Undifferentiated stem cell culture systems

• US 8,940,537 B2
• Filed: 01/25/2012
• Issued: 01/27/2015
• Est. Priority Date: 04/02/2007
• Status: Active Grant

• Current Assignee: Hadasit Medical Research Services & Development Ltd (Hadassah, The Women's Zionist Organization of America, Inc.)
• Original Assignee: Hadasit Medical Research Services & Development Ltd (Hadassah, The Women's Zionist Organization of America, Inc.)
• Inventors: Reubinoff, Benjamin, Steiner, Debora
• Primary Examiner(s): BERTOGLIO, VALARIE E
• Application Number:
  US13/357,948
• Publication Number:
  US 20120122209A1
• Time in Patent Office: 1,098 Days
• Field of Search: None
• US Class Current: 435/384
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• CPC Class Codes: C12N 2501/13 Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2502/1323 Adult fibroblasts
• CPC Class Codes: C12N 2533/52 Fibronectin; Laminin
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696

Abstract

The present disclosure provides methods for maintaining and propagating undifferentiated pluripotent stem cells (SC) in suspension. The methods comprise culturing such SC in a non-adherent culture dish under conditions comprising a basic serum free medium and one or more of a basic medium, a serum replacement, an extra cellular matrix component and a factor supporting expansion of said SC. A specific and preferred culture condition comprise supplementing Neurobasal™ medium with KO serum replacement (KOSR). These conditions allowed for large scale and long term propagation of undifferentiated pluripotent SC. The culture system comprising suspended undifferentiated pluripotent SC were found to have many applications including in methods for directed as well as spontaneous differentiation of the SC into somatic cells. Also disclosed herein is a method of deriving SC, preferably human embryonic SC from human embryos via the formation of cell clusters.

Claims

1. A method for deriving one or more undifferentiated pluripotent cells from a human embryo, comprising:

providing in vitro fertilized embryos;

culturing the embryos to a blastocyte stage;

isolating inner cellular mass (ICM) from the blastocyte; and

culturing the ICM in suspension with a feeder free culture system comprising a basic media, a TGFβ

superfamily factor, and a serum replacement to obtain clusters capable of propagating that comprise the one or more undifferentiated pluripotent cells.

2. The method of claim 1, further comprising isolating the ICM from the blastocyte'"'"'s zona pellucida.

3. The method of claim 1, wherein the ICM is isolated by immunosurgery.

4. The method of claim 3, wherein the immunosurgery comprises lysing and removing at least one trophectoderm cell.

5. The method of claim 1, wherein the clusters comprise compact clusters of uniformly small, packed, substantially transparent cells, free of cystic structures.

6. A method for deriving one or more undifferentiated pluripotent cells comprising:

culturing at least one fertilized embryo to obtain at least one blastocyte;

isolating inner cell mass (ICM) from the one or more of the at least one blastocyte; and

culturing the ICM in suspension with a feeder free culture system comprising a basic media, a TGFβ

superfamily factor, and a serum replacement to obtain clusters capable of propagating that comprise the one or more undifferentiated pluripotent cells.