Posts Tagged ‘stem cells’

Stem Cells Differentiated into Insulin-Producing Cells in Mice

Reported: Irina Robu, PhD

Dr. Douglas Melton team from Harvard University funded in part by NIH’s National Institute of Diabetes and Digestive and Kidney Diseases set out to transform stem cells into beta cells that have the potential to replace damaged beta cells. While scientists have been able to change stem cells into insulin-producing cells, these cells don’t have markers that indicate they are beta cells, and they aren’t responsive to glucose.

Since diabetes is a disorder of elevated blood sugars where the body does not harvest enough insulin to meet where the body does not harvest enough insulin to respond properly to the insulin being made. When blood glucose levels rise, beta cells in the pancreas normally make the hormone insulin. Insulin triggers cells throughout the body to take up sugar from the blood. In type 2 diabetes, the most common form, tissues in the body lose their sensitivity to insulin, and pancreatic beta cells can’t make enough insulin to keep glucose levels in check. In type 1 diabetes, the body’s own immune system attacks and destroys beta cells. High blood glucose levels can lead to heart disease, blindness, and other health problems over time.

One approach to treat diabetes is to replace destroyed beta cells. Transplanted human pancreatic cells from deceased donors have been successfully used to treat people with type 1 diabetes. But this method is restricted by the accessibility of donor cells and the side effects of immunosuppression. The other approach is to develop functioning beta cells from stem cells which have the potential to transform into many different cell types. These cells can grow indefinitely in the laboratory and can differentiate, into any cell type found in the body.
In this experiment, the researchers grew a human embryonic stem cell line and 2 human-induced pluripotent stem cell lines in a culture system that allowed them to produce large numbers of cells. The researchers tested more than 150 combinations of over 70 compounds to figure out a method to produce functional human beta cells from the cultured stem cells which when added in exact combinations over a period of several weeks, they transformed human pluripotent stem cells into beta cells that functioned similarly to normal adult beta cells.

The cultured beta cells had specific markers that were found on normal beta cells which displayed changes in calcium levels when exposed to glucose and packaged insulin into granules. However, when transplanted into mice these cells secreted insulin in response to glucose. However, when the cells were transplanted into diabetic mice, abnormally high blood glucose levels lowered. More work is needed to develop these cells for clinical use. However, at this point they can serve as a useful screening tool for diabetes drugs.





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New Insights into mtDNA, mitochondrial proteins, aging, and metabolic control

Larry H. Bernstein, MD, FCAP, Curator



Newly discovered proteins may protect against age-related illnesses  

The proteins could play a key role in the aging process and the onset of diseases linked to older age

BY Beth Newcomb   APRIL 13, 2016×549.jpg

Pinchas Cohen led a team that identified tiny proteins that appear to play a role in controlling how the body ages. (Photo/Beth Newcomb)

A group of six newly discovered proteins may help to divulge secrets of how we age, potentially unlocking insights into diabetes, Alzheimer’s, cancer and other aging-related diseases.

The tiny proteins appear to play several big roles in our bodies’ cells, from decreasing the amount of damaging free radicals and controlling the rate at which cells die to boosting metabolism and helping tissues throughout the body respond better to insulin. The naturally occurring amounts of each protein decrease with age, leading researchers to believe that they play an important role in the aging process and the onset of diseases linked to older age.

The research team led by Pinchas Cohen, dean of the USC Davis School of Gerontology, identified the tiny proteins for the first time and observed their surprising origin from organelles in the cell called mitochondria and their game-changing roles in metabolism and cell survival. This latest finding builds upon prior research by Cohen and his team that uncovered two significant proteins, humanin and MOTS-c, hormones that appear to have significant roles in metabolism and diseases of aging.

Unlike most other proteins, humanin and MOTS-c are encoded in mitochondria, the structure within cells that produces energy from food, instead of in the cell’s nucleus where most genes are contained.

Key functions

Mitochondria have their own small collection of genes, which were once thought to play only minor roles within cells but now appear to have important functions throughout the body. Cohen’s team used computer analysis to see if the part of the mitochondrial genome that provides the code for humanin was coding for other proteins as well. The analysis uncovered the genes for six new proteins, which were dubbed small humanin-like peptides, or SHLPs, 1 through 6 (the name of this hardworking group of proteins is appropriately pronounced “schlep”).

After identifying the six SHLPs and successfully developing antibodies to test for several of them, the team examined both mouse tissues and human cells to determine their abundance in different organs as well as their functions. The proteins were distributed quite differently among organs, which suggests that the proteins have varying functions based on where they are in the body.

Of particular interest is SHLP 2, Cohen said. The protein appears to have profound insulin-sensitizing, anti-diabetic effects as well as potent neuro-protective activity that may emerge as a strategy to combat Alzheimer’s disease. He added that SHLP 6 is also intriguing, with a unique ability to promote cancer cell death and thus potentially target malignant diseases.

“Together with the previously identified mitochondrial peptides, the newly recognized SHLP family expands the understanding of the mitochondria as an intracellular signaling organelle that communicates with the rest of the body to regulate metabolism and cell fate,” Cohen said. “The findings are an important advance that will be ripe for rapid translation into drug development for diseases of aging.”

The study first appeared online in the journal Aging on April 10. Cohen’s research team included collaborators from the Albert Einstein College of Medicine; the findings have been licensed to the biotechnology company CohBar for possible drug development.

The research was supported by a Glenn Foundation Award and National Institutes of Health grants to Cohen (1P01AG034906, 1R01AG 034430, 1R01GM 090311, 1R01ES 020812) and an Ellison/AFAR postdoctoral fellowship to Kelvin Yen. Study authors Laura Cobb, Changhan Lee, Nir Barzilai and Pinchas Cohen are consultants and stockholders of CohBar Inc.

Feature: The man who wants to beat back aging

By Stephen S. Hall Sep. 16, 201

Nir Barzilai hopes to persuade FDA to bless the proposed anti-aging trial, which is unconventional in its goals and design.

On a blazingly hot morning this past June, a half-dozen scientists convened in a hotel conference room in suburban Maryland for the dress rehearsal of what they saw as a landmark event in the history of aging research. In a few hours, the group would meet with officials at the U.S. Food and Drug Administration (FDA), a few kilometers away, to pitch an unprecedented clinical trial—nothing less than the first test of a drug to specifically target the process of human aging.

“We think this is a groundbreaking, perhaps paradigm-shifting trial,” said Steven Austad, chairman of biology at the University of Alabama, Birmingham, and scientific director of the American Federation for Aging Research (AFAR). After Austad’s brief introductory remarks, a scientist named Nir Barzilai tuned up his PowerPoint and launched into a practice run of the main presentation.

Barzilai is a former Israeli army medical officer and head of a well-known study of centenarians based at the Albert Einstein College of Medicine in the Bronx, New York. To anyone who has seen the ebullient scientist in his natural laboratory habitat, often in a short-sleeved shirt and always cracking jokes, he looked uncharacteristically kempt in a blue blazer and dress khakis. But his practice run kept hitting a historical speed bump. He had barely begun to explain the rationale for the trial when he mentioned, in passing, “lots of unproven, untested treatments under the category of anti-aging.” His colleagues pounced.

“Nir,” interrupted S. Jay Olshansky, a biodemographer of aging from the University of Illinois, Chicago. The phrase “anti-aging … has an association that is negative.”

“I wouldn’t dignify them by calling them ‘treatments,’” added Michael Pollak, director of cancer prevention at McGill University in Montreal, Canada. “They’re products.”

Barzilai, a 59-year-old with a boyish mop of gray hair, wore a contrite grin. “We know the FDA is concerned about this,” he conceded, and deleted the offensive phrase.

Then he proceeded to lay out the details of an ambitious clinical trial. The group—academics all—wanted to conduct a double-blind study of roughly 3000 elderly people; half would get a placebo and half would get an old (indeed, ancient) drug for type 2 diabetes called metformin, which has been shown to modify aging in some animal studies. Because there is still no accepted biomarker for aging, the drug’s success would be judged by an unusual standard—whether it could delay the development of several diseases whose incidence increases dramatically with age: cardiovascular disease, cancer, and cognitive decline, along with mortality. When it comes to these diseases, Barzilai is fond of saying, “aging is a bigger risk factor than all of the other factors combined.”

But the phrase “anti-aging” kept creeping into the rehearsal, and critics kept jumping in. “Okay,” Barzilai said with a laugh when it came up again. “Third time, the death penalty.”

The group’s paranoia about the term “anti-aging” captured both the audacity of the proposed trial and the cultural challenge of venturing into medical territory historically associated with charlatans and quacks. The metformin initiative, which Barzilai is generally credited with spearheading, is unusual by almost any standard of drug development. The people pushing for the trial are all academics, none from industry (although Barzilai is co-founder of a biotech company, CohBar Inc., that is working to develop drugs targeting age-related diseases). The trial would be sponsored by the nonprofit AFAR, not a pharmaceutical company. No one stood to make money if the drug worked, the scientists all claimed; indeed, metformin is not only generic, costing just a few cents a dose, but belongs to a class of drugs that has been part of the human apothecary for 500 years. Patient safety was unlikely to be an issue; millions of diabetics have taken metformin since the 1960s, and its generally mild side effects are well-known.

Finally, the metformin group insisted they didn’t need a cent of federal money to proceed (although they do intend to ask for some). Nor did they need formal approval from FDA to proceed. But they very much wanted the agency’s blessing. By recognizing the merit of such a trial, Barzilai believes,  FDA would make aging itself a legitimate target for drug development.

By the time the scientists were done, the rehearsal—which was being filmed for a television documentary—had the feel of a pep rally. They spoke with unguarded optimism. “What we’re talking about here,” Olshansky said, “is a fundamental sea change in how we look at aging and disease.” To Austad, it is “the key, potentially, to saving the health care system.”

As the group piled into a van for the drive to FDA headquarters, there was more talk about setting precedents and opening doors. So it was a little disconcerting when Austad led the delegation up to the main entrance of FDA—and couldn’t get the door open.   ……

Mitochondrial Peptides Found in a Preclinical Study Seen to Control Cell Metabolism



CohBar, a developer of mitochondria-based therapeutics, announced that preclinical research by its academic collaborators has found small humanin-like peptides (SHLPs) that can control metabolism and cell survival. The findings have implications for age-related diseases such as Alzheimer’s and cancer.

The study, “Naturally Occurring Mitochondrial-derived Peptides are Age-dependent Regulators of Apoptosis, Insulin Sensitivity, and Inflammatory Markers,” was the result of a joint effort between researchers at the University of Southern California (USC) and theInstitute for Aging Research at the Albert Einstein College of Medicine of Yeshiva University. The study was published in the journal Aging.

Researchers discovered the SHLPs by examining the genome of mitochondria with the help of a bioinformatics approach, which identified six peptides. The team then verified the presence of the factors and explored their function in laboratory animals.

CohBar, who have the exclusive license to develop SHLPs into therapeutics, works closely with its academic partners to explore the peptides in preclinical models.

While it was previously believed that mitochondria only have 37 genes, research has revealed that the mitochondrial genome is far more versatile, potentially harboring a multitude of new genes, which can encode peptides acting as cellular signaling factors. The peptides, it has turned out, have shown neuroprotective and anti-inflammatory effects, and act to protect cells in disease-modifying ways in preclinical models of aging.

CohBar’s goal is to bring these peptides to the market as therapies for age-related diseases, such as obesity, type 2 diabetes, cancer, atherosclerosis and neurodegenerative disorders.

“Together with the previously described mitochondrial-derived peptides humanin and MOTS-c, the SHLP family expands our understanding of the role that these peptides play in intracellular signaling throughout the body to regulate both metabolism and cell survival,” Pinchas Cohen, dean of the USC Leonard Davis School of Gerontology, founder and director of CohBar, and the study’s senior author, said in a press release. “These findings further illustrate the enormous potential that mitochondria-based therapeutics could have on treating age-associated diseases like Alzheimer’s and cancer.”

“The pre-clinical evidence continues to confirm that these peptides represent a new class of naturally occurring metabolic regulators,” added Simon Allen, CohBar’s CEO. “They form the foundation of our pipeline of first-in-class treatments for age-related diseases, and we are committed to rapidly advancing them through pre-clinical and clinical activities as we move forward.”

Naturally occurring mitochondrial-derived peptides are age-dependent regulators of apoptosis, insulin sensitivity, and inflammatory markers

Laura J. Cobb1,5, Changhan Lee2, Jialin Xiao2, Kelvin Yen2, Richard G. Wong2, Hiromi K. Nakamura1, ….., Derek M. Huffman4, Junxiang Wan2, Radhika Muzumdar3, Nir Barzilai4 , and Pinchas Cohen2

Mitochondria are key players in aging and in the pathogenesis of age-related diseases. Recent mitochondrial transcriptome analyses revealed the existence of multiple small mRNAs transcribed from mitochondrial DNA (mtDNA). Humanin (HN), a peptide encoded in the mtDNA 16S ribosomal RNA region, is a neuroprotective factor. An in silico search revealed six additional peptides in the same region of mtDNA as humanin; we named these peptides small humanin-like peptides (SHLPs). We identified the functional roles for these peptides and the potential mechanisms of action. The SHLPs differed in their ability to regulate cell viability in vitro. We focused on SHLP2 and SHLP3 because they shared similar protective effects with HN. Specifically, they significantly reduced apoptosis and the generation of reactive oxygen species, and improved mitochondrial metabolism in vitro. SHLP2 and SHLP3 also enhanced 3T3-L1 pre-adipocyte differentiation. Systemic hyperinsulinemic-euglycemic clamp studies showed that intracerebrally infused SHLP2 increased glucose uptake and suppressed hepatic glucose production, suggesting that it functions as an insulin sensitizer both peripherally and centrally. Similar to HN, the levels of circulating SHLP2 were found to decrease with age. These results suggest that mitochondria play critical roles in metabolism and survival through the synthesis of mitochondrial peptides, and provide new insights into mitochondrial biology with relevance to aging and human biology.

Human mitochondrial DNA (mtDNA) is a double-stranded, circular molecule of 16,569 bp and contains 37 genes encoding 13 proteins, 22 tRNAs, and 2 rRNAs. Recent mitochondrial transcriptome analyses revealed the existence of small RNAs derived from mtDNA [1]. In 2001, Nishimoto and colleagues identified humanin (HN), a 24-amino-acid peptide encoded from the 16S ribosomal RNA (rRNA) region of mtDNA. HN is a potent neuroprotective factor capable of antagonizing Alzheimer’s disease (AD)-related cellular insults [2]. HN is a component of a novel retrograde signaling pathway from the mitochondria to the nucleus, which is distinct from mitochondrial signaling pathways, such as the SIRT4-AMPK pathway [3]. HN-dependent cellular protection is mediated in part by interacting with and antagonizing pro-apoptotic Bax-related peptides [4] and IGFBP-3 (IGF binding protein 3) [5].

Because of their involvement in energy production and free radical generation, mitochondria likely play a major role in aging and age-related diseases [68]. In fact, improvement of mitochondrial function has been shown to ameliorate age-related memory loss in aged mice [9]. Recent studies have shown that HN levels decrease with age, suggesting that HN could play a role in aging and age-related diseases, such as Alzheimer’s disease (AD), atherosclerosis, and diabetes. Along with lower HN levels in the hypothalamus, skeletal muscle, and cortex of older rodents, the circulating levels of HN were found to decline with age in both humans and mice [10]. Notably, circulating HN levels were found to be (i) significantly higher in long-lived Ames dwarf mice but lower in short-lived growth hormone (GH) transgenic mice, (ii) significantly higher in a GH-deficient cohort of patients with Laron syndrome, and (iii) reduced in mice and humans treated with GH or IGF-1 (insulin-like growth factor 1) [11]. Age-dependent declines in the circulating HN levels may be due to higher levels of reactive oxygen species (ROS) that contribute to atherosclerosis development. Using mouse models of atherosclerosis, it was found that HN-treated mice had a reduced disease burden and significant health improvements [12,13]. In addition, HN improved insulin sensitivity, suggesting clinical potential for mitochondrial peptides in diseases of aging [10]. The discovery of HN represents a unique addition to the spectrum of roles that mitochondria play in the cell [14,15]. A second mitochondrial-derived peptide (MDP), MOTS-c (mitochondrial open reading frame of the 12S rRNA-c), has also been shown to have metabolic effects on muscle and may also play a role in aging [16].

We further investigated mtDNA for the presence of other MDPs. Recent technological advances have led to the identification of small open reading frames (sORFs) in the nuclear genomes ofDrosophila[17,18] and mammals [19,20]. Therefore, we attempted to identify novel sORFs using the following approaches: 1) in silico identification of potential sORFs; 2) determination of mRNA expression levels; 3) development of specific antibodies against these novel peptides to allow for peptide detection in cells, organs, and plasma; 4) elucidating the actions of these peptides by performing cell-based assays for mitochondrial function, signaling, viability, and differentiation; and 5) delivering these peptides in vivo to determine their systemic metabolic effects. Focusing on the 16S rRNA region of the mtDNA where the humanin gene is located, we identified six sORFs and named them small humanin-like peptides (SHLPs) 1-6. While surveying the biological effects of SHLPs, we found that SHLP2 and SHLP3 were cytoprotective; therefore, we investigated their effects on apoptosis and metabolism in greater detail. Further, we showed that circulating SHLP2 levels declined with age, similar to HN, suggesting that SHLP2 is involved in aging and age-related disease progression.

SHLP2 and SHLP3 regulate the expression of metabolic and inflammatory markers

Epidemiological studies have demonstrated that increased levels of mediators of inflammation and acute-phase reactants, such as fibrinogen, C-reactive protein (CRP), and IL-6, correlate with the incidence of type 2 diabetes mellitus (T2DM) [3436]. In humans, anti-inflammatory drugs, such as aspirin and sodium salicylate, reduce fasting plasma glucose levels and ameliorate the symptoms of T2DM. In addition, anti-diabetic drugs, such as fibrates [37] and thiazolidinediones [38], have been found to lower some markers of inflammation. SHLP2 increased the levels of leptin, which is known to improve insulin sensitivity, but had no effect on the levels of the pro-inflammatory cytokines IL-6 and MCP-1. SHLP3 significantly increased the leptin levels, but also elevated IL-6 and MCP-1 levels, which could explain the lack of an in vivo insulin-sensitizing effect of SHLP3. The mechanism by which SHLPs regulate the expression of metabolic and inflammatory markers remains unclear and needs to be further investigated. Furthermore, SHLPs have different effects on inflammatory marker expression, suggesting differential regulation and function of individual SHLPs.

SHLP2 in aging

Mitochondria have been implicated in increased lifespan in several life-extending treatments [39,40]; however, it is not known whether the relationship is correlative or causative [40]. Additionally, it is well known that hormone levels change with aging. For example, levels of aldosterone, calcitonin, growth hormone, and IGF-I decrease with age. Circulating HN levels decline with age in humans and rodents, specifically in the hypothalamus and skeletal muscle of older rats. These changes parallel increases in the incidence of age-associated diseases such as AD and T2DM. The decline in circulating SHLP2 levels with age (Fig. 6), the anti-oxidative stress function of SHLP2 (Fig. 3C), and its neuroprotective effect (Fig. 6B) indicate that SHLP2 has a role in the regulation of aging and age-related diseases.


By analyzing the mitochondrial transcriptome, we found that sORFs from mitochondrial DNA encode functional peptides. We identified many mRNA transcripts within 13 protein-coding mitochondrial genes [1]. Such previously underappreciated sORFs have also been described in the nuclear genome [41]. The MDPs we describe here may represent retrograde communication signals from the mitochondria to the nucleus and may explain important aspects of mitochondrial biology that are implicated in health and longevity.

Larry, John Walker is working on mt proteins dynamics. His rotor – stator mechanism in ATPase synthase, a ‘complex’ that biologist accepted as energy generator is likely wrong. I was suppose to have met him in Germany few years ago. Energy in biological systems has nothing to do with heat. Heat is an outcome of a reaction, meaning that IR spectra accordingly to wave theory is a source of information memorized in water interference with carbon open systems within protein and glyo-proteins complexes as well as genome space-time outcomes. Physically speaking from a pure perspective of science ATP is highly unstable form of phosphate ‘chains’. It cannot hold energy, it is actually in contrary, it is like a resonator, trapping negativity, thus functioning as space propeller by expanding carbon skeleton of protein ‘machines’ Now, we don’t know what is ‘aging’ in a pure physical sense, except that we observe structural changes in what we call complexes. We we know is that proteins are not stationary structures, but highly dynamic forms of matter, seemingly occupying discrete and relative spaces. A piece of mt ATP ase could be discovered in the nucleus as transcription factor. Our notion of operational space in terms of electro dynamics from a motor – stator perspective is now translated toward defining semi conducting and supracoductive strings. The reality of which is so much more fascinating and beautiful as time progresses overally. There are spaces where time does not change, and there are spaces where time walks, and there are spaces, where time flies, and there are spaces where time runs. Amazing, indeed! The story of aging gets a lot deeper that science could even imagine, probably to roots of immortal energy- spaces. We know that matter is transient, that is nearly all living matter, replenishes of about 3 to 7 weeks.

Take a glass full of some kind of liquid, you know the mass of the glass and the mass of the liquid (say wine, beer, water, or milk) You also know to an approximate reality the composition of both. Now lift the glass full of liquid and let it break on a surface of your choice. Depending on the surface pieces of the glass would travel differential from a center projected by the vertical axis of your hand. What technology does today is recollecting those pieces and modelling them to fit in a form again that would resemble a holding device, a glass. The liquid we don’t know exactly how it spilled due the nature of its absorbancy of both surface physics and physical ‘state’ properties. Thus we can say how much approximate energy we have held thinking of m/z as time flight objectives. Each technology can read 1D and approximate the 2D, absolutely lacking computational methodology for 3D dynamic reality. Many scientists confuse space and volume. Volume is a one dimensional characteristic! So is crystalography! BY taking quantum chemical method computing principles following imaginative rules we could approach 2D, however , that is not enough to define 3D. Time we use as a reference frame of clocks we have invented in order to keep track of a sense to observable ‘change’ . But remember, time is absolute and parallel in continuity while energy is discrete , coming in quantum packages, realization of accumulated information. Information is highly redundant we see, so annotating information is an objective to modern days simulations that could predict outcomes of possible parallel realities we call worlds. One could ‘jump’ from one reality to another through guidance of light and water, but what remains unsolved is why people make mistakes, constantly by accusing in name of greed and power , or disobedience of commandments of the Lord!

On Thu, Apr 21, 2016 at 3:41 AM, Leaders in Pharmaceutical Business Intelligence (LPBI) Group wrote:

> larryhbern posted: “New Insights into mtDNA, mitochondrial proteins, > aging, and metabolic control Larry H. Bernstein, MD, FCAP, Curator LPBI > Newly discovered proteins may protect against age-related illnesses The > proteins could play a key role in the ” >


Metabolic features of the cell danger response
– Mitochondria in Health and Disease

Mitochondrion  Volume 16, May 2014, Pages 7–17     doi:10.1016/j.mito.2013.08.006



  •  The Cell Danger Response (CDR) is defined in terms of an ancient metabolic response to threat.
  •  The CDR encompasses inflammation, innate immunity, oxidative stress, and the ER stress response.
  •  The CDR is maintained by extracellular nucleotide (purinergic) signaling.
  •  Abnormal persistence of the CDR lies at the heart of many chronic diseases.
  •  Antipurinergic therapy (APT) has proven effective in many chronic disorders in animal models

The cell danger response (CDR) is the evolutionarily conserved metabolic response that protects cells and hosts from harm. It is triggered by encounters with chemical, physical, or biological threats that exceed the cellular capacity for homeostasis. The resulting metabolic mismatch between available resources and functional capacity produces a cascade of changes in cellular electron flow, oxygen consumption, redox, membrane fluidity, lipid dynamics, bioenergetics, carbon and sulfur resource allocation, protein folding and aggregation, vitamin availability, metal homeostasis, indole, pterin, 1-carbon and polyamine metabolism, and polymer formation. The first wave of danger signals consists of the release of metabolic intermediates like ATP and ADP, Krebs cycle intermediates, oxygen, and reactive oxygen species (ROS), and is sustained by purinergic signaling. After the danger has been eliminated or neutralized, a choreographed sequence of anti-inflammatory and regenerative pathways is activated to reverse the CDR and to heal. When the CDR persists abnormally, whole body metabolism and the gut microbiome are disturbed, the collective performance of multiple organ systems is impaired, behavior is changed, and chronic disease results. Metabolic memory of past stress encounters is stored in the form of altered mitochondrial and cellular macromolecule content, resulting in an increase in functional reserve capacity through a process known as mitocellular hormesis. The systemic form of the CDR, and its magnified form, the purinergic life-threat response (PLTR), are under direct control by ancient pathways in the brain that are ultimately coordinated by centers in the brainstem. Chemosensory integration of whole body metabolism occurs in the brainstem and is a prerequisite for normal brain, motor, vestibular, sensory, social, and speech development. An understanding of the CDR permits us to reframe old concepts of pathogenesis for a broad array of chronic, developmental, autoimmune, and degenerative disorders. These disorders include autism spectrum disorders (ASD), attention deficit hyperactivity disorder (ADHD), asthma, atopy, gluten and many other food and chemical sensitivity syndromes, emphysema, Tourette’s syndrome, bipolar disorder, schizophrenia, post-traumatic stress disorder (PTSD), chronic traumatic encephalopathy (CTE), traumatic brain injury (TBI), epilepsy, suicidal ideation, organ transplant biology, diabetes, kidney, liver, and heart disease, cancer, Alzheimer and Parkinson disease, and autoimmune disorders like lupus, rheumatoid arthritis, multiple sclerosis, and primary sclerosing cholangitis.

The double face of mitochondrial dysfunction

Dmitry Knorre, Anna Zyrina, and Fedor Severin

pp 420-420

Full text | PDF



Flawed Mitochondrial DNA Could Undermine Stem Cell Therapies

This is a confocal microscopy image of human fibroblasts derived from embryonic stem cells. The nuclei appear in blue, while smaller and more numerous mitochondria appear in red. [Shoukhrat Mitalipov]

Mutations in our mitochondrial DNA tend to be inconspicuous, but they can become more prevalent as we age. They can even vary in frequency from cell to cell. Naturally, some cells will be relatively compromised because they happen to have a higher percentage of mutated mitochondrial DNA. Such cells make a poor basis for stem cell lines. They should be excluded. But how?

To answer this question, a team of scientists scrutinized skin fibroblasts, blood cells, and induced pluripotent stem cells (iPSCs) for mitochondrial genome integrity. When the scientists tested the samples for mitochondrial DNA mutations, the levels of mutations appeared low. But when the scientists sequenced the iPS cell lines, they found higher numbers of mitochondrial DNA mutations, particularly in cells from patients over 60.

The scientists were led by Shoukhrat Mitalipov, Ph.D., director of the Center for Embryonic Cell and Gene Therapy at Oregon Health & Science University, and Taosheng Huang, M.D., a medical geneticist and director of the Mitochondrial Medicine Program at Cincinnati Children’s Hospital. The Mitalipov/Huang-led team also found higher percentages of mitochondria containing mutations within a cell. The higher the load of mutated mitochondrial DNA in a cell, the more compromised the cell’s function.

Since each iPSC line is created from a different cell, each line may contain different types of mitochondrial DNA mutations and mutation loads. To choose the least damaged line, the authors recommend screening multiple lines per patient. “It’s a good idea to check the iPS clones for mitochondrial DNA mutations and make sure you pick a good cell line,” said Dr. Huang.

This recommendation appeared April 14 in the journal Cell Stem Cell, in an article entitled, “Age-Related Accumulation of Somatic Mitochondrial DNA Mutations in Adult-Derived Human iPSCs.” This article holds that mitochondrial genome integrity is a vital readout in assessing the proficiency of patient-derived regenerative products destined for clinical applications.

“We found that pooled skin and blood mtDNA contained low heteroplasmic point mutations, but a panel of ten individual iPSC lines from each tissue or clonally expanded fibroblasts carried an elevated load of heteroplasmic or homoplasmic mutations, suggesting that somatic mutations randomly arise within individual cells but are not detectable in whole tissues,” wrote the article’s authors. “The frequency of mtDNA defects in iPSCs increased with age, and many mutations were nonsynonymous or resided in RNA coding genes and thus can lead to respiratory defects.”

Potential therapies using stem cells hold tremendous promise for treating human disease. However, defects in the mitochondria could undermine the iPS cells’ ability to repair damaged tissue or organs.

“If you want to use iPS cells in a human, you must check for mutations in the mitochondrial genome,” declared Dr. Huang. “Every single cell can be different. Two cells next to each other could have different mutations or different percentages of mutations.”

Prior to the creation of a therapeutic iPS cell line, a collection of cells is taken from the patient. These cells will be tested for mutations. If the tester uses Sanger sequencing, older technology that is not as sensitive as newer next-generation sequencing, any mutation that occurs in less than 20% of the sample will go undetected. But mitochondrial DNA mutations might occur in less than 20% of mitochondria in the pooled cells. As a result, mutation rates have not been well understood. “These mitochondrial mutations are actually hidden,” explained Dr. Mitalipov.

The mitochondrial genome is relatively small, containing just 37 genes, so screening should be feasible using next generation sequencing, Dr. Mitalipov added. “It should be relatively cheap and do-able.”

Dr. Mitalipov also commented on a more general point, the implications of the current study on illuminating the mechanisms of age-related disease: “Pathogenic mutations in our mitochondrial DNA have long been thought to be a driving force in aging and age-onset diseases, though clear evidence was missing. This foundational knowledge of how cells are damaged in the natural process of aging may help to illuminate the role of mutated mitochondria in degenerative disease.”

New Mitalipov paper on stem cell mitochondria: challenge for IPS cell field?

A new paper from Shoukhrat Mitalipov’s lab on stem cell mitochondria points to a pattern whereby induced pluripotent stem (IPS) cells tend to have more problems if they are from older patients.

What does this paper mean for the stem cell field and could it impact more specifically the clinical applications of IPS cells?

Graphical Abstract, Kang, et al. 2016

The new paper Kang, et al is entitled “Age-Related Accumulation of Somatic Mitochondrial DNA Mutations in Adult-Derived Human iPSCs”.

This paper reminds us of the very important realities that mitochondria are key players in stem cell function and that mitochondria have their own genomes that impact that function. A lot of us don’t think about mitochondria and their genome as often as we should.

The paper came to three major scientific conclusions (this from the Highlights section of the paper and also see the graphical abstract for a visual sense of the results overall):

  • Human iPSC clones derived from elderly adults show accumulation of mtDNA mutations
  • Fewer mtDNA mutations are present in ESCs and iPSCs derived from younger adults
  • Accumulated mtDNA mutations can impact metabolic function in iPSCs

Importantly the team looked at IPS cells derived from both blood and skin cells and found that the former were less likely to have mitochondrial mutations.

This study suggests that those teams producing or working with human IPS cells (hIPSCs) should be screening the different lines for mitochondrial mutations. This excellent piece from Sara Reardon on the Mitalipov paper quotes IPS cell expert Jeanne Loring on this very point:

“It’s one of those things most of us don’t think about,” says Jeanne Loring, a stem-cell biologist at the Scripps Research Institute in La Jolla, California. Her lab is working towards using iPS cells to treat Parkinson’s disease, and Loring now plans to go back and examine the mitochondria in her cell lines. She suspects that it will be fairly easy for researchers to screen cells for use in therapies.”

Mitalipov goes further and suggests that his team’s new findings could support the use of human embryonic stem cells (hESC) derived by somatic cell nuclear transfer (SCNT) which would be expected to have mitochondria with fewer mutations. However, as Loring points out in the Reardon article, SCNT is really difficult to successfully perform and only a few labs in the world can do it at present. In that context, working with hIPSC and adding on the additional layer of mitochondrial DNA mutation screening could be more practical.

New York stem cell researcher Dieter Egli, however, is quoted that hIPSC have other differences with hESC as well such as epigenetic differences and he’s quoted in the Reardon piece, “It’s going to be very hard to find a cell line that’s perfect.”

One might reasonably ask both Egli and oneself, “What is a perfect cell line”?

In the end the best approach for use of human pluripotent stem cells of any kind is going to involve a balance between practicality of production and the potentially positive or negative traits of those cells as determined by rigorous validation screening.

With this new paper we’ve just learned more about another layer of screening that is needed. An interesting question is whether adult stem cells such as mesenchymal stromal/stem cells (MSC) also should be screened for mitochondrial mutations. They are often produced from patients who are getting up there in years. I hope that someone will publish on that too.

As to pluripotent cells, I expect that sometimes the best lines, meaning those most perfect for a given clinical application, will be hIPSC (autologous or allogeneic in some instances) and in other cases they may be hESC made from leftover IVF embryos. If SCNT-derived hESC can be more widely produced in an affordable manner and they pass validation as well then those (sometimes called NT-hESC) may also come into play clinically. So far that hasn’t happened for the SCNT cells, but it may over time.   …..

 Age-Related Accumulation of Somatic Mitochondrial DNA Mutations in Adult-Derived Human iPSCs

Eunju Kang, Xinjian Wang, Rebecca Tippner-Hedges, …, Don P. Wolf, Taosheng Huang, Shoukhrat Mitalipov

In Brief Mitalipov, Huang, and colleagues show that human iPSCs derived from older adults carry more mitochondrial DNA mutations than those derived from younger individuals. Defects in metabolic function caused by mtDNA mutations suggest careful screening of hiPSC clones for mutational load before clinical application.


  1. Human iPSC clones derived from elderly adults show accumulation of mtDNA mutations
  2. Fewer mtDNA mutations are present in ESCs and iPSCs derived from younger adults
  3. Accumulated mtDNA mutations can impact metabolic function in iPSCs

Kang et al., 2016, Cell Stem Cell 18, 1–12 May 5, 2016 ª2016 Elsevier Inc.

The genetic integrity of iPSCs is an important consideration for therapeutic application. In this study, we examine the accumulation of somatic mitochondrial genome (mtDNA) mutations in skin fibroblasts, blood, and iPSCs derived from young and elderly subjects (24–72 years). We found that pooled skin and blood mtDNA contained low heteroplasmic point mutations, but a panel of ten individual iPSC lines from each tissue or clonally expanded fibroblasts carried an elevated load of heteroplasmic or homoplasmic mutations, suggesting that somatic mutations randomly arise within individual cells but are not detectable in whole tissues. The frequency of mtDNA defects in iPSCs increased with age, and many mutations were non-synonymous or resided in RNA coding genes and thus can lead to respiratory defects. Our results highlight a need to monitor mtDNA mutations in iPSCs, especially those generated from older patients, and to examine the metabolic status of iPSCs destined for clinical applications.

Induced pluripotent stem cells (iPSCs) offer an unlimited source for autologous cell replacement therapies to treat age-associated degenerative diseases. Aging is generally characterized by increased DNA damage and genomic instability (Garinis et al., 2008; Lombard et al., 2005); thus, iPSCs derived from elderly subjects may harbor point mutations and larger genomic rearrangements. Indeed, iPSCs display increased chromosome aberrations (Mayshar et al., 2010), subchromosomal copy number variations (CNVs) (Abyzov et al., 2012; Laurent et al., 2011), and exome mutations (Johannesson et al., 2014), compared to natural embryonic stem cell (ESC) counterparts (Ma et al., 2014). The rate of mtDNA mutations is believed to be at least 10- to 20-fold higher than that observed in the nuclear genome (Wallace, 1994), and often both mutated and wild-type mtDNA (heteroplasmy) can coexist in the same cell (Rossignol et al., 2003). Large deletions are most frequently observed mtDNA abnormalities in aged post-mitotic tissues such as brain, heart, and muscle (Bender et al., 2006; Bua et al., 2006; Corral-Debrinski et al., 1992; Cortopassi et al., 1992; Mohamed et al., 2006) and have been implicated in aging and diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and diabetes (Larsson, 2010; Lin and Beal, 2006; Petersen et al., 2003; Wallace, 2005). In addition, mtDNA point mutations were reported in some tumors and replicating tissues (Chatterjee et al., 2006; Ju et al., 2014; Michikawa et al., 1999; Taylor et al., 2003). However, the extent of mtDNA defects in proliferating peripheral tissues commonly used for iPSC induction, such as skin and blood, is thought to be low and limited to common non-coding variants (Schon et al., 2012; Yao et al., 2015). Accumulation of mtDNA variants in these tissues with age was insignificant (Greaves et al., 2010; Hashizume et al., 2015). Several point mutations were identified in iPSCs generated from the newborn foreskin fibroblasts, although most of these variants were non-coding, common for the general population, and did not affect their metabolic activity (Prigione et al., 2011). Somatic mtDNA mutations may be under-reported secondary to the level of sample interrogation. …..

Figure 2. mtDNA Mutations in Skin Fibroblasts, Blood, and the iPSCs of a 72-YearOld B Subject (A) Sixteen mutations at low heteroplasmy levels were detected in the DNA of PF, while a panel of ten FiPSC lines carried nine mutations, including four that were homoplasmic. Gray rectangles define the mutations shared between PF and FiPSCs. (B) Venn diagram showing only one mutation in FiPSCs shared with PF. (C) All ten FiPSC lines carried between one and five high-heteroplasmy (>15%) mutations. (D) Mutation distribution in whole blood and BiPSCs was similar to that in PF and FiPSCs. Six mutations at low-heteroplasmy levels were observed in blood, while BiPSC lines displayed 21 mutations, including four over the 80% heteroplasmy level. (E) Venn diagram showing four mutations in BiPSCs shared with whole blood and the 17 novel variants. (F) Distribution of mutations in individual BiPSC lines. See also Figures S2 and S3; Table S1; Table S3, sheet 2; and Table S4, sheet 1   ….

Figure 4. Transmission and Distribution of Somatic mtDNA Mutations to iPSCs (A) A total of 112 mtDNA mutations were discovered in parental cells (PF, CF, and blood) from 11 subjects. Of these, 39 variants (35%) were found in corresponding 130 iPSC lines. Among non-transmitted, transmitted, and novel mutations in iPSCs, comparable percentages of variants (68%, 69%, and 79%, respectively) were coding mutations in protein, rRNA, or tRNA genes. This suggests that most pathogenic mutations do not affect iPSC induction. However, certain coding mutations including in ND3, ND4L, and 14 tRNA genes were not detected in iPSCs, suggesting possible pathogenicity. n, the number of mtDNA mutations. Blue font genes were detected in parental cells. (B–D) A total of 80 high heteroplasmic (>15%) variants were detected in the present study in 130 FiPSC or BiPSC lines from 11 subjects. (B) The majority of these variants (76%) were non-synonymous or frame-shift mutations in protein-coding genes or affected rRNA and tRNA genes. (C) More than half of the mutations (56%) were never reported in a database containing whole mtDNA sequences from 26,850 healthy subjects representing the general human population ( (D) Most mutations (90%) were never reported in a database containing sequences from healthy subjects with corresponding mtDNA haplotypes. freq., frequent. See also Figure S5 and Tables S3 and S4. ….


Mutations will accumulate over age in mitochondrial DNA, however the current study has the difficulty that the authors could not use patient-age-matched controls, in essence they could only compare induced pluripotent stem cells derived from different patients. This could confound the results but the result with higher frequency of mutation in mtDNA in cells reprogrammed from younger patients is interesting but might limit the ability of autologous regenerative therapy in older patients. However reprogramming, although the method not mentioned here although I am assuming by transfection with lentivirus is a rough procedure, involving multiple dedifferentiation steps. Therefore it is very understandable that cells obtained from elderly patients would respond less favorably to such a rough reprogramming regimen, especially if it produced a higher degree of ROS, which has been shown to alter mtDNA. This is why I feel it is more advantageous to obtain a stem cell population from fat cells and forgo the Oct4, htert, reprogramming with lentiviral vectors.


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Colon cancer and organoids

Larry H. Bernstein, MD, FCAP, Curator





Guts and Glory

An open mind and collaborative spirit have taken Hans Clevers on a journey from medicine to developmental biology, gastroenterology, cancer, and stem cells.

By Anna Azvolinsky

Ihave had to talk a lot about my science recently and it’s made me think about how science works,” says Hans Clevers. “Scientists are trained to think science is driven by hypotheses, but for [my lab], hypothesis-driven research has never worked. Instead, it has been about trying to be as open-minded as possible—which is not natural for our brains,” adds the Utrecht University molecular genetics professor. “The human mind is such that it tries to prove it’s right, so pursuing a hypothesis can result in disaster. My advice to my own team and others is to not preformulate an answer to a scientific question, but just observe and never be afraid of the unknown. What has worked well for us is to keep an open mind and do the experiments. And find a collaborator if it is outside our niche.”

“One thing I have learned is that hypothesis-driven research tends not to be productive when you are in an unknown territory.”

Clevers entered medical school at Utrecht University in The Netherlands in 1978 while simultaneously pursuing a master’s degree in biology. Drawn to working with people in the clinic, Clevers had a training position in pediatrics lined up after medical school, but then mentors persuaded him to spend an additional year converting the master’s degree to a PhD in immunology. “At the end of that year, looking back, I got more satisfaction from the research than from seeing patients.” Clevers also had an aptitude for benchwork, publishing four papers from his PhD year. “They were all projects I had made up myself. The department didn’t do the kind of research I was doing,” he says. “Now that I look back, it’s surprising that an inexperienced PhD student could come up with a project and publish independently.”

Clevers studied T- and B-cell signaling; he set up assays to visualize calcium ion flux and demonstrated that the ions act as messengers to activate human B cells, signaling through antibodies on the cell surface. “As soon as the experiment worked, I got T cells from the lab next door and did the same experiment. That was my strategy: as soon as something worked, I would apply it elsewhere and didn’t stop just because I was a B-cell biologist and not a T-cell biologist. What I learned then, that I have continued to benefit from, is that a lot of scientists tend to adhere to a niche. They cling to these niches and are not that flexible. You think scientists are, but really most are not.”

Here, Clevers talks about promoting a collaborative spirit in research, the art of doing a pilot experiment, and growing miniature organs in a dish.

Clevers Creates

Re-search? Clevers was born in Eindhoven, in the south of The Netherlands. The town was headquarters to Philips Electronics, where his father worked as a businessman, and his mother took care of Clevers and his three brothers. Clevers did well in school but his passion was sports, especially tennis and field hockey, “a big thing in Holland.” Then in 1975, at age 18, he moved to Utrecht University, where he entered an intensive, biology-focused program. “I knew I wanted to be a biology researcher since I was young. In Dutch, the word for research is ‘onderzoek’ and I knew the English word ‘research’ and had wondered why there was the ‘re’ in the word, because I wanted to search but I didn’t want to do re-search—to find what someone else had already found.”

Opportunity to travel. “I was very disappointed in my biology studies, which were old-fashioned and descriptive,” says Clevers. He thought medicine might be more interesting and enrolled in medical school while still pursuing a master’s degree in biology at Utrecht. For the master’s, Clevers had to do three rotations. He spent a year at the International Laboratory for Research on Animal Diseases (ILRAD) in Nairobi, Kenya, and six months in Bethesda, Maryland, at the National Institutes of Health. “Holland is really small, so everyone travels.” Clevers saw those two rotations more as travel explorations. In Nairobi, he went on safaris and explored the country in Land Rovers borrowed from the institute. While in Maryland in 1980, Clevers—with the consent of his advisor, who thought it was a good idea for him to get a feel for the U.S.—flew to Portland, Oregon, and drove back to Boston with a musician friend along the Canadian border. He met the fiancé of political activist and academic Angela Davis in New York City and even stayed in their empty apartment there.

Life and lab lessons. Back in Holland, Clevers joined Rudolf Eugène Ballieux’s lab at Utrecht University to pursue his PhD, for which he studied immune cell signaling. “I didn’t learn much science from him, but I learned that you always have to create trust and to trust people around you. This became a major theme in my own lab. We don’t distrust journals or reviewers or collaborators. We trust everyone and we share. There will be people who take advantage, but there have only been a few of those. So I learned from Ballieux to give everyone maximum trust and then change this strategy only if they fail that trust. We collaborate easily because we give out everything and we also easily get reagents and tools that we may need. It’s been valuable to me in my career. And it is fun!”

Clevers Concentrates

On a mission. “Once I decided to become a scientist, I knew I needed to train seriously. Up to that point, I was totally self-trained.” From an extensive reading of the immunology literature, Clevers became interested in how T cells recognize antigens, and headed off to spend a postdoc studying the problem in Cox Terhorst’s lab at Dana-Farber Cancer Institute in Boston. “Immunology was young, but it was very exciting and there was a lot to discover. I became a professional scientist there and experienced how tough science is.” In 1988, Clevers cloned and characterized the gene for a component of the T-cell receptor (TCR) called CD3-epsilon, which binds antigen and activates intracellular signaling pathways.

On the fast track in Holland. Clevers returned to Utrecht University in 1989 as a professor of immunology. Within one month of setting up his lab, he had two graduate students and a technician, and the lab had cloned the first T cell–specific transcription factor, which they called TCF-1, in human T cells. When his former thesis advisor retired, Clevers was asked, at age 33, to become head of the immunology department. While the appointment was high-risk for him and for the department, Clevers says, he was chosen because he was good at multitasking and because he got along well with everyone.

Problem-solving strategy. “My strategy in research has always been opportunistic. One thing I have learned is that hypothesis-driven research tends not to be productive when you are in an unknown territory. I think there is an art to doing pilot experiments. So we have always just set up systems in which something happens and then you try and try things until a pattern appears and maybe you formulate a small hypothesis. But as soon as it turns out not to be exactly right, you abandon it. It’s a very open-minded type of research where you question whether what you are seeing is a real phenomenon without spending a year on doing all of the proper controls.”

Trial and error. Clevers’s lab found that while TCF-1 bound to DNA, it did not alter gene expression, despite the researchers’ tinkering with promoter and enhancer assays. “For about five years this was a problem. My first PhD students were leaving and they thought the whole TCF project was a failure,” says Clevers. His lab meanwhile cloned TCF homologs from several model organisms and made many reagents including antibodies against these homologs. To try to figure out the function of TCF-1, the lab performed a two-hybrid screen and identified components of the Wnt signaling pathway as binding partners of TCF-1. “We started to read about Wnt and realized that you study Wnt not in T cells but in frogs and flies, so we rapidly transformed into a developmental biology lab. We showed that we held the key for a major issue in developmental biology, the final protein in the Wnt cascade: TCF-1 binds b-catenin when b-catenin becomes available and activates transcription.” In 1996, Clevers published the mechanism of how the TCF-1 homolog in Xenopus embryos, called XTcf-3, is integrated into the Wnt signaling pathway.

Clevers Catapults


3DCrypt building and colon cancer.

Clevers next collaborated with Bert Vogelstein’s lab at Johns Hopkins, linking TCF to Wnt signaling in colon cancer. In colon cancer cell lines with mutated forms of the tumor suppressor gene APC, the APC protein can’t rein in b-catenin, which accumulates in the cytoplasm, forms a complex with TCF-4 (later renamed TCF7L2) in the nucleus, and caninitiate colon cancer by changing gene expression. Then, the lab showed that Wnt signaling is necessary for self-renewal of adult stem cells, as mice missing TCF-4 do not have intestinal crypts, the site in the gut where stem cells reside. “This was the first time Wnt was shown to play a role in adults, not just during development, and to be crucial for adult stem cell maintenance,” says Clevers. “Then, when I started thinking about studying the gut, I realized it was by far the best way to study stem cells. And I also realized that almost no one in the world was studying the healthy gut. Almost everyone who researched the gut was studying a disease.” The main advantages of the murine model are rapid cell turnover and the presence of millions of stereotypic crypts throughout the entire intestine.

Against the grain. In 2007, Nick Barker, a senior scientist in the Clevers lab, identified the Wnt target gene Lgr5 as a unique marker of adult stem cells in several epithelial organs, including the intestine, hair follicle, and stomach. In the intestine, the gene codes for a plasma membrane protein on crypt stem cells that enable the intestinal epithelium to self-renew, but can also give rise to adenomas of the gut. Upon making mice with adult stem cell populations tagged with a fluorescent Lgr5-binding marker, the lab helped to overturn assumptions that “stem cells are rare, impossible to find, quiescent, and divide asymmetrically.”

On to organoids. Once the lab could identify adult stem cells within the crypts of the gut, postdoc Toshiro Sato discovered that a single stem cell, in the presence of Matrigel and just three growth factors, could generate a miniature crypt structure—what is now called an organoid. “Toshi is very Japanese and doesn’t always talk much,” says Clevers. “One day I had asked him, while he was at the microscope, if the gut stem cells were growing, and he said, ‘Yes.’ Then I looked under the microscope and saw the beautiful structures and said, ‘Why didn’t you tell me?’ and he said, ‘You didn’t ask.’ For three months he had been growing them!” The lab has since also grown mini-pancreases, -livers, -stomachs, and many other mini-organs.

Tumor Organoids. Clevers showed that organoids can be grown from diseased patients’ samples, a technique that could be used in the future to screen drugs. The lab is also building biobanks of organoidsderived from tumor samples and adjacent normal tissue, which could be especially useful for monitoring responses to chemotherapies. “It’s a similar approach to getting a bacterium cultured to identify which antibiotic to take. The most basic goal is not to give a toxic chemotherapy to a patient who will not respond anyway,” says Clevers. “Tumor organoids grow slower than healthy organoids, which seems counterintuitive, but with cancer cells, often they try to divide and often things go wrong because they don’t have normal numbers of chromosomes and [have] lots of mutations. So, I am not yet convinced that this approach will work for every patient. Sometimes, the tumor organoids may just grow too slowly.”

Selective memory. “When I received the Breakthrough Prize in 2013, I invited everyone who has ever worked with me to Amsterdam, about 100 people, and the lab organized a symposium where many of the researchers gave an account of what they had done in the lab,” says Clevers. “In my experience, my lab has been a straight line from cloning TCF-1 to where we are now. But when you hear them talk it was ‘Hans told me to try this and stop this’ and ‘Half of our knockout mice were never published,’ and I realized that the lab is an endless list of failures,” Clevers recalls. “The one thing we did well is that we would start something and, as soon as it didn’t look very good, we would stop it and try something else. And the few times when we seemed to hit gold, I would regroup my entire lab. We just tried a lot of things, and the 10 percent of what worked, those are the things I remember.”

Greatest Hits

  • Cloned the first T cell–specific transcription factor, TCF-1, and identified homologous genes in model organisms including the fruit fly, frog, and worm
  • Found that transcriptional activation by the abundant β-catenin/TCF-4 [TCF7L2] complex drives cancer initiation in colon cells missing the tumor suppressor protein APC
  • First to extend the role of Wnt signaling from developmental biology to adult stem cells by showing that the two Wnt pathway transcription factors, TCF-1 and TCF-4, are necessary for maintaining the stem cell compartments in the thymus and in the crypt structures of the small intestine, respectively
  • Identified Lgr5 as an adult stem cell marker of many epithelial stem cells including those of the colon, small intestine, hair follicle, and stomach, and found that Lgr5-expressing crypt cells in the small intestine divide constantly and symmetrically, disproving the common belief that stem cell division is asymmetrical and uncommon
  • Established a three-dimensional, stable model, the “organoid,” grown from adult stem cells, to study diseased patients’ tissues from the gut, stomach, liver, and prostate
 Regenerative Medicine Comes of Age   
“Anti-Aging Medicine” Sounds Vaguely Disreputable, So Serious Scientists Prefer to Speak of “Regenerative Medicine”
  • Induced pluripotent stem cells (iPSCs) and genome-editing techniques have facilitated manipulation of living organisms in innumerable ways at the cellular and genetic levels, respectively, and will underpin many aspects of regenerative medicine as it continues to evolve.

    An attitudinal change is also occurring. Experts in regenerative medicine have increasingly begun to embrace the view that comprehensively repairing the damage of aging is a practical and feasible goal.

    A notable proponent of this view is Aubrey de Grey, Ph.D., a biomedical gerontologist who has pioneered an regenerative medicine approach called Strategies for Engineered Negligible Senescence (SENS). He works to “develop, promote, and ensure widespread access to regenerative medicine solutions to the disabilities and diseases of aging” as CSO and co-founder of the SENS Research Foundation. He is also the editor-in-chief of Rejuvenation Research, published by Mary Ann Liebert.

    Dr. de Grey points out that stem cell treatments for age-related conditions such as Parkinson’s are already in clinical trials, and immune therapies to remove molecular waste products in the extracellular space, such as amyloid in Alzheimer’s, have succeeded in such trials. Recently, there has been progress in animal models in removing toxic cells that the body is failing to kill. The most encouraging work is in cancer immunotherapy, which is rapidly advancing after decades in the doldrums.

    Many damage-repair strategies are at an  early stage of research. Although these strategies look promising, they are handicapped by a lack of funding. If that does not change soon, the scientific community is at risk of failing to capitalize on the relevant technological advances.

    Regenerative medicine has moved beyond boutique applications. In degenerative disease, cells lose their function or suffer elimination because they harbor genetic defects. iPSC therapies have the potential to be curative, replacing the defective cells and eliminating symptoms in their entirety. One of the biggest hurdles to commercialization of iPSC therapies is manufacturing.

  • Building Stem Cell Factories

    Cellular Dynamics International (CDI) has been developing clinically compatible induced pluripotent stem cells (iPSCs) and iPSC-derived human retinal pigment epithelial (RPE) cells. CDI’s MyCell Retinal Pigment Epithelial Cells are part of a possible therapy for macular degeneration. They can be grown on bioengineered, nanofibrous scaffolds, and then the RPE cell–enriched scaffolds can be transplanted into patients’ eyes. In this pseudo-colored image, RPE cells are shown growing over the nanofibers. Each cell has thousands of “tongue” and “rod” protrusions that could naturally support rod and cone cells in the eye.

    “Now that an infrastructure is being developed to make unlimited cells for the tools business, new opportunities are being created. These cells can be employed in a therapeutic context, and they can be used to understand the efficacy and safety of drugs,” asserts Chris Parker, executive vice president and CBO, Cellular Dynamics International (CDI). “CDI has the capability to make a lot of cells from a single iPSC line that represents one person (a capability termed scale-up) as well as the capability to do it in parallel for multiple individuals (a capability termed scale-out).”

    Minimally manipulated adult stem cells have progressed relatively quickly to the clinic. In this scenario, cells are taken out of the body, expanded unchanged, then reintroduced. More preclinical rigor applies to potential iPSC therapy. In this case, hematopoietic blood cells are used to make stem cells, which are manufactured into the cell type of interest before reintroduction. Preclinical tests must demonstrate that iPSC-derived cells perform as intended, are safe, and possess little or no off-target activity.

    For example, CDI developed a Parkinsonian model in which iPSC-derived dopaminergic neurons were introduced to primates. The model showed engraftment and enervation, and it appeared to be free of proliferative stem cells.

    • “You will see iPSCs first used in clinical trials as a surrogate to understand efficacy and safety,” notes Mr. Parker. “In an ongoing drug-repurposing trial with GlaxoSmithKline and Harvard University, iPSC-derived motor neurons will be produced from patients with amyotrophic lateral sclerosis and tested in parallel with the drug.” CDI has three cell-therapy programs in their commercialization pipeline focusing on macular degeneration, Parkinson’s disease, and postmyocardial infarction.

    • Keeping an Eye on Aging Eyes

      The California Project to Cure Blindness is evaluating a stem cell–based treatment strategy for age-related macular degeneration. The strategy involves growing retinal pigment epithelium (RPE) cells on a biostable, synthetic scaffold, then implanting the RPE cell–enriched scaffold to replace RPE cells that are dying or dysfunctional. One of the project’s directors, Dennis Clegg, Ph.D., a researcher at the University of California, Santa Barbara, provided this image, which shows stem cell–derived RPE cells. Cell borders are green, and nuclei are red.

      The eye has multiple advantages over other organ systems for regenerative medicine. Advanced surgical methods can access the back of the eye, noninvasive imaging methods can follow the transplanted cells, good outcome parameters exist, and relatively few cells are needed.

      These advantages have attracted many groups to tackle ocular disease, in particular age-related macular degeneration, the leading cause of blindness in the elderly in the United States. Most cases of age-related macular degeneration are thought to be due to the death or dysfunction of cells in the retinal pigment epithelium (RPE). RPE cells are crucial support cells for the rods, cones, and photoreceptors. When RPE cells stop working or die, the photoreceptors die and a vision deficit results.

      A regenerated and restored RPE might prevent the irreversible loss of photoreceptors, possibly via the the transplantation of functionally polarized RPE monolayers derived from human embryonic stem cells. This approach is being explored by the California Project to Cure Blindness, a collaborative effort involving the University of Southern California (USC), the University of California, Santa Barbara (UCSB), the California Institute of Technology, City of Hope, and Regenerative Patch Technologies.

      The project, which is funded by the California Institute of Regenerative Medicine (CIRM), started in 2010, and an IND was filed early 2015. Clinical trial recruitment has begun.

      One of the project’s leaders is Dennis Clegg, Ph.D., Wilcox Family Chair in BioMedicine, UCSB. His laboratory developed the protocol to turn undifferentiated H9 embryonic stem cells into a homogenous population of RPE cells.

      “These are not easy experiments,” remarks Dr. Clegg. “Figuring out the biology and how to make the cell of interest is a challenge that everyone in regenerative medicine faces. About 100,000 RPE cells will be grown as a sheet on a 3 × 5 mm biostable, synthetic scaffold, and then implanted in the patients to replace the cells that are dying or dysfunctional. The idea is to preserve the photoreceptors and to halt disease progression.”

      Moving therapies such as this RPE treatment from concept to clinic is a huge team effort and requires various kinds of expertise. Besides benefitting from Dr. Clegg’s contribution, the RPE project incorporates the work of Mark Humayun, M.D., Ph.D., co-director of the USC Eye Institute and director of the USC Institute for Biomedical Therapeutics and recipient of the National Medal of Technology and Innovation, and David Hinton, Ph.D., a researcher at USC who has studied how actvated RPE cells can alter the local retinal microenvironment.

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3D revolution and tissue repair

Curator: Larry H. Bernstein, MD, FCAP



Berkeley Lab captures first high-res 3D images of DNA segments

DNA segments are targeted to be building blocks for molecular computer memory and electronic devices, nanoscale drug-delivery systems, and as markers for biological research and imaging disease-relevant proteins

In a Berkeley Lab-led study, flexible double-helix DNA segments (purple, with green DNA models) connected to gold nanoparticles (yellow) are revealed from the 3D density maps reconstructed from individual samples using a Berkeley Lab-developed technique called individual-particle electron tomography (IPET). Projections of the structures are shown in the green background grid. (credit: Berkeley Lab)

An international research team working at the Lawrence Berkeley National Laboratory (Berkeley Lab) has captured the first high-resolution 3D images of double-helix DNA segments attached at either end to gold nanoparticles — which could act as building blocks for molecular computer memory and electronic devices (see World’s smallest electronic diode made from single DNA molecule), nanoscale drug-delivery systems, and as markers for biological research and for imaging disease-relevant proteins.

The researchers connected coiled DNA strands between polygon-shaped gold nanoparticles and then reconstructed 3D images, using a cutting-edge electron microscope technique coupled with a protein-staining process and sophisticated software that provided structural details at the scale of about 2 nanometers.

“We had no idea about what the double-strand DNA would look like between the gold nanoparticles,” said Gang “Gary” Ren, a Berkeley Lab scientist who led the research. “This is the first time for directly visualizing an individual double-strand DNA segment in 3D,” he said.

The results were published in an open-access paper in the March 30 edition of Nature Communications.

The method developed by this team, called individual-particle electron tomography (IPET), had earlier captured the 3-D structure of a single protein that plays a key role in human cholesterol metabolism. By grabbing 2D images of an object from different angles, the technique allows researchers to assemble a 3D image of that object.

The team has also used the technique to uncover the fluctuation of another well-known flexible protein, human immunoglobulin 1, which plays a role in the human immune system.
Berkeley Lab | 3-D Reconstructions of Double strand DNA and Gold Nanoparticle Structures

For this new study of DNA nanostructures, Ren used an electron-beam study technique called cryo-electron microscopy (cryo-EM) to examine frozen DNA-nanogold samples, and used IPET to reconstruct 3-D images from samples stained with heavy metal salts. The team also used molecular simulation tools to test the natural shape variations (“conformations”) in the samples, and compared these simulated shapes with observations.

First visualization of DNA strand dynamics without distorting x-ray crystallography

Ren explained that the naturally flexible dynamics of samples, like a man waving his arms, cannot be fully detailed by any method that uses an average of many observations.

A popular way to view the nanoscale structural details of delicate biological samples is to form them into crystals and zap them with X-rays, but that destroys their natural shape, especially fir the DNA-nanogold samples in this study, which the scientists say are incredibly challenging to crystallize. Other common research techniques may require a collection of thousands of near-identical objects, viewed with an electron microscope, to compile a single, averaged 3-D structure. But an averaged 3D image may not adequately show the natural shape fluctuations of a given object.

The samples in the latest experiment were formed from individual polygon gold nanostructures, measuring about 5 nanometers across, connected to single DNA-segment strands with 84 base pairs. Base pairs are basic chemical building blocks that give DNA its structure. Each individual DNA segment and gold nanoparticle naturally zipped together with a partner to form the double-stranded DNA segment with a gold particle at either end.
Berkeley Lab | These views compare the various shape fluctuations obtained from different samples of the same type of double-helix DNA segment (DNA renderings in green, 3D reconstructions in purple) connected to gold nanoparticles (yellow).

The samples were flash-frozen to preserve their structure for study with cryo-EM imaging. The distance between the two gold nanoparticles in individual samples varied from 20 to 30 nanometers, based on different shapes observed in the DNA segments.

Researchers used a cryo-electron microscope at Berkeley Lab’s Molecular Foundry for this study. They collected a series of tilted images of the stained objects, and reconstructed 14 electron-density maps that detailed the structure of individual samples using the IPET technique.

Sub-nanometer images next

Ren said that the next step will be to work to improve the resolution to the sub-nanometer scale.

“Even in this current state we begin to see 3-D structures at 1- to 2-nanometer resolution,” he said. “Through better instrumentation and improved computational algorithms, it would be promising to push the resolution to that visualizing a single DNA helix within an individual protein.”

In future studies, researchers could attempt to improve the imaging resolution for complex structures that incorporate more DNA segments as a sort of “DNA origami,” Ren said. Researchers hope to build and better characterize nanoscale molecular devices using DNA segments that can, for example, store and deliver drugs to targeted areas in the body.

“DNA is easy to program, synthesize and replicate, so it can be used as a special material to quickly self-assemble into nanostructures and to guide the operation of molecular-scale devices,” he said. “Our current study is just a proof of concept for imaging these kinds of molecular devices’ structures.”

The team included researchers at UC Berkeley, the Kavli Energy NanoSciences Institute at Berkeley Lab and UC Berkeley, and Xi’an Jiaotong University in China. This work was supported by the National Science Foundation, DOE Office of Basic Energy Sciences, National Institutes of Health, the National Natural Science Foundation of China, Xi’an Jiaotong University in China, and the Ministry of Science and Technology in China. View more about Gary Ren’s research group here.

Abstract of Three-dimensional structural dynamics and fluctuations of DNA-nanogold conjugates by individual-particle electron tomography

DNA base pairing has been used for many years to direct the arrangement of inorganic nanocrystals into small groupings and arrays with tailored optical and electrical properties. The control of DNA-mediated assembly depends crucially on a better understanding of three-dimensional structure of DNA-nanocrystal-hybridized building blocks. Existing techniques do not allow for structural determination of these flexible and heterogeneous samples. Here we report cryo-electron microscopy and negative-staining electron tomography approaches to image, and three-dimensionally reconstruct a single DNA-nanogold conjugate, an 84-bp double-stranded DNA with two 5-nm nanogold particles for potential substrates in plasmon-coupling experiments. By individual-particle electron tomography reconstruction, we obtain 14 density maps at ~2-nm resolution. Using these maps as constraints, we derive 14 conformations of dsDNA by molecular dynamics simulations. The conformational variation is consistent with that from liquid solution, suggesting that individual-particle electron tomography could be an expected approach to study DNA-assembling and flexible protein structure and dynamics.


World’s smallest electronic diode made from single DNA molecule

Electronic components 1,000 times smaller than with silicon may be possible
By inserting a small “coralyne” molecule into DNA, scientists were able to create a single-molecule diode (connected here by two gold electrodes), which can be used as an active element in future nanoscale circuits. The diode circuit symbol is shown on the left. (credit: University of Georgia and Ben-Gurion University)

Nanoscale electronic components can be made from single DNA molecules, as researchers at the University of Georgia and at Ben-Gurion University in Israel have demonstrated, using a single molecule of DNA to create the world’s smallest diode.

DNA double helix with base pairs (credit: National Human Genome Research Institute)

A diode is a component vital to electronic devices that allows current to flow in one direction but prevents its flow in the other direction. The development could help stimulate development of DNA components for molecular electronics.

As noted in an open-access Nature Chemistry paper published this week, the researchers designed a 11-base-pair (bp) DNA molecule and inserted a small molecule named coralyne into the DNA.*

They found, surprisingly, that this caused the current flowing through the DNA to be 15 times stronger for negative voltages than for positive voltages, a necessary feature of a diode.

Electronic elements 1,00o times smaller than current components

“Our discovery can lead to progress in the design and construction of nanoscale electronic elements that are at least 1,000 times smaller than current components,” says the study’s lead author, Bingqian Xu an associate professor in the UGA College of Engineering and an adjunct professor in chemistry and physics.

The research team plans to enhance the performance of the molecular diode and construct additional molecular devices, which may include a transistor (similar to a two-layer diode, but with one additional layer).

A theoretical model developed by Yanantan Dubi of Ben-Gurion University indicated the diode-like behavior of DNA originates from the bias voltage-induced breaking of spatial symmetry inside the DNA molecule after the coralyne is inserted.

The research is supported by the National Science Foundation.

*“We prepared the DNA–coralyne complex by specifically intercalating two coralyne molecules into a custom-designed 11-base-pair (bp) DNA molecule (5′-CGCGAAACGCG-3′) containing three mismatched A–A base pairs at the centre,” according to the authors.

UPDATE April 6, 2016 to clarify the coralyne intercalation (insertion) into the DNA molecule.

Abstract of Molecular rectifier composed of DNA with high rectification ratio enabled by intercalation

The predictability, diversity and programmability of DNA make it a leading candidate for the design of functional electronic devices that use single molecules, yet its electron transport properties have not been fully elucidated. This is primarily because of a poor understanding of how the structure of DNA determines its electron transport. Here, we demonstrate a DNA-based molecular rectifier constructed by site-specific intercalation of small molecules (coralyne) into a custom-designed 11-base-pair DNA duplex. Measured current–voltage curves of the DNA–coralyne molecular junction show unexpectedly large rectification with a rectification ratio of about 15 at 1.1 V, a counter-intuitive finding considering the seemingly symmetrical molecular structure of the junction. A non-equilibrium Green’s function-based model—parameterized by density functional theory calculations—revealed that the coralyne-induced spatial asymmetry in the electron state distribution caused the observed rectification. This inherent asymmetry leads to changes in the coupling of the molecular HOMO−1 level to the electrodes when an external voltage is applied, resulting in an asymmetric change in transmission.


A stem-cell repair system that can regenerate any kind of human tissue …including disease and aging; human trials next year

UNSW researchers say the therapy has enormous potential for treating spinal disc injury and joint and muscle degeneration and could also speed up recovery following complex surgeries where bones and joints need to integrate with the body (credit: UNSW TV)

A stem cell therapy system capable of regenerating any human tissue damaged by injury, disease, or aging could be available within a few years, say University of New South Wales (UNSW Australia) researchers.

Their new repair system*, similar to the method used by salamanders to regenerate limbs, could be used to repair everything from spinal discs to bone fractures, and could transform current treatment approaches to regenerative medicine.

The UNSW-led research was published this week in the Proceedings of the National Academy of Sciences journal.

Reprogramming bone and fat cells

The system reprograms bone and fat cells into induced multipotent stem cells (iMS), which can regenerate multiple tissue types and has been successfully demonstrated in mice, according to study lead author, haematologist, and UNSW Associate Professor John Pimanda.

“This technique is a significant advance on many of the current unproven stem cell therapies, which have shown little or no objective evidence they contribute directly to new tissue formation,” Pimanda said. “We have taken bone and fat cells, switched off their memory and converted them into stem cells so they can repair different cell types once they are put back inside the body.”

“We are currently assessing whether adult human fat cells reprogrammed into iMS cells can safely repair damaged tissue in mice, with human trials expected to begin in late 2017.”

Advantages over stem-cell types

There are different types of stem cells including embryonic stem (ES) cells, which during embryonic development generate every type of cell in the human body, and adult stem cells, which are tissue-specific, but don’t regenerate multiple tissue types. Embryonic stem cells cannot be used to treat damaged tissues because of their tumor forming capacity. The other problem when generating stem cells is the requirement to use viruses to transform cells into stem cells, which is clinically unacceptable, the researchers note.

Research shows that up to 20% of spinal implants either don’t heal or there is delayed healing. The rates are higher for smokers, older people and patients with diseases such diabetes or kidney disease.

Human trials are planned next year once the safety and effectiveness of the technique using human cells in mice has been demonstrated.

* The technique involves extracting adult human fat cells and treating them with the compound 5-Azacytidine (AZA), along with platelet-derived growth factor-AB (PDGF-AB) for about two days. The cells are then treated with the growth factor alone for a further two-three weeks.

AZA is known to induce cell plasticity, which is crucial for reprogramming cells. The AZA compound relaxes the hard-wiring of the cell, which is expanded by the growth factor, transforming the bone and fat cells into iMS cells. When the stem cells are inserted into the damaged tissue site, they multiply, promoting growth and healing.

The new technique is similar to salamander limb regeneration, which is also dependent on the plasticity of differentiated cells, which can repair multiple tissue types, depending on which body part needs replacing.

Along with confirming that human adult fat cells reprogrammed into iMS stem cells can safely repair damaged tissue in mice, the researchers said further work is required to establish whether iMS cells remain dormant at the sites of transplantation and retain their capacity to proliferate on demand.

Abstract of PDGF-AB and 5-Azacytidine induce conversion of somatic cells into tissue-regenerative multipotent stem cells

Current approaches in tissue engineering are geared toward generating tissue-specific stem cells. Given the complexity and heterogeneity of tissues, this approach has its limitations. An alternate approach is to induce terminally differentiated cells to dedifferentiate into multipotent proliferative cells with the capacity to regenerate all components of a damaged tissue, a phenomenon used by salamanders to regenerate limbs. 5-Azacytidine (AZA) is a nucleoside analog that is used to treat preleukemic and leukemic blood disorders. AZA is also known to induce cell plasticity. We hypothesized that AZA-induced cell plasticity occurs via a transient multipotent cell state and that concomitant exposure to a receptive growth factor might result in the expansion of a plastic and proliferative population of cells. To this end, we treated lineage-committed cells with AZA and screened a number of different growth factors with known activity in mesenchyme-derived tissues. Here, we report that transient treatment with AZA in combination with platelet-derived growth factor–AB converts primary somatic cells into tissue-regenerative multipotent stem (iMS) cells. iMS cells possess a distinct transcriptome, are immunosuppressive, and demonstrate long-term self-renewal, serial clonogenicity, and multigerm layer differentiation potential. Importantly, unlike mesenchymal stem cells, iMS cells contribute directly to in vivo tissue regeneration in a context-dependent manner and, unlike embryonic or pluripotent stem cells, do not form teratomas. Taken together, this vector-free method of generating iMS cells from primary terminally differentiated cells has significant scope for application in tissue regeneration.


First transistors made entirely of nanocrystal ‘inks’ in simplified process

Transistors and other electronic components to be built into flexible or wearable applications; 3D printing planned
Because this process works at relatively low temperatures, many transistors can be made on a flexible backing at once. (credit: University of Pennsylvania)

University of Pennsylvania engineers have developed a simplified new approach for making transistors by sequentially depositing their components in the form of liquid nanocrystal “inks.” The new process open the door for transistors and other electronic components to be built into flexible or wearable applications. It also avoids the highly complex current process for creating transistors, which requires high-temperature, high-vacuum equipment. Also, the new lower-temperature process is compatible with a wide array of materials and can be applied to larger areas.

Transistors patterned on plastic backing

The researchers’ nanocrystal-based field effect transistors were patterned onto flexible plastic backings using spin coating, but could eventually be constructed by additive manufacturing systems, like 3D printers.

Published in the journal Science,  the study was lead by Cherie Kagan, the Stephen J. Angello Professor in the School of Engineering and Applied Science, and Ji-Hyuk Choi, then a member of her lab, now a senior researcher at the Korea Institute of Geoscience and Mineral Resources. Researchers at Korea University Korea’s Yonsei University were also involved.


Kagan’s group developed four nanocrystal inks that comprise the transistor, then deposited them on a flexible backing. (credit: University of Pennsylvania)

The researchers began by dispersing a specific type of nanocrystals in a liquid, creating nanocrystal inks. They developed a library of four of these inks: a conductor (silver), an insulator (aluminum oxide), a semiconductor (cadmium selenide), and a conductor combined with a dopant (a mixture of silver and indium). (“Doping” the semiconductor layer of a transistor with impurities controls whether the device creates a positive or negative charge.)

“These materials are colloids just like the ink in your inkjet printer,” Kagan said, “but you can get all the characteristics that you want and expect from the analogous bulk materials, such as whether they’re conductors, semiconductors or insulators.” Although the electrical properties of several of these nanocrystal inks had been independently verified, they had never been combined into full devices. “Our question was whether you could lay them down on a surface in such a way that they work together to form functional transistors.”

Laying down patterns in layers

Such a process entails layering or mixing them in precise patterns.

First, the conductive silver nanocrystal ink was deposited from liquid on a flexible plastic surface that was treated with a photolithographic mask, then rapidly spun to draw it out in an even layer. The mask was then removed to leave the silver ink in the shape of the transistor’s gate electrode.

The researchers followed that layer by spin-coating a layer of the aluminum oxide nanocrystal-based insulator, then a layer of the cadmium selenide nanocrystal-based semiconductor and finally another masked layer for the indium/silver mixture, which forms the transistor’s source and drain electrodes. Upon heating at relatively low temperatures, the indium dopant diffused from those electrodes into the semiconductor component.

“The trick with working with solution-based materials is making sure that, when you add the second layer, it doesn’t wash off the first, and so on,” Kagan said. “We had to treat the surfaces of the nanocrystals, both when they’re first in solution and after they’re deposited, to make sure they have the right electrical properties and that they stick together in the configuration we want.”

Because this entirely ink-based fabrication process works at lower temperatures than existing vacuum-based methods, the researchers were able to make several transistors on the same flexible plastic backing at the same time.


The inks’ specialized surface chemistry allowed them to stay in configuration without losing their electrical properties. (credit: University of Pennsylvania)

“Making transistors over larger areas and at lower temperatures have been goals for an emerging class of technologies, when people think of the Internet of things, large area flexible electronics and wearable devices,” Kagan said. “We haven’t developed all of the necessary aspects so they could be printed yet, but because these materials are all solution-based, it demonstrates the promise of this materials class and sets the stage for additive manufacturing.”

Because this entirely ink-based fabrication process works at lower temperatures than existing vacuum-based methods, the researchers were able to make several transistors on the same flexible plastic backing at the same time.

3D-printing transistors for wearables

“This is the first work,” Choi said, “showing that all the components, the metallic, insulating, and semiconducting layers of the transistors, and even the doping of the semiconductor, could be made from nanocrystals.”

“Making transistors over larger areas and at lower temperatures have been goals for an emerging class of technologies, when people think of the Internet of things, large area flexible electronics and wearable devices,” Kagan said. “We haven’t developed all of the necessary aspects so they could be printed yet, but because these materials are all solution-based, it demonstrates the promise of this materials class and sets the stage for additive manufacturing.”

The research was supported by the National Science Foundation, the U.S. Department of Energy, the Office of Naval Research, and the Korea Institute of Geoscience and Mineral Resources funded by the Ministry of Science, ICT, and Future Planning of Korea.

Abstract of Exploiting the colloidal nanocrystal library to construct electronic devices

Synthetic methods produce libraries of colloidal nanocrystals with tunable physical properties by tailoring the nanocrystal size, shape, and composition. Here, we exploit colloidal nanocrystal diversity and design the materials, interfaces, and processes to construct all-nanocrystal electronic devices using solution-based processes. Metallic silver and semiconducting cadmium selenide nanocrystals are deposited to form high-conductivity and high-mobility thin-film electrodes and channel layers of field-effect transistors. Insulating aluminum oxide nanocrystals are assembled layer by layer with polyelectrolytes to form high–dielectric constant gate insulator layers for low-voltage device operation. Metallic indium nanocrystals are codispersed with silver nanocrystals to integrate an indium supply in the deposited electrodes that serves to passivate and dope the cadmium selenide nanocrystal channel layer. We fabricate all-nanocrystal field-effect transistors on flexible plastics with electron mobilities of 21.7 square centimeters per volt-second.

Best textile manufacturing methods for creating human tissues with stem cells
Bioengineers determine three best processes for engineering tissues needed for organ and tissue repair
All four textile manufacturing processes and corresponding scaffold (structure) types studied exhibited the presence of lipid vacuoles (small red spheres, right column, indicating stem cells undergoing random differentiation), compared to control (left). Electrospun scaffolds (row a) exhibited only a monolayer of lipid vacuoles in a single focal plane, while meltblown, spunbond, and carded scaffolds (rows b, c, d) exhibited vacuoles in multiple planes throughout the fabric thickness. Scale bars: 100 μm (credit: S. A. Tuin et al./Biomedical Materials)

Elizabeth Loboa, dean of the Missouri University College of Engineering, and her team have tested new tissue- engineering methods (based on textile manufacturing) to find ones that are most cost-effective and can be produced in larger quantities.

Tissue engineering is a process that uses novel biomaterials seeded with stem cells to grow and replace missing tissues. When certain types of materials are used, the “scaffolds” that are created to hold stem cells eventually degrade, leaving natural tissue in its place. The new tissues could help patients suffering from wounds caused by diabetes and circulation disorders, patients in need of cartilage or bone repair, and women who have had mastectomies by replacing their breast tissue. The challenge is creating enough of the material on a scale that clinicians need to treat patients.

Comparing textile manufacturing techniques

Electrospinning experiment: nanofibers are collected into an ethanol bath and removed at predefined time intervals (credit: J. M. Coburn et al./The Johns Hopkins University/PNAS)

In typical tissue engineering approaches that use fibers as scaffolds, non-woven materials are often bonded together using an electrostatic field. This process, called electrospinning (see Nanoscale scaffolds and stem cells show promise in cartilage repair and Improved artificial blood vessels), creates the scaffolds needed to attach to stem cells.

However, large-scale production with electrospinning is not cost-effective. “Electrospinning produces weak fibers, scaffolds that are not consistent, and pores that are too small,” Loboa said. “The goal of ‘scaling up’ is to produce hundreds of meters of material that look the same, have the same properties, and can be used in clinical settings. So we investigated the processes that create textiles, such as clothing and window furnishings like drapery, to scale up the manufacturing process.”

The group published two papers using three industry-standard, high-throughput manufacturing techniques — meltblowing, spunbonding, and carding — to determine if they would create the materials needed to mimic native tissue.

Meltblowing is a technique during which nonwoven materials are created using a molten polymer to create continuous fibers. Spunbond materials are made much the same way but the fibers are drawn into a web while in a solid state instead of a molten one. Carding involves the separation of fibers through the use of rollers, forming the web needed to hold stem cells in place.

Schematic of gilled fiber multifilament spinning and carded scaffold fabrication (credit: Stephen A. Tuin et al./Acta Biomaterialia)

Cost-effective methods

Loboa and her colleagues tested these techniques to create polylactic acid (PLA) scaffolds (a Food and Drug Administration-approved material used as collagen fillers), seeded with human stem cells. They then spent three weeks studying whether the stem cells remained healthy and if they began to differentiate into fat and bone pathways, which is the goal of using stem cells in a clinical setting when new bone and/or new fat tissue is needed at a defect site. Results showed that the three textile manufacturing methods proved as viable if not more so than electrospinning.

“These alternative methods are more cost-effective than electrospinning,” Loboa said. “A small sample of electrospun material could cost between $2 to $5. The cost for the three manufacturing methods is between $.30 to $3.00; these methods proved to be effective and efficient. Next steps include testing how the different scaffolds created in the three methods perform once implanted in animals.”

Researchers at North Carolina State University and the University of North Carolina at Chapel Hill were also involved in the two studies, which were published in Biomedical Materials (open access) and Acta Biomaterialia. The National Science Foundation, the National Institutes of Health, and the Nonwovens Institute provided funding for the studies.

Abstract of Creating tissues from textiles: scalable nonwoven manufacturing techniques for fabrication of tissue engineering scaffolds

Electrospun nonwovens have been used extensively for tissue engineering applications due to their inherent similarities with respect to fibre size and morphology to that of native extracellular matrix (ECM). However, fabrication of large scaffold constructs is time consuming, may require harsh organic solvents, and often results in mechanical properties inferior to the tissue being treated. In order to translate nonwoven based tissue engineering scaffold strategies to clinical use, a high throughput, repeatable, scalable, and economic manufacturing process is needed. We suggest that nonwoven industry standard high throughput manufacturing techniques (meltblowing, spunbond, and carding) can meet this need. In this study, meltblown, spunbond and carded poly(lactic acid) (PLA) nonwovens were evaluated as tissue engineering scaffolds using human adipose derived stem cells (hASC) and compared to electrospun nonwovens. Scaffolds were seeded with hASC and viability, proliferation, and differentiation were evaluated over the course of 3 weeks. We found that nonwovens manufactured via these industry standard, commercially relevant manufacturing techniques were capable of supporting hASC attachment, proliferation, and both adipogenic and osteogenic differentiation of hASC, making them promising candidates for commercialization and translation of nonwoven scaffold based tissue engineering strategies.

Abstract of Fabrication of novel high surface area mushroom gilled fibers and their effects on human adipose derived stem cells under pulsatile fluid flow for tissue engineering applications

The fabrication and characterization of novel high surface area hollow gilled fiber tissue engineering scaffolds via industrially relevant, scalable, repeatable, high speed, and economical nonwoven carding technology is described. Scaffolds were validated as tissue engineering scaffolds using human adipose derived stem cells (hASC) exposed to pulsatile fluid flow (PFF). The effects of fiber morphology on the proliferation and viability of hASC, as well as effects of varied magnitudes of shear stress applied via PFF on the expression of the early osteogenic gene marker runt related transcription factor 2 (RUNX2) were evaluated. Gilled fiber scaffolds led to a significant increase in proliferation of hASC after seven days in static culture, and exhibited fewer dead cells compared to pure PLA round fiber controls. Further, hASC-seeded scaffolds exposed to 3 and 6 dyn/cm2 resulted in significantly increased mRNA expression of RUNX2 after one hour of PFF in the absence of soluble osteogenic induction factors. This is the first study to describe a method for the fabrication of high surface area gilled fibers and scaffolds. The scalable manufacturing process and potential fabrication across multiple nonwoven and woven platforms makes them promising candidates for a variety of applications that require high surface area fibrous materials.

Statement of Significance

We report here for the first time the successful fabrication of novel high surface area gilled fiber scaffolds for tissue engineering applications. Gilled fibers led to a significant increase in proliferation of human adipose derived stem cells after one week in culture, and a greater number of viable cells compared to round fiber controls. Further, in the absence of osteogenic induction factors, gilled fibers led to significantly increased mRNA expression of an early marker for osteogenesis after exposure to pulsatile fluid flow. This is the first study to describe gilled fiber fabrication and their potential for tissue engineering applications. The repeatable, industrially scalable, and versatile fabrication process makes them promising candidates for a variety of scaffold-based tissue engineering applications.

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Brain Biobank and studies of disease structure correlates

Larry H. Bernstein, MD, FCAP, Curator



Unveiling Psychiatric Diseases

Researchers create neuropsychiatric cellular biobank

Image: iStock/mstroz
Image: iStock/mstroz
Researchers from Harvard Medical School and Massachusetts General Hospital have completed the first stage of an important collaboration aimed at understanding the intricate variables of neuropsychiatric disease—something that currently eludes clinicians and scientists.

The research team, led by Isaac Kohane at HMS and Roy Perlis at Mass General, has created a neuropsychiatric cellular biobank—one of the largest in the world.

It contains induced pluripotent stem cells, or iPSCs, derived from skin cells taken from 100 people with neuropsychiatric diseases such as schizophrenia, bipolar disorder and major depression, and from 50 people without neuropsychiatric illness.

In addition, a detailed profile of each patient, obtained from hours of in-person assessment as well as from electronic medical records, is matched to each cell sample.

As a result, the scientific community can now for the first time access cells representing a broad swath of neuropsychiatric illness. This enables researchers to correlate molecular data with clinical information in areas such as variability of drug reactions between patients. The ultimate goal is to help treat, with greater precision, conditions that often elude effective management.

The cell collection and generation was led by investigators at Mass General, who in collaboration with Kohane and his team are working to characterize the cell lines at a molecular level. The cell repository, funded by the National Institutes of Health, is housed at Rutgers University.

“This biobank, in its current form, is only the beginning,” said Perlis, director of the MGH Psychiatry Center for Experimental Drugs and Diagnostics and HMS associate professor of psychiatry. “By next year we’ll have cells from a total of four hundred patients, with additional clinical detail and additional cell types that we will share with investigators.”

A current major limitation to understanding brain diseases is the inability to access brain biopsies on living patients. As a result, researchers typically study blood cells from patients or examine post-mortem tissue. This is in stark contrast with diseases such as cancer, for which there are many existing repositories of highly characterized cells from patients.

The new biobank offers a way to push beyond this limitation.


A Big Step Forward

While the biobank is already a boon to the scientific community, researchers at MGH and the HMS Department of Biomedical Informatics will be adding additional layers of molecular data to all of the cell samples. This information will include whole genome sequencing and transcriptomic and epigenetic profiling of brain cells made from the stem cell lines.

Collaborators in the HMS Department of Neurobiology, led by Michael Greenberg, department chair and Nathan Marsh Pusey Professor of Neurobiology,  will also work to examine characteristics of other types of neurons derived from these stem cells.

“This can potentially alter the entire way we look at and diagnose many neuropsychiatric conditions,” said Perlis.

One example may be to understand how the cellular responses to medication correspond to the patient’s documented responses, comparing in vitro with in vivo. “This would be a big step forward in bringing precision medicine to psychiatry,” Perlis said.

“It’s important to recall that in the field of genomics, we didn’t find interesting connections to disease until we had large enough samples to really investigate these complex conditions,” said Kohane, chair of the HMS Department of Biomedical Informatics.

“Our hypothesis is that here we will require far fewer patients,” he said. “By measuring the molecular functioning of the cells of each patient rather than only their genetic risk, and combining that all that’s known of these people in terms of treatment response and cognitive function, we will discover a great deal of valuable information about these conditions.”

Added Perlis, “In the early days of genetics, there were frequent false positives because we were studying so few people. We’re hoping to avoid the same problem in making cellular models, by ensuring that we have a sufficient number of cell lines to be confident in reporting differences between patient groups.”

The generation of stem cell lines and characterization of patients and brain cell lines is funded jointly by the the National Institute of Mental Health, the National Human Genome Research Institute and a grant from the Centers of Excellence in Genomic Science program.


On C.T.E. and Athletes, Science Remains in Its Infancy

Se Hoon ChoiYoung Hye KimMatthias Hebisch, et al.

Alzheimer’s disease is the most common form of dementia, characterized by two pathological hallmarks: amyloid-β plaques and neurofibrillary tangles1. The amyloid hypothesis of Alzheimer’s disease posits that the excessive accumulation of amyloid-β peptide leads to neurofibrillary tangles composed of aggregated hyperphosphorylated tau2, 3. However, to date, no single disease model has serially linked these two pathological events using human neuronal cells. Mouse models with familial Alzheimer’s disease (FAD) mutations exhibit amyloid-β-induced synaptic and memory deficits but they do not fully recapitulate other key pathological events of Alzheimer’s disease, including distinct neurofibrillary tangle pathology4, 5. Human neurons derived from Alzheimer’s disease patients have shown elevated levels of toxic amyloid-β species and phosphorylated tau but did not demonstrate amyloid-β plaques or neurofibrillary tangles6, 7, 8, 9, 10, 11. Here we report that FAD mutations in β-amyloid precursor protein and presenilin 1 are able to induce robust extracellular deposition of amyloid-β, including amyloid-β plaques, in a human neural stem-cell-derived three-dimensional (3D) culture system. More importantly, the 3D-differentiated neuronal cells expressing FAD mutations exhibited high levels of detergent-resistant, silver-positive aggregates of phosphorylated tau in the soma and neurites, as well as filamentous tau, as detected by immunoelectron microscopy. Inhibition of amyloid-β generation with β- or γ-secretase inhibitors not only decreased amyloid-β pathology, but also attenuated tauopathy. We also found that glycogen synthase kinase 3 (GSK3) regulated amyloid-β-mediated tau phosphorylation. We have successfully recapitulated amyloid-β and tau pathology in a single 3D human neural cell culture system. Our unique strategy for recapitulating Alzheimer’s disease pathology in a 3D neural cell culture model should also serve to facilitate the development of more precise human neural cell models of other neurodegenerative disorders.



Figure 2: Robust increases of extracellular amyloid-β deposits in 3D-differentiated hNPCs with FAD mutations.close

Robust increases of extracellular amyloid-[bgr] deposits in 3D-differentiated hNPCs with FAD mutations.

a, Thin-layer 3D culture protocol. HC, histochemistry; IF, immunofluorescence; IHC, immunohistochemistry. b, Amyloid-β deposits in 6-week differentiated control and FAD ReN cells in 3D Matrigel (green, GFP; blue, 3D6; scale bar, …


Stem Cell-Based Spinal Cord Repair Enables Robust Corticospinal Regeneration


Novel use of EPR spectroscopy to study in vivo protein structure


α-synuclein is a protein found abundantly throughout the brain. It is present mainly at the neuron ends where it is thought to play a role in ensuring the supply of synaptic vesicles in presynaptic terminals, which are required for the release of neurotransmitters to relay signals between neurons. It is critical for normal brain function.

However, α-synuclein is also the primary protein component of the cerebral amyloid deposits characteristic of Parkinson’s disease and its precursor is found in the amyloid plaques of Alzheimer’s disease. Although α-synuclein is present in all areas of the brain, these disease-state amyloid plaques only arise in distinct areas.

Alpha-synuclein protein. May play role in Parkinson’s and Alzheimer’s disease.  © /

Imaging of isolated samples of α-synuclein in vitro indicate that it does not have the precise 3D folded structure usually associated with proteins. It is therefore classed as an intrinsically disordered protein. However, it was not known whether the protein also lacked a precise structure in vivo.

There have been reports that it can form helical tetramers. Since the 3D structure of a biological protein is usually precisely matched to the specific function it performs, knowing the structure of α-synuclein within a living cell will help elucidate its role and may also improve understanding of the disease states with which it is associated.

If α-synuclein remains disordered in vivo, it may be possible for the protein to achieve different structures, and have different properties, depending on its surroundings.

Techniques for determining protein structure

It has long been known that elucidating the structure of a protein at an atomic level is fundamental for understanding its normal function and behavior. Furthermore, such knowledge can also facilitate the development of targeted drug treatments. Unfortunately, observing the atomic structure of a protein in vivo is not straightforward.

X-ray diffraction is the technique usually adopted for visualizing structures at atomic resolution, but this requires crystals of the molecule to be produced and this cannot be done without separating the molecules of interest from their natural environment. Such processes can modify the protein from its usual state and, particularly with complex structures, such effects are difficult to predict.

The development of nuclear magnetic resonance (NMR) spectroscopy improved the situation by making it possible for molecules to be analyzed under in vivo conditions, i.e. same pH, temperature and ionic concentration.

More recently, increases in the sensitivity of NMR and the use of isotope labelling have enabled determinations of the atomic level structure and dynamics of proteins to be determined within living cells1. NMR has been used to determine the structure of a bacterial protein within living cells2 but it is difficult to achieve sufficient quantities of the required protein within mammalian cells and to keep the cells alive for NMR imaging to be conducted.

Electron paramagnetic resonance (EPR) spectroscopy for determining protein structure

Recently, researchers have managed to overcome these obstacles by using in-cell NMR and electron paramagnetic resonance (EPR) spectroscopy. EPR spectroscopy is a technique that is similar to NMR spectroscopy in that it is based on the measurement and interpretation of the energy differences between excited and relaxed molecular states.

In EPR spectroscopy it is electrons that are excited, whereas in NMR signals are created through the spinning of atomic nuclei. EPR was developed to measure radicals and metal complexes, but has also been utilized to study the dynamic organization of lipids in biological membranes3.

EPR has now been used for the first time in protein structure investigations and has provided atomic-resolution information on the structure of α-synuclein in living mammalians4,5.

Bacterial forms of the α-synuclein protein labelled with 15N isotopes were introduced into five types of mammalian cell using electroporation. Concentrations of α-synuclein close to those found in vivo were achieved and the 15N isotopes allowed the protein to be clearly defined from other cellular components by NMR. The conformation of the protein was then determined using electron paramagnetic resonance (EPR).

The results showed that within living mammalian cells α-synuclein remains as a disordered and highly dynamic monomer. Different intracellular environments did not induce major conformational changes.


The novel use of EPR spectroscopy has resolved the mystery surrounding the in vivo conformation of α-synuclein. It showed that α-synuclein maintains its disordered monomeric form under physiological cell conditions. It has been demonstrated for the first time that even in crowded intracellular environments α-synuclein does not form oligomers, showing that intrinsic structural disorder can be sustained within mammalian cells.


  1. Freedberg DI and Selenko P. Live cell NMR Annu. Rev. Biophys. 2014;43:171–192.
  2. Sakakibara D, et al. Protein structure determination in living cells by in-cell NMR spectroscopy. Nature 2009;458:102–105.
  3. Yashroy RC. Magnetic resonance studies of dynamic organisation of lipids in chloroplast membranes. Journal of Biosciences 1990;15(4):281.
  4. Alderson TA and Bax AD. Parkinson’s Disease. Disorder in the court. Nature 2016; doi:10.1038/nature16871.
  5. Theillet FX, et al. Structural disorder of monomeric α-synuclein persists in mammalian cells. Nature 2016; doi:10.1038/nature16531.


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Blood forming precursors in bone marrow

Larry H. Bernstein, MD, FCAP, Curator



Blood stem cells study could pave the way for new cancer therapy



This image shows the formation of blood stem cells inside the embryonic vessel called dorsal aorta. In green is shown secreted molecule called NOGGIN, which plays an important role in this process. The University of Edinburgh

People with leukaemia could be helped by new research that sheds light on how the body produces its blood supply.

Scientists are a step closer to creating blood stem cells that could reduce the need for bone marrow transplants in patients with cancer or blood disorders.

Enabling scientists to grow the stem cells artificially from pluripotent stem cells could also lead to the development of personalised blood therapies, researchers say.

Blood stem cells are found in bone marrow and produce all blood cells in the body. These cells – known as haematopoietic stem cells (HSCs) – help to restore blood supply in patients who have been treated for leukaemia.

Researchers used a mouse model to pinpoint exactly how HSCs develop in the womb. They showed for the first time how three key molecules interact together to generate the cells, which are later found in adult bone marrow.

The discovery could help scientists to recreate this process in the lab, in the hope that HSCs could one day be developed for clinical use.

Scientists say this fundamental understanding of early development may also have an impact on other diseases that affect blood formation and supply.


The research has been published in Nature Communications.

Professor Alexander Medvinsky, of the University of Edinburgh’s MRC Centre for Regenerative Medicine said: “There is a pressing need to improve treatments for diseases like leukaemia and this type of research brings us a step closer to that milestone. The more we understand about how embryos develop these blood stem cells, the closer we come to being able to make them in the lab.”

Céline Souilhol, Christèle Gonneau, Javier G. Lendinez, Antoniana Batsivari, Stanislav Rybtsov, Heather Wilson, Lucia Morgado-Palacin, David Hills, Samir Taoudi, Jennifer Antonchuk, Suling Zhao, Alexander Medvinsky. Inductive interactions mediated by interplay of asymmetric signalling underlie development of adult haematopoietic stem cells. Nature Communications, 2016; 7: 10784 DOI: 10.1038/ncomms10784

During embryonic development, adult haematopoietic stem cells (HSCs) emerge preferentially in the ventral domain of the aorta in the aorta–gonad–mesonephros (AGM) region. Several signalling pathways such as Notch, Wnt, Shh and RA are implicated in this process, yet how these interact to regulate the emergence of HSCs has not previously been described in mammals. Using a combination of ex vivo and in vivo approaches, we report here that stage-specific reciprocal dorso–ventral inductive interactions and lateral input from the urogenital ridges are required to drive HSC development in the aorta. Our study strongly suggests that these inductive interactions in the AGM region are mediated by the interplay between spatially polarized signalling pathways. Specifically, Shh produced in the dorsal region of the AGM, stem cell factor in the ventral and lateral regions, and BMP inhibitory signals in the ventral tissue are integral parts of the regulatory system involved in the development of HSCs.

Haematopoietic stem cells (HSCs) lie at the foundation of the adult haematopoietic system, and give rise to cells of all blood lineages throughout the lifespan of an organism. An important property of adult (definitive) haematopoietic stem cells (dHSCs) is that they are capable of long-term reconstitution of the haematopoietic system upon transplantation into irradiated recipients. In the mouse, such cells develop by embryonic stages E10–E11 in the aorta–gonad–mesonephros (AGM) region1, 2, 3, 4. An ex vivo approach showed that the AGM region has a robust autonomous capacity to generate dHSCs1. The AGM region comprises the dorsal aorta flanked on both sides by the urogenital ridges (UGRs), which contain embryonic rudiments of kidney and mesonephros. HSCs develop in a polarized manner, predominantly in the ventral floor of the dorsal aorta (AoV), more rarely in the dorsal domain of the dorsal aorta (AoD), and are absent in the UGRs2, 5, 6, 7. Localization of dHSCs to the AoV in mouse and human embryos was shown by long-term reconstitution experiments5, 6.

Abundant evidence indicates that during development, a specialized embryonic endothelial compartment known as haematogenic (or haemogenic) endothelium gives rise to haematopoietic stem and progenitors cells7, 8, 9, 10. The haematopoietic programme in various vertebrate models is executed predominantly in the AoV, and is recognized by the expression of essential haematopoietic transcription factors, for example, Runx1 and cKit, and the appearance of clusters of haematopoietic cells budding from the endothelium of the dorsal aorta6, 8, 9, 11, 12, 13, 14.

It is broadly accepted that HSCs develop from the haematogenic endothelium within intra-aortic clusters. This transition involves several consecutive maturation steps of HSC precursors: pro-HSCsright arrowpre-HSC type Iright arrowpre-HSC type IIright arrowdHSC15, 16, 17. All these precursors express endothelial markers, such as vascular-endothelial cadherin (VC) and CD31, and sequentially upregulate haematopoietic surface markers: CD41 (pro-HSCs), CD43 (pre-HSC type I) and finally CD45 (pre-HSC type II). This maturation process occurs in the dorsal aorta between E9 and E11. Specifically, pro-HSCs emerge at E9, pre-HSCs Type I appear at E10 and pre-HSCs type II predominantly at E11. Unlike dHSCs, pre-HSCs cannot reconstitute the adult haematopoietic system by direct transplantation and require prior maturation in an embryonic or neonatal environment15, 16, 17, 18,19.

A number of signalling pathways (Notch, Wnt, retinoic acid, interleukin-3 and inflammatory) have been implicated in HSC development; however, a coherent picture is yet to be elucidated15, 17, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31. HSC precursors (pro-HSCs, pre-HSCs type I and pre-HSCs type II) express cKit17 from early developmental stages. A recent study has shown that the cKit ligand, known as stem cell factor (SCF), is a key regulator driving maturation of these HSC precursors into dHSCs in the AGM region17, which is in agreement with the marked decline of HSC activity in SCF mutant mice32, 33. In the adult, SCF is critically important for HSC maintenance in the bone marrow niche, mainly in the endothelial compartment32. Sonic Hedgehog (Shh) and bone morphogenetic protein 4 (BMP4) pathways are also important mediators; in zebrafish, these two morphogenes are involved in arterial specification and haematopoietic patterning, respectively34,35. In the mouse, subaortic BMP4 and Shh/Indian Hedgehog derived from gut were also proposed to be responsible for HSC development36, 37.

During development, interactions between spatially segregated compartments are essential for tissue patterning and specification, and are often mediated by gradients of secreted molecules38,39, 40. Molecules secreted by distant tissues, such as somites, can influence HSC development in the AGM region41, 42, 43, 44, 45. Developing HSCs are embedded in the complex AGM microenvironment, suggesting that HSC development may require signals derived from different compartments of the AGM region. We sought to test this hypothesis. However, the analysis of HSC development in vivo is significantly hampered by low accessibility of embryos developing in utero, fast maturation of dHSCs, lack of uniquely specific markers for HSC precursors and their low numbers in the AGM region. Therefore, we employed here a robust ex vivo culture system that models HSC development in the embryo in combination with functional HSC analysis using in vivolong-term reconstitution assay15, 16, 17. Specifically, to study interactions between AGM subregions, we took advantage of the in vitro reaggregation system that enables close juxtaposition of cell types15.

We show that interactions between three compartments of the AGM, the AoV, the AoD and the UGRs, are necessary for efficient generation of dHSCs. First, we show that dHSC activity in the isolated E10.5 AoV is limited but can be significantly enhanced by co-culture with the AoD, and that this is mediated at least partly by Shh, secreted dorsally in vivo. Second, while HSC activity in isolated E11.5 AoD is limited, co-culture with a competent AoV microenvironment activates dHSC generation in the AoD. We found that this effect is mediated by SCF, which is secreted abundantly by the AoV stroma in vivo as shown here. Third, we show that downregulation of BMP4 signalling by BMP antagonist Noggin, which is present at high levels in the AoV and especially in intra-aortic clusters as revealed here by in vivo observations, is required for HSC development. Fourth, UGRs, which express high levels of SCF, also enhance HSC development in the dorsal aorta.

Our results based on in vivo observations and ex vivo modelling strongly suggest that juxtaposed, anatomically distinct domains within the AGM region create a complex landscape of interactive signals that underpins HSC development.

Pre-HSCs localize preferentially to the AoV

As dHSCs mature from pre-HSCs, we investigated whether the emergence of dHSC predominantly in the AoV6 is a result of asymmetric (ventralized) distribution of pre-HSCs. Dorsal aortae were separated from UGRs and bisected into AoV and AoD (including notochord) as described previously6 (Supplementary Fig. 1a). The different domains were then directly transplanted into irradiated mice to detect dHSCs. We first confirmed our previous observation that at E11.5 dHSCs appear almost exclusively in the AoV, although some dHSCs were in the AoD and engrafted few recipients at high level (Supplementary Fig. 1b). Limiting dilution analysis showed that dHSCs are approximately four times more frequent in the AoV compared with AoD. UGRs did not contain HSCs in line with previous reports2, 6.

We then investigated the spatial distribution of pre-HSCs type I and pre-HSCs type II in E10.5–E11.5 embryos using the OP9 co-culture system supplemented with Il3+SCF+Flt3 (termed 3GF), which allows pre-HSCs (which do not engraft by direct transplantation) to mature into dHSC that become detectable by long-term repopulation assay as described previously16. Doses of transplanted cells (expressed in embryo equivalents, e.e.) were chosen based on the requirements of individual experiments (explained in Methods section). In these experiments (Fig. 1), the dose injected was high (1–2e.e.) to detect potentially low dHSC numbers in AoD and UGRs.

Figure 1: Localization of pre-HSCs in the AGM region.

Localization of pre-HSCs in the AGM region.

(a) E10.5 AoV, AoD and UGRs were co-aggregated with OP9 and cultured for 5 days, and the formation of dHSCs was then tested by transplantation into irradiated mice (2e.e. per recipient; AoV: six independent experiments; AoD: four independent experiments; UGRs: two independent experiments). Dashed line indicates the cutoff for high-level engraftment (>70% donor chimaerism). (b) E11.5 aortas and UGRs were transplanted after reaggregate culture (Ao: 0.2e.e. per recipient and UGRs: 1e.e. per recipient; two independent experiments). (c,d) Pre-HSCs type I (VC+CD45) (c) or type II (VC+CD45+) (d) sorted from E11.5 AoV and AoD were co-aggregated with OP9 cells and transplanted after culture (1e.e. per recipient; two independent experiments). (ad) Levels of engraftment are plotted, and number of repopulated versus total number of transplanted mice are shown in brackets. Number of embryo equivalents (ee) injected in each experiment are indicated on the graphs. (*P<0.05; ***P<0.005; Mann–Whitney U-test). In all these experiments, tissues were cultured with three growth factors (Flt3I, Il3 and SCF). AGM, aorta–gonad–mesonephros region; Ao, dorsal Aorta; AoV, ventral domain of the dorsal aorta; AoD, dorsal domain of the dorsal aorta; UGRs, urogenital ridges.

We have shown previously that E10.5 AGM region mainly contains type I pre-HSCs, whereas at E11.5, type I and type II pre-HSCs co-exist16. Dissected E10.5 AGM regions co-cultured with OP9 in 3GF for 5 days were transplanted into adult irradiated recipients. Out of 21 recipients that received cultured AoV, 20 showed high levels (>70%) of donor-derived long-term haematopoietic chimerism (Fig. 1a). In contrast, only 7 out of 16 recipients of cultured AoD were repopulated at high levels (>70%), while the remaining recipients showed lower or no repopulation (7 and 2, respectively). Cultured UGRs did not produce dHSCs (Fig. 1a). Thus, we conclude that the E10.5 AoD does contain pre-HSCs but at significantly lower numbers than the AoV.

We then investigated whether pre-HSCs localization changes in E11.5 embryos and found that pre-HSCs were still exclusively localized to the dorsal aorta; UGRs carefully separated from the lateral mesenchyme adjacent to the dorsal aorta did not give any repopulation after culture (Fig. 1b). To establish the location of pre-HSCs within the E11.5 dorsal aorta, cell populations enriched for pre-HSCs type I (VC+CD45) and pre-HSCs type II (VC+CD45+) were sorted from AoV and AoD, and co-cultured with OP9 stromal cells in the presence of 3GF as described previously16. We again were able to detect pre-HSC activity in AoD although at lower levels than in AoV. After maturation ex vivo, pre-HSCs type I from AoV and AoD repopulated 7 of 11 and 2 of 8 recipients, respectively (Fig. 1c). Similarly, cultured pre-HSCs type II from AoV and AoD repopulated 11 out of 12 and 4 out of 10 recipients, respectively (Fig. 1d). In all cases, multilineage engraftment was confirmed (Supplementary Fig. 2). These data show that pre-HSCs are significantly enriched in AoV.

Reciprocal inductive interactions between AoD and AoV

To explore hypothetical interactions between AoD and AoV, we made use of a dissociation–reaggregation system that recapitulates HSC development ex vivo15. This system allowed us to integrate AGM domains in a three-dimensional tissue-like organoid15 and study their interactions in HSC development. To track the origin of dHSCs, AoV and AoD from wild-type (WT) and green fluorescent protein (GFP) embryos with constitutive expression of GFP46 were co-aggregated (termed AoV//AoD co-aggregates) and cultured for 5 days in the presence of 3GF before transplantation (Fig. 2a). Mice transplanted with AoV//AoD co-aggregates can be reconstituted by dHSCs coming from AoD and AoV. The presence of GFP allowed the individual contributions of AoV and AoD to the total repopulation level within the same mouse to be assessed (Fig. 2b,c). This is presented in two separate columns in the graph. Namely, while columns 1 and 3 represent the same recipient mice, the former shows exclusively the contribution of the AoD and the latter shows exclusively the contribution of the AoV into each recipient. To assess the influence of AoD and AoV interaction on HSC development, the repopulation by co-aggregated AoD (column 1) or AoV (column 3) can then be compared with repopulation by independently cultured AoD (column 2) or AoV (column 4). All experiments included reciprocal use of WT and GFP tissues in AoV//AoD co-aggregates, and we observed no difference in repopulation properties between WT and GFP embryos. Homotypic AoV//AoV and AoD//AoD co-aggregates were always used as controls. Note that in these experiments, only 0.2e.e. were injected per recipient, to ensure that the repopulation levels were not saturated and to allow any inductive effects to be revealed.

Figure 2: Inductive interactions between AoV, AoD and UGRs as revealed by an ex vivomodel system.

Inductive interactions between AoV, AoD and UGRs as revealed by an ex vivo model system.

(a) Experimental design: the ventral domain (AoV) and the dorsal domain (AoD) of the aorta, and the urogenital ridges (UGRs) from wild-type (WT) and GFP+ embryos were subdissected, and chimeric reaggregates from tissues of these two genotypes were generated. Left column: to test interactions between AoV and AoD, chimeric AoV//AoD reaggregates were generated and transplanted into irradiated recipients after 4–5 days of culture (b,c). Right column: to test interactions between Ao and UGRs, chimeric Ao//UGR reaggregates were generated and transplanted into irradiated recipients after 4–5 days culture (d). GFP+ and/or GFP− donor-derived long-term repopulation allowed us to conclude whether dHSCs originated from AoV, AoD or UGRs. Accordingly, the tissue of origin of donor dHSCs is indicated below each graph. (b) E10.5 aortas from WT and GFP embryos were used to generate chimeric reaggregates as depicted schematically above plots. The reciprocal combination of WT and GFP tissues was used to generate AoV//AoD reaggregates. The tissue source of dHSCs is shown separately in the leftmost (AoD) and rightmost (AoV) columns as indicated below the plot (0.2e.e. per recipient; two independent experiments). (c) E11.5 aortas from WT and GFP embryos were used to generate chimeric reaggregates. The tissue source of dHSCs is shown separately in the leftmost (AoD) and rightmost (AoV) columns as indicated below the plot (0.2e.e. per recipient; two independent experiments). (d) E11.5 aortas (Ao) and UGRs from WT and GFP embryos were used to generate Ao//UGR chimeric reaggregates. As depicted schematically above the plot, the reciprocal combination of WT and GFP tissues was used to generate Ao//UGR reaggregates. The tissue source of dHSCs is shown separately in left (Ao) and right (UGRs) columns as indicated below the plot (0.01e.e. per recipient; six independent experiments). (e) Reaggregation of WT Ao with UGRs generate more dHSCs than Ao alone (0.05e.e. per recipient; two independent experiments). (be) In all these experiments, tissues were cultured with three growth factors.

Using this approach, we found that the E10.5 AoV generates more dHSCs when combined with AoD than on its own (Fig. 2b, compare two rightmost columns). One day later, E11.5 AoD had no positive influence on dHSC generation by AoV (Fig. 2c, compare two rightmost columns). Conversely, the E11.5 AoD produced more HSCs when reaggregated with the AoV than on its own (Fig. 2c, compare two leftmost columns). This inductive effect of AoV on AoD was not observed at E10.5 (Fig. 2b, compare two leftmost columns). These ex vivo modelling experiments revealed reciprocal stage-specific effects of AoV and AoD on HSC development, which could be explained by the differential release of factors by the two regions and/or by differences in the competency of the target cells to respond to signals.

UGRs enhance HSC development in the dorsal aorta  

SCF expression is involved in polarized HSC development

Figure 3: Involvement of polarized stem cell factor in HSC development.

Involvement of polarized stem cell factor in HSC development.

(a) qRT–PCR on fresh AoV, AoD and UGRs at E10.5 and E11.5 showed high expression levels of stem cell factor (SCF) in AoV and UGRs, compared with AoD (data are mean±s.e.m; *P<0.05, **P<0.01, t-test; three independent experiments). No significant difference was observed between E10.5 and E11.5 expression level in any of the tissues. (b) Expression of SCF-GFP and CD31 determined by immunostaining on thick section (300μm) of SCF-GFP-positive E10.5 AGM region and on SCF-GFP-negative control. Bars, 50μm. (c) Expression of SCF in sorted populations from fresh E10.5–E11.5 AoV (V) and AoD (D) determined by qRT–PCR. Endo, endothelial population (VC+CD45CD43); type I, pre-HSCs type I (VC+CD45CD43+); type II, pre-HSCs type II (VC+CD45+); stroma, stromal population (VCCD45CD43). (*P<0.05, t-test; five independent experiments). (d) E10.5 AoD were cultured as reaggregates in the presence of Il3 and Flt3L with or without SCF and human SCF antagonist (SCF-Rh). (0.5e.e. per recipient; three independent experiments). (e,f) E11.5 AoD (two independent experiments) (e) and E10.5 AoV (two independent experiments) (f) were cultured as explants with or without SCF (no other cytokines); (0.2e.e. per recipient).


Shh signalling enhances dHSC generation


Figure 4: Sonic Hedgehog is a positive modulator of pre-HSC type I.

Sonic Hedgehog is a positive modulator of pre-HSC type I.

(a) Expression level of Sonic Hedgehog (Shh) in E10.5 and E11.5 AGM region determined by qRT–PCR. (data are mean±s.e.m; *P<0.05, t-test; E10.5: three independent experiments and E11.5: two independent experiments). (b) Patched1 and Gli1 expression in endothelial cells (endo: VC+CD45CD43), pre-HSCs type I (I: VC+CD45CD43+) and type II (II: VC+CD45+) sorted from E11.5 AoV and AoD (two independent experiments). (c) E10.5 AoV and AoD explants were cultured in presence of Shh recombinant protein before transplantation (AoV: 0.1e.e. per recipient; two independent experiments and AoD: 0.2e.e. per recipient; three independent experiments). (d) E10.5 AoV and doxycyline-inducible OP9-Shh were co-aggregated and cultured in presence or absence of doxycycline and/or Hedgehog (Hh) antagonist (200nM) before transplantation (0.2e.e. per recipient; two independent experiments). (e) 10.5 AoV and AoD co-aggregated with OP9 were cultured in presence of three growth factors with Hh antagonist before transplantation; (0.2e.e. per recipient; two independent experiments). (f) E11.5 AoV explants were cultured in presence of Shh recombinant protein before transplantation; (0.2e.e. per recipient; two independent experiments). (g): E11.5 AGM reaggregates were cultured in presence of Hh antagonist before transplantation; (0.1e.e. per recipient; two independent experiments). (c,d,f,g) In all these experiments, tissues were cultured without cytokines. Hh anta, Hh antagonist; Dox, doxycycline.


BMP signalling is downregulated in the dHSC lineage

Figure 5: Bone morphogenetic protein signalling is downregulated in dHSC lineage.

Bone morphogenetic protein signalling is downregulated in dHSC lineage.

(a) Expression of bone morphogenetic protein 4 (BMP4) at E10.5 determined by qRT–PCR; (data are mean±s.e.m.; *P<0.05, t-test; three independent experiments). (b) Expression of BMP4 in the E10.5 AGM region determined by immunostaining on frozen sections. Bars, 50μm. Zoomed image shows the subendothelial localization of BMP4 (arrowheads). Bars, 10μm. (c) Expression of phosphorylated-Smad (P-Smad) in the E10.5 AGM region determined by immunostaining on frozen sections. Bars, 50μm. (d) Id genes expression in endothelial cells, pre-HSCs type I and type II directly isolated from E10.5 and E11.5 AoV determined by qRT–PCR. Endo, endothelial population (VC+CD45CD43); type I, pre-HSCs type I (VC+CD45CD43+); type II, pre-HSCs type II (VC+CD45+); stroma, stromal population (VCCD45CD43). (Data are mean±s.e.m.; *P<0.05, **P<0.01; t-test; five independent experiments). (eg) Expression of P-Smad, CD31 and CD45 in the endothelium and haematopoietic clusters of E10.5 dorsal aorta. White arrowheads indicate cells with pre-HSC type II phenotype (CD31+CD45+); green arrows show (CD31+CD45−/low) cells budding out of the dorsal aorta and expressing P-Smad; asterisks indicate CD31+CD45 cells expressing P-Smad within the endothelium. Bars, 10μm. A positive control showing P-Smad staining in the dorsal part of the neural tube can be found in h.


Figure 6: Haematopoietic clusters are exposed to low concentration of BMP4 and high levels of Noggin.

Haematopoietic clusters are exposed to low concentration of BMP4 and high levels of Noggin.

(a) Expression of BMP antagonists at E10.5 determined by qRT–PCR (data are mean±s.e.m.; *P<0.05,***P<0.005; t-test; three independent experiments). (b) Expression of Noggin in the E10.5 AGM region determined by immunostaining on frozen sections. Note the expression of Noggin in the notochord (Nt) as expected. Bar, 50μm. (c) Expression of Noggin and BMP4 in intra-aortic clusters characterized by cKit and CD31 expression. Note that BMP4 is mainly expressed underneath the dorsal aorta (arrowheads), while Noggin is expressed in the cluster (arrows). Bars, 10μm. (d) Expression of Noggin in isolated populations from E10.5 and E11.5 AoV (V) and AoD (D) determined by qRT–PCR. (*P<0.05, t-test; five independent experiments). (e) Model showing downregulation of BMP activity in dHSC lineage. BMP4 is mainly expressed in the ventral mesenchyme, while Noggin is found in haematopoeitic clusters. Accordingly, BMP activity, assessed by the phosphorylation of Smad1,5 and 8 (P-Smad), is high in mesenchymal cells underneath the aortic endothelium and in some endothelial cells (CD31+CD45) of the aortic endothelium and decreases in the haematopoeitic clusters. While some pre-HSC type I cells (CD31+CD45−/low) exhibit BMP signalling at a low level, acquisition of CD45 (shown in red) is accompanied by a complete loss of BMP activity. EC, endothelial cells; MC, mesenchymal cells; I, pre-HSC type I; II, pre-HSC type II.


BMP signalling inhibits HSC development


Interactions between SCF, Shh and BMP signalling pathways

Interplay between SCF, Shh and BMP pathways underpins inductive interactions in the AGM.


We have shown previously that during murine embryo development definitive HSCs emerge predominantly in the ventral domain of the dorsal aorta (AoV)6. This spatially polarized production of HSCs might be explained by different origins of dorsal and ventral endothelium and/or by asymmetric production of key factors involved in HSC development37, 52, 53 and we reasoned that directional inductive interactions between AGM compartments could be involved. Great insight into inductive interactions in various organs has previously been obtained through in vitro modelling39. Here we modelled interactions between AGM domains in a co-culture system, which supports HSC development15. Using this ex vivo system, we demonstrate that at early stages (E10.5) HSC maturation in the AoV region is enhanced by the presence of the AoD. One day later (E11.5), the AoV microenvironment is able to induce HSC development in the AoD, previously thought to be mostly devoid of HSC activity6. We also found that UGRs can enhance HSC production from the dorsal aorta, but cannot generate dHSCs themselves, even under influence of the dorsal aorta. Thus, our data strongly suggest that reciprocal stage-specific inductive AoD//AoV interactions and involvement of UGRs are required for execution of the robust development of HSCs in vivo.

Our data indicate that previously established dorso–ventrally polarized HSC development6 is defined by two main factors. First, our current data show that although the AoD contains pre-HSCs (both type I and type II), their numbers are lower than in AoV, in line with lower intra-aortic cluster formation previously described in mouse AoD6, 13. Second, as shown here, dHSCs can be induced in the AoD by the AoV, and therefore the dHSCs deficiency in AoD cannot be explained solely by asymmetric pre-HSC distribution, but may also be influenced by differences in the microenvironment.

To study this, we focused on SCF, Shh and BMP4, whose expressions are dorso–ventrally polarized in the AGM region36, 47, 49 (and current data). We found that SCF is an inductive signal that is expressed at high levels in the AoV and UGRs, and can stimulate HSC development in isolated AoD, a region which had previously been considered to be mostly devoid of HSC activity. This is in agreement with a key role of SCF in HSC maturation17. We found that the aortic endothelial compartment expresses high levels of SCF, suggesting its important role in HSC development comparable to the bone marrow microenvironment of adult HSCs32. Importantly, we found that the pre-HSC type I population expresses SCF suggesting a positive-autocrine loop, which could promote HSC development.

Shh signalling in zebrafish is required for aortic angioblast migration and subsequent arterial specification of the dorsal aorta34, 54. We found that in mouse Shh stimulates and a Hh antagonist inhibits the development of HSCs at E10.5 but not at E11.5, in keeping with a previous study37. The induction of dHSCs in AoV by AoD is also limited to the E10.5 stage. Since Shh is secreted by the notochord (which is included in AoD-dissected tissue), this stage specificity is likely defined by the predominant presence of pre-HSCs type I at E10.5, which express higher levels of Shh signalling components (Ptch1 and Gli1) compared with pre-HSCs type II. By E11.5, the pre-HSC population is mainly represented by type II cells15. Stage-specific loss of sensitivity to Hh signalling was also described in the developing neural tube55. Notably, the poor ability of AoD to develop HSCs despite abundant presence of Shh can also be explained by lower levels of Ptch1 and Gli1 detected in AoD- compared with AoV-derived pre-HSC type I. Our ex vivo modelling data indicate that AoD-derived Shh is an active inducer of HSC development in the AGM region. This conclusion does not exclude the possibility that Shh secreted by the gut could also reach the dorsal aorta37, although by E10.5 these sites are separated by an extended mesentery.

BMP4 signalling is a key factor involved during differentiation of ventral mesoderm and its further specification into haematopoietic cells. In zebrafish, BMP signalling is clearly required during the patterning of the dorsal aorta and for the emergence of dHSCs in the ventral wall34. Its role in mouse is less clear due to the early lethality of BMP mutants56. Several lines of evidence point to BMP4 as a good candidate regulating HSC development. Indeed, BMP4 is highly expressed in the ventral mesenchyme underneath the dorsal aorta34, 36, 49; some reports suggested its role in controlling dHSC emergence36, 57, 58. However, the in vitro systems used likely assayed the maintenance of dHSCs, rather than their maturation. It was also reported that BMP4 signalling can define their differentiation potential59. BMP4 is also involved in the regulation of essential haematopoietic transcription factors such as Scl/Gata2/Fli1 and Runx1 (refs 60, 61). Here we analysed BMP signalling activity in the dHSC lineage in the AGM region. We show that in vivo the pre-HSC type I to type II transition is accompanied by a downregulation of BMP targets (Id genes). This correlates with our data demonstrating that BMP activity is downregulated in intra-aortic clusters and the observations of others that Runx1 expression is attenuating in the developing HSC lineage60, 62, 63. How is this decrease of BMP activity achieved in vivo, despite the presence of BMP4 in AoV? It has previously been noted that in amphibian embryos several BMP inhibitors are also expressed in AoV34. Similarly, our analysis of the embryo showed high expression of a number of BMP antagonists as well as inhibitory Smad6 and Smad7 in mouse AoV that may counteract BMP4 action in HSC lineage. Furthermore, we found that in the AGM region BMP4 and Noggin are spatially segregated: Noggin being present in haematopoietic clusters and BMP4 being mainly expressed underneath the aortic endothelium. Therefore, maturing HSCs in clusters are exposed to low BMP4 concentration and high concentration of the BMP antagonist Noggin. Furthermore, our qRT–PCR analysis shows that the pre-HSC type I population expresses Noggin, which possibly creates a very effective shield that protects them from BMP4. Accordingly, our ex vivo analysis strongly suggests that downregulation of BMP signalling is functionally important for HSC development in the embryo. Indeed, forced BMP signalling activation by the addition of BMP4, strongly inhibits HSC development, and conversely the addition of Noggin stimulates HSC development in E10.5–E11.5 AGM cultures. These results are in line with recent observation that deletion of Smad4, a common transducer for BMP4/TGFβ signalling, markedly augments the formation of intra-aortic clusters64. Our data do not exclude the possibility that BMP4 is essential for specification of mouse dHSCs at earlier stages, as described in the zebrafish model, where BMP signalling is required for HSC development at stages closer to mouse E8.5 (ref. 34).

Our analysis indicates that all three signalling pathways studied can cooperate for HSC development (Fig. 8c). Notably, the interplay of Shh and BMP pathways is broadly involved in development. For example, counter gradients of polarized Shh and BMP signalling in the developing spinal cord specify neuronal subsets along the dorso–ventral axis65, and the dorsal aorta resembles the neural tube with inverse orientation of Shh- and BMP-secreting centres34. However, we detected an antagonistic relationship between Shh and BMP pathways. At the molecular level, Shh can induce Noggin and Smad6 expression, thus inhibiting BMP4 signalling. In turn, BMP4 suppresses and, accordingly, Noggin enhances Shh signalling. Cooperation between Shh and Noggin has been previously described as critically important for developmental specification of somitic, neural and hair follicle cells66, 67, 68. Our in vitro data suggest that the feed-forward loop Shhright arrowNoggin/Nogginright arrowShh is also involved in HSC development in vivo.

We propose a model where the polarized secreted factors form complex fields of gradients in vivo, which define an effector zone for optimal HSC development in the dorsal aorta and lead to the ventrally shifted appearance of dHSCs (Fig. 8c). Of interest, intra-aortic clusters are abundant in ventro–lateral positions69, which may reflect the position of this zone. The dissection close to such a zone could lead to accidental inclusion of powerful dHSCs in AoD samples observed here. Furthermore, it is possible that spatial segregation of co-operating and spatial overlap of antagonizing factors may also be important for adjustment of HSC development in vivo. Indeed, although the pool of pre-HSCs in the AGM region markedly expands during E9.5–11.5 (Rybtsov et al., submitted), complete maturation of the HSC pool is limited: while the majority of cells reach the pre-HSC type II stage, only one or two dHSCs are generated by the end of E11. Such controlled dynamics of HSC development may be needed to prevent a burst of active haematopoiesis in the AGM region. How exactly HSC maturation dynamics depend on overlapping concentrations of factors requires further analysis. Although ex vivo modelling is a powerful tool to dissect mechanisms of HSC development in vivo, there will likely be some variation in details. For example, spatial polarization in the developing HSC niche may define kinetics of HSC development in vivo.While we have demonstrated spatial polarization in vivo of the factors driving HSC development in our model system, it is currently unclear whether any factors become expressed in a polarized manner within the reaggregates and as such, whether polarization is also a pre-requisite for HSC maturation. Alternatively, if polarization is not required, the entire reaggregate may replicate the optimal zone for HSC development, resulting in massive generation of dHSCs. The distinction between these two scenarios will require further investigation.

In summary, our ex vivo modelling experiments suggest that HSC development in the embryo involves stage-dependent interactions between dorsal, ventral and lateral domains of the AGM region, mediated at least partly by the interplay of SCF, Shh, BMP4 and Noggin. Further detailed analysis will be required to better understand the complexity of the AGM signalling landscape in which HSC development takes place. Such knowledge may lead to development of novel protocols for the generation of definitive HSCs in vitro for clinical applications.

Integrated genomic DNA/RNA profiling of hematologic malignancies in the clinical setting

Jie He1Omar Abdel-Wahab2Michelle K. Nahas1Kai Wang1Raajit K. Rampal3Andrew M. Intlekofer4, et al. 10, 2016

Key Points

  • Novel clinically-available comprehensive genomic profiling of both DNA and RNA in hematologic malignancies.

  • Profiling of 3696 clinical hematologic tumors identified somatic alterations that impact diagnosis, prognosis, and therapeutic selection

The spectrum of somatic alterations in hematologic malignancies includes substitutions, insertions/deletions (indels), copy number alterations (CNAs) and a wide range of gene fusions; no current clinically available single assay captures the different types of alterations. We developed a novel next-generation sequencing-based assay to identify all classes of genomic alterations using archived formalin-fixed paraffin-embedded (FFPE), blood and bone marrow samples with high accuracy in a clinically relevant timeframe, which is performed in our CLIA-certified CAP-accredited laboratory. Targeted capture of DNA/RNA and next-generation sequencing reliably identifies substitutions, indels, CNAs and gene fusions, with similar accuracy to lower-throughput assays which focus on specific genes and types of genomic alterations. Profiling of 3696 samples identified recurrent somatic alterations that impact diagnosis, prognosis and therapy selection. This comprehensive genomic profiling approach has proved effective in detecting all types of genomic alterations, including fusion transcripts, which increases the ability to identify clinically-relevant genomic alterations with therapeutic relevance.

Cohesin Ring Rules Blood Stem Cells, Binds Them to Renewal or Expansion

GEN News

A genome-wide RNAi screen was used to assess the effects of 15,000 genes on the balance between self-renewal and differentiation of human hematopoietic stem cells (HSCs). The screen identified candidate genes whose knockdown maintained the HSC phenotype during culture. Such findings could lead to better protocols to grow these cells outside the body, potentially making bone marrow transplants more available to patients suffering blood cancers, or even identifying novel genes to target during the treatment of leukemia (left and right panels). Four genes in particular implicated cohesin, a ring-like protein complex that binds to the DNA in all of our cells, in the control of self-renewal versus differentiation in HSCs. Deficiency of cohesin causes an increase in self-renewal and a decrease in differentiation of HSCs. [Cell Reports]

Best known for its ability to regulate the separation of sister chromatids during cell division, the cohesin protein complex, a ring-shaped structure, has shown that it has other powers, such as the facilitation of DNA repair and the modification of transcription. And now, according to scientists based at Lund University, there is evidence that the cohesin complex controls the growth of blood stem cells. More to the point, the cohesin complex determines whether blood stem cells self-renew or differentiate.

The new finding is significant because it can help scientists improve the expansion of blood stem cells outside the body, thus increasing the supply of blood stem cells to patients suffering leukemia or hereditary blood disorders. Besides making bone marrow transplant material more available, the new finding could point scientists to new points of attack for the treatment of blood cancer, which is a disruption between blood stem cell multiplication and maturation.

The Lund University scientists, led by Jonas Larsson, presented their results March 17 in the journal Cell Reports, in an article entitled “Genome-wide RNAi Screen Identifies Cohesin Genes as Modifiers of Renewal and Differentiation in Human HSCs.” The article describes how a genome-wide RNA interference (RNAi) screen was performed in primary human CD34+ cells. This screen enabled the scientists to identify candidate genes whose knockdown maintained the HSC phenotype during culture.

“A striking finding was the identification of members of the cohesin complex (STAG2, RAD21, STAG1, and SMC3) among the top 20 genes from the screen,” wrote the authors. “Upon individual validation of these cohesin genes, we found that their knockdown led to an immediate expansion of cells with an HSC phenotype in vitro.”

A similar expansion, the authors added, was observed in vivo following transplantation to immunodeficient mice.

“Transcriptome analysis of cohesin-deficient CD34+ cells showed an upregulation of HSC-specific genes,” the authors continued. This finding, the authors asserted, demonstrates that when cohesin is deficient, transcription shifts to a more stem cell–like pattern.

“The research is unique as the study of so many genes alongside one another is unprecedented,” said Dr. Larsson. “In addition, we have used human blood stem cells, which is difficult in itself as it is requires the gathering of a large amount of material.”

Of the 15,000 genes that were tested, the Lund team found around 20 candidates with a strong capacity to affect the balance of growth in the blood stem cells. What was striking was that four of these 20 genes were physically connected through cooperation in a protein complex.

“The discovery showed that this protein complex is crucial and has an overarching function in the growth of the blood stem cells,” emphasized Dr. Larsson.

The cohesin complex acts as a sort of brace that holds different parts of the DNA strand together in the cell. The researchers believe that this allows the cohesin complex to control access to the “on/off switches” in DNA and to change the impulses the blood stem cells receive from various genes, thereby affecting cell division. The blood stem cell either multiplies or matures to become a specialized cell with other tasks.

Independently of the Lund researchers’ discovery, other research in the field of blood cancer has recently identified mutations in exactly the same four genes in patients with various forms of blood cancer.

“This is incredibly exciting! Together with the results from our study, this indicates that the cohesin genes are directly and crucially significant in the development of blood cancer,” exclaimed the study’s lead author, Ph.D. candidate Roman Galeev. “Our findings entail a new understanding of how the expansion of blood stem cells is controlled. Eventually, this can lead to new ways of affecting the process, either to prevent the development of cancer or to expand the stem cells for transplant.”


  • A genome-wide RNAi screen was performed in primary human CD34+ cells
  • Several cohesin genes were identified as modifiers of renewal and differentiation
  • Cohesin-deficient HSCs show enhanced reconstitution capacity in vivo
  • Cohesin deficiency induces immediate HSC-specific transcriptional programs


To gain insights into the regulatory mechanisms of hematopoietic stem cells (HSCs), we employed a genome-wide RNAi screen in human cord-blood derived cells and identified candidate genes whose knockdown maintained the HSC phenotype during culture. A striking finding was the identification of members of the cohesin complex (STAG2, RAD21, STAG1, andSMC3) among the top 20 genes from the screen. Upon individual validation of these cohesin genes, we found that their knockdown led to an immediate expansion of cells with an HSC phenotype in vitro. A similar expansion was observed in vivo following transplantation to immunodeficient mice. Transcriptome analysis of cohesin-deficient CD34+ cells showed an upregulation of HSC-specific genes, demonstrating an immediate shift toward a more stem-cell-like gene expression signature upon cohesin deficiency. Our findings implicate cohesin as a major regulator of HSCs and illustrate the power of global RNAi screens to identify modifiers of cell fate.

Figure thumbnail fx1

Human hematopoiesis is maintained by a small number of hematopoietic stem cells (HSCs) that are capable of generating all blood cell lineages at an extremely rapid pace for the entire lifespan of a human being (Orkin and Zon, 2008). HSCs have been studied extensively during the last four decades and are probably the best functionally characterized adult stem cells. However, despite this, the regulatory mechanisms that govern different cellular fate options in HSCs have remained incompletely defined. In particular, it has been challenging to understand the molecular basis of the inherent ability of HSCs to self-renew and preserve their undifferentiated state, which has hampered efforts to expand HSCs ex vivo for therapeutic benefit (Dahlberg et al., 2011). Ex vivo expansion of HSCs would allow for critical improvements of bone marrow transplantation procedures in treatment of malignant and inherited hematological diseases (Chou et al., 2010). Defining the genetic and molecular basis of self-renewal of HSCs is thus important to enhance current cell-therapy strategies, but it is also essential in order to better understand mechanisms behind dysregulated hematopoiesis that may cause leukemia. Genes and pathways balancing cell-fate options between renewal and differentiation in stem cells are often key players in cancer development (Orkin and Zon, 2008).

Thumbnail image of Figure 1. Opens large image

Figure 1

Genome-wide RNAi Screen in Primitive Human Hematopoietic Cells Defines Genes and Pathways Associated with Cancer Progression and Cell Proliferation

(A) Overview of the experimental outline for the primary screen. 60 million cord blood-derived CD34+ cells were transduced with a pooled lentiviral library containing 75,000 shRNAs across six transduction replicates in total. A fraction of the cells were isolated after 72 hr, and proviral inserts were deep sequenced to determine the initial library distribution. Following 20 days of culture, CD34+ cells were magnetically isolated and proviral inserts were sequenced again to determine the changes in distribution for all shRNAs.

(B) Relative distribution of shRNAs following 20 days of in vitro culture, ranked from the most enriched to the most depleted. The y axis shows the average enrichment value across six replicate screens.

(C) Gene ontology analysis for all genes represented by multiple shRNAs in the most enriched (10%) fraction.

(D) KEGG pathway analysis showing strong enrichment for cancer-associated pathways among the top-scoring genes.

See also Figure S1 and Table S1.

We report here on the successful development of a genome-wide RNAi screening approach targeted to primary human hematopoietic stem and progenitor cells to define genes and pathways associated with self-renewal and differentiation. Based on findings from the screen, we implicate the cohesin complex as a crucial regulator of cell-fate decisions influencing self- renewal and differentiation in HSCs both in vitro and in vivo.

These efforts represent a genome-wide RNAi screen targeted to primary human HSPCs. The main limiting factor when performing functional screens in primary human cells is cell number. This obviously becomes even more challenging when rare cell subsets, such as stem and progenitor cells, are studied. Through unique access to cord blood with daily deliveries from several local hospitals, we were able to gather the necessary quantities to perform a screen in enriched primary HSPCs with reasonable coverage (300X).


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Cardiomyocytes from mesenchmal stem cells?

Larry H. Bernstein, MD, FCAP, Curator


Introduction: A just published article from the Gladstone Institute establishes that cardiac muscle can be generated from inducible explandable  cardiovascular progenitor cells.  However, while the study has validity, it leaves much to be explained, especially in light of the references to many previous studies to generate cardiomycytes for heart failure.

Skin Cells Opening the Door to the Possibility of Personalized Medicine for Heart Attack Patients



ieCPCs Give Rise to CMs, ECs, and SMCs In Vivo and Improve Cardiac Function after MI

(A–E) Immunofluorescence analyses of RFP and CM (A), EC (B and C), and SMC (D and E) markers in tissue sections collected 2 weeks after transplanting RFP-labeled ieCPCs at passage 10 into infarcted hearts of immunodeficient mice. Scale bars represent 100 μm.

(F and G) Ejection fraction and fractional shortening of the left ventricle (LV) quantified by echocardiography. Results from two independent experiments were shown. D, days; W, weeks.

(H–J) Cardiac fibrosis was evaluated at eight levels (L1–L8) by Masson’s trichrome staining 12 weeks after coronary ligation. The ligation site is marked as X. Sections of representative hearts are shown in (I) with quantification in (J). Scar tissue (%) = (the sum of fibrotic area or length at L1–L8/the sum of LV area or circumference at L1–L8) × 100. Scale bars represent 500 μm.

(K) Quantification of LV circumference of mouse hearts 12 weeks after transplantation of 2nd MEFs or ieCPCs. Data were summarized from 48 sections for each group. Data are mean ± SE. p < 0.05.

“Cardiac progenitor cells could be ideal for heart regeneration,” said senior author Sheng Ding, PhD, a senior investigator at Gladstone. “They are the closest precursor to functional heart cells, and, in a single step, they can rapidly and efficiently become heart cells, both in a dish and in a live heart. With our new technology, we can quickly create billions of these cells in a dish and then transplant them into damaged hearts to treat heart failure.”


Discussion:  The study raises some important questions.

  1. How are the cultured cells different than those used in previous studies?
  2. Cardiomyocytes and fibroblasts are both of mesodermal origin.  What determines which way the stem cell line will differentiate?
  3. What is the difference, if any, between the cell culture environment and the in vivo environment into which they are placed?
  4. There is a difference between chronic hypoxemia with congestive heart failure and acute coronary syndrome.  The experiment performed would be more apt to apply to post-ACS than to chronic heart failure.


Functional heart muscle regenerated in decellularized human hearts

March 11, 2016

A partially recellularized human whole-heart cardiac scaffold, reseeded with human cardiomyocytes derived from induced pluripotent stem cells, being cultured in a bioreactor that delivers a nutrient solution and replicates some of the environmental conditions around a living heart. Credit: Bernhard Jank, MD, Ott Lab, Center for Regenerative Medicine, Massachusetts General Hospital


Massachusetts General Hospital (MGH) researchers have taken some initial steps toward the creation of bioengineered human hearts using donor hearts stripped of components that would generate an immune response and cardiac muscle cells generated from induced pluripotent stem cells (iPSCs), which could come from a potential recipient. The investigators described their accomplishments – which include developing an automated bioreactor system capable of supporting a whole human heart during the recellularization process—earlier this year in Circulation Research.

“Generating functional cardiac tissue involves meeting several challenges,” says Jacques Guyette, PhD, of the MGH Center for Regenerative Medicine (CRM), lead author of the report. “These include providing a structural scaffold that is able to support cardiac function, a supply of specialized cardiac , and a supportive environment in which cells can repopulate the scaffold to form mature tissue capable of handling complex cardiac functions.”

The research team is led by Harald Ott, MD, of the MGH CRM and the Department of Surgery, senior author of the paper. In 2008, Ott developed a procedure for stripping the living cells from a donor organ with a detergent solution and then repopulating the remaining extracellular matrix scaffold with organ-appropriate types of cells. Since then his team has used the approach to generate functional rat kidneys and lungs and has decellularized large-animal hearts, lungs and kidneys. This report is the first to conduct a detailed analysis of the matrix scaffold remaining after decellularization of whole human hearts, along with recellularization of the cardiac matrix in three-dimensional and whole-heart formats.

The study included 73 human hearts that had been donated through the New England Organ Bank, determined to be unsuitable for transplantation and recovered under research consent. Using a scaled-up version of the process originally developed in rat hearts, the team decellularized hearts from both brain-dead donors and from those who had undergone . Detailed characterization of the remaining cardiac scaffolds confirmed a high retention of matrix proteins and structure free of cardiac cells, the preservation of coronary vascular and microvascular structures, as well as freedom from human leukocyte antigens that could induce rejection. There was little difference between the reactions of organs from the two donor groups to the complex decellularization process.

Instead of using genetic manipulation to generate iPSCs from , the team used a newer method to reprogram skin cells with messenger RNA factors, which should be both more efficient and less likely to run into regulatory hurdles. They then induced the  to differentiate into or cardiomyocytes, documenting patterns of gene expression that reflected developmental milestones and generating cells in sufficient quantity for possible clinical application. Cardiomyocytes were then reseeded into three-dimensional matrix tissue, first into thin matrix slices and then into 15 mm fibers, which developed into spontaneously contracting tissue after several days in culture.

The last step reflected the first regeneration of human heart muscle from within a cell-free, human whole-heart matrix. The team delivered about 500 million iPSC-derived cardiomyocytes into the left ventricular wall of decellularized hearts. The organs were mounted for 14 days in an automated bioreactor system developed by the MGH team that both perfused the organ with nutrient solution and applied environmental stressors such as ventricular pressure to reproduce conditions within a living heart. Analysis of the regenerated tissue found dense regions of iPSC-derived cells that had the appearance of immature cardiac muscle tissue and demonstrated functional contraction in response to electrical stimulation.

“Regenerating a whole heart is most certainly a long-term goal that is several years away, so we are currently working on engineering a functional myocardial patch that could replace cardiac tissue damaged due a heart attack or heart failure,” says Guyette. “Among the next steps that we are pursuing are improving methods to generate even more – recellularizing a whole heart would take tens of billions—optimizing bioreactor-based culture techniques to improve the maturation and function of engineered cardiac tissue, and electronically integrating regenerated tissue to function within the recipient’s heart.”

Team leader Ott, an assistant professor of Surgery at Harvard Medical School, adds, “Generating personalized functional myocardium from patient-derived cells is an important step towards novel device-engineering strategies and will potentially enable patient-specific disease modeling and therapeutic discovery. Our team is excited to further develop both of these strategies in future projects.”

Explore further: A tool for isolating progenitor cells from human heart tissue could lead to heart repair

More information: Jacques P. Guyette et al. Bioengineering Human Myocardium on Native Extracellular MatrixNovelty and Significance, Circulation Research (2016). DOI: 10.1161/CIRCRESAHA.115.306874


Stem cell study in mice offers hope for treating heart attack patients

February 15, 2012


Stem cell study in mice offers hope for treating heart attack patients

Cardiac stem cells, pictured here, give hope to patients who have suffered a heart attack. Credit: UCSF

A UCSF stem cell study conducted in mice suggests a novel strategy for treating damaged cardiac tissue in patients following a heart attack. The approach potentially could improve cardiac function, minimize scar size, lead to the development of new blood vessels – and avoid the risk of tissue rejection.

In the investigation, reported online in the journal PLoS ONE, the researchers isolated and characterized a novel type of cardiac stem cell from the tissue of middle-aged mice following a .

Then, in one experiment, they placed the in the culture dish and showed they had the ability to differentiate into cardiomyocytes, or “beating heart cells,” as well as endothelial cells and smooth muscle cells, all of which make up the heart.

In another, they made copies, or “clones,” of the cells and engrafted them in the tissue of other of the same genetic background who also had experienced heart attacks. The cells induced angiogenesis, or blood vessel growth, or differentiated, or specialized, into endothelial and smooth muscle cells, improving .

“These findings are very exciting,” said first author Jianqin Ye, PhD, MD, senior scientist at UCSF’s Translational Cardiac Stem Cell Program. First, “we showed that we can isolate these cells from the heart of middle-aged animals, even after a heart attack.” Second, he said, “we determined that we can return these cells to the animals to induce repair.”

Importantly, the stem cells were identified and isolated in all four chambers of the heart, potentially making it possible to isolate them from patients’ hearts by doing right ventricular biopsies, said Ye. This procedure is “the safest way of obtaining cells from the heart of live patients, and is relatively easy to perform,” he said.

“The finding extends the current knowledge in the field of native cardiac progenitor cell therapy,” said senior author Yerem Yeghiazarians, MD, director of UCSF’s Translational Cardiac Stem Cell Program and an associate professor at the UCSF Division of Cardiology. “Most of the previous research has focused on a different subset of cardiac progenitor cells. These novel cardiac precursor cells appear to have great therapeutic potential.”

The hope, he said, is that patients who have severe heart failure after a heart attack or have cardiomyopathy would be able to be treated with their own cardiac stem cells to improve the overall health and function of the heart. Because the cells would have come from the patients, themselves, there would be no concern of cell rejection after therapy.

The cells, known as Sca-1+ stem enriched in Islet (Isl-1) expressing cardiac precursors, play a major role in cardiac development. Until now, most of the research has focused on a different subset of cardiac progenitor, or early stage, cells known as, c-kit cells.

The Sca-1+ cells, like the c-kit cells, are located within a larger clump of cells called cardiospheres.

The UCSF researchers used special culture techniques and isolated Sca-1+ cells enriched in the Isl-1expressing cells, which are believed to be instrumental in the heart’s development. Since Isl-1 is expressed in the cell nucleus, it has been difficult to isolate them but the new technique enriches for this cell population.

The findings suggest a potential treatment strategy, said Yeghiazarians. “Heart disease, including heart attack and heart failure, is the number one killer in advanced countries. It would be a huge advance if we could decrease repeat hospitalizations, improve the quality of life and increase survival.” More studies are being planned to address these issues in the future.

An estimated 785,000 Americans will have a new heart attack this year, and 470,000 who will have a recurrent attack. Heart disease remains the number one killer in the United States, accounting for one out of every three deaths, according to the American Heart Association.

Medical costs of cardiovascular disease are projected to triple from $272.5 billion to $818.1 billion between now and 2030, according to a report published in the journal Circulation.


Sca-1+ Cardiosphere-Derived Cells Are Enriched for Isl1-Expressing Cardiac Precursors and Improve Cardiac Function after Myocardial Injury

Jianqin Ye , Andrew Boyle , Henry Shih , Richard E. Sievers , Yan Zhang , William Grossman , Harold S. Bernstein , Yerem Yeghiazarians


Endogenous cardiac progenitor cells are a promising option for cell-therapy for myocardial infarction (MI). However, obtaining adequate numbers of cardiac progenitors after MI remains a challenge. Cardiospheres (CSs) have been proposed to have cardiac regenerative properties; however, their cellular composition and how they may be influenced by the tissue milieu remains unclear.

Methodology/Principal Finding

Using “middle aged” mice as CSs donors, we found that acute MI induced a dramatic increase in the number of CSs in a mouse model of MI, and this increase was attenuated back to baseline over time. We also observed that CSs from post-MI hearts engrafted in ischemic myocardium induced angiogenesis and restored cardiac function. To determine the role of Sca-1+CD45 cells within CSs, we cloned these from single cell isolates. Expression of Islet-1 (Isl1) in Sca-1+CD45 cells from CSs was 3-fold higher than in whole CSs. Cloned Sca-1+CD45 cells had the ability to differentiate into cardiomyocytes, endothelial cells and smooth muscle cells in vitro. We also observed that cloned cells engrafted in ischemic myocardium induced angiogenesis, differentiated into endothelial and smooth muscle cells and improved cardiac function in post-MI hearts.


These studies demonstrate that cloned Sca-1+CD45 cells derived from CSs from infarcted “middle aged” hearts are enriched for second heart field (i.e., Isl-1+) precursors that give rise to both myocardial and vascular tissues, and may be an appropriate source of progenitor cells for autologous cell-therapy post-MI.


Incorporation of Mg particles into PDLLA regulates mesenchymal stem cell and macrophage responses

Sandra C. Cifuentes1, Fátima Bensiamar2,3, Amparo M. Gallardo-Moreno3,4, Tim A. Osswald5, José L. González-Carrasco1,3, et al.
J Biomed Materials Res Part A  104(4), pages 866–878, April 2016          

In this work, we investigated a new approach to incorporate Mg particles within a PDLLA matrix using a solvent-free commercially available process. PDLLA/Mg composites were manufactured by injection moulding and the effects of Mg incorporated into PDLLA on MSC and macrophage responses were evaluated. Small amounts of Mg particles (≤1 wt %) do not cause thermal degradation of PDLLA, which retains its mechanical properties. PDLLA/Mg composites release hydrogen, alkaline products and Mg2+ ions without changing pH of culture media. Mg-containing materials provide a noncytotoxic environment that enhances MSC viability. Concentration of Mg2+ ions in extracts of MSCs increases with the increment of Mg content in the composites. Incorporation of Mg particles into PDLLA stimulates FN production, ALP activity, and VEGF secretion in MSCs, an effect mediated by degradation products dissolved from the composites. Degradation products of PDLLA induce an increase in MCP-1, RANTES, and MIP-1α secretion in macrophages while products of composites have minimal effect on these chemokines. Regulation of MSC behavior at the biomaterial’s interface and macrophage-mediated inflammatory response to the degradation products is related to the incorporation of Mg in the composites. These findings suggest that including small amounts of Mg particles into polymeric devices can be a valuable strategy to promote osseointegration and reduce host inflammatory response. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 866–878, 2016.

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