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Human erythroid progenitor cell line comprising HPV E6/E7 operably linked to an inducible promoter and method for producing human enucleated red blood cells UPDATE 実績あり

外国特許コード F190009914
整理番号 08054-US
掲載日 2019年8月26日
出願国 アメリカ合衆国
出願番号 201313745178
公報番号 20140024118
公報番号 8975072
出願日 平成25年1月18日(2013.1.18)
公報発行日 平成26年1月23日(2014.1.23)
公報発行日 平成27年3月10日(2015.3.10)
優先権データ
  • 特願2012-161878 (2012.7.20) JP
発明の名称 (英語) Human erythroid progenitor cell line comprising HPV E6/E7 operably linked to an inducible promoter and method for producing human enucleated red blood cells UPDATE 実績あり
発明の概要(英語) Provided are: a method for producing an immortalized human erythroid progenitor cell line, enabling efficient and stable production of enucleated red blood cells; and a method for producing human enucleated red blood cells from a human erythroid progenitor cell line obtained by the aforementioned production method. An expression cassette capable of inducing expression of HPV-E6/E7 genes in the presence of DOX was introduced into the genomic DNA of blood stem cells. Then, the blood stem cells were cultured in the presence of DOX and a blood growth factor. Thereby, immortalized cell lines of human erythroid progenitor cells were established. Further, it was revealed that culturing the cell lines under a condition where the expression of the HPV-E6/E7 genes was not induced enabled differentiation induction into enucleated red blood cells at a high ratio.
従来技術、競合技術の概要(英語) BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a human erythroid progenitor cell line and a method for producing human enucleated red blood cells. More specifically, the present invention relates to a method for producing immortalized human erythroid progenitor cells (human erythroid progenitor cell line) from human blood stem cells, and a method for producing human enucleated red blood cells from a human erythroid progenitor cell line obtained by the aforementioned production method.
2. Related Background Art
The transfusion medical system has been established based on blood donation, and the system is well organized at present. However, this does not mean the system has no problem at all. One is a problem of relatively fewer donors because of population aging with declining birth rate. There is a concern about shortage of transfusion products in the future. Additionally, at the early stage of infection with hepatitis viruses, AIDS viruses, and the like, it is not always possible to determine that the patient is positive by the inspection. Accordingly, it is difficult to completely identify persons infected with these viruses and to completely eliminate the possibility that an infectious virus carrier is a blood donor. Thus, in the blood donation system from an unspecified number of donors, it is difficult to completely eliminate a risk of infectious diseases in reality. In addition, the cause of recently-defined transfusion related acute lung injury (TRALI) is believed to be an antibody present in a transfusion product. Therefore, if it becomes possible to artificially produce red blood cells from resources, for example, stem cells or immortalized cell lines, with ensured safety, it is likely to solve the existing problems such as risks of infectious diseases and TRALI.
Another reason why the artificial red blood cells production is eagerly desired comes from the necessity of guaranteeing transfusion medicine with very rare blood types. For example, persons having red blood cells without a red blood cell antigen most people in the world have (persons with type Rh-null, type -D-, or the like) need to be transfused with red blood cells not having such an antigen. For persons having such a special blood type, a strategy is conceivable, in which iPS cells are first established from such a person him/herself and blood in need is then artificially produced from the iPS cells thus established.
Now, red blood cells can be produced from pluripotent stem cells such as ES cells and iPS cells; specifically, mature red blood cells (enucleated red blood cells) can be produced in vitro through differentiation of pluripotent stem cells into blood stem cells further to erythroid progenitor cells (International Publication No. WO2009/137629). Moreover, it is also possible to produce enucleated red blood cells from umbilical cord blood-derived blood stem cells or other origins through erythroid progenitor cells (International Publication No. WO2005/118780).
However, in these methods, large amounts of various growth factors and so forth are needed to induce differentiation of pluripotent stem cells or blood stem cells into enucleated red blood cells, requiring high cost. This is because in a case of inducing differentiation of pluripotent stem cells, cells other than blood cells are also included at the early stage after the differentiation induction, and a long culturing period and a large culture volume are needed to obtain a required amount of blood cells. Meanwhile, in a case of using either blood stem cells induced and differentiated from pluripotent stem cells or blood stem cells derived from umbilical cord blood or other origins, when the blood stem cells are induced to differentiate into erythroid cells, blood cells other than erythroid cells are also included at the early stage after the differentiation induction.
Hence, it is necessary for obtaining a required red blood cell volume to culture for a long culturing period and culture in a large. Thus, to produce artificial red blood cells, a development has been demanded in a method that enables efficient and stable production of enucleated red blood cells.
In view of such a circumstance, the present inventors have attempted to establish, as immortalized cell lines having a semi-permanent growth potential, erythroid progenitor cells immediately before differentiation into red blood cells. As a result, by in vitro differentiation-induction manipulation, such immortalized cell lines of erythroid progenitor cells were successfully obtained from mouse ES cells. Further, the present inventors have also revealed that the cell lines are able to stably and efficiently produce enucleated red blood cells (Hiroyama T. et al., “Establishment of mouse embryonic stem cell-derived erythroid progenitor cell lines able to produce functional red blood cells,” PLoS One, Feb. 6, 2008, vol. 3, issue 2, e1544).
特許請求の範囲(英語) [claim1]
1. A method for producing a human erythroid progenitor cell line for producing human enucleated red blood cells, the method comprising the steps of:
introducing into human hematopoietic stem cells an expression cassette comprising E6 and E7 genes of human papillomavirus type 16 operably linked to a promoter responsive to an added external stimulus;
culturing the human hematopoietic stem cells having the expression cassette introduced therein in the presence of the external stimulus and at least one blood growth factor selected from the group consisting of SCF, EPO, TPO and DEX under conditions to produce human erythroid progenitor cells, and
identifying human erythroid progenitor cells in the culture.

[claim2]
2. The method according to claim 1, wherein the human hematopoietic stem cells are cells obtained by inducing differentiation of human pluripotent stem cells expressing a TAL1 gene.

[claim3]
3. A human erythroid progenitor cell line comprising an expression cassette comprising E6 and E7 genes of human papillomavirus type 16 operably linked to a promoter responsive to an added external stimulus, wherein the human erythroid progenitor cell line grows in the presence of the external stimulus and at least one blood growth factor selected from the group consisting of SCF, EPO, TPO and DEX and produces human enucleated red blood cells by culturing in the absence of the external stimulus.

[claim4]
4. A method for producing human enucleated red blood cells, the method comprising the step of culturing the human erythroid progenitor cell line obtained by the method according to any one of claims 1 and 2 or the human erythroid progenitor cell line according to claim 3 in absence of the external stimulus.

[claim5]
5. A method for producing human enucleated red blood cells, the method comprising the steps of:
introducing into human hematopoietic stem cells an expression cassette comprising E6 and E7 genes of human papillomavirus type 16 operably linked to a promoter responsive in response to an added external stimulus;
culturing the human hematopoietic stem cells having the expression cassette introduced therein in the presence of the external stimulus and at least one blood growth factor selected from the group consisting of SCF, EPO, TPO and DEX, thereby inducing the expression of E6 and E7 genes of human papillomavirus type 16 in the cells and obtaining human erythroid progenitor cells; and
culturing the human erythroid progenitor cells in absence of the external stimulus, thereby suppressing the expression of E6 and E7 genes of human papillomavirus type 16 in the cells and inducing the production of human enucleated red blood cells.

[claim6]
6. The method according to claim 5, wherein the human hematopoietic stem cells are cells obtained by inducing differentiation of human pluripotent stem cells expressing a TAL1 gene.

[claim7]
7. A human erythroid stem cell comprising an expression cassette comprising E6 and E7 genes of human papillomavirus type 16 operably linked to a promoter responsive to an added external stimulus.

[claim8]
8. The human erythroid stem cells according to claim 7, wherein the human hematopoietic stem cell is a cell obtained by inducing differentiation of human pluripotent stem cells expressing a TAL1 gene.
  • 発明者/出願人(英語)
  • NAKAMURA Yukio
  • KURITA Ryo
  • RIKEN
国際特許分類(IPC)

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