Maria Margarida Diogo

Scalable Expansion of Human-Induced Pluripotent Stem Cells in Xeno-Free Microcarriers

The expansion of human-induced pluripotent stem cells (hiPSCs) is commonly performed using feeder layers of mouse embryonic fibroblasts or in feeder-free conditions in two-dimensional culture platforms, which are associated with low production yields and lack of process control. Robust large-scale production of these cells under defined conditions has been one of the major challenges to fulfil the large cell number requirement for drug screening applications, toxicology assays, disease modeling and potential cellular therapies. Microcarrier-based systems, in particular, are a promising culture format since they provide a high surface-to-volume ratio and allow the scale-up of the process to stirred suspension bioreactors. In this context, this chapter describes a detailed methodology for the scalable expansion of hiPSCs in spinner flasks and using xeno-free microcarriers to allow further translation to Good Manufacturing Practice (GMP) conditions.

Stem cell culture: mimicking the stem cell niche in vitro

Stem cells have been defined as unspecialized cells with unique properties such as the capacity for prolonged proliferation without loss of potential, and the ability to differentiate into specialized cell types. They have potential in the design of cellular therapies for degenerative diseases and can be used as models for pharmacological and developmental biology research. However, before their enormous potential can be realized, it is crucial to fully understand the cellular and molecular mechanisms governing their self-renewal and differentiation. One of the major objectives in this field is to study the regulation of stem cell function in vivo in tissues where these cells reside – in the endogenous ‘stem cell niche’. The principle behind this, proposed 30 years ago, is that tissues house stem cells within specific locales and stem cell fate is influenced by interactions with several components of their three-dimensional microenvironment, including soluble and immobilized factors, the extracellular matrix (ECM) and signals presented by neighboring cells. It is recognized that this microenvironment presents specific biomechanical signals that strongly influence stem cell behavior. They are often represented as static microenvironments, but stem cell niches are spatially and temporally dynamic, integrating long-term signals with short-term and injury-mediated responses. Thus, the successful application of stem cells depends on a capacity to recreate this complex and dynamic in vivo microenvironment. Studies on stem cell niches in invertebrates and mammals have begun to unravel the regulatory mechanisms involved in these complex cellular interactions, and have inspired stem cell bioengineers to develop biomaterials and technologies that recreate biochemical and structural aspects of in vivo microenvironments as well as microfabricated structures that allow precise control of key niche parameters. These culture platforms are useful for studying stem cell function at a single-cell level and in a high-throughput manner.

Three-Dimensional Cell-Based Microarrays: Printing Pluripotent Stem Cells into 3D Microenvironments

Cell-based microarrays are valuable platforms for the study of cytotoxicity and cellular microenvironment because they enable high-throughput screening of large sets of conditions at reduced reagent consumption. However, most of the described microarray technologies have been applied to two-dimensional cultures, which do not accurately emulate the in vivo three-dimensional (3D) cell-cell and cell-extracellular matrix interactions.Herein, we describe the methodology for production of alginate- and Matrigel-based 3-D cell microarrays for the study of mouse and human pluripotent stem cells on two different chip-based platforms. We further provide protocols for on-chip proliferation/viability analysis and the assessment of protein expression by immunofluorescence.

Purification of Human Induced Pluripotent Stem Cell-Derived Neural Precursors Using Magnetic Activated Cell Sorting

Neural precursor (NP) cells derived from human induced pluripotent stem cells (hiPSCs), and their neuronal progeny, will play an important role in disease modeling, drug screening tests, central nervous system development studies, and may even become valuable for regenerative medicine treatments. Nonetheless, it is challenging to obtain homogeneous and synchronously differentiated NP populations from hiPSCs, and after neural commitment many pluripotent stem cells remain in the differentiated cultures. Here, we describe an efficient and simple protocol to differentiate hiPSC-derived NPs in 12 days, and we include a final purification stage where Tra-1-60+ pluripotent stem cells (PSCs) are removed using magnetic activated cell sorting (MACS), leaving the NP population nearly free of PSCs.

Characteristics of stem cells

Stem cells are characterized by their ability for unlimited or prolonged self-renewal and differentiation into highly distinct cell lineages. Due to these properties they are considered a very attractive source of cells for a wide range of clinical and pharmacological applications. However, significant progress needs to be made to fully realize this enormous potential. Stem cell functions are subject to tightly regulated control mechanisms, so gaining a deeper understanding of these mechanisms is crucial. Only then it will be possible to employ these cells to repair damaged tissue in regenerative medicine and tissue-engineering applications.

Stem cells and regenerative medicine

Stem cell-derived products constitute a promising therapeutic approach for the treatment of a wide range of disorders. Neurodegenerative diseases, like Parkinson’s disease or Huntington’s disease, neurological disorders, cardiac failure and blood disorders – among others – may one day be treated using cellular therapies and regenerative medicine approaches based on stem cells. Furthermore, owing to the potential positive impact on healthcare systems, translation of such stem cell technologies into the clinical arena will bring about social and economic advantages worldwide. In this chapter we provide an overview of this emerging field, and we present several examples of ongoing preclinical and clinical studies.

Stem cell separation

Separation methods have been applied to the stem cell field for many years for the isolation and/or enrichment of rare subpopulations from specific tissues for clinical applications. One typical example is the isolation of CD34 + hematopoietic stem cells (HSCs) or progenitor cells from umbilical cord blood or bone marrow using density gradient centrifugation integrated with magnetic-activated cell sorting (MACS) for the treatment of hematooncological diseases. Since that time many important advances have been made in stem cell research and the therapeutic potential of other adult stem cell populations has been highlighted. Moreover, the breakthrough of human induced pluripotent stem cell (iPSC) derivation through reprograming has also paved the way for the large-scale production of all types of patient-specific cells for regenerative medicine, tissue engineering and drug screening applications, as well as for studies in developmental biology. These are really important breakthroughs but their translation to clinical practice and other applications is delayed by the lack of efficient and high-resolution cell separation techniques. For example, the development of high-resolution methods to separate heterogeneous populations of human mesenchymal stem cells (MSCs) into specific subpopulations is crucial for studying their specific biological and therapeutic features with respect to their clinical role. Also, the depletion of tumorigenic cells from pluripotent stem cell derivatives, such as iPSCs, is essential for safe clinical application. This chapter critically assesses the main cell separation techniques presently available, their basic principles, their advantages and limitations, and examples of their application in the stem cell field. The techniques are grouped according to the basic principles that govern cell separation, related to the main physical, affinity and biophysical characteristics of cells. Novel trends in cell separation are also highlighted, including the use of novel ligands (e.g. aptamers) for affinity targeting of cells, the application of “tag-less” methods to avoid cell labeling, and the use of microfluidics and other microscale devices.

Microscale technologies for stem cell culture

Microscale technologies provide powerful experimental tools for a wide range of relevant applications in stem cell research, including high-throughput screening of large numbers of test samples. Miniaturization increases assay throughput, while reducing reagent consumption and the number of cells required, which makes these platforms attractive for a wide range of applications in drug discovery, toxicology and stem cell research. In this chapter we provide an overview of the emerging technologies that can be used to generate different microscale devices, in addition to highlighting significant advances in the field. This emerging and multidisciplinary approach offers new opportunities for the design and control of stem cells in tissue engineering and cellular therapies, and promises to accelerate drug discovery in the biotechnology and pharmaceutical industries.