Systematic Identification of Barriers to Human iPSC Generation

Abstract

SUMMARY Reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) holds enormous promise for regenerative medicine, but the underlying mechanisms remain poorly understood. We report a systematic dissection of human cellular reprogramming by combining a genome-wide RNAi screen, innovative computational methods, extensive single-hit validation and mechanistic dissection of relevant pathways and networks. We identify novel reprogramming barriers, including genes involved in transcription, chromatin regulation, ubiquitination, dephosphorylation, vesicular transport and cell adhesion. Specific a disintegrin and metalloproteinase (ADAM) proteins are inhibitors of reprogramming, and the disintegrin domain of ADAM29 is necessary and sufficient for this function. Clathrin-mediated endocytosis can be targeted with small molecules and opposes reprogramming by positively regulating TGFβ signaling. Genetic interaction studies of endocytosis or ubiquitination reveal that barrier pathways can act in linear, parallel or feed-forward loop architectures to antagonize reprogramming. Our online resource summarizing these results provides a global view of barriers to human cellular reprogramming.

Coupling shRNA screens with single-cell RNA-seq identifies a dual role for mTOR in reprogramming-induced senescence

“…Multiple efforts have been made to identify the different barriers that inherently limit cellular reprogramming (Qin et al 2014;Sakurai et al 2014;Yang et al 2014). Senescence is one of these cell-intrinsic barriers (Banito et al 2009).…”

“…An inherent technical difficulty of working with primary cells is that once in culture, they eventually undergo replicative or stress-induced senescence (Kuilman et al 2010). To overcome this issue, genetic tricks to blunt senescence (for example, overexpressing hTERT or knocking down p53 expression) have sometimes been used to screen for genes limiting reprogramming (Qin et al 2014), indirectly highlighting that senescence constitutes an intrinsic cellular barrier to iPSC generation (Krizhanovsky and Lowe 2009;Banito and Gil 2010). Key tumor suppressors such as p53, p16 INK4a , or p21 CIP1 control the senescence response to OSKM, and their inhibition increases reprogramming (Banito et al 2009;Hong et al 2009;Kawamura et al 2009;Li et al 2009;Marion et al 2009;Utikal et al 2009).…”

“…The inefficiency of reprogramming has suggested the existence of several barriers impeding iPSC generation. The mechanisms underlying barriers to cellular reprogramming have been investigated previously using functional screens (Qin et al 2014;Sakurai et al 2014;Yang et al 2014;Cheloufi et al 2015). An inherent technical difficulty of working with primary cells is that once in culture, they eventually undergo replicative or stress-induced senescence (Kuilman et al 2010).…”

“…Given the physiological relevance of senescence and that it constitutes an intrinsic barrier for reprogramming, understanding how senescence is regulated during reprogramming is important. Genetic screening is a powerful tool that has been exploited with success to identify genes regulating senescence (Jacobs et al 2000;Rowland et al 2005;Acosta et al 2008;Tordella et al 2016;Wang et al 2016) or cellular reprogramming (Qin et al 2014;Sakurai et al 2014;Yang et al 2014).…”

MicroRNA-mediated regulation of extracellular matrix formation modulates somatic cell reprogramming

“…Despite this progress, there remains only limited information on the mechanisms by which the four transgenes and other cellular factors reprogram MEFs to an undifferentiated or ES-like state. Recent functional genomics studies have provided insight into essential and barrier pathways involved in iPSC generation during various steps of reprogramming (Qin et al 2014;Sakurai et al 2014;Yang et al 2014).…”