Expansion of therapeutic stem cells

Purity and integrity of the reagents used to scale up stem cells for clinical use are of the utmost importance. Removal of all animal or human-serum derived proteins is key since these reagents are notorious for viral contamination as well as varying cell performance between preparation lots. Therefore, replacement of these proteins with highly pure recombinant versions ensures reproducible cell scale up and subsequent in vivo performance of the implanted cells. However, this must be done while keeping the reagents economically feasible. The novel rice-derived rhLIF described in this blog represents just one example of how we addressed a limitation in the current LIF market as other more expensive LIF proteins have high endotoxin contamination that can potentially be problematic if used in clinical preparations of stem cells. By using the ExpressTec expression platform, we have the potential to produce kg quantities of LIF with almost undetectable endotoxin contamination. Other recombinant proteins critical in stem cell culture must follow in order to facilitate the translation of stem cell technology to the clinic.

Although the exact extraction conditions for the rhLIF is proprietary, the link http://www.ventria.com/about-us/our-technology explains the entire expression and purification process. Typically with any ExpressTec-derived protein, a simple aqueous extraction is sufficient to remove the protein from the rice grain for subsequent chromatography.

LIF is a member of the IL-6 cytokine family. It has been identified as a key regulator in the maintenance of stem cell pluripotency. LIF binds to a receptor complex that activates gp130/STAT3-dependent signaling, leading to increased transcription of the stem cell state regulators while simultaneously inhibiting differentiation. See
Burdon et al,. Trends in Cell Biology, 2002. 12(9): p. 432-8 and Chambers., Development, 2009. 136(14): p. 2311-22 for more details into this mechanism. There's also plenty of good reviews on the LIF's role in maintaining stem cell pluripotency. What is even more interesting is the finding of the true naïve phenotype of human stem cells that appear to be LIF-dependent. This is in stark contrast to the bFGF-dependent (but still pluripotent) stem cells. See Buecker., Cell Stem Cell, 2010. 7(5): p. 559-64 for more details. In addition to being a key regulator of pluripotency in mouse embryonic and human naïve stem cells, LIF has been shown to induce stem cell proliferation in various multipotent stem cell systems such as human neural stem cells.

There have been multiple publications on the addition of rhLIF to human neural stem cells in order to enhance NSC proliferation and viability. Therefore, I would recommend adding rhLIF to the medium to see if there is an improvement in the multipotency of your cultures. However, even though rhLIF may improve your cultures, you may have not addressed the core problem. There has been repeated reports of poor multipotency retention and reproducible cell performance in various lots of some of the more popular commercial supplements due to variations in preparation of serum-derived proteins. We have developed an animal component-free neural stem cell supplement that contains multiple ExpressTec-derived proteins. We have found that this novel supplement has competitive cell growth and viability to that of N-2 or B27 supplements and the popular EnStem media. If there is interest in more information for this supplement, contact ralfano@invitria.com.

Traditionally LIF has been supplied to ESCs by secretion directly into the culture media by mitotically-inactivated mouse embryonic fibroblasts (MEFs). However, feeder layers have limitations. Not only are they very labor intensive to prepare, but problems also arise with isolating stem cells due to inefficient separation of the two cell types. Further, variations between MEF preparations can lead to marked differences in stem cell performance. Therefore, the integrity of obtained results from in vitro assays that utilize MEFs and reproducibility between assays presents a challenge for traditional co-culture systems. Given the potential problems and challenges of using MEF-derived LIF, I would recommend using recombinant LIF in stem cell systems. Now the question becomes which LIF to use? Early publications (PMID 8006010) have shown that human LIF is able to bind the LIF receptor on mouse cells. We have also observed that human LIF is active on mESCs in that it can be used to maintain markers of pluripotency. Therefore, the answer of which LIF to use comes down to selecting the most cost-efficient LIF that won't break the bank.