The promise of MSCs

Carolyn Peluso, Ph.D.

 

In 2009, Charla Nash was the victim of a brutal animal mauling that left her without hands and with a face mutilated beyond recognition. Two years later, doctors attempted a face and hand transplant that took a surgical team 20 hours to perform. Sadly, the hand transplants failed almost immediately and had to be removed, but the face transplant succeeded, dramatically improving her quality of life. Her transplant was met with excitement from the medical establishment and the general community, which prompted invitations to appear on Oprah and the Today Show. Her transplant was considered something of a medical marvel and in fact, successful transplants of this kind (also known as a composite tissue allograph or CTA) are something of a rare bird. The first successful hand transplant occurred in 1998 and since then only about 200 patients have undergone the procedure worldwide¹ and with only limited success.  The rarity and poor success rate seems especially low when one considers that 28,000 patients undergo solid organ transplantations (e.g., heart, liver, kidney) with significant success in the just the US every year (United Network for Organ Sharing at www.UNOS.org).

In contrast to solid organ transplants, however, CTAs require the integration of multiple tissue types (i.e., muscle, fat, nerve, and highly-antigenic skin). The complexity of the transplant and the antigenicity of the skin tissue make rejection more likely than solid organ transplants¹. As a result, CTA patients are tied to the same onerous life-long burden of immunosuppressive therapy that solid organ recipients face, but in addition they face an increased risk of rejection complications.  Taking on the transplantation risks make sense for solid organ recipients who cannot survive without the transplant, but CTA recipients are generally healthy, aside from the missing limb². Thus, the procedure is about quality of life, not life-or-death, and the decision to proceed has to be balanced against the dangers of immunosuppressive therapy and rejection complications. Therefore, physicians need a way to reduce the immunological dangers of the CTA in order to offer it to patients on a wider scale; and this is where Mesenchymal Stem Cells (MSCs) may come into play.

MSCs are stromal cells that exist in tissues, such as the bone marrow and adipose and that can differentiate down multiple mesenchymal lineages (i.e., chrodrocytes, osteoblasts, adipocytes). This feature of MSCs has been successfully exploited to facilitate tissue reconstruction in orthopedic procedures³. In addition to their tissue regenerative capacity, however, MSCs exert important influence on both innate and adaptive immunity. For example, MSCs are able to affect innate immune function through various channels that include inhibiting the antigen-presenting function of dendritic cells, regulating HLA-G expression to block Natural Killer cell activity, and down-regulating IFN-g expression⁴. At the same time, MSCs affect adaptive immunity in part by mediating the nitric oxide system, and the expression of cytokines and growth factors⁴.  Clinically, the capacity of MSCs to affect the immune response in the transplant environment has been demonstrated in several studies. One of which, showed, using a randomized kidney transplant trial, that treatment with MSCs lowers the incidence of acute rejection, and opportunistic infection when compared to other immune-modulating therapies (i.e., anti-IL-2 antibodies)^{4, 5}.

Currently, researchers are applying the lessons learned by the solid organ transplant community to the specialized problems of the CTA procedure. For example, investigators need to establish a culture paradigm that optimizes the immune-modulatory potential of the cells, so they can overcome the high antigenicity of the skin graft component of the CTA – a problem not shared by the solid organ transplant community. To this end, researchers have shown in rat and primate models that treatment with MSCs can increase the survival time of skin grafts, although immunosuppression was still necessary and the rate of long-term tolerance was poor⁴. It’s a good first step, but there are still a lot of unanswered questions.  Investigators will need to develop an appropriate method of MSCs application, and they will need to understand how immunosuppressive drugs and MSCs interact so they can limit the negative influence of the former and improve the effectiveness of the later. The path is long, but the goal is in clear sight, and investigators are in active pursuit. It is exciting to think that someday, in the not so distant future, people like Charla Nash will be able to overcome life-altering tissue injuries in a common-place way, without being considered a medical marvel, thanks to MSCs.

1.            Schnider, JT et al. Site-specific immunosuppression in vascularized composite allotransplantation: prospects and potential. Clin Dev Immunol 2013, 495212 (2013).

2.            Antony, AK et al. Composite tissue allotransplantation and dysregulation in tissue repair and regeneration: a role for mesenchymal stem cells. Front Immunol 4, 188 (2013).

3.            Steinert, AF, Rackwitz, L, Gilbert, F, Noth, U & Tuan, RS. Concise review: the clinical application of mesenchymal stem cells for musculoskeletal regeneration: current status and perspectives. Stem Cells Transl Med 1, 237-47 (2012).

4.            Plock, JA, Schnider, JT, Solari, MG, Zheng, XX & Gorantla, VS. Perspectives on the use of mesenchymal stem cells in vascularized composite allotransplantation. Front Immunol 4, 175 (2013).

5.            Tan, J et al. Induction therapy with autologous mesenchymal stem cells in living-related kidney transplants: a randomized controlled trial. JAMA 307, 1169-77 (2012).

Once upon a time . . .

Carolyn Peluso, Ph.D.

. . . in a lab far, far away a postdoc sits nestled in among the test tubes and large, glass sequencing plates. Tapping his pen in time to the soulful sound of the Doobie Brothers, he analyzes 100 base pairs of hard-earned sequencing data, and dreams of an easier way. Years from now, as he tells his graduate students this story, they will unkindly cluck and roll with laughter. That hard-working post-doc of yesteryear was dreaming of next-generation sequencing, but he could never anticipate how it would revolutionize the way we approach cancer research and drug discovery.

Next generation sequencing refers to the high-throughput sequencing techniques that followed first generation Sanger sequencing. These technologies have led to the formation of large-scale sequencing initiatives that have generated a vast amount of actionable data. One such initiative is the Cancer Cell Line Encyclopedia (CCLE). The CCLE is a collaborative effort between Novartis and the Broad Institute that has released mutation data for 1,651 genes for nearly 1,000 cell lines. The CCLE research group used this data set to compare the copy number, expression pattern, and mutation frequency of tumor cell lines with primary tumors and showed that tumor cell lines are reasonably representative of their in vivo counterparts. Additionally, they used the sequencing data to predict that tumor cell lines harboring particular mutations are sensitive to specific classes of drugs¹.

Researchers are using this information, and the data from similar initiatives, to build better models to support basic research, and better platforms for screening potential drug candidates. ATCC is contributing to this effort by generating “sets” of tumor cell lines (the ATCC^® Tumor Cell Panels) that are annotated with mutational data, and arranged by tumor type, such as Pancreatic (TCP-1026™), Lung (TCP-1016™), and Breast (30-4500K™), or by commonly mutated genes like APC, EGFR, and BRAF.

Alone, these tools have the power to accelerate the research, development, and screening phase of drug discovery. The long-term hope, however, is to couple whole-genome sequencing to the transcript and epigenetic information from a single tumor sample. Having such information at their disposal, researchers will be able to develop better classifications for human cancers, and better, more personalized treatments. So, when they stop laughing, those graduate students should take a minute to thank their advisor. The long hours he spent in the lab, struggling for every base pair and thinking about a better way, were setting the stage for them to make huge strides towards a cure for cancer.

1.      Barretina, et al., (2012) Nature 483: 603-607

Choosing the best cell model for the job

Carolyn Peluso, Ph.D.

Our last several blog posts have described the causes of and the solutions to cell line contamination and misidentification. Hopefully, by now you are pretty confident that your cells are exactly what you thought. So on to the next step . . . how do you know that the cell line you have chosen for your experiments is a good model for the hypothesis you are testing?

Many investigators are asking the same question, and for good reason. One group looked at eight commonly used thyroid cancer cell lines, originally derived from thyroid tumors with diverse histological characteristics, representative of their individual states of differentiation. Microarray analysis of the cell lines revealed that all eight assume a similar dedifferentiated phenotype in vitro¹.  Thus, these cell lines may be useful if you are studying poorly differentiated forms of thyroid cancer. However, they may prove misleading if you are looking to answer questions about highly-differentiated thyroid tumors, and you are assuming that they have maintained the phenotypic character of the tumor from which they were derived.  

Another study compared the regulation of the retinoic acid receptor between an immortalized mouse Sertoli cell line (MSC-1) and primary Sertoli cells. They found that the cell line and the primary cells behave in a similar manner, indicating that for these studies at least the cell line is a good model for Sertoli cell function. When they expanded their studies to examine the immune privilege properties of Sertoli cells, they found that this is a property the MSC-1 cells do not share². Once again, this study demonstrates that cell lines are not always a perfect match for the disease or process under examination.

Clearly, some leg work is required when picking a cell line as a model system. First, it is always good to start with cells at early passage number. In general, cell lines are more likely to lose their parent cell character the longer they remain in culture. Second, before beginning a new set of experiments, the cell lines should be tested to ensure that, under normal conditions, the feature of the cell lines you are interested in studying matches up with the relevant primary cell. If you’ve tested that the cell line normally behaves like the primary cell, then alterations in behavior observed during your experiment are likely due to your manipulations and not a quirk of the culture conditions.  Cells grown in culture, and away from their in vivo environment, inevitably lose some of their in vivo character. As long as we appreciate this truth, and are diligent about performing control and proof-of-concept experiments, then cell lines will remain a valuable tool for modeling disease, and will continue to help scientists advance their research.

Next time we will take this discussion a step further, and look at how next generation sequencing is helping investigators generate better model systems for cancer research and drug discovery. So, until next time - we wish you good data and happy culturing,

ATCC Cell Biology

Tools for combating cell line contamination and misidentification.

Carolyn Peluso, Ph.D.

Our last blog post told the story of Stanley Gartler who, in 1966, announced to the founding fathers of cell culture that their cell culture collection was contaminated with HeLa cells. He made this stunning discovery incidentally, while in the process of looking for genetic markers to study cancer. He had already used the electrophoretic variance of glucose-6-phosphate dehydrogenase (G6PD) isotypes to demonstrate the clonal nature of cancer in tissue samples (Linder and Gartler, 1965), but wishing to take his research into cell culture, he began characterizing some commonly used cell lines.  What he discovered was that cell lines purportedly representing a wide range demographically, all carried a G6PD isotype specific to the African-American population. The only viable explanation was that HeLa cells had contaminated and taken over many supposedly independent cell lines.

Dr. Gartler, cleverly used the techniques at hand to identify endemic problems in the cell culture community, and then courageously stood up and tried to solve them. Unfortunately, as we also mentioned in our previous blog post, although Dr. Gartler and others tried to eliminate the problems of cell line cross contamination and misidentification, way back in 1966, they remain with us to this day.

Today, however, we have better tools to combat these problems, we just have to make use of them, and below is a list of some available resources to help researchers do just that.

Isoenzyme analysis: Dr. Gartler’s method of using the differences in the electrophoretic banding patterns of isoenzymes is still relevant. There are kits commercially available that provide the necessary reagents to identify the isotype of enzymes, such as aspartate amino transferase or peptidase B, expressed by the cells in question. These kits also provide a comparison chart, so the researcher can determine the species of the cell, and rule out cross-species contamination.

STR profiling: STR profiling is a PCR based approach that can discriminate the origin of the cell line down to the original donor. It is not surprising, therefore, that it is considered the gold standard in cell authentication techniques. Kits containing primer sets are available, but the data is sometimes difficult to analyze without help from a service, such as is available through ATCC. To learn more, please visit the ATCC Cell Authentication Services Page.

ATCC: Your trusted resource

Your trusted resource: The best way to start any project is with material from a trusted resource. We know you trust your buddy in the neighboring lab, but unless you froze the cells yourself, you have no way of knowing what that cryovial holds. What if, for example, the cells were frozen at high-passage number? Or, what if your neighbor’s advisor got them from his neighbor back when he was post-doc! So many variables can only lead to trouble. Cell repositories, like ATCC, on the other hand, will never lead you astray. They check the lines in their collections regularly to ensure that they are properly identified and free from contamination.  Click here for information on the methods used to authenticate the collection of ATCC cell lines.

Hopefully, we’ve helped you start thinking about the best way to authenticate your cell lines. Don’t forget that we are always here to answer questions, and to help in any way we can, so you can move your research forward.

Until next time, when our blog post will focus on helping you choose the most suitable cell line for your experiments, we wish you good luck, and happy culturing,

ATCC

Online resources:

Searchable STR database:

http://www.atcc.org/CulturesandProducts/CellBiology/STRProfileDatabase/tabid/174/Default.aspx

ATCC Cell Authentication Services Page:

http://www.atcc.org/Services/CellAuthenticationTestingService/tabid/1794/Default.aspx

References:

Characterization and authentication of cancer cell lines: an overview. Reid, YA, Methods Mol Biol. 2011; 731: 35-43. Review

Recommendation of short tandem repeat profiling for authentication human cell lines, stem cells, and tissues. Barallon R, Bauer SR, Butler J, Capes-Davis A, Dirks WG, Elmore E, Furtado M, Kline MC, Kohara A, Los GV, MacLeod RA, Master JR, Nardone M, Nardone RM, Nims RW, Price PJ, Reid YA, Shewale J, Sykes G, Steuer AF, Storts DR, Thomson J, Taraporewala Z, Alston-Roberts C, Kerrigan L.  In Vitro Cell Dev Biol Anim. 2010 Jun; 10(6): 441-8.

Cell line cross-contamination initiative: an interactive reference database of STR profiles covering common cancer cell lines. Dirks WG, MacLeod RA, Nakamura Y, Kohara A, Reid Y, Milch H, Drexler HG, Mizusawa H. Int J Cancer. 2010 Jan; 126(1); 303-4.

Check your cultures! A list of cross-contaminated or mis-identified cell Lines. Capes-Davis A, Theodosopoulos G, Atkin I, Drexler HG, Kohara A, MacLeod RA, Masters JR, Nakamura Y, Reid YA, Reddel RR, Freshney RI. Int J Cancer 2010 Jul; 127(1); 1-8 Review.

Glucose-6-phosphate dehydrogenase mosaicism: utilization as a cell marker in the study of leiomyomas. Linder D, Gartler SM., Science 1965 Oct 1; 150(3692); 67-9

A geneticist walks into a room . . .

 Carolyn Peluso, Ph.D.

Imagine a dimly lit auditorium; it is 1966 and the founding fathers of cell culture fill the audience. It’s a packed room of men in grey suits with thin black ties. One great man of science lounges confidently in a seat towards the back cleaning his horn-rimmed glasses on his jacket, while another great man of science dozes unashamedly in the front row. Into our scene walks the geneticist Stanley Gartler. He makes his way to the podium and announces that the myriad new cell lines they have gathered there to discuss - the cell lines that represent not only an array of cell types and tissues, but the brilliance of the men seated before him . . . are mostly just HeLa cells grown in new media with new labels.

HeLa cells

I like to think of those stoic, staid scientists booing and throwing their slide-rules at the podium (although in reality they just sat there in stunned silence). Nevertheless, the intensity of the scene led one scientist to later remark “He [Gartler] showed up at that meeting with no background or anything else in cell culture and proceeded to drop a turd in the punch bowl.”*

How could this happen? To answer this question, we need to go back to the beginning. Sterile techniques were in their infancy when many of the “new” cell lines were “immortalized.” Couple that with an understandable ignorance of the HeLa cell’s heartiness, and no reliable method to verify the molecular identity of cells, and it isn’t difficult to understand how this situation arose.

It is more difficult, on the other hand, to understand how the problem of misidentified and contaminated cell cultures could persist to the present day - and yet it does. There are several published lists of cell lines that are known to be contaminated (most often with HeLa cells) or misidentified, but that are still in use. In fact, data obtained through the use of misidentified or contaminated cell lines have been used to support clinical trials, grant applications, U.S. patents and publications.

Once again, we find ourselves asking, “how could this happen?” There are many causes, but here are a few likely explanations:

• Using multiple cell lines in the lab or growing cells on a non-human cell feeder layer increases the likelihood of cross-contamination.

• Cells that have been passaged many times can accumulate new mutations, which can contribute to experimental variability.

• Simple human error accounts for many of the contaminated and misidentified cell cultures in use today. Importantly, these errors can be propagated if the contaminated cell line is shared between investigators.

There is no good way to avoid the scenarios described above, but we can avoid the problems associated with them if we stop periodically to authenticate our cell lines. Our next blog post will provide a detailed description of the resources available to help you do just that. Nobody wants to go back to that dimly lit auditorium of 1966, but it will be difficult to make solid progress forward without addressing cell line contamination and misidentification head-on.

*Robert Stevenson, as quoted in The Immortal Life of Henrietta Lacks, Rebecca Skloot, New York: Random House, 2010.