Undifferentiated Stem Cell Culture Systems
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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.
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Citations
65 Claims
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1-38. -38. (canceled)
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39. A method of expanding undifferentiated pluripotent stem cells (SC) in suspension, the method comprises:
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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; andallowing said SC to expand; whereby the expanded SC are maintained in an undifferentiated, pluripotent state. - View Dependent Claims (40, 41, 42, 43, 44, 45, 46)
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- 47. A culture system for expansion of stem cells (SCs) comprising a suspension of undifferentiated stem cells within a basic medium and KOSR.
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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:
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(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. - View Dependent Claims (54, 55, 56, 57, 58, 59, 60)
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61. A method for promoting spontaneous differentiation of SCs into somatic cells, the method comprising:
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(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. - View Dependent Claims (62, 63)
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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.
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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:
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(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.
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Specification