Direct Conversion of Human Fibroblasts into Neural Progenitors Using Transcription Factors Enriched in Human ESC-Derived Neural Progenitors

摘要

class="head no_bottom_margin" id="sec1title">IntroductionCertain progressive, degenerative, and ultimately fatal, neurological disorders, such as Huntington's disease (HD) and Alzheimer's disease (AD), cannot be effectively treated; therefore, there remains a need to elucidate the pathological progress behind these disorders, and further effective clinical interventions (). By taking advantage of pluripotency reprogramming technology, researchers can readily reprogram disease-specific induced pluripotent stem cells (iPSCs) from patients' somatic cells, and subject them to in vitro differentiation for generation of various disease-relevant cell types for disease modeling and drug development (). However, tumorigenic and spontaneous differentiation of iPSCs remains a concern. In addition to iPSCs, induced neurons (iNs), which can be directly converted from fibroblasts (FBs) by defined transcription factors (TFs) (), provide another source of neuronal cells for in vitro disease modeling and drug testing. The advantages of iN technology are that it can provide a fast and simple method for the generation of specific neuronal subtypes, and its use may avoid certain problems, such as uncontrolled cell differentiation and tumor formation, which are associated with hiPSCs. However, the induction of each neuronal subtype requires different combinations of defined factors (), and the yield of such iNs is still too low for meaningful clinical applications. Therefore, developing strategies that allow direct conversion of somatic cells into expandable neural stem cell/progenitor (NSC/NP) populations that possess multiple neural differentiation potentials is an important step toward the generation of patient-specific neural cell types on a scalable level.Previously, it was demonstrated that induced NP (iNPs) can be directly converted from mouse somatic cells by overexpressing various TF combinations (, , ). first demonstrated that expandable iNPs could be generated from FBs via a modified pluripotency reprogramming procedure, and the resulting iNPs were able to differentiate into neurons and glial cells. Subsequently, several studies reported the generation of iNPs through the introduction of neural-enriched factors with/without iPSC factors (, , ), and the resulting iNPs were able to differentiate into all three major neural cell types of the CNS. Meanwhile, reports show that human iNPs can also be converted from somatic cells via the introduction of TFs (, , ). In these studies, several TF combinations, including at least one of the iPS factors, were used for hiNP generation (), and the differentiation propensity of the iNPs described in the aforementioned studies was mainly restricted to CNS neurons.hESCs can be used as an in vitro differentiation model to generate neural phenotypes of various developmental stages, including embryonic NPs (ENPs) populations, and the critical neural genetic factors that contribute to the neural fate acquisition have begun to be uncovered (href="#bib13" rid="bib13" class=" bibr popnode">Hou et al., 2013, href="#bib30" rid="bib30" class=" bibr popnode">Rosa and Brivanlou, 2011, href="#bib35" rid="bib35" class=" bibr popnode">Zhang et al., 2010). Given that hESC-ENP populations possess broad differentiation potential to give rise to both CNS and PNS neural cell types, it may be possible to directly convert FBs into iNPs resembling hESC-ENPs through the use of TFs highly expressed in the hESC-ENP population.Here, we identified a panel of neural TFs (nTFs) highly enriched in hESC-ENPs compared with FBs, through comparative gene expression profiling. We defined two TF combinations, the overexpression of which can efficiently convert human FBs into multipotent iENPs. The iENP populations generated in this manner resemble hESC-ENPs in many respects, including their pattern of proliferation, gene expression profile, and in vitro and in vivo differentiation propensity. Importantly, we found that different combinations of TFs can induce iENP populations with varying proliferative features and regional differentiation preferences. We also demonstrated that neurons derived from AD- and HD-iENPs, recapitulated the major disease pathological features in vitro. Taken together, our results point toward a promising and reproducible strategy for generating iENPs from somatic cells for disease modeling and future clinical intervention.