ES2664194T3 - Método novedoso para la producción de células diferenciadas - Google Patents
Método novedoso para la producción de células diferenciadas Download PDFInfo
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- ES2664194T3 ES2664194T3 ES10817186.9T ES10817186T ES2664194T3 ES 2664194 T3 ES2664194 T3 ES 2664194T3 ES 10817186 T ES10817186 T ES 10817186T ES 2664194 T3 ES2664194 T3 ES 2664194T3
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- 238000004519 manufacturing process Methods 0.000 title abstract description 3
- 238000000034 method Methods 0.000 title description 2
- 210000004027 cell Anatomy 0.000 abstract description 40
- 101000872170 Homo sapiens Polycomb complex protein BMI-1 Proteins 0.000 abstract description 16
- 230000014509 gene expression Effects 0.000 abstract description 10
- 108090000623 proteins and genes Proteins 0.000 abstract description 10
- 210000003593 megakaryocyte Anatomy 0.000 abstract description 5
- 210000003958 hematopoietic stem cell Anatomy 0.000 abstract description 3
- 230000001939 inductive effect Effects 0.000 abstract description 3
- 210000002361 Megakaryocyte Progenitor Cell Anatomy 0.000 abstract description 2
- 108700020796 Oncogene Proteins 0.000 abstract description 2
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 abstract 1
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 abstract 1
- 108091057508 Myc family Proteins 0.000 abstract 1
- 238000012258 culturing Methods 0.000 abstract 1
- 102100033566 Polycomb complex protein BMI-1 Human genes 0.000 description 15
- 238000004458 analytical method Methods 0.000 description 9
- 230000004069 differentiation Effects 0.000 description 7
- VOXZDWNPVJITMN-ZBRFXRBCSA-N 17β-estradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 VOXZDWNPVJITMN-ZBRFXRBCSA-N 0.000 description 6
- 239000013598 vector Substances 0.000 description 6
- 238000012413 Fluorescence activated cell sorting analysis Methods 0.000 description 4
- 230000004913 activation Effects 0.000 description 3
- 210000001671 embryonic stem cell Anatomy 0.000 description 3
- 229960005309 estradiol Drugs 0.000 description 3
- 102000006495 integrins Human genes 0.000 description 3
- 108010044426 integrins Proteins 0.000 description 3
- 101150062914 BMI1 gene Proteins 0.000 description 1
- 108010049003 Fibrinogen Proteins 0.000 description 1
- 102000008946 Fibrinogen Human genes 0.000 description 1
- 101150085741 Hoxa2 gene Proteins 0.000 description 1
- 102100025306 Integrin alpha-IIb Human genes 0.000 description 1
- 102000008607 Integrin beta3 Human genes 0.000 description 1
- 108010020950 Integrin beta3 Proteins 0.000 description 1
- 101100058550 Mus musculus Bmi1 gene Proteins 0.000 description 1
- 108010035030 Platelet Membrane Glycoprotein IIb Proteins 0.000 description 1
- 102100034173 Platelet glycoprotein Ib alpha chain Human genes 0.000 description 1
- 101710107770 Platelet glycoprotein Ib alpha chain Proteins 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 229940012952 fibrinogen Drugs 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 210000001778 pluripotent stem cell Anatomy 0.000 description 1
- 238000000159 protein binding assay Methods 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0641—Erythrocytes
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Abstract
Método para producir células megacariocíticas maduras que comprende: inducir de forma artificial la expresión forzada de un oncogén seleccionado entre los genes de la familia MYC y el gen polycomb BMI1 en células progenitoras hematopoyéticas, células CD34 positivas o células progenitoras megacariocíticas sin poliploidización múltiple y cultivar y hacer crecer las células; y diferenciar las células obtenidas mediante crecimiento en células megacariocíticas maduras.
Description
BMI1, mientras que c-MYC únicamente (vista inferior) hace referencia a los resultados del análisis FACS de las células en las que solamente se introdujo el gen c-MYC. En la figura 11, se muestran los resultados del análisis FACS de las células el día 35 de cultivo, en las que se introdujo el gen c-MYC y el gen BMI1. A hace referencia, de manera esquemática, a las moléculas funcionales específicas de los megacariocitos y B hace referencia a los resultados del análisis FACS. En la figura 12, se muestran los resultados del análisis de la capacidad de crecimiento de las células de expresión de MYC/BMI1. El eje vertical representa la cantidad de células. El eje horizontal representa la cantidad de días después de la introducción de los genes en las células. En la figura 13, se muestra una imagen de plaquetas liberadas de células progenitoras megacariocíticas derivadas de células de expresión de c-MYC/BMI1, según se observa a través de un microscopio electrónico. En la figura 14, se muestran los resultados del análisis FACS de las células el día 105 después de la introducción del gen c-MYC y del gen HOXA2 en células progenitoras hematopoyéticas derivadas de células madre embrionarias (KhES) y el día 27 después de la introducción del gen c-MYC y el gen BCLXL en células progenitoras hematopoyéticas derivadas de células madre embrionarias (KhES). En la figura 15, se muestra una vista para la confirmación de un sistema de regulación de expresión de genes mediante un vector pMX tet off. Se expresó un constructo en el que c-MYC y BMI1 se unieron al vector pMX tet off con 2A en medio en células 293GPG para analizar si la regulación de la expresión de genes funciona o no. A se refiere al constructo y el mecanismo del vector y B se refiere a los resultados del análisis de la expresión de c-MYC en células en un estado en el que se añade o no tetraciclina y βestradiol, mediante la utilización de un citómetro de flujo. El eje horizontal en B representa un nivel de expresión de c-MYC. "293gpg" se refiere a los resultados de las células 293GPG de un control. En la figura 16, se muestran los resultados del análisis de la capacidad de crecimiento y la capacidad de diferenciación de líneas celulares de expresión de vector de regulación de genes. A se refiere a los resultados del análisis de la capacidad de crecimiento de las células de expresión de c-MYC y BMI1 mediante diversos vectores. El eje vertical representa la cantidad de células y el eje horizontal representa la cantidad de días después de la introducción de los genes en las células. B se refiere a los resultados del análisis de las células teñidas con un anticuerpo anti-CD42b (GPIb-alfa) y un anticuerpo anti-CD41a (complejo integrina alfaIIb/beta3) (vista superior) y un anticuerpo antiglicoforina A y un anticuerpo anti-CD41a (vista inferior), mediante la utilización de un citómetro de flujo. Tanto en la vista superior como en la inferior de B, se muestran por separado los resultados de células que expresan forzadamente pMX c-MYC y Dsam BMI1 en el lado izquierdo y en el lado derecho se muestran los resultados de las células que expresan pMX tet off c-MYC 2A BMI1. En la figura 17, se muestra el análisis del grado de multinucleación de una línea celular megacariocítica que expresa pMX tet off c-MYC 2A BMI1 en presencia de β-estradiol. A se refiere a los resultados de las células de un control con solamente un vector (una línea celular que no expresa genes) y B se refiere a los resultados de las células que expresan c-MYC y BMI1. En la figura 18, se muestran los resultados de la realización de ensayos de unión de fibrinógenos en plaquetas derivadas de megacariocitos que expresan de forma forzada c-MYC y BMI1. En la vista superior (plaqueta humana), se muestran los resultados de plaquetas derivadas de sangre periférica humana; en la vista media (pMX tet off c-MYC 2A BMI1), se muestran los resultados de plaquetas derivadas de una línea celular de pMX tet off c-MYC 2A BMI1 en presencia de β-estradiol; y en la vista inferior (pMX Myc Dsam Bmi1), se muestran los resultados de plaquetas derivadas de una línea celular que expresa de forma forzada c-MYC y BMI1 mediante pMX c-MYC y Dsam BMI1. En la figura 19, se muestran los resultados del análisis de la capacidad de activación de integrina de plaquetas producidas a partir de una línea celular megacariocítica en la que se suprimió la expresión de c-MYC y BMI1. En la vista izquierda, se muestra el análisis de la capacidad de activación de integrina en ausencia de ADP mediante la utilización de un citómetro de flujo, mientras que en la vista derecha se muestra un análisis de la capacidad de activación de integrina en presencia de ADP (50 µM) mediante la utilización de un citómetro de flujo. En la figura 20, se muestra una vía de diferenciación desde células madre embrionarias a una línea celular megacariocítica.
Modo de llevar a cabo la invención
[0025] En la presente memoria, se da a conocer un método para la producción de células específicas mediante la inducción de la diferenciación de células que sirven como fuente, donde un oncogén se expresa de forma forzada en células en una fase de diferenciación deseada en un proceso de diferenciación desde las células que sirven como fuente en las células específicas, con el fin de amplificar (o hacer crecer) las células en la fase de diferenciación deseada.
[0026] En el presente documento, "células que sirven como fuente" corresponden a células progenitoras de células diana (células específicas) obtenidas mediante la inducción de la diferenciación y pueden ser cualesquiera células que conserven la capacidad de diferenciación distintas de las células terminalmente diferenciadas. Por ejemplo, las "células que sirven como fuente" pueden ser células madre pluripotentes completamente no diferenciadas o células que están diferenciadas en cierta medida pero todavía conservan
7
Claims (1)
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imagen1
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2009213645 | 2009-09-15 | ||
| JP2009213645 | 2009-09-15 | ||
| PCT/JP2010/065903 WO2011034073A1 (ja) | 2009-09-15 | 2010-09-15 | 分化細胞の新規製造法 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| ES2664194T3 true ES2664194T3 (es) | 2018-04-18 |
Family
ID=43758674
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| ES10817186.9T Active ES2664194T3 (es) | 2009-09-15 | 2010-09-15 | Método novedoso para la producción de células diferenciadas |
| ES16202612T Active ES2725688T3 (es) | 2009-09-15 | 2010-09-15 | Método novedoso para la producción de células diferenciadas |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| ES16202612T Active ES2725688T3 (es) | 2009-09-15 | 2010-09-15 | Método novedoso para la producción de células diferenciadas |
Country Status (13)
| Country | Link |
|---|---|
| US (2) | US9200254B2 (es) |
| EP (2) | EP2500418B1 (es) |
| JP (4) | JP5791191B2 (es) |
| KR (2) | KR101667834B1 (es) |
| CN (2) | CN102712906B (es) |
| AU (1) | AU2010296415B2 (es) |
| BR (2) | BR112012005710B1 (es) |
| CA (1) | CA2774193C (es) |
| DK (2) | DK2500418T3 (es) |
| ES (2) | ES2664194T3 (es) |
| IN (1) | IN2012DN02642A (es) |
| RU (2) | RU2603094C2 (es) |
| WO (1) | WO2011034073A1 (es) |
Families Citing this family (35)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012043651A1 (ja) * | 2010-09-30 | 2012-04-05 | 国立大学法人 熊本大学 | ミエロイド系血液細胞の製造方法 |
| EP2708597B1 (en) * | 2011-05-13 | 2017-11-15 | The University of Tokyo | Polynucleated megakaryocytic cell and method for manufacturing platelets |
| KR102031386B1 (ko) * | 2011-10-03 | 2019-10-11 | 닛산 가가쿠 가부시키가이샤 | 다능성 간세포로부터의 거핵구 및/또는 혈소판의 제조 방법 |
| GB201210857D0 (en) | 2012-06-19 | 2012-08-01 | Cambridge Entpr Ltd | Transcription factor mediated programming towards megakaryocytes |
| CA3177929A1 (en) | 2012-12-21 | 2014-06-26 | Astellas Institute For Regenerative Medicine | Methods for production of platelets from pluripotent stem cells and compositions thereof |
| US20160002599A1 (en) | 2013-02-08 | 2016-01-07 | Kyoto University | Production methods for megakaryocytes and platelets |
| JP6385340B2 (ja) * | 2013-04-12 | 2018-09-05 | 国立大学法人京都大学 | 巨核球の成熟化促進物質 |
| WO2014171486A1 (ja) | 2013-04-17 | 2014-10-23 | 日産化学工業株式会社 | 培地組成物及び当該培地組成物を用いた赤血球の製造方法 |
| WO2014186508A1 (en) * | 2013-05-15 | 2014-11-20 | University Of Rechester | Human extensively self-renewing erythroblasts (esre) |
| US10112977B2 (en) | 2014-06-23 | 2018-10-30 | Toagosei Co., Ltd. | Peptide for inducing multinucleation in cells, and use therefor |
| CA2979173A1 (en) * | 2015-03-09 | 2016-09-15 | Megakaryon Corporation | Method for producing culture containing megakaryocytes, and method for producing platelets using same |
| EP3296390B1 (en) * | 2015-04-14 | 2023-01-04 | Kyoto University | Method for producing stem cell clones suitable for induction of differentiation into somatic cells |
| KR20160129609A (ko) | 2015-04-30 | 2016-11-09 | 삼성전자주식회사 | Ｂｒａｆ 저해제를 포함하는 세포 또는 개체의 노화를 감소시키기 위한 조성물 및 그의 용도 |
| KR102664131B1 (ko) * | 2015-11-02 | 2024-05-10 | 가부시키가이샤 메가카리온 | 왕복 이동 교반 장치를 사용한 혈소판의 제조 방법 |
| EP3409774B1 (en) | 2016-01-29 | 2025-03-19 | Kyoto University | Screening method of platelet production promoter |
| WO2017131230A1 (ja) | 2016-01-29 | 2017-08-03 | 国立大学法人京都大学 | 血小板産生促進剤及びそれを用いた血小板の製造方法 |
| WO2018164040A1 (ja) | 2017-03-06 | 2018-09-13 | 国立大学法人京都大学 | 血小板の製造方法 |
| FR3068366B1 (fr) * | 2017-06-30 | 2023-11-24 | Francais Du Sang Ets | Procede de production de progeniteurs erythroides |
| WO2019009364A1 (ja) | 2017-07-07 | 2019-01-10 | 国立大学法人京都大学 | 血小板の製造方法および製造装置、ならびに血小板の製造装置における運転条件の決定方法 |
| WO2019059234A1 (ja) | 2017-09-19 | 2019-03-28 | 株式会社メガカリオン | 血小板の製造方法、血小板製剤の製造方法、および血液製剤の製造方法 |
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| GB9502022D0 (en) * | 1995-02-02 | 1995-03-22 | Abuljadayel Ilham M S | A method for preparing lymphohaematopoietic progenitor cells |
| US5723333A (en) | 1995-02-10 | 1998-03-03 | Regents Of The University Of California | Human pancreatic cell lines: developments and uses |
| ATE311466T1 (de) | 1995-02-10 | 2005-12-15 | Univ California | Menschliche pancreas zellinien: entwicklungen und anwendungen |
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| JP2006141356A (ja) | 2004-11-24 | 2006-06-08 | Institute Of Physical & Chemical Research | Ｅｓ細胞から樹立された造血幹細胞 |
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| AU2008297024B2 (en) * | 2007-10-31 | 2014-08-28 | Kyoto University | Nuclear reprogramming method |
| CN101492676B (zh) * | 2008-09-16 | 2011-02-16 | 中国科学院广州生物医药与健康研究院 | 用脑膜细胞生成诱导的多能性干细胞的方法及其用途 |
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