Stem Cell Manufacturing

Joaquim M.S. Cabral,Claudia Lobato da Silva,Lucas G. Chase,M. Margardia Diogo

• Editorial: Elsevier
• Año: 2016
• ISBN:
  9780444632661
• Categoría: Medicine
• Categoría: Biotechnology in Medicine

Stem Cell Manufacturing discusses the required technologies that enable the transfer of the current laboratory-based practice of stem cell tissue culture to the clinic environment as therapeutics, while concurrently achieving control, reproducibility, automation, validation, and safety of the process and the product.

The advent of stem cell research unveiled the therapeutic potential of stem cells and their derivatives and increased the awareness of the public and scientific community for the topic. The successful manufacturing of stem cells and their derivatives is expected to have a positive impact in the society since it will contribute to widen the offer of therapeutic solutions to the patients. Fully defined cellular products can be used to restore the structure and function of damaged tissues and organs and to develop stem cell-based cellular therapies for the treatment of cancer and hematological disorders, autoimmune and other inflammatory diseases and genetic disorders.

• Presents the first 'Flowchart' of stem cell manufacturing enabling easy understanding of the various processes in a sequential and coherent manner
• Covers all bioprocess technologies required for the transfer of the bench findings to the clinic including the process components: cell signals, bioreactors, modeling, automation, safety, etc.
• Presents comprehensive coverage of a true multidisciplinary topic by bringing together specialists in their particular area
• Provides the basics of the processes and identifies the issues to be resolved for large scale cell culture by the bioengineer
• Addresses the critical need in bioprocessing for the successful delivery of stem cell technology to the market place by involving professional engineers in sections of the book

Chapter 1

Genetic Engineering in Stem Cell Biomanufacturing

X. Bao, and S.P. Palecek University of Wisconsin-Madison, Madison, WI, United States

Abstract

Human pluripotent stem cells (hPSCs), including human embryonic stem cells and human induced pluripotent stem cells (hiPSCs) offer unprecedented opportunities to study human organogenesis, model human disease, and provide unlimited cell sources for regenerative medicine. To realize their full potential, however, genetic manipulation strategies with high specificity and efficiency are required. Genetic manipulation is an important tool to optimize conditions for direct differentiation of hPSCs toward specific lineages, and to correct genetic mutations for clinical application of patient-specific hiPSCs. This chapter briefly summarizes a variety of genetic manipulation strategies used to generate useful hPSC lines, and reviews their advantages and shortcomings to provide insights on suitable approaches for stem cell biomanufacturing applications.

Keywords

CRISPR/Cas9; Genome editing; Human pluripotent stem cells; TALEN; Zinc-finger nuclease

1.1. Introduction

Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), have the ability to self-renew and can differentiate into any somatic cell type in the adult body [1–4], endowing them as a powerful tool to study human organogenesis, to model human disease, and to provide unlimited cell sources for regenerative medicine. During the past 20 years, rapid methodological developments for creating de novo somatic cells, including neural cells [5–7], cardiomyocytes [8–11], endothelial cells [12–16], hematopoietic cells [17–19], and epithelial cells [20,21], from hPSCs have been made, with a strong focus on applications for drug discovery, safety pharmacology, and cell therapy. While methods for generating specific cell types from hPSCs are continually evolving in the laboratory, significant hurdles remain for generation of robust and consistent high-quality hPSC-derived products at scalable quantities before successful clinical transplantation or commercial translation. In addition, reproducible protocols for specific differentiation of hPSCs are prerequisites to study the fundamental molecular and cellular mechanisms responsible for normal lineage development and disease phenotype.

Genetic engineering of hPSCs herein provides a robust approach to generate high yields of the desired, fully differentiated cell type in a controlled and reproducible manner due to the accessibility to genetic manipulation of hPSCs and their ability to clonally expand after modifications. For example, the incorporation of fluorescent reporter genes under the control of specific promoters enables the fast identification and viable purification of desired cell types during direct differentiation, whereas traditional methods have employed time-consuming immunofluorescence analysis of targeted gene expression, resulting in a significant loss of desirable cells. In addition to directing hPSC differentiation, the importance of genetic manipulation of hPSCs has been extended to broader applications including, but not limited to, labeling and selection of desired lineages [15,22,23], silencing or overexpressing targeted genes [9,16,17], monitoring endogenous signaling activity [8,24,25], reducing or eliminating immunogenicity [26,27], cell tracking in vivo [28,29], and notably, the correction of mutated genes in patient-specific hiPSCs for regenerative therapy [30–33].

Advances in genetic engineering in mammalian cells have opened up new avenues for manipulating the fate and functionality of hPSCs, as well as understanding the regulatory mechanisms responsible for their cellular transformation. This chapter provides a review of different genetic manipulation approaches that have been applied to engineer hPSCs and discusses their strengths and limitations. Gene delivery systems will also be discussed, providing insights on suitable approaches for different applications.

1.2. Genetic Manipulation Approaches in Human Pluripotent Stem Cells

Genetic manipulation of hPSCs is the process of generating genetically modified stem cell lines and their progeny by introducing a foreign gene or silencing an endogenous gene in the host genome. The ability to precisely modify the genome of hPSCs increases their usefulness for both cell-based therapies and fundamental research applications. Currently, genetic manipulation methods applied to hPSCs can be classified into three categories: random integration via transgenic approaches, targeted integration or disruption via knock-in or knock-out approaches, and bacterial artificial chromosome (BAC) introduction. Each of these three approaches has its own strengths and limitations and the selection of a suitable strategy should be determined by experimental conditions or clinical requirements. With recent advances in genetic engineering techniques, it is now possible to suit almost any particular application by using an appropriately selected strategy.

1.2.1. Transgenic Approaches

Transgenic methods involve the random integration of a gene construct that uses a cell- or tissue-specific promoter fragment to drive the expression of an exogenous gene or DNA fragment. Transgenes can be introduced into the hPSC genome via gene transfer methods including transfection, infection, and electroporation, methods which will be reviewed later. Due to its convenience and experimental feasibility, this transgenic approach has been widely used in applications including monitoring the differentiation status of hPSCs with fluorescence reporters, silencing endogenous genes with short hairpin RNA (shRNA), and overexpressing master regulatory genes with constitutively expressing promoters. In 2005, Gerrard et al. [34] generated transgenic hESCs expressing the enhanced green fluorescent protein (eGFP) reporter gene under control of the OCT4 promoter, enabling the identification and selection of pluripotent stem cells from their differentiated pro...