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 —

CELLS.

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.

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Figure 1: Nobel Prize awardees in 2012, reproduced from http://www.nobelprize.org/nobel_prizes/medicine/laureates/2012/

 

 

 

 

 

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).

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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!

REFERENCES

  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.

 

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