On 31 March 2011, ~50 delegates attended a workshop organized by STEMCELL Technologies on implications of standard defined culture conditions for embryonic and induced-pluripotent human stem cells as part of the annual meeting of the UK National Stem Cell Network in York, UK. Researchers from both academia and industry need to develop a better understanding of those implications. Our company wanted to give them a better appreciation of key challenges facing ancillary material suppliers who manufacture standard defined reagents. The workshop fits within the overall UKNSCN meeting, especially given increased interest in robust production of human pluripotent stem cells (hPSCs) for applications such as disease modeling, cell therapies, and drug screening.

On Defined Media

Scientific talks began with an excellent exposition of key issues and challenges in using commercially available media by Lyn Healy from the Health Protection Agency’s UK stem cell bank based at its NIBSC site. Such media make it possible to standardize manufacturing processes for better comparability across laboratories. Defined culture conditions for derivation and expansion of human pluripotent cells have been extensively evaluated and reported by The International Stem Cell Initiative (ISCI) Consortium (1). Different stem cell labs around the world evaluated a range of culture media and determined that those commercially available adequately supported feeder-free, serum-free propagation of human embryonic stem cell (hESC) lines.

Standard defined media systems for hPSC culture have been used recently for derivation and culture of human induced pluripotent stem cells (hiPSCs), but including serum albumin and other human-sourced matrix proteins makes them expensive for routine use. So many researchers continue to maintain hPSCs routinely in defined media that include bovine serum albumin (BSA) and a complex mixture of matrix proteins derived from Engelbreth-Holm-Swarm mouse sarcoma (BD Matrigel from BD Biosciences) that is far from defined (2). Variation inherent in component sources is significant, so extensive quality control is essential for all released batches by their suppliers. Healy described the complexity of defined media that leads to high production costs. “Optimization of defined cell culture medium is an onerous task. Each of 18 components added to a DMEM/F12 base medium has to be sourced and tested before batch testing the whole media for release with appropriate quality control checks in place.”

At present, a universal medium for expansion and maintenance of multiple hESC/hiPSC lines is far from a reality because of inherent differences among cell lines. One primary difference is in integrin signaling, which is evident in attachment activities on different surfaces (3). Effectiveness of various formulations also differs. That may be due to special cell line requirements, either from production of autocrine factors or generation of varied quantities of differentiated derivatives that secrete factors either promoting or inhibiting proliferation. Cell lines also differ in epigenetic adaptation to in vitro culture conditions (4). But a defined standard medium suitable for successful continuous culture of most representative lines could help elucidate their responses to precise signals that control self-renewal and lineage selection. That would also be beneficial in minimizing the variability that influences reproducibility of research results.

When reviewing progress in identifying epigenetic changes and karyotypic abnormalities during hESC culture, the University of Sheffield’s Peter Andrews illustrated the significance of such changes especially in scale-up and expansion of cultures for specific applications. Nonstochastic, cytogenetic changes during long-term hESC culture can increase the probability of their self-renewal and potentially alter their differentiation capacity when they are subjected to strong selection for genetic variation. Such culture-adapted hESCs can undergo both mutation and selection. Andrews described a Monte Carlo simulation model that envisages how alterations in population size, mutation rate, and selection pressure can determine the emergence and spread of mutant hESCs. Simulation approximates the expected rate of culture-adapted hESC generation and accentuates the effect of population size. That is a significant parameter to be considered when devising techniques to minimize emergence of adapted and/ or abnormal cultures during process scale-up. One of Andrews’ conclusions was that maintaining cells in small populations decreases the probability of such events and helps in long-term maintenance of diploid cultures.

He further illustrated culture-induced changes during prolonged hESC passaging by detection of copy-number variations using a high-resolution genomic analysis technology: single nucleotide polymorphism (SNP) comparative genomic hybridization. Andrews’s team detected large changes affecting multiple genes in both early and late-passaged cells (particularly in chromosomes 12 and 17). He added, “Assessment of genetic changes at subkaryotypic level presents another issue: The human population around the world is very diverse, and distinct patterns are observed in different ethnic groups.”

Next month, Part 2 addresses differentiation and the take-home messages of our workshop.

Author Details
Pawanbir Singh, MBBS, MS, PhD is a product manager for STEMCELL Technologies Inc., Rutherford House, Manchester Science Park, Pencroft Way, Manchester, M15 6SZ , United Kingdom; 44-1616-600325, fax 44-1616-6003-81; pawanbir.singh@stemcell.com; www.stemcell.com.

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