Derivation of human induced pluripotent stem cells and Advanced Methods of Image Processing

KRONTORÁD KOUTNÁ, Irena, Pavel ŠIMARA, Pavel MATULA, Lenka TESAŘOVÁ and Barbara ŠALINGOVÁ. Derivation of human induced pluripotent stem cells and Advanced Methods of Image Processing. In CEITEC PhD Retreat, 23-24.4.2015, Valtice Czech Republic. 2015.

Basic information

• Original name: Derivation of human induced pluripotent stem cells and Advanced Methods of Image Processing
• Name in Czech: Příprava lidských indukovaných kmenových buněk a pokročilé metody zpracování obrazu
• Authors: KRONTORÁD KOUTNÁ, Irena (203 Czech Republic, guarantor, belonging to the institution), Pavel ŠIMARA (203 Czech Republic, belonging to the institution), Pavel MATULA (203 Czech Republic, belonging to the institution), Lenka TESAŘOVÁ (203 Czech Republic, belonging to the institution) and Barbara ŠALINGOVÁ (703 Slovakia, belonging to the institution).
• Edition: CEITEC PhD Retreat, 23-24.4.2015, Valtice Czech Republic, 2015.

Other information

• Original language: English
• Type of outcome: Requested lectures
• Field of Study: 10600 1.6 Biological sciences
• Country of publisher: Czech Republic
• Confidentiality degree: is not subject to a state or trade secret
• RIV identification code: RIV/00216224:14330/15:00081082
• Organization unit: Faculty of Informatics
• Keywords (in Czech): iPSC; ESC;zpracování obrazu
• Keywords in English: iPSC; ESC; Image processing

Abstract

Human induced pluripotent stem cells (hiPSCs) possess a great potential for clinical application. Various reprogramming techniques were presented during 7 years of hiPSCs research. Genome non-integrating and completely xeno-free protocols from the first biopsy to stable hiPSCs clones are highly preferable in terms of future clinical application. In CBIA lab we successfully generated hiPSCs using STEMCCA lentivirus, Sendai virus or episomal vectors. Human fibroblasts and CD34+ blood progenitors were used as a source cells and were maintained either on mouse embryonic feeder cells or in feeder-free conditions. The reprogramming efficiency was comparable for all three methods and both cell types, while the best results were obtained in feeder-free conditions. The pluripotency of our hiPSCs was verified by differentiation into all three germ layers and by teratoma assay. In order to study genomic integrity, we monitored DNA damage response (DDR). Image analysis of imunohistochemical or ImunoFISH experiments is very important step of iPSCs evaluation in the context of their genome integrity and stability. In CBIA lab was develop unique software “Acquiarium” for this advanced image analysis. Acquiarium is open source software (GPL) for carrying out the common pipeline of many spatial cell studies using fluorescence microscopy. It addresses image capture, raw image correction, image segmentation, quantification of segmented objects and their spatial arrangement, volume rendering, and statistical evaluation. It is focused on quantification of spatial properties of many objects and their mutual spatial relations in a collection of many 3D images. It can be used for analysis of a collection of 2D images or time lapse series of 2D or 3D images as well. It has a modular design and is extensible via plug-ins. It is a stand-alone, easy to install application written in C++ language. The GUI is written using cross-platform wxWidgets library.

Abstract (in Czech)

Human induced pluripotent stem cells (hiPSCs) possess a great potential for clinical application. Various reprogramming techniques were presented during 7 years of hiPSCs research. Genome non-integrating and completely xeno-free protocols from the first biopsy to stable hiPSCs clones are highly preferable in terms of future clinical application. In CBIA lab we successfully generated hiPSCs using STEMCCA lentivirus, Sendai virus or episomal vectors. Human fibroblasts and CD34+ blood progenitors were used as a source cells and were maintained either on mouse embryonic feeder cells or in feeder-free conditions. The reprogramming efficiency was comparable for all three methods and both cell types, while the best results were obtained in feeder-free conditions. The pluripotency of our hiPSCs was verified by differentiation into all three germ layers and by teratoma assay. In order to study genomic integrity, we monitored DNA damage response (DDR). Image analysis of imunohistochemical or ImunoFISH experiments is very important step of iPSCs evaluation in the context of their genome integrity and stability. In CBIA lab was develop unique software “Acquiarium” for this advanced image analysis. Acquiarium is open source software (GPL) for carrying out the common pipeline of many spatial cell studies using fluorescence microscopy. It addresses image capture, raw image correction, image segmentation, quantification of segmented objects and their spatial arrangement, volume rendering, and statistical evaluation. It is focused on quantification of spatial properties of many objects and their mutual spatial relations in a collection of many 3D images. It can be used for analysis of a collection of 2D images or time lapse series of 2D or 3D images as well. It has a modular design and is extensible via plug-ins. It is a stand-alone, easy to install application written in C++ language. The GUI is written using cross-platform wxWidgets library.

Links

• GBP302/12/G157, research and development project: Name: Dynamika a organizace chromosomů během buněčného cyklu a při diferenciaci v normě a patologii

Investor: Czech Science Foundation