Converting Mesenchymal Stem Cells to Bone Makers


Within human bone, cells called osteoblasts make new bone and without the constant activity of osteoblasts, bone becomes thin and fragile. Osteoblasts are derived from mesenchymal stem cells in the bone marrow. When bones break, orthopedic surgeons try to use growth factors to push more mesenchymal stems and their progeny to become osteoblasts. The growth factor in question is bone morphogen protein (BMP). BMP, however, does not work consistently, and it has some rather nasty side effects (cancer, The specific complications that are drawing the most concern include swelling in the neck and throat, radiating leg pain, and male sterility). Therefore, an alternative method for converting mesenchymal stem cells into osteoblasts is highly desirable.

Kurt Hankenson from the University of Pennsylvania School of Veterinary Medicine has worked on this very problem and described the situation this way, “In the field, we’re always searching for new ways for progenitor cells to become osteoblasts so we became interested in the Notch signaling pathway.” When it comes to BMP, Hankenson said, “it has become clear that BMPs have some issues with safety and efficacy.”

Is there a better way to make bone? There seems to be. A protein called Jagged-1 has been shown by Hankenson’s team to be highly expressed in bone. Jagged-1 is a component of the widely used Notch signaling pathway, which is found in the nervous system and in many other cells as well.

In mouse stem cells, introducing Jagged-1 blocks the progression of mesenchymal stem cells to osteoblasts. This finding has actually hampered osteoblast research for the last two years. Hankenson again, “That had been our operating dogma for a year or two.”

However, as is so often the case in science, you never truly know the result of an experiment until you actually do it. When Jagged-1 was added to human mesenchymal stem cells, the results were very different. Hankenson said, “It was remarkable to find that just putting the cells onto the Jagged-1 ligand seemed sufficient for driving the formation of bone-producing cells.”

From a developmental genetics perspective, this makes perfect sense, since mutations in the Jagged-1 gene cause an inherited disease known as Alagille syndrome which causes liver problems, abnormal metabolisms, and fragile bones that break easily. Also, genome-wide association studies have shown that particular versions of the Jagged-1 gene cause low bone density.

Hankenson and his collaborators are examining ways to manipulate the levels of the Jagged-1 protein in patients with bone problems. To that end, Hankenson is collaborating with Kathleen Loomes of Penn’s Perelman School of Medicine and the Children’s Hospital of Pennsylvania to study pediatric patients with Alagille syndrome to determine if bone abnormalities in these patients are indeed connected to Jagged-1 malfunctions.

Hankenson and his former graduate student Mike Dishowitz started a company called Skelegen through the University of Pennsylvania’s Center for Technology Transfer (CTT) UPstart program. The goal of Skelegen is to develop and improve a system for delivering Jagged-1 to sites that require new bone growth in the hopes of treating bone fractures and other skeletal problems.

See Fengchang Zhu et al., “Pkcdelta is required for Jagged-1 induction of hMSC osteogenic differentiation.” Stem Cells 2013; DOI 10.1002/stem.1353.

Drug Induces Hearing Restoration in Rodents


Fish and birds are able to regenerate their hearing after damage, but mammals are not able to do so, and hearing loss is irreversible in mammals like human beings. However, a new study has shown that the application of a particular drug can activate genes normally expressed during hair cell development. This work resulted from collaboration between researchers at Harvard Medical School, the Massachusetts Eye and Ear Infirmary, and Keio University School of Medicine in Japan. This finding is a first in the field or regenerative medicine.

Hair Cell Regeneration

In the cochlea, small cells known as hair cells convert sound waves into electrical signals that are interpreted by the brain into sounds. If these hair cells are damaged or destroyed by acoustic injury, then a permanent loss of hearing ensues. Such damage is treated with cochlear implants, which are surgically implanted devices that convert sounds to electrical signals.

“Cochlear implants are very successful and have helped a lot of people, but there’s a general feeling among clinicians, scientists, and patients that a biological repair would be preferable,” said Albert Edge, an otologist at Harvard University and the Massachusetts Eye and Ear Infirmary and lead author of the Neuron paper that reports these findings.

In previous work, Edge and his colleagues had shown that inhibiting the Notch signaling pathway was important for hair cells to form properly during fetal development (Jeon, S.J., Fujioka, M., Kim, S.C., and Edge, A.S.B. (2011). Notch signaling alters sensory or neuronal cell fate specification of inner ear stem cells. J. Neurosci. 31, 8351–8358). In their new study, Edge and his colleagues inhibited the Notch signaling pathway to determine, if such inhibition could initiate hair cell regeneration in adult mammals. They used a variety of approaches. In their first experiments, they used different inhibitors to determine their effects on isolated ear tissues. This allowed them to isolate one inhibitor in particular, the ɣ-secretase inhibitor LY411575, that led to increased expression of several molecular markers found in developing hair cells.

LY411575
LY411575

“It was quite a surprise,” said Edge. “We were very excited when we saw that a secretase inhibitor would have any effect at all in an adult animal.”

Next, Edge and his co-workers tested the inhibitor in mice that had hearing damage and reduced hair cell populations as a result of exposure to a loud noise. They tagged cells in the inner ear to follow their fate and discovered that the inhibitor, when applied to the inner ears of the mice, caused supporting cells to differentiate into replacement hair cells. These newly formed hair cells partially restored hearing at low sound frequencies, but not at higher frequencies. This effect lasted for at least three months.

This study examined the effect of the inhibitor when it was given one day after noise damage, which is a time when Notch signaling is naturally increased. This it is possible that a small window of time exists after an acoustic injury during which the drug is effective.

Edge concluded: “The improvement we saw is modest. So we’re now looking at variations of the approach and whether we can use the same drug to treat other types of hearing loss.”

See: Mizutari K, Fujioka M, Hosoya M, Bramhall N, et al. (2013) Notch Inhibition Induces Cochlear Hair Cell Regeneration and Recovery of Hearing after Acoustic Trauma. Neuron 77, 58-69.

Leukemia Gene is a Key Factor for Nerve Cell Differentiation


Research from the laboratory of Pierre Vanderhaeghen from the Universite’ Libre de Bruxelles has provided a new perspective on brain development and neural stem cell biology.

The cerebral cortex is the most complex structure in the brain. It is the seat of such higher cortical functions as consciousness, learning and memory, emotion, motor control, and language. To execute these functions, the cerebral cortex is composed of an array of cortical neurons, and these cells are adversely affected in cases of neurological or even psychiatric disorders.

According to work from Vanderhaeghen’s laboratory, a gene known as BCL6 is a key element in the development of cortical neurons during development. BCL6 acts as a transcription factor, which is to say that it plays a role in gene expression. In the case of BCL6, this gene product prevents gene expression (functions as a repressor). In the immune system, BCL6 is made in antibody-producing cells (B cells) and it controls the response of B cells to a signaling protein called Interleukin 4 (IL-4). IL-4 drives the differentiation of B cells into antibody-making plasma cells and drives the maturation of plasma cells into those that make distinct types of antibodies. Even more interestingly, BCL6 is frequently abnormal in a blood cancer known as diffuse large B cell lymphoma (DLBCL),

Two members of Vanderhaeghen’s lab discovered BCL6 in a search for genes that modulate the production of new nerve cells from mouse embryonic stem cells. If they overexpressed BCL6 in neural stem cells made from mouse embryonic stem cells, these stem cells transformed en mass into cortical neurons. Because BCL6 is normally known for its role in blood cancers (lymphomas), this BCL6-medicated function was a complete surprise.

Because data from overexpression studies is always suspect without verification, Vanderhaeghen and his colleagues used mouse genetics to confirm the role of BCL6 in the production of cortical neurons. Vanderhaeghen’s team made mutant mice embryos that had lost a functional copy of the BCL6 gene. When these mice developed to the fetal stage, it was clear that they had small cerebral cortexes that consisted of far fewer cortical neurons. Therefore, BCL6 overexpression increases cortical neuron production and the absence of it decreases cortical neuron production. This certainly confirms the role of BCL6 in cortical neuron development.

Next, Vanderhaeghen’s lab determined how BCL6 was influencing the development of cortical neurons. A protein that is encoded by the Notch gene are essential in the self-renewal of neural stem cells. BCL6 works with another protein called SIRT1 to repress the Notch pathway, and this repression moves the progeny of neural stem cells to differentiate into cortical neurons.

Because cortical neurons are the main entities affected by neurological and psychiatric disorders, this understanding of cortical neuron development might provide insights into inherited forms of dementia, behavioral problems or other types of neurological problems. Also, Vanderhaeghen’s work bring together three major players involved in cancer BCL6), aging, Alzheimer’s disease, metabolism and diabetes (SIRT1), and brain and heart development and cancer (Notch). Because these three genes were not know to interact with each other prior to this work, Vanderhaeghen’s findings have opened up a new avenue of possible targets for therapies and model systems for understanding stem cell renewal and differentiation.

The Notch Signaling Pathway


Because stem cell differentiation is controlled by signal transduction pathways, some of my readers have suggested that I discuss particular signal transduction pathways. In the previous post, the Notch signaling pathway was mentioned, and this provides a good reason to introduce my readers to it.

To think of signal transduction, one should consider the popular board game, Mouse Trap. When your game icon lands on the mouse trap spot on the game board, you turn a crank, and this crank rotates a vertical gear that is connected to a gear. Once that gear turns, it pushes lever that is braced with a rubber band until it snaps back and hits a swinging boot. The boot kicks over a bucket, which sends a marble down a rickety staircase. At the bottom of the staircase, the marble enters a chute and eventually taps a vertical pole. At the top of this pole is an open hand (palm-up) that supports another marble. The movement of the pole, caused by the tapping of its base by the first marble knocks the second marble free and it falls through a hole in its platform into a bathtub, and then through a hole in the tub onto one end of a seesaw. The propulsion of the seesaw launches a plastic diver on the other end into a round tub that is on the same base as the barbed pole that supports the mouse cage. The tub’s movement shakes the cage free from the top of the pole and the cage falls to trap the mouse.

This machine that traps the mouse is very similar to signal transduction in cells. The signal to catch the mouse (turning the crank), is far removed from the cage that eventually catches the mouse. Also, the act of catching the mouse (the dropping of the mouse cage), requires the prior execution of many other causally linked steps.

Notch signaling begins with a cell surface protein called Notch. Notch has a large region of the protein outside the cell and a small part of it that intersects the cell membrane, and another region that extends into the cell interior. All three of these domains of the Notch protein play an essential role in the function of Notch.

To turn the crank of this mouse trap, Notch must bind to its receptor. The Notch receptor can be a member of the DSL (Delta, Serrate and Lag-2) gene family.  The receptor is found on the surface of another cell. The binding of Notch to its receptor is the action that “turns the crank” on this mouse trap. Notch binding changes the structure of Notch, and it is clipped into two unequal halves by an enzyme that clips proteins at specific sites (the gamma-secretase). The Notch protein is now broken into a portion that remains anchored in the cell membrane, and another regions that remains inside the cell. This portion of the Notch protein is called “Intracellular Notch” or ICN (Wang MM.Int J Biochem Cell Biol.2011 Nov;43(11):1550-62 & D’Souza B, Miyamoto A, Weinmaster G. Oncogene. 2008 Sep 1;27(38):5148-67).

With the cleavage of Notch, the boot has knocked over the bucket and the marble has moved down the rickety staircase to the chute. ICN is able to enter the cell nucleus. There are proteins in the cytoplasm that can bind to ICN and prevent it from doing so, but we will not discuss them at this time (see van Tetering G, Vooijs M. Curr Mol Med. 2011 Jun;11(4):255-69).

Once in the nucleus, ICN teams up with another protein to activate the express of particular genes. Therefore, what began at the cell surface with the binding of the Notch protein by its receptor had culminated in the changes in gene expression in the nucleus. The other proteins that work together with ICN are members of the “CSL” gene family. CSL stands for “CBF1/RBP-Jκ/Suppressor of Hairless/LAG-1.” When ICN combines with CSL the two proteins are converted from inactive proteins into a complex that actives the synthesis of messenger RNAs for specific genes. This rattles the pole that brings the cage down on the mouse’s head (see Kovall RA. Oncogene. 2008 Sep 1;27(38):5099-109).

What are the target genes of Notch signaling? Great question, but the answer is frustrating, since it depends on the cell type. In developing pancreas, once of the target genes of Notch signaling is PTF1a, but in other cell types and tissues, other genes are activated.

In embryonic stem cells and other stem cells as well, the Notch signaling pathway plays a vital role in the differentiation of these cells into various cell types. Notch signaling is also an important component of the pathology of organ failure in many organs and is also a central pathway involved in the onset and maintenance of several different types of cancers.  Understanding its function and how to regulate it is crucial.