Engineering Strategies for Regenerative Medicine

By Tiago G. Fernandes, M. Margardia Diogo and Joaquim M.S. Cabral

Engineering Strategies for Regenerative Medicine considers how engineering strategies can be applied to accelerate advances in regenerative medicine. The book provides relevant and up-to-date content on key topics, including the interdisciplinary integration of different aspects of stem cell biology and technology, diverse technologies, and their applications. By providing massive amounts of data on each individual, recent scientific advances are rapidly accelerating medicine. Cellular, molecular and genetic parameters from biological samples combined with clinical information can now provide valuable data to scientists, clinicians and ultimately patients, leading to the development of precision medicine.

Equally noteworthy are the contributions of stem cell biology, bioengineering and tissue engineering that unravel the mechanisms of disease, regeneration and development.

• Considers how engineering strategies can accelerate novel advances in regenerative medicine
• Takes an interdisciplinary approach, integrating different aspects of research, technology and application
• Provides up-to-date coverage on this rapidly developing area of medicine
• Presents insights from an experienced and cross-disciplinary group of researchers and practitioners with close links to industry

• Language: English
• Publisher: Academic Press
• Release date: Nov 14, 2019
• ISBN: 9780128166703

Book preview

doi:10.1016/j.semarthrit.2016.07.013.

Chapter 1

Pluripotent stem cell biology and engineering

João P. Cotovioa,b; Tiago G. Fernandesa,b; Maria Margarida Diogoa,b; Joaquim M.S. Cabrala,b    a Department of Bioengineering and iBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico (IST), Universidade de Lisboa, Lisbon, Portugal

b The Discoveries Centre for Regenerative and Precision Medicine, Lisbon Campus, Instituto Superior Técnico (IST), Universidade de Lisboa, Lisbon, Portugal

Abstract

Since the isolation and culture of human pluripotent stem cells (hPSCs) in vitro, the use of these cells as a potential tool for research and biomedical applications has been growing. Therefore, it is essential to understand the complexity and the dynamics of such stem cell-derived systems, from the single-cell to the multiorgan level. It has been at the intersection of stem cell biology and engineering that new advances on the study and recreation of niches, tissues, and organ-like structures has been accomplished, mimicking the complexity and architecture of the human body. In this chapter, the main bioengineering strategies used to recreate cellular, multicellular, and multiorgan level systems are discussed. We also highlight how stem cell bioengineering is producing the necessary tools for the development of precision medicine approaches that are expected to have a major impact in the fields of disease modeling, drug discovery, and regenerative medicine.

Keywords

Pluripotent stem cells; Stem cell engineering; Stem cell systems; Bioengineering; Systems biology

1 Stem cells

In 1868 Ernst Haeckel [1], a notable biologist from Germany, came up with the term Stammzelle to describe the unicellular ancestor from which all multicellular organisms evolved, a concept very different from the one existing today. Later, he used the same term to describe the fertilized egg, or the zygote, capable of giving rise to all cell types of an organism [2]. This was the cradle for the English term stem cell (Fig. 1.1) [3]. After previous studies on the continuity of the germ plasm and on the origin of the hematopoietic system, Till and McCulloch proposed in the 1960s what are still today the two gold standard features of stem cells: (1) undifferentiated cells that are capable of self-renewal and (2) the production of specialized progeny through differentiation [4]. To accomplish such attributes, it is now known that stem cells undergo asymmetric cell division, by which the cell divides to generate one stem cell and one differentiating cell. Therefore, it may be added that stem cells are of major importance in the maintenance of homeostasis through a balance between self-renewal and differentiation [5–7].

Fig. 1.1 Stem cell research timeline. Key events and technological breakthroughs in stem cell research. EC , embryonal carcinoma; hESCs , human embryonic stem cells; hiPSCs , human-induced pluripotent stem cells; iPSCs , induced pluripotent stem cells; mESCs , mouse embryonic stem cells.

From conception to death, as cells develop derived from embryonic tissue, they become progressively restricted in their developmental potency, reaching the point when each cell can only differentiate into a single specific cell type. In the beginning, the earliest cells in ontogeny are totipotent, giving rise, in mammals, to all embryonic and extraembryonic tissues, that is, only a totipotent cell can originate an entire organism [8, 9]. Through embryogenesis, when the pluripotent state is reached, a pluripotent stem cell (PSC) can originate all the cells from all the tissues of the body, although the contributions to the extraembryonic membranes or placenta are limited [8]. On the other hand, a multipotent stem cell is restricted to the generation of the mature cell type of its tissue of origin and finally, a unipotent stem cell displays limited developmental potential, giving rise to only a single-cell type [8]. In an adult organism, stem cells can be found in most tissues throughout the body, even within relatively dormant tissues. These stem cells experience low or no division in normal homeostasis, remaining quiescent for extended periods of time. However, these cells can respond efficiently to stimuli upon initiation of homeostasis or injury [10].

Altogether, in both plant and animal kingdoms, the multicellularity of highly regulated tissues is dependent of the generation of new cells for growth and repair. Therefore, biological systems are driven by a balance between cell death and cell proliferation, preserving form and function in tissues. From this point of view, stem cells are the units of the following attributes: development, regeneration, and evolution [9, 11].

1.1 Pluripotent stem cells

Pluripotency can be defined as a transient property of cells within the early embryo, where PSCs have the capacity to form tissues of all three germ layers of the developing embryo and, later, the organs of the adult organism—ectoderm, mesoderm, and endoderm—and still the germ lineage. As previously mentioned, PSCs typically provide little or no contribution to the trophoblast layers of the placenta [8, 12].

The first PSCs to be isolated and investigated in culture were derived from mouse teratocarcinomas—a tumor of germ cell origin that maintain a wide variety of diversely differentiated tissues—known as embryonal carcinoma (EC) cells [8, 13]. Nevertheless, PSCs can be isolated from several sources through development [14], as murine [15, 16] and human blastocyst [17] or even from the postimplantation epiblast [18, 19] or germ line [20, 21]. Also, pluripotency can be recapitulated in vitro by reprogramming somatic cells to become induced pluripotent stem cells (iPSCs) [22–24].

There are specific molecular mechanisms that characterize PSCs anchored by a selected set of core transcription factors essential to establish pluripotency. As part of the core pluripotency transcription factors encoding genes are octamer-binding transcription factor 4 (OCT4), SRY-box 2 (SOX2), and NANOG. In certain circumstances, the loss of SOX2 or NANOG or their substitution can be tolerated [8, 12]. Despite that, PSCs can be classified into different states of pluripotency based on the molecular signatures, with the terms naïve and primed being introduced to describe early and late phases of ontogeny, respectively [25].

Pluripotency can be suggested by such molecular signatures, but only functional assays can reveal the developmental potential of a cell. Functional assays to assess pluripotency include: differentiation into three germ layers in vitro, teratoma formation in vivo, chimaera formation, germline transmission through blastocyst injection, tetraploid complementation, and single-cell chimaera formation [8]. For human pluripotent stem cells (hPSCs), teratoma formation remains the gold standard of functional assays.

1.1.1 Embryonic stem cells

Mammalian embryogenesis starts with a single totipotent cell, the zygote. After the first cell division, the two-cell embryo is composed by two equal blastomeres. In the earlier stages, including two- and four-cell embryos, cells are still considered totipotent. Later, in what is called blastocyst, it is possible to distinguish the extraembryonic trophectoderm (TE) on the outside and the inner cell mass (ICM) [14, 26]. It is in the ICM that pluripotent cells first arise. The ICM cells, cultured in conditions that allow indefinite self-renewal and maintenance of the pluripotent state, are known as embryonic stem cells (ESCs) and were first derived from mouse (mESCs) in 1981 by Martin Evans [15] and Gail Martin [16]. These cultures proved to have all the properties previously established for EC cell cultures, as well as a completely normal karyotype [27]. Only by the year 1998, Thomson derived the first ESC lines from human blastocysts [17], the so-called human embryonic stem cells (hESCs).

Throughout normal development, the amount of ESCs is limited and their existence is constrained in the time course of development, being present for only a short period of time. In contrast, tissue culture allows the generation and maintenance of millions of ESCs indefinitely, preserving their pluripotent state [27].

1.1.2 Induced pluripotent stem cells

The molecular mechanisms by which the genes of the genotype bring about phenotypic effects—the epigenetics concept—was captured by Conrad Waddington [28] in the iconic image of the epigenetic landscape that influences cellular fate during development, analogously to the movement of a marble (Fig. 1.2). Since then, the possibility that cells can change their identity has fascinated scientists [29]. This notion was first suggested by Sir John Gurdon in 1958, establishing that in vivo plasticity of the differentiated state can be induced artificially by directly manipulating cells and their environment [30]. It was demonstrated that the marble can be rolled back to the top of the hill, that is, committed or differentiated cells can be reprogrammed back to a wider developmental potential (dedifferentiation).

Fig. 1.2 Cell fate plasticity. Contemporary version of Waddington landscape depicting an analogy between a marble rolling downhill as development leads undifferentiated cells to a mature state. Cellular reprogramming has shown that it is possible to make the marble roll back to the top of the hill as mature cells can be reprogrammed back to a wider developmental potential.

As the possibility to reprogram cells, not by transplanting their nuclei, but by introducing pluripotency factors into cells became a reality, cells with a gene expression profile and developmental potential similar to ESCs were generated in 2006. This accomplishment was reached using mouse somatic cells together with a cocktail of four transcription factors [23]. The resulting reprogrammed cells were termed iPSCs and were generated after retrovirally introducing four transcription factors encoding genes, OCT4, SOX2, Kruppel-like factor 4 (KLF4) and the MYC proto-oncogene, bHLH transcription factor (MYC)—the Yamanaka factors. After successfully generating mouse iPSCs, in 2007 iPSCs were generated from human fibroblasts, using the same four factors and alternatively NANOG and Lin-28 homolog A (LIN28) instead of KLF4 and MYC [22, 24]. It was the establishment of human induced pluripotent stem cells (hiPSCs). Since then, cellular reprogramming became a robust method to convert differentiated cells to a PSC state [31, 32].

Afterwards, besides the initially used retroviral or lentiviral vectors, nonintegrating methods have been developed and include reprogramming using episomal DNAs, adenovirus, Sendai virus, PiggyBac transposons, minicircles, recombinant proteins, synthetically modified mRNAs, microRNAs (miRNAs), and more recently, small molecules [31]. These new techniques, in addition to lower variability between cell lines, can lead to safer reprogramming of iPSCs and to more suitable cells for clinical applications by avoiding insertional mutagenesis and transgene reactivation.

1.2 Applications of hPSCs

Since the isolation of ESCs from human embryos, the use of PSCs as a potential tool for research and medicine has been growing (Fig. 1.3). Besides that, after finding that somatic cells can revert all the way back to an ESC state through transcription factor activation, manipulation of signaling pathways aiming for cell differentiation has been studied contributing to hiPSCs applications in biomedicine. Accordingly, several protocols have been described for in vitro direct differentiation of neurons, hematopoietic cells, hepatocytes, smooth muscle cells, and cardiomyocytes, among other cell types across the three germ layers [33].

Fig. 1.3 Clinical applications of human induced pluripotent stem cells for precision medicine. After isolation of patient somatic cells, these cells can be cultured and reprogrammed into patient-specific induced pluripotent stem cells (iPSCs). This is a promising cell source for cell therapy as it is possible to silence mutations carried by patient-specific cells using novel gene-editing tools (like CRISPR-Cas9) for the generation of corrected iPSCs that can be used in regenerative medicine approaches. Also, the differentiation of patient-specific iPSCs makes disease modeling and drug screening a possibility.

An obvious application of hPSCs in medicine is in cell therapy. Regenerative medicine strategies based on the use of stem cells to promote regeneration or to replace damaged tissues after cellular transplantation has been shown to successfully induce functional recoveries [31]. In fact, several clinical trials were already established using hPSC-based therapies [34]. In the particular case of hiPSCs, an important advantage of using these cells is the capability to generate autologous differentiated cells, that is, patient-specific cells, theoretically suppressing the risk of immune rejection. For instance, the first clinical study using hiPSC-derived products was performed in 2014 by Masayo Takahashi and Yasuo Kurimoto, in which these two Japanese physicians successfully transplanted autologous retinal pigment epithelium sheets derived from hiPSCs into a woman with macular degeneration [35]. Besides all this progress in hPSC-based therapies, the acquisition of chromosomal aberrations, due to the reprograming process and subsequent culture, represents one of the disadvantages of these cells [36]. Moreover, due to hPSC tumorigenicity, it is critical to ensure that the transplanted product does not contain undifferentiated cells with the potential to generate teratomas [31].

Another important biomedical application of hPSCs is in disease modeling [37]. It is expected that in vitro hPSC-based disease models help to identify the pathological mechanisms underlying human diseases. Both hESCs and hiPSCs have been used for modeling human genetic diseases, establishing isogenic cell lines with novel gene-editing tools (e.g., CRISPR-Cas9), to induce disease-causing mutations or to silence mutations carried by patient-specific cells [38, 39].

Modeling of human diseases is motivated by the necessity of developing novel therapeutic agents allowing the diseases to be treated, alleviated, or cured. Therefore, drug screening and toxicological assays is also considered as a potential application of hPSCs [37]. Animal models have been used in drug screening but differences from the actual human setting lead to an inaccurate forecasting of their effects. Moreover, animal models are not suitable for high-throughput screening of small-molecule libraries [38, 40]. Until now, many drug screens have been conducted using hiPSC-based models and potential drug candidates have been identified. Also, it is not only important to assess efficacy but also toxicity, predicting the likelihood of candidate drugs to cause serious side effects [31]. A specific patient has a specific genetic background and this fact implies different responses to medication for each individual. Accordingly, hiPSC-based drug screening is the key for a personalized therapy, an emerging approach known as precision medicine [40].

Just as new technologies are being developed, the greater will be the potential applicability of hPSCs in the emerging fields of regenerative medicine, disease modeling, and drug screening.

2 Stem cell systems

For a widespread use of stem cells in biomedical applications, it is essential to understand the complexity and the dynamics of the stem cell biological system as well as the information that flows between each layer of it. In this section, a reductionist view of the different layers of complexity of the biological systems is presented (Fig. 1.4), exploring this question from the cell level to the whole organism.

Fig. 1.4 Layers of complexity within biological systems. Different layers of complexity of the biological systems from the cell level to the whole organism and respective engineering approaches. These approaches can help to understand each biological layer, and to a great extent, they can help to create tissues that resemble the in vivo anatomy and physiology of the human body for biomedical applications.

2.1 The cell: The functional unit of the system

The cell by itself can be considered the functional unit of a biological system. Furthermore, within each cell and as a first layer of regulation, there are collections of molecular species that interact and constitute what are called gene regulatory networks (GRNs), a term first coined in sea urchin developmental studies [41]. The GRNs, based on the microenvironmental signals (input), are able to control gene expression levels and, consequently, gene product abundance (output). Thus, the cellular GRN is responsible for the development potential of the cell by regulating its transcriptional activity [42, 43]. For example, the pluripotent state of a PSC is determined by a set of pluripotency transcription factors, namely OCT4, SOX2, and NANOG. These transcription factors are the core of the pluripotency GRN and, therefore, they cooperate and regulate different elements in the genome, including their own promoters [12,