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Selection via pluripotency-related transcriptional screen minimizes the influence of somatic origin on iPSC differentiation propensity.

ABSTRACT: The value of induced pluripotent stem cells (iPSCs) within regenerative medicine is contingent on predictable and consistent iPSC differentiation. However, residual influence of the somatic origin or reprogramming technique may variegate differentiation propensity and confound comparative genotype/phenotype analyses. The objective of this study was to define quality control measures to select iPSC clones that minimize the influence of somatic origin on differentiation propensity independent of the reprogramming strategy. More than 60 murine iPSC lines were derived from different fibroblast origins (embryonic, cardiac, and tail tip) via lentiviral integration and doxycycline-induced transgene expression. Despite apparent equivalency according to established iPSC histologic and cytomorphologic criteria, clustering of clonal variability in pluripotency-related gene expression identified transcriptional outliers that highlighted cell lines with unpredictable cardiogenic propensity. Following selection according to a standardized gene expression profile calibrated by embryonic stem cells, the influence of somatic origin on iPSC methylation and transcriptional patterns was negated. Furthermore, doxycycline-induced iPSCs consistently demonstrated earlier differentiation than lentiviral-reprogrammed lines using contractile cardiac tissue as a measure of functional differentiation. Moreover, delayed cardiac differentiation was predominately associated with upregulation in pluripotency-related gene expression upon differentiation. Starting from a standardized pool of iPSCs, relative expression levels of two pluripotency genes, Oct4 and Zfp42, statistically correlated with enhanced cardiogenicity independent of somatic origin or reprogramming strategy (R(2) ?=?0.85). These studies demonstrate that predictable iPSC differentiation is independent of somatic origin with standardized gene expression selection criteria, while the residual impact of reprogramming strategy greatly influences predictable output of tissue-specification required for comparative genotype/phenotype analyses.

SUBMITTER: Hartjes KA

PROVIDER: S-EPMC4167634 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Selection via pluripotency-related transcriptional screen minimizes the influence of somatic origin on iPSC differentiation propensity.

Hartjes Katherine A KA Li Xing X Martinez-Fernandez Almudena A Roemmich Alexa J AJ Larsen Brandon T BT Terzic Andre A Nelson Timothy J TJ

Stem cells (Dayton, Ohio) 20140901 9

The value of induced pluripotent stem cells (iPSCs) within regenerative medicine is contingent on predictable and consistent iPSC differentiation. However, residual influence of the somatic origin or reprogramming technique may variegate differentiation propensity and confound comparative genotype/phenotype analyses. The objective of this study was to define quality control measures to select iPSC clones that minimize the influence of somatic origin on differentiation propensity independent of t ...[more]

PMID: 24802033

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Project description:BackgroundInduced pluripotent stem cells (iPSCs) offer potential to revolutionize regenerative medicine as a renewable source for islets, dopaminergic neurons, retinal cells, and cardiomyocytes. However, translation of these regenerative cell therapies requires cost-efficient mass manufacturing of high-quality human iPSCs. This study presents an improved three-dimensional Vertical-Wheel® bioreactor (3D suspension) cell expansion protocol with comparison to a two-dimensional (2D planar) protocol.MethodsSendai virus transfection of human peripheral blood mononuclear cells was used to establish mycoplasma and virus free iPSC lines without common genetic duplications or deletions. iPSCs were then expanded under 2D planar and 3D suspension culture conditions. We comparatively evaluated cell expansion capacity, genetic integrity, pluripotency phenotype, and in vitro and in vivo pluripotency potential of iPSCs.ResultsExpansion of iPSCs using Vertical-Wheel® bioreactors achieved 93.8-fold (IQR 30.2) growth compared to 19.1 (IQR 4.0) in 2D (p < 0.0022), the largest expansion potential reported to date over 5 days. 0.5 L Vertical-Wheel® bioreactors achieved similar expansion and further reduced iPSC production cost. 3D suspension expanded cells had increased proliferation, measured as Ki67+ expression using flow cytometry (3D: 69.4% [IQR 5.5%] vs. 2D: 57.4% [IQR 10.9%], p = 0.0022), and had a higher frequency of pluripotency marker (Oct4+Nanog+Sox2+) expression (3D: 94.3 [IQR 1.4] vs. 2D: 52.5% [IQR 5.6], p = 0.0079). q-PCR genetic analysis demonstrated a lack of duplications or deletions at the 8 most commonly mutated regions within iPSC lines after long-term passaging (> 25). 2D-cultured cells displayed a primed pluripotency phenotype, which transitioned to naïve after 3D-culture. Both 2D and 3D cells were capable of trilineage differentiation and following teratoma, 2D-expanded cells generated predominantly solid teratomas, while 3D-expanded cells produced more mature and predominantly cystic teratomas with lower Ki67+ expression within teratomas (3D: 16.7% [IQR 3.2%] vs.. 2D: 45.3% [IQR 3.0%], p = 0.002) in keeping with a naïve phenotype.ConclusionThis study demonstrates nearly 100-fold iPSC expansion over 5-days using our 3D suspension culture protocol in Vertical-Wheel® bioreactors, the largest cell growth reported to date. 3D expanded cells showed enhanced in vitro and in vivo pluripotency phenotype that may support more efficient scale-up strategies and safer clinical implementation.

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Selection via pluripotency-related transcriptional screen minimizes the influence of somatic origin on iPSC differentiation propensity.

Publications

Selection via pluripotency-related transcriptional screen minimizes the influence of somatic origin on iPSC differentiation propensity.

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