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  • C12N5/0012—Cell encapsulation
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  • G01—MEASURING; TESTING
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  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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  • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
  • G01N33/5061—Muscle cells
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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
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  • C12N5/0657—Cardiomyocytes; Heart cells
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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  • C12N2503/02—Drug screening
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  • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
  • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
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  • C12N2513/00—3D culture
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  • C12N2533/00—Supports or coatings for cell culture, characterised by material
  • C12N2533/70—Polysaccharides
  • C12N2533/74—Alginate

Definitions

• the invention relates to the treatment of cardiac diseases, in particular ischemic heart diseases, by the use of specific cardiac tissues obtained from particular cellular microcompartments comprising human cells expressing genes expressed during cardiac differentiation.
• cardiovascular disease and in particular ischemic heart disease (which usually leads to myocardial infarction), is the leading cause of death worldwide (Thomas, H. et al. Global Atlas of Cardiovascular Disease 2000-2016: The Path to Prevention and Control.Glob.Heart 13, 143-163 (2016)).
• hPSC-CMs human pluripotent stem cell-derived cardiomyocytes
• hPSC-CMs human pluripotent stem cell-derived cardiomyocytes
• hPSC-CMs human embryonic stem cells and induced pluripotent stem cells
• cardiomyocytes can be used for other applications, in particular as biological pacemakers for the treatment of sinus node dysfunction (Lee, JH, Protze, SI, Laksman, Z., Backx, PH & Keller, GM Human Pluripotent Stem Cell-Derived Atrial and Ventricular Cardiomyocytes Develop from Distinct Mesoderm Populations. Cell Stem Cell 21, 179-194. e4 (2017)), or to treat congenital heart disease, such as septal anomalies (Devalla, HD & Passier, R. Cardiac differentiation of pluripotent stem cells and implications for modeling the heart in health and disease . Sc/. Transi. Med.
• cardiomyocytes derived from hPSC-CM in cardiac cell therapy currently impossible on a suitable clinical scale.
• production on an industrial scale of cardiac tissues is complex because it is necessary to achieve a compromise between sufficiently mild culture conditions for the survival and proper functioning of the tissues and the constraints of large-volume cultures which inevitably expose the cells to non-physiological stresses (typically hydrodynamic stress in the context of liquid culture in bioreactors).
• the methods for producing cardiomyocytes from hPSCs in particular present the following problems:
• micro-supports still leave the cells exposed to mechanical stresses and which can be difficult to remove
• the object of the invention is therefore to meet all of these needs and to overcome the drawbacks and limitations of the prior art.
• the invention proposes to go through a key developmental intermediate to obtain compacted tissues of human cardiac cells with particular characteristics and in large quantities, suitable for uses in cell therapy.
• the subject of the invention is a cellular microcompartment in three dimensions (3D) successively comprising, organized around at least one lumen:
• At least one inner layer of human cells in the process of cell differentiation into cardiac cells expressing at least one gene chosen from PDGFRa, MESP-1, NKX2-5, GATA4, MEF2C, TBX20, ISL1 and TBX5, said inner layer having a variable thickness;
• the inner layer of human cells and the lumen(s) together form a three-dimensional cellular object. If we measure the smallest and the largest thickness of the inner layer of cells along a segment passing through the geometric center of this cellular object, the ratio between the largest thickness and the smallest thickness is greater than or equal to 2.
• the inner layer thicknesses are measured along the segment passing through the geometric center of the cellular object:
• the invention therefore specifically relates to a cellular microcompartment successively comprising, organized around at least one lumen:
• At least one inner layer of human cells in the process of cell differentiation into cardiac cells expressing at least one gene chosen from PDGFRa, MESP-1, NKX2-5, GATA4, MEF2C, TBX20, ISL1 and TBX5, said inner layer having a variable thickness, the ratio between the largest thickness and the smallest thickness of the internal layer being greater than or equal to 2, the smallest thickness and the largest thickness of the internal layer being the smallest and the largest of the thicknesses of inner layer measured along a segment passing through the geometric center of the cellular object formed by the inner layer and the lumen(s), between the interface of the inner layer and the intermediate layer and the interface of the inner layer and a lumen, and/or between the interface of the inner layer and a lumen and the interface of the inner layer and another lumen,
• the microcompartment according to the invention therefore comprises cells undergoing cell differentiation, the expression of the PDGFRa/MESP1/NKX2-5/GATA4/MEF2C/TBX20/ISL1/TBX5 genes being associated with intermediate stages of cardiac differentiation.
• Such a configuration one or more lumens around which are successively organized a layer of human cells undergoing cardiac differentiation with specific thickness characteristics, a layer of isotonic aqueous solution and at least one hydrogel layer) is new.
• an outer layer of hydrogel and an intermediate layer of isotonic aqueous solution allows a uniform distribution of cells between the microcompartments.
• the homogeneity between the microcompartments is greatly improved by the prior encapsulation of the hPSCs allowing increased yield and quality compared to existing methods.
• this layer of hydrogel makes it possible to avoid fusions of microcompartments which are a major source of unfavorable variability for the phenotypic homogeneity and the survival of the cardiac cells produced in the bioreactor.
• modulation of the WNT pathway used in cardiac differentiation is associated with p-catenin degradation (Lam, ATL et al. Conjoint propagation and differentiation of human embryonic stem cells to cardiomyocytes in a defined microcarrier spinner culture. Stem Cell Res. Ther. 5, 1-15 (2014)), a molecule that plays a role in cell-cell adhesion complexes (Brembeck, FH, Rosârio, M. & Birchmeier, W. Balancing cell adhesion and Wnt signaling , the key role of p-catenin. Curr. Opin. Genet. Dev. 16, 51-59 (2006)).
• the topology of the microcompartment according to the invention makes it possible to protect the cells undergoing cardiac differentiation, despite the fragility of the cell-cell adhesion induced by the modulation of the WNT pathway.
• the microcompartments according to the invention can be used to obtain compacted tissues of specific differentiated human cardiac cells.
• a subject of the invention is therefore also the use of a cell microcompartment according to the invention, to obtain a tissue of cardiac cells expressing cardiac troponin C and preferably also alpha-actinin.
• the invention therefore also relates to compacted cardiac tissues.
• the subject of the invention is a compacted tissue of cardiac human cells expressing cardiac troponin C (that is to say human cells expressing the gene for cardiac troponin C, the alias of the corresponding gene being TNNC1), obtained from from at least one cellular microcompartment as previously described, by a process comprising the compaction of the internal layer of human cells by total or partial disappearance of the light(s).
• the compacted tissue of human cardiac cells according to the invention has a rate of cells expressing cardiac troponin C of at least 50% in number relative to the total number of cells constituting the compacted tissue, even more preferably of at least 60 %, at least 70%, at least 75%, at least 80%, and this rate may be greater than 90%.
• the compacted tissue of human cardiac cells according to the invention has a level of cells expressing alpha-actinin of at least 50% in number relative to the total number of cells constituting the compacted tissue, even more preferably of at least least least 60%, at least 70%, at least 75%, at least 80%, and this rate may be greater than 90%.
• the compacted tissue of human cardiac cells according to the invention has a rate of cells expressing troponin C and alpha-actinin of at least 50% in number relative to the total number of cells constituting the compacted tissue, even more preferably at least 60%, at least 70%, at least 75%, at least 80%, and this rate may be greater than 90%.
• the cardiac tissues according to the invention obtained by a specific process different from those of the prior art, makes it possible to obtain cardiac tissues with a rate of cells expressing cardiac troponin C and/or alpha-actinin of at least 50%, and preferably at least 75%.
• the configuration according to the invention during differentiation allows the transmission of auto/paracrine signals within a protected lumen which allows the cells to self-organize in a biomimetic way of the in vivo structuring.
• This structuring is extremely fragile and requires both mechanical protection and available space, contrary to what is described in Koivisto Janne T. et al. According to the invention, this configuration cannot be implemented either in a confined system or in an unprotected system.
• the invention proposes a controlled structuring of the environment in the form of a protected self-organization, which allows less sensitivity to small variations in the culture system and therefore greater reproducibility.
• the heart tissues according to the invention can be used to regenerate ischemic heart tissue. Also, the invention relates to said tissues for their use in the prevention and/or treatment of pathologies, in particular cardiac pathologies.
• FIG. la is a schematic representation of a sectional view of a cell microcompartment 10 according to the invention, corresponding to the photo represented in figure lb, with an outer layer of hydrogel 12, a layer of isotonic aqueous solution 14, a layer of human cells undergoing cardiac differentiation 16 with greater thickness t2 and a smaller thickness tl, and an internal lumen 18.
• Figure lb is a phase contrast microscopy image of a microcompartment according to the invention taken at 4x magnification which corresponds to the schematic diagram of Figure la.
• FIG. 2a is a schematic representation of a sectional view of a cell microcompartment 10 according to the invention, corresponding to the photo shown in Figure 2b, with an outer layer of hydrogel 12, a layer of isotonic aqueous solution 14, a layer of human cells undergoing cardiac differentiation 16 with a greater thickness t2 and a lesser thickness t1, and two internal lumens 18-1 and 18-2, SI representing the thickness of the layer of aqueous solution isotonic 14.
• Figure 2b is a phase contrast microscopy image of a microcompartment according to the invention taken at 4x magnification which corresponds to the schematic diagram of Figure 2a.
• FIG. 2c is a phase contrast microscopy image of several microcompartments according to the invention, taken at 4x magnification, each microcompartment with different morphologies.
• FIG. 3a is a schematic representation of a sectional view of a cell microcompartment 10 according to the invention, corresponding to the photograph shown in Figure 3b, with an outer layer of hydrogel 12, a layer of isotonic aqueous solution 14, a layer of human cells undergoing cardiac differentiation 16 with a greater thickness t2 and a lesser thickness t1, and two internal lumens 18-1 and 18-1, si representing the thickness of the layer of aqueous solution isotonic 14.
• Figure 3b is a phase contrast microscopy image of a microcompartment according to the invention taken at 4x magnification which corresponds to the schematic diagram of Figure 3a.
• Figure 4a is a schematic representation of a sectional view of a compacted tissue according to the invention, corresponding to the photo shown in Figure 4b, with an outer layer of hydrogel 12, a layer of isotonic aqueous solution 14, a compacted tissue of differentiated cardiac cells 20.
• Figure 4b is a microscopy image at phase contrast of a tissue compacted according to the invention in a microcompartment, taken at a 4x magnification which corresponds to the schematic diagram of Figure 4a.
• FIG. 4c represents phase contrast microscopy images taken at 4x magnification of tissues compacted according to the invention in microcompartments.
• phase contrast microscopy images taken at 4x magnification.
• the image on the left shows the differentiated compacted cardiac tissues in the capsule from encapsulated hiPSCs.
• the image on the right shows the cells obtained by dissociation of cardiac tissues compacted according to the invention.
• the three images in the top row (a, b and c) are images of encapsulated cells.
• the three baseline images (d, e, and f) are images of unencapsulated cells.
• the images in the left column (a and d) represent stem cells induced at the start of differentiation into cardiac cells.
• the images in the middle column (b and e) represent human cells in the process of cellular differentiation into cardiac cells, 3 to 7 days after the initiation of differentiation.
• the images in the right column (c and f) represent differentiated cardiac tissues.
• FIG 8 is a graph which represents the percentage of cells in the tissues (obtained as in Figure 7c and 7f) expressing cardiac troponin C: on the left encapsulated tissues according to the invention (image 7c), on the right tissues not encapsulated (image 7f).
• FIG. 9 is a graph which represents the rate of cellular amplification between the start of differentiation (obtained as in Figure 7a and 7d) in the tissues: on the left according to the invention encapsulated, on the right not encapsulated.
• alginate within the meaning of the invention is meant linear polysaccharides formed from ⁇ -D-mannuronate and ⁇ -L-guluronate, salts and derivatives thereof.
• hydrogel capsule within the meaning of the invention, is meant a three-dimensional structure formed from a matrix of polymer chains, swollen with a liquid and preferably water.
• cell “expressing a gene” within the meaning of the invention is meant a cell which contains at least 5 times more copies of the RNA transcribed from the DNA sequence of the gene concerned in comparison with a pluripotent cell , preferentially 10 times more copies, preferentially 20 times more copies, preferentially 100 times more copies.
• human cells within the meaning of the invention is meant human cells or immunologically humanized non-human mammalian cells. Even when this is not specified, the cells, stem cells, progenitor cells and tissues according to the invention consist of or are obtained from human cells or from immunologically humanized non-human mammalian cells.
• progenitor cell within the meaning of the invention, is meant a stem cell already committed in cell differentiation into cardiac but not yet differentiated cells.
• embryonic stem cell within the meaning of the invention is meant a pluripotent stem cell of a cell derived from the internal cell mass of the blastocyst.
• the pluripotency of embryonic stem cells can be assessed by the presence of markers such as the transcription factors OCT4, NANOG and SOX2 and surface markers such as SSEA3/4, Tra-1-60 and Tra-1-81.
• the embryonic stem cells used in the context of the invention are obtained without destroying the embryo from which they originate, for example using the technique described in Chang et al. (Cell Stem Cell, 2008, 2(2)): 113-117).
• human embryonic stem cells can be excluded.
• pluripotent stem cell or “pluripotent cell” within the meaning of the invention, is meant a cell which has the capacity to form all the tissues present in the entire organism of origin, without however being able to form an entire organism by as such.
• Human pluripotent stem cells may be referred to herein as hPSCs. They may in particular be induced pluripotent stem cells (iPSC or hiPSC for human induced pluripotent stem cells), embryonic stem cells or MUSE cells (for “Multilineage-differentiating Stress Enduring”).
• induced pluripotent stem cell within the meaning of the invention is meant a pluripotent stem cell induced to pluripotency by genetic reprogramming of differentiated somatic cells. These cells are notably positive for pluripotency markers, such as alkaline phosphatase staining and expression of NANOG, SOX2, OCT4 and SSEA3/4 proteins. Examples of methods for obtaining induced pluripotent stem cells are described in the articles Yu et al. (Science 2007, 318 (5858): 1917-1920), Takahashi et al (Cell, 207, 131(5): 861-872) and Nakagawa et al (Nat Biotechnol, 2008, 26(1): 101-106) .
• differentiated cardiac cells within the meaning of the invention is meant cells which exhibit the phenotype of a cardiomyocyte, that is to say expressing specific markers such as TNNC1 (cardiac troponin C gene) and ACTN2 ( alpha actinin gene) and capable of contracting spontaneously in response to an intracellular calcium signal spontaneously (in the case of immature cardiac cells) or following an electrical or chemical stimulation capable of triggering said calcium signal.
• TNNC1 cardiac troponin C gene
• ACTN2 alpha actinin gene
• Fiber diameter of a compacted cardiac tissue according to the invention or of a microcompartment according to the invention is meant the distance “d” comprised between two tangents to said compacted tissue or to said microcompartment, these two tangents being parallel, such that the entire projection of said compacted tissue or of said microcompartment lies between these two parallel tangents.
• variable thickness of the inner layer of human cells in the process of cell differentiation is meant within the meaning of the invention the fact that the inner layer in the same microcompartment does not have the same thickness everywhere.
• implantation or “graft” in the heart within the meaning of the invention, is meant the action of depositing at the level of the heart at a particular location at least one compacted tissue according to the invention.
• the implantation can be carried out by any means, in particular by injection.
• microcompartment or “capsule” within the meaning of the invention, is meant a partially or completely closed three-dimensional structure, containing at least one cell.
• ⁇ culture medium within the meaning of the invention is meant a culture medium animated by internal movements.
• largest dimension of a compacted cardiac tissue according to the invention or of a microcompartment according to the invention within the meaning of the invention is meant the value of the largest Feret diameter of said compacted tissue or of said microcompartment.
• smallest dimension of a compacted cardiac tissue according to the invention or of a microcompartment according to the invention within the meaning of the invention is meant the value of the smallest Feret diameter of said compacted tissue or of said microcompartment.
• tissue or “biological tissue” within the meaning of the invention, is meant the common sense of tissue in biology, that is to say the intermediate level of organization between the cell and the organ.
• a tissue is a set of similar cells of the same origin (usually derived from a common cell lineage, although they may find their origin by association of distinct cell lineages)., grouped into clusters, networks or bundles (fiber ).
• a fabric forms a functional whole, that is to say that its cells contribute to the same function.
• Biological tissues regenerate regularly and are assembled together to form organs.
• tissue or “compacted cardiac tissue” or “compacted tissue of cardiac cells” within the meaning of the invention, is meant a unit of tissue comprising at least one cardiac tissue consisting at least of differentiated cardiac cells.
• the tissue is at least partially compacted, that is to say it is composed mainly of cells, in particular its volume is composed of more than 50% cells, preferentially 75% cells, preferentially 90% cells.
• the tissue may be completely compacted, ie the lights are no longer detectable and/or there is no light.
• the tissues compacted according to the invention can be called microtissues.
• light or “lumen” within the meaning of the invention, is meant a volume of aqueous solution topologically surrounded by cells. Preferably, its content is not in diffusive equilibrium with the volume of convective liquid present outside the microcompartment. cellular
• the invention therefore relates to a cellular microcompartment successively comprising, organized around at least one lumen:
• At least one inner layer of human cells in the process of cell differentiation into cardiac cells expressing at least one gene chosen from PDGFRa, MESP-1, NKX2-5, GATA4, MEF2C, TBX20, ISL1 and TBX5 (known as the "inner layer” ),
• intermediate layer at least one intermediate layer of isotonic aqueous solution
• outer layer at least one outer layer of hydrogel (called “outer layer”.
• the microcompartment according to the invention is a three-dimensional structure therefore comprising at least one internal layer of cells. These cells are living human cells undergoing cell differentiation into cardiac cells. This layer of cells is organized in three dimensions in the microcompartment.
• the human cells undergoing cardiac differentiation present in the microcompartment are cells expressing at least one gene chosen from PDGFRa, MESP-1, NKX2-5, GATA4, MEF2C, TBX20, ISL1 and TBX5. These genes are specific to cardiac cells in the process of differentiation.
• the human cells in the process of cardiac differentiation present in the microcompartment express at least two of these genes.
• human differentiating cardiac cells present in the microcompartment express all of these genes.
• the inner layer of human cells in the process of cellular differentiation into cardiac cells has a variable thickness.
• the inner layer of human cells and the lumen(s) together form a three-dimensional cellular object. If one measures the smallest and the largest thickness of the inner layer of cells along a segment passing through the geometric center of this cellular object (shown 22 in Figures 1a, 2a and 3a), the ratio between the largest greatest thickness and the smallest thickness is greater than or equal to 2, preferably greater than or equal to 5.
• the inner layer thicknesses are measured along the segment passing through the geometric center of the cellular object:
• Figures 1, 2 and 3 represent examples of cellular microcompartments 10 according to the invention, with an outer layer of hydrogel 12, a layer of isotonic aqueous solution 14, one or more internal lumen(s) 18, 18 -1, 18-2, a layer of human cells in the process of cardiac differentiation 16 with a greater thickness t2 and a lesser thickness t1 (the thicknesses being measured along a segment 22 passing through the geometric center of this object cell formed by the layer 16 and the lumen(s) 18, 18-1, 18-2), the ratio t2/tl being well above 2.
• the number of human cells in the process of cell differentiation into cardiac cells of the inner layer is preferably between 1 and 100,000 cells, even more preferably between 50 and 50,000 cells, and in particular between 500 and 25,000 cells.
• Human cells in the process of cell differentiation into cardiac cells of the inner layer have preferentially been obtained from pluripotent stem cells, in particular from human pluripotent stem cells, or possibly from non-pluripotent human cells whose transcriptional profile has been modified. artificially to join that of cardiac progenitors or cardiac cells, typically by forced expression of transcription factors specific for the target cell phenotype.
• the human cells of the inner layer have been obtained from human pluripotent stem cells after contacting with a solution capable of initiating the differentiation of said stem cells.
• the intermediate layer of isotonic aqueous solution preferably contains peptide or peptidomimetic sequences capable of binding to integrins.
• isotonic aqueous solution is meant an aqueous solution having an osmolarity of between 200 and 400 mOsm/L. This layer is located between the inner cell layer and the outer hydrogel layer.
• the intermediate layer may consist of elements which have been added during the manufacture of the microcompartment and/or of elements added in the microcompartment and/or of elements secreted or induced by the other constituents of the microcompartment.
• the intermediate layer may in particular comprise or consist of an extracellular matrix and/or a culture medium. If it comprises extracellular matrix, it may be extracellular matrix secreted by cells of the inner layer and/or by extracellular matrix added at the time of preparation/manufacture of the microcompartment.
• the intermediate layer preferably comprises a mixture of proteins and extracellular compounds necessary for the culture of cells undergoing cardiac differentiation.
• the intermediate layer comprises structural proteins, such as collagen, laminins, entactin, vitronectin, as well as growth factors, such as TGF-beta and/or EGF.
• the intermediate layer can consist of or comprise Matrigel® and/or Geltrex® and/or a hydrogel type matrix of vegetable origin such as modified alginates or of synthetic origin or of poly(N- isopropylacrylamide) and poly(ethylene glycol) (PNIPAAm-PEG) of the Mebiol® type.
• the intermediate layer can form a gel.
• the intermediate layer may optionally contain one or more cells.
• the thickness of the intermediate layer (represented if in FIGS. 1 and 2) is preferably between 30 nm and 300 ⁇ m, even more preferably between 30 nm and 50 ⁇ m.
• the presence of the intermediate layer promotes the structuring according to the invention of the elements in the microcompartment.
• the microcompartment and the inner layer of cells within the microcompartment according to the invention are hollow.
• the microcompartment according to the invention indeed always comprises at least one internal light or lumen which constitutes the hollow part of the microcompartment.
• the lumen contains a liquid, in particular a culture medium (such as for example a RPMI basal medium with a B27 supplement) and/or a fluid secreted by the cells of the inner layer.
• a culture medium such as for example a RPMI basal medium with a B27 supplement
• a fluid secreted by the cells of the inner layer a fluid secreted by the cells of the inner layer.
• the presence of this hollow part allows the cells to have a small diffusive volume whose composition they can control, promoting so-called autocrine/paracrine cellular communication which is in turn favorable to cardiac differentiation.
• the microcompartment according to the invention can comprise several lights, at least two lights.
• This situation has the same advantage vis-à-vis autocrine and paracrine signals as the presence of a single lumen and increases the cells' ability to control the composition of the aqueous solution of the lumen because the cell to volume/cell ratio is then geometrically weaker.
• the stabilization of such a configuration demonstrates the mechanical protection offered by the microcompartment.
• the slot(s) preferably represent(s) between 10% and 90% of the volume of the microcompartment according to the invention.
• the microcompartment includes an outer hydrogel layer.
• the hydrogel used is biocompatible, that is to say it is not toxic to the cells.
• the hydrogel layer must allow the diffusion of oxygen and nutrients to supply the cells contained in the microcompartment and allow their survival.
• the outer layer of hydrogel comprises at least alginate. It may consist exclusively of alginate.
• the alginate may in particular be a sodium alginate, composed of 80% a-L-guluronate and 20%
• the hydrogel layer makes it possible to protect the cells from the external environment, to limit the uncontrolled proliferation of the cells, and their controlled differentiation into cells in the process of cardiac differentiation then into cardiac cells, at least into cardiomyocytes.
• the outer layer is closed or partially closed.
• the microcompartment is therefore closed or partially closed.
• the microcompartment is closed.
• the microcompartment according to the invention can be in any three-dimensional form, that is to say it can have the shape of any object in space.
• the microcompartment according to the invention is in a spherical or elongated shape. He can in particular be in the form of a hollow spheroid, a hollow ovoid, a hollow cylinder or a hollow sphere.
• the external layer of the microcompartment that is to say the hydrogel layer, which gives its size and its shape to the microcompartment according to the invention.
• the diameter of the smallest dimension of the microcompartment according to the invention is between 10 ⁇ m and
• mm preferably between 100 ⁇ m and 700 ⁇ m. It may be between 10 ⁇ m and 600 ⁇ m, in particular between 10 ⁇ m and 500 ⁇ m.
• This smaller dimension is important for the survival of the three-dimensional cardiac tissue which will be obtained from the microcompartment according to the invention, in particular to promote the survival of the cardiac cells within the cardiac tissue and to optimize the reorganization as well as the vascularization of the cardiac tissue after implantation in the heart.
• Its largest dimension is preferably greater than 10 ⁇ m, more preferably between 10 ⁇ m and 1 m, even more preferably between 10 ⁇ m and 50 cm. According to one embodiment, the largest dimension is compatible with the size of the organ and is therefore less than 30 cm (between 10 ⁇ m and 30 cm).
• the microcompartment according to the invention is particularly useful for obtaining a three-dimensional compacted cardiac tissue, consisting of differentiated human cardiac cells.
• microcompartment according to the invention can optionally be frozen in order to be stored.
• the invention also relates to several microcompartments together. Also the invention also relates to a series of cellular microcompartments as described previously comprising at least two cellular microcompartments according to the invention.
• the series of microcompartments according to the invention is in a culture medium, in particular in an at least partially convective culture medium.
• the subject of the invention is a series of cellular microcompartments as described above in a closed enclosure, such as a bioreactor, preferably in a culture medium in a closed enclosure, such as a bioreactor .
• the invention also relates to a process for preparing a microcompartment according to the invention.
• the method consists in producing cellular microcompartments comprising a hydrogel capsule surrounding:
• - differentiated cells intended to undergo in the capsule a reprogramming in the capsule so that they become induced pluripotent stem cells capable of differentiating into cardiac cells, at least into cardiomyocytes.
• the process for preparing a microcompartment according to the invention may comprise at least the implementation of the steps which consist of:
• the total or partial encapsulation in the hydrogel and the contribution of extracellular matrix combined is a means adapted to allow the differentiation of human pluripotent cells towards the cardiac muscle by accumulating several advantages, in particular: favoring a homogeneous distribution of the cells of the lot within the microcompartments, ii) mechanical protection against hydrodynamic stress inflicted by the bioreactor and limitation of unwanted fusions of microcompartments, iii) organization of a microenvironment locally conserving the extracellular matrix elements promoting good cell survival and organization, iv) maintenance a lumen promoting autocrine and paracrine pathways during differentiation.
• any process for the production of cellular microcompartments containing inside a hydrogel capsule at least human cells in the process of cardiac differentiation and an isotonic aqueous solution and possibly the addition of other cells, for example of support can be used.
• a suitable method is in particular described in application WO2018/096277.
• the encapsulation is carried out by co-injection of three solutions:
• an isotonic intermediate solution such as for example a sorbitol solution
• a solution comprising the cells to be encapsulated, culture medium and optionally but preferably extracellular matrix, concentrically via a microfluidic injector which makes it possible to form a jet at the injector outlet consisting of the mixture of the three solutions, said jet fractionating into drops, said drops being collected in a calcium bath which stiffens the hydrogel solution to form the outer layer of each microcompartment, the inner part of each drop being constituted by the solution comprising the encapsulated cells, culture medium and the extracellular matrix.
• the encapsulation is carried out with a device capable of generating hydrogel capsules using a microfluidic chip.
• the device can comprise syringe pumps for several solutions injected concentrically using a microfluidic injector which makes it possible to form a jet which splits into drops then collected in a calcium bath.
• three solutions are loaded onto three syringe pumps: - a hydrogel solution, for example alginate,
• an isotonic intermediate solution such as for example a sorbitol solution
• step b) the solution resulting from step b) comprising iPSCs, culture medium and optionally but preferably extracellular matrix.
• the three solutions are co-injected (injected simultaneously) in a concentric manner thanks to a microfluidic injector or microfluidic chip which makes it possible to form a jet which splits into drops whose outer layer is the hydrogel solution and the heart of the solution comprising the cells to be encapsulated; These drops are collected in a calcium bath which stiffens the alginate solution to form the shell.
• the hydrogel solution is charged with a direct current.
• a grounded ring is placed after the tip in the plane perpendicular to the axis of the jet emerging from the microfluidic injector (coextrusion chip) to generate the electric field.
• the step of producing a cellular microcompartment of the preparation process according to the invention comprises the steps consisting in:
• a culture medium preferably a culture medium containing the growth factors FGF2 and TGF or molecules reproducing its action on the cell, an inhibitor of the Rho kinase pathway or a molecule reproducing its action on the cell, in particular by limiting cell death.
• pluripotent stem cells optionally mixing the pluripotent stem cells with an isotonic aqueous solution, preferably an extracellular matrix,
• the encapsulated cells for the preparation of microcompartments according to the invention are preferably chosen from:
• stem cells capable of differentiating into cardiac cells, at least into cardiomyocytes preferentially embryonic stem cells or induced pluripotent stem cells, very preferentially induced pluripotent stem cells, and/or, * either progenitor cells capable of differentiating into cardiac cells, at least into cardiomyocytes,
• - and/or differentiated cells capable of undergoing reprogramming so that they become induced pluripotent stem cells capable of differentiating into cardiac cells, at least into cardiomyocytes.
• the encapsulated cells can be immuno-compatible with the person intended to receive the differentiated cardiac cells obtained from the microcompartment according to the invention, to avoid any risk of rejection.
• the encapsulated cells have been taken beforehand from the person in whom the compacted cardiac tissues obtained from the microcompartments according to the invention will be implanted.
• the differentiation into cells undergoing cardiac differentiation contained in the microcompartment according to the invention can be carried out by any suitable method. This may include a method known as one of the protocols listed in (Dunn, KK & Palecek, SP Engineering Scalable Manufacturing of High-Quality Stem Cell-Derived Cardiomyocytes for Cardiac Tissue Repair. Front. Med. 5, (2018)).
• the step of inducing cell differentiation of the method according to the invention comprises a step consisting in introducing capsules containing human stem cells capable of being differentiated into human cardiac cells, in a medium of culture containing a WNT pathway activator (such as CHIR99021) for 12 to 72 hours, more preferably 12 to 48 hours.
• a WNT pathway activator such as CHIR99021
• the method may comprise a step which consists in incubating the microcompartments in a culture medium containing an inhibitor of the WNT pathway.
• this step is carried out between 0 and three days after the end of the differentiation induction step (addition of the activator of the WNT pathway), preferably between 12 and 72 hours, in particular between 24 and 48 hours.
• this step consists in incubating the capsules in a culture medium containing an inhibitor of the WNT pathway, preferably for 12 hours to 48 hours, in particular between 24 to 48 hours.
• step (b) 0 to 48 hours after step (a) incubating the capsules in a culture medium containing a WNT pathway inhibitor for 12 hours to 48 hours;
• the culture medium is RPMI with B27 supplement without insulin (during the first 7 days of differentiation) and with insulin (from day 7 of differentiation).
• the microcompartments according to the invention containing cells in the process of cardiac differentiation, are obtained between 2 and 7 days after the start of the induction of differentiation, preferably between 3 and 7 days after the start of the induction of differentiation, even more preferably between 4 and 6 days after the start of differentiation.
• the microcompartment according to the invention appears at the time of addition of the WNT pathway inhibitor or after.
• the light is generated at the time of the formation of the layer of human cells in the process of cardiac differentiation in 3 dimensions, by the cells which multiply and develop.
• the light may contain a liquid and in particular the culture medium used for implementing the method.
• the starting stem cells organize themselves into a layer of stem cells in three dimensions around a light in the microcompartment, then during the differentiation this light disappears, and a second light appears to form the microcompartment according to the invention.
• the method is preferably implemented in a closed enclosure, such as a bioreactor, with a series of microcompartments, even more preferably in a suitable and at least partially convective culture medium.
• the method according to the invention may optionally also comprise: - a step which consists in dissociating the microcompartment or the series of microcompartments to obtain a suspension of cells or a suspension of clusters of cells; elimination of the capsule can be carried out in particular by hydrolysis, dissolution, piercing and/or rupture by any means that is biocompatible, that is to say non-toxic for the cells.
• removal can be achieved using phosphate buffered saline, a divalent ion chelator, an enzyme such as alginate lyase if the hydrogel includes alginate, and/or laser microdissection, and
• Reencapsulation is a suitable means for: i) optimizing the standardization of the size and the homogeneity of the compacted cardiac tissues which will then be obtained, ii) ii) allowing an increase in the cellular amplification obtained from the pluripotent stage, and therefore higher yield.
• the method according to the invention may comprise a step consisting in verifying the phenotype of the cells contained in the microcompartment. This verification can be carried out by identifying the expression by at least part of the cells contained in the microcompartment, of at least one of the following genes PDGFRa, MESP-1, NKX2-5, GATA4, MEF2C, TBX20, ISL1 and TBX5.
• the method according to the invention may comprise a step of freezing the microcompartments according to the invention before their use to pursue differentiation into differentiated cardiac cells and to obtain compacted cardiac tissues.
• the freezing is preferably carried out at a temperature between -190°C and -80°C.
• Thawing can be done in a lukewarm water bath (preferably 37 degrees) so that the cells thaw fairly quickly.
• the microcompartments according to the invention before their use to pursue a differentiation into differentiated cardiac cells and to obtain compacted cardiac tissues can be maintained at more than 4° C. for a limited period of time before their use, preferably between 4° C. and 38° C. .
• microcompartments according to the invention can also be used to pursue differentiation into differentiated cardiac cells and to obtain compacted cardiac tissues, directly after implementation of the method according to the invention, without storage and without freezing.
• the method can continue to obtain a three-dimensional object in the form of a compacted tissue.
• the compacted object generally appears between 2 and 10 days after the addition of the WNT pathway inhibitor, especially between 5 and 7 days. Indeed, the addition of the inhibitor is preliminary to the compaction of the cells which continue to differentiate into cardiac cells.
• the compacted object generally appears between 7 and 14 days after the initiation of differentiation.
• the cells comprise at least in part differentiated human cardiac cells, preferably at least cardiomyocytes.
• the method according to the invention may comprise a step of amplifying the cardiac cells in the microcompartment, and optionally one or more re-encapsulation.
• the resulting compacted heart tissue can be held in the hydrogel capsule. Preferably, it is always surrounded by an isotonic aqueous solution, preferably an extracellular matrix.
• an isotonic aqueous solution preferably an extracellular matrix.
• the compacted tissue is preferably stored in a capsule before use.
• the capsule containing the compacted cardiac tissue can be frozen before removing the hydrogel layer from the capsule.
• the method according to the invention may comprise a step of freezing the capsules containing the compacted cardiac tissues according to the invention before their use. The freezing is preferably carried out at a temperature between -190°C and -80°C.
• the capsules containing the compacted cardiac tissues according to the invention before their use as a graft in the heart can be thawed in a lukewarm water bath (preferably 37 degrees) so that the tissue cells thaw fairly quickly.
• the compacted cardiac tissues according to the invention can be maintained at more than 4° C. for a limited period of time before their use, preferably between 4° C. and 38° C.
• the method according to the invention may comprise a step consisting in verifying the phenotype of the cells contained in the capsule. This verification can be carried out by identifying the expression of cardiac troponin C by the cardiac cells forming the compacted tissue.
• the hydrogel layer of the capsule containing the compacted cardiac tissue according to the invention is removed.
• the elimination of the capsule can be carried out in particular by hydrolysis, dissolution, piercing and/or rupture by any means that is biocompatible, that is to say non-toxic for the cells.
• removal can be achieved using phosphate buffered saline, a divalent ion chelator, an enzyme such as alginate lyase if the hydrogel includes alginate, and/or laser microdissection.
• the compacted cardiac tissue according to the invention is devoid of hydrogel when it is used as a graft, implanted in a heart.
• a subject of the invention is also the cardiac tissue obtained according to the method as described previously.
• a subject of the invention is therefore a compacted tissue of human cardiac cells expressing cardiac troponin C and preferentially alpha-actinin, obtained from at least one cell microcompartment according to the invention.
• the subject of the invention is a compacted tissue of cardiac human cells expressing cardiac troponin C, obtained from at least one cell microcompartment according to the invention, by a process comprising the compaction (compaction called secondary compaction) of the layer of human cells by total or partial disappearance of the light(s) of said microcompartment.
• the compacted tissue of human cardiac cells is obtained by a method as described above.
• the tissue according to the invention is therefore a human tissue comprising at least cells differentiated cardiac cells expressing cardiac troponin C and preferentially alpha-actinin.
• the compacted tissue according to the invention can also contain other cell types.
• the cardiac tissue according to the invention has a rate of cells expressing cardiac troponin C of at least 50% in number relative to the total number of cells constituting the compacted tissue, even more preferably of at least 60%, at least 70%, at least 75%, at least 80%, at least 90%.
• This high rate of cells expressing cardiac troponin C is advantageous for considering uses of the cardiac tissues according to the invention in cell therapy.
• the compacted tissue of human cardiac cells according to the invention has a level of cells expressing alpha-actinin of at least 50% in number relative to the total number of cells constituting the compacted tissue, even more preferably of at least least 60%, at least 70%, at least 75%, at least 80%, and this rate may be greater than 90%.
• This high level of cells expressing alpha-actinin is advantageous for envisaging uses of the cardiac tissues according to the invention in cell therapy.
• the compacted tissue of human cardiac cells according to the invention has a rate of cells expressing troponin C and alpha-actinin of at least 50% in number relative to the total number of cells constituting the compacted tissue, even more preferably at least 60%, at least 70%, at least 75%, at least 80%, and this rate may be greater than 90%.
• This high level of cells expressing both troponin C and alpha-actinin is advantageous for envisaging uses of the cardiac tissues according to the invention in cell therapy.
• this compacted tissue is contractile and has a spontaneous contraction frequency of less than 4 Hz, preferably less than 2 Hz, even more preferably less than 1.7 Hz, in particular less than 1 Hz, and in particular less than 0.5 Hz and possibly be less than 0.25 Hz.
• This tissue contraction frequency is low, which is a great advantage for its implantation in the heart. Indeed such a frequency makes it possible to avoid an arrhythmia at the time of the grafting of the compacted tissue according to the invention in the heart to be treated.
• the average human adult heart rate is between 60 and 100 beats per minute (1 to 1.7 Hz).
• the low frequency of contraction of the compacted cardiac tissues according to the invention reduces the risk of arrhythmia during a transplant of the tissues or cells obtained from these tissues. According to one embodiment, with a spontaneous beat frequency of the tissue according to the invention lower than the heart rate of the patient (recipient), this risk of arrhythmia is further reduced.
• Reduced spontaneous beat frequency is associated with the maturation of human stem cell-derived cardiomyocytes, with the 3D culture environment enhancing cardiomyocyte maturation.
• the encapsulation further reduces the contractile frequency of the compacted cardiac tissue.
• Figure 5 shows that for a given starting cell population, cardiomyocytes differentiated within a microcompartment/capsule (from encapsulated human pluripotent stem cells) have a slower spontaneous beat rate than cardiomyocytes differentiated with the same protocol. (and from the same initial batch of human pluripotent stem cells) but in free suspension culture. Thus, differentiation into cardiomyocytes from encapsulated stem cells decreases the spontaneous contractile rate.
• Human pluripotent stem cells secrete signaling molecules during the process of cardiac differentiation, which generate a specific paracrine microenvironment necessary for successful differentiation (Kempf, H. et al. Bulk cell density and Wnt/TGFbeta signaling regulate mesendodermal patterning of human pluripotent stem cells Nature Communications 7, (2016)).
• the presence of the capsule helps to increase and maintain a local concentration of these paracrine factors, which improves the differentiation phenotype, resulting in the reduction of spontaneous beat frequency.
• the compacted cardiac tissue according to the invention can remain contractile spontaneously for several months. Thus the product is stable over time.
• cardiac differentiation within the microcompartment may be implemented and/or combined with other techniques, such as electrical stimulation and metabolic or hormonal interventions. Combination with such techniques can further reduce the frequency of spontaneous tissue beats. heart compacted according to the invention before the transplant.
• the compacted tissue of human cardiac cells according to the invention can be totally or partially encapsulated in an outer layer of hydrogel.
• the hydrogel capsule may be the original microcompartment of human cells undergoing cardiac differentiation, or it may be a new hydrogel layer if the initial hydrogel layer has been removed , then re-encapsulation at any stage of the process.
• the encapsulation of the compacted tissue of human cardiac cells according to the invention makes it possible to protect the tissue, to maintain the frequency of spontaneous contraction below 4 Hz, preferentially below 2 Hz, even more preferentially below 1 Hz, and in particular below 0 .5Hz. It may be less than 0.25 Hz.
• the mechanism by which the contraction frequency is limited may be linked to the 3D structuring via i) the electrical continuity of the cytoplasms of cardiac cells, ii) and/or the limitation of the quantity of calcium available per cell in the intercellular space of the compacted tissues iii) and/or the mechanical resistance linked to the mechanical continuity of the cytoskeletal elements of the cardiac cells.
• the encapsulation of the cardiac compacted tissue according to the invention also makes it possible to control the size of the compacted tissue which improves the retention, the integration and the cell survival when it is injected into the heart, in particular in comparison with the injections of single cells , which increases the efficiency of cardiac cell therapy with the compacted tissues according to the invention.
• the compacted tissue of human cardiac cells according to the invention is not encapsulated in an outer hydrogel layer.
• the capsule is preferentially removed before use in order to allow the cells of the compacted tissue to implant themselves at the level of the heart after a transplant.
• the compacted tissue of human cardiac cells according to the invention is preferably completely or partially surrounded by a layer of isotonic aqueous solution, such as an extracellular matrix.
• This layer of isotonic aqueous solution is located between the packed tissue of human heart cells and the hydrogel layer when the packed heart tissue is encapsulated.
• the compacted cardiac tissue according to the invention is in three dimensions. It preferably has a spherical or elongated shape. According to a preferred embodiment, the compacted tissue of human heart cells has the shape of a spheroid, ovoid, cylinder or sphere.
• FIGS. 4 An example of compacted fabric according to the invention is represented in FIGS. 4.
• the compacted fabric according to the invention is surrounded by a layer of isotonic aqueous solution and an outer layer of hydrogel.
• it has a diameter or a smaller dimension of between 10 ⁇ m and 1 mm, preferably between 100 ⁇ m and 700 ⁇ m.
• This smaller dimension is important for its survival, in particular for promoting the survival of cardiac cells within the cardiac tissue and optimizing the reorganization as well as the vascularization of the cardiac tissue after implantation in the heart.
• Its largest dimension is preferably greater than 10 ⁇ m, more preferably between 10 ⁇ m and 1 m, even more preferably between 10 ⁇ m and 50 cm. According to one embodiment, the largest dimension is compatible with the size of the organ and is therefore less than 30 cm (between 10 ⁇ m and 30 cm).
• the encapsulation of a controlled number of stem cells and/or the re-encapsulation makes it possible to control the desired size and shape of the cardiac tissues obtained.
• the size of the cardiac tissues according to the invention may vary depending on the therapeutic use envisaged.
• the compacted tissue of human cardiac cells according to the invention can be frozen, to promote its storage.
• the invention allows the production of a large number of quality human cardiac tissues by protecting the tissue units throughout their production by differentiation of pluripotent cells into cardiac cells.
• Figures 7, 8 and 9 show that for a given starting cell population, the tissues obtained within a microcompartment / capsule passing through a phase in the process of differentiation with the presence of at least one light, then a compaction secondary, presents a much higher rate of cells expressing troponin C, in comparison with tissues differentiated with the same protocol (and from the same initial batch of human pluripotent stem cells) but in culture in free suspension.
• differentiation into cardiomyocytes in microcompartments and/or by a process comprising secondary compaction makes it possible to increase the quality of cardiac tissues and therefore improves their possibility of use in cell therapy.
• the compacted tissue of cardiac cells according to the invention can be dissociated into cells.
• the dissociation can be carried out according to conventional methods known to those skilled in the art, in particular using an enzymatic solution making it possible to separate the cells.
• the enzymes used can for example be chosen from trypsin, collagenase, accutase and mixtures thereof.
• the dissociated cells are preferably used in suspension or integrated into a gel such as for example a collagen gel or into a patch.
• the compacted tissue of human cardiac cells according to the invention can be used as such or else to produce a suspension of cardiac cells.
• the compacted tissue of human cardiac cells according to the invention is particularly useful for the production of a suspension of cells (graft cells) which can be implanted in the heart of a human being, in particular for the treatment of cardiac pathologies.
• the shape, size and constitution of the compacted tissue according to the invention promote homogeneous differentiation with an improved yield of cardiac cells within the compacted tissue according to the invention, which can be secondarily dissociated before implantation in the heart.
• the tissue compacted with human cardiac cells according to the invention is also particularly useful for its use as such as a graft which can be implanted in the heart of a human being, in particular for the treatment of cardiac pathologies.
• the shape, size and constitution of the compacted tissue according to the invention allow the survival of cardiac cells within the compacted tissue according to the invention, before implantation and the successful implantation, reorganization and vascularization of the graft once implanted in the heart.
• Another object of the invention is therefore the compacted tissue of human cardiac cells for its use, as such or after dissociation in the form of a suspension of cells, in therapy, in particular in cell therapy, as a medicament, in particular its use in the treatment and/or prevention of a cardiac pathology, in particular in a patient in need thereof, and preferentially in the treatment and/or prevention of an ischemic cardiac disease.
• the dissociated cells obtained from the tissues according to the invention can be used, they exhibit a higher spontaneous contraction frequency than the compacted cardiac tissues.
• the slow spontaneous beating rate of differentiated cardiomyocytes within the capsule is not maintained when the cells are dissociated and cultured under 2D conditions ( Figure 6).
• Treatment means preventive, curative or symptomatic treatment, i.e. any act intended to improve a person's sight temporarily or permanently, and preferably also to eradicate the disease and/or stop or delay the disease progression and/or promote disease regression.
• the compacted tissues of human cardiac cells according to the invention can be used for the treatment of heart diseases in humans, in particular diseases having caused ischemia of at least part of the heart, such as a heart attack for example, to replace damaged areas.
• the treatment consists in implanting, grafting the compacted tissues according to the invention or the cells obtained by their dissociation in the heart, at the level of the ventricles of the hearts, in particular the left ventricle, or integrating them into a patch positioned on said ventricles , ideally between the visceral pericardium and the muscular tissue of the ventricle, or what remains of it in a pathological situation.
• a surgical implantation device suitable for implantation in the heart is very preferably used.
• These may be in particular needles, cannulas or other device making it possible to deposit the compacted tissues according to the invention or the cells obtained by dissociation of the compacted tissues according to the invention, in the heart such as for example those used for the implantation of stents in the arteries or surgical micro implants.
• the implantation can be carried out by direct myocardial injection, in particular by sternotomy or with a device based on a catheter: the compacted cardiac tissues according to the invention or the cells obtained from these tissues (with or without the addition of other cell types) are injected into the mid wall of the patient's left ventricle at one or more locations.
• the implantation can be carried out using an epicardial patch.
• the compacted cardiac tissues according to the invention or the cells obtained from these tissues (with or without the addition of other cell types) are used in the formation of patches. These patches can then be placed on the epicardial surface of the patient's left ventricle, either by sternotomy or by a surgical procedure involving an incision and injection of the patch into the chest cavity.
• tissue compacted according to the invention are implanted.
• the implantation of compacted cardiac tissues according to the invention allows patients suffering from heart diseases, and in particular from ischemic heart diseases, to clinically improve cardiac function, in particular:
• the invention makes it possible to improve the overall health and the quality of life of the patient, while limiting the risk of arrhythmias induced by the transplant.
• the compacted tissues according to the invention can be useful as a model of cardiac tissue in particular:
• the invention also relates to these uses.
• the image in Figure lb is a phase contrast microscopy image of a microcompartment according to the invention taken at 4x magnification. It was taken 5 days after the start of differentiation (8 days after the initial stem cell encapsulation). The steps used to obtain the microcompartment shown in this figure are as follows:
• Encapsulated stem cells were cultured in stem cell culture media (mTeSR1) for 3 days.
• the culture medium was changed from stem cell medium to cardiac differentiation medium containing a molecule activating WNT (CHIR99021).
• the medium is RPMI medium supplemented with B27 without insulin with CHIR99021 This is considered differentiation day 0.
• the medium is RPMI medium with B27 supplement without insulin
• the medium was changed to a cardiac differentiation medium containing a molecule which inhibits the WNT pathway (WNT-C59 or IWR1).
• the medium is RPMI medium supplemented with insulin-free B27 with WNT-C59 or IWR1.
• Human induced pluripotent stem cells were encapsulated in an alginate hydrogel (with extracellular matrix added at the time of encapsulation).
• Encapsulated stem cells were cultured in stem cell culture media (mTeSR1) for 6 days.
• the culture medium was changed from stem cell medium to cardiac differentiation medium containing WNT activating molecule (CHIR99021).
• the medium is RPMI medium supplemented with B27 without insulin with CHIR99021 This is considered differentiation day 0.
• the medium was changed to cardiac differentiation medium without WNT activating molecule.
• the medium is RPMI medium with B27 supplement without insulin.
• the medium was changed to a cardiac differentiation medium containing a molecule which inhibits the WNT pathway (WNT-C59 or IWR1).
• the medium is RPMI medium supplemented with insulin-free B27 with WNT-C59 or IWR1.
• the image in Figure 3b is a phase contrast microscopy image of a microcompartment according to the invention taken at 4x magnification. It was taken 5 days after the start of differentiation (11 days after the initial stem cell encapsulation). The steps used to obtain the microcompartment shown in these figures are the same as cells to obtain Figures 2b and 2c. The difference lies in a larger number of encapsulated stem cells. 4
• Figure 4b and 4c are phase contrast microscopy images taken at 4x magnification of tissues compacted according to the invention in microcompartments.
• the compacted tissues were obtained by continuing the differentiation beyond day 5 (already described in figures 1, 2 and 3):
• the images presented in Figures 4 relate to tissues compacted > 14 days after the start of differentiation.
• Figure 5 shows that for a given starting cell population, cardiomyocytes differentiated within the capsule (from encapsulated hiPSC) have a slower spontaneous beat rate than cardiomyocytes differentiated with the same protocol (and from the same initial batch of hiPSC) but in free-suspension culture.
• Beat frequency in Hz was obtained from a series of phase contrast microscopy images (at a rate of at least 30 frames per second) on a standard tabletop microscope with 4x magnification. Images are phase contrast microscopy images taken at 4x magnification showing encapsulated or free-living stem cells at the start of differentiation (outermost), and final compacted tissues approximately 2 weeks after the start of differentiation (outermost). more internal).
• an intermediate step with a microcompartment according to the invention is presented on differentiation day 5.
• FIG. 6 shows that the slow spontaneous beating rate of differentiated cardiomyocytes in the capsule is slightly increased after the capsule is removed, and greatly increased after the cells have been dissociated and placed in 2D culture.
• the compacted heart tissues have a slower beat frequency than that of the isolated cells obtained by dissociation of said tissues.
• Beat frequency in Hz
• the beat frequency for the heart tissues compacted according to the invention encapsulated and then decapsulated was taken approximately 3 weeks after the start of differentiation.
• Figure 7 shows phase contrast microscopy images taken at 4x magnification.
• the three images in the top line (a, b and c) are images of cells encapsulated according to the invention.
• the three baseline images (d, e, and f) are images of unencapsulated cells.
• the images in the left column (a and d) represent stem cells induced at the start of differentiation into cardiac cells.
• the images in the middle column (b and e) represent human cells in the process of cellular differentiation into cardiac cells, 3 to 7 days after the initiation of differentiation.
• the images in the right column (c and f) represent differentiated cardiac tissues.
• topology is different with and without encapsulation according to the invention. Without encapsulation there is no lumen being differentiated, and the cardiac tissue obtained at the end of differentiation has a very different shape.
• Figure 8 we see that the percentage of cells in the tissues (obtained as in Figure 7c and 7f) expressing cardiac troponin C is greater than 90% under the conditions of the invention while it is 40 % for cardiac tissues obtained under the same conditions but without encapsulation.

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