Stefano Codognotto

Derivation and propagation of human embryonic stem cell lines from frozen embryos in an animal product–free environment

The protocols described here are comprehensive instructions for deriving human embryonic stem (hES) cell lines in xeno-free conditions from cryopreserved embryos. Details are included for propagation, cryopreservation and characterization. Initial derivation is on feeder cells and is followed by adaptation to a feeder-free environment; competent technicians can perform these simplified methods easily. From derivation to cryopreservation of fully characterized initial stocks takes 3–4 months. These protocols served as the basis for standard operating procedures (SOPs), with both operational and technical components, that we set to meet good manufacturing practice (GMP) and UK regulatory body requirements for derivation of clinical-grade cells. As such, these SOPs are currently used in our current GMP compliant facility to derive hES cell lines ab initio, in an animal product–free environment; these lines are suitable for research and potentially for clinical use in cell therapy. So far, we have derived eight clinical-grade lines, which will be freely available to the scientific community after submission/accession to the UK Stem Cell Bank.

Human embryonic and induced pluripotent stem cells in clinical trials

BACKGROUND Human embryonic and induced pluripotent stem cells (hESC and hiPSC) have tremendous potential for clinical implementation. In spite of all hurdles and controversy, clinical trials in treatment of spinal cord injury, macular degeneration of retina, type 1 diabetes and heart failure are already ongoing. SOURCES OF DATA ClinicalTrials.gov database, International Clinical Trials Registry Platform, PubMed and press releases and websites of companies and institutions working on hESC- and iPSC-based cellular therapy. AREAS OF AGREEMENT The initial results from multiple clinical trials demonstrate that hESC-based therapies are safe and promising. AREAS OF CONTROVERSY Are iPSC cells safe in the clinical application? Is there a room for both hESC and iPSC in the future clinical applications? GROWING POINTS Increasing number of new clinical trials. AREAS TIMELY FOR DEVELOPING RESEARCH Development of hESC- and/or iPSC-based cellular therapy for other diseases.

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

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.

Generation of KCL038 clinical grade human embryonic stem cell line

The KCL038 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro assays.

Generation of KCL035 research grade human embryonic stem cell line carrying a mutation in HBB gene.

The KCL035 human embryonic stem cell line was derived from an embryo donated for research that carried a mutation in the HBB gene, which is linked to the β-thalassemia syndrome. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays.

Generation of KCL040 clinical grade human embryonic stem cell line.

The KCL040 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro assays.

Generation of KCL039 clinical grade human embryonic stem cell line

The KCL039 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro assays.

Generation of KCL037 clinical grade human embryonic stem cell line.

The KCL037 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro assays.

Generation of KCL036 research grade human embryonic stem cell line carrying a mutation in the HTT gene

The KCL036 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting one allele of the HTT gene encoding huntingtin (38 trinucleotide repeats; 14 for the normal allele). The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro and in vivo assays.

Generation of KCL034 clinical grade human embryonic stem cell line.

The KCL034 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro assays. The line was also validated for sterility, specific and non-specific human pathogens.