WO2019113139A1 - Eosinophil culture system using human induced pluripotent stem cells - Google Patents

Abstract

Disclosed herein are eosinophil culture systems that contain myeloid cell-forming complexes (MCFCs). The MCFCs may be used to shed MCFC derived cells, including, for example, myeloid progenitor cells, myeloid precursor cells, and combinations thereof. The disclosed MCFC derived cells may be differentiated into eosinophils. Also disclosed are methods of deriving eosinophils from an iPSC cell.

Description

EOSINOPHIL CULTURE SYSTEM USING HUMAN INDUCED PULURIPOTENT

STEM CELLS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of U.S. Provisional Application Serial Number 62/594,818, to Fulkerson, filed December 5, 2017, the contents of which are incorporated in their entirety for all purposes.

BACKGROUND

[0002] Eosinophils are produced in the bone marrow from CD34-expressing eosinophil lineage-committed progenitors, whose levels in the bone marrow are elevated in a variety of human diseases. Differentiation of EoPs to mature eosinophils is a multistage process requiring the coordinated activity of multiple regulators. However, the developmental program that orchestrates eosinophil maturation is not well delineated. Studies focused on EoP maturation are inherently difficult to perform due to the rarity of EoPs in the bone marrow under homeostatic conditions; they comprise -0.05-0.1% of total murine bone marrow cells. As targeting eosinophil production with lineage-specific blockade has clinical potential as treatment for eosinophilia, robust and novel methods are needed to support and encourage studies into eosinophil development. The instant disclosure addresses one or more of the aforementioned needs in the art.

BRIEF SUMMARY

[0003] Disclosed herein are eosinophil culture systems that contain myeloid cell forming complexes (MCFCs). The MCFCs may be used to shed MCFC derived cells, including, for example, myeloid progenitor cells, myeloid precursor cells, and combinations thereof. The disclosed MCFC derived cells may be differentiated into eosinophils. Also disclosed are methods of deriving eosinophils from an iPSC cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0004] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0005] FIG 1 depicts an eosinophil differentiation schematic in human iPSC cultures. Induced stem cells (iPSCs) cultured in feeder-free conditions are initially induced to generate spherical embryoid bodies (EBs). The EBs are culture under conditions to form endothelial- like stromal cells and then myeloid cell-forming complexes that contain CD45+CD34+ hematopoietic progenitor cells. The CD34+ progenitors are subjected to prolonged IL-3 and IL-5 exposure to continuously generate terminally differentiated CDllb+Siglec-8+ eosinophils for 5-6 weeks.

[0006] FIGS 2A-2B. FIG 2 A depicts hematopoietic progenitors generated from human iPSCs. FIG. 2A depicts quantities by week of harvest of CD45+CD34+ hematopoietic progenitors harvested from the supernatants of cultures stimulated with IL-3 or IL-3 and IL-5 at weekly harvests. Quantities are given for one well of a six-well plate (mean ± SEM, n=3 wells per condition per time point). FIG 2B depicts cytospins of sorted CD45+CD34+ cells consistent with hematopoietic progenitors.

[0007] FIGS 3A and 3B. Eosinophils generated from human iPSCs. FIG 3A depicts quantities by week of harvest of CD45+Siglec-8+ eosinophils harvested from the

supernatants of cultures stimulated with IL-3 or IL-3 and IL-5. Quantities are given for one well of a six-well plate (mean ± SEM, n=3 wells per condition per time point). 3B shows that cytospins of sorted CD45+Siglec=8+ cells in top panel are consistent with terminally differentiated human eosinophils. Eosinophil peroxidase activity represented by brown precipitate in generated CD45+Siglec-8+ eosinophils in bottom panel.

[0008] FIG 4 depicts eosinophils generated from three unique human iPSC lines. Representative dot blots showing Siglec-8 surface staining of CD45+ cells generated from iPSCs cultured for hematopoietic and eosinophil differentiation in IL-3 and IL-5 are shown. Each dot blot represents a unique iPSC line. Percentage of gated, live CD45+ cells is shown in the Siglec-8 quadrant. DETAILED DESCRIPTION

[0009] DEFINITIONS

[0010] Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[001 1] As used herein and in the appended claims, the singular forms“a,”“and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to“a method” includes a plurality of such methods and reference to“a dose” includes reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.

[0012] The term“about” or“approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example,“about” may mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively,“about” may mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, for example, within 5-fold, or within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term“about” meaning within an acceptable error range for the particular value should be assumed.

[0013] Applicant has developed an embryoid body-based culture system that yields mature eosinophils starting from induced pluripotent stem cells, which is believed to be useful as a model system to identify critical regulators of eosinophil development, among other uses which will be understood in view of the instant disclosure. [0014] Eosinophils derived from iPS cells via an embryoid body (EB)-based culture system has not been described. Disclosed herein is an induced pluripotent stem cell (iPSC)- derived eosinophil culture system that provides a unique opportunity to test the effect of drugs and/or genetics on eosinophil development. In one aspect, the disclosed culture contains multiple cell types (stromal cells as well as hematopoietic cells) which results in improved differentiation compared to existing differentiation protocols. This culture system is adapted from a hematopoietic differentiation protocol designed to generate neutrophils and monocytes on a large scale (Lachmann N, Stem Cell Reports. 2015). The method of embryoid body (EB) formation and the sequential addition of particular cytokines, IL-3 and then IL-5, to specifically expand the eosinophil lineage are modified for the disclosed culture system. In one aspect, in the disclosed protocol, hematopoietic differentiation of iPSCs takes approximately 4 weeks in culture (FIG 1). Briefly, spherical embryoid bodies may be generated from iPSC colonies, manually transferred onto tissue culture plates and cultivated in media to promote hematopoietic differentiation. Myeloid cell— forming complexes (MCFCs) may be formed and are supported in the culture by endothelial-like stromal cells. In one aspect, MCFCs continuously shed myeloid progenitor and precursor cells for about 4-6 weeks, and these cells further differentiate into eosinophils. MCFCs cultured in IL-3 or IL-3 and IL-5 shed CD45⁺CD34⁺ hematopoietic progenitors that may be recoverable during the first three weeks of the culture (FIG 2A, highest production weeks 2-3). Applicant found that the presence of IL-5 resulted in an increased number of hematopoietic progenitors that are capable of producing eosinophil colonies in semisolid medium (data not shown). The cell morphology of sorted CD45⁺CD34⁺ cells is consistent with hematopoietic progenitors (FIG 2B). Hematopoietic differentiation in the presence of IL-3 or IL-3 and IL-5 resulted in the continuous production of CDl lb⁺Siglec-8⁺ cells over 5 weeks (FIG 3A, highest production weeks 3-5). The cell morphology of the Sigelc-8⁺ cells was consistent with eosinophil peroxidase— containing mature eosinophils (FIG 3B). Applicant has found that the generation of terminally differentiated eosinophils has been successful from multiple iPSC lines (FIG 4).

[0015] Disclosed herein are eosinophil culture systems, that comprising myeloid cell forming complexes (MCFCs) capable of shedding MCFC derived cells. The MCFC derived cells may include, for example, myeloid progenitor cells, myeloid precursor cells, and combinations thereof. The MCFC derived cells may be, in certain aspects, capable of differentiating into eosinophils. In one aspect, the MCFCs may be derived from embryoid bodies, wherein the embryoid bodies are derived from induced pluripotent stem cells (iPSCs). In certain aspects, the iPSCs may be derived from an individual having an eosinophil-related disorder, or an individual known to have a genotype associated with an eosinophil-related disorder.

[0016] Further disclosed are methods of deriving eosinophils from an iPSC cell. The method may comprise the steps of a. culturing an iPSC on a semi-solid medium for a period of time sufficient to form an iPSC monolayer;

b. fragmenting the iPSC monolayer to form a plurality of monolayer fragments; c. culturing the monolayer fragments for a period of time sufficient to form a

spherical embryoid body (EB);

d. contacting the EB with media comprising IL-5, IL-3, or a combination thereof; and

e. culturing the immature myeloid cells in the presence of IL-5, optionally including IL-3, for a period of time sufficient to generate terminally differentiated eosinophils.

[0017] In one aspect, the semi-solid medium may comprise a basement membrane matrix.

[0018] In one aspect, the iPSC cells of step a may be grown to confluence, for example, a confluence of about 70% to about 80%, prior to addition of the EB-media.

[0019] In one aspect, step a may be carried out for a period of time of from about 24 hours to about 150 hours, or from about 48 hours to about 120 hours, or about 72 to about 96 hours.

[0020] In one aspect, step a may be carried out for a period of time of from about 1 day to about 12 days, or from about 2 days to about 10 days, or from about 3 days to about 8 days, or from about 4 days to about 6 days, or about 5 days. [0021] In one aspect, the semi-solid medium containing iPSC cells of step a may be mechanically disrupted to disturb the monolayer into fragments, for example, via mechanical disruption. This may be accomplished be via a needle used to score the iPSC monolayer in a grid.

[0022] In one aspect, the iPSC monolayer of step (b) may be cultured in the presence of EB -media that promotes differentiation of the iPSCs. The EB media may comprise, for example, less than about 10 ng/mL, or less than about 9 ng/mL or less than about 8 ng/mL, or less than about 7 ng/mL, or less than about 6 ng/mL, or less than about 5 ng/mL, or less than about 4 ng/mL, or less than about 3 ng/mL, or less than about 2 ng/mL, or less than about 1 ng/mL, or is substantially free of low basic fibroblast growth factor (bLGL). The EB media may be replaced daily.

[0023] In one aspect, the iPSC monolayer may be contacted with an enzyme during the fragmentation step in an amount and for a period of time sufficient to disrupt the attachment of the monolayer to the culture plates. Exemplary enzymes include, for example, dispase, collagenase, or a combination thereof.

[0024] In one aspect, the fragmentation step may be carried out for a period of time of from about 2 days to about 10 days, or from about 4 days to about 8 days, or from about 5 days to about 6 days.

[0025] In one aspect, the monolayer fragments of step (b) may be cultured in the presence of EB-media. The EB media may comprise, for example, less than about 10 ng/mL, or less than about 9 ng/mL or less than about 8 ng/mL, or less than about 7 ng/mL, or less than about 6 ng/mL, or less than about 5 ng/mL, or less than about 4 ng/mL, or less than about 3 ng/mL, or less than about 2 ng/mL, or less than about 1 ng/mL, or is substantially free of low basic fibroblast growth factor (bLGL). The EB media may be replaced daily.

[0026] In one aspect, the monolayer fragments may be cultured with movement. In one aspect, the movement may be provided by an orbital shaker.

[0027] In one aspect, the EB may be cultured on a surface comprising a gelatin coating. In one aspect, the EB media of step c may be replaced at least once a day. In one aspect, step c may be carried out for a period of time of from about 1 day to about 12 days, or from about 2 days to about 10 days, or from about 3 days to about 8 days, or from about 4 days to about 6 days, or about 5 days. In one aspect, the EB may be cultured in a myeloid media comprising IL-3 in an amount sufficient to promotes hematopoietic differentiation without serum, optionally further comprising IL-5.

[0028] In one aspect, the culture period of step d may be for a period of time sufficient to allow formation of a myeloid-cell-forming-complex (MCFC), for example, wherein the MCFC produces immature myeloid cells expressing CD45, CD34, Siglec8, or combinations thereof. In one aspect, the step (d) may be carried out for a period of time of from about 1 day to about 12 days, or from about 2 days to about 10 days, or from about 3 days to about 8 days, or from about 4 days to about 6 days, or about 5 days. In one aspect, the MCFCs of step (d) are in suspension.

[0029] In one aspect, any of steps a through e may be carried out without the use of a feeder cell.

[0030] In one aspect, IL-3 may be present at an amount of from about 15 to about 40 ng/mL, or from about 20 to about 30 ng/mL or about 25 ng/mL; in one aspect, IL-5 may be present at an amount of from about 10 to about 100 ng/mL, or from about 25 to 75 ng/mL, or about 50 ng/mL. IL-3 may be present at an amount necessary for hematopoietic

differentiation (MCFC formation), and the amount may be empirically determined by one of ordinary skill in the art in view of the inventive concept identified by Applicant herein.

[0031 ] In one aspect, a method of identifying genes or gene polymorphisms that regulate eosinophil development and/or function is disclosed. In this aspect, the eosinophils of the disclosed culture may be used to identify genes and or polymorphisms that relate to or impact eosinophil development and/or function.

[0032] In one aspect, a method of testing a drug intended to affect an eosinophil is disclosed. In this aspect, the method may comprise the step of contacting a drug of interest with the eosinophil culture of claim 1 , further comprising the step of determining an outcome following contact with the drug, wherein the outcome may be selected from eosinophil number, gene expression, eosinophil function, eosinophil phenotype, or a combination thereof. EXAMPLES

[0033] The following non-limiting examples are provided to further illustrate embodiments of the invention disclosed herein. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches that have been found to function well in the practice of the invention, and thus may be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes may be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0034] EB-based eosinophil culture system from induced pluripotent stem cells.

[0035] Human iPSCs are cultured in mTEASR media (StemCElls) using matrigel- coated 6 wells plates. When cells reached 70-80 % confluency, media is changed to embryoid body (EB) -promoting media (EB Media) that can include DMEM, 20% of FBS (Hyclone Defined) and a non-essential amino acid solution or another serum free media that supports EB formation, such as X-VIVO medium (Lonza). EB media is replaced daily for 96 hours. Cells are treated with enzymes, such as dispase or collagenase, to loosen their attachment to the plate, then scored in a grid format using a 25-gauge needle to disturb the monolayer into fragments. Alternatively, the fragments can be generated by plating iPSCs on feeder cells followed by disruption via enzymatic digestion. EB formation is induced by cultivating the fragments in ultra- low binding plates in suspension on an orbital shaker (80 rpm) for 5 days - 7 days. EBs are manually transferred onto gelatin-coated 6-wells plates. Each well contains 15-20 EBs maintained in APEL™2 media ( STEMCELL ) supplemented with IL-3 (Myeloid media) or IL-3 and IL-5 (Eos media) for 7 days to form myeloid-cell-forming-complexes (MCFC). The media is fully replaced after 7 days. Cells in suspension are harvested weekly thereafter. The MCFCs contain CD34-expressing progenitor cells that generate immature eosinophils and other myeloid lineage cells. Cells are analyzed by flow cytometry to detect surface expression of CD45 (hematopoietic marker), CD34 (progenitor cell marker) and Siglec-8 (mature eosinophil marker). The immature myeloid cells can be collected and with continued exposure for an additional 10 days to IL-3 and IL-5 (or IL-5 alone) in IMDM culture media (Gibco) with added FBS and 2-mercaptoethanol to generate terminally

differentiated eosinophils . The EB Media is DMEM (Gibco), 20% FBS (Hyclone Defined), non-essential amino acids or X-VIVO (Lonza) and the Myeloid Media is APEL™2 Medium (STEMCELL), IL-3. For Eos media: APEL™2 Medium (STEMCELL), IL-3and IL-5.

[0036] All percentages and ratios are calculated by weight unless otherwise indicated.

[0037] All percentages and ratios are calculated based on the total composition unless otherwise indicated.

[0038] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0039] The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as“20 mm” is intended to mean “about 20 mm.”

[0040] Every document cited herein, including any cross referenced or related patent or application, is hereby incorporated herein by reference in its entirety unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.

[0041 ] While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications may be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Claims

CLAIMS What is claimed is:

1. An eosinophil culture system comprising myeloid cell- forming complexes (MCFCs), wherein said MCFCs shed MCFC derived cells selected from myeloid progenitor cells, myeloid precursor cells, and combinations thereof, wherein said MCFC derived cells are capable of differentiating into eosinophils, preferably wherein said MCFCs are derived from embryoid bodies, wherein said embryoid bodies are derived from induced pluripotent stem cells (iPSCs).

2. A method of deriving eosinophils from an iPSC cell, comprising the steps of

a. culturing an iPSC on a semi-solid medium for a period of time sufficient to form an iPSC monolayer;

b. fragmenting said iPSC monolayer to form a plurality of monolayer fragments; c. culturing said monolayer fragments for a period of time sufficient to form a spherical embryoid body (EB);

d. contacting said EB with media comprising IL-5, IL-3, or a combination thereof, for a period of time sufficient to allow formation of a myeloid-cell-forming- complex (MCFC) wherein said MCFC produces immature myeloid cells expressing CD45, CD34, Siglec8, or combinations thereof; and

e. culturing said immature myeloid cells in the presence of IL-5, optionally

including IL-3, for a period of time sufficient to generate terminally differentiated eosinophils.

3. The method of claim 2, wherein the semi-solid medium comprises a basement membrane matrix.

4. The method of claim 2 or 3, wherein the iPSC cells of step a are grown to confluence of about 70% to about 80% prior to addition of said EB-media.

5. The method of any of claims 2 through 4, wherein step a is carried out for a period of time of from about 24 hours to about 150 hours, or from about 48 hours to about 120 hours, or about 72 to about 96 hours.

6. The method of any of claims 2 through 5, wherein step a is carried out for a period of time of from about 1 day to about 12 days, or from about 2 days to about 10 days, or from about 3 days to about 8 days, or from about 4 days to about 6 days, or about 5 days.

7. The method of any of claims 2 through 6, wherein said iPSC monolayer of step b is cultured in the presence of EB-media that promotes differentiation of the iPSCs, preferably wherein said EB media may comprise lOng/mL or less of low basic fibroblast growth factor (bFGF), or wherein said EB Media is substantially free of bFGF.

8. The method of any of claims 2 through 7, wherein said iPSC monolayer is contacted with an enzyme in an amount and for a period of time sufficient to disrupt the attachment of the monolayer to the culture plates, preferably dispase, collagense, or a combination thereof.

9. The method of any of claims 2 through 8, wherein said fragmentation step comprises mechanical disruption of said iPSC monolayer, preferably wherein said mechanical disruption comprises scoring of said iPSC monolayer.

10. The method of any of claims 2 through 9, wherein said fragmentation step is carried out for a period of time of from about 2 days to about 10 days, or from about 4 days to about 8 days, or from about 5 days to about 6 days.

11. The method of any of claims 2 through 10, wherein the semi- solid medium containing iPSC cells of step a is mechanically disrupted to disturb the monolayer into fragments, preferably wherein said mechanical disruption is via a needle used to score said iPSC monolayer in a grid.

12. The method of claim any of claims 2 through 11, wherein said monolayer fragments of step b is cultured in the presence of EB media, wherein said EB media may comprise lOng/mL or less of low basic fibroblast growth factor (bFGF), or wherein said EB Media is substantially free of bFGF.

13. The method of any of claims 2 through 12, wherein said monolayer fragments may be cultured with movement, preferably wherein said movement is provided by an orbital shaker.

14. The method of any of claims 2 through 13, wherein said EB media of step c is replaced at least once a day.

15. The method of any of claims 2 through 14, wherein step c is carried out for a period of time of from about 1 day to about 12 days, or from about 2 days to about 10 days, or from about 3 days to about 8 days, or from about 4 days to about 6 days, or about 5 days.

16. The method of any of claims 2 through 15, wherein said EB is cultured on a surface comprising a gelatin coating.

17. The method of any of claims 2 through 16, wherein said EB is cultured in a myeloid media comprising IL-3 in an amount sufficient to promotes hematopoietic differentiation without serum, optionally further comprising IL-5.

18. The method of any of claims 2 through 17, wherein step d is carried out for a period of time of from about 1 day to about 12 days, or from about 2 days to about 10 days, or from about 3 days to about 8 days, or from about 4 days to about 6 days, or about 5 days.

19. The method of any of claims 2 through 18, wherein said MCFCs of step d are in

suspension.

20. The method of any of claims 2 through 19, wherein any of steps a through e do not comprise a feeder cell.

21. A method of identifying genes or gene polymorphisms that regulate eosinophil

development and/or function

22. A method of testing a drug intended to effect an eosinophil, comprising contacting a drug of interest with the eosinophil culture of claim 1, further comprising the step of determining an outcome following contact with said drug, wherein said outcome is selected from eosinophil number, gene expression, eosinophil function, eosinophil phenotype, or a combination thereof.

23. The method of any preceding claim, wherein said iPSC cell is obtained from an

individual having an eosinophilic disorder or known genetic mutation associated with higher than normal eosinophil production.