Here at the Panama College of Cell Science, we have spent a good amount of time on the “Paired Mice ” experiments. Although for our purposes, the seminal paper was issued over 10 years ago in 2005, the importance of the paired mice experiments has yet to be fully appreciated by clinicians, physicians, and clinical researchers all attempting to advance the utility of Adult Stem Cell therapy, and in particular, autologous stem cell therapy (cells harvested from the patient to be treated).
What follows is a nice review, but before we present that, we want to hit the important point to be made to our graduate students: Enzymology techniques are the key to solving the riddle of what stem cell growth factors, activating factors, hormones, cytokines, messenger molecules and other intracellular and extracellular messenger molecules present in plasma and serum can be used to boost adult stem cell therapy many fold.
Column chromatography!! Fractionation and serial collection of eluate from columns loaded with various separation media. That is what is needed, yet for the most part, the scientists in our field of stem cell biology do not seem to consider these tried and true techniques.
Any of our graduate students at the Panama College of Cell Science that may have access to labs, or access to the standard enzymology equipment should definitely consider this line of investigation which is sure to yield positive and useful results: fractionate plasma or serum using column chromatography, locate which fractions have high stem cell stimulatory activity, isolate and purify the material, and then both study it in vitro as well as move toward identifying and describing the molecular structure. Please keep these thought in mind as you proceed on with your studies toward your dissertation.
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OK, now we proceed to a tidy review of the paired mice experiments and why column chromatography is relevant. Most international clinics providing adult stem cell therapy have been concentrating on using a patient’s own stem cells in therapy, how to increase the number of stem cells needed to achieve a therapeutic result, how to multiply the patient derived stem cells to create greater numbers, and what transplant/delivery procedure will achieve the best result.
With the excitement surrounding all the many astounding results seen with transplanted adult stem cells, lesser attention has been given to maybe the most important key to successful therapy—-Growth Factors. That is all the many cytokines, proteins, messenger molecules and the many other intracellular and extracellular substances that contribute to stem cell growth, differentiation, and activation.
[The following is excerpted from a summary authored by Dr. Walter P. Drake
on the Drake Biomedical Institute website]:
1. The Paired Mice Experiments: The Paired Mice experiments demonstrated that serum factors rather than transplanted stem cells caused enhanced muscle regeneration in aged mice. The way the experiment was done, an old mouse is surgically conjoined with a young mouse in such a way that their circulatory systems are mixed, and blood, plasma, and serum flows between them.
The researchers observed that after conjoining, the stem cells in the old mouse were activated to begin dividing again, and that muscle repair in the old mouse was enhanced and became similar to that of young mice.
By marking cells in the young mouse, the experiment further demonstrated that the improvement in the old mouse muscle repair was not due to stem cells from the young mouse, but rather due to circulating blood factors. In addition, it was found that one could replicate the finding in vitro, by just adding plasma from young mice to stem cells of old mice in culture. Irina M. Conboy, Michael J. Conboy, AmyJ. Wagers, Eric R. Girma, Irving L. Weissman, Thomas A. Rando, “Rejuvenation of aged progenitor cells by exposure to a young systemic environment”, Nature 433:760 (2005).
In 2008, part of the same team, in a follow-up work, better identified some of the serum factors involved, noting that serum from the young mice activates the Notch pathway in the older animals, and also results in the deactivation of Transforming growth factor (TGF-β). Carlson, ME, “Imbalance between pSmad3 and Notch induces CDKinhibitors in old muscle stem cells”, Nature 454:528 (2008). In 2014, they further identified the circulating hormone oxytocin, that when injected into aged mice regenerates muscles by activating muscle stem cells. Elabd,C et al “Oxytocin is an age-specific circulating hormone that is necessary for muscle maintenance and regeneration” Nature Commun. 5:4082 (2014)
As summarized in the article “Ageing research: Blood to blood”, [Megan Scudellari,Nature 517:426-429 (2015): “Wyss-Coray, who worked in the room next to Rando’s lab, had previously discovered prominent changes in levels of proteins and growth factors in the blood of ageing humans and people with Alzheimer’s disease. Following up on Rando’s unpublished brain results, he used old–young mouse pairs to show that old mice exposed to young blood did indeed have increased neuron growth, and that young mice exposed to old blood had reduced growth. Plasma alone had the same effects. “We didn’t have to exchange the whole blood,” says Wyss-Coray. “It acts like a drug.” Villeda, SA et al, “The ageing systemic milieu negatively regulates neurogenesis and cognitive function”, Nature 477:90 (2011).
These results can, in summary form, be read at this link:http://www.nature.com/news/ageing-research-blood-to-blood-1.16762
And a nice video discussing the paired mice experiments of Tom Rando can be viewed here at the following Minutes sections: 18.14 minutes to 23.07 minutes:
2. The number of stem cells may remain high as we age. This is a point still being debated. Many studies demonstrate that the number of stem cells in a particular tissue or organ decrease with age. Other data seems to show that with some tissues, we may not lose stem cell numbers as we age, but rather, the regenerative function of the stem cells declines as we age, and that this is due to changes in cellular environment. Take for example this quote from an important review paper:
“Recent evidence supports the model that stem cells in several tissues are largely retained in a quiescent state but can be coaxed back into the cell cycle in response to extracellular cues, even after prolonged periods of dormancy.” Sharpless NE, and DePinho RA, “How stem cells age and why this makes us grow old”, Nature 8:703 (2007).
Bottom line, many international clinics are just implanting more stem cells, and while the results are noteworthy, we could probably do a whole lot better by adding known stem cell activators.
3. Known and unknown Growth Factors. In addition to the few substances mentioned above, we can now find quite a few number of growth factors catalogued, although most of these relate to manipulating embryonic stem cells in culture, which likely may not have any applicability to adult stem cell treatments being implemented now. Respecting adult stem cells, we know that igf (insulin growth factor) and interleukin boost stem cell activity. Dr. Christopher Centeno of the Centeno-Schultz Orthopedic Clinic which treats patients with adult stem cells, believes that growth factors from the patient’s own platelets are required to better expand autologous stem cells for reimplantation to the patient, that is, it works better, even though the exact factors are not identified.
The paired mice experiments are troubling in that they show that serum factors, not the transfer of stem cells themselves, is what caused marked improvement in ability to heal injured muscles in aged mice.
Therefore, in order to really become expert in using adult stem cells for therapeutic use, it would be nice to have an arsenal of cytokines, hormones, small molecules, etc, that could be called upon in specified situations. Not necessarily off the shelf products, which may not be clinically useful unless they could be synthesized to avoid patient contamination. If we knew how to easily isolate the required growth factors and activating factors from a patient’s blood, then those substances could be used in tandem with autologous stem cell transplants to further boost the usefulness of overall stem cell therapy.
It is hoped that our students at the Panama College of Cell Science will be able to focus some attention to this area.