Combination implants and physical stimuli in stem cell research

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Combination implants and physical stimuli in stem
cell research
Outi Hovatta Susanna Narkilahti Sari Ketola Suvi
Haimi Leo Hillman Susanna Miettinen Riitta
Suuronen
Combiokokous 3.4.2006
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Different types of stem cells

• Tissue-derived stem cells or progenitor cells
• ability to differentiate to several (multipotent)
  or one cell type
• blood stem cells, multipotent
• bone marrow, foetal liver, umbilical cord or
  peripheral blood
• mesenchymal stem cells
• found in bone marrow, foetal liver, adipose
  tissue, maybe elsewhere
• multipotent, can differentiate to several cell
  types (bone, cartilage, muscle, other cell types
  such as neurons or liver cells to limited extent)
• MAPC, multipotent adult progenitor cells,
  Verfaillie et al., rare cells
• skin, intestine, muscle, other rapidly renewing
  organs
• nervous tissue, even in adult, but in limited
  numbers
• Embryonic stem cells
• pluripotent, can differentiate to all cell types
  and devide indefinitely
• isolated fromt eh inner cells mass of embryos 5-6
  days after in vitro fertilisation
• specific demands for culture as
  non-differentiated cells

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Stem cell transplantation standard treatment
within the next few years?

• Severe neurological disorders (stroke, injuries,
  Alzheimers and Parkinsons diseases, spinal cord
  injury, MS, ALS, JNCL etc)
• Cardiac failure
• Liver failure
• Diabetes
• Cancers
• Cartilage and bone diseases and injuries

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Cells, biomaterial and physical stimuli needed in
tissue engineering

• Optimal biomaterial
• sufficient physical support for the growth of the
  cells in each tissue and organ
• releases nutrients, growth factors and survival
  factors which the cells need
• disintegrates when the tissue has been integrated
  and is functional, but the products of of
  disintegration do not harm the cells (pH etc)

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Skin produced by tissue engineering is already at
use

• Several products in the market
• a membranous matrix
• Cells keratinocytes, fibroblasts, melanocytes,
  hair bulb cells, blood vessels
• 3D fibrin matrix
• Wide clinical use
• Burns
• Other injuries
• Large tumours

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Cartilage

• autologous chondrocyte transplantation (ACT)
  available since 1994
• healthy chonrocytes (periostium), culture,
  scaffolds
• 3-dimensional using biomaterials
• mesenchymal stem cells biomaterial
• rabbit TMJ (Dr.Mauno) / with Timo Ylikomi
• wide clinical need and applications

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Bone

• bank bone
• engineered bone
• osteoblasts
• mesencymal stem cells
• skull bone defects in Regea
• biomaterial scaffolds
• wide clinical need
• several applications

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Other tissues

• Producing oral tissues
• several plans, bone and mucosa
• will soon start in Regea
• biomaterial needed in all

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Stem cells in cardiac repair

• Satellite cells from skeletal muscle
• arrhytmias, do not function well in cardiac
  muscle
• Cardiomyocytes from mesenchymal stem cells
• do not integrate to the cardiac muscle
• may help in revascularisation
• Cardiomyocytes from embryonic stem cells
• several methods to differentiated (Mummery et al.
  2005)
• functional in animal experiments

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Neural repair

• Clinically extremely important indication
• Adult neural stem cells are not capable of
  correcting major injuries in the body
• Adult neural stem cells very difficult to sample
  for culture
• Cells from olfactory epithelium used with some
  success
• Mesenchymal stem cells with limited potential
• Embryonic stem cells most potential in this
  respect
• differentiation in vitro feasible

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Human ES cells

• First cultures in 1984
• Fishel, Edwards, Evans (Science 1984) (see also
  Edwards, Nature 2001, RBMonline 2002)
• The first permanent hES cell lines
• - Thomson et al. Science 1998
• - Reubinoff et al. Nature Biotechnol 2000
• - Derived using foetal mouse fibroblastas as
  feeder cells
• In 2005, about 250 hES cell lines in the world

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inner cell mass
A blastocyst five days after in vitro
fertilization
hES cell lines can be derived from the inner cell
masses of donated blastocysts
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Human embryonic stem cell lines at Karolinska
University Hospital Huddinge

• A total of 25 permanent lines 2002-2005
• HS181, HS207, HS235, HS237, HS293, HS306, HS346,
  HS351, HS360, HS361, HS362, HS363, HS364, HS366,
  HS368, HS380, HS382, HS386, HS401, HS402, HS415,
  HS420, HS421, HS426, HS429
• 17 additional early lines, which stopped growing
  during the period
• 131 blastocysts have been obtained 2002-2005
• Success dependent on embryo quality
• The good embryos are allways used for patients
  infertility treatment

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The lines HS293-429 (n22) have been derived on
postnatal human skin fibroblasts using serum
replacement containing medium
Inzunza Inzunza J, Gertow K, Strömberg AM
Matilainen E, Blennow E, Skottman H, Wolbank
S, Ährlund-Richter L, Hovatta O. Derivation of
human embryonic stem cell lines in serum
replacement medium using postnatal human
fibroblasts as feeder cells, Stem Cells 23,
544-549, 2005.
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Clinical quality

• GMP
• Animal protein free derivation
• Mechanical isolation of the inner cell mass
• Derivation using GMP-quality human skin
  fibroblasts is a safe option
• human serum to establish skin fibroblast feeder
  cell lines
• human serum containing serum replacement
• Feeder-free derivation
• One team reported succes, but on mouse-derived
  matrix (Klimanskaya et al., Lancet 2005)
• Human matrix would be optimal, but to obtain safe
  cells, it would be necessary to know more about
  the mechhanisms of self-renewal
• High concentration of growth factors and not
  physiological and may drive epigenetic changes
• Enzymatic splitting of the colonies promotes
  selection of chromosomally abnormal cells
  (Henderson et al. Nat Biotechnol 2004)
• Mechanical splitting safer
• If mechanical splitting is used, human rec
  collagenase is safest

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Safety of embryonic stem cells

• The risk of infection has to be eliminated
• EU directive from March 2003
• GMP quality for handling of the cells
• No animal-derived materials in the process
• Non-differentiated ES cells may form teratomas
• Differentiated cells safe in animal experiments

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What is an optimal surface for ES cell growth ?

• A material onto which the cells attach well, grow
  as colonies, which can be taken out mechanically
  
• Transparent, allows follow-up and imaging
• Secretes substances which keep the cells
  non-differentiated
• it is also possible to add the substances to
  culture medium
• conditioned medium from mouse fibroblasta has
  been used, but that one from human fibroblasts
  does not work well (Susanne Ström, submitted 2006)

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Studies in Regea

• Not yet an optimal matrix
• Sarita Ketola tested 3 materials,
• fibroblast conditioned medium
• and non-conditioned medium (immediated
  differentiation)
• did not attach to glass
• not possible to carry out immunohistochemistry on
  the matrix
• when scraped off from the matrix and transferred
  back to the feedr cells, could be imaged, were
  partially differentiated, which can be explained
  by the medium

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Mouse ES cells to neural cells

• Ying, Q. L., and Smith, A. G. (2003). Defined
  conditions for neural commitment and
  differentiation. Methods Enzymol 365, 327-341.
• Ying, Q. L., Stavridis, M., Griffiths, D., Li,
  M., and Smith, A. (2003). Conversion of embryonic
  stem cells into neuroectodermal precursors in
  adherent monoculture. Nat Biotechnol 21, 183-186.

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Human ES cells to neural cells

• Reubinoff, B. E., Itsykson, P., Turetsky, T.,
  Pera, M. F., Reinhartz, E., Itzik, A., and
  Ben-Hur, T. (2001). Neural progenitors from human
  embryonic stem cells. Nat Biotechnol 19,
  1134-1140.
• Reubinoff, B. E., Pera, M. F., Fong, C. Y.,
  Trounson, A., and Bongso, A. (2000). Embryonic
  stem cell lines from human blastocysts somatic
  differentiation in vitro. Nat Biotechnol 18,
  399-404.

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Succesfully used in experimental animals

• Stroke, Parkinsons and Alzheimers diseases,
  spinal cord injury, peripheral nerves, MS, ALS
• Brustle, O., Jones, K. N., Learish, R. D.,
  Karram, K., Choudhary, K., Wiestler, O. D.,
  Duncan, I. D., and McKay, R. D. (1999). Embryonic
  stem cell-derived glial precursors a source of
  myelinating transplants. Science 285, 754-756
• Chiba, S., Ikeda, R., Kurokawa, M. S., Yoshikawa,
  H., Takeno, M., Nagafuchi, H., Tadokoro, M.,
  Sekino, H., Hashimoto, T., and Suzuki, N. (2004).
  Anatomical and functional recovery by embryonic
  stem cell-derived neural tissue of a mouse model
  of brain damage. J Neurol Sci 219, 107-117.
• Horner, P. J., and Gage, F. H. (2000).
  Regenerating the damaged central nervous system.
  Nature 407, 963-970.
• Kim, J. H., Auerbach, J. M., Rodriguez-Gomez, J.
  A., Velasco, I., Gavin, D., Lumelsky, N., Lee, S.
  H., Nguyen, J., Sanchez-Pernaute, R., Bankiewicz,
  K., and McKay, R. (2002). Dopamine neurons
  derived from embryonic stem cells function in an
  animal model of Parkinson's disease. Nature 418,
  50-56

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Differentiation of neural cells

• 1. neural induction in monolayer
• non-differentiated colony of hES cells onto
  laminin, serum-free medium, no RA
• modification from Ying et al.
• DMEM/F12, Neurobasal, N 2, B 27, 1mg/ml HSA, FGF
  
• 2. neural progenitor cells expanded
• 3. expansion
• a) as spheroid bodies in suspension culture
• b) as monolayer on laminin
• 4. differentiation to different cell types
• -no FGF, BDNF, N2, B27

Regea Susanna Narkilahti, Tuomas Huttunen, Tiina
Rajala, Outi Hovatta Karolinska Institutet
Roxana Nat, Agneta Nordberg, Bengt Winblad
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Cell IQ system

• Machine vision informatics, a controlled
  culture system (Chipman Technologies, Tampere,
  Finland)
• Enables detailed follow-up of the developing
  cells in long-term culture

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Function of neural stem cells, derived from hES
cells, in animal models

• Consortium Jari Koistinaho, Seppo Ylä-Herttuala
  ja Outi Hovatta teams
• Integration of the stem cell into the tissue
• The effects of inflammation and apoptosis
• The role of the cytokines
• Can we imporve the vascularization of the drafts
  by trnafection of with the vascular endothelial
  growth factor
• Stroke, spinal cord injuryt and Alzheimers
  disease rat and mouse models

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Spinal cord injury related research

• The promotion of oriented axonal regrowth in the
  injured spinal cord by alginate-based anisotropic
  capillary hydrogels. Prang P, Muller R,
  Eljaouhari A, Heckmann K, Kunz W, Weber T, Faber
  C, Vroemen M, Bogdahn U, Weidner N. Biomaterials
  (2006). Alginate-based highly anisotropic
  capillary hydrogel scaffolds seeded with neural
  progenitor cells were implanted into acute
  cervical spinal cord lesions in adult rats. The
  research displayed induced directed axon
  regeneration across the artificial scaffold.
• Physical and biological performance of a novel
  block copolymer nerve guide. Lietz M, Ullrich A,
  Schulte-Eversum C, Oberhoffner S, Fricke C,
  Müller HW, Schlosshauer B. Wiley InterScience
  (2005). Block copolymers made from trimethylene
  carbonate and e-caprolactone were used as nerve
  guides filled with Schwann cells implanted into
  lesioned spinal cords of adult rats. Promising
  axonal regrowth was observed.

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More spinal cord related

• Multiple-channel scaffolds to promote spinal cord
  axon regeneration. Moore MJ, Friedman JA,
  Lewellyn EB, Mantila SM, Krych AJ, Ameenuddin S,
  Knight AM, Lu L, Currier BL, Spinner RJ, Marsh
  RW, Windebank AJ, Yaszemski MJ. Biomaterials 27
  (2006) 419-429. The effects of scaffold
  architecture, transplanted cells, and locally
  delivered molecular agents on axon regeneration
  were investigated simultaneously. Schwann-cell
  containing scaffolds implanted into transected
  adult rat spinal cords contained regenerating
  axons at one month post-operation
• Freeze-dried agarose scaffolds with uniaxial
  channels stimulate and guide linear axonal growth
  following spinal cord injury. Shula Stokols, Mark
  H. Tuszynski. Biomaterials 27 (2006) 443451.
  Freeze-dried agarose scaffolds with and without
  recombinant Brain-Derived Neurotrophic Factor
  protein were tested on a rat model of spinal cord
  injury. Axons grew through scaffolds in a
  strikingly linear fashion and the regeneration
  was augmented by the BDNF protein.
• Matrix inclusion within synthetic hydrogel
  guidance channels improves specific supraspinal
  and local axonal regeneration after complete
  spinal cord transection. Tsai EC, Dalton PD,
  Shoichet MS, Tator CH. Biomaterials 27 (2006)
  519533. Synthetic hydrogel (pHEMA-MMA) channels
  with different matrix materials were implanted
  into adult Sprague Dawley rats. E.g. collagen and
  fibrin increased the total axon density within
  the channel compared to unfilled channel
  controls.

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Research on peripheral nerve regeneration

• Neural tissue engineering a self-organizing
  collagen guidance conduit. Phillips JB, Bunting
  SC, Hall SM, Brown RA. Tissue Engineering 11
  (2005) 1611-1617. A novel implantable device that
  delivers a tethered aligned collagen guidance
  conduit containing Schwann cells into a
  peripheral nerve injury site. Neural regeneration
  through this device was significantly greater
  than in controls
• Tendon Chitosan Tubes Covalently Coupled With
  Synthesized Laminin Peptides Facilitate Nerve
  Regeneration In Vivo. Suzuki M, Itoh S, Yamaguchi
  I, Takakuda K, Kobayashi H, Shinomiya K, Tanaka
  J. Journal of Neuroscience Research 72 (2003)
  646659. Tendon chitosan tubes having the ability
  to bind peptides covalently coupled with laminin
  peptides were tested in vivo. According to their
  research laminin may effectively assist nerve
  tissue extension.

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Differentiation in vitro

• Topographically modified surfaces affect
  orientation and growth of hippocampal neurons.
  Dowell-Mesfin NM, Abdul-Karim MA, Turner AM,
  Schanz S, Craighead HG, Roysam B, Turner JN,
  Shain W. Journal of Neural Engineering June 2004
  78-90. Neurons from hippocampi of rat embryos
  were grown in vitro on poly-L-lysine-coated
  silicon surfaces. They suggested that
  extracellular matrix topography may contribute to
  cell growth and differentiation.
• Fabrication of nano-structured porous PLLA
  scaffold intended for nerve tissue engineering.
  Yang F, Murugan R, Ramakrishna S, Wang X, Ma YX,
  Wang S. Biomaterials 25 (2004) 1891-1900. A
  nano-fibrous PLLA-polymer scaffold was created
  for in vitro culture of nerve stem cells. The
  cell cultural tests showed that the NSCs could
  differentiate on the nano-structured scaffold and
  the scaffold acted as a positive cue to support
  neurite outgrowth.

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Electric stimulation

• nerve cells developm and grow normally in an
  electric field
• In regea, tested for the firts time on the
  development of nerve cells and cardiomyoblasts
• The nerve cells grow very well, the rest of the
  cells in the colonies disappear

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Tissue derived vs embryonic stem cells

• tissue derived
• No rejection, if own cells
• grow well from many tissues (mesenchymal stem
  cells, skin, cartilage)
• grow slowly from many tissues (neural, heart)
• difficult to isolate (nerve)
• not functional after isolation in many tissues

• embryonic stem cells
• unlimited growth
• can be differentiated to almost any cells
• are immunogenic