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Roy, Bibhas, Luezhen Yuan, Yaelim Lee, Aradhana Bharti, Aninda Mitra, and G. V. Shivashankar. "Fibroblast rejuvenation by mechanical reprogramming and redifferentiation." Proceedings of the National Academy of Sciences 117, no. 19 (April 29, 2020): 10131–41. http://dx.doi.org/10.1073/pnas.1911497117.
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Over the course of the aging process, fibroblasts lose contractility, leading to reduced connective-tissue stiffness. A promising therapeutic avenue for functional rejuvenation of connective tissue is reprogrammed fibroblast replacement, although major hurdles still remain. Toward this, we recently demonstrated that the laterally confined growth of fibroblasts on micropatterned substrates induces stem-cell-like spheroids. In this study, we embedded these partially reprogrammed spheroids in collagen-I matrices of varying densities, mimicking different three-dimensional (3D) tissue constraints. In response to such matrix constraints, these spheroids regained their fibroblastic properties and sprouted to form 3D connective-tissue networks. Interestingly, we found that these differentiated fibroblasts exhibit reduced DNA damage, enhanced cytoskeletal gene expression, and actomyosin contractility. In addition, the rejuvenated fibroblasts show increased matrix protein (fibronectin and laminin) deposition and collagen remodeling compared to the parental fibroblast tissue network. Furthermore, we show that the partially reprogrammed cells have comparatively open chromatin compaction states and may be more poised to redifferentiate into contractile fibroblasts in 3D-collagen matrix. Collectively, our results highlight efficient fibroblast rejuvenation through laterally confined reprogramming, which has important implications in regenerative medicine.
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Bektik, Emre, Yu Sun, Adrienne T. Dennis, Phraew Sakon, Dandan Yang, Isabelle Deschênes, and Ji-Dong Fu. "Inhibition of CREB-CBP Signaling Improves Fibroblast Plasticity for Direct Cardiac Reprogramming." Cells 10, no. 7 (June 22, 2021): 1572. http://dx.doi.org/10.3390/cells10071572.
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Direct cardiac reprogramming of fibroblasts into induced cardiomyocytes (iCMs) is a promising approach but remains a challenge in heart regeneration. Efforts have focused on improving the efficiency by understanding fundamental mechanisms. One major challenge is that the plasticity of cultured fibroblast varies batch to batch with unknown mechanisms. Here, we noticed a portion of in vitro cultured fibroblasts have been activated to differentiate into myofibroblasts, marked by the expression of αSMA, even in primary cell cultures. Both forskolin, which increases cAMP levels, and TGFβ inhibitor SB431542 can efficiently suppress myofibroblast differentiation of cultured fibroblasts. However, SB431542 improved but forskolin blocked iCM reprogramming of fibroblasts that were infected with retroviruses of Gata4, Mef2c, and Tbx5 (GMT). Moreover, inhibitors of cAMP downstream signaling pathways, PKA or CREB-CBP, significantly improved the efficiency of reprogramming. Consistently, inhibition of p38/MAPK, another upstream regulator of CREB-CBP, also improved reprogramming efficiency. We then investigated if inhibition of these signaling pathways in primary cultured fibroblasts could improve their plasticity for reprogramming and found that preconditioning of cultured fibroblasts with CREB-CBP inhibitor significantly improved the cellular plasticity of fibroblasts to be reprogrammed, yielding ~2-fold more iCMs than untreated control cells. In conclusion, suppression of CREB-CBP signaling improves fibroblast plasticity for direct cardiac reprogramming.
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Mueller, Lars, Michael D. Milsom, Kristina Brumme, Chad Harris, Kalindi Parmar, Kaya Zhu, London Wendy, et al. "Mechanisms of Resistance to Reprogramming of Cells Defective In the Fanconi Anemia DNA Repair Pathway." Blood 116, no. 21 (November 19, 2010): 196. http://dx.doi.org/10.1182/blood.v116.21.196.196.
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Abstract Abstract 196 Fanconi anemia (FA), the most common inherited bone marrow failure syndrome, is characterized by progressive loss of hematopoietic stem cells, aplastic anemia, genomic instability and cancer predisposition. Induced pluripotent stem (iPS) cells are a promising source of cells for disease-specific investigations and genetic correction. It has been reported that human FA dermal fibroblasts are resistant to direct reprogramming without prior genetic correction (Raya et al., Nature, 2009), but the mechanism remains unclear. In this study we aimed to define the role of the FA pathway during the transition from fibroblasts to iPS cells in murine cells. We transduced Fanca-/- and wild type (wt) tail-tip fibroblasts with four defined factors (Oct3/4, Klf4, Sox2, c-Myc) and enumerated the number of iPS colonies that were derived from 1×105 cells. We noted a >10-fold decrease in the reprogramming efficiency of Fanca-/- cells compared to wt controls [median (range): wt 132 (0 to 1296) colonies, efficiency 0.328%, n=17; Fanca-/- 4 (0 to 80) colonies, efficiency 0.019%, n=10, p<0.0001, Wilcoxon test]. Despite the markedly reduced reprogramming efficiency, Fanca-/- fibroblasts yielded iPS cells that expressed pluripotency markers (Oct3, Nanog, SSEA-1), gave rise to mature teratomas, and were able to generate chimeric mice. Given the defective DNA repair phenotype of FA cells, we compared baseline and reprogramming-induced DNA damage and senescence in wt and Fanca-/- cells. We observed that double strand DNA (dsDNA) breaks (γH2AX foci) and senescence were significantly increased in Fanca-/- cells four days following the transduction with the reprogramming viruses as compared to wt cells (p<0.0001 and p=0.0012, respectively; Table 1). Because reactive oxygen species (ROS) have been implicated as cellular mediators of DNA damage, senescence and genomic instability in FA cells, we measured the ROS levels during reprogramming and observed a 1.7-fold ROS induction in Fanca-/- fibroblasts. Addition of the ROS scavenger N-acetylcysteine (NAC, 100 μ M) reduced the number of dsDNA breaks in the Fanca-/- cells but failed to increase the reprogramming efficiency, possibly due to off-target toxicity. Therefore, to further assess the impact of oxidative DNA damage on the reprogramming of FA iPS cells, we compared the reprogramming efficiency of Fanca-/- and wt fibroblast that were derived concurrently in normoxic (21% O2) or hypoxic (5% O2) conditions. In both Fanca-/- and wt cells, we observed a significant increase of the reprogramming efficiency under hypoxic conditions (p=0.0098 and p=0.0462, respectively). Complementation of Fanca-/- fibroblasts with the FANCA gene in combination with reprogramming under hypoxic conditions led to a significant rescue of the reprogramming efficiency (p=0.0109, Table 2). This correlated with a significant reduction in senescence and a trend towards decreased dsDNA breaks. These data indicate that oxidative DNA damage engages the FA signaling pathway during the reprogramming process, and acts as a negative physiologic regulator of reprogramming in Fanca-/- cells. Our study implicates the FA pathway as essential to the repair of dsDNA breaks that are induced during the reprogramming process, and provides a plausible mechanism for the reduced efficiency of reprogramming in Fanca-/- cells.Table 1:Increased dsDNA breaks (γH2AX foci) and senescence (β-galactosidase) in Fanca-/- fibroblasts four days post infection with reprogramming viruses.Table 2:Comparison of the number of iPS colonies derived from 1×105 input fibroblasts at 5%O2.GenotypeNumber of Experiments (n)Median (range) number of iPS-like colonies per 1×105 input fibroblastsP-valueFA GFP1468 (0 to 488)0.0018WT GFP18494 (22 to 1812)FA GFP1468 (0 to 488)0.0109FA + FANCA15276 (12 to 1196)WT GFP18494 (22 to 1812)0.1514FA +FANCA15276 (12 to 1196) Disclosures: No relevant conflicts of interest to declare.
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Murry, Charles E., and William T. Pu. "Reprogramming Fibroblasts into Cardiomyocytes." New England Journal of Medicine 364, no. 2 (January 13, 2011): 177–78. http://dx.doi.org/10.1056/nejmcibr1013069.
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Whalley, Katherine. "Reprogramming fibroblasts to OPCs." Nature Reviews Neuroscience 14, no. 6 (May 9, 2013): 380. http://dx.doi.org/10.1038/nrn3512.
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Markov, Glenn J., Thach Mai, Surag Nair, Anna Shcherbina, Yu Xin Wang, David M. Burns, Anshul Kundaje, and Helen M. Blau. "AP-1 is a temporally regulated dual gatekeeper of reprogramming to pluripotency." Proceedings of the National Academy of Sciences 118, no. 23 (June 4, 2021): e2104841118. http://dx.doi.org/10.1073/pnas.2104841118.
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Somatic cell transcription factors are critical to maintaining cellular identity and constitute a barrier to human somatic cell reprogramming; yet a comprehensive understanding of the mechanism of action is lacking. To gain insight, we examined epigenome remodeling at the onset of human nuclear reprogramming by profiling human fibroblasts after fusion with murine embryonic stem cells (ESCs). By assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and chromatin immunoprecipitation sequencing we identified enrichment for the activator protein 1 (AP-1) transcription factor c-Jun at regions of early transient accessibility at fibroblast-specific enhancers. Expression of a dominant negative AP-1 mutant (dnAP-1) reduced accessibility and expression of fibroblast genes, overcoming the barrier to reprogramming. Remarkably, efficient reprogramming of human fibroblasts to induced pluripotent stem cells was achieved by transduction with vectors expressing SOX2, KLF4, and inducible dnAP-1, demonstrating that dnAP-1 can substitute for exogenous human OCT4. Mechanistically, we show that the AP-1 component c-Jun has two unexpected temporally distinct functions in human reprogramming: 1) to potentiate fibroblast enhancer accessibility and fibroblast-specific gene expression, and 2) to bind to and repress OCT4 as a complex with MBD3. Our findings highlight AP-1 as a previously unrecognized potent dual gatekeeper of the somatic cell state.
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Kwon, Erika M., John P. Connelly, Nancy F. Hansen, Frank X. Donovan, Thomas Winkler, Brian W. Davis, Halah Alkadi, et al. "iPSCs and fibroblast subclones from the same fibroblast population contain comparable levels of sequence variations." Proceedings of the National Academy of Sciences 114, no. 8 (February 6, 2017): 1964–69. http://dx.doi.org/10.1073/pnas.1616035114.
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Genome integrity of induced pluripotent stem cells (iPSCs) has been extensively studied in recent years, but it is still unclear whether iPSCs contain more genomic variations than cultured somatic cells. One important question is the origin of genomic variations detected in iPSCs–whether iPSC reprogramming induces such variations. Here, we undertook a unique approach by deriving fibroblast subclones and clonal iPSC lines from the same fibroblast population and applied next-generation sequencing to compare genomic variations in these lines. Targeted deep sequencing of parental fibroblasts revealed that most variants detected in clonal iPSCs and fibroblast subclones were rare variants inherited from the parental fibroblasts. Only a small number of variants remained undetectable in the parental fibroblasts, which were thus likely to be de novo. Importantly, the clonal iPSCs and fibroblast subclones contained comparable numbers of de novo variants. Collectively, our data suggest that iPSC reprogramming is not mutagenic.
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Bruzelius, Andreas, Srisaiyini Kidnapillai, Janelle Drouin-Ouellet, Tom Stoker, Roger A. Barker, and Daniella Rylander Ottosson. "Reprogramming Human Adult Fibroblasts into GABAergic Interneurons." Cells 10, no. 12 (December 8, 2021): 3450. http://dx.doi.org/10.3390/cells10123450.
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Direct reprogramming is an appealing strategy to generate neurons from a somatic cell by forced expression of transcription factors. The generated neurons can be used for both cell replacement strategies and disease modelling. Using this technique, previous studies have shown that γ-aminobutyric acid (GABA) expressing interneurons can be generated from different cell sources, such as glia cells or fetal fibroblasts. Nevertheless, the generation of neurons from adult human fibroblasts, an easily accessible cell source to obtain patient-derived neurons, has proved to be challenging due to the intrinsic blockade of neuronal commitment. In this paper, we used an optimized protocol for adult skin fibroblast reprogramming based on RE1 Silencing Transcription Factor (REST) inhibition together with a combination of GABAergic fate determinants to convert human adult skin fibroblasts into GABAergic neurons. Our results show a successful conversion in 25 days with upregulation of neuronal gene and protein expression levels. Moreover, we identified specific gene combinations that converted fibroblasts into neurons of a GABAergic interneuronal fate. Despite the well-known difficulty in converting adult fibroblasts into functional neurons in vitro, we could detect functional maturation in the induced neurons. GABAergic interneurons have relevance for cognitive impairments and brain disorders, such as Alzheimer’s and Parkinson’s diseases, epilepsy, schizophrenia and autism spectrum disorders.
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Zhou, Huanyu, Matthew E. Dickson, Min Soo Kim, Rhonda Bassel-Duby, and Eric N. Olson. "Akt1/protein kinase B enhances transcriptional reprogramming of fibroblasts to functional cardiomyocytes." Proceedings of the National Academy of Sciences 112, no. 38 (September 9, 2015): 11864–69. http://dx.doi.org/10.1073/pnas.1516237112.
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Conversion of fibroblasts to functional cardiomyocytes represents a potential approach for restoring cardiac function after myocardial injury, but the technique thus far has been slow and inefficient. To improve the efficiency of reprogramming fibroblasts to cardiac-like myocytes (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 reprogrammed mouse embryo fibroblasts displayed spontaneous beating after 3 wk of induction by Akt plus GHMT. Furthermore, addition of Akt1 to GHMT 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 Akt whereas the mitochondrial target of rapamycin complex 1 (mTORC1) and forkhead box o3 (Foxo3a) acted downstream of Akt to influence fibroblast-to-cardiomyocyte reprogramming. These findings provide insights into the molecular basis of cardiac reprogramming and represent an important step toward further application of this technique.
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Esseltine, Jessica L., Qing Shao, Tao Huang, John J. Kelly, Jacinda Sampson, and Dale W. Laird. "Manipulating Cx43 expression triggers gene reprogramming events in dermal fibroblasts from oculodentodigital dysplasia patients." Biochemical Journal 472, no. 1 (October 30, 2015): 55–69. http://dx.doi.org/10.1042/bj20150652.
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We have investigated gene reprogramming events in dermal fibroblasts from oculodentodigital dysplasia (ODDD) patients. Patient fibroblasts contain less functional Cx43 which results in changes in the production of extracellular matrix (ECM)-associated proteins, collagen-I secretion, gel contraction and overall fibroblast function.
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Avagliano, Angelica, Giuseppina Granato, Maria Rosaria Ruocco, Veronica Romano, Immacolata Belviso, Antonia Carfora, Stefania Montagnani, and Alessandro Arcucci. "Metabolic Reprogramming of Cancer Associated Fibroblasts: The Slavery of Stromal Fibroblasts." BioMed Research International 2018 (June 5, 2018): 1–12. http://dx.doi.org/10.1155/2018/6075403.
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Cancer associated fibroblasts (CAFs) are the main stromal cell type of solid tumour microenvironment and undergo an activation process associated with secretion of growth factors, cytokines, and paracrine interactions. One of the important features of solid tumours is the metabolic reprogramming that leads to changes of bioenergetics and biosynthesis in both tumour cells and CAFs. In particular, CAFs follow the evolution of tumour disease and acquire a catabolic phenotype: in tumour tissues, cancer cells and tumour microenvironment form a network where the crosstalk between cancer cells and CAFs is associated with cell metabolic reprogramming that contributes to CAFs activation, cancer growth, and progression and evasion from cancer therapies. In this regard, the study of CAFs metabolic reprogramming could contribute to better understand their activation process, the interaction between stroma, and cancer cells and could offer innovative tools for the development of new therapeutic strategies able to eradicate the protumorigenic activity of CAFs. Therefore, this review focuses on CAFs metabolic reprogramming associated with both differentiation process and cancer and stromal cells crosstalk. Finally, therapeutic responses and potential anticancer strategies targeting CAFs metabolic reprogramming are reviewed.
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Yagi, Masaki, Fei Ji, Jocelyn Charlton, Simona Cristea, Kathleen Messemer, Naftali Horwitz, Bruno Di Stefano, et al. "Dissecting dual roles of MyoD during lineage conversion to mature myocytes and myogenic stem cells." Genes & Development 35, no. 17-18 (August 19, 2021): 1209–28. http://dx.doi.org/10.1101/gad.348678.121.
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The generation of myotubes from fibroblasts upon forced MyoD expression is a classic example of transcription factor-induced reprogramming. We recently discovered that additional modulation of signaling pathways with small molecules facilitates reprogramming to more primitive induced myogenic progenitor cells (iMPCs). Here, we dissected the transcriptional and epigenetic dynamics of mouse fibroblasts undergoing reprogramming to either myotubes or iMPCs using a MyoD-inducible transgenic model. Induction of MyoD in fibroblasts combined with small molecules generated Pax7+ iMPCs with high similarity to primary muscle stem cells. Analysis of intermediate stages of iMPC induction revealed that extinction of the fibroblast program preceded induction of the stem cell program. Moreover, key stem cell genes gained chromatin accessibility prior to their transcriptional activation, and these regions exhibited a marked loss of DNA methylation dependent on the Tet enzymes. In contrast, myotube generation was associated with few methylation changes, incomplete and unstable reprogramming, and an insensitivity to Tet depletion. Finally, we showed that MyoD's ability to bind to unique bHLH targets was crucial for generating iMPCs but dispensable for generating myotubes. Collectively, our analyses elucidate the role of MyoD in myogenic reprogramming and derive general principles by which transcription factors and signaling pathways cooperate to rewire cell identity.
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Jo, Min-Sik, Hyun-Woo Yang, Joo-Hoo Park, Jae-Min Shin, and Il-Ho Park. "Glycolytic reprogramming is involved in tissue remodeling on chronic rhinosinusitis." PLOS ONE 18, no. 2 (February 16, 2023): e0281640. http://dx.doi.org/10.1371/journal.pone.0281640.
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Background Glycolytic reprogramming is a key feature of chronic inflammatory disease. Extracellular matrix (ECM) produced by myofibroblasts plays an important role in tissue remodeling of nasal mucosa in chronic rhinosinusitis (CRS). This study aimed to determine whether glycolytic reprogramming contributes to myofibroblast differentiation and ECM production in nasal fibroblasts. Methods Primary nasal fibroblasts were isolated from the nasal mucosa of patients with CRS. Glycolytic reprogramming was assessed by measuring the extracellular acidification and oxygen consumption rates in nasal fibroblast, with and without transforming growth factor beta 1 (TGF-β1) treatment. Expression of glycolytic enzymes and ECM components was measured by real-time polymerase chain reaction, western blotting, and immunocytochemical staining. Gene set enrichment analysis was performed using whole RNA-sequencing data of nasal mucosa of healthy donors and patients with CRS. Result Glycolysis of nasal fibroblasts stimulated with TGF-B1 was upregulated along with glycolytic enzymes. Hypoxia-inducing factor (HIF)-1α was a high-level regulator of glycolysis, and increased HIF-1α expression promoted glycolysis of nasal fibroblasts, and inhibition of HIF-1α down-regulated myofibroblasts differentiation and ECM production. Conclusion This study suggests that inhibition of the glycolytic enzyme and HIF-1α in nasal fibroblasts regulates myofibroblast differentiation and ECM generation associated with nasal mucosa remodeling.
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Zhang, Lianghui, Asrar B. Malik, and Jalees Rehman. "Reprogramming Fibroblasts to Endothelial Cells." Circulation 130, no. 14 (September 30, 2014): 1136–38. http://dx.doi.org/10.1161/circulationaha.114.012540.
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Choi, Da Hyeon, Kyeong Eun Lee, Jiwon Park, Yoon Jeong Park, Jue-Yeon Lee, and Yoon Shin Park. "Cell-Permeable Oct4 Gene Delivery Enhances Stem Cell-like Properties of Mouse Embryonic Fibroblasts." International Journal of Molecular Sciences 22, no. 17 (August 28, 2021): 9357. http://dx.doi.org/10.3390/ijms22179357.
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Direct conversion of one cell type into another is a trans-differentiation process. Recent advances in fibroblast research revealed that epithelial cells can give rise to fibroblasts by epithelial-mesenchymal transition. Conversely, fibroblasts can also give rise to epithelia by undergoing a mesenchymal to epithelial transition. To elicit stem cell-like properties in fibroblasts, the Oct4 transcription factor acts as a master transcriptional regulator for reprogramming somatic cells. Notably, the production of gene complexes with cell-permeable peptides, such as low-molecular-weight protamine (LMWP), was proposed to induce reprogramming without cytotoxicity and genomic mutation. We designed a complex with non-cytotoxic LMWP to prevent the degradation of Oct4 and revealed that the positively charged cell-permeable LMWP helped condense the size of the Oct4-LMWP complexes (1:5 N:P ratio). When the Oct4-LMWP complex was delivered into mouse embryonic fibroblasts (MEFs), stemness-related gene expression increased while fibroblast intrinsic properties decreased. We believe that the Oct4-LMWP complex developed in this study can be used to reprogram terminally differentiated somatic cells or convert them into stem cell-like cells without risk of cell death, improving the stemness level and stability of existing direct conversion techniques.
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Zhang, Zhentao, Jesse Villalpando, Wenhui Zhang, and Young-Jae Nam. "Chamber-Specific Protein Expression during Direct Cardiac Reprogramming." Cells 10, no. 6 (June 16, 2021): 1513. http://dx.doi.org/10.3390/cells10061513.
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Forced expression of core cardiogenic transcription factors can directly reprogram fibroblasts to induced cardiomyocyte-like cells (iCMs) in vitro and in vivo. This cardiac reprogramming approach provides a proof of concept for induced heart regeneration by converting a fibroblast fate to a cardiomyocyte fate. However, it remains elusive whether chamber-specific cardiomyocytes can be generated by cardiac reprogramming. Therefore, we assessed the ability of the cardiac reprogramming approach for chamber specification in vitro and in vivo. We found that in vivo cardiac reprogramming post-myocardial infarction exclusively induces a ventricular-like phenotype, while a major fraction of iCMs generated in vitro failed to determine their chamber identities. Our results suggest that in vivo cardiac reprogramming may have an inherent advantage of generating chamber-matched new cardiomyocytes as a potential heart regenerative approach.
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Heffernan, Corey, Huseyin Sumer, Luis F. Malaver-Ortega, and Paul J. Verma. "Temporal Requirements of cMyc Protein for Reprogramming Mouse Fibroblasts." Stem Cells International 2012 (2012): 1–12. http://dx.doi.org/10.1155/2012/541014.
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Exogenous expression of Oct4, Sox2, Klf4, and cMyc forces mammalian somatic cells to adopt molecular and phenotypic characteristics of embryonic stem cells, commencing with the required suppression of lineage-associated genes (e.g.,Thy1in mouse). Although omitting cMyc from the reprogramming cocktail minimizes risks of uncontrolled proliferation, its exclusion results in fold reductions in reprogramming efficiency. Thus, the feasibility of substituting cMyc transgene with (non-integrative) recombinant “pTAT-mcMyc” protein delivery was assessed, without compromising reprogramming efficiency or the pluripotent phenotype. Purification and delivery of semisoluble/particulate pTAT-mcMyc maintained Oct4-GFP+colony formation (i.e., reprogramming efficiency) whilst supporting pluripotency by various criteria. Differential repression of Thy1 by pTAT-mcMyc ± Oct4, Sox2, and Klf4 (OSK) suggested differential (and non-additive) mechanisms of repression. Extending these findings, attempts to enhance reprogramming efficiency through a staggered approach (prerepression of Thy1) failed to improve reprogramming efficiency. We consider protein delivery a useful tool to decipher temporal/molecular events characterizing somatic cell reprogramming.
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Pliatska, Maria, Maria Kapasa, Antonis Kokkalis, Alexander Polyzos, and Dimitris Thanos. "The Histone Variant MacroH2A Blocks Cellular Reprogramming by Inhibiting Mesenchymal-to-Epithelial Transition." Molecular and Cellular Biology 38, no. 10 (February 26, 2018): e00669-17. http://dx.doi.org/10.1128/mcb.00669-17.
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ABSTRACT Transcription factor-induced reprogramming of somatic cells to pluripotency is mediated via profound alterations in the epigenetic landscape. The histone variant macroH2A1 (mH2A1) is a barrier to the cellular reprogramming process. We demonstrate here that mH2A1 blocks reprogramming and contributes to the preservation of cell identity by trapping cells at the very early stages of the process, namely, at the mesenchymal-to-epithelial transition (MET). We provide a comprehensive analysis of the genomic sites occupied by the mH2A1 nucleosomes in human fibroblasts and embryonic stem (ES) cells and how they affect the reprogramming of fibroblasts to pluripotency. We have integrated chromatin immunoprecipitation sequencing (ChIP-seq) data with transcriptome sequencing (RNA-seq) data using cells containing reduced levels of mH2A1 and have inferred mH2A1-centered gene-regulatory networks that support the fibroblast and ES cell fates. We found that the exact positions of mH2A1 nucleosomes in regulatory regions of specific network genes with key regulatory roles guarantee the functional robustness of the regulatory networks. Using the reconstructed networks, we can predict and validate several components and their interactions in the establishment of stable cell types by limiting progression to alternative cell fates.
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Muchkaeva, I. A., E. B. Dashinimaev, A. S. Artyuhov, E. P. Myagkova, E. A. Vorotelyak, Y. Y. Yegorov, K. S. Vishnyakova, et al. "Generation of iPS Cells from Human Hair Follice Dermal Papilla Cells." Acta Naturae 6, no. 1 (March 15, 2014): 45–53. http://dx.doi.org/10.32607/20758251-2014-6-1-45-53.
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Dermal papilla (DP) cells are unique regional stem cells of the skin that induce formation of a hair follicle and its regeneration cycle. DP are multipotent stem cells; therefore we supposed that the efficiency of DPC reprogramming could exceed that of dermal fibroblasts reprogramming. We generated induced pluripotent stem cells from human DP cells using lentiviral transfection with Oct4, Sox2, Klf4, and c-Myc, and cultivation of cells both in a medium supplemented with valproic acid and at a physiological level of oxygen (5%). The efficiency of DP cells reprogramming was ~0.03%, while the efficiency of dermal fibroblast reprogramming under the same conditions was ~0.01%. Therefore, we demonstrated the suitability of DP cells as an alternative source of iPS cells.
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Behringer, Richard, Marina Gertsenstein, Kristina Vintersten Nagy, and Andras Nagy. "Reprogramming Mouse Fibroblasts with piggyBac Transposons." Cold Spring Harbor Protocols 2017, no. 10 (October 2017): pdb.prot092627. http://dx.doi.org/10.1101/pdb.prot092627.
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Ahlenius, Henrik, Soham Chanda, Ashley E. Webb, Issa Yousif, Jesse Karmazin, Stanley B. Prusiner, Anne Brunet, Thomas C. Südhof, and Marius Wernig. "FoxO3 regulates neuronal reprogramming of cells from postnatal and aging mice." Proceedings of the National Academy of Sciences 113, no. 30 (July 11, 2016): 8514–19. http://dx.doi.org/10.1073/pnas.1607079113.
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We and others have shown that embryonic and neonatal fibroblasts can be directly converted into induced neuronal (iN) cells with mature functional properties. Reprogramming of fibroblasts from adult and aged mice, however, has not yet been explored in detail. The ability to generate fully functional iN cells from aged organisms will be particularly important for in vitro modeling of diseases of old age. Here, we demonstrate production of functional iN cells from fibroblasts that were derived from mice close to the end of their lifespan. iN cells from aged mice had apparently normal active and passive neuronal membrane properties and formed abundant synaptic connections. The reprogramming efficiency gradually decreased with fibroblasts derived from embryonic and neonatal mice, but remained similar for fibroblasts from postnatal mice of all ages. Strikingly, overexpression of a transcription factor, forkhead box O3 (FoxO3), which is implicated in aging, blocked iN cell conversion of embryonic fibroblasts, whereas knockout or knockdown of FoxO3 increased the reprogramming efficiency of adult-derived but not of embryonic fibroblasts and also enhanced functional maturation of resulting iN cells. Hence, FoxO3 has a central role in the neuronal reprogramming susceptibility of cells, and the importance of FoxO3 appears to change during development.
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Wang, Li, Hong Ma, Peisen Huang, Yifang Xie, David Near, Haofei Wang, Jun Xu, et al. "Down-regulation of Beclin1 promotes direct cardiac reprogramming." Science Translational Medicine 12, no. 566 (October 21, 2020): eaay7856. http://dx.doi.org/10.1126/scitranslmed.aay7856.
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Direct reprogramming of fibroblasts to alternative cell fates by forced expression of transcription factors offers a platform to explore fundamental molecular events governing cell fate identity. The discovery and study of induced cardiomyocytes (iCMs) not only provides alternative therapeutic strategies for heart disease but also sheds lights on basic biology underlying CM fate determination. The iCM field has primarily focused on early transcriptome and epigenome repatterning, whereas little is known about how reprogramming iCMs remodel, erase, and exit the initial fibroblast lineage to acquire final cell identity. Here, we show that autophagy-related 5 (Atg5)–dependent autophagy, an evolutionarily conserved self-digestion process, was induced and required for iCM reprogramming. Unexpectedly, the autophagic factor Beclin1 (Becn1) was found to suppress iCM induction in an autophagy-independent manner. Depletion of Becn1 resulted in improved iCM induction from both murine and human fibroblasts. In a mouse genetic model, Becn1 haploinsufficiency further enhanced reprogramming factor–mediated heart function recovery and scar size reduction after myocardial infarction. Mechanistically, loss of Becn1 up-regulated Lef1 and down-regulated Wnt inhibitors, leading to activation of the canonical Wnt/β-catenin signaling pathway. In addition, Becn1 physically interacts with other classical class III phosphatidylinositol 3-kinase (PI3K III) complex components, the knockdown of which phenocopied Becn1 depletion in cardiac reprogramming. Collectively, our study revealed an inductive role of Atg5-dependent autophagy as well as a previously unrecognized autophagy-independent inhibitory function of Becn1 in iCM reprogramming.
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Suzuki, Yuichiro J., and Nataliia V. Shults. "Antioxidant Regulation of Cell Reprogramming." Antioxidants 8, no. 8 (August 20, 2019): 323. http://dx.doi.org/10.3390/antiox8080323.
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Discovery of induced pluripotent stem cells (iPSCs) has revolutionized regeneration biology, providing further mechanistic insights and possible therapeutic applications. The original discovery by Yamanaka and co-workers showed that the expression of four transcription factors in fibroblasts resulted in the generation of iPSCs that can be differentiated into various cell types. This technology should be particularly useful for restoring cells with limited proliferative capacities such as adult heart muscle cells and neurons, in order to treat diseases affecting these cell types. More recently, iPSCs-mediated cell reprogramming has advanced to new technologies including direct reprogramming and pharmacological reprogramming. Direct reprogramming allows for the conversion of fibroblasts into cardiomyocytes, neurons or other cells by expressing multiple cell type-specific transcription factors without going through the production of iPSCs. Both iPSC-mediated reprogramming as well as direct reprogramming can also be promoted by a combination of small molecules, opening up a possibility for pharmacological therapies to induce cell reprogramming. However, all of these processes have been shown to be affected by reactive oxygen species that reduce the efficacies of reprogramming fibroblasts into iPSCs, differentiating iPSCs into target cells, as well as direct reprogramming. Accordingly, antioxidants have been shown to support these reprogramming processes and this review article summarizes these findings. It should be noted however, that the actions of antioxidants to support cell reprogramming may be through their ROS inhibiting abilities, but could also be due to mechanisms that are independent of classical antioxidant actions.
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McMillan, M. E., A. Grace, N. Andronicos, G. Hinch, and S. Schmoelzl. "281 USE OF SMALL MOLECULES ENHANCES REPROGRAMMING SUCCESS IN BOVINE DERMAL FIBROBLASTS." Reproduction, Fertility and Development 25, no. 1 (2013): 288. http://dx.doi.org/10.1071/rdv25n1ab281.
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Reprogramming of differentiated cells to induced pluripotency holds tremendous potential for livestock reproduction. Here we report use of small molecules to support the generation of stem cell-like colonies from bovine dermal fibroblasts that show characteristics of pluripotent cells. Reprogramming of bovine somatic cells was undertaken by introducing canonical reprogramming factors Oct4, Sox2, Klf4, and c-Myc through a lentiviral drug-inducible polycistronic vector. Several small molecules were tested to support reprogramming. A combination of 3 small molecules (sodium butyrate, PD0325901, and SB431542; NaB-PD-SB) in iPS medium [Minimum Essential Medium Alpha, 20% fetal bovine serum, 1× insulin-transferrin-selenium (ITS), 2 mM Glutamax, 100 µM NEAA, 50 U mol–1 penicillin, 50 mg mL–1 streptomycin, 0.1 mM β-mercaptoethanol, 4 ng mL–1 human leukemia inhibitory factor, and 10 ng mL–1 basic fibroblast growth factor] was found to accelerate the kinetics of the reprogramming process. Colonies appeared at 12 days of culture compared with 21 days in iPS medium alone (P < 0.01, n = 5). Colonies in NaB-PD-SB iPS medium had consistent morphology, with small cells in compact dome formations with well-defined colony borders. Cells were not passaged but were either harvested for mRNA extraction or differentiated into embryoid bodies. Gene expression of the reprogramming factors in stem cell-like colonies was confirmed by qRT-PCR, and further gene expression analysis revealed activation of Nanog and ALPL mRNA expression (P < 0.01, n = 2). Embryoid bodies were analysed for gene expression by quantitative RT-PCR. Silencing of the transgene was not observed in embryoid bodies despite withdrawal of the inducer doxycycline. Expression of the endoderm marker FOXA2, ectoderm markers NES and TUBB3, and mesoderm marker DES was strongly increased in embryoid bodies compared with non-reprogrammed bovine fibroblasts (P < 0.01, n = 2), confirming expression of marker genes for the 3 germ layers.
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Hu, Kejin. "Quick, Coordinated and Authentic Reprogramming of Ribosome Biogenesis during iPSC Reprogramming." Cells 9, no. 11 (November 15, 2020): 2484. http://dx.doi.org/10.3390/cells9112484.
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Induction of pluripotent stem cells (iPSC) by OCT4 (octamer-binding transcription factor 4), SOX2 (SR box 2), KLF4 (Krüppel-Like Factor 4), and MYC (cellular Myelocytomatosis, c-MYC or MYC) (collectively OSKM) is revolutionary, but very inefficient, slow, and stochastic. It is unknown as to what underlies the potency aspect of the multi-step, multi-pathway, and inefficient iPSC reprogramming. Mesenchymal-to-epithelial (MET) transition is known as the earliest pathway reprogrammed. Using the recently established concepts of reprogramome and reprogramming legitimacy, the author first demonstrated that ribosome biogenesis (RB) is globally enriched in terms of human embryonic stem cells in comparison with fibroblasts, the popular starting cells of pluripotency reprogramming. It is then shown that the RB network was reprogrammed quickly in a coordinated fashion. Human iPSCs also demonstrated a more robust ribosome biogenesis. The quick and global reprogramming of ribosome biogenesis was also observed in an independent fibroblast line from a different donor. This study additionally demonstrated that MET did not initiate substantially at the time of proper RB reprogramming. This quick, coordinated and authentic RB reprogramming to the more robust pluripotent state by the OSKM reprogramming factors dramatically contrasts the overall low efficiency and long latency of iPSC reprogramming, and aligns well with the potency aspect of the inefficient OSKM reprogramming.
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Kim, N. H., M. R. Shin, and S. H. Park. "47BOVINE OOCYTE CYTOPLASM SUPPORTS NUCLEAR REMODELING BUT NOT REPROGRAMMING OF MURINE FIBROBLASTS." Reproduction, Fertility and Development 16, no. 2 (2004): 145. http://dx.doi.org/10.1071/rdv16n1ab47.
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Nuclear transfer (NT) is used to elucidate fundamental biological functions such as cell differentiation reversibility and interactions between the cytoplasm and nucleus. In the present study, we compared nuclear and microtubule dynamics in bovine oocytes following NT of bovine and murine fibroblast cells. To clarify the nuclear reprogramming procedures, we additionally examined the expression of development-related genes (Octamer-binding transcription factors, Oct-4; E-cadherin, E-cad) and housekeeping genes (Heat shock protein 70.1, Hsp; Bos taurus apoptosis regulator box-a, Bax; Glucose transporter 1, Glut-1) in bovine embryos that had received nuclei from bovine and murine fibroblast cells. Bovine oocytes were matured in vitro and enucleated after 22h. The oocytes reconstructed with mouse embryonic fibroblast cells or bovine somatic fibroblast cells were cultured in CR1aa media. While the embryos that received nuclei from bovine fibroblast cells developed into blastocysts, those that received nuclei from murine fibroblasts did not develop beyond the 8-cell stage. Similar nuclear and microtubule dynamics were observed in oocytes reconstructed with murine and bovine fibroblast cells. A small microtubule aster-containing γ-tubulin spot was observed in association with decondensed chromatin following NT of mouse fibroblasts. Within 1h of fusion of enucleated, non-activated cytoplasm, most mouse fibroblast nuclei were transformed to premature chromosome condensation (PCC). Randomly arrayed microtubules were tightly associated with PCC and formed meiotic-like microtubular spindles in all cases. Condensed chromosomes were divided into two or three chromatin masses and developed into multiple pronuclear-like structures. Microtubule asters were observed near the pronuclear-like structures during apposition in the cytoplasm. Two poles of the γ-tubulin spot evident at the mitotic metaphase stage are involved in the formation of the astral microtubule spindle for initial mitosis. A number of housekeeping mouse genes (hsp70, bax and glt-1) were abnormally expressed in embryos that had received nuclei from mouse fibroblast cells. However, development-related genes, such as Oct-4 and E-cad, were not expressed. The results collectively suggest that the bovine oocyte cytoplasm supports nuclear remodeling, but not reprogramming of mouse fibroblast cells. Table 1 Relative abundance of mRNA expression (mean±SEM) in mouse and xenonuclear-transferred (X-NT) embryos
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Ostrakhovitch, Elena A., Shin Akakura, and Siamak Tabibzadeh. "Hydrogen sulfide facilitates reprogramming and trans-differentiation in 3D dermal fibroblast." PLOS ONE 15, no. 11 (November 12, 2020): e0241685. http://dx.doi.org/10.1371/journal.pone.0241685.
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The efficiency of cell reprogramming in two-dimensional (2D) cultures is limited. Given that cellular stemness is intimately related to microenvironmental changes, 3D cell cultures have the potential of overcoming this limited capacity by allowing cells to self-organize by aggregation. In 3D space, cells interact more efficiently, modify their cellular topology, gene expression, signaling, and metabolism. It is yet not clear as how 3D culture environments modify the reprogramming potential of fibroblasts. We demonstrate that 3D spheroids from dermal fibroblasts formed under ultra-low attachment conditions showed increased lactate production. This is a requisite for cell reprogramming, increase their expression of pluripotency genes, such as OCT4, NANOG and SOX2, and display upregulated cystathionine-β-synthase (CBS) and hydrogen sulfide (H2S) production. Knockdown of CBS by RNAi suppresses lactic acid and H2S production and concomitantly decreases the expression of OCT4 and NANOG. On the contrary, H2S donors, NaHS and garlic-derived diallyl trisulfide (DATS), promote the expression of OCT4, and support osteogenic trans-differentiation of fibroblasts. These results demonstrate that CBS mediated release of H2S regulates the reprogramming of dermal fibroblasts grown in 3D cultures and supports their trans-differentiation.
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Ma, Yihe, Yumiao Lin, Wenting Huang, and Xusheng Wang. "Direct Reprograming of Mouse Fibroblasts into Dermal Papilla Cells via Small Molecules." International Journal of Molecular Sciences 23, no. 8 (April 11, 2022): 4213. http://dx.doi.org/10.3390/ijms23084213.
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The reprogramming of somatic fibroblasts into alternative cell linages could provide a promising source of cells for regenerative medicine and cell therapy. However, the direct conversion of fibroblasts into other functional cell types is still challenging. In this study, we show that dermal-papilla-cell-like cells (DPC-LCs) can be generated by treating fibroblasts, including L929 mouse fibroblast cell lines and somatic mouse fibroblasts, with small molecules. Based on alkaline phosphatase activity and other molecular markers, different compounds or their combinations are needed for converting the two different fibroblasts into DPC-LCs. Notably, we found that TTNPB alone can efficiently convert primary adult mouse fibroblasts into DPC-LCs. DPC-LCs generated from mouse fibroblasts showed a stronger hair-inducing capacity. Transcriptome analysis reveals that expression of genes associated with a hair-inducing capacity are increased in DPC-LCs. This pharmacological approach to generating functional dermal papilla cells may have many important implications for hair follicle regeneration and hair loss therapy.
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Arnold, Antje, Yahaira M. Naaldijk, Claire Fabian, Henry Wirth, Hans Binder, Guido Nikkhah, Lyle Armstrong, and Alexandra Stolzing. "Reprogramming of Human Huntington Fibroblasts Using mRNA." ISRN Cell Biology 2012 (December 7, 2012): 1–12. http://dx.doi.org/10.5402/2012/124878.
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The derivation of induced pluripotent stem cells (iPS) from human cell sources using transduction based on viral vectors has been reported by several laboratories. Viral vector-induced integration is a potential cause of genetic modification. We have derived iPS cells from human foreskin, adult Huntington fibroblasts, and adult skin fibroblasts of healthy donors using a nonviral and nonintegrating procedure based on mRNA transfer. In vitro transcribed mRNA for 5 factors, oct-4, nanog, klf-4, c-myc, sox-2 as well as for one new factor, hTERT, was used to induce pluripotency. Reprogramming was analyzed by qPCR analysis of pluripotency gene expression, differentiation, gene expression array, and teratoma assays. iPS cells were shown to express pluripotency markers and were able to differentiate towards ecto-, endo-, and mesodermal lineages. This method may represent a safer technology for reprogramming and derivation of iPS cells. Cells produced by this method can more easily be transferred into the clinical setting.
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Jaffer, Sajjida, Pollyanna Goh, Mahnaz Abbasian, and Amit C. Nathwani. "Mbd3 Promotes Reprogramming of Primary Human Fibroblasts." International Journal of Stem Cells 11, no. 2 (November 30, 2018): 235–41. http://dx.doi.org/10.15283/ijsc18036.
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Margariti, A., B. Winkler, E. Karamariti, T. Tsai, L. Zeng, Y. Hu, and Q. Xu. "20 Direct reprogramming fibroblasts into endothelial cells." Heart 97, no. 20 (September 23, 2011): e7-e7. http://dx.doi.org/10.1136/heartjnl-2011-300920b.20.
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Mazumdar, Alekhya, Joaquin Urdinez, Aleksandar Boro, Jessica Migliavacca, Matthias J. E. Arlt, Roman Muff, Bruno Fuchs, Jess Gerrit Snedeker, and Ana Gvozdenovic. "Osteosarcoma-Derived Extracellular Vesicles Induce Lung Fibroblast Reprogramming." International Journal of Molecular Sciences 21, no. 15 (July 30, 2020): 5451. http://dx.doi.org/10.3390/ijms21155451.
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Tumor-secreted extracellular vesicles (EVs) have been identified as mediators of cancer–host intercellular communication and shown to support pre-metastatic niche formation by modulating stromal cells at future metastatic sites. While osteosarcoma, the most common primary malignant bone tumor in children and adolescents, has a high propensity for pulmonary metastases, the interaction of osteosarcoma cells with resident lung cells remains poorly understood. Here, we deliver foundational in vitro evidence that osteosarcoma cell-derived EVs drive myofibroblast/cancer-associated fibroblast differentiation. Human lung fibroblasts displayed increased invasive competence, in addition to increased α-smooth muscle actin expression and fibronectin production upon EV treatment. Furthermore, we demonstrate, through the use of transforming growth factor beta receptor 1 (TGFBR1) inhibitors and CRISPR-Cas9-mediated knockouts, that TGFβ1 present in osteosarcoma cell-derived EVs is responsible for lung fibroblast differentiation. Overall, our study highlights osteosarcoma-derived EVs as novel regulators of lung fibroblast activation and provides mechanistic insight into how osteosarcoma cells can modulate distant cells to potentially support metastatic progression.
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Ghazizadeh, Z., H. Rassouli, H. Fonoudi, M. Alikhani, G. H. Salekdeh, N. Aghdami, and H. Baharvand. "Direct reprogramming of human fibroblasts to a cardiac fate using reprogramming proteins." Cytotherapy 16, no. 4 (April 2014): S39. http://dx.doi.org/10.1016/j.jcyt.2014.01.134.
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Liao, Yanling, Robert Bednarczyk, Shaun Latshaw, and Mitchell S. Cairo. "Reprogramming and Characterization of Cord Blood Derived Stem Cells by Synthetic mRNAs: Potential for Cord Blood Stem Cell Regenerative Therapy." Blood 120, no. 21 (November 16, 2012): 4748. http://dx.doi.org/10.1182/blood.v120.21.4748.4748.
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Abstract Abstract 4748 Background: Recent development of a synthetic mRNA-based technology for reprogramming somatic cells to pluripotency has overcome the challenge faced by DNA-based reprogramming methods (Warren L et al Cell stem cell 2010). This method involves a daily transfection of a five-factor cocktail including modified mRNAs encoding Klf4, cMyc, Oct4, Sox2, and Lin28 (KMOSL) for 17 days. Cord blood is a rich source for stem cells that could be novel sources for reprogramming (Liao/Cairo et al Exp Hem 2010). An important source of cord blood stem cells include unrestricted somatic stem cells (USSCs), which have been demonstrated to have multi-lineage differentiation abilities (Kögler G et al J. Exp Med 2004; Liao/Cairo, ASBMT 2010). Reprogramming cord blood derived USSCs have significant translational applications for a number of pediatric diseases. Goal: To determine the reprogramming efficiency of cord blood derived USSCs utilizing synthetic mRNAs. Method: USSCs were isolated from HUCB in the presence of 30% FBS and 10−7M dexamethasone, and characterized for their stem cell properties. USSCs were further sorted based on the expression of surface marker SSEA4 and reprogrammed using Stemgent mRNA reprogramming system, following daily transfection of KMOSL mRNA cocktail for 4 hours. At the end of each transfection, the culture was replaced by fresh Pluriton Medium supplemented with B18R protein. Human fibroblasts were reprogrammed in parallel as a control. Upon the appearance of iPS-like colonies, DyLight 488 TRA-1–60 antibody was added to the culture to identify iPS cells. The positively stained colonies were then manually picked and expanded in standard ES medium. Expression of ES factors and the formation of teratoma in vivo were used to confirm pluripotency of the derived cells. Results: USSCs share typical MSC cell surface markers and can be distinguished from MSCs based on their expression of d-like 1/preadipocyte factor 1 (DLK-1). 57.46% of USSCs (± 10.97%) displayed stage-specific embryonic antigen-4 (SSEA-4); however USSCs were negative for TRA-1–60. Approximately 33% (± 4.8%) of the USSCs were also stained positive for alkaline phosphatase activity. Moreover, compared to a complete absence of Oct4 and Nanog gene transcription in human fibroblasts, a minimal level of such pluripotency gene expression was detected in USSCs using isoform-specific and intron-spanning primers. Treatment of USSCs with 5-azaCytidine further led to a 10-fold increase in the expression of the Oct4 and Nanog genes in USSCs, but not in fibroblasts. Consistently, the upstream regulatory regions of both Oct4 and Nanog genes exhibited an intermediate level of DNA methylation in USSCs as compared to human ES and fibroblasts. These data suggest the plasticity of USSCs in reprogramming. SSEA4-positive USSCs were then used for daily transfection with mRNA KMOSL cocktail. The iPS-like colonies started to appear in the USSC reprogramming plates on day 11. TRA-1–60 live staining demonstrated that most of the colonies was stained positive for TRA-1–60 and the efficiency of iPS derivation from USSCs was about 0.2%. In comparison, the iPS-like colonies were observed in the fibroblast reprogramming plates on day 15–16 of transfection and the derivation efficiency was five-fold lower than that of USSCs. This demonstrated that USSCs can be reprogramming more efficiently than fibroblasts. The TRA-1–60 positive colonies were manually picked from the USSC reprogramming plates and expanded in ES medium. The colonies were also stained positive for Oct4, Nanog, Sox2 and TRA1–81. The in vivo teratoma assay is now under investigation. We are also investigating the reprogramming of USSCs using a single mRNA factor, in combination with small molecules, including PD0325901 and SB431542. Disclosures: No relevant conflicts of interest to declare.
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Salloum-Asfar, Salam, Sara A. Abdulla, Rowaida Z. Taha, I. Richard Thompson, and Mohamed M. Emara. "Combined Noncoding RNA-mRNA Regulomics Signature in Reprogramming and Pluripotency in iPSCs." Cells 11, no. 23 (November 29, 2022): 3833. http://dx.doi.org/10.3390/cells11233833.
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Somatic cells are reprogrammed with reprogramming factors to generate induced pluripotent stem cells (iPSCs), offering a promising future for disease modeling and treatment by overcoming the limitations of embryonic stem cells. However, this process remains inefficient since only a small percentage of transfected cells can undergo full reprogramming. Introducing miRNAs, such as miR-294 and miR302/3667, with reprogramming factors, has shown to increase iPSC colony formation. Previously, we identified five transcription factors, GBX2, NANOGP8, SP8, PEG3, and ZIC1, which may boost iPSC generation. In this study, we performed quantitative miRNAome and small RNA-seq sequencing and applied our previously identified transcriptome to identify the potential miRNA–mRNA regulomics and regulatory network of other ncRNAs. From each fibroblast (N = 4), three iPSC clones were examined (N = 12). iPSCs and original fibroblasts expressed miRNA clusters differently and miRNA clusters were compared to mRNA hits. Moreover, miRNA, piRNA, and snoRNAs expression profiles in iPSCs and original fibroblasts were assessed to identify the potential role of ncRNAs in enhancing iPSC generation, pluripotency, and differentiation. Decreased levels of let-7a-5p showed an increase of SP8 as described previously. Remarkably, the targets of identifier miRNAs were grouped into pluripotency canonical pathways, on stemness, cellular development, growth and proliferation, cellular assembly, and organization of iPSCs.
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Choi, K. H., D. Son, D. K. Lee, J. N. Oh, S. H. Kim, T. Y. Park, and C. K. Lee. "222 INCOMPLETE REPROGRAMMING OF INDUCED PLURIPOTENT STEM CELLS DERIVED FROM PORCINE FETAL FIBROBLASTS." Reproduction, Fertility and Development 28, no. 2 (2016): 242. http://dx.doi.org/10.1071/rdv28n2ab222.
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Cellular reprogramming of committed cells into a pluripotent state can be accomplished by ectopic expression of genes such as OCT4, SOX2, KLF4, and MYC. However, during reprogramming, it has been verified that failures of reactivating endogenous genes and epigenetic remodelling lead to partially reprogrammed cells exhibiting features similar to those of fully reprogrammed cells. In this study, partially reprogrammed induced pluripotent stem cells (pre-iPSC) were derived from porcine fetal fibroblasts via drug-inducible vector carrying human transcription factors (OCT4, SOX2, KLF4, and MYC). Therefore, this study aimed to investigate characteristics of pre-iPSC and reprogramming mechanisms. The pre-iPSC were stably maintained over an extended period having in vitro differentiation ability into 3 germ layers. The pluripotent state of pre-iPSC was regulated by modulation of culture condition. They showed naive- or primed-like pluripotent state in leukemia inhibitory factor (LIF) or basic fibroblast growth factor (bFGF) supplemented culture conditions respectively. However, pre-iPSC could not be maintained without ectopic expression of transgenes. The cultured pre-iPSC expressed endogenous transcription factors (OCT4 and SOX2) except for NANOG known as gateway into complete reprogramming. In addition, endogenous genes related to mesenchymal-to-epithelial transition (DPPA2, CDH1, EPCAM, and OCLN) were not sufficiently reactivated as measured by qPCR. DNA methylation analysis for promoters of OCT4, NANOG, and XIST showed that epigenetic reprogramming did not occurred in female pre-iPSC. Given the results, we found that expression of exogenous genes could not sufficiently activate the essential endogenous genes and remodel the epigenetic milieu for achieving faithful pluripotency in pig. Accordingly, investigating pre-iPSC could help us to improve and develop reprogramming methods via understanding reprogramming mechanisms in pig. This work was supported by the Next-generation BioGreen 21 Program (PJ0113002015), Rural Development Administration, Republic of Korea.
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Chen, Olivia, and Li Qian. "Direct Cardiac Reprogramming: Advances in Cardiac Regeneration." BioMed Research International 2015 (2015): 1–8. http://dx.doi.org/10.1155/2015/580406.
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Heart disease is one of the lead causes of death worldwide. Many forms of heart disease, including myocardial infarction and pressure-loading cardiomyopathies, result in irreversible cardiomyocyte death. Activated fibroblasts respond to cardiac injury by forming scar tissue, but ultimately this response fails to restore cardiac function. Unfortunately, the human heart has little regenerative ability and long-term outcomes following acute coronary events often include chronic and end-stage heart failure. Building upon years of research aimed at restoring functional cardiomyocytes, recent advances have been made in the direct reprogramming of fibroblasts toward a cardiomyocyte cell fate bothin vitroandin vivo. Several experiments show functional improvements in mouse models of myocardial infarction followingin situgeneration of cardiomyocyte-like cells from endogenous fibroblasts. Though many of these studies are in an early stage, this nascent technology holds promise for future applications in regenerative medicine. In this review, we discuss the history, progress, methods, challenges, and future directions of direct cardiac reprogramming.
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Kang, J. H., S. M. Park, S. Y. Heo, and H. Shim. "290 EFFECT OF EXOGENOUS Oct4 PROTEIN ON DIRECT CONVERSION OF HUMAN FIBROBLASTS INTO NEURAL STEM CELLS." Reproduction, Fertility and Development 25, no. 1 (2013): 292. http://dx.doi.org/10.1071/rdv25n1ab290.
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The generation of neural stem cells (NSC) from somatic cells may provide unlimited source of neuronal cells for autologous transplantation to patients of neurological disorders. Recently, direct conversion of fibroblasts into NSC by epigenetic reprogramming has been reported (Han et al. 2012 Cell Stem Cell 10, 465–472; Thier et al. 2012 Cell Stem Cell 10, 473–479; Ring et al. 2012 Cell Stem Cell 11, 100–109). These reprogrammed cells are referred to as induced neural stem cells (iNSC) and they share the characteristics of NSC in their morphology, molecular marker expressions, and capacity to differentiate into neurons, astrocytes, and oligodendrocytes. One of the procedures to convert fibroblasts into iNSC is restriction of Oct4 activity to the initial phase of reprogramming, while Sox2, Klf4, and c-Myc are constitutively expressed. In the present study, we examined the effect of Oct4 in reprogramming of human fibroblasts into iNSC. Oct4 protein was modified by the addition of poly-arginine protein transduction domain to easily penetrate into the cell membrane. We transduced Oct4 protein, in contrast to the previous reports where the Oct4 gene was virally introduced. First, human fibroblasts were transfected by retroviral vectors carrying the genes encoding Sox2, Klf4, and c-Myc. Then, transfected cells were cultured in ReNcell NSC maintenance medium containing Oct4 protein. After 4 days, Oct4 protein was removed from the medium. With Oct4 protein transduction, 21 flat colonies were formed from 4 × 105 fibroblasts. These colonies were picked and passaged for subculture and later became iNSC. However, in the absence of Oct4 protein, no colonies were obtained from the same number of fibroblasts that were initially plated. Approximately 40 days after transduction of reprogramming factors, cluster of iNSC were obtained. These cells expressed molecular markers of human NSC, including Nestin, Sox2, Pax6, and Blbp. Moreover, these iNSC could differentiate into neurons, astrocytes, and oligodendrocytes in vitro. Results of the present study demonstrate that transduction of exogenous Oct4 protein may be essential to the direct conversion of human fibroblasts into iNSC using a combination of reprogramming factors Sox2, Klf4, and c-Myc.
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Li, Zhenzhen, Chanjun Sun, and Zhihai Qin. "Metabolic reprogramming of cancer-associated fibroblasts and its effect on cancer cell reprogramming." Theranostics 11, no. 17 (2021): 8322–36. http://dx.doi.org/10.7150/thno.62378.
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Basma, Hesham, Yoko Gunji, Shunichiro Iwasawa, Amy Nelson, Maha Farid, Jun Ikari, Xiangde Liu, et al. "Reprogramming of COPD lung fibroblasts through formation of induced pluripotent stem cells." American Journal of Physiology-Lung Cellular and Molecular Physiology 306, no. 6 (March 15, 2014): L552—L565. http://dx.doi.org/10.1152/ajplung.00255.2013.
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Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) eliminates many epigenetic modifications that characterize differentiated cells. In this study, we tested whether functional differences between chronic obstructive pulmonary disease (COPD) and non-COPD fibroblasts could be reduced utilizing this approach. Primary fibroblasts from non-COPD and COPD patients were reprogrammed to iPSCs. Reprogrammed iPSCs were positive for oct3/4, nanog, and sox2, formed embryoid bodies in vitro, and induced teratomas in nonobese diabetic/severe combined immunodeficient mice. Reprogrammed iPSCs were then differentiated into fibroblasts (non-COPD-i and COPD-i) and were assessed either functionally by chemotaxis and gel contraction or for gene expression by microarrays and compared with their corresponding primary fibroblasts. Primary COPD fibroblasts contracted three-dimensional collagen gels and migrated toward fibronectin less robustly than non-COPD fibroblasts. In contrast, redifferentiated fibroblasts from iPSCs derived from the non-COPD and COPD fibroblasts were similar in response in both functional assays. Microarray analysis identified 1,881 genes that were differentially expressed between primary COPD and non-COPD fibroblasts, with 605 genes differing by more than twofold. After redifferentiation, 112 genes were differentially expressed between COPD-i and non-COPD-i with only three genes by more than twofold. Similar findings were observed with microRNA (miRNA) expression: 56 miRNAs were differentially expressed between non-COPD and COPD primary cells; after redifferentiation, only 3 miRNAs were differentially expressed between non-COPD-i and COPD-i fibroblasts. Interestingly, of the 605 genes that were differentially expressed between COPD and non-COPD fibroblasts, 293 genes were changed toward control after redifferentiation. In conclusion, functional and epigenetic alterations of COPD fibroblasts can be reprogrammed through formation of iPSCs.
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Martínez-Ordoñez, Anxo, Samuel Seoane, Leandro Avila, Noemi Eiro, Manuel Macía, Efigenia Arias, Fabio Pereira, et al. "POU1F1 transcription factor induces metabolic reprogramming and breast cancer progression via LDHA regulation." Oncogene 40, no. 15 (March 13, 2021): 2725–40. http://dx.doi.org/10.1038/s41388-021-01740-6.
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AbstractMetabolic reprogramming is considered hallmarks of cancer. Aerobic glycolysis in tumors cells has been well-known for almost a century, but specific factors that regulate lactate generation and the effects of lactate in both cancer cells and stroma are not yet well understood. In the present study using breast cancer cell lines, human primary cultures of breast tumors, and immune deficient murine models, we demonstrate that the POU1F1 transcription factor is functionally and clinically related to both metabolic reprogramming in breast cancer cells and fibroblasts activation. Mechanistically, we demonstrate that POU1F1 transcriptionally regulates the lactate dehydrogenase A (LDHA) gene. LDHA catalyzes pyruvate into lactate instead of leading into the tricarboxylic acid cycle. Lactate increases breast cancer cell proliferation, migration, and invasion. In addition, it activates normal-associated fibroblasts (NAFs) into cancer-associated fibroblasts (CAFs). Conversely, LDHA knockdown in breast cancer cells that overexpress POU1F1 decreases tumor volume and [18F]FDG uptake in tumor xenografts of mice. Clinically, POU1F1 and LDHA expression correlate with relapse- and metastasis-free survival. Our data indicate that POU1F1 induces a metabolic reprogramming through LDHA regulation in human breast tumor cells, modifying the phenotype of both cancer cells and fibroblasts to promote cancer progression.
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Raab, Stefanie, Moritz Klingenstein, Stefan Liebau, and Leonhard Linta. "A Comparative View on Human Somatic Cell Sources for iPSC Generation." Stem Cells International 2014 (2014): 1–12. http://dx.doi.org/10.1155/2014/768391.
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The breakthrough of reprogramming human somatic cells was achieved in 2006 by the work of Yamanaka and Takahashi. From this point, fibroblasts are the most commonly used primary somatic cell type for the generation of induced pluripotent stem cells (iPSCs). Various characteristics of fibroblasts supported their utilization for the groundbreaking experiments of iPSC generation. One major advantage is the high availability of fibroblasts which can be easily isolated from skin biopsies. Furthermore, their cultivation, propagation, and cryoconservation properties are uncomplicated with respect to nutritional requirements and viability in culture. However, the required skin biopsy remains an invasive approach, representing a major drawback for using fibroblasts as the starting material. More and more studies appeared over the last years, describing the reprogramming of other human somatic cell types. Cells isolated from blood samples or urine, as well as more unexpected cell types, like pancreatic islet beta cells, synovial cells, or mesenchymal stromal cells from wisdom teeth, show promising characteristics for a reprogramming strategy. Here, we want to highlight the advantages of keratinocytes from human plucked hair as a widely usable, noninvasive harvesting method for primary material in comparison with other commonly used cell types.
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Nagalingam, Raghu S., Hamza A. Safi, and Michael P. Czubryt. "Gaining myocytes or losing fibroblasts: Challenges in cardiac fibroblast reprogramming for infarct repair." Journal of Molecular and Cellular Cardiology 93 (April 2016): 108–14. http://dx.doi.org/10.1016/j.yjmcc.2015.11.029.
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Wang, Aline Yen Ling. "Application of Modified mRNA in Somatic Reprogramming to Pluripotency and Directed Conversion of Cell Fate." International Journal of Molecular Sciences 22, no. 15 (July 29, 2021): 8148. http://dx.doi.org/10.3390/ijms22158148.
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Modified mRNA (modRNA)-based somatic reprogramming is an effective and safe approach that overcomes the genomic mutation risk caused by viral integrative methods. It has improved the disadvantages of conventional mRNA and has better stability and immunogenicity. The modRNA molecules encoding multiple pluripotent factors have been applied successfully in reprogramming somatic cells such as fibroblasts, mesenchymal stem cells, and amniotic fluid stem cells to generate pluripotent stem cells (iPSCs). Moreover, it also can be directly used in the terminal differentiation of stem cells and fibroblasts into functional therapeutic cells, which exhibit great promise in disease modeling, drug screening, cell transplantation therapy, and regenerative medicine. In this review, we summarized the reprogramming applications of modified mRNA in iPSC generation and therapeutic applications of functionally differentiated cells.
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Capellera Garcia, Sandra, Kishori Dhulipala, Kavitha Siva, Violeta Rayon Estrada, Evelyn Wang, Gregory Hyde, Sofie Singbrant, et al. "Direct Lineage Reprogramming of Murine Fibroblasts to Erythroid Progenitor Cells By Defined Factors." Blood 124, no. 21 (December 6, 2014): 246. http://dx.doi.org/10.1182/blood.v124.21.246.246.
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Abstract Transcription factor-based direct lineage reprogramming is a powerful tool to discover and study factors determining cell lineage fate. We propose that this methodology can be used to define the core transcriptional program directing red blood cell development. The aim of this study was therefore to identify the minimal set of transcription factors that allows direct lineage reprogramming of murine fibroblasts to erythroid progenitor cells. A retrovirus library was created expressing the coding region of 63 transcription factors known to be involved in erythroid and blood development. Adult tail tip fibroblasts were obtained from erythroid lineage tracing mice, which express yellow fluorescent protein (YFP) in cells that have once expressed the erythropoietin receptor (EpoR) gene at any time of their development. Fibroblasts were depleted for hematopoietic lineage markers and passaged at least 3 times prior to transduction with different combinations of reprogramming factors. The readout for erythroid lineage conversion was formation of colonies of round YFP+ (EpoR+) cells, which were further subjected to extensive analyses to determine their resemblance with primary erythroid progenitor cells. Factor-subtraction experiments revealed a combination of 4 transcription factors, Gata1, Tal1, Lmo2 and c-Myc (collectively referred to as GTLM), capable of directly converting fibroblasts to erythroid progenitors in vitro. These induced erythroid progenitors (iEPs) emerged 5 to 8 days after transduction and displayed an erythroid progenitor-like morphology, featuring a characteristic central nucleus, coarse chromatin and deep blue cytoplasm after May Grünwald-Giemsa staining. When cultured in erythroid-promoting conditions, iEPs differentiated to Benzidine-positive normoblast-like cells. Flow cytometric analysis revealed that 28.9% ± 2.2 of live YFP+ cells collected at day 7 co-expressed CD71 and Ter119, and did not express CD45. Bulk GTLM-transduced fibroblasts formed two types of colonies in methylcellulose assays, one with visibly red hemoglobinized cells and one with blast-like cells. Global gene expression analyses showed GTLM expression was generally higher in the hemoglobinized colonies, suggesting that complete reprogramming only occurs when each factor is expressed at a sufficient level. Hierarchical clustering and pairwise comparisons of gene expression data showed that iEP-derived hemoglobinized colonies correlated tightly with definitive erythroid (BFU-E) colonies from bone marrow and fetal liver. As expected, iEP-derived hemoglobinized colonies displayed large-scale downregulation of the fibroblast-specific program and extensive upregulation of genes specific to the erythroid lineage. In contrast to definitive erythroid colonies, iEP-derived hemoglobinized colonies did not upregulate Sox6 and Bcl11a; and predominantly expressed embryonic hemoglobin, similar to primitive erythroid cells. This could mean that additional factors are required to induce definitive erythropoiesis, which we are currently investigating. Importantly, reprogramming was never successful using any combination of three of the GTLM factors, clearly demonstrating that all four factors are needed and that this is the minimal combination of required factors. Reprogramming of p53-null fibroblasts enhanced efficiency, but did not allow reprogramming without c-Myc. Additional experiments demonstrated that other factors known to be important for red cell development, including Klf1, Nfe2 and Myb, were not required and could not substitute for any of the 4 factors to induce erythroid fate. To our knowledge this is the first successful direct conversion of non-hematopoietic cells to the erythroid lineage. Our results suggest that GTLM constitute the core of the erythroid program, capable of inducing expression of other transcriptional regulators such as Klf1, Zfpm1, Gfi1b, Nfe2 and Myb, which are necessary for normal red cell development. We anticipate that GTLM-induced direct erythroid reprogramming can be used as a new platform for understanding, controlling and studying erythroid lineage development and disease. Furthermore, this knowledge could potentially be applied to enhance methods for in vitro production of erythrocytes for personalized transfusion medicine. Disclosures No relevant conflicts of interest to declare.
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Grace, A., M. McMillan, S. Schmoelzl, and G. Hinch. "187 INCREASED EFFICIENCY OF DERIVING BOVINE STEM CELL-LIKE COLONIES USING VALPROIC ACID AND SMALL-MOLECULE COCKTAILS." Reproduction, Fertility and Development 26, no. 1 (2014): 208. http://dx.doi.org/10.1071/rdv26n1ab187.
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Nonviral reprogramming of bovine embryonic and adult fibroblasts was undertaken using electroporation of a polycistronic plasmid carrying human reprogramming factors Oct4, Sox2, Lin28, and Nanog (Addgene Plasmid #20922). Because of difficulties encountered in reliably and reproducibly reprogramming bovine cells to a pluripotent state, several small-molecule combinations have been tested to support reprogramming. Previously, a combination of three molecules, (sodium butyrate, PD0325901 and SB431542; NaB-PD-SB) in induced pluripotent stem (iPS) medium [Minimum Essential Medium Alpha, 20% FBS, 1x insulin-transferrin-selenium (ITS), 2 mM Glutamax (Gibco, Grand Island, NY, USA), 100 μM nonessential amino acids (NEAA), 50 U mol–1 penicillin, 50 mg mL–1 streptomycin, 0.1 mM β-mercaptoethanol, 4 ng mL–1 human leukaemia inhibitory factor, and 10 ng mL–1 basic fibroblast growth factor] was found to accelerate the reprogramming process of bovine iPS cells, with colonies observed at 12 days post-electroporation, as opposed to 21 days without the addition of the small-molecule cocktail. The addition of 1 mM valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, in combination with the other small-molecule cocktail (NaB-PD-SB), was found to improve the reprogramming efficiency of bovine adult and embryonic fibroblasts, with colonies observed at Day 10 post-electroporation, and the average number of colonies present per 10-cm dish, rising from an average of 5 colonies observed in the NaB-PD-SB cocktail to 27 with the addition of VPA. Colonies grown in the presence of VPA had a consistent morphology, forming compact domed colonies consisting of small round cells with well-defined borders. It was observed that colonies growing in the presence of VPA tended to have a better defined border and grew larger in size than those grown in the presence of NaB-PD-SB alone. Colonies began to differentiate after 21 days and were no longer alkaline phosphatase positive after this time. Cells were either harvested for mRNA extraction or differentiated into embryoid bodies (EB). Expression of pluripotency genes, Oct4, Sox2, Klf4, Nanog, and Alkaline Phosphatase were significantly increased in the presence of VPA compared to nonreprogrammed somatic bovine fibroblasts, with expression profiles similar to those grown in the NaB-PD-SB cocktail. Embryoid bodies were analysed for gene expression of different germ layer markers, FOXA2 (endoderm), Nestin and TUBB3 (ectoderm), and Desmin (mesoderm) using quantitative RT–PCR. For both EBs derived from the NaB-PD-SB cocktail and those with VPA, at least one marker from each germ layer was present, demonstrating the potential potency of these cells. At least a 10-fold increase in expression of these was observed in comparison to somatic fibroblast cells. It is apparent that the addition of small molecules can assist in the reprogramming of bovine iPS cells, and addition of VPA to the cocktail results in more consistent putative bovine iPS colonies. Further work is needed to identify the causes for early differentiation of colonies in order to obtain fully reprogrammed pluripotent stem cells.
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Adams, Emma, Rachel McCloy, Ashley Jordan, Kaitlin Falconer, and Iain M. Dykes. "Direct Reprogramming of Cardiac Fibroblasts to Repair the Injured Heart." Journal of Cardiovascular Development and Disease 8, no. 7 (June 22, 2021): 72. http://dx.doi.org/10.3390/jcdd8070072.
Full textAbstract:
Coronary heart disease is a leading cause of mortality and morbidity. Those that survive acute myocardial infarction are at significant risk of subsequent heart failure due to fibrotic remodelling of the infarcted myocardium. By applying knowledge from the study of embryonic cardiovascular development, modern medicine offers hope for treatment of this condition through regeneration of the myocardium by direct reprogramming of fibrotic scar tissue. Here, we will review mechanisms of cell fate specification leading to the generation of cardiovascular cell types in the embryo and use this as a framework in which to understand direct reprogramming. Driving expression of a network of transcription factors, micro RNA or small molecule epigenetic modifiers can reverse epigenetic silencing, reverting differentiated cells to a state of induced pluripotency. The pluripotent state can be bypassed by direct reprogramming in which one differentiated cell type can be transdifferentiated into another. Transdifferentiating cardiac fibroblasts to cardiomyocytes requires a network of transcription factors similar to that observed in embryonic multipotent cardiac progenitors. There is some flexibility in the composition of this network. These studies raise the possibility that the failing heart could one day be regenerated by directly reprogramming cardiac fibroblasts within post-infarct scar tissue.
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Liu, Xiaodong, Jia Ping Tan, Jan Schröder, Asma Aberkane, John F. Ouyang, Monika Mohenska, Sue Mei Lim, et al. "Modelling human blastocysts by reprogramming fibroblasts into iBlastoids." Nature 591, no. 7851 (March 17, 2021): 627–32. http://dx.doi.org/10.1038/s41586-021-03372-y.
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Ricketts, Shea N., and Li Qian. "The heart of cardiac reprogramming: The cardiac fibroblasts." Journal of Molecular and Cellular Cardiology 172 (November 2022): 90–99. http://dx.doi.org/10.1016/j.yjmcc.2022.08.004.
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Sadahiro, Taketaro. "Direct Cardiac Reprogramming ― Converting Cardiac Fibroblasts to Cardiomyocytes ―." Circulation Reports 1, no. 12 (December 10, 2019): 564–67. http://dx.doi.org/10.1253/circrep.cr-19-0104.
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