Stem Cell Implant Is Being Trialled To Cure" Type 1 Diabetes – IFLScience

A groundbreaking attempt to"cure" Type 1 diabetes with stem cells began last week. Embryonic stem cell implants were given to two people, one in the US and one in Canada, with high-risk Type 1 diabetes. The researchers hope that this willhelp the patients manage the condition.

The stem cells, developed by private company ViaCyte, are implanted underneath the patient's forearm, where they take about three months to mature into islet cells. In the pancreas, these cells are responsible for the production of insulin. In people with Type 1 diabetes, these cells are attacked by the bodys own immune system.

If it works, we would call it a functional cure, Paul Laikind of Viacyte told New Scientist. Its not truly a cure because we wouldnt address the autoimmune cause of the disease, but we would be replacing the missing cells.

A smaller implant has already been trialled on 19 people for safety and the company expects to extend the trial to 40 more people later this year, in order to understand both the safety and efficacy of the full-size implant. ViaCyte would like to get preliminary results during the first half of 2018 and to know if the system works between six and12 months later.

Islet transplants have been used to successfully treat patients with unstable, high-risk Type 1 diabetes, but the procedure has limitations, including a very limited supply of donor organs and challenges in obtaining reliable and consistent islet preparations, trial investigator James Shapiro, from the University of Alberta, said in a statement. An effective stem cell-derived islet replacement therapy would solve these issues and has the potential to help a greater number of people.

If a success, the implant will improve the lives of the patients as they wont have to closely monitor their blood levels or inject insulin, but there is a trade-off. They will have to take immunosuppressive drugs, so that their bodies dont attack the newly implanted cells. This iswhy the procedure is targeted atpeople who are at ahigher risk.

Researchers estimate that 140,000 people in Canada and the US are currently suffering from high-risk Type 1 diabetes. The condition can lead to severe episodes of hypoglycemia in the short term and heart disease, stroke, and kidney disease (among others) in thelong term.

[H/T:New Scientist]

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Stem Cell Implant Is Being Trialled To Cure" Type 1 Diabetes - IFLScience

First implants derived from stem cells to ‘cure’ type 1 diabetes – New Scientist

Stem cells have been cultured to treat many different of conditions

Lewis Houghton/Science Photo Library

By Andy Coghlan

Last week, two people with type 1 diabetes became the first to receive implants containing cells generated from embryonic stem cells to treat their condition. The hope is that when blood sugar levels rise, the implants will release insulin to restore them to normal.

About 10 per cent of the 422 million people who have diabetes worldwide have type 1 diabetes, which is caused by the bodys immune system mistakenly attacking cells in the pancreas that make insulin. For more than 15 years, researchers have been trying to find a way to use stem cells to replace these, but there have been several hurdles not least, how to get the cells to work in the body.

Viacyte, a company in San Diego, California, is trying a way to get round this. The firms credit-card-sized implant, called PEC-Direct, contain cells derived from stem cells that can mature inside the body into the specialised islet cells that get destroyed in type 1 diabetes.

The implant sits just below the skin, in the forearm, for example, and is intended to automatically compensate for the missing islet cells, releasing insulin when blood sugar levels get too high.

If it works, we would call it a functional cure, says Paul Laikind, of Viacyte. Its not truly a cure because we wouldnt address the autoimmune cause of the disease, but we would be replacing the missing cells.

A similardevice has already been safety tested in 19 people with diabetes, using smaller numbers of cells. Once implanted, the progenitor cells housed in the device did mature into islet cells, but the trial didnt use enough cells to try to treat the condition.

Now Viacyte has implanted PEC-Direct packages containing the cells into two people with type 1 diabetes. A third person will also get the implant in the near future. Once inside the body, pores in the outer fabric of the device allow blood vessels to penetrate inside, nourishing the islet progenitor cells. Once these cells have matured which should take about three months the hope is that they will be able to monitor sugar levels in the blood, and release insulin as required.

If effective, it could free people with type 1 diabetes from having to closely monitor their blood sugar levels and inject insulin, although they would need to take immunosuppressive drugs to stop their bodies from destroying the new cells.

If successful, this strategy could really change the way we treat type 1 diabetes in the future, says Emily Burns of the charity Diabetes UK. A similar way to treat the condition with pancreas cells from organ donors has been in use for nearly 20 years, successfully freeing recipients from insulin injections, but a shortage of donors limits how many people are able to have this treatment.

This isnt a problem with stem cells. The embryonic stem cells used to make the progenitor cells originally came from a spare early stage embryo donated by a woman who was having IVF. Because embryonic stem cells, and the progenitor cells made from them, can be multiplied in limitless amounts, Laikind says that, if the treatment works, the method would be able to treat everyone who has the condition.

A limitless source of human insulin-producing cells would be a major step forward on the journey to a potential cure for diabetes, says James Shapiro at the University of Alberta, Canada, who has collaborated with Viacyte on this project, and who pioneered the donor pancreas method decades ago. For sure, this will in the end prove to be a durable landmark for progress in diabetes care.

Correction: This article has been updated to clarify that the cells used in this study are derived from stem cells, and to correct the size of the implanted pouch.

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First implants derived from stem cells to 'cure' type 1 diabetes - New Scientist

Lego-Like Brain Balls Could Build a Living Replica of Your Noggin – WIRED

This cerebral organoid, or mini-brain, was grown in a laboratory. It contains a diversity of cell types and internal structures that can make it a good stand-in for an actual brain in experiments. Unpredictable variations and deficiencies have hampered the organoids usefulness in research, but new techniques for creating mini-brains may change that.

Hoffman-Kim lab/Brown University

The human brain is routinely described as the most complex object in the known universe. It might therefore seem unlikely that pea-size blobs of brain cells growing in laboratory dishes could be more than fleetingly useful to neuroscientists. Nevertheless, many investigators are now excitedly cultivating these curious biological systems, formally called cerebral organoids and less formally known as mini-brains. With organoids, researchers can run experiments on how living human brains developexperiments that would be impossible (or unthinkable) with the real thing.

Original story reprinted with permission from Quanta Magazine, an editorially independent publication of the Simons Foundation whose mission is to enhance public understanding of science by covering research developments and trends in mathematics and the physical and life sciences.

The cerebral organoids in existence today fall far short of earning the brain label, mini or otherwise. But a trio of recent publications suggests that cerebral-organoid science may be turning a cornerand that the future of such brain studies may depend less on trying to create tiny perfect replicas of whole brains and more on creating highly replicable modules of developing brain parts that can be snapped together like building blocks. Just as interchangeable parts helped make mass production and the Industrial Revolution possible, organoids that have consistent qualities and can be combined as needed may help to speed a revolution in understanding how the human brain develops.

In 2013 Madeline Lancaster , then of the Austrian Academy of Sciences, created the first true cerebral organoids when she discovered that stem cells growing in a supportive gel could form small spherical masses of organized, functioning brain tissue. Veritable colleges of mini-brains were soon thriving under various protocols in laboratories around the world.

Much to the frustration of impatient experimentalists, however, the mini-brains similarity to the real thing only went so far. Their shrunken anatomies were distorted; they lacked blood vessels and layers of tissue; neurons were present but important glial cells that make up the supportive white matter of the brain were often missing.

Worst of all was the organoids inconsistency: They differed too much from one another. According to Arnold Kriegstein , director of the developmental and stem cell biology program at the University of California, San Francisco, it was difficult to get organoids to turn out uniformly even when scientists used the same growth protocol and the same starting materials. And this makes it very difficult to have a properly controlled experiment or to even make valid conclusions, he explained.

Researchers could reduce the troublesome variability by treating early-stage organoids with growth factors that would make them differentiate more consistently as a less varied set of neurons. But that consistency would come at the expense of relevance, because real brain networks are a functional quilt of cell typessome of which arise in place while others migrate from other brain regions.

For example, in the human cortex, about 20 percent of the neuronsthe ones called interneurons, which have inhibitory effectsmigrate there from a center deeper down in the brain called the medial ganglionic eminence (MGE). An oversimplified organoid model for the cortex would be missing all those interneurons and would therefore be useless for studying how the developing brain balances its excitatory and inhibitory signals.

A stained cross section through one of the cortical organoids created by researchers at the Yale Stem Cell Center shows the organization of various cell types into layers of tissue. The organoid is 40 days old in this image. The blue dots are cell nuclei; the red patches are progenitor cells for neurons; the green patches are differentiated neurons.

Courtesy of Yangfei Xiang

Deliverance from those problems may have arrived with recent results from three groups. They point toward the possibility of an almost modular approach to building mini-brains, which involves growing relatively simple organoids representative of different developing brain regions and then allowing them to connect with one another.

The most recent of those results was announced two weeks ago in Cell Stem Cell by a group based at the Yale Stem Cell Center. In the first stage of their experiments, they used human pluripotent stem cells (some derived from blood, others from embryos) to create separate organoid replicas of the cortex and MGE. The researchers then let mixed pairs of the ball-shaped organoids grow side by side. Over several weeks, the pairs of organoids fused. Most important, the Yale team saw that, in keeping with proper brain development, inhibitory interneurons from the MGE organoid migrated into the cortical organoid mass and began to integrate themselves into the neural networks there, exactly as they do in the developing fetal brain.

Earlier this year, teams from the Stanford University School of Medicine and the Austrian Academy of Sciences published reports on similar experiments in which they too developed cortical and MGE organoids and then fused them. The three studies differ significantly in their detailssuch as how the researchers coaxed stem cells to become organoids, how they nurtured the growing organoids, and what tests they ran on the derived cells. But they all found that the fused organoids yielded neural networks with a lifelike mix of excitatory neurons, inhibitory neurons and supporting cells, and that they could be developed more reliably than the older types of mini-brain organoids.

To Kriegstein, all three experiments beautifully illustrate that the cells in organoids will readily transform into mature, healthy tissue if given the opportunity. Once you coax the tissue down a particular developmental trajectory, it actually manages to get there very well on its own with minimal instruction, he said. He believes that specialized organoids could bring a new level of experimental control to neuroscientists explorations: Scientists could probe different brain organoids for information about development within subregions of the brain and then use that combined or fused platform to study how these cells interact once they start migrating and encountering each other.

In-Hyun Park , an associate professor of genetics who led the Yale study, is hopeful that organoids might already be useful in preliminary investigations of the developmental roots of certain neuropsychiatric conditions, such as autism and schizophrenia. Evidence suggests that in these conditions, Park said, there seems to be an imbalance between excitatory and inhibitory neural activity. So those diseases can be studied using the current model that weve developed.

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Kriegstein cautions, however, that no one should rush to find clinical significance in organoid experiments. What we really lack is a gold standard of human brain development to calibrate how well these organoids are mimicking the normal condition, he said.

Whatever applications organoid research may eventually find, the essential next steps will consist of learning how to produce organoids that are even more true to life, according to Park. He has also not given up hope that it will eventually be possible to create a mini-brain in the laboratory that is a more complete and accurate stand-in for what grows in our head. Maybe doing so will involve a more complex fusion of organoid subunits, or maybe it will demand a more sophisticated use of growth media and chemicals for directing the organoid through its embryonic stages. There should be an approach to generating a human brain organoid that is composed of forebrain plus midbrain plus hindbrain all together, Park said.

Jordana Cepelewicz contributed reporting to this article.

Original story reprinted with permission from Quanta Magazine , an editorially independent publication of the Simons Foundation whose mission is to enhance public understanding of science by covering research developments and trends in mathematics and the physical and life sciences.

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Lego-Like Brain Balls Could Build a Living Replica of Your Noggin - WIRED

Amniotic sac in a dish: Stem cells form structures that may aid of infertility research – Phys.Org

August 8, 2017 The PASE, or post-implantation amniotic sac embryoid, is a structure grown from human pluripotent stem cells that mimics many of the properties of the amniotic sac that forms soon after an embryo implants in the uterus wall. The structures could be used to study infertility. Credit: University of Michigan

The first few weeks after sperm meets egg still hold many mysteries. Among them: what causes the process to fail, leading to many cases of infertility.

Despite the importance of this critical stage, scientists haven't had a good way to explore what can go wrong, or even what must go right, after the newly formed ball of cells implants in the wall of the human uterus.

But a new achievement using human stem cells may help change that. Tiny lab-grown structures could give researchers a chance to see what they couldn't before, while avoiding ethical issues associated with studying actual embryos.

A team from the University of Michigan reports in Nature Communications that they have coaxed pluripotent human stem cells to grow on a specially engineered surface into structures that resemble an early aspect of human development called the amniotic sac.

The cells spontaneously developed some of the same structural and molecular features seen in a natural amniotic sac, which is an asymmetric, hollow ball-like structure containing cells that will give rise to a part of the placenta as well as the embryo itself. But the structures grown at U-M lack other key components of the early embryo, so they can't develop into a fetus.

It's the first time a team has grown such a structure starting with stem cells, rather than coaxing a donated embryo to grow, as a few other teams have done.

"As many as half of all pregnancies end in the first two weeks after fertilization, often before the woman is even aware she is pregnant. For some couples, there is a chronic inability to get past these critical early developmental steps, but we have not previously had a model that would allow us to explore the reasons why," says co-senior author Deborah Gumucio, Ph.D. "We hope this work will make it possible for many scientists to dig deeper into the pathways involved in normal and abnormal development, so we can understand some of the most fascinating biology on earth." Gumucio is the Engel Collegiate Professor of Cell & Developmental Biology at Michigan Medicine, U-M's academic medical center.

A steady PASE

The researchers have dubbed the new structure a post-implantation amniotic sac embryoid, or PASE. They describe how a PASE develops as a hollow spherical structure with two distinct halves that remain stable even as cells divide.

One half is made of cells that will become amniotic ectoderm, the other half consists of pluripotent epiblast cells that in nature make up the embryonic disc. The hollow center resembles the amniotic cavity - which in normal development eventually gives rise to the fluid-filled sac that protects and cushions the fetus during development.

Gumucio likens a PASE to a mismatched plastic Easter egg or a blue-and-red Pokmon ball - with two clearly divided halves of two kinds of cells that maintain a stable form around a hollow center.

The team also reports details about the genes that became activated during the development of a PASE, and the signals that the cells in a PASE send to one another and to neighboring tissues. They show that a stable two-halved PASE structure relies on a signaling pathway called BMP-SMAD that's known to be critical to embryo development.

Gumucio notes that the PASE structures even exhibit the earliest signs of initiating a "primitive streak", although it did not fully develop. In a human embryo, the streak would start a process called gastrulation. That's the division of new cells into three cell layersendoderm, mesoderm and ectodermthat are essential to give rise to all organs and tissues in the body.

Collaboration provides the spark

The new study follows directly from previous collaborative work between Gumucio's lab and that of the other senior author, U-M mechanical engineering associate professor Jianping Fu, Ph.D.

In the previous work, reported in Nature Materials, the team succeeded in getting balls of stem cells to implant in a special surface engineered in Fu's lab to resemble a simplified uterine wall. They showed that once the cells attached themselves to this substrate, they began to differentiate into hollow cysts composed entirely of amnion - a tough extraembryonic tissue that holds the amniotic fluid.

But further analysis of these cysts by co-first authors of the new paper Yue Shao, Ph.D., a graduate student in Fu's lab, and Ken Taniguchi, a postdoctoral fellow in Gumucio's lab, revealed that a small subset of these cysts were stably asymmetric and looked exactly like early human or monkey amniotic sacs.

The team found that such structures could also grow from induced pluripotent stem cells (iPSCs)cells derived from human skin and grown in the lab under conditions that give them the ability to become any type of cell, similar to how embryonic stem cells behave. This opens the door for future work using skin cells donated by couples experiencing chronic infertility, which could be grown into iPSCs and tested for their ability to form proper amniotic sacs using the methods devised by the team.

Important notes and next steps

Besides working with genetic and infertility specialists to delve deeper into PASE biology as it relates to human infertility, the team is hoping to explore additional characteristics of amnion tissue.

For example, early rupture of the amnion tissue can endanger a fetus or be the cause of a miscarriage. The team also intends to study which aspects of human amnion formation also occur in development of mouse amnion. The mouse embryo model is very attractive as an in vivo model for investigating human genetic diseases.

The team's work is overseen by a panel that monitors all work done with pluripotent stem cells at U-M, and the studies are performed in accordance with laws regarding human stem cell research. The team ends experiments before the balls of cells effectively reach 14 developmental days, the cutoff used as an international limit on embryo researcheven though the work involves tissue that cannot form an embryo. Some of the stem cell lines were derived at U-M's privately funded MStem Cell Laboratory for human embryonic stem cells, and the U-M Pluripotent Stem Cell Core.

Explore further: Team uses stem cells to study earliest stages of amniotic sac formation

More information: Yue Shao et al, A pluripotent stem cell-based model for post-implantation human amniotic sac development, Nature Communications (2017). DOI: 10.1038/s41467-017-00236-w

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Amniotic sac in a dish: Stem cells form structures that may aid of infertility research - Phys.Org

BioTech Marketing and market opportunity for Stem Cells – Checkbiotech.org (press release)

The global market for stem cells has been estimated at USD 12 billion in 2016and is projected to reach USD 26.6 billion by 2021, at a CAGR of 13.7% during the forecast period 2016to 2021. A stem cell is an undifferentiated cell that has the potential to develop into any type of cell in the body.

Regenerative medicine is the major application of stem cells and other areas are neurology, orthopedics, oncology, cardiology, hematology and others (diabetes, injuries, and wounds). Another prominent application of stem cells is drug discovery and development. The end-users of this market are usually hospitals, cell banks, clinical research laboratories and academic institutes.

Global Stem Cell Marketing Market Dynamics

The global stem cells market is one of the most promising markets in the field of life sciences at present and is forecasted to grow even more in the coming years as stem cells enable cost-effective treatment of many conditions that currently have poor or no treatment.

Drivers

Some of the factors driving the global stem cells market are:

Restraints

While the global stem cells market has ample scope for growth, there are some factors restraining it as well. These include:

The market for stem cells is segmented on the basis of cell types and technology. The cells type segment includes adult stem cells, human embryonic stem cells, induced pluripotent stem cells, rat neural stem cells and very small embryonic-like stem cells. Adult stem cells are again divided into hematopoietic stem cells, mesenchymal stem cells, neuronal stem cells, dental stem cells and umbilical cord cells. The adult stem cells hold the highest share in the global stem cells market, while the market share of induced pluripotent stem cells is expected to grow in the coming years. The technology segment is divided into stem cell acquisition, stem cell production, stem cell cryopreservation, and stem cell expansion sub-segments.

Based on geography, the global market for stem cells is segmented into North America, Europe, Asia-Pacific and Rest of the World. The global stem cells market is dominated byNorth America, followed byEurope, the estimated market share of which is more than 25% as per a recent study. With 30% of the market, the USA holds the majority of share. However, due to increasing awareness among the public and advances in technologies, the market in the Asia-Pacific is expected to grow at a high rate.

Many players in this market are trying to expand their product portfolio in order to top the global market. While some companies are entering into the market by acquisitions, top companies are expanding their growth in this market by acquiring other companies. Few companies have adopted product innovation and new product launches as their key business strategy to ensure their dominance in this market.

Some of the key players in the market are:

Key Deliverables in the Study

If you are in need of BioTechnology marketing or Stem Cell Marketing call 972-800-6670

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BioTech Marketing and market opportunity for Stem Cells - Checkbiotech.org (press release)

Stem Cell Training and Top Protocols using Human Umbilical Cell Tissue – Checkbiotech.org (press release)

What is HUCT?

Human Umbilical Cell Tissue is stem cells extracted from the umbilical cord, placenta and cord blood from healthy mothers who have been prequalified and deliver through a c-section. HUCT are captured at the earliest point the umbilical cord, placenta and cord blood are ready for utilization in its earliest stages making them some of the best stem cells available for therapy purposes.

Using HUCT stem cells aids in healing otherwise deficient human cells, tissues and organs. In regenerative medicine, the use of cell replacement approaches which usually requires stem cells is a primary therapy. Current research and scientific studies have shown that stem cells can replace bone, fat, cartilage, heart tissue and muscle. Use of stem cells from HUCT has much potential for helping to heal or reduce the severity of many disease states. The umbilical cord serves as a conduit of nutrients for a fetus. Oxygenated nutrient rich blood is carried from the placenta to the fetus until the baby is born. This cord blood within the tissue is rich in primitive stem cells, growth factors and immune cells that are nave as they have to be compatible for the baby and mother. Moreover, the use of allogeneic cord blood has been used for decades in greater than 500 patients with diseases such as Hurlers syndrome, Duchenne muscular dystrophy and Krabbes disease. Most importantly it is safe. These otherwise discarded tissues are an excellent source of high-quality stem cells and growth factors which may be used in regenerative medicine. Utilizing HUCT from the newborn after birth increases the potency and effectiveness of the cells because it is a known fact as we age the number of stem cells greatly diminishes making the HUCT protocol a very popular solution.

Stem cells that are a derivative from umbilical cord tissue are clinically the least invasive and safest method of removal available. Alternative options include extraction from embryonic tissue that is derived from embryos, bone marrow which can be extracted by aspiration and is usually painful, and adipose tissue which can be surgically extracted through liposuction under general anesthesia.

The human umbilical cord is a reliable source of mesenchymal stem cells (HUCMSC). Unlike bone marrow stem cells, HUCMSCs have a comparatively painless collection procedure and faster self-regenerative properties. Different derivation protocols may provide different amounts and populations of stem cells. Stem cell populations have also been identified in other compartments of the umbilical cord, such as the cord lining, perivascular tissue, and Whartons jelly. The use of HUCMSCs are noncontroversial sources compared to embryonic stem cells. They can differentiate into the three germ layers that promote tissue repair, moderate immune response, and anti-cancer properties. Additionally, they are considered more beneficial autologous or allogeneic agents for the treatment of malignant and nonmalignant solid and soft type cancers. Other alternative benefits from HUCT include correction of corneal epithelial defects, burn and diabetic wound ulcer treatment modalities, better osteogenesis capabilities, rescue of liver fibrosis, and hyperglycemia in the diabetic population. In comparison, embryonic stem cells (ESCs) can differentiate into almost all tissues in the human body and are thus labeled as pluripotent. However, the use of ESCs generated from embryos has raised ethical concerns. Furthermore, the clinical applications of therapies derived from embryonic stem cells have been criticized because of the possibility of forming tumors by integrated oncogenes and suppression of cells that disrupt tumor formation. This alternative portrays HUCT as a better, safer choice for clinical applications, efficiency, and safety precautions. Regarding the therapeutic principles of HUCT, effective storage banking systems and protocols should be established immediately.

Isolation by enzymatic digestion Type I collagenase, or collagenase type A, is extensively used for the isolation of mesenchymal-like cells from the cord tissue to facilitate the degradation of matrix ground substance and shortens the time required for the isolation process. The time required for tissue digestion ranges from 30 minutes to 16 hours depending on the quantity/concentration of enzyme and duration of treatment with digesting reagents. Filtration of the digested material through 70100m pore sized cell strainers facilitates the removal of any unwanted tissue debris. Isolation by explants culture The principle of the method is generally described as fine chopping of the Whartons jelly sections of the cord tissue, after excision of the blood vessels, with a scalpel, plating of the fine fragments in sterile culture plates or Petri dishes, and culturing of these with low-glucose DMEM, supplemented with foetal bovine serum (10-20% v/v), L-glutamine and antibiotics/antimycotics. Cryopreservation methods for UCT and hMSCs isolated from UCT: Slow cooling and Vitrification Cryopreservation of cord tissue and/or cells extracted from the tissue represents an important stage to overcome in the view of the therapeutic use of stem cells. HUCT cryopreservation should be able to maintain the cellular metabolism in a dormancy state for an indefinite period of time. Use of defined culture media supplemented with high amounts of foetal bovine serum and 7% 10% (v/v) dimethyl sulfoxide (DMSO) or glycerol and freeze the cells gradually (eg. 10C/min) and keep them between -135C and -196C. After rapid thawing at 37C, viability rates of over 50% can be achieved.

The samples should be frozen for a period of time ranging from 5 to 78 days. The slow cooling protocol was more efficient than the vitrification, for cryopreservation of umbilical cord tissue, because it has caused fewer changes in the structure of tissue (edema and degeneration of the epithelium) and despite the significant decrease cell viability compared to fresh samples, the ability of cell proliferation in vitro is greatly preserved. Cryopreservation of small fragments of tissue from the umbilical cord and to obtain viable cells capable of proliferation in vitro after thawing, contribute to the creation of a frozen tissue bank. In vitro differentiation To successfully differentiated lineages using a variety of cell culture techniques and reagents for in vitro differentiation of Adipocytes, Chondrocytes, Osteocytes, Cardiomyocytes, Skeletal myocytes, Neuronal/glial precursors, Dopaminergic neurons, and Endothelial cells. Independently of their origin, the adipogenic potential of HUCT MSCs is inversely related to the length of in vitro culture, and sharply declines when HUCT MSCs become senescent. Contrary, prolonged culturing increases their osteogenic differentiation. In vitro expansion of HUCT MSCs should, therefore, be performed with limited passaging, to avoid changes in their differentiation ability. Gradual shortening of the telomeres during a cells life continues until the presence of critically short telomeres triggers a senescence pathway, which results in proliferation arrest. Because of that, a normal human cell can only divide 50 to 100 times in vitro conditions; HUCT MSCs are no exception. UC blood HUCT MSCs, however, have slightly longer telomeres than other MSCs, and thus can be cultured for longer periods before they senesce. Proliferation arrest in HUCT MSCs results in their senescence, which is described by the appearance of large senescent cells with a flat shape, circumscribed nuclei, and increased lysosome compartment. These morphological changes are not restricted to the senescent stage only, but represent continuous alterations in the course of HUCT MSCs long-term culture. Immunophenotyping of HUCT MSCs Characterisation of HUCT MSCs is generally accomplished by flow cytometry analysis of surface markers. Stro-1 has been identified as a marker for cells that can differentiate into multiple mesenchymal lineages. CD9/CD90/CD166 triple positive subpopulation of HUCT MSCs showed multipotency for chondrogenic, osteogenic and adipogenic differentiation providing a basis for identification of HUCT MSCs. It has been indicated that positive expression of CD166 is indicative of multipotency in HUCT MSCs. Expression levels of CD90 and CD105 are maintained over sequential passages and they can be important for validating cultures of HUCT MSCs intended for therapy. A good indication of HUCT MSC identity can be reached by expression of CD90, CD105 and CD166 and lack of expression of CD34 and CD45 as a minimum set of surface markers. Variability in MSCs extraction from HUCT There are ways of minimizing variations between lots produced, by controlling process parameters, and by screening the raw materials that will be in contact with the cells and cell source. There are other noncontrollable parameters such as the source of the cells, which represents a real challenge for regenerative medicine applications. Each cell extraction method is produced with cells from a different patient/donor, with intrinsic characteristics that result in variations of cell growth patterns and differentiation. It is, therefore, necessary to develop extraction methods that are suitable to real world applications. It is necessary to map the operating environment and assess risk factors before empirically determining the effect on the process. This will be particularly critical for processes using primary tissue or cell sources where the biological variation at input is likely to be high. Regulated therapeutic products will require characterized and risk assessed manufacturing processes. This fits the philosophy of process control industry tools such as quality by design (QbD) and Six Sigma, represent approaches to understanding process operating space and risks of associated variables. Extraction of hMSCs from umbilical cord tissue via enzymatic digestion. 200-400 mg cord slices from multiple cords, both fresh and frozen, with the purpose to screen different methods for an assessment of method success with a view to downstream standardization of the isolation and expansion of mesenchymal stem cells from the HUCT aided in the development of this protocol.

2 hours enzyme digestion of cord tissue with 3 different enzymatic solutions: 9 (200 400 mg) slices of cord tissue were digested in 3 ml of enzymatic solution/each; solutions obtained after digestion were filtered through 40 m cell strainers and centrifuged at 1500 rcf for 10 min/each; all cord tissue is kept frozen through this initial process.

4 hours enzyme digestion of cord tissue with 3 different enzymatic solutions: 9 (200 400 mg) slices of cord tissue were digested in 3 ml of enzymatic solution/each; solutions obtained after digestion were filtered through 70 m cell strainers and centrifuged at 1500 rcf for 10 min/each; all cord tissue still remains frozen.

18 hours enzyme digestion of cord tissue with 3 different enzymatic solutions: 9 (200 400 mg) slices of cord tissue were digested in 3 ml of enzymatic solution/each; solutions obtained after digestion were filtered through 70 m cell strainers and centrifuged at 1500 rcf for 10 min/each; all cord tissue still remains frozen.

2, 4 and 18 hours enzyme digestion of cord tissue with 3 different enzymatic solutions: 18 (200 400 mg) slices of cord tissue were digested in 3 ml of enzymatic solution/each; solutions obtained after digestion were filtered through 70 m cell strainers and centrifuged at 1500 rcf for 10 min/each; all cord tissue is fresh (2 days old).

2, 4 and 18 hours enzyme digestion of cord tissue with 3 different enzymatic solutions: 18 (200 400 mg) slices of cord tissue were digested in 5 ml of enzymatic solution/each; solutions obtained after digestion were filtered through 100 m cell strainers and centrifuged at 500 rcf for 10 min/each; cord tissue is frozen.

2, 4 and 18 hours enzyme digestion of cord tissue with 3 different enzymatic solutions: 9 (200 400 mg) slices of cord tissue were digested in 5 ml of enzymatic solution/each; solutions obtained after digestion were filtered through 100 m cell strainers; no centrifugation for slices digested with 2 of the enzymatic solutions and 1000 rcf centrifugation speed for slices digested with the 3rd type of enzymatic solution; cord tissue is frozen.

2, 4 and 18 hours enzyme digestion of cord tissue with 3 different enzymatic solutions: 9 (200 400 mg) slices of cord tissue were digested in 5 ml of enzymatic solution/each; solutions obtained after digestion were filtered through 100 m cell strainers; no centrifugation for slices digested with 2 of the enzymatic solutions and 1000 rcf centrifugation speed for slices digested with the 3rd type of enzymatic solution; cord tissue is fresh (not frozen).

18 hours enzyme digestion of cord tissue with cord banks method and reagents: (200 400 mg) slices of cord tissue were digested in 5 ml of enzymatic solution/each; solutions obtained after digestion were filtered through 100 m cell strainers and diluted with 3ml of culture media, before being plated in T25 flasks; no centrifugation; cord tissue is frozen.

a) Vials containing cryopreserved 200-400 mg cord tissue slices are defrosted by placing them in a 37C water bath for 3-5 minutes, or until only a trace of ice remains b) After, cryovials are transferred to a Class II biological safety cabinet (BSC) and the cord sections are removed from cryovials with a sterile aspirator stripette and placed in a Petri dish containing DPBS + 1% antibiotic/antimycotic v/v (PSA) in it. c) Individual slices are transferred to a fresh, sterile Petri dish, and chopped up into fine fragments (1-2mm3 ), with the aid of a scalpel and forceps. The fragments are then placed into a 15 ml centrifuge tube, with the aid of a scalpel and forceps. d) Cord slice fragments are enzymatically digested for 2h, 4h or 18h at 37C, with the following enzymatic solutions: A. Collagenase type I, (in serum free growth Medium A, 3-5ml/slice), 300 CDU/ml; B. Collagenase type I, 300 CDU/ml + hyaluronidase, 1mg/ml (in serum free growth Medium A, 3-5ml/slice); C. Collagenase type I, 300 CDU/ml for 1, 3 or 17 1/2 h, depending on the digestion period, followed by trypsin-EDTA 0.25% for a further 30 min (both enzyme solutions are prepared in serum free growth Medium A, 3-5ml/slice) e) Upon completion of digestion, tubes containing slices digested with enzymatic solutions A and B, are treated as follows: 5.1 Diluted 50% with serum free growth Medium A. 5.2 Filtered through a 40m, a 70m, or a 100m cell strainer; squeezing remaining tissue fragments with the forceps to aid cell release after filtration. 5.3 Centrifuged cell suspension at 1500 rcf for 10 min, 500 rcf for 10 min, or no centrifugation. 5.3.1 In the case of no centrifugation, an appropriate amount of FBS (final concentration 10%) os added to the suspension before filtration and 2ml of fresh growth media to wash the cell strainer with. 5.3.2 For methods that involved centrifugation the supernatant is discarded and the pellet re-suspended in 5ml of fresh growth Medium A; 5.4 Count cells by using a disposable haemocytometer (20l of cell suspension and 20l of trypan blue); and seeded at 104 cell/cm2 , in an appropriately sized culture vessel. f) Upon completion of digestion, tubes containing slices digested with method C, are treated according to the protocol below: 6.1 Diluted 50% with serum free growth Medium A.Centrifuged at 1500 rcf for 10 min, 500 rcf, or 1000 rcf. 6.2 Discarded supernatant and re-suspended pellet in 3-5ml of 0.25% trypsin-EDTA; 6.3 Replaced in the incubator for another 30 minutes. 6.4 After 30 min took tubes out and added 0.3-0.5ml of FBS/each tube to stop the enzyme action. 6.6 Diluted 50% with serum free growth Medium A. 6.5 Filtered through a 40m, a 70m, or a 100m cell strainer; squeezing remaining tissue fragments with the forceps to aid cell release after filtration. 6.7 Centrifuged at 1500 rcf for 10 min, 500 rcf, or 1000 rcf. 6.8 Discarded supernatant and re-suspended pellet in 5ml of fresh Medium A. 6.8.1 Counted cells by using a disposable haemocytometer (20l of cell suspension and 20l of trypan blue); and seeded at 104 cell/cm , in an appropriately sized culture vessel. g) All culture vessels are incubated at 37C and 5% CO2 in a humidified incubator. h) First media change is performed after 48h and every 3 days thereafter until cells have reached 80-85% confluence.

Protocol for enzymatic digestion of fresh cord tissue Procedure: a. For processing, cord sections are removed from tubes, inside a BSC, with sterile forceps and positioned on sterile prep trays; the outside of the cord is wiped with alcohol wipes (also held with sterile forceps to avoid touching cord surface). The remaining cord blood is squeezed from the cord by pressing the blunt edge of a sterile scalpel along the length of the cord. b. The cord tissue sections are then placed in a Petri dish with DPBS and 1% PSA in it. Swirled contents to wash. If the saline water is really cloudy with blood, the wash step is repeated. c. Cord sections are cut into 200-400 mg slices (approximately 2-4mm thick, depending on the thickness of the cord), and placed into separate Petri dishes with fresh DPBS and 1% PSA, to wash. The slices are then placed in separate Petri dishes with warm, serum free Media A. Each slice is weighed in a pre-weighed sterile, closed container; only slices that weigh approximately 300 mg are used, in order to maintain consistency. d. Each slice, is placed on a separate, sterile Petri dish, and chopped up in fine fragments (1-2mm). The fragments from each slice are placed in individual 15 ml centrifuge tubes. e. Cord fragments are enzymatically digested for 2h, 4h or 18h at 37C, with the following enzymatic solutions: A. Collagenase type I, (in serum free growth Medium A, 3-5ml/slice), 300 CDU/ml; B. Collagenase type I, 300 CDU/ml + hyaluronidase, 1mg/ml (in serum free growth Medium A, 3-5ml/slice); Mesenchymal Stem Cell E, C. Collagenase type I, 300 CDU/ml for 1, 3 or 17 1/2 h, depending on the digestion period, followed by trypsin-EDTA 0.25% for a further 30 min (both enzyme solutions being prepared in serum free growth Medium A, 3-5ml/slice); f. For tubes containing slices digested with methods A and B, after digestion time had finished, refer to previous protocol, step e-g. For tubes containing slices digested with method C, after digestion time has finished refer to previous protocol, step f. g. All culture vessels are incubated at 37C and 5% CO2, in a humidified incubator. h. First media change is performed after 48h and every 3 days thereafter until cells reached 80-85% confluence.

Protocol for enzymatic digestion of fresh and frozen cord tissue with the cord banks method a. Take each UCT slice, placed on separate, sterile Petri dish, and chop it up into fine tissue fragments (1-2mm3 ), which are then placed in individual 15 ml centrifuge tubes; b. Fragments from each slice are digested for 18h with: 2.1 collagenase type I (AMS Biotechnology Ltd, UK) 5ml/slice/tube; enzyme solution is prepared in serum free growth Medium B, at a concentration of 0.075% (750 CDU/ml) enzymatic solution A (used by cord blood bank); 2.2 collagenase type I (Sigma Aldrich, UK) 5ml/slice/tube; enzyme solution is prepared in serum free growth Medium B, at a concentration of 0.075% (750 CDU/ml) enzymatic solution B. c. Upon completion of digestion: Filter all digested slices through 100m cell strainer in 50 ml tubes; squeezing remaining tissue fragments with the forceps to aid cell release after filtration. 0.5ml FBS (provided by cord blood bank) + 3ml growth Media B are added to the suspension resulted from a slice digested with enzymatic solution A, through the cells strainer; this action served two purposes, releasing the remaining cells on the strainer and dilution of suspension. d. 0.5ml FBS (provided by cord blood bank) + 3ml growth Media B is added to the suspension, resulted from a slice digested with enzymatic solution B, through the cells strainer. 0.5ml FBS + 3ml growth Media B is added to the suspension resulted from a slice digested with enzymatic solution A, through the cells strainer. 3.3 The cell suspension obtained after filtration and dilution is seeded in T25 culture flasks. e. All culture vessels incubate at 37C and 5% CO2, in a humidified incubator. f. After 48h, culture flasks are removed from the incubator, spent media containing dead cells and extracellular matrix, left over from the digestion process, is aspirated and a wash with warm DPBS is performed. After washing the surface of the cell culture, fresh, warm (37C), growth Media B is added. Media change was performed after that every 3 days until cells reached 80-85% confluence.

Stem Cell References: Conconi MT, Burra P, Di Liddo R et al., 2006. CD105(+) cells from Whartons jelly show in vitro and in vivo myogenic differentiative potential. Int J Mol Med, 18, pp. 1089 1096 Ding, D., Chang, Y., Shyu, W., & Lin, S. (2015). Human Umbilical Cord Mesenchymal Stem Cells: A New Era for Stem Cell Therapy. Cell Transplantation, 24(3), 339-347. Fan CG, Zhang Q, Zhou J 2011. Therapeutic Potentials of Mesenchymal Stem Cells Derived from Human Umbilical Cord. Stem Cell Rev., 7(1), pp. 195-207. Fazzina, R., Mariotti, A., Procoli, A., Fioravanti, D., Iudicone, P., Scambia, G., & Bonanno, G. (2015). A new standardized clinical-grade protocol for banking human umbilical cord tissue cells. Transfusion, 55(12), 2864-2873. doi:10.1111/trf.13277 Izadpanah R, Kaushal D, Kriedt C, et al., 2008. Long-term in vitro expansion alters the biology of adult mesenchymal stem cells. Cancer Res., 68, pp. 4229-4238. Lu LL, Liu YJ, Yang SG et al., 2006. Isolation and characterization of human umbilical cord mesenchymal stem cells with hematopoiesis-supportive function and other potentials. Haematologica, 91, pp. 10171026. Petsa A, et al., 2009. Effectiveness of protocol for the isolation of Whartons jelly stem cells in large-scale applications. In Vitro Cell. Dev. Biol. Animal, The Society for In Vitro Biology. Thomas RJ, Hourd P, Williams DJ, 2008. Application of process quality engineering techniques to improve the understanding of the in vitro processing of stem cells for therapeutic use. Journal of Biotechnology, 136, pp. 148155.

This is an informational page designed to help collect information and share information. It is not intended to provide medical advice nor is it qualified by any government source. Stem Cells are not FDA approved but the labs and tissue banks should be in the US. Author is not a doctor and we do not provide medical advice on this page, if you need medical assistance call a professional or in the US dial 911 for an emergency.

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Stem Cell Training and Top Protocols using Human Umbilical Cell Tissue - Checkbiotech.org (press release)

Israeli Scientists Develop First Haploid Human Stem Cells – NoCamels – Israeli Innovation News (press release) (blog)

Israeli scientists have developed the first haploid human stem cells, a discovery that will change our understanding of human genetics and medical research.

Already being used to predict whether people are resistant to chemotherapy drugs, the finding earned Igo Sagi, a PhD student at the Hebrew University of Jerusalem, the 2017 Kaye Innovation Award.

The long-sought haploid

Most of the cells in our body are diploid, which means they carry two sets of chromosomes (the structure in which DNA is contained) one chromosome from each parent. Haploid cells, in contrast, contain only a single set of chromosomes.

Scientists have long been trying to develop haploid stem cells. It is an important area of research, as embryonic stem cells are able to grow into any cell in the human body; this makes them extremely useful for treatment of diseases.

Haploid cells in particular are a powerful discovery, as they allow for a much better understanding of the human genetic makeup. For example, in diploid cells, it is difficult to identify the effects of mutations in one chromosome because the other copy is normal and provides a backup. Haploid cells dont have this limitation.

SEE ALSO: Five Israeli Biotech Companies Using Stem Cells To Change The Face Of Medicine

Up until now, scientists have only succeeded in creating haploid embryonic stem cells in animals such as mice, rats, and monkeys. The research conducted by Igo Sagi was the first time anyone was able to successfully isolate and maintain human haploid embryonic stem cells. These haploid stem cells were able to turn into many other cell types, such as brain, heart, and pancreas, while still retaining a single set of chromosomes.

The benefits are immense. Professor Nissim Benvenisty, who worked with Sagi on the research, explained: It will aid our understanding of human development for example, why we reproduce sexually instead of from a single parent. It will make genetic screening easier and more precise, by allowing the examination of single sets of chromosomes. And it is already enabling the study of resistance to chemotherapy drugs, with implications for cancer therapy.

SEE ALSO: Biological Breakthrough: Researchers Succeed In Creating Human Egg and Sperm Cells In Lab

Haploid Human Embryonic Stem Cells

Diagnosis of Chemotherapy Resistance

Based on this research, Yissum, the Technology Transfer arm of the Hebrew University, launched the company NewStem. The company is currently developing a diagnostic kit that can predict resistance to chemotherapy drugs. The large library of human haploid stem cells they are amassing will allow them to provide therapeutic and reproductive products, as well as personalized medication.

The haploid stem cells were developing have the potential to change the face of medical research as they hold a pivotal role in regenerative medicine, disease therapy and cancer research, revealed CEO of NewStem, Ayelet Dilion-Mashiah.

The research was conducted by Igo Sagi, a doctoral student at the Hebrew University of Jerusalem, along with Professor Nissim Benvenisty, Director of the Azrieli Center for Stem Cells and Genetic Research at the Hebrew University. The Kaye Innovation Awards at the Hebrew University of Jerusalem have been awarded annually since 1994 with the goal of encouraging academics to develop innovative methods and inventions with good commercial potential.

Photo:Azrieli Center for Stem Cells and Genetic Research at Hebrew University

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Israeli Scientists Develop First Haploid Human Stem Cells - NoCamels - Israeli Innovation News (press release) (blog)

Early gene-editing holds promise for preventing inherited diseases – The Jerusalem Post

The secret to healing what ails you lies within your own DNA. (photo credit:DREAMSTIME)

Scientists have, for the first time, corrected a disease-causing mutation in early-stage human embryos using gene editing.

The technique, which uses the CRISPR- Cas9 system, corrected the mutation for a heart condition at the earliest stage of embryonic development so that the defect would not be passed on to future generations.

It could pave the way for improved in vitro fertilization outcomes as well as eventual cures for some thousands of diseases caused by mutations in single genes.

The breakthrough and accomplishment by American and Korean scientists, was recently explained in the journal Nature. Its a collaboration between the Salk Institute, Oregon Health and Science University and South Koreas Institute for Basic Science.

Thanks to advances in stem cell technologies and gene editing, we are finally starting to address disease-causing mutations that impact potentially millions of people, said Prof. Juan Carlos Izpisua Belmonte of Salks gene expression lab and a corresponding author of the paper. Gene editing is still in its infancy, so even though this preliminary effort was found to be safe and effective, it is crucial that we continue to proceed with the utmost caution, paying the highest attention to ethical considerations.

Though gene-editing tools have the power to potentially cure a number of diseases, scientists have proceeded cautiously partly to avoid introducing unintended mutations into the germ line (cells that become eggs or sperm).

Izpisua Belmonte is uniquely qualified to speak on the ethics of genome editing because, as a member of the Committee on Human Gene Editing at the US National Academies of Sciences, Engineering and Medicine, he helped author the 2016 roadmap Human Genome Editing: Science, Ethics and Governance.

Hypertrophic cardiomyopathy is the most common cause of sudden death in otherwise healthy young athletes, and affects approximately one in 500 people. It is caused by a dominant mutation in the MYBPC3 gene, but often goes undetected until it is too late. Since people with a mutant copy of the MYBPC3 gene have a 50% chance of passing it on to their own children, being able to correct the mutation in embryos would prevent the disease not only in affected children but also in their descendants.

The researchers generated induced pluripotent stem cells from a skin biopsy donated by a male with Hypertrophic cardiomyopathy and developed a gene-editing strategy based on CRISPR-Cas9 that would specifically target the mutated copy of the MYBPC3 gene for repair. The targeted mutated MYBPC3 gene was cut by the Cas9 enzyme, allowing the donors cells own DNA -repair mechanisms to fix the mutation during the next round of cell division by using either a synthetic DNA sequence or the non-mutated copy of MYBPC3 gene as a template.

Using IVF techniques, the researchers injected the best-performing gene-editing components into healthy donor eggs that are newly fertilized with donors sperm. All the cells in the early embryos are then analyzed at single-cell resolution to see how effectively the mutation was repaired.

They were surprised by the safety and efficiency of the method. Not only were a high percentage of embryonic cells get fixed, but also gene correction didnt induce any detectable off-target mutations and genome instability major concerns for gene editing.

The researchers also developed an effective strategy to ensure the repair occurred consistently in all the cells of the embryo, as incomplete repairs can lead to some cells continuing to carry the mutation.

Even though the success rate in patient cells cultured in a dish was low, we saw that the gene correction seems to be very robust in embryos of which one copy of the MYBPC3 gene is mutated, said Jun Wu, a Salk staff scientist and one of the authors.

This was in part because, after CRISPR- Cas9 mediated enzymatic cutting of the mutated gene copy, the embryo initiated its own repairs. Instead of using the provided synthetic DNA template, the team surprisingly found that the embryo preferentially used the available healthy copy of the gene to repair the mutated part.

Our technology successfully repairs the disease-causing gene mutation by taking advantage of a DNA repair response unique to early embryos, said Wu.

The authors emphasized that although promising, these are very preliminary results and more research will need to be done to ensure no unintended effects occur.

Our results demonstrate the great potential of embryonic gene editing, but we must continue to realistically assess the risks as well as the benefits, they added.

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Early gene-editing holds promise for preventing inherited diseases - The Jerusalem Post

In a first, scientists rid human embryos of a potentially fatal gene mutation by editing their DNA – Los Angeles Times

Using a powerful gene-editing technique, scientists have rid human embryos of a mutation responsible for an inherited form of heart disease thats often deadly to healthy young athletes and adults in their prime.

The experiment marks the first time that scientists have altered the human genome to erase a disease-causing mutation not only from the DNA of the primary subject but from the genes of his or her progeny as well.

The controversial procedure, known as germ-line editing, was conducted at Oregon Health and Science University in Portland using human embryos expressly created for the purpose. It was reported Wednesday in the journal Nature.

Scientists ultimate goal is to fix gene mutations that lead to debilitating or fatal diseases, and to prevent the propagation of those mutations to future generations. Study leader Shoukhrat Mitalipov, a biologist at OHSU, said the new findings might correct genetic variants that can cause breast and ovarian cancer, cystic fibrosis and muscular dystrophy in those who inherit them.

But others fret that the technique may be used for less noble purposes, such as creating designer babies with desired traits like green eyes, an athletic build or an aptitude for mathematics.

In the United States, the Food and Drug Administration currently forbids any use of germ-line editing outside of a research setting.

But recent history has shown that people who want access to such techniques can find people willing to perform them in venues where theyre able to do so, said Jeffrey Kahn, who directs Johns Hopkins Universitys Berman Institute of Bioethics.

It will happen whether we discuss it or not, and we need to talk about these things before they happen, said Kahn. Thats now.

The new research comes less than six months after the National Academies of Science, Engineering and Medicine recommended that scientists limit their trials of human germ-line editing to diseases that could not be treated with reasonable alternatives at least for the time being.

In a bid to make the experiment relevant to the real-life problems faced by parents who carry disease-causing mutations, the researchers focused on a gene variant that causes inherited hypertrophic cardiomyopathy.

In this condition, a parent who carries one normal and one mutated copy of the MYBPC3 gene has a 50-50 chance of passing the faulty copy on to his or her offspring. If the child inherits the mutation, his or her heart muscle is likely to grow prematurely weak and stiff, causing heart failure and often early death.

In diseases in which one parent carries a gene like this, a couple will often seek the assistance of fertility doctors to minimize the risk of passing the mutation on to a child. A womans eggs and mans sperm meet in a lab using in vitro fertilization. Then specialists inspect the resulting embryos, cull the ones that have inherited an unwanted mutation, and transfer unaffected embryos into a womans uterus to be carried to term.

In the new research, researchers set out to test whether germ-line gene editing could make the process of choosing healthy embryos more effective and efficient by creating more of them.

It could. The targeted correction of a disease-causing gene carried by a single parent can potentially rescue a substantial portion of mutant human embryos, thus increasing the number of embryos available for transfer, the study authors reported.

The fix was made possible by a system known as CRISPR-Cas9, which has been sweeping through biology labs because it greatly simplifies the gene-editing process. It uses a small piece of RNA and an enzyme to snip out unwanted DNA and, if desired, replace it with something better.

If the process is found to be safe for use in fertility clinics, it could potentially decrease the number of cycles needed for people trying to have children free of genetic disease, said Dr. Paula Amato, a coauthor and professor of obstetrics and gynecology at Oregon Health and Science University.

The team encountered several scientific surprises along the way. Long-feared effects of germ-line editing, including collateral damage to off-target genetic sequences, scarcely materialized. And mosaicism, a phenomenon in which edited DNA appears in some but not all cells, was found to be minimal.

Mitalipov called these exciting and surprising moments. But he cautioned that there is room to improve the techniques for producing mutation-free embryos. Clinical trials would have to wait until the DNA editing showed a near-perfect level of efficiency and accuracy, he said, and could be limited by state and federal regulations.

There is still a long road ahead, said Mitalipov, who heads the Center for Embryonic Cell and Gene Therapy at OHSU.

Oregon Health & Science University

Human embryos developing into blastocysts after being injected with a gene-correcting enzyme and sperm carrying a mutation for a potentially fatal disease of the heart muscle.

Human embryos developing into blastocysts after being injected with a gene-correcting enzyme and sperm carrying a mutation for a potentially fatal disease of the heart muscle. (Oregon Health & Science University)

Oregon Health & Science University

Individual blastomeres within the early embryos two days after the co-injection. Each new cell in the developing embryos was uniformly free of the disease-causing mutation.

Individual blastomeres within the early embryos two days after the co-injection. Each new cell in the developing embryos was uniformly free of the disease-causing mutation. (Oregon Health & Science University)

Biologists, fertility doctors and ethicists have long anticipated that scientists would one day manipulate the DNA of human embryos. Now that the milestone has been reached, it drew a mix of praise and concern from experts in genetic medicine.

Dr. Richard O. Hynes, who co-chaired the National Academies report issued in February, called the new study very good science that advances the understanding of genetic repair on many fronts. Hynes, who was not involved with the research effort, said he was pleasantly surprised by the Oregon-based teams clever modifications and their outcomes.

Its likely to become feasible, technically not tomorrow, not next year, but in some foreseeable time. Less than a decade, Id say, said Hynes, a biologist and cancer researcher at MIT and the Howard Hughes Medical Institute.

UC Berkeley molecular and cell biologist Jennifer Doudna, one of pioneers of the CRISPR-Cas9 gene-editing system, said the new research highlights a prospective use of gene editing for one inherited disease and offers some insights into the process. But she questioned how broadly the experiments results would apply to other inherited diseases.

Doudna also said she does not believe using germ-line editing to improve efficiency at fertility clinics meets the criteria laid out by the National Academies of Sciences, which urged that the technology be explored only in cases in which its needed essentially as a last resort.

Already, 50% of embryos would be normal, she said. Why not just implant those?

Doudna said she feared that the new findings will encourage people to proceed down this road before the scientific and ethical implications of germ-line editing have been fully considered.

A large group of experts concluded that clinical use should not proceed until and unless theres broad societal consensus, and that just hasnt happened, she said. This study underscores the urgency of having those debates. Because its coming.

Kristyna Wentz-Graff/Oregon Health & Science University

Study leader Shoukhrat Mitalipov with coauthors Hong Ma, left, and Nuria Marti-Gutierrez.

Study leader Shoukhrat Mitalipov with coauthors Hong Ma, left, and Nuria Marti-Gutierrez. (Kristyna Wentz-Graff/Oregon Health & Science University)

The study authors a multinational team of geneticists, cardiologists, fertility experts and embryologists from OHSU, the Salk Institute in La Jolla, and labs in South Korea and China tested a number of innovations in an effort to improve the safety, efficiency and fidelity of gene editing. And most yielded promising results.

After retrieving eggs from 12 healthy female volunteers, the researchers simultaneously performed two steps that had never been combined in a lab: fertilizing the eggs with sperm and introducing the CRISPR-Cas9 repair machinery.

The resulting embryos took up the gene-editing program so efficiently and uniformly that, after five days of incubation, 72.4% of the 58 embryos tested were free of the MYBPC3 mutation. By comparison, when there was no attempt at gene editing, just 47.4% of embryos were free of the mutation responsible for the deadly heart condition.

The researchers believe their method prompted the embryos to rely on the healthy maternal copy of the gene as a model for fixing the MYBPC3 mutation, and not a repair template that used DNA from the sperm donors normal version of the gene. Only one of the 42 embryos used the introduced template for repair. The scientists contrasted this process to stem cells, which do use repair templates.

The embryos cells divided normally as they matured to the blastocyst stage, the point at which they would usually be ready for transfer to a womans uterus. After extensive testing, the embryos were used to make embryonic stem-cell lines, which are stored in liquid nitrogen and can be used in future research.

Researchers also noted that genetic mosaicism a concern raised by earlier experiments in gene-editing was virtually absent from 41 of the 42 embryos that were free of the disease-causing mutation.

MITs Hynes said such findings offer important insights into how human embryos grow, develop and respond to anomalies, and will help families facing infertility and inherited illnesses.

Human embryogenesis is clearly different from that of a mouse, which we know a lot about, Hynes said. That needs to be studied in human embryos, and theres no other way to do it.

At the same time, he downplayed fears that embryologists would soon tinker with such attributes as looks, personality traits and intelligence in human children.

Were not looking at designed babies around the corner not for a long time, he said.

melissa.healy@latimes.com

@LATMelissaHealy

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UPDATES:

3:50 p.m.: This story has been updated with comments from Jeffrey Kahn of Johns Hopkins Universitys Berman Institute of Bioethics.

This story was originally published at 10 a.m.

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In a first, scientists rid human embryos of a potentially fatal gene mutation by editing their DNA - Los Angeles Times

Cloning/Embryonic Stem Cells – National Human Genome …

Cloning/Embryonic Stem Cells

The term cloning is used by scientists to describe many different processes that involve making duplicates of biological material. In most cases, isolated genes or cells are duplicated for scientific study, and no new animal results. The experiment that led to the cloning of Dolly the sheep in 1997 was different: It used a cloning technique called somatic cell nuclear transfer and resulted in an animal that was a genetic twin -- although delayed in time -- of an adult sheep. This technique can also be used to produce an embryo from which cells called embryonic stem (ES) cells could be extracted to use in research into potential therapies for a wide variety of diseases.

Thus, in the past five years, much of the scientific and ethical debate about somatic cell nuclear transfer has focused on its two potential applications: 1) for reproductive purposes, i.e., to produce a child, or 2) for producing a source of ES cells for research.

The technique of transferring a nucleus from a somatic cell into an egg that produced Dolly was an extension of experiments that had been ongoing for over 40 years. In the simplest terms, the technique used to produce Dolly the sheep - somatic cell nuclear transplantation cloning - involves removing the nucleus of an egg and replacing it with the diploid nucleus of a somatic cell. Unlike sexual reproduction, during which a new organism is formed when the genetic material of the egg and sperm fuse, in nuclear transplantation cloning there is a single genetic "parent." This technique also differs from previous cloning techniques because it does not involve an existing embryo. Dolly is different because she is not genetically unique; when born she was genetically identical to an existing six-year-old ewe. Although the birth of Dolly was lauded as a success, in fact, the procedure has not been perfected and it is not yet clear whether Dolly will remain healthy or whether she is already experiencing subtle problems that might lead to serious diseases. Thus, the prospect of applying this technique in humans is troubling for scientific and safety reasons in addition to a variety of ethical reasons related to our ideas about the natural ordering of family and successive generations.

Several important concerns remain about the science and safety of nuclear transfer cloning using adult cells as the source of nuclei. To date, five mammalian species -- sheep, cattle, pigs, goats, and mice -- have been used extensively in reproductive cloning studies. Data from these experiments illustrate the problems involved. Typically, very few cloning attempts are successful. Many cloned animals die in utero, even at late stages or soon after birth, and those that survive frequently exhibit severe birth defects. In addition, female animals carrying cloned fetuses may face serious risks, including death from cloning-related complications.

An additional concern focuses on whether cellular aging will affect the ability of somatic cell nuclei to program normal development. As somatic cells divide they progressively age, and there is normally a defined number of cell divisions that can occur before senescence. Thus, the health effects for the resulting liveborn, having been created with an "aged" nucleus, are unknown. Recently it was reported that Dolly has arthritis, although it is not yet clear whether the five-and-a-half-year-old sheep is suffering from the condition as a result of the cloning process. And, scientists in Tokyo have shown that cloned mice die significantly earlier than those that are naturally conceived, raising an additional concern that the mutations that accumulate in somatic cells might affect nuclear transfer efficiency and lead to cancer and other diseases in offspring. Researchers working with clones of a Holstein cow say genetic programming errors may explain why so many cloned animals die, either as fetuses or newborns.

The announcement of Dolly sparked widespread speculation about a human child being created using somatic cell nuclear transfer. Much of the perceived fear that greeted this announcement centered on the misperception that a child or many children could be produced who would be identical to an already existing person. This fear is based on the idea of "genetic determinism" -- that genes alone determine all aspects of an individual -- and reflects the belief that a person's genes bear a simple relationship to the physical and psychological traits that compose that individual. Although genes play an essential role in the formation of physical and behavioral characteristics, each individual is, in fact, the result of a complex interaction between his or her genes and the environment within which he or she develops. Nonetheless, many of the concerns about cloning have focused on issues related to "playing God," interfering with the natural order of life, and somehow robbing a future individual of the right to a unique identity.

Several groups have concluded that reproductive cloning of human beings creates ethical and scientific risks that society should not tolerate. In 1997, the National Bioethics Advisory Commission recommended that it was morally unacceptable to attempt to create a child using somatic cell nuclear transfer cloning and suggested that a moratorium be imposed until safety of this technique could be assessed. The commission also cautioned against preempting the use of cloning technology for purposes unrelated to producing a liveborn child.

Similarly, in 2001 the National Academy of Sciences issued a report stating that the United States should ban human reproductive cloning aimed at creating a child because experience with reproductive cloning in animals suggests that the process would be dangerous for the woman, the fetus, and the newborn, and would likely fail. The report recommended that the proposed ban on human cloning should be reviewed within five years, but that it should be reconsidered "only if a new scientific review indicates that the procedures are likely to be safe and effective, and if a broad national dialogue on societal, religious and ethical issues suggests that reconsideration is warranted." The panel concluded that the scientific and medical considerations that justify a ban on human reproductive cloning at this time do not apply to nuclear transplantation to produce stem cells. Several other scientific and medical groups also have stated their opposition to the use of cloning for the purpose of producing a child.

The cloning debate was reopened with a new twist late in 1998, when two scientific reports were published regarding the successful isolation of human stem cells. Stem cells are unique and essential cells found in animals that are capable of continually reproducing themselves and renewing tissue throughout an individual organism's life. ES cells are the most versatile of all stem cells because they are less differentiated, or committed, to a particular function than adult stem cells. These cells have offered hope of new cures to debilitating and even fatal illness. Recent studies in mice and other animals have shown that ES cells can reduce symptoms of Parkinson's disease in mouse models, and work in other animal models and disease areas seems promising.

In the 1998 reports, ES cells were derived from in vitro embryos six to seven days old destined to be discarded by couples undergoing infertility treatments, and embryonic germ (EG) cells were obtained from cadaveric fetal tissue following elective abortion. A third report, appearing in the New York Times, claimed that a Massachusetts biotechnology company had fused a human cell with an enucleated cow egg, creating a hybrid clone that failed to progress beyond an early stage of development. This announcement served as a reminder that ES cells also could be derived from embryos created through somatic cell nuclear transfer, or cloning. In fact, several scientists believed that deriving ES cells in this manner is the most promising approach to developing treatments because the condition of in vitro fertilization (IVF) embryos stored over time is questionable and this type of cloning could overcome graft-host responses if resulting therapies were developed from the recipient's own DNA.

For those who believe that the embryo has the moral status of a person from the moment of conception, research or any other activity that would destroy it is wrong. For those who believe the human embryo deserves some measure of respect, but disagree that the respect due should equal that given to a fully formed human, it could be considered immoral not to use embryos that would otherwise be destroyed to develop potential cures for disease affecting millions of people. An additional concern related to public policy is whether federal funds should be used for research that some Americans find unethical.

Since 1996, Congress has prohibited researchers from using federal funds for human embryo research. In 1999, DHHS announced that it intended to fund research on human ES cells derived from embryos remaining after infertility treatments. This decision was based on an interpretation "that human embryonic stem cells are not a human embryo within the statutory definition" because "the cells do not have the capacity to develop into a human being even if transferred to the uterus, thus their destruction in the course of research would not constitute the destruction of an embryo." DHHS did not intend to fund research using stem cells derived from embryos created through cloning, although such efforts would be legal in the private sector.

In July 2001, the House of Representatives voted 265 to 162 to make any human cloning a criminal offense, including cloning to create an embryo for derivation of stem cells rather than to produce a child. In August 2002, President Bush, contending with a DHHS decision made during the Clinton administration, stated in a prime-time television address that federal support would be provided for research using a limited number of stem cell colonies already in existence (derived from leftover IVF embryos). Current bills before Congress would ban all forms of cloning outright, prohibit cloning for reproductive purposes, and impose a moratorium on cloning to derive stem cells for research, or prohibit cloning for reproductive purposes while allowing cloning for therapeutic purposes to go forward. As of late June, the Senate has taken no action. President Bush's Bioethics Council is expected to recommend the prohibition of reproductive cloning and a moratorium on therapeutic cloning later this summer.

Prepared by Kathi E. Hanna, M.S., Ph.D., Science and Health Policy Consultant

Last Reviewed: April 2006

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