IntroductionOne of the challenges facing stem cell research and Cell Therapy applications today is the characterization and investigation of stem cell and differentiated populations. There are several options currently available for this type of investigation, for example, rt-PCR, western blot, immunochemistry, and flow cytometry. However all these methods are invasive, which means that valuable cells are lost during the process of characterization. Recently, we published a Poster Article on “Predicting Differentiation and Characterizing Pluripotent Stem Cells Using Non-invasive Multi-analyte Luminex® Assays.” The article discusses a non-invasive method for cell characterization during differentiation. This method utilizes the flexibility of Proteome Profiler™ Antibody Arrays and Luminex® Assays to assess the expression of multiple analytes in cell culture supernates. Because this method uses cell culture supernates it is non-invasive and enables the detection of changes in cytokine expression throughout the progression of stem cell differentiation. We received a good deal of interest from readers on this method, and so during this week’s Ask the Expert session, Dr. Joy Aho, Manager of Product Development for the Stem Cell and Cell Culture Department at R&D Systems, will be answering your questions regarding using Luminex Technology to monitor stem cell differentiation without using any invasive methods, including using this technology to assess secretion profiles and how those differences may provide a method for predicting differentiation success and cell line specific optimization of differentiation protocols. Dr. Aho received a Ph.D. in Molecular, Cellular, Developmental Biology and Genetics from the University of Minnesota – Twin Cities and has over 10 years of experience developing media, reagents, and differentiation kits for stem cells. For more information on this technology, please view Dr. Aho’s webinar “How to Non-invasively Monitor Stem Cell Differentiation using Luminex Technology,” now available on demand. As stem cell research moves closer toward therapeutic application, there is a recognized need to increase the quality and consistency of pluripotent stem cells and their derivatives. While variability in the efficiency of stem cell differentiation can undermine data interpretation and slow research progress, the ability to quickly monitor changes in cell phenotype without sacrificing yield continues to be a challenge. In this webinar, Richard Feurstenberg and Joy Aho discuss Luminex technology, its power as a non-invasive tool for monitoring stem cell differentiation, and its utility for troubleshooting and optimizing stem cell culturing and differentiation conditions.
Current methods to track the efficiency and success of stem cell differentiation require the allotment of a specific well or wells of cells that are stained for stage-specific markers along the differentiation process. The non-invasive methods utilized by Proteome Profiler Arrays and Luminex Assays enable researchers to be able to monitor the same stem cell population throughout the differentiation protocol, while preserving the ability to use those same cells for downstream application once they have been fully differentiated. In addition, using a pre-identified secretory profile for successful differentiation, researchers may be able push cells that aren’t differentiating efficiently into a more successful trajectory by manipulating cell culture media so that it reflects the conditions observed in successfully differentiating cells.
Most certainly. These techniques are applicable across a variety of different stem cells and differentiation protocols. All cells are secreting protein all of the time and it’s just a matter of using these methodologies to identify particular proteins of interest. From that point, for you can build secretory signatures across the differentiation time course of your stem cell of interest.
Do you have a protocol for predicting differentiation or is this something that needs to be developed by the lab using it?
We do not currently have a protocol for predicting successful differentiation. This in part due to our observation that the profile of successful differentiation is dependent on the downstream differentiated cell of interest. For example, we find that hepatocytes and cardiomyocytes have unique secretory profiles when differentiated from iPSCs. We suggest that each lab independently determine the secretory profile for their differentiated cell type of interest. This can be done qualitatively using the Proteome Profiler Arrays, followed by the more quantitative Luminex Assays.
Using your method, can I confirm that my cells are still pluripotent without damaging them after reprogramming and during expansion.
In order to non-invasively monitor pluripotency, you would first need to determine the signature secretory profile of your pluripotent stem cells. I’d suggest first assessing the secretory profile of your cells with the qualitative Proteome Profile XL Cytokine Array. You can compare your results with the data presented in the webinar, for which we show similar secretory profiles across iPS cell lines. As an alternate, there are pluripotent live-cell staining kits available that allow you to visually confirm pluripotency in live cells using fluorescence without out affecting proliferation or stemness.
I am looking for a simpler way to analyze my undifferentiated and differentiating iPSC population for biomarker expression and germ layer specific markers to confirm pluripotency. Any suggestions?
The Proteome Profiler Arrays would be a good option for you. R&D Systems has a Human Pluripotent Stem Cell Array that detects 15 pluripotent and germ layer-specific markers simultaneously. This array requires cell lysates rather than cell culture supernatants, which are used for non-invasive monitoring of stem cells. R&D also offers a number of pluripotent and germ layer staining kits which can be viewed here.
Have you seen any effects of serum in the media or CD media having an impact on your ability to study the secretion profiles?
You will definitely detect various growth factors/cytokines from the media you are using, especially if you are culturing your cells with growth factors/cytokines. We would recommend running samples of media alone as a control to ensure that the proteins you are detecting through the arrays and/or Luminex are due to secretion by cells and not from the media.
Do you have any customers who have been able to use this method to improve their differentiation success rate?
The use of non-invasive techniques, such as Luminex and Proteome Profiler Arrays, to monitor stem cell differentiation is relatively new. Our initial studies have focused on profiling differentiation of iPSCs into various lineages. We are currently working in-house and with collaborators on ways in which this technology could identify growth factors or cytokines that could be added to improve differentiation success. We are interested in more collaborative opportunities to explore the boundaries of these platforms for predicting and optimizing stem cell differentiation.
We currently use the Luminex for assays, but I am confused about how the proteome profiler works in conjunction or when you use each?
The Proteome Profiler Arrays do not require any specialized equipment and are an easy screening solution for researchers that do not have access to a Luminex machine. Additionally, the Arrays are ideal for researchers who are interested in first qualitatively screening for changes in the secretome on a large panel of analytes. Using the initial Proteome Profiler data, one can identify candidate analytes that can be more quantitatively investigated using Luminex.
We are still undergoing this process to find signatures that will correlate with differentiation success. We have done up to 3 replicates at this point and are focusing on molecules that were seen both in the Array and confirmed via Luminex. We found very consistent results. It will be important to then test these analytes across more cell lines as well as different protocols to confirm our findings.
My work involves generating iPSCs from patient cells. One aspect requires identification of the cells with the best differentiation potential. Do you think there is a way to use this technique to assist?
We believe this technique could be used in that manner. We are currently working to correlate the expression pattern of iPSCs and the robustness of their differentiation to expand the potential uses for this technique. In the meantime, we would recommend trying our Human Pluripotent Functional Identification Kit, which provides reagents and protocols to assess for tri-lineage differentiation of pluripotent stem cells in 5 days.
In your study, what method are you using to confirm differentiation? Are you measuring marker expression at all stages and/or function?
We have used this technology for iPSC-derived hepatocytes and iPSC-derived cardiomyocytes. For hepatocytes, we used our StemXVivo Hepatocyte Differentiation Kit to differentiate, which we have verified for expression of stage-specific markers as well as for function, including albumin secretion, urea secretion, and p450 activity. Hepatocyte verification data can be viewed here. For cardiomyocytes, we used our StemXVivo Cardiomyocyte Differentiation Kit to differentiate, which we have verified for stage specific marker expression, contractility, response to known regulators of cardiomyocyte contractility, and response to cardiotoxic agents. Cardiomyocyte verification data can be viewed here.
We do not have specific analyte panels for transdifferentiation or reprogramming. However, we have a large list of analytes at our disposal, as wells as preassembled analyte panels. We suggest screening your cell culture supernates with a broader panel of analytes first. This would help you identify signature changes in protein secretion that could be used to track the success of future reprogramming or trandifferentiation experiments.
No. In addition to human we have analytes available for mouse and rat. This list can be viewed here.