Early onset Parkinsons might begin in the womb: Prevention a possibility – The New Daily

An intriguing experiment has led researchers to conclude that people who develop early-onset Parkinsons disease between the age of 21 and 50 may have been born with abnormal brain cells that go undetected for decades.

These disordered cells allow gradual accumulation of the -synuclein protein that forms abnormal deposits in the brain, and dysregulated lysosomal proteins that ordinarily play a role in clearing abnormal proteins from cells.

The researchers from Cedars-Sinai Medical Center say they are investigating an FDA approved skin cancer drug they believe might help correct these abnormalities before they become symptomatic.

In other words, they suggest that early-onset Parkinsons the form of the disease that Michael J. Fox was diagnosed with at the age of 29 may be treatable or even prevented. Its an astonishing claim.

To perform the study, the research team generate pluripotent stem cells master cells that can potentially produce any cell or tissue the body needs to repair itself from blood cells of three patients with young-onset Parkinsons disease.

The patients were aged 30-39 and had no known familial history of the disease and no Parkinsons disease mutations.

When generated in the laboratory, these master cells called induced pluripotent stem cells (iPSCs). In their experiment, the Cedars-Sinai researchers described this process as taking adult blood cells back in time to a primitive embryonic state.

The team used the stem cells to produce dopamine neurons from each patient and then cultured them in a dish and analysed the neurons functions.

In Parkinsons patients, brain neurons that make dopamine a neurotransmitter that works to coordinate muscle movement become impaired or die.

Our technique gave us a window back in time to see how well the dopamine neurons might have functioned from the very start of a patients life, said Dr Clive Svendsen, PhD, director of the Cedars-Sinai Board of Governors Regenerative Medicine Institute, and the studys senior author.

According to a statement from Cedars-Sinai, the researchers detected two key abnormalities in the dopamine neurons in the dish:

Dr Svendsen said the experiment allowed the researchers to see the very first signs of young-onset Parkinsons.

It appears that dopamine neurons in these individuals may continue to mishandle alpha-synuclein over a period of 20 or 30 years, causing Parkinsons symptoms to emerge.

The investigators went further, using their iPSC to test a number of drugs that might reverse the lab-born abnormalities.

They found that that one drug, PEP005 already approved by the Food and Drug Administration for treating pre-cancers of the skin reduced the elevated levels of alpha-synuclein in both the dopamine neurons in the dish and in laboratory mice.

The drug also countered another abnormality they found in the patients dopamine neurons elevated levels of an active version of an enzyme called protein kinase C. However, the role of this enzyme version in Parkinsons is not clear.

The drug PEP005 is only available in gel form and the researchers plans to investigate how it might be delivered to the brain to potentially treat or prevent young-onset Parkinsons.

In Parkinsons disease, the symptoms including slowness of movement, rigid muscles, tremors, loss of balance and impaired mood control get worse over time. In most cases, the exact cause of neuron failure is unclear, and there is no known cure.

Just about every week, a new insight into the disease is published. Last week, The New Daily reported on new research that found living less than 50 metres from a major road or less than 150 metres from a highway has been linked to significantly higher incidence of dementia and Parkinsons disease.

In 2018, we published an exciting Australian study that suggested subject to clinical testing the inflammation of the brain that causes so much of the progressive damage in Parkinsons disease (PD) could be halted by taking a single pill each day.

Both these studies might eventually prove to be correct. But its a long wait for the more than 10 million sufferers worldwide and their families.

This latest study could be a game-changer. But it could just as easily wither on the vine. Still, better to take heart than not.

Most patients are 60 or older when they are diagnosed, about 10 per cent are between 21 and 50 years old. .

Young-onset Parkinsons is especially heartbreaking because it strikes people at the prime of life, said Dr Michele Tagliati, director of the Movement Disorders Program, vice chair and professor in the Department of Neurology at Cedars-Sinai, and co-author of the study.

This exciting new research provides hope that one day we may be able to detect and take early action to prevent this disease in at-risk individuals.

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Early onset Parkinsons might begin in the womb: Prevention a possibility - The New Daily

Global Stem Cell Banking Market Analysis, Trends, and Forecasts 2019-2025 – ResearchAndMarkets.com – Business Wire

DUBLIN--(BUSINESS WIRE)--The "Stem Cell Banking - Market Analysis, Trends, and Forecasts" report has been added to ResearchAndMarkets.com's offering.

The global market for Stem Cell Banking is projected to reach US$9.9 billion by 2025, driven by their growing importance in medicine given their potential to regenerate and repair damaged tissue.

Stem cells are defined as cells with the potential to differentiate and develop into different types of cells. Different accessible sources of stem cells include embryonic stem cells, fetal stem cells, peripheral blood stem cells, umbilical cord stem cells, mesenchymal stem cells (bmMSCs) and induced pluripotent stem cells. Benefits of stem cells include ability to reverse diseases like Parkinsons by growing new, healthy and functioning brain cells; heal and regenerate tissues and muscles damaged by heart attack; address genetic defects by introducing normal cells; reduce mortality among patients awaiting donor organs for transplant by regenerating healthy cells and tissues as an alternative to donated organs. While currently valuable in bone marrow transplantation, stem cell therapy holds huge potential in treating a host of common chronic diseases such as diabetes, heart disease (myocardial infarction), Parkinsons disease, spinal cord injury, arthritis, and amyotrophic lateral sclerosis. The technology has the potential to revolutionize public health.

The growing interest in regenerative medicine which involves replacing, engineering or regenerating human cells, tissues or organs, will push up the role of stem cells. Developments in stem cells bioprocessing are important and will be key factor that will influence and help regenerative medicine research move into real-world clinical use. The impact of regenerative medicine on healthcare will be comparable to the impact of antibiotics, vaccines, and monoclonal antibodies in current clinical care. With global regenerative medicine market poised to reach over US$45 billion 2025, demand for stem cells will witness robust growth.

Another emerging application area for stem cells is in drug testing in the pharmaceutical field. New drugs in development can be safely, accurately, and effectively be tested on stem cells before commencing tests on animal and human models. Among the various types of stem cells, umbilical cord stem cells are growing in popularity as they are easy and safe to extract. After birth blood from the umbilical cord is extracted without posing risk either to the mother or the child. As compared to embryonic and fetal stem cells which are saddled with safety and ethical issues, umbilical cord is recovered postnatally and is today an inexpensive and valuable source of multipotent stem cells. Until now discarded as waste material, umbilical cord blood is today acknowledged as a valuable source of blood stem cells. The huge gap between newborns and available cord blood banks reveals huge untapped opportunity for developing and establishing a more effective banking system for making this type of stem cells viable for commercial scale production and supply. Umbilical cord and placenta contain haematopoietic blood stem cells (HSCs). These are the only cells capable of producing immune system cells (red cells, white cells and platelet).

HSCs are valuable in the treatment of blood diseases and successful bone marrow transplants. Also, unlike bone marrow stem cells, umbilical cord blood has the advantage of having 'off-the-shelf' uses as it requires no human leukocyte antigen (HLA) tissue matching. Developments in stem cell preservation will remain crucial for successful stem cell banking. Among the preservation technologies, cryopreservation remains popular. Development of additives for protecting cells from the stresses of freezing and thawing will also be important for the future of the market. The United States and Europe represent large markets worldwide with a combined share of 60.5% of the market. China ranks as the fastest growing market with a CAGR of 10.8% over the analysis period supported by the large and growing network of umbilical cord blood banks in the country. The Chinese government has, over the years, systematically nurtured the growth of umbilical cord blood (UCB) banks under the 'Developmental and Reproductive Research Initiation' program launched in 2008. Several hybrid public-private partnerships and favorable governmental licensing policies today are responsible for the current growth in this market.

Companies Mentioned

Key Topics Covered:

I. METHODOLOGY

II. EXECUTIVE SUMMARY

1. MARKET OVERVIEW

2. FOCUS ON SELECT PLAYERS

3. MARKET TRENDS & DRIVERS

4. GLOBAL MARKET PERSPECTIVE

III. MARKET ANALYSIS

GEOGRAPHIC MARKET ANALYSIS

UNITED STATES

CANADA

JAPAN

CHINA

EUROPE

FRANCE

GERMANY

ITALY

UNITED KINGDOM

REST OF EUROPE

ASIA-PACIFIC

REST OF WORLD

IV. COMPETITION

V. CURATED RESEARCH

For more information about this report visit https://www.researchandmarkets.com/r/9b2ra3

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Global Stem Cell Banking Market Analysis, Trends, and Forecasts 2019-2025 - ResearchAndMarkets.com - Business Wire

The Future of Antivenom May Involve Mini Lab-Grown Snake Glands – Smithsonian.com

For the first time, scientists have grown miniature, venom-producing glands in the lab using coral snake embryos, according to a news study published in the journal Cell. Why might researchers want to create artificial venom glands, you ask?

The project was initially aimed to establish proof-of-concept more than anything else. Three graduate students at the Hubrecht Institute in the Netherlands had wondered: If lab-grown organs could be made that acted like mouse and human tissues, would it work for other animals, like reptiles?

Luckily, they were working in molecular geneticist Hans Clevers lab. Clevers is a prominent expert in stem cell research who pioneered research on the lab-grown organ imitationscalled organoidsa decade ago. Since then, researchers have created miniature human kidneys, livers, and brains in petri dishes.

On Fridays, members of the Clevers Lab are allowed to work on unstructured projects. To put their question to the test, Clevers students Yorick Post, Jens Puschhof, and Joep Beumer, would need a source of reptilian stem cells. As it happened, one of the researchers knew a guy: a snake breeder who could supply them with fertilized eggs, as STAT News Andrew Joseph reports.

They started with the egg of a Cape coral snake, removing the embryos venom glands and placing them in a dish. Then, they followed nearly the same protocol as they did with human cells, giving the cells ample supply of growth-inducing chemicals and storing them at a comfortable temperatureabout 89 degrees Fahrenheit, about ten degrees lower than the temperature used for human cells.

Soon, the plates held one-millimeter-long white blobs producing dangerous venom. With the organoids alive and well, the researchers told Clevers what theyd done, Leslie Nemo at Discover reports. If theyd told him beforehand, he would have told them it probably wouldnt work, Clevers tells the Atlantics Ed Yong. The chemicals they used were designed for human stem cells, and very little was known about stem cells in snakes. Still, the researchers were able to grow organoids from nine species of snakes.

Its a breakthrough, University of Costa Rica snake venom toxicologist Jos Mara Gutirrez, who was not involved in the study, tells Erin Malsbury at Science magazine. This work opens the possibilities for studying the cellular biology of venom-secreting cells at a very fine level, which has not been possible in the past, Malsbury says.

By looking closely at the organoids, Clevers team gained new insight into how multiple kinds of cells work together to produce the specific mixture of toxins and proteins that results in fully-developed venom.

Venomous snake bites kill between 81,000 and 138,000 people every year, according to the World Health Organization, and cause three times as many amputations and disabilities. The antidote to a snakebite is an antivenom, but each of thousands of venomous snakes have a different biteeach requiring a unique treatment. Even snakes of the same species can produce a slightly different venoms if they live in different regions.

Right now, antivenoms are produced using much the same process as was invented in the 19th century: a live snake is milked for its venom, that venom is injected into a horse. Horses have been used for antivenom production for years because of their docile nature and big veins, as Douglas Main wrote for Popular Mechanics in 2016. They are first injected with adjuvant, which stimulates their immune system to produce enough antibodies to neutralize the venom. Then, researchers take a sample of their blood and separate the antivenom from other component of blood, like plasma, in a centrifuge.

Clevers now hopes to create a bank of dozensand eventually thousandsof organoids from dangerous snakes and other reptiles that could aid in the effort to manufacture effective antivenoms.

"We could just sample one tissue once, and we have a source of [that snakes] venom for eternity," Clevers tells Discover.

Clevers is working with the Dutch biologist Freek Vonk, who he calls the Dutch Steve Irwin, to get samples of the snake species he hopes to include in the venom gland biobank. (Vonk works at Naturalis Biodiversity Center in Leiden and also has some excellent Dutch science tunes available on Spotify.)

With venom from organoids more easily available, the hope is to skip the horse in the antitoxin-production process. Researchers could instead use the organoid-produced venom to test an array of molecules for neutralizing abilities.

It will be interesting to see how the cost of producing venom using this system compares to the cost of purchasing venom milked from live snakes, since cost of antivenom is a key impediment to its wider use in countries where snakebite is a huge issue, like India and Nigeria, as Bangor University molecular zoologist Anita Malhotra tells the Atlantic.

Antivenoms made from lab-grown venom glands are likely years away, but the organoids could also be a big step for studying toxin production in more detail than previously possible. With the cells isolated from the rest of the snake, researchers might be able to look at how they can produce toxic chemicals without damaging themselves, for example.

Clevers tells Discover, We do the most interesting work when we dont have a proposal and just try things.

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The Future of Antivenom May Involve Mini Lab-Grown Snake Glands - Smithsonian.com

Fighting cancer with every step to Patagonia – Essex News Daily

Photo Courtesy of Michael MankowichAbove, Michael Mankowich and his wife, Kathleen, in Patagonia

NUTLEY, NJ When Nutley resident Michael Mankowichs lower back started to bother him, he figured it was a souvenir from his earlier athletic days. Mike, 58, had been a top-notch wrestler at 132 pounds at Long Islands Commack North High School. Hed been an all-American, in fact, as well as a two-time all-Ivy, three-time New York state champ and three-time EIWA tournament placer as a wrestler at Cornell University. An old wrestlers injury was all it was, he figured, a physical reminder of a quick takedown of an opponent 40 years long forgotten.

But the pain did not go away.

Mike began to see a doctor and a chiropractor, and eventually he got an MRI. The news he received at Memorial Sloan Kettering Cancer Center in February 2017 was not good. He was diagnosed with multiple myeloma, a cancer that attacks the blood plasma cells responsible for creating disease-fighting antibodies.

They figured it out quickly at Sloan, he said recently, seated with his wife, Kathleen, in their Rutgers Place home. I kept it from Kathleen.

With this news, he became withdrawn, and his wife realized something was wrong. Mike told her what he had learned, and, as so often happens when a couple puts their heads together, they found some reason for hope: multiple myeloma is a blood disease in the bone marrow and, as such, does not metastasize.

Thats where all the action takes place, in the bone marrow, Mike said. You have to keep your chin up.

For treatment, he became part of a six-month chemotherapy clinical study. Mike was glad to be in the study, because most multiple myeloma patients go on chemotherapy for three months and then undergo a stem-cell transplant. He, however, would not.

A stem-cell transplant blows out the immune system, he said.

Kathleen, an administrative coordinator at Felician University School of Nursing, said her husband, a real estate management employee, did not break stride and never missed the commute to New York City during the clinical study.

A member of Nutley High Schools Class of 1976, Kathleen got on the computer.

When your spouse is diagnosed with an incurable cancer, you do a bit of research, she said.

She discovered the Multiple Myeloma Research Foundation website and learned it was founded 30 years earlier by a woman named Kathy Giusti, who was living with the disease.

That gave me hope, Kathleen said.

She also learned about a collaboration between MMRF and CURE Media Group called Moving Mountains for Multiple Myeloma, or MM4MM.

This collaboration promotes endurance events, undertaken by multiple myeloma patients, to places like Mount Fuji, Mount Kilimanjaro and Iceland. The treks raise money for research, as well as public awareness about the disease. A patient selected to participate in one of these exotic treks had to raise funds, but the trip itself was underwritten by Celgene, a pharmaceutical company headquartered in Summit.

Mike was interested and applied in November 2018 for a spot on a team going to Patagonia. He was interviewed and accepted on condition of raising $10,000 for MMRF research. He suggested that Kathleen accompany him, and they eventually raised $30,000 through social media and by asking friends, family and neighbors.

The online MMRF page devoted to Mikes fundraising shows a photograph of him with his arms around Kathleen and their daughter, Mary, a Class of 2020 NHS student.

In a letter featured on the page, Mike informs the reader that MMRF is one of the worlds leading private funders of myeloma research, with 10 new treatments approved by the Food and Drug Administration.

In August 2019, Mike and Kathleen were flown to Oregon to meet their teammates and to get a taste of what was in store for them in Patagonia. According to the MM4MM website: Each team is carefully selected, representing a microcosm of the myeloma community patients, caregivers, health care professionals and clinical trials managers, as well as representatives from our pharma partners, from CURE Magazine and the MMRF to emphasize the collaboration necessary to drive toward cures.

The foundation sent the group to Mount Hood, Mike said. It was the first time we met. What a great group of people. There were around 15 from all over the country, and there was one other couple, but no one else from New Jersey.

Four other multiple myeloma patients were in the group, he said. he team climbed for nine hours and then headed home.

To prepare for the trip to Patagonia, a region containing part of the Andes mountain range, Mike and Kathleen began a regime of long walks. For instance, theyd walk from Nutley to South Orange and went hiking in New Yorks Harriman State Park.

The MMRF website described the journey as one of arduous adventure: This team will traverse Patagonia crossing over glaciers, through deep valleys, and ascending challenging peaks. This is a powerful and life-changing experience, as the team overcomes challenges, pushes beyond perceived limits and honors loved ones and friends living with multiple myeloma.

For the trek, the team flew to El Calafate, Argentina. As the team embarked on different climbs, documentary filmmakers accompanied them.

The hiking was physically difficult, Mike said. We hiked in rain and incredible winds. In one particular hike, as soon as you felt the winds, you hit the ground. I was surprised nobody got hurt. Some of those slopes were pretty steep. But the scenery was unworldly, and there were condors.

Both Mike and Kathleen agreed that the most memorable sight was La Condorera, which their itinerary described as a nearly vertical massif, offering a home to one of the greatest concentrations of endangered condors in the world. A massif is a group of mountains standing apart from other mountains.

It was a difficult hike, Kathleen said. Youre ready to pass out getting to the top. But its so worth it. The panorama is a view of glaciers and condors. It was spectacular.

Mike and Kathleen returned home on Nov. 16, but there were no goodbyes at the airport. The team had grown so incredibly close that everyone felt they would be seeing each other again, a feeling grounded in the knowledge that multiple myeloma can be challenged and hopefully, one day, defeated.

Our goal in all of this is that you can have multiple myeloma and still do incredible things, Kathleen said.

Its an incentive to other patients to get out there and enjoy their lives, Mike said. And find a cure for multiple myeloma. I have a little bias. I have it.

FEATURED, MOBILE

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New Gene Therapy Successfully Sends Six Patients With Rare Blood Disorder Into Remission – IFLScience

Six patients with a rare blood disease are now in remission thanks to a new gene therapy. The condition, known as X-CGD, weakens the immune system leaving the body vulnerable to a range of nasty infections and shortens a persons lifespan. It is normally treated using bone marrow transplants, but matching donors to patients can be tricky and time-consuming and the procedure comes with risks.

A team led by UCLA recently treated nine people with the disease and six successfully went into remission, allowing them to stop other treatments. All six patients are doing well and havent suffered any adverse effects.

X-CGD is a form of chronic granulomatous disease (CGD). People with CGD have an inherited mutation in one of five genes involved in helping their immune system attack invading microbes with a burst of chemicals. This means that CGD sufferers have weaker immune systems than healthy people, so they have a greater risk of getting infections. These infections can be life-threatening, particularly if they affect the bones or cause abscesses in vital organs.

X-CGD is the most common type of CGD and only affects males. It is caused by a mutation in a gene on the X-chromosome. Current treatments are limited to targeting the actual infections with antibiotics as well as bone marrow transplants. Bone marrow contains stem cells that develop into white blood cells, so bone barrow from a healthy donor can provide a CGD patient with healthy white blood cells that can help their body to fend off disease.

However, bone marrow transplants are far from ideal. The patient has to be matched to a specific donor, and the body can reject the implanted bone marrow. That means that following a transplant, the patient needs to take anti-rejection drugs for at least six months.

For their new treatment, researchers removed blood cell-forming stem cells from the patients themselves and genetically modified them so that they no longer carried the unwanted mutation. Then, the edited stem cells were returned to their bodies, ready to produce healthy new infection-fighting white blood cells.

This is the first time this treatment has been used to try to correct X-CGD. The researchers followed up with the nine patients but sadly, two passed away within three months of the treatment. Its important to note that their deaths were not a result of the treatment but of rather severe infections that they had been suffering from for a long time. The remaining seven were followed for 12 to 36 months all remain free from infections related to their condition, and six have been able to stop taking preventative antibiotics entirely. The results are reported in Nature Medicine.

None of the patients had complications that you might normally see from donor cells and the results were as good as youd get from a donor transplant or better, said Dr Donald Kohn, a member of theEli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLAand a senior author of the paper.

Whats more, four new patients have also been treated since the initial research was conducted. None experienced any adverse reactions and all remain infection-free. Now, the team plans to conduct a bigger clinical trial to further test the safety and efficacy of their new treatment, with the hopes that it may one day become available to the masses.

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New Gene Therapy Successfully Sends Six Patients With Rare Blood Disorder Into Remission - IFLScience

The Asia Pacific human microbiome market is expected to reach US$ 207.81 Mn in 2025 from US$ 41.73 in 2017 – Yahoo Finance

The market is estimated to grow with a CAGR of 22. 8% from 2018-2025. The growth of the market is driven by the factors such as rising chronic disease due to change in lifestyle and growing interest in human microbiome treatment approach.

New York, Jan. 30, 2020 (GLOBE NEWSWIRE) -- Reportlinker.com announces the release of the report "Asia Pacific Human Microbiome Market to 2025 - Regional Analysis and Forecasts by Product, Disease, Application, and Country" - https://www.reportlinker.com/p05764187/?utm_source=GNW Whereas, stringent regulatory environment and lack of awareness about human microbiome science is likely to have a negative impact on the growth of the market in the coming years.

Probiotics, prebiotics dietary supplements and foods that contain live microbes have been studied thoroughly to assess their effects on human health.The Gut Health Congress was held in Hong Kong Asia in 2018, the conference explore in detail of diet & personalised nutrition, gastrointestinal microbiome and several case studies with regards to clinical studies, diagnostics studies, treatment methods, biomarker developments, molecular therapy and gastrointestinal diseases.

Also, the 5th Microbiome R&D and Business Collaboration Congress was held in Taiwan, Asia in March 2019, the conference focused on recent developments in gut microbiome, skin microbiome, infant, women and oral health, therapeutics, microbiome and diet.Also, many companies are designing and developing many microbiome therapies. Thus, the increasing focus on human microbiome therapies is the prime factor driving the growth of human microbiome market in the coming years.

Japan is anticipated to lead the adoptions of Human microbiome across the Asia Pacific region through the forecast period.Researchers from Japan are using the outcomes of studies on centenarians in the country to try and produce new products that will replicate the beneficial aspects of their microbiota.

The goal of the collaboration is to solve few of the major technological hurdles in advancing stem cell research. Moreover, Cykinso (Tokyo) received the US$ 2.3 million (270 million yen) funds from the Regional Health Care Industry Support Fund, develop and sell Mykinso or a test kit for intestinal flora. The company plans to use the funds for business development purposes, which include using the data collected from the intestinal flora tests to develop a system for offering nutritional guidance. Thus, the investments and the initiatives taken by the government are likely to propel the growth of the market in the forecast period.

Exhibit: Rest Of Asia Pacific Human microbiome Market Revenue and Forecasts to 2027 (US$ Bn)

ASIA PACIFIC HUMAN MICROBIOME- MARKET SEGMENTATIONBy ProductProbioticsFoodsPrebioticsMedical FoodsDiagnostic DeviceDrugsSupplementsASIA PACIFIC HUMAN MICROBIOME- MARKET SEGMENTATIONBy DiseaseObesityDiabetesAutoimmune DisordersCancerMental DisordersOthersASIA PACIFIC HUMAN MICROBIOME- MARKET SEGMENTATIONBy ApplicationTherapeuticsDiagnostics

By CountryU.S.CanadaMexico

Companies MentionedEnteromeMicroBiome Therapeutics, LLCRebiotix Inc.Yakult Honsha Co., Ltd.Osel Inc.Vedanta Biosciences, Inc.Metabiomics CorporateSynthetic Biologics, Inc.DuPontBiomX Ltd.

Reasons to BuySave and reduce time carrying out entry-level research by identifying the growth, size, leading players and segments in the human microbiome market.Highlights key business priorities in order to assist companies to realign their business strategies.The key findings and recommendations highlight crucial progressive industry trends in the global human microbiome market, thereby allowing players across the value chain to develop effective long-term strategies.Develop/modify business expansion plans by using substantial growth offering developed and emerging markets.Scrutinize in-depth global market trends and outlook coupled with the factors driving the market, as well as those hindering it.Enhance the decision-making process by understanding the strategies that underpin security interest with respect to client products, segmentation, pricing and distribution.Read the full report: https://www.reportlinker.com/p05764187/?utm_source=GNW

About ReportlinkerReportLinker is an award-winning market research solution. Reportlinker finds and organizes the latest industry data so you get all the market research you need - instantly, in one place.

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The Asia Pacific human microbiome market is expected to reach US$ 207.81 Mn in 2025 from US$ 41.73 in 2017 - Yahoo Finance

The 5 Best Traits Of Micropreneurs, The Smallest Of Small Business Owners – Forbes

Even though I've taken a full-time role writing content for a fintech company, I'm still running my content agency part-time. I will always be an entrepreneur at heartnay, a micropreneur, which I've written about many times.

A micropreneur (or microbusiness) is one that operates on a very small scale, with no more than five employees. We micropreneurs are a breed all our own, and there's plenty to admire about us. So let's pat ourselves on the back, shall we?

Whether youre a micropreneur yourself or thinking about hiring or partnering with one, here are the ... [+] key traits that make us so successful as entrepreneurs.

Whether youare a micropreneur yourself or are thinking about hiring or partnering with one, here are some of our best traits:

When there's no one around to help you solve a problem, what do you do? Solve it, of course. Micropreneurs rely on themselves togit-'er-done, and that makes us strong. It's funnynow that I'm working with an extraordinary team of people in my new job, I realize how long I've been problem-solving on my own. I've gotta say, it's kind of amazing to find people whom I can also trust to help find a solution as good as (or better than) what I would have come up with on my own. And the fact that I've been doing that solo for so many years makes me a great asset to the team.

The drawback to this trait:I guess in my personal life, this isn't always an asset. Sometimes my friends just want to vent about a problem they're having, and I'm already on top of trying to solve it!

Once a micropreneur, always a micropreneur. I don't know one person who has owned a business, shut it down, and never started something new. I myself can count at least five businesses I've started (going back to college when I launched Snazzy Baskets, a custom gift basket brand that didn't make it long). I know in my heart I will start more businesses in the future; it's exciting to wonder what they'll be centered on.

It's like our brains are wired to find opportunities. Saying we're opportunistic isn't accurate; it's more that we find gaps in existing solutions or come up with new and better ways to do things. And that is what makes for the innovation that the world turns on.

The drawback to this trait:We are never, ever satisfied. There's always a better way, and looking for it can be exhausting (see #1).

Other Articles FromAllBusiness.com:

Ask 100 micropreneurs how they manage their daily tasks, and you'll get 100 answers. Maybe 102. That's because we don't prescribe to how others do things; we need to forge our own paths. For me, my day consists of constantly being pinged by Google Calendar tasks, as well as Alexa shouting reminders to me from the kitchen. Sometimes, just for fun, I'll write things on paper.

I love that we micropreneur types are unique and that we don't take the path most traveled for anything we do. I love hearing how other business owners manage things and sometimes modify their solutions.

The drawback to this trait: Ever heard the phrase "Don't reinvent the wheel"? Well ... we can't help doing exactly that, over and over.

Being a micropreneur doesn't mean we are always isolated (though, yes, it sometimes does). We don't need guidance, which makes us uber-productive in our home offices away from other humans. But when we are part of a team, we also thrive. We're like the kid in your school group project who essentially carried the slackers. Because we have such high expectations for our own work, we apply the same diligence when we're working with others.

The drawback to this trait: It's probably hard to have such a go-getter on a team for those who don't operate the same way. It can be easy for us to dominate a project. It's the Type A in us coming out.

So, on a personal note, I'm single. I have engaged in more dating app conversations than I care to count, and despite advice telling me not to ask this (apparently, it's a very clich thing to do), I actually like asking what people do for a living. Because I'm genuinely curious.

Ooh, you're an engineer in the aerospace industry? What sort of technologies are we launching into space?

A doctor involved in stem cell research? Tell me more!

I like understanding what people do and what attracted them to that role. I'm the same with my marketing clients: I want to know what makes their businesses tick so I can make it shine through words.

The drawback to this trait: Again, I think a saying communicates it all: "Curiosity killed the cat." When we spend so much time being curious or going down a research rabbit hole (I know about those), we are less productive.

Micropreneurs are entrepreneurs, certainly, but they're also creatures of their own design. If you are a micropreneur, what other qualities do you love about yourself?

RELATED:Take the 4-Week Micropreneur Challenge to Bring Your Small Business to the Next Level

This article was originally published on AllBusiness. See all articles by Susan Guillory.

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The 5 Best Traits Of Micropreneurs, The Smallest Of Small Business Owners - Forbes

Stem Cell Banking Outsourcing Market Competitive Research And Precise Outlook 2019 To 2025 – NY Telecast 99

The Stem Cell Banking Outsourcing Market Perspective, Comprehensive Analysis along with Major Segments and Forecast, 2019-2025. The Stem Cell Banking Outsourcing market report is a valuable source of data for business strategists. It provides the industry overview with market growth analysis with a historical & futuristic perspective for the following parameters; cost, revenue, demands, and supply data (as applicable). The report explores the current outlook in global and key regions from the perspective of players, countries, product types and end industries. This Stem Cell Banking Outsourcing Market study provides comprehensive data that enhances the understanding, scope, and application of this report.

Top Companies in the Global Stem Cell Banking Outsourcing MarketCCBC, CBR, ViaCord, Esperite, Vcanbio, Boyalife, LifeCell, Crioestaminal, RMS Regrow, Cordlife Group, PBKM FamiCord, cells4life, Beikebiotech, StemCyte, Cryo-cell, Cellsafe Biotech Group, PacifiCord, Americord, Krio, Familycord, Cryo Stemcell.

Stem Cell Banking refers to the human stem cell transplantation for the purpose, with acquisition, processing, preservation and provides the ability to differentiate stem cell storage bank, has been called the life bank.

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The Stem Cell Banking Outsourcing market can be divided based on product types and its sub-type, major applications and Third Party usage area, and important regions.

This report segments the global Stem Cell Banking Outsourcing Market on the basis ofTypesare:Umbilical Cord Blood Stem Cell, Embryonic Stem Cell, Adult Stem Cell, Other

On The basis Of Application, the Global Stem Cell Banking Outsourcing Market is Segmented into:Diseases Therapy, Healthcare

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Regions are covered by Stem Cell Banking Outsourcing Market Report 2019 To 2025.

North America, Europe, China, Japan, Southeast Asia, India.North America (USA, Canada and Mexico).Europe (Germany, France, UK, Russia and Italy).Asia-Pacific (China, Japan, Korea, India and Southeast Asia).

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-Detailed overview of Stem Cell Banking Outsourcing Market-Changing Stem Cell Banking Outsourcing market dynamics of the industry-In-depth market segmentation by Type, Application etc.-Historical, current and projected Stem Cell Banking Outsourcing market size in terms of volume and valueRecent industry trends and developments-Competitive landscape of Stem Cell Banking Outsourcing Market-Strategies of key players and product offerings-Potential and niche segments/regions exhibiting promising growth.

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Free country Level analysis for any 5 countries of your choice.Free Competitive analysis of any 5 key market players.Free 40 analyst hours to cover any other data point.

In this study, the years considered to estimate the market size of Stem Cell Banking Outsourcing are as follows:

History Year: 2014-2018Base Year: 2018Estimated Year: 2019Forecast Year 2019 to 2025

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Stem Cell Banking Outsourcing Market Competitive Research And Precise Outlook 2019 To 2025 - NY Telecast 99

Global Stem Cell Reconstructive Market 2019 Revenue, Opportunity, Forecast and Value Chain 2024 – Science of Change

TheGlobalStem Cell ReconstructiveMarket Growth 2019-2024begins with a market overview and covers market research data that is relevant for new market entrants or established players. The report comprehensively prepared with main focus on the segmentation, competitive landscape, geographical growth, market forecast (2019 to 2024) and major market dynamics including drivers, restraints, and opportunities. The report throws light on key production, revenue, and consumption trends. Key strategies of the companies operating in the market along with a detailed analysis of the competition and leading companies of the globalStem Cell Reconstructivemarket has been highlighted in this report. Additionally, a business overview, revenue share, and SWOT analysis of the leading players in the market have been provided in the report.

For each manufacturer covered, this report analyzes its manufacturing sites, capacity, production, ex-factory price, revenue, and market share in the global market. The followingManufacturersare covered:Osiris Therapeutics, NuVasive, Cytori Therapeutics, Takeda (TiGenix), Cynata, Celyad, Medi-post, Anterogen, Molmed

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Global Stem Cell Reconstructive Market 2019 Revenue, Opportunity, Forecast and Value Chain 2024 - Science of Change

Single-cell analysis reveals different age-related somatic mutation profiles between stem and differentiated cells in human liver – Science Advances

INTRODUCTION

Genome integrity is critically important for cellular function. Evidence has accumulated that loss of genome integrity and the increasingly frequent appearance of various forms of genome instability, from chromosomal aneuploidy to base substitution mutations, are hallmarks of aging (1, 2). However, thus far, of all mutation types, only chromosomal alterations could readily be studied directly during in vivo aging using cytogenetic methods (3). Because of their small size, random nature, and low abundance, most somatic mutations are difficult to detect, except in single cells or in clonal lineages (4). In the past, using transgenic reporters, mutations have been found to accumulate with age in a tissue-specific manner (5). However, this approach does not allow a genome-wide, direct analysis of somatic mutations in human primary cells. More recently, using single-cell whole-genome sequencing (WGS), somatic mutations were found to accumulate with age in human neurons (6) and B lymphocytes (7). Others also reported increased somatic mutations in human primary cells isolated from intestine, colon, and liver, albeit in clones propagated from human tissue-specific stem cells (8), which may not be representative of the differentiated cells that ultimately provide tissue function. Nevertheless, together, these studies confirmed that mutations in different somatic cell types of humans accumulate with age.

Here, we present single-cell genome-wide somatic mutation profiles of differentiated human liver hepatocytes as compared with adult liver stem cells (LSCs). Human liver is of particular interest for studying genome instability because of its high metabolic activity and its role in detoxification of xenobiotics, which makes this organ the most important target for genotoxicity in the body. In humans, accumulation of de novo mutations could contribute to the observed age-related loss of liver function, most notably a severe reduction in metabolic capacity, and multiple pathologies, including fatty liver disease, cirrhosis, hepatitis, infections, and cancer (9, 10). Our results indicate high spontaneous mutation frequencies in differentiated hepatocytes that significantly increase with age. By contrast, mutation frequencies in adult LSCs, defined as the cells that give rise to clonal outgrowths, were fairly low. In differentiated hepatocytes, a considerable number of mutations were found in functional parts of the genome. These results indicate that the human liver is subject to a high burden of genotoxicity and that adult stem cells are a critical component in maintaining overall genome integrity within a tissue.

The quantitative detection of de novo somatic mutations in single cells after whole-genome amplification (WGA) and WGS remains a challenge because of the high chance of errors. Here, we used a well-validated, highly accurate method, single-cell multiple displacement amplification (single-cell MDA, or SCMDA) (11), to analyze somatic mutations in single primary hepatocytes from human donors varying in age between 5 months and 77 years. These cells were isolated shortly after death through perfusion of whole livers from healthy human individuals after informed consent by the donors family (Lonza Walkersville Inc.). Cell viability was higher than 80% and, after Hoechst staining, individual, diploid hepatocytes were isolated via fluorescence-activated cell sorting (FACS) into individual polymerase chain reaction (PCR) tubes (fig. S1A). In total, we sequenced four single hepatocytes and bulk genomic liver DNA for each of 12 human donors (table S1). Each cell was subjected to our recently developed procedure for WGA and WGS (11, 12). Somatic single-nucleotide variants (SNVs) in single cells were identified relative to bulk genomic DNAs at a depth of 20 using VarScan2, MuTect2, and HaplotypeCaller with certain modifications (Materials and Methods and table S2). Overlapping mutations from this tricaller procedure were exclusively considered for further analysis. The results were essentially confirmed by using two alternative variant callers: SCcaller (11) and LiRA (Linked Read Analysis) (13).

After adjusting for genomic coverage, the number of SNVs per cell for 48 hepatocytes from 12 donors was found to vary between 357 and 5206 with four extreme outliers of 20,557 to 37,897 SNVs per cell excluded from the statistical model (Fig. 1A and table S2). The number of mutations per cell was found to increase significantly with the age of the donor (P = 1.22 109), with median values of 1222 855 SNVs per cell in the young group (36 years, n = 21 cells), and 4054 1168 SNVs per cell in the aged group (46 years, n = 23 cells), excluding the four outliers (Fig. 1A). The median number of mutations per cell in hepatocytes from the youngest donor was in the same range as what we recently reported for primary human fibroblasts from young donors, i.e., 1027 and 926 SNVs per cell from the 5-month-old and 6-year-old donors, respectively (11, 12). However, during aging, mutation levels increased over the same age range up to 2.5 times higher than in our previously analyzed human B lymphocytes (7) or human neurons analyzed by others (6) (fig. S2A).

(A) SNV levels in individual differentiated hepatocytes. The y axis on the left indicates the number of mutations per cell, and the y axis on the right indicates mutation frequency per base pair. The median values with SDs among four cells of each subject are indicated. Data indicate an exponential increase in mutation frequency with donor age (R = 0.892, P = 1.16 106). bp, base pair. (B) SNV levels in LSC-derived parent clones (red) and their kindred cells (light green) from three young donors. The Venn diagrams indicate the fraction of SNVs detected in the parent clones (collectively for each individual; n = 3) that were also detected in the kindred LSCs. The bars indicate the median mutation frequencies in clones (red) and kindred single cells (light green). (C) Comparison of SNV levels in differentiated hepatocytes (dark green dots; n = 24 from six donors) and LSCs (light green; n = 10 from three donors), all within the young donor group 36 years. Mutation frequencies were corrected for the estimated number of cell divisions. (D) SNV levels in LSCs and differentiated hepatocytes from the same participants, corrected for the estimated number of cell divisions.

At this stage, we were interested in the possible cause of the high mutation frequencies in the four outlier cells. Three of the four outliers with the highest SNV levels revealed multiple mutations in genes involved in DNA repair (table S3) (14), which could conceivably underlie the observed accelerated mutation accumulation in these cells. Of note, individual outlier cells with high mutation levels have been detected in other tissues (6, 7).

Together, these findings indicate that the liver is prone to high levels of de novo somatic mutations, which could possibly be related to its major role in the metabolization and detoxification of xenobiotics.

The mutation frequencies observed in human hepatocytes from older subjects were higher than those previously found in human neurons and B lymphocytes (6, 7). They were also higher than the mutation frequencies reported for stem cellderived liver organoids (fig. S2B) (8). It is critically important to validate the results obtained with single-cell mutation analysis to rule out possible amplification artifacts. In our previous studies on human primary fibroblasts, we validated single-cell data by also analyzing unamplified DNA from clones derived from cells in the same population (11). Here, we generated liver-specific clones from young donors by plating the prepurified hepatocyte cell suspensions in selective medium for LSC expansion (Materials and Methods). Under these conditions, the differentiated hepatocytes died within 5 to 7 days, while the residential LSCs could be propagated without differentiation. The latter was confirmed using biomarker analysis (Materials and Methods and fig. S1B) (15, 16). In addition, we obtained from a commercial source one sample of human postnatal LSCs from a 1-year-old donor at passage 9 (approximately 27 population doublings), which were expanded and also grown into clones in the same way.

LSC clones could be established only from young individuals, i.e., hepatocyte samples from the 1-year-old, 5-month-old, and 18-year-old participants. This is in keeping with observations that resident stem cell properties change with age, with a general reduction in proliferative capacity and increased cellular senescence (17).

Both LSC clones and kindred single cells derived from the young individuals were processed and subjected to WGS, as described above for differentiated hepatocytes. We then tested for the fraction of mutations called in the clones that were also found in the single cells derived from them. As shown in the Venn diagrams (Fig. 1B and fig. S3, A and B), most of these mutations were indeed confirmed in the single cells. This is very similar to what we previously reported for human single fibroblasts and clones derived from the same population of cells (11), which underscores the validity of our single-cell mutation detection method, also in liver cells. Of note, most of the mutations found in the single cells, but not in their parental clones, are likely to be also real. These are likely either mutations missed during variant calling in the clone or de novo mutations arising in the individual cells during clone culture and expansion.

Once we confirmed the validity of our single-cell data, we directly compared mutation frequencies between the single cells defined as LSCs and differentiated hepatocytes, both from the young donor group. Previous studies have provided evidence for lower spontaneous mutation frequencies in stem as compared with differentiated cells (18, 19). For this comparison to be valid, we compared mutation frequencies per cell division in both cell types. This was necessary because the number of cell divisions is a major factor in causing base substitution mutations through replication errors. We first estimated the number of cell divisions that had occurred in human somatic cells of the young age group since the zygote, as described previously (20) (Materials and Methods). We then added, only to the LSCs, the estimated additional numbers of cell divisions during culture (Materials and Methods). The results show that, on a per cell division basis, somatic mutation frequencies were indeed lower in the LSCs than in the differentiated hepatocytes (about twofold), i.e., 11 SNVs versus 21 SNVs per cell per mitosis, respectively (P = 1.26 104, two-tailed Students t test) (Fig. 1C and table S2). A reduced mutation rate in LSCs could explain the fairly modest age-related increase reported previously for stem cellderived organoids (figs. S2B and S3C) (8). The tendency of differentiated hepatocytes to accumulate mutations to a much higher level than stem cells is further confirmed by the significantly higher cell-to-cell variation among the former (P = 1.42 103, Levenes test; Fig. 1, C and D). These observations are in keeping with the idea that stem cells are superior to differentiated cells in preserving their genome integrity, possibly through an enhanced capability to prevent or repair DNA damage (21, 22).

Next, we analyzed the mutational spectra in LSCs and differentiated hepatocytes. In differentiated hepatocytes, the most common mutation types were GC-to-AT transitions and GC-to-TA transversions (Fig. 2A and fig. S4, A and B). These mutations are known to be induced by oxidative damage (23), which itself has often been considered as a main driver of aging and age-related diseases (24). However, the most rapidly increased mutation type with age was the AT-to-GC transition (P = 2.16 1010, two-tailed Students t test; table S4 for Pearsons 2 test). This mutation can be caused by mispairing of hydroxymethyluracil (5-hmU), another common oxidative DNA lesion. Alternatively, AT-to-GC mutations are induced by mutagenic alkyl-DNA adducts formed as a result of thymine residue alkylation (25, 26). Notably, certain minor alkyl-pyrimidine derivatives can escape repair, accumulate during aging, and lead to mutations much later (26, 27).

(A) Relative contribution of the indicated six mutation types to the point mutation spectrum for the five indicated liver sample groups. Data are represented as the mean relative contribution of each mutation type in sample groups of young and aged differentiated hepatocytes (21 cells from six donors 36 years, and 23 cells from six donors 46 years), adult LSC-derived parent clones and their kindred single cells separately, and a group of outlier cells (n = 4). (B) Three mutational signatures (L1, L2, and L3) were de novo identified by non-negative matrix factorization analysis from the somatic mutations in the different groups in (A). (C) Contributions of signatures L1, L2, and L3 to all SNVs in young and aged hepatocytes, young LSCs, and outlier cells.

Mutation spectra of the LSCs and LSC clones revealed a lower fraction of GC-to-AT transitions as compared with differentiated hepatocytes from the young group (Fig. 2A and figs. S3D and S4, A and B). This could be due to the virgin state of these cells, not participating in metabolizing xenobiotics, which is associated with oxidative DNA damage. However, we cannot rule out that, instead, the altered spectrum is related to in vitro culturing, which may alter the ratio of GC-to-AT transitions and GC-to-TA transversions. In the human LSCs derived from clones, the relative frequency of the GC-to-AT transition mutations is slightly, albeit significantly, increased as compared with the parent clones themselves (P = 7.43 104, two-tailed Students t test; table S4 for Pearsons 2 test; Fig. 2A and fig. S4A). Kindred single LSCs, which were derived from parent LSC clones, representing the original LSCs, have undergone multiple rounds of cell division with ample opportunity for replication errors, for example, as a consequence of ambient oxygen to which these cells have been inevitably exposed during subculture. Hence, this would suggest that cell culture has the opposite effect of what we observed from the stem cell versus differentiated cell difference, i.e., increasing rather than decreasing the fraction of GC-to-AT transitions.

To analyze mutation spectra more precisely, we performed non-negative matrix factorization (Materials and Methods) to extract three de novo mutation signatures, signatures L1, L2, and L3, from the mutation spectra of the four groups of human liver cells analyzed, i.e., combined LSCs and clones collectively, differentiated hepatocytes from young participants, differentiated hepatocytes from aged participants, and the four combined outlier cells. We compared these signatures to the COSMIC (Catalogue Of Somatic Mutations in Cancer) signatures described for various human tumors (Fig. 2B and table S5). Signature L1 substantially increased in differentiated hepatocytes from the aged group as compared with hepatocytes and LSCs from young individuals (Fig. 2C). This signature highly correlated with the liver-specific and age-associated mutation signature A dominant in human organoids of liver-specific origin in the aforementioned organoid study (8), as well as with COSMIC signature SBS5, strongly associated with aging (fig. S4C and table S5) (28, 29). Signature L2, with its increased level of oxidative GC > TA transversions, dominated the mutation spectrum of both LSCs and differentiated hepatocytes from young donors (Fig. 2C) and was significantly reduced in cells from the aged donors. Signature L2 highly correlated with COSMIC signatures SBS18 and SBS36, known to be associated not only with oxidative stress (fig. S4C and table S5) but also with proliferation signature C (table S5), found in all in vitro propagated cell types in the aforementioned organoid study (8). Since this signature was dominant in the LSCs, it possibly reflects the stem/progenitor-like origin of hepatocytes and remains dominant in differentiated hepatocytes of the young individuals (Fig. 2C). Signature 3, dominant in the outlier cells, highly correlated with COSMIC signature SBS5, the aging signature, but also correlated with SBS6 and SBS1, signatures associated with DNA mismatch repair deficiency (29).

The above analysis was confirmed when we, instead of extracting de novo signatures from our four groups of liver cell mutation spectra, tested which of the reference COSMIC signatures could be found in these groups (fig. S4C).

Next, we analyzed the distribution of the somatic mutations in human liver cells across the genome. After pooling all mutations of the 21 differentiated cells from the young and the 23 differentiated cells from the old individuals, excluding the four outliers, the large majority of mutations distributed randomly across the genome in both groups (Fig. 3A). We then tested the possibility that during aging, mutations in functionally relevant sequences were selected against, as we previously observed for age-related mutation accumulation in B lymphocytes (7). Here, the functional liver genome was defined as the transcribed liver exome, using available data on gene expression levels in 175 previously described total liver samples [Genotype-Tissue Expression (GTEx) Consortium] (30), and its regulatory regions, identified as promoters of active genes or open chromatin regions, e.g., transcription factor binding regions, identified by ATAC (Assay for Transposase-Accessible Chromatin) sequencing in total liver tissue (ENCODE) (31). Of note, since the databases used were from whole liver, these definitions would not necessarily apply to LSCs or other subpopulations. However, it is reasonable to assume that whole liver is a good surrogate even for those fairly rare liver-specific cells.

(A) Circos diagram of genomic SNV distribution in four groups: pooled LSCs, young and aged hepatocytes, and outlier cells. (B) SNV levels in the functional genome and genome overall in differentiated hepatocytes (left) and in LSCs (right) as a function of age. Each data point represents the ratio of the number of mutations per cell to the median number of mutations of the four cells from the 5-month-old subject. Mutations in the functional genome are shown in red and those in the genome overall in blue. (C) Mutation frequency per base pair in the transcribed part of the liver genome (red) and the nontranscribed part (blue) in differentiated hepatocytes (left) and LSCs (right) as a function of age.

The ratio of total to functional SNVs in differentiated hepatocytes was found to remain about 1 across the different age levels (P = 0.5134, Wilcoxon signed-rank test, two tailed) (Fig. 3B), indicating no selection against deleterious somatic mutations in low-proliferating hepatocyte populations during aging. By contrast, the same ratio in pooled adult LSCs was about 2 and significantly different from that in differentiated hepatocytes (P = 5.34 104, Wilcoxon signed-rank test, two tailed). This suggests selection against deleterious mutations during the cell proliferation cycles that gave rise to these stem cells. It also suggests that LSCs may have an increased capacity to protect their genome simply by remaining quiescent. We also compared mutation frequencies in transcribed versus untranscribed liver cell genes. Transcribed liver genes were defined as genes with expression values 1 transcripts per kilobase per million (TPM), while nontranscribed genome included all sequences with expression values <1 TPM in liver tissue (GTEx) (30). The results indicated a significantly lower number of SNVs affecting transcribed liver genes than nontranscribed genes across all donor ages (P = 7.21 108, Wilcoxon signed-rank test, two tailed) as well as in the LSCs and clones (P = 7.63 106, Wilcoxon signed-rank test, two tailed) (Fig. 3B), suggesting active transcription-coupled repair in normal human liver (32).

Somatic mutations have long been implicated as a cause of aging (33, 34). However, thus far, it has not been possible to test this hypothesis directly because of a lack of advanced methods to analyze random somatic mutagenesis in vivo, which requires high-throughput sequencing of single cells. Using our advanced single-cell sequencing method, we show that the number of somatic base substitution mutations in normal human liver significantly increases with age, reaching as much as 3.3 times more mutations per cell in aged humans than in young individuals. Of note, the numbers of mutations in aged liver are significantly higher than what has previously been reported for aged human liver organoids (fig. S2B) (8) and also higher than recent results reported for aged human neurons (fig. S2A) and B cells (7). Since we essentially ruled out that many of these mutations are artifacts of the amplification system, the most likely cause of this high mutagenic activity in the human liver is the high metabolic and detoxification activity in this organ, which is known to be associated with genotoxicity (35).

Out of 48 hepatocyte cells analyzed, 4 cells revealed extremely elevated mutation loads, over 10 times exceeding SNV levels in age-matched normal hepatocytes even from the same subject. These outliers have also been observed in the only two studies of somatic mutations in human tissues in vivo using a single-cell WGS approach (6, 7). Of the four outliers observed in this present study, multiple de novo SNVs were found to reside in DNA repair genes, strongly suggesting that these mutations were responsible for mutator phenotypes similar to what has been shown for cancers (36). While we cannot know when the mutations that gave rise to rapid mutation accumulation in these cells occurred, this may have been fairly recently, with imminent death of the cells likely. On average, almost 60 nonsynonymous mutations in the functional exome of these cells were found, suggesting a likely functional effect (table S6). However, since we could not longitudinally follow mutation loads in the same single cells, our data do not allow any conclusions on the cause and effect of the observed mutations.

Somatic mutation frequencies in normal differentiated hepatocytes were found to be much higher than in residential LSCs. This means that in vitro clonal surrogates for cells do not always accurately represent the mutation loads of in vivo differentiated cells, which makes predictions of a functional impact of somatic mutations from these clonal data difficult. While we do not know the mechanism(s) of reduced spontaneous mutation loads in stem as compared with differentiated cells, such evidence has also been reported by others (18, 19), and it is possible that stem cells have superior genome maintenance systems as compared with their differentiated counterparts. However, a caveat in this respect is that the LSCs that we enriched for may not in fact be the LSCs giving rise to most of the differentiated hepatocytes. Hence, we cannot be sure that a direct comparison between a stem cell and differentiated cells derived from this stem cell was in fact made.

Another important question is the possible functional impact of random somatic mutagenesis on the aging phenotype. While from our current data we cannot conclude direct cause-and-effect relationships, our observation that the functional part of the genome accumulated numerous mutations suggests that aging-related cellular degeneration and death could at least, in part, be due to somatic mutations. While the occurrence of no more than 11 nonsynonymous mutations in the transcribed exome of liver hepatocytes from humans in their 70s suggests a minor contribution of changes in the protein-coding part of the genome, the well over 100 de novo mutations in gene regulatory sequences may point toward an important role for stochastic gene expression changes in age-related loss of organ function and increased disease incidence. These mutations could possibly increase transcriptional noise, a molecular phenotype that appears characteristic for cells from aged individuals (3739).

Last, while in our current work only base substitution mutations were analyzed, other types of mutations are likely to occur as well. The frequency of most of these mutations, e.g., small insertions and deletions, copy number variation, and genome structural variation, is likely to be much lower than the frequencies of base substitutions observed to rise to thousands of mutations per cell. However, their effects are possibly much larger since they affect a larger part of the genome and, when in exomes, almost always lead to loss of function. It is conceivable that, taken together, de novo mutations could have serious effects on the function of human somatic cells in vivo above and beyond their causal relevance in liver cancer.

Frozen human hepatocyte samples were purchased from Lonza Walkersville Inc. Whole livers for hepatocyte isolation were obtained with the informed consent of families of registered organ donors. The obtained liver organs were rejected for transplant due to either lack of a donor match or morphological alterations (e.g., tearing and hematoma). All 12 selected hepatocyte donors were healthy participants of various age, gender, and ethnicity (table S1) without any liver cancer or other liver pathology history. These cells had been isolated using a gold standard, two-step liver/liver lobe perfusion procedure. Cells were suspended in 2 to 5 ml of media and counted with Trypan blue to estimate viability (higher than 80%), and frozen in dimethyl sulfoxide/liquid nitrogen (www.lonza.com). One specimen of frozen human neonatal LSCs from a 1-year-old donor was purchased from Kerafast Inc. (www.kerafast.com). These cells had been derived by the Sherley laboratory (Boston, MA, USA) and characterized to confirm their stem cell identity (4042).

After thawing, hepatocyte suspensions were used to collect single hepatocytes into individual 0.2-ml PCR tubes with 2.5 l of phosphate-buffered saline (PBS) by means of FACS (FACSAria, Becton Dickenson). Selection of the target hepatocyte population was based on the large cell size of hepatocytes (forward-scatter/side-scatter parameters) along with the additional fluorescence staining for DNA content and cell viability. Briefly, bulk hepatocyte suspension samples were prior stained according to the manufacturers protocol with the viable DNA-binding dye Hoechst 33342 (Life Technologies) to discriminate cells with a standard diploid chromosome set and LIVE/DEAD Cell Vitality Assay Kit C12 Resazurin/SYTOX Green (Thermo Fisher Scientific) to select viable healthy cells. Typical FACS layout is shown in fig. S1A. Upon sorting, tubes with single cells were frozen on dry ice and kept at 80C until use.

Neonatal LSCs of passage 9 (one passage corresponds to approximately three cell population doublings for these cells according to the manufacturers protocol) from the 1-year-old donor were purchased from Kerafast Inc. The commercial LSCs were cultured in polarization media [Dulbeccos modified Eagles medium, 10% dialyzed fetal bovine serum (Invitrogen), 1.5 mM xanthosine (Sigma), 1 penicillin/streptomycin, epidermal growth factor human (20 ng/ml; Invitrogen), transforming growth factor human recombinant (0.5 ng/ml Sigma)] according to the manufacturers protocol (Kerafast Inc.) (4042). These cells served as controls to characterize de novo isolated and polarized LSCs.

Additional LSC cultures were isolated and polarized and characterized from the bulk commercial hepatocyte suspensions (Lonza Walkersville Inc.) from young donors using previously described protocols with specific modifications (15, 16) combined with the aforementioned Kerafast protocol for neonatal LSCs. Briefly, bulk suspension hepatocytes (0.5 106 to 1 106 of cells) were transferred to polarization media as described for the neonatal LSCs and cultured on cell-adhesive 12-well plates for 5 to 7 days. Then, all nonattached hepatocytes were removed, and fresh media were added to the small remaining population of attached progenitor cells. After 1 to 1.5 weeks of culture and media changes, attached cells symmetrically divided, growing to mixed clonal populations of polarized adult LSCs. These cultures were frozen at early passage (p = 3 to 5) until further use. Only LSCs from donors of younger age (22 years) could be isolated in this way.

Phenotypes of the polarized cells were analyzed for the presence of specific surface stem cell and epithelial progenitor cell epitopes, e.g. EpCAM (epithelial cell adhesion molecule), Lgr5, CD90, CD29, CD105, and CD73, upon staining with antibodies by means of multicolor flow cytometry analysis (LSRII, Becton Dickinson) as recommended previously (15, 16, 43, 44). Characteristic FACS profiles and specific phenotypes for commercial LSCs (control) and two manually isolated and polarized LSC lineages are shown in fig. S1B.

Single-cell derived parent clones and their kindred single cells were prepared and collected using CellRaft arrays (Cell Microsystems) as described previously (11). Briefly, an LSC suspension was plated on a CellRaft array consisting of 12,000 individual portable rafts for single cells at the required density of 5000 cells per array. After 4 to 8 hours, individual LSCs were elongated and attached to the array surface locating on individual rafts. After attachment, the medium with floating cells was replaced, and single-cell positions were marked and tracked during the following 7 to 10 days to detect dividing cells and growing individual single-cell derived clones. Once the colony/clone reached confluence on the raft (8 to 10 cells per raft), it was dislocated from the array with a positioned automatic needle and transferred with a magnetic wand to a 96-well plate. Upon reaching confluence, single-cell derived clones were trypsinized and subsequently transferred to 24-well plates, then 12-well plates, 6-well plates, and, lastly, 10-cm plates to reach a total amount of 1.5 106 to 3 106 cells per parent clone. Together, the process of establishing a clone from a single cell took about 25 to 30 days.

Individual single cells from the parent clones were collected, also using CellRafts, and transferred to a 0.2-ml PCR tube containing 2.5 l of PBS. The presence of a single raft was observed under a magnifying glass. Upon single-cell collection, tubes were fast frozen on dry ice and kept on 80C until further use.

Single hepatocytes from each subject were subjected to WGA using our modified procedure of low-temperature cell lysis and DNA denaturation followed by MDA as described (11). As positive and negative controls for WGA, we used 1 ng of human genomic DNA and DNA-free PBS solution, respectively. Resultant MDA products were purified using AMPureXP beads (Beckman Coulter), and the amplified DNA concentration was measured with the Qubit High Sensitivity dsDNA kit (Invitrogen Life Sciences). To verify sufficient and uniformly amplified single-cell MDA products, we performed the eight-target locus-dropout test as described previously (11). Selected confirmed samples (four single-cell MDA products per subject) were further subjected to library preparation and WGS.

Human bulk genomic DNA was collected from total cell suspensions using the DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturers protocol. LSC clonederived DNA was extracted from clones of at least 1.5 106 to 2.5 106 cells in a similar way. DNA concentration was quantified with the Qubit High Sensitivity dsDNA kit (Invitrogen Life Sciences), and DNA quality was evaluated by 1% agarose gel electrophoresis.

The libraries for Illumina next-generation WGS were generated from 0.2 to 0.4 g of genomic DNA, clone-derived bulk DNA, and single-cell MDA DNA human samples using the NEBNext Ultra II FS DNA Library Prep Kit for Illumina (New England BioLabs). The libraries were sequenced with 2 350base pair paired-end reads on an Illumina HiSeq X Ten sequencing platform by Novogene Inc.

Next-generation WGS at a minimal depth of 20X base coverage was performed on four individual mature hepatocytes per human subject (12 human subjects, 48 single cells in total) (table S2). Bulk DNA from two or three LSC-derived clones and MDA products from three to four corresponding kindred single cells per donor (three donors, eight parent clones, and 10 kindred single LSCs) were sequenced similarly.

For all samples, adapter and low-quality reads were trimmed by Trim Galore (version 0.3.7). Quality checks were performed before and after read trimming by FastQC (version 0.11.4). The trimmed reads were aligned to the human reference genome (GRCh37 with decoy) by BWA mem (version 0.7.10) (45). Duplications were removed using samtools (version 0.1.19) (46). The known indels and single-nucleotide polymorphism (SNPs) were collected from the 1000 Genomes Project (phase 1) and Single Nucleotide Polymorphism Database (dbSNP) (build 144). Then, the reads around known indels were locally realigned, and their base quality scores were recalibrated on the basis of known indels and SNVs, both via the Genome Analysis Toolkit (GATK, version 3.5.0) (47).

Somatic mutations between each single cell and the corresponding bulk and between each clone and corresponding bulk were identified using three different variant callers: VarScan2 (48), MuTect2 (49), and HaplotypeCaller (47). To obtain high-quality mutation calls and avoid high false-positive rates in individual callers, we applied a comprehensive procedure in filtering. First, we only considered mutations on autosomes. Then, we considered mutations with a GATK phred-scaled quality score of at least 30 and excluded mutations overlapping with known SNPs from dbSNP. Furthermore, we required a minimum base depth of 20X and filtered mutations with variant-supporting reads in bulk. Moreover, mutations present in at least two cells in each individual were also removed to further exclude potential germline mutations. The mutations present in all three variant callers were considered as true de novo mutations. Last, considering that amplification errors and/or nonuniform coverage could induce false-positive mutations in no more than one-eighth of the reads, we used a binomial distribution to filter these potential false-positive mutations, which excluded most mutations present in 25% of the reads or less. To further check the power of the used pipeline in filtering amplification errors, we also called the somatic mutations using our alternative, the SCcaller tool (11) and the LiRA pipeline (13) (figs. S2A and S3B).

The frequency of somatic SNVs per cell was estimated after normalizing genomic coveragefrequency of somatic SNVs per cell=#somatic SNVssurveyed genometotal size of genome

As the reads were aligned to the haploid reference genome, the frequency of somatic SNVs per base pair was calculated by dividing the frequency of somatic SNVs per cell by genome size and ploidy of the genome (ploidy = 2)frequency of somatic SNVs per base pair=frequency of somatic SNVs per celltotal size of genome*ploidy of genome

The surveyed genome per single cell/clone was calculated as the number of nucleotides with read mapping quality 20 and position coverage 20X.

The outliers of the hepatocytes were defined using Tukeys range test: Four cells were defined as extreme outliers as their frequencies were higher than Q3 + 3 * IQR, where Q3 is the third quartile of the frequencies and IQR is the interquartile range. The outlier cells were excluded from the statistical model.

For the LSC-differentiated hepatocyte comparison, the absolute de novo mutation frequencies were corrected for the number of cell divisions undergone since the zygote (table S2). We used 45.1 as the number of developmental mitoses (20) and assumed a subsequent turnover rate of one cell division per year, based on empirical evidence from rodents (50, 51). In total, 45.5, 46.3, and 61.6 cell divisions were estimated for both LSCs and differentiated hepatocytes from 5-month-old, 1-year-old, and 18-year-old individuals, respectively. For LSCs from 5-month-old, 1-year-old, and 18-year-old individuals, we then added, respectively, an estimated 33, 41.7, and 33 cell divisions during the enrichment process of stem cells, and 21.9, 24.5, and 21.9 cell divisions associated with clonal outgrowth of the single LSCs.

To determine the overlap between SNVs called in the clones and the single cells derived from them, genome coverage in the clone was normalized to that in its kindred single cell. Mutations found in a single cell and appearing in at least 1 read in the parent clone were considered as overlapping. When there were no variant-supporting reads in the clone, the mutation was determined as kindred cell specific. This assignment left some mutations with an unknown status more likely to be de novo mutations arising in the individual cells during clone culture and expansion.

The identified mutations in all individuals were pooled into four groups: LSC cells/clones from young donors, hepatocytes from young and aged donors, and outlier hepatocytes. The integrated spectra of six mutation types in each group were plotted using the R package MutationalPatterns (52). Using non-negative matrix factorization (NMF) decomposition in the same package, we revealed group-specific mutational signatures as well as de novo identified three signatures in normal human liver cells. To identify the potential origin of the mutational spectra, the group mutational signatures and newly revealed signatures to the published signatures associated with liver-specific organoids and various cancer tissues. Three tissue-specific organoid signatures were obtained from a recent study (8); 67 cancer mutation signatures were downloaded from the latest version 3 of the COSMIC database (https://cancer.sanger.ac.uk/cosmic/signatures/SBS/) (28, 29). The cosine similarity between newly identified and published signatures was calculated for comparisons (table S5).

All reported mutations were annotated based on the gene definitions of GRCh37.87. Mutations were further extracted from the functional genome, including transcribed genes, promoters, and open chromatin regions. The nonsynonymous and synonymous mutations were identified by analysis of variance (ANOVA) (53), while damaging and tolerated mutations were checked by SIFT (54) and PROVEAN (55). When damaging (Sorting Intolearnt From Tolerant, SIFT) or deleterious (Protein Variation Effect Analyzer, PROVEAN), the mutation was marked as damaging, and when tolerated (SIFT) and neutral (PROVEAN), a tolerated mutation.

The open chromatin regions were identified by ENCODE transcription factor binding regions in whole genome and ATAC sequencing data in the functional genome in liver tissue samples. Raw ATAC sequencing data were downloaded from ENCODE (experiment name: ENCSR373TDL) (31). The adapter and low-quality ATAC sequencing reads were filtered using Trim Galore (version 0.3.7). Clean reads were aligned to the human reference genome (GRCh37) with Bowtie2 (version 2.2.3; option: -X 2000). Duplicated reads were removed with the Picard tool (version 1.119). Open chromatin regions were determined by MACS2 (version 2.1.1; option: callpeak -g hs --nomodel --shift 100 --extsize 200) (56).

Gene expression levels for total human liver tissue were obtained from GTEx (https://gtexportal.org/) (30). We defined the transcribed genes as those with expression level 1 TPM in all samples. Also, we separated the transcribed and nontranscribed genome by TPM 1 and < 1 in all samples, respectively.

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Single-cell analysis reveals different age-related somatic mutation profiles between stem and differentiated cells in human liver - Science Advances