Stem Cell Treatments to Improve Blood Flow in Angina Patients


Angina pectoris is defined as chest pain or discomfort that results from poor blood flow through the blood vessels in the heart and is usually activated by activity or stress.

In Los Angeles, California, physicians have initiated a double-blind, multicenter Phase III clinical trial that uses a patient’s own blood-derived stem cells to restore circulation to the heart of angina patients.

This procedure utilizes state-of-the-art imaging technology to map the heart and generate a three-dimensional image of the heart. These sophisticated images will guide the physicians as they inject stem cells into targeted sites in the heart.

This is a double-blinded study, which means that neither the patients nor the researcher will know who is receiving stem-cell injections and who is receiving the placebo.

The institution at which this study is being conducted, University of Los Angeles (UCLA), is attempting to establish evidence for a stem cell treatment that might be approved by the US Food and Drug Administration for patients with refractory angina. The subjects in this study had received the standard types of care but did not receive relief. Therefore by enrolling in this trial, these patients had nothing to lose.

Dr. Ali Nasir, assistant professor of cardiology at the David Geffen School of Medicine and co-principal investigator of this study, said: “We’re hoping to offer patients who have no other options a treatment that will alleviate their severe chest pain and improve their quality of life.”

Before injecting the stem cells or the placebo, the team examined the three-dimensional image of the heart and ascertained the health of the heart muscle and voltage it generated. Damaged areas of the heart fail to produce adequate quantities of voltage and show low levels of energy.

Jonathan Tobis, clinical professor of cardiology and director of interventional cardiology research at Geffen School of Medicine, said: “We are able to tell by the voltage levels and motion which area of the [heart] muscle is scarred or abnormal and not getting enough blood and oxygen. We then targeted the injections to the areas just adjacent to the scarred and abnormal heart muscle to try to restore some of the blood flow.”

What did they inject? The UCLA team extracted bone marrow from the pelvic bones and isolated CD34+ cells. CD34 refers to a cell surface protein that is found on bone marrow stem cells and mediates the adhesion of bone marrow stem cells to the bone marrow matrix. It is found on the surfaces of hematopoietic stem cells, placental cells, a subset of mesenchymal stem cells, endothelial progenitor cells, and endothelial cells of blood vessels. These are not the only cells that express this cell surface protein, but it does list the important cells for our purposes. Once the CD34+ cells were isolated, the were injected into the heart through a catheter that was inserted into a vein in the groin.

CD34

The team hopes that these cells (a mixture of mesenchymal stem cells, hematopoietic stem cells, and endothelial progenitor cells) will stimulate the growth of new blood vessels (angiogenesis) in the heart, and improve blood flow and oxygen delivery to the heart muscle.

“We will be tracking patients to see how they’re doing,” said William Suh MD, assistant clinical professor of medicine in the division of cardiology at Geffen School of Medicine.

The goal of this study is to enroll 444 patients nation-wide, of which 222 will receive the stem cell treatment, 111 will receive the placebo, and 111 who will be given standard heart care.

Reprogramming cell in tissue repair


Outrageous data – transfecting adult cells with the Lin28a/b genes induce a kind of fetal state where they can increase responsiveness to glucose in laboratory animals, and resist obesity and prevent diabetes. This is remarkable. Read it for yourself here.

Leaders in Pharmaceutical Business Intelligence (LPBI) Group

Reprogramming Cell in Tissue Repair

Reporter and Curator: Larry H Bernstein, MD, FCAP

This is a novel concept in regenerative medicine that needs attention.

Lin28 enhances tissue repair by reprogramming cellular metabolism

Shyh-Chang N, Zhu H, Yvanka de Soysa T, Shinoda G, Seligson M T, Tsanov K M, Nguyen L, Asara J M, Cantley L C and Daley G Q.

Stem Cell Transplantation Program,Boston Children’s Hospital and Dana Farber Cancer Institute, Boston; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School; Harvard Stem Cell Institute;
Manton Center for Orphan Disease Research; Howard Hughes Medical Institute; Department of Medicine, Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, MA 02115.

Cell.  7 Nov 2013; 155(4):778-792.    http://dx.doi.org/10.1016/j.cell.2013.09.059.

Lin28 overview

Copyright © 2013 Elsevier Inc.  PMID:     23561442     PMCID:     PMC3652335

Abstract

In recent years, the highly conserved Lin28 RNA-binding proteins have emerged as factors that define stemness in several tissue lineages. Lin28 proteins repress let-7 microRNAs and influence mRNA translation, thereby regulating the self-renewal of mammalian embryonic stem cells. Subsequent discoveries revealed that Lin28a and Lin28b are also important in organismal growth and metabolism, tissue development, somatic reprogramming, and cancer. In this review, we discuss the Lin28 pathway and its regulation, outline its roles in stem cells, tissue development, and pathogenesis, and examine the ramifications for re-engineering mammalian physiology.

Figure 1. Overview of Molecular Mechanisms Underlying Lin28 Function. From: Lin28: Primal…

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Human neural stem cells could meet the clinical problem of critical limb ischemia – Science Codex


Neural stem cell lines show remarkable efficacy in animal models of limb ischemia (limbs that lose oxygen delivery to them). Enjoy.

Leaders in Pharmaceutical Business Intelligence (LPBI) Group

See on Scoop.itCardiovascular and vascular imaging

Human neural stem cells could meet the clinical problem of critical limb ischemia
Science Codex
New research has shown human neural stem cells could improve blood flow in critical limb ischemia through the growth of new vessels.

See on www.sciencecodex.com

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Stem Cell Therapy for Patients with Ischemic Cardiomyopathy


A medical research group from Miami Miller School of Medicine has examined the safety of transendocardial stem cell injections with a patient’s own bone marrow stem cells in patients with ischemic cardiomyopathy.

Ischemic cardiomyopathy is the most common type of “dilated cardiomyopathy,” which is a fancy way of saying that the heart enlarges in its failing struggle to supply the body with blood. The enlarged heart has more heart muscle to feed with oxygen, but because the heart enlarges faster than the blood vessels remodel, large portions of the enlarged heart are left without adequate blood supply, and the result is and oxygen deficit, also known as “ischemia.” In patients with ischemic cardiomyopathy, the heart’s ability to pump blood is decreased because the heart’s main pumping chamber, the left ventricle, is enlarged, dilated and weak. Usually, heart ischemia also results from coronary artery disease and heart attacks.

The symptoms of ischemic CM include shortness of breath, swelling of the legs and feet (edema), Fatigue (feeling overly tired), inability to exercise, or carry out activities as usual, angina (chest pain or pressure that occurs with exercise or physical activity and can also occur with rest or after meals), weight gain, cough and congestion related to fluid retention, palpitations or fluttering in the chest due to abnormal heart rhythms (arrhythmia), dizziness or light-headedness, and fainting (caused by irregular heart rhythms, abnormal responses of the blood vessels during exercise, without apparent cause).

Clearly an effective regenerative treatment of ischemic cardiomyopathy (ICM) would address of the needs of some of these patients. Bone marrow transplants into the heart have been tested as treatments and the stem cells were directly injected into the heart muscle (see Williams AR, et al., Circ Res. 2011;108(7):792-796; and Losordo DW, et al., Circ Res. 2011;109(4):428-436). Both of these studies, however used mononuclear cells from bone marrow. Mononuclear cells refer to white blood cells from bone marrow and it includes a wide variety of stem cells, progenitor cells, and other mature white blood cells, but excludes red blood cells or platelets, which have no nuclei.

In order to determine if mesenchymal stem cells were also safe for this type of treatment, Alan W. Haldman and his colleagues from the laboratory of Joshua M. Hare tested 65 patients who suffered from ICM and compared injection of mesenchymal stem cells (n = 19) with placebo (n = 11) and bone marrow mononuclear cells (n = 19). Patients were followed up to one year after their procedures.

To measure serious adverse effects of the procedure, all patients were evaluated at 30 days post-procedure. Severe adverse effects includes death, heart attack, stroke, hospitalization for worsening heart failure, perforation of rupture of the heart, tamponade (compression of the heart due to a collection of fluid around it), or sustained ventricular arrhythmias.

None of the patients in this study showed any severe adverse events up to day 30, and up to 1 year after the procedure, 31.6% of the bone marrow mononuclear and mesenchymal stem cell groups had some sort of serious adverse event, and 38.1% of the placebo group had serious adverse events.

Over one year, the Minnesota Living with Heart Failure score, which is a measure of the quality of life of a heart patient, improved with the mesenchymal stem cell and bone marrow cells but not with the placebo. Also, the 6-minute walk distance increased in the mesenchymal stem cell group, but none of the other groups when the baseline time was compared with the six-month and 12-month trials.

Patients in the mesenchymal stem cell group exhibited a significant increase in 6-minute walk distance when 6-month and 12-month time points were compared to baseline in a repeated measures model (P = .03). No significant difference was observed for patients in the bone marrow cell group (P = .73) or in the placebo group (P = .25). Data markers represent means; error bars, 95% CIs. Analysis of variance (ANOVA) was conducted with repeated measures.aWithin group, P<.05.bWithin group, P<.01.
Patients in the mesenchymal stem cell group exhibited a significant increase in 6-minute walk distance when 6-month and 12-month time points were compared to baseline in a repeated measures model (P = .03). No significant difference was observed for patients in the bone marrow cell group (P = .73) or in the placebo group (P = .25). Data markers represent means; error bars, 95% CIs. Analysis of variance (ANOVA) was conducted with repeated measures.aWithin group, P

Also, the size of the heart scar showed greater shrinkage in the mesenchymal stem cell group than in the other groups.

Significant reduction in scar size as the percentage of left ventricular mass for patients treated with mesenchymal stem cells (MSCs) and those in the placebo group who underwent serial magnetic resonance imaging. Repeated measures of analysis of variance model P values: treatment group, P=.99; time, P=.007; treatment group × time, P=.22. Data markers represent means; error bars, 95% CIs. Analysis of variance (ANOVA) was conducted with repeated measures.aWithin group, P<.05 vs baseline.bWithin group, P<.01 vs baseline.
Significant reduction in scar size as the percentage of left ventricular mass for patients treated with mesenchymal stem cells (MSCs) and those in the placebo group who underwent serial magnetic resonance imaging. Repeated measures of analysis of variance model P values: treatment group, P=.99; time, P=.007; treatment group × time, P=.22. Data markers represent means; error bars, 95% CIs. Analysis of variance (ANOVA) was conducted with repeated measures.aWithin group, P

And if a more visual way to view this would help, here is the heart of one particular patient.  Notice the shrinkage in the red area, which represents the scarred area, after one year.

A, Short-axis views of the basal area of a patient’s heart, with delayed tissue enhancement delineated at the septal wall. Delayed tissue enhancement corresponds to scarred tissue and is depicted brighter than the nonscarred tissue (automatically detected and delineated with red using the full width at half maximum technique). The red, green, and white lines demarcating the endocardial, epicardial contours, and borders of the segments, respectively, were drawn manually. Twelve months after injection of mesenchymal stem cells, scar mass was reduced from 30.85 g at baseline to 21.17 g at 12 months. B, Long-axis 2-chamber views of the same heart with delayed tissue enhancement delineated at the anterior and inferior wall, as well as the entire apex. At baseline and at 12 months after injection of mesenchymal stem cells, the delayed tissue enhancement receded in the midinferior and basal anterior walls (see Interactive of representative cardiac MRI cine sequences).
A, Short-axis views of the basal area of a patient’s heart, with delayed tissue enhancement delineated at the septal wall. Delayed tissue enhancement corresponds to scarred tissue and is depicted brighter than the nonscarred tissue (automatically detected and delineated with red using the full width at half maximum technique). The red, green, and white lines demarcating the endocardial, epicardial contours, and borders of the segments, respectively, were drawn manually. Twelve months after injection of mesenchymal stem cells, scar mass was reduced from 30.85 g at baseline to 21.17 g at 12 months. B, Long-axis 2-chamber views of the same heart with delayed tissue enhancement delineated at the anterior and inferior wall, as well as the entire apex. At baseline and at 12 months after injection of mesenchymal stem cells, the delayed tissue enhancement receded in the midinferior and basal anterior walls (see Interactive of representative cardiac MRI cine sequences).

The authors concluded from this study that these “results provide the basis for larger studies to provide definitive assessment of safety and to assess efficacy of this new therapeutic approach.”  Mesenchymal stem cells might certainly provide a way to treat ICM patients.  Also, if the patient’s bone marrow is of poor quality as a result of their poor health, then mesenchymal stem cells from a donor might provide healing for these patients.  For now, I say, “bring on the larger trials!!”

Artificial Skin Created Using Umbilical Cord Stem Cells


Major burn patients usually must wait weeks for artificial skin to be grown in the laboratory to replace their damaged skin, buy a Spanish laboratory has developed new protocols and techniques that accelerate the growth of artificial skin from umbilical cord stem cells. Such laboratory-grown skin can be frozen and stored in tissue banks and used when needed.

Growing skin in the laboratory requires the acquisition of keratinocytes, those cells that compose the skin and the mucosal covering inside our mouths.  Keratinocytes can be cultured in the laboratory, but they have a long cell cycle, which means that they take a really long time to divide.  Consequently, cell cultures of keratinocytes tend to take a very long time to grow.

Keratinocytes in culture
Keratinocytes in culture

As they grow, the keratinocytes respond to connective tissue underneath them to receive the cues that tell them how to connect with each other and form either skin or oral mucosa.  In patients with severe burns, however, the underlying connective tissue is also often damaged.  Therefore, finding a way to not only accelerate the growth of cultured keratinocytes, but also to provide the underlying structure that directs the cells to form a proper epithelium is essential.

Remember that severe burn patients are living on borrowed time.  Without a proper skin covering, water loss is severe and dehydration is a genuine threat.  Also, infection is another looming threat.  Therefore, the treatment of a burn patient is a race against time.

Because umbilical cord stem cells grow quickly and effectively in culture, they might be able to differentiate into keratinocytes and form the structures associated with oral mucosa and skin.

University of Granada researchers used a new type of epithelial covering to grow their artificial skin in addition to a biomaterial made of fibrin (the stiff, cable-like protein that forms clots) and agarose to provide the underlying connective tissue. In case you might need a refresher, an epithelium refers to a layer of cells that have distinct connects with each other and form a discrete layer. Epithelia can form single or multiple layers and can be composed of long, skinny cells, short, flat cells, or boxy cells.  An epithelium is a membrane-like tissue composed of one or more layers of cells separated by very little intervening substances.  Epithelia cover most internal and external surfaces of the body and its organs.

Previous work from this same research group showed that stem cells from Wharton’s jelly (connective tissue within the umbilical cord), could be converted into epithelial cells. This current study confirms and extends this previous work and applies it to growing skin, and oral mucosa.

“Creating this new type of skin suing stem cells, which can be stored in tissue banks, mains that it can be used instantly when injuries are caused, and which would bring the application of artificial skin forward many weeks,” said Antonio Campos, professor of histology and one of the authors of this study.

By growing the Wharton’s jelly stem cells on their engineered matrix in a three-dimensional culture system, Campos and his colleagues saw that the stem cells stratified (formed layers), and expressed a bunch of genes that are peculiar to skin and other types of epithelia that cover surfaces (e.g., cytokeratins 1, 4, 8, and 13; plakoglobin, filaggrin, and involucrin).  When examined with an electron microscope, the cells had truly formed the kinds of tight connections and junctions that are so common to skin epithelia.

Electron microscopy analysis of controls and three-dimensional bioactive models of H-hOM and H-hS. SEM images (top) corresponding to N-hOM and N-hS controls showed a tight superficial layer of flat polygonal cells with desquamation signs in which cells were covering the entire surface, whereas samples kept in vitro for 2 weeks showed immature differentiation patterns, and samples implanted in vivo for 40 days tended to resemble the structure of the native control tissues, with flattened cells and evident signs of desquamation. Scale bars = 50 μm. TEM samples (bottom) were analyzed after 40 days of in vivo implantation and demonstrated that in vivo-implanted tissues were mature and well-differentiated, with numerous intercellular junctions, abundant cell organelles, and a collagen-rich stroma. Scale bars = 1 μm. Abbreviations: H-hOM, heterotypical human oral mucosa; H-hS, heterotypical human skin; N-hOM, native human oral mucosa; N-hS, native human skin; SEM, scanning electron microscopy; TEM, transmission electron microscopy.
Electron microscopy analysis of controls and three-dimensional bioactive models of H-hOM and H-hS. SEM images (top) corresponding to N-hOM and N-hS controls showed a tight superficial layer of flat polygonal cells with desquamation signs in which cells were covering the entire surface, whereas samples kept in vitro for 2 weeks showed immature differentiation patterns, and samples implanted in vivo for 40 days tended to resemble the structure of the native control tissues, with flattened cells and evident signs of desquamation. Scale bars = 50 μm. TEM samples (bottom) were analyzed after 40 days of in vivo implantation and demonstrated that in vivo-implanted tissues were mature and well-differentiated, with numerous intercellular junctions, abundant cell organelles, and a collagen-rich stroma. Scale bars = 1 μm. Abbreviations: H-hOM, heterotypical human oral mucosa; H-hS, heterotypical human skin; N-hOM, native human oral mucosa; N-hS, native human skin; SEM, scanning electron microscopy; TEM, transmission electron microscopy.

The authors conclude the article with this statement: “All these findings support the idea that HWJSCs could be useful for the development of human skin and oral mucosa tissues for clinical use in patients with large skin and oral mucosa injuries.”  Think of it folks – new skin for burn patients, quickly, safely and ethically.

Now back to reality – this is exciting, but it is a a pre-clinical study.  Larger animals studies must show the efficacy and safety of this protocol before human trials can be considered, but you must admit that it looks exciting; and without killing any embryos.

See I. Garzón, et al., Stem Cells Trans MedAugust 2013 vol. 2 no. 8625-632.

Human Fat Contains Multilineage Differentiating Stress Enduring Cells With Great Potential for Regenerative Medicine


A collaboration between American and Japanese scientists has discovered and characterized a new stem cell population from human fat that do not cause tumors and can differentiate into derivatives from ectoderm, mesoderm, and endoderm.

Multilineage Differentiating Stress-Enduring or Muse cells are found in bone marrow and the lower layers of the skin (dermis). Muse cells are a subpopulation of mesenchymal stem cells, and even express a few mesenchymal stem cell-specific genes (e.g., CD105, a cell-surface protein specific to mesenchymal stem cells). However, Muse cells also express cell surface proteins normally found in embryonic stem cells (e.g., stage-specific embryonic antigen-3, SSEA-3). Additionally, Muse cells have the ability to self-renew, and differentiate into cell types from all three embryonic germ layers, ectoderm (which forms skin and brain), mesoderm, (which forms muscle, bone, kidneys, gonads, heart, blood vessels, adrenal glands, and connective tissue), and endoderm (which forms the gastrointestinal tract and its associated tissues). Finally, Muse cells can home to damaged sites and spontaneously differentiate into tissue-specific cells as dictated by the microenvironment in which the cells find themselves.

A new publication by Fumitaka Ogura and others from Tohoku University Graduate School of Medicine in Sendai, Japan and Saleh Heneidi from the Medical College of Georgia (Augusta, Georgia), and Gregorio Chazenbalk from the David Geffen School of Medicine at UCLA has shown that Muse cells also exist in human fat.

The source of cells came from two places: commercially available fat tissue and freshly collected fat from human subjects, collected by means of liposuction. After growing these cells in culture, the mesenchymal stem cells and Muse cells grew steadily over the 3 weeks. Then the Dezawa research group used fluorescence-activated cell sorting (FACS) to isolate from all these cells those cells that express SSEA-3 on their cell surfaces.

FACS uses antibodies conjugated to dyes that can bind to specific cell proteins. Once the antibodies bind to cells, the cells are sluiced through a small orifice while they are illuminated by the laser. The laser activates the dyes if the cell fluoresces, one door opens and the other closes. The cell goes to one test tube. If the cell does not fluoresce, then the door stay shut and another door opens and the cell goes into a different test tube.  In this way, cells with a particular cell-surface protein are isolated from other cells that do not have that cell-surface protein.

Fluorescent-Activated Cell Sorting
Fluorescent-Activated Cell Sorting

In addition to expression SSEA-3, the fat-based Muse cells expressed other mesenchymal stem cell-specific cell-surface proteins (CD29, CD90), but they did not express proteins usually thought to be diagnostic for fat-based mesenchymal stem cells (MSCs) such as CD34 and CD146.  Muse cells also expressed pluripotency genes (Nanog, Oct3/4, PAR4, Sox2, and Tra-1-81).  The Muse cells grew in small clusters and some cell expressed ectodermal-specific genes (neurofilament, MAP2), others expressed mesodermal-specific genes (smooth muscle actin, NKX2) and endodermal-specific genes (alpha-fetoprotein, GATA6).  These data suggested that the cultured Muse cells were poised to form either ectoderm, mesodermal, or endodermal derivatives.

When transplanted into mice with non-functional immune systems, the Muse cells never formed any tumors or disrupted the normal structure of the nearly tissues.  When placed in differentiating media, fat-derived Muse cells differentiated into cells with neuron-like morphology that expressed neuron-specific genes (Tuj-1), liver cells, and fat.  When compared with Muse cells from bone marrow or skin, the fat-derived Muse cells were better at making bone, fat, and muscle, but not as good as bone marrow Muse cells at making neuronal cell types, but not as good at making glial cells.  Many of these assays were based on gene expression experiments and not more rigorous tests.  Therefore, the results of these experiments might be doubtful until they are corroborated by more rigorous experiments.

These cells are expandable and apparently rather safe to use.  More work needs to be done in order to fully understand the full regenerative capacity of these cells and protocols for handling them must also be developed.  However, hopefully pre-clinical experiments in rodents will give way to larger animal experiments.  If these are successful, then maybe human trials come next.  Here’s to hoping.

A protocol for direct reprogramming of fibroblasts into motor neurons


A very efficient protocol for directly reprogramming skin-based fibroblasts into neurons is provided at this link. The induced neurons have all the electrophysiological characteristics of normal neurons and because no embryonic stage is passed through, these cells are safer than iPSCs. The problem is making enough of them for therapeutic purposes. At this point, the iPSCs really have it hands down.

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Adult Stem Cells Help Build Human Blood Vessels in Engineered Tissues


University of Illinois researchers have identified a protein expressed by human bone marrow stem cells that guides and stimulates the construction of blood vessels.

Jalees Rehman, associate professor of cardiology and pharmacology at the University of Illinois at Chicago College of Medicine and lead author of this paper, said: “Some stem cells actually have multiple jobs.”

As an example, stem cells from bone marrow known as mesenchymal stem cells can form bone, cartilage, or fat, but they also have a secondary role in that they support other cells in bone marrow.

Rehman and others have worked on developing engineered tissues for use in cardiac patients, and they noticed that mesenchymal stem cells were crucial for organizing other cells into functional stem cells.

Workers from Rehman’s laboratory mixed mesenchymal stem cells from human bone marrow with endothelial cells that line the inside of blood vessels. The mesenchymal stem cells elongated and formed a kind of scaffold upon which the endothelial cells adhered and organized to form tubes.

“But without the stem cells, the endothelial cells just sat there,” said Rehman.

When the cell mixtures were implanted into mice, blood vessels formed that were able to support the flow of blood. Then Rehman and his colleagues examined the genes expressed when their stem cells and endothelial cells were combined. They were aided in this venture by two different bone marrow stem cell lines, one of which supported the formation of blood vessels, and the other of which did.

Their microarray experiments showed that the vessel-supporting mesenchymal stem cells expressed high levels of the SLIT3 protein. SLIT3 is a blood vessel-guidance protein that directs blood vessel-making cells to particular places and induces them to make blood vessels. The cell line that do not stimulate blood vessel production made little to no SLIT3.

Rehman commented, “This means that not all stem cells are created alike in terms of their SLIT3 production and their ability to encourage blood vessel formation.”

Rehman continued: “While using a person’s own stem cells for making blood vessels is ideal because it eliminates the problem of immune rejection, it might be a good idea to test a patient’s stem cells to make sure they are good producers of SLIT3. If they aren’t, the engineered vessels may not thrive or even fail to grow.

Mesenchymal stem cells injections are being evaluated in clinical trials to see if their can help grow blood vessels and improve heart function in patients who have suffered heart attacks.

So far, the benefits of stem cell injection have been modest, according to Rehman. Evaluating the gene and protein signatures of stem cells from each patient may allow for a more individualized approach so that every patient receives mesenchymal stem cells that are most likely to promote blood vessel growth and cardiac repair. Such pre-testing might substantially improve the efficacy of stem cell treatments for heart patients.

The Mechanism Behind Blood Stem Cell Longevity


The blood stem cells that live in bone marrow divide and send their progeny down various pathways that ultimately produce red cells, white cells and platelets. These “daughter” cells must be produced at a rate of about one million cells per second in order to constantly replenish the body’s blood supply.

A nagging question is how these stem cells to persist for decades even though their progeny last for days, weeks or months before they need to be replaced. A study from the University of Pennsylvania has uncovered one of the mechanisms, and these cellular mechanisms allow these stem cells to keep dividing in perpetuity.

Dennis Discher and his colleagues in the Department of Chemical and Biomolecular Engineering in the School of Engineering and Applied Science found that a form of a protein called “myosin,” the motor protein that allow muscles to contract, helps bone marrow stem cells divide asymmetrically. This asymmetric cell division helps one cell remains a stem cell while the other cell becomes a daughter cell. Discher’s findings might provide new insights into blood cancers, such as leukemia, and eventually lead to ways of growing transfusable blood cells in a laboratory.

The participants in this study were members of the Discher laboratory, which include lead author Jae-Won Shin, Amnon Buxboim, Kyle R. Spinler, Joe Swift, Dave P. Dingal, Irena L. Ivanovska and Florian Rehfeldt. Discher collaborated with researchers at the Univ. de Strasbourg, Lawrence Berkeley National Laboratory and Univ. of California, San Francisco. This paper was published in Cell Stem Cell.

“Your blood cells are constantly getting worn out and replaced,” Discher said. “We want to understand how the stem cells responsible for making these cells can last for decades without being exhausted.”

Presently, scientists understand the near immortality of hematopoietic stem cells (HSCs) as a result of their asymmetric cell division, although how this asymmetric cell division enables stem cell longevity was unknown. To ferret out this mechanism, Discher and his coworkers analyzed all of the genes expressed in the stem cells and compared them with the genes expression in their more rapidly dividing progeny. Those proteins that only went to one side of the dividing cell might play a role in partitioning other key factors responsible for keeping one of the cells a stem cell and the other a progeny cell.

One of the proteins that showed a distinct expression pattern was the motor protein myosin II, which has two forms, myosin A and myosin B. Myosin II is the protein that enables the body’s muscles to contract, but in nonmuscle cells also it used during cell division. During the last phase of cell division, known as cytokinesis, myosin II helps cleave and close off the cell membranes as the cell splits apart.

“We found that the stem cell has both types of myosin,” Shin said, “whereas the final red and white blood cells only had the A form. We inferred that the B form was key to splitting the stem cells in an asymmetric way that kept the B form only in the stem cell.”

With these myosins as their top candidate, Discher and others labeled key proteins in dividing stem cells with different colors and put them under the microscope.

“We could see that the myosin IIB goes to one side of the dividing cell, which causes it to cleave differently,” Discher said. ”It’s like a tug of war, and the side with the B pulls harder and stays a stem cell.”

The researchers then performed in vivo tests using mice that had human stem cells injected into their bone marrow. By genetically inhibiting myosin IIB production, Shin and others saw the stem cells and their early progeny proliferating while the amount of downstream blood cells dropped.

“Because the stem cells were not able to divide asymmetrically, they just kept making more of themselves in the marrow at the expense of the differentiated cells,” Discher said.

HSC cell division mechanism

Discher and his team then used a drug that temporarily blocked both myosin A and myosin B. They observed that myosin inhibition increased the prevalence of non-dividing stem cells, blocking the more rapid division of progeny.

Discher believes that these findings could eventually help regrow blood stem cells after chemotherapy treatments for blood cancers or even grow blood products in the lab. Finding a drug that can temporarily shut down only the B form of myosin, while leaving the A form alone, would allow the stem cells to divide symmetrically and make more of themselves without preventing their progeny from dividing themselves.

“Nonetheless, the currently available drug that blocks both the A and B forms of myosin II could be useful in the clinic,” Shin said, “because donor bone marrow cultures can now easily be enriched for blood stem cells, and those are the cells of interest in transplants. Understanding the forces that stem cells use to divide can thus be exploited to better control these important cells.”

A Molecular Switch that Causes Stem Cell Aging


A study from the Cincinnati Children’s Hospital Medical Center, in collaboration with the University of Ulm in Germany has discovered a molecular switch that causes the aging of blood stem cells. This same work suggests a therapeutic strategy to delay stem cell aging.

Hematopoietic stem cells (HSCs) reside in the bone marrow and make all the red and white blood cells that populate the bloodstream. Proper HSC function is absolutely vital to the ongoing production of different types of blood cells that allow the immune system to fight infections and organs to receive adequate quantities of oxygen.

Hartmut Geiger from the Cincinnati Children’s Hospital Medical Center and the University of Ulm was the senior researcher on this project. Dr. Geiger said, “Although there is a large amount of data showing that blood stem cell function declines during aging, the molecular processes that cause this remain largely unknown. This prevents rational approaches to attenuate stem cell aging. This study puts us significantly closer to that goal through novel findings that show a distinct switch in a molecular pathway is very critical to the aging process.”

The pathway to which Dr. Geiger referred is the Wnt signaling pathway, which plays a foundational role in animal development, cell-cell communication, tissue generation, and is also involved in the pathology of various diseases.

Crystal structure of XWnt8
Crystal structure of XWnt8

Analysis of mouse models and cultured HSCs showed that under normal conditions, Wnt signaling in HSCs occurred through the so-called “canonical” Wnt signaling pathway. The canonical Wnt signaling pathway utilizes the typical components of Wnt signaling that were first identified in the fruit fly and then isolated and characterized in vertebrates (shown below).

Canonical Wnt signaling

However, Wnt proteins can also signaling through other, distinct signal transduction pathways, and these types of pathways are collectively known as “noncanonical” Wnt signaling pathway. In aging HSCs, a switch from canonical Wnt signaling to noncanonical Wnt signaling marked the onset of HSC aging.  See below for one example of non-canonical Wnt signaling.

Non-canonical Wnt signaling

To test this observation, Geiger’s group overexpressed Wnt5 in HSCs (a Wnt protein known to induced signaling through noncanonical Wnt signaling pathways), and immediately, the HSCs began to show the signs of aging.

One of the targets of Wnt5 signaling is a protein called Cdc42, which influences the cytoskeleton of cells.  Therefore, Geiger and his crew asked if Cdc42 was activated in those HSCs that overexpressed Wnt5.  The answer to this question was a clear “yes.”  Then they treated cultured HSCs with a molecule that inhibited Cdc42 activity.  This treatment reversed the aging process in HSCs.

To test their hypothesis in a living animal, Geiger and others removed a copy of the Wnt5 gene from HSCs in laboratory mice.  Mice that lacked functional Wnt5 protein in HSCs, showed rejuvenation of the aged HSCs.  Mice that lacked both copies of the Wnt5 gene showed a delayed aging process in their HSCs.

Even though this study has definitely made an important contribution to understanding HSC aging, more work is needed before a therapeutic strategy is in place.

Physical Cues Push Mature Cells into Induced Pluripotent Stem Cells


Bioengineers from the laboratory of Song Li at UC Berkeley have used physical cues to help push mature cells to de-differentiate into embryonic-like cells known as induced pluripotent stem cells.

Essentially, Li and his coworkers grew skin fibroblasts from human skin and mouse ears on surfaces with parallel grooves 10 micrometers apart and 3 micrometers high, in a special culture medium. This procedure increased the efficiency of reprogramming of these mature cells four-fold when compared to cells grown on a flat surface. Growing cells in scaffolds of nanofilbers aligned in parallel had similar effects.

Li’s study could significantly advance the protocols for making induced pluripotent stem cells (iPSCs). Normally iPSCs are made by genetically engineering adult cells so that they overexpress four different genes: Oct-4, Sox-2, Klf-4, and c-Myc. To put these genes into the cells, genetically modified viruses are used, or plasmids (small circles of DNA). Initially, Shinya Yamanaka, the scientist who invented iPSCs, and his co-workers used retroviruses that contained these four genes. When fibroblasts were infected with these souped-up retroviruses, the viruses inserted their viral DNA into the genomes of the host cells and expressed these genes.

retrovirus_life_cycle

Shinya Yamanaka won the Nobel Prize for this work in Physiology or Medicine in 2012 for this work. Unfortunately, retroviruses and can cause insertional mutations when they integrate into the genome (Zheng W., et al., Gene. 2013 Apr 25;519(1):142-9), and for this reason they are not the preferred way of making iPSCs. There are other viral vectors that do not integrate into the genome of the host cell (e.g., Sendai virus; see Chen IP, et al., Cell Reprogram. 2013 Dec;15(6):503-13). There are also techniques that use plasmids, which encode the four genes but do not integrate into the genome of the host cell. Finally, synthetic messenger RNAs that encode these four genes have also been used to make iPSCs (Tavernier G,, et al., Biomaterials. 2012 Jan;33(2):412-7).

The use of physical cues to make iPSCs may replace the need for gene overexpression, just as the use of particular chemicals can replace the need for particular genes (Zhu, S. et al. Cell Stem Cell 7, 651–655 (2010); Li, Y. et al. Cell Res. 21, 196–204 (2011)). If physical cues can replace the need for the overexpression of particular genes, then this discovery could revolutionize iPSC derivation; especially since the overexpression of particular genes in mature cells tends to cause genome instability in cells (Doris Steinemann, Gudrun Göhring, and Brigitte Schlegelberger. Am J Stem Cells. 2013; 2(1): 39–51).

“Our study demonstrates for the first time that the physical features of biomaterials can replace some of these biochemical factors and regulate the memory of a cell’s identity,” said study principal investigator Song Li, UC Berkeley, Professor of bioengineering. “We show that biophysical signals can be converted into intracellular chemical signals that coax cells to change.”

a, Scanning electron micrograph of PDMS membranes with a 10 μm groove width. All grooves were fabricated with a groove height of 3 μm. b, The top row shows phase contrast images of flat and grooved PDMS membranes with various widths and spacings. The bottom row shows fibroblast morphology on various PDMS membranes. Images are fluorescence micrographs of the nucleus (DAPI, blue) and actin network (phalloidin, green; scale bars, 100 μm). c, Reprogramming protocol. Colonies were subcultured and expanded or immunostained and quantified by day 12–14. d, Fluorescence micrograph showing the morphology of iPSC colonies generated on flat and grooved membranes (scale bar, 1 mm). Groove dimensions were 10 μm in width and spacing, denoted as 10 μm in this and the rest of the figures. Double-headed arrow indicates microgroove orientation of alignment. e, Reprogramming efficiency of fibroblasts transduced with OSKM and cultured on PDMS membranes with flat or grooved microtopography. The number of biological replicates, n, used for this experiment was equal to 6. Groove width and spacing were varied between 40, 20 and 10 μm. Differences of statistical significance were determined by a one-way ANOVA, followed by Tukey’s post-hoc test. * indicates significant difference (p<0.05) compared with the control flat surface. f, Reprogramming efficiency in fibroblasts transduced with OSK (n = 4). *p<0.05 (two-tailed, unpaired t-test) compared with the control flat surface. Error bars represent one standard deviation. g, Immunostaining of a stable iPSC line expanded from colonies generated on 10 μm grooves. These cells express mESC-specific markers Oct4, Sox2, Nanog and SSEA-1 (scale bar, 100 μm). h, The expanded iPSCs in g were transplanted into SCID mice to demonstrate the formation of teratomas in vivo (scale bar, 50 μm).
a, Scanning electron micrograph of PDMS membranes with a 10 μm groove width. All grooves were fabricated with a groove height of 3 μm. b, The top row shows phase contrast images of flat and grooved PDMS membranes with various widths and spacings. The bottom row shows fibroblast morphology on various PDMS membranes. Images are fluorescence micrographs of the nucleus (DAPI, blue) and actin network (phalloidin, green; scale bars, 100 μm). c, Reprogramming protocol. Colonies were subcultured and expanded or immunostained and quantified by day 12–14. d, Fluorescence micrograph showing the morphology of iPSC colonies generated on flat and grooved membranes (scale bar, 1 mm). Groove dimensions were 10 μm in width and spacing, denoted as 10 μm in this and the rest of the figures. Double-headed arrow indicates microgroove orientation of alignment. e, Reprogramming efficiency of fibroblasts transduced with OSKM and cultured on PDMS membranes with flat or grooved microtopography. The number of biological replicates, n, used for this experiment was equal to 6. Groove width and spacing were varied between 40, 20 and 10 μm. Differences of statistical significance were determined by a one-way ANOVA, followed by Tukey’s post-hoc test. * indicates significant difference (p<0.05) compared with the control flat surface. f, Reprogramming efficiency in fibroblasts transduced with OSK (n = 4). *p

To boost the efficiency of mature cell reprogramming, scientists also use a chemical called valproic acid, which dramatically affects global DNA structure and expression.

“The concern with current methods is the low efficiency at which cells actually reprogram and the unpredictable long-term effects of certain imposed genetic or chemical manipulations,” said the lead author of this study Timothy Downing. “For instance, valproic acid is a potent chemical that drastically alters the cell’s epigenetic state and can cause unintended changes inside the cell. Given this, many people have been looking at different ways to improve various aspects of the reprogramming process.”

This new study confirms and extends previous studies that showed that mechanical and physical cues can influence cell fate. Li’s group showed that physical and mechanical cues can not only affect cell fate, but also the epigenetic state and cell reprogramming.

a, Scanning electron micrograph of nanofibres showing fibre morphology in aligned and random orientations (scale bar, 20 μm). Confocal fluorescence micrograph of fibroblasts cultured on nanofibres (DAPI (blue) and phalloidin (green) staining; scale bar, 100 μm). b, Western blotting analysis for fibroblasts cultured on random and aligned nanofibres for three days. c, Fibroblasts were transduced with OSKM and seeded onto nanofibre surfaces, followed by immunostaining for Nanog expression (red) at day 12. Nuclei were stained with DAPI in blue; scale bar, 500 μm. d, Quantification of colony numbers in c (n = 5). *p<0.05 (two-tailed, unpaired t-test) compared with the control surface with random nanofibres. e, Fibroblasts were micropatterned into single-cell islands of 2,000 μm2 area with a CSI value of 1 (round) or 0.1 (elongated). After 24 h, cells were immunostained for AcH3, H3K4me2 or H3K4me3 (in green). Phalloidin staining (red) identifies the cell cytoskeleton for cell shape accuracy. The white arrowhead indicates the location of the nucleus (scale bars, 20 μm). f, Quantification of fluorescence intensity in e (n = 34, 20 and 34 for AcH3, H3K4me2 and H3K4me3, respectively). *p<0.05 (two-tailed, unpaired t-test) compared with the circular micropatterned cells (CSI = 1). Error bars represent one standard deviation.
a, Scanning electron micrograph of nanofibres showing fibre morphology in aligned and random orientations (scale bar, 20 μm). Confocal fluorescence micrograph of fibroblasts cultured on nanofibres (DAPI (blue) and phalloidin (green) staining; scale bar, 100 μm). b, Western blotting analysis for fibroblasts cultured on random and aligned nanofibres for three days. c, Fibroblasts were transduced with OSKM and seeded onto nanofibre surfaces, followed by immunostaining for Nanog expression (red) at day 12. Nuclei were stained with DAPI in blue; scale bar, 500 μm. d, Quantification of colony numbers in c (n = 5). *p

“Cells elongate, or example, as they migrate throughout the body,” said Downing, who is a research associate in Li’s lab. “In the case of topography, where we control the elongation of a cell by controlling the physical microenvironment, we are able to more closely mimic what a cell would experience in its native physiological environment. In this regard, these physical cues are less invasive and artificial to the cell and therefore less likely to cause unintended side effects.”

Li and his colleagues are studying whether growing cells on grooved surfaces eventually replace valproic acid and even replace other chemical compounds in the reprogramming process.

“We are also studying whether biophysical factors could help reprogram cells into specific cell types, such as neurons,” said Jennifer Soto, a UC Berkeley graduate student in bioengineering who was also a co-author on this paper.

Timothy Downing, et al., Nature Materials 12, 1154–1162 (2013).  

Induced Pluripotent Stem Cells Recapitulate ALS in Culture and Suggest New Treatment


Induced pluripotent stem cells are made from the adult cells of an individual by means of genetic engineering techniques. After introducing four different genes into adult cells, some of the cells de-differentiate to form cells that grow indefinitely in culture and have most of the characteristics of embryonic stem cells. However, if iPSCs are made from a patient who suffers from a genetic disease, then those stem cells will have the same mutation as the patient, and any derivatives of those iPSCs will show the same behaviors and pathologies of the tissues from the patient. This strategy is called the “disease in a dish” model and it is being increasingly used to make seminal discoveries about diseases and treatment strategies.

Scientists from Cedars-Sinai Regenerative Medicine Institute have used iPSC technology to study Lou Gehrig’s disease, and their research has provided a new approach to treat this horrific, debilitating disease.

Because I have previously written about Lou Gehrig’s disease or Amyotrophic Lateral Sclerosis (ALS), I will not describe it further.

Cedar Sinai scientists isolated skin scrapings from each patient and used the skin fibroblasts from each sample to make iPSCs. According to Dhruv Sareen, the director of the iPSC facility and faculty research scientist with the Department of Biomedical Sciences and the first author on this article, skins cells of patients who have ALS were converted into motor neurons that retained the genetic defects of the disease, thanks to iPSC technology. Then they focused on gene called C9ORF72, which was found to be the most common cause of familial ALS and frontotemporal lobar disease, and is even responsible for some cases of Alzheimer’s and Parkinson’s disease.

Mutations in a gene that has the very non-descriptive name “chromosome 9 open reading frame 72” or C9ORF72 for short seems to play a central role in the onset of Lou Gehrig’s disease. Mutations in C9orf72 have been linked with familial frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). FTD is a brain disorder that typically leads to dementia and sometimes occurs in tandem with ALS.

Mutations in C9ORF72 result from the expansion of a hexanucleotide repeat GGGGCC. When the C9ORF72 gene is replicated, the enzyme that replicates DNA (DNA polymerase) has a tendency to slip when comes to this stretch of nucleotides and this polymerase slip causes the hexanucleotide GGGGCC sequence to wax and wane (expand and shrink). Normally, there are up to 30 repeats of this GGGCC sequence, but in people with mutations in C9ORF72, this GGGGCC repeat can occur many hundreds of times. Massive expansions of the GGGGCC repeat interferes with normal expression of the protein made by C9ORF72. The presence of messenger RNAs (mRNAs) with multiple copies of GGGGCC in the nucleus and cytoplasm is toxic to the cell, since it gums up protein synthesis, RNA processing and other RNA-dependent functions. Also the lack of half of the C9ORF72 protein contributes to the symptoms of this conditions.

Robert Baloh, director of Cedars-Sinai’s Neuromuscular Division and the lead researcher of this research project, said, “We think this buildup of thousands of copies of the repeated sequence GGGGCC in the nucleus of patient’s cells may become toxic by altering the normal behavior of other genes in the motor neurons. Because our studies supported the toxic RNA mechanism theory, we used to small segments of genetic material called antisense oligonucleotides – ASOs – to block the buildup and degrade the toxic RNA. One ASO knocked down overall C9ORF72 levels. The other knocked down the toxic RNA coming from the gene without suppressing overall gene expression levels. The absence of potentially toxic RNA, and no evidence of detrimental effect on the motor neurons, provides a strong basis for using this strategy to treat patients suffering from these diseases.”

Baloh continued: “In a sense, this represents the full spectrum of what we are trying to accomplish with patient-based stem cell modeling. It gives researchers the opportunity to conduct extensive studies of a disease’s genetic and molecular makeup and develop potential treatments in the laboratory before translating them into patient trials.”

Researchers from another institution recently began a phase one clinical trial that used a similar ASO strategy to treat ALS caused by a different mutation. No safety issues were reported in this clinical trial.

Clive Svendsen, director of the Regenerative Medicine Institute and one of the authors, has investigated ALS for more than a decade, said, “ALS may be the cruelest, most severe neurological disease, but I believe the stem cell approach used in this collaborative effort holds the key to unlocking the mysteries of the and other devastating disorders. Within the Regenerative Medicine Institute, we are exploring several other stem cell-based strategies in search of treatments and cures.”

ALS affects 30,000-50,000 people in the US alone, but unlike other neurodegenerative diseases, it is almost always fatal within three to five years.

How Neural Stem Cells Become Neurons and Glia


How do neural stem cells differentiate into neurons or glia? A new paper from researchers at the University of California, Los Angeles (UCLA) seeks to explain this very phenomenon.

Neurons serve as the conductive cells of the nervous system. They transmit electrochemical signals from one neuron to another and provide signals to muscles, glands, and so on. They are responsible for consciousness, thought, learning and memory, and personality.

Despite their immense utility, neurons are not the only cells in the nervous system. Glial cells or just glia support neurons, hold them in place, and supply neurons with oxygen and nutrients and protect them from pathogens.

Glial Cells

When mouse neural stem cells were grown in culture, Wange Lu, associate professor of biochemistry and molecular biology at the Keck School of Medicine, and his colleagues came upon a protein called SMEK1 that promotes the differentiation of neural stem and progenitor cells. SMEK1 also keeps neural stem cells in check by preventing them from dividing uncontrollably.

When Lu and others took a more detailed look at the role of SMEK1, they discovered that it does not work alone, but in concert with a protein called Protein Phosphatase 4 (PP4) to suppress the function of a third protein called PAR3. PAR3 discourages the birth of new neurons (neurogenesis), and PAR3 inhibition leads to the differentiation of neural stem progenitor cells into neurons and glia.

“These studies reveal the mechanisms of how the brain keeps the balance of stem cells and neurons when the brain is formed,” said Wange. “If this process goes wrong, it leads to cancer, or mental retardation or other neurological diseases.”

Neural stem and progenitor cells offer tremendous promise as a future treatment for neurodegenerative disorders, and understanding their differentiation is the first step towards co-opting the therapeutic potential of these cells. This could offer new treatments for patients who suffer from Alzheimer’s, Parkinson’s and many other currently incurable diseases.

This work is interesting. It was published in Cell Reports 5, 593–600, November 14, 2013. My only criticism of some of the thinking in this paper is that neural stem cell lines are usually made from aborted fetuses. I realize that some of these neural stem cell lines come from medical abortions in which the baby had already died, but many of them come from aborted babies. If we are going to use neural stem cells for therapeutic purposes, then we should make them from induced pluripotent stem cells and take them from aborted babies.

Possible Treatment for Brain Disorders


Tuberous Sclerosis is a rare genetic disease that causes the growth of tumors in the brain and other vital organs and may also lead to other conditions such as autism, epilepsy, and cognitive impairment; all of which result from the abnormal generation of neurons.

Tuberous sclerosis is also called tuberous sclerosis complex or TSC, and it is a rare genetic disease that affects multiple organ systems. TSC causes the growth of benign tumors in the brain and on other vital organs such as the kidneys, heart, eyes, lungs, and skin. TSC typically affects the central nervous system and results in a combination of symptoms including seizures, developmental delay, behavioral problems, skin abnormalities, and kidney disease.

TSC affects as many as 25,000 to 40,000 individuals in the United States and about 1 to 2 million individuals worldwide. The estimated prevalence of this disease is one in 6,000 newborns, and it occurs in all races and ethnic groups, and in both genders.

TSC derives its name from the characteristic tuber or potato-like nodules in the brain. These growths calcify with age and become hard or sclerotic.

Many TSC patients show evidence of the disorder in the first year of life. However, clinical features can be subtle initially, and many signs and symptoms take years to develop. As a result, TSC can be unrecognized or misdiagnosed for years.

TSC is caused by defects, or mutations, on two genes-TSC1 and TSC2. Only one of the genes needs to be affected for TSC to be present. The TSC1 gene, discovered in 1997, is on chromosome 9 and produces a protein called Hamartin. The TSC2 gene, discovered in 1993, is on chromosome 16 and produces the protein Tuberin. These proteins combine to form a complex that suppresses cell growth by preventing activation of a master control protein called mTOR. Loss of regulation of mTOR occurs in cells lacking either Hamartin or Tuberin, and this leads to abnormal differentiation and development, and to the generation of enlarged cells, as are seen in TSC brain lesions.

Since Tuberous Sclerosis affect stem cell activity, scientists at Clemson University are examining how neurons are formed from neural stem cells and this research is vital to providing a treatment to Tuberous Sclerosis, which affects how neurons are formed in the brain.

David M Feliciano, assistant professor of biological sciences at Clemson University, said: “Current medicine is directed at inhibiting the mammalian target of rapamycin (mTOR), a common feature within these tumors that have abnormally high activity. However, current treatments have severe side effects, like due to mTOR’s many functions and playing an important role in cell survival, growth and migration.”

mTOR pathway

Feliciano continued: “Neural stem cells generate the primary communicating cells of the brain called neurons through the process of neurogenesis, yet how this is orchestrated is unknown.”

Neural stem cells lie at the very heart of brain development and repair, and alterations in the ability of these cells to self-renew and differentiate can have profound consequences for brain function at any stage of life, according to researchers.

In order to further elucidate the regulation of neurogenesis, Feliciano and his team delivered small pieces of DNA into the neural stem cells of the new-born mice. The team used electroporation to introduce the DNA into the mouse cells, and these small pieces of DNA allowed Feliciano’s team to express and control specific components of the mTOR pathway.

By using these tools, Feliciano and others showed that Increasing the activity of the mTOR pathway cause the neural stem cells to make more neurons at the expense of self-renewal. Increasing mTOR activity caused upregulation of 4E-BP2. 4E-BP2, also known as Eukaryotic translation initiation factor 4E-binding protein 2, binds to a component of the protein synthesis machinery and inhibits its function. Mice that lack functional EIF4EBP2 exhibit autism-like symptoms, including poor social interaction, altered communication and repetitive behaviors.

This work suggests that 4E-BP2 might be a new target for the treatment of TSC and that targeting this protein might cause fewer side effects than targeting mTOR. Future experiments hope to identify those proteins that are made due to the activation of this pathway in neural tissues.

Stem Cells and Society, charting a course for the future


Stem Cell Transplant Repairs the Damage that Results from Inflammatory Bowel Disease


A source of stem cells from the digestive tract can repair a type of inflammatory bowel disease when transplanted into mice has been identified by British and Danish scientists.

This work resulted from a collaboration between stem cell scientists at the Wellcome Trust-Medical Research Council/Cambridge Stem Cell Institute at Cambridge University, and the Biotech Research and Innovation Centre (BRIC) at the University of Copenhagen, Denmark. This research paves the way for patient-specific regenerative therapies for inflammatory bowel diseases such as ulcerative colitis.

All tissues in out body probably contain a stem cell population of some sort, and these tissue-specific stem cells are responsible for the lifelong maintenance of these tissues, and, ultimately, organs. Organ-specific stem cells tend to be restricted in their differentiation abilities to the cell types within that organ. Therefore, stem cells from the digestive tract will tend to differentiate into cell types typically found in the digestive tract, and skin-based stem cells will usually form cell types found in the skin.

When this research team examined developing intestinal tissue in mouse fetuses, they discovered a stem cell population that differed from the adult stem cells that have already been described in the gastrointestinal tract. These new-identified cells actively divided and could be grown in the laboratory over a long period of time without terminally differentiating into adult cell types. When exposed to the right conditions, however, these cells could differentiate into mature intestinal tissue.

Fordham_CellStemCell_GraphicalAbstract

Could these cells be used to repair a damaged bowel? To address this question, this team transplanted these cells into mice that suffered from a type of inflammatory bowel disease, and within three hours the stem cells has attached to the damaged areas of the mouse intestine. integrated into the intestine, and contributed to the repair of the damaged tissue.

“We found that the cells formed a living plaster (British English for a bandage) over the damaged gut,” said Jim Jensen, a Wellcome Trust researcher and Lundbeck Foundation fellow, who led the study. “They seemed to response to the environment they had been placed in and matured accordingly to repair the damage. One of the risks of stem cell transplants like this is that the cells will continue to expand and form a tumor, but we didn’t see any evidence of that with this immature stem cell population from the gut.”

Because these cells were derived from fetal intestines, Jensen and his team sought to establish a new source of intestinal progenitor cells.  Therefore, Jensen and others isolated cells with similar characteristics from both mice and humans, and  made similar cells similar cells by reprogramming adult human cells in to induced pluripotent stem cells (iPSCs) and growing them in the appropriate conditions.  Because these cells grew into small spheres that consisted of intestinal tissue, they called these cells Fetal Enterospheres (FEnS).

Established cultures of FEnS expressed lower levels of Lgr5 than mature progenitors and grew in the presence of the Wnt antagonist Dkk1 (Dickkopf).  New cultures can be induced to form mature intestinal organoids by exposure to the signaling molecule Wnt3a. Following transplantation in a model for colon injury, FEnS contributed to regeneration of the epithelial lining of the colon by forming epithelial crypt-like structures that expressed region-specific differentiation markers.

“We’ve identified a source of gut stem cells that can be easily expanded in the laboratory, which could have huge implications for treating human inflammatory bowel diseases. The next step will be to see whether the human cells behave in the same way in the mouse transplant system and then we can consider investigating their use in patients,” Jensen said.

Stem-Based Treatment of Stoke


When blood flow to the brain ceases as the result of a blood clot, trauma, or injury, the brain suffers from a shortage of oxygen. Such an incident is known as a stroke and it can result in the death of neurons and the loss of those functions to which the dead neurons contributed. Treatment for stroke is largely supportive, but regenerative treatments that replace the dead neurons would be the most ideal treatment.

A research consortium at Lund University in Lund, Sweden has found that neurons made from induced pluripotent stem cells integrate into the brains of mice that had suffered strokes. This experiment takes a closer step towards the development of a regenerative treatment for strokes.

Strategies for stem cell-based regenerative therapy in neurodegenerative diseases.
Strategies for stem cell-based regenerative therapy in neurodegenerative diseases.

In the aftermath of a stroke, nerve cells in the brain die. At the Lund Stem Cell Center, the research groups of Zaal Kokaia and Olle Lindvall teamed up to develop a stem cell-based method to treat stroke patients.

After a stroke, the cerebal cortex tends to take the bulk of the damage and neuron loss from the cerebral cortex underlies many of the symptoms following a stroke, such a paralysis and speech problems. The method developed by the Lund Institute scientists should make it possible to generate nerve cells for transplantation from the patient’s own skin cells.

Transient-Ischemic-Attack

First, the Lund team isolated skin fibroblasts from the afflicted mice and used genetic engineering techniques to convert them into induced pluripotent stem cells (iPSCs), which have many of the differentiation capabilities of embryonic stem cells. These iPSC lines were differentiated into cortical neurons, which tend to populate the cerebral cortex. However, transplanting fully differentiated neurons into the brain tend to not work terribly well because the mature neurons are unable to divide and have poor abilities to connect with other cells. Therefore, the neuron progenitor cells that will give rise to cortical neurons are a better candidate for transplantation.

After generating long-term self-renewing neuroepithelial-like stem cells from iPSCs in the laboratory, the Lund group scientists showed that these stem cells could give rise to neural progenitors that expressed the types of genes found in mature cortical neurons. When these neural progenitor cells were transplanted into rats that had suffered strokes, two months after transplantation, the cortically fated cells showed less proliferation and more efficient differentiation into mature neurons with the right shape, size, and structure of cortical neurons and expressed the same proteins as cortical neurons. These tranplanted cells also extended more axons than those cells that were not fated to form cortical neurons. Transplantation of both the cortical neuron-fated and non-cortical neuron-fated cells caused recovery of the impaired function in the stepping test in comparison to controls. At 5 months after stroke, there was no tumor formation and the grafted cells had all the electrophysiological properties of mature neurons and showed full evidence that they had integrated into the existing neural circuitry.

These results are very promising and represent a very early but important step towards a stem cell-based treatment for stroke in patients. Further experimental studies are necessary if these experiments are to be translated into the clinic in a responsible way.

Scientists Generate “Mini-kidney” Structures from Human Stem Cells


Kidney Disease represents a major and unsolved health issue worldwide. Once damaged by disease, kidneys rarely recover their original level of function, and this highlights the urgent need for better knowledge of kidney development and physiology.

Now, a team of researchers led by scientists at the Salk Institute for Biological Studies has developed a novel platform to study kidney diseases. This new platform should open new avenues for the future application of regenerative medical strategies to restore kidney function.

For the first time, the Salk researchers have generated three-dimensional kidney structures from human stem cells. These findings were reported November 17, 2013 in Nature Cell Biology, and they suggest new ways to study the development and diseases of the kidneys and to discover and test new drugs that target human kidney cells.

Scientists had created precursors of kidney cells using stem cells as recently as this past summer, but the Salk team was the first to coax human stem cells into forming three-dimensional cellular structures similar to those found in our kidneys.

“Attempts to differentiate human stem cells into renal cells have had limited success,” says senior study author Juan Carlos Izpisua Belmonte, a professor in Salk’s Gene Expression Laboratory and holder of the Roger Guillemin Chair. “We have developed a simple and efficient method that allows for the differentiation of human stem cells into well-organized 3D structures of the ureteric bud (UB), which later develops into the collecting duct system.”

The Salk findings demonstrate for the first time that pluripotent stem cells capable of differentiating into the many cells and tissue types that make up the body can be induced to differentiate into those cells found in the ureteric bud, which is an early developmental structure of the kidneys. Furthermore, these same cells can differentiate further into three-dimensional structures in organ cultures. Ureteric bud cells form the early stages of the human urinary and reproductive organs during development and later develop into a conduit for urine drainage from the kidneys. Izpisua Belmonte’s research group accomplished this with both human embryonic stem cells and induced pluripotent stem cells (iPSCs), human cells from the skin that have been reprogrammed into their pluripotent state.

Kidney development

After generating iPSCs that demonstrated pluripotent properties and were able to differentiate into mesoderm, the embryonic germ cell layer from which the kidneys develop, the Salk Institute team used growth factors known to be essential during the natural development of our kidneys to culture both iPSCs and embryonic stem cells.  The combination of signals from these growth factors, molecules that guide the differentiation of stem cells into specific tissues, committed the cells to become progenitors that exhibit clear characteristics of renal cells in only four days.

The researchers then guided these cells to further differentiate into organ structures similar to those found in the ureteric bud by culturing them with kidney cells from mice. This demonstrated that the mouse cells were able to provide the appropriate developmental cues to allow human stem cells to form three-dimensional structures of the kidney.

Izpisua Belmonte’s team also tested their protocol on iPSCs from a patient clinically diagnosed with polycystic kidney disease (PKD), a genetic disorder characterized by multiple, fluid-filled cysts that can lead to decreased kidney function and kidney failure. They found that their methodology could produce kidney structures from patient-derived iPSCs.

Polycystic kidneys
Polycystic kidneys

Because of the many clinical manifestations of the disease, neither gene- nor antibody-based therapies are realistic approaches for treating PKD. The Salk team’s technique might help circumvent this obstacle and provide a reliable platform for pharmaceutical companies and other investigators studying drug-based therapeutics for PKD and other kidney diseases.

“Our differentiation strategies represent the cornerstone of disease modeling and drug discovery studies,” says lead study author Ignacio Sancho-Martinez, a research associate in Izpisua Belmonte’s laboratory. “Our observations will help guide future studies on the precise cellular implications that PKD might play in the context of kidney development.”

New 3D Method Used to Grow Miniature Pancreas


Researchers from the University of Copenhagen, in collaboration with an international team of investigators, have successfully developed an innovative three-dimensional method to grow miniature pancreas from progenitor cells. The future goal of this research is to utilize this model system to fight against diabetes. This research was recently published in the journal Development.

The new method allows the cell material from mice to grow vividly in picturesque tree-like structures.
The new method allows the cell material from mice to grow vividly in picturesque tree-like structures.

The new method takes cell material from mice and grows them in vividly picturesque tree-like structures.  The cells used were mouse embryonic pancreatic progenitors, and they were grown in a compound called Matrigel with accompanying cocktails of growth factors.  In vitro maintenance and expansion of these pancreatic progenitors requires active Notch and FGF signaling, and therefore, this culture system recapitulated the in vivo conditions that give rise to the pancreas in the embryo.

Professor Anne Grapin-Botton and her team at the Danish Stem Cell Centre, in collaboration with colleagues from the Ecole Polytechnique Fédérale de Lausanne in Switzerland, have developed a three-dimensional culture method that takes pancreatic cells and vigorously expands them. This new method allows the cell material from mice to grow vividly into several distinct picturesque, tree-like structures. The method offers tremendous long-term potential in producing miniature human pancreas from human stem cells. Human miniature pancreas organoids would be valuable as models to test new drugs fast and effectively, without the use of animal models.

“The new method allows the cell material to take a three-dimensional shape enabling them to multiply more freely. It’s like a plant where you use effective fertilizer, think of the laboratory like a garden and the scientist being the gardener,” says Anne Grapin-Botton.

In culture, pancreatic cells neither thrive nor develop if they are alone. A minimum of four pancreatic cells, growing close together is required for these cells to undergo organoid development.

“We found that the cells of the pancreas develop better in a gel in three-dimensions than when they are attached and flattened at the bottom of a culture plate. Under optimal conditions, the initial clusters of a few cells have proliferated into 40,000 cells within a week. After growing a lot, they transform into cells that make either digestive enzymes or hormones like insulin and they self-organize into branched pancreatic organoids that are amazingly similar to the pancreas,” adds Anne Grapin-Botton.

The scientists used this system to discover that the cells of the pancreas are sensitive to their physical environment, and are influenced by such seemingly insignificant factors as the stiffness of the gel and contact with other cells.

An effective cellular therapy for diabetes is dependent on the production of sufficient quantities of functional beta-cells. Recent studies have enabled the production of pancreatic precursors but efforts to expand these cells and differentiate them into insulin-producing beta-cells have proved a challenge.

“We think this is an important step towards the production of cells for diabetes therapy, both to produce mini-organs for drug testing and insulin-producing cells as spare parts. We show that the pancreatic cells care not only about how you feed them but need to be grown in the right physical environment. We are now trying to adapt this method to human stem cells,” adds Anne Grapin-Botton.

The Benefits of Repeated Mesenchymal Stem Cell Treatments to the Heart


Mesenchymal stem cells have the ability to improve the heart after a heart attack. However can repeated administrations of mesenchymal stem cells cause an increased benefit to the heart after a heart attack?

A collaborative research project between the Royal Adelaide Hospital, the University of Adelaide in South Australia, and the Mayo Clinic in Rochester, Minnesota has administered mesenchymal stem cells multiple times to rodents after a heart attack to determine if administering these stem cells multiple times after a heart attack increases the performance of the heart.

The experimental procedure was relatively straight-forward. Three groups of mice were evaluated by means of cardiac magnetic resonance imaging (MRI). Then all three were given heart attacks by tying off the left anterior descending artery. Immediately after the heart attack, two groups were injected with one million mesenchymal stem cells into the heart. The third group was injected with ProFreeze (a cryopreservation solution). One week later, a second set of heart MRIs were taken, and the first and third group of mice received injections of ProFreeze and the third group received another one million mesenchymal stem cells. All animals were given two more heart MRIs one week later and two weeks after that. One month after the initial heart attacks, the mice were euthanized and their hearts were sectioned and examined.

Those mice that did not receive injections of mesenchymal stem cells showed a precipitous drop in their heart performance. The ejection fraction (average percent of blood pumped from the heart) dropped from around 60% to about 20% and then stayed there. Those mice treated with one round of mesenchymal stem cells (MSCs) after their ejection fractions drop from 60% to about 35% after one week, and then stayed there. Those animals that received two shots of MSCs have their ejection fractions drop from around 60% to about 41%. Thus the administration of a second round of MSCs did significantly increase the performance of the heart.

The heart also shows tremendous structural improvements as a result of MSC transplantation. These improvements are even more dramatic in those mice that received two doses of MSCs. The mass of the heart and the thickness of the walls of the heart are greater in those animals that received two MSC doses, than those that received only one dose. Secondly, the size of the heart scar is smallest in those animals that received two doses of MSCs. Third, the density of blood vessels was MUCH higher in the animals that received two MSC doses. Also, the tissue far from the infarction in those animals that had received two doses of MSCs showed twice the density of blood vessels per cubic millimeter of heart tissue than those animals that had only received one injection of MSCs. Therefore, additional transplantations of MSCs increase blood vessel density, decrease the size of the heart scar and increase the thickness of the walls of the heart.

MSCs have the capacity to heal the heart after a heart attack. The degree to which they heal the heart differs from patient to patient, but additional treatments have the capacity to augment the healing capacities of these cells.  Also, in this experiment, the mice received someone else’s MSCs.  This is known as “allogeneic” transplantation, and it is an important concept, since older patients, diabetic patients, or those who have had a heart attack typically have MSCs that do not perform well.  Therefore to receive MSCs from a donor is a way around this problem.

The problem with this experiment is that it was done in mice, and they were injected directly into the heart tissue. Such a procedure is almost certainly impractical for human patients. Instead, intracoronary delivery is probably more practical, but here again, repeated releasing cells into the coronary arteries increases the risk of clogging them. Therefore, it is probably necessary to administer the second dose of MSCs some time after the first dose. To calibrate when to administer the second dose, large animal experiments will be required.

Thus, while this experiment looks interesting and hopeful, more work is required to make this usable in humans.  It does, however, establish the efficacy of repeated allogeneic MSC transplantations, which is an important feature of these experiments.