Browsing by Subject "Cellular Reprogramming"
Now showing 1 - 4 of 4
- Results Per Page
- Sort Options
Item Building a Methodological Framework for Cell Fate Engineering(August 2021) Li, Boxun; Xu, Jian; Hon, Gary C.; Banaszynski, Laura; Cleaver, Ondine; Munshi, NikhilCell fate engineering has become an area of intense research in the last fifteen years. A useful framework of cell fate engineering should include three pillars: the discovery of new cell fate-reprogramming cocktails of factors, the evaluation of engineered cells, and the revelation of underlying molecular mechanisms. One major challenge has been the lack of a scalable screening approach in vitro for the performance of reprogramming cocktails. This limits the speed of discovering new cocktails that can efficiently reprogram diverse cell types. Such new cocktails are needed to unleash the full applicational potential of engineered cells in regenerative medicine, disease modeling, and drug discovery. Another challenge is that despite the advantages of in vivo reprogramming, such as more efficient and mature fate conversion, the underlying gene programs, and thereby the molecular mechanisms, have been largely unknown. This is in large part due to the difficulty of specifically isolating and analyzing reprogrammed cells, without contamination from their endogenous counterparts. To address these, in this thesis, I first develop Reprogram-seq, a method that screens thousands of transcription factor cocktails for their reprogramming performance by single-cell perturbation screens. Reprogram-seq found a cocktail of three factors that efficiently and functionally reprograms fibroblasts to epicardial-like cells. Thus, Reprogram-seq accelerates rational cell fate engineering. Next, I performed single-cell transcriptomic analysis of in vivo neurogenesis induced in astrocytes by a novel reprogramming factor, DLX2. This is enabled by a lineage tracer that highly specifically tracks all cells reprogrammed from astrocytes. My analysis reveals that DLX2 induces a neural stem cell-like behavior, transitioning from quiescence to activation, proliferation, and neurogenesis. Gene regulatory network analysis and mouse genetics identify and confirm key nodes mediating DLX2-dependent fate reprogramming. Therefore, this study dissects the gene programs of in vivo reprogramming with single-cell transcriptomics and paves the way for applying Reprogram-seq in vivo. Together, my thesis research has demonstrated that single-cell omic technologies accelerate the discovery of new reprogramming cocktails, streamline the transcriptional evaluation of engineered cells, and dissect gene programs that underlie reprogramming, contributing to all three pillars of the framework. I expect these methodologies to be generalizable to and useful for other cell fate engineering scenarios.Item Molecular Dissection of Hand2 During the Formation of Pacemaker-Like Myocytes During Direct Reprogramming(2019-03-06) Fernandez-Perez, Antonio; Zhang, Chun-Li; Cleaver, Ondine; Olson, Eric N.; Munshi, NikhilDirect reprogramming of one cell type into another has great promise for regenerative medicine, disease modeling, and lineage specification. Currently, the conversion of fibroblasts into induced cardiomyocytes (iCM) by Gata4, Mef2c, and Tbx5 (GMT) represents an important avenue for generating de novo cardiac myocytes. Recent evidence has shown that iCM formation and diversity can be enhanced by the addition of Hand2 to GMT (GHMT). These four transcription factors give rise to a heterogenous CM population, consisting of atrial (iAM), ventricular (iVM), and pacemaker myocytes (iPM). However, the molecular mechanisms that drive this plastic fate conversion remain poorly understood. Although chromatin and single-cell studies in GMT-iCM have shown the existence of a set of temporal steps that orchestrate iCM formation, little is known about how Hand2 enhances this process. In the present study, we seek to characterize these Hand2-dependent mechanisms. We hypothesize that Hand2 regulates a discrete pacemaker regulatory network that becomes active during GHMT-iCM reprogramming. To test this, we compared the transcriptional and genomic profiles of fibroblasts, GMT, GHMT, and endogenous mouse Pacemaker cells. We observe similar chromatin landscape and gene expression profiles between Hand2-iPM and endogenous sinoatrial node (SAN), however several known key PM pathways are not active. Activation of these networks further enhances iCM-iPM fo Moreover, we show that Hand2 enhances chromatin accessibility in regions related to sarcomere function and electrical coupling, as well as promoting the closing of regions related to alternative fates. Utilizing integrative genomics between ATAC-seq and RNA-seq datasets, we identify the desmosome machinery as an important feature of iPM formation. In parallel, we define a novel Hand2 domain region that regulates cardiac subtype diversity. Taken together, our results showcase Hand2-dependent mechanisms for iPM formation and gives insight into the improvement of future iPM engineering.Item Molecular Regulation of Direct Cardiac Reprogramming(2017-08-14) Zhou, Huanyu; Zhang, Chun-Li; Hill, Joseph A.; Cleaver, Ondine; Olson, Eric N.A heart attack (also known as myocardial infarction, MI) happens when the flow of blood to the heart is blocked. A massive heart attack can kill billions of cardiomyocytes. The heart has limited regenerative potential because adult mammalian cardiomyocytes cannot proliferate, therefore lost cardiomyocytes cannot be replaced. This causes permanent heart damage and results in decreased contraction properties to a large portion of the heart muscle. Therapeutic treatments for heart attack patients have improved dramatically over the past decades. However, due to the inability to replenish lost cardiomyocytes, heart failure is still the primary cause of death in the world. Cardiac fibroblasts (CFs) constitute ~50% of the cells in the heart and form scar tissue following heart injury. Reprogramming CFs to induced-cardiomyocytes (iCMs) by forced expression of cardiac specific transcription factors holds promise for enhancing cardiac repair by reducing scar tissue while simultaneously generating new cardiomyocytes. However, low efficiency as well as a lack of understanding of molecular mechanism of the reprogramming process have significantly hampered its clinical application. The two goals of my PhD study were 1) to optimize the cardiac reprogramming protocol by increasing the efficiency; and 2) to decipher molecular mechanisms of cardiac reprogramming using the information obtained from the optimization process. To improve the efficiency of reprogramming fibroblasts to iCMs by cardiac transcription factors [Gata4, Hand2, Mef2c, and Tbx5 (GHMT)], we screened 192 protein kinases and discovered that Akt/protein kinase B dramatically accelerates and amplifies this process in three different types of fibroblasts (mouse embryo, adult cardiac, and tail tip). Approximately 50% of the reprogrammed mouse embryo fibroblasts displayed spontaneous beating after 3 weeks of induction by AKT1 plus GHMT (AGHMT). Furthermore, AGHMT evoked a more mature cardiac phenotype for iCMs, as seen by enhanced polynucleation, cellular hypertrophy, gene expression, and metabolic reprogramming. Insulin-like growth factor 1 (IGF1) and phosphoinositol 3-kinase (PI3K) acted upstream of AKT1 whereas the mitochondrial target of rapamycin complex 1 (mTORC1) and forkhead box o3a (Foxo3a) acted downstream of AKT1 to influence fibroblast-to-cardiomyocyte reprogramming. Addition of AGHMT converted 50% of mouse embryo fibroblasts to beating cardiomyocytes. However, only 1% of adult fibroblasts displayed spontaneous beating after three weeks of induction by AGHMT. This indicates that there are "barriers" in adult fibroblasts that hinder cardiac reprogramming. We continued to optimize methods for reprogramming fibroblasts to cardiomyocytes in vitro and in vivo. To identify additional regulators of this reprogramming process, we carried out an unbiased screen of ~1,100 open reading frames (ORFs) encoding transcription factors and cytokines for the ability to enhance reprogramming by AGHMT in adult tail-tip fibroblasts (ATTFs). One of the strongest activators of cardiac reprogramming was Krüppel-Type Zinc-Finger Transcription Factor 281 (ZNF281). Adding ZNF281 in AGHMT converted ~30% of ATTFs to iCMs which is comparable to AGHMT reprogrammed MEFs. We showed that ZNF281 enhanced cardiac reprogramming by associating with GATA4 on cardiac enhancers and by inhibiting inflammatory signaling, which antagonizes cardiac reprogramming. Our findings not only identify AKT1 and ZNF281 as robust and efficient activators of adult cardiac reprogramming, but also provide new insights into the molecular mechanisms underlying direct cardiac reprogramming.Item Small Molecules Modulate Chromatin Accessibility to Promote NEUROG2-Mediated Fibroblast-to-Neuron Reprogramming(2016-07-12) Smith, Derek Kurtis; Johnson, Jane E.; Kim, Tae-Kyung; Olson, Eric N.; Zhang, Chun-LiThe activity of pro-neural signaling molecules and transcription factors is sufficient to induce the transdifferentiation of lineage-restricted fibroblasts into functional neurons; however, a mechanistic model of the immediate-early events that catalyze this conversion has not been well defined. We utilized a high-efficiency reprogramming system of NEUROG2, forskolin (F), and dorsomorphin (D) to characterize the genetic and epigenetic events that initiate an acquisition of neuronal identity in fetal human fibroblasts. NEUROG2 immediately activates a neurogenic program, but is only sufficient to impart a functional identity in the presence of FD. These small molecules promote NEUROG2 and CREB1 co-transcription, induce SOX4 expression, and promote SOX4-dependent chromatin remodeling. Genome-wide occupancy analysis revealed that SOX4 targets numerous SWI/SNF complex subunits and co-binds with NEUROG2 to enhance the expression of diverse neurogenic factors. The overexpression of SWI/SNF chromatin remodeling factors or treatment with small molecules that modify chromatin accessibility enhanced NEUROG2-mediated neuronal reprogramming of adult human skin fibroblasts. This work represents the first comprehensive mechanism for the immediate events that catalyze neuronal transdifferentiation.