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Incomplete Transgene Silencing Identified as the Downfall of iPSCs

“Comparative Analysis of Targeted Differentiation of Human Induced Pluripotent Stem Cells (hiPSCs) and Human Embryonic Stem Cells Reveals Variability Associated With Incomplete Transgene Silencing in Retrovirally Derived hiPSC Lines”

From Stem Cells Translational Medicine

Multiple studies have been published on the comparison of human induced pluripotent stem cells (iPSCs) to human embryonic stem cells (hESCs) to analyse whether iPSCs are a suitable replacement for hESCs in a therapeutic context. However, many of these studies have focused on the undifferentiated phenotype, non-directed differentiation (embryoid bodies or teratomas), or their differentiation toward a single specific lineage (Boulting et alHu et alOsafune et al and Zhang et al). However now, in a study from the laboratory of Timo Otonkoski from the University of Helsinki, Finland, published in Stem Cells Translational Medicine, researchers have taken advantage of protocols optimized for endoderm, mesoderm, and ectoderm differentiation, to make a mass comparison of differentiation efficiency.   Interestingly, no systematic differences were observed, with the main difference arising in one cell line with incomplete transgene silencing (Toivonen, Ojala and Hyysalo et al).

hiPSCs lines utilised were either independently established using retroviral infection of OCT4, SOX2, c-MYC, and KLF4 (OSKM) or OCT4, SOX2, NANOG, and LIN28 (OSNL) (Hay et al, Hussein et al and Lahti et al) (hiPSC1, 2 and 4) or separately characterized (hiPSC3 and 5). Analysis of transgene silencing in each cell line found that apart from constant expression of exogenous KLF4 in hiPSC4, transgenes were silenced and were not induced by differentiation, except for the long-term RPE differentiation protocol in which all retrovirally derived hiPSC lines reactivated exogenous OCT4 mRNA expression. Additionally, RPE differentiation of hiPSC1 led to an increase in exogenous LIN28 and NANOG mRNA levels. However, hiPSC5, which was generated using a Sendai-virus mediated protocol, exhibited no increase in transgene expression during RPE differentiation.

Studies of definitive endoderm differentiation utilised a hepatocyte differentiation protocol (Hay et al). Initial stages direct cells to committed definitive endoderm (DE) cells and at this stage all cells analysed had a similar morphology and grew in homogenous monolayers and had upregulated SOX17 and HHEX and downregulated OCT4, a process slower in hiPSCs compared to hESCs. 90% of cells expressed FOXA2 and CXCR4+ cells made up between 65% and 96% between all the lines suggesting that the hESC and hiPSC lines used in this study differentiated into definitive endoderm stage with equal efficiency. Further differentiation towards hepatocyte-like cells (HLCs), led to a change of morphology from a spiky shape to a polygonal shape, at day 21 of differentiation all cultures contained foci exhibiting features of human hepatocytes and expressed AFP and Albumin, except for hiPSC4. Differences in differentiation, measured by Albumin expression and a secretion assay, were not significant between iPSCs and hESCs

Mesoderm differentiation was assessed through cardiomyocyte differentiation (Passier et al). All hiPSC and hESCs yielded beating cardiomyocytes although the efficiency was variable between cell line and on repeat of the protocol with the same lines, and these cells expressed α-actinin, Troponin T, connexin-43, and MHC in immunocytochemical stainings. Additionally, cardiomyocytes were deemed functional after assessment of electrical activity using a microelectrode array (MEA) platform. Overall, while hESC1 differentiation was most efficient, iPSC1 and 4 had the least efficient cardiac differentiation and the lowest number of beating areas. NANOG and OCT4 expression descended during differentiation, while SOX17 expression was highest at day 3. However, while BRA was also highest at day 3 for hESCs, iPSCs expressed BRA maximally at day 6, suggesting a slower differentiation rate.

Neural differentiation (ectoderm) as neurospheres demonstrated clear differences between cell lines; hiPSC3 formed unwanted cystic structures, hiPSC2 produced relatively fast-growing and less firm neurospheres while hiPSC4 neurosphere formation was weaker than that in the other lines. OCT4 downregulation was stronger for hESCs, as was the increase in neural precursor cell marker Musashi and neural marker NF-68, when compared to hiPSCs. Qualitative analysis found hESC3 and hiPSC3 to produce very pure neuronal populations at 8 weeks, whereas hiPSC4 was weakest for neural differentiation, and correlating to this, the highest levels of MAP-2-positive cells were detected within hESC3- and hiPSC3-derived cultures whereas in hiPSC4-derived cultures only single cells positive for MAP-2 could be detected. Finally RPE (ectoderm) differentiation was assessed (Vaajasaari et al) by monitoring the appearance of the first pigmented cells emerging in the cultures. hESC lines produced pigmented cells on average 2 days earlier than hiPSC lines (day 13 for hESCs), with the highest level observed in hESC3 and hiPSCS2 and the lowest in hESC1 and hiPSC1, although a groupwise comparison showed no significant difference in the efficiency. OCT4 decreased during differentiation but was higher for hiPSCs while the expression of differentiation markers (MITF, BEST1, and RLBP1) increased in all cell lines during differentiation, with hESCs expressing more MITF at day 52

Overall, while some hESCs displayed variable propensities for differentiation (hESCs1 and 3), none of the induced pluripotent stem cell (iPSC) lines showed such preferential differentiation capacity. Differences within the hiPSCs were only obvious for iPSC4 which did not silence KLF4 and during RPE differentiation where transgenes were reactivated; suggesting that retrovirally created iPSCs may be hampered in their differentiation propensities due to their transgenes, and also suggesting that iPSC technology moves towards other reprogramming techniques such as polycistronic minicircle vectors. PiggyBac transposons, and modified mRNA-based or protein transduction-based methods.

References

  • Boulting GL, et al. (2011) A functionally characterized test set of human induced pluripotent stem cells. Nat Biotechnol 29:279–286.
  • Hay DC, et al. (2008) Highly efficient differentiation of hESCs to functional hepatic endoderm requires ActivinA and Wnt3a signaling. Proc Natl Acad Sci USA 105:12301–12306.
  • Hu BY, et al. (2010) Neural differentiation of human induced pluripotent stem cells follows developmental principles but with variable potency. Proc Natl Acad Sci USA 107:4335–4340.
  • Hussein SM, et al. (2011) Copy number variation and selection during reprogramming to pluripotency. Nature 471:58–62.
  • Lahti AL, et al. (2012) Model for long QT syndrome type 2 using human iPS cells demonstrates arrhythmogenic characteristics in cell culture. Dis Model Mech 5:220–230.
  • Osafune K, et al. (2008) Marked differences in differentiation propensity among human embryonic stem cell lines. Nat Biotechnol 26:313–315.
  • Passier R, et al. (2005) Increased cardiomyocyte differentiation from human embryonic stem cells in serum-free cultures. Stem Cells 23:772–780.
  • Vaajasaari H, et al. (2011) Toward the defined and xeno-free differentiation of functional human pluripotent stem cell-derived retinal pigment epithelial cells. Mol Vis 17:558–575.
  • Zhang J, et al. (2009) Functional cardiomyocytes derived from human induced pluripotent stem cells. Circ Res 104:e30–e41.

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.