WO2015153586A1 - Traitements utilisant des cellules souches pluripotentes pour les rétinopathies ischémiques - Google Patents

Traitements utilisant des cellules souches pluripotentes pour les rétinopathies ischémiques Download PDF

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WO2015153586A1
WO2015153586A1 PCT/US2015/023553 US2015023553W WO2015153586A1 WO 2015153586 A1 WO2015153586 A1 WO 2015153586A1 US 2015023553 W US2015023553 W US 2015023553W WO 2015153586 A1 WO2015153586 A1 WO 2015153586A1
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ipsc
vascular progenitors
derived
progenitors
hipsc
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Elias T. Zambidis
Gerard LUTTY
Tea Soon Park
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The Johns Hopkins University
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
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Definitions

  • the present invention relates to the field of stem cells. More specifically, the present invention provides methods and compositions for using pluripotent stem cells to treat ischemic retinopathies.
  • the human retina's high metabolism makes it uniquely reliant on an intact, functional vasculature.
  • Photoreceptors consume the highest amount of oxygen per gram of tissue in the body, and thus require a continuous source of oxygenated blood.
  • the human ophthalmic artery supplies 20% of its blood to the retina and ⁇ 80% to the choroid and uveal tract. If either the retinal or choroidal vasculature becomes compromised, neurons in affected ischemic areas rapidly die. Following branch vein occlusion and during the course of diabetic retinopathy, ischemia results in retinal pericyte and endothelial cell (EC) death. This leads to acellular vascular segments, rapid death of retinal neurons, secondary inflammation, and further retinal damage from subsequent compensatory neovascularization.
  • EC endothelial cell
  • the regeneration of retinal capillaries with cellular therapies could reverse the ischemic death of retinal neurons, and potentially ameliorate or prevent end-stage blindness in disorders such as diabetic retinopathy and branched vein occlusion.
  • vascular progenitors VPs
  • retinal photoreceptors could be differentiated synchronously from patient-specific human induced pluripotent stem cells for comprehensive regeneration of the damaged retina.
  • cord blood cells offer an especially attractive universal donor source for generating human induced pluripotent stem cells, because they carry few somatic mutations, and they can more efficiently generate nonviral, clinically relevant pluripotent stem cell lines that could theoretically be assembled to create a human leukocyte antigen-defined stem cell bank via worldwide networks of existing repositories.
  • embryonic VP population differentiated from human induced pluripotent stem cells that can functionally integrate long-term into ischemia-damaged mouse retinal vasculature.
  • the present invention provides methods for treating ischemic conditions.
  • the ischemic conditions are ischemic eye diseases.
  • a method for treating ischemic retinopathy in a subject comprising administering a therapeutically effective amount of human induced pluripotent stem cells (hiPSC)-derived vascular progenitors.
  • the hiPSC-derived vascular progenitors comprise cord blood induced pluripotent stem cells (CB-iPSC)-derived vascular progenitors.
  • the CB-iPSC-derived vascular progenitors comprise nonintegrated CB-iPSC-derived vascular progenitors.
  • the CB-iPSC-derived vascular progenitors comprise one or more of CD31+CD146- vascular progenitors; CD31+CD146+ vascular progenitors; CD31-CD146- vascular progenitors; and CD31-CD146+ vascular progenitors.
  • the CB-iPSC-derived vascular progenitors comprise one or more of CD31+CD146- vascular progenitors; and CD31+CD146+ vascular progenitors.
  • the CB-iPSC-derived vascular progenitors comprise CD31+CD146+ vascular progenitors.
  • the ischemic retinopathy is branch vein occlusion. In other embodiments, the ischemic retinopathy is diabetic retinopathy.
  • the therapeutically effective amount of hiPSC-derived vascular progenitors are administered by injection via orbital sinus.
  • the present invention also provides a method for treating diabetic retinopathy in a subject comprising the step of administering a therapeutically effective amount of
  • the present invention also provides a method for treating branch vein occlusion in a subject comprising the step of administering a therapeutically effective amount of CD31+CD146+ vascular progenitors via the orbital sinus.
  • a method for treating ischemic retinopathy in a subject comprises administering a therapeutically effective amount of nonintegrated CB-iPSC-derived vascular progenitors.
  • the present invention also provides methods for the generation of novel vascular progenitors from high fidelity human pluripotent stem cells and the use of such vascular progenitors to treat ischemic retinopathy.
  • the methods for the generation of novel vascular progenitors are described herein.
  • FIG. 1 Efficient generation of embryonic VP populations from hPSCs.
  • A Schema for vascular differentiation (diff) and expansion of VP.
  • C Percentage Dil-Ac-LDL uptake (mean ⁇ SEM) of FACS-purified and EGM2-expanded populations differentiated from 2 hESC lines (gold), 3 CB-iPSC lines (red), and 6 fibroblast-iPSC lines (green). Each data point represents an independent, replicate experiment.
  • D In vitro Matrigel assays of purified and expanded populations.
  • E Representative Matrigel plugs consisted of vascular structures formed by indicated CB-iPSC-6.2 hEB-derived populations, and immunostained with anti-CD31 (brown). Scale bars, 100 um.
  • F Measurements in Matrigel plug sections: Left, percentages of blood vessels >30 ⁇ diameter per microscopic field (mean ⁇ SEM); Right, total number of blood vessels per microscopic field (mean ⁇ SEM) (2- tailed t tests; *P ⁇ 0.05; ** P ⁇ 0.01).
  • AM indicates adaptation medium; CB, cord blood; Dil- Ac-LDL, Dil-acetylated low-density lipoprotein; EGM2, endothelial growth medium-2;
  • FACS fluorescence-activated cell sorting
  • hEB human embryoid body
  • hESC human embryonic stem cell
  • hPSC human pluripotent stem cell
  • iPSC induced pluripotent stem cell
  • LDM liquid differentiation medium
  • MM methylcellulose medium
  • SEM standard error of the mean
  • VEGF vascular endothelial growth factor
  • VP vascular progenitor
  • B Phase-contrast image of FACS-purified, expanded CD31+CD146+ VP cells differentiated from CB-iPSC-6.2, with Ulex europaeus agglutinin (UEA) and DAPI costaining (C), and with Dil-Ac-LDL uptake staining (D).
  • E Percentage Dil-Ac-LDL uptake (meaniSEM) of expanded CD31+CD146+ VPs from individual differentiations of hESC (gold), CB-iPSC (red), and fibroblast-iPSC (green). Mann- Whitney tests: * P ⁇ 0.05.
  • F TEM image CB-iPSC-derived VPs forming vascular tubes in collagen gel via cooperating endothelial and pericytic-like cells; all border on lumens and are potential ECs in apparent bifurcation.
  • G Representative surface marker analyses of FACS-purified/expanded hPSC- derived CD31+CD146+ VPs and HUVECs.
  • CB indicates cord blood; Dil-Ac-LDL, Dil- acetylated low-density lipoprotein; FACS, fluorescence-activated cell sorting; fibro, fibroblast; hEB, human embryoid body; hESC, human embryonic stem cell; hPSC, human pluripotent stem cell; HUVEC, human umbilical vein endothelial cell; iPSC, induced pluripotent stem cell; L, lumen; n, nuclei; SEM, standard error of the mean; TEM, transmission electron microscopy; and VP, vascular progenitor.
  • Dil-Ac-LDL Dil- acetylated low-density lipoprotein
  • FACS fluorescence-activated cell sorting
  • fibro fibroblast
  • hEB human embryoid body
  • hESC human embryonic stem cell
  • hPSC human pluripotent stem cell
  • HUVEC human umbilical vein endothelial cell
  • FIG. 3 Expression signatures of hPSC-derived VPs.
  • B Heatmap-dendrogram of Illumina expression array data of the vascular lineage-specific genes indicated.
  • ANOVA indicates analysis of variance
  • CB cord blood
  • Dil-Ac-LDL Dil-acetylated low- density lipoprotein
  • Fib fibroblast
  • H hematopoietic-specific genes
  • hEB human embryoid body
  • hESC human embryonic stem cell
  • HMVEC human microvascular endothelial cell
  • hPSC human pluripotent stem cell
  • HUVEC human umbilical vein endothelial cells
  • iPSC induced pluripotent stem cell
  • P pluripotency-specific genes
  • Q-RT-PCR quantitative realtime polymerase chain reaction
  • VASCULAR vascular lineage-specific genes
  • VP vascular progenitor
  • vWF von Willebrand factor
  • FIG. 4 Senescence and DNA damage sensitivity of expanded VPs.
  • A Representative ⁇ -galactosidase senescence staining in hPSC classes; legend symbols are as before. Scale bars, 100 um.
  • CB indicates cord blood; fibro, fibroblast; hESC, human embryonic stem cell; iPSC, induced pluripotent stem cell; SEM, standard error of the mean; and VP, vascular progenitor.
  • FIG. 5 In vivo migration, homing, and engraftment of luciferase-transgenic VP cells into I/R-damaged mouse retina.
  • A Experimental design for quantifying human VP engraftment into NOD-SCID mouse retinas. Left, Anatomy of mouse eye indicating I/R location at anterior chamber and site of human cell injections into vitreous body. Right, Timeline of in vivo engraftment analysis.
  • B Representative immunofluorescent retinal sections of I/R-damaged eyes injected with hESC-luciferase-transgenic (green)
  • CD31+CD146+ VP left
  • CD31+CD146- right cells at 3 days post-injection.
  • CD31+CD146+ cells readily migrated into deep retinal layers, whereas CD31+CD146- cells remained primarily in vitreous.
  • D CD31+CD146+ hESC-VP injected to I/R-damaged (injury, left) and normal eye (no injury, right) demonstrating that cells do not migrate into retinal layers without injury signals.
  • hESC indicates human embryonic stem cell
  • ILM internal limiting membrane
  • iPSC induced pluripotent stem cell
  • I/R ischemia-reperfusion
  • NOD-SCID nonobese diabetic-severe combined immunodeficient
  • ONH optic nerve head
  • PR photoreceptor
  • VB vitreous body
  • VP vascular progenitor
  • FIG. 6 In vivo engraftment of CB-iPSC-VP into retinal vasculature. Luciferase- transgenic human VPs were injected directly into the eye (intra vitreal; A through H), or systemically intravenously (orbital sinus; I). G, Transgenic CB-iPSC-VP (green) homed to both damaged capillaries (arrows) and larger blood vessels (retinal flat mounts). C, Damaged host vessels lacked murine ECs (lack of blue signal from anti-mouse CD31), but their Coll IV+ basement membranes remained intact (red).
  • FIG. 7 Quantification of human VP engraftment into murine retinal vasculature.
  • A Representative experiments (from Tables 1 and 2) demonstrating CB-iPSC-VP (green) engrafting into murine vessels following orbital sinus (OS) or tail-vein (TV) injections. Scale bars, 20 ⁇ .
  • B Representative experiments showing detection of CB-iPSC-VP injected via orbital sinus (left) or tail vein (right). Retinas were harvested at post-injection day 7, and whole flat-mount retinas were scanned and human cells were quantified in superficial (near vitreous body, red) and deep retinal vasculatures (blue) layers.
  • C Representative experiments (from Tables 1 and 2) demonstrating CB-iPSC-VP (green) engrafting into murine vessels following orbital sinus (OS) or tail-vein (TV) injections. Scale bars, 20 ⁇ .
  • B Representative experiments showing detection of CB-iPSC-VP injected via orbital sinus (
  • FIG. 8 Diagram showing use of myeloid progenitors for clinically safe hiPSC-based therapies.
  • FIG. 9. Summary of experimental design for reprogramming of somatic target cells.
  • FIG. 10. Flow cytometry results of stem cell markers over time in cord blood cells.
  • FIG. 11 Generation of non-integrated 7F episomal hiPSC from norma! adult hair follicle-derived keratinocytes.
  • Colonies with hESC-like morphology, and expressing pluripotency markers e.g., SSEA4, Tra- 1-60/81, CD90, OCT4, NANOG, SOX2
  • pluripotency markers e.g., SSEA4, Tra- 1-60/81, CD90, OCT4, NANOG, SOX2
  • KER-iPSC were further subcloned, and confirmed for lack of integrated episomal. sequences by RT-PCR.
  • FIG. 12 Generation of non-integrated 4F and 7F episomal CB-iPSC lines.
  • H&E stains of cystic teratomas obtained from a representative CB- iPSC line 6-8 weeks following injection into NOD/SCID mice illustrate well-differentiated ceil lineages of all three germ layers, including regions containing neural rosettes, pigmented retinal epithelium, glandular epithelium, fetal intestinal structures, cartilage, striated muscle, and hyalinized bone.
  • FIG. 13 Stromal-primed CBiPSC have been shown to differentiate to functional photoreceptors with superior efficiencies.
  • FIG. 14 Stromal-primed CBiPSC lack the lineage skewing typical of most human pluripotent stem cells.
  • FIG. 15. Procedure and pictures of differentiation procedure of pluripotent stem cells into vascular progenitors.
  • FIG. 16 Murine model for retinal ischemic disease.
  • FIG. 17 Representative experiments showing detection of CB-iPSC-VP injected via orbital sinus. Retinas were harvested at post-injection day 7, and whole flat-mount retinas were scanned and human cells were quantified in superficial (near vitreous body, red) and deep retinal vasculatures (blue) layers. Representative quantification of a retinal engraftment experiment demonstrated that systemic injections attracted higher numbers of homing CD31+CD146+ VPs into damaged deep retinal blood vessels (OS>TV).
  • CB indicates cord blood; Coll IV, collagenase type IV; iPSC, induced pluripotent stem cell; and VP, vascular progenitor.
  • FIG. 18. Table showing characteristics of primed vs. naive pluripotent states.
  • FIG. 19. Summary of research groups who have generated human mESC-like pluripotent states.
  • FIG. 20 Procedure for generating non-integrated blood-derived iPSC lines with ground state na ' ive pluripotency.
  • FIG. 21 Images and flow cytometry showing na ' ive human pluripotent state possess augmented VP differentiation capacities.
  • FIG. 22 Generation of cGMP-grade bank of HLA-defined universal donor N-hiPSCs for vascular regenerative medicine.
  • FIG. 23 Most common HLA allele haplotypes among NMDP donors.
  • the human retinal vasculature arises from embryonic angioblasts that differentiate into vascular endothelial cells and pericytes during fetal development but lose their regenerative ability during adult life.
  • vascular progenitors VP
  • hiPSC patient-specific human induced pluripotent stem cells
  • This discovery has wide utility for autologous regeneration of blood vessels in ischemic eye diseases including ischemic diabetic retinopathy (DR).
  • DR ischemic diabetic retinopathy
  • Pluripotent stem cells offer unrivaled advantages over adult cell therapies.
  • diseases such as DR may ultimately require not only repair of the retinal vascular niche, but also regeneration of multiple other damaged cell types (e.g., beta islet cells;
  • hiPSC-based therapies could revitalize adult donor progenitors, which may have been rendered defective in their adult diseased environment, by reprogramming them back to a pristine, non-diseased embryonic state.
  • this approach will rely on the derivation of high-quality patient-specific hiPSCs with superior differentiation capacities.
  • these high quality hiPSC generate human embryonic VP with augmented capacity for regenerating the retinal vasculature in a humanized NOD-SCID ischemia/reperfusion (I/R) model 7. More interestingly, these hiPSC lines were recently shown to be capable of generating functionally mature photoreceptors with unsurpassed efficiency in a three-dimensional retinal cup differentiation system. See International Application No. PCT/US2015/011701.
  • Efficient generation of functional vascular progenitors in parallel with differentiation of retinal photoreceptors from the same patient-specific hiPSC line could be used to comprehensively treat ischemic eye diseases including diabetic retinopathy.
  • Such hiPSC- based therapies could potentially revitalize adult vascular progenitors, which are often rendered defective in their diabetic environment, by reprogramming them back to a pristine, non-diseased embryonic state.
  • vascular progenitors from human induced pluripotent stem cells (hiPSCs) has great potential for treating vascular disorders such as ischemic retinopathies.
  • hiPSCs human induced pluripotent stem cells
  • long-term in vivo engraftment of hiPSC-derived VPs into the retina has not yet been reported. This goal may be limited by the low differentiation yield, greater senescence, and poor proliferation of hiPSC-derived vascular cells.
  • VPs a repertoire of viral-integrated and nonintegrated fibroblast and cord blood (CB)-derived hiPSC lines and tested their capacity for homing and engrafting into murine retina in an ischemia-reperfusion model.
  • VPs from human embryonic stem cells and hiPSCs were generated with an optimized vascular differentiation system. Fluorescence-activated cell sorting purification of human embryoid body cells differentially expressing
  • endothelial/pericytic markers identified a CD31+CD146+ VP population with high vascular potency.
  • Episomal CB-induced pluripotent stem cells iPSCs
  • iPSCs Episomal CB-induced pluripotent stem cells
  • CB- iPSC-VPs maintained expression signatures more comparable to human embryonic stem cell VPs, expressed higher levels of immature vascular markers, demonstrated less culture senescence and sensitivity to DNA damage, and possessed fewer transmitted reprogramming errors.
  • Luciferase transgene-marked VPs from human embryonic stem cells, CB-iPSCs, and fibroblast-iPSCs were injected systemically or directly into the vitreous of retinal ischemia- reperfusion-injured adult nonobese diabetic-severe combined immunodeficient mice. Only human embryonic stem cell- and CB-iPSC-derived VPs reliably homed and engrafted into injured retinal capillaries, with incorporation into damaged vessels for up to 45 days.
  • VPs generated from CB-iPSCs possessed augmented capacity to home, integrate into, and repair damaged retinal vasculature.
  • hESC lines HI WA01
  • H7 WA07
  • H9 WA09
  • ES03 ES03
  • day 8 human embryoid body cells were disaggregated by using collagenase type IV (1 mg/mL, Sigma- Aldrich, St Louis, MO), and plated onto fibronectin (10 ⁇ g/mL, Life Technologies, Grand Island, NY)-coated plates in endothelial growth medium-2 (EGM2, Lonza, Walkersville, MD) supplemented with 25 ng/mL vascular endothelial growth factor (VEGF)165 (Peprotech, Rocky Hill, NJ).
  • endothelial growth medium-2 ECM2, Lonza, Walkersville, MD
  • VEGF vascular endothelial growth factor
  • adherent human embryoid body-derived cells were treated with 0.05%
  • Ocular Ischemic Reperfusion Injury and Human Cell Injections Four- to 6-week old male nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice (Jackson Laboratory, Bar Harbor, ME) were subjected to high intraocular pressure to induce retinal ischemia reperfusion (I/R) injury. Mice were deeply anesthetized by intraperitoneal injection of ketamine/xylazine (50 mg/kg ketamine+10 mg/kg xylazine in 0.9% NaCl).
  • the pupils were dilated with 2.5% phenylephrine hydrochloride ophthalmic solution (AK-DILATE, Akorn, Buffalo Glove, IL) followed by 0.5% tetracaine hydrochloride ophthalmic topical anesthetic solution (Phoenix Pharmaceutical, St. Joseph, MO).
  • the anterior chamber of the eye was cannulated under microscopic guidance (OPMI VISU 200 surgical microscope; Zeiss, Gottingen, Germany) with a 30-gauge needle connected to a silicone infusion line providing balanced salt solution (Alcon Laboratories, Fort Worth, TX), avoiding injury to the corneal endothelium, iris, and lens.
  • Retinal ischemia was induced by raising intraocular pressure of cannulated eyes to 120 mm Hg for 90 minutes by elevating the saline reservoir. Ischemia was confirmed by iris whitening and loss of the retinal red reflex. Anesthesia was maintained with 2 doses of 50 intramuscular ketamine (20 mg/mL) during the 90 minutes. The needle was subsequently withdrawn, the intraocular pressure was normalized, and reperfusion of the retinal vessels was confirmed by the reappearance of the red reflex. The contralateral eye of each animal served as a nonischemic control. Antibiotic ointment (Bacitracin zinc and Polymyxin B sulfate, AK-Poly-Bac, Akron) was applied topically.
  • FACS-purified human VPs were injected into either the vitreous body (50 000 cells in 1 ⁇ /eye), the orbital sinus (100 000 cells in 2 eye) by using a microinjector (PLI-100, Harvard Apparatus, HoUiston, MA), or into the tail vein (300 000 cells/100 per mouse).
  • BMSC bone marrow mesenchymal stromal cells
  • Sp- CB-iPSC colonies were manually subcloned at ⁇ 3 weeks post-nucleofection in hESC medium supplemented with 10-20 ng/mL of bFGF.
  • Non-integrated 7F episomal fibroblast- iPSC (7ta) and lentivirally-generated 4F fibroblast-iPSC lines IMR90-1, IMR90-43
  • hESC and hiPSC Culture All hESC and hiPSC lines used in these studies were maintained and expanded in undifferentiated states on irradiated MEF, as previously described5, 6. MEF were derived from mouse embryos (E12.5-13.5) generated from CF1 (Charles River, Wilmington, MA) and DR4 (The Jackson Laboratory, Bar Harbor, ME) mouse matings. High quality, early passage ( ⁇ P2) MEF were used for iPSC generation and thawing of hESC/iPSC.
  • hPSC Human pluripotent stem cells
  • hESC medium comprising of DMEM/F12 supplemented with 20% Knockout Serum Replacer (KSR), 0.1 mM MEM-non-essential amino acid (MEM-NEAA), 1 mM L-glutamine, 0.1 mM beta- mercaptoethanol, and 4-20 ng/mL FGF-2 (all Life Technologies, Grand Island, NY) (Table 3).
  • KSR Knockout Serum Replacer
  • MEM-NEAA MEM-non-essential amino acid
  • FGF-2 all Life Technologies, Grand Island, NY
  • Spontaneously differentiating colonies were routinely removed manually from cultures by micropipette- ablation using an inverted microscope (EVOS, Life Technologies) in a laminar flow cabinet (MidAtlantic Diagnostics Inc., Mount Laurel, NJ).
  • Pluripotent stem cells were passaged in bulk enzymatically every 6-7 days using a solution of collagenase type-IV (1 mg/mL in DMEM/F12, 5 min, 37°C, Sigma, St. Louis, MO), or manually dissected using a 10 ⁇ , micropipette (Eppendorf, Hamburg, Germany) under EVOS microscope.
  • Luciferase Transgene+ hPSC Lines A humanized luciferase cDNA optimized for expression was acquired from pGL4.13 (Promega), and subcloned into lentivector construct pWPT (Addgene) containing a modified EF-1 alpha promoter and a puromycin selection cassette for stable, constitutive expression in hESC/hiPSC. High titer lentiviral supernatants were prepared via standard methods.
  • hESC and hiPSC Prior to lentiviral transduction, hESC and hiPSC were plated onto Matrigel pre-coated plate (BD Biosciences, 1 : 100, 1 hr at 37°C) in MEF conditioned medium (CM) supplemented with 10 ng/ml FGF-2.
  • CM MEF conditioned medium
  • pluripotent stem cells were -20-30% confluent (2nd day of passage)
  • medium was replaced with 1 mL fresh CM containing high titer luciferase lentivirus and diluted polybrene (final concentration 6 ⁇ g/mL, Life Technologies), incubated for 8 hours, and then replaced with fresh CM, and changed daily until hESC/hiPSC were -70-80% confluent (5th day of passage).
  • Stably-integrated hESC/hiPSC clones were selected with puromycin (0.5-1.0 ⁇ g/mL) for 2 days, and puromycin-resistant colonies were expanded on DR4-MEF (9th day).
  • Stable luciferase transgene+ hESC/hiPSC clones were routinely maintained in hESC medium containing 0.5 ⁇ g/mL puromycin until vascular differentiation.
  • VP Vascular Progenitors
  • hEB differentiation Hemato-endothelial differentiations of hEB with BMP4, VEGF, FGF2/heparin (BVF2H) were performed essentially as described7, 8 which is modified and optimized from our original method5.
  • hESC and hiPSC lines with >95%> undifferentiated morphology were expanded on gelatinized 6-well tissue culture plates to 80-90%) confluency.
  • Culture medium was switched to adaptation medium (AM, Table 5) for one day prior to hEB generation.
  • day 1 see FIG. 1A
  • hPSC were treated with 2 mg/mL dispase (Sigma Aldrich) in DMEM/F12 for 5 min at 37°C.
  • DMEM/F12 DMEM/F12
  • a cell scraper Sarstedt, Newton, NC
  • hPSC clumps were resuspended in an ample amount of DMEM/F12 medium.
  • Cell suspensions were centrifuged (300 g, 5 min, room
  • hEB clumps were collected in ample amount of phosphate buffer saline (PBS, Life Technologies), centrifuged at 300 g for 5 min at room temperature, washed once more in PBS, and re- suspended in liquid differentiation medium (LDM, Table 7). The hEB/LDM suspension was returned to the original ultra-low attachment plate, and further incubated in semi-hypoxic conditions. hEB were collected every 2-3 days, centrifuged at 200g, 1 min and re-suspended in fresh LDM.
  • PBS phosphate buffer saline
  • LDM liquid differentiation medium
  • hEB clumps were collected using a pi 000 Pipetman following gentle disaggregation in MEF medium, centrifuged 200g, 5 min, then plated (-1x105 cells/well) onto fibronectin (10 ⁇ g/mL/well)- coated plates in endothelial growth medium 2 (EGM2, Lonza) supplemented with 25 ng/mL VEGF.
  • day 1 cells were replenished with fresh EGM2 supplemented with 25 ng/mL VEGF and the medium was changed every 2 days.
  • Adherent hEB cells were harvested from EGM2 cultures for FACS purification of VP populations with 0.05% trypsin- EDTA (Life Technologies) 4-5 days later.
  • EGM2-cultured hEB cells were washed once in PBS, and enzymatically digested with 0.05% trypsin-EDTA (5 min, 37°C). Enzymatic activity was neutralized using MEF medium, and the cell suspension was filtered through a 40 ⁇ cell- strainer (Fisher Scientific, Pittsburgh, PA) to eliminate large cell clusters.
  • FIX & PERM reagents Life Technologies
  • All data files were subsequently analyzed offline using Flowjo analysis software (Tree Star Inc., Ashland, OR).
  • TEM Transmission Electron Microscopy
  • Tubes grown in either collagen or Matrigel in 6 well or 96 well tissue culture plates were fixed at room temperature in 2.5% paraformaldehyde/2%gluteraldehyde in 0.1M cacodylate buffer, pH 7.4 for a minimum of 3 hours then stored in fix overnight at 4°C. They were washed in the culture dishes with 0.05M cacodylate buffer, pH 7.4 to remove the fix. The gel drops were teased from the wells using a small spatula. Areas containing tubes were dissected out and placed in small vials of 0.05M cacodylate buffer. The tubes were post fixed for 1.5 hours in 1% osmimum in 0.05M cacodylate, pH 7.4 and washed in cacodylate buffer.
  • Dehydration was accomplished using a grade series of ethyl alcohols followed by lhr en bloc staining with l%uranyl acetate in absolute ethanol. Propylene oxide washes were used to enhance resin infiltration. Final embedding was done in LX112 resin (Ladd Research Industries, Inc., Williston, VT). The blocks were polymerized at 60°C for 48 hours.
  • One micron semi-thin and 72nm ultrathin sections were prepared using a Leica UC7 (Leica Microsystems, Wetzlar, Germany). Sections were imaged with a Hitachi H7600 TEM at 80KV (Gaithersburg, MD) and a side mount AMT CCD camera (Woburn, Mass).
  • Dil-Acetylated Low Density Lipoprotein (Dil-Ac-LDL) Uptake FACS-purified populations based on expression of CD31 and CD146 were expanded for 5-7 days prior to Dil-Ac-LDL (Life Technologies, Cat No. L-3484) uptake assay. Each population was plated on fibronectin pre-coated 6-well plates (1-1.5 x 105 cells/well), and cultured in EGM2 until they reached 60 to 70% confluency. Medium was then switched to fresh EGM2
  • Matrigel was mixed with 5 x 105 cells in a final volume of 200 ⁇ , and the mixture was subcutaneously injected into the mid- lower abdominal region of immunodeficient NOD- SCID mice. After 14 days, the animals were euthanized and dissected to remove the Matrigel plugs. For the purpose of orientation, the abdominal skeletal muscle was removed intact with each Matrigel plug. Plugs were paraffin-embedded, sectioned, and stained with a polyclonal rabbit anti-CD31 (Abeam, Massachusetts, USA) followed by goat anti-mouse horseradish peroxidase secondary antibody and streptavidin horseradish peroxidase.
  • eyes were enucleated and fixed in 2% paraformaldehyde in TBS for 60 min at room temperature. After removing cornea and lens, the retina was carefully separated from the choroid and sclera and permeabilized via incubation with 0.1% Triton-X-100 in TBS solution for 15 min at 4°C.
  • a goat anti-rabbit Cy3 secondary antibody (111-165-003, Jackson Immuno Research, 1 :200) was used to detect collagen IV primary antibody, and a goat anti-rat Alexafluor-647 secondary antibody (A21247, Invitrogen, 1 :200) was used to detect the anti- CD31 primary antibody.
  • Human cells were detected using anti-luciferase-FITC antibody (1 :10, Abeam, Cambridge, MA, Cat no: ab34506, Lot no: 831617 or 765206). After washing in TBS, the flatmount retinas were observed with a confocal microscope LSM510 Meta (Carl Zeiss Inc., Thornwood, NY) in the Wilmer Eye Institute Imaging Core Facility.
  • TUNEL method Retinas for TUNEL analysis were fixed in 2% paraformaldehyde overnight at 40C, washed and incubated with Rabbit anti-collagen IV as described above, and goat anti rabbit-Alexifluor 647 was used as the second step antibody.
  • the retinas were cryopreserved as described above. Eight mm cryosections were processed for detection of TUNEL-positive cells using the In situ Death Detection kit with TM R red indicator (Roche Applied Science, Mannheim, Germany), as suggested by the manufacturer. TUNEL+ cells were detected with a 710 LSCM microscope in relationship to retinal blood vessels.
  • Senescence Assays FACS-purified populations were plated onto 6-well tissue culture plates pre-coated with fibronectin (10 ⁇ g/mL), and passaged in EGM2 3-4 times up to 30 days. Prior to the analysis, cells were plated onto fibronectin coated 12-well plate and expanded to -70-80% confluency. Cells were fixed in 2% paraformaldehyde and stained for ⁇ -galactosidase expression per manufacturer's protocol (Cell Signaling Technology, Danvers, MA) to detect senescent cells. Senescence-positive cells stained blue and were enumerated with inverted microscopy and Nikon NS3.1 software.
  • Protein lysates were separated with SDS-polyacrylamide gel electrophoresis (PAGE) with 4-20% TGX precast gel (Bio-Rad, Hercules, CA) and transferred onto a PVDF membrane. After blocking with 5% BSA in TBS-T, membranes were probed with primary and secondary antibodies. Enhanced chemiluminescence (ECL; GE Healthcare, Pittsburgh, PA) was used for detection of immunoblotted proteins. The antibodies used were monoclonal anti-p53 (1 : 1000) (Cell Signaling Technology, Danvers, MA), anti-P-Actin (1 :2000) (Abeam, Cambridge, MA), and anti-RAD51(1 :200) (Santa Cruz Biotechnology, Santa Cruz, CA). Western blot band densities were quantified using the ImageJ software (NIH).
  • CD31+CD146-, CD31+CD146+, CD31-CD146-, and CD31-CD146+ Four populations were FACSpurified from adherent cells (CD31+CD146-, CD31+CD146+, CD31-CD146-, and CD31-CD146+), further expanded in EGM2 medium, and analyzed for surface coexpression of established hematoendothelial, mesenchymal, pericytic, and smooth muscle cell markers.
  • CD31+ vascular- endothelial populations for high coexpression of CD 146+ enriched a putative VP population that coexpressed mesenchymal stem cell markers (CD44, CD90, CD 105, the
  • C-KIT hematoendothelial progenitor marker CD117
  • a-SMA positive smooth muscle
  • CD31+CD146+ VP generated 2 discrete cellular phenotypes: an endothelial progenitor celllike (CD105hiCD144+CD140b-) population, and a pericytic-like
  • CD31 -CD 105 dimCD 144-CD 140b+ CD31 -CD 105 dimCD 144-CD 140b+ population.
  • Dil-Ac-LDL Matrigel tube-forming and Dil-acetylated low-density lipoprotein
  • CD31+CD146- and CD31+CD146+ populations both formed organized microtubes in Matrigel (and with capillary-like lumens in collagen gels), CD31+CD146+ VPs formed larger diameter and more extensively branching vascular tubes (FIG. ID).
  • CB-iPSCs Generated CD31+CD146+ VPs With Higher Efficiency Than Fibroblast- iPSC.
  • 18 hPSC lines derived via various methods; this included 4 hESC lines (HI, H7, H9, ES03), 4 viral fibroblast-iPSC lines (IMR90-1, IMR90-4, HUF3, HUF5), 4 7F-episomal (7F-E) fibroblast iPSC lines (7ta, WT2, WT4, SF-iPSC 6.1), and 6 episomal CB-iPSC lines (7F-E: 6.2, 6.13, 19.11; 4F-episomal (4F-E): E5C3, E12C5, E17C6).
  • CB-iPSC As a group produced significantly (PO.01) higher frequencies of CD31+CD146+ VP cells than fibroblast-iPSC (FIG. 2A).
  • These hiPSC- derived VP populations displayed morphologies similar to neonatal EC (e.g., human umbilical vein endothelial cells [HUVECs]), and readily took up Dil-Ac-LDL and
  • Purified CB-iPSC-VPs expanded for several weeks also maintained higher expressions of VP markers (e.g., CD31, CD 146, KDR [VEGFR2], CD90, and CD 144 [VE-Cadherin]), and stably maintained their capacity for generating branching, sprouting endothelial-pericytic microvascular tubes with capillary-like lumens in collagen gels (FIG. 2F and 2G).
  • VP markers e.g., CD31, CD 146, KDR [VEGFR2], CD90, and CD 144 [VE-Cadherin]
  • vascular lineage-specific genes A focused expression analysis of vascular lineage-specific genes by microarray revealed that VPs generated from CB-iPSC shared more congruence in their vascular expression signatures39 (Table 10) with hESC-VPs than did VPs from fibroblast-iPSC (FIG. 3B and 3C).
  • CB-iPSC-VP populations expressed higher transcript levels of endothelial-specific, perivascular/ pericytic-specific genes (e.g., PDGFRb [CD 140b]), and adhesion/ migration-specific genes (e.g., integrin a5) than other hPSC classes (FIG. 3B, Table 9).
  • VPs differentiated from hESC and CB-iPSC expressed higher transcript levels of immature endothelial progenitor markers such as TIE1 and TIE2, and lower expressions of mature endothelial transcripts (e.g., von
  • CB-iPSC-VP Demonstrated Reduced Senescence and Sensitivity to DNA Damage.
  • Previous studies with standard viral fibroblast-iPSC lines reported significantly diminished and highly variable directed differentiation to the vascular-endothelial lineage.29 For example, differentiations of fibroblast-iPSC were characterized by poor growth and expansion of vascular-endothelial cells, with high rates of apoptosis and early senescence.
  • purified VP differentiated from hESC, fibroblast-iPSC, and CB-iPSC were expanded in EGM2 for up to 30 days ( ⁇ 4 passages) and senescent cells were quantified via ⁇ -galactosidase activity (FIG.
  • CB-iPSC-derived VP protein levels of RAD51 which also plays an important role in mediating repair from double stranded DNA damage following irradiation, was also more comparable to hESC-derived VPs (data not shown).
  • luciferase+ human VP cells were injected directly into the vitreous body of NOD-SCID recipient eyes 2 days following I/R injury, and human cell engraftment in murine retina was evaluated at 3, 7, 14, 21, and 45 days later with antiluciferase immuno fluorescent staining (FIG. 5A).
  • hPSC-derived CD31+CD146- cells failed to migrate efficiently into I/R-damaged retina and home to blood vessels, and instead remained in the vitreous body, or adherent to the superficial layer of retina adjacent to vitreous (FIG. 5B, right).
  • CD31+CD146+ VPs migrated efficiently through the deep retinal layers, and homed and incorporated readily into blood vessels (FIG. 5B, left; FIG. 5D, left).
  • the migration distance and cell numbers of these CD31+CD146+ and CD31+CD146- populations were
  • CD31+CD146+ cells not only migrated longer distances into the deeper retinal layers ( ⁇ 5-fold; ⁇ 0.05), but also higher numbers of cells were detected in comparison with CD31+CD146- cells ( ⁇ 5-fold; ⁇ 0.05).
  • CD31+CD146+ VP cells expressed higher levels of CXCR4 than
  • CD31+CD146- cells before vitreal injections An analysis of migration of CXCR4+
  • CD31+CD146+ VP cells into I/R-damaged eyes versus normal noninjured control eyes revealed that homing to retinal vessels depended on vascular damage: CD31+CD146+ VP stayed adjacent to the internal limiting membrane in eyes without injury signals (FIG. 5D, right; Tables 1 and 2).
  • FIG. 5E shows that robust homing of CD31+CD146+ VPs from both hESC and CB-iPSC into the retinal vasculatures was observed as early as 3 days following vitreal injection (FIG. 5E, Tables 1 and 2).
  • CD31+CD146+ VPs from fibroblast-iPSC poorly homed and engrafted into retinal vessels in I/R-damaged eyes in comparison with CB-iPSC (Tables 1 and 2).
  • n number of ind ⁇ ident experifnefifs
  • 3 ⁇ 4B fiat done
  • R cells engraied refei blood vesseis ⁇ , vascu!s progenitor
  • + 1-2 cells detected sngrafterf per r icrcscopiG field
  • ++ >2 cdk detected engrafted per microscopic ieid.
  • CB-iPSC-VPs Efficiently Engrafted Into Damaged Retinal Blood Vessels After Local and Systemic Injection for at Least 45 Days.
  • retinal vessel damage increases over time following I/R injury.
  • the retinal vasculature was stained with an antibody specific to mouse anti-CD31 , and the vascular basement membrane was labeled with a murine anticollagen type IV antibody.
  • This method demonstrated that blood vessel branches lost viable ECs as early as 7 days post-I/R with the formation of acellular collagen tubelike capillary structures4. This damage was more severe in capillaries and veins presumably owing to their higher collapsible or compressible nature under high intraocular pressure in comparison with arteries.
  • VPs injected into vitreous body initially assumed ab luminal (pericytic) positions at early post-injection days 3 and 7 (FIG. 5E, arrows; FIG. 6A through 6C, arrows). Cryopreservation and sectioning of these retinas demonstrated stable enwrapping of the retinal blood vessels by human VP cells (FIG. 6D and 6F, arrows).
  • mice were systemically perfused before the collection of retinas.
  • CB-iPSC-VPs were clearly detected engrafting into both luminal endothelial and abluminal pericytic locations (FIG. 6G). Although hESC-VPs could be found
  • CB-iPSC-VPs consistently demonstrated more specific engraftment into blood vessels (Tables 1 and 2).
  • CB-iPSC-VPs appeared to favor venous engraftment (blood vessels with larger diameter) than arteries (blood vessels with smaller diameter and more rigid walls), suggesting again that these cells preferentially migrated in response to injury signals.
  • human cells were observed primarily in luminal locations in murine host retinal capillaries (FIG. 6H, FIG). Chimeric capillaries on both linear, and branch point vascular segments, as well, were detected, suggesting an injury- induced vasculogenesis, but that had no short-term impact on I/R-degenerated neuronal viability.
  • CB-iPSC-VP via orbital sinus or tail vein injection resulted in robust engraftment in endothelial or luminal cell positions that could be detected for at least 45 days (FIG.s 61 and 7, Tables 1 and 2).
  • Intravenously injected human cells were detected and quantified in both the superficial capillary layer and the deeper retinal vascular networks, as well (FIG. 7).
  • CB-iPSC-VPs still homed to damaged retinal vessels in greater numbers ( ⁇ 2.4- fold for orbital sinus and ⁇ 1.6 fold for tail vein) in damaged eyes in comparison with uninjured normal eyes. This long-term engraftment is, to our knowledge, the most durable yet reported for injected hiPSC-derived vascular cells.
  • hPSC-derived embryonic VP population that can integrate long-term into ischemia-damaged mouse retinal vasculature.
  • This study provides a preclinical model for evaluating the potential of patient-specific hiPSC-VP therapies for vascular degenerative disorders such as diabetic retinopathy and branch vein occlusion. Both ocular disorders progress to end-stage death of retinal neurons and subsequent pathological neovascularization. If VPs could be used to repopulate acellular retinal capillaries and regenerate viable blood vessels, areas of ischemia could be reperfused, potentially avoiding the end-stage blinding complications.
  • Such novel vascular therapies will require the efficient nonviral reprogramming of accessible somatic donor cells (e.g., from skin or blood) that can generate hiPSCs with superior vascular differentiation potential.
  • accessible somatic donor cells e.g., from skin or blood
  • Nonintegrated patient- specific hiPSCs could be used to simultaneously generate both retinal neurons and VPs for treating a variety of blinding disorders.
  • hiPSCs Although hESCs and hiPSCs share high molecular similarity, hiPSCs generally possess more variable directed differentiation potencies than hESCs.27-29,31 Incomplete reprogramming and retention of donor-specific epigenetic memory have been proposed as etiologies for poor hiPSC-differentiation potencies, including to vascular-endothelial lineages. In these studies, we found that HF CB-iPSCs derived at very high efficiencies31,32 possessed significantly augmented vascular potency in comparison with fibroblast-hiPSCs derived via standard methods.
  • CB-iPSCs generated CD31+CD146+ VPs that were more akin molecularly to those generated from hESCs, and with significantly fewer aberrant hiPSC-specific genes expressed that are likely attributable to transmitted reprogramming errors.
  • previous studies demonstrated high senescence in vascular lineage cells generated from fibroblast- derived hiPSCs,29 CB-iPSC-VPs expanded more robustly in culture, possessed lower rates of culture senescence, and demonstrated more resistance to DNA damage than fibroblast- iPSCs.
  • CB-iPSC-VPs may possess advantages in survival, migration, and homing to damaged tissues in comparison with fibroblast-iPSC-VPs.
  • Derivation methods with more effective reprogramming capacities may greatly improve the final functional pluripotency of hiPSCs, including to the vascular lineage.
  • Further studies that explore the role of epigenetic memory will ultimately confirm if HF CB-iPSCs will have greater clinical utility for generating multiple transplantable lineages (e.g., neural, vascular, retinal pigmented epithelium) for comprehensive regenerative therapy of blinding ocular diseases.
  • CB-iPSC-derived CD31+CD146+ VPs One important aspect of CB-iPSC-derived CD31+CD146+ VPs was their efficient capacity to home to injured vessels. Despite damage to ischemic acellular capillaries, the basement membrane shared by EC and pericytes in ischemic retinal capillaries is normally spared.
  • stromal-derived factor- 1 (SDF-l)/CXCR4-mediated migration.40
  • SDF-l stromal-derived factor- 1
  • CXCR4 the receptor for hypoxia-inducible SDF-1
  • angioblasts migrate to the inner retina, they continue to express CXCR4 until they differentiate into the ECs that line patent retinal vessel lumens.
  • SDF-1 is prominently localized to the innermost portion of retina during retinal vascular development and displays a gradient toward the outer retina.44 Retinal angioblasts also expressed VEGFR-2/KDR and C-KIT similar to these embryonic VPs.
  • hPSC-derived CD31+CD146+ VP migration, homing, and engraftment in our I/R injury model likely recapitulate the hypoxic events during retinal development, when
  • VPs When directly injected into vitreous, VPs homed to injured blood vessels and engrafted primarily in pericytic positions on the outside of host collagenous vascular tubes. In contrast, VPs delivered intravenously engrafted primarily in endothelial positions. This behavior is reminiscent of early studies in developing retinal vasculature that suggested position in reference to the basement membrane determines the developmental fate of VPs.45
  • the future aim of selectively delivering hiPSC-derived ECs or pericytes (or both) by targeting different routes to the damaged retina could have value in regenerating vascular segments in the diabetic retina where pericytes die before ECs before yielding acellular capillaries.
  • this model establishes an important tool for evaluating the further development of clinically relevant hiPSC-based regenerative therapies for the treatment of ischemic retinopathies.
  • FC Sow c tornefey
  • F.&CS fuoresoent a iated cdt sotting
  • VP embryonic vascular progenitors
  • N ground state naive
  • DR rodent diabetes retinopathy
  • Photoreceptors are nourished by retinal and choroidal vasculatures, and are highly susceptible to ischemia.
  • Pathological retinal changes in diabetic retinopathy include acellular vascularization, inflammation, proliferative neovascularization; BM thickening; and loss of pericytes.
  • ischemic retinopathy Limited sources of cell therapy for ischemic retinopathy are available and include, endothelial progenitor cells purified from peripheral blood and cord blood, mesenchymal stem cells, and CD34+ cells from bone marrow. Myeloid progenitors are ideal donors for clinically safe hiPSC-based therapies. See FIGS. 8-10; Park et al, 7(8) PLOS ONE e42838 (2012).
  • BMSC priming dramatically increases episomal reprogramming efficiency.
  • non-integrated reprogramming requires 7F (SOX2, OCT4, KLF4, MYC, NANOG, LIN28, SV40 T antigen), and efficiency ⁇ 0.001%.
  • BMSC-primed CD34+ hematopoetic progenitors require only 4F (SOX2, OCT4, KLF4, MYC), and efficiency is about 5 - 20% per total input cells.
  • transfection efficiency of CD34+ cells is 10 - 20%> / total input cells, and >50%> reprogramming efficiencies are achieved with transgene -purified populations. See FIG. 11.
  • BMSC-primed CB- iPSC rapidly achieved a high-fidelity state of pluripotency at early passage. See FIG. 12; Park et al, 7(8) PLOS ONE e42838 (2012); and Park, Huo, Zimmerlin et al, in preparation.
  • hiPSC human induced pluripotent stem cells
  • experiments are conducted using a murine model for retinal ischemic disease. See FIG. 16.
  • the present inventors have shown the in vivo homing, migration, and engraftment of luciferase-transgenic VP cells.
  • FIG. 17 Park et al., 129 CIRCULATION 359-72 (2014).
  • Sp-CB-iPSC-derived angioblasts engrafted efficiently in vivo into damaged retinal veins and capillaries.
  • CD31+CD146+ cells from sp-CB-iPSC engrafted primarily on capillaries and veins, which are the most damaged ocular blood vessels in I/R.
  • CD31+CD146+ cells appear to have a predilection for engrafting in a pericytic or ab luminal location when injected intravitreally.
  • Comparisons of various injection methods are currently in progress (e.g., intra- vitreal, orbital sinus, and tail-vein).
  • Functional tests sinum fluorescein angiograms also are in progress.
  • Novel non-integrated blood-derived iPSC lines with ground state na ' ive pluripotency are generated.
  • the present inventors In research aim 2, the present inventors expect to determine the potential of embryonic VP from reprogrammed diabetic blood for re-vascularizing degenerating retinal neurons and improving visual function in a rodent diabetes retinopathy (DR) model.
  • the present inventors generate a cGMP-grade bank of HLA-defmed universal donor N-hiPSCs for vascular regenerative medicine. See FIG. 23.
  • non- integrated sp-CB-iPSC in cGMP conditions are derived. See FIG. 24.
  • Human iPSC with high-fidelity epigenetic signatures can generate embryonic angioblast-like CD31+CD146+CXCR4+ VP that possess augmented abilities to functionally regenerate the vasculature of diseased ischemic retina.
  • the primary aim is to determine the potential of embryonic VP generated from reprogrammed diabetic blood cells for
  • human hematopoietic progenitors are an epigenetically malleable and highly accessible donor source for generating hiPSC with superior re- differentiation potential.
  • Our ultimate goal is to use hiPSC-based technology to reprogram a diabetic patient's own blood cells to self-renewing hiPSCs that can provide unlimited supplies of autologous (or HLA-matched) embryonic VP and photoreceptors for a comprehensive regeneration of the diseased adult diabetic retina.
  • hiPSC-based technology to reprogram a diabetic patient's own blood cells to self-renewing hiPSCs that can provide unlimited supplies of autologous (or HLA-matched) embryonic VP and photoreceptors for a comprehensive regeneration of the diseased adult diabetic retina.
  • high fidelity myeloid-hiPSC derived under conventional as well as unpublished second generation ground state na ' ive pluripotent conditions We will explore the vascular therapeutic potential of diabetic hiPSC lines derived via newly emerging technologies such as stimulus-activated acquisition

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Abstract

La présente invention concerne le domaine des cellules souches. Plus précisément, la présente invention concerne des procédés et des compositions pour l'utilisation de cellules souches pluripotentes dans le but de traiter des rétinopathies ischémiques. On pourrait utiliser, pour traiter d'une manière exhaustive des rétinopathies ischémiques, une production efficace de progéniteurs vasculaires fonctionnels en parallèle avec une différenciation des photorécepteurs rétiniens, à partir de la même lignée de hiPSC spécifique de patient. Ces traitements à base de hiPSC pourraient potentiellement revitaliser les progéniteurs vasculaires adultes, qui sont souvent rendus défectueux dans leur environnement diabétique, par leur reprogrammation à un état embryonnaire vierge non affecté par la maladie.
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KR101991624B1 (ko) * 2018-04-11 2019-06-20 서울대학교산학협력단 당뇨병 개체 유래의 형질전환체, 이의 제조방법 및 이를 포함하는 세포치료제
CN115003794A (zh) * 2019-08-28 2022-09-02 安斯泰来再生医药协会 治疗血管疾病的组合物和方法

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KR101991624B1 (ko) * 2018-04-11 2019-06-20 서울대학교산학협력단 당뇨병 개체 유래의 형질전환체, 이의 제조방법 및 이를 포함하는 세포치료제
CN115003794A (zh) * 2019-08-28 2022-09-02 安斯泰来再生医药协会 治疗血管疾病的组合物和方法

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