Current Applications of Pharmaceutical Biotechnology by Unknown

Author:Unknown
Language: eng
Format: epub
ISBN: 9783030404642
Publisher: Springer International Publishing

4.3 Purification of hPS Cell-Derived Products

The major risk of the application of hPS cell-derived products resides in the possibility of teratoma formation due to an uncontrolled proliferation of a small number of pluripotent cells that remain in the final cell therapy product. In order to prevent this, clinical-scale purification of hPS cell derivatives for cell-based therapies needs to be addressed [96, 97].

Fluorescent-activated cell sorting (FACS) is probably the most widely used and more accurate method for purification of cells. This technique involves labeling the target cells with an antibody linked to a fluorescent molecule and has demonstrated efficiencies of separation above 95% [98]. For example, Kikuchi et al. included a sorting step at day 12 of neural induction of hPS cells that allowed the selection of more than 90% CORIN-positive cells, which would further differentiate into midbrain dopaminergic neurons [37]. In this study, on day 26 of differentiation, there were less than 1% of cells expressing pluripotency markers. FACS sorting of O4-positive oligodendrocyte progenitors led to an enriched cell population containing more than 93% of these cells that upon animal engraftment did not originate teratomas [38].

A widely used example of a target antigen for cell purification is SSEA-1, because this surface marker is absent from hPS cells [99]. Since the efficiency of the differentiation protocol of hES cells into CVPs only led to approximately 64% of efficiency, Menasche et al. integrated within the production pipeline a purification step taking advantage of this marker [51, 100]. The purification strategy consists in the depletion of hPS cells in culture by magnetically labeling cells with SSEA-1 and then using magnetic activated cell sorting (MACS) to select SSEA-1-positive cells, which had lost their pluripotency. Furthermore, the final product, which was required to contain less than 0.1% of undifferentiated cells, was also tested for the expression of Nanog.

For the treatment of Stargardt’s macular dystrophy (SMD) and dry-age-related macular degeneration (AMD), hES cell-derived retinal pigment epithelium (RPE) cells were purified by exposure to type IV collagenase followed by manual isolation of RPE using a glass pipette [101]. Still, long-term safety studies in preclinical models have not detected any uncontrolled cell proliferation, indicating that no teratomas were generated upon implantation of hES cell-derived RPE [102]. An approach to address spinal cord injury by using hES cell-derived OP cells has demonstrated that a final product containing up to 5% of undifferentiated hES cells did not lead to teratoma formation [103]. However, the OP cell differentiation protocol has an efficiency that leads to less than 1% of hES cells in the final product.

Metabolic purification of hPS cell-derived products has also been focused in the last few years, mainly due to its reduced cost and high technical feasibility, which relies on the supplementation of the culture medium with components that are already present in most culture media formulation. Furthermore, these methods can be easily integrated within bioreactor culture systems without the need for cell singularization, contrary to methods like FACS or MACS. One good example of the application of these methods is for hPSC-derived cardiomyocyte purification.

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