Cost-Effective Master Cell Bank Validation of Multiple Clinical-Grade Human Pluripotent Stem Cell Lines From a Single Donor

Abstract

Standardization guidelines for human pluripotent stem cells are still very broadly defined, despite ongoing clinical trials in the U.S., U.K., and Japan. The requirements for validation of human embryonic (hESCs) and induced pluripotent stem cells (iPSCs) in general follow the regulations for other clinically compliant biologics already in place but without addressing key differences between cell types or final products. In order to realize the full potential of stem cell therapy, validation criteria, methodology, and, most importantly, strategy, should address the shortfalls and efficiency of current approaches; without this, hESC-and, especially, iPSC-based therapy will not be able to compete with other technologies in a cost-efficient way. We addressed the protocols for testing cell lines for human viral pathogens and propose a novel strategy that would significantly reduce costs. It is highly unlikely that the multiple cell lines derived in parallel from a tissue sample taken from one donor would have different profiles of endogenous viral pathogens; we therefore argue that samples from the Master Cell Banks of sibling lines could be safely pooled for validation. We illustrate this approach with tiered validation of two sibling clinical-grade hESC lines, KCL033 and KCL034 (stage 1, sterility; stage 2, specific human pathogens; and stage 3, nonspecific human pathogens). The results of all tests were negative. This cost-effective strategy could also be applied for validation of Master Cell Banks of multiple clinical-grade iPSC lines derived from a single donor. STEM CELLS

MicroRNAs in spent blastocyst culture medium are derived from trophectoderm cells and can be explored for human embryo reproductive competence assessment

“…The hESC line KCL034 was cultured as previously described elsewhere (24)(25)(26). Smaller (approximately 10-50 cells) and larger (approximately 500-1,000 cells) clumps of hESCs were microdissected free of feeders, transferred directly to lysis buffer, and snap-frozen in liquid nitrogen.…”

Induced Pluripotent Stem Cell Differentiation and Three-Dimensional Tissue Formation Attenuate Clonal Epigenetic Differences in Trichohyalin

“…Since the focus of our work was keratinocyte differentiation culminating in the stratum corneum derived epidermal permeability barrier, we concentrated on the epidermal differentiation complex (EDC) on chromosome 1, which contains multiple genes involved in epidermal cornification [11][12][13][14][15][16][17][18][19]. One of our clinical grade lines, KCL034, was used as a control in most analyses [20].…”

Hurdles to clinical translation of human induced pluripotent stem cells

“…Nonspecific viral contaminant screening uses embryonated chicken eggs and costs ~$12,000 per cell line. Devito et al have shown that samples from a single donor of human ESCs can be batched to reliably screen for infectious agents (57). They estimate that a bank pooled from 100 donors with three cell lines per donor could save approximately $18 million dollars in the clinical validation process.…”

“…A sizable fraction of the development cost comes from cGMP requirements. For example, in addition to genetic testing, it is necessary to screen for many infectious agents, including mycoplasma, intracellular bacteria, and viral contaminants (57). Some of the screenings require cultures as long as 28 days and transmission electron microscopy for testing.…”