Search

EP-4735582-A1 - LIPOTOXIC MEDIA

EP4735582A1EP 4735582 A1EP4735582 A1EP 4735582A1EP-4735582-A1

Abstract

The invention provides culture media for cultivating cardiac tissue, methods of inducing a diabetic cardiomyopathy disease phenotype in cardiac tissue, diseased cardiac tissue, and kits. An exemplary culture medium for cultivating cardiac tissue includes 100-300 pM palmitate, 100-300 pM oleate, and 0.5-15 nM endothelin-1.

Inventors

  • SOROTA, Stephen
  • FERIC, Nicole
  • REKHTER, MARK, DAVID
  • GEARHART, Jamie
  • MARUKIAN, Svetlana

Assignees

  • Valo Health, Inc.

Dates

Publication Date
20260506
Application Date
20240626

Claims (20)

  1. Claims 1. A culture medium for cultivating cardiac tissue, wherein the culture medium comprises: 100-300 µM palmitate; 100-300 µM oleate, and 0.5-15 nM endothelin-1.
  2. 2. A culture medium according to claim 1, wherein the culture medium is for inducing a diabetic cardiomyopathy disease phenotype in the cultivated cardiac tissue.
  3. 3. A culture medium according to any preceding claim, wherein the culture medium comprises 180-220 µM palmitate.
  4. 4. A culture medium according to claim 3, wherein the culture medium comprises 200 µM palmitate.
  5. 5. A culture medium according to any preceding claim, wherein the culture medium comprises 180-220 µM oleate.
  6. 6. A culture medium according to claim 5, wherein the culture medium comprises 200 µM oleate.
  7. 7. A culture medium according to any preceding claim, wherein the culture medium comprises 1-5 nM endothelin-1.
  8. 8. A culture medium according to any preceding claim, wherein the culture medium comprises 1 nM or 5 nM endothelin-1.
  9. 9. A culture medium according to claim 1, wherein the culture medium comprises: 180-220 µM palmitate; 180-220 µM oleate, and 0.5-10 nM endothelin-1.
  10. 10. A culture medium according to claim 1, wherein the culture medium comprises: 200 µM palmitate; 200 µM oleate, and 1-5 nM endothelin-1.
  11. 11. A culture medium according to any preceding claim, wherein the culture medium does not comprise cortisol.
  12. 12. A culture medium according to any preceding claim, further comprising StemPro-34 serum-free base medium.
  13. 13. A culture medium according to any preceding claim, further comprising one or more of: (i) GlutaMAX, optionally 0.1-2% (v/v) GlutaMAX, and optionally 1% (v/v) GlutaMAX; (ii) HEPES, optionally 10-30 mM HEPES, and optionally 20 mM HEPES; (iii) Penicillin-streptomycin, optionally 0.1-2% Penicillin-streptomycin, and optionally 1% Penicillin-streptomycin; (iv) Transferrin solution, optionally 0.05-0.25 mg/mL Transferrin solution, and optionally 0.15 mg/mL Transferrin solution; (v) Ascorbic Acid solution, optionally 0.1-0.4 mg/mL Ascorbic Acid solution, and optionally 0.256 mg/mL Ascorbic Acid solution; (vi) StemPro-34 nutrient supplement, optionally 1-4% (v/v) StemPro-34 nutrient supplement, and optionally 2.6% (v/v) StemPro-34 nutrient supplement.
  14. 14. A culture medium according to claim 13, comprising: (i) 0.1-2% (v/v) GlutaMAX; (ii) 10-30 mM HEPES; (iii) 0.1-2% Penicillin-streptomycin; (iv) 0.05-0.25 mg/mL Transferrin solution; (v) 0.1-0.4 mg/mL Ascorbic Acid solution; (vi) 1-4% (v/v) StemPro-34 nutrient supplement.
  15. 15. A method of inducing a diabetic cardiomyopathy disease phenotype in cardiac tissue, wherein the method comprises: cultivating cardiac tissue in the culture medium according to any one of claims 1-14.
  16. 16. A method according to claim 15, wherein the cardiac tissue is cultivated in the culture medium for 3-80 days.
  17. 17. A method according to claim 16, wherein the cardiac tissue is cultivated in the culture medium for 21-35 days.
  18. 18. A method according to any one of claims 15-17, further comprising a step of cultivating pluripotent stem cells in a differentiation medium to provide cardiomyocytes.
  19. 19. A method according to claim 18, wherein the pluripotent stem cells are human induced pluripotent stem cells.
  20. 20. A method according to claim 18 or claim 19, wherein the pluripotent stem cells are autologous.

Description

P603581PC00 Lipotoxic media Field of the Invention The present invention relates to a culture medium for cultivating cardiac tissue, a method of inducing a diabetic cardiomyopathy disease phenotype in cardiac tissue, diseased cardiac tissue produced by the method, a diseased cardiac tissue, and a kit. Background to the Invention Heart failure currently affects more than 64 million people globally, and it only seems to be increasing in prevalence. In the United States alone, health experts are projecting a 46% increase in the prevalence of heart failure by 2030. Among all cases of heart failure, heart failure with preserved ejection fraction (HFpEF) accounts for around 50% of cases and the percentage of heart failure patients with HFpEF has been increasing recently. Due to its status as a global epidemic, along with the heavy burden of symptoms imposed by HFpEF, there is a pressing need to identify drug candidates that may treat and prevent HFpEF. HFpEF is typically associated with other comorbidities such as diabetes, obesity, and metabolic syndrome, with approximately 65% of HFpEF patients having at least one of these diseases. A key feature of metabolic disorders that are presumed to lead to HFpEF is hyperlipidemia, a condition in which abnormally high amounts of lipids and fatty acids circulate in the blood. Hyperlipidemia induces a negative effect on cellular metabolism, leading to higher fatty acid oxidation and lower glucose oxidation. Elevated lipids contribute to insulin resistance which is associated with elevated serum glucose and insulin levels. In addition, lipids such as diacylglycerols and ceramides accumulate in cardiac cells over time, impacting cell morphology and function, which can lead to a condition called diabetic cardiomyopathy. Diabetic cardiomyopathy may be formally defined as cardiac dysfunction in the absence of any other risk factors, like hypertension, ischemic heart disease, and/or valvular dysfunction. In a clinical setting, patients with diabetic cardiomyopathy may initially present as asymptomatic but with fibrosis, increased ventricular stiffness, and diastolic dysfunction. This disease may later progress to include left ventricular hypertrophy, worsening diastolic dysfunction, and HFpEF. In extreme cases, heart failure with reduced ejection fraction (HFrEF) may be observed as well. Several 2D in vitro models of diabetic cardiomyopathy exist (Geraets et al. "Human embryonic stem cell-derived cardiomyocytes as an in vitro model to study cardiac insulin resistance," Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, vol. 1864, no. 5, Part B, pp. 1960-1967, 2018; Granéli et al. "Diabetic Cardiomyopathy Modelling Using Induced Pluripotent Stem," Stem Cell Reviews and Reports, vol. 15, pp. 13-22, 2019). However, these models do not provide a good representation of diabetic cardiomyopathy. There is a need to provide improved models of diabetic cardiomyopathy which can be used to better understand lipotoxic mechanisms and for drug discovery and development. Summary of the Disclosure According to a first aspect of the disclosure, there is provided a culture medium for cultivating cardiac tissue, wherein the culture medium comprises: 100-300 µM palmitate; 100-300 µM oleate, and 0.5-15 nM endothelin-1. The inventors have developed a lipotoxic culture medium that can be used to cultivate cardiac tissue to induce a diabetic cardiomyopathy phenotype. Advantageously, the cultured tissue can be used as a three-dimensional (3D) in vitro or ex vivo model that recapitulates key features of diabetic cardiomyopathy and can therefore be used to better understand lipotoxic mechanisms and can be used in drug discovery and development. It could not have been expected or anticipated from the prior art that the combination of palmitate, oleate and endothelin-1, and particularly at the concentrations recited, could be used to induce a diabetic cardiomyopathy phenotype in cardiac tissue. In addition, it could not have been expected or anticipated from the prior art that endothelin-1, when combined with palmitate and oleate, would induce a diabetic cardiomyopathy phenotype in cardiac tissue. The term “culture medium”, as used herein, refers to solutions that contain factors and nutrients including, for example, growth factors, energy sources, amino acids, and organic and inorganic salts, which are used for the maintenance and growth of cells in ex vivo or in vitro culture. Culture media are often buffered to an approximately neutral pH (e.g., a pH from about pH 6.6 to about pH 7.8) and can be supplemented with one or more antibiotics to prevent the growth of a bacterial and/or fungal contaminants. Any suitable basal culture medium may be used, for example, StemPro™-34 serum free medium, MEM, DMEM, RPMI 1640, Advanced MEM, BME, Neurobasal medium, cardiomyocyte selective medium, sodium bicarbonate buffered Medium 199, myocyte growth medium, cardiomyocyte growth medium, cardiomyocyte ma