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PHILADELPHIA – The evolution of adaptations for life on land have long puzzled biologists – are feathers descendents of dinosaur scales, how did arms and legs evolve from fins, and from what ancient fish organ did the lung evolve?
Biologists have known that the co-development of the cardiovascular and pulmonary systems is a recent evolutionary adaption to life outside of water, coupling the function of the heart with the gas exchange function of the lung. And, the lung is one of the most recent organs to have evolved in mammals and is arguably the most vital for terrestrial life.
The coordinated maturation of the cells of these two systems is illustrated during embryonic development, when the primitive lung progenitor cells protrude into the primitive cardiac progenitor cells as the two organs develop in parallel to form the cardiopulmonary circulation. However, little is known about the molecular cues guiding this simultaneous development, and how a common progenitor cell for both organs may influence the pathology of such related diseases as pulmonary hypertension.
In a new paper published this week online in Nature, a team from the Perelman School of Medicine, University of Pennsylvania, shows that the pulmonary vasculature, the blood vessels that connect the heart to the lung, develops even in the absence of the lung. Mice in which lung development is inhibited still have pulmonary blood vessels, which revealed to the researchers that cardiac progenitors, or stem cells, are essential for cardiopulmonary co-development.
The Penn team, led by Edward E. Morrisey, PhD, professor of Medicine and Cell and Developmental Biology and scientific director of the Penn Institute for Regenerative Medicine, identified a population of multi-potent CardioPulmonary mesoderm Progenitor cells they named CPPs. The CPPs can be distinguished from many other early embryonic cells by the expression of a well-studied signaling molecule Wnt2.
“We asked if these progenitor cells are capable of generating both heart and lung derivatives,” says Morrisey. “Our data show that Wnt2-positive cells exist prior to lung development and help coordinate lung and heart co-development by generating cell types in both tissues.”
The issue of how the lung develops and connects to the cardiovascular system has intrigued the Morrisey lab for many years. “It’s pretty obvious to anyone who has looked at the anatomy of most terrestrial animals that the heart and lung are intimately linked. This is even reflected in clinical medicine where in many places, including the Perelman School of Medicine, the Division of Cardiovascular Medicine was once referred to as the Division of Cardiopulmonary Medicine,” notes Morrisey.
The Morrisey lab began with a couple of simple questions: how do the lung and heart co-develop and what are the critical signals that regulate this process? The breakthrough in this work occurred when the team characterized the expression pattern of the Wnt2 gene.
“Wnt2 is expressed in a unique place in the early embryo -- exactly in between the early heart and foregut tube, where the lung will arise from.” This allowed the researchers to create a model system in mice, whose cardiopulmonary anatomy is very similar to humans, and ask whether Wnt2-positive cells could coordinate heart and lung co-development.
Using cell lineage tracing analysis, they showed that Wnt2 cells generate single clones that, in turn, generate both heart and lung tissue, including cardiomyocytes and blood vessel cells such as vascular smooth muscle. Indeed, CPPs are capable of generating the vast majority of early embryonic cell types in the heart and lung. These studies also showed that the different cell lineages within the lung are related. For example, vascular smooth muscle and airway smooth muscle share a common progenitor cell in the lung.
The development of CPPs is regulated by the expression of another well-known protein called hedgehog, which is required for proper connection of the pulmonary vasculature to the heart. These studies show that hedgehog, which is also expressed by early lung progenitor cells, helps to promote CPPs to differentiate into the smooth muscle component of the pulmonary vasculature.
Together, these studies identify a novel population of multi-potent cardiopulmonary progenitors that coordinate heart and lung co-development, which is required for adaptation to terrestrial existence.
The finding that CPPs coordinate lung and heart co-development also has important implications for diseases that affect both organs, such as pulmonary hypertension. It is unclear whether pulmonary hypertension is primarily a lung disease or whether there are also intrinsic defects in the heart or cardiovascular system. The identification of CPPs could provide important insight into pulmonary hypertension and other diseases by identifying a common progenitor cell for both organs. Future studies will focus on whether CPPs exist in the adult cardiopulmonary system and whether they play a role in the response of the lung and heart to injury or disease.
Co-authors are Tien Peng, Ying Tian, Cornelis J. Boogerd, Min Min Lu, Rachel S. Kadzik, Kathleen M. Stewart, all from Penn, and Sylvia M. Evans, University of California, San Diego.
This work was funded by the Heart, Lung and Blood Institute (HL110942, HL100405, HL087825, HL117649) and the American Heart Association Jon DeHaan Myogenesis Center.
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Markers Discovered in Initial Stages of Disease Could be Used for Early Detection, Treatment, Prognosis
PHILADELPHIA — Pancreatic cancer carries a dismal prognosis. According to the National Cancer Institute, the overall five-year relative survival for 2003-2009 was 6 percent.
Still, researchers and clinicians don’t have a non-invasive way to even detect early cells that portent later disease. ‘There’s no PSA test for pancreatic cancer,’ they say, and that’s one of the main reasons why pancreatic cancer is detected so late in its course.
They have been searching for a human-cell model of early-disease progression. Now, Perelman School of Medicine, University of Pennsylvania scientists have used stem-cell technology to create a research cell line from a patient with advanced pancreatic ductal adenocarcinoma (PDAC).
“It is the first example using induced pluripotent stem [iPS] cells to model cancer progression directly from a solid tumor, and the first human cell line that can model pancreatic cancer progression from early to invasive stages,” says Zaret, also the associate director of the Penn Institute for Regenerative Medicine.
“We were able to predict the appearance of cellular features and protein markers in the intermediate stages of pancreatic cancer that are not evident in the terminal stages. This has given us new perspectives into what this deadly type of cancer looks like – something no one has seen before in human cells. Our analysis revealed known molecular networks that are activated during PDAC progression, as well as a new molecular network that is activated during the intermediate stages. This could provide a fresh outlook on biomarkers for early stages of the disease.”
A Leap of Faith
Zaret and first author Jungsun Kim, PhD, a postdoctoral associate in the Zaret lab, hypothesized that if human PDAC cells were reprogrammed back to pluripotent cells and then allowed to re-differentiate into pancreatic tissue, they might undergo the early stages of cancer. To do this, they created the PDAC pluripotent cells and indeed found that they recapitulated the early to intermediate stages of pancreatic cancer. They then isolated the cells at the early stage, cultured the cells in vitro, and identified the secreted and released proteins that might serve as early-stage biomarkers of disease progression.
There’s one caveat, though, says Zaret. “Using the iPS method, we could only get a cancer cell line from one patient to reprogram, meaning this work is representative of one individual’s cancer,” noting that his close collaboration with John Hoffman, a surgeon from the Fox Chase Cancer Center (FCCC) was key in order to get fresh cancer cells for the reprogramming. They tried this method with cells from nine human tumors in total. However, as Zaret points out, there are many examples of where a single human cell line has served as a highly useful model for human disease.
“Our iPS-like cells exhibited pluripotency, like other stem cells, but when they differentiated after we injected them into the immunodeficient mice, they preferentially developed into early-stage pancreatic cancer cells,” says Zaret. He explains that the approach is another example of using iPS cells from human patients to model disease, by capturing the genome of an affected individual. However, this is a first in solid tumor cancers, whereas many other labs have developed these types of cell lines for neurological and cardiovascular disorders.
The visual characteristics of these cancer cells -- as they progress from early- to late-stage cancer -- are typical of what is seen in cells analyzed from cancer patients in the clinic now. In the early stage, pancreatic ducts have lesions with cells of an abnormal shape, and express characteristic proteins as measured by stains. Over time, some of these aberrant cells may grow excessively, lose their ductal characteristics, and become invasive.
When the human PDAC iPS cells are grown as lesions in the mouse, they secrete or release proteins corresponding to protein networks expressed during the progression of human pancreatic cancer, namely molecules centered on a trio of key proteins: HNF4, integrin, and TGFbeta.
“We propose to look in the blood of potential pancreatic cancer patients or relatively early-stage patients for the biomarkers we found downstream of these molecular networks, to see if they are present in people,” says Zaret.
The Making of a Model
This approach allows human cells to be studied directly, as opposed to examining characteristics of pancreatic cancer progression in an animal model and then having to assess whether the findings apply to humans. The cells harvested from the cancer patient were reprogrammed using the four Yamanaka factors carried in a lentivirus, which was adapted by the Zaret lab.
To see what the reprogramming did at a genetic level, the team compared the genomic structure of the early iPS pancreatic cancer cell line to original tumor cells from which the cell line was isolated. They found 23 chromosomal aberrations in the primary tumor cells as compared to 20 of the same chromosomal aberrations in the PDAC iPS line, demonstrating that the PDAC iPS line was derived from the original tumor cells.
By contrast, no chromosomal abberations were seen in cells taken from the margin of the tumor in a cancer-free part of the pancreas from the patient, as well as from an iPS line that the Zaret group made from the margin cells.
These sets of comparisons allowed the team to directly observe the changes in the pancreatic cancer cell line versus a normal cell line derived from the same human genetic background.
“We understand that the pancreatic cancer field has been dogged by searching for unique markers in the blood that detect disease early, and we hope that this live-cell progression approach will gives us a new way to see early molecular markers for pancreatic cancer,” says Zaret.
“Since we can detect released protein markers of at least three different networks from the early-stage human lesions in our model, we think that looking for blood markers of the simultaneous activation the three pathways, instead of a single marker, could provide better leverage in detection. The new model could also be used to test drugs that block the intermediate stages of the disease. We would also like to know how we got lucky with this one cell line, so that the iPS technology can be adapted to model other human cancers.”
The research was funded by the National Institute for General Medical Sciences through an NIH Merit award (R37GM36477)
Other co-authors are R. Katherine Alpaugh, Andrew D. Rhim, Maximilian Reichert, Ben Z. Stanger, Emma E. Furth, Antonia R. Sepulveda, Chao-Xing Yuan, Kyoung-Jae Won, Greg Donahue, and Jessica Sands, all from Penn, and Andrew A. Gumbs, from FCCC.
By David Brown, The Washington Post
By Robb Stein and Michaeleen Doucleff, NPR
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Mutation Causing Wrong-Way Plumbing Explains One Type of Blue-Baby Syndrome
PHILADELPHIA — Total anomalous pulmonary venous connection (TAPVC), one type of “blue baby” syndrome, is a potentially deadly congenital disorder that occurs when pulmonary veins don’t connect normally to the left atrium of the heart. This results in poorly oxygenated blood throughout the body, and TAPVC babies are born cyanotic — blue-colored — from lack of oxygen.
TAPVC is usually detected in newborns when babies are blue despite breathing normally. Life-threatening forms of the disorder are rare – about 1 in 15,000 live births. A closely related, but milder disorder, partial anomalous pulmonary venous connection (PAPVC), in which only some of the pulmonary veins go awry, is found in as many as 1 in 150 individuals.
Now, researchers have found that a mutation in a key molecule active during embryonic development makes the plumbing between the immature heart and lungs short-circuit, disrupting the delivery of oxygenated blood to the brain and other organs. The mutation ultimately causes blood to flow in circles from the lungs to the heart’s right side and back to the lungs.
Senior author Jonathan A. Epstein, MD, chair of the Department of Cell and Developmental Biology, at the Perelman School of Medicine, University of Pennsylvania, and colleagues from The Children’s Hospital of Philadelphia, describe in Nature Medicine, that a molecule called Semaphorin 3d (Sema3d) guides the development of endothelial cells and is crucial for normal development of pulmonary veins. It is mutations in Sema3d that cause embryonic blood vessels to hook up in the wrong way.
Epstein is also the William Wikoff Smith professor and scientific director of the Penn Cardiovascular Institute. Karl Degenhardt, MD, PhD, assistant professor at The Children’s Hospital of Philadelphia; Manvendra K. Singh, PhD, an instructor of Cell and Developmental Biology at Penn; and Haig Aghajanian, a graduate student in Cell and Molecular Biology at Penn are the co-first authors on the paper.
Physicians thought that TAPVC occurred when the precursor cells of the pulmonary vein failed to form at the proper location on the embryonic heart atrium. However, analysis of Sema3d mutant embryos showed that TAPVC occurs despite normal formation of embryonic precursor veins.
In these embryos, the maturing pulmonary venous plexus, a tangle of vessels, does not connect just with properly formed precursor veins. In the absence of the Sema3d guiding signal, endothelial tubes form in a region that is not normally full of vessels, resulting in aberrant connections. Normally, Sema3d provides a repulsive cue to endothelial cells in this area, establishing a boundary.
Sequencing of Sema3d in individuals affected with anomalous pulmonary veins identified a point mutation that adversely affects Sema3d function in humans. The mutation causes Sema3d to lose its normal ability to repel certain types of cells to be able to guide other cells to grow in the correct place. When Sema3d can’t keep developing veins in their proper space, the plumbing goes haywire.
Since it’s already known that semaphorins guide blood vessels and axons to grow properly, the authors surmise that Sema3d could be used for anti-angiogenesis therapies for cancer, to treat diabetic retinopathy, or to help to grow new blood vessels to repair damaged hearts or other organs.
Daniele Massera, Qiaohong Wang, Jun Li, Li Li, Connie Choi, Amanda D. Yzaguirre, Lauren J. Francey, Emily Gallant, Ian D. Krantz, and Peter J. Gruber are co-authors.
This work was supported by the National Institutes of Health (NIH 5K12HD043245-07, NIH T32 GM07229, and NIH UO1 HL100405).
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Tweaking Gene Expression to Repair Lungs
PHILADELPHIA — Lung diseases such as asthma and chronic obstructive pulmonary disease (COPD) are on the rise, according to the American Lung Association and the National Institutes of Health.
“A healthy lung has some capacity to regenerate itself like the liver,” notes Ed Morrisey, Ph.D., professor of Medicine and Cell and Developmental Biology and the scientific director of the Penn Institute for Regenerative Medicine in the Perelman School of Medicine, University of Pennsylvania. “In COPD, these reparative mechanisms fail.”
Morrisey is looking at how epigenetics controls lung repair and regeneration. Epigenetics involves chemical modifications to DNA and its supporting proteins that affect gene expression. Previous studies found that smokers with COPD had the most significant decrease in one of the enzymes controlling these modifications, called HDAC2.
“HDAC therapies may be useful for COPD, as well as other airway diseases,” he explains. “The levels of HDAC2 expression and its activity are greatly reduced in COPD patients. We believe that decreased HDAC activity may impair the ability of the lung epithelium to regenerate.”
Using genetic and pharmacological approaches, they showed that development of progenitor cells in the lung is specifically regulated by the combined function of two highly related HDACs, HDAC/1 and /2. Morrisey and colleagues published their findings in this week’s issue of Developmental Cell.
By studying how HDAC activity, as well as other epigenetic regulators, controls lung development and regeneration, they hope to develop new therapies to alleviate the unmet needs of patients with asthma and COPD.
HDAC1/2 deficiency leads to a loss of expression of the key transcription factor, a protein called Sox2, which in turn leads to a block in airway epithelial cell development. This is affected in part by deactivating a repressor of expression (derepressing) of two other proteins, Bmp4 and the tumor suppressor Rb1 - targets of HDAC1/2.
In the adult lung, loss of HDAC1/2 leads primarily to increased expression of inhibitors of cell proliferation including the proteins Rb1, p16, and p21. This results in decreased epithelial proliferation in lung injury and inhibition of regeneration.
Together, these data support a critical role for HDAC-mediated mechanisms in regulating both development and regeneration of lung tissue. Since HDAC inhibitors and activators are currently in clinical trials for other diseases, including cancer, such compounds could be tested in the future for efficacy in COPD, acute lung injury, and other lung diseases that involve defective repair and regeneration, says Morrisey.
This work was funded by the National Heart, Lung and Blood Institute (HL071589, HL087825, HL100405, HL110942) and the Lung Repair and Regeneration consortium, funded by the NHLBI.
By studying the first 48 hours of stem cell reprogramming—in which adult somatic cells are converted into induced pluripotent stem cells (iPSCs)—researchers have identified some barriers to the process and one potential way to boost speed and efficiency early on. For now, the process is not very efficient: Only about one out of every 1000 human somatic cells wind up becoming iPSCs, and the process of conversion can take about a month. “[The finding] was thrilling, and we didn’t expect it at all,” said lead investigator Kenneth Zaret, PhD, associate director of the Penn Institute for Regenerative Medicine and Professor of cell and development, in BioTechniques News. Click here for full article.
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Penn Study Decodes Molecular Mechanisms Underlying Stem Cell Reprogramming
PHILADELPHIA — Fifty years ago, UK researcher John Gurdon demonstrated that genetic material from non-reproductive cells could be reprogrammed into an embryonic state when transferred into an egg. In 2006, Kyoto University researcher Shinya Yamanaka expanded on those findings by expressing four proteins in mouse somatic cells to rewind their genetic clocks, converting them into embryonic-like stem cells called induced pluripotent stem cells, or iPS cells.
In early October, Gurdon and Yamanaka were awarded the 2012 Nobel Prize in Physiology or Medicine for their discoveries. Now, thanks to some careful detective work by a team of scientists led by Kenneth Zaret, PhD, at the Perelman School of Medicine, University of Pennsylvania, researchers can better understand just how iPS cells form – and why the Yamanaka process is so inefficient, an important step to work out for regenerative medicine. Zaret is associate director of the Penn Institute for Regenerative Medicine and professor of Cell and Developmental Biology.
The findings, which appear in the Nov. 22 issue of the journal Cell, uncover cellular impediments to iPS cell development that, if overcome, could dramatically improve the efficiency and speed of iPS cell generation.
“These studies provide detailed insights into how reprogramming factors interact with the chromatin of differentiated cells and start them down the path toward becoming stem cells,” said Susan Haynes, PhD, National Institute of General Medical Sciences, which partially funded the work. “Dr. Zaret’s work also identified a major structural roadblock in the chromatin that the factors must overcome in order to bind DNA. This knowledge will help improve the efficiency of reprogramming, which is important for any future therapeutic applications.”
Human iPS cells are generated by expressing four DNA-binding proteins – Oct4, Sox2, Klf4, and c-Myc (O, S, K, and M) – in human non-reproductive, or somatic cells, such as skin cells. These factors have generated intense interest in the stem cell and medical communities, not least because they offer the promise of embryonic stem cells with none of the messy ethical and moral dilemmas. Just as significantly, patient-specific iPS cells from individuals with genetic disorders can be used to study disease origin and to develop drugs for a range of conditions such as Huntington’s and Parkinson’s diseases.
Yet, the process of generating iPS cells is highly inefficient. It can take a month to fully reprogram somatic cells into iPS cells, and as few as one in 10,000 cells that take up the four factors will successfully convert. What’s more, some studies indicate that, for all their plasticity, iPS cells are not precisely equivalent to embryonic stem cells. Zaret, with Penn postdoctoral fellow Abdenous Soufi, PhD, and bioinformatician Greg Donahue, PhD, decided to find out why.
The team analyzed the destination in the human genome of the four reprogramming factors 48 hours after the initiation of iPS cell reprogramming and compared those locations to four cell types: the starting cell population; the fully reprogrammed iPS cells; cells nearing the end of the reprogramming process (pre-iPS); and embryonic stem cells.
They found that at 48 hours the factors tended to bind gene regulatory elements called enhancers, far removed from the genes they regulate, rather than the target genes themselves. That suggests that O, S, and K serve as “pioneer factors” that open closed chromatin structures on the DNA itself, facilitating the reprogramming process by making target sections of the genome available to be read by messenger RNA.
The team also found large regions of the genome that were “refractory” to the binding of reprogramming factors at 48 hours, but which were eventually activated in, and are in fact required, for the formation of iPS cells.
“Basically, large chunks of the human genome were physically resisting these factors from entering,” Zaret explained. “That provided some understanding that you’ve got to overcome the binding impediment to get these factors to their final destination.”
These refractory sequences tended to be chemically marked with a histone modification called H3K9me3. When the team blocked the enzymes that create that modification, they “significantly accelerated” the reprogramming process.
According to Zaret, these findings reveal genetic roadblocks that slow and impede the iPS cell reprogramming process, as well as factors that may underlie the subtle differences between iPS and embryonic stem cells. They also suggest a potential workaround to these issues, by adding inhibitors of H3K9me3.
But the findings also reveal a normal cellular mechanism that cells may be using to repress genes that are contrary to the cell’s biology, Zaret said. “We went into this thinking we were going to learn something about the mechanism of conversion to pluripotency, but at the end of the day we ended up discovering new ways that cells control gene expression by shutting down parts of their genome.”
The study was supported by the NIGMS (Grants R37GM36477 and P01GM099134).
IRM Outreach Director, Dr. Jamie Shuda describes in the Penn Current, her new course in stem cell ethics and education. “Stem Cell Science in Schools: History, Ethics, and Education,” will give undergraduate students a better understanding what stem cells are and how they apply to their lives.
For full details, visit the Penn Current (click here).
|March 6, 2012|
CONTACT: Karen Kreeger
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Penn Medicine Science Educator Recognized by Society for Developmental Biology
PHILADELPHIA — Jamie Shuda, EdD, director of life science outreach at the University of Pennsylvania's Institute for Regenerative Medicine (IRM), and coordinator of life science education at the Netter Center for Community Partnerships also at Penn, along with Steve Farber, PhD, Investigator, Embryology Department, Carnegie Institution for Science, Baltimore, have been awarded the Hamburger Outstanding Educator Prize from the Society for Developmental Biology (SBD).
Shuda and Farber run Project BioEYES, a K-12 science education program that provides classroom-based, hands-on learning using live zebrafish to teach about how cells and animals develop. The program is located within the Perelman School of Medicine, Penn; the Carnegie Institution; Notre Dame University in South Bend, IN; and Monash University in Melbourne, Australia, among others, and reaches over 9,000 students per year.
"I am honored that the Society for Developmental Biology has chosen me and Dr. Farber as the 2012 recipients of the Viktor Hamburger prize," says Shuda. "Project BioEYES exemplifies how scientists and educators can come together to teach cutting edge, exciting science to students of all ages. Collaboration across disciplines is greatly supported by Penn and the IRM and it is wonderful that the university is being recognized for their public engagement. Viktor Hamburger was a pioneer in both science and teaching and I hope our education programs inspire more scientists just like him."
With over 10 years of experience in public education, Dr. Shuda has worked with teachers, students, and university staff to develop innovative science curricula. Her research focuses on the role informal science education plays in developing an effective science curriculum in K-12 schools and the characteristics of successful university and community partnerships to enhance science education at the undergraduate level. At the University of Pennsylvania, Dr. Shuda teaches Stem Cell Science in Schools: History, Ethics, and Education, which provides university and high school students with the opportunity to learn the science of stem cells while becoming deeply engaged with social and ethical issues relevant to everyday life. Dr. Shuda holds an MS.Ed and teaching certification from Drexel University and an Ed.D in education policy from Temple University.
Established in 2002 by the SDB Board of Directors in honor of Dr. Viktor Hamburger and sponsored by the Professional Development and Education Committee, this Hamburger award recognizes individuals who have made outstanding contributions to developmental biology education. The recipients deliver a lecture at the Education Symposium of the SDB Annual Meetings.