Human Embryonic Stem Cells
Human embryonic stem (hES) cells are pluripotent cells derived from the inner cell mass of a blastocyst.
These cells can either self-renew, thereby maintaining their pluripotency, or differentiate into all three germ layers depending upon the culture conditions. Induced pluripotent stem (iPS) cells, which are similar in potential to hES cells, have been generated by infecting adult cells. iPS cells, like hES cells, can form all three germ layers as well as self-renew. Tremendous hope is associated with the potential application of hES and iPS cells in cell therapy and regenerative medicine because of their ability to differentiate into multiple, clinically useful cell types. Defined culture conditions are essential to realizing the potential of hES and iPS cells.
A culture environment for hES cells consisting of both a serum-free, defined medium, and a cell culture surface specifically qualified for hES cells saves researchers time and resources normally spent qualifying reagents. BD Biosciences, StemCell Technologies, and the WiCell™ Research Institute have established a strategic collaboration to develop optimized, feeder-independent cell culture environments for hES cell research, including serum-free, defined media and qualified surfaces. BD Matrigel™ Matrix, coupled with a variety of culture media, has been widely accepted as an alternative substrate to feeder-dependent culture of hES cells1-4, and BD Matrigel Matrix has been used to culture iPS cells5-6. BD Matrigel Matrix is a reconstituted basement membrane isolated from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma.
StemCell Technologies has commercially developed and optimized WiCell™ Research Institute’s mTeSR™1 medium formulation to standardize feeder-independent hES cell culture. mTeSR1 is complete, defined and serum-free, and has been designed to maintain and expand hES cells in an undifferentiated state when used with BD Matrigel hESC-qualified Matrix as a substrate (Figure 1).
An alternative surface for hES cell culture is BD™ Laminin/Entactin Complex High Concentration. BD Laminin/Entactin Complex High Concentration (Figure 2), with a purity greater than or equal to 90%, is a more defined surface that can support undifferentiated hES cell growth. Unlike BD Matrigel hESC-qualified Matrix, this surface is not specifically qualified for maintenance of undifferentiated hES cells.
Figure 1. Human Embryonic Stem Cells Cultured on BD Matrigel hESC-qualified Matrix
A) Phase contrast images of H9 colonies grown on mouse embryonic fibroblast (MEF) feeder layer in hES media (left), BD Matrigel hESC-qualified Matrix in MEF-conditioned media (middle), or mTeSR™1 maintenance media (right). Images were taken at 4x magnification.
B) Flow cytometry analysis of H9 cells cultured on BD Matrigel hESC-qualified Matrix coated surface in mTeSR1 maintenance media. Cells were probed with the following antibodies: Tra-1-60 PE (Cat. No. 560193), Tra-1-81 PE (Cat. No. 560161), SSEA-4 PE (Cat. No. 560128) and Oct3/4 PE (Cat. No. 560186) compared to isotype control. Percent positive is indicated. Cells were run on a BD FACSCalibur™ system and the data was analyzed with CellQuest software.
Figure 2. BD Laminin/Entactin Complex High Concentration for hES Cell Culture
A)Phase contrast images of H9 cells grown on BD Matrigel hESC-qualified Matrix (left) and BD Laminin/Entactin Complex High Concentration (right) in mTeSR1 maintenance media. Images were taken at 4x magnification.
B) Flow cytometry analysis of H9 cells cultured on BD Laminin/Entactin Complex High Concentration (red line) and BD Matrigel hESC-qualified Matrix coated surface (green line) in mTeSR1 maintenance media. Cells were probed with the following antibodies: SSEA-4 PE (Cat. No. 560128) and Oct3/4 PE (Cat. No. 560186) compared to isotype control (black line). Cells were run on a BD FACSCalibur™ system and the data was analyzed with CellQuest software. Both surfaces supported undifferentiated expansion of hESC, H9.
C) G banding chromosome analysis. Karyotype analysis of H9 cells grown on BD Laminin/Entactin Complex High Concentration in mTeSR1 media for 26 passages. Cells maintained normal karyotype under these culture conditions.
References
Xu C, Inokuma MS, Denham J, Golds K, Kundu P, Gold JD, Carpenter MK. (2001) Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol. 19:971.
Amit M, Shariki C, Margulets V, Itskovitz-Eldor J. (2004) Feeder layerand serum-free culture of human embryonic stem cells. Biol Reprod. 70:837.
Ludwig TE, Bergendahl V, Levenstein ME, Yu J, Probasco MD, Thomson JA. (2006) Feeder-independent culture of human embryonic stem cells. Nat Methods. 3(8):637.
Ludwig TE, Levenstein ME, Jones JM, Berggren WT, Mitchen ER, Frane JL, Crandall LJ, Daigh CA, Conard KR, Piekarczyk MS, Llanas RA, Thomson JA. (2006) Derivation of human embryonic stem cells in defined conditions. Nat Biotechnol. 24(2):185.
Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 131:1.
Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA. (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science. 381(5858):1917.
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