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Basic principles

Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s P rogrammes at the University of Pécs and at the University of Debrecen Identification number : TÁMOP-4.1.2-08/1/A-2009-0011.

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Basic principles

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  1. Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat theUniversity of Pécs and at the University of Debrecen Identificationnumber: TÁMOP-4.1.2-08/1/A-2009-0011

  2. Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat theUniversity of Pécs and at the University of Debrecen Identification number: TÁMOP-4.1.2-08/1/A-2009-0011 Dr. Judit Pongrácz Threedimensionaltissuecultures and tissueengineering–Lecture 1 Basic principles

  3. Aim of the course • To provide theoretical background knowledge to complex tissue cultures

  4. Course contents

  5. What is tissue engineering? • Tissue engineering was previously known as a subfield of engineering and biomaterials, but having grown in scale and significance tissue engineering has become a discipline of its own. • Tissue engineering aims to regenerate or recreate specific tissues or full organs with maintained or recreated biological function (bone, cartilage, blood vessels, bladder, skin, etc). • To achieve the aims tissue engineering uses a combination of techniques including cell culture, engineering bio-materials, biochemical and biophysical methods.

  6. Basic principles of tissue engineering Cells from a biopsy or resection Grafting Biopsy Generation of a graft Culture in 3D conditions (scaffold based or scaffold free) Expanded cell culture Monolayer cell culture

  7. 2D tissue cultures Adherent Non-Adherent Trypsin No trypsin Suspension Pelleting Re-suspension Seeding

  8. 3D tissue cultures No diffusion Diffusion of nutrients and oxygen Apoptotic death Secondary necrosis Healthy tissue Necrotic tissue

  9. Engineering tissues In vitro In vivo Blood cell regeneration (stimulation of stem cell proliferation and differentiation) Cell injection into injured tissues (myocardiocytes) Large injury regeneration (bio-absorbable scaffolds with or without growth factors) • Tissue regeneration (tissue proliferation and regeneration on artificial scaffold) • Bio-artificial organs (liver, skin, pancreas)

  10. Bioreactor • Bioreactorsareneededtoavoidnecrosis Acid Antifoam Base Substrate Spinnerflask bioreactor Industrialbioreactor Peristaltic pumps Foam Heatervessel T ProcessContoller pH Pump Waterin pO2 Counterpressure valve Safetyvalve Water out Q valve Electromagnetic valveforcooling Air Q Drive

  11. Potential uses of cell and tissue replacement therapy • Stroke • Traumatic brain injury • Alzheimer’s disease • Parkinson’s disease • Missing teeth • Wound healing • Bone marrow transplantation • Spinal cord injury • Osteoarthritis • Rheumatoid arthritis • Crohn’s disease • Baldness • Blindness • Deafness • Myocardial infarction • Muscular dystrophy • Diabetes • Multisite cancers • Amyotrophic lateral sclerosis

  12. Cells in tissue engineering • Stem cells • Embryonic • Cord blood • Bone marrow or adipoid tissue derived • Adult, tissue specific • Mature differentiated cells • Tissue specific

  13. Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat theUniversity of Pécs and at the University of Debrecen Identification number: TÁMOP-4.1.2-08/1/A-2009-0011 Dr. Judit Pongrácz Threedimensionaltissuecultures and tissueengineering – Lecture 2 Stem cells (1)

  14. Stem cells • Totipotent • Pluripotent • Multipotent

  15. Stemcelltypes Fertilized oocyte 8 cellembryo Blastocyst Culturedstemcells Neuralcells Bloodcells Totipotent Pluripotent Brain Neuralcells Bonemarrow Bloodcells Multipotent Multipotent

  16. Types of stem cell replications I Parentalstemcell Self-renewal Asymmetric, replicating, differentiatingdivision Self-renewed daughtercell Daughtercell Symmetric, replicatingdivision Symmetric, differentiatingdivision

  17. Types of stem cell replications II Proliferationcapacity Differentiationcapacity Asymmetric division Amplificationof cell numbers Differentiationvia progenitor cells Stem cell Terminally differentiated cells Proliferatingcompartment

  18. Sources of Stem Cells • Adult (somatic) stem cells (ASC) • Adult multipotent stem cells • Embryonic stem cells

  19. Epiblaststemcells(EpiSC) I • Epiblast is a tissuethatformsatalaterstagethan ES cells. The epiblastsformsafterthedevelopingembryoimplantsintotheuterus.

  20. Epiblaststemcells(EpiSC)II Embryonic germcells (EG) Extraembryonic endoderm (XEN) cells Trophoblast stemcells (TS) Embryonic stemcells (ESC) EpiSCs IPS cells Primitive endoderm Trophectoderm (TE) Epiblast • Somaticcelllineages • Endoderm • Mesoderm • Ectoderm Dedifferentiation and reprogramming Fertilization Primordial germcells (GSC) Morula Lateblastocyst

  21. Location of multipotentadultsomaticstemcellswasidentified Formerlyidentifiedin: Brain Recentlystemcellshavebeenidentifiedinpracticallyalltissues Skin Bonemarrow

  22. Adult (somatic) stem cells (ASC) • The microenvironment where stem cells live are called stem cell niches: ASC in:

  23. The adult stem cell niche • Stem cellniches defined as microenvironments:

  24. Marrow stem cells (MSCs) • Hematopoietic stem cells • Mesenchymal stem cells • Bone Marrow Stromal Stem cells • Endothelial Progenitor cells

  25. Hematopoietic stem cells(HSC) I • ThyloLin-Sca-1+ • CD4 and Mac-1 based fractionation resolved three populations and only the Lin-Mac-1-CD4- population was highly enriched for long-term reconstituting HSCs • c-kit+Sca-1+ThyloFlk-2- long-term repopulating HSC and c-kit+Sca-1+ThyloFlk-2+ short-term repopulating HSC • Long term repopulating HSCs in mice c-kit+ThyloLin-(Flk-2-)Sca-1+

  26. Hematopoieticstemcells(HSC) II • In human samplesCD34 antigen(functionunknown) is expressed • Additionalmarkers: CD38, CDE90 and CD133 • Thy+CD34+ long-termculturesgeneratingbothmyeloid and lymphoidlineages • Thy-CD34+ no long-termculturesgeneratingbothmyeloid and lymphoidlineages

  27. Hematopoietic stem cells(HSC) III Multipotential hematopoietic stem cell (Hemocytoblast) Hematopoiesis in Humans Commonmyeloid progenitor Common lymphoid progenitor Myeloblast Proerythroblast (Pronormblast) Lymphoblast Megakaryoblast B. promyelocyte N. promyelocyte E. promyelocyte Monoblast Basophilic erythroblast Promegakaryoblast Prolymphocyte Polychromatic erythroblast B. myelocyte N. myelocyte E. myelocyte Promonocyte Orthochromatic erythroblast(Normoblast) B. metamyelocyte N. metamyelocyte E. metamyelocyte Small lymphocyte Megakaryocyte Natural killer cell Polychromatic erythrocyte(Reticulocyte) B. band N. band E. band T lymphocyte B lymphocyte Monocyte Thrombocytes Erythrocyte Basophil Neutrophil Eosinophil Mast cell Macrophage Myeloid dendritic cell Plasma cell Lymphoid dendritic cell

  28. Mesenchymal stem cells (MSC)I • Lacking hematopoietic markers (CD2, CD3, CD4, CD8, Mac-1/CD11b, CD14, CD15, CD19, CD20, B220, CD45, Thy1 and myeloperoxidase) • Express: LDL receptor, alkaline phosphatase, smooth muscle actin, type IV collage, laminin, factor VII, MUC18, CD29, CD44, CD49A-F, CD51, CD73, CD105, CD106, CD166+

  29. Mesenchymal stem cells(MSC)II • MSCs can directly be isolated from bone marrow based on NGFR (CD271), SSEA-1, SSEA-4, CD140b , CD340 (HER-2), CD349 (Frizzled9) • Purification is based on: CD29+, CD44+, CD73+, CD105+, CD106+, CD166+ and lack of hematopoietic receptors

  30. Endothelial progenitor cells (EPC) • Adult EPCs CD34+ or Flk-1+ (VEFR-2) • Additional markers: CD31, Tie2 and E-selectin, eNOS, LDLR, VEGFR-1,-2

  31. Bone marrow progenitor cells • HSC and EPC lineages follow similar temporal and spatial development • HSC and EPC shared markers: VEGFR-2 (Flk-1), SCL/Tal1, Runx1 • Existence of the hemangioblast

  32. Ontogeny of tissue lineages in bone marrow Hemangioblast (Flk-1+VEcadherin+CD45-) Meso-Angioblast HSCs Osteoblasts MSCs EPCs Stromal Cells Pericytes SMCs Lymphoid progenitors Myeloid progenitors Monocytes ECs T-lineage B-lineage

  33. Functional interdependency of bone marrow stem cells Array of matrix proteins, adhesion molecules, cytokines is expressed by HSC MSC EPC

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