subretinal-implantation-rpe-on-carrier-minipigs-guidelines-for

Subretinal Implantation of RPE on a Carrier in Minipigs: Guidelines for Preoperative Preparations, Surgical Techniques, and Postoperative Care

Degenerative disorders of the retina (including age-related macular degeneration), which originate primarily at or within the retinal pigmented epithelial ...

Czech Academy of Sciences

The technique for fabrication of soft porous hydrogels, in which both the size and the orientation of inner pores can be controlled, was developed. Three-dimensional hydrophilic gels based on poly[2-hydroxyethyl methacrylate] are designed as scaffolds for regeneration of soft tissues, e.g., nerve tissue. Anisotropic macropores of the size ranging from 10 to 50 microm were formed (1) by using a porogen-leaching method with a solid organic porogen, (2) by phase-separation during gelation in solvent-nonsolvent mixture, or (3) by combination of solid porogen elimination and phase-separation. As a porogen, poly(L-lactide) fibers were applied and consequently washed away under mild conditions to obtain desired spatial orientation of pores. Highly water-swollen polymer gels were characterized with high pressure (low vacuum) scanning electron microscopy (AquaSEM). The morphology of voids remaining after removing the solid PLLA porogen (the macropores) was clearly shown.

Poly(HEMA) hydrogels with controlled pore architecture for tissue regeneration applications.

Preparation of soft poly(amino acid) hydrogels containing biomimetic cell-adhesive peptides was investigated. Covalently crosslinked gels were formed by radical co-polymerization of methacryloylated macromonomer poly[N(5)-(2-hydroxyethyl)-L-glutamine-stat-L-alanine-stat-methacryloyllysine] with 2-hydroxyethyl methacrylate (HEMA) as minor co-monomer. Hydrogels carrying biomimetic peptides were prepared by using methacryloylated peptides, such as methacryloyl-GGGRGDSG-OH and methacryloyl-GGGYIGSR-OH, as additional monomers in the polymerization mixture. Mechanical stability and swelling in water of the hydrogels obtained for different solid:water and polypeptide:HEMA ratios were evaluated. The microporosity of gels (5-20 microm), dependent on the polyHEMA phase separation in water, was followed by low-vacuum SEM. The effect of biomimetic modification of hydrogels with RGDS and YIGSR peptides on the seeding efficiency of porcine mesenchymal stem cells (MSCs) was studied in vitro. While unmodified hydrogels showed very low cell adhesion, due to their highly hydrophilic nature, the incorporation of adhesive peptides significantly improved the adhesion and viability of seeded cells.

Synthetic poly(amino acid) hydrogels with incorporated cell-adhesion peptides for tissue engineering.

Neural precursor cells (NSCs) hold great potential to treat a variety of neurodegenerative diseases and injuries to the spinal cord. However, current delivery techniques require an invasive approach in which an injection needle is advanced into the spinal parenchyma to deliver cells of interest. As such, this approach is associated with an inherent risk of spinal injury, as well as a limited delivery of cells into multiple spinal segments. Here, we characterize the use of a novel cell delivery technique that employs single bolus cell injections into the spinal subpial space. In immunodeficient rats, two subpial injections of human NSCs were performed in the cervical and lumbar spinal cord, respectively. The survival, distribution, and phenotype of transplanted cells were assessed 6-8 months after injection. Immunofluorescence staining and mRNA sequencing analysis demonstrated a near-complete occupation of the spinal cord by injected cells, in which transplanted human NSCs (hNSCs) preferentially acquired glial phenotypes, expressing oligodendrocyte (Olig2, APC) or astrocyte (GFAP) markers. In the outermost layer of the spinal cord, injected hNSCs differentiated into glia limitans-forming astrocytes and expressed human-specific superoxide dismutase and laminin. All animals showed normal neurological function for the duration of the analysis. These data show that the subpial cell delivery technique is highly effective in populating the entire spinal cord with injected NSCs, and has a potential for clinical use in cell replacement therapies for the treatment of ALS, multiple sclerosis, or spinal cord injury.

Spinal parenchymal occupation by neural stem cells after subpial delivery in adult immunodeficient rats.

Gene silencing with virally delivered shRNA represents a promising approach for treatment of inherited neurodegenerative disorders. In the present study we develop a subpial technique, which we show in adult animals successfully delivers adeno-associated virus (AAV) throughout the cervical, thoracic and lumbar spinal cord, as well as brain motor centers. One-time injection at cervical and lumbar levels just before disease onset in mice expressing a familial amyotrophic lateral sclerosis (ALS)-causing mutant SOD1 produces long-term suppression of motoneuron disease, including near-complete preservation of spinal α-motoneurons and muscle innervation. Treatment after disease onset potently blocks progression of disease and further α-motoneuron degeneration. A single subpial AAV9 injection in adult pigs or non-human primates using a newly designed device produces homogeneous delivery throughout the cervical spinal cord white and gray matter and brain motor centers. Thus, spinal subpial delivery in adult animals is highly effective for AAV-mediated gene delivery throughout the spinal cord and supraspinal motor centers.

Spinal subpial delivery of AAV9 enables widespread gene silencing and blocks motoneuron degeneration in ALS.

We generated new in vitro model for sporadic form of amyotrophic lateral sclerosis by reprogramming isolated skin fibroblasts into iPSCs. Fibroblasts were reprogrammed with commercially available synthetic polycistronic, self-replicating RNA vector. As verified by FISH, an early passages of a new iPSC line showed mosaic karyotype (cells with normal and abnormal karyotype 46,XY,t(2;14)(q13;p12) were present), while late passages contained only cells with abnormal karyotype. New iPSCs differentiated into all three germ layers and formed a teratoma in nude mice. Our iPSC line represents a new model for therapy testing and drug development in the field of ALS research.

Generation of ORIONi001-A induced pluripotent stem cell line for in vitro modeling of sporadic form of amyotrophic lateral sclerosis.

Second-order spinal cord excitatory neurons play a key role in spinal processing and transmission of pain signals to the brain. Exogenously induced change in developmentally imprinted excitatory neurotransmitter phenotypes of these neurons to inhibitory has not yet been achieved. Here, we use a subpial dorsal horn-targeted delivery of AAV (adeno-associated virus) vector(s) encoding GABA (gamma-aminobutyric acid) synthesizing-releasing inhibitory machinery in mice with neuropathic pain. Treated animals showed a progressive and complete reversal of neuropathic pain (tactile and brush-evoked pain behavior) that persisted for a minimum of 2.5 months post-treatment. The mechanism of this treatment effect results from the switch of excitatory to preferential inhibitory neurotransmitter phenotype in dorsal horn nociceptive neurons and a resulting increase in inhibitory activity in regional spinal circuitry after peripheral nociceptive stimulation. No detectable side effects (e.g., sedation, motor weakness, loss of normal sensation) were seen between 2 and 13 months post-treatment in naive adult mice, pigs, and non-human primates. The use of this treatment approach may represent a potent and safe treatment modality in patients suffering from spinal cord or peripheral nerve injury-induced neuropathic pain.

Precision spinal gene delivery-induced functional switch in nociceptive neurons reverses neuropathic pain.

We present here a new iPS cell line for modeling sporadic form of ALS. Cell line was generated by reprogramming skin fibroblasts isolated with explant culture technology from skin biopsy, donated by ALS patient. For reprogramming, polycistronic self-replicating RNA vector was used and derived iPS cells were characterized by immunocytochemistry and FACS (pluripotent factors expression), karyotyping, STR fingerprinting analysis and in vitro differentiation assay. New cell line showed normal (46, XY) karyotype and differentiated in vitro into cells from three germ layers. STR analysis proved the origin and originality of the cell line.

Article	Year
Poly(HEMA) hydrogels with controlled pore architecture for tissue regeneration applications. Journal of materials science. Materials in medicine\| PubMed ID: 17619953	2008
Synthetic poly(amino acid) hydrogels with incorporated cell-adhesion peptides for tissue engineering. Journal of tissue engineering and regenerative medicine\| PubMed ID: 20084624	2010
Spinal parenchymal occupation by neural stem cells after subpial delivery in adult immunodeficient rats. Stem cells translational medicine\| PubMed ID: 31800978	2020
Spinal subpial delivery of AAV9 enables widespread gene silencing and blocks motoneuron degeneration in ALS. Nature medicine\| PubMed ID: 31873312	2020
Generation of ORIONi001-A induced pluripotent stem cell line for in vitro modeling of sporadic form of amyotrophic lateral sclerosis. Stem cell research\| PubMed ID: 32919353	2020
Precision spinal gene delivery-induced functional switch in nociceptive neurons reverses neuropathic pain. Molecular therapy : the journal of the American Society of Gene Therapy\| PubMed ID: 35524407	2022
In vitro modeling of amyotrophic lateral sclerosis with induced pluripotent stem cell technology-derived cell line ORIONi002-A. Stem cell research\| PubMed ID: 35907349	2022

Hana Studenovska

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