Efficient neuronal in vitro and in vivo differentiation after immunomagnetic purification of mESC derived neuronal precursors

The cellular heterogeneity that is generated during the differentiation of pluripotent stem cells into specific neural subpopulations represents a major obstacle for experimental and clinical progress. To address this problem we developed an optimized strategy for magnetic isolation of PSA-NCAM positive neuronal precursors from embryonic stem cells (ESCs) derived neuronal cultures. PSA-NCAM enrichment at an early step of the in vitro differentiation process increased the number of ES cell derived neurons and reduced cellular diversity. Gene expression analysis revealed that mainly genes involved in neuronal activity were over-represented after purification. In vitro derived PSA-NCAM⁺ enriched precursors were characterized in vivo through grafting into the forebrain of adult mice. While unsorted control cells 40 days post graft gave rise to a mixed population composed of immature precursors, early postmitotic neurons and glial cells, PSA-NCAM⁺ enriched cells differentiated predominantly into NeuN positive cells. Furthermore, PSA-NCAM enriched population showed efficient migration towards the olfactory bulb after transplantation into the rostral migratory stream of the forebrain neurogenic system. Thus, enrichment of neuronal precursors based on PSA-NCAM expression represents a general and straightforward approach to narrow cellular heterogeneity during neuronal differentiation of pluripotent cells.     

Synaptically-competent neurons derived from canine embryonic stem cells by lineage selection with EGF and Noggin

Pluripotent stem cell lines have been generated in several domestic animal species; however, these lines traditionally show poor self-renewal and differentiation. Using canine embryonic stem cell (cESC) lines previously shown to have sufficient self-renewal capacity and potency, we generated and compared canine neural stem cell (cNSC) lines derived by lineage selection with epidermal growth factor (EGF) or Noggin along the neural default differentiation pathway, or by directed differentiation with retinoic acid (RA)-induced floating sphere assay. Lineage selection produced large populations of SOX2+ neural stem/progenitor cell populations and neuronal derivatives while directed differentiation produced few and improper neuronal derivatives. Primary canine neural lines were generated from fetal tissue and used as a positive control for differentiation and electrophysiology. Differentiation of EGF- and Noggin-directed cNSC lines in N2B27 with low-dose growth factors (BDNF/NT-3 or PDGFαα) produced phenotypes equivalent to primary canine neural cells including 3CB2+ radial progenitors, MOSP+ glia restricted precursors, VIM+/GFAP+ astrocytes, and TUBB3+/MAP2+/NFH+/SYN+ neurons. Conversely, induction with RA and neuronal differentiation produced inadequate putative neurons for further study, even though appropriate neuronal gene expression profiles were observed by RT-PCR (including Nestin, TUBB3, PSD95, STX1A, SYNPR, MAP2). Co-culture of cESC-derived neurons with primary canine fetal cells on canine astrocytes was used to test functional maturity of putative neurons. Canine ESC-derived neurons received functional GABA(A)- and AMPA-receptor mediated synaptic input, but only when co-cultured with primary neurons. This study presents established neural stem/progenitor cell populations and functional neural derivatives in the dog, providing the proof-of-concept required to translate stem cell transplantation strategies into a clinically relevant animal model.     

TGF‐beta signalling in the adult neurogenic niche promotes stem cell quiescence as well as generation of new neurons

Members of the transforming growth factor (TGF)‐β family govern a wide range of mechanisms in brain development and in the adult, in particular neuronal/glial differentiation and survival, but also cell cycle regulation and neural stem cell maintenance. This clearly created some discrepancies in the field with some studies favouring neuronal differentiation/survival of progenitors and others favouring cell cycle exit and neural stem cell quiescence/maintenance. Here, we provide a unifying hypothesis claiming that through its regulation of neural progenitor cell (NPC) proliferation, TGF‐β signalling might be responsible for (i) maintaining stem cells in a quiescent stage, and (ii) promoting survival of newly generated neurons and their functional differentiation. Therefore, we performed a detailed histological analysis of TGF‐β1 signalling in the hippocampal neural stem cell niche of a transgenic mouse that was previously generated to express TGF‐β1 under a tetracycline regulatable Ca‐Calmodulin kinase promoter. We also analysed NPC proliferation, quiescence, neuronal survival and differentiation in relation to elevated levels of TGF‐β1 in vitro and in vivo conditions. Finally, we performed a gene expression profiling to identify the targets of TGF‐β1 signalling in adult NPCs. The results demonstrate that TGF‐β1 promotes stem cell quiescence on one side, but also neuronal survival on the other side. Thus, considering the elevated levels of TGF‐β1 in ageing and neurodegenerative diseases, TGF‐β1 signalling presents a molecular target for future interventions in such conditions.     

PRIMORDIAL GERM CELL-DERIVED MOUSE EMBRYONIC GERM (EG) CELLSIN VITRORESEMBLE UNDIFFERENTIATED STEM CELLS WITH RESPECT TO DIFFERENTIATION CAPACITY AND CELL CYCLE DISTRIBUTION

Embryonic germ (EG) cells of line EG-1 derived from mouse primordial germ cells were investigated for theirin vitrodifferentiation capacity. By cultivation as embryo-like aggregates EG-1 cells differentiated into cardiac, skeletal muscle and neuronal cells accompanied by the expression of tissue-specific genes and proteins as shown by RT-PCR analysis and indirect immunofluorescence. In comparison to embryonic stem (ES) cells of line D3 the efficiency of differentiation into cardiac and muscle cells was comparatively low, whereas spontaneous neuronal differentiation was more efficient than in D3 cells. Furthermore, the distribution of cell cycle phases as a parameter for the differentiation state was analysed in undifferentiated EG cells and ES cells and compared to data obtained for embryonic carcinoma (EC) cells of line P19 and differentiated, epithelioid EPI-7 cells. Flow cytometric analysis revealed similar cell cycle phase distributions in EG, EC and ES cells. In contrast, the somatic differentiated EPI-7 cells showed a longer G₁-phase and shorter S- and G₂/M-phases. Together, our results demonstrate that the differentiation state and capacity of EG cellsin vitroresemble that of totipotent ES cells.     

Pluripotency of male germline stem cells

The ethical issues and public concerns regarding the use of embryonic stem (ES) cells in human therapy have motivated considerable research into the generation of pluripotent stem cell lines from non-embryonic sources. Numerous reports have shown that pluripotent cells can be generated and derived from germline stem cells (GSCs) in mouse and human testes during in vitro cultivation. The gene expression patterns of these cells are similar to those of ES cells and show the typical self-renewal and differentiation patterns of pluripotent cells in vivo and in vitro. However, the mechanisms underlying the spontaneous dedifferentiation of GSCs remain to be elucidated. Studies to identify master regulators in this reprogramming process are of critical importance for understanding the gene regulatory networks that sustain the cellular status of these cells. The results of such studies would provide a theoretical background for the practical use of these cells in regenerative medicine. Such studies would also help elucidate the molecular mechanisms underlying certain diseases, such as testicular germ cell tumors.     

Two Pore Channel 2 Differentially Modulates Neural Differentiation of Mouse Embryonic Stem Cells

Nicotinic acid adenine dinucleotide phosphate (NAADP) is an endogenous Ca²⁺ mobilizing nucleotide presented in various species. NAADP mobilizes Ca²⁺ from acidic organelles through two pore channel 2 (TPC2) in many cell types and it has been previously shown that NAADP can potently induce neuronal differentiation in PC12 cells. Here we examined the role of TPC2 signaling in the neural differentiation of mouse embryonic stem (ES) cells. We found that the expression of TPC2 was markedly decreased during the initial ES cell entry into neural progenitors, and the levels of TPC2 gradually rebounded during the late stages of neurogenesis. Correspondingly, TPC2 knockdown accelerated mouse ES cell differentiation into neural progenitors but inhibited these neural progenitors from committing to neurons. Overexpression of TPC2, on the other hand, inhibited mouse ES cell from entering the early neural lineage. Interestingly, TPC2 knockdown had no effect on the differentiation of astrocytes and oligodendrocytes of mouse ES cells. Taken together, our data indicate that TPC2 signaling plays a temporal and differential role in modulating the neural lineage entry of mouse ES cells, in that TPC2 signaling inhibits ES cell entry to early neural progenitors, but is required for late neuronal differentiation.     

Formation and preservation of cortical layers in slice cultures

During cortical development, neurons generated at the same time in the ventricular zone migrate out into the cortical plate and form a cortical layer (Berry and Eayrs, 1963, Nature 197:984–985; Berry and Rogers, 1965, J. Anat. 99:691–709). We have been studying both the formation and maintenance of cortical layers in slice cultures from rat cortex. The bromodexyuridine (BrdU) method was used to label cortical neurons on their birthday in vivo. When slice cultures were prepared from animals at different embryonic and postnatal ages, all cortical layers that have already been established in vivo remained preserved for several weeks in vitro. In slice cultures prepared during migration in the cortex, cells contiuned to migrate towards the pial side of the cortical slice, however, migration ceased after about 1 week in culture. Thus, cortical cells reached their final laminar position only in slice cultures from postnatal animals, whereas in embryonic slices, migrating cells became scattered throughout the cortex. Previous studies demonstrated that radial glia fibers are the major substrate for migrating neurons (Rakic, 1972, J. Comp. Neurol. 145:61–84; Hatten and Mason, 1990, Experientia 46:907–916). Using antibodies directed against the intermediate filament Vimentin, radial glial cells were detected in all slice cutures where cell migration did occur. Comparable to the glia development in vivo, radial glial fibers disappeared and astrocytes containing the glia fibrillary-associated protein (GFAP) differentiated in slice cultures from postnatal cortex, after the neurons have completed their migration. In contrast, radial glial cells were detected over the whole culture period, and very few astrocytes differentiated in embryonic slices, where cortical neurons failed to finish their migration. The results of this study indicate that the local environment is sufficient to sustain the layered organization of the cortex and support the migration of cortical neurons. In addition, our results reveal a close relationship between cell migration and the developmental status of glial cells. © 1992 John Wiley & Sons, Inc.     

BMP‐4 inhibits neural differentiation of murine embryonic stem cells

Members of the transforming growth factor‐β superfamily, including bone morphogenetic protein 4 (BMP‐4), have been implicated as regulators of neuronal and glial differentiation. To test for a possible role of BMP‐4 in early mammalian neural specification, we examined its effect on neurogenesis in aggregate cultures of mouse embryonic stem (ES) cells. Compared to control aggregates, in which up to 20% of the cells acquired immunoreactivity for the neuron‐specific antibody TuJ1, aggregates maintained for 8 days in serum‐free medium containing BMP‐4 generated 5‐ to 10‐fold fewer neurons. The action of BMP‐4 was dose dependent and restricted to the fifth through eighth day in suspension. In addition to the reduction in neurons, we observed that ES cell cultures exposed to BMP‐4 contained fewer cells that were immunoreactive for glial fibrillary acidic protein or the HNK‐1 neural antigen. Furthermore, under phase contrast, cultures prepared from BMP‐4–treated aggregates contained a significant proportion of nonneuronal cells with a characteristic flat, elongated morphology. These cells were immunoreactive for antibodies to the intermediate filament protein vimentin; they were rare or absent in control cultures. Treatment with BMP‐4 enhanced the expression of the early mesodermal genes brachyury and tbx6 but had relatively little effect on total cell number or cell death. Coapplication of the BMP‐4 antagonist noggin counteracted the effect of exogenous BMP‐4, but noggin alone had no effect on neuralization in either the absence or presence of retinoids. Collectively, our results suggest that BMP‐4 can overcome the neuralizing action of retinoic acid to enhance mesodermal differentiation of murine ES cells. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 271–287, 1999     

Clonal analysis for elucidating the lineage potential of embryonic NG2+ cells

Background aimsThe widespread NG2-expressing neural progenitors in the central nervous system (CNS) are considered to be multifunctional cells with lineage plasticity, thereby possessing the potential for treating CNS diseases. Their lineages and functional characteristics have not been completely unraveled. The present study aimed to disclose the lineage potential of clonal NG2⁺ populations in vitro and in vivo.MethodsTwenty-four clones from embryonic cerebral cortex-derived NG2⁺ cells were induced for oligodendrocyte, astrocyte, neuronal and chondrocyte differentiation. The expression profiles of neural progenitor markers chondroitin sulfate proteoglycan 4 (NG2), platelet-derived growth factor-α receptor (PDGFαR); nestin and neuronal cell surface antigen (A2B5) were subsequently sorted on cells with distinct differentiation capacity. Transplantation of these NG2⁺ clones into the spinal cord was used to examine their lineage potential in vivo.ResultsIn vitro differentiation analysis revealed that all the clones could differentiate into oligodendrocytes, and seven of them were bipotent (oligodendrocytes and astrocytes). Amazingly, one clone exhibited a multipotent capacity of differentiating into not only neuronal–glial lineages but also chondrocytes. These distinct subtypes were further found to exhibit phenotypic heterogeneity based on the examination of a spectrum of neural progenitor markers. Transplanted clones survived, migrated extensively and differentiated into oligodendrocytes, astrocytes or even neurons to integrate with the host spinal cord environmentConclusionsThese results suggest that NG2⁺ cells contain heterogeneous progenitors with distinct differentiation capacities, and the immortalized clonal NG2⁺ cell lines might provide a cell source for treating spinal cord disorders.     

Retinoic acid directs neuronal differentiation of human pluripotent stem cell lines in a non-cell-autonomous manner

Differentiation of human embryonic stem (ES) cells and embryonal carcinoma (EC) cells provides an in vitro model to study the process of neuronal differentiation. Retinoic acid (RA) is frequently used to promote neural differentiation of pluripotent cells under a wide variety of culture conditions. Through systematic comparison of differentiation conditions we demonstrate that RA induced neuronal differentiation of human ES and EC cells requires prolonged RA exposure and intercellular communication mediated by high cell density. These parameters are necessary for the up-regulation of neural gene expression (SOX2, PAX6 and NeuroD1) and the eventual appearance of neurons. Forced over-expression of neither SOX2 nor NEUROD1 was sufficient to overcome the density dependency of neuronal differentiation. Furthermore, inhibition of GSK3β activity blocked the ability of RA to direct cell differentiation along the neural lineage, suggesting a role for appropriately regulated WNT signalling. These data indicate that RA mediated neuronal differentiation of human EC and ES cell lines is not a cell autonomous program but comprises of a multi-staged program that requires intercellular input.     

Immortalization of neural precursors when telomerase is overexpressed in embryonal carcinomas and stem cells

The DNA repair enzyme telomerase maintains chromosome stability by ensuring that telomeres regenerate each time the cell divides, protecting chromosome ends. During onset of neuroectodermal differentiation in P19 embryonal carcinoma (EC) cells three independent techniques (Southern blotting, Q-FISH, and Q-PCR) revealed a catastrophic reduction in telomere length in nestin-expressing neuronal precursors even though telomerase activity remained high. Overexpressing telomerase protein (mTERT) prevented telomere collapse and the neuroepithelial precursors produced continued to divide, but deaggregated and died. Addition of FGF-2 prevented deaggregation, protected the precursors from the apoptotic event that normally accompanies onset of terminal neuronal differentiation, allowed them to evade senescence, and enabled completion of morphological differentiation. Similarly, primary embryonic stem (ES) cells overexpressing mTERT also initiated neuroectodermal differentiation efficiently, acquiring markers of neuronal precursors and mature neurons. ES precursors are normally cultured with FGF-2, and overexpression of mTERT alone was sufficient to allow them to evade senescence. However, when FGF-2 was removed in order for differentiation to be completed most neural precursors underwent apoptosis indicating that in ES cells mTERT is not sufficient allow terminal differentiation of ES neural precursors in vitro. The results demonstrate that telomerase can potentiate the transition between pluripotent stem cell and committed neuron in both EC and ES cells.     

The double inhibition of endogenously produced BMP and Wnt factors synergistically triggers dorsal telencephalic differentiation of mouse ES cells

Embryonic stem (ES) cells are becoming a popular model of in vitro neurogenesis, as they display intrinsic capability to generate neural progenitors that undergo the known steps of in vivo neural development. These include the acquisition of distinct regional fates, which depend on growth factors and signals that are present in the culture medium. The control of the intracellular signaling that is active at different steps of ES cell neuralization, even when cells are cultured in chemically defined medium, is complicated by the endogenous production of growth factors. However, this endogenous production has been poorly investigated so far. To address this point, we performed a high‐throughput analysis of the expression of morphogens during mouse ES cell neuralization in minimal medium. We found that during their neuralization, ES cells increased the expression of members of Wnt, Fibroblast Growth Factor (FGF), and BMP families. Conversely, the expression of Activin/Nodal and Shh ligands was low in early steps of neuralization. In this experimental condition, neural progenitors and neurons generated by ES cells expressed a gene expression profile that was consistent with a midbrain identity. We found that endogenous BMP and Wnt signaling, but not FGF signaling, synergistically affected ES cell neural patterning, by turning off a profile of dorsal/telencephalic gene expression. Double BMP and Wnt inhibition allowed neuralized ES cells to sequentially activate key genes of cortical differentiation. Our findings are consistent with a novel synergistic effect of Wnt and BMP endogenous signaling of ES cells in inhibiting a cortical differentiation program. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 66–79, 2015     

Lineage of radial glia in the chicken optic tectum.

In many parts of the central nervous system, the elongated processes of radial glial cells are believed to guide immature neurons from the ventricular zone to their sites of differentiation. To study the clonal relationships of radial glia to other neural cell types, we used a recombinant retrovirus to label precursor cells in the chick optic tectum with a heritable marker, the E. coli lacZ gene. The progeny of the infected cells were detected at later stages of development with a histochemical stain for the lacZ gene product. Radial glia were identified in a substantial fraction of clones, and these were studied further. Our main results are the following. (a) Clones containing radial glia frequently contained neurons and/or astrocytes, but usually not other radial glia. Thus, radial glia derive from a multipotential progenitor rather than from a committed radial glial precursor. (b) Production of radial glia continues until at least embryonic day (E) 8, after the peak of neuronal birth is over (approximately E5) and after radial migration of immature neurons has begun (E6-7). Radial glial and neuronal lineages do not appear to diverge during this interval, and radial glia are among the last cells that their progenitors produce. (c) As they migrate, many cells are closely apposed to the apical process of their sibling radial glia. Thus, radial glia may frequently guide the migration of their clonal relatives. (d) The population of labelled radial glia declines between E15 and E19-20 (just before hatching), concurrent with a sharp increase in the number of labelled astrocytes. This result suggests that some tectal radial glia transform into astrocytes, as occurs in mammalian cerebral cortex, although others persist after hatching. To reconcile the observations that many radial glia are present early, that radial glia are among the last offspring of a multipotential stem cell, and that most clones contain only a single radial glial cell, we suggest that the stem cell is, or becomes, a radial glial cell.     

REST regulates the pool size of the different neural lineages by restricting the generation of neurons and oligodendrocytes from neural stem/progenitor cells

REST is a master repressor of neuronal genes; however, whether it has any role during nervous system development remains largely unknown. Here, we analyzed systematically the role of REST in embryonic stem cells and multipotent neural stem/progenitor (NS/P) cells, including neurogenic and gliogenic NS/P cells derived from embryonic stem (ES) cells or developing mouse embryos. We showed that REST-null ES cells remained pluripotent and generated teratomas consisting of the three germ layers. By contrast, multipotent NS/P cells lacking REST displayed significantly reduced self-renewal capacity owing to reduced cell cycle kinetics and precocious neuronal differentiation. Importantly, although early-born neurogenic NS/P cells that lack REST were capable of differentiating to neurons and glia, the neuronal and oligodendrocytic pools were significantly enlarged and the astrocytic pool was shrunken. However, gliogenic NS/P cells lacking REST were able to generate a normal astrocytic pool size, suggesting that the shrinkage of the astrocytic pool generated from neurogenic NS/P cells lacking REST probably occurs by default. Microarray profiling of early-born NS/P cells lacking REST showed upregulation of neuronal as well as oligodendrocytic genes, specifically those involved in myelination. Furthermore, chromatin immunoprecipitation analyses showed that some of the upregulated oligodendrocytic genes contain an RE1 motif and are direct REST targets. Together, our data support a central role for REST during neural development in promoting NS/P cell self-renewal while restricting the generation and maturation of neurons and oligodendrocytes.     

Embryonic stem cell-derived neural progenitors as non-tumorigenic source for dopaminergic neurons

AIM:To find a safe source for dopaminergic neurons,we generated neural progenitor cell lines from human embryonic stem cells.METHODS:The human embryonic stem(hES)cell line H9 was used to generate human neural progenitor(HNP)cell lines.The resulting HNP cell lines were differentiated into dopaminergic neurons and analyzed by quantitative real-time polymerase chain reaction and immunofluorescence for the expression of neuronal differentiation markers,including beta-III tubulin(TUJ1)and tyrosine hydroxylase(TH).To assess the risk of teratoma or other tumor formation,HNP cell lines and mouse neuronal progenitor(MNP)cell lines were injected subcutaneously into immunodeficient SCID/beige mice.RESULTS:We developed a fairly simple and fast protocol to obtain HNP cell lines from hES cells.These cell lines,which can be stored in liquid nitrogen for several years,have the potential to differentiate in vitro into dopaminergic neurons.Following day 30 of differentiation culture,the majority of the cells analyzed expressed the neuronal marker TUJ1 and a high proportion of these cells were positive for TH,indicating differentiation into dopaminergic neurons.In contrast to H9 ES cells,the HNP cell lines did not form tumors in immunodeficient SCID/beige mice within 6 mo after subcutaneous injection.Similarly,no tumors developed after injection of MNP cells.Notably,mouse ES cells or neuronal cells directly differentiated from mouse ES cells formed teratomas in more than 90%of the recipients.CONCLUSION:Our findings indicate that neural progenitor cell lines can differentiate into dopaminergic neurons and bear no risk of generating teratomas or other tumors in immunodeficient mice.     

Differentiation of monkey embryonic stem cells into neural lineages

Embryonic stem (ES) cells are self-renewing, pluripotent, and capable of differentiating into all of the cell types found in the adult body. Therefore, they have the potential to replace degenerated or damaged cells, including those in the central nervous system. For ES cell-based therapy to become a clinical reality, translational research involving nonhuman primates is essential. Here, we report monkey ES cell differentiation into embryoid bodies (EBs), neural progenitor cells (NPCs), and committed neural phenotypes. The ES cells were aggregated in hanging drops to form EBs. The EBs were then plated onto adhesive surfaces in a serum-free medium to form NPCs and expanded in serum-free medium containing fibroblast growth factor (FGF)-2 before neural differentiation was induced. Cells were characterized at each step by immunocytochemistry for the presence of specific markers. The majority of cells in complex/cystic EBs expressed antigens (alpha-fetal protein, cardiac troponin I, and vimentin) representative of all three embryonic germ layers. Greater than 70% of the expanded cell populations expressed antigenic markers (nestin and musashi1) for NPCs. After removal of FGF-2, approximately 70% of the NPCs differentiated into neuronal phenotypes expressing either microtubule-associated protein-2C (MAP2C) or neuronal nuclear antigen (NeuN), and approximately 28% differentiated into glial cell types expressing glial fibrillary acidic protein. Small populations of MAP2C/NeuN-positive cells also expressed tyrosine hydroxylase (approximately 4%) or choline acetyltransferase (approximately 13%). These results suggest that monkey ES cells spontaneously differentiate into cells of all three germ layers, can be induced and maintained as NPCs, and can be further differentiated into committed neural lineages, including putative neurons and glial cells.