Stamceller och ingenjörskonst

Transkript

1 Stamceller och ingenjörskonst Anna Herland Ana Teixeira lab Cell and Molecular Biology

2 Innehåll Introduktion av labbet Vad är stamceller? Stamceller, forskning och utveckling, till vilken nytta? Stamcellsnicher Stamceller och materialinteraktioner Studier av stamceller i mikrostrukturer Stamceller och tissue engineering Forskningsprojekt 1: Scaffolds for dopaminergic differentiation Forskningsprojekt 2: Control of neural stem cell maintenance through voltage activated growth factor presentation

3 The stem cell engineering group: Forskningsområden CNS Development Cancer Stem Cell Therapy Gemensamma nämnare: Förstå och manipulera interaktion mellan stamceller och deras mikromiljö.

4 Stamcellers karaktäristik Icke Specialiserad cell Självförnyelse Symmetrisk/assymetrisk cell delning Differentieringspotential Stamceller Symmetrisk Aymmetrisk Stamceller + Stamceller Differentierade, Specialiserade celler

5 Stamceller under utvecklingen Specialization Zygote Blastocyst Fetus Adult Potency Totipotency Pluripotency Multipotency Embryonic SC Fetal SC Adult and neonatal SC Slide courtesy of Goncalo Castelo- Branco

6 Embryonala Stamceller in Vivo Morula - totipotent, gives rise to both placenta and embryo. Blastocyst - pluripotent, the inner cell mass gives rise to the embryo, the outer to placenta. The inner cell mass = Embryonic Stem Cells.

7 Embryonala stamceller: in vitro Tekniker för embryonala stamceller Embryon från IVF Terapeutisk kloning Nuclear reprogramming (induced pluripotent cells, ips)

8 Neuronala stamceller Mer begränsade i sin differentieringspotential, normalt tripotenta Neuroner, Astrocyter, Oligodendrocyter Neurala stamceller uttrycker det intermediära filament proteinet nestin Lendhal U. Et al., Cell 1990;60:

9 Stamceller, forskning och utveckling - till vilken nytta? Förstå normal och icke-normal utveckling Till läkemedelsutveckling Stamcellsterapi: Återskapa Ersätta Förbättra pancreatic cells blood cells nerve cells muscle cells liver cells

10 Stamcellsterapi: gammal och framtida teknologi Stamcellsterapi för hematopoetiska stamceller är en fullständig säker och nödvändig behandligsform (sen 1950-talet) Terapi baserat på ESC eller ipsc är ännu osäker ESCs bildar teratomas In vitro differentieringsprotokoll är mycket grovhuggna Det är en mycket fin linje mellan stamceller och tumörceller Given the nature of pluripotent cells and their innate capacity to form teratomas, there is a particular concern for the potential tumorigenicity of hescs and induced pluripotent stem cells or their differentiated derivatives. Guidelines for the Clinical Translation of Stem Cells, ISSCR, December 2008.

11 Kontrollera stamcellens mikromiljö In vivo niche Targets to control in vitro and in vivo Sprading, A et al. Nature, Physical factors Mechanical support Substrate topography Spatial organization Chemical factors Extracellular matrix comp. Growth factors Cellular interactions Biodegradability Inhibition of tumor formation

12 Stamcellsdifferentiering Efterliknar normal embryonal utveckling i tid och signalsubstanser/morfogen Mus, days Människa, days

13 Den fysikaliska mikromiljön: Dimensionsbegränsad kontroll av celler Chen C.S, et al. Geometric control of life and Death, Science 1997.

14 Den fysikaliska mikromiljön: Celler känner av nanostrukturer Cells elongate along nanopatterns 500nm Nanoscale topography of the human corneal epithelial basement membrane Nanopatterns direct cytoskeletal organization Teixeira A.I., Journal of Cell Science 2003

15 Den fysikaliska mikromiljön: Biomekanisk kontroll av stamceller Engler, A., et al. Cell, 2006

16 Den kemiska mikromiljön: Gradienter kontrollerar stamceller in vivo Park YJ et al StemCells 2009

17 Den kemiska mikromiljön: Kombinatorisk mikrofluidik Kombinatorisk mikrofluidik har potential att förbättra stam cells differentieringsprotokoll Morfogen/läkemedel Gradienter Temporal upplösning

18 Stamcellsterapi i CNS Potential therapy for: Neurodegenerative diseases (Parkinson s, Alzheimer s). Tissue loss from stroke, tumor mass removal. Spinal cord injury Stem cell based strategies to replace damaged brain tissue: Stem cell transplantation. Stimulation of endogenous neurogenesis. Protection of existing functional cells. Stem cells as drug delivery agents. The is only a very fine line between stem cells and tumor cells Many naturally occurring CNS tumors express neural stem cell specific genes (CD133, nestin, NG2). Cancer stem cells. Transplantation of embryonic stem cells in mouse striatum caused tumor formation in 20% of the animals. Björklund et al., PNAS (2002).

19 Clinical trials using stem cell therapy in the CNS Stem Cells Inc. Adult NSC (HuCNS-SC) Treatment of Batten disease (barin disorder in children, lack of lyzosomal enzyme PPT1) Pelizaeus-Merzbacher Disease (PMD), myelination disorder in children NeuralStem ALS ReNeuron Genetically modified neural stem cell of fetal origin Stroke

20 Parkinson s disease Current incidence is 1% of people above 65 years. It is rare in people younger than 40 years. PD is a chronic neurodegenerative disease, which involves primarily the selective loss and atrophy of the dopaminergic neurons of the substantia nigra (SN). SN dopaminergic neurons are part of the basal ganglia, a group of brain nuclei important for motor control, and are specialized in the synthesis, storage and release of the neurotransmitter dopamine. Degeneration of SN dopaminergic neurons leads to impaired dopamine release in the dorsal striatum (caudate and putamen), which results in excessive inhibitory inputs to the thalamus and the prefrontal motor cortex.

21 Parkinson s disease Main symptoms of Parkinson s disease are: akinesia (inability to initiate movements) bradykinesia (slowness of movement) muscular rigidity (lack of facial expression) tremor unstable posture The Michael J. Fox Foundation for Parkinson s research At later stages of the disease, other neurons degenerate, such as dopaminergic neurons in the ventral tegmental area, leading to posture dysfunction, depression, cognitive and sensorial deficits. Causes for the degeneration and neuronal cell death are unknown, except for a few cases in which genetic and pharmacological evidence was found.

22 Stem Cell therapy in Parkinson s Disease Neuronal transplant in PD Lindvall O., Nature Neuroscience 2000;3: Ectopic transplantation of fetal cells, 6 fetuses per treatment Recovery of function by restoration of Dopaminergic transmission in the striatum, first treatments 1990 Reproducible and reliable cell source is essential - stem cells?

23 Stem cell culture for dopaminergic neurons ES cell line NesE-Lmx1a developed in J Ericsson and T Perlmann labs, A WT ES cell line and control: Andersson et al Cell 2006 and Friling et al PNAS 2009 NesE-eGFP: 15-20% dopamin. neurons NesE-Lmx1a: 75-95% dopamin. neurons Transplanted cells re-innervated lesioned striatum, after 4 weeks. Transplanted cells had poor survival Tumor formation/ cell overgrowth observed in vitro and in vivo. Friling S. et al., PNAS 2009

24 Tissue engineering in the stem cell field - 3 D cell scaffolds In vivo aims of 3D scaffolds, improving cellular integration: Simple reconstitution of tissue providing a 3D support Mimicking ECM Local delivery of growth factors, small molecules etc In vitro aims of 3D scaffolds Mimicking in vivo like 3D environments Understanding stem cell behavior in 3D environment Khademhosseini, Ali et al. PNAS, 2006.

25 Active tumor suppressive cell scaffolds Adapted from Lindvall O et al. Nat Med, 2004.

26 Active release can be achieved through conjugated polymers Active release through changing the electronic state (doping) of the polymer: REDUCTION or OXIDATION, i.e a voltage pulse Release of charge compensating ions, drugs Contact with e.g deep brain stimulating electrode R R R S S S S S R R Example of conjugated polymer n Electroactivation Doped polymer Undoped polymer Positively doped polymer segment Negative charge compensating counter-ion

27 Scaffold materials and geometries Materials Synthetic materials (PLLA, PGA, etc) Natural materials (Collagen, Matrigel) Recombinant protein materials (Self assembling peptides) Geometries Porous structures Fibrous structures Hydrogel materials Park KI, et al. Nat Biotechnol 2002

28 Scaffold geometries - techniques Particle leaching method Phase transition method Polymer solution Casting Drying Defined particles Scaffold Leaching Levenberg PNAS 2003 Tomita et al, Stem cells 2005

29 Non-vowen structures Non-vowen structures of biodegradable mono/multi filaments A wide range of products commercially available BMS used as a supplier of biodegradable fells

30 3D scaffolds show good cell invasion and differentiation Cryo-secetioned polymer scaffold Good cell survival Differentiation is comparable to 2D Fewer cells of unknown phenotype Pore size is crucial

31 Non-vowen materials have good scaffold properties, (but are difficult to image) PLLA 89 mg/cc Non-vowen materials show good cell survival and differentiation Autofluorescence and stiffness makes imaging difficult Confocal stacks of differentiated ES cells Tuj Fluorescence micrograph of neuroblastoma cells Tuj Synap

32 Nyckelbudskap Stamceller har stora möjligheter för framtida behandlingsformer och läkemedelsutveckling Ökad kunskap om stamcellers interaktioner med sin mikromiljö är fundamentalt för vidare utveckling Framgångsrik stamcellsterapi kommer vara beroende av tissue engineering Mikromönstring/mikrofluidik har potential att ta stamcellsforskning till en ny nivå

33 Kontakt Anna Herland, CMB, Karolinska Institutet Mobil: Duktiga, kreativa exjobbare sökes!!

34 Ana Teixeira, Neuroscience, KI Vanessa Lundin, Neuroscience, KI Acknowledgements Magnus Berggren, ITN, LiU Edwin Jager Kristin Persson Funding: Johan Ericson, CMB, KI Zhanna Alekseenko Elisabeth Andersson Thomas Perlmann, CMB, KI Ola Hermanson and his lab, Neuroscience, KI