Establishment of transgene-free induced pluripotent stem cells reprogrammed from human stem cells of apical papilla for neural differentiation

Abstract

IntroductionInduced pluripotent stem cells (iPSCs) are a potent cell source for neurogenesis. Previously we have generated iPSCs from human dental stem cells carrying transgene vectors. These exogenous transgenes may affect iPSC behaviors and limit their clinical applications. The purpose of this study was to establish transgene-free iPSCs (TF-iPSCs) reprogrammed from human stem cells of apical papilla (SCAP) and determine their neurogenic potential.MethodsA single lentiviral 'stem cell cassette' flanked by the loxP site (hSTEMCCA-loxP), encoding four human reprogramming factors, OCT4, SOX2, KLF4, and c-MYC, was used to reprogram human SCAP into iPSCs. Generated iPSCs were transfected with plasmid pHAGE2-EF1α-Cre-IRES-PuroR and selected with puromycin for the TF-iPSC subclones. PCR was performed to confirm the excision of hSTEMCCA. TF-iPSC clones did not resist to puromycin treatment indicating no pHAGE2-EF1α-Cre-IRES-PuroR integration into the genome. In vitro and in vivo analyses of their pluripotency were performed. Embryoid body-mediated neural differentiation was undertaken to verify their neurogenic potential.ResultsTF-SCAP iPSCs were generated via a hSTEMCCA-loxP/Cre system. PCR of genomic DNA confirmed transgene excision and puromycin treatment verified the lack of pHAGE2-EF1α-Cre-IRES-PuroR integration. Transplantation of the TF-iPSCs into immunodeficient mice gave rise to teratomas containing tissues representing the three germ layers -- ectoderm (neural rosettes), mesoderm (cartilage and bone tissues) and endoderm (glandular epithelial tissues). Embryonic stem cell-associated markers TRA-1-60, TRA-2-49 and OCT4 remained positive after transgene excision. After neurogenic differentiation, cells showed neural-like morphology expressing neural markers nestin, βIII-tubulin, NFM, NSE, NeuN, GRM1, NR1 and CNPase.ConclusionsTF-SCAP iPSCs reprogrammed from SCAP can be generated and they may be a good cell source for neurogenesis.

Human iPSC Glial Mouse Chimeras Reveal Glial Contributions to Schizophrenia

“…Fibroblasts were isolated from each sample; from these, 11 new independent hiPSC lines were derived from 8 patient samples (5 juvenile-onset schizophrenia patients and 3 healthy gender-matched and age-analogous controls (Table S1). iPSCs were generated using excisable floxed polycistronic hSTEMCCA lentivirus (Somers et al, 2010; Zou et al, 2012) encoding Oct4, Sox2, Klf4 and c-Myc (Takahashi et al, 2007; Welstead et al, 2008). All lines were initially characterized and validated as pluripotent using global transcriptome profiling by RNA sequencing to assess pluripotent gene expression, as well as immunostaining for Oct4, Nanog, and SSEA4.…”

Transgene-free induced pluripotent dental stem cells for neurogenic differentiation

“…To date the production of sufficient numbers of functional neurons or glial cells from non-neural stem/progenitor cells is difficult to achieve. A recent study by George Huang and colleagues published in Stem Cell Research and Therapy used stem cells from the dental apical papilla (SCAP) [1]. These neural-crest-derived dental cells express typical neural cell markers such as β-III-tubulin and are closely related to the neural-crest-derived cells of the peripheral nervous system.…”

“…Moreover, the use of SCAP for the regeneration of nervous tissues is also problematic, because numbers of stem cells are limited in dental apical papillae. Huang and colleagues' new publication has tackled this problem with transgene-free (TF) induced pluripotent stem cells (iPSCs) from SCAP [1]. …”

“…Although these dental iPSCs had typical characteristics of ESCs, they did also have unfavorable features. For example, most of the frozen-down dental iPSCs did not survive after thawing or the dental iPSCs underwent massive cell death after differentiation toward mesenchymal cell lineages with an ESC standard differentiation protocol [1,7]. …”

Integration-Free Reprogramming of Lamina Propria Progenitor Cells

“…This has been achieved via a variety of approaches, including nonintegrating adenoviral vectors (Stadtfeld et al 2008), delivery of factors via PiggyBac transposition (Woltjen et al 2009), episomal vectors incorporating the Epstein-Barr virus OriP and EBNA sequences (Yu et al 2009), vectors based on the Sendai virus (Fusaki et al 2009), and direct reprogramming through transient mRNA (Warren et al 2010). An approach based on a lentiviral stem cell cassette with the cre/loxP system to excise the cassette following reprogramming has been described (Somers et al 2010) and utilized for the reprogramming of apical papilla stem cells (Zou et al 2012).…”