Designing a living mammalian cell

HJ421

Living cells continue to attract the attention of researchers for understanding their origin, design and function. Recent advances in molecular science, biochemical devices and computational tools have generated renewed interest in unravelling the secret of formation of living cells. It is unclear how primitive life forms originated from non-living molecules. Multi-directional approaches have yielded deeper insight into this question. From a top-down approach, a wealth of invaluable information has been obtained concerning molecular design and genome coding in many types of cells. On the other hand, assemblies of complex structures with living attributes starting from simple inanimate molecules have also been attempted. Synthetic biologists have achieved significant milestones in designing and creating a living bacterial cell by transplanting a chemically synthesized genome, but overcoming the limitations of bacterial design and constructing a minimal mammalian cell with multiple sub-cellular organelles remains a future dream. In this paper, we give a brief account of progress made and major challenges faced in designing a functional mammalian cell in the laboratory. Integration of knowledge from genome science, molecular engineering and computational technologies, in particular, shows promise. Success in designing an artificial mammalian cell would open a plethora of new opportunities for biomolecular engineering, shed light on evolution, and assist with the diagnosis and therapy of disease.


Introduction
The modern world has witnessed many scientific discoveries leading to advanced technologies and innovations in the service of humankind. Over the years, remarkable progress has been made in science, engineering and technology related to the living world, thereby unlocking many biological secrets. However, a frequently asked question concerns how life originated on the Earth or elsewhere (1) from inanimate molecules. It is commonly thought that certain non-living molecules spontaneously assemble into unique structures capable of acquiring the complexity and specificity observed in living cells. Cellular complexity enormously increases from bacteria to mammals and from single-celled to multicellular organisms. Cell biotechnologists have long been striving to emulate nature in designing a living cell starting with simple non-living chemical entities in the laboratory but have remained puzzled by a host of practical hurdles.

The major components of living cells are lipids, proteins, DNA, RNA, water and a few other macromolecules that consist primarily of elements like carbon, hydrogen, nitrogen, and oxygen acquired from the environment. In a living cell, non-living molecules spontaneously assemble in a complex fashion into structures that begin functioning like a living entity (Figure 1). However, the underlying technology of assembly of non-living matter is complex and in most cases beyond our present level of understanding.

 

HJ421_1Figure 1 | Designing a mammalian cell.

 

In this mini review, we aim to outline the basic steps to create artificial cells and present an account of important milestones achieved from recent experiments. We also point out new prospects on the horizon, as well as technical limitations at our present level of achievement. Finally, we emphasise that orchestrated research efforts are needed for designing and creating a living mammalian cell.

Points of discussion
The essential feature of a living cell is the formation of a localized, unique molecular assembly capable of undergoing division, replication and evolution. An intriguing question is how certain molecules assemble or associate to acquire the features of living entities. How are biomolecules, such as lipid-based membranes and segments of genetic material, synthesized and assembled in such a manner that the characteristics of life emerge? How might we achieve this process in the laboratory? The simplest strategy is to follow what is called the bottom-up approach. The essential steps are synthesizing basic building block molecules (lipids, DNA, RNA and most other proteins can all be synthesized artificially) and packaging these metabolic components inside a physical container of lipid vesicles/membranes. A designed entity with the minimum capability to function as a living cell requires meeting the criteria of regulation of chemical processes and regeneration.

A potential alternative method is the top-down approach, which aims to create a living cell from an existing real cell by progressive restructuring to simpler forms and reprogramming the genetic makeup of the experimental cell accordingly (2). The top-down method helps to understand the minimal essential molecular requisites for a living cellular assembly. It appears possible that the bottom-up and top-down approaches will meet in the middle to overcome the limitations and challenges faced in creating an artificial living cell.

In the past few decades, many attempts have been made by researchers all over the globe to biochemically synthesize basic components of living cells. A study by Luisi et al. of ETH, Zurich, Switzerland has shown artificial synthesis of self-replicating lipid vesicles and polymerization of amino acids into proteins on the vesicular surfaces (3). Another significant result showed thermodynamically controlled peptide binding polymerization reactions on the synthesized lipid vesicles (4). Furthermore, intracellular protein (ferritin) encapsulation inside lipid vesicles revealed the spontaneous formation of protein-rich vesicles (3), suggesting possible accumulation of solutes inside primitive cells. These results open new pathways to synthesise some of the essential cellular biomolecules necessary for creating a synthetic cell. Hanczyc et al. from Harvard University have demonstrated the formation of lipid vesicles that can be catalyzed by encapsulated clay particles with RNA adsorbed on their surfaces (5). This study demonstrated the biochemical control of the synthetic lipid vesicles. More notably, a model of a simple dividing artificial cell (protocell) having an integrated metabolic, genetic and container system was developed (6). These remarkable discoveries have stimulated research for creation of a complete functional biological entity in future.

Current status of progress
The endeavour of constructing a living cell has led to the emergence of a new discipline of synthetic biology. The aim is to create or modify living biological systems by drawing on natural biological principles and engineering techniques.

The task of designing an artificial cell can be simplified into three steps. The first step requires synthesis of basic biomolecules that are essential components of the cell. The next is to build synthetic genetic material to control the properties of regeneration, replication and evolution of the constructed cell. The final step consists of successful integration of synthesized genetic material, chemical reaction system and enclosing outer assembly into a functional entity. It is significant to note that scientists have already succeeded in achieving significant milestones in producing lipids using light energy and forming self-replicating RNA-containing lipid vesicles (4,5,7). It was also demonstrated that lipid vesicles presented specialized catalytic surfaces for polymerization of amino acids into proteins. More significantly, Hanczyc et al. showed that the formation of lipid vesicles was catalyzed by encapsulated clay particles with RNA adsorbed on their surfaces, suggesting catalysis of lipid vesicle formation and RNA polymerization by encapsulated clay (5). Moreover, a recent experimental demonstration of formation and spontaneous overcrowding of liposomes in situ provides the basis of the formation of cellular structure and evolution of metabolism in the scheme of origin of life on the Earth (8).

The availability of powerful computers, information technology and the tools of bioinformatics have improved our understanding and helped to digitize genomic information. Using digitization of genomic information, Gibson et al. made a landmark progress in creating a synthetic functional genome in a living cell. The authors reported designing and synthesizing a 1.08 Mbp Mycoplasma mycoides genome (named JCVI-syn1.0) from the computerized genome sequence information which was successfully transplanted into a recipient cell (M. capricolum) to create a new M. mycoides cell controlled by the artificial chromosome (9). It was a remarkable achievement, leading cell biotechnologists one step further towards the target of decoding the secret of life and creating a synthetic organism. Here, the synthesized genetic material from outside the cell was incorporated into a living cellular assembly. However, the greater objective that remains to be achieved is the successful integration of synthetic genetic material and synthetic cellular entities capable of automonous self-replication and regeneration.

One obvious limitation of current progress is the availability of external genetic material and a means to appropriately pack it inside an artificial bacterial cell. It is hoped that modern scientific tools using chemistry and thermodynamics will aid in providing the stabilized energy required to assemble these biochemical entities during the integration to a living cell. Furthermore, the use of supercomputers has enabled digitization and successive transplantation of very complex genome sequences with specific information. Already, powerful computational methods and simulation experiments have demonstrated the spontaneous formation and reproduction of cell-like structures (10). In addition, nanobiotechnology has enabled us to develop nanosize tools and devices that offer enormous new prospects for designing nanomachines plantable inside a basic artificial living cell to assist the construction of a pseudo-mammalian cell in the near future.

Summary and future prospects
Extensive research has enabled us to overcome some key challenges in designing a synthetic living cell. The noteworthy discovery that synthesized genetic material combined with bacteria-derived materials can produce a self-replicating cell with regeneration capacity and control over cellular mechanisms has stimulated further research in synthetic biology. However, the successful integration of synthesized cellular entities and genetic material is yet to be achieved. The combined use of tools of chemistry, nano-bioscience, and increasingly rapid computational processing has enormous potential to unravel the secret of transition between nonliving and living. Significantly, new knowledge of induced pluripotent stem (iPS) cells—specifically, demonstration of a normal adult skin cell being induced to become a pluripotent cell (11)—brings to light the potential to design a pluripotent synthetic living mammalian cell – a truly remarkable achievement in science with huge potential as a beneficial tool for medicine. An iPS cell can be transformed into a variety of phenotypes of cells found in the body which can be persuaded to produce or utilize critical external biomolecules. So in the near future, laboratory designed functional mammalian cells will hopefully open many new vistas into molecular engineering by making nanomachines for mimicking normal living cells, understanding the refined steps of evolution of living organisms and possibly curing illnesses by designing the desired cells ready for replacement of defective/malfunctioning cells.H

Acknowledgements
Mr. Prabodha Kumar Meher and Ms. Prerna Sharma were recipients of Senior Research Fellowships from the Board of Research in Nuclear Sciences (BRNS), Department of Atomic Energy, Govt. of India, though this work was an outcome of the encouragement for free thinking on basic questions of life by the mentor (KPM) beyond the scope of the doctoral research. Nevertheless, authors acknowledge the base for thinking provided by the initial BRNS support for their research careers.

Authors declare no conflicts of interest.

About the authors
Mr. Prabodha Kumar Meher and Ms. Prerna Sharma are Senior Research Fellows of the Board of Research in Nuclear Sciences (BRNS), Department of Atomic Energy, Govt. of India, funded Project and Doctoral research scholars at Nehru Gram Bharati University, Allahabad. Prof. Kaushala Prasad Mishra is a Senior Scientist and Ex-Head of Radiation Biology & Health Science Division, Bhabha Atomic Research Center (BARC), Mumbai. He is Vice-President of the Asian Association of Radiation Research and Founder President of the Society of Radiation Research, India.

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