WO2018081616A1 - Bioengineering of injectable encapsulated aggregates of pluripotent stem cells for therapy of myocardial infarction - Google Patents

Bioengineering of injectable encapsulated aggregates of pluripotent stem cells for therapy of myocardial infarction Download PDF

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WO2018081616A1
WO2018081616A1 PCT/US2017/058837 US2017058837W WO2018081616A1 WO 2018081616 A1 WO2018081616 A1 WO 2018081616A1 US 2017058837 W US2017058837 W US 2017058837W WO 2018081616 A1 WO2018081616 A1 WO 2018081616A1
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stem cells
cells
differentiated
acm
encapsulated
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PCT/US2017/058837
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French (fr)
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Xiaoming He
Shuting Zhao
Zhaobin XU
Zhenguo Liu
Noah Weisleder
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Ohio State Innovation Foundation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5036—Polysaccharides, e.g. gums, alginate; Cyclodextrin
    • C—CHEMISTRY; METALLURGY
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    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0012—Cell encapsulation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0657—Cardiomyocytes; Heart cells
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells

Definitions

  • MI myocardial infarction
  • CMs cardiomyocytes
  • SCT Stem cel l therapy
  • Various types of stem cells have been investigated exhibiting both advantages and disadvantages. To date, only pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), are well accepted to be capable of differentiating into functional CMs.
  • PSCs pluripotent stem cells
  • ESCs embryonic stem cells
  • iPSCs induced pluripotent stem cells
  • the retention of single (i.e., dissociated) stem cells in the infarct zone delivered in suspension has been dismal (often less than -10% within a few hours to a few days post injection).
  • the retained cells can die of the hostile MI microenvironment that could be exacerbated by the implanted cell s to trigger immune reactions.
  • the presence of macrophages together with the cytokines secreted by them in the first few days after MI creates a strong proinflammatory environment resulting in chemo-attraction of more immune cells and damage to the transplanted stem cells.
  • Temporary systemic immunosuppression for a few days has been proposed to mitigate immune rejection to the implanted stem cells to improve their survival.
  • a micromatrix comprising encapsulated aggregated pre-differentiated stem cells the method comprising a) microencapsulating a stem cell, wherein the microcapsule comprises a permissive liquid core and a hydrogel shell; b) expanding the microencapsulated stem cells for at least 1 day; wherein the microencapsulated stem cells proliferate and form aggregates; c) pre-differentiating the microencapsulated stem cell s into early stage cardiac lineage cell s; d) releasing the aggregates of pre-differentiated microencapsulated stem cells from the core-shell microcapsules; and e) encapsulating the released pre-differentiated stem cells in a micromatrix.
  • a micromatrix compri sing encapsulated aggregated pre-differentiated stem ceils of any preceding aspect, wherein the stem cell comprises an embryonic stem cell, induced plunpotent stem cells, extraembryonic fetal stem cells, amniotic stem ceils, or adult stem.
  • a micromatrix comprising encapsulated aggregated pre-differentiated stem cells of any preceding aspect, wherein the stem cel l is microencapsulated via coaxial eiectrospray.
  • a micromatrix comprising encapsulated aggregated pre-differentiated stem cells of any preceding aspect, wherein hydrogel shell is a semipermeable alginate hydrogel shell.
  • a micromatrix comprising encapsulated aggregated pre-differentiated stem cell s of any preceding aspect, wherein the microencapsulated stem cells are expanded for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 days (such as, for example, culturing the microencapsulated stem ceils in a media comprising fibroblast growth factor (FGF) and bone morphogeny e protein.
  • FGF fibroblast growth factor
  • a micromatrix comprising encapsulated aggregated pre-differentiated stem ceils of any preceding aspect, wherein the pre- differentiated microencapsulated stem cells comprise first heart field (FHF) cells, second heart field (SHF) cells, or epicardium derived cells (EPDC). 11. Also disclosed herein are methods of making a micromatrix comprising encapsulated aggregated pre-differentiated stem cells of any preceding aspect, wherein the pre-differentiated microencapsulated stem cells are released from the core-shell microcapsules by dissolving the hydrogen using an isotonic solution of sodium.
  • FHF first heart field
  • SHF second heart field
  • EPDC epicardium derived cells
  • a micromatrix comprising encapsulated aggregated pre-differentiated stem ceils of any preceding aspect, wherein the released pre-differentiated stem cells of step e are encapsulated in a micromatrix by soaking the released aggregates in a solution comprising chitosan and/or alginate (such as, for example, oxidized alginate); wherein the solution comprises a pH of about 6 to about 8 (for example, about 6.7 to about 7.2). 13.
  • micromatrix of encapsulated aggregates of stem cells wherein the stem cells are pre-differentiated stem cells; and wherein the stem cells are encapsulated by an integral with a micromatrix of alginate and chitosan.
  • encapsulated aggregates of stem cells of any- preceding aspect wherein the encapsulated aggregate of stem cells comprises about 1500 stem ceils.
  • encapsulated aggregates of stem cells of any preceding aspect wherein the encapsulated aggregate of stem cells is an early stage cardiac lineage multi-potent stem cell.
  • ischemic event such as, for example, arterial embolism, venous embolism, thromboembolism, pulmonary embolism, traumatic injur ⁇ ', atherosclerosis, myocardial infarction, thoracic outlet syndrome, tachycardia, hypotension, tourniquet, or surgery
  • administering to the subject the encapsulated aggregate of stem cells of any- preceding aspect, 17.
  • Figure 1 shows the bioinspired approach for preparing pluripotent stem cells to implant by injectable delivery.
  • Figure 1 A shows a schematic illustration of the multi-step procedure to prepare the totipotent-pluripotent stem cells for implantation in the uterus wall, including proliferation to form a microscale ceil aggregate in zona pellucida (i.e., morula), pre- differentiation of morula into trophoblast cells and inner cell mass in the zona pellucida, hatching out of the zona pellucida, and re-encapsulation in the trophoblast before implantation during early embryo development in the female reproductive system.
  • zona pellucida i.e., morula
  • pre- differentiation of morula into trophoblast cells and inner cell mass in the zona pellucida hatching out of the zona pellucida
  • re-encapsulation in the trophoblast before implantation during early embryo development in the female reproductive system.
  • FIG. IB shows a schematic illustration of the bioinspired procedure for producing 3D microscale constaicts of murine embryonic stem cells (mESCs) together with real images, showing the analogy between the bioinspired approach and the aforementioned natural procedure.
  • the bioinspired approach mimics the natural procedure phenomenologically rather than mechanistically.
  • Scale bar 100 ⁇
  • Figure 2 shows the pre-differentiation of the mESC aggregates into the early cardiac stage.
  • Figure 2A shows microarray data showing significantly increased expression of mesoderm and cardiac marker genes and significantly decreased expression of piuripotency marker genes in the aggregated cells after pre-differentiation.
  • Figure 2B shows flow cytometry data showing successful pre-differentiation of the mESC aggregates with diminished expression of piuripotency protein makers (OCT-4 and NANOG).
  • Figure 2C shows flow cytometry data showing early cardiac pre-differentiation with significantly increased expression of cardiac specific protein marker (cTnT) and the early cardiac protein marker (NKX2.5). 22.
  • Figure 3 shows the characterization of the aggregates pre-differentiated to the early cardiac stage.
  • Figure 3 A shows SEM images showing successful encapsulation of the pre- differentiated cell aggregates with the alginate-chitosan micromatrix (ACM) both outside (first two columns) and inside (third column) the aggregates. Scale bars; 5 ⁇ .
  • Figure 3B shows confocal fluorescence micrographs of the middle plane of the ACM-A showing micromatrix inside the aggregates indicated by labeling alginate in the ACM with FITC to show up with green fluorescence. Scale bar: 50 um.
  • Figure 4 shows therapy of myocardial infarction by injecting A CM -encap ulated pre- differentiated aggregates.
  • Figure 4A shows a schematic illustration of surgical ligation (X) of the LAD at its proximal location and implantation of samples by intramyocardial injection at three different locations to create large-area myocardial infarction (MI).
  • Figure 4B shows typical gross images of a heart with no MI and MI hearts with five different treatments showing granulomas in single cell (Single) and Bare-A treated mice (arrows), scale bar: 3 mm.
  • Figure 4C shows quantitative data of cumulative granuloma occurrence in both wild type (WT) and CARD9 knockout (K.O) mice, showing treatments with single, Bare-A, Bare-A-KO have significantly higher occurrence of granuloma than the other treatments including ACM-A.
  • the animal number (n) was 10, 31, 29, 38, 31, 24, and 9 for No MI, Saline, Single, Bare-A, ACM-A, ACM, and Bare-A-KO, respectively. *, p ⁇ 0.05 (Chi-square test).
  • Figure 4D shows typical micrograph of granuloma with immunofluorescence staining of F4/80 (for macrophage, red) and CD3 (for T cells, red) showing many immune cells within a loose matrix in the granuloma collected at 28 days after injected with Bare-A. The nuclei are stained blue. Scale bar: 20 urn.
  • Figure 4F shows the survival of WT MI mice at 28 days after injection, showing the ACM-A treatment can maintain a significantly higher animal survival than all the other treatments.
  • the animal number (n) was 31, 29, 38, 31, and 24 for Saline, Single, Bare-A, ACM-A, and ACM, respectively.
  • *, p ⁇ 0,05 one-way ANOVA.
  • Figure 5 shows cardiac regeneration in situ with the ACM-encapsulated pre- differentiated aggregates.
  • Figure 5 A shows low-magnification sagittal micrographs of Masson' s trichrome stained tissue sections (top row) and zoom-in views of the left ventricular wall (bottom row) showing extensive fibrosis in the MI hearts treated with saline, materials alone (i.e., ACM), single cells, and Bare- A while it is minimal with the ACM-A treatment. Scale bar: 2 mm (top row) and 00 ⁇ (bottom row).
  • Figure 5C shows micrographs of sectioned and H&E stained tissue
  • FIG. 5D shows immunohistochemically stained tissue from the MI zone of hearts treated with the ACM-A showing positive staining of CM (cTnl, connexin 43, and a-actinin, in red) markers co-localized with injected GFP cells. The striated pattern of the cardiac tissue with green fluorescence is evident. Scale bar: 10 ⁇ . All tissues were harvested on day 28 after intramyocardial injection.
  • Figure 6 shows a schematic illustration of the setup for coaxial electrospray.
  • Figure 6A shows a schematic overview of the setup for coaxial electrospray to encapsulate murine embryonic stem cells (mESCs) in core-shell microcapsules.
  • Figure 6B shows a zoom-in cross- sectional view of the coaxial needle.
  • the core solution (green) with live mESCs and the shell solution of alginate (gray) are pumped through the inner and outer lumen in the coaxial needle, respectively, resulting in the formation of concentric drops at the needle tip.
  • FIG. 7 shows the biodegradation of the micromatrix of alginate and chitosan in vitro.
  • DIC differential interference contrast
  • FOG 7B ACM-encapsulated
  • FIG. 7C Typical differential interference contrast (DIC) and fluorescence images showing the morphology and high cell viability of bare (FIG, 7B) and ACM-encapsulated (FIG 7C) pre- differentiated aggregates cultured in petri dishes for 15 min, 1 day, and 3 days.
  • Scale bar 100 um.
  • Bare-A Bare pre- differentiated aggregates.
  • ACM-A ACM-encapsulated, pre-differentiated aggregates.
  • Figure 8 shows the biodegradation of the micromatrix of alginate and chitosan in vivo.
  • Figure 8A shows IVIS images showing successful labeling non-oxidized (NonOxi) alginate and oxidized (Oxi) alginate (by default and used for making the alginate-chitosan micromatrix or AC : in this study) with indocyanine green (ICG).
  • Figure 8B shows ICG fluorescence in the MI heart of mice injected with Bare-A, NonOxi- ACM-A-ICG made of ICG labeled non-oxidized alginate, and ACM-A-ICG made of ICG labeled oxidized alginate.
  • NonOxi-ACM-A-ICG and ACM-A-ICG is similar to the
  • Figure 9 shows the cardiac functional data from PV loop measurements at 28 days after surgery.
  • Figure 9A shows the maximum and minimum rate of pressure change (dP/dt).
  • Figure 9B shows the cardiac output and stroke volume.
  • Figure 10 shows the cardiac functional data from PV loop measurements at 28 days after surgery.
  • Figure 10A shows the isovolumic relaxation constant (Tau).
  • Figure 10B shows the left ventricle (LV) end-diastolic volume (LVED), and LV end-systolic volume (LVES).
  • FIG. 1 1 shows antibody staining of green fluorescent protein.
  • the green fluorescent protein (GFP) was stained in tissue from the infarct zone of heart treated with ACM-A made of GPF positive cells.
  • the extensive co-localization of the green fluorescence with the antibody staining of GFP (in red) indicates the green fluorescence is indeed from the implanted cells with GFP expression.
  • the cell nuclei were stained with DAP I (blue).
  • DIC differential interference contrast. Scale bar: 100 urn.
  • Ranges can be expressed herein as from “about” one particular value, and/or to "about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “ 10" is disclosed, then “about 10" is also disclosed.
  • micromatrix comprising encapsulated aggregated pre-differentiated stem cells For example, if a particular micromatrix comprising encapsulated aggregated pre-differentiated stem cells is disclosed and discussed and a number of modifications that can be made to a number of molecules including the micromatrix comprising encapsulated aggregated pre-differentiated stem cells are discussed, specifically contemplated is each and every combination and permutation of micromatrix comprising encapsulated aggregated pre-differentiated stem cells and the modifications that are possible unless specifically indicated to the contrary.
  • A-D a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B- D, B-E, B ⁇ F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions.
  • the microenvironment can be hostile due to the presence of pro-inflammatory immune cells such as macrophages which secrete cytokines at the site of injury and reduce engraftment of the transplanted cells.
  • pro-inflammatory immune cells such as macrophages which secrete cytokines at the site of injury and reduce engraftment of the transplanted cells.
  • injection of stem cells at 4-7 days after MI may help to improve the survival of the implanted/retained cells.
  • significant injury to the infarcted myocardium would accumulate during the 4-7 days of delay. Therefore, early treatment to minimize the injury or pathological development after Ml is desired.
  • the disclosure herein provides novel compositions and treatments that do not suffer the limitations of previous methods and overcome obstacles posed by the microenvironment.
  • microcapsule the effects of the microenvironment on the stem ceils can be minimized allowing for early delivery of stem cells and avoiding further damage to the tissue at the site of injur ⁇ '. This also has the benefit of extended release. Accordingly, in one aspect, disclosed herein are micromatrixes of encapsulated aggregates of stem cells; wherein the stem cells are encapsulated by and integral with a micromatrix.
  • the micromatrix can be comprised on an material suitable for production of a matrix and supporting the encapsulated cells.
  • the micromatrix can be formed through the combination of a positively charged marcromolecule (for example, Poly-l-lysine or chitosan) and a negatively charged macromolecule (for example, alginate or hyaluronan).
  • a positively charged marcromolecule for example, Poly-l-lysine or chitosan
  • a negatively charged macromolecule for example, alginate or hyaluronan.
  • the stem cells can be pre-differentiated to avoid teratoma formation. Accordingly, disclosed herein are micromatrixes of encapsulated aggregates of stem cells;
  • stem cells are pre-differentiated stem cells; and wherein the stem cells are encapsulated by an integral with a micromatrix of alginate (for example oxidized alginate) and chitosan.
  • alginate for example oxidized alginate
  • the micromatrix of encapsulated aggregates of stem cells comprises pre-differentiated cells that are differentiated to facilitate their use in repairing tissue damage and minimize the potential for teratoma formation. Therefore, for cardiac related ischemias it is advantageous to pre-differentiate the PSCs into the early cardiac stage lineage cells rather than into mature cardiomyocytes for implantation into the heart.
  • This approach utilizes the native chemical, mechanical, and electrical cues in the heart to further guide the pre-differentiated cells (at the early cardiac stage) into mature cardiomyocytes with similar electromechanical properties to the native CMs.
  • the pre- differentiated cells can be an early stage cardiac lineage multi-potent stem cell such as, for example first heart field (FHF) cells, second heart field (SHF) cells, and/or epicardium derived cells (EPDC).
  • FHF first heart field
  • SHF second heart field
  • EPDC epicardium derived cells
  • the disclosed aggregates of pre-differentiated stem cells can comprise a large number of ceils.
  • the aggregates can comprise at least 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, or 2500 cells/ aggregate construct.
  • the aggregate can comprise about 1500 cells.
  • the encapsulated aggregates of stem cells of any preceding aspect wherein the encapsulated aggregate of stem cells comprises about 1500 stem cells. 43.
  • the alginate chitosan matrix will degrade over time releasing the aggregates of pre-differentiated stem cells as the degradation occurs. The gradual release of cells insures a continual population of pre-differentiated ceils over the course of degradation.
  • the micromatrix of encapsulated aggregates of stem cells comprises an alginate chitosan matrix (ACM) that completely degrades in about 24hrs, 36hrs, 48hrs, 60 hrs, 72 hrs, 84hrs, 96hrs, 5 days, 6 days, or 7 days. In one aspect, the degradation takes at least 2, 3, 4, 5, 6, or 7 days. 44.
  • the preparation of aggregates of pre-diff erentiated stem cells in a matrix overcomes the limitations to previous stem cell based therapeutics of ischemic injury.
  • Prior to the present disclosure no micromatrix of encapsulated aggregates of pre-diff erentiated cells has existed.
  • the preparation of such cells is unique to the disclosed cells.
  • a micromatrix comprising encapsulated aggregated pre- diff erentiated stem cells
  • a) microencapsulating a stem cell wherein the microcapsule comprises a permissive liquid core and a hydrogel shell
  • expanding the microencapsulated stem cells for at least 1 day wherein the microencapsulated stem ceils proliferate and form aggregates
  • stem cell refers to any multipotent, pluripotent, or totipotent progenitor ceil capable of self-renewal including, but not limited to embryonic stem cell, induced pluripotent stem cells, extraembryonic fetal stem cells, amniotic stem cells (including cells stem cells obtained from the cord blood), and adult stem cells (including, but not limited to hematopoietic stem cells, endothelial stem cells, intestinal stem cells, mammary stem cells, neural stem cells, and mesenchymal stem ceils).
  • a micromatrix comprising encapsulated aggregated pre-differentiated stem cells of any preceding aspect, wherein the stem cell comprises an embryonic stem cell, induced pluripotent stem cells, extraembryonic fetal stem cells, amniotic stem cells, or adult stem.
  • the microcapsule can be formed (i.e., the
  • microencapsulation via any means known in the art including, but not limited to coaxial electrospray, oil-aqueous solution emulsion, pan coating, air-suspension coating, centrifugal coating, vibrational nozzle, spray-drying, ionotropic gelation, coacervation-phase separation, interfacial polycondensation, and interfacial cross-linking.
  • the core of the microcapsule which will contain the stem ceils, can comprise any aqueous solution suitable for the propagation of cells including, but not limited to saline, collagen, hyaluronan, fibrin, laminin, ceil culture media (such as, for example, Minimal essential media (MEM), Eagles' minimal essential medium (EMEM), Dulbecco's Modified Eagle Media (DMEM), Ham's Nutrient Mixtures (Ham' F-10, and Ham's F-12), Roswell Park Insitute Medium (RPMI), Iscove's Modified Dulbecco's Medium and (IMDM)), Hanks' balanced salt solution, Earle's balanced salt solution, and Dulbecco's phosphate-buffered saline.
  • the core can be a hydrogel.
  • the shell of the microcapsule can comprise any material from which a shell may be formed.
  • the shell can be a hydrogel material comprising alginate, collagen, silicone, polyvinyl alcohol (PVA), sodium poiyacrylate, polyethylene oxide, polyethylene glycol, polyvinylpyrrolidone, poly-methyl methacrylate, and any co-polymer or ter-polymers thereof.
  • the shell can be a semipermeable alginate hydrogel shell.
  • the hydrogel shell can comprise materials found in the zona pellucida of an embryo.
  • the core-shell To expand the starting stem cell population and facilitate the pre-differentiation of the stem cells to early stage multi-potent cells of a committed lineage, the core-shell
  • microencapsulated stem cells can be expanded for at least 60, 12, 18 hours, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11. 12, 13, 14, 15, 16. 17, 18, 19. 20, 21, 22, 23, 24. 25, 26, 27. 28, 29, or 30 davs.
  • the stem cells can be cultured in core comprising fibroblast growth factor (FGF), tbx l 8, Oct4, Sox2, Klf4, cMyc, NRG, IGF, Notch 1, wingless- related integration site (Wnt) protein (including, but not limited to Wntl, Wnt2, Wnt3, Wnt4, Wnt5, Wnt6, Wnt7A, Wnt7b, WntSA, WntSB, Wnt9A, Wnt.9B, Wntl OA, WntlOB, Wntl 1 and Wntl 6), ⁇ -catenin and/or bone morphogenic protein (BMP) such as, for example BMP-4.
  • BMP bone morphogenic protein
  • a micromatrix comprising encapsulated aggregated pre-differentiated stem cells of an preceding aspect, wherein the microencapsulated stem cells are expanded for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days in a microcapsule core comprising a media comprising FGF and BMP-4.
  • a micromatrix comprising encapsulated aggregated pre-differentiated stem cells comprising a) microencapsulating a stem cell, wherein the microcapsule comprises a permissive liquid core and a hydrogel shell, b) expanding the microencapsulated stem cells for at least 1 day; wherein the microencapsulated stem cells proliferate and form aggregates: d) releasing the aggregates of pre-differentiated microencapsulated stem cells from the core-shell microcapsules, and e) encapsulating the released pre-differentiated stem cells in a micromatrix.
  • the disclosed herein are methods of making a micromatrix comprising encapsulated aggregated pre-differentiated stem cells can be used to provide a micromatrix of aggregated pre-differentiated cells of any cell lineage that would be of interest given the application.
  • the pre-differentiated can comprise a bone-marrow derived stem cells, adipose derived stem cells, or any organ specific stem cells such as neural stem cells or cardiac stem cells.
  • the stem cells can be pre-difTerentiated to early- stage cardiac lineage multipotent cells such as, for example, first heart field (FHF) cells, second heart field (SHF) cells, or epicardium derived cells (EPDC),
  • FHF first heart field
  • SHF second heart field
  • EPDC epicardium derived cells
  • the pre-differentiated stem cell must be released from the core- shell microcapsule. This can be accomplished by any means suitable to dissolved the shell of the microcapsule and will depend on the composition of the shell.
  • disclosed herein are methods of making a micromatrix comprising encapsulated aggregated pre- differentiated stem cells of any preceding aspect, wherein the pre-differentiated
  • microencapsulated stem cells are released from the core-shell microcapsules by dissolving the hydrogel using an isotonic solution of sodium.
  • the aggregates of pre-differentiated stem cells must be re-encapsulated in a micromatrix.
  • methods of making a micromatrix comprising encapsulated aggregated pre-differentiated stem cells of any preceding aspect, wherein the released pre-differentiated stem cells of step e are encapsulated in a micromatrix by soaking the released aggregates in a solution comprising chitosan and/or alginate (such as, for example, oxidized alginate).
  • the solution comprising alginate and chitosan can be at any physiological relevant pH for the formation the micromatrix and continued viability of the pre-differentiated stem cells.
  • the pH can be any pH between about 6 and about 8, more preferably between about 6.5 and about 7.5, most preferably between about 6.7 and about 7.2.
  • the pH can be 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7,3, 7,4, 7,5, 7,6, 7.7, 7.8, 7.9, or 8.0,
  • disclosed herein are methods of making a micromatrix comprising encapsulated aggregated pre- differentiated stem cells of any preceding aspect, wherein the released pre-differentiated stem cells of step e are encapsulated in a micromatrix by soaking the released aggregates in a solution comprising chitosan and/or alginate; wherein the solution comprises a pH of about 6.7 to about 7.2. 55.
  • the micromatrix encapsulated pre- differentiated stem cells can be used to treat damaged tissue resulting from an ischemic event
  • Treating include the administration of a composition with the intent or purpose of partially or completely preventing, delaying, curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving, stabilizing, mitigating, and/or reducing the intensity or frequency of one or more a diseases or conditions, a symptom of a disease or condition, or an underlying cause of a disease or condition.
  • Treatments according to the invention may be applied preventively, prophyiactically, pailatively or remediaily. Prophylactic treatments are
  • Prophylactic administration can occur for day(s) to years prior to the manifestation of symptoms of an infection.
  • the ischemic event can be any ischemic event where tissue damage results from the change in blood flow including, but not limited to embolism (such as, for example, arterial embolism, venous embolism, thromboembolism, pulmonary embolism), traumatic injury, atherosclerosis, myocardial infarction, thoracic outlet syndrome, tachycardia, hypotension, tourniquet, and surgery. It is understood and herein contemplated that the ischemia is not limited to cardiac ischemia, but can include acute and chronic brain ischemia (including transient ischemic attacks), acute limb ischemia, and bowel ischemia.
  • ischemic event such as, for example, arterial embolism, venous embolism, thromboembolism, pulmonary embolism, traumatic injury, atherosclerosis, myocardial infarction, thoracic outlet syndrome, tachycardia, hypotension, tourniquet, or surgery
  • ischemic event such as, for example, arterial embolism, venous embolism, thromboembolism, pulmonary embolism, traumatic injury, atherosclerosis, myocardial infarction, thoracic outlet syndrome, tachycardia, hypotension, tourniquet, or surgery
  • the encapsulated aggregate of stem cells disclosed herein are methods treating damage cause by or reducing the severity of any preceding aspect, wherein the ischemic event comprises a myocardial infarction and wherein the stem cells are pre-dif erentiated to an early stage cardiac lineage multi-potent stem cell.
  • compositions can also be administered in vivo in a
  • pharmaceutically acceptable carrier a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • the carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.
  • the material s may be in solution, suspension (for example, incorporated into
  • microparticles, liposomes, or cells may be targeted to a particular cell type via antibodies, receptors, or receptor ligands.
  • the following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et ai., Bioconjugate Chen?., 2:447-451 , (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et a!., Br. J. Cancer, 58:700-703, (1988); Senter, et al,, Bioconjugate Chem., 4:3-9, (1993); Battelli, et al ., Cancer Immunol. Immunoiher., 35 :421-425, (1992); Pietersz and McKenzie, Immunolog.
  • Vehicles such as "stealth” and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo.
  • the following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al.. Cancer Research, 49:6214- 6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1 104: 179-187, (1992)).
  • receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intraceliuiariy, or are degraded in iysosomes.
  • the internalization pathways serve a vari ety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation.
  • compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malomc acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, triaikyl and aryl amines and substituted ethanol amines.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid
  • organic acids such as formic acid, acetic acid, prop
  • composition refers to an amount that is effective to achieve a desired therapeutic result.
  • a desired therapeutic result is the control of type I diabetes.
  • a desired therapeutic result is the control of obesity.
  • Therapeutically effective amounts of a given therapeuti c agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject. The term can also refer to an amount of a therapeutic agent, or a rate of deliver)' of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect, such as pain relief.
  • the precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the agent and/or agent formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of agent in the tonnulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art.
  • a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years.
  • Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art.
  • the dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms of the disorder are effected.
  • the dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like.
  • the dosage will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art.
  • the dosage can be adjusted by the individual physician in the event of any counterindi cations.
  • Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.
  • Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.
  • guidance in selecting appropriate doses for antibodies can be found in the literature on therapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J., (1985) ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp. 365-389.
  • a dose of about lxlO 4 , 2x10 4 , 3xl0 4 , 4xl0 4 , 5xl0 4 , 6x I 0 4 , 7xl0 4 , 8xl0 4 , 9xl0 4 , Ix O 5 , 2xl0 5 , 3xl0 5 , 4xl0 5 , 5xl0 5 , 6xl0 5 , 7xl0 5 , 8xl0 5 , 9xl0 5 , or IxlO 6 pre-differentiated cells can admi ni stered to th e subj ect ,
  • compositions may be administered orally, parenteraily (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, intramyocardial injection, transdermally, extracorporeally, topically or the like, including topical intranasal administration or
  • topical intranasal administration means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise deliver ⁇ ' by a spraying mechanism or droplet mechanism, or through aerosoiization of the nucleic acid or vector.
  • Administration of the compositions by inhalant can be through the nose or mouth via deliveiy by a spraying or droplet mechanism. Deliveiy can also be directly to any area of the respirator ⁇ ' system (e.g., lungs) via intubation.
  • compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.
  • Parenteral administration of the composition is generally characterized by injection.
  • Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions.
  • a more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein.
  • compositions including micromatrixes, can be used therapeutically in combination with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier ed. A.R. Gennaro, Mack Publishing Company, East on, PA 1995.
  • an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic.
  • the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution.
  • the pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5.
  • Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.
  • compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.
  • compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice.
  • compositions may also include one or more active ingredients such as antimicrobial agents, anti -inflammatory agents, anesthetics, and the like.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringers, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti -oxidants, chelating agents, and inert gases and the like. 70.
  • Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. 71.
  • compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable,
  • the disclosed micromatrixes may be administered concurrently or in combination with a therapeutic agent useful in the treatment of damage resulting from ischemia.
  • a therapeutic agent refers to any composition that has a beneficial biological effect, Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition (e.g., an ischemia such as, for example, myocardial infarction).
  • the terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like.
  • therapeutic agent or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically
  • micromatrixes can be used for the therapeutic treatment of damage to tissue caused by an ischemic event.
  • the micromatrixes can be administered to the subject during, or 1, 2, 3, 4,5 ,6, 7 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24hrs, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 45, 60, 75, 90 days, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, 2, 3, 4, 5, 6, 7, 8, 9 , or 10 years after the ischemic event.
  • methods treating damage cause by an ischemic event comprising administering to the subject a micromatrix of
  • encapsulated aggregates of pre-differentiated stem cells wherein the encapsulated aggregate of stem cells is administered to the subject within 7 days following the ischemic event.
  • ischemia can be induced medicinally or mechanically.
  • risk factors for ischemia can be sufficiently significant to warn of an immediate likelihood of ischemia (such as, embolism or myocardial infarction).
  • the disclosed micromatrixes can be administered prophylactically.
  • the micromatrixes can be administered during or 1, 2. 3, 4,5 ,6, 7 8, 9, 10. 11, 12, 13, 14, 15. 16, 17, 18. 19, 20, 21, 22, 23.
  • ischemic event 24hrs, 2, 3, 4, 5, 6, or 7 days before the ischemic event.
  • methods of reducing tissue damage caused by an ischemic event in a subject comprising administering to the subject a micromatrix of encapsulated aggregates of pre-differentiated stem ceils, wherein the encapsulated aggregate of stem cells is administered to the subject within about 1, 2, 3, or 4 days prior to the ischemic event.
  • the approach for preparing PSCs for implantation by injectable delivery to treat MI is inspired by the multi-step procedure used by nature to prepare totipotent-pluripotent stem cells for implantation into the uterus.
  • This natural procedure includes the following steps (Fig. la): proliferation of the totipotent-pluripotent stem cells in the miniaturized permissive core enclosed in the semipermeable shell (known as zona pellucida) of pre-hatching embryos into a
  • mESCs single (i .e., dissociated) murine ESCs
  • Fig. 6 coaxial electrospray technology
  • the mESCs proliferate to form one single aggregate of 128.9 ⁇ 1 7.4 ⁇ consisting of 1450 ⁇ 381 ceils per aggregate with high (>90%) viability in each microcapsule in 7 days.
  • the core-shell architecture of the microcapsules mimics the physical configuration of pre-hatching embryo with a hydrogel-like shell (i.e., zona pellucida) and a permissive core, the native home of stem cells from the totipotent to pluripotent stage.
  • a hydrogel-like shell i.e., zona pellucida
  • a permissive core the native home of stem cells from the totipotent to pluripotent stage.
  • the aggregates were pre-differentiated into the early cardiac stage inside the microcapsules.
  • the pre-differentiated aggregates were released from the core-shell microcapsules by dissolving the alginate hydrogel shell using an isotonic solution of sodium citrate and further re-encapsulated within a micromatrix for implantation. This does not significantly affect their size and integrity (see the images in Fig.
  • RNA microarray data showing that the expression of cardiac marker genes was significantly higher in the pre-differentiated mESCs than the cells before pre-differentiation.
  • the expression of other typical genes that regulate heart and mesoderm development were also up-regulated, while the pluripotency marker genes were down-regulated in the pre-differentiated ceils.
  • microarray analysis was validated with the conventional method of analyzing gene expression using quantitative reverse transcription polymerase chain reaction (qRT-PCR) to quantify the expression of the early cardiac gene marker Nkx2.5. Both the microarray and qRT-PCR analyses show that the expression of the N ⁇ kx2.5 gene was significantly up-regulated by 4-5 times after pre- differentiation (Fig. 2a). Further flow cytometry analyses were conducted for typical
  • pluripotency protein markers OCT-4 and NANOG, Fig. 2b
  • cardiac specific protein makers including cardiac troponin T (cTnT) and NKX2.5 (Fig. 2c).
  • the aggregates were released from the core- shell microcapsules using isotonic sodium citrate solution, the released aggregates were re- encapsulated in an aiginate-ehitosan micromatrix (ACM) by soaking the aggregates first in chitosan (0.4% w/v in saline) and then in a solution (0.15% w/v in saline) of oxidized alginate.
  • ACM aiginate-ehitosan micromatrix
  • bare single cells (Single), saline, and materials alone (i.e., ACM) for treating MI intramyocardial injection of 2x 0 5 cells/animal in a total of 20 ⁇ saline into the peri-infarct zone (injected at 3 different injection sites with equal volume, Fig. 4a) were conducted using a 28 gauge needle. This was done within 5 minutes after the anterior wall of the left ventricle turned pale following permanent ligation of the left anterior descending artery (L AD) at its proximal location, to create a large-area MI in 8-12 week old male C57BL6/J mice.
  • L AD left anterior descending artery
  • mice are either wild-type (WT) with a normal immune system or caspase-recruitment domain 9 (Card9) knockout (KO) with deficient macrophage function.
  • WT wild-type
  • Card9 knockout KO
  • This immediate injection of therapeutic cells is desired to minimize the damaging effect of MI.
  • All surgical procedures were conducted under aseptic conditions.
  • the treatments with Bare-A and single cells differentiate themselves from the others with the formation of a large mass of tissue that grows out of the heart (indicated by arrows in Fig. 4b).
  • the Bare-A treated MI mice started to die at as early as 2 days post injection while mortality was not observed in the ACM-A treatment group until 7 days after injection (Fig. 4f). Nearly 40% of the Bare- A treated MI mice died during the first 4 days, a known time frame for acute immune reaction to implanted cells. Interestingly, while WT mice treated with single cells have a higher occurrence of granuloma, the overall size of granuloma is smaller than that of Bare- A treatment (Fig. 4b). This is due to the reduced cell survival/retention and can explain the moderate animal mortality in the early time frame for the single cell treatment, compared to the Bare-A treatment.
  • Macrophages are known to regulate acute inflammation and remodeling in response to cardiac injury.
  • Bare-A was injected into the MI hearts of Card9 KO mice.
  • Granuloma was observed in only -33% of the KO mice (Fig. 4c).
  • the ACM-A treatment also significantly improves the cardiac function as indicated by ejection traction measured by pressure-volume (PV) loops (Fig, 4g) and electrocardiography, compared to saline, Bare-A, and ACM treatments. All groups with cells (Single, Bare-A, and ACM-A) can partially restore the maximum and minimum rate of pressure change in the left ventricle (Fig. 9a).
  • the ACM-A treatment significantly improves the stroke volume and cardiac output compared to the saline and ACM treatments (Fig. 9b), the time constant of isovolumic pressure relaxation (tau) compared to saline treatment (Fig.
  • the ACM-A treatment significantly reduces fibrosis (Fig. 5a-b) compared to all other treatments at 28 days after injection. This contributes to the high survival of the WT MI mice with the ACM-A treated mice (Fig. 4f).
  • all treated hearts are markedly larger than no-MI hearts, as a result of pathological hypertrophy in response to MI, trying to restore the stroke volume compromised by MI.
  • the hearts from mice treated with ACM-A suffered less hypertrophy than mice with the other treatments, indicating that the ACM- A treatment facilitates the restoration of cardiac function of mice with ML
  • the heart tissue harvested from the MI mice implanted with ACM-A prepared using niESCs constitutiveiy expressing green fluorescent protein (GFP) using the bioinspired approach was examined. It was observed that extensive green fluorescence is evident in the MI zone (Fig. 5c) at 28 days post injection. Moreover, this green fluorescence co-localizes with the antibody staining of GFP expressed in live ceils (Fig.
  • CMs including cardiac troponin I (cTnl), connexin 43, and a-actinin in the green fluorescent CM-like cells derived from the implanted cells (Fig. 5d).
  • the green fluorescent CMs integrated seamlessly with the neighboring native CMs without green fluorescence (Fig. 5c-d), indicating the micromatrix was biodegraded allowing for timely integration of the implanted cell-derived CMs with the native cardiac tissue.
  • Microcapsules with a core-shell structure resemble the native physical configuration of pre-hatching embryos, the native home of totipotent-pluri potent stem cells.
  • the miniaturized and semi -closed culture in the core- shell microcapsules can better maintain the sternness of both pluripotent and multipotent stem cells compared to conventional culture in homogeneous liquid (medium) or hydrogel. This is crucial to the expansion of stem cells with high quality and purity in vitro for the application of stem cell-based medicine, including but not limited to cardiac tissue regeneration for treating MI.
  • the remaining steps of this bioinspired approach (including early cardiac pre- differentiation, dissolving the alginate hydrogel shell to release the pre-differentiated aggregates, and re-encapsulation in ACM) mimic the phenomena of the processes of pre-differentiation, zona hatching, and re-encapsulation in the trophoblast rather than the exact biological mechanisms of processes that nature uses to prepare totipotent-pluripotent stem cells for implantation into the uterus wall .
  • Biological processes of this natural procedure are tightly regulated and cell-induced and mediated.
  • the bioinspired approach is based on phenomenologically mimicking the natural procedure and does provide multiple advantages in preparing PSCs for implantation to treat MI and other diseases as detailed below.
  • the bioinspired approach enables injectable delivery of cells with much improved efficiency.
  • the contemporary practice of scaffold engineering is to make a scaffold first and then seed ceils into the scaffold, which is extremely difficult (if not impossible) to utilize for making microscale constructs (i.e., ACM-A) with densely packed cells as shown in Fig, 3.
  • the bioinspired approach resolves this challenge by making microscale cell aggregates first and then forming a micromatrix within and over the aggregates (Fig. l b).
  • An important advantage of this inverse (and bioinspired) scaffold engineering method is that it does not significantly change the construct size, which allows for intramyocardial injection of the constaicts for minimally invasive delivery.
  • the Card9 KO mice with compromised macrophage function were used to determine whether macrophage-mediated events are the dominating mechanism of the immune responses observed.
  • the occurrence of granuloma is reduced, but not completely eliminated in the Card9 KO mice, indicating that T cells also play a significant role in the immune responses to the bare aggregates and single cells.
  • the occurrence of granuloma can be significantly reduced to 0, indicating the exceptional capability of immunoisolation by the ACM.
  • the biodegradable ACM temporarily isolates the encapsulated cells from contacting the host cells including macrophages, T cells, and possibly N cells to achieve a temporary immunoisolation (or "local immunosuppression") effect.
  • This contributes to the significantly improved animal survival ( 80° ⁇ > after 28 days of injection) and elimination of granuloma in WT mice with the ACM-A treatment (Fig. 4c and f).
  • animal survival for the saline control at day 28 after surgery was -48%, which is consistent with the literature for WT mice with a large-area MI. This long-term impact of temporary immunosuppressi on/isolation was also recently reported elsewhere.
  • the pre-differentiated cells at the early cardiac lineage can release factors to facilitate angiogenesis to promote their survival.
  • the heart always beats, which can induce some (albeit insufficient alone) degree of fluid flow in the interstitial space of the cardiac tissue to improve the survival of the implanted ceils migrating into the infarct zone.
  • pre-differentiated mESCs were used in mouse MI model (i.e., allogeneic model).
  • teratoma can form in mouse models injected with pluripotent stem cells including the Rl mESCs used in this study in 3-4 weeks.
  • the aforementioned observation indicates that teratoma formation can be eliminated by using the bioinspired method to prepare the mESCs for injectable deliver ⁇ ' with early rather than mature cardiac pre-differentiation before implantation.
  • the data show that ACM encapsulation of pre-differentiated microscale PSC aggregates significantly improves cell retention/survival after intramyocardial injection, and provides a temporary "local immunosuppression" for the ceils to minimize cell injury and early animal death due to acute immune reactions.
  • the implanted cells pre-differentiated to the early- cardiac stage have a superb capability of regenerating cardiac tissue in situ via further direct differentiation guided by the cardiac-inducive chemical, mechanical, and electrical cues in the heart into mature cardiomyocytes. This significantly reduces fibrosis and enhances cardiac function, which ultimately contributes to the significantly improved animal survival.
  • This study indicates that the bioinspired engineering approach can be valuable to facilitate clinical applications of SCT for treating MI and possibly many other degenerative diseases.
  • mice Male wild-type (WT) C57BL6/J mice (Jackson Laboratory, Bar Harbor, ME, USA) of 8- 12 weeks, and age-matched Card9 KO mice with C57BL6/J background (from Dr. Xin Lin's laboratory at Department of Molecular and Cellular Oncology and Department of Immunology, University of Texas MD Anderson Cancer Center, Houston, TX, USA) were housed at constant temperature (22 ⁇ 2 °C) with a 12-hr light/dark cycle. Mice were given standard lab chow and water ad libitum. All investigations in this study conform to the Guide for the Care and Use of Laboratory Animals published by the US National institutes of Health and approved by the Institutional Animal Care and Use Committee at The Ohio State University.
  • Sodium alginate plant cell culture tested and low viscosity, -240 kDa was purchased from Sigma (St. Louis, MO, USA) and purified by washing in chloroform and charcoal and dialyzing against deionized water, followed by freeze-drying. The oxidization of alginate was performed by mixing sodium periodate (2mM) with 1% (w/v) purified alginate for 24 hr in the dark at room temperature, and then using an equivalent amount of ethylene glycol to stop the reaction, followed by 24 hr of dialysis against deionized water with three water changes.
  • ICG and I f K labeled alginate (with and without oxidization) was prepared by dissolving alginate or oxidized alginate (1.8% w/v) in 2-(N-morpholino) ethanesulfonic (MES) acid buffer (pH 4.7) and mixing with 9 mM l-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and 9 mM N-hydroxysulfosuccinimide (Sulfo-NHS).
  • MES 2-(N-morpholino) ethanesulfonic acid buffer
  • EDC 9 mM l-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride
  • Sulfo-NHS 9 mM N-hydroxysulfosuccinimide
  • chitosan of 80 kDa ( ⁇ 95% deacetylation) was obtained from Weikang Biological Products Co. Ltd (Shanghai, China). The chitosan was dissolved in saline buffered with acetic acid at pH 6.7 and filtered through a 0.22 ⁇ ⁇ filter before use.
  • the primary antibodies including ab47003 (to cTnL rabbit polyclonal), ab 18061 (to ot-actinin, mouse monoclonal, clone 0.T.02), ab45932 (to cTnT, rabbit polyclonal), and abl 1370 (to connexin 43/GJA1, rabbit polyclonal) were purchased from Abeam (Cambridge, MA, USA).
  • N X2.5 sc376565, mouse monoclonal, clone A-3
  • CD3 sc20047, for T cells, mouse monoclonal, clone PC3-188A
  • fibronectin sc9068, rabbit polyclonal
  • F4/80 sc25830, for macrophages, rabbit polyclonal
  • GFP GFP
  • All secondary antibodies were purchased from Life Technologies. All other materials were purchased from Sigma unless specifically mentioned otherwise,
  • Rl mESCs with and without GFP were purchased from ATCC (Manassas, VA, USA).
  • the GFP Rl cells express GFP constitutiveiy with the plasmid pEYFP from Clontech Laboratories, Inc. (Mountain View, CA, USA).
  • the mESCs were cultured in feeder-free medium made of Knockout ® ' DMEM supplemented with 15% Knockout* 1 serum (Life Technologies), 1000 U ml "1 leukemia inhibitory factor, 4 mM 1-glutamine, 0.1 M 2- mercaptoethanol, 10 ,ug ml "1 gentamicin, 100 U ml "1 penicillin, and 100 ⁇ g mi "1 streptomycin in gelatin coated tissue culture flasks with daily medium change.
  • mESCs To encapsulate the mESCs in microcapsules with a liquid core and hydrogel shell, they were detached from the flasks using Ix trypsin/EDTA, washed by phosphate buffered saline (PBS), and then suspended at 5 x 10 6 ml "1 in 0,25 M aqueous mannitol solution supplemented with 1% (w/v) sodium carboxymethyl cellulose as the core solution.
  • PBS phosphate buffered saline
  • the coaxial electrosprav system including coaxial needle, pumps, voltage generator, and collecting bath i s shown in Fig, 6. Before experiments, the coaxial needle, collection beaker, and connection tubes were autociaved to ensure sterility. All solutions used for electrosprav were filtered using 0.22 ⁇ filters.
  • the core solution was pumped through the inner lumen (28G) of the coaxial needle at 47 ⁇ min "1 , while the shell solution consisting of 2% purified sodium alginate (w/v) in 0.25 M aqueous mannitol solution was pushed through the outer lumen (21G) at 60 ⁇ min "1 . Concentric drops generated by the core and shell flows are then broken up into
  • microdropiets under a 1.8 kV electrostatic field and finally sprayed into the gelling solution made of 100 mM calcium chloride in deionized water.
  • the distance between the needle tip and the top surface of CaCb solution in the gelling bath was 5.5 mm.
  • the high viscosities of core and shell solutions as well as the instant gelling kinetics of sodium alginate in the 100 mM CaCl 2 solution ensure negligible mixture between the two aqueous solutions and therefore the formation of core-shell microcapsules.
  • the resultant microcapsules were 3 15 ⁇ 31 ⁇ in outer diameter 29 .
  • mESC aggregate After 7-day culture in mESC medium, one integrated mESC aggregate was formed in the core of each microcapsule.
  • cell aggregates were cultured in cardiac induction medium with regular DMEM supplemented with 25 ng ml '1 BMP-4, 5 ng ml "1 bFGF, 100 U ml "1 penicillin, and 100 mg 1 streptomycin for 3 days in the core-shell microcapsules to prevent attachment on the culture plate.
  • RNA isolation Following the manufacture's instruction with the RNeasy Plus Mini Kit (Qiagen). The quality of the extracted RNA was then examined by its A Azaj, A260/230 and 28S/18S ribosomal RNA (rRNA) ratio.
  • the microarray was conducted using the ClariomTM D assays for mouse (Affymetrix, Santa Clara, CA, USA) on GENECHIP® Hybridization Oven 645 and analyzed using the AFFYMETRIX® Transcriptome Analysis Console (TAC) Software by technicians in The Genomics Shared Resource in The Ohio State University Comprehensive Cancer Center.
  • cDNAs complementary DNAs
  • NKX2.5 protein For inimunostaining of NKX2.5 protein, the mESC aggregates before (as control) and after pre-differentiation were either dissociated using lx trypsin/EDTA into single cells or kept as intact aggregates for fixation using 4% paraformaldehyde for 1 hr at room temperature. The fixed single cells or aggregates were incubated first with 3% bovine serum albumin (BSA, to block non-specific binding) in PBS with 0.
  • BSA bovine serum albumin
  • This protocol was also used for other antibodies including QCT-4 (1 :200 primary antibody dilution), NANOG (1 :200 primary antibody dilution), and cTnT (1 : 100 primary antibody dilution). This is the protocol suggested by the manufacturers (Abeam and Santa Cruz) for the primary and secondary antibodies used for immunohistochemical staining and flow cytometry.
  • the aggregate-laden microcapsules were collected from each culture dish by pipetting with a 15 ml serological pipette into a 15 ml centrifuge tube. Due to gravity, the aggregate-laden microcapsules sink down at the bottom of the collection tube in 5 min. The supernatant was then removed using 5 ml serological pipettes and the aggregate-laden microcapsules were rinsed with 5 ml of PBS. The latter was done by gently tilting the tube for mixing and by allowing the microcapsules to sink down at the bottom of the tube as a result of gravity before removing the supernatant by pipetting.
  • a total of 1 ml of 55 tnM isotonic sodium citrate was then added into the tube to dissolve the alginate hydrogel shell of the microcapsules in 30 s, followed by removing the supernatant containing sodium citrate and dissolved alginate by pipetting.
  • the aggregates were then washed using 5 ml of PBS in the same way as that aforementioned for washing the aggregate-laden microcapsules. Unlike trypsin or collagenase, the process of dissolving alginate hydrogel using sodium citrate is quick and gentle and it does not affect the integrity of aggregates.
  • alginate and chitosan solutions were dissolved in saline (0.9%) and with a pH at 7.2 and 6.7 (this is necessary to dissolve chitosan), respectively.
  • the size (diameter) of aggregates was determined by measuring the average diameter at three different locations on each aggregate with a 120-degree interval.
  • Chitosan is a positively charged polymer that can be attracted to the cells in the aggregate because their plasma membrane is negatively charged. The same electrostatic interaction applies to the complexation of alginate with chitosan and vice versa (alginate is negatively charged).
  • Alginate and chitosan are both naturally derived polymers with high biocompatibility and can gradually degrade into nontoxic oligosaccharide and glucosamine (amino sugar), respectively .
  • the materials alone (ACM) control was achieved by- processing alginate microbeads of similar size to the cell aggregates in the same way as that for forming the ACM within the cell aggregates.
  • the ACM encapsulated aggregates were then used for either in vitro studies to determine the existence and degradation of ACM or in vivo transplantation into mice.
  • the aggregates encapsulated in ACM of oxidized or regular alginate were plated on gelatin-coated dishes and maintained in regular DMEM with 20% FBS. The ratio of attached to total aggregates were calculated to determine the percentage of aggregate attachment.
  • Non-encapsulated aggregates were studies in the same way to serve as control.
  • the mESC aggregates were encapsulated in ACM with FITC-labeled alginate using the aforementioned procedure and the encapsulated aggregates were further stained for nuclei using 5 ⁇ Hoechst 33342 for 15 min before examination using an Olympus FV1000 confocal microscope.
  • -150 ⁇ microbeads made of 2% (w/v) oxidized alginate were generated by electrospray and then soaked in chitosan and alginate solutions in the same way as preparing the ACM encapsulated pre-differentiated aggregates to serve as the materials alone control.
  • cells were cultured at 37 °C in a humidified 5% C0 2 incubator.
  • nanoindentation was performed using an integrated system consisting of an Olympus 1X81 inverted optical microscope and an Asylum MFP-3D Bio atomic force microscope (AFM).
  • AFM Bio atomic force microscope
  • the cantilever was kept at the same temperature as the mESC aggregates prior to obtaining force curves.
  • the thermal tuning method was used to determine the cantilever's spring constant.
  • Multiple mESC aggregates were then suspended in PBS and allowed to settle onto the glass bottom of petri dishes (Fluorodish, World Precision Instruments, Inc.). Individual aggregates were identified and positioned underneath the cantilever tip using the inverted optical microscope.
  • Force versus indentation curves were then generated using the deflection data from the contact point between the sample surface and the cantilever tip.
  • Colloidal probes (2 ⁇ diameter polystyrene beads) were chosen for their low spring constant (0.32 N m "1 ) and spherical tip geometry, which are commonly used for indenting soft biological samples.
  • Force curves at a distance of 4 ⁇ were acquired at 0.5 Hz to reduce the viscoelastic effects that can occur when using larger approach velocities.
  • a relative trigger force of no more than 10 nN was also used to ensure that indentations did not produce any unwanted effects from the underlying substrate, as determined by preliminary testing over a wide range of indentation depths.
  • the resulting curves were then fit using a Hertz model built into the AFM software. Multiple force curves (at least 10) were taken for each sample in order to ensure that a consistent elastic modulus was produced. The resulting data sets were then combined and evaluated using statistical analysis for comparison,
  • mice were initially anesthetized by 3% isoflurane inhalation, intubated with a 20G intravenous catheter, and ventilated with a mixture of 02 (0.3 1 min-1) and 1.5-2% isoflurane (tidal volume of 250 ⁇ and 120 breaths min-1) with a mouse respirator (Harvard Apparatus, Holliston, MA, USA). Animals were placed in a right lateral decubitus position and a left thoracotomy was then performed through the left fourth intercostal space by cutting pectoralis muscles transversely to expose the thoracic cage. After removal of the pericardium, the left anterior descending artery was visualized and a 6-0 silk ligature was placed approximately 1 mm from origin of the vessel. The ligature was confirmed to be successful when the anterior wall of the left ventricle turned pale.
  • Triphenyl tetrazolium chloride (TTC) staining was conducted at 24 hr after surgery to confirm the ligation.
  • TTC Triphenyl tetrazolium chloride
  • -200 ⁇ of 0% Phthalo Blue were slowly injected into the aorta to stain the heart.
  • the heart were then rapidly collected and washed in 30 mM KC1 to cease the heart beating and allow for more consistent sectioning.
  • the heart was then frozen down for at least 4 hr at -20 °C before cutting them into slices of 1 mm.
  • the heart tissue slices were incubated with 2% TTC at 37 °C for 40 min. Further fixation of the stained slices with 10% formaldehyde overnight was performed, to increase the contrast between the infarct area and the normal tissue.
  • Fig. 4a Injection of cell aggregates and other control groups was performed as illustrated in Fig. 4a.
  • the single cells were obtained by dissociating Bare- A with Ix trypsin/EDTA, similarly to detaching 2D cultured cells.
  • the number of cells in each aggregate was determined by dissociating 50 aggregates using trypsin and counting the dissociated cells manually.
  • the same ceils with GFP were used for tracking cell differentiation in vivo. Mice randomly received a total of 20 ⁇ ! saline or saline containing single cells, Bare- A, ACM- A, or ACM via three injections given at three different sites in the periphery of infarcted tissue using a 28G needle within 5 minutes after the anterior wall of the left ventricle turned pale.
  • the size (diameter) of the aggregates in this study was measured to be 123.3 ⁇ 12.4 ⁇ before ACM encapsulation and 126.9 ⁇ 1 1.0 Liffi after ACM-encapsulation. Therefore, adding 3 times standard deviation (or 3 sigma) to the average equals -160 ⁇ , which is still much smaller than the inner diameter (184 ⁇ ) of a 28G syringe needle. Assuming normal distribution of the size, there is only 0.15% possibility for the cell aggregates to be bigger than 160 ⁇ by the three-sigma rule. In addition, the cell aggregates are soft and deformable, which can help to push them through the syringe needle. Indeed, there was no difficulty during injection of the cell aggregates during the experiments.
  • injection volumes of 20-30 ⁇ were used in other studies with mouse models, and the animal survival is not significantly different from that reported in the literature for the treatment with saline.
  • saline, ACM- A, and ACM treatments in 20 ⁇ of injection volume no granuloma formation was observed. Therefore, the effect of a total of 20 ⁇ of injection volume on granuloma formation and animal survival is insignificant in this study.
  • the level of anesthesia end-tidal concentration of isoflurane: 1-2%) was adjusted according to the surgical stimulation, by monitoring signs of movement. As soon as the anesthesia was stopped, the animals woke up immediately (normally in -1-2 min).
  • mice Sham operations were performed on 10 mice without LAD occlusion.
  • 4-6 mice were randomly assigned to the six groups including saline, single ceils (Single), Bare-A, ACM-A, and ACM.
  • the data of heart rate was plotted and showed no significant difference among different groups before versus after surgery.
  • 29 or more mice received treatment with saline, single cell, Bare-A, or ACM- A, and 24 mice were treated with ACM (materials alone).
  • the sample size was chosen to ensure adequate power to detect the difference between the saline control and ACM-A groups.
  • Mice were sacrificed by C0 2 exposure at 28 days, and heart samples were harvested for further analyses. No blind method is involved for the animal study. 105.
  • mice were attached with three electrical leads (lead I: right front foot, lead II: left rear foot, and lead III: left front foot) connected to the Vevo 2100 High-Resolution in vivo Imaging System for recording the electrocardiogram and heartbeat during the whole
  • a Millar (Houston, TX, US A) tip conductance catheter (Model SPR-893, 1.4 Fr.) was inserted into the right carotid artery, and further advanced into the left ventricle (LV). Baseline zero reference was obtained by placing the sensor in isotonic saline. After recording the basal hemodynamic parameters, a series of PV loops were generated using an AD Instruments (Colorado Springs, CO, USA) Power-Lab system connected to the Millar catheter. All the measurement and characterization were determined from the PV loop data using ChartPro Software (AD
  • the cryo-sectioning was conducted as follows: 10 locations were chosen from the apex to the ligation site with an even interval, and then 2-3 sections of 10 ⁇ in thickness ( 7) were cut at each location. All the resultant 20-30 sections were further stained with DAPI for visualizing nuclei and then examined under a Zeiss (Oberkochen, Germany) Axio Observer.Zl microscope with fluorescence capability. GFP positive cells (A) were counted on each section and averaged among the 20-30 sections. The length of the infarct zone (Z) was measured using a ruler. The retention/survival rate (RR) was calculated as follows:
  • mice were harvested on day 28 post treatments, fixed in 10% neutral buffered formalin for 24 hr at 4 °C, and embedded in paraffin. Both transverse and sagittal sections of 5 ⁇ thick were then cut using a microtome.
  • H&E staining sections were stained in hematoxylin 2 for 8 minutes and then washed by water, dipped in 1% acid alcohol and 1% ammonium hydroxide, stained in Eosin Y for 1 min, dehydrated with graded alcohols, and mounted on slides.
  • Masson ' s Tri chrome staining sample sections were fixed using Bouin' s Fixative overnight at room temperature and then stained in Weigert' s Iron hematoxylin for 10 minutes.
  • the fixed and paraffin-embedded heart tissues were cut into 5 um of sections. All sections were deparaffmized and hydrated in decreasing concentrations of ethanol. Antigen retrieval reagent (HK080-9K, BioGenex, Fremont, CA, USA) was used according to the manufacturer's instructions.
  • the heart sections were incubated with rabbit anti-GFP primary antibody (1 :200 dilution) for 3 h at room temperature in blocking buffer (2% BS A in PBS). Following washing with PBS, tissue sections were further incubated with goat anti-rabbit (H+L) Alexa 568 secondary antibody (Life Technologies, A-11011, 1 :500 dilution) for 1 h at room temperature.
  • the cell nuclei were visualized by staining the tissue with Vectashield Mounting Medium with DAPI (H-1500, Vector Laboratories, Buriingame, CA, LISA). Images for GFP staining were taken using an Olympus FluoViewTM FV1000 confocal Microscope.
  • Hiroi Y et al. Tbx5 associates with Nkx2-5 and synergist! cally promotes cardiomyocyte differentiation, Nat Genet 28, 276-280 (2001).
  • Hirt MN Hansen A, Eschenhagen T. Cardiac tissue engineering: state of the art. Circ Res 1 14, 354-367 (2014).
  • Proinflammatory protein CARD9 is essential for infiltration of monocytic fibroblast precursors and cardiac fibrosis caused by Angiotensin II infusion. Am J Hyper tens 24, 701-707 (201 1).
  • Zhang W et al. A novel core-shell microcapsule for encapsulation and 3D culture of embryonic stem cells. Journal of Materials Chemistry B 1, 1002-1009 (2013). Zhao S, et al Coaxial electrosprav of liquid core-hydrogel shell microcapsules for encapsulation and miniaturized 3D culture of pluripotent stem cells. Integrative Biology 6, 874-884 (2014).
  • GATGGGAAAGCTCCCACTATG SEQ ID NO: 1
  • GAGACACCAGGCTACGTCAATA SEQ ID NO: 2

Abstract

Disclosed are compositions and methods relating to micromatrices of encapsulated aggregates of pre-differentiated stem cells.

Description

This application claims the benefit of priority to U.S. Provisional Application
62/413,462, filed October 27, 2016, which is incorporated by reference herein in its entirety.
IL STATEMENT REGARDING FEDERA LLY SPONSORED RESEARCH OR
DEVELOPMENT
This study was supported by grant numbers CBET-1 154965 and CBET- 1605425 awarded by the National Science Foundation and grants number R01 AR063084, R01HL094650, R01HL124122, R01EB012108, R01AI123661 , and R01CA206366 awarded by the National Institutes of Health. The government has certain rights in the invention.
IIL BACKGROUND
1 . Myocardial infarction (MI) is a leading cause of death globally. Thi s is due, in part, to the fact that the human heart has a very limited capacity of self-repair, and that there is no clinical treatment targeting the loss of cardiomyocytes (CMs) following MI. Stem cel l therapy (SCT) has been explored as a promising option for regenerating cardiac tissue, including CMs, to treat MI. Various types of stem cells have been investigated exhibiting both advantages and disadvantages. To date, only pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), are well accepted to be capable of differentiating into functional CMs. However, the delivery of stem cel ls needs significant further improvement regardless of which types of stem cells are used.
2. The retention of single (i.e., dissociated) stem cells in the infarct zone delivered in suspension has been dismal (often less than -10% within a few hours to a few days post injection). In addition, the retained cells can die of the hostile MI microenvironment that could be exacerbated by the implanted cell s to trigger immune reactions. The presence of macrophages together with the cytokines secreted by them in the first few days after MI, creates a strong proinflammatory environment resulting in chemo-attraction of more immune cells and damage to the transplanted stem cells. Temporary systemic immunosuppression for a few days has been proposed to mitigate immune rejection to the implanted stem cells to improve their survival.
However, systemic immunosuppression could induce severe complications to patients including infection and possible cancer occurrence. Lastly, it has been reported that surviving PSCs can form teratomas, consisting of cells of all the three different lineages (i.e., ectoderm, mesoderm,
..... i ..... and endoderm), in the heart. However, attempts to address this issue via the implantation of mature cardiomyocytes has been reported to cause an electromechanical mismatch with the host cardiomyocytes.
3. What are needed are new treatments and methodologies that can avoid the issues of retention loss, inflammation, teratoma formation, and rejection.
IV. SUMMARY
4. Disclosed are methods and compositions related to micromatrixes of encapsulated aggregates of pre-differentiated stem cells.
5. Disclosed herein are methods of making a micromatrix comprising encapsulated aggregated pre-differentiated stem cells the method comprising a) microencapsulating a stem cell, wherein the microcapsule comprises a permissive liquid core and a hydrogel shell; b) expanding the microencapsulated stem cells for at least 1 day; wherein the microencapsulated stem cells proliferate and form aggregates; c) pre-differentiating the microencapsulated stem cell s into early stage cardiac lineage cell s; d) releasing the aggregates of pre-differentiated microencapsulated stem cells from the core-shell microcapsules; and e) encapsulating the released pre-differentiated stem cells in a micromatrix.
6. In one aspect, disclosed herein are methods of making a micromatrix compri sing encapsulated aggregated pre-differentiated stem ceils of any preceding aspect, wherein the stem cell comprises an embryonic stem cell, induced plunpotent stem cells, extraembryonic fetal stem cells, amniotic stem ceils, or adult stem.
7. Also disclosed herein are methods of making a micromatrix comprising encapsulated aggregated pre-differentiated stem cells of any preceding aspect, wherein the stem cel l is microencapsulated via coaxial eiectrospray.
8. In one aspect, disclosed herein are methods of making a micromatrix comprising encapsulated aggregated pre-differentiated stem cells of any preceding aspect, wherein hydrogel shell is a semipermeable alginate hydrogel shell.
9. In one aspect, disclosed herein are methods of making a micromatrix comprising encapsulated aggregated pre-differentiated stem cell s of any preceding aspect, wherein the microencapsulated stem cells are expanded for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 days (such as, for example, culturing the microencapsulated stem ceils in a media comprising fibroblast growth factor (FGF) and bone morphogeny e protein. 10. In one aspect, disclosed herein are methods of making a micromatrix comprising encapsulated aggregated pre-differentiated stem ceils of any preceding aspect, wherein the pre- differentiated microencapsulated stem cells comprise first heart field (FHF) cells, second heart field (SHF) cells, or epicardium derived cells (EPDC). 11. Also disclosed herein are methods of making a micromatrix comprising encapsulated aggregated pre-differentiated stem cells of any preceding aspect, wherein the pre-differentiated microencapsulated stem cells are released from the core-shell microcapsules by dissolving the hydrogen using an isotonic solution of sodium.
12, In one aspect, disclosed herein are methods of making a micromatrix comprising encapsulated aggregated pre-differentiated stem ceils of any preceding aspect, wherein the released pre-differentiated stem cells of step e are encapsulated in a micromatrix by soaking the released aggregates in a solution comprising chitosan and/or alginate (such as, for example, oxidized alginate); wherein the solution comprises a pH of about 6 to about 8 (for example, about 6.7 to about 7.2). 13. Disclosed herein are micromatrix of encapsulated aggregates of stem cells wherein the stem cells are pre-differentiated stem cells; and wherein the stem cells are encapsulated by an integral with a micromatrix of alginate and chitosan.
14. In one aspect, disclosed herein are encapsulated aggregates of stem cells of any- preceding aspect, wherein the encapsulated aggregate of stem cells comprises about 1500 stem ceils.
15. Also disclosed herein are encapsulated aggregates of stem cells of any preceding aspect, wherein the encapsulated aggregate of stem cells is an early stage cardiac lineage multi-potent stem cell.
16. In one aspect, disclosed herein are methods of treating damage caused by or reducing the severity of an ischemic event (such as, for example, arterial embolism, venous embolism, thromboembolism, pulmonary embolism, traumatic injur}', atherosclerosis, myocardial infarction, thoracic outlet syndrome, tachycardia, hypotension, tourniquet, or surgery) in a subject comprising administering to the subject the encapsulated aggregate of stem cells of any- preceding aspect, 17. Also disclosed herein are methods treating damage cause by or reducing the severity of an ischemic event of any preceding aspect, wherein the ischemic event comprises a myocardial infarction and wherein the stem cells are pre-differentiated to an early stage cardiac lineage multi-potent stem cell.
18. In one aspect, disclosed herein are methods treating damage cause by or reducing the severity of an ischemic event of any preceding aspect, wherein the encapsulated aggregate of stem cells is administered to the subject within 7 days of the ischemic event.
V. BRIEF DESCRIPTION OF THE DRAWINGS
19. The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and together with the description illustrate the disclosed compositions and methods. 20. Figure 1 shows the bioinspired approach for preparing pluripotent stem cells to implant by injectable delivery. Figure 1 A shows a schematic illustration of the multi-step procedure to prepare the totipotent-pluripotent stem cells for implantation in the uterus wall, including proliferation to form a microscale ceil aggregate in zona pellucida (i.e., morula), pre- differentiation of morula into trophoblast cells and inner cell mass in the zona pellucida, hatching out of the zona pellucida, and re-encapsulation in the trophoblast before implantation during early embryo development in the female reproductive system. Figure IB shows a schematic illustration of the bioinspired procedure for producing 3D microscale constaicts of murine embryonic stem cells (mESCs) together with real images, showing the analogy between the bioinspired approach and the aforementioned natural procedure. The bioinspired approach mimics the natural procedure phenomenologically rather than mechanistically. Scale bar: 100 μηι
21. Figure 2 shows the pre-differentiation of the mESC aggregates into the early cardiac stage. Figure 2A shows microarray data showing significantly increased expression of mesoderm and cardiac marker genes and significantly decreased expression of piuripotency marker genes in the aggregated cells after pre-differentiation. Figure 2B shows flow cytometry data showing successful pre-differentiation of the mESC aggregates with diminished expression of piuripotency protein makers (OCT-4 and NANOG). Figure 2C shows flow cytometry data showing early cardiac pre-differentiation with significantly increased expression of cardiac specific protein marker (cTnT) and the early cardiac protein marker (NKX2.5). 22. Figure 3 shows the characterization of the aggregates pre-differentiated to the early cardiac stage. Figure 3 A shows SEM images showing successful encapsulation of the pre- differentiated cell aggregates with the alginate-chitosan micromatrix (ACM) both outside (first two columns) and inside (third column) the aggregates. Scale bars; 5 μτη. Figure 3B shows confocal fluorescence micrographs of the middle plane of the ACM-A showing micromatrix inside the aggregates indicated by labeling alginate in the ACM with FITC to show up with green fluorescence. Scale bar: 50 um. Figure 3C shows the elastic modulus of the pre- differentiated aggregates is significantly increased after ACM encapsulation. The modulus was determined by atomic force microscopy (AFM) nanoindentation. Error bars represent standard deviation (s.d., n= 3). *, p < 0.05 (Student two-tailed t test)
23. Figure 4 shows therapy of myocardial infarction by injecting A CM -encap ulated pre- differentiated aggregates. Figure 4A shows a schematic illustration of surgical ligation (X) of the LAD at its proximal location and implantation of samples by intramyocardial injection at three different locations to create large-area myocardial infarction (MI). Figure 4B shows typical gross images of a heart with no MI and MI hearts with five different treatments showing granulomas in single cell (Single) and Bare-A treated mice (arrows), scale bar: 3 mm. Figure 4C shows quantitative data of cumulative granuloma occurrence in both wild type (WT) and CARD9 knockout (K.O) mice, showing treatments with single, Bare-A, Bare-A-KO have significantly higher occurrence of granuloma than the other treatments including ACM-A. The animal number (n) was 10, 31, 29, 38, 31, 24, and 9 for No MI, Saline, Single, Bare-A, ACM-A, ACM, and Bare-A-KO, respectively. *, p < 0.05 (Chi-square test). Figure 4D shows typical micrograph of granuloma with immunofluorescence staining of F4/80 (for macrophage, red) and CD3 (for T cells, red) showing many immune cells within a loose matrix in the granuloma collected at 28 days after injected with Bare-A. The nuclei are stained blue. Scale bar: 20 urn. Figure 4E shows data of implanted cells (expressing green fluorescence protein, GFP) retained and survived in the heart after 28 days showing a significantly higher cell survival with the ACM-A treatment than all the other treatments. The cell survival was quantified by counting cells with green fluorescence in the heart from the apex to the point of ligation. Error bars represent s.d. (n=3). *,p < 0.05 (one-way ANOVA). Figure 4F shows the survival of WT MI mice at 28 days after injection, showing the ACM-A treatment can maintain a significantly higher animal survival than all the other treatments. The animal number (n) was 31, 29, 38, 31, and 24 for Saline, Single, Bare-A, ACM-A, and ACM, respectively. *, p < 0,05 (one-way ANOVA). Figure 4G shows ejection fraction measured by PV loops showing the ACM-A treatment significantly improves the heart function after MI, compared to saline, Bare-A, and ACM treatments. Error bars represent s.d. (n=4 for Single and n=3 for other groups). *, p < 0.05 (one-way ANOVA) 24, Figure 5 shows cardiac regeneration in situ with the ACM-encapsulated pre- differentiated aggregates. Figure 5 A shows low-magnification sagittal micrographs of Masson' s trichrome stained tissue sections (top row) and zoom-in views of the left ventricular wall (bottom row) showing extensive fibrosis in the MI hearts treated with saline, materials alone (i.e., ACM), single cells, and Bare- A while it is minimal with the ACM-A treatment. Scale bar: 2 mm (top row) and 00 μιη (bottom row). Figure 5B shows quantitative analysis showing the ACM-A treatment significantly reduces fibrosis in the MI heart. The n=3 and *, p < 0.05 (oneway ANOVA). Figure 5C shows micrographs of sectioned and H&E stained tissue
(Morphology) in the MI zone of heart treated with the ACM-A showing green fluorescent CM- like cells (GFP) and their seamless integration with the neighboring non -fluorescent native host tissue (Merged). Scale bar: 100 μτη. Figure 5D shows immunohistochemically stained tissue from the MI zone of hearts treated with the ACM-A showing positive staining of CM (cTnl, connexin 43, and a-actinin, in red) markers co-localized with injected GFP cells. The striated pattern of the cardiac tissue with green fluorescence is evident. Scale bar: 10 μηι. All tissues were harvested on day 28 after intramyocardial injection.
25. Figure 6 shows a schematic illustration of the setup for coaxial electrospray. Figure 6A shows a schematic overview of the setup for coaxial electrospray to encapsulate murine embryonic stem cells (mESCs) in core-shell microcapsules. Figure 6B shows a zoom-in cross- sectional view of the coaxial needle. The core solution (green) with live mESCs and the shell solution of alginate (gray) are pumped through the inner and outer lumen in the coaxial needle, respectively, resulting in the formation of concentric drops at the needle tip. The drops are then broken up into microscale droplets under an open electrostatic field (that is, with no electric current), and sprayed into the gelling bath that contains divalent calcium cations for instant gelling of alginate to form the hydrogel shell of the resultant microcapsules. 26. Figure 7 shows the biodegradation of the micromatrix of alginate and chitosan in vitro. Figure 7A shows quantitative data showing alginate-chitosan micromatrix (ACM) encapsulation slows down the attachment of the pre-differentiated cells from their aggregates when cultured in tissue culture petri dishes. Error bars represent s.d. (n=3). **, p < 0,01 (Student's two-tailed t test). Typical differential interference contrast (DIC) and fluorescence images showing the morphology and high cell viability of bare (FIG, 7B) and ACM-encapsulated (FIG 7C) pre- differentiated aggregates cultured in petri dishes for 15 min, 1 day, and 3 days. Scale bar: 100 um. Cells gradually detached from ACM-encapsulated aggregates and attached on petri dish as a result of the gradual degradation of the oxidized alginate in the ACM. Bare-A: Bare pre- differentiated aggregates. ACM-A: ACM-encapsulated, pre-differentiated aggregates.
27. Figure 8 shows the biodegradation of the micromatrix of alginate and chitosan in vivo. Figure 8A shows IVIS images showing successful labeling non-oxidized (NonOxi) alginate and oxidized (Oxi) alginate (by default and used for making the alginate-chitosan micromatrix or AC : in this study) with indocyanine green (ICG). Figure 8B shows ICG fluorescence in the MI heart of mice injected with Bare-A, NonOxi- ACM-A-ICG made of ICG labeled non-oxidized alginate, and ACM-A-ICG made of ICG labeled oxidized alginate. No fluorescence was observed in the hearts treated with Bare-A on days 0 (I h), 1, and 3. The fluorescence in the hearts treated with ACM-A-ICG reduced over time and disappeared on day 3, indicating the ACM gradually degraded over three days in vivo in the heart. The degradation of NonOxi- ACM-A-ICG is slower than that of ACM-A-ICG and the fluorescence was still observable on day 3 in the heart. Figure 8C shows ICG fluorescence in mice subcutaneously injected with Bare-A, NonOxi -ACM-A-ICG, and ACM-A-ICG. The trend of degradation for the
subcutaneously injected NonOxi-ACM-A-ICG and ACM-A-ICG is similar to the
intramyocardialiy injected NonOxi -ACM-A-ICG and ACM-A-ICG. The former degrades slower than the latter.
28. Figure 9 shows the cardiac functional data from PV loop measurements at 28 days after surgery. Figure 9A shows the maximum and minimum rate of pressure change (dP/dt). Figure 9B shows the cardiac output and stroke volume. The data show that the treatments with cells (Single, Bare-A, and ACM-A) significantly improve the heart function of mice with MI, compared to the saline and ACM treatments with no cell. Error bars represent s.d. (n=4 for Single and n==3 for all other groups), *, ? < 0.05 (one-way ANOVA)
29. Figure 10 shows the cardiac functional data from PV loop measurements at 28 days after surgery. Figure 10A shows the isovolumic relaxation constant (Tau). Figure 10B shows the left ventricle (LV) end-diastolic volume (LVED), and LV end-systolic volume (LVES). The data indicates ACM-A improves heart function by consistently maintaining a nearly intact isovolumic relaxation with minimal enlargement of the left ventricle (LV), compared to the other treatments. Error bars represent s.d. (n=4 for Single and n= 3 for all other groups). *, p < 0,05 (one-way ANOVA)
30. Figure 1 1 shows antibody staining of green fluorescent protein. The green fluorescent protein (GFP) was stained in tissue from the infarct zone of heart treated with ACM-A made of GPF positive cells. The extensive co-localization of the green fluorescence with the antibody staining of GFP (in red) indicates the green fluorescence is indeed from the implanted cells with GFP expression. The cell nuclei were stained with DAP I (blue). DIC: differential interference contrast. Scale bar: 100 urn.
VL DETAILED DESCRIPTION
31. Before the present compounds, compositions, articles, devices, and/or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods or specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the puipose of describing particular embodiments only and is not intended to be limiting.
A. Definitions
32. As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.
33. Ranges can be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as "about" that particular value in addition to the value itself. For example, if the value " 10" is disclosed, then "about 10" is also disclosed. It is also understood that when a value is disclosed that "less than or equal to" the value, "greater than or equal to the value" and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value "10" is disclosed the "less than or equal to 10"as well as "greater than or equal to 10" is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point " 10" and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.
34. In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:
35. 'Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.
36. Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.
B. Compositions and Methods
37. Disclosed are the components to be used to prepare the disclosed compositions as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular micromatrix comprising encapsulated aggregated pre-differentiated stem cells is disclosed and discussed and a number of modifications that can be made to a number of molecules including the micromatrix comprising encapsulated aggregated pre-differentiated stem cells are discussed, specifically contemplated is each and every combination and permutation of micromatrix comprising encapsulated aggregated pre-differentiated stem cells and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B- D, B-E, B~F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods. 38. Ischemic events such as myocardial infarction result in severe tissue damage that will not repair in the subject in which the event occurred. To combat the damage associated with ischemia, many new treatments have been attempted including the use of pluripotent stem cells. However, delivery of stem cells in tissue-engineered constructs in the form of a macro-scale (up to a few centimeters) hydrogel, porous scaffold, or cell sheet/patch while this may improve cell retention is not a viable solution. There is significant cell death inside the macro-scale constructs due to the limited diffusion length of oxygen in vivo (less than -150 μηι), and it can require multiple surgeries to overcome the diffusion limit of oxygen in vivo for using ceil sheets/patches less than -150 μτη thick as ID microscale stem cell constructs,
39. Moreover, following ischemic events (such as, for example, myocardial infarction), the microenvironment can be hostile due to the presence of pro-inflammatory immune cells such as macrophages which secrete cytokines at the site of injury and reduce engraftment of the transplanted cells. Injection of stem cells at 4-7 days after MI may help to improve the survival of the implanted/retained cells. However, significant injury to the infarcted myocardium would accumulate during the 4-7 days of delay. Therefore, early treatment to minimize the injury or pathological development after Ml is desired.
40. The disclosure herein provides novel compositions and treatments that do not suffer the limitations of previous methods and overcome obstacles posed by the microenvironment. By- encapsulating stem cells in a micromatrix rather than a porous scaffold or shell-core
microcapsule, the effects of the microenvironment on the stem ceils can be minimized allowing for early delivery of stem cells and avoiding further damage to the tissue at the site of injur}'. This also has the benefit of extended release. Accordingly, in one aspect, disclosed herein are micromatrixes of encapsulated aggregates of stem cells; wherein the stem cells are encapsulated by and integral with a micromatrix. The micromatrix can be comprised on an material suitable for production of a matrix and supporting the encapsulated cells. In one aspect, the micromatrix can be formed through the combination of a positively charged marcromolecule (for example, Poly-l-lysine or chitosan) and a negatively charged macromolecule (for example, alginate or hyaluronan). For example, in one aspect disclosed herein are micromatrixes of encapsulated aggregates of stem cells; wherein the stem cells are encapsulated by and integral with a micromatnx, wherein the micromatrix comprises alginate (for example oxidized alginate) and chitosan. In one aspect, the stem cells can be pre-differentiated to avoid teratoma formation. Accordingly, disclosed herein are micromatrixes of encapsulated aggregates of stem cells;
wherein the stem cells are pre-differentiated stem cells; and wherein the stem cells are encapsulated by an integral with a micromatrix of alginate (for example oxidized alginate) and chitosan.
41. It is understood and herein contemplated that the micromatrix of encapsulated aggregates of stem cells comprises pre-differentiated cells that are differentiated to facilitate their use in repairing tissue damage and minimize the potential for teratoma formation. Therefore, for cardiac related ischemias it is advantageous to pre-differentiate the PSCs into the early cardiac stage lineage cells rather than into mature cardiomyocytes for implantation into the heart. This approach utilizes the native chemical, mechanical, and electrical cues in the heart to further guide the pre-differentiated cells (at the early cardiac stage) into mature cardiomyocytes with similar electromechanical properties to the native CMs. Accordingly, in one aspect, the pre- differentiated cells can be an early stage cardiac lineage multi-potent stem cell such as, for example first heart field (FHF) cells, second heart field (SHF) cells, and/or epicardium derived cells (EPDC).
42, The disclosed aggregates of pre-differentiated stem cells can comprise a large number of ceils. In one aspect, the aggregates can comprise at least 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, or 2500 cells/ aggregate construct. For example, the aggregate can comprise about 1500 cells. Thus, in one aspect disclosed herein are encapsulated aggregates of stem cells of any preceding aspect, wherein the encapsulated aggregate of stem cells comprises about 1500 stem cells. 43. It is understood and herein contemplated that the alginate chitosan matrix will degrade over time releasing the aggregates of pre-differentiated stem cells as the degradation occurs. The gradual release of cells insures a continual population of pre-differentiated ceils over the course of degradation. In one aspect, the micromatrix of encapsulated aggregates of stem cells comprises an alginate chitosan matrix (ACM) that completely degrades in about 24hrs, 36hrs, 48hrs, 60 hrs, 72 hrs, 84hrs, 96hrs, 5 days, 6 days, or 7 days. In one aspect, the degradation takes at least 2, 3, 4, 5, 6, or 7 days. 44. The preparation of aggregates of pre-diff erentiated stem cells in a matrix overcomes the limitations to previous stem cell based therapeutics of ischemic injury. Prior to the present disclosure no micromatrix of encapsulated aggregates of pre-diff erentiated cells has existed. Thus, the preparation of such cells is unique to the disclosed cells. Thus, in one aspect, disclosed herein are methods of making a micromatrix comprising encapsulated aggregated pre- diff erentiated stem cells comprising a) microencapsulating a stem cell, wherein the microcapsule comprises a permissive liquid core and a hydrogel shell; b) expanding the microencapsulated stem cells for at least 1 day; wherein the microencapsulated stem ceils proliferate and form aggregates; c) pre-differentiating the microencapsulated stem cells into early stage cardiac lineage cells; d) releasing the aggregates of pre-diff erentiated microencapsulated stem cells from the core-shell microcapsules; and e) encapsulating the released pre-differentiated stem cells in a micromatrix.
45. As used herein, "stem cell" refers to any multipotent, pluripotent, or totipotent progenitor ceil capable of self-renewal including, but not limited to embryonic stem cell, induced pluripotent stem cells, extraembryonic fetal stem cells, amniotic stem cells (including cells stem cells obtained from the cord blood), and adult stem cells (including, but not limited to hematopoietic stem cells, endothelial stem cells, intestinal stem cells, mammary stem cells, neural stem cells, and mesenchymal stem ceils). Thus, in one aspect, disclosed herein are methods of making a micromatrix comprising encapsulated aggregated pre-differentiated stem cells of any preceding aspect, wherein the stem cell comprises an embryonic stem cell, induced pluripotent stem cells, extraembryonic fetal stem cells, amniotic stem cells, or adult stem.
46. To form a core-shell microcapsule, the microcapsule can be formed (i.e., the
microencapsulation) via any means known in the art including, but not limited to coaxial electrospray, oil-aqueous solution emulsion, pan coating, air-suspension coating, centrifugal coating, vibrational nozzle, spray-drying, ionotropic gelation, coacervation-phase separation, interfacial polycondensation, and interfacial cross-linking.
47. The core of the microcapsule, which will contain the stem ceils, can comprise any aqueous solution suitable for the propagation of cells including, but not limited to saline, collagen, hyaluronan, fibrin, laminin, ceil culture media (such as, for example, Minimal essential media (MEM), Eagles' minimal essential medium (EMEM), Dulbecco's Modified Eagle Media (DMEM), Ham's Nutrient Mixtures (Ham' F-10, and Ham's F-12), Roswell Park Insitute Medium (RPMI), Iscove's Modified Dulbecco's Medium and (IMDM)), Hanks' balanced salt solution, Earle's balanced salt solution, and Dulbecco's phosphate-buffered saline. In some aspect, the core can be a hydrogel.
48. The shell of the microcapsule can comprise any material from which a shell may be formed. In one aspect, the shell can be a hydrogel material comprising alginate, collagen, silicone, polyvinyl alcohol (PVA), sodium poiyacrylate, polyethylene oxide, polyethylene glycol, polyvinylpyrrolidone, poly-methyl methacrylate, and any co-polymer or ter-polymers thereof. In one aspect, the shell can be a semipermeable alginate hydrogel shell. In another aspect, the hydrogel shell can comprise materials found in the zona pellucida of an embryo.
49. To expand the starting stem cell population and facilitate the pre-differentiation of the stem cells to early stage multi-potent cells of a committed lineage, the core-shell
microencapsulated stem cells can be expanded for at least 60, 12, 18 hours, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11. 12, 13, 14, 15, 16. 17, 18, 19. 20, 21, 22, 23, 24. 25, 26, 27. 28, 29, or 30 davs.
50. To direct the stem cells to differentiate towards a particular lineage it is understood and herein contemplated that growth factor supplements can be added to the microcapsule core that will facilitate a direction of differentiation. For example, to pre-differentiate stem cells into early stage multipotent cardiac lineage cells, the stem cells can be cultured in core comprising fibroblast growth factor (FGF), tbx l 8, Oct4, Sox2, Klf4, cMyc, NRG, IGF, Notch 1, wingless- related integration site (Wnt) protein (including, but not limited to Wntl, Wnt2, Wnt3, Wnt4, Wnt5, Wnt6, Wnt7A, Wnt7b, WntSA, WntSB, Wnt9A, Wnt.9B, Wntl OA, WntlOB, Wntl 1 and Wntl 6), β-catenin and/or bone morphogenic protein (BMP) such as, for example BMP-4.
Accordingly, in one aspect, disclosed herein are methods of making a micromatrix comprising encapsulated aggregated pre-differentiated stem cells of an preceding aspect, wherein the microencapsulated stem cells are expanded for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days in a microcapsule core comprising a media comprising FGF and BMP-4.
51. In one aspect, it is understood and herein contemplated that where a muiti -potent stem cell, or stem cell already committed to a particular lineage or an organ specific lineage stem cell is used in the core of the core-shell macromoiecule, further differentiation, depending on the application, could be avoided while propagation and aggregate formation would continue to take place during culture. Accordingly, in one aspect, disclosed herein are methods of making a micromatrix comprising encapsulated aggregated pre-differentiated stem cells comprising a) microencapsulating a stem cell, wherein the microcapsule comprises a permissive liquid core and a hydrogel shell, b) expanding the microencapsulated stem cells for at least 1 day; wherein the microencapsulated stem cells proliferate and form aggregates: d) releasing the aggregates of pre-differentiated microencapsulated stem cells from the core-shell microcapsules, and e) encapsulating the released pre-differentiated stem cells in a micromatrix. 52. As noted herein, the disclosed herein are methods of making a micromatrix comprising encapsulated aggregated pre-differentiated stem cells can be used to provide a micromatrix of aggregated pre-differentiated cells of any cell lineage that would be of interest given the application. For example, the pre-differentiated can comprise a bone-marrow derived stem cells, adipose derived stem cells, or any organ specific stem cells such as neural stem cells or cardiac stem cells. In an cardiac or ischemic application, the stem cells can be pre-difTerentiated to early- stage cardiac lineage multipotent cells such as, for example, first heart field (FHF) cells, second heart field (SHF) cells, or epicardium derived cells (EPDC), Thus in one aspect disclosed herein are methods of making a micromatrix comprising encapsulated aggregated pre-differentiated stem cells, wherein in the pre-differentiated stem cells are FHF, SHF, or EPDC cells. 53 , To form the micromatrix, the pre-differentiated stem cell must be released from the core- shell microcapsule. This can be accomplished by any means suitable to dissolved the shell of the microcapsule and will depend on the composition of the shell. In one aspect, disclosed herein are methods of making a micromatrix comprising encapsulated aggregated pre- differentiated stem cells of any preceding aspect, wherein the pre-differentiated
microencapsulated stem cells are released from the core-shell microcapsules by dissolving the hydrogel using an isotonic solution of sodium.
54. Once released from the microcapsule, the aggregates of pre-differentiated stem cells must be re-encapsulated in a micromatrix. In one aspect, disclosed herein are methods of making a micromatrix comprising encapsulated aggregated pre-differentiated stem cells of any preceding aspect, wherein the released pre-differentiated stem cells of step e are encapsulated in a micromatrix by soaking the released aggregates in a solution comprising chitosan and/or alginate (such as, for example, oxidized alginate). The solution comprising alginate and chitosan can be at any physiological relevant pH for the formation the micromatrix and continued viability of the pre-differentiated stem cells. For example, the pH can be any pH between about 6 and about 8, more preferably between about 6.5 and about 7.5, most preferably between about 6.7 and about 7.2. In one aspect, the pH can be 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7,3, 7,4, 7,5, 7,6, 7.7, 7.8, 7.9, or 8.0, Thus, in one aspect, disclosed herein are methods of making a micromatrix comprising encapsulated aggregated pre- differentiated stem cells of any preceding aspect, wherein the released pre-differentiated stem cells of step e are encapsulated in a micromatrix by soaking the released aggregates in a solution comprising chitosan and/or alginate; wherein the solution comprises a pH of about 6.7 to about 7.2. 55. It is understood and herein contemplated that the micromatrix encapsulated pre- differentiated stem cells can be used to treat damaged tissue resulting from an ischemic event or reduce ischemic damage that a subject is in risk of experiencing.
56. As used herein "Treat," "treating," "treatment," and grammatical variations thereof as used herein, include the administration of a composition with the intent or purpose of partially or completely preventing, delaying, curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving, stabilizing, mitigating, and/or reducing the intensity or frequency of one or more a diseases or conditions, a symptom of a disease or condition, or an underlying cause of a disease or condition. Treatments according to the invention may be applied preventively, prophyiactically, pailatively or remediaily. Prophylactic treatments are
administered to a subject prior to onset (e.g., before obvious signs of cancer), during early onset (e.g., upon initial signs and symptoms of cancer), or after an established development of cancer. Prophylactic administration can occur for day(s) to years prior to the manifestation of symptoms of an infection.
57. The ischemic event can be any ischemic event where tissue damage results from the change in blood flow including, but not limited to embolism (such as, for example, arterial embolism, venous embolism, thromboembolism, pulmonary embolism), traumatic injury, atherosclerosis, myocardial infarction, thoracic outlet syndrome, tachycardia, hypotension, tourniquet, and surgery. It is understood and herein contemplated that the ischemia is not limited to cardiac ischemia, but can include acute and chronic brain ischemia (including transient ischemic attacks), acute limb ischemia, and bowel ischemia. Accordingly, in one aspect, disclosed herein are methods of treating damage caused by or reducing the severity of an ischemic event (such as, for example, arterial embolism, venous embolism, thromboembolism, pulmonary embolism, traumatic injury, atherosclerosis, myocardial infarction, thoracic outlet syndrome, tachycardia, hypotension, tourniquet, or surgery) in a subject comprising
administering to the subject the encapsulated aggregate of stem cells disclosed herein. For example, in one aspect disclosed herein are methods treating damage cause by or reducing the severity of any preceding aspect, wherein the ischemic event comprises a myocardial infarction and wherein the stem cells are pre-dif erentiated to an early stage cardiac lineage multi-potent stem cell.
58. As described above, the compositions can also be administered in vivo in a
pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.
59, The material s may be in solution, suspension (for example, incorporated into
microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et ai., Bioconjugate Chen?., 2:447-451 , (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et a!., Br. J. Cancer, 58:700-703, (1988); Senter, et al,, Bioconjugate Chem., 4:3-9, (1993); Battelli, et al ., Cancer Immunol. Immunoiher., 35 :421-425, (1992); Pietersz and McKenzie, Immunolog.
Reviews, 129:57-80, (1992), and Roffler, et al ,, Biochem. Pharmacol, 42:2062-2065, (1991 )). Vehicles such as "stealth" and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al.. Cancer Research, 49:6214- 6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1 104: 179-187, (1992)). In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intraceliuiariy, or are degraded in iysosomes. The internalization pathways serve a vari ety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracel lular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).
60. Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malomc acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, triaikyl and aryl amines and substituted ethanol amines.
61. "Therapeutically effective amount" or "therapeutically effective dose" of a composition (e.g. a composition comprising an agent) refers to an amount that is effective to achieve a desired therapeutic result. In some embodiments, a desired therapeutic result is the control of type I diabetes. In some embodiments, a desired therapeutic result is the control of obesity. Therapeutically effective amounts of a given therapeuti c agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject. The term can also refer to an amount of a therapeutic agent, or a rate of deliver)' of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect, such as pain relief. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the agent and/or agent formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of agent in the tonnulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art. In some instances, a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years.
62. Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms of the disorder are effected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindi cations. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. For example, guidance in selecting appropriate doses for antibodies can be found in the literature on therapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J., (1985) ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp. 365-389. In one aspect, a dose of about lxlO4, 2x104, 3xl04, 4xl04, 5xl04, 6x I 04, 7xl04, 8xl04, 9xl04, Ix O5, 2xl05, 3xl05, 4xl05, 5xl05, 6xl05, 7xl05, 8xl05, 9xl05, or IxlO6 pre-differentiated cells can admi ni stered to th e subj ect ,
63. The compositions may be administered orally, parenteraily (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, intramyocardial injection, transdermally, extracorporeally, topically or the like, including topical intranasal administration or
administration by inhalant. As used herein, "topical intranasal administration" means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise deliver}' by a spraying mechanism or droplet mechanism, or through aerosoiization of the nucleic acid or vector. Administration of the compositions by inhalant can be through the nose or mouth via deliveiy by a spraying or droplet mechanism. Deliveiy can also be directly to any area of the respirator}' system (e.g., lungs) via intubation. The exact amount of the compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.
64. Parenteral administration of the composition, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein.
65. The compositions, including micromatrixes, can be used therapeutically in combination with a pharmaceutically acceptable carrier. 66, Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, East on, PA 1995, Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.
67. Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH, The compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.
68. Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice.
Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, anti -inflammatory agents, anesthetics, and the like.
69, Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringers, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti -oxidants, chelating agents, and inert gases and the like. 70. Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. 71. Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable,
72. Other molecules which do not have a specific pharmaceutical function, but which may be used for tracking changes within cellular chromosomes or for the delivery of diagnostic tools for example can be delivered in ways similar to those described for the pharmaceutical products,
73. In one aspect, the disclosed micromatrixes may be administered concurrently or in combination with a therapeutic agent useful in the treatment of damage resulting from ischemia. "Therapeutic agent" refers to any composition that has a beneficial biological effect, Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition (e.g., an ischemia such as, for example, myocardial infarction). The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the terms "therapeutic agent" is used, then, or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically
acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc. 74. The disclosed micromatrixes can be used for the therapeutic treatment of damage to tissue caused by an ischemic event. In one aspect, the micromatrixes can be administered to the subject during, or 1, 2, 3, 4,5 ,6, 7 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24hrs, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 45, 60, 75, 90 days, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, 2, 3, 4, 5, 6, 7, 8, 9 , or 10 years after the ischemic event. Thus, in one aspect, disclosed herein are methods treating damage cause by an ischemic event comprising administering to the subject a micromatrix of
encapsulated aggregates of pre-differentiated stem cells, wherein the encapsulated aggregate of stem cells is administered to the subject within 7 days following the ischemic event.
75. It is further understood that in some instances such as surgery, ischemia can be induced medicinally or mechanically. Additionally, risk factors for ischemia can be sufficiently significant to warn of an immediate likelihood of ischemia (such as, embolism or myocardial infarction). In such instances as surgery or significant risk of ischemia, the disclosed micromatrixes can be administered prophylactically. For example, the micromatrixes can be administered during or 1, 2. 3, 4,5 ,6, 7 8, 9, 10. 11, 12, 13, 14, 15. 16, 17, 18. 19, 20, 21, 22, 23.
24hrs, 2, 3, 4, 5, 6, or 7 days before the ischemic event. Accordingly, disclosed herein are methods of reducing tissue damage caused by an ischemic event in a subject comprising administering to the subject a micromatrix of encapsulated aggregates of pre-differentiated stem ceils, wherein the encapsulated aggregate of stem cells is administered to the subject within about 1, 2, 3, or 4 days prior to the ischemic event.
C. Examples
76. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric. a) RESULTS
(1) Preparing PSCs for implantation by injection to treat Ml.
77. The approach for preparing PSCs for implantation by injectable delivery to treat MI is inspired by the multi-step procedure used by nature to prepare totipotent-pluripotent stem cells for implantation into the uterus. This natural procedure includes the following steps (Fig. la): proliferation of the totipotent-pluripotent stem cells in the miniaturized permissive core enclosed in the semipermeable shell (known as zona pellucida) of pre-hatching embryos into a
multicellular aggregate known as morula, pre-differentiation of the aggregated cells into trophoblast cells and inner cell mass, hatching out of zona pellucida and re-encapsulation within the trophoblast, and implantation into the uterus wall. Because the experimental strategy shares some similarity with the basic steps of the early embryonic development and includes proliferation, pre-differentiation, zona hatching, and re-encapsulation of the natural procedure, referred to herein as the 'bioinspired' approach, (illustrated in Fig. lb). First, approximately 21 ± 4 single (i .e., dissociated) murine ESCs (mESCs) were microencapsulated in the permissive liquid core of microcapsules with a semipermeable alginate hydrogel shell produced using the coaxial electrospray technology (Fig. 6) for culture. The mESCs proliferate to form one single aggregate of 128.9 ± 1 7.4 μιη consisting of 1450 ± 381 ceils per aggregate with high (>90%) viability in each microcapsule in 7 days. The core-shell architecture of the microcapsules mimics the physical configuration of pre-hatching embryo with a hydrogel-like shell (i.e., zona pellucida) and a permissive core, the native home of stem cells from the totipotent to pluripotent stage. After forming the mESC aggregates, the aggregates were pre-differentiated into the early cardiac stage inside the microcapsules. Afterward, the pre-differentiated aggregates were released from the core-shell microcapsules by dissolving the alginate hydrogel shell using an isotonic solution of sodium citrate and further re-encapsulated within a micromatrix for implantation. This does not significantly affect their size and integrity (see the images in Fig. lb) and enables injectable delivery (mtramyocardial injection) of the pre-differentiated aggregates as a result of their miniaturized dimension. More data on characterizing the early cardiac pre- differentiation of the mESC aggregates and re-encapsulation of the pre-differentiated aggregates within a micromatrix are given below.
(2) Pre-differentiating mESC aggregates to early cardiac stage.
78. To minimize the issue of teratoma associated with using PSCs in their pluripotent state for tissue regeneration, the mESC aggregates were pre-differentiated for 3 days before implantation using BMP-4 and bFGF to specifically direct the cells towards an early cardiac stage. Successful pre-differentiation is confirmed by RNA microarray data (Fig, 2a) showing that the expression of cardiac marker genes was significantly higher in the pre-differentiated mESCs than the cells before pre-differentiation. The expression of other typical genes that regulate heart and mesoderm development were also up-regulated, while the pluripotency marker genes were down-regulated in the pre-differentiated ceils. The microarray analysis was validated with the conventional method of analyzing gene expression using quantitative reverse transcription polymerase chain reaction (qRT-PCR) to quantify the expression of the early cardiac gene marker Nkx2.5. Both the microarray and qRT-PCR analyses show that the expression of the N ~ kx2.5 gene was significantly up-regulated by 4-5 times after pre- differentiation (Fig. 2a). Further flow cytometry analyses were conducted for typical
pluripotency protein markers (OCT-4 and NANOG, Fig. 2b), and cardiac specific protein makers including cardiac troponin T (cTnT) and NKX2.5 (Fig. 2c). These together with immunohistostaining analy sis of the early cardiac protein marker (N X2.5) confirmed that the aggregated ceils were successfully directed into the early cardiac lineage after pre- differentiation. This is because the pluripotency markers in the cells after pre-differentiation are negligible, while the expression of cardiac specific markers (particularly the early cardiac marker) is significantly increased after the early cardiac pre-differentiation treatment. (3) Re-encapsislaiisig the pre-dif erentiated aggregates.
79. After the early cardiac pre-differentiation, the aggregates were released from the core- shell microcapsules using isotonic sodium citrate solution, the released aggregates were re- encapsulated in an aiginate-ehitosan micromatrix (ACM) by soaking the aggregates first in chitosan (0.4% w/v in saline) and then in a solution (0.15% w/v in saline) of oxidized alginate. The oxidized alginate degrades much faster than the regular alginate in the core-shell microcapsules. This soaking procedure was repeated once. As shown in the first two columns of Fig. 3 a, individual cells on the surface of bare pre-differentiated aggregate (Bare-A, top) are clearly visible while the aggregate with ACM encapsulation (ACM- A, bottom) is covered with materials. Moreover, as shown in the 3rd column of Fig. 3a for cut-open aggregates, an extensive micromatrix of materials that cover the ceils is visible inside the ACM-A (bottom), while it is minimal with visible individual cells inside the Bare-A (top). The micromatrix inside the ACM-A is visualized by confocal fluorescence microscopy (Fig. 3b) showing green fluorescence throughout the ACM-A, due to labeling the alginate in the ACM (only for this experiment) with a green fluorescent probe fluorescein isothiocyanate (FITC). Furthermore, the elastic modulus of ACM-A determined by atomic force microscopy (AFM) nanoindentation is significantly higher than that of Bare-A (Fig, 3c).
80. The degradability of the micromatrix in vitro was studied by culturing the Bare-A versus ACM-A and observing their attachment in petri dishes. As shown in Fig. 7a-b, ceils detached from nearly all the Bare-A, spread out, and attached to the bottom surface of the petri dish after 1 day of culture. This occurred to 1.5, 35.4, and 71.7% of the ACM-A after 1, 2, and 3 days of culture, respectively (Fig, 7a and c), as a result of the gradual degradation of the micromatrix. Experiments were conducted to study the micromatrix degradation in vivo by labeling both oxidized (by default) and non-oxidized (NonOxi) alginate with indocyanine green (ICG, for non-invasive near infrared imaging as shown Fig. 8a) to form the ACM-A-ICG and NonOxi - ACM-A-ICG, respectively. After intramyocardial injection into the peri-infarct zone of mice with MI (see Fig, 4a), the ACM-A-ICG fluorescence was observed to gradually decrease and disappear over three days (Fig. 8b). This indicates that the ACM degrades and diffuses away within 3 days after intramyocardial injection, which is slightly faster than its in vitro degradation due to the different microenvironment in the MI heart compared to the culture medium in petri dish. In contrast, the fluorescence of NonOxi- ACM-A-ICG stays over three days after intramyocardial injection because the non-oxidized (i.e., regular) alginate degrades much slower than the oxidized alginate. A similar trend was observed for the degradation rate when the ACM-A-ICG and NonOxi-ACM-A-ICG were subcutaneously injected into mice (Fig. 8c).
These in vitro and in vivo degradation data indicate that the ACM used in this study is biodegradable and provides a temporary micromatrix for the pre-differentiated cell aggregates.
81. It worth noting that the aforementioned procedure for pre-differentiating the aggregated ceils, releasing the pre-differentiated aggregates out of the microcapsules, and re-encapsulating them in ACM to form the A CM- A does not affect the high cell viability or the size and integrity of the aggregates (Fig. lb and Fig. 7b-c). The size (diameter) of aggregates was determined to be 128.9 ± 17.4, 123.3 ± 12.4, and 126.9 ± 1 1 .0 μνα before releasing them from microcapsules, after releasing from microcapsules (for Bare-A), and after re-encapsulating them in ACM (for ACM- A), respectively.
(4) Injecting the re-encapsulated aggregates for therapy of MI,
82. To investigate the safety and efficacy of the ACM-A as compared to Bare-A, bare single cells (Single), saline, and materials alone (i.e., ACM) for treating MI, intramyocardial injection of 2x 05 cells/animal in a total of 20 μΐ saline into the peri-infarct zone (injected at 3 different injection sites with equal volume, Fig. 4a) were conducted using a 28 gauge needle. This was done within 5 minutes after the anterior wall of the left ventricle turned pale following permanent ligation of the left anterior descending artery (L AD) at its proximal location, to create a large-area MI in 8-12 week old male C57BL6/J mice. The mice are either wild-type (WT) with a normal immune system or caspase-recruitment domain 9 (Card9) knockout (KO) with deficient macrophage function. This immediate injection of therapeutic cells is desired to minimize the damaging effect of MI. All surgical procedures were conducted under aseptic conditions. The treatments with Bare-A and single cells differentiate themselves from the others with the formation of a large mass of tissue that grows out of the heart (indicated by arrows in Fig. 4b). This kind of large grown-out tissue was observed in 60 and 57% of WT mice with the bare single cell (n=29) and Bare-A (n=38) treatment, respectively. In stark contrast, it was not detected in any of the WT mice treated with ACM-A (n=31), ACM (n=24), or saline (n=31) (Fig. 4c).
83. Further microscopic examination of the grown-out tissue revealed that it is a typical granuloma consisting of immune cells (macrophages and T cells) and fibroblasts within a loose tissue matrix (Fig. 4d, at 28 days post injection), indicating the outgrowth is a result of immune reactions. This kind of severe immune reactions can also lead to the clearance of transplanted cells by macrophages in vivo, because cell retention/survival in the heart is significantly lower for the single cell (7.3+4.5%, consistent with that reported in the literature for single cell injection) and Bare- A (17.6+6.3%) treatments than the ACM-A treatment (47,0+5.6%) at 28 days post injection (Fig. 4e). Furthermore, the Bare-A treated MI mice started to die at as early as 2 days post injection while mortality was not observed in the ACM-A treatment group until 7 days after injection (Fig. 4f). Nearly 40% of the Bare- A treated MI mice died during the first 4 days, a known time frame for acute immune reaction to implanted cells. Interestingly, while WT mice treated with single cells have a higher occurrence of granuloma, the overall size of granuloma is smaller than that of Bare- A treatment (Fig. 4b). This is due to the reduced cell survival/retention and can explain the moderate animal mortality in the early time frame for the single cell treatment, compared to the Bare-A treatment.
84. Macrophages are known to regulate acute inflammation and remodeling in response to cardiac injury. To further understand this role of macrophages, Bare-A was injected into the MI hearts of Card9 KO mice. Granuloma was observed in only -33% of the KO mice (Fig. 4c). These data indicate that maerophage-medi ated acute immune reactions contribute to the occurrence of granuloma and possibly the early death of WT mice with MI treated with Bare-A.
85. The ACM-A treatment also significantly improves the cardiac function as indicated by ejection traction measured by pressure-volume (PV) loops (Fig, 4g) and electrocardiography, compared to saline, Bare-A, and ACM treatments. All groups with cells (Single, Bare-A, and ACM-A) can partially restore the maximum and minimum rate of pressure change in the left ventricle (Fig. 9a). The ACM-A treatment significantly improves the stroke volume and cardiac output compared to the saline and ACM treatments (Fig. 9b), the time constant of isovolumic pressure relaxation (tau) compared to saline treatment (Fig. 10a), and the left ventricle (LV) end- systolic and end-diastolic volumes compared to Bare-A and ACM treatments (Fig. 10b). In addition, these functional parameters for the single cell and Bare-A treatments exhibit large variations, probably due to the occurrence of granuloma and strong immune responses in more than half of the mice with the two treatments (Fig. 4c).
(5) Cardiac regeneration in situ of the injected aggregates.
86. Furthermore, the ACM-A treatment significantly reduces fibrosis (Fig. 5a-b) compared to all other treatments at 28 days after injection. This contributes to the high survival of the WT MI mice with the ACM-A treated mice (Fig. 4f). In addition, all treated hearts are markedly larger than no-MI hearts, as a result of pathological hypertrophy in response to MI, trying to restore the stroke volume compromised by MI. However, the hearts from mice treated with ACM-A suffered less hypertrophy than mice with the other treatments, indicating that the ACM- A treatment facilitates the restoration of cardiac function of mice with ML To find out if the significantly improved survival and the therapeutic benefit with the ACM-A treatment are partially a result of cardiac regeneration by the implanted cells in situ, the heart tissue harvested from the MI mice implanted with ACM-A prepared using niESCs constitutiveiy expressing green fluorescent protein (GFP) using the bioinspired approach was examined. It was observed that extensive green fluorescence is evident in the MI zone (Fig. 5c) at 28 days post injection. Moreover, this green fluorescence co-localizes with the antibody staining of GFP expressed in live ceils (Fig. 11), indicating that the implanted cells survived, detached from the aggregates, and migrated into the MI zone. The implanted cells (expressing GFP) appear to be similar to CM with evident striations, indicating further differentiation of the implanted cells (with early cardiac pre-differentiation) into more mature CMs under the in situ cardiac inductive chemical, mechanical, and electrical cues in the heart after implantation. This is further supported by the expression of three typical markers of CMs including cardiac troponin I (cTnl), connexin 43, and a-actinin in the green fluorescent CM-like cells derived from the implanted cells (Fig. 5d). Moreover, the green fluorescent CMs integrated seamlessly with the neighboring native CMs without green fluorescence (Fig. 5c-d), indicating the micromatrix was biodegraded allowing for timely integration of the implanted cell-derived CMs with the native cardiac tissue. b) DISCUSSION
87. In this study, is reported a bioinspired approach as illustrated in Fig. 1 to prepare PSCs for implantation. Microcapsules with a core-shell structure resemble the native physical configuration of pre-hatching embryos, the native home of totipotent-pluri potent stem cells. Probably because of the biomimetic nature, the miniaturized and semi -closed culture in the core- shell microcapsules can better maintain the sternness of both pluripotent and multipotent stem cells compared to conventional culture in homogeneous liquid (medium) or hydrogel. This is crucial to the expansion of stem cells with high quality and purity in vitro for the application of stem cell-based medicine, including but not limited to cardiac tissue regeneration for treating MI. However, the remaining steps of this bioinspired approach (including early cardiac pre- differentiation, dissolving the alginate hydrogel shell to release the pre-differentiated aggregates, and re-encapsulation in ACM) mimic the phenomena of the processes of pre-differentiation, zona hatching, and re-encapsulation in the trophoblast rather than the exact biological mechanisms of processes that nature uses to prepare totipotent-pluripotent stem cells for implantation into the uterus wall . Biological processes of this natural procedure are tightly regulated and cell-induced and mediated. The bioinspired approach is based on phenomenologically mimicking the natural procedure and does provide multiple advantages in preparing PSCs for implantation to treat MI and other diseases as detailed below.
88. First, the bioinspired approach enables injectable delivery of cells with much improved efficiency. The contemporary practice of scaffold engineering is to make a scaffold first and then seed ceils into the scaffold, which is extremely difficult (if not impossible) to utilize for making microscale constructs (i.e., ACM-A) with densely packed cells as shown in Fig, 3. In stark contrast, the bioinspired approach resolves this challenge by making microscale cell aggregates first and then forming a micromatrix within and over the aggregates (Fig. l b). An important advantage of this inverse (and bioinspired) scaffold engineering method is that it does not significantly change the construct size, which allows for intramyocardial injection of the constaicts for minimally invasive delivery. Moreover, using the ACM-A densely packed with -1,500 cells in each aggregate for delivery is much more efficient than the contemporary method of sparsely encapsulating only 2-5 cells in -100 μιη hydrogel microcapsules (without a core- shell structure) for injection into the heart. 89. Secondly, the bioinspired approach minimizes immune responses to implanted cells. Severe immune responses were observed after implanting Bare-A and bare single ceils as demonstrated by the formation of granuloma containing many macrophages and T cells (Fig. 4b- d). CARD9 signaling plays an essential role in immune response due to its critical involvement in the function of macrophages, neutrophils, and monocytes that are important for the acute phase of inflammation. Therefore, the Card9 KO mice with compromised macrophage function were used to determine whether macrophage-mediated events are the dominating mechanism of the immune responses observed. The occurrence of granuloma is reduced, but not completely eliminated in the Card9 KO mice, indicating that T cells also play a significant role in the immune responses to the bare aggregates and single cells. With the ACM encapsulation, the occurrence of granuloma can be significantly reduced to 0, indicating the exceptional capability of immunoisolation by the ACM.
90. Interestingly, few T cells or macrophages were observed in the heart treated with ACM- A at 4 weeks even though the micromatrix (i.e., ACM) was degraded within three days (Fig. 7 and 8). Due to the porous nature of the ACM as shown in Fig. 3a, cells in the ACM-A after implantation can still communicate with the microenvironment in the host via soluble factors and gradually adapt to it. As a result, minimal immune responses are evoked after the cells are exposed with the gradual degradation of the ACM. Accordingly, it is understood that the biodegradable ACM temporarily isolates the encapsulated cells from contacting the host cells including macrophages, T cells, and possibly N cells to achieve a temporary immunoisolation (or "local immunosuppression") effect. This contributes to the significantly improved animal survival ( 80° <> after 28 days of injection) and elimination of granuloma in WT mice with the ACM-A treatment (Fig. 4c and f). Of note, animal survival for the saline control at day 28 after surgery was -48%, which is consistent with the literature for WT mice with a large-area MI. This long-term impact of temporary immunosuppressi on/isolation was also recently reported elsewhere. For example, temporary systemic immunosuppression for up to 6 days was shown to mitigate immune rejection to bare pluripotent stem cells and their derivatives in the long term. The study shows that the impact of temporary immunoisolation for 3 days can last over 80 days in a T cell-based therapy of leukemia.
91. Due to the minimized immune response to the implanted cells in ACM-A as well as the size of the aggregates (-125 μτη) preventing the cells from being carried away by blood perfusion, significantly improved cell retention and survival was observed with the ACM-A treatment at 4 weeks after implantation (Fig. 4c). Furthermore, apparent engraftment of the implanted cells was observed in the infarct region as shown in Fig. 5c-d although the injection was done at the peri -infarct zone. These data indicate that cells pre-differentiated to the early- cardiac stage have migration potential, in contrast to mature cardiomyocytes with very limited migration potential. Furthermore, unlike mature cardiomyocytes that have a very specific role of beating in the heart, the pre-differentiated cells at the early cardiac lineage can release factors to facilitate angiogenesis to promote their survival. In addition, the heart always beats, which can induce some (albeit insufficient alone) degree of fluid flow in the interstitial space of the cardiac tissue to improve the survival of the implanted ceils migrating into the infarct zone. In addition, pre-differentiated mESCs were used in mouse MI model (i.e., allogeneic model). This also contributes to the improved engraftment when compared with xenograft models reported in the literature, where the human ESCs (hESCs) derived mature cardiomyocytes have more difficulty integrating with the guinea pig or rat model since they are from different species. It is worth noting that BMP-4 and bFGF have been used as the only two chemicals for specific cardiac (rather than the primitive mesoderm ) differentiation of the pluripotent stem ceils including the Rl mESCs used in this study. Electrospray was also used to encapsulate the Rl mESCs in core- shell microcapsules for proliferation before cardiac differentiation. Thus, after the 3-day incubation in culture medium with BMP-4 and bFGF, it can take 4-1 1 more days for the pre- differentiated cel ls to mature further into beating cardiomyocytes when they are cultured in the base culture medium without BMP-4 and bFGF. 92. No teratoma formation was observed from these implanted cells retained and survived in the hearts of 98 WT mice treated with the pre-differentiated ceils including ACM- A (31), Bare- A (38), and single cells (29) at 4 weeks after intramyocardial injection. Tissue slides from the all the in vivo studies were carefully evaluated by an American Board of Pathology certified anatomic/clinical pathologist with extensive experience in evaluating both human and animal tissues without and with various diseases including cardiovascular diseases and cancer. It has been reported that teratoma can form in mouse models injected with pluripotent stem cells including the Rl mESCs used in this study in 3-4 weeks. The aforementioned observation indicates that teratoma formation can be eliminated by using the bioinspired method to prepare the mESCs for injectable deliver}' with early rather than mature cardiac pre-differentiation before implantation.
93. In summary, the data show that ACM encapsulation of pre-differentiated microscale PSC aggregates significantly improves cell retention/survival after intramyocardial injection, and provides a temporary "local immunosuppression" for the ceils to minimize cell injury and early animal death due to acute immune reactions. The implanted cells pre-differentiated to the early- cardiac stage have a superb capability of regenerating cardiac tissue in situ via further direct differentiation guided by the cardiac-inducive chemical, mechanical, and electrical cues in the heart into mature cardiomyocytes. This significantly reduces fibrosis and enhances cardiac function, which ultimately contributes to the significantly improved animal survival. This study indicates that the bioinspired engineering approach can be valuable to facilitate clinical applications of SCT for treating MI and possibly many other degenerative diseases. c) METHODS
(1) Animals and materials,
94. Male wild-type (WT) C57BL6/J mice (Jackson Laboratory, Bar Harbor, ME, USA) of 8- 12 weeks, and age-matched Card9 KO mice with C57BL6/J background (from Dr. Xin Lin's laboratory at Department of Molecular and Cellular Oncology and Department of Immunology, University of Texas MD Anderson Cancer Center, Houston, TX, USA) were housed at constant temperature (22 ± 2 °C) with a 12-hr light/dark cycle. Mice were given standard lab chow and water ad libitum. All investigations in this study conform to the Guide for the Care and Use of Laboratory Animals published by the US National institutes of Health and approved by the Institutional Animal Care and Use Committee at The Ohio State University.
95. Sodium alginate (plant cell culture tested and low viscosity, -240 kDa) was purchased from Sigma (St. Louis, MO, USA) and purified by washing in chloroform and charcoal and dialyzing against deionized water, followed by freeze-drying. The oxidization of alginate was performed by mixing sodium periodate (2mM) with 1% (w/v) purified alginate for 24 hr in the dark at room temperature, and then using an equivalent amount of ethylene glycol to stop the reaction, followed by 24 hr of dialysis against deionized water with three water changes. ICG and I f K labeled alginate (with and without oxidization) was prepared by dissolving alginate or oxidized alginate (1.8% w/v) in 2-(N-morpholino) ethanesulfonic (MES) acid buffer (pH 4.7) and mixing with 9 mM l-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and 9 mM N-hydroxysulfosuccinimide (Sulfo-NHS). After stirring for 2 hr at room temperature, 2 mM FITC (Sigma) or ICG amine (AAT Bioquest, Sunnyvale, CA, USA) was added to the solution and stirred for 1 8 hr, followed by dialysis against distilled water for 24 hr and freeze- drying to obtain the dry polymers of ICG or FITC labeled alginate (or oxidized alginate).
Pharmaceutical grade chitosan of 80 kDa (~ 95% deacetylation) was obtained from Weikang Biological Products Co. Ltd (Shanghai, China). The chitosan was dissolved in saline buffered with acetic acid at pH 6.7 and filtered through a 0.22 μ ι filter before use. The primary antibodies including ab47003 (to cTnL rabbit polyclonal), ab 18061 (to ot-actinin, mouse monoclonal, clone 0.T.02), ab45932 (to cTnT, rabbit polyclonal), and abl 1370 (to connexin 43/GJA1, rabbit polyclonal) were purchased from Abeam (Cambridge, MA, USA). The primary antibodies for N X2.5 (sc376565, mouse monoclonal, clone A-3), CD3 (sc20047, for T cells, mouse monoclonal, clone PC3-188A), fibronectin (sc9068, rabbit polyclonal), and F4/80 (sc25830, for macrophages, rabbit polyclonal) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The primary antibody for GFP (cs2956, rabbit monoclonal, clone D5.1) was purchased from Cell Signaling (Danvers, MA, USA). All secondary antibodies were purchased from Life Technologies. All other materials were purchased from Sigma unless specifically mentioned otherwise,
(2) Cell culture and encapsulation in core-shell microcapsules, 96. Rl mESCs with and without GFP were purchased from ATCC (Manassas, VA, USA). The GFP Rl cells (ATCC® SCRC-1033™) express GFP constitutiveiy with the plasmid pEYFP from Clontech Laboratories, Inc. (Mountain View, CA, USA). The mESCs were cultured in feeder-free medium made of Knockout®' DMEM supplemented with 15% Knockout*1 serum (Life Technologies), 1000 U ml"1 leukemia inhibitory factor, 4 mM 1-glutamine, 0.1 M 2- mercaptoethanol, 10 ,ug ml"1 gentamicin, 100 U ml"1 penicillin, and 100 μg mi"1 streptomycin in gelatin coated tissue culture flasks with daily medium change. To encapsulate the mESCs in microcapsules with a liquid core and hydrogel shell, they were detached from the flasks using Ix trypsin/EDTA, washed by phosphate buffered saline (PBS), and then suspended at 5 x 106 ml"1 in 0,25 M aqueous mannitol solution supplemented with 1% (w/v) sodium carboxymethyl cellulose as the core solution. The coaxial electrosprav system including coaxial needle, pumps, voltage generator, and collecting bath i s shown in Fig, 6. Before experiments, the coaxial needle, collection beaker, and connection tubes were autociaved to ensure sterility. All solutions used for electrosprav were filtered using 0.22 μιη filters. All the devices including pumps, syringes, cables of the voltage generator were wiped with 70% alcohol and then put in a biosafety cabinet/laminar flow hood designed for cell handling. The voltage generator was left outside the hood. The ceil encapsulation experiments were performed inside the hood to avoid
contamination. In brief, the core solution was pumped through the inner lumen (28G) of the coaxial needle at 47 μΐ min"1, while the shell solution consisting of 2% purified sodium alginate (w/v) in 0.25 M aqueous mannitol solution was pushed through the outer lumen (21G) at 60 μΐ min"1. Concentric drops generated by the core and shell flows are then broken up into
microdropiets under a 1.8 kV electrostatic field and finally sprayed into the gelling solution made of 100 mM calcium chloride in deionized water. The distance between the needle tip and the top surface of CaCb solution in the gelling bath was 5.5 mm. The high viscosities of core and shell solutions as well as the instant gelling kinetics of sodium alginate in the 100 mM CaCl2 solution ensure negligible mixture between the two aqueous solutions and therefore the formation of core-shell microcapsules. The resultant microcapsules were 3 15 ± 31 μτη in outer diameter29. After encapsulation, all the resultant cell-laden microcapsules were washed with 0.5 M mannitol solution and cultured in mESC medium with daily medium change. Al l cells were cultured at 37 °C in a humidified 5% C02 incubator.
(3) Pre-differentiation of mESC aggregates.
97. After 7-day culture in mESC medium, one integrated mESC aggregate was formed in the core of each microcapsule. For pre-differentiation of the mESC aggregates towards mesoderm and further the early cardiac lineage, cell aggregates were cultured in cardiac induction medium with regular DMEM supplemented with 25 ng ml'1 BMP-4, 5 ng ml"1 bFGF, 100 U ml"1 penicillin, and 100 mg 1 streptomycin for 3 days in the core-shell microcapsules to prevent attachment on the culture plate. To characterize gene expression after cardiac induction, aggregates were released from the microcapsules by dissolving the alginate hydrogel shell using 55 mM sodium citrate for 30 s and then washed by PBS for 3 min. The pre-differentiated aggregates together with undifferentiated ones (as control) were then homogenized and used for RNA isolation following the manufacture's instruction with the RNeasy Plus Mini Kit (Qiagen). The quality of the extracted RNA was then examined by its A Azaj, A260/230 and 28S/18S ribosomal RNA (rRNA) ratio. The microarray was conducted using the Clariom™ D assays for mouse (Affymetrix, Santa Clara, CA, USA) on GENECHIP® Hybridization Oven 645 and analyzed using the AFFYMETRIX® Transcriptome Analysis Console (TAC) Software by technicians in The Genomics Shared Resource in The Ohio State University Comprehensive Cancer Center. A complete list all genes investigated with the Clariom™ D assays can be found online. For qRT-PCR studies, the synthesis of complementary DNAs (cDNAs) was conducted using the iScriptTM cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) and quantitative PGR analysis was performed using a Bio-Rad CFX96 real time PCR machine. In brief, relative gene expression was calculated using the AACt method built in the Bio-Rad software and the expression of the early cardiac gene Nkx2.5 (F: 5!-GATGGGAAAGCTCCCACTATG-3' (SEQ ID NO: 1) and R: 5'-GAGACACCAGGCTACGT CAATA-3') (SEQ ID NO: 2) was studied.
98. For inimunostaining of NKX2.5 protein, the mESC aggregates before (as control) and after pre-differentiation were either dissociated using lx trypsin/EDTA into single cells or kept as intact aggregates for fixation using 4% paraformaldehyde for 1 hr at room temperature. The fixed single cells or aggregates were incubated first with 3% bovine serum albumin (BSA, to block non-specific binding) in PBS with 0. 1 % TritonX-100 (for permeabilization of the membrane within cells) for 30 min at room temperature and then with NKX2.5 primary antibody (1 :200 dilution) overnight at 4 °C, washed three times using PBS, incubated with secondary antibody (1 :200 dilution) for 1 hr at room temperature. The samples were further washed three times using PBS before microscopic examination using an Olympus FV1000 confocal microscope and flow cytometry analysis using a BD (Franklin Lakes, NJ, USA) LSR-II Flow Cytometer and FlowJo software. This protocol was also used for other antibodies including QCT-4 (1 :200 primary antibody dilution), NANOG (1 :200 primary antibody dilution), and cTnT (1 : 100 primary antibody dilution). This is the protocol suggested by the manufacturers (Abeam and Santa Cruz) for the primary and secondary antibodies used for immunohistochemical staining and flow cytometry.
(4) Re-encapsulation of the pre-differentiated aggregates.
99. After aggregates formed after 7-day culture, the aggregate-laden microcapsules were collected from each culture dish by pipetting with a 15 ml serological pipette into a 15 ml centrifuge tube. Due to gravity, the aggregate-laden microcapsules sink down at the bottom of the collection tube in 5 min. The supernatant was then removed using 5 ml serological pipettes and the aggregate-laden microcapsules were rinsed with 5 ml of PBS. The latter was done by gently tilting the tube for mixing and by allowing the microcapsules to sink down at the bottom of the tube as a result of gravity before removing the supernatant by pipetting. A total of 1 ml of 55 tnM isotonic sodium citrate was then added into the tube to dissolve the alginate hydrogel shell of the microcapsules in 30 s, followed by removing the supernatant containing sodium citrate and dissolved alginate by pipetting. The aggregates were then washed using 5 ml of PBS in the same way as that aforementioned for washing the aggregate-laden microcapsules. Unlike trypsin or collagenase, the process of dissolving alginate hydrogel using sodium citrate is quick and gentle and it does not affect the integrity of aggregates. For ACM encapsulation of the resultant ceil aggregates, alginate and chitosan solutions were dissolved in saline (0.9%) and with a pH at 7.2 and 6.7 (this is necessary to dissolve chitosan), respectively. The size (diameter) of aggregates was determined by measuring the average diameter at three different locations on each aggregate with a 120-degree interval. Chitosan is a positively charged polymer that can be attracted to the cells in the aggregate because their plasma membrane is negatively charged. The same electrostatic interaction applies to the complexation of alginate with chitosan and vice versa (alginate is negatively charged). Alginate and chitosan are both naturally derived polymers with high biocompatibility and can gradually degrade into nontoxic oligosaccharide and glucosamine (amino sugar), respectively . The materials alone (ACM) control was achieved by- processing alginate microbeads of similar size to the cell aggregates in the same way as that for forming the ACM within the cell aggregates. The ACM encapsulated aggregates were then used for either in vitro studies to determine the existence and degradation of ACM or in vivo transplantation into mice. In brief, the aggregates encapsulated in ACM of oxidized or regular alginate were plated on gelatin-coated dishes and maintained in regular DMEM with 20% FBS. The ratio of attached to total aggregates were calculated to determine the percentage of aggregate attachment. Non-encapsulated aggregates were studies in the same way to serve as control.
100. For morphological characterization of the bare versus ACM encapsulated pre- differentiated aggregates using scanning electron microscopy (SEM), the aggregates were fixed using 2.5% glutaraidehyde in phosphate buffer overnight. After the standard procedures of dehydration in ethanol and drying using hexamethyldisilazane, the samples were sputter coated using a Cressington 108 sputter coater at 17 niA for 120 s before examination using an FEI Nova NanoSEM 400 scanning electron microscope. For visualization of ACM, the mESC aggregates were encapsulated in ACM with FITC-labeled alginate using the aforementioned procedure and the encapsulated aggregates were further stained for nuclei using 5 μΜ Hoechst 33342 for 15 min before examination using an Olympus FV1000 confocal microscope. In addition, -150 μηι microbeads made of 2% (w/v) oxidized alginate were generated by electrospray and then soaked in chitosan and alginate solutions in the same way as preparing the ACM encapsulated pre-differentiated aggregates to serve as the materials alone control. For all in vitro studies, cells were cultured at 37 °C in a humidified 5% C02 incubator.
101. To quantify the elastic modulus of mESC aggregates, nanoindentation was performed using an integrated system consisting of an Olympus 1X81 inverted optical microscope and an Asylum MFP-3D Bio atomic force microscope (AFM). To reduce any potential transient thermal effects on cantilever deflection, the cantilever was kept at the same temperature as the mESC aggregates prior to obtaining force curves. After calibrating the cantilever sensitivity, the thermal tuning method was used to determine the cantilever's spring constant. Multiple mESC aggregates were then suspended in PBS and allowed to settle onto the glass bottom of petri dishes (Fluorodish, World Precision Instruments, Inc.). Individual aggregates were identified and positioned underneath the cantilever tip using the inverted optical microscope. Force versus indentation curves were then generated using the deflection data from the contact point between the sample surface and the cantilever tip. Colloidal probes (2 μτη diameter polystyrene beads) were chosen for their low spring constant (0.32 N m"1) and spherical tip geometry, which are commonly used for indenting soft biological samples. Force curves at a distance of 4 μιη were acquired at 0.5 Hz to reduce the viscoelastic effects that can occur when using larger approach velocities. A relative trigger force of no more than 10 nN was also used to ensure that indentations did not produce any unwanted effects from the underlying substrate, as determined by preliminary testing over a wide range of indentation depths. The resulting curves were then fit using a Hertz model built into the AFM software. Multiple force curves (at least 10) were taken for each sample in order to ensure that a consistent elastic modulus was produced. The resulting data sets were then combined and evaluated using statistical analysis for comparison,
(5) Surgical procedure and intramyocardial injection.
102. To perform permanent ligation of LAD, mice were initially anesthetized by 3% isoflurane inhalation, intubated with a 20G intravenous catheter, and ventilated with a mixture of 02 (0.3 1 min-1) and 1.5-2% isoflurane (tidal volume of 250 μΐ and 120 breaths min-1) with a mouse respirator (Harvard Apparatus, Holliston, MA, USA). Animals were placed in a right lateral decubitus position and a left thoracotomy was then performed through the left fourth intercostal space by cutting pectoralis muscles transversely to expose the thoracic cage. After removal of the pericardium, the left anterior descending artery was visualized and a 6-0 silk ligature was placed approximately 1 mm from origin of the vessel. The ligature was confirmed to be successful when the anterior wall of the left ventricle turned pale.
103. Triphenyl tetrazolium chloride (TTC) staining was conducted at 24 hr after surgery to confirm the ligation. In brief, -200 μΐ of 0% Phthalo Blue were slowly injected into the aorta to stain the heart. The heart were then rapidly collected and washed in 30 mM KC1 to cease the heart beating and allow for more consistent sectioning. The heart was then frozen down for at least 4 hr at -20 °C before cutting them into slices of 1 mm. The heart tissue slices were incubated with 2% TTC at 37 °C for 40 min. Further fixation of the stained slices with 10% formaldehyde overnight was performed, to increase the contrast between the infarct area and the normal tissue.
104. Injection of cell aggregates and other control groups was performed as illustrated in Fig. 4a. The single cells were obtained by dissociating Bare- A with Ix trypsin/EDTA, similarly to detaching 2D cultured cells. The number of cells in each aggregate was determined by dissociating 50 aggregates using trypsin and counting the dissociated cells manually. For tracking cell differentiation in vivo, the same ceils with GFP were used. Mice randomly received a total of 20 μ! saline or saline containing single cells, Bare- A, ACM- A, or ACM via three injections given at three different sites in the periphery of infarcted tissue using a 28G needle within 5 minutes after the anterior wall of the left ventricle turned pale. The size (diameter) of the aggregates in this study was measured to be 123.3 ± 12.4 μπι before ACM encapsulation and 126.9 ± 1 1.0 Liffi after ACM-encapsulation. Therefore, adding 3 times standard deviation (or 3 sigma) to the average equals -160 μιη, which is still much smaller than the inner diameter (184 μηι) of a 28G syringe needle. Assuming normal distribution of the size, there is only 0.15% possibility for the cell aggregates to be bigger than 160 μηι by the three-sigma rule. In addition, the cell aggregates are soft and deformable, which can help to push them through the syringe needle. Indeed, there was no difficulty during injection of the cell aggregates during the experiments. In addition, injection volumes of 20-30 μΐ were used in other studies with mouse models, and the animal survival is not significantly different from that reported in the literature for the treatment with saline. Moreover, with saline, ACM- A, and ACM treatments in 20 μΐ of injection volume, no granuloma formation was observed. Therefore, the effect of a total of 20 μΐ of injection volume on granuloma formation and animal survival is insignificant in this study. After injection, the chest wall and skin were closed. The level of anesthesia (end-tidal concentration of isoflurane: 1-2%) was adjusted according to the surgical stimulation, by monitoring signs of movement. As soon as the anesthesia was stopped, the animals woke up immediately (normally in -1-2 min). Sham operations were performed on 10 mice without LAD occlusion. On each day of conducting surgery, 4-6 mice were randomly assigned to the six groups including saline, single ceils (Single), Bare-A, ACM-A, and ACM. The data of heart rate was plotted and showed no significant difference among different groups before versus after surgery. In total, 29 or more mice received treatment with saline, single cell, Bare-A, or ACM- A, and 24 mice were treated with ACM (materials alone). The sample size was chosen to ensure adequate power to detect the difference between the saline control and ACM-A groups. In brief, it was estimated that the saline group had a survival rate of -35% from pilot studies and the literature, the hypothetical survival rate of ACM-A from pilot studies was 80%, the probability of type I error (a) = 0.05, the power (1 - β) = 0.8, and ratio of sample sizes is 1, which gives sample size required for each group to be at least 24. Mice were sacrificed by C02 exposure at 28 days, and heart samples were harvested for further analyses. No blind method is involved for the animal study. 105. The in vivo degradation of ACM were studied by encapsulating cell aggregates with ACM labeled by ICG and then injecting them into hearts of animals with the same surgical procedure as aforementioned, Bare-A and NonOxi-ACM-A-ICG were also injected as negative and positive controls, respectively. Hearts were collected at days 0 (1 hr), 1, and 3 and the ICG fluorescence was monitored using a PerkinElmer (Waltham, MA, USA) IVIS imaging instrument with excitation at 780 nm and an 831 nm filter. Subcutaneous injection was also performed with injection of Bare-A, NonOxi-ACM-A-ICG, and ACM-A-ICG, and the ICG fluorescence was observed noninvasively on days 0 (1 h), 1 , and 3 ,
(6) Characterization of cardiac function in vivo.
106. Post-surgery mice were anesthetized with 2% isoflurane in 100% oxygen. The chest area was shaved and ultrasound coupling gel was liberally applied to the left chest wall. Two- dimensional and M-mode echocardiographic images were recorded and analyzed by a Vevo 2100 High -Resolution in vivo Imaging System with a MS400 transducer (Visual Sonics, Toronto, ON, Canada). Images were obtained in a parasternal short and long axis view. At the same time, the limbs of mice were attached with three electrical leads (lead I: right front foot, lead II: left rear foot, and lead III: left front foot) connected to the Vevo 2100 High-Resolution in vivo Imaging System for recording the electrocardiogram and heartbeat during the whole
echocardiography examination process. 107. Since the data from echocardiography can be subject to a lot of noise and provide inconclusive data, cardiac function was further determined by hemodynamic studies using PV loop measurements as the gold standard for quantifying cardiac function. In brief, a mouse PV loop analysis system was used for the assessment of cardiac function. Briefly, 28 days after left coronary ligation, all mice with no MI and treated with saline, single cells, Bare-A, ACM-A, and ACM were anesthetized with 2% isoflurane and oxygen at a flow rate of 0.4 1 min'1. A Millar (Houston, TX, US A) tip conductance catheter (Model SPR-893, 1.4 Fr.) was inserted into the right carotid artery, and further advanced into the left ventricle (LV). Baseline zero reference was obtained by placing the sensor in isotonic saline. After recording the basal hemodynamic parameters, a series of PV loops were generated using an AD Instruments (Colorado Springs, CO, USA) Power-Lab system connected to the Millar catheter. All the measurement and characterization were determined from the PV loop data using ChartPro Software (AD
Instruments).
(7) Histomorphological and immunostaining analyses.
108. Gross examination of the distribution of cells with GFP in MI hearts treated with saline, single ceils, Bare-A, ACM-A, and ACM was done by Zeiss MosaiX tiling and stitching bright- field and green fluorescence images taken using the same exposure time. To estimate the retention and survival of injected cells, a method similar to that reported elsewhere was used with slight modification. Briefly, hearts injected with GFP cells (Single, Bare-A, and ACM-A) and treatments without cells (saline and ACM) were harvested at 4 weeks after the injection and then embedded in OCT (Sakura Finetek, Torrance, CA, USA) to freeze at -80 °C for 1 hr before cryo-sectioning. The cryo-sectioning was conducted as follows: 10 locations were chosen from the apex to the ligation site with an even interval, and then 2-3 sections of 10 μπι in thickness ( 7) were cut at each location. All the resultant 20-30 sections were further stained with DAPI for visualizing nuclei and then examined under a Zeiss (Oberkochen, Germany) Axio Observer.Zl microscope with fluorescence capability. GFP positive cells (A) were counted on each section and averaged among the 20-30 sections. The length of the infarct zone (Z) was measured using a ruler. The retention/survival rate (RR) was calculated as follows:
Figure imgf000038_0001
where Nmjected is 0.2 x 10°. From Figure 5a, it can be seen that the infarct zone is from the apex to the site of ligation. Therefore, the retention rate calculation was based on this area. The left ventricular wall of mice with no MI and without any treatment was checked in the same way, and did not observe any green fluorescence.
109. For histomorphological analysis, hearts were harvested on day 28 post treatments, fixed in 10% neutral buffered formalin for 24 hr at 4 °C, and embedded in paraffin. Both transverse and sagittal sections of 5 μηι thick were then cut using a microtome. For H&E staining, sections were stained in hematoxylin 2 for 8 minutes and then washed by water, dipped in 1% acid alcohol and 1% ammonium hydroxide, stained in Eosin Y for 1 min, dehydrated with graded alcohols, and mounted on slides. For Masson ' s Tri chrome staining, sample sections were fixed using Bouin' s Fixative overnight at room temperature and then stained in Weigert' s Iron hematoxylin for 10 minutes. After rinsing with water for 10 min, the sections were dipped in Biebrich Scarlet Acid Fuchsin for 2 min and then in 5% Phosphomolybdic/Phosphotungsic acid for 15 min, followed by dipping for 5 min in 2.5% Aniline blue and 1% Glacial Acetic Acid, respectively. The sections were further dehydrated with graded alcohols and mounted on slides. For slides stained with Masson's tri chrome, ImagePro 6,2 software was used to calculate the percentage of area that has increased collagen content indicating fibrosis of the cardiac tissue.
1 10. Immunofluorescence staining of macrophages and T cells in granulomas of single cell and bare-A treated mouse hearts was performed. In brief, the granuloma tissues were taken from the hearts and then cryo-sectioned into 5 um of sections using a Leica CM 1510S Cryostat and fixed with 4% paraformaldehyde (PFA). Sections were immunostained against specific markers including F4/80 (for macrophages, 1 : 100 dilution), CDS (for T cells, 1 :50 dilution), fibronectin (for fibroblasts, 1 :200 dilution), as well as their corresponding secondary antibodies (1 :200 dilution). Before final examination, cell nuclei were stained with 5 μΜ Hoechst for 15 min at room temperature. The images were captured with an Olympus FV1000 confocal microscope.
1 1 1. To determine the capacity of in situ differentiation for cardiac regeneration of the cells in ACM-A as well as their capability of integration with the host tissue, heart samples collected at
28 days post intramyocardial injection were cryo-sectioned into 10 um of sections using the Leica CM1510S Cryostat and fixed with 4% paraformaldehyde (PFA). Sections were immunostained against cardiac specific markers including cTnl (1 :200 dilution), a-actinin (1 :200 dilution), connexin 43 (1 :200 dilution), as well as their corresponding secondary antibodies (1 :200 di lution), according to the manufacturer' s instructions. Before final examination, cell nuclei were also stained with 5 μΜ Hoechst for 15 min at room temperature. The images were captured using an Olympus F VI 000 confocal microscope. 112. For GFP staining, the fixed and paraffin-embedded heart tissues were cut into 5 um of sections. All sections were deparaffmized and hydrated in decreasing concentrations of ethanol. Antigen retrieval reagent (HK080-9K, BioGenex, Fremont, CA, USA) was used according to the manufacturer's instructions. The heart sections were incubated with rabbit anti-GFP primary antibody (1 :200 dilution) for 3 h at room temperature in blocking buffer (2% BS A in PBS). Following washing with PBS, tissue sections were further incubated with goat anti-rabbit (H+L) Alexa 568 secondary antibody (Life Technologies, A-11011, 1 :500 dilution) for 1 h at room temperature. The cell nuclei were visualized by staining the tissue with Vectashield Mounting Medium with DAPI (H-1500, Vector Laboratories, Buriingame, CA, LISA). Images for GFP staining were taken using an Olympus FluoView™ FV1000 confocal Microscope.
(8) Statistical analysis.
1 13. Student' s two-tailed t-test assuming equal variance was performed using Microsoft® Excel to determine the statistical significance for in vitro studies assuming normal distribution and equal variances, including flow cytometry, AFM nanoindentation, and qRT-PCR. Each experiment was independently repeated at least 3 times. Data are presented as mean ± standard deviation (s.d.) unless specifically indicated otherwise. The fold change of genes in microarray study was analyzed using the Affymetrix® Transcriptome Analysis Console (TAC) Software. The survival curve was plotted and analyzed using Prism (v6.0, GraphPad software, San Diego, CA). To determining the statistical significance of granuloma formation among the different groups, a chi-square test was performed using Prism. For the analysis of in vivo results, unpaired t-test or ANOVA was conducted using Prism. The data analyzed by ANOVA follows normal distribution and each sample was independent and random with similar variances between different groups. In all cases, a p value less 0.05 was considered to be statistically significant.
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GATGGGAAAGCTCCCACTATG (SEQ ID NO: 1 ) GAGACACCAGGCTACGTCAATA (SEQ ID NO: 2)

Claims

VII. CLAIMS What is claimed is:
1. A method of making a micromatrix comprising encapsulated pre-differentiated stem cells the method comprising: a) microencapsulating a stem cell, wherein the microcapsule comprises a permissive liquid core and a hydrogel shell; b) expanding the microencapsulated stem cells for at least 1 days; wherein the microencapsulated stem cells proliferate and form aggregates; c) pre-differentiating the microencapsulated stem cells into early stage cardiac-lineage cells. d) releasing the aggregates of pre-differentiated microencapsulated stem cells from the core-shell microcapsules; and e) encapsulating the released pre-differentiated stem cells in a micromatrix,
2. The method of claim 1, wherein the stem cell comprises an embryonic stem cell, induced pluripotent stem cells, extraembryonic fetal stem cells, amniotic stem cells, or adult stem ceils.
3. The method of claim 1, wherein the stem cell is microencapsulated via coaxial electrospray.
4. The method of claim 1 , wherein hydrogel shell is a semipermeable alginate hydrogel shell.
5. The method of claim 1, wherein the permissive core comprises an aqueous mannitol solution
6. The method of claim 1, wherein the microencapsulated stem ceils are expanded for at least 7 days,
7. The method of claim 1, wherein the pre-differentiation step comprises culturing the microencapsulated stem cells in a media comprising fibroblast growth factor (FGF) and bone morphogenic protein (BMP).
8. The method of claim 1, wherein the pre-differentiated microencapsulated stem cells comprise first heart field (FHF) cells, second heart field (SHF) cells, or epicardium derived ceils
9. The method of claim 1, wherein the pre-differentiated microencapsulated stem cells are released by dissolving the hydrogel using an isotonic solution of sodium citrate.
10. The method of claim 1, wherein the released pre-differentiated stem cells of step e are encapsulated by soaking the released aggregates in a solution comprising chitosan or alginate; wherein the solution comprises a pH of about 67 to about 8
11. The method of claim 1, wherein the micromatix comprises a matrix of alginate and chitosan.
12. The method of claim 10, wherein the alginate is oxidized alginate.
13. A micromatrix of encapsulated aggregate of stem cells wherein the stem cells are pre- differentiated stern cells, and wherein the stem cells are encapsulated by an integral with a micromatrix of alginate and chitosan.
14. The encapsulated aggregate of stem cells of claim 13, wherein the encapsulated aggregate of stem cells comprises about 1500 stem cells.
15. The encapsulated aggregate of stem cells of claim 13, wherein the encapsulated aggregate of stem ceils is an early stage cardiac lineage stem cell.
16. A method of treating damage caused by an ischemic event or reducing the severity of an ischemic event in a subject comprising administering to the subject the encapsulated aggregate of stem cells of claim 13.
17. The method of claim 16, wherein the ischemic event comprises an arterial embolism, venous embolism, thromboembolism, pulmonary embolism, traumatic injury, atherosclerosis, myocardial infarction, thoracic outlet syndrome, tachycardia, hypotension, tourniquet, or surgery,
18. The method of claim 17, wherein the ischemic event comprises a myocardial infarction and wherein the stem cells are pre-differentiated to an early stage cardiac lineage multi-potent stem cell.
19. The method of claim 6, wherein the encapsulated aggregate of stem cells is administered to the subject within 7 days of the ischemic event.
PCT/US2017/058837 2016-10-27 2017-10-27 Bioengineering of injectable encapsulated aggregates of pluripotent stem cells for therapy of myocardial infarction WO2018081616A1 (en)

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CN117363482A (en) * 2023-12-06 2024-01-09 中国医学科学院北京协和医院 Method for combined culture of different kinds of organoids

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CN114602397A (en) * 2022-03-11 2022-06-10 江南大学 Multi-cavity microsphere based on electrospraying method and preparation method thereof
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