AN IN VITRO METHOD OF DIFFERENTIATING A HUMAN PLURIPOTENT STEM CELL POPULATION INTO A CARDIOMYOCYTE CELL POPULATION

Abstract

The current invention relates to a method of differentiation of human pluripotent stem cells into a human stem-cell derived population of cardiomyocytes. The method comprises the use of specific combination of steps and compounds to induce and/or promote differentiation. The method also comprises steps directed to further maturation of the cardiomyocytes obtained with the method of the invention. Also provided are kits for use in a method of differentiation as well as cell populations obtainable with the method disclosed.

Claims

1. An in vitro method of differentiating a human pluripotent stem cell population into a cardiomyocyte cell population, the method comprising b) contacting the human pluripotent stem cell population with XAV-939 and a second compound wherein the compound is IWP-L6 or C59 in an aqueous media.

2. The method of claim 1 wherein the method further comprises a) contacting, prior to step b), the human pluripotent stem cell population with at least a Wnt-signaling agonist in an aqueous media.

3. The method of claim 1, wherein the method further comprises contacting the human pluripotent stem cell population with IGF, preferably long range IGF in an aqueous media.

4. The method of claim 1, wherein the method further comprises c) contacting, after step b), the human pluripotent stem cell population with an aqueous media not comprising XAV-939, said second compound, and/or said Wnt-signaling agonist, preferably wherein the aqueous media comprises IGF.

5. The method of claim 1, wherein the second compound is IWP-L6.

6. The method of claim 1, wherein the media is essentially serum-free, and preferably wherein the media comprises lipids and one or more trace elements, and optionally insulin, transferrin and selenium.

7. The method of claim 1, wherein step b) is initiated at least 1 and up to 7 days after initiation of step a) and/or wherein said step b) is concluded within 35 days, preferably within 14 days, more preferably within 5 days, even more preferably within 3 days, most preferably within 2 days after its initiation.

8. The method of claim 1, wherein step c) is concluded within 35 days, preferably within 28 days, more preferably within 21 days, even more preferably within 14 days, most preferably within 10 days after its initiation.

9. The method of claim 1, wherein the concentration XAV-939 is 0.1-20 microM in the aqueous media; the concentration C-59 is 0.05-0.50 microM in the aqueous media; the concentration IWP-L6 is 0.01-15 microM in the aqueous media; and/or the concentration IGF is 0.01-10 ng/ml in the aqueous media.

10. The method of claim 1, wherein the second compound is IWP-L6 and wherein the ratio XAV-939:IWP-L6 is between 1:1 and 1:0.001, preferably between 1:0.1 and 1:0.01, more preferably between 1:0.2 and 1:0,8, even more preferably about 1:0.05.

11. The method of claim 1, wherein the concentration XAV-939 with which the human pluripotent stem cell population are contacted in step b) is a concentration, the increase of which will not further stimulate differentiation of the human pluripotent stem cell population in the absence of the second compound.

12. The method of claim 1, wherein the human pluripotent stem cell population is a population of embryonic stem cells or a population of induced pluripotent stem cells.

13. The method of claim 1, wherein prior to step a) or step b) the human pluripotent stem cell population is cultured in a stem cell maintenance media.

14. The method of claim 1, wherein the method further comprises, d) culturing the cardiomyocyte cell population in an aqueous media for maturing the cardiomyocyte cell population, preferably wherein the aqueous media comprises at least glucose, lipids, carnitine, creatine, taurine, optionally thyroid hormone or analogs, and optionally insulin, transferrin and selenium, preferably wherein prior to step d) the cells have been harvested and frozen before cultivating in step d).

15. The method of claim 1, wherein the method further comprises the step of verifying the presence of cardiomyocytes after step b), step c) and/or d).

16. The method of claim 15, wherein at least 60%, 70%, 80%, 90% or 95% of the cells are determined to be cardiomyocytes.

17. The method of claim 1, wherein the media of step b) does not contain vitamin A and/or derivative thereof.

18. The method of claim 1, wherein the media of step a), b) and/or c) does not contain vitamin A and/or derivative thereof.

19. A kit for differentiating a human pluripotent stem cell population into a cardiomyocyte cell population the kit comprising a) XAV939; a second compound wherein the compound is IWP-L6 or C59; an aqueous media; and optionally IGF, preferably wherein the kit further comprises a Wnt-signaling agonist, preferably CHIR-99021; or b) a first aqueous media comprising a Wnt-signaling agonist, preferably CHIR-99021; a second aqueous media comprising XAV-939 and the second compound wherein the compound is IWP-L6 or C59, preferably IWP-L6; and a third aqueous media that does not comprise XAV939, IWP-L6, C59 and a Wnt-signaling agonist, preferably wherein the kit further comprises a fourth aqueous media, wherein the fourth aqueous media is for maturation of in vitro obtained stem-cell derived cardiomyocytes.

20. A cardiomyocyte cell population obtained with the method of claim 1, preferably wherein the cardiomyocyte cell population resembles a human (fetal) ventricular cell population or wherein at least 70%, 80%, 90% or 95% of the cells express mlc2v.

21. An in vitro method of differentiating a human pluripotent stem cell population into a cardiomyocyte cell population wherein at least 70, 80%, 90% or 95% of the cells express mlc2v, as defined in claim 14.

22. An in vitro human cardiomyocyte cell population wherein at least 80%, 90% or 95% of the cells express mlc2v.

Description

RESULTS

[0166] FIG. 1 shows an example schematic overview of an embodiment of the method of the invention.

[0167] FIG. 2 shows exemplary results of experiments studying the effects of wnt inhibitors on the differentiation of the stem cells. For the data in the figure, NKX2-5-GFP reporter stem cells were cultured on feeders in KOSR/bFGF supplemented DMEM-F12 (Amit, M. et al. (2004) Biol Reprod 70, 837-45.). NKX-GFP reporter hESC and iPSC cells have been described previously. These cells express the fluorescent protein GFP under the control of the endogenous NKX2-5 promoter. GFP+ve cells mark cardiomyocytes (Elliott D A et al. Nat. Methods, 8 (2011), pp. 1037-1040; van den Berg C W et al., Development. 2015 Jul. 24. pii: dev.123810. [Epub ahead of print]). To start differentiation, cells were dissociated using Accutase and resuspended in mTesR (StemCell Technologies; Ludwig T E et al. Nat Methods 3: 637-46, 2006) at a concentration of 60,000/ml cells, the cell suspension was seeded onto Matrigel coated 12-well plates. Cells were maintained for 4 days before onset of differentiation. Cells were treated for 2 days with 5 microMolar CHIR99021, followed by 2 days culture in the presence of IWP-L6/XAV-939 (or C59/IWPL-6), next the cells were cultured for 10 days in the differentiation medium in the absence of the Wnt inhibiting small molecules. Percentage of cardiomyocytes was determined at day 14 of differentiation using flow cytometry for detection/quantification of GFP positive cells. The combination of IWP-L6 and XAV is the most effective. It has been observed that measurement of GFP expression cells gives an underestimation of percentage TNNT2 cells.

[0168] FIG. 3 shows exemplary results studying the effects of wnt inhibitors on the differentiation of the stem cells. Here, NKX2-5-GFP reporter stem cells were cultured on feeders in KOSR/FGF supplemented DMEM-F12. To start differentiation, cells were dissociated using Accutase and resuspended in mTesR at a concentration of 60,000/ml cells, the cell suspension was seeded onto Matrigel coated 12-well plates. Cells were maintained for 4 days before onset of differentiation. Cells were treated for 2 days with 5 microMolar CHIR99021, followed by 2 days culture in the presence of specified wnt inhibiting small molecules, next the cells were cultured for 10 days in differentiation medium. Percentage of cardiomyocytes was determined at day 14 of differentiation using flow cytometry for detection/quantification of GFP positive cells. The results show that reduction of IWP-L6 from 2 microMolar to 0.25 microMolar increases the differentiation efficiency even further. It has been observed that measurement of GFP expression cells gives an underestimation of percentage TNNT2 cells.

[0169] FIG. 4 shows titration of XAV-939 and the effects on differentiation. Two hIPSC lines were cultured on feeders in KOSR (InvitroGen)/FGF supplemented DMEM-F12. To start differentiation, cells were dissociated using Accutase and resuspended in mTesR at a concentration of 60,000/ml cells, the cell suspension was seeded onto Matrigel coated 12-well plates. Cells were maintained for 4 days before onset of differentiation. Cells were treated for 2 days with 5 microMolar CHIR99021, followed by 2 days culture in the presence of specific Wnt inhibiting small molecules, next the cells were cultured for 10 days in differentiation medium. Percentage of cardiomyocytes (FIG. 4A; % TNNT2 positive cells at day 14) was determined at day 14 of differentiation using flow cytometry for the pan-cardiac marker Troponin-T (TNNT-2). Data confirms that XAV939 at 5 microMolar is an optimal concentration, further increase of concentration reduces differentiation efficiency. Addition of IWP-L6 at 0.25 microMolar further improves the differentiation even further. Presence and absence or concentration have no significant effect on the total number of cells (FIG. 4B; cell count at day 14, in million cells/well).

[0170] FIG. 5 shows the effect of wnt inhibitors on performance of the differentiation system. Human IPSC lines were cultured on feeders in KOSR/FGF supplemented DMEM-F12. To start differentiation, cells were dissociated using Accutase and resuspended in mTesR at a concentration of 60,000/ml cells, the cell suspension was seeded onto Matrigel coated 12-well plates. Cells were maintained for 4 days before onset of differentiation. Cells were treated for 2 days with 5 microMolar CHIR99021, followed by 2 days culture in the presence of specific wnt inhibiting small molecules, next the cells were cultured for 10 days in differentiation medium. Percentage of cardiomyocytes was determined at day 14 of differentiation using flow cytometry for the pan-cardiac marker Troponin-T (TNNT-2). Here, IWP-L6 alone gives a differentiation efficiency of about 70% this is further increased by the addition of XAV939.

[0171] FIG. 6 shows the differentiation performance using different hiPSC maintenance systems. A panel of up to 2 hiPSC lines was used to test performance of the cardiac differentiation system. Cells were maintained on feeders (Amit, M. et al. (2004). Biol Reprod 70, 837-45.), L7 culture medium (Lonza; www.lonza.com/products-services/bio-research/stem-cells/pluripotent-stem-cells/pluripotent-stem-cells-and-media/l7-hipsc-reprogramming-and-hpsc-culture-system/l7-culture-system.aspx) or Essential 8 culture medium (Thermo Fisher Scientific; Chen G. et al. Nat Methods 8(5):424-429, 2011; www.thermofisher.com/order/catalog/product/A1517001;

tools.thermofisher.com/content/sfs/manuals/feeder free_PSCs_in_essential8_medium.pdf). Cells from all three maintenance systems were pre-cultured for 4 days prior to differentiation. Cells were treated for 2 days with 5 microMolar CHIR99021, 2 days with a combination of 5 microMolar XAV939 and 0.25 microMolar IWP-L6 followed by 10 days culture in differentiation medium. Percentage of cardiomyocytes was determined at day 14 of differentiation using flow cytometry for the pan-cardiac marker Troponin-T (TNNT-2).

[0172] Surprisingly, despite the wide range of hIPSC maintenance methods tested the results are highly consistent between experimental conditions confirming the robustness of the method (PLM 2 is NCRM1).

[0173] FIG. 7 shows hPSC-CM differentiated from (A) PLM line 1 and (B) NCRM-1 hiPSC. hIPSC lines were cultured on feeders in KOSR/FGF supplemented DMEM-F12. To start differentiation, cells were dissociated using Accutase and resuspended in mTesR at a concentration of 60,000/ml cells, the cell suspension was seeded onto Matrigel coated 12-well plates. Cells were maintained for 4 days before onset of differentiation. Cells were treated for 2 days with 5 microMolar CHIR99021, followed by 2 days culture in the presence of specific wnt inhibiting small molecules IWPL-6/XAV939, next the cells were cultured for 10 days in differentiation medium. Cells were dissociated at day 14 and cryopreserved. Cells were thawed and replated in maturation medium as disclosed herein and cultured for 7 days (day 14+7). Cardiomyocytes were characterized using flow cytometry with the pan-cardiac marker Troponin-T (TNNT-2) and the ventricular marker MLC2v.

[0174] Cells are predominantly of the ventricular subtype as shown by expression of ventricular marker Mlc2v. For PLM line 1, an efficiency of 97.85% of TNNT2+ cells was obtained and 82% of this population was Mlc2v %+. For NCRM-1, an efficiency of 80.10% of TNNT2+ cells was obtained and 82% of this population was 75.05% Mlc2v %+.

[0175] Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.

[0176] While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the inventions following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims.

[0177] All references cited herein, including journal articles or abstracts, published or corresponding patent applications, patents, or any other references, are entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited references. Additionally, the entire contents of the references cited within the references cited herein are also entirely incorporated by references./nlp

[0178] Reference to known method steps, conventional methods steps, known methods or conventional methods is not in any way an admission that any aspect, description or embodiment of the present invention is disclosed, taught or suggested in the relevant art.

[0179] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein), readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.