Strategies for rapidly mapping proviral integration sites and assessing cardiogenic potential of nascent human induced pluripotent stem cell clones.
Recent methodological advances have improved the ease and efficiency of generating human induced pluripotent stem cells (hiPSCs), but this now typically results in a greater number of hiPSC clones being derived than can be wholly characterized. It is therefore imperative that methods are developed which facilitate rapid selection of hiPSC clones most suited for the downstream research aims. Here we describe a combination of procedures enabling the simultaneous screening of multiple clones to determine their genomic integrity as well as their cardiac differentiation potential within two weeks of the putative reprogrammed colonies initially appearing. By coupling splinkerette-PCR with Ion Torrent sequencing, we could ascertain the number and map the proviral integration sites in lentiviral-reprogrammed hiPSCs. In parallel, we developed an effective cardiac differentiation protocol that generated functional cardiomyocytes within 10 days without requiring line-specific optimization for any of the six independent human pluripotent stem cell lines tested. Finally, to demonstrate the scalable potential of these procedures, we picked 20 nascent iPSC clones and performed these independent assays concurrently. Before the clones required passaging, we were able to identify clones with a single integrated copy of the reprogramming vector and robust cardiac differentiation potential for further analysis.
Department of Cardiology, Leiden University Medical Center, Leiden, The Netherlands; Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands.
Cheryl
C
Dambrot
Leiden Genome Technology Center, Center for Human and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands.
Henk P J
HP
Buermans
BioTalentum Ltd., Gödöllö, Hungary; Molecular Animal Biotechnology Laboratory, Szent Istvan University, Gödöllö, Hungary.
Eszter
E
Varga
Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands.
Georgios
G
Kosmidis
Pluriomics B.V., Leiden, The Netherlands.
Karin
K
Langenberg
Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands.
Simona
S
Casini
Murdoch Childrens Research Institute, The Royal Children's Hospital, Parkville, VIC, Australia.
David A
DA
Elliott
BioTalentum Ltd., Gödöllö, Hungary; Molecular Animal Biotechnology Laboratory, Szent Istvan University, Gödöllö, Hungary.
Andras
A
Dinnyes
Department of Cardiology, Leiden University Medical Center, Leiden, The Netherlands.
Douwe E
DE
Atsma
Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands; Netherlands Proteomics Institute, Utrecht, The Netherlands.
Christine L
CL
Mummery
Pluriomics B.V., Leiden, The Netherlands. Electronic address: stefan.braam@pluriomics.com.
Stefan R
SR
Braam
Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands; Netherlands Proteomics Institute, Utrecht, The Netherlands. Electronic address: r.p.davis@lumc.nl.
Richard P
RP
Davis
D015139:Blotting, Southern; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D065150:Cellular Reprogramming; D003782:Dental Pulp / Q000166:cytology / Q000378:metabolism; D020405:Dermis / Q000166:cytology / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D057166:High-Throughput Screening Assays; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000378:metabolism; D011533:Proviruses / Q000235:genetics*; D016662:Virus Integration / Q000235:genetics*
Cardiomyocytes; Cell differentiation; High-throughput DNA sequencing; Human induced pluripotent stem cell