Centre For Regenerative Medicine Research Articles

An interview with Valerie Wilson.

Valerie Wilson is Professor of Early Embryo Development and a group leader at the Centre for Regenerative Medicine at the University of Edinburgh, UK. Val's lab works on the development of the vertebrate head-to-tail axis, focusing on the differentiation potential of axial progenitors. In 2022, she was awarded the British Society for Developmental Biology's (BSDB) Waddington Medal, which recognises outstanding individuals who have made major contributions to UK developmental biology. After her award lecture, we spoke to Val about her career and heard her thoughts on the past, present and future of the field.

Resilience and Mayo Clinic team up

The pharmaceutical manufacturing and services firm National Resilience and the Mayo Clinic have announced a partnership to develop biologic therapies for rare diseases. Resilience will collaborate with Mayo’s Center for Regenerative Medicine in Rochester, Minnesota, where the partners plan to build process and analytical development laboratories for cell therapies and other modalities with the aim of advancing therapeutic candidates to early-stage clinical trials. Resilience and Mayo hope to attract biotechnology firms interested in collaborations and the sponsorship of clinical trials.

Nucleic‐Acid Mediated Expression of Cardiopoietic Markers in Human Umbilical Cord‐Derived Mesenchymal Stem Cells

The management of patients with heart failure focuses on halting the progression of disease. To date, no therapeutic approach has been able to restore cardiac function to its original baseline state. Cell therapies, such as cardiopoietic (CP) stem cells, have shown to be promising platforms to restore cardiac function. The CP platform enhances the cardioregenerative potency of mesenchymal stem cells (MSCs). CP cells are characterized by the robust expression and nuclear translocation of cardiac transcription factors such Mesoderm Posterior Basic Helix‐Loop‐Helix Transcription Factor 1 (MESP1), early cardiac transcription factor Nkx2.5, myocyte enhancer factor 2C (Mef2C), and zinc finger‐containing transcription factor GATA4. Clinical trials using CP cells showed signs of safety and efficacy in improving cardiac function in patients with heart failure. However, the costs, time, and complexities associated with the manufacturing process have precluded the adoption of the therapy as a large‐scale clinical practice. In response, we adopted a microencapsulated‐modified‐messenger RNA (M3RNA) platform as a cost‐effective and time‐saving alternative to induce high expression of cardiopoietic markers. We hypothesized that the transfection of human umbilical cord‐derived mesenchymal stem cells (hUC‐MSCs) with M3RNA encoding mesodermal and/or cardiac transcription factor(s) will induce the expression of cardiopoietic markers (MESP1, Nkx2.5, Mef2C, and GATA4). hUC‐MSCs were transfected with M3RNA encoding Brachyury (this protein plays a key role in cardiomyocyte differentiation) and MESP1, Nkx2.5, Mef2C, and GATA4 using the transfection reagent Lipofectamine STEM. Protein expression was measured and quantified using fluorescence microscopy and flow cytometry. hUC‐MSCs transfected with M3RNA encoding mesodermal and/or cardiac transcription factor(s) demonstrated rapid protein expression at 4 hours post‐transfection that reached peak levels between 24 hours and 48 hours post‐transfection. Immunocytochemistry demonstrated similar morphology between transfected and non‐transfected cells. Thus, hUC‐MSCs can be transfected with M3RNA with high efficiency to produce rapid and predictable protein expression in vitro. We here demonstrate a simple, cost‐effective, and time‐saving approach to induce the expression of cardiopoietic markers in UC‐MSCs using M3RNA, which can serve as a highly translatable platform to circumvent many burdens associated with current strategies and development of cell‐based therapies for future clinical trials and practice.Support or Funding InformationMayo Clinic Center for Regenerative Medicine, Van Cleve Cardiac Regenerative Medicine Program, Marriott Foundation, Michael S. and Mary Sue Shannon Family, NIH NHLBI HL134664, Mayo Clinic Regenerative Sciences Post‐Baccalaureate Training Program

An interview with Sally Lowell.

Sally Lowell is a Wellcome Trust Senior Research Fellow at the University of Edinburgh, UK. Her research group, which is based in the Centre for Regenerative Medicine, studies cell fate decisions in stem cells, focussing on how communication and interactions between cells can influence these decisions. Sally, who has been a long-serving committee member of the British Society for Developmental Biology, recently joined the Board of Directors at The Company of Biologists. We caught up with Sally to find out more about her career and her research, and why she's excited about her role at the Company.

Evolving Visions for Global Stem Cell Institutes

Evolving Visions for Global Stem Cell Institutes

In Response

We thank Drs Ariza and Raffan for their interest in our recent publication regarding the impact of older red blood cell (RBC) transfusion on the incidence of postoperative acute kidney injury (AKI) after orthotopic liver transplantation (OLT).1,2 In this study, we found that transfusion of older RBCs significantly increased the risk of postoperative AKI in liver transplant recipients. Due to the poor coagulation function of patients with end-stage liver disease, transfusion of fresh frozen plasma (FFP) is common during OLT. However, recent studies indicated that it is a risk factor for AKI after liver transplantation.3–5 Our study is a single center, retrospective, observational study. In retrospective observational studies, there are often baseline differences between patient populations. As expected, we found that there were indeed some baseline differences between the 2 groups including the amount of transfused FFP (see Table 1). In this regard, we used the inverse probability of treatment weighting technique. After this statistical adjustment, the baseline differences including the amount of transfused FFP between the 2 groups were eliminated (see Table 3); however, the differences in the incidence of AKI and severe AKI remained significant (see Table 4). In addition, we performed sensitivity analyses by excluding patients over 60 years of age, with a Model for End-Stage Liver Disease score <10, or anhepatic phase <60 minutes. The sensitivity analyses confirmed that receiving older RBCs was a significant risk factor for postoperative AKI and severe AKI (see Table 5). AKI is one of the most common postoperative complications of OLT. We agree with Drs Ariza and Raffan that many factors contribute to the development of AKI after OLT.1 However, please note that the focus of our study was to investigate the association between the age of transfused RBCs and post-OLT AKI, which has not been reported before. Other known or unknown factors related to postoperative AKI in liver transplant recipients apparently warrant further investigation. Regarding Ariza and Raffan’s comment on the preservation method of RBC units, we also think that more studies are needed to develop better methods of blood storage. Yue Wang, MDShaanxi Provincial Center for Regenerative Medicine and Surgical EngineeringXi'an, ChinaInstitute of Advanced Surgical Technology and EngineeringXi'an, ChinaDepartment of Hepatobiliary SurgeryFirst Affiliated HospitalXi'an Jiaotong UniversityXi'an, China Rongqian Wu, MD, PhDShaanxi Provincial Center for Regenerative Medicine and Surgical EngineeringXi'an, ChinaInstitute of Advanced Surgical Technology and EngineeringXi'an, China[email protected]

Topical Inhibition of the Electron Transport Chain Can Stimulate the Hair Cycle

Abstract While many signaling pathways have been implicated in control of activation of adult hair follicle stem cells and the hair cycle, less is known about cell intrinsic mechanisms of stem cell control. We previously identified lactate production as a key cell intrinsic regulator of hair follicle stem cell activity, suggesting that cellular metabolism is important in stem cell activation. Others have used transgenic methods to suggest that transgenic blockade of the Electron Transport Chain (ETC) leads to degeneration of the hair follicle. Here, we show that pharmacological abrogation of ETC activity, as opposed to complete ablation of ETC, can promote hair cycle activation without significant cell toxicity. Furthermore, metabolic data suggest that ETC inhibition leads to increased pyruvate accessibility for the Ldh enzyme and therefore increased lactate production, which we previously showed can promote hair cycle activation. Finally, this type of ETC inhibition can even be used to accelerate the hair cycle in aged mice. These results point toward an unexpected and safe method to promote hair follicle stem cell activation.

An interview with Jayaraj Rajagopal.

Jayaraj (Jay) Rajagopal is a Principal Investigator at the Center for Regenerative Medicine at Massachusetts General Hospital and an Associate Professor of Medicine at Harvard Medical School. A Howard Hughes Medical Institute Faculty Scholar, his lab works on the development and regeneration of the lung. He uses stem cell and animal models to develop novel insights that hopefully will provide inspiration for therapies to help treat human lung disease. He was awarded the Dr Susan Lim Award for Outstanding Young Investigator at the 2017 International Society for Stem Cell Research (ISSCR) meeting in Boston (MA,USA), where we met him to talk about how a fish tank started a life-long fascination with the lung, the transition to running his own lab, and his optimism for the future of both basic stem cell research and its clinical translation.

2018

OBJECTIVES/SPECIFIC AIMS: Translating conventional and regenerative medicine strategies from the research laboratory into the clinic is a complex process that can delay bringing novel therapies to the patient. Navigating the increasingly complex regulation surrounding cell-based and combination product technologies is a major challenge for the translational biomedical scientist. To this end, Mayo Clinic created a new position, the “Translational Integrator,” as part of the cGMP Biomaterials Facility in the Center for Regenerative Medicine. METHODS/STUDY POPULATION: The Translational Integrator educates investigators about FDA standards and regulatory pathways; determines where the product is on the translational spectrum; works to understand the science behind the product; determines what additional studies may be needed; supports investigators in preparing for FDA communications and submissions; and educates researchers about institutional resources and funding mechanisms needed to move their product into manufacturing and trials. A primary objective is to meet investigators at an early stage in product development to avoid conducting potentially redundant work to meet regulatory requirements. RESULTS/ANTICIPATED RESULTS: Robust training in clinical and translational research methodology enables the integrator to facilitate the collaboration necessary between investigators, clinicians, institutional resources, regulators and funders to move products towards FDA IND/IDE approval and first-in-human trials. It is an iterative process using technology/translational readiness criteria, project management and review by subject matter experts that is highly interactive and customized to each project. Current projects include topics in orthopedic surgery and ENT. In creating and refining this position, several key lessons have been learned. DISCUSSION/SIGNIFICANCE OF IMPACT: First, the Translational Integrator must undergo constant reflection and assessment of investigator needs, which requires flexibility and understanding that their role may change in the context of each product. Second, the support that the Translational Integrator provides can shift the mindset of the investigator from being averse to engaging in the translational process to eager to move their product forward. Finally, for the investigator who does not personally want to move their work into first-in-human trials, establishing connections to intellectual property generation and licensing may support movement of their findings into patients.

Interview with Darrell Kotton.

Dr Kotton is the David Seldin Professor of Medicine and the founding Director of the Center for Regenerative Medicine of Boston University and Boston Medical Center. He conducts basic stem cell research as an NIH-funded Principle Investigator in the Kotton Laboratory (focused on stem cell biology and gene therapy related to lung injury and repair). In addition, he attends in the Medical Intensive Care Unit, the Pulmonary Consultation Service, the Alpha-1 Center and the Pulmonary Outpatient Clinic, all at Boston Medical Center.

Mesenchymal Stem Cell‐Derived Extracellular Vesicles Improve the Renal Microvasculature in Coexisting Metabolic Syndrome and Renovascular Disease in Swine

BackgroundSuperimposition of the metabolic syndrome (MetS) on renovascular disease (RVD) amplifies intrarenal microvascular loss. Extracellular vesicles (EVs) released from adipose tissue‐derived mesenchymal stem/stromal cells (MSCs) may mediate their paracrine effect, but their efficacy to protect the renal microcirculation in RVD complicated by MetS remains unclear. Using a novel swine model, we tested the hypothesis that EVs would attenuate microvascular loss in MetS+RVD.MethodsFour groups of pigs (n=7 each) were studied after 16 weeks of diet‐induced MetS and unilateral RVD (MetS+RVD), MetS+RVD treated 4 weeks earlier with a single intra‐renal delivery of pre‐labeled EVs harvested from autologous MSCs, and Lean and MetS Sham controls. Stenotic‐kidney renal blood flow (RBF) and glomerular filtration rate (GFR) were measured in‐vivo (fast CT), whereas EV characteristics, renal microvascular architecture (micro‐CT), and injury pathways ex‐vivo.ResultsmRNA sequencing of EVs and their parent MSCs revealed that EVs were enriched with pro‐angiogenic genes, including vascular endothelial growth factor (VEGF)‐A, VEGF–C, and Von Willebrand Factor (vWF)‐A Domain Containing‐1 (VWA)‐1 (Figure A). In addition, protein expression of VEGF and vWF was higher in EVs compared to MSCs (Figure B). EVs were detected in the stenotic‐kidney ex‐vivo internalized by tubular and endothelial cells. EVs restored renal expression of the pro‐angiogenic factors VEGF, Notch‐1, and Notch ligand delta‐like‐4 (DLL4) and improved cortical microvascular density (Figure C–D). Tubulointerstitial fibrosis increased and RBF and GFR decreased in MetS+RVD vs. MetS, but were restored in MetS+RVD+EV (Figure E–F).ConclusionsIntra‐renal delivery of MSC‐derived EVs bearing pro‐angiogenic properties in swine MetS+RVD restored stenotic‐kidney microcirculation, and in turn its hemodynamics and function. Our study suggests a novel therapeutic potential for MSC‐derived EVs in restoring renal function in chronic experimental MetS+RVD.Support or Funding InformationThis study was partly supported by NIH grant numbers DK73608, DK104273, HL123160, and DK102325, DK106427, the Mayo Clinic Center for Regenerative Medicine, and the Mayo Clinic Mary Kathryn and Michael B. Panitch Career Development Award.

Primary cilia are present on human blood and bone marrow cells and mediate Hedgehog signaling.

Primary cilia are nonmotile, microtubule-based organelles that are present on the cellular membrane of all eukaryotic cells. Functional cilia are required for the response to developmental signaling pathways such as Hedgehog (Hh) and Wnt/β-catenin. Although the Hh pathway has been shown to be active in leukemia and other blood cancers, there have been no reports describing the presence of primary cilia in human blood or leukemia cells. In the present study, we show that nearly all human blood and bone marrow cells have primary cilia (97-99%). In contrast, primary cilia on AML cell lines (KG1, KG1a, and K562) were less frequent (10-36% of cells) and were often shorter and dysmorphic, with less well-defined basal bodies. Finally, we show that treatment of blood cells with the Hh pathway ligand Sonic Hedgehog (SHh) causes translocation of Smoothened (SMO) to the primary cilia and activation of Hh target genes, demonstrating that primary cilia in blood cells are functional and participate in Hh signaling. Loss of primary cilia on leukemia cells may have important implications for aberrant pathway activation and response to SMO inhibitors currently in clinical development.

Corrigendum: Adipose-derived Stem Cell Conditioned Media Extends Survival time of a mouse model of Amyotrophic Lateral Sclerosis.

Scientific Reports 5: Article number: 16953; published online: 20 November 2015; updated: 15 April 2016. In this Article, an additional affiliation for Keith L. March was omitted. The correct affiliation is listed below: Krannert Institute of Cardiology and the Indianapolis VA Center for regenerative medicine

Surface Topography During Neural Stem Cell Differentiation Regulates Cell Migration and Cell Morphology

We sought to determine the contribution of scaffold topography to the migration and morphology of neural stem cells by mimicking features of scaffolds found in vivo. We mimicked two types of CNS scaffolds encountered by neural stem cells during development in vitro by constructing different diameter electrospun polycaprolactone (PCL) fiber mats, a substrate that we have shown to be topographically similar to brain scaffolds. We compared the effects of large fibers (made to mimic blood vessel topography) with those of small diameter fibers (made to mimic radial glial process topography) on the migration and differentiation of neural stem cells. Neural stem cells showed differential migratory and morphological reactions with laminin in different topographical contexts. We demonstrate, for the first time, that neural stem cell biological responses to laminin are dependent on topographical context. Topography‐induced cell morphology changes were inhibited by nocodazole, but not by cytochalasin‐D or ROCK‐inhibitor, suggesting that microtubule function is necessary for topography‐induced morphology changes. We propose that the physical structure of distinct scaffolds induces unique signaling cascades that regulate migration and morphology in embryonic neural progenitor cells.Support or Funding InformationBS acknowledges Pelotonia Foundation Undergraduate Research Fellowship. AS acknowledges The Ohio State University/Metro High School Graduate Teaching Assistant Fellowship. The project described was supported by Award Number Grant 8UL1TR000090‐05, from the National Center for Advancing Translational Sciences. The content is solely the responsibility of the authors and does not necessarily represent official views of the National Center for Advancing Translational Sciences or the National Institutes of Health. This work was sponsored by and represents activity of The Ohio State University Center for Regenerative Medicine and Cell Based Therapies (regenerativemedicine.osu.edu).

Mesoporous nanocarriers with internal cavity for the delivery of siRNA for bone regeneration

Event Abstract Back to Event Mesoporous nanocarriers with internal cavity for the delivery of siRNA for bone regeneration Jungju Kim1, 2, Tsendmaa Bold1, 2, Jeong-Hyun Ryu1, 2, Roman Perez1, 2 and Hae-Won Kim1, 2, 3 1 dankook university, Tissue Regeneration Engineering, Korea 2 dankook university, Nanobiomedical Science & BK21 PLUS NBM Global Research Center for Regenerative Medicine, Korea 3 dankook university, Biomaterials Science, College of Dentistry, Korea Introduction: Nanocarriers have great interest in the field of tissue engineering for the delivery of drugs and genes. Among other issues, the loading of those molecules in sufficient amount and the sustained release are considered of special importance for therapeutic efficacy of the delivery system. For this purpose, we developed a mesoporous silica nanocarrier with an internal cavity to allow high loading capacity and sustained delivery of small interference RNA (siRNA). Here, we delivered siRNA to downregulate Plekho-1 gene, which has been shown to significantly enhance bone regeneration. We investigated the ability to load and release the siRNA as well as the cellular interaction and osteogenic potential. Materials and Methods: Apatite nanorods were used as sacrifice core templates which were then sheathed with mesoporous silica through a sol-gel reaction. Apatite was then selectively removed by an acid treatment. The surface of the nanocarriers was aminated with APTES for proper siRNA loading. The nanocarriers were characterized by transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy (EDX), Fourier transform infrared spectroscopy (FT-IR), and Brunauer Emmett Teller (BET). The loading and delivery of the siRNA was quantified in the range of 10 to 100 µg/ml. In order to observe cellular interactions, pre-osteoblastic cells were cultured with the siRNA-loaded nanocarriers at the different siRNA concentrations. The cell viability, intracellular uptake capacity, gene silencing efficiency, and bone-related gene expressions were investigated. Results and Discussion: Mesoporous and internally-hollowed silica nanocarriers were produced well, as observed by TEM images, which presenting an internal cavity of ~20 nm. Compared to the dense nanocarriers, the surfaces area and pore volume of developed nanocarriers. Due to the enhanced surface area and pore volume, the siRNA loading capacity increased 5-fold. The unique pore morphology and characteristics allowed sustained release of the siRNA from the developed nanocarriers for up to 18 days, which was significantly different from the dense nanocarriers which completely released the cargo after only 3 days. Cellular viability of the developed nanocarriers was high for the studied concentrations, allowing therefore high doses of the nanocarriers and the loading of siRNA to be delivered. The siRNA-loaded nanocarriers were successfully taken up by the pre-osteoblastic cells, with a cellular uptake ~90%, and silenced the expression of the target gene by 80%, which subsequently induced an upregulation of osteoblasts-related genes. Conclusion: Mesoporous nanocarriers with an internal cavity showed the capacity of high loading and sustained release of siRNA molecules. The siRNA-delivering nanocarriers successfully downregulated Plekho-1 target genes while upregulating osteogenic genes in vitro. Further in vivo study remains to investigate the practical efficacy of the nanocarriers for bone regeneration.

The Thymus: A Forgotten, But Very Important Organ.

Medical science seems to be on the threshold of a revolution: It seems possible that in twenty years, doctors will be able to replace organs in the human body like parts in a car. This is thanks to the recent achievement of a team from the Medical Research Council Center for Regenerative Medicine in Edinburgh, Scotland - the group of researchers tried to regenerate the thymus gland in mice. The thymus gland is an essential organ for the development of the immune system, but very few people have any idea that it exists. In the literature and also in people's awareness, the fact is often that the thymus controls and harmonizes the entire immune system and the immune functioning of the organism. It is the primary donor of cells for the lymphatic system, much as bone marrow is the cell donor for the cardiovascular system. It is within the thymus that progenitor cells are created and then undergo maturation and differentiation into mature T cells. The thymus gland is located in the mediastinum, behind the sternum. It is composed of two identical lobes. Each lobe is divided into a central medulla and a peripheral cortex. The thymus is at its largest and most active during the neonatal and pre-adolescent periods. After this period the organ gradually disappears and is replaced by fat. In elderly individuals the thymus weighs 5 g. The aim of this work is to shed new light on this important immune defense organ, whose function is not confined to the destruction of nonfunctional T cells.

Identification of the origin of circulating endothelial progenitor cells

Identification of the origin of circulating endothelial progenitor cells

Impact of a six-week olive oil supplementation in healthy adults on urinary proteomic biomarkers of coronary artery disease, chronic kidney disease and diabetes

Impact of a six-week olive oil supplementation in healthy adults on urinary proteomic biomarkers of coronary artery disease, chronic kidney disease and diabetes

Mesenchymal Stem Cell Survival after Intraspinal Transplantation

Following spinal cord hemisection at C2 (SH), removal of descending excitatory drive to phrenic motoneurons results in ipsilateral diaphragm muscle paralysis with partial spontaneous recovery of rhythmic phrenic activity over time. Brain‐derived neurotrophic factor (BDNF) plays an important role in neuroplasticity following SH. Intraspinal transplantation of mesenchymal stem cells engineered to produce BDNF‐GFP (BDNF‐MSCs) at the time of injury promotes functional recovery of all animals, despite low levels of MSC survival at 14 days post‐SH. The goal of the present study was to determine the survival and localization of BDNF‐MSCs after intraspinal transplantation. BDNF‐MSCs were injected intraspinally at C4 (2 sites, total 2 x 105 cells in 4 µl) in adult male Sprague‐Dawley rats. After one and three days, the cervical spinal cord was fixed, excised, and cut longitudinally. Survival of transplanted BDNF‐MSCs was confirmed in all animals at 1 day post‐transplantation (n=5) and in five of 6 animals at 3 days post‐transplantation. BDNF‐MSCs remained localized at the site of injection in the white matter at 1 and 3 days, with an average volume of 1.1 ± 0.3 µl. There was no localization of MSCs in the vicinity of phrenic motoneurons identified by retrograde labeling with cholera toxin subunit B. Understanding the time course of stem cell survival after transplantation is important in defining windows of opportunity during which the beneficial effects of BDNF on respiratory function after spinal cord injury are executed.Supported by HL96750 and Mayo Clinic Center for Regenerative Medicine.

Letter

Department of Nanobiomedical Science and BK21PLUS, NBM Global Research Center for Regenerative Medicine, Department of Rehabilitation Medicine, College of Medicine, Dankook University, Cheonan, Republic of Korea The manuscript submitted does not contain information about medical device(s)/drug(s). No funds were received in support of this work. No relevant financial activities outside the submitted work.