Differential Immunogenicity of Cells Derived from Induced Pluripotent Stem Cells

Induced pluripotent stem cell (iPSC) technology has raised the possibility that patient-specific pluripotent stem cells may become a renewable source of a patient’s own cells for regenerative therapy without the concern of immune rejection. However, the immunogenicity of autologous human iPSC (hiPSC)-derived cells is not well understood.

Using a humanized mouse model (denoted Hu-mice) with a functional human immune system, Yang Xu and his colleagues from UC San Diego has shown that most teratomas or tumors formed by human iPSCs were readily recognized by immune cells and rejected. However, when these human iPSCs were differentiated into smooth muscle cells or retinal pigmented epithelial cells, the results were rather different. Human iPSC-derived smooth muscle cells appear to be highly immunogenic, but human iPSC-derived retinal pigment epithelial (RPE) cells are tolerated by the immune system, even when transplanted outside the eye.


When Xu and others examined these results more closely, they discovered that this differential immunogenicity is due to the abnormal expression of cell surface proteins in hiPSC-derived Smooth Muscle Cells, but not in hiPSC-derived RPEs.

These findings support the feasibility of developing hiPSC-derived RPEs for treating macular degeneration. They also show that iPSC lines must be individually screened to determine if their derivatives are recognized by the patient’s immune system as foreign.

These results were published in Cell Stem Cell.

Transplanted Mesenchymal Stem Cells Prevent Bladder Scarring After Spinal Cord Injury

A collaborative research effort between laboratories from Canada and South Korea have shown that a cultured mesenchymal stem cell line called B10 can differentiate into smooth muscle cells and improve bladder function after a spinal cord injury.

Spinal cord injury can affect the lower portion of the urinary tract. Overactive bladder, urinary retention, and increased bladder thickness and fibrosis (bladder scarring) can result from spinal cord injuries. Human mesenchymal stem cells (MSCs) can differentiate under certain conditions into smooth muscle. For this reason, MSCs have therapeutic potential for patients who have suffered from spinal cord injuries.

Seung U. Kim and his colleagues from Gachon University Gil Hospital in Inchon, South Korea have made an immortalized human mesenchymal stem cell line by transfecting primary cell cultures of fetal human bone marrow mesenchymal stem cells with a retroviral vector that contains the v-myc oncogene. This particular cells line, which they called HM3.B10 (or B10 for short), grows well in culture and can also differentiates into several different cell types.

In this present study, which was published in the journal Cell Transplantation, Kim and his colleagues and collaborators injected B10 hMSCs directly into the bladder wall of mice that had suffered a spinal cord injury but were not treated showed no such improvement.

“Human MSCs can secrete growth factors,” said study co-author Seung U. Kim of the Division of Neurology at the University of British Columbia Hospital, Vancouver, Canada. “In a previous study, we showed that B 10 cells secrete various growth factors including hepatocyte growth factor (HGF) and that HGF inhibits collagen deposits in bladder outlet obstructions in rats more than hMSCs alone. In this study, the SCI control group that did not receive B10 cells showed degenerated spinal neurons and did not recover. The B10-injected group appeared to have regenerated bladder smooth muscle cells.”

Four weeks after the initial spinal cord injury, the mice in the B10-treated group received injections of B10 cells transplanted directly into the bladder wall. Kim and his team used magnetic resonance imaging (MRI) to track the transplanted B10 cells. The injected B10 cells had been previously labeled with fluorescent magnetic particles, which made them visible in an MRI.

“HGF plays an essential role in tissue regeneration and angiogenesis and acts as a potent antifibrotic agent,” explained Kim.

These experiments also indicated that local stem cell injections rather than systemic, intravenous infusion was the preferred method of administration, since systemic injection caused the hMSCs get stuck largely in the blood vessels of the lungs instead of the bladder.

The ability of the mice to void their bladders was assessed four weeks after the B10 transplantations. MRI analyses clearly showed strong signals in the bladder as a result of the labeled cells that had been previously transplanted. Post-mortem analyses of the bladders of the transplanted group showed even more pronounced differences, since the B10-injected animals had improved smooth muscle cells and reduced scarring.

These results suggest that MSC-based cell transplantation may be a novel therapeutic strategy for bladder dysfunction in patients with SCI.

“This study provides potential evidence that an human [sic] stable immortalized MSC line could be useful in the treatment of spinal cord injury-related problems such as bladder dysfunction.” said Dr. David Eve, associate editor of Cell Transplantation and Instructor at the Center of Excellence for Aging & Brain Repair at the University of South Florida. “Further studies to elucidate the mechanisms of action and the long-term effects of the cells, as well as confirm the optimal route of administration, will help to illuminate what the true benefit of these cells could be.”

Making Heart Muscle from Skeletal Muscle Stem Cells

Several experiments in animals and a few clinical trials in human patients have shown that implanting skeletal muscle cells isolated from muscle biopsies into the heart after a heart attack can help the heart to some degree, but the implanted skeletal muscle cells do not integrate into the existing heart muscle mass and the skeletal muscle cells do not differentiate into heart muscle cells.

Experiments like those mentioned above utilized muscle satellite cells. Muscle satellite cells are a resident stem cell population that respond to muscle damage and divide to form skeletal muscle cells form new muscle. Satellite cells are a perfect example of a unipotent stem cell, which is to say a cell that makes one type of terminally differentiated cell type.

Skeletal muscles, however, have another cell population called muscle-derived stem cells or MDSCs. MDSCs express an entirely different set of cell surface proteins than satellite cells, and have the capacity to differentiate into skeletal muscle, smooth muscle, bone, tendon, nerve, endothelial and hematopoietic cells. MDSCs grow well in culture, tolerate low oxygen conditions quite well, and show excellent regenerative potential.

Other laboratories have managed to culture MDSCs in collagen and produce beating heart muscle cells. Others have observed MDSCs forming a proper myocardium under certain conditions. Several studies have established the ability to MDSCs to treat laboratory animals that have suffered a heart attack. The most recent work from Sekiya and others has established that cell sheets made from MDSCs can reduce dilation of the left ventricle, increased capillary density, and promoted recovery without causing erratic heat beat patterns.

Despite their obvious efficacy. MDSCs remain difficult to isolate in high enough numbers to therapeutic purposes. None of the cell surface molecules sported by MDSCs are unique to those cells. Therefore, getting clean cultures of MDSCs remains a challenge. Still, these cells represent some of the best hopes for regenerative medicine in the heart. These cells do form heart muscle cells and heal ailing hearts. They can be grown in bioreactors to high numbers and can also be combined with engineered materials to shore up a damaged heart and mediate its regeneration. While the use of MDSCs is still in its infancy, the promise certainly is there.

Using Stem Cells to Make Two Different Building Blocks of Blood Vessels

A research team from Johns Hopkins University has discovered methods that use stem cells to make two different types of tissues that help construct blood vessels.

Even though many experiments have used a variety of stem cells to make blood vessels in living laboratory animals and human patients, making blood vessels in the laboratory from scratch has been a colossal headache. Because patients with vascular diseases need new blood vessels, being able to grow blood vessels in the laboratory for clinical use is an important step in tissue engineering.

Sharon Gerecht, an assistant professor of chemical and biomolecular engineering, led this research team. Through this work, they hope to provide material that can be used to treat patients with diabetes, heart disease, and patients with other types of vascular illnesses.

In an interview, Gerecht said: “That’s our goal: to give doctors a new tool to treat patients who have problems in the pipelines that carry blood through their bodies. Finding our how to steer these stem cells into becoming critical building blocks to make these blood vessel networks is an important step.”

Gerecht and her team focused on smooth muscle cells (SMCs). SMCs are found in the walls of blood vessels and by contracting or relaxing, they regulate the diameter of blood vessels, the rates of blood flow and blood pressure. They are two main types of SMCx: synthetic and contractile. Synthetic SMCs migrate through surrounding tissue and continue to divide. They primarily support newly-formed blood vessels. Contractile smooth muscle cells remain in place and stabilize the growth of new blood vessels. Contractile SMCs also control blood pressure.

To make SMCs in the laboratory, Gerecht and her colleagues used embryonic stem cells and induced pluripotent stem cells. in earlier work, Gerecht’s team differentiated pluripotent stem cells into SMC-like cells that were close to SMCs, but not completely like them. However, by modifying their protocol, Gerecht’s team were able to differentiate pluripotent stem cells into synthetic SMCs. This modified protocol included high concentrations of growth factors and serum, but they also modified their protocol further and were able to induce pluripotent stem cells to differentiate into contractile SMCs.

“When we added more of the growth factor and serum, the stem cells turned into synthetic smooth muscle cells,” Gerecht said. “When we provided a much small er amount of these materials, they became contractile smooth muscle cells.”

This ability to make one type or another type of SMC in the laboratory could be critical in developing new blood vessel networks, since SMCs are such a vital part of blood vessels. Gerecht sad as much when she noted that when you are “building a pipeline to carry blood, you need the contractile cells to provide structure and stability.” Gerecht continued” “But in working with very small blood vessels, the migrating synthetic smooth muscle cells can be more useful.”

This work also carries additional bonuses, since cancer cells induce the formation of small blood vessels to nourish the growing tumor. The current work could also help researchers understand how blood vessels are formed and stabilized in tumors, which could be useful in the treatment of cancer.

Gerecht concluded: “We still have a lot more research to do before we can build complete new blood vessels networks in the lab, but our progress in controlling the fate of these stem cells appears to be a big step in the right direction.”

See M. Wanjare, Cardiovascular Research 2012; DOI: 10.1093/cvr/cvs315.

Making Cardiovascular Progenitor Cells from Induced Pluripotent Stem Cells

In fetal heart, stem cells known as cardiovascular progenitor cell (CPC) differentiates into smooth muscle cells for blood vessels, blood vessel wall cells, and heart muscle cells. Making CPCs from stem cells has proven to be rather difficult because CPCs do not express any known surface molecules that distinguishes them from other cell types. Therefore, if you want to differentiate pluripotent stem cells into CPCs, determining that you have made CPCs is very difficult.

This problem has been addressed by an international research team led by a team from Stuttgart, Germany who have discovered cell surface molecules that allow the identification and isolation of CPCs. With this knowledge, it will be possible to derive CPCs from induced pluripotent stem cells, which can be implanted into damaged hearts, differentiate into heart-specific cell types and integrate into the heart.

Heart attacks are the most frequent cause of death in the developed world. The cause of a heart attack is usually a clogged coronary vessel, which prevents sufficient blood flow through the heart and kills off heart tissue as a result of ischemia. There are some 17 million people who die from cardiovascular disease each.

Heart muscle cells (cardiomyocytes) do not have the ability to regenerate sufficiently after a heart attack. A heart attack causes a huge loss of cells and further impairs blood supply through the heart. This causes the heart to deteriorate further. To fix the heart, new heart muscle cells are required to replace to dead ones.

This now seems to be a distinct possibility. A research team led by Dr. Katja Schenke-Layland from the Frauhofer Institute for Interfacial engineering and Biotechnology IGD in Stuttgart, in collaboration with Dr. Ali Nasar from the University of California and Dr. Robb MacLean from the University of Washington in Seattle have used cultured CPCs to make heart muscle cells.

To identify CPCs, two proteins of the surfaces of CPCs were identified; a receptor called Flt1 and another called Flt4. By exploiting these two surface proteins, scientists can identify and isolate CPCs from a culture of differentiating pluripotent stem cells. To exploit this new finding, these groups, made induced pluripotent stem cells (iPSCs) from a mouse strain that expressed a green fluorescent protein. They then used skin cells from these mice to make iPSCs.

Japanese stem cell researcher Shinya Yamanaka won the Nobel Prize this year for the discovery of iPSCs. To make iPSCs, adult cells are genetically engineered with four different genes and these genes de-differentiate the adult cells to a pluripotent stem cells state.

The iPSCs made from the green fluorescent mice were then differentiated into CPCs. They were able to isolate and identify CPCs by means of capturing all the cells that made Flt1 and Flt4.

According to Schenke-Layland, “Using our newly established cell surface markers, we could detect and isolate the Flt1- and Flt4-positive CPCs in culture. When we cultured the isolated mouse CPCs then in vitro, they actually developed – as well as the embryonic stem cell-derived progenitor cells – into endothelial cells, smooth muscle cells and more interestingly into functional heart muscle cells.”

To determine if these iPSC-derived CPCs could integrate into a living heart, they injected them into the hearts of living mice. 28 days later, the noticed that the injected hearts were loaded with green fluorescent cells that had differentiated into beating heart muscle that were fully integrated into the heart muscle tissue of the heart.

The next step is to determine if these CPCs can help heal a heart after a heart attack. Bone marrow-derived stem cells have been used to help heal the hearts of heart attack patients, and to date, these stem cells are safe, but only seem to help most people just little, even though they seem to help some patients more than others. However, iPSC-derived CPCs could potentially heal the heart to a greater degree.

According to Schenke-Layland, “We are currently focusing on research with human iPS cells. If we can show that cardiovascular progenitor cells can be derived from human iPS cells that have the ability to mature into functional heart muscle, we will have discovered a truly therapeutic solution for heart attack patients.”

See “Characterization and Therapeutic Potential of INduced Pluripotent Stem Cell-Derived Cardiovascular Progenitor Cells;” Ali Nasar et al: PLoS ONE, 2012; 7 (10): e45603 DOI: 10.1371/journal.pone.0045603.

Fat-Based Stem Cells from Liposuction Can Make Blood Vessels

Adipose tissue, otherwise known as fat, contains a stem cell population. This stem cell population consists of mesenchymal stem cells (MSCs). MSCs have the ability to form cartilage, bone, fat, or smooth muscle rather readily, but the efficiency with which different MSC populations forms these cells types differs dramatically.

To that end, MSCs can be used to form blood vessels, since they form endothelial cells (the cell types that line the inside of blood vessels). The beauty of this capacity is that millions of patients with cardiovascular disease need small-diameter vessel grafts for those procedures that require the rerouting of blood around blocked arteries.

Blood vessels grown in the laboratory from fat-derived MSCs can help solve this problem, since they are made from living tissue and not artificial materials. Blood vessels made from artificial materials (for example, Rayon), tend to promote the formation of blood clots.

The lead author of this work is Matthias Nollert, who is associate professor at the University of Oklahoma School of Chemical, Biological and Materials Engineering in Norman, Oklahoma. His commented on this work: “Current small-diameter vessel grafts carry an inherent risk of clotting, being rejected or otherwise failing to function normally. Our engineered blood vessels have good mechanical properties and we believe they will contract normally when exposed to hormones. They also appear to prevent the accumulation of blood platelets – a component in blood that causes arteries to narrow.

For this study, adult stem cells derived from fat were differentiated into smooth muscle cells in the laboratory and they seeded onto a very thin layer of collagen (a protein found in tendons, ligaments, basement membranes, and many other places). Once the stem cells multiplied, they were rolled into tubes that matched the diameter of small blood vessels. Then after growing in culture for three-four weeks, they formed usable blood vessels.

This technique has the potential to become an “off-the-shelf” technique to make replacement vessels for vascular surgery, according the Nollert. Nollert and his group hope to have a working prototype to test in laboratory animals within the next six months.

Cultured Smooth Muscle Cells are Formed from Stem Cells

Laboratory research needs tissue as a model system. Smooth muscle is found in the urogenital system, circulatory system, digestive system, and respiratory systems of the human body. Various diseases affect smooth muscle and being able to work on cultured smooth muscle would greatly advance the ability of medical researchers to find treatments for smooth muscle disorders.

To address this need, Cambridge University scientists have devised a protocol for generating different types of vascular smooth muscle cells (SMCs) using cells from patients’ skin. This work could lead to new treatments and better screening for cardiovascular disease.

The Cambridge group used embryonic stem cells and reprogrammed skin cells. Skin cells were turned into induced pluripotent skin cells (iPSCs), which were then differentiated into SMCs. They found that they could create all the major vascular smooth muscle cells in high purity using iPSCs. This technique can also be scaled up to produce clinical-grade SMCs.

The scientists created three subtypes of SMCs from these different types of stem cells. They also showed that various SMC subtypes responded differently when exposed to substances that cause vascular diseases. They concluded that differences in the developmental origin play a role in the susceptibility of SMCs to various diseases. Furthermore, the developmental origin of specific SMCs might part some role in determining where and when common vascular diseases such as aortic aneurysms or atherosclerosis originate.

Alan Colman MD, Principle Investigator of the Institute of Medical Biology at Cambridge University, said: “This is a major advance in vascular disease modeling using patient-derived stem cells. The development of methods to make multiple, distinct smooth muscle subtypes provides tools for scientists to model and understand a greater range of vascular diseases in a culture dish than was previously available.”