Laser-Assisted Photoablation of Human Pluripotent Stem Cells from Differentiating Cultures

Abstract

Due to their pluripotency and their self-renewal capacity, human pluripotent stem cells (hPSC) provide fascinating perspectives for biomedical applications. In the long term, hPSC-derived tissue-specific cells will constitute an important source for cell replacement therapies in non-regenerative organs. These therapeutic approaches, however, will critically depend on the purity of the in vitro differentiated cell populations. In particular, remaining undifferentiated hPSC in a transplant can induce teratoma formation. In order to address this challenge, we have developed a laser-based method for the ablation of hPSC from differentiating cell cultures. Specific antibodies were directed against the hPSC surface markers tumor related antigen (Tra)-1-60 and Tra-1-81. These antibodies, in turn, were targeted with nanogold particles. Subsequent laser exposure resulted in a 98,9 +/- 0,9% elimination of hPSCs within undifferentiated cell cultures. In order to study potential side effects of laser ablation on cells negative for Tra-1-60 and Tra-1-81, hPSC were mixed with GFP-positive hPSC-derived neural precursors (hESCNP) prior to ablation. These studies showed efficient elimination of hPSC while co-treated hESCNP maintained their normal proliferation and differentiation potential. In vivo transplantation of treated and untreated mixed hPSC/hESCNP cultures revealed that laser ablation can dramatically reduce the risk of teratoma formation. Laser-assisted photothermolysis thus represents a novel contact-free method for the efficient elimination of hPSC from in vitro differentiated hPSC-derived somatic cell populations.

Important factors for cell-membrane permeabilization by gold nanoparticles activated by nanosecond-laser irradiation

“…This has been investigated for use in stem-cell differentiation and tissue engineering. 34 Cells that are differentiating in an unpreferred direction can be eliminated with higher laser energy. This elimination is very selective at the cellular level, 35 while differentiation could be driven additionally by optically controlled permeabilization for uptake of cytokines, for example.…”

Clinical‐scale purification of pluripotent stem cell derivatives for cell‐based therapies

“…Critically, because this energy does not diffuse away from targeted cells, non‐specific cell death is avoided. Nanogold particles functionalized with anti‐Tra‐1‐60 and Tra‐1‐81 antibodies were used to eliminate almost all hPSCs within undifferentiated cultures and post‐laser exposure mixtures did not generate teratomas after injection into severe combined immunodeficient (SCID) mice [40]. Despite these promising results and the fact that this approach permits dissociation‐free depletion, photothermolysis of hPSCs has not emerged as a widely used method.…”

Two ways to inactivate the Ki‐67 protein—Fragmentation by nanoparticles, crosslinking with fluorescent dyes

“…This enables a nanoparticle‐assisted laser inactivation (NALI). The use of AuNP and short laser pulses with wavelengths at the plasmon absorption band provide spatial and temporal selectivity of the physical effects . The underlying mechanism of destruction and accompanying physical phenomena, for example, vapor bubble formation, depend on the irradiation parameters and the nanoparticles properties.…”