Perfusion cultures of human embryonic stem cells

Human embryonic stem cells (hESC) are self-renewing pluripotent cells capable of differentiating into cells representative of all three embryonic germ layers. Hence, they hold great potential for regenerative medicine. However, significant cell numbers are required to fulfill their potential therapeutic applications. In this study, perfusion with supplemented conditioned media (SCM), produced by mouse embryonic fibroblasts (MEF), was adopted to improve cell densities of hESC cultures. Perfusion enhanced hESC numbers by 70% compared to static conditions, on both organ culture dish (OCD) and petridish cultures. All cultures maintained healthy expression of the pluripotent marker, Oct-4 transcription factor. In vivo, perfused hESC formed teratomas in severe combined immunodeficiency (SCID) mice models that represent the three embryonic germ layers. When SCM was produced with lower concentrations of MEF, hESC densities and Oct-4 levels were reduced. Hence, perfusion with SCM is a potential feeding method for scale-up production of hESC.

[1]  Steve Oh,et al.  Expansion of pluripotent human embryonic stem cells on human feeders , 2004, Biotechnology and bioengineering.

[2]  J. Itskovitz‐Eldor,et al.  Human Feeder Layers for Human Embryonic Stem Cells1 , 2003, Biology of reproduction.

[3]  J. Itskovitz‐Eldor,et al.  Controlled, Scalable Embryonic Stem Cell Differentiation Culture , 2004, Stem cells.

[4]  D. Kaufman,et al.  Multilineage Differentiation from Human Embryonic Stem Cell Lines , 2001, Stem cells.

[5]  M. Rao,et al.  Enrichment of Neurons and Neural Precursors from Human Embryonic Stem Cells , 2001, Experimental Neurology.

[6]  Smadar Cohen,et al.  Bioreactor cultivation enhances the efficiency of human embryoid body (hEB) formation and differentiation , 2004, Biotechnology and bioengineering.

[7]  J. Thomson,et al.  Embryonic stem cell lines derived from human blastocysts. , 1998, Science.

[8]  J F Elliott,et al.  Clinical outcomes and insulin secretion after islet transplantation with the Edmonton protocol. , 2001, Diabetes.

[9]  J. Itskovitz‐Eldor,et al.  Differentiation of Human Embryonic Stem Cells into Insulin‐Producing Clusters , 2004, Stem cells.

[10]  Shang-Tian Yang,et al.  Culturing and differentiation of murine embryonic stem cells in a three-dimensional fibrous matrix , 2004, Cytotechnology.

[11]  J. Itskovitz‐Eldor,et al.  Feeder Layer- and Serum-Free Culture of Human Embryonic Stem Cells1 , 2004, Biology of reproduction.

[12]  J. D. Yang,et al.  Achievement of high cell density and high antibody productivity by a controlled-fed perfusion bioreactor process. , 2000, Biotechnology and bioengineering.

[13]  Irving L. Weissman,et al.  Stem cells [14] , 1991 .

[14]  J. Itskovitz‐Eldor,et al.  Cardiovascular potential of embryonic stem cells. , 2004, The anatomical record. Part A, Discoveries in molecular, cellular, and evolutionary biology.

[15]  F. Gage,et al.  Human embryonic stem cells express an immunogenic nonhuman sialic acid , 2005, Nature Medicine.

[16]  H. Schöler,et al.  Formation of Pluripotent Stem Cells in the Mammalian Embryo Depends on the POU Transcription Factor Oct4 , 1998, Cell.

[17]  Chunhui Xu,et al.  Feeder-free growth of undifferentiated human embryonic stem cells , 2001, Nature Biotechnology.

[18]  D K Robinson,et al.  Industrial choices for protein production by large-scale cell culture. , 2001, Current opinion in biotechnology.