A Living Patch for Damaged Hearts

Duke University scientists have constructed a three-dimensional human heart muscle patch that behaves much like natural heart muscle tissue. This advance could be used to either treat heart attack patients or to test new heart medicines.

This “heart patch” was grown in the laboratory from human cells, and the procedures used in this research overcame two large roadblocks. First the patch conducts electrochemical impulses at the same speed as normal adult human heart tissue and it contracts to the same degree as normal human heart tissue. In the past, heart tissue patches have conducted electrochemical impulses too slowly and contracted weakly.

The cell source used by the Duke University team were human embryonic stem cells. Thus, the heart patch would not be appropriate for human patients, since it would be rejected by the patient’s immune system. However, the procedures used in this research could also be applied to heart muscle cells made from induced pluripotent stem cells.

Nenad Bursac, associate professor of biomedical engineering at Pratt Engineering, said, “The structural and functional properties of these 3-D tissue patches surpass all previous reports for engineered human heart muscle. This is the closest man-made approximately of native human heart tissue to date.” Bursac also said that the approach does not involve genetic manipulation of the cells.

Bursac continued: “In past studies, human stem cell-derived cardiomyocytes (that is, heart muscle cells) were not able to both rapidly conduct electrical activity and strongly contract as well as normal cardiomyocytes. Through optimization of a three-dimensional environment for cell growth, we were able to ‘push’ cardiomyocytes to reach unprecedented levels of electrical and mechanical maturation.”

The rate of functional maturation is a procedural issue that has very practical implications. If clinicians want to make a heart patch for a patient, the time required to make the heart patch is important, since a heart patch that takes too long to make is of no clinical use to heart patients. In the developing human, it takes about nine months for the newborn heart to develop and an additional five years to reach adult levels of function. These heart patches, however, were grown in about 1 month. And, according to Brusac, further work should shorten the time required to make such a heart patch.

Bursac commented: “It would take us about five to six weeks starting from pluripotent stem cells to grow a highly functional heart patch. When someone has a heart attack, a portion of the heart muscle dies. Our goal would be to implant a patch of new and functional heart tissue at the site of the injury as rapidly after heart attack as possible. Using a patient’s own cells to generate pluripotent stem cells would add further advantage in that there would likely be no immune system reaction, since the cells in the patch would be recognized by the body as self.”

Bursac added that besides using these heart patches in patients, the patches could also be used in the laboratory to test new heart medicines and to model heart pathologies.

“Tests of trials of new drugs can be expensive and time-consuming.  Instead of, or along with testing drugs on animals, the ability to test on actual, functioning human tissue may be more predictive of the drugs’ effects and help determine which drugs should go into further studies.”

Some drug tests are conducted on two-dimensional sheets of heart cells, but according to Bursac, the three-dimensional culture of heart muscle cells provides a more realistic model system for drug testing.  Engineered heart tissues from patients who suffer from cardiac diseases could be used as a model to study that disease and test and explore potential therapies.

Even though Bursac used a particular embryonic stem cell line, but his co-workers also were able to replicate these results with two other embryonic stem cell lines.  Bursac also wants to test his heart muscle patches in animals to determine how well they integrate into the host heart tissue and how well they conduct electrical signals.

Silk and Cellulose as Scaffolds for Stem Cell-Mediated Cartilage Repair

When two bones come together, they grind each other into oblivion. This results in inflammation, joint swelling and pain, and scar tissue accumulation, which eventually results in the immobilization of the joint. To prevent this, bone are capped at their ends with a layer of hyaline cartilage that acts as a shock absorber. However, cartilage regenerates poorly and the wear and tear on cartilage, particularly at the knee, causes it to degenerate. The loss of the cartilage cap at the end of long bones causes osteoarthritis . The only way to mitigate the damage of osteoarthritis is to replace the knee with a prosthetic knee-joint.

Stem cells can make a significant contribution to the regeneration of lost cartilage. The Centeno/Schultz group near Denver, Colorado has been using bone marrow-derived mesenchymal stem cells to treat patients for over a decade with positive results. However, finding a way to grow large amounts of cartilage in culture that is the right shape for transplantation has proven difficult.

One way to mitigate this issue is the use of scaffolds for the cartilage-making cells that pushes them into a three-dimensional arrangement that forces them to make cartilage that mimics the cartilage found in a living organism. However a problem with scaffolds is finding the right material for the scaffold.

A recent publication has formed scaffolds from naturally occurring fibers such as cellulose and silk. By blending silk and cellulose fibers together, researchers at the University of Bristol have made a very inexpensive and easily manufactured scaffold for cartilage production.

Silk scaffold
Silk scaffold

When mixed with stem cells, cartilage and silk coax connective tissue-derived stem cells to differentiate into chondrocytes or cartilage-making cells. In the silk/cellulose scaffold, the chondrocytes secrete the extracellular matrix molecules characteristic of joint-specific cartilage.

Wael Kafienah, lead author of this work from the University of Bristol’s School of Cellular and Molecular Medicine, said, “The blend seems to provide complex chemical and mechanical cues that induce stem cell differentiation into preliminary form of chondrocytes without need for biochemical induction using expensive soluble differentiation factors. Kafienah continued: “This new blend can cut the cost for health providers and makes progress towards effective cell-based therapy for cartilage repair a step closer.”

To make the blended silk/cellulose scaffolds, Kafienah and his colleagues used ionic fluids, which effectively dissolve polymers like cellulose and silk, but are also much more environmentally benign in comparison to the organic solvents normally used to process silk and cellulose.

Presently, the U of Bristol team to trying to fabricate three-dimensional scaffolds that can be safely and easily implanted into patients for future clinical studies. Before human clinical studies are commenced, however, they must first be extensively tested in animals and also, the nature of the interactions between the scaffold and the stem cells that drive the cells to form cartilage must be better understood.

Skin Cells Used to Make Personalized Bone Substitutes

Patient-specific bone substitutes have been produced by a team of scientists from the New York Stem Cell Foundation. Darja Marolt and Giuseppe Maria de Peppo from the New York Stem Cell Foundation (NYSCF) led the study that demonstrated that customizable, three-dimensional bone grafts that can be produced on-demand for patients from their own cells.

Marolt and de Peppo and their co-workers used skin grafts from their patients to isolate skin fibroblasts that were reprogrammed to induced pluripotent stem cells (iPSCs). Because iPSCs are made from the patient’s own cells, they have the same profile of cell surface proteins as the patient’s own tissues. Therefore, they are very unlikely to be rejected by the patient’s immune system. Also, iPSCs have the ability to differentiate into any cell type found in the adult body, and therefore, can be used to form bone cells.

iPS cells

After deriving iPSCs from patient skin cells, de Peppo, Marolt, and colleagues coaxed the cells to form osteoblasts (the cells that form bone), and seeded them onto a scaffold that mimicked three-dimensional bone structure. These structures were grown in a bioreactor that fed the cells oxygen and nutrients.

According to Marolt, “Bone is more than a hard mineral composite, it is an active organ that constantly remodels. Blood vessels shuttle important nutrients to healthy cells and remove waste; nerves provide connection to the brain; and, bone marrow cells form new blood and immune cells.”

Previous studies have demonstrated that cells from other sources also possess bone-forming potential. However, these same studies have revealed serious shortcomings of the clinical potential of such cells. A patient’s own bone marrow stem cells can form bone and cartilaginous tissue, but not the accompanying underlying vasculature and nerve compartments. Also, embryonic stem cell derived bone may prompt an immune rejection. Therefore, the use of iPSCs can overcome many of these limitations.

As de Peppo noted: “No other research group has published work on creating fully viable functional three-dimensional bone substitutes from humans iPS cells. These results bring us closer to achieving our ultimate goal, to develop the most promising treatments.”

Since bone injuries and defects are often treated with bone grafts that are taken from other parts of the body or a tissue bank. Alternatively, synthetic alternatives can also be used, but none of these possibilities provide the means for complex reconstruction and they may also be rejected by the immune system, or fail to integrate with surrounding connective tissue. n the case of trauma patients who suffer from shrapnel wounds or vehicular injuries , the traditional treatments provide only limited functional and cosmetic improvements.

To access the integrity of the bioreactor-made bone, the NYSCF team implanted them into animals. Implantation of undifferentiated iPSCs formed tumors, but transplantation of the iPSC-derived bone produced no tumors, but also produced grafts that effectively integrated into the bones, connective tissues and blood vessels of the animals.

Susan Solomon, CEO of NYSCF, said of this work, “Following from these findings, we will be able to create tailored bone grafts, on demand, for patients without any immune rejection issues. She continued: “it is the best approach to repair devastating damage or defects.”

The therapeutic relevance of this work aside, these adaptive bone substitutes can also serve as models for bone development and various bone pathologies. Such bone exemplars could serve as models for drug testing and drug development.

Isolating Mammary Gland Stem Cells

Female mammary glands are home to a remarkable population of stem cells that grow in culture as small balls of cells called “mammospheres.” Clayton and others were able to identify these stem cells in 2004 (Clayton, Titley, and Vivanco, Exp Cell Res 297 (2004): 444-60), and Max Wicha’s laboratory at the University of Michigan showed that a signaling molecule called Sonic Hedgehog and a Polycomb nuclear factor called Bmi-1 are necessary for the self-renewal of normal and cancerous mammary gland stem cells (Lui, et al., Cancer Res June 15, 2006 66; 606). The biggest problem with mammary gland stem cells is isolating them from the rest of the mammary tissue.

Mammary gland stem cells or MaSCs are very important for mammary gland development and during the induction of breast cancer. Getting cultures of MsSCs is really tough because the MaSCs share cell surface markers with normal cells and they are also quite few in number.

Gregory Hannon and his co-workers at Cold Spring Harbor Laboratory used a mouse model to identify a novel cell surface protein specific to MaSCs. By exploiting this unusual marker, Hannon and his team were able to isolate MaSCs from mouse mammary glands of rather high purity.

Camila Do Santos, the paper’s first author, said that “We are describing a marker called Cd1d.” Cd1d is also found on the surfaces of cells of the immune system, but is specific to MaSCs in mammary tissue. Additionally, MaSCs divide slower than the surrounding cells. Do Santos and her colleagues used this feature to visually isolate MaSCs from cultured mammary cells.

They used a mouse strain that expresses a green glowing protein in its cells and then made primary mammary cultures from these green glowing mice. After shutting of the expression of the green glowing protein with doxycycline, the cultured cells divided, and diluted the quantity of green glow protein in the cells. This caused them to glow less intensely. However, the slow-growing MaSCs divided much more slowly and glowed much more intensely. Selecting out the most intensely glowing cells allowed Dos Santos and her colleagues to enrich the culture for MaSCs.

“The beauty of this is that by stopping GFP expression, you can directly measure the number of cell divisions that have happened since the GFP was turned off,” said Dos Santos. She continued: “The cells that divide the least will carry GFP the longest and are the ones we characterized.”

Using this strategy, Dos Santos and others selected stem cells from the mammary glands in order to examine their gene expression signature. They also confirmed that by exploiting Cd1d expression in the MaSCS, in combination with other techniques, they could enhance the purity of the cultures several fold.

Hannon added, “With this advancement, we are now able to profile normal and cancer stem cells at a very high degree of purity, and perhaps point out which genes should be investigated as the next breast cancer drug targets.”

Will we be able to use these cell for therapeutic purposes some day?  Possibly, but at this time, more must be known about them and MaSCs must be better characterized.