Stem Cell Assay Market: Snapshot

Stem cell assay refers to the procedure of measuring the potency of antineoplastic drugs, on the basis of their capability of retarding the growth of human tumor cells. The assay consists of qualitative or quantitative analysis or testing of affected tissues and tumors, wherein their toxicity, impurity, and other aspects are studied.

With the growing number of successful stem cell therapy treatment cases, the global market for stem cell assays will gain substantial momentum. A number of research and development projects are lending a hand to the growth of the market. For instance, the University of Washingtons Institute for Stem Cell and Regenerative Medicine (ISCRM) has attempted to manipulate stem cells to heal eye, kidney, and heart injuries. A number of diseases such as Alzheimers, spinal cord injury, Parkinsons, diabetes, stroke, retinal disease, cancer, rheumatoid arthritis, and neurological diseases can be successfully treated via stem cell therapy. Therefore, stem cell assays will exhibit growing demand.

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Another key development in the stem cell assay market is the development of innovative stem cell therapies. In April 2017, for instance, the first participant in an innovative clinical trial at the University of Wisconsin School of Medicine and Public Health was successfully treated with stem cell therapy. CardiAMP, the investigational therapy, has been designed to direct a large dose of the patients own bone-marrow cells to the point of cardiac injury, stimulating the natural healing response of the body.

Newer areas of application in medicine are being explored constantly. Consequently, stem cell assays are likely to play a key role in the formulation of treatments of a number of diseases.

Global Stem Cell Assay Market: Overview

The increasing investment in research and development of novel therapeutics owing to the rising incidence of chronic diseases has led to immense growth in the global stem cell assay market. In the next couple of years, the market is expected to spawn into a multi-billion dollar industry as healthcare sector and governments around the world increase their research spending.

The report analyzes the prevalent opportunities for the markets growth and those that companies should capitalize in the near future to strengthen their position in the market. It presents insights into the growth drivers and lists down the major restraints. Additionally, the report gauges the effect of Porters five forces on the overall stem cell assay market.

Global Stem Cell Assay Market: Key Market Segments

For the purpose of the study, the report segments the global stem cell assay market based on various parameters. For instance, in terms of assay type, the market can be segmented into isolation and purification, viability, cell identification, differentiation, proliferation, apoptosis, and function. By kit, the market can be bifurcated into human embryonic stem cell kits and adult stem cell kits. Based on instruments, flow cytometer, cell imaging systems, automated cell counter, and micro electrode arrays could be the key market segments.

In terms of application, the market can be segmented into drug discovery and development, clinical research, and regenerative medicine and therapy. The growth witnessed across the aforementioned application segments will be influenced by the increasing incidence of chronic ailments which will translate into the rising demand for regenerative medicines. Finally, based on end users, research institutes and industry research constitute the key market segments.

The report includes a detailed assessment of the various factors influencing the markets expansion across its key segments. The ones holding the most lucrative prospects are analyzed, and the factors restraining its trajectory across key segments are also discussed at length.

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Global Stem Cell Assay Market: Regional Analysis

Regionally, the market is expected to witness heightened demand in the developed countries across Europe and North America. The increasing incidence of chronic ailments and the subsequently expanding patient population are the chief drivers of the stem cell assay market in North America. Besides this, the market is also expected to witness lucrative opportunities in Asia Pacific and Rest of the World.

Global Stem Cell Assay Market: Vendor Landscape

A major inclusion in the report is the detailed assessment of the markets vendor landscape. For the purpose of the study the report therefore profiles some of the leading players having influence on the overall market dynamics. It also conducts SWOT analysis to study the strengths and weaknesses of the companies profiled and identify threats and opportunities that these enterprises are forecast to witness over the course of the reports forecast period.

Some of the most prominent enterprises operating in the global stem cell assay market are Bio-Rad Laboratories, Inc (U.S.), Thermo Fisher Scientific Inc. (U.S.), GE Healthcare (U.K.), Hemogenix Inc. (U.S.), Promega Corporation (U.S.), Bio-Techne Corporation (U.S.), Merck KGaA (Germany), STEMCELL Technologies Inc. (CA), Cell Biolabs, Inc. (U.S.), and Cellular Dynamics International, Inc. (U.S.).

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Stem Cell Assay Market Competitive Analysis and Forecast 2017-2025 - Monroe Scoop

Cardiac Stem Cells Comments Off on Stem Cell Assay Market Competitive Analysis and Forecast 2017-2025 – Monroe Scoop | March 9th, 2020

This story originally appeared onStockhead.

As with the early medical cannabis plays, a cluster of ASX-listed stocks has wasted little time attaching itself to the c word. Were talking of course about the coronavirus COVID-19 but sadly not another c word: cure.

Or not yet.

According to broker Morgans daily tally, the virulent bug has so far infected 95,332 people, with 38,564 current cases (6,883 of them critical).

Of the remaining 56,768 cases with an outcome, 53,483 recovered and 6,883 achieved a definitive performance indicator. They died.

Okay, a circa 7 per cent mortality rate or even a 1 or 2 per cent rate is nothing to sneeze at, so to speak. But we do wish breathless TV reporters would cease referring to it as the deadly virus, but that would be like asking them to stop referring to a horror smash rather than a sad everyday road accident.

While were on it, we also implore folk to stop hoarding toilet paper: after all, its the coronavirus, not the Caroma-virus.

Named after its crown-like shape but not the Royal Family per se, the common coronavirus is responsible for past pestilences including Severe Acute Respiratory Syndrome (SARS) and Middle Eastern Respiratory Syndrome (MERS).

The virus may indeed fizzle out, as the earlier SARS plague did.

But for the time being, we need the best and brightest minds in the labs to come up with a treatment or more likely a vaccine.

There are some promising developments overseas, which your columnist will return to if he hasnt succumbed as well (he did shake hands with someone who went to a Chinese restaurant a couple of weeks back).

Among the local biotechs and we use the term loosely theres been no lack of endeavour in linking their efforts to the virus.

But to be fair, in some cases investors did it for them.

Take Biotron (ASX:BIT), which was an obvious subject of attention given the company is focused on developing antiviral drugs for HIV and hepatitis.

Biotron also has a program for pan respiratory viruses and mentioned corona in a June 2019 presentation. Some punters latched on to the fact that it wasnt referring to a 1970s Toyota or Mexican beer and the Hot Copper pundits were off and running.

Biotron CEO Dr Michelle Miller has been more circumspect.

Yes, she says, the company has some good advanced compounds to work on, but the reality is that theres nothing that would be ready to fight the current outbreak.

Dr Miller says while the companys work on pan respiratory viruses continues, theres not much to add at this stage.

Uscom (ASX:UCM) shares went on a run after the company reported increased orders for its haemodynamic monitoring devices in China.

Uscom stands for Ultra-Sonic Cardiac Output Monitors.

The Uscom 1A device is a non-invasive diagnostic that monitors cardiovascular functions, using Doppler ultrasound to detect abnormalities.

Chinese health authorities have recommended Uscom 1A as a monitoring device for severe coronavirus cases, while international guidelines also suggest using the device for paediatric sepsis.

Uscom reported that in the first five weeks of 2019, Chinese sales orders rose 124 per cent, from 17 units to 38 units.

Uscom chief Professor Rob Phillips says the company is well positioned with the virus, but notes that Uscom is not a coronavirus story as such: fatalities from cardiovascular pulmonary failure result from conditions such as pneumonia.

Happily for Uscom, the outbreak comes as the company hones-in on the Chinese market with a new direct sales model.

The molecular diagnostics house has a suite of approved tests that cover gastro-enteric strains, flavivirus/alphavirus, sexually-transmitted diseases and drum roll respiratory pathogens.

Genetic Signatures (ASX:GSS) Easyscreen tests cover pan coronaviruses, which until now has not been able to distinguish COVID-19 from, say, SARS.

But thats all changed, with the company introducing a supplementary test that does just that. Management is fast-tracking a validation program to obtain the data required for international regulatory approvals as rapidly as possible.

However, Genetic Signatures cant be accused of beating up its prospects: management says while the bug presents significant opportunities, the outcome of the emerging pandemic is uncertain.

While the early-stage coronavirus is detected by a blood test, chest x-rays are then used to gauge the severity of the illness and assess fluid in the lungs.

Micro-X (ASX:MX1) is all about developing lightweight and portable x-ray machines for medical applications, as well as other purposes such as defence and airports.

The companys first product, Carestream DRX Revolution Nano is approved in the US and Europe.

In mid-February the company said it had procured orders for $780,000 of machines from governments of two Asian countries, in response to the coronavirus threat. This week, another $1m of orders, all marked for urgent delivery, flooded in.

While these are terrible circumstances with the coronavirus spreading so quickly, we are pleased that our equipment will soon be able to assist medical teams with their responses in affected countries, Micro-X CEO Peter Rowland says.

Why waste a crisis? No fewer than four ASX stocks are capitalising on demand for hand and surface sanitisers to halt the bug in the first place.

Antimicrobial solutions house Zoono Group (ASX:ZNO) proclaims that its impressively-monikered Z-71 Microbe Shield, as used in its hand sanitisers, kills COVID-19 99.99 percent of the time.

Zoono is selling into China via a tie up with Eagle Health (ASX:EHH), which manufactures and distributes product into 26 provinces.

Aeris Environmental (ASX:AEI) goes one step better, claiming its Aeris Active product kills influenza and noroviruses in 99.999 percent of cases.

For those remaining 0.001 percent, bad luck and dont buy a lottery ticket.

Interestingly, that announcement did not refer specifically to the coronavirus. But earlier, Aeris announced the Singapore National Environment Agency had listed Aeris Active as one of the general disinfectants effective against the virus.

Meanwhile, fruit juice maker Food Revolution Group (ASX:FOD) has turned from filling its bottles with squeezed oranges to stuffing them with alcohol-based hand sanitiser under the Sanicare brand.

Who would have thought? The swift repositioning results from a 1,260sqm upgrade at the companys plant at Mill Park in outer Melbourne, which enables all sorts of gels, powders, oils and cosmetics to be bottled.

Mainstream sanitiser products such as Dettol and Lysol (made by multinational Reckitt and Benckiser) are flying off the shelves.

But is a good scrub with soap and water just as effective? Australian National University microbiologist Professor Peter Collignon opines theres little difference between hand washing and the alcohol-based sanitisers.

One is just more convenient than the other and contains alcohol, he says. You can put it in your pocket and dont have to be near a sink or basin to use it.

So whos actually tackling the disease? Offshore, theres a conga line of developers having a crack at a vaccine.

In Israel, scientists at the Galilee Research Institute claim to be on the cusp of finalising a product that is capable of getting regulatory assent within 90 days.

Thats what you call fast-track approval.

According to the Jerusalem Post, the same team of scientists has been developing a prophylactic against infectious bronchitis virus, which affects poultry.

The effectiveness of the vaccine has been proven in pre-clinical trials carried out at the countrys Veterinary Institute.

In the US, Gilead Sciences plans to recruit 1,000 patients with coronavirus for a clinical trial to test its experimental anti-viral drug remdesivir (as used to tackle Ebola virus).

With the backing of the World Health Organisation, the drug is also being trialed in China.

Maryland-based, Nasdaq-listed Novavax says it is cloning the coronavirus to develop a vaccine, in the same way it developed one for MERS in 2013.

Novavax is looking at several vaccine candidates for animals and hopes to find one for human testing by the end of May.

Our previous experience working with other coronaviruses, including both MERS and SARS, allowed us to mobilise quickly, Novavax CEO Stanley Eck said.

Fellow Nasdaq minnow Moderna has shipped an experimental vaccine to the National Institute of Allergy and Infectious Diseases for testing.

Backed by billionaire hedge fund founder Jim Simons, Long Island-based private outfit Codagenix expects to have a vaccine ready for animal testing in four to six weeks, with one suitable for testing about six weeks later.

The Codagenix know-how is based on recoding the genomes of viruses to render them harmless. The technique is not exactly unknown, as its been used to eradicate polio and small pox.

And who can forget Australias very own Relenza anti-influenza Biota, which became Alpharetta Georgias Nabi, changed its name to Aviragen and then was subsumed as a sub-division of San Franciscos Vaxart, popping its head above the parapet to also claim an anti-viral program for COVID-19.

The South China Morning Post reports that a 65-year-old woman on her COVID-19 deathbed walked out of Chinas Kunming Hospital after being given a stiff shot of mesenchymal stem cells (MSCs).

Two trials are also underway to test the therapy against pneumonia, at a Beijing Military Hospital and Zhongnan Hospital of Wuhan University (yep, in the coronavirus capital).

Could the excitement rub-off on our ASX-listed plays Mesoblast (ASX:MSB), Cynata Therapeutics (ASX:CYP), Orthocell (ASX:OCC) and Regeneus (ASX:RGS)?

Cynatas Dr Ross Macdonald says the reports look authentic; and he believes that MSCs could be an effective adjunct in managing patients with serious issues pertaining to COVID-19.

This is not because MSCs are inherently anti-viral or can act as a vaccine, but more because they have shown benefit in major pathologies associated with infection, he says.

Cynata, we stress, has not mentioned coronavirus in its dispatches and nor has any of the other non-China MSC plays or not yet anyway.

But still, what decent CEO would not give his company a plug?

The clear advantage of (Cynatas) Cymerus technology (is) the ability to make large quantities of consistent, robust MSCs without having to find gazillions of donors, Dr Macdonald says.

Your columnist stresses that the coronavirus influence on the sector is not all positive, with some biotechs likely to be affected by supply or other disruptions.

In mid-February, Cochlear (ASX:COH) quickly stepped off the mark by announcing its earnings for the 2019-20 year were likely to come in at $270-290m, compared with the previously guided $290-300m.

The reason is that hospitals in China and Hong Kong have delayed cochlear implant procedures to avoid the risk of infection.

The aforementioned Uscom notes that with labs preoccupied with the virus, short-term revenues are less predictable. In other words, the coronavirus is a distraction as well as an opportunity.

IDT Australias (ASX:IDT) Dr David Sparling told Biotech Daily that his company had no direct supply chain exposure to China at all, and was doubtful that even the companys gowns and protective gear had much to do with the Middle Kingdom.

Editors note: Dr. Tim Boreham, who wrote this article for Stockhead, is one of Australias best-known small cap analysts and business journalists.

If you throw enough money and resources at tackling a disease you will get a result, right?

Er, not quite: cures for well-researched ailments such as Alzheimers disease, multiple sclerosis and an array of cancers remain elusive.

But when youve got an ailment that is crippling the global economy, the imperative to find a solution is somewhat more intensive.

Our best guess is that like SARS and MERS, COVID-19 will hang around for years to come, but the ill-effects will be made more tolerable with an effective vaccine and/or improved immunity over time.

In other words, it will become just another disease in the pantheon of maladies blighting humanity.

In the race for a cure, Gileads Remdesivir looks interesting, given it has been used before.

As for the opportunists in the sanitiser game, the surge in demand means tangible revenue gains and good on them.

But lets be clear: theyre hardly breaking new ground technology-wise and their gains will only be short term as other suppliers enter the market.

As for a cure, or lack of one, we suggest that investors hedge their bets with an exposure to the funeral stocks Invocare (ASX:IVC) and Propel Funeral Partners (ASX:PFP).

After all, theyre the last people to let you down.

Stockheadcovers emerging ASX companies and investment opportunities. Get daily stock updates atStockhead.

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The Aussie Biotech Companies Trying To Make A Buck From Coronavirus - D'Marge

Cardiac Stem Cells Comments Off on The Aussie Biotech Companies Trying To Make A Buck From Coronavirus – D’Marge | March 9th, 2020

Steven Hodi Jr., the i3 Centers other PI, and director of Melanoma Center and the Center for Immuno-Oncology at Dana-Farber, and professor of medicine at Harvard Medical School (HMS), is leading the clinical cancer vaccine trial. He has been at the forefront of developing cancer immunotherapies using immune checkpoint inhibitors, a class of drugs able to re-activate tumor-destroying T cells that are muted in the tumor microenvironment. The funding for this center provides a unique opportunity to unite key investigators for translating fundamental advancements in immunology and biomedical engineering into highly synergistic approaches to improve the treatments for cancer patients, said Hod

Using both in vivo and ex vivo biomaterials-based approaches, the i3 Center aims to boost tumor-specific activities of cytotoxic T cells, by boosting different stages of the normal process by which T cells develop, and acquire anti-cancer activity. T cells normal development starts in the bone marrow where hematopoietic stem cells generate T cell progenitor cells. These migrate to the thymus to differentiate into nave T cells, which then travel further to lymph nodes. There, they encounter cancer-derived antigens presented to them by specialized antigen-presenting cells (APCs) that can activate T cells to recognize and eliminate cancer cells.

In relation to adoptive T cell therapies in which T cells are given to patients to fight their cancers, one team at the i3 Center will be led by Dana-Farber researchers Catherine J. Wu and Jerome Ritz, who along with Mooney, will develop and test biomaterials that can better mimic normal APCs in activating and directing the function of patient-derived T cells outside the human body, prior to their transplantation. Wu is chief of the Division of Stem Cell Transplantation and Cellular Therapies, and Ritz is executive director of the Connell and OReilly Families Cell Manipulation Core Facility at Dana-Farber.

We need to make efforts to enhance the ability of theimmune systemto recognizetumor cells. One directionmylaboratoryis taking makes use of innovative biomaterialsto help us to efficiently expandpolyclonaltumor-specificfunctionally-effectiveT cellsex vivoin a way that can be readily translated to theclinical setting. In our studies, we are currently focusing on melanoma and acute myeloid leukemia, said Wu, whose research interests include understanding the basis of effective human anti-tumor responses, including the identification and targeting of the tumor-specific antigens.

A second project explores the use of DNA origami, biocompatible nanostructures composed of DNA, to create cancer vaccines. DNA origami could provide significant advantages in presenting tumor-specific antigens and immune-enhancing adjuvants to APCs because the concentrations, ratios, and geometries of all components can be modulated with nano-scale precision to determine configurations that are more effective than other vaccination strategies. The project will be run by Wyss Institute Core Faculty member William Shih, Derin Keskin, lead immunologist at Dana-Farbers Translational Immunogenomics Lab, and Mooney.

In a third project, David Scadden, professor at Harvards Department of Stem Cell and Regenerative Biology, will collaborate with Mooney to build on their previous work. They will engineer biomaterials that recreate key features of the normal hematopoietic stem cell niche in the bone marrow. Such implantable biomaterials could help rapidly amplify T cell progenitor cells, and enhance T cell-mediated anti-cancer immunity. Scadden also is the Gerald and Darlene Jordan Professor of Medicine at Harvard University, and co-director of the Harvard Stem Cell Institute.

The i3 Centers investigators anticipate that it will stimulate additional cross-disciplinary concepts and research, due to the culture of continuous interactions, sharing of findings, data and samples between all investigators, as well strong biostatistical expertise provided by Donna Neuberg, a senior biostatistician broadly involved with exploring immune-modulating cancer interventions at the Dana-Farber.

This new i3 Center for cancer immunotherapy innovation really embodies how the Wyss Institute with its unparalleled capabilities in bioengineering and serving as a site for multidisciplinary collaboration, and can liaise with clinicians and researchers at our collaborating institutions to confront major medical problems and bring about transformative change, said Wyss Founding Director Donald Ingber. He is also theJudah Folkman Professor of Vascular Biologyat HMS and the Vascular Biology Program at Boston Childrens Hospital, and Professor of Bioengineering at SEAS.

Excerpt from:
NIH-funded i3 Center formed to advance cancer immunotherapy - Harvard Gazette

Bone Marrow Stem Cells Comments Off on NIH-funded i3 Center formed to advance cancer immunotherapy – Harvard Gazette | March 8th, 2020

Dr. Meng-Lun Hsieh and Dr. Andrew Dean Shubin were married March 7 at the Warwick Melrose Hotel in Dallas. The Rev. Beth Dana, a Unitarian Universalist minister, officiated.

The bride, 33, who goes by Meng, is a fourth-year medical school student at Michigan State University, from which she also received a doctorate in biochemistry. She graduated from Williams College.

She is a daughter of Huey-Jen Liaw and Jyh-Cheng Hsieh of San Diego. The brides father is a research scientist at Sheatech, a biotech company in San Diego. Her mother, a stay-at-home parent, was a history teacher at Taibei High School, a private school in Taipei.

The groom, also 33, is a second-year general surgery resident at the University of Texas Southwestern Medical Center in Dallas. He graduated magna cum laude from the University of Washington, and received a doctorate in biomedical engineering from the University of Rochester, from which he also received a medical degree.

He is a son of Carol E. Shubin and Andy N. Shubin of Vancouver, Wash. His father retired as a photography teacher at Shahala Middle School in Vancouver. The grooms mother retired as a special-education teacher at Burton Elementary School in Vancouver.

The couple met a decade ago at the National Institutes of Health in Bethesda, Md., as post-baccalaureate research fellows, she focusing on a virus that infects bacteria, and he on skin stem cells. In April 2016, they met again at a conference for physician scientists in Chicago, and began dating long-distance.

Original post:
Meng Hsieh, Andrew Shubin - The New York Times

Skin Stem Cells Comments Off on Meng Hsieh, Andrew Shubin – The New York Times | March 8th, 2020

There are only two northern white rhinos left on the planet, and theyre both female. Unless scientists can make a dramatic breakthrough, the entire species will die with those two individuals.

In a nondescript building just north of San Diego, California, the fight to save the northern white rhino is coming down to the wire. However, the battleground here looks less like a scene from a wildlife documentary and more akin to something out of a science fiction novel.

At the San Diego Zoo Institute for Conservation Research, an army of scientists armed with liquid nitrogen, microscopes, and ultrasound machines is working around the clock to create an unprecedented first in the conservation world: they are looking to turn frozen rhino skin cells into baby rhinos.

Its not just the science that is groundbreaking, but also the team looking to save this species. Composed mostly of women, the lab is a rarity in a field traditionally dominated by men.

Find out more about Call to Earth and the extraordinary people working for a more sustainable future

The first step in this conservation effort began more than four and a half decades ago in 1975 when scientists established the institutes Frozen Zoo. In a small room measuring no more than 36 square meters the skin cells of more than 10,000 individuals across 1,100 species sit in giant steel tanks suspended in time, frozen in liquid nitrogen.

Among the collection are the skin samples of 12 northern white rhinos. These are vital to the groups efforts because there is such a small gene pool of living northern whites.

The population has been decimated by poachers, who target rhinos because of the belief in parts of Asia that their horns can cure various ailments. The two surviving females both live under guard at the Ol Pejeta Conservancy in Kenya. Even though embryos have been produced in an Italian lab using eggs extracted from the pair, any future descendants from this kind of embryo would carry the genes of those two females.

That may not be enough genetic diversity to maintain a stable population. The hope is that the skin samples of those 12 individuals at the Frozen Zoo contain enough diversity to sustain the northern white species long-term.

The arduous task for these scientists is to create a rhino population from those samples.

Marlys Houck is curator of the Frozen Zoo. She graduated high school in 1979, the same year the Frozen Zoo froze its very first northern white rhino skin cell. She later joined the institute to work on the rhino project.

I was hired specifically to try to make the cells of the rhinos grow better because they were one of the most difficult to grow cell lines, she told CNN.

Since then, shes figured out how to successfully grow and freeze the skin cells of the northern white.

The impact of this work is not lost on her. Were losing species so rapidly, she said. One of the things we can do is save the living cells of these animals before its too late.

Were at the forefront of science today, she added. If we do everything right these cells should be here 50 years from now being used for purposes that we cant even imagine today.

Marisa Korody is one of the four scientists tasked with turning these frozen cells into new life. They have to reprogram the frozen skin cells into pluripotent stem cells. In laymans terms, Korody explains that stem cells can become any cell type in the body if theyre given the right signals.

Read: Former war zones turn into wildlife paradise

The aim is to ultimately turn the stem cells into sperm and eggs. The ambitious feat has only been achieved in animals by Japanese scientists. While Korody and her team have looked to that research as a road map, she admits that doing the same with rhinos is uncharted territory. We dont really know what twists and turns we need to take in order to get from A to B, she said.

They havent even figured out how to do this in humans, she added. We have as much information as we possibly can about humans. We have a fraction of that for rhinos.

Korody says being at the forefront of this kind of science has been a dream job. This was really the first project thats trying to apply this type of science to conservation as a whole, she said.

She may spend most of her time at work looking through the lens of a microscope, but her mind is always on the final goal for the rhinos: We want to be able to put them back into the wild one day and have them living free.

Because the remaining two female northern white rhinos cant carry a pregnancy, even if the team can create embryos, the last obstacle is finding rhinos who can carry them to term.

The woman tasked with that job is Barbara Durrant. As the director of reproductive sciences, shes spent four years studying the reproductive systems of six female southern white rhinos at the institutes sister facility, the Nikita Kahn Rhino Rescue Center.

Though the rhinos at the center are a different species, Durrant says they are the closest relative to the northern white. The aim is to eventually have them be surrogates for northern white embryos.

On any given day, Durrant can be found conducting ultrasounds to help her understand each rhinos distinct reproductive cycle. In 2019, two of the centers females gave birth to southern white babies. Both were conceived via artificial insemination, giving Durrant and the teams working on the rhino project hope for the future.

Durrant believes one reason the project works so well is because there are so many women involved. Women are naturally collaborative with each other, she said. Because we have so many obstacles along the way and challenges and setbacks, we support each other and we have sympathy for each other.

Read: Rare bird brought back from extinction in the wild

Houck says women tend to be naturally nurturing. The cells are living little organisms that were growing and tending almost every day, and I think women are drawn to taking care of something and growing it into something more.

Its wonderful leading a team of women, and I really think theyre changing the world, she added. People are going to look back and see it was this amazing group of women who quietly, unrecognized, work at this and just get better and better.

Read more here:
Meet the women racing to save the northern white rhino from extinction - KTVZ

Skin Stem Cells Comments Off on Meet the women racing to save the northern white rhino from extinction – KTVZ | March 8th, 2020

EVERY 20 minutes, someone in the UK is diagnosed with a form of blood cancer, such as leukaemia or lymphoma. It's the fifth most common cancer, and the third biggest cause of cancer deaths.

Yet warning signs of blood cancers can be so unlike those of other cancers, that it's often diagnosed at a very late stage. Research by the blood cancer charity Bloodwise (bloodwise.org.uk) found more than a third of people with blood cancer had to visit their GP three or more times with symptoms before being referred to hospital. This makes it the worst performing cancer in terms of early diagnosis, the charity says.

Why is it so difficult to spot? Well, blood cancers, which stop blood stem cells from working normally and can make you weak and prone to infections, have three main types with many different variations. These variations have numerous diverse symptoms, which can often be mistaken for other less serious conditions.

"Not all signs of blood cancer are easily identifiable, or are associated with typical symptoms of cancer, such as a lump or abnormal mole," explains haematologist Dr Manos Nikolousis, a medical consultant with the blood cancer charity DKMS (dkms.org.uk).

"Blood cancer often presents in ways which are most commonly associated with unrelated and less serious illnesses, like a cold or flu. In other circumstances, patients notice a change in their body which they can't quite put their finger on."

One of the treatments for blood cancer is a stem cell transplant that restores blood-forming stem cells in patients who've had theirs destroyed by very high doses of chemotherapy and/or radiotherapy. But Nikolousis points out that only one in three blood cancer patients who need a transplant will find a matching blood stem cell donor in their family. The remaining two-thirds have to rely on an unrelated donor, which significantly reduces their chance of finding a crucial match.

"The more people who register as potential blood stem cell donors, the better the chances of finding a donor for someone in need," stresses Nikolousis, who points out that potential donors, who should be healthy and aged between 17-55, just need to sign up with DKMS online.

The charity will send a screening kit to obtain a swab from inside the donor's cheeks. If they then prove to be a match for someone, 90 per cent of donations are done by collecting blood and harvesting stem cells from it. The process is simple and could potentially save lives.

What else do you need to know about blood cancer? Here, Nikolousis outlines some blood cancer symptoms and warning signs...

1. Back pain

Musculoskeletal pain in muscles, joints, tendons, bones or structures that support the limbs, neck or back.

2. Bruising or bleeding

One of the most common symptoms associated with blood cancer is bruising, and/or bleeding. The frequency and lasting impact of bruising can be a key warning sign of blood cancer, so it's important to book an appointment with your GP if this develops.

3. Fatigue

Unexplained and persistent tiredness is one of the biggest telltale signs of blood cancer. People who have cancer-related fatigue find it incredibly challenging to complete simple tasks that we tend to take for granted.

4. Swollen lymph nodes/glands

The lymph nodes are small lumps of tissue that contain white blood cells. When inflamed, they can be felt as lumps under the skin; most commonly in the neck, armpit or groin area.

5. Headaches

There may be new headaches that feel different to headaches previously experienced. They're likely to occur frequently and be severe and long-lasting. They also often appear alongside other physical symptoms associated with blood cancer.

6. Stomach discomfort

Persistent abdominal discomfort, presenting as a sharp pain or a sense of feeling full.

7. Numbness in the hands or feet

This can be described as a feeling of pins and needles/numbness in the feet that moves up to the legs, or from fingers into the arms.

8. Heart palpitations

This can feel like a fluttering, a sudden thump or a fast pounding sensation in the chest. It can also be felt in the neck or ear when lying down.

9. Loss of concentration

There may be a prolonged or recurring inability to concentrate or focus, and people may describe this as feeling mentally drained or dizzy.

10. Sleep problems

Blood cancer patients may have continuous trouble falling asleep or staying asleep. This might result in a restless night's sleep causing a feeling of extreme tiredness.

11. Itchy skin

Persistent, irritable and itchy skin. This may be experienced all over the body, or in isolated spots. Again, this symptom often appears alongside other physical symptoms associated with blood cancer.

Get it checked

These symptoms are very common and don't automatically mean you have cancer. But if you notice any unusual or ongoing changes, it's always best to see your doctor and get checked out sooner rather than later.

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Blood cancer symptoms everybody needs to know - The Irish News

Skin Stem Cells Comments Off on Blood cancer symptoms everybody needs to know – The Irish News | March 8th, 2020

Note to editors and reporters: Live coverage on NASA Television of the SpaceX CRS-20 cargo launch carrying this experiment is scheduled at 8:30 p.m. EST, 11:30 p.m. PST March 6 and will be replayed twice on March 7. Coverage of the rendezvous with the International Space Station will be at 5:30 a.m. EST Monday, March 8, with installation at 8:30 a.m. All times are subject to change due if weather or launch conditions are unfavorable

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Space exploration can take a toll on the human heart. Astronauts are at risk for changes in their cardiac function and rhythm. To learn how microgravity and other physical forces in space exact their effects on heart muscle, a Tissue Chips in Space project has now been packed and is awaiting launch to the International Space Station.

The experimental equipment consists of small, compact devices, a little bit larger than cell phone cases. The holders contain a row of tiny, 3-D globs of beating heart tissue grown from pluripotent stem cells, generated from human adult cells. The heart muscle tissue is supported between two flexible pillars that allow it to contract freely, in contrast to the rigid constraints of a Petri dish.

The devices also house a novel invention from the University of Washington. It automatically senses and measures the contractions of the heart tissues, and reduces the amount of time the astronauts will need to spend conducting this study.

The flexible pillars contain tiny magnets, explained UW graduate student Ty Higashi, one of the inventors. When the muscle tissue contracts, the position of the embedded magnets changes, and the motion can be detected by a sensor, he said. That information is then sent down to a laboratory on Earth.

This model will recapitulate, on a miniature scale, what might be happening to the architecture and function of heart muscle cells and tissues in astronauts during a space mission.

The project head is Deok-Ho Kim, a professor in bioengineering, who recently joined the Johns Hopkins University faculty in Baltimore. He and co-investigator, Nathan Sniadecki, a professor in mechanical engineering, began this study two years at the UW Medicine Institute for Stem Cell and Regenerative Medicine (ISCRM). Jonathan Tsui, a postdoc in bioengineering, Ty Higashi, a graduate student in mechanical engineering , and other members of the UW project team, continue the cross-country collaboration in Seattle. The team is working with several NASA and National Institutes of Health groups, and researchers at other universities, on this effort.

Sniadecki said that each of the tissues heading to the International Space Center contain about a half million heart cells.

They act like a full tissue, he explained. They contract, they beat and you can actually see them physically shorten in the dish. Were actually able to see little heart beats from these tissues.

The SpaceX shuttle delivering this scientific payload is expected to leave from Cape Canaveral no earlier than 8:50 p.m. PST (11:50 p.m. EST) Friday, March 6. The exact departure schedule depends on the weather and other factors.

Once on board, the experiment will run for 30 days before being returned to Earth for further analysis. A related space-based experiment will follow skyward later, to see if medications or mechanical interventions can offset what the heart muscle endures during extended space missions.

The space program is looking at ways to travel longer and farther, Sniadecki said. To do so, they need to think about protecting their crews. Having treatments or drugs to protect astronauts during their travel would make long term space travel possible.

Guarding against cardiac problems would be especially critical during space travel at distances never attempted before, such as a mission to Mars, said Sniadecki. This opportunity to really kind of push the frontier for space travel is every engineers dream.

He added, We also hope to gather information that will help in preventing and treating heart muscle damage in people generally, as well as in understanding how aging changes heart muscle.

Microgravity is known to speed up aging, and likely influence other cell or tissue properties. Because aging is accelerated in space, studies on the International Space Station is a way to more quickly assess this process over weeks, instead of years.

I think the medicine side of it is extremely helpful on Earth, too, because what we discover could potentially lead to treatments for counteracting aging, Sniadecki said.

This space medicine research project is funded by the National Center for Advancing Translational Sciences and the National Institute of Biomedical Imaging and Bioengineering. This heart tissue study is part of the national Tissue Chips in Space program.

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3D beating heart tissue experiment heads to Space Station - UW Medicine Newsroom

Cardiac Stem Cells Comments Off on 3D beating heart tissue experiment heads to Space Station – UW Medicine Newsroom | March 8th, 2020

CAPE CANAVERAL, Fla. SpaceX is preparing for its fifth launch of the year: a resupply mission to the International Space Station (ISS). The mission, which is scheduled to launch Friday (March 6) at 11:50 p.m. EST (0450 GMT on March 7), will bring a bevy of science material to the astronauts living and working in the orbiting laboratory.

This flight, dubbed CRS-20, marks the 20th and final mission for SpaceX under the company's first commercial cargo resupply services contract with NASA. Perched atop a Falcon 9 rocket will sit a cargo Dragon capsule filled with more than 4,300 lbs. (1,950 kilograms) of supplies, including more than 2,100 lbs. (950 kg) of science equipment.

The scientific cargo will support a host of experiments across Expeditions 62 and 63, focusing on a range of topics, from biological sciences (growing human heart cells in space), to water conservation methods, to particle-foam manufacturing and the addition of a new research platform on the ISS.

You can watch SpaceX's Dragon launch livehere on Space.com, courtesy of SpaceX, beginning at about 11:30 p.m. EST (0430 GMT), courtesy of NASA TV. You can alsowatch the launch directly from SpaceX here, beginning at 11:35 p.m. EST (0435 GMT).

Video: What's flying to the space station on SpaceX's CRS-20 mission?Related: SpaceX Dragon cargo ship launching tonight. How to watch live.

In its never-ending quest to create the best athletic shoe, Adidas has turned its sights to the International Space Station. The sportswear company has developed a performance midsole an additional shoe layer between the insole (next to your feet) and the sole (what touches the ground) that will enhance comfort.

To create its midsole, Adidas uses a process called particle foam molding, in which thousands of small pellets are blasted into a mold so they fuse together. To streamline the process and create the best shoe it can, Adidas is going to try this process in microgravity. The experiment, dubbed Adidas BOOST (Boost Orbital Operations on Spheroid Tessellation), will look at how the particles fuse together in space.

By removing gravity from the process, the team can take a closer look at individual pellet motion and location. The results of this investigation could show that the space station is a good platform for testing out new manufacturing methods and could lead to more-efficient means of packing and cushioning materials.

Related: Adidas launching new sneakers inspired by historic NASA spacesuits

Delta Faucet Co., a manufacturer of shower heads and other bathroom hardware, is launching a payload on CRS-20 that will seek to better understand how water droplets form. The company will use that knowledge to build a better shower head that lines up with Delta's ultimate goal: creating the sensation of increased pressure while using less water.

Conserving water is incredibly important, but one of the biggest drawbacks is that eco-friendly, low-flow shower heads do not perform as well as their less environmentally friendly counterparts. Users complain that the water pressure feels so low it's difficult to rinse off properly, which can result in longer showers and, ultimately, more water usage.

To help mitigate this issue, Delta has created a unique shower head, called the H2Okinetic, that controls the size and the speed of the water droplets with the help of an oscillating chip. That chip creates a better shower experience by breaking up the water flow into bigger droplets and shooting them out faster, giving the illusion of more water.

Related: Showering in space: Astronaut home video shows off 'hygiene corner'

"Water is a precious commodity," Garry Marty, principal engineer at Delta Faucet, said during a prelaunch briefing on Thursday (March 5). "We are trying to create a shower head to keep our customers happy while using less water."

He went on to explain that once the water leaves the pipes, it essentially doesn't have any pressure. What you're feeling are the droplets. With this new shower head, Delta Faucet is able to control the size and speed on each drop, revolutionizing the way a shower device delivers a shower.

"Lower-flow showers aren't really great to be under," Marty said. "But the more we understand, the more we can improve."

Marty added that, someday, humanity will be living on the moon or Mars and will need a way to take a shower. The lessons learned from this research go beyond conserving water and user experience, he said; it has implications for the space industry as well. But for now, the bigger concern is to better understand the fundamentals of water droplet formation.

Heart disease is the No. 1 cause of death in the U.S. A team of researchers from Emory University in Atlanta, led by Chunhui Xu, are sending an experiment up to the space station to explore how effectively stem cells can be turned into heart muscle cells.

The data collected could lead to new therapies and even speed up the development of new drugs that can better treat heart disease.

The microgravity environment found on the space station is known to have a profound effect on cell growth. Through this research, the team aims to understand the impact microgravity has on cardiac precursors (cardiac cells created from stem cells) and how effectively they produce cardiac muscle cells, called cardiomyocytes.

Related: Heart cells beat differently in microgravity, may benefit astronauts

Ground-based research shows that when cells are grown under simulated microgravity conditions, the production rate of cardiomyocytes is greater than if they were grown under the effects of gravity. By sending the experiment to the space station, Xu and her team will be able to determine if their results are accurate.

"Our goal is to help make stem cell-based therapy more readily available," Xu said during the briefing. "If successful, the demand for it will be tremendous, because heart disease is the No. 1 killer in America."

In order to have a successful therapy, Xu said that the team will need to produce a large number of high-quality cardiomyocytes. To do that, the researchers need to first understand the mechanisms behind cell transformation.

Bartolomeo is a new research platform that will be installed on the exterior of the space station. Placed outside the European Columbus module, this science balcony will host as many as 12 research experiments at one time.

Built by Airbus, the platform will enable researchers to conduct more experiments on the station's exterior. During a prelaunch briefing, NASA and Airbus explained that Bartolomeos potential uses include Earth observation, robotics, materials science and astrophysics.

"All of your [research] dreams can come true with Bartolomeo," said Andreas Schuette, program manager of Bartolomeo at Airbus.

And parking spots on the washing machine-sized platform are all-inclusive, which means that researchers can pay one price to launch, install, operate and even return to Earth. By working directly with agencies like NASA, ESA, and SpaceX, Airbus is able to offer a cost-effective means of conducting research on the space station.

The company is also working with the United Nations in an effort to entice those who wouldn't otherwise be able to afford to send payloads into space, Schuette told Space.com. The duo have teamed up with the United Nations Office for Outer Space (UNOOSA) to make that happen. (The agency works to make space more accessible.)

If all goes as scheduled, the Dragon will arrive at the International Space Station on Monday (March 9) at approximately 6 a.m. EDT (1000 GMT). From there, NASA astronauts Jessica Meir and Drew Morgan will use the station's Canadarm2 robotic arm to capture and attach the spacecraft, before beginning the unloading process.

Follow Amy Thompson on Twitter @astrogingersnap. Follow us on Twitter @Spacedotcom or Facebook.

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SpaceX Dragon to launch heart cell experiment and more to space station tonight - Space.com

Cardiac Stem Cells Comments Off on SpaceX Dragon to launch heart cell experiment and more to space station tonight – Space.com | March 8th, 2020

This new report by Eon Market Research, titled Global Autologous Stem Cell and Non-Stem Cell Based Therapies Market 2020 Research Report, 2015 2025 offers a comprehensive analysis of Autologous Stem Cell and Non-Stem Cell Based Therapies industry at a global as well as regional and country level. Key facts analyzed in this report include the Autologous Stem Cell and Non-Stem Cell Based Therapies market size by players, regions, product types and end industries, history data 2014-2018 and forecast data 2020-2025. This report primarily focuses on the study of the competitive landscape, market drivers and trends, opportunities and challenges, risks and entry barriers, sales channels, distributors in global Autologous Stem Cell and Non-Stem Cell Based Therapies market.

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Global Autologous Stem Cell and Non-Stem Cell Based Therapies Market Provides An In-Depth Insight Of Sales Analysis-US STEM CELL, INC. - Fashion...

Cardiac Stem Cells Comments Off on Global Autologous Stem Cell and Non-Stem Cell Based Therapies Market Provides An In-Depth Insight Of Sales Analysis-US STEM CELL, INC. – Fashion… | March 8th, 2020

Painless non surgical skin tightening procedures are now available and very popular for people who don't want to go through surgery in order to look better. Call LifeGaines in Boca Raton for more information about "Thread Lifts."

Boca Raton, Florida--(Newsfile Corp. - March 5, 2020) - Thread Lifts are a simple, painless procedure used for skin tightening on the face, neck, or anywhere else on the body. As popular and effective as Botox is, it simply doesn't have the ability to lift the skin. In the past, there hasn't been a great way to produce lifting results without surgery.

Call LifeGaines to inquire about this new method of skin tightening in the South Florida area. Call 561-295-9007.

To view an enhanced version of this image, please visit:https://orders.newsfilecorp.com/files/6848/53186_lifegaines_orig.jpg

Non-surgical aesthetics are in high demand and ThreadLifts, new to the United States, have the ability to produce skin that is instantly lifted and tightened.

This procedure uses no cuts or incisions, only injections. Threads are needles that are pre-loaded with PDO thread. The whole needle is inserted in the tissue at the sub-dermal level, along the surface of the skin and then the needle is pulled out. Threads can be used nearly anywhere on the body, but they are especially effective on the neck and jawline.

What is PDO?

Polydioxanone (PDO) sutures have been used for surgical procedures for many decades. It is one of the safest materials to implant in the body. PDO is completely dissolvable and your skin fully absorbs it within 4 to 6 months, leaving no scar tissue behind. This is especially effective when it's used together with chemical peels, Botox, and fillers to effect a patient's entire facial structure, remove sunspots and other conditions.

It is a great way to rejuvenate and restore youthful contours to brows, cheeks, jowls and the neck area. They are also effective on the breasts, buttocks and upper arms, areas that are prone to sagging due to weight loss, aging, pregnancy and childbirth or poor muscle tone.

Thread Lifts are a great way to rejuvenate and restore youthful contours to brows, cheeks, jowls and the neck area. Results from threads generally last between 12 months to several years depending on the area that has been treated, how many threads are used and what kinds of threads are used in each needle.

Amy Steffey, is a Licensed Nurse Practitioner with LifeGaines Medical and Aesthetics, and with Thread Liftsprocedure, she helps Boca Raton residents regain their confidence with rejuvenating procedures and body contouring.

Amy Steffey works at LifeGaines, which is one of the most highly respected Age Management Medical teams in South Florida. Age Management Medicine pioneer Dr. Richard Gaines is the founder of LifeGaines, and he has years of experience specializing in Hormone Replacement Therapy, Sexual Wellness, Platelet-rich Plasma, Stem Cells, Aesthetics, and Advanced Age Management protocols.

LifeGaines is located at 3785 N Federal Hwy #150, Boca Raton, FL 33431.

Call 561-295-9007 Today to Schedule a Consultation with Amy Steffey at LifeGaines Medical & Aesthetics Center in Boca Raton.

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people-who-dont-want-to-do-surgery.jpg People who don't want to do surgery could consider a non-surgical way of tightening skin. Call LifeGaines to inquire about this new method of skin tightening in the South Florida area. Call 561-295-9007.

To view the source version of this press release, please visit https://www.newsfilecorp.com/release/53186

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Non Surgical Aesthetics Are in High Demand, So Thread Lifts Are a New Way to Achieve Instant Skin Tightening - Yahoo Finance

Skin Stem Cells Comments Off on Non Surgical Aesthetics Are in High Demand, So Thread Lifts Are a New Way to Achieve Instant Skin Tightening – Yahoo Finance | March 6th, 2020

1 Molecular missing link: Research explains why some creams cause a skin rash

Allergic skin reactions can be caused by many different chemical compounds found in skincare creams, cosmetics and other topical consumer products, but how they trigger the reaction has remained somewhat mysteriousuntil now.

New research that suggests the way some chemicals displace natural fat-like molecules known as lipids in skin cells may explain how many common ingredients trigger allergic contact dermatitis.

The breakthrough could help stem soaring cases of rashes, lumps, blisters, itchy eyes and facial swellings. It has been dubbed the molecular missing link because it might have brought a new way to treat the condition.

Currently, the only way to stop allergic contact dermatitis is to identify and avoid coming into contact with the chemical that causes the reaction.

Most allergies are attributed to proteins or synthetically produced peptide antigens that set off the immune system.

A new study from the Hamamatsu University School of Medicine has discovered that docosahexaenoic acid (DHA) could potentially be an important component in creating more efficient lip care products.

The university collaborated with Kose Corporations research laboratories in order to better understand the molecular profile of the lip area.

The study highlighted it was especially important to study the lip in closer detail as it was one of the major targets of cosmetics.

Its important to under the molecular profile specific to human lips to discover the intrinsic ingredients for lip cosmetics.

To gain a better understanding, the team aimed to map out the human lip using imaging mass spectrometry to gather insight into its lipid distribution.

Researchers at Kao Corporation have developed a method to predict odour intensity, a skill previously dependent on the experience of perfumers.

The development of the technology was the result of a project by the firms Sensory Science Research Laboratory, which compiled a database of 314 commonly used fragrance ingredients.

The database of olfaction characteristics allowed scientists to develop a method that can predict the odour intensity based on the concentration of perfumery raw materials (PRMs) present in a gas sample.

Data were obtained from an evaluation testing performed by 18 perfumers and researchers, who scored the intensity of samples emitted from a fragrance diluter with different gas concentrations.

Based on evaluations of those results, the team managed to visualise the relationship between gas concentration and odour intensity.

Singapore biotech start-up Insectta is rearing black soldier flies on its urban farm to produce a more sustainable and purer chitosan for the cosmetics industry.

The company, which claims to have the first insect farm in Singapore, raises black soldier flies that feed on food waste before being converted into viable materials such as chitosan.

It is a useful material that has many functions. For cosmetics, it has antioxidant, antimicrobial and wound-healing properties. Additionally, it can enhance penetration into skin.

Chitosan is conventionally sourced from shrimp and crab. However, the company believes using insects is cleaner and more sustainable.

These flies are not pests, they don't bite. They are native to Singapore and not an invasive species. They feed on food waste which would otherwise go into the incinerator. We're trying to promote a circular economy, said Chua Kai Ning, chief marketing officer of Insectta.

The flies on the farm, which are housed in a small room, can consume around 7.5 tonnes of food waste a month.

Experts in Australia have been urging the public to carry on using sunscreen even though a widely publicised study has shown chemicals can be absorbed from some products into the bloodstream.

US Food and Drug Administration scientists found that sunscreen users might be taking in more of the active ingredients into our blood, far beyond regulatory thresholds.

They tested six of the main active ingredients in sunscreen lotions, sprays and pumps, revealing quantities of sunscreen chemicals in the blood high enough for the products to have to undergo additional FDA safety studies.

This happens when formulations surpass a threshold that requires them to be taken for further testing. The chemicals studied were avobenzone, oxybenzone, octocrylene, ecamsule, homosalate, octisalate and octinoxate.

The researchers stress the findings do not mean that sunscreens are unsafe, merely that more research is needed. The FDA will now conduct more research to determine the maximum levels of sunscreen ingredients that are safe to use.

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Skin science: Top five stories on cosmetics science and formulation - CosmeticsDesign-Asia.com

Skin Stem Cells Comments Off on Skin science: Top five stories on cosmetics science and formulation – CosmeticsDesign-Asia.com | March 6th, 2020

Buyers looking to own a private, well-stocked fishing pond or enjoy the simplicity of a quiet paddle across 18 acres of tranquil water will find all of that and a 5,619-square-foot custom contemporary home at 355 Pond St. in Uxbridge.

Set back from the road on more than 56 scenic acres, the 10-room home, on the market for $1.1 million, offers unique upgrades such as cedar paneled ceilings and 14-karate-gold plated fixtures in the master bath.

The house also features a 2,400-square-foot, wraparound deck that overlooks Lee Pond.

Owner Dr. Charles A. Vacanti said his favorite feature of the property is its pond, which is stocked upstream along Emerson Brook by two fishing clubs.

Both of their ponds drain into our 18-acre pond, he said. It may be the best fishing hole in New England.

Listing agent Gary Smith of Mendon Area Real Estate called the pond and its surrounding property beautiful, while his favorite feature in the home is the spectacular family room.

The sunken combination family and dining room share a wood burning stove with a two-story stone surround. The cathedral ceilings are paneled in cedar and hold skylights and fans. Light from two panels of floor to ceiling glass brightens this room that gives access to the deck.

The modern, eat-in kitchen holds a center island, custom-built cabinetry, granite countertops and large pantry.

The upstairs hallway is an open loft overlooking the family/dining room.

The master suite offers scenic views through a wall of glass with a door that opens to a balcony. The master bath has marble flooring, a cathedral ceiling with skylights, custom built closet and cabinets, a spa hot tub, and large stall shower.

Also upstairs are an office and gym space.

Dr. Vacanti and his wife, Linda, bought the property in 1998.

Since then, the couple has replaced all 27 skylights, all external doors and windows, and the roof. The installation of solar panels, Dr. Vacanti said, has reduced annual utility costs to nearly zero.

Dr. Vacanti is widely known for his research work in stem cell and tissue engineering. While at the University of Massachusetts Medical School in 1996, he grew new cartilage in the shape of a human ear under the skin of a lab mouse. The Vacanti Mouse was part of research on organ generation and the first demonstration of growing new tissue from cells in a lab.

As for the property, Smith called it a beautiful high and dry pasture that just begs for equestrian use, a corporate retreat, Bed and Breakfast, or Rod and Gun Club.

The property is minutes from Route 146.

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House of the Week: Uxbridge property is a sportsmans paradise - Worcester Telegram

Skin Stem Cells Comments Off on House of the Week: Uxbridge property is a sportsmans paradise – Worcester Telegram | March 6th, 2020

Technology Networksrecently had the pleasureof speaking with Bruce Steel, CEOEquillium, Inc. to learn more about how they are leveraging their comprehensive understanding of immunobiology to develop novel treatments for inflammatory and autoimmune disorders.Bruce provides insight on the various indications the company is currently developing treatments for, elaborates on the clinical progress of their initial product candidate EQ001 (itolizumab), and explains the role CD6 plays in autoimmunity and how it can be targeted therapeutically.

Laura Lansdowne (LL): How can immunobiology be harnessed to develop therapeutics?Bruce Steel (BS): The role of the immune system is to defend the body against foreign organisms and cells, including cancerous cells, and in doing so, it must distinguish accurately between self- and non-self-entities a process called tolerance. Autoimmunity is an immune response directed against the bodys own healthy cells and tissues and is the underlying process in many inflammatory diseases. Autoimmunity results from a loss of tolerance caused in part by an imbalance in the relationship between effector T cells and regulatory T cells. Therefore, developing therapeutics, such as EQ001 (itolizumab), that target these critical regulators of immune activation pathways has the potential dramatically improve the lives of patients with severe autoimmune and inflammatory disorders.LL: What indications are you currently developing treatments for?BS: We select target indications based on three primary criteria: strong scientific rationale for why itolizumab has potential to be a best-in-class therapeutic approach, areas of high unmet medical need where there are little or no treatments available today, and indications where we believe there is an attractive future commercial opportunity. Today we have ongoing clinical trials with itolizumab in acute graft-versus-host disease (GVHD), uncontrolled asthma and lupus/lupus nephritis.

While these three indications are our initial areas of focus, we believe itolizumab has the potential to be developed in other areas such as transplant science, systemic autoimmunity, pulmonary, neurologic, gastrointestinal, renal, vascular, ophthalmic and dermatologic disorders.We licensed itolizumab from our partner Biocon Limited who developed the drug and received regulatory approval in India for the treatment of psoriasis.LL: Can you tell us more about the mechanism of your drug candidate EQ001 (itolizumab)?BS: Itolizumab is a clinical-stage, first-in-class monoclonal antibody that selectively targets the CD6-ALCAM pathway, which plays a central role in modulating the activity and trafficking of effector T cells that drive a number of immune-inflammatory diseases.LL: What role does CD6 play in autoimmunity?BS: CD6 is a novel co-stimulatory receptor that uniquely modulates T cell activity and trafficking. It is a key checkpoint in regulating effector T cells that are central to autoimmune responses. CD6 binds activated leukocyte cell adhesion molecule (ALCAM). ALCAM is expressed on both antigen-presenting cells and tissue including the skin, gut, lung and kidney.In preclinical studies, blockade of CD6 with itolizumab leads to reduction in effector T cell activation and proliferation. Additionally, inhibiting the binding of ALCAM to CD6 with itolizumab modulates lymphocyte trafficking and reduces effector T cell infiltration into inflamed tissues.

Our work with CD6 builds upon the research conducted from researchers at the Dana-Farber Cancer Institute, our partner Biocon, and other leading academic centers. Today there are numerous peer reviewed publications related to the novel CD6 checkpoint receptor and targeting the CD6-ALCAM pathway.LL: Can you elaborate on the clinical progress of itolizumab?BS: Itolizumab is currently being studied in three different indications: acute graft-versus-host disease (aGVHD), uncontrolled moderate to severe asthma and lupus/lupus nephritis. This is an important catalyst year for Equillium as we expect initial data from all three programs in 2H 2020.

Each of these studies will allow us to understand the safety of itolizumab in these different disease areas and understand what potential dose we will carry forward. This is important as we consider making a larger investment in Phase II studies to advance the program.Bruce Steel was speaking with Laura Elizabeth Lansdowne, Senior Science Writer for Technology Networks.

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Exploiting Immunobiology To Treat Severe Autoimmune and Inflammatory Disorders - Technology Networks

Skin Stem Cells Comments Off on Exploiting Immunobiology To Treat Severe Autoimmune and Inflammatory Disorders – Technology Networks | March 6th, 2020

Avoid close contact with sick patients. Stay home if youre feeling unwell. Scrub your hands with soap and water for at least 20 seconds and for goodness sake, stop touching your face.

By now, youve probably heard or seen the advice from the Centers for Disease Control and Prevention (CDC) for staving off COVID-19, the viral epidemic ricocheting across the globe. Most cases of the disease are mild, triggering cold-like symptoms including fever, fatigue, dry cough and shortness of breath. The death rate appears to be lowabout two or three percent, perhaps much less. But the virus responsible, called SARS-CoV-2, is a fearsomely fast spreader, hopping from person to person through the droplets produced by sneezes and coughs. Since COVID-19 was first detected in Chinas Hubei province in December 2019, nearly 100,000 confirmed cases have been reported worldwide, with many more to come.

To curb the virus spread, experts stress the importance of hand hygiene: keeping your hands clean by regularly lathering up with soap and water, or, as a solid second choice, thoroughly rubbing them down with an alcohol-based sanitizer. That might sound like simple, even inconsequential advice. But such commonplace practices can be surprisingly powerful weapons in the war against infectious disease.

[Washing your hands] is one of the most important ways to interrupt transmission of viruses or other pathogens, says Sallie Permar, a physician and infectious disease researcher at Duke University. It can have a major impact on an outbreak.

In the strictest sense of the word, viruses arent technically alive. Unlike most other microbes, which can grow and reproduce on their own, viruses must invade a host such as a human cell to manufacture more of themselves. Without a living organism to hijack, viruses cant cause illness. Yet viral particles are hardy enough to remain active for a while outside of the host, with some staying infectious for hours, days or weeks. For this reason, viruses can easily spread unnoticed, especially when infected individuals dont always exhibit symptomsas appears to be the case with COVID-19.

Researchers are still nailing down the details of exactly how SARS-CoV-2 is transmitted and how resilient it is outside the body. Because the virus seems to hang out in mucus and other airway fluids, it almost certainly spreads when infected individuals cough or sneeze. Released into the air, infectious droplets can land on another person or a frequently touched surface like a doorknob, shopping cart or subway seat. The virus can also transfer through handshakes after someone carrying the virus sneezes or coughs into their hand.

After that, its a short trip for the virus from hand to head. Researchers estimate that, on average, humans touch their faces upwards of 20 times an hour, with about 44 percent of these encounters involving eyes, mouths and nosessome of the quickest entry points into the bodys interior.

Breaking this chain of transmission can help stem the spread of disease, says Chidiebere Akusobi, an infectious disease researcher at Harvards School of Public Health. Sneezing or coughing into your elbow can keep mucus off your mitts; noticing when your hand drifts towards your face can help you reduce the habit.

All this public-health-minded advice boils down to a game of keep away. To actually infect a person, viruses must first get inside the body, where they can infect living cellsso if one lands on your hands, the best next move is to remove or destroy it.

The most important step to curbing infection may be hand-washing, especially before eating food, after using the bathroom and after caring for someone with symptoms. Its simply the best method to limit transmission, says Kellie Jurado, a virologist at the University of Pennsylvanias Perelman School of Medicine. You can prevent yourself from being infected as well as transmitting to others.

According to the CDC, you should wet your handsfront and backwith clean, running water; lather up with soap, paying mind to the easily-forgotten spaces between your fingers and beneath your nails; scrub for at least 20 seconds; then rinse and dry. (Pro tip: If counting bores you or youre sick of the birthday song, try the chorus of these popular songs to keep track.)

Done properly, this process accomplishes several virus-taming tasks. First, the potent trifecta of lathering, scrubbing and rinsing physically removes pathogens from your skin, says Shirlee Wohl, a virologist and epidemiologist at Johns Hopkins University.

In many ways, soap molecules are ideal for the task at hand. Soap can incapacitate SARS-CoV-2 and other viruses that have an outer coating called an envelope, which helps the pathogens latch onto and invade new cells. Viral envelopes and soap molecules both contain fatty substances that tend to interact with each other when placed in close proximity, breaking up the envelopes and incapacitating the pathogen. Basically, the viruses become unable to infect a human cell, Permar says.

Alcohol-based hand sanitizers also target these vulnerable viral envelopes, but in a slightly different way. While soap physically dismantles the envelope using brute force, alcohol changes the envelopes chemical properties, making it less stable and more permeable to the outside world, says Benhur Lee, a microbiologist at the Icahn School of Medicine at Mount Sinai. (Note that alcohol here means a chemical like ethanol or isopropyl alcoholnot a beverage like vodka, which contains only some ethanol.)

Alcohol also can penetrate deep into the pathogens interior, wreaking havoc on proteins throughout the virus. (Importantly, not all viruses come with outer envelopes. Those that dont, like the viruses that cause HPV and polio, wont be susceptible to soap, and to some extent alcohol, in the same way.)

Hand sanitizers made without alcohollike some marketed as baby-safe or naturalwont have the same effect. The CDC recommends searching for a product with at least 60 percent alcohol contentthe minimum concentration found to be effective in past studies. (Some water is necessary to unravel the pathogens proteins, so 100 percent alcohol isnt a good option.)

As with hand-washing, timing matters with sanitizers. After squirting a dollop onto your palm, rub it all over your hands, front and back, until theyre completely drywithout wiping them off on a towel, which could keep the sanitizer from finishing its job, Jurado says.,

But hand sanitizers come with drawbacks. For most people, using these products is less intuitive than hand-washing, and the CDC notes that many people dont follow the instructions for proper application. Hand sanitizers also dont jettison microbes off skin like soap, which is formulated to lift oily schmutz off surfaces, Akusobi says.

Soap emulsifies things like dirt really well, he says. When you have a dirty plate, you dont want to use alcoholthat would help sterilize it, but not clean it.

Similarly, anytime the grit is visible on your hands, dont grab the hand sanitizer; only a full 20 seconds (or more) of scrubbing with soapy water will do. All told, hand sanitizer should not be considered a replacement for soap and water, Lee says. If I have access to soap and water, I will use it.

Technically, it is possible to overdo it with both hand-washing and hand sanitizing, Akusobi says. If your skin is chronically dry and cracking, thats no good. You could be exposing yourself to other infections, he says. But it would take a lot to get to that point.

In recent weeks, hand sanitizers have been flying off the shelves, leading to shortages and even prompting some retailers to ration their supplies. Some people have begun brewing up hand sanitizers at home based on online recipes.

Many caution against this DIY approach, as the end products cant be quality controlled for effectiveness, uniformity or safety, says Eric Rubin, an infectious disease researcher at Harvards School of Public Health. On average, one would imagine that [a homemade sanitizer] would not work as well, so it would be a mistake to rely on it, he says.

As more information on SARS-CoV-2 and COVID-19 emerges, experts stress the importance of awareness. Even as the news changes and evolves, peoples vigilance shouldnt.

Do the small things you need to do to physically and mentally prepare for whats next, Wohl says. But dont panic. That never helps anybody.

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Why Is Washing Your Hands So Important, Anyway? - Smithsonian

Skin Stem Cells Comments Off on Why Is Washing Your Hands So Important, Anyway? – Smithsonian | March 6th, 2020

A new population of stem cells that can generate bone has been revealed by researchers, which they say could have implications in regenerative medicine.

A population of stem cells with the ability to generate new bone has been newly discovered by a group of researchers at the University of Connecticut (UConn) School of Dental Medicine, US.

The researchers present a new population of cells that reside along the vascular channels that stretch across the bone and connect the inner and outer parts of the bone.

This is a new discovery of perivascular cells residing within the bone itself that can generate new bone forming cells, said lead investigator Dr Ivo Kalajzic. These cells likely regulate bone formation or participate in bone mass maintenance and repair.

Stem cells for bone have long been thought to be present within bone marrow and the outer surface of bone, serving as reserve cells that constantly generate new bone or participate in bone repair. Recent studies have described the existence of a network of vascular channels that helped distribute blood cells out of the bone marrow, but no research has proved the existence of cells within these channels that have the ability to form new bones.

In this study, Kalajzic and his team are the first to report the existence of these progenitor cells within cortical bone that can generate new bone-forming cells osteoblasts that can be used to help remodel a bone.

To reach this conclusion, the researchers observed the stem cells within an ex vivo bone transplantation model. These cells migrated out of the transplant and began to reconstruct the marrow cavity and form new bone.

While this study shows there is a population of cells that can help aid formation, more research needs to be done to determine the cells potential to regulate bone formation and resorption, say the scientists.

According to the authors of the study: we have identified and characterised a novel stromal lineagerestricted osteoprogenitor that is associated with transcortical vessels of long bones. Functionally, we have demonstrated that this population can migrate out of cortical bone channels, expand and differentiate into osteoblasts, therefore serving as a source of progenitors contributing to new bone formation.

The results are published inSTEM CELLS.

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Stem cells that can grow new bone discovered by researchers - Drug Target Review

Bone Marrow Stem Cells Comments Off on Stem cells that can grow new bone discovered by researchers – Drug Target Review | March 6th, 2020

Researchers from UConn School of Dental Medicine have recently discovered a group of stem cells that help in generating a new bone. In regards with this, Dr Ivo Kalajzic, professor of reconstructive sciences, stated that, this newly discovered perivascular stem cells that reside in the bone itself have capability of generating the bone and these cells are highly instrumental in repair & mass maintenance of the bone along with its formation.

Since ages, it has been thought that stem cells only reside in bone marrow and exterior surface of the bone stores the cells that continuously generate new bone or repair the bone. Postdoctoral individuals Dr Sierra Root and Dr Natalie Wee, and collaborators at Harvard, Maine Medical Research Center, and the University of Auckland also were part of this study along with Dr Ivo Kalajzic and confirmed that these new cluster of cells residing in the vascular channels that range across the bone and serve as connection between inner and outer part of the bone is capable of generating a new bone.

This team is also pioneer in bringing forward a study that says existence of these progenitor cells inside cortical bone not only generates a new bone but also help remodeling of the bone. The conclusion was made after these researchers observed that these stem cells within an ex vivo bone transportation model migrated out of the transplant and started manufacturing a new bone marrow cavity along with completely new bone.

In order to establish this, more research needs to done as it will definitely turn out wonderful to the field of medical science and mankind.

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Stem Cells that will aid new bone generation discovered as per latest research - Medical Herald

Bone Marrow Stem Cells Comments Off on Stem Cells that will aid new bone generation discovered as per latest research – Medical Herald | March 6th, 2020

Stem cell research has been the subject of discussion and heated debate for many years. Much of the social and political drama surrounding stem cells is the result of misunderstanding what stem cells are, where they come from, and what they can do for those with injuries and diseases.

Working from a common set of facts is a great way to dispel controversy, however. Whether we fall into the pro-choice or pro-life camp, it is more than evident that supporting stem cell research, including the development of stem cell therapies, is very much a pro-life position to take.

Stem cells function essentially like raw materials for the body. Depending on instructions from the body (or researchers in laboratories), stem cells can become many other types of cells with specialized functions.

The daughters of stem cells either become new stem cells (self-renewal) or they become more specialized cells for use in specific areas of the body (differentiation). These specialized cells include brain cells, heart muscle cells, bone cells, blood cells and others.

There are several reasons why stem cells are the focus of some of the most important medical science research today:

This last avenue of medical research stem cell therapies is the most consequential as well as the most controversial, depending on your point of view. Understanding stem cell therapy and its divisiveness requires understanding where stem cells come from in medical research and why they have considerable palliative potential.

Stem cells come from one of these three sources:

Embryonic stem cells are the most controversial as well as the most important type of stem cells right now. Thanks to a low-information electorate and gross misinformation from within the government, embryonic stem cells remain mired in needless debate.

Despite the rhetoric, these cells arent harvested from slain newborns. Instead, they are carefully gathered from blastocysts. Blastocysts are three-to-five-day-old embryos comprised of around 150 cells. According to some religious-political arguments, blastocysts are potential human beings, and therefore deserve legal protection.

Embryonic stem cells are the most valuable in medical research because they are fully pluripotent, which means they are versatile enough to become any type of cell the body requires to heal or repair itself.

Adults have limited numbers of stem cells in a variety of bodily tissues, including fat and bone marrow. Unlike pluripotent embryonic stem cells, adult stem cells have more limits on the types of cells they can become.

However, medical researchers keep uncovering evidence that adult stem cells may be more pliable than they originally believed. There is reason to believe cells from adult bone marrow may eventually help patients overcome heart disease and neurological problems. However, adult stem cells are more likely than embryonic stem cells to show abnormalities and environment-induced damage, including cell replication errors and toxins.

The newest efforts in stem cell research involve using genetic manipulation to turn adult stem cells into more versatile embryonic variants. This could help side-step the thorny abortion controversy, but its also not clear at present whether these altered stem cells may bring unforeseen side-effects when used in humans.

More research is required to fully understand the medical potential of perinatal stem cells. However, some scientists believe they may in time become a viable replacement for other types of stem cells. Perinatal stem cells come from amniotic fluid and umbilical cord blood.

Using a standard amniocentesis, doctors can extract umbilical cord mesenchymal stem cells, hematopoietic stem cells, amniotic membrane and fluid stem cells, amniotic epithelial cells and others.

Among other things, stem cell therapy is the next step forward for organ transplants. Instead of waiting on a transplant waiting list, patients may soon be able to have new organs grown from their very own stem cells.

Bone marrow transplants are one of the best-known examples of stem cell therapy. This is where doctors take bone marrow cells and induce them to become heart muscle cells.

Stem cell-based therapies hold significant promise across a wide range of medical conditions and diseases. With the right approach, stem cells show the potential to:

As the FDA notes, there is a lot of hype surrounding stem cell therapy. Much of it is warranted, but some of it deserves caution.

According to the FDA, stem cells have the potential to treat diseases or conditions for which few treatments exist. The FDA has a thorough investigational process for new stem cell-based treatments. This includes Investigational New Drug Applications (IND) and conducting animal testing.

However, the FDA notes that not every medical entity submits an IND when they bring a new stem cell therapy to market. It is vital that patients seek out only FDA-reviewed stem cell therapies and learn all they can about the potential risks, which include reactions at the administration site and even the growth of tumors.

The FDA submitted a paper, Clarifying Stem-Cell Therapys Benefits and Risks, to the New England Journal of Medicine in 2017. Its goal is to help patients fully understand what theyre getting themselves into.

For now, a great deal more research is required before we begin deploying stem cell therapies on a larger scale. The only FDA-approved stem cell therapies on the market today involve treating cancer in bone marrow and blood. Some clinics claim their therapy delivers miracle-like cures for everything from sports injuries to muscular dystrophy, but there just isnt enough evidence yet to take them at face value.

Unfortunately, the religious and political climate makes this evidence difficult to achieve. In some parts of the United States, the hostility toward stem cell researchers and medical practitioners has reached dangerous new levels.

Republicans in Ohio and Georgia want to make it illegal for doctors to perform routine procedures on ectopic pregnancies. This condition is life-threatening for the mother and involves the removal of a nonviable embryo from the fallopian tube.

These laws wouldnt just outlaw ectopic pregnancy surgery in the name of potential human life. It would, in fact, require women to undergo a reimplantation procedure after the ectopic pregnancy is corrected by a physician. If this procedure was actually medically possible, it would be dangerous and unnecessary. Thankfully, it doesnt exist outside the nightmarish imaginations of some of the more extreme Christian lawmakers and Planned Parenthood demonstrators.

Acquiring embryonic stem cells from ectopic pregnancies would seem to be the least controversial way to go about it. Unfortunately, even that small step toward medical progress sees itself hampered by reactionary politics.

No matter how theyre acquired, however, the 150 or so cells in blastocysts are packed with medical potential. Its clear that further exploration down this road will unlock unprecedented scientific progress. It will also, almost certainly, save many times more potential life than even the most outlandish estimates of what the achievement will cost us to achieve. Abortions today are rarer and safer than ever, and the vast majority occur within eight weeks of conception.

The medical community is poised for a revolution here, using these and other nonviable embryos and blastocysts. But realizing that potential requires, among other things, that we collectively make peace with modern medicine and family planning.

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Despite Pro-Life Claims, Stem Cell Therapy Has Very Real Benefits and Should Be Accessible - Patheos

Bone Marrow Stem Cells Comments Off on Despite Pro-Life Claims, Stem Cell Therapy Has Very Real Benefits and Should Be Accessible – Patheos | March 6th, 2020

INTRODUCTION

In recent years, organoids have emerged as powerful tools for basic research, drug screening, and tissue engineering. The organoids formed in vitro show many features of the structural organization and the functional hallmarks of adult or embryonic anatomical structures (1). In addition, the formation of organoids alleviates the need to perform animal studies and provides an attractive platform for robust quantitative studies on the mechanisms regulating organ homeostasis and tissue repair in vivo (1). The formation of organoids usually starts with populations of stem cells. They are therefore expected to be heterogeneous because pluripotent stem cells [induced pluripotent stem cells (pscs) or embryonic stem cells] have been shown to dynamically and stochastically fluctuate from ground to differentiated state (2). In the same vein, LGR5+ intestinal stem cells are reported to contain several distinct populations (3). As such, the formation of organoids involves the inherent capacity of these heterogeneous populations to self-sort and self-pattern to form an organized three-dimensional (3D) architecture (4). However, the rules underlying organoid formation as well as the contribution of intrinsic population heterogeneity to the organoid self-assembly remain poorly understood (5). Consequently, there is a need for novel quantitative approaches at the single-cell level to reliably understand the mechanisms of spatial tissue patterning in 3D organoids, for which microfluidic and quantitative image analysis methods are well suited.

In this work, we use mesenchymal progenitors, alternatively named mesenchymal stromal cells (MSCs), which constitute a self-renewing population with the ability to differentiate into adipocytes, chondrocytes, and osteoblasts (5). Although human MSCs (HMSCs) express high levels of undifferentiation markers (e.g., CD105, CD44, CD73), they constitute a heterogeneous population of cells that exhibit considerable variation in their biophysical properties and epigenetic status, as well as the basal level of expression of genes related to differentiation, immunoregulation, and angiogenesis (6, 7). Nonetheless, their aggregation leads to the formation of highly cohesive 3D spherical structures [which we designate hereafter as mesenchymal bodies (MBs)] with improved biological activities in comparison to 2D cultures (8). However, little is known on how HMSCs self-organize or whether the intrinsic heterogeneity of the population regulates MB formation and individual cell functions in 3D.

The self-aggregation of HMSCs into MBs can recapitulate the early stages of mesenchymal condensation, and it promotes the secretion of paracrine molecules taking part in the process of ossification (9). During mesenchymal condensation in vivo, mesenchymal progenitors self-aggregate and form dense cell-cell contacts that lead to the initiation of bone organogenesis through endochondral (necessitating a chondrogenic intermediate) and intramembranous (direct osteogenic differentiation) ossification (10). In addition, the formation of these 3D MBs in vivo is associated with the secretion of important paracrine molecules such as prostaglandin E2 (PGE2) and vascular endothelial growth factor (VEGF), which participate in the recruitment of endogenous osteoblasts, osteoclasts, and blood vessels, leading to the initiation/restoration of bone homeostasis (11, 12). In these two ossification processes, the induction of nuclear factor B (NF-B) target genes, such as cyclooxygenase-2 (COX-2), and their downstream products (e.g., PGE2 and VEGF) plays a critical role as developmental regulators of ossification and bone healing (13). However, while mesenchymal condensation is critical for bone organogenesis, there is still a limited understanding on how the cellular spatial organization within 3D MBs regulates the individual cells endocrine functions (14).

In the present work, we interrogate the influence of phenotypic heterogeneity within a population of stem cells on the mechanisms of self-assembly and functional patterning within 3D organoids using HMSCs as a model of heterogeneous progenitor cell population. This is performed using a novel microfluidic platform for high-density formation of mensenchymal bodies, combined with the analysis of individual cells by quantitative image analysis. Our study reveals that the progenitor cell population self-assembles in a developmentally hierarchical manner. We also find that the structural arrangement in mensenchymal bodies is linked with the functional patterning in 3D, through a modulation of the activity of regulatory molecular signaling at a local scale. This study demonstrates the interplay between cell size and differentiation status, which mediates cellular spatial rearrangement in 3D, leading to the regionalized activation of unique biological functions while forming aggregates.

HMSCs are known to constitute a heterogeneous population (6, 7). In this study, fetal HMSCs were derived from the Whartons jelly of the umbilical cord (UC). UC-derived HMSCs are considered to be more primitive than HMSCs derived from adult bone marrow because of their higher proliferative capacity, their ability to form colony-forming unitfibroblast, as well as their lower degree of basal commitment (15). To examine the cellular diversity within the population, HMSCs were first characterized by their expression of membrane markers. Most of the HMSC population consistently expresses CD73, CD90, CD105, and CD146, but not CD31 (an endothelial cell marker), CD34 (a hematopoietic cell marker), CD14 (an immune cell marker), or human leukocyte antigenDR (HLA-DR) (a type of major histocompatibility complex II) (Fig. 1, A to F, and fig. S1, A to C). However, a deeper analysis of the flow cytometric data shows that the HMSC population contains cells of heterogeneous size [coefficient of variation (CV) = 33 to 37%] (Fig. 1, G and I), having a broad distribution in the expression of CD146 (Fig. 1F). Of note, the CD146 level of expression was linked to the size of the cells: The highest levels of CD146 were found for the largest cells (Fig. 1, H and J). Similar correlations with cell size were also observed for CD73, CD90, and CD105 (fig. S1, D to F). In addition, upon specific induction, the HMSC population used in this study successfully adopted an adipogenic (Fig. 1K), an osteogenic (Fig. 1L), or a chondrogenic (Fig. 1M) phenotype, demonstrating their mesenchymal progenitor identity.

(A) Percentage of positive cells for CD31, CD73, CD90, CD105, and CD146 (n = 3). Representative histograms of the distribution of the CD31 (B), CD73 (C), CD90 (D), CD105 (E), and CD146 (F) level of expression are shown. (G) Representative histogram of the forward scatter (FSC) distribution. (H) Correlation between cell size [FSC and side scatter (SSC)] and the level of CD146 expression. (I) Representative histogram of the cell projected area distribution. (J) Representative histogram of the size distribution of the CD146dim, CD146int, and CD146bright (ImageSteam analysis). (K) Representative images of hMSCs differentiated toward adipogenic lineage (Oil Red O staining). (L) Representative images of UC-hMSCs differentiated toward osteogenic lineage in (Alizarin Red S staining). (M) Representative images of UC-hMSCs differentiated toward chondrogenic lineage (Alcian Blue staining in 2D and cryosectioned micromass cultures). Scale bars, 50 m. The images were acquired using a binocular. FITC-A, fluorescein isothiocyanateA; APC-A, allophycocyanin-A.

To interrogate contribution of cellular heterogeneity (i.e., in terms of size and levels of CD marker expression) in the self-organization of HMSCs in 3D, MBs were formed at high density on an integrated microfluidic chip. This was done by encapsulating cells into microfluidic droplets at a density of 380 cells per droplet, with a CV of 24% (fig. S2, A and B). The drops were then immobilized in 250 capillary anchors in a culture chamber, as previously described (Fig. 2, A and B) (16). The loading time for the microfluidic device was about 5 min, after which the typical time for complete formation of MBs was about 4 hours (movie S1), as obtained by measuring the time evolution of the projected area (Fig. 2, C and D) and circularity of individual MBs (Fig. 2E and movie S2). The protocol resulted in the formation of a single MB per anchor (fig. S2C) with an average diameter of 158 m (Fig. 2F), when starting with a seeding concentration of 6 106 cells ml1. The diameter of the aggregates can easily be tuned by modulating the concentration of cells in the seeding solution (fig. S2, A and B). In addition, the complete protocol yielded the reproducible formation of a high-density array of fully viable MBs ready for long-term culture (for the images of the individual fluorescent channel, see Fig. 2G and fig. S2D), as described previously (16). Of interest, the CV of the MB diameter distribution was lower than the CV of the individual cell size and of the cell number in droplets (CV MB diameter = 13.3%, CV cell number per drop = 24%, and CV cell size = 35%), which demonstrates that the production of MBs leads to more homogeneous size conditions, compared with the broad heterogeneity in the cell population.

(A) Chip design. Scale bar, 1 cm. (B) Schematized side view of an anchor through the MB formation and culture protocol. (C) Representative time lapse of an MB formation. Scale bar, 100 m. (D and E) Measurement of the time evolution of the projected area (D) and circularity of each aggregate (E). n = 120 MBs. (F) Distribution of the MB diameter normalized by the mean of each chip (n = 10,072 MBs). (G) Top: Representative images of MBs after agarose gelation and oil-to-medium phase change. Bottom: The same MBs are stained with LIVE/DEAD. Scale bar, 100 m. (H) Representative images of MBs formed in the presence of EDTA, an N-cadherin, or a CD146-conjugated blocking antibody (Ab) (the red color shows the position of the CD146 brightest cells, and the dilution of the antibody was 1/100 and remain in the droplet for the whole experiment). Scale bar, 100 m. The images were acquired using a wide-field microscope.

To gain insight into the cellular components required to initiate the self-organization of HMSCs in 3D, the MB formation was disrupted by altering cell-cell interactions. This was first performed by adding EDTA, a chelating agent of the calcium involved in the formation of cadherin junctions, to the droplet contents. Doing so disrupted the MB formation, as shown in Fig. 2H, where the projected area of the cells increased and the circularity decreased in the presence of EDTA compared with the controls, as previously reported (17). The role of N-cadherins among different types of cadherins was further specified by adding a blocking antibody in the droplets before MB formation. This also led to a disruption of the MB formation, demonstrating that N-cadherin homodimeric interactions are mandatory to initiate the process of HMSC aggregation. CD146 [melanoma cell adhesion molecule (M-CAM)] plays important dual roles: as an adhesion molecule (that binds to Laminin 411) (18) and a marker of the commitment of HMSCs (19). We, thus, interrogate its contribution to MB formation. The addition of a CD146-conjugated blocking antibody also disrupted the formation of the MB (Fig. 2H), demonstrating that cell-cell interactions involving CD146 are also required during MB formation, as reported with other cell types (18). Of note, the brightest signal from the CD146-stained cells was located in the core of the cellular aggregates (Fig. 2H), suggesting that HMSCs self-organize relatively to their degree of commitment.

We found that the population of HMSCs constituted of cells of broad size and expressing different levels of undifferentiated markers [i.e., CD90, CD73, CD105, and CD146 are known to be down-regulated upon differentiation; (20)] and that the cells are capable of self-organizing cohesively in 3D. To better understand how the heterogeneous cells organized within the MBs, we measured how the different cell types composing the population self-assembled spatially in 3D by investigating the role of CD146. For this purpose, the CD146dim and CD146bright cells were separated from the whole HMSC population by flow cytometry (Fig. 3, A and B). The cells were then reseeded on a chip for the MB formation after fluorescently labeling the brighter and/or the dimmer CD146 populations. Image analysis revealed that the CD146bright cells were mostly located in the center of the cellular aggregates, while CD146dim cells were found at the boundaries of the MBs (Fig. 3, C to E, figs. S3A and S5A for confocal images, and movie S1). This organization was stable for a 3-day culture (fig. S3B).

(A) Representative dot plot of the hMSC population separation based on the level of CD146: The CD146dim constitutes 20% of the population expressing the highest levels of CD146; the CD146bright constitutes the 20% of the population expressing the lowest levels of CD146. (B) Fluorescence signal distribution in the CD146dim and CD146bright populations after cell sorting. (C) After cell sorting, the CD146bright or the CD146dim was stained with Vybrant Dil (red) or Vybrant DiO (green), remixed together and allowed to form MBs. Representative images of the CD146bright and CD146dim within the MBs. Scale bar, 100 m (n = 185 MBs). (D) The position of the CD146bright and CD146dim was quantified by correlating the fluorescence signal of the different stained cells as a function of their radial position within the MBs, after the staining of individual population with Vybrant Dil (CD146bright) = 500 and CD146dim (MBs; CD146bright = 85). Error bars show the SD. (E) Schematized representation of the structural organization of MBs. (F and G) RT-qPCR analysis of the relative RUNX-2, CEBP/, and SOX-9 expression to glyceraldehyde-3-phosphate dehydrogenase (GADPH) [Ct (cycle threshold) (D) and relative RNA expression (E)] in the CD146bright and CD146dim populations (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001.

As we found that the CD146bright cells were larger than the CD146dim cells, the cells from the HMSC population were also separated on the basis of their relative size (a parameter that also discriminates the CD90, CD105, and CD73bright from the CD90, CD105, and CD73dim cells; fig. S1, D to F). After reseeding on the chip, the MBs were composed of large cells in the core, while the smallest cells were located at the boundaries, as expected from the previous experiments (fig. S3A). Moreover, we found that the speed of self-assembly of each population is not related to the rearrangement of CD146dim and CD146bright cells in 3D, because the mixing of dissociated cells or the fusion of aggregates made each population give rise to the same structural organization (21). It is well established that CD146bright defines the most undifferentiated HMSCs (20). The heterogeneity in level of commitment between the two subpopulations was therefore checked by reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis to quantify differences in the expression of differentiation markers. The analysis showed that the CD146dim cells expressed higher levels of osteogenic differentiation markers (i.e., RUNX-2) than the CD146bright cells (Fig. 3, F and G).

The level of RUNX-2 expression was also quantified at the protein level using immunocytochemistry and image analysis of the MBs on the microfluidic device by developing a layer-by-layer description of the MBs. This mapping was constructed by estimating the boundaries of each cell in the image from a Voronoi diagram, built around the positions of the cell nuclei stained with 4,6-diamidino-2-phenylindole (DAPI) (Fig. 4A) (22). These estimates were then used to associate the fluorescence signal from each cell with one of the concentric layers (Fig. 4B). Such a mapping provides better resolution for discriminating the spatial heterogeneity of protein expression than simply assigning a fluorescence signal to a defined radial coordinate (fig. S4). Moreover, the reliability of the measurements by quantitative image analysis was confirmed by performing several control experiments. In particular, we verified (i) the specificity of the fluorescence labeling, (ii) the absence of limitation for antibody diffusion, and (iii) the absence of the light path alteration in the 3D structure (fig. S5 and Materials and Methods). Consistent with the qPCR data, we found that HMSCs located at the boundaries of the MBs expressed higher levels of the protein RUNX-2 than the cells located in the core (see Fig. 4, C and D, and fig. S7 for individual experiments).

(A and B) The detection of nuclei within MBs enables the construction of a Voronoi diagram (A) that allows the identification of concentric cell layers (B) within the MBs. (C and D) Representative image (C) and quantitative analysis (D) (error bars represent the SD) of RUNX-2 staining within the cell layers of the MB (Nchips = 3 and nMBs = 458). N.S., nonsignificant. (E to H) Quantitative analysis (E) and representative images (F to H) of N-cadherin staining after methanol/acetone (F) (Nchips = 3 and nMBs = 405), after PFA/Triton X-100 fixation and permeabilization (G) (Nchips = 3 and nMBs = 649), and F-actin staining with phalloidin (H) (Nchips = 3 and nMBs = 421). Scale bars, 20 m. The images were acquired using a wide-field microscope. ***P < 0.001. (I) Schematized representation of the structural organization of MBs.

Thus, as CD146 defines the most undifferentiated and clonogenic cells as well as regulates the trilineage differentiation potential of HMSCs, the results indicate that HMSCs self-organize within MBs based on their initial commitment. The most undifferentiated and largest cells are found in the core (r/R < 0.8), while more differentiated cells positioned in the outer layers of the MBs (r/R > 0.8) (Fig. 4, C to E). In addition, these data reveal that HMSCs are conditioned a priori to occupy a specific location within the MBs.

The commitment of HMSCs is known to regulate their level of CD146 expression and the type of cell-cell adhesion molecules (23), which plays a fundamental role in the structural cohesion of the MBs (Fig. 2H). For this reason, we interrogated the organization of cell-cell junctions after the MB formation through measurements of the N-cadherin and F-actin fluorescence signal distribution. Two different protocols were used to discriminate several forms of N-cadherin interactions. First, paraformaldehyde (PFA) fixation and Triton X-100 permeabilization were used, because they were reported to retain in place only the detergent-insoluble forms of N-cadherin. Alternatively, ice-cold methanol/acetone fixation and permeabilization enabled the detection of all forms of N-cadherins (26). The results show a higher density of total N-cadherins in the core of the MBs (Fig. 4, E and F), while a higher density of F-actin was found in the cell layers located near the edge of the MBs (Fig. 4, E and H). The pattern of F-actin distribution was not related to the agarose gel surrounding the MBs (fig. S5B). These results are consistent with the theories of cell sorting in spheroids that postulate that more adhesive cells (i.e., expressing more N-cadherin or CD146) should be located in the core, while more contractile cells (i.e., containing denser F-actin) are located at the edge of the MBs (24). Moreover, our observations are in accordance with recent results demonstrating that HMSCs establishing higher N-cadherin interactions show reduced osteogenic commitment than HMSCs making fewer N-cadherin contacts, potentially through the modulation of Yap/Taz signaling and cell contractility (23).

In contrast, the most triton-insoluble forms of N-cadherins were located at the boundaries of the HMSC aggregates (Fig. 4, E and G), at the same position as the cells containing the denser F-actin. These results demonstrate that different types of cellular interactions were formed between the core and the edges of the MBs, which correlated with the degree of cell commitment that apparently stabilize the adherens junctions (Fig. 4I) (25).

We found above that the degree of commitment was linked with the pattern of HMSC self-organization in MBs (i.e., formation of adherens junctions), which may also regulate their paracrine functions (26). We therefore interrogated the functional consequences of the cellular organization in MBs by investigating the distribution of VEGF- and PGE2-producing cells.

The specific production of COX-2, VEGF, and two other molecules regulating bone homeostasis such as tumor necrosis factorinducible gene 6 (TSG-6) (27) and stanniocalcin 1 (STC-1) (28) was evaluated by RT-qPCR analysis. An increased transcription (20- to 60-fold) of these molecules was measured in 3D in comparison to the monolayer culture (Fig. 5, A and B). Consistent with this observation, while a very limited level of secreted PGE2 and VEGF was measured by enzyme-linked immunosorbent assay (ELISA) in 2D culture, they were significantly increased (by about 15-fold) upon the aggregation of HMSCs in 3D (Fig. 5C). In addition, to interrogate the specific role of COX-2 [the only inducible enzyme catalyzing the conversion of arachidonic acid into prostanoids; (29)] in PGE2 and VEGF production, indomethacin (a pan-COX inhibitor) was added to the culture medium. Indomethacin abrogated the production of PGE2, and it significantly decreased VEGF secretion (Fig. 5C), which suggests an intricate link between COX-2 expression and the secretion of these two molecules (30, 31).

(A and B) RT-qPCR analysis of the relative TSG-6, COX-2, STC-1, and VEGF expression to GADPH (Ct) (A) and relative RNA expression (B) in the 3D and 2D populations (n3D = 3 and n2D = 3). (C) Quantification by ELISA of the PGE-2 and VEGF secreted by hMSCs cultivated in 2D, as MBs or as MBs treated with indomethacin (nchips = 3 and n2D = 3). (D and E) Representative image (D) and quantitative analysis (E) of COX-2 (Nchips = 13 and nMBs = 2936) and (F) VEGF-A (Nchips = 3 and nMBs = 413) staining within the cell layers of the MBs (error bars represent the SD). Scale bars, 50 m. The images were acquired using a wide-field microscope *P < 0.05; ***P < 0.001; a and b: P < 0.05. (G) Schematized representation of the structural organization of MBs.

To further interrogate the link between the COX-2 and the VEGF-producing cells, their location was analyzed by quantitative image analysis at a layer-by-layer resolution. These measurements showed significantly higher levels of COX-2 in the first two layers, compared with the successive layers of the MBs (Fig. 5, D and E), with a continuous decrease of about 40% of the COX-2 signal between the edge and the core. This pattern of COX-2 distribution was not affected by the MB diameter (fig. S7B). Similar observations were made with VEGF (Fig. 5, D and F), demonstrating that cells at the boundaries of the MBs expressed both COX-2 and VEGF (Fig. 5G). Taken with the measurements of Fig. 5C, these results imply that COX-2 acts as an upstream regulator of PGE2 and VEGF secretion. Conversely, oxygen deprivation was unlikely to occur within the center of the MBs because no hypoxic area was detected through the whole MBs (fig. S5). Consequently, it is unlikely that hypoxia-inducible factor1 (HIF-1) signaling mediates the increase in VEGF expression at the boundaries of the MBs. Note that finding the link between these three molecules requires the 3D format, because the molecules are not detected in 2D. Here, the combination of population-scale measurements (Fig. 5C) and cell layer analysis (Fig. 5, E and F) provides strong evidence for this pathway.

Because variations of COX-2 and adherens junction distribution are colocalized within the MBs (Figs. 4, E to G, and 5, D and E), the results point to a link between the quality of cell-cell interactions and the spatial distribution of the COX-2high cells in 3D. The mechanisms leading to the spatial patterning of COX-2 expression in the MBs were therefore explored using inhibitors of the signaling pathways related to anti-inflammatory molecule production and of the molecular pathways regulating the structural organization (table S3): (i) 4-N-[2-(4-phenoxyphenyl)ethyl]quinazoline-4,6-diamine (QNZ) that inhibits NF-B, a critical transcription factor regulating the level of COX-2 expression (32); (ii) N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) that inhibits the canonical Notch pathway, modulating cell-cell interactions and several differentiation pathways; (iii) Y-27632 (Y27) that inhibits ROCK involved in the bundling of F-actin (i.e., formation of stress fibers) to assess the role of actomyosin organization; and (iv) cytochalasin D (CytoD) that inhibits the polymerization of actin monomers.

While the addition of DAPT had virtually no effect on the ability of the cells to form MBs, Y27 led to MBs with more rounded cells, and both QNZ and CytoD strongly interfered with the MB formation process (Fig. 6, A to C). The results indicate that NF-B activation and the promotion of actin polymerization are critical signaling steps initiating the process of MB formation by HMSCs.

(A) Representative images of MBs formed 1 day after the droplet loading. Scale bar, 100 m. Inhibitors are added to the culture medium before the MB formation. (B and C) Quantitative analysis of the aggregates projected area (B) and shape index (C) in the presence of the different inhibitors. Red lines represent the mean value for each condition. (D and E) Representative images (D) (contrast is adjusted individually for a better visualization of the pattern; scale bar, 100 m; the images were acquired using a wide-field microscope) and quantitative analysis (E) of the COX-2 fluorescence signal intensity normalized by the control value with the different inhibitors. For these longer culturing times, QNZ and CytoD are only added during the phase change to allow the MB formation. Small dots represent one MB. Large dots represent the average normalized COX-2 fluorescence signal per chip. Each color corresponds to a specific chip. *P < 0.05. (F) Estimation of inhibitor effect in the cell layers with the COX-2 signal normalized by the control value. Control: Nchips = 11 and nMBs = 2,204; QNZ: Nchips = 6 and nMBs = 1215; DAPT: Nchips = 3 and nMBs = 658; Y27: Nchips = 4 and nMBs = 709; CytoD: Nchips = 3 and nMBs = 459. *P < 0.05; **P < 0.01; ***P < 0.001. (G) Proposed mechanisms regulating the MB formation and the patterning of their biological functions. (i) Regulation of the formation of MBs. (ii and iii) Spatial patterning of hMSC biological properties within MBs.

To assess the role of NF-B and actin polymerization in the pattern and the level of COX-2 expression in the MBs, QNZ and CytoD were added 1 day after the cell seeding, once the MBs were completely formed. In contrast, Y27 and DAPT were included in the initial droplets and maintained in the culture medium for the whole culture period. Typical images showing the COX-2 signal in these different conditions are shown in Fig. 6D (see also fig. S7 for quantification of the individual experiments). Of note, none of the inhibitors had an effect on Casp3 activation, indicating that they do not induce apoptosis at the concentration used in this study (fig. S8). The levels of COX-2 expression in MBs, after 3 days in culture, were significantly reduced with QNZ, also decreasing after the addition of CytoD (Fig. 6E). By contrast, Y27 and DAPT had no effect on the levels of COX-2 expression. As a consequence, the results demonstrate that a sustained NF-B activity after the MB formation is required to promote COX-2 expression. Moreover, the induction of actin polymerization in MBs constitutes a mandatory step to initiate COX-2 production.

To get a deeper understanding on the local regulation of these signaling pathways, we analyzed at the single-cell resolution the distribution of COX-2 within the MBs. The spatial mapping revealed that the COX-2 fluorescence intensity was mostly attenuated at the edge of the MBs treated with CytoD and QNZ, while more limited change in the pattern of its expression was observed in the presence of Y27 and even less so with DAPT (Fig. 6F). Consequently, the results revealed a strong link between cell phenotype, the capability to form functional adherens junctions, and the local regulation of NF-B and actin polymerization leading to the increased expression of PGE2 and VEGF that are mediated by COX-2 in 3D (Fig. 6G). Together, the results indicate that in 3D cell aggregates, the spatial organization has some implications on the specific activation of signaling pathways, resulting in local functional heterogeneity.

Understanding the mechanisms of the formation and the spatial tissue patterning within organoids requires a characterization at single-cell level in 3D. In this study, we used a novel microfluidic and epifluorescence imaging technology to obtain a precise quantitative mapping of the structure, the position, and the link with individual cell functions within MBs. The image analysis provided quantitative data that were resolved on the scale of the individual cells, yielding measurements on 700,000 cells in situ within over 10,000 MBs.

While the microfluidic technology developed here is very efficient for high-density size-controlled MB formation, the method is prone to some limitations. Chief among them, the cultivation in nanoliter-scale drops may subject the cells to nutrient deprivation and by-product accumulation under static culture conditions. This limits the duration of the culture to a few days, depending on the cell type and droplet size. To overcome this limitation, it is possible to continuously perfuse the chip with fresh culture medium after performing the oil-aqueous phase exchange, as we demonstrated previously (16). Alternatively, it is also possible to maintain the cells in liquid droplets (without using a hydrogel) by resupplying culture medium through the fusion of additional drops at later times. This operation requires, however, a new design of the anchors and additional microfluidic steps (21).

The second major drawback of the method emerges from the large distance between the MBs and the microscope objective, which requires the use of very large working distance objectives. This compounds the difficulty of applying different confocal techniques by limiting the fluorescence intensity of the images, which, in turn, reduces the throughput when 3D image stacks are required. Although we have shown above that wide-field imaging can be used to obtain spatial mappings of spheroid structure and cell functions, true single-cell measurements will need to overcome the limitations on imaging in the future.

A Voronoi segmentation was used to categorize the cells into concentric layers, starting from the edge of the MBs and ending with the cells in the central region (22), which allowed us to measure variations in the structural organization and in the protein expressions on a layer-by-layer basis within the 3D cultures. The MBs were found to organize into a core region of undifferentiated cells, surrounded by a shell of committed cells. This hierarchical organization results from the spatial segregation of an initially heterogeneous population, as is generally the case for populations of pluripotent and somatic stem cells (2, 3, 33). The process of aggregation of HMSCs obtained within a few hours takes place through different stages (Fig. 6G): The first steps of the aggregation of MBs are mediated by N-cadherin interactions. In parallel, NF-B signaling is activated, promoting cell survival by preventing anoikis of suspended cells (34, 35). At later stages, the formation of polymerized F-actin and, to a lesser extent, stress fibers mediates the MB compaction, mainly at the edge of the MBs where the cellular commitment helps the stabilization of adherens junctions. The formation of adherens junctions facilitates the cohesion of the 3D structure, probably through the enhanced - and -catenin availability in the CD146dim/RUNX-2+ cells (36, 37, 38), which are recruited in the CCC complexes of the adherens junctions to promote the stable coupling of the F-actin to the N-cadherin (39), which become more insoluble to Triton X-100 than unbounded N-cadherins.

A functional phenotype that correlates with this hierarchical segregation is an increase in endocrine activity of the cells located at the boundaries of the MBs. COX-2 expression is increased in the outer layers of the MBs, which also contain more functional adherens junctions as well as a sustained NF-B activity in this region. The promoter of COX-2 contains RUNX-2 and NF-B cis-acting elements (40). While RUNX-2 is required for COX-2 expression in mesenchymal cells, its level of expression does not regulate the levels of COX-2 (40). The increased COX-2 expression is, in turn, due to the unbundled form of F-actin (i.e., a more relaxed form of actin, in comparison to the dense stress fibers observed in 2D) near the edge of the MBs, which was reported to sustain NF-B activity (41) and to down-regulate COX-2 transcriptional repressors (42). Therefore, NF-B has a high activity in the outer layers of the MB, where it locally promotes COX-2 expression.

These results show that the 3D culture format may provide some insights to understand the mesenchymal cell behavior in vivo, because we found that the expression of key bone regulatory molecules is spatially regulated as a function of the structural organization of the MBs. The 3D structure obtained here recalls some of the conditions found at the initial steps of intramembranous ossification that occurs after mesenchymal condensation (i.e., no chondrogenic intermediate was found in the MBs). In the developing calvaria, the most undifferentiated mesenchymal cells (e.g., Sca-1+/RUNX-2 cells) are located in the intrasutural mesenchyme, which is surrounded by an osteogenic front containing more committed cells (e.g., Sca-1/RUNX-2+ cells) (43, 44). Similarly, we observed that undifferentiated HMSCs (i.e., CD146bright/RUNX-2 HMSCs) were surrounded by osteogenically committed cells (i.e., CD146dim/RUNX-2+ HMSCs), which also coexpressed pro-osteogenic molecules, namely, COX-2 and its downstream targets, PGE2 and VEGF. While the link between COX-2 and PGE2 is well established, there is also evidence that COX-2 can induce the production of VEGF in different cell types, e.g., colon cancer cells (45), prostate cancer cells (46), sarcoma (47), pancreatic cancer cells (48), retinal Mller cells (49), gastric fibroblasts (50), skin or lung fibroblasts (51). In these cases, the mechanism for VEGF production through COX-2 induction is thought to be linked to PGE-2, either in an autocrine/paracrine manner (52) or in an intracrine manner (53).

Beyond HMSCs, spatial organization related to the level of differentiation and cell size has been documented in growing embryoids and organoids, with more committed cells being positioned in the outer layers (54, 55, 56). Our results show that a similar hierarchical structure can also be obtained through the aggregation of a mixed population of adult progenitors. This suggests that cell sorting, based on the size and commitment, plays a dominant role in organizing stem cell aggregates. This data-driven approach of combining high-throughput 3D culture and multiscale cytometry (16) on complex biological models can be applied further for getting a better understanding of the equilibria that determine the structure and the function of cells within multicellular tumor spheroids, embryoid bodies, or organoids.

HMSCs derived from the Whartons jelly of the UC (HMSCs) [American Type Culture Collection (ATCC) PCS-500-010, LGC, Molsheim, France] were obtained at passage 2. Four different lots of HMSCs were used in this study (lot nos. 60971574, 63739206, 63516504, and 63739206). While the lots were not selected a priori, we found consistent results for COX-2 and CD146 distribution within MBs. HMSCs from the different lots were certified for being CD29, CD44, CD73, CD90, CD105, and CD166 positive (more than 98% of the population is positive for these markers) and CD14, CD31, CD34, and CD45 negative (less than 0.6% of the population is positive for these markers) and to differentiate into adipocytes, chondrocytes, and osteocytes (ATCC, certificates of analysis). HMSCs were maintained in T175 cm2 flasks (Corning, France) and cultivated in a standard CO2 incubator (Binder, Tuttlingen, Germany). The culture medium was composed of modified Eagles medium (-MEM) (Gibco, Life Technologies, Saint Aubin, France) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco) and 1% (v/v) penicilin-streptomycin (Gibco). The cells were seeded at 5 103 cells/cm2, subcultivated every week, and the medium was refreshed every 2 days. HMSCs at passage 2 were first expanded until passage 4 [for about five to six population doublings (PDs)], then cryopreserved in 90% (v/v) FBS/10% (v/v) dimethyl sulfoxide (DMSO), and stored in a liquid nitrogen tank. The experiments were carried out with HMSCs at passages 4 to 11 (about 24 to 35 PDs, after passage 2).

HMSCs were harvested by scrapping or trypsinization from T175 cm2 flasks. Then, the cells were incubated in staining buffer [2% FBS in phosphate-buffered saline (PBS)], stained with a mouse anti-human CD146Alexa Fluor 647 (clone P1-H12, BD Biosciences), a mouse anti-human CD31Alexa Fluor 488 (BD Biosciences, San Jose, CA) antibody, a mouse anti-human CD105Alexa Fluor 647 (BD Biosciences, San Jose, CA), a mouse anti-human CD90fluorescein isothiocyanate (FITC) and a mouse anti-human CD73allophycocyanin (APC) (Miltenyi Biotec, Germany), a CD14-APC (Miltenyi Biotec), a CD34-FITC (BioLegend), and an HLA-DRAPC (BD Biosciences).

The percentages of CD73-, CD90-, CD105-, CD146-, CD31-, CD34-, and HLA-DRpositive cells were analyzed using a FACS LSRFortessa (BD Biosciences, San Jose, CA) or an ImageStream (Amnis) flow cytometer. To validate the specificity of the antibody staining, the distributions of fluorescently labeled cells were compared to cells stained with isotype controls: mouse immunoglobulin G1 (IgG1), k-PE-Cy5 (clone MOPC-21, BD Biosciences), and mouse IgG2a K isotype control FITC (BD Biosciences, San Jose, CA). Alternatively, HMSCs were sorted on the basis of their level of expression of CD146 or their size [forward scatter (FSC) and side scatter (SSC)] using a FACSAria III (BD Biosciences, San Jose, CA).

To induce adipogenic differentiation, UC-HMSCs were seeded at 1 104 cells/cm2 in culture medium. The day after, the culture medium was switched to StemPro Adipogenesis Differentiation medium (Life Technologies) supplemented with 10 M rosiglitazone (Sigma-Aldrich) for 2 weeks. To visualize the differentiated adipocytes, the cells were stained with Oil Red O (Sigma-Aldrich). As a control, UC-HMSCs were maintained in culture medium for 2 weeks and stained with Oil Red O, as above.

To induce osteogenic differentiation, UC-HMSCs were seeded at 5 103 cells/cm2 in culture medium. The day after, the culture medium was switched to StemPro Osteogenesis Differentiation medium (Life Technologies) supplemented with 2-nm bone morphogenetic protein 2 (BMP-2) (Sigma-Aldrich) for 2 weeks. To visualize the differentiated osteoblasts, the cells were stained with Alizarin Red S (Sigma-Aldrich). As a control, UC-HMSCs were maintained in culture medium for 2 weeks and stained with Alizarin Red S, as above.

To induce chondrogenic differentiation, UC-HMSCs were seeded at 1 106 cells/ml in a 15-ml conical tube to promote micromass culture. The medium consisted of StemPro Chondrogenic Differentiation medium (Life Technologies). After 3 weeks in culture, the pellets were fixed and cryosectioned and then stained for Alcian Blue 8GX (Sigma-Aldrich). As a control, UC-HMSCs were maintained in 2D using culture medium for 3 weeks and stained with Alcian Blue, as above.

The color images were acquired using a binocular (SMZ18, Nikon) equipped with a camera (D7500, Nikon).

Standard dry-film soft lithography was used for the flow-focusing device (top of the chip) fabrication, while a specific method for the fabrication of the anchors (bottom of the chip) was developed. For the first part, up to five layers of dry-film photoresist consisting of 50-m Eternal Laminar E8020, 33-m Eternal Laminar E8013 (Eternal Materials, Taiwan), and 15-m Alpho NIT215 (Nichigo-Morton, Japan) negative films were successively laminated using an office laminator (PEAK pro PS320) at a temperature of 100C until the desired channel height, either 135, 150, 165, or 200 m, was reached. The photoresist film was then exposed to ultraviolet (Lightningcure, Hamamatsu, Japan) through a photomask of the junction, the channels, and the culture chamber boundaries. The masters were revealed after washing in a 1% (w/w) K2CO3 solution (Sigma-Aldrich). For the anchor fabrication, the molds were designed with RhinoCAM software (MecSoft Corporation, LA) and were fabricated by micromilling a brass plate (CNCMini-Mill/GX, Minitech Machinery, Norcross). The topography of the molds and masters was measured using an optical profilometer (Veeco Wyco NT1100, Veeco, Mannheim, Germany).

For the fabrication of the top of the chip, poly(dimethylsiloxane) [PDMS; SYLGARD 184, Dow Corning, 1:10 (w/w) ratio of curing agent to bulk material] was poured over the master and cured for 2 hours at 70C. For the fabrication of the bottom of the chip, the molds for the anchors were covered with PDMS. Then, a glass slide was immersed into uncured PDMS, above the anchors. The mold was lastly heated on a hot plate at 180C for 15 min. The top and the bottom of the chip were sealed after plasma treatment (Harrick, Ithaca). The chips were filled three times with Novec Surface Modifier (3M, Paris, France), a fluoropolymer coating agent, for 30 min at 110C on a hot plate.

HMSCs were harvested with TrypLE at 60 to 70% confluence, and a solution containing 6 105 cells in 70 l of medium was mixed with 30 l of a 3% (w/v) liquid low-melting agarose solution (i.e., stored at 37C) (Sigma-Aldrich, Saint Quentin Fallavier, France) diluted in culture medium containing gentamicin (50 g/ml; Sigma-Aldrich) (1:3, v/v), resulting in a 100-l solution of 6 106 cells/ml in 0.9% (w/v) agarose.

HMSCs and agarose were loaded into a 100-l glass syringe (SGE, Analytical Science, France), while Fluorinert FC-40 oil (3M, Paris, France) containing 1% (w/w) PEG-di-Krytox surfactant (RAN Biotechnologies, Beverly, USA) was loaded into a 1- and 2.5-ml glass syringes (SGE, Analytical Science). Droplets of cell-liquid agarose were generated in the FC-40 containing PEG-di-Krytox, at the flow-focusing junction, by controlling the flow rates using syringe pumps (neMESYS Low-Pressure Syringe Pump, Cetoni GmbH, Korbussen, Germany) (table S1). After complete loading, the chips were immersed in PBS, and the cells were allowed to settle down and to organize as MBs overnight in the CO2 incubator. Then, the agarose was gelled at 4C for 30 min, after which the PEG-di-Krytox was extensively washed in flushing pure FC-40 in the culture chamber. After washing, cell culture medium was injected to replace the FC-40. All flow rates are indicated in table S1. Further operations were allowed by gelling the agarose in the droplets, such that the resulting beads were retained mechanically in the traps rather than by capillary forces (Fig. 2G). This step allowed the exchange of the oil surrounding the droplets by an aqueous solution, for example, to bring fresh medium for long-term culture, chemical stimuli, or the different solutions required for cell staining.

For the live imaging of the MB formation, the chips were immersed in PBS and then were incubated for 24 hours in a microscope incubator equipped with temperature, CO2, and hygrometry controllers (Okolab, Pozzuoli, Italy). The cells were imaged every 20 min.

2D cultures or MBs were washed in PBS and incubated with a 5 M NucView 488 caspase-3 substrate (Interchim, Montluon, France) diluted in PBS. After washing with PBS, HMSCs were fixed with a 4% (w/v) PFA (Alpha Aesar, Heysham, UK) for 30 min and permeabilized with 0.2 to 0.5% (v/v) Triton X-100 (Sigma-Aldrich) for 5 min. The samples were blocked with 5% (v/v) FBS in PBS for 30 min and incubated with a rabbit polyclonal antiCOX-2 primary antibody (ab15191, Abcam, Cambridge, UK) diluted at 1:100 in 1% (v/v) FBS for 4 hours. After washing with PBS, the samples were incubated with an Alexa Fluor 594conjugated goat polyclonal anti-rabbit IgG secondary antibody (A-11012, Life Technologies, Saint Aubin, France) diluted at 1:100 in 1% (v/v) FBS for 90 min. Last, the cells were counterstained with 0.2 M DAPI for 5 min (Sigma-Aldrich) and then washed with PBS.

The same protocol was used for the staining of VEGF-Aexpressing cells using a rabbit anti-human VEGF-A monoclonal antibody (ab52917, Abcam, Cambridge, UK), which was revealed using the same secondary antibody as above. RUNX-2positive cells were similarly stained using a mouse anti-human RUNX-2 monoclonal antibody (ab76956, Abcam, Cambridge, UK), which was revealed using an Alexa Fluor 488 goat anti-mouse IgG2a secondary antibody (A-21131, Life Technologies, Saint Aubin, France), both diluted at 1:100 in 1% (v/v) FBS.

To measure potential induction of hypoxia within the core of the MBs, the cells were stained with Image-iT Red Hypoxia Reagent (Invitrogen) for 3 hours and then imaged using a fluorescence microscope. As a positive control, the chips containing the MBs were immersed into PBS, incubated overnight in an incubator set at 37C under 3% O2/5% CO2, and lastly imaged as above.

To interrogate the contribution of signaling related to anti-inflammatory molecule production (COX-2 and NF-B) or molecular pathways regulated by the cell structural organization (Notch, ROCK, and F-actin), several small molecules inducing their inhibition were added to the culture medium (table S1). For all the conditions, the final concentration of DMSO was below 0.1% (v/v) in the culture medium.

The cell viability was assessed using LIVE/DEAD staining kit (Molecular Probes, Life Technologies). The MBs were incubated for 30 min in PBS containing 1 M calcein AM and 2 M ethidium homodimer-1, in flushing 100 l of the solution. The samples were then washed with PBS and imaged under a motorized fluorescence microscope (Nikon, France).

For the detection of the functional forms of N-cadherins (i.e., the N-cadherins closely linked to the actin network, which are PFA insoluble), the MBs were fixed with a 4% (w/v) PFA (Alpha Aesar, Heysham, UK) for 30 min and permeabilized with 0.2 to 0.5% (v/v) Triton X-100 (Sigma-Aldrich) for 5 min. Alternatively, the aggregates were incubated for 5 min with 100% cold methanol followed by 1 min with cold acetone, for the detection of total N-cadherins (i.e., the PFA-soluble and PFA-insoluble forms).

Then, the samples were blocked with 5% (v/v) FBS in PBS for 30 min and incubated with a rabbit polyclonal antiN-cadherin primary antibody (ab18203, Abcam, Cambridge, UK) diluted at 1:100 in 1% (v/v) FBS for 4 hours. After washing with PBS, the samples were incubated with an Alexa Fluor 594conjugated goat polyclonal anti-rabbit IgG secondary antibody (A-11012, Life Technologies, Saint Aubin, France) diluted at 1:100 in 1% (v/v) FBS for 90 min. Last, the cells were counterstained with 0.2 M DAPI for 5 min (Sigma-Aldrich) and then washed with PBS.

For the quantification of the polymerized form of actin (F-actin), the MBs were first fixed with a 4% (w/v) PFA (Alpha Aesar, Heysham, UK) for 30 min and permeabilized with 0.2 to 0.5% (v/v) Triton X-100 (Sigma-Aldrich) for 5 min. The samples were then blocked with a 5% (v/v) FBS solution and incubated for 90 min in a 1:100 phalloidinAlexa Fluor 594 (Life Technologies) diluted in a 1% (v/v) FBS solution. The cells were then counterstained with 0.2 M DAPI for 5 min (Sigma-Aldrich) and then washed with PBS.

To ensure the specificity of the antibody to COX-2 and N-cadherin, control UC-HMSCs were permeabilized, fixed, and incubated only with the secondary antibody (Alexa Fluor 594conjugated goat polyclonal anti-rabbit IgG), as above. The absence of fluorescence signal indicated the specific staining for intracellular COX-2 and N-cadherin.

Next, to validate that the distribution of the fluorescence intensity was not related to any antibody diffusion limitation, the MBs were fixed and permeabilized, as above. For this assay, the MBs were not subjected to any blocking buffer. The cells were incubated for 90 min with the Alexa Fluor 594conjugated goat polyclonal anti-rabbit IgG secondary antibody (A-11012, Life Technologies, Saint Aubin, France) diluted at 1:100 in 1% (v/v) FBS. Then, the cells were counterstained for DAPI, as above. Last, the MBs were collected from the chip, deposed on a glass slide, and imaged.

For the analysis of COX-2 expression by flow cytometry, the total MBs were recovered from the chip. The MBs were then trypsinized and triturated to obtain single-cell suspension. UC-HMSCs were stained for COX-2, as above. The percentage of COX-2positive cells was quantified on 5 103 dissociated UC-HMSCs using a Guava easyCyte Flow Cytometer (Merck Millipore, Guyancourt, France). The results were compared to the fluorescence intensity distribution obtained by image analysis.

To interrogate the influence of the MB opacity in the COX-2 and N-cadherin fluorescence signals, the samples were treated by the Clear(T2) method after immunostaining (57). Briefly, the MBs were incubated for 10 min in 25% (v/v) formamide/10% (w/v) polyethylene glycol (PEG) (Sigma-Aldrich), then for 5 min in 50% (v/v) formamide/20% (w/v) PEG, and lastly for 60 min in 50% (v/v) formamide/20% (w/v) PEG, before their imaging. The fluorescence signal distribution was compared with the noncleared samples.

The MBs were collected from the chip and then fixed using PFA, as above. The MBs were incubated overnight in a 30% sucrose solution at 4C. Then, the sucrose solution was exchanged to O.C.T. medium (optimal cutting temperature; Tissue-Tek) in inclusion molds, which were slowly cooled down using dry ice in ethanol. The molds were then placed at 80C. On the day of the experiments, the O.C.T. blocks were cut at 7 m using a cryostat (CM3050 S, Leica). The cryosections were placed on glass slides (SuperFrost Plus Adhesion, Thermo Fisher Scientific), dried at 37C, and rehydrated using PBS. The cryosections were permeabilized and stained for COX-2, as above. The slides were lastly mounted in mounting medium containing DAPI (Fluoromount-G, Invitrogen).

All the images used for the quantitative analysis were taken using a motorized wide-field microscope (Ti, Eclipse, Nikon), equipped with a CMOS (complementary metal-oxide semiconductor) camera (ORCA-Flash4.0, Hamamatsu) and a fluorescence light-emitting diode source (Spectra X, Lumencor). The images were taken with a 10 objective with a 4-mm working distance (extra-long working distance) and a 0.45 numerical aperture (NA) (Plan Apo , Nikon).

For control experiments, images were taken using a motorized (Ti2, Nikon) confocal spinning disc microscope equipped with lasers (W1, Yokogawa) and the same camera and objective as above. Alternatively, the samples were imaged with a multiphoton microscope (TCS SP8 NLO, MP, Leica). The objective was an HCX PL APO CS 10, 0.40 NA, working distance of 2.2 mm (Leica).

All immunostained samples were counterstained with DAPI, and most of the images (i.e., for N-cadherin, COX-2, VEGF-A, and F-actin) were taken using red light excitation that is known to penetrate deeper into the 3D objects than dyes emitting at lower weight length (e.g., DAPI, FITC). For wide-field microscopy, the focal plane was defined as the area containing the maximal number of DAPI-stained nuclei covering the focal area, while z stacks were taken for the whole in-focus planes containing DAPI-stained nuclei using spinning discs and two-photon confocal microscopy.

Wide-field imaging is sensitive for the emission of fluorescence from inside and outside the focal plane (i.e., from the out-of-focus upper and bottom planes of the spheroids) (58). Consistently, more DAPI signal from nuclei is emitted from the core than in the edges of MBs using epifluorescence microscopy (fig. S5I). We confirmed that our interpretation of the signal distribution from epifluorescence images was consistent with confocal and two-photon microscopy by comparing with images taken from the median z plane and the maximal z projection (fig. S5, N to P).

Consequently, the results unambiguously demonstrate that even if there are more cells in the z plane of the middle area of the MBs, the contribution of the out-of-focus signal from N-cadherin, COX-2, VEGF-A, and F-actin staining in this area of the MBs is minimal using wide-field imaging. Because of the higher throughput of wide-field microscopy, this method was chosen to quantitatively analyze the distribution of these immunolabeled proteins within MBs.

The culture supernatants of six-well plates were collected, while the total medium content of the chip was recovered by flushing the culture chamber with pure oil. A PGE2 human ELISA kit (ab133055, Abcam, Cambridge, UK) was used for the quantification of PGE2 concentration in the culture supernatant, following the manufacturers instructions. Briefly, a polynomial standard curve of PGE2 concentration derived from the serial dilution of a PGE2 standard solution was generated (r2 > 0.9). The absorbance was measured using a plate reader (Chameleon, Hidex, Finland).

A VEGF-A human ELISA kit (Ab119566, Abcam, Cambridge, UK) was used for the quantification of VEGF-A concentration in the culture supernatant of 2D cultures or from the chips. A linear standard curve of VEGF-A concentration derived from the serial dilution of a VEGF-A standard solution was generated (r2 > 0.9). The absorbance was measured using a plate reader (Chameleon, Hidex, Finland).

The total MBs of a 3-day culture period were harvested from the chips, as described above. Alternatively, cells cultured on regular six-well plates were recovered using trypsin after the same cultivation time; CD146dim and CD146bright isolated cells were immediately treated for RNA extraction after sorting. The total RNA of 1 104 cells were extracted and converted to complementary DNA (cDNA) using SuperScript III CellsDirect cDNA Synthesis System (18080200, Invitrogen, Life Technologies), following the manufacturers instructions. After cell lysis, a comparable quality of the extracted RNA was observed using a bleach agarose gel, and similar RNA purity was obtained by measurement of the optical density at 260 and 280 nm using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Wilmington, DE) between total RNA preparations from 2D and on-chip cultures.

The cDNA was amplified using a GoTaq qPCR Master Mix (Promega, Charbonnieres, France) or a FastStart Universal SYBR Green Master Mix (containing Rox) (Roche) and primers (Life Technologies, Saint Aubin, France or Eurofins Scientific, France) at the specified melting temperature (Tm) (table S2) using a MiniOpticon (Bio-Rad) or a QuantStudio 3 (Thermo Fisher Scientific) thermocycler. As a negative control, water and total RNA served as template for PCR. To validate the specificity of the PCR, the amplicons were analyzed by dissociation curve and subsequent loading on a 2.5% (w/v) agarose gel and migration at 100 V for 40 min. The PCR products were revealed by ethidium bromide (Sigma-Aldrich) staining, and the gels were imaged using a transilluminator. The analysis of the samples not subjected to reverse transcription (RT) indicated negligible genomic DNA contamination (i.e., <0.1%), while no amplification signal was observed for the water template (no template control). The amount of TSG-6, COX-2, STC-1, VEGF-A, RUNX-2, CEBP-, and SOX-9 transcripts was normalized to the endogenous reference [glyceraldehyde-3-phosphate dehydrogenase (GADPH)], and the relative expression to a calibrator (2D cultures) was given by 2Ct calculation. At least five biological replicates of 2D and on-chip cultures were analyzed by at least duplicate measurements. The standard curves for GADPH, TSG-6, COX-2, and STC-1 were performed using a five serial dilution of the cDNA templates and indicated almost 100% PCR efficiency.

The image analysis allowed us to perform a multiscale analysis (16) of the MBs. For each chip, single images of the anchors were acquired automatically with the motorized stage of the microscope. The analysis was conducted on a montage of the detected anchors using a custom MATLAB code (r2016a, MathWorks, Natick, MA). Two distinct routines were used: one with bright-field detection and one for the fluorescence experiments.

For the bright field-detection described previously (16), the cells were detected in each anchor as pixels with high values of the intensity gradient. This allowed for each cell aggregate to compute morphological parameters such as the projected area A and the shape index SI that quantifies the circularity of an objectSI=4APwhere P is the perimeter. Shape index values range from 0 to 1, with 1 being assigned for perfect disk.

The MB detection with fluorescence staining (DAPI/Casp3/COX-2, DAPI/phalloidin, DAPI/N-cadherin, or LIVE/DEAD) was performed as described previously (16). First, morphological data were extracted at the MB level, such as the equivalent diameter of the MBs or the shape index. Also, the mean fluorescence signal of each MB was defined as the subtraction of the local background from the mean raw intensity.

At the cellular level, two different methods were used, both relying on the detection of the nuclei centers with the DAPI fluorescence signal. On the one hand, each cell location could be assigned to a normalized distance from the MB center (r/R) to correlate a nuclear fluorescence signal with a position in the MB, as previously described (16). On the other hand, the cell shapes inside the MBs were approximated by constructing Voronoi diagrams on the detected nuclei centers. Basically, the edges of the Voronoi cells are formed by the perpendicular bisectors of the segments between the neighboring cell centers. These Voronoi cells were used to quantify the cellular cytoplasmic signal (COX-2, F-actin and N-cadherin, VEGF and RUNX-2). In detail, to account for the variability of the cytoplasmic signal across the entire cell (nucleus included), the fluorescence signal of a single cell was defined as the mean signal of the 10% highest pixels of the corresponding Voronoi cell.

Image processing was also used to get quantitative data on 2D cultures, as previously described (16). Last, different normalization procedures were chosen in this paper. When an effect was quantified compared with a control condition, the test values were divided by the mean control value, and the significance was tested against 1. For some other data, the values were simply normalized by the corresponding mean at the chip level to discard the interchip variation from the analysis.

*P < 0.05; **P < 0.01; ***P < 0.001; NS, nonsignificant. Details of each statistical test and P values can be found in table S4.

Acknowledgments: C. Frot is gratefully acknowledged for the help with the microfabrication, and F. Soares da Silva is gratefully acknowledged for the help in flow cytometry. The group of Biomaterials and Microfluidics (BMCF) of the Center for Innovation and Technological Research as well as the Center for Translational Science (CRT)Cytometry and Biomarkers Unit of Technology and Service (CB UTechS is also acknowledged for the access to the microfabrication and flow cytometry platform at the Institut Pasteur). Funding: The research leading to these results received funding from the European Research Council (ERC) grant agreement 278248 Multicell. Author contributions: S.S., C.N.B., and A.C. conceived the experiments. S.S. performed the experiments. R.F.-X.T. wrote the image processing code and performed the image analysis. R.F.-X.T., S.S., G.A., and A.B. performed the image and data analyses. S.S., C.N.B., and A.C. discussed the results and wrote the manuscript. All authors discussed the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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Two-year-old Regann Moore lights up as she watches videos on her iPad at home on Thursday, Feb. 20, 2020. Moore has a rare disease known as Krabbe Disease and received a life-saving stem cell donation less than a month after being born.(Photo: Nathan Papes/Springfield News-Leader)

Soon after the News-Leader published a story about 2-year-old Regann Moore,a Springfield child whose life was saved thanks to a newborn screening test, someone tweeted the story toMissouri State Rep. Becky Ruth.

"I bawled my eyes out," Ruth said. "I just cried."

She cried because she knew Regann is alive thanks to the death of Ruth's grandson, Brady.

"I cry and smile when I see these children," Ruth said. "We are always so thankful. For us, we see Brady's death wasn't in vain. His legacy lives on by helping save the lives of other children."

More: Springfield child with rare, deadly disease continues to amaze doctors, family

Regann, who is 2 now, was diagnosed right after she was born withKrabbe Disease, a rare metabolic disorder that must be diagnosed at birth and treated as soon as possible with a stem cell donation.

The newborn screening is important because babies with Krabbe Disease appear healthy at birth. Signs something is wrong usually don't appear until it's too late for treatment to be effective.

That is what happened to Brady in 2009. He wasn't diagnosed with the disease until he was 4.5 months old too late for treatment.

Brady died 10 days before his first birthday.

Brady Cunningham died of Krabbe Disease just before his first birthday.(Photo: Courtesy of the Cunningham family)

That's why Ruth and her family fought to get lawmakers on board with making sure all newborns in Missouri are screened for Krabbe Disease.

TheBrady Alan Cunningham Newborn Screening Act was passed in 2009 and screening began in 2012. Ruthsaid her family was OK with the three-year lag because they realized the lab needed time to become equipped to test for the disease.

Missouri is one of just a few states that do the newborn screening.

Brady's law also includes screening for Pompe, Fabry, Gauche and Niemann-Pick diseases. Since then, SCID, MPS I, MPS II and SMA diseases are screened, as well.

Ruth became a state representative in 2015and said newborn screening is her passion.

Her experience with getting Brady's law passed is what led her to seek office.

"It showed me what just a regular everyday person can do and what a differenceyou can make," Ruth said. "People a lot of times complain about politicians and the legislature, but we also do very good things here."

Ruth said her family knows of another child with Krabbe Disease who was saved thanks to newborn screening and a stem cell transplant.

That child is now 4. Ruth said her family and that child's family have a "strong connection."Ruth said shehopes to someday meet Regann's family.

Brady Cunningham was born in 2008. His family is from Campbell in southeast Missouri.

Bradyappeared healthy at birth and was not tested for Krabbe Disease.

Ruth said he started having health problems after about a month and a half. Brady went through "a myriad of diagnoses," Ruth recalled, including acid reflux and seizures.

"Finally my daughter took him to Children's Hospital in St. Louis," she said. "They promised her he wouldn't leave without a diagnosis."

Missouri State Rep. Becky Ruth was moved to tears after reading about Regann Moore, a Springfield child whose life was saved thanks to newborn screening for Krabbe Disease. Ruth and her family encouraged Missouri lawmakers to make sure all Missouri babies are tested for the deadly disease after her grandson, Brady, died from it.(Photo: Submitted by Becky Ruth)

Three weeks later, Brady was diagnosed with Krabbe Disease, which rapidly destroys the nervous system.

"We were told there was nothing they could do," she said. "It was one of the worst days of all of our lives."

Brady was 4.5 months old when he was diagnosed. In order for a stem cell donation to have any chance of being effective, babies must have the transplant within the first month of their life.

Regann, the Springfield child, was given a stem cell donation thanks to an umbilical cord donation.

Thediseaseaffects about one in every 100,000 people in the United States.

"They are missing an enzyme that helps keep their nervous system intact," said Dr. Shalini Shenoy, Regann's transplant doctor. "Because this is missing, they have degeneration of the brain and nervous system. And if you let it progress, it is fatal very early."

Without the stem cell donation, babies die within the first few months, Shenoy said.

"You can't change someone's genetic makeup," Shenoy said. "But when you put stem cells into their bone marrow from somebody else who is normal, some of these cells migrate into their brain and into their nervous system and supply what they are lacking themselves."

It takes some time for the transplant to begin working for the transplanted cells to "settle down" and begin making the missing enzyme, Shenoy said.

"Because of that, the earlier you transplant a Krabbe patient, the more you will be able to rescue them," she said. "You want to catch them before too much damage is done. Once there's a lot of nerve damage, it's not reversible. If I saw a Krabbe patient two months after they were born or four months after they were born when they already had major problems, it's unlikely I'd be able to rescue them too much."

Since the screening and the stem cell transplant treatment are both relatively recent medical advancements, Shenoy said it's anybody's guess what the future will hold for children who, like Regann, were successfully treated with a stem cell transplant early on.

Ferrell Moore holds his two-year-old daughter Regann Moore at their home on Thursday, Feb. 20, 2020. Regann has a rare disease known as Krabbe Disease and received a life-saving stem cell donation less than a month after being born.(Photo: Nathan Papes/Springfield News-Leader)

Regann can't stand on her own or walk yet. But her family is determined to make that happen. She cannot talk but is learning sign language to communicate.

She has regular visits with speech and occupational therapists.

Regann's dad Ferrell Moore got to take her to the circus recently, something the little girl seemed to enjoy.

"She is the joy of my life," Ferrell Moore said. "When I come home, it couldn't be any better to see her and how happy she is to see me."

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'His legacy lives on': Grandmother who helped create newborn screening law tells history of bill - News-Leader

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A unique molecular structureevident in induced pluripotent stem cells taken from people with young-onset Parkinson's diseasesuggests that the defects may be present throughout patients' lives, and that they could therefore be used as diagnostic markers.

Induced pluripotent stem cells (iPSCs) taken from patients with young-onset Parkinson's disease (YOPD) and grown into dopamine-producing neurons displayed a molecular signature that was corrected in vitro, as well as in the mice striatum, by a drug already approved by the US Food and Drug Administration (FDA), a study published in the January 27 online edition of Nature Medicine found.

Although the patients had no known genetic mutations associated with PD, the neurons grown from their iPSCs nonetheless displayed abnormally high levels of soluble alpha-synucleina classic phenotype of the disease, but one never before seen in iPSCs from patients whose disease developed later in life. Surprisingly, for reasons not yet understood, the cells also had high levels of phosphorylated protein kinase C-alpha (PKC).

In addition, the cells also had another well-known hallmark of PD: abnormally low levels of lysosomal membrane proteins, such as LAMP1. Because lysosomes break down excess proteins like alpha-synuclein, their reduced levels in PD have long been regarded as a key pathogenic mechanism.

When the study team tested agents known to activate lysosomal function, they found that a drug previously approved by the FDA as an ointment for treating precancerous lesions, PEP005, corrected all the observed abnormalities in vitro: it reduced alpha-synuclein and PKC levels while increasing LAMP1 abundance. It also decreased alpha-synuclein production when delivered to the mouse striatum.

Unexpectedly, however, PEP005 did not work by activating lysosomal function; rather, it caused another key protein-clearing cellular structure, the proteasome, to break down alpha-synuclein more readily.

The findings suggest that the defects seen in the iPSCs are present throughout patients' lives, and that they could therefore be used as diagnostic markers. Moreover, the drug PEP005 should be considered a potentially promising therapeutic candidate for YOPD and perhaps even for the 90 percent of PD patients in whom the disease develops after the age of 50, according to the study's senior author, Clive Svendsen, PhD, director of the Cedars-Sinai Board of Governors Regenerative Medicine Institute and professor of biomedical sciences and medicine at Cedars-Sinai.

These findings suggest that one day we may be able to detect and take early action to prevent this disease in at-risk individuals, said study coauthor Michele Tagliati, MD, FAAN, director of the movement disorders program and professor of neurology at Cedars-Sinai Medical Center.

But the study still raises questions regarding the biological mechanisms, and certainly does not warrant off-label prescribing of PEP005 at this time, said Marco Baptista, PhD, vice president of research programs at the Michael J. Fox Foundation, who was not involved with the study.

Repurposing PEP005 is a long way away, Dr. Baptista said. This is not something that neurologists should be thinking about prescribing or recommending to their patients.

Accumulation of alpha-synuclein has been seen in iPSC-derived dopaminergic cultures taken from patients with known genetic defects, but such defects account for only about 10 percent of the PD population. In those without known mutations, on the other hand, no defects in iPSC-derived dopamine-producing neurons have been seen. Until now, however, such studies had been conducted only in patients who had developed PD after age 50.

My idea was why to look in young-onset patients, said Dr. Svendsen.

The idea paid off more richly than he expected. We were shocked to find a very, very prominent phenotype, a buildup of alpha-synuclein, in the neurons of these patients who are genetically normal, Dr. Svendsen said. None of the controls had a buildup of synuclein, and all but one of the early PD patients had a twofold increase in it.

The signature is so consistent, he said, that it offers a natural model that can be interrogated to further understand its workings.

Because high levels of PKC were also seen, Dr. Svendsen said, We picked a bunch of drugs known to reduce PKC. We found one, PEP005, which is actually extracted from the milkweed plant, and it completely reduced synuclein levels almost to normal in dopaminergic neurons. And it also increased dopamine levels in those cells, so we got two for one.

After observing the effects of PEP005 in vitro, We put it into the mouse brain and found it reduced synuclein in vivo, Dr. Svendsen said. But we had to infuse it right into the brain. We're now trying to work out how to get it across the blood-brain barrier more efficiently.

To determine how PEP005 lowers cellular levels of alpha-synuclein, his group tested whether it was activating the lysosome, but found to their surprise that it did not do this until after the synuclein had already been degraded.

Then we asked whether it could be the proteosome, which also breaks down proteins but normally doesn't break down synuclein, Dr. Svendsen said. But when we applied PEP005, it did activate the proteasome. So we think that might be the mechanism.

Because the drug is currently applied externally, Dr. Svendsen said, the next step will be to see if it crosses the blood-brain barrier when applied to the skin of mice, and whether that results in a lowering of synuclein levels in dopaminergic neurons.

Justin Ichida, PhD, the Richard N. Merkin assistant professor of stem cell biology and regenerative medicine at the USC Keck School of Medicine, said the findings are quite important in the field. The potential diagnostic tools they made could be important in clinical care. And identifying a drug that may very effectively reverse the disease in neurons is a very important discovery.

He wondered, however, whether the increase in alpha-synuclein is truly specific to Parkinson's neurons or if it would also be seen in iPSC neurons from patients with Alzheimer's disease or amyotrophic lateral sclerosis.

I wonder if alpha-synuclein accumulating is a sign of PD in a dish or is a consequence of neurodegeneration or impaired protein degradation in general, Dr. Ichida said. That's a key question if you want to use this molecular signature as a diagnostic tool.

He also questioned if proteins other than alpha-synuclein, such as tau, would also be seen to accumulate in the iPSCs of YOPD patients.

If one of the protein-clearance mechanisms in the cell is working poorly, you would imagine that other things would also accumulate, Dr. Ichida said.

In response, Dr. Svendsen said that while some proteins other than alpha-synuclein were reported in the paper at increased levels, We did not look at tau specifically, but are in the process of looking right now. It could be that synuclein and some other proteins are somehow altered to evade them from being degraded by the lysosome, or that there is a general lysosomal problem.

Patrik Brundin, MD, PhD, director of the Center for Neurodegenerative Science and Jay Van Andel Endowed Chair at Van Andel Research Institute in Grand Rapids, MI, called the paper very interesting and thought-provoking. If these findings hold up, they could shift our understanding of young-onset PD. They imply that there is a strong genetic component that has not been picked up in prior genetic studies.

Dr. Brundin said he would like to see the results replicated in another lab using different sets of reagents. It is so intriguing and rather unexpected that one wonders if the observations really apply, as the study states, to 95 percent of all YOPD.

He also questioned whether all the young-onset PD patients are similar. Clearly the iPSCs studied here are not monogenetic PD, so they must be very diverse genetically and still all have the same alpha-synuclein change.

Dr. Brundin also asked why the abnormalities seen in YOPD neurons have not previously been seen in older cases of PD. Is there a specific cutoff regarding age-of-onset when these purposed genetic differences apply? he asked.

Dr. Svendsen responded: We don't know why the YO have this phenotype or exactly what the cut off is. We have, however, looked at one adult-onset case that did not show this phenotype. Also, one of our YO cases did not show this phenotype. Thus some patients even with early onset may not have it. We are currently testing many more cases from older-onset patients.

Dr. Brundin also wanted to know whether non-dopaminergic neurons have the same deficits described in the study.

We don't know which neurons specifically have the protein deficit as we cannot do single-cell proteomics, Dr. Svendsen answered. It could be a little in all cells or a lot in a small set. Immunocytochemistry is not quantitative but showed that it is more likely a general increase in synuclein and not specific to dopaminergic neurons.

While the findings in iPSCs suggest that the abnormal levels of alpha-synuclein must be present at birth, Dr. Brundin said, I do not know how to reconcile the present findings with genetic data.

The absence of previously described mutations in the YOPD patients means only that more work must be done to uncover the genetic underpinnings, Dr. Svendsen said.

We're just at the tip of the iceberg with understanding the genome, he said. It's such a bizarrely complex beast. Perhaps there are a thousand different proteins interacting to stop the synuclein from being degraded. In 10 years, we probably will be clever enough to see it. We know it must be there. Now the genome guys will go after it.

Dr. Baptista from the Michael J. Fox Foundation said he agreed with the view that there must be genetic alterations underpinning the defects seen in the iPSCs.

Just because we call something non-genetic could simply reflect the current ignorance of the field, he said. I think the discoveries are simply difficult to make.

He added that he wished that the main comparator in the study was not healthy controls, and that there were more older-onset iPSCs to compare against YOPD patients' samples.

Dr. Svendsen said it could be that the iPSCs from older-onset patients might yet be found with additional study to display abnormalities similar to those seen in YOPD.

Right now we only see it in young onset, he said. We may need to leave the cultures longer to see in the older-onset patients. We are doing those experiments now.

Drs. Tagliati and Svendsen disclosed that an intellectual patent is pending for diagnostic and drug screening for molecular signatures of early-onset Parkinson's disease. Dr. Ikeda is a co-founder of AcuraStem Inc. Dr. Brundin has received commercial support as a consultant from Renovo Neural, Inc., Lundbeck A/S, AbbVie, Fujifilm-Cellular Dynamics International, Axial Biotherapeutics, and Living Cell Technologies. He has also received commercial support for research from Lundbeck A/S and Roche and has ownership interests in Acousort AB and Axial Biotherapeutics. Dr. Baptista had no disclosures.

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