| The Living Dead (of the iPSC world): Autopsy donor-derived iPSCs |
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From Neuroscience Letters In vitro disease modelling approaches largely involve the use of immortalised cell lines that have been genetically altered in order to induce a disease phenotype. While these systems provide valuable information, such models are unable to represent complex human disease aetiology and are therefore not always physiologically relevant. As induced pluripotent stem cells (iPSCs) retain the genetic profile of the somatic donor cell of origin, iPSCs represent an important additional option for disease modelling in vitro. This approach offers many advantages over previous methods, for example the non-invasive study of neurological or neurodegenerative conditions that are ordinarily impossible premortem, or certainly risk some cognitive or functional impairment if undertaken. Yet one major complication of this approach is that the effectiveness of iPSC disease models in vitro relies entirely upon the accuracy of the premortem diagnosis. In fact, most premortem diagnoses of neurological disease made from clinical criteria are not definite and can only be confirmed following postmortem histopathological analysis, so it would be advantageous if we could generate iPSC from post-mortem tissues. A study from Arizona, USA, by Hjelm et al.1 has demonstrated that iPSCs can indeed be generated from autopsy-derived fibroblasts. Creating iPSC lines from dead human tissues may be viewed by some as a little macabre, hence our reference to the 1974 film “The living dead” in the title, but in reality this development opens another avenue for iPSC-based disease modelling following a definite postmortem diagnosis. This work was based upon another study published over ten years ago by Meske et al.2 which demonstrated that cultures of human dermal fibroblasts can be established within 48 hours postmortem from autopsy donors of up to 99 years of age, yet no reports had emerged which described the generation of iPSCs from these cells. Hjelm et al.1 sought to do exactly this, from 19 donors aged between 72 and 97 years. Of these subjects only one 75 year old male could be considered an experimental control, having been cleared of major neurological and neuropathological conditions. First the authors investigated whether the location of the skin biopsy affected primary fibroblast culture proliferation levels. To do this they generated 30 primary fibroblast lines from biopsies taken from the arm, torso and leg of three autopsy donors between 3 and 7 hours postmortem, and performed cell counts after the first passage. Interestingly, significantly increased proliferation was observed in fibroblast cultures originating from the arm, compared with the leg or torso which displayed similar proliferation rates. Surprisingly, autopsy donor age (spanning an impressive 25 year range) was not found to significantly alter fibroblast proliferation rate. One autopsy donor-derived fibroblast line was chosen to test iPSC induction via lentiviral transduction of the Yamanaka factors (Oct3/4, Sox2, Klf4, c-Myc) under feeder-free conditions. Multiples iPSC clones were generated from this line, which were expanded and then frozen before further expansion prior to characterisation and differentiation experiments. Western blot and immunocytochemical analysis confirmed the expression and correct location of the Yamanaka factors within these clones. The authors state that further iPSC lines were generated from other fibroblast lines derived from multiple donors, however they focus only on the iPSC derivatives of this chosen line in their study. Genotype concordance and copy number variation analysis using Affymetrix SNP Array confirmed chromosomal integrity of the resulting iPSC clones and their autopsy donor-derived fibroblastic origin. For loss of pluripotency experiments, neural induction of iPSC was initiated by the culture of floating embryoid bodies (EBs) for 5 days in human embryonic stem cell media in the absence of FGF and then in neural medium supplemented with EGF plus FGF for another 5 days. Cells were then seeded onto Matrigel and allowed to differentiate further in neural medium. Confocal microscopy revealed the emergence of GFAP+ astrocytes and neuronal cells identified by their co-expression of neuron-specific beta III tubulin and Neurexin IV after 14 days of differentiation, and a small number of MOG+ cells marking putative oligodendrocytes by day 35. The authors, however, do not elaborate upon the proportions of these cell types in their neural-induced cultures. Reports indicate that cellular senescence is a barrier to reprogramming and thus may limit the potential for iPSC therapies in elderly patients.3,4 Given that senescent cells accumulate as an organism ages, it is very interesting that autopsy donor age had no significant effect on fibroblast proliferation in this study and may indicate that a short postmortem interval may be a stronger determining factor than donor age for iPSC generation from autopsy-derived somatic cells (also see another related article on the Stem Cell portal ‘You are only as old as the factors you are reprogrammed by’). This study is an interesting proof-of-principle that iPSCs can be generated from autopsy donor-derived fibroblasts and paves the way for the use of iPSC-based disease models which afford researchers greater certainty of the model under scrutiny, overall increasing the power of this system. Although the mechanisms underlying the increased levels of proliferation observed in fibroblast cultures originating from the arm was not discussed, nor the transduction efficiency of iPSC generation from autopsy-derived tissues, the yield of iPSCs from this additional resource is an important result. The ability to use a system that is backed by a definite diagnosis increases the statistical power and physiological relevance of in vitro disease models, and will likely accelerate disease research efforts and aid us in our pursuit to develop suitable treatments.
References 1. Hjelm et al. Induction of pluripotent stem cells from autopsy donor-derived somatic cells. Neurosci Lett. 2011. 20;502(3):219-24. 2. Meske et al. Culture of autopsy-derived fibroblasts as a tool to study systemic alterations in human neurodegenerative disorders such as Alzheimer's disease – methodological investigations. J Neural Transm. 1999. 106;5-6:537-548. 3. Banito et al. Senescence impairs successful reprogramming to pluripotent stem cells. Genes Dev. 2009. 23: 2134–2139. 4. Marion et al. A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity. Nature 2009. 460:1149–1153.
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