Effect of Biophysical Cues on Cell Reprogramming
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摘要
Cell reprograming technologies have broad applications in cell therapy,disease modeling and drug screening.Direct reprogramming is the process of converting from one cell type into a very distantly related cell type.In this direct conversion process,cells do not proceed through a pluripotent stage,which can be time-consuming and challenging due to spontaneous differentiation.This method also offers the advantage of circumventing the teratoma potential that is associated with using iPSCs.Previous works have demonstrated that with the use of genetic manipulation,fibroblasts can be directly converted into other cell types,including neurons,cardiomyocytes,blood cell progenitors,and hepatocytes.It is well known that the microenvironment can directs cell fate,and in turn cells interact with or remodel their niches.Accumulative evidence suggests that biophysical factors such as the microtopography and mechanical property of cell adhesive substrates regulate a variety of cellular functions such as migration,proliferation and differentiation,which in turn can modulate wound healing,tissue remodeling and tumor growth,but there are limited number of studies on the roles of biophysical cues in cell reprogramming~([1]).Passive topographical cues offer a simple and effective method to improve reprogramming efficiency without the need for biochemical manipulations.Our previous study has demonstrated that somatic cells cultured on the parallel microgrooves,which can replace the effects of small-molecule epigenetic modifiers and significantly improve the iPSCs reprogramming efficiency.The mechanism relies on the mechanomodulation of the cells'epigenetic state,specifically,an increase of histone H3 acetylation and H3K4 methylation~([2]).Additionally,in cardiomyocytes reprogramming study,culturing the fibroblasts on microgrooved substrate enhances the expression of cardiomyocyte genes by day 2 and improves the yield of partially reprogrammed cells at day 10.By combining microgrooved substrate with an optimized culture protocol,the conversion from fibroblasts to cardiomyocytes is increased through genetic changes and structural organization of sarcomeres~([3]).Besides biomaterial topography,recent studies have demonstrated the effects of matrix stiffness on cell reprogramming.For example,a decrease of substrate stiffness can improve the iPSCs reprogramming efficiency,while an intermediate stiffness can significantly enhance the efficiency of neuronal reprogramming ~([4]).Further analysis suggests that intracellular biomechanical changes play an important role in reprogramming process.Cells interact with the biophysical factors in the microenvironment through an"inside-out"and"outside-in"feedback loop,which is mediated by focal adhesions and cytoskeleton ~([5]).Therefore,we investigated the role of the intracellular mechanical structure in cell reprogramming.We showed,for the first time,that the mechanical property of cells was modulated during the early phase of reprogramming as determined by atomic force microscopy(AFM)and high-throughput quantitative deformability cytometry(q-DC).We observed that cell stiffness increased by day 1 during reprogramming process,which was followed by a pronounced decrease within a few days.Examination of actin cytoskeleton showed that actin assembled into a network with a cage-like structure around the nucleus by day 1,but this structure along with the majority of the cytoskeleton gradually disappeared,coinciding with the changes in intracellular mechanical property.Furthermore,inhibition of actin contractility by using small molecules significantly altered the reprogramming efficiency.These findings provide new insights into the mechanisms of how biophysical cues modulate cell fate.In any given physiological microenvironment,cells may experience various of biophysical inputs,which,as we show,may affect cell phenotype changes.
Cell reprograming technologies have broad applications in cell therapy,disease modeling and drug screening.Direct reprogramming is the process of converting from one cell type into a very distantly related cell type.In this direct conversion process,cells do not proceed through a pluripotent stage,which can be time-consuming and challenging due to spontaneous differentiation.This method also offers the advantage of circumventing the teratoma potential that is associated with using iPSCs.Previous works have demonstrated that with the use of genetic manipulation,fibroblasts can be directly converted into other cell types,including neurons,cardiomyocytes,blood cell progenitors,and hepatocytes.It is well known that the microenvironment can directs cell fate,and in turn cells interact with or remodel their niches.Accumulative evidence suggests that biophysical factors such as the microtopography and mechanical property of cell adhesive substrates regulate a variety of cellular functions such as migration,proliferation and differentiation,which in turn can modulate wound healing,tissue remodeling and tumor growth,but there are limited number of studies on the roles of biophysical cues in cell reprogramming~([1]).Passive topographical cues offer a simple and effective method to improve reprogramming efficiency without the need for biochemical manipulations.Our previous study has demonstrated that somatic cells cultured on the parallel microgrooves,which can replace the effects of small-molecule epigenetic modifiers and significantly improve the iPSCs reprogramming efficiency.The mechanism relies on the mechanomodulation of the cells'epigenetic state,specifically,an increase of histone H3 acetylation and H3K4 methylation~([2]).Additionally,in cardiomyocytes reprogramming study,culturing the fibroblasts on microgrooved substrate enhances the expression of cardiomyocyte genes by day 2 and improves the yield of partially reprogrammed cells at day 10.By combining microgrooved substrate with an optimized culture protocol,the conversion from fibroblasts to cardiomyocytes is increased through genetic changes and structural organization of sarcomeres~([3]).Besides biomaterial topography,recent studies have demonstrated the effects of matrix stiffness on cell reprogramming.For example,a decrease of substrate stiffness can improve the iPSCs reprogramming efficiency,while an intermediate stiffness can significantly enhance the efficiency of neuronal reprogramming ~([4]).Further analysis suggests that intracellular biomechanical changes play an important role in reprogramming process.Cells interact with the biophysical factors in the microenvironment through an"inside-out"and"outside-in"feedback loop,which is mediated by focal adhesions and cytoskeleton ~([5]).Therefore,we investigated the role of the intracellular mechanical structure in cell reprogramming.We showed,for the first time,that the mechanical property of cells was modulated during the early phase of reprogramming as determined by atomic force microscopy(AFM)and high-throughput quantitative deformability cytometry(q-DC).We observed that cell stiffness increased by day 1 during reprogramming process,which was followed by a pronounced decrease within a few days.Examination of actin cytoskeleton showed that actin assembled into a network with a cage-like structure around the nucleus by day 1,but this structure along with the majority of the cytoskeleton gradually disappeared,coinciding with the changes in intracellular mechanical property.Furthermore,inhibition of actin contractility by using small molecules significantly altered the reprogramming efficiency.These findings provide new insights into the mechanisms of how biophysical cues modulate cell fate.In any given physiological microenvironment,cells may experience various of biophysical inputs,which,as we show,may affect cell phenotype changes.
Cell reprograming technologies have broad applications in cell therapy,disease modeling and drug screening.Direct reprogramming is the process of converting from one cell type into a very distantly related cell type.In this direct conversion process,cells do not proceed through a pluripotent stage,which can be time-consuming and challenging due to spontaneous differentiation.This method also offers the advantage of circumventing the teratoma potential that is associated with using iPSCs.Previous works have demonstrated that with the use of genetic manipulation,fibroblasts can be directly converted into other cell types,including neurons,cardiomyocytes,blood cell progenitors,and hepatocytes.It is well known that the microenvironment can directs cell fate,and in turn cells interact with or remodel their niches.Accumulative evidence suggests that biophysical factors such as the microtopography and mechanical property of cell adhesive substrates regulate a variety of cellular functions such as migration,proliferation and differentiation,which in turn can modulate wound healing,tissue remodeling and tumor growth,but there are limited number of studies on the roles of biophysical cues in cell reprogramming~([1]).Passive topographical cues offer a simple and effective method to improve reprogramming efficiency without the need for biochemical manipulations.Our previous study has demonstrated that somatic cells cultured on the parallel microgrooves,which can replace the effects of small-molecule epigenetic modifiers and significantly improve the iPSCs reprogramming efficiency.The mechanism relies on the mechanomodulation of the cells'epigenetic state,specifically,an increase of histone H3 acetylation and H3K4 methylation~([2]).Additionally,in cardiomyocytes reprogramming study,culturing the fibroblasts on microgrooved substrate enhances the expression of cardiomyocyte genes by day 2 and improves the yield of partially reprogrammed cells at day 10.By combining microgrooved substrate with an optimized culture protocol,the conversion from fibroblasts to cardiomyocytes is increased through genetic changes and structural organization of sarcomeres~([3]).Besides biomaterial topography,recent studies have demonstrated the effects of matrix stiffness on cell reprogramming.For example,a decrease of substrate stiffness can improve the iPSCs reprogramming efficiency,while an intermediate stiffness can significantly enhance the efficiency of neuronal reprogramming ~([4]).Further analysis suggests that intracellular biomechanical changes play an important role in reprogramming process.Cells interact with the biophysical factors in the microenvironment through an"inside-out"and"outside-in"feedback loop,which is mediated by focal adhesions and cytoskeleton ~([5]).Therefore,we investigated the role of the intracellular mechanical structure in cell reprogramming.We showed,for the first time,that the mechanical property of cells was modulated during the early phase of reprogramming as determined by atomic force microscopy(AFM)and high-throughput quantitative deformability cytometry(q-DC).We observed that cell stiffness increased by day 1 during reprogramming process,which was followed by a pronounced decrease within a few days.Examination of actin cytoskeleton showed that actin assembled into a network with a cage-like structure around the nucleus by day 1,but this structure along with the majority of the cytoskeleton gradually disappeared,coinciding with the changes in intracellular mechanical property.Furthermore,inhibition of actin contractility by using small molecules significantly altered the reprogramming efficiency.These findings provide new insights into the mechanisms of how biophysical cues modulate cell fate.In any given physiological microenvironment,cells may experience various of biophysical inputs,which,as we show,may affect cell phenotype changes.
引文
[1] Wong SY,Soto J,Li S. Biophysical regulation of cell reprogramming. Current Opinion in Chemical Engineering. 2017 Feb 28; 15:95-101.
[2] Downing T,Soto J,Morez C,Houssin T,Yuan F,Chu J,Fritz A,Patel S,Schaffer D,Li S. Biophysical regulation of epigenetic state and cell reprogramming.Nature materials. 2013 Dec; 12(12):1154.
[3] Sia J,Yu P,Srivastava D,Li S. Effect of biophysical cues on reprogramming to cardiomyocytes. Biomaterials. 2016 Oct 1; 103:1-11.
[4] Caiazzo M,Okawa Y,Ranga A,Piersigilli A,Tabata Y,Lutolf MP. Defined three-dimensional microenvironments boost induction of pluripotency. Nature materials. 2016 Mar; 15(3):344.
[5] Discher DE,Janmey P,Wang YL. Tissue cells feel and respond to the stiffness of their substrate. Science. 2005 Nov 18; 310(5751):1139-43.
[2] Downing T,Soto J,Morez C,Houssin T,Yuan F,Chu J,Fritz A,Patel S,Schaffer D,Li S. Biophysical regulation of epigenetic state and cell reprogramming.Nature materials. 2013 Dec; 12(12):1154.
[3] Sia J,Yu P,Srivastava D,Li S. Effect of biophysical cues on reprogramming to cardiomyocytes. Biomaterials. 2016 Oct 1; 103:1-11.
[4] Caiazzo M,Okawa Y,Ranga A,Piersigilli A,Tabata Y,Lutolf MP. Defined three-dimensional microenvironments boost induction of pluripotency. Nature materials. 2016 Mar; 15(3):344.
[5] Discher DE,Janmey P,Wang YL. Tissue cells feel and respond to the stiffness of their substrate. Science. 2005 Nov 18; 310(5751):1139-43.