Patent Application
20250051727
A field of the invention concerns cardiac organoids and processes for making the same.
It is known to cultivate cardiac cells in order to obtain layers of these cells which adhere to a cultivation vessel surface. The trademark Matrigel (Corning Life Sciences and BD Bioscience) is known to designate a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells.
Richards et al., Biomaterials 112-123 (2017) describe the fabrication of cardiac organoids by depositing cardiomyocytes, cardiac ventricular fibroblast cells, umbilical vein endothelial cells (HUVEC), human adipose-derived microvascular endothelial cells (HAMEC), and human adipose-derived stem cells (hADSC) in agarose moulds to first generate spherical microtissues which are then assembled to spheroids.
Elliott et al. (2011) “NKX2-5 (eGFP/w) hESCs for isolation of human cardiac progenitors and cardiomyocytes”, Nature methods 8 (12), S. 1037-1040. DOI: 10.1038/nmeth.1740 describe the generation of hESC3 NKX2.5-eGFP cells.
US 2017/0198256 A1 describes that hiPSC were cultivated and harvested as single cells or cell clumps, which were combined with a liquid, cross-linkable PEG fibrinogen precursor solution to generate a statistical distribution of single hiPSC or clumps of hiPSC in the cross-linkable solution, which was cross-linked in forms to give cylinders of 6 mm width and 200 μm thickness. After cultivating these cylinders for 3 days, medium was changed to induce differentiation of the hiPSC, generating cardiac myocytes.
US 2016/0271183 A1 describes the differentiation of human embryonic stem cells in suspension in medium containing Matrigel with BMP4, Rho kinase inhibitor, activin-A and IWR-1 to cardiomyocytes.
WO 2018/035574 A1 describes a 96-well culture plate with two spaced-apart upright posts in each well. Seeded progenitor cells in cardiac cell maturation medium shall form contracting tissue attached to both posts.
Pashmforoush et al., Nkx2-5 Pathways and Congenital Heart Disease: Loss of Ventricular Myocyte Lineage Specification Leads to Progressive Cardiomyopathy and Complete Heart Block, Cell 2004, 117, 373-386, describe the in vivo structural defects for genetic defects of NKX2.5.
Ng E S, Azzola L, Bruveris F F, Calvanese V, Phipson B, Vlahos K, et al., Differentiation of human embryonic stem cells to HOXA+ hemogenic vasculature that resembles the aorta-gonad-mesonephros. Nature Biotechnology 2016, 34 (11), describe the hematopoietic cell-specific gene RUNX1C (Runt-related transcription factor 1c.
Guibentif C, Rönn R E, Böiers C, Lang S, Saxena S, Soneji S, et al. Single-Cell Analysis Identifies Distinct Stages of Human Endothelial-to-Hematopoietic Transition. Cell Reports 2017, 19, 10-19, describe the hematopoietic cell-specific gene WAS of the Wiskott-Aldrich syndrome.
Kasahara et al., “Progressive atrioventricular conduction defects and heart failure in mice expressing a mutant Csx/Nkx2.5 homeoprotein”, 2001; J. Clin. Invest. 108:189-201, describe that mouse embryos and neonatal mice with a ventricle-restricted NKX2.5-KO contain cardiomyocytes with well-organized sarcomeres similar to wildtype mouse cardiomyocytes.
Davis et al., “Targeting a GFP reporter gene to the MIXL1 locus of human embryonic stem cells identifies human primitive streak-like cells and enables isolation of primitive hematopoietic precursors”, Blood 2008, 111:1876-1884 describes the HES3 MIXL1-GFP reporter cell line.
Anderson D J et al. “NKX2-5 regulates human cardiomyogenesis via a HEY2 dependent transcriptional network”, Nature Communications 2018, 9:1373 describe generation of the human embryonic stem cell line HES3 NKX2.5eGFP/eGFP.
A preferred embodiment is a cardiac organoid which has a first layer that forms an inner part and has cavities, which first layer is at least in part surrounded by a second layer including endothelial cells and cardiomyocytes, which second layer is at least in part surrounded by a third layer including cardiomyocytes and epicardial cells, which third layer is at least in part surrounded by a fourth layer including fibroblast cells.
The invention is now described in greater detail by way of examples with reference to the figures, which show in
A preferred process produces cardiac organoids using pluripotent stem cells (PSC), preferably human PSC, to the cardiac organoids obtainable by the process, and to a process for analysing the activity of a test compound on cardiac cells by exposing the PSC during the process for their production to the test compound and/or exposing at least one cardiac organoid obtainable by the process for their production to the test compound, and analysing the organoid, e.g. in comparison to PSC and/or a cardiac organoid treated in parallel but in the absence of the test compound.
The processes and the cardiac organoid have the advantage of being available on the basis of cultivated pluripotent stem cells, and have the advantage of providing adjoining layers including or consisting of different cardiac cells, optionally including non-cardiac cells, which layers are grown by a purely in vitro process, and to provide an in vitro model of adjoining cardiac tissue layers, optionally including non-cardiac cells. These tissue layers have been found to be well-structured and to contain cell types, which layers and cell types are typical of the early embryonic heart. The cardiac organoids can be kept in cell culture medium in static or agitated, e.g. shaken, culture vessels, e.g. without directional fluid flow, without blood flow, and freely suspended, i.e. without mechanical fixation.
A preferred process produces cardiac tissue in an in vitro process, which cardiac tissue shall form at least two layers including or consisting of different cells, which can be cardiac cells, optionally in combination with other cell types.
A preferred in vitro process for producing a cardiac organoid and by the cardiac organoid obtainable by the process includes or consists of the following order of steps of
As an alternative, PSC can be replaced by omnipotent or totipotent stem cells. Accordingly, in the process the stem cells can optionally be at least pluripotent stem cells. Generally preferred, the PSC are generated without use of a human embryo. Preferably, PSC are human PSC (hPCS), e.g. induced PSC (iPSC), e.g. generated from a mammalian cell sample, e.g. a blood or tissue sample, especially a human induced PSC (hiPSC), or an embryonic stem cell line (ESC), which preferably is non-human, or a human embryonic stem cell line (hESC). Generally, the PSC or ESC are not generated using a human embryo.
Cultivated pluripotent stem cells (PSC) preferably are characterized by presence of the markers Tra-1-60 and SSEA4.
Preferably, the cultivated pluripotent stem cells (PSC) provided in step a) are single cells in suspension.
The first vessel preferably has a low-attachment surface, preferably an ultra-low attachment surface which prevents attachment of the PSC to the vessel. The first vessel can e.g. be contained in a microtiter plate, e.g. in a 96-well plate. The second vessel preferably has a low-attachment surface, preferably an ultra-low attachment surface which prevents attachment of the PSC to the vessel. The second vessel may have a flat or U-shaped bottom. The first vessel and the second vessel can have the same shape and/or surface properties.
Generally, the U-shaped bottom of the first and second culture vessels can be a tapering concave bottom, e.g. round or stepped or having angled surfaces, e.g. having a concave pyramid shape or V-shape.
The first medium preferably is E8 medium, having the composition of DMEM/F12, L-ascorbic acid-2-phosphate magnesium (64 mg/L), sodium selenium (14 μg/L), FGF2 (100 μg/L), insulin (19.4 mg/L), NaHCO3 (543 mg/L) and transferrin (10.7 mg/L), TGFB1 (2 μg/L) or NODAL (100 μg/L). Osmolarity of all media was adjusted to 340 mOsm at pH7.4, in order to provide for pluripotency of the ESC. Preferably, the first medium is supplemented with ROCK inhibitor, e.g. 10 μM ROCK inhibitor (Rho-associated-kinase inhibitor).
The volume of each medium can be in the range of 100 μL to 300 μL, especially 150 μL to 250 μL for every 5000 cells.
The centrifugation in step b), e.g. twice at 300× g in a centrifuge temperature-controlled to 4° C., serves to localize the PSC on the bottom of the first vessel, e.g. to collect the PSC. Following the centrifugation, the first vessel is placed in an incubator essentially without moving the first medium in relation to the first vessel, in order to maintain the PSC localized close together. The first medium remains in the first vessel, also in step c).
The incubation of the PSC localized on the bottom of the first vessel under cell culture conditions and without agitation in step c). This incubation, e.g. for 24 h, results in the PSC to form one aggregate of cells.
Removing the first medium from the aggregate of cells in step d) serves to minimize or avoid the transfer of first medium into the hydrogel that is positioned in the U-shaped bottom of a second vessel in step e), when transferring the aggregate of cells into the hydrogel in step f). Transferring the aggregate of cells in step e) can e.g. be done using a pipette having a wide opening in order to avoid shearing the aggregate of cells.
The hydrogel is for cell culture, e.g. is permeable for dissolved oxygen and permeable for medium components and preferably permeable for metabolic products, e.g. permeable for diffusion. Preferably, the hydrogel is on the basis of an extracellular matrix of basal membranes of animal cells. The hydrogel can be on the basis of laminin and/or entactin and/or collagen and/or fibronectin and/or PEG, optionally on the basis of PEG (polyethylene glycol), e.g. in combination with fibronectin. The hydrogel preferably has a gelatinous structure. A preferred hydrogel is Matrigel.
The hydrogel, e.g. Matrigel, preferably has a protein concentration of 9.7 to 10.1 mg/mL. In step e), preferably a volume of 15 to 30 μL, preferably 20 μL Matrigel is positioned onto the U-shaped bottom of a second vessel and the aggregate of PSC is positioned into the Matrigel for embedding the aggregate of PSC within the Matrigel. The second vessel preferably has a low-attachment surface, preferably an ultra-low-attachment surface. The second vessel can have the same specifications as the first vessel. Preferably, the hydrogel is not cross-linkable, e.g. does not contain reactive groups that cross-link during the process, and accordingly, the process preferably is devoid of a step of cross-linking the hydrogel. Less preferred, the hydrogel may be cross-linkable, e.g. contain cross-linkable groups, and the process includes a step of cross-linking the hydrogel.
The incubation of step f), e.g. for 45 to 60 min under cell culture conditions, serves to solidify the hydrogel prior to adding the second culture medium in step g).
In step g), the second medium can have the same composition as the first medium, e.g. medium E8. The volume of the second medium in step g) can be 80 to 150 μL, e.g. 100 μL. The incubation of step g) is for at least 1 day or at least two days, preferably up to 3 days, preferably for 36 to 60 h.
Removal of the second medium and adding the third medium containing a first differentiation factor in step i) serves to activate the WNT pathway. The first differentiation factor having activity to induce the WNT pathway preferably is an inhibitor of GSK3beta (glycogen synthase kinase 3 beta), and preferably has no effect or cross-reactivity on CDKs (cyclin-dependent kinases). A preferred first differentiation factor is CHIR99021 (CHIR, 6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2 pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile), e.g. at 7.5 μM, e.g. using 200 μL of the third medium per second vessel. The third medium preferably is RPMImedium containing B27 supplement without insulin and it additionally contains the first differentiation factor.
Preferably, after step h), the third medium containing the first differentiation factor is removed in step i), preferably at 24 h after adding this third medium in step j), and a fourth cell culture medium is added, which does not contain a differentiation factor, e.g. no first differentiation factor, and incubating under cell culture conditions, e.g. for 1d. When the process includes step i), this is presently considered to reduce the WNT activation induced the first differentiation factor. The fourth medium preferably is free from insulin, e.g. RPMI medium containing B27 supplement but without insulin. Generally preferably, in the process, especially in step h), only a first differentiation factor having activity to activate the WNT pathway only is added, e.g. contained in a third cell culture medium, and the process, especially in step h), is devoid of adding a differentiation factor having activity to 4587*143202D1 SUBSTITUTE SPECIFICATION M1077U'S-DIN activate another differentiation pathway than the WNT pathway, e.g. devoid of adding BMP4, Rho kinase inhibitor, activin-A and IWR-1.
In step j), the medium is removed from the cell aggregate and a fifth cell culture medium is added, which contains a second differentiation factor which inhibits the WNT2 pathway, and preferably the fifth medium contains no insulin. The fifth medium can be RPMI medium containing B27 supplement but without insulin. The second differentiation factor preferably is an inhibitor of the WNT pathway activator Porcupine, e.g. IWP2 (Inhibitor of WNT Production-2, CAS No. 686770-61-6), e.g. at a concentration of 5 μM, e.g. using 100 μL to 300 μL, e.g. up to 200 μL medium, per second vessel.
In step l), preferably after 2 d incubation of step k), the fifth medium is removed and a sixth medium is added to the agglomerate, which medium does not contain a first nor a second inhibitor. The sixth medium preferably contains no insulin. The sixth medium can e.g. be RPMI medium containing B27 supplement without insulin. In step k), the volume of the sixth medium can e.g. be 100 μL to 300 μL, e.g. 150 to 250 μL, preferably 200 μL per second vessel. RPMI medium is RPMI1640 (Inorganic Salts: Calcium nitrate*4H2O (0.1 g/L), magnesium sulfate (0.04884 g/L), potassium chloride (0.4 g/L), sodium bicarbonate (2 g/L), sodium chloride (6 g/L), sodium phosphate dibasic (0.8 g/L); Amino Acids: L-alanyl-L-glutamine (0 g/L), L-arginine (0.2 g/L), L-asparagine (0.05 g/L), L-aspartic acid (0.02 g/L), L-cystine*2HCl (0.0652 g/L), L-glutamic acid (0.02 g/L), glycine (0.01 g/L), L-histidine (0.015 g/L), hydroxy-L-proline (0.02 g/L), L-isoleucine (0.05 g/L), L-leucine (0.05 g/L), L-lysine*HCI (0.04), L-methionine (0.015 g/L), L-phenylalanine (0.015 g/L), L-proline (0.02 g/L), L-serine (0.03 g/L), L-threonine (0.02 g/L), L-tryptophan (0.005 g/L), L-tyrosine*2Na*2H2O (0.02883 g/L), L-valine (0.02 g/L); Vitamins: D-biotin (0.0002 g/L), choline chloride (0.003 g/L), folic acid (0.001 g/L), myo-inositol (0.035 g/L), niacinamide (0.001 g/L), p-aminobenzoic acid (0.001 g/L), D-phantothenic acid (hemicalcium) (0.00025 g/L), pyridoxine*HCl (0.001 g/L), riboflavin (0.0002 g/L), thiamine*HCI (0.001 g/L), vitamin B12 (0.000005 g/L);
D-glucose (2 g/L), glutathione (0.001 g/L), phenol red*Na (0.0053 g/L); L-Glutamine (0.3 g/L), sodium bicarbonate (0 g/L)).
In step l), the sixth medium is removed and replaced by a seventh medium which contains insulin, e.g. RPMI medium containing B27 supplement with insulin added.
In step m) the seventh medium of step l) is replaced by fresh cell culture medium, preferably fresh seventh medium, which preferably contains added insulin.
Preferably, the incubation in steps c) to m), preferably in steps c) to h), are under static conditions, e.g. without shaking or moving of the vessel. Optionally, the incubation starting from step m) are with mild agitation, e.g. under orbital shaking, preferably in a larger volume of medium and in a vessel for cell culture with orbital shaking.
The process for producing the cardiac organoids has the advantage that only one type of cells, namely PSC, e.g. iPSC or ESC, can be used in the process and that during the process the PSC, e.g. iPSC or ESC, differentiate and self-organize into a three-dimensional structure which includes adjoining layers including or consisting of different cardiac cell types, e.g. without mechanically manipulating cells into a specific layered structure, and without initially providing different cardiac cell types.
Herein, the gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells is generally referred to as Matrigel.
Generally, all media can contain anti-bacterial agents, e.g. penicillin and/or streptomycin for the prevention of bacterial contaminations.
For analysing the effect of a test compound, the test compound can be added in any of the steps of the process. For analysing the effect of a test compound, the test compound can be added to the medium in at least one of steps e) to m, e.g. in at least one of steps i) to m), preferably after a total of 10 to 13 days after step i).
The process for producing the cardiac organoids has the advantage of reproducibly generating cardiac organoids of one structure, e.g. having an outer diameter of 0.5 to 5 mm or up to 3 or up to 2.5 mm, e.g. of 1.2 to 3 mm or up to 2.5 mm. The structure of the cardiac organoids contains or consists of a first layer, including or essentially consisting of endothelial cells and foregut endoderm cells, which first layer forms an inner part and has cavities, which preferably include hemogenic endothelium and/or foregut endoderm cells, a second layer directly adjacent the first layer and surrounding only a portion of the first layer, the second layer forming dense myocardium, optionally also endocardium, including or essentially consisting of NKX2.5-positive cardiomyocytes, and opposite the first layer a third layer directly adjacent to the second layer only and surrounding only a portion of the second layer, which third layer forms epicardium and includes or essentially consists of epicardial cells and cardiomyocytes, has blood vessel-like structures and forms a loose tissue from epicardial cells and cardiomyocytes, wherein the third layer can be covered by a directly adjacent fibroblast layer. Therein, preferably the first layer completely surrounds the cavity or cavities, i.e. the first layer is circumferentially closed, and the second layer surrounds the first layer only partially, leaving a portion of the first layer non-covered from another layer. The third layer surrounds the second layer only partially, e.g. the third layer covers the second layer up to a ring-shaped section of the second layer, which ring-shaped section of the second layer is non-covered, which ring-shaped section is adjacent to the non-covered portion of the first layer. Hemogenic endothelium can e.g. be localized in the inner part of the cardiac organoids, especially at least a portion of inner cavities, e.g. in only a portion of inner cavities with another portion of cavities being devoid of hemogenic endothelium, which can be foregut endoderm cells. The portion of cavities having hemogenic endothelium can have a layer of hemogenic endothelium lining the cavities, with the inner part being formed of foregut endoderm cells. The portion of cavities having no hemogenic endothelium are only formed of foregut endoderm cells. The inner part of the cardiac organoids can be formed of foregut endoderm cells, wherein separate cavities can be formed of foregut endoderm cells, be lined with hemogenic endothelium, and/or contain blood vessels.
The structure of the cardiac organoids therefore includes or consists of, e.g. in one perspective, at least one non-covered portion of the first layer surrounded by a ring-shaped section of the second layer, wherein the second layer is covered by the adjacent third layer opposite the first layer, and the ring-shaped section of the second layer is not covered by the third layer. The structure of the cardiac organoids preferably has at least one surface area which is formed by a non-covered portion of the first layer, by a ring-shaped section of the second layer directly adjacent the first non-covered portion of the first layer, and directly adjacent to the ring-shaped section of the second layer, the third layer which covers and surrounds the second layer, the third layer bordering on the ring-shaped section of the second layer, and preferably a fourth layer, e.g. of loose cardiac fibroblasts, only adjacent the third layer. Optionally, the first layer in its part directly under its non-covered portion contains less or no cavities than its portions which are covered by the second layer. In the structure of the cardiac organoids, the at least one surface area can be formed by the ring-shaped section of the second layer, which encloses the non-covered portion of the first layer and, opposite the non-covered portion of the first layer, by the third layer surrounding the ring-shaped section of the second layer, wherein the third layer preferably covers the second layer completely and borders on the ring-shaped section of the second layer.
As the process for producing the cardiac organoids reproducibly generates the cardiac organoids having this structure, the effect of a test compound added during the process or to cardiac organoids having a pre-determined size, can be analysed, e.g. by determining structural differences to cardiac organoids produced without the addition of the test compound.
The structure of the cardiac organoids has the advantage of allowing direct observation of all the layers from the perspective directed onto the non-covered portion of the first layer and/or onto the ring-shaped section of the second layer. Accordingly, the invention provides the cardiac organoids having the specific structure, which cardiac organoids are available by the process. Further, the invention provides analytical processes in which a compound to be tested is added to at least one of the media in at least one of the steps of the process of producing the cardiac organoid, or in which a compound to be tested is added to a cardiac organoid produced by the process, e.g. the cardiac organoid having a structure described herein, followed by analysis of the cardiac organoid.
Optionally, a section of the cardiac organoids, e.g. at least one of its layers, can be provided for use as a tissue implant for implantation into a patient, e.g. provided as a tissue implant, e.g. as a replacement or filler for a defect tissue.
Optionally, the pluripotent stem cells originate from a patient, e.g. from a tissue sample taken from a patient. The patient can be diagnosed to have a disease or can be suspected of having a disease, and the process for producing the cardiac organoids can be used to analyse the effect of a test compound onto the cardiac organoids, e.g. during or following their development to a pre-determined size, wherein the test compound can be a pharmaceutical compound for use in the treatment of the disease. Such a process can be used to analyse the effect of the pharmaceutical compound prior to administration of the compound to the patient. Alternatively or additionally, cardiac organoids produced according to the process of the invention from pluripotent stem cells that originate from a patient can be used to produce a cardiac organoid for use of at least one of its layers as a replacement for a defect tissue in the patient.
Optionally, the PSC may have a genetic aberration, e.g. a genetic defect selected from an aberrant, e.g. dysfunctional gene, which is present heterozygously or homozygously. The genetic aberration can e.g. be a dysfunctional gene selected from the genes of the group NKX2.5, a dysfunction of which at least homozygously results in congenital heart disease with structural defects including muscle overgrowth and conduction defects in the heart, a dysfunction of a factor such as GATA4 or TBX5, which lead to congenital heart defects.
Analyses could demonstrate that genetic aberrations of the PSC used in the process result in structural aberrations of cardiac organoids in comparison to cardiac organoids produced from PSC without genetic aberrations, i.e. from genetically wild-type, or healthy, PSC.
The PSC preferably are a cell line, e.g. the hESC HES3.
Preferred embodiments of the invention will now be discussed with respect to experiments and drawings. Broader aspects of the invention will be understood by artisans in view of the general knowledge in the art and the description of the experiments that follows.
The step i) of replacing the third medium for a fourth medium that is free of a differentiation factor, is made on day 1 and incubation in the fourth medium is for 2 days until day 3 (d3). In the alternative, step i) can be performed with an incubation of 1 to 2 days, preferably 24 h under cell culture conditions, and the third medium is subsequently directly removed and exchanged for the fifth medium containing an inhibitor of the WNT pathway, IWP2 at 5 μM, 200 μL medium per second vessel in step j). Incubation was for 2 days until day 5 (d5), when in step k) the fifth medium was removed and replaced by a sixth medium which is free from a differentiation factor and preferably contains no insulin, and incubation is for 2 days until day 7 (d7). The sixth medium is replaced by a seventh medium, which is generally free from differentiation factors and preferably contains insulin, e.g. B27 supplement, followed by incubation for at least 1 day, e.g. for up to 3 days, until day 10 (d10) in step l). Following step l), the sixth medium is exchanged for fresh medium, preferably containing insulin, in order to maintain viability, e.g. every day to every 2 days in step m).
In
The process was performed for steps a) to n) as described with reference to
These PSC were cultivated on cell culture flasks (preferably Geltrex-coated) in E8 medium and detached from the flasks to provide the PSC in suspension. In step a), 5000 PSC suspended in 100 μL of the first medium, which was E8 medium (DMEM/F12, L-ascorbic acid-2-phosphate magnesium (64 mg/L), sodium selenium (14 μg/L), FGF2 (100 μg/L), insulin (19.4 mg/L), NaHCO3 (543 mg/L) and transferrin (10.7 mg/L), TGFβ1 (2 μg/L) or NODAL (100 μg/L), osmolarity adjusted to 340 mOsm at pH7.4) supplemented with 10 μM ROCK inhibitor (Y-27632, catalogue No. 72302, Stemcell Technologies)), were carefully dispensed into wells of the microtiter plate. Centrifugation of step b) was twice for 3 min at 300× g with the centrifuge cooled to 4° C. The centrifuged first vessels each contained one agglomerate on the U-shaped bottom and were carefully placed into a cell culture incubator in order to avoid disturbance of the cells localized at the U-shaped bottom. Incubation of step c) without agitation was for 24 h, resulting in the formation of an aggregate of the cells. For embedding in Matrigel (thawed on ice, 9.7 to 10.1 mg/mL protein content) as the exemplary hydrogel, 20 μL Matrigel were positioned in each well of a microtiter plate, the wells of which forming second vessels, which plate was of the same ultra-low attachment quality as the first. As preferred, the medium was removed from the cell aggregates contained in the first vessels, and the cell aggregates were singly and carefully transferred into the Matrigel droplets contained in the second vessels. It was found that for the transfer of a cell aggregate, a pipette set to approx. 3 μL and equipped with a cut tip having a large opening could be used. The second vessel, i.e. the second microtiter plate, was re-positioned into the incubator for 45 to 60 min, which allowed for a solidification of the Matrigel. Then, 100 μL E8 medium but without ROCK inhibitor was dispensed into each second vessel and the second vessel was incubated for 2 days without agitation according to step h). After the incubation of step h), on day 0, differentiation was started by completely removing the E8 medium and adding 200 μL RPMI medium supplemented with B27 supplement without insulin and containing 7.5 μM CHIR99021 into each second vessel, and incubated for exactly 24 h according to step h). Then, this third medium was exchanged for a fourth medium consisting of RPMI medium with B27 supplement without insulin, and incubation was for 2d. Then, the fourth medium was removed and exchanged for 200 μL RPMI medium with B27 supplement without insulin and containing 5 μM IWP2 as the second differentiation factor in each second vessel according to step k), with incubation for 48 h. Subsequently, the medium was exchanged every 2 days for RPMI medium with B27 supplement containing insulin.
It was generally observed that the cardiac organoids start to regularly contract after approx. 11 days of the process (day 7).
Further,
The electron micrograph of
After 14 days of the process, i.e. on day 10, cardiac organoids produced according to Example 1 were transferred to wells of a 12-well plate containing PBS (phosphate-buffered saline, pH 7.4), the PBS was removed by suction and the Matrigel was dissolved by adding Cell Recovery Solution, obtained from Corning, according to the manufacturer's instructions. Then, the organoid was gently washed with PBS, PBS was removed, and 2 mL RPMI medium with B27 supplement and containing insulin was added into each well, then the plate was incubated further, with exchange of the medium for fresh medium every 3 to 4 days. The organoid shown in
As an exemplary test compound, Thalidomide was used to test its influence on the development of cardiac organoids during the production process. Cardiac organoids were produced according to Example 1, and on day 1 (d1), i.e. after the incubation for 1 day in step i), 80 μg/mL Thalidomide was added to the culture medium. Micrographs of 7 cardiac organoids treated in parallel with Thalidomide (80 g/mL Thalidomide) are shown in
A comparison of the micrographs of the Thalidomide-treated cardiac organoids to the Control makes it clear that Thalidomide results in the absence of any NKX2.5-eGFP-positive cells on the outer layer, and that the myocardial ring of the Thalidomide-treated cardiac organoids is substantially thinner than in Controls. The average diameter of the myocardial ring in Controls was 276 μm, with Thalidomide it was 165 μm, which result is also shown in
This result on the exemplary test compound shows that the known effect of Thalidomide to cause also cardiac malformations during embryo development is also seen in the development of the cardiac organoids in the process according to the invention. This shows that the process for producing cardiac organoids is a suitable in vitro assay for the effect of test compounds, e.g. in respect of teratogenic effects.
As an example for PSC that carry a genetic aberrations, a human embryonic stem cell line HES3 NKX2.5eGFP/eGFP was used, in which both NKX2.5 genes are dysfunctional (knock-out, NKX2.5-KO) and which expresses no functional NKX2.5 protein, but which expresses eGFP (enhanced green fluorescent protein) as a marker under the control of the NKX2.5 promoter. This cell line was produced as described by Anderson D J et al. “NKX2-5 regulates human cardiomyogenesis via a HEY2 dependent transcriptional network”, Nature Communications 2018, 9:1373.
For comparison (Control), cardiac organoids were produced by the same process from a cell line that expressed normal levels of NKX2.5 protein and in addition contains the expression cassette encoding eGFP under the NKX2.5 promoter.
Cardiac organoids from the genetically aberrant, NKX2.5-KO PSC, and from the Control were produced by the process of the invention, as e.g. described in Example 1.
These results, which are strongly reminiscent of both the muscle overgrowth and the lack of myocardial tissue compaction that was described for NKX2.5 defects in mouse and in human, show that cardiac organoids produced according to the invention from PSC that have a genetic aberration closely resemble the development of hearts observed in vivo and show structural aberrations resembling structural aberrations observed in vivo for the genetic aberration.
Cardiac organoids were produced by the process of the invention, using two different reporter cell lines, in which formation of hematopoietic cells derived from hemogenic endothelium is revealed by expression of GFP as a reporter, the expression of which is controlled by the hematopoietic cell-specific marker genes RUNX1C (Runt-related transcription factor 1c), cell line HES3 RUNX1C-GFP, or WAS (Wiskott-Aldrich Syndrome), iPS cell line WAS-GFP.
The fluorescence microscopic of
The fluorescence microscopic picture of
For both these cell lines, the expression of GFP in the inner part of the cardiac organoids shows that hemogenic endothelium is contained in the inner part of the cardiac organoids during the process of the invention. This indicates that the cardiac organoids produced by the process of the invention in their inner part contain hemogenic endothelium, which developed from the PSC originally used in the process. Hemogenic endothelium is known to be the origin of hematopoiesis.
Cardiac organoids were produced as described in Example 1 and analysed for presence of endothelial cells. Paraffin-embedded cardiac organoids were stained with an endothelial cell-specific anti-CD31 antibody (CD31, Clone JC70A obtained from Agilent, Dako; secondary antibody: Cy3-AffiniPure Donkey Anti-Mouse IgG (H+L) obtained from Jackson ImmunoResearch Laboratories).
Positive staining of endothelial cells (represented by bright white stained cells) supports the finding that cardiac organoids of the invention contain endothelium-lined cavities resembling blood vessel-like structures.
Initial tests using immuno-staining of the endothelial marker vascular endothelial cadherin (VE-cadherin or CD144) by the labelled anti-CD144 antibody human CD144 antibody BMS158 obtained from eBioscience were later found to not only detect blood-vessel like structures, but to also give false-positive signals.
Cardiac organoids were produced from NKX2.5-KO PSC and wild-type PSC (control) as described in Example 1.
Further, cardiac organoids were produced according to the method of the invention from the HES3 MIXL1-GFP reporter cell line. In this cell line, previously described by Davis et al. Blood 2008, GFP is expressed under the control of the MIXL1 gene promoter demarcating mesendodermal progenitor cells. These cardiac organoids of the invention demonstrate that the ring-like structure, which is typical for cardiac organoids generated with the HES3 NKX2.5-eGFP reporter cell line (see also
While specific embodiments of the present invention have been shown and described, it should be understood that other modifications, substitutions and alternatives are apparent to one of ordinary skill in the art. Such modifications, substitutions and alternatives can be made without departing from the spirit and scope of the invention, which should be determined from the appended claims. Various features of the invention are set forth in the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 18161619.4 | Mar 2018 | EP | regional |
This application is a divisional of and claims priority under 35 U.S.C. §§ 120 and 121 from prior pending U.S. application Ser. No. 16/980,200, which was filed on Sep. 11, 2020, which was a U.S. National Phase under 35 U.S.C. § 371 of International Application No. PCT/EP2019/054225, filed Feb. 20, 2019, which claims priority to European Application No. 18161619.4, filed Mar. 13, 2018. The applications in this paragraph are incorporated by reference herein.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16980200 | Sep 2020 | US |
| Child | 18734856 | US |
