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Methods for identifying factors for differentiating definitive endoderm

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Methods for identifying factors for differentiating definitive endoderm


Disclosed herein are methods of identifying one or more differentiation factors that are useful for differentiating cells in a cell population comprising definitive endoderm cells into cells which are capable of forming tissues and/or organs that are derived from the gut tube.
Related Terms: Endoderm

Inventors: Kevin Allen D'Amour, Alan D. Agulnick, Susan Eliazer, Emmanuel E. Baetge
USPTO Applicaton #: #20120276624 - Class: 435347 (USPTO) - 11/01/12 - Class 435 
Chemistry: Molecular Biology And Microbiology > Animal Cell, Per Se (e.g., Cell Lines, Etc.); Composition Thereof; Process Of Propagating, Maintaining Or Preserving An Animal Cell Or Composition Thereof; Process Of Isolating Or Separating An Animal Cell Or Composition Thereof; Process Of Preparing A Composition Containing An Animal Cell; Culture Media Therefore >Two Or More Cell Types, Per Se, In Co-culture

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The Patent Description & Claims data below is from USPTO Patent Application 20120276624, Methods for identifying factors for differentiating definitive endoderm.

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RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 12/476,570, entitled METHODS FOR IDENTIFYING FACTORS FOR DIFFERENTIATING DEFINITIVE ENDODERM, filed Jun. 2, 2009, which is a continuation of U.S. patent application Ser. No. 11/165,305, entitled METHODS FOR IDENTIFYING FACTORS FOR DIFFERENTIATING DEFINITIVE ENDODERM, filed Jun. 23, 2005, which is a continuation-in-part of U.S. patent application Ser. No. 11/115,868, entitled PDX1 EXPRESSING ENDODERM, filed Apr. 26, 2005, now abandoned, which claims priority under 35 U.S.C. §119(e) as a nonprovisional application of U.S. Provisional Patent Application No. 60/566,293, entitled PDX1 EXPRESSING ENDODERM, filed Apr. 27, 2004; U.S. Provisional Patent Application No. 60/587,942, entitled CHEMOKINE CELL SURFACE RECEPTOR FOR THE ISOLATION OF DEFINITIVE ENDODERM, filed Jul. 14, 2004; and U.S. Provisional Patent Application No. 60/586,566, entitled CHEMOKINE CELL SURFACE RECEPTOR FOR THE ISOLATION OF DEFINITIVE ENDODERM, filed Jul. 9, 2004; U.S. patent application Ser. No. 11/165,305 is also a continuation-in-part of U.S. patent application Ser. No. 11/021,618, entitled DEFINITIVE ENDODERM, filed Dec. 23, 2004, now U.S. Pat. No. 7,510,876, issued Mar. 31, 2009, which claims priority under 35 U.S.C. §119(e) as a nonprovisional application to U.S. Provisional Patent Application No. 60/587,942, entitled CHEMOKINE CELL SURFACE RECEPTOR FOR THE ISOLATION OF DEFINITIVE ENDODERM, filed Jul. 14, 2004; and U.S. Provisional Patent Application No. 60/586,566, entitled CHEMOKINE CELL SURFACE RECEPTOR FOR THE ISOLATION OF DEFINITIVE ENDODERM, filed Jul. 9, 2004. and U.S. Provisional Patent Application No. 60/532,004, entitled DEFINITIVE ENDODERM, filed Dec. 23, 2003. The disclosure of each of the foregoing priority applications is incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a text file entitled CYTHERA45CP1C2.TXT, created Jul. 9, 2012, which is 5.55 KB in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the fields of medicine and cell biology. In particular, the present invention relates the identification of factors that are useful for differentiating definitive endoderm cells into other cell types.

BACKGROUND

Human pluripotent stem cells, such as embryonic stem (ES) cells and embryonic germ (EG) cells, were first isolated in culture without fibroblast feeders in 1994 (Bongso et al., 1994) and with fibroblast feeders (Hogan, 1997). Later, Thomson, Reubinoff and Shamblott established continuous cultures of human ES and EG cells using mitotically inactivated mouse feeder layers (Reubinoff et al., 2000; Shamblott et al., 1998; Thomson et al., 1998).

Human ES and EG cells (hESCs) offer unique opportunities for investigating early stages of human development as well as for therapeutic intervention in several disease states, such as diabetes mellitus and Parkinson\'s disease. For example, the use of insulin-producing β-cells derived from hESCs would offer a vast improvement over current cell therapy procedures that utilize cells from donor pancreases for the treatment of diabetes. However, presently it is not known how to generate an insulin-producing β-cell from hESCs. As such, current cell therapy treatments for diabetes mellitus, which utilize islet cells from donor pancreases, are limited by the scarcity of high quality islet cells needed for transplant. Cell therapy for a single Type I diabetic patient requires a transplant of approximately 8×108 pancreatic islet cells. (Shapiro et al., 2000; Shapiro et al., 2001a; Shapiro et al., 2001b). As such, at least two healthy donor organs are required to obtain sufficient islet cells for a successful transplant. Human embryonic stem cells offer a source of starting material from which to develop substantial quantities of high quality differentiated cells for human cell therapies.

Two properties that make hESCs uniquely suited to cell therapy applications are pluripotence and the ability to maintain these cells in culture for prolonged periods. Pluripotency is defined by the ability of hESCs to differentiate to derivatives of all 3 primary germ layers (endoderm, mesoderm, ectoderm) which, in turn, form all somatic cell types of the mature organism in addition to extraembryonic tissues (e.g. placenta) and germ cells. Although pluripotency imparts extraordinary utility upon hESCs, this property also poses unique challenges for the study and manipulation of these cells and their derivatives. Owing to the large variety of cell types that may arise in differentiating hESC cultures, the vast majority of cell types are produced at very low efficiencies. Additionally, success in evaluating production of any given cell type depends critically on defining appropriate markers. Achieving efficient, directed differentiation is of great importance for therapeutic application of hESCs.

In order to use hESCs as a starting material to generate cells that are useful in cell therapy applications, it would be advantageous to overcome the foregoing problems. Additionally, it would be beneficial to identify factors which promote the differentiation of precursor cells derived from hESCs to cell types useful for cell therapies.

SUMMARY

OF THE INVENTION

Embodiments of the present invention relate to methods of identifying one or more differentiation factors that are useful for differentiating cells in a cell population comprising PDX1-positive (PDX1-expressing) endoderm cells and/or PDX1-negative endoderm cells (endoderm cells which do not significantly express PDX1), such as definitive endoderm cells, into cells that are useful for cell therapy. For example, some embodiments of the methods described herein relate to methods of identifying factors capable of promoting the differentiation of definitive endoderm cells into cells which are precursors for tissues and/or organs which include, but are not limited to, pancreas, liver, lungs, stomach, intestine, thyroid, thymus, pharynx, gallbladder and urinary bladder. In some embodiments, such precursor cells are PDX1-positive endoderm cells. In other embodiments, such precursor cells are endoderm cells that do not significantly express PDX1.

In some embodiments of the methods described herein, cell cultures or cell populations of definitive endoderm cells are contacted or otherwise provided with a candidate (test) differentiation factor. In preferred embodiments, the definitive endoderm cells are human definitive endoderm cells. In more preferred embodiments, the human definitive endoderm cells are multipotent cells that can differentiate into cells of the gut tube or organs derived therefrom.

In other embodiments of the methods described herein, cell cultures or cell populations of PDX1-positive endoderm cells are contacted or otherwise provided with a candidate differentiation factor. In preferred embodiments, the PDX1-positive endoderm cells are human PDX1-positive endoderm cells. In certain embodiments, the human PDX1-positive endoderm cells are PDX1-positive foregut/midgut endoderm cells. In more preferred embodiments, the human PDX1-positive endoderm cells are PDX1-positive foregut endoderm cells. In other preferred embodiments, the human PDX1-positive endoderm cells are PDX1-positive endoderm cells of the posterior portion of the foregut. In especially preferred embodiments, the human PDX1-positive foregut endoderm cells are multipotent cells that can differentiate into cells, tissues or organs derived from the anterior portion of the gut tube.

As related to the methods described herein, the candidate differentiation factor may be one that is known to cause cell differentiation or one that is not known to cause cell differentiation. In certain embodiments, the candidate differentiation factor can be a polypeptide, such as a growth factor. In some embodiments, the growth factor includes, but is not limited to, FGF10, FGF4, FGF2, Wnt3A or Wnt3B. In other embodiments, the candidate differentiation factor can be a small molecule. In particular embodiments, the small molecule is a retinoid compound, such as retinoic acid. Alternatively, in some embodiments, the candidate differentiation factor is not a retinoid, is not a foregut differentiation factor or is not a member of the TGFβ superfamily. In other embodiments, the candidate differentiation factor is any molecule other than a retinoid compound, a foregut differentiation factor, or a member of the TGFβ superfamily of growth factors, such as activins A and B. In still other embodiments, the candidate differentiation factor is a factor that was not previously known to cause the differentiation of definitive endoderm cells.

Additional embodiments of the methods described herein relate to testing candidate differentiation factors at a plurality of concentrations. For example, a candidate differentiation factor may cause the differentiation of definitive endoderm cells and/or PDX1-positive endoderm cells only at concentrations above a certain threshold. Additionally, a candidate differentiation factor can cause the same cell to differentiate into a first cell type when provided at a low concentration and a second cell type when provided at a higher concentration. In some embodiments, the candidate differentiation factor is provided at one or more concentrations ranging from about 0.1 ng/ml to about 10 mg/ml.

Prior to or at approximately the same time as contacting or otherwise providing the cell culture or cell population comprising definitive endoderm cells and/or PDX1-positive endoderm cells with the candidate differentiation factor, at least one marker is selected and evaluated so as to determine its expression. This step may be referred to as the first marker evaluation step. Alternatively, this step may be referred to as determining expression of a marker at a first time point. The marker can be any marker that is useful for monitoring cell differentiation, however, preferred markers include, but are not limited to, sex determining region Y-box 17 (SOX17), pancreatic-duodenal homeobox factor-1 (PDX1), albumin, hepatocyte specific antigen (HAS), prospero-related homeobox 1 (PROX1), thyroid transcription factor 1 (TITF1), villin, alpha fetoprotein (AFP), cytochrome P450 7A (CYP7A), tyrosine aminotransferase (TAT), hepatocyte nuclear factor 4a (HNF4a), CXC-type chemokine receptor 4 (CXCR4), von Willebrand factor (VWF), vascular cell adhesion molecule-1 (VACM1), apolipoprotein A1 (APOA1), glucose transporter-2 (GLUT2), alpha-1-antitrypsin (AAT), glukokinase (GLUKO), and human hematopoietically expressed homeobox (hHEX) and CDX2.

After sufficient time has passed since contacting or otherwise providing cell culture or cell population comprising definitive endoderm cells and/or PDX1-positive endoderm cells with the candidate differentiation factor, the expression of the at least one marker in the cell culture or cell population is again evaluated. This step may be referred to as the second marker evaluation step. Alternatively, this step may be referred to as determining expression of a marker at a second time point. In preferred embodiments, the marker evaluated at the first and second time points is the same marker.

In some embodiments of the methods described herein, it is further determined whether the expression of the at least one marker at the second time point has increased or decreased as compared to the expression of this marker at the first time point. An increase or decrease in the expression of the at least one marker indicates that the candidate differentiation factor is capable of promoting the differentiation of the definitive endoderm cells and/or the PDX1-positive endoderm cells. Sufficient time between contacting or otherwise providing a cell culture or cell population comprising definitive endoderm cells and/or PDX1-positive endoderm cells with the candidate differentiation factor and determining expression of the at least one marker at the second time point can be as little as from about 1 hour to as much as about 10 days. In some embodiments, the expression of the at least one marker is evaluated multiple times subsequent to contacting or otherwise providing the cell culture or cell population comprising definitive endoderm cells and/or PDX1-positive endoderm cells with the candidate differentiation factor. In certain embodiments, marker expression is evaluated by Q-PCR. In other embodiments, marker expression is evaluated by immunocytochemistry.

Additional embodiments of the present invention relate to a method of identifying a differentiation factor capable of promoting the differentiation of PDX1-negative definitive endoderm cells to PDX1-positive foregut endoderm cells. In such methods, PDX1-negative definitive endoderm cells are contacted with a candidate differentiation factor and it is determined whether PDX1 expression in the cell population after contact with the candidate differentiation factor has increased as compared to PDX1 expression in the cell population before contact with the candidate differentiation factor. An increase in the PDX1 expression in the cell population indicates that the candidate differentiation factor is capable of promoting the differentiation of PDX1-negative definitive endoderm cells to PDX1-positive foregut endoderm cells. In some embodiments, PDX1 expression is determined by quantitative polymerase chain reaction (Q-PCR). Some embodiments of the foregoing method further comprise the step of determining expression of the HOXA13 and/or the HOXC6 gene in the cell population before and after contact with the candidate differentiation factor. In some embodiments, the candidate differentiation factor is a small molecule, for example, a retinoid, such as RA. In others, the candidate differentiation factor is a polypeptide, for example, a growth factor, such as FGF-10.

Still other embodiments of the present invention relate to a method of identifying a differentiation factor capable of promoting the differentiation of PDX1-positive foregut endoderm cells. In such methods, PDX1-positive foregut endoderm cells are contacted with a candidate differentiation factor and it is determined whether expression of a marker in the population is increased or decreased after contact with the candidate differentiation factor as compared to the expression of the same marker in the population before contact with the candidate differentiation factor. An increase or decrease in the expression of the marker indicates that the candidate differentiation factor is capable of promoting the differentiation of PDX1-positive foregut endoderm cells. In some embodiments, marker expression is determined by Q-PCR. In some embodiments, the candidate differentiation factor is a small molecule, for example, a retinoid, such as RA. In others, the candidate differentiation factor is a polypeptide, for example, a growth factor, such as FGF-10.

Yet other embodiments of the present invention relate to cells differentiated by the methods described herein. Such cells include but are not limited to precursors of the pancreas, liver, lungs, stomach, intestine, thyroid, thymus, pharynx, gallbladder and urinary bladder. In some embodiments, the cells may be terminally differentiated. Other embodiments described herein relate to cell cultures and/or cell populations comprising the above-described cells.

In certain jurisdictions, there may not be any generally accepted definition of the term “comprising.” As used herein, the term “comprising” is intended to represent “open” language which permits the inclusion of any additional elements. With this in mind, additional embodiments of the present inventions are described with reference to the numbered paragraphs below:

1. A method of identifying a differentiation factor capable of promoting the differentiation of human definitive endoderm cells in a cell population comprising human cells, said method comprising the steps of: (a) obtaining a cell population comprising human definitive endoderm cells; (b) providing a candidate differentiation factor to said cell population; (c) determining expression of a marker in said cell population at a first time point; (d) determining expression of the same marker in said cell population at a second time point, wherein said second time point is subsequent to said first time point and wherein said second time point is subsequent to providing said cell population with said candidate differentiation factor; and (e) determining if expression of the marker in said cell population at said second time point is increased or decreased as compared to the expression of the marker in said cell population at said first time point, wherein an increase or decrease in expression of said marker in said cell population indicates that said candidate differentiation factor is capable of promoting the differentiation of said human definitive endoderm cells.

2. The method of paragraph 1, wherein said human definitive endoderm cells comprise at least about 10% of the human cells in said cell population.

3. The method of paragraph 1, wherein human feeder cells are present in said cell population and wherein at least about 10% of the human cells other than said feeder cells are definitive endoderm cells.

4. The method of paragraph 1, wherein said human definitive endoderm cells comprise at least about 90% of the human cells in said cell population.

5. The method of paragraph 1, wherein said human feeder cells are present in said cell population and wherein at least about 90% of the human cells other than said feeder cells are definitive endoderm cells.

6. The method of paragraph 1, wherein said human definitive endoderm cells differentiate into cells, tissues or organs derived from the gut tube in response to said candidate differentiation factor.

7. The method of paragraph 1, wherein said human definitive endoderm cells differentiate into pancreatic precursor cells in response to said candidate differentiation factor.

8. The method of paragraph 7, wherein said marker is selected from the group consisting of pancreatic-duodenal homeobox factor-1 (PDX1), homeobox A13 (HOXA13) and homeobox C6 (HOXC6).

9. The method of paragraph 1, wherein said human definitive endoderm cells differentiate into liver precursor cells in response to said candidate differentiation factor.

10. The method of paragraph 9, wherein said marker is selected from the group consisting of albumin, prospero-related homeobox 1 (PROX1) and hepatocyte specific antigen (HSA).

11. The method of paragraph 1, wherein said human definitive endoderm cells differentiate into lung precursor cells in response to said candidate differentiation factor.

12. The method of paragraph 11, wherein said marker is thyroid transcription factor 1 (TITF1).

13. The method of paragraph 1, wherein said human definitive endoderm cells differentiate into intestinal precursor cells in response to said candidate differentiation factor.

14. The method of paragraph 13, wherein said marker is selected from the group consisting of villin and caudal type homeobox transcription factor 2 (CDX2).

15. The method of paragraph 1, wherein said first time point is prior to providing said candidate differentiation factor to said cell population.

16. The method of paragraph 1, wherein said first time point is at approximately the same time as providing said candidate differentiation factor to said cell population.

17. The method of paragraph 1, wherein said first time point is subsequent to providing said candidate differentiation factor to said cell population.

18. The method of paragraph 1, wherein expression of said marker is increased.

19. The method of paragraph 1, wherein expression of said marker is decreased.

20. The method of paragraph 1, wherein expression of said marker is determined by quantitative polymerase chain reaction (Q-PCR).

21. The method of paragraph 1, wherein expression of said marker is determined by immunocytochemistry.

22. The method of paragraph 1, wherein said marker is selected from the group consisting of pancreatic-duodenal homeobox factor-1 (PDX1), homeobox A13 (HOXA13) and homeobox C6 (HOXC6).

23. The method of paragraph 1, wherein said marker is selected from the group consisting of albumin, prospero-related homeobox 1 (PROX1) and hepatocyte specific antigen (HSA).

24. The method of paragraph 1, wherein said marker is selected from the group consisting of villin and caudal type homeobox transcription factor 2 (CDX2).

25. The method of paragraph 1, wherein said marker is thyroid transcription factor 1 (TITF1).

26. The method of paragraph 1, wherein said differentiation factor comprises a foregut differentiation factor.

27. The method of paragraph 1, wherein said differentiation factor comprises a small molecule.

28. The method of paragraph 1, wherein said differentiation factor comprises a retinoid.

29. The method of paragraph 1, wherein said differentiation factor comprises retinoic acid.

30. The method of paragraph 1, wherein said differentiation factor comprises a polypeptide.

31. The method of paragraph 1, wherein said differentiation factor comprises a growth factor.

32. The method of paragraph 1, wherein said differentiation factor comprises FGF-10.



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stats Patent Info
Application #
US 20120276624 A1
Publish Date
11/01/2012
Document #
13544870
File Date
07/09/2012
USPTO Class
435347
Other USPTO Classes
International Class
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Drawings
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