Controlling genes with your thoughts

Researchers have constructed the first gene network that can be controlled by our thoughts. Scientists have developed a novel gene regulation method that enables thought-specific brainwaves to control the conversion of genes into proteins (gene expression). The inspiration was a game that picks up brainwaves in order to guide a ball through an obstacle course.

 

It sounds like something from the scene in Star Wars where Master Yoda instructs the young Luke Skywalker to use the force to release his stricken X-Wing from the swamp: Marc Folcher and other researchers from the group led by Martin Fussenegger, Professor of Biotechnology and Bioengineering at the Department of Biosystems (D-BSSE) in Basel, have developed a novel gene regulation method that enables thought-specific brainwaves to control the conversion of genes into proteins -- called gene expression in technical terms.

Thoughts control a near-infrared LED, which starts the production of a molecule in a reaction chamber.

Credit: Martin Fussenegger et al., Copyright ETH Zurich

"For the first time, we have been able to tap into human brainwaves, transfer them wirelessly to a gene network and regulate the expression of a gene depending on the type of thought. Being able to control gene expression via the power of thought is a dream that we've been chasing for over a decade," says Fussenegger.

 

A source of inspiration for the new thought-controlled gene regulation system was the game Mindflex, where the player wears a special headset with a sensor on the forehead that records brainwaves. The registered electroencephalogram (EEG) is then transferred into the playing environment. The EEG controls a fan that enables a small ball to be thought-guided through an obstacle course.

 

Wireless Transmission to Implant

 

The system, which the Basel-based bioengineers recently presented in the journal Nature Communications, also makes use of an EEG headset. The recorded brainwaves are analysed and wirelessly transmitted via Bluetooth to a controller, which in turn controls a field generator that generates an electromagnetic field; this supplies an implant with an induction current.

 

A light then literally goes on in the implant: an integrated LED lamp that emits light in the near-infrared range turns on and illuminates a culture chamber containing genetically modified cells. When the near-infrared light illuminates the cells, they start to produce the desired protein.

 

Thoughts Control Protein Quantity

 

The implant was initially tested in cell cultures and mice, and controlled by the thoughts of various test subjects. The researchers used SEAP for the tests, an easy-to-detect human model protein which diffuses from the culture chamber of the implant into the mouse's bloodstream.

 

To regulate the quantity of released protein, the test subjects were categorised according to three states of mind: bio-feedback, meditation and concentration. Test subjects who played Minecraft on the computer, i.e. who were concentrating, induced average SEAP values in the bloodstream of the mice. When completely relaxed (meditation), the researchers recorded very high SEAP values in the test animals. For bio-feedback, the test subjects observed the LED light of the implant in the body of the mouse and were able to consciously switch the LED light on or off via the visual feedback. This in turn was reflected by the varying amounts of SEAP in the bloodstream of the mice.

 

New Light-sensitive Gene Construct

 

"Controlling genes in this way is completely new and is unique in its simplicity," explains Fussenegger. The light-sensitive optogenetic module that reacts to near-infrared light is a particular advancement. The light shines on a modified light-sensitive protein within the gene-modified cells and triggers an artificial signal cascade, resulting in the production of SEAP. Near-infrared light was used because it is generally not harmful to human cells, can penetrate deep into the tissue and enables the function of the implant to be visually tracked.

 

The system functions efficiently and effectively in the human-cell culture and human-mouse system. Fussenegger hopes that a thought-controlled implant could one day help to combat neurological diseases, such as chronic headaches, back pain and epilepsy, by detecting specific brainwaves at an early stage and triggering and controlling the creation of certain agents in the implant at exactly the right time.

 

Date: November 11, 2014

Source: ETH Zurich

Bio-inspired bleeding control: Synthesized platelet-like nanoparticles created

Stanching the free flow of blood from an injury remains a holy grail of clinical medicine. Controlling blood flow is a primary concern and first line of defense for patients and medical staff in many situations, from traumatic injury to illness to surgery. If control is not established within the first few minutes of a hemorrhage, further treatment and healing are impossible.

 

Artist's rendering of synthetic platelets.

Credit: Peter Allen illustration

At UC Santa Barbara, researchers in the Department of Chemical Engineering and at Center for Bioengineering (CBE) have turned to the human body's own mechanisms for inspiration in dealing with the necessary and complicated process of coagulation. By creating nanoparticles that mimic the shape, flexibility and surface biology of the body's own platelets, they are able to accelerate natural healing processes while opening the door to therapies and treatments that can be customized to specific patient needs.Taking a cue from the human body’s own coagulation processes, researchers have synthesized platelet-like nanoparticles that can do more than clot blood.

 

"This is a significant milestone in the development of synthetic platelets, as well as in targeted drug delivery," said Samir Mitragotri, CBE director, who specializes in targeted therapy technologies. Results of the researchers' findings appear in the current issue of the journal ACS Nano.

The process of coagulation is familiar to anyone who has suffered even the most minor of injuries, such as a scrape or paper cut. Blood rushes to the site of the injury, and within minutes the flow stops as a plug forms at the site. The tissue beneath and around the plug works to knit itself back together and eventually the plug disappears.

 

But what we don't see is the coagulation cascade, the series of signals and other factors that promote the clotting of blood and enable the transition between a free-flowing fluid at the site and a viscous substance that brings healing factors to the injury. Coagulation is actually a choreography of various substances, among the most important of which are platelets, the blood component that accumulates at the site of the wound to form the initial plug.

 

"While these platelets flow in our blood, they're relatively inert," said graduate student researcher Aaron Anselmo, lead author of the paper. As soon as an injury occurs, however, the platelets, because of the physics of their shape and their response to chemical stimuli, move from the main flow to the side of the blood vessel wall and congregate, binding to the site of the injury and to each other. As they do so, the platelets release chemicals that "call" other platelets to the site, eventually plugging the wound.

 

But what happens when the injury is too severe, or the patient is on anti-coagulation medication, or is otherwise impaired in his or her ability to form a clot, even for a modest or minor injury?

That's where platelet-like nanoparticles (PLNs) come in. These tiny, platelet-shaped particles that behave just like their human counterparts can be added to the blood flow to supply or augment the patient's own natural platelet supply, stemming the flow of blood and initiating the healing process, while allowing physicians and other caregivers to begin or continue the necessary treatment. Emergency situations can be brought under control faster, injuries can heal more quickly and patients can recover with fewer complications.

 

"We were actually able to render a 65 percent decrease in bleeding time compared to no treatment," said Anselmo.

 

According to Mitragotri, the key lies in the PLNs' mimicry of the real thing. By imitating the shape and flexibility of natural platelets, PLNs can also flow to the injury site and congregate there. With surfaces functionalized with the same biochemical motifs found in their human counterparts, these PLNs also can summon other platelets to the site and bind to them, increasing the chances of forming that essential plug. In addition, and very importantly, these platelets are engineered to dissolve into the blood after their usefulness has run out. This minimizes complications that can arise from emergency hemostatic procedures.

 

"The thing about hemostatic agents is that you have to intervene to the right extent," said Mitragotri. "If you do too much, you cause problems. If you do too little, you cause problems."

 

These synthetic platelets also let the researchers improve on nature. According to Anselmo's investigations, for the same surface properties and shape, nanoscale particles can perform even better than micron-size platelets. Additionally, this technology allows for customization of the particles with other therapeutic substances -- medications, therapies and such -- that patients with specific conditions might need.

 

"This technology could address a plethora of clinical challenges," said Dr. Scott Hammond, director of UCSB's Translational Medicine Research Laboratories. "One of the biggest challenges in clinical medicine right now -- which also costs a lot of money -- is that we're living longer and people are more likely to end up on blood thinners. When an elderly patient presents at a clinic, it's a huge challenge because you have no idea what their history is and you might need an intervention."

With optimizable PLNs, physicians would be able to strike a finer balance between anticoagulant therapy and wound healing in older patients, by using nanoparticles that can target where clots are forming without triggering unwanted bleeding. In other applications, bloodborne pathogens and other infectious agents could be minimized with antibiotic-carrying nanoparticles. Particles could be made to fulfill certain requirements to travel to certain parts of the body -- across the blood-brain barrier, for instance -- for better diagnostics and truly targeted therapies.

 

Additionally, according to the researchers, these synthetic platelets cost relatively less, and have a longer shelf life than do human platelets -- a benefit in times of widespread emergency or disaster, when the need for these blood components is at its highest and the ability to store them onsite is essential.

 

Further research into PLNs will involve investigations to see how well the technology and synthesis can scale up, as well as assessments into the more practical matters involved in translating the technology from the lab to the clinic, such as manufacturing, storage, sterility and stability as well as pre-clinical and clinical testing.

 

Date: November 13, 2014

Source: University of California - Santa Barbara

      

Diabetes: β cells at last

The race to generate β cells from stem cells has taken another big turn. We can already generate definitive endoderm from human embryonic stem cells and functional insulin-producing cells from transplanted pancreatic progenitors. Now, differentiating glucose-responsive insulin-producing cells in vitro that function like adult human β cells has been achieved.

 

Intensive insulin therapy can considerably reduce the incidence and severity of complications associated with type 1 diabetes mellitus; however, only the replacement of lost β cells (islet transplantation) can restore physiological control of blood levels of glucose and could potentially cure patients with diabetes mellitus.¹ Unfortunately, the scarcity of islet donors has restricted the number of patients who have been able to receive this therapy. In clinical practice, two or three donors are usually required for a transplantation procedure in a single patient. The discovery of human pluripotent stem cells (hPSC) was, therefore, readily adopted as a potential means of providing cell therapy to millions of patients with type 1 diabetes mellitus or advanced type 2 diabetes mellitus, as it was assumed that an unlimited number of β cells could be generated upon their differentiation. This hope, as shown by early studies in the field, did not sufficiently consider the complexity of incorporating features of pancreatic development into a model of pluripotent stem cell differentiation in vitro. Despite the difficulties, the development of a therapy for diabetes mellitus based on stem cells (Figure 1) has now reached another milestone with the publication of a study by Pagliuca and colleagues.² In this study, the researchers demonstrated that functional β cells can be obtained in vitro from hPSC that were sequentially treated with a total of 11 extracellular growth and differentiation factors for 4–5 weeks. These findings are valuable for developing cell-based therapies for diabetes mellitus, and might also provide a platform for disease modelling and for screening thousands of small molecules (with potential effects on human β-cell function, proliferation or survival) for the development of antidiabetic drugs.

 

Figure 1: β-cell differentiation from pluripotent stem cells.

The third most important milestone (differentiating glucose-responsive insulin-producing cells) has just been set in this process. Pagliuca and colleagues achieved in vitro differentiation of functional β cells using a scalable 3D culture system.

As in the majority of previous studies in the field,^{3, 4, 5, 6, 7, 8} the study by Pagliuca and co-workers built on the knowledge gathered from examining pancreatic development in model organisms. Pagliuca et al. also followed the fairly safe strategy of modulating the extracellular microenvironment to induce cell differentiation rather than introducing DNA encoding transcription factors inside the hPSC. They tested 150 different combinations of factors that had to be added sequentially to their cultures to induce proper cell differentiation.

 

Previous studies have indicated that insulin-producing cells derived in vitro from hPSC are polyhormonal, are similar to fetal β cells and do not respond to glucose challenge.^{3, 4, 5, 6} However, the stem-cell-derived β (SCβ) cells described in this new study display the major and specific characteristics of a genuine adult β cell (including calcium flux, glucose-responsiveness and expression of a single hormone—insulin). The SCβ cells also expressed several β-cell differentiation markers (PDX1, NKX6.1 and ZnT8) and had ultrastructural morphological features similar to those of adult human β cells. In addition, in a mouse model of diabetes mellitus, transplantation of SCβ cells quickly restored and maintained normal blood levels of glucose.

“...the importance of this breakthrough will depend on the reproducibility of its findings...”

The thorough characterization of the SCβ cells in the paper by Pagliuca and associates is more comprehensive than that reported for insulin-producing cells derived from hPSC that were glucose responsive upon culture in a 3D matrix during the maturation stage of in vitro differentiation.⁷ Thus, the work by Pagliuca and colleagues is an important technical and biological breakthrough in the field of pancreatic β-cell differentiation from hPSC in vitro. The research has unravelled the minimal conditions required to efficiently turn hPSC-derived pancreatic progenitor cells into endocrine progenitor cells that are amenable to acquiring a phenotype very close to that of genuine islet β cells. These conditions could only be poorly defined in previous in vivo experiments, wherein hPSC-derived pancreatic progenitors required 3–4 months to mature into β cells, which contrasts with the 4–5 weeks required for SCβ to mature in vitro.⁸ The study has also consolidated the central position that a good understanding of tissue and organ developmental biology occupies in the race for generating different cell types of interest from hPSC. However, despite the mention that the protocol works on a total of four cell lines in the authors' laboratory, the importance of this breakthrough will depend on the reproducibility of its findings by other investigators using different hPSC lines.

 

The SCβ cells are phenotypically very close, but not transcriptionally identical, to primary β cells. In addition to the strategies highlighted by the authors to tackle this issue, it will be of interest to examine whether this discrepancy fades with time spent in culture or upon SCβ cell transplantation in vivo, given that the reported protocol is still a big reduction of the 13–15 weeks required for islet development in the human embryo. Nevertheless, the SCβ cells are obtained at a sufficiently high efficiency (33%) in the current system so that any future moves towards clinical implementation would probably not face challenges with mass production. As the SCβ cells could respond to several glucose challenges and their transplantation in mice resulted in detectable levels of human C-peptide and normalization of blood glucose levels within 2 weeks, they might represent an optimal candidate for testing in clinical trials in patients with diabetes mellitus. However, important questions need to be solved before reaching this step.

 

Many laboratories around the world are working toward the goal of using genuine or 'surrogate' insulin-producing cells as a basis for treating patients with type 1 diabetes mellitus and advanced type 2 diabetes mellitus. The current paper is very important in this context; however, it will undoubtedly be some time before these results are translated into treatment for patients with diabetes mellitus. For example, the FDA has just approved the first clinical trial using a product derived from embryonic stem cells that consists of pancreatic progenitor cells delivered in a macro-encapsulation device; however, this cell product was developed on the basis of research that was published 8 years ago.³

 

An advantage of the product developed by Pagliuca and co-workers is that it consists of fairly pure β cells. However, some encapsulation technology to prevent graft rejection will also be needed. Cellular grafts encapsulated in biomaterials often trigger a process that begins with inflammation and ends with the implant being surrounded by macrophages and fibrotic tissue. This reaction to a foreign body is absent in transplantation experiments using immune-deficient animals,² but might severely compromise the survival and function of encapsulated cells in patients.⁹ For this reason (and others, such as the lack of direct vascularization of the encapsulated cells in a graft of sufficient size for use in humans), islet transplantation in combination with encapsulation has not yet been successful in clinical trials. Encapsulation is also needed to prevent the risk of outgrowth of teratoma cells or cells containing potentially oncogenic mutations.¹⁰ The presence of such cells actually needs to be ruled out by more sensitive techniques than those used by Pagliuca et al., even though mice examined 18 weeks after transplantation were declared to be free of tumours. Furthermore, the SCβ cells might function differently in the human microenvironment. Therefore, caution is needed when we talk of a therapeutic breakthrough for diabetes mellitus.

 

Josué K. Mfopou^1, & Luc Bouwens^1,

Nature Reviews Endocrinology | News and Views

Nature Reviews Endocrinology

doi:10.1038/nrendo.2014.200

Published online 11 November 2014