As an Irishman, I am well versed in roundabouts, we love them. Entering the roundabout, going around it and then hoping to get off it when its your exit. If you don’t exit, you just keep going round and around and around, well you get the drift. It reminds me of that great scene in National Lampoon’s European vacation where the Griswalds enter an English roundabout and just keep going around in circles for hours. If you don’t know what I am talking about, then check it out, I can wait.
Ok, welcome back, you enjoy the video, of course you did, how could you not. Now, I know you must be wondering, Tom what are you on about, just get to the point. Did you hit your head on a Montreal construction cone, not once have you mentioned dinosaurs. Well I am taking a break from dinosaurs today. And the reason for my earlier meander about roundabouts, is because I am going to talk a little about the unending circle of iPSC quality control (QC), which in my mind is just like any major roundabout in Dublin or London. Always moving in a circular fashion as we check and recheck our cells to ensure we are working with top-notch iPSCs. And the reason for this, your stem cell culture is the core of all your experiments, have a bad cell or a bad batch, and you will be having one bad week, month or even year. Trust me, I know all too well this feeling which is why our group, and in particular Carol (I talked about her in my last blog) and her fellow iPSC specialist Narges Abdian have gone to great lengths to implement our roundabout of iPSC quality control, which I encourage you all to adopt or to share with me ideas on what you do in your group or ideas we could take on board.
I will only loosely explain it here, but I do encourage you to check out our poster on this. Carol presented this at the recent International Society for Stem Cell Research (ISSCR) meeting in LA at the end of June. For those who missed her poster, we are making it available for all to see, to read and to start a dialog over the importance of quality control.
For us, we take several steps to ensure that when we make a new line or receive a line from elsewhere that it checks all the boxes of a “bona fide” good line to be used in experiments. These steps include:
- Check your iPSC is pluripotent: We image cells for common pluripotency markers and also check for these markers by qPCR. You can also look at the morphology of your cells, and if they don’t look like an iPSC, then you have big problems. But if you pass this first step, now it is on to Step 2.
- Does your cell grow well: For this we do a combination of live cell imaging in two different stem cell medias (mTESR and Essential 8) and check for cell growth and cell viability through a combination of live cell imaging and crystal violet staining. Some lines grow well on both medias, but there are times when a line might prefer one line versus another, so that’s important to keep in mind.
- Is the genome of the cell stable: For this you can look at total chromosome number, along with genetic duplications that often arise. These can often confer selective growth advantages for your cell-line so its important to check this when working with any cell-line. You can also do other methods like whole-genome sequencing to check the entire genetic sequence of your cell-line, and this is currently in progress for many of our control iPSCs and the lines they were derived from, so this data we will soon have for a number of our lines being made available.
- Can my stem cell form other cells: Obviously when you are working with a stem cell, you might want a specific cell type. So, if your cell is good, you can always go ahead and try your SOP for generating the cell-line you want. But another way to test the differentiation potential is through a trilineage test in which you promote the formation of endoderm, mesoderm and ectoderm.
There you have it, our roundabout of iPSC QC; our little circle of life. We will be adding more to this process over the next month, as we are in the preparation phase of a manuscript on iPSC QC. More data is being added so we will return to this roundabout in due time.
You will also notice from the poster we have made and profiled 10 healthy control lines relative to cells from other sources. Yes, you can now access these iPSCs through the MNI C-BIG and requests for the lines can be made. Other lines are entering their freezers in coming weeks and months from our Parkinson’s disease and ALS cohorts, and I will keep you all updated as they become available for access under the open science principles of the MNI.
Next week, I start to outline how we make and work with neurons from patient-derived iPSCs. Got to run, this is my exit, or I will be on this roundabout forever.