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Variability Necessitates More iPSCs

Original article from STEM CELLS Translational Medicine

“Variability in the Generation of Induced Pluripotent Stem Cells - Importance for Disease Modeling”

One of the most interesting uses of induced pluripotent stem cells (iPSCs), alongside potential immunologically matched cell replacement therapy, is the modelling of complex human diseases (Vitale et al, 2011). However, diseases with undefined genetic components such as idiopathic Parkinson disease (Soldner et al) raise questions about the effectiveness of iPSCs as disease models. However comparisons between multiple patients and controls to identify disease-specific attributes that are independent of individual differences may overcome this problem. Such variability includes both genetic and epigenetic mechanisms and many laboratories are moving to attempt to maximise the efficiency and efficacy of iPSC generation and standardise any production protocols in order to minimise any identified variations so that disease-associated changes can be observed more clearly (Boulting et al and Saha and Jaenisch). Now, in a study published in the September edition of Stem Cell Translational Medicine researchers from the laboratory of Alan Mackay-Sim have derived 18 human iPSC lines from 8 individuals in order to determine the variability in the generation, selection, and characterization of these iPSCs (Vitale et al, 2012), finding low inter-individual and interclonal variability but also reprogramming instability.

All iPSCs were generated from primary dermal fibroblasts taken from 4 healthy patients and 4 aged matched patients with schizophrenia with an age range of 21 to 51 using lentiviral transduction of OCT4, SOX2, KLF4, and cMYC. 18 lines in total were made from these 8 patients; all showed human embryonic stem cell (hESC)-like morphology, most were genetically stable, cells expressed many endogenous pluripotency-associated markers and grew normally for up to 50 passages. Teratoma formation assays demonstrated that 11 of the lines formed teratomas efficiently, suggesting that 11 of the lines could be called “true” iPSCs (from criteria laid out in Maherali and Hochedlinger). However, of the 11 lines, the exogenous OCT4 transgene was not completely silenced in one line, while another did not have endogenous OCT4 expression. Additionally, three clones from one donor presented with the same abnormal karyotype (a balanced reciprocal translocation involving chromosomes 9 and 12, and a pericentric inversion in one chromosome 20) which upon re-evaluation of the donor fibroblasts was observed in 53% of the cells, suggesting the abnormality was not due to the reprogramming process. Three other lines presented with a decrease in SSEA4+ cells with increased passage number, although were initially identified as true iPSCs.

Gene expression analysis was undertaken using SSEA4+ cells from 15 of the 18 lines; 2 presented SSEA4- and one cell line was lost due to failure to recover after freezing. Analysis of the expression plots obtained demonstrated that, as expected, the fibroblast donor cells clustered apart from the iPSCs and hESCs. iPSCs and hESCs clustered together although two distinct clusters of iPSCs were observed which could be distinguished by the proportion of SSEA4+ cells. Of the clones expressing high SSEA4, those from the same donor clustered together tightly, while passage number seemed not to affect this correlation.

Further investigation of the SSEA4-high and SSEA4-low iPSCs found a similar cellular morphology and expression of SOX2 and cMYC, but found that OCT4 and NANOG were lower in SSEA4-low iPSCs, while KLF4 demonstrated a range of expression throughout the iPSCs. Genome wide transcriptional analysis found 2,826 probes to be significantly different; 952 enriched in the SSEA4-high and 904 in the SSEA4-low. Using a study which brought together multiple expression profiles of pluripotent cells to give a test which could assess the pluripotent state of a cell by its transcriptional output (Muller ­­et al), SSEA4-high cells were deemed to fully reprogrammed pluripotent cells, while the SSEA4-low cells were deemed to be partially reprogrammed. However, the authors note that 3 lines deemed by this assay to be fully reprogrammed could form only one or two germ layers in the teratoma assay. Finally, comparisons of the iPSCs generated from cells of schizophrenic donors with control iPSCs found no obvious differences at the transcriptional level. Scores generated on the expression of 307 key genes were highly similar and the different iPSC lines were not segregated.

Taken together these results demonstrate the difficulty in defining successfully reprogrammed iPSCs and also in defining similar cell lines for inter-individual or intergroup comparisons, as is necessary for disease modelling. This observation is underlined by another study which compared 16 iPSC lines, of which only 7 were deemed to be fully pluripotent although they expressed many pluripotency-associated markers and many could differentiate towards neuronal cells (Boulting et al). The results also suggest that a large cohort of clones in a similar stable state will be required to make observations between disease- and control-iPSCs which may prove to  be difficult for complex genetic diseases.



Boulting GL et al.
A functionally characterized test set of human induced pluripotent stem cells.
Nat Biotechnol 2011;29:279 –286.

Maherali N, Hochedlinger K.
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Cell Stem Cell 2008;3: 595–605.

Muller FJ et al.
A bioinformatic assay for pluripotency in human cells.
Nat Methods 2011;8:315–317.

Saha K, Jaenisch R.
Technical challenges in using human induced pluripotent stem cells to model disease.
Cell Stem Cell 2009;5:584– 595.

Soldner F et al.
Parkinson’s disease patient-derived induced pluripotent stem cells free of viral reprogramming factors.
Cell 2009;136:964 –977.

Vitale AM, et al.
Induced pluripotent stem cells: A new technology to study human diseases.
Int J Biochem Cell Biol 2011;43:843– 846.

Vitale, AM. et al.
Variability in the Generation of Induced Pluripotent Stem Cells - Importance for Disease Modeling.
Stem Cells Translational Medicine. 2012;1:641-650


STEM CELLS correspondent Stuart P Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.