A High-Throughput Method as a Diagnostic Tool for HIV Detection in Patient-Specific Induced Pluripotent Stem Cells Generated by Different Reprogramming Methods

Abstract

Induced pluripotent stem cells (iPSCs) provide a unique opportunity for generation of patient-specific cells for use in translational purposes. We aimed to compare iPSCs generated by different reprogramming methods regarding their reprogramming efficiency, pluripotency capacity, and the possibility to use high-throughput PCR-based methods for detection of human pathogenic viruses. iPSCs from skin fibroblasts (FB), peripheral blood mononuclear cells (PBMCs), or mesenchymal stem cells (MSCs) were generated by using three different reprogramming systems including chromosomal integrating and nonintegrating methods. Reprogramming efficiencies were in accordance with the literature, indicating that the parental cell type and the reprogramming method play a major role for the reprogramming efficiencies (FB: STEMCCA: 1.30±0.18, Sendai virus: 1.37±0.01, and episomal plasmids: 0.04±0.02; PBMCs: Sendai virus: 0.002±0.001, episomal plasmids: 0) but result in the same characteristics of pluripotency. We found the highest reprogramming efficiencies for MSC with 3.32±1.2 by using episomal plasmids. Since GMP standard working procedures and screening units need virus contamination-free cell lines, we studied HIV-1 contamination in the generated iPSCs. We used the high-throughput cobas® 6800/8800 system, which is normally used for detection of HIV-1 in plasma of patients, and found that footprint-free reprogramming methods as episomal plasmids and Sendai virus are useful for the described virus detection method. This fast, cost-effective, robust, and reliable assay demonstrates the feasibility to use high-throughput PCR-based methods for detection of human pathogenic viruses in ps-iPSC lines that were generated with nongenome integrating reprogramming methods.

Pluripotent Stem Cell-Derived Mesenchymal Stem Cells Show Comparable Functionality to Their Autologous Origin

“…However, they could represent an important alternative source for functional MSCs. MSCs are well-established somatic cell sources for reprogramming into iPSCs with high reprogramming efficiencies [ 22 , 27 ]. Vice versa, iPSCs have been successfully differentiated into functional MSCs (iP-MSCs) with high efficiency [ 28 ], which might potentially serve as an ideal source for homogeneous and high-quality MSCs.…”

Hochdurchsatzanalyse zur Identifizierung von Virusinfektionen in ps-iPSCs

“…Unserer Arbeitsgruppe ist es gelungen, die in-house generierten iPSC-Linien mittels einer Hochdurchsatzmethode am Cobas® 6800/8800 (Roche) auf humanpathogene Viren zu untersuchen [ 8] . Unsere Arbeitsgruppe fokussiert sich auf die Herstellung von iPSC-Linien von Patienten, die unter mono-bzw.…”

Oxidative DNA Damage Signalling in Neural Stem Cells in Alzheimer’s Disease

“…Importantly, these screenings used various iPSCs from skin fibroblasts, peripheral blood mononuclear cells (PBMCs), or mesenchymal stem cells generated by various reprogramming systems, both integrating and nonchromosomally integrating. The cell type of origin and the reprogramming method play an important role in process efficiency [37]. Another example of the use of iPSC to diagnose disease was a cell-based phenotypic diagnostic test for identifying long QT syndrome to identify pathogenic variants [38].…”