Regenerative MedicineVol. 7, No. 6 News & ViewsFree AccessResearch Highlights: Highlights from the latest articles in regenerative medicineThomas Bollenbach, Amber E Kerstetter-Fogle, Charles C King, Paolo Madeddu & Eleftherios SachlosThomas BollenbachOrganogenesis, Preclinical Research & Development, 150 Dan Road, Canton, MA 02021, USA. Search for more papers by this authorEmail the corresponding author at tbollenbach@organo.com, Amber E Kerstetter-FogleCase Western Reserve University, Center for Translational Neuroscience, Department of Neurological Surgery, Cleveland, OH 44106, USA. Search for more papers by this authorEmail the corresponding author at aek20@case.edu, Charles C KingPediatric Diabetes Research Center, University of California, San Diego, 9500 Gilman Drive, 0721, La Jolla, CA 92093-0721, USA. Search for more papers by this authorEmail the corresponding author at chking@ucsd.edu, Paolo MadedduSchool of Clinical Sciences, University of Bristol, Level 7, Bristol Royal Infirmary, Upper Maudlin Street, Bristol, BS2 8HW, UK. Search for more papers by this authorEmail the corresponding author at madeddu@yahoo.com & Eleftherios SachlosStem Cell & Cancer Research Institute, McMaster University, ON, Canada. Search for more papers by this authorEmail the corresponding author at sachlos@mcmaster.caPublished Online:20 Nov 2012https://doi.org/10.2217/rme.12.93AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInReddit Evaluation of: Nugent HM, Ng Y-S, White D, Groothius A, Kanner G, Edelman ER. Ultrasound-guided, percutaneous delivery of tissue-engineered endothelial cells to the adventitia of stented arteries controls the response to vascular injury in a porcine model. J. Vasc. Surg. doi: 10.1016/j.jvs.2012.03.002 (2012) (Epub ahead of print).Restenosis, or blood vessel renarrowing, occurs after damage to the intima, the monolayer of smooth muscle cells that lines the vessel’s luminal surface and is, ironically, a common adverse event associated with balloon angioplasty and other vascular procedures intended to treat vessel stenosis in the first place. Although placement of stents is often performed as part of an angioplasty, the stent does not prevent the cascade of events, including thrombosis, inflammation and endothelial cell proliferation that lead to hyperplasia, which is the ultimate cause of vessel restenosis.In recent years, research has focused on the regulation of intimal hyperplasia by endothelial cells within the adventitia, or outermost layer of large vessels, and in the vasa vasorum, the smaller vessels that supply blood to veins and arteries. This research spawned the hypothesis that supplementing the adventitial endothelium with a tissue-engineered construct containing endothelial cells would allow control over intimal hyperplasia. This hypothesis was tested by embedding allogeneic porcine aortic endothelial (PAE) cells within an absorbable gelatin sponge, which was implanted in an open surgical procedure into the perivascular space surrounding vessels in a porcine carotid artery angioplasty model [1]. In this configuration, the PAE-containing construct decreased the restenosis index by 54% versus control.Since many vascular interventions involve percutaneous rather than open surgical procedures, it was of interest to re-engineer the construct, while maintaining efficacy. In the study by Nugent et al., allogeneic PAE cells were cultured on gelatin particles for percutaneous delivery to the perivascular space surrounding femoral arteries in a stent-induced injury model. Proliferation rates of PAE cells, and cell-based assays designed to test for endothelial cell phenotype and for their ability to inhibit inflammatory and thrombotic gene expression, were first performed to confirm that PAE cells cultured on gelatin particles retained the same functional properties as those cultured in gelatin foam. Ultrasound-guided needles were then used to inject the PAE/matrix to the perivascular space of injured vessels, which were examined histomorphometrically after 90 days. The stented segments of arteries treated with PAE/matrix had significantly higher luminal areas, higher luminal diameters, smaller intimal areas, and overall, were 33–50% less occluded than noninjected or gelatin particle-injected controls. Furthermore, histopathology revealed a decrease in the number of leukocytes associated with the intima of PAE-treated vessels, suggesting an effect on the inflammatory phase of the injury response.Although the exact mechanism of action of the PAE/matrix on intimal hyperplasia has not been determined, it is thought that paracrine factors are involved, and that the effect is delivered by the construct within the first few weeks post-injection, since neither the allogeneic cells nor the matrix persist beyond this time frame. Overall, the data show the power of allogeneic cells in providing control over the intimal healing process. Whether this allogeneic cell therapy translates well to the clinic remains to be seen, and is the subject of ongoing clinical research.– Written by Thomas BollenbachReference1 Nugent HM, Edelman ER. Endothelial implants provide long-term control of vascular repair in a porcine model of arterial injury. J. Surg. Res.99(2),228–234 (2001).Crossref, Medline, CAS, Google ScholarEvaluation of: Ju P, Zhang S, Yeap Y, Feng Z. Induction of neuronal phenotypes from NG2+ glial progenitors by inhibiting epidermal growth factor receptor in mouse spinal cord injury. Glia 60(11), 1801–1814 (2012).Generation of new neurons after spinal cord injury (SCI) is a problem that has been studied for a number of years and is yet to be resolved. Transplantation of neural stem stems, differentiated neurons and the like, fail to survive and therefore fail to create functional recovery over time due to their suboptimal integration and downstream consequences. Researchers have begun to seek out the ‘reprogramming’, per se, of endogenous cells that are highly proliferative after SCI. In the CNS, these highly proliferative cells after injury include macrophages/microglia, astrocytes and NG2 cells (thought to be oligodendrocyte progenitor cells). These glia have demonstrated that they have neurogenic potential through induction paradigms in culture. Recent work has pointed to the role of NG2 as a stem cell after CNS injury and the neurogenic potential of these cells is elucidated in the highlighted review by Ju et al.The authors of the study looked at the potential of NG2 cells to generate neurons through EGF receptor (EGFR) inhibition. EGFR signaling is known to promote gliogenesis in the CNS and may enhance neuronal differentiation of stem cells after inhibition. By making use of primary NG2 glial progenitors and cell lines, Ju et al. demonstrated that inhibition of EGFR signaling through PD168393 and siRNA generate neurons as demonstrated through morphological, immunohistochemical and PCR evidence. The investigators utilized a contusion model to generate a mouse SCI and implanted either EGFR inhibitor (PD168393) or phosphate buffered saline-soaked matrix to assess whether endogenous cells in the SCI could be induced to differentiate into neurons. Locomotor improvements were witnessed in animals treated with the EGFR inhibitor after 4 weeks. To ascertain the levels of inhibition they quantified the number of phosphorylated EGFRs in the region of the SCI after 14 days. They verified that the phosphorylated EGFR signal was greatly reduced after only 3 days of treatment with PD168393. Interestingly, the number of cells expressing phosphorylated EGFR and NG2 increased after the initial injury and were inhibited with PD168393. Ju et al. then analyzed the cells that were proliferating in response to the SCI by utilizing the S-phase marker, bromodeoxyuridine (BrdU). The authors established that more than half of the proliferative cells were NG2 cells. However, none of the BrdU+ cells labeled mature neurons. After inhibition of EGFR signaling with PD168393, a portion of the NG2+/BrdU+ cells were also labeled with neuronal marker, NeuN. There were also cells that had low or no expression of NG2 but were labeled with BrdU and NeuN. Crucially, the researchers validated other neuronal markers to be expressed with NG2 and BrdU including MAP2 and β-tubulin III. Additional characterization of the lesion treated with PD168393 demonstrated that a significant number of BrdU+ cells were also labeled GFAP-expressing cells; nevertheless, these cells were never observed to coexpress NeuN. The mechanism of the locomotor recovery after EGFR inhibition was further studied in this article. Ju et al. used PD168393 treatment to determine that there are a small percentage of NG2+/BrdU+ cells which generate motor and sensory neurons by labeling with SMI32 and GABA, correspondingly. Notably, the direct evidence for the role of EGFR in neurogenesis after injury was assessed using transplantation experiments. The researchers generated NG2 progenitors and determined that the cells treated with PD168393 were able to express motor and sensory neuronal markers. On the other hand, none of the GFP-labeled NG2 exogenous transplanted cells treated with phosphate buffered saline were able to express neuronal markers. Mechanistically, they resolved the signaling to be mediated by Ras and MEK in addition to ERK1/2 and p90RSK, without affecting other kinases. Interestingly, inhibitors of Ras and MEK were also able to induce neuronal morphology, mRNA, and protein levels, in culture, similar to P168393.This work has demonstrated how we must look at harnessing the cells that remain in the injury after SCI or traumatic brain injury. If we are able to increase proliferation and enhance differentiation of endogenous cells within the injury, we may be able to treat SCI patients with downstream activators of neurogenesis. Ju et al. have eloquently shown the role of Ras/MEK in mediating EGFR signaling in NG2 cells to produce functional neurons. It will be very interesting to observe the long-term effects of treatment with EGFR inhibitors and downstream effectors. Furthermore, it will be interesting to look at EGFR inhibition in NG2 cells in other models of degeneration of the CNS, such as multiple sclerosis or Alzheimer’s disease.– Written by Amber E Kerstetter-FogleEvaluation of: Faustman DL, Wang L, Okubo Y et al. Proof-of-concept, randomized, controlled clinical trial of bacillus-Calmette–Guerin for treatment of long-term Type 1 diabetes. PLoS One 7(8), e41756 (2012).Selective destruction of autoimmune T lymphocytes (T cells) that target insulin-secreting β-cells could potentially provide a therapeutic avenue to treat Type 1 diabetes. Boosting the innate immunity through selective exposure to pathogens is a potential mechanism to achieve this goal. The administration of bacillus Calmette–Guerin (BCG), which is known to induce TNF expression through an innate immune response that selectively targets and destroys insulin-autoreactive T cells, is the underlying hypothesis driving this proof-of-principle study. In mice, BCG immunization-induced production of the cytokines TNF-α, INFγ and IL-4 by splenocytes and increased expression of Fas, Fas ligand, and TNF receptors on T cells, which led to Tcell apoptosis [1]. Previous work by the Faustman laboratory had established that a subpopulation of CD8+ cells in patient blood was vulnerable to TNF [2]. Adjuvent therapy, such as BCG, in humans was found to be effective in a small population of prediabetic patients; however, expanded clinical trials have cast doubt on the clinical efficacy of this approach. In the study by Faustman et al., BCG was administered to patients with long-term Type 1 diabetes in a double-blind, placebo-controlled trial. Blood samples were monitored over 20 weeks for immune and pancreatic function. Patients treated with BCG, and one control patient diagnosed with acute Epstein–Barr virus infection (another trigger of innate immunity), had increased numbers of dead insulin-autoreactive T cells as measured by flow cytometry. Additionally, regulatory T-cell numbers increased in the BCG-treated subjects. Antibody levels against glutamic acid decarboxylase, the tyrosine phosphatase IA–2A and the zinc transporter, ZnT8A, were measured in placebo and BCG-treated populations. In two out of the three BCG-treated subjects, glutamic acid decarboxylase levels sustainably changed, one increased and one decreased, but the third did not change from the baseline. For the other islet-specific autoantibodies, only ZnT8A decreased in one subject with statistical significance. C-peptide levels significantly and temporarily rose in two out of the six BCG-treated subjects as well as in the Epstein–Barr virus-infected placebo subject, which was suggestive of a brief functional improvement in βcell function. Taken together, these results are consistent with BCG inducing a TNF expression that modulates the innate immune response, targets insulin-autoreactive T cells and transiently increases C-peptide levels. The results provide a foundation upon which expanded clinical trials will determine whether sustained increases in C-peptide levels can be reproducible in a larger population of patients with Type 1 diabetes.– Written by Charles C KingReferences1 Qin H, Chaturvedi P, Singh B. In vivo apoptosis of diabetogenic T cells in NOD mice by IFN-gamma/TNF-alpha. Int. Immunol.16(12),1723–1732 (2004).Crossref, Medline, CAS, Google Scholar2 Ban L, Zhang J, Wang L, Kuhtreiber W, Burger D, Faustman DL. Selective death of autoreactive T cells in human diabetes by TNF or TNF receptor 2 agonism. Proc. Natl Acad. Sci. USA.102(36),13644–13649 (2008).Crossref, Google ScholarEvaluation of: Yaniz-Galende E, Chen JQ, Chemaly ER et al. Stem cell factor gene transfer promotes cardiac repair after myocardial infarction via in situ recruitment and expansion of c-kit+ cells. Circ. Res. doi:10.1161/CIRCRESAHA.111.263830 (2012) (Epub ahead of print).Regeneration of the heart was once considered a remote possibility, mainly inferred from calculation of cardiomyocyte number and ventricular mass during aging. In fact, due to the continuous loss of cardiomyocytes, the heart would be expected to shrink unless new cardiomyocytes are concomitantly generated by a pool of resident cardiac stem cells. The demonstration of adult human heart renewal provides new hope for the development of regenerative strategies to rescue cardiac disease by either transplanting exogenous cardiac stem cells or boosting the regenerative potential of endogenous stem cells.The ongoing SCIPIO clinical trial using intracoronary infusion of autologous c-kit+ cardiac stem cells showed preliminary evidence of functional improvement in patients with ischemic cardiomyopathy [1]. Moreover, injection of ex vivo expanded autologous cardiospheres (which contain a significant amount of c-kit+ cells) were also demonstrated to improve clinical parameters in patients with heart failure [2].A recent Circulation Research article from Yaniz-Galende et al. investigates the capability of recruiting c-kit+ cells through modulation of local signaling pathways by gene transfer of the c-kit ligand, stem cell factor (SCF) [3]. SCF exists in two isoforms, a soluble and a membrane-bound form. While the former leads to rapid and transient activation, autophosphorylation and fast degradation of c-kit, stimulation with the membrane-associated form leads to more sustained activation by preventing receptor–ligand complex internalization. Hence, the authors decided to apply gene therapy with recombinant adenoviruses expressing the membrane-bound form of SCF to rescue cardiac function in rats with acute myocardial infarction (aMI). They used sham operated rats and aMI rats given adenoviruses containing β-gal and GFP as controls.Results of this elegant study indicate a positive effect of SCF gene therapy on infarct size, cardiac MRI indexes of left ventricular function, cardiac progenitor cell recruitment, fibrosis and cardiomyocyte cell cycle activation. Fractional shortening and ejection fraction were both improved at 3 months after gene therapy and myocardial infarction, suggesting a durable effect of SCF. This could be attributed to preservation of the area at risk, as denoted by reduced cardiomyocyte fibrosis and stimulation of reactive responses by resident T cells. Notably, an increased number of cardiomyocytes were found to be cycling in hearts given SCF gene therapy. Likewise, the number of c-kit+ cells was increased, indicating that SCF recruits and probably helps the expansion of this pool of stem cells.Molecular mechanisms behind cardiomyocyte renewal may comprise the Wnt signaling pathway, according to data showing β-catenin accumulation and induction of Notch1, HoxB4 and cyclin D1; however, use of inhibitors of canonical and noncanonical Wnt signaling pathways should have been used to confirm the causative role of Wnt in activation of the regenerative process.There are obvious advantages of using SCF gene therapy for the promotion of cardiomyogenesis as compared with transplantation of exogenous cardiac stem cells. The latter approach requires cell expansion, hence, it cannot be applied to patients with aMI. However, it is not clear if there is an optimal time window for activation of cardiac stem cells in the setting of an aMI. The study from Yaniz-Galende et al. indicates the prolonged effect of acute intramyocardial injection of SCF gene therapy [3]. Systemic administration of the transgene using cardiotropic adeno-associated viral vectors encoding for SCF under a α-sarcomeric actin promoter can improve the practicality and duration of the transgene expression.There are important caveats to be considered on a translational perspective. First, the study did not address the optimal therapeutic dosage of SCF. This factor is already increased in the infarcted heart, hence, it is not clear how much SCF needs to be further augmented to achieve therapeutic effects and what the threshold is for adverse reactions to occur. Second, risk factors not considered here might depress the responsiveness to SCF stimulation. Third, the effect of SCF seems to be pleiotropic and the relative contribution of stem cells in functional and anatomical end points remains controversial. Fourth, no attention was given to reparative angiogenesis. Finally, an improved survival rate was noted, but not interpreted, at the early post-myocardial infarction stage, which may suggest protection of SCF from arrhythmias.In conclusion, SCF therapy provides a new means to sustain cardiac function in a rodent model of myocardial infarction. Initially designed to support cardiac stem cell expansion, the gene therapy approach resulted in a series of pleiotropic effects, indicating that SCF might induce unpredicted responses on a spectrum of cardiac cells. An alternative explanation is that c-kit+ cells release paracrine factors in response to SCF, indirectly influencing other cardiac cells. In vitro studies, expressional analyses on c-kit+ and c-kit- cells from SCF-injected hearts and use of c-kit tyrosine kinase inhibitors could answer these intriguing questions.– Written by Paolo MadedduReferences1 Bolli R, Chugh AR, D’Amario D et al. Cardiac stem cells in patients with ischaemic cardiomyopathy (SCIPIO): initial results of a randomised Phase 1 trial. Lancet378,18471–1857 (2011).Crossref, Google Scholar2 Makkar RR, Smith RR, Cheng K et al. Intracoronary cardiosphere-derived cells for heart regeneration after myocardial infarction (CADUCEUS): a prospective, randomised Phase 1 trial. Lancet379,895–904 (2012).Crossref, Medline, Google Scholar3 Yaniz-Galende E, Chen JQ, Chemaly ER et al. Stem cell factor gene transfer promotes cardiac repair after myocardial infarction via in situ recruitment and expansion of c-kit+ cells. Circ. Res. doi:10.1161/CIRCRESAHA.111.263830 (2012) (Epub ahead of print).Medline, Google ScholarEvaluation of: Zheng Y, Chen J, Craven M et al.In vitro microvessels for the study of angiogenesis and thrombosis. Proc. Natl Acad. Sci. USA 109(24), 9342–9347 (2012).The inability to generate functional microvascular networks (µVNs) capable of perfusing tissue engineering scaffolds remains a major obstacle in the realization of engineered 3D tissues for regenerative medicine. Now, Zheng et al. have reinvigorated scaffold-based tissue engineering approaches by developing a soft lithography collagen gel molding technique that is able to predefine lumen structures that, when seeded with human umbilical vein endothelial cells (HUVECs), generate endothelium while the bulk scaffold structure can be seeded with supportive cells. Scaffold fabrication involved gelling a collagen type I solution seeded with or without perivascular cells on a PDMS stamp molded into a simple jail-bar pattern. Additional molding structures were assembled on diagonally opposite corners in order to generate dedicated inlet and outlet channels. A second flat collagen gel 70 µm in height sandwiched the two gel layers together, and pressure was applied via a hermetic plexiglass device to seal the jail-bar lumens enveloped in a collagen matrix. The lumen cross-sections were reported to be 100 × 100 µm. HUVECs were seeded by perfusion through the lumens, while medium flow was gravity-driven. The HUVECs were shown to organize into an endothelium resembling a venule-like cobblestone pattern with an elliptical cross-section. The endothelium’s permeability was investigated using FITC-dextran and fluorescein and permeability coefficients were calculated to be five-times higher than reported mammalian venules in vivo. Nonetheless, this µVN system was used to model elements of angiogenesis in vitro. In an attempt to mimic the proangiogenic environment found in ischemic tissue or solid tumors, medium containing VEGF, bFGF and PMA was perfused through the µVN, and an increase in endothelial sprouts, along with increased permeability, was observed after 1 week of treatment. These responses were further measured in the presence of perivascular cells in order to study endothelial–pericyte interactions, while perfusion with whole blood provided insights into leukocyte and platelet interactions under normal and prothrombotic states. In summary, the technology developed by the authors is a powerful new tool to study blood vessel interactions with multiple cell phenotypes including stem cells. How these µVNs will be scaled up into multiple planes still remains to be demonstrated, but at least we are one step closer to engineering tissue that may fulfill the promise of regenerative medicine.– Written by Eleftherios SachlosFinancial & competing interests disclosureT Bollenbach is an employee of Organogenesis Inc. P Madeddu is a Full Professor at the University of Bristol. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.FiguresReferencesRelatedDetails Vol. 7, No. 6 Follow us on social media for the latest updates Metrics History Published online 20 November 2012 Published in print November 2012 Information© Future Medicine LtdPDF download