Direct Reprogramming Rather than iPSC-Based Reprogramming Maintains Aging Hallmarks in Human Motor Neurons

Abstract

In vitro generation of motor neurons (MNs) is a promising approach for modeling motor neuron diseases (MNDs) such as amyotrophic lateral sclerosis (ALS). As aging is a leading risk factor for the development of neurodegeneration, it is important to recapitulate age-related characteristics by using MNs at pathogenic ages. So far, cell reprogramming through induced pluripotent stem cells (iPSCs) and direct reprogramming from primary fibroblasts are two major strategies to obtain populations of MNs. While iPSC generation must go across the epigenetic landscape toward the pluripotent state, directly converted MNs might have the advantage of preserving aging-associated features from fibroblast donors. In this study, we confirmed that human iPSCs reset the aging status derived from their old donors, such as telomere attrition and cellular senescence. We then applied a set of transcription factors to induce MNs from either primary fibroblasts or iPSC-derived neural progenitor cells. The results revealed that directly reprogrammed MNs, rather than iPSC-derived MNs, maintained the aging hallmarks of old donors, including extensive DNA damage, loss of heterochromatin and nuclear organization, and increased SA-β-Gal activity. iPSC-derived MNs did not regain those aging memories from old donors. Collectively, our study indicates rejuvenation in the iPSC-based model, as well as aging maintenance in direct reprogramming of MNs. As such, the directly reprogrammed MNs may be more suitable for modeling the late-onset pathogenesis of MNDs.

Mitochondrial Aging Defects Emerge in Directly Reprogrammed Human Neurons due to Their Metabolic Profile

“…Several groups have previously shown that iPSC reprogramming results in clonal cell lines with rejuvenated mitochondria (Lapasset et al, 2011; Prigione et al, 2011; Suhr et al, 2010) and that iPSC-derived neurons lack age-related phenotypes in general (Huh et al, 2016; Mertens et al, 2015; Miller et al, 2013; Tang et al, 2017). To confirm these observations with regard to our readouts and our present cohort, we analyzed iPSC-derived neurons from three of our donors (29, 43, and 71 years of age; Figure S3A), and consistent with previous data, we observed no age-dependent transcriptional differences in mitochondrial or OXPHOS gene expression as determined by RNA sequencing (RNA-seq) (Figure S3B).…”

“…Therefore, to investigate and link the specific susceptibility of neurons to aging and mitochondrial dysfunction, a human neuronal model that integrates known and as-yet-unknown mitochondrial aging phenotypes would be desirable. Directly converted induced neurons (iNs) from old human donor-derived fibroblasts possess important features of cellular aging, including global transcriptomic changes, nuclear pore defects, and DNA methylation, rendering them a valuable tool for the study of age-related neurodegenerative diseases (Huh et al, 2016; Mertens et al, 2015; Tang et al, 2017). Here, we specifically assessed endogenous human age-dependent mitochondrial aging phenotypes in iNs using our previously established iN system (Mertens et al, 2015).…”

Next‐generation disease modeling with direct conversion: a new path to old neurons

“…We expect such criteria to become more and more popular, extending our knowledge of cell identity, and that they will eventually largely replace classical means of neuronal characterization. The rejuvenation effect of iPSC reprogramming is a major drawback when attempting to model late-onset diseases [17,157,158], and artificial induction of the factor age by overexpressing progerin [151], shortening of telomeres [159], or exposing cells to age-related stressors [12,160] is an upcoming and widely used strategy to elicit a relevant phenotype in iPSC models for neurodegenerative diseases [12,148,[161][162][163]. In vitro generation of patient-specific neurons from iPSCs for modeling diseases of the brain has evolved into an integral part of neuroscience [144][145][146], and employing human iPSC technology to investigate aspects of age-related neurodegenerative diseases in a patient-specific genetic context at a cellular level has yielded important human neuron-specific insights [147][148][149][150].…”

“…In vitro generation of patient-specific neurons from iPSCs for modeling diseases of the brain has evolved into an integral part of neuroscience [144][145][146], and employing human iPSC technology to investigate aspects of age-related neurodegenerative diseases in a patient-specific genetic context at a cellular level has yielded important human neuron-specific insights [147][148][149][150]. 2) [12,17,157]. 2) [10,[151][152][153][154][155][156].…”

“…Tang et al [157] have generated induced motor neurons, as well as iPSC-derived motor neurons from three young (0-3 years) and three old (53-71 years) healthy donors, as well as from four familial ALS patients carrying SOD1 or FUS mutations. Although it is a new concept, harnessing the power of combining iPSC differentiation and direct iN conversion has already been published.…”