Novel Topical Mesenchymal Stem Cell Transplant Paradigm to Treat Organ Diseases

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Novel Topical Mesenchymal Stem Cell Transplant Paradigm to Treat Organ Diseases by Dr. Ping Kuen Lam, Dr. Kevin K. W. Wang, Dr. Paul BS Lai & Dr. Wai S. Poon Mesenchymal Stem Cells as Organ Treatment Therapy

Somatic stem cells attract great scientific and public interest and have real appeal for tissue repair and regeneration. Hematopoietic cells and mesenchymal stem cells are the two major adult stem cells. Mesenchymal Stem Cells (MSCs) were initially found in bone marrow by Friedenstein and his colleagues. Subsequent work showed that MSCs are also present in adipose tissue, placenta, etc. MSCs were characterized as adherent, clonogenic and fibroblastic in appearance. They express CD105, CD73 and CD90 and are negative for CD45, CD34 and CD14. They are a heterogeneous population of multipotent adult stem cells with capacities of prolonged self-renewal and differentiation potential into mature cells of various lineages. Unlike the protocols using embryonic stem cells in regenerative medicine is hindered by ethnical issue and risk of tumor formation, clinical application of autologous MSCs is safe and feasible. In fact, MSCs express human leukocyte antigen (HLA) major histocomplex class (MHC) I and are negative for MHC II and Fas ligand, which enable an allogeneic transplant without immunosuppression. It was initially believed that MSCs differentiated into respective cells of injured tissue, integrated into the disease organ with subsequent restoration of tissue function. More recent data suggest that the therapeutic effects are mediated by paracrine and endocrine actions, resulting in inhibition of inflammation and apoptosis, and also stimulation of mitosis, angiogenesis, proliferation, differentiation of resident tissue-specific precursor stem cells and tissue matrix remodeling. Thus, MSCs have advantages over pharmacological agents that usually target a single pathophysiological cascade of a disease, whereas MSCs work through multiple mechanisms of immunological, inflammatory, vascular and regenerative pathways. The recruitment of MSCs to the injured tissue is the prerequisite for cell therapy. Systemic infusion is the most commonly used administration in animal studies and clinical trials of MSCs. Injured and inflammatory tissues express specific receptors or ligands that facilitate trafficking of MSCs. On the other hand, expression of chemokine and extracellular matrix receptors on the surface of MSCs may account for their homing capacity. It is likely that intravenously infused MSCs preferentially home to the site of injury, probably via SDF1/CXCR4 pathway. However, most of the infused MSCs could be trapped by pulmonary capillaries because of their larger mean

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diameter. The retention of large amount of MSCs in normal somatic organs may cause unfavorable complications. Delivery of infused MSCs to the brain is further hindered by the blood brain barrier unless it is severely damaged to allow the passage of MSCs. The threat of arterial embolism and occlusion also limit intra-arterial administration of MSCs. Direct injection of MSCs through Hamilton syringe into vascular organs like brain and liver, is associated with high risk of bleeding. A fatality report in the Telegraph newpaper prompted us to revisit the primary consideration of safety when injecting stem cells directly into somatic organs - an 18-month old baby died after receiving a therapeutic intracerebral injection of stem cells [1]. Moreover, repeated trituration of MSC suspension to avoid cell clumps before implantation through Hamilton syringe may inevitably damage the cell membrane, and hence affects cell viability and integration into host tissue. Therefore, an effective and minimally invasive transplantation method is highly desirable.

Topical Application of MSCs as a Disruptive Technology

Tissue engineering technology is commonly adopted for transplantation of cultured epidermal skin graft to burn wounds and chronic wounds. By borrowing the concept from the tissue engineering technology, we topically applied MSCs which were derived from GFP-transgenic Sprague-Dawley (SD) rats to the surface of somatic organs such as brain, liver, etc. immediately after experimental models of traumatic brain injury (TBI) and hepatic ischemia-reperfusion injury (IRI), respectively [2, 3]. TBI was induced by driving a 3mm diameter tip of an electromagnetically controlled cortical impact device over the exposed parietal cortex of an anesthetized SD rat. On the other hand, IRI was induced by clamping portal vein and hepatic artery for 30 minutes. The MSCs were fixed in a position by a thin layer of fibrin (Baxter). Few days after topical application, immunohistochemistry staining using anti-GFP antibody showed the presence of some GFP-positive cells in the cerebral cortex (Figure 1) and liver parenchyma (Figure 2). These cells expressed the markers of neuron, GFAP and hepatocytes respectively. Compared with the controls (TBI/IRI without any treatment), neurogenesis and liver regeneration were significantly enhanced by the topical MSCs in the respective animals. No GFP-positive cells were found in other organs including the lung. The cellular and molecular

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FEATURES mechanism leading to the trafficking of topical MSCs remains to be determined. As topical MSCs did not pass through the bloodstream, the mechanism might differ from the chemokine gradient theory proposed for systemically infused MSCs. Homing of topical MSCs was not observed in organs without injury. Fibrin has long been used as a hemostatic sealant in surgery. It can be utilized as a vehicle for the engraftment of cultured keratinocytes onto the burns and chronic wounds. Experiments showed the topical application without fibrin failed to firmly hold the transplanted MSCs onto the recipient organ surface. Subsequently, the MSCs shed off from the target organs. Fibrin enhances the attachment of MSCs on the recipient surface of somatic organs and provides a three-dimensional template for cellular proliferation of MSCs before it is completely absorbed.

potential (Schematic 1). Compared with systemic infusion method, topical application does not raise the concerns of possible complications caused by retention of MSCs in normal organs, such as arterial embolism and occlusion. Although only a small amount of topical MSC homed to the injured site, the biological roles played by the MSCs residing on the surface of diseased organ should not be ruled out. If these MSCs release paracrine and/or endocrine factors, topical application could offer an additional therapeutic potential because these trophic factors would be delivered to the site close to the target organ directly. Projecting into the future, topical transplantation of MSC and other stem cell types could be added into the expanding batteries of cell therapy strategies in treating various organ injuries or organ diseases [4].

Figure 1

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Figure 2 Topical GFP-MSCs migrated into the cerebral cortex (Figure 1) and liver parenchyma (Figure 2) (Both IHC X400)

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Beneficial Effects of MSC

Topical application offers a direct and highly efficient delivery of MSCs to the target organs. As a large amount of MSCs can be topically applied on the surface of somatic organs by a single minimally invasive procedure, this method offers a great therapeutic www.asiabiotech.com

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Mendick R, Alasdair P: Baby death scandal at stem cell clinic which treats hundreds of British patents a year. The Telegraph, 23 October 2010. http://www.telegraph.co.uk/ news/worldnews/germany/8500233/Europe-largest.8 2010 Lam PK, Ng CF, To KF, Ng SS, Mak TW, Chan ES, Lo AW, Lai FM, Poon WS, Lai PB. Topical application of mesenchymal stem cells to somatic organs--a preliminary report. Transplantation. 2011 Jul 27;92(2):e9-11. PMID: 21747276 Lam PK, Lo AW, Wang KK, Lau HC, Leung KK, Li KT, Lai PB, Poon WS. Transplantation of mesenchymal stem cells to the brain by topical application in an experimental traumatic brain injury model. J Clin Neurosci. 2013 Feb;20(2):306-9. PMID: 23219830 Wu X1, Ren J, Li J. Fibrin glue as the cell-delivery vehicle for mesenchymal stromal cells in regenerative medicine. Cytotherapy. 2012 May;14(5):555-62. PMID: 22175911

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FEATURES About the Authors Ping Kuen Lam, PhD received his PhD from The Chinese University of Hong Kong in 1999 in the area of tissue engineering and wound healing. He is the Honorary Research Fellow of Department of Surgery, and General Manager of the Chow Tai Fook-Cheng Yu Tung Surgical Stem Cell Surgical Research Center, Faculty of Medicine, The Chinese University of Hong Kong. His current research focuses on translational stem cell research as well as clinical trials of adult stem cells. He and his colleagues found that topically applied mesenchymal stem cells can migrate from the recipient surface of somatic organs into the injured parenchyma in various rodent models.

Paul B.S. Lai, MD graduated from the Chinese University of Hong Kong. He received his training as a specialist hepato-biliary and pancreatic surgeon in Hong Kong and Edinburgh. He is currently the Honorary Chief of Service of the Department of Surgery at the Prince of Wales Hospital and Professor & Chief, Division of Hepato-biliary and Pancreatic Surgery, the Chinese University of Hong Kong. Prof. Lai has been involved in basic and clinical research in surgery and significant amount of work has been focused on liver cancer. Prof. Lai is also the Cluster Coordinator for Surgical Services at the New Territories East Cluster of Hospital Authority and the Director of the Office of Medical Education, Faculty of Medicine of CUHK. He is currently the Censor-in-Chief of the College of Surgeons of Hong Kong and Chief Editor of the College’s official journal – Surgical Practice. He has been the Director of the Surgical Outcome Monitoring and Improvement Program (SOMIP) in the Hospital Authority since 2014. Prof. Lai has also developed an interest in surgical outcome research, and he has launched full-coverage surgical outcome audit for all surgical procedures since 2010 at the Department of Surgery in the Prince of Wales Hospital.

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Wai S. Poon, FRCS received his undergraduate medical education at Glasgow University (1973-8), general surgical training at the City and University Hospitals, Nottingham, U.K. (1980-1982), neurosurgical training at Glasgow's Institute of Neurological Sciences (1983-6), experimental cerebral ischaemia at Glasgow University’s Wellcome Surgical Institute with Jim McCulloch (1986) and experimental neuro-oncology at Harvard Medical School's Massachusetts General Hospital (1990-1). Dr Poon is currently the Chair Professor and Chief of the Division of Neurosurgery and Director of Brain Tumour Centre, Department of Surgery, Prince of Wales Hospital, the Chinese University of Hong Kong. He is active in undergraduate and postgraduate surgical education, and specialist training in neurosurgery, as Chairman of the Specialty Board in Neurosurgery. His clinical interests include pituitary surgery and surgical management of Parkinson's disease; research interests include clinical and experimental head injury, hyponatraemia, telemedicine, neurorehabilitation and neuro-oncology. He is author and co-author of more than 360 peer-reviewed articles.

Kevin K.W. Wang, PhD is currently at the Departments of Psychiatry and Neuroscience of University of Florida McKnight Brain Institute as the Director of the Program for Neurotrauma, Neuroproteomics & Biomarkers Research /Associate Professor. Dr. Kevin Wang is internationally recognized for his original contributions to the fields of traumatic brain injury (TBI)-linked proteolytic enzymes, therapeutic targets, neuroproteomics/ systems biology, biomarker discovery and validation. The clinical diagnostic utility for two TBI protein biomarkers during the acute phase of brain injury has now been confirmed in peer-reviewed journals. These TBI diagnostic biomarker tests are now moving forward to FDA-approval seeking pivotal clinical study. His current research directions include studying mechanisms for CNS injury and substance abuse-induced brain perturbation using systems biology approach. He published more than 200 peer-reviewed papers, reviews and book chapters and co-authored eight US patents and co-edited four books. Dr. Wang is also past President (2011-12) and current Councilor (2013 - present) of the National Neurotrauma Society (USA).

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