# US 10,119,122 B2 · Differentiation and expansion of endothelial cells from pluripotent stem cells and the in vitro formation of vasculature like structures > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,119,122 B2 - Document ID: US10119122B2 - Status: Granted - Kind: B2 - Application number: 14/897,619 - Filed: 2014-06-10 - Published: 2016-04-28 - Granted: 2018-11-06 - Earliest claimed: 2013-06-10 - Assignees: Academisch Ziekenhuis Leiden - Applicants: Academisch Ziekenhuis Leiden - Inventors: Christine Lindsay Mummery; Valeriya Viktorovna Orlova - Web page: https://patentgrep.com/patent/US10119122B2 ## CPC - C12N 5/0691: {Vascular smooth muscle cells; 3D culture thereof, e.g. models of blood vessels} - C12N 5/069: {Vascular Endothelial cells} - G01N 33/5061: {Muscle cells} - G01N 33/5064: {Endothelial cells} - C12N 2500/90: Serum-free medium, which may still contain naturally-sourced components - C12N 2501/15: Transforming growth factor beta (TGF-β) - C12N 2501/155: Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor - C12N 2501/16: Activin; Inhibin; Mullerian inhibiting substance - C12N 2501/165: Vascular endothelial growth factor [VEGF] - C12N 2501/415: Wnt; Frizzeled - C12N 2501/727: Kinases (EC 2.7.) - C12N 2503/06: Screening or testing on artificial skin - C12N 2506/45: from artificially induced pluripotent stem cells - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract The disclosure is concerned among others with means and methods for obtaining endothelial cells and to means and methods for in vitro cell culture comprising endothelial cells and pericytes and/or smooth muscle cells derived from the pericytes. The endothelial cells or the pericytes and/or smooth muscle cells, or both, are preferably derived from in vitro differentiated pluripotent stem cells. ## Claims ### Claim 1 A method for producing an in vitro cell culture comprising endothelial cells derived from in vitro differentiated pluripotent stein cells and smooth muscle cells derived from in vitro differentiated pluripotent stem cells or derived from pericytes, the method comprising the following steps: (a) culturing pluripotent stem cells in defined medium comprising ActivinA, BMP4, VEGF, and a canonical WNT ligand or GSK3 inhibitor, to produce a culture comprising differentiated cells; (b) culturing the cells obtained in step (a) in defined medium containing VEGF and a TGF{beta} signaling inhibitor to produce endothelial cells; and (c) culturing the endothelial cells obtained in step (b) with smooth muscle cells derived from in vitro differentiated pluripotent stem cells or derived from pericytes. ### Claim 2 (depends on 1) The method according to claim 1, further comprising: collecting the cells produced in step (b) and obtaining therefrom a collection of cells that comprises more than 90% endothelial cells. ### Claim 3 (depends on 1) The method according to claim 1, further comprising: collecting the cells produced in step (b) and obtaining therefrom a collection of cells that comprises more than 90% pericytes. ### Claim 4 (depends on 2) The method according to claim 2, wherein said collection of cells is obtained by separating the cells of step (b) on the basis of CD31 expression. ### Claim 5 (depends on 3) The method according to claim 3, further comprising: providing the endothelial cells with a cell storage medium and storing the endothelial cells at a temperature of −70° C. or less. ### Claim 6 (depends on 2) The method according to claim 2, further comprising: culturing said endothelial cells together with smooth muscle cells. ### Claim 7 A method of producing an in vitro cell culture comprising endothelial cells and smooth muscle cells, wherein the endothelial cells are able to integrate into a vascular network in vivo, the method comprising: (a) culturing pluripotent stem cells in a defined medium comprising ActivinA, BMP4, VEGF, and a canonical WNT ligand or GSK3 inhibitor, to produce a culture comprising differentiated cells; (b) culturing the cells of step (a) in defined medium containing VEGF and a TGF(beta) signaling inhibitor to produce endothelial cells; and (c) culturing the endothelial cells obtained in step (b) with smooth muscle cells. ### Claim 8 (depends on 2) The method according to claim 2, wherein the collection of cells comprises more than 90% endothelial cells based on the expression of CD31, CD34 or VECadherin. ### Claim 9 (depends on 3) The method according to claim 3, wherein the collection of cells comprises more than 90% pericytes based on the expression of CD31, CD34 or VECadherin. ### Claim 10 (depends on 4) The method according to claim 4, wherein separating the cells of step (b) on the basis of CD31 expression is by means of beads comprising a CD31 binding antibody. ### Claim 11 A method for producing an in vitro cell culture comprising purified endothelial cells and purified smooth muscle cells, wherein the cell culture comprises a capillary network comprising the endothelial cells and the smooth muscle cells, the method comprising: deriving endothelial cells from in vitro differentiated induced pluripotent stem cells (iPSCs) and purifying the endothelial cells, deriving smooth muscle cells from in vitro differentiated induced pluripotent stem cells or from pericytes that are derived from in vitro differentiated induced pluripotent stem cells and purifying the smooth muscle cells, and culturing the purified endothelial cells together with the purified smooth muscle cells. ### Claim 12 (depends on 11) The method according to claim 11, wherein the endothelial cells and the smooth muscle cells are derived from induced pluripotent stem cells (iPSCs) generated from skin biopsy fibroblasts or blood outgrowth endothelial cells. ### Claim 13 (depends on 11) The method according to claim 11, wherein the endothelial cells are able to integrate into a vascular network in vivo. ### Claim 14 (depends on 11) The method according to claim 11, wherein the endothelial cells have a genetic defect that in vivo exhibits a phenotype in vasculature. ### Claim 15 (depends on 11) The method according to claim 11, wherein the smooth muscle cells have a genetic defect that in vivo exhibits a phenotype in vasculature. ### Claim 16 (depends on 11) The method according to claim 11, further comprising: isolating the endothelial cells and the smooth muscle cells, and cryopreserving the endothelial cells and the smooth muscle cells. ### Claim 17 (depends on 11) The method according to claim 11, further comprising: screening the cell culture for angiogenic and anti-angiogenic compounds.