Stem Cell-Based Skin Graft for Severe Burns


Severe wounds are typically treated with full thickness skin grafts. Some new work by researchers from Michigan Tech and the First Affiliated Hospital of Sun Yat Sen University in Guangzhou, China might provide a way to use a patient’s own stem cells to make split thickness skin grafts (STSG). If this technique pans out, it would eliminate the needs for donors and could work well for large or complicated injury sites.

This work made new engineered tissues were able to capitalize on the body’s natural healing power. Dr. Feng Zhao at Michigan Tech and her Chinese colleagues used specially engineered skin that was “prevascularized, which is to say that Zhao and other designed it so that it could grow its own veins, capillaries and lymphatic channels.

This innovation is a very important one because on of the main reasons grafted tissues or implanted fabricated tissues fail to integrate into the recipient’s body is that the grafted tissue lacks proper vascular support. This leads to a condition called graft ischemia. Therefore, getting the skin to form its own vasculature is vital for the success of STSG.

STSG is a rather versatile procedure that can be used under unfavorable conditions, as in the case of patients who have a wound that has been infected, or in cases where the graft site possess less vasculature, where the chances of a full thickness skin graft successfully integrating would be rather low. Unfortunately, STSGs are more fragile than full thickness skin grafts and can contract significantly during the healing process.

In order to solve the problem of graft contraction and poor vascularization, Zhao and others grew sheets of human mesenchymal stem cells (MSCs) and mixed in with those MSCs, human umbilical cord vascular endothelial cells or HUVECs. HUVECs readily form blood vessels when induced, and growing mesenchymal stem cells tend to synthesize the right cocktail of factors to induce HUVECs to form blood vessels. Therefore this type of skin is truly poised to form its own vasculature and is rightly designated as “prevascularized” tissue.

Zhao and others tested their MSC/HUVEC sheets on the tails of mice that had lost some of their skin because of burns. The prevascularized MSC/HUVEC sheets significantly outperformed MSC-only sheets when it came to repairing the skin of these laboratory mice.

When implanted, the MSC/HUVEC sheets produced less contracted and puckered skin, lower amounts of inflammation, a thinner outer skin (epidermal) thickness along with more robust blood microcirculation in the skin tissue. And if that wasn’t enough, the MSC/HUVEC sheets also preserved skin-specific features like hair follicles and oil glands.

The success of the mixed MSC/HUVEC cell sheets was almost certainly due to the elevated levels of growth factors and small, signaling proteins called cytokines in the prevascularized stem cell sheets that stimulated significant healing in surrounding tissue. The greatest challenge regarding this method is that both STSG and the stem cell sheets are fragile and difficult to harvest.

An important next step in this research is to improve the mechanical properties of the cell sheets and devise new techniques to harvest these cells more easily.

According to Dr. Zhao: “The engineered stem cell sheet will overcome the limitation of current treatments for extensive and severe wounds, such as for acute burn injuries, and significantly improve the quality of life for patients suffering from burns.”

This paper can be found here: Lei Chen et al., “Pre-vascularization Enhances Therapeutic Effects of Human Mesenchymal Stem Cell Sheets in Full Thickness Skin Wound Re-pair,” Theranostics, October 2016 DOI: 10.7150/ thno.17031.

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VM202 is a Safe, Beneficial Treatment for Limb Ischemia


The Korean biotechnology company ViroMed Co., Ltd. has announced the publication of a Phase 2 study that evaluated their VM202 product in patients with critical limb ischemia. This study involved 52 patients in the United States and showed that VM202 is not only safe, but also produced significant clinical benefits.

VM202 is a plasmid (small circle of DNA) that encodes the human hepatic growth factor (HGF) gene. When injected into muscles, VM202 is readily taken up by nearby cells that then quickly synthesize the two isoforms of HGF. Heightened HGF concentrations can treat ischemic cardiovascular diseases by inducing the formation of new blood vessels (angiogenesis). These new collateral vessels increase blood flow and tissue perfusion in the sick tissue, which effectively treats any tissue ischemia.

VM202

Severe obstruction of the arteries that feed the extremities (hands, feet and legs) is the cause of critical limb ischemia (CLI). The term “ischemia” refers to the starvation of a tissue for oxygen. The lack of sufficient blood flow to an organ or tissue can cause severe pain and even skin ulcers, sores, or gangrene. CLI-induced pain can awaken the patient during the night, and, therefore, is called “rest pain.” Rest pains often occur in the leg and is usually temporarily relieved by dangling the leg over the bed or getting up and walking.

CLI does not improve on its own. It is a severe condition that requires immediate by a vascular surgeon or vascular specialist.

Look at the right side of these angiograms and you will see that a vessel is obstructed and blood is not flowing through it. This is an example of Critical Limb Ischemia.
Look at the right side of these angiograms and you will see that a vessel is obstructed and blood is not flowing through it. This is an example of Critical Limb Ischemia.

In this Phase 2 study, patients were divided into three groups, one of which received a placebo treatment, the second of which received a low-dose treatment VM202, and a third group that received a high-dose of VM202.

Both patient groups that received VM202 showed improvement compared to the placebo group, but patients in the higher-dose group showed significantly better ulcer healing and higher tissue oxygen levels than the placebo group. For example, 62 percent of the ulcers healed in patients treated with high-dose VM202 compared to only 11 percent of ulcers in patients who were treated with the placebo. Also, 71 percent of patients who received the high-dose VM202 showed improved oxygen concentrations in their tissues, compared to only 33 percent of patients who were treated with the placebo.

Emerson C. Perin, Director of the Stem Cell Center at the Texas Heart Institute and the principal investigator of this Phase 2 study, said: “These positive results are exciting, and VM202 shows great promise for treating patients with this debilitating disease who often have limited therapeutic options. We are looking forward to conducting a phase III trial to better understand the potential of this novel approach, especially in treating non-healing ulcers, which is a serious symptom that often leads to amputation because of the lack of medical therapies available.”

ViroMed has already been granted an IND or Investigational New Drug approval by the USFDA to initiate a Phase 3 study in diabetic patients who suffer from non-chronic ischemic foot ulcers. This study will enroll 300 subjects who will be divided into a VM202 group and a placebo group. The treatment regiment will mimic that of this smaller Phase 2 study and will only follow patients for seven months. This time, ViroMed is interested in determining if VM202 helps wound closure, which will constitute the primary efficacy endpoint on this new study.

Godspeed ViroMed!!

First Clinical Trial for Genetically Engineered Stem Cell Treatment for Pulmonary Arterial Hypertension


A Canadian research team has published the results of the world’s first clinical trial of a genetically enhanced stem cell therapy for pulmonary arterial hypertension (PAH).

PAH is a rare and deadly disease that mainly affects young women, and is characterized by very high blood pressure in those arteries that supply blood to the lungs. Some cases of PAH are caused by mutations in the BMPR2 gene, but in many cases the cause remains unknown. Currently, PAH patients are treated with combination of various drug and oxygen. Drug treatments include blood vessel dilators, such as epoprosternol (Flolan or the inhaled form known as iloprost or Ventavis), endothelin receptor antagonists, such as bosentan (Tracleer) or ambrisentan (Letaris), sildenafil (Viagra) or tadalafil (Cialis), high doses of calcium channel blockers, anticoagulants, and diuretics. Such treatments can improve symptoms and exercise capacity (at best), but they cannot repair the blood vessel damage to the lungs or cure the disease.

This new study, entitled “Endothelial NO-Synthase Gene-Enhanced Progenitor Cell Therapy for Pulmonary Arterial Hypertension: the PHACeT Trial“ was published in the journal Circulation Research, and was coauthored lead investigator Duncan J. Stewart of the Ottawa Hospital Research Institute, and his collaborators.

The paper describes PAH as a progressive and eventually lethal disease that is characterized by eventual loss of functional lung microvasculature. This paper also argues that cell-based therapies offer the possibility of repairing and regenerating the lung microcirculation. The paper also reports that stem-cell therapy has shown promise in a pre-clinical evaluation that utilized experimental models of PAH.

This trial was a phase 1, dose-escalating clinical study whose goal was to test the tolerability, feasibility, and side-effects of a genetically-enhanced stem cell therapy to repair and regenerate lung blood vessels in PAH patients. Seven PAH patients who volunteered for this study underwent a blood cell selection process known as apheresis in order to harvest a certain population of white blood cells from their blood. These white blood cells were grown in the laboratory under special conditions that specifically selected for stem-like cells called endothelial progenitor cells (EPCs). These EPCs were genetically engineered to produce greater amounts of nitric oxide synthase, which makes the signaling molecule, nitric oxide (NO), a natural substance that widens blood vessels and is essential for efficient vascular repair and regeneration. These genetically enhanced cells were then injected directly into the lung circulation of the patient from whom there were originally harvested.

Of these seven patients, five were female and two were male, and all seven patients received treatment from December 2006 to March 2010. Continued observation and follow-up exams of these patients showed that the cell infusion procedure was well tolerated, and, on the whole, these patients showed a trend towards improvement in total pulmonary resistance (TPR) over the three-day delivery period. However, there was one serious adverse event (death) that occurred immediately after discharge in a patient who had severe, end-stage disease.

These investigators concluded that delivery of EPCs overexpressing eNOS was tolerated in PAH patients, and also produced evidence of short-term improvements, associated with long-term benefits in functional and quality-of-life assessments. However, they caution that future studies will be needed in order to further establish the efficacy of this therapy.

It must be noted that this study was not designed to rigorously assess the benefits the stem cell therapy versus a placebo. However, this research group observed improved blood flow in the lungs of patients during days following the therapy, and enhanced ability to exercise and better quality of life for up to six months after the therapy. Once again, I must provide the caveat that since this was not a double-blinded, placebo-controlled study, it is no possible to determine for sure if these observed effects were due to the cells or to psychological effects.

The therapy was generally well-tolerated, but one patient who had very severe and disease and signs of poor prognosis died one day after treatment. As unfortunate as this is, it is an expected outcome, given how sick the patient was and given their declining condition prior to treatment.

“Pulmonary arterial hypertension is a deadly and incurable disease that often strikes people in the prime of their life,” says the Circulation Research paper’s senior author Dr. Duncan Stewart, a practicing cardiologist and Executive Vice-President of Research at The Ottawa Hospital, and a professor of medicine at the University of Ottawa. “We desperately need new therapies for this disease, and regenerative medicine approaches have shown great promise in laboratory models and in clinical trials for other conditions.”

“This trial shows that genetically-enhanced stem cell therapy is a promising treatment approach for pulmonary arterial hypertension,” observes Dr. Stewart. “Although this is an important start, we will need to do larger studies to establish whether this therapy can produce important and durable benefits for people suffering from this challenging disease.”

Dr. Stewart is also the lead researcher of the first clinical trial in the world of a genetically-enhanced stem cell therapy for heart attack.

Artifical Blood Vessels Made From Thermoplastic Polyurethane Polymers


Wherever we find some of the worse medical events – heart attacks, strokes, pulmonary embolisms, we find blocked blood vessels. Obstructed blood vessels are a lurking time bomb in our bodies and they usually have to be replaced. Blood vessel replacement requires cutting another blood vessel from another part of the body or the implantation of artificial vascular prostheses.

A new option might emerge in the future, however. Vienna University of Technology, in collaboration with the Vienna Medical University developed artificial blood vessels that were fabricated from specialized elastomer material that have excellent mechanical properties. After implantation, these artificial blood vessels are dissolved and replaced by the body’s own blood vessels. At the end of the healing process, natural, fully functional blood vessels are once again in place. The technique works quite well in tissue cultures systems, but now it has been shown to successfully regenerate blood vessels in laboratory animals, specifically rats.

Atherosclerotic vascular disorders, in which blood vessels are obstructed by cholesterol-filled plaques, are one of the most common causes of death in industrialized countries. Typically, patients are treated with a bypass operation, and for such procedures, blood vessels are extirpated from another part of the patient’s body and used to replace the damaged vessel. This creates a new wound and a new area of the body with less than optimal blood supply that must heal. Also, the transplanted vessel rarely has the properties necessary to thrive in its new location.

This new strategy to replace diseased blood vessels is the result of a fruitful collaboration between Vienna University of Technology (or TU Wien, which is short for Technische Universität Wien) and the Medical University of Vienna. Hopefully the success of this research will cause artificially manufactured vessels to be used more frequently in future.

To make an artificial blood vessel, the most important thing is to start with the right material. The material must be compatible with body tissue, and pliable enough to be formed into a small diameter tube that is not easily blocked by blood clots.

Extensive work at TU Wien has resulted in the development of new polymers. “These are so-called thermoplastic polyurethanes,” explains Robert Liska from the Institute of Applied Synthetic Chemistry of TU Wien.  “By selecting very specific molecular building blocks we have succeeded in synthesizing a polymer with the desired properties.”

In order to generate artificial blood vessels from their thermoplastic polyurethanes, TU Wien materials scientists spun polymer solutions in an electrical field. This allowed them to form very fine threads and that could be wound into a spool. “The wall of these artificial blood vessels is very similar to that of natural ones,” says Heinz Schima of the Medical University of Vienna. The thermoplastic polyurethanes form a polymer fabric that is slightly porous and allows a small amount of blood to leak through it. This also enriches the blood vessel wall with growth factors, which encourages the migration of endothelial progenitor cells. Martina Marchetti-Deschmann at TU Wien studied the interaction between the thermoplastic polyurethane material and blood by using spatially resolved mass spectrometry.

This new technology has already proven to successfully form functional blood vessels in rats. “The rats’ blood vessels were examined six months after insertion of the vascular prostheses,” says Helga Bergmeister of MedUni Vienna. “We did not find any aneurysms, thromboses or inflammation. Endogenous cells had colonized the vascular prostheses and turned the artificial constructs into natural body tissue.” In fact, the body’s own blood vessel-forming tissues re-grew significantly faster than expected, which shortened the degradation period of the plastic tubes and their replacement with the body’s own endothelial cells. TU Wein and the Medical University of Vienna are making further adaptations to the material.

A few more preclinical trials are necessary before the artificial blood vessels can be used in human clinical trials. However, based on the results so far, the research team is very confident that the new method will prove itself for use in humans in a few years’ time.

This project was recently awarded PRIZE prototype funding from Austria Wirtschaftsservice (AWS).

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.

Mesenchymal Stem Cells from Neonatal Thymus Helps Make New Blood Vessels


The thymus is an organ that sits over the top of the heart and it plays a pivotal role in the development of T-lymphocytes. The thymus is a very durable organ that can readily regenerate if it is injured. This regenerative ability is largely due to it high level of vascularization (lots of blood vessels). This vascularization is due to a robust population of resident mesenchymal stem cells that supports blood vessel formation in the damaged thymus. The process of blood vessel formation is called “angiogenesis.” The angiogenic potential of these thymus-based mesenchymal stem cells might hold excellent potential for regenerative therapies.

Thymus_lg

As it turns out, neonatal surgeries tend to generate thymus tissue that is usually thrown out as medical waste. Ming-Sing Si from Mott’s Children Hospital in Ann Arbor, Michigan and colleagues isolated mesenchymal stem cells from these surgically-derived neonatal thymuses and tested their ability to stimulate blood vessels in an experimental setting.

Discarded thymus tissue was obtained from the University of Michigan, and this tissue was minced, degraded with enzymes, and cultured. The mesenchymal stem cells (MSCs) moved from the thymus tissue onto the culture dishes. These thymus-based MSCs grew like gangbusters in culture and could be passaged over 30 times.

Discarded human neonatal thymus tissue is a source of mesenchymal stromal cells (MSCs). (A): Discarded human neonatal thymus tissue during pediatric cardiac surgery. (B): Minced thymus tissue prior to plating. (C): Cells migrating from thymus tissue fragments during explant culture at 10 days. (D): Clonogenicity of thymus MSCs at 2 weeks (representative of 7 donors). (E): Colony-forming efficiency of thymus MSCs. (F): Averaged cumulative population doubling of thymus MSCs (n = 4) over 9 weeks of culture. Abbreviation: CFU-F, fibroblastic colony-forming unit.
Discarded human neonatal thymus tissue is a source of mesenchymal stromal cells (MSCs). (A): Discarded human neonatal thymus tissue during pediatric cardiac surgery. (B): Minced thymus tissue prior to plating. (C): Cells migrating from thymus tissue fragments during explant culture at 10 days. (D): Clonogenicity of thymus MSCs at 2 weeks (representative of 7 donors). (E): Colony-forming efficiency of thymus MSCs. (F): Averaged cumulative population doubling of thymus MSCs (n = 4) over 9 weeks of culture. Abbreviation: CFU-F, fibroblastic colony-forming unit.

When these thymus-based MSCs were combined with human umbilical vein endothelial cells, within one day, the cells formed an extensive network of blood vessels.

Thymus mesenchymal stromal cells (MSCs) cooperate with human umbilical vein endothelial cells (HUVECs) to form a network in a two-dimensional angiogenesis assay. (A): Monolayer appearance of HUVECs after 48 hours of culture on fibrin hydrogel. (B): Thymus MSCs clustered together after 24 hours of culture on fibrin hydrogel. (C): Combining HUVECs with thymus MSCs (2:1) resulted in the appearance of interconnected tubules at 24 hours. Scale bars = 100 μm. Results are representative of two independent experiments.
Thymus mesenchymal stromal cells (MSCs) cooperate with human umbilical vein endothelial cells (HUVECs) to form a network in a two-dimensional angiogenesis assay. (A): Monolayer appearance of HUVECs after 48 hours of culture on fibrin hydrogel. (B): Thymus MSCs clustered together after 24 hours of culture on fibrin hydrogel. (C): Combining HUVECs with thymus MSCs (2:1) resulted in the appearance of interconnected tubules at 24 hours. Scale bars = 100 μm. Results are representative of two independent experiments.

Gene expression studies showed that culturing thymus MSCs with human umbilical vein endothelial cells (HUVECs) caused the HUVECs to express a variety of blood vessel-specific genes.  These thymus-based MSCs were also able to induce blood vessels if the cells were wadded up into a ball (spheroids).

To top it all off, Si and others implanted thymus-based MSCs underneath the skin of nude mice.  They used hydrogels with no cells, hydrogels plus HUVECs, hydrogels plus thymus-based MSCs, and hydrogels with thymus-based MSCs plus HUVECs.  The control implants and the HUVEC implants showed no blood vessels.  HUVECs make very good blood vessels, but they have to be directed to do so.  Both the thymus-based MSCs and the MSCs plus HUVECs showed extensive integration into the host tissue with lots of blood vessels.

Thymus mesenchymal stromal cells (MSCs) incite angiogenesis in vivo. Fibrin constructs without spheroids (control) or with 500 spheroids with 600 human umbilical vein endothelial cells (HUVECs) per spheroid, 200 thymus MSCs per spheroid, or 600 HUVECs plus 200 thymus MSCs per spheroid were generated (n = 3 per group) and were implanted subcutaneously for 14 days in NOD-SCID mice. Explanted constructs were photographed (edges traced in A–D) and processed for histology. (A): Controls did not manifest local reaction. (B): HUVEC constructs appeared avascular. (C): Thymus MSC constructs were integrated and caused increased adjacent vascularization. (D): HUVEC plus thymus MSC constructs were integrated and surrounded by a host vascular response and appeared to have vessels within. (E–H): Construct hematoxylin and eosin staining. Scale bars = 50 μm. (E): Avascular tissue invasion of control construct. Scale bar = 100 μm. (F): HUVEC construct with adjacent cellularity and vascularity between panniculus carnosus muscle layer (∗) and construct. “Ghost” (†) of the prior locations of spheroid and necrotic spheroid (‡) were present in internal regions of all constructs with spheroids. (G): Thymus MSC construct with increased adjacent cellularity and vascularity. (H): HUVEC plus thymus MSC construct with increased vascularization within the construct. (I): Manual measurement of vessel density demonstrates significant differences by two-way analysis of variance. Control and HUVEC constructs had minimal adjacent vascularization. Thymus MSC constructs promoted the greatest adjacent response, whereas HUVEC plus thymus MSC constructs contained the greatest vessel density within the construct. (J, K): Immunohistochemical staining with human-specific CD31 monoclonal antibody revealed that only constructs with HUVEC plus thymus MSCs contained CD31-positive luminal structures with blood cells. Scale bar = 20 μm. Abbreviations: C, controls; H, human umbilical vein endothelial cell constructs; T, thymus mesenchymal stromal cell construct.
Thymus mesenchymal stromal cells (MSCs) incite angiogenesis in vivo. Fibrin constructs without spheroids (control) or with 500 spheroids with 600 human umbilical vein endothelial cells (HUVECs) per spheroid, 200 thymus MSCs per spheroid, or 600 HUVECs plus 200 thymus MSCs per spheroid were generated (n = 3 per group) and were implanted subcutaneously for 14 days in NOD-SCID mice. Explanted constructs were photographed (edges traced in A–D) and processed for histology. (A): Controls did not manifest local reaction. (B): HUVEC constructs appeared avascular. (C): Thymus MSC constructs were integrated and caused increased adjacent vascularization. (D): HUVEC plus thymus MSC constructs were integrated and surrounded by a host vascular response and appeared to have vessels within. (E–H): Construct hematoxylin and eosin staining. Scale bars = 50 μm. (E): Avascular tissue invasion of control construct. Scale bar = 100 μm. (F): HUVEC construct with adjacent cellularity and vascularity between panniculus carnosus muscle layer (∗) and construct. “Ghost” (†) of the prior locations of spheroid and necrotic spheroid (‡) were present in internal regions of all constructs with spheroids. (G): Thymus MSC construct with increased adjacent cellularity and vascularity. (H): HUVEC plus thymus MSC construct with increased vascularization within the construct. (I): Manual measurement of vessel density demonstrates significant differences by two-way analysis of variance. Control and HUVEC constructs had minimal adjacent vascularization. Thymus MSC constructs promoted the greatest adjacent response, whereas HUVEC plus thymus MSC constructs contained the greatest vessel density within the construct. (J, K): Immunohistochemical staining with human-specific CD31 monoclonal antibody revealed that only constructs with HUVEC plus thymus MSCs contained CD31-positive luminal structures with blood cells. Scale bar = 20 μm. Abbreviations: C, controls; H, human umbilical vein endothelial cell constructs; T, thymus mesenchymal stromal cell construct.

These MSCs show low expression of human leukocyte antigen class I, which, translated, means that these cells are unlikely to be recognized by the patient’s immune system.  Therefore, these cells could be donated to patients whose resident MSCs are of poor quality or do not have enough of their own MSCs for therapeutic processes.

This paper shows that discarded neonatal thymus contains large numbers of resident MSCs that can be isolated and cultured by a standard explant method.  These MSCs have all the characteristics of traditional MSCs, but have more robust growth characteristics in culture.  These thymus MSCs also possess outstanding proangiogenesis qualities that should be further tested and considered as promoters of tissue and organ regeneration in tissue engineering strategies.

Stem Cells from Fat Improve Blood Vessel Responses after Injury


When tissues are injured, the blood vessels that feed them are often shocked and damaged as well. “Vasoactivity” refers the ability of blood vessels to dilate or constrict. When tissues are harmed, blood vessels tend to shrink in order to squelch blood loss at the site of damage. This same response, however, and deprive the damaged tissues of much-needed oxygen and lead to “ischemia,” which is the insufficient supply of blood and oxygen to an organ.

James B. Hoying and his colleagues at the University of Louisville in Kentucky used the “stromal vascular fraction” or SVF from fat in order to treat damaged blood vessels to determine if they could mitigate the decrease in vasoactivity as a result of injury.

The SVF refers to the stem fraction from fat after the fat has been minced, digested with enzymes, and centrifuged (it’s more complicated than that, but this is a short summary). The cells that remain include mesenchymal stromal cells, growth factors, immune cells, pre-fat cells and fat cells, blood-cell-making stem cells, and blood vessel-making cells (endothelial cells). The SVF, therefore, contains a cocktail of cell types and growth factors that are available for regenerative medicine.

Hoying and his team discovered that when fluorescent SVF cells were injected into a laboratory mouse, they cells distributed to a variety of tissues. Further and more detailed examinations showed that these cells were finding their ways into organs and tissues because they traveled through the circulatory system and could be found in the walls of blood vessels.

Next, the composition of the SVF was examined. About 25% of the cells in the SVF were endothelial cells, 22% were various types of blood cells, 20% were “CD11b” cells, which means that these cells had a protein called CD11b on their cell surfaces. That protein was formerly canned “Mac-1” and is was normally found on the surfaces of phagocytic cells called macrophages. Therefore, this CD11b faction could very well be macrophages, but other cell types have this protein on their surfaces as well.

Macrophages

Next, Hoying and others injected these SVF-derived cells into the large leg vein (saphenous) of the leg. Such injections consistently caused these vessels to relax and dilate. Secondly, the SVF-derived cells caused the vessels to relax in a CD11b-dependent manner. In other words, the more CD11b cells there were in the SVF preparation, the greater the amount of vasoactivity they induced. If fractions were depleted of their CD11b, they could not induce vasoactivity.

When Hoying and others examined the SVF-treated vessels, they saw CD11b+ cells lining the inner layer of the vessels. Thus these cells were getting right up against the inside of the vessel and signaling to the underlying smooth muscle to relax.

Finally, Hoying and others clamped the saphenous veins of laboratory mice. Such clamping will induce tissue ischemia and inflammation in the vessels. Can SVF cells calm the inflammation and make the vessels more vasoactive? The answer is an unqualified yes.  See below.  The veins from SVF-treated animals show signficantly greater dilation than those from untreated or CD11b-depleted SVF-treated animals.

SVF cells relax vasomotor tone in inflamed saphenous arteries. (A): Schematic of the experimental plan involving the cell treatment of locally inflamed (cuffed) saphenous arteries of mice injected with syngeneic adipose SVF cells constitutively expressing luciferase and GFP reporter transgenes or SVF cells depleted of CD11b+ cells. Also shown is a gross view and a histological cross-section of a cuffed saphenous artery. (B): Hematoxylin and eosin-stained histological cross-sections of normal (noncuffed) and cuffed mouse saphenous arteries untreated or injected with SVF cells or SVF-11bΔ cells. Rightmost panels: Higher magnification images of the adjacent images. Scale bars = 25 μm in the left and right columns and 100 μm in the middle column. (C): Lumen diameters of untreated (n = 9) and cell-injected cuffed saphenous arteries measured from histological sections. Cell treatments included complete SVF cell isolates (C + SVF, n = 7) or SVF isolates depleted of CD11b+ cells (C + SVF-11bΔ, n = 7). Data are shown as the mean ± SEM; ∗, p < .05, determined by one-way analysis of variance. (D): Visualization of luciferase-positive SVF cells within histological paraffin sections of cuffed saphenous arteries from untreated, SVF-injected, and SVF-11bΔ-injected mice via immunostaining for luciferase. Brown stain indicates positive luciferase immune-staining and the presence of SVF cells. Tissues were harvested 1 week after cell delivery. Scale bars = 100 μm. Abbreviations: C, cuff; GFP, green fluorescent protein; PE, polyethylene; SVF, stromal vascular fraction; SVF-11bΔ, CD11b+ cell-depleted adipose SVF cells.
SVF cells relax vasomotor tone in inflamed saphenous arteries. (A): Schematic of the experimental plan involving the cell treatment of locally inflamed (cuffed) saphenous arteries of mice injected with syngeneic adipose SVF cells constitutively expressing luciferase and GFP reporter transgenes or SVF cells depleted of CD11b+ cells. Also shown is a gross view and a histological cross-section of a cuffed saphenous artery. (B): Hematoxylin and eosin-stained histological cross-sections of normal (noncuffed) and cuffed mouse saphenous arteries untreated or injected with SVF cells or SVF-11bΔ cells. Rightmost panels: Higher magnification images of the adjacent images. Scale bars = 25 μm in the left and right columns and 100 μm in the middle column. (C): Lumen diameters of untreated (n = 9) and cell-injected cuffed saphenous arteries measured from histological sections. Cell treatments included complete SVF cell isolates (C + SVF, n = 7) or SVF isolates depleted of CD11b+ cells (C + SVF-11bΔ, n = 7). Data are shown as the mean ± SEM; ∗, p < .05, determined by one-way analysis of variance. (D): Visualization of luciferase-positive SVF cells within histological paraffin sections of cuffed saphenous arteries from untreated, SVF-injected, and SVF-11bΔ-injected mice via immunostaining for luciferase. Brown stain indicates positive luciferase immune-staining and the presence of SVF cells. Tissues were harvested 1 week after cell delivery. Scale bars = 100 μm. Abbreviations: C, cuff; GFP, green fluorescent protein; PE, polyethylene; SVF, stromal vascular fraction; SVF-11bΔ, CD11b+ cell-depleted adipose SVF cells.

This an interesting and exciting finding not only because of the ability of these fat-based cells to maintain vasoactivity even under pro-inflammatory conditions, but because it is the macrophage cell population that is doing the work.  In most stem preparations, macrophages are excluded.  This paper shows that macrophages have greater therapeutic capabilities than previously thought, and should also be tested for sanative properties.