Synthetic biology for improving cell fate decisions and tissue engineering outcomes

Abstract

Synthetic biology is a relatively new field of science that combines aspects of biology and engineering to create novel tools for the construction of biological systems. Using tools within synthetic biology, stem cells can then be reprogrammed and differentiated into a specified cell type. Stem cells have already proven to be largely beneficial in many different therapies and have paved the way for tissue engineering and regenerative medicine. Although scientists have made great strides in tissue engineering, there still remain many questions to be answered in regard to regeneration. Presented here is an overview of synthetic biology, common tools built within synthetic biology, and the way these tools are being used in stem cells. Specifically, this review focuses on how synthetic biologists engineer genetic circuits to dynamically control gene expression while also introducing emerging topics such as genome engineering and synthetic transcription factors. The findings mentioned in this review show the diverse use of stem cells within synthetic biology and provide a foundation for future research in tissue engineering with the use of synthetic biology tools. Overall, the work done using synthetic biology in stem cells is in its early stages, however, this early work is leading to new approaches for repairing diseased and damaged tissues and organs, and further expanding the field of tissue engineering.

Summary

• Stem cells serve as a repair system for the body and hold tremendous promise for replacing and/or regenerating tissues damaged by disease and injury.

• Stem cells tightly control their gene expression to either self-renew, or differentiate into more specialized cell types.

• Genetic circuits built by synthetic biologists offer a platform technology to regulate gene expression patterns in stem cells to drive their cell fate.

• Coupling synthetic biology with stem cells will help to facilitate more robust tissue engineering outcomes.

Introduction

Synthetic biology offers a platform of technologies to unlock the potential to engineer cells as custom-made therapies. A cell’s ability to proliferate, migrate, or differentiate is regulated by various mechanisms including environmental factors, in addition to the level of activation and silencing of various genes including transcription factors. In recent years, synthetic biology has significantly advanced the design of genetic circuits to offer unique expression patterns in mammalian cells [1–16]. This is particularly exciting for engineering stem cells since dynamic control of gene expression patterns is thought to improve differentiation outcomes, which play an important role in the fields of cell therapy, tissue engineering, and regenerative medicine. This review provides a brief introduction to stem cells and synthetic biology. We also describe studies that have implemented synthetic biology in stem cells and discuss their impact on tissue engineering and regenerative medicine.

Stem cells

Stem cells serve as a repair system for the body and therefore hold tremendous promise for replacing and/or regenerating tissues damaged by disease and injury. Stem cells are defined as cells that have the capacity to differentiate into more mature cells, in addition to having the ability to self-renew (Figure 1). They also have many levels of cell potency, which is defined as the cell’s ability to differentiate into other cell types. A greater cell potency indicates a larger number of cell types that a stem cell can become. When cells terminally differentiate, they exit the cell cycle and mature into specialized functional cells of tissues (Figure 1) [17].

Figure 1. Stem cells.

Stem cells are defined as cells that can differentiate into more specialized cells and also self-renew themselves (yellow and light blue cells). Stem cells have many levels of cell potency, which is the cell’s ability to differentiate into other cell types. A greater cell potency indicates a larger number of cell types that a stem cell can become (e.g. the yellow stem cell has more potency than the light blue stem cell). As cells differentiate, they lose their potency. When cells terminally differentiate, they exit the cell cycle and mature into specialized functional cells of tissues.

Pluripotent stem cells

With the exception of the bacteria that make up the gut microbiome, all cells in the human body originate from embryonic stem (ES) cells [18,19]. One of the first major events during embryonic development of vertebrate animals, is the specification of the three embryonic germ layers: ectoderm, mesoderm, and endoderm [20–22]. These germ layers give rise to multipotent stem cells that have the potential to differentiate into specific tissue lineages (Figure 2). The discovery that pluripotent ES cells can be harvested from the inner cell mass of an early stage pre-implanted embryo, be grown in culture in the laboratory and have the capacity to generate any cell type in the body, offered a controversial promise that these cells could be used as replacement cells and/or for generating organs and tissues for treating many different diseases and injuries. However, the more recent discovery that just 4 transcription factors can override previously made cell fate decisions to become pluripotent (Figure 2) has circumvented much of the controversy surrounding pluripotent stem cell research [23–31]. Moreover, iPS cells provide patient-specific models of disease that can be used for drug screening [32–38]. These induced pluripotent stem (iPS) cells are powerful tools that have opened the door to an unlimited source of any type of human cell needed for many therapeutic purposes.

Figure 2. Pluripotent stem cells.

Pluripotent stem cells are undifferentiated cells with the potential to become any cell in the body. These special stem cells are able to form all three germ layer cells: the mesoderm (green cell), endoderm (yellow cell), and ectoderm (pink cell). These cell types are multipotent stem cells. They are somewhat differentiated, but still have the capabilities to differentiate into a more specific cell type. Once multipotent stem cells further differentiate, they are known as terminally differentiated and can no longer self-renew. Induced pluripotent stem cells (iPS cells), start out as terminally differentiated cells, most often skin cells from the ectoderm. The skin cells are then reprogrammed using transcription factors to revert them back into pluripotent stem cells, hence the name ‘induced’ pluripotent stem cells.

Adult stem cells

Adult stem cells are multipotent stem cells that are found among differentiated cells in a tissue or organ. As stem cells, they can self-renew themselves and differentiate into other specialized cell types of tissues or organs (Figure 2), however they have relatively restricted potency; that is, their ability to differentiate into specific cell types is generally limited to the type of tissue in which the adult stem cell resides. Therefore, it is believed that the primary role of adult stem cells is to maintain and repair the tissue in which they are found. The bone marrow has been a site of extensive research because of the two adult stem cells located within it: hematopoietic stem cells and mesenchymal stem cells. Hematopoietic stem cells (HSCs) are responsible for making all of the cells of the blood system, that is, all of the immune cells in addition to the red blood cells and platelets [39–45]. Mesenchymal stem cells (MSCs) were originally identified as cells capable of differentiating into mature cells of several mesenchymal tissues such as bone, muscle, tendon, cartilage, and fat [46–54]; however more recently, studies have shown that MSCs also play a supporting role when growing HSCs in culture, in addition to having unique immunomodulatory properties that make them an important cell type for the repair of damaged and diseased tissues [51,55–67]. Indeed, progress with enlisting adult stem cells for the purpose of regenerating tissues has been a primary focus for many scientists in the stem cell community because these cells are more likely to select the necessary gene expression patterns that are consistent with the more specialized cells.

Altogether, both pluripotent and adult stem cells continue to enhance our understanding of development, disease and drug screening, and provide the cells for tissue replacement, and regeneration [68–76]. Despite these advances, challenges that exist with using stem cells for therapeutic utilization include obtaining large enough numbers of cells for particular applications, producing homogeneous cell populations when needed, along with proper cell and tissue patterning, morphogenesis, and the directed differentiation of these cells to desired tissue outcomes.

Synthetic biology

Synthetic biologists use engineering approaches to build genetic circuits that enable the predictable programming of new functions into cells. This is done by rationally designing individual gene expression parts to engineer and assemble functional genetic devices to perform defined functions inside or outside of a cell [77–91]. These functions may include variations in sensing of environmental conditions [92,93], light [94–102], as well as producing custom proteins and/or changing the expression level of proteins [6,14,103,104].

By piecing together individual functional gene regulatory units, synthetic biologists create genetic circuits that are capable of reprogramming cells. Traditionally, the design of genetic circuits included a trigger element that was usually a small molecule, or different wavelengths of light that act on regulator proteins within the circuit (Figure 3A). For example, the bacterial repressor proteins, LacI and TetR have been extensively used and shown to offer inducible transcriptional control by placing the proteins’ binding domains within promoter regions (Figure 3B). By fusing these regulatory proteins to either a transcriptional repressor domain (e.g. KRAB), or an activator domain (e.g. VP16 or VP64), they can function to repress gene expression or activate gene expression, respectively. The effects of the LacI and the TetR regulatory proteins can be reversed by adding the small molecule inducers, isopropyl β-d-1-thiogalactopyranoside (IPTG) and tetracycline, respectively (Figure 3C). For example, in the case of the LacI repressor proteins, the LacI proteins bind to lacO binding sites that have been placed in the promoter that regulates a downstream gene of interest (GOI). When LacI repressor proteins bind, the proteins block the binding sites for RNA polymerase, the enzyme responsible for starting transcription, resulting in the repression of the downstream gene. Upon adding IPTG, the LacI repressor proteins undergo a conformational change and can no longer bind to the lacO binding sites, enabling RNA polymerase to bind to the DNA and transcribe the GOI. Genetic parts like the LacI and TetR systems have been regularly used for the construction of genetic circuits.

Figure 3. Building genetic circuits.

(A) Genetic circuits are built by assembling genetic parts that can be induced by either (i) small molecules or (ii) light. (B) Traditional approaches to building genetic circuits consist of implementing repressor proteins (blue circles) that are transcribed and translated and bind to their respective binding sites (blue squares) that are placed in or close to the promoter. When the repressor proteins bind to their respective binding sites, preventing RNA polymerase from sitting down and transcribing the downstream gene of interest (GOI, green box). (C) Adding the inducer small molecule, IPTG, binds to the LacI repressor proteins, causing a conformational change in their structure, and they can no longer bind to their binding sites. This opens up the DNA strand for RNA polymerase to bind, and transcription of the downstream GOI ensues. (D) The genetic toggle switch was built by assembling genetic parts to create a genetic circuit. Here, each promoter is inhibited by the repressor that is transcribed by the opposing promoter. The bistability of the toggle switch arises from the mutually inhibitory arrangement of the repressor genes, and the state of the switch can be flipped by adding the appropriate inducer.

The inception of synthetic biology was put forth with the introduction of the genetic toggle switch [103] and the repressilator [104] in bacteria where individual regulatory genetic parts were assembled into genetic circuits to reprogram cells with novel functions. The genetic toggle switch behaves as an on/off switch, and the repressilator implements oscillations of gene expression in cells. Specifically, the genetic toggle switch is a bistable switch that has two stable equilibrium states where each promoter is inhibited by the repressor that is transcribed by the opposing promoter. The bistability of the toggle switch arises from the mutually inhibitory arrangement of the repressor genes, and the state of the switch can be flipped by adding the appropriate inducer (Figure 3D). Synthetic biologists also demonstrated that it is possible to use genetic parts to build gene circuits for programming cells to perform Boolean logic operations upon induction with small molecules that endow cells to perform programmed decision-making capabilities [86,105–111]. Small molecule gene expression systems are the most common genetic parts that synthetic biologists use to engineer gene circuits. Their assembly and function have been extensively reviewed elsewhere [11,16,112–119].

Synthetic biologists have also demonstrated that prokaryotic genetic parts (e.g. repressors and their respective binding sites) can be used to build genetic circuits that function in mammalian cells. However, mammalian cells are more difficult than bacteria to engineer due to the complexity of eukaryotic cell signaling. Despite this challenge, synthetic biologists have successfully engineered genetic circuits to function in mammalian cells by combining prokaryotic and eukaryotic genetic parts [6,120], and constructing daisy-chain interconnections of individual transcription control circuits [121,122] that program cells to display tight levels of gene control that can be used to reprogram the cells to perform specific functions.

Genome engineering and controlling cell fate

Many of the genetic circuits built utilize transcription factors and their respective binding sites. Transcription factors are proteins that regulate gene transcription by binding to specific areas of DNA within the cellular genome and play an important role in stem cell identity and differentiation. They translate intracellular and extracellular signals into dynamic patterns of gene expression that drive stem cell differentiation and define cellular phenotype [90,123,124]. Modeling the natural gene expression patterns during development, it has been demonstrated that the overexpression of specific transcription factors can reprogram cells to obtain desired cellular outcomes [125]. A classic example of this is the reprogramming of adult cells into iPS cells through the overexpression of Oct4, Sox2, Klf4, and cMyc transcription factors [27,28]. While the overexpression of these transcription factors has paved the way for producing tissue-specific cell types, the differentiation of iPS cells into desired adult cell types and tissues remains difficult due to epigenetic memory, and the requirement of spatial and temporal control of gene expression patterns of lineage-specific transcription factors [31].

DNA recombinases

Site-specific DNA recombinase technology was the first widely used technique for engineering the mammalian genome. This technology is based on recombinase enzymes that selectively recognize two target sections of DNA, that have been previously inserted, cutting out the DNA between these target sections, and gluing the strands back together [126]. This approach has been used by molecular biologists for years, specifically for the generation of transgenic and knockout mice. On the other hand, synthetic biologists have used site-specific DNA recombinase technology to build genetic circuits capable of programming cells as memory devices for recording cellular events, performing mathematical computations, in addition to higher-order disease identification [127–134]. Using DNA recombinase technology as a platform, Boolean Logic and Arithmetic through DNA Excision (BLADE) technology was created. BLADE was shown to function in mammalian cells and has six programmed inputs to allow for 16 logic operations [135]. This type of programmed circuitry in cells can be used to custom build cells for targeted therapeutic applications and directing stem cell differentiation.

CRISPR

More recently, it was discovered that DNA nucleases could be programmed to bind to and cleave DNA sequences without the recognition sequences already present in the genome that is required for site-specific DNA recombinase technology. Zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR) are powerful genetic tools that enable precise genetic modifications by inducing targeted DNA breaks that stimulate cellular DNA repair mechanisms that can result in deletions or insertions of the original DNA sequence [136–141]. An alternative to double-stranded breaks in the DNA, targeted genome editing can be done using a D10A nickase Cas endonuclease, cytidine deaminase, and a DNA uracil glycosylase inhibitor [142]. Altogether, these components are capable of introducing a mutation into the DNA by converting cytosine to thymine. To report base editing activity within a cell population, Transient Reporter for Editing Enrichment (TREE) was developed [143], an assay that allows for the real-time, florescent-based identification and isolation of base-edited cell populations. This was done by engineering a blue florescent protein (BFP) variant to switch and express green fluorescent protein (GFP) after the targeted modification of converting a cytosine to thymine occurs. TREE was demonstrated to work in human iPS cells, which enabled the enrichment of edited cells. Since the discovery of CRISPR, methods for improving the targeting of Cas9 to specific locations in the genome continue to advance this technology [144–147].

Synthetic transcription factors

Engineering synthetic transcription factors (Syn-TFs) is a novel approach for targeting endogenous cellular networks within cells to modulate gene expression at specific locations within the genome by binding to DNA sequences. These Syn-TFs are made using DNA domains of one of several well-established genetic parts known for their ability to target specific sequences, such as zinc finger (ZF) proteins, transcription activator-like effectors (TALEs), and the clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9 system where the Cas9 protein has been engineered to remove the nuclease activity (dCas9) [119,136,148–150]. These proteins can be engineered to target genomic regulatory elements that either suppress or activate expression at a specific DNA sequence by fusing gene regulator domains to the synthetic transcription factors. Syn-TFs have been instrumental in studying endogenous DNA sequences to better understand the epigenetic landscapes of cells, for reprogramming cells to obtain desired cell types [151], and to study the fundamental genomic molecular mechanisms that dictate health and behavior of cells.

Synthetic biology, stem cells, and tissue engineering

Efforts in synthetic biology are currently underway to engineer stem cells with genetic circuits that dynamically control gene expression for the purpose of driving stem cell fate decisions. Recently, a new genetic circuit was engineered by assembling genetic parts from the bacterial LacI repressor system and the bread mold, Neurospora crassa, that functions as an orthogonal genetic switch in mammalian cells [120]. Specifically, the metabolic cluster of regulatory genes, called the Q system, was optimized and moved to mammalian cells. In this genetic circuit, upon the QF transcription factor binding to its QUAS binding sites, gene expression of the downstream gene is turned on. This genetic switch was demonstrated to dynamically control gene expression in pluripotent stem cells. Additional studies have been reported of genetic circuits improving cell fate decisions by engineering a band-pass filter to dynamically control three key transcription factors in pancreatic progenitor stem cells to enable the timely coordination of their expression to produce a homogeneous population of cells that produce insulin [152]. Another study emphasized the need for temporal regulation of gene expression during cell fate decisions with the pulsing of a key transcription factor [153]. In this study, the early pulsing of the transcription factor, Gata6, in iPS cells initiated the formation of a complex three-dimensional multicellular tissue construct that displayed liver tissue phenotypes. All in all, to improve cell fate outcomes, studies demonstrate the need to dynamically control transcription factors throughout differentiation. Having the tools and understanding for directing cell fate decisions will enable the translation of stem cell therapies to clinical practices.

The primary goal of tissue engineering is to repair and regenerate diseased and damaged tissues. While remarkable progress has been made in understanding tissue repair and regeneration from endogenous adult stem cells, it still remains unexplained why mammals have a tendency for imperfect healing and scarring rather than regeneration [154]. One significant difference between embryonic tissue development and repairing injured and diseased tissue is that inflammation is usually present with a disease and after an injury. Studies have shown that the immune system plays a central role in tissue repair and regeneration [155,156]. While an immune system response to tissue injury is essential in determining the recovery time and the outcome of the healing process, a prolonged inflammatory environment for stem cells can drastically change the outcome of the tissue healing process. For example, a prolonged exposure to an inflammatory environment can lead to incomplete healing and scar formation, whereas a tightly controlled immune response will result in complete regeneration where the tissue has total repair and restoration of function. To address this, a synthetic promoter was designed and built to contain five NF-κB recognition sequences and was placed upstream of the gene that encodes for interleukin-1 receptor antagonist (IL-1Ra), an anti-inflammatory protein [157]. In cells, the NF-κB pathway naturally controls the transcription of potent inflammatory cytokines that are responsible for activating inflammatory responses (Figure 4A). An iPS cell line with the synthetic promoter was created and was shown to possess self-regulating and attenuating inflammation properties in vitro with the controlled release of anti-inflammatory molecules. Specifically, iPS cells were virally transduced with the engineered synthetic inducible promoter driving the expression of IL-1Ra, and differentiated into chondrocytes, the cells that make cartilage tissue (Figure 4B). Repairing cartilage is difficult since it is not vascularized, leading to a lack of natural regeneration capabilities, along with a harsh inflammatory response that usually accompanies damaged and diseased tissues. The resulting engineered cartilage tissue is therefore endowed with the ability to sense and respond to inflammatory stimuli by producing anti-inflammatory proteins in an autoregulatory fashion. This study suggests that when the engineered cells are implanted in an animal, it will have the capacity to sense and respond to inflammatory stimuli and lead to improved tissue regeneration (Figure 4C).

Figure 4. Engineered cartilage.

(A) The natural cellular response to inflammatory cytokines, like IL-1. IL-1 binds to the IL-1 receptor, which activates the NF-κB pathway that turns on the transcription of potent inflammatory cytokines that are responsible for activating inflammatory responses (red background). (B) Engineered cells with a synthetic promoter containing five NF-κB recognition sequences upstream of interleukin-1 receptor antagonist (IL-1Ra) was integrated into the genome of iPS cells (dotted line in DNA). In these engineered cells, when IL-1 proteins bind to their receptors, rather than inflammatory cytokines being produced, these cells produce IL-1Ra, a protein that blocks the IL-1 receptors from binding to the IL-1 protein and therefore prevents inflammatory cytokines from being produced (green background). (C) These engineered cells can be implanted into the knee of an animal experiencing inflammation (red background) to aide in the immune response by sensing and responding to inflammatory stimuli and producing anti-inflammatory proteins in an autoregulatory fashion (green background).

Conclusions

Stem cells serve as a repair system for the body and therefore hold tremendous promise for replacing and/or regenerating tissues damaged by disease and injury. However, the molecular mechanisms of tissue repair have yet to be realized, precluding the development of go-to clinical therapies. Furthermore, the complex relationship between tissue regeneration and the immune system during wound healing has further delayed clinical advances. A primary goal in synthetic biology is to engineer genetic circuits that exert fine control over cell behavior by endowing cells with complex functions like sensing and responding. Recent studies have capitalized on the utility of using synthetic biology approaches to drive stem cell differentiation into desired lineages, and to engineer synthetic tissues that mediate the inflammatory environment for improved tissue repair.

Abbreviations

BFP

blue florescent protein

BLADE

Boolean Logic and Arithmetic through DNA Excision

CRISPR

clustered regulatory interspaced short palindromic repeats

ES

embryonic stem

GFP

green fluorescent protein

GOI

gene of interest

HSC

hematopoietic stem cell

iPS

induced pluripotent stem

IPTG

isopropyl β-D-1-thiogalactopyranoside

MSC

mesenchymal stem cell

Syn-TFs

synthetic transcription factors

TALEs

transcription activator-like effectors

ZF

zinc finger proteins