NEW REPROGRAMMING MARKERS REVEAL A DETOUR ON THE PATH TO iPS CELLS By James O’Malley PhD student, MRC Centre for Regenerative Medicine, The University of Edinburgh www.crm.ed.ac.uk The discovery of pluripotent embryonic stem (ES) cells has long been touted as one of the most important developments in modern science. By growing these cells in the appropriate conditions, researchers are aiming to generate cells required by patients and also examine differences between healthy and diseased cells. However, the use of ES cells is somewhat contentious as they are derived from fertilised eggs, and it is not yet possible to create patient specific ES cells. Induced pluripotent stem cells (iPS cells, see ‘what are stem cells’ box on page 3), which can be made from a patient’s own skin cells, have addressed these issues and have proved an invaluable tool for investigating the causes of and solutions to many genetic diseases. The mystery of iPS cells Despite the numerous studies and huge interest iPS cells have attracted, the exact mechanism by which a differentiated cell, with a defined and limited role in the body, undergoes the transformation into an iPS cell, with its almost unlimited potential, largely remains a mystery. By An overview of the reprogramming process by studying the different changes and transitions which differentiated cells are transformed into that occur during this transformation, which is iPS cells by reprogramming factors. At present termed “reprogramming”, scientists aim to little is understood about how differentiated improve their understanding and the safety of cells become iPS cells.
this technology. However, while it is generally easy to grow and study large numbers of differentiated and iPS cells, studying the intermediate stages of reprogramming is difficult. This is due to three major constraints, namely the low efficiency of most methods used to generate iPS cells, an asynchronous behaviour of the cells during the reprogramming process and a lack of technology to identify the cells that are turning into iPS cells. Marking out the reprogramming pathway A number of recent studies have attempted to address these issues by using so‐called “markers”. These are proteins found on the surface of cells. Different types of cells have different markers, and they have been used in other areas of research to isolate specific cells, for example to identify the different types of cells found in blood. However the previous set of markers used in most reprogramming studies were not totally accurate Using new markers we discovered that differentiated cells follow a defined reprogramming pathway during their transformation into iPS cells. How does our system work? Unlike iPS cell colonies in which all cells are similar, colonies made during reprogramming contain many different types of cells, as shown in green and red in A. Our markers ‐ ICAM1 and CD44 ‐ showed that all iPS cells have a similar profile, while reprogramming cells are mixed (B). C shows how we isolated one population by using cell sorting. After a few days we saw that some of these cells had changed their markers and had moved closer to iPS cells, as outlined by the white arrow in D. We repeated this analysis for each population we had identified, and saw that cells always followed the same route as they became more similar to iPS cells. This allowed us to create a pathway to trace the movement of cells undergoing reprogramming, depicted in E. in their ability to differentiate between successfully reprogrammed cells and those that actually fail to become an iPS cell. Therefore, our lab set out to identify new, more accurate markers of the intermediate stages of reprogramming. By using two new marker proteins we had identified – ICAM1 and CD44 – we identified 6 different groups, or populations, of cells undergoing reprogramming. Using a technique called cell sorting, we isolated these different populations from a mixed batch of cells undergoing reprogramming. We discovered that each population we had identified gave rise to a different number of iPS cells and corresponded to a different stage of the reprogramming process. Next we monitored how cells moved from one stage to the next, allowing us to trace a route from differentiated cells to iPS cells. This was an important finding as it proved that cells do not move towards an iPS cell state in a random manner, but that they actually appear to follow a defined reprogramming ‘pathway’. A detour on the road to iPS cells Now that we could accurately identify how cells moved along this path, we tried to determine what factors were required to move from one point to the next by looking at their gene expression: the genes turned on and off at each stage. To our surprise, reprogramming was not simply a loss of differentiated cell gene The pathway from differentiated cells to iPS cells is not as simple as one might think. We found that cells appear to take a detour to an intermediate state during reprogramming. expression and a gain of stem cell gene expression or reversal of the differentiation process. Instead, we found cells showed signatures of an unrelated intermediate cell type before becoming iPS cells. This was a key outcome of our work because if cells are veering away from the most direct route to an iPS cell state, it may explain why the efficiency of reprogramming is so low. Or, alternatively, there may be a major unknown obstacle forcing cells to make a detour on their way to becoming iPS cells. Where will this pathway lead? The marker system we have developed can be used to help investigate methods to increase the efficiency of the reprogramming process. Moving forward, we are attempting to understand why cells are taking this detour during reprogramming, and if there are things we can do to prevent this from happening. These strategies may increase or decrease the efficiency of reprogramming; this will provide us with vital clues about the requirements and limitations of current reprogramming methods. Our findings can be used to accelerate the drive towards the use of iPS cells for patients by increasing the accuracy and efficiency of the reprogramming process. What are Stem Cells? Stem cells were first identified in the 1960s to describe a type of cell with the ability to give rise to a number of other cell types, in a process called differentiation. In addition to this, stem cells also have the capacity to self‐renew, the process by which they can produce exact copies of themselves. This is a fundamental system to maintain our body and has also become an important research tool with the potential to study numerous diseases and, ultimately, generate specific cells and tissues for treatments One way to think about stem cells is to divide them into three categories: 1. Adult or tissue stem cells: found only in specific areas of the body; 2. Embryonic stem (ES) cells: grown in the laboratory from the early embryo; 3. Induced pluripotent stem (iPS) cells, or ‘reprogrammed’ stem cells: similar to embryonic stem cells but made from adult specialised cells using a laboratory technique discovered by Japanese scientist Prof Shinya Yamanaka in 2006. Tissue or adult stem cells are multipotent and sometimes even unipotent, they can only give rise to limited, more tissue‐specific cells. For example, multipotent blood stem cells can only make the specialised cells found in your blood and unipotent germline stem cells in males can only make one type of cell, sperm cells. ES cells are pluripotent, meaning they have the capacity to produce every type of cell in the body. The 'reprogrammed' iPS cells behave identically to the pluripotent ES cells and share the capacity to make every type of cell in the body. These cells, unlike ES cells, do not require the use of embryos. More information can be found at: www.crm.ed.ac.uk Publication details O’Malley J, Skylaki S, Iwabuchi KA, Chantzoura E, Ruetz T, Johnsson A, Tomlinson SR, Linnarsson S and Kaji K. 2013. High resolution analysis with novel cell‐surface markers identifies routes to iPS cells. Nature, epub ahead of print 02 June 2013, doi: 10.1038/nature12243.