Search

CN-119421945-B - Maturation medium compositions and methods for human cardiac organoids maturation

CN119421945BCN 119421945 BCN119421945 BCN 119421945BCN-119421945-B

Abstract

Provided herein are maturation media and methods for maturing early embryo human cardiac organoids into mature human cardiac organoids. The maturation medium comprises a cell growth medium comprising a medium supplement comprising one or more fatty acids, triiodothyronine (T3) growth hormone, insulin, one or more antioxidants, sugar and carnitine, one or more additional fatty acids, additional carnitine or creatine, and additional T3 growth hormone. The maturation medium may also comprise one or more additional sugars, additional antioxidants, and growth factors. The method comprises contacting an early embryo human cardiac organoid with one or more maturation media to produce a mature human cardiac organoid.

Inventors

  • Etor Aguirre
  • Brett Fulmert

Assignees

  • 密歇根州立大学董事会

Dates

Publication Date
20260512
Application Date
20230719
Priority Date
20220722

Claims (20)

  1. 1. A maturation medium comprising: a cell growth medium; B-27TM media supplement; one or more additional fatty acids, wherein the maturation medium comprises a total of 20 μΜ to 80 μΜ palmitic acid, 20 μΜ to 80 μΜ oleic acid and 10 μΜ to 60 μΜ linoleic acid; An additional carnitine or creatine, wherein the additional carnitine or creatine is present in an amount of 60 to 200 μΜ, and/or wherein the total amount of carnitine or creatine present in the maturation medium is 60 to 200 μΜ; An additional triiodothyronine hormone, wherein the additional triiodothyronine hormone is present in an amount of 10 nM to 60 nM, and/or wherein the total amount of triiodothyronine hormone present in the maturation medium is 10 nM to 60 nM; An additional sugar, wherein the additional sugar is present in an amount of 1mM to 10 mM, and/or wherein the total amount of sugar present in the maturation medium is 1mM to 10 mM, and An additional antioxidant, wherein the additional antioxidant is present in an amount of 0.1mM to 1mM, and/or wherein the total amount of antioxidants present in the maturation medium is 0.1mM to 1mM, the additional antioxidant comprising ascorbic acid, glutathione, lipoic acid, uric acid, carotenes, tocopherols or panthenol.
  2. 2. The maturation medium of claim 1, wherein the cell growth medium comprises RPMI medium, DMEM, a derivative of DMEM selected from the group consisting of IMDM and ADMEM, or a combination thereof.
  3. 3. The maturation medium of claim 1, wherein the additional carnitine comprises L-carnitine, acetyl-L-carnitine, propionyl-L-carnitine, or a combination thereof.
  4. 4. The maturation medium of claim 1 wherein the additional sugar comprises fructose, galactose, or glucose.
  5. 5. The maturation medium of claim 4 wherein the additional sugar comprises glucose from 2 mM to 6 mM.
  6. 6. The maturation medium of claim 1 wherein the additional antioxidant comprises 0.1 mM to 1mM ascorbic acid.
  7. 7. The maturation medium of claim 1 further comprising a growth factor selected from the group consisting of IGF-1 and IGF-2.
  8. 8. The maturation medium of claim 7 comprising 10 ng/mL to 100 ng/mL IGF-1 or IGF-2.
  9. 9. The maturation medium of claim 1, wherein the maturation medium does not comprise extracellular matrix material and/or exogenous retinoic acid.
  10. 10. A method for maturing an early embryo human cardiac organoid into a mature human cardiac organoid, the method comprising contacting the early embryo human cardiac organoid with a maturation medium comprising: a cell growth medium; B-27TM media supplement; One or more additional fatty acids, wherein the maturation medium comprises 20 μΜ to 80 μΜ palmitic acid, 20 μΜ to 80 μΜ oleic acid and 10 μΜ to 60 μΜ linoleic acid; An additional carnitine or creatine, wherein the additional carnitine or creatine is present in an amount of 60 to 200 μΜ, and/or wherein the total amount of carnitine or creatine present in the maturation medium is 60 to 200 μΜ; An additional triiodothyronine hormone, wherein the additional triiodothyronine hormone is present in an amount of 10 nM to 60 nM, and/or wherein the total amount of triiodothyronine hormone present in the maturation medium is 10 nM to 60 nM; An additional sugar, wherein the additional sugar is present in an amount of 1mM to 10 mM, and/or wherein the total amount of sugar present in the maturation medium is 1mM to 10 mM, and An additional antioxidant, wherein the additional antioxidant is present in an amount of 0.1mM to 1mM, and/or wherein the total amount of antioxidants present in the maturation medium is 0.1mM to 1mM, the additional antioxidant comprising ascorbic acid, glutathione, lipoic acid, uric acid, carotenes, tocopherols or panthenol.
  11. 11. The method of claim 10, wherein the cell growth medium comprises RPMI medium, DMEM, a derivative of DMEM selected from the group consisting of IMDM and ADMEM, or a combination thereof.
  12. 12. The method of claim 10, wherein the additional carnitine comprises L-carnitine, acetyl-L-carnitine, propionyl-L-carnitine, or a combination thereof.
  13. 13. The method of claim 10, wherein the additional sugar comprises fructose, galactose, or glucose.
  14. 14. The method of claim 13, wherein the additional sugar comprises glucose of 2 mM to 6 mM.
  15. 15. The method of claim 10, wherein the additional antioxidant comprises 0.1 mM to 1 mM ascorbic acid.
  16. 16. The method of claim 10, wherein the maturation medium further comprises a growth factor selected from the group consisting of IGF-1 and IGF-2.
  17. 17. The method of claim 16, wherein the maturation medium further comprises 10 ng/mL to 100 ng/mL of IGF-1 or IGF-2.
  18. 18. The method of claim 10, wherein the early embryo human cardiac organoid is formed from the differentiation of hiPSC and is contacted with the maturation medium at day 20 after the zeroth day of the start of the hiPSC differentiation.
  19. 19. The method of claim 10, wherein -Contacting the early embryo human cardiac organoid with the maturation medium from day 20 to day 26, wherein the maturation medium further comprises a growth factor, and Contacting said early embryonic human cardiac organoid with said maturation medium from day 26 to day 30, wherein said maturation medium does not comprise growth factors, and Wherein the maturation medium is changed at day 26.
  20. 20. The method of claim 19, wherein the growth factor is IGF-1.

Description

Maturation medium compositions and methods for human cardiac organoids maturation Cross-reference to related patent applications This patent application claims the benefit of U.S. provisional patent application No. 63/391,452, filed on 7.22, 2022, and U.S. provisional patent application No. 63/432,565, filed on 12.14, 2022. The entire contents of each of the above-referenced patent applications are hereby incorporated by reference. Government rights The invention was carried out with government support under HL135464 and HL151505 awarded by the National institutes of health (National Institute of Health) and the National Heart and blood institute (Lung and Blood Institute). The government has certain rights in this invention. Technical Field The present disclosure relates to maturation media and methods and models for high throughput production of mature human cardiac organoids, such as fetal-like human cardiac organoids, which can be further matured into more adult-like human cardiac organoids. Background Cardiovascular disease (cardiovascular disease, CVD), a condition involving the heart and blood vessels, is a leading cause of death worldwide, leading to an estimated 1790 tens of thousands of deaths each year 1. Laboratory models of the heart are used to better understand the etiology and mechanism of CVD in more detail. Several model systems were used to study CVD, ranging from primary and induced pluripotent stem cell (induced pluripotent stem cell, iPSC) derived cardiomyocyte cultures to animal models and 3D culture systems, such as spheroids and engineered heart tissue 2-7. However, many of these systems fail to fully reproduce the complex nature of the human heart 8,9 for a variety of reasons, including the lack of endogenous extracellular matrix (extracellular matrix, ECM) and cardiac cell types other than cardiomyocytes, as well as lack of physiological morphology and cellular organization. In addition, animal models have unique non-human physiological, metabolic, electrophysiological, and pharmacokinetic characteristics that often do not accurately predict human-related responses 8,9. Thus, these systems and methods are not suitable for comprehensive study and simulation of human disease and physiology. The introduction of human-related models is crucial for the discovery of efficient, clinically transferable CVD solutions. In the last decade, advances in human induced pluripotent stem cells (human induced pluripotent stem cell, hiPSC) 10-12 and organoid 13,14 technology have enabled advanced technology to better model and study human systems with increasing accuracy. Recently, methods for producing human cardiac organoids from pluripotent stem cells have been reported. Because of their cellular complexity and physiological relevance, these methods enable studies of human heart development and disease 15-19 in dishes (dish) to an unprecedented extent. However, these systems still fail to reproduce important aspects of late-embryonic human heart and human heart development (e.g., anterior-posterior model build-up (anticoron-posterior patterning), coronary angiogenesis) and lack important cell populations that contribute to cardiac architecture (e.g., neural crest). Furthermore, the metabolic shift from glycolysis to fatty acid oxidation is a vital step in the later stages of heart development, preparing the heart for increased energy expenditure, and inducing transcriptional regulation and stimulating physiological maturation 28,30,95-97,141. Efforts have been made to mimic these phenomena in vitro with cardiomyocytes and engineered heart tissue, and the beneficial effects 7,20,21,23,142 of altering glucose concentration and adding fatty acids have been found. However, these systems are simple models and do not have the high physiological complexity observed in human cardiac organoids. In general, there is an urgent need to develop more sophisticated and accurate in vitro model systems for studying human cardiac development and disease pathology. Disclosure of Invention This section provides a general summary of the disclosure, and is not a comprehensive disclosure of its full scope or all of its features. Provided herein are maturation media comprising a cell growth medium comprising a media supplement comprising one or more fatty acids, triiodothyronine (T3) growth hormone, insulin, one or more antioxidants, sugar, and carnitine. The maturation medium further comprises one or more additional fatty acids, additional carnitine or creatine and additional T3 growth hormone. In another embodiment, a method for maturing an early embryo human cardiac organoid into a mature human cardiac organoid is provided. The method comprises contacting an early embryo human cardiac organoid with maturation medium. The maturation medium comprises a cell growth medium comprising a medium supplement comprising one or more fatty acids, triiodothyronine (T3) growth hormone, insulin, one or more antioxidants, sugar, and