Transfer of Synthetic Human Chromosome into Human Induced Pluripotent Stem Cells for Biomedical Applications

Abstract

AlphoidtetO-type human artificial chromosome (HAC) has been recently synthetized as a novel class of gene delivery vectors for induced pluripotent stem cell (iPSC)-based tissue replacement therapeutic approach. This HAC vector was designed to deliver copies of genes into patients with genetic diseases caused by the loss of a particular gene function. The alphoidtetO-HAC vector has been successfully transferred into murine embryonic stem cells (ESCs) and maintained stably as an independent chromosome during the proliferation and differentiation of these cells. Human ESCs and iPSCs have significant differences in culturing conditions and pluripotency state in comparison with the murine naïve-type ESCs and iPSCs. To date, transferring alphoidtetO-HAC vector into human iPSCs (hiPSCs) remains a challenging task. In this study, we performed the microcell-mediated chromosome transfer (MMCT) of alphoidtetO-HAC expressing the green fluorescent protein into newly generated hiPSCs. We used a recently modified MMCT method that employs an envelope protein of amphotropic murine leukemia virus as a targeting cell fusion agent. Our data provide evidence that a totally artificial vector, alphoidtetO-HAC, can be transferred and maintained in human iPSCs as an independent autonomous chromosome without affecting pluripotent properties of the cells. These data also open new perspectives for implementing alphoidtetO-HAC as a gene therapy tool in future biomedical applications.

Human AlphoidtetO Artificial Chromosome as a Gene Therapy Vector for the Developing Hemophilia A Model in Mice

“…The metaphase spread was prepared as previously described [20,21,25]. Exponentially growing HAC-carrying CHO or iPSCs were treated for 4 or 12 h at 37 • C with 0.1 µg/mL Colcemid (Sigma-Aldrich) in a 5% CO 2 incubator.…”

“…The slides were consequently dehydrated with 70%, 90%, and 100% ethanol. A hybridization solution containing 10 M Tris-HCl pH 7.4, 70% formamide (Sigma-Aldrich, St. Louis, MO, USA), 5% dextran sulfate, 10 ng tetO PNA-FITC (Panagen Company, Bethel, PA, USA), and 10 ng telomere PNA-TRITS (Panagen Company), was added onto each slide [20]. The slides were covered with coverslips and treated at 80 • C for 3 min and then for 2-6 h at RT in darkness.…”

“…One of the developed alphoid tetO -HAC features is the multiplied tetracycline operator (tet-O) sequence that can bind tet-repressor fusion proteins, leading to a conditional inhibition of the kinetochore function and subsequent HAC loss in dividing cells [14][15][16]. The DNA sequence of the alphoid tetO -HAC vector has been shown to exclude any cryptic transcripts [17,18] and demonstrates structural integrity during gene loading and transfer into different host cells [15,[19][20][21]. The alphoid tetO -HAC features a unique loxP gene acceptor site designed for Cre recombinase-mediated insertion of full-length genes in Chinese hamster ovary (CHO) cells, from which the HAC can be transferred into various recipient cells [22].…”

“…The alphoid tetO -HAC still requires further functional investigation, particularly with respect to the improvement of its mitotic stability. The HAC was also shown to be unstable in some human cell lines and induced pluripotent stem cells (iPSCs) [20,24]. Apart from this, the alphoid tetO -HAC has become an attractive gene delivery vector that can be stably maintained in murine embryonic stem cells (ESCs) and their derivatives throughout mouse ontogeny [25] and can also be maintained as a 47th chromosome in human iPSCs [20].…”

“…The HAC was also shown to be unstable in some human cell lines and induced pluripotent stem cells (iPSCs) [20,24]. Apart from this, the alphoid tetO -HAC has become an attractive gene delivery vector that can be stably maintained in murine embryonic stem cells (ESCs) and their derivatives throughout mouse ontogeny [25] and can also be maintained as a 47th chromosome in human iPSCs [20]. The HAC-based gene therapy approach consists of an ex vivo genetic modification step of the autologous patient-derived stem cells, and consequent infusion of the modified cells into a patient [26,27].…”