A Powerful Tool For Repairing Damaged Hearts


A new report from Johns Hopkins University researchers indicates that a particular stem cells that helps build mouse hearts can self-renew. This discovery, which might very well apply to humans as well, could potentially open inroads to use these cells to repair hearts damaged by disease, or, perhaps, even grow new heart tissue for transplantation.

This study is slated for publication in the journal eLife. Chulan Kwon, Ph.D., an assistant professor of cardiology and member of the Institute for Cell Engineering at the Johns Hopkins University School of Medicine and his team, found that during heart formation, these so-called cardiac progenitor cells or CPCs proliferate, but do not differentiate into heart cells in an embryonic structure known as the second pharyngeal arch. This insight into the biology of CPCs may contribute to better understanding of how to prevent and treat congenital heart defects.

Kwon noted that, “Our finding that CPCs are self-renewing—that they can keep dividing to form new CPCs—means they might eventually be maintained in a dish and used to make specific types of heart cells.”

Kwon continued: “Growing such cells in a dish would be an enormous step toward better treatment for heart disease.”

Kwon’s laboratory initially tackled the elucidating the contribution of two genes, Numb and Numbl, in the CPC biology. Other studies have shown that these two genes are required to guide stem and progenitor cells to their fully mature, specialized functions. Numb and Numbl are highly conserved in mice and humans (i.e. the proteins encoded by these genes in mice and humans are nearly identical). This conservation indicates that Numb and Numbl are probably doing something very important the lives of CPCs.

As a first step, Kwon and others made loss-function mutations in Numb and Numbl. The results were striking. According to Kwon, embryos that lacked functional Numb and Numbl protein, “failed to develop normal hearts and died at an early stage of development, showing us that Numb and Numbl are needed for CPCs to build the heart.”

With the crucial role of Numb and Numbl in the lives of CPCs in mind, Kwon and his colleagues tried to determine the location of CPCs in the developing embryo. For these experiments, they used mouse embryonic stem cells that lacked functional Numb and Numbl, and expressed a glowing red protein in all CPC cells. Such a glowing red protein would instantly give away the CPCs’ location. These embryonic stem cells have the ability to integrate into a growing embryo, but the absence of functional Numb and Numble proteins in these cells prevents them from growing into a viable embryo.

Next, Kwon’s group injected these engineered embryonic stem cells into viable mouse embryos at the blastocysts stage. The blastocyst stage forms early during mammalian development, and it consists of the two cell populations that will form the embryo (inner cell mass cells) and the placenta (trophoblast cells). “The normal cells in these blastocysts compensated for those that lacked Numb and Numbl, allowing the resulting embryos to survive,” Kwon says.

Once these chimeric embryos began to grow, Kwon’s group examined them for red-glowing cells. They found the glowing red cells in the second pharyngeal arch, which is known for forming parts of the neck and face. Kwon says their study is the first to identify the second pharyngeal arch as the home of the CPCs.

pharyngeal_arches

 

The cells of the second pharyngeal arch go on to form the stapes in the middle ear and the stapedius muscle that attaches to the stapes.

Pharyngeal_arch_cartilages

Additionally, Kwon’s group cultured second pharyngeal arch cells with CPCs. They discovered that the cultured CPCs self-renewed without developing into specialized heart cells. This is potentially an important step toward using CPCs to treat heart disease.

The next step, he says, is to direct the laboratory-grown CPCs to form new heart tissue that could be used to regenerate disease-damaged heart tissue. “Eventually, we might even be able to deliver cells to damaged hearts to repair heart disease,” Kwon says.

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Mouse Blood Cells Reprogrammed into Blood Cell Stem Cells


Boston Children’s Hospital researchers have directly reprogrammed mature blood cells from mice into blood-forming hematopoietic stem cells by using a cocktail of eight different transcription factors.

These reprogrammed cells have been called induced hematopoietic stem stem cells or iHSCs. These cells have all the hallmarks of mature mouse HSCs and they have the capacity to self-renew and differentiate into all the blood cells that circulate throughout the body.

These findings are highly significant from a clinical perspective because they indicate that it might be entirely possible to directly reprogram a patient’s existing, mature blood cells into a hematopoietic stem cell for transplantation purposes. Such a procedure, known as hematopoietic stem cells transplantation or HSCT, is a common treatment for patients whose bone marrow has suffered irreparable damage due to environmental causes (heavy metals, chloramphenicol, etc) or disease (cancer). The problem with HSCT is finding a proper match for the patient and then procuring clinically significant quantities of high-quality bone marrow for HSCT.

Derrick J. Rossi, an investigator in the Program in Cellular and Molecular Medicine at Boston Children’s Hospital and Assistant Professor in the Department of Stem Cell & Regenerative Biology, explained: “HSCs comprise only about one in every 20,000 cells in the bone marrow. If we could generate autologous (a patient’s own) HSCs from other cells, it could be transformative for transplant medicine and for our ability to model diseases of blood development.”

Rossi and his collaborators have screened genes that are expressed in 40 different types of blood progenitor cells in mice. This screen identified 36 different genes that control the expression of the other genes. These 36 genes encode so-called “transcription factors,” which are proteins that bind to DNA and turn gene express on or shut it off.

Blood cell production tends to go from the stem cells to progeny cells called progenitor cells that can still divide to some limited extent, and to effector cells that are completely mature and, in most cases, do not divide (the exception is lymphocytes, which expand when activated by specific foreign substances called antigens).

Further work by Rossi and others identified six transcription factors (Hlf, Runx1t1, Pbx1, Lmo2, Zfp37, and Prdm5) of these 36 genes, plus two others that were not part of their original screen (N-Myc and Meis1) that could robustly reprogram myeloid progenitor cells or pro/pre B lymphocytes into iHSCs.

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To put these genes into these blood cells, Rossi and others uses souped-up viruses that introduced all either genes under the control of sequences that only allowed expression of these eight genes in the presence of the antibiotic doxycycline. Once these transfected cells were transplanted into mice, the recipient mice were treated with doxycycline, and the implanted cells formed HSCs.

When this experiment utilized mice that were unable to make their own blood cells, because their bone marrow had been wiped out, the implanted iHSCs reconstituted the bone marrow and blood cells of the recipient mice.

To further show that this reconstituted bone marrow was normal, high-quality bone marrow, Rossi and others used these recipient mice as bone marrow donors for sibling mice whose bone marrow had been wiped out. This experiment showed that the mice that had received the iHSCs had bone marrow that completely reconstituted the bone marrow of their siblings. This established that the iHSCs could completely reestablish the bone marrow of another mouse.

Thus Rossi and others had established that iHSCs could in fact created HSCs from progenitor cells, but could they do the same thing with mature blood cells that were not progenitor cells? Make that another yes. When Rossi and others transfected their eight-gene cocktail into mature myeloid cells, these mature cells also made high-quality iHSCs.

Rossi noted that no one has been able to culture HSCs in the laboratory for long periods of time. A few laboratories have managed expand HSCs in culture, but only on a limited basis for short periods of time (see Aggarwal R1, Lu J, Pompili VJ, Das H. Curr Mol Med. 2012 Jan;12(1):34-49).  In these experiments, Rossi used his laboratory mice as living culture systems to expand his HSCs, which overcomes the problems associated with growing these fussy stem cells in culture.

Gene expression studies of his iHSCs established that, from a gene expression perspective, the iHSCs were remarkably similar to HSCs isolated from adult mice.

This is certainly an exciting advance in regenerative medicine, but it is far from being translated into the clinic.  Can human blood progenitor cells also be directly reprogrammed using the same cocktail?  Can mature myeloid cells be successfully reprogrammed?  Will some non-blood cell be a better starting cell for iHSC production in humans?  As you can see there are many questions that have to be satisfactorily answered before this procedure can come to the clinic.

Nevertheless, Rossi and his team has succeeded where others have failed and the results are remarkable.  HSCs can be generated and transplanted with the use of only a few genes.  This is certainly the start of what will hopefully be a fruitful regenerative clinical strategy.

On a personal note, my mother passed about almost a decade ago after a long battle with myelodysplastic syndrome, which is a pre-leukemic condition in which the bone marrow fails to make mature red blood cells.  Instead the bone marrow fills up with immature red blood cells and the patient has to survive on blood transfusions.

The reason for this condition almost certainly stems from defective HSCs that do not make normal progeny.  Therefore the possibility of using a patient’s own cells to make new HSCs that can repopulate the bone marrow is a joyful discovery for me to read about, even though it is some ways from the clinic at this point.

Skin Tissue Grown From Human Stem Cells


A research team from King’s College, London, in collaboration with the San Francisco Veteran Affairs Medical Center has succeeded in growing the epidermal layer of skin in culture, this cultured skin has many of the mechanical and biological properties of actual human skin.

The outermost layer of the skin, known as the epidermis forms a protective barrier between the external environment and the body. It protects against water loss and prevents the entry of microorganisms.

Tissue engineers have been able to grow skin cells (keratinocytes) in culture, but getting them to organize into an organ that resembles biological skin has proven rather difficult. However, the ability to test drugs on cultured skin that greatly mimics human skin has been the goal of such research for several years.

For this present project, keratinocytes were made from induced pluripotent stem cells that were derived from skin cells obtained from biopsies. These keratinocytes made from induced pluripotent stem cells (iPSCs) were very similar to keratinocytes made from embryonic stem cells and primary keratinocytes isolated from skin biopsies.

To form a three-dimensional structure like skin, the keratinocytes were cultured in a high-to-low humidity environment and they assembled into a layer structure that looked like human skin. When this cultured skin was compared with skin made from embryonic stem cell-derived keratinocytes or from keratinocytes isolated from skin biopsies, there were no significant structural differences.

Scientists hope to use this cultured skin to study congenital skin diseases like ichthyosis (characterized by dry, flaky skin) or atopic dermatitis. Growing large quantities of skin in culture will also allow drugs and cosmetics to be effectively tested for safety without the use of expensive and sometimes highly variable animal models.

This technology would also allow different laboratories to grow skin from different ethnic groups that have distinct types of skin with variable biological properties.

Mesenchymal Stem Cells Reduce Scarring of Intervertebral Discs and Facilitate Healing


Intervertebral disc degeneration causes substantial back pain and associated pain that shoots down the legs (radiculopathy). Back issues associated with bad intervertebral discs are a leading cause of disability. Such disability costs employers millions of dollars of lost man and woman power and employees extensive loss of wages. Chronic back pain can also seriously compromise the quality of life and presents a large societal burden.

To date, surgery is the only effective treatment option, but surgical interventions sometimes leave patients worse off than before. Thus there is presently no effective intervention for this disease.

However, in a recent paper, Victor Y.L. Leung and his colleagues from the University of Hong Kong and several other institutions as well have used human mesenchymal stem cells from bone marrow to treat damaged intervertebral discs in rabbits. The results, published in the journal Stem Cells, are quite hopeful

Leung and others discovered that by puncturing the intervertebral discs of rabbits with a syringe needle, they could induce damage to the disc that mimics disc degeneration in humans.

Next, they implanted human bone marrow-derived mesenchymal stem cells (MSCs) into the damaged discs. Such implantations prevented scarring of the disc in the center of the disc. The center of the disc, the nucleus pulposus, is more gel-like than the surrounding annulus fibrosus. Scarring of the nucleus pulposus stiffens it and prevents it from moving with stress. An inability to bend with stress causes the disc to become brittle with time and herniate. However, implantation of mesenchymal stem cells preserved the mechanical properties of the disc and benefitted overall spinal function.

By looking more deeply at the mechanism by which mesenchymal stem cells preserve disc function, Leung and others showed that MSCs suppress abnormal deposition of collagen I in the nucleus pulposus. Since collagen is made during scarring, suppression of collagen I synthesis suppressed scarring. Secondly, implanted MSCs decreased the expression of two molecules that promote the synthesis of collagen I. By suppressing the expression of MMP12 and HSP47, the implanted MSCs also reduced collagen aggregation and maintained the microarchitecture of the disc and its mechanical properties.

This  study supports the ability of MSCs to stimulate resident stem cell activities and disc healing. The implanted MSCs seem to do so by means of down-regulating collagen  fibril formation. This provides the basis for the MSC‐based disc therapies.

Researchers Transplant Regenerated Esophagus


Paolo Macchiarini and a research consortium from the Karolinska Institutet in Stockholm, Sweden have made a tissue engineered scaffold for the esophagus from esophagi that were extracted from rats.

After the cells were stripped from the rat esophagi, bone marrow mesenchymal stromal cells were seeded onto the decellularized esophagi and grown in a perfusion bioreactor. A variety of experiments demonstrated that these mesenchymal stromal cells differentiated into esophageal epithelial cells and smooth muscle cells. Macchiarini and his group used several gene expression and functional assays to confirm that these cells had in fact differentiated into these esophageal-specific cell types.

Next, Maccharini and others transplanted these esophagi into rats. The transplanted rats survived 14 days after the transplantations and ate and gain weight. Because the cells used to reconstitute the esophagi came from the rats into which they were transplanted, immunosuppressive drugs were neither used nor needed.

When the recipients of the transplanted esophagi were sacrificed after fourteen days, tissue examinations showed that all the major cell and tissue components of the esophagus including the inside covering of the esophagus (epithelium), muscle fibers, nerves, and blood vessels had nicely regenerated.

This successful bioproduction and transplantation of a tissue-engineered esophagus represents a significant step towards the clinical application of bioengineered esophagi.

Think of it: children and adults with tumors, congenital malformations of the esophagus of traumatic injuries to the esophagus may have new hope and possibilities because of advances in tissue engineering like this.

Meta Study Shows that Mesenchymal Stem Cells Promote Healing in Animal Models of Stroke


Two scientists from my alma mater, UC Irvine, have examined experiments that treated stroke with bone marrow-derived stem cells. Their analysis has shown that infusions of these stem cells trigger repair mechanisms and limit inflammation in the brains of stroke patients.

UC Irvine neurologist Dr. Steven Cramer and biomedical engineer Weian Zhao identified 46 studies that examined the use of a specific type of bone marrow stem cells called mesenchymal stromal cells to treat stroke. Mesenchymal stromal cells are a type of multipotent adult stem cells that are found in many locations in the body. The best-known examples of mesenchymal stem cells are from bone marrow. When purified from whole bone marrow and used to treat stroke in animal models of stroke, Cramer and Zhao found that mesenchymal stromal cells (MSCs) were significantly better than control therapy in 44 of the 46 studies that were examined.

Further data culling of these studies showed that functional recovery from stroke were robust regardless of the MSC dosage or the time when MSCs were administered relative to the onset of the stroke, or the method of administration (whether introduced directly into the brain or injected via a blood vessel).

“Stroke remains a major cause of disability, and we are encouraged that the preclinical evidence shows [MSCs’] efficacy with ischemic stroke,” said Cramer, a professor of neurology and leading stroke expert. “MSCs are of particular interest because they come from bone marrow, which is readily available, and are relatively easy to culture. In addition, they already have demonstrated value when used to treat other human diseases.”

Another theme of these studies is that MSCs do not differentiate into brain-specific. MSCs have the capacity to differentiate into bone, cartilage and fat cells. “But they do their magic as an inducible pharmacy on wheels and as good immune system modulators, not as cells that directly replace lost brain parts,” he said.

In an earlier Cramer and Zhao examined the mechanism by which MSCs promote brain repair after stroke. These cells have the ability to home to the damages areas in the brain and release chemicals that stimulate healing. By releasing their cornucopia of healing-promoting molecules, MSCs orchestrate blood vessel creation to enhance circulation, the protection of moribund cells on the verge of death, and the growth of existing brain cells. Additionally, when MSCs reach the bloodstream, they settle in those parts of the body that control the immune system and they suppress the inflammatory response that can augment tissue damage. In this way, MSCs foster an environment more conducive to brain repair.

“We conclude that MSCs have consistently improved multiple outcome measures, with very large effect sizes, in a high number of animal studies and, therefore, that these findings should be the foundation of further studies on the use of MSCs in the treatment of ischemic stroke in humans,” said Cramer, who is also clinical director of the Sue & Bill Gross Stem Cell Research Center.

Long-Term Survival of Transplanted Human Neural Stem Cells in Primate Brains


A Korean research consortium has transplanted human neural stem cells (hNSCs) into the brains of nonhuman primates and ascertained the fate of these cells after being inside the brains of these animals for 22 and 24 months. They discovered that the implanted hNSCs had not only survived, but differentiated into neurons and never caused any tumors.

This important study is slated to be published in the journal Cell Transplantation.

To properly label the hNSCs so that they were detectable inside the brains of the animals, Lee and others loaded them with magnetic nanoparticles to enable them to be followed by magnetic resonance imaging (MRI). Also, they did not use immunosuppressants when they transplanted their hNSCs into the animals. This study is the first to examine the long-term survival and differentiation of hNSCs without the need for immunosuppression.

“Stroke is the fourth major cause of death in the US behind heart failure, cancer, and lower respiratory disease,” said study co-author Dr. Seung U. Kim of University of British Columbia Hospital’s department of neurology in Canada. “While tissue plasminogen activator (tPA) treatment within three hours after a stroke has shown good outcomes, stem cell therapy has the potential to address the treatment needs of those stroke patients for whom tPA treatment was unavailable or did not help.”

Based on the ability of hNSCs to differentiate into a variety of types of nerves cells, Lee and his colleagues thought that these cells have remarkable potential to treat damaged brain tissue and replace what was lost after a stroke, head injury or other type of brain trauma. Cell regeneration therapy can potentially treat brain-specific diseases like Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), spinal cord injury and stroke.

Dr. Kim and colleagues in Korea grafted magnetic particle-labeled hNSCs into the brains of laboratory primates and evaluated their performance to assess their survival and differentiation over 24 months. Of particular interest was determining their ability to differentiate into neurons and to determine whether the cells caused tumors.

“We injected hNSCs into the frontal lobe and the putamen of the monkey brain because they are included in the middle cerebral artery (MCA) territory, which is the main target in the development of the ischemic lesion in animal stroke models,” commented Dr. Kim. “Thus, research on survival and differentiation of hNSCs in the MCA territory should provide more meaningful information to cell transplantation in the MCA occlusion stroke model.”

Lee’s team said that they chose NSCs for transplantation because the existence of multipotent NSCs “has been known in developing rodents and in the human brain with the properties of indefinite growth and multipotent potential to differentiate” into the three major CNS cell types – neurons, astrocytes and oligodendrocytes.

“The results of this study serve as a proof-of-principle and provide evidence that hNSCs transplanted into the non-human primate brain in the absence of immunosuppressants can survive and differentiate into neurons,” wrote the researchers. “The study also serves as a preliminary study in our planned preclinical studies of hNSC transplantation in non-human primate stroke models.”

“The absence of tumors and differentiation of the transplanted cells into neurons in the absence of immunosuppression after transplantation into non-human primates provides hope that such a therapy could be applicable for use in humans.” said Dr. Cesar V. Borlongan, Prof. of Neurosurgery and Director of the Center of Excellence for Aging & Brain Repair at the University of South Florida. “This is an encouraging study towards the use of NSCs to treat neurodegenerative disorders”.