UTMB Galveston Physicians Build Lungs in Laboratory


As a bioreactor bubbles and whirls a pair of living lungs slowly takes shape. Is this a scene from Shelly’s Frankenstein? No. Instead it is an everyday occurrence in a laboratory at the University of Texas Medical Branch (UTMB) Galveston National Laboratory. The star of this “lung show” is a little pig named “Harry.” Little Harry has the distinction of being the first patient to be surgically implanted with a laboratory-built lung, and both the doctor and patient are doing just fine.

Dr. Joan Nichols of UTMB’s Galveston National Laboratory put it this way: “We build lungs here.” Nichols continued: “That’s pretty much what it’s become in the last six months or so, is a little factory to build lungs.”

The lung is a uniquely sculpted organ. Therefore, UTMB medical researchers required a pattern or scaffold upon which they could build lungs. They began with lungs acquired from dead animals and humans and spent more than a year perfecting protocols to isolate the lungs without damaging them and then remove all the cells. After the decellularization process, Nichols and her group were left with nothing but the elastic protein structure that serves as the skeleton of the lung. These lung skeletons were then incubated in a bioreactor that constant bathes the lung tissue in fresh culture medium and oxygen with lung cells extracted from living creatures. The lungs cells adhered to the lung skeleton and grew until they thoroughly covered it to create a new pair of lungs.

Bioengineered lungs
Bioengineered Lungs in a Bioreactor

 

Dr. Joaquin Cortiella, the director of UTMB’s Laboratory of Regenerative and Nano Medicine, likened this entire process to engineering a building: “You basically have a scaffold and then you build on top of that to create the building.”

Nichols and her colleagues have used this procedure to create both animal and human lungs. A freshly minted set of pig lungs were transplanted into Harry the pig. Harry’s healthy recovery from lung transplantation surgery indicates that this procedure experiment and potentially opens up the prospect of implanting new laboratory-built lungs into people.

“It’s the first time it’s ever been done, where we’ve taken a lung and it’s inside of the lung cavity of this pig,” Cortiella said.

Bioengineered lungs could vastly expand the number of organs available for those patients who require transplants, and this is especially the case in children, according to Cortiella. Cortiella’s experience in pediatric medicine is his main motivation for taking on this research project; he sometimes had to watch babies die from lung ailments.

“I also have lung disease,” Cortiella said. “I have pulmonary fibrosis. Breathing is a difficult thing for me at times. And so, for me, I appreciate the fact that there are not enough lungs out there to give to everybody who needs them.

“And so, if we develop something that can actually be tailor-made for somebody – or at least, have something available that we can transplant into people that are on the waiting list – the less people will die waiting for them,” he said.

Harry the pig is doing well, but in order to get a fuller picture of how well the bioengineered lung interacted with the rest of the Harry’s body, he will have to be euthanized for more detailed tissue examinations.

Previously, research groups have attempted to use synthetic materials instead of lung skeletons derived from living lungs. Unfortunately, none of the synthetic materials that were tried provided adequate structural support to make a living lung. Nichols thinks that doctors may eventually be able to make replacement organs with 3-D printers.

“Someday?” she said. “Someday, we are going to use these techniques to bio-engineer organs for people that need them.”

Investigational “CART” Cells, A Personalized Cellular Cancer Therapy is Well Tolerated By Patients


Chimeric Antigen Receptor T cells or CART cells are genetically modified versions of a patients’ own immune cells that expressed molecules that specifically bind tumor cells and mark them for destruction.  A host of animal experiments have demonstrated the safety and effectiveness of CART cells for treating tumors, but getting a therapy to work in animals is different than getting it to work in human patients.

CAR-Engineered_T-Cell_Adoptive_Transfer

Thus, the recent news that patients treated with CART cells made from their own T cells are tolerating them well is very welcome news.  Equally welcome is the news that the infused CART cells successfully traveled to those tumors they were designed to attack in an early-stage trial for mesothelioma and pancreatic and ovarian cancers at the Perelman School of Medicine at the University of Pennsylvania. Data from these trials adds to an already growing body of research that shows that CAR T cell technology shows remarkable promise for fighting tumors.  These interim results will be presented at the American Association for Cancer Research (AACR) Annual Meeting 2015, April 18-22.

“The goal of this phase I trial was to study the safety and feasibility of CART-meso cells in patients with mesothelin-expressing tumors,” says Janos L. Tanyi, MD, PhD, an assistant professor of Gynecologic Oncology. “We found no major adverse events associated with the treatment, which suggests that the patients tolerated it very well. But importantly, the T cells successfully targeted the patients’ tumor sites and survived in the blood stream for up to 28 days.”

The data that Tanyi will present at this conference will consist of scans and measurements acquired from five different patients; two of whom are suffering from ovarian cancer, two who have epithelial mesothelioma, and one with pancreatic cancer.  All five patients agreed to received the new investigational CART cell therapy.  Significantly, all the patients who received this therapy had cancers that stopped responding to conventional treatments.

CAR T cells are made from each patient’s T lymphocytes that are extracted from blood by a process known as “apheresis.”  T lymphocytes are isolated from the blood cells by cell sorting and then genetically modified to secrete a special protein that identifies and attacks tumor cells.  In this case, the cells were genetically engineered to target those cancer cells that express a protein called Mesothelin on their surfaces.  The engineered protein secreted by the engineered T cells could identify and kill them the tumor cells.  Even though Mesothelin is also found on the surfaces of the pleura (membranes that surround the lungs), the peritoneum (the lining that surrounds the abdominal cavity), and the pericardium (the scar that surrounds the heart),a variety of tumors express Mesothelin at such high levels that they are much more likely to be attacked by the CAR T cells that the normal tissues.

The preliminary results suggest the T cells did not attack normal tissues, but these patients must be followed up annually for 15 years in order to more closely observe the persistence of the CART-meso cells, their potential antitumor activity, and to better characterize their safety profiles.  Because the CAR T cells to not last indefinitely in the bloodstream, their ability to attack normal tissue should, theoretically at least, be minimal.

3D Printer Makes the Tiniest Human Organs Ever Made


Through the use of 3-D printers, mini human organs can be made in all kinds of shapes and sizes. A new experiment by tissue engineers from Wake Forest University has made tiny beating hearts that beat in sync, and another pulsing heart that fused with a spherical, liver.

These printed, mini-organs were by Anthony Atala and his team at the Wake Forest Institute for Regenerative Medicine in Winston-Salem, North Carolina. They represent the first step in developing an entire human body on a chip. The mini-hearts were made by reprogramming human skin cells into heart cells, which were then clumped together in a cell culture. A 3-D printer was then used to give them the desired shape and size, which in this case was a sphere of tissue with a diameter of 0.25 millimeters.

The development of these miniature organs was motivated by a desire to make model systems that mimic the function of life-size organs. Eventually, such a system could create mini-organs that could be linked up to form an entire organ system that could be used to test new treatments or probe the effects of chemicals and viruses.

The production of these mini-organs could potentially serve an alternative to animal testing, which is usually rather costly and doesn’t always produce results that are applicable to humans.

Further work on these mini-organs could also discover ways to expand these organs and make them life-size that they can be used for organ transplants.

3-D Printing to Make Replacement Body Parts


Advances in three-dimensional (3D) printing have produced a swell of interest in artificial organs that are designed to replace, or even enhance, human tissues.

3-D printed organs

At the Inside 3D Printing conference in New York on April 15–17, 2015, researchers from academia and industry are gathering to discuss the growing interest in using three-dimensional (3D) printing to make replacement body parts. Although surgeons are already using 3D-printed metal and plastic implants to replace bones, researchers are looking ahead to printing organs using cells as “ink.”  All the structures shown here were all 3D printed at Wake Forest Baptist Medical Center in Winston-Salem, North Carolina, and include a rudimentary proto-kidney (top left), complete with living cells.

Printed organs, such as a prototype outer ear that was developed by researchers at Princeton University in New Jersey and Johns Hopkins University in Baltimore, Maryland, will be featured at the conference.  This ear is printed from a range of materials: a hydrogel to form an ear-shaped scaffold, cells that will grow to form cartilage, and silver nanoparticles to form an antenna (see M.S. Mannoor et al. Nano Lett. 13, 2634−2639; 2013. This is just one example of the increasing versatility of 3D printing.

This New York meeting, which is being advertised as the largest event in the industry, will provide exposure for a whole world of devices and novelties. But it will also feature serious discussions on the emerging market for printed body parts.

The dream of bioprinting is to print organs that can be used for transplant. For example, at the Wake Forest Baptist Medical Center in Winston-Salem, North Carolina, researchers are developing a 3D-printed kidney. The project is in its early stages and the kidney is far from functional and some doubt that researchers will ever be able to print such a complex organ. Perhaps a more achievable near-term goal might be to print sheets of kidney tissue that could be grafted onto existing kidneys.

Printed replacement for skull

Printed structures made of hard metal or polymers are already on the market for people in need of an artificial hip, finger bone or facial reconstruction. This skull implant (grey) was made by Oxford Performance Materials of South Windsor, Connecticut, and was approved by US regulators in 2013. It is made of a polymer meant to encourage bone growth, to aid integration of the implant into the surrounding skeleton. The company also sells implants for facial reconstruction and for replacing small bones in the feet and hands.

3-D printed lung tree

One of the key advantages of using 3D printing for surgical implants is the opportunity to model the implant to fit the patient. This airway splint (shown on the right branch of the model trachea) was designed by researchers at the University of Michigan in Ann Arbor to fit an infant with a damaged airway. The splint was made out of a material that is gradually absorbed by the body as the airway heals. The research team benefited from the concentration of 3D-printing expertise that has built up in Michigan because of the US automobile industry, which uses the technology for printing prototypes and design samples.

The business of 3-D printing also includes titanium replacement hip joints, which can be tailored to fit individual people, and made-to-order polymer bones to reconstruct damaged skulls and fingers. Printed body parts brought in US $537 million last year, up about 30% on the previous year, says Terry Wohlers, president of Wohlers Associates, a business consultancy firm in Fort Collins, Colorado, that specializes in 3D printing.

3-D printed prosthetics

3D printing can also be used to generate cheap — and creative — prostheses.  A prosthetic hand can cost thousands of dollars, which is a burdensome expense when fitting it to a growing child.  Jon Schull founded a company called e-NABLE that provides free printed prosthetics to those in need, harnessing the efforts of hundreds of volunteers who own consumer-grade 3D printers. “When people get tired of printing Star Wars figurines, they give us a call,” he says.  The cost of materials for a printed prosthesis is about US $35.

3-D animal prosthesis

Also, 3-D printed prostheses are not just for humans.  For example, a duck named Buttercup was born with its left foot turned backwards.  The Feathered Angels Waterfowl Sanctuary in Arlington, Tennessee, arranged for the fowl to receive a new foot, complete with a bendable ankle.  Also in the an eagle, a box turtle and a handful of dogs also have been fitted with 3-D printed prostheses.

Scientists are looking ahead to radical emerging technologies that use live cells as ‘ink’, assembling them layer-by-layer into rudimentary tissues, says Jennifer Lewis, a bioengineer at Harvard University in Cambridge, Massachusetts. Bioprinting firm Organovo of San Diego, California, already sells such tissues to researchers aiming to test experimental drugs for toxicity to liver cells. The company’s next step will be to provide printed tissue patches to repair damaged livers in humans, says Organovo’s chief executive, Keith Murphy.

Lewis hesitates to say that 3D printing will ever yield whole organs to relieve the shortage of kidneys and livers available for transplant. “I would love for that to be true,” she says. “But these are highly complicated architectures.”

Induced Pluripotent Stem Cells from Bone Cancer Patients Provide Crucial Insights into the Genesis of Bone Cancer


A team of Mount Sinai researchers have utilized induced pluripotent stem cells (iPSCs) to elucidate the genetic changes that seem to convert a well-known anti-cancer signaling gene into a driver bone cancers. When it comes to bone cancers, the survival rate has not improved in 40 years despite advances in treatment. Since this study might provide new targets and suggest new strategies for attacking such cancers. it represents a welcome addition to the cancer literature.

This study, which was published in the journal Cell, revolves around iPSCs, which were discovered in 2006 by Nobel laureate Shinya Yamanaka. iPSCs use genetic engineering and cell culture techniques to reprogram mature, adult cells to become like embryonic stem cells. These iPSCs are “pluripotent,” which means that they are able to differentiate into any adult cell type and can also divide in culture indefinitely.

For therapeutic purposes, iPSCs can be derived from a patient’s own cells, differentiated into the cells the patient needs to be replaced, and then implanted into the patient’s body to augment tissue healing or even organ reconstruction. Since iPSCs can be successfully differentiated into heart muscle, nerve cells, bone, and other cell types, they have the potential advance the field of regenerative medicine by leaps and bounds.

iPSCs have already made their presence known in the clinic by serving as model systems for research and diagnosis. The new Mount Sinai study used iPSCs to construct an accurate model of a genetic disease “in a dish.” The culture dishes contain self-renewing patient-specific iPSCs or a specific cell line that enable in-depth study diseases that are driven by each person’s genetic differences. When matched with patient records, iPSCs and iPSC-derived target cells have the ability to help physicians predict a patient’s prognosis and whether or not a given drug will be effective for him or her.

In this study, skin cells from healthy patients and patients with a genetic disease called Li-Fraumeni syndrome were isolated and reprogrammed into patient-specific iPSC lines. These iPSCs were then differentiated into bone-making cells (osteoblasts), which are the cells where particular rare and common bone cancers start. Li-Fraumeni syndrome greatly predisposes patients to a variety of cancers in several different types of tissues.

The patient-derived osteoblasts were then tested for their tendencies to become tumor cells and to make bone. This particular bone cancer model did a better job of recapitulating the characteristics of bone cancer than previously used mouse or cellular models.

LFS iPSCs for stem cell production

“Our study is among the first to use induced pluripotent stem cells as the foundation of a model for cancer,” said lead author Dung-Fang Lee, PhD, a postdoctoral fellow in the Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai. “This model, when combined with a rare genetic disease, revealed for the first time how a protein known to prevent tumor growth in most cases, p53, may instead drive bone cancer when genetic changes cause too much of it to be made in the wrong place.”

The Mount Sinai disease model research uses a simple fact of human life as its basis: human genes undergo mutations at a certain rate that tends to increase as we age, and the formation of new mutations in relentless and constant. Some mutations make no difference, a few some confer advantages, and others cause disease. Beyond inherited mutations that contribute to cancer risk, the combination of random, accumulated DNA changes in our cells as we age also increase our cancer risk.

The current study focused on those genetic pathways involved in Li-Fraumeni Syndrome or LFS, a rare genetic disease that causes high risk for many cancers in affected families. Osteosarcoma (bone cancer) is a common cancer observed in LFS patients and many of them are diagnosed before the age of 30. Additionally, osteosarcoma is the most common type of bone cancer in all children, and after leukemia, the second leading cause of cancer death for them.

 

Importantly, about 70 percent of LFS families have a mutation in their copy of a genes called TP53, which encodes the p53 protein. P53 is a “the tumor suppressor,” which means that it functions to preserve the integrity of the genome and keep the rate of cell division in check. Common forms of osteosarcoma, which are driven by somatic or inherited mutations, have also been closely linked by past studies to defects in p53 when mutations interfere with the ability of the protein to function properly.

p53
Crystal Structure of p53 protein bound to DNA

 

Rare genetic diseases like LFS provide excellent model systems because they tend to result from a change in a single gene, instead of the diverse and overlapping mutations observed in common diseases, and, in this case, more common, non-inherited bone cancers. The LFS-iPSC based modeling highlights the contribution of p53 alone to osteosarcoma.

By analyzing iPSC lines, and bone cancer driven by p53 mutations in LFS patients, the Munt Sinai research team showed, for the first time, that the LFS bone cancer results from an overactive p53 gene. Too much p53 in osteoblasts dampens the function of a gene, H19, and a related protein, decorin, that would otherwise help stem cells differentiate into normal osteoblasts.

The inability of cells to differentiate makes them vulnerable to genetic mistakes that drive cancer, since more “stemness” means a tendency toward rapid, abnormal growth, like that observed in tumors. One tragic feature of osteosarcoma is the rapid, error-prone production of weaker bone by cancerous bone-making cells, where a young person surprisingly breaks a bone to reveal undiagnosed, advanced cancer.

Dung-Fang Lee and his colleagues discovered that the H19 gene seems to control a network of interconnected genes that fine-tune the balance between cell growth and resistance to growth. Decorin is a protein that is part of connective tissue like bone, but that also plays a signaling role, interacting with growth factors to slow the rate that cells divide and multiply, unless turned off by too much p53.

“Our experiments showed that restoring H19 expression hindered by too much p53 restored “protective differentiation” of osteoblasts to counter events of tumor growth early on in bone cancer,” said co-author, Ihor Lemischka, PhD, Director of The Black Family Stem Cell Institute within the Icahn School of Medicine. “The work has implications for the future treatment or prevention of LFS-associated osteosarcoma, and possibly for all forms of bone cancer driven by p53 mutations, with H19 and p53 established now as potential targets for future drugs.”

A New Way to Prepare Fat-Based Stem Cells to Treat Wounds


An Italian laboratory headed by Dr. Raposio at the University of Parma has designed a simple and fast technique for preparing fat-based stem cells for use in the clinic.

Fat contains an alternative source of mesenchymal stem cells with characteristics similar to those found in bone marrow, but the fat-based stem cells are easier to isolate and have been shown to be effective enhancers of wound healing.

Raposio and his colleagues used fat contributed by liposuction patients. Each patient provided about 80 cubic centimeters of fat in liposuction procedures that were collected under anesthesia. Once the cells from this fat were isolated, they were mixed with platelet-rich plasma (PRP) that had been previously collected. Mixing PRP with stem cells enhances the capabilities of the fat-based stem cells and generates a concoction called “e-PRP.”  This simple procedure that consisted of fat collection, stem cell collection and mixing the cells with PRP to make e-PRP quickly made a produce that was ready for grafting onto wounds on the skin.

Detailed analyses of the cells isolated from the fat showed that they consisted of about 50,000 fat-based mesenchymal stem cells or ASCs. They represented about 5% of all cells in the sample. The remaining cells were blood-derived cell and blood vessel-making endothelial cells.

The significance of this procedure lies in the fact that most of the protocols used to isolate stem cells from fat take about two hours and require animal-derived reagents. However, the number of ASCs isolated with this new procedure is sufficient for application to wounds without the need of expanding the cells in culture. Also, this new procedure does not require serum or animal-derived reagents, and it takes only 15 minutes.

Thus this method of ASC isolation is innovative, feasible, and represents an advance in the stem cell-based treatment of chronic wounds.

Amniotic Fluid Stem Cells Make Robust Blood Vessel Networks


The growth of new blood vessels in culture received in new boost from researchers at Rice University and Texas Children’s Hospital who used stem cells from amniotic fluid to promote the growth of robust, functional blood vessels in healing hydrogels.

These results were published in the Journal of Biomedical Materials Research Part A.

Engineer Jeffrey Jacot thinks that amniotic fluid stem cells are valuable for regenerative medicine because of their ability to differentiate into many other types of cells, including endothelial cells that form blood vessels. Amniotic fluid stem cells are taken from the discarded membranes in which babies are encased in before birth. Jacot and others combined these cells with an injectable hydrogel that acted as a scaffold.

In previous experiments, Jacot and his colleagues used amniotic fluid cells from pregnant women to help heal infants born with congenital heart defects. Amniotic fluids, drawn during standard tests, are generally discarded but show promise for implants made from a baby’s own genetically matched material.

“The main thing we’ve figured out is how to get a vascularized device: laboratory-grown tissue that is made entirely from amniotic fluid cells,” Jacot said. “We showed it’s possible to use only cells derived from amniotic fluid.”

Researchers from Rice, Texas Children’s Hospital and Baylor College of Medicine combined amniotic fluid stem cells with a hydrogel made from polyethylene glycol and fibrin. Fibrin is the proteins formed during blood clots, but it is also used for cellular-matrix interactions, wound healing and angiogenesis (the process by which new vessels are made). Fibrin is widely used as a bioscaffold but it suffers from low mechanical stiffness and is degraded rapidly in the body. When fibrin was combined with polyethylene glycol, the hydrogel became much more robust, according to Jacot.

Additionally, these groups used a growth factor called vascular endothelial growth factor to induce the stem cells to differentiate into endothelial cells. Furthermore, when induced in the presence of fibrin, these cells infiltrated the native vasculature from neighboring tissue to make additional blood vessels.

When mice were injected with fibrin-only hydrogels, thin fibril structures formed. However if those same hydrogels were infused with amniotic fluid stem cells that had been induced with vascular endothelial growth factor, the cell/fibrin hydrogel concoctions showed far more robust vasculature.

In similar experiments with hydrogels seeded with bone marrow-derived mesenchymal cells, once again, vascular growth was observed, but these vessels did not have the guarantee of a tissue match. Interestingly, seeding with endothelial cells didn’t work as well as the researchers expected, he said.

Jacot and others will continue to study the use of amniotic stem cells to build biocompatible patches for the hearts of infants born with birth defects and for other procedures.