Development and production of good manufacturing practice grade human embryonic stem cell lines as source material for clinical application

AbstractFrom 2006 to 2011, Roslin Cells Ltd derived 17 human embryonic stem cells (hESC) while developing (RCM1, RC-2 to -8, -10) and implementing (RC-9, -11 to -17) quality assured standards of operation in a facility operating in compliance with European Union (EU) directives and United Kingdom (UK) licensure for procurement, processing and storage of human cells as source material for clinical application, and targeted to comply with an EU Good Manufacturing Practice specification. Here we describe the evolution and specification of the facility, its operation and outputs, complementing hESC resource details communicated in Stem Cell Research Lab Resources

Enabling consistency in pluripotent stem cell-derived products for research and development and clinical applications through material standards

There is a need for physical standards (reference materials) to ensure both reproducibility and consistency in the production of somatic cell types from human pluripotent stem cell (hPSC) sources. We have outlined the need for reference materials (RMs) in relation to the unique properties and concerns surrounding hPSC-derived products and suggest in-house approaches to RM generation relevant to basic research, drug screening, and therapeutic applications. hPSCs have an unparalleled potential as a source of somatic cells for drug screening, disease modeling, and therapeutic application. Undefined variation and product variability after differentiation to the lineage or cell type of interest impede efficient translation and can obscure the evaluation of clinical safety and efficacy. Moreover, in the absence of a consistent population, data generated from in vitro studies could be unreliable and irreproducible. Efforts to devise approaches and tools that facilitate improved consistency of hPSC-derived products, both as development tools and therapeutic products, will aid translation. Standards exist in both written and physical form; however, because many unknown factors persist in the field, premature written standards could inhibit rather than promote innovation and translation. We focused on the derivation of physical standard RMs. We outline the need for RMs and assess the approaches to in-house RM generation for hPSC-derived products, a critical tool for the analysis and control of product variation that can be applied by researchers and developers. We then explore potential routes for the generation of RMs, including both cellular and noncellular materials and novel methods that might provide valuable tools to measure and account for variation. Multiparametric techniques to identify "signatures" for therapeutically relevant cell types, such as neurons and cardiomyocytes that can be derived from hPSCs, would be of significant utility, although physical RMs will be required for clinical purposes

An Improved Technique for Chromosomal Analysis of Human ES and iPS Cells

Prolonged in vitro culture of human embryonic stem (hES) cells can result in chromosomal abnormalities believed to confer a selective advantage. This potential occurrence has crucial implications for the appropriate use of hES cells for research and therapeutic purposes. In view of this, time-point karyotypic evaluation to assess genetic stability is recommended as a necessary control test to be carried out during extensive ‘passaging’. Standard techniques currently used for the cytogenetic assessment of ES cells include G-banding and/or Fluorescence in situ Hybridization (FISH)-based protocols for karyotype analysis, including M-FISH and SKY. Critical for both banding and FISH techniques are the number and quality of metaphase spreads available for analysis at the microscope. Protocols for chromosome preparation from hES and human induced pluripotent stem (hiPS) cells published so far appear to differ considerably from one laboratory to another. Here we present an optimized technique, in which both the number and the quality of chromosome metaphase spreads were substantially improved when compared to current standard techniques for chromosome preparations. We believe our protocol represents a significant advancement in this line of work, and has the required attributes of simplicity and consistency to be widely accepted as a reference method for high quality, fast chromosomal analysis of human ES and iPS cells

Tissue Engineering in Oral and Maxillofacial Surgery : From Lab to Clinics

Regenerative medicine aims at the functional restoration of tissue malfunction, damage or loss, and can be divided into three main approaches. Firstly, the cell-based therapies, where cells are administered to re-establish a tissue either directly or through paracrine functions. Secondly, the often referred to as classical tissue engineering, consisting of the combined use of cells and a bio-degradable scaffold to form tissue. Thirdly, there are material-based approaches, which have made significant advances which rely on biodegradable materials, often functionalized with cellular functions (De Jong et al. 2014). In 1993, Langer and Vacanti, determined tissue engineering as an “interdisciplinary field that applies the principles of engineering and the life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function”. They published this definition in Science in 1993. Tissue engineering has been classically thought to consist of three elements: supporting scaffold, cells and regulating factors such as growth factors (Fig. 1). Depending on the tissue to be regenerated, all three vary. Currently, it is known, that many other factors may have an effect on the outcome of the regenerate. These include factors enabling angiogenesis, physical stimulation, culture media, gene delivery and methods to deliver patient specific implants (PSI) (Fig. 2). During the past two decades, major obstacles have been tackled and tissue engineering is currently being used clinically in some applications while in others it is just taking its first baby steps.Peer reviewe

Cardiac regeneration: different cells same goal

Cardiovascular diseases are the leading cause of mortality, morbidity, hospitalization and impaired quality of life. In most, if not all, pathologic cardiac ischemia ensues triggering a succession of events leading to massive death of cardiomyocytes, fibroblast and extracellular matrix accumulation, cardiomyocyte hypertrophy which culminates in heart failure and eventually death. Though current pharmacological treatment is able to delay the succession of events and as a consequence the development of heart failure, the only currently available and effective treatment of end-stage heart failure is heart transplantation. However, donor heart availability and immunorejection upon transplantation seriously limit the applicability. Cardiac regeneration could provide a solution, making real a dream of both scientist and clinician in the previous century and ending an ongoing challenge for this century. In this review, we present a basic overview of the various cell types that have been used in both the clinical and research setting with respect to myocardial differentiation

Screening ethnically diverse human embryonic stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage

The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines, from 38 laboratories worldwide, for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphism (SNP) analysis revealed that they included representatives of most major ethnic groups. Most lines remained karyotypically normal, but there was a progressive tendency to acquire changes on prolonged culture, commonly affecting chromosomes 1, 12, 17 and 20. DNA methylation patterns changed haphazardly with no link to time in culture. Structural variants, determined from the SNP arrays, also appeared sporadically. No common variants related to culture were observed on chromosomes 1, 12 and 17, but a minimal amplicon in chromosome 20q11.21, including three genes expressed in human ES cells, ID1, BCL2L1 and HM13, occurred in >20% of the lines. Of these genes, BCL2L1 is a strong candidate for driving culture adaptation of ES cells

Rapid establishment of the European Bank for induced Pluripotent Stem Cells (EBiSC):The Hot Start experience

A fast track “Hot Start” process was implemented to launch the European Bank for Induced Pluripotent Stem Cells (EBiSC) to provide early release of a range of established control and disease linked human induced pluripotent stem cell (hiPSC) lines. Established practice amongst consortium members was surveyed to arrive at harmonised and publically accessible Standard Operations Procedures (SOPs) for tissue procurement, bio-sample tracking, iPSC expansion, cryopreservation, qualification and distribution to the research community. These were implemented to create a quality managed foundational collection of lines and associated data made available for distribution. Here we report on the successful outcome of this experience and work flow for banking and facilitating access to an otherwise disparate European resource, with lessons to benefit the international research community. eTOC: The report focuses on the EBiSC experience of rapidly establishing an operational capacity to procure, bank and distribute a foundational collection of established hiPSC lines. It validates the feasibility and defines the challenges of harnessing and integrating the capability and productivity of centres across Europe using commonly available resources currently in the field