I’m on Life Report Podcast


Josh Brahm runs the Life Report podcast and is one of the nicest guys on the planet. Josh invited me to his podcast at the end of last year to talk about my book and stem cell research in general.  The editing of that exchange has become available.

The interview is here, and the bonus discussion is here. Josh is a very good interviewer.  I think you’ll enjoy it.

Micro-Grooved Surfaces Influence Stem Cell Differentiation


Martin Knight and his colleagues from the Queen Mary’s School of Engineering and Materials Science and the Institute of Bioengineering in London, UK have shown that growing adult stem cells on micro-grooved surfaces disrupts a particular biochemical pathway that specified the length of a cellular structure called the “primary cilium.” Disruption of the primary cilium ultimately controls the subsequent behavior of these stem cells.

Primary cilia are about one thousand times narrower than a human hair. They are found in most cells and even though they were thought to be irrelevant at one time, this is clearly not the case.

Primary Cilium

The primary cilium acts as a sensory structure that responds to mechanical and chemical stimuli in the environment, and then communicates that external signal to the interior of the cell.  Most of the basic research on this structure was done using a single-celled alga called Chlamydomonas.

Martin Knight and his team, however, are certain that primary cilia in adult stem cells play a definite role in controlling cell differentiation.  Knight said, “Our research shows that they [primary cilia] play a key role in stem cell differentiation.  We found it’s possible to control stem cell specialization by manipulating primary cilia elongation, and that this occurs when stem cells are grown on these special grooved surfaces.”

When mesenchymal stromal cells were grown on grooved surfaces, the tension inside the cells was altered, and this remodeled the cytoskeleton of the cells.  Cytoskeleton refers to a rigid group of protein inside of cells that act as “rebar.” for the cell.  If you have ever worked with concrete, you will know that structural use of concrete requires the use of reinforcing metal bars to prevent the concrete from crumbling under the force of its own weight.  In the same way, cytoskeletal proteins reinforce the cell, give it shape, help it move, and help it resist shear forces.  Remodeling of the cytoskeleton can greatly change the behavior of the cell.

The primary cilium is important for stem cell differentiation.  Growing mesenchymal stromal cells on micro-grooved surfaces disrupts the primary cilium and prevents stem cell differentiation.  This simple culture technique can help maintain stem cells in an undifferentiated state until they have expanded enough for therapeutic purposes.

Once again we that there are ways to milk adult stem cells for all they are worth.  Destroying embryos is simply not necessary to save the lives of patients.

Adult Stem Cell Research Has Defeated Embryonic Stem Cells for Funding Priorities


Mallory Quigley from LifeNews has written an article on a report by the Charlotte Lozier Institute, which analyzes funding trends in stem cell research in the state of Maryland. Funding for non-embryonic stem cells greatly outnumbers funding for embryonic stem cells. Read the article here.

Bmi1 Controls Adult Stem Cell “Stemness”


Stem cell scientists from the laboratory of Ophir Klein at UC San Francisco have discovered a new role for a protein called Bmi1 that might give clues as to how to get adult stem cells to regenerate organs.

Ophir Klein, the director of the Craniofacial and Mesenchymal Biology Program and chairman of the Division of Craniofacial Anomalies at UC San Francisco, said “Scientists have known that Bmi1 is a central control switch within the adult stem cells of many tissues, including the brain, blood, lung and mammary gland. Bmi1 also is a cancer-causing gene that becomes reactivated in cancer cells.”

Crystal structure of the BMI1 protein
Crystal structure of the BMI1 protein

All stem cells are somewhat immature in comparison to their adult counterparts. Stem cells also have the capacity to divide almost indefinitely and generate specialized cells. Bmi1 acts as a molecular switch that, if pushed in one direction, drives stem cells to proliferate and grow, but if pushed in the opposite direction, keeps cell proliferation in check. Research from Klein’s lab now suggests that Bmi1 might prevent the progeny of stem cells from differentiating into the wrong cell types in the wrong location.

Downstream targets of Bmi1
Downstream targets of Bmi1

This new discovery suggests that manipulation of Bmi1 and other regulatory molecules might be some of the steps included in laboratory recipes to turn specialized cell development on and off to create new cell-based treatments for tissue lost to injury, disease, or aging.

Also, the dual role of Bmi1 in pathological settings might be intriguing. Cancers are, in many cases, driven by adult stem cells that behave abnormally. If these stem cells could be differentiated, then their growth would slow. Possibly, inactivating Bmi1 in tumor stem cells might be one strategy.

In these experiments, Klein and his colleagues examined those adult stem cells found in the large incisors of mice. Unlike humans, these teeth grow continuously and are, therefore, an attractive model for stem cell research. Klein explained, “There is a large population of stem cells, and the way the daughter cells of the stem cells are produced is easy to track – it’s if they are on a conveyor belt.” Early in life, human beings possess a stem cell population that similarly drive tooth development, but they become inactive after the adult teeth are fully formed during early childhood.

Mouse mandible showing  the large, paired incisors
Mouse mandible showing the large, paired incisors

In the current study, postdoctoral research fellows Brian Biehs and Jimmy Hu showed that at the base of the growing mouse incisor there is a stem cell population that actively expresses Bmi1. In these cells, Bmi1 suppressed a set of genes called Hox genes. When activated, the Hox genes trigger the development of specific cell types and body structures.

In the mouse incisor, Bmi1 keeps these stem cells in their stem cell state and prevent them from differentiating prematurely or inappropriately. “This new knowledge is useful in a fundamental way for understanding how cell differentiating is controlled and may help us manipulate stem cells to get them to do what we want to do,” said Klein.

As they state in the abstract of their paper: “As Hox gene upregulation has also been reported in other systems when Bmi1 is inactivated our findings point to a general mechanism whereby BMI1-mediated repression of Hox genes is required for the maintenance of adult stem cells and for prevention of inappropriate differentiation.”

Thus this finding from the Klein lab may provide a vital clue for the manipulation of adult stem cells and, perhaps, cancer cells.

Stress-Resistant Stem Cells From Fat


During liposuction patients lose a fat cells, fat-based mesenchymal stem cells, and now, according to new results from UCLA scientists, stress-enduring stem cells.

This new stem cell population has been called a Multi-lineage Stress-Enduring Adipose Tissue or Muse-AT stem cells. UCLA scientists found Muse-AT stem cells by accident when a particular machine in the laboratory malfunctioned, killing all the cells found in cells from human liposuction, with the exception on the Muse-AT stem cells.

Gregorio Chazenbalk from the UCLA Department of Obstetrics and Gynecology and his research team discovered, after further tests on Muse-AT stem cells, that they not only survive stress, but might be activated by it.

The removal of Muse-AT stem cells from the human body by means of liposuction revealed cells that express several embryonic stem cell-specific proteins (SSEA3, TR-1-60, Oct3/4, Nanog and Sox2). Furthermore, Muse-AT stem cells were able to differentiate into muscle, bone, fat, heart muscle, liver, and neuronal cells. Finally, when Chazenbalk and his group examined the properties of Muse-AT stem cells, they discovered that these stem cells could repair and regenerate tissues when transplanted back into the body after having been exposed to cellular stress.

Muse-ATs express pluripotent stem cell markers. Immunofluorescence microscopy demonstrates that Muse-AT aggregates, along with individual Muse-AT cells, express characteristic pluripotent stem cell markers, including SSEA3, Oct3/4, Nanog, Sox2, and TRA1-60. Comparatively, ASCs (right panel) derived from the same lipoaspirate under standard conditions (see above, [16] were negative for these pluripotent stem cell markers. Nuclei were stained with DAPI (blue). Original magnification, 600 X. doi:10.1371/journal.pone.0064752.g002
Muse-ATs express pluripotent stem cell markers.
Immunofluorescence microscopy demonstrates that Muse-AT aggregates, along with individual Muse-AT cells, express characteristic pluripotent stem cell markers, including SSEA3, Oct3/4, Nanog, Sox2, and TRA1-60. Comparatively, ASCs (right panel) derived from the same lipoaspirate under standard conditions (see above, [16] were negative for these pluripotent stem cell markers. Nuclei were stained with DAPI (blue). Original magnification, 600 X.
doi:10.1371/journal.pone.0064752.g002
“This population of cells lies dormant in the fat tissue until it is subjected to very harsh conditions. These cells can survive in conditions in which usually cancer cells can survive. Upon further investigation and clinical trials, these cells could prove a revolutionary treatment option for numerous diseases, including heart disease, stroke and for tissue damage and neural regeneration,” said Chazenbalk.

Purifying and isolating Muse-AT stem cells does not require the use of a cell sorter or other specialized, high-tech machinery. Muse-AT stem cell can grow in liquid suspension, where they grow as small spheres or as adherent cells that pile on top of each other to form aggregates, which is rather similar to embryonic stem cells and the embryoid bodies that they form.

Isolation and morphologic characterization of Muse-ATs. (A) Schematic of Muse-AT isolation and activation from their quiescent state by exposure to cellular stress. Muse-AT cells were obtained after 16 hours, with incubation with collagenase in DMEM medium without FCS at 4°C under very low O2 (See Methods). (B) FACS analysis demonstrates that 90% of isolated cells are both SSEA3 and CD105 positive. (C) Muse-AT cells can grow in suspension, forming spheres or cell clusters as well as individual cells (see red arrows) or (D) Muse-AT cells can adhere to the dish and form cell aggregates. Under both conditions, individual Muse-AT cells reached a diameter of approximately 10µm and cell clusters reached a diameter of up to 50µm, correlating to stem cell proliferative size capacity. doi:10.1371/journal.pone.0064752.g001
Isolation and morphologic characterization of Muse-ATs.
(A) Schematic of Muse-AT isolation and activation from their quiescent state by exposure to cellular stress. Muse-AT cells were obtained after 16 hours, with incubation with collagenase in DMEM medium without FCS at 4°C under very low O2 (See Methods). (B) FACS analysis demonstrates that 90% of isolated cells are both SSEA3 and CD105 positive. (C) Muse-AT cells can grow in suspension, forming spheres or cell clusters as well as individual cells (see red arrows) or (D) Muse-AT cells can adhere to the dish and form cell aggregates. Under both conditions, individual Muse-AT cells reached a diameter of approximately 10µm and cell clusters reached a diameter of up to 50µm, correlating to stem cell proliferative size capacity.
doi:10.1371/journal.pone.0064752.g001

We have been able to isolate these cells using a simple and efficient method that takes about six hours from the time the fat tissue is harvested,” said Chazenbalk. “This research offers a new and exciting source of fat stem cells with pluripotent characteristics, as well as a new method for quickly isolating them. These cells also appear to be more primitive than the average fat stem cells, making them potentially superior sources for regenerative medicine.”

Embryonic stem cells and induced pluripotent stem cells are the two main sources of pluripotent stem cells. However, both of these stem cells have an uncontrolled capacity for differentiation and proliferation, which leads to the formation of undesirable teratomas, which are benign tumors that can become teratocarcinomas, which are malignant tumors. According to Chazenbalk, little progress has been made in resolving this defect (I think he overstates this).

Muse-AT stem cells were discovered by a research group at Tokohu University in Japan and were isolated from skin and bone marrow rather than fat (see Tsuchiyama K, et al., J Invest Dermatol. 2013 Apr 5. doi: 10.1038/jid.2013.172). The Japanese group showed that Muse-AT stem cells do not form tumors in laboratory animals. The UCLA group was also unable to get Muse-AT stem cells to form tumors in laboratory animals, but more work is necessary to firmly establish that these neither form tumors nor enhance the formation of other tumors already present in the body.

Chazenbalk also thought that Muse-AT stem cells could provide an excellent model system for studying the effects of cellular stress and how cancer cells survive and withstand high levels of cellular stress.

Chazenbalk is understandable excited about his work, but other stem cells scientists remain skeptical that this stem cells population has the plasticity reported or that these cells are as easily isolated as Chazenbalk says.  For a more skeptical take on this paper, see here.

Adult Stem Cells to Cure Diabetes?


Type 1 diabetics must inject themselves with insulin on a daily basis in order to survive. Without these shots, they would die.

Insulin injection

In most cases, type 1 diabetics have diabetes because their immune systems have attacked their insulin-producing cells and have destroyed them. However, a recent study at the University of Missouri has revealed that the immune system-dependent damage to the pancreas in type 1 diabetics goes beyond direct damage to the insulin-producing cells in the pancreas, The immune response also destroys blood vessels that feed tissues within the pancreas. This finding could provide the impetus for a cure that includes a combination of drugs and stem cells.

Habib Zaghouani and his research team at the University of Missouri School of Medicine discovered that “type 1 diabetes destroys not only insulin-producing cells but also blood vessels that support them,” explained Zaghouani. “When we realized how important the blood vessels were to insulin production, we developed a cure that combines a drug we created with adult stem cells from bone marrow. The drug stop the immune system attack, and the stem cells generate new blood vessels that help insulin-producing cells to multiply and thrive.”

Type 1 diabetes or juvenile diabetes, can lead to numerous complications, including cardiovascular disease, kidney damage, nerve damage, osteoporosis and blindness. The immune response that leads to type 1 diabetes attacks the pancreas, and in particular, the cell clusters known as the islet of Langerhans or pancreatic islets. Pancreatic islets contain several hormone-secreting cells types, but the one cell type in particular attacked by the immune system in type 1 diabetics are the insulin-secreting beta cells.

Pancreatic islets
Pancreatic islets

Destruction of the beta cells greatly decreases the body’s capability to make insulin, and without sufficient quantities of insulin, the body’s capability to take up, utilize and store sugar decelerates drastically, leading to mobilization of fats stores, the production of acid, wasting of several organs, excessive water loss, constant hunger, thirst, urination, acidosis (acidification of the blood), and eventually coma and death if left untreated.

The immune system not only destroys the beta cells, it also causes collateral damage to small blood vessels (capillaries) that carry blood to and from the pancreatic islets. This blood vessel damage led Zaghouani to examine ways to head this off at the pass and heal the resultant damage.

In previous studies, Zaghouani and others developed a drug against type 1 diabetes called Ig-GAD2. Treatment with this drug stops the immune system from attacking beta cells, but, unfortunately too few beta cells survived the onslaught from the immune system to reverse the disease. In his newest study, Zaghouani and his colleagues treated non-obese diabetic (NOD) with Ig-GAD2 and then injected bone marrow-based stem cells into the pancreas in the hope that these stem cells would differentiate into insulin-secreting beta cells.

“The combination of Ig-GAD2 and bone marrow [stem] cells did result in production of new beta cells, but not in the way we expected,” explained Zaghouani. “We thought the bone marrow [stem] cells would evolve directly into beta cells. Instead, the bone marrow cells led to growth of new blood vessels, and it was the new blood vessels that facilitated reproduction of the new beta cells. In other words, we discovered that to cure type 1 diabetes, we need to repair the blood vessels that allow the subject’s beta cells to grow and distribute insulin throughout the body.”

Zaghouani would lie to acquire a patent for his promising treatment and hopes to translate his preclinical research discovery from mice to larger animals and then to humans. In the meantime, his research continues to be funded by the National Institutes of Health and the University of Missouri.

Improving Cartilage Production By Stem Cells


To repair cartilage, surgeons typically take a piece of cartilage from another part of the injured joint and patch the damaged area, this procedure depends on damaging otherwise healthy cartilage. Also, such autotransplantation procedures are little protection against age-dependent cartilage degeneration.

There must be a better way. Bioengineers want to discover more innovative ways to grow cartilage from patient’s own stem cells. A new study from the University of Pennsylvania might make such a wish come true.

This research, comes from the laboratories of Associate professors Jason Burdick and Robert Mauck.

“The broad picture is trying to develop new therapies to replace cartilage tissue, starting with focal defects – things like sports injuries – and then hopefully moving toward surface replacement for cartilage degradation that comes with aging. Here, we’re trying to figure the right environment for adult stem cells to produce the best cartilage,” said Burdick.

Why use stem cells to make cartilage? Mauck explained, “As we age, the health and vitality of cartilage cells declines so the efficacy of any repair with adult chondrocytes is actually quite low. Stem cells, which retain this vital capacity, are therefore ideal.”

Burdick and his colleagues have long studied mesenchymal stem cells (MSCs), a type of adult stem cell found in bone marrow and many other tissues as well that can differentiate into bone, cartilage and fat. Burdick’s laboratory has been investigating the microenvironmental signals that direct MSCs to differentiate into chondrocytes (cartilage-making cells).

chondrocytes
chondrocytes

A recent paper from Burdick’s group investigated the right conditions for inducing fat cell or bone cell differentiation of MSCs while encapsulated in hydrogels, which are polymer networks that simulate some of the environmental conditions as which stem cells naturally grow (see Guvendiren M, Burdick JA. Curr Opin Biotechnol. 2013 Mar 29. pii: S0958-1669(13)00066-9. doi: 10.1016/j.copbio.2013.03.009). The first step in growing new cartilage is initiating cartilage production or chondrogenesis. To do this, you must convince the MSCs to differentiate into chondrocytes, the cells that make cartilage. Chondrocytes secrete the spongy matrix of collagen and acidic sugars that cushion joints. One challenge in promoting MSC differentiation into chondrocytes is that chondrocyte density in adult tissue is rather low. However, cartilage production requires that the chondrocytes be in rather close proximity.

Burdick explained: “In typical hydrogels used in cartilage tissue engineering, we’re spacing cells apart so they’re losing that initial signal and interaction. That’s when we started thinking about cadherins, which are molecules that these cells used to interact with each other, particularly at the point they first become chondrocytes.”

Desmosomes can be visualized as rivets through the plasma membrane of adjacent cells. Intermediate filaments composed of keratin or desmin are attached to membrane-associated attachment proteins that form a dense plaque on the cytoplasmic face of the membrane. Cadherin molecules form the actual anchor by attaching to the cytoplasmic plaque, extending through the membrane and binding strongly to cadherins coming through the membrane of the adjacent cell.
Desmosomes can be visualized as rivets through the plasma membrane of adjacent cells. Intermediate filaments composed of keratin or desmin are attached to membrane-associated attachment proteins that form a dense plaque on the cytoplasmic face of the membrane. Cadherin molecules form the actual anchor by attaching to the cytoplasmic plaque, extending through the membrane and binding strongly to cadherins coming through the membrane of the adjacent cell.

In order to simulate this microenvironment, Burdick and his collaborators and colleagues used a peptide sequence that mimics these cadherin interactions and bound them to the hydrogels that were then used to encapsulate the MSCs.

According to Mauck, “While the direct link between cadherins and chondrogenesis is not completely understood, what’s known is that if you enhance these interactions early during tissue formation, you can make more cartilage, and, if you block them, you get very poor cartilage formation. What this gel does is trick the cells into think it’s got friends nearby.”

See L Bian, et al., PNAS 2013; DOI:10.1073/pnas.1214100110.