U.S. patent application number 13/808809 was filed with the patent office on 2013-11-28 for method for stem cell differentiation in vivo by delivery of morphogenes with mesoporous silica and corresponding pharmceutical active ingredients.
This patent application is currently assigned to Nanologica AB. The applicant listed for this patent is Alfonso E. Garcia-Bennett, Elena Nickolaevna Kozlova. Invention is credited to Alfonso E. Garcia-Bennett, Elena Nickolaevna Kozlova.
Application Number | 20130315962 13/808809 |
Document ID | / |
Family ID | 44534302 |
Filed Date | 2013-11-28 |
United States Patent
Application |
20130315962 |
Kind Code |
A1 |
Garcia-Bennett; Alfonso E. ;
et al. |
November 28, 2013 |
METHOD FOR STEM CELL DIFFERENTIATION IN VIVO BY DELIVERY OF
MORPHOGENES WITH MESOPOROUS SILICA AND CORRESPONDING PHARMCEUTICAL
ACTIVE INGREDIENTS
Abstract
A pharmaceutical active ingredient for cell differentiation to
alleviate cell and cell-related deficiencies in mammals comprising
porous silica containing a releasable agent capable of contributing
to a cell environment conducive for stem cell differentiation in
co-implanted stem cells and/or in endogenous stem cells.
Inventors: |
Garcia-Bennett; Alfonso E.;
(Stockholm, SE) ; Kozlova; Elena Nickolaevna;
(Stockholm, SE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Garcia-Bennett; Alfonso E.
Kozlova; Elena Nickolaevna |
Stockholm
Stockholm |
|
SE
SE |
|
|
Assignee: |
Nanologica AB
Stockholm
SE
|
Family ID: |
44534302 |
Appl. No.: |
13/808809 |
Filed: |
July 6, 2011 |
PCT Filed: |
July 6, 2011 |
PCT NO: |
PCT/EP11/61377 |
371 Date: |
July 23, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61361741 |
Jul 6, 2010 |
|
|
|
Current U.S.
Class: |
424/400 ;
424/93.7; 514/234.2; 514/559 |
Current CPC
Class: |
C12N 2500/46 20130101;
A61K 38/1709 20130101; A61K 38/185 20130101; C12N 2501/385
20130101; A61P 21/00 20180101; A61K 9/1611 20130101; C12N 2500/38
20130101; A61K 35/545 20130101; C12N 2533/00 20130101; C12N 2506/08
20130101; A61K 9/5115 20130101; A61K 31/203 20130101; C12N 2533/14
20130101; A61K 31/00 20130101; C12N 5/0619 20130101; A61P 25/00
20180101; C12N 2506/02 20130101; C12N 2501/41 20130101; A61K 35/30
20130101; A61K 47/02 20130101; A61K 31/5377 20130101; C12N 2501/998
20130101; A61K 38/1709 20130101; A61K 2300/00 20130101; A61K 35/545
20130101; A61K 2300/00 20130101; A61K 35/30 20130101; A61K 2300/00
20130101; A61K 38/185 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/400 ;
514/234.2; 514/559; 424/93.7 |
International
Class: |
A61K 47/02 20060101
A61K047/02; A61K 35/30 20060101 A61K035/30; A61K 35/54 20060101
A61K035/54; A61K 31/5377 20060101 A61K031/5377; A61K 31/203
20060101 A61K031/203 |
Claims
1. A pharmaceutical active ingredient for cell differentiation to
alleviate cell and cell-related deficiencies in mammals comprising
porous silica containing a releasable agent capable of contributing
to a cell environment conducive for stem cell differentiation in
co-implanted stem cells and/or in endogenous stem cells.
2. A pharmaceutical active ingredient as described in claim 1,
wherein said porous silica has a surface area higher than 200
m.sup.2/g and a pore size between 1.5-50 nm.
3. A pharmaceutical active ingredient as described in claim 1
wherein said porous silica has average particle size and/or sizes
in the range between 50-5000 nm.
4. A pharmaceutical active ingredient as described in claim 1
wherein the porous silica particles have a particle shape
comprising of spheres or rod-shaped particles.
5. A pharmaceutical active ingredient as described in claim 4
wherein the porous silica particles are in the form of
substantially spherical particles having a size range of 200-500
nm.
6. A pharmaceutical active ingredient according to claim 1, wherein
said releasable agent capable of contributing to a cell environment
conducive for stem cell differentiation in co-implanted stem cells
and/or in endogenous stem cells is 1-60% of the total weight of the
pharmaceutical active ingredient.
7. A pharmaceutical active ingredient according to claim 6, wherein
said releasable agent capable of contributing to a cell environment
conducive for stem cell differentiation in co-implanted stem cells
and/or in endogenous stem cells is 1-45 wt % of the total weight of
the pharmaceutical active ingredient.
8. A pharmaceutical active ingredient according to claim 1, wherein
said releasable agents is capable of forcing co-implanted cells to
become postmitotic, in case when transplanted cells are or may be
pre-mitotic.
9. A pharmaceutical active ingredient according to claim 1, wherein
said co-implanted stem cells are selected from the group consisting
of regional stem cells, embryonic stem (ES) cells, neural crest
stem cells, neural stem cells from brain and spinal cord,
mesenchymal stem cells, endothelial stem cells, endodermal stem
cells, induced pluripotent stem (iPS) cells.
10. A pharmaceutical active ingredient according to claim 1,
wherein said releasable agent is selected from the group consisting
of secreted growth factors and morphogens, including, but not
limited to fibroblast growth factors (FGFs), Wnts, transforming
growth factor (TGF)-beta family members, Hedgehog (hh) proteins,
retinoic acid, vascular endothelial growth factor (VEGF), Dickkopf
(Dick)-1, insulin, Activin, SDF-1/CXCL12), pleiotrophin (PTN),
insulin-like growth factor 2 (IGF2), ephrin B1 (EFNB1), cAMP,
Semaphorins, Slits, Netrins, NCAM, L1-CAM, NGF, BDNF, NT3, NT4/5,
GDNF, Artemin, Persephin, CNTF, LIF, Oncostatin M, Cardiotrophin 1,
CDNF/MANF.
11. A pharmaceutical active ingredient according claim 1, wherein
the activity of the porous silica containing releasable agents is
able to provide simultaneous or independent effects on
co-implanted/endogenous stem cells.
12. A delivery system for delivery of a pharmaceutical active
ingredient in mammals, comprising a pharmaceutical active
ingredient according to claim 1, and stem cells.
13. A delivery system for delivery of a pharmaceutical active
ingredient in mammals according to claim 12, wherein said stem
cells are selected from the group consisting of regional stem
cells, embryonic stem (ES) cells, neural crest stem cells, neural
stem cells from brain and spinal cord, mesenchymal stem cells,
endothelial stem cells, endodermal stem cells, induced pluripotent
stem (iPS) cells.
14. A method of treating cell and cell-related deficiencies in a
mammal, comprising the consecutive steps: (a) preparation of a
pharmaceutical active ingredient for cell differentiation,
long-term survival and axonal growth to alleviate cell and
cell-related deficiencies in mammals comprising porous silica
containing a releasable agent capable of delivering factors
conducive for differentiation/survival/tumor suppression, axonal
growth and optionally additional suppressor(s) or activator(s) with
the cell; (b) transplantation of said cells to said mammal, and (c)
activation or suppression of releasable factors (s) by regulation
of pore size, surface chemistry, lipophilicity and dissolving
properties of the porous silica in co-implanted or endogenous stem
cells.
15. The method according to claim 14, wherein the cells for
implantation are selected from the group consisting of regional
stem cells, embryonic (ES) stem cells, neural crest stem cells,
neural stem cells from brain and spinal cord, mesenchymal stem
cells, endothelial stem cells, endodermal stem cells, iPS
cells.
16. The method according to claim 14, wherein the releasable agents
are selected from the group consisting of secreted growth factors
or their peptide mimetic analogs, guidance molecules and
morphogens, including, but not limited to, fibroblast growth
factors (FGFs), Wnts, transforming growth factor (TGF)-beta family
members, and Hedgehog (hh) proteins, retinoic acid, VEGF, Dkk1,
insulin, Activin, SDF-1/CXCL12), pleiotrophin (PTN), insulin-like
growth factor 2 (IGF2), ephrin B1 (EFNB1), cAMP, Semaphorins,
Slits, Netrins, NCAM, L1-CAM, NGF, BDNF, NT3, NT4/5, GDNF, Artemin,
Persephin, CNTF, LIF, Oncostatin M, Cardiotrophin 1, CDNF/MANF.
17. The method according to claim 14 for treating degenerative
disorders including but not limited to Alzheimer's disease,
Parkinson's disease, Amyotrophic lateral sclerosis, Spinal muscular
atrophy, Stroke, Traumatic brain or spinal cord injury, Multiple
sclerosis, Diabetes type 1 and 2, Muscular dystrophies,
Cardiomyopathies, and Age-related macular degeneration.
18. A pharmaceutical active ingredient as described in claim 2
wherein said porous silica has average particle size and/or sizes
in the range between 50-5000 nm.
19. A pharmaceutical active ingredient as described in claim 2
wherein the porous silica particles have a particle shape
comprising of spheres or rod-shaped particles.
20. A pharmaceutical active ingredient as described in claim 3
wherein the porous silica particles have a particle shape
comprising of spheres or rod-shaped particles.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to pharmaceutical active
ingredients comprising sets composed of stem cells and porous
silica, preferably mesoporous silica, containing a defined set of
differentiation factors for desired differentiation of different
types of cells, and to a method of enhancing survival and control
differentiation of transplanted stem cells for regenerative
medicine by providing said sets. In particular, the pharmaceutical
active ingredients and methods of the invention are preferably used
for controlling differentiation of embryonic stem cells (ESCs) and
induced pluripotent stem cells (iPSCs); in general, they are also
applicable for other stem cells, e.g. tissue-specific stem cells
and mesenchymal stem cells.
BACKGROUND OF THE INVENTION
[0002] Experimental stem cell research has achieved enormous
progress during the past few years in generating desired types of
cells in vitro, which can be used in regenerative medicine. These
in vitro protocols have confirmed that extrinsic factors/morphogens
may induce expression of specific transcription factors (TFs),
which establish molecular codes for the identity of stem cell
differentiation. The identification of extrinsic factors which
transduce their effects through TF codes in stem cell
differentiation and exploration of this information for the
development of delivery systems for these factors to the
transplanted stem cells may result in translation of in vitro
protocols to in vivo applications. A method to control stem cell
differentiation after transplantation by controlled expression of
TFs in the transplanted cells using drug-inducible regulation
systems is described in patent application WO2008/002250, having
title "Improved Stem Cells For Transplantation And Methods For
Production Thereof".
[0003] Stem cell transplantation is an attractive strategy for
replacement of specific cells that are permanently lost or
non-functional as a result of injury or disease. Transplanted stem
cells can also promote tissue repair through trophic and cell
protective effects, i.e. without replacing the specific cells that
have been lost by injury or disease. While such "unspecific"
effects may be beneficial and important, the ultimate goal of stem
cell transplantation still remains to replace the damaged or
diseased cells with fully functional cells of the same type. The
environment that transplanted stem/progenitor cells will meet is
predominantly adult and marked by pathological responses. For cell
replacement therapy to be successful, it is important to understand
these responses and how they can either be modified to provide a
host environment which is compatible with long term survival and
the desired differentiation or how this environment can be changed,
so that the transplanted cells will be unaffected by negative
external stimuli.
[0004] The results from numerous in vitro and in vivo experiments
have convincingly demonstrated that the timely expression of
certain cell-intrinsic factors, transcription factors, during
normal development or during stem cell differentiation in vitro can
be sufficient to induce and in some cases to guide differentiation
of stem cells. Using the tetracycline gene regulation system to
induce the expression of the key transcription factor Runx1 in
Sox10 expressing neural crest stem cells we achieved specific
differentiation of nociceptor neurons in vitro and in vivo after
transplantation (Aldskogius H, Berens C, Kanaykina N, et al.
Regulation of boundary cap neural crest stem cell differentiation
after transplantation. Stem Cells 2009; 27:1592-603). In order to
achieve such a timely expression of cell-intrinsic and
transcription factors it is hence desired to achieve the kinetic
release of gene regulating molecules through the use of a delivery
vehicle.
[0005] Another possibility to promote tissue protection and/or stem
cell differentiation after transplantation is to create a suitable
environment for transplanted cells. This can be achieved by
co-transplantation of supporting cells or the use of osmotic
minipumps that provide substances for improved survival,
differentiation and function of transplanted cells.
Co-transplantation of neural crest stem cells with pancreatic
islets showed beneficial effects for both islets and stem cells
with improved insulin secretion, increased proliferation of
beta-cells and advanced differentiation of neural crest stem cells
in the vicinity of islets (Olerud J, Kanaykina N, Vasylovska S, et
al. Neural crest stem cells increase beta cell proliferation and
improve islet function in co-transplanted murine pancreatic islets.
Diabetologia 2009; 52:2594-601. Erratum in: Diabetologia. 2010;
53:396. Vasilovska, S [corrected to Vasylovska, S]).
[0006] As for the delivery, porous particles have been developed
for pharmaceutical drug delivery due to their potential to control
(delay) drug release, enhance drug dissolution, promote drug
permeation across the intestinal cell wall (bioavailability) and
improve drug stability under the extreme environment of the
gastro-intestinal tract when administered orally (Vallhov H,
Gabrielsson S, Stromme M, et al. Mesoporous silica particles induce
size dependent effects on human dendritic cells. Nano Lett 2007;
7:3576-82; Fadeel B, Garcia-Bennett A E. Better safe than sorry:
Understanding the toxicological properties of inorganic
nanoparticles manufactured for biomedical applications. Adv Drug
Deliv Rev 2010; 62:362-74).
[0007] External Factors for Cell Differentiation
[0008] Nearly all developmental decisions during embryogenesis are
regulated by a relatively small number of families of secreted
growth factors and morphogens, including fibroblast growth factors
(FGFs) (Bottcher R T, Niehrs C. Fibroblast growth factor signaling
during early vertebrate development. Endocr Rev 2005; 26:63-77),
Wnts (Logan C Y, Nusse R. The Wnt signaling pathway in development
and disease. Annu Rev Cell Dev Biol 2004; 20:781-810), transforming
growth factor (TGF)-beta family members (Massague J. TGF-beta
signal transduction. Annu Rev Biochem 1998; 67:753-91), and
Hedgehog (hh) proteins (McMahon A P, Ingham P W, Tabin C J.
Developmental roles and clinical significance of hedgehog
signaling. Curr Top Dev Biol 2003; 53:1-114). The first clues to
the molecular nature of the organizer's (signals from a region of
dorsal mesoderm) inductive influence came from studies of a
receptor for TGF-beta superfamily members, and Noggin, a secreted
factor expressed by the organizer (Hemmati-Brivanlou A, Melton D A,
A truncated activin receptor inhibits mesoderm induction and
formation of axial structures in Xenopus embryos. Nature 1992;
359:609-14; Smith W C, Harland R M. Expression cloning of noggin, a
new dorsalizing factor localized to the Spemann organizer in
Xenopus embryos. Cell 1992; 70:829-40; Lamb T M, Knecht A K, Smith
W C, et al. Neural induction by the secreted polypeptide noggin.
Science 1993; 262:713-8). Embryonic stem (ES) cells differentiate
to neural stem cells based on FGF4/Notch treatment followed by the
FGF/epidermal growth factor (EGF) treatment are described in
http://www.cscr.cam.ac.uk/asmith.html. The stem cell signaling
network, and specifically the Wnt, Notch, FGF, and BMP signaling
cascades, are implicated in the regulation of the balance for
neural stem cells, progenitor cells, and differentiated neural
cells (Israsena N, Hu M, Fu W, et al. The presence of FGF2
signaling determines whether beta-catenin exerts effects on
proliferation or neuronal differentiation of neural stem cells. Dev
Biol 2004; 268:220-31; Akai J, Halley P A, Storey K G.
FGF-dependent Notch signaling maintains the spinal cord stem zone.
Genes Dev 2005; 19:2877-87).
[0009] Sonic Hedgehog (Shh)
[0010] Shh plays a prominent role in the patterning of the
developing neural tube (Lee K J, Jessell T M. The specification of
dorsal cell fates in the vertebrate central nervous system Annu Rev
Neurosci 1999; 22:261-94; Tanabe Y, Jessell T M. Diversity and
pattern in the developing spinal cord. Science 1996; 274:1115-23.
Review. Erratum in: Science 1997; 276:21). Ectopic application of
Shh is sufficient to induce formation of motor neurons in the
dorsal neural tube (Ericson J, Morton S, Kawakami A, et al. Two
critical periods of Sonic Hedgehog signaling required for the
specification of motor neuron identity. Cell 1996; 87:661-73;
Ericson J, Muhr J, Placzek M, et al. Sonic hedgehog induces the
differentiation of ventral forebrain neurons: a common signal for
ventral patterning within the neural tube. Cell 1995; 81:747-56.
Erratum in: Cell 1995; 82:following 165) and in culture (Hu B Y,
Zhang S C. Differentiation of spinal motor neurons from pluripotent
human stem cells. Nat Protoc 2009; 4:1295-304). Shh both patterns
cell fates along the dorso-ventral axis of the spinal cord and
regulates cell number through its effects on proliferation and
programmed cell death (Ericson J, Morton S, Kawakami A, et al. Two
critical periods of Sonic Hedgehog signaling required for the
specification of motor neuron identity. Cell 1996; 87:661-73;
Oppenheim R W, Homma S, Marti E, et al. Modulation of early but not
later stages of programmed cell death in embryonic avian spinal
cord by sonic hedgehog. Mol Cell Neurosci 1999; 13:348-61). Shh
promotes neural crest proliferation (Fu M, Lui V C, Sham M H,
Pachnis V, et al. Sonic hedgehog regulates the proliferation,
differentiation, and migration of enteric neural crest cells in
gut. J Cell Biol 2004; 166:673-84) and survival (Ahlgren S C,
Bronner-Fraser M Inhibition of sonic hedgehog signaling in vivo
results in craniofacial neural crest cell death. Curr Biol 1999;
9:1304-14; Ahlgren S C, Thakur V, Bronner-Fraser M. Sonic hedgehog
rescues cranial neural crest from cell death induced by ethanol
exposure. Proc Natl Acad Sci USA 2002; 99:10476-81) in addition to
regulating neural crest motility (Testaz S, Jarov A, Williams K P,
et al. Sonic hedgehog restricts adhesion and migration of neural
crest cells independently of the Patched-Smoothened-Gli signaling
pathway. Proc Natl Acad Sci USA 2001; 98:12521-6). Furthermore Shh
promotes both cell proliferation and programmed cell death (PCD) of
early dorsal root ganglion (DRG) cells thus regulating DRG cell
number, the distribution of sensory phenotypes and sensory path
finding (Guan W, Wang G, Scott S A, et al. Shh influences cell
number and the distribution of neuronal subtypes in dorsal root
ganglia. Dev Biol 2008; 314:317-28).
[0011] Retinoic Acid (RA)
[0012] Retinoic acid is another signaling molecule with pronounced
effect on differentiation and survival of developing vertebrate CNS
neurons (Maden, M. Retinoid signalling in the development of the
central nervous system. Nat Rev Neurosci 2002; 3:843-53; Appel B,
Eisen J S. Retinoids run rampant: multiple roles during spinal cord
and motor neuron development. Neuron 2003; 40:461-4). Retinoic acid
can stimulate both neurite number and neurite length (Maden, M.
Role and distribution of retinoic acid during CNS development. Int
Rev Cytol 2001; 209:1-77); and is implicated in the regeneration of
injured peripheral nerve (Zhelyaznik N, Schrage K, McCaffery P, et
al. Activation of retinoic acid signaling after sciatic nerve
injury: up-regulation of cellular retinoid binding proteins. Eur J
Neurosci 2003; 18:1033-40). In embryonic DRG neurons, RARb2
mediates neurite outgrowth induced by retinoic acid (Corcoran J,
Shroot B, Pizzey J, et al. The role of retinoic acid receptors in
neurite outgrowth from different populations of embryonic mouse
dorsal root ganglia. J. Cell Sci 2000; 113:2567-74). Recently was
demonstrated that induced expression of RAR-beta2 in adult DRG
neurons is sufficient to drive growth of their axons through the
non-permissive interface between the dorsal root and the spinal
cord, the dorsal root transitional zone (DRTZ) and into the spinal
cord (Wong L F, Yip P K, Battaglia A, et al. Retinoic acid receptor
beta2 promotes functional regeneration of sensory axons in the
spinal cord. Nat Neurosci 2006; 9:243-50).
[0013] Wnt
[0014] Another group of molecules implicated in orchestrating
embryogenesis are the Wnt family members. FZD1, FZD2, FZD3, FZD4,
FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, LRP5, LRP6, and ROR2 are
transmembrane receptors transducing Wnt signals based on
ligand-dependent preferentiality for caveolin- or clathrin-mediated
endocytosis. Wnt signals are transduced to canonical pathway for
cell fate determination, and to non-canonical pathways for
regulation of planar cell polarity, cell adhesion, and motility.
Thus for canonical Wnt signaling cascade MYC, CCND1, AXIN2, FGF20,
WISP1, JAG1, DKK1 and glucagon are target genes, while CD44,
vimentin and STX5 are target genes of non-canonical Wnt signaling
cascades.
[0015] The target genes of Wnt signaling cascades are dependent on
the expression profile of transcription factors and epigenetic
status. Wnt signaling cascades associated with Notch, FGF, BMP and
Hedgehog signaling cascades regulate the balance of mesenchymal
stem cells, hematopoietic stem cells, and intestinal stem cells and
their progenitor cells. Wnt3, Wnt5A and Wnt10B are expressed in
undifferentiated human embryonic stem cells, whereas Wnt6, Wnt8B
and Wnt10B are expressed in endoderm precursor cells. Wnt6 is
expressed in the intestinal crypt region for stem or progenitor
cells. TNF/alpha-Wnt10B signaling maintains homeostasis of adipose
tissue and gastrointestinal mucosa with chronic inflammation.
Recombinant Wnt protein or Wnt mimetic (circular peptide, small
molecule compound, or RNA aptamer) in combination with Notch
mimetic, FGF, and BMP (Katoh M. WNT signaling in stem cell biology
and regenerative medicine. Curr Drug Targets 2008; 9:565-70) open a
new window to mesoporous silica application in regulation of stem
cell differentiation. The Wnt signaling pathway is critically
important for organogenesis and the development of the body plan.
Beta-catenin/TCF7L2-dependent Wnt signaling (the canonical pathway)
is involved in pancreas development, islet function, and insulin
production and secretion. The glucoincretin hormone glucagon-like
peptide-1 and the chemokine stromal cell-derived factor-1 modulate
canonical Wnt signaling in beta-cells which is obligatory for their
mitogenic and cytoprotective actions. The transcription factor
TCF7L2 is particularly strongly associated with a risk for diabetes
and appears to be fundamentally important in both canonical Wnt
signaling and beta-cell function.
[0016] An inhibitory role for Wnts is shown in a repressive
function for Wnt signaling in mouse neural induction. Mouse mutants
lacking effectors of b-catenin-dependent Wnt signaling, such as
Wnt3a, display increased neural tissue and even ectopic neural
tubes (Yoshikawa Y, Fujimori T, McMahon A P, et al. Evidence that
absence of Wnt-3a signaling promotes neutralization instead of
paraxial mesoderm development in the mouse. Dev Biol 1997;
183:234-42). Similarly, mutation of the Wnt co-receptors Lrp5 and
Lrp6 results in an expansion of the anterior neuroectoderm (Kelly O
G, Pinson K I, Skarnes W C. The Wnt co-receptors Lrp5 and Lrp6 are
essential for gastrulation in mice. Development 2004; 131:2803-15).
Consistent with this negative regulatory role, loss of Dickkopf
(Dick), a Wnt inhibitor, prevents forebrain development
(Mukhopadhyay M, Shtrom S, Rodriguez-Esteban C, et al. Dickkopf1 is
required for embryonic head induction and limb morphogenesis in the
mouse. Dev Cell 2001; 1: 423-34).
[0017] Bone Morphogenetic Protein (BMP)
[0018] FIG. 1 schematically shows in vitro neural induction. Many
in vivo neural inducers that act as inhibitors of BMPs, Nodal and
Wnt signaling also promote ES cell differentiation to committed
neural cells. In contrast, RA, which promotes neural induction in
ESCs, is not known to be important for neural induction in vivo
(Gaulden J, Reiter J F. Neur-ons and neur-offs: regulators of
neural induction in vertebrate embryos and embryonic stem cells.
Hum Mol Genet 2008; 17:R60-6).
[0019] Fibroblast Growth Factors (FGFs)
[0020] In support of the involvement of FGFs in neural induction,
FGFs can act cooperatively with BMP inhibition to promote Xenopus
neural induction (Reversade B, Kuroda H, Lee H, et al. Depletion of
Bmp2, Bmp4, Bmp7 and Spemann organizer signals induces massive
brain formation in Xenopus embryos. Development 2005; 132:3381-92).
One possible molecular mechanism for this functional cooperation is
through MAPK pathway convergence on BMP signaling through the
differential phosphorylation of Smad1, an important BMP effector
(Pera E M, Ikeda A, Eivers E, et al. Integration of IGF, FGF, and
anti-BMP signals via Smad1 phosphorylation in neural induction.
Genes Dev 2003; 17:3023-8). GSK3, a component and inhibitor of the
Wnt pathway, promotes additional phosphorylation and degradation of
Smad1 after a priming phosphorylation by MAPK, which may similarly
explain the anti-neuralizing properties of Wnts (Fuentealba L C,
Eivers E, Ikeda A, et al. Integrating patterning signals: Wnt/GSK3
regulates the duration of the BMP/Smad1 signal. Cell 2007;
131:980-93). However, mutation of the MAPK phosphorylation site of
Smad1 does not overtly abrogate neural induction in mice,
suggesting that this interaction is not essential for the effects
of FGFs on neural induction (Aubin J, Davy A, Soriano P. In vivo
convergence of BMP and MAPK signaling pathways: impact of
differential Smad1 phosphorylation on development and homeostasis.
Genes Dev 2004; 18:1482-94). Another possibility is that early FGF
signals downregulate expression of BMPs in the prospective neural
domain, allowing neural differentiation to proceed (Wilson S I,
Graziano E, Harland R. An early requirement for FGF signalling in
the acquisition of neural cell fate in the chick embryo. Curr Biol
2000; 10:421-9; Furthauer M, Van Celst J, Thisse C, et al. Fgf
signalling controls the dorsoventral patterning of the zebrafish
embryo. Development 2004; 131:2853-64). A third possibility is that
early FGF signaling promotes neural fate through a parallel,
BMP-independent mechanism (Delaune, E., Lemaire, P. and
Kodjabachian, L. Neural induction in Xenopus requires early FGF
signalling in addition to BMP inhibition. Development 2005; 132:
299-310; Linker C, Stern CD. Neural induction requires BMP
inhibition only as a late step, and involves signals other than FGF
and Wnt antagonists. Development 2004:131:5671-81). One BMP
independent mechanism may involve Wnts which, as described above,
can inhibit neural induction during gastrulation stages (Wilson S
I, Rydstrom A, Trimborn T, et al. (2001) The status of Wnt
signaling regulates neural and epidermal fates in the chick embryo.
Nature 2001; 411:325-30). However, addition of multiple FGFs, BMP
antagonists and Wnt antagonists to the chick embryonic epiblast is
not sufficient to induce the expression of the neural marker Sox2,
suggesting that still other pathways regulate neural induction
(Linker C, Stern CD. Neural induction requires BMP inhibition only
as a late step, and involves signals other than FGF and Wnt
antagonists. Development 2004:131:5671-81).
Maintenance and Enhancement of Axonal Outgrowth of Differentiated
Stem Cells In Vivo
[0021] Different sets of cues are implicated in the guidance of
axonal growth: the "canonical" guidance cues--Netrins, Slits,
Semaphorins, and Ephrins (O'Donnell M, Chance R K, Bashaw G J. Axon
growth and guidance: receptor regulation and signal transduction.
Annu Rev Neurosci. 2009; 32:383-412), the morphogens of the
Hedgehog, BMP, and Wnt families, and the cell adhesion molecules
such as N-cadherin, NCAM, and LI-CAM (Skaper S D, Moore S E, Walsh
F S. Cell signalling cascades regulating neuronal growth-promoting
and inhibitory cues. Prog Neurobiol. 2001 December; 65(6):593-608).
A combination of these cues working at different ranges exerts fine
directional control for axon.
[0022] Whereas initial neuronal differentiation of implanted stem
cells can be achieved in vivo by exposing them to the appropriate
factors described above, optimal survival and efficient axonal
outgrowth of these cells is also dependent on the early presence of
specific trophic factors in their environment (Lindsay R M, Wiegand
S J, Altar C A, DiStefano P S. Neurotrophic factors: from molecule
to man. Trends Neurosci. 1994 May; 17(5):182-90). These factors
signal through specific receptors expressed by target neurons and
regulated their expression of survival and axon outgrowth promoting
genes. Thus, the delivery of trophic factors to in vivo implanted
stem cells will significantly improve their long term survival and
functional integration. The relevant trophic factors include the
neurotrophin family (nerve growth factor (NGF), brain-derived
neurotrphic factor (BDNF), neurotrophin (NT)3 and NT4/5), the
TGF-beta-related family of growth factors (glial cell line-derived
neurotrophic factor (GDNF), artermin and persephin) (Airaksinen M
S, Saarma M. The GDNF family: signalling, biological functions and
therapeutic value. Nat Rev Neurosci. 2002 May; 3(5):383-94), the
cytokines ciliary neurotrophic factor (CNTF), leukemia inhibitory
factor (LIF), cardiotrophin 1 and oncostatin M (Murphy M, Dutton R,
Koblar S, Cheema S, Bartlett P. Cytokines which signal through the
LIF receptor and their actions in the nervous system. Prog
Neurobiol. 1997 August; 52(5):355-78), and the cerebral dopamine
neurotrophic factor and astrocyte-derived neurotrophic factor
(CDNF/MANF) family (Lindholm P, Saarma M. Novel CDNF/MANF family of
neurotrophic factors. Dev Neurobiol. 2010 April; 70(5):360-71).
BRIEF DESCRIPTION OF THE INVENTION
[0023] Small molecules that function as agonists or antagonists of
cellular receptors comprise some of the most valuable therapeutic
agents and molecular probes. Their controlled long-term delivery to
injured, diseased or transplanted tissues can have critical effects
on cell differentiation and tissue repair in the target areas.
According to the present invention mesoporous silicas with
controlled particle size have been prepared using a variety of
methods, for example using anionic amino acid-derived amphiphiles
and alkoxy silane costructure-directing agents (CSDAs), denoted
AMS-n mesoporous silicas (Shunai Che, Alfonso E. Garcia-Bennett,
Toshiyuki Yokoi, Kazutami Sakamoto, Hironobu Kunieda, Osamu
Terasaki, Takashi Tatsumi. A novel anionic surfactant templating
route for synthesizing mesoporous silica with unique structure;
Nature Materials, 2003, 2, 801.). Mesoporous materials are used
according to the present invention to control stem cell
differentiation after transplantation. We evaluated how mesoporous
particles, including nanoparticles with well-defined porosity,
control the release of the Sonic Hedgehog (Shh) agonist
purmorphamin (Pur) and retinoic acid (RA) over extended periods of
time affecting stem cell survival, migration and differentiation.
We showed that delivery of RA and Pur from AMS-n is effective in
promoting neuronal differentiation from embryonic stem cells and
regional neural crest stem cells in vitro and in vivo. These
findings indicate that porous silica-mediated delivery of cell
differentiation factors is a useful and advantageous approach in
transplantation and tissue repair strategies.
[0024] Thus, according to the present invention, we have used a new
approach--the mesoporous silica delivery system for induced
differentiation of stem cells in vitro and in vivo after
transplantation with extrinsic factors. The combination of both
methods--intrinsic and extrinsic factors will facilitate the
development of in vivo protocols for controlled and reproducible
stem cell differentiation that may be translated to clinical
application.
[0025] As better shown hereinafter, according to the present
invention, porous particles can be employed for controlled delivery
of differentiation factors to obtain the desired type of cells from
stem cell transplants. In the following description, we will also
see the effect of mesoporous on stem cell survival, glial scar
formation and migration of stem cells.
[0026] To explore the utility of this delivery technology for stem
cell differentiation, mesoporous silica with controlled particle
and pore size have been prepared using a variety of pore forming
templates including surfactants and non-surfactant molecules, for
controlled release of purmorphamine, a Sonic Hedgehog (Shh) agonist
and retinoic acid (RA) over extended time periods after
co-transplantation with bNCSCs, mouse and human embryonic stem
cells. The creation of such system, which will provide an in vitro
and/or in vivo environment favorable for stem cell survival and
differentiation, has great potential for use in developmental
biology and stem cell transplantation strategies.
[0027] We anticipate that timely delivery of extracellular
substances, such as fibroblast growth factors (FGFs), Wnts,
transforming growth factor (TGF)-beta family members, and Hedgehog
(hh) proteins to transplanted stem/progenitor cells, which are
pre-differentiated in vitro before transplantation, can lead to the
development of in vivo protocols for controlled stem cell
differentiation.
SUMMARY OF THE INVENTION
[0028] The present invention relates to a method to stimulate the
survival and differentiation of transplanted stem cells by delivery
of defined external factors from mesoporous silica. The present
invention is, among others, suitable for transplantation of human
ESCs and iPSCs, human tissue-specific stem cells and mesenchymal
stem cells in a clinic, as well as for experimental systems with
corresponding stem cells from other species.
[0029] The present inventors have found that human ESCs can be
forced to differentiate to neurons by local delivery of the sonic
hedgehog agonist purmorphamine (Pur) and retinoic acid (RA) from
mesoporous nanoparticles. This approach may therefore be useful for
improving initial survival of transplanted stem cells, as well as
for achieving desired differentiation of these cells and maintain
their long term viability.
[0030] According to one aspect, the present invention provides a
method of enhancing cell-survival during implantation of stem
cells.
[0031] According to one embodiment, said method relates to
enhancement of the survival of implanted stem cells during and
after their differentiation from stem cells to fully functional
cells for replacing lost of non-functional host cells. Said
enhancement may be achieved by one of the following ways:
[0032] a) delivery from mesoporous silica of survival and
differentiation factors specific for implanted stem cells and the
desired derivatives,
[0033] b) delivery from mesoporous silica of survival and
differentiation factors specific for co-implanted neural crest stem
cells and their desired derivatives which are aimed to provide
trophic support and functional reinnervation of stem cells used for
cell replacement therapy. According to another aspect of the
invention, the above-mentioned method of enhancing cell-survival of
stem cells during implantation may also be used as a therapeutic
method for treating patients with disorders for which cell
replacement therapy is required. Said treatment may be achieved by
one of the following ways:
[0034] c) by transplanting ESCs/iPSCs or other stem cells,
producing desired progenitor cells either before or after
transplantation to patients with disorders in which cells are
permanently lost or non-functional.
[0035] d) by transplanting ECSs/iPSCs, producing desired cells
either before or after transplantation to patients with disorders
in which cells are permanently lost or non-functional together with
neural crest stem cells. Said therapeutic method has the potential
to produce neurotrophic support and specific innervation from the
differentiated bNCSCs of newly differentiated replaced cells.
[0036] According to a further aspect of the invention, the
therapeutic method may be directed to patients requiring organs and
tissues to be reinnervated after transplantation (cardiac
transplants, pancreatic islet transplants, liver transplants etc.)
or newly created organs/tissues from stem/progenitor cells of
different sources, including somatic cell nuclear transfer, single
cell embryo biopsy, arrested embryos, altered nuclear transfer and
reprogramming somatic cells. Said method comprises using mesoporous
silica for delivery of survival and differentiation factors
for:
[0037] e) transplanted stem cells,
[0038] f) in vitro pre-differentiated stem/progenitor cells for
sensory neuron subtypes or autonomic neuron subtypes, or glial cell
subtypes,
[0039] g) for co-transplanted neural crest stem cells.
[0040] According to yet another aspect, the present invention
relates to kits for use with the above-mentioned therapeutic
methods.
[0041] According to one embodiment, the kit is devised for
co-implantation of stem cells with mesoporous silica containing
controlled delivery of survival and differentiation factors for the
generation of desired type of cells for cell replacement therapy.
These cells include, but are not limited to:
[0042] i) cardiomyocytes,
[0043] ii) skeletal muscle cells,
[0044] iii) insulin producing beta-cells,
[0045] iv) retinal photoreceptors,
[0046] v) midbrain dopaminergic neurons,
[0047] vi) spinal motorneurons,
[0048] vii) glutamatergic neurons
[0049] viii) GABAergic neurons
[0050] ix) oligodendrocytes
[0051] x) astrocytes
[0052] According to another embodiment, the kit is devised, for
example, for a method of reinnervation and trophic support of
organs after transplantation or organs/tissues either created from
stem/progenitor cells of different sources, or transplanted from
organ/tissue donors.
[0053] Said kit comprises, in addition to stem cells, one or more
of the following cell types in combination with mesoporous silica
containing survival and differentiation factors for:
[0054] xi) co-transplanted neural crest stem cells,
[0055] xii) in vitro pre-differentiated stem/progenitor cells for
sensory neuron subtypes or autonomic neuron subtypes, or glial cell
subtypes.
[0056] The above methods and kits may alternatively comprise cells
derived from animals, as these methods and kits may be used for the
corresponding veterinary purposes.
[0057] The present invention relates also to a pharmaceutical
active ingredient for cell differentiation to alleviate cell and
cell-related deficiencies in mammals which comprises porous silica
containing releasable agents capable of contributing to a cell
environment conducive for stem cell differentiation in co-implanted
stem cells and/or in endogenous stem cells.
[0058] Preferably said porous silica is characterized by a surface
area higher than 200 m.sup.2/g and a pore size between 1.5-50
nm.
[0059] According to a preferred embodiment, the porous silica
particle have a particle shape comprising of spheres, or rod-shaped
particles. The porous silica has preferably average particle size
and/or sizes in the range between 50-5000 nm and more preferably it
is in the form of substantially spherical particles having a size
range of 200-500 nm.
[0060] In the active ingredient of the invention the releasable
agent capable of contributing to a cell environment conducive for
stem cell differentiation in co-implanted stem cells and/or in
endogenous stem cells is preferably 1-60% of the total weight of
the pharmaceutical active ingredient containing silica, and more
preferably between 10-45 wt %.
[0061] Further, the present invention relates to a pharmaceutical
active ingredient for elimination of undifferentiated co-implanted
stem cells with the potential for tumor formation in a mammal,
comprising in vitro produced porous silica containing releasable
agents capable of forcing co-implanted cells to become
postmitotic.
[0062] Preferably, the in vitro produced porous silica has the
above-mentioned chracteristics in terms of surface area and
porosity.
[0063] According to a preferred embodiment, the co-implanted stem
cells which are combined with mesoporous silica are chosen from the
group consisting of regional stem cells, embryonic stem (ES) cells,
neural crest stem cells, neural stem cells from brain and spinal
cord, mesenchymal stem cells, endothelial stem cells, endodermal
stem cells, induced pluripotent stem (iPS) cells.
[0064] Preferably, the releasable agent(s) are selected from the
group consisting of secreted growth factors and morphogens,
including, but not limited to fibroblast growth factors (FGFs),
Wnts, transforming growth factor (TGF)-beta family members,
Hedgehog (hh) proteins, retinoic acid, vascular endothelial growth
factor (VEGF), Dickkopf (Dkk)-1, insulin, Activin, SDF-1/CXCL12),
pleiotrophin (PTN), insulin-like growth factor 2 (IGF2), ephrin B1
(EFNB1), and cAMP.
[0065] The present invention relates also to a delivery system for
delivery of a pharmaceutical active ingredient in mammals,
comprising a pharmaceutical active ingredient for cell
differentiation to alleviate cell and cell-related deficiencies in
mammals, said pharmaceutical active ingredient comprising porous
silica containing a releasable agent capable of contributing to a
cell environment conducive for stem cell differentiation in
co-implanted stem cells and/or in endogenous stem cells, and stem
cells.
[0066] Preferably, said cells are selected from the group
consisting of regional stem cells, embryonic (ES) stem cells,
neural crest stem cells, neural stem cells from brain and spinal
cord, mesenchymal stem cells, endothelial stem cells, endodermal
stem cells, iPS cells. Examples of releasable agents include, but
are not limited to, secreted growth factors and morphogens,
including, but not limited to, fibroblast growth factors (FGFs),
Wnts, transforming growth factor (TGF)-beta family members, and
Hedgehog (hh) proteins, retinoic acid, VEGF, Dkk1, insulin,
Activin, SDF-1/CXCL12), pleiotrophin (PTN), insulin-like growth
factor 2 (IGF2), and ephrin B1 (EFNB 1), cAMP
DETAILED DESCRIPTION OF THE INVENTION
Methods for Loading Survival and Differentiation Factors into
Mesoporous Silica
[0067] The present invention includes a method of loading survival
enhancing and differentiation factors into mesoporous particles
whereby the porous silica material, be it a solvent extracted or
calcined material (see Rambabu Atluri, Niklas Hedin, Alfonso E.
Garcia-Bennett. Hydrothermal Phase transformations of cubic
mesoporous solid AMS-6. Chemistry of Materials, 2008, 20 (12),
3857-3866.) is mixed with the desired amount of survival enhancing
and differentiation factors in a solvent that will dissolve or
partially dissolve the aforementioned factors. The mixture may be
stirred, centrifuged, spray dried, or filtered after periods
between 0.5 hours and 2 days at temperatures between 0-80 degrees
Celsius. The recovered solid if the sample is stirred typically
contains between 20-49 wt % of factors within the pores of the
silica particle, but may contain higher amounts if the loading
process is repeated several times.
[0068] Sources of Donor Cells for Stem Cell Transplantation
[0069] We show the proof-of-principle on the example of ESCs and
neural stem cells to differentiate towards neuronal phenotype under
effect of extrinsic factors delivered with AMS. However we
anticipate that differentiation of different types of cells can be
generated from different sources of stem cells with this method. We
therefore briefly describe in the following section different
sources of stem cells that are considered for cell replacement
therapy.
[0070] Embryonic stem (ES) cells are capable of generating all cell
types in the organism and show great promise for cell replacement
therapy in clinical applications. ESCs have been isolated as
homogenous cell lines, they can be expanded and modified to meet
the needs of the patient using standardized and optimized
protocols.
[0071] The recent discovery of induced pluripotent stem (iPS) cells
offers the possibility to generate patient-specific cells, which
can be implanted without ethical or immunological complications.
Several laboratories have demonstrated that a limited set of less
than four transcription factors is sufficient for dedifferentiation
of most somatic cell types to the undifferentiated iPS state
(Takahashi K, Yamanaka S. Induction of pluripotent stem cells from
mouse embryonic and adult fibroblast cultures by defined factors.
Cell 2006; 126:663-76; Takahashi K, Tanabe K, Ohnuki M, et al.
Induction of pluripotent stem cells from adult human fibroblasts by
defined factors. Cell 2007; 131:861-72; Huangfu D, Osafune K, Maehr
R, et al. Induction of pluripotent stem cells from primary human
fibroblasts with only Oct4 and Sox2. Nat Biotechnol 2008;
26:1269-75; Carey B W, Markoulaki S, Hanna J, et al. Reprogramming
of murine and human somatic cells using a single polycistronic
vector. Proc Natl Acad Sci USA 2009; 106:157-62. Erratum in: Proc
Natl Acad Sci USA 2009; 106:5449. Proc Natl Acad Sci USA 2009;
106:11818; Carey B W, Markoulaki S, Beard C, et al. Single-gene
transgenic mouse strains for reprogramming adult somatic cells. Nat
Methods 2010; 7:56-9; Silva J, Nichols J, Theunissen T W, et al.
Nanog is the gateway to the pluripotent ground state. Cell 2009;
138:722-37). The initial protocol of reprogramming adult cells to
obtain iPSCs raised concerns of its therapeutic applicability due
to the expression of the c-myc proto-oncogene, but newer, improved
protocols for iPS cell production show that pluripotency can be
achieved without any genetic alteration of the adult cell (Zhou H,
Wu S, Joo J Y, et al. Generation of induced pluripotent stem cells
using recombinant proteins. Cell Stem Cell 2009; 4:381-4. Erratum
in: Cell Stem Cell 2009; 4:581). Recent studies have shown that
generation of differentiated cells from iPSCs occurs at a slower
rate than for ESCs (Hu B Y, Zhang S C. Directed differentiation of
neural-stem cells and subtype-specific neurons from hESCs. Methods
Mol Biol 2010; 636:123-37), possibly due to a genetic difference in
a developmentally important region of chromosome 12qF1 in the mouse
(Stadtfeld M, Apostolou E, Akutsu H, et al. Aberrant silencing of
imprinted genes on chromosome 12qF1 in mouse induced pluripotent
stem cells. Nature 2010; 465:175-8).
[0072] Tissue-specific stem/progenitor cells are attractive source
for transplantation since they are already committed to some extent
to differentiation towards the desired cell type(s). These cells
were found in the human thyroid, with an intrinsic ability to
generate thyroidal cells and the potential to produce non-thyroidal
cells (Fierabracci A, Puglisi M A, Giuliani L, et al.
Identification of an adult stem/progenitor cell-like population in
the human thyroid. J Endocrinol 2008; 198:471-87); in the adult
human pancreas (Puglisi M A, Guilani L, Fierabracci A 2008
Identification and characterization of a novel expandable adult
stem/progenitor cell population in the human exocrine pancreas. J
Endocrinol Invest 2008; 31:563-72) in human skeletal muscle
(Alessandri G, Pagano S, Bez A, et al. Isolation and culture of
human muscle-derived stem cells able to differentiate into myogenic
and neurogenic cell lineages. Lancet 2004; 364:1872-83) human and
murine heart (Messina E, De Angelis L, Frati G, et al. Isolation
and expansion of adult cardiac stem cells from human and murine
heart. Circulation Res 2004; 95:911-21) and human bladder
(Fierabracci A, Caione P, Di Giovine M, et al. Identification and
characterisation of adult stem/progenitor cells in the human
bladder (Bladder spheroids): perspectives of application in
pediatric surgery. Ped Surg Int 2007; 23 837-9). However, their
potential for regenerative medicine is questionable since these
cells are not always easily accessible and they are often poorly
viable when obtained from adult tissue.
[0073] Mesenchymal stem cells (MSCs) can be easily harvested from
e.g. bone marrow, adipose tissue, umbilical cord blood or amnion,
are able to differentiate to a variety of mesodermal as well as
non-mesodermal cell types in vitro (Franco Lambert A P, Fraga
Zandonai A, Bonatto D, et al. Differentiation of human
adipose-derived adult stem cells into neuronal tissue: does it
work? Differentiation 2009; 77:221-8; Meirelles Lda S, Nardi NB.
Methodology, biology and clinical applications of mesenchymal stem
cells. Front Biosci 2009; 14:4281-98 09) and can be used for
autologous transplantation. MSCs have the ability to modify immune
processes (Uccelli A, Moretta L, Pistola V. Mesenchymal stem cells
in health and disease. Nat Rev Immunol 2008; 8:726-36; Sadan O,
Melamed E, Offen D. Bone-marrow-derived mesenchymal stem cell
therapy for neurodegenerative diseases. Expert Opin Biol Ther 2009;
9:1487-97) and were used for transplantation in an undifferentiated
state with the intention of promoting tissue repair rather than for
the purpose of cell replacement (Kim S U, de Vellis J. Stem
cell-based cell therapy in neurological diseases: a review. J
Neurosci Res 2009; 87:2183-200; Abdallah B M, Kassem M. The use of
mesenchymal (skeletal) stem cells for treatment of degenerative
diseases: current status and future perspectives. J Cell Physiol
2009; 218:9-12). MSC transplantation has received considerable
attention in the treatment of graft-versus-host disease and showed
promising results in clinical trials (Kebriaei P, Isola L, Bahceci
E, et al. Adult human mesenchymal stem cells added to
corticosteroid therapy for the treatment of acute graft-versus-host
disease. Biol Blood Marrow Transplant 2009; 15:804-11; Picinich S
C, Mishra P J, Mishra P J, et al. The therapeutic potential of
mesenchymal stem cells. Cell- & tissue-based therapy. Expert
Opin Biol Ther 2007; 7:965-73).
Current State of Stem Cell Transplantation as Cell Replacement
Therapy for Major Disorders
[0074] There is a long list of medical conditions which are
potential therapeutic targets for stem cell transplantation. For
some of these disorders no treatment is yet able to significantly
change the course of the disease; for others treatment exists, but
does not cure the disease or is insufficient. Here we describe
disorders with huge impact on the affected individuals and on
society, and where treatment is generally thought to benefit
strongly in the long run from cell replacement therapy by stem cell
transplants. Stem cell transplantation in animal models of these
disorders has been and still is the subject of intense research
providing evidence, to some degree, of structural repair and
functional improvement. Moreover, clinical trials with stem cell
transplantation have already been carried out or are in preparation
and early results show that this approach is feasible and safe, but
therapeutic benefits are so far inconsistent (reviewed in Trounson
A. New perspectives in human stem cell therapeutic research. BMC
Medicine 2009; 7:2). P 1. Myocardial Disease
[0075] Ischemic heart disease is the most common cause of death in
the Western world. Cardiomyocytes are the contracting elements of
the heart and the intrinsic capacity of the heart to replace these
cells is very limited. Transplantation of stem cells that can
replace lost cardiomyocytes is therefore an attractive option to
treat this condition. Cardiomyocytes have been generated in vitro
from a wide range of stem/progenitor cells, including iPSCs (Gai H,
Leung E L, Costantino P D, et al. Generation and characterization
of functional cardiomyocytes using induced pluripotent stem cells
derived from human fibroblasts. Cell Biol Int. 2009; 33:1184-93;
Kuzmenkin A, Liang H, Xu G, et al. Functional characterization of
cardiomyocytes derived from murine induced pluripotent stem cells
in vitro. FASEB J 2009; 23:4168-80 Pfannkuche K, Liang H, Hannes T,
et al. Cardiac myocytes derived from murine reprogrammed
fibroblasts: intact hormonal regulation, cardiac ion channel
expression and development of contractility. Cell Physiol Biochem
2009; 24:73-86), ESCs (Beqqali A, van Eldika W, Mummery C, et al.
Human stem cells as a model for cardiac differentiation and disease
Cell Mol Life Sci 2009; 66:800-13; Steel D, Hyllner J, Sartipy P.
Cardiomyocytes derived from human embryonic stem
cells--characteristics and utility for drug discovery. Curr Opin
Drug Discov Dev 2009; 12:133-40), hematopoietic progenitor/stem
cells, MSCs (Choi S C, Shim W J, Lim D S. Specific monitoring of
cardiomyogenic and endothelial differentiation by dual
promoter-driven reporter systems in bone marrow mesenchymal stem
cells. Biotechnol Lett 2008; 30:835-43; Antonitsis P,
Ioannidou-Papagiannaki E, Kaidoglou A, et al. Cardiomyogenic
potential of human adult bone marrow mesenchymal stem cells in
vitro. Thorac Cardiovasc Surg 2008; 56:77-82; Ge D, Liu X, Li L, et
al. Chemical and physical stimuli induce cardiomyocyte
differentiation from stem cells. Biochem Biophys Res Commun 2009;
381:317-21; Gwak S J, Bhang S H, Yang H S, et al. In vitro
cardiomyogenic differentiation of adipose-derived stromal cells
using transforming growth factor-beta1. Cell Biochem Funct 2009;
27:148-54), and cardiomyocyte progenitor cells (Smits A M, van
Vliet P, Metz C H, et al. Human cardiomyocyte progenitor cells
differentiate into functional mature cardiomyocytes: an in vitro
model for studying human cardiac physiology and pathophysiology.
Nat Protoc 2009; 4:232-43).
[0076] An additional source for repair of myocardial contractility
are skeletal myoblasts (Nomura T, Ueyama T, Ashihara E, et al.
Skeletal muscle-derived progenitors capable of differentiating into
cardiomyocytes proliferate through myostatin-independent TGF-beta
family signaling. Biochem Biophys Res Commun 2008; 365:863-9),
although they do not seem to be able to differentiate to
cardiomyocytes (Reinecke H, Poppa V, Murry C E. Skeletal muscle
stem cells do not transdifferentiate into cardiomyocytes after
cardiac grafting. J Mol Cell Cardiol 2002; 34:241-9; Leobon B,
Garcin I, Menasche P, et al. Myoblasts transplanted into rat
infarcted myocardium are functionally isolated from their host.
Proc Natl Acad Sci USA 2003; 100:7808-11). Intravascular delivery
or cardiac transplants of multipotent or pre-differentiated
cardiogenic cells from these stem cell sources have been shown to
promote cardiac structural repair and functional restoration in
animal models of myocardial injury (Fukushima S, Coppen S R, Lee J,
et al. Choice of cell-delivery route for skeletal myoblast
transplantation for treating post-infarction chronic heart failure
in rat. PLoS One 2008; 3:e3071; Hendry S L 2nd, van der Bogt K E,
Sheikh A Y, et al. Multimodal evaluation of in vivo magnetic
resonance imaging of myocardial restoration by mouse embryonic stem
cells. J Thorac Cardiovasc Surg 2008; 136:1028-37; Matsuura K,
Honda A, Nagai T, et al. Transplantation of cardiac progenitor
cells ameliorates cardiac dysfunction after myocardial infarction
in mice. J Clin Invest 2009; 119:2204-17; Jin J, Jeong S I, Shin Y
M, et al. Transplantation of mesenchymal stem cells within a
poly(lactide-co-epsilon-caprolactone) scaffold improves cardiac
function in a rat myocardial infarction model. Eur J Heart Fail
2009; 11:147-53; Okura H, Matsuyama A, Lee CM, et al.
Cardiomyoblast-like cells differentiated from human adipose
tissue-derived mesenchymal stem cells improve left ventricular
dysfunction and survival in a rat myocardial infarction model.
Tissue Eng Part C Methods 2010; 16:417-25).
[0077] The encouraging results from this experimental research have
prompted several clinical trials in patients with myocardial
disease, using different types of progenitor/stem cells (Segers V
F, Lee R T. Stem-cell therapy for cardiac disease. Nature 2008;
451:937-42; Joggerst S J, Hatzopoulos A K Stem cell therapy for
cardiac repair: benefits and barriers. Exp Rev Molec Med Epub 2009
Jul. 8; 11:e20; Piepoli M F. Transplantation of progenitor cells
and regeneration of damaged myocardium: more facts or doubts?
Insights from experimental and clinical studies. J Cardiovasc Med
2009; 10:624-34).
[0078] Protocol for Cardiomyocytes
[0079] Cardiomyocyte progenitors were generated from hESC embryoid
bodies treated with Activin A, BMP4 or with those 2+Wnt3 and bFGF.
The progenitors express Nkx2.5, Tbx5/20, Gata-4, Mef2c and Hand1/2.
Their differentiation to functional cardiomyocytes in vitro can be
promoted with VEGF and Dkk1 (Vidarsson H, Hyllner J, Sartipy P.
Differentiation of human embryonic stem cells to cardiomyocytes for
in vitro and in vivo applications. Stem Cell Rev 2010;
6:108-20).
[0080] 2. Skeletal Muscle Disorders
[0081] Muscular dystrophies include a large number of inherited
disorders characterized by severe and progressive degeneration of
skeletal muscle fibers, resulting in serious disability and often
early death. In view of the genetic basis of this disorder,
transplantation of stem cells which are able to form functional
muscle fibers is an attractive approach to cure these disorders.
Myotubes formation can be achieved from regional stem cells (muscle
satellite cells) by activation of transcription factor Pax7 under
the influence of FGF and HGF, followed by transcription factors
MyoD and MyoG (Yablonka-Reuveni Z, Day K, Vine A, et al. Defining
the transcriptional signature of skeletal muscle stem cells. J Anim
Sci 2008; 86(14 Suppl):E207-16). Retinoic acid appears to play a
critical role in the generation of muscle progenitor stage by
activating beta-catenin and inhibiting BMP (Kennedy K A, Porter T,
Mehta V et al. Retinoic acid enhances skeletal muscle progenitor
formation and bypasses inhibition by bone morphogenetic protein 4
but not dominant negative beta-catenin. BMC Biol 2009; 7:67).
[0082] Embryonic stem cells (human, mouse), bone marrow-associated
stem cells (human, mouse), stem cells from the hematopoetic or
vascular system (human, mouse), adipose tissue derived stem cells
(human) and skeletal muscle-associated precursor cells (human,
mouse) have been delivered intramuscularly (mostly) or
intravascularly to mouse models of muscular dystrophies (Darabi R,
Perlingeiro R C. Lineage-specific reprogramming as a strategy for
cell therapy. Cell Cycle 2008; 7:1732-7; Quattrocelli M, Cassano M,
Crippa S et al. Cell therapy strategies and improvements for
muscular dystrophy. Cell Death Differ. 2009 Oct. 30 [Epub ahead of
print]). Promising results have been obtained with several, but not
all of these approaches. Important limiting factors include
extensive early death of grafted cells, failure of grafted cells to
differentiate properly and integrate functionally with host muscle
tissue. Several clinical trials have been undertaken with
transplantation of myogenic stem cells, but with limited success
(Tedesco F S, Dellavalle A, Diaz-Manera J et al. Repairing skeletal
muscle: regenerative potential of skeletal muscle stem cells. J
Clin Invest 2010; 120:11-9). Results from recent experimental
studies using ESCs are promising in terms of survival and potential
for functional improvement in mouse muscular dystrophy models
(Darabi R, Gehlbach K, Bachoo R M, et al. Functional skeletal
muscle regeneration from differentiating embryonic stem cells. Nat
Med 2008; 14:134-43; Darabi R, Baik J, Clee M, et al. Engraftment
of embryonic stem cell-derived myogenic progenitors in a dominant
model of muscular dystrophy. Exp Neurol 2009; 220:212-6).
[0083] Transplantation of stem cells for repair of dysfunctional
muscle have been tried with some success in patients with stress
urinary incontinence, a common disorder characterized by reduced
tone in pelvic muscle regulation bladder emptying (Nikolaysky D,
Chancellor M B. Stem cell therapy for stress urinary incontinence.
Neurourol Urodyn 2010; 29 Suppl 1:S36-41).
[0084] Protocol for Skeletal Muscle Fibers
[0085] Mesodermal progenitors able to generate muscle fibers
express Pax3 and Pax 7. Pax3 acts as a master regulator to
determine a myogenic lineage (Darabi R, Gehlbach K, Bachoo R M, et
al. Functional skeletal muscle regeneration from differentiating
embryonic stem cells. Nat Med 2008; 14:134-43; Darabi R, Santos F
N, Perlingeiro R C. The therapeutic potential of embryonic and
adult stem cells for skeletal muscle regeneration. Stem Cell Rev
2008; 4:217-25). An essential target for Pax3 appears to be FGF
signaling through FRGR4 (Lagha M, Kormish J D, Rocancourt D, et al.
Pax3 regulation of FGF signaling affects the progression of
embryonic progenitor cells into the myogenic program. Genes Dev
2008; 22:1828-37; Lagha M, Sato T, Bajard L, et al. Regulation of
skeletal muscle stem cell behavior by Pax3 and Pax7. Cold Spring
Harb Symp Quant Biol 2008; 73:307-15) by FGF8, which in combination
with Shh promote expression of myogenic regulatory factors (MRFs)
myf5 and myoD (Hammond C L, Hinits Y, Osborn D P et al. Signals and
myogenic regulatory factors restrict Pax3 and pax7 expression to
dermomyotome-like tissue in zebrafish. Dev Biol 2007; 302:504-21).
Skeletal muscle fibers were also generated from hESCs via
generation of multipotent mesenchymal stem cells (Stavropoulos M E,
Mengarelli I, Barberi T. Differentiation of multipotent mesenchymal
precursors and skeletal myoblasts from human embryonic stem cells.
Curr Protoc Stem Cell Biol 2009;Chapter 1:Unit 1F.8). Embryonic
skeletal muscle cells express NCAM and are FACS sorted after
incubation with anti-NCAM. Sorted cells differentiate to
spontaneously twitching muscle cells in vitro and long-term
survival after transplantation to mice with toxin-induced muscle
damage. No functional data are presented. Retinoic acid appears to
play a critical role in the generation of muscle progenitors by
activating beta-catenin and inhibiting BMP (Kennedy K A, Porter T,
Mehta V et al. Retinoic acid enhances skeletal muscle progenitor
formation and bypasses inhibition by bone morphogenetic protein 4
but not dominant negative beta-catenin. BMC Biol 2009; 7:67).
[0086] 3. Insulin Producing Beta-Cells
[0087] Type 1 diabetes is characterized by an autoimmune mediated
loss of insulin producing .beta.-cells in the pancreatic islets of
Langerhans. Today, transplantation of either the entire pancreas or
of isolated islets has become a treatment of choice for selected
patients with diabetes mellitus (Frank A M, Barker C F, Markmann J
F Comparison of whole organ pancreas and isolated islet
transplantation for type 1 diabetes. Adv Surg 2005; 39:137-63; Ryan
E A, Bigam D, Shapiro A M. Current indications for pancreas or
islet transplant. Diabetes Obes Metab 2006; 8:1-7). However,
long-term results after islet transplantation are disappointing
with adequate graft function seen in less than 10% of the patients
after five years (Ryan E A, Paty B W, Senior P A, et al. Five-year
follow-up after clinical islet transplantation. Diabetes 2005;
54:2060-9). Furthermore, the number of patients in need of new
.beta.-cells far outnumbers the limited access to islet tissue for
transplantation. Transplantation of stem cells to replace the lost
.beta.-cells is therefore an attractive therapy for long-term
treatment of type 1 diabetes and also for some cases of type 2
diabetes. Insulin producing .beta.-cells have been generated from
several sources, including ESCs (Baharvand H, Jafary H, Massumi M,
et al. Generation of insulin-secreting cells from human embryonic
stem cells. Dev Growth Differ 2006; 48:323-32; Schroeder I S,
Rolletschek A, Blyszczuk P, et al. Differentiation of mouse
embryonic stem cells to insulin-producing cells. Nat Protoc 2006;
1:495-507; Marchand M, Schroeder I S, Markossian S, et al. Mouse ES
cells over-expressing the transcription factor NeuroD1 show
increased differentiation towards endocrine lineages and
insulin-expressing cells. Int J Dev Biol 2009; 53:569-78;
Evans-Molina C, Vestermark G L, Mirmira R G. Development of
insulin-producing cells from primitive biologic precursors. Curr
Opin Organ Transplant 2009; 14:56-63; Van Hoof D, D'Amour K A,
German M S. Derivation of insulin-producing cells from human
embryonic stem cells. Stem Cell Res 2009; 3:73-87), stem/progenitor
cells from the exocrine pancreas (Demeterco C, Hao E, Lee S H, et
al. Adult human beta-cell neogenesis? Diabetes Obes Metab 2009; 11
Suppl 4:46-53; Noguchi H, Oishi K, Ueda M, et al. Establishment of
mouse pancreatic stem cell line. Cell Transplant 2009; 18:563-71;
Mato E, Lucas M, Petriz J, et al. Identification of a pancreatic
stellate cell population with properties of progenitor cells: new
role for stellate cells in the pancreas. Biochem J 2009;
421:181-91), biliary ducts (Nagaya M, Kubota S, Isogai A, et al.
Ductular cell proliferation in islet cell neogenesis induced by
incomplete ligation of the pancreatic duct in dogs. Surg Today
2004; 34:586-92), MSCs from various sources (Parekh V S, Joglekar M
V, Hardikar A A. Differentiation of human umbilical cord
blood-derived mononuclear cells to endocrine pancreatic lineage.
Differentiation 2009; 78:232-40; Xie Q P, Huang H, Xu B, et al.
Human bone marrow mesenchymal stem cells differentiate into
insulin-producing cells upon microenvironmental manipulation in
vitro. Differentiation 2009; 77:483-91; Kajiyama H, Hamazaki T S,
Tokuhara M, et al. Pdx1-transfected adipose tissue-derived stem
cells differentiate into insulin-producing cells in vivo and reduce
hyperglycemia in diabetic mice. Int J Dev Biol 2009; 54:699-705),
from iPSCs derived from fibroblasts of patients with diabetes type
1 (Maehr R, Chen S, Snitow M, et al. Generation of pluripotent stem
cells from patients with type 1 diabetes. Proc Natl Acad Sci USA
2009; 106:15768-73). Human ESCs have been converted to beta-cells
capable of synthesizing insulin through a stepwise procedure of
transcriptional regulation that mimics the normal development of
beta-cells (D'Amour K A, Agulnick A D, Eliazer S, et al. Efficient
differentiation of human embryonic stem cells to definitive
endoderm. Nat Biotechnol. 2005; 23:1534-41; D'Amour K A, Bang A G,
Eliazer S, et al. Production of pancreatic hormone-expressing
endocrine cells from human embryonic stem cells. Nat Biotechnol
2006; 24:1392-401). Exocrine pancreatic cells were also shown to
give rise to insulin producing 13-cells by transcriptional
reprogramming with a specific combination of the three
transcription factors Ngn3 (also known as Neurog3), Pdx1 and Mafa
(Zhou Q, Brown J, Kanarek A, et al. In vivo reprogramming ofadult
pancreatic exocrine cells to beta-cells. Nature 2008;
455:627-32).
[0088] Transplantation of ESCs to diabetic mice also improved
glucose homeostasis indirectly by promoting endogenous .beta.-cell
neogenesis (Kodama M, Takeshita F, Kanegasaki S, et al. Pancreatic
endocrine and exocrine cell ontogeny from renal capsule
transplanted embryonic stem cells in streptozocin-injured mice. J
Histochem Cytochem 2008; 56:33-44). Human ESCs pre-differentiated
to committed pancreatic endoderm developed into functional
beta-cells after transplantation to immune-compromised mice (Kroon
E, Martinson L A, Kadoya K, et al. Pancreatic endoderm derived from
human embryonic stem cells generates glucose-responsive
insulin-secreting cells in vivo. Nat Biotechnol 2008; 26:443-52).
Interestingly, treatment of the recipients with the beta-cell toxin
streptozotocin destroyed their endogenous beta-cell population, but
the grafted cells were protected and provided a functional source
of insulin. Thus, although efficient and reproducible replacement
of lost .beta.-cells in type 1 diabetes have still not been fully
achieved with stem cell transplants (reviewed in Trounson A. New
perspectives in human stem cell therapeutic research. BMC Medicine
2009; 7:2), promising steps in this direction have been taken.
[0089] Protocol for Beta-Cells
[0090] Protocol for generating insulin producing beta-cells from
hESCs involve stepwise lineage restriction generating in sequence:
definitive endodermal cells (Activin+Wnt3), primitive foregut
endoderm (FGF10+KAAD-cyclopamine), posterior foregut endoderm
(RA+FGF10+KAAD-cyclopamine), pancreatic endoderm and endocrine
precursors (Extendin-4), and hormone producing cells (IGF1+HGF).
Transcription factor profiles are: Sox17, CER, FoxA2, and the
cytokine receptor CXCR4 (definitive endodermal cells), Hnf1B, Hnf4A
(primitive foregut endoderm), Pdx1, Hnf6, H1xB9 (posterior foregut
endoderm), Nkx6.1, Nkx2.2, Ngn3, Pax4 (pancreatic endoderm and
endocrine precursors). (D'Amour K A, Bang A G, Eliazer S, et al.
Production of pancreatic hormone-expressing endocrine cells from
human embryonic stem cells. Nat Biotechnol 2006; 24:1392-401; Kroon
E, Martinson L A, Kadoya K, et al. Pancreatic endoderm derived from
human embryonic stem cells generates glucose-responsive
insulin-secreting cells in vivo. Nat Biotechnol 2008;
26:443-52).
[0091] An improved protocol has been suggested which focuses on
regulating the key signaling pathways form ESC to insulin producing
beta-cells (Champeris Tsaniras S, Jones P M. Generating pancreatic
beta-cells from embryonic stem cells by manipulating signaling
pathways. J Endocrinol. 2010 April 12. [Epub ahead of print]).
According to this protocol the principles are: mesendoderm
(stimulating Wnt and nodal pathways, down-regulating
phosphatidylinositol 3-kinase pathway (PI3K)), definitive endoderm
(remove Wnt), posterior foregut (down-regulate Wnt), pancreatic
endoderm (block Shh), beta-cell precursors (block Notch),
beta-cells (block PI3K, stimulate Shh).
[0092] 4. Retinal Disease
[0093] Age-related macular degeneration is associated with the loss
of photoreceptors and a common cause of blindness or severe visual
impairment in the aging Western population. Efficient treatment for
this disorder is currently lacking. Stem cells have been identified
and characterized in several locations of the adult mammalian eye,
as well as the molecular pathways leading to their differentiation
to different cell types (Locker M, Borday C, Perron M. Stemness or
not stemness? Current status and perspectives of adult retinal stem
cells. Curr Stem Cell Res Ther 2009; 4:118-30). However, recruiting
these cells in vivo to replace lost photoreceptors has so far been
unsuccessful. On the other hand, photoreceptors or retinal ganglion
cells have been generated from iPSCs, ESCs and retinal
stem/progenitor cells in vitro (Mayer E J, Carter D A, Ren Y et al.
Neural progenitor cells from postmortem adult human retina. Br J
Ophthalmol 2005; 89:102-6; Zhao B, Allinson S L, Ma A et al.
Targeted cornea limbal stem/progenitor cell transfection in an
organ culture model. Invest Ophthalmol Vis Sci 2008; 49:3395-40;
Hirami Y, Osakada F, Takahashi K et al. Generation of retinal cells
from mouse and human induced pluripotent stem cells. Neurosci Lett
2009; 458:126-31; Osakada F, Jin Z B, Hirami Y et al. In vitro
differentiation of retinal cells from human pluripotent stem cells
by small-molecule induction. J Cell Sci 2009; 122(Pt 17):3169-79).
Following transplantation, these cells are able to integrate into
the retinal network, differentiate to functional photoreceptors and
help preserve or restore visual function in experimental model of
retinal degeneration (Klassen H. Transplantation of cultured
progenitor cells to the mammalian retina. Expert Opin Biol Ther
2006; 6:443-51; Klassen H, Schwartz P H, Ziaeian B et al. Neural
precursors isolated from the developing cat brain show retinal
integration following transplantation to the retina of the
dystrophic cat. Vet Ophthalmol 2007; 10:245-53; Gias C, Jones M,
Keegan D et al. Preservation of visual cortical function following
retinal pigment epithelium transplantation in the RCS rat using
optical imaging techniques. Eur J Neurosci 2007; 25:1940-8 Pinilla
I, Cuenca N, Sauve Y et al. Preservation of outer retina and its
synaptic connectivity following subretinal injections of human RPE
cells in the Royal College of Surgeons rat. Exp Eye Res 2007;
85:381-92; Wang S, Girman S, Lu B et al. Long-term vision rescue by
human neural progenitors in a rat model of photoreceptor
degeneration. Invest Ophthalmol Vis Sci 2008; 49:3201-6; Chen F K,
Uppal G S, MacLaren R E et al. Long-term visual and microperimetry
outcomes following autologous retinal pigment epithelium choroid
graft for neovascular age-related macular degeneration. Clin Exp
Ophthalmol 2009; 37:275-85). Results from preclinical studies have
verified the safety of these protocols Francis P J, Wang S, Zhang Y
et al. Subretinal transplantation of forebrain progenitor cells in
nonhuman primates: survival and intact retinal function. Invest
Ophthalmol Vis Sci 2009; 50:3425-31; Lu B, Malcuit C, Wang S et al.
Long-term safety and function of RPE from human embryonic stem
cells in preclinical models of macular degeneration. Stem Cells
2009; 27:2126-35) and clinical trials in patients with age-related
macular degeneration are in preparations (Coffey P. Interview:
stemming vision loss with stem cells: seeing is believing. Regen
Med 2009; 4:505-7). The experimental basis for successful clinical
trials is thus promising. However, tools to optimize survival and
differentiation of stem/progenitor transplants when they hosted by
retinal tissue undergoing a chronic degenerative process, might
still be needed.
[0094] Protocol for Retinals Cells
[0095] Various types of retinal cells were generated from hESCs
(Lamba D A, Karl M O, Ware C B, et al. Efficient generation of
retinal progenitor cells from human embryonic stem cells. Proc Natl
Acad Sci USA 2006; 103:12769-74; Reh T A, Lamba D, Gust J.
Directing human embryonic stem cells to a retinal fate. Methods Mol
Biol 2010; 636:139-53). Embryoid bodies were produced and
thereafter treated with IGF1, Noggin (BMP inhibitor) and Dkk1 (Wnt
inhibitor). This forces hESCs to adopt a retinal progenitor
phenotype, expressing Pax6 and Chx10. Exposing these progenitors to
N-(N-(3,5-difluorophenacetyl)-1-alanyl)-S-phenylglycine t-butyl
ester (DAPT), a blocker of Notch signaling, the progenitor cells
will undergo neuronal differentiation). A similar protocol was used
to generate retinal cells from human iPSCs (Lamba D A, McUsic A,
Hirata R K, et al. Generation, purification and transplantation of
photoreceptors derived from human induced pluripotent stem cells.
PLoS One 2010; 5:e8763). Decision to undergo photoreceptor
differentiation is under the control of transcription factor Blimp1
(Brzezinski J A 4th, Lamba D A, Reh T A. Blimp1 controls
photoreceptor versus bipolar cell fate choice during retinal
development. Development 2010; 137:619-29).
[0096] 5. Parkinson's Disease
[0097] In Parkinson's disease (PD), the dopamine-releasing neurons
in the substantia nigra are gradually lost, resulting in the
progressive and severely disabling motor dysfunction which is the
hallmark of this disease. Previous studies in experimental animal
models of PD have shown that dopamine release can be restored and
motor dysfunction reversed by transplantation of embryonic neurons
into the striatum (Lindvall O, Kokaia Z. Prospects of stem cell
therapy for replacing dopamine neurons in Parkinson's disease.
Trends Pharmacol Sci 2009; 30:260-8; Lindvall O, Kokaia Z. Stem
cells in human neurodegenerative disorders--time for clinical
translation? J Clin Invest 2010; 120:29-40). Clinical trials with
human embryonic dopaminergic (DA) neurons initially provided
encouraging results, but later follow-up evaluations indicate only
limited success (Schwarz J. Developmental perspectives on human
midbrain-derived neural stem cells. Parkinsonism Relat Disord 2007;
13 Suppl 3:S466-8).
[0098] DA neurons have been generated in vitro from iPSCs, ESCs,
MSCs and regional stem/progenitor cells. In vitro
pre-differentiated cells were subsequently grafted into the
striatum and found to partially reverse PD-like symptoms in animal
models (Rodriguez-Gomez J A, Lu J Q, Velasco I, et al. Persistent
dopamine functions of neurons derived from embryonic stem cells in
a rodent model of Parkinson disease. Stem Cells 2007; 25:918-28;
Cho M S, Lee Y E, Kim J Y, et al. Highly efficient and large-scale
generation of functional dopamine neurons from human embryonic stem
cells. Proc Natl Acad Sci USA 2008; 105:3392-7; Parish C L,
Castelo-Branco G, Rawal N, et al. Wnt5a-treated midbrain neural
stem cells improve dopamine cell replacement therapy in
parkinsonian mice. J Clin Invest 2008; 118:149-60; Sanchez-Pernaute
R, Lee H, Patterson M, et al. Parthenogenetic dopamine neurons from
primate embryonic stem cells restore function in experimental
Parkinson's disease. Brain 2008; 131(Pt 8):2127-39). However, the
mechanism(s) responsible for symptom reversal are not fully
understood, since functional improvement in an animal model of PD
was shown to occur from human neural progenitor cell transplants
without differentiation to DA neurons (Hovakimyan M, Haas S J,
Schmitt O, et al. Mesencephalic human neural progenitor cells
transplanted into the adult hemiparkinsonian rat striatum lack
dopaminergic differentiation but improve motor behaviour. Cells
Tissues Organs 2008; 188:373-83). Encouraging results in an animal
model of PK were recently reported with striatal transplants of
ESCs, which gave rise to an abundance of functional DA neurons
after forced expression of the TF Lmx1a (Friling S, Andersson E,
Thompson L H, et al. Efficient production of mesencephalic dopamine
neurons by Lmx1a expression in embryonic stem cells. Proc Natl Acad
Sci USA 2009; 106:7613-8).
[0099] Protocol for Dopaminergic Neurons
[0100] Protocol for ESC differentiation to DA neurons include
overexpression of the transcription factor Nurr1 followed by their
exposure to Shh, FGF-8 and ascorbic acid (Lee S H, Lumelsky N,
Studer L, Auerbach J M, McKay R D. Efficient generation of midbrain
and hindbrain neurons from mouse embryonic stem cells. Nat
Biotechnol. 2000 June; 18(6):675-9; Kriks S, Studer L. Protocols
for generating ES cell-derived dopamine neurons. Adv Exp Med Biol.
2009; 651:101-11; Lindvall O, Kokaia Z. Prospects of stem cell
therapy for replacing dopamine neurons in Parkinson's disease.
Trends Pharmacol Sci. 2009 May; 30(5):260-7.). Alternatively to the
combination of stromal cell-derived factor 1 (SDF-1/CXCL12),
pleiotrophin (PTN), insulin-like growth factor 2 (IGF2), and ephrin
B1 (EFNB1). This combination applied to hESCs induces their
differentiation to TH-positive neurons in vitro, expressing
midbrain specific markers, including Engrailed 1, Nurr1, Pitx3, and
dopamine transporter (DAT), and capable of generating action
potentials and forming functional synaptic connections (Vazin T,
Becker K G, Chen J, et al. A novel combination of factors, termed
SPIE, which promotes dopaminergic neuron differentiation from human
embryonic stem cells. PLoS One 2009; 4:e6606).
[0101] 6. Motor Neuron Disease
[0102] Amyotrophic lateral sclerosis (ALS), spinal bulbar muscular
atrophy (or Kennedy's disease), spinal muscular atrophy (SMA) and
spinal muscular atrophy with respiratory distress 1 are
neurodegenerative disorders leading to loss of motor neurons and
death of the patient. There is currently no treatment that can
significantly halt or delay the disease progression. The
pathogenesis of these disorders are incompletely known, but
compromised function in surrounding astrocytes and/or microglia
have been implicated. Stem cell based therapy with replacement of
lost motor neurons as well as of replacement of dysfunctional
astrocytes is therefore considered (Mazzini L, Vercelli A, Ferrero
I, Mareschi K, Boido M, Servo S, Oggioni G D, Testa L, Monaco F,
Fagioli F. Stem cells in amyotrophic lateral sclerosis: state of
the art. Expert Opin Biol Ther. 2009 October; 9(10):1245-58;
Papadeas S T, Maragakis N J. Advances in stem cell research for
Amyotrophic Lateral Sclerosis. Curr Opin Biotechnol. 2009 October;
20(5):545-51. Epub 2009 Oct. 12. Review. PubMed PMID: 19819686.).
For disorders such as ALS which only affects adults, the
possibility to restore lost neuromuscular connections by motor
neuron replacement is problematic given the long distances from the
spinal cord to the target muscles. Using stem cells to generate
astrocytes for neuroprotection may therefore be the most rational
approach in this disorder. However, for SMA, which affects infants
or children, replacing lost motor neurons is an attractive
strategy.
[0103] Protocol for Motor Neurons
[0104] Motor neurons were generated from human ESCs using neural
differentiation medium, treatment with RA (Pax6 expressing
primitive neuroepithelial cells), RA+Shh (Pax6/Sox1 expressing
neuroepithelial cells, which gradually start to express the motor
neuron progenitor marker Olig2). Reducing RA+Shh concentration
promotes the emergence of motor neurons expressing HB9 and Islet1.
The addition of brain-derived neurotrophic factor (BDNF),
glial-derived neurotrophic factor (GDNF), insulin-like growth
factor-1 (IGF1), and cAMP promotes process outgrowth (Hu B Y, Du Z
W, Zhang S C. Differentiation of human oligodendrocytes from
pluripotent stem cells. Nat Protoc 2009; 4:1614-22; Hu B Y, Weick J
P, Yu J, et al. Neural differentiation of human induced pluripotent
stem cells follows developmental principles but with variable
potency. Proc Natl Acad Sci USA 2010; 107:4335-40).
[0105] 7. Stroke, Brain Injury and Spinal Cord Injury
[0106] Stroke is a leading cause of lifelong disability and death
in the western world. Traumatic brain injury is a leading cause of
death and long-term disability in young adults in the western
world. Spinal cord injury is less frequent than traumatic brain
injury but usually affects young individuals and results in serious
disability and reduced quality of life for the patients. Stem cell
transplantation is an attractive strategy in all these conditions,
both in terms of achieving early neuroprotection, and in restoring
lost functions during the rehabilitation phase (Bliss T M, Andres R
H, Steinberg G K. Optimizing the success of cell transplantation
therapy for stroke. Neurobiol Dis 2010; 37:275-83; Lindvall O,
Kokaia Z. Stem cells in human neurodegenerative disorders--time for
clinical translation? J Clin Invest 2010; 120:29-40; Orlacchio A,
Bernardi G, Orlacchio A, et al. Stem cells: an overview of the
current status of therapies for central and peripheral nervous
system diseases. Curr Med Chem 2010; 17:595-608; Ronaghi M, Erceg
S, Moreno-Manzano V, et al. Challenges of stem cell therapy for
spinal cord injury: human embryonic stem cells, endogenous neural
stem cells, or induced pluripotent stem cells? Stem Cells 2010;
28:93-9). Thus, the choice of stem cells and desired derivatives
may be different depending on the stage of the disorder. The fact
that these conditions results in loss of different glial and
neuronal cell types presents additional challenges.
[0107] For stroke and traumatic brain injury, restoring function in
local neural circuitry may be the most relevant. The basic
components of these circuitries are glutamatergic and GABAergic
neurons, as well as cholinergic neurons for selected circuitry
mediating cognitive functions. Glutamatergic neurons also form
major parts of the descending motor projection pathways from the
cerebral cortex to the brain stem and spinal cord. Implantation of
GABAergic neurons have shown promising therapeutic results in
experimental models of epilepsy which is a common sequelae of
traumatic brain injury.
[0108] Spinal cord injuries are often contusion injuries which lead
to loss of white matter oligodendrocytes and myelin and hence
conduction failure in affected ascending and descending pathways.
Restoring myelin competent oligodendrocytes is therefore a prime
objective after spinal cord injury. Additional targets for cell
replacement in this condition are glutamatergic neurons, which can
form descending connections across the lesion site and thereby
restore lost motor and autonomic functions below the injury
site.
[0109] Protocol for Glutamatergic Neurons
[0110] Glutamatergic neurons can be generated from mouse ESCs in
vitro by producing cell aggregates which are then treated for 8
days with RA. This results in the Pax6 expressing cells radial
glial cells, which after additional culturing in N2 followed by
"complete" medium results in ca 95% glutamate neurons (Bibel M,
Richter J, Lacroix E, et al. Generation of a defined and uniform
population of CNS progenitors and neurons from mouse embryonic stem
cells. Nat Protoc 2007; 2:1034-43).
[0111] Protocol for GABAergic Neurons
[0112] GABAergic neurons were generated from mouse ESCs by exposing
embryoid bodies (EBs) for 3 days to all-trans-RA. After subsequent
culture in serum-free neuronal induction medium, comprising
Neurobasal medium supplemented with B27, bFGF and EGF ca 95% GABA
neurons developed (Chatzi C, Scott R H, Pu J, et al. Derivation of
homogeneous GABAergic neurons from mouse embryonic stem cells. Exp
Neurol 2009; 217:407-16).
[0113] Protocol for Oligodendrocytes
[0114] Oligendrocyte precursors (OPCs) capable of developing to
mature myelinating oligodendrocytes were generated from human (h)
ESCs (Hu B Y, Du Z W, Zhang S C. Differentiation of human
oligodendrocytes from pluripotent stem cells. Nat Protoc 2009;
4:1614-22). hESCs are first directed toward the neuroectoderm fate
under a chemically defined condition in the absence of growth
factors for 2 weeks and express neuroectoderm transcription
factors, including Pax6 and Sox1. Next hESCs are exposed to the
caudalizing factor retinoic acid (RA) and the ventralizing
morphogen Shh for 10 d to begin expression of Olig2. To prevent the
differentiation to motoneurons and promote the generation of OPCs,
cells are cultured with we use FGF2 for 10 d. By day 35, the Olig2
progenitors co-express NkxX2.2 and no longer give rise to
motoneurons. The co-expression of Olig2 and Nkx2.2 reflects a stage
prior to human OPCs ("pre-OPCs). These are finally cultured in a
glia medium containing triiodothyronine (T3), neurotrophin 3 (NT3),
PDGF, cAMP, IGF-1 and biotin, which individually or synergistically
can promote the survival and proliferation of the hESC derived
OPCs, for another 8 weeks to generate OPCs. These OPCs are bipolar
or multipolar, express Olig2, Nkx2.2, Sox10 and PDGFR.alpha.,
become motile and are able to differentiate to competent
oligodendrocytes.
[0115] There is also a simpler protocol for generating OPCs from
mouse ESCs (Jiang P, Selvaraj V, Deng W. Differentiation of
embryonic stem cells into oligodendrocyte precursors. J Vis Exp.
2010; pii:1960).
EXAMPLES
Example 1
Induced Differentiation of Transplanted Human Embryonic Stem Cells
with Shh and Retinoic Acid Delivered with Mesoporous Silica
[0116] Background:
[0117] Embryonic stem cells (ESCs) differentiate into motor
neurons, establish functional synapses with muscle fibers, and
acquire physiological properties characteristic of embryonic motor
neurons when cultured with sonic hedgehog (Shh) agonist and
retinoic acid (RA) (Wichterle H, Lieberam I, Porter J A, et al.
Directed differentiation of embryonic stem cells into motor
neurons. Cell 2002; 110:385-97; Miles G B, Yohn D C, Wichterle H,
et al. Functional properties of motoneurons derived from mouse
embryonic stem cells. J Neurosci 2004; 24:7848-58). ESC-derived
motorneurons transplanted into the developing chick neural tube
projected axons toward muscles, received synaptic input, and
developed electrophysiological properties similar to endogenous
motor neurons (Soundararajan P, Miles G B, Rubin L L, et al.
Motoneurons derived from embryonic stem cells express transcription
factors and develop phenotypes characteristic of medial motor
column neurons. J Neurosci 2006; 26:3256-68; De Marco Garcia and
Jessel, 2008). These results show that ESCs after Shh and RA
treatment readily differentiate to functional motor neurons in
vitro and can be subjected to transplantation.
[0118] Motor neurons were also generated in vitro by activation of
transcription factors (TFs) Olig2 and HB9 in the presence of Shh
and RA (Zhang X, Cai J, Klueber K M, Guo Z, et al. Role of
transcription factors in motoneuron differentiation of adult human
olfactory neuroepithelial-derived progenitors. Stem Cells 2006;
24:434-42). In developmental studies it was shown that Nkx6.1 and
Isl1 TFs govern the differentiation of stem cells to motor neurons
of the lateral motor column (LMC motor neurons) (De Marco Garcia N
V, Jessell T M. Early motor neuron pool identity and muscle nerve
trajectory defined by postmitotic restrictions in Nkx6.1 activity.
Neuron 2008; 57:217-31), which innervate the muscles of the
limbs.
[0119] Material and Methods:
[0120] Here we use human (h) ESCs and guide their differentiation
to motor neurons in the dorsal root ganglion (DRG) cavity of adult
recipient rats by administration of extrinsic factors (Shh agonist
and RA delivered with mesoporous nanoparticles). The hESCs in our
experiment expressed green fluorescent protein (GFP).
[0121] AMS-Silica
[0122] The mesoporous silica was prepared as previously described
(Garcia-Bennett et al., 2008). Loading with RA was performed by
adding 250 mg of RA to 500 mg of AMS-6 silica in 20 ml of ethanol
and left at room temperature for 30 minutes before filtering and
drying as a powder.
[0123] The loading with PUR was performed by adding 5 mg of PUR to
500 mg of AMS-6 silica. The loading was performed in a mixture of
DMSO/ethanol, and at room temperature. After loading the sample was
filtered and washed with H2O, the dried for a short period. To each
transplants was added mesoporous silica containing 25 .mu.g of RA
and 150 .mu.g of purmorphamine. The neurospheres were mixed in the
Eppendorf with the nanoparticles and then transplanted to the DRG
cavity.
[0124] Material:
[0125] After 2 months the mice were perfused via the left ventricle
with cold 4% formaldehyde (w/v) and 14% saturated picric acid (v/v)
in 0.15M phosphate buffer, postfixed for 4 hs, and stored overnight
in cold phosphate buffer containing 15% sucrose for cryoprotection.
14 .mu.m cryosections were cut and analysed in a Nikon fluorescence
microscope. The untreated hESCs showed mixed morphology with
typical teratoma formation, whereas treated transplants
demonstrated a developed morphology of neuronal cells with extended
processes. FIG. 2 shows hESC transplants 2 months after
transplantation to the DRG cavity of adult nu/nu mice. FIG. 2A
shows untreated transplants, FIG. 2B shows transplants with
AMS+RA+Purmorphamine.
[0126] The immunostaining confirmed the presence of beta-tubulin
(bTUB) positive cells (neuronal marker) in treated transplants and
some of the cells expressed HB9 transcription factor--the marker
for motor neuronal differentiation (see FIG. 3.).
[0127] FIG. 3A shows how bTUB (indicated with the arrow) is
expressed in some GFP-expressing hESCs. FIG. 3B shows how HB9
(indicated with stars) is expressed in some GFP-expressing
cells.
[0128] We were thus able to induce differentiation of
undifferentiated ESCs towards motor neurons in vivo by RA and Shh
delivered with mesoporous silica.
Example 2
Differentiation of Neural Crest Stem Cells Toward Neuronal
Phenotype In Vitro by RA Delivered with Mesoporous Silica
[0129] Background:
[0130] The RA receptor RAR-beta2 is expressed in dorsal root
ganglion (DRG) neuron subtypes. It was shown that retinoid
signaling has a role in neurite outgrowth in vivo (Corcoran J,
Shroot B, Pizzey J, et al. The role of retinoic acid receptors in
neurite outgrowth from different populations of embryonic mouse
dorsal root ganglia. J. Cell Sci 2000; 113:2567-74; Dmetrichuk J M,
Spencer G E, Carlone R L. Retinoic acid-dependent attraction of
adult spinal cord axons towards regenerating newt limb blastemas in
vitro. Dev Biol 2005; 281:112-20) by demonstrating that in a
peripheral nerve crush model there is less sensory neurite
outgrowth in RAR-beta null compared to wild-type mice. In vitro
experiments identified sonic hedgehog (Shh) as a downstream target
of the RAR-beta2 signaling pathway since it is expressed in the
injured DRG of wild-type but not RAR-beta null mice and that Shh
alone cannot induce neurite outgrowth but potentiates RAR-beta2
signaling in this process (So P L, Yip P K, Bunting S, et al.
Interactions between retinoic acid, nerve growth factor and sonic
hedgehog signalling pathways in neurite outgrowth. Dev Biol. 2006;
298:167-75).
[0131] We culture boundary cap neural crest stem cells (bNCSCs) as
neurospheres. These cells have the potential produce neurons and
glial cells in vitro and can be induced to produce specific type of
neurons in vivo by conditional activating of key transcription
factors for nociceptor neuron differentiation (Aldskogius H, Berens
C, Kanaykina N, et al. Regulation of boundary cap neural crest stem
cell differentiation after transplantation. Stem Cells 2009;
27:1592-603).
[0132] Material and Methods:
[0133] bNCSCs were isolated in a semiclonal fashion from EGFP
embryos on embryonic day (E)11, as described previously
(Hjerling-Leffler J, Marmigere F, Heglind M, et al. The boundary
cap: a source of neural crest stem cells that generate multiple
sensory neuron subtypes. Development 2005; 132:2623-32). Briefly,
the DRGs along with boundary caps were mechanically separated from
the isolated spinal cord and mechano-enzymatically dissociated
using Collagenase/Dispase (1 mg/ml) and DNase (0.5 mg/ml) for 30
minutes at room temperature. Cells were plated at
0.5-1.times.10.sup.5 cells/cm.sup.2 in N2 medium containing B27
(Gibco, Grand Island, N.Y., http://www.invitrogen.com) as well as
EGF and basic fibroblast growth factor (R&D Systems,
Minneapolis, http://www.rndsystems.com; 20 ng/ml, respectively).
After 12 hours, nonadherent cells were removed together with half
of the medium before adding fresh medium. The medium was changed
every other day (50% of the medium replaced with fresh medium)
until neurospheres could be observed after approximately 2 weeks of
culture.
[0134] Here we performed differentiation assay of bNCSCs in vitro
under three conditions--bNCSC alone, bNCSC+RA and bNCSC+AMS-RA.
[0135] Cells were plated at a density of 1.2.times.10.sup.3 cells
on a poly-D-lysine (50 .mu.g/ml)/laminin (20 ng/ml)-coated
coverslip and maintained in Dulbecco's modified Eagle's
medium-F12/neurobasal medium supplemented with N2, B27, 0.1 mM
nonessential amino acids and 2 mM sodium pyruvate. To each well
were added AMS with 24 ng of RA for 3 days.
[0136] After 3 days the cultures were fixed and immunolabeling was
performed as described previously (Kozlova E N. Differentiation and
migration of astrocytes in the spinal cord following dorsal root
injury in the adult rat. Eur J Neurosci 2003; 17:782-90). Primary
antibodies were anti-bIII-tubulin (bTUB; mouse monoclonal; Covance,
Princeton, N.J., http://www.covance.com, 1:500; anti-glial
fibrillary acidic protein (GFAP; rabbit polyclonal; DAKO, Glostrup,
Denmark, http://www.dako.com; 1:1,000). Secondary antibodies
(Jackson Immunoresearch Laboratories, West Grove, Pa.,
http://www.jacksonimmuno.com) were diluted in PBS with 0.3% Triton
X-100 and 0.1% sodium azide: AMCA-conjugated donkey anti-rabbit and
anti-mouse, Cy3-conjugated donkey anti-mouse).
[0137] The AMS were added to the cultures and the time-window for
their presence before dissolving in the culture medium was
established. We also analyzed their contact with the stem cells.
After 3 and 7 days the cultures were fixed and processed for
immunohistochemistry. After staining 10 photographs were taken from
each slide and the ratios of neurons and glial cells were
calculated in all types of experiments. We also calculated the
neurite length per cell as a measure of the level of neuronal
differentiation (Kozlova E N. Differentiation and migration of
astrocytes in the spinal cord following dorsal root injury in the
adult rat. Eur J Neurosci 2003; 17:782-90).
[0138] Results:
[0139] We found that AMS have a strong affinity to the stem cells
and tightly attach neurospheres during first minutes/hours after
placing to the culture dish. The differentiation of neurospheres
was not hampered and the cells in spite on their close contact with
the particles successfully spread on the surface and differentiated
(see FIG. 4).
[0140] In FIG. 4, left columns show eGFP-expressing NCSCs
neurospheres cultured with the AMS particles (the eGFP spheres look
perforated. On the faze-contrast pictures the particles are seen as
dark three-angles--second column). On the right columns the NCSCs
are cultures without particles. In both cases the spheres
differentiated and spread on the surface. The amount of particles
reduced during first week.
[0141] We then compared if RA delivered to the NCSCs will have
similar effect on their differentiation compare to RA delivered
with AMS (AMS-RA). RA and AMS-RA both induced neuronal
differentiation (FIG. 5A). Based on the calculation of neurite
lengths differentiation of neurons in RA treated cultures increased
up to 37% and in AMS-RA treated cultures up to 18% compared to
non-treated neurospheres and the neuron/glia ratio in RA-treated
cultures increased 3.4 times and in AMS-RA 3.9 times compared to
neurospheres-alone (FIG. 5B).
[0142] FIG. 5A shows in vitro differentiation assay of bNCSCs
cultured without special treatment (upper panel), with RA (middle
panel) and with AMS-RA (lower panel). The level of neuronal
differentiation in the middle and lower panels is higher compared
to the upper panel (untreated cells), whereas differentiation of
glial cells is strongly reduced in RA and AMS-RA treated cultures.
First column--eGFP(bNCSCs), second column GFAP(glial marker), third
column bTUB (neuronal marker). The graph of FIG. 5B shows increased
neuro/glial ration in the RA-treated cultures.
[0143] Furthermore we performed RT-PCR analysis of bNCSCs cultured
alone (BC), NCSCs cultures with AMS, with AMS-RA and with AMS-Purm
(purmorphomine--the agonist of Shh) and found specific expression
of transcription factors in response to activation of RA and Shh
(FIGS. 6A and 6B) (Guan W, Wang G, Scott S A, et al. Shh influences
cell number and the distribution of neuronal subtypes in dorsal
root ganglia. Dev Biol 2008; 314:317-28). As shown in the figures,
the RT-PCR analysis showed specific expression of Ngn2 in the
AMS-RA treated cultures (A) and Patch1 in response to AMS Purm
treated cultures.
[0144] We thus show that RA delivered with mesoporous silica has a
similar effect as direct administration of RA on neuronal
differentiation and neurite outgrowth in vitro of bNCSCs.
Example 3
Differentiation and Migration of NCSCs in the Presence of AMS Under
the Kidney Capsule
[0145] Background:
[0146] We previously showed that NCSCs transplanted under the
kidney capsule of one pole of the kidney extensively migrate
towards co-transplanted pancreatic islets placed in the opposite
pole of the same kidney (Olerud J, Kanaykina N, Vasylovska S, et
al. Neural crest stem cells increase beta cell proliferation and
improve islet function in co-transplanted murine pancreatic islets.
Diabetologia 2009; 52:2594-601. Erratum in: Diabetologia. 2010;
53:396. Vasilovska, S [corrected to Vasylovska, S]; Kozlova E N,
Jansson L. Differentiation and migration of neural crest stem cells
are stimulated by pancreatic islets. Neuroreport 2009; 20:833-8).
The purpose of these previous studies was to develop a new protocol
for improved outcome after transplantation of pancreatic islets.
Transplantation of pancreatic islets is an established therapy in
selected patients with type 1 diabetes. The survival of
transplanted islets is however insufficient and ways to improve
their survival need to be developed. Co-transplanted NCSCs, which
are able to secrete trophic factors and have potential to
re-innervate transplanted islets may be a useful approach to
improve the clinical outcome from islet transplantation. Our
results showed that bNCSCs migrate towards islets, strongly promote
their function and increase proliferation of beta-cells. However
transplanted bNCSCs did not differentiate to functional neurons and
did not re-innervate transplanted islets. Thus the development of
protocols for NCSC differentiation in vivo is an important
objective. The use of AMS containing morphogens may facilitate
differentiation of bNCSCs in vivo after transplantation. Before
embarking on long-term experiments we investigated whether AMS may
have a negative effect on survival of bNCSCs grafted under the
kidney capsule and their migration towards pancreatic islets
grafted to the opposite pole of the same kidney.
[0147] Material and Methods:
[0148] We transplanted AMS-RA in one pole of the kidney together
with bNCSCs and pancreatic islests in the opposite pole of the
kidney (see FIG. 7).
[0149] FIG. 7 is an overview of kidney with three different
combinations of transplants: [0150] left: NCSCs (white circles)
transplants located in the low pole of the kidney. NCSCs do not
migrate from their location; [0151] middle: NCSCs migrate towards
Islets (oval) which are transplanted on another side of the kidney;
[0152] right: the reduced migration of NCSCs (small circles)
towards Islets and increased differentiation of NCSCs in the
initial location when they were co-transplanted with AMS (black
triangle).
[0153] The islets were collected from transgenivc mice containing
red fluorescent protein (RFP) and bNCSCs were prepared from the
eGFP transgenic mice. The transplantation was performed as
previously described (Olerud J, Kanaykina N, Vasylovska S, et al.
Neural crest stem cells increase beta cell proliferation and
improve islet function in co-transplanted murine pancreatic islets.
Diabetologia 2009; 52:2594-601. Erratum in: Diabetologia. 2010;
53:396. Vasilovska, S [corrected to Vasylovska, S]; Kozlova E N,
Jansson L. Differentiation and migration of neural crest stem cells
are stimulated by pancreatic islets. Neuroreport 2009; 20:833-8)
and after one month mice were perfused with fixative, their kidneys
collected, postfixed for 4 hours, stored overnight in cold
phosphate buffer containing 15% sucrose. The next day serial 14
.mu.m cryostat sections were prepared through the whole organ.
[0154] Results:
[0155] AMS did not negatively affect survival of bNCSCs nor their
migration towards pancreatic islets (FIG. 7, FIG. 8).
[0156] FIG. 8 shows eGFP-expressing bNCSC under the kidney capsule:
[0157] A: migrated bNCSCs (left column) towards RFP-expressing
pancreatic islets (middle column) in the other pole of the kidney.
[0158] B: left--bNCSC transplanted without AMS, [0159]
right--bNCSCs co-transplanted with AMS.
[0160] Based on the morphology of NCSCs appears that NCSC
co-transplanted with particles are more differentiated compare to
NCSCs transplanted alone (FIG. 8).
Example 4
Investigation Whether AMS Influences Glial Scar Formation after
Brain Injury
[0161] Background:
[0162] Glial scar formation after injury to the brain or spinal
cord represents a major cause for the inability of damaged axons to
regenerate in the CNS. The consequences of these injuries are
therefore permanent loss of functions which were served by the
damaged neuronal systems. To reduce glial scar formation is
important in order to promote axonal regeneration and restore lost
functions.
[0163] We previously showed that glial scar formation in the brain
connected with the expression of the calcium-binding protein
Mts1/S100A4 which is produced specifically in white matter
astrocytes. Down-regulation of Mts1/S100A4 in astrocytes reduced
glial scar formation and increased their motility whereas
up-regulation of Mts1/S100A4 expression resulted in increased glial
scar formation (Fang Z, Duthoit N, Wicher G, et al. Intracellular
calcium-binding protein S100A4 influences injury-induced migration
of white matter astrocytes. Acta Neuropathol 2006; 111:213-9).
[0164] Here we investigate whether AMS and AMS+RA affect the extent
of glial scar formation one week after injury to the cerebral
cortex.
[0165] Material and Methods:
[0166] We made an injury of a defined diameter and depth with a xx
gauge needle in the frontal cerebral cortex of adult rats. In some
of these cases implanted AMS or AMS-RA in the cavity. One week
after injury the rats were perfused (see example 1), the brains
were removed, postfixed, cryoprotected and cryosectioned in the
coronal plane. The staining was performed for bTUB--neuronal marker
(the same as above), glial fibrillary acidic protein (GFAP)--glial
marker (see Example 2) and Mts1/S100A4 white matter astrocytes
marker (gift from Lukanidin, 1:700, rabbit polyclonal),
ED1--microglial marker (monoclonal, 1:500).
[0167] Results:
[0168] Our in vitro data showed that AMS and AMS-RA reduces glial
differentiation (FIG. 5). We thus were curious if AMS affects glial
scar formation after cortical injury. The pilot experiment shows
that glial response in the brain to the injury treated or not
treated with AMS and AMS-RA differs from the response in untreated
animals (see FIG. 9).
[0169] FIG. 9 shows coronal sections through the rat cerebral
cortex one week after a focal injury.
[0170] Upper panel--the injury without treatment;
[0171] middle panel--the injury+AMS;
[0172] lower panel--the injury+AMS-RA.
[0173] GFAP (glial marker) first column, OX42 (microglial marker)
second column, Hoechst nuclear staining third column. Note the
increased microglial reaction (second column) and reduced
astroglial reaction (first column) in the treated injury sites
(middle and lowest panels).
[0174] Our pilot experiments show that AMS decreases glial scar
formation and induces microglial reaction in the injured areas.
Further experiments will show if reduced glial scar formation is a
direct effect of AMS or is a consequence of increased microglial
reaction.
Example 5
[0175] We estimated the length of axons in the transplants treated
with mesoporous particles of type NLAB-Silica(200) with pore size
distribution centered on 200 .ANG. and disordered pore structure,
loaded with mimetics compared to non-treated transplants. The
length of axons was calculated with the previously described method
(Ronn L C, Ralets I, Hartz B P, Bech M, Berezin A, Berezin V,
Moller A, Bock E. A simple procedure for quantification of neurite
outgrowth based on stereological principles. J Neurosci Methods.
2000 Jul. 31; 100(1-2):25-32). The length of axons in mimetic
treated transplants was 5 times greater than in untreated
transplants (data not shown).
[0176] For evaluation of transplant size and neural stem/progenitor
cell survival the HB9-EGFP expressing cells were analysed on every
5th section.
[0177] The NIH software ImageJ (Rasband, 1997, available at
http://rsb.info.nih.gov/ij) was used to measure transplant areas.
The transplant volume estimate was calculated according to the
formula A=TK[.SIGMA.(S1 to Sn)], where T is the thickness of the
section (T=12 .mu.m), K is the number of sections between the
measured areas (K=50) and S is the area of the transplant on the
sections from 1 to N. The evaluation demonstrated that the volume
of mimetic-treated transplants was significantly increased
(3.times., p<0.5).
[0178] The release properties of factors loaded into mesoporous
material NLAB-Silica (200) were as follows:
[0179] GDNF release in vivo: (Kirik D, Georgievska B, Rusenblad C,
Bjorklund A. Delayed infusion of GDNF promotes recovery of motor
function in the partial lesion model of Parkinson's disease. EJN,
2001, v13, p. 1589-99) 0.25 .mu.g/.mu.l for 2 weeks infusion rate
0.5 .mu.l per hour), i.e. 6 .mu.g/day of GDNF. This release rate
was achieved during 14 days. CNTF release in vivo: (Kelleher M O,
Myles L M, Al-Abri R K, Glasby M A The use of Ciliary neurotrophic
factor to promote recovery after peripheral nerve injury by
delivering it at the site of the cell body. Acta Neurochir (Wien)
2006, v146, p. 55-61) 100 .mu.g/ml for 4 weeks 2.5 .mu.l per hour,
i.e. 6 .mu.g/day of CNTF. This release rate was achieved during 14
days. FIGS. 10 and 11 show the structural and textural properties
of NLAB-Silica (200), including SEM image (FIG. 10) of the
disordered pore structure, and nitrogen adsorption isotherm and
pore size distribution (FIG. 11). The volume of transplants treated
with NLAB-Silica (200) loaded with peptide mimics (see FIG. 12B)
was about 4 times larger than untreated ones (see FIG. 12A), and
HB9-EGFP cells in treated transplants were on an average 8 times
larger compared to cells in untreated transplants.
* * * * *
References