Engineered human cardiac tissue
AssigneesMurdoch Childrens Research Institute, The Council of the Queensland Institute of Medical Research
InventorsEnzo Porello, Christian Brizard, James Edward Hudson, Ellen Frances Mathieson, Andrew Laskary, Liam Reynolds
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Abstract
The present application relates to methods for the generation of engineered human pluripotent stem cell-derived cardiac tissue, engineered cardiac tissue produced by such methods and methods for their use.
Claims83
A method for producing engineered human cardiac tissue comprising endothelial cells, the method comprising culturing a cell suspension comprising a population of human pluripotent stem cell (hPSC) culture-derived cardiomyocytes and non-cardiomyocyte cells in the presence of a FGFR1 agonist and a PDGFR agonist for a time and under conditions sufficient to produce engineered human cardiac tissue comprising endothelial cells.
A method for producing engineered human cardiac tissue, the method comprising the steps of:
- i) mixing a cell suspension comprising a population of human pluripotent stem cell (hPSC) culture-derived cardiomyocytes and non-cardiomyocyte cells with a flowable hydrogel composition, wherein said cell suspension is in a serum-free medium;
- ii) loading the flowable hydrogel composition comprising said cell suspension into a mold and incubating the hydrogel for a time and under conditions sufficient to solidify the hydrogel;
- and
- iii) culturing the hydrogel from step ii) in serum free medium;
- wherein the population of human pluripotent stem cell (hPSC) culture-derived cardiomyocytes and non-cardiomyocyte cells is contacted with an FGFR1 agonist and a PDGFR agonist to thereby provide engineered human cardiac tissue, wherein said tissue comprises cardiomyocytes, stromal cells/fibroblasts and endothelial cells.
The method of any one of the preceding claims, wherein said cell suspension comprises about 70% cardiomyocytes and about 30% to about 50% non-cardiomyocyte cells.
The method of any one of the preceding claims, wherein said cell suspension comprises about 50 to about 75% cardiomyocytes and about 25 to about 50% non-cardiomyocyte cells.
The method of any one of the preceding claims, wherein said cell suspension is derived from a single differentiation process.
The method of any one of the preceding claims, wherein the engineered human cardiac tissue further comprises vascular smooth muscle cells and cardiac progenitor cells.
The method of any one of the preceding claims, wherein the engineered human cardiac tissue comprises:
- at least about 30 % cardiomyocytes;
- about 35% or less stromal cells/fibroblasts;
- and
- about 30% or less endothelial cells.
The method of any one of the preceding claims, wherein the engineered human cardiac tissue comprises:
- about 30 to about 70 % cardiomyocytes;
- about 10 to about 35% stromal cells/fibroblasts;
- about 5 to about 30% endothelial cells;
- up to about 15% vascular smooth muscle cells;
- and
- up to about 10 % cardiac progenitor cells.
The method of any one of the preceding claims, wherein the FGFR1 agonist is selected from FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23.
The method of any one of the preceding claims, wherein the PDGFR agonist is selected from PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC or PDGF-DD.
The method of any one of the preceding claims, wherein the serum free medium comprises about 0.5-100 ng/ml human PDGF-BB and about 0.5-100 ng/ml human FGF2.
The method of any one of the preceding claims wherein the serum free medium comprises about 10 ng/ml human PDGF-BB and about 10 ng/ml human FGF2.
The method of any one of the preceding claims, wherein said serum free medium comprises a basal medium comprising albumin and transferrin.
The method of any one of the preceding claims, wherein a population of about 3% to about 10% of the cardiomyocytes in said engineered human cardiac tissue are proliferative.
The method of any one of the preceding claims, wherein said engineered human cardiac tissue displays glycolytic metabolism.
The method of any one of the preceding claims, wherein said engineered human cardiac tissue displays log2 relative gene expression of >2 for PKM, when normalized to TUBA1A expression.
The method of any one of the preceding claims, wherein said engineered human cardiac tissue has tolerance to hypoxia.
The method of any one of the preceding claims, wherein said engineered human cardiac tissue yields a lactate/pyruvate ratio of at least 1:1 in cell culture medium obtained following 20 h of culture under hypoxic conditions.
The method of any one of the preceding claims, wherein said engineered human cardiac tissue displays downregulation of ALDH1, AGMO, DPYD, GPX3, CYP1B1 and PLIN5.
The method of any one of the preceding claims, wherein the cardiomyocytes are MMP1+.
The method of any one of the preceding claims, wherein the fibroblasts are MMP1+, EMP1+, FOXD1+, RAB27B+, NR2F1+, F2RL1+, SPP1+, TMEM158+, PTHLH+, PHLDA2+, MALL+, MYCT1+, DUSP4+, PLAU+, TMEM156+, CD274+, MMP10+, ARRDC4+, RFX8+, MLPH+, THBD+, HHEX+, VGF+, OTULINL+, IL33+, CA12+, C6orf141+, MFSD2A+ and/or CARD10+.
The method of any one of the preceding claims, wherein said engineered human cardiac tissue exhibits contractile activity of less than 10 mN/mm2.
The method of any one of the preceding claims, hPSCs are induced to differentiate towards cardiac linage by the steps of:
- a) culturing hPSCs in a basal medium comprising an effective amount of BMP4, Activin A, FGF, a GSK3-inhibitor, a serum-free supplement comprising albumin and transferrin minus insulin for about 72 h wherein the medium is replaced with fresh medium daily;
- b) culturing the cells obtained in step a) in a basal medium comprising an effective amount of an inhibitor of the Wnt-signaling pathway and a serum-free supplement as in a) for about 72 h wherein the medium is replaced with fresh medium daily,
- c) culturing the cells obtained in step b) in a basal medium comprising an effective amount of an inhibitor of the Wnt-signaling pathway and a serum-free supplement comprising albumin and transferrin including insulin for a period of about 7 days wherein the medium is replaced with fresh medium after on day 2 and day 4 of said period of about 7 days;
- d) culturing the cells obtained in step c) in a basal medium comprising a serum-free supplement comprising albumin, transferrin and insulin for a period of about 72 h;
- wherein the basal medium used in each of steps a)-d) has a calcium concentration of less than about 1.2 mM;
- to thereby provide a cell suspension comprising a population of human pluripotent stem cell (hPSC) culture-derived cardiomyocytes and non-cardiomyocyte cells.
The method of any one of the preceding claims, wherein the engineered human cardiac tissue is in the form of a tissue patch.
The method of any one of the preceding claims, wherein said hPSCs are iPSCs.
The method of any one of the preceding claims, wherein said hPSCs are embryonic stem cells.
Engineered human cardiac tissue comprising at least about 30% hPSC-derived cardiomyocytes, about 35% or less hPSC-derived stromal cells/fibroblasts and up about 30% or less hPSC-derived endothelial cells, wherein the aforementioned cells are located throughout a hydrogel composition.
Engineered human cardiac tissue comprising hPSC-derived cardiomyocytes, hPSC-derived stromal cells/fibroblasts and hPSC-derived endothelial cells, wherein the aforementioned cells are derived from a single differentiation process, wherein the aforementioned cells are located throughout a hydrogel composition.
Dependencies are inferred from the claim text.