Current and Future Innovations in Stem Cell Technologies

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, classified 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 first 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 difficult 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

影响当前和未来干细胞技术创新的一个重要概念是良好生产规范(GMP)。这由控制药品生产过程的药品监管机构(如FDA)实施的正式法规进行管理。使用干细胞作为治疗剂已经需要专门的药物法规，即先进治疗药物产品(ATMPs)。与化学合成药物不同的是，最终产品可以通过化学分析来确定，atmp是复杂的生物生命体，其整个生产过程来确定最终产品。简单来说，在干细胞的整个生命周期中，每一个在制造过程中接触到干细胞的试剂都会成为最终产品的组成部分。因此，在“现实世界”中，干细胞制造过程中使用的试剂的质量和一致性对于下游临床应用至关重要，即使该项目仍处于研发或临床前阶段。GMP已成为影响干细胞研究和应用的各个方面的主导概念。研究人员和临床开发人员都受益于gmp聚焦的干细胞技术创新，提供稳定的生长特性和高质量的结果。

The Stem Cell Workflow

在体外干细胞培养的各个方面都取得了克服过去挑战的重大进展。这包括在组织培养基、细胞外基质、3D合成细胞培养塑料、生长因子、解离酶、低温保存剂和分化技术方面的进展。干细胞体外培养的工作流程不是一个线性的过程，而是一个连续的循环，可以分为6个步骤:1)组织培养塑料的细胞外基质包覆;2)组培瓶再生/播种;3)培养箱中细胞培养物的扩增;4)培养基变化;5)亚文化或将一个烧瓶传递给多个烧瓶;6)干细胞培养物超低温保存。干细胞工作流程如图1所示。

Components of the Stem Cell Workflow

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 flasks 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. 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 fibroblast 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 fibroblasts 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 benefit from recombinantly expressed extracellular matrix containing laminin-511 fragments that provides highly efficient 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 defined 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 insignificant 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 defined 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.

Subculture

干细胞在培养容器中进行细胞分裂。随着它们的扩张，它们最终会生长出自己的家园，必须被再次培养到单独的烧瓶中，为进一步的生长提供空间。通常的做法是使用一种消化酶将干细胞从培养表面释放出来。从牛体内分离的胰蛋白酶在组织培养实验室中很常见。今天的产品的进展使用在玉米中表达的胰蛋白酶，在室温溶液中稳定。胶原酶是一种可替代的解离试剂，对各种细胞温和而高效，既可用于动物，也可用于GMP级，这再次实现了稳健的一致培养条件，并消除了对动物衍生产品的依赖，这些产品本身是可变的。

Cryopreservation

The stem cells harvested from cultures can be frozen and stored (or ‘cryopreserved’) safely for several decades. When required, the cryopreserved stem cells may be defrosted, revived and expanded in culture providing a renewable source of stem cells. During cryopreservation of stem cells, it is critical to prevent cell death and changes in genotype/phenotype. Today’s cryopreservation media can maintain consistent high cell viability after thawing; maintaining cell pluripotency, normal karyotype and proliferation even after long term cell storage. Traditionally, the cryopreservation process involved a rate-controlled freezer or a specialised container to freeze the cells at -1ºC/min. Advances in cryopreservation agents have removed the need for rate-controlled freezing. The process is now simple - you just place the stem cell suspension into a -80ºC freezer. Moreover, cryopreservation agents are available in GMP grade and with no animal-derived ingredients.

Differentiation

The power of stem cells lies in their ability both to self-renew and to differentiate into specialised cell types. The process of differentiation removes the stem cells from the workflow towards applications. Directed differentiation of stem cells into specific cell types enables the number of applications to grow. A typical differentiation protocol uses stepwise changes in culture medium, cytokines, growth factors and extracellular matrix over several weeks to direct the stem cells into a particular lineage and fate. Today, innovative technologies use genetic reprogramming factors that rapidly (< 1 week) differentiate stem cells into mature cell phenotypes. This advance significantly reduces time to experiment and increases manufacturing capacity for differentiated cell types.

Table 1. Advances in Stem Cell Technologies.
Description Area of Innovation Examples of Innovative Products
Extracellular Matrix Recombinant Laminin Expressed in CHO and Silkworm iMatrix-511
Culture Medium No medium change required over the weekend, GMP grade, animal free StemFit Medium
Growth Factors Recombinant, GMP grade, animal free StemFit Purotein
Dissociation Reagents Trypsin enzyme recombinantly expressed in maize. Collagenase & Neutral Protease expressed in Clostridium histolyticum TrypLE
Collagenase NB
Neutral Protease NB
Cryopreservation Rate-controlled freezing not required. GMP grade, animal free and available for clinical use. Suitable for all cell types. STEM-CELLBANKER
Differentiation Rapid directed differentiation through genetic reprogramming Quick-Skeletal Muscle
Quick-Endothelium
Quick-Neuron

Future Technologies and Applications

Disease Modelling

成熟电池类型的可再生能源具有无限的应用前景。使用分化干细胞的一个令人兴奋的创新领域是疾病建模。在过去，对器官或组织疾病状态的研究仅限于使用体内动物模型;然而，分化的干细胞打开了在体外特定细胞类型中创造疾病状态的机会。此外，目前的技术可以在实验室中生成类器官或“微型器官”。疾病特异性诱导多能干细胞也可用于体外建立疾病模型，这是疾病研究和药物开发的宝贵工具，而不需要体内动物模型。理论上，任何组织都有可能在体外培育出来。在一个激动人心的干细胞疾病建模例子中，来自日本京都CiRA的Takayama博士成功地在类器官和未分化多能干细胞中模拟了SARS-CoV-2的生命周期(Takayama, 2020) (Sano, 2021)(图2)。在另一个例子中，骨骼肌分化试剂盒被用于从干细胞生产骨骼肌肌管，以创建体外疾病模型(图3)。骨骼肌的多能干细胞模型也已成功用于开发治疗杜氏肌营养不良的新疗法(Moretti, 2020)。

Farming Meat in a Dish

Promising progress is being made to create meat in the laboratory or what is commonly called ‘cultured meat’. Environmental concerns are driving the need for more sustainable meat production over traditional farming methods. Stem cell research in itself is reducing the need for the use of animals across multiple aspects as highlighted here. Producing cultured meat is straightforward in principle but faces many challenges in practice, for example maintaining the correct environment and stimuli for cultured cells to produce meat with the correct consistency and characteristics of the animal derived product. Stem cell cultures are expanded at scale in bioreactors and differentiated into skeletal muscle cells. These can be structured, using an edible scaffold for example, or used unstructured as the raw material to produce meat products (Figure 4). Tools and technologies are readily available to achieve this goal: expansion and differentiation of stem cells is highly efficient. However, a key consideration is the cost of goods. Current technologies are too costly but these are pioneering times and research is moving at an exciting pace.

Conclusions

The promise and potential of stem technologies to advance biology, medicine and food production can only be fulfilled if stem cell culture conditions are consistent, and accessible to research scientists and commercial operations alike. Exciting advances across multiple aspects of the stem cell workflow have streamlined processes to deliver products that are fully defined and animal-free. Furthermore, clinical translation of stem cell therapies and drug discovery are accelerated by the availability of GMP compliant reagents. The foundations are set for a bright future of discoveries and applications emerging from stem cell technologies.

The Authors

Dr William Hadlington-Booth is the business unit manager for stem cell technologies and the extracellular matrix at AMSBIO. Erik Miljan, PhD, is a pioneer in the development of cellular therapies for a range of degenerative and disease conditions. He holds a PhD in biochemistry from Hong Kong University. For further information please contact:
William@amsbio.com

References

Moretti, A. F., et al. (2020). Somatic gene editing ameliorates skeletal and cardiac muscle failure in pig and human models of Duchenne muscular dystrophy. Nature Medicine, 26, 207–214.
Takahashi K., et al. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. . Cell, 126, 663-676.
Takayama, K. (2020). In Vitro and Animal Models for SARS-CoV-2 research. Trends in Pharmacological Sciences, 41. 513-517.
Sano, E., et al. (2021). Modeling SARS-CoV-2 infection and its individual differences with ACE2-expressing human iPS cells. Iscience, 24(5), 102428.