One of the challenges in clinical translation of cell-replacement therapies is the definition of optimal cell generation and storage/recovery protocols which would permit a rapid preparation of cell-treatment products for patient administration. Besides, the availability of injection devices that are simple to use is critical for potential future dissemination of any spinally targeted cell-replacement therapy into general medical practice. Here, we compared the engraftment properties of established human-induced pluripotent stem cells (hiPSCs)-derived neural precursor cell (NPCs) line once cells were harvested fresh from the cell culture or previously frozen and then grafted into striata or spinal cord of the immunodeficient rat. A newly developed human spinal injection device equipped with a spinal cord pulsation-cancelation magnetic needle was also tested for its safety in an adult immunosuppressed pig. Previously frozen NPCs showed similar post-grafting survival and differentiation profile as was seen for freshly harvested cells. Testing of human injection device showed acceptable safety with no detectable surgical procedure or spinal NPCs injection-related side effects.

• Klíčová slova
• cryopreservation, human induced pluripotent stem cells (hiPSCs), human injection device, immunosuppressed adult pig, neural precursor cells (NPCs), spinal cord,
• MeSH
• buněčná diferenciace fyziologie   MeSH
• dospělí MeSH
• genetické vektory genetika   MeSH
• indukované pluripotentní kmenové buňky * fyziologie   transplantace   MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• mícha MeSH
• mozek MeSH
• nervové kmenové buňky * fyziologie   transplantace   MeSH
• odběr biologického vzorku metody   MeSH
• odběr tkání a orgánů metody   MeSH
• prasata MeSH
• přeprogramování buněk * genetika   fyziologie   MeSH
• přežívání štěpu fyziologie   MeSH
• spinální injekce * škodlivé účinky   přístrojové vybavení   metody   MeSH
• transplantace kmenových buněk * škodlivé účinky   přístrojové vybavení   metody   MeSH
• virus Sendai MeSH
• výsledek terapie MeSH
• zvířata MeSH
• Check Tag
• dospělí MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The critical requirements in developing clinical-grade human-induced pluripotent stem cells-derived neural precursors (hiPSCs-NPCs) are defined by expandability, genetic stability, predictable in vivo post-grafting differentiation, and acceptable safety profile. Here, we report on the use of manual-selection protocol for generating expandable and stable human NPCs from induced pluripotent stem cells. The hiPSCs were generated by the reprogramming of peripheral blood mononuclear cells with Sendai-virus (SeV) vector encoding Yamanaka factors. After induction of neural rosettes, morphologically defined NPC colonies were manually harvested, re-plated, and expanded for up to 20 passages. Established NPCs showed normal karyotype, expression of typical NPCs markers at the proliferative stage, and ability to generate functional, calcium oscillating GABAergic or glutamatergic neurons after in vitro differentiation. Grafted NPCs into the striatum or spinal cord of immunodeficient rats showed progressive maturation and expression of early and late human-specific neuronal and glial markers at 2 or 6 months post-grafting. No tumor formation was seen in NPCs-grafted brain or spinal cord samples. These data demonstrate the effective use of in vitro manual-selection protocol to generate safe and expandable NPCs from hiPSCs cells. This protocol has the potential to be used to generate GMP (Good Manufacturing Practice)-grade NPCs from hiPSCs for future clinical use.

• Klíčová slova
• brain grafting, human-induced pluripotent stem cells (hiPSCs), immunodeficient rat, manual selection, neural precursor cells (NPCs), spinal cord grafting,
• MeSH
• buněčná diferenciace MeSH
• indukované pluripotentní kmenové buňky * MeSH
• krysa rodu Rattus MeSH
• leukocyty mononukleární MeSH
• lidé MeSH
• nervové kmenové buňky * MeSH
• neurony metabolismus   MeSH
• virus Sendai genetika   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Human multipotent neural stem cells could effectively be used for the treatment of a variety of neurological disorders. However, a defining signature of neural stem cell lines that would be expandable, non-tumorigenic, and differentiate into desirable neuronal/glial phenotype after in vivo grafting is not yet defined. Employing a mass spectrometry approach, based on selected reaction monitoring, we tested a panel of well-described culture conditions, and measured levels of protein markers routinely used to probe neural differentiation, i.e. POU5F1 (OCT4), SOX2, NES, DCX, TUBB3, MAP2, S100B, GFAP, GALC, and OLIG1. Our multiplexed assay enabled us to simultaneously identify the presence of pluripotent, multipotent, and lineage-committed neural cells, thus representing a powerful tool to optimize novel and highly specific propagation and differentiation protocols. The multiplexing capacity of this method permits the addition of other newly identified cell type-specific markers to further increase the specificity and quantitative accuracy in detecting targeted cell populations. Such an expandable assay may gain the advantage over traditional antibody-based assays, and represents a method of choice for quality control of neural stem cell lines intended for clinical use.

• Klíčová slova
• Cell line characterization, Mass spectrometry, Neural differentiation, Neural stem cell, Protein marker, Selected reaction monitoring,
• MeSH
• biologické markery MeSH
• buněčná diferenciace * MeSH
• buněčné linie MeSH
• buněčný rodokmen genetika   MeSH
• hmotnostní spektrometrie MeSH
• imunohistochemie MeSH
• lidé MeSH
• nervové kmenové buňky cytologie   metabolismus   MeSH
• neuroglie MeSH
• neurony MeSH
• stanovení celkové genové exprese MeSH
• vývojová regulace genové exprese MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• biologické markery MeSH

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.

• Klíčová slova
• glia limitans formation from grafted neural precursors, human-specific mRNA sequencing, immunodeficient rat, neuraxial neural precursor migration, subpial stem cell injection,
• MeSH
• krysa rodu Rattus MeSH
• nervové kmenové buňky metabolismus   MeSH
• parenchymatická tkáň cytologie   metabolismus   MeSH
• potkani Sprague-Dawley MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• MeSH
• amyotrofická laterální skleróza genetika   patofyziologie   terapie   MeSH
• atrofie MeSH
• degenerace nervu genetika   patofyziologie   terapie   MeSH
• Dependovirus metabolismus   MeSH
• interneurony patologie   MeSH
• lidé MeSH
• malá interferující RNA aplikace a dávkování   MeSH
• messenger RNA genetika   metabolismus   MeSH
• mícha diagnostické zobrazování   patologie   patofyziologie   MeSH
• motorické evokované potenciály MeSH
• motorické neurony patologie   MeSH
• myši inbrední C57BL MeSH
• myši transgenní MeSH
• pia mater patologie   patofyziologie   MeSH
• prasata MeSH
• primáti MeSH
• progrese nemoci MeSH
• regulace genové exprese MeSH
• sbalování proteinů MeSH
• superoxiddismutasa 1 genetika   metabolismus   MeSH
• technika přenosu genů * MeSH
• umlčování genů * MeSH
• vývoj svalů MeSH
• zánět patologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• malá interferující RNA MeSH
• messenger RNA MeSH
• superoxiddismutasa 1 MeSH

BACKGROUND: A well-characterized method has not yet been established to reproducibly, efficiently, and safely isolate large numbers of clinical-grade multipotent human neural stem cells (hNSCs) from embryonic stem cells (hESCs). Consequently, the transplantation of neurogenic/gliogenic precursors into the CNS for the purpose of cell replacement or neuroprotection in humans with injury or disease has not achieved widespread testing and implementation. METHODS: Here, we establish an approach for the in vitro isolation of a highly expandable population of hNSCs using the manual selection of neural precursors based on their colony morphology (CoMo-NSC). The purity and NSC properties of established and extensively expanded CoMo-NSC were validated by expression of NSC markers (flow cytometry, mRNA sequencing), lack of pluripotent markers and by their tumorigenic/differentiation profile after in vivo spinal grafting in three different animal models, including (i) immunodeficient rats, (ii) immunosuppressed ALS rats (SOD1G93A), or (iii) spinally injured immunosuppressed minipigs. RESULTS: In vitro analysis of established CoMo-NSCs showed a consistent expression of NSC markers (Sox1, Sox2, Nestin, CD24) with lack of pluripotent markers (Nanog) and stable karyotype for more than 15 passages. Gene profiling and histology revealed that spinally grafted CoMo-NSCs differentiate into neurons, astrocytes, and oligodendrocytes over a 2-6-month period in vivo without forming neoplastic derivatives or abnormal structures. Moreover, transplanted CoMo-NSCs formed neurons with synaptic contacts and glia in a variety of host environments including immunodeficient rats, immunosuppressed ALS rats (SOD1G93A), or spinally injured minipigs, indicating these cells have favorable safety and differentiation characteristics. CONCLUSIONS: These data demonstrate that manually selected CoMo-NSCs represent a safe and expandable NSC population which can effectively be used in prospective human clinical cell replacement trials for the treatment of a variety of neurodegenerative disorders, including ALS, stroke, spinal traumatic, or spinal ischemic injury.

• Klíčová slova
• Amyotrophic lateral sclerosis (ALS), Bioinformatic tools to study xenografts, Human embryonic stem cell (hESC), Neural stem cell (NSC), Spinal cord, Spinal traumatic injury,
• MeSH
• buněčné linie MeSH
• lidé MeSH
• multipotentní kmenové buňky cytologie   MeSH
• nervové kmenové buňky cytologie   MeSH
• průtoková cytometrie * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Effective in vivo use of adeno-associated virus (AAV)-based vectors to achieve gene-specific silencing or upregulation in the central nervous system has been limited by the inability to provide more than limited deep parenchymal expression in adult animals using delivery routes with the most clinical relevance (intravenous or intrathecal). Here, we demonstrate that the spinal pia membrane represents the primary barrier limiting effective AAV9 penetration into the spinal parenchyma after intrathecal AAV9 delivery. We develop a novel subpial AAV9 delivery technique and AAV9-dextran formulation. We use these in adult rats and pigs to show (i) potent spinal parenchymal transgene expression in white and gray matter including neurons, glial and endothelial cells after single bolus subpial AAV9 delivery; (ii) delivery to almost all apparent descending motor axons throughout the length of the spinal cord after cervical or thoracic subpial AAV9 injection; (iii) potent retrograde transgene expression in brain motor centers (motor cortex and brain stem); and (iv) the relative safety of this approach by defining normal neurological function for up to 6 months after AAV9 delivery. Thus, subpial delivery of AAV9 enables gene-based therapies with a wide range of potential experimental and clinical utilizations in adult animals and human patients.

• Publikační typ
• časopisecké články MeSH

Expression of doublecortin (DCX), a 43-53 kDa microtubule binding protein, is frequently used as (i) an early neuronal marker to identify the stage of neuronal maturation of in vivo grafted neuronal precursors (NSCs), and (ii) a neuronal fate marker transiently expressed by immature neurons during development. Reliable identification of the origin of DCX-immunoreactive cells (i.e., host vs. graft) requires detailed spatial and temporal mapping of endogenous DCX expression at graft-targeted brain or spinal cord regions. Accordingly, in the present study, we analyzed (i) the time course of DCX expression in pre- and postnatal rat and porcine spinal cord, and (ii) the DCX expression in spinally grafted porcine-induced pluripotent stem cells (iPS)-derived NSCs and human embryonic stem cell (ES)-derived NSCs. In addition, complementary temporospatial GFAP expression study in porcine spinal cord was also performed. In 21-day-old rat fetuses, an intense DCX immunoreactivity distributed between the dorsal horn (DH) and ventral horn was seen and was still present in the DH neurons on postnatal day 20. In animals older than 8 weeks, no DCX immunoreactivity was seen at any spinal cord laminae. In contrast to rat, in porcine spinal cord (gestational period 113-114 days), DCX was only expressed during the pre-natal period (up to 100 days) but was no longer present in newborn piglets or in adult animals. Immunohistochemical analysis was confirmed with a comparable expression profile by western blot analysis. Contrary, the expression of porcine GFAP started within 70-80 days of the pre-natal period. Spinally grafted porcine iPS-NSCs and human ES-NSCs showed clear DCX expression at 3-4 weeks postgrafting. These data indicate that in spinal grafting studies which employ postnatal or adult porcine models, the expression of DCX can be used as a reliable marker of grafted neurons. In contrast, if grafted neurons are to be analyzed during the first 4 postnatal weeks in the rat spinal cord, additional markers or grafted cell-specific labeling techniques need to be employed to reliably identify grafted early postmitotic neurons and to differentiate the DCX expression from the neurons of the host.

• MeSH
• časové faktory MeSH
• doménové proteiny doublecortinu MeSH
• druhová specificita MeSH
• embryonální kmenové buňky metabolismus   transplantace   MeSH
• indukované pluripotentní kmenové buňky metabolismus   transplantace   MeSH
• krysa rodu Rattus MeSH
• kultivované buňky MeSH
• lidé MeSH
• mícha růst a vývoj   metabolismus   MeSH
• neurogeneze fyziologie   MeSH
• neuropeptidy biosyntéza   MeSH
• potkani Wistar MeSH
• prasata MeSH
• protein doublecortin MeSH
• proteiny asociované s mikrotubuly biosyntéza   MeSH
• těhotenství MeSH
• transplantace kmenových buněk trendy   MeSH
• vývojová regulace genové exprese * MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DCX protein, human MeSH Prohlížeč
• Dcx protein, rat MeSH Prohlížeč
• doménové proteiny doublecortinu MeSH
• neuropeptidy MeSH
• protein doublecortin MeSH
• proteiny asociované s mikrotubuly MeSH