Expansion of therapeutic stem cells

Introduction

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. Please submit your questions about the information in the blog, LIF itself, the application of LIF, or animal component-free stem cell media.

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Question 1

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 … Continued

Question 2

Can you explain how you extract the LIF from the rice.

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.

Question 3

Can you explain more about LIF’s role in stem cell culture its importance?

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 … Continued

Question 4

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 … Continued

Question 5

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 … Continued