Patent Application
20200199541
Blood vessels are prone to various diseases called vascular diseases. Disorders in the network of blood vessels can cause a range of health problems which can be severe or prove fatal. Such diseases can be due to environmentally causes pathogenesis or due to developmental defects.
Goodwin (Microvasc Res. 2007; 74(2-3): 172-183) describes an in vitro angiogenesis assay in order to assess the activity of agents that affect angiogenesis in the pathogenesis of many dissases.
Duffy et al. (European Cells and Materials 21, 2011: 15-30) describes in vitro vascularisation of collagen-glycosaminoglycan scaffolds in a surface adherent 2D culture.
Nakagami et al. (Hypertension 2006; 48:112-119) provide a method of vascularization by cell-matrix interaction using matrigel that forced embryonic stem cells into development of sprouting blood vessels containing endothelial and vascular smooth muscle cells.
Kusuma et al. (PNAS 110(31), 2013: 12601-12606), Gerecht-Nir et al. (Laboratory Investigation 83(12), 2003: 1811-1820), WO2007/140340 A2, WO2014/145871 A1, US 2014/273220 A1 and WO2017/015415 A1 describe the formation of vascular structures out of isolated early vascular cells in an engineered matrix. Before introduction into the matrix, the cells may be derived from human pluripotent stem cells that differentiated to early vascular cells and then individualized (by trypsinization and/or 40 μm mesh filtration). The individual cells grow in the matrix and assemble there to form vascular networks. The goal in these papers was to provide self-assembling cells that are useful in regenerative medicine.
WO2011/115974 A1 relates to a device to form 2D cultured vascular networks on a surface.
Shen et al. (Cell Research 13 (5) (2003): 335-341) describes the formation of enginerred blood vessels from adult rabit smooth muscle cells and differentiated endothelial cells from mouse.
Still, previous vascular models lack sufficient similarity with natural in vivo formed vascular networks and there is a need for improved, more life-like vascular models.
Therefore, it is a goal of the present invention to provide improved vascular models, in addition also models that allow broader varieties of uses, such as disease models and testing in screening procedures.
The present invention provides a method of generating an artificial blood vessel organoid, comprising providing stem cells capable of vascular differentiation, stimulating mesoderm differentiation in said stem cells, stimulating vascular differentiation in said stem cells, developing a cell aggregate from said stem cells, embedding said cell aggregate in a collagenous 3D matrix and stimulating vascular differentiation of the aggregate in said collagenous 3D matrix.
In a closely related aspect, the invention provides a method of generating an artificial blood vessel organoid, comprising embedding vascular stem cells in a collagenous 3D matrix comprising 10%-50% laminin, 20%-70% collagen I, and/or 2%-30% collagen IV and stimulating vascular differentiation of said stem cells in said collagenous 3D matrix.
Such methods can be used to provide a blood vessel organoid, that forms a further aspect of the invention. In particular, the invention provides an artificial blood vessel organoid culture comprising an interconnected network of vascular capillaries, said capillaries comprising endothelium and a basal membrane with peri-vascular pericytes, (i) wherein said organoid is produced by a method of the invention and/or (ii) wherein the capillaries are embedded in an artificial 3D matrix comprising a hydrogel with collagen and/or (iii) wherein the organoid culture comprises 40 to 1000 blood vessels as counted by counting individual vessels and vessels between capillary intersections. All three characteristics (i), (ii) and (iii) are hallmarks of the invention, that can be individually required by an inventive artificial blood vessel organoid culture or in combination.
The invention further provides a method of providing a non-human animal model with human vascular capillaries, wherein said human capillaries comprise endothelium and a basal membrane with perivascular pericytes, comprising the steps of introducing a human blood vessel organoid of the invention into a non-human animal and letting said organoid grow its vascular capillaries.
The invention also relates to a non-human animal model comprising such an artificial blood vessel organoid culture, e.g. as an insert. Furthermore, a non-human animal model with human vascular capillaries is provided, wherein said human capillaries comprise endothelium and a basal membrane with perivascular pericytes.
The invention further relates to the use of the inventive culture or non-human animal model or the method in generating them as model of a pathology, e.g. diabetes, wherein the cells in the method, the organoid or the organoid in the non-human animal model are subject to pathogenesis to develop said pathology, e.g. hyperglycemia or destruction of pancreatic beta-cells in case of diabetes.
The invention further provides a method of screening a candidate chemical compound for influencing a pathogenesis or a pathology comprising administering said candidate chemical compound to a culture or non-human animal model or during generation of said culture or non-human animal model according to any aspect of the invention and monitoring for physiological differences in said culture or animal model as compared to said culture or animal model without administration of the candidate chemical compound.
The invention has provided new treatment models for diabetes. Accordingly, the invention provides a use of a Notch3 activation pathway inhibitor (such as a gamma-secretase inhibitor, a Notch3 inhibitor, DLL4 inhibitor or a combination thereof) in the treatment or prevention of a thickened capillary basement membrane, such as in diabetic vasculopathy, occlusive angiopathy, altered vascular permeability, tissue hypoxia, heart disease, stroke, kidney disease, blindness, impaired wound healing or chronic skin ulcers. Also provided is a Notch3 activation pathway inhibitor (e.g. gamma-secretase inhibitor, a Notch3 inhibitor, DLL4 inhibitor) for use in such a treatment or prevention; or the use of a Notch3 activation pathway inhibitor (e.g. gamma-secretase inhibitor, a Notch3 inhibitor, DLL4 inhibitor) for use in manufacturing a medicament or pharmaceutical composition for such a treatment or prevention.
Finally, the invention provides a kit suitable for the generation of an artificial blood vessel organoid according to any inventive method, comprising (i) a Wnt agonist or a GSK inhibitor; (ii) a vascular differentiation factor selected from VEGF, a FGF, a BMP; (iii) a collagenous 3D matrix.
All embodiments of the invention are described together in the following detailed description and all preferred embodiments relate to all embodiments, aspects, methods, organoids, animal models, uses and kits alike. E.g. kits or their components can be used in or be suitable for inventive methods. Any component used in the described methods can be in the kit. Inventive organoids are the results of inventive methods or can be used in inventive methods and uses. Preferred and detailed descriptions of the inventive methods read alike on suitability of resulting or used organoids or animal models of the inventions. All embodiments can be combined with each other, except where otherwise stated.
The present invention provides a method of generating an artificial blood vessel organoid. Such artificial organoids are in vitro grown but highly resemble in vivo capillary structures. An organoid is a miniaturized and simplified version of an organ produced in vitro in three dimensions that shows realistic micro-anatomy. They are derived from one or a few cells from a tissue, embryonic stem cells or induced pluripotent stem cells, which can self-organize in three-dimensional culture owing to their self-renewal and differentiation capacities.
The inventive organoids derived from human stem cells recapitulate the structure and function of human blood vessels. 3D blood vessel organoids from embryonic and induced pluripotent stem cells are provided. These blood vessel organoids contain endothelium, perivascular pericytes, and basal membranes, and self-assemble into lumenized interconnected capillary networks. Human blood vessel organoids transplanted into mice form a perfused human vascular tree, including human arterioles and venules. Intriguingly, exposure of blood vessel organoids to hyperglycemia and inflammatory cytokines in vitro induced thickening of the basal membrane and transcriptional changes in endothelial cells, mimicking the microvascular changes in diabetic patients. A drug screen uncovered a y-secretase inhibitor that attenuated this “diabetic” vasculopathy in blood vessel organoids. Blood vessel organoids can be used to generate disease models for drug discovery, as we have shown by identifying y-secretase as a potential therapeutic target for diabetic vasculopathy, which affects hundreds of millions of patients.
The method of generating such organoids comprises the steps of providing stem cells capable of vascular differentiation, stimulating mesoderm differentiation in said stem cells, stimulating vascular differentiation in said stem cells, developing a cell aggregate from said stem cells, embedding said cell aggregate in a collagenous 3D matrix and stimulating vascular differentiation of the aggregate in said collagenous 3D matrix.
Stem cells capable of vascular differentiation are for example pluripotent stem cells. Pluripotent stem cells may be derived from embryonic stem cells or they may be induced pluripotent stem cells (iPS). iPS are preferred.
The stem cells are differentiated into a mesodermal-vascular route. Differentiation can be achieved by contacting cells with a tissue (mesodermal/vascular) specific growth or differentiation factor. The cells may then develop into the desired tissue. Such a tissue specific growth or differentiation factor may be a mesoderm and/or a vascular differentiation factor, preferably used at different stages of the inventive method. This will determine the development into the respective type of cellular tissue in later development. The cells will thereby transit from pluripotent to multipotent cells. Other tissue types shall then be not or only by a return to a pluripotent status be possible again. Usually not all cells are differentiated to the selected tissue type. It usually sufficient when about 50% or more or at least 55% or at least 60%, or at least 65% or at least 70% or at least 75% of the cells initiate differentiation towards the selected tissue type (in particular mesoderm) and transform to reduce their differentiation potential by multipotent cell with the respective tissue destiny (%-values as fractions of the cell amount) at first. Of course, this differentiation destiny only applies for the cells that are not returned to a un- or less differentiated state by use of artificial growth and dedifferentiation stimuli. Clearly, even somatic cells can be returned to pluripotent cells and this is not meant when defining a differentiated state herein. Preferably, no factors are introduced to the cells that would return the cells to pluripotent cells once the mesoderm or vascular differentiation is initiated.
The inventive organoids can be obtained from culturing pluripotent stem cells. In principle, the cells may also be totipotent, if ethical reasons allow.
A “totipotent” cell can differentiate into any cell type in the body, including the germ line following exposure to stimuli like that normally occurring in development. Accordingly, a totipotent cell may be defined as a cell being capable of growing, i.e. developing, into an entire organism.
The cells used in the methods according to the present invention are preferably not totipotent, but (strictly) pluripotent.
In a particular preferred embodiment, the cells of the present invention (including all further embodiments related thereto), are pluripotent.
A “pluripotent” stem cell is not able of growing into an entire organism, but is capable of giving rise to cell types originating from all three germ layers, i.e., mesoderm, endoderm, and ectoderm, and may be capable of giving rise to all cell types of an organism. Pluripotency can be a feature of the cell per see, e.g. in certain stem cells, or it can be induced artificially. E.g. in a preferred embodiment of the invention, the pluripotent stem cell is derived from a somatic, multipotent, unipotent or progenitor cell, wherein pluripotency is induced. Such a cell is referred to as induced pluripotent stem cell herein. The somatic, multipotent, unipotent or progenitor cell can e.g. be used from a patient, which is turned into a pluripotent cell, that is subject to the inventive methods. Such a cell or the resulting organoid culture can be studied for abnormalities, e.g. during organoid culture development according to the inventive methods. A patient may e.g. suffer from a vascular disorder. Characteristics of said disorder can be reproduced in the inventive organoids and investigated.
A “multipotent” cell is capable of giving rise to at least one cell type from each of two or more different organs or tissues of an organism, wherein the said cell types may originate from the same or from different germ layers, but is not capable of giving rise to all cell types of an organism.
In contrast, a “unipotent” cell is capable of differentiating to cells of only one cell lineage.
A “progenitor cell” is a cell that, like a stem cell, has the ability to differentiate into a specific type of cell, with limited options to differentiate, with usually only one target cell. A progenitor cell is usually a unipotent cell, it may also be a multipotent cell.
With decreasing differentiation capabilities, stem cells differentiate in the following order: totipotent, pluripotent, multipotent, unipotent. During development of the inventive organoid, stem cells differentiate from pluripotent (also totipotent cells are possible) into multipotent mesoderm, vascular or endothelial stem cells, further into unipotent stem cells of endothelial cells and pericytes.
Preferably, the stem cell is from a vertebrate, such as a mammal, reptile, bird, amphibian or fish. In particular preferred are land-living vertebrates. Possible are non-human animals and humans. Especially preferred are mammals for all aspects and embodiments of the invention, such as mouse, cattle, horses, cats, dogs, non-human primates; human cells are most preferred. The non-human animal model comprising the organoid may be selected from the same animals. The stem cells and the animal model may not be the same organism.
Differentiation of stem cells has become a standard technique in the art. For example, differentiation and growth factors to form vascular grafts are disclosed in WO2016/094166 A1. Such growth factors can also be used according to the invention as differentiation factors.
The inventive method comprises a step of inducing mesoderm differentiation. There are differentiation stimuli, that drive differentiation specifically into one direction (e.g. mesoderm) and those that drive unspecific differentiation with mesoderm being among several other differentiation routes. Such unspecific differentiation can be achieved by serum, such as FBS (fetal bovine serum) as used in Gerecht-Nir et al. (see background section). Unspecific differentiation may lead do various germ layer being present, including ectoderm, including neuroectoderm and endoderm.
According to a preferment of the invention specific mesoderm differentiation is performed, such as by a mesoderm specific differentiation factor. Alternatively, but less preferred, mesoderm can be selected from the differentiated cells. Selection can be combined with specific differentiation stimulation. Selection of cells is not desired because it would require isolation and individualization of cells. According to the invention, such individualization is disadvantageous because the cells should form or start forming an aggregate at this stage. Preferably, the cells after mesoderm stimulation have at least 50%, preferably at least 60%, even more preferred at least 70% or even at least 80%, of its cells in mesoderm differentiation. Preferably, mesoderm differentiation comprises treating the stem cells with a Wnt agonist or a GSK inhibitor, preferably CHIR99021. Wnt agonist or a GSK inhibitor achieves a high rate of mesoderm differentiation. A Wnt agonist may be a Wnt stimulator like CHIR99021
The stem cells are also treated by vascular differentiation. Vascular differentiation is continuously or repeatedly stimulated in the inventive method, in particular within the 3D matrix but also before that aggregate of cells is introduced into the 3D matrix, when when the cells are forming said aggregate.
Vascular differentiation may comprise an endothelial differentiation and results in small capillary or capillary precursor formation. In early stages of the method, e.g. before the 3D matrix treatment, such endothelial/vascular differentiation may not lead to the same well-defined and life-like capillaries that will alter form in the 3D matrix.
As with mesoderm differentiation, preferably the vascular differentiation is a specific vascular differentiation, with preferably at least 50%, preferably at least 60%, even more preferred at least 70% or even at least 80%, of its cells in vascular differentiation. Preferably vascular differentiation in said stem cells comprises treating the stem cells with a VEGF and/or a FGF and/or a BMP and/or low oxygen conditions of 12% (v/v) or less atmospheric oxygen. VEGF, FGF, BMP and low oxygen may be combined. A preferred VEGF is VEGF-A. A preferred FGF is FGF-2. A preferred BMP is BMP4. Low oxygen conditions are 12% (v/v) or less atmospheric oxygen, i.e. oxygen in a gas phase supplied to the cells. The gas phase is preferably at atmospheric pressure. Preferably the oxygen content is even less, preferably 10% or less, more preferred 8% or less, e.g. 6% or less (all % in v/v). The oxygen content is preferably 2% or more, e.g. 2% to 12% (all % in v/v). Preferably, the cells are cultured in a medium with VEGF in a concentration of 10 ng/ml to 50 ng/ml, preferably about 30 ng/ml. Preferably, the cells are cultured in a medium with FGF in a concentration of 10 ng/ml to 50 ng/ml, preferably about 30 ng/ml. Preferably, the cells are cultured in a medium with BMP in a concentration of 10 ng/ml to 50 ng/ml, preferably about 30 ng/ml.
The stem cells before introduction into the 3D matrix are forming the aggregate of cells. Preferably these cells are in suspension culture that allows such aggregation. This means that the stem cells that are treated for mesoderm and/or vascular differentiation are already in small aggregates. Such aggregates are usually small to be able to be suspended in suspension culture in a liquid culture medium, without a stable 3D matrix.
After the differentiation, before embedding the differentiated stem cells, now in an aggregate, in to the 3D matrix usually at least 30%, preferably at least 40%, e.g. about 50% of the cells of the aggregate are endothelial cells. Preferably at least 20%, e.g. 30% of the cells of the aggregate are pericytes. Together, preferably at least 60%, preferably at least 70%, e.g. about 80% of the cells are vascular cells.
Once the aggregates are embedded in the 3D matrix, the inventive method also comprises vascular differentiation of the aggregate in said 3D matrix. Preferably also this vascular differentiation comprises specific vascular differentiation. Especially preferred vascular differentiation of the aggregate comprises treating cells of the aggregate with a VEGF and/or a FGF. A preferred VEGF is VEGF-A. A preferred FGF is FGF-2. Preferably, the aggregates in the matrix are cultured in a medium with VEGF in a concentration of 60 ng/ml to 150 ng/ml, preferably about 100 ng/ml. Preferably, the aggregates in the matrix are cultured in a medium with FGF in a concentration of 60 ng/ml to 150 ng/ml, preferably about 100 ng/ml.
The cell aggregate that formed from the stem cells after culturing, especially during mesoderm and/or vascular differentiation, is embedded into the 3D matrix. This aggregate that is embedded into the 3D matrix preferably has a size of at least 30 cells or at least 50 cells, preferably of at least 100 cells, especially preferred at least 300 cells, e.g. about 1000 cells. Preferably the size is less than 100000 cells, e.g. less than 30000 cells. The cell aggregate should have an established size but not too large to suffer from low stability in liquid suspension cultures. An aggregate is an accumulation of cells attached to each other, in particular by intercellular bonds and intercellular connections.
Preferably, said aggregate is embedded in the collagenous 3D matrix at day 7 to 15 from the start of aggregate formation. In this time, the aggregate usually has a suitable size and differentiation status. A preferred time-line is shown in
Embedding the cells into the 3D matrix can be performed by any method known in the art. A preferred method is fluidizing 3D matrix material and solidifying or gelling the 3D matrix around the aggregate of cells.
The 3D matrix is a collagenous matrix, it has preferably at least 50 wt.-% collagen. Collagen includes collagen I, collagen II, collagen III and collagen IV. Collagen I and collagen IV are most preferred. Preferably the at least 50% is composed of collagen I or collagen IV, most preferably a mixture of collagen I and collagen IV.
The aggregate is cultured in the three dimensional (3D) matrix. A 3D matrix is distinct from 2D cultures, such as 2D cultures in a dish on a flat surface. A “3D culture” means that the culture can expand in all three dimensions without being blocked by a one-sided wall (such as a bottom plate of a dish). Such a culture, preferably including the 3D matrix, is preferably in suspension. The 3D matrix may be a gel, especially a rigid stable gel, which results in further expansion of growing cell culture/tissue and differentiation. The gel may be a hydrogel. A suitable 3D matrix according to the invention comprises collagen. More preferably the 3D matrix comprises extracellular matrix (ECM) or any component thereof selected from collagen, lam inin, entactin, and heparin-sulfated proteoglycan or any combination thereof. Extra-cellular matrix may be from the Engelbreth-Holm-Swarm tumor or any component thereof such as laminin, collagen, preferably type 4 collagen, entactin, and optionally further heparan-sulfated proteoglycan or any combination thereof. Such a matrix is Matrigel. Matrigel is known in the art (U.S. Pat. No. 4,829,000) and has been used to model 3D heart tissue previously (WO 01/55297 A2) or neuronal tissue (WO 2014/090993). Preferably the matrix comprises laminin, collagen and entactin, preferably in concentrations 20%-85% laminin, 3%-50% collagen and sufficient entactin so that the matrix forms a gel, usually 0.5%-10% entactin. Laminin may require the presence of entactin to form a gel if collagen amounts are insufficient for gel forming. A Matrigel-rich matrix may comprise a concentration of at least 3.7 mg/ml containing in parts by weight about 50%-85% laminin, 5%-40% collagen IV, optionally 1%-10% nidogen, optionally 1%-10% heparan sulfate proteoglycan and 1%-10% entactin. According to the invention, the collagen content is preferably increased, in particular preferred by collagen I. A particularly preferred matrix of the present invention in all embodiments comprises 10%-50% laminin, 20%-70% collagen I, and/or 2%-30% collagen IV; preferably further 0.5%-10% nidogen, 0.5%-10% heparan sulfate proteoglycan, and/or 0.5%-10% entactin (all wt.-%). These percentages relate to the solid, proteinaceous components only, i.e. not liquid components like water, which may me the major component in a hydrogel. Matrigel's solid components usually comprise approximately 60% laminin, 30% collagen IV, and 8% entactin. The 3D matrix may be a mixture of Matrigel and collagen, e.g. a mixture 2:1 to 1:3, preferably of about 1:1 of matrigel:collagen I. All %-values given for the matrix components are in wt.-%. These values may vary, e.g. by +/−30% depending on the source. Entactin is a bridging molecule that interacts with laminin and collagen. Such matrix components can be added in step r). These components are also preferred parts of the inventive kit. The 3D matrix may further comprise growth factors, such as any one of EGF (epidermal growth factor), FGF (fibroblast growth factor), NGF, PDGF, IGF (insulin-like growth factor), especially IGF-1, TGF-β, tissue plasminogen activator. The 3D matrix may also be free of any of these growth factors.
In general, the 3D matrix is a three-dimensional structure of a biocompatible matrix. It preferably comprises collagen, gelatin, chitosan, hyaluronan, methylcellulose, laminin and/or alginate. The matrix may be a gel, in particular a hydrogel. Organo-chemical hydrogels may comprise polyvinyl alcohol, sodium polyacrylate, acrylate polymers and copolymers with an abundance of hydrophilic groups. Hydrogels comprise a network of polymer chains that are hydrophilic, sometimes found as a colloidal gel in which water is the dispersion medium. Hydrogels are highly absorbent (they can contain over 99 wt.-% water) natural or synthetic polymers. Hydrogels also possess a degree of flexibility very similar to natural tissue, due to their significant water content. It is possible that the three dimension-al matrix, or its components, especially ECM or collagen, still remains in the produced tissue culture. Preferably the 3D matrix is a collagenous matrix, preferably it contains type I and/or type IV collagen.
Preferably the 3D matrix is a hydrogel. The matrix, in particular the hydrogel may have a viscoelastic storage modulus G′ of 10 to 30. The storage modulus in viscoelastic materials measure the stored energy, representing the elastic portion, and the energy dissipated as heat, representing the viscous portion. A method to determine the storage modulus, which can be used according to the invention, e.g. by rheometer, is given in Angui ano et al. PLoS ONE 2017, 12(2): e0171417.
Preferably the collagenous 3D matrix comprises 10%-50% laminin, 20%-70% collagen I, and/or 2%-30% collagen IV; preferably further 0.5%-10% nidogen, 0.5%-10% heparan sulfate proteoglycan, and/or 0.5%-10% entactin (all wt.-o). Matrigel usually comprises 50%-85% laminin, 5%-40% collagen IV, 1%-10% nidogen, 1%-10% heparan sulfate proteoglycan and 1%-10% entactin (solid, proteinaceous components only).
The invention also provides a method of generating an artificial blood vessel organoid, comprising embedding vascular stem cells in a collagenous 3D matrix comprising 10%-50% laminin, 20%-70% collagen I, and/or 2%-30% collagen IV and stimulating vascular differentiation of said stem cells in said collagenous 3D matrix. All aspects and preferred embodiments discussed so far also apply to this method, that also forms an independent aspect of the invention. It has been shown herein, that such a 3D matrix results in very favourable vascular networks that are reminiscent of in vivo vascular networks. In particular, such networks have large lumen and are capable of incorporation into model animals with connection to the circulatory system of the model animals. Preferably the vascular stem cells are generated by differentiating mesodermal stem cells into vascular stem cells, preferably wherein the mesodermal stem cells have been obtained by stimulating mesodermal differentiation in pluripotent stem cells, in particular, as mentioned above. All of these aspects are combinable with the above description.
In all embodiments and aspects of the invention, preferably the cells of the aggregate are cultured in said 3D matrix for at least 5 days, preferably for at least 7 days. Culturing in the 3D matrix may be for 5 to 60 days or more, preferably for at least 10 days.
The 3D matrix itself may be suspended in suspension culture.
In the 3D matrix, the aggregates form vascular networks, comprising an endothelium, formed by endothelial cells, surrounded by perivascular pericytes forming a basal membrane, which is further described in the following. The self-assembly of vascular networks usually occurs through sprouting angiogenesis by sprouting of vessels into the matrix.
The invention further provides an artificial blood vessel organoid culture comprising an interconnected network of vascular capillaries, said capillaries comprising endothelium and a basal membrane with perivascular pericytes, (i) wherein said organoid is produced by a method of the invention and/or (ii) wherein the capillaries are embedded in an artificial 3D matrix comprising a hydrogel with collagen and/or (iii) wherein the organoid culture comprises 40 to 1000 blood vessels as counted by counting individual vessels and vessels between capillary intersections. All three characteristics (i), (ii) and (iii) are hallmarks of the invention, that can be individually required by an inventive artificial blood vessel organoid culture or in combination. The organoid may still comprise the 3D matrix or parts of it (ii). The same as described above for the 3D matrix in the method applies to the organoid.
The organoid is considered an artificial tissue. “Artificial” means that it is grown in vitro and has certain characteristics of artificial cultures, like size, consistency, shape and cell organization. The shape may be irregular and different from natural occurring tissues and cell organization may differ due to size restrains. In particular, “artificial” excludes naturally occurring tissues and organs and their parts, e.g. natural tissue slices. The 3D matrix may be still in the culture and/or the organoid may have the shape determined by growth in such a matrix. E.g. the organoid may be obtainable by growth in a 3D matrix, especially those as described above. The artificial organoid culture is in particular not a culture of an in vivo developed vascular system or a tissue sample thereof.
The number of blood vessels in the organoid is surprisingly high and has not been achieved before in an artificial culture (iii). Preferably, the organoid culture comprises at least 40, even more preferred at least 60, at least 100, at least 200 or at least 300 or more blood vessels as counted by counting individual vessels and vessels between capillary intersections. The upper value of 1000 capillaries is a result of a usual organoid that still has a small manageable size, e.g. for screening methods in cell culture well plates, but of course even larger sizes and capillary numbers are possible by continuing the organoid cultivation. Capillary numbers are counted as is usually in the field, i.e. by counting individual vessels and vessels between capillary intersections. The number may be inferred from counting in a small portion of the organoid and extrapolating the number to the entire organoid.
Preferably, the vascular capillaries of the artificial blood vessel organoid culture have an average diameter of from 1 μm to 30 μm, preferably 5 μm to 20 μm. Such large diameters and volumes of the capillaries allow perfusion in a circulatory animal model. Preferably, the average diameter of vascular capillaries is at least 1 μm, more preferred at least 2 μm, even more preferred at least 3 μm, at least 4 μm, at least 5 μm, at least 6 pm or more.
The artificial blood vessel organoid preferably has a size of 100 μm to 10 mm in its longest dimension. Preferred is as size of 250 μm to 10 mm or 500 μm to 5 mm. This size is of the organoid itself, i.e. the culture comprising the entire vascular network, preferably the organoid is still in the 3D matrix.
Such a size, especially of about 1-2 mm, makes the organoids manageable for cell culture well plates, such as 96 well plates, that may be used for large-scale testing purposes. The organoids are stable and can endure physical stress that allows transportation, e.g. by pipette and they are thus suitable for routine lab handling or automated processing in a screening robot.
The artificial blood vessel organoid may be provided in form of a globular body, e.g. in particular with the shortest dimension being not less than 20% of the longest dimension, in particular not less than 30% or not less than 40% of the longest dimension. Preferably the volume of the artificial blood vessel organoid is at least 1×106 μm3, in particular preferred at least 2×106 μm3, at least 4×106 μm3, at least 6×106 μm3, at least 8×106 μm3, at least 10×106 μm3, at least 15×106 μm3 and/or sizes of at least 250 μm, especially preferred at least 350 μm.
The organoids may also be provided in discs, which may be suspended in a free floating environment for easy handling.
The presence of sufficient perivascular pericytes in the inventive artificial organoid was particularly surprising and shows that the inventive vascular network in the organoid has achieved in vivo characteristics. Perivascular pericytes support the endothelial cells. The ration of endothelial cells and perivascular pericytes may vary, dependent on organoid culturing time. Preferably, the ratio of endothelial cells to perivascular pericytes in the artificial blood vessel organoid culture is between 100:1 to 1:10. Preferably, the ratio is 50:1 to 1:5, or 25:1 to 1:4 or 10:1 to 1:3 or 5:1 to 1:2. Usually, in young organoids, the endothelial cells are in excess, in older organoids the ratio may be about 1:1: or even result in a pericyte excess in relation to endothelial cells.
Preferably the vascular capillaries of the artificial blood vessel organoid culture comprise mature endothelial cells. The mature endothelial cells may be reactive to TNF-alpha by responding by ICAM-1 expression. Preferably the vascular capillaries of the artificial blood vessel organoid culture comprise mature pericytes. Maturity of the pericytes may be detected by determining expression markers of mature pericytes.
The endothelial cells may be surrounded by a basal membrane (also referred to as basement membrane). The basal membrane may comprise collagen IV, fibronectin and/or laminin; it may be rich in collagen IV. Basal membrane thickness may be a marker for health of the capillaries and may be determined as an indicator in screening or other testing methods. The basal membrane of the vascular capillaries of the artificial blood vessel organoid culture may have a thickness in the range of 0.1 μm to 3 μm, preferably 0.3 μm to 2.5 μm, dependent on the size of the capillaries. Preferably the average thickness of the basal membrane of the vascular capillaries of the artificial blood vessel organoid is 0.3 μm to 2.5 μm, preferably 0.6 μm to 2.1 μm, especially preferred 0.8 μm to 1.8 μm, most preferred about 1.2 μm.
“About” means in this case +/−30%.
A further hallmark of the invention is that the organoids develop venules and arterioles as found in an in vivo vascular tree.
The invention further provides a method of providing a non-human animal model with human vascular capillaries, wherein said human capillaries comprise endothelium and a basal membrane with perivascular pericytes, comprising the steps of introducing a human blood vessel organoid of the invention, in particular as described above, into a non-human animal and letting said organoid grow its vascular capillaries. Preferably, said human organoid is introduced onto or into the kidney of the non-human animal. The invention also provides a non-human animal model comprising an inserted artificial blood vessel organoid culture according to the invention. As said, an advantage of the inventive organoid is its versatility and in vivo-like structure of vascular capillaries. It is possible to study the behaviours of these capillaries in vivo by introduction into a non-human animal.
Animal models to study and investigate vascular diseases, such as diabetes have been known in the art (e.g. WO 2015/044339 A1). These models usually are based on an animal that has a genetic alteration that gives rise to a diseased state. However, such mutations also alter the study premise and potentially the alters not just disease occurrence but also the response to any tested treatment options. Therefore, there is a need to study life-like situations. Especially preferred is a model of human vascular systems. The present invention achieves that goal by providing an organoid that is suitable for implantation in a test animal. As described above, the inventive organoid, that is grown in vitro has a high resemblance of vascular networks formed in vivo, however, in an artificial and controllable environment (e.g. still in the 3D matrix instead of connective tissue). Among the properties of the inventive vascular system, that can now be introduced in a non-human anima, are capillaries with endothelium and a basal membrane with perivascular pericytes. Therefore, the invention also provides a non-human animal model with human vascular capillaries, wherein said human capillaries comprise endothelium and a basal membrane with perivascular pericytes. All these kinds of animal models are described together and each preferred or further embodiment reads on all inventive animal models.
The invention now allows studying human vascular capillaries in non-human animals. Therefore, preferably the organoid is from human cells, i.e. having human vascular capillaries, in a non-human animal. The non-human animal is preferably a vertebrate, e.g. a mammal, reptile, bird, amphibian or fish. In particular preferred are land-living vertebrates. Especially preferred are mammals for all aspects and embodiments of the invention, such as mouse, cattle, horses, cats, dogs, non-human primates. Of course, also any vertebrate may be used as source for the organoid and hence the vascular capillaries. But of course, the remarkable advantages are associated with human organoids giving that it is also possible to create non-human animals as model organisms without having to use an organoid. Prefearly, the non-human animal is immunocompromised in order to avoid organoid rejection.
Contrary to previous non-human animals comprising human endothelial cells as summarized in the background section, the present invention not only introduces human cells into non-human animals but introduces fully developed capillary systems of a human into the non-human animal. I.e. human endothelial is investigated in the surrounding of human basal membrane and human pericytes in a structure reminiscent of human capillary trees, in particular a capillary system comprising venules and arterioles.
Preferably the vascular capillaries of the artificial blood vessel organoid culture or the human vascular capillaries in the animal model are perfused by the blood circulatory system of the non-human animal. As said, it is one of the advantages of the invention that the capillaries of the organoid are capable to connect to the vascular system of the non-human animal model. Such connections will form once implanted into a suitable location in the animal. A very reactive location is the kidney membrane but other locations are suitable as well as is known in the art for tissue transplant and trans-grafting studying techniques. Other organs may be used, such as any organ in the abdominal cavity or by subcutaneous transplantation. In certain cases, a given location may require further stimulation of capillary growth, such as by supplying growth factors, e.g. in a suitable matrix, like a hydrogel or sponge.
The inventive artificial blood vessel organoid culture can also be used as a research tool to study the effects of any external (e.g. drugs or other stimuli) or internal (mutations) influences on growth and activity of cells in the organoid. Therefore, in an additional aspect, the invention provides a method of investigating a developmental vascular tissue effect, e.g. a defect, in particular a developmental defect, comprising (i) decreasing or increasing the expression in a gene of interest in a cell at any stage during the inventive method or to the developed (finished) organoid or animal model, or (ii) administering a candidate chemical compound of interest to a cell during development of the organoid at any stage during the inventive method or to the developed (finished) organoid or animal model. A gene of interest can be a gene, that is suspected to be essential or detrimental when active during the development healthy vascular tissue. Preferred genes are genes that are associated with a disease, for example genes that are causative agents in genetic diseases. Methods to decrease or increase expression in a gene are well known in the art, and include knock-out, knock-down methods or mutagenesis (especially RNA interference, anti-sense inhibition, shRNA silencing, mutagenesis by CRISPR-Cas, etc.), or introductions of transgenes (e.g. knock-in), respectively. Such decrease or increases can be conditional, e.g. by introducing a genetic construct with inducible promoters and/or conditional knock-out or knock-downs or knock-ins. The introduction of conditional mutations of essential genes or introductions of lethal genes are possible by using suitable conditional mutation vectors, e.g. comprising a reversible gene trap. Conditional mutations preferably facilitate reversible mutations, which can be reversed to a gene-active or inactive, respectively, state upon stimulation, e.g. as in the double-Flex system (WO 2006/056615 A1; WO 2006/056617 A1; WO 2002/88353 A2; WO 2001/29208 A1). Mutations can either be random or site-directed at specific genes. Thus in preferred embodiments of the invention, reversible mutations are introduced into the pluripotent stem cells, either by random (forward) or site directed (reverse) mutagenesis. Suitable vectors comprising insertion cassette with a reversible mutations. Mutations can be switched on or off at any stage of the inventive method. Vectors or other nucleic acids can be introduced into cells with any method known in the art, e.g. electroporation. It is of course also possible to provide cells having a given mutation. Such cells can be isolated from a patient, followed by a step of inducing pluripotent stem cell status, and letting the cells develop into the inventive tissue, e.g. by the method described above. The patient may have a particular disease of interest, especially a vascular defect or capillary deformity. Candidate chemical compounds are further explained below with regard to candidate therapeutic potential. However, any candidate compound can also be assayed for any desired effect to the cells, capillaries or the entire organoid. Preferred candidate compounds are small organic molecules.
In any method or culture or non-human animal model of the invention, preferably the blood vessels or capillaries are subjected to pathogenesis and said organoid or human animal model is a model of a pathology.
The inventive capillary formation process may be subject to a disorder; alternatively, a pathological state may be induced in the organoid as such, e.g. in a culture, or as implant in the animal model.
Induction of a pathology for research purposes is known in the art and may be exposure to harmful compounds or pathogens, a disadvantageous diet or mechanical stress or injury or a combination of these (e.g. as disclosed in US 2010/124533 A1). Pathogens include microorganisms, in particular bacteria or fungi and viruses. A pathology may also be the result of a genetic disorder or malfunction.
Pathogenesis may comprise hyperglycaemia and/or inflammation. Both can be found in diabetes, for example. In particular preferred, the pathology is diabetes. Inflammation can comprise exposure to or induction of one or more inflammatory cytokines, preferably TNF-alpha and/or IL-6. Hyperglycaemia means increased glucose levels reminiscent of diabetes type 2. Such glucose levels may e.g. be at least 50 mM, preferably at least 70 mM. Example conditions to induce diabetes are 75 mM D-Glucose+1 ng/mL TNF-α+ing/mL IL-6 for 1-2.5 weeks. Diabetic changes in the vessels of the organoids include thickening of basement membrane (increased collagen type IV, fibronectin, laminin, perlecan), reduced vessel growth and endothelial/pericyte death. In an animal model, diabetes may also be induced by selecting an animal with organic causes of diabetes, such as a deficiency of pancreatic beta-cells; or by causing pancreatic beta-cell insufficiency. Beta-cell insufficiency may be caused by autoimmune destruction, such as in diabetes type 1, or by chemical toxicity, e.g. induced by streptozotocin.
The prevalence of autoimmune type 1 and especially of type 2 diabetes mellitus (T2D) is increasing constantly, resulting in a global epidemic of already more than 420 million patients. There are various risk factors for T2D, such as obesity, aging, nutritional states and physical inactivity, in addition to genetic pre-dispositions in different populations. The consequences of high blood glucose include damaged blood vessels, leading to arteriosclerosis and chronic diabetic microangiopathies. The structural hallmark of diabetic microangiopathy is the thickening of the capillary basement membranes due to increased expression and deposition of extracellular matrix proteins, in particular of type IV collagen. These changes lead to occlusive angiopathy, altered vascular permeability, or tissue hypoxia, resulting in complications such as heart disease, strokes, kidney disease, blindness, impaired wound healing, chronic skin ulcers, or amputations. Such conditions can be studies in the inventive organoid, e.g. but not necessarily in an animal model.
Although diabetic microvascular changes can occur in dogs, hamsters, or monkeys, no single experimental animal model displays all the clinical features of the vascular changes seen in human patients. Moreover, these vascular changes are insufficiently recapitulated in previous human in vitro cell culture models. Thus, a comprehensive understanding of the vascular changes affecting hundreds of millions of diabetes patients, which cause life-altering morbidities and ever increasing mortalities, is still lacking. The inventive 3D human blood vessel organoids that exhibit morphological features and molecular signatures of bona fide human microvasculature. These human 3D blood vessels can grow vascular trees in vivo in non-human animals, like mice. Importantly, these organoids can be used to model diabetic microangiopathy and to screen for pathways that could be targeted to protect from “diabetes”-induced vascular damage.
The invention further relates to a method of screening a candidate chemical compound for influencing a pathogenesis or a pathology comprising administering said candidate chemical compound to a culture or non-human animal model or during generation of said culture or non-human animal model according to any aspect and embodiment of the invention and monitoring for physiological differences in said culture or animal model as compared to said culture or animal model without administration of the candidate chemical compound. The stem cells used in the method, organoid or non-human animal model used for screening may have or is developing the pathology or is subject to pathogenesis as mentioned above. A method of testing or screening a candidate compound for influencing properties, modification and development of vascular capillaries and their networks is provided, said method comprises contacting cells or a organoid or the animal in a method of any one of the invention with the candidate compound or contacting an organoid of the invention with the candidate compound and maintaining said contacted organoid in culture or in vivo, and observing any changes, such as developmental changes, in the capillaries of the organoid as compared to said organoid without contacting by said candidate compound, including changes (like physiological changes or gene expression changes) in developed or developing capillaries of the organoid.
The contacting step is a treating step of cells to be developed into the inventive organoid or of the organoid or its precursor cell aggregates. The candidate compound may be a small organic molecule, such as molecules with a mass of 100 Da to 5000 Da. Other candidate compounds may be biomolecules such as proteins, nucleic acids or carbohydrates. Further candidate compounds may be bulk chemicals such as solvents, like ethanol—of course used in concentrations generally viable for cells—or polymers. The treatment should be in a concentration wherein a specific effect of the compound can be expected. Various concentrations may be tested in parallel. Usually concentration of candidate compounds is 1 ng/ml to 100 mg/ml, e.g. of 100 ng/ml to 1 mg/ml.
Also provided is a method of screening or testing a candidate therapeutic agent suitable for treating a pathology in the organoid of interest, comprising providing an organoid of the invention, e.g. by performing the inventive differentiation method and administering the candidate agent to said cells at any stage during the method (as above), preferably at all stages, or to the pathology-affected organoid. As above, a change in the vascular network of the organoid is observed as compared to without such candidate agent. Such a change may be e.g. in the thickness of the basal membrane, as e.g. observed in case of diabetes.
This method has been employed in the diabetes model mentioned above. Accordingly, the Notch3 activation pathway, in particular gamma-secretase and its pathways was identified as an ameliorating agent suitable for the treatment of diabetes. Accordingly, the invention also provides the use of a Notch3 activation pathway inhibitor (such as a gamma-secretase inhibitor, a Notch3 inhibitor, DLL4 inhibitor or a combination thereof) in the treatment or prevention of a thickened capillary basement membrane, such as in diabetic vasculopathy, occlusive angiopathy, altered vascular permeability, tissue hypoxia, heart disease, stroke, kidney disease, blindness, impaired wound healing or chronic skin ulcers.
Example Notch3 activation pathway inhibitors, in particular inhibitors of gamma-secretase, Notch3, or DLL4, are inhibitory antibodies and binding partners of gamma-secretase, Notch3, or DLL4. An antibody includes any functional equivalents and derivatives thereof, including antibody fragments such as Fab, F(ab)2, Fv, single chain antibodies (scAb), nanobodies or like camelid antibodies, or an antibody anti gen binding domain. Antibodies specifically binding gamma-secretase, Notch3, or DLL4, are encompassed by the invention. The antibodies may be produced by immunization with full-length protein, soluble forms of the protein, or a fragment thereof. The antibodies of the invention may be polyclonal or monoclonal, or may be recombinant antibodies, such as chimeric antibodies wherein the murine constant regions on light and heavy chains are replaced by human sequences, or CDR-grafted antibodies wherein only the complementary determining regions are of murine origin. Antibodies of the invention may also be human antibodies prepared, for example, by immunization of transgenic animals capable of producing human antibodies (WO 93/12227). The antibodies are useful for detecting gamma-secretase, Notch3, or DLL4 in biological samples, thereby allowing the identification of cells or tissues which produce the protein in addition, antibodies which bind to gamma-secretase, Notch3, or DLL4 (and block inter-action with other binding compounds) have therapeutic use as gamma-secretase, Notch3, or DLL4 inhibitor. Particularly preferred are anti-gamma-secretase antibodies. A preferred anti-Notch3-antibody is tarextumab. Abcam anti-DLL4 antibodies are for example ab7280, ab176876, ab183532.
Further inhibitors of these components may be any (physiological) binding partner, such as receptors or ligands, that sequester gamma-secretase, Notch3, or DLL4 and thus reduce biological activity. Binding partners are preferably binding proteins. An example ligand for Notch 3 is recombinant soluble DLL4 protein. Such a binding partner (which is preferably not cross-linked to a substrate or membrane, e.g. crosslinked on a plate) will bind to the corresponding Notch receptor without activation. Thus recombinant DLL4 protein can act as an inhibtitor since it is not presenteted on a cell surface, such as an endothel cell surface (Scehnet et al. Blood 2007 109(11): 4753-4760; Noguera-Troise et al. Nature 2006 444(7122): 1032-1037). Prefearbly an such binding protein is provided in soluble form, without being immobilized on a solid durface or on a cell membrane, especially also not in complex with another protein. Suc soluble forms bind to the respective target (such as gamma-secretase, Notch3, or DLL4) but fail to activate the signalling cascade but instead inhibit it by blocking the target. Binding proteins may also be provided as sequestering or masking agents that bind the target and mask its effect due to complex formation that prevents binding of activating signalling molecules.
Further Notch3 activation pathway inhibitors, in particular gamma-secretase, Notch3, or DLL4 inhibitors, are small molecule inhibitors. Small molecules are usually small organic compounds having a size of 5000 Dalton or less, e.g. 2500 Dalton or less, or even 1000 Dalton or less. A small molecule inhibitor inhibits the activity of gamma-secretase, Notch3, or DLL4 on diabetic organoids as can be easily tested by the methods disclosed herein. Example gamma-secretase inhibitors are Semagacestat, Avagacestat, R04929097, DAPT, LY3039478 (Crenigacestat), LY411575, Dehydroxy-LY411575, LY 450139, MK-0752, IMR-1, Dibenzazepine, PF-03084014 (Nirogacestat), L-685,458, FLI-06, NGP 555, Flurbiprofen and Sulindac.
Further inhibitors are inhibitory nucleic acids, like siRNA, shRNA or sgRNA (in combination with CRISPR-Cas). RNA interference (RNAi) is a mechanism to suppress gene expression in a sequence specific manner. RNA interference (RNAi) is highly effective methodology for suppression of specific gene function in eukaryotic cells. When applied to cells and organisms, RNAi entails the degradation of target mRNA upon transfection of short interfering RNA (siRNA) oligos or short-hairpin RNA (shRNA) encoding vectors. Various methods of RNAi have been described and are generally known for the altering gene expression in plant cells, drosophila and human melanoma cells as is described for example in US 2002/0162126 and US 2002/0173478. The siRNA for use in the methods and compositions of the invention are selected to target a desired molecule of the gamma-secretase, Notch3, or DLL4 signalling pathway or combinations of such molecules. In this manner they are targeted to various RNAs corresponding to a target gene. It is understood by one of skill in the art that the siRNA as herein described may also include altered siRNA that is a hybrid DNA/RNA construct or any equivalent thereof, double-stranded RNA, microRNA (miRNA), as well as siRNA forms such as siRNA duplications, small hairpin RNA (shRNA) in viral and non-viral vectors and siRNA or shRNA in carriers. There exists several methods in the art for inhibiting gene expression using RNAi such as described for example in WO 02/055692, WO 02/055693, EP 1 144 623 B1 and WO 03/074654. By using a siRNA therapy any cellular factor can be targeted and inhibited for the inventive gamma-secretase, Notch3, or DLL4 antagonizing and inhibiting therapy. Therefore, any such compound can be used as a gamma-secretase, Notch3, or DLL4 inhibitor.
Also provided are inhibitors in form of encoding nucleic acids. The inhibitory nucleic acid, antibody or binding partner (e.g. receptor or ligand) may be encoded on nucleic acid that expresses said inhibitors in a cell, thereby exhibiting the inhibitory action.
The inhibitor is usually administered in a therapeutically effective amount, an amount that reduces gamma-secretase, Notch3, or DLL4 activity to significantly decrease diabetic morphology. Preferably the gamma-secretase, Notch3, or DLL4 activity is reduced by at least 25%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70% by at least 80% or by at least 90% as compared to amounts without treatment (but otherwise similar conditions). In preferred embodiments this reduction equates to gamma-secretase, Notch3, or DLL4 intracellular levels.
The inhibitor may be provided in a pharmaceutical composition. Pharmaceutical compositions or formulations for therapeutic or prophylactic use may comprise a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant. The invention also provides for pharmaceutical compositions comprising a therapeutically effective amount of a gamma-secretase, Notch3, or DLL4 inhibitor. The term “therapeutically effective amount” means an amount which provides a therapeutic effect for a specified condition and route of administration. The composition may be in a liquid or lyophilized form and comprises a diluent (Tris, acetate or phosphate buffers) having various pH values and ionic strengths, solubilizer such as Tween or Polysorbate, carriers such as human serum albumin or gelatin, preservatives such as thimerosal or benzyl alcohol, and antioxidants such as ascorbic acid or sodium metabisulfite. Selection of a particular composition will depend upon a number of factors, including the condition being treated, the route of administration and the pharmacokinetic parameters desired. siRNA formulations are preferably administered in liposome formulations.
The invention further provides the use of an artificial blood vessel organoid according to the invention, such as obtained from the invetive culture, preferably with a hydrogel with collagen, as an implant in a tissue replacement therapy. A therapy of using the invetive organoid may comprise placing the artificial blood vessel organoid into a wound and letting said artificial blood vessel organoid culture integrate into the wound. The use as an implant can comprise placing the organoid into a subject to be treated, in particular at a position that requires connective tissue regrowth. Sich regrowth can be stalled, e.g. due to diseases that impair regrowth, like diabetes, or medication or other therapies, such as in a chemotherapoy or radiation, e.g. as in a chermotherapy or radiation accidents. A wound is preferably treated. Such a wound may be a chronic wound, in particular a wound that fails to close in 30 days or 60 days or even 90 days. A chronic wound can be due to the above circumstances, diseases, medication or therapy. In particular, the wound is a diabetic wound, such as a diabetic foot ulcer, or a burn, such as a third degree burn. The wound may comprise a skin wound. A skin wound may comprise damage to both epidermal and dermal layers. Any wound ,including a skin wound may comprise trauma to the underlying muscles, bones, and tendons. The therapy may comprise wound cleaning, in particular removal of dead tissue to ease regrowth.
In such a therapy, one or more organoids, the number depending on the size of the wound, are placed into the volume to be treated, such as a wound, and the organoids are allowed to integrate with the tissue surrounding the volume. Said volume is preferably surrounded by tissue of the patient in at least 50%, preferably at least 75%, of the one or more organoids surface area facing the outside of said one or more organoids (not counting internal surface area in case of more than one organoids that face other organoids). This means that the volume is mostly internal and able to integrate within the subject. Woundas may be internal or open wounds. Even open woundas may have such a volume facing an open surface, such as a skin wound.
Letting the organoid integrate into the wound usually comprises regenerative processes that are improved by the prsesnce of the blood vessels of the inventive organoid. Said blood vessels are able to connect with the circulatory system of the patient and improve oxygenation of damaged tissue and by concequence its regeneration.
In other to avoid immunereactions against the organoid and its cells, the cells of the organoid are preferably of the same organism as the patient (preferably both human, or both of the same non-human animanl, preferably mammal) and are a MHC match to the patient. To quickly provide such a fitting organoid, it is possible to create a organoid library with various documented MHC-types. Its organoids can be quickly provided to a patient.
Further provided is a kit of compounds and substances. The kit may comprise means to perform any of the inventive methods. Of course, not all substances need to be included since some are standard chemicals or usually available. Nevertheless, preferably the core substances are provided. In other kits, rarer substances are provided. The inventive kits or their substances may be combined. Components in the kit are usually provided in separate containers, such as vials or flasks. Containers may be packaged together. Preferably the kit comprises a manual or instructions for performing the inventive methods or their steps.
Provided is a kit suitable for the generation of an artificial blood vessel organoid according to any inventive method. The kit may comprise (i) a Wnt agonist or a GSK inhibitor; (ii) a vascular differentiation factor selected from VEGF, preferably VEGF-A, a FGF, preferably FGF-2, a BMP, preferably BMP4; (iii) a collagenous 3D matrix, preferably comprising 10%-50% laminin, 20%-70% collagen I, and/or 2%-30% collagen IV (all wt.-%).
Preferably, the kit comprises a 3D matrix as described above, or their components to generate such a 3D matrix. Matrix components may be provided in solid state, such as lyophilized state to be reconstituted to the matrix, e.g. by hydration. Any matrix or their components as described above may be included in the kit. Preferably the matrix is a hydrogel, especially a collagenous hydrogel as described above. The kit may comprise such reconstitutable (preferably collagenous) components. Further preferred matrix components are carbohydrates, in particular in polymeric form (polysaccharides). A preferred polysaccharide is agarose.
Any kit may further comprise cell growth nutrients, preferably DMEM/F12, knock-out serum replacement (KOSR) medium, Glutamax, or essential amino acids and/or non-essential amino acids (NEAA), or any combination thereof. Any compound mentioned in the examples can be included in the kit.
It is contemplated that any method or product described herein can be implemented with respect to any other method or product described herein and that different embodiments may be combined.
The kit may further comprise instructions for performing the inventive method. Such instructions may be in printed form or in a computer-readable format on a suitable data carrier.
The claims originally filed are contemplated to cover claims that are multiply dependent on any filed claim or combination of filed claims. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or product of the invention, and vice versa. Any embodiment discussed with respect to a particular condition can be applied or implemented with respect to a different condition. Furthermore, compositions and kits of the invention can be used to achieve methods of the invention.
“Comprising” is understood as an open-ended term, i.e. allowing further components or steps of substance. “Consisting of” is understood of a closed term without any further components or steps of substance.
Throughout this application, the term “about” may be used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value or in a set value may refer to ±10%.
The present invention is further defined in the following preferred embodiments and definitions, that are all combinable with the above detailed description:
The present invention is further exemplified by the following figures and examples, without being limited to these particular embodiments of the invention.
Human stem cells and differentiation into vascular organoids. All experiments presented were done in either the human iPS cell line NC8 (Pripuzova et al. Stem Cell Res. 14, 323-338 (2015)) or the human embryonic stem cell (ESC) line H9 (Thomson et al. Science 282, 1145-1147 (1998)). All stem cells were cultured under chemically defined, feeder-free conditions as previously described (Chen et al. Nat. Methods 8, 424-9 (2011)). For differentiation, H9 ESCs or NC8 iPS cells were disaggregated using 0.5 mM EDTA for 2 min and subsequently incubated with 0.1% Stempro Accutase (Life Technologies) for 3 min. 2×105 cells were resuspended in differentiation media (DMEM:F12 medium, 20% KOSR, Glutamax, NEAA; all from Gibco) including 50 μM Y-27632 (Calbiochem) and plated into one well of an ultra-low attachment surface 6 well plate (Corning) for cell aggregation. Cell aggregates were treated on day 3 with 12 μM CHIR99021 (Tocris) and on days 5, 7 and 9 BMP4 (30 ng/mL, Stemcell Tech.), VEGF-A (30ng/mL, Peprotech), and FGF-2 (30 ng/mL, Miltenyi) were added. On day 11, cells were switched to media containing VEGF-A (30ng/mL), FGF-2 (30 ng/mL) and SB43152 (10 μM) to balance the endothelial/pericyte ratio. The resulting cell aggregates were embedded on day 13 in Matrigel:Collagen I (1:1) gels and overlaid with differentiation media containing 100 ng/mL VEGF-A and 100 ng/ml FGF-2. This differentiation medium was changed every 2nd to 3rd day. Around day 18 vascular networks were established and either directly analysed or networks from individual cell aggregates were cut out from gels and further cultured in 96 well low attachment plates (Sumilon, PrimeSurface 96U) as free floating vascular organoids for up to 3 months.
Reprogramming and characterization of human iPSCs. Human dermal fibroblasts (ATCC) and blood samples were reprogrammed as previously described (Agu et al. Stem cell reports 5, 660-71 (2015)). To check chromosomal integrity, multiplex-fluorescence in situ hybridization (M-FISH) was performed as described by Agu et al. For Genotyping, sample preparation was performed according to Infinium HTS Protocol Guide as recommended by Illumina Inc. The genotyping was performed using the Illumina Infinium PsychArray-24 BeadChip scanned with the Illumina iScan systems according to manufacturer's instruction. Genotypes were called using Illumina GenomeStudio (Illumina, San Diego, Calif., USA) with Genotyping software (Module 2.0.1), excluding samples with call rate <0.995. For analysis we applied the default settings by Illumina, the InfiniumPsychArray-24v1-1 Al manifest and Infinium PsychArray-24v1-1 Al ClusterFile cluster files. The CNV analysis and plotting were performed by using the bcftools cnv.
Immunocytochemistry. Vascular networks in Collagen I:Matrigel gels were fixed for 20min and free floating vascular organoids fixed for lh with 4% PFA at room temperature (RT) and blocked with 3% FBS, 1% BSA, 0.5% Triton, and 0.5% Tween for 2 h at RT on a shaker. Of note, vascular organoids are more stable than the initially formed vascular networks in 3D gels and therefore could be used for standard immunohistochemistry procedures. Primary antibodies were diluted 1:100-1:200 in blocking buffer and incubated over night at 4° C. The following antibodies were used in these study: anti-CD31 (DAKO, M082329), anti-VE-Cadherin (Santa Cruz, sc-9989), anti-ICAM-1 (Sigma, HPA002126), anti-PDGFR-β (CST, 31695), anti-SMA (Sigma, A2547), anti-Calponin (Abcam, AB46794), anti-Collagen Type IV (Merck AB769), anti-Laminin (Merck, 19012), anti-MYH11 (Sigma HPA014539). After 3×10 min washes in PBS-T (0.05% Tween) the samples were incubated with the corresponding secondary antibodies from Life Technologies: Alexa Fluor 555 donkey-anti-mouse (A31570), Alexa Fluor 647 donkey-anti-rabbit (A31573), Alexa Fluor 488 donkey-anti-goat (A11055), Alexa Fluor 488 donkey-anti-sheep (A11015) at 1:250 in blocking buffer for 2h at room temperature. After 3 20 min washes in TBST the samples were counterstained with DAPI. The samples were mounted (DAKO S302380), dried overnight and imaged subsequently with a Zeiss 780 Laser Scanning Microscope. Vascular organoid transplantation. Vascular organoids were transplanted under the kidney capsule of 12-15 weeks old NSG mice. All surgical procedures were done accordingly to the Austrian law and ethical approval. Mice were imaged using MRI to monitor the transplant over time. To test perfusion of the human blood vessel implants, mice were injected i.v. with either FITC-Dextran (1.25mg/mouse, Invitrogen D1822) or anti-human CD31-Alexa 647 (2pg/mouse, BD 558094). Excised transplants were fixed with 4% PFA for 2 h at RT and stained as a whole as described for the vascular organoids above or processed for immunohistochemistry or standard H&E histology. To distinguish between the endogenous mouse and transplanted human vasculature, a specific anti-human CD31 antibody (DAKO, M082329) was used and to visualize the murine blood vessels, we used a specific anti-mouse CD31 antibody (Abcam, AB56299). To exclude possible cross-reactivity, these antibodies were tested on both human and mouse control sections, validating specificity. Samples were imaged with a Zeiss 780 Laser Scanning Microscope.
MRI imaging. MRI was performed on a 15.2 T Bruker system (Bruker BioSpec, Ettlingen Germany) with a 35 mm quadrature birdcage coil. Before imaging, the tail line was inserted for delivery of contrast agent (30-gauge needle with silicon tubing). All animals (N=3) were anaesthetised with isoflurane (4% induction, maintenance with 1.5%). During imaging, respiration was monitored and isoflurane levels were adjusted if breathing was <50 or >80 breaths per minute. Mice were kept warm with water heated to 37° C. circulated using a water pump. For anatomical localization and visualization of the implant a multi-slice multi-echo (MSME) spin echo sequence was used (repetition time (TR)/echo time (TE)=3000/5.8−81.18 ms, 14 echoes, 117 μm2 in-plane resolution, 0.5 mm slice thickness, number of experiments [NEX]=1). A pre-bolus injection of 0.05 ml of 0.01 mol/l gadolinium-based contrast agent (Magnevist, Berlex) was injected in order to correct for the contrast agent leakage. Dynamic susceptibility contrast (DSC) perfusion MRI was collected using fast imaging with steady-state precession (FISP) with 500.6 ms temporal resolution (1 slice; TR/TE=500/1.7 ms; flip-angle=5 degrees; 468×468 μm2 in-plane resolution; 1-mm slice thickness; NEX=2; 360 repetitions) following tail-vein injection of 0.05 mL of 0.25 mol/L Magnevist. DSC data were used to calculate perfusion, relative blood volume (rBV), mean transit time (MTT) and leakage (K2). Processing was done offline using ImageJ (National Institutes of Health; rsbweb.nih.gov/ij/), and the DSCoMAN plugin (Duke University, dblab.duhs.duke.edu/wysiwyg/downloads/DSCoMAN 1.0.pdf). The analysis consisted of truncating the first 5 time points in the DSC-MRI time series to ensure steady-state magnetization, calculating the pre-bolus signal intensity (S0) on a pixel-wise basis, converting the truncated DSC-MRI time series to a relaxivity-time curve (ΔR2*(t)=−(1/TE)ln(S(t)/S0) is dynamic signal intensity curve, and correcting for the gadolinium leakage (K2) as described previously (Boxerman et al. Am. J. Neuroradiol. 27, 859-867 (2006)).
Modeling diabetic vasculopathy in human vascular organoids. Established endothelial networks in vascular organoids were cultured in a non-diabetic control medium (17 mM Glucose) or diabetic medium (75 mM Glucose, in the presence of absence of human TNFα (1 ng/mL, Invitrogen PHC3011) and/or IL-6 (1 ng/mL, Peprotech 200-06)) for up to 3 weeks before the basement membrane was investigated by Collagen type IV immunostaining and electron microscopy. D-Mannitol was used in non-diabetic media to control for hyperosmotic effects. For basement membrane quantifications, acquired z-stacks were analysed and the thickness of ColIV coats around luminal structures were measured using ImageJ software. For drug treatment, organoids were exposed to diabetic medium (75 mM Glucose, 1 ng/mL human TNFα, and 1 ng/mL IL-6) in the presence or absence of the following drugs: 2,4-Thiazolidinedione (5 mM, Abcam ab144811), Metformin (5 mM, Abcam ab120847), Acarbose (80 μg/mL, Sigma A8980), Nateglindine (100 μM, Sigma N3538), Diphenyleneiodonium (10 μM), Glimepiride (30 nM, Sigma G2295), Pioglitazone (10 μM, Sigma E6910). The following small molecule inhibitors were used: N-Acetyl-L-cysteine (500 μM, Sigma, A7250), CHIR99021 (10 μM, Tocris 4423), Goe6976 (100nM, Merck U51365250), MK2206 (10 μM, EubioS1078), QNZ (10 μM, Eubio S4902), 5B203580 (10 μM, Eubio S1076), SCH772984 (500 nM, Eubio S7101), 5P600125 (10 μM, Eubio 51460) Y-27632 (10 μM, Calbiochem 688000), DAPT (25 μM, Sigma D5942), 5B431542 (10 μM, Abcam ab120163).
FACS analysis of vascular organoids. Non-diabetic and diabetic vascular organoids were disaggregated using 25 μg/mL Hyaluroniduase (Worthington), 3 U/mL Dispase (Gibco), 2 U/mL Liberase (Roche) and 100 U DNAse (Stemcell Tech) in PBS for 45-60 min at 37° C. Subsequently, single cells were stained with the following antibodies: anti-CD31 (BD, 558094), anti-PDGFR-β (BD, 558821), anti-CD90 (Biolegend, 328117), anti-CD45 (ebioscience, 11-0459-41), and anti-CD73 (BD 742633). DAPI staining was used to exclude dead cells. A BD FACS Aria III was used for cell sorting and a BD FACS LSR Fortessa II for cell analysis.
Genome editing using CRISPR/Cas9. A mammalian expression vector expressing Cas9 from S. pyogenes with a 2A-Puro cassette13 (Addgene Plasmid: #62988;) was cut with BbSI (Thermo Fisher ER1011) shortly after the U6 promoter. Subsequently the plasmid was re-ligated introducing sgRNAs for either Neurogenic locus notch homolog protein 3 (Notch3) or Delta-like protein 4 (D114). The following primers were used for sgRNA annealing: Notch3: forward, caccgGCCACTATGTGAGAACCCCG (SEQ ID NO: 7); reverse, aaacCGGGGTTCTCACATAGTGGCc (SEQ ID NO: 8); D114: forward, caccg-CAGGAGTTCATCAACGAGCG (SEQ ID NO: 9); reverse, aaacCGCTCGTT-GATGAACTCCTGc (SEQ ID NO: 10). sgRNA plasmids were verified by
Sanger sequencing and used for electroporation of iPSCs (NC8) with the 4D-Nucleofector System (Lonza). 2 μg plasmid DNA were transfected using the P3 Primary Cell 4D-Nucleofector Kit. Transfected NC8 cells were seeded on Matrigel coated 6-well plates in Essential 8 Media (Gibco) containing 50 μM Y27632 (Calbiochem) and cultured for 24 hours before Puromycin treatment (0.2 μg/ml) for 48 hours. Remaining cells were cultivated until colony formation could be observed and single colonies were further expanded for genotyping with Sanger sequencing. Knock-out cell lines were verified by Western Blot or immunofluorescence staining.
Modeling diabetic vasculopathy in human vascular organoids in vivo. Immunodeficient NSG mice carrying human vascular organoid transplants were daily i.p. injected with 40 mg/kg Streptozotocin (STZ) (Merck, 572201) for 5 consecutive days. Every day, STZ was freshly dissolved in citrate buffer (pH4.6) and immediately used. Diabetes was confirmed (blood glucose >300 mg/dL) by measuring non-fasting glucose using the OneTouch UltraEasy system (Lifetouch, AW 06637502C). DAPT (Selleckchem 52215) was dissolved in ethanol and injected with 90% cornoil for 5 consecutive days at 5 mg/kg with 2 days of no treatment per week. Anti-Notch3 blocking antibodies (R&D AF1559) were injected 3 time/week at lmg/kg. For quantifying vessel leakage, the FITC-Dextran+ area was measured and normalized to the area of perfused human blood vessels (hCD31+) using the FIJI software. This ratio was then further normalized to control non-diabetic mice. Permeability of long-term DAPT treated vessels were measured after 2 days of treatment stop to avoid measuring acute effects of DAPT on vessel permeability.
Next Generation Sequencing and qRT-PCR analysis. Vascular networks of non-diabetic and diabetic vascular organoids were disaggregated using 25 μg/mL Hyaluroniduase (Worthington), 3 U/mL Dispase (Gibco), 2 U/mL Liberase (Roche) and 100 U DNAse (Stemcell Tech) in PBS for 45-60 min at 37° C. Subsequently, single cells were stained for CD31 expression (BD 558094) and DAPI negative (=alive cells) were FACS sorted using an FACS Aria III machine. The CD31 positive, DAPI negative endothelial cells were directly sorted into Trizol LS buffer (Invitrogen) and processed further to RNA isolation. For RNA Seq mRNA was enriched by poly-A enrichment (NEB) and sequenced on a Illumnia HiSeq2500. For qRT-PCR analysis, total RNA was extracted from whole vascular organoids using Trizol (Invitrogen) and cDNA was synthesized using the iscript cDNA synthesis kit (Biorad), performed with a SYBR Green master mix (Thermo) on a Biorad CFX real time PCR machine. All data were first normalized to GAPDH and then compared to the non-diabetic control samples. The following primers were used:
Bioinformatic analysis. RNA-seq reads were aligned to the human genome (GRCh38/hg38) using Tophat v2.0.10 and bowtie2/2.1.0, gene- and transcript-level abundance estimation in TPM, FPKM and expected counts was performed with RSEM v1.2.25, aligned reads were counted with HTSeq v0.6.1p1 and differential expression analysis was carried out using DESeq2 v1.10.1, with FDR threshold of 0.05. Enrichr (Kuleshov et al. Nucleic Acids Res. 44, W90-7 (2016)) was used to identify GO terms of up-regulated genes. The expression profile similarity search server-CellMontage v2 (cellmontage2.cira.kyoto-u.ac.jp; Fujibuchi et al. Bioinformatics 23, 3103-3104 (2007)), was used to classify the expression profiles of iPS.EC cells with respect to normal cell types. The average iPS.EC cells transcripts relative abundance in TPM was compared to the 2919 pre-processed human gene expression datasets. Based on the obtained results iPS.EC are most similar to endothelial cells—the 75 datasets with highest correlation are all derived from endothelial cells—55 stem from vein endothelial cells, 13 from microvascular endothelial cell, 5 from artery endothelial cell, 2 from lymphatic vessel endothelial cell, with correlation coefficients ranging from 0.69 to 0.64 and p-values ranging from 5.38e-2212 to 6.39e-1828. To compare iPS.EC expression profiles with published human tissue RNA-seq data (Lonsdale et al. Nat. Genet. 45, 580-5 (2013)), we combined our and available expression profiles as previously described (Danielsson et al. Brief. Bioinform. 16, 941-949 (2015).), using log-transformed F/RPKM values for 2338 tissue specific genes (Cavalli et al. Genome Biol. 12, R101 (2011)), removing sequencing study batch effects using ComBat, and inspecting sample clustering in a correlation heat map. Expression profiles of iPS.EC are found to cluster with those of GTEx arterial samples.
Skin samples from type II diabetes and normo-glycemic control patients. Surgical samples of human skin were taken from T2D and non-diabetic patients. Non-necrotic, healthy skin was taken from leg amputates. Leg amputations of T2D patients were necessary because of diabetic foot syndrome. Leg amputations of non-diabetic patients were performed as a consequence of accidents, venous ulcerations or other vascular diseases not related to T2D. The present study was approved by the local ethics committee and all included patients gave their informed consent (no. 449/2001; 81/2008). Of note, we included dermis that was isolated at the maximal possible distance of any ulcers or necroses of the leg amputates. The details of patient collectives are shown in the following Table 1.
Immunohistochemistry in patient skin. Human skin material was either cryofixed in Geltol and stored at −80° C., or embedded in paraffin after 4% paraformaldehyde fixation. 2-5 μm sections were cut and used for subsequent immunofluorescence or immunohistochemical stainings. Paraffin sections were dewaxed, hydrated, and heat induced antigen retrieval was performed. Antigenicity was retrieved by microwaving (3×5 minutes, 620 W) or by heating the sections in an autoclave (60 minutes) in 10 mM citrate buffer (pH 6.0). Cryosections were stored at −20° C., thawed and dried at time of use and fixed in ice-cold acetone for 20 minutes. This was followed by incubation with indicated primary antibodies and visualized by a biotin-streptavidin-horseradish peroxidase dase method, or by using fluorescently labelled secondary antibodies.
Human patient-derived endothelial cell preparations. Four T2D and six non-diabetic patients were analyzed. For ex vivo preparations of BECs (spell out), a mechanical and enzymatic micropreparation protocol was used, including the use of Dispase I (Roche Inc., #210455) as previously described11. The resulting single cell suspensions were blocked with 1×PBS-1% FCS and incubated with anti-CD31, anti-CD45 and anti-podoplanin antibodies in a three-step procedure with intervening washing steps. For antibodies see section above. Subsequently, cells were subjected to cell sorting using a FACStar Plus (Becton Dickinson). Total CD31+ podoplanin endothelial cells were separated, re-analyzed, twice pelleted (200 g), lysed in RLT buffer (Qiagen; #74104) and further processed for RNAseq.
Electron Microscopy. Vessel organoids were fixed using 2.5% glutaraldehyde in 0.1M sodium phosphate buffer, pH 7.2. for 1 h at room temperature. For electron microscopy of dermal blood vessel from diabetic and non-diabetic leg amputates, human skin was fixed in 4% PFA and 0.1% glutaraldehyde and embedded in Lowicryl-K4M. Samples were then rinsed with the same buffer, post-fixed in 1% osmium tetroxide in ddH2O, dehydrated in a graded series of acetone and embedded in Agar 100 resin. For electron microscopy of dermal blood vessel from diabetic and non-diabetic leg amputates, human skin was fixed in 4% PFA and 0.1% glutaraldehyde and embedded in Lowicryl-K4M. 70-nm sections were cut and post-stained with 2% uranyl acetate and Reynolds lead citrate. Sections were examined with an FEI Morgagni 268D (FEI, Eindhoven, The Netherlands) operated at 80 kV. Images were acquired using an 11 megapixel Morada CCD camera (Olympus-SIS).
Rodent models of diabetes. Rodent models used in this study and according references are listed in Table 2. Controls were either age matched WT animals or untreated strains as indicated in the Table. Sections of paraffin embedded skin samples of all rodent models were stained HE and PAS to visualize vessel morphology.
Statistics. If not otherwise stated, all values are presented as means±SEM. Statistical analyses were performed using GraphPad Prism. All statistical tests used are described in the figure legends. P<0.05 was accepted as statistically significant.
Capillaries are composed of endothelial cells that form the inner lining of the wall and pericytes that are embedded within a surrounding basement membrane. Human endothelial cells have been previously derived from human embryonic stem cells (hESCs) and from induced pluripotent stem cells (iPSC) (James et al. Nat Biotechnol 28, 161-6 (2010); Patsch et al. Nat. Cell Biol. 17, 994-1003 (2015)). Moreover, perivascular cells such as vascular smooth muscle cells can be generated from human ESCc and iPSCs (Cheung et al. Nat. Biotechnol. 30, 165-73 (2012)). However, to investigate mechanisms of complex vascular diseases, there is a need for highly sophisticated models that resembles all features of a human microvasculature such as a lumenized endothelium, endothelial-pericyte interactions and the formation of a common basement membrane and is applicable to high throughput drug screening. We therefore set out to establish 3D-human blood vessel organoids from hESC and iPS cells.
To accomplish this, we developed a multistep protocol to modulate signaling pathways involved in mesoderm development and blood vessel specification (
Next, we verified that the CD31+ blood vessel organoids recapitulated features of human blood vessels in vivo. To profile gene expression, we disaggregated the organoids, sorted for CD31+ endothelial cells and performed RNAseq. The gene expression profile of CD31+ endothelial cells from our 3D cultures indeed clustered most closely with gene expression profiles (GTEx) of human blood vessels (
To further improve and standardize these in vitro microvasculatures for drug screening approaches and, we developed free-floating 3D organoid cultures in a 96 microwell format (
Next, we assessed the cellular composition of the vascular networks and free-floating organoids using FACS. Both contained PDGFR-β+ pericytes as well as CD31+ endothelium, to varying extents. The remaining cells were primarily CD90+ CD73+ mesenchymal stem like cells and a minor population of CD90−CD45+ haematopoitic cells (
Thus, we have established self-organizing 3D human blood vessel organoids from hESCs and iPSCs that exhibit the morphological features and molecular signatures of bona fide human microvasculature.
To test whether the blood vessel organoids can form functional blood vessels in vivo, we differentiated hiPSCs and hESCs into blood vessel organoids in vitro and transplanted them under the kidney capsule of immunodeficient host mice. The human organoids, because of their compact structure, could reproducibly be transplanted and indeed grew and survived in the mouse environment, in some cases for more than 6 months. We stained organoid and kidney tissue with human-specific anti-CD31, which showed that human vasculature had been established beside endogenous mouse blood vessels (
Importantly, histological sectioning showed that these human blood vessels had specified into arterioles, capillaries and venules (
Diabetes is a major cause of blindness, kidney failure, heart attacks, stroke or lower limb amputation; in large parts because of marked changes in blood vessels, defined by expansion of the basal membrane. Such structural changes due to diabetic microangiopathy have been observed in the human kidney or muscle biopsies. To confirm diabetic microvascular changes in humans, we examined the dermal skin microvasculature in surgical specimens of normo-glycemic individuals and type 2 diabetic (T2D) patients. The clinical characteristics including age, sex, body-mass index (BMI), serum creatinine levels, and years of disease are shown in Table 1. In dermal blood vessels of normo-glycemic controls, the CD31+ capillary endothelium was surrounded by a thin basal membrane, as determined by collagen IV and by PAS staining (
Although diabetic microangiopathy is evident in the skin of diabetic patients, it has not been observed previously in various rodent models of diabetes. We thus performed a comprehensive evaluation of the skin microvasculature of multiple genetic and environment-induced mouse and rat models of diabetes. However we failed to detect increased basement membranes indicative of dermal vasculopathy in any of these models, including leptin and leptin receptor mutant ob/ob and db/db mice, streptozotocin-treated mice, doxycycline induced knock-out of the insulin receptor in mice, high fat diet, high fat and glucose in LDLR mutant mice, or Zucker diabetic fatty rats, carrying a leptin receptor mutation, and streptozotocin treated rats (
Given that rodent models of diabetes do not recapitulate the dermal vasculopathy present in diabetic patients, we wanted to test if we could model this crucial phenotype in our human blood vessel organoids. To accomplish this, we cultured our 3D blood vessel organoids in medium with elevated glucose and monitored expansion of the basement membrane as detected by collagen IV, similarly to the human skin samples. Intriguingly, hyperglycemia resulted in a significant increase in collagen IV in human blood vessel organoids (
Having engineered an in vitro organoid model of human diabetic vasculopathy, we next wanted to identify a drug that could block the thickening and expansion of the basal membrane in human blood vessel organoids treated with high glucose/TNFα/IL6. To this end, we first tested multiple approved drugs that are currently used in the clinic to treat diabetes. However, none of the medicines that we tested, namely metformin, pioglitazone, glimepiride, acarbose, nateglindine, thiazolidinedione, or diphenyleneiodonium, had any effect on the high glucose/TNFα/IL6-induced thickening of the basal blood vessel membrane in blood vessel organoids (
We next screened diabetic blood vessel organoids with small molecule inhibitors of various common signaling and downstream pathways, namely GSK3, PKC, AKT, NFkB, ROS, p38-MAPK, JNK, ROCK, and ERK. We found that none of these inhibitors had any significant effect on collagen IV expansion and thickening of the capillary basement membrane (
γ-secretase is an enzyme that can cleave multiple different receptors to activate distinct signaling pathways, including Notch. To identify the molecular DAPT target involved in protection from our experimental diabetic blood vessel changes, we blocked the Notch ligands Jaggedl, D111, and D114, as well as Notch1 and Notch3, all of which are prominently expressed in blood vessels. Inhibition of Jagged1, D111, as well as Notch1 had no apparent effect on the “diabetic” changes in our free-floating organoids; however, blockade of D114 as well as Notch3 significantly rescued from basal membrane thickening (
Blood vessels contribute to the development of essentially all organ systems and have critical roles in multiple diseases ranging from strokes to heart attacks or cancer. Because of their importance, multiple cell systems have been developed to study blood vessel biology during development and disease, including the use of classic endothelial cell lines such as HUVEC cells. Moreover, endothelial cells and pericytes have each been developed from human stem cells.
We have now developed a robust and reproducible system to grow bona fide human capillaries from hESCs and iPSCs. These blood vessel organoids fulfill all previously defined criteria for human organoids. Intriguingly, blood vessel organoids transplanted into immunodeficient mice resulted in connections between the human blood vessels and the mouse circulatory system, as demonstrated by dextran perfusion, antibody injection, as well as MRI of blood flow revealing perfusion and leakage rates comparable to endogenous mouse organs. Connecting the human vasculature to the mouse circulatory system shows distinct vascular trees. Most importantly, the transplanted human organoids further develop in vivo into arterioles and venules, thus forming a true vascular tree, which has never been previously demonstrated. Therefore, these organoids could also be used to develop more complex, multi-lineage organoids, for instance form blood vessels with cardiomyocytes, or attempt to develop blood vessels in brain or liver organoids. They could also be used to study rare vascular diseases using patient-derived iPSCs.
The global prevalence of diabetes has nearly doubled in the last three decades, with a current estimate of about 420 million diabetic patients and many more with pre-diabetes, resulting in often long-term morbidities and enhanced mortality. Diabetes is a major cause of blindness, kidney failure, heart attacks, stroke and lower limb amputation, in many cases as a consequence of blood vessel pathologies such as massive thickening of the basement membrane that result in insufficient tissue oxygenation, impaired cell trafficking, or rupture of the vessels. One can model certain aspects of diabetic blood vessel changes in the retina and kidney of rodents, though until now, there was no single model that displays all the clinical features of diabetic blood vessel changes seen in human. Moreover, dermal microvascular changes do not occur in any of the diabetic rodent models we have studied, making it vital to develop novel systems to identify pathways and potential drug targets that are operational in these microvascular changes. To demonstrate the utility of our blood vessel organoids, we exposed them to a “diabetic cocktail” containing high glucose, IL6, and TNFα, that resulted in the marked expansion of the collagenous basal membrane and gene expression profiles resembling microvascular changes observed in the dermal capillaries of T2D patients.
We tested a number of current anti-diabetic medicines, as well as small molecule inhibitors of multiple common signaling pathways, but only few had any effect on the expansion of the basal membranes in diabetic organoids. Intriguingly, we found that that the γ-secretase inhibitor DAPT nearly completely prevented the thickening of the basal membrane in our organoid culture. γ-secretase inhibitors have already undergone clinical trials for Alzheimer's disease and are currently being tested as cancer treatment. These drugs can be repurposed for the treatment of diabetic vasculopathies in humans. Importantly, these data provide a proof-of-principle that the human blood vessel organoid model for diabetic microvasculopathy is a useful screening tool to discover novel drugs that alleviate microvascular changes.
γ-secretase inhibitors have already undergone clinical trials for Alzheimer's disease and y-secretase inhibitors as well as Notch2/3 blockers are currently being tested as cancer treatments. Furthermore, inhbiting the Notch pathway by y-secretase inhibitors reduced diabetes-induced glomerulosclerosis and podocyte loss by apoptosis in diabetic rats. These drugs can therefore be repurposed for the treatment of diabetic vasculopathies in humans. Importantly, these data provide a proof-of-principle that the human blood vessel organoids and our in vivo diabetic microvasculopathy model could be a useful screening tool to develop novel drugs that alleviate microvascular changes in diabetes.
| Number | Date | Country | Kind |
|---|---|---|---|
| 17176335.2 | Jun 2017 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/065949 | 6/15/2018 | WO | 00 |
