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Before there will be blood: Pro-inflammatory signaling plays surprising role in creation of hematopoietic stem cells

By Stem Cell Medicine

8 hours ago In this multiple confocal analysis of transverse sections from transgenic zebrafish embryos, vasculature is labeled by red fluorescence, NF-kB protein complex that regulates inflammation by green fluorescence and nuclei by blue fluorescence. The arrowhead indicates a potential hematopoietic stem cell emerging in the dorsal aorta with high expression of NF-kB. The image at bottom right combines all channels. Credit: UC San Diego School of Medicine

Hematopoietic stem cells (HSCs) give rise to all blood and immune cells throughout the life of vertebrate organisms, from zebrafish to humans. But details of their genesis remain elusive, hindering efforts to develop induced pluripotent stem cell (iPSC) replacements that might address a host of blood disorders.

In a paper published Nov. 20 in the journal Cell, researchers at the University of California, San Diego School of Medicine describe the surprising and crucial involvement of a pro-inflammatory signaling protein in the creation of HSCs during embryonic development, a finding that could help scientists to finally reproduce HSCs for therapeutic use.

"The recent breakthrough of induced pluripotency has made the concept of patient-specific regenerative medicine a reality," said principal investigator David Traver, PhD, professor in the Department of Cellular and Molecular Medicine. "The development of some mature cell lineages from iPSCs, such as cardiac and neural, has been reasonably straightforward, but not with HSCs. This is likely due, at least in part, to not fully understanding all of the factors used by the embryo to generate HSCs. We believe the discovery that pro-inflammatory cues are important in vivo will help us recapitulate instruction of HSC fate in vitro from iPSCs."

Traver and colleagues specifically looked at the role of a cytokine (a type of cell signaling protein) called tumor necrosis factor alpha or TNF, which plays a pivotal role in regulating systemic inflammation and immunity. The work extended previous research by Spanish biologist Victoriano Mulero, who had reported that TNF was important in the function of the embryonic vascular system and that in animal models where TNF function was absent, blood defects resulted.

The Cell paper's first author Raquel Espin-Palazon, a postdoctoral researcher in Traver's lab and a former colleague of Mulero's, determined that TNF was required for the emergence of hematopoietic stem cells during embryogenesis in zebrafish a common animal model.

Traver said the finding was completely unexpected because HSCs emerge relatively early in embryonic formation when the developing organism is considered to be largely sterile and devoid of infection.

"Thus, there was no expectation that pro-inflammatory signaling would be active at this time or in the blood-forming regions," Traver said. "Equally surprising, we found that a population of embryonic myeloid cells, which are transient cells produced before HSCs arise, are the producers of the TNF needed to establish HSC fate. So it turns out that a small subset of myeloid cells that persist for only a few days in development are necessary to help generate the lineal precursors of the entire adult blood-forming system."

The newly discovered role of TNF in HSC development mirrors a parallel discovery regarding interferon gamma (INFg), another cytokine and major mediator of pro-inflammatory signaling, highlighting multiple inputs for inflammatory signaling in HSC emergence. Traver said the crucial roles of TNF and INFg in HSC emergence are likely similar in humans because of the highly conserved nature of HSC development across vertebrate evolution.

Explore further: New blood: Tracing the beginnings of hematopoietic stem cells

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Before there will be blood: Pro-inflammatory signaling plays surprising role in creation of hematopoietic stem cells

Before There Will Be Blood

By Stem Cell Medicine

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Newswise Hematopoietic stem cells (HSCs) give rise to all blood and immune cells throughout the life of vertebrate organisms, from zebrafish to humans. But details of their genesis remain elusive, hindering efforts to develop induced pluripotent stem cell (iPSC) replacements that might address a host of blood disorders.

In a paper published Nov. 20 in the journal Cell, researchers at the University of California, San Diego School of Medicine describe the surprising and crucial involvement of a pro-inflammatory signaling protein in the creation of HSCs during embryonic development, a finding that could help scientists to finally reproduce HSCs for therapeutic use.

The recent breakthrough of induced pluripotency has made the concept of patient-specific regenerative medicine a reality, said principal investigator David Traver, PhD, professor in the Department of Cellular and Molecular Medicine. The development of some mature cell lineages from iPSCs, such as cardiac and neural, has been reasonably straightforward, but not with HSCs. This is likely due, at least in part, to not fully understanding all of the factors used by the embryo to generate HSCs. We believe the discovery that pro-inflammatory cues are important in vivo will help us recapitulate instruction of HSC fate in vitro from iPSCs.

Traver and colleagues specifically looked at the role of a cytokine (a type of cell signaling protein) called tumor necrosis factor alpha or TNFa, which plays a pivotal role in regulating systemic inflammation and immunity. The work extended previous research by Spanish biologist Victoriano Mulero, who had reported that TNFa was important in the function of the embryonic vascular system and that in animal models where TNF function was absent, blood defects resulted.

The Cell papers first author Raquel Espin-Palazon, a postdoctoral researcher in Travers lab and a former colleague of Muleros, determined that TNFa was required for the emergence of hematopoietic stem cells during embryogenesis in zebrafish a common animal model.

Traver said the finding was completely unexpected because HSCs emerge relatively early in embryonic formation when the developing organism is considered to be largely sterile and devoid of infection.

Thus, there was no expectation that pro-inflammatory signaling would be active at this time or in the blood-forming regions, Traver said. Equally surprising, we found that a population of embryonic myeloid cells, which are transient cells produced before HSCs arise, are the producers of the TNFa needed to establish HSC fate. So it turns out that a small subset of myeloid cells that persist for only a few days in development are necessary to help generate the lineal precursors of the entire adult blood-forming system.

The newly discovered role of TNFa in HSC development mirrors a parallel discovery regarding interferon gamma (INFg), another cytokine and major mediator of pro-inflammatory signaling, highlighting multiple inputs for inflammatory signaling in HSC emergence. Traver said the crucial roles of TNFa and INFg in HSC emergence are likely similar in humans because of the highly conserved nature of HSC development across vertebrate evolution.

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Before There Will Be Blood

Scientists find that SCNT derived cells and IPS cells are similar

By Stem Cell Medical Center

8 hours ago

A team led by New York Stem Cell Foundation (NYSCF) Research Institute scientists conducted a study comparing induced pluripotent stem (iPS) cells and embryonic stem cells created using somatic cell nuclear transfer (SCNT). The scientists found that the cells derived from these two methods resulted in cells with highly similar gene expression and DNA methylation patterns. Both methods also resulted in stem cells with similar amounts of DNA mutations, showing that the process of turning an adult cell into a stem cell introduces mutations independent of the specific method used. This suggests that both methods of producing stem cells need to be further investigated before determining their suitability for the development of new therapies for chronic diseases.

The NYSCF Research Institute is one of the only laboratories in the world that currently pursues all forms of stem cell research including SCNT and iPS cell techniques for creating stem cells. The lack of laboratories attempting SCNT research was one of the reasons that the NYSCF Research Institute was established in 2006.

"We do not yet know which technique will allow scientists to create the best cells for new cellular therapies," said Susan L. Solomon, NYSCF CEO and co-founder. "It is critical to pursue both SCNT and iPS cell techniques in order to accelerate research and bring new treatments to patients."

While both techniques result in pluripotent stem cells, or cells that can become any type of cell in the body, the two processes are different. SCNT consists of replacing the nucleus of a human egg cell or oocyte with the nucleus of an adult cell, resulting in human embryonic stem cells with the genetic material of the adult cell. In contrast, scientists create iPS cells by expressing a few key genes in adult cells, like a skin or blood cell, causing the cells to revert to an embryonic-like state. These differences in methods could, in principle, result in cells with different properties. Advances made earlier this year by NYSCF Research Institute scientists that showed that human embryonic stem cells could be derived using SCNT revived that debate.

"Our work shows that we now have two methods for the generation of a patient's personal stem cells, both with great potential for the development of treatments of chronic diseases. Our work will also be welcome news for the many scientists performing basic research on iPS cells. It shows that they are likely working with cells that are very similar to human embryonic stem cells, at least with regard to gene expression and DNA methylation. How the finding of mutations might affect clinical use of stem cells generated from adult cells is the subject of an ongoing debate," said Dr. Dieter Egli, NYSCF Senior Research Fellow, NYSCF - Robertson Investigator, Assistant Professor in Pediatrics & Molecular Genetics at Columbia University, and senior author on the paper.

The study, published today in Cell Stem Cell, compared cell lines derived from the same sources using the two differing techniques, specifically contrasting the frequency of genetic coding mutations seen and measuring how closely the stem cells matched the embryonic state through the analysis of DNA methylation and of gene expression patterns. The scientists showed that both methods resulted in cell types that were similar with regard to gene expression and DNA methylation patterns. This suggested that both methods were effective in turning a differentiated cell into a stem cell.

The scientists also showed that cells derived using both SCNT and iPS techniques showed similar numbers of genetic coding mutations, implying that neither technique is superior in that regard. A similar number of changes in DNA methylation at imprinted genes (genes that are methylated differentially at the maternal versus the paternal allele) were also found. It is important to note that both types of techniques led to cells that had more of these aberrations than embryonic stem cells derived from an unfertilized human oocyte, or than embryonic stem cells derived from leftover IVF embryos. These findings suggest that a small number of defects are inherent to the generation of stem cells from adult differentiated cells and occur regardless of the method used.

Explore further: Some stem cell methods closer to 'gold standard' than others

Researchers around the world have turned to stem cells, which have the potential to develop into any cell type in the body, for potential regenerative and disease therapeutics. Now, for the first time, researchers ...

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Scientists find that SCNT derived cells and IPS cells are similar

Scientists create Parkinson's disease in a dish

By Stem Cell Medical Center

PUBLIC RELEASE DATE:

6-Nov-2014

Contact: David McKeon dmckeon@nyscf.org 212-365-7440 New York Stem Cell Foundation @nyscf

New York, NY (November 6, 2014) - A team of scientists led by The New York Stem Cell Foundation (NYSCF) Research Institute successfully created a human stem cell disease model of Parkinson's disease in a dish. Studying a pair of identical (monozygotic) twins, one affected and one unaffected with Parkinson's disease, another unrelated Parkinson's patient, and four healthy control subjects, the scientists were able to observe key features of the disease in the laboratory, specifically differences in the patients' neurons' ability to produce dopamine, the molecule that is deficient in Parkinson's disease. In addition, the scientists also identified a potential strategy for developing novel therapies for Parkinson's disease.

Attributed to a combination of genetic and nongenetic factors, Parkinson's disease has no completely effective therapy or cure. Parkinson's disease is moderately heritable, but the mechanisms of this inheritance are not well understood. While genetic forms of the disease exist, sporadic forms are far more common.

"The unique scenario of identical twins, one with this disease and one without, allowed our scientists an unprecedented look into the mechanisms of Parkinson's disease," said Susan L. Solomon, NYSCF Chief Executive Officer. "Advanced stem cell research techniques allow us to push the boundaries of science and see what actually goes wrong at the cellular level, step by step during the disease process."

DNA mutations resulting in the production of a specific enzyme called glucocerebrosidase (GBA) have been linked to a five-fold greater risk of developing Parkinson's disease; however, only 30% of individuals with this mutation have been shown to develop Parkinson's disease by the age of 80. This discordance suggests that multiple factors contribute to the development of Parkinson's disease, including both genetic and non-genetic factors. To date, there has been no appropriate model to identify and test multiple triggers leading to the onset of the disease.

In this study, published today in Cell Reports, a set of identical twins, both with a GBA mutation, provided a unique opportunity to evaluate and dissect the genetic and non-genetic contributions to the development of Parkinson's disease in one twin, and the lack of disease in the other. The scientists made induced pluripotent stem (iPS) cells from skin samples from both twins to generate a cellular model of Parkinson's in a dish, recapitulating key features of the disease, specifically the accumulation of -synuclein and dopamine deficiency.

Upon analyzing the cell models, the scientists found that the dopamine-producing neurons from both twins had reduced GBA enzymatic activity, elevated -synuclein protein levels, and a reduced capacity to synthesize and release dopamine. In comparison to his unaffected brother, the neurons generated from the affected twin produced less dopamine, had higher levels of an enzyme called monoamine oxidase B (MAO-B), and poor ability to connect with each other. Treating the neurons with molecules that lowered the activity of MAO-B together with overexpressed GBA normalized -synuclein and dopamine levels in the cell models. This suggests that a combination therapy for the affected twin may be possible by simultaneously targeting these two enzymes.

"The subject of Parkinson's disease discordant twins gave us an incredible opportunity to utilize stem cell models of disease in a dish to unlock some of the biological mechanisms of disease," said Dr. Scott Noggle, NYSCF Vice President, Stem Cell Research and The NYSCF - Charles Evans Senior Research Fellow for Alzheimer's Disease. "Working with these various different groups and scientists added to the depth and value of the research and we hope our findings will be applicable to other Parkinson's disease patients and other neurodegenerative disorders."

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Scientists create Parkinson's disease in a dish

Direct generation of neural stem cells could enable transplantation therapy

By Stem Cell Medical Center

8 hours ago by Nicole Giese Rura

Induced neural stem cells (iNSCs) created from adult cells hold promise for therapeutic transplantation, but their potential in this capacity has been limited by failed efforts to maintain such cells in the desirable multi-potent NSC state without continuous expression of the transcription factors used initially to reprogram them.

Now, Whitehead Institute scientists have created iNSCs that remain in the multi-potent state without ongoing expression of reprogramming factors. This allows the iNSCs to divide repeatedly to generate cells in quantities sufficient for therapy.

"Therapeutically, it's important to make neural stem cells because they can self-renew and make lots of cells," says Whitehead Institute Founding Member Rudolf Jaenisch, who is also a professor of biology at MIT. "If you just make mature neurons, which has been done by others, you never get enough cells."

To make iNSCs via direct lineage conversion researchers use viruses to insert a cocktail of transcription factors into the genome of mouse adult skin cells. A drug triggers these transcription factors to turn on genes active in neural stem cells. This direct conversion, known as transdifferentiation, bypasses the step of pushing the cells first through an embryonic stem-cell-like state.

In previous research, iNSCs remained addicted to the drug and reprogramming transcription factors; if either the drug or the factors was removed, the cells revert to skin cells.

"If the reprogramming factors are still active, it's horrible for the cells," says John Cassady, a scientist in Jaenisch's lab. "The cells would be unable to differentiate and the resulting cells would not be therapeutically useful."

In a paper published online this week in the current issue of the journal Stem Cell Reports, Cassady and other Whitehead scientists describe how they prevented the cells' relapse without keeping the reprogramming factors active. First, the cells were grown in a special medium that selects for neural stem cells. Then, the drug is removed. Instead of reverting into skin cells, the iNSCs remain in a multi-potent state that can differentiate into neurons and glia cells. Cassady also refined the reprogramming cocktail to contain eight transcription factors, which produces iNSCs that are transcriptionally and epigenetically similar to mouse neural stem cells.

Cassady notes that a random sample of skin cells can contain neural precursor cells, which can more easily make the transition to iNSCs. To eliminate the possibility that his method might actually rely on cells having this sort of "head start", Cassady converted fully mature immune system cells called B-lymphocytes, which have a very specific genetic marker, to iNSCs. The resulting cells had the profile of their new identity as iNSCs, yet retained their B-lymphocyte genetic marker, showing that Cassady's method could indeed convert non-neural cells to iNSCs.

Although promising, all of the work to date has been conducted in mouse cells. According to Cassady, researchers should next test this protocol in human cells to see if it can successfully produce the cell populations necessary for therapeutic use.

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Direct generation of neural stem cells could enable transplantation therapy

Stem cell transplants may help reduce seizures, study says

By Uncategorized

New research from McLean Hospital and the Harvard Stem Cell Institute has shown that stem cell therapy reduces seizures in mice.

Researchers used an animal model to transplant seizure-inhibiting, human embryonic stem cell-derived neurons into the brains of mice that had a common form of epilepsy. Half of the mice that received the transplanted neurons no longer had seizures, while the other half experienced a significant drop in seizure frequency.

The transplanted neurons integrated into the mouse brains and began to receive neuronal activity. The neurons then released GABA, an inhibitory response that reversed the electrical hyperactivity that causes seizure.

Previous studies showed increasing inhibition in the epileptic brain can help control the seizure and also a lot of anti-epilepsy drugs are mimicking this GABA, so many of them worked by binding to the GABA receptors, researcher Sangmi Chung, assistant professor of psychiatry at Harvard, told FoxNews.com.

Researchers initially set out to test the functionality of human neurons, but later decided to test their effect on epilepsy because it is such a devastating disease. About 30 percent of people do not respond to seizure drugs and one out of 26 people will be affected by seizures in their lifetime, Chung said.

Over 65 million people worldwide are affected by epileptic seizures, which can cause convulsions, loss of consciousness and other neurological symptoms. Patients are treated with anti-seizure drugs, and may choose to have a portion of their brain removed.

Because mouse cells mature more quickly than human cells within weeks instead of years it was unclear how long a stem cell transplant in a human would take before becoming effective, Chung noted.

If we compare it with the mouse [model], we believe it will be years, not weeks, she said.

However, the study found that, even without full maturation, the cells integrated into the epileptic mouse brains, receive signals and release GABA, therefore preventing seizures.

I think its really good news in terms of transplantation even maturing, not fully mature [cells] still work, Chung said.

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Stem cell transplants may help reduce seizures, study says

Transplant of stem-cell-derived dopamine neurons shows promise for Parkinson's disease

By Stem Cell Treatment

PUBLIC RELEASE DATE:

6-Nov-2014

Contact: Mary Beth O'Leary moleary@cell.com 617-397-2802 Cell Press @CellPressNews

Parkinson's disease is an incurable movement disorder that affects millions of people around the world, but current treatment options can cause severe side effects and lose effectiveness over time. In a study published by Cell Press November 6th in Cell Stem Cell, researchers showed that transplantation of neurons derived from human embryonic stem cells (hESCs) can restore motor function in a rat model of Parkinson's disease, paving the way for the use of cell replacement therapy in human clinical trials.

"Our study represents an important milestone in the preclinical assessment of hESC-derived dopamine neurons and provides essential support for their usefulness in treating Parkinson's disease," says senior study author Malin Parmar of Lund University.

Parkinson's disease is caused, in part, by the death of neurons that release a brain chemical called dopamine, leading to the progressive loss of control over dexterity and the speed of movement. Currently available drug and surgical treatment options can lose effectiveness over time and cause serious side effects such as involuntary movements and psychiatric problems. Meanwhile, another approach involving the transplantation of human fetal cells has produced long-lasting clinical benefits; however, the positive effects were only seen in some individuals and can also cause involuntary movements driven by the graft itself. Moreover, the use of tissue from aborted human fetuses presents logistical issues such as the limited availability of cells, hampering the effective translation of fetal tissue transplantation as a realistic therapeutic option.

To rigorously assess an alternative hESC-based treatment approach, Parmar and lead study author Shane Grealish of Lund University transplanted hESC-derived dopamine neurons into brain regions that control movement in a rat model of Parkinson's disease. The transplanted cells survived the procedure, restored dopamine levels back to normal within five months, and established the correct pattern of long-distance connections in the brain. As a result, this therapy restored normal motor function in the animals. Importantly, the hESC-derived neurons show efficacy and potency similar to fetal neurons when transplanted in the rat model of Parkinson's disease, suggesting that the hESC-based approach may be a viable alternative to the approaches that have already been established with fetal cells in Parkinson's patients.

In a related Forum article published in the same issue, Roger Barker of Addenbrooke's Hospital and the University of Cambridge laid out the roadmap for taking stem-cell-derived dopamine neurons to the clinic for treating Parkinson's disease. "This involves understanding the history of the whole field of cell-based therapies for Parkinson's disease and some of the mistakes that have happened," he says. "It also requires a knowledge of what the final product should look like and the need to get there in a collaborative way without being tempted to take shortcuts, because a premature clinical trial could impact negatively on the whole field of regenerative medicine."

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Cell Stem Cell, Grealish et al.: "Human ESC-derived dopamine neurons show preclinical efficacy and potency similar to fetal neurons when grafted in a rat model of Parkinson's disease."

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Transplant of stem-cell-derived dopamine neurons shows promise for Parkinson's disease

Stem cells help doctors restore womans smile, regenerating bone to hold dental implants

By Stem Cell Doctors

Durham, NC (PRWEB) November 05, 2014

Half of all traumatic injuries to the face result in a loss of teeth and the surrounding tissue and bone that once supported them, which in turn makes these types of injuries very debilitating and difficult to treat. But in a new study published in the latest issue of STEM CELLS Translational Medicine, doctors at the University of Michigan School of Dentistry (UMSoD), Ann Arbor, have found a new way to regenerate a patients jawbone through the use of stem cells.

The procedure, done under local anesthesia, significantly speeds up the healing time relative to that of traditional bone grafting while allowing a patient to experience only a minimal amount of pain.

Part of a larger clinical trial, the findings highlighted in this issue focus on a 45-year-old woman missing seven front teeth plus 75 percent of the bone that once supported them, the result of a blow to her face five years earlier. She was left with severe functional and cosmetic deficiencies, since the missing bone made it impossible for her to have dental implant-based teeth replacements.

Darnell Kaigler, DDS, MS, PhD, an assistant professor of dentistry in the Department of Periodontics and Oral Medicine, was a lead member of the study team. "In small jawbone defects of the mouth created after teeth were extracted, we have placed gelatin sponges populated with stem cells into these areas to successfully grow bone."

Since the sponge material is soft, it does not work in larger areas. Thus, he and his team of researchers decided to try b-tricalcium phosphate (b-TCP) as a scaffold upon which to place the cells instead. "For treating larger jawbone defects, it is important to have a scaffold material that is rigid and more stable to support bone growth," he explained.

They then placed the b-TCP scaffold, which had been seeded with a mixed population of bone marrow-derived autologous stem and progenitor cells 30 minutes prior to treatment at room temperature, into the defective area of the patients mouth during a procedure that requires only local anesthesia. Four months later, 80 percent of her missing jawbone had been regenerated, allowing them to proceed with placing oral implants that supported a dental prosthesis to once again give her a complete set of teeth.

Study team member Sharon Aronovich, DMD, FRCD(C), a clinical assistant professor of dentistry in the Department of Oral and Maxillofacial Surgery at the UMSoD, said, I am very grateful to all the patients and researchers that participated in this study. Thanks to everyone's efforts, we are one step closer to providing patients with a minimally invasive option for implant-based tooth replacement.

As the first report to describe a cell therapy for craniofacial trauma reconstruction, this research serves as the foundation for expanded studies using this approach, said Anthony Atala, M.D., Editor-in-Chief of STEM CELLS Translational Medicine and director of the Wake Forest Institute for Regenerative Medicine.

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Stem cells help doctors restore womans smile, regenerating bone to hold dental implants

Ladera Ranch teen trying new strategies in her miraculous cancer fight

By Stem Cell Doctors

After four surgeries, six kinds of chemotherapy, stem cell transplants, 12 radiation treatments and antibody treatments, Katie Hawley beat cancer. Twice.

Katie, then 9, learned she had cancer in 2009, when doctors found an-egg sized, stage 3 tumor in her stomach, the cause of her pain and nausea. The rare tumor strikes fewer than five out of every 1 million children each year, according to the National Institute of Health.

In my heart, I hoped and believed she would beat this and live to 100, said Mary Kay Hawley, Katies mom. Yet, that quiet voice would whisper the nightmarish words, She may not make it to her 10th birthday. With that I became her court jester I wanted to give her the world.

After she endured a yearlong battle with ganglio neuroblastoma, doctors found no evidence of cancer cells left in her body. For more than two years, the Ladera Ranch girl lived like a normal kid again aside from scans every three months.

Until the day before Valentines Day last year, when one of her routine CT scans showed the cancer had returned with a vengeance, spreading to her skull, hips and legs.

I was terrified that I was going to lose her, Hawley said. She fell to her knees and begged God to spare her daughter. I prayed until I had peace.

Katie, now a freshman at San Juan Hills High, was sent to undergo an experimental, strong chemotherapy treatment in San Francisco. She had a 33 percent chance of improvement, 33 percent chance the disease would stay the same and 33 percent chance it would get worse. There was a 1 percent chance the treatment would kill the cancer cells, Hawley said.

Katie turned out to be the 1 percent and had a scan clear of cancer in June last year.

KEEPING THE CANCER AT BAY

To stop the cancer from returning, Katie was put on an aggressive, 10-month, in-home chemotherapy with Accutane treatments, making her extremely sick, tired and depressed, Hawley said. She took 14 pills a day, went to the hospital twice a week for blood tests and checkups and continued with CT scans every three months to check that the cancer hadnt returned.

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Ladera Ranch teen trying new strategies in her miraculous cancer fight

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