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METHOD OF MANUFACTURING DIFFERENTIATED PLURIPOTENT STEM CELL

Foreign code F150008107
Posted date Feb 9, 2015
Country WIPO
International application number 2013JP079311
International publication number WO 2014069479
Date of international filing Oct 29, 2013
Date of international publication May 8, 2014
Priority data
  • P2012-237734 (Oct 29, 2012) JP
Title METHOD OF MANUFACTURING DIFFERENTIATED PLURIPOTENT STEM CELL
Abstract The present invention allows a TET1 protein to be more stably expressed in human differentiated pluripotent stem cells than in the past by, inter alia , substituting the second amino acid from the amino terminal of a TET1 protein with a different amino acid. Furthermore, it is possible to quickly eliminate the expression of, inter alia , NANOG, which is an inhibitor of differentiation induction, and promote the expression of factors related to differentiation induction by introducing a variant TET1 protein to a differentiated pluripotent stem cell. The present invention provides a method for manufacturing differentiated pluripotent stem cells with increased differentiation performance, and a substance that is useful to said method.
Outline of related art and contending technology BACKGROUND ART
Differentiation of the pluripotent stem cells, a plurality of systems with self-different from the ability to change types of cells (pluripotent differentiation) and, even after cell division to a cell having a self-similar capability so as to produce the daughter cells as the ability (ability to self-replicate) the cells comprising a defined, embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells) and the like fall within this definition is a cell that is considered to be. Then, since it has such ability, and the like ES cells or iPS cells, in the field of regenerative medicine and drug discovery development plays an important role is strongly expected.
However, recently, mouse and human pluripotent stem cell differentiation pluripotent stem cells differentiation, the basic properties are different from each other, can be differentiated with respect to, the mouse pluripotent stem cell differentiation human pluripotent stem cell differentiation that inferior have been reported.
With respect to the mouse pluripotent stem cell differentiation, terminally differentiated pluripotent experiment can be evaluated more accurately. For example, mouse ES cells were injected into the lumen of the blastocyst, inner cell mass cell are mixed so that, in the ES cell-derived cell is an embryo and be of a normal development, this is referred to as chimera formation. For mouse ES cells, in this way is equivalent to the time of occurrence of inner cell mass occurs whether or not the synchronization of the progress of, an indicator of good pluripotent differentiation. In this case, also occur along with the progression of, a portion of these ES cells can be recruited germline also known. This germ cell migration (germ line transmission) occur, ES cell-derived genetic information is passed on to the next generation means and, in this sense in a broader sense to indicate compliance of normal cells as possible. Also referred to as "ultimate" further demonstrated that there are pluripotent differentiation. Mouse cell division of the fertilized egg after temporary inhibition and cultured dishes, five (tetraploid) 4 is formed of a blastocyst. The 4 diploid blastocyst inner cell mass is contained, appears at first glance normal nature, subsequent embryogenesis is does not proceed. For this reason, it is the 4 diploid blastocyst in the uterus of a pseudo-pregnant mother mouse to be returned to the individual is not obtained. However, this 4 to diploid blastocysts, differentiation into a pluripotent stem cell having, chimera formation is performed in the above, in this case of injected cells which has a normal karyotype, can be one that is conducive to. In this manner, in a single generation, differentiation pluripotent stem cells are injected into individuals from occurs. This method is generally referred to as diploid complement (tetraploid-complementation) 4 assay, mice were born at this time, named after the injected mouse derived cells referred to as all-iPSC. In this way, in mice, it is possible to verify pluripotent differentiation and as a premise, a number of accepted by far, the differentiation of the pluripotent mouse iPS cells in a strict sense is shown.
On the other hand, with regard to human pluripotent stem cell differentiation, it will be appreciated, ethical above from the perspective of the validation is cannot be used in humans. As a substitute method is often used for the formation of a teratoma. Transplanting stem cells subcutaneously into nude mouse and, rest on the blood circulation in the mouse host and that the cells were differentiated for organizing and "random". The pieces of tumor grow, out of the pathology specimen, in, searching for endoderm, once pluripotent differentiation are shown. However, teratoma in the evaluation of pluripotent differentiation by large that there is a problem. It is, in this method, even if the source of the stem cells of the differentiation efficiency even in poor, confirmed the tissue into the three germ layers in a stage of differentiation pluripotent and point determined. This is, does not proceed at all of the tumor generated in the undifferentiated cells remain in many cases this is ignored and also pluripotent differentiation will be evaluated. Teratoma formation, the original stem cell population present in several true if the differentiation of pluripotent cells, and other cells may be of any kind at all, no evaluation method for this is not the quantitativeness. For example, mouse iPS cells that may be fabricated using c-Myc is made with the, differentiating into three germ layers in teratoma of it is assumed that the, chimeric formation is carried out, in most cases, the cell is derived from a naturally occurring tumor iPS of the sheet of paper. As the best, actually induced to differentiate with individually three germ layers, a method may be considered for evaluation. This is, dependent on the differentiation protocol, quantitative experiment also expected to be difficult.
Further, with regard to human pluripotent stem cell differentiation, more underlying problems arise. It is, to differentiate into all cell lineages with the potential to equal the ideal human pluripotent stem cell differentiation can be employed with. Reportedly osafune et al., 2 gene expression level of human ES cells are the same as one which does not exist, individual ES cell lines in response to it, any particular inductive differentiation into germ layers biased, and thus, human ES cells, points in the quantitative, having sufficient "pluripotent differentiation" is not suggested (non-patent document 1).
In addition, from the mouse ES cell was established, human ES cells become established takes time until the year 17. This is because, in the establishment of mouse ES cells become established human ES method could not be nothing but. Mouse ES cell self-replication of culture of the leukemia inhibitory factor (hereinafter also referred to as' LIF ') is required, human ES cells in a medium added LIF cannot be replicated. Required for maintenance of human ES cells Thomson et al. and which is found, and activin/Nodal and FGF (Activin) (non-patent document 2) was. Further, while the mouse ES cells via JAK/STAT signal, the signal passes through the SMAD2/3 human ES/iPS cells in both the differentiation of the different signals system with each other via the pluripotent stem cells have been found to replicate autonomously. In addition, in human pluripotent stem cell differentiation, intracellular signals via the SMAD2/3, and related to the perspective of the elevated expression of NANOG predominates. On the other hand, the signal SMAD2/3, in the middle of the mammal that generates the signal transduction pathways required for endoderm differentiation and, in fact, with the aim to induce the differentiation of these cells in order to provide a lineage of high concentration using ActivinA. Therefore differs from the purpose, frequently used in the present human differentiation pluripotent stem cell self-replication signals, in the middle of the same cells to differentiate into ectodermic system overlaps with the signal.
Human ES cells with the mouse ES cells, established from a blastocyst is the same as that point. However, either from the difference in culture conditions described above, their nature is different. And the findings so far, mouse ES cells of the inner cell mass of blastocysts by following the gene expression profile is found, human ES cells, blastocysts developed slightly formed in the primitive ectoderm epi blast mean that they are similar in most properties. Then, epi blast mouse epiblast stem cells (EpiSC) directly from the human ES cell lines were established at the same culture conditions in response to (non-patent document 3 and 4), 2 pluripotent differentiation in consideration of increase becomes divided into two. 1 Mouse ES cells and mouse iPS cells one represented by the differentiation of a pluripotent blastocyst having a stem cell, human ES cells one another 1, human iPS cells, such as a stem cell mouse EpiSC-epi blast, and the nature of the former (naive) naive, latter property is referred to as prime (primed) (non-patent document 5) adapted to.
As mentioned above, the differentiation of pluripotent stem cells are also prime naive-type differentiation pluripotent stem cells having an ability to differentiate into three germ layers together, if transplanted into a nude mouse teratoma is formed is the same as that in the transition. However, the differentiation of pluripotent stem cells are naive-type, developmental stage in a fertilized egg (blastocyst) close to the initial state and therefore reflect the nature of the, or the like are easily established and, further, the cells are derived from an individual completely can be generated to such a degree that has complete differentiation pluripotent. On the other hand, prime type differentiation of the pluripotent stem cells, generated from the stage of blastocyst reflect the nature of the epi blast and advanced to the forefront, and the like is not easy to establish. In addition, in the cells, as described above, there is bias in pluripotent differentiation has also proved to be (non-patent document 1). Further, prime type differentiation of the pluripotent stem cells, or germ cell migration ability of chimera formation does not have the ability, in terms of differentiated pluripotent differentiation including, a human having a prime-of-like pluripotent stem cell differentiation, differentiation-of-naive mice than pluripotent stem cells have been found to be inferior.
In the development of practical use of regenerative medicine and drug discovery, a wide variety of cells, tissues, and organs can be provided by the ability to differentiate a high to such a degree, the human pluripotent stem cell differentiation can be sought. Therefore, the prime-of-such as a human having the ability to differentiate pluripotent stem cell differentiation is improved is strongly demanded, such methods have been developed in has not been set.
Scope of claims (In Japanese)請求の範囲 [請求項1]
 分化多能性幹細胞の製造方法であって、
 下記(a)~(c)からなる群から選択される少なくとも一つの分子を、分化多能性幹細胞又は該細胞に誘導するための体細胞に導入する工程を含む方法
(a)TET1タンパク質
(b)TET1タンパク質をコードする核酸
(c)TET1タンパク質をコードする核酸が挿入されているベクター。

[請求項2]
 前記TET1タンパク質は、変異型TET1タンパク質であって、正常型TET1タンパク質と比較して安定性が向上しているタンパク質である、請求項1に記載の製造方法。

[請求項3]
 前記変異型TET1タンパク質は、そのアミノ末端から2番目のアミノ酸残基が正常型TET1タンパク質のアミノ末端から2番目のアミノ酸残基とは異なるTET1タンパク質である、請求項2に記載の製造方法。

[請求項4]
 前記変異型TET1タンパク質は、さらにジオキシゲナーゼ領域を欠失しているTET1タンパク質である、請求項2又は3に記載の製造方法。

[請求項5]
 前記TET1タンパク質は、配列番号:2、4又は6に記載のアミノ酸配列からなるタンパク質である、請求項1に記載の製造方法。

[請求項6]
 変異型TET1タンパク質であって、正常型TET1タンパク質と比較して安定性が向上しているタンパク質。

[請求項7]
 アミノ末端から2番目のアミノ酸残基が正常型TET1タンパク質のアミノ末端から2番目のアミノ酸残基とは異なる、請求項6に記載のタンパク質。

[請求項8]
 さらにジオキシゲナーゼ領域が欠失している、請求項7に記載のタンパク質。

[請求項9]
 配列番号:4又は6に記載のアミノ酸配列からなるタンパク質。

[請求項10]
 請求項6~9のうちのいずれか一項に記載のタンパク質をコードする核酸。

[請求項11]
 請求項10に記載の核酸が挿入されているベクター。

[請求項12]
 下記(a)~(c)からなる群から選択される少なくとも一つの分子が導入されている、分化多能性幹細胞又は該細胞に誘導するための体細胞
(a)TET1タンパク質
(b)TET1タンパク質をコードする核酸
(c)TET1タンパク質をコードする核酸が挿入されているベクター。

  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • SAITAMA MEDICAL UNIVERSITY
  • Inventor
  • KATO Hidemasa
  • MORIYAMA Yosuke
  • HIRAKI Keiko
  • OKUDA Akihiko
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JP KE KG KN KP KR KZ LA LC LK LR LS LT LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN TD TG

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