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

Yu Tang,Tong Zang,Meng-Lu Liu,Chun-Li Zhang

doi:10.3389/fnmol.2017.00359

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.

Highlights

Motor neuron diseases (MNDs) are a heterogeneous group of neurologic disorders characterized by the selective loss of motor neurons (MNs) in spinal cord and motor cortex (Robberecht and Philips, 2013; Winner et al, 2014), including amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA)
Our results indicate that age-associated features are maintained in directly converted Fib-iMNs but not induced pluripotent stem cells (iPSCs)-MNs, suggesting that Fib-iMNs may be better suited to studying late-onset pathogenesis of motor neuron diseases (MNDs)
Three clones were successfully derived from each fibroblast line and displayed hallmarks of pluripotency as determined by Alkaline Phosphatase (AP) staining as well as markers for pluripotency such as OCT4, SOX2, and SSEA4 (Figures 1B,C). Quantitative real-time PCR (qRT-PCR) analysis using primers specific for retroviral transcripts revealed gradual silencing of all four retroviruses in established iPSCs (Figure 1D)

Summary

Introduction

Motor neuron diseases (MNDs) are a heterogeneous group of neurologic disorders characterized by the selective loss of motor neurons (MNs) in spinal cord and motor cortex (Robberecht and Philips, 2013; Winner et al, 2014), including amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). Since it is rarely possible to obtain diseased cells directly from patients, in vitro generation of MNs becomes an attractive approach for modeling and potentially treating MNDs. So far, two major cell reprogramming strategies have been applied to generate MNs. The usage of human induced pluripotent stem cells (iPSCs) derived from primary fibroblasts. IPSCs from patients with ALS harbor the patient’s complex genetic makeup, and can be subsequently differentiated toward MNs, albeit with varied conversion efficiencies (Chen et al, 2014; Kiskinis et al, 2014). Neurons can be directly reprogrammed in vitro from primary fibroblasts by introducing specific transcription factors or microRNAs (Masserdotti et al, 2016). We employed four transcription factors including NGN2, SOX11, ISL1, and LHX3 in combination with a cocktail of small molecules to yield functional Fib-iMNs from both human fetal and adult fibroblasts with high efficiencies (Liu et al, 2013, 2016)

Methods

Results

Conclusion