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.I...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.展开更多
目的 探讨STAT活化抑制蛋白2(protein inhibitor of the activated STAT2, PIAS2)基因在前列腺癌中的表达水平及其与临床病理特征的关系,以及PIAS2对前列腺癌脂质代谢重编程的潜在作用。方法 采用癌症基因图谱(The Cancer Genome Atlas ...目的 探讨STAT活化抑制蛋白2(protein inhibitor of the activated STAT2, PIAS2)基因在前列腺癌中的表达水平及其与临床病理特征的关系,以及PIAS2对前列腺癌脂质代谢重编程的潜在作用。方法 采用癌症基因图谱(The Cancer Genome Atlas Program,TCGA)数据库和人类蛋白图谱(Human Protein Atas,HPA)免疫组化分析显示PIAS2蛋白的表达情况;采用qRT-PCR、Western bolt及免疫组化检测前列腺癌组织和周围癌旁组织中PIAS2的表达,并分析其与患者临床病理特征的关系;慢病毒感染前列腺癌细胞株PC-3M,稳定敲低PIAS2,通过超高效液相色谱质谱技术(UPLC-MS)进行脂质组学分析。结果 TCGA分析显示PIAS2在前列腺癌组织的表达高于癌旁组织,HPA免疫组化分析显示PIAS2蛋白在前列腺癌组织高表达;PIAS2 mRNA和蛋白在前列腺癌中的表达量相较于癌旁组织均有显著提升,差异有统计学意义(P<0.05);免疫组织化学显示PIAS2蛋白表达主要定位在前列腺的细胞核,且前列腺癌组织的AOD值显著高于癌旁组织:临床病理参数研究结果显示,PIAS2与Gleason评分、TNM分期具有相关性(P<0.05),而与患者年龄、PSA及淋巴结转移之间相关性无统计学意义(P > 0.05);UPLC-MS分析提示,敲降PIAS2影响10种脂类变化,与shNC组相比,shPIAS2组的磷脂酰胆碱和磷脂酰乙醇胺的含量下调,磷脂酰肌醇、磷脂酰丝氨酸、二酰基甘油和三酰基甘油的含量上调。结论 PIAS2蛋白的表达量在前列腺癌组织中明显升高,提示PIAS2与前列腺癌的发生发展有关,其发病机制可能与前列腺癌的脂质代谢异常相关。展开更多
基金supported in part by a grant from the National Institute of Health ( HL121450)UCLA Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research Innovation Award
文摘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.
