Finding a Key to Unlock Blocked Differentiation in Microrna-Deficient Embryonic Stem Cells

This aids goal to use stem cells in therapy, where an important hurdle is efficient differentiation.

The more than 200 different types of human cells have the same DNA but express different ensembles of genes. Each cell type was derived from embryonic stem cells, which are called pluripotent stem cells because they can differentiate to all those different cell fates.

One very active area of biology is cells that mimic these fountainhead embryonic stem cells, cells that are called induced pluripotent stem cells, or iPSCs. With genetic and biochemical tricks, researchers can reverse a differentiated cell — such as a skin fibroblast — into a pluripotent state.

Such iPSCs have the potential to create tissue for regenerative medicine, such as repair heart attacks, create models of human disease or make cells that enable drug screening. But future progress with iPSCs needs a much greater understanding of the basic biology of pluripotency and differentiation.

“For the goal of using stem cells in therapy, the most important step is differentiation from iPSCs,” said Rui Zhao, Ph.D., an assistant professor of biochemistry and molecular genetics at the University of Alabama at Birmingham. “We need to be able to differentiate the iPSCs into a disease-relevant cell type at high efficiency and high purity.”

In a study published in Stem Cell Reports, Zhao and colleagues have partly solved a long-unanswered basic question about stem cells — why are pluripotent stem cells that have mutations to block the production of microRNAs unable to differentiate?

Zhao and colleagues, including co-corresponding author Kitai Kim, Ph.D., of the Sloan Kettering Institute, have found a key that allows those microRNA-deficient pluripotent stem cells to differentiate into neural cells, including subtypes with markers for dopaminergic, glutamatergic and GABAergic neurons

“For many years, we did not know why these cells did not differentiate,” Zhao said. The answer for neural cell differentiation in the microRNA-deficient cells turned out to be simple — a single microRNA or a single protein.

In the Stem Cell Reports study, Zhao and colleagues show that a microRNA-302 mimic — delivered by a specially constructed lentivirus — was sufficient to enable neural differentiation of mouse embryonic stem cells that lacked Dgcr8, a vital gene for the processing of the more than 2,000 microRNAs in cells.

When they examined gene expression profiles in the differentiated cells, they saw changes in many gene sets regulated by p53, also known as tumor suppressor p53. This tumor suppressor has been called “the guardian of the genome” because of its many roles in preventing DNA damage and cancer.

Zhao, Kim and colleagues showed that microRNA-302 acted to reduce p53 expression in the microRNA-deficient embryonic stem cells by binding to the 3′ untranslated region of p53 mRNA.

They further showed that direct inhibition of p53 with the simian virus large T antigen or short hairpin RNA, or even deleting the p53 gene itself, allowed embryonic stem cells or iPSCs to proceed to neural differentiation without the need for microRNA-302. Thus, the differentiation barrier that prevents the neuronal lineage specification from microRNA-deficient stem cells is expression of p53.

The keys to unlock the paths cells to other cell lineage specifications from microRNA-deficient embryonic stem are still unknown, Zhao says.

Prostaglandin E1 Inhibits Leukemia Stem Cells

Targeting leukemia stem cells in combination with standard chemotherapy may improve treatment for chronic myeloid leukemia

Two drugs, already approved for safe use in people, may be able to improve therapy for chronic myeloid leukemia (CML), a blood cancer that affects myeloid cells, according to results from a University of Iowa study in mice.

CML is a relatively common cancer. The American Cancer Society estimates that in 2017 there will be about 8,950 new cases and about 1,080 people will die of the disease.

In its initial, chronic stage, CML is relatively easy to treat. Drugs called tyrosine kinase inhibitors (TKIs) are generally successful at controlling the cancer. However, patients need to continue the expensive treatment for their lifetime. In some cases, even with that treatment, the cancer can progress to a more advanced stage that is no longer controlled.

One reason for this, explains Hai-Hui (Howard) Xue, MD, PhD, UI professor of microbiology and immunology, is that there are two kinds of tumor cells—bulk leukemia cells that can be killed by TKI drugs, and a subset of cells called leukemia stem cells, which are resistant to TKIs and to chemotherapy.

“A successful treatment is expected to kill the bulk leukemia cells and at the same time get rid of the leukemic stem cells. Potentially, that could lead to a cure,” says Xue, who is senior author of the study published in the September issue of the journal Cell Stem Cell as the cover story.

With that goal in mind, Xue and his team joined forces with Chen Zhao, MD, PhD, UI assistant professor of pathology, and used their understanding of CML genetics to look for small molecules or drug compounds that might be able to eradicate the leukemia stem cells.

Focusing on two proteins known as transcription factors, the researchers showed that genetically removing the two transcription factors, Tcf1 and Lef1, in mice is sufficient to prevent leukemia stem cells from persisting. Importantly, this genetic alteration did not affect normal hematopoietic (blood) stem cells.

Next the researchers used an informatics method called connectivity maps to identify drugs or small molecules that can replicate the gene expression pattern that occurs when the two transcription factors are removed. This screening test identified a drug called prostaglandin E1 (PGE1).

The team tested a combination of PGE1 and the TKI drug called imatinib in a mouse model of CML. The mice lived longer than control mice; 30 percent lived longer than 80 days compared to mice treated with only imatinib, all of which died within 60 days.

The team also looked at a different mouse model of CML, where human CML cells were transplanted into an immunocompromised mouse. When the mice received no treatment or were treated with imatinib alone, the human leukemia stem cells propagated and grew to relatively large numbers. In contrast, when the animals were treated with a combination of imatinib and PGE1, those numbers were greatly reduced, and mice did not develop leukemia.

“The results are a pleasant surprise,” says Xue who also is a member of Holden Comprehensive Cancer Center at the UI. “We do these kinds of genetic studies all the time—looking at transcription factors and what they do. This is a good opportunity to connect what we do at the bench to something that could be useful clinically.”

Investigating how the PGE1 works to suppress the leukemia stem cells, the team found that the effect relies on a critical interaction between PGE1 and its receptor EP4. They then tested the effect of a second drug molecule called misoprostol, which also interacts with EP4, and showed that misoprostol also has the ability to combine with TKI and significantly reduce the number of leukemia stem cells.

Both PGE1 and misoprostol are currently approved by the FDA for use in people. PGE1 is an injectable drug that is used to treat erectile dysfunction. Misoprostol is a pill that is used to treat ulcers.

“We would like to be able to test these compounds in a clinical trial,” Xue says. “If we could show that the combination of TKI with PGE1, or misoprostol, can eliminate both the bulk tumor cells and the stem cells that keep the tumor going, that could potentially eliminate the cancer to the point where a patient would no longer need to depend on TKI.”

Prostaglandin E1 Inhibits Leukemia Stem Cells Targeting leukemia stem cells in combination with standard chemotherapy may improve treatment for chronic myeloid leukemia

Two drugs, already approved for safe use in people, may be able to improve therapy for chronic myeloid leukemia (CML), a blood cancer that affects myeloid cells, according to results from a University of Iowa study in mice.

CML is a relatively common cancer. The American Cancer Society estimates that in 2017 there will be about 8,950 new cases and about 1,080 people will die of the disease.

In its initial, chronic stage, CML is relatively easy to treat. Drugs called tyrosine kinase inhibitors (TKIs) are generally successful at controlling the cancer. However, patients need to continue the expensive treatment for their lifetime. In some cases, even with that treatment, the cancer can progress to a more advanced stage that is no longer controlled.

One reason for this, explains Hai-Hui (Howard) Xue, MD, PhD, UI professor of microbiology and immunology, is that there are two kinds of tumor cells—bulk leukemia cells that can be killed by TKI drugs, and a subset of cells called leukemia stem cells, which are resistant to TKIs and to chemotherapy.

“A successful treatment is expected to kill the bulk leukemia cells and at the same time get rid of the leukemic stem cells. Potentially, that could lead to a cure,” says Xue, who is senior author of the study published in the September issue of the journal Cell Stem Cell as the cover story.

With that goal in mind, Xue and his team joined forces with Chen Zhao, MD, PhD, UI assistant professor of pathology, and used their understanding of CML genetics to look for small molecules or drug compounds that might be able to eradicate the leukemia stem cells.

Focusing on two proteins known as transcription factors, the researchers showed that genetically removing the two transcription factors, Tcf1 and Lef1, in mice is sufficient to prevent leukemia stem cells from persisting. Importantly, this genetic alteration did not affect normal hematopoietic (blood) stem cells.

Next the researchers used an informatics method called connectivity maps to identify drugs or small molecules that can replicate the gene expression pattern that occurs when the two transcription factors are removed. This screening test identified a drug called prostaglandin E1 (PGE1).

The team tested a combination of PGE1 and the TKI drug called imatinib in a mouse model of CML. The mice lived longer than control mice; 30 percent lived longer than 80 days compared to mice treated with only imatinib, all of which died within 60 days.

The team also looked at a different mouse model of CML, where human CML cells were transplanted into an immunocompromised mouse. When the mice received no treatment or were treated with imatinib alone, the human leukemia stem cells propagated and grew to relatively large numbers. In contrast, when the animals were treated with a combination of imatinib and PGE1, those numbers were greatly reduced, and mice did not develop leukemia.

“The results are a pleasant surprise,” says Xue who also is a member of Holden Comprehensive Cancer Center at the UI. “We do these kinds of genetic studies all the time—looking at transcription factors and what they do. This is a good opportunity to connect what we do at the bench to something that could be useful clinically.”

Investigating how the PGE1 works to suppress the leukemia stem cells, the team found that the effect relies on a critical interaction between PGE1 and its receptor EP4. They then tested the effect of a second drug molecule called misoprostol, which also interacts with EP4, and showed that misoprostol also has the ability to combine with TKI and significantly reduce the number of leukemia stem cells.

Both PGE1 and misoprostol are currently approved by the FDA for use in people. PGE1 is an injectable drug that is used to treat erectile dysfunction. Misoprostol is a pill that is used to treat ulcers.

“We would like to be able to test these compounds in a clinical trial,” Xue says. “If we could show that the combination of TKI with PGE1, or misoprostol, can eliminate both the bulk tumor cells and the stem cells that keep the tumor going, that could potentially eliminate the cancer to the point where a patient would no longer need to depend on TKI.”

Fetal membranes may help transform regenerative medicine

A new review looks at the potential of fetal membranes, which make up the amniotic sac surrounding the fetus during pregnancy, for regenerative medicine.

Fetal membranes have been used as biological bandages for skin grafts as well as for serious burns. They may also have numerous other applications because they contain a variety of stem cells, which might be used to treat cardiovascular and neurological diseases, diabetes, and other medical conditions.

“The fetal membranes have been used successfully in medical applications for over a century, but we continue to discover new properties of these membranes,” said Dr. Rebecca Lim, author of the STEM CELLS Translational Medicine review. “The stem cell populations arising from the fetal membranes are plentiful and diverse, while the membrane itself serves as a unique biocompatible scaffold for bioengineering applications.”

CRI Scientists Discover Vitamin C Regulates Stem Cell Function and Suppresses Leukemia Development

Not much is known about stem cell metabolism, but a new study from the Children’s Medical Center Research Institute at UT Southwestern (CRI) has found that stem cells take up unusually high levels of vitamin C, which then regulates their function and suppresses the development of leukemia.

“We have known for a while that people with lower levels of ascorbate (vitamin C) are at increased cancer risk, but we haven’t fully understood why. Our research provides part of the explanation, at least for the blood-forming system,” said Dr. Sean Morrison, the Director of CRI.

The metabolism of stem cells has historically been difficult to study because a large number of cells are required for metabolic analysis, while stem cells in each tissue of the body are rare. Techniques developed during the study, which was published in Nature, have allowed researchers to routinely measure metabolite levels in rare cell populations such as stem cells.

The techniques led researchers to discover that every type of blood-forming cell in the bone marrow had distinct metabolic signatures – taking up and using nutrients in their own individual way. One of the main metabolic features of stem cells is that they soak up unusually high levels of ascorbate. To determine if ascorbate is important for stem cell function, researchers used mice that lacked gulonolactone oxidase (Gulo) – a key enzyme that most mammals, including mice but not humans, use to synthesize their own ascorbate.

Loss of the enzyme requires Gulo-deficient mice to obtain ascorbate exclusively through their diet like humans do. This gave CRI scientists strict control over ascorbate intake by the mice and allowed them to mimic ascorbate levels seen in approximately 5 percent of healthy humans. At these levels, researchers expected depletion of ascorbate might lead to loss of stem cell function but were surprised to find the opposite was true – stem cells actually gained function. However, this gain came at the cost of increased instances of leukemia.

“Stem cells use ascorbate to regulate the abundance of certain chemical modifications on DNA, which are part of the epigenome,” said Dr. Michalis Agathocleous, lead author of the study, an Assistant Instructor at CRI, and a Royal Commission for the Exhibition of 1851 Research Fellow. “The epigenome is a set of mechanisms inside a cell that regulates which genes turn on and turn off.  So when stem cells don’t receive enough vitamin C, the epigenome can become damaged in a way that increases stem cell function but also increases the risk of leukemia.”

This increased risk is partly tied to how ascorbate affects an enzyme known as Tet2, the study showed. Mutations that inactivate Tet2 are an early step in the formation of leukemia. CRI scientists showed that ascorbate depletion can limit Tet2 function in tissues in a way that increases the risk of leukemia.

These findings have implications for older patients with a common precancerous condition known as clonal hematopoiesis. This condition puts patients at a higher risk of developing leukemia and other diseases, but it is not well understood why certain patients with the condition develop leukemia and others do not. The findings in this study might offer an explanation.

“One of the most common mutations in patients with clonal hematopoiesis is a loss of one copy of Tet2. Our results suggest patients with clonal hematopoiesis and a Tet2 mutation should be particularly careful to get 100 percent of their daily vitamin C requirement,” Dr. Morrison said. “Because these patients only have one good copy of Tet2 left, they need to maximize the residual Tet2 tumor-suppressor activity to protect themselves from cancer.”

Researchers in the Hamon Laboratory for Stem Cell and Cancer Biology, in which Dr. Morrison is also appointed, intend to use the techniques developed as part of this study to find other metabolic pathways that control stem cell function and cancer development. They also plan to further explore the role of vitamin C in stem cell function and tissue regeneration.

Dr. Morrison is a Professor of Pediatrics at UT Southwestern, a Cancer Prevention and Research Institute of Texas (CPRIT) Scholar in Cancer Research, and a Howard Hughes Medical Institute (HHMI) Investigator. He also holds the Mary McDermott Cook Chair in Pediatric Genetics at UT Southwestern and the Kathryne and Gene Bishop Distinguished Chair in Pediatric Research at Children’s Research Institute at UT Southwestern.

CRI and UTSW co-authors include Dr. Zhiyu Zhao, Assistant Professor at CRI and of Pediatrics at UT Southwestern; Dr. Weina Chen, Associate Professor of Pathology at UT Southwestern; Dr. Corbin Meacham, Dr. Rebecca Burgess, and Dr. Malea Murphy, postdoctoral researchers; and Dr. Ralph DeBerardinis, Associate Professor at CRI, of Pediatrics, and in the Eugene McDermott Center for Human Growth & Development. Dr. DeBerardinis, who holds the Joel B. Steinberg, M.D. Chair in Pediatrics and is a Sowell Family Scholar in Medical Research at UTSW, also is the Director of CRI’s Genetic and Metabolic Disease Program and Chief of the Division of Pediatric Genetics and Metabolism at UTSW.

The National Institutes of Health, HHMI, CPRIT, and donors to the Children’s Medical Center Foundation supported this work.

Scientists create stem cell therapy for lung fibrosis conditions

A team of scientists from the UNC School of Medicine and North Carolina State University (NCSU) has developed promising research towards a possible stem cell treatment for several lung conditions, such as idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), and cystic fibrosis — often-fatal conditions that affect tens of millions of Americans.

In the journal Respiratory Research, the scientists demonstrated that they could harvest lung stem cells from people using a relatively non-invasive, doctor’s-office technique. They were then able to multiply the harvested lung cells in the lab to yield enough cells sufficient for human therapy.

In a second study, published in the journal Stem Cells Translational Medicine, the team showed that in rodents they could use the same type of lung cell to successfully treat a model of IPF – a chronic, irreversible, and ultimately fatal disease characterized by a progressive decline in lung function.

The researchers have been in discussions with the FDA and are preparing an application for an initial clinical trial in patients with IPF.

“This is the first time anyone has generated potentially therapeutic lung stem cells from minimally invasive biopsy specimens,” said co-senior author of both papers Jason Lobo, MD, an assistant professor of medicine at UNC and medical director of lung transplant and interstitial lung disease.

Co-senior author Ke Cheng, PhD, an associate professor in NCSU’s Department of Molecular Biomedical Sciences and the UNC/NCSU Joint Department of Biomedical Engineering, said, “We think the properties of these cells make them potentially therapeutic for a wide range of lung fibrosis diseases.”

These diseases of the lung involve the buildup of fibrous, scar-like tissue, typically due to chronic lung inflammation. As this fibrous tissue replaces working lung tissue, the lungs become less able to transfer oxygen to the blood. Patients ultimately are at risk of early death from respiratory failure. In the case of IPF, which has been linked to smoking, most patients live for fewer than five years after diagnosis.

The two FDA-approved drug treatments for IPF reduce symptoms but do not stop the underlying disease process. The only effective treatment is a lung transplant, which carries a high mortality risk and involves the long-term use of immunosuppressive drugs.

Scientists have been studying the alternative possibility of using stem cells to treat IPF and other lung fibrosis diseases. Stem cells are immature cells that can proliferate and turn into adult cells in order to, for example, repair injuries. Some types of stem cells have anti-inflammatory and anti-fibrosis properties that make them particularly attractive as potential treatments for fibrosis diseases.

Cheng and Lobo have focused on a set of stem cells and support cells that reside in the lungs and can be reliably cultured from biopsied lung tissue. The cells are called lung spheroid cells for the distinctive sphere-like structures they form in culture. As the scientists reported in an initial paper in 2015, lung spheroid cells showed powerful regenerative properties when applied to a mouse model of lung fibrosis. In their therapeutic activity, these cells also outperformed other non-lung-derived stem cells known as mesenchymal stem cells, which are also under investigation to treat fibrosis.

In the first of the two new studies, Lobo and his team showed that they could obtain lung spheroid cells from human lung disease patients with a relatively non-invasive procedure called a transbronchial biopsy.

“We snip tiny, seed-sized samples of airway tissue using a bronchoscope,” Lobo said. “This method involves far less risk to the patient than does a standard, chest-penetrating surgical biopsy of lung tissue.”

Cheng and his colleagues cultured lung spheroid cells from these tiny tissue samples until they were numerous enough – in the tens of millions – to be delivered therapeutically. When they infused the cells intravenously into mice, they found that most of the cells gathered in the animals’ lungs.

“These cells are from the lung, and so in a sense they’re happiest, so to speak, living and working in the lung,” Cheng said.

In the second study, the researchers first induced a lung fibrosis condition in rats. The condition closely resembled human IPF. Then the researchers injected the new cultured spheroid cells into one group of rats. Upon studying this group of animals and another group treated with a placebo, the researchers saw healthier overall lung cells and significantly less lung inflammation and fibrosis in the rats treated with lung spheroid cells.

“Also, the treatment was safe and effective whether the lung spheroid cells were derived from the recipients’ own lungs or from the lungs of an unrelated strain of rats,” Lobo said. “In other words, even if the donated stem cells were ‘foreign,’ they did not provoke a harmful immune reaction in the recipient animals, as transplanted tissue normally does.”

Lobo and Chen expect that when used therapeutically in humans, lung spheroid cells initially would be derived from the patient to minimize any immune-rejection risk. Ultimately, however, to obtain enough cells for widespread clinical use, doctors might harvest them from healthy volunteers, as well as from whole lungs obtained from organ donation networks. The stem cells could later be used in patients as-is or matched immunologically to recipients in much the same way transplanted organs are typically matched.

“Our vision is that we will eventually set up a universal cell donor bank,” Cheng said.

Cheng, Lobo, and their teams are now planning an initial study of therapeutic lung spheroid cells in a small group of IPF patients and expect to apply later this year for FDA approval of the study. In the long run, the scientists hope their lung stem cell therapy will also help patients with other lung fibrosis conditions of which there are dozens, including COPD, cystic fibrosis, and fibro-cavernous pulmonary tuberculosis.

Identification of PTPRZ as a drug target for cancer stem cells in glioblastoma

Glioblastoma is the most malignant brain tumor with high mortality. Cancer stem cells are thought to be crucial for tumor initiation and its recurrence after standard therapy with radiation and temozolomide (TMZ) chemotherapy. Protein tyrosine phosphatase receptor type Z (PTPRZ) is an enzyme that is highly expressed in glioblastoma, especially in cancer stem cells.

The research group of Professor Masaharu Noda and Researcher Akihiro Fujikawa of the National Institute for Basic Biology (NIBB) showed that the enzymatic activity of PTPRZ is requisite for the maintenance of stem cell properties and tumorigenicity in glioblastoma cells. PTPRZ knockdown strongly inhibited tumor growth of C6 glioblastoma cells in a mouse xenograft model. In addition, the research team discovered NAZ2329, an allosteric inhibitor of PTPRZ, in collaboration with ASUBIO Pharma Co. Ltd.. NAZ2329 efficiently suppressed stem cell-like properties of glioblastoma cells in culture, and tumor growth in C6 glioblastoma xenografts. These results indicate that pharmacological inhibition of PTPRZ is a promising strategy for the treatment of malignant gliomas.

Stem Cells May Be The Key To Staying Strong In Old Age

University of Rochester Medical Center researchers have discovered that loss of muscle stem cells is the main driving force behind muscle decline in old age in mice. Their finding challenges the current prevailing theory that age-related muscle decline is primarily caused by loss of motor neurons. Study authors hope to develop a drug or therapy that can slow muscle stem cell loss and muscle decline in the future.

As early as your mid 30’s, the size and strength of your muscles begins to decline. The changes are subtle to start — activities that once came easily are not so easy now — but by your 70’s or 80’s, this decline can leave you frail and reliant on others even for simple daily tasks. While the speed of decline varies from person to person and may be slowed by diet and exercise, virtually no one completely escapes the decline.

“Even an elite trained athlete, who has high absolute muscle strength will still experience a decline with age,” said study author Joe Chakkalakal, Ph.D., assistant professor of Orthopaedics in the Center for Musculoskeletal Research at URMC.

Chakkalakal has been investigating exactly how muscle loss occurs in aging mice in order to figure out how humans might avoid it.

In a study, published today in eLife, Chakkalakal and lead author Wenxuan Liu, Ph.D., recent graduate of the Biomedical Genetics Department at URMC, define a new role for stem cells in the life long maintenance of muscle. All adults have a pool of stem cells that reside in muscle tissue that respond to exercise or injury — pumping out new muscle cells to repair or grow your muscles. While it was already known that muscle stem cells die off as you age, Chakkalakal’s study is the first to suggest that this is the main driving factor behind muscle loss.

To better understand the role of stem cells in age-related muscle decline, Chakkalakal and his team depleted muscle stem cells in mice without disrupting motor neurons, nerve cells that control muscle. The loss of stem cells sped up muscle decline in the mice, starting in middle, rather than old age. Mice that were genetically altered to prevent muscle stem cell loss maintained healthier muscles at older ages than age-matched control mice.

At the same time, Chakkalakal and his team did not find evidence to support motor neuron loss in aging mice. Very few muscle fibers had completely lost connection with their corresponding motor neurons, which questions the long-held and popular “Denervation/Re-innervation” theory. According to the theory, age-related muscle decline is primarily driven by motor neurons dying or losing connection with the muscle, which then causes the muscle cells to atrophy and die.

“I think we’ve shown a formal demonstration that even for aging sedentary individuals, your stem cells are doing something,” said Chakkalakal. “They do play a role in the normal maintenance of your muscle throughout life.”

Chakkalakal is building on this discovery and searching for a drug target that will allow him to maintain the muscle stem cell pool and stave off muscle degeneration as long as possible and he hopes this discovery will help move the field forward.