyst stage of embryos from mammalian sources. An embryoid body can be formed from embryonic stem cells derived through any technique generally known in the art, including but not limited to somatic cell nuclear transfer or the reprogramming of somatic cells to yield induced pluripotent stem cells. [0098] As used herein, "mammal," for the purposes of treatments, refers to any animal classified as a mammal, including human, domestic and farm animals, nonhuman primates, and zoo, sports, or pet animals, such as, but not limited to, dogs, horses, cats, and cows.

[0099] As used herein, "mesenchymal stem cell" or "MSC" refers herein to a multipotent cell capable of differentiating into cells that compose adipose, bone, cartilage, and muscle tissue.

[00100] As used herein, "mesenchymal lineage cell" refers to a multipotent cell that is capable of differentiating into one or more cells that compose adipose, bone, cartilage, and muscle tissue, and, may or may not express the surface protein markers that are commonly associated with mesenchymal stem cells.

[00101] As used herein, "mesenchymal cell population" refers to a population of cells that comprises one or more of mesenchymal stem cells, mesenchymal lineage cells, osteoblasts, adipocytes, and chondrocytes. In such a population, the osteoblasts, adipocytes, and

chondrocytes can be immature, mature, or a combination thereof.

[00102] As used herein, "mesenchymal stem cell-like" refers to cells derived from a stem cell population that are morphologically similar to mesenchymal stem cells and express cell surface markers typical of mesenchymal stem cells. Although mesenchymal stem cell-like cells may have some overlapping characteristics (e.g. similar morphology) with mesenchymal stem cells, they are not identical to mesenchymal stem cells for at least the fact that mesenchymal stem cell-like cells are not directly derived from bone marrow aspirate. Mesenchymal stem cells are derived directly from bone marrow aspirate.

[00103] As used herein, "stem cell" refers to any self-renewing totipotent, pluripotent cell or multipotent cell or progenitor cell or precursor cell that is capable of differentiating into multiple cell types. [00104] As used herein, "totipotent" refers cells that can differentiate and give rise to all cells types in an organism, plus the extraembryonic, or placental, cells.

[00105] As used herein, "pluripotent" refers to cells that can differentiate and give rise to all of the cell types that make up an organism, except for the extraembryonic, or placental, cells.

[00106] As used herein, "multipotent" refers to cells that can develop into more than one cell type, but are more limited than pluripotent cells in the cell types that they can develop into.

[00107] As used interchangeably herein, "subject," "individual," or "patient" refers to a vertebrate organism.

[00108] As used herein, "substantially pure cell population" refers to a population of cells having a specified cell marker characteristic and differentiation potential that is about 50%, preferably about 75-80%, more preferably about 85-90%, and most preferably at least about 95% of the cells making up the total cell population. Thus, a "substantially pure cell population" refers to a population of cells that contain fewer than about 50%, preferably fewer than about 20-25%, more preferably fewer than about 10-15%, and most preferably fewer than about 5% of cells that do not display a specified marker characteristic and differentiation potential under designated assay conditions.

[00109] As used herein, "pre-differentiation" refers to the process by which precursor or progenitor cells (e.g., pluripotent stem cells) differentiate into intermediate cell types, e.g., mesenchymal stem cells, which have the potential to differentiate further to final effector cells (e.g. chondrocytes).

[00110] As used herein, "therapeutic" refers to treating, healing, and/or ameliorating a disease, disorder, condition, or side effect, or to decreasing in the rate of advancement of a disease, disorder, condition, or side effect. The term also includes within its scope enhancing normal physiological function, pallative treatment, and partial remediation of a disease, disorder, condition or side effect.

[00111] The terms "treating" and "treatment" as used herein refer generally to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof such as of OA, and/or may be therapeutic in terms of a partial or complete cure of a disease, condition, symptom, or adverse effect attributed to the disease. The term "treatment" as used herein covers any treatment of OA or other cartilage disorder in a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., mitigating or ameliorating the disease and/or its symptoms or conditions. The term "treatment" as used herein refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.

[00112] As used herein, "preventative" refers to hindering or stopping a disease or condition before it occurs, even if undiagnosed, or while the disease or condition is still in the sub-clinical phase.

[00113] As used herein, "active agent" refers to a substance, compound, or molecule, which is biologically active or otherwise induces a biological or physiological effect on a subject to which it is administered to.

[00114] As used herein, "pharmaceutically acceptable carrier" refers to diluent, adjuvant, excipient, or vehicle with which an active agent, chondrocytes of the present disclosure, or composition containing chondrocytes of the present disclosure is administered in conjunction with and that is approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals and/or humans.

[00115] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[00116] Cell types:

[00117] Exemplary cell types can include, for example, Neural; Neural progenitor cells;

GABA-ergic, glutamatergic, and DA neurons; Mesenchymal; Mesenchymal stem cells;

Adipocytes; Cardiac; Cardiomyocytes; Hematopoetic; Megakaryocytes; Platelets

[00118] Exemplars of suitable surfaces for culture vessels:

[00119] Vitronetin, E-cadherin, Coming® Synthemax® II or Matrigel for iPSCs; poly-L- ornithine/lamin or Matrigel coating for neural lineage; Collagen I or Matrigel coating for Hepatocyte; Gelatin, Collagen I or Matrigel coating for Adipocytes.

[00120] In one aspect, exemplary method for dedifferentiating or reprogramming somatic cell can include the use of any one or more of a synthetic mRNA reprogramming factor selected from Oct4, Sox2, Klf4, cMyc, Nanog, and Lin28 and a transactivation domains whereby the somatic cell is reprogrammed or de-differentiated. Methods and compositions for IPSC modulation are described in USSN 13/893,166 and USSN 14/292,317, the contents of which are hereby incorporated by reference.

[00121] In certain embodiments protocols for suspension cells, low attachment plate can be used for suspension cultures.

[00122] In certain embodiments, the environmental conditions such as oxygen concentration can be modulated for optimal induction condition. Some cells are more like to stay in low oxygen, like iPSCs, NPCs, Hepatocyte progenitors. Some cells are more like to stay in high oxygen, like Neurons. As used herein, suitable oxygen levels can range from about 2.5 % to 10 %, including about 5 %.

[00123] In certain embodiments, processes and methods of selection of desired cells or enhancement of their percentage confluence in the overall cell culture population are provided.

[00124] Cardiomyocyte are selected in a selection solution to kill the non-cadiomyocytes and enrich the cadiomyocytes. This will improve the purity of the cells generated by our cadiomyocyte differentiation protocols.

[00125] In certain embodiments, methods of cryopreserve are provided. Some of the differentiated cells need to be cryopreserved for optimal storage. 10% BSA or HSA, or 50% FBS with 10%DMSO in culture medium can be used. The cell numbers can be optimized for the further application.

[00126] Re-culture differentiated cells methods are also provided. Some of the differentiated cells need to be cryopreserved, then shipped out and be re-cultured in outside facility. These cells included NPCs, Spontaneous differentiated Neurons, Astrocytes,

Hepatocytes, Adipocytes, MSCs and Cadiomyocytes. Cells can be re-cultured in most of the culture vessels, like T75 flask, T25 flask, 6-well plate, 96-well plate. Cells can be re-cultured in different cell density due to the different application. For example, for quick neuron

differentiation assays, the cells are seeded at very low density, such as 1000 per well of 96 well plate; for hepatocytes, the cells are seeded at very high density, like 10^Λ5 per well of 96 well plate to achieve more functional mature hepatocytes.

[00127] In certain embodiments, the present disclosure also provided methods for managing physical stress during handling throughout the differentiation process. Certain types of cells during the differentiation are very small, like iPSCs and NPCs. These small cells are very sensitive to centrifuge force. For maintenance, these cells can culture them as colonies and dissociate them as cluster, instead of single cells; for differentiation, if single cells are necessary, one can end the dissociation prior to the cell detaching, remove the dissociation solution, let the solution left further dissociate the cells. This protocol is commonly used in cell culture. iPSCs and NPCs are very sensitive to centrifuge force are not strengthened by other groups. Some types of cells during the differentiation are very sticky, like iPSCs, NPCs, endoderm stage. Some types of cells have lots of branches, like most of the neurons and some astrocytes. These cells are very sensitive to sheer force. When handle these cells a 10ml pipet is used to avoid use any small tips. And to avoid pipet the cells up and down repeatedly.

EXAMPLES:

[00128] 1. ADIPOCYTES:

[00129] Exemplary protocol for Inducing Adipocytes Differentiation

[00130] * Stagel can proceed to stage 2 or cryopreservation.

[00131] Day 0

[00132] Ultra-low attachment cell culture vessels (Coming) were used at the first stage. 6- well plate was used in our experiments, but all the other types are applicable.

[00133] iPSCs which were prepared and ready for this experiment were dissociated with

0.5mM EDTA (Sigma Aldrich) in DPBS (Fisher Scientific) from the original plates. Ready means there were enough typical iPSCs colonies with sharp edge, compact cells, not overgrown. After adding the EDTA solution, the plates were incubated at 37°C for 10 mins. The concentration range of EDTA is from 0.1 mM to 1 mM, the range of incubation time is from 1 min to 20 mins. Lower concentration of EDTA need longer incubation time.

[00134] This EDTA solution were called non-enzyme cell dissociation solution which was much better than enzyme based dissociation solution, like Trypsin (Life Technology),

Accutase (Life Technology) or Triple (Life Technology), if single cells are not necessary for next step. Chemicals in the same family of EDTA might all work. Even though quite a few experienced people knew this, it's still a small trick to many other people. Actually some companies use this trick to package some expensive reagents and lots of people buy them.

[00135] With this protocol, iPSCs colonies in the original plates were dissociated into small clusters. These small clusters were cultured in the ultra-low attachment plates with E8 medium (Stemcell Technology) supplemented with 5uM rock inhibitor Y27632 (Reagents Direct). E8 medium can be replaced with any iPSCs/ESCs maintenance medium, like mTeSR^TI (Stemcell Technology), Stemline ®(Sigma Aldrich) or L7™(Lonza). The concentration range of Y27632 was 5uM to 15uM as tested. Rock inhibitor will help the cells survival after dissociation or cryopreservation.

[00136] This suspension culture system is critical if one needs to scale up this protocol.

Most will prefer to use attached monolayer. But with the suspension one can achieve a much better manufacture scale. In the future, this stage can be further adapt to any suitable automatic suspension culture system.

[00137] Day 1

[00138] The small clusters formed iPSC spheres floating in the medium on the second day. If the spheres are healthy, they will have clear sharp edges. Then the experiment started.

[00139] Centrifuged the plates at 50g for 2 mins. This part is also a small trick. Most people will transfer the culture into tubes, then wait about 10 mins until the cells down to the bottom when they handle suspension iPSCs culture. Because the centrifuge force usually used, like 400g or even 200g will damage the spheres a bit. But without centrifuge, the cell pellets in the bottom are loose and very easy to be agitated. With a simple 50 g centrifuge for 2 mins, the most of the spheres will attach to the bottom firmly enough, yet the force won't affect the spheres at all, which can be very convenient for medium change. Removed the old medium with pipet, then add the ADS1 medium, which is the combination of

[00140] Basal medium can be selected from DMEM/F12, RPMI, DMEM and ACDF. In one embodiment DMEM/F12 (Fisher Scientific) is used.

[00141] N2 (Life Technology), which is a reagent commercialized by Life Technology. Any other cell culture supplements, like B27 (Life Technology), KnockOut Serum Replacement (Life Technology), they will all be applicable here.

[00142] 1% BSA (Life Technology) or HSA (Irvine Scientific)

[00143] lmM Glutamine (Life Technology), or GlutaMAX™ (Life Technology) as product protocol

[00144] NEAA (Life Technology) as product protocol [00145] Lipid Mixture (Sigma Aldrich) as product protocol

[00146] 280ug/ml L-Ascorbic Acid (Sigma Aldrich). The concentration range was from

0. lug/ml to 400ug/ml. 500ug/ml showed some toxicity.

[00147] 20ng/ml bFGF. The concentration range was from 5ng/ml to lOOng/ml;

[00148] 7.5uM CHIR99021. The concentration range was from 2.5uM to 15uM. Other chemicals in the GSK-3 inhibitor family worked either.

[00149] * optimize the complete new recipe for ADS1 medium.

[00150] D2

[00151] Because of the toxicity from the treatments, some cells died and suspended in the medium as single cells. Changed medium with the same protocol as Dl.

[00152] D3 [00153] Because of the toxicity from the treatments, more cells died and suspended in the medium as single cells. If there were enough healthy spheres left, experiment proceeded to stage 2 or Cryopreservation.

[00154] The cells at this stage were transfected with PPARg or C/EBP mRNAs to induce fast transition to adipocytes.

[00155] For stage 2, the following protocols are used

[00156] Commercial cell culture vessels were used in stage 2. For example, 12-well plate was used in our studies but all the other types are applicable.

[00157] Plates were pre-coated with Matrigel (BD Biosciences). Collagen I (Sigma Aldrich) worked either.

[00158] Collected the spheres in 15 ml tubes by centrifuge at 50g for 2 mins. Dissociated the cells with Triple Dissociation Solution (Life Technology). Incubate at 37°C for 4 mins. Shake gently until most of the cells are dissociate into single cells or small clusters (-10 cells). Seeded the cells on the Matrigel (BD Biosciences) or Collagen I (Sigma Aldrich) coating at 5x10^Λ5 per well of 12-well plate in ADS2 medium supplemented with 5uM rock inhibitor Y27632 (Reagents Direct). Part of the cells won't attached because of the toxicity of the treatment on Dl and D2.

[00159] Basal medium, can be selected from DMEM/F 12, RPMI, DMEM and ACDF.

DMEM/F12 (Fisher Scientific) is used.

[00160] N2 (Life Technology), which is a reagent commercialized by Life Technology. Any other cell culture supplements, like B27 (Life Technology), KnockOut Serum Replacement (Life Technology), they will all be applicable here.

[00161] 1% BSA (Life Technology) or HSA (Irvine Scientific) [00162] lmM Glutamine (Life Technology), or GlutaMAX™ (Life Technology) as product protocol

[00163] NEAA (Life Technology) as product protocol

[00164] Lipid Mixture (Sigma Aldrich) as product protocol

[00165] 280ug/ml L-Ascorbic Acid (Sigma Aldrich). The concentration range was from 0. lug/ml to 400ug/ml. 500ug/ml showed some toxicity.

[00166] 20ng/ml bFGF. The concentration range was from 5ng/ml to l OOng/ml;

[00167] ΙΟΟηΜ dexamethasone. The concentration range was from InM to luM;

[00168] 500uM IBMX. The concentration range was from l OOuM to lmM;

[00169] lOuM Rosiglitazone. The concentration range was from 0. luM to 20uM;

[00170] 60uM Indomethacin. The concentration range was from lOuM to l OOuM;

[00171] Optional: 2nM T3. The concentration range was from InM to Ι ΟηΜ.

[00172] * In the future one might optimize complete new recipe for ADS2 medium.

[00173] For cryopreservation, an exemplary protocol is described below.

[00174] Cells are collected as cell spheres in 15 ml tubes by centrifuge at 50g for 2 mins.

Dissociated the cells with Triple Dissociation Solution (Life Technology). Incubate at 37°C for 4 mins. Shake gently until most of the cells are dissociate into single cells or small clusters (-10 cells). Counting the live cell numbers by Countess (Life Technology). Cryopreserved lxl 0^Λ6 live cell per vial in 10% DMSO 10% BSA/HSA ADMl medium. These cells can be thawed and re-seeded in one well of 6-well plate pre-coated with Matrigel (BD Biosciences) or Collagen I (Sigma Aldrich) in ADS2 medium supplemented with 5uM rock inhibitor Y27632 (Reagents Direct), which proceeded to stage 2. [00175] D4

[00176] Changed medium with the same protocol as D3.

[00177] D5

[00178] Switch medium to Adipo medium.

[00179] Basal medium, is selected from DMEM/F12, RPMI, DMEM and ACDF.

Currently we used DMEM/F12 (Fisher Scientific).

[00180] N2 (Life Technology), which is a reagent commercialized by Life Technology. Any other cell culture supplements, like B27 (Life Technology), KnockOut Serum Replacement (Life Technology), they will all be applicable here.

[00181] 1% BSA (Life Technology) or 1% HSA (Irvine Scientific) or optional 2.5%-5%

FBS

[00182] lmM Glutamine (Life Technology), or GlutaMAX™ (Life Technology) as product protocol

[00183] NEAA (Life Technology) as product protocol [00184] Lipid Mixture (Sigma Aldrich) as product protocol

[00185] 280ug/ml L- Ascorbic Acid (Sigma Aldrich). The concentration range was from

0. lug/ml to 400ug/ml. 500ug/ml showed some toxicity.

[00186] 20ng/ml bFGF. The concentration range was from 5ng/ml to lOOng/ml;

[00187] * In the future we might optimize complete new recipe for HDS3 medium.

[00188] EXEMPLE 2. ASTROCYTES

[00189] Exemplary Protocol for Inducing Astrocyte Differentiation [00190] Astrocytes Differentiation

[00191] Table Astro-1

[00192] DO

[00193] Commercial cell culture vessels were used in this experiment. 6-well plate was used type in our protocol, but all the other types are applicable.

[00194] Plates were pre-coated with poly-L-omithine (PLO)/laminin (Sigma Aldrich),

Matrigel (BD Biosciences) or Fibronectin (Sigma Aldrich).

[00195] Neural Progenitor Cells (NPCs) which were prepared and ready for this experiment were dissociated with 0.5mM EDTA (Sigma Aldrich) in DPBS (Fisher Scientific) mixed with Triple (Life Technology) by 3: 1 from the original plates. Ready means there were enough NPCs growing as thin plateau, but not overgrown. After adding the dissociation solution, the plates were incubated at 37°C for 10 mins. The final concentration range of EDTA is from 0.1 mM to 0.25 mM, the final concentration range of Triple is from ¼ to ½, the range of incubation time is from 1 min to 10 mins.

[00196] With this protocol, NPCs plateau in the original plates were dissociated into single cells or small clusters. 5x10^Λ5 cells were seeded into one well of 6-well plate pre-coated with PLO/laminin coating in NBM medium supplemented with 5uM rock inhibitor Y27632

(Reagents Direct). The cell number ranged from about 50000 to 500000. In another embodiment, the cell number ranged from 50000 to 800000. The concentration range of Y27632 was 5uM to 15uM as tested. Rock inhibitor will help the cells survival after dissociation or cryopreservation.

[00197] Basal medium, can be selected from DMEM/F 12, RPMI, DMEM and ACDF.

Currently we used DMEM/F12 (Fisher Scientific).

[00198] N2 (Life Technology), which is a reagent commercialized by Life Technology. Any other cell culture supplements, like B27 (Life Technology), KnockOut Serum Replacement (Life Technology), they will all be applicable here.

[00199] 1% BSA (Life Technology) or HSA (Irvine Scientific)

[00200] lmM Glutamine (Life Technology), or GlutaMAX™ (Life Technology) as product protocol

[00201] NEAA (Life Technology) as product protocol [00202] Lipid Mixture (Sigma Aldrich) as product protocol

[00203] 280ug/ml L-Ascorbic Acid (Sigma Aldrich). The concentration range was from

0. lug/ml to 400ug/ml. 500ug/ml showed some toxicity.

[00204] 20ng/ml bFGF. The concentration range was from 5ng/ml to l OOng/ml;

[00205] * In the future we might optimize complete new recipe for NBM medium.

[00206] Dl

[00207] The next day, if the cells attached well, experiment proceeded to transfection. Transfected the cells with Ngn2 mRNA at a dose of l OOng per well with Stemgent Transfection Reagent (Stemgent) in 1ml of DMEM/F12 basal medium supplemented with 200ng/ml B18R. mRNA dose ranged from 10-400 ng per well. Most of the transfection regents in the market were applicable. Two hours after the transfection, switched the medium to NBM medium.

[00208] Transfected the cells with Ngn3, Ngn2, NeuroDl, or other neurogenic factor mRNA alone or in combinations

[00209] D2

[00210] Second transfection, same protocol as Dl .

[00211] The transfection repeats varied from one day to four days, which depend on the differentiation process, until expected differentiation achieved.

[00212] D3

[00213] Switch medium to Astro medium.

[00214] Basal medium, can be selected from DMEM/F 12, RPMI, DMEM and ACDF.

Currently we used DMEM/F12 (Fisher Scientific).

[00215] N2 (Life Technology), which is a reagent commercialized by Life Technology. Any other cell culture supplements, like B27 (Life Technology), KnockOut Serum Replacement (Life Technology), they will all be applicable here.

[00216] 1% BSA (Life Technology) or HSA (Irvine Scientific), optional 2.5% FBS (Life

Technology)

[00217] lmM Glutamine (Life Technology), or GlutaMAX™ (Life Technology) as product protocol

[00218] NEAA (Life Technology) as product protocol [00219] Lipid Mixture (Sigma Aldrich) as product protocol [00220] 280ug/ml L-Ascorbic Acid (Sigma Aldrich). The concentration range was from

0. lug/ml to 400ug/ml. 500ug/ml showed some toxicity.

[00221] 50ng/ml bFGF. The concentration range was from 20ng/ml to lOOng/ml;

[00222] * In the future we might optimize complete new recipe for Astro medium.

[00223] Change medium very single day. Typically cells will grows up and reach confluent in four days.

[00224] When cells grew to 90% confluent, dissociated the cells with Triple (Life Technology) and split the cells by 1 :3, seeded 1χ10^Λ5 cells in one well of 6-well plate on PLO/laminin coating with Astro medium. These astrocytes can be maintained for at least 6 passages in Astro medium. For storage, these cells can be cryopreserved in 10% DMSO Astro medium. For thawing and re-culture, 90% of the cyropreserved cells can attached and grow in Astro medium with 5uM rock inhibitor Y27632 (Reagents Direct).

[00225] Example 3. HEPATOCYTES

[00226] Exemplary Protocol for Inducing Hepatocyte Differentiation

[00227] Hepatocytes Differentiation

[00228] Table hepato-1

[00229] Day 0

[00230] Ultra-low attachment cell culture vessels (Coming) were used at the first stage. For example, 6-well plate was used type in our experiments, but all the other types are applicable.

[00231] iPSCs which were prepared and ready for this experiment were dissociated with

0.5mM EDTA (Sigma Aldrich) in DPBS (Fisher Scientific) from the original plates. Ready means there were enough typical iPSCs colonies with sharp edge, compact cells, not overgrown. After adding the EDTA solution, the plates were incubated at 37°C for 10 mins. The

concentration range of EDTA is from 0.1 mM to 1 mM, the range of incubation time is from 1 min to 20 mins. Lower concentration of EDTA need longer incubation time.

[00232] This EDTA solution was called non-enzyme cell dissociation solution which was much better than enzyme based dissociation solution, like Trypsin (Life Technology), Accutase (Life Technology) or Triple (Life Technology), if single cells are not necessary for next step. Chemicals in the same family of EDTA might all work. Even though quite a few experienced people knew this, it's still a small trick to many other people. Actually some companies use this trick to package some expensive reagents and lots of people buy them.

[00233] With this protocol, iPSCs colonies in the original plates were dissociated into small clusters. These small clusters were cultured in the ultra-low attachment plates with E8 medium (Stemcell Technology) supplemented with 5uM rock inhibitor Y27632 (Reagents Direct). E8 medium can be replaced with any iPSCs/ESCs maintenance medium, like mTeSR™ (Stemcell Technology), Stemline ®(Sigma Aldrich) or L7™(Lonza). The concentration range of Y27632 was 5uM to 15uM as tested. Rock inhibitor will help the cells survival after dissociation or cryopreservation.

[00234] This suspension culture system is critical if one wants to scale up this protocol.

[00235] Day 1

[00236] The small clusters formed iPSC spheres floating in the medium on the second day. If the spheres are healthy, they will have clear sharp edges. Then the experiment started.

[00237] Centrifuged the plates at 50g for 2 mins. This part is also a small trick. Most people will transfer the culture into tubes, then wait about 10 mins until the cells down to the bottom when they handle suspension iPSCs culture. Because the centrifuge force usually used, like 400g or even 200g will damage the spheres a bit. But without centrifuge, the cell pellets in the bottom are loose and very easy to be agitated. With a simple 50 g centrifuge for 2 mins, the most of the spheres will attach to the bottom firmly enough, yet the force won't affect the spheres at all, which can be very convenient for medium change. Removed the old medium with pipet, then add the HDS1 medium, which is the combination of [00238] Basal medium, can be selected from DMEM/F 12, RPMI, DMEM and ACDF.

Currently we used DMEM/F 12 (Fisher Scientific).

[00239] B27 minus insulin (Life Technology), which is a reagent commercialized by Life

Technology. Any other cell culture supplements, like N2 (Life Technology), KnockOut Serum Replacement (Life Technology), if can minus insulin, they will all be applicable here.

[00240] 1% BSA (Life Technology) or HSA (Irvine Scientific).

[00241] lmM Glutamine (Life Technology), or GlutaMAX™ (Life Technology) as product protocol.

[00242] NEAA (Life Technology) as product protocol. [00243] Lipid Mixture (Sigma Aldrich) as product protocol.

[00244] 280ug/ml L-Ascorbic Acid (Sigma Aldrich). The concentration range was from

0. lug/ml to 400ug/ml. 500ug/ml showed some toxicity.

[00245] 20ng/ml bFGF. The concentration range was from 5ng/ml to lOOng/ml;

[00246] 7.5uM CHIR99021. The concentration range was from 2.5uM to 15uM. Other chemicals in the GSK-3 inhibitor family worked either.

[00247] Optional, lOuM LY294002. The concentration range was from 5uM to 20uM.

Other chemicals in PI3K inhibitor family worked either.

[00248] * optimize complete new recipe for HDS1 medium.

[00249] D2

[00250] Because of the toxicity from the treatments, some cells died and suspended in the medium as single cells. Changed medium with the same protocol as Dl.

[00251] D3 [00252] Because of the toxicity from the treatments, more cells died and suspended in the medium as single cells. If there were enough healthy spheres left, experiment proceeded to stage 2.

[00253] Commercial cell culture vessels were used in stage 2. 12-well plate was used in our experiments, but all the other types are applicable.

[00254] Plates were pre-coated with Matrigel (BD Biosciences). Collagen I (Sigma Aldrich) worked either.

[00255] Collected the spheres in 15 ml tubes by centrifuge at 50g for 2 mins. Dissociated the cells with Triple Dissociation Solution (Life Technology). Incubate at 37°C for 4 mins. Shake gently until most of the cells are dissociate into single cells or small clusters (-10 cells). Seed the cells on the precoated coating at 5χ10^Λ5 per well of 12-well plate in HDS2 medium supplemented with 5uM rock inhibitor Y27632 (Reagents Direct). Part of the cells won't attached because of the toxicity of the treatment on Dl and D2.

[00256] Basal medium, we selected within DMEM/F12, RPMI, DMEM and ACDF.

Currently we used DMEM/F12 (Fisher Scientific).

[00257] N2 (Life Technology), which is a reagent commercialized by Life Technology. Any other cell culture supplements, like B27 (Life Technology), KnockOut Serum Replacement (Life Technology), they will all be applicable here.

[00258] 1% BSA (Life Technology) or HSA (Irvine Scientific)

[00259] lmM Glutamine (Life Technology), or GlutaMAX™ (Life Technology) as product protocol

[00260] NEAA (Life Technology) as product protocol

[00261] Lipid Mixture (Sigma Aldrich) as product protocol [00262] 280ug/ml L-Ascorbic Acid (Sigma Aldrich). The concentration range was from

0. lug/ml to 400ug/ml. 500ug/ml showed some toxicity.

[00263] 20ng/ml bFGF. The concentration range was from 5ng/ml to lOOng/ml;

[00264] * In the future we might optimize complete new recipe for HDS2 medium.

[00265] Twelve hours later, if the cells attached well, experiment proceeded to transfection. Transfected the cells with Soxl7 mRNA at a dose of lOOng per well with Stemgent Transfection Reagent (Stemgent) in 1ml of DMEM/F12 basal medium supplemented with 200ng/ml B18R. mRNA doze range from 10~200ng per well. Most of the tranfection regents in the market were applicable. Two hours after the transfection, switched the medium to HDS2 medium.

[00266] D4

[00267] Because of the Soxl7 mRNA transfection, the cell morphology greatly changed.

Second Soxl7 mRNA transfection was performed following the same protocol of D3.

[00268] The range of Soxl7 mRNA transfection repeated is at as least as 1. One D4, if morphology changed to endoderm and hepatocyte progenitor cells had been great and even, one can skip the second transfection and moved on directly to stage 3; if morphology change hadn't been as great or as even as expected after second transfection, one can perform extra one more or two more transfection. In one embodiment, transfected the cells with Fox2A, Soxl7, GATA4,HNF4a, or other mesoendoderm promoting factor mRNAs.

[00269] D5

[00270] Switch medium to HDS3 medium, incubate the cells in 5% oxygen. The range of the oxygen is 2.5% to 10% [00271] Basal medium, we selected within DMEM/F12, RPMI, DMEM and ACDF.

Currently we used DMEM/F12 (Fisher Scientific).

[00272] N2 (Life Technology), which is a reagent commercialized by Life Technology. Any other cell culture supplements, like B27 (Life Technology), KnockOut Serum Replacement (Life Technology), they will all be applicable here.

[00273] 1% BSA (Life Technology) or HSA (Irvine Scientific)

[00274] lmM Glutamine (Life Technology), or GlutaMAX™ (Life Technology) as product protocol

[00275] NEAA (Life Technology) as product protocol [00276] Lipid Mixture (Sigma Aldrich) as product protocol

[00277] 280ug/ml L-Ascorbic Acid (Sigma Aldrich). The concentration range was from

0. lug/ml to 400ug/ml. 500ug/ml showed some toxicity.

[00278] 1 % DMSO (Sigma Altrich) The concentration range was from 0.5% to 2%;

[00279] * In the future we might optimize complete new recipe for HDS3 medium.

[00280] D6

[00281] Some hepatocyte progenitor cells already appeared. Changed medium with the same protocol as D5.

[00282] D7

[00283] Switch medium to HPCM medium.

[00284] Basal medium, we selected within DMEM/F12, RPMI, DMEM and ACDF.

Currently we used DMEM/F12 (Fisher Scientific). [00285] N2 (Life Technology), which is a reagent commercialized by Life Technology. Any other cell culture supplements, like B27 (Life Technology), KnockOut Serum Replacement (Life Technology), they will all be applicable here.

[00286] 1% BSA (Life Technology) or HSA (Irvine Scientific)

[00287] lmM Glutamine (Life Technology), or GlutaMAX™ (Life Technology) as product protocol

[00288] NEAA (Life Technology) as product protocol [00289] Lipid Mixture (Sigma Aldrich) as product protocol

[00290] 280ug/ml L-Ascorbic Acid (Sigma Aldrich). The concentration range was from

0. lug/ml to 400ug/ml. 500ug/ml showed some toxicity.

[00291] 1% DMSO (Sigma Altrich) The concentration range was from 0.5% to 2%;

[00292] lOng/ml EGF. The concentration range was from lng/ml to 20ng/ml;

[00293] 10 mM nicotinamide. The concentration range was from lmM to 20mM;

[00294] 100 nM dexamethasone. The concentration range was from InM to 200nM;

[00295] Optional 20ng/ml bFGF. The concentration range was from 5ng/ml to lOOng/ml;

[00296] When cells grew to 90% confluent, dissociated the cells with Triple (Life Technology) and split the cells by 1 :3, seeded the cells on Matrigel pre-coated or Collagen I pre- coated plate. These hepatocyte progenitor cells can be maintained for at least 6 passages in HPCM medium. For storage, these cells can be cryopreserved in 10% DMSO HPCM medium. For thawing and re-culture, 90% of the cryo-preserved cells can attached and grow in HPCM medium with 5uM rock inhibitor Y27632 (Reagents Direct).

[00297] 4. MSCs [00298] Example 4. Exemplary Protcol for Inducing MSC Differentiation

[00299] MSCs Differentiation

[00300] Table MSC-1

[00301] Day 0

[00302] Commercial cell culture vessels (Corning) were used at the first stage. 6-well plate was used in our experiments, but all the other types are applicable. Plates were pre-coated with Matrigel (BD Biosciences). As alternatives, E-cadherin (Primorigen), Synthemax® II (Corning), Vitrogectin (Primorigen) or feeder cells, all worked in this stage.

[00303] iPSCs which were prepared and ready for this experiment were dissociated with

Triple (Life Technology) from the original plates. Ready means there were enough typical iPSCs colonies with sharp edge, compact cells, not overgrown. After adding the Triple dissociation solution, the plates were incubated at 37°C for 2 mins. The range of incubation time is from 1 min to 10 mins. [00304] With this protocol, iPSCs colonies in the original plates were dissociated into single cells or small clusters, lxl 0^Λ6 cells were in one well of pre-coated 6-well plate with E8 medium (Stemcell Technology) supplemented with 5uM rock inhibitor Y27632 (Reagents Direct). E8 medium can be replaced with any iPSCs/ESCs maintenance medium, like mTeSR™ (Stemcell Technology), Stemline ®(Sigma Aldrich) or L7™(Lonza). The concentration range of Y27632 was 5uM to 15uM as we tested. Rock inhibitor will help the cells survival after dissociation or cryopreservation.

[00305] Stage 1: Day 1-Day 9

[00306] The next day, cells reached 90% confluent. Change medium to MSCD. Change medium every day and keep culture the cells at least 6 days, mostly 9 days, not beyond 12 days, depending on the cell growth condition.

[00307] Basal medium, we selected within DMEM/F12, RPMI, DMEM and ACDF.

Currently we used DMEM/F12 (Fisher Scientific).

[00308] N2 (Life Technology), which is a reagent commercialized by Life Technology. Any other cell culture supplements, like B27 (Life Technology), KnockOut Serum Replacement (Life Technology), they will all be applicable here.

[00309] 2.5% FBS (Life Technology)

[00310] lmM Glutamine (Life Technology), or GlutaMAX™ (Life Technology) as product protocol

[00311] NEAA (Life Technology) as product protocol [00312] Lipid Mixture (Sigma Aldrich) as product protocol

[00313] 280ug/ml L-Ascorbic Acid (Sigma Aldrich). The concentration range was from

0. lug/ml to 400ug/ml. 500ug/ml showed some toxicity. [00314] 5ng/ml bFGF. The concentration range was from 5ng/ml to lOng/ml.

[00315] Stage 2 : Day 10-Day 18

[00316] Commercial cell culture vessels (Corning) were used at stage 2. 6-well plate was used in our experiments, but all the other types are applicable. Plates were pre-coated with Matrigel (BD Biosciences). As alternatives, E-cadherin (Primorigen), Synthemax® II (Corning), Vitrogectin (Primorigen) or feeder cells, all worked in this stage.

[00317] Cells produced from stage 1 were dissociated with Triple (Life Technology) from the original plates. After adding the Triple dissociation solution, the plates were incubated at 37°C for 2 mins. The range of incubation time is from 1 min to 10 mins.

[00318] With this protocol, cells produced from stage 1 were dissociated into single cells or small clusters, lxl 0^Λ6 cells were seeded in one well of pre-coated 6-well plate in MSCD medium supplemented with 5uM rock inhibitor Y27632 (Reagents Direct). The concentration range of Y27632 was 5uM to 15uM as we tested. Rock inhibitor will help the cells survival after dissociation or cryopreservation.

[00319] Cells at stage 2 grew slower than stage 1. So we don't need to change medium every day. Usually we changed medium every three days and keep culture the cells at least 6 days, mostly 9 days, not beyond 12 days, depending on the cell growth condition.

[00320] Stage 3 : Day 19-Day 22

[00321] Commercial cell culture vessels (Corning) were used at stage 2. 6-well plate was used in our experiments, but all the other types are applicable. Plates were pre-coated with Matrigel (BD Biosciences). [00322] Cells produced from stage 2 were dissociated with Triple (Life Technology) from the original plates. After adding the Triple dissociation solution, the plates were incubated at 37°C for 2 mins. The range of incubation time is from 1 min to 10 mins.

[00323] With this protocol, cells produced from stage 2 were dissociated into single cells or small clusters. 5x10^Λ5 cells were seeded in one well of pre-coated 6-well plate in MSCM medium supplemented with 5uM rock inhibitor Y27632 (Reagents Direct). The concentration range of Y27632 was 5uM to 15uM as we tested. Rock inhibitor will help the cells survival after dissociation or cryopreservation.

[00324] Change Medium every two days.

[00325] MSCM medium recipe:

[00326] Basal medium, we selected within DMEM/F12, RPMI, DMEM and ACDF.

Currently we used DMEM/F12 (Fisher Scientific).

[00327] N2 (Life Technology), which is a reagent commercialized by Life Technology. Any other cell culture supplements, like B27 (Life Technology), KnockOut Serum Replacement (Life Technology), they will all be applicable here.

[00328] 10% FBS (Life Technology)

[00329] lmM Glutamine (Life Technology), or GlutaMAX™ (Life Technology) as product protocol

[00330] NEAA (Life Technology) as product protocol [00331] Lipid Mixture (Sigma Aldrich) as product protocol

[00332] 280ug/ml L- Ascorbic Acid (Sigma Aldrich). The concentration range was from

0. lug/ml to 400ug/ml. 500ug/ml showed some toxicity.

[00333] 20ng/ml bFGF. The concentration range was from 5ng/ml to lOng/ml. [00334] Stage 4: Expansion

[00335] Dissociated the cells with Triple (Life Technology). After adding the Triple dissociation solution, the plates were incubated at 37°C for 2 mins. The range of incubation time is from 1 min to 10 mins. Then split the cells in one well of 6-well plate into one T25 flask without coating.

[00336] When cells grew to 90% confluent, dissociated the cells with Triple (Life Technology) and split the cells by 1 :3 again. These cells can be maintained for at least 6 passages in MSCM medium. For storage, these cells can be cryopreserved in 10% DMSO MSCM medium. For thawing and re-culture, 90% of the cryo-preserved cells can attached and grow in MSCM medium with 5uM rock inhibitor Y27632 (Reagents Direct).

[00337] EXAMPLE 5. Neurons

[00338] Exemplary Method of Inducing Neuron Differentiation

[00339] Neurons Differentiation

[00340] Commercial cell culture vessels were used in this experiment. 6-well plate or 96- well plate were the mostly used types in our protocol, but all the other types are applicable.

[00341] Plates were pre-coated with poly-L-omithine (PLO)/laminin (Sigma Aldrich),

Matrigel (BD Biosciences) or Fibronectin (Sigma Aldrich).

[00342] Neural Progenitor Cells (NPCs) which were prepared and ready for this experiment were dissociated with 0.5mM EDTA (Sigma Aldrich) in DPBS (Fisher Scientific) mixed with Triple (Life Technology) by 3: 1 from the original plates. Ready means there were enough NPCs growing as thin plateau, but not overgrown. After adding the dissociation solution, the plates were incubated at 37°C for 10 mins. As we had tried, the final concentration range of EDTA is from 0.1 mM to 0.25 mM, the final concentration range of Triple is from ¼ to ½, the range of incubation time is from 1 min to 10 mins.

[00343] With this protocol, NPCs plateau in the original plates were dissociated into single cells or small clusters. 100K cells were seeded into one well of 6-well plate pre-coated with PLO/laminin in NM medium supplemented with 5uM rock inhibitor Y27632 (Reagents Direct). The cell numbers ranged from 50k to 500k. The concentration range of Y27632 was 5uM to 15uM as we tested. Rock inhibitor will help the cells survival after dissociation or cryopreservation. NPCs will spontaneously differentiate into neurons in 6 days.

[00344] Basal medium, we selected within DMEM/F12, RPMI, DMEM and ACDF.

Currently we used DMEM/F12 (Fisher Scientific).

[00345] N2 (Life Technology), which is a reagent commercialized by Life Technology. Any other cell culture supplements, like B27 (Life Technology), KnockOut Serum Replacement (Life Technology), they will all be applicable here.

[00346] 1% BSA (Life Technology) or HSA (Irvine Scientific)

[00347] lmM Glutamine (Life Technology), or GlutaMAX™ (Life Technology) as product protocol

[00348] NEAA (Life Technology) as product protocol [00349] Lipid Mixture (Sigma Aldrich) as product protocol

[00350] 280ug/ml L-Ascorbic Acid (Sigma Aldrich). The concentration range was from

0. lug/ml to 400ug/ml. 500ug/ml showed some toxicity.

[00351] * In the future we might optimize complete new recipe for NM medium. [00352] For storage, these cells can be cryopreserved in 10% DMSO 50% FBS or 10%

BSA NM medium. For thawing and re-culture, 90% of the cyropreserved cells can attached and grow in NM medium with 5uM rock inhibitor Y27632 (Reagents Direct).

[00353] EXAMPLE 6. NPCs

[00354] Exemplary Method for Inducing NPCs Differentiation

[00355] Table NPC-1

[00356] DO

[00357] Commercial cell culture vessels were used in this experiment. 6-well plate was used in our protocol, but all the other types are applicable. Plates were pre-coated with Matrigel (BD Biosciences). As altematives, E-cadherin (Primorigen), Synthemax® II (Corning), Vitrogectin (Primorigen) or feeder cells, all worked in this stage. [00358] iPSCs which were prepared and ready for this experiment were dissociated with

Triple (Life Technology) from the original plates. Ready means there were enough typical iPSCs colonies with sharp edge, compact cells, not overgrown. After adding the Triple dissociation solution, the plates were incubated at 37°C for 2 mins. The range of incubation time is from 1 min to 10 mins.

[00359] With this protocol, iPSCs colonies in the original plates were dissociated into single cells or small clusters, lxl 0^Λ6 cells were in one well of pre-coated 6-well plate with E8 medium (Stemcell Technology) supplemented with 5uM rock inhibitor Y27632 (Reagents Direct). E8 medium can be replaced with any iPSCs/ESCs maintenance medium, like mTeSR™ (Stemcell Technology), Stemline ®(Sigma Aldrich) or L7™(Lonza). The concentration range of Y27632 was 5uM to 15uM as we tested. Rock inhibitor will help the cells survival after dissociation or cryopreservation.

[00360] Stage 1 of the exemplary protocol: (Day 1-Day 3)

[00361] The next day, cells reached 90% confluent. Change medium to NDS1. Change medium every day.

[00362] Basal medium, we selected within DMEM/F12, RPMI, DMEM and ACDF.

Currently we used DMEM/F12 (Fisher Scientific).

[00363] N2 (Life Technology), which is a reagent commercialized by Life Technology. Any other cell culture supplements, like B27 (Life Technology), KnockOut Serum Replacement (Life Technology), they will all be applicable here.

[00364] 1% BSA (Life Technology) or HSA (Irvine Scientific)

[00365] lmM Glutamine (Life Technology), or GlutaMAX™ (Life Technology) as product protocol

[00366] NEAA (Life Technology) as product protocol [00367] Lipid Mixture (Sigma Aldrich) as product protocol

[00368] 280ug/ml L-Ascorbic Acid (Sigma Aldrich). The concentration range was from

0. lug/ml to 400ug/ml. 500ug/ml showed some toxicity.

[00369] luM PD03205901. The concentration range was from 0. luM to lOuM;

[00370] luM LDN193189. The concentration range was from O. luM to lOuM;

[00371] lOuM SB431542. The concentration range was from 5uM to 20Um.

[00372] Stage 2 of the exemplary protocol: (Day 4-Day 9)

[00373] Change medium to NDS 1. Change medium every day.

[00374] Basal medium, we selected within DMEM/F12, RPMI, DMEM and ACDF.

Currently we used DMEM/F12 (Fisher Scientific).

[00375] N2 (Life Technology), which is a reagent commercialized by Life Technology. Any other cell culture supplements, like B27 (Life Technology), KnockOut Serum Replacement (Life Technology), they will all be applicable here.

[00376] 1% BSA (Life Technology) or HSA (Irvine Scientific)

[00377] lmM Glutamine (Life Technology), or GlutaMAX™ (Life Technology) as product protocol

[00378] NEAA (Life Technology) as product protocol [00379] Lipid Mixture (Sigma Aldrich) as product protocol

[00380] 280ug/ml L-Ascorbic Acid (Sigma Aldrich). The concentration range was from

0. lug/ml to 400ug/ml. 500ug/ml showed some toxicity.

[00381] 20ng/ml bFGF. The concentration range was from 5ng/ml to lOOng/ml; [00382] luM LDN193189. The concentration range was from 0.1 uM to lOuM;

[00383] lOuM SB431542. The concentration range was from 5uM to 20uM.

[00384] Stage 3: Expansion

[00385] Dissociated the cells with 0.05mM EDTA in DBPS. After adding the EDTA solution, incubated for 30 mins. This protocol will selectively release NPC regions as NPC clusters. Collect the clusters further dissociated with EDTA/Triple (3: 1). After adding the solution, incubated for 10 mins. This protocol will break the clusters into single cells or small cluster. Seeded the NPCs from one well of 6-well plate into one well of 12-well plate pre- coated with poly-L-ornithine (PLO)/laminin (Sigma Aldrich) with NBM mediums. Usually cell will reach confluent in three to six days. Cells will grow up and reach confluent quickly, then form thin neural plateau regionally. Then dissociated the cells with EDTA/Triple (3: 1) with the same protocol, split the cells by 1 :3. This NPCs can be expanded in NBM mediums for at least 25 passages. Suspension culture at low oxygen will significantly increase the expansion efficiency, which can be helpful for large scale production.

[00386] EXAMPLE 7. EXEMPLARY CARDIAC DIFFERENTIATION

PROTOCOL

[00387] Exemplary Protocol for Induction: Cardiac Differentiation protocol and reagents

[00388] Chemical defined protocol

[00389] Reference: Chemically defined generation of human cardiomyocytes, Burridge et al 2014, Nature Methods, 11 :855-860

CD3+CHIR CD3+C59 CD3 medium only CDM3L (no D-glucose CD3+insulin

5mM Lactic acid

Exemplary Schematic of optimized chemically defined cardiac differentiation protocol.

[00390] H9 hESCs are maintained in 5% O2, 5% CO2 incubator until cells are around 80- 90% confluent and ready to passage.

[00391] On day 4, cells are digested and plated on Matrigel coated 12-well plates. Cells are aspirated and spend medium removed; cells are washed once with PBS-Ca²⁺-Mg²⁺, 1 ml of TrypLE is added to each well of 6 well plate, and incubated in 37 degree for 5 minutes. During incubation, mTeSR-E8 medium is prepared with 10 μΜ of Rock inhibitor Y27632.

[00392] After 5 minutes digestions, the cells are collected and diluted each in 1 ml of TrypLE with 4 ml of DMEM/F-12, then centrifuged at 200g for 5 minutes. Cells are resuspended in mTeSR-E8 medium with Rock inhibitor and counted.

[00393] Cells are plated at 100,000-200,000 cells/ well of 12-well plate with mTeSR-E8 containing Rock inhibitor.

[00394] The media is changed in the 12 well plate with 2 ml of fresh mTeSR-E8 for the next three days.

[00395] On day 0, CD3+ medium is prepared according to the information below,

CHIR9902 (GSK3 ) inhibitor at final concentration of 6-12 μΜ is added to the CD3+ medium and the cell culture medium is replaced with 2 ml of CD3+ with CHIR. [00396] On day 2, CD3+ medium is prepared, C59 (Wnt inhibitor) is added at final concentration of 2 μΜ , and CHIR containing medium is replaced with C59 medium.

[00397] On day 4, the medium is replaced with with CD3+ medium, and the medium is changed every other day till day 10. The beating cardiomyocyte-like cells can be observed from day 7-10.

[00398] On day 10, selection medium according to the instructions below is prepared, the

CD3+ medium is replaced with CD3L selection medium. From day 10-day 20, the medium is replaced every other day with fresh selection medium.

[00399] On day 20, the medium is switched back to CD3+ medium, with Insulin added

(Insulin can help cell survival after selection at 10μg/ml).

[00400] Medium for cardiac differentiation:

[00401] L-Ascrobic acid 2-phosphate (AA), Sigma, A8960; Recombinant human serum albumin (HSA), Sigma, A0237; Insulin, Life Tech, 12585-014; Wnt-C59(C59), Selleckchem, S7037.

[00402] Differentiation medium-RPMI with HSA and AA— CDM3+: to a 500ml bottle of RPMI is added a 2 ml aliquot of HSA and one 2 ml aliquot of AA, add one 5 ml aliquot of P/S, the mixture is mixed well. The medium can last in 4 degree for 10 days, or make 50ml aliquots and store in minus 20 degrees C.

Recombinant

Human Serum 125 mg/ml 10g 80m 500 ug/m Albumin (HSA)

L-Ascrobic acid

53,2385

2-phosphate 5g )3,917ml 213 ug/m

mg/mi

(AA)

[00403] Medium for cardiac cell selection and enrichment:

[00404] Selection medium— RPMI without glucose with HSA, AA and lactate— CDM3-

[00405] RPMI 1640 with L-Glu and without glucose, Life Tech, 11879-020; IM HEPES buffer Life Tech 156300-80, Lactic acid

[00406] To a 500 ml bottle of RPMI without glucose is added one 2 ml aliquot of HSA and one 2 ml aliquot of AA. A IM lactate stock solution is prepared: to a 9 ml of HEPES buffer is added 1ml of lactic acid. This solution can be kept at 4 degree for up to 30 days. 2.5 ml of IM lactate stock is added to the bottle of RPMI without glucose with HSA and AA and one 5 ml aliquot of P/S is added and the mixture is mixed well.

[00407] Immunofluorescence

[00408] Cardiomyocytes can be dissociated with TrypLE and re-plated on suitable laminin-coated cell-imaging surfaces such as 8-well glass chamber slides.

[00409] Mouse anti-sarcomeric alpha actinin Sigma A7811

[00410] Rabbit anti-NKX2.5 Santa Cruz sc-14033

[00411] 1) cells are washed once with PBS+ [00412] 2) cells are fixed with 4% PFA for 30min at room temperature

[00413] 3) cells are washed once with PBS+

[00414] 4) Cells are permeabilized with PBS-0.3% TritonX-100 for lOmin at room temperature.

[00415] 5) Cells are blocked with blocking buffer (3% BSA in PBS) for 30min at room temperature.

[00416] 6) primary antibodies in immunofluorescence blocking solution is prepared:

[00417] Mouse anti-sarcomeric a-actinin is at 1 : 100

[00418] Rabbit anti-NKX2.25 is at 1 : 100

[00419] To the reaction aspirated blocking solution and added primary antibody mixture. Incubated overnight at 4C in a humidified chamber.

[00420] 7) cells are washed three times for 5min at room temperature with PBS+.

[00421] 8) Prepared secondary antibodies and DNA stained in blocking solution.

[00422] Aspirated PBS+ and add secondary antibody mixture. Incubated for 2h in the dark, in a humidified chamber.

[00423] 9) cells are washed three times for 5min at room temperature with PBS+.

[00424] 10) cells are mounted and maintained at 4C in the dark until ready to perform immunofluorescence analysis.

[00425] EXAMPLE 8. EXEMPLARY Muscle Cell Differentiation

[00426] Stagel can proceed to stage 2 or cryopreservation as needed.

[00427] Day 0 [00428] Ultra-low attachment cell culture vessels (Coming) were used at the first stage. 6- well plate was used in our experiments, but all the other types are applicable.

[00429] iPSCs which were prepared and ready for this experiment were dissociated with

[00430] This EDTA solution were called non-enzyme cell dissociation solution which was much better than enzyme based dissociation solution, like Trypsin (Life Technology), Accutase (Life Technology) or Triple (Life Technology), if single cells are not necessary for next step. Chemicals in the same family of EDTA might all work. Even though quite a few experienced people knew this, it's still a small trick to many other people. Actually some companies use this trick to package some expensive reagents and lots of people buy them.

[00431] With this protocol, iPSCs colonies in the original plates were dissociated into small clusters. These small clusters were cultured in the ultra-low attachment plates with E8 medium (Stemcell Technology) supplemented with 5uM rock inhibitor Y27632 (Reagents Direct). E8 medium can be replaced with any iPSCs/ESCs maintenance medium, like mTeSR™ (Stemcell Technology), Stemline ®(Sigma Aldrich) or L7™(Lonza). The concentration range of Y27632 was 5uM to 15uM as tested. Rock inhibitor will help the cells survival after dissociation or cryopreservation.

[00432] This suspension culture system is an important factor to scale up this protocol.

[00433] Day 1

[00434] The small clusters formed iPSC spheres floating in the medium on the second day. If the spheres are healthy, they will have clear sharp edges. Then the experiment started.

[00435] Centrifuged the plates at 50g for 2 mins. This part is also a small trick. Most people will transfer the culture into tubes, then wait about 10 mins until the cells down to the bottom when they handle suspension iPSCs culture. Because the centrifuge force usually used, like 400g or even 200g will damage the spheres a bit. But without centrifuge, the cell pellets in the bottom are loose and very easy to be agitated. With a simple 50 g centrifuge for 2 mins, the most of the spheres will attach to the bottom firmly enough, yet the force won't affect the spheres at all, which can be very convenient for medium change. Removed the old medium with pipet, then add the AD SI medium.

[00436] Basal medium can be selected from DMEM/F12, RPMI, DMEM and ACDF. In one embodiment DMEM/F12 (Fisher Scientific) is used.

[00437] N2 (Life Technology), which is a reagent commercialized by Life Technology. Any other cell culture supplements, like B27 (Life Technology), KnockOut Serum Replacement (Life Technology), they will all be applicable here.

[00438] 1% BSA (Life Technology) or HSA (Irvine Scientific)

[00439] lmM Glutamine (Life Technology), or GlutaMAX™ (Life Technology) as product protocol

[00440] NEAA (Life Technology) as product protocol [00441] Lipid Mixture (Sigma Aldrich) as product protocol [00442] 280ug/ml L-Ascorbic Acid (Sigma Aldrich). The concentration range was from

0. lug/ml to 400ug/ml. 500ug/ml showed some toxicity.

[00443] 20ng/ml bFGF. The concentration range was from 5ng/ml to lOOng/ml;

[00444] 7.5uM CHIR99021. The concentration range was from 2.5uM to 15uM. Other chemicals in the GSK-3 inhibitor family worked either.

[00445] Optimized for ADS 1 medium.

[00446] D2

[00447] Because of the toxicity from the treatments, some cells died and suspended in the medium as single cells. Changed medium with the same protocol as Dl.

[00448] D3

[00449] Because of the toxicity from the treatments, more cells died and suspended in the medium as single cells. If there were enough healthy spheres left, experiment proceeded to stage 2 or Cryopreservation.

[00450] The cells at this stage were transfected with MyoD or other muscle lineage factor mRNA to induce fast transition.

[00451] EXAMPLE 9. Exemplary protocol for induction of differentiation using

Endoderm cells/tissues

[00452] The exemplary induction protocol using "Endoderm" derived cells/tissue comprises the following combination of steps and parameters using Soxl7 mRNA to derive pluripotent stem cell towards all the cell types.

[00453] Table Endo-1 I Stage 1 I Stage 2 Duration 2 days 2-6 days

Starting Cells iPSCs »>

Ultra-low attachment plate /

Culture Vessels flask Culture Plate/flask

Coating NO Matrigel / Collagen I

Dissociation EDTA TrypLE

1χ10^Λ6~1χ10^Λ7 cells per ml

Seeding Density medium

Medium and Main

Components MEMa, DMEM/F12, DMEM MEMa, DMEM/F12, DMEM

B27-insulin, +10-50uM Insulin B27-insulin, +10-50uM Insulin / l/5x

/ l/5x KSR KSR

2.5-10 uM CHIR.99021 Sox 17 mRNA transfection

The resultant cell culture and induction are shown in FIG 1-4 for stage 1 and 5-8 for stage 2.

[00454] EXAMPLE 10. Exemplary protocol for induction of differentiation using Hepatic cells/tissues

[00455] Hepatic cells are one type of endoderm that can be made after endoderm induction. The exemplary induction protocol using "hepatic" derived cells/tissue comprises the following combination of steps and parameters:

[00456] Table Hepato-2

Stage 1 Stage 2 Stage 3

Duration 3-5 days 3-5 days ~ months

Starting Cells Endoderm »> »>

Culture Vessels Culture Plate/flask Culture Plate/flask Culture Plate/flask

Coating Matrigel / Collagen I Matrigel / Collagen I Matrigel / Collagen I

Dissociation TrypLE TrypLE TrypLE

1χ10^Λ5~1χ10^Λ6 cells

Seeding Density per ml medium »> »>

Medium and Main

Components HD1 HD2 HM

MEMa, DMEM/F12, MEMa, DMEM/F12,

DMEM DMEM

B27 / KSR B27 / KSR

DMSO / Hex mRNA 5-20 ng/ml KGF/FGF10

transfection (about lOng/ml)

5-20 ng/ml BMP4

(about lOng/ml)

10 mM nicotinamide 100 nM dexamethasome

HNF4a mRNA

transfection

Optional:

5-20 ng/ml EGF

(optimized lOng/ml)

5-20 ng/ml bFGF

(optimized lOng/ml)

[00457] The resultant cell culture and induction are shown in FIG 9-11 (stage 1) FIG 12

(Stage 2)

[00458] EXAMPLE 11. Exemplary protocol for induction of differentiation using Cardiac cells/ tissues

[00459] Applicant surprisingly discovered that Induction of Cardiac cell/tissue was possible using the present protocol as the induction was rather unexpected as cells origin is mesoderm, however, mesoderm cells/ lineage taken from close proximity to endoderm can make these cells using an mRNA scheme as described herein.

[00460] The exemplary induction protocol using "cardiac" derived cells/tissue comprises the following combination of steps and parameters:

[00461] Table cardiac- 1

EGF 5~20ng/Ml

[00462] The resultant cell culture and induction are shown in FIG 43.

[00463] EXAMPLE 12. Exemplary protocol for induction of differentiation using Lung e.g. Lung Epithelial cells/tissues

[00464] The exemplary induction protocol using "lung" derived cells/tissue (e.g. lung epithelium) comprises the following combination of steps and parameters:

[00465] Table lung-1

[00466] The resultant cell culture and induction are shown in FIG 13-14 (stage 3) and FIG

15-16 (stage 4)

[00467] EXAMPLE 13. Exemplary protocol for induction of differentiation using Pancreatic cells / tissues

[00468] The exemplary induction protocol using "pancreatic" derived cells/tissue comprises the following combination of steps and parameters: Pancreatic (e.g. beta island) cell

[00469] Table pancreatic- 1

[00470] PDl medium was used in both Stage 1 and 2, which is MCBD131 supplemented with 8 mM Glucose or DMEM/F12.

[00471] PD2 medium was used in Stage 3, which is MCBD131 supplemented with 12 mM Glucose or DMEM High Glucose [00472] Before we start Stage 1 , we can optionally use 10-50ng/ml KGF pre-treatment for

1-3 days in a medium same as PD1 except 15% KSR.

[00473] Preferable to co-transfect, PDX1, SOX9, Ptfla, MNX1 in Stage 1

[00474] Preferable to co-transfect, NKX6.1, NKX2.2, Pax6, Pax4, Arx, Isll, NeuronDl,

Ngn3, RFX6, Mafa in Stage2 and Stage3

[00475] The resultant cell culture and induction are shown in FIG 17-21 (Stage 1); FIG

22-24 (stage 2) and FIG 25-28 (stage 3).

Claims

Claims:

1. A method for inducing differentiation of stem cell into cell or tissue -type specific precursor cells comprising the steps of : providing starting cells C obtained from a subject; culturing cells under suitable growth conditions whereby specific differentiation is induced; and

whereby the pluripotent state or progenitor state of stem cells or progenitor cells towards a specific lineage or tissue cell type is induced.

2. The method of claim 1 wherein the method comprises the steps of : culturing cell at seeding or plating density "S" by plating cells in a vessel and/or on a coated solid support comprising one or more coating agent selected from anone or more of the coating agents listed in Examples 1-13.

3. The method of claim 2 wherein "S" is a cell density set forth in the protocols of Examples 1-3 and can range between any of two values "S" set forth therein.

4. The method of claims 2-3 wherein the method comprises the steps of :

culturing cells for a time duration " T" ranging between any of the two values as set forth in Examples 1-13 therein.

5. The method of claims 2-4 wherein the method comprises:

culturing cell by maintaining cells in the presence of medium "M" comprising one or more of the "M" ingredients listed in the protocols from Examples 1-13.

6. The method of claims 2-5 wherein the method comprises:

dissociating cells during passaging by contacting the cells with dissociating agent "D" comprising one or more of the ingredients as set forth in Examples 1-13.

7. A cell obtained the method of claim 1.

8. A composition for treating disease, disorder or malformation comprising the cell of claim 7.

9. A method of treating disease, disorder, and/or malformation comprising administering into the subject in need thereof the cell/and/or the composition of claim 8.

10. The method of claim 9 wherein the cell is derived from the recipient subject.

11. The method of claim 1 wherein the starting cells are harvested from a body fluid or tissue.

12. The method of claim 1 1 wherein the starting cells are harvested from the recipient.