Abstract
Human pluripotent stem cells hold great promise for their practical and scientific potentials. To improve understanding of self-renewal and differentiation, we previously reported a defined serum-free medium hESF9 could generate and maintain human induced pluripotent stem cells (iPSCs) in serum- and feeder-free culture conditions using retroviral vectors. To avoid the unpredictable side effects associated with retrovirus integration, we report here the successful generation of hiPSCs from dental pulp cells with a non-integrating replication-defective and persistent Sendai virus (SeVdp) vector expressing four key reprogramming genes. We found that hESF9 medium in combination with fibronectin are effective for generating and maintaining hiPSCs with SeVdp (KOSM). Using this system, pluripotent and self-renewing hiPSCs could be easily and stably generated and propagated. With this system, we successfully generated hiPSCs from cleidocranial dysplasia (CCD) caused by a heterozygous germ-line mutation of runt-related protein2 (RUNX2), which has an important role in the differentiation of osteoblasts and maturation of chondrocytes. This is the first report of the establishment of CCD-specific iPSCs. The cartilage in the teratomas of CCD-iPSCs showed abnormalities. These CCD-iPSCs would be beneficial to clarify the molecular mechanism and for development of medical applications. Moreover, it brings new pathophysiological role of RUNX2 in the differentiation of the human chondrocytes and osteocytes.Electronic supplementary materialThe online version of this article (doi:10.1007/s11626-015-9968-x) contains supplementary material, which is available to authorized users.
Highlights
The generation of induced pluripotent stem cells by expression of exogenous defined factors provides valuable tools for transplantation therapies and regenerative medicine using patient-specific stem cells and for understanding the mechanisms of human diseases
The SeVdp vector can express up to four exogenous genes simultaneously at a fixed ratio, and it can be erased quickly by interfering vectorencoded RNA polymerase. These characteristics are ideal for generating high-quality, exogenous gene-free induced pluripotent stem cells (iPSCs), and we demonstrated that SeVdp installed with Yamanaka’s four factors could reprogram human tissue cells (Nishimura et al 2011; Nakanishi and Otsu 2012)
dental pulp cells (DPCs) were harvested in 0.05% trypsin-0.02% ethylenediamine tetraacetic acid (EDTA) in Ca2+and Mg2+-free phosphate-buffered saline (CMF-PBS), and the trypsin was inactivated with 0.1% soybean trypsin inhibitor
Summary
The generation of induced pluripotent stem cells (iPSCs) by expression of exogenous defined factors provides valuable tools for transplantation therapies and regenerative medicine using patient-specific stem cells and for understanding the mechanisms of human diseases. DNA-integrative retroviral and lentiviral vectors have been used widely because of the stable expression of transgenes owing to chromosomal insertion of the vector genome (Takahashi et al 2007; Yu et al 2007; Lowry et al 2008). Silencing the expression of exogenous transgenes is indispensable for maintaining pluripotency (Zhou and Zeng 2013), and genome-integrating viral vectors can produce insertional mutations, which may influence differentiation potential while reactivation of the cMyc oncogene may lead to tumorigenesis (Okita et al 2007). For these reasons, more efficient and safer reprogramming methods have been explored to generate iPSCs carrying no exogenous genetic material
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