London: In a major study, researchers have found that Covid-19 patients with cardiovascular comorbidities or risk factors are more likely to develop heart complications while hospitalised, and more likely to die from the virus.

According to the study, published in the journal PLOS ONE, it is crucial for clinicians working with cardiovascular patients to understand the clinical presentation and risk factors for Covid-19 infection in this group.

"For most people, the Novel Coronavirus Disease 2019 (Covid-19) causes mild illness, however, it can generate severe pneumonia and lead to death in others," said study authors from the Magna Graecia University in Italy.

At the time they were admitted to the hospital, 12.89 per cent of the patients had cardiovascular comorbidities, 36.08 per cent had hypertension and 19.45 per cent had diabetes.

The findings showed that cardiovascular complications were documented during the hospital stay of 14.09 per cent of Covid-19 patients.

According to the researchers, the most common of these complications were arrhythmias or palpitations; significant numbers of patients also had myocardial injury.

Myocardial injury is considered acute if there is a rise and fall of cardiac troponin concentrations exceeding biological and analytical variation.

When the researchers analysed the data, they found that pre-existing cardiovascular comorbidities or risk factors were significant predictors of cardiovascular complications, but age and gender were not.

The study showed that both age and pre-existing cardiovascular comorbidities or risk factors were significant predictors of death.

"Cardiovascular complications are frequent among Covid-19 patients and might contribute to adverse clinical events and mortality," the study author concluded.

See the original post:
Covid-19 patients with heart problems more likely to die: Study - ETHealthworld.com

Cardiac Stem Cells Comments Off on Covid-19 patients with heart problems more likely to die: Study – ETHealthworld.com | August 16th, 2020

Global Stem Cell Reconstructive Marketwas valued US$ XX Mn in 2019 and is expected to reach US$ XX Mn by 2027, at a CAGR of 24.5% during a forecast period.

Market Dynamics

The Research Report gives an in-depth account of the drivers and restraints in the stem cell reconstructive market. Stem cell reconstructive surgery includes the treatment of injured or dented part of body. Stem cells are undifferentiated biological cells, which divide to produce more stem cells. Growing reconstructive surgeries led by the rising number of limbs elimination and implants and accidents are boosting the growth in the stem cell reconstructive market. Additionally, rising number of aged population, number of patients suffering from chronic diseases, and unceasing development in the technology, these are factors which promoting the growth of the stem cell reconstructive market. Stem cell reconstructive is a procedure containing the use of a patients own adipose tissue to rise the fat volume in the area of reconstruction and therefore helping 3Dimentional reconstruction in patients who have experienced a trauma or in a post-surgical event such as a mastectomy or lumpectomy, brain surgery, or reconstructive surgery as a result of an accident or injury. Stem cell reconstructive surgeries are also used in plastic or cosmetic surgeries as well. Stem cell and regenerative therapies gives many opportunities for development in the practice of medicine and the possibility of an array of novel treatment options for patients experiencing a variety of symptoms and conditions. Stem cell therapy, also recognised as regenerative medicine, promotes the repair response of diseased, dysfunctional or injured tissue using stem cells or their derivatives.

The common guarantee of all the undifferentiated embryonic stem cells (ESCs), foetal, amniotic, UCB, and adult stem cell types is their indefinite self-renewal capacity and high multilineage differentiation potential that confer them a primitive and dynamic role throughout the developmental process and the lifespan in adult mammal.However, the high expenditure of stem cell reconstructive surgeries and strict regulatory approvals are restraining the market growth.

The report study has analyzed revenue impact of covid-19 pandemic on the sales revenue of market leaders, market followers and disrupters in the report and same is reflected in our analysis.

Global Stem Cell Reconstructive Market Segment analysis

Based on Cell Type, the embryonic stem cells segment is expected to grow at a CAGR of XX% during the forecast period. Embryonic stem cells (ESCs), derived from the blastocyst stage of early mammalian embryos, are distinguished by their capability to distinguish into any embryonic cell type and by their ability to self-renew. Owing to their plasticity and potentially limitless capacity for self-renewal, embryonic stem cell therapies have been suggested for regenerative medicine and tissue replacement after injury or disease. Additionally, their potential in regenerative medicine, embryonic stem cells provide a possible another source of tissue/organs which serves as a possible solution to the donor shortage dilemma. Researchers have differentiated ESCs into dopamine-producing cells with the hope that these neurons could be used in the treatment of Parkinsons disease. Upsurge occurrence of cardiac and malignant diseases is promoting the segment growth. Rapid developments in this vertical contain protocols for directed differentiation, defined culture systems, demonstration of applications in drug screening, establishment of several disease models, and evaluation of therapeutic potential in treating incurable diseases.

Global Stem Cell Reconstructive Market Regional analysis

The North American region has dominated the market with US$ XX Mn. America accounts for the largest and fastest-growing market of stem cell reconstructive because of the huge patient population and well-built healthcare sector. Americas stem cell reconstructive market is segmented into two major regions such as North America and South America. More than 80% of the market is shared by North America due to the presence of the US and Canada.

Europe accounts for the second-largest market which is followed by the Asia Pacific. Germany and UK account for the major share in the European market due to government support for research and development, well-developed technology and high healthcare expenditure have fuelled the growth of the market. This growing occurrence of cancer and diabetes in America is the main boosting factor for the growth of this market.

The objective of the report is to present a comprehensive analysis of the Global Stem Cell Reconstructive Market including all the stakeholders of the industry. The past and current status of the industry with forecasted market size and trends are presented in the report with the analysis of complicated data in simple language. The report covers all the aspects of the industry with a dedicated study of key players that includes market leaders, followers and new entrants. PORTER, SVOR, PESTEL analysis with the potential impact of micro-economic factors of the market has been presented in the report. External as well as internal factors that are supposed to affect the business positively or negatively have been analysed, which will give a clear futuristic view of the industry to the decision-makers.

The report also helps in understanding Global Stem Cell Reconstructive Market dynamics, structure by analysing the market segments and projects the Global Stem Cell Reconstructive Market size. Clear representation of competitive analysis of key players by Application, price, financial position, Product portfolio, growth strategies, and regional presence in the Global Stem Cell Reconstructive Market make the report investors guide.Scope of the Global Stem Cell Reconstructive Market

Global Stem Cell Reconstructive Market, By Sources

Allogeneic Autologouso Bone Marrowo Adipose Tissueo Blood Syngeneic OtherGlobal Stem Cell Reconstructive Market, By Cell Type

Embryonic Stem Cell Adult Stem CellGlobal Stem Cell Reconstructive Market, By Application

Cancer Diabetes Traumatic Skin Defect Severe Burn OtherGlobal Stem Cell Reconstructive Market, By End-User

Hospitals Research Institute OthersGlobal Stem Cell Reconstructive Market, By Regions

North America Europe Asia-Pacific South America Middle East and Africa (MEA)Key Players operating the Global Stem Cell Reconstructive Market

Osiris Therapeutics NuVasives Cytori Therapeutics Takeda (TiGenix) Cynata Celyad Medi-post Anterogen Molmed Baxter Eleveflow Mesoblast Ltd. Micronit Microfluidics TAKARA BIO INC. Tigenix Capricor Therapeutics Astellas Pharma US, Inc. Pfizer Inc. STEMCELL Technologies Inc.

Global Stem Cell Reconstructive Market Request For View Sample Report Page : @https://www.maximizemarketresearch.com/request-sample/54688

About Us:

Maximize Market Research provides B2B and B2C market research on 20,000 high growth emerging technologies & opportunities in Chemical, Healthcare, Pharmaceuticals, Electronics & Communications, Internet of Things, Food and Beverages, Aerospace and Defense and other manufacturing sectors.

Contact info:Name: Vikas GodageOrganization: MAXIMIZE MARKET RESEARCH PVT. LTD.Email: sales@maximizemarketresearch.comWebsite:www.maximizemarketresearch.com

More:
Global Stem Cell Reconstructive Market- Industry Analysis and Forecast (2020-2027) - Good Night, Good Hockey

Cardiac Stem Cells Comments Off on Global Stem Cell Reconstructive Market- Industry Analysis and Forecast (2020-2027) – Good Night, Good Hockey | August 16th, 2020

New York, Aug. 13, 2020 (GLOBE NEWSWIRE) -- Reportlinker.com announces the release of the report "Cell Isolation/Cell Separation Market Research Report by Product, by Cell Type, by Cell Source, by Technique, by Application, by End User - Global Forecast to 2025 - Cumulative Impact of COVID-19" - https://www.reportlinker.com/p05913776/?utm_source=GNW

The Global Cell Isolation/Cell Separation Market is expected to grow from USD 6,356.88 Million in 2019 to USD 14,485.68 Million by the end of 2025 at a Compound Annual Growth Rate (CAGR) of 14.71%.

Market Segmentation & Coverage:This research report categorizes the Cell Isolation/Cell Separation to forecast the revenues and analyze the trends in each of the following sub-markets:

Based on Product, the Cell Isolation/Cell Separation Market studied across Consumables and Instruments. The Consumables further studied across Beads, Disposables, and Reagents, Kits, Media, and Sera. The Instruments further studied across Centrifuges, Filtration Systems, Flow Cytometers, and Magnetic-Activated Cell Separator Systems.

Based on Cell Type, the Cell Isolation/Cell Separation Market studied across Animal Cells and Human Cells. The Human Cells further studied across Differentiated Cells and Stem Cells.

Based on Cell Source, the Cell Isolation/Cell Separation Market studied across Adipose Tissue, Bone Marrow, and Cord Blood/Embryonic Stem Cells.

Based on Technique, the Cell Isolation/Cell Separation Market studied across Centrifugation-Based Cell Isolation, Filtration-Based Cell Isolation, and Surface Marker-Based Cell Isolation.

Based on Application, the Cell Isolation/Cell Separation Market studied across Biomolecule Isolation, Cancer Research, In Vitro Diagnostics, Stem Cell Research, and Tissue Regeneration & Regenerative Medicine.

Based on End User, the Cell Isolation/Cell Separation Market studied across Biotechnology & Biopharmaceutical Companies, Hospitals & Diagnostic Laboratories, and Research Laboratories & Institutes.

Based on Geography, the Cell Isolation/Cell Separation Market studied across Americas, Asia-Pacific, and Europe, Middle East & Africa. The Americas region surveyed across Argentina, Brazil, Canada, Mexico, and United States. The Asia-Pacific region surveyed across Australia, China, India, Indonesia, Japan, Malaysia, Philippines, South Korea, and Thailand. The Europe, Middle East & Africa region surveyed across France, Germany, Italy, Netherlands, Qatar, Russia, Saudi Arabia, South Africa, Spain, United Arab Emirates, and United Kingdom.

Company Usability Profiles:The report deeply explores the recent significant developments by the leading vendors and innovation profiles in the Global Cell Isolation/Cell Separation Market including Beckman Coulter Inc. (Subsidiary of Danaher Corporation), Becton, Dickinson and Company, Bio-Rad Laboratories, Inc., GE Healthcare, Merck KGaA, Miltenyi Biotec, Pluriselect Life Science Ug (Haftungsbeschrnkt) & Co. Kg, Stemcell Technologies, Inc., Terumo Bct, and Thermo Fisher Scientific, Inc..

FPNV Positioning Matrix:The FPNV Positioning Matrix evaluates and categorizes the vendors in the Cell Isolation/Cell Separation Market on the basis of Business Strategy (Business Growth, Industry Coverage, Financial Viability, and Channel Support) and Product Satisfaction (Value for Money, Ease of Use, Product Features, and Customer Support) that aids businesses in better decision making and understanding the competitive landscape.

Competitive Strategic Window:The Competitive Strategic Window analyses the competitive landscape in terms of markets, applications, and geographies. The Competitive Strategic Window helps the vendor define an alignment or fit between their capabilities and opportunities for future growth prospects. During a forecast period, it defines the optimal or favorable fit for the vendors to adopt successive merger and acquisition strategies, geography expansion, research & development, and new product introduction strategies to execute further business expansion and growth.

Cumulative Impact of COVID-19:COVID-19 is an incomparable global public health emergency that has affected almost every industry, so for and, the long-term effects projected to impact the industry growth during the forecast period. Our ongoing research amplifies our research framework to ensure the inclusion of underlaying COVID-19 issues and potential paths forward. The report is delivering insights on COVID-19 considering the changes in consumer behavior and demand, purchasing patterns, re-routing of the supply chain, dynamics of current market forces, and the significant interventions of governments. The updated study provides insights, analysis, estimations, and forecast, considering the COVID-19 impact on the market.

The report provides insights on the following pointers:1. Market Penetration: Provides comprehensive information on the market offered by the key players2. Market Development: Provides in-depth information about lucrative emerging markets and analyzes the markets3. Market Diversification: Provides detailed information about new product launches, untapped geographies, recent developments, and investments4. Competitive Assessment & Intelligence: Provides an exhaustive assessment of market shares, strategies, products, and manufacturing capabilities of the leading players5. Product Development & Innovation: Provides intelligent insights on future technologies, R&D activities, and new product developments

The report answers questions such as:1. What is the market size and forecast of the Global Cell Isolation/Cell Separation Market?2. What are the inhibiting factors and impact of COVID-19 shaping the Global Cell Isolation/Cell Separation Market during the forecast period?3. Which are the products/segments/applications/areas to invest in over the forecast period in the Global Cell Isolation/Cell Separation Market?4. What is the competitive strategic window for opportunities in the Global Cell Isolation/Cell Separation Market?5. What are the technology trends and regulatory frameworks in the Global Cell Isolation/Cell Separation Market?6. What are the modes and strategic moves considered suitable for entering the Global Cell Isolation/Cell Separation Market?Read the full report: https://www.reportlinker.com/p05913776/?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.

__________________________

Originally posted here:
Cell Isolation/Cell Separation Market Research Report by Product, by Cell Type, by Cell Source, by Technique, by Application, by End User - Global...

Bone Marrow Stem Cells Comments Off on Cell Isolation/Cell Separation Market Research Report by Product, by Cell Type, by Cell Source, by Technique, by Application, by End User – Global… | August 16th, 2020

Mitochondria are small organelles that provide the energy critical for each cell in our body, in particular in the high fuel-consuming brain. In this week's edition ofScience, a Belgian team of researchers led by Pierre Vanderhaeghen (VIB-KU Leuven, ULB) finds that mitochondria also regulate a key event during brain development: how neural stem cells become nerve cells. Mitochondria influence this cell fate switch during a precise period that is twice as long in humans compared to mice. The seminal findings highlight an unexpected function for mitochondria that may help explain how humans developed a bigger brain during evolution, and how mitochondrial defects lead to neurodevelopmental diseases.

Our brains are made up of billions of incredibly diverse neurons. They first arise in the developing brain when stem cells stop self-renewing and differentiate into a particular type of neuron. This process, called neurogenesis, is precisely regulated to give rise to the enormous complex structure that is our brain. It is thought that small differences in the way neural stem cells generate neurons are at the origin of the dramatic increase in the size and complexity of our brain.

To gain insight in this complex process, prof. Pierre Vanderhaeghen (VIB-KU Leuven, ULB) and his colleagues examined the mitochondria, small organelles that provide energy in every cell in the body, including the developing brain.

"Diseases caused by defects in mitochondria lead to developmental problems in many organs, in particular the brain," explains Vanderhaeghen, a specialist in stem cell and developmental neurobiology. "We used to think that this was related to the crucial function of mitochondria to provide energy to the cells, but this is only part of the story: recent work in stem cells suggests that mitochondria have a direct influence on organ development. We have tested whether and how this could be the case in the brain."

Ryohei Iwata, a postdoctoral researcher in the Vanderhaeghen lab, developed a new method to watch mitochondria in great detail as the neural stem cells are 'caught in the act' to become neurons. "We found that shortly after stem cells divide, the mitochondria in daughter cells destined to self-renew will fuse, while those in daughter cells that become neurons show high levels of fission instead," says Ryohei Iwata.

But this was not just a coincidence: indeed, the researchers could show that increased mitochondrial fission in fact promotes differentiation to a neuronal fate, while mitochondrial fusion after mitosis redirects daughter cells towards self-renewal.

"We found that the influence of mitochondrial dynamics on cell fate choice is limited to a very specific time window, right after cell division," says Pierre Casimir, a PhD student in Vanderhaeghen's lab. "Interestingly, the restricted time window is twice as long in humans compared to mice."

"Previous findings were primarily focused on fate decision of neural stem cells before they divide, but our data reveal that cell fate can be influenced for a much longer period, even after neural stem cell division," says Vanderhaeghen. This may have interesting implications in the emerging field of cell reprogramming, where scientists try to convert non-neuronal cells directly in neuronal cells for therapeutic purposes for instance.

"Since this period of plasticity is much longer in human cells compared to mouse cells, it is tempting to speculate that it contributes to the increased self-renewal capacity of human progenitor cells, and thus to the uniquely developed brain and cognitive abilities of our species. It is fascinating to think that mitochondria, small organelles that have evolved in cells more than a billion years ago, might have contributed to the recent evolution of the human brain."

Reference:Iwata, R., Casimir, P., & Vanderhaeghen, P. (2020). Mitochondrial dynamics in postmitotic cells regulate neurogenesis. Science, 369(6505), 858862. https://doi.org/10.1126/science.aba9760

Link:
To Become a Nerve Cell, Timing Is of the Essence - Technology Networks

Skin Stem Cells Comments Off on To Become a Nerve Cell, Timing Is of the Essence – Technology Networks | August 15th, 2020

Efforts to get the two to breed had not worked.

He was the equivalent of a 70-year-old man, so of course you dont expect the sperm to be all that good, said John Payne of the Borneo Rhino Alliance (BORA), who has campaigned for about four decades to save Malaysias rhinos.

It was obvious that, to increase the chances of success, one should get sperm and eggs from the rhinos in Indonesia. But right till today, Indonesia is still not keen on this.

ACROSS THE BORDER

Indonesias environment ministry disputed accusations of cross-border rivalry as a reason why Malaysias rhinos died out, saying talks continue on ways to work with conservationists in the neighboring southeast Asian nation.

Because this is part of diplomatic relations, the implementation must be in accordance with the regulation of each country, said Indra Exploitasia, the ministrys director for biodiversity conservation.

The Malaysian scientists plan to use cells from the dead rhinos to produce sperm and eggs that will yield test-tube babies to be implanted into a living animal or a closely related species, such as the horse.

The plan is similar to one for the African northern white rhinoceros, which number just two. Researchers in that effort reported some success in 2018 in producing embyronic stem cells for the southern white rhino.

But the process is still far from producing a whole new animal, say Thomas Hildebrandt and Cesare Galli, the scientists leading the research.

Link:
Scientists hope to bring Malaysian rhinoceros back from extinction with stem cell technology - National Post

Skin Stem Cells Comments Off on Scientists hope to bring Malaysian rhinoceros back from extinction with stem cell technology – National Post | August 15th, 2020

Image: Steve Wrubel/Parfums Christian Dior By Chloe Pek August 14, 2020

The beauty expert counts Bella Hadid and Kim Kardashian amongst her clientele

Sign up for our newsletters to get all our top stories delivered.

You've Successfully Subscribed

When it comes to facial massages, celebrity facialist Joanna Czech has that magic touch. Counting Hollywood and runway bigwigs like Bella Hadid, Kim Kardashian, Jennifer Aniston, Cate Blanchett, Kate Winslet, Liam Neeson and more amongst her star-studded list of clients, Joannas skincare treatments are highly coveted internationally.

Now, the skincare expert is adding another credential to her portfoliojoining the House of Dior as a skincare ambassador and lending her expertise in developing the Dior skincare techniques international training.

In an interview via Zoom, Czech told us she had her reservations about joining Dior Skincare at first. I mean, theyre famous for make-up and fashion. I couldnt put my name next to fashion skincareIm very particular about that. Then, I heard about how the Capture Totale range stimulates cell energy, so that changed everything and my skin as well.

Czech, who originally planned to go to medical school in her schooling years, fell in love with skincare when she enrolled in the Aesthetics Institute and never looked back. However, she has remained inquisitive and fascinated with science. This is evident from her holistic approach to beauty, which combines both traditional techniques and cutting-edge technology.

See also: The Best Beauty Launches In August 2020

Adenosine triphosphate (ATP, the main carrier of energy for cellular activities) is responsible for the very first mitosis of cells. It is human physiology that production of ATP drastically drops at around seven years old, and the energy keeps slowing down. So any treatment or product that would stimulate cell energy is fascinating to me.

Culminated from Diors decades-long research into stem cells, the Capture Totale line is infused with a regenerative floral complex of Madagascan longozo, Chinese peony, white lily, and Chinese jasmine, which help to re-energise stem cells rather than replenish them.

Dont believe a product that says it contains stem cells because the stem cells are not alive within the product. Only stem cells that are directly re-injectedand most likely come from your bone marrowworks, Joanna explained, debunking one of the most popular beauty fads in recent years.

With the expert on the line, we took the opportunity to ask our burning questions about maskneand skincare misconceptions.

What is a skincare philosophy that you live by?

Respect, support and protect. This goes for skincare, how we treat ourselves and others.

Your all-time favourite Dior Skincare product and why?

The Capture Totale C.E.L.L. Energy Super Potent Serum because it contains the most concentrated version of the cell energising complex and acetylated hyaluronic acid. It creates hydrated, plump and radiant skin. If you are consistent, you see results in days. My skin has never looked better.

A common skincare mistake many people make?

In my opinion, its using toner. Thats a misconception because still, many people use a toner as the second step of cleansing as opposed to the first step of treating the skin, and this is from my experience of talking with clients.

They put toner or micellar water on a cotton pad and they keep wiping and seeing more make-up. If you see more make-up on your cotton pad, that means you need to go back into washing.

Toner is very often misunderstood or skipped, and it shouldnt be. I cant imagine, for instance, applying a serum on my face without applying toner first. There is no way the efficacy of the product will be the same if you have not applied a toner. Depending on the toner, they offer hydration and sometimes micro-exfoliation, but mainly they are used to maintain the pH of the skin. The optimal pH for our skin is 5.5, and many factors from our diet to lifestyle, and even washing our face can throw the skins pH off the scale, so it's very important to balance it back.

See also: Lancme's Celebrity Make-Up Artist Lisa Eldridge & Neelofa Share 5 Beauty Tips

With face masks becoming part of everyday life, maskne has become a real problem. How can we prevent these breakouts?

When you wear a mask, it creates a micro-climate and we keep breathing carbon dioxide back and forth, so there is not enough of anti-bacterial oxygen getting into the skin. There is sometimes too much moisture happening, so we will get super hydrated initially, and then get quite dehydrated right after. Thats when you will experience eczema and redness.

What I recommend is keeping the skin as clean as possible before wearing the mask, with just a balancing toner, and protecting balm or healing ointments to lubricate areas where the mask could potentially irritate the skin. Very often, its on the nose bridge, as well as on the side and behind the ears.

For less reactivity, I wouldnt go through with the whole routine, but if you have to, I would advise starting your routine earlier so the products are on your skin for at least 30-40 minutes. If you will be stepping out shortly, reduce the routine and skip some steps. But no matter what, never forget about your SPF because the friction of the mask could also get rid of our stratum corneum and create little scabs, causing discolourations.

Then, as soon as you arrive home, take the mask off, wash your face, and again balance your skin with toner and use your serums.

What are your tips for soothing breakouts or eczema caused by wearing masks?

Even with microscopic breakouts, I would continue using any product that is hydrating because sometimes we misunderstand we have a breakout and then we try to use benzoyl peroxide, or everything that is dehydrating. No, your skin would be producing even more sebum. So keep hydrating your skin with soothing ingredients like colostrum and hyaluronic acid.

Your skincare routine?

My morning routine is very brief: cleanser, toner, a serum and then there is moisturiser. For my night time routine, my very first step is getting into the shower when I get home. I begin with massaging my body with my shower gel and silicone gloves under the shower, then I apply products like multi-vitamin oils and sometimes micro-exfoliating toners all over my body.

Then, I go to my face. I usually dont wear any make-up, so I start right away with my cleanser with some massaging movements and I remove it with a linen washcloth, followed by a toner. My favourite way of applying toner is the sponge techniqueinitially, you spread the product on your face, and then you press and release. When you press, your skin microscopically opens and when you release, the skin grasps whatever is on the surface.

After the toner, I use my serum. Ive been using the Capture Totale C.E.L.L. Energy Super Potent Serum since September. If you have weaker areas like forehead lines and nasolabial folds, these are the areas I would concentrate longer on, followed by an eye cream and moisturiser. Thats usually my five-step basic routine.

About twice a week, I do facial masks, one of those quick ones because Im the kind of New Yorker who only has five seconds for myself. But no matter how busy or tired I am at night, I would never forget about my skincare routine. Your skin is 60 per cent more potent to absorb everything during relaxation and rejuvenation time. So if you dont take care of your skin at night, you might as well forget doing anything in the morning. Twenty-five per cent of our immune system is within our skin, and we can improve that percentage of our health with products chosen for your skin condition and consistent skincare.

See also: Sulwhasoo's Ginseng To Achieve Skin As Flawless As South Korean Superstar Song Hye Kyo's

You work with many notable clientswhats the most common skincare problem celebrities deal with and treatments that they request for?

Celebrities have exactly the same problems as we do. The only one little difference is that celebrities tend to wear more make-up and more often than some of us, usually under the heat of theatrical or film lights, so they need a lot of hydration and rebalancing. Ive been called to the movie set many times to help soothe their skin with algae masks, or any cooling and hydrating treatments.

Their needs are equal to ours. They want to work on their facial contours and ensure their skin is as evenly textured as possible so their makeup looks perfect, so I would offer some mild exfoliation, perhaps micro-currents for targeted muscle stimulation, and maybe manual massaging to stimulate blood flow to create the sort of rosy healthy oxygenated looking skin. Many people call it a red carpet facial but I call them ordinary facial because every woman wants the samesmooth hydrated skin with nice cheekbones, beautiful jawlines, and thats what really works.

See also: Beauty Talk With Aznita Azman, Founder Of Nita Cosmetics

The rest is here:
Dior Skincare Ambassador Joanna Czech On Her Self-Care Routine And How To Prevent Maskne - Tatler Malaysia

Skin Stem Cells Comments Off on Dior Skincare Ambassador Joanna Czech On Her Self-Care Routine And How To Prevent Maskne – Tatler Malaysia | August 15th, 2020

DUBLIN--(BUSINESS WIRE)--The "Global Autologous Cell Therapy Market: Growth, Trends and Forecasts (2020-2025)" report has been added to ResearchAndMarkets.com's offering.

The Global Autologous Cell Therapy market is anticipated to grow at a CAGR of 15.9% during the forecast period.

The major factors attributing to the growth of the autologous cell therapy market are the rising incidence of chronic diseases such as autoimmune diseases, cancer, blood disorder, and others.

A rise in the population suffering from chronic diseases is also propelling the demand for market growth. In 2018, as per the AARDA (American Autoimmune Related Diseases Association) statistics, around 50 million Americans have an autoimmune disease, and this number is expected to rise in the future.

As per the CDC (Centers for Disease Control and Prevention) estimates Sickel Cell Disease (SCD) affects around 100,000 Americans annually - and there are few more factors which are playing crucial roles in taking the autologous cell therapy market to the next level, among them one is on-going drug developments for new applications which are expected to further propel the growth of the autologous cell therapy market.

Key Market Trends

Bone Marrow Segment Expected to Hold the Largest Market Share

Bone marrow transplant is a technique for replacing damaged and destroyed cells with new stem cells in the bone marrow. Bone marrow is the most commonly used for autologous cell therapy as it can benefit individuals with a range of cancer (malignant) and non-cancer (benign) diseases and will drive the market during the forecast period.

As per the statistics from Globocan 2018, worldwide 18,078,957 individuals have cancer. Asia remains the leading contributor in the rising incidence of cancer with a reported share of 48.4% followed by Europe, North and Latin America, Africa, and Oceania with a share of 23.4%, 13.2% and 7.8%, 5.8%, and 1.4% respectively.

North America Dominates the Market and is Expected to do Same Over the Forecast Period

North America is expected to dominate the overall autologous cell therapy market, throughout the forecast period. This is owing to factors such as the rising incidence of chronic diseases such as cancer, blood disorder, autoimmune diseases, and other diseases and the availability of advanced healthcare infrastructure among the major factors.

In North America, the United States holds the largest market share owing to the factors such as increasing number of population suffering from cancer and other chronic diseases, along with the rising geriatric population and developments related to stem cell therapy and rising demand for biotechnological practices in the country, is anticipated to further drive the demand in this region.

Competitive Landscape

The autologous cell therapy market is moderately competitive and consists of several major players. In terms of market share, few of the major players are currently dominating the market. And some prominent players are vigorously making acquisitions and joint ventures with the other companies to consolidate their market positions across the globe.

Some of the companies which are currently dominating the market are Vericel Corporation, Pharmicell Co. Inc., Holostem Terapie Avanzate S.r.l., Lineage Cell Therapeutics Inc., and Opexa Therapeutics.

Key Topics Covered

1 INTRODUCTION

1.1 Study Deliverables

1.2 Study Assumptions

1.3 Scope of the Study

2 RESEARCH METHODOLOGY

3 EXECUTIVE SUMMARY

4 MARKET DYNAMICS

4.1 Market Overview

4.2 Market Drivers

4.2.1 Rising Incidence of Chronic Diseases

4.2.2 Emphasis Increasingly on Drug Development for New Applications

4.3 Market Restraints

4.3.1 Systemic Immunological Reactions Possibility

4.3.2 Expensive Practise, Product and High Capital Investment

4.4 Porter's Five Force Analysis

5 MARKET SEGMENTATION

5.1 By Therapy

5.1.1 Autologous Stem Cell Therapy

5.1.2 Autologous Cellular Immunotherapies

5.2 By Application

5.2.1 Oncology

5.2.2 Musculoskeletal Disorder

5.2.3 Blood Disorder

5.2.4 Autoimmune Disease

5.2.5 Others

5.3 By Source

5.3.1 Bone Marrow

5.3.2 Epidermis

5.3.3 Others

5.4 By End User

5.4.1 Hospitals

5.4.2 Research Centers

5.4.3 Others

5.5 Geography

5.5.1 North America

5.5.2 Europe

5.5.3 Asia-Pacific

5.5.4 Middle-East and Africa

5.5.5 South America

6 COMPETITIVE LANDSCAPE

6.1 Company Profiles

6.1.1 Vericel Corporation

6.1.2 Pharmicell Co. Inc.

6.1.3 Holostem Terapie Avanzate S.r.l.

6.1.4 Lineage Cell Therapeutics, Inc.

6.1.5 Opexa Therapeutics

6.1.6 BrainStorm Cell Therapeutics

6.1.7 Sangamo Therapeutics

7 MARKET OPPORTUNITIES AND FUTURE TRENDS

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

Continue reading here:
World Autologous Cell Therapy Industry 2020-2025 with Vericel, Pharmicell, Holostem Terapie Avanzate, Lineage Cell Therapeutics and Opexa Therapeutics...

Bone Marrow Stem Cells Comments Off on World Autologous Cell Therapy Industry 2020-2025 with Vericel, Pharmicell, Holostem Terapie Avanzate, Lineage Cell Therapeutics and Opexa Therapeutics… | August 15th, 2020

Companies are exhorting expectant parents to protect their baby from the medical evils that lie ahead. But are claims of benefits overblown?

By: Mikkael A. Sekeres

My patient, a man in his 70s, sat a few feet away from me in a clinic room at our cancer center. His wife was by his side, both literally and emotionally she was his touchstone, his connection to the normal life he led before his leukemia diagnosis. I noticed they tended to wear outfits that even complemented each other, as if their sartorial choices had harmonized and become intertwined along with their affection over the 40 years of their marriage. Their choice for the day: grey sweatshirts declaring their allegiance to the hapless Cleveland Browns.

He had weathered the slings and arrows of the chemotherapy we used to treat his cancer during a five-week hospital stay, and now was in a tenuous remission. We talked about next steps in his treatment, which ranged from giving him a break, to more chemotherapy, to considering the most aggressive intervention we could offer a bone marrow transplant.

The phrase bone marrow transplant was a bit of a misnomer, though. While we could wipe out any residual leukemia in his bone marrow with high-dose chemotherapy and replace his fresh bone marrow from a healthy person, we may not be able to find a good bone marrow match. Another potential option: We could use umbilical cord blood from a newborn, which is rich in the stem cells normally found in the bone marrow, and which recent studies have shown may not need to match as closely as is necessary for a marrow donor. Hearing this, my patients wife interjected.

Our daughter is pregnant, and her due date is next month. She started, glancing at my patient as he nodded his head in agreement. She wanted us to ask if she should save the babys cord blood in case he needs it for a transplant.

I explained to them that the babys cord blood was unlikely to be a close enough match to my patient, as my patients daughter would only be a half-match for him, and her baby less than that. My patient then asked me a question I have been hearing more and more over the years: Should my daughter save the cord blood in case our grandbaby needs it, in case he or she develops cancer?

Brochures for these companies line Plexiglas display cases in obstetrics offices, with pamphlets exhorting nervous, expectant parents to protect their baby from the medical evils that lie ahead.

Indeed, in the U.S., the practice of storing umbilical cord blood is steadily on the rise. Banking cord blood in case a bone marrow transplant is needed in the future is appealing on so many levels. The umbilical cord attaching the developing fetus to its mothers placenta is rich in those juicy bone marrow stem cells that are so effective at making the blood components. Coming from an infant at the time of birth, they should be uncorrupted by cancer (emphasis on the should, as well see in a moment). Cord blood is also easy to collect: At the time of delivery, after the cord is cut, the remaining blood in that cord is milked out into a collection bag. That bag is then kept in a freezer until the time comes, if ever, when it is needed and can be infused as a transplant.

The cost for using commercial cord blood banking companies, however, can be substantial. Upfront charges with whats called an enrollment fee can range from $1,500 to $3,500. On top of that, a yearly storage fee is assessed, with the total amount for 18 to 20 years of storage cresting $5,000 in some cases.

Brochures for these companies line Plexiglas display cases in obstetrics offices, with pamphlets exhorting nervous, expectant parents to protect their baby from the medical evils that lie ahead. What better source for a transplant than a childs own, pure stem cells, harvested at a time years before that child ever developed cancer? But cost aside, is the effort even worth it for the risk that a child may one day develop a cancer and need a future transplant?

To answer this question, we need to take a couple of things into consideration. First, what is the likelihood of a child developing a cancer, and then needing a transplant to treat that cancer? A study conducted by the Center for International Blood and Marrow Transplant Research attempted to figure this out. They first identified the cancers for which transplantation could potentially be needed. For people aged 0 to 19 years (the length of time a cord blood would be kept banked) leukemia was the most common, followed by lymphoma, neuroblastoma, brain tumors, and sarcomas. Cancer in children and adolescents are rare all told, the incidence rate in the United States for all of these cancers combined is about 12 per 100,000 children per year. Its horrible if its your child who develops cancer, but pediatric cancer is still an uncommon event.

Its horrible if its your child who develops cancer, but pediatric cancer is still an uncommon event.

The next conclusion is based on the likelihood that these cancers would not be eradicated by chemotherapy and/or radiation therapy and would require an allogeneic transplant that is, one that uses stem cells taken from a genetically matched donor and the assumption that everyone could identify a sibling or brother from another mother transplant and was healthy enough to undergo the procedure. The authors estimated that the incidence rate of transplant for children and adolescents was a little over 2 per 100,000 per year in the United States during their first two decades of life. Analyzed another way, the probability a child will need a transplant by the time he or she reaches age 20 is 0.04 percent.

The lifetime chance of getting struck by lightning is similar, at about 1 in 3,000, or 0.033 percent.

Would you pay thousands of dollars for a medication right now, in the event that sometime in your life you may be struck by lightning, and that medication may help you survive the lightning strike?

Seems excessive to me.

A second way of determining the value of cord blood banking in case a child develops cancer is to consider whether that cord blood is really as pure as we think. The most common childhood cancer through age 19 is leukemia, with an annual incidence rate of 4.7 per 100,000 children in the United States. Could it be possible that the leukemia was present at some small level even at birth, years before the child was diagnosed with leukemia?

One approach to studying this would be to screen every newborn for leukemia. Given the incidence rate of childhood leukemia, this would mean subjecting over 21,000 babies to a blood test for every case of future leukemia identified.

Its difficult to justify that type of monumental screening effort to answer a research question about the origins of leukemia. A more reasonable approach would be to identify children who have leukemia, and try to determine whether they had it when they were born.

But how to go about obtaining a blood sample from a birth that occurred years earlier? A group of clever scientists from the United Kingdom and Germany thought the answer might be found in something called Guthrie cards. Robert Guthrie was a microbiologist working at the Roswell Park Cancer Institute in Buffalo, New York, in the 1950s when his niece was diagnosed with phenylketonuria (PKU), an inherited deficiency in the enzyme necessary to metabolize the amino acid phenylalanine. If caught early enough, an infants diet can be modified so that the effects of the deficiency are minimized. If not, the condition can lead to developmental defects and mental disability.

Guthries niece was not so lucky.

This, and having a child of his own with cognitive delays, motivated Guthrie to devote his career to detecting preventable childhood diseases. He developed a test for PKU that could be performed when a drop of blood from a finger prick or heel stick was applied to filter paper on a card. It was successfully piloted in Newark in 1960, and by 1963, 400,000 infants had been tested in 29 states. Testing spread around the country, and across the pond.

And hospital laboratories kept those Guthrie cards for years after a child was born.

Could it be possible that the leukemia was present at some small level even at birth, years before the child was diagnosed with leukemia?

The scientists found three children with acute lymphocytic leukemia (more common in children than AML, whereas the opposite is true in adults) who had the same chromosome mutation associated with their leukemias a translocation of chromosomes 4 and 11. After obtaining permission from the parents of these children, the scientists then searched laboratory repositories to find the Guthrie cards stored there from when the children were born. They used a PCR-specific lab test for this translocation on the dried blood still remaining on the childrens Guthrie cards, and were able to detect the chromosome abnormality for all three children from a blood drop obtained months or years before the leukemia was diagnosed. In another, similar study, the same group of scientists was able to detect chromosome evidence of leukemia in 9 of the 12 Guthrie cards obtained from children who diagnosed with leukemia between two and five years later.

The leukemia was there all along, even prior to birth in these children, waiting years in some cases to rear its ugly head. And if the leukemia was measurable on a genetic level in their blood, it was almost certainly present in their cord blood. Banking cord blood from these children would have preserved those juicy, healthy stem cells, but also probably cells already corrupted by genetic abnormalities that would lead to leukemia again, if the cells were re-infused into a child as a transplant years later.

Getting back to the question: Is the cost and effort of banking cord blood worth it for the risk that a child may one day develop a cancer and need a future transplant?

I didnt think so when my three children were born.

But I did have their cord blood collected and I donated it to be stored for use through the Be The Match program, in case a complete stranger needs it. So that one day, my children could be the brothers from another mother, or sister from another mister me being the mister!

And so that one day, my patients wont have to forego potentially curative treatments for their leukemias because they cant find an adequate donor.

Mikkael Sekeres is the Director of the Leukemia Program at the Cleveland Clinic and the author of When Blood Breaks Down: Life Lessons from Leukemia, from which this article is adapted.

See the original post:
The Fallacy of Banking Umbilical Cord Blood for Your Baby - The MIT Press Reader

Bone Marrow Stem Cells Comments Off on The Fallacy of Banking Umbilical Cord Blood for Your Baby – The MIT Press Reader | August 15th, 2020

Latest released the research study onGlobal Hematopoietic Stem Cell Transplantation Market, offers a detailed overview of the factors influencing the global business scope.Hematopoietic Stem Cell TransplantationMarket research report shows the latest market insights, current situation analysis with upcoming trends and breakdown of the products and services. The report provides key statistics on the market status, size, share, growth factors of theHematopoietic Stem Cell Transplantation Market. The study covers emerging players data, including: competitive landscape, sales, revenue and global market share of top manufacturers.

Top players in Global Hematopoietic Stem Cell Transplantation Market are:

Gilead Sciences Inc. (United States)

Thermo Fisher Scientific (United States)

PromoCell (Germany)

CellGenix Technologie Transfer GmbH (Germany)

Cesca Therapeutics Inc.(United States)

R&D Systems (United States)

Genlantis (United States)

Lonza Group Ltd.(Switzerland)

TiGenix N.V.(Belgium)

ScienCell Research Laboratories (United States)

Regen Biopharma Inc. (United States)

China Cord Blood Corp (Hong Kong)

CBR Systems Inc. (United States)

Free Sample Report + All Related Graphs & Charts @: https://www.advancemarketanalytics.com/sample-report/69543-global-hematopoietic-stem-cell-transplantation-market-1

Brief Overview on Hematopoietic Stem Cell Transplantation

Despite the increasing availability of smart antineoplastic therapies in recent years, Hematopoietic stem cell transplantation (HSCT) remains an optimal treatment modality for many hematologic malignancies. HSCT is one of a range of therapeutic options which is available to patients suffering from various diseases. It is a widely accepted treatment for many life-threatening diseases. The treatment is available to patients who suffer from refractory or relapsing neoplastic disease and non-neoplastic genetic disorders, as well as from chronic bone marrow failure. Hematopoietic stem cells are young or immature blood cells which are found to be living in bone marrow. These blood cells when matures in bone marrow very few enters into bloodstream. These cells that enter bloodstream are called as peripheral blood stems cells. Hematopoietic stem cells transplantation is the replacement of absent, diseased or damaged hematopoietic stem cells due to chemotherapy or radiation, with healthy hematopoietic stem cells.

Recent Development in Global Hematopoietic Stem Cell Transplantation Market:

In January 2019, Paul-Ehrlich Institute discovered important surface molecules supporting hematopoietic recovery after transplantation of blood stem cells. The protein C receptor on hematopoietic stem cell improves stem cell transplantation. Transplantation of blood stem cells (HSCTs) is an important treatment for the patients with hematopoietic disorders

Keep yourself up-to-date with latest market trends and changing dynamics due to COVID Impact and Economic Slowdown globally. Maintain a competitive edge by sizing up with available business opportunity in Hematopoietic Stem Cell Transplantation Market various segments and emerging territory.

Market Drivers

Market Trend

Market Challenges

Market Restraints:

Market Opportunities:

Region Included are: North America, Europe, Asia Pacific, Oceania, South America, Middle East & Africa

Country Level Break-Up: United States, Canada, Mexico, Brazil, Argentina, Colombia, Chile, South Africa, Nigeria, Tunisia, Morocco, Germany, United Kingdom (UK), the Netherlands, Spain, Italy, Belgium, Austria, Turkey, Russia, France, Poland, Israel, United Arab Emirates, Qatar, Saudi Arabia, China, Japan, Taiwan, South Korea, Singapore, India, Australia and New Zealand etc.

Enquire for customization in Report @: https://www.advancemarketanalytics.com/enquiry-before-buy/69543-global-hematopoietic-stem-cell-transplantation-market-1

Strategic Points Covered in Table of Content of Global Hematopoietic Stem Cell Transplantation Market:

Chapter 1: Introduction, market driving force product Objective of Study and Research Scope the Global Hematopoietic Stem Cell Transplantation market

Chapter 2: Exclusive Summary the basic information of the Global Hematopoietic Stem Cell Transplantation Market.

Chapter 3: Displaying the Market Dynamics- Drivers, Trends and Challenges of the Global Hematopoietic Stem Cell Transplantation

Chapter 4: Presenting the Global Hematopoietic Stem Cell Transplantation Market Factor Analysis Porters Five Forces, Supply/Value Chain, PESTEL analysis, Market Entropy, Patent/Trademark Analysis.

Chapter 5: Displaying the by Type, End User and Region 2013-2020

Chapter 6: Evaluating the leading manufacturers of the Global Hematopoietic Stem Cell Transplantation market which consists of its Competitive Landscape, Peer Group Analysis, BCG Matrix & Company Profile

Chapter 7: To evaluate the market by segments, by countries and by manufacturers with revenue share and sales by key countries in these various regions.

Chapter 8 & 9: Displaying the Appendix, Methodology and Data Source

Finally, Global Hematopoietic Stem Cell Transplantation Market is a valuable source of guidance for individuals and companies.

Data Sources & Methodology

The primary sources involve the industry experts from the Global Hematopoietic Stem Cell Transplantation Market including the management organizations, processing organizations, analytics service providers of the industrys value chain. All primary sources were interviewed to gather and authenticate qualitative & quantitative information and determine the future prospects.

In the extensive primary research process undertaken for this study, the primary sources Postal Surveys, telephone, Online & Face-to-Face Survey were considered to obtain and verify both qualitative and quantitative aspects of this research study. When it comes to secondary sources Companys Annual reports, press Releases, Websites, Investor Presentation, Conference Call transcripts, Webinar, Journals, Regulators, National Customs and Industry Associations were given primary weightage.

Get More Information: https://www.advancemarketanalytics.com/reports/69543-global-hematopoietic-stem-cell-transplantation-market-1

What benefits does AMA research study is going to provide?

Definitively, this report will give you an unmistakable perspective on every single reality of the market without a need to allude to some other research report or an information source. Our report will give all of you the realities about the past, present, and eventual fate of the concerned Market.

Thanks for reading this article; you can also get individual chapter wise section or region wise report version like North America, Europe or Asia.

About Author:

Advance Market Analytics is Global leaders of Market Research Industry provides the quantified B2B research to Fortune 500 companies on high growth emerging opportunities which will impact more than 80% of worldwide companies revenues.

Our Analyst is tracking high growth study with detailed statistical and in-depth analysis of market trends & dynamics that provide a complete overview of the industry. We follow an extensive research methodology coupled with critical insights related industry factors and market forces to generate the best value for our clients. We Provides reliable primary and secondary data sources; our analysts and consultants derive informative and usable data suited for our clients business needs. The research study enables clients to meet varied market objectives a from global footprint expansion to supply chain optimization and from competitor profiling to M&As.

Contact Us:

Craig Francis (PR & Marketing Manager)AMA Research & Media LLPUnit No. 429, Parsonage Road Edison, NJNew Jersey USA 08837Phone: +1 (206) 317 1218[emailprotected]

Connect with us athttps://www.linkedin.com/company/advance-market-analyticshttps://www.facebook.com/AMA-Research-Media-LLP-344722399585916https://twitter.com/amareport

View post:
Hematopoietic Stem Cell Transplantation Market is Stunning Worldwide Gaining Revolution in Eyes of Global Exposure - Owned

Bone Marrow Stem Cells Comments Off on Hematopoietic Stem Cell Transplantation Market is Stunning Worldwide Gaining Revolution in Eyes of Global Exposure – Owned | August 15th, 2020

Journalists received not one but three announcements this week from American Gene Technologies (AGT) touting the most promising potential cure for HIV in the world. But such claims amount to unjustified hype, advocates say. The experimental therapy has not yet been tested in humans andif it worksit could be years before its ready for clinical use.

AGT just received clearance from the Food and Drug Administration (FDA) to start the first Phase I human clinical trial of its genetically modified T-cell product, dubbed AGT103-T, which the company is developing in collaboration with researchers at the National Institute of Allergy and Infectious Disease.

From its research, AGT believes a cure is attainable and is now taking the significant step of testing in humans, the company announced in a press release. Added AGT founder and CEOJeff Galvin, I am confidentAGT103-Twill be an important step toward an eventual cure for HIV.

But advocates say such claims are not only premature, they are also harmful in giving people with HIV the false impression that a cure is around the corner.

AGTs public relations strategy preys on the emotions of people living with HIV and has a deleterious effect on the understanding of the cure field overall, Seattle advocate Michael Louella told POZ. They make their outrageous comments, and these are then picked up and believed to be certain truth. Any attempt to promote a more nuanced and better-grounded understanding of gene therapy or the clinical process becomes impossible.

Although HIV can be suppressed indefinitely with combination antiretroviral therapy, it has proved exceedingly difficult to cure because a so-called reservoir of latent virus can remain hidden from the drugs in resting immune cells. Only two people appear to have been cured after bone marrow transplants from donors with HIV-resistant stem cellsa procedure far too dangerous for people who dont have life-threatening blood cancer.

Nonetheless, researchers are exploring numerous cure strategies, ranging from flushing HIV out of resting cells to genetically engineering immune cells to make them resistant to the virus. Most experts expect that a combination approach will likely be needed to maintain durable control of HIV after stopping antiretroviral therapythe definition of a functional cure.

AGTs process involves collecting immune cells from a patient and selecting those cells that target HIV antigens. A harmless lentivirus vector is then used to insert genes into the HIV-specific CD4 T cells that disable CCR5 receptorswhich most strains of HIV use to enter cellsas well as genes involved in HIV replication. The genetically modified CD4 cells are then reinfused back into the same patient in a single dose. The entire process takes 11 days.

The company said it expects the approach will provide durable control of genetically diverse strains of HIV, including those that use a different receptor (known as CXCR4) to enter cells. The experimental therapy should work to remove infected cells from the body and decrease or eliminate the need for lifelong antiretroviral treatment, AGT claims.

Another company, Sangamo BioSciences, previously reported promising results from early studies using a different gene therapy technique (a zinc finger nuclease) to edit out CCR5 receptors from T cells. Although it did not cure HIV, some study participants saw a reduction in the size of their viral reservoir and a long-term increase in CD4 counts. More recently, Chinese researcher He Jiankui used yet another technique (CRISPR-Cas9) to disable the CCR5 gene in human embryos in an effort to protect them from HIV.

AGTs approach not only uses a different gene-editing method to disable CCR5, but it also selects CD4 T cells that target HIV and protects them from destruction by the virus, thereby helping the selected cells survive and avoiding the wasted effort of modifying cells that wont attack HIV.

A recent medical journal report described preclinical studies of the approach, which showed that it is feasible to manufacture the modified HIV-specific CD4 T cells. AGT claims that in laboratory studies, the product demonstrates the ability to clear itself of HIV when challenged with the virus and HIV-infected human cells. The company has not yet reported results from studies of the experimental therapy in animals.

These findings were used to support AGTs investigational new drug application to the FDA to allow the company to proceed with a Phase I study in human volunteers, which will be conducted in Baltimore and Washington, DC. Eligible participants must have been on antiretroviral therapy for one to three years, have an undetectable viral load, have a stable CD4 count above 500 and may not have any AIDS-defining conditions.

AGT expects to enroll the first participant in September, with the first infusion of genetically modified T cells to be administered in December. The company said it expects initial data by the end of the year.

But this will be far too soon to determine whether the altered T cells persist in the body or whether they can maintain long-term viral suppression after antiretroviral therapy is discontinued.

Saying AGT believes there is a high likelihood that participants in the upcoming trial will be cured is beyond outrageous and completely undermines informed consent because its an unethical inducement to participate [in trials], Richard Jefferys of the Treatment Action Group told POZ.

And its not based on a shred of evidence. To my knowledge, theres no humanized mouse data, no macaque dataits all theory, he continued. "I would hope that they pause to reconsider their PR strategy and broaden their consultation with stakeholders, including community-based advocates.

Click here for more news about HIV cure research.

Originally posted here:
Gene Therapy Cure Claims Are Premature, Advocates Say - POZ

Bone Marrow Stem Cells Comments Off on Gene Therapy Cure Claims Are Premature, Advocates Say – POZ | August 15th, 2020

INTRODUCTION

In recent years, a number of growth factors have been tested in clinical trials for a variety of therapeutic applications including bone regeneration and neovascularization of ischemic tissues. Despite early promising results, the results obtained in larger phase 2 trials have often not shown the expected benefit to patients (1, 2), with some having marked adverse effects (35). The Infuse bone graft, which consists of recombinant human bone morphogenetic protein-2 (rhBMP-2) soaked onto a collagen sponge at a dosage of 1.5 mg/ml, has received Food and Drug Administration approval for certain spinal, dental, and trauma indications and is in widespread clinical use. However, major complications and adverse effects have increasingly been attributed because of the off-label use of the product (3, 4). Clinically, the current delivery vehicle for BMP-2 is a collagen powder or sponge that has been shown to result in a large initial burst release, which contrasts with the expression profile observed during normal fracture repair where BMP expression increases until day 21, suggesting a need for slower and more sustained growth factor release profile (6, 7). Furthermore, because of the short half-life of the growth factor and the harsh fracture environment (5), supraphysiological dosages of BMP-2 are being delivered to elicit bone regeneration, which has been linked to adverse effects such as heterotopic ossification. Therefore, there is a clear clinical need to develop alternative strategies to deliver single or multiple growth factors to the site of injury with sustainable and physiologically relevant dosages such that repair is induced without these adverse effects.

A number of growth factors have been shown to be expressed at different phases of fracture healing, including vascular endothelial growth factor (VEGF) and BMPs. The coupled relationship in bone healing, both physical and biochemical, between blood vessels and bone cells has long been recognized (8, 9). During fracture healing, VEGF is released directly after injury and predominately drives the formation of the fracture hematoma (9). Inhibition of VEGF has been shown to disrupt the repair of fractures and large bone defects (1012). Despite this, VEGF delivery alone is often not sufficient to heal critically sized bone defects, which may be due to suboptimal dosing or the timing of VEGF release. Furthermore, VEGF does not appear to drive progenitor cell differentiation toward the chondrogenic or osteogenic lineage; therefore, combination therapies with BMPs have been developed in an attempt to accelerate the regeneration of large bone defects (9, 1318). During normal fracture healing, VEGF expression peaks around day 5/10 (19, 20) and then decreases, whereas BMP-2 expression increases constantly until day 21, suggesting a need for delivery systems that support the early release of VEGF and the sustained release of BMP-2 (6, 7, 19, 20). To this end, composite polymer systems have been used to deliver VEGF and BMP-2 in a sequential fashion (1518). The timed release of VEGF/BMP-2 was found to enhance ectopic bone formation (1618); however, in an orthotopic defect, no significant benefit was observed (17, 18). This may be due to the high dose of VEGF used in these studies (18), which has previously been shown to disrupt osteogenesis as a result of abnormal angiogenesis and vascular structure (8), or due to suboptimal growth factor release profiles from these constructs. This suggests that novel strategies are required for delivering low-dosage VEGF and BMP-2, with tight temporal control, to enhance vascularization and subsequent bone formation in orthotopic defects. Nanoparticles such as hydroxyapatite (HA) and laponite are known to be osteoinductive and have previously been shown to facilitate the adsorption and immobilization of proteins such as VEGF and BMP-2 because of the strong attraction between the nanoparticles and the growth factor (2123). This motivates the integration of these nanoparticles into regenerative implants to enable tight temporal control over the rate at which encapsulated growth factors are released into damaged tissue.

Processes such as angiogenesis are regulated not only by the temporal presentation of growth factors but also by spatial gradients of morphogens that regulate chemotactic cell migration. Using microfluidic devices (24, 25) or three-dimensional (3D) culture models (26, 27), it has been demonstrated that endothelial cell migration is mediated by gradients in VEGF. However, it is unclear whether incorporating gradients of VEGF into tissue-engineered scaffolds will enhance angiogenesis in vivo. Here, we used emerging multiple-tool biofabrication techniques (28) to deliver VEGF and BMP-2 with distinct spatiotemporal release profiles to enhance the regeneration of critically sized bone defects. To tune the temporal release of these morphogens from 3D printed constructs, we functionalized alginate-based bioinks with different nanoparticles known to bind these regulatory factors. Both the spatial position and temporal release of growth factor from the 3D printed implant determined its therapeutic potential. By slowing the release of BMP-2, it was possible to enhance bone formation in vivo within predefined positions of the implant. Furthermore, introducing spatial gradients of VEGF into 3D printed implants enhanced vascularization in vivo compared to controls homogenously loaded with the same total amount of growth factor. We also demonstrate accelerated large bone defect healing, with minimal ectopic bone formation, using 3D printed implants containing a spatial gradient of VEGF and spatially localized BMP-2.

To produce a printable bioink, various weight concentrations of methylcellulose were first added to RGD -irradiated alginate. Print fidelity (as measured by the filament spreading ratio) improved by increasing the methylcellulose content [see fig. S1 (A and B)]; however, the capacity to print multiple layers of material worsens because of the overly adhesive nature of the ink. For these reasons, a weight concentration of 2:1 (w/w) alginate to methylcellulose was chosen for all bioinks, as it substantially increased the print fidelity while allowing multiple layers of material to be accurately deposited.

To tune the temporal release profile of growth factor (here, VEGF), clay nanoparticles (22, 23, 29) or hydroxyapatite nanoparticles (nHA) (21) were added to the alginate-methylcellulose bioink. Adding methylcellulose to the alginate to produce a printable ink significantly increased the release of VEGF compared to that observed from alginate only [see fig. S1 (C and D)]. The addition of laponite, a clay-based nanoparticle, markedly slowed the release of VEGF (see fig. S1C), while the incorporation of nHA only had a small effect on growth factor release, producing a slightly more gradual release profile (see fig. S1D). This blend (alginate, methylcellulose, and nHA) will hereafter be referred to as the vascular bioink, as it allowed for the near complete release of VEGF over 10 days, mimicking that observed during normal fracture healing (19, 20). No laponite was included in this vascular bioink.

To demonstrate the utility of this vascular bioink, two strategies were compared to print implants containing a spatial gradient of VEGF (see fig. S1E). In the first, VEGF (100 ng/ml) was printed into the central 5-mm core of constructs 8 mm in diameter and 4 mm high, with a VEGF-free bioink used to print the periphery of the construct. In the second, VEGF (80 ng/ml) was printed into the center of the construct, and VEGF (20 ng/ml) was printed around the periphery of the implant. Control constructs containing a homogenous distribution of VEGF were also printed. One hour after printing, clear spatial differences in VEGF localization were observed in both gradient constructs, while roughly the same amount of protein was detected in the core and periphery of the homogenous VEGF control (see fig. S1F). Fourteen days after printing, a spatial gradient still existed in the construct that initially had all VEGF loaded into its central region, with no gradient observed in the other groups (see fig. S1G). This demonstrates that spatial gradients of growth factor can be maintained within constructs for at least 14 days after printing.

We next sought to assess whether depositing spatial gradients of VEGF within 3D printed polycaprolactone (PCL) implants would accelerate vascularization of the constructs in vivo. To this end, Homogenous VEGF, Gradient VEGF, and No VEGF constructs were implanted subcutaneously in the back of mice (see Fig. 1A), where the total amount of growth factor (25 ng) within the two VEGF-containing implants was constant. Two weeks after implantation, histological analysis of hematoxylin and eosin (H&E)stained samples revealed the presence of vessels in the Homogenous VEGF and Gradient VEGF groups; however, there were no obvious vessels present in the No VEGF group (see Fig. 1B). These vessels appeared mature, complete with smooth muscle actin (-SMA) and von Willebrand factor (vWF)stained walls and perfused with erythrocytes (see fig. S2A). The Homogenous VEGF constructs had vessels predominantly located in the periphery of the scaffold, with little to none present within the center of the scaffold. On the other hand, vessels were present both in the periphery and in the center of the Gradient VEGF group. Four weeks after implantation, all three experimental groups had mature vessels present (see Fig. 1C and fig. S2B). Similar to the Homogeneous VEGF group, the No VEGF group had vessels predominantly located in the periphery of the constructs, with little to none present within the center of the construct. When quantified, at both 2 and 4 weeks, there were significantly more vessels present in the Gradient VEGF group compared to both the Homogenous VEGF and No VEGF group (see Fig. 1D). There was significantly more vessels present in the periphery of the Gradient VEGF constructs at both 2 and 4 weeks in vivo compared to the other two experimental groups [see Fig. 1 (E and F)]. There was also a trend toward a larger number of vessels present in the center of the Gradient VEGF construct at 4 weeks compared to No VEGF (P = 0.09) and Homogenous VEGF (P = 0.1) groups (see Fig. 1F).

(A) Schematic of the 3D printed scaffold and experimental groups. Construct design (4 mm in diameter, 5 mm in height). H&E-stained sections of the three experimental groups at (B) 2 and (C) 4 weeks in vivo. Images were taken at 20. Arrows denote vessels. (D) Total number of vessels of the experimental groups at 2 and 4 weeks in vivo. Number of vessels present in the center versus the periphery at (E) 2 and (F) 4 weeks in vivo. **P < 0.01. Error bars denote SDs (n = 8 animals; n = 5 slices per animal). FBS, fetal bovine serum; pen/strep, penicillin/streptomycin.

Recognizing that a slower and more sustained release of BMP-2 could be beneficial for promoting osteogenesis (6, 7), we next sought to compare bone formation in vivo within implants with temporally distinct growth factor release profiles. To the base alginate-methylcellulose bioink (here termed the Fast BMP-2 Release bioink), laponite at varying w/w ratios of laponite to alginate were compared to determine the optimum ratio to generate a Slow BMP-2 Release bioink (see fig. S3). As there was little difference in the growth factor release profile from the different groups, a 6:1 alginate:laponite w/w ratio was chosen to minimize the amount of laponite in the bioink. The addition of laponite markedly slowed the in vitro release of BMP-2 from the bioink, resulting in a reasonable constant release of growth factor from day 7 to day 35 (see Fig. 2C). The addition of laponite also had no significant effect on the degradation rate of the bioink (Fig. 2B).

(A) Schematic of the experimental groups. Construct design (4 mm in diameter, 5 mm in height). MEM, alpha minimum essential medium. (B) Degradation of the two bioinks. (C) Cumulative release of BMP-2 of the fast release bioink versus the slow release bioink. (D) 3D reconstructions of the CT data for each group at 8 weeks. (E) CT analysis on total mineral deposition of each of the groups after 8 weeks in vivo. (F) CT analysis on the location of mineral deposition of each of the groups after 8 weeks in vivo. ***P < 0.001; error bars denote SDs (n = 8 animals). (G) Goldners trichromestained sections of both groups after 8 weeks in vivo. Images were taken at 20. White arrows denote developing bone tissue, and black arrows denote blood vessels. (H) Quantification of the amount of new bone formation per total area. Error bars denote SDs; **P < 0.01 (n = 8 animals, n = 6 slices per animal).

To assess whether slow and sustained release of BMP-2 would enhance ectopic bone formation in vivo, Fast BMP-2 Release (laponite) and Slow BMP-2 Release (+laponite) bioinks were mixed with bone marrowderived mesenchymal stem cells (BMSCs), deposited within 3D printed scaffolds, and then implanted subcutaneously in the back of mice (see Fig. 2A). Seeding these bioinks with MSCs was used to test their potential for promoting osteogenesis in an ectopic location. BMP-2 was specifically localized around the periphery of the implant. This pattern of growth factor presentation was chosen to test the capacity of the printed implants to spatially localize bone formation in vivo (note that the geometry of the implant is the same as that which will be used in the segmental defect study below, with the BMP-2 localized to the periphery of the implant such that bone would only form along the cortical shaft of the damaged limb rather than throughout). Eight weeks after implantation, there was significantly more mineral within the Slow BMP-2 Release group compared to the Fast BMP-2 Release group [see Fig. 2 (D and E)]. Microcomputed tomography (CT) reconstructions revealed that the mineral was preferentially deposited around the periphery of the constructs where the BMP-2 was localized [see Fig. 2 (D and F)]. Histological staining further verified this finding, with positive staining for new bone seen predominantly in the periphery of both groups (see Fig. 2G, denoted by white arrows). Quantification revealed that the Slow BMP-2 Release constructs had significantly more new bone formation per total area of construct (see Fig. 2H).

We next sought to assess whether the delayed release of BMP-2 from printed constructs containing spatial gradients in VEGF would enhance angiogenesis and bone formation within critically sized bone defects. To this end, VEGF gradient only, BMP-2 gradient only, and Composite (VEGF+BMP-2 gradient) constructs were printed and implanted in a 5-mm rat femoral defect (see Fig. 3A) and compared to an empty defect.

(A) Schematic of the 3D printed experimental groups including key features of the developed bioinks and the segmental defect procedure. Construct design (4 mm in diameter, 5 mm in height). (B) CT angiography representative images of vessel diameter. Red arrows denote leaky blood vessels denoted by pools of contrast agent. Quantification on (C) total vessel volume, (D) average vessel diameter, and (E) connectivity for all groups after 2 weeks in vivo. *P < 0.05 and **P < 0.01; error bars denote SDs (n = 9 animals). (F) Immunohistochemical staining of nuclei (blue), vWF (red), and SMA (green) of the experimental groups at 2 weeks after implantation. Images were taken at 40 and 63. Yellow arrows denote vessels with SMA and vWF dual staining; white arrows denote slightly less mature vessels with only vWF positive staining.

Two weeks after implantation, CT angiography was used to quantify and visualize the early vascular network that had formed within the defect site. 3D reconstructions revealed that vascular networks had formed in all four experimental groups (see Fig. 3B). When quantified, there was a significant increase in vessel volume in the Composite group compared to the VEGF gradient group (see Fig. 3C). There was also a significant increase in average vessel thickness in the BMP-2 gradient and Composite groups compared to the VEGF gradient group (see Fig. 3D). Although there was no significant difference in the connectivity of the vessels, there was a trend (P = 0.1) toward increased connectivity in the Composite group compared to the VEGF gradient group (see Fig. 3E). 3D reconstructions also revealed the presence of primitive immature blood vessels depicted by large globules of contrast agent (denoted by the red arrows in Fig. 3B). There appeared to be fewer primitive blood vessels present in the Composite group than the other three experimental groups. This was further verified by SMA and vWF staining, which revealed a larger number of vessels with only positive vWF-stained walls in the Empty and VEGF gradient groups (see Fig. 3F, denoted by white arrows). On the other hand, there were predominately more mature vessels with SMA and vWF-stained walls in both the BMP-2 gradient and Composite groups (see Fig. 3F, denoted by yellow arrows). Note that the differences in angiogenesis seen between the VEGF gradient and Composite groups (same amount of VEGF in both groups) could at least partially be explained by looking at the VEGF release profile from both groups (see fig. S4). The addition of the osteoinductive ink around the implant periphery significantly reduced the VEGF release rate from construct into the media, with a more linear release of growth factor over time.

Two weeks after surgery, defects within the Empty group were filled with a fibrous tissue (see Fig. 4A). In contrast, positive staining for cartilage and new bone deposition was observed in the BMP-2 gradient and Composite groups, suggesting that new bone was forming at least partially via endochondral ossification. When quantified, there was a trend toward increased cartilage development (red staining in Safranin O images) in both the BMP-2 gradient (P = 0.12) and Composite (P = 0.18) groups compared to the Empty (see Fig. 4B). No significant differences in bone formation was observed between any of the groups at week 2; however, the CT reconstructions showed mineralized calluses beginning to form in the BMP-2 gradient and Composite groups, which was less evident in the Empty and VEGF gradient groups [see Fig. 4 (C and D)].

(A) H&E- and Safranin Ostained sections of all groups after 2 weeks in vivo. Images were taken at 20. DB denotes cartilage undergoing endochondral ossification to become developing bone, and B denotes positive new bone tissue. Quantification of the amount of (B) bone formation and (C) developing bone per total area. Error bars denote SDs (n = 9 animals). (D) CT reconstructed images of the defect site.

Next, CT analysis was used to visualize and quantify bone formation within the defects at 4, 8, 10, and 12 weeks after implantation. Compared to the Empty group, there were significantly higher levels of new bone formation in the Composite group as early as 8 weeks after implantation [see Fig. 5 (A and B)]. A consistent pattern of healing was observed in the Composite group, with bone forming down through the PCL scaffold framework (see Fig. 5A and fig. S5). After 10 weeks of implantation, significantly higher levels of bone formation was observed in the BMP-2 gradient and Composite groups compared to the Empty group. By 12 weeks, all three experimental groups contained significantly higher levels of new bone compared to the Empty group. Twelve weeks after implantation, bone density mapping revealed that the new bone formed in the experimental groups consisted of a dense cortical-like bone present around the periphery of defect, comparable to the adjacent native bone (1200 mg HA/cm3) (see Fig. 5C). Quantitative densitometry analysis revealed no significant difference in the average density (mg HA/cm3) of the new bone that did form between any of the groups over the 12 weeks (see Fig. 5D).

(A) Reconstructed in vivo CT analysis of bone formation in the defects. (B) Quantification of total bone volume (mm3) in the defects at each time point. (C) Representative images of CT bone densities in the defects at 12 weeks halfway through the defect (scale bar, 1 mm throughout). (D) Average bone density (mg HA/cm3) in the defects at each time point. (E) Outline of ROI bone volume analysis including definitions of core, annulus, and heterotopic regions. (F) Total bone volume (mm3) in each region at 12 weeks. **P < 0.01, ***P < 0.001, and ****P < 0.0001; error bars denote SDs (n = 9 animals).

To assess the levels of heterotopic bone formation, region of interest (ROI) bone volume analysis was performed on the week 12 reconstructions. The total bone volume was quantified in the core, annulus, and heterotopic regions of the defect (see Fig. 5E). In all three experimental groups, bone preferentially formed in the annulus of the defect, with little ectopic bone formation (see Fig. 5F). All three experimental groups had significantly higher total bone volume in the annulus of the defect compared to the Empty annulus, with the highest total bone volume present in the Composite group.

We next sought to assess the nature of new bone tissue being formed using histological staining. Goldners trichrome staining revealed predominantly fibrous tissue formation, similar to what was seen previously at 2 weeks, in the Empty group (see Fig. 6A). There was positive staining for new bone, complete with marrow cavities, in all three experimental groups at 12 weeks after implantation. When quantified, there was significantly more bone found in all three experimental groups compared to the Empty group (see Fig. 6B). There were also significantly higher amounts of bone marrow present in the Composite group compared to the Empty group (see Fig. 6C). As observed in the CT 3D reconstructions, it is clear that the bone is forming down through the PCL scaffold framework, specifically in the Composite group. Safranin O staining revealed the presence of cartilage in all three experimental groups after 12 weeks, demonstrating that bone is continuing to develop via endochondral ossification. When quantified, there was significantly more cartilage present in the Composite group compared to all other groups at this time point (see Fig. 6D).

(A) Goldners trichrome and Safranin Ostained sections of all groups after 12 weeks in vivo. Images were taken at 20. BM denotes bone marrow. PCL denotes areas where the PCL frame was. DB denotes cartilage undergoing endochondral ossification to become new bone, and B denotes positive bone tissue. Quantification of the amount of (B) bone formation, (C) bone marrow, and (D) developing bone per total area. Error bars denote SDs. *P < 0.05, **P < 0.01, and ****P < 0.0001 (n = 9 animals).

Despite the tremendous potential of growth factor delivery, the results obtained in larger clinical trials have not always shown the expected benefit to patients (2), with some studies reporting marked adverse effects (35). The reasons for this are multifaceted, from the delivery methods to the supraphysiological dosages needed to elicit a therapeutic effect and the costs and adverse effects attached to these high doses. This study presents a novel alternative approach for spatiotemporally controlled delivery of growth factors. We developed a range of nanoparticle-functionalized bioinks to precisely control the temporal release of growth factors from 3D printed implants. Using multiple tool biofabrication techniques, we were able to print constructs containing spatiotemporal gradients of growth factors, which allowed for controlled tissue regeneration without the need for supraphysiological dosages. Specifically, the appropriate patterning of VEGF enhanced angiogenesis in vivo and, when coupled with defined BMP-2 localization and release kinetics, enhanced large bone defect healing with little heterotopic bone formation.

Alginate hydrogels are commonly used for bone tissue engineering, with a number of studies demonstrating the bone regeneration potential of RGD functionalized and -irradiated alginate (3033), making it a promising base bioink for the 3D bioprinting of osteogenic implants. However, one drawback to using RGD -irradiated alginate as a bioink is its low viscosity. It is imperative when printing multilayered structures that the bioink have appropriate rheological properties to prevent collapsing or sagging of the printed structure. The addition of methylcellulose to alginate-based bioinks was found to have a significant effect on both printability and the rate of growth factor release. The addition of methylcellulose has previously been shown to substantially increase the print fidelity of an alginate base bioink (22, 34, 35), although typically using higher concentrations than the one used in this study. Adding methylcellulose also accelerated the rate of growth factor release. This was previously seen with albumin release from alginate-methylcellulose beads (36). Such a polymeric network is at least partially defined by physical entanglements between the alginate or methylcellulose chains. As methylcellulose is characterized by high swellability, when the alginate/methylcellulose bioink is exposed to the medium, it swells rapidly, resulting in accelerated growth factor release from the bioink. The addition of methylcellulose may also have neutralized the charge on the alginate, which would also influence growth factor release kinetics. In contrast, the addition of nanoparticles, and, in particular, laponite, slowed the release of growth factor from the inks. Both nHA and laponite have previously been shown to facilitate with the adsorption and immobilization of VEGF within a hydrogel due to the strong attraction between the nanoparticles and the growth factor (2123). The stronger association between growth factors and laponite can be linked to the physiochemical properties of these particles (22, 29). These disc-shaped particles [typically 25 nm in diameter and 1 nm in thickness (37)] are characterized by a highly negatively charged face and a positively charged rim (22), with a zeta potential of 61 mV (as determined by the manufacturer). This allowed the positively charged growth factors such as VEGF to form strong electrostatic bonds with the negatively charged face of the nanoparticles (22). In contrast, the nHA nanoparticles used in this study, which we have previously shown to have a zeta potential of around 5 mV (38), would form a slightly weaker electrostatic bond with the VEGF. The addition of laponite to bioinks has also previously been shown to influence their mechanical properties (37). While we did not directly assess whether the addition of laponite influenced the stiffness of our ink, we did observe that it had no effect on their degradability, and on the basis of w/w ratio used in this study, we do not believe it had marked effects on mechanical properties such as matrix stiffness. Previous studies have shown that when using high concentrations of alginate (similar to that used in this study), the addition of laponite does not markedly affect the rheological properties of the bioink (37). However, future studies should investigate the overall mechanical properties of a bioink, as this may also influence its osteogenic potential (39). A potential limitation of laponite is that the strong electrostatic bond can limit the amount of growth factor released from a delivery system in the short-medium term (22). In this study, by tuning the ratio of laponite to alginate, it was possible to engineer bioinks that released most of their loaded protein over 35 days. Therefore, using specifically selected nanoparticles, it is possible to develop bioinks that support growth factor release profiles spanning days to weeks.

Using multiple-tool biofabrication, we demonstrated that distinct growth factor gradients can be established and maintained over time and that incorporating these gradients into printed implants can enhance sprouting angiogenesis in vivo. The process of sprouting angiogenesis begins with the selection of a distinct site on the mother vessel where sprout formation is initiated. This distinct site is referred to as the tip cell, and as the new sprout elongates, branches, and connects with other sprouts, the selection process for the tip cell is constantly reiterated (40). Previous studies have shown in the early postnatal retinas that tip cell migration depends on a gradient of VEGF-A and its proliferation is regulated by its concentration (40, 41). Therefore, the increase in vessel infiltration observed in VEGF gradient implants can possibly be attributed to tip cell migration and proliferation toward the areas of high VEGF concentration (40, 41). In contrast, when VEGF was homogenously distributed within the implant, there was less of a chemotactic effect, resulting in lower levels of vessel infiltration into the center of the construct.

When this bioprinting strategy was used to deliver both growth factors within a large bone defect, there was a significant increase in vessel infiltration within implants containing both a VEGF gradient and BMP-2 compared to those containing VEGF alone. Although it has been shown that delivery of BMP-2 alone can enhance new blood vessel formation within bone defects (42, 43), previous studies have not reported a benefit to delivering both growth factors to the defect site (17, 18). The finding that the laponite-functionalized bioink around the periphery of the implant was slowing the release of VEGF from the implant may partially explain the higher levels of vessel infiltration observed within the composite implant, with the slower VEGF release profile being perhaps more conducive to angiogenesis within the orthotopic environment. Somewhat unexpectedly, despite enhancing overall levels of bone formation, VEGF delivery alone did not increase early vessel infiltration into the implant. Note that orthotopic hematomas, generated by the surgical procedure, would have provided all defects with a source of endogenous chemotactic, angiogenic, and mitogenic growth factors (17). This may have mitigated the effect that an implant containing a VEGF gradient alone had on early angiogenesis.

3D printed implants containing spatial gradients of VEGF, coupled with defined BMP-2 localization, enhanced large bone defect healing with little heterotopic bone formation. Critically, this increase in bone healing was achieved using very low concentrations of exogenous growth factors. The concentration of VEGF used in this study was substantially less (80 to 160 times less) than previous studies (17, 18). Achieving therapeutic benefits with these low concentrations of growth factors is important for multiple reasons, not least of which is the observation that high concentrations of VEGF have been previously shown to disrupt osteogenesis as the result of abnormal angiogenesis and vascular structure (8). Furthermore, the concentrations of BMP-2 used here are at least an order of magnitude lower than that used previously to repair similar sized defects in a rat femoral defect model (28, 31). Repair in these studies is typically associated with a substantial amount of heterotopic bone formation (28, 31). Directly comparing to previous work in our lab, which used a clinically relevant BMP-2 dose in the same defect model (28), the results from this study exhibited substantially less heterotrophic bone formation [10% versus 50% (28) of total bone volume]. Although we did not observe full bone bridging after 12 weeks, new bone was still being formed via the process of endochondral ossification at 12 weeks, suggesting that regeneration was still proceeding. Allowing some level of physiological loading earlier in the healing process would likely have further accelerated regeneration (44). Together, the results from this study demonstrate the potential of 3D printing morphogen gradients for controlled tissue regeneration (with minimal heterotopic bone formation) without the need of supraphysiological dosages.

The translation of tissue engineering concepts from bench to bedside is a challenging, expensive, and time-consuming process. Numerous products have not made it past phase 2 trials, as they have not shown the expected benefit in patients (1, 2), while others have been associated with marked adverse effects (35). Here, we describe a previously unidentified approach for spatiotemporally defined growth factor delivery and demonstrate a potential clinical utility in the regeneration of large bone defects or the increased vascularization of any 3D printed construct. Proof-of-concept studies in small animals established the potential of these growth factor loaded bioinks for inducing enhanced angiogenesis and bone regeneration without the need for supraphysiological dosages. The benefit of this precise localization of growth factors in both time and space is that it allows for tightly controlled angiogenesis and new tissue formation, thereby reducing off-target effects. It is envisioned that this platform technology could be applied to the controlled regeneration of numerous different tissue types.

This study was designed to test whether the delayed release of BMP-2 from bioprinted constructs containing spatial gradients in VEGF will first enhance vascularization and sequentially enhance orthotopic bone regeneration. All animal experiments were conducted in accordance with the recommendations and guidelines of The Health Products Regulatory Authority, the competent authority in Ireland responsible for the implementation of Directive 2010/63/EU on the protection of animals used for scientific purposes in accordance with the requirements of the Statutory Instrument no. 543 of 2012. Subcutaneous mouse experiments were carried out under license (AE 19136/P069), and the rat femoral defect experiments were carried out under license (AE19136/P087) approved by The Health Products Regulatory Authority and in accordance with protocols approved by the Trinity College Dublin Animal Research Ethics Committee. The n for rodent models were based on the predicted variance in the model and was powered to detect 0.05 significance. For the subcutaneous surgeries, constructs were implanted in a balanced manner, such that each group contained an implant placed at each of the subcutaneous locations and samples for both surgical procedures were randomly distributed across the operated animals. For the rat surgeries, three rats from the empty group died from unforeseen complications and so were removed from the n number at the 12-week time point. One rat from the BMP-2 gradient group at 12-week time point was also removed, as it was deemed a statistical outlier using the Grubbs test.

Lowmolecular weight sodium alginate (58,000 g/mol) was prepared by irradiating sodium alginate (196, 000 g/mol; Protanal LF 20/40, Pronova Biopolymers, Oslo, Norway) at a gamma dose of 50,000 gray, as previously described (45). RGD-modified alginate was prepared by coupling the GGGGRGDSP to the alginate using standard carbodiimide chemistry. All bioinks were prepared by dissolving the RGD -irradiated alginate in growth medium, which consisted of alpha minimum essential medium (MEM) (GlutaMAX; Gibco, Biosciences, Ireland), 10% fetal bovine serum (FBS) (EU Thermo Fisher Scientific), penicillin (100 U/ml; Sigma-Aldrich), and streptomycin (100 g/ml; Sigma-Aldrich) (pen-strep) to make up a final concentration of 3.5% (w/v).

3D bioplotter from RegenHU (3DDiscovery) was used to evaluate the printability of the generated bioinks. The printability of varying the w/w ratio (2:1, 1:1, and 1:2) of methylcellulose to alginate was evaluated by measuring the spreading ratio as previously described (39)Spreading Ratio=Printed Filament DiameterActual Needle Diameter

To establish whether increasing the viscosity of the bioink influences growth factor release, methylcellulose (Sigma-Aldrich) was also added at ratio of 1:2 (w/w) to a 3.5% alginate solution of RGD -irradiated alginate. To establish whether the addition of clay-based particles to the bioink could further tailor the growth factor release profile of the bioinks, a 3.5% RGD -irradiated alginate solution was made, and either methylcellulose (2:1) (w/w) or a combination of both methylcellulose and laponite (Laponite XLG, BYK Additives & Instruments, UK) (6:3:1) (w/w) was added.

To establish whether the addition of nHA to the alginate would facilitate the adsorption and immobilization of growth factors within the hydrogel due to their strong electrostatic attraction between nHAs, three bioinks were tested (21). nHAs were prepared following a previously described protocol (46). A 3.5% RGD -irradiated alginate solution was made, and either methylcellulose (1:2) (w/w) or a combination of methylcellulose and nHA (2:1:2) (w/w) particles was added.

For all the growth factor release studies, VEGF (100 ng/ml; Gibco Life Technologies, Gaithersburg, MD, USA) was added to the solutions using dual-syringe approach, before precross-linking with 60 mM CaSO4 to make the bioinks as previously described (39). All constructs were cultured in growth medium in normoxic conditions, and media from each sample were changed bi-weekly. For VEGF release study, medium samples were taken (days 0, 3, 5, and 10) and snap-frozen at 80C. Hydrogels were also snap-frozen at 80C on day 0 to quantify the concentration of growth factor present in the constructs directly after printing.

To demonstrate the utility of the vascular bioink, two strategies were compared to print implants containing a spatial gradient of VEGF. The vascular bioink was prepared, cross-linked with 60 mM CaSO4, and printed to generate three experimental groups: (i) Homogenous VEGF. Bioink loaded with VEGF (100 ng/ml) was used to print constructs 8 mm in diameter and 4 mm high. (ii) Gradient 1. Bioink loaded with VEGF (100 ng/ml) was used to print a central 5-mm core with a VEGF-free bioink printed around the periphery of the 8-mm-diameter construct. (iii) Gradient 2. VEGF (80 ng/ml) was printed into the core, and VEGF (20 ng/ml) was printed into the periphery. Postprinting constructs were cross-linked again in a bath of 100 mM CaCl2 for 1 min. Constructs were cultured in growth medium in normoxic conditions for 14 days in vitro. The center and periphery of each construct were separated by coring out the center from the periphery of the scaffold and then snap-frozen at 80C, 1 hour after printing, and after 14 days in vitro.

To investigate whether the addition of laponite can tailor the growth factor release profile over a long culture period, a base bioink (Fast BMP-2 Release) and a laponite bioink (Slow BMP-2 Release) were compared. For both growth factor release profiles, a dual-syringe approach was used to deliver BMP-2 (200 ng/ml; PeproTech, UK) to the solutions before precross-linking with 60 mM CaSO4 to make the bioinks. These were printed into a 100 mM CaCl2 soak agarose mold to generate final constructs of 6 mm by 6 mm high. In addition to comparing the growth factor release profile of the two bioinks, the degradation rate of the bioinks was also investigated. These scaffolds were cultured in normoxic conditions for up to 35 days and media from each sample were changed weekly. For BMP-2 release study, medium samples were taken (days 0, 5, 7, 14, 21, and 35) and snap-frozen at 80C. Printed hydrogels were also snap-frozen at 80C on day 0 to quantify the concentration of growth factor present in the constructs directly after printing. For the degradation study, samples were washed and snap-frozen at 80C and each time point (days 0, 5, 7, 14, and 21). Samples were lyophilized by placing the samples in a freeze dryer (FreeZone Triad, Labconco, Kansas City, USA). Each sample was then weighed using an analytical balance (Mettler Toledo, XS205).

An enzyme-linked immunosorbent assay was used to quantify the levels of VEGF and BMP-2 (Bio-Techne, MN, USA) released by the alginates. The alginate samples were depolymerized with 1 ml of citrate buffer (150 mM sodium chloride, 55 mM sodium citrate, and 20 mM EDTA in H2O) for 15 min at 37C. The cell culture media and depolymerized alginate samples were analyzed at the specific time points detailed above. Assays were carried out as per the manufacturers protocol and analyzed on a microplate reader at a wavelength of 450 nm.

BMSCs were obtained from the femur of a 4-month-old porcine donor as previously described (47). All expansion was conducted in normoxic conditions, expanded in growth medium where the medium was changed twice weekly. Cells were used at the end of passage 3.

A 3D bioplotter from RegenHU (3DDiscovery) was used to print all of the scaffolds. Using a 30-gauge needle, constructs of 4 mm 5 mm high with both lateral and horizontal porosity and a fiber spacing of 1.2 mm were printed with PCL (Cappa, Perstop). The printing parameters of the PCL were as follows: temperature of thermopolymer tank (69C), temperature of thermopolymer head (72C), pressure (1 bar), screw speed (30 rpm), and feed rate (3 mm/s). Scaffolds were sterilized using ethylene oxide sterilization before hydrogel printing.

For the VEGF gradient study, the vascular bioink was prepared, cross-linked with 60 mM CaSO4, and printed within the PCL framework to generate three experimental groups: (i) No VEGF, bioink not loaded with VEGF; (ii) Homogenous, bioink loaded with VEGF (100 ng/ml) deposited (25 ng per construct) throughout the construct; and (iii) Gradient, bioink loaded with VEGF (500 ng/ml) deposited in the center (25 ng per construct) and VEGF-free bioink deposited on the outside (see Fig. 1A). Postprinting constructs were cross-linked again in a bath of 100 mM CaCl2 for 1 min.

For the BMP-2 release study, both a fast and slow release bioink were prepared and using the dual syringe approach, porcine MSCs were (2 106/ml) mixed to both bioinks to have an overall seeding density of 500 105 porcine MSCs/construct before being cross-linked with 60 mM CaSO4. Both bioinks were printed within the PCL framework to generate two experimental groups: (i) Fast release, fast release bioink loaded with BMP-2 (2 g/ml; 0.5 g per construct) deposited only in the periphery with the fast release bioink not loaded with BMP-2 in the center; and (ii) Slow release, slow release bioink loaded with BMP-2 (2 g/ml; 0.5 g per construct) deposited only in the periphery with the fast release bioink not loaded with BMP-2 in the center (see Fig. 2A). Postprinting constructs were cross-linked again in a bath of 100 mM CaCl2 for 1 min.

For the rat femoral defect, the vascular bioink, the osteoinductive bioink, and a base bioink (3.5% RGD -irradiated alginate and 1.75% methylcellulose) were prepared, cross-linked with 60 mM CaSO4, and printed within the PCL framework to generate three experimental groups: (i) VEGF Gradient, the vascular bioink loaded with VEGF (500 ng/ml) in the center of the implant and base bioink in the periphery; (ii) BMP-2 gradient, the osteoinductive bioink loaded with BMP-2 (10 g/ml) in the implant periphery (2 g per construct), with the base bioink in the center; and (iii) Composite (VEGF+BMP-2), the osteoinductive bioink in the periphery with the vascular bioink in the center (see Fig. 3A). Postprinting constructs were cross-linked again in a bath of 100 mM CaCl2 for 1 min.

Subcutaneous surgeries were performed on 20 8-week-old female BALB/c OlaHsd-Foxn 1nu nude mice (12 mice for the VEGF gradient study and 8 for the BMP-2 gradient study) (Envigo, Oxon, UK) as previously described (47). Scaffolds were 3D printed the morning of surgeries and implanted that day. Constructs were implanted in a balanced manner, such that each group contained an implant placed at each of the two subcutaneous locations and samples were randomly distributed across the operated animals.

For the rat segmental surgery, 72 12-week-old F344 Fischer male rats (Envigo, Oxon, UK) were anesthetized in an induction box using a mix of isoflurane and oxygen, initially at a flow rate of isoflurane of 5 liters/min to induce, followed by ~3 liters/min to maintain anesthesia. Once anesthetized, the animal was transferred to a heating plate that was preheated to 37C and preoperative analgesia was provided by buprenorphine (0.03 mg/ml). Surgical access to the femur was achieved via an anterolateral longitudinal skin incision and separation of the hindlimb muscles, the vastus lateralis, and biceps femoris. The femoral diaphysis was exposed by circumferential elevation of attached muscles, and the periosteum was removed. Before the creation of the defect, a PEEK plate was fixed to the anterolateral femur and was held in position using a clamp. Holes were created in the femur with a surgical drill using the plate as a template. Screws were then inserted into the drill holes in the femur to maintain the fixation plate in position. A 5-mm segmental defect was created using an oscillating surgical saw under constant irrigation with sterile saline solution. In the test groups, a scaffold was placed in the defect after a thorough washout of the surgical site. In the case of the empty defect group, the gap between bone ends was left empty. Soft tissue was accurately readapted with absorbable suture material. Closure of the skin wound was achieved using suture material and tissue glue.

Eight weeks after surgery, the BMP-2 gradient scaffolds were extracted and incubated in paraformaldehyde for 24 hours before being imaged via CT scans on a MicroCT42 (Scanco Medical, Brttisellen, Switzerland) as previously described (47).

Two weeks after surgery, 24 rats underwent a vascular perfusion protocol developed by Daly et al. (28). Briefly, the rat was sacrificed using CO2 asphyxiation, and the thoracic cavity was opened to insert a 20-gauge needle through the left ventricle of the heart. The inferior cava was cut and solutions of heparin (25 U/ml), and then, phosphate-buffered saline (PBS) was perfused through the vasculature using a peristaltic pump (Masterflex, Cole-Parmer, Vernon Hills, IL, USA) until the vasculature system was completely flushed clear. A solution of 10% formalin was then perfused for 5 min. Animals received a final perfusion of 20- to 25-ml radiopaque contrast agent MICROFIL (Flow Tech, Carver, MA, USA) and were left at 4C overnight. Explants were extracted and incubated in PBS for 24 hours before being imaged via CT scans on a MicroCT42 (Scanco Medical, Brttisellen, Switzerland) at 70 kVp, 113 A, and a 10-m voxel size. The volume of interest (VOI) was determined by positioning a 5-mm circle around the cross section of the femur with an overall length of 6.26 mm. MICROFIL has the same threshold as bone mineral, and therefore, to segment perfused vasculature from mineralized tissue within each construct, two scans were analyzed: calcified construct versus decalcified construct. The calcified constructs were scanned and postprocessed using a threshold value that accurately depicted both the mineral content and the vessel volume by visual inspection of the 2D grayscale tomograms (Scanco Medical MicroCT42). Noise was removed using a low-pass Gaussian filter (sigma = 1.2, support = 2), and a global threshold of 210 was applied. Next, samples were decalcified in EDTA (15 weight %, pH 7.4) for 2 weeks with the decalcification solution replaced daily (decalcified constructs). After 2 weeks, these decalcified constructs were scanned using the same settings and postprocessed at the same threshold as the calcified constructs to determine mineral content. Mineralized tissue content was determined by subtracting the bone volume of the decalcified scans from the calcified scans. Next, the decalcified scans were postprocessed at a threshold of 99 that accurately depicted just the vessel volume upon visual inspection of the 2D grayscale tomograms.

CT scans were performed on the rats using a Scanco Medical vivaCT 80 system (Scanco Medical, Bassersdorf, Switzerland). Rats (n = 9) were scanned at 4, 8, 10, and 12 weeks after surgery to assess defect bridging and bone formation within the defect. First, anesthesia was induced in an induction box using a mix of isoflurane and oxygen, initially at a flow rate of isoflurane of 5 liters/min to induce, followed by ~3 liters/min to maintain anesthesia. Next, the rats were placed inside the vivaCT scanner, and anesthesia was maintained by isoflurane-oxygen throughout the scan. Next, a radiographic scan of the whole animal was used to isolate the rat femur. The animals femur was aligned parallel to the scanning field of view to simplify the bone volume assessments. Scans were performed using a voltage of 70 kVp and a current of 113 A. A Gaussian filter (sigma = 0.8, support = 1) was used to suppress noise, and a global threshold of 210 was applied. A voxel resolution of 35 m was used throughout. 3D evaluation was carried out on the segmented images to determine bone volume and density and to reconstruct a 3D image. Bone volume and bone density in the defects were quantified by measuring the total quantity of mineral in the central 130 slices of the defect. To differentiate regional differences in bone formation, three VOIs were created. Concentric 2 mm, 4 mm, and 10 mm were aligned with the defect and used to encompass bone formation. The VOIs were aligned using untreated native bone along the femur. The core bone volume was quantified from the inner 2-mm VOI. The annular bone volume was quantified by subtracting the 2-mm VOI from the 4-mm VOI. Ectopic bone volume was quantified by subtracting the 4-mm VOI from the 10-mm VOI. The bone volume percentages for each region were then calculated by dividing the corresponding bone volume (i.e., bone volume in the annulus) by the total bone volume in the defect. The bone volume and densities were then quantified using scripts provided by Scanco.

For segmental defect samples, all constructs that were not being processed for vascular-CT imaging, were decalcified in Decalcifying Solution-Lite (Sigma-Aldrich) for 1 week before tissue processing. Once decalcified, all samples were dehydrated and embedded in paraffin using an automatic tissue processor (Leica ASP300, Leica). All samples were sectioned with a thickness of 8 m using a rotary microtome (Leica Microtome RM2235, Leica). Sections were stained with H&E for vessel infiltration, Safranin O to assess sulphated glycosaminoglycans (sGAG) content, and Goldners trichrome for bone formation. Quantitative analysis was performed on multiple H&E-stained slices, whereby vessels (positive staining for endothelium and erythrocytes present within the lumen), were counted on separate sections taken throughout each construct and averaged for each construct. Safranin O sections were evaluated for new developing bone (positive sGAG content). Massons trichromestained sections were evaluated for new bone formation. The percentage of developing bone, new bone, and marrow per total area of construct was measured in separate sections with the Deconvolution ImageJ plugin.

Immunofluorescence analysis was used to detect -SMA and vWF as previously described (47). Briefly, following blocking step, sections were then incubated overnight at +4C with goat polyclonal -SMA (1:250; ab21027, Abcam) in PBS with 3% of donkey serum (w/v) and 1% bovine serum albumin (BSA). After three washing steps with PBS containing 1% w/v BSA, the sections were incubated with Alexa Fluor 488 donkey anti-goat secondary antibody (1:200; ab150129, Abcam) for 1 hour at room temperature in the dark. The samples were washed three times in PBS with 1% w/v BSA, and the slides were then incubated overnight at +4C with rabbit polyclonal vWF antibody (1:200; ab6994, Abcam) in PBS with 3% of donkey serum (w/v) and 1% BSA (all from Sigma-Aldrich). After three washing steps with PBS and 1% w/v BSA, the sections were incubated with Alexa Fluor 647 donkey anti-rabbit secondary antibody (1:200; ab150075, Abcam) for 1 hour at room temperature in the dark. Last, samples were washed three times with PBS and 1% w/v BSA, and the sections were mounted using 4,6-diamidino-2-phenylindole mounting media (Sigma-Aldrich). Fluorescence emission was detected using a confocal laser scanning microscopy (Olympus FluoView 1000).

Results were expressed as means SD. Statistics was performed using the following variables: (i) When there were two groups and one time point, a standard two-tailed t test was performed. (ii) When there were more than two groups and one time point, a one-way analysis of variance (ANOVA) was performed. (iii) When there were more than two groups and multiple time points, a two-way ANOVA was performed. All analyses were performed using GraphPad (GraphPad Software, La Jolla, CA, USA; http://www.graphpad.com). For all comparisons, the level of significance was P 0.05.

Acknowledgments: We thank the staff at the Bioresources Unit in Trinity College Dublin for veterinary assistance and technical support. Funding: This publication has emanated from research supported by a research grant from the European Research Council (ERC) under grant no. 647004, the Irish Research Council (GOIPD/2016/324), and NIHs NIAMS grant R01AR063194. Author contributions: F.E.F. was responsible for technical design, development of bioinks, performing all animal surgeries, performing vessel perfusion, all CT scans, data interpretation, histological analysis, and drafting the paper. P.P. assisted with the rat surgeries and assisted with the vessel perfusions. L.H.A.v.D. assisted with CT analyses and CT scans. J.N. and D.C.B. assisted with all animal surgeries. J.-Y.S. and E.A. developed the RGD -irradiated alginate. D.J.K. conceived and helped design the experiments, oversaw the collection of results and data interpretation, and finalized the paper. Competing interests: Research undertaken in the laboratory of D.J.K. at Trinity College Dublin is part-funded by Johnson & Johnson. The authors declare no other 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.

Read this article:
3D bioprinting spatiotemporally defined patterns of growth factors to tightly control tissue regeneration - Science Advances

Bone Marrow Stem Cells Comments Off on 3D bioprinting spatiotemporally defined patterns of growth factors to tightly control tissue regeneration – Science Advances | August 15th, 2020

Mystified by the need for defibrillation to save a 10-year-old from drowning, Michael Ackerman, M.D., Ph.D., vowed to dig for answers. That pivotal case during a Mayo Clinic pediatric cardiology residency was the catalyst for Dr. Ackermans career in genetic sleuthing of inherited sudden cardiac death syndromes. With help from the Center for Regenerative Medicine Biotrust, Dr. Ackermans team reprograms cell lines to zero in on precise causes and possible treatments for genetic heart disorders that increase the risk of sudden cardiac death. His research and practice focus on inherited conditions like long QT syndrome (LQTS), catecholaminergic polymorphic ventricular tachycardia (CPVT) and Brugada syndrome (BrS) along with heart muscle diseases such as hypertrophic cardiomyopathy (HCM).

Working with the Center for Regenerative Medicine has opened up a whole new investigative arm to our lab. It is bench to bedside research. We take cells from a blood sample from my patients and then reprogram those cells to become cardiac cells. This research effort has been a powerful tool in gene discovery to prove beyond a shadow of a doubt when a monogenetic variant is indeed the cause of a sudden cardiac death syndrome, says Dr. Ackerman.

Reprogramming cells to identify disease-causing mutations

Reprogramming a patients cells is like a step back in time to when the cells were initially forming in the mothers womb. At that time, cells were dividing and could become any type of cell or tissue in the body. Reprogrammed cells, known as induced pluripotent stem cells, can be redirected to become new heart cells. Dr. Ackermans team uses these patient-specific cell lines to create a disease in a dish model and investigate whether genetic mutations are causing the patients genetic heart disease such as long QT syndrome.

Once we think weve found the root cause of disease, we then go to the patients cell line. We ask, does it show in the dish, in that patients re-engineered heart cells, a prolonged QT cellular phenotype? If it does, then we edit out and correct that variant of interest and at the cellular level test whether the abnormality disappears, says Dr. Ackerman.

Dr. Ackermans team then introduces that genetic variant into normal, healthy cells. If those cells produce a long QT phenotype, they have proof that exact genetic variant is the cause.

Using this disease in a dish model and other genetic sleuthing strategies, Dr. Ackermans team has discovered six of the 17 known genes that cause long QT syndrome. And, they have recently described two entirely new syndromes. One is triadin knockout syndrome, a heart arrhythmia that could lead to cardiac arrest in children during exercise. The second is an autosomal recessive genetic mechanism for calcium release channel deficiency syndrome, prevalent within Amish communities. That key discovery solved the mystery of why so many Amish children were dying suddenly during ordinary childhood play. The disease in a dish model is also useful for discovering new therapies. After creating the patients disease in a dish, Dr. Ackermans team tests potential new drug compounds to see if they could be effective.

We are developing a new gene therapy for the most common genetic subtype of long QT syndrome.With this model, the gene therapy vector is essentially curing the diseased long QT phenotype in the dish, says Dr. Ackerman.

Almost quit research

Dr. Ackerman began medical and graduate school at Mayo Clinic in 1988, where he worked in a research lab next to then fellow trainee, Andre Terzic, M.D., Ph.D., who now is director of Mayo Clinic Center for Regenerative Medicine. Initially not seeing the relevance to patient care, Dr. Ackerman finished his Ph.D. and left research vowing to never, ever return. True to his mentors predictions that youll be back, Mike, Dr. Ackerman felt the pull back to research to address unmet medical needs of his patients.He joined Mayo Clinics faculty in 2000 as one of the first genetic cardiologists with a goal of establishing a practice for patients at risk of sudden cardiac death from genetic heart diseases. Dr. Ackerman now directs the Mayo Clinic Windland Smith Rice Genetic Heart Rhythm Clinic and the Mayo Clinic Windland Smith Rice Sudden Death Genomics Laboratory.

Dr. Ackermans return to research has provided many answers for patients, with over 600 peer-reviewed publications that have occurred since that time 23 years ago when Dr. Ackerman and his team first solved that 10-year-old boys near fatal drowning. It was a mutation in the gene causing type 1 long QT syndrome.

Dr. Ackerman is one of the innovators the Center for Regenerative Medicine collaborates with as it seeks to be a global leader and trusted destination for regenerative care driven by research and education.

###

Tags: Brugada syndrome, Center for Regenerative Medicine Biotrust, hypertrophic cardiomyopathy, long Q T syndrome, Mayo Clinic Center for Regenerative Medicine, Michael Ackerman, People, Research, Stem cell research, sudden cardiac death

Read the rest here:
Using stem cells to find causes and treatments to prevent ...

Cardiac Stem Cells Comments Off on Using stem cells to find causes and treatments to prevent … | August 15th, 2020

Global Stem Cell Therapy Market is expected to reach an approximate CAGR of 10.3% during the forecast period. The use of stem cell for treating medical conditions is referred to as stem cell therapy. Stem cells are undifferentiated cells and differentiate into specialized cell types.

This ability of stem cells to differentiate into cells of interest is used to treat diseases like diabetes, heart disease, hematopoietic disorders (for example leukemia, thalassemia, and others), degenerative disorders (osteoarthritis, Alzheimers disease, Parkinsons disease, chronic renal failure, congestive cardiac failure,) and others.

Some of the key players are Osiris Therapeutics, Inc. (US), MEDIPOST Co., Ltd. (South Korea), Anterogen Co., Ltd. (South Korea), Pharmicell Co., Ltd. (South Korea), Holostem Terapie Avanzate S.r.l. (Italy), JCR Pharmaceuticals Co., Ltd. (Japan), NuVasive, Inc. (US), RTI Surgical, Inc. (US), and AlloSource (US), Thermo Fisher Scientific are some of the key players operating in the global stem cell therapy market.

Avail Sample copy For this Report at: https://www.marketresearchfuture.com/sample_request/6422

Global Stem Cell Therapy Market, by Technique,

Global Stem Cell Therapy Market, by Product Type

Global Stem Cell Therapy Market, by Application

Global Stem Cell Therapy Market, by End-User

Geographically, Americas is the largest in the market owing to the increasing prevalence of heart diseases and growing healthcare expenditure. According to the Centers for Disease Control and Prevention in November 2017, report every year 735,000 Americans have a heart problem. Such a high number of heart patients in the Americas drives the market growth in this region.

Brows Full Report at: https://www.marketresearchfuture.com/reports/stem-cell-therapy-market-6422

Europe (UK, Belgium, France, and Netherlands) is the second largest global stem cell therapy market during the forecast period. The increasing occurrence of stroke, cancer, and osteoarthritis drives the market in this region. According to Anthony Nolan organization 2017, annual review 1.4million people register for donating stem cell in 2017. Also, more than 2,200 searches for a lifesaving stem cell transplant were made in 2017 by UK people. Such a high demand for Stem cell transplantation in this region promotes the market.

Asia-Pacific was projected to be the fastest growing region for the global stem cell therapy market in 2017. The market is expected to witness growth owing to the rising prevalence of smoking in this region.

According to the American Cancer Society, Inc 2018, report China 48.9%, India 16.2%, Japan 11.2% accounts of cancer cases in this region. Such a high cancer rate in this region favors the stem cell therapy market in this region.

The Middle East and Africa accounts for the least share due to low per capita income and lack of availability of well-trained healthcare professionals. However, the rising oncology and technology both at the hospital level and in the community are expected to influence the market in a positive way.

Enquire Discount for this Report at: https://www.marketresearchfuture.com/check-discount/6422

Key factors responsible for the market growth are the rising awareness for therapeutic application of stem cells in disease management, rising research for stem cell therapy applications, development of advanced genetic analysis techniques, increasing public-private investments for stem cell research, growing research in identification of new stem cell lines, and new developments in stem cell banking infrastructure are driving the growth of the global stem cell therapy market. Stem cells are used in the treatment of Alzheimers by replacing the diseased cells with stem cells

About Market Research Future:

At Market Research Future (MRFR), we enable our customers to unravel the complexity of various industries through our Cooked Research Report (CRR), Half-Cooked Research Reports (HCRR), Raw Research Reports (3R), Continuous-Feed Research (CFR), and Market Research & Consulting Services.

Contact:

Market Research Future

+1 646 845 9312

Email:[emailprotected]

Continue reading here:
Stem Cell Therapy Market Application Growth, Technology, Trends and Key Players Developments on Regional Industry Size Till 2023 - eRealty Express

Cardiac Stem Cells Comments Off on Stem Cell Therapy Market Application Growth, Technology, Trends and Key Players Developments on Regional Industry Size Till 2023 – eRealty Express | August 15th, 2020

"You start to feel like there's this temptation of fate," she said. "Once your allusion of permanence is shattered, you feel like anything could happen."

But it was exactly this that made her want to go through with it. After planning so many funerals, going to the hospital to give bone marrow that would help a young boy and his family seemed the right thing to do.

Losing time with her own relatives made her adamant about the ability to help give more time to someone else.

In June, she underwent physical tests and surgery to extract the bone marrow. She felt mostly OK like she had fallen on ice and "got out of laundry for a few days." She knows she can't speak for all donors, but for her, it was a fairly swift recovery.

She thought of the child's family. She remembered her four children at age 7.

"I remember how little they were," she said. "You just want to protect them."

She doesn't know anything more about the boy, and DMKS can't release more information because of privacy laws. His family can reach out to her, but Leone says she's not expecting any communication because she is sure they have plenty going on with him undergoing treatment.

But she isn't seeking gratitude. In fact, she feels she has been given a gift. The thought that perhaps she is able to help is a bright spot in a tough year.

Go here to see the original:
Chicago mom of 4 donates bone marrow to 7-year-old boy she doesn't know. 'You just want to protect them.' - Herald & Review

Bone Marrow Stem Cells Comments Off on Chicago mom of 4 donates bone marrow to 7-year-old boy she doesn’t know. ‘You just want to protect them.’ – Herald & Review | August 13th, 2020

New Jersey, United States,- Verified Market Researchhas recently published an extensive report on the Stem Cell Therapy Market to its ever-expanding research database. The report provides an in-depth analysis of the market size, growth, and share of the Stem Cell Therapy Market and the leading companies associated with it. The report also discusses technologies, product developments, key trends, market drivers and restraints, challenges, and opportunities. It provides an accurate forecast until 2027. The research report is examined and validated by industry professionals and experts.

The report also explores the impact of the COVID-19 pandemic on the segments of the Stem Cell Therapy market and its global scenario. The report analyzes the changing dynamics of the market owing to the pandemic and subsequent regulatory policies and social restrictions. The report also analyses the present and future impact of the pandemic and provides an insight into the post-COVID-19 scenario of the market.

Global Stem Cell Therapy Market was valued at USD 117.66 million in 2019 and is projected to reach USD 255.37 million by 2027, growing at a CAGR of 10.97% from 2020 to 2027.

The report further studies potential alliances such as mergers, acquisitions, joint ventures, product launches, collaborations, and partnerships of the key players and new entrants. The report also studies any development in products, R&D advancements, manufacturing updates, and product research undertaken by the companies.

Leading Key players of Stem Cell Therapy Market are:

Competitive Landscape of the Stem Cell Therapy Market:

The market for the Stem Cell Therapy industry is extremely competitive, with several major players and small scale industries. Adoption of advanced technology and development in production are expected to play a vital role in the growth of the industry. The report also covers their mergers and acquisitions, collaborations, joint ventures, partnerships, product launches, and agreements undertaken in order to gain a substantial market size and a global position.

1.Stem Cell Therapy Market, By Cell Source:

Adipose Tissue-Derived Mesenchymal Stem Cells Bone Marrow-Derived Mesenchymal Stem Cells Cord Blood/Embryonic Stem Cells Other Cell Sources

2.Stem Cell Therapy Market, By Therapeutic Application:

Musculoskeletal Disorders Wounds and Injuries Cardiovascular Diseases Surgeries Gastrointestinal Diseases Other Applications

3.Stem Cell Therapy Market, By Type:

Allogeneic Stem Cell Therapy Market, By Application Musculoskeletal Disorders Wounds and Injuries Surgeries Acute Graft-Versus-Host Disease (AGVHD) Other Applications Autologous Stem Cell Therapy Market, By Application Cardiovascular Diseases Wounds and Injuries Gastrointestinal Diseases Other Applications

Regional Analysis of Stem Cell Therapy Market:

A brief overview of the regional landscape:

From a geographical perspective, the Stem Cell Therapy Market is partitioned into

North Americao U.S.o Canadao MexicoEuropeo Germanyo UKo Franceo Rest of EuropeAsia Pacifico Chinao Japano Indiao Rest of Asia PacificRest of the World

Key coverage of the report:

Other important inclusions in Stem Cell Therapy Market:

About us:

Verified Market Research is a leading Global Research and Consulting firm servicing over 5000+ customers. Verified Market Research provides advanced analytical research solutions while offering information enriched research studies. We offer insight into strategic and growth analyses, Data necessary to achieve corporate goals, and critical revenue decisions.

Our 250 Analysts and SMEs offer a high level of expertise in data collection and governance use industrial techniques to collect and analyze data on more than 15,000 high impact and niche markets. Our analysts are trained to combine modern data collection techniques, superior research methodology, expertise, and years of collective experience to produce informative and accurate research.

Contact us:

Mr. Edwyne Fernandes

US: +1 (650)-781-4080UK: +44 (203)-411-9686APAC: +91 (902)-863-5784US Toll-Free: +1 (800)-7821768

Email: [emailprotected]

See the article here:
Stem Cell Therapy Market Size by Top Companies, Regions, Types and Application, End Users and Forecast to 2027 - Bulletin Line

Bone Marrow Stem Cells Comments Off on Stem Cell Therapy Market Size by Top Companies, Regions, Types and Application, End Users and Forecast to 2027 – Bulletin Line | August 13th, 2020

Jakafi may offer a survival benefit for patients with myelofibrosis and an increased number of circulating blasts, a recent study found.

While the presence of circulation blasts in the blood is considered an important factor in patient prognosis, the impact of bone marrow blasts on survival is not as well defined. To better understand the connection between the amount of blasts found in the blood and bone marrow together, all in regard to patient prognosis, researchers performed a retrospective analysis of 1,316 patients with myelofibrosis, a type of myeloproliferative neoplasm (MPN).

These patients (median age, 66 years), who all presented to the University of Texas MD Anderson Cancer Center in Houston, Texas, from July 1984 and 2018, had to have available circulation blasts in the blood and bone marrow percentages to be included in the analysis. Survival was noted as the time from the date of referral to the date of last follow-up or death, whichever came first. The median follow-up was 27 months.

Among the total, 700 (53%) had 0% circulation blasts in the blood and less than 5% had bone marrow blasts. Of the remaining patients who had 1% or greater circulation blasts in the blood, the range was as follows:

The researchers also found that higher percentages of circulating blasts in the blood had a negative correlation with hemoglobin and platelets, but a positive correlation with white blood cells, age and the presence of symptoms, among other factors.

Out of the total group, 523 patients (44%) received the JAK1/JAK2 inhibitor Jakafi. The authors noted that patients who received this treatment and also had 10% or less blasts, regardless of whether they were in the blood or bone marrow, saw a superior overall survival rate compared to those with similar disease features who did not receive Jakafi.

The studys authors went on to conclude that patients who have circulating blasts in the blood of 4% or more have an unfavorable prognosis; however, Jakafi offers a significant survival benefit to patients with circulating blasts in the blood of 10% or less, making a combination approach to treatment vital in improving the outcomes of patients with myelofibrosis.

Go here to see the original:
Jakafi May Offer Survival Benefit in Subset of Patients with Myelofibrosis - Curetoday.com

Bone Marrow Stem Cells Comments Off on Jakafi May Offer Survival Benefit in Subset of Patients with Myelofibrosis – Curetoday.com | August 13th, 2020

Unlike other CAR T-cell therapies, clinical success was not associated with significant complications from therapy, said Dr. Jonathan Serody. This means this treatment should be available to patients in a clinic setting and would not require patients to be hospitalized, which is critical in our current environment.

Results from the parallel phase 1 and phase 2 studies also demonstrated that the CAR T-cell therapy was safe and did not produce any serious or severe side effects.

Researchers from the UNC Lineberger Comprehensive Cancer Center and Baylor College of Medicine administered anti-CD30 CAR T cells to 41 patients with relapsed or refractory Hodgkin lymphoma. All patients underwent lymphodepletion with bendamustine alone, bendamustine and fludarabine, or cyclophosphamide and fludarabine prior to the anti-CD30 CAR T-cell therapy.

Measuring safety was the primary goal of the two parallel studies.

The overall response rate, or the percentage of partial or complete responses to therapy, among 37 evaluable patients was 62%. Thirty-four of the patients received fludarabine-based lymphodepletion 17 of which received it with bendamustine, and the other half received it with cyclophosphamide. Two of these patients were considered to be complete response at infusion and maintained the response, so they were not included in final analysis.

The overall response rate among the remaining patients was 72%, with 59% of patients achieving a complete response. After a median follow-up of 533 days, researchers identified the one-year progression free survival rate to be 36% and the one-year overall survival rate to be 94%.

This is particularly exciting because the majority of these patients had lymphomas that had not responded well to other powerful new therapies, said senior study author Dr. Barbara Savoldo, professor in the Department of Microbiology and Immunology at the UNC School of Medicine, in a press release.Patients within the study had received a median of seven previous lines of therapy that included checkpoint inhibitors and autologous or allogeneic stem cell therapies, therapies known to be powerful but also tend to come with a host of side effects.

However, treatment with the anti-CD30 CART cells demonstrated a favorable safety profile. Although 10 patients developed cytokine release syndrome, all cases were considered minor.

Patients who received fludarabine-containing lymphodepletion were the only participants in the study to have a response to the anti-CD30 CAR T-cell therapy.

Although CD30 CAR T (cells) showed modest activity in (Hodgkin lymphoma) when infused without lymphodepletion, robust clinical responses were achieved when these cells were infused in hosts lymphodepleted with fludarabine-containing regimens, the authors wrote.

The activity of this new therapy is quite remarkable and while we need to confirm these findings in a larger study, this treatment potentially offers a new approach for patients who currently have very limited options to treat their cancer, said Dr. Jonathan Serody, director of the bone marrow transplant and cellular therapy program at UNC Lineberger Comprehensive Cancer Center, in the release. Additionally, unlike other CAR T-cell therapies, clinical success was not associated with significant complications from therapy. This means this treatment should be available to patients in a clinic setting and would not require patients to be hospitalized, which is critical in our current environment.

Follow this link:
Novel CAR T-Cell Therapy Shows Promise in Advanced Hodgkin Lymphoma - Curetoday.com

Bone Marrow Stem Cells Comments Off on Novel CAR T-Cell Therapy Shows Promise in Advanced Hodgkin Lymphoma – Curetoday.com | August 13th, 2020

Poverty, Government apathy and Covid-19 induced-lockdown restricting travel proved fatal for little Kishan, a 11-year-old boy suffering from Aplastic anemia, a life-threatening blood disorder condition in which the bone marrow and stem cells do not produce enough blood cells

Facing severe financial constraints and waiting timely medical aid, first at Safdarjung Hospital and then AIIMS, both Government hospitals in Delhi, Kishans life was cut short in March this year amid Covid-19 pandemic.

However, Kishans is not a lone case. Dr Nita Radhakrishnan, paediatric haemato-oncologist at Super Speciality Paediatric Hospital, Noida, Uttar Pradesh says that as the deadly Coronavirus captured the attention of the nation in the most unprecedented manner, the non-Covid patients particularly those with the Aplastic anemia have suffered the most in the crisis.

She gave instances of her two teenage patients who succumbed to blood disorder in the Covid catastrophe. Manish (name change), a 17-year-old was suffering with on-and-off fever, gum bleeding, and melena for three months, he came to us in December last year just when Coronavirus had started spreading its tentacles from China to other parts of the world.

The boy was diagnosed with severe Aplastic anemia and was recommended requisite treatment like regular hospital visit for red cell transfusion before he could be given bone marrow transplant (BMT), a life saving treatment.

However, while the family was not able to visit our hospital in Noida due to the covid-lockdown, no blood products were available at the hospital near to the patients locality. In want of blood, Manish could not survive more days.

13-year-old Suresh (name change) too faced similar fate. While Government funds could not be sanctioned for his BMT in time the boy could not visit the Noida hospital for further follow-up due to travel restrictions. Two weeks later, Suresh died due to hemorrhage at his native place, lamented the doctor.

These are just two reported cases from the NCR hospital located near the countrys capital. Several have gone unreported. The Government has no policy nor any long-term plan for such patients.

The prognosis of severe aplastic anemia in our country is dismal. The incidence of 46 per million population of childhood aplastic anemia in India and other Asian countries is higher than what is observed in the West, explains Dr Radhakrishnan. The scenario is gloomy for the patients afflicted with the disease as they need blood transfusion almost every 20 days.

A significant proportion of patients of aplastic anemia (around 30 per cent) die before any definitive treatment is initiated. A study by AIIMS based on a recent series of patients follow-up showed that out of 1501 patients diagnosed over last seven years, only 303 ie 20 per cent received the definitive treatment modalities through either BMT or IST with ATG and cyclosporine, says Dr Radhakrishnan in her case report Aplastic anemia: Non-COVID casualties in the Covid-19 era, published in the latest edition of Indian Journal of Palliative Care.

The doctors have sought urgent intervention. Dr Radhakrishnan says that as we await the peak of Covid-19 in our country and possibly secondary and tertiary waves thereafter, patients with aplastic anemia who are the sickest among all hematological illnesses would benefit greatly from urgent intervention from the Government to ensure timely treatment.

Those suffering with Aplastic anemia, there is mostly delay in diagnosis, delay in initiation of treatment due to monetary constraints, non-inclusion of the disease under government schemes such as Ayushman Bharat and NHM and delay in sanction of money from other Government schemes such as Rashtriya Arogya Nidhi, Chief Minister and Prime Ministers relief fund often due to lack of proper documents, she added.

Delay means, risk of contracting fungal infections and increase in drug-resistant bacterial infections increase which further hamper the treatment, point out Dr Ravi Shankar and Dr Savitri Singh in the study.

Though the Union Health Ministry, after few days of lockdown period, issued directions for continuing treatment for essential health services including reproductive and maternal health services, newborn care, severe malnutrition, and NCDs including cancer care, palliative care, dialysis, and care of disabled, unfortunately those with Aplastic anemia got ignored.

This despite of the fact that these patients are at the highest risk of death following a break in the treatment of few weeks, notes Dr Radhakrishnan.

Because of the closure of offices and absence of staff, during the lockdown period, there was delay in sanction of usual grants due to the lockdown of offices and inability in generating documents such as income certificate from the tehsils.

For instance, Suresh and Manish, both our patients received the Government grant after around 34 months of applying for the same. But both had died before they could reach the hospital for treatment, lamented the hematologist.

View post:
Covid-19 Impact: Patients with aplastic anemia at receiving end - Daily Pioneer

Bone Marrow Stem Cells Comments Off on Covid-19 Impact: Patients with aplastic anemia at receiving end – Daily Pioneer | August 13th, 2020

INTRODUCTION

The ability to regenerate complex body parts varies considerably in the animal kingdom. While planarian and hydra are able to regenerate their entire bodies, many avian and mammalian species mostly stop at the wound healing stage without a reparative regeneration process (1). This disparity may result from complexity differences among organisms by nature, yet it leaves us the hope that we may learn from highly regenerative species to improve our own regenerative potential.

Zebrafish is known for its ability to regenerate multiple complex body structures (2). Among regenerable tissues, the caudal fin serves as a great model due to its faithful and rapid regeneration, ease of manipulation, and relatively low complexity. A key step in regeneration is the formation of the blastema, a layer of proliferative and undifferentiated cells that accumulates between the wound site and the wound epidermis following initial wound closure. This step occurs in response to appendage loss and is one of the key features that separates regenerative systems from nonregenerative systems. At later stages of regeneration, the blastema further proliferates and differentiates to regenerate the missing complex structures.

However, the molecular signatures of blastemal cell state transitions during regeneration in zebrafish remain elusive. The state of a cell can be represented by its collective gene expression profile, which has only been measured in bulk for all genes or in specific lineages of cells for a subset of genes during caudal fin regeneration. Prior work has shown that both proliferation of progenitors and dedifferentiation of adult lineage cells contribute to the blastema (38). Progenitors respond to injury cue and proliferate as in normal development. Cells derived from mature adult lineages, however, lose their lineage-specific markers while obtaining progenitor-like markers when they proliferate. Neither type of cell gains multipotency, but rather, they proliferate and regenerate with lineage restrictions. The limited resolution and throughput of these approaches have prevented a more systematic understanding of blastema cells. The advent of single-cell transcriptomic technologies promises to reveal signals masked at the bulk tissue level (9), granting us an opportunity to define and monitor cellular state transition in regenerating fin at an unprecedented resolution.

In this study, we generated single-cell transcriptomic maps of regenerating fin tissue. These maps allowed us to separate the contribution from different cell types and track the transcriptomic dynamics in cell state transitions during regeneration. By comparing with the profiles obtained from uninjured fin tissue, we identified cell types involved in regeneration. We demonstrated the activation of cell cyclerelated programs shared across cell types as well as cell typespecific programs. Furthermore, we defined the heterogeneity in both epithelial and blastemal populations and their functional relations to the regeneration process.

To better understand cell type involvement in fin regeneration, we characterized single-cell transcriptional landscapes for both preinjury and regenerating caudal fin tissues using the 10x Genomics platform (see Materials and Methods and table S1) (9). We sampled regenerating fins from 1, 2, and 4 days post-amputation (dpa) time points to interrogate the stages of blastema formation, outgrowth, and maintenance (Fig. 1A). Fin samples were collected from multiple fish to control for individual variation while at the same position along the proximal-distal axis to avoid positional effects. To establish the transcriptional ground states for each cell type in the fin tissue, we first focused on cells collected from the preinjury time point. Via an unsupervised clustering of 4134 cells, we identified epithelial cells (epcam and cdh1), hematopoietic cells (mpeg1.1 and cxcr3.2), and mesenchymal cells (msx1b and twist1a) (fig. S1, A and B) (1014). Epithelial cells are from three transcriptionally distinct subgroups, representing the superficial (krt4), intermediate (tp63), and basal layers (tp63 and krtt1c19e) of the epithelium (fig. S1, A and B) (15, 16).

(A) General experimental design. Zebrafish caudal fin tissues at preinjury and 1/2/4 dpa stages were collected. (B) Clustering assignments for caudal fin cells collected from each stage. Uniform Manifold Approximation and Projection (UMAP) axes were calculated from the integrated cells dataset as in (C). (C) Clustering assignments for caudal fin cells collected from both preinjury and regenerating stages. Cells were plotted on UMAP axes. Color coding is the same as in (E). (D) Percentage distribution of the major cell types captured in caudal fin, grouped by their stage of collection. Color coding is the same as in (E). (E) Differential expressions of the key marker genes by the identified major cell types. Color gradient: normalized relative expression level. Dot size: percentage of cells in the cluster that express the specified gene.

To determine whether the same cell types existed in the regenerating stages, we performed analysis using two different approaches: (i) Cells from each stage were clustered independently, and (ii) cells from both uninjured fins and injured fins were integrated through the anchoring approach (see Materials and Methods; Fig. 1, B, C, and E; and table S2) (17). For both approaches, we regressed out cell cycle effects before principal components analysis (PCA). Agreement between cluster assignments was measured using Hubert and Arabies adjusted Rand index (ARI). An average ARI of 0.86 (preinjury, 0.86; 1 dpa, 0.85; 2 dpa, 0.90; and 4 dpa, 0.83) indicated that clustering results generated using the two approaches were highly consistent. Cell types identified in the preinjury cells presented consistently across all regenerating stages, suggesting that regenerating fins contain the same cell types as the preinjury fins.

New regenerates are built up by the proliferation and migration of cells located at a number of fin segments away from the amputation plane (2). In response to injury cues, these cells gained the ability to detach from local tissue, enter cell cycle, and migrate toward the wound site while undergoing transcriptional reprogramming. We computationally separated S phase, G2-M phase, and G1-phase cells based on the expression level of cell cyclerelated genes and performed clustering analysis using only S phase cells (see Materials and Methods and fig. S2A). In this cycling cell population, we identified epithelial, mesenchymal, and hematopoietic cell groups as before (Fig. 2, A to C, and table S3). Our data support a model in which cells likely keep their original identities during proliferation.

(A) Cell type clustering of S phase cells plotted onto UMAP axes calculated by S phase cell only. Cells are colored by the general cell types merged from major cells types in Fig. 1B. (B) Stage distribution of S phase cells. Cells were plotted on the same UMAP axes as in (A) and colored by stage when the cells were collected. (C) Relative expression levels of the top 30 differentially expressed genes from each cluster of only S phase cells. (D) Venn diagrams of numbers of genes shared between the cell cycleactivated genetic programs. Left, included all genes; right, included only cell cyclerelated genes (see Materials and Methods).

Next, we asked whether different regenerating cell types exhibited similar or distinct cell cycle regulations. To this end, we identified genes up-regulated in S phase cells compared to G1 phase cells in each cell type, respectively (logFC, >0.25; minimum percentage, >10%). Of the 1098 differentially expressed genes, 161 were shared across all three groups of comparisons (Fig. 2D and table S4). Of these shared genes, at least 54 genes were related to cell cycle regulation, underscoring a shared program governing cell cycle reentry (criteria described in Materials and Methods). In contrast, hundreds of genes differentially highly expressed in S phase exhibited cell typespecific pattern, of which dozens were related to cell cycle (Fig. 2D). We next evaluated the degree of conservation of these enriched genes by asking what fraction did not have human orthologs that had been curated in the Metascape database (18). Twenty-five percent of genes in the epithelial cellspecific group had no human ortholog, whereas all shared groups had at most 15% genes without a human ortholog, suggesting that enriched genes shared by cell types were more evolutionarily conserved (fig. S2C).

Some cell typespecific S-G1 enriched genes were also expressed in a cell typespecific manner regardless of their cell cycle phases: For example, psmb8a and psmb9a shared similar epithelial-hematopoietic enrichments (fig. S2D). The human homologs of these genes (PSMB8 and PSMB9) encode 5i and 1i subunits of the immunoproteasome (19). Together with 2i and PA28 subunits of the proteasome, they turn the proteasome into immunoproteasome and take part in immune response (20). Immunoproteasome digests peptides more efficiently, promoting antigen presentation by a major histocompatibility complex (MHC) class I molecule. Although they did not pass the differential expression criteria in the S-G1 comparison, zebrafish psmb10, psme1, and psme2 shared a differential expression signature similar to that of psmb8a and psmb9a, suggesting that zebrafish might use the same group of subunits for the assembly of immunoproteasomes that might help increase immune responses during regeneration, especially in epithelial and hematopoietic cells (fig. S2, D and E). In addition, we found three genes that shared the same expression signature with the immunoproteasome subunits (psmb13a, psmb12, and psma6l) (fig. S2E) without known human or mouse homologs, suggesting that they might form zebrafish-specific proteasomes with functional relevance to regeneration (19).

Consistent with current knowledge, we observed three transcriptionally distinct subgroups in the preinjury epcam+ epithelial cells, representing the superficial, intermediate, and basal layers of the adult zebrafish epithelium (Fig. 3A and fig. S1B) (15, 16). By integrating cells from all stages during regeneration, we found clusters of cells that corresponded to all three layers of the epithelium after injury (Fig. 1, B and C). In addition, we captured a rare agr2+ population (referred to as mucosal-like epithelium herein) that was too small to be clustered by itself in the preinjury stage (Fig. 1E). These cells shared general epithelial features with the other epithelial layers but exhibited higher expression of a unique set of 200 genes. We examined the expression distribution of the orthologs of these genes in human tissues (The Human Protein Atlas, http://proteinatlas.org/) (21). Among the top 30 genes with human orthologs, 11 showed enriched expressions in proximal digestive or gastrointestinal tract and another 11 in bone marrow of blood lineages, suggesting that this population is analogous to cells in the mucosa in mammalian systems (table S2). In zebrafish, agr2 is required for the differentiation of the mucosal-producing goblet cells in the intestinal epithelium (22). To confirm the cell typespecific expression pattern of this gene in the fin tissue, we performed in situ hybridization on agr2 in both uninjured and regenerating fin tissues (see Materials and Methods). agr2 transcripts are scattered within the epithelium regardless of the sample collection stage and reflect a round morphology of the cell expressing it (fig. S3, A, C, E, and G to I). A proportion of agr2+ cells overlap with positive dark blue staining of Alcian blue in serial sections, suggesting that these cells are mucous cells that are known to exist in the caudal fin epithelium (fig. S3, B, D, and F) (23).

(A) Diagram of the stratified adult zebrafish epithelium. (B) Differential expressions of claudin family and keratin family genes in epithelial subgroups shown as a dot plot. Known epithelial markers krt4, fn1b, tp63, and krtt1c19e were included for comparison. Cells were first grouped by major cell types and then separated into preinjury and regenerating stages. Darkness of dot color: relative expression level. Dot size: percentage of cells in the cluster that express the specified gene. (C) In situ hybridization targeting krt1-19d, cldna, cldn1, and krt4 of 4-dpa fin tissues. Brown dots indicate positive RNA signals from target genes, while pale blue blocks represent hematoxylin-stained cell nuclei. Zoomed-in views are presented. Original images can be found in fig. S4. All epithelial layers are above the black dotted lines. (D) Clustering assignment of epithelial cells plotted on UMAP axes calculated with only epithelial cells. Cells are colored by their epithelial layer identity as in (A). (E) The same UMAP visualization as in (D), with cells colored by stage of collection. Arrows connect the groups of comparison, with a direction from preinjury stage to regenerating stages (1, 2, and 4 dpa). Numbers next to the green triangle: number of genes up-regulated in regenerating stage. Numbers next to the red triangle: number of genes down-regulated in regenerating stage. (F) Clustered GO enrichment for genes up-regulated in regenerating basal, intermediate, and superficial epithelial cells comparing to their preinjury counterparts. GTPase, guanosine triphosphatase; ER, endoplasmic reticulum; PKN, protein kinases N; snRNP, small nuclear ribonucleoprotein.

Although the same three-layer classification of epithelial cells could be defined when cells from regenerating stages were integrated with the preinjury cells, the expression of the commonly used layer-specific marker genes changed dramatically during regeneration: Superficial epithelial marker krt4 expanded into basal and intermediate layers of the epithelium, the intermediate layer marker fn1b was also highly expressed in the basal layer, and the basal epithelial marker krtt1c19e was barely detectable in the postinjury cell populations (Fig. 3B) (15, 16). To better understand the molecular features of the epithelial populations, we identified genes significantly more highly expressed in epithelial cells than in hematopoietic and mesenchymal cells and found that cell-cell junction genes ranked high in the list. Among these, genes from the claudin and keratin families were detected at a ratio 20-fold higher than that in randomly selected detectable genes (2 test, P value of <0.0001). We focused on expression patterns of all claudin and keratin genes in zebrafish and found that cldne, cldnf, krt1-19d, and krt17 labeled the superficial cluster; cldnh labeled the mucosal-like cluster; cldna, krt93, and krt94 labeled the intermediate cluster; and cldn1 and cldni labeled the basal cluster (Fig. 3B). Claudin genes are expressed in a tissue-specific manner in zebrafish and are generally considered to be the proteins responsible for regulating the paracellular permeability in the vertebrate epithelium (24). Their differential expression signature in both uninjured and regenerating tissues suggests that they might play important roles in maintaining the permeability in each epithelial population. On the other hand, the expression of keratin genes displayed less restriction across the three layers relative to claudin genes but stronger dependence on regenerating states (Fig. 3B). The differential expression signature suggests that they might perform epithelial subtyperelated function in regeneration. To verify their expression pattern, we performed RNA in situ hybridization targeting the known marker krt4 and new candidates, including krt1-19d, cldna, and cldn1 (Fig. 3C) as well as cldne, krt94, and cldni (fig. S4, A to H). Comparing with the known marker krt4, these genes exhibited more restricted expression patterns in epithelial layers, better representing the molecular signatures of different epithelial populations in the fin tissue regardless of regeneration status (Fig. 3, B and C).

The three epithelial layers were present across the regeneration stages albeit with varying proportions (Fig. 1D). The proportion of basal epithelial cells peaked at 2 dpa, reaching up to 42%, whereas the superficial layer epithelial cells decreased from 27 to 6% at 2 dpa (the coefficient of variations of cell proportions between biological replicates is below 15%). The observed compositional change of the two epithelial populations is consistent with a previous finding that the initial migration of superficial layer cells to the new regenerates is followed by replenishment by basal epithelial cells (25). This basal replenishment was also reflected in the two-dimensional Uniform Manifold Approximation and Projection (UMAP) calculation from only epithelial cells, in which preinjury cells were separated by their respective layers, whereas regenerating cells were closer in the projection space (Fig. 3, D and E). Superficial layer cells from before and after injury stages were in juxtaposition to each other, consistent with our knowledge that this layer of epithelial cells directly migrates to and covers the wound site (25). On the other hand, basal layer cells from before and after injury stages were more distantly separated, suggesting more dramatic changes between resting and regenerating basal epithelial cells.

To understand the mechanisms of epithelium regeneration, we compared the transcriptome between preinjury and regenerating cells for the three epithelial layers. Basal layer cells up-regulated 1271 genes and down-regulated 198 genes during regeneration; both were the highest numbers across all comparisons (numbers of differentially expressed genes were from Wilcoxon rank sum test, adjusted P value of < 0.01; Fig. 3E). We performed gene ontology (GO) enrichment analysis on genes up-regulated in the regenerating stage by layer and found both common and layer-specific programs associated with regeneration (18). All three layers were enriched for oxidative phosphorylation (dre00190), proteasome (dre03050), and cell redox homeostasis (GO:0045454). While basal and intermediate layer cells could be regulated by Rho guanosine triphosphatasemediated Wnt signaling for extracellular matrix organization and actin filament depolymerization, respectively (R-DRE-195258, R-DRE-5625740, R-DRE-195721, GO:0030198, and GO:0030042), superficial layer cells showed enrichment mainly for general transcriptional and translational regulations (Fig. 3F). When comparing the expression profiles between regenerating superficial epithelial and basal epithelial, we saw enrichment for antigen presentation and apoptosis features in the superficial layer (table S5). In addition, the superficial layer contained the lowest proportion of cells in S phase or G2-M phase, further supporting that superficial layer epithelium was most likely maintained by migration and proliferation from other layers (fig. S2B).

Subcluster identification within regenerating basal epithelial cells revealed two subgroups that represented different functionalities during regeneration, one labeled by distally distributed fgf24, while the other by proximally distributed lef1 (fig. S5, A to C) (26, 27). We compared expression profiles between group I (distal) and group II (proximal) cells and found that their suggested functionalities were consistent with their expected roles in regeneration: The distal subgroup (or distal wound epidermis) up-regulated genes associated with extracellular matrix degradation, and the proximal subgroup (or proximal wound epidermis) up-regulated genes associated with organization of extracellular matrix, skeletal system development, and negative regulation of locomotion (fig. S5, D and E). In addition, the increase of proximal cell proportion was accompanied by the decrease of distal cell proportion, suggesting that basal layer epithelium become gradually active in promoting blastema proliferation and differentiation during the initial regeneration process (fig. S5C). To confirm the distribution of these cells, we performed RNA in situ hybridization targeting two candidate genes, stmn1b and sema3b, one from each cluster. The expression of stmn1b was first observed at the basal layer of the wound epidermis at 1 dpa but diminished as regeneration proceeded (fig. S4, I to K). On the contrary, sema3b showed expression at later stages and was enriched in the relatively proximal portion of the basal layer epithelium (fig. S4, L to N). The expression dynamics of these two genes matched the predicted proportion changes of the two clusters (fig. S5C). While sema3b was more restricted to the basal layer, stmn1b showed low expression levels in the intermediate layer as well, potentially suggesting that this subpopulation could be labeling cells transitioning from the basal layer to the other layers of epithelium.

We next performed subcluster analysis within the hematopoietic cluster and found four subpopulations (Fig. 4, A to C and table S6). The first three populations were enriched for the macrophage marker mpeg1.1, with the cluster H1 being M1-like (lgals2+ and lcp1+) and the cluster H3 M2-like (ctsc+ and lgmn+) (Fig. 4D) (12). We speculated that the cluster H2 represented a transition state between M1-like and M2-like or a state before the macrophages differentiate toward M1-like or M2-like. From 1 to 4 dpa, the proportion of M1-like macrophages remained at a constant level, while that of M2-like macrophages expanded (Fig. 4B), potentially suggesting a shift in the function of macrophages in the new regenerates from pro-inflammatory toward anti-inflammatory as regeneration proceeded. Macrophages are important for proper blastema proliferation (28). The change in the proportions of M1/M2-like macrophage in our data matched with that observed in the larvae fin, suggesting that the adults followed a similar rule for immune cell recruitment after injury.

(A) Subcluster assignments of the hematopoietic cells. Cells were plotted on UMAP axes. The same color code is used for (B) to (D). (B) Proportion of subgroups of hematopoietic cells. (C) Expression enrichment of the top 30 differentially expressed genes in the four subclusters within hematopoietic cluster shown as a heatmap. (D) Expression distribution of genes associated with macrophage activation grouped by subclusters. Expression levels were log normalized by Seurat. y axis: cluster identity. z axis: cell density. (E) Expressions of pigment cell markers gch2 and mlpha in the hematopoietic population.

The cluster H4 enriched for genes including mlpha and gch2, both well-characterized markers for the chromatophore lineages in zebrafish (Fig. 4E) (29). Chromatophores are derived from neural crest lineage, yet here, they clustered with macrophages that were from hematopoietic lineage. One possibility is that this clustering result could be driven by features related to antigen presentation via MHC class II, a feature of pigment cells based on studies using human melanocytes (30). The proportion of this cluster decreased as regeneration started, agreeing with the known pattern of fin stripe recovery after amputation (Fig. 4B) (31).

To understand the component and function of the cells in the mesenchymal cell cluster before and during fin regeneration, we focused on genes enriched in this cluster and found previously identified blastema marker genes that are required for fin regeneration, including the muscle segment homeobox family members msx1b and msx3 and the insulin-like growth factor signaling ligand igf2b (logFC, >0.25; minimum percentage, >25%; and adjusted P value of <1 105, as listed in table S1) (2, 13). The mesenchymal cluster expressed these genes nearly exclusively, confirming their blastema identity in regenerating stages (fig. S6A). In addition, we found key genes involved in zebrafish bone development and regeneration: twist1a, the transcription factor that controls the skeletal development by regulating the expression of runx2 (14); cx43, the gap junction protein required for building the fin ray up to the right length; and hapln1a and serpinh1b, two genes downstream of cx43 (32, 33). By performing conserved marker analysis using Seurat, we found that msx1b and twist1a were also among the markers conserved across all stages, underscoring shared features that existed between regenerating and preinjury mesenchymal cells (maximum P values across stages: 4.72 1010 and 2.84 109 for msx1b and twist1a, respectively). This theme of building and supporting bone tissues in mesenchymal cells was not only reflected by a handful of genes: GO analysis of all the detected up-regulated genes in this cluster revealed significant enrichment of genes associated with GO terms, including fin regeneration (GO:0031101) and skeletal system development (GO:0001501) (fig. S6B). When more stringent criteria for differential expression were used, genes were also significantly enriched for GO terms, including skeletal system morphogenesis (GO:0048705) and extracellular matrix organization (GO:0030198) (fig. S6C).

Previous work has shown that blastema comprises bone cells and non-bone cells but has not defined the cell types and the regeneration process of each type (23, 34, 35). To better understand the regeneration process by cell type, we performed clustering analysis within the mesenchymal cluster and identified nine distinct subgroups (Fig. 5A and fig. S6D). Of the two preinjury subgroups, M-2 represented the mature bone lineage, which was enriched for expressions of bglap, mgp, and sost (fig. S6E) (36, 37). Comparing to M-2, cluster M-1 presented low expression levels of bglap, mgp, and sost and high expression levels of a group of other genes, including fhl1a, fhl2a, and tagln (fig. S6E). Mammalian orthologs of these genes are required for chondrogenesis and osteogenesis, leading us to speculate that cluster M-1 could represent the supporting non-bone cell lineage in the preinjury state (38, 39).

(A) Subclustering assignments of mesenchymal cells shown on UMAP axes. Cells are colored by their cluster assignments and connected by Slingshot-reconstructed trajectories. Lineage 1: 1-2-3-4; lineage 2: 1-2-3-5-6; lineage 3: 1-2-3-5-7-8; lineage 4: 1-2-3-5-9. (B) By-lineage highlighting of mesenchymal cells. Cells with colors other than gray represent the cells included in each corresponding lineage in (A). (C) Expression distribution of genes labeling cell lineages and cell states in mesenchymal cells. Gene feature plots were connected by estimated lineages using the same lineage color code as in (A). (D to G) In situ hybridization targeting the tnfaip6 gene in (D) preinjury, (E) 1-dpa, and [(F) and (G)] 4-dpa fin tissues. Brown dots indicate positive RNA signals from target genes, while pale blue blocks represent hematoxylin-stained cell nuclei. A zoomed-in view for the region inside the focused rectangle is provided within (D). (G) Zoomed-in view for the region highlighted by a rectangle in (F). Dotted lines indicate the amputation plane. All scale bars, 100 m.

The remaining seven populations came from regenerates. Pseudotime analysis via Slingshot (40) suggested that these subgroups formed four trajectories, all initiated from the tnfaip6+ cluster (M-3), which was composed mainly of 1-dpa cells (Fig. 5, B and C, and fig. S6D). tnfaip6 was ranked top by an adjusted P value in the differentially expressed genes labeling the regeneration initiation cluster and was also expressed exclusively in the mesenchymal cluster (Fig. 5C and fig. S6A). The mammalian ortholog of this gene is required for proliferation and proper differentiation of mesenchymal stem cells (MSCs) and balances the mineralization via osteogenesis inhibitions (41). The expression of tnfaip6 in the postinjury zebrafish fin suggested that it could also be required in the early stages of regeneration for promoting mesenchymal proliferation. To confirm the expression pattern of tnfaip6, we performed RNA in situ hybridization for uninjured and regenerating fin tissues targeting this gene (Fig. 5, D and E). In the uninjured fin, tnfaip6 was expressed in a segmental pattern, presumably enriching at joints between bone segments. At 1 dpa, tnfaip6 was expressed not only near the bony rays but also in the cavity, showing a general activation in the mesenchymal population. As regeneration proceeded from 1 to 4 dpa, mesenchymal cells divided into cdh11+ (M-4) and tph1b+ (M-5) branches, with the latter further divided into mmp13a+ (M-6), tagln+ (M-7), and vcanb+ (M-9) branches (Fig. 5C and fig. S6D). The mmp13+ (M-6) cluster maintained a high-level tnfaip6 expression, whereas all other branches had a lower but detectable tnfaip6 expression. This was consistent with the observation we made from in situ hybridization at 4 dpa targeting tnfaip6: the broad expression in the mesenchymal population and segmental enrichments similar to that in the uninjured fin (Fig. 5, F and G).

The four trajectories initiated from the tnfaip6+ cluster revealed four putative lineages representing bone and non-bone cells in the blastema. cdh11+ lineage 1 specifically expressed runx2 and osterix/sp7, which are the key transcription factors regulating osteogenesis (fig. S6E) (42). Mammalian ortholog of cdh11 could induce Sp7-dependent bone and cartilage formation in vivo, suggesting that the cdh11+ branch in the blastema represented the regenerating osteoblasts (43). Genes highly expressed at the end of this lineage (M-4) compared to the initiation point (M-3) were associated with bone mineralization and skeletal system development, further supporting their bone cell identity (table S7).

Mesenchymal cells outside the osteoblast branch shared enrichment for tph1b and aldh1a2 expressions at 2 dpa, followed by and1 expression at 4 dpa (Fig. 5C and fig. S6F). These three genes had been suggested to label joint fibroblasts, fibroblast-derived blastema cells, and actinotrichia-forming cells in the blastema, respectively (34, 35, 44). However, their expression signatures implied that instead of labeling separate populations in the blastema, they might be labeling different states of the same non-osteoblastic cells at the early stage of fin regeneration.

Upon 4 dpa, these non-osteoblastic cells diverged into three groups (Fig. 5C and fig. S6D). To understand this separation, we performed differential expression analysis for each branch between cells at the end of the lineage tree (lineage 1, M-4; lineage 2, M-6; lineage 3, M-7 and M-8; and lineage 4, M-9) and cells in the initiation cluster (M-3). Genes highly expressed at the lineage end points were included for GO analysis for functional predictions (logFC, >0.25; minimum percentage of >25%; and adjusted P value of <0.01). These three lineages were also associated with skeletal system development or extracellular matrix organizations as were the bone cell lineage; however, the association was driven by a nearly completely different set of genes (table S7). Unlike the osteoblast lineage, none of these three non-bone cell lineages showed enrichment for bone mineralization, suggesting that these cells might indirectly contribute to bone formation. In lineage 2, top differentially expressed genes mmp13a and ogn both have mammalian orthologs that are associated with bone formation (Fig. 5C and fig. S6F) (45, 46). In addition, this lineage presented up-regulation of DLX family genes, especially dlx5a, suggesting the reactivation of fin outgrowthrelated developmental programs during regeneration (fig. S6F and table S7) (47). Lineages 3 and 4 both enriched for estrogen response and expressed the retinoic acid (RA) synthesis gene aldh1a2. However, only lineage 3 displayed up-regulation of the RA-degrading enzyme cyp26b1 (fig. S6F and table S7). The cyp26b1high-aldh1a2low pattern helped to reduce RA levels in the blastema, promoting redifferentiation of the osteoblasts (44). The differentiation-promoting signature was also reflected in the enrichment of genes, including col6a1 and tagln, whose mammalian orthologs are essential for bone formation (fig. S6F and table S7) (39, 48). These genes were also enriched in the preinjury non-bone cell population, suggesting a connection between this subset of the non-bone cells and their preinjury counterparts (Fig. 5C and fig. S6F). Top up-regulated genes in lineage 4, on the other hand, were main contributors of the extracellular matrix, including and1/2, loxa, and vcanb (35, 49, 50). Enriched expression of these genes suggested that this lineage could be responsible for creating and organizing the fibrous environment. Together, the various non-osteoblastic cells could potentially work collaboratively with the osteoblasts in creating the environment for bone tissue regeneration.

Genes that had been suggested to label progenitors contributing to fin regeneration (mmp9 and cxcl12a) and several orthologs of known mammalian MSC markers (lrrc15, prrx1a/b, and pdgfra) (6, 7, 51, 52) were expressed almost exclusively in the mesenchymal cluster (fig. S6A). Consistent with the observations made in the lineage-tracing study, the mmp9 expression was associated with the regenerating bone cell lineage (lineage 1; Fig. 5B and fig. S6E) (7). However, mmp9 was detected only in a small portion of the mesenchymal cells and was highly expressed in the basal epithelium cells at similar proportions. On the other hand, we observed coenrichment of cxcl12a (previously known as sdf-1) and orthologs of the known mammalian MSC markers in the preinjury population (fig. S6E). cxcl12a-expressing cells in zebrafish were found to carry osteogenic, adipogenic, and chondrogenic characteristics in vitro like MSCs would do and contributed to the mesenchyme of the newly developing bony rays during fin regeneration (6, 53). The coenrichment pattern suggested that some of the preinjury cxcl12a-expressing cells could be MSCs in the fin tissue, which contribute to fin regeneration.

Zebrafish caudal fin is a unique regeneration system to model the injury response and regeneration of vertebrate appendages despite being a simple structure without muscular and adipose tissues. Major components of the regenerating caudal fin are epithelial cells covering the wound site and blastemal cells producing the connective tissue and bone matrices. Early studies established that actively proliferating blastema is the key to regeneration. Formed by cell migration and proliferation, this layer of cells continues in outgrowth and differentiation, rebuilding the complex body structure. Despite efforts in understanding its importance, basic questions regarding the formation of blastema remained: (i) Which type of cells contributes to the blastema and (ii) how do they shape the regeneration process?

Using single-cell transcriptomes, we defined cell types in both preinjury and postinjury fin tissues. Although regenerating cells were drastically different from their preinjury counterparts, both stage-specific and integrated clustering analysis revealed the same major cell type compositions in the fin tissues regardless of their time of collection. Common cell types detected include epithelial cells from all three layers, hematopoietic cells, and mesenchymal cells. Our data lay a foundation for lineage-targeted analysis to investigate the role of epithelial layers and subtypes in fin regeneration.

For each cell type to be a consistent component in the regenerated fin, cell cycle entry is required. We found that both common and unique cell cycle programs activated in the regenerating fin, with the shared ones appearing to be more evolutionarily conserved than the unique ones. Among the genes showing cell typespecific S phase enrichment, several immunoproteasome subunits also showed a clear cell typespecific expression. We speculated that the increasing level of immunoproteasome subunits in epithelial and hematopoietic cells specifically might accelerate antigen processing and presentation, which could be important for immune cell recruitment and tumor necrosis factorinduced blastemal proliferation (54).

Epithelial cells were the most abundant cell type in the profiled fins and could be clustered into four different subgroups, including the three layers in the adult fish epithelium and the mucosal-like cells within the intermediate layer. However, markers labeling these layers did not perform well in separating cell groups when only regenerating cells were considered. An unbiased differential expression test suggested that some members of the krt and cldn families were expressed in specific layers more consistently throughout regeneration. RNA in situ hybridization targeting cldne, krt1-19d, cldna, krt94, cldni, and cldn1 confirmed their exclusive layer-specific expression pattern, underscoring their potential to serve as markers for the distinct epithelial layers during regeneration. Our epithelium-specific analysis suggested that basal layer epithelial cells proliferate and could be the main source for replenishing the other two layers of the epithelium, similar to findings in a previous study based on genetic lineage tracing in zebrafish and echoing findings made using the axolotl limb regeneration model (25, 55). We observed higher apoptosis and lower proliferation features in the superficial epithelial layer compared to the other layers. At the same time, we observed transition patterns in gene expression, connecting the basal to the intermediate and the superficial layer during regeneration.

The behavior of mucosal-like cells during regeneration had been rarely reported for zebrafish in literature. We found in this study that this group of cells was an integral part of the regeneration process. Enrichment of foxp1b in this population and enrichment of foxp4 in basal and intermediate epithelial cells supported that zebrafish foxp homologs could be involved in regulating agr2 expression as does the Fox family in mice and, furthermore, the mucin production in the epithelium during regeneration (Fig. 1E) (56). The protein encoded by amphibian homologs of agr2, nAG (from newts) and aAG (from axolotl), are necessary and sufficient for salamander limb regeneration (57, 58). They are expressed in both dermal glands and the nerve sheaththe pattern of which has also been recovered from single-cell RNA sequencing (scRNA-seq) analysis (55). Regeneration deficiencies caused by denervation before amputation can be rescued by the ectopic expression of nAG. Although we do not have data supporting the nerve sheath expression pattern, as shown for the amphibian models, we hypothesize that agr2 could similarly mediate neuronal signals in zebrafish during regeneration.

Macrophages are critical players in the zebrafish caudal fin regeneration (28, 54). We observed subgroups of the mpeg1.1+ macrophage population in the regenerating fin tissue, resembling M1 and M2 macrophages in mammalian systems. However, we were not able to recover other immune cell population in the hematopoietic cells. This could potentially be due to the systematic bias against certain cell types during tissue dissociation and droplet incorporation in the microfluidic device. The same bias might also explain why we were not able to recover some other known players in the regenerating fin tissue, including neurons and endothelial cells (4). Increasing the number of cells sampled for scRNA-seq or performing scRNA-seq on sorted hematopoietic lineage cells would help to better understand the involvement of these populations in the regeneration process.

The expression profiles of mesenchymal cells captured from the postinjury stages resembled those of blastema in histology studies. We found four connected but distinct lineages representing both bone and non-bone cells in the blastema. All four lineages initiated from one cluster mostly consisted of 1-dpa cells and enriched for the tnfaip6 expression. A similar scenario has been observed in the axolotl limb regeneration model. By using scRNA-seq on a lineage-labeled axolotl model, Gerber et al. (58) found that connective tissue cells funnel into a progenitor state at initiation. Whether the cluster identified in our study represented a shared cell origin for the blastema or a shared state across mesenchymal cell types in the initial blastema-formation stage requires further investigation. High proportion of epithelial population in the fins could also hamper the discovery of relatively rare population with multipotency. Finer dissection before single-cell profiling might help in future study designs in capturing these populations.

While the bone cell lineage has been well studied in the regenerating fin, non-bone cells had been labeled by different markers and given different names and their intercorrelations left to be clarified. We found that tph1b, aldh1a2, and and1/and2 genes were shared among the non-bone cell lineages and could be labeling states instead of types of blastemal cells during regeneration. Meanwhile, differential analysis revealed similar enrichment for bone formation in all lineages yet distinct associations with reactivation of developmental programs, RA signaling, and collagen metabolism, underscoring their collaborative and complementary roles in the regeneration process.

Our scRNA-seq data also provided more details about the fish system we are working with. For all sample collections, we used the transgenic strain Tg(sp7:EGFP)b1212, which specifically labels osteoblast lineage in the fish (59). It was reported that green fluorescence signal could be detected in the fish skin after 72 hours post-fertilization. This ectopic expression, however, does not interfere with confocal imaging of skeletal structures of fish at any stage due to the fact that they lie in different planes of focus. What these cells are and why they expressed the transgene were unclear. In this study, we obtained a holistic view of the transgene expression pattern in the fin region regardless of whether that was associated with the cell type of interest, i.e., osteoblasts in this context. Unsupervised clustering on the expression profiles from single fin cells suggested that green fluorescent protein (GFP) is not only expressed in the mesenchymal but also highly enriched in the superficial layer epithelium (table S2). A closer examination of this classic reporter gene construct revealed that the regulatory region of sp7 used for the construction of the transgene did not exactly represent the endogenous sp7 regulatory region. Tg(sp7:EGFP)b1212 was generated from bacterial artificial chromosome transgenesis using CH73-243G6 as the backbone, which did not contain the first exon of sp7 according to the annotation of the current genome assembly (chr6:58630884-58720045 and GRCz10), leading to the usage of a regulatory sequence different from the endogenous version. Whether this usage difference contributed to the ectopic expression pattern of the transgene requires further study. This finding points to the potential of using single-cellbased approaches in reporter line validation and more thorough analysis of the transgene behavior.

All zebrafish were used in accordance with protocol no. 20190041 approved by the Washington University Institutional Animal Care and Use Committee. Wild-type and Tg(sp7:EGFP) strains are maintained under standard husbandry in the Washington University Fish Facility, with the system water temperature at 28.5C and a day-night cycle controlled as 14-hour light/10-hour dark. For fin amputation, we anesthetized 1-year-old fish with MS-222 (0.16 g/liter) in the system water and then removed the distal half of their caudal fin with sterilized razor blades. The fish were then sent back to circulating water system for recovery. We collected regenerating fin tissue from 39 fish by doing secondary fin amputation at the primary cutting plane with the same anesthesia and recovery procedures.

Collected fin tissues were digested by Accumax (Innovative Cell Technologies), filtered through 40-m cell strainers, and washed with 1 Dulbeccos phosphate-buffered saline (DPBS)0.04% bovine serum albumin to generate single-cell suspensions. Libraries were constructed from these cell suspensions following the instruction of the Chromium Single Cell Gene Expression Solution 3 v2 (10x Genomics) and were subsequently sequenced on HiSeq2500 (Illumina) with read lengths of 26 + 75 (Read1 + Read2). Raw reads were processed by Cell Ranger (10x Genomics) with default parameters for read tagging, alignment to zebrafish reference genome (GRCz10), and feature counting based on Ensembl release 91 (cellranger count). EGFP sequence was added into the reference genome as a separate chromosome for mapping reads from the reporter gene.

We performed unsupervised clustering using Seurat v3.0 following the procedure of normalization (SCTransform), highly variable gene detection, dimensional reduction (principal components analysis), and cells clustering (Louvain clustering at resolutions from 0.1 to 0.6) (17). For integrating the four stages in finding conserved cell types, we used the anchoring approach provided by Seurat v3. Cell clustering was based on the top principal components that account for most of the cell-cell variances. The same set of principal components was used in UMAP calculation for visualization as well.

We found differentially expressed genes in each cluster by comparing the expression profiles of them with those of the rest of the cells using Wilcoxon rank sumbased approach with the criteria of log fold change more than 0.25 and a minimum cell percentage of 0.25. The same criteria were applied to all pairwise comparisons, unless stated otherwise. We made functional connections between the list of differentially expressed genes and the type of cell that they most likely represent by testing for GO term enrichment (18) and manual curation by searching The Zebrafish Information Network database and PubMed. Certain cell clusters were taken as independent samples for secondary clustering following the same unsupervised clustering procedures.

We calculated the by-cell average expression level of a set of S phase or G2-M phase markers suggested by Seurat that are detected in our zebrafish dataset and normalized by subtracting aggregated expression of control genes. Although G1 phase cells are also within cell cycle, they are hardly separable from G0 cells. To avoid false-positive labeling for active cycling cells, we set stringent thresholds and only included cells with |S.score G2M.score| > 0.1 in the S or G2-M group, while cells with both S.score and G2M.score below zero as G1. Other cells were not included in this part of the analysis. Differentially expressed genes were also identified by Wilcoxon rank sumbased approach. These differentially expressed genes were considered to be cell cycle related if they were in the list of genes associated with R-DRE-1640170 Cell Cycle and/or cycling marker genes used for cell cycle phase score calculations.

We collected uninjured and regenerating fin tissues from casper (nacrew2/w2;roya9/a9) fish and fixed in 4% paraformaldehyde overnight (60). Fixed tissues were subsequently submerged in 10% sucrose in 1 PBS, 20% sucrose in 1 PBS, and 30% sucrose in 1 PBS for 4 hours each. After sucrose exchange, tissues were embedded in Optimal Cutting Temperature (O.C.T.) compound (Fisher Healthcare Tissue-Plus) and snap frozen on dry ice. The frozen tissue blocks were then processed into 15-m sections on a Leica CM1950 cryostat. We performed RNA in situ hybridization targeting krt4, cldne, krt1-19d, cldna, krt94, cldni, cldn1, agr2, sema3b, stmn1b, and tnfaip6 for mRNA detection using an RNAscope kit (Advanced Cell Diagnostics, Hayward, CA, USA). Alcian blue/periodic acidSchiff (PAS) staining was subsequently performed on the same section or separately on a consecutive serial section following the manufacturers protocol (Newcomer Supply). Microscopic images were taken by ZEISS Axio Observer.

Cell trajectories were constructed using Slingshot v1.3.1 (40). Through initial subclustering and cell type identifications, we found one subcluster with high epcam expression, potentially a doublet cell contamination from the major cell type classifications. We removed this group of cells from all downstream analysis within the mesenchymal cluster. We used UMAP embedding and subclustering assignments as input for the Slingshot calculation.

We performed nonparametric Wilcoxon rank sum test to identify differentially expressed genes across cell groups as implemented in Seurat. P values were adjusted by all features in the dataset using Bonferroni correction.

See the article here:
Cellular diversity of the regenerating caudal fin - Science Advances

Bone Marrow Stem Cells Comments Off on Cellular diversity of the regenerating caudal fin – Science Advances | August 13th, 2020

Some skin, eggs and tissue samples are all that remain of Malaysia's last rhino, Iman, who died last November after years of failed breeding attempts.

Now scientists are pinning their hopes on experimental stem cell technology to bring back the Malaysian variant of the Sumatran rhinoceros, making use of cells from Iman and two other dead rhinos.

"I'm very confident," molecular biologist Muhammad Lokman Md Isa told Reuters in his laboratory at the International Islamic University of Malaysia.

"If everything is functioning, works well and everybody supports us, it's not impossible."

The smallest among the world's rhinos, the Sumatran species was declared extinct in the wild in Malaysia in 2015. Once it had roamed across Asia, but hunting and forest clearance reduced its numbers to just 80 in neighboringIndonesia.

Iman, 25, died in a nature reserve on Borneo island, following massive blood loss caused by uterine tumors, within six months of the death of Malaysia's last male rhino, Tam.

Efforts to get the two to breed had not worked.

"He was the equivalent of a 70-year-old man, so of course you don't expect the sperm to be all that good," said John Payne of the Borneo Rhino Alliance (BORA), who has campaigned for about four decades to save Malaysia's rhinos.

"It was obvious that, to increase the chances of success, one should get sperm and eggs from the rhinos inIndonesia. But right till today,Indonesiais still not keen on this."

Across the border

Indonesia's environment ministry disputed accusations of cross-border rivalry as a reason why Malaysia's rhinos died out, saying talks continue on ways to work with conservationists in the neighboring southeast Asian nation.

"Because this is part of diplomatic relations, the implementation must be in accordance with the regulation of each country," said Indra Exploitasia, the ministry's director for biodiversity conservation.

The Malaysian scientists plan to use cells from the dead rhinos to produce sperm and eggs that will yield test-tube babies to be implanted into a living animal or a closely related species, such as the horse.

The plan is similar to one for the African northern white rhinoceros, which number just two. Researchers in that effort reported some success in 2018 in producing embryonic stem cells for the southern white rhino.

But the process is still far from producing a whole new animal, say Thomas Hildebrandt and Cesare Galli, the scientists leading the research.

And even if it worked, the animals' lack of genetic diversity could pose a threat to long-term survival, Galli told Reuters.

Indonesian scientist Arief Boediono is among those helping in Malaysia, hoping success will provide lessons to help his country's rhinos.

"It may take five, 10, 20 years, I don't know," Arief added. "But there has already been some success involving lab rats in Japan, so that means there is a chance."

Japanese researchers have grown teeth and organs such as pancreas and kidneys using embryonic stem cells from rats and mice in efforts to grow replacement human organs.

For now, however, Iman's hide will be stuffed and put on display alongside Tam in a Borneo museum.

Your premium period will expire in 0 day(s)

Subscribe to get unlimited access Get 50% off now

See original here:
Back from the dead? Stem cells give hope for revival of Malaysia's extinct rhinos - The Jakarta Post - Jakarta Post

Skin Stem Cells Comments Off on Back from the dead? Stem cells give hope for revival of Malaysia’s extinct rhinos – The Jakarta Post – Jakarta Post | August 13th, 2020