The full realization of the therapeutic potential of stem cells has only recently come into the forefront of regenerative medicine. Promising in vivo results have fueled the enthusiasm among basic researchers and their clinical colleagues and thus have widened the scope of stem cell application in human disease but major scientific and regulatory challenges exist and must be addressed in order to both facilitate the “bench to bedside” process of this nascent technology as well as enhance safety of the final cell product. One potential key to advancing stem cell therapies is described in the recent benchmark publication entitled “Application of recombinant human leukemia inhibitor factor (LIF) produced in rice (Oryza sativa L.) for maintenance of mouse embryonic stem cells” by Youngblood et al. This paper describes how we expressed the human LIF protein (rhLIF) in rice grain using a plant-based expression platform (ExpressTec) and demonstrated a 97% purity of the protein. Subsequent biochemical analysis of the rhLIF showed similar biochemical characteristics as compared to rhLIF derived from E. coli.
One challenge to realizing the potential of stem cell therapies is the expansion of a homogeneous, self-renewing population of stem cells in a regulatory-friendly optimized cell culture medium that is economically feasible for scale up operations. The removal of serum and animal-derived proteins that are critical for the support of stem cell division, high cell viability, and maintenance of pluripotency represents a major hurdle in the formulation of such a medium. Although recombinant versions of these serum proteins such as human albumin and transferrinare the obvious immediate solution, addition of the recombinant versions of these components can prove costly due to the combination of relatively high protein concentrations required in medium formulations and the volumes of growth medium needed to produce a clinically relevant dose of stem cells per patient. Human blood-derived proteins used in xeno-free growth media do not eliminate the risk of transmitting new or emerging blood-borne pathogens to the recipient patient. Further, protein lot recalls, poor supply reliability, and performance variability of blood-derived proteins can further complicate clinical expansion protocols.
Harvesting the benefits of the ExpressTec production platform
Plants have emerged as powerful production platforms for the expression of fully functional recombinant mammalian proteins. These expression systems have demonstrated the ability to produce complex glycoproteins in a cost-efficient manner at large scale. From a safety stand point, plant-derived recombinant proteins are considered totally animal-free since production is void of human or animal products and thus risk of pathogen contamination is minimal.
ExpressTec is a proprietary rice (Oryza sativa L.)-based expression system that has been developed by Ventria Bioscience Inc. It has been used to produce and market several recombinant proteins including recombinant human serum albumin, transferrin, and lactoferrin under the InVitria Cell Culture Division. The full InVitria animal component-free product portfolio can be viewed here. Due to the production scale and low operational costs, ExpressTec has been shown to produce some of the most cost-effective recombinant proteins on the market. Combined with minimal risk of serum-derived pathogen contamination, ExpressTec-derived proteins make for an attractive alternative to circumvent current market limitations.
Making a case for plant biologics in stem cell media: rice-derived human leukemia inhibitory factor
Leukemia inhibitory factor (LIF), a member of the IL-6 cytokine family, is known to be a critical stem cell factor in a variety of different human and mouse stem cell systems. LIF has been shown to be the primary driver in the maintenance of the true naïve phenotype from the more differentiated bFGF-dependent, albeit pluripotent state, in human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSCs). In addition, LIF has been shown to enhance the proliferation of various human multipotent stem cell types. Mouse ESCs (mESCs) resemble the naïve phenotype of hESC and iPSCs in that mESCs require LIF signaling for retention of cell proliferation in the pluripotent state as withdrawal of LIF signaling results in rapid cessation of cell proliferation and differentiation.
These characteristics result in recombinant LIF being used extensively in both mESC and various human stem cell media systems. Inclusion of LIF in cell culture medium is a costly addition, estimated to be the majority of cost for mESC culture media. Though the cause varies between different expression platforms, low yields and laborious purification schemes are suspected to be the cause of the high cost of rhLIF. Further, the importance of the molecule and subsequent wide usage further exacerbates current market prices of recombinant LIF. These characteristics of LIF presented an opportunity for InVitria to provide a proof of principal to demonstrate success of plant-based biologics in stem cell systems.
As described in the publication Youngblood et al., the ExpressTec-made rhLIF was found to have 1-2 logs lower endotoxin contamination compared to the next best expression system available. Extensive analysis of currently marketed LIF protein release specifications revealed that the rice-derived rhLIF was the lowest endotoxin specification on the market. Specific activity of ExpressTec-made rhLIF was found to be slightly higher than currently marketed E. coli-derived rhLIF in standard M1 cell differentiation studies. This and other data shows that ExpressTec-made rhLIF is highly pure and biologically active.
The question remained as to whether ExpressTec-made rhLIF would be able to sustain stem cells in the undifferentiated state in vitro. We chose to exploit mESCs since these cells are extremely sensitive to LIF signaling for the maintenance of pluripotency. C57BL/6-derived mESCs were cultured in the presence of the ExpressTec-made rhLIF, rhLIF produced in E. coli, and mouse rLIF. Doubling time and cell viability were examined among the different LIF treatments in cells grown for 6 days. We could detect no difference in the doubling time or cell viability among the cells grown in the different LIF treatments over multiple subcultures. Further, the pluripotency markers Pou5f1 (Oct4), Nanog, and Zpf42 (Rex1) were analyzed after 10 passages. The mRNA levels of the transcription factors Pou5f1 (Oct4) and Nanog were found to be indistinguishable between the ExpressTec-made rhLIF and E. coli-derived rhLIF. Cells grown in the rice-derived rhLIF demonstrated slightly elevated Zfp42 (Rex1). After 12 passages, the cell surface marker of pluripotency SSEA-1 was found to be equal among the LIF treatment groups.
Taken together, these studies demonstrate that ExpressTec-made rhLIF exhibits indistinguishable activity in stem cell culture systems in promoting cell proliferation and maintenance of the pluripotent state when compared to rhLIF produced in E. coli. Further, the large scale production and straightforward processing to yield an rhLIF of exceptional purity and the lowest endotoxin on the market enables InVitria to offer this product at an exceptionally reduced cost in comparison to alternatives. This translates into a product that is well suited for researchers as well as commercial manufacturing of regenerative medicine.