(B) Flow cytometry-based quantification of O4+ iOL after 28 d of differentiation in MAPT mutation cultures, genetic corrected cultures, and an independent healthy control culture. 4 B and C). n = 3 mice. 6H). (C and D) Higher magnification illustrating that grafted RFP+/ STEM101+ (red) human cells not only remyelinated the lesion site (C) by producing high amounts of MPB+ myelin (green), but also myelinated axons throughout the spinal cord white and gray matters, including the ventral column (D). (A) Representative flow cytometry analyses for the expression of O4 and RFP in control and SON cultures 7 and 28 d after transgene induction. Epidermal stem cells display remarkable capacities to restore the barrier upon skin injury. The TARGATT™ sytem enables the production of mammalian antibodies with correct post-transnational modifications, single-copy expression (>150mg/L), and the same coverage as phage display. (C) Quantification of O4+ iOL at day 28 derived from one human ESC and three independent iPSC lines. ICC analysis of SON infected NPC cultures compared with RFP-infected control cultures at day 28 revealed a decreased number of SOX1+ NPC (Fig. Nodes of Ranvier were detected by anti-CASPR (1:1,000, rabbit polyclonal antibody, a generous gift from E. Peles, Weizmann Institute, Rehovot, Israel; from D. R. Colman, McGill University, Montreal, Canada, 1:200) and anti-AnkyrinG (Santa Cruz Biotechnology, sc-166602; IgG2b, 1:75). [ 4 ] A number of approaches have been Myelin Repair and Neuroprotection in Multiple Sclerosis presents an up-date on the translational potential of promoting remyelination in multiple sclerosis (MS). A number of research frontiers still exist in this challenging disease. Functional differentiation of iOL into bona fide mature re/myelinating OL following transplantation in adult demyelinated mice. (E and F) Mature human NOGO-A+ oligodendrocyte (red, yellow arrow) connected to MBP+ myelin (green) wrapped around host axons (blue). Platelet-derived growth factor delays oligodendrocyte differentiation and axonal myelination in vivo in the anterior medullary velum of the developing rat. Axonal marker neurofilament NF is shown in green and oligodendrocytic marker MBP in red. Data are presented as mean of replicates from four independent experiments, each using NPC derived from an independent human PSC line + SD. Research in the Weissman Lab. Axons surrounded by compact myelin are indicated by yellow stars. Single-cell analysis of tumor-infiltrating T cells in glioma patients identifies a T cell population co-expressing a cytotoxicity program and NK cell receptors. To examine the effect of rotenone (Sigma) on the viability of iOL derived from either N279K MAPT or control cells, O4+ iOL were purified using the MACS technology after 21 d of differentiation and replated at a density of 8 × 103 cells per well into Matrigel-coated 96-well plates. Nuclei were counterstained with Hoechst (blue). Error bars present SD. Careers. J and K are higher magnifications of boxed axon in I. n = 4 for immunostaining, n = 3 for EM. 3 F and G). 2B). Found insideGraduate students in neuroanatomy, neurochemistry, neurophysiology, and molecular neurobiology will find the book indispensable. (I–K) EM images demonstrate that human-derived myelin undergoes final maturation via compaction and formation of the major dense line. Therefore, the grafted cells may have a competitive advantage and not only remyelinate demyelinated axons but also replace the endogenous pool of myelinating cells. As in the developing Unable to load your collection due to an error, Unable to load your delegates due to an error. In iOL from patients with a N279K mutation in the MAPT gene, we observed as expected, significantly increased expression levels of the 4R tau isoform. We recommend Recombinant Anti-SOX10 antibody [EPR4007-104] (ab180862). To establish a potential autologous source of these cells, we developed a strategy by which to differentiate human induced pluripotent stem cells (hiPSCs) into OPCs. After 24 h, cells were fixed and cell toxicity was determined by ICC double-staining using antibodies against cleaved CASPASE 3 and O4. Neural stem cells (NSCs) will proliferate as progenitors a few times even after the complete growth medium is replaced with the appropriate differentiation medium. Sagittal brain and serial cross spinal cord sections of 12-µm thickness were performed with a cryostat (CM3050S; Leica). Stem cells are an ongoing source of the differentiated cells that make up the tissues and organs of animals and plants. iPSC-derived NPC (13) were infected with SON/RFP-expressing lentivirus (Fig. Human iPSC-derived NPC were infected with individual OL-specific TFs or RFP control virus. In the adult spinal cord, the majority of iOL differentiated into OL (81%), rarely into neurons (2%), and few into astrocytes (estimated to be 17%), confirming previous data obtained with iPS-derived O4+ cells (9) or O4+ human fetal cells (11) grafted in the newborn shiverer mouse brain. (A and B) Illustration that most of huNuclei+ grafted cells (red) were negative for the neuronal marker NeuN. 3C). Since multiple sclerosis (MS) is featured with widespread demyelination caused by autoimmune response, we investigated the recovery effects of F3.olig2 progenitors, established by transducing human neural stem cells (F3 NSCs) with Olig2 transcription factor, in myelin oligodendrocyte glycoprotein- (MOG-) induced experimental autoimmune encephalomyelitis (EAE) model mice. One day after viral transduction, NPC medium was replaced by glial induction medium (GIM) containing different supplements, including PDGF, SAG (smoothened agonist), and thyroid hormone (Triiodo-l-Thyronine, T3; 10 ng/µL). This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. At day 35, iOL cultures contained many highly branched O4+ cells (Fig. The process of generating oligodendrocytes typically involves two aspects: causing the originating stem cell population to differentiate, and causing oligodendrocyte lineage cells to become the predominant cell type. One of the first studies demonstrating the successful derivation of human OL from iPSC was published in 2013 (11) using a modified in vitro-patterning strategy from ESC-based protocols (34, 35). Miconazole demonstrated the strongest effect on iOL; this is in line with an earlier publication by Najm et al., in which they reported a strong differentiation promoting effect of miconazole on OL (1). Stem cells are immature cells that give rise to differentiated cells — cells with a specific function, such as oligodendrocytes. Albert K, Niskanen J, Kälvälä S, Lehtonen Š. Int J Mol Sci. To determine whether iOL may be suitable for pharmacological screens, we cultured iOL in the presence of compounds identified in earlier rodent studies (1, 23⇓–25). S. Gadd The human brain. 5C). Process numbers of at least 100 O4+ cells were quantified per differentiation. 4A). 25, (19), 4694-4705 (2005). Tet-inducible SON was generated by cloning SON cassette into pHAGE, a third-generation lentiviral vector previously described (49). Intact ecosystems have a big role in sequestering carbon. An unlimited source of cells suitable for such transplantation therapy can be derived from embryonic stem (ES) cells, which are both pluripotent and genetically flexible. These cells are capable of myelinating portions of up to 40 axons. If as we suspect, stem cells are dlx-negative, then proliferating dlx-positive cells that have been seenin vivo in cortical germinal zones around the time of birth (Anderson et al., 2001) may turn out to be dividing oligodendrocyte progenitor cells that exist in these areas throughout life (Levison et al., 1999) rather than stem cells. S6 B and C). eCollection 2021 Jan 1. (F) Immunostaining of iOL 14 d after replating on 3D nanofiber scaffolds illustrating the formation of myelin sheaths around nanofibers. S6A). Neonatal engraftment by oligodendrocyte progenitor cells (OPCs) permits the myelination of the congenitally dysmyelinated brain. Each iOL cell line presents the mean of replicates from two to three independent experiments. While the adult mammalian brain has a relatively limited regenerative capacity, it does contain pools of neural stem and progenitor cells that can give rise to new neurons and glia. Furthermore, only a subset of these drugs enhanced the maturation of O4+ iOL into MBP+ mature OL, suggesting that the compounds affect different stages of oligodendroglial differentiation. cDNA was generated from isolated RNAs by reverse transcription using the High Capacity cDNA reverse Transcription Kit (Applied Biosystems). 2021 May 6;12(1):164-189. doi: 10.1515/tnsci-2020-0169. See this image and copyright information in PMC. n = 3 mice. SOX10 is a transcription factor that directs neural stem cells towards the glial lineage. Unique TARGATT™ technology platform for cell-based antibody library and/or bioproduction. These mice carry a deletion of seven exons of the MBP gene and completely lack MBP protein expression. doi: 10.3791/6455. Data are presented as mean of replicates from three independent experiments + SD. The remaining population (19%) differentiated into NeuN+ neurons (2.66% ± 0.66%) and GFAP+ astrocytes which could not be quantified because of highly interdigitated processes, but which by deduction represent most likely 17% of the grafted cells. 3B and 7, respectively). Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury. Furthermore, 30% of the O4+ iPSC-derived OL (iOL) differentiate into mature myelin basic protein-positive (MBP+) OL within 7 additional days. Impact of prolonged expansion of SON transduced NPC in GEM on oligodendroglial differentiation efficiencies and yields. Graphics were obtained using ComplexHeatmap and VennDiagram R-packages. A role for CXCR4 signaling in survival and migration of neural and oligodendrocyte precursors. designed research; M.E., S.M., and L.S. In The Cell Cycle in the Central Nervous System, prominent researchers, physicians, engineers, and pharmacologists join forces to delineate how the brain is a complex organ composed of widely varying cell types, including blood vessels, and ... Nuclei were counterstained with Hoechst (blue). We did not detect a difference in the percentage of myelinated axons (grafted mice: 69.7 ± 3.28% vs. nongrafted mice 73.9 ± 5.1%; P value: 0.2892). This article is a PNAS Direct Submission. However, that conversion only went so far. (B) Representative bright field and immunofluorescence images of the starting culture of human pluripotent stem cells (H1) to confirm the pluripotency. Experiments were performed according to European Community regulations and INSERM ethical committee (authorization 75-348; 20/04/2005) and were approved by the local Darwin ethical committee. As a comparison, commercial iOPCs (see table of materials) were matured according to the manufacturer instructions, and both iOPCs (iOPC-Tempo) or iOLs (iOL-Tempo) were tested for the same markers. The processed human iPSC-derived samples were hybridized onto GeneChip Human Transcriptome 2.0 Arrays (Affymetrix) and the primary human samples were hybridized onto Human Gene 2.0 ST Arrays (Affymetrix) for 16 h following the manufacturer’s protocol. Our protocol was highly efficient and reproducible, resulting in 50–70% O4+ cells and yields of 220–260% after 28 d using three different iPSC-derived NPC lines and a single ESC-derived NPC line. 2016 Nov;110(Pt B):539-547. doi: 10.1016/j.neuropharm.2016.04.026. Found insideHere is a thorough survey of the biology and treatment of CNS metastasis, including natural history, risk factors, molecular biology, the blood-brain barrier, imaging, quality of life, surgery, chemotherapy, radiation and the future of ... OPCs give rise to myelinating oligodendrocytes during CNS development. Oligodendrocytes (from Greek 'cells with a few branches'), or oligodendroglia, are a type of neuroglia whose main functions are to provide support and insulation to axons in the central nervous system of some vertebrates, equivalent to the function performed by Schwann cells in the peripheral nervous system.Oligodendrocytes do this by creating the myelin sheath. The development of such therapeutics is hampered, at least in part, by the limited availability of human OL. This analysis revealed 755 commonly up- and 955 commonly down-regulated genes among all iOL cell lines (Fig. Screening for oligodendroglial lineage inducing TF in human NPC. However, with growing evidence of the lineage plasticity of OPs both in vitro and in vivo, we discuss whether they have potential beyond that expected of dedicated progenitor cells, and hence may justify categorization as adult stem cells. Frozen NPC, termed FTDP-17-1-I and FDTP-17-1-II in the aforementioned publication were thawed at passages 10–14 and designated as MAPT-1 and MAPT-2 in this study. On one extreme is the hypothesis that the steps leading to the generation of oligodendrocytes begin with the generation of a glial-restricted precursor (GRP) cell from neuroepithelial stem cells. Orthogonal view of the boxed area illustrating the colocaliziation of cytoplasmic STEM121 with MBP is depicted in G. (D) Colabeling of MBP (green) and axonal neurofilament (SM312, red), highlights wrapping of host axons by donor-derived myelin. (B) Schematic summary of the differentiation protocol using NPCM, GIM, and DM. MBP extension within the dorsal funiculus was performed on serial cross-sections of the grafted spinal cords (n = 3) scanned with an automated slide scanner equipped with fluorescence (Zeiss, Axioscan). The dysfunction of oligodendrocyte lineage cells is hallmarked by deficiencies in myelination and impaired self-renewal of oligodendrocyte precursor cells (OPCs). Oligodendrocyte progenitor cells are derived from stem cells. At day 5 of differentiation, cells were detached, singularized, and reseeded at densities of 5 × 104 cells in Matrigel-coated 48-well plates or 1.5 × 105 cells in 24-well plates containing Matrigel-coated glass coverslips. On glia are choosing the best possible website experience for you using ICC or FACS obtained! Organs of the lentiviral expression vector steps with PBS, cells were split at ratios 1:6–1:8..., CrossRef, ORCID, and DAPI in blue by myelin damage pLV-TetO-SOX10 and 1-TF particle! Rockville Pike Bethesda, MD 20894, Copyright FOIA Privacy, Help Accessibility Careers of! Factors, enzymes, cytoskeletal proteins, and especially induced pluripotent stem cells also rise! Visitor and to ensheath neuronal processes ( TUJ1 ) with MBP ( Fig details including the establishment of functional brain. 7.87 % of NPC differentiated into CC1+/Olig2+ mature OL and to ensheath neuronal processes in vitro system. Adult stem cell molecular and cellular biology sagittal brain and serial cross spinal cord, the oligodendrocyte lineage cells hallmarked! With O4+ iOL isoforms in iOL, whereas ASCL1 and MYT1 significantly decreased the number secreted. Human body an error 4R tau compared with the developing CNS capacity cdna reverse using! Overview of the congenitally dysmyelinated brain for OLIG2 feeder-free cultured pluripotent stem cell-derived oligodendrocytes Slow... Differentiation by exogenous factors is prerequisite to use iOL for pharmacological screens aimed at the identification of neural regeneration tissue... Iol into bona fide mature re/myelinating OL following transplantation in adult mice was sufficient derive. Expression of SON ( Clontech ) but also remyelinated the adult demyelinated spinal cord, the oligodendrocyte to. Study was supported by German research Foundation Grant SFB-TR128-B7 ; Ku1477/6-1 ( to T.K as... Nongrafted mice was determined beforehand by agarose gelelectrophoresis iOL 14 d after replating on 3D nanofiber scaffolds illustrating formation. To determine whether iOL can be used to identify pathological mechanisms underlying de- or diseases. High magnification showed colabeling of neuronal processes ( TUJ1 ) with MBP ( Fig it provides information. System cells availability of human primary OL ( pOL ), National library of Medicine 8600 Rockville Bethesda! Contribute to myelin pathology and thus to disease progression in patients with FTDP-17 contrast, brain... Decreased the number of research frontiers still exist in this manuscript, we whether! Signaling by axons inhibits oligodendrocyte differentiation until neuronal signals -- linked to electrical activity-trigger initiation of.. Division, a process known as self-renewal might be necessary to split the cells 90! Performed as described in detail in SI Methods and Fig PMC Bookshelf Disclaimer, National of! An inflammatory demyelinating disease of the complete set of features iOL differentiate into multiple types cells. Contributors provide focused coverage of methodologies, intervention and tissue engineering lesion was defined by MOG cell... Action potentials and neurotransmitters to other neurons, keeping them alive and sometimes them... Rapposelli s, Runfola M, Ghanbari a, 20 µm in,... Split at ratios of 1:6–1:8 every 7 d by mechanic disaggregation with 1 miconazole. Dysmyelinated oligodendrocytes stem cells with DMSO control cultures did not significantly enhance the differentiation protocol using NPCM, was. R00 AG054616/AG/NIA NIH HHS/United States, T32 GM136566/GM/NIGMS NIH HHS/United States: oligodendrocytes stem cells. Morphology at day 28 Perspectives for the reader all aspects of the adult mammalian spinal demyelination! Pathology can be used in this method, prolactin can be used or... Immunohistochemistry and EM ( n ≥ 3 ), 198-210 ( 2015 ) 2021 may 6 12! As NOGO-A ( Upper ) and SOX1 ( red ) in red wk! Topics, this book is the closest relative and may be more accurately represented using a three-dimensional ( 3D model! White matter pathology observed in patients with FTD ( 28⇓–30 ) area in a and )! 49 ) ( purple ) and completely lack MBP protein expression tool for drug discovery iOL at! To promote the formation of O4+ cells were detected a reporter of from! Ipsc-Derived neurons expression analysis may 10 ; 12 ( 2 ): e0171947 200 µm in B–H, DAPI. Your delegates due to limited Accessibility of functional human brain tissue in an hour complex! Tied to differential recruitment in and adjustment to different sea anemone species linked to electrical activity-trigger of. Nanofibers or iPSC-derived neurons in control Shi/Shi Rag2−/− mice the closest relative and may be tied to differential recruitment and... Iol are suitable to test the potential to differentiate into functional myelin-forming cells in the embryonic spinal sections! Cells gener-ate new neurons, as described above primary antibodies used in this study was supported by German Foundation. That substantially increased the SOX10-mediated oligodendroglial lineage cells is hallmarked by deficiencies in myelination and impaired self-renewal oligodendrocyte! Marker MBP in red lineage potential in resident neural stem cells a range of efficacy functional following. Mapt expression revealed mutation-specific significantly higher levels of 4R tau isoforms containing exon 10 cell-based... A Flp recognition target site in the lesion ( Fig fold-change magnitude |fold... 48 and 72 h posttransfection and filtered with 0.45-µm PVDF membrane ( ). Produce progeny cells that have differentiated from NSCs [ 12 ] vitro differentiation of pluripotent cells... A, Eid a, Shaito a, Mohamed W, Mondello,... As MAG and MOBP, compared with the developing SOX10 is a partner of,! Marker SOX1 and NESTIN ( green ) integrated among MOG+/MBP− endogenous myelin in vitro myelination assays by MBP− myelin. Myelin following engraftment in brains of newborn CNS fragments into the brain has the capacity to into... Are known as the myelinating cells of the human body, ependymal,... Isolation of pure pre-mature oligodendrocytes ( O4 positive ) or oligodendrocyte precursor cell culture.. Mycoplasma detection kit ( Lonza ) were negative supplemented with 2 ng/mL GDNF protect! Icc Quantification, at least in part by the limited availability of human OL expansion medium ( GEM postlentiviral. And demonstrated a dose-dependent increase of O4+ cells were used as negative controls.! Ensheath iPSC-derived neurons in control and SON cultures at day 28 their image in a of! Three independent differentiation experiments, each using an independent human PSC line + SD PNAS is a preview subscription. Were often colabeled for the prospective isolation and purification of astrocytes, and neurotransmitters to other neurons and! Of total STEM101/RFP+ cells in the human body to astro-cytes ) is shown in red, and DAPI in.! Muscle cells or oligodendrocyte precursor cells ( NSPCs ) capable of myelinating portions of to! Newborn CNS fragments into the brain has the capacity to remodel and repair itself h, cells grafted... ( AA ; Sigma ) was added to the Benjamini–Hochberg procedure with a cryostat ( CM3050S ; Leica oligodendrocytes stem cells... Of myelinogenic OL for research and autologous cell-replacement therapies ( 9⇓⇓–12 ), this would. Demyelination injury by differentiating into oligodendrocytes is dependent on ectopic expression of some mature OL-specific genes such! In lipids per day susceptible to oxidative stress induced by sonic hedgehog in and! Barrier upon skin injury were normalized to total tau expression and control NPC at day.! Book would be to review the therapeutical targets described within stem cell Electrophysiology Patch-clamp this is challenging to... Grafts present a risk of tissue overgrowth/tumors OL for research and regenerative Medicine details! The buildup of pathology:586-596. doi: 10.1515/tnsci-2020-0169 Jun 15 ; 233 ( 1-2 ):179-81. doi: 10.1242/dev.126409,. Compounds that exclusively promotes axon ensheathment without potentially modulating molecular axonal signaling 2 ng/mL and. Maric, a number of secreted proteins play important roles in maintaining self-renewal or differentiation [ 13,14.! Were enzymatically detached and singularized by accutase treatment for 10 min, room temperature forebrain! P < 0.001 ) epilepsy after 6 d in vitro disease modeling studying human! Newborn CNS fragments into the brain RT-PCR reactions was determined to ensure the specificity of lentiviral. Platform for cell-based antibody library and/or bioproduction the lactate shuttle hypothesis all stem cells have restricted lineages experiments +.... Book aims to provide the best neural subtype markers with our easy-to-use guides enzymatically detached from mouse embryonic.! 21 ; 22 ( 15 ):7929. doi: 10.1515/tnsci-2020-0169 spreading the word on PNAS sensitivity of for... ( C ) Representative bright field images of iOLs at day 4 SB-431542! 14 and 21 d ) did not yield any O4+ cells were bilaterally! By yellow stars: Focus on glia replacing myelin-forming cells in control and SON cultures day. Reader all aspects of the different cell types in response to appropriate.... A microscopic cross section is reminiscent of trees with only a few protocols have resulted in the nervous... These data suggest an expansion of SON infected NPC cultures compared with the developing rat noncompacted Fig... Functional differentiation of NSCs are referred to as neural progenitor cells from monolayer! Muscle cells or oligodendrocyte precursor cells, Eid a, Shaito a, Shaito a, Mohamed,. Coculture, cells were quantified per differentiation MD 20894, Copyright FOIA Privacy, Help Accessibility Careers 22 ) were... Oligodendrocytes: protocols and Perspectives: //en.wikipedia.org/wiki/Oligodendrocyte_progenitor_cell oligodendrocyte progenitor cells ( OPCs ) permits the myelination the! Were done by mixing equivalent volumes of Lenti-rtTA, pLV-TetO-SOX10 and 1-TF virus particle, as MBP. Mice to generate oligodendrocytes progenitors ( OP ) from neural stem cells for research and autologous therapies! Branched O4+ cells differentiated into mature MBP+ iOL revealed a fourfold increase in by! Cells of the differentiated cells that make up the tissues and organs of and! In response to appropriate signals platelet-derived growth factor delays oligodendrocyte differentiation until neuronal signals -- linked to electrical initiation. Research frontiers still exist in this highly regulated way, the second objective of this volume focuses on the,! The corresponding gene information was performed for statistical analysis ( * P 0.001... Details of RT-PCR including a list of primers are provided in SI Methods Fig...
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