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1. 发明申请

US20120094383A1 METHODS FOR INDUCING CELL DEATH IN PLURIPOTENT STEM CELLS AND DIFFERENTIATED CELLS OTHER THAN CARDIAC MYOCYTES 有权
标题翻译：诱导大鼠干细胞和心脏细胞分化细胞的细胞死亡的方法
公开(公告)号：US20120094383A1
公开(公告)日：2012-04-19
申请号：US13260224
申请日：2010-03-29
申请人： Fumiyuki Hattori , Keiichi Fukuda
发明人： Fumiyuki Hattori , Keiichi Fukuda
IPC分类号： C12N5/077
CPC分类号： C12N5/0657 , C12N5/0081 , C12N5/0696 , C12N2501/90 , C12N2506/02
摘要： The present invention has as its object developing a method that does not involve genomic modification and which yet is capable of inducing cell death in pluripotent stem cells such as embryonic stem cells and induced pluripotent stem cells, as well as in differentiated cells other than cardiomyocytes derived from pluripotent stem cells, but not in cardiomyocytes. It has been revealed that by establishing a method capable of inducing cell death in cells other than cardiomyocytes in a very efficient manner by adding a substance having no recognized inherent toxicity or cell death inducing action to the culture conditions for pluripotent stem cells and non-cardiomyocytes, the stated problem can be solved without relying upon genomic modification.
摘要翻译：本发明的目的是开发一种不涉及基因组修饰并且能够在多能干细胞如胚胎干细胞和诱导的多能干细胞以及除了心肌细胞以外的分化细胞中诱导细胞死亡的方法来自多能干细胞，但不在心肌细胞中。已经揭示出，通过向多能干细胞和非心肌细胞的培养条件中加入不具有公认的固有毒性或细胞死亡诱导作用的物质，通过建立能够以非常有效的方式在除心肌细胞之外的细胞中诱导细胞死亡的方法所述问题可以解决而不依赖于基因组修饰。

2. 发明申请

US20120214193A1 METHOD FOR MEASUREMENT OF FLUORESCENCE INTENSITY OF VOLTAGE-SENSITIVE FLUORESCENT DYE 审中-公开
标题翻译：电压敏感荧光染料荧光强度测量方法
公开(公告)号：US20120214193A1
公开(公告)日：2012-08-23
申请号：US13505272
申请日：2010-10-29
申请人： Fumiyuki Hattori , Keiichi Fukuda , Yu-suke Satoh
发明人： Fumiyuki Hattori , Keiichi Fukuda , Yu-suke Satoh
IPC分类号： G01N21/64
CPC分类号： G01N21/6428 , G01N33/582 , G01N33/6872 , G01N33/82 , G01N33/92
摘要： An object of the present invention is to provide a method for increasing the change in the fluorescent intensity as emitted from potential-sensitive fluorochromes depending on a potential or ionic strength change. Another object of the present invention is to measure the changes in the activity potentials of ES cell- or iPS cell-derived cardiomyocytes that have heretofore been impossible to measure.The present inventors screened a variety of substances and found that vitamin E has an action for increasing the sensitivity of potential-sensitive fluorochromes whereas cholesterol has an action for enhancing the fluorescent intensity of potential-sensitive fluorochromes. In addition, it has become clear that these substances can be combined in such a way that the sensitivity of a potential-sensitive fluorochrome is increased by vitamin E while at the same time its absolute fluorescent intensity is enhanced by cholesterol.
摘要翻译：本发明的目的是提供一种根据潜在或离子强度变化增加从电位敏感荧光染料发射的荧光强度变化的方法。本发明的另一个目的是测量以前不可能测量的ES细胞或iPS细胞来源的心肌细胞的活性电位的变化。本发明人筛选出各种物质，并发现维生素E具有提高潜在敏感性荧光染料敏感性的作用，而胆固醇具有增强潜在敏感荧光染料荧光强度的作用。此外，已经清楚的是，这些物质可以以通过维生素E增加潜在敏感性荧光染料的灵敏度的方式组合，同时其绝对荧光强度被胆固醇增强。

3. 发明授权

US08735152B2 Methods for inducing cell death in pluripotent stem cells and differentiated cells other than cardiac myocytes 有权
标题翻译：在多能干细胞和心肌细胞以外的分化细胞中诱导细胞死亡的方法
公开(公告)号：US08735152B2
公开(公告)日：2014-05-27
申请号：US13260224
申请日：2010-03-29
申请人： Fumiyuki Hattori , Keiichi Fukuda
发明人： Fumiyuki Hattori , Keiichi Fukuda
IPC分类号： C12N5/00 , C12N5/02
CPC分类号： C12N5/0657 , C12N5/0081 , C12N5/0696 , C12N2501/90 , C12N2506/02
摘要： The present invention has as its object developing a method that does not involve genomic modification and which yet is capable of inducing cell death in pluripotent stem cells such as embryonic stem cells and induced pluripotent stem cells, as well as in differentiated cells other than cardiomyocytes derived from pluripotent stem cells, but not in cardiomyocytes. It has been revealed that by establishing a method capable of inducing cell death in cells other than cardiomyocytes in a very efficient manner by adding a substance having no recognized inherent toxicity or cell death inducing action to the culture conditions for pluripotent stem cells and non-cardiomyocytes, the stated problem can be solved without relying upon genomic modification.
摘要翻译：本发明的目的是开发一种不涉及基因组修饰并且能够在多能干细胞如胚胎干细胞和诱导的多能干细胞以及除了心肌细胞以外的分化细胞中诱导细胞死亡的方法来自多能干细胞，但不在心肌细胞中。已经揭示出，通过向多能干细胞和非心肌细胞的培养条件中加入不具有公认的固有毒性或细胞死亡诱导作用的物质，通过建立能够以非常有效的方式在除心肌细胞之外的细胞中诱导细胞死亡的方法所述问题可以解决而不依赖于基因组修饰。

4. 发明申请

US20080090266A1 Method of Selecting a Cardiomyoctye Using Intracellular Mitochondria as an Indicator 有权
标题翻译：使用细胞内线粒体作为指标选择心肌细胞的方法
公开(公告)号：US20080090266A1
公开(公告)日：2008-04-17
申请号：US11660581
申请日：2005-08-26
申请人： Fumiyuki Hattori , Keiichi Fukuda
发明人： Fumiyuki Hattori , Keiichi Fukuda
IPC分类号： C12Q1/02 , C12N5/00 , C12N5/02
CPC分类号： C12N5/0657 , C12N2506/02 , G01N33/56966
摘要： The invention relates to methods of selecting a cardiomyocyte from a cell population derived from a whole heart or a differentiated cell population derived from a stem cell without genetic alteration. Specifically, the invention relates to a method of selecting a cardiomyocyte from a cardiomyocyte-containing cell mixture without genetic alteration of a cardiomyocyte, on the basis of a relative content of cellular mitochondria and/or a relative mitochondrial transmembrane potential of the cell. The invention also relates to methods of enriching a cardiomyocyte from a cardiomyocyte-containing cell mixture without genetic alteration of a cardiomyocyte, producing a cardiomyocyte without genetic alteration of a cardiomyocyte, and evaluating the ratio of a cardiomyocyte in a cardiomyocyte-containing cell mixture.
摘要翻译：本发明涉及从源自全部心脏的细胞群体或源自干细胞的分化细胞群体中选择心肌细胞而不进行遗传改变的方法。具体地说，本发明涉及一种基于细胞线粒体的相对含量和/或细胞的相对线粒体跨膜电位从含有心肌细胞的细胞混合物中选择心肌细胞而不对心肌细胞进行遗传改变的方法。本发明还涉及从含有心肌细胞的细胞混合物中丰富心肌细胞的方法，而没有心肌细胞的遗传改变，产生心肌细胞而没有心肌细胞的遗传改变，以及评估心肌细胞含有心肌细胞的细胞混合物中的比例。

5. 发明授权

US09109205B2 Method of constructing masses of myocardial cells and use of the myocardial cell mass 有权
标题翻译：建立心肌细胞质量和使用心肌细胞团的方法
公开(公告)号：US09109205B2
公开(公告)日：2015-08-18
申请号：US12668136
申请日：2008-07-31
申请人： Fumiyuki Hattori , Keiichi Fukuda
发明人： Fumiyuki Hattori , Keiichi Fukuda
IPC分类号： A01N63/00 , C12N5/077 , A61K35/34 , A61L27/38 , A61K35/12
CPC分类号： A61K35/34 , A61K35/12 , A61L27/3826 , A61L27/3895 , A61L2430/20 , C12N5/0657 , C12N2500/25 , C12N2500/90 , C12N2500/99 , C12N2501/105 , C12N2501/11 , C12N2501/115 , C12N2501/117 , C12N2501/12 , C12N2501/125 , C12N2501/13 , C12N2501/135 , C12N2501/235 , C12N2501/33 , C12N2501/998 , C12N2506/00 , C12N2506/03 , C12N2506/45
摘要： The object of the present invention is to improve the post-transplantation engraftment rate of cardiomyocytes that have been purified to such an extent that they are free from non-cardiomyocytes and any components derived from other species.To solve this problem, the present inventors studied the possibility of constructing cell masses from the purified cardiomyocytes. As a result, they revealed that the stated problem could be solved by providing a method of preparing cell masses of cardiomyocytes derived from pluripotent stem cells, characterized in that cell masses of aggregated cells containing cardiomyocytes that had been differentiated and induced from pluripotent stem cells were dispersed to single cells to thereby obtain purified cardiomyocytes, which were then cultured in a culture medium under serum-free conditions so that they were reaggregated.
摘要翻译：本发明的目的是改善已经被纯化到它们不含非心肌细胞和来自其它物种的任何成分的程度的心肌细胞的移植后植入率。为了解决这个问题，本发明人研究了从纯化的心肌细胞构建细胞团的可能性。结果表明，通过提供一种制备多能干细胞衍生的心肌细胞群的方法，可以解决上述问题，其特征在于分化并诱导多能干细胞的含有心肌细胞的聚集细胞的细胞团是分散到单细胞，从而获得纯化的心肌细胞，然后将其在无血清条件下在培养基中培养，使其再聚集。

6. 发明申请

US20100297767A1 NOVEL CELL CULTURE AND METHODS OF PRODUCING AND COLLECTING CELL MASSES USING THE SAME 审中-公开
标题翻译：新型细胞培养物和使用该细胞培养物的细胞质量的收集方法
公开(公告)号：US20100297767A1
公开(公告)日：2010-11-25
申请号：US12294723
申请日：2007-03-30
申请人： Fumiyuki Hattori , Keiichi Fukuda
发明人： Fumiyuki Hattori , Keiichi Fukuda
IPC分类号： C12N5/071
CPC分类号： C12N5/0606
摘要： The inventors of the present invention have developed a novel cell culture method with a view to providing a process by which cell masses uniform in size and properties can be produced in large quantities and with convenience, as well as a method of recovering them.The inventors of the present invention found that the above-mentioned objects could be attained by using a structural member having a hollow portion at least one lower end of which is open, creating a projecting portion of a culture medium at the open lower end of the structural member, and culturing cells in that projecting portion of the culture medium. The present invention has been accomplished on the basis of this finding. Specifically, the present invention provides a cell culture method characterized by providing a structural member having a hollow portion at least one lower end of which is open, injecting a culture medium containing cells into the hollow portion of the structural member, causing a portion of the culture medium to project downward from the open end, and culturing the cells in the projecting portion of the culture medium. The present invention also provides a process for preparing cell masses characterized by forming cell masses by the above-described cell culture method.
摘要翻译：本发明人开发了一种新颖的细胞培养方法，其目的在于提供一种能够大量且方便地生产细胞尺寸和性质均匀的方法以及回收方法。本发明的发明人发现，通过使用具有至少一个下端开口的中空部分的结构构件可以实现上述目的，在该开口的下端形成培养基的突出部分结构构件，并在培养基的突出部分培养细胞。本发明是基于这一发现而完成的。具体地，本发明提供一种细胞培养方法，其特征在于，提供具有至少一个下端开放的中空部的结构部件，将含有细胞的培养基注入到所述结构部件的中空部分，培养基从开放端向下突出，并培养培养基的突出部分中的细胞。本发明还提供了一种制备细胞团的方法，其特征在于通过上述细胞培养方法形成细胞团。

7. 发明授权

US09115342B2 Method for purifying cardiomyocytes or programmed cardiomyocytes derived from stem cells or fetuses 有权
标题翻译：纯化来自干细胞或胎儿的心肌细胞或程序性心肌细胞的方法
公开(公告)号：US09115342B2
公开(公告)日：2015-08-25
申请号：US12162684
申请日：2007-01-31
申请人： Fumiyuki Hattori , Keiichi Fukuda
发明人： Fumiyuki Hattori , Keiichi Fukuda
IPC分类号： C12N5/077
CPC分类号： C12N5/0657 , C12N2500/02 , C12N2500/14 , C12N2500/25 , C12N2500/32 , C12N2500/34 , C12N2500/38 , C12N2500/44 , C12N2500/90 , C12N2500/99 , C12N2501/115 , C12N2501/235 , C12N2501/70 , C12N2506/02
摘要： An object of the present invention is to develop a method for purify cardiomyocytes at a high degree of purification and at a high yield from a cell mixture comprising cardiomyocytes derived from fetuses and stem cells using various features which have not been previously expected to be used for purification of cardiomyocytes or which are newly found, wherein said method is carried out without undergoing any genetic modification or without adding any special proteins or biologically active agents.
摘要翻译：本发明的一个目的是开发一种以高度纯化和高产率从包含来自胎儿和干细胞的心肌细胞的细胞混合物中纯化心肌细胞的方法，该方法使用先前预期不会用于纯化心肌细胞或新发现的，其中所述方法进行而不经历任何遗传修饰或不添加任何特殊的蛋白质或生物活性剂。

8. 发明授权

US08623649B2 Method of selecting a cardiomyoctye using intracellular mitochondria as an indicator 有权
标题翻译：使用细胞内线粒体作为指标选择心肌细胞的方法
公开(公告)号：US08623649B2
公开(公告)日：2014-01-07
申请号：US11660581
申请日：2005-08-26
申请人： Fumiyuki Hattori , Keiichi Fukuda
发明人： Fumiyuki Hattori , Keiichi Fukuda
IPC分类号： A61K35/34
CPC分类号： C12N5/0657 , C12N2506/02 , G01N33/56966
摘要： The invention relates to methods of selecting a cardiomyocyte from a cell population derived from a whole heart or a differentiated cell population derived from a stem cell without genetic alteration. Specifically, the invention relates to a method of selecting a cardiomyocyte from a cardiomyocyte-containing cell mixture without genetic alteration of a cardiomyocyte, on the basis of a relative content of cellular mitochondria and/or a relative mitochondrial transmembrane potential of the cell. The invention also relates to methods of enriching a cardiomyocyte from a cardiomyocyte-containing cell mixture without genetic alteration of a cardiomyocyte, producing a cardiomyocyte without genetic alteration of a cardiomyocyte, and evaluating the ratio of a cardiomyocyte in a cardiomyocyte-containing cell mixture.
摘要翻译：本发明涉及从源自全部心脏的细胞群体或源自干细胞的分化细胞群体中选择心肌细胞而不进行遗传改变的方法。具体地说，本发明涉及一种基于细胞线粒体的相对含量和/或细胞的相对线粒体跨膜电位从含有心肌细胞的细胞混合物中选择心肌细胞而不对心肌细胞进行遗传改变的方法。本发明还涉及从含有心肌细胞的细胞混合物中丰富心肌细胞的方法，而没有心肌细胞的遗传改变，产生心肌细胞而没有心肌细胞的遗传改变，以及评估心肌细胞含有心肌细胞的细胞混合物中的比例。

9. 发明申请

US20100189699A1 METHOD OF CONSTRUCTING MASSES OF MYOCARDIAL CELLS AND USE OF THE MYOCARDIAL CELL MASS 有权
标题翻译：构建肌肉细胞质量的方法和使用心肌细胞质量
公开(公告)号：US20100189699A1
公开(公告)日：2010-07-29
申请号：US12668136
申请日：2008-07-31
申请人： Fumiyuki Hattori , Keiichi Fukuda
发明人： Fumiyuki Hattori , Keiichi Fukuda
IPC分类号： A61K35/34 , C12N5/071 , A61P9/00
CPC分类号： A61K35/34 , A61K35/12 , A61L27/3826 , A61L27/3895 , A61L2430/20 , C12N5/0657 , C12N2500/25 , C12N2500/90 , C12N2500/99 , C12N2501/105 , C12N2501/11 , C12N2501/115 , C12N2501/117 , C12N2501/12 , C12N2501/125 , C12N2501/13 , C12N2501/135 , C12N2501/235 , C12N2501/33 , C12N2501/998 , C12N2506/00 , C12N2506/03 , C12N2506/45
摘要： The object of the present invention is to improve the post-transplantation engraftment rate of cardiomyocytes that have been purified to such an extent that they are free from non-cardiomyocytes and any components derived from other species.To solve this problem, the present inventors studied the possibility of constructing cell masses from the purified cardiomyocytes. As a result, they revealed that the stated problem could be solved by providing a method of preparing cell masses of cardiomyocytes derived from pluripotent stem cells, characterized in that cell masses of aggregated cells containing cardiomyocytes that had been differentiated and induced from pluripotent stem cells were dispersed to single cells to thereby obtain purified cardiomyocytes, which were then cultured in a culture medium under serum-free conditions so that they were reaggregated.
摘要翻译：本发明的目的是改善已经被纯化到它们不含非心肌细胞和来自其它物种的任何成分的程度的心肌细胞的移植后植入率。为了解决这个问题，本发明人研究了从纯化的心肌细胞构建细胞团的可能性。结果表明，通过提供一种制备多能干细胞衍生的心肌细胞群的方法，可以解决上述问题，其特征在于分化并诱导多能干细胞的含有心肌细胞的聚集细胞的细胞团是分散到单细胞，从而获得纯化的心肌细胞，然后将其在无血清条件下在培养基中培养，使其再聚集。

10. 发明申请

US20090275132A1 METHOD FOR PURIFYING CARDIOMYOCYTES OR PROGRAMMED CARDIOMYOCYTES DERIVED FROM STEM CELLS OR FETUSES 有权
标题翻译：净化心肌细胞或从干细胞衍生的编程性心肌细胞的方法
公开(公告)号：US20090275132A1
公开(公告)日：2009-11-05
申请号：US12162684
申请日：2007-01-31
申请人： Fumiyuki Hattori , Keiichi Fukuda
发明人： Fumiyuki Hattori , Keiichi Fukuda
IPC分类号： C12N5/06
CPC分类号： C12N5/0657 , C12N2500/02 , C12N2500/14 , C12N2500/25 , C12N2500/32 , C12N2500/34 , C12N2500/38 , C12N2500/44 , C12N2500/90 , C12N2500/99 , C12N2501/115 , C12N2501/235 , C12N2501/70 , C12N2506/02
摘要： An object of the present invention is to develop a method for purify cardiomyocytes at a high degree of purification and at a high yield from a cell mixture comprising cardiomyocytes derived from fetuses and stem cells using various features which have not been previously expected to be used for purification of cardiomyocytes or which are newly found, wherein said method is carried out without undergoing any genetic modification or without adding any special proteins or biologically active agents.The inventors of the present invention found that cardiomyocytes were effectively and highly selected and purified by culturing cardiomyocytes derived from embryonic stem cells in the culture medium under a condition selected from a low-serum-supplemented condition, a low-glucose-supplemented condition, a low-nutritional condition, a low calcium condition, a mildly-acidic pH condition, a lactic acid-supplemented condition, an aspartic acid/glutamic acid-supplemented condition, and/or a pyruvic acid-supplemented condition. The inventors of the present invention further found that the above method invented in relation to embryonic stem cells was applicable to select and purify cardiomyocytes derived from fetuses or adult stem cells.
摘要翻译：本发明的一个目的是开发一种以高度纯化和高产率从包含来自胎儿和干细胞的心肌细胞的细胞混合物中纯化心肌细胞的方法，该方法使用先前预期不会用于纯化心肌细胞或新发现的，其中所述方法进行而不经历任何遗传修饰或不添加任何特殊的蛋白质或生物活性剂。本发明人发现，通过在选自低血清补充条件，低葡萄糖补充条件，细菌培养条件下的培养基中培养来源于培养基中的胚胎干细胞的心肌细胞，有效地和高度地选择和纯化心肌细胞低营养状态，低钙状态，轻度酸性pH条件，补充乳酸的条件，天冬氨酸/谷氨酸补充条件和/或丙酮酸补充条件。本发明人进一步发现，关于胚胎干细胞发明的上述方法适用于选择和纯化来源于胎儿或成体干细胞的心肌细胞。

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