The canonical Wnt cascade has emerged as a critical regulator of stem cells. In many tissues, activation of Wnt signalling has also been associated with cancer. This has raised the possibility that the tightly regulated self-renewal mediated by Wnt signalling in stem and progenitor cells is subverted in cancer cells to allow malignant proliferation. Insights gained from understanding how the Wnt pathway is integrally involved in both stem cell and cancer cell maintenance and growth in the intestinal, epidermal and haematopoietic systems may serve as a paradigm for understanding the dual nature of self-renewal signals.

Wnt signalling in stem cells and cancer

The complexity of the human brain has made it difficult to study many brain disorders in model organisms, highlighting the need for an in vitro model of human brain development Here we have developed a human pluripotent stem cell-derived three-dimensional organoid culture system, termed cerebral organoids, that develop various discrete, although interdependent, brain regions These include a cerebral cortex containing progenitor populations that organize and produce mature cortical neuron subtypes Furthermore, cerebral organoids are shown to recapitulate features of human cortical development, namely characteristic progenitor zone organization with abundant outer radial glial stem cells Finally, we use RNA interference and patient-specific induced pluripotent stem cells to model microcephaly, a disorder that has been difficult to recapitulate in mice We demonstrate premature neuronal differentiation in patient organoids, a defect that could help to explain the disease phenotype Together, these data show that three-dimensional organoids can recapitulate development and disease even in this most complex human tissue

/pdf/cerebral-organoids-model-human-brain-development-and-3wt90buxu3.pdf

Cerebral organoids model human brain development and microcephaly

Haematopoietic stem cells (HSCs) have the ability to renew themselves and to give rise to all lineages of the blood; however, the signals that regulate HSC self-renewal remain unclear. Here we show that the Wnt signalling pathway has an important role in this process. Overexpression of activated beta-catenin expands the pool of HSCs in long-term cultures by both phenotype and function. Furthermore, HSCs in their normal microenvironment activate a LEF-1/TCF reporter, which indicates that HCSs respond to Wnt signalling in vivo. To demonstrate the physiological significance of this pathway for HSC proliferation we show that the ectopic expression of axin or a frizzled ligand-binding domain, inhibitors of the Wnt signalling pathway, leads to inhibition of HSC growth in vitro and reduced reconstitution in vivo. Furthermore, activation of Wnt signalling in HSCs induces increased expression of HoxB4 and Notch1, genes previously implicated in self-renewal of HSCs. We conclude that the Wnt signalling pathway is critical for normal HSC homeostasis in vitro and in vivo, and provide insight into a potential molecular hierarchy of regulation of HSC development.

A role for Wnt signalling in self-renewal of haematopoietic stem cells

Human and mouse embryonic stem cells (HESCs and MESCs, respectively) self-renew indefinitely while maintaining the ability to generate all three germ-layer derivatives. Despite the importance of ESCs in developmental biology and their potential impact on tissue replacement therapy, the molecular mechanism underlying ESC self-renewal is poorly understood. Here we show that activation of the canonical Wnt pathway is sufficient to maintain self-renewal of both HESCs and MESCs. Although Stat-3 signaling is involved in MESC self-renewal, stimulation of this pathway does not support self-renewal of HESCs. Instead we find that Wnt pathway activation by 6-bromoindirubin-3'-oxime (BIO), a specific pharmacological inhibitor of glycogen synthase kinase-3 (GSK-3), maintains the undifferentiated phenotype in both types of ESCs and sustains expression of the pluripotent state-specific transcription factors Oct-3/4, Rex-1 and Nanog. Wnt signaling is endogenously activated in undifferentiated MESCs and is downregulated upon differentiation. In addition, BIO-mediated Wnt activation is functionally reversible, as withdrawal of the compound leads to normal multidifferentiation programs in both HESCs and MESCs. These results suggest that the use of GSK-3-specific inhibitors such as BIO may have practical applications in regenerative medicine.

Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor

Glial cells were long considered end products of neural differentiation, specialized supportive cells with an origin very different from that of neurons. New studies have shown that some glial cells—radial glia (RG) in development and specific subpopulations of astrocytes in adult mammals—function as primary progenitors or neural stem cells (NSCs). This is a fundamental departure from classical views separating neuronal and glial lineages early in development. Direct visualization of the behavior of NSCs and lineage-tracing studies reveal how neuronal lineages emerge. In development and in the adult brain, many neurons and glial cells are not the direct progeny of NSCs, but instead originate from transit amplifying, or intermediate, progenitor cells (IPCs). Within NSCs and IPCs, genetic programs unfold for generating the extraordinary diversity of cell types in the central nervous system. The timing in development and location of NSCs, a property tightly linked to their neuroepithelial origin, appear to be the key determinants of the types of neurons generated. Identification of NSCs and IPCs is critical to understand brain development and adult neurogenesis and to develop new strategies for brain repair.

The glial nature of embryonic and adult neural stem cells

Transgenic mice expressing a stabilized β-catenin in neural precursors develop enlarged brains with increased cerebral cortical surface area and folds resembling sulci and gyri of higher mammals. Brains from transgenic animals have enlarged lateral ventricles lined with neuroepithelial precursor cells, reflecting an expansion of the precursor population. Compared with wild-type precursors, a greater proportion of transgenic precursors reenter the cell cycle after mitosis. These results show that β-catenin can function in the decision of precursors to proliferate or differentiate during mammalian neuronal development and suggest that β-catenin can regulate cerebral cortical size by controlling the generation of neural precursor cells.

https://science.sciencemag.org/content/sci/297/5580/365.full.pdf

Regulation of Cerebral Cortical Size by Control of Cell Cycle Exit in Neural Precursors

Cleavage orientation and the asymmetric inheritance of notchl immunoreactivity in mammalian neurogenesis

beta-Catenin can function in the decision of neural precursors to proliferate or differentiate during mammalian neuronal development and may regulate cerebral cortical size by controlling the generation of neural precursor cells. Mice expressing high levels of a stabilized beta-catenin transgene in neural precursors develop enlarged brains with expanded precursor populations, increased cerebral cortical surface area, and folds resembling sulci and gyri of higher mammals present at birth. Here we report the effects in adult mice expressing lower levels of the same stabilized beta-catenin transgene in neural precursors. Adult transgenic animals develop enlarged forebrains with thin cerebral cortices with increased surface area, expanded subventricular zones with subcortical aggregations of neurons and enlarged, distorted hippocampi. The brains from transgenic mice also show apparent arrest of neuronal migration and dramatic disorganization of the layering of the cerebral cortex. These findings suggest that beta-catenin can cause expansion of the precursor pool resulting in increased neuronal production and greater brain size and suggest a crucial role for beta-catenin in neuronal migration and cortical lamination.

Increased Neuronal Production, Enlarged Forebrains and Cytoarchitectural Distortions in β-Catenin Overexpressing Transgenic Mice

We previously demonstrated that chemokine receptors are expressed by neural progenitors grown as cultured neurospheres. To examine the significance of these findings for neural progenitor function in vivo, we investigated whether chemokine receptors were expressed by cells having the characteristics of neural progenitors in neurogenic regions of the postnatal brain. Using in situ hybridization we demonstrated the expression of CCR1, CCR2, CCR5, CXCR3, and CXCR4 chemokine receptors by cells in the dentate gyrus (DG), subventricular zone of the lateral ventricle, and olfactory bulb. The pattern of expression for all of these receptors was similar, including regions where neural progenitors normally reside. In addition, we attempted to colocalize chemokine receptors with markers for neural progenitors. In order to do this we used nestin-EGFP and TLX-LacZ transgenic mice, as well as labeling for Ki67, a marker for dividing cells. In all three areas of the brain we demonstrated colocalization of chemokine receptors with these three markers in populations of cells. Expression of chemokine receptors by neural progenitors was further confirmed using CXCR4-EGFP BAC transgenic mice. Expression of CXCR4 in the DG included cells that expressed nestin and GFAP as well as cells that appeared to be immature granule neurons expressing PSA-NCAM, calretinin, and Prox-1. CXCR4-expressing cells in the DG were found in close proximity to immature granule neurons that expressed the chemokine SDF-1/CXCL12. Cells expressing CXCR4 frequently coexpressed CCR2 receptors. These data support the hypothesis that chemokine receptors are important in regulating the migration of progenitor cells in postnatal brain.

/pdf/chemokine-receptor-expression-by-neural-progenitor-cells-in-29b45rv9k2.pdf

Chemokine receptor expression by neural progenitor cells in neurogenic regions of mouse brain

http://web.stanford.edu/group/skmlab/resources/chenn.pdf

Anjen Chenn

Papers

Regulation of Cerebral Cortical Size by Control of Cell Cycle Exit in Neural Precursors

Cleavage orientation and the asymmetric inheritance of notchl immunoreactivity in mammalian neurogenesis

Increased Neuronal Production, Enlarged Forebrains and Cytoarchitectural Distortions in β-Catenin Overexpressing Transgenic Mice

Chemokine receptor expression by neural progenitor cells in neurogenic regions of mouse brain

Intrinsic Polarity of Mammalian Neuroepithelial Cells