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Suppose you want to convert the skin cell into a pluripotent stem cell. I know there are a few genes that were identified for this purpose (a recent Nobel Prize). But apart from finding such genes, it also seems to involve maintaining the right environment, such that some critical receptor doesn't get triggered and alter the entire expression. It also seem to involve ensuring gene expression stability after the change has occurred. Could someone add to this? What are the broad paradigm ideas in stem cell engineering?
The roles of the reprogramming factors Oct4, Sox2 and Klf4 in resetting the somatic cell epigenome during induced pluripotent stem cell generation
Current reprogramming technology, pioneered by Takahashi and Yamanaka 1, was built on several seminal advances in the field of developmental biology. First, nuclear transfer experiments demonstrated that a somatic cell nucleus could be epigenetically reset to an early developmental state . Second, cell culture conditions were developed that allowed for the isolation and culture of pluripotent cells, termed embryonic stem (ES) cells, from the inner cell mass of the human and mouse blastocyst [3,4]. Finally, study of these cells and of early embryonic development led to the identification of factors that were ultimately able to reprogram mouse embryonic fibroblasts (MEFs) to the iPS cell state when ectopically expressed, albeit at low frequency.
Reprogramming of somatic cells is a multistep process that culminates in the expression of pluripotency genes such as Nanog. Although morphological changes occur at early and intermediate stages of reprogramming, pluripotency gene expression is only induced during the late stage and indicates faithful reprogramming.
The core reprogramming cocktail, consisting of the transcription factors Oct4, Sox2 and Klf4 can be augmented by the addition of factors that enhance the efficiency of iPS cell generation
Its a multistep process in which much research is still needed,
The frequency with which somatic cells convert to iPS cells is typically below 1%. Therefore, much effort has gone into improving reprogramming.
specially to increase efficiency of iPS cell generation
The ability of cells to pass through the cell cycle has also been shown to be an important determinant of reprogramming efficiency. Knockdown or gene deletion of p53, p21 or proteins expressed from the Ink4/Arf locus allows cells undergoing reprogramming to avoid the activation of cell cycle checkpoints and cellular senescence, leading to greater iPS cell formation ,24-27].
Gene expression changes during reprogramming
First and foremost, stem cell therapies are cell therapies that, in the US, are regulated by such agencies as FACT (foundation for the accreditation cell therapies), CAP (college of American pathologists) and the FDA (food & drug administration). Manufacturing must be compliant under GTP (good tissue practice) and GMP (good manufacturing practice) standards. Even the labeling of products is moving to the harmonized system ISBT128, managed by ICCBBA (International Council for Commonality in Blood Banking Automation). The take-home point here is that everything is tightly regulated, and produced under rigorous standards of safety, conformity and documentation. Not everyone is FACT accredited, but the benefits are becoming of increasing importance, especially to insurance providers (ref). The therapies are costly to manufacture, to be certain. The two FDA-approved CAR-T immunotherapies YESCARTA and Kymriah run around $500,000.
So you have a number of types of stem cell therapy. Bone marrow transplantation is considered a stem cell therapy, and then you have therapies that involve human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC).
In terms of hiPSC, groups have been successful both in culturing with transcription factors such as Oct4, Sox2, Klf2 and so forth, and in using sendai virus to deliver these transcription factors directly within the cell (thermofisher's Cytotune®-iPS Sendai Reprogramming Kit, youtube video).
We can also see, however, that stem cells can be obtained elsewhere and don't necessarily need to be induced. You can source hESC from embryos and cord blood, and hematopoietic stem cells (HSC) can be mobilized from the marrow with a drug cocktail (like plerixafor and GM-CSF) and harvested with apheresis.
So just for a real-world example of what you're thinking: Papers on the production of glucose sensitive insulin-secreting ß cells from hESC using carefully controlled culture conditions have been published (ref), and companies have taken advantage of this technology. Subdural implants using this system are in clinical trials right now. They produce the ß cells from hESC by controlling the cultures:
The transcription factors along the bottom of the figure, which I've pasted together from two sources, shows what transcription factors should be active at that stage. The identity of the final product can be surmised from a combination of what transcription factors should and should not be active. It's also important to realize that there will be off-target cells, but that the process should be validated to ensure on-target cells stay within an acceptable range as defined during pre-clinical development.
Manufacturers would also need to test for the purity, sterility, endotoxin and potency of their cells. Potency assays have become something of a prerogative for the FDA, since anyone can produce a cell, but how can they say it's effective or does what it's marketed to do? An example potency assay might be two cultures, one with glucose and one without, does one secrete more insulin than the other? You'd pick this assay because the proposed mechanism of action for ß cells is they respond to glucose, and secrete insulin.
For sterility, they sell some good kits like BacT alert aerobic and anaerobic test bottles, mycoplasma testing kits, you can also do culture plates and gram stain. Products that don't exhibit 100% sterility can't be infused, and so it generally follows that exceptional releases can be made for many other issues by a physician, but contamination will stop a cell therapy product dead in its tracks.
You're also right in that cell therapies which involve a gene modification step need to look at such things as transduction efficiency, live virus titer, expression of molecules related to the transduction and so forth. The sendovirus kit referenced above is a fair pick because as they demonstrate, after several passages the non-integrating virus titer is negligible. For human administration, in the CAR-T space, the FDA has required that studies where CAR-T have been infused follow patients for 15 years after treatment to study the effect of infusing virally-transduced vectors. I don't think that there's a clear answer as to whether the virus is an issue. You're also right, however, that it's necessary to test the stability of the transduced gene(s) , especially through passaging, holding and cryopreservation. All of this is nicely outlined here.
What Is Biomedical Engineering?
Biomedical engineering is the application of the principles and problem-solving techniques of engineering to biology and medicine. This is evident throughout healthcare, from diagnosis and analysis to treatment and recovery, and has entered the public conscience though the proliferation of implantable medical devices, such as pacemakers and artificial hips, to more futuristic technologies such as stem cell engineering and the 3-D printing of biological organs.
Engineering itself is an innovative field, the origin of ideas leading to everything from automobiles to aerospace, skyscrapers to sonar. Biomedical engineering focuses on the advances that improve human health and health care at all levels.
Biomedical engineers differ from other engineering disciplines that have an influence on human health in that biomedical engineers use and apply an intimate knowledge of modern biological principles in their engineering design process. Aspects of mechanical engineering, electrical engineering, chemical engineering, materials science, chemistry, mathematics, and computer science and engineering are all integrated with human biology in biomedical engineering to improve human health, whether it be an advanced prosthetic limb or a breakthrough in identifying proteins within cells.
There are many subdisciplines within biomedical engineering, including the design and development of active and passive medical devices, orthopedic implants, medical imaging, biomedical signal processing, tissue and stem cell engineering, and clinical engineering, just to name a few.
Cell & Tissue Engineering
Cell and tissue engineering centers on the application of physical and engineering principles to understand and control cell and tissue behavior. Cellular engineering focuses on cell-level phenomena, while tissue engineering and regenerative medicine seek to generate or stimulate new tissue for disease treatment.
Two areas in which the department has established special leadership are cellular mechanobiology, which focuses on understanding the interaction and conversion between force-based and biochemical information in living systems, and stem cell engineering, which includes platforms to expand, implant, and mobilize stem cells for tissue repair and replacement.
Faculty working in cell & tissue engineering:
Dean A. Richard Newton Memorial Professor, Bioengineering
Senior Faculty Scientist, Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory
Director, Berkeley Synthetic Biology Institute
CEO/CSO, DOE Systems Biology Knowledgebase
PI and Co-Director, ENIGMA SFA
The Arkin Lab’s research focuses on the systems and synthetic biology of microorganisms. They are experts in theory, computation and experiments surrounding the modeling of biological systems at the molecular and population level and have developed a number of genome scale technologies with which we can rapidly assess the genomic function of uncharacterized microorganisms. The lab’s models span the deterministic and stochastic analysis of both homogeneous and spatially distributed systems.
Professor, Mechanical Engineering
Theory and applications of solid mechanics to traditional materials and biomaterials.
Our work has been focused on establishing new paradigms in multi-tissue stem cell aging, rejuvenation and regulation by conserved morphogenic signaling pathways. One of our goals is to define pharmacology for enhancing maintenance and repair of adult tissues in vivo. The spearheaded by us heterochronic parabiosis and blood apheresis studies have established that the process of aging is reversible through modulation of circulatory milieu. Our synthetic biology method of choice focuses on bio-orthogonal non-canonical amino acid tagging (BONCAT) and subsequent identification of age-imposed and disease-causal changes in mammalian proteomes in vivo. Our drug delivery reg medicine projects focus on CRISPR/Cas9 based therapeutics for more effective and safer gene editing.
Professor in Residence, Department of Bioengineering
Professor and Chair, Bioengineering and Therapeutic Sciences, UCSF
Dr. Desai’s lab focuses in the area of biomedical micro and nanotechnology for therapeutic delivery. Professor Desai’s research spans multiple disciplines including materials engineering, cell biology, tissue engineering, and drug delivery.
Purnendu Chatterjee Chair in Engineering Biological Systems, Bioengineering
Faculty Scientist, Lawrence Berkeley National Laboratory
The Fletcher Lab develops diagnostic technologies and studies mechanical regulation of membrane and cytoskeleton organization in the context of cell motility, signaling, and host-pathogen interactions. We specialize in development of optical microscopy, force microscopy, and microfluidic technologies to understand fundamental organizational principles through both in vitro reconstitution and live cell experiments. Recent work includes investigating the mechano-biochemistry of branched actin network assembly with force microscopy, studying membrane deformation by protein crowding and oligomerization with model membranes, and reconstituting spindle scaling in encapsulated cytoplasmic extracts. The long-term goal of our work is to understand and harness spatial organization for therapeutic applications in cancer and infectious diseases.
Jan Fandrianto Professor, Bioengineering
Professor, Materials Science & Engineering
Research in the Healy Lab emphasizes the relationship between materials and the cells or tissues they contact. The research program focuses on the design and synthesis of bioinspired materials that actively direct the fate of mammalian cells, and facilitate regeneration of damaged tissues and organs. Major discoveries from his laboratory have centered on the control of cell fate and tissue formation in contract with materials that are tunable in both their biological content and mechanical properties. Professor Healy also has extensive experience with human stem cell technologies, microphysiological systems, drug delivery systems, and novel bioconjugate therapeutics.
Adjunct Professor, Bioengineering
Assistant Professor, Bioengineering
The Hsu Lab aims to understand and manipulate the genetic circuits that control brain and immune cell function to improve human health. We explore the rich biological diversity of nature to create new molecular technologies, perturb complex cellular processes at scale, and develop next-generation gene and cell therapies. To do this, our group draws from a palette of experimental and computational techniques including CRISPR-Cas systems, single cell genomics, engineered viruses, brain organoids, and pooled genetic screens.
Current interests include 1) inventing novel approaches for editing the postmitotic genome, 2) developing engineered vehicles for therapeutic macromolecule delivery, and 3) leveraging library screens and brain organoids to interrogate human neuroscience at scale.
Chancellors Professor, Bioengineering
Chancellors Professor, Mechanical Engineering
Biomechanics of cortical and trabecular bone design of spine prostheses bone fracture and osteoporosis tissue engineering of bone.
Chancellor’s Professor and Chair, Bioengineering
Professor of Chemical and Biomolecular Engineering
Professor in Residence, Bioengineering and Therapeutic Sciences, UCSF
Faculty Scientist, Biological Systems and Engineering, LBNL
Our lab seeks to understand and engineer mechanical and other biophysical communication between cells and materials. In addition to investigating fundamental aspects of this problem with a variety of micro/nanoscale technologies, we are especially interested in discovering how this signaling regulates tumor and stem cell biology in the central nervous system. Recent directions have included: (1) Engineering new tissue-mimetic culture platforms for biophysical studies, molecular analysis, and screening (2) Exploring mechanobiological signaling systems as targets for limiting the invasion of brain tumors and enhancing stem cell neurogenesis and (3) Creating new biomaterials inspired by cellular structural networks.
Class of 1941 Endowed Professor of Bioengineering and Materials Science and Engineering
My laboratory is interested in understanding structure-property relationships in biological materials and in using this information to design biologically inspired materials for use in healthcare. Fundamental studies include single molecule and bulk biophysical studies of biointerfacial and bulk mechanochemical phenomena in biological materials, whereas our applied studies the design and synthesis of novel biomaterials for tissue repair and regeneration.
Professor, Mechanical Engineering
Faculty Scientist, Lawrence Berkeley National Lab
Our research program is focused on understanding cell mechanobiology and molecular mechanisms involved in human disease, in particular cardiovascular dysfunctions, brain and neurological disorders, and cancer.
Our laboratory is focused on developing new materials for drug delivery and molecular imaging.
Professor, Mechanical Engineering
Lawrence Talbot Professor, Mechanical Engineering
Characterization of structural evolution in medical grade ultra high molecular weight poliethylene due to sterilization: the implications for total joint replacements.
Professor Emeritus, Bioengineering
Professor Emeritus, Medicine, UCSF
The research focus is on hand and arm biomechanics and the design of workplace tools and tasks in order to improve productivity and the quality of work while preventing upper extremity fatigue and injury. The lab has studied designs of tablets, gesture interfaces, keyboards, mice, pipettors, touch screens, dental tools, construction drills, chairs, and agricultural tools. Funding is primarily from NIH and CDC but also from Hewlett-Packard, Microsoft, BART, Logitech, and Herman-Miller.
Professor Emeritus, Bioengineering
Professor of the Graduate School, Mechanical Engineering
Bioelectronic devices, biotransport, medical imaging, electrical impedance tomography.
Professor, Chemical & Biomolecular Engineering, Bioengineering, and Molecular & Cell Biology
Director, Berkeley Stem Cell Center
Regenerative Biology & Tissue Engineering
Organ malformation, damage and failure are the most common causes of human morbidity and mortality. It is estimated yearly that more than 35,000 infants will be born with organ malformations, 100,000 people will have their limbs amputated, 5,000 people waiting for organs will die without receiving a transplant, and 1 million people will die from organ disease. The goal of the Regenerative Biology and Tissue Engineering Theme (RBTE) is to develop the knowledge base and technologies that are needed to replace or regenerate human tissues and organs and thereby solve these severe health issues and contribute to improving life quality and welfare.
To achieve this goal, RBTE scientists are pursuing three related avenues of research. First, RBTE scientists are using stem cell and developmental approaches to investigate how organs form and regenerate. Second, RBTE scientists are using the knowledge gained from stem cell and developmental studies to regenerate tissues and organs in a series of model and non-model organisms. Third, RBTE scientists are translating their research from model and non-model organisms into humans. These three research avenues are discussed in more detail below. With this multi-faceted approach, RBTE scientists are making major strides toward developing regenerative technologies that will positively impact the lives of the many people suffering from organ malformation, damage and failure.
Stem Cell and Developmental Biology: An understanding of the cellular and developmental processes underlying organ and tissue growth provides a critical and necessary foundation for organ and tissue regeneration. Organ regeneration is a complex process that requires the simultaneous replacement of bones, skeletal muscles and tendons, and the re-growth of nerves, blood vessels and skin. Therefore, before organs can be regenerated, the developmental basis of all of these events must be understood. To pursue this goal, RBTE scientists are utilizing a diversity of research approaches that fall into two broad categories: stem cell research and developmental biology. In regard to stem cell research, RBTE scientists are studying cell fate determination. In this research, RBTE scientists are investigating the signals that transform an undifferentiated cell into one that is tissue or organ specific. In regard to developmental biology, RBTE scientists are investigating the cellular and molecular processes that underpin normal organ and tissue development in model and non-model organisms.
Regeneration in Animal Models: RBTE scientists are applying the knowledge gained from research in stem cell and developmental biology to regenerate tissues and organs in many model and non-model animals. Animals being studied include frogs, mice, rats, rabbits, pigs, and opossums. This research forms an essential foundation for organ and tissue regeneration in humans. Specifically, research in animal models can provide significant insights into the cellular and molecular processes that underpin organ and tissue regeneration. RBTE scientists are taking two major approaches to regenerate tissues in animal models. In the first, RBTE scientists are comparing the cellular and molecular processes operating in tissues and organs that are capable of regeneration with the processes operating in tissues and organs that are not. In this way, RBTE scientists are able to identify specific processes that underpin organ and tissue regeneration. In the second approach, RBTE scientists are applying the principles of bioengineering and materials science to tissue and organ regeneration. In this innovative approach, RBTE scientists are using engineered scaffolds, tissues, bioactive molecules, and cells to enhance organ and tissue regeneration with great success.
Translational Research on Human Regeneration: The ultimate goal of regenerative biology is to successfully regenerate tissues and organs in humans. To achieve this goal, RBTE scientists have established active research collaborations with medical doctors at several institutions, including the Mayo Clinic, University of Illinois at Chicago, Northwestern University, and Carle Hospital. Through these collaborations, RBTE scientists are translating the knowledge gained from stem cell, developmental, and regenerative research in model and non-model animals to organ and tissue regeneration in humans. These translational efforts involve the use of biodegradable scaffolds in combination with growth factors and adult stem cells as treatments for large bone and/or cartilage trauma or disease. Areas of focus include the regeneration of bone in the skull, spine, long bones and digits, and of cartilage in the face, trachea, and joints. The methodology employed could reduce or even eliminate the need for bone harvest and grafting procedures, decreasing the number of patient surgeries.
How to Engineer Biology
Because biology is the result of evolution and not human development, bringing engineering principles to it is guaranteed to fail. Or so goes the argument behind the &ldquoGrove fallacy,&rdquo first invoked by drug industry observer Derek Lowe in a critique of Intel CEO Andy Grove in 2007. After being diagnosed with prostate cancer, Grove found himself frustrated by what he described as the &ldquolack of real output&rdquo in pharma especially as compared to the drive of Moore&rsquos Law in his own industry.
This was a naive and invalid criticism from Silicon Valley outsiders, Lowe argued, because &ldquomedical research is different [and harder] than semiconductor research&rdquo&mdashand &ldquothat&rsquos partly because we didn&rsquot build them. Making the things [like semiconductors] from the ground up is a real advantage when it comes to understanding them, but we started studying life after it had a few billion years head start.&rdquo So the very idea of engineering biology by nature is doomed to fail, he further wrote, given that &ldquobillions of years of evolutionary tinkering have led to something so complex and so strange that it can make the highest human-designed technology look like something built with sticks.&rdquo
But we&rsquove seen incredible advances in the world of biology and tech the last few years, from AI diagnosing cancer more accurately than humans do, to editing genes with CRISPR. So is it still true that the idea of bringing an engineering mindset to bio is another case of starry-eyed technologist solutionism?
It is absolutely true that we&rsquore very much in the process of discovering biology, still untangling the &ldquotechnical debt&rdquo of evolution. Just when one thinks one understands the biology, another layer of the onion appears. It&rsquos also dangerously easy to break biology, with far greater consequences than broken code&mdasheven single-point mutations can lead to disease, and extremely small quantities of certain chemicals can have disastrous side effects. Many of the failures of medicine and especially drug design stem from the complexity and unpredictability of biology.
But the fact that we are still discovering biology doesn&rsquot mean that we can&rsquot design. We can engineer the tools we use to manage biology.
In fact, we have been building and designing tools to control, augment, replace or enhance biology as long as humanity itself has existed&mdashwhether it&rsquos taming the jungle to build habitable villages halting and containing infection making advanced prosthetics for people who lost their limbs making synthetic drugs to replace defective parts or now, even creating functionality that nature never had. We can do this because we empirically learned the properties of those materials, then iterated, designed, and built new structures with them. There is no reason why we should not continue be able to do so for our medicines and bodies.
The only question is how we get there. If discovery is the systematic exploration of ideas and concepts with the goal of understanding the world around us, then design is the mainstay of engineering&mdashwhere concepts learned in the scientific arena are employed to build everything around us in a repeatable, less time-consuming and more predictable manner.
The way things are right now, we design bridges, but we discover drugs. This is not without cost: Billion-dollar bridges, which we have learned how to design through trial and error, practice and well-tried engineering principles, rarely fail&mdashwhereas billion-dollar drug failures are routine, not to mention costly. With design, however, we can plan and progress very systematically along a roadmap and make incremental innovations along the way. Borrowing from engineering, here are principles that allow us to overcome the so-called Grove fallacy and harness biology.
PRINCIPLE 1: LEGO-LIKE BUILDING BLOCKS
Biology has a hierarchical nature: amino acids are made from atoms proteins from amino acids and so on&mdashall coming together to make cells, which make tissue, which leads to organs, which leads to organisms, and then niches, then complete ecosystems. Evolution is the ultimate algorithm. And the ability to facilitate evolution more rapidly to respond better to selective pressure (meta-evolution), has led to mechanisms that reinforce this modularity. A great deal of the hierarchy is known, so if you want to engineer cellular machinery, the parts are proteins if you want to engineer tissue, the parts are cells and onward at higher scales.
This isn&rsquot just hypothetical. There are numerous successful examples of this already, from the engineering of myosin (proteins that walk along cell microtubule highways for transport) to CAR-T therapies, where by identifying two key protein modules (the &ldquoLegos&rdquo in this case) and bringing them together we can engineer patients&rsquo immune cells and thus treat their cancers. Researchers and entrepreneurs used this fundamental aspect of biology to program cells, basically curating from all proteins in the cell a limited set that &ldquoplays well with each other&rdquo and the rest of the cell to use as a set of Legos for building genetic circuits.
Once we identify the Legos in biology and their properties, we can engineer them and even mix and match them to design novel functionality.
PRINCIPLE 2: REPEATABILITY AND REPRODUCIBILITY
Irreproducibility is a major crisis in modern biology, especially when it comes to publishing papers and being unable to replicate results. But reproducibility is a critical hallmark of an engineering-based approach to biology it is, by definition, impossible to engineer a process without reproducibility.
One of the principle causes of irreproducibility in biology is the pre&ndashIndustrial Revolution, bespoke (literally, hand-crafted) nature of biological experiments even today. This makes most experiments more art than science. But modern technology makes the process of doing biology much more reproducible, from problems of consistency in reagents to re-running and debugging issues. Robotics is one of the most obvious ways, with exact motions now done with the precision of a machine and directed by software.
Machine learning plays a huge part too. The identification of biomarkers (chemical substances we can measure and then target) for disease is currently driven by discovery via a bespoke, one-off process&mdashso the discovery of PSA for prostate cancer, for instance, does not suggest a biomarker for ovarian cancer. Introducing machine learning into the process, however, can turn this handcrafting into assembly-line production. Furthermore, we&rsquore teaching the machine how to fish, allowing not just for reproducibility but for the improvement of accuracy over time, thanks to inputs from additional raw data and identification of complex patterns that humans are incapable of seeing.
A number of companies are already doing this. Apple&rsquos latest watch has been heralded as a the &ldquoiPhone moment&rdquo where consumer wearables might &ldquomorph into medical grade devices.&rdquo Entrepreneurs applying deep learning to medicine can use AI/ML and labeled data from generic Apple watch pulse data streams to accurately and precisely identify atrial fibrillation. But more notably&mdashwhere biology becomes engineering&mdashis the ability to take the very same process they use to detect heart disease and then predict patient illness in many other areas as well (hypertension, sleep apnea and type 2 diabetes).
Such machine-learning driven companies&mdashwhich can also detect cancer early or identify biomarkers linked to longevity&mdashhave all engineered a kind of &ldquofactory line.&rdquo Given the right input ingredients, they can now mass-produce many tests in a predictable, precise and repeatable manner. It&rsquos yet another way the Grove fallacy is false. The massive advances in computer chips (Moore&rsquos Law), storage (&ldquoKryder's law&rdquo), and genomics&mdashall exponential decreases in cost, 1,000 times over a decade&mdashcome merely from 30% improvement year over year. In biology, better reproducibility plus gradual improvement over time plus greater accuracy all add up to even more massive advancements, because even a little goes a very long way.
PRINCIPLE 3: TESTING AND PROCESS ENGINEERING
Testing is the ability to understand exactly where a given product/diagnostic/drug stands, and while the need for testing is obvious, how to test and what metrics to measure success are not. So, the choice and engineering of key performance indicators (KPIs) is critically important here without this guiding compass, a project could go in the wrong direction.
KPIs are used all the time in engineering, and in all businesses, as a way to define and measure success (or at the very least, progress). But &ldquotraditional&rdquo biology experiments and drug development haven&rsquot used the concept, since biology was conceptually driven by discovery: how can you assign a KPI when you don&rsquot know what you&rsquoll discover? Now, a new wave of bio startups&mdashdrawing on engineering and computer science&mdashare identifying KPIs for measuring molecules synthesized to protein expression, numbers of cells screened, and much more.
The critical part is determining what the right KPIs are, and in engineering biology, there are few precedents, so this can be challenging. But much like in medicine more broadly, the basic principle is: what can be measured can be improved, and those improvements can have huge payoffs. In fact, the evolution from subjective intuition to objective measure is itself another indicator of moving from discovery (biology) to design (engineering), and fits more into Grove&rsquos worldview.
PRINCIPLE 4: BORROWING FROM OTHER DISCIPLINES
An obvious approach to bring engineering into biology is to apply existing engineering disciplines&mdashmaterials, chemical, electrical, mechanical, and so on&mdashin the biological realm. Until recently, the ability to quantitatively test and iterate biology was greatly limited. But the rise of numerous, novel quantitative measurements of biology&mdashi.e., big data sets in biology&mdashhas opened the door to incorporating other engineering approaches.
For example, there are also companies out there using mechanical engineering principles to bring simulations to predicting the outcomes of surgeries, so that treatments can be engineered, instead of discovering through trial-and-error on patients. By applying the materials-science based engineering technology he learned in solar cell materials design to food, James Rogers used techniques from nanoscience to create nanoscopic barriers that protect fruits and vegetables from spoilage. This isn&rsquot biomimicry, but a way of borrowing the fundamentals of engineering from other disciplines to harness biology.
PRINCIPLE 5: REINVENTING THE PROCESS ITSELF
Engineers at NASA in 1962 could perhaps imagine going to the moon, but how would they even start? The short answer: By breaking the problem down into parts, and then breaking the process down into steps. And then by having versions (to borrow from the analogy of software). It wasn&rsquot Apollo 1 that went to the moon, but Apollo 11.
Such &ldquobig hairy audacious goals&rdquo (aka BHAGs) are daunting, seemingly impossible aspirations. The key here is the ability to think long-term, to project and to plan forward&mdashmuch as one does when designing any other engineering-based roadmap. So, the Apollo team, like most engineers, broke things down into more doable steps of engineering put together, those steps created the stuff of dreams.
The challenge in biology lies in breaking down the problem into steps and often reinventing the process itself. But once the desire to consistently improve performance (what Grove was suggesting in the first place) moves biology from bespoke, artisanal approaches to designed, scalable processes, even seemingly modest performance increases can make a difference. A 1 percent increase performed weekly, for instance, would lead to almost doubling in a year and a tripling in two years, and in biology, such improvements could have huge impact.
Andy Grove did not found Intel, but he was there early and was deeply influenced not just by manufacturing methods for management but by &ldquothe law&rdquo (that the number of transistors in a circuit doubles every couple years) proposed by Intel co-founder Gordon Moore. However, Moore&rsquos isn&rsquot a law of physics but of economics and arose from an engineering push that continued across different technologies, different teams, different decades. It&rsquos a law of will, imposed by man, not nature.
In biology, we&rsquove already surpassed Moore&rsquos Law the cost of genomics has come down over a million-fold in two decades. Why can&rsquot we carry this process to other areas in bio as well? The question now isn&rsquot whether this is possible in biology or not, as the Grove fallacy argued, but how to do it, given where we are in engineering biology today.
The views expressed are those of the author(s) and are not necessarily those of Scientific American.
ABOUT THE AUTHOR(S)
Vijay Pande, PhD, is a general partner at Andreessen Horowitz where he leads the firm's investments in companies at the cross section of biology and computer science including areas such as the application of computation, Machine Learning, and Artificial Intelligence broadly into Biology and Healthcare as well as the application of novel transformative scientific advances. Pande is also an Adjunct Professor of Bioengineering at Stanford University previously, he was the Henry Dreyfus Professor of Chemistry and Professor of Structural Biology and of Computer Science there.
Scaffold design and manufacture
As the field of tissue engineering progresses, the need for novel scaffold structures and reproducible fabrication techniques has become of paramount importance. The use of biodegradable polymers, such as poly lactic acid (PLA), has become widespread, but the manner in which these polymers are processed and the additives used at the time of manufacture allows the final properties of the scaffold to be tailored.
Some of the scaffold types discussed include: high-pressure CO2 foamed scaffolds, injectable scaffolds, novel custom scaffolds and how these can be further modified using growth factors, zonation of materials and plasma polymerization deposition.
Poly-hydroxyl acids such as PLA and poly lactic-co-glycolic acid (PLGA) have been extensively used for tissue engineering procedures, as these materials bulk-degrade by hydrolysis, providing a controllable drug release and degradation profile to match tissue in-growth. With careful use of molecular weights, cross links and side chains, materials can be produced with tailor-made properties making them ideal for use in tissue engineering matrices. Furthermore, poly-hydroxyl acid materials also have a long history of in vivo usage as degradable sutures, drug delivery devices and biodegradable surgical components.
Injectable materials for tissue engineering/regenerative medicine
A scaffold developed for orthopaedic use is ‘Injectabone’, a novel biodegradable, particulate, scaffold system which can be injected into a site of bone trauma (Hamilton et al. 2006). The scaffold forms via the use of two types of PLGA microparticles. Type 1 is a temperature-sensitive PLGA/polyethylene glycol (PEG) composite that acts as an adhesive for the type II PLGA particles. The dynamics of this scaffold type allows injection at room temperature and solidification at body temperature allowing for a non-invasive delivery system for treatment of non-union bone defects.
Microparticles are small enough to be delivered by syringe and can be used as an injectable scaffold by incorporating temperature and mositure sensitive or adherent systems. These versatile subunits can be produced using droplet formation of solvents (Suciati et al. 2006) or by spraying (Hao et al. 2004 Whitaker et al. 2005). Setting of a microparticle slurry was initially performed using the attraction between biotin in one set of beads and avidin in another. Furthermore, live cells could be incorporated into this system such that scaffolds could be injected containing evenly distributed cells. The range of applications can be increased with the incorporation of various drugs and surface modifications.
Growth factor incorporation into scaffolds
In addition to scaffolds being used as a support for cell growth they can simultaneously be used as a vehicle for drug delivery. In theory, the scaffolds can be used to deliver growth factors/drugs to the sites of repair, thus expediting the recovery process. Owing to the kinetics and complexity of biological growth factor release, the process has required extensive investigation. One of the major issues is maintaining the conformation and function of proteins during the process of scaffold manufacture. However, once this issue has been solved many more complications lie ahead, including the control of growth factor release to match the kinetics of physiological processes, as well as the independent release of many factors at different stages.
Recently, vascular endothelial growth factor (VEGF), a peptide growth factor, has been incorporated into PLA scaffolds to provide a controlled release of angiogenic signals from a scaffold (Kanczler et al. 2007). Release of bioactive VEGF was confirmed using the in vitro human umbilical vascular endothelial cell (HUVEC) assay and in vivo chick allantoic membrane (CAM) angiogenesis assay. It was demonstrated that the VEGF retained its angiogenic properties and encouraged vascularization of the PLA scaffold.
Growth factors can also be attached to the surface of scaffolds following manufacture through the use of functional groups to chemically attach the proteins and/or drugs. Chen et al. (2006) used this method to attach basic firoblastic growth factors (bFGF) to the surface of alginate beads via an –NH functional group. This scaffold provided a microenvironment permissive for the growth and differentiation of human neuronal stem cells prior to their use in tissue engineering procedures.
The function of growth factor incorporation can be further enhanced by zoning, offering an interesting way of controlling tissue integration and development, which potentially allows the regionalized release of proteins to act on specific cell populations or initiate physiological processes, i.e. angiogenesis, at particular sites throughout scaffolds. This system has been demonstrated by Suciati et al. 2006, in which PLA/PEG microparticles were loaded with proteins such as horseradish peroxidase, trypsin or BMP-2. These particles were then sintered to form distinct layers. These scaffolds could maintain release over a period of up to 30 days, with the BMP-2 loaded particles able to initiate zonal osteogenic differentiation of responsive C2C12 cells in vitro.
An alternative to growth factor incorporation is to integrate DNA plasmids encoding a gene and mammalian promoter into the polymer transfection with the DNA programmes the cells to produce their own growth factors. Once optimized, changing the inserted gene to alter the growth factor produced would allow a range of factors to be produced however, uptake rates and toxicity are still major issues to this promising technique (Heyde et al. 2007).
Supercritical carbon dioxide processing of polymers
Processing of polymers into reticulated tissue engineering scaffolds often requires organic solvents and a method to provide pores, such as inclusion of salt granules, which are later removed by leaching, or by addition of blowing or foaming agents. Organic solvents, used in scaffold fabrication, such as dichloromethane, also often interact with many sensitive structural motifs found in peptide drugs, and can leave toxic residues behind (the upper FDA limit for DCM residues is only 600 parts per million).
Supercritical CO2 forms a phase between liquid and gas ( Fig. 3 ) that is able to penetrate many polymers and plasticize them. Evaporation results in solidification of the polymer and can be controlled to fuse separate bubble nucleation points, providing a reticulated and interconnected scaffold with a high strength to weight ratio ( Fig. 4 ). Supercritical CO2 is also able to incorporate peptide drugs with minimal damage (Kanczler et al. 2007) if exposed briefly it is sufficiently inert to incorporate living cells by plasticizing a scaffold around cells (Ginty et al. 2006). The use of CO2 is not without limitations, as careful control of the supercritical foaming process is key to the correct formation of interconnected chamber structures and the use of this process requires quality control of the scaffolds produced. However, the structures produced are architecturally very strong and the ability easily to incorporate otherwise sensitive peptide drugs is a major advantage.
Clinical treatments that introduce living cellular material into a patient. They may engraft in the body, leading to long-term replacement of damaged or missing tissue, or stimulate endogenous repair and promote endogenous viability.
(ESC). A type of pluripotent stem cell, derived from the inner cell mass of the developing embryo, that is responsible for giving rise to all of the cells in the developing fetus but not the extra-embryonic tissues.
A minimal and miniaturized organ that is developed from a suspension of stem cells in vitro. These stem cells undergo division and self-organization to give rise to a 3D structure that mimics the anatomy of organs in the body. Thus, organoids can serve as models for understanding organ development and for modelling disease states.
A cell’s identity based on its expression of genetic, proteomic and epigenetic markers but also in terms of its functional abilities. Cell fate determines a cell’s self-renewal ability, proliferative ability, differentiation potential, survival and motility.
A form of cellular signalling in which secreted chemicals bind to receptors on the same cell. By contrast, juxtacrine and paracrine signalling induce responses in neighbouring cells, either through direct contact (juxtacrine) or secreted chemicals (paracrine).
(ECM). A collection of extracellular molecules, including proteins, proteoglycans and polysaccharides, that supports the growth of nearby cells by providing biomechanical and biochemical cues. It enables cell adhesion and cell–cell communication.
(GRNs). A set of genes and their direct and indirect regulatory interactions with one another. GRNs are akin to decision-making computational circuits that serve to process input signals and generate robust outputs in cell behaviour.
Interaction patterns that recur more frequently than in randomized networks — for example, negative autoregulation (or ‘autorepression’) and the feedforward loop.
The in vivo microenvironments in which stem cells reside that regulate their homeostasis and fate choices.
The process by which developing organisms acquire their structure and shape.
Probabilistic models that relate the dependencies of the expression of a set of genes on one another through a directed graph.
Models of gene regulatory networks that can predict gene expression outcomes given the initial state of genes in the network as well as the derivation of steady-state gene expression status.
Artificial neural networks
Networks composed of nodes, which can be genes, that process and transmit information. The output of each node is a nonlinear function of a sum of its regulatory inputs.
Ordinary differential equations
A mathematical framework capturing gene expression dynamics as a function of the presence of regulators and the rate of change of mRNA and/or protein concentration due to production and degradation.
The process of analysing a system to uncover underlying design rules to create representations of the system at higher levels of abstraction (inverse of forward engineering).
The iterative process by which a system is designed, prototyped, tested and further optimized from a model (the classical engineering design process).
Technology that enables transfer of miniature ‘islands’ of extracellular matrix proteins to enforce control of the shape and size of adherent cells either as single cells or cell colonies.
The characteristic of a cell that makes it a stem cell. That is, the ability to self-renew and differentiate to specify to different cell types.
Vessels in which biological species, such as stem cells and their progeny, are grown, maintained and manipulated in a controlled environment (pH, oxygen and media change) for cell manufacturing pipelines.
Utilization of printing techniques ranging from inkjet printers to 3D printers to combine cells, biomaterials, extracellular matrix, growth factors, etc. to fabricate complex tissue surrogates in vitro.
A process during embryogenesis in which cell fates are allocated or ‘patterned’ as a function of space and time.
Signalling molecules, typically soluble chemicals, for which the asymmetric distribution in a developing tissue gives rise to fate patterning and morphogenesis.
Stem Cells and Functional Tissue Laboratory
The Laboratory for Stem Cells and Functional Tissue Engineering, directed by Prof. Gordana Vunjak-Novakovic, is well-known for tissue engineering of functional human grafts using stem cells in conjunction with biomaterial scaffolds custom-designed to mimic the native tissue matrix and advanced bioreactors. The cells are employed as actual &ldquoarchitects&rdquo of the tissue, the scaffold serves as a template for tissue formation, and the bioreactor provides a controlled environment for functional tissue assembly. A &ldquobiomimetic&rdquo approach to tissue engineering is pursued, where the design of scaffolds and bioreactors are inspired by the native developmental milieu, in order to direct the cells to differentiate into the right phenotype and form the right tissues.
Biology-Inspired Engineering and Engineering-Inspired Biology
Biology has been an important inspiration for developments in all aspects (e.g. designs) of engineering (e.g., robots), i.e., biology-inspired engineering (BIE). Bio-inspired attachment systems, bio-inspired sensors, bio-inspired materials, etc. have enabled robots to produce robust and comparable behaviors .
Biology has been an important inspiration for developments in all aspects (e.g. designs) of engineering (e.g., robots), i.e., biology-inspired engineering (BIE). Bio-inspired attachment systems, bio-inspired sensors, bio-inspired materials, etc. have enabled robots to produce robust and comparable behaviors to their biological counterparts. Owing to the biologically comparable behaviors, interdisciplinary biologists tend to flip the approach, i.e., engineering-inspired biology (EIB) whereby engineering systems and principles are utilized to initiate and test new hypotheses in biological research. For example, robots have been used as tools to investigate and test animal functions. However, the mutual inspirations and enhancements between biology and engineering remain an open question in many types of research.
This Research topic welcomes articles from the fields of bionic science and engineering including, but not limited to, the following topics: Bio-inspired robotics, bio-inspired sensors and actuators, bio-inspired energy storage, bio-inspired flexible electronics, bio-inspired solar cells, biomimetic engineering, embodied artificial intelligence and neurorobotics, functional morphology, musculoskeletal systems, biomechanics, biological interface and functionalization, biomimetic materials, biomimetic structures and mechanics, biomimetic surfaces, biomimetic designs and methodologies, bionic fluid, biological systems, and brain-inspired computing and neuromorphic systems.
Keywords: Bio-inspired flexible electronics, Bio-inspired robotics, Biomimetic engineering, Bionic fluid, Musculoskeletal systems
Important Note: All contributions to this Research Topic must be within the scope of the section and journal to which they are submitted, as defined in their mission statements. Frontiers reserves the right to guide an out-of-scope manuscript to a more suitable section or journal at any stage of peer review.
Most of the efforts of biological research, both in studying healthy organisms as well as disease states, is focused either on studying the whole live cell (or organism), or on studying isolated reactions between few purified and well defined components in vitro. Most biological processes are not isolated events of interaction between few components, but rather complex interconnected networks built of often multifunctional nodes (proteins acting on many targets). The in vitro studies give results that are less relevant to natural biology – since an experiment with a few purified components does not acknowledge the vast complexity of a natural system. On the other hand, live cell studies are notoriously hard to reproduce and interpret, due to the variability between live subjects, as well as due to the inherent complexity of biology (cross-talk between the studied process and other pathways, or background signal from unrelated processes is often present).
Synthetic minimal cells deliver a solution bridging the existing gap between in vitro and live cell research: use synthetic minimal cells to investigate multicomponent gene pathways, combining the advantages of in vitro systems with the relevancy and complexity approaching that of whole cell studies.
Reading and controlling cells is the core purpose of modern synthetic biology, and the overarching goal of all biomedical studies. Both studying mechanisms of most diseases, as well as investigating healthy cellular processes, is currently done as either in vitro or live cell experiments. In vitro research methods are easy to use, cheap and efficient way to obtain information about behavior of specific, well defined protein or nucleic acid complexes or single enzymes, or to characterize small molecule interactions between metabolites or drugs and their specific biological targets. However, since life is structured in complexes that involve many components organized in precise 3D assemblies, the in vitro experiments often only deliver information about small snapshot of this complex, natural system. Studies of live cells allow to obtain truly biologically relevant information about complex pathways, but at significantly higher cost, and with results that are harder to interpret and often less reproducible. The variability between live cell subjects and the underlying intricacy of interconnected biological networks constantly interacting with each other makes signal measured in live cell experiments often more noisy and the experiment itself difficult to design.
Synthetic minimal cells offer a platform that allows studying complex genetic pathways, while keeping the complexity of the system at a level that still allows us understanding fully what the system contains and how to engineer it. Our research focuses on building tools for general use in many areas of synthetic biology, as well as studying some specific cases of complex biological processes, both healthy and diseased, that are not accessible by studying natural complex cells.
Research with Jonas Salk and Christiaan Barnard
Developed Polio Vaccine
J. Supramol. Struct 18233 (1979)
Lanza (with Salk)
Work with Christiaan Barnard
Performed the World’s First Heart Transplant
New England Journal of Medicine 307 1275 (1982)
Lanza (with Barnard & Cooper)
JAMA 249 1746 (1983)
Lanza (with Barnard, Cooper & Cassidy)
American Heart Journal 107 8 (1984)
Lanza (with Barnard, Cooper & Boyd)