Bone Marrow Mesenchymal Stem Cells Spontaneously Make Cartilage After Blockage of VEGF Signaling


Bone marrow-derived mesenchymal stem cells (MSCs) can be induced to make cartilage by incubating the cells with particular growth factors.  Unfortunately, batches of MSCs show respectable variability from patient-to-patient.  Therefore the growth factor-dependent method suffers from poor efficacy, limited reproducibility from batch-to-batch, and the cell types that are induced are not always terribly stable.  Finding a better way to make cartilage would certainly be a welcome addition to regenerative treatments,

Cartilage that coats the ends of bones is known as articulate cartilage, and articular cartilage lacks blood vessels.  Therefore, is it possible that inhibiting blood vessel formation could conveniently push MSCs to differentiate into cartilage-making chondrocytes?

A new paper by Ivan Martin and Andrea Basil from the University Hospital Basel and their colleagues have used this very strategy to induce cartilage formation in MSCs from bone marrow.

Martin and others isolated MSCs from bone marrow aspirates from human donors.  These cultured human MSCs were then genetically engineered with modified viruses to express a receptor for soluble vascular endothelial growth factor (VEGF) that binds this growth factor, but fails to induce any intracellular signals.  Such a receptor that binds the growth factor but does not induce any biological effects is called a “decoy receptor,” and decoy receptors efficiently sequester or vacuum up all the endogenous VEGF.  VEGF is the major blood vessel-inducing growth factor and it is heavily expressed during development, by cancer cells, and during healing.

After expressing the decoy VEGF receptor in these human MSCs, these genetically engineered cells were grown on collagen sponges and then implanted in immunodeficient mice.  If the implanted MSCs were not genetically engineered to express decoy VEGF receptors, they induced for formation of vascularized fibrous tissue.  However, the implantation of genetically engineered MSCs that expressed the decoy VEGF receptor efficiently and reproducibly differentiated into chondrocytes and formed hyaline cartilage. This is significant because headline cartilage is the very type of cartilage found at articular surfaces where the ends of bones come together to form joints.

In vivo chondrogenesis. Histological staining with Safranin-O for glycosaminoglycans and immunohistochemistry for type II collagen of engineered tissue generated by naïve (control) or sFlk-1 MSCs after 4 (A) or 12 (B) weeks in vivo. Fluorescence staining with DAPI (in blue) and a specific anti-human nuclei antibody (in red) of constructs generated by control or sFlk-1 MSCs after 4 (A) or 12 (B) weeks in vivo. Scale bar = 100 µm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; MSC, bone marrow-derived mesenchymal stromal/stem cell.
In vivo chondrogenesis. Histological staining with Safranin-O for glycosaminoglycans and immunohistochemistry for type II collagen of engineered tissue generated by naïve (control) or sFlk-1 MSCs after 4 (A) or 12 (B) weeks in vivo. Fluorescence staining with DAPI (in blue) and a specific anti-human nuclei antibody (in red) of constructs generated by control or sFlk-1 MSCs after 4 (A) or 12 (B) weeks in vivo. Scale bar = 100 µm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; MSC, bone marrow-derived mesenchymal stromal/stem cell.

This articular cartilage was quite stable and showed no signs of undergoing the chondrocytes enlargement found in terminally differentiated cartilage that is ready to form bone.  This stability was maintained for up to 12 weeks.

In vivo cartilage stability. Immunohistochemistry for type X collagen, BSP, and MMP-13 on sections of hypertrophic cartilage generated in vitro by MSCs (as a positive control) and on sections of the cartilaginous constructs generated in vivo by sFlk1 MSCs 12 weeks after implantation. Scale bar = 50 µm. Abbreviations: BSP, bone sialoprotein; MMP-13, metalloproteinase-13; MSC, bone marrow-derived mesenchymal stromal/stem cell.
In vivo cartilage stability. Immunohistochemistry for type X collagen, BSP, and MMP-13 on sections of hypertrophic cartilage generated in vitro by MSCs (as a positive control) and on sections of the cartilaginous constructs generated in vivo by sFlk1 MSCs 12 weeks after implantation. Scale bar = 50 µm. Abbreviations: BSP, bone sialoprotein; MMP-13, metalloproteinase-13; MSC, bone marrow-derived mesenchymal stromal/stem cell.

Why did inhibition of VEGF signaling induce cartilage?  Inhibition of angiogenesis induced low oxygen tensions, which activated a growth factor called transforming growth factor-β.  Activation of the TGF-beta pathway robustly enhanced the formation of articular cartilage.

In vitro chondrogenesis at different oxygen tensions. Histological staining with Safranin-O and immunohistochemistry for type II collagen on constructs generated in vitro by naïve MSC cultured with (A) or without (B) TGFβ3 supplementation at 2% or 20% of oxygen tension. Scale bar = 50 µm. Expression levels of the mRNA for type II and X collagen, Gremlin-1, IHH TGFβ1 were quantified in pellets generated by naïve bone marrow-derived mesenchymal stromal/stem cells (C, D) cultured in the two different oxygen tensions. ∆Ct values were normalized to expression of the GAPDH housekeeping gene, and results are shown as mean ± SD (n = 6 samples/group from 3 independent experiments). ∗, p < .05, ∗∗∗, p < .001. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IHH, Indian hedgehog; TGFβ, transforming grown factor-β.
In vitro chondrogenesis at different oxygen tensions. Histological staining with Safranin-O and immunohistochemistry for type II collagen on constructs generated in vitro by naïve MSC cultured with (A) or without (B) TGFβ3 supplementation at 2% or 20% of oxygen tension. Scale bar = 50 µm. Expression levels of the mRNA for type II and X collagen, Gremlin-1, IHH TGFβ1 were quantified in pellets generated by naïve bone marrow-derived mesenchymal stromal/stem cells (C, D) cultured in the two different oxygen tensions. ∆Ct values were normalized to expression of the GAPDH housekeeping gene, and results are shown as mean ± SD (n = 6 samples/group from 3 independent experiments). ∗, p < .05, ∗∗∗, p < .001. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IHH, Indian hedgehog; TGFβ, transforming grown factor-β.

Cartilage formation from MSCs was induced by blocking VEGF-mediated angiogenesis.  These results represent a remarkable advance in cartilage formation that can be used for regenerative treatments.  This cartilage formation was spontaneous and efficient and if it can be carried out with VEGF-inhibiting drugs rather than genetic engineering techniques, then we might have a transferable technique for making cartilage in the laboratory to treat osteoarthritis and other joint-based maladies.  Clinical trials will be required, but this is certainly an auspicious start.

Pretreatment of Mesenchymal Stem Cells with Melatonin Improves Their Healing Properties in Animals with Strokes


The transplantation of mesenchymal stem cells or MSCs as they as affectionately known, does indeed benefit patients who have had a stroke. Unfortunately, the benefits of MSC transplantation if is limited by inability of these cells to survive after they are implanted into a low-oxygen environment. When a person suffers from a stroke, a blood vessel that feeds the brain has been blocked, and this blockage results in the death of particular cells in the brain. The affected areas of the brain, however, have been deprived of oxygen, and the transplantation of new cells into these areas can result in the prompt death of the implanted cells.

Fortunately, previous studies have revealed that pretreatment of the implanted cells with the hormone melatonin can increase the survival of MSCs that were implanted into kidneys that suffered oxygen deprivation. Therefore, could melatonin pretreatment also improve MSC survival in the case of strokes?

A new study by Guo-Yuan Yang and his colleagues at the Med-X Research Institute in Shanghai, China has examined the effects of melatonin pretreatment on the survival of MSCs that were implanted into the brains of laboratory animals that suffered a stroke.

In a nutshell, Yang and his colleagues showed that melatonin pretreatment greatly increased survival of cultured MSCs when these cells were subjected to low-oxygen conditions. Then when they went whole hog and transplanted their melatonin-pretreated MSCs into the brains of animals that had suffered a stroke, they once again observed that these cells survived at a substantially higher rate than their untreated counterparts. Melatonin-pretreated MSCs also further reduced bleeds into the brain (infarction) and improved the behavioral outcomes of the laboratory animals.

When Yang’s group examined the molecules secreted by the melatonin-treated MSCs, they discovered that the melatonin-pretreated MSCs made a lot more blood-vessel-promoting proteins (such as vascular endothelial growth factor or VEGF), and nerve cell-promoting molecules. Not surprisingly, the rats implanted with melatonin-pretreated MSCs shows significantly more new blood vessels formed, new neurons formed, and better looking brains in general.

Melatonin treatment increased the levels of two signaling molecules, p-ERK1/2, in MSCs. These particular signaling molecules are linked to higher survival rates. When Yang and his crew blocked melatonin signaling by treating cells with as drug called luzindole, these positive effects were reversed and when another drug called U0126, which prevents ERK from becoming phosphorylated was also applied to the cells, it completely reversed the protective effects of melatonin.

These results show that melatonin improves MSC survival and function. Furthermore, melatonin does this by activating the ERK1/2 signaling pathway. Therefore, mesenchymal cells pretreated by melatonin may represent a viable approach to enhance the beneficial effects of stem cell therapy for strokes, and maybe other conditions too? Well shall see. Stay tuned…..

A Genetic Recipe To Convert Stem Cells into Blood


University of Wisconsin at Madison Stem Cell researchers led by Igor Slukvin discovered two genetic programs that can convert pluripotent stem cells into the wide array of white and red blood cells found in human blood (pluripotent means “capable of developing into more than one organ or tissue and not fixed as to potential development).

This research has ferreted out the actual pathway used by the developing human body to make blood-based cells at the early stages of development.

During embryonic development, blood formation, which includes the formation of blood cells and blood vessels from the same progenitor cell; a cell called a hemangioblast. This begins in week three of development in the extraembryonic mesoderm or the primary embryonic umbilical sac, which is also known as the yolk sac. Also, the connecting stalk and chorion contain blood islands as well. These blood islands are rich in particular growth factors such as vascular endothelial growth factor (VEGF) and placental growth factor (PIGF). The blood islands form clusters with two cell populations; peripheral cells (angioblasts) that form the endothelial cells that form vessels. These networks of vessels extend and fuse together to form a robust a network. The cores of the blood islands (hemocytoblasts) form blood cells. Initially all vessels (arteries and veins) look the same. Blood formation occurs later in week 5, and occurs throughout the embryonic mesenchyme (connective tissue), and then moves to the liver, and then the spleen, and then bone marrow.

Embryonic red blood cells
Embryonic red blood cells

Hematopoietic stem cells (HSCs), the stem cells that form the blood cells, form from the wall of the aorta, which is the major blood vessel in the embryo. In the aortic wall, cells called hemogenic endothelial cells bud off progenitor cells that become HSCs.

A course of transcription factors have now been identified by Slukvin and his team as the triggers that switch these cells into HSCs. Two groups of transcriptional regulators can induce distinct developmental programs from pluripotent stem cells. The first developmental program, directed by the transcription factors ETV2 and ​GATA2, the pan-myeloid pathway, switches cells into the myeloid lineage (the myeloid lineage includes red blood cells, platelets, neutrophils, macrophages, basophils and eosinophils). The second developmental pathway, directed by the transcription factors GATA2 and ​TAL1, directs cells into the erythro-megakaryocytic pathway. In either cases, these transcription factors directly convert human pluripotent stem cells into an endothelium, which subsequently transform into blood cells with pan-myeloid or erythro-megakaryocytic potential.

Hematopoietic_and_stromal_cell_differentiation

In Slukvin’s laboratory, treatment of either ETV2 and ​GATA2 or GATA2 and ​TAL1 induced cells to make the complete range of human blood cells. Slukvin said of these experiments, “This is the first demonstration of the production of different kinds of cells from human pluripotent stem cells using transcription factors.” Transcription factors bind to DNA at specific sites and regulate gene expression.

Slukvin continued: “By overexpressing just two transcription factors, we can, in the laboratory dish, reproduce the sequence of events we see in the embryo.”

Slukvin and his co-workers showed that his technique produced blood cells by the millions. For every million stem cells, it was possible to produce 30 million blood cells.

Slukvin and his colleagues did not use viruses to genetically modify these stem cells. Instead they used modified RNA to induce overexpression of these transcription factors. Such a technique avoids genetic modification of cells and is inherently safer.

“You can do it without a virus, and genome integrity is not affected,” said Slukvin.  This technique might also work to differentiate pluripotent stem cells into other cell types, such as pancreatic beta cells, brain-specific cells, or liver cells.

Despite these successes, Slukvin says that the “Holy grail” of hematopoietic research is to differentiate pluripotent stem cells into HSCs.  Since HSC transplants are used to treat multiple myeloma and other types of blood-based cancers as well, making HSCs in the laboratory remains a significant goal and challenge as well.

“We still don’t know how to do that,” said Slukin, “but our new approach to making blood cells will give us an opportunity to model their development in a dish and identify novel hematopoietic stem cell factors.”

Sheets of Heart Muscle Cells Made from Induced Pluripotent Stem Cells Increases Blood Vessel Density in Infarcted Heart


Induced pluripotent stem cells (iPSCs) are made from adult cells by means of genetic engineering techniques that introduce specific genes into adult cells. These genes express their proteins and they push the adult cell to de-differentiate into cells that, in many ways, resemble embryonic stem cells. These de-differentiated cells can form all the cell types, and they can potentially be used for regenerative medicine.

In particular, the heart can experience the death of heart muscle cells after a heart attack, and replacement of dead heart muscle cells can return the heart to its full potential. To this end, iPSCs and embryonic stem cells (ESCs) can be differentiated into heart muscle cells and transplanted into the damaged heart. Such experiments have been done and they do improve heart function, but what is the best way to apply the heart muscle cells? Should they be injected into the heart wall? Should they be applied on sheets?

Hidetoshi Matsumoto and colleagues in the laboratory of Jun Yamashita at Kyoto University has made mouse iPSCs, and differentiated them into heart muscle cells that were grown in sheets. These sheets were laid over the heart of athymic rats that had suffered a heart attack (athymic mice do not have a thymus gland and therefore are unable to reject transplanted, foreign tissue). The results were telling not for what they did to the heart, but how they did it.

Matsumoto made sheets of heart muscle, sheets of endothelial cells, which make blood vessels, and sheets of mural cells, which are the smooth muscle cells that control the diameter of blood vessels. He also made sheets that contained combinations of cells and sheets with all three cell types.

The sheets that contained all three cell types improved heart function, but after four weeks, no transplanted cells could be detected. How then did these sheets improve heart function? The answer was in a vast increase in the density of blood vessels. When the sheets were applied, they quickly showed a vast increase in the expression of those genes that induce the formation of blood vessels. Thus, even though the cells did not survive, the blood vessels they induced endured and improved heart function.

When cell sheets consisting only of endothelial cells or endothelial cells and mural cells were applied, only slight improvements in heart function were observed and the vast increase in blood vessel density did not ensue. Therefore, heart muscle cells are necessary to induce the formation of new blood vessels.

These results are very similar to those of Yoon et al, who used genetically engineered bone marrow cells to treat rodent hearts that had experienced a heart attack. When Yoon and others depleted these populations of either endothelial or mural cells, the implanted cells failed to improve heart function (Yoon et al., Circulation 2010 121: 2001-11).

Thus. iPSC-derived heart muscle sheets might very improve heart function after a heart attack, but they might do so without actually integrating into the heart.

On a closing note, it seems to me that preconditioning these cells to survive in the hostile environment of the infarcted heart might improve the survival of these cells, and therefore, and their ability to improve heart function. This might be an experiment for future researchers.

See Matsumoto et al, Stem Cells 2012 30:1196-1205.

Rejuvenating Aged Stem Cells With a Fountain-Of-Youth Cocktail


Stem cell researchers from the laboratory of Ren-Ke Li at the University of Toronto have discovered a cocktail that can kick old, lagging stem cells in the backside and renew their regenerative capacities.

Donated bone marrow stem cells are transplanted into patients with leukemia, or diseases that compromise bone marrow function. Unfortunately, even though such therapies save hundreds to thousands of lives every year, some of these patients die or become horribly ill because the patient rejects some of the cells in the donated bone marrow. To reduce the risk of bone marrow rejection, stem cells treatments have used stem cells from the patient’s own body. Unfortunately, such a strategy is unusable in older patients, since their stem cell function has been vitiated by the ravages of age. If there is a way to beef up the stem cell function of an older patient, why then, this protocol would definitely be preferred.

Ren-Ke Li, professor in the Division of Cardiovascular Surgery and a member of the Institute for Biomaterials and Biomedical Engineering at the University of Toronto, Canada and his colleague Milica Radisic, an associate professor of chemical engineering have designed a unique micro-environment that allows heart tissue to grow from stem cells donated by elderly patients.

This micro-environment utilizes a porous scaffold made of collagen (the protein found in scar tissue), and embedded in this scaffold are two growth factors (vascular endothelial growth factor and basic fibroblast growth factor). Radisic and Li and their co-worked seeded these scaffolds with stem cells taken from younger (~50 years old) and older donors (~75 years old) and then used them to repair the left ventricles of rats with damaged hearts.

The scaffolds without growth factors and seeded with stem cells from older donors did not repair the hearts very well, but those scaffolds without growth factors and seeded with stem cells from younger donors did a good job of repairing the hearts. When the scaffolds impregnated with growth factors were seeded with stem cells from older donors, the patches did a much better job of repairing the hearts; they did as good a job of facilitating heart repair and those scaffolds seeded with stem cells from younger patients.

Patch Morphology 28 Days After Implantation In Vivo(A) Representative images of rat hearts show the outer border of the patches depicted by the yellow dotted line. (B) Patch area was quantified using computerized planimetry. The patch area increased in all groups from the original size of 39 mm2(red dotted line) at the time of SVR. Patch area in the Old group was significantly larger after implantation than that in the other groups. The addition of cytokines significantly prevented patch expansion. (C) Representative images of heart slices stained with Masson's trichrome. Arrows indicate patch thickness. (D) Patch thickness was quantified using computerized planimetry. The patches in the Old group were significantly thinner than patches in the Young and Young + GF groups. Cytokine enhancement did not significantly increase patch thickness for old or young cells. *p < 0.05, **p < 0.01 vs. Old; Old n = 5, Young n = 8, Old + GF n = 6, Young + GF n = 8. GF = growth factor.
Patch Morphology 28 Days After Implantation In Vivo(A) Representative images of rat hearts show the outer border of the patches depicted by the yellow dotted line. (B) Patch area was quantified using computerized planimetry. The patch area increased in all groups from the original size of 39 mm2(red dotted line) at the time of SVR. Patch area in the Old group was significantly larger after implantation than that in the other groups. The addition of cytokines significantly prevented patch expansion. (C) Representative images of heart slices stained with Masson’s trichrome. Arrows indicate patch thickness. (D) Patch thickness was quantified using computerized planimetry. The patches in the Old group were significantly thinner than patches in the Young and Young + GF groups. Cytokine enhancement did not significantly increase patch thickness for old or young cells. *p < 0.05, **p < 0.01 vs. Old; Old n = 5, Young n = 8, Old + GF n = 6, Young + GF n = 8. GF = growth factor.

When Li and his team tracked the molecular changes in the stem cells grown on the scaffolds, they found that these cells acted like younger stem cells. In Li’ words: “We saw certain aging factors turned off.” The levels of two molecules in particular, p16 and RGN were reduced in the older stem cells grown on the growth factor-containing scaffolds, which turned back the clock in these cells and returned them to a more robust and healthy state.

Li and Radisic hope to experiment with their micro-environment in order to make it as effective as possible. According to Li, “We can create much better tissues which can then be used to repair defects such as aneurysms.” Li also thinks that these cells could be used to repair the heart after a heart attack.

See Kai Kang, et al., Aged Human Cells Rejuvenated by Cytokine Enhancement of Biomaterials for Surgical Ventricular Restoration,” Journal of the American College of Cardiology 2012 60(21): 2237 DOI: 10.1016/j.jacc.2012.08.985.

Growth Factors to Heal the Heart


When the heart suffers a heart attack, local areas of the heart experience cell death as a result of blockage in a coronary vessel. The cell death is followed by local inflammation which causes further cell death and produces a heart scar. This produces a situation in which a portion of the heart does not contract and also does not conduct impulses to beat. Can this dead heart tissue live again?

Several experiments have used stem cells to refurbish the dead heart tissue, and a variety of different stem cells can clearly produce new heart cells that help the heart beat better. Can growth factors that stimulate cell growth and division do a similar job?

Just injecting growth factors into the bloodstream will not do because the growth factors will not spend any appreciable time in or around the heart cells. Is there another way to do it? Yes. The answer is hydrogels.

Hydrogels are semi-solid materials that can be made and in which the growth factors can be embedded. The hydrogels are gradually degraded while they release growth factors into the heart tissue. The slow but stead release of various growth factors can induce the heart to heal itself.

Works from the laboratory of Michael E. Davis at Georgia Institute of Technology and Emory University School of Medicine in Atlanta, Georgia have published a paper in PLoS ONE describing this very strategy. Using rats that had suffered heart attacks, Davis and his group applied a polyethylene glycol-based hydrogel laced with two growth factors, hepatic growth factor (HGF) and vascular endothelial growth factor (VEGF) to the hearts of these animals.

There were no immediate effects to the application of these hydrogels as determined by electrocardiograms. However, with the passage of time, some remarkable changes to the hearts of these rats were observed. Three weeks after the application of hydrogels to rat hearts, animals treated hydrogel material only, injected with growth factors only showed no significant improvement over those rats that were not injected with anything. But those rats whose hearts had been injected with hydrogels laced with VEGF showed a 50% increase in blood vessel density and those injected with hydrogel imbued with HGF and VEGF showed a 100% increase in blood vessel density. These same rats also showed a huge reduction in the size of the heart scar (41.5 % vs 13.9% fibrosis), and also showed significant increased in heart function after three weeks.

Why did these growth factors work so well? Several experiments conducted by Davis’ group showed that the stem cell population in the heart, the cardiac progenitor cells or CPCs, were pitched into overdrive by the growth factors, In short, in the presence of these two growth factors, the cells went nuts. They went to area where the hydrogel had been applied and made new heart muscle cells and blood vessels.

Therefore, these two growth factors can be applied to the heart to elicit healing within the heart after a heart attack. The hydrogels keep the growth factors there and release them slowly so tat they can perform their healing magic.

Hopefully this experiment will lead to preclinical studies in larger animals (pigs and sheep), and then, hopefully, clinical trials in human patients.  See Salimath AS, et al., PLoS ONE 2012 7(11) e50980.