Introducing cells with a lot of potential

I felt it was time for a “cell” post here. No pain, no surface electrodes or imaging data —


Not just simple cells however, but the most amazing cells on earth: induced pluripotent stem cells.

With the original publication in 2007 1 describing the reprogramming of adult human terminally differentiated somatic cells into induced pluripotent stem cells (hiPSCs) this fascinating technique has since initiated further innumerable  ground-breaking  research and two involved pioneers have finally been awarded the Nobel Prize in Physiology or Medicine in 2012.



Figure 1: Nobel Prize awardees in 2012, reproduced from






Briefly, via transfection with lenti-, Sendai-, retroviruses, minicircles, piggyBac, miRNA, mRNA or episomal plasmids that carry different pluripotency-associated transcription factors (the most commonly used combination of reprogramming factors consists of c-myc, SOX2, OCT4 and KLF4, also named ‘Yamanaka factors’) this reprogramming technique initiates a cascade of erasure and remodelling of epigenetic marks that turn previously fully differentiated somatic cells into so called induced pluripotent stem cells. These pluripotent cells display features similar to true human embryonic stem cells (hESCs) and can be generated readily from adult human skin cells, or subsequently most other human fully differentiated tissues (see Figure 2 for a schematic).


Figure 2: Scheme of induced pluripotent stem cell generation: Somatic cells from adults can be cultured and reprogrammed to induced pluripotent stem cells by transient transfection with exogenous pluripotency factors. These factors are thought to change the epigenetic landscape of transfected cells towards silencing of differentiation-associated genes and enhanced transcription of endogenous pluripotency genes. Figure reproduced from 3.

Dependent on the technique used for reprogramming (miRNA, episomal plasmids, viral transfection, etc.) and the adult cell type starting the process with (fibroblasts, blood cells, renal epithelial cells from urine, etc.), the reprogramming process can take around 2-8 weeks and is variably efficient. Upon introduction of reprogramming factors, the cells reprogrammed to 100% will start forming colonies of pluripotent stem cells slowly. Given their enormous proliferative capacity these gain significant growth advantage over the non- or only partially reprogrammed cells in the dish. They can then be isolated based on expression of surface markers or reporter genes, their morphology or based on media and surface conditions that additionally select for their growth. Extensive validation and rigorous quality control of iPSC clones is necessary. This includes differentiation capacity in vitro and in vivo (even though nowadays less common), expression of endogenous pluripotency markers, silencing of exogenous transcription factors and exclusion of chromosomal abnormalities or transgene integration.

The iPSC clones can then be differentiated into almost any cell type of the human body – isn’t this magic? – presupposed the existence of efficient differentiation protocols that suppress pluripotency and guide the cells to their desired fate. Depending on cell type and individual hypotheses of the respective studies, generated cells can be analysed using classical cellular phenotyping assays investigating apoptosis, cell cycle, cell metabolism, membrane texture, cell migration, cell growth, nuclear or cytoplasmic foci, cell shape, cytoskeletal reorganisation, neurite outgrowth, mitochondrial mass, mitochondrial membrane potential, ROS production, protein localisation, expression and quantification, etc.  The sky is the limit, really (and your cell culture skills, patience, assay optimisation endurance and money, obviously…).

Neuronal cells have been amongst the earliest cell types to be differentiated from hESCs 4 5 and hiPSCs 6 7 with the help of efficient and robust differentiation protocols. Approaches include co-culture with neural inducing feeder cells, in vitro generation of suspension based three-dimensional embryoid bodies exposed to retinoids in a stage specific manner or direct pharmacological inhibition of transforming growth factor beta 1 (TGF-beta)- and bone morphogenetic protein (BMP)-signalling performed on a monolayer of confluent stem cells – a process the field calls ‘dual SMAD inhibition’. All outlined procedures reliably initiate ‘neural induction’ and efficient ‘neural conversion’ representing important early checkpoints in any protocol that generates neurons from iPSCs. The resulting neural precursor cells can be “patterned“ (second step) by application or absence of developmentally rationalised extrinsic cues, and finally be terminally differentiated (third step) towards region-specific glial and neuronal subtypes.

Interestingly, unless differentiated iPSC-derived neurons are “forced“ to age (e.g. via progerin expression 8), this technology models human developmental processes and diseases 9 10 in a fetal system 11. An ever-growing number of studies nonetheless confirm mutant hiPSC-derived neurons obtained from patients with inherited disease – including adult-onset disorders – do successfully recapitulate central cellular pathomechanisms despite their fetal characteristics 12-18. Furthermore, in addition to confirming known and uncovering novel cellular and molecular pathomechanisms for a range of sporadic and inherited disorders 13 16, human iPSCs have also been used to screen for novel potential therapeutics successfully 19-21 and have been helpful in dissecting genetic and non-genetic factors driving neuronal degeneration 18.

As introduced here, the ground-breaking discovery of efficient reprogramming of adult human fibroblasts into iPSCs in 2007 1 initiated an unprecedented paradigm shift for regenerative medicine with even more unmet potential for neurology and neurodegeneration. Ever since, only nine years have passed. We are all very excited and curious for the next nine years to come!


  1. Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007;131(5):861-72.
  2. Tabar V, Studer L. Pluripotent stem cells in regenerative medicine: challenges and recent progress. Nature reviews Genetics 2014;15(2):82-92.
  3. Yamanaka S, Blau HM. Nuclear reprogramming to a pluripotent state by three approaches. Nature 2010;465(7299):704-12.
  4. Reubinoff BE, Itsykson P, Turetsky T, et al. Neural progenitors from human embryonic stem cells. Nature biotechnology 2001;19(12):1134-40.
  5. Zhang SC, Wernig M, Duncan ID, et al. In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nature biotechnology 2001;19(12):1129-33.
  6. Chambers SM, Fasano CA, Papapetrou EP, et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nature biotechnology 2009;27(3):275-80.
  7. Shi Y, Kirwan P, Livesey FJ. Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks. Nature protocols 2012;7(10):1836-46.
  8. Miller JD, Ganat YM, Kishinevsky S, et al. Human iPSC-based modeling of late-onset disease via progerin-induced aging. Cell stem cell 2013;13(6):691-705.
  9. Lancaster MA, Renner M, Martin CA, et al. Cerebral organoids model human brain development and microcephaly. Nature 2013;501(7467):373-9.
  10. Marchetto MC, Carromeu C, Acab A, et al. A model for neural development and treatment of Rett syndrome using human induced pluripotent stem cells. Cell 2010;143(4):527-39.
  11. Patani R, Lewis PA, Trabzuni D, et al. Investigating the utility of human embryonic stem cell-derived neurons to model ageing and neurodegenerative disease using whole-genome gene expression and splicing analysis. J Neurochem 2012;122(4):738-51.
  12. Dimos JT, Rodolfa KT, Niakan KK, et al. Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science 2008;321(5893):1218-21.
  13. Israel MA, Yuan SH, Bardy C, et al. Probing sporadic and familial Alzheimer’s disease using induced pluripotent stem cells. Nature 2012;482(7384):216-20.
  14. Koch P, Breuer P, Peitz M, et al. Excitation-induced ataxin-3 aggregation in neurons from patients with Machado-Joseph disease. Nature 2011;480(7378):543-6.
  15. Lee G, Papapetrou EP, Kim H, et al. Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs. Nature 2009;461(7262):402-6.
  16. Sanchez-Danes A, Richaud-Patin Y, Carballo-Carbajal I, et al. Disease-specific phenotypes in dopamine neurons from human iPS-based models of genetic and sporadic Parkinson’s disease. EMBO molecular medicine 2012;4(5):380-95.
  17. Schondorf DC, Aureli M, McAllister FE, et al. iPSC-derived neurons from GBA1-associated Parkinson’s disease patients show autophagic defects and impaired calcium homeostasis. Nature communications 2014;5:4028.
  18. Woodard CM, Campos BA, Kuo SH, et al. iPSC-derived dopamine neurons reveal differences between monozygotic twins discordant for Parkinson’s disease. Cell reports 2014;9(4):1173-82.
  19. Yang YM, Gupta SK, Kim KJ, et al. A small molecule screen in stem-cell-derived motor neurons identifies a kinase inhibitor as a candidate therapeutic for ALS. Cell stem cell 2013;12(6):713-26.
  20. Bellin M, Marchetto MC, Gage FH, et al. Induced pluripotent stem cells: the new patient? Nature reviews Molecular cell biology 2012;13(11):713-26.
  21. Cooper O, Seo H, Andrabi S, et al. Pharmacological rescue of mitochondrial deficits in iPSC-derived neural cells from patients with familial Parkinson’s disease. Science translational medicine 2012;4(141):141ra90.
  22. Xie YZ, Zhang RX. Neurodegenerative diseases in a dish: the promise of iPSC technology in disease modeling and therapeutic discovery. Neurological sciences : official journal of the Italian Neurological Society and of the Italian Society of Clinical Neurophysiology 2015;36(1):21-7.


New England vs Montreal: Data Sharing in 2016

Research parasites are all the rage these days.

This last week, two stories about data sharing caught my eyes. And even thought they have emerged from just about 350 miles apart, the attitudes involved could not be further from each other.

The first story that caught my eye was the editors of the New England Journal of Medicine summarising what they think about data sharing. They talk about it like self-assured conscientious capitalists describe their idea of communism:

“The aerial view of the concept of data sharing is beautiful. […] The moral imperative to honor their collective sacrifice is the trump card that takes this trick. However, many of us who have actually conducted clinical research […] have concerns about the details.”

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A Sensitivity Problem in Pain Imaging: #cingulategate

Screen Shot 2015-12-31 at 1.03.04 AM

I know that the #cingulategate horse has been beaten to death, but I thought it would be worth putting it here, just in case someone had managed to miss it. It all started with a recent paper in PNAS by Lieberman and Eisenberger1 (L&E) that (in my opinion) makes some egregious statements about the specificity of the anterior midcingulate cortex based on Neurosynth – a nifty and useful meta-analysis software, ideal for hypothesis forming. Tal Yarkoni (TY) – the creator of Neurosynth – published a blogpost discussing the various problems with the paper.

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Pain and Pleasure: Towards a mechanism of neuropathic pain chronification

There is no doubt that pain is one of the most important systems for survival. Even though we all feel pain, surprisingly little is known about pain mechanisms. Indeed, there are many outstanding fundamental questions in pain research, such as where acute pain is encoded in the brain, how pain competes with other processes. To further complicate matters, chronic pain is very different from acute or experimental pain. Read more

It’s not what it looks like: High Frequency Oscillations

Following on from the last post on baseline shifts, this is the second post on a few things we don’t normally consider when talking about standard EEG measurements. I had the idea for this one after a few excellent talks by Liset Menendez de la Prida at ISWP7 earlier this year.

Fast and furious (or is it)

Fast stuff on the EEG is difficult to see for a number of reasons: i) We usually filter raw EEG signal to make it look neater and often exclude high-gamma range signal. ii) The signal we measure on the scalp itself is already attenuated by passing through different tissues, making fast activity appear less sharp and prominent. This is true even for ECOG when compared to direct LFP recordings (which is becoming more relevant now that microelectrodes are being used more and more in patients with epilepsy). iii) Higher frequencies have a lower power – usually fast fluctuations are a lot smaller than bigger shifts on the EEG and seem to pale in comparison, when visually analysing the EEG.

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It’s not what it looks like: Baseline Shifts

Cook et al. 2013
The implantable device from Cook et al. 2013

I’ve just come back from the fantastic IWSP7: Epilepsy Mechanisms, Prediction and Control conference in Melbourne. Having apparently outgrown the initial meetings’ focus on seizure prediction, this year covered all aspects from computational models, intracranial devices, to imaging in epilepsy. For those who don’t know – Melbourne is a great place for such a conference, since Mark Cook and colleagues have managed a couple of years ago to pull off a clinical trial of implantable intracranial recording devices designed for long-term ambulatory recordings, in addition to the potential for responsive neuromodulation. The set-up can be seen on the right (an image the conference conveners seemed to love), and was a first in the world of seizure prediction. [1]

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A Wolf in Sheep’s Clothing


I spent the day yesterday in clinic, seeing a few fantastic kids, all with a rare epilepsy syndrome that is known to cause a lot of cognitive and learning problems. This is exactly what I worry about most in childhood epilepsies – the effect of the seizures and the epilepsy on learning and development.

So as odd as it may sound, making a diagnosis of one of the more ‘benign’ epilepsy syndromes can be reassuring for me as a clinician. If I have a child with typical childhood absence epilepsy in clinic, I know that there is a good chance we will get the seizures under control, and that after puberty many patients will become seizure free. Yet ‘benign’ in medicine is always a double edged sword: Whilst all the above may be true, it turns out childhood absence epilepsy in some ways is not a harmless condition without any lasting effects. 
It’s Purple Day – aka Epilepsy Awareness Day. So yes – let’s get talking about Epilepsy! It’s great to see so many people, differently affected by epilepsy join the discussion on twitter and give a public face to the condition.

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Moving on from the Seizure Reservoir

One of the most challenging and puzzling issues for both patients and clinicians is the apparent unpredictability of seizures. Beyond a few general statements of things that increase your chance of having a seizure, it is difficult (impossible) to pinpoint, why a seizure happens at exactly the time that it does. The issue becomes even more intriguing when there is not even a focal ‘epileptogenic’ zone – as for children with idiopathic generalised epilepsy, whose brains will look completely normal on brain scans, but will suffer apparently unprovoked seizures again and again. 

Richardson (2011)
Drawing from Richardson MP (2011) J Progr Biophys Mol Biol, 105:5-13, originally from Lennox (1941) Science and Seizures, New Light on epilepsy and Migraine. Harper Bros, NY

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Let’s talk about death

When making a new diagnosis of epilepsy, I often encounter fear – I speak to parents who have seen their child having a seizure in front of them, becoming unresponsive and shaking uncontrollably. And from their faces I can see that they were worried about one thing above all: Is my child going to die?

In the majority of cases the answer is clearly no. Seizures themselves very rarely cause mortality, and other than the rare sudden unexplained death in epilepsy (SUDEP), or patients with significant neurodevelopmental disabilities and life-limiting comorbidities, we rarely see deaths in our paediatric patients with epilepsy.

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