Cells differentiated from immunoengineered pluripotent cells

Abstract

The present invention provides generally acceptable "off-the-shelf" low immunogenic pluripotent cells and their differentiated cardiac, endothelial, neuronal, islet or retinal pigment cells. Such low immune cells are useful for treating a patient in need thereof. The cells lack major immune antigens that trigger an immune response and are engineered to avoid phagocytosis and endocytosis.

Description

[0001] This application is a divisional application of CN201980056481.9.

[0002] I. Cross-Reference to Related Applications

[0003] This application claims priority to U.S. Provisional Application Nos. 62 / 698,965, filed on July 17, 2018, 62 / 698,973, filed on July 17, 2018, 62 / 698,978, filed on July 17, 2018, 62 / 698,981, filed on July 17, 2018, and 62 / 698,984, filed on July 17, 2018, the entire contents of which are incorporated herein by reference in their entirety.

[0004] II. Field of the Invention

[0005] Regenerative cell therapy is an important potential treatment for regenerating injured organs and tissues. Due to the low availability of organs for transplantation and the long waiting time associated with it, the possibility of regenerating tissues by transplanting readily available cell lines into patients is understandably attractive. Regenerative cell therapy has shown promising preliminary results in animal models (e.g., after myocardial infarction) for repairing damaged tissues after transplantation. However, the tendency of the transplant recipient's immune system to reject allogeneic material greatly reduces the potential efficacy of the therapeutic agent and reduces the possible positive effects surrounding such a therapeutic agent.

[0006] Therefore, the present invention provides generally acceptable "off-the-shelf" low immunogenic pluripotent cells and their differentiated cardiac, endothelial, neuronal, pancreatic islet or retinal pigment cells. Such low immunogenic cells are used to treat patients in need. The cells lack the main immune antigens that trigger the immune response and are engineered to avoid phagocytosis.

[0007] III. Background of the Invention

[0008] Regenerative cell therapy is an important potential treatment for regenerating injured organs and tissues. Due to the low availability of organs for transplantation and the long waiting time associated with it, the possibility of regenerating tissues by transplanting readily available cell lines into patients is understandably attractive. Regenerative cell therapy has shown promising preliminary results in animal models (e.g., after myocardial infarction) for repairing damaged tissues after transplantation. However, the tendency of the transplant recipient's immune system to reject allogeneic material greatly reduces the potential efficacy of the therapeutic agent and reduces the possible positive effects surrounding such a therapeutic agent.

[0009] Autologous induced pluripotent stem cells (iPSCs) theoretically constitute an unlimited source of cells for patient-specific cell-based organ repair strategies. However, their generation brings technical and manufacturing challenges and is a long process that conceptually prevents any acute treatment. From a manufacturing perspective, allogeneic iPSC-based therapies are easier and allow the generation of high-quality cell products that have been strictly screened and standardized. However, due to their allogeneic origin, such cell products will experience rejection. By reducing or eliminating the antigenicity of the cells, generally acceptable cell products can be produced. Since pluripotent stem cells can differentiate into any cell type of the three germ layers, the potential applications of stem cell therapy are extensive. Ex vivo or in vivo differentiation can be performed by transplanting progenitor cells that continue to differentiate and mature in the organ environment of the transplant site. Ex vivo differentiation allows researchers or clinicians to closely monitor the program and ensure that appropriate cell populations are produced before transplantation.

[0010] However, in most cases, undifferentiated pluripotent stem cells are avoided in clinical transplantation therapy because they tend to form teratomas. Instead, such therapies tend to use differentiated cells (e.g., transplanting stem cell-derived cardiomyocytes into the myocardium of patients with heart failure). The clinical application of such pluripotent cells or tissues would benefit from a "safety feature" that controls the growth and survival of cells after their transplantation.

[0011] The art seeks stem cells that can generate cells for regeneration or replacement of diseased or defective cells. Pluripotent stem cells (PSCs) can be used because they rapidly multiply and differentiate into many possible cell types.

[0012] To date, preclinical success of PSC-based approaches has been achieved only in immunosuppressed or immunodeficient models, or when the cells are encapsulated and protected from attack by the host immune system. However, systemic immunosuppression used in allogeneic organ transplantation is not suitable for regenerative approaches. Immunosuppressive drugs have severe side effects and significantly increase the risk of infection and malignancy.

[0013] There is a need in the art for cells generated from low immunogenic pluripotent stem cells that can be used in regenerative medicine.

[0014] IV. Summary of the Invention

[0015] In some aspects, provided herein are isolated, engineered hypoimmune cardiac, endothelial, neuronal, pancreatic islet or retinal pigment cells differentiated from hypoimmune pluripotent stem cells (HIP cells). HIP cells have, for example, reduced or eliminated endogenous beta-2 microglobulin (B2M) gene activity, reduced or eliminated endogenous class II transactivator (CIITA) gene activity and increased CD47 expression.

[0016] In some embodiments, HIP cells are human engineered induced pluripotent stem cells (human engineered iPSC), B2M genes are human B2M genes, CIITA genes are human B2M genes, and increased CD47 expression is caused by introducing at least one copy of human CD47 genes into the iPSC under the control of a promoter. In certain embodiments, HIP is mouse engineered induced pluripotent stem cells (mouse engineered iPSC), B2M genes are mouse B2M genes, CIITA genes are mouse B2M genes, and increased CD47 expression is caused by introducing at least one copy of human CD47 genes into the iPSC under the control of a promoter. In some cases, the elimination of B2M gene activity is caused by the clustered regularly spaced short palindromic repeats (CRISPR) / Cas9 reaction of two alleles of the B2M gene. In some cases, the elimination of CIITA gene activity is caused by the CRISPR / Cas9 reaction of two alleles of the CIITA gene.

[0017] In some embodiments, the method further comprises a suicide gene activated by a triggering agent that induces HIP cell death. In some embodiments, the suicide gene is the herpes simplex virus thymidine kinase (HSV-tk) gene, and the triggering agent is ganciclovir. In some cases, the HSV-tk gene encodes a protein having at least 90% sequence identity to SEQ ID NO: 4. In other cases, the HSV-tk gene encodes a protein comprising the amino acid sequence of SEQ ID NO: 4.

[0018] In other embodiments, the suicide gene is an E. coli cytosine deaminase (CD) gene and the trigger is 5-fluorocytosine (5-FC). In some cases, the CD gene encodes a protein having at least 90% sequence identity to SEQ ID NO: 5. In other cases, the CD gene encodes a protein comprising the amino acid sequence of SEQ ID NO: 5.

[0019] In other embodiments, the suicide gene encodes an inducible caspase 9 protein and the trigger is a chemical inducer of dimerization (CID). In some cases, the inducible caspase 9 protein has at least 90% sequence identity to SEQ ID NO: 6. In other cases, the inducible caspase 9 protein comprises the amino acid sequence of SEQ ID NO: 6. In some cases, the CID is compound AP1903.

[0020] In some embodiments, the isolated hypoimmune cardiac cell is selected from the group consisting of a cardiomyocyte, a nodal cardiomyocyte, a conducting cardiomyocyte, a working cardiomyocyte, a cardiomyocyte precursor, a cardiomyocyte progenitor cell, a cardiac stem cell, and a cardiac muscle cell.

[0021] In some aspects, a method of treating a patient with a cardiac condition or disease is provided herein. The method comprises administering a composition comprising a therapeutically effective amount of any isolated, engineered low-immunity cardiac cell population described herein. In some embodiments, the composition further comprises a therapeutically effective carrier.

[0022] In some embodiments, administration comprises implantation into cardiac tissue of a patient, intravenous injection, intraarterial injection, intracoronary injection, intramuscular injection, intraperitoneal injection, intramyocardial injection, transendocardial injection, transepicardial injection, or infusion.

[0023] In some embodiments, the cardiac condition or disease is selected from pediatric cardiomyopathy, age-related cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, chronic ischemic cardiomyopathy, peripartum cardiomyopathy, inflammatory cardiomyopathy, other cardiomyopathies, myocarditis, myocardial ischemia-reperfusion injury, ventricular dysfunction, heart failure, congestive heart failure, coronary artery disease, end-stage heart disease, atherosclerosis, ischemia, hypertension, restenosis, angina, rheumatic heart disease, arterial inflammation, or cardiovascular disease.

[0024] In some aspects, the present invention provides a method for producing a low-immunity cardiac cell population from a low-immunity pluripotent cell (HIP cell) population by in vitro differentiation, wherein endogenous beta-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity have been eliminated in the HIP cells, and CD47 expression has been increased. The method includes: (a) culturing the HIP cell population in a culture medium containing a GSK inhibitor; (b) culturing the HIP cell population in a culture medium containing a WNT antagonist to produce a cardiac progenitor cell population; and (c) culturing the cardiac progenitor cell population in a culture medium containing insulin to produce a low-immunity cardiac cell population.

[0025] In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some cases, the concentration of the GSK inhibitor is about 2 μM to about 10 μM. In some embodiments, the WNT antagonist is IWR1, a derivative thereof, or a variant thereof. In some cases, the concentration of the WNT antagonist is about 2 μM to about 10 μM.

[0026] In some embodiments, the method further comprises culturing the population of cardiac precursor cells of step (c) in a medium comprising a trigger if the HIP cells comprise a suicide gene, wherein the trigger is ganciclovir if the suicide gene is a herpes simplex virus thymidine kinase (HSV-tk) gene, the trigger is 5-fluorocytosine (5-FC) if the suicide gene is an Escherichia coli cytosine deaminase (CD) gene, or the trigger is a chemical inducer of dimerization (CID) if the suicide gene encodes an inducible caspase 9 protein. In certain embodiments, the method further comprises culturing the population of hypoimmune cardiac cells of step (d) in a medium comprising a trigger if the HIP cells comprise a suicide gene, wherein the trigger is ganciclovir if the suicide gene is a herpes simplex virus thymidine kinase (HSV-tk) gene, the trigger is 5-fluorocytosine (5-FC) if the suicide gene is an Escherichia coli cytosine deaminase (CD) gene, or the trigger is a chemical inducer of dimerization (CID) if the suicide gene encodes an inducible caspase 9 protein.

[0027] In some embodiments, the method further comprises isolating the population of hypoimmune cardiac cells from non-cardiac cells. In other embodiments, the method further comprises cryopreserving the isolated population of hypoimmune cardiac cells.

[0028] In some embodiments, the isolated, engineered immunocompromised endothelial cells are selected from the group consisting of capillary endothelial cells, vascular endothelial cells, aortic endothelial cells, brain endothelial cells, and kidney endothelial cells.

[0029] In some aspects, provided herein is a method of treating a patient suffering from a vascular condition or disease. In some embodiments, the method comprises administering a composition comprising a therapeutically effective amount of an isolated, engineered population of immunocompromised endothelial cells.

[0030] The method comprises administering a composition comprising a therapeutically effective amount of any of the isolated, engineered low-immunity endothelial cell populations described herein. In some embodiments, the composition further comprises a therapeutically effective carrier. In some embodiments, administration comprises implantation into a patient's cardiac tissue, intravenous injection, intraarterial injection, intracoronary injection, intramuscular injection, intraperitoneal injection, intramyocardial injection, transendocardial injection, transepicardial injection, or infusion.

[0031] In some embodiments, the vascular condition or disease is selected from vascular injury, cardiovascular disease, vascular disease, ischemic disease, myocardial infarction, congestive heart failure, hypertension, ischemic tissue damage, limb ischemia, stroke, neuropathy, and cerebrovascular disease.

[0032] In some aspects, the present invention provides a method for producing a low immune endothelial cell population from a low immune multipotent cell (HIP cell) population by in vitro differentiation, wherein endogenous β-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity have been eliminated, and CD47 expression in HIP cells has been increased. The method includes: (a) culturing a HIP cell population in a first culture medium comprising a GSK inhibitor; (b) culturing a HIP cell population in a second culture medium comprising VEGF and bFGF to produce an endothelial progenitor cell population; and (c) culturing an endothelial progenitor cell population in a third culture medium comprising a ROCK inhibitor and an ALK inhibitor to produce a low immune endothelial cell population.

[0033] In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some cases, the concentration of the GSK inhibitor is about 1 μM to about 10 μM. In some embodiments, the ROCK inhibitor is Y-27632, a derivative thereof, or a variant thereof. In some cases, the concentration of the ROCK inhibitor is about 1 μM to about 20 μM. In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or a variant thereof. In some cases, the concentration of the ALK inhibitor is about 0.5 μM to about 10 μM.

[0034] In some embodiments, the first culture medium comprises 2 μM to about 10 μM CHIR-99021. In some embodiments, the second culture medium comprises 50 ng / ml VEGF and 10 ng / ml bFGF. In other embodiments, the second culture medium further comprises Y-27632 and SB-431542. In various embodiments, the third culture medium comprises 10 μM MY-27632 and 1 μM SB-431542. In certain embodiments, the third culture medium further comprises VEGF and bFGF. In certain cases, insulin is absent from the first culture medium and / or the second culture medium.

[0035] In some embodiments, the method further comprises isolating the immunocompromised endothelial cell population from the non-endothelial cells. In some embodiments, the method further comprises cryopreserving the isolated immunocompromised endothelial cell population.

[0036] In some embodiments, the isolated hypoimmune dopaminergic neuron is selected from the group consisting of a neuronal stem cell, a neuronal progenitor cell, an immature dopaminergic neuron, and a mature dopaminergic neuron.

[0037] In some aspects, a method for treating a patient suffering from a neurodegenerative disease or condition is provided herein. In some embodiments, the method includes administering a composition comprising any one of the isolated low-immunity dopaminergic neuron populations of a therapeutically effective amount. In some embodiments, the composition further comprises a therapeutically effective carrier. In some embodiments, the isolated low-immunity dopaminergic neuron population is on a biodegradable support. Administration may include transplantation or injection. In some embodiments, the neurodegenerative disease or condition is selected from Parkinson's disease, Huntington's disease, and multiple sclerosis.

[0038] In certain aspects, the present invention provides a method for producing a low-immunity dopaminergic neuron population from a low-immunity pluripotent cell (HIP cell) population by in vitro differentiation, wherein endogenous β-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity have been eliminated, and CD47 expression in HIP cells has been increased. In some embodiments, the method includes (a) culturing a HIP cell population in a first culture medium to produce an immature dopaminergic neuron population, wherein the first culture medium comprises one or more factors selected from the following: sonic hedgehog factor (SHH), BDNF, EGF, bFGF, FGF8, WNT1, retinoic acid, GSK3β inhibitor, ALK inhibitor, and ROCK inhibitor; and (b) culturing an immature dopaminergic neuron population in a second culture medium different from the first culture medium to produce a dopaminergic neuron population.

[0039] In some embodiments, the GSKβ inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some cases, the concentration of the GSKβ inhibitor is about 2 μM to about 10 μM. In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or a variant thereof. In some cases, the concentration of the ALK inhibitor is about 1 μM to about 10 μM. In some embodiments, the first culture medium and / or the second culture medium is free of animal serum.

[0040] In some embodiments, the method further comprises culturing the immature dopaminergic neuron population of step (a) in a medium comprising a triggering agent if the HIP cells comprise a suicide gene, wherein if the suicide gene is a herpes simplex virus thymidine kinase (HSV-tk) gene, the triggering agent is ganciclovir, if the suicide gene is an Escherichia coli cytosine deaminase (CD) gene, the triggering agent is 5-fluorocytosine (5-FC), or if the suicide gene encodes an inducible caspase 9 protein, the triggering agent is a chemical inducer of dimerization (CID).

[0041] In some embodiments, the method further comprises separating the hypoimmune dopaminergic neuron population from the non-dopaminergic neurons.

[0042] In some embodiments, the isolated immunocompromised islet cells are selected from the group consisting of islet progenitor cells, immature islet cells, and mature islet cells.

[0043] In some aspects, a method for treating a patient with diabetes is provided herein. The method includes administering a composition comprising a therapeutically effective amount of any of the isolated low-immunity islet cell populations described herein. In some embodiments, the composition further comprises a therapeutically effective carrier. In some embodiments, the isolated low-immunity islet cell population is on a biodegradable scaffold. In some cases, administration includes transplantation or injection.

[0044] In some aspects, the present invention provides a method for generating a low immunogenic islet cell population from a low immunogenic pluripotent cell (HIP cell) population by in vitro differentiation, wherein endogenous β-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity have been eliminated, and CD47 expression in the HIP cells has been increased. The method comprises: (a) culturing a HIP cell population in a first culture medium to produce an immature islet cell population, wherein the first culture medium comprises one or more factors selected from the group consisting of insulin-like growth factor (IGF), transforming growth factor (TGF), fibroblast growth factor (EGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), sonic hedgehog (SHH) and vascular endothelial growth factor (VEGF), transforming growth factor β (TGFβ) superfamily, bone morphogenetic protein 2 (BMP2), bone morphogenetic protein 7 (BMP7), GSK3β inhibitor, ALK inhibitor, BMP type 1 receptor inhibitor and retinoic acid; and (b) culturing the immature islet cell population in a second culture medium different from the first culture medium to produce a hypoimmune islet cell population.

[0045] In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some cases, the concentration of the GSK inhibitor is about 2 μM to about 10 μM. In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or a variant thereof. In some cases, the concentration of the ALK inhibitor is about 1 μM to about 10 μM. In some embodiments, the first culture medium and / or the second culture medium is free of animal serum.

[0046] In some embodiments, the method further comprises culturing the immature islet cell population of step (a) in a medium comprising a trigger if the HIP cells comprise a suicide gene, wherein if the suicide gene is a herpes simplex virus thymidine kinase (HSV-tk) gene, the trigger is ganciclovir, if the suicide gene is an Escherichia coli cytosine deaminase (CD) gene, the trigger is 5-fluorocytosine (5-FC), or if the suicide gene encodes an inducible caspase 9 protein, the trigger is a chemical inducer of dimerization (CID).

[0047] In certain embodiments, the method further comprises culturing the islet cell population of step (b) in a medium comprising a trigger if the HIP cells comprise a suicide gene, wherein if the suicide gene is a herpes simplex virus thymidine kinase (HSV-tk) gene, the trigger is ganciclovir, if the suicide gene is an Escherichia coli cytosine deaminase (CD) gene, the trigger is 5-fluorocytosine (5-FC), or if the suicide gene encodes an inducible caspase 9 protein, the trigger is a chemical inducer of dimerization (CID).

[0048] In some embodiments, the method further comprises separating the low immunity islet cell population from the non-islet cells. In some embodiments, the method further comprises cryopreserving the separated low immunity islet cell population.

[0049] In some embodiments, the isolated immunocompromised RPE cells are selected from the group consisting of RPE progenitor cells, immature RPE cells, mature RPE cells, and functional RPE cells.

[0050] In some aspects, a method for treating a patient with an ocular condition is provided herein. The method includes administering a composition comprising any of the isolated low-immunity RPE cell groups described herein for a therapeutically effective amount. In some embodiments, the composition also includes a therapeutically effective carrier. In some embodiments, the isolated low-immunity RPE cell group is on a biodegradable support. In certain embodiments, administration includes transplantation or injection to the patient's retina. In some embodiments, the ocular condition is selected from wet macular degeneration, dry macular degeneration, juvenile macular degeneration, Leber congenital amaurosis, retinitis pigmentosa and retinal detachment.

[0051] In some aspects, there is provided herein a method for producing a low-immunity retinal pigment epithelium (RPE) cell group from a low-immunity multipotent cell (HIP cell) group by in vitro differentiation, wherein endogenous beta-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity have been eliminated, and the CD47 expression in the HIP cells has been increased. The method includes: (a) culturing a HIP cell group in a first culture medium to produce an RPE precursor cell group, wherein the first culture medium comprises any one selected from the following factors: activin A, bFGF, BMP4 / 7, DKK1, IGF1, noggin, BMP inhibitor, ALK inhibitor, ROCK inhibitor and VEGFR inhibitor; and (b) culturing an RPE precursor cell group in a second culture medium different from the first culture medium, to produce a low-immunity RPE cell group.

[0052] In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or a variant thereof. In some cases, the concentration of the ALK inhibitor is about 2 μM to about 10 μM. In some embodiments, the ROCK inhibitor is Y-27632, a derivative thereof, or a variant thereof. In some cases, the concentration of the ROCK inhibitor is about 1 μM to about 10 μM.

[0053] In some embodiments, the first culture medium and / or the second culture medium is devoid of animal serum.

[0054] In some embodiments, the method further comprises culturing the RPE precursor cell population of step (a) in a culture medium comprising a trigger if the HIP cells comprise a suicide gene, wherein if the suicide gene is a herpes simplex virus thymidine kinase (HSV-tk) gene, the trigger is ganciclovir, if the suicide gene is an Escherichia coli cytosine deaminase (CD) gene, the trigger is 5-fluorocytosine (5-FC), or if the suicide gene encodes an inducible caspase 9 protein, the trigger is a chemical inducer of dimerization (CID).

[0055] In some embodiments, the method further comprises culturing the colony of the RPE cells of step (b) in a culture medium comprising a trigger if the HIP cells comprise a suicide gene, wherein if the suicide gene is a herpes simplex virus thymidine kinase (HSV-tk) gene, the trigger is ganciclovir, if the suicide gene is an Escherichia coli cytosine deaminase (CD) gene, the trigger is 5-fluorocytosine (5-FC), or if the suicide gene encodes an inducible caspase 9 protein, the trigger is a chemical inducer of dimerization (CID).

[0056] In some embodiments, the method also includes separating low immunity RPE cell group and non-RPE cell.In some embodiments, the method also includes freezing and preserving the low immunity RPE cell group separated.

[0057] V. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 Elispot results of mouse B2m- / -Ciita- / -CD47 tg iPSCs incubated with mouse NK cells (approximately 95% NK cells and 5% macrophages) are shown.

[0059] Figure 2 Elispot results of human B2M- / -CIITA- / -CD47 tg iPSCs incubated with human NK cells (approximately 95% NK cells and 5% macrophages) are shown.

[0060] Figure 3 Elispot results of mouse B2m- / -Ciita- / -CD47 tg iPSCs incubated with human NK cells (approximately 95% NK cells and 5% macrophages) are shown.

[0061] Figure 4 Elispot results of human B2M- / -CIITA- / -CD47 tg iPSCs incubated with mouse NK cells (approximately 95% NK cells and 5% macrophages) are shown.

[0062] Figure 5 Shown are the results of a phagocytosis assay of firefly luciferase-labeled human B2M- / -CIITA- / -CD47tg iPSCs co-cultured with human macrophages.

[0063] Figure 6 Shown are the results of a phagocytosis assay of firefly luciferase-labeled mouse B2m- / -Ciita- / -CD47tg iPSCs co-cultured with mouse macrophages.

[0064] Figure 7 Shown are the results of a phagocytosis assay of firefly luciferase-labeled human B2M- / -CIITA- / -CD47tg iPSCs co-cultured with mouse macrophages.

[0065] Figure 8 Shown are the results of a phagocytosis assay of firefly luciferase-labeled mouse B2m- / -Ciita- / -CD47tg iPSCs co-cultured with human macrophages.

[0066] Fig. 9 A diagram of the differentiation method is provided.

[0067] Fig.10 Shows the results of the experiment on Matrigel just before differentiation. TM Human iPSCs cultured on (magnification 100 times).

[0068] Fig.11 Shown are cells at day 2 of differentiation before medium change (magnification 100X).

[0069] Fig.12 Shown are cells at day 3 of differentiation before medium change (magnification 100X).

[0070] Fig.13 Shown are cells at day 5 of differentiation before medium change (magnification 100X).

[0071] Fig.14 Shown are cells at day 7 of differentiation before medium change (magnification 100X).

[0072] Fig.15 Shown are cells at day 9 of differentiation before medium change (magnification 100X).

[0073] Fig.16 : Differentiation and enrichment of HIP-CM cells as shown by rtPCR.

[0074] Fig.17 It was shown that 28 days after transplantation, hiCMs were not rejected and did not migrate into other organs.

[0075] Fig.18 Histopathology and trichrome staining of recipient hearts 28 days after myocardial infarction are shown. Infarct size as well as left ventricular dimensions were significantly reduced in allogeneic recipients of hiCMs.

[0076] Fig.19A A detailed PV circuit analysis is shown, demonstrating significant improvements in left ventricular parameters.

[0077] Fig.19B hiCMs were shown to restore cardiac function as measured by ejection fraction (EF, the ratio of the amount of blood ejected from a ventricle per beat to the amount of blood in that ventricle at end-diastole) and stroke volume (SV, the amount of blood ejected by the ventricle in a single contraction).

[0078] Fig.19C hiCM was shown to restore cardiac function as measured by ventricular stroke work (SW, the work performed by the left ventricle to eject the stroke volume into the aorta) and cardiac output (CO, the volume of blood pumped by the ventricle per unit time).

[0079] Fig. 20Shown are the results of analyses in which wild-type or B2M- / -CIITA- / -CD47 tg hiCMs were transplanted intramuscularly into allogeneic humanized mice.

[0080] Fig.21 It was shown that hypoimmune hiCM cells also survive after transplantation following myocardial infarction.

[0081] Fig. 22 Mouse hypo-IPSC colonies on MEFs before split are shown (magnification 100X).

[0082] Fig.23 Shown are ESC mouse hypo-IPSCs on gelatin just prior to differentiation (magnification 100X).

[0083] Fig.24 Shown are cells on day 2 of differentiation before the differentiation medium was changed from 5 μM CHIR to 2 μM CHIR (magnification 100X).

[0084] Fig.25 Shown are cells on day 4 of EC differentiation prior to medium change (magnification 100X).

[0085] Fig.26 EC cells at day 7 of differentiation are shown (magnification 100X).

[0086] Fig. 27 Cells after MACS sorting day 12 to day 14 and before MACS sorting are shown (magnification 100X).

[0087] Fig.28 rtPCR is shown, which demonstrates that EC cells derived from both miPSCs and HIP cells displayed differentiated gene expression profiles, including VE-cadherin expression, whereas parental cells did not.

[0088] Fig.29 Bioluminescence analysis of wild-type and hypoimmunogenic endothelial cells transplanted in a mouse model of hindlimb ischemia following removal of the femoral artery (A. femoralis) is shown. BLI values ​​for all animals are plotted. The highly immunogenic wt mECs were rejected within 15 days, showing a decrease in BLI signal over time, whereas all B2M- / -CIITA- / -CD47 tg transplants survived.

[0089] Fig.30 :The inhibitory effect of Cd47 overexpression on NK cell killing was evaluated. - / - Ciita - / - miPSC or B2m - / - Ciita- / - Cd47tg miPSC-miECs were subjected to IFN-γ Elispot using NK cells. - / - Ciita - / - miPSC derivatives are susceptible to NK cell killing.

[0090] Fig.31 Shows B2M - / - CIITA - / - CD47 tg hiPSCs were successfully differentiated into corresponding hiEC derivatives. rtPCR showed that all derivatives lost pluripotency gene expression.

[0091] Fig.32 : Wild-type or B2M- / -CIITA- / -CD47 tg hiEC grafts were injected into allogeneic humanized NSG-SGM3 mice. IFN-γ Elispot was performed 5 days later. Low immune cells did not elicit an IFN-γ response, while wild-type did.

[0092] VI. Detailed Description of the Invention

[0093] A. Introduction

[0094] The present invention provides for the generation of cardiac cells derived from (differentiated from) hypo-immunogenic pluripotent (HIP) cells, and eventual transplantation into patients in need thereof.

[0095] As described in PCT / US18 / 13688, low immunogenic pluripotent (HIP) cells lack major immune antigens that can trigger immune responses and are engineered to avoid phagocytosis. This allows the derivation of "ready-made" cell products for the production of specific tissues and organs. The benefits of being able to use human allogeneic HIP cell derivatives in human patients bring significant benefits, including the ability to avoid long-term adjuvant immunosuppressive therapy and drug use commonly seen in allogeneic transplants. Since cell therapy can be used without the need for individual treatment for each patient, it also provides significant cost savings.

[0096] It has been shown that cell products produced from autologous cell sources can become subject to immune rejection with few or even one antigenic mutation. Therefore, autologous cell products are not inherently non-immunogenic. In addition, since cell engineering and quality control are very labor-intensive and costly, autologous cells may not be easily used for acute treatment options. Therefore, there is a need in the art for a universal cell source such as HIP cells that can be differentiated into a variety of generally acceptable cells for the treatment of various human diseases.

[0097] This application is related to International Application No. PCT / US18 / 13688, filed on January 14, 2018, and U.S. Provisional Application No. 62 / 445,969, filed on January 13, 2017, the entire disclosure of which (especially the examples, figures, figure descriptions, and descriptions of generating low immunogenic pluripotent stem cells and differentiating such cells into other cell types) is incorporated herein by reference.

[0098] B. Definition

[0099] The term "pluripotent cell" refers to a cell that can self-renew and proliferate while maintaining an undifferentiated state and can be induced to differentiate into a specialized cell type under appropriate conditions. As used herein, the term "pluripotent cell" encompasses embryonic stem cells and other types of stem cells, including fetal, amniotic or somatic stem cells. Exemplary human stem cell lines include the H9 human embryonic stem cell line. Other exemplary stem cell lines include those available through the National Institutes of Health HumanEmbryonic Stem Cell Registry and the Howard Hughes Medical Institute HUES collection (described in Cowan, CA et al., New England J.Med.350:13. (2004), which is incorporated herein by reference in its entirety)

[0100] As used herein, "pluripotent stem cells" have the potential to differentiate into any of the three germ layers: endoderm (e.g., gastric junction, gastrointestinal tract, lung, etc.), mesoderm (e.g., muscle, bone, blood, urogenital tissue, etc.), or ectoderm (e.g., epidermal tissue and nervous system tissue). As used herein, the term "pluripotent stem cell" also encompasses "induced pluripotent stem cells" or "iPSCs," which are a type of pluripotent stem cell derived from non-pluripotent cells. Examples of parental cells include somatic cells that have been reprogrammed to induce a pluripotent undifferentiated phenotype in various ways. Such "iPS" or "iPSC" cells can be produced by inducing the expression of certain regulatory genes or by applying certain proteins exogenously. Methods for inducing iPS cells are known in the art and are further described below. (See, e.g., Zhou et al., Stem Cells 27(11):2667-74 (2009); Huangfu et al., Nature Biotechnol. 26(7):795 (2008); Woltjen et al., Nature 458(7239):766-770 (2009); and Zhou et al., Cell Stem Cell 8:381-384 (2009); each of which is incorporated herein by reference in its entirety). The generation of induced pluripotent stem cells (iPSCs) is summarized below. As used herein, "hiPSCs" are human induced pluripotent stem cells, while "miPSCs" are mouse induced pluripotent stem cells.

[0101] "Pluripotent stem cell characteristics" refers to the cell characteristics that distinguish pluripotent stem cells from other cells. The ability to differentiate into offspring of cell types that jointly prove the characteristics associated with the cell lineages from all three germinal layers (endoderm, mesoderm and ectoderm) under appropriate conditions is a pluripotent stem cell characteristic. The expression or non-expression of certain combinations of molecular markers is also a pluripotent stem cell characteristic. For example, human pluripotent stem cells express at least several and in some embodiments all markers in the following non-limiting list: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49 / 6E, ALP, Sox2, E-cadherin, UTF-1, Oct4, Rex1 and Nanog. The cell morphology associated with pluripotent stem cells is also a pluripotent stem cell characteristic. As described herein, cells do not need to be reprogrammed into endoderm progenitor cells and / or hepatocytes through pluripotency.

[0102] As used herein, "multipotent" or "multipotent cell" refers to a cell type that can produce a limited number of other specific cell types. For example, induced multipotent cells are capable of forming endoderm cells. In addition, multipotent blood stem cells can differentiate themselves into several types of blood cells, including lymphocytes, monocytes, neutrophils, etc.

[0103] As used herein, the term "oligopotent" refers to the ability of an adult stem cell to differentiate into only a few different cell types. For example, a lymphoid or myeloid stem cell is capable of forming cells of the lymphoid or myeloid lineage, respectively.

[0104] As used herein, the term "unipotent" refers to the ability of a cell to form a single cell type. For example, spermatogonial stem cells are only able to form sperm cells.

[0105] As used herein, the term "totipotency" refers to the ability of a cell to form a whole organism. For example, in mammals, only fertilized eggs and first division stage blastomeres are totipotent.

[0106] As used herein, "non-pluripotent cells" refers to mammalian cells that are not pluripotent cells. Examples of such cells include differentiated cells and progenitor cells. Examples of differentiated cells include, but are not limited to, cells from tissues selected from bone marrow, skin, skeletal muscle, adipose tissue, and peripheral blood. Exemplary cell types include, but are not limited to, fibroblasts, hepatocytes, myoblasts, neurons, osteoblasts, osteoclasts, and T cells. The starting cells for generating induced multipotent cells, endoderm progenitor cells, and hepatocytes can be non-pluripotent cells.

[0107] Differentiated cells include, but are not limited to, multipotent cells, oligopotent cells, unipotent cells, progenitor cells, and terminally differentiated cells. In certain embodiments, less potent cells are considered "differentiated" relative to more potent cells.

[0108] "Somatic cells" are cells that form the body of an organism. Somatic cells include cells that make up organs, skin, blood, bones, and connective tissues in an organism, but not reproductive cells.

[0109] The cell can be from, for example, a human or non-human mammal. Exemplary non-human mammals include, but are not limited to, mice, rats, cats, dogs, rabbits, guinea pigs, hamsters, sheep, pigs, horses, cattle, and non-human primates. In some embodiments, the cell is from an adult or a non-human mammal. In some embodiments, the cell is from a newborn, an adult, or a non-human mammal.

[0110] As used herein, the term "subject" or "patient" refers to any animal, such as a domestic animal, a zoo animal, or a human. A "subject" or "patient" can be a mammal, such as a dog, a cat, a bird, a livestock, or a human. Specific examples of "subjects" and "patients" include, but are not limited to, individuals (particularly humans) suffering from diseases or conditions related to the liver, heart, lungs, kidneys, pancreas, brain, nervous tissue, blood, bones, bone marrow, etc.

[0111] Mammalian cells can be from humans or non-human mammals. Exemplary non-human mammals include, but are not limited to, mice, rats, cats, dogs, rabbits, guinea pigs, hamsters, sheep, pigs, horses, cows, and non-human primates (e.g., chimpanzees, macaques, and apes).

[0112] As used herein, "hypoimmunogenic pluripotent cells", "hypoimmune pluripotent cells" or "HIP cells" refer to pluripotent cells that retain their pluripotent characteristics and cause a reduced immune rejection response when transferred into an allogeneic host. In a preferred embodiment, HIP cells do not cause an immune response. Therefore, "hypoimmunogenicity" or "hypoimmunity" refers to an immune response that is significantly reduced or eliminated compared to the immune response of the parental (i.e., "wild type" or "wt") cells before immunoengineering as outlined herein. In many cases, HIP cells are immunologically silent but still retain pluripotent capabilities. Determination of HIP characteristics is outlined below.

[0113] The "HLA" or "human leukocyte antigen" complex is a gene complex that encodes the major histocompatibility complex (MHC) proteins in humans. These cell surface proteins that make up the HLA complex are responsible for regulating the immune response to antigens. In humans, there are two types of MHC, class I and class II, "HLA-I" and "HLA-II". HLA-I includes three proteins, HLA-A, HLA-B and HLA-C, which present peptides from the inside of the cell, while the antigens presented by the HLA-I complex attract killer T cells (also known as CD8+T- cells or cytotoxic T cells). HLA-I proteins are associated with beta-2 microglobulin (B2M). HLA-II includes five proteins, HLA-DP, HLA-DM, HLA-DOB, HLA-DQ and HLA-DR, which present antigens to T lymphocytes from the outside of the cell. This stimulates CD4+ cells (also known as T helper cells). It should be understood that the use of "MHC" or "HLA" is not meant to be limiting, as it depends on whether the gene is from human (HLA) or mouse (MHC). Therefore, as it relates to mammalian cells, these terms can be used interchangeably herein.

[0114] "Gene knockout" herein refers to the process of inactivating a specific gene in the host cell in which it resides, resulting in the non-production of the protein of interest or the formation of an inactivated form. As will be appreciated by those skilled in the art and described further below, this can be accomplished in many different ways, including removing the nucleic acid sequence from the gene, or interrupting the sequence with other sequences, changing the reading frame, or changing the regulatory components of the nucleic acid. For example, all or part of the coding region of the gene of interest can be removed or replaced by a "nonsense" sequence, all or part of a regulatory sequence such as a promoter can be removed or replaced, a translation initiation sequence can be removed or replaced, etc.

[0115] "Gene knock-in" herein refers to the process of increasing genetic function to a host cell. This results in an increase in the level of the encoded protein. As will be appreciated by those skilled in the art, this can be achieved in several ways, including adding one or more other copies of the gene to the host cell or changing the regulatory components of the endogenous gene, thereby increasing the expression of the protein. This can be achieved by modifying the promoter, adding different promoters, adding enhancers or modifying other gene expression sequences.

[0116] "β-2 microglobulin" or "β2M" or "B2M" protein refers to the human β2M protein having the amino acid and nucleic acid sequences shown below; the accession number of the human gene is NC_000015.10:44711487-44718159.

[0117] The "CD47 protein" protein refers to the human CD47 protein having the amino acid and nucleic acid sequences shown below; the accession number of the human gene is NC_000003.12:108043094-108094200.

[0118] The "CIITA protein" protein refers to the human CIITA protein having the amino acid and nucleic acid sequences shown below; the accession number of the human gene is NC_000016.10:10866208-10941562.

[0119] In the context of cells, "wild type" refers to cells found in nature. However, as used herein, in the context of pluripotent stem cells, it also means iPSCs that may contain nucleic acid changes that lead to pluripotency but have not undergone the gene editing procedures of the present invention to obtain low immunogenicity.

[0120] As used herein, "syngeneic" refers to the genetic similarity or identity of the host organism and the cell transplant wherein immunological compatibility exists; eg, no immune response is generated.

[0121] "Allogeneic" in this context refers to the genetic dissimilarity between the host organism and the cell transplant, in which an immune response is generated.

[0122] "B2M- / -" herein refers to a diploid cell having an inactivated B2M gene in both chromosomes. As described herein, this can be accomplished in a variety of ways.

[0123] "CIITA- / -" herein refers to a diploid cell having an inactivated CIITA gene in both chromosomes. As described herein, this can be accomplished in a variety of ways.

[0124] As used herein, "CD47 tg" (standing for "transgenic") or "CD47+") refers to a host cell that expresses CD47 and, in certain instances, has at least one additional copy of the CD47 gene.

[0125] "Oct polypeptide" refers to any naturally occurring member of the Octamer family of transcription factors or a variant thereof that maintains transcription factor activity, which is similar to the most relevant naturally occurring family member (within at least 50%, 80% or 90% of the activity), or a polypeptide that contains at least the DNA binding domain of a naturally occurring family member and may also contain a transcriptional activation domain. Exemplary Oct polypeptides include Oct-1, Oct-2, Oct-3 / 4, Oct-6, Oct-7, Oct-8, Oct-9, and Oct-11. Oct3 / 4 (referred to herein as "Oct4") contains a POU domain, which is a 150 amino acid sequence conserved in Pit-1, Oct-1, Oct-2, and uric-86. (See Ryan, AK & Rosenfeld, MG, Genes Dev. 11: 1207-1225 (1997), which is incorporated herein by reference in its entirety). In some embodiments, the variant has at least 85%, 90% or 95% amino acid sequence identity over its entire sequence compared to a naturally occurring Oct polypeptide family member (e.g., those listed above or those listed in Genbank Accession No. NP-002692.2 (human Oct4) or NP-038661.1 (mouse Oct4). Oct polypeptides (e.g., Oct3 / 4 or Oct 4) can be from humans, mice, rats, cattle, pigs, or other animals. Typically, proteins of the same species will be used with the cell species being manipulated. Oct polypeptides can be pluripotent factors that can help induce pluripotency in non-pluripotent cells.

[0126] "Klf polypeptide" refers to any naturally occurring member of the Krüppel-like factor (Klf) family (which is a zinc finger protein containing an amino acid sequence similar to the Drosophila embryonic patterning regulator Krüppel), or a variant of a naturally occurring member that retains similar activity (at least 50%, 80% or 90% of the activity) to the most related naturally occurring family member, or a polypeptide that comprises at least the DNA binding domain of a naturally occurring family member and may also comprise a transcriptional activation domain. (See Dang, DT, Pevsner, J. & Yang, VW, Cell Biol. 32: 1103-1121 (2000), which is incorporated herein by reference in its entirety). Exemplary Klf family members include Klf1, Klf2, Klf3, Klf-4, Klf5, Klf6, Klf7, Klf8, Klf9, Klf10, Klf11, Klf12, Klf13, Klf14, Klf15, Klf16, and Klf17. Klf2 and Klf-4 were found to be factors capable of generating iPS cells in mice, and the related genes Klf1 and Klf5 also do so, albeit with reduced efficiency. (See Nakagawa et al., Nature Biotechnology 26: 101-106 (2007), which is incorporated herein by reference in its entirety). In some embodiments, the variant has at least 85%, 90%, or 95% amino acid sequence identity over its entire sequence with a naturally occurring Klf polypeptide family member (e.g., those listed above or those listed in GenBank Accession Nos. CAX16088 (mouse Klf4) or CAX14962 (human Klf4)). Klf polypeptides (e.g., Klf1, Klf4, and Klf5) can be from humans, mice, rats, cows, pigs, or other animals. Typically, the same species of protein will be used with the cell species being manipulated. Klf polypeptides can be pluripotency factors. Expression of a Klf4 gene or polypeptide can help induce pluripotency in a starting cell or starting cell population.

[0127] "Myc polypeptide" refers to any natural member of the Myc family. (See, e.g., Adhikary, S. & Eilers, M., Nat. Rev. Mol. Cell Biol. 6: 635-645 (2005), which is incorporated herein by reference in its entirety). It also includes variants that maintain similar transcription factor activity (i.e., within at least 50%, 80%, or 90% of the activity) compared to the most relevant naturally occurring family member. It also includes polypeptides that contain at least the DNA binding domain of a naturally occurring family member and may also contain a transcriptional activation domain. Exemplary Myc polypeptides include, for example, c-Myc, N-Myc, and L-Myc. In some embodiments, the variant has at least 85%, 90%, or 95% amino acid sequence identity over its entire sequence compared to naturally occurring Myc polypeptide family members (e.g., those listed above or those listed in GenBank Accession No. CAA25015 (human Myc)). Myc polypeptides (e.g., c-Myc) can be from humans, mice, rats, cattle, pigs, or other animals. Typically, the same class of protein will be used with the cell class being manipulated.The Myc polypeptide may be a pluripotency factor.

[0128] "Sox polypeptide" refers to any naturally occurring member of the SRY-related HMG-box (Sox) transcription factor, characterized by the presence of a high mobility group (HMG) domain or a variant thereof that maintains similar transcription factor activity (i.e., within at least 50%, 80% or 90% of the activity) compared to the most related naturally occurring family member. It also includes polypeptides that contain at least the DNA binding domain of a naturally occurring family member and may also contain a transcriptional activation domain. (See, e.g., Dang, DT et al., Int. J. Biochem. Cell Biol. 32: 1103-1121 (2000), which is incorporated herein by reference in its entirety). Exemplary Sox polypeptides include, e.g., Sox1, Sox-2, Sox3, Sox4, Sox5, Sox6, Sox7, Sox8, Sox9, Sox10, Sox11, Sox12, Sox13, Sox14, Sox15, Sox17, Sox18, Sox-21, and Sox30. Sox1 has been shown to generate iPS cells with similar efficiency to Sox2, and the genes Sox3, Sox15, and Sox18 have also been shown to generate iPS cells, although with slightly lower efficiency than Sox2. (See Nakagawa, et al., Nature Biotechnology 26: 101-106 (2007), which is incorporated herein by reference in its entirety). In some embodiments, the variant has at least 85%, 90%, or 95% amino acid sequence identity over its entire sequence compared to a naturally occurring Sox polypeptide family member (e.g., those listed above or those listed in GenBank Accession No. CAA83435 (human Sox2)). The Sox polypeptide (e.g., Sox1, Sox2, Sox3, Sox15, or Sox18) can be from a human, mouse, rat, cow, pig, or other animal. Typically, proteins of the same species will be used with the cell species being manipulated. The Sox polypeptide can be a pluripotent factor. As described herein, the SOX2 protein has found specific use in the generation of iPSCs.

[0129] Herein, "differentiated low immunogenic pluripotent cells" or "differentiated HIP cells" or "dHIP cells" refer to iPS cells that have been engineered to have low immunogenicity (e.g., by knocking out B2M and CIITA and knocking in CD47) and subsequently differentiated into cell types for ultimate transplantation into a subject. Thus, for example, HIP cells can be differentiated into hepatocytes ("dHIP hepatocytes"), β-like pancreatic cells or pancreatic islet organoids ("dHIP β cells"), endothelial cells ("dHIP endothelial cells"), etc.

[0130] In the context of two or more nucleic acids or polypeptide sequences, the term "identity" percentage refers to two or more sequences or subsequences of identical nucleotides or amino acid residues with a specified percentage when comparing and aligning with respect to maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to those skilled in the art) or by visual inspection. Depending on the application, the "identity" percentage may be present in the region of the sequence being compared, such as in a functional domain, or alternatively in the total length of the two sequences to be compared. For sequence comparison, a sequence is usually used as a reference sequence for comparison with a test sequence. When using a sequence comparison algorithm, test sequence and reference sequence are input into a computer, subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. Then, the sequence comparison algorithm calculates the sequence identity percentage of the test sequence relative to the reference sequence based on the specified program parameters.

[0131] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., see below).

[0132] The BLAST algorithm is one example of an algorithm suitable for determining percent sequence identity and sequence similarity and is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ).

[0133] "Inhibitors," "activators," and "modulators" affect the function or expression of biologically relevant molecules. The term "modulator" includes inhibitors and activators. They can be identified using in vitro and in vivo assays for the expression or activity of a target molecule.

[0134] "Inhibitors" are agents that, for example, inhibit expression or binding of a target molecule or protein. They may partially or completely block stimulation or have protease inhibitor activity. They may reduce, decrease, prevent or delay activation, including inactivation, desensitization or downregulation of the described target protein activity. Modulators may be antagonists of a target molecule or protein.

[0135] "Activators" are agents that, for example, induce or activate the function or expression of a target molecule or protein. They can bind to, stimulate, increase, turn on, activate or promote the activity of a target molecule. Activators can be agonists of a target molecule or protein.

[0136] "Homologues" are biologically active molecules that are similar to reference molecules at the nucleotide sequence, peptide sequence, function or structural level. Homologues can include sequence derivatives that have a certain percentage of identity with the reference sequence. Therefore, in one embodiment, homologous or derived sequences have at least 70% sequence identity. In a specific embodiment, homologous or derived sequences have at least 80% or 85% sequence identity. In a specific embodiment, homologous or derived sequences have at least 90% sequence identity. In a specific embodiment, homologous or derived sequences have at least 95% sequence identity. In a more specific embodiment, homologous or derived sequences have at least 50, 55, 60, 65, 70, 75, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity. Homologous or derived nucleic acid sequences can also be defined by their ability to remain bound to a reference nucleic acid sequence under high stringency hybridization conditions. A homologue having structural or functional similarity to a reference molecule may be a chemical derivative of the reference molecule. Methods for detecting, generating and screening structural and functional homologues and derivatives are known in the art.

[0137] "Hybridization" generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of homology required between the probe and the hybridizable sequence, the higher the relative temperature that can be used. As a result, it is concluded that higher relative temperatures will tend to make the reaction conditions more stringent, while lower temperatures are less stringent. For additional details and explanations of the stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers (1995), the entire contents of which are incorporated herein by reference.

[0138] The "stringency" of a hybridization reaction is readily determined by one of ordinary skill in the art and is generally an empirical calculation that depends on probe length, washing temperature, and salt concentration. Typically, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures.

[0139] "Stringent conditions" or "high stringency conditions" as defined herein can be identified by: (1) using low ionic strength and high temperature for washing, for example, 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C; (2) using a denaturing agent, such as formamide, during hybridization at 42°C, for example, 50% (v / v) formamide with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer, pH 6.5, with 750 mM sodium chloride, 75 mM sodium citrate; or (3) using 50% formamide, 5x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Hybridization was performed overnight at 42°C in a solution of Denhardt's solution, sonicated salmon sperm DNA (50 μl / ml), 0.1% SDS and 10% dextran sulfate, washed in 0.2x SSC (sodium chloride / sodium citrate) at 42°C for 10 minutes, and then washed in a high stringency wash solution consisting of 0.1x SSC containing EDTA at 55°C for 10 minutes.

[0140] It is intended that each maximum numerical limitation given throughout this specification includes each lower numerical limitation, just as if these lower numerical limitations were expressly written herein. Each minimum numerical limitation given throughout this specification will include each higher numerical limitation, just as if these higher numerical limitations were expressly written herein. Each numerical range given throughout this specification will include each narrower numerical range that falls within such wider numerical range, just as if these narrower numerical ranges were expressly written herein.

[0141] As used herein, the term "modification" refers to a change that physically distinguishes a modified molecule from a parent molecule. In one embodiment, the amino acid changes in the CD47, HSVtk, EC-CD or iCasp9 variant polypeptides prepared according to the methods described herein distinguish them from the corresponding parents (e.g., wild-type proteins, naturally occurring mutant proteins, or other engineered proteins that do not include modifications of such variant polypeptides) that are not modified according to the methods described herein. In another embodiment, the variant polypeptide includes one or more modifications that distinguish the function of the variant polypeptide from the unmodified polypeptide. For example, the amino acid changes in the variant polypeptide affect its receptor binding profile. In other embodiments, the variant polypeptide comprises a substitution, deletion or insertion modification or a combination thereof. In another embodiment, the variant polypeptide includes one or more modifications that increase its affinity for the receptor compared to the affinity of the unmodified polypeptide.

[0142] In one embodiment, variant polypeptide comprises one or more substitutions, insertions or deletions relative to a corresponding native or parent sequence. In certain embodiments, variant polypeptide comprises 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, 30, 31-40, 41 to 50 or 51 or more modifications.

[0143] "Episomal vector" herein refers to a genetic vector that can exist and replicate autonomously in the cytoplasm of a cell; for example, it is not integrated into the genomic DNA of the host cell. Many episomal vectors are known in the art and are described below.

[0144] In the context of a gene, "knockout" refers to a host cell with the knockout that does not produce a functional protein product of the gene. As described herein, knockout can be produced in a variety of ways by removing all or part of the coding sequence, introducing a frameshift mutation so that no functional protein (truncated or nonsense sequence) is produced, removing or changing a regulatory component (e.g., a promoter) so that the gene is not transcribed, preventing translation by binding to mRNA, etc. Typically, knockout is achieved at the genomic DNA level so that the offspring of the cell also permanently carry the knockout.

[0145] In the context of a gene, "knock-in" means that a host cell with the knock-in has more functional protein with activity in the cell. As described herein, knock-in can be accomplished in a variety of ways, typically by introducing at least one copy of a protein-encoding transgene (tg) into a cell, although this can also be accomplished by replacing regulatory components, for example, by adding a constitutive promoter to an endogenous gene. Typically, knock-in techniques result in the integration of additional copies of the transgene into the host cell.

[0146] VII. Low immunogenic pluripotent cells

[0147] The present invention provides compositions and methods for generating mouse and human HIP cells by starting with wild-type cells, rendering them pluripotent (eg, making induced pluripotent stem cells or iPSCs), and then generating HIP cells from the iPSC population.

[0148] Provided herein is a low immunogenic pluripotent (HIP) stem cell, comprising: one or more changes that inactivate two alleles of the endogenous B2M gene; one or more changes that inactivate two alleles of the endogenous CIITA gene; and one or more changes that cause increased CD47 gene expression in human HIP stem cells; wherein the first natural killer (NK) cell response caused by human HIP stem cells is lower than the second NK cell response caused by induced pluripotent stem cells (iPSC), the induced pluripotent stem cells including the B2M and CIITA changes, but not including the increased CD47 gene expression, wherein the first and second NK cell responses are measured by measuring the IFN-γ level of NK cells incubated in vitro with human HIP or iPSCs containing B2M and CIITA changes but not including increased CD47 gene expression. HIP stem cells can be mouse HIP stem cells. In some embodiments, HIP stem cells are human HIP stem cells.

[0149] Due to reduced HLA-I function, reduced HLA-II function, and reduced sensitivity to NK cell killing, the low immunogenic pluripotent cells may be less susceptible to rejection when transplanted into a subject.

[0150] In some embodiments, the low immunogenic pluripotent cells have reduced or absent beta-2 microglobulin expression. In a preferred embodiment, the gene encoding beta-2 microglobulin is eliminated or knocked out. In a more preferred embodiment, the beta-2 microglobulin has at least 90% (e.g., 91%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 1. In a more preferred embodiment, the beta-2 microglobulin protein has the sequence of SEQ ID NO: 1.

[0151] In some embodiments, HLA-I function is reduced by reducing HLA-A protein expression. In a preferred embodiment, the gene encoding the HLA-A protein is eliminated or knocked out. In some embodiments, HLA-I function is reduced by reducing HLA-B protein expression. In a preferred embodiment, the gene encoding the HLA-B protein is eliminated or knocked out. In some embodiments, HLA-I function is reduced by reducing HLA-C protein expression. In a preferred embodiment, the gene encoding the HLA-C protein is eliminated or knocked out. In another embodiment, the low immunogenic pluripotent cells do not contain HLA-I function.

[0152] In some embodiments, the low immunogenic pluripotent cells have reduced or lack CIITA protein expression. In a preferred embodiment, the gene encoding the CIITA protein is eliminated or knocked out. In a more preferred embodiment, the CIITA protein has at least 90% (e.g., 91%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with SEQ ID NO: 2. In a more preferred embodiment, the CIITA protein has the sequence of SEQ ID NO: 2.

[0153] In some embodiments, HLA-II function is reduced by reducing HLA-DP protein expression. In a preferred embodiment, the gene encoding the HLA-DP protein is eliminated or knocked out. In some embodiments, HLA-II function is reduced by reducing HLA-DR protein expression. In a preferred embodiment, the gene encoding the HLA-DR protein is eliminated or knocked out. In some embodiments, HLA-II function is reduced by reducing HLA-DQ protein expression. In a preferred embodiment, the gene encoding the HLA-DQ protein is eliminated or knocked out. The present invention provides low immunogenic pluripotent cells without HLA-II function.

[0154] The present invention provides low immunogenic pluripotent cells with reduced sensitivity to macrophage phagocytosis or NK cell killing. The reduced sensitivity is due to increased expression of CD47 protein. In some embodiments, the increased CD47 expression is caused by modification of the endogenous CD47 gene site. In other embodiments, the increased CD47 expression is derived from the CD47 transgene. In a preferred embodiment, the CD47 protein has at least 90% (e.g., 91%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with SEQ ID NO: 3. In a more preferred embodiment, the CD47 protein has the sequence of SEQ ID NO: 3.

[0155] In another embodiment of the method, the increased expression of a protein that reduces the sensitivity of pluripotent cells to macrophage phagocytosis is caused by modification of an endogenous locus. In a preferred embodiment, the endogenous locus encodes a CD47 protein. In another embodiment, the increased protein expression is caused by expression of a transgene. In a preferred embodiment, the transgene encodes a CD47 protein. In a more preferred embodiment, the CD47 protein has at least 90% (e.g., 91%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 3. In a more preferred embodiment, the CD47 protein has the sequence of SEQ ID NO: 3.

[0156] In some embodiments, the level of CD47 protein in HIP cells is higher than the level in corresponding pluripotent stem cells, such as embryonic stem cells or induced pluripotent stem cells. In some cases, the level of mouse CD47 protein in mouse HIP cells is higher (e.g., at least 0.5 times, at least 1.0 times, at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 8 times or more) than the level in corresponding mouse pluripotent stem cells. In some cases, the level of human CD47 protein in human HIP cells is higher (e.g., at least 0.5 times, at least 1.0 times, at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 8 times or more) than the level in corresponding human pluripotent stem cells.

[0157] Another embodiment of the method further comprises expressing a suicide gene activated by a trigger that causes death of pluripotent or differentiated progeny cells with low immunogenicity. In a preferred embodiment, the suicide gene is the herpes simplex virus thymidine kinase gene (HSV-tk), and the trigger is ganciclovir. In a more preferred embodiment, the HSV-tk gene encodes a protein having at least 90% (e.g., 91%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 4. In a more preferred embodiment, the HSV-tk gene encodes a protein having the sequence of SEQ ID NO: 4.

[0158] In another embodiment of the method, the suicide gene is an E. coli cytosine deaminase gene (EC-CD), and the trigger is 5-fluorocytosine (5-FC). In a preferred embodiment, the EC-CD gene encodes a protein having at least 90% (e.g., 91%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO: 5. In a more preferred embodiment, the EC-CD gene encodes a protein having the sequence of SEQ ID NO: 5.

[0159] In another embodiment of the method, the suicide gene encodes an inducible caspase protein and the trigger is a specific chemical inducer of dimerization (CID). In a preferred embodiment of the method, the gene encodes an inducible caspase comprising a sequence identity to SEQ ID NO: 6 of at least 90% (e.g., 91%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%). In a more preferred embodiment, the gene encodes an inducible caspase comprising the sequence of SEQ ID NO: 6. In a more preferred embodiment, the CID is AP1903.

[0160] A. Methods for Genetic Alteration

[0161] The present invention includes modifying the nucleic acid sequence in the cell or under the cell-free condition to produce the method for pluripotent cells and HIP cells. Exemplary techniques include homologous recombination, knock-in, ZFN (zinc finger nuclease), TALEN (transcription activator-like effector nuclease), CRISPR (clustered regularly spaced short palindromic repeats) / Cas9 and other site-specific nuclease technologies. These technologies enable double-stranded DNA breaks to be performed at the desired gene locus. These controlled double-stranded breaks promote homologous recombination at specific gene locus points. The process focuses on the specific sequence of the nucleic acid molecule (such as chromosome) targeted by endonuclease, and the endonuclease recognizes and combines the sequence and induces double-strand breaks in the nucleic acid molecule. Double-strand breaks are repaired by fallible non-homologous end joining (NHEJ) or by homologous recombination (HR).

[0162] As will be appreciated by those skilled in the art, a number of different techniques may be used to engineer the pluripotent cells of the invention, as well as to engineer iPSCs to become hypoimmunogenic as described herein.

[0163] Typically, these techniques can be used alone or in combination. For example, in the production of HIP cells, CRISPR technology can be used to reduce the expression of active B2M and / or CIITA proteins in engineered cells, and viral technology (e.g., lentivirus) can be used to knock in CD47 functionality. In addition, as will be appreciated by those skilled in the art, although one embodiment sequentially utilizes a CRISPR step to knock out B2M, then knocks out CIITA by a CRISPR step, and finally knocks in CD47 functionality using a lentivirus, these genes can be manipulated in different orders using different techniques.

[0164] As discussed more fully below, transient expression of reprogramming genes is typically performed to generate / induce pluripotent stem cells.

[0165] a. CRISPR technology

[0166] In one embodiment, cells are manipulated using clustered regularly interspaced short palindromic repeats / Cas ("CRISPR") technology known in the art. CRISPR can be used to generate starting iPSCs or generate HIP cells from iPSCs. There are a large number of CRISPR-based technologies, see, for example, Doudna and Charpentier, Science doi: 10.1126 / science.1258096, which is incorporated herein by reference. CRISPR technology and kits are commercially available.

[0167] b.TALEN technology

[0168] In some embodiments, the HIP cells of the present invention are prepared using the transcription activator-like effector nuclease (TALEN) method. TALEN is a restriction enzyme combined with a nuclease that can be engineered to bind to and cut virtually any desired DNA sequence. TALEN kits are commercially available.

[0169] c. Zinc finger technology

[0170] In one embodiment, cells are manipulated using zinc finger nuclease technology. Zinc finger nucleases are artificial restriction enzymes created by fusing zinc finger DNA binding domains to DNA cleavage domains. Zinc finger domains can be engineered to target specific desired DNA sequences, which enables zinc finger nucleases to target unique sequences in complex genomes. By exploiting endogenous DNA repair mechanisms, these agents can be used to precisely alter the genomes of higher organisms, similar to CRISPR and TALEN.

[0171] d. Virus-based technologies

[0172] There are a variety of viral technologies that can be used to generate the HIP cells of the present invention (as well as for the original generation of iPSCs), including but not limited to the use of retroviral vectors, lentiviral vectors, adenoviral vectors, and Sendai viral vectors. Episomal vectors for generating iPSCs are described below.

[0173] e. Gene downregulation using interfering RNA

[0174] In other embodiments, the genes encoding proteins used in HLA molecules are downregulated by RNAi technology. RNA interference (RNAi) is a process in which RNA molecules inhibit gene expression, usually by causing degradation of specific mRNA molecules. Two types of RNA molecules, microRNA (miRNA) and small interfering RNA (siRNA), are core to RNA interference. They bind to target mRNA molecules and increase or decrease their activity. RNAi helps cells defend against parasitic nucleic acids, such as nucleic acids from viruses and transposons. RNAi also affects development.

[0175] sdRNA molecules are a class of asymmetric siRNAs that contain a guide (antisense) strand of 19-21 bases. They contain 5' phosphate, 2'Ome or 2'F modified pyrimidines and six thiophosphates (phosphotioate) at the 3' position. They also contain a sense strand that contains a sterol moiety conjugated to 3', 2 thiophosphates (phosphotioate) at the 3' position, and 2'Ome modified pyrimidines. Both strands contain 2'Ome purines, wherein the length of the continuous segment of unmodified purines does not exceed 3. sdRNA is disclosed in U.S. Patent No. 8,796,443, which is incorporated herein by reference in its entirety.

[0176] For all these techniques, well-known recombinant techniques are used to produce recombinant nucleic acids as outlined herein. In certain embodiments, recombinant nucleic acids (not encoding desired polypeptides, such as CD47, or disrupting sequences) can be operably linked to one or more regulatory nucleotide sequences in an expression construct. The regulatory nucleotide sequence will generally be suitable for the host cell and the subject to be treated. For a variety of host cells, various types of suitable expression vectors and suitable regulatory sequences are known in the art. Typically, one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader sequences or signal sequences, ribosome binding sites, transcription start and stop sequences, translation start and stop sequences, and enhancers or activation sequences. Constitutive or inducible promoters known in the art are also contemplated. These promoters may be naturally occurring promoters or hybrid promoters that combine elements of more than one promoter. The expression construct may be present in cells of an episome such as a plasmid, or the expression construct may be inserted into a chromosome. In a specific embodiment, the expression vector includes a selectable marker gene to allow selection of transformed host cells. Some embodiments include an expression vector comprising a nucleotide sequence encoding a variant polypeptide operably linked to at least one regulatory sequence. Regulatory sequences used herein include promoters, enhancers and other expression control elements. In certain embodiments, the expression vector is designed to select the host cell to be transformed, the specific variant polypeptide desired to be expressed, the copy number of the vector, the ability to control the copy number, or the expression of any other protein encoded by the vector, such as an antibiotic marker.

[0177] Examples of suitable promoters include, for example, promoters from the following genes: the ubiquitin / S27a promoter of hamsters (WO 97 / 15664), the simian vacuolating virus 40 (SV40) early promoter, the adenovirus major late promoter, the mouse metallothionein-I promoter, the long terminal repeat region of Rous sarcoma virus (RSV), the mouse mammary tumor virus promoter (MMTV), the Moloney murine leukemia virus long terminal repeat region, and the early promoter of human cytomegalovirus (CMV). Examples of other heterologous mammalian promoters are actin, immunoglobulin, or heat shock promoters. In some embodiments, the elongation factor 1-alpha promoter is used.

[0178] In other embodiments, promoters for mammalian host cells can be obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published July 5, 1989), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and simian virus 40 (SV40). In other embodiments, heterologous mammalian promoters are used. Examples include actin promoters, immunoglobulin promoters, and heat shock promoters. The early and late promoters of SV40 are conveniently obtained in the form of SV40 restriction fragments, which also contain the replication origin of the SV40 virus. Fiers et al., Nature 273: 113-120 (1978). The immediate early promoter of human cytomegalovirus can be conveniently obtained as a HindIII E restriction fragment. Greenaway, PJ et al., Gene 18: 355-360 (1982). The foregoing references are incorporated herein by reference in their entirety.

[0179] B. Generation of pluripotent cells

[0180] The present invention provides a method for producing non-immunogenic pluripotent cells from pluripotent cells. Therefore, the first step is to provide pluripotent stem cells.

[0181] The generation of mouse and human pluripotent stem cells (commonly referred to as iPSC: mouse cells are miPSC or human cells are hiPSC) is well known in the art. As will be appreciated by those skilled in the art, there are a variety of methods for generating iPCS. The initial induction is accomplished from mouse embryos or adult fibroblasts using viral introduction of four transcription factors, namely Oct3 / 4, Sox2, c-Myc and Klf4: see Takahashi and Yamanaka Cell 126: 663-676 (2006), the entire contents of which (particularly the techniques outlined therein) are incorporated herein by reference. Since then, many methods have been developed: for review, see Seki et al., World J. Stem Cells 7(1): 116-125 (2015), and Lakshmipathy and Vermuri, editors, Methods in Molecular Biology: Pluripotent Stem Cells, Methods and Protocols, Springer 2013, the entire contents of which are expressly incorporated herein by reference, especially the methods for generating hiPSCs (see, e.g., Chapter 3 of the latter reference).

[0182] In general, iPSC is produced by transient expression (usually introduced using episomal vectors) of one or more "reprogramming factors" in host cells. Under these conditions, a small amount of cells are induced to become iPSC (usually, the efficiency of this step is very low because selection markers are not used). Once cells are "reprogrammed" and become pluripotent, they will lose the episomal vector and use endogenous genes to produce factors. Such a loss of episomal vectors results in cells referred to as "zero footprint" cells. This is desirable because the less genetic modification (especially in the genome of the host cell), the better. Therefore, it is preferred that the resulting hiPSC does not have permanent genetic modification.

[0183] As also understood by those skilled in the art, the number of reprogramming factors that can be used or used can vary. Typically, when using fewer reprogramming factors, the efficiency of converting cells into a pluripotent state and, for example, "pluripotency" is reduced, for example, fewer reprogramming factors can result in incomplete pluripotency but can only be able to differentiate into cells of fewer cell types.

[0184] In some embodiments, a single reprogramming factor OCT4 is used. In other embodiments, two reprogramming factors OCT4 and KLF4 are used. In other embodiments, three reprogramming factors OCT4, KLF4 and SOX2 are used. In other embodiments, four reprogramming factors OCT4, KLF4, SOX2 and c-Myc are used. In other embodiments, 5, 6 or 7 reprogramming factors selected from SOKMNLT: SOX2, OCT4 (POU5F1), KLF4, MYC, NANOG, LIN28 and SV40L T antigen can be used.

[0185] Typically, these reprogramming factor genes are provided on episomal vectors, as known in the art and commercially available. For example, ThermoFisher / Invitrogen sells a Sendai virus reprogramming kit for zero-footprint generation of hiPSCs, see catalog number A34546. ThermoFisher also sells an EBNA-based system, see catalog number A14703.

[0186] Additionally, there are many commercially available hiPSC cell lines: see e.g., Episomal hiPSC cell line, K18945, which is a zero-footprint, viral integration-free human iPSC cell line (see also Burridge et al., 2011, supra).

[0187] Typically, as known in the art, iPSCs are made from non-pluripotent cells such as CD34+ umbilical cord blood cells, fibroblasts, etc. by transiently expressing reprogramming factors as described herein.

[0188] For example, despite the omission of C-Myc, the use of only Oct3 / 4, Sox2, and Klf4 generated successful iPSCs albeit with reduced reprogramming efficiency.

[0189] Typically, iPSCs are characterized by the expression of certain factors, including KLF4, Nanog, OCT4, SOX2, ESRRB, TBX3, c-Myc, and TCL1. For the purposes of the present invention, the new or increased expression of these factors can be through induction or regulation of endogenous loci or from expression of transgenes.

[0190] For example, murine iPSCs can be generated using the method of Diecke et al., Sci Rep. 2015, Jan. 28; 5: 8081 (doi: 10.1038 / srep08081) (incorporated herein by reference in its entirety, particularly for methods and reagents for generating miPSCs). See also, for example, Burridge et al., PLoS One, 2011 6 (4): 18293, which is incorporated herein by reference in its entirety, particularly for the methods outlined therein.

[0191] In some cases, the pluripotency of the cells is measured or confirmed as outlined herein, for example, by assaying for reprogramming factors as generally shown in PCT / US18 / 13688 or by performing a differentiation reaction as outlined herein and in the Examples.

[0192] C. Generation of low immunogenic pluripotent cells

[0193] The present invention relates to the generation, manipulation, growth and transplantation of hypoimmunogenic cells into patients as defined herein.The generation of HIP cells from pluripotent cells is performed using as few as three genetic alterations, resulting in minimal disruption of cell activity, but rendering the cells immunologically silent.

[0194] As discussed herein, one embodiment utilizes the reduction or elimination of the protein activity of MHC I and II (HLA I and II when the cell is human). This can be accomplished by altering the genes encoding its components. In one embodiment, CRISPR is used to disrupt the coding region or regulatory sequence of the gene. In another embodiment, interfering RNA technology is used to reduce gene translation. A third change is a change in a gene such as CD47 that regulates sensitivity to macrophage phagocytosis, and this is typically a gene "knock-in" using viral technology.

[0195] Further descriptions of hypoimmunogenic pluripotent cells (HIP cells) can be found in International Application No. PCT / US18 / 13688, filed on January 14, 2018, and U.S. Provisional Application No. 62 / 445,969, filed on January 13, 2017, the entire disclosures of which (particularly the examples, figures, descriptions of the figures, and descriptions of generating hypoimmunogenic pluripotent stem cells and differentiating such cells into other cell types) are incorporated herein by reference.

[0196] In some cases, where CRISPR is used for genetic modification, hiPSC cells may be used that contain a Cas9 construct that enables efficient editing of the cell line: see, e.g., the human episomal Cas9 iPSC cell line A33124 from Life Technologies.

[0197] 1. HLA-I reduction

[0198] The HIP cells of the present invention include a reduction in MHC I function (HLA I when the cells are derived from human cells).

[0199] As will be appreciated by those skilled in the art, reduction of function can be achieved in a variety of ways, including removal of nucleic acid sequences from a gene, interruption of the sequence with other sequences or alteration of regulatory components of the nucleic acid. For example, all or a portion of the coding region of a gene of interest can be removed or replaced by a "nonsense" sequence, a frameshift mutation can be performed, all or a portion of a regulatory sequence such as a promoter can be removed or replaced, a translation initiation sequence can be removed or replaced, etc.

[0200] As will be appreciated by those skilled in the art, successful reduction of MHC I function (HLA I when the cells are derived from human cells) in pluripotent cells can be measured using techniques known in the art and as described below; for example, FACS techniques using labeled antibodies that bind to the HLA complex; for example, using commercially available HLA-A, B, C antibodies that bind to the alpha chain of the human major histocompatibility HLA class I antigen.

[0201] B2M Change

[0202] In one embodiment, the reduction of HLA-I activity is accomplished by disrupting the expression of the beta-2 microglobulin gene (whose human sequence is disclosed herein) in pluripotent stem cells. This change is generally referred to herein as a gene "knockout," and in the HIP cells of the present invention, it is accomplished on both alleles in the host cell. Typically, the technique for performing both disruptions is the same.

[0203] A particularly useful embodiment uses CRISPR technology to disrupt genes. In some cases, CRISPR technology is used to introduce small deletions / insertions into the coding region of a gene so that no functional protein is produced, which is usually the result of a frameshift mutation, which leads to the generation of a stop codon, resulting in a truncated non-functional protein.

[0204] Therefore, a useful technique is to use a CRISPR sequence designed to target the B2M gene in mice or the coding sequence of the B2M gene in people.After gene editing, the transfected iPSC culture is dissociated into single cells.Single cells are expanded to full-size colonies, and CRISPR editing is tested by screening for the presence of abnormal sequences from the CRISPR cleavage site.Choose clones with deletions in both alleles.As shown by PCR, such clones do not express B2M, and as shown by FACS analysis, such clones do not express HLA-I (e.g., see Examples 1 and 6).

[0205] The determination of whether the B2M gene has been inactivated is known and described herein. In one embodiment, the determination is a Western blot of cell lysates probed with antibodies to the B2M protein. In another embodiment, recombinase polymerase amplification (RPA) or reverse transcriptase polymerase chain reaction (RT-PCR) confirms the presence of the inactivation change.

[0206] In addition, the cells can be tested to confirm that the HLA I complex is not expressed on the cell surface. This can be determined by FACS analysis using antibodies against one or more HLA cell surface components as discussed above.

[0207] Notably, other published results were poor when attempting to silence the B2M gene on both alleles. See, e.g., Gornalusse et al., Nature Biotech. Doi / 10.1038 / nbt.3860).

[0208] 2. HLA-II reduction

[0209] In addition to the reduction of HLA I, the HIP cells of the present invention also lack MHC II function (HLA II when the cells are derived from human cells).

[0210] As will be appreciated by those skilled in the art, the reduction of function can be achieved in a variety of ways, including removing the nucleotide sequence from the gene, adding the nucleotide sequence to the gene, destroying the reading frame, interrupting the sequence with other sequences or changing the regulating components of the nucleic acid. In one embodiment, all or part of the coding region of the gene of interest can be removed or replaced by a "nonsense" sequence. In another embodiment, regulating sequences such as promoters can be removed or replaced, can remove or replace translation initiation sequences etc.

[0211] Successful reduction of MHC II function (HLA II when the cells are derived from human cells) in pluripotent cells or their derivatives can be measured using techniques known in the art, such as Western blotting using antibodies against the protein, FACS technology, RPA technology, RT-PCR technology, etc.

[0212] CIITA Changes

[0213] In one embodiment, the reduction of HLA-II activity is accomplished by disrupting the expression of the CIITA gene (whose human sequence is shown herein) in pluripotent stem cells. This change is generally referred to as a gene "knockout" herein, and in the HIP cells of the present invention, it is accomplished on both alleles in the host cell.

[0214] The mensuration of whether the CIITA gene has been inactivated is known and described in this article. In one embodiment, the mensuration is the Western blot of the cell lysate detected with the antibody for CIITA protein. In another embodiment, recombinase polymerase amplification (RPA) or reverse transcriptase polymerase chain reaction (RT-PCR) confirm the existence of the inactivation change.

[0215] In addition, cells can be tested to confirm that the HLA II complex is not expressed on the cell surface. Again, this assay is performed as known in the art (see PCT / US18 / 13688). Fig.21 , e.g. PCT / US18 / 13688), and is typically performed using Western blot or FACS analysis based on commercial antibodies that bind to human HLA class II HLA-DR, DP, and most DQ antigens, as described below.

[0216] A particularly useful embodiment uses CRISPR technology to destroy the CIITA gene. CRISPR is designed to target the coding sequence of the CIITA gene in mice or the CIITA gene in people (an essential transcription factor for all MHC II molecules). After gene editing, the transfected iPSC culture is dissociated into single cells. They are amplified into full-size colonies and tested for successful CRISPR editing by screening for the presence of abnormal sequences from the CRISPR cutting site. As determined by PCR, clones with deletions do not express CIITA, and as determined by FACS analysis, they do not express MHC II / HLA-II.

[0217] 3. Decreased macrophage phagocytosis and / or NK cell killing

[0218] In addition to the usual use of B2M and CIITA knockouts to reduce HLA I and II (or MHC I and II), the HIP cells of the present invention have reduced sensitivity to macrophage phagocytosis and NK cell killing. The generated HIP cells "escape" immune macrophages and innate pathways due to one or more CD47 transgenes.

[0219] In PCT / US18 / 13688 Fig.14 A-14C and 34A-34C show the ability of HIP cells and cells derived from HIP cells to evade or escape NK cell killing and / or macrophage phagocytosis, and the contents of the patent application, especially the drawings, descriptions of the drawings and examples, are incorporated herein by reference. For example, Fig.14 B-14C shows that mouse HIP cells (e.g., B2m- / -Ciita- / -CD47 transgenic mouse iPSC) cannot induce CD107a expression of NK cells, and therefore do not cause NK cell response. In addition, it is shown that such mouse HIP cells do not induce activation of NK cells or release of IFNγ. When NK cells are incubated with differentiated cells (e.g., endothelial cells, smooth muscle cells, and cardiomyocytes) derived from HIP cells, NK cell response is not induced (see, e.g., Figures 34A-34C of PCT / US18 / 13688).

[0220] Increased CD47 expression

[0221] In some embodiments, reduced macrophage phagocytosis and NK cell killing sensitivity are caused by increased CD47 on the surface of HIP cells. As will be appreciated by those skilled in the art, this is accomplished in several ways using "knock-in" or transgenic technology. In some cases, increased CD47 expression is caused by one or more CD47 transgenes.

[0222] Thus, in some embodiments, one or more copies of the CD47 gene are added to HIP cells under the control of an inducible or constitutive promoter, wherein a constitutive promoter is preferred. In some embodiments, a lentiviral construct is used as described herein or known in the art. As known in the art, the CD47 gene can be integrated into the genome of a host cell under the control of a suitable promoter.

[0223] The HIP cell line was generated from B2M- / -CIITA- / -iPSCs. Cells containing a lentiviral vector expressing CD47 were selected using the blasticidin marker. The CD47 gene sequence was synthesized and the DNA was cloned into the lentiviral pLenti6 / V5 plasmid with blasticidin resistance (Thermo Fisher Scientific, Waltham, MA).

[0224] In some embodiments, the expression of the CD47 gene can be increased by changing the regulatory sequence of the endogenous CD47 gene, for example, by exchanging the endogenous promoter for a constitutive promoter or a different inducible promoter. This operation can usually be done using known techniques (e.g., CRISPR).

[0225] Once altered, the presence of sufficient CD47 expression can be determined using known techniques, such as those described in the Examples, such as Western blots using anti-CD47 antibodies, ELISA assays, or FACS assays. Typically, "sufficient" herein refers to an increase in the expression of CD47 on the surface of HIP cells, which silences NK cell killing and / or macrophage phagocytosis. The natural expression levels on the cells are too low to protect them from NK cell lysis after their MHC I is removed.

[0226] 4. Suicide Gene

[0227] In some embodiments, the present invention provides low immunogenic pluripotent cells (HIP cells) comprising "suicide genes" or "suicide switches". These are incorporated to act as "safety switches", which can cause the death of low immunogenic pluripotent cells when the low immunogenic pluripotent cells grow and divide in an undesirable manner. The "suicide gene" ablation method includes a suicide gene in a gene transfer vector encoding a protein, which only causes cell killing when activated by a specific compound. The suicide gene can encode an enzyme that selectively converts a non-toxic compound into a highly toxic metabolite. The result is the specific elimination of cells expressing the enzyme. In some embodiments, the suicide gene is a herpes virus thymidine kinase (HSV-tk) gene, and the trigger is ganciclovir. In other embodiments, the suicide gene is the Escherichia coli cytosine deaminase (EC-CD) gene and the trigger is 5-fluorocytosine (5-FC) (Barese et al., Mol. Therap. 20(10): 1932-1943 (2012), Xu et al., Cell Res. 8:73-8 (1998), both of which are incorporated herein by reference in their entireties).

[0228] In other embodiments, the suicide gene is an inducible caspase. The inducible caspase protein comprises at least a portion of a caspase protein capable of inducing apoptosis. In one embodiment, a portion of the caspase is exemplified in SEQ ID NO: 6. In a preferred embodiment, the inducible caspase is iCasp9. It comprises the sequence of a human FK506 binding protein FKBP12 with an F36V mutation, which is linked to a gene encoding human caspase 9 through a series of amino acids. FKBP12-F36V binds to the small molecule dimer AP1903 with high affinity. Therefore, the suicide function of iCasp9 in the present invention is triggered by the administration of a chemical inducer of dimerization (CID). In some embodiments, CID is the small molecule drug AP1903. Dimerization leads to rapid induction of apoptosis. (See WO2011146862; Stasi et al., N. Engl. J. Med 365;18 (2011); Tey et al., Biol. Blood Marrow Transplant. 13:913-924 (2007), each of which is herein incorporated by reference in its entirety).

[0229] 5. Determination of HIP Cell Phenotype and Pluripotency Retention

[0230] Once HIP cells are generated, they can be assayed for reduced immunogenicity and / or retention of pluripotency as generally described herein and in the Examples.

[0231] For example, using PCT / US18 / 13688 Fig.13 and Fig.15 Multiple techniques exemplified measure low immunogenicity. These techniques include transplantation into an allogeneic host and monitoring the growth of HIP cells that evade the host immune system (e.g., teratomas). HIP derivatives are transduced to express luciferase, which can then be tracked using bioluminescent imaging. Similarly, the host animal is tested for T cell and / or B cell responses to HIP cells to confirm that HIP cells do not cause an immune response in the host animal. T cell function is assessed by Elispot, ELISA, FACS, PCR, or mass spectrometry (CYTOF). FACS or luminex are used to assess B cell responses or antibody responses. Additionally or alternatively, the ability of cells to avoid innate immune responses such as NK cell killing can be measured, as generally shown in 14A-14C of PCT / US18 / 13688. NK cell lysis activity (e.g., in vitro or in vivo) is assessed. Fig.15 shown).

[0232] Similarly, retention of pluripotency is tested in a variety of ways. In one embodiment, pluripotency is determined by expression of certain pluripotency-specific factors, as generally described herein and in PCT / US18 / 13688. Fig.29 Additionally or alternatively, the HIP cells are differentiated into one or more cell types as an indication of pluripotency.

[0233] D. Preferred Embodiments of HIP Cells

[0234] Provided herein are pluripotent stem cells ("HIP cells") with low immunogenicity that exhibit pluripotency but do not cause a host immune response when transplanted into an allogeneic host, such as a human patient (as HIP cells or as a differentiation product of HIP cells).

[0235] In one embodiment, human pluripotent stem cells (hiPSC) are made to have low immunogenicity by: a) destruction of the B2M gene at each allele (e.g., B2M- / -), b) destruction of the CIITA gene at each allele (e.g., CIITA- / -), and c) overexpression of the CD47 gene (CD47+, for example, by introducing one or more additional copies of the CD47 gene or activating genomic genes). This makes the hiPSC population B2M- / -CIITA- / -CD47tg. In a preferred embodiment, the cells are non-immunogenic. In another embodiment, the HIP cells are made non-immunogenic B2M- / -CIITA- / -CD47tg as described above, but are further modified by including an inducible suicide gene that is induced to kill cells in vivo when needed.

[0236] E. Maintenance of HIP cells

[0237] Once generated, HIP cells can be maintained in an undifferentiated state as is known for maintaining iPSCs. For example, HIP cells are cultured on Matrigel using a medium that prevents differentiation and maintains pluripotency.

[0238] In some embodiments, the HIP cells are cryopreserved. Before differentiation into different cell types, the cells can be cryopreserved. In other words, before differentiation, the HIP cells described herein are thawed and cultured, and then differentiated. In other embodiments, the HIP cells are not cryopreserved before differentiation. In some embodiments, the differentiated HIP cells are cryopreserved before administration to a patient. In other embodiments, the differentiated HIP cells are not cryopreserved before administration to a patient.

[0239] F. Differentiation of HIP cells

[0240] The present invention provides HIP cells that are differentiated into different cell types for subsequent transplantation into a subject. As will be appreciated by those skilled in the art, the method for differentiation depends on the desired cell type using known techniques. The cells are differentiated in suspension and then placed in a gel matrix form such as matrigel, gelatin or fibrin / thrombin form to promote cell survival. As is known in the art, differentiation is usually measured by assessing the presence of cell-specific markers.

[0241] In some embodiments, HIP cells are differentiated into hepatocytes to address the loss of hepatocyte function or cirrhosis. There are many techniques that can be used to differentiate HIP cells into hepatocytes: see, for example, Pettinato et al., doi:10.1038 / spre32888, Snykers et al., Methods Mol Biol 698:305-314 (2011), Si-Tayeb et al., Hepatology 51:297-305 (2010) and Asgari et al., Stem Cell Rev (:493-504 (2013), all of which are expressly incorporated herein by reference in full (particularly for the methods and reagents for differentiation). Differentiation is measured as known in the art, usually by assessing the presence of hepatocyte-related and / or specific markers (including but not limited to albumin, alpha-fetoprotein and fibrinogen). Differentiation can also be measured functionally, such as ammonia metabolism, LDL storage and absorption, ICG absorption and release, and glycogen storage.

[0242] In some embodiments, the hiPS cells are differentiated into β-like cells or pancreatic islet organoids for transplantation to treat type 1 diabetes (T1DM). The cell system is a promising method for solving T1DM, see, for example, Ellis et al., doi / 10.1038 / nrgastro.2017.93, which is incorporated herein by reference. In addition, Pagliuca et al. reported a method for successfully differentiating β cells from hiPSC (see doi / 10.106 / j.cell.2014.09.040, incorporated herein by reference in its entirety (particularly the methods and reagents for large-scale production of functional human β cells from human pluripotent stem cells outlined therein). In addition, Vegas et al. showed that human β cells were produced from human pluripotent stem cells and then encapsulated to avoid host immune rejection; (doi: 10.1038 / nm.4030, incorporated herein by reference in its entirety (particularly the methods and reagents for large-scale production of functional human β cells from human pluripotent stem cells outlined therein)).

[0243] As is known in the art, differentiation is typically determined by assessing the presence of β cell-related or specific markers, including but not limited to insulin. Differentiation can also be measured functionally, such as measuring glucose metabolism, see generally Muraro et al., doi: 10.1016 / j.cels.2016.09.002, which is hereby incorporated by reference in its entirety (particularly for the biomarkers outlined therein).

[0244] Once dHIP β cells are generated, they can be transplanted (either as a cell suspension or within a gel matrix as discussed herein) into the portal vein / liver, omentum, gastrointestinal mucosa, bone marrow, muscle, or subcutaneous pockets.

[0245] In some embodiments, HIP cell differentiation becomes retinal pigment epithelium (RPE) to solve the visual impairment disease of eyes.Use Kamao et al., Stem Cell Reports2014:2:205-18 (incorporated herein by quoting the full text (particularly for the method and reagent for differentiation technique and reagent summarized therein)) technology outlined in human pluripotent stem cell differentiation into RPE cell; Also referring to Mandai et al., doi:10.1056 / NEJMoa1608368, it is also incorporated herein by the technology integral body for producing RPE cell sheet and being transplanted into patient.

[0246] As known in the art, differentiation can usually be determined by assessing the presence of RPE-related and / or specific markers or by functional measurements.See, for example, Kamao et al., doi: 10.1016 / j.stemcr.2013.12.007, which is incorporated herein by reference as a whole (particularly for the markers summarized in the first paragraph of the results section).

[0247] In some embodiments, hiPS cells are differentiated into cardiomyocytes to address cardiovascular disease. Techniques for differentiating hiPSCs into cardiomyocytes are known in the art and are discussed in the Examples. Differentiation can be determined as known in the art, typically by assessing the presence of cardiomyocyte-related or specific markers or by functional measurements; see, e.g., Loh et al., doi: 10.1016 / j.cell.2016.06.001, which is incorporated herein by reference in its entirety (particularly for methods of differentiating stem cells including cardiomyocytes).

[0248] In some embodiments, HIP cells are differentiated into endothelial colony forming cells (ECFCs) to form new blood vessels to address peripheral arterial disease. Techniques for differentiating endothelial cells are known. See, for example, Prasain et al., doi: 10.1038 / nbt.3048, which is incorporated herein by reference in its entirety (particularly for methods and reagents for producing endothelial cells from human pluripotent stem cells, and for transplantation techniques). Differentiation can be determined as known in the art, typically by assessing the presence of endothelial cell-related or specific markers or by functional measurements.

[0249] In some embodiments, HIP cells are differentiated into thyroid progenitor cells and thyroid follicular organoids that can secrete thyroid hormone to address autoimmune thyroiditis. Techniques for differentiating thyroid cells are known in the art. See, for example, Kurmann et al., doi: 10.106 / j.stem.2015.09.004, which is incorporated herein by reference in its entirety (particularly for methods and reagents for producing thyroid cells from human pluripotent stem cells and for transplantation techniques). Differentiation is determined as known in the art, typically by assessing the presence of thyroid cell-related or specific markers or by functional measurements.

[0250] VIII. Hypoimmune cardiac cells derived from HIP cells

[0251] In some embodiments, the heart cells are derived from the HIP cells described herein. For example, human heart cells can be produced by differentiating human HIP cells. Similarly, mouse heart cells can be produced by differentiating mouse HIP cells. Such heart cells are low-immunity heart cells.

[0252] Useful methods for differentiating induced or embryonic pluripotent stem cells into cardiac cells are described in, for example, US20170152485, US20170058263, US20170002325, US20160362661, US20160068814, US9062289, US7897389, and US7452718.

[0253] Other methods for generating cardiac cells from induced or embryonic pluripotent stem cells are described, for example, in Xu et al., Stem Cells and Development, 2006, 15(5):631-9, Burridge et al., Cell Stem Cell, 2012, 10:16-28, and Chen et al., Stem Cell Res, 2015, 15(2):365-375.

[0254] In various embodiments, HIP cells (e.g., mouse HIP cells and human HIP cells) can be cultured in a medium containing a BMP pathway inhibitor, a WNT signaling activator, a WNT signaling inhibitor, a WNT agonist, a WNT antagonist, a Src inhibitor, an EGFR inhibitor, a PCK activator, a cytokine, a growth factor, a cardiotropic agent, a compound, or the like.

[0255] WNT signaling activators include but are not limited to CHIR99021. PCK activators include but are not limited to PMA. WNT signaling inhibitors include but are not limited to compounds selected from KY02111, SO3031 (KY01-1), SO2031 (KY02-1) and SO3042 (KY03-1) and XAV939. Src inhibitors include but are not limited to A419259. EGFR inhibitors include but are not limited to AG1478.

[0256] Non-limiting examples of agents for generating cardiac cells from iPSCs include activin A, BMP-4, Wnt3a, VEGF, soluble Frizzled, cyclosporin A, angiotensin II, phenylephrine, ascorbic acid, dimethyl sulfoxide, 5-aza-2'-deoxycytidine, and the like.

[0257] Cells of the present invention can be cultured on a surface, such as a synthetic surface, to support and / or promote the differentiation of HIP cells into cardiac cells. In some embodiments, the surface comprises a polymeric material, which includes but is not limited to a homopolymer or copolymer of one or more selected acrylate monomers. Non-limiting examples of acrylate monomers and methacrylate monomers include tetra(ethylene glycol) diacrylate, dimethacrylate glyceride, 1,4-butanediol dimethacrylate, poly(ethylene glycol) diacrylate, di(ethylene glycol) dimethacrylate, tetra(ethylene glycol) dimethacrylate, 1,6-hexanediol propoxylate diacrylate, neopentyl glycol diacrylate, trimethylolpropane benzoate diacrylate, trimethylolpropane ethoxylate (1EO / OH) methyl, tricyclo[5.2.1.02,6]decane-dimethanol diacrylate, neopentyl glycol ethoxylate diacrylate and trimethylolpropane triacrylate. Acrylates were synthesized as known in the art or obtained from commercial suppliers such as Polysciences, Inc., Sigma Aldrich, Inc., and Sartomer, Inc.

[0258] The polymeric material may be dispersed on the surface of the support material. Useful support materials suitable for culturing cells include ceramic substances, glass, plastic, polymers or copolymers, any combination thereof or a coating of one material on another. In some cases, the glass includes soda-lime glass, pyrex glass, viscose glass, quartz glass, silicon, or derivatives of these, etc.

[0259] In some cases, the plastic or polymer comprising the dendrimer comprises poly(vinyl chloride), poly(vinyl alcohol), poly(methyl methacrylate), poly(vinyl acetate-maleic anhydride), poly(dimethylsiloxane) monomethacrylate, cycloolefin polymers, fluorocarbon polymers, polystyrene, polypropylene, polyethyleneimine or derivatives thereof, etc. In some cases, the copolymer comprises poly(vinyl acetate-maleic anhydride copolymer), poly(styrene-maleic anhydride copolymer), poly(ethylene-acrylic acid copolymer) or derivatives thereof, etc.

[0260] The engineered cardiac cells of the present invention include but are not limited to cardiomyocytes, nodular cardiomyocytes, conductive cardiomyocytes, working cardiomyocytes, cardiomyocyte precursors, cardiomyocyte progenitors, cardiac stem cells and cardiac muscle cells. In some embodiments, cardiomyocyte precursors refer to cells that can produce offspring including mature (terminal) cardiomyocytes (in the absence of dedifferentiation or reprogramming). Cardiomyocyte precursor cells can usually be identified using one or more markers selected from GATA-4, Nkx2.5 and MEF-2 transcription factor families. In some cases, cardiomyocytes refer to immature cardiomyocytes or mature cardiomyocytes expressing one or more markers (sometimes at least 3 or 5 markers) in the following list: cardiac troponin I (cTnI), cardiac troponin T (cTnT), sarcomeric myosin heavy chain (MHC), GATA-4, Nkx2.5, N-cadherin, β1-adrenergic receptor (β1-AR), ANF, MEF-2 transcription factor family, creatine kinase MB (CK-MB), myoglobin or atrial natriuretic factor (ANF). In some embodiments, the engineered cardiac cells exhibit spontaneous periodic contractile activity. In some cases, when in the presence of appropriate Ca 2+ When cardiac cells are cultured in a suitable tissue culture environment with a balanced concentration of electrolytes, the cells can be observed to contract along one axis of the cell in a periodic manner and then release from the contraction without having to add any other components to the culture medium. In some embodiments, the cardiac cells are hypoimmune cardiac cells.

[0261] The efficacy of cardiac cells prepared as described herein can be evaluated in an animal model for cardiac cryoinjury, which causes 55% of the left ventricular wall tissue to become scar tissue without treatment (Li et al., Ann. Thorac. Surg. 62: 654, 1996; Sakai et al., Ann. Thorac. Surg. 8: 2074, 1999, Sakai et al., J. Thorac. Cardiovasc. Surg. 118: 715, 1999). Successful treatment can reduce the area of ​​scar, limit the extension of scar, and improve the function of the heart as measured by systolic, diastolic and formed blood pressure. Cardiac injury can also be modeled using embolic coils at the distal end of the left anterior descending artery (Watanabe et al., Cell Transplant. 7: 239, 1998), and the effect of treatment can be evaluated by histology and cardiac function.

[0262] In some embodiments, the cardiac cells of through engineering approaches (for example, low immunity cardiac cells) are administered to patients in need thereof, such as human patients. Cardiac cells can be administered to patients suffering from the following diseases: pediatric cardiomyopathy, age-related cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, chronic ischemic cardiomyopathy, peripartum cardiomyopathy, inflammatory cardiomyopathy, other cardiomyopathy, myocarditis, myocardial ischemic reperfusion injury, ventricular dysfunction, heart failure, congestive heart failure, coronary artery disease, advanced heart disease, atherosclerosis, ischemia, hypertension, restenosis, angina, rheumatic heart disease, arterial inflammation or cardiovascular disease. In some cases, the patient suffers from myocardial infarction. In particular cases, the patient is undergoing coronary artery bypass surgery.

[0263] Engineered cardiac cells can be transplanted into the patient using well-known surgical techniques for transplanting tissue and / or isolated cells into the heart. In some embodiments, cells are introduced into the patient's cardiac tissue by injection (e.g., intramyocardial injection, intracoronary injection, transendocardial injection, transepicardial injection, percutaneous injection), infusion, and implantation.

[0264] Administration (delivery) of the engineered cardiac cells includes, but is not limited to, subcutaneous or parenteral, including intravenous, intraarterial (e.g., intracoronary), intramuscular, intraperitoneal, intramyocardial, transendocardial, transepicardial, intranasal administration, as well as intrathecal and infusion techniques.

[0265] In some embodiments, cardiac drugs are also administered to patients to whom engineered cardiac cells are administered. Illustrative examples of cardiac drugs suitable for combination therapy include, but are not limited to, growth factors, polynucleotides encoding growth factors, angiogenic agents, calcium channel blockers, hypotensive agents, antimitotic agents, positive inotropes, antiatherosclerotic agents, anticoagulants, beta blockers, antiarrhythmics, anti-inflammatory drugs, vasodilators, thrombolytics, cardiac glycosides, antibiotics, antivirals, antifungals, drugs that inhibit protozoa, nitrates, angiotensin converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists, brain natriuretic peptide (BNP); antineoplastic agents, steroids, and the like.

[0266] The therapeutic effect according to the method of the present invention can be monitored in a variety of ways. For example, an electrocardiogram (ECG) or a holter monitor can be used to determine the therapeutic effect. ECG is a measurement of heart rhythm and electrical impulses, and is a very effective and non-invasive method for determining whether therapy has improved or maintained, prevented or slowed down the degradation of electrical conduction in the subject's heart. The use of a holter monitor (a portable ECG that can be worn for a long time to monitor heart abnormalities, arrhythmias, etc.) is also a reliable method for evaluating the therapeutic effect. ECG or nuclear studies can be used to determine the improvement of ventricular function.

[0267] As will be appreciated by those skilled in the art, differentiated HIP derivatives are transplanted using techniques known in the art that depend on the cell type and the ultimate use of these cells. Typically, the differentiated HIP cells of the present invention are transplanted intravenously or by injection at a specific location in a patient. When transplanted at a specific location, the cells may be suspended in a gel matrix to prevent the cells from dispersing when fixed.

[0268] IX. Endothelial cells derived from HIP cells

[0269] In some embodiments, the endothelial cells are derived from the HIP cells described herein. For example, human endothelial cells can be produced by differentiating human HIP cells. Similarly, mouse endothelial cells can be produced by differentiating mouse HIP cells. Such endothelial cells are low-immunity endothelial cells.

[0270] Useful methods for differentiating induced or embryonic pluripotent stem cells into endothelial cells are described, for example, in US 2004 / 0009589, WO 2011 / 090684 and WO 2012 / 006440.

[0271] In various embodiments, HIP cells (e.g., mouse HIP cells and human HIP cells) can be cultured in a medium containing a GSK3 inhibitor, an ALK inhibitor, a BMP pathway inhibitor, a ROCK inhibitor, a WNT signaling activator, a WNT signaling inhibitor, a WNT agonist, a WNT antagonist, a Src inhibitor, an EGFR inhibitor, a PCK activator, a cytokine, a growth factor, an endothelial cell differentiation compound, an endothelial cell promoting compound, and the like.

[0272] WNT signaling activators (e.g., GSK3 inhibitors) include but are not limited to CHIR-99021. PCK activators include but are not limited to PMA. WNT signaling inhibitors include but are not limited to compounds selected from KY02111, SO3031 (KY01-1), SO2031 (KY02-1), SO3042 (KY03-1), and XAV939. Src inhibitors include but are not limited to A419259. EGFR inhibitors include but are not limited to AG1478.

[0273] Non-limiting examples of agents for generating endothelial cells from iPSCs include activin A, BMP-4, Wnt3a, VEGF, soluble Frizzled, cyclosporin A, angiotensin II, phenylephrine, ascorbic acid, dimethyl sulfoxide, 5-aza-2'-deoxycytidine, and the like.

[0274] Cells of the present invention can be cultured on surfaces such as synthetic surfaces to support and / or promote HIP cell differentiation into low immune endothelial cells. In some embodiments, the surface comprises a polymeric material, which includes but is not limited to a homopolymer or copolymer of one or more selected acrylate monomers. Non-limiting examples of acrylate monomers and methacrylate monomers include tetra(ethylene glycol) diacrylate, dimethacrylate glyceride, 1,4-butylene glycol dimethacrylate, poly(ethylene glycol) diacrylate, di(ethylene glycol) dimethacrylate, tetra(ethylene glycol) dimethacrylate, 1,6-hexanediol propoxylate diacrylate, neopentyl glycol diacrylate, trimethylolpropane benzoate diacrylate, trimethylolpropane ethoxylate (1EO / OH) methyl, tricyclo[5.2.1.02,6]decane-dimethanol diacrylate, neopentyl glycol ethoxylate diacrylate and trimethylolpropane triacrylate. Acrylates were synthesized as known in the art or obtained from commercial suppliers such as Polysciences, Inc., Sigma Aldrich, Inc., and Sartomer, Inc.

[0275] In some embodiments, endothelial cells can be plated on a polymer matrix. In some cases, the polymer matrix is ​​biodegradable. Suitable biodegradable substrates are well known in the art and include collagen-GAG, collagen, fibrin, PLA, PGA and PLA / PGA copolymers. Other biodegradable materials include poly (anhydrides), poly (hydroxy acids), poly (orthoesters), poly (propyl fumarate), poly (caprolactone), polyamides, polyamino acids, polyacetals, biodegradable polycyanoacrylates, biodegradable polyurethanes and polysaccharides.

[0276] Non-biodegradable polymers can also be used. Other non-biodegradable but still biocompatible polymers include polypyrrole, polyaniline, polythiophene, polystyrene, polyester, non-biodegradable polyurethane, polyurea, poly-(ethylene vinyl acetate), polypropylene, polymethacrylate, polyethylene, polycarbonate and poly-(ethylene oxide). The polymer matrix can be formed in any shape, for example, as a particle, sponge, pipe, ball, line, spiral line, capillary network, film, fiber, net or sheet. The polymer matrix can be modified to include natural or synthetic extracellular matrix materials and factors.

[0277] The polymeric material may be dispersed on the surface of the support material. Useful support materials suitable for culturing cells include ceramic substances, glass, plastic, polymers or copolymers, any combination thereof or a coating of one material on another. In some cases, the glass includes soda-lime glass, pyrex glass, viscose glass, quartz glass, silicon, or derivatives of these, etc.

[0278] In some cases, the plastic or polymer comprising the dendrimer includes poly(vinyl chloride), poly(vinyl alcohol), poly(methyl methacrylate), poly(vinyl acetate-maleic anhydride), poly(dimethylsiloxane) monomethacrylate, cycloolefin polymers, fluorocarbon polymers, polystyrene, polypropylene, polyethyleneimine or derivatives thereof, etc. In some cases, the copolymer includes poly(vinyl acetate-maleic anhydride copolymer), poly(styrene-maleic anhydride copolymer), poly(ethylene-acrylic acid copolymer) or derivatives thereof, etc.

[0279] The engineered endothelial cells of the present invention may express one or more endothelial cell markers. Non-limiting examples of such markers include VE-cadherin (CD144), ACE (angiotensin converting enzyme) (CD143), BNH9 / BNF13, CD31, CD34, CD54 (ICAM-1), CD62E (E-selectin), CD105 (endothelin), CD146, Endocan (ESM-1), Endoglyx-1, Endothelial Mucin, Eotaxin-3, EPAS1 (endothelial PAS domain protein 1), Factor VIII-related antigen, FLI-1, Flk-1 (KDR, VEGFR-2), FLT-1 (VEGFR- 1), GATA2, GBP-1 (guanylate binding protein-1), GRO-α, HEX, ICAM-2 (intercellular adhesion molecule 2), LM02, LYVE-1, MRB (magic roundabout), nucleolin, PAL-E (pathoanatomical Leiden endothelium), RTK, sVCAM-1, TALI, TEM1 (tumor endothelial marker 1), TEM5 (tumor endothelial marker 5), TEM7 (tumor endothelial marker 7), thrombomodulin (TM), CD141), VCAM-1 (vascular cell adhesion molecule-1) (CD106), VEGF (vascular endothelial growth factor), vWF (von Willebrand factor), ZO-1, endothelial cell selective adhesion molecule (ESAM), CD102, CD93, CD184, CD304, and DLL4.

[0280] Endothelial cells include, but are not limited to, endothelial progenitor cells, capillary endothelial cells, arterial endothelial cells, venous endothelial cells, lymphatic endothelial cells, other vascular endothelial cells, aortic endothelial cells, blood-brain endothelial cell barrier, cardiac endothelial cells, renal endothelial cells, and liver endothelial cells. Atkins et al., Arteriosclerosis, Thrombosis, and Vascular Biology, 2011, 31: 1476-1484 and US5,980,088 describe the types and characteristics of different endothelial cells. In some embodiments, the isolated, engineered endothelial cells of the present invention are selected from vascular endothelial cells, brain endothelial cells, renal endothelial cells, and aortic endothelial cells. In a preferred embodiment, the endothelial cells are capillary endothelial cells.

[0281] In some embodiments, engineered endothelial cells are genetically modified to express exogenous genes encoding proteins of interest, such as, but not limited to, enzymes, hormones, receptors, ligands, or drugs that can be used to treat a disorder / condition or improve the symptoms of a disorder / condition. Standard methods for genetically modifying endothelial cells are described in, for example, US5,674,722.

[0282] Such endothelial cells can be used to provide constitutive synthesis and delivery of polypeptides or proteins that can be used to prevent or treat diseases. In this way, polypeptides are secreted directly into the bloodstream of an individual or other areas of the body (e.g., the central nervous system). In some embodiments, endothelial cells can be modified to secrete insulin, coagulation factors (e.g., factor VIII or von Willebrand factor), alpha-1 antitrypsin, adenosine deaminase, tissue plasminogen activator, interleukins (e.g., IL-1, IL-2, IL-3), etc.

[0283] In certain embodiments, the engineered endothelial cells can be modified in a manner that improves their performance in the context of an implanted graft. Non-limiting illustrative examples include secreting or expressing thrombolytic agents to prevent intraluminal clot formation, secreting inhibitors of smooth muscle proliferation to prevent luminal stenosis due to smooth muscle hypertrophy, and expressing and / or secreting endothelial cell mitogens or autocrine factors to stimulate endothelial cell proliferation and improve the extent or duration of endothelial cell lining of the transplant lumen.

[0284] In some embodiments, engineered endothelial cells are used to deliver therapeutic levels of secretory products to specific organs or limbs. For example, vascular implants lined with endothelial cells engineered (transduced) in vitro can be transplanted into specific organs or limbs. The secretory products of the transduced endothelial cells will be delivered to the perfused tissue at high concentrations, thereby achieving the desired effect on the targeted anatomical location.

[0285] In other embodiments, engineered endothelial cells are genetically modified to contain genes that disrupt or inhibit angiogenesis when expressed by endothelial cells in vascularized tumors. In some cases, endothelial cells may also be genetically modified to express any of the selective suicide genes described herein, which allows negative selection of transplanted endothelial cells after tumor treatment is completed.

[0286] In some embodiments, the engineered endothelial cells are applied to patients who need them, such as human patients.Endothelial cells can be applied to patients with the following diseases or conditions, such as but not limited to cardiovascular disease, vascular disease, peripheral vascular disease, ischemic disease, myocardial infarction, congestive heart failure, peripheral vascular obstructive disease, stroke, reperfusion injury, limb ischemia, neuropathy (such as peripheral neuropathy or diabetic neuropathy), organ failure (such as liver failure, renal failure, etc.), diabetes, rheumatoid arthritis, osteoporosis, vascular damage, tissue damage, hypertension, angina pectoris and myocardial infarction caused by coronary artery disease, renal vascular hypertension, renal artery stenosis caused by renal failure, lower limb claudication, etc. In certain embodiments, the patient suffers from or is suffering from transient ischemic attack or stroke, which may be caused by cerebrovascular disease in some cases.In some embodiments, engineered endothelial cells are applied to treat tissue ischemia, such as the situation occurring in atherosclerosis, myocardial infarction and limb ischemia, and repair injured blood vessels.In some cases, cells are used for the bioengineering of transplants.

[0287] For example, engineered endothelial cells can be used in cell therapy for the repair of ischemic tissue, the formation of blood vessels and heart valves, the engineering of artificial blood vessels, the repair of damaged blood vessels, and the formation of blood vessels in inducing engineered tissue (e.g., before transplantation). In addition, endothelial cells can be further modified to deliver agents to target and treat tumors.

[0288] In a specific embodiment, a method for repairing or replacing a tissue in need of vascular cells or vascularization is provided herein. The method involves administering a composition comprising isolated endothelial cells to a human patient in need of such treatment to promote vascularization in such tissue. The tissue in need of vascular cells or vascularization can be cardiac tissue, liver tissue, pancreatic tissue, kidney tissue, muscle tissue, neural tissue, bone tissue, etc., which can be damaged tissue and is characterized by excessive cell death, tissue at risk of damage, or artificially engineered tissue.

[0289] In certain embodiments, engineered endothelial cells are used to improve prosthetic implants (e.g., blood vessels made of synthetic materials such as Dacron and Gortex) used in revascularization surgery. For example, prosthetic arterial grafts are often used to replace diseased arteries that irrigate vital organs or limbs. In other embodiments, engineered endothelial cells are used to cover the surface of prosthetic heart valves to reduce the risk of emboli by making the valve surface less thrombogenic.

[0290] The well-known surgical techniques for transplanting tissue and / or separated cells into blood vessels can be used to transplant engineered endothelial cells into the patient. In some embodiments, cells are introduced into the patient's cardiac tissue by injection (e.g., intramyocardial injection, intracoronary injection, transendocardial injection, transepicardial injection, percutaneous injection), infusion, transplantation, and implantation.

[0291] Administration (delivery) of the engineered endothelial cells includes, but is not limited to, subcutaneous or parenteral, including intravenous, intraarterial (e.g., intracoronary), intramuscular, intraperitoneal, intramyocardial, transendocardial, transepicardial, intranasal administration, as well as intrathecal and infusion techniques.

[0292] As will be appreciated by those skilled in the art, differentiated HIP derivatives are transplanted using techniques known in the art that depend on the cell type and the ultimate use of these cells. Typically, the differentiated HIP cells of the present invention are transplanted intravenously or by injection at a specific location in a patient. When transplanted at a specific location, the cells may be suspended in a gel matrix to prevent the cells from dispersing when fixed.

[0293] X. Hypoimmune dopaminergic neurons derived from HIP cells

[0294] In some embodiments, dopaminergic (DA) neurons are derived from HIP cells described herein. For example, human DA neurons can be generated by differentiating human HIP cells. Similarly, mouse DA neurons can be generated by differentiating mouse HIP cells. Such DA neurons are low-immune DA neurons.

[0295] Useful methods for differentiating pluripotent stem cells into DA neurons are described, for example, in U.S. Pat. Nos. 9,968,637 and 7,674,620, the entire disclosures of which, including the specifications, are incorporated herein by reference. Other methods for generating DA cells from human pluripotent stem cells can be found, for example, in Kim, J.-H., et al., Nature, 2002, 418, 50-56; LM, et al., PNAS, 2002, 99(4), 2344-2349; Grow, DA, et al., Stem Cells Transl Med. 2016, 5(9): 1133-44 and Cho, MS, et al., PNAS, 2008, 105: 3392-3397.

[0296] The term "dopaminergic neuron" refers to a neuronal cell expressing tyrosine hydroxylase (TH), the rate-limiting enzyme for dopamine synthesis. Preferably, dopaminergic neurons secrete the neurotransmitter dopamine and have little or no expression of dopamine-β-hydroxylase. Dopaminergic neurons may express one or more of the following: neuron-specific enolase (NSE), 1-aromatic amino acid decarboxylase, vesicular monoamine transporter 2, dopamine transporter, Nurr-1, and dopamine 2 receptor (D2 receptor). Dopaminergic neurons include neuronal stem cells, neuronal progenitor cells, immature dopaminergic neurons, and mature dopaminergic neurons.

[0297] The term "neural stem cell" refers to a subset of multipotent cells that have been partially differentiated along the neural cell pathway and express some neural markers, including, for example, nestin. Neural stem cells can differentiate into neurons or glial cells (e.g., astrocytes and oligodendrocytes). The term "neural progenitor cell" refers to a cultured cell that expresses FOXA2 and low levels of β-tubulin, but does not express tyrosine hydroxylase (i.e., has a FOXA2+ / β-tubulin LO / TH- phenotype). Such neural progenitor cells have the ability to differentiate into a variety of neuronal subtypes after culturing appropriate factors (e.g., those described herein); Especially a variety of dopaminergic neuronal subtypes.

[0298] HIP cells and DA neurons derived from HIP cells can be cultured in a growth medium. Exemplary growth medium include, but are not limited to, human embryonic stem cell medium (hESC medium), Dulbecco's modified Eagle's medium mammalian cell culture medium (DMEM), Ham's F12 medium, Neurobasal medium, and TM (ThermoFisher), knockout serum replacement (KOSR), minimal essential medium Eagle-α modified (Alpha MEM), knockout DMEM (KO-DMEM), N-2 (ThermoFisher), MS-5 stromal cell culture medium, etc.

[0299] Useful additives for promoting differentiation, growth, expansion, maintenance and / or maturation of DA neurons include, but are not limited to, Wnt1, fibroblast growth factor 2 (FGF2), FGF8, FGF8a, sonic hedgehog (SHH), brain-derived neurotrophic factor (BDNF), transforming growth factor alpha (TGF-α), TGF-β3, interleukin 1β (IL1β), glial cell line-derived neurotrophic factor (GDNF), GSK-3 inhibitors (e.g., CHIR-99021), TGF-β inhibitors (e.g., SB-431542), B-27 supplement, doxorubicin, morphamine, noggin, retinoic acid, cAMP, ascorbic acid, GlutaMax TM , neurorankin, knockout serum replacement, N-acetylcysteine, c-kit ligand, modified forms thereof, mimetics thereof, analogs thereof, and variants thereof. In some embodiments, DA neurons are differentiated in the presence of one or more factors that activate or inhibit the WNT pathway, NOTCH pathway, SHH pathway, BMP pathway, FGF pathway, TGFβ pathway, etc. Differentiation protocols and detailed descriptions thereof are provided, for example, in U.S. Pat. Nos. 9,968,637 and 7,674,620, Kim, J.-H., et al., Nature, 2002, 418, 50-56; LM, et al., PNAS, 2002, 99(4), 2344-2349; Grow, DA, et al., Stem Cells Transl Med. 2016, 5(9): 1133-44, and Cho, MS, et al., PNAS, 2008, 105: 3392-3397, the full disclosures of which including detailed descriptions of the invention, examples, methods, online methods and results are incorporated herein by reference.

[0300] To characterize and monitor DA differentiation and assess DA phenotype, the expression of any number of molecular and genetic markers can be assessed. For example, the presence of genetic markers can be determined by various methods known to those skilled in the art. The expression of molecular markers can be determined by quantitative methods, such as, but not limited to, qPCR-based assays, immunoassays, immunocytochemical assays, immunoblot assays, and the like.

[0301] Exemplary markers of DA neurons include TH, β-tubulin, paired box protein (Pax6), insulin gene enhancer protein (ISL1), nestin, diaminobenzidine (DAB), G protein-activated inwardly rectifier potassium channel 2 (GIRK2), microtubule-associated protein 2 (MAP-2), nuclear receptor-related 1 protein (NURR1), dopamine transporter (DAT), forkhead box protein A2 (FOXA2), FOX3, doublecortin, and LIM homeobox transcription factor 1-β (LMX1B), among others.

[0302] DA neurons can also be evaluated based on cell electrophysiological markers. The electrophysiology of the cells can be evaluated using assays known to those skilled in the art. For example, whole cell and perforated patch clamp, assays for detecting electrophysiological activity of cells, assays for measuring the magnitude and duration of action potentials of cells, and functional assays for detecting dopamine production by DA cells.

[0303] In some embodiments, DA neuron differentiation is characterized by spontaneous rhythmic action potentials and high frequency action potentials with spike frequency adaptation upon injection of depolarizing current. In other embodiments, DA differentiation is characterized by the production of dopamine. The level of dopamine produced is calculated by measuring the width of the action potential when it reaches half of its maximum amplitude (spike half-maximum width).

[0304] In some embodiments, DA neurons derived from HIP cells are administered to a patient, such as a human patient, to treat a neurodegenerative disease or condition. In some cases, the neurodegenerative disease or condition is selected from Parkinson's disease, Huntington's disease, and multiple sclerosis. In other embodiments, DA neurons are used to treat or alleviate one or more symptoms of a neuropsychiatric disorder, such as attention deficit hyperactivity disorder (ADHD), Tourette syndrome (TS), schizophrenia, psychosis, and depression. In other embodiments, DA neurons are used to treat patients with damaged DA neurons.

[0305] Differentiated DA neurons can be transplanted intravenously or by injection at a specific location in a patient. In some embodiments, differentiated DA cells are transplanted into the substantia nigra (particularly in or near the compact area), ventral tegmental area (VTA), caudate nucleus, putamen, nucleus accumbens, subthalamic nucleus, or any combination thereof of the brain to replace DA neurons whose degeneration leads to Parkinson's disease (PD). Differentiated DA cells can be injected into the target area as a cell suspension. Alternatively, differentiated DA cells can be embedded in a supporting matrix or scaffold (when contained in such a delivery device). In some embodiments, the scaffold is biodegradable. In other embodiments, the scaffold is non-biodegradable. The scaffold may comprise natural or synthetic (artificial) materials.

[0306] General principles of therapeutic dosage forms of cell compositions are found in Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, G. Morstyn & W. Sheridaneds, Cambridge University Press, 1996, and Hematopoietic Stem Cell Therapy, E. Ball, J. Lister & P. ​​Law, Churchill Livingstone, 2000 (specifically incorporated herein by reference). In some embodiments, differentiated DA neurons are provided in the form of a pharmaceutical composition. Delivery of DA neurons can be achieved by using a suitable vehicle, such as, but not limited to, liposomes, microparticles, or microcapsules. In other embodiments, differentiated DA neurons are administered in a pharmaceutical composition comprising an isotonic excipient. The pharmaceutical composition is prepared under sufficiently sterile conditions for human administration.

[0307] As will be appreciated by those skilled in the art, differentiated HIP derivatives are transplanted using techniques known in the art that depend on the cell type and the ultimate use of these cells. Typically, the differentiated HIP cells of the present invention are transplanted intravenously or by injection at a specific location in a patient. When transplanted at a specific location, the cells may be suspended in a gel matrix to prevent the cells from dispersing when fixed.

[0308] XI. Low-immunity pancreatic islet cells derived from HIP cells

[0309] In some embodiments, islet cells (also referred to as pancreatic β cells) are derived from HIP cells described herein. For example, human islet cells can be produced by differentiating human HIP cells. Similarly, mouse islet cells can be produced by differentiating mouse HIP cells. Such islet cells are low-immunity islet cells.

[0310] In some embodiments, the islet cells are derived from the HIP cells described herein.Useful methods for differentiating pluripotent stem cells into islet cells are described, for example, in US Pat. No. 9,683,215, US Pat. No. 9,157,062, and US Pat. No. 8,927,280.

[0311] In some embodiments, the islet cells produced by the methods disclosed herein secrete insulin. In some embodiments, the islet cells exhibit at least two characteristics of endogenous islet cells, such as, but not limited to, insulin secretion in response to glucose and expression of beta cell markers.

[0312] Exemplary β cell markers or β cell progenitor markers include, but are not limited to, c-peptide, Pdx1, glucose transporter 2 (Glut2), HNF6, VEGF, glucokinase (GCK), prohormone convertase (PC1 / 3), Cdcp1, NeuroD, Ngn3, Nkx2.2, Nkx6.1, Nkx6.2, Pax4, Pax6, Ptfla, Isl1, Sox9, Sox17, and FoxA2.

[0313] In some embodiments, the isolated islet cells produce insulin in response to an increase in glucose. In various embodiments, the isolated islet cells secrete insulin in response to an increase in glucose. In some embodiments, the cells have different morphologies, such as cobblestone cell morphology and / or a diameter of about 17 μm to about 25 μm.

[0314] In some embodiments, the method of differentiating HIP cells into low-immunity pancreatic islet cells comprises culturing HIP stem cells in a culture medium comprising FGF10. In some cases, the culture medium comprises one or more differentiation factors selected from the following: keratinocyte growth factor (KGF), epidermal growth factor (EGF); transforming growth factor-α (TGFα), transforming growth factor-β (TGFβ), hepatocyte growth factor (HGF), Wnt3a, activin A, Nodal, KAAD-CYC, basic fibroblast growth factor (bFGF), nicotinamide, indolatam V, HDAC inhibitors, IDE1 and IDE2. In some embodiments, HIP cells are differentiated into pancreatic islet cells by culturing the cells in a medium comprising one or more of the following factors: insulin-like growth factor (IGF), transforming growth factor (TGF), fibroblast growth factor (EGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), sonic hedgehog (SHH) and vascular endothelial growth factor (VEGF), transforming growth factor β (TGFβ) superfamily, bone morphogenetic protein 2 (BMP2), bone morphogenetic protein 7 (BMP7), GSK3β inhibitor, ALK inhibitor, BMP1 receptor inhibitor, retinoic acid, and any combination thereof.

[0315] In some embodiments, a population of hypoimmune pluripotent stem cells (HIP cells) may be contacted or exposed to one or more compounds of formula (I) alone, such as IDE1 or IDE2 as described herein, and in other embodiments, a population of pluripotent stem cells may be contacted with at least one additional agent simultaneously with (e.g., in combination with) or before the pluripotent cells are contacted with a compound of formula (I) as disclosed in US Pat. No. 8,927,280.

[0316] In some embodiments, additional compounds used in combination with compounds of formula (I) as disclosed in US Pat. No. 8,927,280 may include, but are not limited to, agents of transforming growth factor-β (TGFβ) family members (e.g., Nodal or activin A), fibroblast growth factor (FGF) family members (e.g., FGF10), Wnt growth factor family members (e.g., Wnt3a), bone morphogenetic proteins (BMPs), and / or members of the AKT / PI3K pathway. The definition and details of the TGF-β3 / BMP pathway are disclosed in the prior art, e.g., Kawabata M. and Miyazono K., J. Biochem. (Tokyo), 125, 9-16 (1999); Wrana JL, Miner. Electrolyte Metab., 24, 120-130 (1998) and Markowitz SD, and Roberts AB, Cytokine Growth Factor Rev., 7, 93-102 (1996). In some embodiments, the pluripotent stem cells may be exposed to at least one other compound or factor (including but not limited to cyclopamine, TGF family members (TGF-α, activin A, activin B, TGF-β-1, TGF-β-3), exendin 4, nicotinamide, n-butyrate, DMSO, all-trans retinoic acid, GLP-I, bone morphogenetic proteins (BMP-2, BMP-5, BMP-6, BMP-7), insulin-like growth factor (IGF-I, IGF-II), fibroblast growth factor (FGF7, FGF10, bFGF, FGF4), other growth factors (EGF, beta-cellulose, growth hormone, HGF), other hormones (prolactin, cholecystokinin, gastrin I, placental lactogen), TGF-β family antagonists (noggin, follistatin, tenascin), IBMX, wortmannin, dexamethasone, Reg, INGAP, cAMP or cAMP activator (forskolin) and / or extracellular matrix components (laminin, fibronectin)) in combination with a compound of formula (I), such as IDE1 and / or IDE2.

[0317] In some embodiments, HIP cells are contacted with at least one histone deacetylase (HDAC) inhibitor (e.g., class I / II HDAC inhibitor) to differentiate the cells into pancreatic islet cells. Histone deacetylase (HDAC) is a class of enzymes that remove acetyl groups from eN-acetyllysine on histones. Exemplary HDACs include those class I HDACs: HDAC1, HDAC2, HDAC3, HDAC8; and class II HDACs: HDAC4, HDAC5, HDAC6, HDAC7A, HDAC9, HDAC10. Type I mammalian HDACs include: HDAC1, HDAC2, HDAC3, HDAC8, and HDAC11. Type II mammalian HDACs include: HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, and HDAC1.

[0318] Many structural classes of HDAC negative regulators (e.g., HDAC inhibitors) have been developed, for example, small molecular weight carboxylates (e.g., less than about 250 amu), hydroxamic acids, benzamides, epoxyketones, cyclic peptides, and hybrid molecules. (See, e.g., Drummond DC, Noble CO, Kirpotin DB, Guo Z, Scott GK, et al. (2005) Clinical development of histone deacetylase inhibitors as anticancer agents. Annu Rev Pharmacol Toxicol 45:495-528 (including specific examples therein), the entire contents of which are incorporated herein by reference). Non-limiting examples of type I / II HDAC negative regulators include: suberoylanilide hydroxamic acid (SAHA (e.g., MK0683, vorinostat) and other hydroxamic acids), BML-210, deptine (e.g., (-)-deptine), HC toxin, Nullscript (4-(1,3-dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-N-hydroxybutyramide), phenylbutyric acid (e.g., sodium phenylbutyrate) and valproic acid ((VPA) and other short chain fatty acids), Scriptaid, suramin sodium, trichostatin A (TSA), APHA compound 8, Apicidin, sodium butyrate, pivaloyloxymethylbutyrate (Pivanex, AN-9), Trapoxin B, Chlamydocin, desphenolphthalein (also known as FR901228 or FK228), benzamide (e.g., CI-994 (i.e., N-acetyl benzophenone) and MS-27-275), MGCD0103, NVP-LAQ-824, CBHA (m-carboxycinnamic acid dihydroxamic acid), JNJ16241199, Tubacin, A-161906, proxamide, oxartanamide, 3-Cl-UCHA (i.e., 6-(3-chlorophenylureido) hexanoic acid hydroxamic acid), AOE (2-amino-8-oxo-9,10-epoxydecanoic acid), CHAP31, CHAP 50, IDE1 and IDE2. Other inhibitors include, for example, dominant negative forms of HDAC (e.g., catalytically inactive forms), siRNA inhibitors of HDAC, and antibodies that specifically bind to HDAC.Inhibitors are commercially available, for example, from BIOMOL International, Fukasawa, Merck Biosciences, Novartis, Gloucester Pharmaceuticals, Aton Pharma, Titan Pharmaceuticals, Schering AG, Pharmion, MethylGene, and Sigma Aldrich. In some embodiments, IDE1 or IDE2 are preferred histone deacetylase inhibitors.

[0319] Differentiation of HIP cells can be achieved by contacting (e.g., covering) a monolayer of HIP cells with one or more components of an extracellular matrix (ECM). In some embodiments, the HIP cell layer is contacted with an extracellular matrix component, which is one or more of the following: laminin, such as laminin 1; collagen, such as collagen IV; entactin; heparin sulfate proteoglycans; nestin. The extracellular matrix component can be a basement membrane-derived substance, such as a basement membrane laid by cells, such as tumor cells, such as Engelbreth-Holm-Swarm (EHS) tumor cells. In some embodiments, the extracellular matrix component is Matrigel. TM , which is commercially available from Becton-Dickenson. The extracellular component may also include: one or more growth factors, one or more matrix metalloproteinases (MMPs), such as MMP-2, MMP-3, and combinations thereof.

[0320] The HIP cells can be cultured in the presence of the extracellular matrix or one or more components of the extracellular matrix for at least 1, 2, 3, 5, 7, 10, 12, 14, 16, 18, 21, 25, 28, 30, 35, 40, 42, 48, 50 days or more.

[0321] In some embodiments, HIP-derived islet cells can be administered to patients in need thereof, such as human patients. In some cases, the patient suffers from a disease, disorder or condition that can be treated with such cells. In other words, as is well known in the art, administration of HIP-derived islet cells can reduce or alleviate at least one adverse effect or symptom associated with insulin metabolism. In some embodiments, the patient suffers from a disease characterized by insufficient insulin activity, which can include a disease of abnormal glucose utilization due to abnormal insulin function. Abnormal insulin function can include insulin production (e.g., expression and / or transport through organelles, such as insulin deficiency due to, for example, loss of β cells); secretion (e.g., impairment of insulin secretion response); the form of the insulin molecule itself (e.g., primary, secondary or tertiary structure); the effect of insulin on target cells (e.g., insulin resistance in body tissues including peripheral tissues); and any abnormality or impairment in the response of target cells to insulin.

[0322] The common method of administering islet cells to a subject, particularly a human subject, is described herein. For example, islet cells can be administered to a subject by injecting or implanting the islet cells into a target site of the subject. In addition, the cells can be inserted into a delivery device that helps introduce the cells by injecting or implanting the cells into a subject. Such a delivery device includes a tube, such as a catheter for injecting cells and fluid into the body of a recipient subject. In a preferred embodiment, the tube also has a needle, such as a syringe, by which the cells of the present invention can be introduced into the subject at a desired position. Islet cells can be inserted into such a delivery device, such as a syringe, in different forms. For example, when the cells are included in such a delivery device, they can be suspended in a solution or embedded in a supporting matrix.

[0323] As used herein, the term "solution" includes a pharmaceutically acceptable carrier or diluent in which the cells of the present invention remain viable. Pharmaceutically acceptable carriers and diluents include saline, buffered aqueous solutions, solvents and / or dispersion media. The use of such carriers and diluents is well known in the art. The solution is preferably sterile and fluid to the extent that the cells and solution can pass through a syringe. In some embodiments, the solution is stable under production and storage conditions and is preserved by using, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. to resist the contamination of microorganisms such as bacteria and fungi. Such a solution can be prepared by incorporating the islet cells described herein into a pharmaceutically acceptable carrier or diluent and then filtering and sterilizing.

[0324] The support matrix in which the islet cells can be incorporated or embedded includes a matrix that is compatible with the receptor and degrades into a product that is harmless to the receptor. Natural and / or synthetic biodegradable matrix is ​​an example of such a matrix. Natural biodegradable matrix includes plasma clots and collagen matrix derived from mammals, for example. Synthetic biodegradable matrix includes synthetic polymers, such as polyanhydrides, polyorthoesters and polylactic acid. Other examples of synthetic polymers and methods for incorporating or embedding cells in the matrix are known in the art. See, for example, U.S. Patent No. 4,298,002 and U.S. Patent No. 5,308,701. The matrix provides support and / or protection for fragile pancreatic cells in vivo.

[0325] As will be appreciated by those skilled in the art, differentiated HIP derivatives are transplanted using techniques known in the art that depend on the cell type and the ultimate use of these cells. Typically, the differentiated HIP cells of the present invention are transplanted intravenously or by injection at a specific location in a patient. When transplanted at a specific location, the cells may be suspended in a gel matrix to prevent the cells from dispersing when fixed.

[0326] XII. Hypoimmune retinal pigment epithelial (RPE) cells derived from HIP cells

[0327] In some embodiments, retinal pigment epithelium (RPE) cells are derived from HIP cells as described herein. For example, human RPE cells can be produced by differentiation human HIP cells. Similarly, mouse RPE cells can be produced by differentiation mouse HIP cells. Such RPE cells are low immunity RPE cells.

[0328] The HIP cells described herein can be differentiated into retinal pigment epithelial (RPE) cells, including RPE progenitor cells, immature RPE cells, mature RPE cells, and functional RPE cells.

[0329] Useful methods for differentiating embryonic pluripotent stem cells into RPE cells are described, for example, in US Pat. Nos. 9,458,428 and 9,850,463, which are incorporated herein by reference in their entireties, including specifications. Other methods for generating RPE cells from human embryonic or induced pluripotent stem cells can be found in, for example, Lamba et al., PNAS, 2006, 103(34):12769-12774; Mellough et al., Stem Cells, 2012, 30(4):673-686; Idelson et al., Cell Stem Cell, 2009, 5(4):396-408; Rowland et al., Journal of Cellular Physiology, 2012, 227(2):457-466, Buchholz et al., Stem Cells Trans Med, 2013, 2(5):384-393 and da Cruz et al., Nat Biotech, 2018, 36:328-337.

[0330] The term "RPE" cell refers to a pigmented retinal epithelial cell whose genetic expression profile is similar or substantially similar to that of a natural RPE cell. Such pluripotent stem cell-derived RPE cells, when grown to confluence on a planar substrate, may have the polygonal, planar sheet morphology of a natural RPE cell.

[0331] HIP cells and RPE cells derived from HIP cells can be cultured in a growth medium. Exemplary growth medium includes, but is not limited to, X-VIVO 10 TM (Lonza Biosciences), X-VIVO 15 TM (Lonza Biosciences), MTESR2 TM (Stem Cell Technologies), NUTRISTEM TM (StemGent) and HESCGRO TM (Millipore). Lonza X-VIVO 10 TM Supplemented with 5-40% xeno-free knockout serum replacement (XF-KOSR TM , Invitrogen), MX-302 (Iscove's modified Dulbecco's medium (IMDM) with B-27 supplement), Essential8 TMCulture medium, Dulbecco's modified Eagle's medium mammalian cell culture medium (DMEM), Ham's F12 medium, Iscove's modified Dulbecco's medium (IMDM), minimum essential medium Eagle (MEM), Roswell Park Memorial Institute medium 1640 (RPMI-1640), MCDB medium, etc.

[0332] Useful additives for promoting differentiation, growth, expansion, maintenance and / or maturation of RPE cells include, but are not limited to, BMP signaling inhibitors (e.g., LDN-193189, dorsomorphin, tenascin, cerburus and noggin), WNT signaling inhibitors (e.g., Dickkopf-related protein (DKK1), IWP-2, IWP-3, IWP-4, XAV939, secreted frizzled-related proteins (SFRP1 and SFRP2) and Wnt inhibitory factor 1 (WIF-1)), FGF signaling inhibitors (e.g., SU5402, AZD4547 and PD173074), insulin-like growth factor (IGF1), nicotinamide, benzoic acid, 3-aminobenzoic acid, 6-aminonicotinamide, inhibitors of poly (ADP-ribose) polymerase (PARP) (e.g., 3-aminobenzamide, enibaribacil (BSI)), and / or 201), olaparib (AZD-2281) and rucaparib (AG014699, PF-01367338), veliparib (ABT-888), CEP 9722, MK 4827 and BMN-673), ROCK inhibitors (e.g., Y-27632, thiazovivin, GSK429286A and fasudil), basic fibroblast growth factor 1 (bFGF1), FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, SUN13837, F2A4-K-NS, trichostatin A, activin A, activin AB, activin B, BMP-4, BMP-7, TGF-β1, vasoactive intestinal peptide (VIP), forskolin, rolipram, B-27 supplement, knockout serum replacement, N2 supplement, taurine, progesterone, vitamin A, blebbistatin, modified forms thereof, mimetics thereof, analogs thereof and variants thereof. Differentiation protocols and detailed descriptions thereof are provided in, for example, U.S. Pat. Nos. 9,458,428 and 9,850,463 and da Cruz et al., Nat Biotech, 2018, 36:328-337, the entire disclosures of which, including detailed descriptions of the invention, examples, methods, online methods, and results, are incorporated herein by reference.

[0333] In some embodiments, the low-immunity RPE cells are differentiated, proliferated or fixed on a cell culture substrate. Exemplary cell culture substrates are commonly used substrates, such as matrigel. TM (Corning Life Sciences), mouse embryonic fibroblast feeder cell layers, human embryonic fibroblasts, human fallopian tube epithelial or human foreskin fibroblast feeder layers. Xeno-free substrates may also be used, such as, but not limited to, Synthemax TM (Corning Life Sciences), CELLstart TM (Invitrogen), GELstart TM (Invitrogen) and StemAdhere TM (Primorigen). Additional cell culture substrates may include one or more of the following: purified human vitronectin, recombinant human vitronectin, recombinant human fibronectin (e.g.; Takara Bio), purified human laminin, recombinant laminin, recombinant laminin 511, recombinant laminin 521, poly-D-lysine, etc.

[0334] In certain embodiments, RPE cells are differentiated, propagated or fixed on biocompatible substrates such as synthetic substrates. Non-limiting substrates include polymer substrates, polyester films, polyethylene terephthalate (PET) films, poly-(DL-lactic acid-co-glycolic acid) (PLGA) films, porous polytetrafluoroethylene (ePTFE) films, polycaprolactone films, electrostatic spinning artificial scaffolds produced by methyl methacrylate and poly-(ethylene glycol) etc. Exemplary substrates are described in, for example, U.S. Patent number 8,808,687, and its disclosure is incorporated herein by reference in its entirety. Illustrative examples of suitable materials for the substrate include, but are not limited to, parylene polypropylene, polyimide, glass, nitinol, polyvinyl alcohol, polyvinyl pyrrolidone, collagen, chemically treated collagen, polyethersulfone (PES), poly(glycerol sebacate) PGS, poly(styrene-isobutyl-styrene), polyurethane, ethyl vinyl acetate (EVA), polyethyl ether ketone (PEEK), Kynar (polyvinylidene fluoride; PVDF), polytetrafluoroethylene (PTFE), polymethyl methacrylate (PMMA), Pebax, acrylic, polyolefins, polydimethylsiloxane (PDMS) and other silicone elastomers, polypropylene, hydroxyapatite, titanium, gold, silver, platinum, other metals and alloys, ceramics, plastics, and mixtures or combinations thereof. Other suitable materials for constructing the substrate include, but are not limited to, poly-para-xylylene (e.g., parylene, including but not limited to parylene A, parylene AM, parylene C, ammonia-treated parylene, polydopamine-treated parylene C), poly(lactic acid) (PLA), polyethylene vinyl acetate, poly(lactic-co-glycolic acid) (PLGA), poly(D,L-lactide), poly(D,L-lactide-co-trimethylene carbonate), collagen, heparinized collagen, denatured collagen, modified collagen (e.g., silicone with gelatin), other cell growth matrices (e.g., SYNTHEMAX TM ), poly(caprolactone), poly(glycolic acid) and / or other polymers, copolymers or block copolymers, poly(caprolactone) containing cyclic arginine-glycine-asparagine, cyclic or linear arginine-glycine-aspartic acid, mixtures of polycaprolactone and polyethylene glycol (PCL-PEG), thermoplastic polyurethanes, silicone-modified polyether urethanes, poly(urethane carbonate) or polyimides. Exemplary thermoplastic polyurethanes are polymers or copolymers that may include aliphatic polyurethanes, aromatic polyurethanes, polyurethane hydrogel-forming materials, hydrophilic polyurethanes or combinations thereof. Non-limiting examples include elastomers (poly(ether urethanes)), such as Elasthane TM 80A,Lubrizol,Tecophilic. TM ,PellethaneTM ,Carbothane TM ,Tecothane TM ,Tecoplast TM and Estane TM The silicone-modified polyether urethane may include Carbosil TM 20 or Pursil TM 2080A and the like. Poly(urethane carbonate) may include Bionate TM 80A or similar polymer.

[0335] In some embodiments, the substrate is biodegradable. In other embodiments, the substrate is non-biodegradable. In specific embodiments, the substrate comprises one or more biodegradable components and one or more non-biodegradable components.

[0336] In order to characterize and monitor RPE differentiation and assess RPE phenotype, the expression of any number of molecules and genetic markers can be assessed. For example, the presence of genetic markers can be determined by a whole host of methods known to those skilled in the art. The expression of molecular markers can be determined by quantitative methods, such as but not limited to mensuration based on qPCR, immunoassay, immunocytochemistry assay, immunoblotting assay, etc.

[0337] Exemplary markers for RPE cells include paired box protein (Pax6), Rax homeobox protein (Rax), LIM / homeobox protein 2 (Lhx2), homeobox protein SIX3, tyrosinase (TYR), microphthalmia-associated transcription factor (MITF), cellular retinal binding protein (CRALBP), trypsin 1 (cationic trypsinogen, TYRP1), trypsin 2 (anionic trypsinogen, TYRP2), melanin precursor protein (PMEL17), silver locus protein homolog (SILV), ceh-10 homeodomain-containing homolog (Chx10), bestrophin-1 (BEST), and retinal pigment epithelium-specific 65 kDa protein (RPE65).

[0338] RPE cells can also be assessed according to cell physiological markers and morphological markers.Immunocytochemistry and electron microscope can be used to determine the form of cells.Functional assays known to those skilled in the art can be used to assess RPE cells.For example, pigment epithelium derived factor (PEDF) secretion characteristic spectrum analysis, rod outer segment (ROS) phagocytosis assay, for trans-retinol is converted into 11-cis retinol assay, for determining the polarized secretion of growth factor and for detecting the tightly connected assay producing electrical barrier can be used to characterize the RPE cells derived from HIP cells.

[0339] In some embodiments of differentiation, pluripotent stem cell experience neural induction and express any other molecule, physiology or morphological marker of one or more retinal progenitor cell markers Pax6, Rax, Lhx2, Six3 or neural induction.In other embodiments, differentiated cell experience RPE specialization and / or form rosette structure.Along with cell continues to differentiate, rosette structure may be flattened into immature RPE cell layer or thin sheet.The layer of immature RPE cell can include the planar cell with polygon and / or hexagonal shape.

[0340] In some embodiments, low immunity RPE is implanted in patients who need it. RPE cells can be implanted in patients with macular degeneration or patients with damaged RPE cells. In some embodiments, the patient suffers from age-related macular degeneration (AMD), early AMD, moderate AMD, late AMD, non-vascular age-related macular degeneration, dry macular degeneration (dry age-related macular degeneration), wet macular degeneration (wet age-related macular degeneration, juvenile macular degeneration (JMD) (such as Stargardt disease, Best disease and juvenile retinoschisis), Leber congenital amaurosis or retinitis pigmentosa. In other embodiments, the patient suffers from retinal detachment.

[0341] RPE cells can be fixed on any substrate as described herein, to produce the RPE patch that can be transplanted into the patient in need thereof.The patch comprising one or more layers of RPE cells can be applied or delivered to eye tissue by surgical operation.In some cases, the patch is delivered to neural retina or subretinal space.In certain embodiments, the patch is delivered endoscopically, by catheter-based method, intravascular, intramuscular or by other means known in the art for specific eye tissue.Stereoscopic biomicroscopy, fundus photography can be used, spectral domain optical coherence tomography (SD-OCT) and other methods recognized by those skilled in the art determine the placement of the patch.

[0342] As will be appreciated by those skilled in the art, differentiated HIP derivatives are transplanted using techniques known in the art that depend on the cell type and the ultimate use of these cells. Typically, the differentiated HIP cells of the present invention are transplanted intravenously or by injection at a specific location in a patient. When transplanted at a specific location, the cells may be suspended in a gel matrix to prevent the cells from dispersing when fixed.

[0343] In order to more fully understand the invention described herein, the following examples are presented. It should be understood that these examples are for illustrative purposes only and should not be interpreted as limiting the present invention in any way.

[0344] VIII. Examples

[0345] Example 1: Generation of mouse induced pluripotent stem cells

[0346] The methods described herein were adapted from Diecke et al., Sci Rep, 2015, 8081.

[0347] Mouse tail tip fibroblasts were dissociated and isolated using type IV collagenase (Life Technologies, Grand Island, NY, USA) and maintained in a humidified incubator at 37°C, 20% O2 and 5% CO2 with Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), L-glutamine, 4.5 g / L glucose, 100 U / mL penicillin and 100 μg / mL streptomycin.

[0348] Then, 1 × 10 cells were transfected with a novel codon-optimized mini-intron plasmid (co-MIP) (10–12 μM DNA) expressing four reprogramming factors, Oct4, KLF4, Sox2, and c-Myc, using the Neon transfection system. 6 After transfection, fibroblasts were plated on a mouse embryonic fibroblast (MEF) feeder layer and maintained in fibroblast culture medium with the addition of sodium butyrate (0.2 mM) and 50 μg / mL ascorbic acid.

[0349] When ESC-like colonies appeared, the culture medium was changed to mouse iPSC culture medium containing DMEM, 20% FBS, L-glutamine, non-essential amino acids (NEAA), β-mercaptoethanol and 10 ng / mL leukemia inhibitory factor (LIF). After 2 passages, mouse iPSCs were transferred to 0.2% gelatin-coated plates and further expanded. For each passage, iPSCs were sorted for the mouse pluripotent marker SSEA-1 using magnetic activated cell sorting (MACS).

[0350] The isolated mouse iPSCs can be used to generate mouse low immunogenic iPSCs according to the above methods.

[0351] Example 2: Generation of human induced pluripotent stem cells

[0352] Gibco is derived from CD34+ umbilical cord blood using a three-plasmid, seven-factor (SOKMNLT: SOX2, OCT4 (POU5F1), KLF4, MYC, NANOG, LIN28, and SV40L T antigen) EBNA-based episomal system TMHuman episomal iPSC cell line (Catalog No. A18945, ThermoFisher). The iPSC cell line is considered to be zero footprint because there is no integration in the genome from the reprogramming event. It has been shown to be free of all reprogramming genes. The protocol for thawing, culturing and passage of human iPSC is provided in the product manual.

[0353] The pluripotency of human iPSCs can be determined by in vivo teratoma assays and in vitro pluripotency gene expression assays (eg, PCR and arrays) or by fluorescent staining for pluripotency markers.

[0354] Gibco TM Human episomal iPSC cell lines have normal karyotype and endogenous expression of pluripotent markers such as OCT4, SOX2 and NANOG (as shown in RT-PCR) and OCT4, SSEA4, TRA-1-60 and TRA-1-81 (as shown in ICC). Whole genome expression and epigenetic signature spectrum analysis show that the episomal hiPSC cell line is molecularly indistinguishable from the human embryonic stem cell line (Quintanilla et al., PloS One, 2014, 9 (1): e85419). In directed differentiation and teratoma analysis, these hiPSCs retain their differentiation potential for ectoderm, endoderm and mesoderm lineages (Burridge et al., PLoS One, 2011, 6 (4): e18293). In addition, vascular, hematopoietic, neural and cardiac lineages were derived with robust efficiency (Burridge et al., supra).

[0355] Table 1. Illustrative protocols for culturing human iPSCs (eg, Cas9 iPSCs)

[0356]

[0357] The isolated human iPSCs can be used to generate human hypoimmunogenic iPSCs (HIP cells) according to the methods described above.

[0358] Example 3: Low immunogenic pluripotent cells are less sensitive to NK cell killing and macrophage phagocytosis.

[0359] This example was performed to evaluate the ability of low immunogenic pluripotent cells (e.g., mouse b2m- / -ciita- / -CD47 tgiPSCs and human B2M- / -CIITA- / -CD47 tg iPSCs) to evade immune innate response pathways.

[0360] Specifically, an enzyme-linked immunospot (Elispot) assay was performed. NK cells were co-cultured with mouse HIP cells or human HIP cells (mouse B2m- / -Ciita- / -CD47 tg iPSC or human B2M- / -CIITA- / -CD47 tg iPSC), and the release of IFNγ was measured (e.g., using an Elispot plate reader to measure the frequency of innate IFNγ spots. In some instances, CD47 was blocked by using an anti-CD47 antibody.

[0361] Mouse B2m- / -Ciita- / -CD47 tg iPSCs co-cultured with mouse NK cells, such as approximately 95% NK cells and 5% macrophages, failed to stimulate NK cell activation ( Figure 1 ). In the Elispot assay, mouse B2m- / -Ciita- / -iPSCs triggered IFNγ release from NK cells, whereas mouse B2m- / -Ciita- / -CD47 tg iPSCs did not. Blocking CD47 (e.g., using anti-CD47 antibodies) had no effect on mouse B2m- / -Ciita- / -iPSCs. However, CD47 blockade completely abolished the protective effect possessed by B2m- / -Ciita- / -CD47tg iPSCs. YAC-1 cells, which are known to activate NK cells and thus release IFNγ, were used as a control.

[0362] Human B2M- / -CIITA- / -CD47 tg iPSCs co-cultured with human NK cells also failed to stimulate NK cell activation. Figure 2 Human B2M- / -CIITA- / -iPSCs were shown to trigger IFNγ release from NK cells in an Elispot assay, whereas human B2M- / -CIITA- / -CD47 tg iPSCs did not. Blockade of CD47 had no effect on human B2M- / -CIITA- / -iPSCs, but it did eliminate the protective effect possessed by human B2M- / -CIITA- / -CD47 tg iPSCs. K562 cells, which are known to activate NK cells and thus release IFNγ, were used as a control.

[0363] Figure 3 Elispot results of mouse B2m- / -Ciita- / -CD47 tg iPSCs incubated with human NK cells (approximately 95% NK cells and 5% macrophages) are shown. Mouse B2m- / -Ciita- / -iPSCs and mouse B2m- / -Ciita- / -CD47 tg iPSCs triggered IFNγ release from human NK cells. Blockade of CD47 had no effect on NK cell responses. YAC-1 cells elicited strong IFNγ release from human NK cells and were used as a control.

[0364] Figure 4 Elispot results of human B2M- / -CIITA- / -CD47 tg iPSCs incubated with mouse NK cells (approximately 95% NK cells and 5% macrophages) are shown. Human B2M- / -CIITA- / -iPSCs and human B2M- / -CIITA- / -CD47tg iPSCs triggered IFNγ release from mouse NK cells. Blockade of CD47 had no effect on NK cell responses. Human K562 cells caused strong IFNγ release from mouse NK cells and were used as a control.

[0365] Macrophage phagocytosis assays were also performed to determine whether the HIP cells of the present invention were susceptible to macrophage phagocytosis. Briefly, the HIP cells described herein were labeled with firefly luciferase and co-cultured with macrophages. The viability or presence of the HIP cells was analyzed by luciferase reporter gene assays.

[0366] Figure 5 The results of phagocytosis assays of firefly luciferase-labeled human B2M- / -CIITA- / -CD47tg iPSCs co-cultured with human macrophages are shown. When incubated with macrophages, the viability signal of human B2M- / -CIITA- / -iPSCs decreased significantly. On the other hand, the viability signal of human B2M- / -CIITA- / -CD47 tg iPSCs did not change in the presence of human macrophages. TritonX-100, which kills all HIP cells, was used as a control. Blockade of CD47 eliminates the protective features of human B2M- / -CIITA- / -CD47 tg iPSCs and makes them susceptible to phagocytosis or elimination by macrophages.

[0367] Figure 6 Shown are the results of a phagocytosis assay of firefly luciferase-labeled mouse B2m- / -Ciita- / -CD47tg iPSCs co-cultured with mouse macrophages.

[0368] When incubated with macrophages, the viability signal of mouse B2m- / -Ciita- / -iPSCs decreased significantly. In contrast, the viability signal of mouse B2m- / -Ciita- / -CD47 tg iPSCs did not change in the presence of mouse macrophages. TritonX-100, which kills all HIP cells, was used as a control. Blockade of CD47 eliminates the protective features of mouse B2M- / -CIITA- / -CD47 tg iPSCs and makes them susceptible to phagocytosis or elimination by macrophages. TritonX-100, which kills all HIP cells, was used as a control.

[0369] Figure 7The results of phagocytosis assays of firefly luciferase-labeled human B2M- / -CIITA- / -CD47tg iPSCs co-cultured with mouse macrophages are shown. When co-cultured with mouse macrophages, the viability signals of both human B2M- / -CIITA- / -iPSCs and human B2M- / -CIITA- / -CD47 tg iPSCs were significantly reduced. TritonX-100, which kills all HIP cells, was used as a control.

[0370] Figure 8 The results of phagocytosis assays of firefly luciferase-labeled mouse B2m- / -Ciita- / -CD47tg iPSCs co-cultured with human macrophages are shown. The viability signals of both mouse B2m- / -Ciita- / -iPSCs and mouse B2m- / -Ciita- / -CD47 tg iPSCs were significantly reduced when co-cultured with human macrophages. TritonX-100, which kills all HIP cells, was used as a control.

[0371] The results presented here demonstrate that mouse B2m- / -Ciita- / -CD47tg iPSCs and human B2M- / -CIITA- / -CD47tg iPSCs are able to evade innate immune responses, such as NK cell activation and macrophage phagocytosis.

[0372] Example 4: Generation of cardiomyocytes from human induced pluripotent stem cells

[0373] Provided herein is a method for producing human HIP cell-derived cardiomyocytes (CM). In an exemplary embodiment, human HIP cells are differentiated into an in vitro monolayer of cardiomyocytes. The exemplary procedures described herein are adapted from Sharma et al., J Vis Exp, 2015, 97, doi: 10.3791 / 52628, which is incorporated herein by reference in its entirety (and in particular for the technique of differentiating cells).

[0374] Human HIP cells were plated on diluted Matrigel (356231, Corning) in 6-well plates and maintained in Essential 8Flex medium (Thermo Fisher). The medium was changed every 24 hours.

[0375] After the cells reached 90%-100% confluence, differentiation began ( Fig.10), the CM differentiation medium on day 0 was replaced with 5 mL of RPMI1640 (Gibco, catalog number 61870) containing 2% B-27-insulin (Gibco, catalog number A1895601) and 2 μM-8 μM CHIR-99021 (Selleck Chem, catalog number S1263). In some cases, the concentration of CHIR-99021 was 6 μM. There was no medium change between day 0 and day 2 of differentiation.

[0376] On the second day ( Fig.11 ), the CM differentiation medium was replaced with RPMI1640 containing 2% B-27-insulin but without CHIR-99021. Be careful not to agitate the cells.

[0377] On Day 3 ( Fig.12 ), the CM differentiation medium was replaced with RPMI1640 containing 2% B-27-insulin and 5 μL IWR1 (WNT inhibitor, Selleck Chem, catalog number S7086). Be careful not to agitate the cells. After changing the medium, the cells were kept undisturbed in a 37°C incubator for 48 hours. There was no medium change between days 3 and 5 of differentiation.

[0378] On the 5th day ( Fig.13 ), the CM differentiation medium was changed back to RPMI1640 medium containing 2% B-27-insulin and incubated for 48 hours. The CM differentiation medium on day 5 was the same as that on day 2. After changing the medium, the cells were left undisturbed in a 37°C incubator for 48 hours.

[0379] On Day 7 ( Fig.14 ), the CM differentiation medium was replaced with RPMI1640 containing B27 plus insulin (Gibco, catalog number 17504044), and replaced with the same medium every other day thereafter. At about day 8 to day 10, spontaneous beating of cardiomyocytes was first seen.

[0380] After 10 days of differentiation ( Fig.15 ), more than 50% of the cell area was beating. Branching structures were very obvious. In some cases, the cells were terminally differentiated and no longer proliferating. Because the cells were beating, the culture of cardiomyocytes was maintained in CM differentiation medium on day 7. The medium was replaced every other day.

[0381] In some instances, when the differentiated cells do not beat or if there is a small beating area, the cells are subjected to glucose starvation. Glucose starvation loosens the non-cardiac cells on the culture plate. On the 10th day of differentiation, the culture medium is replaced with a purified culture medium (Gibco, catalog number (Cat. No.) 11879) comprising RPMI1640 without glucose, which contains B-27 plus insulin (Gibco, catalog number (Cat. No.) 17504044). The cells are maintained in purified culture medium for 3 days (until the 13th day of differentiation).

[0382] On day 13, the medium was changed to the CM differentiation medium from day 7 to maintain the cardiomyocytes in culture. In some examples, the purification procedure (glucose starvation) was repeated on day 14 so that the medium was changed to the CM differentiation medium on day 17. The remaining cells were highly purified cardiomyocytes.

[0383] The isolated and purified cardiomyocytes were maintained in CM differentiation medium on day 7. The medium was changed every other day. In some examples, approximately 1×10 6 Cardiomyocytes were plated in a 6-well plate.

[0384] The beating cardiomyocytes are frozen in freezing medium and stored in liquid nitrogen. In some cases, the freezing medium includes 90% heat-inactivated FCS and 10% DMCO, or a xeno-free equivalent. After thawing, the cardiomyocytes continue to beat.

[0385] Fig. 9 A diagram of the differentiation method is provided. Fig.10 Shows the results of the experiment on Matrigel just before differentiation. TM Human iPSCs cultured on (magnification 100x). Fig.11 Cells at day 2 of differentiation before medium change are shown (magnification 100x). Cells remain in a monolayer with approximately 10% floating cells. Before medium change, the differentiation medium is light yellow. Fig.12 Cells at day 3 of differentiation before medium change are shown (magnification 100 times). Cells remain in a monolayer with approximately 20% floating cells. The color of the medium is less yellow compared to day 2 of differentiation. Some cyst-like structures are visible in the monolayer. Fig.13 Shown are cells at day 5 of differentiation before media change (magnification 100X). Cells remain in a monolayer with approximately 15% floating cells. Cardiomyocytes appear above other cell clusters. Fig.14 Cells at day 7 before medium change are shown (magnification 100X). Cells remain in a monolayer with approximately 15% floating cells. Branch-like structures are visible. Fig.15Shown are cells at day 9 of differentiation before media change (magnification 100X). Beating cardiomyocytes are visible and appear darker in the photograph.

[0386] Example 5: Differentiation of mouse HIP cells into induced cardiomyocytes

[0387] mHIP cells were differentiated into murine induced cardiomyocytes (hmiCM). Before differentiation, mHIP cells and miPSCs were passaged twice in gelatin-coated culture dishes to remove feeder cells. On day 0, differentiation was started at 80,000 cells / mL in uncoated 10 cm plates in IMEM / Ham F12 (3:1, all from Corning) + 0.5% N2-supplement, 1% B27 retinoic acid, 0.05% BSA, 1% pen-strep, 1% glutamine (Gibco), 5mg / mL ascorbic acid and 40nL / mL MTG (all from Sigma-Aldrich) for 2 days. On the second day, cells were transferred to 10 cm plates containing 0.5% N2-supplement, 1% B27 retinoic acid, 0.05% BSA, 1% pen-strep, 1% glutamine (all from Gibco), 5mg / mL ascorbic acid and 40nL / mL MTG (Sigma-Aldrich) in IMEM / Ham F12 (3:1, all from Corning) and placed for 2 days. On the fourth day, cells were plated in 6-well plates coated with gelatin in SP34 medium (R&D Systems) containing 1% glutamine, 50μg / mL ascorbic acid, 5ng / mL VEGF, 500ug / mL hFGFb and 25ng / mL hFGF10. On the 7th day, the medium was replaced with SP34 medium containing 1% glutamine and 50ug / mL ascorbic acid, and replaced every other day. Cell beating began around day 11-14 and their functionality was confirmed.

[0388] Cells were enriched by separation using MACS using magnetic microbeads (Miltenyi) coated with anti-CD15 mAb for negative selection according to the manufacturer's protocol. The flow-through containing enriched hiCMs and miCMs was replated and used for different assays. Differentiation of Gata4 and Mhy6 was confirmed by rtPCT ( Fig.16 ). Gata4 forward primer (SEQ ID NO: 7 is:

[0389] 5'-CTGTCATCTCACTATGGGCA-3'

[0390] The reverse primer (SEQ ID NO: 8) is:

[0391] 5'-CCAAGTCCGAGCAGGAATTT-3'

[0392] Mhy6 forward primer (SEQ ID NO: 9) is

[0393] 5'-ATCATTCCCAACGAGCGAAAG-3'

[0394] The reverse primer (SEQ ID NO: 10) is

[0395] 5'-AAGTCCCCATAGAGAATGCGG-3'

[0396] Differentiation was also confirmed by immunofluorescence (IF). The primary antibodies were against α-sarcomeric actin (EA-53, Abcam) or troponin I (ab47003, Abcam), followed by the corresponding secondary antibodies conjugated with AF488 or AF555 (Invitrogen). (Data not shown.)

[0397] Example 6: Long-term survival of transplanted hypo-mCMs in allogeneic recipients

[0398] hiCMs and miCMs were transplanted into the infarct border zone of allogeneic recipient mice (BALB / c). The cell line is luciferase(+) producing luc(+) CMs that were tracked in vivo by bioluminescence (BLI).

[0399] HIP cells and iPSCs were transduced to express luciferase. Cells were transduced to express Fluc. One hundred thousand mHIP cells or miPSCs were plated in gelatin-coated 6-well plates and incubated overnight at 37°C and 5% CO2. The next day, the medium was changed and a vial of Fluc lentiviral particles (GenTarget, San Diego, CA) expressing the luciferase II gene under the reengineered EF1a promoter was added to 1.5 ml of medium. After 36 hours, 1 ml of medium was added. After another 24 hours, a complete medium change was performed. After 2 days, luciferase expression was confirmed by adding D-luciferin (Promega, Madison, WI). The fluorescence was measured using an IVIS200 (Perkin Elmer Waltham, MA) at maximum photon s -1 cm -2 The signal was quantified in steradian.

[0400] hiCMs were not rejected and did not migrate to other organs 28 days after transplantation ( Fig.17). After allogeneic transplantation, hiCMs evaded immune recognition and showed longitudinal survival. Because the injections were performed in the myocardium, the transplanted cells were optically mapped. hiCMs resulted in “supercell” engraftment, but miCMs did not. (Data not shown.)

[0401] Optical Mapping:

[0402] Whole heart Langendorff preparation. Four to five weeks after surgery, mice were euthanized by cervical dislocation. The heart was rapidly excised and placed in ice-cold modified Tyrode's solution, which consisted (in mmol / L) of 93 NaCl, 20 NaHCO3, 1 Na2HPO4, 1 MgSO4, 5 KCl, 1.8 CaCl2, 20 sodium acetate, 20 glucose. The heart was fixed to a cannula through the aorta and perfused retrogradely at 9 ml / min at 37°C using the same Tyrode's solution (pH maintained at 7.4 by bubbling with a 95% O2 / 5% CO2 gas mixture). The perfusate was then switched to a Tyrode's solution containing 10 mmol / L 2,3-butanedione monoxime (BDM) and 10 μmol / L blebbistatin (Enzo Life Sciences, Exeter, United Kingdom) to inhibit contraction and minimize motion artifacts. The heart was placed horizontally in a custom-made plexiglass chamber to allow imaging of the left ventricle (LV). Throughout the experiment, a pseudo electrocardiogram (ECG) was monitored using 2 Ag / AgCl disk electrodes placed close to the heart and a reference electrode placed in the perfusion bath. Two platinum electrodes connected to an isolated stimulator were placed on the right atrial (RA) appendage to enable pacing of the heart through the physiological endocardial to epicardial conduction pathway with a cycle length of 200 ms (5 Hz).

[0403] Optical measurements were performed as described in detail previously (Kelly et al., Circ. Arrhythmia Electrophysiol 6:809-17 (2013), which is incorporated herein by reference in its entirety). Briefly, the heart was loaded with a single dose of 50 μL of 2 mM pressure-sensitive dye (di-4-ANEPPS) within 10 minutes. Wide-field single-photon epifluorescence recordings were performed from the LV using a CardioCMOS-SM128 camera (Redshirt Imaging, Decatur, GA) with a 590 nm long-pass emission filter. Excitation was provided by 470 nm LED light. Image resolution was 128 × 128 pixels / frame and recorded at a frame rate of 2.5 kHz. Two-photon (2P) laser scanning microscopy (2PLSM) was performed using a Zeiss LSM 510NLO upright microscope (Carl Zeiss, Jena, Germany) equipped with a Ti: Sapphire 690–1080 nm tunable laser (Chameleon Ultra II, Coherent, Santa Clara, CA). These 2P measurements provide a high degree of depth resolution, enabling identification of discrete tissue layers that exhibit electrical activity (Rubart, 2004). Di-4-ANEPPS was excited at 920 nm, and the emitted light was collected by two dual-base PMT detectors at 510-560 nm and 590-650 nm, respectively, enabling ratiometric measurements. Line scans were performed in the direction of cells observed on the epicardial surface, with a scan time of 0.39 ms for short scans and 1.93 ms for long scans. Line scans were started after the trigger pulse arrived and synchronized with the electrical stimulation pulses used to pace the heart.

[0404] Sequential widefield and 2P voltage recordings were performed in the myocardium away from the infarct scar in the NZ, in the BZ at the edge of the visible scar, adjacent to the NZ, and in the IZ towards the center of the scar. Widefield electromapping using a 10× / 0.3NA objective (Carl Zeiss, Jena, Germany) was first used to identify the remaining areas of electrical activity within the BZ and IZ. Widefield electromapping was then repeated using a 40× / 0.8NA objective (Carl Zeiss, Jena, Germany) on areas showing electrical signals to further localize the areas of electrical activity. Finally, 2P excitation was used in frame scan mode to image the structures in the optical plane, and then the electrical signals were recorded using line scan mode at discrete depths below the epicardial surface. A series of recordings was performed, starting at 50 μm below the surface and at increasing depths (50-100 μm steps) until the signal-to-noise ratio (S / N) became too low to resolve clear action potential (AP) signals. The ability to visualize clear structures decreases at deeper layers; the maximum depth from which a discernible image can be obtained depends on the region, but the electrical signals from line scan recordings can always be recorded outside the layer where the structure can be imaged. Therefore, it is not possible to identify the source of electrical activity directly from images of deeper tissue structures. A subset of the analyses was performed using a modified vertical 2P laser scanning device (Intelligent Imaging Innovations; Denver, CO) using a pair of high-sensitivity GaAsP PMT detectors and a combination of FluoVolt and Rhod2AM excited at 840 nm. These were used to verify the findings in the higher sensitivity settings.

[0405] Determining the origin of electrical activity in scars. To determine the cellular origin of electrical activity in scars, intracellular Ca 2+ was performed in areas where voltage signals were measured by pre-loading the myocardium with Fura-2 / AM. 2+ If the signal is caused by residual myocytes, one would expect the Ca2+ in response to electrical stimulation to be 2+ However, if the voltage signal is caused by abnormal myocytes or other cellular entities (i.e., fibroblasts or myofibroblasts), CaTs may not be generated in response to electrical stimulation (Chilton et al., 2007).

[0406] The Tyrode solution used for these experiments was supplemented with 1mmol / L probenecid to block the anion transporter that excretes Fura-2, thereby improving the retention of the dye in the cell. (Di Virgilio et al., Biochem. J. 256: 959-63 (1988), the entire text is incorporated herein by reference). Fura-2 / AM was made into a 1mmol / L stock solution in DMSO-Pluronic acid F-127 (25% w / v). A single dose of 100 μL of the dye was injected into the bubble trap in the perfusion line to allow dilution of the dye and slow down the loading into the heart. Due to low fluorescence signal or time-dependent loss, an additional single dose of the dye was injected if necessary. Fura-2 / AM is 2P excited at 760nm, and the fluorescence emission is guided by a short-pass 650nm dichroic mirror and collected at 510-560nm. After any voltage signal is detected on the same focal plane and using the same scan line and 1.93ms scan time, Ca is immediately performed. 2+ Measurement.

[0407] Data analysis and interpretation. Widefield voltage signals were averaged across a 3 × 3 pixel array. 2P voltage and Ca signals were processed using custom written software. 2+ The software uses information from the exact cycle length timing and line scan rate to align and generate the average voltage or Ca signal from 25 consecutive stimuli (5 s recording). 2+ Trajectories. AP signal characteristics were analyzed from averaged traces. These measurements included the 10–90% upward rise time (TRise) and the AP duration from 50% activation to 50, 75, and 90% repolarization (APD50, APD75, and APD90, respectively). Activation time was determined as the time the AP arrived at that point in the LV wall relative to the time of stimulation.

[0408] S / N was calculated as the peak amplitude of the entire trace after a single stimulation pulse (signal) divided by the peak amplitude of the trace during diastole (noise) (Supplementary Figure S1). An S / N value of 1 indicates no signal beyond the baseline noise. Based on observation of individual traces and corresponding S / N values, S / N > 1.4 was considered to be a meaningful transient signal (voltage or Ca 2+ All traces with S / N>1.4 were further examined to exclude artifact signals generated by motion or noise spikes. All data are expressed as mean ± standard error. Student's t-test was used to compare several groups of data.

[0409] 28 days after myocardial infarction, histopathology and trichrome staining of recipient hearts showed a significant reduction in infarct size as well as left ventricular size in allogeneic recipients of hiCMs. Infarct length was measured from 25 slides each with a gap of 150 μm (3 sections of 5 μm per slide every 10th slide; slides 1-250). We identified α-sarcomeric positive donor cardiomyocytes and immature vascular structures in recipients receiving hiCM cells. In contrast, no cells were detected for allogeneic miCMs ( Fig.18 ).

[0410] Detailed PV circuit analysis showed significant improvements in left ventricular parameters. IT demonstrated not only survival of the allogeneic cells but also that they engrafted and functionally restored the heart after myocardial infarction ( Fig.19A ). PV circuit analysis of infarcted hearts receiving injections of miCM (green) or hiCM “supercells” (red).

[0411] The following parameters showed that hiCM restored cardiac function: Ejection fraction (EF) is the ratio of the amount of blood ejected from the ventricle per beat to the amount of blood in that ventricle at the end of diastole. Stroke volume (SV) is the amount of blood ejected by the ventricle in a single contraction. It is the difference between the end-diastolic volume and the end-systolic volume. ( Fig.19B ). Ventricular stroke work (SW) is defined as the work performed by the left ventricle in ejecting the stroke volume into the aorta. Cardiac output (CO) is defined as the volume of blood pumped by the ventricle per unit time. ( Fig.19C ). The end-systolic pressure-volume relationship (ESPVR) describes the maximum pressure that the ventricle can generate for any given cardiac chamber volume. The ESPVR is relatively insensitive to changes in preload, afterload, and heart rate. This makes it an index of improved systolic function compared to other hemodynamic parameters such as ejection fraction, cardiac output, and stroke volume.

[0412] Myocardial infarction results in reduced cardiac pump function with increased volume and decreased pressure in PV circuit analysis. When HIP cardiomyocytes are injected after myocardial infarction, changes in pressure and volume are prevented, indicating regeneration and impaired remodeling of the heart.

[0413] Example 7: Differentiation of human HIP cells into hiCMs

[0414] Human HIP cells are differentiated into hypoimmune cardiomyocytes. hHIP cells are plated on diluted matrigel (356231, Corning) in 6-well plates and maintained in Essential 8Flex culture medium (Thermo Fisher). Differentiation begins at 90% confluence, and the culture medium is replaced with 5mL of RPMI1640 containing 2% B-27-insulin (Gibco) and 6μM CHIR-99021 (Selleckchem). After 2 days, the culture medium is replaced with RPMI-1640 containing 2% B-27-insulin without CHIR. On the 3rd day, 5uL IWR1 is added to the culture medium for another two days. On the 5th day, the culture medium is changed back to RPMI-1640 containing 2% B-27-insulin culture medium and placed for 48 hours. On the 7th day, the culture medium is replaced with RPMI-1640 containing B27 plus insulin (Gibco), and replaced with the same culture medium every 3 days thereafter.

[0415] Purification of cardiomyocytes was performed on day 10 after differentiation. Briefly, the medium was changed to low glucose medium and maintained for 3 days. On day 13, the medium was changed back to RPMI-1640 containing B27 plus insulin. The procedure was repeated again on day 14.

[0416] The differentiated phenotype was confirmed by rtPCR for troponin (cTNT, data not shown). The forward primer (SEQ ID NO: 11) was:

[0417] 5'-GAGGCACCAAGTTGGGCATGAACG A-3',

[0418] The reverse primer (SEQ ID NO: 12) is:

[0419] 5'-GGCAGCGGAAGAGGATGCTGAA'

[0420] The differentiation phenotype was also confirmed by immunofluorescence (IF) staining. The primary antibodies were directed against α-sarcomeric actin (EA-53, Abcam) and troponin I (ab47003, Abcam), followed by the corresponding secondary antibodies conjugated with AF488 or AF555 (Invitrogen). The nuclei were stained with DAPI. Imaging was performed using a Leica SP5 laser confocal microscope (Leica).

[0421] (Data not shown.)

[0422] Spontaneous beating of cardiomyocytes was first seen around differentiation day 10. Cardiomyocytes displayed a beating rate similar to that of human cardiac tissue and were sensitive to cardiac stimulation (via isoproterenol) and cardiac block (via verapamil).

[0423] Example 8: hiCM cells survive allogeneic transplantation in humanized mice

[0424] Low-immunity cardiomyocytes survive transplantation into allogeneic humanized mice (humanized NSG-SGM3 mice (18-30 weeks) were purchased from Jackson Laboratories (Sacramento, CA). NSG-SGM3 mice transplanted with human CD34+ hematopoietic stem cells develop multi-lineage human immune cells and display a functional human immune system that exhibits T-cell-dependent immune responses without donor cell immune reactivity against the host. Animals were randomly assigned to experimental groups. The percentage of CD3+ cells in the human CD45+ cell population in each animal was evaluated, and the CD3 percentage was never significantly different between WT and B2M- / -CIITA- / -CD47 tg groups (Deuse et al., Nat Biotech 37(3):252-258 (2019), which is incorporated herein by reference in its entirety).

[0425] Wild-type or B2M- / -CIITA- / -CD47 tg hiCMs transplanted intramuscularly into allogeneic humanized mice confirmed the mouse data in the additional humanized mouse model. Cardiomyocytes were tracked longitudinally by bioluminescence (BLI). All wt hiCM grafts were rejected over time (5 animals). All five B2M- / -CIITA- / -CD47 tg hiCM grafts survived permanently ( Fig. 20 ). After myocardial infarction, low-immunity hiCM cells also survived transplantation ( Fig.21 ).

[0426] Example 9: Generation of endothelial cells from mouse induced pluripotent stem cells

[0427] Provided herein are methods for generating human iPSC-derived endothelial cells (ECs).In an exemplary embodiment, murine iPSCs (eg, murine HIP cells) can be differentiated into an in vitro monolayer of endothelial cells.

[0428] The protocol involves using 6-well plates or 10 cm dishes. The cells are trypsinized using standard trypsin. However, the trypsinized cells are not centrifuged. Instead, the trypsinized colonies are resuspended in culture medium in a volume 3 times greater than the volume of trypsin used.

[0429] Prior to differentiation (approximately 12 days), mouse embryonic fibroblasts (MEFs) were thawed and plated at 1 × 10 cells / well on gelatin-coated plates using MEF medium. 6 MEF (6-well plate). Thaw 1 vial of iPSC and transfer to three 6-wells on MEF. Change ESC medium every day. Split colonies on MEF at 1:3-1:6 in ESC medium (about 8 days after thawing). Observe NT-ESC colonies on MEF before splitting (magnification 100 times). Split colonies from MEF to gelatin at 1:3-1:6 in ESC medium (about 4 days after the first split; also called day -2).

[0430] On days 0 to 2, cells were 70-85% confluent to begin the differentiation protocol. EC differentiation medium #1 included RPMI and B-27 supplement-insulin (Life Technologies) and 5 μM CHIR-99021 (Selleck Chemicals, Houston, TX, USA) for 2 days. Approximately 2.5 ml of medium was used per 6-well plate or 10 cm plate. No medium was changed between days 0-2. The cells were left in the incubator untouched.

[0431] On days 2 to 4, observe the cell culture morphology. Change the culture medium to EC Differentiation Medium #2, which contains RPMI and B-27 Supplement-Insulin, and contains 2 μM CHIR-99021. Take care to avoid agitating the cells.

[0432] Endothelial cell (EC) differentiation medium #3 containing RPMI medium-insulin was used from day 4 to day 7. Medium changes were performed on day 4 and day 6. Undifferentiated cell clusters remained floating and initial EC colonies emerged.

[0433] EC colonies continued to grow to confluence in the plates from day 7 to day 17. Endothelial cell (EC) differentiation medium #3 contained EC medium (Lonza, Benicia, California) supplemented with VEGF, FGF, ROCK inhibitor, SB431542, and Y-27632. During EC differentiation, the medium was changed approximately every other day (e.g., on days 7, 9, 11, and 13).

[0434] Materials and reagents

[0435] Trypsin: Gibco, catalog number 12605-010.

[0436] Gelatin coating: 0.1% Gelatin Solution, Catalog No. ES-006-B. The surface of the plate was covered with the solution and the coated plate was stored at 37°C until use.

[0437] MEF medium: DMEM+glutamax+sodium pyruvate with 4.5 g / L glucose (Gibco), 15% FBS, NEAA, and 1% penicillin / streptomycin (P / S).

[0438] EC culture medium for Day 0-Day 2: RPMI with B-27 supplement-insulin (Life Technologies Cat. No. A1895601, 5 μM CHIR-99021 (Selleck Chemicals, Houston, TX, USA., Cat. No. S2924) and 1% P / S.

[0439] EC medium for Day 2-Day 4: RPMI with B-27 supplement-insulin (Life Technologies Cat. No. A1895601, 2 μM CHIR-99021 ((Selleck Chemicals, Houston, TX, USA., Cat. No. S2924) and 1% P / S.

[0440] For day 0-day 2 culture medium, CHIR stock solution (10 mM) was diluted to 5 μM (1:2000) in culture medium. For day 2-day 4 culture medium, CHIR stock solution was diluted to 2 μM (1:5000) in culture medium.

[0441] EC medium from day 4 to day 7 (RPMI medium-insulin): RPMI and B-27 supplement-insulin with 50 ng / mL vascular endothelial growth factor (VEGF; R&D Systems, Minneapolis, MN, USA), 10 ng / mL basic fibroblast growth factor (FGFb; R&D Systems), 10 μM Y-27632 (ROCK inhibitor) (Sigma-Aldrich, Saint Louis, MO, USA), and 1 μM SB 431542 (Sigma-Aldrich) for 3 days.

[0442] For EC medium from day 7 to day 14 (EC medium from Lonza): EGM-2 SingleQuots medium (or CC-3162 EGM™-2 Bullet Kit™, EBM™-2 plus SingleQuots™ growth supplement, 500 ml), 10 μM Y-27632 (Sigma-Aldrich, Saint Louis, MO, USA), 1 μM SB 431542 (Sigma-Aldrich), 25 ng / ml VEGF and 2 ng / ml FGF.

[0443] CD31+ cells were sorted and selected from mouse EC derived from pluripotent stem cells. Magnetic bead sorting methods were performed, including MACS. For example, CD31 microbeads (Miltenyi, catalog number 130-097-418) were used for positive selection of CD31+ cells. Cells were observed after day 12-day 14 and before MACS sorting (magnification 100 times). EC colonies were 80%-90% confluent and there were undifferentiated cell clusters. Spindle-shaped long cells represent mesenchymal differentiated cells.

[0444] An illustrative protocol for MACS selection of CD31+ cells is as follows.

[0445] 1. Wash cells once with PBS

[0446] 2. Trypsin: 0.5 ml / well; incubate at 37°C for 5 minutes

[0447] 3. Stop trypsin digestion with culture medium (culture medium from day 7-14), 1 ml / well

[0448] 4. Using a 1000 μl Eppendorf pipette: Resuspend the well to dissolve clumps

[0449] 5. Use a yellow 100 μm mesh filter in a 50 ml falcon tube to remove clumps and collect cells in a 50 ml tube

[0450] 6. Centrifuge the 50 ml tube at 300g for 5 minutes at 4°C

[0451] 7. Observe the precipitate in the 50 ml tube. Large precipitate = about 90 Mio cells

[0452] 8. For every 10 Mio cells: 90 μl MACS buffer + 10 μl beads

[0453] 9. Remove the supernatant to obtain the stem cell pellet

[0454] 10. Use a 1000 μl Eppendorf pipette to resuspend the pellet in MACS buffer.

[0455] 11. Add 200 μl of yellow Eppendorf beads to step 10 and resuspend.

[0456] 12. Vortex (very short)

[0457] 13. Incubate at 4°C (refrigerator) for 30 minutes

[0458] 14. Wash with 1-2 ml MACS buffer per 10 Mio cells by adding the buffer to a 50 ml tube.

[0459] 15. Centrifuge at 300 g for 5 minutes at 4°C

[0460] 16. Place the MidiMACS column in the magnet. Add 3 ml of MACS buffer to the column (as "priming"). Below the column: 50 ml Falcon tube

[0461] 17. Remove the supernatant from the cell pellet (the pellet needs to be dry)

[0462] 18. Add 500 μl of MACS buffer on top of the pellet (independent of pellet size, always use 500 μl)

[0463] 19. Add 500 μl (cells + MACS buffer) to the column and wait until it passes through the column.

[0464] 20. Add 3 ml of pure MACS buffer to the column and wait until it passes through the column: do this 3 times (washing step)

[0465] 21. Remove the column from the magnet and place it in a 15 ml falcon tube

[0466] 22. Add 5 ml of MACS buffer to the column and use a "stamp" or "plunger": press 5 ml into a 15 ml falcon tube to elute the CD31+ cells

[0467] 23. Centrifuge the cells at 300g for 5 minutes at 4°C

[0468] 24. Form a pellet (about 5-10 Mio if large): plate into 1×6-well plate (gelatin coated)

[0469] 25. Culture medium: EC differentiation medium for day 7-day 14. Do not move the plates until day 3 after MACS; do not remove them from the incubator before then.

[0470] 26. Maintain the differentiation medium from days 7-14 until day 5.

[0471] 27. On the 5th day after MACS sorting, the culture medium was replaced with: Lonza EGM-2 SingleQuots medium (CC-3162 EGMTM-2 BulletKitTM, EBMTM-2 plus SingleQuotsTM of growth supplement, 500 ml) plus 10 μM Y-27632.

[0472] 28. Change the medium every other day. Maintain cells on gelatin-coated plates.

[0473] 29. When necessary, split the cells 1:3 using trypsin.

[0474] 30. To characterize ECs, perform immunocytochemistry, LDL assays and / or tube formation assays according to methods known to those skilled in the art.

[0475] Fig. 22 NT-ESC colonies on MEFs before splitting are shown (magnification 100X). Fig.23 Shown are NT-ESCs on gelatin just prior to differentiation (magnification 100X). Fig.24 Cells at day 2 of differentiation are shown before the differentiation medium was changed from 5 μM CHIR to 2 μM CHIR (magnification 100X). CHIR induces mesodermal differentiation. Non-mesodermal cells form clumps and can be seen floating. Fig.25 Shown are cells at day 4 of EC differentiation prior to medium change (magnification 100x). EC colonies appear beneath floating undifferentiated cell clusters. Fig.26 EC cells at day 7 of differentiation are shown (magnification 100X). EC colonies are visible and becoming more confluent. Only a few undifferentiated cell clusters are visible. Fig. 27 Cells after day 12 to 14 and before MACS sorting are shown (magnification 100X).

[0476] Example 10 - Differentiation of HIP cells into murine endothelial cells (miEC)

[0477] HIP iPSC and miPSC are differentiated into endothelial cells (miEC), but only HIP cell-derived miEC survive long-term in allogeneic hosts. HIP and miPSC are plated on gelatin in 6-well plates and maintained in mouse iPSC culture medium. After the cells reach 60% confluence, differentiation begins, and the culture medium is replaced with RPMI-1640 containing 2% B-27-insulin (all from Gibco) and 5 μM CHIR-99021 (Selleckchem, Munich, Germany). On the second day, the culture medium was replaced with a reducing medium: RPMI-1640 containing 2% B-27-insulin (all from Gibco) and 2 μM CHIR-99021 (Selleckchem). From day 4 to day 7, cells were exposed to RPMI-1640EC medium, i.e., RPMI-1640 containing 2% B-27-insulin plus 50 ng / mL mouse vascular endothelial growth factor (mVEGF; R&D Systems, Minneapolis, MN), 10 ng / mL mouse basic fibroblast growth factor (mFGFb; R&D Systems), 10 μM Y-27632 (Sigma-Aldrich, Saint Louis, MO), and 1 μM SB 431542 (Sigma-Aldrich). Endothelial cell clusters were visible starting on day 7, and cells were maintained in EGM-2 SingleQuots medium (Lonza) plus 10% FCS hi (Gibco), 25 ng / mL mVEGF, 2 ng / mL mFGFb, 10 μM Y-27632 (Sigma-Aldrich), and 1 μM SB431542. The differentiation process was completed after 21 days and undifferentiated cells were separated during the differentiation process. For purification, cells were MACS purified using anti-CD15 mAb-coated magnetic microbeads (Miltenyi, Auburn, CA) for negative selection according to the manufacturer's protocol.

[0478] Endothelial cell differentiation was confirmed by rtPCR and immunofluorescence. Highly purified HIP and miEC from the flow-through were cultured in EGM-2 SingleQuots medium plus supplements and 10% FCS hi. Cells were passaged 1:3 using TrypLE every 3 to 4 days. PCR was performed as described above. The following primers were used: VE-cadherin forward primer (SEQ ID NO: 13): 5'-GGATGCAGAGGCTCACAGAG-3', reverse primer (SEQ ID NO: 14): 5'-CTGGCGGTTCACGTTGGACT-3'. EC cells from both miPSCs and HIP cells showed differentiated gene expression profiles, including VE-cadherin expression, while parental cells did not ( Fig.28 ).

[0479] Their phenotypes were also confirmed by immunofluorescence (IF) for CD31 (ab28364, Abcam) and VE-cadherin (sc-6458, Santa Cruz Biotechnology, Santa Cruz, CA). Briefly, cells were fixed with 4% paraformaldehyde in PBS for 15 minutes. The cell membrane was permeabilized with permeabilization solution (ASB-0102, Applied StemCell), then treated with blocking solution (ASB-0103, Applied StemCell), and incubated with a primary antibody. For visualization, cells were incubated with a secondary antibody (Invitrogen) conjugated with AF488 or AF555. After staining the nuclei with DAPI, images were obtained and analyzed using a Leica SP5 laser confocal microscope (Leica). EC cells from miPSC and HIP cells were positive for CD31 and VE-cadherin staining (data not shown).

[0480] Tube formation was also confirmed by immunofluorescence assay. 5 miECs were stained with 5 μM CFSE and 0.1 μg / mL Hoechst (both from Thermo Fisher) for 10 min at room temperature and plated on 10 mg / mL undiluted Matrigel (356231, Corning, Corning, NY) in 24-well plates. After 48 h, tube formation was observed by IF (data not shown).

[0481] Example 6 - Survival of mHIP cell-derived endothelial cells after allogeneic transplantation

[0482] Allogeneic hypo-mECs survive in vivo in the hindlimb ischemia model. Grafts of Fluc+wt or B2M- / -CIITA- / -CD47 tg mECs were transplanted into allogeneic mice after removal of the femoral artery and tracked longitudinally by BLI. Fifteen animals were used per group.

[0483] For bioluminescent imaging (BLI), D-luciferin firefly potassium salt (375 mg / kg; Biosynth AG, Staad, Switzerland) was dissolved in PBS (pH 7.4) (Gibco, Invitrogen) and injected intraperitoneally (250 μL per mouse) into anesthetized mice. Animals were imaged using the IVIS200 system (Xenogen). The bioluminescence of the region of interest (ROI) was quantified in units of maximum photons / second / square centimeter / steradian (p / s / cm2 / sr). The maximum signal from the ROI was measured using LivingImage software (MediaCybernetics). Mice were monitored at day 0, day 1, and every other day until day 30 and every 10 days thereafter.

[0484] BLI values ​​for all animals are plotted. The highly immunogenic wt mECs were rejected within 15 days, showing a decrease in BLI signal over time, whereas all B2M- / -CIITA- / -CD47 tg grafts survived ( Fig.29 ).

[0485] Example 11 - mHIP cell-derived EC cells evade immune response in vitro

[0486] HIP cell-derived endothelial cells do not elicit IFN-γ or natural killer responses in vitro.

[0487] Elispot assay. For the one-way Elispot assay, recipient splenocytes were isolated from the spleen 5 days after cell injection and used as reactive cells. Donor cells were treated with mitomycin (50 μg / mL for 30 minutes) and used as stimulator cells. One hundred thousand stimulator cells were mixed with 1×10 6 Receptor-reactive splenocytes were incubated together for 24 h, and the frequency of IFN-γ and IL-4 spots was counted using an Elispot plate reader.

[0488] Donor-specific antibodies. Serum from recipient mice was decomplemented by heating to 56°C for 30 minutes. Equal amounts of serum and cell suspension (5×10 6 / mL) and incubated at 4° C. for 45 minutes. Cells were labeled with FITC-conjugated goat anti-mouse IgM (Sigma-Aldrich) and analyzed by flow cytometry (BD Bioscience).

[0489] Elispot in vitro assay of mouse NK cells. NK cells were isolated from fresh BALB / c spleens 18 h after injection of poly I:C (150 ng Poly I:C in 200 μL sterile saline, intraperitoneally (ip), Sigma-Aldrich). After erythrocyte lysis, the cells were purified by magnetic bead sorting coated with anti-CD49b mAb and used as reactive cells. The cell population was >99% CD3- and contained NK cells (>90%) and other cells, including myeloid cells (<10%). Using the Elispot principle, NK cells were co-cultured with B2m- / -Ciita- / -or B2m- / -Ciita- / -Cd47 tg miPSCs in the presence of IL-2 (1 ng / mL, Peprotech, Rocky Hill, NJ), and their IFN-γ release was measured. YAC-1 cells (Sigma-Aldrich) were used as a positive control. Stimulator cells treated with mitomycin (50 μg / mL for 30 min) were incubated with NK cells (1:1) for 24 h, and the frequency of IFN-γ spots was counted using an Elispot plate reader.

[0490] Five days after injection of miECs derived from wt miPSCs into C57BL / 6 or BALB / c recipients, splenocytes were recovered for IFN-γ Elispot assays (25th to 75th percentile bins with median, whiskers min-max, 6 animals per group, two-tailed Student's t-test). IFN-γ responses were much more robust in all allogeneic recipients. Mean fluorescence (MFI) of IgM bound to wild-type miPSC-derived miECs incubated with recipient serum after 5 days (25th to 75th percentile bins with median, whiskers min-max, 6 animals per group, two-tailed Student's t-test). IgM responses were significantly stronger in all allogeneic recipients ( Fig.30 ).

[0491] Similarly, the source from B2m - / - Ciita - / -miECs derived from Cd47 tg miPSCs were injected into C57BL / 6 or BALB / c recipients and subjected to IFN-γ Elispot treatment 5 days later (25th to 75th percentile bins with median, whiskers min-max, 6 animals per group, two-tailed Student’s t-test). - / - Ciita - / - Mean fluorescence (MFI) of miEC-bound IgM of Cd47tg miPSCs (25th to 75th percentile bins with median, whiskers min-max, 6 animals per group, two-tailed Student's t-test). There were no measurable IFN-γ responses or IgM responses in allogeneic recipients. To assess the inhibitory effect of Cd47 overexpression on NK cell killing, B2m-derived NK cells were used to - / - Ciita - / - miPSC or B2m - / - Ciita - / - Cd47tg miPSCs and miECs were subjected to IFN-γ Elispot using NK cells (25th to 75th percentile bins with median, min-max, 6 independent experiments, ANOVA with Bonferroni post hoc test). - / - Ciita - / - miPSC derivatives are sensitive to NK cell killing ( Fig.30 ).

[0492] Example 12 - Endothelial cell morphology derived from HIP cells

[0493] Endothelial cells derived from HIP cells showed typical EC morphology. - / - Ciita - / - Cd47tg miEC grafts were transplanted subcutaneously into allogeneic BALB / c mice. These low immunogenic derivatives further matured in vivo in the allogeneic recipients or changed their morphology over time.

[0494] Eight hundred thousand B2m- / -Ciita- / -Cd47 tg miECs in a 1:1 dilution of Matrigel (Corning) were injected into allogeneic BALB / c mice. Matrigel plugs were recovered at multiple time points up to 56 days and fixed in 4% paraformaldehyde in PBS containing 1% glutaraldehyde for 24 hours. The samples were dehydrated, embedded in paraffin, and cut into 5 μm thick sections. For histopathology, the sections were stained with hematoxylin and eosin (Carl Roth) and images were taken with an inverted optical microscope. Immunofluorescence staining was used to prove the origin of the cells. The sections were rehydrated and antigen retrieval and blocking were performed. The samples were incubated with antibodies against luciferase (ab21176), VE-cadherin (SC-6458) and the corresponding secondary antibodies (Invitrogen) conjugated to AF488 or AF555. Cell nuclei were counterstained with DAPI, and images were captured using a Leica SP5 laser confocal microscope (Leica, Wetzlar, Germany).

[0495] For co-staining experiments of miECs and immune cells, primary antibodies against VE-cadherin (SC-6458, Sigma) and CD3 (ab16669, Abcam) were used, followed by corresponding secondary antibodies conjugated with AF488 or AF555 (Invitrogen). Premature formation of blood vessels was observed. Data not shown.

[0496] Transplanted miECs began to organize into round structures around day 14 and formed primitive blood vessels containing erythrocytes around week 3 (data not shown).

[0497] Doppler perfusion (Periscan PIMII) (PERIMED Ltd., Italy) of cells taken from animals of Example 6 demonstrated neovascularization and rescue of the limb in the hypo-EC group (data not shown).

[0498] Example 13 - Differentiation of human HIP cells into endothelial cells

[0499] Human HIP cells are differentiated into hiEC cells. Wild-type hiPSC and human HIP cells are plated on diluted matrigel (356231, Corning) in 6-well plates and maintained in Essential 8Flex culture medium (Thermo Fisher). Differentiation begins at 60% confluence, and the culture medium is replaced with RPMI-1640 containing 2% B-27-insulin (all from Gibco) and 5 μM CHIR-99021 (Selleckchem). On the 2nd day, the culture medium is replaced with a reducing medium: RPMI-1640 containing 2% B-27-insulin (all from Gibco) and 2 μM CHIR-99021 (Selleckchem). From day 4 to day 7, cells were exposed to RPMI-1640EC medium, i.e., RPMI-1640 containing 2% B-27-insulin plus 50 ng / mL human vascular endothelial growth factor (VEGF; R&D Systems), 10 ng / mL human basic fibroblast growth factor (FGFb; R&D Systems), 10 μM Y-27632 (Sigma-Aldrich), and 1 μM SB 431542 (Sigma-Aldrich). Endothelial cell clusters were visible from day 7, and cells were maintained in EGM-2 SingleQuots medium (Lonza) plus 10% FCS hi (Gibco), 25 ng / mL VEGF, 2 ng / mL FGFb, 10 μM Y-27632 (Sigma-Aldrich), and 1 μM SB431542 (Sigma-Aldrich). The differentiation process was completed after 14 days, and undifferentiated cells were separated during the differentiation process. For purification, cells were treated with 20 μM PluriSln-1 (StemCell Technologies, Vancouver, BC, Canada) for 48 hours. Highly purified ECs were cultured in EGM-2 SingleQuots medium (Lonza) plus supplements and 10% FCS hi (Gibco). Cells were passaged 1:3 every 3 to 4 days using TrypLE Express.

[0500] IF staining was performed as described above to confirm its phenotype. Primary antibodies against CD31 (ab28364, Abcam) and VE-cadherin (sc-6458, Santa Cruz Biotechnology) were used, followed by the corresponding secondary antibodies conjugated to AF488 or AF555 (Invitrogen). Nuclei were stained with DAPI. Imaging was performed using a Leica SP5 laser confocal microscope (Leica). PCR for VE-cadherin was performed as described above (forward (SEQ ID NO: 15): 5'-AAGATGCAGAGGCTCATG-3', reverse primer (SEQ ID NO: 16): 5'-CATGAGCCTCTGCATCTT-3').

[0501] wt hiPSC (a) and B2M - / - CIITA - / - CD47 tg hiPSCs (b) were successfully differentiated into the corresponding hiEC derivatives. EC cells from both hiPSCs and HIP cells showed differentiated gene expression profiles, including CDH5 expression, while parental cells did not. By confocal immunofluorescence, miECs stained positive for CD31 and VE-cadherin. All derivatives lost expression of their pluripotency genes (representative images of two independent experiments) ( Fig.31 and data not shown).

[0502] Example 14 - hiEC cell survival in humanized mice

[0503] Human HIP cells survive after transplantation into mice with a humanized immune system. Humanized NSG-SGM3 mice (18-30 weeks) were purchased from Jackson Laboratories (Catalog No. SMG3-CD34, Sacramento, CA). NSG-SGM3 mice transplanted with human CD34+ hematopoietic stem cells (HSE) developed multi-lineage human immune cells. They demonstrated a functional human immune system that showed T cell-dependent immune responses without donor cell immunoreactivity against the host. Animals were randomly assigned to experimental groups. The percentage of CD3+ cells in the human CD45+ cell population in each animal was assessed, and the CD3 percentage was never significantly different between the wild type and B2M- / -CIITA- / -CD47 tg groups. All humanized NSG-SGM3 mice were HLA-A typed and the number of mismatches with cell transplants was calculated. In the Elispot assay with the hiEC group, there were always 2 mismatches.

[0504] Wild-type or B2M- / -CIITA- / -CD47tg hiEC grafts were injected into allogeneic humanized NSG-SGM3 mice. IFN-γElispot was performed 5 days later (mean ± sd, 3 animals per group, two-tailed Student's t-test). Background spot frequencies in naive mice are shown (mean ± sd, 4 animals per group, two-tailed Student's t-test). 1, MFI of IgM bound to either hiEC incubated with recipient serum after 5 days (mean ± sd, 3 animals per group, two-tailed Student's t-test). Background fluorescence in naive mice is shown (mean ± sd, 3 animals per group, two-tailed Student's t-test). IFN-γ Elispot using human NK cells was performed with B2M- / -CIITA- / -hiECs or B2M- / -CIITA- / -CD47 tg hiECs (bins with 25th to 75th percentile of median, min-max, 6 independent experiments, ANOVA with Bonferroni post hoc test). Fig.32 )

[0505] All publications and patent documents disclosed or cited herein are incorporated herein by reference in their entirety. The foregoing description has been given for purposes of illustration and description only. This description is not intended to limit the invention to the precise form disclosed.

[0506] It is intended that the scope of the invention be defined by the claims appended hereto.

[0507] Informal Sequence Listing

[0508] SEQ ID NO: 1—Human beta-2-microglobulin

[0509] MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDI

[0510] SEQ ID NO: 2—human CIITA protein, 160 amino acids N-terminal

[0511] MRCLAPRPAGSYLSEPQGSSQCATMELGPLEGGYLELLNSDADPLCLYHFYDQMDLAGEEEIELYSEPDTDTINCDQFSRLLCDMEGDEETREAYANIAELDQYVFQDSQLEGLSKDIFKHIGPDEVIGESMEMPAEVGQKSQKRPFPEELPADLKHWKP

[0512] SEQ ID NO: 3 - Human CD47 Protein

[0513] MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVE

[0514] SEQ ID NO: 4 - Herpes Simplex Virus Thymidine Kinase (HSV-tk) Protein

[0515] MASYPCHQHASAFDQAARSRGHSNRRTALRPRRQQEATEVRLEQKMPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWQVLGASETIANIYTTQHRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHVGGEAGSSHAPPPALTLIFDRHPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGALPEDRHIDRLAKRQRPGERLDLAMLAAIRRVYGLLANTVRYLQGGGSWWEDWGQLSGTAVPPQGAEPQSNAGPRPHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFILDYDQSPAGCRDALLQLTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN

[0516] SEQ ID NO: 5 - Escherichia coli Cytosine Deaminase (CD) Protein

[0517] MSNNALQTIINARLPGEEGLWQIHLQDGKISAIDAQSGVMPITENSLDAEQGLVIPPFVEPHIHLDTTQTAGQPNWNQSGTLFEGIERWAERKALLTHDDVKQRAWQTLKWQIANGIQHVRTHVDVSDATLTALKAMLEVKQEVAPWIDLQIVAFPQEGILSYPNGEALLEEALRLGADVVGAIPHFEFTREYGVESLHKTFALAQKYDRLIDVHCDEIDDEQSRFVETVAALAHHEGMGARVTASHTTAMHSYNGAYTSRLFRLLKMSGINFVANPLVNIHLQGRFDTYPKRRGITRVKEMLESGINVCFGHDDVFDPWYPLGTANMLQVLHMGLHVCQLMGYGQINDGLNLITHHSARTLNLQDYGIAAGNSANLIILPAENGFDALRRQVPVRYSVRGGKVIASTQPAQTTVYLEQPEAIDYKR

[0518] SEQ ID NO: 6 - truncated human caspase-9 protein

[0519] GFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS

Claims

1. A low-immunogenic neural stem cell, comprising: i. one or more genetic alterations that reduce the surface expression of functional class I human leukocyte antigen (HLA-I) complexes compared to wild-type cells of the same type of low immunogenic cells, and One or more genetic alterations that reduce surface expression of functional class II human leukocyte antigen (HLA-II) complexes compared to said wild-type cells; and ii. One or more genetic alterations that increase the surface expression of functional CD47 protein compared to said wild-type cells.

2. The low immunogenic neural stem cell of claim 1, wherein the neural stem cell expresses nestin. The low immunogenic neural stem cell of claim 1 , wherein the low immunogenic neural stem cell is differentiated from a pluripotent stem cell.

4. The low immunogenic neural stem cell of any one of claims 1 to 3, wherein the one or more genetic alterations that reduce the expression of a functional HLA-I complex comprise one or more genetic alterations that reduce the expression of a beta-2 microglobulin (B2M) gene. The low-immunogenic neural stem cell according to claim 4 , wherein the expression of the B2M gene has been eliminated.

6. The low immunogenic neural stem cell of any one of claims 1 to 3, wherein the one or more genetic changes that reduce the expression of a functional HLA-II complex comprise one or more genetic changes that reduce the expression of a class II transactivator (CIITA) gene.

7. The low-immunogenic neural stem cell according to claim 6, wherein the expression of the CIITA gene has been eliminated.

8. The low immunogenic neural stem cell of claim 1, wherein expression of the B2M gene has been eliminated and expression of the CIITA gene has been eliminated, and the one or more genetic alterations that increase surface expression of functional CD47 protein include an introduced CD47 transgene under the control of an inducible or constitutive promoter.

9. The low immunogenic neural stem cell of any one of claims 1 to 3, wherein the one or more genetic alterations that reduce the expression of a functional HLA-I complex comprise one or more genetic alterations that reduce the expression of one or more HLA-I genes compared to said wild-type cells.

10. The low immunogenic neural stem cell of claim 9, wherein the one or more genetic alterations that reduce the expression of a functional HLA-I complex comprise one or more genetic alterations that knock out the expression of one or more of an HLA-A gene, an HLA-B gene, and an HLA-C gene.

11. The low immunogenic neural stem cell of any one of claims 1 to 3, wherein the one or more genetic alterations that reduce the expression of a functional HLA-II complex comprise one or more genetic alterations that reduce the expression of one or more HLA-II genes compared to said wild-type cells.

12. The low immunogenic neural stem cell of claim 11, wherein the one or more genetic alterations that reduce the expression of a functional HLA-II complex comprise one or more genetic alterations that knock out the expression of one or more of the HLA-DP gene, the HLA-DR gene, and the HLA-DQ gene.

13. The low immunogenic neural stem cell of any one of claims 1 to 3, wherein the one or more genetic alterations that increase surface expression of functional CD47 protein comprise an introduced CD47 transgene under the control of an inducible or constitutive promoter.

14. The low immunogenic neural stem cell of any one of claims 1 to 3, wherein one or more of the genetic alterations are introduced into the cells using zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR) / nucleases, retroviral vectors, lentiviral vectors, adenoviral vectors, Sendai virus vectors, or a combination thereof.

15. The low immunogenic neural stem cell according to any one of claims 1 to 3, wherein the low immunogenic cell is a human cell.

16. The low immunogenic neural stem cell according to any one of claims 1 to 3, wherein the low immunogenic cell comprises a safety switch comprising a suicide gene.

17. The low immunogenic neural stem cell of claim 16, wherein the suicide gene: i. is the herpes simplex virus thymidine kinase (HSV-tk) gene, ii. is the Escherichia coli cytosine deaminase (EC-CD) gene, or iii. Encodes an inducible caspase protein.

18. The low immunogenic neural stem cell according to any one of claims 1 to 3, wherein the pluripotent stem cell is an embryonic stem cell or an induced pluripotent stem cell (iPSC).

19. Low immunogenic glial cells differentiated from the low immunogenic neural stem cells of claim 1.

20. The low immunogenic glial cell of claim 19, wherein the low immunogenic glial cell is an astrocyte or an oligodendrocyte.

21. A low immunogenic glial cell comprising: i. one or more genetic alterations that reduce the surface expression of functional class I human leukocyte antigen (HLA-I) complexes compared to wild-type cells of the same type of low immunogenic cells, and One or more genetic alterations that reduce surface expression of functional class II human leukocyte antigen (HLA-II) complexes compared to said wild-type cells; and ii. One or more genetic alterations that increase the surface expression of functional CD47 protein compared to said wild-type cells.

22. The low immunogenic glial cell of claim 21, wherein the low immunogenic glial cell is an astrocyte or an oligodendrocyte.

23. The low immunogenic glial cell of claim 21, wherein the expression of the B2M gene has been eliminated.

24. The low immunogenic glial cell of claim 21, wherein the expression of the CIITA gene has been eliminated.

25. The low immunogenic glial cell of claim 21, wherein expression of the B2M gene has been eliminated and expression of the CIITA gene has been eliminated, and the one or more genetic alterations that increase surface expression of functional CD47 protein include an introduced CD47 transgene under the control of an inducible or constitutive promoter.

26. The low immunogenic glial cell of claim 21, wherein the one or more genetic alterations that reduce expression of a functional HLA-I complex comprise one or more genetic alterations that reduce expression of one or more HLA-I genes compared to said wild-type cells.

27. The low immunogenic glial cell of claim 26, wherein the one or more genetic alterations that reduce the expression of a functional HLA-I complex comprise one or more genetic alterations that knock out the expression of one or more of an HLA-A gene, an HLA-B gene, and an HLA-C gene.

28. The low immunogenic glial cell of claim 21, wherein the one or more genetic alterations that reduce expression of a functional HLA-II complex comprise one or more genetic alterations that reduce expression of one or more HLA-II genes compared to said wild-type cells.

29. The low immunogenic glial cell of claim 28, wherein the one or more genetic alterations that reduce the expression of a functional HLA-II complex comprise one or more genetic alterations that knock out the expression of one or more of the HLA-DP gene, the HLA-DR gene, and the HLA-DQ gene.

30. The low immunogenic glial cell of claim 21, wherein the one or more genetic alterations that increase surface expression of functional CD47 protein comprise an introduced CD47 transgene under the control of an inducible or constitutive promoter.

31. The low immunogenic glial cells of claim 21, wherein the low immunogenic cells are human cells.

32. The low immunogenic glial cell of claim 21, wherein the low immunogenic glial cell comprises a safety switch comprising a suicide gene.

33. The low immunogenic glial cell of claim 32, wherein the suicide gene: i. is the herpes simplex virus thymidine kinase (HSV-tk) gene, ii. is the Escherichia coli cytosine deaminase (EC-CD) gene, or iii. Encodes an inducible caspase protein.

34. The low immunogenic glial cell of claim 21, wherein the low immunogenic glial cell is differentiated from a pluripotent stem cell.

35. The low immunogenic glial cell of claim 34, wherein the pluripotent stem cell is an embryonic stem cell or an induced pluripotent stem cell (iPSC).

36. Use of the low immunogenic glial cell population of claim 19 or 20 in the preparation of a medicament for use in a method of treating a patient suffering from a neurodegenerative disease or condition, the method comprising administering the low immunogenic glial cell population to the patient.

37. The use of claim 36, wherein the medicament further comprises a therapeutically effective carrier.

38. The use of claim 36, wherein the low immunogenic glial cell population is on a biodegradable scaffold.

39. The use of claim 36, wherein administration comprises implantation or injection.

40. The use of claim 36, wherein the neurodegenerative disease or condition is Huntington's disease.

41. Use of the low immunogenic glial cell population of any one of claims 21 to 35 in the preparation of a medicament for use in a method of treating a patient suffering from a neurodegenerative disease or condition, the method comprising administering the low immunogenic glial cell population to the patient.

42. The use of claim 41, wherein the medicament further comprises a therapeutically effective carrier.

43. The use of claim 41, wherein the low immunogenic glial cell population is on a biodegradable scaffold.

44. The use of claim 41, wherein administration comprises implantation or injection.

45. The use of any one of claims 41 to 44, wherein the neurodegenerative disease or condition is Huntington's disease.

46. ​​A hypoimmune cardiac cell differentiated from a hypoimmune pluripotent stem cell (HIP cell), comprising: i. one or more genetic alterations that reduce the surface expression of functional class I human leukocyte antigen (HLA-I) complexes compared to wild-type cells of the same type of low immunogenic cells, and One or more genetic alterations that reduce surface expression of functional class II human leukocyte antigen (HLA-II) complexes compared to said wild-type cells; and ii. One or more genetic alterations that increase the surface expression of functional CD47 protein compared to said wild-type cells.

47. Hypoimmune endothelial cells differentiated from hypoimmune pluripotent stem cells (HIP cells), comprising: i. one or more genetic alterations that reduce the surface expression of functional class I human leukocyte antigen (HLA-I) complexes compared to wild-type cells of the same type of low immunogenic cells, and One or more genetic alterations that reduce surface expression of functional class II human leukocyte antigen (HLA-II) complexes compared to said wild-type cells; and ii. One or more genetic alterations that increase the surface expression of functional CD47 protein compared to said wild-type cells.

48. Hypoimmune dopaminergic neurons (DN) differentiated from hypoimmune pluripotent stem cells (HIP cells), comprising: i. one or more genetic alterations that reduce the surface expression of functional class I human leukocyte antigen (HLA-I) complexes compared to wild-type cells of the same type of low immunogenic cells, and One or more genetic alterations that reduce surface expression of functional class II human leukocyte antigen (HLA-II) complexes compared to said wild-type cells; and ii. One or more genetic alterations that increase the surface expression of functional CD47 protein compared to said wild-type cells.

49. A hypoimmune retinal pigment epithelium (RPE) differentiated from a hypoimmune pluripotent stem cell (HIP cell), comprising: i. one or more genetic alterations that reduce the surface expression of functional class I human leukocyte antigen (HLA-I) complexes compared to wild-type cells of the same type of low immunogenic cells, and One or more genetic alterations that reduce surface expression of functional class II human leukocyte antigen (HLA-II) complexes compared to said wild-type cells; and ii. One or more genetic alterations that increase the surface expression of functional CD47 protein compared to said wild-type cells.

50. Low immunogenic dopaminergic neurons (DN) differentiated from the low immunogenic neural stem cells of claim 1.

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