Abstract Reprogramming of fibroblasts to cardiomyocytes offers exciting potential in cell therapy and regenerative medicine, but has low efficiency. We hypothesize that physical cues may positively ...affect the reprogramming process, and studied the effects of periodic mechanical stretch, substrate stiffness and microgrooved substrate on reprogramming yield. Subjecting reprogramming fibroblasts to periodic mechanical stretch and different substrate stiffness did not improve reprogramming yield. On the other hand, culturing the cells on microgrooved substrate enhanced the expression of cardiomyocyte genes by day 2 and improved the yield of partially reprogrammed cells at day 10. By combining microgrooved substrate with an existing optimized culture protocol, yield of reprogrammed cardiomyocytes with striated cardiac troponin T staining and spontaneous contractile activity was increased. We identified the regulation of Mkl1 activity as a new mechanism by which microgroove can affect reprogramming. Biochemical approach could only partially recapitulate the effect of microgroove. Microgroove demonstrated an additional effect of enhancing organization of sarcomeric structure, which could not be recapitulated by biochemical approach. This study provides insights into new mechanisms by which topographical cues can affect cellular reprogramming.
Here, we show that as human embryonic stem cells (ESCs) exit the pluripotent state,
NANOG can play a key role in determining lineage outcome. It has previously been reported that BMPs induce ...differentiation of human ESCs into extraembryonic lineages. Here, we find that FGF2, acting through the MEK-ERK pathway, switches BMP4-induced human ESC differentiation outcome to mesendoderm, characterized by the uniform expression of
T (brachyury) and other primitive streak markers. We also find that MEK-ERK signaling prolongs
NANOG expression during BMP-induced differentiation, that forced
NANOG expression results in FGF-independent BMP4 induction of mesendoderm, and that knockdown of
NANOG greatly reduces
T induction. Together, our results demonstrate that FGF2 signaling switches the outcome of BMP4-induced differentiation of human ESCs by maintaining
NANOG levels through the MEK-ERK pathway.
► FGF2 switches BMP4-induced differentiation outcome to mesendoderm ► Blocking the MEK-ERK blocks the FGF2-mediated lineage switch ► MEK-ERK signaling prolongs
NANOG expression during BMP4-induced differentiation ► Forced
NANOG expression results in FGF2-independent BMP4 induction of mesendoderm
BACKGROUND:Reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells in situ represents a promising strategy for cardiac regeneration. A combination of 3 cardiac transcription ...factors, Gata4, Mef2c, and Tbx5 (GMT), can convert fibroblasts into induced cardiomyocyte-like cells, albeit with low efficiency in vitro.
METHODS:We screened 5500 compounds in primary cardiac fibroblasts to identify the pathways that can be modulated to enhance cardiomyocyte reprogramming.
RESULTS:We found that a combination of the transforming growth factor-β inhibitor SB431542 and the WNT inhibitor XAV939 increased reprogramming efficiency 8-fold when added to GMT-overexpressing cardiac fibroblasts. The small molecules also enhanced the speed and quality of cell conversion; we observed beating cells as early as 1 week after reprogramming compared with 6 to 8 weeks with GMT alone. In vivo, mice exposed to GMT, SB431542, and XAV939 for 2 weeks after myocardial infarction showed significantly improved reprogramming and cardiac function compared with those exposed to only GMT. Human cardiac reprogramming was similarly enhanced on transforming growth factor-β and WNT inhibition and was achieved most efficiently with GMT plus myocardin.
CONCLUSIONS:Transforming growth factor-β and WNT inhibitors jointly enhance GMT-induced direct cardiac reprogramming from cardiac fibroblasts in vitro and in vivo and provide a more robust platform for cardiac regeneration.
Ectopic expression of combinations of transcription factors (TFs) can drive direct lineage conversion, thereby reprogramming a somatic cell’s identity. To determine the molecular mechanisms by which ...Gata4, Mef2c, and Tbx5 (GMT) induce conversion from a cardiac fibroblast toward an induced cardiomyocyte, we performed comprehensive transcriptomic, DNA-occupancy, and epigenomic interrogation throughout the reprogramming process. Integration of these datasets identified new TFs involved in cardiac reprogramming and revealed context-specific roles for GMT, including the ability of Mef2c and Tbx5 to independently promote chromatin remodeling at previously inaccessible sites. We also find evidence for cooperative facilitation and refinement of each TF’s binding profile in a combinatorial setting. A reporter assay employing newly defined regulatory elements confirmed that binding of a single TF can be sufficient for gene activation, suggesting that co-binding events do not necessarily reflect synergy. These results shed light on fundamental mechanisms by which combinations of TFs direct lineage conversion.
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•Integrated scRNA-, ATAC-, and ChIP-seq analyses to interrogate cardiac reprogramming•Gata4, Mef2c, and Tbx5 both facilitate and limit one another's ability to bind to DNA•Mef2c and Tbx5 bind to inaccessible chromatin and promote its remodeling•Context-specific cooperative mechanisms guide cardiac reprogramming
Srivastava and colleagues integrate multiple (epi)genomic assays to dissect the mechanisms by which transcription factors function independently and combinatorially to initiate cell fate transitions. Heterogeneous binding relationships between Gata4, Mef2c, and Tbx5 highlight the context-specific mechanisms that dictate cardiac reprogramming.
Fibroblast growth factor (FGF), transforming growth factor (TGF)/Nodal, and Insulin/insulin‐like growth factor (IGF) signaling pathways are sufficient to maintain human embryonic stem cells (ESCs) ...and induced pluripotent stem cells in a proliferative, undifferentiated state. Here, we show that only a few FGF family members (FGF2, FGF4, FGF6, and FGF9) are able to sustain strong extracellular‐signal‐regulated kinase (ERK) phosphorylation and NANOG expression levels in human ESCs. Surprisingly, FGF1, which is reported to target the same set of receptors as FGF2, fails to sustain ERK phosphorylation and NANOG expression under standard culture conditions. We find that the failure of FGF1 to sustain ES is due to thermal instability of the wild‐type protein, not receptor specificity, and that a mutated thermal‐stable FGF1 sustains human ESCs and supports both differentiation and reprogramming protocols. STEM CELLS 2012; 30:623–630
Phosphorylation regulates human OCT4 Brumbaugh, Justin; Hou, Zhonggang; Russell, Jason D ...
Proceedings of the National Academy of Sciences - PNAS,
05/2012, Letnik:
109, Številka:
19
Journal Article
Recenzirano
Odprti dostop
The transcription factor OCT4 is fundamental to maintaining pluripotency and self-renewal. To better understand protein-level regulation of OCT4, we applied liquid chromatography–MS to identify 14 ...localized sites of phosphorylation, 11 of which were previously unknown. Functional analysis of two sites, T234 and S235, suggested that phosphorylation within the homeobox region of OCT4 negatively regulates its activity by interrupting sequence-specific DNA binding. Mutating T234 and S235 to mimic constitutive phosphorylation at these sites reduces transcriptional activation from an OCT4-responsive reporter and decreases reprogramming efficiency. We also cataloged 144 unique phosphopeptides on known OCT4 interacting partners, including SOX2 and SALL4, that copurified during immunoprecipitation. These proteins were enriched for phosphorylation at motifs associated with ERK signaling. Likewise, OCT4 harbored several putative ERK phosphorylation sites. Kinase assays confirmed that ERK2 phosphorylated these sites in vitro, providing a direct link between ERK signaling and the transcriptional machinery that governs pluripotency.
The treatment of myocardial infarction is a major challenge in medicine due to the inability of heart tissue to regenerate. Direct reprogramming of endogenous cardiac fibroblasts into functional ...cardiomyocytes via the delivery of transcription factor mRNAs has the potential to regenerate cardiac tissue and to treat heart failure. Even though mRNA delivery to cardiac fibroblasts has the therapeutic potential, mRNA transfection in cardiac fibroblasts has been challenging. Herein, we develop an efficient mRNA transfection in cultured mouse cardiac fibroblasts via a polyarginine-fused heart-targeting peptide and lipofectamine complex, termed C-Lipo and demonstrate the partial direct reprogramming of cardiac fibroblasts towards cardiomyocyte cells. C-Lipo enabled the mRNA-induced direct cardiac reprogramming due to its efficient transfection with low toxicity, which allowed for multiple transfections of Gata4, Mef2c, and Tbx5 (GMT) mRNAs for a period of 2 weeks. The induced cardiomyocyte-like cells had α-MHC promoter-driven GFP expression and striated cardiac muscle structure from α-actinin immunohistochemistry. GMT mRNA transfection of cultured mouse cardiac fibroblasts via C-Lipo significantly increased expression of the cardiomyocyte marker genes, Actc1, Actn2, Gja1, Hand2, and Tnnt2, after 2 weeks of transfection. Moreover, this study provides the first direct evidence that the stoichiometry of the GMT reprogramming factors influence the expression of cardiomyocyte marker genes. Our results demonstrate that mRNA delivery is a potential approach for cardiomyocyte generation.
We have developed and implemented a sequence identification algorithm (inSeq) that processes tandem mass spectra in real-time using the mass spectrometer's (MS) onboard processors. The inSeq ...algorithm relies on accurate mass tandem MS data for swift spectral matching with high accuracy. The instant spectral processing technology takes ~16 ms to execute and provides information to enable autonomous, real-time decision making by the MS system. Using inSeq and its advanced decision tree logic, we demonstrate (i) real-time prediction of peptide elution windows en masse (~3 min width, 3,000 targets), (ii) significant improvement of quantitative precision and accuracy (~3x boost in detected protein differences), and (iii) boosted rates of posttranslation modification site localization (90% agreement in real-time vs. offline localization rate and an approximate 25% gain in localized sites). The decision tree logic enabled by inSeq promises to circumvent problems with the conventional data-dependent acquisition paradigm and provides a direct route to streamlined and expedient targeted protein analysis.
Pluripotency, the ability of a cell to differentiate and give rise to all embryonic lineages, defines a small number of mammalian cell types such as embryonic stem （ES） cells. While it has been ...generally held that pluripotency is the product of a transcriptional regulatory network that activates and maintains the expression of key stem cell genes, accumulating evidence is pointing to a critical role for epigenetic processes in establishing and safeguarding the pluri-potency of ES cells, as well as maintaining the identity of differentiated cell types. In order to better understand the role of epigenetic mechanisms in pluripotency, we have examined the dynamics of chromatin modifications genome- wide in human ES cells （hESCs） undergoing differentiation into a mesendodermal lineage. We found that chromatin modifications at promoters remain largely invariant during differentiation, except at a small number of promoters where a dynamic switch between acetylation and methylation at H3K27 marks the transition between activation and silencing of gene expression, suggesting a hierarchy in cell fate commitment over most differentially expressed genes. We also mapped over 50 000 potential enhancers, and observed much greater dynamics in chromatin modifications, especially H3K4mel and H3K27ac, which correlate with expression of their potential target genes. Further analysis of these enhancers revealed potentially key transcriptional regulators of pluripotency and a chromatin signature indicative of a poised state that may confer developmental competence in hESCs. Our results provide new evidence supporting the role of chromatin modifications in defining enhancers and plnripotency.
NANOG is a divergent homeobox protein and a core component of the transcriptional circuitry that sustains pluripotency and self-renewal. Although NANOG has been extensively studied on the ...transcriptional level, little is known regarding its posttranslational regulation, likely due to its low abundance and challenging physical properties. Here, we identify eleven phosphorylation sites on endogenous human NANOG, nine of which mapped to single amino acids. To screen for the signaling molecules that impart these modifications, we developed the multiplexed assay for kinase specificity (MAKS). MAKS simultaneously tests activity for up to ten kinases while directly identifying the substrate and exact site of phosphorylation. Using MAKS, we discovered site-specific phosphorylation by ERK2 and CDK1/CyclinA2, providing a putative link between key signaling pathways and NANOG.
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•We report 11 phosphorylation sites (9 localized) on endogenous, human NANOG•We introduce a multiplexed assay to identify kinases that modify a given substrate•We demonstrate that NANOG is directly phosphorylated by ERK2 and CDK1 in vitro•We connect cell-cycle- and growth-factor-mediated signaling to NANOG
NANOG is a core pluripotency factor. Here, Brumbaugh et al. show that NANOG is heavily phosphorylated on the N terminus and many of these sites are proline directed. To place these modifications in the context of signaling pathways, they developed the multiplexed assay for kinase specificity (MAKS) and show that NANOG is directly phosphorylated by ERK2 and CDK1 in vitro.
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