A series of fluorescence microscopy images detail the blinking behavior of the teams nanoparticle buckyswitches. Credit: Nano Letters 17 (6) pp. 38963901

Visualizing biological cells under a microscope was just made clearer, thanks to research conducted by graduate student Yifei Jiang and principal investigator Jason McNeill of Clemson University's department of chemistry.

With the help of Rhonda Powell and Terri Bruce of Clemson's Light Imaging Facility, the team was able to develop a nanoparticle "switch" that fluoresces to sharpen the resolution of microscopic images that depict small cellular structures. As recently published in Nano Letters, this switch improves upon an imaging method that won the 2014 Nobel Prize in Chemistry.

Because cellular structures emit light at wavelengths smaller than 400-700 nanometers on the electromagnetic spectrum, they often appear blurred through a light microscope. This constraint is referred to as the diffraction limit, and it occurs because of the wave properties of light. As light passes around structures within biological cells, it diffracts, or bends, to a point that light microscopes cannot clearly resolve. The 2014 prize-winning imaging method - single molecule localization microscopy - was invented to surpass this limitation.

"Single molecule localization microscopy is based on molecular 'photoswitches' - fluorescent molecules that you can turn on and off, like a light switch, to beat the diffraction limit," McNeill said. "With this imaging method, the sample is imaged one fluorescent molecule at a time and a computer is used to construct an image that is much sharper than what you could get with a regular light microscope."

The catch, however, is that the fluorescence provided by photoswitches is dim at best, with only a slight improvement in image resolution. Single molecule localization microscopy also requires specialized equipment that can be expensive to obtain.

Cue the "buckyswitch" - the Clemson researchers' enhanced version of a photoswitch. This new type of nanoparticle retains the photoswitch's on-off capability, but is 10 times brighter and easier to use. It also allows microscopes to capture images up to the terapixel level. (That's the equivalent of one trillion pixels, or one million megapixels.)

"These nanoparticles are the first photoswitches to achieve precision down to approximately 1 nanometer, which greatly improves the resolution of super-resolution imaging," Jiang said. "Also, our method only requires one excitation light source, where conventional super-resolution techniques require two lasers; thus, we have simplified the microscope setup."

Jiang assembled the buckyswitch out of a fluorescent, semiconducting conjugated polymer complexed with a chemical derivative of buckminsterfullerene: a soccer-ball-shaped form of carbon.

"The hard part of making a fluorescent nanoparticle that you can turn on and off is that there are lots of areas emitting fluorescence at once," McNeill said. "In the case of fluorescent conjugated polymer, there are dozens or hundreds of chain segments. You can try to make a lot of little switches for each segment, but it's hard to get them all to switch off at the same time. You can't get them synchronized."

By adding the derivative of buckminsterfullerene, called PCBM, to the making of buckyswitches, a "master switch" is formed that regulates the atomic charge of the polymer's segments, thus synchronizing fluorescence. PCBM is able to seize electrons from the polymer segment, giving the segment an overall positive charge. This positive charge reduces the fluorescence of nearby segments, which has a domino effect that turns off fluorescence in the entire nanoparticle.

Bruce - whose background traverses the topics of chemical engineering, applied biology, cell biology, and experience in teaching and industry - likens this imaging method to the view of a suspension bridge at night.

"The wires of the bridge are often illuminated, and when you are standing far away from the bridge, the lights look like one continuous 'rope' of light, instead of individual bulbs. However, if you can make the bulbs blink - such that only every other bulb is 'on' at any time - your eyes can discern the individual bulbs from far away," Bruce said. "The basis for super-resolution microscopy lies in the ability to make fluorescent labels 'blink' just like the lights on the bridge. The work that Dr. McNeill's lab is doing is vital for the advancement of this technology because it focuses on making those individual blinks much brighter, so that our current photon detectors can actually see the blinks. If we can see the blinks with a camera or other photon detector, we can map where the blink occurs, and create an image where we can discern two points of light that are within 10-20 nanometers of one another."

Once the buckyswitch was synthesized, Jiang tested it in E. coli, but not before developing a unique growth media for the bacteria. Typically, E. coli is grown in media that is autofluorescent, meaning that it naturally emits light. Without the proper media, the buckyswitch's fluorescence would be obscured by background light, something that Powell underlined.

"A study like the one Yifei conducted required very little background fluorescence, so I researched media components that would be less likely to be autofluorescent and prepared a 'recipe' for a non-conventional, less autofluorescent nutrient media for bacteria culture," said Powell, who studied both biological sciences and microbiology at Clemson before becoming the research lab manager of the Clemson Light Imaging Facility. Powell and Bruce also worked to provide Jiang with the E. coli for the study.

After all of the necessary components were squared away, Jiang attached the nanoparticle buckyswitches to the surface of E. coli. As hoped, the buckyswitches emitted small flashes of light, which allowed the researchers to determine their precise positions. They then pieced together each flash of light to reconstruct the shape of the E. coli, yielding a super-resolution image.

"We hope this breakthrough will eventually be able to help researchers tackle difficult problems in biology, leading to breakthroughs in the understanding and treatment of disease," the Clemson team said.

The team designed the buckyswitches to work with standard fluorescent microscopes and free software that's available online, making the technology inexpensive and accessible for labs worldwide.

Their publication, titled "Improved Superresolution Imaging Using Telegraph Noise inOrganic Semiconductor Nanoparticles," is featured in the June 14 issue of Nano Letters.

Explore further: Background suppression for super-resolution light microscopy

More information: Yifei Jiang et al, Improved Superresolution Imaging Using Telegraph Noise in Organic Semiconductor Nanoparticles, Nano Letters (2017). DOI: 10.1021/acs.nanolett.7b01440

Journal reference: Nano Letters

Provided by: Clemson University

Excerpt from:
Researchers illuminate the field of microscopy with nanoparticle 'buckyswitch' - Phys.Org

CRISPR-Cas9 gene editing is based on a tactic bacteria developed to protect themselves from viruses.

The Cas9 protein gloms onto a targeted piece of DNA (purple) before cutting it. Bacteria developed Cas9 as aweapon to kill viruses by snipping their DNA, but some viruses came up witha defense, called anti-CRISPR, that inactivates Cas9. The guide RNA (orange)directs Cas9 to its target. (Fuguo Jiang image)

Research now shows that the countermeasure viruses came up with inhibitory proteins referred to as anti-CRISPRs can be used to improve CRISPR-Cas9 as a gene-therapy tool, decreasing off-target gene editing that could cause unwanted side effects.

In a study reported online this week in the journal Science Advances, researchers from UC Berkeley and UC San Francisco show that recently discovered anti-CRISPR proteins decrease off-target effects by as much as a factor of four, acting like a kill switch to disable CRISPR-Cas9 after its done its job.

The study demonstrated that one particular anti-CRISPR protein called AcrIIA4 reduced by four-fold the off-target effects of a CRISPR-Cas9 molecule that uses a guide RNA to find, snip and replace the mutated hemoglobin gene responsible for sickle cell disease. It does this without significantly reducing the desired on-target gene-editing.

Unexpected mutations can arise as a result of off-target gene editing, but our paper like many others shows that off-target effects can be modulated and it is not as serious as people might think, said UC Berkeley postdoctoral fellow Jiyung Jenny Shin, from the lab of Jacob Corn at the Innovative Genomics Institute and one of three first authors of the paper.

In her experiments on human cells in culture, Shin found that delivering CRISPR-Cas9 and then, several hours later, the anti-CRISPR protein, was the most effective way to reduce off-target effects. The protein mimics DNA, glomming onto Cas9, the enzyme that actually cuts the double-stranded DNA, and preventing further cutting.

Even after six hours of effective CRISPR, inserting anti-CRISPR decreases off-target effects by more than two-fold compared to on-target effects, Shin said. Therapeutically, you could treat a patient with CRISPR first, and then treat with anti-CRISPR at a later time and decrease off-target effects.

The researcher who discovered AcrIIA4, Joseph Bondy-Denomy of UC San Francisco, foresees these anti-CRISPR proteins becoming a standard part of CRISPR gene therapy, given along with CRISPR-Cas9 to disable gene editing after a fixed period of time to prevent random off-target cutting.

This Cas9 inhibitor could be encoded on the same piece of DNA as Cas9, for example, precisely timed to turn Cas9 off after the gene editing is done, instead of letting Cas9 linger in the cell and risk off-target effects, said Bondy-Denomy, who is also a co-author of the paper.

Anti-CRISPR bindingThe team included researchers in the lab of Jennifer Doudna, one of the inventors of CRISPR-Cas9 gene editing, who determined how the anti-CRISPR protein binds to the CRISPR-Cas9 complex. Using cryo-electron microscopy, they found that anti-CRISPR essentially mimics DNA, tricking CRISPR-Cas9 into binding with it, and then never letting go.

The anti-CRISPR protein (red on right) mimics DNA, binding in the site where the cutting enzyme Cas9 typically grips the target DNA (left) before it cuts. But the anti-CRISPR protein doesnt let go, essentially killing Cas9s gene editing ability. (Fuguo Jiang image?

The CRISPR inhibitor targets a spot on the Cas9 protein that is so essential for Cas9s function that it cannot operate to cut DNA when its bound by the anti-CRISPR.

Last year, Bondy-Denomy reported finding four anti-CRISPR proteins used by attacking viruses to inactivate the version of the Cas9 protein found in the bacterium Listeria monocytogenes. Two of these also inhibited the Cas9 protein most commonly used by researchers, which is adapted from the bacterium Streptococcus pyogenes and is referred to as SpyCas9. Another team found three other anti-CRISPR proteins that work against a different but promising Cas9 protein adapted from the bacterium Neisseria meningitidis.

The current study looked at the effect of one of the proteins from Listeria, AcrIIA4, on SpyCas9 loaded with a guide RNA that homes in on complementary DNA to bind and cut.

Research at UC Berkeley and elsewhere suggests that CRISPR-Cas9 constantly feints with the cells DNA repair system: as the enzyme cuts at its target site, the cell repairs the DNA, and CRISPR-Cas9 cuts again, repeating this vicious cycle until a mutation arises in the DNA that prevents enzyme binding, at which point the CRISPR-Cas9 molecule moves on to find another binding site.

The current work from the Corn and Doudna labs now suggests that adding an anti-CRISPR after Cas9 has successfully edited a target gene would prevent unintended damage to other portions of a genome.

The ability to turn Cas9 gene editing off is just as important as the ability to turn it on, said Corn, scientific director for biomedicine of the IGI and a UC Berkeley assistant adjunct professor of molecular and cell biology. Imagine if you had an electric razor with no off-switch! For eventual therapeutic applications, it is critical to be able to precisely control when and where gene editing is active. The anti-CRISPR proteins offer opportunities to completely turn off Cas9 as well as fine-tune its activity.Jennys data suggests that there is an ideal time window for letting Cas9 do its job and then turning it off after that amount of time has passed, Bondy-Denomy said. We can actually use the anti-CRISPR proteins as tools to figure out what that time window is, that is, for any one cell type with any one guide RNA sequence, how long we want Cas9 to be active in the cell.

Shin and postdoctoral fellows Fuguo Jiang and Jun-Jie Liu are the three first authors of the paper, which was also co-authored by Benjamin Rauch of UCSF and postdoc Nicolas Bray, researcher Seung Hyun Baik and professor Eva Nogales, in addition to Corn and Doudna, of IGI and UC Berkeleys Department of Molecular and Cell Biology. Doudna and Nogales are Howard Hughes Medical Institute investigators.

The work was supported in part by HHMI, the Li Ka Shing Foundation, the Heritage Medical Research Institute and the National Institute on Aging.

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Anti-CRISPR proteins decrease off-target side effects of CRISPR-Cas9 - UC Berkeley

(Ames)--Dry weather forces plants to save energy by reducing their growth rate, but its not as if a plant can consult a rain gauge or weather report. So how do they know when to ease up on growth?

Yanhai Yin, a professor of genetics, development and cell biology and a Plant Sciences Institute Faculty Scholar, has spent years charting the genetic mechanisms that govern plant stress response and growth to answer that question.

Yin and his colleagues recently published an article in the peer-reviewed academic journal The Plant Cell focusing on WRKYs (pronounced workies), a family of proteins named for the several critical amino acids of which theyre composed. The new paper shows how WRKYs govern both stress response and growth in plants, making the proteins of particular interest to plant breeders and crop growers eager for varieties that will withstand dry conditions.

They are important regulators for the balance of drought response and growth, Yin said. They are very promising targets for plant breeding.

The paper describes how researchers in Yins lab managed to cross Arabidopsis plants in such a manner as to eliminate, or knock out, three different WRKYs genes. The resulting plants showed dramatically less growth than normal but were more drought tolerant. Arabidopsis is a small flowering plant often used as a model in experiments.

Jianai Chen and Trevor Nolan, graduate assistants in Yins lab, contributed to the paper, as well as Mingcai Zhang and Zhaohu Li of China Agricultural University, who are long-time collaborators with Yin.

Much of Yins research has centered on a plant protein known as BES1, an important switch in plant genomes regulated by a plant steroid called brassinosteroid that influences thousands of other genes. Yin said WRKYs and BES1 work together to promote plant growth under normal conditions.

Previous studies have shown that WRKYs also help to govern bacterial response in plants as well. Yin said his future studies will tease out how growth, drought tolerance and bacterial response interact with one another. Such efforts could lead to crops with genetics better suited to withstand many of the most pressing challenges producers face.

As the paper hints, harnessing the versatility of WRKYs to breed more resilient crops would make rain gauges look primitive by comparison.

Excerpt from:
Iowa State University plant scientists explore the balance between plant growth and drought response - KMAland

By Arthur Hirsch

Sure, you know how old you are, but what about your cells? Are they the same age? Are they older, younger? Why does it matter?

Led by scientists at Johns Hopkins University, a team of researchers is reporting progress in developing a method to accurately determine the functional age of cells, a step that could eventually help clinicians evaluate and recommend ways to delay some health effects of aging and potentially improve other treatments, including skin graft matching and predicting prospects for wound healing.

The more accurate system could eventually enable clinicians to see aging in cells before a patient experiences age-related health decline, or help clinicians produce more successful skin grafts by matching cell characteristics of the donor and the graft site.

In the current issue of Nature Biomedical Engineering, lead author Jude M. Phillip, who conducted this research while completing his doctorate in chemical and biomolecular engineering at Johns Hopkins, reports success in creating a system that considers a wide array of cellular and molecular factors in one comprehensive aging assessment.

These results show that the biophysical qualities of cells, such as cell movements and structural features, make better measures of functional age than other factors, including cell secretions and cell energy.

The multidisciplinary team of engineers and clinicians examined dermal cells from just underneath the surface of the skin taken from both males and females between the ages of 2 and 96.

The researchers from Johns Hopkins, Yale University, and the National Cancer Institute of the National Institutes of Health hoped to devise a system that, through computational analysis, could take the measure of various factors of cellular and molecular functions. From that information, they hoped to determine the biological age of individuals more accurately using their cells, in contrast to previous studies, which makes use of gross physiology, or examining cellular mechanisms such as DNA methylation.

"We combined some classic biomolecular hallmarks of aging, and sought to further elucidate the role of biophysical properties of aging cells, all in one study," said Phillip, now a post-doctoral fellow at Weill Cornell Medicine.

Researchers trying to understand aging have, up until now, focused on factors such as tissue and organ function and on molecular-level studies of genetics and of epigenetics, meaning heritable traits that are not traced to DNA. The level in betweencellshas received relatively little attention, the researchers wrote.

This research was meant to correct for that omission by considering the biophysical attributes of cells, including such factors as the cells' ability to move, maintain flexibility, and structure. This focus emerges from the understanding that changes associated with aging at the physiological levelsuch as diminished lung capacity, grip strength, and mean pressure in the arteries"tend to be secondary to changes in the cells themselves, thus advocating the value of cell-based technologies to assess biological age," the research team wrote.

Older cells, for example, are more rigid and do not move as well as younger cells, which, among other consequences, most likely contributes to the slower wound healing commonly seen in older people.

For example, older cells are more rigid and do not move as well as younger cells, which, among other consequences, most likely contributes to the slower wound healing commonly seen in older people, said Denis Wirtz, the senior author and Johns Hopkins' vice provost for research. Wirtz and Phillip conducted their research in the Johns Hopkins Institute for NanoBioTechnology.

From the analysis, they were able to stratify individuals' samples into three groups: those whose cells roughly reflected their chronological age, those whose cells were functionally older, and those whose cells were functionally younger. The results also showed that the so-called biophysical factors of cells determined a more accurate measure of age than biomolecular factors such as cell secretions, cell energy, and the organization of DNA.

Phillip explained that this better accuracy from the biophysical factors most likely results from the orchestration of many biomolecular factors. He compared it to the more complete picture you get looking at a forest from a distance without binoculars.

"With binoculars you can see details about the individual trees, the color and shapes of the leaves, the roughness of the bark, the type of tree, but without the binoculars you can now see the density of the trees, and whether there is a barren plot, or a group or dying trees," Phillip said. "This is something you may miss with the binoculars, unless you are looking at the correct spot."

The more accurate system could eventually enable clinicians to see aging in cells before a patient experiences age-related health decline. This in turn could allow doctors to recommend treatments or changes in life habits, such as exercise or diet changes, Wirtz said. Phillip said the work could potentially help clinicians produce more successful skin grafts by matching cell characteristics of the donor and the graft site. Other potential applications range from toxicology screening for cosmetics and topical therapeutics to predicting progression of some age-related diseases.

The researchers acknowledge that the system needs further testing with a larger cell sample, but the results are robust and encouraging. Conducted along with clinicians such as Jeremy Walston, the Raymond and Anna Lublin Professor of Geriatric Medicine, and co-director of the Biology of Healthy Aging program at the Johns Hopkins School of Medicine, this work promises to allow clinicians to measure a person's health in the present and the future.

"It opens the door to finally be able to track how a person is doing at the cellular level," Wirtz said.

Added Phillip: "This platform is also more than just a cellular age predictor; it has the ability to do so much more in terms of assessing an individual's cellular health."

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How old are your cells? New method determines cell age more accurately, could help elderly patients - The Hub at Johns Hopkins

When the Japanese researcher Shinya Yamanaka managed to reprogram adult cells into an embryonic-like state to yield induced pluripotent stem cells (iPSCs), this was supposed to herald a revolution in regenerative medicine. But 10 years after their discovery, a therapeutic breakthrough is still outstanding.

The overall stem cell therapy field has failed today to show a very clear cut clinical benefit, told me Georges Rawadi, VP for Business Development at Celyad. The field now needs some significant success to attract attention.

Even though investors prefer placing their bets on the hot T cell therapies these days, some stem cell technologies such as iPSCs are starting to get traction as big industry players are exploring the territory. Last year, Bayer and Versant threw $225M into the pot to launch BlueRock Therapeutics, a regenerative medicine company that plans to develop iPSC-based therapies. A year before, Fujifilm spent $307M to acquire the iPSC company Cellular Dynamics.

Although a big success story is still lagging behind, recent advances in the field argue that stem cells indeed have the potential to translate into effective therapies for currently intractable diseases. Heres an overview of what biotechs stem cells are up to!

Stem cell treatment is not a new concept hematopoietic stem cells (HSCs) were described as early as the 1960s and bone marrow transplants have been used to treat blood cancer for decades.

The reason that we get excited about stem cell therapies comes from our experience with the hematopoietic stem cells. If you want to see what a mature stem cell therapy is like, you only need to look at bone marrow transplantation explained James Peyer, Managing Partner at Apollo Ventures, who has a Ph.D. in stem cell biology.

According to Peyer, the hematopoietic stem cell field is one of the most active areas in the stem cell world right now, mainly fueled by our advances in the gene editing space. Tools like CRISPR and TALEN allow for the genetic modification of a patients own bone marrow stem cells, which can then be expanded and returned to the patient for the correction of a genetic defect.

Last year, regulators gave green light to one of the first therapies of this kind. Strimvelis, developed by GSK, consists of an ex vivo stem cell gene therapy to treat patients with the very rare type of Severe Combined Immunodeficiency (SCID). Using the patients own cells avoids the risk of graft versus host disease (GvHD), which still affects around 30% of people receiving a bone marrow transplant.

Small wonder that the CRISPR companies, CRISPR Therapeutics, Editas, and Intellia are all active in this field, with preclinical programs in a number hematological diseases.

To date, the most prominent stem cells in the clinic are mesenchymal stem cells (MSCs), which are moving through more than 300 registered clinical trials for a wide array of diseases. These cells are able to form a variety of tissues including bone, cartilage, muscle or fat, and can be readily harvested from patients or donors for use in autologous or allogeneic therapies.

While MSCs have deluded the biotech scene with good safety profiles in clinical trials, their actual regenerative potential remains controversial, and there have been a great number of clinical failures, which many blame on a lack of demonstrated mechanisms of action.

As Peyer explained, The problem here is that, as opposed to other adult stem cells, the MSC has been unclearly defined. We know roughly what it does but we dont fully understand the molecular mechanisms driving these cells. On top of being unclearly defined, the regenerative powers of MSCs have been massively over-claimed in the past.

Another reason for the lack of clinical benefit has also been attributed to the use of undifferentiated MSCs, as Rawadi explained to me. The Belgian biotech Celyad, which has been pioneering cell therapy in the cardiovascular space, is using bone-marrow derived autologous MSCs and differentiates them into cardiomyocyte precursors to produce new heart muscle in patients with heart failure.

Although the company missed its primary endpoint in a phase III trial last year, Celyad has staked out a patient subpopulation that showed significant improvement. Its technology still has the confidence of the FDA, which just handed out a Fast Track designation and Celyad is now planning a refined Phase III trial.

One of Celyads major competitors, Australian Mesoblast, is forging ahead using allogeneic MSCs with Phase III programs in heart failure, chronic low back pain (CLBP) due to disc degeneration, as well as a range of inflammatory conditions including GvHD and rheumatoid arthritis.

Although the ability of MSCs to regenerate tissues remains questionable, the Mesoblasts approach hinges on a body of evidence showing that MSCs can suppress inflammation and mobilize endogenous repair mechanisms through indirect effects on immune cells.

Indeed, the first-ever approved stem cell therapy, Prochymal, also depends on this mechanism. Prochymal was developed by US-based Osiris Therapeutics and in 2012 received Canadian approval to treat acute GvHD. But after Sanofi opted to shelve its partnership with Osiris prior to FDA approval, the biotech sold out its off-the-shelf stem cell platform to Mesoblast in a $100M deal.

In Belgium, companies like TiGenix and Promethera are also banking on the immunomodulatory properties of MSCs. The companies are developing treatments for patients with Crohns disease and liver diseases, respectively.

The ultimate hope for stem cell therapies has been to regenerate damaged or diseased tissues as found in diabetes, heart failure or blindness. Holostem Terapie Avanzate, a spin-off from the University of Modena and Reggio Emilia was the first company to move towards this goal.

Building on 20 long years of research, the biotech has developed Holoclar, the first and only autologous stem cell therapy (apart from bone marrow transplants) to enter the European market. Holoclar is based on limbal stem cells, located in a part of the eye called the limbus, which can be used to restore eyesight in patients that have lost sight due to burn injuries.

Meanwhile, UK-based Reneuron is developing off-the-shelf therapies that aim to restore the cognitive function of patients following a stroke. Backed by no other than Neil Woodford, the company recently raised an impressive 100M to advance its lead therapy to the market.

The biotechs fetal-derived neural stem cell line CTX was able to significantly reduce the disability of post-stroke patients in a Phase II trial and ReNeuron is now planning to push its candidate into pivotal trials.

A major question in the space a decade ago was safety. Today, theres been a lot of trials done that show that safety is not an issue. I think safety is kind of off the table but efficacy is still a question mark. And thats what were trying to deliver now, Olav Helleb, CEO of ReNeuron, told me.

While neural stem cells and other tissue-specific stem cells are able to regenerate the cells of a particular tissue, Embryonic Stem Cells (ESCs) and their engineered counterparts, iPSCs, are capable of making every cell type in the body, a property known as pluripotency. Pluripotent stem cells can also expand indefinitely in culture and their identification unlocked massive expectations for these cells to transform the regenerative medicine field.

Yet, these cells come with significant challenges associated with the safety of the final preparation. Apart from ethical issues surrounding ESCs, today, a lot of companies have been cautious about using these cells for therapy, because undifferentiated pluripotent cells can drive tumor formation, explained Rawadi. Since ESCs can, in principle, form every cell type, they can lead to the formation of teratomas.

A major reason for the fairly slow progress in the field is based on the difficulties of directing a pluripotent cell to exactly the cell type that is needed for cell therapy. We can readily drive the cells from the undifferentiated state to the differentiated state. However, getting those cells to pause anywhere in the middle of this continuum to yield progenitor cells is incredibly challenging, Peyer explained. Another challenge, he says, is to engraft the cells in the right place to enable them to become fully integrated.

Besides initial hurdles, companies like US-based Asterias or ViaCyte are now running the first Phase I/II trials with ESC-derived cells to treat patients with spinal cord injuries and to restore the beta cells in type I diabetes. So far, the eye has been the the dominant organ for many of the first human clinical trials with pluripotent stem cells, where the cells are assessed in diseases such as age-related macular degeneration (AMD) to restore the loss of the retinal epithelium.

Deriving retinal epithelium from pluripotent cells is relatively easy and in fact, researchers in Japan are now running the very first clinical trial using donor-derived iPSCs to treat patients with AMD. For reasons of safety and standardization, the trial is based on an allogeneic approach. However, since this doesnt offer an exact genetic match, allogeneic therapies raise the prospect of immune rejection, an issue that has been plaguing the use of ESCs.

But the scientists in Japan have contended that iPSC banks could potentially solve this problem. The team in Japan is currently establishing an iPSC bank, consisting of HLA-characterized cell lines from 5-10 different donors, which should match 3050% of Japans population.

Such haplobanks have the benefits of allogeneic cell therapy, namely cost-effectiveness and standardization, but you still have matching immune systems, Peyer agrees.

For now, this remains a vision for the future, but the potential seems enormous. As Julian Howell, CMO of ReNeuron, told me, iPSCs have still got an awful long way to go. For the iPSC program running in Japan, they recently acknowledged that it took about $1.5M and 6 months to treat each patient. Its a great idea but its still got some way to go before it reaches the scale that could get into the clinic.

Images via nobeastsofierce,Natali_ Mis,vchal/ Shutterstock

Original post:
Regenerating the Body With Stem Cells Hype or Hope? - Labiotech.eu (blog)

Irvine, Calif., July 11, 2017 To paraphrase the classic poem, no hair is an island entire of itself.

Instead, University of California, Irvine scientists have discovered that all hairs can communicate with each other and grow in coordination across the entire body. This is regulated by a single molecular mechanism that adjusts by skin region to ensure efficient hair growth so no bald patches form and enable distinct hair densities in different body areas.

Study co-leader Maksim Plikus is an assistant professor of developmental & cell biology. Daniel A. Anderson / UCI

In animals, this regulatory process is important for survival in the wild. In humans, these findings could lead to new ways of addressing both balding and unwanted hair growth and further understanding of how regions of faster and slower regeneration work in coordination in other fast-renewing tissues, such as the intestines and bone marrow.

For the study, the researchers used the first mouse model of poor hair growth to analyze human-like hair behavior that leads to baldness. Their results appear in eLife, an open-access journal focusing on the life and biomedical sciences. UCIs Maksim Plikus, assistant professor of developmental & cell biology, and Qing Nie, professor of mathematics, led the effort. Ji Won Oh from Plikus lab and Qixuan Wang from Nies lab contributed equally to this work.

How skin regions communicate

The researchers focused on the interaction of the Wnt signaling pathway, which is important in embryonic development and regeneration, and bone morphogenetic proteins, which are hair growth inhibitory factors.

Study co-leader Qing Nie is professor of mathematics. UCI

While previous studies have shown that Wnt-BMP signals regulate hair growth in certain body areas, it was not known how different skin regions communicate with one another to coordinate hairs across their borders. By combining expertise in mathematical modeling from Nies lab and expertise in skin studies from Plikus lab, Wnt-BMP regulation was found to be ubiquitous across all skin.

In analogy with languages spoken in two neighboring countries, it was unclear how the back skin talks with the belly skin to coordinate the tasks of growing hairs, Plikus said. We showed that although different signaling dialects may exist between belly and back skin, for instance, all hairs can understand one another through the use of similar words and sentences.

The roots of hair growth problems

A breakdown of this complex signaling could uncover the roots of human hair growth irregularities and point to solutions.

For example, common male pattern baldness affects frontal and crown regions but not the back of the head. In adult humans, messaging among scalp hairs appears to stop, and every hair follicle is thought to grow independently.

If communication between nonbalding and balding regions can be reactivated, hair growth signals can then start spreading across the entire head skin, preventing regional baldness, Plikus said.

Just like scalp skin can show hair growth deficiency, skin in other body sites such as the face, arms and legs can often show excessive hair growth that can be cosmetically undesirable, he added. Our findings suggest that increased signaling crosstalk among hair follicles could be one major reason for this.

Whats next?

Plikus said that Wnt and BMP signaling activities can be regulated pharmacologically. Our study identified the types of Wnt-BMP signaling levels that are very favorable for hair growth and the types that prevent it, he said. It provides the road map for optimizing Wnt-BMP levels to achieve enhanced hair growth.

He added that the findings point toward additional signaling factors besides Wnt and BMP positively correlated with robust hair growth. Studying these will be the researchers next step.

Nie noted that laboratory experiments can be insufficient to study complex biological functions, such as hair growth across the entire skin. In such cases, mathematical modeling can greatly assist in the discovery process, he said. Our new mathematical model predicted details of signaling communications between hairs, otherwise difficult to reveal with standard biological experiments alone.

Scientists from UCI, the University of Southern California and Indiana University, as well as research centers in Australia, Poland, South Korea and China, contributed to the study, which received support from the National Institutes of Health, the National Science Foundation, the Edward Mallinckrodt Jr. Foundation, the Pew Charitable Trusts and UCIs Center for Complex Biological Systems.

About the University of California, Irvine: Founded in 1965, UCI is the youngest member of the prestigious Association of American Universities. The campus has produced three Nobel laureates and is known for its academic achievement, premier research, innovation and anteater mascot. Led by Chancellor Howard Gillman, UCI has more than 30,000 students and offers 192 degree programs. Its located in one of the worlds safest and most economically vibrant communities and is Orange Countys second-largest employer, contributing $5 billion annually to the local economy. For more on UCI, visit http://www.uci.edu.

Media access: Radio programs/stations may, for a fee, use an on-campus ISDN line to interview UCI faculty and experts, subject to availability and university approval. For more UCI news, visit news.uci.edu. Additional resources for journalists may be found at communications.uci.edu/for-journalists.

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UCI study sheds light on regulation of hair growth across the entire body - UCI News

July 11, 2017 NASA immunologist Hawley Kunz performs a microgravity evaluation of the pipetting steps necessary for terrestrial purification of cells. Credit: NASA

Just like early explorers, NASA Twins Study investigators are venturing into new territory. Conducting human omics research on twin astronauts as part of the One Year Mission that took place aboard the International Space Station is one such venture. As technology evolves so does the research. NASA is evaluating more efficient and innovative research techniques to prepare for the journey to Mars.

Innovative thinking could improve the way biological samples are processed and transported from space back to research labs on Earth for future studies. This thinking was prompted by researchers in NASA's Human Research Program (HRP) and Twins Study investigators at Johns Hopkins Medicine.

Freshly isolated samples yield better results than cells isolated from frozen samples returned to Earth from the orbiting laboratory. Pipetting fresh samples at ambient temperature and performing cell isolation on the space station also eliminates the need for expeditious transportation logistics, and allows for more frequent sampling. Once cells are isolated, the samples can be viably frozen and return on any transfer vehicle at any time for further analysis.

On an aircraft that is used as a parabolic flight analog to create short periods of simulated microgravity, Twins Study Investigators Dr. Andrew Feinberg and Lindsay Rizzardi of Johns Hopkins Medicine tested a theory that liquids could be transferred safely in microgravity using a pipettor, which is a slender, graduated measurement tube. Previously researchers thought transferring biological fluids in space could pose risks to precisely controlling the sample.

"This analog demonstrated that pipetting of open fluids is relatively simple and easily controlled and that all fluid transfer steps associated with centrifugation can be replicated in microgravity," Feinberg said. "When dealing with genetic material, research requires precise transfer of liquids among different types of tubes in order to purify DNA, RNA or protein from biological samples to perform molecular analyses."

Coinciding with the fluid transfer research was cell isolation research being conducted by NASA immunologists Brian Crucian, Clarence Sams, Hawley Kunz and NASA astronaut and molecular biologist Kate Rubins. NASA researchers tested terrestrial protocols for cell purification in microgravity using the parabolic flight analog. They found that cell isolation and purification could both be performed in microgravity. Rubins also confirmed some of these findings in space. They published their research with Feinberg and Rizzardi in the July 2016 issue of NPJ Microgravity.

Crucian said, "Laboratory procedures for isolating and purifying cells typically require sensitive gradient centrifugation, careful extraction of isolated cells, and general open pipetting of liquids for washing and transferring the isolated cells."

Being able to transfer fluids and isolate cells in space is significant for a variety of reasons. Mars is a challenging distance from Earth if diagnostics are ever needed of a crewmember. Enabling astronauts to conduct more human research independently could help diagnose an illness more quickly, possibly saving a life in a medical emergency.

As NASA prepares for its journey to Mars, the way researchers handle and processes biological samples in space could change. The protocols validated by the Johns Hopkins and NASA investigators demonstrate that standard cell isolation protocols may indeed be performed in space, something which may enable certain types of genetic, or 'omics', research onboard the space station. Molecular biology technologies such as hand-held sequencers continue to evolve pushing the boundaries of scientific research. HRP will continue to adapt its methodologies to support novel research that protects and ensures the safety of future crews on long-duration missions while opening the door for innovative opportunities.

Explore further: Study examines effects of spaceflight on immune system

More information: Lindsay F Rizzardi et al, Evaluation of techniques for performing cellular isolation and preservation during microgravity conditions, npj Microgravity (2016). DOI: 10.1038/npjmgrav.2016.25

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Squeezing innovation out of the NASA Twins study: Pipetting and cell isolation in space - Phys.Org

Our article is written for a bioscience audience and offers guidance on best practice in chemical probe selection, evaluation and use.

I have previously postedabout how small molecule chemical probes of high quality are crucial for investigating the function of proteins in cells and organisms and also for validating them (or not) as drug targets. This applies to all areas of biomedical research see articles by Stephen Fryeand Mark Bunnage and colleagues.

My colleague Julian Blagg and I have today published a Perspective on choosing and using chemical probes in the journal Cancer Cellthat is written specifically with the audience of biologists in mind.

There are numerous examples of how the use of fit-for-purpose chemical probeshas led to important discoveries in biomedical research. The value of chemical probes is particularly well demonstrated in the field of cancer research.

A good case in point is the relatively recent rapid growth in our understanding of the biology and pharmacology of bromodomains that was triggered by the discovery of potent chemical probes such as JQ1and I-BETand their closely matched inactive partner compounds used as controls.

Yet in my earlier blogI also drew attention to how loose standards in the selection and use of chemical probes are leading to serious errors in biomedical research studies.

To be effective as chemical probes, small molecule agents need to be cell permeable and to bind potently (i.e. strongly at low concentrations) to the desired protein target and modulate its function in the cell as measured by direct target interaction and appropriate downstream biomarker changes. And they also need to bind selectively, meaning that they dont interact with and modulate other cellular targets or more realistically that they only affect an acceptable number of additional relevant proteins.

However, use of poorly selective, or otherwise unsuitably flawed even frankly dreadful chemical compounds is widespread. This is sloppy science and contributes to what is often referred as a crisis in the reproducibility and robustness of biological findings.

Moreover, recent calculations have suggested that spending $150 on a poor quality, out-of-date chemical compound from a vendor catalogue, instead of buying a high quality chemical probe, can cost the scientific community billions of dollars. There are at least200 historic compoundsthat are often used and should be replaced with better probes.

So misuse of chemical probes is wasting scientists time and money and in many instances is undoubtedly leading to delays in the discovery of much needed medicines.

I explained in my previous posthow, in a Commentary article in the journal Nature Chemical Biology by Arrowsmith et alpublished in August 2015, an international panel of chemical biology scientists (of which I was a member) had issued a call-to-arms aimed at eliminating the use of substandard research tools in biomedical research and promoting best practice. That call was linked with the launch of a new community-based, 'TripAdvisor-style' online resource available at the Chemical Probes Portal.

The non-profit Portal works by offering online expert annotation and recommendations for use of chemical probes for particular molecular targets. These are provided by a Scientific Advisory Board (for full disclosure I am a member of this and a Board Director) with about 400 probes assessed to date.

Theres no doubt that great progress has been made in discovering high quality tools for cancer biology and target validation. Unfortunately two years on now from the publication of the Arrowsmith et al paper and the initial launch of the Chemical Probes Portal it is abundantly obvious that bad practice in the selection and use of chemical probes is still very widespread in biomedical research, including numerous, continuing high profile examples in cancer.

Its clear that biologists commonly choose chemical probes based on querying search engines such as Google which will lead them to vendor catalogues that provide variable levels of information, do not prioritize probes based on quality, and sometimesrecommend the same compound as a probe for different protein targets.

Alternatively, use of search engines like Google Scholar will return as top hits publications that are the most highly cited, but that also describe the oldest chemical probes. Such searches are less likely to find the best, usually more recent tools. For example, when Chemical Probes Portal staff looked at 10 compounds, randomly selected from the 200 no longer recommended historical probes listed on the Portal website, they found that since 2016 these past-their-sell-by date reagents have been used in 2,090 publications.

A specific illustration is the still very frequent use of one of the above historical compounds, LY294002 an initially valuable early inhibitor of the phosphoinositide 3-kinase lipid kinases (PI3 kinase) that was originally described in 1994. Although a usefulpathfinder probe,LY294002 exhibits only weak, micromolar potency for PI3 kinases and through chemoproteomic studies it was subsequently found to be active against numerous members of the PI3 kinase family, and also other unrelated proteins including bromodomains.

LY249002 has been cited in over 30,000 publications; moreover despite its poor potency and selectivity and its supersedence by severalsuperior compounds as chemical probes for PI3K, a recent search for LY294002 on Google Scholar returned 1,190 publications for the year 2016 alone and this now outdated and flawed probecontinues to be sold by many commercial vendors.

Its clear then that we need to find a way make things change and especially influence behaviour of biological research community which is the main user group for chemical probes. This is why Julian Blagg and I have written our Perspectivein way that we hope will get the message out to biologists.

As we say in the Abstract of the Perspective:

Small-molecule chemical probes or tools have become progressively more important in recent years as valuable reagents to investigate fundamental biological mechanisms and processes causing disease, including cancer. Chemical probes have also achieved greater prominence alongside complementary biological reagents for target validation in drug discovery. However, there is evidence of widespread continuing misuse and promulgation of poor-quality and insufficiently selective chemical probes, perpetuating a worrisome and misleading pollution of the scientific literature. We discuss current challenges with the selection and use of chemical probes, and suggest how biologists can and should be more discriminating in the probes they employ.

Despite the efforts so far within the chemical biology community, we point out that we have been guilty of: largely preaching to the choir [meaning chemical biology specialists] and failing to connect to a really critical audience: namely, the wider cancer biology community who rely upon small-molecule tool compounds, often in harness with biological reagents, to interrogate cancer cell biology and who frequently draw important and highly impactful biological interpretations, whether correct or misleading, from such studies.

A topical example that we highlight in our Perspectiveis the initially erroneous discovery and validation of the proposed target MTH1 in cancer. MTH1 has a role in breaking down damaged metabolites called nucleotides in cells and thus preventing them from being incorporated into DNA and was first published as a cancer target in very high profile publications in the journal Nature.

Small molecule agents that were originally used to validate MTH1 include compounds TH287 and TH588 as well as S-crizotinib. We discuss in our Perspectivethe elegant publicationfrom AstraZeneca scientists that identifies three different chemical series of potent and highly selective chemical probes that clearly inhibit MTH1 in cancer cells but despite this have no therapeutic effect on cancer cells.

Furthermore, the same article shows that neither small interfering RNA (siRNA) reagents that deplete MTH1 nor CRISPR-mediated removal of MTH1 had any beneficial effect of cancer cells, pointing to off-target activity with the original siRNA reagent as well the chemical compounds used.

Furthermore, the blogger Derek Lowehas just a couple of days ago updated this story by discussing a new publication from researchers at Bayerwho discovered BAY-707, yet another highly potent and selective inhibitor of MTH1, and found it to have no therapeutic effect on cancers cells. Hence at this stage the balance of opinion strongly indicates that MTH1 is not a valid target for cancer treatment.

One piece of evidence in the AstraZeneca study that was particularly critical in invalidating the initial chemical probes was the demonstration that both TH287 and S-crizotinib killed cancer cell lines lacking MTH1 and subsequent protein screening workshowed that the binding of TH287 and TH588 to tubulin is responsible for their cytotoxic effects.

We provide in the Perspectiveseveral other examples of how the close integration of orthogonal chemical and biological tools can be very powerful, as in the case of studies on SWI/SNF chromatin remodelling complex components, the transcription factor HIF2 and the Jumonji family of histone lysine demethylases. In addition, we describe cautionary tales of the problems arising with uncritical use of claimed chemical probes for proteins including poly ADP ribose polymerases (PARPs; for which a flawed PARP compound progressed to the clinic and failed); the molecular chaperone HSP70; KRAS-regulated autophagy; and pan-steroid receptor co-activators.

We discusshow a gold-standard test to validate the functional on-target response to a chemical probe is to demonstrate reversal of the cellular effects of a proposed small molecule probe compound by mutation in the protein target that abrogates compound binding.

Another useful technique is to engineer the target to interact with chemical probes not recognized by the natural (so-called) wild-type protein. An additional approach now becoming common is to determine the effects of the chemical probe in cells where the proposed protein target has been removed by CRISPR technology.

We point outthat although Small molecules are from Mars, biological tools are from Venus, they are nevertheless part of the same overall universe, providing orthogonal and complimentary approaches to understanding biology and target validation a very powerful, multidisciplinary and essential toolkit for modern biomedical research.

Also in our Perspectivewe highlight and explain an important aspect of target binding selectivity that is rarely articulated in discussion of chemical probes that it is absolutely to be expected that most small molecules will generally interact with multiple biological targets in cells and organisms. By contrast, biological reagents, for example siRNA oligonucleotides and antibodies, are intrinsically more likely than small molecules to bind selectively to the desired biological target as a result of the greater breadth, complexity and thus specificity of their combined intermolecular interactions.

Of course there are also major problems with the use of insufficiently selective biological reagents and greater rigour in their use is important too as elegantly discussed recently by Bill Kaelin but biologists need to be even more critical in their use of small molecule probes because their smaller size and lower complexity means that at least some degree promiscuity is likely to be the rule rather than the exception. This tendency can be mitigated by careful design and optimization of the probe but even then rigorous and broad experimental testing for selectivity is essential.

Indeed, we strongly advisethe maxim of caveat emptor let the buyer beware! when choosing and using chemical probes for biological exploration and target validation.

In discussing the challenge of selectivity, we illustrate how the off-target effects can range from an interaction with one or two proteins perhaps but necessarily related to the target of interest through binding to tens of other targets, all the way to the extreme end of unacceptability where compounds are frequent hitters or chemical impostersthat have totally unacceptable features like indiscriminate chemical reactivity, aqueous insolubility and self-aggregation that make them worthless for biological research.

Hard to believe, but there are even isolated examples of vendors supplying the incorrect chemical compound and routine checking for evidence of authenticity is advisable. Related to this, in our Perspectivewe call for further efforts in the community to eliminate the especially egregious behaviour of publishing biological results without disclosing compound structures which of course means that the suitability of a probe cannot be assessed, nor can the claims be independently checked. Reviewers of submitted papers and grant applications as well as journal editors should be especially vigilant about this.

We recognize that for many if not most biologists these considerations of the selectivity of chemical probes are not part of their training or expertise. They may not have ready access to advice from chemical biology or medicinal chemistry colleagues. And they may find articles in the specialist chemical biology literature off-putting and full of jargon as most scientific disciplines are.

So in our Perspective we provide what we hope will be useful tools for biologists using chemical probes. Firstly, we include as Box 1a Glossary so that that any specialist terminology that cannot really be avoided is not too much of a turn-off.

We provide in Box 2a comparison of the desired selectivity profiles of chemical probes with those of approved drugs making the point that in comparison to drugs, chemical probes generally need to be even more selective than drugs so that probes can be used with confidence to modulate the intended target of interest.

In Box 3we summarize the factors that determine the fitness and quality of chemical probes and in Figure 2we present an overview of Dos and Donts for their selection and use. In particular, we strongly recommend taking a routinely sceptical approach, including the use of orthogonal chemical and biological reagents; the use of at least two different chemical series (chemotypes) of probe along with inactive control compounds; demonstration of potency and selectivity; and obtaining evidence for selective target engagement and modulation in cells (e.g. using thePharmacological Audit Trail).

We advise (on page 13 of the Perspective) strongly against a common and dangerous practice, which is to expose cells with ever increasing concentrations of a chemical probe until a desired cell effect (phenotype), usually cell death, is seen and then attributing this phenotype to the specific effect of the probe on the protein target under investigation.

Higher probe concentrations increase the likelihood of off-target effects and the general range that should not be exceeded is 10-20 micromolar to minimize non-specific effects. Accompanying biomarker evidence of target modulation is also important.

Alongside the general guidance provided in our Cancer Cell Perspective, we strongly recommend the use of the Chemical Probes Portalfor expert advice and ratings for specific probes and targets.

We likenthe provision of advice on the selection and use of chemical probes to ensuring the biological researcher avoids being equipped with the equivalent of a defective global positioning/satellite navigation system, as illustrated in the cartoon below:

Download a larger version of Professor Julian Blagg's cartoon (PDF, 51KB)

Caption: The right way and wrong way with chemical probes

We finish the Perspectivewith the following strong new call-to-action:

We need to maximize the promise and minimize the peril of chemical probes and this requires the broad research community to use high-quality chemical probes that have been critiqued with equivalent rigor to biological reagents. It is time to put our house in order and biologists as well as chemists have an important responsibility to do so.

Im grateful to my colleague and joint senior co-author of our Cancer Cell PerspectiveProfessor Julian Blaggfor his excellent collaboration and insights. We developed the content of the Perspective very much in partnership. I also thank Julian for drafting the cartoon illustration.

In addition, I thank many colleagues and collaborators for helpful discussions and input, including the anonymous reviewers of the Perspective, and those in the field whose outstanding work we have built upon.

Excerpt from:
Call to bioscientists: choose and use your chemicaI probes very carefully - The Institute of Cancer Research (blog)

July 10, 2017 by Jade Boyd Researchers from Rice University and Baylor College of Medicine have shown they initiate a process called tubulogenesis that is crucial to the formation of blood-transporting capillaries. In microscopic images taken a different times during a weeklong experiment, researchers tracked the changes in cells (green) and cell nuclei (orange) using fluorescent markers. Credit: Jeff Fitlow/Rice University

In their work toward 3-D printing transplantable tissues and organs, bioengineers and scientists from Rice University and Baylor College of Medicine have demonstrated a key step on the path to generate implantable tissues with functioning capillaries.

In a paper published online in the journal Biomaterials Science, a team from the laboratories of Rice bioengineer Jordan Miller and Baylor College of Medicine biophysicist Mary Dickinson showed how to use a combination of human endothelial cells and mesenchymal stem cells to initiate a process called tubulogenesis that is crucial to the formation of blood-transporting capillaries.

The work is an important step with fragile endothelial cells (ECs) made from "induced pluripotent stem cells," or iPSCs, a type of cell that can potentially be made from the cells of any human patient. Because iPSCs can be patient-specific, researchers hope to find ways of using them to generate tissues and replacement organs that can be transplanted without risk of rejection by a patient's immune system. But the fragility of endothelial cells during laboratory growth has limited the utilization of this critical cell type, which is found in all vasculature.

"Our work has important therapeutic implications because we demonstrate utilization of human cells and the ability to live-monitor their tubulogenesis potential as they form primitive vessel networks," said study lead author Gisele Calderon, a graduate student in Miller's Physiologic Systems Engineering and Advanced Materials Laboratory.

"We've confirmed that these cells have the capacity to form capillary-like structures, both in a natural material called fibrin and in a semisynthetic material called gelatin methacrylate, or GelMA," Calderon said. "The GelMA finding is particularly interesting because it is something we can readily 3-D print for future tissue-engineering applications."

Tissue engineering, also known as regenerative medicine, is a field aimed at integrating advances in stem cell biology and materials science to grow transplantable replacement tissues and organs. While tissue engineers have found dozens of ways to coax stems cells into forming specific kinds of cells and tissues, they still cannot grow tissues with vasculaturecapillaries and the larger blood vessels that can supply the tissues with life-giving blood. Without vascularization, tissues more than a few millimeters in thickness will die due to lack of nutrients, so finding a way to grow tissues with blood vessels is one of the most sought-after advances in the field.

Miller, who earned his Ph.D. at Rice in 2008, has studied vascularization in tissue engineering for more than 14 years. During his postdoctoral studies at the University of Pennsylvania, he also became heavily involved in the open-source 3-D printing movement, and his work at Rice combines both.

"Ultimately, we'd like to 3-D print with living cells, a process known as 3-D bioprinting, to create fully vascularized tissues for therapeutic applications," said Miller, assistant professor of bioengineering. "To get there, we have to better understand the mechanical and physiological aspects of new blood-vessel formation and the ways that bioprinting impacts those processes. We are using 3-D bioprinting to build tissues with large vessels that we can connect to pumps, and are integrating that strategy with these iPS-ECs to help us form the smallest capillaries to better nourish the new tissue."

Each of the trillions of living cells in the human body are constantly supplied with oxygen and nutrients by tiny blood vessels known as capillaries. Measuring just a few thousandths of a millimeter in diameter, some capillaries are so narrow that individual blood cells must squeeze through them in single-file. Capillaries are made entirely from networks of endothelial cells, the type of cell that lines the inner surface of every blood vessel in the human body.

In the process of tubulogenesisthe first step to making capillariesendothelial cells undergo a series of changes. First, they form small, empty chambers called vacuoles, and then they connect with neighboring cells, linking the vacuoles together to form endothelial-lined tubes that can eventually become capillaries.

"We expect our findings will benefit biological studies of vasculogenesis and will have applications in tissue engineering to prevascularize tissue constructs that are fabricated with advanced photo-patterning and three-dimensional printing," said Dickinson, the Kyle and Josephine Morrow Chair in Molecular Physiology and Biophysics at Baylor College of Medicine and adjunct professor of bioengineering at Rice.

In the study, Calderon, Rice undergraduate Patricia Thai and colleagues investigated whether commercially available endothelial cells grown from iPSCs had tubulogenic potential. The test examined this potential in two types of semisolid gelsfibrin and GelMA. Finally, the researchers also investigated whether a second type of stem cell, human mesenchymal stem cells, could improve the likelihood of tubulogenesis.

Calderon said fibrin was chosen for the experiment because it's a natural material that's known to induce tubulogenesis for wound healing. As such, the researchers expected endothelial cells would be induced to form tubules in fibrin.

Calderon said the first step in the experiments was to develop a third-generation lentivirus reporter to genetically modify the cells to produce two types of fluorescent protein, one located only in the nucleus and another throughout the cell. This permanent genetic modification allowed the team to noninvasively observe the cell morphology and also identify the action of each individual cell for later quantitative measurements. Next, the cells were mixed with fibrin and incubated for a week. Several times per day, Calderon and Thai used microscopes to photograph the growing samples. Thanks to the two fluorescent markers, time-lapse images revealed how the cells were progressing on their tubulogenic odyssey.

Calderon conducted advanced confocal microscopy at the Optical Imaging and Vital Microscopy Core facility at Baylor College of Medicine. Calderon and Thai then used an open-source software called FARSIGHT to quantitatively analyze the 3-D growth patterns and development character of the tubulogenenic networks in each sample. In fibrin, the team found robust tubule formation, as expected. They also found that endothelial cells had a more difficult time forming viable tubules in GelMA, a mix of denatured collagen that was chemically modified with methacrylates to allow rapid photopolymerization.

Over several months and dozens of experiments the team developed a workflow to produce robust tubulogenesis in GelMA, Calderon said. This involved adding mesenchymal stem cells, another type of adult human stem cell that had previously been shown to stabilize the formation of tubules.

Miller said that while clinical applications of 3-D bioprinting are expected to advance rapidly over the next few decades, even small tissue samples with working capillary networks could find use much more quickly for laboratory applications like drug testing.

"You could foresee using these three-dimensional, printed tissues to provide a more accurate representation of how our bodies will respond to a drug," Miller said. "Preclinical human testing of new drugs today is done with flat two-dimensional human tissue cultures. But it is well-known that cells often behave differently in three-dimensional tissues than they do in two-dimensional cultures. There's hope that testing drugs in more realistic three-dimensional cultures will lower overall drug development costs. And the potential to build tissue constructs made from a particular patient represents the ultimate test bed for personalized medicine. We could screen dozens of potential drug cocktails on this type of generated tissue sample to identify candidates that will work best for that patient."

Explore further: Giving blood to artificial organs

More information: G. A. Calderon et al. Tubulogenesis of co-cultured human iPS-derived endothelial cells and human mesenchymal stem cells in fibrin and gelatin methacrylate gels, Biomater. Sci. (2017). DOI: 10.1039/C7BM00223H

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Houston team one step closer to growing capillaries - Phys.Org

July 10, 2017 Nanonet Force Microscropy (NFM) can measure the contractile inside-out forces of a single cell attached to multiple fibers. Shown here are f-actin (red), paxillin (green), and the nucleus (blue). Scale bar = 20 micron. Credit: Abinash Padhi, STEP Lab, Virginia Tech

Researchers from Virginia Tech and the University of Pittsburgh have collaborated to employ a novel nanoscale fibrous system that can measure the tiny forces exerted by and upon individual cells with extreme precision. The team hopes that this platform, which investigators call nanonet force microscopy (NFM), will provide new knowledge about smooth muscle cell biology that could have implications for treating cardiovascular disease, which is still a leading cause of death in the United States.

The results of investigations on cells using this platform appear in the "Forces" issue of the journal Molecular Biology of the Cell, in the article "Nanonet Force Microscopy for Measuring Forces in Single Smooth Muscle Cells of Human Aorta," published July 7, 2017.

The main goal of this current study, said Julie Phillippi, assistant professor at the University of Pittsburgh Department of Cardiothoracic Surgery whose laboratory provided healthy human patient smooth muscle cells for the study, was to quantify forces that healthy cells experience in various conditions of stress. The fibrous nanonet itself was designed in the mechanical engineering laboratory of Amrinder Nain, associate professor at Virginia Tech and member of the American Society for Cell Biology. Forces measured using NFM, Nain said, include forces exerted by the cells themselves and forces exerted by the environment on the cells. "Everything in nature exerts and experiences a physical force," said Nain. "This platform measures both simultaneously."

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Phillippi said that previous work tested the mechanical strength of whole aortic tissue and understanding the single cell biomechanics is vitally important. Single-cell studies provide insight into the proteins involved in the fleeting so-called focal adhesions that most cells make as they move around their microenvironment. The NFM assembly aims to mimic, in as physiologically relevant a way as possible, what cells endure within the collagen fibers of the extracellular matrix (ECM)the matrix that supports cell growth in living things. Tweaking the artificial matrix by changing fiber diameter, density, and spacing in a controlled and repeatable manner, as well as using cells from diseased patients at different disease severities, will allow Phillippi and Nain to simulate the conditions experienced by cells in many realistic situations.

"We have looked very closely at how the collagen and elastin fibers in the ECM are arranged and the micro-architecture and everything points to these microstructural defects in the ECM contributing to the weakening of the aortic walls and the ballooning of the vessel," said Phillippi. "What we don't know is, are these ECM proteins arranged that way from birth or is it something that happens over time? Or is it both? What role do the cells play? This engineered platform will allow us to answer some of those questions." Furthermore, Nain said, NFM could reveal the heterogeneity of cells taken from the same patient or from different patients with the same disease state down to the single-cell resolution.

Next steps for Phillippi and Nain include testing cells from the Pittsburgh team's large repository of aortic specimens from patients, collected in collaboration with Thomas Gleason, Chief of the Division of Cardiac Surgery, University of Pittsburgh, to establish a database of baseline forces for many types of cells that researchers and clinicians can use to diagnose and treat disease. "The platform gives us the ability to create in vitro disease models with multiple layers of sophistication," said Phillippi.

In a broader context, the ability to achieve precise control on fiber diameter, spacing, and orientation to mimic native fibrous environments, will allow NFM to interrogate the push and pulls in a cell's journey in developmental, disease, and repair biology.

Explore further: Cancer cells 'talk' to their environment, and it talks back

More information: Alexander Hall et al. Nanonet force microscopy for measuring forces in single smooth muscle cells of the human aorta, Molecular Biology of the Cell (2017). DOI: 10.1091/mbc.E17-01-0053

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Bonobos have a reputation for being the peaceful, free-loving hippies of the primate world. But, researchers reporting in Current Biology on July 10 have discovered that despite friendly relations between the sexes, particular ...

To keep blood and oxygen flowing throughout their bodies, most animals depend on a beating heart. But researchers reporting in Current Biology on July 10 have discovered that sea spiders use a strange alternative: they move ...

When Jeremy Goldbogen, an assistant professor of biology at Stanford University, affixed recording devices to humpback whales, it was with the hope of learning more about how the animals move in their natural environment ...

When does a (typically) vegetarian caterpillar become a cannibalistic caterpillar, even when there is still plenty of plant left to eat?

Why did life on Earth change from small to large when it did? Researchers from the University of Cambridge and the Tokyo Institute of Technology have determined how some of the first large organisms, known as rangeomorphs, ...

Birds such as parrots and crows have been using touchscreen technology as part of an international research study examining whether the ways in which animals respond to new things influences how eager they are to explore.

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Nanoscale forces measured in aortic smooth muscle cells tell story ... - Phys.Org