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Bone marrow mesenchymal stem cells

Bone marrow mesenchymal stem cells. Orthodox plasticity Progenitor cells of the skeletal tissues Unorthodox plasticity Astrocytes Azizi et al., 1998 Neurons Woodbury et al., 2000 Myocytes Orlic et al., 2001 Hepatocytes Lagasse et al., 2001 Myelination of spinal cord Akiyama et al., 2002.

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Bone marrow mesenchymal stem cells

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  1. Bone marrow mesenchymal stem cells • Orthodox plasticity Progenitor cells of the skeletal tissues • Unorthodox plasticity AstrocytesAzizi et al., 1998 NeuronsWoodbury et al., 2000 MyocytesOrlic et al., 2001 HepatocytesLagasse et al., 2001 Myelination of spinal cordAkiyama et al., 2002 • Rapidly dividing • Obtained from adult bone marrow • Differentiation to glial cells • Potential for implantation in PHB conduits

  2. Mesenchymal stem cell Proliferation Osteogenesis Chondrogenesis Adipogenesis Commitment Transitory osteoblast Transitory chondrocyte Preadipocytes Lineage progression Osteoblast Chondrocyte Adipocytes Differentiation Maturation FAT CARTILAGE BONE Mesenchymal lineages

  3. Osteogenic differentiation Alizarin Red x20 Differentiation medium; 10mM -glycophosphate, 100g/ml ascorbic acid, 10-7 M dexamethasone for 3 weeks

  4. Stem cells culture Aspiration and centrifugation of marrow from adult long bones Plated and haematopoietic non-adherent cells removed through washings uMSCs passaged, split and replated (T2 to T8) Differentiation with PDGF, FGF and GGF for 2 wks

  5. Stem cell differentiationin vitro with GGF Schwann cells uMSC D7 post differentiation D14 post differentiation

  6. S100 Undifferentiated MSCs Schwann Cells Differentiated MSCs P75

  7. 33µm DRG functional assay SC, uMSC and dMSC seeded onto 1µm pores insert Filter inserted into DRG cultures DRG dissected and processed to single cell suspension Co-culture 24h + ß-tubulin staining DRG cells seeded onto coverslips 3) Neurite density 1) % of sprouting DRG 2) Neurite length

  8. DRG + stem cells co-culture uMSC BS medium dMSC SC

  9. PHB conduit + stem cells 4 * 3 2 PHB 1 Axon Regeneration (mm) 0 Con dMSC SC dMSC + GFP 2 weeks

  10. Bioengineered nerve conduits • Schwann cells More experimental information available Likely to be used clinically in near future • Stem cells Define characterisation & stability Differentiated vs. undifferentiated Clinical use to be in far future

  11. Tissue engineering Material science Cellular biology Construct for transplantation Surgery Pharmacology Engineering

  12. Biomaterial scaffold • Antigenicity & compatibility • Source of material synthetic polymer (silicone) animals (collagen) bacterial (PHB) plants (alginate) • Biodegradable / resorbable scaffold in the regeneration microenvironment • Surface modification ECM molecules, growth factors • Toxicology assessment

  13. Stem cells • Capable of rapid proliferation and multi-lineage differentiation = potential for tumours ? • Limited immunogenicity of undifferentiated cells = maintained in differentiated cells ? • Possibility to have “ready to use” cultured cells or cell lines for implantation = cell banks ? Differentiated cells Progenitor cell Multipotent progenitors (adult) Totipotent progenitors (embryonic)

  14. Transplant of cells • Autologous vs. allogeneic • Genetic modification / marking of cells • Embryonic stem cells primary culture, cell lines ethical problems feeder layer • Adult stem cells bone marrow mesenchymal stem cells (MSC) adipose derived stem cells (ADSC) olfactory ensheathing cells (OEC) tissue endogenous (muscle, brain, skin, retina …) • Growth factors used for differentiation of stem cells

  15. The Human Tissue Act (2004)What is it? • The Human Tissue Act (2004) is the legislative framework for the collection, storage and use of human tissue (currently interpreted as anything containing cells) from the living or deceased. • It replaces the Human Tissue Act 1961, the Anatomy Act 1984 and the Human Organ Transplants Act 1989.

  16. What is in the Act? • The HT Act identifies scheduled purposes that are lawful to perform under certain conditions • Scheduled purposes exempt from consent (tissue for diagnosis) • Scheduled purposes requiring consent • Scheduled purposes that require a licence from the regulatory body • It provides for the creation of a regulatory body for implementation of the Act (The Human Tissue Authority). • Effective from 1st September 2006

  17. Scheduled Purposes requiring consent • Anatomical examination • Determining the cause of death • Obtaining scientific or medical information (i.e. drug efficacy, medical treatment) about a living or deceased person which may be relevant to any other person • Transplantation • Research in connection with disorders or functioning of the human body • Public display

  18. Scheduled purposes requiring a licence • Storage of anatomical specimens anatomical examination • Post-mortem examination, removal and storage of relevant material • Tissue for transplantation • Storage of material from a living person for research (other than for a specific ethically approved project) or for human application • Public display of a body or material from a deceased person

  19. PRIMARY PURPOSE:RESEARCH Is it stored for a specific ethically-approved research project? Yes No Isconsent required? Is a licence required? Is consent required? Is a licence required? Yes, unless material is obtained from a living person and is anonymised No Yes Yes Research and the Human Tissue ActWhat do you need to have in place?

  20. The Licensing Structure The University of Manchester Licence Holder The University of Manchester (Professor Maynard Case) Designated Individual for the Scheduled Purpose Professor Giorgio Terenghi (Research) Professor Alan North (Anatomy) Person Designated as a person to whom the licence applies Persons working under the licence

  21. What material is subject to HTA? • Any material consisting of or containing cells of immediate human origin. • Any other material collected for DNA analysis (including hair and nails) What is not covered by the Act? • Hair, nails and gametes (except for DNA analysis) • Cell lines • Embryos • Tissue from individual died more than 100 years ago

  22. A typical Human Tissue project A researcher at the University would like to collect tissues in collaboration with colleagues at a NHS Trust for a research project. Six steps are identified that need to be followed.

  23. The six steps • Informed consent taken from the donors • Notification of the Designated Individual at the University (the recipient of the tissue) • Notification of the Designated Individual at the NHS Trust (the provider of the tissue) • Transfer Agreement put in place between Trust and University • Written evidence made available that proves staff are appropriately trained and qualified to perform the tasks they are responsible for. • Clear records kept of the location of the tissue and of its disposal, exhaustion or transfer

  24. Tissue engineering construct • Cell preparation in Clean Rooms GMP and ISO 9000 • Sterility of biomaterial scaffold autoclave ethylene oxide UV irradiation • Delivery to patient double bagging of construct surgery • Non-invasive monitoring of successful grafting

  25. Clean Room commissioning • Construction and surface finishes • Air supply and flow measurements Differential pressure HEPA filter • Microbiological monitoring settle and contact plates • GMP training for staff

  26. Staff training seminar • Contamination • Standards and monitoring • Class II cabinets • Garments • Gloves, masks and wipers • Sterile consumables • Cleaning the clean rooms

  27. Contamination • Airborne particles particle concentration lab, office, clean rooms • Human sources skin flakes, hair, airborne droplets • Clothing fibres • Dust and spores

  28. Standards & monitoring • Environmental monitoring monthly settle plates, surface swabs, finger dabs • Active air sampling quarterly hand-held vs continuous monitoring • Disinfectant testing six-monthly before and after use • Physical monitoring quarterly airborne particle counts, air velocity measurements, pressure differential monitoring, laminar flow cabinets • Annual quality audit

  29. Science to patient translation The future looks bright …. but full of rules and regulations

  30. Nerve Regeneration Group Sue Shawcross Paul Kingham Cristina Mantovani Daljeet Mahay Stephanie Armstrong Adam Reid Alex Hamilton Jenny Caddick & Chris West

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