The Ideal Recipe for Cartilage from Stem Cells

Researchers at Case Western Reserve and Harvard University will use a 5-year, $2-million NIH grant to build a microfactory that bangs out the optimal formula for joint cartilage. Such an end product could one day potentially benefit many of the tens of thousands of people in the United States who suffer from cartilage loss or damage.

Joint cartilage or articular cartilage caps the ends of long bones and bears the loads, absorbs shocks and, in combination with lubricating synovial fluid, helps knees, hips, shoulders, and other joints to smoothly bend, lift, and rotate. Unfortunately, this tissue has little capacity to regenerate, which means that there is a critical need for new therapeutic strategies.

Artificial substitutes cannot match real cartilage and attempts to engineer articular cartilage have been stymied by the complexities of directing stem cells to differentiate into chondrocytes and form the right kind of cartilage.

Stem cells are quite responsive to the environmental cues presented to them from their surroundings. What this research project hopes to determine are those specific cue that drive stem cells to differentiate into chondrocytes that make the right kind of cartilage with the right kind of microarchitecture that resembles natural, articular cartilage. To do this, they will engage in a systematic study of the effects of cellular micro-environmental factors that influence stem cell differentiation and cartilage formation.

Bone marrow- and fat-derived mesenchymal stem cells have been differentiated into cartilage-making chondrocytes in the laboratory. These two stem cell populations are distinct, however, and required different conditions in order to drive them to differentiate into chondrocytes. This research group, however, has designed new materials with unique physical properties, cell adhesive capabilities, and have the capacity to deliver bioactive molecules.

By controlling the presentation of these signals to cells, independently and in combination with mechanical cues, this group hopes to identify those most important cues for driving cells to differentiate into chondrocytes.

Ali Khademhosseini specializes in microfabrication and micro-and nano-scale technologies to control cell behavior. He and his team will develop a microscale high-throughput system at his laboratory that will accelerate the testing and analysis of materials engineered in another laboratory.

This research cooperative hopes to test and analyze more than 3,000 combinations of factors that may influence cell development, including differentiation, amounts of biochemicals, extracellular matrix properties, compressive stresses, and more. Khademhosseini and his colleagues hope to begin testing comditions identified from these studies in animal models by the of the grant term.

Scientists Reprogram Adult Skin Cells to Make Mini Kidneys

Japanese and Australian researchers have used induced pluripotent stem cell (iPSC) technology to reprogram human skin cells to make the most mature human kidneys yet to be grown in a culture. These mini kidneys have hundreds of filtering units (nephrons) and blood vessels and appear to be developing just as kidneys would in an embryo.

“The short-term goal is to actually use this method to make little replicas of the developing kidney and use that to test whether drugs are toxic to the kidney,” said lead researcher Professor Melissa Little, of the Murdoch Children’s Research Institute. “Ultimately we hope we might be able to scale this up so we can … maybe bioengineer an entire organ.”

In other previous research, Professor Little and her co-workers generated cells that self-organized into the nephrons and collecting ducts needed for the kidney to filter blood and produce urine. They used a precise combination of called growth factors to direct embryonic stem cells to develop into the different cell types.

In the journal Nature, Professor Little and her collaborators report they have made a developing kidney from a type of skin cell called a fibroblast. Little and her team reprogrammed adult fibroblasts to become “induced pluripotent stem cells,” which act like embryonic stem cells, and can become any cell in the body. By adopting their growth factor recipe, Little and others were able to grow these cells into larger and more complex, three-dimensional kidneys than previously made.

“These kidneys have something like 10 or 12 different cell types in them … all from the one starting stem cell,” said Professor Little. “What we had previously were little flat structures over the surface of a dish … Now we have an organoid that is about 5-6 millimetres across, has about 100 filtering units in it, and is starting to form blood vessels. It’s starting to mature and the cell types are starting to do more of the functions of the final kidney.”

Scientists in Little’s laboratory demonstrated that the genes expressed in the mini kidneys as they formed faithfully recapitulated the expression of those same genes in a developing kidney in a first trimester embryo.

“It is actually mirroring what is happening in human development,” said Professor Little.

Little and her group also found that the laboratory-grown kidney was damaged when it was treated with known renal toxins. Little suggested that the iPSCs cells they had created were functioning as a kidney, but further tests would be required to demonstrate that.

It might be possible to use these bioengineered kidneys to test the renal toxicity of drugs. Likewise, the production of mini kidneys using cells from kidney patients might provide a way to study inherited forms of kidney disease.

“You can take a fibroblast [from someone with inherited kidney disease], make a stem cell out of it, generate a little kidney and use that as our model for their disease,” said Professor Little.

Perhaps most exciting, laboratory-generated kidneys might one day provide rejection-free transplants for patients, and gene editing could be used to fix the genetic defect that caused an inherited kidney disease.

Professor Jamie Davies of the University of Edinburgh, who was not involved with this work, but commented on it for Nature, emphasized this was not a full-fledged, functional kidney. “The structure’s fine-scale tissue organization is realistic, but it does not adopt the macro-scale organization of a whole kidney. For example, it is not ‘plumbed’ into a waste drain, and it lacks large-scale features that are crucial for kidney function, such as a urine-concentrating medulla region. There is a long way to go until clinically useful transplantable kidneys can be engineered, but [this] protocol is a valuable step in the right direction.”

Davies also mentioned that these mini kidneys had the potential to replace “poorly predictive” animal drug safety tests, and called on researchers to team up with toxicologists to test the potential of their system.

Transplantation of Unique, Newly Discovered Stem Cells May Lead to Promising Stroke Therapy

Stroke treatments have seen some remarkable advances in the past few years. Stem cell treatments for stroke have even seen some successes in clinical trials, showing that stem cell transplantation aimed at neural repair after a stroke is a possible way to ameliorate the effects of stroke.

Now, collaboration between teams of American and Japanese researchers has shown that a newly-identified stem cell has the ability to successfully treat stroke in rats. When administered to rats who have suffered from an experimentally-induced stroke, MUSE or multilineage-differentiating stress-enduring cells induced the regeneration of neurons and resulted in “significant improvements in neurological and motor functions” compared to control groups that were not transplanted with MUSE cells. MUSE cells also do not cause tumors.

The study has increased the number of therapeutic arrows in the quiver of neurologists and neuroscientists and lengthens the list of cells that might one day be considered for human clinical trials if continued pre-clinical tests prove successful. Future clinical studies aimed at regenerating neurological and motor function in patients who have suffered ischemic stroke.

The paper describing this study appeared in a recent issue of Stem Cells (Sept. 2015).

“Muse cells are unique stem cells that are able to self-renew and display high-efficiency for differentiating into neuron-like cells,” explained lead author Dr. Cesar V Borlongan, Distinguished Professor and Vice-Chairman for Research at the University of South Florida (USF) College of Medicine Department of Neurosurgery and Brain Repair and Director of USF’s the Center of Excellence for Aging and Brain Repair. “Unlike mesenchymal stem cells (MSCs) that have previously been used in stem cell transplantation in stroke-related clinical trials, in the present study Muse cells were found to possess functional characteristics of neurons as they attain the attributes of the host microenvironment. When MUSEcells were transplanted into to the brains of rats modeled with stroke, they attained neuronal characteristics.”

MUSE cells are found in many different tissues, including bone marrow, skin and fat. Since these cells can be derived from dermal fibroblasts (a type of connective tissue cell that provides the structural framework for animal tissues and plays a critical role in wound healing), they can be accessed with relative ease, without the need for the painful, invasive procedures required for obtaining other kinds of stem cells. Furthermore, while some stem cells used in stem cell transplantation studies have been found to cause cancer, MUSE cells do not produce tumors and exhibit exceptional tissue repair potential when introduced into the blood stream.

Some researchers think that fetal stem cells might be better candidates for replacing lost neural circuitry. The main reason in favor of fetal stem cells is that they preferentially differentiate into neuronal cells. However, the accessibility to fetal stem cells is limited and, like embryonic stem cells, the immaturity of these cells may present safety issues, such as tumor development. Additionally, the use of fetal and embryonic stem cells has many ethical difficulties to say the least. Since MUSE cells can be derived from adult tissue rather than fetal or embryonic tissue, the ethical quandaries associated with using them is minimal.

Not only do MUSE cells also have the practical advantage of being non-tumorigenic, they are readily accessed commercially and can also be easily collected from patient skin biopsies. MUSE cells also do not have to be “induced,” or genetically manipulated in order to be used, since they already display inherent stem cell properties after isolation. MUSE cells also spontaneously home toward the stroke-damaged sites.

“Ours is the first study to show that human skin fibroblast-derived Muse cells can have neuron-like function, possess an inherent ability to assume ‘stemness’ properties, and to readily differentiate into neural-lineage cells after integration into the stroke brain,” said co-lead author Dr. Mari Dezawa, Department of Stem Cell Biology and Histology, Tohoku University Graduate School of Medicine in Sendai, Japan. “Our results show that Muse cells are a feasible and promising source for cell-based approaches to ischemic stroke therapy.”

Clincal Trial Validates Stem Cell-Based Treatments of Sickle Cell Disease in Adults

Santosh Saraf and his colleagues at the University of Illinois have used a low-dose irradiation/alemtuzumab plus stem cell transplant procedure to cure patients of sickle-cell disease. 12 adult patients have been cured of sickle-cell disease by means of a stem cell transplantation from a healthy, tissue-matched donor.

This new procedure obviates the need for chemotherapy to prepare the patient to receive transplanted cells and offers the possibility of curing tens of thousands of adults from sickle-cell disease.

Sickle cell disease is an inherited disease that primarily affects African-Americans born in the United States. The genetic lesion occurs in the beta-globin gene that causes hemoglobin molecules to assemble into filaments under low-oxygen conditions. These hemoglobin filaments deform red blood cells and cause them to plug small capillaries in tissues, causing severe pain, strokes and even death.

Fortunately, a bone marrow transplant from a healthy donor can cure sickle-cell disease, but few adults undergo such a procedure because the chemotherapeutic agents that are given to destroy the patient’s bone marrow leaves from susceptible to diseases, unable to make their own blood cells, and very weak and sick.

Fortunately, a gentler procedure that only partially ablate the patient’s bone marrow was developed at the National Institutes of Health ()NIH) in Bethesda, Maryland. Transplant physicians there have treated 30 patients, with an 87% success rate.

In the Phase I/II clinical trial at the University of Illinois, 92% of the patients treated with this gentler procedure that was developed at the NIH.

Approximately 90% of the 450 patients who received stem cells transplants for sickle-cell disease have been children. However, chemotherapy has been considered too risky for adult patients who are often weakened far more than children by it.

Adult sickle-cell patients live an average of 50 years with a combinations of blood transfusions and pain medicines to manage the pain crisis. However, their quality of life can be quite low. Now, with this chemotherapy-free procedure, adults with sickle-cell disease can be cured of their disease within one month of their transplant. They can even go back to work or school and operate in a pain-free fashion.

In the new procedure, patients receive immunosuppressive drugs just before the transplant, with a very low dose of whole body radiation. Alemtuzumab (Campath, Lemtrada) is a monoclonal antibody that binds to the CD52 glycoprotein on the surfaces of lymphocytes and elicits their destruction, but not the hematopoietic stem cells that gives rise to them.  Next, donor cells from a healthy a tissue-matched sibling or donor are transfused into the patient. Stem cells from the donor home to the bone marrow and produce healthy, new blood cells in large quantities. Patients must continue to take immunosuppressive drugs for at least a year.

In the University of Illinois trial, 13 patients between the ages of 17-40 were given transplants from the blood of a healthy, tissue-matched sibling. Donors must be tested for human leukocyte antigen (HLA) markers on the surfaces of cells. Ten different HLA markers must match between the donor and the recipient for the transplant to have the best chance of evading rejection. Physicians have transplanted two patients with good HLA matches, to their donor, but had a different blood type than the donor. In many cases, the sickle cells cannot be found in the blood after the transplant.

In all 13 patients, the transplanted cells successfully engrafted into the bone marrow of the patients, but one patient failed to follow the post-transplant therapy regimen and reverted to the original sickle-cell condition.

One year after the transplantation, the 12 successfully transplanted patients had normal hemoglobin concentrations in their blood and better cardiopulmonary function. They also reported significantly less pain and improved health and vitality,

For of the patients were able to stop post-transplantation immunotherapy, without transplant rejection or other complications.

“Adults with sickle-cell disease can be cured with chemotherapy – the main barrier that has stood in the way for so long,” said Damiano Rondelli, Professor of Medicine and Director of the Stem Cell Transplantation Program at the University of Illinois. “Our data provide more support that this therapy is safe and effective and prevents patients from living shortened lives, condemned to pain and progressive complications.”

These data were published in the journal Biology of Blood and Marrow Transplantation, 2015; DOI 10.1016/j.bbmt.2015.08.036.

Rebooting Pancreatic Cells Can Normalize Blood Sugar Levels in Diabetic Mice

Type 1 diabetes results from the inability of the endocrine portion of the pancreas to secrete sufficient quantities of the hormone insulin. The cells that make insulin, beta cells, have been destroyed. Consequently, type 1 diabetics must inject themselves with insulin routinely in order to stay alive. Is there a better way?

A new strategy suggests that maybe pancreatic cells can be “rebooted” to produce insulin and that sure reprogramming could potentially help people with type 1 diabetes manage their blood sugar levels without the need for daily injections. This therapeutic approach is simpler and potentially safer than giving people stem cells that have been differentiated into pancreatic beta cells.

Philippe Lysy at the Cliniques Universitaires Saint Luc, which is part of the Catholic University of Louvain in Belgium, and his colleagues have reprogrammed pancreatic duct cells extracted from dead donors who were not diabetic at the time of death. The duct cells do not produce insulin, but they have a natural tendency to grow and differentiate into specific types of cells.

Lysy and his team grew the cells in the laboratory and encouraged them to become insulin-producing cells by exposing them to fatty particles. These fatty particles are absorbed into the cells after which they induce the synthesis of the MAFA transcription factor. MAFA acts as a genetic “switch” that binds to DNA and activates insulin production.

Implantation of these altered cells into diabetic mice showed that the cells were able to secrete insulin in a way that controls blood sugar levels. “The results are encouraging,” says Lysy.

Lysy’s colleague, Elisa Corritore, reported these results at this week’s annual meeting of the European Society for Pediatric Endocrinology in Barcelona, Spain. Lysy and others are preparing to submit their results for publication.

This work, if it continues to pan out, might lead to the harvesting of pancreatic ducts from deceased donors and converted in bulk into insulin-making cells. Such “off-the-shelf” cells could then be transplanted into people with type 1 diabetes to compensate for their inability to make their own insulin.

“We would hope to put the cells in a device under the skin that isolates them from the body’s immune system, so they’re not rejected as foreign,” says Lysy. He says devices like this are already being tested for their ability to house insulin-producing cells derived from stem cells.

Previous attempts to get round this problem have included embedding insulin-producing cells in a seaweed derivative prior to transplantation in order to keep them from being destroyed by the recipient’s immune system.

Lysy thinks that since insulin-producing cells originate from pancreatic tissue, they have an inherently lower risk of becoming cancerous after the transplant. This has always been a worry associated with tissues produced from embryonic stem cells, since these have the capability to form tumors if any are left in their original state in the transplanted tissue.

The basic premise of the work looks solid, says Juan Dominguez-Bendala, director of stem cell development for Translational Research at the University of Miami Miller School of Medicine’s Diabetes Research Institute in Florida. “However, until a peer-reviewed manuscript is published and all the details of the work become available to the scientific community, it is difficult to judge if this advance represents a meaningful leap in the state of the art.”

Lysy expects it will take between three and five years before the technique is ready to be tested in human clinical trials.

Porous Hydrogels Boost Stem Cell-Based Bone Production

Regenerative medicine relies upon the ability to isolate, manipulate, and exploit stem cells from our own bodies or from the bodies of stem cell donors. A present obstacle to present therapeutic strategies is the poor survival of implanted stem cells. There are also worries of about properly directing the differentiation of transplanted stem cells. After all, if implanted stem cells do not differentiate into the terminal cell types you want to be replaced, the use of such cells seems pointless.

To address this problem, David Mooney from the Wyss Institute and his colleagues have designed a three-dimensional system that might keep transplanted stem cells alive and happy, ready to heal.

Mooney’s group has adopted a strategy based on the concept of stem cell “niches.”. In our bodies, stem cells have particular places where they live. These stem cell-specific microenvironments provide unique support systems for stem cells and typically include extracellular matrix molecules to which stem cells attach.

Mooney and others have identified chemical and physical cues that act in concert to promote stem cell growth and survival. The chemical cues found in stem cell niches are relatively well-known but the physical and mechanical properties are less well understood at the present time.

Stem cells in places like bone, cartilage, or muscle, when cultured in the laboratory, display particular mechanical sensitivities and they must rest on a substrate with a defined elasticity and stiffness in order to proliferate and mature. As you might guess, reproducing the right physical properties in the laboratory is no mean feat. However, several laboratories have used hydrogels to generate the right combination of chemical and physical properties.

Mooney and his colleagues have made two hydrogels with very different properties. A stable, “bulk” gel is filled with small bubbles of a pore-making molecule called a “porogen,” which degrades quickly and leaves porous cavities in its wake. When the bulk hydrogel is combined with extracellular matrix molecules from stem cell niches and filled with tissue-specific stem cells, and the porogen, Mooney and his team can make an artificial bone-forming stem cell niche. The porous cavities in the hydrogel, in combination with the chemical signals, drive the stem cells to grow, and divide while expanding into the open spaces in the gel. Then the cell move from the hydrogel to form mineralized bone.

In small animal experiments, Mooney and his colleagues showed that a porous hydrogel with the correct chemical and elastic properties provides better bone regeneration than transplanting stem cells alone. The maturing stem cells deployed by the hydrogel also induce neighboring stem cell populations to contribute to the bone repair, which further amplifies their regenerative effects.

This study provides the first demonstration that adjusting the physical properties of a biomaterial can not only help deliver stem cells but also tune the behavior of those cells in a living organism. Even though Mooney has primarily focused on bone formation, he and his group believe that the hydrogel concept can be broadly applied to other regenerative process as well.

This work was published in Nature Materials 2015; DOI: 10.1038/nmat4407.