DAVID GRANOVSKY

Posts Tagged ‘scientist’

DISCOVERY OF PRIMARY CILIA IN STEM CELLS

In ALL ARTICLES, SCIENCE & STEM CELLS on January 7, 2013 at 10:40 am
“…since stem cells are now being more routinely used for regenerative medicine such as repair of severed spinal cord (Lu et al. 2012), it behooves us to better learn the molecular mechanisms that keeps these invaluable cells in an undifferentiated state so that we can harness their full therapeutic potential.”
Discovery of Primary Cilia in Stem Cells

Author: Aashir Awan, PhD

The primary cilium is organelle that has garnered much attention in the field of cell biology during the last 15 years. It is a slender, solitary hair-like organelle that extends 5-10 uM from each mammalian cell (in the G0 cell cycle state) that is microtubule-based (9 outer doublets arranged in a circular fashion) and dependent on a process called Intraflagellar Transport (IFT). IFT is the bidirectional movement of motors (kinesin-2 in the anterograde and dynein-2 in the retrograde direction) responsible for the assembly and maintenance of the cilium (Pedersen et al., 2006).

Until this time, it had been labeled a ‘vestigial’ organelle not worthy of research. Yet, a breakthrough into the sensory role of the primary cilium came in 2000 based on Dr. Rosenbaum’s research on Chlamydomonas and the motile cilium or flagella. Along with Dr. George Whitman’s group, they were able to show the importance of Tg737 (IFT88) protein to the pathology of polycystic kidney disease in mouse (Pazour et al., 2000). Since then, research into the primary cilium has exploded and has been linked to diverse pathologies (collectively known as ciliopathies) such as

  • retinitis pigmentosa,
  • hydrocephaly,
  • situs inversus,
  • ovarian and pancreatic cancers among others (Nielsen et al., 2008; Edberg et al., 2012). Also, various
  • signal transduction pathways have been found to be coordinated by the primary cilia such as hedgehog, wnt, PDGF among others (Veland et al., 2008).

Thus, in 2006, the Christensen lab at the University of Copenhagen (Denmark) with the collaboration of Dr. Peter Satir’s group at Albert Einstein College of Medicine (Bronx, NY) began to investigate whether the human embryonic stem cells (hESCs) possess primary cilium and then to begin preliminary molecular dissections of the role that this organelle could play in the proliferation and differentiation profiles of these pluripotent cells. The Albert Einstein group, due to NIH restrictions, had to work with two federally-sanctioned cell lines. Working with the Laboratory of Reproductive Biology at RigsHospital, the Danish side had access to in-house derived stem cell lines from discarded blastocysts. The advantage for the Danish side was obvious since these newer cell lines hadn’t undergone as many passages as the NIH cell lines and were thus more robust. To begin preliminary characterizations of these lines, some basic hallmarks of hESCs (Bernhardt et al., 2012) had to be localized to the nucleus such as the transcription factor (TF) Oct4 (Fig. 1).

In addition, a single primary cilium can be seen denoted by the acetylated tubulin staining emanating from each cell in the micrographs. Also, the base of the cilium is marked by the presence of pericentrin and centrin which demarcate the centriole.

Fig1 Fig. 1 Primary cilia stained with anti-acetylated tubulin (tb, red) are indicated by arrows and undifferentiated stem cells are identified by nuclear colocalization of OCT-4 (green) and DAPI (dark blue) in the merged image (light blue). A primary cilium (tb, red, arrow) in undifferentiated hESCs emerges from one of the centrioles (asterisks) marked with anti-centrin (centrin, green). Inset shows anti-pericentrin localization to base of cilia (Pctn, green).

Together, the three labs were the first to discover primary cilia in stem cells while other groups have since then confirmed these findings (Kiprilov et  al. 2008; Han et al. 2008). Attention was then to characterize different signal transduction pathways in the stem cell cilium. Since the hedgehog pathway has been shown to be important for differentiation and proliferation (Cerdan and Bhatia, 2012), the groups characterized this signal pathway in these cells using immunofluorescence, electron microscopy and qPCR techniques. One particularly interesting experiment to show that the hedgehog pathway was functional in these cells was to add the hedgehog agonist, SAG (Smoothened agonist), and then to isolate the cells for immunofluorescence at different times.

Gradually, one can see the appearance of the smoothened protein into the cilium as indicated by increasing intensity of the immunofluorescence staining. Conversely, patched levels in the cilium, decreased. This is a hallmark of hedgehog activation (Fig. 2).
Fig. 2 copiaFig. 2 Immunofluorescence micrographs of hESC showing smoothened (green), acetylated tubulin (red) and DAPI (blue). The micrographs from left to right represents SAG treatments at t = 0, 1 and 4 hours.

However, an additional interesting observation was made concerning these stem cells. An important characteristic for stem cells is the presence of certain transcription factors which render these cells in the pluripotent or undifferentiated state. These include Oct4, Sox2, and Nanog whose localization had been observed in the nucleus as expected for other TFs.

However, the Danish groups curiously found a subpopulation of stem cells where these TFs were additionally localized to the primary cilium (Fig. 3). This had never been observed or investigated before.  Additionally, proper negative controls were  carried out to exclude this phenomenon from being an artifact (e.g. bleed through).
Fig. 3 copia Fig. 3 Stem cell markers (Sox2, Nanog, and Oct4) localizing to the nucleus and the primary cilia (arrows) of hESC line LRB003. This and the previous figure show shifted overlay images whereby the green and red channels have been slightly shifted so that the red channel doesn’t swamp out the intensity of the green channels.

Thus, it raises an intriguing possibility that perhaps the primary cilia plays a previously uncharacterized role in the differentiation/proliferation state of the hESCs via possible modifications of these TFs perhaps analogous to the processing of the Gli transcription factors (Hui and Angers, 2011). Another curious observation is that the subpopulation of cells whose primary cilia are positive for these TFs always occur in clusters which might hint at its mechanistic explanation.  In conclusion, since stem cells are now being more routinely used for regenerative medicine such as repair of severed spinal cord (Lu et al. 2012), it behooves us to better learn the molecular mechanisms that keeps these invaluable cells in an undifferentiated state so that we can harness their full therapeutic potential.

REFERENCES

Awan A, Oliveri RS, Jensen PL, Christensen ST, Andersen CY. 2010 Immunoflourescence and mRNA analysis of human embryonic stem cells (hESCs) grown under feeder-free conditions. Methods Mol Biol. 584:195-210.

Bernhardt M, Galach M, Novak D, Utikal J. 2012 Mediators of induced pluripotency and their role in cancer cells – current scientific knowledge and future perspectives. Biotechnol J. 7:810-821.

Cerdan C, Bhatia M. 2010 Novel roles for Notch, Wnt and Hedgehog in hematopoesis derived from human pluripotent stem cells. Int J Dev Biol. 54:955-963.

Han YG, Spassky N, Romaguera-Ros M, Garcia-Verdugo JM, Aguilar A, Schneider-Maunoury S, Alvarez-Buylla A. 2008 Hedgehog signaling and primary cilia are required for the formation of adult neural stem cells.Nat Neurosci. 11:277-284.

Hui CC, Angers S. 2011 Gli proteins in development and disease. Annu Rev Cell Dev Biol. 27:513-537.

Kiprilov EN, Awan A, Desprat R, Velho M, Clement CA, Byskov AG, Andersen CY, Satir P, Bouhassira EE, Christensen ST, Hirsch RE 2008 Human embryonic stem cells in culture possess primary cilia with hedgehog signaling machinery. J Cell Biol. 2008 180:897-904.

Lu P, Wang Y, Graham L, McHale K, Gao M, Wu D, Brock J, Blesch A, Rosenzweig ES, Havton LA, Zheng B, Conner JM, Marsala M, Tuszynski MH. 2012 Long-distance growth and connectivity of neural stem cells after severe spinal cord injury. Cell 150:1264-73.

Nielsen SK, Møllgård K, Clement CA, Veland IR, Awan A, Yoder BK, Novak I, Christensen ST. 2008 Characterization of primary cilia and Hedgehog signaling during development of the human pancreas and in human pancreatic duct cancer cell lines. Dev Dyn. 237:2039-52.

Pazour GJ, Dickert BL, Vucica Y, Seeley ES, Rosenbaum JL, Witman GB, Cole DG. 2000 Chlamydomonas IFT88 and its mouse homologue, polycystic kidney disease gene tg737, are required for assembly of cilia and flagella. J Cell Biol 151: 709-18.

Pedersen LB, Veland IR, Schrøder JM, Christensen ST. 2008 Assembly of primary cilia. Dev Dyn. 237:1993-2006.

Veland IR, Awan A, Pedersen LB, Yoder BK, Christensen ST. 2009 Primary cilia and signaling pathways in mammalian development, health and disease. Nephron Physiol. 111: 39-53.

Discovery of Primary Cilia in Stem Cells.

//

Babies Can Be Treated With Adult Stem Cells, Even in the Womb | LifeNews.com

In VICTORIES & SUCCESS STORIES on February 13, 2011 at 9:40 am

This is AWESOME!

They’ve been trying to treat fetuses in the womb with stem cells for awhile now (adult stem cells, NOT embryonic!  embryonic can cause tumors!)  In the past, they tried to use the baby’s own stem cells…with mixed results.

“But despite the fact that the immature immune system of an unborn baby can tolerate donor transplants, with little risk of graft rejection, most previous attempts to transplant blood stem cells into a human fetus have been unsuccessful…”

BUT

“When transplants were matched to the mother rather than the fetus, the transplants were accepted!…Transplanting stem cells harvested from the mother makes sense because the mother and her developing fetus are prewired to tolerate each other.”

IMAGINE!

‘in-the-womb treatments for “everything from neurological disorders to muscular disorders before birth.”’


Two recent stories are exciting about the possibility of treating young children, even in the womb, with adult stem cells. One study shows that cardiac adult stem cells can be isolated from young children with heart problems, even as young as one day old…

MORE BAD NEWS ABOUT AVAILABLE US TREATMENTS FOR HEART DISEASE

In VICTORIES & SUCCESS STORIES on February 12, 2011 at 9:41 am

MORE BAD NEWS ABOUT AVAILABLE US TREATMENTS FOR HEART DISEASE

Most Americans with the biggest risks for heart disease are not doing enough to control these risks, and the fragmented U.S. healthcare system is partly to blame, federal health officials said on Tuesday…

U.S. health system not helping heart disease: CDC

THE COST OF NOT PURSUING ADULT STEM CELL TREATMENTS IN THE USA

In VICTORIES & SUCCESS STORIES on February 9, 2011 at 9:40 am
COST BY DISEASE # OF PATIENTS COST PER YEAR
TYPE 1 DIABETES 5.8 MILLION PATIENTS $125.1 BILLION
STROKE 5.7 MILLION PATIENTS $62.7 BILLION
HEART FAILURE 5.2 MILLION PATIENTS $50.7 BILLION
SPINAL CORD INJURY 250,000 PATIENTS $37.2 BILLION
PARKINSON’S DISEASE 650,000 PATIENTS $23 BILLION

TOTAL COST THAT ADULT STEM CELLS COULD SAVE THE UNITED STATES

$250 BILLION PER YEAR


Stem cells may hold secret to multiple spinal injury repair

In VICTORIES & SUCCESS STORIES on February 8, 2011 at 9:40 am

CATCH UP!  They’ve been treating SCI patients successfully with stem cells for years now!

Stem cells may hold secret to multiple spinal injury repair

21 January 2011

Scientists may soon be able to repair multiple aspects of a spinal cord injury in humans, using adult stem cells.

A Case Western Reserve University School of Medicine study involving rats showed that use of multipotent adult progenitor cells (mapc) could prevent neuron retraction, promote new neuron re-growth and reduce inflammation in spinal injury.

Researchers will no doubt be hopeful that the development of such treatment can contribute significantly to reducing the costs of spinal injury treatment, which can range between $500,000 (£312,000) and $3 million (£1.9 million) in the US.

Commenting on the findings, published in the Journal of Neuroscience, university professor Jerry Silver explained: “Using preclinical models of spinal cord injury, we found that MAPC can both dynamically regulate macrophages, which cause inflammatory damage, and stimulate neuron growth simultaneously.

“Our results demonstrate that MAPC convey meaningful therapeutic benefits after spinal cord injury.”

Meanwhile, recent research conducted at Otago University suggests that cell tissue extracted from the nose could be transplanted to the site of spinal cord injury.

Scientists are hopeful the procedure could overcome blocks that prevent nerve cells regenerating following damage.

News by Serious Law. Expert legal assistance for those affected by spinal injury

Posted by Timothy Walters

arguendo > Super Stemmys, a stem cell story

In VICTORIES & SUCCESS STORIES on April 15, 2010 at 7:08 pm

Super Stemmys, a stem cell story

“We need more children’s books like this.”

Posted by Jimalakirti in Books, Critical Thinking, General

at 11:00 am on Saturday, 10 April 2010

http://www.the-scientist.com/blog/display/57276/

Stem cells to save the day! Or the heart, at least. That’s the plot of a new children’s book on adult (or repair) stem cells, published by theRepair Stem Cell Institute(RSCI) — a Dallas- and Bangkok-based public affairs company that provides interested patients with contact information for stem cell treatment centers around the world.

“It’s a nice idea,” said cell biologist Mahendra Rao ofLife Technologies,a California-based biotechnology company. “I think it’s good to tell kids about all current events, [including] technological breakthroughs,” and “it’s a nice book for kids [with] illustrations [that] are nice and a logical flow to it.”

(The-scientist.com, April 8, 2010)

Comments (1)

Comment by Jimalakirti at 11:11 am on 10 April 2010 at

The subject of this book is a little off–topic for us. But, since it is about science education (and we are all about science education), it is worth while to look at this attempt to introduce 6th graders to some basic concepts about stem-cell research and use.

The question that dogs nearly every attempt to use a story to teach children about scientific subjects is that the story oversimplifies, or is too limited in scope, or some such objection. These objections are out of bounds if the story doesn’t distort the science or represent bad science. A story has its own needs and to ignore the needs of a story for children in order to get a bit more complicated science into it is disastrous.

A children’s book cannot be a complete science course. It is enough that the science be basically accurate, so that the reader will gain some familiarity with basic concepts in a format that helps the reader to understand and remember. Then, someday, when the student encounters the subject in a class or in a more advanced book, she can recognize it and have a bit of a head start on people who have never heard of it (or have been taught that it is evil meddling in god’s business).

We need more children’s books like this.

via arguendo > Super Stemmys, a stem cell story.

FRC Blog » Adult Stem Cells for Vascular Grafts

In VICTORIES & SUCCESS STORIES on April 13, 2010 at 6:39 pm

Researchers have used adult stem cells to create functional blood vessels that can function for bypass surgeries. The work was presented at the American Heart Association’s Arteriosclerosis, Thrombosis and Vascular Biology Annual Conference 2010. For many people undergoing bypass surgery, blocked arteries are replaced using another vessel from a different part of the body. But many patients don’t have a suitable replacement vessel and synthetic grafts are used. However, the synthetic vessels often become clogged within a couple of years.

Dr. Stephen McIlhenny and his group at Thomas Jefferson University Hospital in Philadelphia have used adult stem cells from fat tissue to create functional blood vessels. Testing the process in rabbits, they grew adult stem cells on human vein scaffolds in the lab. Grafts were prepared using adult stem cells from each test rabbit, then the graft was put back into the individual rabbits so they received grafts containing their own cells, removing the risk of transplant rejection. After eight weeks, rabbits receiving the customized grafts fared better than those receiving synthetic grafts.

Dr. McIlhenny said

“We found that using the stem cells as a coating prevented clotting and thickening of the graft wall. I would say those grafts were significantly better. Potentially, patients requiring bypass surgery could receive optimized grafts that would reduce their future complications.”

McIlhenny’s group has previously done work growing smooth muscle from adult stem cells for vascular grafts, and developed a method to prevent shearing of the adult stem cells from the scaffold.

via FRC Blog » Adult Stem Cells for Vascular Grafts.

Magnetic attraction of stem cells creates more potent treatment for heart attack

In VICTORIES & SUCCESS STORIES on April 13, 2010 at 6:20 pm

NASA spent 3 billion on developing a zero grav pen for use in in space…the Russians used a pencil.
Likewise, as the biotech industry spends hundreds of millions on smart/nanotechnology devices to send medicines and cells to specific sites or organs…Cedars-Sinai uses a magnet and iron dust.

Sometimes, old school is definitely the way to go. -dg

http://repairstemcell.files.wordpress.com/2009/04/heart2.jpg?w=800&h=600Magnetic Attraction of Stem Cells Creates More Potent Treatment for Heart Attack

ScienceDaily (Apr. 10, 2010) — Researchers at the Cedars-Sinai Heart Institute have found in animals that infusing cardiac-derived stem cells with micro-size particles of iron and then using a magnet to guide those stem cells to the area of the heart damaged in a heart attack boosts the heart’s retention of those cells and could increase the therapeutic benefit of stem cell therapy for heart disease.

via Magnetic attraction of stem cells creates more potent treatment for heart attack.

How can you heal a broken heart? | ScienceBlog.com

In VICTORIES & SUCCESS STORIES on April 12, 2010 at 4:22 pm

More stuff we already know…but we certainly appreciate all new trials/studies that reiterate the benefits of stem cell therapy on the heart.

How can you heal a broken heart?

Montréal, Québec, Canada, April 12, 2010 – Some patients with heart muscles seriously affected by coronary heart disease may soon be able to benefit from an innovative treatment. Researchers at the Research Centre of the Centre hospitalier de l’Université de Montréal (CRCHUM), in collaboration with the Maisonneuve-Rosemont Hospital (MRH) are evaluating the safety, feasibility and efficacy of injecting stem cells into the hearts of patients while they are undergoing coronary bypass surgery. These stem cells could improve healing of the heart and its function.

The IMPACT-CABG (implantation of autologous CD133+ stem cells in patients undergoing coronary artery bypass grafting) protocol evaluates this experimental procedure, which is destined for patients suffering from ischemic heart disease, in which the blood supply to the heart is decreased and associated with heart failure. These patients undergo open-heart coronary bypass surgery, performed by the medical team to improve perfusion of the heart muscle. A few weeks ago, the first patient received progenitor CD133+ stem cells isolated from his bone marrow and enriched at the Cell Therapy Laboratory of the MRH, and has been doing very well ever since. Already, improvement has been noted in the contraction capacity of his heart, which has improved its ability to pump blood…

Full story: How can you heal a broken heart? | ScienceBlog.com.

First Ever Multicellular Animals Found In Oxygen-Free Environment | Popular Science

In OFF THE BEATEN PATH on April 7, 2010 at 3:48 pm

First Ever Multicellular Animals Found In Oxygen-Free Environment

Unnamed Loriciferan:   via Nature

By Stuart Fox Posted 04.06.2010 at 5:30 pm

In the 236 years since oxygen was identified as a life-giving necessity, no scientist anywhere has discovered a multicellular animal capable of living without the stuff. Until now. Researchers from the Polytechnic University of Marche in Ancona, Italy, have discovered three new species that live their entire life in an anoxic pit beneath the Mediterranean Sea. This discovery drastically revises science’s understand of where animals can thrive.Prior to this discovery, the only organisms capable of life in oxygen-free environments were viruses and bacteria.

via First Ever Multicellular Animals Found In Oxygen-Free Environment | Popular Science.

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