WO2019025826A1 - Methods for producing endothelial cells and vascular endothelial constructs derived from induced human pluripotent stem cells - Google Patents

Methods for producing endothelial cells and vascular endothelial constructs derived from induced human pluripotent stem cells Download PDF

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WO2019025826A1
WO2019025826A1 PCT/HU2018/050034 HU2018050034W WO2019025826A1 WO 2019025826 A1 WO2019025826 A1 WO 2019025826A1 HU 2018050034 W HU2018050034 W HU 2018050034W WO 2019025826 A1 WO2019025826 A1 WO 2019025826A1
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expression
cells
population
fold
cell
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PCT/HU2018/050034
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French (fr)
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Gábor Földes
Edit GARA
Béla MERKELY
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Semmelweis Egyetem
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
    • A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells

Definitions

  • the invention relates to a method of providing CD31+ cells suitable for forming 3D vascular construct, said cells expressing an increased amount of angiogenesis related and cell-matrix adhesion related proteins and having an arterial phenotype.
  • Endothelial derivatives of human pluripotent stem cells can be new sources of cells for regenerative therapies in cardiovascular diseases as well as means for drug toxicity and efficacy testing.
  • Tissue-specific variations and phenotypic heterogeneity of endothelial cells (EC) from different sources can however be a challenging part for their translation into the clinic.
  • EC-EC endothelial cells
  • hESC Human embryonic stem cells
  • hESC-derived cardiovascular cells with mesenchymal stem cells (MSC) may enhance the beneficial effects of hESC derivatives.
  • MSC mesenchymal stem cells
  • the first clinical trials with hESC-derivatives in cardiovascular diseases are launched (Transplantation of Human embryonic Stem Cell-derived Progenitors in Severe Heart Failure (ESCORT, NCT02057900)).
  • iPSC human induced pluripotent stem cells
  • 3D culture conditions have been used to improve endothelial differentiation efficiency (Orlova et al., 2014) or to model in vivo conditions with higher fidelity (EP 2580319).
  • Ikuno et al. have developed a differentiation method comprising stage-specific stimulation with VEGF and cAMP combined with the elimination of non-responder cells at early EC stage, showing very high efficiency.
  • the method achieves induction and maintenance of an early stage EC population, which may be modified to take up an arterial or venous profile (Ikuno et al. 2017).
  • the authors suggest their method for 3D tissue engineering and regenerative medicine.
  • WO 2016/011283 describes endothelial cells derived from iPSC, wherein the differentiation occurs in the presence of leptin and the cells are further cultured on a scaffold to obtain microvessels.
  • the endothelial cells in the microvessel express one or more of CD31, VE-cadherin, von Willebrand factor, KLF-2 and KLF-4.
  • WO 2014/200340 describes a method to produce a cell culture of endothelial cells and smooth muscle cells, wherein the cells are obtained by culturing pluripotent stem cells in a medium containing ActivinA, BMP4, VEGF and a canonical WNT ligand or GSK3 inhibitor and another medium containing VEGF and a TGFb signalling inhibitor in 2D culture.
  • WO 2017/011748 recommends overexpression of Sirtuin-1 to regulate cell senescence, and thereby enhance iPSC-EC performance in a 2D culture.
  • PSC-derived EC should meet many requirements to be used in generating vascular models for drug screening and vascular grafts for implantation.
  • a three-dimensional vascular construct comprising PSC-EC on a scaffold is highly desirable in drug screening tests, for studying the genetics and molecular pathways of vascular differentiation and vasculogenesis, as well as for implantable vascular grafts for the treatment of vascular dis- eases.
  • PSC-EC should also be capable of potent angiogenesis and to readily adhere to natural or synthetic matrices.
  • Arterial endothelial cells would be preferable in many clinical scenarios, as arterial endothelial cells are more atheroprotective and venous endothelial cells are more atheroprone (Conway et al., 2010).
  • angiogenesis related and cell-matrix adhesion related proteins ADAMTSl, ANG (angiogenin), ANGPT1 (angiopoietin 1), ARTN (artemin), COL18A1 (collagen type XVIII alpha 1), DPP4 (dipeptidyl peptidase-4), (ECGF1, endothelial cell growth factor 1), EGF (epidermal growth factor), PROKR2 (prokineticin receptor 2), ENG (endoglin), FGF1 (acidic fibroblast growth factor), FGF2 (basic fibroblast growth factor), HGF (hepatocyte growth factor), IGFBP1 (insulin-like growth factor binding protein 1), IGFBP3 (insulin-like growth factor binding protein 3), ILIB (interleukin 1 beta), LEP (leptin), CCL2 (chemokine (C-C motif) ligand 2), PDGFA (platelet-derived growth factor sub
  • arterial phenotype is indicated by an increased arterial marker level compared to the corresponding arterial marker level in a population of human umbilical vein endothelial cells (HUVEC population) cultured under 2D culture conditions, wherein said arterial marker level is selected from the group consisting: EphrinB2 EphB4 mRNA ratio, wherein the EphrinB2/EphB4 mRNA ratio in the cells obtained in ii) is at least 2-fold higher than the EphrinB2/EphB4 mRNA ratio in said HUVEC population, expression of Notch2, wherein the expression of Notch2 is at least 4-fold higher in the cells obtained in ii) than the expression of Notch2 in said HUVEC population, expression of EphrinB2, wherein the expression of EphrinB2 is at least 2-fold higher in the cells obtained in iii) than the expression of EphrinB2 in said HUVEC population.
  • EphrinB2 EphB4 mRNA ratio wherein the Ephr
  • the cells are cul- tured for at least 2 days, more preferably for at least 3 days and even more preferably for at least 4 days and most preferably for more than 5 days in ii).
  • the one or more protein(s) selected from ANG, TYMP, COL18A1, ADAMTSl, ADAM10, ADAM9, IGFBP3, LEP, IGFBP1, ENG, ECGF1, PLG, ANGPT1, FGF2, EGF, TIMPl, ILIB, HGF, CCL2, SERPINEl, TIMP3, FGFl, PDGFA.
  • the expression of said one or more protein(s) is at least 15 fold increased.
  • angiogenesis related proteins ANG, ANGPTl, ARTN, DPP4, EGF, ENG, FGFl, FGF2, ILIB, LEP, PDGFA, CCL2, PLG, CD9, LAP3 is increased in said 3D cultured CD31+ cells obtained in ii) compared to the expression of said protein(s) in a population of human coronary arterial endothelial cells (HCAEC population) cultured under 2D culture conditions wherein said increase is at least 10-fold, and
  • one or more protein(s) selected from the group consisting of cell-matrix adhesion related proteins ADAMTSl, COL18A1, ECGF1, HGF, IGFBP1, IGFBP3, TIMPl, TIMP4, TYMP, ADAM9, ADAMIO, SERPINEl, TIMP3, PIGF, PSPN, PROKR2 is increased in said 3D cultured CD31+ cells obtained in ii) compared to the expression of said protein(s) in a population of human coronary arterial endothelial cells (HCAEC population) cultured under 2D culture conditions, wherein said increase is at least 10-fold.
  • HCAEC population human coronary arterial endothelial cells
  • the one or more of angiogenesis related protein(s) is/are selected from the group consisting of ANGPTl, FGF2, EGF, ILB1, PLG, CCL2, FGFl, PDGFA, and
  • cell-matrix adhesion related protein(s) is/are selected from the group consisting of TIMPl, HGF, SERPINEl, TIMP3.
  • the one or more protein(s) in iii) is selected from ANGPTl, FGF2, EGF, TIMPl, ILIB,
  • the expression of said one or more pro- tein(s) is at least 15 fold increased.
  • the expression of at least one, preferably at least two, more preferably at least three angiogenesis- related proteins selected from PF4, vasohibin (VASH1), and VEGF-A is at least 3-fold, preferably at least 4- fold higher than in a population of HCAEC cultured under 2D culture conditions,
  • the expression of at least one, preferably at least two, more preferably at least three, more preferably at least four, even more preferably at least five cell-matrix adhesion proteins selected from AREG, CD59, MMP9, urokinase, TIMP2 is at least 3 -fold higher than in a population of HCAEC cultured under 2D culture conditions
  • the EphrinB2/EphB4 mRNA ratio is at least 2-fold higher than the EphrinB2/EphB4 mRNA ratio in said HUVEC population and/or the expression of Notch2 is at least 4-fold higher and/or the expression of EphrinB2 is at least 2-fold higher than the expression of Notch2 and/or the expression of EphrinB2, respectively, in a HUVEC population cultured under 2D culture conditions.
  • iii) is carried out at a point in time when expression of at least one angiogenesis-related protein selected from ACVR1B, angiopoietin-2 (ANGPT2), CCL3, CSF2, CXCL-16, FGF4, HB-EGF, IGFBP2,
  • IL-8, NRG-1, PDGFB, platelet factor 4 (PF4), PLG, PTX3, serpin Fl, TGFB1, vasohibin, VEGF-A, VEGF-C is increased at least 10 4 fold in the cells obtained in ii) as compared to the expression of the at least one angiogenesis-related protein in a population of human pluripotent stem cells.
  • iii) is carried out at a point in time when
  • angiogenesis-related protein selected from ACVR1B, angiopoietin-2,
  • CCL3, CSF2, CXCL-16, FGF4, HB-EGF, IGFBP2, IL-8, NRG-1, PDGFB, platelet factor 4 (PF4), PLG, PTX3, serpin Fl, TGFB1, vasohibin, VEGF-A, VEGF-C is increased at least 5 fold, preferably at least 10 fold, more preferably about 5-21 fold in the cells obtained in ii) as compared to the expression of the at least one angiogenesis-related protein in HCAEC population cultured under 2D culture conditions,
  • MMP9, serpin b5, THBS1, thrombospondin 2 (THBS2), TIMP2, urokinase is increased at least 5 fold, preferably at least 10 fold, more preferably about 5-21 fold in the cells obtained in ii) as compared to the expression of the at least one cell-matrix adhesion protein in HCAEC population cultured under 2D culture conditions.
  • iii) is carried out at a point in time when
  • vasohibin and/or VEGFA is increased at least 8 fold, preferably at least 10 fold in the cells obtained in ii) as compared to the expression of PF4, vasohibin and/or VEGFA, respectively, in a HCAEC population cultured under 2D culture conditions,
  • the expression of at least one, preferably at least two, preferably at least three, more preferably at least four, most preferably five proteins selected from AREG, CD59, MMP9, urokinase, TIMP2 is increased at least 3 fold, preferably at least 5 fold, more preferably at least 10 fold, most preferably at least 50 fold in the cells obtained in ii) as compared to the expression of the corresponding protein(s) in a HCAEC population cultured under 2D culture conditions and
  • the EphrinB2 EphB4 mRNA ratio in the cells obtained in ii) is at least 2-fold higher than the EphrinB2 EphB4 mRNA ratio in said HUVEC population and/or the expression of Notch2 is at least 4-fold higher and/or the expression of EphrinB2 is at least 2-fold higher in the cells obtained in ii) than the expression of Notch2 and/or the expression of EphrinB2, respectively, in a population of human umbilical vein endothelial cells (HUVEC population) cultured under 2D culture conditions and/or the level of dll4 in said 3D cultured CD31+ cells is increased at least 4 fold, preferably at least 5 fold, more preferably at least 10 fold compared to undifferentiated human pluripotent stem cells.
  • HUVEC population human umbilical vein endothelial cells
  • the starting population of CD31+ cells is obtainable, preferably produced by a method comprising
  • pluripotent stem cells media preferably mTeSRl
  • growth factors and small molecules comprise: foetal bovine serum, human epidermal growth factor, vascular endothelial growth factor, human fibroblast growth factor B, insulin growth factor R3, hydrocortisone, heparin, ascorbic acid, antibiotic,
  • a) + b) are carried out within 4 days and d) is initiated on day 4.
  • c) is carried out one day before d).
  • EBM2 medium 500 ml
  • 2% FBS 10 ml
  • human epidermal growth factor 500 ⁇
  • vascular endothelial growth factor 500 ⁇
  • human fibroblast growth factor B 500 ⁇
  • insulin growth factor R3 500 ⁇
  • hydrocortisone 200 ⁇
  • heparin 500 ⁇
  • ascorbic acid 500 ⁇
  • amphotericin B/gentamycin 500 ⁇ .
  • EBs are seeded in d) onto 0.5% gelatine coated 24-well plates or T25 flasks.
  • d) is carried out for 8 or 9 days.
  • 0.25% Trypsin and 0.03% EDTA is used in e).
  • the cultures are centrifuged with 1100 RPM for 5 min in f) and pellets are re- suspended in PBS, containing 1% FBS (FACS buffer).
  • FBS FACS buffer
  • cells are counted before sorting.
  • cells are labeled in g) with anti-CD31 antibody, conjugated with Alexa Fluor 488 fluorophore (1 :20 dilution).
  • normoxia (21% 0 2 ) is maintained during culture.
  • 0.1-1%, preferably 0.5% gelatinised flasks and plates are used.
  • ii) is carried out in a bioreactor spinner flask at 35°C to 38°C, 75-90% humidity, about 21% 0 2 , with steering at 70-75 RPM and with addition of fresh medium every other day, preferably wherein the temperature is about 37°C to 37.5°C and the humidity is about 85%.
  • the medium is EGM2.
  • ii) is carried out in a density of 25-75000 endothelial cells/0.5cm 2 culturing 3D scaffold, preferably wherein the density is about 50000 endothelial cells/0.5cm 2 culturing 3D scaffold.
  • the culturing 3D scaffold is a decellularised human extracellular biomatrix, preferably derived from human aorta.
  • the antibiotics is amphotericin B or gentamycin.
  • the detergent is 0.1% sodium dodecyl sulphate and sodium azide and preferably the concentration of the antibiotics is 1%.
  • the method further comprises seeding the cells isolated in iii) on a 3D matrix, preferably wherein the 3D matrix is suitable for forming a 3D vascular endothelial construct for use in a 3D vascular assay or the extracellular biomatrix is suitable for forming an implantable vascular graft.
  • CD31+ cells for forming a 3D vascular endothelial construct are provided, wherein the cells are capable of adhering to a 3D matrix, capable of angiogenesis and have an arterial phenotype, obtained by the method according to any one of points 1-14.
  • a method for providing a CD31+ cell population for forming a 3D vascular endothelial construct comprising
  • pluripotent stem cells media preferably mTeSRl
  • growth factors and small molecules comprise: foetal bovine serum, human epidermal growth factor, vascular endothelial growth factor, human fibroblast growth factor B, insulin growth factor R3, hydrocortisone, heparin, ascorbic acid, antibiotic,
  • the expression of at least one, preferably at least two, more preferably at least three angiogenesis- related proteins selected from PF4, vasohibin, and VEGF-A is at least 3 -fold, preferably at least 4-fold higher than in a population of HCAEC cultured under 2D culture conditions,
  • the expression of at least one, preferably at least two, more preferably at least three, more preferably at least four, even more preferably at least five cell-matrix adhesion proteins selected from AREG, CD59, MMP9, urokinase, TIMP2 is at least 3 -fold higher than in a population of a HCAEC cultured under 2D culture condi- tions, - the EphrinB2/EphB4 mRNA ratio is at least 2-fold higher than the EphrinB2 EphB4 mRNA ratio in a HUVEC population cultured under 2D culture conditions and/or the expression of Notch2 is at least 4-fold higher and/or the expression of EphrinB2 is at least 2-fold higher than the expression of Notch2 and/or the expression of EphrinB2, respectively, in the HUVEC population and/or the level of dll4 in said 3D cultured CD31+ cells is increased at least 4 fold, preferably at least 5 fold, more preferably at least 10
  • A) + B) are carried out within 4 days and D) is initiated on day 4.
  • C) is carried out one day before D).
  • the following medium is used in C): EBM2 medium (500 ml), 2% FBS (10 ml), human epidermal growth factor (500 ⁇ ), vascular endothelial growth factor (500 ⁇ ), human fibroblast growth factor B (500 ⁇ ), insulin growth factor R3 (500 ⁇ ), hydrocortisone (200 ⁇ ), heparin (500 ⁇ ), ascorbic acid (500 ⁇ ), amphotericin B/gentamycin (500 ⁇ ).
  • EBs are seeded in D) onto 0.5% gelatine coated 24-well plates or T25 flasks.
  • D) is carried out for 8 or 9 days.
  • 0.25% Trypsin and 0.03% EDTA is used in E).
  • the cultures are centrifuged with 1100 RPM for 5 min in F) and pellets are re-suspended in PBS, containing 1% FBS (FACS buffer).
  • FACS buffer 1% FBS
  • cells are counted before sorting.
  • cells are labeled in G) with anti-CD31 antibody, conjugated with Alexa Fluor 488 fluorophore (1 :20 dilution).
  • normoxia (21% 0 2 ) is maintained during culture.
  • 0.1-1%, preferably 0.5% gelatinised flasks and plates are used.
  • a preferred embodiment is the method according to point 16, further comprising
  • N) isolating the CD31+ cells obtained in M) from the culturing 3D scaffold at a point in time when the expression of one or more protein(s) selected from ADAMTS1, ANG, ANGPT1, ARTN, COL18A1, DPP4, ECGF1, EGF, PROKR2, ENG, FGF1, FGF2, HGF, IGFBP1, IGFBP3, IL1B, LEP, CCL2, PDGFA, PIGF, PLG, PSPN, SERPINE1, TIMP1, TIMP4, TYMP, ADAM9, ADAMIO, CD9, LAP3, TIMP3 is increased in the cells obtained in M) as compared to the expression of said protein(s) in a HCAEC population cultured under 2D culture conditions, wherein said increase is at least 10-fold and
  • the cells show arterial phenotype indicated by an EphrinB2/EphB4 mRNA ratio which is at least 2- fold higher than the EphrinB2/EphB4 mRNA ratio in a HUVEC population cultured under 2D culture conditions and/or an expression of Notch2 which is at least 4-fold higher and/or an expression of EphrinB2 which is at least 2-fold higher than the expression of Notch2 and/or the expression of EphrinB2, respectively, in the HUVEC population.
  • the cells are cultured for at least 2 days, more preferably for at least 3 days and even more preferably for at least 4 days and most preferably for more than 5 days in M).
  • the one or more protein(s) selected from ANG, TYMP, COL18A1, ADAMTS1, ADAMIO, ADAM9, IGFBP3, LEP, IGFBP1, ECGF1, ENG, PLG, ANGPTl, FGF2, EGF, TIMP1, IL1B, HGF, CCL2, SERPINEl, TIMP3, FGF1, PDGFA.
  • the expression of said one or more protein(s) is at least 15 fold increased.
  • the expression of one or more protein(s) selected from the group consisting of cell-matrix adhesion related proteins ADAMTS1, COL18A1, ECGF1, HGF, IGFBP1, IGFBP3, TIMP1, TIMP4, TYMP, ADAM9, ADAMIO, SERPINE1, TIMP3, PIGF, PSPN, PROKR2 is increased in said 3D cultured CD31+ cells obtained in M) compared to the expression of said protein(s) in a population of human coronary arterial endothelial cells (HCAEC population) cultured under 2D culture conditions, wherein said increase is at least 10-fold
  • the cells show arterial phenotype indicated by an EphrinB2/EphB4 mRNA ratio which at least 2-fold higher than the EphrinB2 EphB4 mRNA ratio in a HUVEC population cultured under 2D culture conditions and/or an expression of Notch2 which is at least 4-fold higher and/or an expression of EphrinB2 which is at least 2-fold higher than the expression of Notch2 and/or the expression of EphrinB2, respectively, in the HUVEC population.
  • HCAEC population human coronary arterial endothelial cells
  • the expression of one or more protein(s) selected from the group consisting of cell-matrix adhesion related proteins TIMP1, HGF, SERPINEl, TIMP3 is increased in said 3D cultured CD31+ cells obtained in M) compared to the expression of said protein(s) in a population of human coronary arterial endothelial cells (HCAEC population) cultured under 2D culture conditions, wherein said increase is at least 10-fold,
  • the cells show arterial phenotype indicated by an EphrinB2/EphB4 mRNA ratio which at least 2-fold higher than the EphrinB2 EphB4 mRNA ratio in a HUVEC population cultured under 2D culture conditions and/or an expression of Notch2 which is at least 4-fold higher and/or an expression of EphrinB2 which is at least 2-fold higher than the expression of Notch2 and/or the expression of EphrinB2, respectively, in the HUVEC population.
  • the one or more protein(s) in N) is selected from ANGPTl, FGF2, EGF, TIMP1, IL1B, PLG, HGF, CCL2, SERPINEl, TIMP3, FGFl, PDGFA.
  • CCL3, CSF2, CXCL-16, FGF4, HB-EGF, IGFBP2, IL-8, NRG-1, PDGFB, platelet factor 4 (PF4), PLG, PTX3, serpin Fl, TGFB1, vasohibin, VEGF-A, VEGF-C is increased at least 5 fold, preferably at least 10 fold, more preferably about 5-21 fold in the cells obtained in M) as compared to the expression of the at least one angiogenesis-related protein in a HCAEC population cultured under 2D culture conditions,
  • urokinase is increased at least 5 fold, preferably at least 10 fold, more preferably about 5-21 fold in the cells obtained in M) as compared to the expression of the at least one cell-matrix adhesion protein in a HCAEC population cultured under 2D culture conditions.
  • the EphrinB2 EphB4 mRNA ratio in the cells obtained in M) is at least 2-fold higher than the EphrinB2 EphB4 mRNA ratio in a HUVEC population cultured under 2D culture conditions and/or the expression of Notch2 is at least 4-fold higher and/or the expression of EphrinB2 is at least 2-fold higher in the cells obtained in M) than the expression of Notch2 and/or the expression of EphrinB2, respectively, in the HUVEC population and/or level of dll4 in said 3D cultured CD31+ cells is increased at least 4 fold, preferably at least 5 fold, more preferably at least 10 fold compared to undifferentiated human pluripotent stem cells.
  • N is carried out at a point in time when the expression of PF4, vasohibin and/or VEGFA is increased at least 8 fold, preferably at least 10 fold in the cells obtained in M) as compared to the expression of PF4, vasohibin and/or VEGFA, respectively, in a HCAEC population cultured under 2D culture conditions,
  • the expression of at least one, preferably at least two, preferably at least three, more preferably at least four, most preferably five proteins selected from AREG, CD59, MMP9, urokinase, TIMP2 is increased at least 3 fold, preferably at least 5 fold, more preferably at least 10 fold, most preferably at least 50 fold in the cells obtained in M) as compared to the expression of the corresponding protein(s) in a HCAEC population cultured under 2D culture conditions and
  • the EphrinB2 EphB4 mRNA ratio in the cells obtained in M) is at least 2-fold higher than the EphrinB2 EphB4 mRNA ratio in a HUVEC population cultured under 2D culture conditions and/or the expression of Notch2 is at least 4-fold higher and/or the expression of EphrinB2 is at least 2-fold higher in the cells obtained in M) than the expression of Notch2 and/or the expression of EphrinB2, respectively, in the HUVEC population and/or the level of dll4 in said 3D cultured CD31+ cells is increased at least 4 fold, preferably at least 5 fold, more preferably at least 10 fold compared to undifferentiated human pluripotent stem cells.
  • the antibiotics is amphotericin B or gentamycin.
  • the detergent is 0.1% sodium dodecyl sulphate and sodium azide and preferably the concentration of the antibiotics is 1%.
  • a CD31+ cell population for forming a 3D vascular en- dothelial construct is provided, wherein the population is capable of adhering to 3D matrix and has an arterial phenotype, obtained by the method according to any one of points 16 to 28.
  • a CD31+ cell population for forming a 3D vascular endothelial construct is provided, wherein the population is capable of adhering to 3D matrix and is derived from hiPSC, characterized in that
  • the expression of at least one, preferably at least two, more preferably at least three angio genesis- related proteins selected from PF4, vasohibin, and VEGF-A in the CD31+ cells is at least 3-fold, preferably at least 4-fold higher in the CD31+ cells than in a population of HCAEC cultured under 2D culture conditions,
  • the expression of at least one, preferably at least two, more preferably at least three, more preferably at least four, even more preferably at least five cell-matrix adhesion proteins selected from AREG, CD59, MMP9, urokinase, TIMP2 in the CD31+ cells are at least 3-fold higher than in a population of HCAEC cultured under 2D culture conditions,
  • the EphrinB2 EphB4 mRNA ratio in the CD31+ cells is at least 2-fold higher than the EphrinB2 EphB4 mRNA ratio in a HUVEC population cultured under 2D culture conditions and/or the expression of Notch2 in the CD31 cells is at least 4-fold higher and/or the expression of EphrinB2 in the CD31+ cells is at least 2-fold high- er than the expression of Notch2 and/or the expression of EphrinB2, respectively, in the HUVEC population and/or the level of dll4 in said 3D cultured CD31+ cells is increased at least 4 fold, preferably at least 5 fold, more preferably at least 10 fold compared to undifferentiated human pluripotent stem cells.
  • CD31+ cell population of point 30 wherein additionally the expression of one or more protein(s) selected from ADAMTSl, ANG, ANGPT1, ARTN, COL18A1, DPP4, ECGF1, EGF, PROKR2, ENG, FGF1, FGF2, HGF, IGFBP1, IGFBP3, IL1B, LEP, CCL2, PDGFA, PIGF, PLG, PSPN, SERPINE1, TIMPl, TIMP4, TYMP, ADAM9, ADAMIO, CD9, LAP3,TIMP3 is increased in said CD31+ cells as compared to the expression of said protein(s) in said HCAEC population, wherein said increase is at least 10-fold.
  • protein(s) selected from ADAMTSl, ANG, ANGPT1, ARTN, COL18A1, DPP4, ECGF1, EGF, PROKR2, ENG, FGF1, FGF2, HGF, IGFBP1, IGFBP3, IL1B, LEP, CCL2, PDGFA, PI
  • CD31+ cell population of point 30 wherein additionally the expression of one or more angiogenesis-related protein(s) selected from the group consisting of ANG, ANGPT1, ARTN, DPP4, EGF, ENG, FGF1, FGF2, IL1B, LEP, PDGFA, CCL2, PLG, CD9, LAP3, and
  • the expression of one or more cell-matrix adhesion protein(s) selected from the group consisting of ADAMTSl, COL18A1, ECGF1, HGF, IGFBP1, IGFBP3, TIMPl, TIMP4, TYMP, ADAM9, ADAMIO, SERPINEl, TIMP3, PIGF, PSPN, PROKR2 is increased in said 3D cultured CD31+ cells obtained in ii) compared to the expression of said protein(s) in a population of human coronary arterial endothelial cells (HCAEC population) cultured under 2D culture conditions, wherein said increase is at least 10-fold.
  • Preferred is the CD31+ cell population of point 32, wherein the additional one or more angiogenesis- related protein(s) is/are selected from the group consisting of ANGPT1, FGF2, EGF, ILB 1, PLG, CCL2, FGF1, PDGFA, and
  • the additional one or more cell-matrix adhesion related protein(s) is/are selected from the group consist- ing of TIMP1, HGF, SERPINE1, TIMP3.
  • CD31+ cell population of point 31 wherein the additional one or more protein(s) is/are selected from ANGPT1, FGF2, EGF, TIMP1, IL1B, PLG, HGF, CCL2, SERPINE1, TIMP3, FGF1, PDGFA.
  • the 3D vascular assay is selected from antiplatelet Rantes assay, a vasoactive assay, an in vitro signaling assay, a permeability assay.
  • the CD31+ cell popula- tion used in the 3D vascular assay is seeded on a 3D matrix and is suitable for forming 3D vascular endothelial construct for use in a 3D vascular assay, such as on a decellularised porcine intestinal extracellular matrix scaffold or a decellularised human extracellular matrix scaffold, such as the decellularised human extracellular biomatrix defined in point 25 or 26.
  • the 3D matrix is an extracellular biomatrix.
  • the 3D scaffold is an extracellular biomatrix scaffold.
  • the iPSC-derived cells are derived or obtained from iPSC or an iPSC line reprogrammed by viral or non-viral delivery of transcription factors.
  • the iPSC-derived cells are derived or obtained from iPSC or an iPSC line for which the following (transcription) factors have been used in the reprogramming method: Oct4, Sox2, Nanog and optionally or preferably LIN28.
  • the iPSC-derived cells are derived or obtained from iPSC or an iPSC line of mesodermal origin, preferably of mesenchymal origin, preferably of connective tissue origin, in particular of lung origin.
  • the iPSC-derived cells are derived or obtained from iPSC or an iPSC line of mesodermal origin, preferably of epithelial origin, preferably of endothelial origin.
  • the iPSC-derived cells are derived or obtained from iPSC or an iPSC line for which the following (transcription) factors have been used in the reprogramming method: Oct4, Sox2, Nanog and optionally or preferably LIN28, wherein the iPSC or the iPSC cell line is of mesodermal origin and preferably it has been reprogrammed by a retroviral method.
  • the culturing 3D scaffold in i), ii) and/or in L) is a decellularised porcine intestinal extracellular matrix scaffold.
  • FIG. 1 Expressions of endothelial markers. Bar graphs show changes in endothelial gene expression levels of arterial (EphrinB2, Notchl, Notch2) (A-C), venous (EphB4) (D) and common (CD31, VE-Cadherin) (E, F) endothelial markers. Fold changes in mRNA levels in human embryonic stem cell-derived endothelial cells (hESC-EC), human induced pluripotent stem cell-derived endothelial cells (hiPSC-EC) and human umbili- cal vein endothelial cells (HUVEC) are normalized to those in undifferentiated hPSC.
  • hESC-EC human embryonic stem cell-derived endothelial cells
  • hiPSC-EC human induced pluripotent stem cell-derived endothelial cells
  • HAVEC human umbili- cal vein endothelial cells
  • FIG. 2 Angiogenesis and cell-matrix adhesion protein profiling in 2D and 3D endothelial cultures.
  • Heat map diagram shows expression of angiogenesis-related proteins before and after the recellularisation procedure (2D and 3D cultures, respectively) with human embryonic stem cell-derived endothelial cells (hESC-EC) and human induced pluripotent stem cell-derived endothelial cells (hiPSC-EC).
  • hESC-EC human embryonic stem cell-derived endothelial cells
  • hiPSC-EC human induced pluripotent stem cell-derived endothelial cells
  • Proteomics in hESC-EC and hiPSC- EC are represented as fold changes versus those in control HCAEC.
  • n 4 technical replicates from 2 independent experiments.
  • Array membranes for proteomics were visualized by chemiluminescent detection; pixel densities were quantified by ImageJ software. Numbers of cells were equalized in each experimental setting; passages between 3 and 5 were
  • FIG. 3 In vivo conditioning of hPSC-EC induces expression of arterial, venous and common endothelial marker genes.
  • Grouped bar graphs show expression of arterial (EphrinB2, Notchl, Notch2), venous (EphB4) and common (CD31) endothelial marker genes at two weeks after subcutaneous transplantation of human em- bryonic stem cell-derived endothelial cells (hESC-EC) and human induced pluripotent stem cell-derived endothelial cells (hiPSC-EC) in athymic rats.
  • Data are presented as mean ⁇ SEM. Fold changes in normalised mRNA levels are showed in log(10) scale. mRNA levels are normalised to those in pre-implanted control cells.
  • n 3 biological replicates of endothelial implantation from differentiation from hESC, 3 technical replicates at each point. * indicates p ⁇ 0.05, ** indicates p ⁇ 0.01, one-way ANOVA with Tukey post-hoc test
  • FIG. 4 Endothelial marker expressions during differentiation and specification.
  • Radar graph shows endothelial markers during human embryonic stem cell-derived endothelial cells (hESC-EC), human induced pluripotent stem cell-derived endothelial cells (hiPSC-EC) differentiation in three-dimensional (3D) specification compared to expression in human umbilical vein endothelial cells (HUVEC). Changes in mRNA levels of arterial (EphrinB2, Notchl, Notch2, D114) and venous marker genes (EphB4, FLT4) are shown.
  • FIG. 5 Antiplatelet activities of endothelial cells in 3D culture on vascular wall.
  • A Schematic figure shows experimental set used for antiplatelet assay. 1. Platelet rich plasma was incubated with endothelial cells in 2D culture; 2. with endothelial cells on decellularised vessel wall in 3D culture; 3. alone on decellularised matrix and 4. platelet rich plasma alone.
  • FIG. 1 Bar graph shows changes in Rantes chemokine levels measured from platelet rich plasma after incubating developed vascular cells (human embryonic stem cell-derived endo- thelial cells (hESC-EC), human induced pluripotent stem cell-derived endothelial cells (hiPSC-EC), human umbilical vein endothelial cells (HUVEC)), acellular and recellularised vascular constructs.
  • vascular cells human embryonic stem cell-derived endothelial cells (hESC-EC), human induced pluripotent stem cell-derived endotheli
  • FIG. 7 Vasoactive activities of hPSC-EC in 3D culture and isolated vessel platform.
  • Vasoactive effects of human embryonic stem cell-derived endothelial cells hESC-EC
  • human induced pluripotent stem cell- derived endothelial cells hiPSC-EC
  • human umbilical vein endothelial cells HUVEC
  • X axis refers to time
  • Y axis refers to force (positive changes on y axis means decreased force and vasodilatation).
  • FIG. 8 3D culture modulates arterial-venous endothelial marker expression of hPSC-EC.
  • A Acellular biomatrices were seeded with human embryonic stem cell-derived endothelial cells (hESC-EC) and human induced pluripotent stem cell-derived endothelial cells (hiPSC-EC) and stained with anti-human CD31 antibody and Hoechst.
  • B, C Bar graphs show changes in arterial (Notchl, Notch2, EphrinB2, D114), venous (EphB4) and common (VE-Cadherin and CD31) endothelial markers in hESC-EC (B) and hiPSC-EC (C) after recellu- larisation.
  • FIG. 9 Expression pattern of endothelial markers in endothelial cells from different sources.
  • Heat map diagrams show normalised mRNA expression of arterial (Notchl, Notch2, EphrinB2, D114), venous (EphB4) and general (CD31, VE-Cad) endothelial marker genes.
  • Gene expression levels were normalised to (A) those in human pluripotent stem cells (hPSC), (B) human coronary arterial endothelial cells (HCAEC) and (C) HUVEC.
  • hESC-EC Human embryonic stem cell-derived endothelial cells
  • hiPSC-EC human induced pluripotent stem cell-derived endothelial cells
  • HCAEC human coronary arterial endothelial cells
  • HUVEC human microvascular endothelial cells
  • FIG. 10 Angiogenesis and cell-matrix adhesion protein profiling in endothelial cultures.
  • Scatter dot diagrams show expression levels of angiogenesis (A) related and cell-matrix interaction (B) related proteins of human embryonic stem cell-derived endothelial cells (hESC-EC) and human induced pluripotent stem cell- derived endothelial cells (hiPSC-EC), expression are compared to those in human coronary arterial endothelial cells (HCAEC).
  • HCAEC human coronary arterial endothelial cells
  • FIG. 11 Angiogenesis and cell-matrix adhesion protein profiling in 2D and 3D endothelial cultures.
  • A Functional association protein network diagrams for hESC-EC (left panel) and hiPSC-EC (right panel) are gen- erated by Ingenuity pathway analysis.
  • B Scatter dot diagrams show expression levels of angiogenesis related and cell-matrix interaction related proteins of human embryonic stem cell-derived endothelial cells (hESC-EC) and human induced pluripotent stem cell-derived endothelial cells (hiPSC-EC), 2D and 3D cell culture conditions are compared.
  • hESC-EC human embryonic stem cell-derived endothelial cells
  • hiPSC-EC human induced pluripotent stem cell-derived endothelial cells
  • Figure 12 In vivo characterisation of endothelial cells.
  • Human embryonic stem cell-derived endothelial cells hESC-EC
  • human induced pluripotent stem cell-derived endothelial cells hiPSC-EC
  • A and take up ac-LDL (B). Both cell types form tubes on Matrigel (C).
  • Immunocyto- chemistry shows positive staining for anti-CD31 (D) endothelial and anti-EphrinB2 (E) and D114 (F) markers.
  • FIG. 13 Immunocytochemistry and microscopy analysis of cells derived by Harding et al. (2017) protocol.
  • A, B Immunocytochemistry with endothelial CD31 marker (green; ⁇ ) and mesenchymal marker FSP1 (red; ⁇ ). Nuclear staining Hoechst is also shown (blue; ⁇ ), lOx magnification.
  • C Automated high content analysis to assess endothelial purity. Objects are identified by three-channel fluorescent imaging and compart- mental analysis bioassays.
  • D High content plot to show high heterogeneity of population.
  • FIG. 14 Real PCR assessment of undifferentiated hiPSC (IMR line) and differentiated endothelial culture by our and Harding et al. (2017) protocols. mRNA levels of endothelial markers (CD31, VE-cadherin), arterial markers (Notchl/2, D114, EphrinB2) and venous markers (EphB4) were quantitated. One-way ANOVA was used for statistics. * indicates p ⁇ 0.05, ** indicates pO.01, *** indicates pO.001.
  • FIG. 15 Proteome profiling.
  • A Proteome prolifer blot, developed.
  • B Comparison of 54 angiogenic factors.
  • C Heat map presentation of Harding et al. (2017) protocol (indicated by AH prot) versus our protocol in 2D and 3D.
  • Figure 16 Schematic illustration of the protocol used to produce CD31+ cells and 3D vascular construct on acellular biomatrices.
  • hiPSC-EC show high abundance of angiogenesis- and cell adhesion-related proteome along with an arterial phenotype.
  • 2D culture of the cells resulted in an increased expression of angio- genesis- and cell adhesion-related proteins as compared to HCAEC 2D culture.
  • the cells provided by the method according to the invention are particularly suitable for forming a 3D vascular endothelial construct, for use in 3D vascular assays and for use in implantable grafts.
  • Their arterial phenotype is characterized by and may be measured by increased arterial marker levels, such as e.g. EphrinB2/EphB4 mRNA ratio, expression of Notch2, expression of EphrinB2 and expression of dll4.
  • EphrinB2/EphB4 mRNA ratio expression of Notch2
  • EphrinB2 EphrinB2
  • the 2D and 3D hiPSC-EC culture show a dominant increase mainly in the expression of arterial rather than venous markers, confirming the presence of an arterial dominance in the endothelial culture.
  • Arterial to venous mRNA ratio increased from day 5 of differentiation and remained similar in sorted CD31+ cells, show- ing arterial dominancy in the expanding culture.
  • differentiated hiPSC-EC show less than mature endo- thelial phenotype in 2D culture, some of the markers of a functional endothelial phenotype are already present, i.e. hiPSC-EC cultures show cobblestone pattern, take up ac-LDL, show tube formation activity, and are stained positive with anti-CD31 antibody.
  • hiPSC-EC When compared to hESC-EC obtained under the same culture conditions, the expression of endothelial markers in hiPSC-EC is lower than in hESC-EC (FIG 1), while hiPSC-EC according to the invention show increased levels of angiogenesis- and cell adhesion-related proteins.
  • hiPSC-EC generated in 2D culture conditions
  • 3D cell culture systems were used.
  • hiPSC-EC could not only adhere to decellularised extracellular biomatrices, remained viable and functional during recellularisation, but underwent further maturation.
  • levels of many angiogenesis-related proteins e.g. angiopoietin 1 and 2, endoglin, FGFs
  • FGFs endoglin
  • collagen XVIII, MMP8, MMP9, TIMP1, MCP-1, ADAMTSl showed robust increase in 3D culture, thereby increasing adhesive capacity of cells upon reseeding of matrices.
  • Factors involved in the regulation of platelet aggregation or fibrinolytic pathway e.g. TIMP4, plasminogen
  • TIMP4 plasminogen
  • Arterial endothelial derivatives of hiPSC develop mature endothelial characteristics and functional behaviour in 3D cultures. Arterial fate can be modulated even on differentiated endothelial cells during expansion in 2D cultures and on seeding cells on biomatrices for 3D cultures. Further, the markedly increased levels of angiogenesis- and adhesion-related proteins indicate, that hiPSC derivatives are better prepared to form a 3D endothelial construct than hESC derivatives, while having a favourable arterial phenotype.
  • hiPSC-EC were able to reduce platelet activation, as shown by decreased Rantes levels in platelet rich plasma upon incubation of vascular constructs (FIG 5,6). 3D culture conditions further enhanced antiplatelet effects of hiPSC-EC (FIG 5,6). Antiplatelet effect and regulation of clot formation are specific tasks for arterial endothelial cells; proteome profiling and antiplatelet assay together emphasize the presence of functional arterial hiPSC-EC in the culture. An improved antiplatelet activity is favourable when considering use of cells for vascular tissue engineering. Direct (acute) regulation of myogenic tone from hiPSC-EC is another key property of vascular grafts for clinical use. It has been shown by using rat aortic rings as an in vitro isolated vessel platform that acute reseeding of vascular surfaces with secretome of hiPSC-EC according to the invention had marked direct vasoactive effect. (FIG 7)
  • cultured under two-dimensional culture conditions and “cultured under three- dimensional culture conditions” refer to conditions appropriate for culturing the desired cell type and promoting the expression of angiogenesis-related proteins, cell-matrix adhesion proteins, common, venous and arterial endothelial markers in case of endothelial cells.
  • A. Endothelial differentiation of human pluripotent stem cells 1. Maintenance of human induced pluripotent stem cells on adherent plates in pluripotent stem cells medium (preferably in mTeSRl). Observing stem cell colonies daily in a phase contrast light microscope. Mechanical removal spontaneously differentiated cells from culture. Regular passage of stem cell colonies via Versene- based dissociation of clamps and reseeding stem cells on larger dish surface at confluency of 65-70% (1-1.5M cells/9.6 cm 2 ). Proliferation (by colony formation assay and immunocytochemistry with anti-Ki67 proliferation marker antibodies) and chromosome stability are regularly checked.
  • Additional factors comprise: 2% batch-tested foetal bovine serum, human epidermal growth factor, vascular endothelial growth factor, human fibroblast growth factor B, insulin growth factor R3, hydrocortisone, heparin.
  • detergent solution for 72 hours.
  • the detergent is 0.1% sodium dodecyl sulphate and sodium azide.
  • two-dimensional (2D) culture refers to conventional adherent tissue culture involving growing cells on (coated) solid flat surfaces as 2D monolayers. Cells are adhering to an artificial plastic or glass substrate and are growing side by side. Protocols using scaffold-free method (e.g. suspension culture of EBs) are also termed as 2D culture conditions in the present disclosure.
  • CD31+ cells according to the invention are obtainable by a method comprising the steps described herein below.
  • hiPSC may be seeded into a feeder-free medium capable of supporting endothelial differentiation and growth, preferably containing VEGF, such as EGM2, containing an endothelial basal medium, e.g. EBM2 media with SingleQuot supplements.
  • VEGF vascular endothelial growth factor
  • EGM2 endothelial basal medium
  • the endothelial basal medium preferably contains essential and non-essential amino acids, vitamins, trace minerals, organic compounds, and inorganic salts and lacks hormones, growth factors or other proteins.
  • Embryoid bodies are generated by mechanically breaking the hiPSC colonies, with a standard sized cell culture scraper. EBs are then cultured for 4 days in suspension in low-adherent plates in a medium capable of supporting endothelial differentiation and growth, preferably containing VEGF, such as EGM2.
  • VEGF vascular endothelial growth factor
  • the differentiation protocol may include extra growth factors and morphogens in a serum-free, feeder-free, complete medium suitable for iPSC, such as the mTeSRl media (e.g. Activin A, FGF-2, VEGFi 65 and BMP4).
  • a serum-free medium suitable for hematopoietic stem cell expansion such as the Stemline II complete media containing e.g. FGF-2, VEGFi 65 and BMP4, differentiating cells are then maintained in the serum-free medium suitable for hematopoietic stem cell expansion (e.g. Stemline II media).
  • EBs are then plated on gelatinized plates in a cell-free medium capable of supporting endothelial differentiation and growth, preferably containing VEGF, such as EGM2, and cultured in a monolayer.
  • VEGF vascular endothelial growth factor
  • Normoxic (21% 0 2 ) conditions are maintained throughout culturing.
  • CD31+ cells are selected and collected after approximately 12 or 13 days of culture, followed by seeding onto collagen coated plates in a medium capable of supporting endothelial differentiation and growth, compris- ing endothelial cell growth medium, hematopoietic stem cell expansion medium, preferably supplemented with antibiotics (e.g. EGM2, Stemline II and P/S media) coated plates.
  • a medium capable of supporting endothelial differentiation and growth comprising endothelial cell growth medium, hematopoietic stem cell expansion medium, preferably supplemented with antibiotics (e.g. EGM2, Stemline II and P/S media) coated plates.
  • antibiotics e.g. EGM2, Stemline II and P/S media
  • Differentiated EC cells are normally utilized for experiments and seeding of biomatrices between passages 3-5 and may be from passage 1.
  • 2D culture of the cells resulted in an increased expression of angiogenesis- and cell adhesion-related pro- teins as compared to HCAEC 2D culture.
  • the expression of angiogenesis-related proteins ACVR1B, ANGPT2, CCL3, CXCL16, FGF4, CSF2, HBEGF, IGFBP2, IL8, NRG1, PDGFB, PF4, PLG, PTX3, SERPINF1, TGFB 1, VASH1, VEGFA, VEGFC and cell-matrix adhesion related proteins AREG, GDNF, CCL2, MMP9, urokinase (PLAU), THBS1, THBS2, CD59, TIMP2, SERPINB5 in cells according to the invention is at least 2-fold high- er than the expression of the corresponding protein in HCAEC cultured under 2D conditions.
  • HCAEC and HUVEC may be maintained in a medium capable of supporting endothelial growth (such as EGM2) and may be grown as monolayer culture according to the instructions of the provider of the medium and/or the cells.
  • EGM2 endothelial growth
  • a method exemplifying the appropriate culture conditions is described in the Examples.
  • a 3D cell culture is an artificially -created environment in which biological cells are permitted to grow or interact with their surroundings in all three dimensions. 3D cell culture allows cells in vitro to grow in all directions, similar to how they would in vivo.
  • Tissue engineered three-dimensional cell culture systems may be used to study survival, viability and proliferation of pluripotent stem cell-derived endothelial cells.
  • hESC-EC and hiPSC-EC according to the invention cultured under 2D conditions may be seeded on bioscaffolds and cultured by using a bioreactor system.
  • Decellularised vascular scaffold obtained from human aorta
  • decellularised scaffold derived from porcine small intestinal submucosa (CorMatrix) were used to demonstrate that hiPSC-EC according to the invention are capable of recellularising the matrix, remain viable and undergo further maturation on the matrix.
  • the bioscaffold suitable to form a three-dimensional vascular endothelial construct according to the invention may be any material comprising extracellular matrix components to provide structural and mechanical properties for further development of the cells seeded thereon.
  • the properties of the matrix may be optimized to mimic those of a tissue to be grafted or to be tested.
  • Decellularised human extracellular matrix (derived from aorta) and the decellularised scaffold derived from porcine small intestinal submucosa were shown to be appropriate as 3D scaffold.
  • Other cells involved in vasculogenesis may be co-cultured with the cells under both 2D and 3D conditions and seeded onto a bioscaffold to produce a three-dimensional vascular endothelial construct.
  • Such cells may comprise human primary or hiPSC-derived vascular smooth muscle cells, primary or hiPSC-derived pericytes, other mesenchymal cells and fibroblasts.
  • Cells according to the invention are characterized by a robust increase in arterial (EphrinB2, Notchl, Notch2 and D114) and venous (EphB4 and FLT4) endothelial markers during differentiation and a dominant increase in arterial rather than venous markers (EphrinB2, Notchl, Notch2 vs EphB4 mRNA levels).
  • EphrinB2, Notchl, Notch2 vs EphB4 mRNA levels endothelial markers during differentiation and a dominant increase in arterial rather than venous markers.
  • mRNA levels of arterial, venous and common endothelial marker genes all increase markedly after 3D bioreactor culturing vs 2D cultures.
  • D114 and Notch2 key signals of arterial fate and sprouting angiogenesis, respectively, were both increased in hiPSC-EC showing a more profound increase of Notch2 level.
  • Abundant secretion of several proteins in hiPSC-EC may be measured; their protein secretome patterns are comparable to those in control human arterial endothelial cells. (FIG 2)
  • angiogenesis and hematopoietic soluble receptor pro- teome profiling measurement may be carried out on commercially available array kits, such as the R&D System Proteome Profiler Human Angiogenesis Array Kit (ARY007) and Human Soluble Receptor Array Kit Hematopoietic Panel (ARYOl 1). Both cell surface supernatant and cell lysates may be used in these studies.
  • the levels of angiogenesis-related proteins ACVR1B, angiopoietin-2, CCL3, CSF2, CXCL-16, FGF4, HB-EGF, IGFBP2, IL-8, NRG-1, PDGFB, platelet factor 4 (PF4), PLG, PTX3, serpin Fl, TGFB l, vasohibin, VEGF-A, VEGFC in hiPSC-EC are at least 2-fold higher than the expression of the same proteins in HCAEC cultured under 2D conditions.
  • the levels of angiogenesis-related proteins PF4, SERPINF1, VASH1 and VEGFA are at least 3 -fold higher than the expression of the same proteins in HCAEC.
  • the levels of adhesion-related proteins AREG, CCL2, CD59, GDNF, MMP9, serpin b5, THBSl, thrombospondin 2, TIMP2, urokinase are at least 2-fold higher than the expression of the same proteins in HCAEC cultured under 2D conditions.
  • the levels of MMP9, CD59, TIMP2, urokinase are at least 3 -fold higher than the expression of the same proteins in HCAEC.
  • Cells and vascular constructs according to the invention may be analyzed using standard immunocyto- chemistry and immuno histochemistry using commercially available antibodies and methods known in the art.
  • vWF von Willebrand Factor
  • D114 delta like 4 staining
  • hiPSC-EC according to the invention are negative for haematopoietic marker CD45.
  • High content automated microscopy analyses proved high intensity of arterial EphrinB2 and D114 markers as compared to control arterial endothelial cell HCAEC.
  • hiPSC-EC form capillary -like structures in Matrigel tube formation assay both after 2D culture and upon seeding and culturing on a biomatrix (FIG 12)
  • Endothelial cells may be identified by their high-level metabolism of acetylated low density lipoprotein (Ac-LDL).
  • Ac-LDL acetylated low density lipoprotein
  • cultures are incubated with l,r-dioctadecyl-3,3,3',3'-tetramethyl- indocarbocyanine perchlorate labelled with fluorescent probe (Dil-Ac-LDL, ThermoFisher Scientific) and im- aged by fluorescence microscopy.
  • hiPSC-EC take up Ac-LDL both after 2D culture and upon seeding and cultur- ing on a biomatrix (FIG 12).
  • a cobblestone morphology in culture is characteristic for endothelial cells.
  • hiPSC-EC according to the invention show this characteristic morphology both after 2D culture and upon seeding and culturing on a biomatrix. (FIG 12)
  • hiPSC-EC are able to fulfill therapeutic expectations only when they possess sufficient functional activity to support the physiological mechanisms of the vascular system.
  • Endothelial cells should provide an antithrombotic surface to line vessel walls.
  • Antiplatelet effect and regulation of clot formation is an essential task for arterial endothelial cells.
  • Anticlotting function may be investigated by Rantes (CCL5) chemokine ELISA assay. Rantes is secreted from activated platelets, thus its level refers to clotting status in platelet rich plasma. Secretion of Rantes chemokine is measured in platelet rich plasma after incu- bating cells or vascular constructs to be tested.
  • 3D culture conditions enhance anticlotting effects of hiPSC-EC.
  • Rantes is also related to angiogenic mechanisms, its modulation by hiPSC-EC emphasizes their strong angiogenic characteristics.
  • Antiplatelet effects of hiPSC-EC according to the invention are comparable to control HUVEC. Rantes levels are similar in 2D samples and in 3D scaffolds alone (aorta slices or CorMatrix), suggesting that acellular biomatrices are not thrombogenic in short-term experiments in vitro. Antiplatelet effects of hiPSC-EC according to the invention are higher in 3D-cultured cells than in 2D-cultured cells. (FIG 5 and 6)
  • Direct (acute) regulation of myogenic tone from hPSC-EC should be another key property of vascular grafts for clinical use.
  • Acute reseeding of vascular surfaces with hiPSC-EC according to the invention had marked direct vasoactive effect.
  • the vasoactive function of hiPSC-EC may be assessed by using an isolated vessel platform.
  • aortic rings may be isolated and basal tension of the vessels, precontraction, viability of endothelium and smooth muscle cells may be verified and vasoactive function of stem cell-derived endothelial cells may be tested.
  • Viability of the endothelium may be assessed by its endothelium-dependent vasodilative response to e.g. acetylcholine.
  • Viability of the smooth muscle cells may be assessed as endothelium-independent vasodilative response to e.g. sodium-nitroprusside.
  • Conditioned medium of endothelial cells has vasodilative effects on rat aortic rings.
  • hiPSC-EC according to the invention and HUVEC supernatants significantly dilate vessels pre-constricted with phenylephrine. (FIG 7)
  • 3D vascular assays provide a platform for testing barrier function, immune response, vasoactive effects, antithrombotic surface, secretory activity, cell death and angiogenesis in in vitro cultured EC. 3D vascular assays are particularly useful in modelling human vascular responses to various pathological and drug induced effects.
  • 3D culture is inevitable to develop functional tissue engineered vascular grafts.
  • Proper endothelial char- acteristics are cornerstones of clinical translation. These include antiplatelet, vasoactive, permeability and signal transduction assays.
  • Rantes assay was developed and applied on our engineered 3D vascular constructs. Rantes assay provid- ed us with a platform to study antiplatelet effects of 3D cultured hiPSC-EC. Antiplatelet functional activity of hiPSC-EC significantly increased in 3D culture.
  • Endothelial cells affect vasomotion and tone of vasculature. These can only be investigated in 3D culture, as 2D culture lacks a dimension of vasoconstriction or vasodilatation.
  • 3D vasoactivity assay in which direct effects and endothelial responses can be measured on the level of force developed or dropped via vascular smooth muscle constriction or dilatation (respectively).
  • selective inhibition of major endothelial signalling pathways e.g. the NO-cGMP, reveals mechanism of action of vascular activity.
  • 3D vascular constructs can be exposed to any cardiovascular drugs or other medications (for instance chemotherapeutic drugs) in order to assess signal transduction or drugs related endothelial effects - both beneficial effects and disadvantageous side effects.
  • cardiovascular drugs or other medications for instance chemotherapeutic drugs
  • Endothelial permeability may be characterised by labelling 3D vascular construct with fluorescence dyes and dextran compounds.
  • a normal range of endothelial permeability can be calculated by Dextran molecular size and common endothelial permeability rate. Measuring the intensity of fluorescence dies on the other side of the exposure (technically culturing the graft on insert plates) allows quantification of increased permeability (endothelial injury) after selected treatments (e.g. exposure to chemotherapeutic drugs).
  • the invention provides a population of human iPSC-derived CD31+ cells with arterial dominancy, wherein the expression of angiogenesis related proteins, cell-matrix adhesion proteins and arterial gene markers are increased.
  • Cultured CD31+ cell population useful for forming a three-dimensional vascular endothelial construct, wherein the cells of the population are derived from human induced pluripotent stem cells and
  • the population of cells possesses angiogenic potential indicated by an increased expression of one or more angiogenesis-related protein(s)
  • the population of cells is capable of adhering to an extracellular biomatrix, indicated by an increased expression of one or more cell-matrix adhesion protein(s)
  • the population shows arterial phenotype indicated by an increased arterial/venous marker ratio.
  • Cultured CD31+ cell population useful for forming a 3D vascular endothelial construct, wherein the cell population is derived from human induced pluripotent stem cells and
  • the cell population possesses angiogenic potential indicated by an increased expression of at least one angiogenesis-related protein
  • the cell population is capable of adhering to an extracellular biomatrix, indicated by an increased expres- sion of at least one cell-matrix adhesion protein, and
  • the cell population shows arterial phenotype indicated by an increased arterial/venous marker ratio.
  • the at least one angiogenesis-related protein is selected from ACVR1B (activin receptor type-IB), angiopoietin-2, CCL3 (chemokine (C-C motif) ligand 3), CSF2 (colony stimulating factor 2), CXCL-16 (chem- okine (C-X-C-motif) ligand 16), FGF4 (fibroblast growth factor 4), HB-EGF (heparin binding epidermal growth factor-like growth factor), IGFBP2 (insulin-like growth factor binding protein 2), IL-8 (interleukin 8), NRG-1 (neuregulin 1), PDGFB (platelet derived growth factor subunit B), platelet factor 4 (PF4), PLG (plasminogen), PTX3 (pentraxin 3), serpin Fl, TGFB1 (transforming growth factor beta 1), vasohibin, VEGF-A, VEGF-C (vascular endothelial growth factor A and C), and said at least one angiogenesis-related protein shows increased expression
  • the at least one cell-matrix adhesion protein is selected from AREG (amphiregulin), CCL2 (chemokine (C-C motif) ligand 2), CD59, GDNF (glial cell derived neurotrophic factor), MMP9 (matrix metallopeptidase 9), serpin b5, THBS1 (thrombospondin 1), thrombospondin 2, TIMP2 (tissue inhibitor of metalloproteinases 2), urokinase, and said at least one cell-matrix adhesion protein shows increased expression in said CD31+ cell population as compared to the expression of said protein(s) in a HCAEC population cultured under 2D culture conditions,
  • the increased arterial/venous marker ratio is selected from increased EphrinB2 EphB4 mRNA ratio, increased expression of Notch2 and increased expression of EphrinB2 in said CD31+ cell population, as compared to the EphrinB2/EphB4 mRNA ratio, expression of Notch2 and expression of EphrinB2, respectively, in a population of human umbilical vein endothelial cells (HUVEC population) cultured under 2D culture conditions.
  • HAVEC population human umbilical vein endothelial cells
  • the cell population possesses angiogenic potential indicated by an increased expression of at least one angiogenesis-related protein selected from ACVR1B, angiopoietin-2 (ANGPT2), CCL3, CSF2, CXCL-16, FGF4, HB-EGF, IGFBP2, IL-8, NRG-1, PDGFB, platelet factor 4 (PF4), PLG, PTX3, serpin Fl, TGFB1, vasohibin (VASHl), VEGF-A, VEGF-C and said at least one angiogenesis-related protein shows increased expression in said CD31+ cell population as compared to the expression of said protein(s) in a population of human coronary arterial endothelial cells (HCAEC population) cultured under 2D culture conditions, the expression of said protein(s) in said CD31+ cell population being at least 2-fold higher than the expression of said protein(s) in said HCAEC population;
  • angiogenesis-related protein selected from ACVR1B, angiopoietin-2 (ANGPT2),
  • the cell population is capable of adhering to an extracellular bio matrix, indicated by an increased expression of one or more cell-matrix adhesion protein(s) selected from AREG, CCL2, CD59, GDNF, MMP9, serpin b5, THBS1, thrombospondin 2, TIMP2, urokinase (PLAU) and said at least one cell-matrix adhesion protein shows increased expression in said CD31+ cell population as compared to the expression of said pro- tein(s) in a population of human coronary arterial endothelial cells (HCAEC population) cultured under 2D culture conditions, the expression of said protein(s) in said CD31+ cell population being at least 2-fold higher than the expression of said protein(s) in said HCAEC population;
  • HCAEC population human coronary arterial endothelial cells
  • the CD31+ cell population shows arterial phenotype indicated by an increased arterial/venous marker ratio selected from increased EphrinB2/EphB4 mRNA ratio, increased expression of Notch2 and increased expression of EphrinB2, as compared to the EphrinB2 EphB4 mRNA ratio, expression of Notch2 and expres- sion of EphrinB2, respectively, in a population of human umbilical vein endothelial cells (HUVEC population) cultured under 2D culture conditions.
  • HAVEC population human umbilical vein endothelial cells
  • the expression of at least two, particularly at least three, more particularlyat least four or even more particularly at least five angiogenesis-related proteins selected from ACVR1B, angiopoietin-2, CCL3, CSF2, CXCL-16, FGF4, HB-EGF, IGFBP2, IL-8, NRG-1, PDGFB, platelet factor 4 (PF4), PLG, PTX3, serpin Fl, TGFBl, vasohibin, VEGF-A, VEGF-C is increased in said CD31+ cell population as compared to the expression of said proteins in a HCAEC population cultured under 2D culture conditions.
  • the expression of at least two, particularly at least three, more particularly at least four or even more particularly at least five angiogenesis-related proteins selected from angiopoietin-2, CCL3, PDGFB, PF4, serpin Fl, TGFBl, vasohibin, VEGF-A, and VEGF-C is increased in said CD31+ cell population as compared to the expression of said proteins in a HCAEC population cultured under 2D culture conditions.
  • the expression of at least two, preferably at least three angiogenesis-related proteins selected from PF4, vasohibin and VEGF-A is increased in said CD31+ cell population as compared to the expression of said proteins in a HCAEC population cultured under 2D culture conditions.
  • the expression of the at least one or the at least two, particularly the at least three, more particularly the at least four or even more particularly the at least five angiogenesis-related pro- tein(s) is at least 3 -fold or at least 4-fold higher in said CD31+ cell population than in said HCAEC population.
  • the expression of at least two, particularly at least three, more particularly at least four or even more particularly at least five cell-matrix adhesion proteins selected from AREG, CCL2, CD59, GDNF, MMP9, serpin b5, THBS1, thrombospondin 2, TIMP2, urokinase is increased in said CD31+ cell population as compared to the expression of said proteins in a HCAEC population cultured under 2D culture conditions.
  • the expression of at least one or at least two, particularly at least three, more particularly at least four or even more particularly at least five cell-matrix adhesion protein(s) selected from AREG, MMP9, CD59, urokinase, TIMP2 is increased in said CD31+ cell population as compared to the expression of said protein(s) in a HCAEC population cultured under 2D culture conditions.
  • the expression of the at least one or preferably the at least two, more preferably the at least three, more preferably the at least four or even more preferably the at least five cell- matrix adhesion protein(s) is at least 3-fold higher in said CD31+ cell populations than in said HCAEC popula- tion.
  • the expression of at least one or at least two, in particular at least three angiogenesis-related protein(s) selected from PF4, vasohibin, and VEGF-A is at least 3 -fold, preferably at least 4-fold higher in said CD31+ cell population than in said HCAEC population
  • At least one or at least two, particularly at least three, more particularly at least four or even more particularly at least five cell-matrix adhesion protein(s) selected from AREG, CD59, MMP9, urokinase, TIMP2 is at least 3-fold higher in said CD31+ cell population than in said HCAEC population.
  • the expression of EphrinB2 in said CD31+ cell population is increased as compared to a HUVEC population cultured under 2-dimensional culture conditions.
  • the population shows arterial phenotype indicated by increased EphrinB2 EphB4 mRNA ratio, as compared to the EphrinB2 EphB4 mRNA ratio in a HUVEC population cultured under 2D culture conditions, and indicated by increased expression of Notch2 and increased expression of EphrinB2 in said CD31+ cell population, as compared to the expression of Notch2 and the expression of EphrinB2, respectively in said HUVEC population.
  • the EphrinB2/EphB4 mRNA ratio in said CD31+ cell population is at least 2-fold higher than the EphrinB2/EphB4 mRNA ratio a HUVEC population cultured under 2-dimensional culture conditions.
  • the EphrinB2 EphB4 mRNA ratio in said CD31+ cell population is at least 2-fold higher than the EphrinB2/EphB4 mRNA ratio in a HUVEC population cultured under two- dimensional culture conditions and/or the expression of Notch2 is at least 4-fold higher and/or the expression of EphrinB2 is at least 2-fold higher in said CD31+ cell population than the expression of Notch2 and/or EphrinB2, respectively in said HUVEC population.
  • the expression of at least one or at least two, in particular at least three angiogenesis-related protein(s) selected from PF4, vasohibin, and VEGF-A is at least 3 -fold, in particular at least 4-fold higher in said CD31+ cell population than in said HCAEC population
  • the expression of at least one or at least two, particularly at least three, more particularly at least four or even more particularly at least five cell-matrix adhesion protein(s) selected from AREG, CD59, MMP9, urokinase, TIMP2 is at least 3-fold higher in said CD31+ cell population than in said HCAEC population
  • EphrinB2 in said CD31+ cell population is increased as compared to a HUVEC population cultured under 2D culture conditions.
  • the expression of said at least one or at least two, particularly at least three, more particularly at least four or even more particularly at least five angiogenesis-related protein(s) in said CD31+ cell population is at least 10-fold higher, in particular at least 15-fold higher than the expression of said protein(s) in said HCAEC population;
  • the expression of said at least one or at least two, particularly at least three, more particularly at least four or even more particularly at least five cell-matrix adhesion protein(s) in said CD31+ cell population is at least 10-fold higher, in particular at least 15-fold higher than the expression of said protein(s) in said HCAEC population.
  • ADAMTSl ANG
  • ANGPT1 angiopoietin 1
  • ARTN artemin
  • COL18A1 collagen type XVIII alpha 1
  • DPP4 dipeptidyl peptidase-4
  • ECGF1 EGF (epidermal growth factor)
  • PROKR2 prokineticin receptor 2
  • ENG endoglin
  • FGF1 acidic fibroblast growth factor
  • FGF2 basic fibroblast growth factor
  • HGF hepatocyte growth factor
  • IGFBP1 insulin-like growth factor binding protein 1
  • IGFBP3 insulin-like growth factor binding protein 3
  • IL1B interleukin 1 beta
  • LEP laeptin
  • CCL2 chemokine (C-C motif) ligand 2)
  • ECGF1 endo- thelial growth factor 1; TYMP (thymidine phosphorilase)
  • PDGFA chemokine (C-C motif) ligand 2
  • ECGF1 end
  • the expression of one or more protein(s) selected from ANG, TYMP, COL18A1, ADAMTS1, ADAM10, ADAM9, IGFBP3, LEP, IGFBP1, ENG, ECGF1, PLG, ANGPT1, FGF2, EGF, TIMP1, IL1B, HGF, CCL2, SERPINE1, TIMP3, FGF1, PDGFA is increased in said CD31+ cell population as com- pared to the expression of said protein(s) in said HCAEC population, said increase being at least 10-fold, in particular at least 15 -fold higher.
  • the expression of one or more protein(s) selected from ANGPT1, FGF2, EGF, TIMP1, IL1B, PLG, HGF, CCL2, SERPINE1, TIMP3, FGF1, PDGFA is increased in said CD31+ cell population as compared to the expression of said protein(s) in said HCAEC population, said increase being at least 10-fold, in particular at least 15 -fold higher.
  • the CD31+ cell population is cultured on a decellularised extracellular matrix bioscaffold, preferably on a decellularised human vascular bioscaffold or on a decellularised porcine intestinal extracellular matrix scaffold.
  • the decellularised human vascular bioscaffold is derived from human aorta.
  • the CD31+ cell population is cultured for a period of time on a decellularised extracellular matrix bioscaffold, wherein the period of time is preferably at least 1 day, at least 2 days, more preferably at least 3 days and even more preferably at least 4 days.
  • the culturing on the decellularised extracellular matrix bioscaffold is initiated after an increased expression of at least one angiogenesis-related protein together with an increased expression of at least one cell-matrix adhesion protein can be detected in the CD31+ cell population as compared to the expression of the at least one angiogenesis-related protein and the at least one cell-matrix adhesion protein, respectively, in a HCAEC population cultured under 2D culture conditions
  • the cell population shows arterial phenotype indicated by an increased arterial/venous marker ratio in said CD31+ cell population as compared to the arterial/venous marker ratio in a HCAEC population cultured under 2D culture conditions.
  • the culturing on the decellularised extracellular matrix bioscaffold is initiated when the cells show the characteristics described in any one of the numbered paragraphs I-XIV above.
  • a three-dimensional vascular endothelial construct comprising
  • an extracellular matrix bioscaffold preferably a decellularised human vascular bioscaffold or a decellularised porcine intestinal extracellular matrix scaffold, the decellularised human vascular bioscaffold being preferably derived from human aorta
  • CD31+ cell population defined in any one of the numbered paragraphs I-XIV above or in the claims, wherein the cells form a monolayer on the bioscaffold.
  • a method for providing a three-dimensional vascular endothelial construct having an arterial phenotype comprising
  • a decellularised extracellular matrix bioscaffold preferably a decellularised human vascular bioscaffold or a decellularised porcine intestinal extracellular matrix scaffold, the decellularised human vascular bioscaffold being preferably derived from human aorta,
  • the iPSC-derived cells are derived or obtained from iPSC or an iPSC line reprogrammed by viral or non-viral delivery of transcription factors.
  • the iPSC-derived cells are derived or obtained from iPSC or an iPSC line for which the following (transcription) factors have been used in the reprogramming method: Oct4, Sox2, Nanog and optionally or preferably LIN28.
  • the iPSC-derived cells are derived or obtained from iPSC or an iPSC line of mesodermal origin, preferably of mesenchymal origin, preferably of connective tissue origin, in particular of lung origin.
  • the iPSC-derived cells are derived or obtained from iPSC or an iPSC line of mesodermal origin, preferably of epithelial origin, preferably of endothelial origin.
  • the iPSC-derived cells are derived or obtained from iPSC or an iPSC line reprogrammed by a viral method, preferably a retroviral method, in particular by a lentiviral method.
  • the iPSC-derived cells are derived or obtained from iPSC or an iPSC line for which the following (transcription) factors have been used in the reprogramming method: Oct4, Sox2, Nanog and optionally or preferably LIN28, wherein the iPSC or the iPSC cell line is of mesodermal origin and preferably it has been reprogrammed by a retroviral method.
  • Human pluripotent stem cell cultures were carried out on H7 hESC line ⁇ WiCell Research Institute Bank, Madison, USA) and hiPSC line (IMR 90-4) from ReproCELL or IMR 90-4 from WiCell. Following the supplier's protocols, human PSC were maintained in their undifferentiated state in feeder cell-free conditions on Matrigel-coated ⁇ BD Biosciences, San Jose, USA) six-well plates, in mTeSRl medium ⁇ StemCell Technologies, Vancouver, Canada). Stem cell cultures were fed daily by complete media change and main- tained at 37°C, 5% C0 2 and 21% 0 2 .
  • Reprocell hiPSC line was generated by lentiviral reprogramming (OCT4, KLF4, SOX2, cMyc) using myocytes as a starting cell type. Cells were maintained in ReproFF medium.
  • H7 human embryonic stem cells h7 hESCs
  • IMR 90-4 induced pluripotent stem cells IMR 90-4 hiPSCs
  • WiCell Bank Wi, USA.
  • Cells were cultured in six-well plates (Falcon, Corning, USA) using 2 ml per well of mTeSRl complete medium (Stem Cell Technologies INC, UK) and incubated at 37°C and 5% C0 2 with the culture medium being changed daily. Cells were subcultured using a 1 :3 or 1 :6 ratio when 70-80% confluency was reached (about every 4 to 6 days).
  • DMEM medium Prior to seeding the cells, plates were coated with a 1 :30 diluted Matrigel solution (Becton Dickinson-BD, UK) in knockOut DMEM medium (Gibco, UK) and incubated for at least 30 minutes at 37°C and 5% C0 2 .
  • the cell passaging protocol included a washing step with phosphate buffer saline solution without calcium and magnesium (PBS w/o Ca-Mg) (Gibco, UK) prior to the detachment of cells which was performed using 1 ml per well of Versene solution (0.048 mM, 0.2 gr EDTA) (Gibco, UK) and incubation for 5 min at 37°C.
  • PBS w/o Ca-Mg phosphate buffer saline solution without calcium and magnesium
  • umbilical cords were collected in sterile conditions. Umbilical cords were cannulated at both sides with 3 side syringe taps and were perfused with collagenase B solution (Sigma Aldrich, 2.5mg/ml). Umbilical cords were filled with collagenase B and incubated for 30 min at 37°C. After 30 min the umbilical cords were washed out with PBS solution (three times). All enzyme and PBS solution were collected during the washing into 15ml tubes. Then the collected samples were centrifuged (1100 RPM, 10 min).
  • the cell pellets were re-suspended in EGM2 medium and plated onto gelatin-coated plates or flask (T25 and T75 cell culture flasks).
  • Gelatin was prepared with general PBS (phosphate buffer saline) and used in 0.05% concentration.
  • the endothelial cells were grown as monolayer culture, adhesion contact inhibition developed monolayer culture. Endothelial cells were passaged (1 :3 surface, at 70-75% confluency), as cells grew on the whole surface of the flasks. Enzymatic passaging was performed with trypsin-EDTA (TE) digestion. To enable activation of the enzyme, endothelial cultures were first washed in Mg 2+ and Ca 2+ rich PBS.
  • Endothelial cultures were incubated with TE at 37°C for 3 mins. Fetal bovine serum was added to inhibit further enzymatic activity. To obtain pellet of supernatants tubes were centrifuged at 1100 RPM, for 5mins. Endothelial cell pellets were resuspended in EGM2 medium and cultured on gelatin-coated flasks up to passage 6. For cryopreservation 10% DMSO, 10% foetal bovine serum and 80% EGM2 medium is used. As HCAEC, commercially available cell cultures (e.g. Lonza) was used and cultured as described to those with HUVEC.
  • HCAEC commercially available cell cultures (e.g. Lonza) was used and cultured as described to those with HUVEC.
  • the endothelial differentiation was performed in 1. normoxic (21% 0 2 ) conditions, and 2. in the presence of additional VEGF165 (10 ng/ml, Peprotech). After 13 days cells were stained with anti-human CD31-Alexa Fluor 488 fluorescence dye-labelled antibody. Cells were sorted using a FACS Aria II cell sorter (BD Biosciences) and further expanded in EGM2 medium. 3.
  • one differentiation protocol included morphogens: Activin A (10 ng/ml, R&D systems, Minnesota, USA), FGF-2 (10 ng/ml, Peprotech, New Jersey, USA), VEGF165 (10 ng/ml, R&D systems) and BMP4 (10 ng/ml, R&D systems) in mTeSRl media. After 24 hours media was removed and replaced with Stemline II complete media (Sigma-Aldrich, St. Louis, USA) containing FGF-2, VEGF165 and BMP4 (all at 10 ng/ml). Endothelial cells for 3D culture studies were differentiated with monolayer in normoxic protocol.
  • hPSC were maintained in ultra-low attachment plates in order to form embryoid bodies (simulating the blastocyst stadium of the developing embryo).
  • EBs were developed by mechanically breaking the hESC colonies as described before. EBs were cultured for four days in suspension in ultra-low attachment plates either in normoxic or hypoxic conditions. After four days, the EBs were seeded onto 0.5% gelatine (Sigma-Aldrich) coated 24-well plates or T25 flasks. After 13 days in EGM2 medium, CD31 positive endothelial cells were sorted from differentiating culture by fluorescence activated cell sorting method (FACS).
  • FACS fluorescence activated cell sorting method
  • Endothelial differentiation colonies were seeded into EGM2 medium containing EBM2 media (Lonza CC-3156, Basel, Switzerland) and SingleQuot supplements (Lonza CC-4176): growth factors, peptides, hor- mones and antibiotics.
  • EBM2 media Litrigel, Matrigel, and SingleQuot supplements.
  • SingleQuot supplements Litrigel, and antibiotics.
  • stem cells were maintained on Matrigel in monolayer.
  • the differentiation protocol included further VEGF165 supplementation (1 ng/ml Peprotech).
  • the first step included the detachment of hPSCs from the maintenance culture by performing a washing step with PBS w/o Ca-Mg prior to addition of versene solution, as previously described.
  • Cell suspension was centrifuged at 1200 rpm for 5 min leading to the formation of cell pellet and supernatant, with the supernatant being aspirated and the cell pellet being resuspended in 1 ml mTeSRl.
  • the number of cells obtained was calculated according to the trypan blue exclusion method, using a 0.4% trypan blue solution (Gibco) and a Neubauer hemocytometer (Hauser Scientific, Bright-Line, USA) under the phase contrast microscope.
  • the volume of cell suspension containing 10 5 cells was calculated and diluted in the appropriate volume to seed every well of a 24-well plate with 0.5 ml mTeSRl medium containing lOng/ml of Rho- associated protein kinase (ROCK) inhibitor (Gibco, UK).
  • ROCK Rho- associated protein kinase
  • the 24-well plates Prior to seeding, the 24-well plates (Greiner bioOne, UK) were coated with 1:30 matrigel/ KnockOut DMEM solution. After seeding, cell cultures were incubated for two days (Day -2) at 37°C and 5% C0 2 .
  • the media was replaced with 1ml mTeSRl me- dium per well containing the growth factors Activin A (R&D systems, UK, #338-AC), bFGF (R&D systems, UK, #4114-TC), VEGF165 (Peprotech, UK, #100-20) and BMP4 (R&D systems, UK, #314-PB/CF) all at a concentration of 10 ng/ml, in order to initiate the differentiation.
  • Activin A R&D systems, UK, #338-AC
  • bFGF R&D systems, UK, #4114-TC
  • VEGF165 Peprotech, UK, #100-20
  • BMP4 R&D systems, UK, #314-PB/CF
  • RNA samples were collected on day 0, day 5 and day 12 to monitor changes in expression profile sorted cells for pluripotent, endothelial and mesenchymal/fibroblast genes during differentiation.
  • FACS fluorescence activated cell sorting
  • the cells in medium were transferred to a 50 ml falcon tube (Falcon, Corning, USA) and the wells were rinsed with another volume of 10% FBS/Stemline II medium to collect any remaining cells. Collected cells were dispersed by gentle pipetting and filtered using a 70 ⁇ strainer (Falcon) to eliminate clumps and debris. After this, cells were counted (trypan blue exclusion) to make a rough estimation about their number and viability. Suspended cells were centrifuged at 1200 rpm for 5 min and then were resuspended in 130 ⁇ of FACS/blocking buffer 1% Fetal Bovine Serum (FBS) (Gibco, UK) in PBS w/o Ca-Mg.
  • FBS Fetal Bovine Serum
  • Cell samples were prepared including an unstained control (10 ⁇ cell suspension in 500 ⁇ FACS buffer), single stained controls (10 ⁇ cell suspension in 100 ⁇ FACS buffer) and the full stained sample to be sorted (100 ⁇ cell suspension).
  • fluorescent antibodies for anti- human CD31 AlexaFluor 488-conjugated (BD, UK, #557703) and NRP-1 APC-conjugated (Miltenyi Biotec, UK, #130-090-900) were added at a dilution rate of 1 :20 and 1 : 11, respectively up to a 1 10 7 cells/ FACS buffer concentration.
  • samples were mixed using a vortex in low speed for 30 seconds and then incubated at 4°C for 25 minutes in the dark.
  • Sorted CD31 / NP-1 positive cells were collected in sterile polystyrene round bottom tubes (BD) containing 1ml of 40% Stemline II medium, 40% of full supplemented endothelial growth factor medium-2 (EGM-2) (EBM-2 plus all SingleQuot supplements) (Lonza, UK), 20% FBS and 1% Penicillin/Streptomycin solution (P/S) (Gibco, UK). A small proportion of the sorted cells were reanalyzed to check sorting efficiency and purity. 5000 cells were seeded per well onto 24-well plates coated with collagen IV (Sigma- Aldrich, UK, #C7521) and 1 ml of 50% EGM-2 medium, 50% Stemline II medium and 1% P/S.
  • EGM-2 endothelial growth factor medium-2
  • P/S Penicillin/Streptomycin solution
  • hPSC-ECs hPSC-derived endothelial cells
  • EGM-2 full medium was replaced every two days until cells reached 80% confluency.
  • cells could be used for setting up an experiment, further expanded in collagen IV coated T25 flasks at a 1 :3 ratio or could be cryopreserved.
  • hESC-EC and hiPSC-EC spinner flask bioreactors were used. Endothelial cells were seeded on decellularised human aortic samples or on decellularised porcine intestinal mucosal matrix (Cormatrix, Roswell, GA). Human aortic rings and wall segments were collected from heart and vascular sur- gery operations (heart transplantation and aortic aneurysm surgery) and stored at the Biobank of the Heart and Vascular Centre, Semmelweis University, Budapest (approved by ETT TUKEB 7891/2012/EKU regional ethics committee).
  • Aortic samples were decellularised, using detergent solution containing 2% sodium-dodecyl- sulphate and 0.05% sodium-azide in PBS (all from Sigma-Aldrich). Detergent solution was changed every 6 hours upon total 72 hours of detergent wash.
  • the decellularised aortic samples were sliced into 300 ⁇ thin slices by microtome (Campden Instruments Lafayette, USA). Decellularised samples were washed in sterile PBS with 2% penicillin/streptomycin for further 48 hours and then preconditioned in EGM2 medium. Some slices were homogenized to test DNA/RNA content (isolation described later). Decellularisation was confirmed by diminishing all DNA and RNA components, measured by Nanodrop. For recellularisation, the decellularised aortic slices were cut for standard size (0.3 cm 2 ) and 10 6 cells per well of 96-well plates were seeded.
  • CorMatrix® (CorMatrix ECM Technologies, Roswell, USA) is a commercially available ECM for heart and vascular surgery.
  • CorMatrix® is derived from porcine small intestinal submucosa. During the manufacturing process CorMatrix® is decellularised, thus only ECM proteins, such as collagen, elastin and adhesion proteins remain. Clinical use of CorMatrix® has already received FDA approval for vascular and cardiac repair.
  • CorMatrix® is preconditioned in EGM2 media and antibiotics (Penicillin Streptomycin) at 37°C before endothelial cell seeding.
  • CorMatrix® is stored and manipulated in sterile conditions. As CorMatrix® is designed as a cardiac patch vascular stamps can be engineered.
  • ECM ECM Preconditioning of ECM was performed in the same incubator system as used for cell maintenance. Keeping the ECM in physiological conditions enabled softening of the tissue and cellular growth factors could settle inside the matrix components in biologically active form.
  • the endothelial cells were seeded onto aortic pieces cut for standard size (0.3cm 2 ) and 10 6 cells/sample were seeded.
  • the aortic slices were mounted into the bottom of 96-well plates and endothelial cells were seeded on top, thus endothelial cells proliferate mainly on the top luminal surface of the aortic samples, resulting in a monolayer endothelial culture. Fresh medium was added every other day.
  • the small bioreactor system composed of scale-up spinning flask (Corning, Basel, Switzerland), endothelial growth media, endothelial cells and extracellular matrix as a carrier for endothelial growth (50000 endothelial cells/ 0.5cm 2 matrix).
  • the small bioreactor system was kept under physiologic conditions (37°C, 85% humidity, 21% 0 2 ) for endothelial growth.
  • a magnetic stirrer is integrated into the spinning flask which enables shaking together endothelial cells and the scaffolds.
  • Bioscaffolds along with hPSC-EC were seeded into the spinning flasks. Every other day fresh media was added to the flasks.
  • Duration of reseeding for 5 days was optimized in pilot dose- and duration finding experiments (using 3 to 5 days protocols).
  • endothelial re-cellularisation vital imaging Calcein AM, Thermo Fisher Scientific
  • nuclear imaging with Hoechst-33342 or DAPI staining were performed.
  • Confo- cal microscopy and PCR analyses were performed to verify endothelial seeding on biomatrices. Endothelial proliferation on the 3D vascular constructs were also visualised by microscopy.
  • AlexaFluor 546 anti-rabbit antibody raised in donkey was used (Thermo Fischer Scientific, Walthman, Massachusetts USA A10040, dilution 1/400). Cells were washed between incubations with PBS. Cell nuclei were counterstained with DAPI (Thermo Fischer Scientific D21490, dilution 1/5000). Plates were stored in PBS at 4°C prior to imaging using a Zeiss Observer microscope or Cellomics VTi HCS ArrayScanner (Thermo Fisher Scientific, Waltham, USA), as described earlier (Merkely et al., 2015). Some samples were treated with secondary antibody only in order to determine levels of non-specific background staining.
  • Matrigel tube formation and Ac-LDL assays Matrigel tube formation assay was performed on endothelial cells as described previously (Merkely et al., 2015). To identify endothelial cells based on their increased metabolism of Ac-LDL, l,r-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (Dil-Ac-LDL) labelled with fluorescent probe (Thermo Fischer Scientific) was used. Cells were incubated with 10 ⁇ g/ml Dil- Ac-LDL for 4 hours at 37°C and subsequently examined by fluorescence microscopy.
  • Dil-Ac-LDL l,r-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate
  • fluorescent probe Thermo Fischer Scientific
  • Proteome profiling Proteome profiling measurements were carried out on Proteome Profiler Human Angiogenesis Array Kit (R&D System ARY007) and Human Soluble Receptor Hematopoietic Array Kit (R&D System ARY011). Sample preparation and experimental setup followed product guide. Pixel densities of the resulted chemiluminescent signals were analysed by ImageJ software. As our primary aim was to investigate arterial-like endothelial function in hESC-EC and hiPSC-EC, HCAEC and HMVEC were selected as control endothelial cells.
  • Antiplatelet assay To characterise antiplatelet function of hPSC-EC, levels of Rantes/CCL5 chemokine were measured in antiplatelet assay on 3D vascular constructs in vitro. In this set of experiments 1. 2D endothelial cultures, 2. engineered 3D constructs and 3. acellular biomatrices alone were incubated with platelet rich plasma (4.) of healthy adults. The levels of Rantes/CCL5 were measured from platelet rich plasma with ELISA assay (R&D Systems DRNOOB). To prepare platelet rich plasma, blood samples were collected in trisodium- citrate tubes. Tubes were shaken gently immediately after blood sampling to enhance dilution of anticoagulant in blood.
  • the vasoactive function of hPSC-EC was assessed by using an isolated vessel platform (Radovits et al., 2013).
  • the in vitro organ bath experiments allowed us studying vasoactive characteristics of hPSC-EC in an angio-myograph system (Radnoti Glass Technology). Briefly, aortic rings of young, adult Sprague-Dawley rats (250-350g, Charles-River) were used. The animals received general housing: room temperature and 12 hours' light/dark cycles, standard laboratory diet, free access for food and water. All animal care and handling fulfilled the Guide for Care and use of Laboratory Animals, published by U.S. National Institutes of Health. The experimental protocols were approved by the Animal Use and Care Committee of Semmelweis University, Budapest.
  • aortic rings were applied into triangles of stainless steel and positioned between two arms of isometric force transducers.
  • Each glass chambers contained 30 ml of pre-warmed (37°C) Krebs solution, supplied with 95% 0 2 and 5% C0 2 .
  • Basal tension was set at 2g of force, followed by incubation for 60 minutes, Krebs was changed at 30 min; to reach maximum plateau of contraction, vessels were treated with KC1 (80mM). Vessels were precontracted with phenylephrine (10 "6 M, Sig- ma-Aldrich).
  • Viability of the endothelium was assessed by its endothelium-dependent vasodilative response to acetylcholine (Ach) (10 "9 -10 "4 M, Sigma-Aldrich). Viability of the smooth muscle cells was assessed as endothe- lium-independent vasodilatative response to sodium-nitroprussid (SNP) (10 "10 -10 "5 M) (Sigma-Aldrich); supernatant of 10 6 hPSC-ECs (5ml) were added to each bath. Data from contraction force were registered, digitalised and stored on LabChart7 (Powerlab).
  • SNP sodium-nitroprussid
  • NanoSPECT/CT and PET/MRI systems which are in vivo high-resolution dual modality small animal imaging platforms to acquire whole-body images.
  • albumin By radiolabelled albumin, a local significant increase in perfusion was detected at the grafted sites at 2 weeks after subcutaneous implantation of hESC-EC and hiPSC, suggesting the functional incorporation of cells into the microvasculature.
  • PET/MRI for detecting angiogenesis with 68Gallium-NOTA-RGD2 peptide (for ⁇ 3 integrin) showed an increased level of signal on the site of Matrigel plugs.
  • GPDH Hs02758991_gl Human glyceraldehyde 3-phosphate dehydrogenase
  • PCR was performed with real-time PCR instrument (Applied Biosystem, StepOnePlus) and the relative expressions were determined by AACt method, a convenient way to calculate relative gene expression levels between different samples in that it directly uses the threshold cycles (CTs) generated by the qPCR system for calculation.
  • CTs threshold cycles
  • Proteomics expression statistics used the z score method to calculate standard deviations from the mean for each data point in a normal population of data set.
  • the method and cells according to Harding et al. (2017) were compared with the method and cells according to the invention.
  • the experiment was performed using a research grade human induced pluripotent line (IMR).
  • IMR human induced pluripotent line
  • the method described by Harding et al. (2017) showed very low efficiency (FIG 14).
  • Both CD31 and VE-cadherin endothelial markers were significantly higher in the cell population obtained by the method of the invention.
  • Arterial Notch ligand dll4 showed upregulation as compared to undifferentiated stem cells in the cell population obtained by the method of the invention, while no such change has been detected in the other population (FIG 14).
  • Levels of most of the angiogenic factors were markedly lower in the cells from Harding protocol (FIG 15), making these cells unsuitable for angiogenic assays and for vascular endothelial constructs.
  • Yoder MC Differentiation of pluripotent stem cells into endothelial cells. Curr Opin Hematol 2015 May;22(3):252-7.

Abstract

The invention relates to a method of providing CD31+ cells suitable for forming 3D vascular construct, said cells expressing an increased amount of angiogenesis related and cell-matrix adhesion related proteins and having an arterial phenotype.

Description

METHODS FOR PRODUCING ENDOTHELIAL CELLS AND VASCULAR ENDOTHELIAL CONSTRUCTS DERIVED FROM INDUCED HUMAN PLURIPOTENT STEM CELLS
FIELD OF THE INVENTION
The invention relates to a method of providing CD31+ cells suitable for forming 3D vascular construct, said cells expressing an increased amount of angiogenesis related and cell-matrix adhesion related proteins and having an arterial phenotype.
BACKGROUND OF THE INVENTION
Endothelial derivatives of human pluripotent stem cells (hPSC) can be new sources of cells for regenerative therapies in cardiovascular diseases as well as means for drug toxicity and efficacy testing. Tissue-specific variations and phenotypic heterogeneity of endothelial cells (EC) from different sources (Nolan et al., 2013) can however be a challenging part for their translation into the clinic. Earlier studies aimed at improving function of hPSC-EC and enhancing their angiogenic activity (Merkely et al., 2015; Chatterjee et al., 2016; Yoder 2015). Human embryonic stem cells (hESC) have emerged as one of the most promising sources of new cardiac cells for transplantation because of their capacity to efficiently undergo directed differentiation into genuine cardiomyocytes and supportive endothelial cells. A number of research groups have successfully isolated cardiomyocytes or cardiac progenitor cells from differentiating ESC cultures (Wang et al., 2006; Zandstra et al., 2003). Recent preclinical data suggest that co-transplantation of hESC-derived cardiovascular cells with mesenchymal stem cells (MSC) may enhance the beneficial effects of hESC derivatives. MSC may support the engraftment and survival of implanted hESC-derivatives via anti-inflammatory and immunosuppressive effects (Rubach et al., 2014). The first clinical trials with hESC-derivatives in cardiovascular diseases are launched (Transplantation of Human embryonic Stem Cell-derived Progenitors in Severe Heart Failure (ESCORT, NCT02057900)).
Besides ESC, human induced pluripotent stem cells (hiPSC) are another possibilities in basic research, trans- lational medicine and therapy. High yields of iPSC-derived EC have been achieved by several differentiating protocols using two or three-dimensional (3D) culture conditions (Ikuno et al. 2017). 3D culture conditions have been used to improve endothelial differentiation efficiency (Orlova et al., 2014) or to model in vivo conditions with higher fidelity (EP 2580319).
Other studies have used clear differentiation protocols to induce iPSC to differentiate into arterial and ve- nous endothelial subpopulations (Sriram et al., 2015, Ikuno et al. 2017). Others have managed to differentiate arterial endothelial cells from mouse pluripotent stem cells (Kang et al., 2016).
Ikuno et al. have developed a differentiation method comprising stage-specific stimulation with VEGF and cAMP combined with the elimination of non-responder cells at early EC stage, showing very high efficiency. The method achieves induction and maintenance of an early stage EC population, which may be modified to take up an arterial or venous profile (Ikuno et al. 2017). The authors suggest their method for 3D tissue engineering and regenerative medicine.
WO 2016/011283 describes endothelial cells derived from iPSC, wherein the differentiation occurs in the presence of leptin and the cells are further cultured on a scaffold to obtain microvessels. The endothelial cells in the microvessel express one or more of CD31, VE-cadherin, von Willebrand factor, KLF-2 and KLF-4.
WO 2014/200340 describes a method to produce a cell culture of endothelial cells and smooth muscle cells, wherein the cells are obtained by culturing pluripotent stem cells in a medium containing ActivinA, BMP4, VEGF and a canonical WNT ligand or GSK3 inhibitor and another medium containing VEGF and a TGFb signalling inhibitor in 2D culture.
WO 2017/011748 recommends overexpression of Sirtuin-1 to regulate cell senescence, and thereby enhance iPSC-EC performance in a 2D culture.
PSC-derived EC should meet many requirements to be used in generating vascular models for drug screening and vascular grafts for implantation. A three-dimensional vascular construct comprising PSC-EC on a scaffold is highly desirable in drug screening tests, for studying the genetics and molecular pathways of vascular differentiation and vasculogenesis, as well as for implantable vascular grafts for the treatment of vascular dis- eases. Besides being able to function as mature EC in vitro and in vivo, PSC-EC should also be capable of potent angiogenesis and to readily adhere to natural or synthetic matrices. Arterial endothelial cells would be preferable in many clinical scenarios, as arterial endothelial cells are more atheroprotective and venous endothelial cells are more atheroprone (Conway et al., 2010).
Harding et al. (2017) have described a protocol to differentiate hiPSCs into endothelial cells with high purities without cell sorting. However, in our hands, this protocol had a very low efficiency and reproducibility, and the levels of arterial markers (such as dll4) and angiogenic factors remained moderate. There is thus still a need for a method for providing CD31+ cells useful in 3D vascular assays and for forming three-dimensional vascular endothelial constructs which may be used as implantable grafts.
SHORT DESCRIPTION OF THE INVENTION L A method for providing a CD31+ cell population for forming a three-dimensional (3D) vascular endothelial construct, wherein the cells of the population are capable of angiogenesis, capable of adhering to a 3D matrix and have an arterial phenotype,
said method comprising
i) seeding a starting population of CD31+ cells on a decellularised 3D scaffold used as a culturing 3D scaffold, wherein the starting population of CD31+ cells are derived from human induced pluripotent stem cells and was cultured under 2 dimensional (2D) culture conditions,
ii) culturing the seeded starting population of CD31+ cells of i) on the culturing 3D scaffold for at least one day to provide 3D cultured CD31+ cells
and
iii) isolating the 3D cultured CD31+ cells obtained in ii) from the culturing 3D scaffold at a point in time when
the expression of one or more protein(s) selected from the group consisting of angiogenesis related and cell-matrix adhesion related proteins ADAMTSl, ANG (angiogenin), ANGPT1 (angiopoietin 1), ARTN (artemin), COL18A1 (collagen type XVIII alpha 1), DPP4 (dipeptidyl peptidase-4), (ECGF1, endothelial cell growth factor 1), EGF (epidermal growth factor), PROKR2 (prokineticin receptor 2), ENG (endoglin), FGF1 (acidic fibroblast growth factor), FGF2 (basic fibroblast growth factor), HGF (hepatocyte growth factor), IGFBP1 (insulin-like growth factor binding protein 1), IGFBP3 (insulin-like growth factor binding protein 3), ILIB (interleukin 1 beta), LEP (leptin), CCL2 (chemokine (C-C motif) ligand 2), PDGFA (platelet-derived growth factor subunit A), PIGF (phosphatidylinositol-glycan biosynthesis class F protein), PLG, PSPN (persephin), SERPINEl, TIMPl (tissue inhibitor of metalloproteinases 1), TIMP4 (tissue inhibitor of metalloproteinases 4), TYMP, ADAM9 (disintegrin and metalloproteinase domain-containing protein 9), ADAM10 (disintegrin and metalloproteinase domain-containing protein 10), CD9, LAP3 (leucine aminopeptidase 3), TIMP3 (tissue inhibitor of metalloproteinases 3) is increased in said 3D cultured CD31+ cells obtained in ii) as compared to the expression of said protein(s) in a population of human coronary arterial endothelial cells (HCAEC population) cultured under 2D culture conditions, wherein said increase is at least 10- fold and
arterial phenotype is indicated by an increased arterial marker level compared to the corresponding arterial marker level in a population of human umbilical vein endothelial cells (HUVEC population) cultured under 2D culture conditions, wherein said arterial marker level is selected from the group consisting: EphrinB2 EphB4 mRNA ratio, wherein the EphrinB2/EphB4 mRNA ratio in the cells obtained in ii) is at least 2-fold higher than the EphrinB2/EphB4 mRNA ratio in said HUVEC population, expression of Notch2, wherein the expression of Notch2 is at least 4-fold higher in the cells obtained in ii) than the expression of Notch2 in said HUVEC population, expression of EphrinB2, wherein the expression of EphrinB2 is at least 2-fold higher in the cells obtained in iii) than the expression of EphrinB2 in said HUVEC population. In preferred embodiments the cells are cul- tured for at least 2 days, more preferably for at least 3 days and even more preferably for at least 4 days and most preferably for more than 5 days in ii). Preferably, the one or more protein(s) selected from ANG, TYMP, COL18A1, ADAMTSl, ADAM10, ADAM9, IGFBP3, LEP, IGFBP1, ENG, ECGF1, PLG, ANGPT1, FGF2, EGF, TIMPl, ILIB, HGF, CCL2, SERPINEl, TIMP3, FGFl, PDGFA. Preferably, the expression of said one or more protein(s) is at least 15 fold increased.
2. Preferably, in iii)
the expression of one or more protein(s) selected from the group consisting of angiogenesis related proteins ANG, ANGPTl, ARTN, DPP4, EGF, ENG, FGFl, FGF2, ILIB, LEP, PDGFA, CCL2, PLG, CD9, LAP3is increased in said 3D cultured CD31+ cells obtained in ii) compared to the expression of said protein(s) in a population of human coronary arterial endothelial cells (HCAEC population) cultured under 2D culture conditions wherein said increase is at least 10-fold, and
the expression of one or more protein(s) selected from the group consisting of cell-matrix adhesion related proteins ADAMTSl, COL18A1, ECGF1, HGF, IGFBP1, IGFBP3, TIMPl, TIMP4, TYMP, ADAM9, ADAMIO, SERPINEl, TIMP3, PIGF, PSPN, PROKR2 is increased in said 3D cultured CD31+ cells obtained in ii) compared to the expression of said protein(s) in a population of human coronary arterial endothelial cells (HCAEC population) cultured under 2D culture conditions, wherein said increase is at least 10-fold.
3. Preferably, in iii) the one or more of angiogenesis related protein(s) is/are selected from the group consisting of ANGPTl, FGF2, EGF, ILB1, PLG, CCL2, FGFl, PDGFA, and
the one or more of cell-matrix adhesion related protein(s) is/are selected from the group consisting of TIMPl, HGF, SERPINEl, TIMP3.
4. Preferably, the one or more protein(s) in iii) is selected from ANGPTl, FGF2, EGF, TIMPl, ILIB,
PLG, HGF, CCL2, SERPINEl, TIMP3, FGFl, PDGFA. Preferably, the expression of said one or more pro- tein(s) is at least 15 fold increased.
5. Preferably, in the starting CD31+ cell population at the time of i)
- the expression of at least one, preferably at least two, more preferably at least three angiogenesis- related proteins selected from PF4, vasohibin (VASH1), and VEGF-A is at least 3-fold, preferably at least 4- fold higher than in a population of HCAEC cultured under 2D culture conditions,
- the expression of at least one, preferably at least two, more preferably at least three, more preferably at least four, even more preferably at least five cell-matrix adhesion proteins selected from AREG, CD59, MMP9, urokinase, TIMP2 is at least 3 -fold higher than in a population of HCAEC cultured under 2D culture conditions, - the EphrinB2/EphB4 mRNA ratio is at least 2-fold higher than the EphrinB2/EphB4 mRNA ratio in said HUVEC population and/or the expression of Notch2 is at least 4-fold higher and/or the expression of EphrinB2 is at least 2-fold higher than the expression of Notch2 and/or the expression of EphrinB2, respectively, in a HUVEC population cultured under 2D culture conditions.
6. Preferably, iii) is carried out at a point in time when expression of at least one angiogenesis-related protein selected from ACVR1B, angiopoietin-2 (ANGPT2), CCL3, CSF2, CXCL-16, FGF4, HB-EGF, IGFBP2,
IL-8, NRG-1, PDGFB, platelet factor 4 (PF4), PLG, PTX3, serpin Fl, TGFB1, vasohibin, VEGF-A, VEGF-C is increased at least 104 fold in the cells obtained in ii) as compared to the expression of the at least one angiogenesis-related protein in a population of human pluripotent stem cells.
7. More preferably, iii) is carried out at a point in time when
the expression of at least one angiogenesis-related protein selected from ACVR1B, angiopoietin-2,
CCL3, CSF2, CXCL-16, FGF4, HB-EGF, IGFBP2, IL-8, NRG-1, PDGFB, platelet factor 4 (PF4), PLG, PTX3, serpin Fl, TGFB1, vasohibin, VEGF-A, VEGF-C is increased at least 5 fold, preferably at least 10 fold, more preferably about 5-21 fold in the cells obtained in ii) as compared to the expression of the at least one angiogenesis-related protein in HCAEC population cultured under 2D culture conditions,
the expression of at least one cell-matrix adhesion protein selected from AREG, CCL2, CD59, GDNF,
MMP9, serpin b5, THBS1, thrombospondin 2 (THBS2), TIMP2, urokinase is increased at least 5 fold, preferably at least 10 fold, more preferably about 5-21 fold in the cells obtained in ii) as compared to the expression of the at least one cell-matrix adhesion protein in HCAEC population cultured under 2D culture conditions.
8. Even more preferably, iii) is carried out at a point in time when
the expression of PF4, vasohibin and/or VEGFA is increased at least 8 fold, preferably at least 10 fold in the cells obtained in ii) as compared to the expression of PF4, vasohibin and/or VEGFA, respectively, in a HCAEC population cultured under 2D culture conditions,
the expression of at least one, preferably at least two, preferably at least three, more preferably at least four, most preferably five proteins selected from AREG, CD59, MMP9, urokinase, TIMP2 is increased at least 3 fold, preferably at least 5 fold, more preferably at least 10 fold, most preferably at least 50 fold in the cells obtained in ii) as compared to the expression of the corresponding protein(s) in a HCAEC population cultured under 2D culture conditions and
the EphrinB2 EphB4 mRNA ratio in the cells obtained in ii) is at least 2-fold higher than the EphrinB2 EphB4 mRNA ratio in said HUVEC population and/or the expression of Notch2 is at least 4-fold higher and/or the expression of EphrinB2 is at least 2-fold higher in the cells obtained in ii) than the expression of Notch2 and/or the expression of EphrinB2, respectively, in a population of human umbilical vein endothelial cells (HUVEC population) cultured under 2D culture conditions and/or the level of dll4 in said 3D cultured CD31+ cells is increased at least 4 fold, preferably at least 5 fold, more preferably at least 10 fold compared to undifferentiated human pluripotent stem cells.
9. Highly preferably, the starting population of CD31+ cells is obtainable, preferably produced by a method comprising
a) providing human induced pluripotent stem cells on adherent plates in pluripotent stem cells media, preferably mTeSRl,
b) mechanical dissociation of stem cell colonies to develop embryoid bodies (EBs) and culturing said EBs in suspension on low adherent culture dishes,
c) mesodermal induction via administration of growth factors and small molecules in culture media, wherein said growth factors and small molecules comprise: foetal bovine serum, human epidermal growth factor, vascular endothelial growth factor, human fibroblast growth factor B, insulin growth factor R3, hydrocortisone, heparin, ascorbic acid, antibiotic,
d) seeding and culturing EBs onto gelatine coated plates or flasks,
e) trypsinising the cultures obtained in d),
f) centrifuging the trypsinised cultures and re-suspending pellets in PBS, counting cells,
g) labelling the cells with a label suitable for fluorescence activated cell sorting (FACS),
h) sorting CD31 positive endothelial cells by using FACS,
i) culturing and expanding CD31-positive cells in gelatinised flasks,
j) maintaining cells in EGM2 medium, feeding on every other day with EGM2 medium,
k) 1 :3 surface passaging cells on every 3-6 day according to confluency (up to 80%) and proliferation rate.
In a preferred embodiment, a) + b) are carried out within 4 days and d) is initiated on day 4. In a pre- ferred embodiment, c) is carried out one day before d). In a preferred embodiment the following medium is used in c): EBM2 medium (500 ml), 2% FBS (10 ml), human epidermal growth factor (500 μΐ), vascular endothelial growth factor (500 μΐ), human fibroblast growth factor B (500 μΐ), insulin growth factor R3 (500 μΐ), hydrocortisone (200 μΐ), heparin (500 μΐ), ascorbic acid (500 μΐ), amphotericin B/gentamycin (500 μΐ). In a preferred embodiment, EBs are seeded in d) onto 0.5% gelatine coated 24-well plates or T25 flasks. In a preferred embod- iment, d) is carried out for 8 or 9 days. In a preferred embodiment, 0.25% Trypsin and 0.03% EDTA is used in e). In a preferred embodiment, the cultures are centrifuged with 1100 RPM for 5 min in f) and pellets are re- suspended in PBS, containing 1% FBS (FACS buffer). Preferably, cells are counted before sorting. Preferably, cells are labeled in g) with anti-CD31 antibody, conjugated with Alexa Fluor 488 fluorophore (1 :20 dilution). In preferred embodiments normoxia (21% 02) is maintained during culture. In preferred embodiments, 0.1-1%, preferably 0.5% gelatinised flasks and plates are used.
10. Preferably, ii) is carried out in a bioreactor spinner flask at 35°C to 38°C, 75-90% humidity, about 21% 02, with steering at 70-75 RPM and with addition of fresh medium every other day, preferably wherein the temperature is about 37°C to 37.5°C and the humidity is about 85%. Preferably, the medium is EGM2.
11. Preferably, ii) is carried out in a density of 25-75000 endothelial cells/0.5cm2 culturing 3D scaffold, preferably wherein the density is about 50000 endothelial cells/0.5cm2 culturing 3D scaffold.
12. Preferably, the culturing 3D scaffold is a decellularised human extracellular biomatrix, preferably derived from human aorta.
13. In preferred embodiments the decellularised human extracellular biomatrix is produced by a method comprising:
- harvesting aortic samples from a human, - decelullarising said aortic samples by
- washing the samples in detergent solution for 60-80 hours, preferably for about 72 hours,
- washing the aortic samples in PBS + antibiotics for 60-80 hours, preferably for about 72 hours to provide decellularised aortic samples,
- preconditioning the acellular aortic samples in EGM2 media prior to cell seeding. In a preferred embodiment the antibiotics is amphotericin B or gentamycin. Preferably the detergent is 0.1% sodium dodecyl sulphate and sodium azide and preferably the concentration of the antibiotics is 1%.
14. In preferred embodiments the method further comprises seeding the cells isolated in iii) on a 3D matrix, preferably wherein the 3D matrix is suitable for forming a 3D vascular endothelial construct for use in a 3D vascular assay or the extracellular biomatrix is suitable for forming an implantable vascular graft.
15. CD31+ cells for forming a 3D vascular endothelial construct are provided, wherein the cells are capable of adhering to a 3D matrix, capable of angiogenesis and have an arterial phenotype, obtained by the method according to any one of points 1-14.
16. According to another aspect of the invention, a method for providing a CD31+ cell population for forming a 3D vascular endothelial construct is provided, wherein the CD31+ cell population is capable of adhering to a 3D matrix, and has an arterial phenotype, the method comprising
A) providing human induced pluripotent stem cells on adherent plates in pluripotent stem cells media, preferably mTeSRl,
B) mechanical dissociation of stem cell colonies to develop embryoid bodies (EBs) and culturing said EBs in suspension on low adherent culture dishes,
C) mesodermal induction via administration of growth factors and small molecules in culture media, wherein said growth factors and small molecules comprise: foetal bovine serum, human epidermal growth factor, vascular endothelial growth factor, human fibroblast growth factor B, insulin growth factor R3, hydrocortisone, heparin, ascorbic acid, antibiotic,
D) seeding and culturing EBs onto gelatine coated plates or flasks,
E) trypsinising the cultures obtained in D),
F) centrifuging the trypsinised cultures, and re-suspending pellets in PBS, counting cells,
G) labelling the cells with a label suitable for fluorescence activated cell sorting (FACS),
H) sorting CD31 positive endothelial cells by using FACS,
I) culturing and expanding CD31-positive cells in gelatinised flasks,
J) maintaining cells in EGM2 medium, feeding on every other day with EGM2 medium,
K) 1:3 surface passaging cells on every 3-6 day according to confluency and proliferation rate, wherein in the cells obtained in J)
- the expression of at least one, preferably at least two, more preferably at least three angiogenesis- related proteins selected from PF4, vasohibin, and VEGF-A is at least 3 -fold, preferably at least 4-fold higher than in a population of HCAEC cultured under 2D culture conditions,
- the expression of at least one, preferably at least two, more preferably at least three, more preferably at least four, even more preferably at least five cell-matrix adhesion proteins selected from AREG, CD59, MMP9, urokinase, TIMP2 is at least 3 -fold higher than in a population of a HCAEC cultured under 2D culture condi- tions, - the EphrinB2/EphB4 mRNA ratio is at least 2-fold higher than the EphrinB2 EphB4 mRNA ratio in a HUVEC population cultured under 2D culture conditions and/or the expression of Notch2 is at least 4-fold higher and/or the expression of EphrinB2 is at least 2-fold higher than the expression of Notch2 and/or the expression of EphrinB2, respectively, in the HUVEC population and/or the level of dll4 in said 3D cultured CD31+ cells is increased at least 4 fold, preferably at least 5 fold, more preferably at least 10 fold compared to undifferentiated human pluripotent stem cells. In a preferred embodiment, A) + B) are carried out within 4 days and D) is initiated on day 4. In a preferred embodiment, C) is carried out one day before D). In a preferred embodiment the following medium is used in C): EBM2 medium (500 ml), 2% FBS (10 ml), human epidermal growth factor (500 μΐ), vascular endothelial growth factor (500 μΐ), human fibroblast growth factor B (500 μΐ), insulin growth factor R3 (500 μΐ), hydrocortisone (200 μΐ), heparin (500 μΐ), ascorbic acid (500 μΐ), amphotericin B/gentamycin (500 μΐ). In a preferred embodiment, EBs are seeded in D) onto 0.5% gelatine coated 24-well plates or T25 flasks. In a preferred embodiment, D) is carried out for 8 or 9 days. In a preferred embodiment, 0.25% Trypsin and 0.03% EDTA is used in E). In a preferred embodiment, the cultures are centrifuged with 1100 RPM for 5 min in F) and pellets are re-suspended in PBS, containing 1% FBS (FACS buffer). Preferably, cells are counted before sorting. Preferably, cells are labeled in G) with anti-CD31 antibody, conjugated with Alexa Fluor 488 fluorophore (1 :20 dilution). In preferred embodiments normoxia (21% 02) is maintained during culture. In preferred embodiments, 0.1-1%, preferably 0.5% gelatinised flasks and plates are used.
17. A preferred embodiment is the method according to point 16, further comprising
L) seeding the cells obtained in K) on a decellularised 3D scaffold used as a culturing 3D scaffold, M) culturing the CD31+ cells seeded in L) on the culturing 3D scaffold for at least one day to provide 3D cultured CD31+ cells
and
N) isolating the CD31+ cells obtained in M) from the culturing 3D scaffold at a point in time when the expression of one or more protein(s) selected from ADAMTS1, ANG, ANGPT1, ARTN, COL18A1, DPP4, ECGF1, EGF, PROKR2, ENG, FGF1, FGF2, HGF, IGFBP1, IGFBP3, IL1B, LEP, CCL2, PDGFA, PIGF, PLG, PSPN, SERPINE1, TIMP1, TIMP4, TYMP, ADAM9, ADAMIO, CD9, LAP3, TIMP3 is increased in the cells obtained in M) as compared to the expression of said protein(s) in a HCAEC population cultured under 2D culture conditions, wherein said increase is at least 10-fold and
and the cells show arterial phenotype indicated by an EphrinB2/EphB4 mRNA ratio which is at least 2- fold higher than the EphrinB2/EphB4 mRNA ratio in a HUVEC population cultured under 2D culture conditions and/or an expression of Notch2 which is at least 4-fold higher and/or an expression of EphrinB2 which is at least 2-fold higher than the expression of Notch2 and/or the expression of EphrinB2, respectively, in the HUVEC population. In preferred embodiments the cells are cultured for at least 2 days, more preferably for at least 3 days and even more preferably for at least 4 days and most preferably for more than 5 days in M). Pref- erably, the one or more protein(s) selected from ANG, TYMP, COL18A1, ADAMTS1, ADAMIO, ADAM9, IGFBP3, LEP, IGFBP1, ECGF1, ENG, PLG, ANGPTl, FGF2, EGF, TIMP1, IL1B, HGF, CCL2, SERPINEl, TIMP3, FGF1, PDGFA. Preferably, the expression of said one or more protein(s) is at least 15 fold increased.
18. Preferred is the method according to point 17, wherein the cells are isolated in N) at a point in time when
the expression of one or more protein(s) selected from the group consisting of angiogenesis related pro- teins ANG, ANGPTl, ARTN, DPP4, EGF, ENG, FGFl, FGF2, IL1B, LEP, PDGFA, CCL2, PLG, CD9, LAP3, and
the expression of one or more protein(s) selected from the group consisting of cell-matrix adhesion related proteins ADAMTS1, COL18A1, ECGF1, HGF, IGFBP1, IGFBP3, TIMP1, TIMP4, TYMP, ADAM9, ADAMIO, SERPINE1, TIMP3, PIGF, PSPN, PROKR2 is increased in said 3D cultured CD31+ cells obtained in M) compared to the expression of said protein(s) in a population of human coronary arterial endothelial cells (HCAEC population) cultured under 2D culture conditions, wherein said increase is at least 10-fold
and the cells show arterial phenotype indicated by an EphrinB2/EphB4 mRNA ratio which at least 2-fold higher than the EphrinB2 EphB4 mRNA ratio in a HUVEC population cultured under 2D culture conditions and/or an expression of Notch2 which is at least 4-fold higher and/or an expression of EphrinB2 which is at least 2-fold higher than the expression of Notch2 and/or the expression of EphrinB2, respectively, in the HUVEC population.
19. Even more preferred is the method according to point 18, wherein the cells are isolated in N) at a point in time when
the expression of one or more protein(s) selected from the group consisting of angiogenesis related proteins ANGPTl, FGF2, EGF, ILBl, PLG, CCL2, FGFl, PDGFAis increased in said 3D cultured CD31+ cells obtained in M) compared to the expression of said protein(s) in a population of human coronary arterial endothelial cells (HCAEC population) cultured under 2D culture conditions, wherein said increase is at least 10-fold, and
the expression of one or more protein(s) selected from the group consisting of cell-matrix adhesion related proteins TIMP1, HGF, SERPINEl, TIMP3 is increased in said 3D cultured CD31+ cells obtained in M) compared to the expression of said protein(s) in a population of human coronary arterial endothelial cells (HCAEC population) cultured under 2D culture conditions, wherein said increase is at least 10-fold,
and the cells show arterial phenotype indicated by an EphrinB2/EphB4 mRNA ratio which at least 2-fold higher than the EphrinB2 EphB4 mRNA ratio in a HUVEC population cultured under 2D culture conditions and/or an expression of Notch2 which is at least 4-fold higher and/or an expression of EphrinB2 which is at least 2-fold higher than the expression of Notch2 and/or the expression of EphrinB2, respectively, in the HUVEC population.
20. The method according to point 17, wherein the one or more protein(s) in N) is selected from ANGPTl, FGF2, EGF, TIMP1, IL1B, PLG, HGF, CCL2, SERPINEl, TIMP3, FGFl, PDGFA.
21. The method according to any one of point 17 to 20, wherein N) is carried out at a point in time when the expression of at least one angiogenesis-related protein selected from ACVRIB, angiopoietin-2,
CCL3, CSF2, CXCL-16, FGF4, HB-EGF, IGFBP2, IL-8, NRG-1, PDGFB, platelet factor 4 (PF4), PLG, PTX3, serpin Fl, TGFB1, vasohibin, VEGF-A, VEGF-C is increased at least 5 fold, preferably at least 10 fold, more preferably about 5-21 fold in the cells obtained in M) as compared to the expression of the at least one angiogenesis-related protein in a HCAEC population cultured under 2D culture conditions,
the expression of at least one cell-matrix adhesion protein selected from AREG, CCL2, CD59, GDNF, MMP9, serpin b5, THBSl, thrombospondin 2, TIMP2, urokinase is increased at least 5 fold, preferably at least 10 fold, more preferably about 5-21 fold in the cells obtained in M) as compared to the expression of the at least one cell-matrix adhesion protein in a HCAEC population cultured under 2D culture conditions. Preferably, the EphrinB2 EphB4 mRNA ratio in the cells obtained in M) is at least 2-fold higher than the EphrinB2 EphB4 mRNA ratio in a HUVEC population cultured under 2D culture conditions and/or the expression of Notch2 is at least 4-fold higher and/or the expression of EphrinB2 is at least 2-fold higher in the cells obtained in M) than the expression of Notch2 and/or the expression of EphrinB2, respectively, in the HUVEC population and/or level of dll4 in said 3D cultured CD31+ cells is increased at least 4 fold, preferably at least 5 fold, more preferably at least 10 fold compared to undifferentiated human pluripotent stem cells.
22. The method according to any one of point 17 to 21, wherein N) is carried out at a point in time when the expression of PF4, vasohibin and/or VEGFA is increased at least 8 fold, preferably at least 10 fold in the cells obtained in M) as compared to the expression of PF4, vasohibin and/or VEGFA, respectively, in a HCAEC population cultured under 2D culture conditions,
the expression of at least one, preferably at least two, preferably at least three, more preferably at least four, most preferably five proteins selected from AREG, CD59, MMP9, urokinase, TIMP2 is increased at least 3 fold, preferably at least 5 fold, more preferably at least 10 fold, most preferably at least 50 fold in the cells obtained in M) as compared to the expression of the corresponding protein(s) in a HCAEC population cultured under 2D culture conditions and
the EphrinB2 EphB4 mRNA ratio in the cells obtained in M) is at least 2-fold higher than the EphrinB2 EphB4 mRNA ratio in a HUVEC population cultured under 2D culture conditions and/or the expression of Notch2 is at least 4-fold higher and/or the expression of EphrinB2 is at least 2-fold higher in the cells obtained in M) than the expression of Notch2 and/or the expression of EphrinB2, respectively, in the HUVEC population and/or the level of dll4 in said 3D cultured CD31+ cells is increased at least 4 fold, preferably at least 5 fold, more preferably at least 10 fold compared to undifferentiated human pluripotent stem cells.
23. The method according to any one of points 17 to 22, wherein M is carried out in a bioreactor spinner flask at 35°C to 38°C, 75-90% humidity, about 21% 02, with steering at 70-75 RPM and with addition of fresh medium every other day, preferably wherein the temperature is about 37°C to 37.5°C and the humidity is about 85%.
24. The method according to any one of points 17 to 23, wherein L) is carried out in a density of 25- 75000 endothelial cells/0.5cm2 culturing 3D scaffold, preferably wherein the density is about 50000 endothelial cells/0.5cm2 culturing 3D scaffold.
25. The method according to any one of points 17 to 24, wherein the culturing 3D scaffold is a decellularised human extracellular biomatrix, preferably derived from human aorta.
26. The method according to point 25, wherein the decellularised human extracellular biomatrix is produced by a method comprising:
- harvesting aortic samples from a human,
- decelullarising said aortic samples by
- washing the samples in detergent solution for 60-80 hours, preferably for about 72 hours,
- washing the aortic samples in PBS + antibiotics for 60-80 hours, preferably for about 72 hours to provide decellularised aortic samples,
- preconditioning the decellularised aortic samples in EGM2 media prior to cell seeding. In a preferred embodiment the antibiotics is amphotericin B or gentamycin. Preferably the detergent is 0.1% sodium dodecyl sulphate and sodium azide and preferably the concentration of the antibiotics is 1%. 27. The method according to point 16 further comprising seeding the cells obtained in K) on a 3D matrix, preferably wherein the extracellular biomatrix is suitable for forming 3D vascular endothelial construct for use in a 3D vascular assay or the extracellular biomatrix is suitable for forming an implantable vascular graft.
28. The method according to any one of points 17 to 26, further comprising seeding the cells isolated in N) on 3D matrix, preferably wherein the extracellular biomatrix is suitable for forming 3D vascular endothelial construct for use in a 3D vascular assay or the extracellular biomatrix is suitable for forming an implantable vascular graft.
29. According to another aspect of the invention, a CD31+ cell population for forming a 3D vascular en- dothelial construct is provided, wherein the population is capable of adhering to 3D matrix and has an arterial phenotype, obtained by the method according to any one of points 16 to 28.
30. According to yet another aspect of the invention, a CD31+ cell population for forming a 3D vascular endothelial construct is provided, wherein the population is capable of adhering to 3D matrix and is derived from hiPSC, characterized in that
- the expression of at least one, preferably at least two, more preferably at least three angio genesis- related proteins selected from PF4, vasohibin, and VEGF-A in the CD31+ cells is at least 3-fold, preferably at least 4-fold higher in the CD31+ cells than in a population of HCAEC cultured under 2D culture conditions,
- the expression of at least one, preferably at least two, more preferably at least three, more preferably at least four, even more preferably at least five cell-matrix adhesion proteins selected from AREG, CD59, MMP9, urokinase, TIMP2 in the CD31+ cells are at least 3-fold higher than in a population of HCAEC cultured under 2D culture conditions,
- the EphrinB2 EphB4 mRNA ratio in the CD31+ cells is at least 2-fold higher than the EphrinB2 EphB4 mRNA ratio in a HUVEC population cultured under 2D culture conditions and/or the expression of Notch2 in the CD31 cells is at least 4-fold higher and/or the expression of EphrinB2 in the CD31+ cells is at least 2-fold high- er than the expression of Notch2 and/or the expression of EphrinB2, respectively, in the HUVEC population and/or the level of dll4 in said 3D cultured CD31+ cells is increased at least 4 fold, preferably at least 5 fold, more preferably at least 10 fold compared to undifferentiated human pluripotent stem cells.
31. Preferred is the CD31+ cell population of point 30, wherein additionally the expression of one or more protein(s) selected from ADAMTSl, ANG, ANGPT1, ARTN, COL18A1, DPP4, ECGF1, EGF, PROKR2, ENG, FGF1, FGF2, HGF, IGFBP1, IGFBP3, IL1B, LEP, CCL2, PDGFA, PIGF, PLG, PSPN, SERPINE1, TIMPl, TIMP4, TYMP, ADAM9, ADAMIO, CD9, LAP3,TIMP3 is increased in said CD31+ cells as compared to the expression of said protein(s) in said HCAEC population, wherein said increase is at least 10-fold.
32. Preferred is the CD31+ cell population of point 30, wherein additionally the expression of one or more angiogenesis-related protein(s) selected from the group consisting of ANG, ANGPT1, ARTN, DPP4, EGF, ENG, FGF1, FGF2, IL1B, LEP, PDGFA, CCL2, PLG, CD9, LAP3, and
the expression of one or more cell-matrix adhesion protein(s) selected from the group consisting of ADAMTSl, COL18A1, ECGF1, HGF, IGFBP1, IGFBP3, TIMPl, TIMP4, TYMP, ADAM9, ADAMIO, SERPINEl, TIMP3, PIGF, PSPN, PROKR2 is increased in said 3D cultured CD31+ cells obtained in ii) compared to the expression of said protein(s) in a population of human coronary arterial endothelial cells (HCAEC population) cultured under 2D culture conditions, wherein said increase is at least 10-fold. 33. Preferred is the CD31+ cell population of point 32, wherein the additional one or more angiogenesis- related protein(s) is/are selected from the group consisting of ANGPT1, FGF2, EGF, ILB 1, PLG, CCL2, FGF1, PDGFA, and
the additional one or more cell-matrix adhesion related protein(s) is/are selected from the group consist- ing of TIMP1, HGF, SERPINE1, TIMP3.
34. Preferred is the CD31+ cell population of point 31, wherein the additional one or more protein(s) is/are selected from ANGPT1, FGF2, EGF, TIMP1, IL1B, PLG, HGF, CCL2, SERPINE1, TIMP3, FGF1, PDGFA.
35. Use of the CD31+ cell population according to point 15 or according to any one of points 29 to 34 is provided,
for forming a 3D vascular endothelial construct, wherein the population is capable of adhering to 3D matrix and has an arterial phenotype.
36. Use of the CD31+ cell population according to point 15 or according to any one of points 29 to 35 is also provided, in a 3D vascular assay. Preferably, the 3D vascular assay is selected from antiplatelet Rantes assay, a vasoactive assay, an in vitro signaling assay, a permeability assay. Preferably, the CD31+ cell popula- tion used in the 3D vascular assay is seeded on a 3D matrix and is suitable for forming 3D vascular endothelial construct for use in a 3D vascular assay, such as on a decellularised porcine intestinal extracellular matrix scaffold or a decellularised human extracellular matrix scaffold, such as the decellularised human extracellular biomatrix defined in point 25 or 26.
In preferred embodiments the 3D matrix is an extracellular biomatrix. In preferred embodiments the 3D scaffold is an extracellular biomatrix scaffold.
The iPSC-derived cells are derived or obtained from iPSC or an iPSC line reprogrammed by viral or non-viral delivery of transcription factors.
In highly preferred embodiments the iPSC-derived cells are derived or obtained from iPSC or an iPSC line for which the following (transcription) factors have been used in the reprogramming method: Oct4, Sox2, Nanog and optionally or preferably LIN28.
In highly preferred embodiments the iPSC-derived cells are derived or obtained from iPSC or an iPSC line of mesodermal origin, preferably of mesenchymal origin, preferably of connective tissue origin, in particular of lung origin.
In highly preferred embodiments the iPSC-derived cells are derived or obtained from iPSC or an iPSC line of mesodermal origin, preferably of epithelial origin, preferably of endothelial origin.
In highly preferred embodiments the iPSC-derived cells are derived or obtained from iPSC or an iPSC line for which the following (transcription) factors have been used in the reprogramming method: Oct4, Sox2, Nanog and optionally or preferably LIN28, wherein the iPSC or the iPSC cell line is of mesodermal origin and preferably it has been reprogrammed by a retroviral method. In certain embodiments the culturing 3D scaffold in i), ii) and/or in L) is a decellularised porcine intestinal extracellular matrix scaffold.
SHORT DESCRIPTION OF THE DRAWINGS
Figure 1 Expressions of endothelial markers. Bar graphs show changes in endothelial gene expression levels of arterial (EphrinB2, Notchl, Notch2) (A-C), venous (EphB4) (D) and common (CD31, VE-Cadherin) (E, F) endothelial markers. Fold changes in mRNA levels in human embryonic stem cell-derived endothelial cells (hESC-EC), human induced pluripotent stem cell-derived endothelial cells (hiPSC-EC) and human umbili- cal vein endothelial cells (HUVEC) are normalized to those in undifferentiated hPSC. Human ESC-EC, hiPSC- EC and HUVEC were used for these experiments between passages 3-5. Data are presented as mean ± SEM, n=3 biological replicates of endothelial differentiation from hPSC. Normalised mRNA levels are showed in log (10) scale. * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, **** indicates pO.0001, One-way ANOVA and Tukey post-hoc tests. Histograms (G) show intensity of arterial EphrinB2 and D114 markers in hESC-EC, hiPSC-EC and human coronary artery endothelial cells (HCAEC). Cellular fluorescence signals were quantitated by high content microscopy; arterial expression pattern of hPSC-EC was compared with arterial control HCAEC.
Figure 2 Angiogenesis and cell-matrix adhesion protein profiling in 2D and 3D endothelial cultures. Heat map diagram shows expression of angiogenesis-related proteins before and after the recellularisation procedure (2D and 3D cultures, respectively) with human embryonic stem cell-derived endothelial cells (hESC-EC) and human induced pluripotent stem cell-derived endothelial cells (hiPSC-EC). Proteomics in hESC-EC and hiPSC- EC are represented as fold changes versus those in control HCAEC. n=4 technical replicates from 2 independent experiments. Array membranes for proteomics were visualized by chemiluminescent detection; pixel densities were quantified by ImageJ software. Numbers of cells were equalized in each experimental setting; passages between 3 and 5 were used.
Figure 3 In vivo conditioning of hPSC-EC induces expression of arterial, venous and common endothelial marker genes. Grouped bar graphs show expression of arterial (EphrinB2, Notchl, Notch2), venous (EphB4) and common (CD31) endothelial marker genes at two weeks after subcutaneous transplantation of human em- bryonic stem cell-derived endothelial cells (hESC-EC) and human induced pluripotent stem cell-derived endothelial cells (hiPSC-EC) in athymic rats. Data are presented as mean ± SEM. Fold changes in normalised mRNA levels are showed in log(10) scale. mRNA levels are normalised to those in pre-implanted control cells. n=3 biological replicates of endothelial implantation from differentiation from hESC, 3 technical replicates at each point. * indicates p<0.05, ** indicates p<0.01, one-way ANOVA with Tukey post-hoc test.
Figure 4 Endothelial marker expressions during differentiation and specification. Radar graph shows endothelial markers during human embryonic stem cell-derived endothelial cells (hESC-EC), human induced pluripotent stem cell-derived endothelial cells (hiPSC-EC) differentiation in three-dimensional (3D) specification compared to expression in human umbilical vein endothelial cells (HUVEC). Changes in mRNA levels of arterial (EphrinB2, Notchl, Notch2, D114) and venous marker genes (EphB4, FLT4) are shown.
Figure 5 Antiplatelet activities of endothelial cells in 3D culture on vascular wall. (A) Schematic figure shows experimental set used for antiplatelet assay. 1. Platelet rich plasma was incubated with endothelial cells in 2D culture; 2. with endothelial cells on decellularised vessel wall in 3D culture; 3. alone on decellularised matrix and 4. platelet rich plasma alone. (B) Bar graph shows changes in Rantes chemokine levels measured from platelet rich plasma after incubating developed vascular cells (human embryonic stem cell-derived endo- thelial cells (hESC-EC), human induced pluripotent stem cell-derived endothelial cells (hiPSC-EC), human umbilical vein endothelial cells (HUVEC)), acellular and recellularised vascular constructs. Rantes levels are in correlation with platelet clotting activity. Data are presented as mean ± SEM. n=3 biological replicates for antiplatelet assay, 5 biological replicates for vasoactive assay, 3 technical replicated at each point. * indicates p<0.05 and ** indicates p<0.01, one-way ANOVA with Dunnett post-hoc test.
Figure 6 Antiplatelet activity of endothelial cells in 3D culture on Cormatrix. Bar graph shows changes in Rantes chemokine levels measured from platelet rich plasma after incubating developed vascular cells (human embryonic stem cell-derived endothelial cells (hESC-EC), human induced pluripotent stem cell-derived endothelial cells (hiPSC-EC), human umbilical vein endothelial cells (HUVEC)), acellular Cormatrix and recel- lularised non-vascular constructs. Rantes levels are in correlation with platelet clotting activity. Data are pre- sented as mean ± SEM. n=3 biological replicates for antiplatelet assay, 5 biological replicates for vasoactive assay, 3 technical replicated at each point. ** indicates p<0.01, one-way ANOVA with Dunnett post-hoc test.
Figure 7 Vasoactive activities of hPSC-EC in 3D culture and isolated vessel platform. Vasoactive effects of human embryonic stem cell-derived endothelial cells (hESC-EC), human induced pluripotent stem cell- derived endothelial cells (hiPSC-EC) and human umbilical vein endothelial cells (HUVEC) were tested in vitro in isolated vessel water-bath system. (A) Original traces showing vasoactive effects of hESC-EC (top), hiPSC- EC (middle) and HUVEC (bottom) supernatant on isolated rat aortic rings. Changes are reflecting force changes in vessel wall. X axis refers to time, Y axis refers to force (positive changes on y axis means decreased force and vasodilatation). (B) Bar graph shows vasoactive effects of conditioned medium from hESC-EC, hiPSC-EC, and HUVEC compared to phenylephrine (PE)-induced vascular tone and acetylcholine (ACh) induced vascular tone. Changes in vascular tone are compared to mechanically set basal tone; fold changes are presented on bar diagram. Data are presented as mean ± SEM. n=3 biological replicates for antiplatelet assay, 5 biological replicates for vasoactive assay, 3 technical replicated at each point. * indicates p<0.05, ** indicates pO.01, *** indicates pO.001 and **** indicates pO.0001, one-way ANOVA with Tukey post-hoc test.
Figure 8 3D culture modulates arterial-venous endothelial marker expression of hPSC-EC. (A) Acellular biomatrices were seeded with human embryonic stem cell-derived endothelial cells (hESC-EC) and human induced pluripotent stem cell-derived endothelial cells (hiPSC-EC) and stained with anti-human CD31 antibody and Hoechst. (B, C) Bar graphs show changes in arterial (Notchl, Notch2, EphrinB2, D114), venous (EphB4) and common (VE-Cadherin and CD31) endothelial markers in hESC-EC (B) and hiPSC-EC (C) after recellu- larisation. (D) Principal component analyses (PCA) plot for gene expression data showing the parallel activation of arterial or venous markers. The two first principal components are plotted with the proportion of variance explained by each component printed next to the axes labels. Human ESC-EC, hiPSC-EC were used for these experiments between passages 3-5. Control endothelial cell were cultured in 2D. Data are presented as mean ± SEM, n=3 biological replicates. Please note log (10) scale on y axis (B, C). **** indicates pO.0001, one-way ANOVA and Tukey post-hoc tests.
Figure 9 Expression pattern of endothelial markers in endothelial cells from different sources. Heat map diagrams show normalised mRNA expression of arterial (Notchl, Notch2, EphrinB2, D114), venous (EphB4) and general (CD31, VE-Cad) endothelial marker genes. Gene expression levels were normalised to (A) those in human pluripotent stem cells (hPSC), (B) human coronary arterial endothelial cells (HCAEC) and (C) HUVEC. Human embryonic stem cell-derived endothelial cells (hESC-EC), human induced pluripotent stem cell-derived endothelial cells (hiPSC-EC), human coronary arterial endothelial cells (HCAEC), HUVEC and human microvascular endothelial cells (HMVEC) were analysed.
Figure 10 Angiogenesis and cell-matrix adhesion protein profiling in endothelial cultures. Scatter dot diagrams show expression levels of angiogenesis (A) related and cell-matrix interaction (B) related proteins of human embryonic stem cell-derived endothelial cells (hESC-EC) and human induced pluripotent stem cell- derived endothelial cells (hiPSC-EC), expression are compared to those in human coronary arterial endothelial cells (HCAEC). Proteomics in hESC-EC and hiPSC-EC are represented as fold changes versus those in control HCAEC. n=4 technical replicates from 2 independent experiments.
Figure 11 Angiogenesis and cell-matrix adhesion protein profiling in 2D and 3D endothelial cultures. (A) Functional association protein network diagrams for hESC-EC (left panel) and hiPSC-EC (right panel) are gen- erated by Ingenuity pathway analysis. (B) Scatter dot diagrams show expression levels of angiogenesis related and cell-matrix interaction related proteins of human embryonic stem cell-derived endothelial cells (hESC-EC) and human induced pluripotent stem cell-derived endothelial cells (hiPSC-EC), 2D and 3D cell culture conditions are compared.
Figure 12 In vivo characterisation of endothelial cells. Human embryonic stem cell-derived endothelial cells (hESC-EC) and human induced pluripotent stem cell-derived endothelial cells (hiPSC-EC) show cobblestone pattern in vitro (A) and take up ac-LDL (B). Both cell types form tubes on Matrigel (C). Immunocyto- chemistry shows positive staining for anti-CD31 (D) endothelial and anti-EphrinB2 (E) and D114 (F) markers.
Figure 13 Immunocytochemistry and microscopy analysis of cells derived by Harding et al. (2017) protocol. (A, B) Immunocytochemistry with endothelial CD31 marker (green; ^ ) and mesenchymal marker FSP1 (red; ^ ). Nuclear staining Hoechst is also shown (blue; Π ), lOx magnification. (C) Automated high content analysis to assess endothelial purity. Objects are identified by three-channel fluorescent imaging and compart- mental analysis bioassays. (D) High content plot to show high heterogeneity of population.
Figure 14 Real PCR assessment of undifferentiated hiPSC (IMR line) and differentiated endothelial culture by our and Harding et al. (2017) protocols. mRNA levels of endothelial markers (CD31, VE-cadherin), arterial markers (Notchl/2, D114, EphrinB2) and venous markers (EphB4) were quantitated. One-way ANOVA was used for statistics. * indicates p<0.05, ** indicates pO.01, *** indicates pO.001.
Figure 15 Proteome profiling. (A) Proteome prolifer blot, developed. (B) Comparison of 54 angiogenic factors. (C) Heat map presentation of Harding et al. (2017) protocol (indicated by AH prot) versus our protocol in 2D and 3D.
Figure 16 Schematic illustration of the protocol used to produce CD31+ cells and 3D vascular construct on acellular biomatrices.
DETAILED DESCRIPTION OF THE INVENTION
hiPSC-EC according to the invention show high abundance of angiogenesis- and cell adhesion-related proteome along with an arterial phenotype. 2D culture of the cells resulted in an increased expression of angio- genesis- and cell adhesion-related proteins as compared to HCAEC 2D culture. The cells provided by the method according to the invention are particularly suitable for forming a 3D vascular endothelial construct, for use in 3D vascular assays and for use in implantable grafts. Their arterial phenotype is characterized by and may be measured by increased arterial marker levels, such as e.g. EphrinB2/EphB4 mRNA ratio, expression of Notch2, expression of EphrinB2 and expression of dll4. In the context of the description the name or abbreviation of the name of a gene may refer to the mRNA or protein coded by the gene and the name or abbreviation of the name of a protein may refer to the gene encoding the protein.
The 2D and 3D hiPSC-EC culture show a dominant increase mainly in the expression of arterial rather than venous markers, confirming the presence of an arterial dominance in the endothelial culture. Arterial to venous mRNA ratio increased from day 5 of differentiation and remained similar in sorted CD31+ cells, show- ing arterial dominancy in the expanding culture. Although differentiated hiPSC-EC show less than mature endo- thelial phenotype in 2D culture, some of the markers of a functional endothelial phenotype are already present, i.e. hiPSC-EC cultures show cobblestone pattern, take up ac-LDL, show tube formation activity, and are stained positive with anti-CD31 antibody. When compared to hESC-EC obtained under the same culture conditions, the expression of endothelial markers in hiPSC-EC is lower than in hESC-EC (FIG 1), while hiPSC-EC according to the invention show increased levels of angiogenesis- and cell adhesion-related proteins.
To further test and improve the functional properties, survival, viability and proliferation of hiPSC-EC generated in 2D culture conditions, 3D cell culture systems were used. hiPSC-EC, according to the invention, could not only adhere to decellularised extracellular biomatrices, remained viable and functional during recellularisation, but underwent further maturation. Levels of many angiogenesis-related proteins (e.g. angiopoietin 1 and 2, endoglin, FGFs) were markedly higher when hiPSC-EC were cultured on 3D vascular biomatrix (FIG 2). Cell-matrix adhesion related proteins (e.g. collagen XVIII, MMP8, MMP9, TIMP1, MCP-1, ADAMTSl) showed robust increase in 3D culture, thereby increasing adhesive capacity of cells upon reseeding of matrices. Factors involved in the regulation of platelet aggregation or fibrinolytic pathway (e.g. TIMP4, plasminogen) were also activated. Surprisingly, the increase in levels of the measured proteins in hiPSC-EC is markedly higher than in hESC-EC cultured by the same protocol.
3D culture of hiPSC-EC on acellular biomatrices increased expression of arterial, venous and common endothelial marker genes. Arterial endothelial derivatives of hiPSC develop mature endothelial characteristics and functional behaviour in 3D cultures. Arterial fate can be modulated even on differentiated endothelial cells during expansion in 2D cultures and on seeding cells on biomatrices for 3D cultures. Further, the markedly increased levels of angiogenesis- and adhesion-related proteins indicate, that hiPSC derivatives are better prepared to form a 3D endothelial construct than hESC derivatives, while having a favourable arterial phenotype. It is to be noted that in vivo conditioning (implantation in athymic nude rat) of hPSC-EC also induces expression of arterial, venous and common endothelial marker genes and EphB4 expression has been found to increase significantly in hESC-EC, while the arterial/venous marker ratio remained characteristic of an arterial pheno- type in hiPSC-EC (FIG 3).
hiPSC-EC were able to reduce platelet activation, as shown by decreased Rantes levels in platelet rich plasma upon incubation of vascular constructs (FIG 5,6). 3D culture conditions further enhanced antiplatelet effects of hiPSC-EC (FIG 5,6). Antiplatelet effect and regulation of clot formation are specific tasks for arterial endothelial cells; proteome profiling and antiplatelet assay together emphasize the presence of functional arterial hiPSC-EC in the culture. An improved antiplatelet activity is favourable when considering use of cells for vascular tissue engineering. Direct (acute) regulation of myogenic tone from hiPSC-EC is another key property of vascular grafts for clinical use. It has been shown by using rat aortic rings as an in vitro isolated vessel platform that acute reseeding of vascular surfaces with secretome of hiPSC-EC according to the invention had marked direct vasoactive effect. (FIG 7)
Cell culture
The expressions "cultured under two-dimensional culture conditions" and "cultured under three- dimensional culture conditions" refer to conditions appropriate for culturing the desired cell type and promoting the expression of angiogenesis-related proteins, cell-matrix adhesion proteins, common, venous and arterial endothelial markers in case of endothelial cells.
A. Endothelial differentiation of human pluripotent stem cells 1. Maintenance of human induced pluripotent stem cells on adherent plates in pluripotent stem cells medium (preferably in mTeSRl). Observing stem cell colonies daily in a phase contrast light microscope. Mechanical removal spontaneously differentiated cells from culture. Regular passage of stem cell colonies via Versene- based dissociation of clamps and reseeding stem cells on larger dish surface at confluency of 65-70% (1-1.5M cells/9.6 cm2). Proliferation (by colony formation assay and immunocytochemistry with anti-Ki67 proliferation marker antibodies) and chromosome stability are regularly checked.
2. Mechanical dissociation of stem cell colonies in order to develop embryoid bodies and culture them in suspension on low adherent culture dishes. This stage mimics the blastocyst stage of the early embryo.
3. Mesodermal induction via administration of additional growth factors and small molecules in culture media. Additional factors comprise: 2% batch-tested foetal bovine serum, human epidermal growth factor, vascular endothelial growth factor, human fibroblast growth factor B, insulin growth factor R3, hydrocortisone, heparin. (EBM2 medium 500 ml; Foetal bovine serum 10 ml; Human epidermal growth factor 500μ1; Vascular endothelial growth factor 500 μΐ; Human fibroblast growth factor B 500 μΐ; Insulin growth factor R3 500 μΐ; Hydrocortisone 200 μΐ; Heparin 500 μΐ; Ascorbic acid 500 μΐ; Amphotericin B/Gentamycin 500 μΐ.)
4. At day four, seeding EBs onto 0.5% gelatine (e.g. Sigma Aldrich) coated 24-well plates or T25 flasks.
5. At day 13, trypsinising differentiated cultures (0.25% Trypsin and 0.03% EDTA, Life Technologies), centrifuging and re-suspending pellets (1100 RPM, 5 min) in PBS, containing 1% FBS (FACS buffer). Counting cells before FACS. Sorting CD31 positive endothelial cells by fluorescence activated cell sorting method (FACS): labelling differentiating cells with anti-CD31 antibody, conjugated with Alexa Fluor 488 fluorophore (1:20 dilution, e.g. BD Biosciences, Minneapolis, USA). As control, IgG isotype antibodies are used to match to the primary CD31 antibody; cell sorting e.g. on a FACS Aria II Cell Sorter (e.g. BD Biosciences).
6. Culturing and expansion of CD31-positive endothelial cells in 0.5% gelatinised flasks. Maintaining cells in EGM2 medium, feeding on every other day with fresh EGM2 medium. Endothelial cells proliferate in monolayer. Passages (1:3 surface) on every 3-6 day according to confluency (up to 80%) and proliferation rate.
B. Development of acellular human extracellular biomatrices (3D scaffold)
1. Use of human aortic wall pieces or ring specimens from clinical cardiovascular biobank.
2. Upon harvest storage of aortic samples in liquid nitrogen.
3. For decellularisation samples being thawed on room temperature.
4. Washing aortic samples in detergent solution for 72 hours. Preferably the detergent is 0.1% sodium dodecyl sulphate and sodium azide.
5. Washing aortic samples in PBS + 1% antibiotics for 72 hours.
6. Cutting aortic pieces into 300 μπι thin slices with a vibrating microtome system (e.g. Campden Instruments, Lafayette, USA 7000 smz).
7. For quality control, homogenising slice materials to perform DNA/RNA isolation and quantitation. 8. 48-h preconditioning acellular slices in EGM2 media prior to cell seeding.
3D culture protocol for developing tissue engineered vascular grafts
1. Seeding endothelial cells onto acellular biomatrices for 3D, vascular tissue engineering (e.g. 50,000 endothelial cells/0.5 cm2 matrix). (Biomatrices e.g. from B. decellularisation protocol, described above).
2. Culturing 3D endothelial constructs in bioreactor spinner flask (e.g. Corning, Basel, Switzerland) at 37°C, 85% humidity, 21% 02. Steering media (EGM2) with a magnetic impeller at 70-75 RPM. Fresh medium every other day.
3. Vital and nuclear imaging with Calcein AM (1 : 1000, e.g. Thermo Scientific) and Hoechst-33342 staining (e.g. Thermo Scientific). Confocal microscopy and real time PCR analysis of endothelial markers CD31, VE-cadherin and eNOS (including preamplification if required) to verify endothelial seeding on biomatrices. (FIG15)
The steps defined in the above protocols may be optimized to the aims and needs of the experiment or measurement to be carried out. The skilled person finds ample guidance for such optimization in the art, e.g. in Laco et al. 2018.
Two-dimensional culture conditions ofhiPSC-EC and endothelial differentiation
The term "two-dimensional (2D) culture" refers to conventional adherent tissue culture involving growing cells on (coated) solid flat surfaces as 2D monolayers. Cells are adhering to an artificial plastic or glass substrate and are growing side by side. Protocols using scaffold-free method (e.g. suspension culture of EBs) are also termed as 2D culture conditions in the present disclosure.
The CD31+ cells according to the invention are obtainable by a method comprising the steps described herein below.
hiPSC may be seeded into a feeder-free medium capable of supporting endothelial differentiation and growth, preferably containing VEGF, such as EGM2, containing an endothelial basal medium, e.g. EBM2 media with SingleQuot supplements. The endothelial basal medium preferably contains essential and non-essential amino acids, vitamins, trace minerals, organic compounds, and inorganic salts and lacks hormones, growth factors or other proteins.
Embryoid bodies (EBs) are generated by mechanically breaking the hiPSC colonies, with a standard sized cell culture scraper. EBs are then cultured for 4 days in suspension in low-adherent plates in a medium capable of supporting endothelial differentiation and growth, preferably containing VEGF, such as EGM2.
To force differentiation into mesodermal lineage the differentiation protocol may include extra growth factors and morphogens in a serum-free, feeder-free, complete medium suitable for iPSC, such as the mTeSRl media (e.g. Activin A, FGF-2, VEGFi65 and BMP4). After 24 hours the media may be removed and replaced with a serum-free medium suitable for hematopoietic stem cell expansion, such as the Stemline II complete media containing e.g. FGF-2, VEGFi65 and BMP4, differentiating cells are then maintained in the serum-free medium suitable for hematopoietic stem cell expansion (e.g. Stemline II media).
EBs are then plated on gelatinized plates in a cell-free medium capable of supporting endothelial differentiation and growth, preferably containing VEGF, such as EGM2, and cultured in a monolayer.
Normoxic (21% 02) conditions are maintained throughout culturing.
CD31+ cells are selected and collected after approximately 12 or 13 days of culture, followed by seeding onto collagen coated plates in a medium capable of supporting endothelial differentiation and growth, compris- ing endothelial cell growth medium, hematopoietic stem cell expansion medium, preferably supplemented with antibiotics (e.g. EGM2, Stemline II and P/S media) coated plates. The medium is replaced after 2 days and again after 3 days with a medium containing an increasing portion of endothelial cell growth medium.
Differentiated EC cells are normally utilized for experiments and seeding of biomatrices between passages 3-5 and may be from passage 1.
2D culture of the cells resulted in an increased expression of angiogenesis- and cell adhesion-related pro- teins as compared to HCAEC 2D culture. The expression of angiogenesis-related proteins ACVR1B, ANGPT2, CCL3, CXCL16, FGF4, CSF2, HBEGF, IGFBP2, IL8, NRG1, PDGFB, PF4, PLG, PTX3, SERPINF1, TGFB 1, VASH1, VEGFA, VEGFC and cell-matrix adhesion related proteins AREG, GDNF, CCL2, MMP9, urokinase (PLAU), THBS1, THBS2, CD59, TIMP2, SERPINB5 in cells according to the invention is at least 2-fold high- er than the expression of the corresponding protein in HCAEC cultured under 2D conditions.
2D culture conditions of HCAEC and HUVEC
HCAEC and HUVEC may be maintained in a medium capable of supporting endothelial growth (such as EGM2) and may be grown as monolayer culture according to the instructions of the provider of the medium and/or the cells. A method exemplifying the appropriate culture conditions is described in the Examples.
Three-dimensional culture system
A 3D cell culture is an artificially -created environment in which biological cells are permitted to grow or interact with their surroundings in all three dimensions. 3D cell culture allows cells in vitro to grow in all directions, similar to how they would in vivo.
There is a clear need to perform in vivo comparability testing with stem cell derivatives; however, this may not be suitable to describe changes in hPSC-EC phenotype. Therefore, 3D in vitro alternative approaches may be used to obtain key information on the functional ability of cells. Tissue engineered three-dimensional cell culture systems may be used to study survival, viability and proliferation of pluripotent stem cell-derived endothelial cells. hESC-EC and hiPSC-EC according to the invention cultured under 2D conditions may be seeded on bioscaffolds and cultured by using a bioreactor system.
Bioscaffolds
Decellularised vascular scaffold (obtained from human aorta) and decellularised scaffold derived from porcine small intestinal submucosa (CorMatrix) were used to demonstrate that hiPSC-EC according to the invention are capable of recellularising the matrix, remain viable and undergo further maturation on the matrix. The bioscaffold suitable to form a three-dimensional vascular endothelial construct according to the invention may be any material comprising extracellular matrix components to provide structural and mechanical properties for further development of the cells seeded thereon. The properties of the matrix may be optimized to mimic those of a tissue to be grafted or to be tested. Decellularised human extracellular matrix (derived from aorta) and the decellularised scaffold derived from porcine small intestinal submucosa were shown to be appropriate as 3D scaffold.
The expression of angiogenesis and cell-matrix related proteins further increased under 3D culture conditions. The increase in levels of the measured proteins in hiPSC-EC is markedly higher than in hESC-EC cultured by the same protocol. (Table 1)
Table 1 Protein expression upon 3D culture
Protein fold change of expression vs 2D HCAEC
hESC-EC hiPSC-EC
ACVR1B 1.48 15.40
ADAMTS1 1.63 15.32
ANG 1.80 15.33
ANGPT1 2.59 16.15
ANGPT2 3.14 17.51 Protein fold change of expression vs 2D HCAEC hESC-EC hiPSC-EC
AREG 4.10 17.86
ARTN 4.63 17.75
CCL3 3.50 18.02
COL18A1 3.54 15.81
DPP4 3.87 15.53
PROKR2 1.68 15.70
ENG 1.62 16.41
FGF1 1.62 15.59
FGF2 1.52 15.88
FGF4 1.96 16.70
GDNF 2.59 17.29
CSF2 3.28 17.64
HBEGF 3.84 17.47
HGF 3.35 17.82
IGFBP1 3.12 17.99
IGFBP2 3.06 18.10
IGFBP3 4.06 17.82
IL1B 1.25 16.31
IL8 1.29 16.27
LEP 0.55 16.01
CCL2 1.30 16.00
MMP9 2.54 16.20
NRGl 2.91 16.98
ECGF1 3.01 17.73
PDGFA 2.97 18.00
PDGFB 2.58 17.37
PF4 3.14 17.94
PIGF 3.41 17.49
PLAU 4.26 18.31
PLG 1.38 16.55
PSPN 2.64 16.50
SERPINB5 1.30 16.25
SERPINE1 2.82 16.22
SERPINF1 2.53 16.47
TGFB 1 2.85 17.24
THBS1 2.65 18.10
THBS2 2.84 17.96 Protein fold change of expression vs 2D HCAEC
hESC-EC hiPSC-EC
TIMP1 3.52 17.47
TIMP4 4.56 17.53
TYMP 1.94 17.35
VASH1 2.045 18.62
VEGFA 2.44 16.75
VEGFC 2.847 16.60
ADAM9 2.28 16.517
ADAM10 2.72 16.71
CD9 2.95 16.73
CD59 2.87 16.82
LAP3 3.49 17.82
TIMP2 4.22 18.64
TIMP3 4.64 18.47
Other cells involved in vasculogenesis may be co-cultured with the cells under both 2D and 3D conditions and seeded onto a bioscaffold to produce a three-dimensional vascular endothelial construct. Such cells may comprise human primary or hiPSC-derived vascular smooth muscle cells, primary or hiPSC-derived pericytes, other mesenchymal cells and fibroblasts.
Endothelial characterisation
Arterial, venous and common endothelial markers
Arterial, venous and common endothelial marker (EphrinB2, Notchl, Notch2, D114, EphB4, VE- Cadherin, CD31) mRNA levels measured in hiPSC-EC according to the invention and hESC-EC in 2D and 3D culture are shown in FIG 8. 3D culture enhances the expression of these markers and especially arterial markers EphrinB2, Notchl, Notch2, D114 in hiPSC-EC much more radically than the corresponding levels in hESC-EC.
Cells according to the invention are characterized by a robust increase in arterial (EphrinB2, Notchl, Notch2 and D114) and venous (EphB4 and FLT4) endothelial markers during differentiation and a dominant increase in arterial rather than venous markers (EphrinB2, Notchl, Notch2 vs EphB4 mRNA levels). After maintenance and expansion of cells in 2D cultures, both arterial (EphrinB2, Notchl, Notch2 and D114) and ve- nous (EphB4) markers show a more robust upregulation compared to those in undifferentiated hiPSC lines. In parallel, common endothelial marker genes, CD31 and VE-cadherin show a further increase in hiPSC-EC. At this phase, increase in arterial markers is still significantly higher than those in venous markers in hiPSC-EC. The expression of another venous marker FLT4 is less abundant in hiPSC-EC populations.
mRNA levels of arterial, venous and common endothelial marker genes all increase markedly after 3D bioreactor culturing vs 2D cultures. D114 and Notch2, key signals of arterial fate and sprouting angiogenesis, respectively, were both increased in hiPSC-EC showing a more profound increase of Notch2 level. Abundant secretion of several proteins in hiPSC-EC may be measured; their protein secretome patterns are comparable to those in control human arterial endothelial cells. (FIG 2)
It was found that during in vivo conditioning general endothelial and specific arterial and venous endo- thelial marker gene expressions were all altered in the cells according to the invention. mRNA levels of all endothelial genes were increased at least 10-fold, compared to those in 2D culture in vitro. In hESC-EC significant increase was observed in venous EphB4 expression, indicating a shift towards a more venous phenotype, while arterial dominancy characteristic of hiPSC-EC further developed in vivo. In hiPSC-EC the endothelial marker CD31 showed robust increase upon in vivo conditioning. Furthermore, arterial to venous ratio in hiPSC maintained the pattern seen in vitro (FIG 3,4).
Angiogenesis- and adhesion-related proteins
To identify protein production of endothelial cells angiogenesis and hematopoietic soluble receptor pro- teome profiling measurement may be carried out on commercially available array kits, such as the R&D System Proteome Profiler Human Angiogenesis Array Kit (ARY007) and Human Soluble Receptor Array Kit Hematopoietic Panel (ARYOl 1). Both cell surface supernatant and cell lysates may be used in these studies.
The fold changes in the levels of angiogenesis- and adhesion-related proteins of hiPSC-EC according to the invention and hESC-EC cultured under the same conditions as compared to HCAEC are shown in FIG 2.
The levels of angiogenesis-related proteins ACVR1B, angiopoietin-2, CCL3, CSF2, CXCL-16, FGF4, HB-EGF, IGFBP2, IL-8, NRG-1, PDGFB, platelet factor 4 (PF4), PLG, PTX3, serpin Fl, TGFB l, vasohibin, VEGF-A, VEGFC in hiPSC-EC are at least 2-fold higher than the expression of the same proteins in HCAEC cultured under 2D conditions.
The levels of angiogenesis-related proteins PF4, SERPINF1, VASH1 and VEGFA are at least 3 -fold higher than the expression of the same proteins in HCAEC.
The levels of adhesion-related proteins AREG, CCL2, CD59, GDNF, MMP9, serpin b5, THBSl, thrombospondin 2, TIMP2, urokinase are at least 2-fold higher than the expression of the same proteins in HCAEC cultured under 2D conditions. The levels of MMP9, CD59, TIMP2, urokinase are at least 3 -fold higher than the expression of the same proteins in HCAEC.
The difference between protein levels in hESC-EC 2D vs 3D culture conditions and hiPSC-EC 2D vs 3D culture conditions is shown in Fig 11.
Other endothelial markers
Cells and vascular constructs according to the invention may be analyzed using standard immunocyto- chemistry and immuno histochemistry using commercially available antibodies and methods known in the art.
Cells according to the invention are positive for von Willebrand Factor (vWF), CD31 and delta like 4 (D114) staining (FIG 12). hiPSC-EC according to the invention are negative for haematopoietic marker CD45. High content automated microscopy analyses proved high intensity of arterial EphrinB2 and D114 markers as compared to control arterial endothelial cell HCAEC. (FIG 12)
Tube formation
hiPSC-EC according to the invention form capillary -like structures in Matrigel tube formation assay both after 2D culture and upon seeding and culturing on a biomatrix (FIG 12)
Ac-LDL uptake
Endothelial cells may be identified by their high-level metabolism of acetylated low density lipoprotein (Ac-LDL). To test Ac-LDL uptake, cultures are incubated with l,r-dioctadecyl-3,3,3',3'-tetramethyl- indocarbocyanine perchlorate labelled with fluorescent probe (Dil-Ac-LDL, ThermoFisher Scientific) and im- aged by fluorescence microscopy. hiPSC-EC according to the invention take up Ac-LDL both after 2D culture and upon seeding and cultur- ing on a biomatrix (FIG 12).
Morphology
A cobblestone morphology in culture is characteristic for endothelial cells. hiPSC-EC according to the invention show this characteristic morphology both after 2D culture and upon seeding and culturing on a biomatrix. (FIG 12)
Antiplatelet activity
hiPSC-EC are able to fulfill therapeutic expectations only when they possess sufficient functional activity to support the physiological mechanisms of the vascular system. During tissue engineering with the aim of developing therapeutic applications, vascularisation and endothelialisation are among the main considerations. Endothelial cells should provide an antithrombotic surface to line vessel walls. Antiplatelet effect and regulation of clot formation is an essential task for arterial endothelial cells. Anticlotting function may be investigated by Rantes (CCL5) chemokine ELISA assay. Rantes is secreted from activated platelets, thus its level refers to clotting status in platelet rich plasma. Secretion of Rantes chemokine is measured in platelet rich plasma after incu- bating cells or vascular constructs to be tested.
3D culture conditions enhance anticlotting effects of hiPSC-EC. As Rantes is also related to angiogenic mechanisms, its modulation by hiPSC-EC emphasizes their strong angiogenic characteristics.
Antiplatelet effects of hiPSC-EC according to the invention are comparable to control HUVEC. Rantes levels are similar in 2D samples and in 3D scaffolds alone (aorta slices or CorMatrix), suggesting that acellular biomatrices are not thrombogenic in short-term experiments in vitro. Antiplatelet effects of hiPSC-EC according to the invention are higher in 3D-cultured cells than in 2D-cultured cells. (FIG 5 and 6)
Vasoactive effects
Direct (acute) regulation of myogenic tone from hPSC-EC should be another key property of vascular grafts for clinical use. Acute reseeding of vascular surfaces with hiPSC-EC according to the invention had marked direct vasoactive effect.
The vasoactive function of hiPSC-EC may be assessed by using an isolated vessel platform. For example, aortic rings may be isolated and basal tension of the vessels, precontraction, viability of endothelium and smooth muscle cells may be verified and vasoactive function of stem cell-derived endothelial cells may be tested. Viability of the endothelium may be assessed by its endothelium-dependent vasodilative response to e.g. acetylcholine. Viability of the smooth muscle cells may be assessed as endothelium-independent vasodilative response to e.g. sodium-nitroprusside.
Conditioned medium of endothelial cells (24h collection) has vasodilative effects on rat aortic rings. hiPSC-EC according to the invention and HUVEC supernatants significantly dilate vessels pre-constricted with phenylephrine. (FIG 7)
3D vascular assays
3D vascular assays provide a platform for testing barrier function, immune response, vasoactive effects, antithrombotic surface, secretory activity, cell death and angiogenesis in in vitro cultured EC. 3D vascular assays are particularly useful in modelling human vascular responses to various pathological and drug induced effects.
3D culture is inevitable to develop functional tissue engineered vascular grafts. Proper endothelial char- acteristics are cornerstones of clinical translation. These include antiplatelet, vasoactive, permeability and signal transduction assays.
Antiplatelet Rantes assay
Rantes assay was developed and applied on our engineered 3D vascular constructs. Rantes assay provid- ed us with a platform to study antiplatelet effects of 3D cultured hiPSC-EC. Antiplatelet functional activity of hiPSC-EC significantly increased in 3D culture.
Vasoactive assay
Endothelial cells affect vasomotion and tone of vasculature. These can only be investigated in 3D culture, as 2D culture lacks a dimension of vasoconstriction or vasodilatation. Here we described a 3D vasoactivity assay, in which direct effects and endothelial responses can be measured on the level of force developed or dropped via vascular smooth muscle constriction or dilatation (respectively). Additionally, selective inhibition of major endothelial signalling pathways, e.g. the NO-cGMP, reveals mechanism of action of vascular activity.
In vitro signalling assay
3D vascular constructs can be exposed to any cardiovascular drugs or other medications (for instance chemotherapeutic drugs) in order to assess signal transduction or drugs related endothelial effects - both beneficial effects and disadvantageous side effects.
Permeability assay
Endothelial permeability may be characterised by labelling 3D vascular construct with fluorescence dyes and dextran compounds. A normal range of endothelial permeability can be calculated by Dextran molecular size and common endothelial permeability rate. Measuring the intensity of fluorescence dies on the other side of the exposure (technically culturing the graft on insert plates) allows quantification of increased permeability (endothelial injury) after selected treatments (e.g. exposure to chemotherapeutic drugs).
Certain aspects and embodiments are detailed in the following paragraphs.
The invention provides a population of human iPSC-derived CD31+ cells with arterial dominancy, wherein the expression of angiogenesis related proteins, cell-matrix adhesion proteins and arterial gene markers are increased.
Cultured CD31+ cell population, useful for forming a three-dimensional vascular endothelial construct, wherein the cells of the population are derived from human induced pluripotent stem cells and
the population of cells possesses angiogenic potential indicated by an increased expression of one or more angiogenesis-related protein(s)
and optionally
the population of cells is capable of adhering to an extracellular biomatrix, indicated by an increased expression of one or more cell-matrix adhesion protein(s)
and/or
the population shows arterial phenotype indicated by an increased arterial/venous marker ratio.
Cultured CD31+ cell population, useful for forming a 3D vascular endothelial construct, wherein the cell population is derived from human induced pluripotent stem cells and
the cell population possesses angiogenic potential indicated by an increased expression of at least one angiogenesis-related protein,
the cell population is capable of adhering to an extracellular biomatrix, indicated by an increased expres- sion of at least one cell-matrix adhesion protein, and
the cell population shows arterial phenotype indicated by an increased arterial/venous marker ratio.
I. Cultured CD31+ cell population, useful for forming a 3-dimensional (3D) vascular endothelial construct, wherein the cell population is derived from human induced pluripotent stem cells and
the at least one angiogenesis-related protein is selected from ACVR1B (activin receptor type-IB), angiopoietin-2, CCL3 (chemokine (C-C motif) ligand 3), CSF2 (colony stimulating factor 2), CXCL-16 (chem- okine (C-X-C-motif) ligand 16), FGF4 (fibroblast growth factor 4), HB-EGF (heparin binding epidermal growth factor-like growth factor), IGFBP2 (insulin-like growth factor binding protein 2), IL-8 (interleukin 8), NRG-1 (neuregulin 1), PDGFB (platelet derived growth factor subunit B), platelet factor 4 (PF4), PLG (plasminogen), PTX3 (pentraxin 3), serpin Fl, TGFB1 (transforming growth factor beta 1), vasohibin, VEGF-A, VEGF-C (vascular endothelial growth factor A and C), and said at least one angiogenesis-related protein shows increased expression in said CD31+ cell population as compared to the expression of said protein(s) in a population of human coronary arterial endothelial cells (HCAEC population) cultured under 2D culture conditions;
the at least one cell-matrix adhesion protein is selected from AREG (amphiregulin), CCL2 (chemokine (C-C motif) ligand 2), CD59, GDNF (glial cell derived neurotrophic factor), MMP9 (matrix metallopeptidase 9), serpin b5, THBS1 (thrombospondin 1), thrombospondin 2, TIMP2 (tissue inhibitor of metalloproteinases 2), urokinase, and said at least one cell-matrix adhesion protein shows increased expression in said CD31+ cell population as compared to the expression of said protein(s) in a HCAEC population cultured under 2D culture conditions,
the increased arterial/venous marker ratio is selected from increased EphrinB2 EphB4 mRNA ratio, increased expression of Notch2 and increased expression of EphrinB2 in said CD31+ cell population, as compared to the EphrinB2/EphB4 mRNA ratio, expression of Notch2 and expression of EphrinB2, respectively, in a population of human umbilical vein endothelial cells (HUVEC population) cultured under 2D culture conditions.
II. Cultured CD31+ cell population, useful for forming a three-dimensional vascular endothelial construct, wherein the cell population is derived from human iPSC and
(i) the cell population possesses angiogenic potential indicated by an increased expression of at least one angiogenesis-related protein selected from ACVR1B, angiopoietin-2 (ANGPT2), CCL3, CSF2, CXCL-16, FGF4, HB-EGF, IGFBP2, IL-8, NRG-1, PDGFB, platelet factor 4 (PF4), PLG, PTX3, serpin Fl, TGFB1, vasohibin (VASHl), VEGF-A, VEGF-C and said at least one angiogenesis-related protein shows increased expression in said CD31+ cell population as compared to the expression of said protein(s) in a population of human coronary arterial endothelial cells (HCAEC population) cultured under 2D culture conditions, the expression of said protein(s) in said CD31+ cell population being at least 2-fold higher than the expression of said protein(s) in said HCAEC population;
(ii) the cell population is capable of adhering to an extracellular bio matrix, indicated by an increased expression of one or more cell-matrix adhesion protein(s) selected from AREG, CCL2, CD59, GDNF, MMP9, serpin b5, THBS1, thrombospondin 2, TIMP2, urokinase (PLAU) and said at least one cell-matrix adhesion protein shows increased expression in said CD31+ cell population as compared to the expression of said pro- tein(s) in a population of human coronary arterial endothelial cells (HCAEC population) cultured under 2D culture conditions, the expression of said protein(s) in said CD31+ cell population being at least 2-fold higher than the expression of said protein(s) in said HCAEC population;
(iii) the CD31+ cell population shows arterial phenotype indicated by an increased arterial/venous marker ratio selected from increased EphrinB2/EphB4 mRNA ratio, increased expression of Notch2 and increased expression of EphrinB2, as compared to the EphrinB2 EphB4 mRNA ratio, expression of Notch2 and expres- sion of EphrinB2, respectively, in a population of human umbilical vein endothelial cells (HUVEC population) cultured under 2D culture conditions.
III. In certain embodiments the expression of at least two, particularly at least three, more particularlyat least four or even more particularly at least five angiogenesis-related proteins selected from ACVR1B, angiopoietin-2, CCL3, CSF2, CXCL-16, FGF4, HB-EGF, IGFBP2, IL-8, NRG-1, PDGFB, platelet factor 4 (PF4), PLG, PTX3, serpin Fl, TGFBl, vasohibin, VEGF-A, VEGF-C is increased in said CD31+ cell population as compared to the expression of said proteins in a HCAEC population cultured under 2D culture conditions.
In certain embodiments the expression of at least two, particularly at least three, more particularly at least four or even more particularly at least five angiogenesis-related proteins selected from angiopoietin-2, CCL3, PDGFB, PF4, serpin Fl, TGFBl, vasohibin, VEGF-A, and VEGF-C is increased in said CD31+ cell population as compared to the expression of said proteins in a HCAEC population cultured under 2D culture conditions.
IV. In certain embodiments the expression of at least two, preferably at least three angiogenesis-related proteins selected from PF4, vasohibin and VEGF-A is increased in said CD31+ cell population as compared to the expression of said proteins in a HCAEC population cultured under 2D culture conditions.
V. In certain embodiments the expression of the at least one or the at least two, particularly the at least three, more particularly the at least four or even more particularly the at least five angiogenesis-related pro- tein(s) is at least 3 -fold or at least 4-fold higher in said CD31+ cell population than in said HCAEC population.
VI. In certain embodiments the expression of at least two, particularly at least three, more particularly at least four or even more particularly at least five cell-matrix adhesion proteins selected from AREG, CCL2, CD59, GDNF, MMP9, serpin b5, THBS1, thrombospondin 2, TIMP2, urokinase is increased in said CD31+ cell population as compared to the expression of said proteins in a HCAEC population cultured under 2D culture conditions.
VII. In preferred embodiments the expression of at least one or at least two, particularly at least three, more particularly at least four or even more particularly at least five cell-matrix adhesion protein(s) selected from AREG, MMP9, CD59, urokinase, TIMP2 is increased in said CD31+ cell population as compared to the expression of said protein(s) in a HCAEC population cultured under 2D culture conditions.
VIII. In preferred embodiments the expression of the at least one or preferably the at least two, more preferably the at least three, more preferably the at least four or even more preferably the at least five cell- matrix adhesion protein(s) is at least 3-fold higher in said CD31+ cell populations than in said HCAEC popula- tion.
IX. In a highly preferred embodiment
the expression of at least one or at least two, in particular at least three angiogenesis-related protein(s) selected from PF4, vasohibin, and VEGF-A is at least 3 -fold, preferably at least 4-fold higher in said CD31+ cell population than in said HCAEC population
and the expression of at least one or at least two, particularly at least three, more particularly at least four or even more particularly at least five cell-matrix adhesion protein(s) selected from AREG, CD59, MMP9, urokinase, TIMP2 is at least 3-fold higher in said CD31+ cell population than in said HCAEC population.
X. In a preferred embodiment the expression of EphrinB2 in said CD31+ cell population is increased as compared to a HUVEC population cultured under 2-dimensional culture conditions.
XL In preferred embodiments the population shows arterial phenotype indicated by increased EphrinB2 EphB4 mRNA ratio, as compared to the EphrinB2 EphB4 mRNA ratio in a HUVEC population cultured under 2D culture conditions, and indicated by increased expression of Notch2 and increased expression of EphrinB2 in said CD31+ cell population, as compared to the expression of Notch2 and the expression of EphrinB2, respectively in said HUVEC population.
XII. In certain embodiments the EphrinB2/EphB4 mRNA ratio in said CD31+ cell population is at least 2-fold higher than the EphrinB2/EphB4 mRNA ratio a HUVEC population cultured under 2-dimensional culture conditions.
XIII. In preferred embodiments the EphrinB2 EphB4 mRNA ratio in said CD31+ cell population is at least 2-fold higher than the EphrinB2/EphB4 mRNA ratio in a HUVEC population cultured under two- dimensional culture conditions and/or the expression of Notch2 is at least 4-fold higher and/or the expression of EphrinB2 is at least 2-fold higher in said CD31+ cell population than the expression of Notch2 and/or EphrinB2, respectively in said HUVEC population.
XIV. In a highly preferred embodiment
the expression of at least one or at least two, in particular at least three angiogenesis-related protein(s) selected from PF4, vasohibin, and VEGF-A is at least 3 -fold, in particular at least 4-fold higher in said CD31+ cell population than in said HCAEC population
and
the expression of at least one or at least two, particularly at least three, more particularly at least four or even more particularly at least five cell-matrix adhesion protein(s) selected from AREG, CD59, MMP9, urokinase, TIMP2 is at least 3-fold higher in said CD31+ cell population than in said HCAEC population
and the expression of EphrinB2 in said CD31+ cell population is increased as compared to a HUVEC population cultured under 2D culture conditions.
Preferably,
(i) the expression of said at least one or at least two, particularly at least three, more particularly at least four or even more particularly at least five angiogenesis-related protein(s) in said CD31+ cell population is at least 10-fold higher, in particular at least 15-fold higher than the expression of said protein(s) in said HCAEC population;
and/or
(ii) the expression of said at least one or at least two, particularly at least three, more particularly at least four or even more particularly at least five cell-matrix adhesion protein(s) in said CD31+ cell population is at least 10-fold higher, in particular at least 15-fold higher than the expression of said protein(s) in said HCAEC population.
Preferably, additionally the expression of one or more protein(s) selected from ADAMTSl, ANG (angiogenin), ANGPT1 (angiopoietin 1), ARTN (artemin), COL18A1 (collagen type XVIII alpha 1), DPP4 (dipeptidyl peptidase-4), ECGF1, EGF (epidermal growth factor), PROKR2 (prokineticin receptor 2), ENG (endoglin), FGF1 (acidic fibroblast growth factor), FGF2 (basic fibroblast growth factor), HGF (hepatocyte growth factor), IGFBP1 (insulin-like growth factor binding protein 1), IGFBP3 (insulin-like growth factor binding protein 3), IL1B (interleukin 1 beta), LEP (leptin), CCL2 (chemokine (C-C motif) ligand 2), ECGF1 (endo- thelial growth factor 1; TYMP (thymidine phosphorilase)), PDGFA (platelet-derived growth factor subunit A), PIGF (phosphatidylinositol-gly can biosynthesis class F protein), PSPN (persephin), SERPINE1, TIMP1 (tissue inhibitor of metalloproteinases 1), TIMP4 (tissue inhibitor of metalloproteinases 4), ADAM9 (disintegrin and metalloproteinase domain-containing protein 9), ADAM10 (disintegrin and metalloproteinase domain- containing protein 10), CD9, LAP3 (leucine aminopeptidase 3), TIMP3 (tissue inhibitor of metalloproteinases 3) is increased in said CD31+ cell population as compared to the expression of said protein(s) in said HCAEC population, said increase being at least 10-fold, preferably at least 15-fold higher.
Preferably, the expression of one or more protein(s) selected from ANG, TYMP, COL18A1, ADAMTS1, ADAM10, ADAM9, IGFBP3, LEP, IGFBP1, ENG, ECGF1, PLG, ANGPT1, FGF2, EGF, TIMP1, IL1B, HGF, CCL2, SERPINE1, TIMP3, FGF1, PDGFA is increased in said CD31+ cell population as com- pared to the expression of said protein(s) in said HCAEC population, said increase being at least 10-fold, in particular at least 15 -fold higher.
More preferably, the expression of one or more protein(s) selected from ANGPT1, FGF2, EGF, TIMP1, IL1B, PLG, HGF, CCL2, SERPINE1, TIMP3, FGF1, PDGFA is increased in said CD31+ cell population as compared to the expression of said protein(s) in said HCAEC population, said increase being at least 10-fold, in particular at least 15 -fold higher.
In a preferred embodiment the CD31+ cell population is cultured on a decellularised extracellular matrix bioscaffold, preferably on a decellularised human vascular bioscaffold or on a decellularised porcine intestinal extracellular matrix scaffold. Preferably, the decellularised human vascular bioscaffold is derived from human aorta. In certain embodiments the CD31+ cell population is cultured for a period of time on a decellularised extracellular matrix bioscaffold, wherein the period of time is preferably at least 1 day, at least 2 days, more preferably at least 3 days and even more preferably at least 4 days.
In preferred embodiments the culturing on the decellularised extracellular matrix bioscaffold is initiated after an increased expression of at least one angiogenesis-related protein together with an increased expression of at least one cell-matrix adhesion protein can be detected in the CD31+ cell population as compared to the expression of the at least one angiogenesis-related protein and the at least one cell-matrix adhesion protein, respectively, in a HCAEC population cultured under 2D culture conditions
and
the cell population shows arterial phenotype indicated by an increased arterial/venous marker ratio in said CD31+ cell population as compared to the arterial/venous marker ratio in a HCAEC population cultured under 2D culture conditions.
In preferred embodiments the culturing on the decellularised extracellular matrix bioscaffold is initiated when the cells show the characteristics described in any one of the numbered paragraphs I-XIV above.
A three-dimensional vascular endothelial construct is provided, comprising
- an extracellular matrix bioscaffold, preferably a decellularised human vascular bioscaffold or a decellularised porcine intestinal extracellular matrix scaffold, the decellularised human vascular bioscaffold being preferably derived from human aorta
- a CD31+ cell population defined in any one of the numbered paragraphs I-XIV above or in the claims, wherein the cells form a monolayer on the bioscaffold.
A method for providing a three-dimensional vascular endothelial construct having an arterial phenotype is provided, the method comprising
- seeding a CD31+ cell population as defined in any one of the numbered paragraphs I-XIV above or in the claims on a decellularised extracellular matrix bioscaffold, preferably a decellularised human vascular bioscaffold or a decellularised porcine intestinal extracellular matrix scaffold, the decellularised human vascular bioscaffold being preferably derived from human aorta,
- optionally seeding other cells involved in vasculogenesis on the bioscaffold,
- culturing the seeded cells on the decellularised extracellular matrix bioscaffold.
The iPSC-derived cells are derived or obtained from iPSC or an iPSC line reprogrammed by viral or non-viral delivery of transcription factors.
In highly preferred embodiments the iPSC-derived cells are derived or obtained from iPSC or an iPSC line for which the following (transcription) factors have been used in the reprogramming method: Oct4, Sox2, Nanog and optionally or preferably LIN28.
In highly preferred embodiments the iPSC-derived cells are derived or obtained from iPSC or an iPSC line of mesodermal origin, preferably of mesenchymal origin, preferably of connective tissue origin, in particular of lung origin.
In highly preferred embodiments the iPSC-derived cells are derived or obtained from iPSC or an iPSC line of mesodermal origin, preferably of epithelial origin, preferably of endothelial origin.
In highly preferred embodiments the iPSC-derived cells are derived or obtained from iPSC or an iPSC line reprogrammed by a viral method, preferably a retroviral method, in particular by a lentiviral method.
In highly preferred embodiments the iPSC-derived cells are derived or obtained from iPSC or an iPSC line for which the following (transcription) factors have been used in the reprogramming method: Oct4, Sox2, Nanog and optionally or preferably LIN28, wherein the iPSC or the iPSC cell line is of mesodermal origin and preferably it has been reprogrammed by a retroviral method.
EXAMPLES
Methods
Human pluripotent stem cell cultures. Experiments were carried out on H7 hESC line {WiCell Research Institute Bank, Madison, USA) and hiPSC line (IMR 90-4) from ReproCELL or IMR 90-4 from WiCell. Following the supplier's protocols, human PSC were maintained in their undifferentiated state in feeder cell-free conditions on Matrigel-coated {BD Biosciences, San Jose, USA) six-well plates, in mTeSRl medium {StemCell Technologies, Vancouver, Canada). Stem cell cultures were fed daily by complete media change and main- tained at 37°C, 5% C02 and 21% 02. Cell cultures were observed daily; mechanical or enzymatic passaging was made every 4-10 days by scraping or enzymatic/chemical detachment with dispase or Versene {StemCell Technologies). Chromosomal stability, gene expressions of pluripotency markers were assessed as described earlier (Merkely et al 2015). Reprocell hiPSC line was generated by lentiviral reprogramming (OCT4, KLF4, SOX2, cMyc) using myocytes as a starting cell type. Cells were maintained in ReproFF medium.
H7 human embryonic stem cells (h7 hESCs) and IMR 90-4 induced pluripotent stem cells (IMR 90-4 hiPSCs) are commercially available by WiCell Bank (Wi, USA). Cells were cultured in six-well plates (Falcon, Corning, USA) using 2 ml per well of mTeSRl complete medium (Stem Cell Technologies INC, UK) and incubated at 37°C and 5% C02 with the culture medium being changed daily. Cells were subcultured using a 1 :3 or 1 :6 ratio when 70-80% confluency was reached (about every 4 to 6 days). Prior to seeding the cells, plates were coated with a 1 :30 diluted Matrigel solution (Becton Dickinson-BD, UK) in knockOut DMEM medium (Gibco, UK) and incubated for at least 30 minutes at 37°C and 5% C02. The cell passaging protocol included a washing step with phosphate buffer saline solution without calcium and magnesium (PBS w/o Ca-Mg) (Gibco, UK) prior to the detachment of cells which was performed using 1 ml per well of Versene solution (0.048 mM, 0.2 gr EDTA) (Gibco, UK) and incubation for 5 min at 37°C. After incubation, moderate tapping of the plate caused the colonies to dislodge. Then cells were resuspended in fresh mTeSRl medium, re-plated in the matrigel- coated plates and finally incubated at 37°C and 5% C02 until they reached the desired confluency. Only blunt- end pipette tips were used in order to avoid the excessive break of hPSCs aggregates which is important to the colony forming ability after re-plating. Cultures were checked under the microscope daily in order to monitor their growth and detect any signs of contamination, cell death or spontaneous differentiation.
HCAEC and HUVEC culture
For the fresh isolation of HUVEC, umbilical cords were collected in sterile conditions. Umbilical cords were cannulated at both sides with 3 side syringe taps and were perfused with collagenase B solution (Sigma Aldrich, 2.5mg/ml). Umbilical cords were filled with collagenase B and incubated for 30 min at 37°C. After 30 min the umbilical cords were washed out with PBS solution (three times). All enzyme and PBS solution were collected during the washing into 15ml tubes. Then the collected samples were centrifuged (1100 RPM, 10 min). The cell pellets were re-suspended in EGM2 medium and plated onto gelatin-coated plates or flask (T25 and T75 cell culture flasks). Gelatin was prepared with general PBS (phosphate buffer saline) and used in 0.05% concentration. The endothelial cells were grown as monolayer culture, adhesion contact inhibition developed monolayer culture. Endothelial cells were passaged (1 :3 surface, at 70-75% confluency), as cells grew on the whole surface of the flasks. Enzymatic passaging was performed with trypsin-EDTA (TE) digestion. To enable activation of the enzyme, endothelial cultures were first washed in Mg2+ and Ca2+ rich PBS. Endothelial cultures were incubated with TE at 37°C for 3 mins. Fetal bovine serum was added to inhibit further enzymatic activity. To obtain pellet of supernatants tubes were centrifuged at 1100 RPM, for 5mins. Endothelial cell pellets were resuspended in EGM2 medium and cultured on gelatin-coated flasks up to passage 6. For cryopreservation 10% DMSO, 10% foetal bovine serum and 80% EGM2 medium is used. As HCAEC, commercially available cell cultures (e.g. Lonza) was used and cultured as described to those with HUVEC.
Endothelial differentiation and 2D culture.
The endothelial differentiation was performed in 1. normoxic (21% 02) conditions, and 2. in the presence of additional VEGF165 (10 ng/ml, Peprotech). After 13 days cells were stained with anti-human CD31-Alexa Fluor 488 fluorescence dye-labelled antibody. Cells were sorted using a FACS Aria II cell sorter (BD Biosciences) and further expanded in EGM2 medium. 3. To force differentiation into mesodermal lineage one differentiation protocol included morphogens: Activin A (10 ng/ml, R&D systems, Minnesota, USA), FGF-2 (10 ng/ml, Peprotech, New Jersey, USA), VEGF165 (10 ng/ml, R&D systems) and BMP4 (10 ng/ml, R&D systems) in mTeSRl media. After 24 hours media was removed and replaced with Stemline II complete media (Sigma-Aldrich, St. Louis, USA) containing FGF-2, VEGF165 and BMP4 (all at 10 ng/ml). Endothelial cells for 3D culture studies were differentiated with monolayer in normoxic protocol.
In the embryoid body method, hPSC were maintained in ultra-low attachment plates in order to form embryoid bodies (simulating the blastocyst stadium of the developing embryo). EBs were developed by mechanically breaking the hESC colonies as described before. EBs were cultured for four days in suspension in ultra-low attachment plates either in normoxic or hypoxic conditions. After four days, the EBs were seeded onto 0.5% gelatine (Sigma-Aldrich) coated 24-well plates or T25 flasks. After 13 days in EGM2 medium, CD31 positive endothelial cells were sorted from differentiating culture by fluorescence activated cell sorting method (FACS). Endothelial differentiation colonies were seeded into EGM2 medium containing EBM2 media (Lonza CC-3156, Basel, Switzerland) and SingleQuot supplements (Lonza CC-4176): growth factors, peptides, hor- mones and antibiotics. In monolayer method, stem cells were maintained on Matrigel in monolayer. At this point, the differentiation protocol included further VEGF165 supplementation (1 ng/ml Peprotech).
Protocol 3
Differentiation culture
The first step included the detachment of hPSCs from the maintenance culture by performing a washing step with PBS w/o Ca-Mg prior to addition of versene solution, as previously described. Cell suspension was centrifuged at 1200 rpm for 5 min leading to the formation of cell pellet and supernatant, with the supernatant being aspirated and the cell pellet being resuspended in 1 ml mTeSRl. Afterwards, the number of cells obtained was calculated according to the trypan blue exclusion method, using a 0.4% trypan blue solution (Gibco) and a Neubauer hemocytometer (Hauser Scientific, Bright-Line, USA) under the phase contrast microscope. After cell counting, the volume of cell suspension containing 105 cells (seeding density) was calculated and diluted in the appropriate volume to seed every well of a 24-well plate with 0.5 ml mTeSRl medium containing lOng/ml of Rho- associated protein kinase (ROCK) inhibitor (Gibco, UK). Prior to seeding, the 24-well plates (Greiner bioOne, UK) were coated with 1:30 matrigel/ KnockOut DMEM solution. After seeding, cell cultures were incubated for two days (Day -2) at 37°C and 5% C02. After two days (Day 0), the media was replaced with 1ml mTeSRl me- dium per well containing the growth factors Activin A (R&D systems, UK, #338-AC), bFGF (R&D systems, UK, #4114-TC), VEGF165 (Peprotech, UK, #100-20) and BMP4 (R&D systems, UK, #314-PB/CF) all at a concentration of 10 ng/ml, in order to initiate the differentiation. After 24 hours (Day 1), the medium was replaced with 1 ml of Stemline II Hematopoietic stem cell medium (Sigma-Aldrich, UK) containing bFGF, VEGF165 and BMP4, again at a concentration of lOng/ml (Differentiation medium). On day 3 and 5 one ml of fresh differen- tiation medium was added to the culture while from day 6 till day 8 1.5 ml was used daily. Finally, from day 8 till day 11 the medium was replaced also daily with 2 ml of fresh differentiation media. Pictures of the cell cultures were taken regularly in order to monitor the differentiation process and detect any signs of contamination or cell death. RNA samples were collected on day 0, day 5 and day 12 to monitor changes in expression profile sorted cells for pluripotent, endothelial and mesenchymal/fibroblast genes during differentiation.
Sample preparation for fluorescence activated cell sorting (FACS)
At day 12 of differentiation, cells were detached from the culture plates in order to isolate those that have differentiated out of the total population through fluorescence activated cell sorting (FACS). This included the aspiration of differentiation medium, a washing step with PBS w/o Ca-Mg followed by addition of ΙΟΟμΙ/well of pre- warmed 0.05 M EDTA/trypsin solution (Gibco, UK) and incubation at 37°C for 3 minutes maximum. After incubation, moderate tapping of the plate caused the colonies to dislodge and 5 parts of 10% FBS/Stemline II medium was added immediately in each well to deactivate trypsin (5: 1 ratio). After gentle stirring, the cells in medium were transferred to a 50 ml falcon tube (Falcon, Corning, USA) and the wells were rinsed with another volume of 10% FBS/Stemline II medium to collect any remaining cells. Collected cells were dispersed by gentle pipetting and filtered using a 70 μπι strainer (Falcon) to eliminate clumps and debris. After this, cells were counted (trypan blue exclusion) to make a rough estimation about their number and viability. Suspended cells were centrifuged at 1200 rpm for 5 min and then were resuspended in 130 μΐ of FACS/blocking buffer 1% Fetal Bovine Serum (FBS) (Gibco, UK) in PBS w/o Ca-Mg. Cell samples were prepared including an unstained control (10 μΐ cell suspension in 500 μΐ FACS buffer), single stained controls (10 μΐ cell suspension in 100 μΐ FACS buffer) and the full stained sample to be sorted (100 μΐ cell suspension). Next, fluorescent antibodies for anti- human CD31 AlexaFluor 488-conjugated (BD, UK, #557703) and NRP-1 APC-conjugated (Miltenyi Biotec, UK, #130-090-900) were added at a dilution rate of 1 :20 and 1 : 11, respectively up to a 1 107 cells/ FACS buffer concentration. After addition of the antibodies, samples were mixed using a vortex in low speed for 30 seconds and then incubated at 4°C for 25 minutes in the dark. Following the incubation, cells were resuspended in another volume of PBS w/o Ca-Mg and centrifuged again at 1200 rpm for 5 minutes to wash the excess of the antibodies that did not conjugate. Finally, each sample was resuspended in 0.5ml of the 1% FBS/PBS FACS buffer and filtered again through a 20 μπι strainer (BD, UK) to form a single cell suspension and avoid clogging the FACS analyser. Cell samples were kept in the dark at 4°C until the scheduled time for sorting which was performed on a FACS Aria analyser (BD, UK). In order to sort the CD31/NP-1 positive cells, single stained controls were used for distinguishing specific from non-specific binding of antibodies on cell surface (auto- fluorescence) and set up the analysis gates. Unstained sample was used as a negative control and for the elimination of samples auto-fluorescence.
Collection and culture of CD31 / NP-1 positive cells
Sorted CD31 / NP-1 positive cells were collected in sterile polystyrene round bottom tubes (BD) containing 1ml of 40% Stemline II medium, 40% of full supplemented endothelial growth factor medium-2 (EGM-2) (EBM-2 plus all SingleQuot supplements) (Lonza, UK), 20% FBS and 1% Penicillin/Streptomycin solution (P/S) (Gibco, UK). A small proportion of the sorted cells were reanalyzed to check sorting efficiency and purity. 5000 cells were seeded per well onto 24-well plates coated with collagen IV (Sigma- Aldrich, UK, #C7521) and 1 ml of 50% EGM-2 medium, 50% Stemline II medium and 1% P/S. This medium was replaced with 1 ml of 75% EGM-2, 25% Stemline II and 1% P/S media after two days (day 14) and with 100% EGM-2 and 1% P/S after three more days (day 16). At this stage sorted cells were considered as Passage 0 cells and from now on will be referred as hPSC-derived endothelial cells (hPSC-ECs). After reaching 80% confluency (around day 19-20), hPSC-ECs were subcultured (Passage 1) by trypsinisation as previously described and then were counted. 10,000-12,000 cells/cm2 were seeded onto a collagen IV coated T25 flask (25 cm2 surface area) (Greiner bio One, UK) with 5 ml of EGM-2 medium. After this point, EGM-2 full medium was replaced every two days until cells reached 80% confluency. When confluent, cells could be used for setting up an experiment, further expanded in collagen IV coated T25 flasks at a 1 :3 ratio or could be cryopreserved.
3D culture
For 3D culture of hESC-EC and hiPSC-EC spinner flask bioreactors (Corning) were used. Endothelial cells were seeded on decellularised human aortic samples or on decellularised porcine intestinal mucosal matrix (Cormatrix, Roswell, GA). Human aortic rings and wall segments were collected from heart and vascular sur- gery operations (heart transplantation and aortic aneurysm surgery) and stored at the Biobank of the Heart and Vascular Centre, Semmelweis University, Budapest (approved by ETT TUKEB 7891/2012/EKU regional ethics committee). Aortic samples were decellularised, using detergent solution containing 2% sodium-dodecyl- sulphate and 0.05% sodium-azide in PBS (all from Sigma-Aldrich). Detergent solution was changed every 6 hours upon total 72 hours of detergent wash. The decellularised aortic samples were sliced into 300μπι thin slices by microtome (Campden Instruments Lafayette, USA). Decellularised samples were washed in sterile PBS with 2% penicillin/streptomycin for further 48 hours and then preconditioned in EGM2 medium. Some slices were homogenized to test DNA/RNA content (isolation described later). Decellularisation was confirmed by diminishing all DNA and RNA components, measured by Nanodrop. For recellularisation, the decellularised aortic slices were cut for standard size (0.3 cm2) and 106 cells per well of 96-well plates were seeded.
CorMatrix® (CorMatrix ECM Technologies, Roswell, USA) is a commercially available ECM for heart and vascular surgery. CorMatrix® is derived from porcine small intestinal submucosa. During the manufacturing process CorMatrix® is decellularised, thus only ECM proteins, such as collagen, elastin and adhesion proteins remain. Clinical use of CorMatrix® has already received FDA approval for vascular and cardiac repair. CorMatrix® is preconditioned in EGM2 media and antibiotics (Penicillin Streptomycin) at 37°C before endothelial cell seeding. CorMatrix® is stored and manipulated in sterile conditions. As CorMatrix® is designed as a cardiac patch vascular stamps can be engineered.
Preconditioning of ECM was performed in the same incubator system as used for cell maintenance. Keeping the ECM in physiological conditions enabled softening of the tissue and cellular growth factors could settle inside the matrix components in biologically active form.
Slicing the decellularised aortic samples allowed purification of debris and extra connective tissue, thus cellular reseeding was more efficient. Furthermore, this technique enables detailed characterisation of the engineered construct (e.g. even confocal scanning and z-stack microscope is challenging on larger tissues).
Reseeding
Monolayer technique in plates
For recellularising aortic slices, the endothelial cells were seeded onto aortic pieces cut for standard size (0.3cm2) and 106cells/sample were seeded. The aortic slices were mounted into the bottom of 96-well plates and endothelial cells were seeded on top, thus endothelial cells proliferate mainly on the top luminal surface of the aortic samples, resulting in a monolayer endothelial culture. Fresh medium was added every other day.
Bioreactor system
The small bioreactor system composed of scale-up spinning flask (Corning, Basel, Switzerland), endothelial growth media, endothelial cells and extracellular matrix as a carrier for endothelial growth (50000 endothelial cells/ 0.5cm2 matrix). The small bioreactor system was kept under physiologic conditions (37°C, 85% humidity, 21% 02) for endothelial growth. A magnetic stirrer is integrated into the spinning flask which enables shaking together endothelial cells and the scaffolds. Bioscaffolds along with hPSC-EC were seeded into the spinning flasks. Every other day fresh media was added to the flasks.
Duration of reseeding for 5 days was optimized in pilot dose- and duration finding experiments (using 3 to 5 days protocols). After endothelial re-cellularisation vital imaging (Calcein AM, Thermo Fisher Scientific) and nuclear imaging with Hoechst-33342 or DAPI staining (Thermo Fisher Scientific) were performed. Confo- cal microscopy and PCR analyses were performed to verify endothelial seeding on biomatrices. Endothelial proliferation on the 3D vascular constructs were also visualised by microscopy.
Immunostaining. Cells were washed in PBS and fixed immediately in 4% paraformaldehyde for 10 minutes at room temperature. Cells were permeabilized with 0.2% Triton X-100 for 10 minutes and blocked with 4% FBS in PBS for lh. For specific antigen staining, cells were incubated with anti-CD31 human primary monoclonal antibodies (Abeam 24590/28364, dilution 1/100), anti-EphrinB2 human primary antibody (Abeam 75868, dilution 1/100) and anti-D114 human primary antibody (Abeam 7280, diluted 1/100), overnight at 4°C. For secondary staining AlexaFluor 546 anti-rabbit antibody raised in donkey was used (Thermo Fischer Scientific, Walthman, Massachusetts USA A10040, dilution 1/400). Cells were washed between incubations with PBS. Cell nuclei were counterstained with DAPI (Thermo Fischer Scientific D21490, dilution 1/5000). Plates were stored in PBS at 4°C prior to imaging using a Zeiss Observer microscope or Cellomics VTi HCS ArrayScanner (Thermo Fisher Scientific, Waltham, USA), as described earlier (Merkely et al., 2015). Some samples were treated with secondary antibody only in order to determine levels of non-specific background staining. For immunohistochemistry, paraformaldehyde-fixed tissue samples of recellularised bioscaffold and the implantation area from later described in vivo studies were embedded in paraffin. After deparaffinising and permeabilisation 3-5μπι thin slices were stained with anti-CD31 human primary monoclonal antibody (Abeam 28364, dilution 1/100) and anti-human EphrinB2 (Abeam 75868, dilution 1/100) and corresponding secondary antibody, respectively.
Matrigel tube formation and Ac-LDL assays. Matrigel tube formation assay was performed on endothelial cells as described previously (Merkely et al., 2015). To identify endothelial cells based on their increased metabolism of Ac-LDL, l,r-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (Dil-Ac-LDL) labelled with fluorescent probe (Thermo Fischer Scientific) was used. Cells were incubated with 10 μg/ml Dil- Ac-LDL for 4 hours at 37°C and subsequently examined by fluorescence microscopy.
Proteome profiling. Proteome profiling measurements were carried out on Proteome Profiler Human Angiogenesis Array Kit (R&D System ARY007) and Human Soluble Receptor Hematopoietic Array Kit (R&D System ARY011). Sample preparation and experimental setup followed product guide. Pixel densities of the resulted chemiluminescent signals were analysed by ImageJ software. As our primary aim was to investigate arterial-like endothelial function in hESC-EC and hiPSC-EC, HCAEC and HMVEC were selected as control endothelial cells.
Antiplatelet assay. To characterise antiplatelet function of hPSC-EC, levels of Rantes/CCL5 chemokine were measured in antiplatelet assay on 3D vascular constructs in vitro. In this set of experiments 1. 2D endothelial cultures, 2. engineered 3D constructs and 3. acellular biomatrices alone were incubated with platelet rich plasma (4.) of healthy adults. The levels of Rantes/CCL5 were measured from platelet rich plasma with ELISA assay (R&D Systems DRNOOB). To prepare platelet rich plasma, blood samples were collected in trisodium- citrate tubes. Tubes were shaken gently immediately after blood sampling to enhance dilution of anticoagulant in blood. Samples were centrifuged (1000 RPM, 7 min, 24°C), supernatant resulted in platelet rich plasma (PRP). Then samples were further centrifuged (4000 RPM, 30 min, 15°C), supernatant resulted in platelet poor plasma (PPP). In PRP platelets were counted, samples were used in 300 g/L concentration for antiplatelet functional assay. To dilute PRP, PPP were used; samples were incubated with PRP for 30 min at 37°C on slow rate stirrer. Finally, PRP was collected and centrifuged (4000 RPM, 15 min). ELISA measurements were performed from supernatant. Isolated vessel platform. The vasoactive function of hPSC-EC was assessed by using an isolated vessel platform (Radovits et al., 2013). The in vitro organ bath experiments allowed us studying vasoactive characteristics of hPSC-EC in an angio-myograph system (Radnoti Glass Technology). Briefly, aortic rings of young, adult Sprague-Dawley rats (250-350g, Charles-River) were used. The animals received general housing: room temperature and 12 hours' light/dark cycles, standard laboratory diet, free access for food and water. All animal care and handling fulfilled the Guide for Care and use of Laboratory Animals, published by U.S. National Institutes of Health. The experimental protocols were approved by the Animal Use and Care Committee of Semmelweis University, Budapest. The animals received general anaesthesia with the use of intraperitoneal injection of sodium-pentobarbital (60mg/kg) solution. Ascending part of thoracic aorta was isolated and imme- diately put into 4°C Krebs solution (118mM NaCl, 4.7mM KC1, 1.2mM KH2P04, 1.2mM MgS04, 1.77mM CaCl2, 25mM NaHC03, 11.4mM glucose; pH 7.4). Fat and connective tissue debris were cut off from the peri- adventitial region and the aortic samples were cut into 4mm thin rings. After preparation, aortic rings were applied into triangles of stainless steel and positioned between two arms of isometric force transducers. Each glass chambers contained 30 ml of pre-warmed (37°C) Krebs solution, supplied with 95% 02 and 5% C02. After setting up basal tension of the vessels, precontraction, viability of endothelium and smooth muscle cells were verified and vasoactive function of stem cell-derived endothelial cells were tested. Basal tension was set at 2g of force, followed by incubation for 60 minutes, Krebs was changed at 30 min; to reach maximum plateau of contraction, vessels were treated with KC1 (80mM). Vessels were precontracted with phenylephrine (10"6 M, Sig- ma-Aldrich). Viability of the endothelium was assessed by its endothelium-dependent vasodilative response to acetylcholine (Ach) (10"9-10"4M, Sigma-Aldrich). Viability of the smooth muscle cells was assessed as endothe- lium-independent vasodilatative response to sodium-nitroprussid (SNP) (10"10-10"5M) (Sigma-Aldrich); supernatant of 106 hPSC-ECs (5ml) were added to each bath. Data from contraction force were registered, digitalised and stored on LabChart7 (Powerlab).
In vivo transplantation. Small animal transplantation experiments were performed as described previ- ously (Merkely et al., 2015). Human ESC-EC, hiPSC-EC and control endothelial HUVEC were transplanted subcutaneously into 3 months old, male, athymic nude rats (Charles River, Sulzfeld, Germany Crl: NIH- Foxnlrnu). Animals were housed in microbe-free conditions, care and handling fulfilled the Guide for Care and Use of Laboratory Animals, published by U.S. National Institutes of Health, experimental protocols were approved by the Animal Use and Care Committee of Semmelweis University. 106 endothelial cells/plug were implanted at four sites of abdominal subcutaneous tissue of rats in Matrigel under general anaesthesia with sodium-pentobarbital (60mg/kg). After two weeks animals were euthanized with supramaximal dose of sodium- pentobarbital and all implantation sites were harvested and analysed. Live endothelial cells were re-isolated and re-cultured. Samples were stored for RNA isolation in TriReagent (Sigma-Aldrich) at -80°C.
We used a NanoSPECT/CT and PET/MRI systems which are in vivo high-resolution dual modality small animal imaging platforms to acquire whole-body images. By radiolabelled albumin, a local significant increase in perfusion was detected at the grafted sites at 2 weeks after subcutaneous implantation of hESC-EC and hiPSC, suggesting the functional incorporation of cells into the microvasculature. Using PET/MRI for detecting angiogenesis with 68Gallium-NOTA-RGD2 peptide (for ανβ3 integrin) showed an increased level of signal on the site of Matrigel plugs.
Polymerase chain reaction Total RNA was isolated from all samples with the RNeasy Mini Kit {Qiagen, Hilden, Germany Kit No.74104), according to manufacturer's protocols. RNA concentration was measured by spectrophotometer NanoDrop 2000c {Thermo Scientific). cDNA was synthesized from extracted RNA using the High Capacity cDNA Transcription Kit {Thermo Fischer Scientific Kit No. 4368814), according to manufacturer's protocols. For quantifying mRNA levels of arterial, venous and common endothelial marker genes TaqMan Gene Expression Assays (Notchl Hs00384907_CE, Notch2 Hs00247288_CE, EphrinB2 Hs00341124_CE, D114 Hs00184092_ml, EphB4 Hs01822537_cn, CD31 PECAM1 Hs00169777_ml, Angiopoietin-2 HsO 1048043 ml, VE-Cadherin Hs00170986_ml, FLT4 Hs646917 all from Applied Biosystems) were used. Human glyceraldehyde 3-phosphate dehydrogenase (GAPDH Hs02758991_gl) was used as endogenous housekeeping gene. The PCR was performed with real-time PCR instrument (Applied Biosystem, StepOnePlus) and the relative expressions were determined by AACt method, a convenient way to calculate relative gene expression levels between different samples in that it directly uses the threshold cycles (CTs) generated by the qPCR system for calculation.
Statistical analysis Statistical analysis was carried out using Graphpad Prism 5 software. Data are presented as the mean ± standard error of mean (SEM). Data were analysed by using one-way ANOVA following Tukey post-hoc test or two-way ANOVA where appropriate. In all cases significance was taken as p<0.05. All experiments were performed as at least three biological replicates from endothelial differentiation.
Proteomics expression statistics used the z score method to calculate standard deviations from the mean for each data point in a normal population of data set.
Comparative examples
The method and cells according to Harding et al. (2017) were compared with the method and cells according to the invention. The experiment was performed using a research grade human induced pluripotent line (IMR). In lack of any information on e.g. cell seeding density, the method described by Harding et al. (2017) showed very low efficiency (FIG 14). Both CD31 and VE-cadherin endothelial markers were significantly higher in the cell population obtained by the method of the invention. Arterial Notch ligand dll4 showed upregulation as compared to undifferentiated stem cells in the cell population obtained by the method of the invention, while no such change has been detected in the other population (FIG 14). Levels of most of the angiogenic factors were markedly lower in the cells from Harding protocol (FIG 15), making these cells unsuitable for angiogenic assays and for vascular endothelial constructs.
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Claims

1. A method for providing a CD31+ cell population for forming a three-dimensional (3D) vascular endothelial construct, wherein the cells of the population are capable of angiogenesis, capable of adhering to a 3D matrix and have an arterial phenotype,
said method comprising
i) seeding a starting population of CD31+ cells on a decellularised 3D scaffold used as a culturing 3D scaffold, wherein the starting population of CD31+ cells is derived from human induced pluripotent stem cells and was cultured under 2 dimensional (2D) culture conditions,
ii) culturing the seeded starting population of CD31+ cells of i) on the culturing 3D scaffold for at least one day to provide 3D cultured CD31+ cells, and
iii) isolating the 3D cultured CD31+ cells obtained in ii) from the culturing 3D scaffold at a point in time when
the expression of one or more protein(s) selected from the group consisting of angiogenesis related and cell-matrix adhesion related proteins ADAMTSl, ANG, ANGPT1, ARTN, COL18A1, DPP4, ECGF1, EGF, PROKR2, ENG, FGF1, FGF2, HGF, IGFBP1, IGFBP3, IL1B, LEP, CCL2, PDGFA, PIGF, PLG, PSPN, SERPINE1, TIMP1, TIMP4, TYMP, ADAM9, ADAMIO, CD9, LAP3, TIMP3 is increased in said 3D cultured CD31+ cells obtained in ii) compared to the expression of said protein(s) in a population of human coronary arterial endothelial cells (HCAEC population) cultured under 2D culture conditions, wherein said increase is at least 10-fold and
arterial phenotype is indicated by an increased arterial marker level compared to the corresponding arterial marker level in a population of human umbilical vein endothelial cells (HUVEC population) cultured under 2D culture conditions, wherein said arterial marker level is selected from the group consisting of: EphrinB2 EphB4 mRNA ratio, wherein the EphrinB2/EphB4 mRNA ratio in the cells obtained in ii) is at least 2-fold higher than the EphrinB2/EphB4 mRNA ratio in said HUVEC population, expression of Notch2, wherein the expression of Notch2 is at least 4-fold higher in the cells obtained in ii) than the expression of Notch2 in said HUVEC population, expression of EphrinB2, wherein the expression of EphrinB2 is at least 2-fold higher in the cells obtained in ii) than the expression of EphrinB2 in said HUVEC population.
2. The method according to claim 1, wherein
the expression of one or more protein(s) selected from the group consisting of angiogenesis related proteins ANG, ANGPTl, ARTN, DPP4, EGF, ENG, FGF1, FGF2, IL1B, LEP, PDGFA, CCL2, PLG, CD9, LAP3is increased in said 3D cultured CD31+ cells obtained in ii) compared to the expression of said protein(s) in a population of human coronary arterial endothelial cells (HCAEC population) cultured under 2D culture conditions, wherein said increase is at least 10-fold and
the expression of one or more protein(s) selected from the group consisting of cell-matrix adhesion related proteins ADAMTSl, COL18A1, ECGF1, HGF, IGFBP1, IGFBP3, TIMP1, TIMP4, TYMP, ADAM9, ADAMIO, SERPINEl, TIMP3, PIGF, PSPN, PROKR2 is increased in said 3D cultured CD31+ cells obtained in ii) compared to the expression of said protein(s) in a population of human coronary arterial endothelial cells (HCAEC population) cultured under 2D culture conditions, wherein said increase is at least 10-fold.
3. The method according to claim 2, wherein the one or more of angiogenesis related protein(s) is/are selected from the group consisting of ANGPTl, FGF2, EGF, ILB 1, PLG, CCL2, FGF1, PDGFA, and the one or more of cell-matrix adhesion related protein(s) is/are selected from the group consisting of TIMP1, HGF, SERPINE1, TIMP3.
4. The method according to claim 1, wherein the one or more protein(s) in iii) is selected from the group consisting of angiogenesis related and cell-matrix adhesion related proteins ANGPT1, FGF2, EGF, TIMP1, IL1B, PLG, HGF, CCL2, SERPI E1, TIMP3, FGF1, PDGFA.
5. The method according to any one of the preceding claims, wherein in the starting CD31+ cell population at the time of i)
- the expression of at least one, preferably at least two, more preferably at least three angiogenesis- related proteins selected from PF4, vasohibin, and VEGF-A is at least 3 -fold, preferably at least 4-fold higher in said starting CD31+ cell population than in a population of HCAEC cultured under 2D culture conditions,
- the expression of at least one, preferably at least two, more preferably at least three, more preferably at least four, even more preferably at least five cell-matrix adhesion proteins selected from AREG, CD59, MMP9, urokinase, TIMP2 is at least 3-fold higher in said starting CD31+ cell population than in a population of HCAEC cultured under 2D culture conditions,
- the EphrinB2 EphB4 mRNA ratio in said starting CD31+ cell population is at least 2-fold higher than the EphrinB2/EphB4 mRNA ratio in said HUVEC population and/or the expression of Notch2 is at least 4-fold higher and/or the expression of EphrinB2 is at least 2-fold higher in said starting CD31+ cell population than the expression of Notch2 and/or the expression of EphrinB2, respectively, in a HUVEC population cultured under 2D culture conditions.
6. The method according to any one of the preceding claims, wherein iii) is carried out at a point in time when expression of at least one angiogenesis-related protein selected from ACVR1B, angiopoietin-2, CCL3, CSF2, CXCL-16, FGF4, HB-EGF, IGFBP2, IL-8, NRG-1, PDGFB, platelet factor 4 (PF4), PLG, PTX3, serpin Fl, TGFB 1, vasohibin, VEGF-A, VEGF-C is increased at least 104 fold in the cells obtained in ii) as compared to the expression of the at least one angiogenesis-related protein in a population of human pluripotent stem cells.
7. The method according to any one of the preceding claims, wherein iii) is carried out at a point in time when
the expression of at least one angiogenesis-related protein selected from ACVRIB, angiopoietin-2, CCL3, CSF2, CXCL-16, FGF4, HB-EGF, IGFBP2, IL-8, NRG-1, PDGFB, platelet factor 4 (PF4), PLG, PTX3, serpin Fl, TGFB 1, vasohibin, VEGF-A, VEGF-C is increased at least 5 fold, preferably at least 10 fold, more preferably about 5-21 fold in the cells obtained in ii) as compared to the expression of the at least one angiogenesis-related protein in a HCAEC population cultured under 2D culture conditions,
the expression of at least one cell-matrix adhesion protein selected from AREG, CCL2, CD59, GDNF, MMP9, serpin b5, THBS1, thrombospondin 2, TIMP2, urokinase is increased at least 5 fold, preferably at least 10 fold, more preferably about 5-21 fold in the cells obtained in ii) as compared to the expression of the at least one cell-matrix adhesion protein in a HCAEC population cultured under 2D culture conditions.
8. The method according to any one of the preceding claims, wherein iii) is carried out at a point in time when
the expression of PF4, vasohibin and/or VEGFA is increased at least 8 fold, preferably at least 10 fold in the cells obtained in ii) as compared to the expression of PF4, vasohibin and/or VEGFA, respectively, in a HCAEC population cultured under 2D culture conditions, the expression of at least one, preferably at least two, preferably at least three, more preferably at least four, most preferably five proteins selected from AREG, CD59, MMP9, urokinase, TIMP2 is increased at least 3 fold, preferably at least 5 fold, more preferably at least 10 fold, most preferably at least 50 fold in the cells obtained in ii) as compared to the expression of the corresponding protein(s) in a HCAEC population cultured under 2D culture conditions.
9. The method according to any one of the preceding claims, wherein the starting population of CD31+ cells is obtainable by a method comprising
a) providing human induced pluripotent stem cells on adherent plates in pluripotent stem cells medium, preferably mTeSRl,
b) mechanical dissociation of stem cell colonies to develop embryoid bodies (EBs) and culturing said
EBs in suspension on low adherent culture dishes,
c) mesodermal induction via administration of additional growth factors and small molecules in culture media, wherein said growth factors and small molecules comprise: foetal bovine serum, human epidermal growth factor, vascular endothelial growth factor, human fibroblast growth factor B, insulin growth factor R3, hydrocortisone, heparin, ascorbic acid, antibiotic,
d) seeding and culturing EBs onto gelatine coated plates or flasks,
e) trypsinising the cultures obtained in d),
f) centrifuging the trypsinised cultures, and re-suspending pellets in PBS, counting cells,
g) labelling the cells with a label suitable for fluorescence activated cell sorting (FACS),
h) sorting CD31 positive endothelial cells by using FACS,
i) culturing and expanding CD31-positive cells in gelatinised flasks,
j) maintaining cells in EGM2 medium, feeding on every other day with EGM2 medium,
k) 1:3 surface passaging cells on every 3-6 day according to confluency and proliferation rate.
10. The method according to any one of the preceding claims, wherein ii) is carried out in a bioreactor spinner flask at 35°C to 38°C, 75-90% humidity, 21% 02, with steering at 70-75 RPM and with addition of fresh medium every other day, preferably wherein the temperature is about 37°C to 37.5°C and the humidity is about 85%.
11. The method according to any one of the preceding claims, wherein ii) is carried out in a density of 25- 75000 endothelial cells/0.5cm2 culturing 3D scaffold, preferably wherein the density is about 50000 endothelial cells/0.5cm2 culturing 3D scaffold.
12. The method according to any one of the preceding claims, wherein the culturing 3D scaffold is a decellularised human extracellular biomatrix, preferably derived from human aorta.
13. The method according to claim 12, wherein the decellularised human extracellular biomatrix is produced by a method comprising:
- harvesting aortic samples from a human,
- decellularising said aortic samples by
- washing the samples in detergent solution for 60-80 hours, preferably for about 72 hours,
- washing the aortic samples in PBS + antibiotics for 60-80 hours, preferably for about 72 hours to provide decellularised aortic samples,
- preconditioning the decellularised aortic samples in EGM2 media prior to cell seeding.
14. The method according to any one of the preceding claims, further comprising seeding the cells isolated in iii) on a 3D matrix, preferably wherein the 3D matrix is suitable for forming 3D vascular endothelial construct for use in a 3D vascular assay or the 3D matrix is suitable for forming an implantable vascular graft.
15. A CD31+ cell population for forming a 3D vascular endothelial construct, wherein the cells of the population are capable of adhering to a 3D matrix, capable of angiogenesis and have an arterial phenotype, obtained by the method according to any one of the preceding claims.
16. A method for providing a CD31+ cell population for forming a 3D vascular endothelial construct, wherein the population is capable of adhering to a 3D matrix, capable of angiogenesis and has an arterial phenotype, comprising
A) providing human induced pluripotent stem cells on adherent plates in pluripotent stem cells medium, preferably mTeSRl,
B) mechanical dissociation of stem cell colonies to develop embryoid bodies (EBs) and culturing said EBs in suspension on low adherent culture dishes,
C) mesodermal induction via administration of additional growth factors and small molecules in culture media, wherein said growth factors and small molecules comprise: foetal bovine serum, human epidermal growth factor, vascular endothelial growth factor, human fibroblast growth factor B, insulin growth factor R3, hydrocortisone, heparin, ascorbic acid, antibiotic,
D) seeding and culturing EBs onto gelatine coated plates or flasks,
E) trypsinising the cultures obtained in D),
F) centrifuging the trypsinised cultures, and re-suspending pellets in PBS, counting cells,
G) labelling the cells with a label suitable for fluorescence activated cell sorting (FACS),
H) sorting CD31 positive endothelial cells by using FACS,
I) culturing and expanding CD31 -positive cells in gelatinised flasks,
J) maintaining cells in EGM2 medium, feeding on every other day with EGM2 medium,
K) 1 :3 surface passaging cells on every 3-6 day according to confluency and proliferation rate wherein in the cells obtained in J)
- the expression of at least one, preferably at least two, more preferably at least three angiogenesis- related proteins selected from PF4, vasohibin, and VEGF-A is at least 3 -fold, preferably at least 4-fold higher than in a population of HCAEC cultured under 2D culture conditions,
- the expression of at least one, preferably at least two, more preferably at least three, more preferably at least four, even more preferably at least five cell-matrix adhesion proteins selected from AREG, CD59, MMP9, urokinase, TIMP2 is at least 3 -fold higher than in a population of HCAEC cultured under 2D culture conditions,
- the EphrinB2/EphB4 mRNA ratio is at least 2-fold higher than the EphrinB2 EphB4 mRNA ratio in a HUVEC population cultured under 2D culture conditions and/or the expression of Notch2 is at least 4-fold high- er and/or the expression of EphrinB2 is at least 2-fold higher than the expression of Notch2 and/or the expression of EphrinB2, respectively, in the HUVEC population.
17. The method according to claim 16, further comprising
L) seeding the cells obtained in K) on a decellularised 3D scaffold used as a culturing 3D scaffold M) culturing the CD31+ cells seeded in L) on the culturing 3D scaffold for at least one day to provide 3D cultured CD31+ cells, and N) isolating the CD31+ cells obtained in M) from the culturing 3D scaffold at a point in time when the expression of one or more protein(s) selected from ADAMTS1, ANG, ANGPTl, ARTN, COL18A1, DPP4, ECGF1, EGF, PROKR2, ENG, FGF1, FGF2, HGF, IGFBP1, IGFBP3, IL1B, LEP, CCL2, PDGFA, PIGF, PLG, PSPN, SERPINE1, TIMP1, TIMP4, TYMP, ADAM9, ADAMIO, CD9, LAP3, TIMP3 is increased in the cells obtained in M) as compared to the expression of said protein(s) in a HCAEC population cultured under 2D culture conditions, wherein said increase is at least 10-fold,
and the cells show arterial phenotype indicated by an EphrinB2/EphB4 mRNA ratio which at least 2-fold higher than the EphrinB2 EphB4 mRNA ratio in a HUVEC population cultured under 2D culture conditions and/or an expression of Notch2 which is at least 4-fold higher and/or an expression of EphrinB2 which is at least 2-fold higher than the expression of Notch2 and/or the expression of EphrinB2, respectively, in the HUVEC population.
18. The method according to claim 17, wherein the cells are isolated in N) at a point in time when the expression of one or more protein(s) selected from the group consisting of angiogenesis related proteins ANG, ANGPTl, ARTN, DPP4, EGF, ENG, FGF1, FGF2, IL1B, LEP, PDGFA, CCL2, PLG, CD9, LAP3 is increased in said 3D cultured CD31+ cells obtained in M) compared to the expression of said protein(s) in a population of human coronary arterial endothelial cells (HCAEC population) cultured under 2D culture conditions, wherein said increase is at least 10-fold, and
the expression of one or more protein(s) selected from the group consisting of cell-matrix adhesion related proteins ADAMTS1, COL18A1, ECGF1, HGF, IGFBP1, IGFBP3, TIMP1, TIMP4, TYMP, ADAM9, ADAMIO, SERPINEl, TIMP3, PIGF, PSPN, PROKR2 is increased in said 3D cultured CD31+ cells obtained in M) compared to the expression of said protein(s) in a population of human coronary arterial endothelial cells (HCAEC population) cultured under 2D culture conditions, wherein said increase is at least 10-fold,
and the cells show arterial phenotype indicated by an EphrinB2/EphB4 mRNA ratio which at least 2-fold higher than the EphrinB2/EphB4 mRNA ratio in a HUVEC population cultured under 2D culture conditions and/or an expression of Notch2 which is at least 4-fold higher and/or an expression of EphrinB2 which is at least 2-fold higher than the expression of Notch2 and/or the expression of EphrinB2, respectively, in the HUVEC population.
19. The method according to claim 18, wherein the cells are isolated in N) at a point in time when the expression of one or more protein(s) selected from the group consisting of angiogenesis related pro- teins ANGPTl, FGF2, EGF, ILB 1, PLG, CCL2, FGF1, PDGFA, and
the expression of one or more protein(s) selected from the group consisting of cell-matrix adhesion related proteins TIMP1, HGF, SERPINEl, TIMP3 is increased in said 3D cultured CD31+ cells obtained in M) compared to the expression of said protein(s) in a population of human coronary arterial endothelial cells (HCAEC population) cultured under 2D culture conditions, wherein said increase is at least 10-fold
and the cells show arterial phenotype indicated by an EphrinB2/EphB4 mRNA ratio which at least 2-fold higher than the EphrinB2 EphB4 mRNA ratio in a HUVEC population cultured under 2D culture conditions and/or an expression of Notch2 which is at least 4-fold higher and/or an expression of EphrinB2 which is at least 2-fold higher than the expression of Notch2 and/or the expression of EphrinB2, respectively, in the HUVEC population.
20. The method according to claim 17, wherein the one or more protein(s) in N) is selected from ANGPT1, FGF2, EGF, TIMP1, IL1B, PLG, HGF, CCL2, SERPINE1, TIMP3, FGF1, PDGFA.
21. The method according any one of claims 17 to 20, wherein N) is carried out at a point in time when the expression of at least one angiogenesis-related protein selected from ACVR1B, angiopoietin-2,
CCL3, CSF2, CXCL-16, FGF4, HB-EGF, IGFBP2, IL-8, NRG-1, PDGFB, platelet factor 4 (PF4), PLG, PTX3, serpin Fl, TGFB 1, vasohibin, VEGF-A, VEGF-C is increased at least 5 fold, preferably at least 10 fold, more preferably about 5-21 fold in the cells obtained in M) as compared to the expression of the at least one angiogenesis-related protein in a HCAEC population cultured under 2D culture conditions,
the expression of at least one cell-matrix adhesion protein selected from AREG, CCL2, CD59, GDNF, MMP9, serpin b5, THBSl, thrombospondin 2, TIMP2, urokinase is increased at least 5 fold, preferably at least 10 fold, more preferably about 5-21 fold in the cells obtained in M) as compared to the expression of the at least one cell-matrix adhesion protein in a HCAEC population cultured under 2D culture conditions.
22. The method according to any one of claims 17 to 21, wherein N) is carried out at a point in time when the expression of PF4, vasohibin and/or VEGFA is increased at least 8 fold, preferably at least 10 fold in the cells obtained in M) as compared to the expression of PF4, vasohibin and/or VEGFA, respectively, in a HCAEC population cultured under 2D culture conditions,
the expression of at least one, preferably at least two, preferably at least three, more preferably at least four, most preferably five proteins selected from AREG, CD59, MMP9, urokinase, TIMP2 is increased at least 3 fold, preferably at least 5 fold, more preferably at least 10 fold, most preferably at least 50 fold in the cells obtained in M) as compared to the expression of the corresponding protein(s) in a HCAEC population cultured under 2D culture conditions.
23. The method according to any one of claims 17 to 22, wherein M) is carried out in a bioreactor spinner flask at 35°C to 38°C, 75-90% humidity, 21% 02, with steering at 70-75 RPM and with addition of fresh medium every other day, preferably wherein the temperature is about 37°C to 37.5°C and the humidity is about 85%.
24. The method according to any one of claims 17 to 23, wherein L) is carried out in a density of 25- 75000 endothelial cells/0.5cm2 culturing 3D scaffold, preferably wherein the density is about 50000 endothelial cells/0.5cm2 culturing 3D scaffold.
25. The method according to any one of claims 17 to 24, wherein the culturing 3D scaffold is a decellularised human extracellular biomatrix, preferably derived from human aorta.
26. The method according to claim 25, wherein the decellularised human extracellular biomatrix is pro- duced by a method comprising:
- harvesting aortic samples from a human,
- decellularising said aortic samples by
- washing the samples in detergent solution for 60-80 hours, preferably for about 72 hours,
- washing the aortic samples in PBS + antibiotics for 60-80 hours, preferably for about 72 hours to provide decellularised aortic samples,
- preconditioning the decellularised aortic samples in EGM2 media prior to cell seeding.
27. The method according to claim 16 further comprising seeding the cells obtained in K) on a 3D matrix, preferably wherein the 3D matrix is suitable for forming 3D vascular endothelial construct for use in a 3D vascular assay or the 3D matrix is suitable for forming an implantable vascular graft.
28. The method according to any one of claims 17 to 26, further comprising seeding the cells isolated in N) on a 3D matrix, preferably wherein the 3D matrix is suitable for forming 3D vascular endothelial construct for use in a 3D vascular assay or the 3D matrix is suitable for forming an implantable vascular graft.
29. A CD31+ cell population for forming a 3D vascular endothelial construct, wherein the cells of the population are capable of adhering to a 3D matrix, capable of angiogenesis and have an arterial phenotype, obtained by the method according to any one of claims 16 to 28.
30. A CD31+ cell population for forming a 3D vascular endothelial construct, wherein the population is capable of adhering to a 3D matrix, capable of angiogenesis, has an arterial phenotype and is derived from hiPSC, characterized in that
- the expression of at least one, preferably at least two, more preferably at least three angiogenesis- related proteins selected from PF4, vasohibin, and VEGF-A in the CD31+ cells is at least 3-fold, preferably at least 4-fold higher than in a population of HCAEC cultured under 2D culture conditions,
- the expression of at least one, preferably at least two, more preferably at least three, more preferably at least four, even more preferably at least five cell-matrix adhesion proteins selected from AREG, CD59, MMP9, urokinase, TIMP2 in the CD31+ cells is at least 3-fold higher than in a population of HCAEC cultured under 2D culture conditions,
- the EphrinB2 EphB4 mRNA ratio in the CD31+ cells is at least 2-fold higher than the EphrinB2 EphB4 mRNA ratio in a HUVEC population cultured under 2D culture conditions and/or the expression of Notch2 in the CD31+ cells is at least 4-fold higher and/or the expression of EphrinB2 in the CD31+ cells is at least 2-fold higher than the expression of Notch2 and/or the expression of EphrinB2, respectively, in the HUVEC population.
31. The CD31+ cell population of claim 30, wherein additionally the expression of one or more protein(s) selected from ADAMTSl, ANG, ANGPT1, ARTN, COL18A1, DPP4, EGF, PROKR2, ENG, FGF1, FGF2, HGF, IGFBP1, IGFBP3, IL1B, LEP, CCL2, ECGF1, PDGFA, PIGF, PLG, PSPN, SERPINE1, TIMP1, TIMP4, TYMP, ADAM9, ADAM10, CD9, LAP3,TIMP3 is increased in said CD31+ cells as compared to the expression of said protein(s) in said HCAEC population, wherein said increase is at least 10-fold.
32. The CD31+ cell population of claim 30, wherein additionally the expression of one or more angiogen- esis-related protein(s) selected from the group consisting of ANG, ANGPT1, ARTN, DPP4, EGF, ENG, FGF1, FGF2, IL1B, LEP, PDGFA, CCL2, PLG, CD9, LAP3, and
the expression of one or more cell-matrix adhesion protein(s) selected from the group consisting of ADAMTSl, COL18A1, ECGF1, HGF, IGFBP1, IGFBP3, TIMP1, TIMP4, TYMP, ADAM9, ADAMIO, SERPINEl, TIMP3, PIGF, PSPN, PROKR2 is increased in said 3D cultured CD31+ cells obtained in ii) compared to the expression of said protein(s) in a population of human coronary arterial endothelial cells (HCAEC population) cultured under 2D culture conditions, wherein said increase is at least 10-fold.
33. The CD31+ cell population of claim 32, wherein the additional one or more angiogenesis-related pro- tein(s) is/are selected from the group consisting of ANGPTl, FGF2, EGF, ILB 1, PLG, CCL2, FGF1, PDGFA, and the additional one or more cell-matrix adhesion related protein(s) is/are selected from the group consisting of TIMP1, HGF, SERPINEl, TIMP3.
34. The CD31+ cell population of claim 31, wherein the additional one or more protein(s) is/are selected from ANGPTl, FGF2, EGF, TIMP1, IL1B, PLG, HGF, CCL2, SERPINEl, TIMP3, FGF1, PDGFA.
35. Use of the CD31+ cell population according to claim 15 or according to any one of claims 29 to 34, for forming a 3D vascular endothelial construct.
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