Recent advances in cellular reprogramming have enabled the generation of embryoniclike
cells from virtually any cell of the body. These inducible pluripotent stem cells
(iPSCs) are capable of indefinite self-renewal while maintaining the ability to
differentiate into all cell types. Nowhere will this technology have a greater impact than
in the ability to generate disease and patient-specific cell lines. Here we explore the
capacity of human iPSCs reprogrammed from peripheral blood endothelial progenitor
cells lines to differentiate into atrial myocytes for the study of patient specific atrial
physiology.
Methods and Results: Late outgrowth endothelial progenitor cells (EPCs) cultured from
clinical blood samples provided a robust cell source for genetic reprogramming.
Transcriptome analysis hinted that EPCs would be comparatively more amenable to
pluripotent reprogramming than the traditional dermal fibroblast. After 6 passages,
EPCs were transduced with a doxycycline inducible lentivirus system encoding human
transcription factors OCT4, SOX2, KLF4 and Nanog to permit differentiation after
removal of doxycycline. The high endogenous expression of key pluripotency transcripts
enhanced the ease of iPSC generation as demonstrated by the rapid emergence of typical
iPSC colonies. Following removal of doxycycline, genetically reprogrammed EPC-iPSC
colonies displayed phenotypic characteristics identical to human embryonic stem cells
and expressed high levels of the pluripotent markers SSEA-4, TRA1-60 and TRA1-81.
After exposure to conditions known to favor atrial identity, EPC- iPSC differentiating
into sheets of beating cardiomyocytes that expressed high levels of several atrial-specific
expressed genes (CACNA1H, KCNA5, and MYL4).
Conclusions: EPCs provide a stable platform for genetic reprogramming into a
pluripotent state using a doxycycline conditional expression system that avoids reexpression
of oncogenic/pluripotent factors. Human EPC-derived iPSC can be
differentiated into functional cardiomyocytes that express characteristic markers of atrial
identity.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OOU.#10393/31158 |
| Date | 30 May 2014 |
| Creators | Jambi, Majed |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Thèse / Thesis |
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