WO2011026221A1

WO2011026221A1 - Methods of culturing embryoid bodies

Info

Publication number: WO2011026221A1
Application number: PCT/CA2010/001334
Authority: WO
Inventors: Mickie Bhatia; Eleftherios Sachlos
Original assignee: McMaster University
Current assignee: McMaster University
Priority date: 2009-09-01
Filing date: 2010-08-31
Publication date: 2011-03-10
Anticipated expiration: 2012-03-01

Abstract

The present disclosure provides a method of generating immobilized embryoid bodies (iEBs) comprising plating multipotent or pluripotent stem cells in a predetermined adhesive location in a well; and culturing the cells under conditions that allow formation of embryoid bodies; wherein the embryoid bodies produced are immobilized to the predetermined adhesive location in the well. Also provided are methods of high throughput screening using the methods described herein.

Description

Title: METHODS OF CULTURING EMBRYOID BODIES Cross Reference to Related Applications

[0001] This application claims the benefit of priority of copending U.S. provisional application 61/238,796 filed on September 1 , 2009, the contents of which are incorporated herein by reference in their entirety.

Field of the disclosure

[0002] The disclosure relates to improved methods of culturing embryoid bodies and uses thereof. The methods are particularly useful for high throughput screening platforms.

Background of the disclosure

[0003] Pluripotent and multipotent stem cells distinguish themselves from other cell types by their ability to 1 ) self-renew and 2) differentiate into specialized cell types. Differentiation of stem cells is of particular interest to drug discovery research due to the potential of using these cells in identifying novel therapeutic compounds. Methods have been developed to coerce stem cells to differentiate into multiple specialized cells types including blood progenitors, cardiomyocytes, hepatocytes, neurons and pancreatic islet cells. Such differentiated cell types are expected to behave as reliable in vitro models by resembling native tissue states more than immortalized cell lines of the same tissue type. With truly reliable in vitro models, it will be possible to identify more compounds with potential therapeutic benefit at an earlier stage of the drug development process and reduce the drug attrition rate.

[0004] One method frequently employed to differentiate stem cells is culturing them in three dimensional clusters known as embryoid bodies (EBs). These EBs are created by detaching stem cells from the Petri dish in clusters and transferring them to non-adhesive plates (Fig 1 (B)). The stem cell clusters then form balls of cells that start interacting with each other in ways that are still not completely understood but which eventually lead to differentiation into multiple cell types. It is presumed that the 3D nature of the EB is important in recapitulating the early stages of embryonic development. For this reason, EBs remain the most reliable technique used to differentiate stem cells into multiple cell types.

[0005] However, floating EBs create several technical challenges if one wishes to use them in high throughput screening (HTS) platforms. For example, the liquid medium exchanges required to culture adherent cells in HTS microtiter plates would result in the EBs being aspirated from the well.

Summary of the disclosure

[0006] The present inventors have developed improvements in methods for stem cell differentiation that allow use of them in high throughput systems.

[0007] Accordingly, the present application provides a method of generating immobilized embryoid bodies (iEBs) comprising

(a) plating multipotent or pluripotent stem cells in a predetermined adhesive location in a well; and (b) culturing the cells under conditions that allow formation of embryoid bodies; wherein the embryoid bodies produced in (b) are immobilized to the predetermined adhesive location in the well.

[0008] In one embodiment, the well is a microwell. In another embodiment, the well comprises an adhesive layer patterned onto a non- adhesive surface in the predetermined location.

[0009] The present application also provides a well or a plurality of wells comprising immobilized embryoid bodies. In one embodiment, the well is a microwell. In another embodiment, the well comprises an adhesive layer patterned onto a non-adhesive surface in the predetermined location.

[0010] Also provided herein are methods and uses of the cultures of immobilized embryoid bodies or the wells comprising said embryoid bodies for screening analysis, such as high throughput screening. [0011] Further provided herein are methods and uses of the cultures of immobilized embryoid bodies or the wells comprising said embryoid bodies for producing differentiated cells.

[0012] Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the disclosure are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

Brief description of the drawings

[0013] The disclosure will now be described in relation to the drawings in which:

[0014] Figure 1 shows suspension of embryoid bodies (EBs) versus immobilized EBs (iEBs).

[0015] Figure 2 shows hematopoietic output of suspension EBs. The frequency of CD34+ and CD45+ cells is recorded at 16.2%.

[0016] Figure 3 shows flow cytometry analysis of hematopoietic cells derived from iEBs. The frequency of CD34+ and CD45+ cells is recorded at 48.2%.

[0017] Figure 4 shows a schematic of the various stages of cell differentiation.

Detailed description of the disclosure

[0018] The present application provides a method of generating immobilized embryoid bodies (iEBs) comprising

(a) plating multipotent or pluripotent stem cells in a predetermined adhesive location in a well; and

(b) culturing the cells under conditions that allow formation of embryoid bodies; wherein the embryoid bodies produced in (b) are immobilized to the predetermined adhesive location in the well.

[0019] The term "stem cell" as used herein refers to a cell that has the ability for self-renewal and has the ability to differentiate into a diverse range of cell types.

[0020] The term "pluripotent stem cell" as used herein refers to a stem cell that can give rise to cells of multiple cell types. The term "multipotent stem cell" as used herein refers to a stem cell that can give rise to many but limited types of cells. [0021] In one embodiment, the stem cell is an embryonic stem cell. In another embodiment, the stem cell is an induced pluripotent stem cell.

[0022] The cells may be a multipotent or pluripotent stem cell derived from any organism, including, without limitation, members of the animal and plant kingdom. In one embodiment, the cells are derived or obtained from an animal, such as a human or mouse.

[0023] The term "embryoid body" as used herein refers to a three- dimensional cluster or aggregate derived from multipotent or pluripotent stem cells.

[0024] The phrase "formation of embryoid bodies" as used herein refers to the formation of three dimensional clusters or aggregates of cells in culture. Conditions which allow formation of embryoid bodies are known in the art, including, without limitation: (1 ) use of ultra-low adhesion culture dishes which inhibit cell adhesion and promote self-aggregation; (2) hanging-drop formation which utilizes gravitational and droplet surface tension forces to confine and promote cellular aggregation; and (3) forged aggregation using centrifugation techniques.

[0025] In one embodiment, the method further comprises culturing the immobilized embryoid bodies under conditions to allow differentiation. [0026] The term "differentiation" as used herein refers to the process by which a less specialized cell, such as a stem cell, becomes a more specialized cell type, such that it is committed to a specific lineage. Conditions for differentiation of multipotent or pluripotent stem cells are readily known in the art. For example culturing EBs in the presence of serum and several growth factors and cytokines increases hematopoietic progenitor output, for example, SCF, Flt3 ligand. IL-3, IL-6, G-CSF and BMP4 (see Chadwick KK et al., Blood. 2003 Aug 1 ;102(3):906-15). To produce cardiomyocyte; cells may be treated with 5-aza-2'-deoxycytidin or co-cultured with visceral endoderm-like cells (Xu et al. 2002; Mummery et al. 2003; and Kehat et al. 2004).). To produce neural cells, cells may be treated with FGF2, Retinoic acid (RA), noggin or medium supplements like N2 or B27 (Reubinoff et al. 2000; Zhang et al. 2001 ; Ben-Hur et al. 2004; and Aharonowiz et al. 2008). To produce hepatocytes, cells may be treated with aFGF, hematocyte growth factor (HGF), oncostatin (OSM) and Dex treatment (Rambhatla et al. 2003; and Lavon et al. 2004). To produce pancreatic cells, cells may be treated with activin and RA, Nicitinamide, HGF and glucagons-like peptide I (Lumelsky et al. 2001 ; and Assady et al. 2001).

[0027] In one embodiment, the well is a microwell. In another embodiment, the microwell is on a microtiter plate.

[0028] The term "a predetermined adhesive location" as used herein refers to one or more specific adhesive locations in the well where the cells are plated. In one embodiment, the well comprises an adhesive layer patterned at one or more predetermined locations onto a non-adhesive surface. In such an embodiment, the plated cells adhere to the patterned adhesive layer or layers but do not spread to the non-adhesive surface. Adhesive layers include, without limitation, matrigel, which is a basement membrane analogue, laminin, fibronectin, the collagens (ie type I, IV, III, etc), vitronectin, elastin to name a few biological molecules. Non-biological surfaces include, without limitation, plasma treated polystyrene. In an embodiment, the non-adhesive surface comprises repellent plastic or low adhesion plates. In another embodiment, the non-adhesive surface is created by treating plates with agents that convert the polarity of the tissue culture surface, such as pluronic. Pluronic F68 (BASF) is a block copolymer based on ethylene oxide and propylene oxide.

[0029] Chemical or biological compounds are typically added to the media used to culture EBs. EBs are grown in suspension and considerable care is exercised when exchanging the medium due to the risk of aspirating the EBs and destroying them. On the other hand, microtiter plates are central to the high throughput screening (HTS) process because they permit multiple experiments with different compounds in small volumes to be conducted. Suspension EBs cannot be cultured in microtiter plates because the robotic system would aspirate them from the wells. The methods described herein immobilize EBs to the well, for example, on a microtiter plate and permit the addition of chemical or biological compounds to or alteration of conditions in each well. Furthermore, it is recognized that differentiation is a multistep process and multiple compounds or conditions can be added, changed and/or removed at different stages of the iEB culture in order to trigger and analyze different responses during differentiation (see Figure 4). Suspension EBs are only usually treated with a single compound or single dose of a cocktail of compounds.

[0030] Accordingly, in another embodiment, there is provided a method of screening of embryoid bodies comprising

(a) preparing a plurality of immobilized embryoid bodies (iEBs) by the methods described herein;

(b) treating the plurality of iEBs with a test agent or agent(s) or culturing the plurality of iEBs under test conditions;

(c) subjecting the iEBs of (b) to comparative analysis.

[0031] The phrase "comparative analysis" as used herein refers to a comparison of the treated cells to a control. The term "control" as used herein refers to an untreated sample or samples (negative control) or a sample or samples known to have a particular effect or known to be indicative of a particular level of differentiation or treatment (positive control). The term "control" also includes a pre-determined standard or value.

[0032] The comparative analysis includes, without limitation, detecting cell surface molecules indicative of various differentiation states, detecting particular gene or protein expression levels, and analyzing cell morphology, in comparison to control. In one embodiment, CD45, CD34 and CD31 cell markers are detected to indicate the hematopoietic lineage. The ability to form hematopoieitic colonies of the erythryoid (CFU-E or BFU-E), macrophage (CFU-M) and granulocyte (CFU-G) lineages or combinations of these lineages is a hallmark of stem cell or progenitor with hematopoietic potential. In one embodiment, other markers are detected that are indicative of other lineages. Stem cells differentiate into numerous lineages, with each lineage characterized by a unique set of markers useful for detecting a particular lineage (See for example Regenerative Medicine. Department of Health and Human Services. August 2006. Available from the National Institutes of Health (</info/scireport/2006report.htm>) and online at http://stemcells.nih.gov/info/scireport/appendixe.asp).

[0033] In an embodiment, the screening is high throughput screening. The term "high throughput screening" as used herein refers to automated in vitro testing of the effect of agents or conditions on cells and such screening is typically performed with the aid of computer or robot-controlled processes.

[0034] The term "plurality" as used herein refers to more than 1 , 5, 10, 50, 75, 100, 1000, 1500, 2000, 3000, or more than 5000.

[0035] In one embodiment, each well contains one immobilized embryoid body (iEB). In such an embodiment, the plurality of iEBs comprises a plurality of wells. In another embodiment, each well contains more than one iEB at the predetermined locations. In an embodiment, the wells are contained on a microtiter plate. [0036] In one embodiment, the test agent is a chemical, biological or other substance that is being tested for its effect on triggering different responses during differentiation of the embryoid bodies into specific cell types. Examples include, without limitation, organic and inorganic compounds, hydrocarbons, growth factors, cytokines, extracellular matrix components, cell-culture conditioned media, live or dead cells, tissues and portions thereof, test proteins, lipids, carbohydrates, peptides, and nucleic acids.

[0037] Examples of test conditions include, without limitation, environmental conditions such as temperature, pH and oxygen pressure and physical conditions, such as cell-cell interactions.

[0038] In another embodiment, the test agent or agents or test conditions are added, altered and/or removed at various stages of differentiation.

[0039] In yet another embodiment, the screening is used to identify compounds useful in regenerative therapy, for example, for regenerating blood. In a further embodiment, the test agent is a chemical or drug and the screening is used as a primary screen, or as a secondary pharmacology and toxicology evaluation screen for the chemical or drug. In yet a further embodiment, the screening is useful in gene expression profiling. Gene expression profiling provides information indicative of differentiated state. Compounds capable of inducing differentiation are identified by their ability to upregulate or downregulate characteristic genes indicative of a specific differentiated state eg. neuronal or hematopoietic. Differentiation of the cells towards a certain lineage can be defined based on the expression of a specific marker by immunostaining of cells or by transcriptional or translational profiling of one or multiple genes.

[0040] The present disclosure also provides a well or a plurality of wells comprising immobilized embryoid bodies. In one embodiment, the well is a microwell. In another embodiment, the microwell is on a microtiter plate. [0041] The disclosure also includes the use of the immobilized embryoid bodies to produce differentiated cells. Also included herein is a method of producing differentiated cells comprising preparing immobilized embryoid bodies as described herein and culturing the immobilized embryoid bodies under differentiation culture conditions. One of skill in the art can determine the appropriate differentiation culture conditions for preparing particular cells some of which are described above. Differentiated cells include without limitation, hematopoietic cells (such as blood cells), cardiomyocytes, neural cells, hepatocytes and pancreatic cells.

[0042] In one embodiment, the present disclosure provides a method of producing hematopoietic cells comprising preparing immobilized embryoid bodies as described herein and culturing the immobilized embryoid bodies under hematopoietic differentiation conditions, such as media containing serum and supplemented with cytokines including SCF, Flt3 ligand, IL-3, IL-6, G-CSF and BMP4. Hematopoietic cells include, without limitation, hematopoietic stem cells, hematopoietic progenitor cells or their derivatives. In one embodiment, the derivatives are blood cells.

[0043] In one embodiment, other conditions and associated methods known to a person of skill in the art for the differentiation of other lineages can be used to culture the immobilized embryoid bodies described herein. For example, stem cells can be coerced to differentiate to a specific lineage using either defined differentiation culture media, specialized extracellular matrices, treating with soluble molecules, co-culturing with supportive cell types and/or using mechanical stimulation as known in the art.

[0044] The above disclosure generally describes the present disclosure. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely for the purpose of illustration and are not intended to limit the scope of the disclosure. Changes in form and substitution of equivalents are contemplated as circumstances might suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

[0045] The following non-limiting examples are illustrative of the present disclosure: Examples

[0046] The present application provides a method to generate immobilized EBs (iEBs) in microtiter plates. This process employed surface patterning to constrain the growth surface of the stem cells and force them to aggregate as 3D clusters. Specifically, an adhesive layer of Matrigel, a basement membrane analogue commonly used in stem cell culture, was patterned onto a non-adhesive surface (option of using repellent plastic/low adhesion plates or treating plates with pluronic or other agents that convert the polarity of tissue culture surfaces) creating areas to which stem cells selectively attached. As the stem cells proliferated, they were unable to migrate onto the non-adhesive areas and consequently were forced to aggregate laterally. This led to packing of the cells and triggering of EB formation (Fig 1 C). The iEBs were cultured in KO-DMEM with 20% fetal bovine serum, 1 mM L-glutamine, 1 % non-essential amino acids, 0.1 mM beta- mercaptoethanol supplemented with hematopoietic cytokines including SCF, Flt-3 ligand, IL-3, IL-6, G-CSF and BMP4 for at least 14 days at 37°C and 5% C0₂.

[0047] It was found that these iEBs have increased hematopoietic differentiation potential. The hematopoietic output of both suspension EBs (figure 2) and iEBs (figure 3) was measured by staining for CD34 and CD45 and analyzed with a flow cytometer. The higher frequency of CD34+ and CD45+ in iEBs means that this method of differentiating stem cells could potentially be used for regenerative medicine applications which aim to produce blood.

[0048] While the present disclosure has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the disclosure is not limited to the disclosed examples. To the contrary, the disclosure is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[0049] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

REFERENCES

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2) Assady S, Maor G, Amit M, Itskovitz-Eldor J, Skorecki KL, Tzukerman M. Insulin production by human embryonic stem cells. Diabetes. 2001 Aug;50(8):1691-7.

3) Ben-Hur T, Idelson M, Khaner H, Pera M, Reinhartz E, Itzik A, Reubinoff BE. Transplantation of human embryonic stem cell-derived neural progenitors improves behavioral deficit in Parkinsonian rats. Stem Cells. 2004;22(7): 1246-55.

4) Kehat I, Khimovich L, Caspi O, Gepstein A, Shofti R, Arbel G, Huber I, Satin J, Itskovitz-Eldor J, Gepstein L. Electromechanical integration of cardiomyocytes derived from human embryonic stem cells. Nat Biotechnol. 2004 Oct;22(10): 1282-9. Epub 2004 Sep 26.

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Claims

Claims:

1. A method of generating immobilized embryoid bodies (iEBs) comprising

(a) plating multipotent or pluripotent stem cells in a predetermined adhesive location in a well; and (b) culturing the cells under conditions that allow formation of embryoid bodies;

wherein the embryoid bodies produced in (b) are immobilized to the predetermined adhesive location in the well.

2. The method of claim 1 , wherein the multipotent or pluripotent stem cell comprises an embryonic stem cell or an induced pluripotent stem cell.

3. The method of claim 1 or 2, wherein the well comprises an adhesive layer patterned at one or more predetermined locations onto a non-adhesive surface.

4. The method of claim 3, wherein the adhesive layer comprises matrigel. 5. The method of claim 3 or 4, wherein the non-adhesive surface comprises repellent plastic or low adhesion plates.

6. The method of claim 3 or 4, wherein the non-adhesive surface has been created by treating plates with an agent that converts the polarity of the tissue culture surface. 7. The method of claim 6, wherein the agent is pluronic.

8. A method of screening of embryoid bodies comprising

(a) preparing a plurality of immobilized embryoid bodies (iEBs) by the method of any one of claims 1 to 7;

(b) treating the plurality of iEBs with a test agent or agents or culturing the iEBs under test conditions;

(c) subjecting the treated iEBs to comparative analysis.

9. The method of claim 8, wherein each well contains one immobilized embryoid body (iEB).

10. The method of claim 8, wherein each well contains more than one iEB at the predetermined locations. 1 1 . The method of any one of claims 8 to 10, wherein the wells are contained on a microtiter plate.

12. The method of any one of claims 8 to 1 1 , wherein the screening is high throughput screening.

13. The method of any one of claims 8 to 12, wherein the test agent is a chemical, biological or other substance that is being tested for its effect on triggering different responses during differentiation of the embryoid bodies into specific cell types.

14. The method of any one of claims 8 to 12, wherein the test conditions are environmental or physical conditions. 15. The method of any one of claims 8 to 14, wherein the test agent or agents or test conditions are added and/or removed at various stages of differentiation. 6. The method of any one of claims 8 to 15, for identifying compounds or conditions useful in regenerative therapy. 17. The method of claim 16, wherein the regenerative therapy comprises blood.

18. The method of any one of claims 8 to 17, wherein the comparative analysis comprises detecting cell surface molecules indicative of various differentiation states. 19. The method of any one of claims 8 to 17, wherein the comparative analysis comprises detecting levels of apoptotic markers or cell debris.

20. The method of any one of claims 8 to 17, wherein the comparative analysis comprises detecting cell death or cell toxicity.

21. The method of any one of claims 8 to 17, wherein the comparative analysis comprises gene expression profiling. 22. A well or plurality of wells comprising immobilized embryoid bodies prepared according to a method of any one of claims 1 to 7.

23. The well or plurality of wells according to claim 22 wherein the wells are microwells.

24. The well or plurality of wells according to claim 23 wherein the microwells are on a microtiter plate.

25. A method of producing differentiated cells comprising:

a) preparing immobilized embryoid bodies according to the method of any one of claims 1 to 7; and

b) culturing the immobilized embryoid bodies under differentiation culture conditions to produce differentiated cells.

26. The method according to claim 25 wherein the differentiated cells are hematopoietic cells.

27. The method according to claim 26 wherein the hematopoietic cells are hematopoietic stem cells, hematopoietic progenitor cells or their derivatives. 28. The method of claim 27, wherein the derivatives are blood cells.