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Combining hiPSC and CRISPR/ Cas9 technologies for studying cardiac disease and development

2016-07-11 13:15:19

Medizinische Klinik  March 7 2017



Lisa K. Dreizehnter, Dr. Jürgen Ruland
, Dr. Karl-Ludwig Laugwitz



Abstract


Reprogramming of somatic cells into human induced pluripotent stem cells (hiPSCs) by
expression of a defined set of transcription factors allows patient- specific generation of
hiPSC lines. By differentiation of patient hiPSCs into cardiomyocytes (CMs) in vitro,
disease mechanisms and pathogenic pathways can be investigated. Two varying heterozygous
de novo point mutations on the CALM1 gene were previously identified as long
QT syndrome (LQTS)- causing mutations by whole genome sequencing of two patients. hiPSC lines were generated of patient’s skin fibroblasts, characterized and differentiated
into patient- specific CMs. hiPSC- CMs of one patient were further analysed. Electrophysiological
examinations confirmed (AP) prolongation. Incomplete Ca2+
- dependent
inactivation (CDI) of Cav1.2 channels was identified as underlying cause of the mutationinduced
electrical abnormalities



Introduction


hESC research raises many ethical and legal issues since it requires derivation of cells
from blastocysts or embryos 14. Derivation of patient-specific ESCs for disease modelling
is difficult 15. The discovery by Takahashi and Yamanaka in 2006, that somatic cells can
be induced to become pluripotent stem cells, known as induced pluripotent stem cells
(iPSCs), revolutionised stem cell research and led the path to a new level of disease modelling
15
. One year after the first report on mouse iPSC generation by ectopic overexpression
of a defined set of transcription factors in mouse fibroblasts 16, Yamanaka and
Takahashi reported the generation of human iPSCs (hiPSCs) by transfection of adult fibroblasts
with the same set of transcription factors 15. Adult human dermal fibroblasts
were transduced with retroviruses containing OCT3/4, SOX2, KLF4, and c-MYC and
cultured on proliferation- deficient mouse embryonic fibroblasts (MEFs). Thorough characterizations
showed expression of ESC marker genes OCT3/4, SOX2, NANOG, GDF3,
REX1, FGF4, ESG1, DPPA2, DPPA4, and hTERT by qRT PCR at levels similar to those
of hESCs. Expression of ESC specific surface antigens SSEA-3, SSEA-4, TRA-1-60,
TRA-1-81 and TRA-2-49/6E was demonstrated. Embryonic body (EB) formation proved
the capacity of the cells to differentiate into endodermal, ectodermal and mesodermal
lineages 15. Reprogramming human cells was a ground breaking discovery and opened up
a completely new field of research in disease modelling, drug discovery, therapy and
many more 17. Shinya Yamanaka received 2012 the Nobel Prize in Physiology and Medicine
for his work 18



In this study, our aim was to eliminate an entire chromosome in human cells to generate normal disomic cells from trisomic cells. Previous research has indicated that XO mice lacking the Y chromosome can be created using a pair of loxP sites in an inverted orientation. When male mice carrying the Y chromosome containing inverted loxP sites are mated with females carrying a Cre gene, chromosome loss is induced by recombination which is mediated by Cre present between the loxP sites on sister chromatids during embryogenesis. Moreover, targeted chromosome elimination has been achieved in mouse embryonic stem-somatic hybrid cells using a Cre-inverted loxP system that included a cassette consisting of green fluorescent protein (GFP) and drug-resistant genes bracketed by a pair of inverted loxP sites. To adapt this system to human cells, we developed a modified cassette containing two inverted loxP sites, in which a counter selectable gene HSV-TK was added to efficiently select cells lacking the targeted chromosome. This cassette was integrated into the target site by homologous recombination using the clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins 9 (CRISPR/Cas9) nickase system.





Empire Genomic's Custom RP-11 Probes were used in this publication.



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