Proteomics, Genomics & Microarrays
Current and Future Innovations in Stem Cell Technologies Erik Miljan, PhD, and William Hadlington Booth, PhD Stem Cells 101
Every cell type in the body that makes up organs and tissues arose from a more primitive cell type called a stem cell. Stem cells are the foundation of living organisms, with the unique ability to self-renew and differentiate into specialised cell types. There are three different types of stem cell, classifi ed by the number of specialised cell types they can produce: i) pluripotent stem cells (e.g. embryonic stem cells) can generate any specialised cell type; ii) multipotent stem cells (e.g. mesenchymal stem cells) are able to generate multiple, but not all, specialised cell types; and, iii) unipotent stem cells (e.g. epidermal stem cells that produce skin) give rise to only one cell type. It was long believed that stem cell differentiation into specialised cell types only occurs in one direction. There have been many exciting advances in stem cell biology, most notable the discovery of induced pluripotent stem cells (iPSCs) that demonstrated a mature differentiated specialised cell can be reverted to a primitive pluripotent stem cell (Takahashi K, 2006). This discovery transformed our understanding of stem cell biology enabling exciting and substantial advances in stem cell tools, technologies and applications. This article focuses on pluripotent stem cells, as they offer the most promising future applications.
Working with Stem Cells in Lab - Past and Present
To harness the power of stem cells, they must fi rst be maintained in vitro tissue culture. Culture expansion of stem cells is tricky because they must be maintained in an undifferentiated state and not permitted to differentiate into other cell types until desired. In short, if stem cells are not dividing in log phase growth, they are differentiating. Historically, pluripotent stem cells were notoriously diffi cult to work with in the lab largely because of the of inherent variability of reagents derived from animal tissues.
GMP: The Future is About Process Resilience
An important concept affecting current and future innovations in stem cell technologies is Good Manufacturing Practice (‘GMP’). This is governed by formal regulations administered by drug regulatory agencies (for example the FDA) that control the manufacture processes of medicines. The use of stem cells as therapeutic agents has necessitated specialised drug regulations known as Advanced Therapeutic Medicinal Products (ATMPs). Unlike chemically synthesised medicines where the fi nal product can be defi ned through chemical analysis, ATMPs are complex biological living entities whereby the entire manufacturing process defi nes the fi nal product. In simple terms, every reagent that touches the stem cells in the manufacturing process throughout the entire lifetime of the stem cell becomes a component of the fi nal product. As such, in the ‘real world’ the quality and consistency of the reagents used in a stem cell manufacturing process is paramount for downstream clinical applications, even if the project is still in the R&D or preclinical phase. Once reserved for clinical applications, GMP has become a dominating concept that affects all aspects of stem cell research and applications. Researchers and clinical developers benefi t alike from GMP-focused innovations in stem cell technologies that deliver consistent growth properties and high-quality results.
The Stem Cell Workfl ow
Signifi cant advances that overcome the challenges of the past have been made in all aspects of in vitro stem cell culture. These include advances in tissue culture medium, extracellular matrix, 3D synthetic cell culture plastic, growth factors, dissociation enzymes, cryopreservation agents and differentiation technologies. The workfl ow to culture stem cells in vitro is not a linear process but rather a continuous circle that can be broken down into 6 steps: 1) Extracellular Matrix coating of tissue culture plasticware; 2) Revival/seeding of tissue culture fl asks; 3) Expansion of the cell culture in an incubator; 4) Culture medium change; 5) Subculture or “passaging” one fl ask to many; and 6) Cryopreservation of the stem cell culture. The stem cell workfl ow is shown in Figure 1.
Components of the Stem Cell Workfl ow Extracellular Matrix
The art of culturing stem cells is a lot easier today than in the past. Stem cells grow as adherent cultures on the surface of tissue culture fl asks or dishes (image shown in Figure 1, Step 3). For the stem cells to adhere to the surface it must be coated with extracellular matrix.
Figure 1. Synergy of the stem cell workfl ow and applications of stem cells.
In the early days, it was an effort to maintain stem cells in culture because the cultures needed to be grown on a ‘feeder layer’ of fi broblast cells. The requirement for a second cell culture combined with the stem cell culture is laborious to set up and severely limited experiments and applications (due to the contaminating fi broblasts mixed with the stem cells). Extracellular matrix isolated from mouse tumours removed the need for feeder layer cultures but can be variable in consistency and contain contaminants. Today, researchers benefi t from recombinantly expressed extracellular matrix containing laminin-511 fragments that provides highly effi cient adherence of a broad range of cell types and is easy to use (with only 1 hour coating time required that saves time and cost). Exceptional pluripotent stem cell adherence is achieved with laminin-511 fragments. The recombinant extracellular matrix laminin-511 is expressed in mammalian cell culture (e.g. CHO cells) or insect culture (e.g. silkworm) that eliminates the need for animal derived products in the extracellular matrix. Alternatively, synthetic 3D plastic scaffolds (e.g. Alvetex) are also available that offer a rigid defi ned matrix that is non-biological.
Culture Medium
Early stem cell culture media required the medium to be replenished daily. This means 7 days a week in the lab tending to the stem cell cultures. Optimisation of tissue culture medium composition enables cultures to be maintained over the weekend without a medium change, enabling feeder-free, weekend-free stem cell culture. This may sound insignifi cant but does have a huge impact on the lifestyle of researchers working with stem cells. Unlike early tissue culture media, the composition of the culture media are fully defi ned and contain no animal derived products. Removal of animal-derived products offers important advantages by removing variability inherent in animal-derived products and guaranteeing consistent cell growth. Furthermore, animal-free formulations eleminate the risk of infection arising from the animal product (e.g. TSE risk). Growth factors are a critical component of the culture medium to maintain the stem cells in an undifferentiated state. Products available on the market contain growth factors that are expressed and isolated from barley.
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