Stem Cell Therapy Might Improve Brain Function of Traumatic Brains Injury Patients


Accidents happen and sometimes really bad accidents happen; especially if they injure your head.  Traumatic brain injuries or TBIs can result from automobile accidents, explosions or other events that result from severe blows to the head.  TBIs  an adversely affect a patient and his/her family for long periods of time.  TBI patients can experience cognitive deficits that prevent them from thinking or speaking straight, and sensory deficits that prevent them from seeing, hearing or smelling properly.  Psychological problems can also result.  Essentially, TBIs represent a major challenge for modern medicine.

According to data from the Centers for Disease Control (CDC), 1.7 million Americans suffer from TBIs each year (of varying severity).  Of these, 275,000 are hospitalized for their injuries and approximately 52,000 of these patients die from their injuries.  In fact, TBIs contribute to one-third of all injury-related deaths in the United States each year.  More than 6.5 million patients are burdened by the deleterious effects of TBIs, and this leads to an economic burden of approximately $60 billion each year.

Currently, treatments for TBI are few and far between.  Neurosurgeons can use surgery to repair damaged blood vessels and tissues, and diminish swelling in the brain.  Beyond these rather invasive techniques, the options for clinicians are poor.

A new study by Charles S. Cox, professor of Pediatric Surgery and co-director of the Memorial Hermann Red Duke Trauma Institute, and his colleagues suggest that stem cell treatments might benefit TBI patients.  The results of this study were published in the journal Stem Cells.

This study enrolled 25 TBI patients.  Five of them received no treatment and served as controls, but the remaining 20 received gradually increasing dosages of their own bone marrow stem cells.  The harvesting, processing and infusion of the bone marrow cells occurred within 48 hours of injury.  Functional and cognitive results were measured with standard tests and brain imaging with magnetic resonance imaging and diffusion tensor imaging.

This work is an extension of extensive preclinical work done by Cox and his coworkers in laboratory animals and a phase I study that established that such stem cell transplantation are safe for human patients.  The implanted stem cells seem to quell brain inflammation and lessen the damage to the brain by the TBI.

Despite the fact that those TBI patients who received the stem cell treatments had greater degrees of brain damage, the treatment group showed better structural preservation of the brain and better functional outcomes than the control group.  Of particular interest was the decrease in indicators of inflammation as a result of the bone marrow cell-based infusions.

Cox said of this trial, “The data derived from this trial moves beyond just testing safety of this approach.”  He continued:  “We now have a hint of a treatment effect that mirrors our pre-clinical work, and were are now pursuing this approach in a phase IIb clinical trial sponsored by the Joint Warfighter Program within the US Army Medical Research Acquisition Activity, as well as our ongoing phase IIb pediatric severe TBI clinical trial; both using the same autonomous cell therapy.”

This an exciting study, but it is a small study.  While the safety of this procedure has been established, the precise dosage and long-term benefits will require further examination.  However it is a fine start to what may become the flowering of new strategies to treat TBI patients.

Targeting EGFL6 Protein Halts Growth and Spread of Ovarian Cancer


Dr. Ronald J. Buckanovich, professor of hematology/oncology and gynecologic oncology at the University of Michigan Medical School, and his colleagues have identified a protein that help ovarian cancer cells multiply and spread to other organs.  When he and his coworkers inhibited this protein with an antibody they were able to stop the spread of ovarian cancer cold.

The EGFL6 or epidermal growth factor like 6 precursor protein, which is also known as MAEG, maps to human Xp22 chromosome.  The EGFL6 protein is expressed primarily in fetal tissues and during early development (see Yeung G., et al., (1999) Genomics 62, 304307; and Buchner G., et al., (2000) Genomics 65, 1623).  The expression of MAEG has also been detected in several tissues, including the dermis of the trunk, hair follicles, and the mesenchyme of the cranio-facial region (see Buchner G., and others, (2000) Mech. Dev. 98, 179182).  EGFL6 protein has been proposed as a possible biomarker in ovarian cancer (Buckanovich R. J., and others, (2007) J. Clin. Oncol. 25, 852861).

In this paper, which appeared in Cancer Research, Buckanovich and others amplified the expression of EGFL6 in ovarian cancer cells.  Increased EGFL6 expression stimulated cancer growth some two-three times.  This effect was observed in cultured ovarian cancer cells and in a mouse model of ovarian cancer.  Conversely, elimination of EGFL6 greatly reduced ovarian cancer growth, decreasing the rate of growth some four-fold.

EGFL6 specifically acts in cancer stem cells.  To review, in tumors, not all cancer cells are the same.  Inside malignant tumors or even among circulating cancerous cells (as in the case of leukemia) there are usually a variety of different types of cancer cells.  The stem cell theory of cancer proposes that among cancerous cells, a small population among them act as stem cells that reproduce themselves and sustain the cancer.  Cancer stem cells, therefore, are like normal stem cells that renew and sustain our organs and tissues.  Therefore, cancer cells that are not stem cells can certainly adversely affect health, but they cannot sustain the cancer long-term.  Therefore, cancer stem cells fuel the growth and spread of cancers and also are often resistant to chemotherapy and radiation treatments.

Further experiments by Buckanovich and his colleagues showed that EGFL6 cause cancer stem cells to divide asymmetrically so that the one of the daughter cells remains a cancer stem cell while the other daughter cell is a cancer cell that can affect the patient but cannot sustain the cancer. This asymmetric cell division also generates a good deal of diversity among cancer cells.

Buckanovich noted: “What this means is that the stem cell population remains stable.  But the daughter cells, which can have a burst of growth, multiply, and allow the cancer to grow.”.

EGFL6 does more than just promote cancer cell proliferation.  EGFL6 is also a promoter of cancer stem cell migration.  When blood vessels were engineered to express EGFL6, tumor metastasis (spread) was even more robust.

Treatment of tumor-afflicted mice with an antibody that specifically binds to EGFL6 and inactivates it caused a 35% reduction in cancer stem cells and significantly reduced overall tumor growth.  Additionally, the antibody also prevented tumor metastasis.

Buckanovich thinks that targeting EGFL6 might be a potential therapy for women with stage 3 ovarian cancer.  Such a treatment might control the growth and spread of ovarian cancers.

Dr. Buckanovich added: “The bigger implication is for women at high risk of ovarian cancer.  These patients could be treated before cancer develops, potentially blocking cancer from developing or preventing it from spreading.  If cancer did develop, it could be diagnosed at an early stage, which would improve patient outcomes.”.

The next step for Buckanovich and his colleagues is developing an antibody that can properly work in human cancer patients.

Patient-Specific Heart Muscle Cells Before the Baby Is Born


Prenatal ultrasound scans can detect congenital heart defects (CHDs) before birth. Some 1% of all children born per year have some kind of CHD. Most of these children will require some kind of rather invasive, albeit life-saving surgery but an estimated 25% of these children will die before their first birthday. This underscores the need for netter therapies of children with CHDs.

To that end, Shaun Kunisaka from C.S. Mott Children’s Hospital in Ann Arbor, Michigan and his colleagues have used induced pluripotent stem cell (iPSC) technology to make patient-specific heart muscle cells in culture from the baby’s amniotic fluid cells. Because these cells can be generated in less than 16 weeks, and because the amniotic fluid can be harvested at about 20-weeks gestation, this procedure can potentially provide large quantities of heart muscle cells before the baby is born.

In this paper, which was published in Stem Cells Translational Medicine, Kunisaki and others collected 8-10 milliliter samples of amniotic fluid at 20 weeks gestation from two pregnant women who provided written consent for their amniocentesis procedures. The amniotic fluid cells from these small samples were expanded in culture, and between passages 3 and 5, cells were selected for mesenchymal stem cell properties. These amniotic fluid mesenchymal stem cells were then infected with genetically engineered non-integrating Sendai viruses that caused transient expression of the Oct4, Sox2, Klf4, and c-Myc genes in these cells. The transient expression of these four genes drove the cells to dedifferentiate into iPSCs that were then grown and then differentiated into heart muscle cells, using well-worked out protocols that have become rather standard in the field.

Not only were the amniotic fluid mesenchymal stem cells very well reprogrammed into iPSCs, but these iPSCs also could be reliably differentiated into cardiomyocytes (heart muscle cells, that is) that had no detectable signs of the transgenes that were used to reprogram them, and, also, had normal karyotypes. Karyotypes are spreads of a cell’s chromosomes, and the chromosome spreads of these reprogrammed cells were normal.

As to what kinds of heart muscle cells were made, these cells showed the usual types of calcium cycling common to heart muscle cells. These cells also beat faster when they were stimulated with epinephrine-like molecules (isoproterenol in this case). Interestingly, the heart muscle cells were a mixed population of ventricular cells that form the large, lower chambers of the heart, atrial cells, that form the small, upper chambers of the heart, and pacemaker cells that spontaneously form their own signals to beat.

This paper demonstrated that second-trimester human amniotic fluid cells can be reliably reprogrammed into iPSCs that can be reliably differentiated into heart muscle cells that are free of reprogramming factors. This approach does have the potential to produce patient-specific, therapeutic-grade heart muscle cells for treatment before the child is even born.

Some caveats do exist. The use of the Sendai virus means that cells have to be passaged several times to rid them of the viral DNA sequences. Also, to make these clinical-grade cells, all animal produces in their production must be removed. Tremendous advances have been made in this regard to date, but those advancements would have to be applied to this procedure in order to make cells under Good Manufacturing Practices (GMP) standards that are required for clinical-grade materials. Finally, neither of these mothers had children who were diagnosed with a CHD. Deriving heart muscle cells from children diagnosed with a CHD and showing that such cells had the ability to improve the function of the heart of such children is the true test of whether or not this procedure might work in the clinic.

Stem Cell-Derived Smooth Muscle Cells Help Restructure Urethral Sphincter Muscles in Rats


Stress urinary incontinence affects 25%-50% of the female population and is defined as the leakage of the bladder upon exertion. The exertions that can cause the bladder to leak can be as simple as laughing, coughing, sneezing, hiccups, yelling, or even jumping up and down. Stress urinary incontinence costs Americans some $12 billion a year and also causes a good deal of embarrassment and compromises quality of life. Unsurprisingly, stress urinary incontinence also is associated with an increased incidence of anxiety, stress, and depression.

In most cases of stress urinary incontinence, injury to the internal sphincter muscles of the urethra or to the nerves that innervate these muscles (both smooth and voluntary muscles) significantly contribute to the condition. Conservative management of stress urinary incontinence can work at first, but can fail later on. The other option is corrective surgery that reconstructs the urethral sphincter and increases urethral support. However, even though such surgeries can and often do work, recurrence of the incontinence is rather common. Is there a better way?

Yan Wen from Stanford University School of Medicine and colleagues and collaborators from College of Medicine of Case Western Reserve in Cleveland, Ohio, Southern Medical University in Guangzhou, China, and Montana State University have used a novel stem cell-based technique to treat laboratory Rowett nude rats that had a surgically-induced form of stress urinary incontinence. While the results are not overwhelming, they suggest that a stem cell-based approach might be a step in the right direction.

Wen and others used a human embryonic stem cell line called H9 and two different types of induced pluripotent stem cell lines to make, in culture, human smooth muscle progenitor cells (pSMCs). Fortunately, protocols for differentiating pluripotent stem cells into smooth muscle cells is well worked out and rather well understood. These pSMCs were also tagged with a firefly luciferase gene that allowed visualization of the cells after implantation.

Six groups of rats were treated in various ways. The first group had stress urinary incontinence and were only treated with saline solutions. The second group of animals also had stress urinary incontinence and were treated with cultured human pSMCs that were derived from human bladders. The third group of animals also had stress urinary incontinence and were treated with pSMCs made from H9 human embryonic stem cells. The next two groups also had stress urinary incontinence and were treated with two different induced pluripotent stem cell lines; one of which was induced with a retroviral vector and the second of which was made with episomal DNA. Both lines were originally derived from dermal fibroblasts. The final group of rats did not have stress urinary incontinence and were used as a control group.

The cells were introduced into the mice by means of injections into the urethra under anesthesia. Two million cells were introduced in each case, three weeks after the induction of stress urinary incontinence. All animals were examined five weeks after the cells were injected into the animals.

Because the cells were tagged with firefly luciferase, the animals could be given an injection of luciferin, which is the substrate for luciferase. Luciferase catalyzes a reaction with luciferin, and the cells glow. This glow is easily detected by means of a machine called the Xenogen Imaging System. Such experiments showed that the injected cells did not survive terribly well, and by 9 days after the injections, they were usually not detectable. Two rats that had been injected with retrovirally-induced induced pluripotent stem cell-derived pSMCs lasted until 35 days after injection, but these rats were the exception and not the rule.

Did the cells integrate into the urethral sphincter by the signal is too low to be detected using luciferase? The answer to this question was certainly yes, but the amount of integration was nothing to write home about. Small patches of cells showed up in the urethra sphincters that expressed human gene products, and therefore, had to be derived from the injected cells.

In vivo survival of transplanted pSMCs in RNU rats. (A): The RV-iPSC pSMCs were periurethrally injected into the rats and monitored with BLI. (B): At day 12, a small number of the transplanted cells were detected in the proximal rat urethra. The transplanted human cells were determined by positive staining of HuNuclei and smoothelin. (C): Gene expression of human ERV-3 in rat urethras 5 weeks after cell transplantation. Y-axis on left shows the scale for ERV-3 copy numbers from tissue samples. Each human cell contains one copy of ERV-3 transcript; hence, the number of copies is equal to the number of cells. Y-axis on right shows the ERV-3 copy numbers of the standard cell samples. The cell numbers in the standard graph are 0.5 × 103, 1 × 103, 2 × 103, 4 × 103, 8 × 103, and 10 × 103 (red dots). ERV-3 amplifications in all pSMC-treated groups were very low. Abbreviations: BLI, bioluminescent imaging; bSMC, bladder smooth muscle cell; DAPI, 4′,6-diamidino-2-phenylindole; Epi, episomal plasmid; ERV-3, endogenous retrovirus group 3; H&E, hematoxylin and eosin; Hu, human; HuNuclei, human nuclei; iPSC, induced pluripotent stem cell; pSMCs, smooth muscle progenitor cells; RNU, Rowett Nude; RV, retrovirus vector.
In vivo survival of transplanted pSMCs in RNU rats. (A): The RV-iPSC pSMCs were periurethrally injected into the rats and monitored with BLI. (B): At day 12, a small number of the transplanted cells were detected in the proximal rat urethra. The transplanted human cells were determined by positive staining of HuNuclei and smoothelin. (C): Gene expression of human ERV-3 in rat urethras 5 weeks after cell transplantation. Y-axis on left shows the scale for ERV-3 copy numbers from tissue samples. Each human cell contains one copy of ERV-3 transcript; hence, the number of copies is equal to the number of cells. Y-axis on right shows the ERV-3 copy numbers of the standard cell samples. The cell numbers in the standard graph are 0.5 × 103, 1 × 103, 2 × 103, 4 × 103, 8 × 103, and 10 × 103 (red dots). ERV-3 amplifications in all pSMC-treated groups were very low. Abbreviations: BLI, bioluminescent imaging; bSMC, bladder smooth muscle cell; DAPI, 4′,6-diamidino-2-phenylindole; Epi, episomal plasmid; ERV-3, endogenous retrovirus group 3; H&E, hematoxylin and eosin; Hu, human; HuNuclei, human nuclei; iPSC, induced pluripotent stem cell; pSMCs, smooth muscle progenitor cells; RNU, Rowett Nude; RV, retrovirus vector.

The exciting part about these results, however, was that when Wen and others examined the rat urethral sphincters for the presence of things like elastin and other proteins that make for a healthy urethral sphincter, there was a good deal of elastin, but it was not human elastin but rat elastin. Therefore, this elastin synthesis was INDUCED by the implanted cells even though it was not made by the implanted cells. Instead, the implanted cells seemed to signal to the native cells to beef up their own production of sphincter-specific gene products, which made from a better sphincter. This was not the case in animals that received injections of human pSMCs derived from human bladders.

Assessment of elastin fibers in the proximal urethra of the rat. (A): Representative images of cross-section of proximal urethra with Weigert’s Resorcin-Fuchsin’s elastin and van Gieson’s collagen staining. Elastic fiber shown as dark blue, collagen as red pink, and other tissue elements as yellow. Scale bars = 100 µm. (B): Quantification of elastin fibers was assessed using Image-Pro Plus software and expressed as a percentage of the arbitrary ROIs. Each bar represents the mean value ± SEM. Abbreviations: bSMC, human bladder smooth muscle cells; H9-pSMC, surgery plus H9-pSMC injection; Hu-bSMC, surgery plus injection of human bladder smooth muscle cells; pSMCs, smooth muscle progenitor cells; Pure control, no surgery and no treatment; ROIs, regions of interest; Sham saline, surgery plus saline injection.
Assessment of elastin fibers in the proximal urethra of the rat. (A): Representative images of cross-section of proximal urethra with Weigert’s Resorcin-Fuchsin’s elastin and van Gieson’s collagen staining. Elastic fiber shown as dark blue, collagen as red pink, and other tissue elements as yellow. Scale bars = 100 µm. (B): Quantification of elastin fibers was assessed using Image-Pro Plus software and expressed as a percentage of the arbitrary ROIs. Each bar represents the mean value ± SEM. Abbreviations: bSMC, human bladder smooth muscle cells; H9-pSMC, surgery plus H9-pSMC injection; Hu-bSMC, surgery plus injection of human bladder smooth muscle cells; pSMCs, smooth muscle progenitor cells; Pure control, no surgery and no treatment; ROIs, regions of interest; Sham saline, surgery plus saline injection.

Because these mice were sacrificed five weeks after the injections, Wen and others could not assess the urethral function of these animals. Therefore, it is uncertain if the improved tissue architecture of the urethral sphincter properly translated into improved function even though it is reasonable to assume that it would. Having said that, it is possible that the experiments that detected the presence of increased amounts of elastin and collagen in the sphincters of these rats was complicated by the presence of bladder tissue in the preparations. Since bladder tissue was included in all trials of this experiment, it is unlikely that bladder tissue is the sole cause of increase elastin and collagen in the stem cell-treated rats. Secondly, rat regenerative properties may not properly match the regenerative properties in older human patients. Here again, unless such an experiment is attempted in larger animal models and then in human patients, we will never know if this procedure is viable for regenerative treatments in the future.

For now, it is an interesting observation, and perhaps a promising start to might someday become a viable regenerative treatment for human patients.

This paper appeared in Stem Cells Translational Medicine, vol 5, number 12, December 2016, pp. 1719-1729.

Bone Marrow Mesenchymal Stem Cells Spontaneously Make Cartilage After Blockage of VEGF Signaling


Bone marrow-derived mesenchymal stem cells (MSCs) can be induced to make cartilage by incubating the cells with particular growth factors.  Unfortunately, batches of MSCs show respectable variability from patient-to-patient.  Therefore the growth factor-dependent method suffers from poor efficacy, limited reproducibility from batch-to-batch, and the cell types that are induced are not always terribly stable.  Finding a better way to make cartilage would certainly be a welcome addition to regenerative treatments,

Cartilage that coats the ends of bones is known as articulate cartilage, and articular cartilage lacks blood vessels.  Therefore, is it possible that inhibiting blood vessel formation could conveniently push MSCs to differentiate into cartilage-making chondrocytes?

A new paper by Ivan Martin and Andrea Basil from the University Hospital Basel and their colleagues have used this very strategy to induce cartilage formation in MSCs from bone marrow.

Martin and others isolated MSCs from bone marrow aspirates from human donors.  These cultured human MSCs were then genetically engineered with modified viruses to express a receptor for soluble vascular endothelial growth factor (VEGF) that binds this growth factor, but fails to induce any intracellular signals.  Such a receptor that binds the growth factor but does not induce any biological effects is called a “decoy receptor,” and decoy receptors efficiently sequester or vacuum up all the endogenous VEGF.  VEGF is the major blood vessel-inducing growth factor and it is heavily expressed during development, by cancer cells, and during healing.

After expressing the decoy VEGF receptor in these human MSCs, these genetically engineered cells were grown on collagen sponges and then implanted in immunodeficient mice.  If the implanted MSCs were not genetically engineered to express decoy VEGF receptors, they induced for formation of vascularized fibrous tissue.  However, the implantation of genetically engineered MSCs that expressed the decoy VEGF receptor efficiently and reproducibly differentiated into chondrocytes and formed hyaline cartilage. This is significant because headline cartilage is the very type of cartilage found at articular surfaces where the ends of bones come together to form joints.

In vivo chondrogenesis. Histological staining with Safranin-O for glycosaminoglycans and immunohistochemistry for type II collagen of engineered tissue generated by naïve (control) or sFlk-1 MSCs after 4 (A) or 12 (B) weeks in vivo. Fluorescence staining with DAPI (in blue) and a specific anti-human nuclei antibody (in red) of constructs generated by control or sFlk-1 MSCs after 4 (A) or 12 (B) weeks in vivo. Scale bar = 100 µm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; MSC, bone marrow-derived mesenchymal stromal/stem cell.
In vivo chondrogenesis. Histological staining with Safranin-O for glycosaminoglycans and immunohistochemistry for type II collagen of engineered tissue generated by naïve (control) or sFlk-1 MSCs after 4 (A) or 12 (B) weeks in vivo. Fluorescence staining with DAPI (in blue) and a specific anti-human nuclei antibody (in red) of constructs generated by control or sFlk-1 MSCs after 4 (A) or 12 (B) weeks in vivo. Scale bar = 100 µm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; MSC, bone marrow-derived mesenchymal stromal/stem cell.

This articular cartilage was quite stable and showed no signs of undergoing the chondrocytes enlargement found in terminally differentiated cartilage that is ready to form bone.  This stability was maintained for up to 12 weeks.

In vivo cartilage stability. Immunohistochemistry for type X collagen, BSP, and MMP-13 on sections of hypertrophic cartilage generated in vitro by MSCs (as a positive control) and on sections of the cartilaginous constructs generated in vivo by sFlk1 MSCs 12 weeks after implantation. Scale bar = 50 µm. Abbreviations: BSP, bone sialoprotein; MMP-13, metalloproteinase-13; MSC, bone marrow-derived mesenchymal stromal/stem cell.
In vivo cartilage stability. Immunohistochemistry for type X collagen, BSP, and MMP-13 on sections of hypertrophic cartilage generated in vitro by MSCs (as a positive control) and on sections of the cartilaginous constructs generated in vivo by sFlk1 MSCs 12 weeks after implantation. Scale bar = 50 µm. Abbreviations: BSP, bone sialoprotein; MMP-13, metalloproteinase-13; MSC, bone marrow-derived mesenchymal stromal/stem cell.

Why did inhibition of VEGF signaling induce cartilage?  Inhibition of angiogenesis induced low oxygen tensions, which activated a growth factor called transforming growth factor-β.  Activation of the TGF-beta pathway robustly enhanced the formation of articular cartilage.

In vitro chondrogenesis at different oxygen tensions. Histological staining with Safranin-O and immunohistochemistry for type II collagen on constructs generated in vitro by naïve MSC cultured with (A) or without (B) TGFβ3 supplementation at 2% or 20% of oxygen tension. Scale bar = 50 µm. Expression levels of the mRNA for type II and X collagen, Gremlin-1, IHH TGFβ1 were quantified in pellets generated by naïve bone marrow-derived mesenchymal stromal/stem cells (C, D) cultured in the two different oxygen tensions. ∆Ct values were normalized to expression of the GAPDH housekeeping gene, and results are shown as mean ± SD (n = 6 samples/group from 3 independent experiments). ∗, p < .05, ∗∗∗, p < .001. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IHH, Indian hedgehog; TGFβ, transforming grown factor-β.
In vitro chondrogenesis at different oxygen tensions. Histological staining with Safranin-O and immunohistochemistry for type II collagen on constructs generated in vitro by naïve MSC cultured with (A) or without (B) TGFβ3 supplementation at 2% or 20% of oxygen tension. Scale bar = 50 µm. Expression levels of the mRNA for type II and X collagen, Gremlin-1, IHH TGFβ1 were quantified in pellets generated by naïve bone marrow-derived mesenchymal stromal/stem cells (C, D) cultured in the two different oxygen tensions. ∆Ct values were normalized to expression of the GAPDH housekeeping gene, and results are shown as mean ± SD (n = 6 samples/group from 3 independent experiments). ∗, p < .05, ∗∗∗, p < .001. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IHH, Indian hedgehog; TGFβ, transforming grown factor-β.

Cartilage formation from MSCs was induced by blocking VEGF-mediated angiogenesis.  These results represent a remarkable advance in cartilage formation that can be used for regenerative treatments.  This cartilage formation was spontaneous and efficient and if it can be carried out with VEGF-inhibiting drugs rather than genetic engineering techniques, then we might have a transferable technique for making cartilage in the laboratory to treat osteoarthritis and other joint-based maladies.  Clinical trials will be required, but this is certainly an auspicious start.

Cultured Skin-Based Stem Cells Regenerate Hair Follicles and Sebaceous Glands


It has been over ten years since the development of cultured skin substitutes or CSSs. CSSs consist of cultured epidermis from the patients and dermal fibroblasts that can form a good epidermal layer. However, CSSs do not form hair follicles or sebaceous glands which makes for mighty dry skin. Therefore, it is highly preferable to make at skin substitute that can form such epidermal appendages.

Fortunately the skin possesses several types of stem cells for use in regenerative medicine. Epidermal stem cells (Epi-SCs) in the basal layer of the epidermis that constantly provide new cells to the epidermis (the uppermost layer of the skin). Unfortunately, adult Epi-SCs cannot form hair follicles, but they can if they are combined with embryonic or newborn dermal cells. Dermal papilla (DP) cells can induce hair follicles, but the availability of DP cells has greatly limited work with them. Adult dermal skin also contains multipotent SKPs or skin-derived precursors. Injection of SKPs underneath the skin of mice, leads to the induction of new hair follicles (Biernaskie et al., 2009, Cell Stem Cell 5:610-623). This suggests that SKPs might be applicable in a clinical setting to induce hair follicles in cultured skin substitutes.

A new paper by Yaojiong Wu and coworker from the Shenzhen Key Laboratory of Health Sciences and Technology, in collaboration with Edward E. Tredget from University College Dublin has successfully induced the growth of hair follicles and sebaceous glands in a cultured skin substitute. They combined cultured Epi-SCs and SKPs, from mice and humans, and embedded them into a hydrogel. These stem cell-embedded hydrogels were then implanted into immunodeficient mice with substantial skin wounds.

The results were remarkable. The implants able to induce the formation of new epidermis with hair follicles. Furthermore, when Epi-SCs and SKPs taken from human scalps were used in these experiments, they worked just as well as those taken from mice.

Hair neogenesis with cultured epidermal stem cells (Epi-SCs) and skin-derived precursors (SKPs). (A): Putative epidermal stem cells residing in the basal layer of neonatal mouse epidermis expressed CD49f (red) in immunofluorescence stain, and mature keratinocytes in the top layers of the epidermis expressed cytokeratin (CK)6 (green). Nuclei were stained with 4′,6-diamidino-2-phenylindole. (B–E): Cultured Epi-SCs derived from neonatal mice (B) were positive for CD49f (C) and CK15 (D) in immunofluorescence stain; fluorescence-activated cell sorting analysis of the Epi-SCs indicated high levels of surface CD29 and CD49f (E). (F): The expression level of CD49f decreased progressively upon successive passages (P) in culture as determined by immunofluorescence analysis (in relation to the fluorescence intensity of P0 cells). Triple wells were used for each of the above experiments, and each experiment was repeated three times with similar results (∗, p < .05; ∗∗, p < .01; ∗∗∗, p < .001). (G): Hair genesis of cultured Epi-SCs in different passages. Cultured Epi-SCs derived from neonatal mice in different passages (P0 to P5) were implanted into excisional wounds in nude mice in combination with freshly isolated neonatal dermal cells (fresh D) in Matrigel; dermal cells alone or freshly isolated neonatal epidermal cells plus dermal cells (fresh E+D) were used as controls. Hair shafts generated 20 days posttransplant were counted (n = 6; ∗, p < .05; ∗∗∗, p < .001). (H–J): SKPs derived from neonatal mice in spheroid culture (H) expressed nestin, fibronectin (I), and BMP6 (J) in immunofluorescence analysis. (K): Hair genesis of SKPs in different passages. SKPs in P0 to P5 were implanted into excisional wounds in nude mice in combination with freshly isolated neonatal mouse epidermal cells (fresh E), and freshly isolated neonatal mouse epidermal cells alone or in combination with freshly isolated neonatal mouse dermal cells (fresh E+D) were used as controls. Twenty days posttransplant, hairs generated were counted (n = 6; ∗, p < .05; ∗∗, p < .01; ∗∗∗, p < .001). (L-N): Cultured Epi-SCs and SKPs in hair genesis. Combinations of cultured neonatal mouse Epi-SCs (P0 to P3) and SKPs (P0 to P3) were engrafted into excisional wounds in nude mice, and the number of hairs generated were counted 20 days posttransplant (n = 3, ∗, p < .05). (L). A representative image of hairs generated 20 days after a transplantation of P1 Epi-SCs and SKPs (M). Immunofluorescence analysis of the skin tissue with hair genesis showed densely populated hair follicles and sebaceous glands (N). Scale bars = 50 μm. Abbreviations: BM, basement membrane; BMP6, bone morphogenetic protein 6; CK, cytokeratin; DAPI, 4′,6-diamidino-2-phenylindole; Derm, dermis; Epi, epidermis; Epi-SC, epidermal stem cells; FITC, fluorescein isothiocyanate; fresh D, freshly isolated neonatal dermal cells; fresh D+E, freshly isolated neonatal epidermal cells plus dermal cells; HF, hair follicle; HS, hair shafts; P, passage; PE, phycoerythrin.
Hair neogenesis with cultured epidermal stem cells (Epi-SCs) and skin-derived precursors (SKPs). (A): Putative epidermal stem cells residing in the basal layer of neonatal mouse epidermis expressed CD49f (red) in immunofluorescence stain, and mature keratinocytes in the top layers of the epidermis expressed cytokeratin (CK)6 (green). Nuclei were stained with 4′,6-diamidino-2-phenylindole. (B–E): Cultured Epi-SCs derived from neonatal mice (B) were positive for CD49f (C) and CK15 (D) in immunofluorescence stain; fluorescence-activated cell sorting analysis of the Epi-SCs indicated high levels of surface CD29 and CD49f (E). (F): The expression level of CD49f decreased progressively upon successive passages (P) in culture as determined by immunofluorescence analysis (in relation to the fluorescence intensity of P0 cells). Triple wells were used for each of the above experiments, and each experiment was repeated three times with similar results (∗, p < .05; ∗∗, p < .01; ∗∗∗, p < .001). (G): Hair genesis of cultured Epi-SCs in different passages. Cultured Epi-SCs derived from neonatal mice in different passages (P0 to P5) were implanted into excisional wounds in nude mice in combination with freshly isolated neonatal dermal cells (fresh D) in Matrigel; dermal cells alone or freshly isolated neonatal epidermal cells plus dermal cells (fresh E+D) were used as controls. Hair shafts generated 20 days posttransplant were counted (n = 6; ∗, p < .05; ∗∗∗, p < .001). (H–J): SKPs derived from neonatal mice in spheroid culture (H) expressed nestin, fibronectin (I), and BMP6 (J) in immunofluorescence analysis. (K): Hair genesis of SKPs in different passages. SKPs in P0 to P5 were implanted into excisional wounds in nude mice in combination with freshly isolated neonatal mouse epidermal cells (fresh E), and freshly isolated neonatal mouse epidermal cells alone or in combination with freshly isolated neonatal mouse dermal cells (fresh E+D) were used as controls. Twenty days posttransplant, hairs generated were counted (n = 6; ∗, p < .05; ∗∗, p < .01; ∗∗∗, p < .001). (L-N): Cultured Epi-SCs and SKPs in hair genesis. Combinations of cultured neonatal mouse Epi-SCs (P0 to P3) and SKPs (P0 to P3) were engrafted into excisional wounds in nude mice, and the number of hairs generated were counted 20 days posttransplant (n = 3, ∗, p < .05). (L). A representative image of hairs generated 20 days after a transplantation of P1 Epi-SCs and SKPs (M). Immunofluorescence analysis of the skin tissue with hair genesis showed densely populated hair follicles and sebaceous glands (N). Scale bars = 50 μm. Abbreviations: BM, basement membrane; BMP6, bone morphogenetic protein 6; CK, cytokeratin; DAPI, 4′,6-diamidino-2-phenylindole; Derm, dermis; Epi, epidermis; Epi-SC, epidermal stem cells; FITC, fluorescein isothiocyanate; fresh D, freshly isolated neonatal dermal cells; fresh D+E, freshly isolated neonatal epidermal cells plus dermal cells; HF, hair follicle; HS, hair shafts; P, passage; PE, phycoerythrin.

Additionally, when the ability of Epi-SCs to differentiate into sebaceous glands was examined, Wu and others showed that Epi-SCs can form the precursors to sebaceous glands, sebocytes. Additionally, the oils secreted by these Epi-SC-derived sebocytes were chemically similar to sebaceous glands from native skin.

Thus, a combination of Epi-SCs and SKPs from human or mouse skin were sufficient to generate newly formed hair follicles and functional sebaceous glands. These results provide knowledge that is potentially transferable to clinical applications for regenerating damaged skin.

Fat-derived cells Enhance the Bone-Forming Capacity of Hypertrophic Cartilage Matrices


Treating particular bone defects or injuries present a substantial challenges for clinicians. The method of choice usually involves the use of an “autologous” bone graft (“autologous” simply means that the graft comes from the patient’s own bone). However, autologous bone grafts have plenty of limitations. For example, if a patient has a large enough bone defect, there is no way the orthopedist and take bone from a donor site without causing a good deal of risk to the donor site. Even with small bone grafts, so-called “donor site morbidity” remains a risk. Having said that, plenty of patients have had autologous bone grafts that have worked well, but larger bone injuries or defects are not treatable with autologous bone grafts.

The answer: bone substitute materials. Bone substitute materials include tricalcium phosphate, hydroxyapatite, cement, ceramics, bioglass, hydrogels, polylactides, PMMA or poly(methy methacrylate) and other acrylates,, and a cadre of commercially available granules, blocks, pastes, cements, and membranes. Some of these materials are experimental, but others do work, even if do not work every time. The main problem with bone substitute materials is that, well, they are not bone, and, therefore lack the intrinsic ability to induce the growth of new bone (so-called osteoinductive potential) and their ability to integrate into new bone is also a problem at times.

We must admit that a good deal of progress has been made in this area and it’s a good thing too. Many of our fabulous men and women-at-arms have returned home with severe injuries from explosives and wounds from large-caliber weapons that have shattered their bones. These courageous men and women have been the recipient of these technologies. However, the clinician is sometimes left asking herself, “can we do better?”

A new paper from the laboratories of Ivan Martin and Claude Jaquiery from the University Hospital of Basel suggests that we can. This paper appeared in Stem Cells Translational Medicine and describes the use of a hypertrophic cartilage matrix that was seeded with cells derived from the stromal vascular faction of fat to not only make bone in the laboratory, but to also heal skull defects in laboratory animals. While this work benefitted laboratory animals, it was performed with human cells and materials, which suggests that this technique, if it can be efficiently and cheaply scaled up, might be usable in human patients.
The two lead authors of this paper, Atanas Todorov and Matthias Kreutz and their colleagues made hypertrophic cartilage matrices from human bone marrow mesenchymal stem cells (from human donors) that were induced to make cartilage. Fortunately, protocols have been very well worked out and making cartilage plugs with chondrocytes that are enlarged (hypertrophic) is something that has been successfully done in many laboratories. After growing the mesenchymal stem cells in culture, half a million cells were induced to form cartilage with dexamethasone, ascorbic acid 2-phosphate, and the growth factor TGF-beta1. After three weeks, the cartilage plugs were subjected to hypertrophic medium, which causes the cartilage cells to enlarge.

Chondrocyte enlargement is a prolegomena to the formation of bone and during development, many of our long bones (femur, humerus, fibula, radius, etc.), initially form as cartilage exemplars that are replaced by bone as the chondrocytes enlarge. Ossification begins when a hollow cylinder forms in the center of the bone (known as the periosteal collar). The underlying chondrocytes degenerate and die, thus releasing the matrix upon which calcium phosphate crystals accrete. The primary ossification center commences with the calcification of the central shaft of the bone and erosion of the matrix by the invasion of a blood vessel. The blood vessels bring osteoprogenitor cells that differentiate into osteoblasts and begin to deposit the bone matrix.

Next, Todorov and his crew isolated the stromal vascular fraction from fat that was donated by 12 volunteers who had fat taken from them by means of liposuction. The fat is then minced, digested with enzymes, centrifuged, filtered and then counted. This remaining fraction is called the stromal vascular fraction or SVF, and it consists of a pastiche of blood vessel-forming cells, mesenchymal stem cells, and bone-forming cells (and probably a few other cells types too). These SVF cells were seeded onto the hypertrophic cartilage plugs and used for the experiments in this paper.

First, the SVF-seeded plugs were used to grow bone in laboratory rodents. The cartilage plugs were implanted into the backs for nude mice. Different cartilage plugs were used that had been seeded with gradually increasing number of SVF cells. The implanted plugs definitely made ectopic bone, but the amount of bone they made was directly proportional to the number of SVF cells with which they had been seeded. Staining experimental also showed that some of the newly-grown bone came from the implanted SVF cells.

Ectopic bone formation. Grafts based on devitalized hypertrophic cartilage pellets were embedded in fibrin gel without or with stromal vascular fraction cells from adipose tissue and implanted subcutaneously in nude mice. (A): Representative hematoxylin and eosin, Masson-Tri-Chrome, and Safranin-O (Saf-O) staining and in situ hybridization for human ALU sequences (dark blue = positive) after 12 weeks in vivo. Saf-O stainings are blue-green because of lack of glycosaminoglycans and counterstaining with fast green. Osteoid matrix and bone marrow are visible. Scale bars = 200 µm. (B): Stainings for metalloproteinase (MMP)13 and MMP9, as well as for the N-terminal neoepitope at the major MMP cleavage site (DIPEN) after 12 weeks in vivo (red/pink = positive). Scale bars = 50 µm. +, osteoid matrix; ⋆, bone marrow. Abbreviations: ALU, Arthrobacter luteus; H&E, hematoxylin and eosin; Masson, Masson’s trichrome; MMP, metalloproteinase; Saf-O, Safranin-O; SVF, stromal vascular fraction.
Ectopic bone formation. Grafts based on devitalized hypertrophic cartilage pellets were embedded in fibrin gel without or with stromal vascular fraction cells from adipose tissue and implanted subcutaneously in nude mice. (A): Representative hematoxylin and eosin, Masson-Tri-Chrome, and Safranin-O (Saf-O) staining and in situ hybridization for human ALU sequences (dark blue = positive) after 12 weeks in vivo. Saf-O stainings are blue-green because of lack of glycosaminoglycans and counterstaining with fast green. Osteoid matrix and bone marrow are visible. Scale bars = 200 µm. (B): Stainings for metalloproteinase (MMP)13 and MMP9, as well as for the N-terminal neoepitope at the major MMP cleavage site (DIPEN) after 12 weeks in vivo (red/pink = positive). Scale bars = 50 µm. +, osteoid matrix; ⋆, bone marrow. Abbreviations: ALU, Arthrobacter luteus; H&E, hematoxylin and eosin; Masson, Masson’s trichrome; MMP, metalloproteinase; Saf-O, Safranin-O; SVF, stromal vascular fraction.

In the second experiment, Todorov and Kreutz used these SVF-seeded cartilage plugs to repair skull lesions in rats. Once again, the quantity of bone produced was directly proportional to the number of SVFs seeded into the cartilage matrices prior to implantation. In both experiments, the density of SVF cells positively correlates with the bone-forming cells in the grafts and the percentage of SVF-derived blood vessel-forming cells correlates with the amount of deposited mineralized matrix.

Bone repair capacity. Devitalized hypertrophic cartilage pellets were embedded in fibrin gel without or with stromal vascular fraction (SVF) cells from adipose tissue and implanted in rat calvarial defects. (A): Mineralized volume quantified by microtomography (n = 9 grafts assessed per group). (B): Bone area assessed in histological sections, expressed as percentage of total defect area (n = at least 24 sections assessed per group). ∗∗∗∗, p < .0001. (C, D): Representative three-dimensional microtomography reconstructions (left) and hematoxylin/eosin (H&E) staining (right) of the calvarial defects filled with devitalized grafts, implanted without (C) or with (D) activation by SVF cells after 4 weeks. Dotted circles indicate the defect borders (4 mm diameter). Scale bars = 500 µm. (E): Microtomography (left) and H&E staining (middle and right) displaying the bridging between hypertrophic matrix and bone of the calvarium, or the fusion of single pellets (right) in activated grafts. White bar = 850 µm; black bars = 500 µm. Dotted lines indicate the edge of the calvarium. (F): In situ hybridization for Arthrobacter luteus sequences showing the presence of human cells (dark blue, positive) in the explants. Scale bar = 200 µm. Abbreviations: C, calvarium; dev, fibrin gel without stromal vascular fraction; dev + SVF, fibrin gel with stromal vascular fraction; P, hypertrophic matrix; SVF, stromal vascular fraction.
Bone repair capacity. Devitalized hypertrophic cartilage pellets were embedded in fibrin gel without or with stromal vascular fraction (SVF) cells from adipose tissue and implanted in rat calvarial defects. (A): Mineralized volume quantified by microtomography (n = 9 grafts assessed per group). (B): Bone area assessed in histological sections, expressed as percentage of total defect area (n = at least 24 sections assessed per group). ∗∗∗∗, p < .0001. (C, D): Representative three-dimensional microtomography reconstructions (left) and hematoxylin/eosin (H&E) staining (right) of the calvarial defects filled with devitalized grafts, implanted without (C) or with (D) activation by SVF cells after 4 weeks. Dotted circles indicate the defect borders (4 mm diameter). Scale bars = 500 µm. (E): Microtomography (left) and H&E staining (middle and right) displaying the bridging between hypertrophic matrix and bone of the calvarium, or the fusion of single pellets (right) in activated grafts. White bar = 850 µm; black bars = 500 µm. Dotted lines indicate the edge of the calvarium. (F): In situ hybridization for Arthrobacter luteus sequences showing the presence of human cells (dark blue, positive) in the explants. Scale bar = 200 µm. Abbreviations: C, calvarium; dev, fibrin gel without stromal vascular fraction; dev + SVF, fibrin gel with stromal vascular fraction; P, hypertrophic matrix; SVF, stromal vascular fraction.

This is not the first time scientists have proposed the use of cartilage plugs to induce the formation of new bone. Van der Stok and others and Bahney and colleagues were able to repair segmental bone defects in laboratory rodents. Is this technique transferable to human patients? Possibly. Hypertrophic cartilage is relatively easy to make and it is completely conceivable that hypertrophic cartilage wedges can be sold as “off-the-shelf” products for bone treatments. SVF can also be derived from the patient or can be derived from donors.

Furthermore, the protocols in this paper all used human cells and grew the products in immunodeficient rats and mice. Therefore, in addition to scaling this process up, we have a potentially useful protocol that might very well be adaptable to the clinic.

The efficacy of this technique must be confirmed in larger animal model system before human trials can be considered. Hopefully human trials are in the future for this fascinating technique.

Fat-Based Stem Cell Treatment Suggests a New Way to Slow Scarring in Scleroderma Patients


Scleroderma is an autoimmune disease that causes chronic scarring of the skin and internal organs. The deposition of massive quantities of collagen decrease the pliability and elasticity of the skin, lungs, and blood vessels. As you might guess, the prognosis of scleroderma patients is quite poor and this disease causes a good deal of suffering and morbidity.

Treatments options usually include steroids, and other drugs that suppress the immune system, all of which have severe side effects.

New research from scientists at the Hospital for Special Surgery in New York City and other collaborating institutions, led by Dr. Teresa T. Lu, may have identified a new mechanism in operation during the onset and maintenance of scleroderma. This work was published in the Journal of Clinical Investigation.

In this study, scleroderma patients were shown to possess diminished numbers of “adipose-derived stromal cells” (ADSCs) in the layer of fat that underlies the upper layers of the skin. These fatty tissues are referred to as “dermal white adipose tissue.” The loss of these dermal white adipose tissue ADSCs tightly correlates with the onset of scarring in two different mouse model systems that recapitulate scleroderma in laboratory mice. These observations may show that ADSC loss contributes to scarring of the skin.

Why do these ADSCs die? Lu and her coworkers discovered that ADSC survival depends on the presence of particular molecules secreted by immune cells called “dendritic cells.” Skin-based dendritic cells secrete a molecule called lymphotoxin B. Although this molecule is called a toxin, it is required for ADSC survival. In laboratory mice that suffered from a scleroderma-like disease, artificial stimulation of the lymphotoxin B receptor in ADSCs amplified and eventually restored the numbers of ADSCs in the skin. Could stimulating ADSCs in this manner help treat scleroderma patients?

According the Dr. Lu, the administrating author of this publication, injecting “ADSCs is being tried in scleroderma; the possibility of stimulating the lymphotoxin B pathway to increase the survival of these stem cells is very exciting.” Dr. Lu continued, “By uncovering these mechanisms and targeting them with treatments, perhaps one day we can better treat the disease.”

Lu also thinks that a similar strategy that targets stem cells from other tissues might provide a treatment for other rheumatological conditions – such as systemic lupus erythematosis and rheumatoid arthritis. Additionally, bone and cartilage repair might also benefit from such a treatment strategy.

In the coming years, Dr. Lu and her colleagues hope to test the applicability of this work in human cells. If such a strategy works in human cells, then the next stop would be trial in human scleroderma patients. The success of such a treatment strategy would be a welcome addition to the treatment options for scleroderma patients, but only if this treatment is shown to be proven safe and effective.

“Improving ADSC therapy would be a major benefit to the field of rheumatology and to patients suffering from scleroderma,” said Lu.

The Amino Acid Valine Helps Maintain Hematopoietic Stem Cell Niches


Hematopoietic stem cells (HSCs) populate our bone marrow and divide throughout our lifetimes to provide the red and white blood cells we need to live. However, during normal, healthy times, only particular HSCs are hard at work dividing and making new blood cells. The remaining HSCs are maintained in a protective dormant state. However, in response to blood loss or physiological stress of some sort, dormant HSCs must wake from their “slumbers” and begin dividing to make the needed blood cells. Such conditions, it turns out, can cause HSCs to experience a good deal of damage to their genomes. A paper that was published in Nature last year by Walter Dagmar and colleagues (Vol 520: pp. 549) showed that repeatedly subjecting mice to conditions that required the activation of dormant HSCs (in this case they injected the mice with polyinosinic:polycytidylic acid or pI:pC to mimic a viral infection and induce a type I interferon response) resulted in the eventual collapse of the bone marrow’s ability to produce new blood cells. The awakened HSCs accumulated such large quantities of DNA damage, that they were no longer able to divide and produce viable progeny. How then can HSCs maintain the integrity of their genomes while still dividing and making new blood cells?

The answer to this question is not completely clear, but a new paper in the December 2 edition of Science magazine provides new insights into HSC physiology and function. This paper by Yuki Taya and others, working in the laboratories of Hiromitsu Nakauchi at the Institute for Stem Cell Biology and Regenerative Medicine at Stanford University School of Medicine, and Satoshi Yamazaki from the University of Tokyo, has shown that amino acid metabolism plays a vital role in HSC maintenance. As it turns out, the amino acid concentrations in bone marrow are approximately 100-fold higher than the concentrations of these same amino acids in circulating blood. Taya and others reasoned that such high amino acid concentrations must exist for reasons other than protein synthesis. Therefore, they designed dietary regimens that depleted mice for specific amino acids. Sure enough, when mice were fed valine-depleted diets, the HSCs of those mice lost their ability to repopulate the bone marrow.

Valine
Valine

After only two weeks of valine depletion, several nooks and crannies of the bone marrow – so-called stem cell “niches” – were devoid of HSCs. The bone marrow of such mice was easily reconstituted with HSCs from donor mice without the need for radiation or chemical ablation treatments.

Taya and others found that vascular endothelial stromal cells in the bone marrow secrete valine and that this secreted valine (which, by the way, is a branched-chain amino acid) is integral for maintaining HSC niches.

The excitement surrounding this finding is plain, since using harsh chemicals or radiation to destroy the bone marrow (a procedure known as “myeloablation”) causes premature ageing, infertility, lousy overall health, and other rather unpleasant side effects. Therefore, finding a “kinder, gentler” way to reconstitute the bone marrow would certainly be welcomed by patients and their physicians. However, valine depletion, even though it does not affect sterility, did cause 50% of the mice to die once valine was restored to the diet. This is due to a phenomenon known as the “refeeding effect” which has also been observed in human patients. Such side effects could probably be prevented by gradually returning valine to the diet. Taya and others also showed that cultured human HSCs required valine and another branched-chain amino acid, leucine. Since both leucine and valine are metabolized to alpha-ketoglutatate, which is used as a substrate for DNA-modifying enzymes, these amino acids might exert their effects through epigenetic modifications to the genome.

Alpha-ketoglutarate
Alpha-ketoglutarate

More work is needed in this area, but the Taya paper is a welcomed finding to a vitally important field.