Non-integrating episomal plasmid-based reprogramming of human amniotic fluid stem cells into induced pluripotent stem cells in chemically defined conditions

Abstract

Amniotic fluid stem cells (AFSC) represent an attractive potential cell source for fetal and pediatric cellbased therapies. However, upgrading them to pluripotency confers refractoriness toward senescence, higher proliferation rate and unlimited differentiation potential. AFSC were observed to rapidly and efficiently reacquire pluripotency which together with their easy recovery makes them an attractive cell source for reprogramming. The reprogramming process as well as the resulting iPSC epigenome could potentially benefit from the unspecialized nature of AFSC. iPSC derived from AFSC also have potential in disease modeling, such as Down syndrome or b-thalassemia. Previous experiments involving AFSC reprogramming have largely relied on integrative vector transgene delivery and undefined serumcontaining, feeder-dependent culture. Here, we describe non-integrative oriP/EBNA-1 episomal plasmidbased reprogramming of AFSC into iPSC and culture in fully chemically defined xeno-free conditions represented by vitronectin coating and E8 medium, a system that we found uniquely suited for this purpose. The derived AF-iPSC lines uniformly expressed a set of pluripotency markers Oct3/4, Nanog, Sox2, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 in a pattern typical for human primed PSC. Additionally, the cells formed teratomas, and were deemed pluripotent by PluriTest, a global expression microarray-based insilico pluripotency assay. However, we found that the PluriTest scores were borderline, indicating a unique pluripotent signature in the defined condition. In the light of potential future clinical translation of iPSC technology, non-integrating reprogramming and chemically defined culture are more acceptable.

Evaluation of Therapeutic Oligonucleotides for Familial Amyloid Polyneuropathy in Patient-Derived Hepatocyte-Like Cells

“…We routinely observed first iPSC-like colony formation around day 10 after reprogramming of the FAP patient derived UCs using plasmids that encode EBV elements as also reported by others [49,50]. The similarity of iPSCs and UCs with regard to their epithelial state might therefore accelerate reprogramming as compared to fibroblasts that typically form iPSC-like colonies around week 3 to week 4 [49].…”

Cardiac Restoration Stemming From the Placenta Tree: Insights From Fetal and Perinatal Cell Biology

“…Hence, the quest is now on defining the most suitable cell to efficiently induce iPS from and since amniotic fluid stem cells are immature fetal progenitors endowed with some degree of instrinsic pluripotency and active expression of embryonic genes including OCT4, NANOG, and SOX2 (Loukogeorgakis and De Coppi, 2017 ), they represent an ideal candidate. Recent studies have reported that murine and human AFSC can be reprogrammed into iPS more easily than adult stem cells by applying either transgene-free approaches, like chemical defined conditions via stimulation with the histone deacetylase inhibitor valproic acid (Moschidou et al, 1953 ), as well as non-integrating methods by episomal plasmid, transposon system, sendai virus or mRNA delivery by lipofection (Jiang et al, 2016 ; Slamecka et al, 2016 ; Bertin et al, 2017 ; Velasquez-Mao et al, 2017 ); notably the obtained AFSC-iPS have been proven capable of functional cardiac differentiation, thus providing important impact for future cardiac regenerative therapy and specific relevance for the treatment of neonatal cardiac congenital disease (Jiang et al, 2016 ; Velasquez-Mao et al, 2017 ). Indeed, hAFSC can be effortlessly harvested during prenatal diagnosis, treated by gene therapy and iPS technology to derived healthy myocardial and cardiovascular cells to be then processed by tissue engineering approaches so to provide cardiac grafts developed in parallel with gestation and promptly implanted in utero or at birth.…”

Global trends in clinical trials involving pluripotent stem cells: A systematic multi-database analysis

“…However, when considering the increasing number of trials on iPSCs, a future switch towards interventional iPSC trials might just be a matter of time. In particular the introduction of non-integrating transgene-free reprogramming approaches may further support the clinical translation of the iPSC technology as the issue of in situ carcinogenicity of iPSCs due to remaining viral particles may be solved by these technologies (51).…”