Improved Cryopreservation of Human Induced Pluripotent Stem Cell (iPSC) and iPSC-derived Neurons Using Ice-Recrystallization Inhibitors.

Human induced pluripotent stem cells (iPSCs) and iPSC-derived neurons (iPSC-Ns) represent a differentiated modality toward developing novel cell-based therapies for regenerative medicine. However, the successful application of iPSC-Ns in cell-replacement therapies relies on effective cryopreservation. In this study, we investigated the role of ice recrystallization inhibitors (IRIs) as novel cryoprotectants for iPSCs and terminally differentiated iPSC-Ns. We found that one class of IRIs, N-aryl-D-aldonamides (specifically 2FA), increased iPSC post-thaw viability and recovery with no adverse effect on iPSC pluripotency. While 2FA supplementation did not significantly improve iPSC-N cell post-thaw viability, we observed that 2FA cryopreserved iPSC-Ns re-established robust neuronal network activity and synaptic function much earlier compared to CS10 cryopreserved controls. The 2FA cryopreserved iPSC-Ns retained expression of key neuronal specific and terminally differentiated markers and displayed functional electrophysiological and neuropharmacological responses following treatment with neuroactive agonists and antagonists. We demonstrate how optimizing cryopreservation media formulations with IRIs represents a promising strategy to improve functional cryopreservation of iPSCs and post-mitotic iPSC-Ns, the latter of which have been challenging to achieve. Developing IRI enabling technologies to support an effective cryopreservation and an efficiently managed cryo-chain is fundamental to support the delivery of successful iPSC-derived therapies to the clinic.

Intrinsic base substitution patterns in diverse species reveal links to cancer and metabolism.

Analyses of large-scale cancer sequencing data have revealed that mutagenic processes can create distinctive patterns of base substitutions, called mutational signatures. Interestingly, mutational patterns resembling some of these signatures can also be observed in normal cells. To determine whether similar patterns exist more generally, we analyzed large data sets of genetic variation, including mutations from 7 model species and single nucleotide polymorphisms in 42 species, totaling >1.9 billion variants. We found that base substitution patterns for most species closely match single base substitution (SBS) mutational signature 5 in the Catalog of Somatic Mutations in Cancer (COSMIC) database. SBS5 is ubiquitous in cancers and also present in normal human cells, suggesting that similar patterns of genetic variation across so many species are likely due to conserved biochemistry. We investigated the mechanistic origins of the SBS5-like mutational pattern in Saccharomyces cerevisiae, and show that translesion DNA synthesis and sugar metabolism are directly linked to this form of mutagenesis. We propose that conserved metabolic processes in cells are coupled to continuous generation of genetic variants, which can be acted upon by selection to drive the evolution of biological entities.

Analyses of mutational patterns induced by formaldehyde and acetaldehyde reveal similarity to a common mutational signature.

Formaldehyde and acetaldehyde are reactive small molecules produced endogenously in cells as well as being environmental contaminants. Both of these small aldehydes are classified as human carcinogens, since they are known to damage DNA and exposure is linked to cancer incidence. However, the mutagenic properties of formaldehyde and acetaldehyde remain incompletely understood, at least in part because they are relatively weak mutagens. Here, we use a highly sensitive yeast genetic reporter system featuring controlled generation of long single-stranded DNA regions to show that both small aldehydes induced mutational patterns characterized by predominantly C/G → A/T, C/G → T/A, and T/A → C/G substitutions, each in similar proportions. We observed an excess of C/G → A/T transversions when compared to mock-treated controls. Many of these C/G → A/T transversions occurred at TC/GA motifs. Interestingly, the formaldehyde mutational pattern resembles single base substitution signature 40 from the Catalog of Somatic Mutations in Cancer. Single base substitution signature 40 is a mutational signature of unknown etiology. We also noted that acetaldehyde treatment caused an excess of deletion events longer than 4 bases while formaldehyde did not. This latter result could be another distinguishing feature between the mutational patterns of these simple aldehydes. These findings shed new light on the characteristics of 2 important, commonly occurring mutagens.

Electrophysiological- and Neuropharmacological-Based Benchmarking of Human Induced Pluripotent Stem Cell-Derived and Primary Rodent Neurons.

Human induced pluripotent stem cell (iPSC)-derived neurons are of interest for studying neurological disease mechanisms, developing potential therapies and deepening our understanding of the human nervous system. However, compared to an extensive history of practice with primary rodent neuron cultures, human iPSC-neurons still require more robust characterization of expression of neuronal receptors and ion channels and functional and predictive pharmacological responses. In this study, we differentiated human amniotic fluid-derived iPSCs into a mixed population of neurons (AF-iNs). Functional assessments were performed by evaluating electrophysiological (patch-clamp) properties and the effect of a panel of neuropharmacological agents on spontaneous activity (multi-electrode arrays; MEAs). These electrophysiological data were benchmarked relative to commercially sourced human iPSC-derived neurons (CNS.4U from Ncardia), primary human neurons (ScienCell™) and primary rodent cortical/hippocampal neurons. Patch-clamp whole-cell recordings showed that mature AF-iNs generated repetitive firing of action potentials in response to depolarizations, similar to that of primary rodent cortical/hippocampal neurons, with nearly half of the neurons displaying spontaneous post-synaptic currents. Immunochemical and MEA-based analyses indicated that AF-iNs were composed of functional glutamatergic excitatory and inhibitory GABAergic neurons. Principal component analysis of MEA data indicated that human AF-iN and rat neurons exhibited distinct pharmacological and electrophysiological properties. Collectively, this study establishes a necessary prerequisite for AF-iNs as a human neuron culture model suitable for pharmacological studies.