Using Fat Stem Cells to Treat a Deadly Cancer


Johns Hopkins University researchers have reported the successful use of stem cells derived from human body fat to deliver biological treatments directly to the brains of mice suffering from the most common and aggressive form of brain tumor. Such treatments significantly extended the lives of these cancer-stricken animals.

These experiments offer proof-of-principle that such a technique would work in human patients after surgical removal of brain cancers called glioblastomas. This technique provides a way to find and destroy any remaining cancer cells in those areas of the brain that are difficult to reach. Glioblastoma cells represent a challenge for cancer treatments, since they are quite sprightly, and can migrate across the entire brain, hide out and establish new tumors. Consequently, the cure rates for glioblastoma are notoriously low.

In the mouse experiments conducted by the Johns Hopkins group, investigators used mesenchymal stromal cells (MSCs) from fat tissue. Fat-based MSCs have a mysterious ability to sniff out cancer and other damaged cells. After genetically modifying the MSCs so that they secreted a protein called bone morphogenetic protein 4 (BMP4), these MSCs were injected into the brains of mice that suffered from glioblastomas. BMP-4 is a small, secreted protein that plays essential regulatory roles in embryonic development, but also has a demonstrated tumor suppression function.

Study leader Alfredo Quinones-Hinojosa, M.D., a professor of neurosurgery, oncology and neuroscience at the Johns Hopkins University School of Medicine and his colleagues published the results of this experiment in the journal Clinical Cancer Research. According to their results, those mice that were treated with the BMP-4-secreting fat-based MSCs had significantly less tumor growth and spread. In general the cancers in these animals were less aggressive and had fewer migratory cancer cells compared to mice that didn’t get the treatment. Also, the stem cell-treated mice survived significantly longer (an average of 76 days, compared to 52 days in the untreated mice).

“These modified mesenchymal stem cells are like a Trojan horse, in that they successfully make it to the tumor without being detected and then release their therapeutic contents to attack the cancer cells.”

Standard treatments for glioblastoma include chemotherapy, radiation and surgery. Unfortunately, even a combination of all three rarely leads to more than 18 months of survival after diagnosis. Discovering new ways to seek and destroy straggling glioblastoma cells that other treatments can’t get is a long-sought goal, says Quinones-Hinojosa. However, he also cautions that years of additional studies are needed before human trials of fat-derived MSC therapies could begin.

Quinones-Hinojosa also treated brain cancer patients at Johns Hopkins Kimmel Cancer Center, and he and his co-workers were greatly encouraged that the genetically-engineered stem cells let loose into the brain in his experiments did not transform themselves into new tumors.

These latest findings build on research published in March 2013 by Quinones-Hinojosa and his team, which demonstrated that harvesting MSCs from fat was much less invasive and less expensive than getting them from bone marrow (PLoS One, March 2013).

Ideally, he says, if MSCs work as a cancer treatment, a patient with a glioblastoma would have some adipose tissue (fat) removed from any number of locations in the body a short time before surgery. Afterwards, these fat-derived MSCs would be isolated and manipulated in the laboratory so that they would secrete BMP4. Then, after surgeons removed whatever parts of the brain tumor they could get to, they would deposit these BMP-secreting cells into the brain in the hopes that they would seek out and destroy the left-over cancer cells.

 

Vascular Progenitors Made from Induced Pluripotent Stem Cells Repair Blood Vessels in the Eye Regardless of the Site of Injection


Johns Hopkins University medical researchers have reported the derivation of human induced-pluripotent stem cells (iPSCs) that can repair damaged retinal vascular tissue in mice. These stem cells, which were derived from human umbilical cord-blood cells and reprogrammed into an embryonic-like state, were derived without the conventional use of viruses, which can damage genes and initiate cancers. This safer method of growing the cells has drawn increased support among scientists, they say, and paves the way for a stem cell bank of cord-blood derived iPSCs to advance regenerative medical research.

In a report published Jan. 20 in the journal Circulation, Johns Hopkins University stem cell biologist Elias Zambidis and his colleagues described laboratory experiments with these non-viral, human retinal iPSCs, that were created generated using the virus-free method Zambidis first reported in 2011.

“We began with stem cells taken from cord-blood, which have fewer acquired mutations and little, if any, epigenetic memory, which cells accumulate as time goes on,” says Zambidis, associate professor of oncology and pediatrics at the Johns Hopkins Institute for Cell Engineering and the Kimmel Cancer Center. The scientists converted these cells to a status last experienced when they were part of six-day-old embryos.

Instead of using viruses to deliver a gene package to the cells to turn on processes that convert the cells back to stem cell states, Zambidis and his team used plasmids, which are rings of DNA that replicate briefly inside cells and then are degraded and disappear.

Next, the scientists identified and isolated high-quality, multipotent, vascular stem cells that resulted from the differentiation of these iPSC that can differentiate into the types of blood vessel-rich tissues that can repair retinas and other human tissues as well. They identified these cells by looking for cell surface proteins called CD31 and CD146. Zambidis says that they were able to create twice as many well-functioning vascular stem cells as compared with iPSCs made with other methods, and, “more importantly these cells engrafted and integrated into functioning blood vessels in damaged mouse retina.”

Working with Gerard Lutty, Ph.D., and his team at Johns Hopkins’ Wilmer Eye Institute, Zambidis’ team injected these newly iPSC-derived vascular progenitors into mice with damaged retinas (the light-sensitive part of the eyeball). The cells were injected into the eye, the sinus cavity near the eye or into a tail vein. When Zamdibis and his colleagues took images of the mouse retinas, they found that the iPSC-derived vascular progenitors, regardless of injection location, engrafted and repaired blood vessel structures in the retina.

“The blood vessels enlarged like a balloon in each of the locations where the iPSCs engrafted,” says Zambidis. Their vascular progenitors made from cord blood-derived iPSCs compared very well with the ability of vascular progenitors derived from fibroblast-derived iPSCs to repair retinal damage.

Zambidis says that he has plans to conduct additional experiments in diabetic rats, whose conditions more closely resemble human vascular damage to the retina than the mouse model used for the current study, he says.

With mounting requests from other laboratories, Zambidis says he frequently shares his cord blood-derived iPSC with other scientists. “The popular belief that iPSCs therapies need to be specific to individual patients may not be the case,” says Zambidis. He points to recent success of partially matched bone marrow transplants in humans, shown to be as effective as fully matched transplants.

“Support is growing for building a large bank of iPSCs that scientists around the world can access,” says Zambidis, although large resources and intense quality-control would be needed for such a feat. However, Japanese scientists led by stem-cell pioneer Shinya Yamanaka are doing exactly that, he says, creating a bank of stem cells derived from cord-blood samples from Japanese blood banks.

An Easy Way to Make Retinal Pigment Epithelium from Pluripotent Stem Cells


Age-related macular degeneration is the leading cause of irreversible vision loss and blindness among the aged in industrialized countries. One of the earliest events associated with age-related macular degeneration (AMD) is damage to the retinal pigmented epithelium (RPE), which lies just behind the photoreceptor cells in the retinal. The RPE serves several roles in visual function, including absorption of stray light, formation of blood retina barrier, transport of nutrients, secretion of growth factors, isomerization of retinol, and daily clearance of shed outer photoreceptor outer segments. RPE cell death and dysfunction is associated with both wet (neovascular) and dry (atrophic) forms of AMD.

How then do we make RPE cells from stem cells in order to treat AMD? In previous experiments, scientists have used RPEs made from human embryonic stem cells to treat two patients with inherited eye diseases. The results from these experiments were underwhelming to say the least. Also, the derivation of RPEs from embryonic stem cells was tedious and laborious. Is there a better way?

Make that a yes. A paper in Stem Cells Translational Medicine from Donald Zack’s laboratory at Johns Hopkins University School of Medicine describes a simple and highly scalable process for deriving RPEs from human pluripotent stem cells.

To begin with, the cells were plated at relatively high densities (20,000 cells / cm square centimeter) in a medium called TeSR1. This medium can support the growth of human pluripotent stem cells and can also keep them undifferentiated without the use of animal feeder cell lines. SInce there are no feeder cells to make, the cultivation of these cells is much simpler than before and the variability from culture to culture decreases.

After five days of growth, the cells grew to a monolayer (the cells had grown and spread throughout the culture dish) and were transferred to a 5% carbon dioxide and 20% oxygen incubator. Three days later, they were transferred to Delbecco’s Modified Eagle Medium with F12 supplement or DMEM/F12. This culture medium supports stem cell differentiation. The cells grew and differentiated, for about 25 days, but RPEs were easily visible because they make loads of dark pigment. Once the dark colonies appeared, the cells were allowed to grow another 25 days. The cells were transferred into Delbecco’s Medium with enzymes to pull the cells apart from each other for four hours, then, after pipetting them vigorously, the cells were centrifuged, and suspended in a cell detachment solution called Accumax.

The separated cells were filtered and plated on specially coated plates, and cultured in “RPE medium.” This is a mixture of several different culture media that favors the survival and growth of RPEs. Because RPE colonies were easily seen with their dark pigments, they were specifically picked and passaged. The result was extremely clean RPE cultures from pluripotent stem cells.

Differentiation of hPSCs into RPE. (A): Schematic view of the differentiation process. (B): Kinetics of marker expression of differentiating hESCs and hiPSCs as measured by quantitative real-time polymerase chain reaction. d1 denotes the time at which the cells were transferred to DM. Error bars represent standard deviation of biological replicates. (C): Morphology of hPSCs after 50 days in DM, with arrowheads indicating representative pigmented colonies. Scale bar = 5 mm. (D, E): Flow cytometric analysis of the expression of RPE65 by differentiating hPSCs after 50 days in DM. The profile of cells stained with the anti-RPE65 antibody is shown in red, and the isotype control is displayed in black. Only a minority of cells were RPE65-positive, which is in accordance with the limited number of pigmented colonies obtained in (C). Abbreviations: d, day of the experiment; DM, differentiation medium; hESC, human embryonic stem cell; hiPSC, human induced pluripotent stem cell; hPSC, human pluripotent stem cell; P1, first passage; P2, second passage; RPE, retinal pigment epithelium; w, week.
Differentiation of hPSCs into RPE. (A): Schematic view of the differentiation process. (B): Kinetics of marker expression of differentiating hESCs and hiPSCs as measured by quantitative real-time polymerase chain reaction. d1 denotes the time at which the cells were transferred to DM. Error bars represent standard deviation of biological replicates. (C): Morphology of hPSCs after 50 days in DM, with arrowheads indicating representative pigmented colonies. Scale bar = 5 mm. (D, E): Flow cytometric analysis of the expression of RPE65 by differentiating hPSCs after 50 days in DM. The profile of cells stained with the anti-RPE65 antibody is shown in red, and the isotype control is displayed in black. Only a minority of cells were RPE65-positive, which is in accordance with the limited number of pigmented colonies obtained in (C). Abbreviations: d, day of the experiment; DM, differentiation medium; hESC, human embryonic stem cell; hiPSC, human induced pluripotent stem cell; hPSC, human pluripotent stem cell; P1, first passage; P2, second passage; RPE, retinal pigment epithelium; w, week.

The cells were subjected to a battery of tests: flow cytometry, Western blotting, Immunostaining and so on. These cells passed with flying colors and they are clearly RPE cells that express RPE-specific genes, have RPE-specific proteins on their cell surfaces, and even snuggle up to photoreceptors and recycle their terminal segments.  The final functional test came from a transplantation experiment in which human RPEs made from human pluripotent stem cells were transplanted behind the retinas of mice with impaired immune systems.  The cells, as you can see in the figures below, integrated beautifully, and were also highly functional, as indicated by the rhodopsin-positive vesicles in the implanted RPE cells.   No tumors were seen in any of the laboratory animals implanted with the stem cell-derived RPEs.

Transplantation of human pluripotent stem cell (hPSC)-RPE cells into the subretinal space of albinos NOD-scid mice. (A): Fundus photograph of an injected eye, 1 week post-transplantation. Note the numerous pigmented clusters formed by the transplanted hPSC-RPE cells. (B): Confocal micrograph showing the presence of rhodopsin-positive material (yellow arrows) within the cell membrane of carboxyfluorescein diacetate succinimidyl ester-labeled hPSC-RPE cells, 1 week after subretinal injection. Scale bars = 10 μm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; ONL, outer nuclear layer; OS, outer segment; RPE, retinal pigment epithelium.
Transplantation of human pluripotent stem cell (hPSC)-RPE cells into the subretinal space of albinos NOD-scid mice. (A): Fundus photograph of an injected eye, 1 week post-transplantation. Note the numerous pigmented clusters formed by the transplanted hPSC-RPE cells. (B): Confocal micrograph showing the presence of rhodopsin-positive material (yellow arrows) within the cell membrane of carboxyfluorescein diacetate succinimidyl ester-labeled hPSC-RPE cells, 1 week after subretinal injection. Scale bars = 10 μm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; ONL, outer nuclear layer; OS, outer segment; RPE, retinal pigment epithelium.

This new procedure is able to make RPEs from pluripotent stem cells in a simple and highly scalable way.  If human induced pluripotent stem cells could be used with this protocol, and there seems little reason that should not be highly possible, then such cells could be easily used for human clinical trials.

Tissue Engineers Use New Biomaterial to Repair Knee Cartilage


Tissue engineers from Johns Hopkins University School of Medicine’s Translational Tissue Engineering Center (TTEC) have used a biomaterial to stimulate and facilitate the growth of new cartilage in human patients.

An illustration of the cartilage repair surgical procedure. A mini-incision exposes the cartilage defect (top left-hand panel), and any dead tissue is removed from the edges. (B) The adhesive is then applied to the base and walls of the defect, followed by microfracture. (C) Lastly, the hydrogel solution is injected into the defect. (D) Bleeding from the microfracture holes is trapped in and around the hydrogel.Science Translational Medicine/AAAS
An illustration of the cartilage repair surgical procedure. A mini-incision exposes the cartilage defect (top left-hand panel), and any dead tissue is removed from the edges. (B) The adhesive is then applied to the base and walls of the defect, followed by microfracture. (C) Lastly, the hydrogel solution is injected into the defect. (D) Bleeding from the microfracture holes is trapped in and around the hydrogel.
Science Translational Medicine/AAAS

This was a rather small study that only examined 15 patients. All 15 patients had cartilage defects and were scheduled to undergo “microfracture surgery.” Microfracture surgery uses a drill to bore tiny holes in the bone. These small holes allow bone marrow stem cells to leak into the joint space and make new bone and cartilage. In this study, hydrogel scaffolding was troweled into the wound to in order to support and nourish the healing process. The results from this study were published in the Jan. 9 issue of Science Translational Medicine. According to the authors, this study is a proof of concept trial that paves the way for larger trials to test the hydrogel’s safety and effectiveness.

“Our pilot study indicates that the new implant works as well in patients as it does in the lab, so we hope it will become a routine part of care and improve healing,” says Jennifer Elisseeff, the Jules Stein Professor of Ophthalmology and director of the Johns Hopkins University School of Medicine’s TTEC. Cartilage damage results from overuse, injury, disease or faulty genes. Microfracture surgery is a standard of care for cartilage repair, but when holes in cartilage are caused by joint injuries, microfracture surgery often either fails to stimulate new cartilage growth or grows cartilage that is less hardy than the original tissue

To address this problem, tissue engineers, such as Elisseeff, have postulated that the bone marrow mesenchymal stem cells need a nourishing scaffold on which to grow in order to make the right type of cartilage and enough of it. Unfortunately, demonstrating the clinical value of hydrogels has been slow, difficult, and expensive. By experimenting with various materials, Elisseeff and her colleagues have developed a promising hydrogel, and an adhesive that sticks the hydrogel to the bone.

After testing the combination for several years in the lab and in goats, the hydrogel seemed ready for human trials. Elisseeff and her group collaborated with orthopedic surgeons to conduct their first clinical study. 15 patients with holes in the cartilage of their knees received a hydrogel and adhesive implant along in combination with microfracture surgery. In order to compare the efficacy of their hydrogel, another three patients were treated with microfracture surgery alone. After six months, it was clear that the hydrogel implants had caused no major problems. Furthermore, magnetic resonance imaging of these patient’s knees showed that patients with implants had new cartilage filling an average 86% of their defects in their knees, and patients that had received only microfracture surgery had an average of 64% of their tissue replaced. Patients with the implant also reported a greater decrease in knee pain in the six months following surgery, according to the investigators.

As the trial continues, more patients have enrolled. This clinical trial is presently managed by a company called Biomet. These data from this trial is part of an effort to earn European regulatory approval for the device.

Elisseeff and her team have begun developing a next-generation implant in which the hydrogel and adhesive will be combined in a single material. Elisseeff and others are also interested in technologies for joint lubrication that reduce joint pain and inflammation