In Vitro Recapitulation of Developmental Transitions in Human Neural Stem Cells
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Page 1 of 185 Ostermann, Ladewig et al., 1 Running title: NS cells from NES cells In vitro recapitulation of developmental transitions in human neural stem cells Laura Ostermann1*, Julia Ladewig1,5,6,7*, Franz-Josef Müller2, Jaideep Kesevan1, Jignesh Tailor3, Austin Smith3,4, Oliver Brüstle1#, Philipp Koch1,5,6,7# (1) Institute of Reconstructive Neurobiology, University of Bonn School of Medicine & University Hospital Bonn, Bonn, Germany (2) Department of Psychiatry and Psychotherapy, Centre for Integrative Psychiatry, Kiel, Germany (3) Wellcome Trust - Medical Research Council Stem Cell Institute, University of Cambridge, Cambridge, United Kingdom (4) Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom (5) Central Institute of Mental Health, University of Heidelberg, Medical Faculty Mannheim, Mannheim, Germany (6) HITBR Hector Institute for Translational Brain Research gGmbH, Heidelberg, Germany (7) German Cancer Research Center (DKFZ), Heidelberg, Germany * these authors contributed equally to the study # To whom correspondence should be addressed: Prof. Dr. Philipp Koch (M.D.) Hector Institute for Translational Brain Research Central Institute of Mental Health University of Heidelberg / Medical Faculty Mannheim Square J5 68159 Mannheim Phone: +49(0)621 17036711 Email: [email protected] Prof. Dr. Oliver Brüstle (M.D.) Institute of Reconstructive Neurobiology Life&Brain Center University of Bonn Medical Faculty Sigmund-Freud-Str. 25 53127 Bonn Phone: +49 (0)228 6885 500 Fax.: +49 (0)228 6885 501 Email: [email protected] Author Contributions Laura Ostermann, Franz-Josef Müller, Jaideep Kesevan: collection and assembly of data, data analysis and interpretation, manuscript writing. Jignesh Tailor, Austin Smith: provision of study material, data analysis and interpretation. Oliver Brüstle: financial support, data analysis and interpretation, manuscript writing. Julia Ladewig and Philipp Koch: Conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing. All authors approved the manuscript. Key words: stem cell biology, neural stem cells, neuroepithelial stem cells, radial glia stem cells, regional identity Page 2 of 185 Ostermann, Ladewig et al., 2 ABSTRACT During nervous system development, early neuroepithelial stem (NES) cells with a highly polarized morphology and responsiveness to regionalizing morphogens give rise to radial glia (RG) cells, which generate region-specific neurons. Recently, stable neural cell populations reminiscent of NES cells have been obtained from pluripotent stem cells and the fetal human hindbrain. Here we explore whether these cell populations, similar to their in vivo counterparts, can give rise to neural stem (NS) cells with radial glia-like properties and whether region-specific NS cells can be generated from NES cells with different regional identity. In vivo RG cells are thought to form from NES cells with the onset of neurogenesis. We therefore cultured NES cells temporarily in differentiating conditions. Upon re-initiation of growth factor treatment, cells were found to enter a developmental stage reflecting major characteristics of radial glia-like NS cells. These NES cell-derived NS cells exhibited a very similar morphology and marker expression as compared to primary NS cells generated from human fetal tissue, indicating that conversion of NES cells into NS cells recapitulates the developmental progression of early NES cells into RG cells observed in vivo. Importantly, NS cells generated form NES cells with different regional identities exhibited stable region-specific transcription factor expression and generated neurons appropriate for their positional identity. Page 3 of 185 Ostermann, Ladewig et al., 3 INTRODUCTION Neural stem cells (NSC) represent a self-renewing, multipotent somatic stem cell population of the central nervous system (CNS). They serve as source for the different cell types of the brain including neurons, astrocytes and oligodendrocytes 1, 2. During early embryonic development the neuroectoderm is generated from the ectodermal layer and folds into the neural tube, which initially consists of a single layer of rapidly and symmetrically dividing neuroepithelial stem cells (NES cells). In this context, the term ‘neuroepithelial’ accounts for the fact, that these cells exhibit multiple epithelial characteristics. For instance, they are highly polarized spanning from the apical to the basal side of the neural tube. As other epithelial tissues, they form adherens and tight junctions at their apical pole 3. As from the initial pool of neuroepithelial cells all the diverse regional brain identities along the anterior- posterior and dorso-ventral axis are generated, neuroepithelial cells are highly responsive to extrinsic patterning signals and morphogens 4, 5. At the onset of neurogenesis and following extensive proliferation, NES cells transform into radial glia (RG) cells, the major stem cell population of the embryonic and fetal brain 2, 3, 6. RG cells maintain several aspects of NES cells such as their self-renewing capacity, their multipotentiality as well as their apical-basal polarity. They exhibit long radial processes, which span through the developing brain while staying attached to the pial surface and the luminal side 7, 8. During neurogenesis, these radial processes serve as guiding tracks for radially migrating neurons 3, 8. The term radial ‘glia’ refers to the fact that RG cells share many immunohistochemical characteristics with glial cells such as the expression of the astrocyte specific glutamate transporter (GLAST). During the transition of NES cells into RG cells, RG cells get progressively specified and their responsiveness to regionalizing morphogens diminishes. As a consequence, RG cells are regionally determined and consistently give rise to neuronal progeny of their region of origin 2, 9-11. In the last decades, various strategies for the isolation and in vitro expansion of different neural stem and progenitor populations have been described using free-floating aggregates Page 4 of 185 Ostermann, Ladewig et al., 4 called neurospheres or monolayer culture systems 12-15. Similar to their in vivo counterpart, in vitro cultured neural stem and progenitor cells are defined by their ability to self-renew and their multipotent differentiation capacity. In vitro neural stem cells, however, rely on the addition of growth factors such as fibroblast growth factor (FGF) or epithelial growth factor (EGF) and the extent to which in vitro propagated NSCs correlate to NSCs found in vivo remains controversial 12. In the human system, two distinct expandable and well-characterized neural stem/progenitor cell populations have recently been established. Human NS cells isolated from embryos beyond Carnegie stage 17 share many aspects of radial glia such as a bipolar morphology or expression of typical radial glia-associated markers including brain lipid binding protein (BLBP), GLAST or 3CB2 16. Using pluripotent stem cells as a starting population, we established a population of long-term self-renewing neuroepithelial stem cells (lt-NES cells; 14, 17). These cells are characterized by their continued ability to form rosette-like structures in vitro and, in the presence of morphogens, can be guided towards distinct regional identities 14. Interestingly, lt-NES cells could also be generated from primary human brain tissue when applying early fetal brain sources before Carnegie stage 17, indicating that lt-NES cells do have an in vivo correlate 18. Thus, isolation and propagation of the two major types of neural stem/progenitor cells, NES- and RG-like NS cells, has been established from primary human brain tissue. How these two populations hierarchically correlate in development and whether lt-NES cells can be converted into NS cells in vitro has, however, not been determined. Here we explored the potential of pluripotent and primary NES cells to give rise to radial glia- like NS cells in vitro. Using partially differentiating conditions to induce neurogenesis and subsequent re-initiation of proliferation with growth factors we were able to establish a multipotent cell population, which exhibits features of RG cells and shows high similarity to primary fetal brain-derived RG-like NS cells. These NES cell-derived NS cells could be expanded for multiple passages and expressed classic radial glia markers, while NES cell markers were down-regulated. Importantly, NS cells generated from forebrain or spinal cord- Page 5 of 185 Ostermann, Ladewig et al., 5 patterned NES cells retain their regional phenotype during multiple passages of in vitro proliferation and upon differentiation. Page 6 of 185 Ostermann, Ladewig et al., 6 MATERIAL AND METHODS Cell culture Lt-NES generated from human embryonic stem cells (hESCs) and primary brain tissue- derived NES cells were maintained in neural stem cell medium containing DMEM/F12, N2 supplement (1:100; both Life Technologies), 1,6 g/L glucose, 10 ng/mL FGF2 (R&D Systems), 10 ng/mL EGF (R&D Systems), 1:1000 B27 and penicillin-streptomycin (1:100, Invitrogen) on poly-L-ornithine /laminin (both Sigma Aldrich) precoated plastic dishes. For the derivation of NS cells, NES cells were induced to differentiate by growth factor withdrawal on Geltrex (Life Technologies) coated dishes in neural differentiation medium containing DMEM/12 (N2 supplement; 1:50) and Neurobasal (B27 supplement; 1:100) mixed at a 1:1 ratio. Medium was changed every other day. After 4