Undifferentiated Stem Cell Culture Systems

• US 20100093091A1
• Filed: 09/30/2009
• Published: 04/15/2010
• Est. Priority Date: 04/02/2007
• Status: Abandoned Application

• 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: Steiner, Debora, Reubinoff, Benjamin
• Application Number:
  US12/570,457
• Publication Number:
  US 20100093091A1
• Time in Patent Office: Days
• US Class Current: 435/377
• 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

1-38. -38. (canceled)

39. A method of expanding undifferentiated pluripotent stem cells (SC) in suspension, the method comprises:

preparing a culture system comprising a non adherent culture dish, a basic medium;

a serum replacement, a factor that promote maintenance of pluripotency, an extracellular matrix component and suspended therein said SC; and

allowing said SC to expand;

whereby the expanded SC are maintained in an undifferentiated, pluripotent state.

40. The method of claim 39, wherein said culture system comprises a basic medium;

a Knockout™

serum replacement (KOSR) and undifferentiated SC suspended therein and the method comprises incubating said SC in said culture system for a period of time allowing said cells to expand, the expanded cells being in an undifferentiated pluripotent state.

41. The method of claim 39, wherein said undifferentiated SCs are derived from SC colonies cultivated on a feeder layer or in a feeder free adherent culture system.

42. The method of claim 39, wherein the undifferentiated SCs in said suspension are in form of free floating cells, free floating clusters of cells or free floating aggregates of cells.

43. The method of claim 39, wherein said basic medium is Neurobasal™

.

44. The method of claim 39, wherein said culture system comprises one or more of the following:

a member of FGF family;

a extracellular matrix (ECM) component;

an antibacterial agent;

nonessential amino acids;

a TGFβ

superfamily factor;

a neurotrophin;

nicotinamide (NA);

a bone morphogenic protein (BMP) antagonist;

or

a serum free medium supplement.

45. The method of claim 44, wherein said ECM component is selected from fibronectin, laminin and gelatin;

said antibacterial agent is selected from penicillin and streptomycin;

said TGFβ

superfamily factor is selected from activin A;

said BMP antagonist is selected from noggin, chordin; and

said serum free medium supplement is selected from Nutridoma-CS or TCH™

.

46. The method of claim 39, for one or more of large-scale expansion and long term cultivation of undifferentiated pluripotent SCs in suspension.

47. A culture system for expansion of stem cells (SCs) comprising a suspension of undifferentiated stem cells within a basic medium and KOSR.

48. The culture system of claim 47, wherein said suspension of stem cells comprises free floating cells, free floating clusters of cells or free floating aggregates of cells.

49. The culture system of claim 47, wherein said the basic medium is Neurobasal™

.

50. The culture system of claim 47, comprising one or more of the following:

a member of FGF family;

an extracellular matrix (ECM) component;

an antibacterial agent;

non-essential amino acids

a TGFβ

superfamily factor;

a neurotrophin;

nicotinamide (NA);

a bone morphogenic protein (BMP) antagonist;

or

a serum free medium supplement.

51. The culture system of claim 47, wherein said FGF member is FGF-2, ECM component is selected from fibronectin, laminin and gelatin;

said antibacterial agent is selected from penicillin and streptomycin;

said TGFβ

superfamily factor is activin A;

said BMP antagonist is selected from noggin, chordin, or gremlin, said neurotrophin is selected from BDNF, NT3, NT4; and

said a serum free medium supplement is selected from Nutridoma-CS or TCH™

.

52. The culture system of claim 47, wherein said SCs are pluripotent, human embryonic SCs (hESCs).

53. A method for directing differentiation of SCs into a selected population of somatic cells from a culture system of SCs in suspension, the method comprising:

(a) providing a suspension of undifferentiated SCs obtainable from a culture system comprising a basic medium and Knockout™

serum replacement (KOSR); and

(b) incubating said undifferentiated SCs in a culture system that supports directed differentiation of SCs into the selected fate of somatic cells.

54. The method of claim 53, wherein said SCs are hESC.

55. The method of claim 53, wherein the SCs are incubated in a culture system that directs differentiation into somatic cells are selected from mesodermal, endodermal or ectodermal cells.

56. The method of claim 55, wherein said selected population of somatic cells consists essentially of a single population of differentiated somatic cells.

57. The method of claim 55, wherein said population of somatic cells consists essentially of neural precursor or neural stem cells.

58. The method of claim 53, wherein the culture system directing differentiation comprises a basic media, FGF2 and/or noggin.

59. The method of claim 58, wherein said selected population of somatic cells consists essentially of dopaminergic neuronal cells

60. The method of claim 59, wherein said culture system supporting differentiation of neural precursor cells into dopaminergic neuronal cells comprises at least one of sonic hedgehog (SHH), a fibroblast growth factor (FGF), or a member of the Wnt family followed by final differentiation in the presence of at least one of FGF, member of the Wnt family and survival factors.

61. A method for promoting spontaneous differentiation of SCs into somatic cells, the method comprising:

(a) providing a suspension of undifferentiated pluripotent SCs obtainable from a culture system comprising a basic medium and KOSR;

(b) incubating said undifferentiated SCs in culture system that support spontaneous differentiation of SCs into the somatic cells.

62. The method of claim 61, wherein said somatic cells comprise a mixture or any one of mesodermal cells, ectodermal cells, endodermal cells and combination of same.

63. The method of claim 61, wherein said culture system that supports spontaneous differentiation of SCs into somatic cells comprises a basic medium of DMEM supplemented by FCS 20%.

64. A method of expanding pluripotent stem cells in an undifferentiated state, the method comprising incubating undifferentiated pluripotent SCs in a culture system comprising a non-adherent culture dish, a basic serum free medium, a serum replacement, an extra cellular matrix component and a factor supporting expansion of SC in an undifferentiated state.

65. A method for deriving cells from a human embryo the cells exhibiting a morphological characteristic of a human embryonic stem cell, the method comprising:

(a) providing in vitro fertilized embryos;

(b) culturing said embryos to a blastocyte stage;

(c) isolating from said blastocyte inner cell mass (ICM);

(d) culturing said ICM in suspension with a feeder free culture system comprising a basic media and a serum replacement until clusters capable of propagating are formed;

wherein said clusters comprise cells exhibiting at least one morphological characteristic of a SC.