Novel technologies for embryonic stem cell and induced pluripotent stem cell research

David Welch, Ph.D.
Business Manager, Primary and Stem Cell Systems, Invitrogen

Abstract

The relatively new discovery that pluripotent human cells can be derived from somatic cells through the expression of exogenous genes has generated great excitement and promise in the fields of biology, drug discovery and cell therapy. Unlike embryonic stem (ES) cells, induced pluripotent stem (iPS) cells do not present any cultural or ethical barriers for use, are derived from somatic cells that are plentiful, and can potentially overcome immune rejection issues when used for cell therapy.

The most common cell types used for the creation of iPS cells are fibroblasts and keratinocytes derived from skin.  Although the majority of studies to date have utilized fibroblasts for reprogramming efforts, more recently it has been demonstrated that epidermal keratinocytes can be reprogrammed with higher frequency than fibroblasts.  EpiLife Basal Medium more than doubles the in vitro lifespan of human keratinocytes when compared to classic growth media.  When combined with one of our single-addition growth supplements, EpiLife forms a complete growth medium that supports rapid proliferation of human keratinocytes with culture lifespans of greater than 50 population doublings even in samples from adult donors.

Initial iPS cell experiments used retroviral and lentiviral technologies to introduce reprogramming factors into somatic cells. Retroviruses and lentiviruses are randomly integrating viruses that are extremely efficient at transfecting multiple cell types and provide high levels of short-term expression of the required transgenes. Invitrogen's ViraPower™ HiPerform™ iPS cell Lentiviral products deliver reprogramming factors at high titer and high expression to convert somatic cells into iPS cells. In addition, recent experiments with BacMam technology for iPSC research will be discussed.

Culture conditions for the selection and expansion of both mouse and human iPS cells are the same as those used for ES cell maintenance. In order to use iPS cells and ES cells in a clinical setting, the cells must be cultured in an environment that is free from poorly defined components.  Moving towards a fully defined, animal origin-free culture system remains the primary goal of hES cell researchers headed for the clinic.  StemPROâ hESC SFM is a serum-free and feeder-free medium for the culture of hES cells, and it is formulated to meet the unique requirements of hES cells to support expansion, retention of undifferentiated stem cell phenotype, and long term karyotypic stability over extended (>70) passages.  KnockOut™ SR XenoFree is the first xeno-free medium formulated for the derivation, growth, expansion, and cryopreservation of human iPS and ES cells and can be adapted for use with or without human feeder cells. These formulations will facilitate the transition of human iPS cells from the lab bench to clinical applications by mitigating the risks associated with contaminating animal-derived pathogens and possible immune rejection by the patient.