Yuanyuan Wu

Oligodendrocyte and astrocyte development in rodents: An in situ and immunohistological analysis during embryonic development†

Lineally related multipotent neuroepithelial cells (NEP), neuronal restricted precursors (NRP), and glial restricted precursors (GRP) have been identified in the spinal cord. To determine the sequence of differentiation and identify lineage and stage‐specific markers, we have examined the spatiotemporal expression of established glial markers during rodent embryonic development and within fetal cell culture. In this report, we show that proliferating stem cells in the developing neural tube do not express any glial markers at E10.5. By E11, however, glial precursors have begun to differentiate and at least two regions of the ventral neural tube containing glial precursor cells can be distinguished, an Nkx2.2/Neurogenin 3 (Ngn3) domain and a platelet‐derived growth factor receptor alpha (PDGFRα)/Olig2/Sox10 domain. Radial glia, as identified by RC1 immunoreactivity, develop in concert with other glial precursors and can be distinguished by their morphology, spatial distribution, and antigen expression. Astrocytes as assessed by glial fibrillary acidic protein (GFAP) immunoreactivity are first detected at E16. A novel dorsal domain of CD44 immunoreactivity that can be distinguished from the more ventral glial precursor domains can be detected as early as E13.5. GLIA 40:25–43, 2002. Published 2002 Wiley‐Liss, Inc.

Motoneurons and oligodendrocytes are sequentially generated from neural stem cells but do not appear to share common lineage-restricted progenitors in vivo.

Olig gene expression is proposed to mark the common progenitors of motoneurons and oligodendrocytes. In an attempt to further dissect the in vivo lineage relationships between motoneurons and oligodendrocytes, we used a conditional cell-ablation approach to kill Olig-expressing cells. Although differentiated motoneurons and oligodendrocytes were eliminated, our ablation study revealed a continuous generation and subsequent death of their precursors. Most remarkably, a normal number of oligodendrocyte precursors are formed at day 12 of mouse development, after all motoneuron precursors have been killed. The data presented herein supports a sequential model in which motoneuron and oligodendrocyte precursors are sequentially generated in vivo from neuroepithelial stem cells, but do not share a common lineage-restricted progenitor.

Hes1 but not Hes5 regulates an astrocyte versus oligodendrocyte fate choice in glial restricted precursors

To determine the role of Hes genes in the differentiation process of neuroepithelial (NEP) cells to glial restricted precursor cells (GRPs) and subsequently GRPs to oligodendrocytes and astrocytes, we have examined the effects of Hes1 and Hes5 on glial differentiation. We find that both Hes1 and Hes5 are expressed by GRPs and that Hes1 can drive GRPs to an astrocyte cell fate at the expense of oligodendrocyte differentiation. Overexpression of Hes1 in GRPs results in the up‐regulation of the astrocyte markers glial fibrillary acidic protein and CD44 and the down‐regulation of oligodendrocyte markers myelin proteolipid protein/DM20, GalC, and CNPase. Transcription factors involved in oligodendrocyte differentiation, such as Nkx2.2, Olig1, and Mash1, are also down‐regulated in Hes1‐overexpressing cells. The effect of Hes1 on gliogenesis is stage‐specific as Hes1 does not direct NEP cells to an astrocytic fate. In contrast to Hes1, Hes5 does not promote astrocyte differentiation. Instead, it inhibits both astrocyte and oligodendrocyte differentiation. Overexpression of Notch1 has an effect on gliogenesis similar to that of Hes1 and the mRNA levels of Hes1 are up‐regulated in cells overexpressing Notch1, suggesting that Notch1 could be an upstream activator of Hes1. Developmental Dynamics 675–689, 2003.

Hoxc10 and Hoxd10 regulate mouse columnar, divisional and motor pool identity of lumbar motoneurons

A central question in neural development is how the broad diversity of neurons is generated in the vertebrate CNS. We have investigated the function of Hoxc10 and Hoxd10 in mouse lumbar motoneuron development. We show that Hoxc10 and Hoxd10 are initially expressed in most newly generated lumbar motoneurons, but subsequently become restricted to the lateral division of the lateral motor column (lLMC). Disruption of Hoxc10 and Hoxd10 caused severe hindlimb locomotor defects. Motoneurons in rostral lumbar segments were found to adopt the phenotype of thoracic motoneurons. More caudally the lLMC and dorsal-projecting axons were missing, yet most hindlimb muscles were innervated. The loss of the lLMC was not due to decreased production of motoneuron precursors or increased apoptosis. Instead, presumptive lLMC neurons failed to migrate to their normal position, and did not differentiate into other motoneurons or interneurons. Together, these results show that Hoxc10 and Hoxd10 play key roles in establishing lumbar motoneuron columnar, divisional and motor pool identity.

piggyBac mediates efficient in vivo CRISPR library screening for tumorigenesis in mice

Significance Because genome-wide CRISPR/Cas9 libraries are mostly constructed in lentiviral vectors, direct in vivo screening has not been possible as a result of low efficiency in delivery. Here, we examined the piggyBac (PB) transposon as an alternative vehicle to deliver a guide RNA (gRNA) library for in vivo screening. Through hydrodynamic tail vein injections, we delivered a PB-CRISPR library into mouse liver. Rapid tumor formation could be observed in less than 2 mo. By sequencing analysis of PB-mediated gRNA insertions, we identified corresponding genes mediating tumorigenesis. Our results demonstrate that PB is a simple and nonviral choice for efficient in vivo delivery of CRISPR libraries for phenotype-driven screens. CRISPR/Cas9 is becoming an increasingly important tool to functionally annotate genomes. However, because genome-wide CRISPR libraries are mostly constructed in lentiviral vectors, in vivo applications are severely limited as a result of difficulties in delivery. Here, we examined the piggyBac (PB) transposon as an alternative vehicle to deliver a guide RNA (gRNA) library for in vivo screening. Although tumor induction has previously been achieved in mice by targeting cancer genes with the CRISPR/Cas9 system, in vivo genome-scale screening has not been reported. With our PB-CRISPR libraries, we conducted an in vivo genome-wide screen in mice and identified genes mediating liver tumorigenesis, including known and unknown tumor suppressor genes (TSGs). Our results demonstrate that PB can be a simple and nonviral choice for efficient in vivo delivery of CRISPR libraries.

Isolation of Neural Stem and Precursor Cells from Rodent Tissue

Isolation and characterization of neural stem cells and lineage-specific progenitors provide important information for central nervous system development study and regenerative medicine. We describe methods for dissection of rodent embryonic spinal cords by enzymatic separation, and isolation and enrichment (or purification) of neuronal and glial precursors at different developing stages by fluorescence-activated cell sorting.

Human neural precursor cells – an in vitro characterization

Abstract We have compared the properties of human neural precursors isolated from fetal tissue with progenitor and precursor cells identified from rodent fetal tissue and human excretory/secretory (ES) cells. We have identified multipotent human neuroepithelial precursor cells (hNEPs) that are fibroblast growth factor dependent, grow in adherent culture, and differentiate into neurons, astrocytes, and oligodendrocytes in mass and clonal cultures. A subset of these multipotent cells express an antigen recognized by the AC133/2 antibody. hNEPs appear similar to rodent-derived NEP cells, and unlike other human multipotent precursor cell populations, do not require Leukemia Inhibitory Factor (LIF) or Epithelial Growth Factor (EGF) for their survival. hNEPs constitute a small fraction of the cells present at any stage examined and three additional dividing populations can be identified based on expression of epitopes recognized by E-NCAM, A2B5 and CD44. E-NCAM+ cells co-express neuronal markers and can differentiate into multiple classes of neurons. Two types of A2B5+ cells can be distinguished: a small neuronal population that co-expresses E-NCAM immunoreactivity and a larger glial population that is E-NCAM negative. CD44+ cells do not express neuronal markers or oligodendrocytic markers but co-express astrocytic markers and likely represent an astrocyte precursor cell. Dividing E-NCAM+, A2B5+ and CD44+ cells can be identified in differentiating human ES cell cultures and the properties of these cells appear similar to cells present in fetal tissue.

Barriers for Deriving Transgene‐Free Pig iPS Cells with Episomal Vectors

To date no authentic embryonic stem cell (ESC) line or germline‐competent‐induced pluripotent stem cell (iPSC) line has been established for large animals. Despite this fact, there is an impression in the field that large animal ESCs or iPSCs are as good as mouse counterparts. Clarification of this issue is important for a healthy advancement of the stem cell field. Elucidation of the causes of this failure in obtaining high quality iPSCs/ESCs may offer essential clues for eventual establishment of authentic ESCs for large animals including humans. To this end, we first generated porcine iPSCs using nonintegrating replicating episomal plasmids. Although these porcine iPSCs met most pluripotency criteria, they could neither generate cloned piglets through nuclear transfer, nor contribute to later stage chimeras through morula injections or aggregations. We found that the reprogramming genes in iPSCs could not be removed even under negative selection, indicating they are required to maintain self‐renewal. The persistent expression of these genes in porcine iPSCs in turn caused differentiation defects in vivo. Therefore, incomplete reprogramming manifested by a reliance on sustained expression of exogenous‐reprogramming factors appears to be the main reason for the inability of porcine iPSCs to form iPSC‐derived piglets. Stem Cells 2015;33:3228–3238

Efficient germ-line transmission obtained with transgene-free induced pluripotent stem cells

Significance Using a single, nonintegrating episome, containing an optimized assembly of reprogramming factors and positive/negative selection markers, we generated germ-line–competent induced pluripotent stem (iPS) cells. To ensure that the iPS cells were transgene-free (i.e., were independent of exogenous reprogramming factors to achieve and maintain their pluripotent ground state) required the inclusion on the episome more that the classical four (POU5F1/OCT4, KLF4, SOX2, and cMYC) reprogramming factors. Also critical for the transgene-free iPS cells exhibiting competency for germ-line transmission was the requirement for growth in 2i medium. Induced pluripotent stem (iPS) cells hold great promise for regenerative medicine. To overcome potential problems associated with transgene insertions, efforts have been directed over the past several years to generate transgene-free iPS cells by using non-viral-vector approaches. To date, however, cells generated through such procedures have had problems producing reproductively competent animals, suggesting that their quality needed further improvement. Here we report the use of optimized assemblies of reprogramming factors and selection markers incorporated into single plasmids as nonintegrating episomes to generate germ-line–competent iPS cells. In particular, the pMaster12 episome can produce transgene-free iPS cells that, when grown in 2i medium, recapitulate good mouse ES cells, in terms of their competency for generating germ-line chimeras.

Fine-Tuning of iPSC Derivation by an Inducible Reprogramming System at the Protein Level

Summary Induced pluripotent stem cells (iPSCs) generated from somatic cells by ectopic expression of reprogramming factors, e.g., POU5F1 (OCT4), KLF4, and SOX2, have great potential for regenerative medicine. However, before they can be used in a clinical setting, the mechanism of reprogramming needs to be better understood. Here, by engineering reprogramming factors to a destabilizing protein domain, we achieved inducible generation of mouse and pig iPSCs. Stability of the fusion protein was precisely regulated by the addition of the cell-permeable small molecule trimethoprim (TMP) in a dose-dependent manner. With these tools, we found that during the early and middle stages of reprogramming, exogenous OCT4 or KLF4 could be omitted, whereas exogenous SOX2 expression at early and middle stages was required for successful reprogramming. Our TMP reprogramming system is useful for defining the stoichiometry and temporal requirements of transcription factors for reprogramming.