Culturing hPSCs – From FBS to a Completely Defined Culture System
The ability of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) to proliferate continuously and form derivatives of all three germ layers makes them attractive to researchers to use for study of basic biology, drug discovery and cell therapies. The culture of these human pluripotent stem cells (hPSCs) has evolved at a rapid pace since the first cell lines were isolated in 1998 (Thomson et al., Science 1998). The first cell lines were cultured in DMEM-F12 basal medium containing 10% fetal bovine serum (FBS) and on inactivated mouse embryonic fibroblasts (MEFs). While this method worked well initially, the potential xenogeneic contamination needed to be addressed to allow the field to move towards clinical applications. There is significant variability between different batches of MEFs and across different labs. Also, because MEFs are labor intensive to generate, the potential for large-scale generation of hPSCs is limited. The FBS also represents an undefined, animal-derived component in the culture system which can be highly variable from batch to batch. The nature of these undefined culture systems led to variability in experimental results and difficulty for labs world-wide to compare results also making potential therapeutic applications unattainable. These reasons motivated the relatively rapid transition towards feeder-independent, animal-free, more defined culture medium and matrix. Additionally, the need for higher regulatory compliance and standardization has led many companies to continue developing culture media and matrices for hPSCs.
To begin with, FBS was replaced with a serum-free commercial product called Knockout™ Serum Replacement (KSR) by Life Technologies™ (Amit et al., Dev Biol 2000). The aim was to reduce the batch variability of FBS and because of its commercial availability, allow multiple labs worldwide to use the same material to culture their hPSCs. The next step was to remove the direct contact between the MEFs and hPSCs. This was accomplished by utilizing MEF-conditioned hPSC culture medium on Matrigel™ matrix from BD Biosciences (Xu et al., Nat Biotech 2001). While this removed the direct contact with the feeder cells, the factors being secreted by the MEFs that allow for the maintenance of undifferentiated hPSCs was still not well understood. From here, focus shifted to better defining the components of the culture medium required to maintain hPSCs in a pluripotent state. This led to a number of publications for defined culture media formulation that eliminated the requirement for MEFs for both conditioning the media and as a culture matrix (Vallier et al., J Cell Sci 2005; Lu et al. PNAS 2006; Ludwig et al., Nature Methods 2006; Wang et al., Blood 2007).
The commercialization of several feeder-independent culture media has allowed for the advancement of research in field through standardization of reagents and culture protocols. mTeSR™1 (STEMCELL Technologies, Inc) and StemPro® hESC SFM (Life Technologies, Inc) were the initial offers from industry. Both formulations contain Bovine Serum Albumin (BSA) and have been shown to be effective at maintaining pluripotency of hPSCs. Of these, mTeSR™1 (STEMCELL Technologies, Inc) with Matrigel™ is the most widely used culture system for derivation and propagation and has been extensively published. While these systems have proven to be robust, there are still challenges and limitations to overcome when looking at potential therapeutic applications of hPSCs. Media containing BSA is still considered to be of animal origin and culturing cells on Matrigel™ or other similar matrices like Life Technologies’ Geltrex™ pose hurdles to meet regulatory compliance standards.
Ludwig et al published in 2006 in Nature Biotechnology a completely defined, animal-component free medium and culture matrix for derivation and expansion of hESCs. This was an exciting proof of principle publication demonstrating a completely humanized culture system could be successful and robust. However, challenges with sourcing low-cost materials particularly for the culture matrix made this unrealistic for basic research labs or large-scale cultures. The culture medium contains Human Serum Albumin (HSA) instead of BSA to remove the animal protein component. This animal-protein free formulation was commercialized by STEMCELL Technologies, Inc. in 2010, initially for use with Matrigel™. More recently, the StemAdhere™ Defined Matrix for hPSC was introduced to the market by the same company. This defined, recombinant matrix was developed and manufactured by Primorigen Biosciences, Inc. and is animal-protein free alternative to Matrigel™ allowing for long-term culture of hPSCs. In combination, StemAdhere™ and TeSR™2 make a fully defined, animal-protein free culture system. Also available from Biological Industries is NutriStem™ hESC SFM, which is a proprietary xeno-free, defined formulation containing HSA for culture of hPSCs optimized for use on Matrigel™ only.
A recent publication by Chen et al (Nature Methods, 2011), examined the TeSR formulation to elucidate the essential factors in this complex formulation required for hPSC maintenance. In addition to the components of the DMEM-F12 basal medium, TeSR contains 18 different components. It is known that there are huge differences between batches of albumins in their ability to support expansion of undifferentiated hPSCs. The commercial media available (StemPro® hESC SFM, mTeSR™1, TeSR™2) are extensively quality controlled and batch tested for their intended purpose to maintain expansion of hPSCs but how each batch of albumin used in the media affects downstream differentiation can vary widely for example. Also, the biological function of albumin to bind lipids and act as a detoxifier means that the media is not truly defined as the albumin has bound materials that are contributing to the composition of the media. This led to examining the TeSR™ formulation in the absence of albumin. It was found that when β-mercaptoethanol was removed from the formulation, albumin was no longer required. It appears the albumin was counteracting the toxic effects of the reducing agent. The other 16 media components in TeSR were re-examined in the absence of albumin and β-mercaptoethanol resulting in the new, simplified media containing the “Essential 8” components (E8). A recombinant vitronectin variant, labeled VTN-NC was found to be an effective matrix for use with E8 to form a completely defined culture system. Derivation of several hiPSC lines from dermal fibroblasts was successful in this new E8/VTN-NC system as well as supporting the expansion of the cells. This was important to show that new hiPSC lines could be generated and maintained in completely animal and albumin-free conditions. E8 is being commercially developed by both Life Technologies™ (Essential 8™) and STEMCELL Technologies, Inc (TeSR™-E8™).
This most recent publication by Chen et al demonstrates the field is striving to simplify and define culture methods by minimizing xeno-contamination during derivation and subsequent propagation. Approaches such as these are no doubt being applied to downstream differentiation methodologies for hPSCs. As the field moves towards cell therapies using hiPSC-derived cells, companies like STEMCELL Technologies, Inc. and Life Technologies™ will need to continue to attain higher regulatory compliance through raw material sourcing and manufacturing methods (GMP).