# US 2019/0316082 A1 · Media for culturing stem cells > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2019/0316082 A1 - Document ID: US20190316082A1 - Status: Published application - Kind: A1 - Application number: 16/454,168 - Filed: 2019-06-27 - Published: 2019-10-17 - Earliest claimed: 2005-08-29 - Assignees: Technion Research & Development Foundation Limited - Applicants: Technion Research & Development Foundation Limited - Inventors: Michal AMIT; Joseph ITSKOVITZ-ELDOR - Web page: https://patentgrep.com/patent/US20190316082A1 ## CPC - C12N 5/0606: {Pluripotent embryonic cells, e.g. embryonic stem cells [ES] (embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696)} - C12N 5/0018: {Culture media for cell or tissue culture (media for specific animal cell type C12N5/06)} - C12N 5/0037: {Serum-free medium, which may still contain naturally-sourced components} - C12N 5/0043: {Medium free of human- or animal-derived components} - C12N 5/0056: {Xeno-free medium} - C12N 5/0602: {Vertebrate cells} - C12N 5/0603: {Embryonic cells (production of embryos, nuclear transfer A01K67/027); Embryoid bodies} - C12N 2500/90: Serum-free medium, which may still contain naturally-sourced components - C12N 2500/98: Xeno-free medium and culture conditions - C12N 2501/115: Basic fibroblast growth factor (bFGF, FGF-2) - C12N 2501/15: Transforming growth factor beta (TGF-β) - C12N 2501/23: Interleukins [IL] - C12N 2501/2306: Interleukin-6 (IL-6) - C12N 2506/02: from embryonic cells - C12N 2533/52: Fibronectin; Laminin - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract Well-defined, xeno-free culture media which comprise a TGF-beta isoform or the chimera formed between IL6 and the soluble IL6 receptor (IL6RIL6), which are capable of maintaining stem cells, and particularly, human embryonic stem cells, in an undifferentiated state are provided. Also provided are cell cultures comprising the culture media and the stem cells and methods of expanding and deriving embryonic stem cells in such well-defined, xeno-free culture media. In addition, the present invention provides methods of differentiating ESCs or EBs formed therefrom for the generation of lineage specific cells. ## Claims ### Claim 1 A culture medium comprising an IL6RIL6 chimera and basic fibroblast growth factor (bFGF), wherein said IL6RIL6 chimera comprises amino acids 112-355 of GenBank Accession No. AAH89410 wherein said culture medium is capable of maintaining said human pluripotent stem cell in an undifferentiated state for at least 7 passages. ### Claim 2 (depends on 1) The culture medium of claim 1, wherein said IL6RIL6 chimera is provided at a concentration of at least 25 ng/ml. ### Claim 3 (depends on 1) The culture medium of claim 1, wherein said IL6RIL6 chimera is provided at a concentration of at least 200 ng/ml. ### Claim 4 (depends on 1) The culture medium of claim 1, wherein said culture medium is serum replacement-free. ### Claim 5 (depends on 1) The culture medium of claim 1, wherein said culture medium comprises serum replacement. ### Claim 6 (depends on 1) The culture medium of claim 1, wherein said culture medium is devoid of animal contaminant. ### Claim 7 A cell culture comprising a human pluripotent stem cell and a culture medium, said culture medium comprising an IL6RIL6 chimera, wherein said IL6RIL6 chimera comprises amino acids 112-355 of GenBank Accession No. AAH89410, wherein said culture medium is capable of maintaining said human pluripotent stem cell in an undifferentiated state. ### Claim 8 (depends on 7) The cell culture of claim 7, wherein said culture medium further comprises basic fibroblast growth factor (bFGF). ### Claim 9 (depends on 7) The cell culture of claim 7, wherein said IL6RIL6 chimera is provided at a concentration of at least 25 ng/ml. ### Claim 10 (depends on 7) The cell culture of claim 7, wherein said IL6RIL6 chimera is provided at a concentration of at least 200 ng/ml. ### Claim 11 (depends on 7) The cell culture of claim 7, wherein said culture medium is capable of expanding said human pluripotent stem cells in an undifferentiated state for at least 7 passages. ### Claim 12 (depends on 7) The cell culture of claim 7, wherein said culture medium comprises serum or serum replacement. ### Claim 13 (depends on 7) The cell culture of claim 7, wherein said culture medium is serum replacement-free. ### Claim 14 (depends on 7) The cell culture of claim 7, wherein said culture medium is devoid of animal contaminant. ### Claim 15 (depends on 7) The cell culture of claim 7, wherein the human pluripotent stem cell is a human embryonic stem cell. ### Claim 16 A method of expanding and maintaining human pluripotent stem cells in an undifferentiated state, the method comprising culturing the human pluripotent stem cells in a culture medium which comprises an IL6RIL6 chimera, wherein said IL6RIL6 chimera comprises amino acids 112-355 of GenBank Accession No. AAH89410, said culture medium is capable of maintaining the human pluripotent stem cells in an undifferentiated state, thereby expanding and maintaining the embryonic stem cells in the undifferentiated state. ### Claim 17 (depends on 16) The method of claim 16, wherein said culture medium further comprises basic fibroblast growth factor (bFGF). ### Claim 18 (depends on 1) A method of deriving a human embryonic stem cell line, the method comprising: (a) obtaining a human embryonic stem cell from a pre-implantation stage blastocyst, post-implantation stage blastocyst and/or a genital tissue of a fetus; and (b) culturing said human embryonic stem cell in the culture medium of claim 1, thereby deriving the embryonic stem cell line. ### Claim 19 (depends on 16) A method of generating lineage-specific cells from pluripotent stem cells, the method comprising: (a) culturing the pluripotent stem cells according to the method of claim 16, to thereby obtain expanded, undifferentiated embryonic stem cells; and (b) subjecting said expanded, undifferentiated pluripotent stem cells to culturing conditions suitable for differentiating and/or expanding lineage specific cells; thereby generating the lineage-specific cells from the pluripotent stem cells. ### Claim 20 (depends on 16) A method of generating embryoid bodies from pluripotent stem cells, the method comprising: (a) culturing the pluripotent stem cells according to the method of claim 16, to thereby obtain expanded, undifferentiated embryonic stem cells; and (b) subjecting said expanded, undifferentiated pluripotent stem cells to culturing conditions suitable for differentiating said pluripotent stem cells to embryoid bodies; thereby generating the embryoid bodies from the pluripotent stem cells. ### Claim 21 (depends on 16) A method of generating lineage-specific cells from pluripotent stem cells, the method comprising: (a) culturing the pluripotent stem cells according to the method of claim 16, to thereby obtain expanded, undifferentiated pluripotent stem cells; (b) subjecting said expanded, undifferentiated pluripotent stem cells to culturing conditions suitable for differentiating said expanded, undifferentiated pluripotent stem cells to embryoid bodies; and (c) subjecting cells of said embryoid bodies to culturing conditions suitable for differentiating and/or expanding lineage specific cells; thereby generating the lineage-specific cells from the pluripotent stem cells.