- Cool Tool – PRIME-XV® T Cell CDM – First Commercially Available Chemically-defined, Animal-component-free Medium for T Cell CulturePosted 2 days ago
- Increasing Protein Production with Novel Cell-Ess Titer Boost without Affecting the Metabolic ProfilePosted 7 days ago
- Continuous Processing Optimization with Smarter ToolsPosted 1 week ago
- Cool Tool – Generation of Neural Stem Cells from AlphaSTEM Cultured Pluripotent Stem CellsPosted 1 week ago
- Synergizing Transient and Stable Protein Expression for Accelerated Biotherapeutic DevelopmentPosted 1 week ago
- Cell Culture Dish Top Ten Ask the Expert Sessions and Podcasts of 2016Posted 2 weeks ago
- A Look at the Current State of Continuous BioprocessingPosted 2 weeks ago
- Cool Tool – Biomek i-Series – Next Generation Automated Workstations Specifically Designed to Meet Evolving WorkflowsPosted 2 weeks ago
- Filling Industry Gaps with Dedicated Cell Therapy Fluid Transfer SetsPosted 3 weeks ago
- Cell Culture Monitoring – A Critical Component for Quality by Design in Cell TherapyPosted 3 weeks ago
A Discussion about the Expansion of Therapeutic Stem Cells using plant-based biologics
We recently finished our Ask the Expert discussion on Expansion of Therapeutic Stem Cells and the use of innovative plant based biologics in this task. The result was an interesting discussion about the use of LIF (leukemia inhibitory factor) in stem cell culture. Other topics included maintaining stem cells in an undifferentiated state and the use of a plant-based expression platform for rhLIF production.
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 blog “Planting the seed: plant-based biologics for the expansion of therapeutic stem cells.” The blog discusses how human LIF protein (rhLIF) was expressed in rice grain using a plant-based expression platform (ExpressTec) and demonstrated a 97% purity of the protein. The rhLIF was then used in stem cells to promote cell proliferation and maintenance of the pluripotent state. This blog served as the basis for our Ask the Expert discussion.
This Ask the Expert Session was Sponsored by InVitria and was hosted by Randall Alfano, Ph.D. Randall Alfano, Cell Culture Scientist, joined InVitria in 2012. He currently develops animal free proteins and supplements for various cell systems including stem cells. He has over 6 years’ experience in recombinant protein expression and purification as well as medium development for CHO-based biomanufacturing and stem cells. Randall was awarded his Ph.D. in 2009 from the Texas A&M College of Medicine Health Science Center in Cell Biology.
Below is a sneak peek of the discussion. For a full transcript of the discussion, please see – Ask the Expert –Expansion of Therapeutic Stem Cells.
How important is having an ultra-pure rhLIF for the advancement of stem cell therapy?
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.
I am having trouble maintaining my human neural stem cells in an undifferentiated state. Do you think adding LIF would help?
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.
Can you explain the different applications for mouse vs. human vs. recombinant human LIF. When would I need to use each?
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.