Abstract
Clinically compliant human embryonic stem cells (hESCs) should be developed in adherence to ethical standards, without risk of contamination by adventitious agents. Here we developed for the first time animal-component free and good manufacturing practice (GMP)-compliant hESCs. After vendor and raw material qualification, we derived xeno-free, GMP-grade feeders from umbilical cord tissue, and utilized them within a novel, xeno-free hESC culture system. We derived and characterized three hESC lines in adherence to regulations for embryo procurement, and good tissue, manufacturing and laboratory practices. To minimize freezing and thawing, we continuously expanded the lines from initial outgrowths and samples were cryopreserved as early stocks and banks. Batch release criteria included DNA-fingerprinting and HLA-typing for identity, characterization of pluripotency-associated marker expression, proliferation, karyotyping and differentiation in-vitro and in-vivo. These hESCs may be valuable for regenerative therapy. The ethical, scientific and regulatory methodology presented here may serve for development of additional clinical-grade hESCs.
Highlights
IntroductionMost of the reported human embryonic stem cells (hESCs) lines worldwide are not ideal for use in clinical trials
Human ESC lines [1],[2] holds promise for disease modeling, basic scientific research, drug development, toxicity studies, and may serve as an unlimited renewable source of cells for transplantation therapy.Most of the reported human embryonic stem cells (hESCs) lines worldwide are not ideal for use in clinical trials
Foreskin was obtained during surgical circumcision, tissues from aborted fetuses were collected from first trimester terminations of pregnancy and umbilical cord was harvested during elective cesarean sections
Summary
Most of the reported hESC lines worldwide are not ideal for use in clinical trials. They were developed without adherence to Good Manufacture Practices (GMPs), using animal-derived researchgrade reagents, which may infect the cells with animal pathogens. In order to use hESC derivatives for clinical applications, the hESCs (clinical-grade hESCs) should ideally be developed under stringent ethical guidelines, from traceable and tested donors, preferably in an animal-free, GMPgrade culture system. Following 3–4 weeks of differentiation as embryoid bodies (EBs), immunofluorescence staining showed cells expressing beta-tubulin III (neuronal marker, ectoderm), muscle actin (mesoderm) and sox-17 (endoderm; Figure 4M-O and Figures S1, S2, S3M–O). Hematoxylin-eosin stained sections showed neural rosettes (ectoderm), cartilage (mesoderm) and columnar glandular epithelium with goblet cells (endoderm; Figure 4P–R and Figures S1, S2, S3P–R)
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