Patients with chronic diseases are putting their hope into new discoveries from regenerative medicine, based on mesenchymal stem cell (MSC) research. As promising as some published outcomes appear, they may not be reproducible due to poor handling and inadequate authentication of cell cultures. Meticulous validation of cell identity is critical for working with human mesenchymal stem cells and due to the needed effort, regrettably, this is very often overlooked.
Dr. Hagen Wieland from the development department at PromoCell said, “The isolation of MSCs from human tissue such as bone marrow, umbilical cord, cartilage or fat tissue per se isn’t a big deal. After dissolving the tissue in a selected buffer, you can quite easily extract active MSCs from this ‘cell soup’ – at least you think that’s what you have.”
It is not possible to obtain only the multipotent stem cells alone, regardless of which method has been used, since mesenchymal stem cells are highly heterogeneous. Separation based on the ability of MSCs to stick to plastic surfaces, for example, might lead to the formation of MSCs together with their progenitor cells (Kuznetsov, et al., J. Bone Miner Res. 1997). Moreover, researchers might end up with fibroblasts, or MSC derivatives that are not multipotent.
This is the starting point of a real MSC study. Researchers need to cultivate, differentiate, and characterize the cells before even working on their own research. Therefore, they have created different protocols for every step. Based on the media and serums used, in which the ingredients can vary from one lot to another, results may differ considerably.
Dr. Wieland said, “Until now, there have not been any guidelines or regulations. Scientists have had no choice but to use their own judgment. For every paper that revealed interesting results, another group would come up with quite contrary data. Some experiments were simply not reproducible due to variations in cell handling and protocols. MSC research has been a real hodgepodge for years!”
PromoCell’s stem cell specialist: Dr. Hagen Wieland works at the R&D department at PromoCell. His never-ending job is to explore and develop new stem cell culture systems. He and his team also write user-friendly application notes.
How Paperwork Helps Stop Wasting Money and Time
A recent study points to the fact that in the United States alone, 28 billion dollars are spent every year on experiments that are irreproducible. Furthermore, the same analysis signifies that over half of all preclinical studies are not reproducible (Freedman, et al., PLoS Biol. 2015). The intent of all of these irreproducible analyses was to find life-saving cures and therapies, but instead, time and money have been wasted.
For this reason, at least in MSC research, more and more journals ask for certification that researchers have confirmed the identity of the cells used in the experiments. The same applies to several institutions that sponsor research projects. Therefore, the paperwork of protocols describing stem cell isolation and characterization should be included if researchers want to seek financial assistance.
The U.S. National Institution of Health recently incorporated authentication recommendations in their guidelines to support preclinical research reporting. Initiatives of this kind are aimed at increasing comparability and reproducibility.
The Multipotent Potential of Human Mesenchymal Stem Cells
Over the years, scientific attention on human mesenchymal stem cells has been increasing. “Each year, there are 4,000 to 5,000 publications about MSCs on PubMed. It is a consistent upward trend and I don’t believe it is going to stop. Quite the opposite, I expect this field of research to grow,” stated Dr. Wieland, who works with blood and stem cells at PromoCell.
MSCs are well known because of their exceptional characteristics. They were first isolated from bone marrow in the 1980s and they enable scientists to extract the regenerative cells from different tissues of the human body. Due to this, any ethical problems that could come with embryonic stem cells are eliminated, and MSCs become easily accessible.
MSCs separate into distinct cells in the body, which make structures such as cartilage, adipose tissue, and bones. In contrast to their embryonic siblings, having limited differentiation routes might look like a drawback but it actually provides an excellent opportunity for MSCs. They form strong tissue, unlike pluripotent cells that tend to develop into tumors. Moreover, almost no markers are found on the surface of the mesenchymal stem cells, which signifies that they are not immunogenic. MSCs even possess a down-modulating function, which is a great advantage considering regenerative therapies and graft versus host disease. Many doctors put their efforts into developing combinational therapies by co-culturing MSCs with hematopoietic stem cells to improve the success rates of bone marrow transplants.
Is this too good to be true? A larger number of clinical trials with MSCs, presently around 720, are in good agreement. “When you think of typical lifestyle-related diseases, such as arthritis, prolapsed discs, or osteoporosis—all of the affected cell lineages lie within the MSCs differentiation spectrum—and there have been promising studies that can help develop therapies to regenerate damaged tissue. The heterogeneity of MSCs in the body is mirrored by the variety of applications in biomedical research.”
Mesenchymal stem cells adhere to plastic surfaces, but this is not the only criteria they have to fulfill. The ISCT defined two more in 2006 to make research more comparable and replicable.
Make three out of one: 1. Depending on their microenvironment, mesenchymal stem cells differentiate into three lineages: Chondrocytes—cartilage is built of these spheroid cells.
2. Adipocytes—the cells with accumulated intracellular lipid vesicles are commonly known as “fat cells.”
3. Osteoblasts—these mineralized cells with extracellular calcium deposits form bones.
The number of common diseases registered for MSC-based cell therapy (Ullah, et al., Biosci Rep. 2015).
Authentication is Indispensable for Scientific Success
With regards to heterogeneity, what is the way to perform more reproducible and comparable cell-based experiments? Authentication is the keyword. To this end, the International Society for Cellular Therapy (ISCT), which is an association that supports the translation of cellular therapies, has set three criteria that MSCs must meet:
(1) The cells have to grow adherently
(2) they must exhibit specific surface markers - the classification determinant (CD) markers
(3) they must exhibit the potential to differentiate into three lineages
To fulfill these criteria, researchers have to conduct many analyses, which is a crucial process, but also laborious and tedious, as it can take up to four weeks. It has to be repeated each time when isolating new cells, only to identify that, eventually, there might not even be enough cells to conduct an experiment.
Wouldn’t it be great if somebody else did all the testing for you?
Dr. Wieland has a thorough knowledge of CD markers on mesenchymal stem cells. His day-to-day work involves developing, establishing, and optimizing stem cells systems for research purposes. “We have seen many times that testing only for markers isn’t sufficient. To have a solid scientific basis, all ISCT criteria are crucial. This is only one reason why quality management is a standardized and sophisticated procedure that we have been working on for years.” Stem cells are only one part of the system, and growth factors and media are the other part. “Actually, no one exactly knows the factors that make MSCs expand and differentiate. But we do know that consistency is essential. This is one reason why our proliferation and differentiation media always have the same ingredients, ones that we have tested and found to work best. It is a little bit like the Coca Cola formula.”
The three minimal criteria for defining multipotent human MSCs: (1) MSCs must express CD105, CD73, and CD90, and lack expression of CD45, CD34, CD14 or CD11b, CD79α or CD19, and HLA-DR surface molecule; (2) MSCs must be plastic-adherent when maintained in standard culture (3) and have the ability to differentiate into adipocytes, osteoblasts, and chondrocytes in vitro (criteria proposed by the International Society for Cellular Therapy).
If anyone ever attempted to create his or her own Coke as a child, they will know what Dr. Wieland is referring to. “With the view to saving money, some laboratories mix their own media and run through all the steps of MSC isolation with some sort of identification. That is one way to do it, but there are alternatives. Along with the actual MSCs, authentication certificates and detailed application notes, we provide a complete toolbox for the scientists. Through this support, we’re telling them ‘We’ve done all the preparatory work for you. You can rely on it, and concentrate on your actual research’.”
MSC Research is More Than a “One-Hit Wonder”
Similar to many other researchers, Dr. Wieland realizes the difficulties faced by many of his colleagues. “Currently, researchers are under an increasing pressure to publish, while at the same time, there is less and less money available. It is a dilemma. Still, you shouldn’t try to reduce costs by trying to isolate your MSCs by yourself. In the end, you might not only lose time but even more money.”
Regardless of the difficulties, Dr. Wieland has trust in the forthcoming MSC research. “This is not one of these ‘one-hit wonders’ we sometimes observe in science. It shows consistent progress. In addition to stem cell research, scientists use MSCs, or the differentiated cell types, in diabetes, biomaterials, and cancer research. There are even trials on using MSCs to support the regeneration of heart muscle after myocardial infarction. I’m sure there is still a lot to come. Just imagine what would happen if everyone could pick the red candy right from the start ...”
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