Stem Cell Treatments for Aortic Aneurysms


The aorta is the largest blood vessel in our bodies and it emerges from the left ventricle of the heart, takes a U-turn, and swings down toward the legs (descending or dorsal aorta). There are several branches of the aorta as it sharply turns that extend towards the head and upper extremities.

Aorta structure

Sometimes, as a result of inflammation of the aorta or other types of problems, the elastic matrix that surrounds and reinforces the aorta breaks down.  This weakens the wall of the aorta and it bulges out.  This bulge is called an aortic aneurysm and it is a dangerous condition because the aneurysm can burst, which will cause the patient to bleed to death.

Aortic Aneurysm

If an aneurysm is discovered through medical imaging techniques, drugs are given to lower blood pressure and take some of the pressure off the aorta.  Also, drugs that prevent further degradation of the elastic matrix are also used.  Ultimately, for large or fast-growing aneurysms, surgical repair of the aorta is necessary.  For aneurysms of the abdominal aorta, a surgical procedure called abdominal aortic aneurysm open repair is the “industry standard.”  For this surgery, the abdomen is cut open, and the aneurysm is repaired by the use of a long cylinder-like tube called a graft.  Such grafts are made of different materials that include Dacron (textile polyester synthetic graft) or polytetrafluoroethylene (PTFE, a nontextile synthetic graft).  The surgeon sutures the graft to the aorta, and connects one end of the aorta at the site of the aneurysm to the other end.

A “kinder, gentler” way to fix an aneurysm is to use a procedure called endovascular aneurysm repair (EVAR).  EVAR uses these devices called “stents” to support the wall of the aorta.  A small insertion is made in the groin and the collapsed stent is inserted through the large artery in the leg.  Then the stent, which is long cylinder-like tube made of a thin metal framework and covered with various materials such as Dacron or polytetrafluoroethylene (PTFE), is inserted into the aneurysm.  Once in place, the stent-graft will be expanded in a spring-like fashion to attach to the wall of the aorta and support it.  The aneurysm will eventually shrink down onto the stent-graft.

In some cases, the patient is too weak for surgery, and is not a candidate for EVAR.  A much better option would be to non-surgically repair the elastic support framework that surrounds the aorta, and stem cells are candidates for such repair.

To repair the elastic mesh work that surrounds the wall of the aorta, smooth muscle cells that secrete the protein “elastin” must be introduced into the wall of the aorta.  Also, using the patient’s own stem cells offers a better strategy at this point, since this circumvents such issues as immune rejection of implanted tissues and so on.  The sources of stem cells for smooth muscle cells include bone marrow stem cells, fat-based stem cells, and stem cells from peripheral blood.  All three of these stem cell sources have problems with finding enough cells in the body and expanding them to high enough numbers in order to properly treat the aneurysm.

Fortunately, the use of induced pluripotent stem cells, which are made from a patient’s mature cells and have many, though not all of the characteristics of embryonic stem cells, can provide large quantities of elastin-secreting smooth muscle cells.  Also, one laboratory in particular has reported differentiating human induced pluripotent stem cells into smooth muscle cells (Lee TH, Song SH, Kim KL, et al. Circ Res 106:120–128).  While there are challenges to making functional elastin, there are possibilities that many of these can be overcome.

Ideal characteristics and expected roles of iPSCs and differentiated SMC-like derivatives for treating AAAs. Shown are several of the necessary properties for expansion/differentiation in culture, delivery to the AAA, and elastogenesis within the tunica media microenvironment. Abbreviations: AAA, abdominal aortic aneurysm; ECM, extracellular matrix; Eln, elastin; iPSC, induced pluripotent stem cell; LOX, lysyl oxidase; MMPs, matrix metalloproteinases; SMC, smooth muscle cell; TNFα, tumor necrosis factor-α.
Ideal characteristics and expected roles of iPSCs and differentiated SMC-like derivatives for treating AAAs. Shown are several of the necessary properties for expansion/differentiation in culture, delivery to the AAA, and elastogenesis within the tunica media microenvironment. Abbreviations: AAA, abdominal aortic aneurysm; ECM, extracellular matrix; Eln, elastin; iPSC, induced pluripotent stem cell; LOX, lysyl oxidase; MMPs, matrix metalloproteinases; SMC, smooth muscle cell; TNFα, tumor necrosis factor-α.

In addition to induced pluripotent stem cells, other laboratories have examined umbilical cord mesenchymal stem cells and their ability to decrease the inflammation within the aorta that leads to aneurysms.  The researchers discovered that all the indicators of inflammation decreased, but the synthesis of new elastin was not examined.  However, a Japanese laboratory used mouse mesenchymal stem cells from bone marrow and found that not only did these cells shut down enzymes that tend to degrade elastin, but also initiated new elastin synthesis in culture.  The same study also showed that MSCs implanted into the vessel walls of an aorta that was experiencing an aneurysm stabilized the aneurysm by inhibiting the elastin-degrading enzymes, and increasing the elastin content of the vessel wall.  This had the net effect of stabilizing the aneurysms and preventing them from growing further (see Hashizume R, Yamawaki-Ogata A, Ueda Y, et al. J Vasc Surg 54:1743–1752).  

These experiments show that stem cell treatments for abdominal aneurysms are feasible and would definitely be a much-needed nonsurgical treatment option for the high-risk elderly demographic, which is rapidly growing in the developed world.

For more information on this interesting topic, see Chris A. BashuraRaj R. Raob and Anand Ramamurthia. Perspectives on Stem Cell-Based Elastic Matrix Regenerative Therapies for Abdominal Aortic Aneurysms.  Stem Cells Trans Med June 2013 vol. 2 no. 6 401-408.

New Approach for Corneal Stem Cell Treatments


More than 8 million people worldwide suffer from corneal blindness; a form of blindness that results from cloudiness of the outermost covering of the eye, the cornea.

Usually, the cornea copes quite well with minor injuries or scrapes and scratches. If the cornea is scratched, healthy cells slide over quickly and patch the injury before infection occurs and vision is not adversely affected. However, if the scratch penetrates the cornea more deeply, then the healing process takes longer and can result in greater pain, blurred vision, tearing, redness, and extreme sensitivity to light. Such scratches may require professional treatment. Even deeper scratches can also cause corneal scarring, which results in a haze on the cornea that can greatly impair vision, and the patient might require a corneal transplant.

Alternatively, corneal stem cells can help heal a damaged cornea; especially in those cases where the cornea has been damaged to the point where the native stem cell population has suffered irreparable damage (e.g., chemical burns, eye infections, or cases where the patient was born with a corneal stem cell deficiency).

A feasible treatment for such cases is a corneal stem cell transplant from another eye or from cultured corneal stem cells. Unfortunately, this procedure has not yet been standardized to date.

Fortunately, researchers at the Eye Program at the Cedar-Sinai Regenerative Medicine Institute have designed a fast, new procedure for preparing human amniotic membrane to use as a scaffold for corneal stem cells. The membrane provides a foundation that supports the growth of stem cells that can be grafted onto the cornea.

To date, a standardized method does not exist for the preparation of amniotic membranes for culturing corneal stem cells. Many methods use chemicals and may leave behind amniotic cells and membrane components.

This new procedure, however, takes less than one minute and ensures complete amniotic cell removal and preservation of amniotic membrane components, and, as an added bonus, supports the overall growth of various stem and tissue cells.

“We believe that this straightforward and relatively fast procedure would allow easier standardization of amniotic membrane as a valuable stem cell support and improve the current standard of care in corneal stem cell transplantation,” said the lead author of this work Alexander Ljubimov, the director of the Eye Program at the Cedar-Sinai Regenerative Medicine Institute. “This new method may provide a better method for researchers, transplant corneal surgeons, and manufacturing companies alike.”

The amniotic membrane has several beneficial properties for corneal stem cells culturing and use in corneal transplantations. For this reason it is an attractive framework for the growth and culture of corneal stem cells and for corneal transplantations.

The new method for amniotic membrane preparation will provide a fast way to create scaffolds for cell expansion and might potentially streamline clinical applications of cell therapies.

Priming Cocktail for Cardiac Stem Cell Grafts


Approximately 700,000 Americans suffer a heart attack every year and stem cells have the potential to heal the damage wrought by a heart attack. Stem cells therapy has tried to take stem cells cultured in the laboratory and apply them to damaged tissues.

In the case of the heart, transplanted stem cells do not always integrate into the heart tissue. In the words of Jeffrey Spees, Associate Professor of Medicine at the University of Vermont, “many grafts simply didn’t take. The cells would stick or would die.”

To solve this problem, Spees and his colleagues examined ways to increase the efficiency of stem cell engraftment. In his experiments, Spees and others used mesenchymal stem cells from bone marrow. Mesenchymal stem cells are also called stromal cells because they help compose the spider web-like filigree within the bone marrow known as “stroma.” Even though the stroma does not make blood cells, it supports the hematopoietic stem cells that do make all blood cells.  Here is a picture of bone marrow stroma to give you an idea of what it looks like:

Immunohistochemistry-Paraffin: Bone marrow stromal cell antigen 1 Antibody [NBP2-14363] Staining of human smooth muscle shows moderate cytoplasmic positivity in smooth muscle cells.
Immunohistochemistry-Paraffin: Bone marrow stromal cell antigen 1 Antibody [NBP2-14363] Staining of human smooth muscle shows moderate cytoplasmic positivity in smooth muscle cells.
Stromal cells are known to secrete a host of molecules that protect injured tissue, promote tissue repair, and support the growth and proliferation of stem cells.

Spees suspected that some of the molecules made by bone marrow stromal cells could enhance the engraftment of stem cells patches in the heart. To test this idea, Spees and others isolated proteins from the culture medium of bone marrow stem cells grown in the laboratory and tested their ability to improve the survival and tissue integration of stem cell patches in the heart.

Spees tenacity paid off when he and his team discovered that a protein called “Connective tissue growth factor” or CTGF plus the hormone insulin were in the culture medium of these stem cells. Furthermore, when this culture medium was injected into the heart prior to treating them with stem cells, the stem cell patches engrafted at a higher rate.

“We broke the record for engraftment,” said Spees. Spees and his co-workers called their culture medium from the bone marrow stem cells “Cell-Kro.” Cell-Kro significantly increases cell adhesion, proliferation, survival, and migration.

Spees is convinced that the presence of CTGF and insulin in Cell-Kro have something to do with its ability to enhance stem cell engraftment. “Both CTGF and insulin are protective,” said Spees. “Together they have a synergistic effect.”

Spees is continuing to examine Cell-Kro in rats, but he wants to take his work into human trials next. His goal is to use cardiac stem cells (CSCs) from humans, which already have a documented ability to heal the heart after a heart attack. See here, here, and here.

“There are about 650,000 bypass surgeries annually,” said Spees. “These patients could have cells harvested at their first surgery and banked for future application. If they return for another procedure, they could then receive a graft of their own cardiac progenitor cells, primed in Cell-Kro, and potentially re-build part of their injured heart.”

New Model for Kidney Regeneration


Harvard Stem Cell Institute Kidney Diseases Program Leader Benjamin Humphreys has examined tissue regeneration in the kidney. His interest in kidney regeneration has occupied a major part of his career, but some of his more recent work resulted from his skepticism of a particular theory of kidney regeneration.

The kidney stem cell repair model postulates that scattered throughout the kidney are small stem cell populations and are activated after the kidney is injured to repair it. This theory, however, conflicts with another view of kidney regeneration. Namely that after injury, the cells of the kidney dedifferentiate into more primordial versions of themselves and proliferate, after which they differentiate into the various tissues of the kidney.

Humphreys and his colleagues now have evidence that strongly suggests that all the cells of the kidney have the capacity to divide after injury and contribute to kidney regeneration.

Their evidence comes in the form of experiments in mice in which the cells of the kidney were genetically tagged, and then the kidneys were injured to determine what cells contributed to the regeneration of the kidney.

The tagging in this experiment is complicated, but quite technically brilliant. The kidney is composed of myriads of tiny functional units called nephrons. Each nephron is fed by a tiny knot of blood vessels called a glomerulus.  The structure of a nephron is shown below.  

Nephron-image

The blood supply to the kidney comes from branches off the descending aorta knows as renal arteries.  After entering the kidneys, the renal arteries branch multiple times until they become tiny vessels that feed into each nephron known as afferent arterioles.  The afferent arterioles forms a dense network of knot-like vessels that form the glomerulus and the portion of the nephron that interacts with the glomerulus is known as the Bowman’s capsule..  The blood vessels of the glomerulus are very special because they are exceptionally porous.  However, the Bowman’s capsule has a series of cells with foot-like extensions that coat the glomerulus called “podocytes.”  An especially beautiful picture of podocytes wrapped around a glomerular vessel is shown below.

normal-kidney-podocyte

The podocytes cover the pores of the glomerulus and only allows water and things dissolved in water through the pores.  Proteins do not make it through – they are too heavily charged.  Cells also do not make it through – they are too big.  But water, sodium ions, potassium ions, hydrogen ions, some drugs, metabolites, waste products, and things like that all make it through the podocyte-guarded pores.  For this reason, if you have excessive protein or some blood cells in your urine, it is usually an indication that something is wrong.  

Now, rest of the tubing attached to the nephron serve to reabsorb all the things you do not want to get rid of and not absorb all the things you do want to get rid of.  The amount of water you eliminate depends on your degree of hydration and is controlled by a hormone called antidiuretic hormone, which is release by the posterior lobe of your pituitary gland when you are dehydrated.  In the presence of ADH, the posterior tubing reabsorbs more water, and in lower concentrations of this hormone, it reabsorbs far less.  

Now that we know something about the kidney, here’s how Humphreys and others genetically marked the kidneys of their mice.  The sodium-dependent inorganic phosphate transporter (SLC34a1) is only expressed in mature proximal tubule cells.  Tetsuro Kusaba, the first author on this paper, and his colleagues inserted a CreERT2 cassette into this gene.  If you are lost at this point all you need to remember is this: the CreERT2 cassette is inserted into a gene that is ONLY expressed in specific kidney cells.  The Cre gene encodes a recombinase that clips out specific bits of DNA from a chromosome.  Kusaba and others crossed these engineered mice with another strain of mice that had the gene for a bright red dye inserted into another gene, but this dye could not be expressed because another piece of DNA was in the way.  When these hybrid mice were fed a drug called tamoxifen, it activated the expression of the Cre protein, but only in the proximal tubule cells of the kidney and this Cre protein clipped out the piece of DNA that was preventing the red dye gene from being expressed.  Therefore, these mice had a particular part of their nephrons, the proximal tubules glowing bright red.  This is a stroke of shear genius and it genetically marks these cells specifically and strongly.  

Next, Kusaba and colleagues used unilateral ischemia reperfusion injury (IRI) to damage the kidneys.  In IRI, the blood supply is stopped to one kidney but not the other for a short period of time (26 minutes).  This causes cell death and kidney damage.  The other kidney is not damaged and serves as a control for the experiment.  

Examination of the damaged kidneys showed that  red-glowing cells were found in areas other than the proximal tubules.  The only way these cells could have ended up in these places was if the differentiated cells divided and helped repair the damaged parts of the nephrons.  

Other research groups have seen similar results, but interpreted them as evidence of stem cell populations in the kidney.  However, Humphreys groups discovered something even more fascinating.  These “stem cell-markers” in the kidney are actually markers of kidney damage and regeneration and all cells in the kidney express them.  In Humphreys words, “What was really interesting is when we looked at the appearance and expression patterns of these differentiated cells, we found that they expressed the exact same ‘stem cell markers’ that these other groups claimed to find in their stem cell populations.  And so, if a differentiated cell is able to express a ‘stem cell marker’ after injury, then what our work shows is that that’s an injury marker – is doesn’t define a stem cell.”  

Indeed, several genes that have been taken to be signs of a kidney stem cell population (CD133, CD24, vimentin, and KIM-1) were expressed in red-glowing cells.  A stem cell population should not be fully differentiated and therefore, should not be able to express the red dye.  However, red-glowing cells clearly expressed these found genes after injury.  This rather definitely shows that it is the fully differentiated cells that are doing the regeneration in the kidney and not a resident stem cell population.  This does not prove that there is no resident stem cell population in the kidney, but only that the lion’s share of the regeneration is done by differentiated cells, and that under these conditions, no stem cell population was detected.  

This new interpretation of kidney repair suggests that cells can reprogram themselves in a way that resembles the way mature cells are chemically manipulated to revert to an induced pluripotent state.  

See Tetsuro Kusaba, Matthew Lalli, Rafael Kramann, Akio Kobayashi, and Benjamin D. Humphreys. Differentiated kidney epithelial cells repair injured proximal tubule. PNAS (October 14, 2013); doi:10.1073/pnas.1310653110.  

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.”

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.

Porous Material Helps Deliver Molecules to Stem Cell-Derived Cells


A Swedish group has successfully tested a new porous material that allows for the efficient delivery of key molecules to transplanted cells that have been derived from stem cells. Such a material can dramatically improve the way stem cell-based treatments for neurodegenerative diseases.

This research project included a collaboration between Danish, Swedish and Japanese laboratories, and it tested a new type of porous material that efficiently delivers key molecules to transplanted cells derived from stem cells in an animal model.

Mesoporous silica loaded with differentiation factors induce motor neuron differentiation in vitro. (A): Top: Scanning and transmission (inset) electron micrographs of Meso. Scale bars = 200 nm (main panel) and 50 nm (inset). Bottom: CNTF with the Cintrofin motif shown in magenta and GDNF with the Gliafin motif shown in magenta. Amino acid residues are numbered according to UniProtKB entry nos. P26441 (Cintrofin) and Q07731 (Gliafin). (B): Differentiating motor neurons (MNs) extended numerous bTUB-labeled neurites (red) on poly-D-lysine (PDL)/laminin-coated coverslips after direct administration of CNTF and GDNF or treatment with MesoMim. Neurite formation was absent from MN precursors exposed to unloaded Meso. Scale bar = 75 μm. (C): Quantitative analysis of neurite length from MNs on PDL/laminin-coated coverslips after direct administration of CNTF and GDNF, treatment with MesoMim, or treatment with unloaded Meso. Results from 7–10 experiments are expressed as mean ± SEM, and the MesoMim group is set at 100%. Direct and MesoMim administration of the factors induced a significantly greater extent of neurite outgrowth compared with the unloaded Meso group; ***, p  .05). (D): HB9-GFP+ MNs expressed the MN markers ChAT and Isl1 in a 3-day differentiation assay after treatment with CNTF and GDNF or MesoMim but not in the absence of factors. Scale bar = 25 μm. (E, F): Almost all GFP+ cells expressed Isl1 (E) and ChAT (F) after treatment with CNTF and GDNF or MesoMim. Abbreviations: bTUB, β-tubulin; ChAT, choline acetyltransferase; CNTF, ciliary neurotrophic factor; D, day; GDNF, glial cell line-derived neurotrophic factor; GFP, green fluorescent protein; Isl1, Islet 1; Meso, mesoporous silica; MesoMim, mesoporous silica loaded with peptide mimetics; Rel., relative.
Mesoporous silica loaded with differentiation factors induce motor neuron differentiation in vitro. (A): Top: Scanning and transmission (inset) electron micrographs of Meso. Scale bars = 200 nm (main panel) and 50 nm (inset). Bottom: CNTF with the Cintrofin motif shown in magenta and GDNF with the Gliafin motif shown in magenta. Amino acid residues are numbered according to UniProtKB entry nos. P26441 (Cintrofin) and Q07731 (Gliafin). (B): Differentiating motor neurons (MNs) extended numerous bTUB-labeled neurites (red) on poly-D-lysine (PDL)/laminin-coated coverslips after direct administration of CNTF and GDNF or treatment with MesoMim. Neurite formation was absent from MN precursors exposed to unloaded Meso. Scale bar = 75 μm. (C): Quantitative analysis of neurite length from MNs on PDL/laminin-coated coverslips after direct administration of CNTF and GDNF, treatment with MesoMim, or treatment with unloaded Meso. Results from 7–10 experiments are expressed as mean ± SEM, and the MesoMim group is set at 100%. Direct and MesoMim administration of the factors induced a significantly greater extent of neurite outgrowth compared with the unloaded Meso group; ***, p < .001. No statistically significant differences were observed between groups with direct or MesoMim administration of the factors (p > .05). (D): HB9-GFP+ MNs expressed the MN markers ChAT and Isl1 in a 3-day differentiation assay after treatment with CNTF and GDNF or MesoMim but not in the absence of factors. Scale bar = 25 μm. (E, F): Almost all GFP+ cells expressed Isl1 (E) and ChAT (F) after treatment with CNTF and GDNF or MesoMim. Abbreviations: bTUB, β-tubulin; ChAT, choline acetyltransferase; CNTF, ciliary neurotrophic factor; D, day; GDNF, glial cell line-derived neurotrophic factor; GFP, green fluorescent protein; Isl1, Islet 1; Meso, mesoporous silica; MesoMim, mesoporous silica loaded with peptide mimetics; Rel., relative.

This potentially versatile and widely applicable strategy for the efficient differentiation and functional integration of stem cell derivatives upon transplantation, and it can serve as a foundation for improving stem cell-based neurodegenerative protocols, for example, Parkinson’s disease.

Alfonso Garcia-Bennett of Stockholm University, one of the lead authors of this study, said: “We are working to provide standard and reproducible methods for the differentiation and implementation of stem cell therapies using this type of approach, which coupled material science with regenerative medicine.”

Garcia-Bennett continued: “We demonstrated that delivering key molecules for the differentiation of stem cells in vivo with these particles enabled not only robust functional differentiation of motor neurons from transplanted embryonic stem cells but also improved their long-term survival.”

This research group is already working together with two companies to speed up the commercialization of a standard differentiation kit that will allow other scientists and clinicians to reproduce their work in their own laboratories.