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US20130315962A1 - Method for stem cell differentiation in vivo by delivery of morphogenes with mesoporous silica and corresponding pharmceutical active ingredients - Google Patents

Method for stem cell differentiation in vivo by delivery of morphogenes with mesoporous silica and corresponding pharmceutical active ingredients Download PDF

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US20130315962A1
US20130315962A1 US13/808,809 US201113808809A US2013315962A1 US 20130315962 A1 US20130315962 A1 US 20130315962A1 US 201113808809 A US201113808809 A US 201113808809A US 2013315962 A1 US2013315962 A1 US 2013315962A1
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stem cells
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Alfonso E. Garcia-Bennett
Elena Nickolaevna Kozlova
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Nanologica AB
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    • A61K38/18—Growth factors; Growth regulators
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Definitions

  • 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.
  • 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.
  • 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.
  • 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]).
  • 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).
  • FGFs fibroblast growth factors
  • Wnts Logan C Y, Nusse R. The Wnt signaling pathway in development and disease. Annu Rev Cell Dev Biol 2004; 20:781-810
  • TGF transforming growth factor
  • 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 (
  • the presence of FGF2 signaling determines whether beta-catenin exerts effects on proliferation or neuronal differentiation of neural stem cells.
  • 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.
  • 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).
  • PCD programmed cell death
  • 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.
  • retinoic acid signaling after sciatic nerve injury up-regulation of cellular retinoid binding proteins. Eur J Neurosci 2003; 18:1033-40).
  • 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).
  • 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.
  • 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.
  • 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.
  • BMP Bone Morphogenetic Protein
  • 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.
  • 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).
  • FGFs Fibroblast Growth Factors
  • 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).
  • BMP inhibition Xenopus neural induction
  • 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.
  • 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).
  • 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).
  • CDNF/MANF cerebral dopamine neurotrophic factor and astrocyte-derived neurotrophic factor
  • 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.
  • CSDAs alkoxy silane costructure-directing agents
  • 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.
  • porous particles can be employed for controlled delivery of differentiation factors to obtain the desired type of cells from stem cell transplants.
  • differentiation factors for controlled delivery of differentiation factors to obtain the desired type of cells from stem cell transplants.
  • 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.
  • 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.
  • FGFs fibroblast growth factors
  • Wnts Wnts
  • TGF-beta family members transforming growth factor-beta family members
  • 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.
  • 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.
  • 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.
  • the present invention provides a method of enhancing cell-survival during implantation of stem cells.
  • 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:
  • 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:
  • Said therapeutic method has the potential to produce neurotrophic support and specific innervation from the differentiated bNCSCs of newly differentiated replaced cells.
  • 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:
  • the present invention relates to kits for use with the above-mentioned therapeutic methods.
  • 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.
  • stem cells include, but are not limited to:
  • 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.
  • 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:
  • 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.
  • 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.
  • said porous silica is characterized by a surface area higher than 200 m 2 /g and a pore size between 1.5-50 nm.
  • 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.
  • 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 %.
  • 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.
  • the in vitro produced porous silica has the above-mentioned chracteristics in terms of surface area and porosity.
  • 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.
  • ES embryonic stem
  • iPS induced pluripotent stem
  • 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.
  • FGFs fibroblast growth factors
  • Wnts transforming growth factor
  • TGF-beta family members Hedgehog (hh) proteins
  • VEGF vascular endothelial growth factor
  • Dkk Dickkopf
  • PDN insulin-like growth factor 2
  • EFNB1 ephrin B1
  • 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.
  • 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.
  • ES embryonic
  • ES embryonic 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.
  • 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
  • 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.
  • the porous silica material be it a solvent extracted or calcined material (see Rambabu Atluri, Niklas He
  • 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.
  • iPS induced pluripotent stem
  • 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.
  • MSCs Mesenchymal stem cells
  • bone marrow e.g. bone marrow, adipose tissue, umbilical cord blood or amnion
  • adipose tissue e.g. bone marrow, adipose tissue, umbilical cord blood or amnion
  • mesodermal e.g. bone marrow, adipose tissue, umbilical cord blood or amnion
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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).
  • 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
  • skeletal muscle-associated precursor cells human, mouse
  • 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.
  • MRFs myogenic regulatory factors
  • 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).
  • Type 1 diabetes is characterized by an autoimmune mediated loss of insulin producing ⁇ -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).
  • 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.
  • 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).
  • Ngn3 also known as Neurog3
  • Pdx1 also known as Neurog3
  • Mafa Zahou Q, Brown J, Kanarek A, et al. In vivo reprogramming ofadult pancreatic exocrine cells to beta-cells. Nature 2008; 455:627-32).
  • 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 ⁇ -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.
  • 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).
  • definitive endodermal cells Activin+Wnt3
  • FGF10+KAAD-cyclopamine primitive foregut endoderm
  • RA+FGF10+KAAD-cyclopamine posterior foregut endoderm
  • pancreatic endoderm and endocrine precursors Extendin-4
  • IGF1+HGF hormone producing cells
  • 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).
  • 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).
  • PI3K phosphatidylinositol 3-kinase pathway
  • definitive endoderm remove Wnt
  • posterior foregut down-regulate Wnt
  • pancreatic endoderm block Shh
  • beta-cell precursors block Notch
  • beta-cells block PI3K, stimulate Shh.
  • 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.
  • 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.
  • Neural precursors isolated from the developing cat brain show retinal integration following transplantation to the retina of the dystrophic cat.
  • 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.
  • Blimp1 transcription factor 1 (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).
  • Parkinson's disease 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).
  • DA neurons have been generated in vitro from iPSCs, ESCs, MSCs and regional stem/progenitor cells.
  • pre-differentiated cells were subsequently grafted into the striatum and found to partially reverse PD-like symptoms in animal models (Rodriguez-Gómez 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.
  • 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.).
  • stromal cell-derived factor 1 SDF-1/CXCL12
  • PDN pleiotrophin
  • IGF2 insulin-like growth factor 2
  • EFNB1 ephrin B1
  • 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.
  • 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.
  • BDNF brain-derived neurotrophic factor
  • GDNF glial-derived neurotrophic factor
  • IGF1 insulin-like growth factor-1
  • cAMP cAMP promotes process outgrowth (Hu B Y, Du Z W, Zhang S C. Differentiation of human oligodendrocytes from pluripotent stem cells.
  • 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?
  • Stem cells an overview of the current status of therapies for central and peripheral nervous system diseases.
  • 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.
  • Glutamatergic neurons 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.
  • 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).
  • 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).
  • Oligendrocyte precursors 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.
  • RA caudalizing factor retinoic acid
  • OPCs 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 ⁇ , become motile and are able to differentiate to competent oligodendrocytes.
  • T3 triiodothyronine
  • NT3
  • Embryonic stem cells 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).
  • Sh sonic hedgehog
  • RA retinoic acid
  • 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).
  • h human 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).
  • 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.
  • 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.
  • the neurospheres were mixed in the Eppendorf with the nanoparticles and then transplanted to the DRG cavity.
  • 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.
  • 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 .).
  • bTUB beta-tubulin
  • 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.
  • 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.
  • DRG dorsal root ganglion
  • boundary cap neural crest stem cells bNCSCs
  • bNCSCs boundary cap neural crest stem cells
  • 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).
  • bNCSCs were isolated in a semiclonal fashion from EGFP embryos on embryonic day (E)11, as described previously (Hjerling-Leffler J, Marmigère 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 a density of 1.2 ⁇ 10 3 cells on a poly-D-lysine (50 ⁇ 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.
  • 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).
  • 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 ).
  • 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.
  • RA and AMS-RA both induced neuronal differentiation ( FIG. 5A ).
  • AMS-RA both induced neuronal differentiation
  • FIG. 5B 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 ).
  • 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.
  • the graph of FIG. 5B shows increased neuro/glial ration in the RA-treated cultures.
  • RA delivered with mesoporous silica has a similar effect as direct administration of RA on neuronal differentiation and neurite outgrowth in vitro of bNCSCs.
  • AMS containing morphogens may facilitate differentiation of bNCSCs in vivo after transplantation.
  • 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.
  • FIG. 7 is an overview of kidney with three different combinations of transplants:
  • 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.
  • 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 ⁇ m cryostat sections were prepared through the whole organ.
  • AMS did not negatively affect survival of bNCSCs nor their migration towards pancreatic islets ( FIG. 7 , FIG. 8 ).
  • FIG. 8 shows eGFP-expressing bNCSC under the kidney capsule:
  • NCSC co-transplanted with particles are more differentiated compare to NCSCs transplanted alone ( FIG. 8 ).
  • 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.
  • FIG. 9 shows coronal sections through the rat cerebral cortex one week after a focal injury.
  • GFAP glial marker
  • OX42 microglial marker
  • 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).
  • HB9-EGFP expressing cells were analysed on every 5th section.
  • the NIH software ImageJ (Rasband, 1997, available at http://rsb.info.nih.gov/ij) was used to measure transplant areas.
  • 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 ⁇ g/ ⁇ l for 2 weeks infusion rate 0.5 ⁇ l per hour), i.e. 6 ⁇ 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.
  • 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.

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US20140248696A1 (en) * 2013-03-01 2014-09-04 Wisconsin Alumni Research Foundation Methods of maintaining, expanding, and diffrentiating neuronal subtype specific progenitors
US11319527B2 (en) 2013-03-01 2022-05-03 Wisconsin Alumni Research Foundation Methods of maintaining, expanding, and differentiating neuronal subtype specific progenitors
US11993652B2 (en) 2013-12-20 2024-05-28 Fred Hutchinson Cancer Center Tagged chimeric effector molecules and receptors thereof
WO2016176652A2 (fr) 2015-04-29 2016-11-03 Fred Hutchinson Cancer Research Center Cellules souches modifiées et leurs utilisations
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CN113454456A (zh) * 2019-04-15 2021-09-28 纳诺洛吉卡股份公司 用于治疗、预防和/或延缓神经退行性疾病以及神经元和神经胶质退化的空多孔颗粒

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