Niche-Independent Symmetrical Self-Renewal of a Mammalian Tissue Stem Cell

Abstract

Pluripotent mouse embryonic stem (ES) cells multiply in simple monoculture by symmetrical divisions. In vivo, however, stem cells are generally thought to depend on specialised cellular microenvironments and to undergo predominantly asymmetric divisions. Ex vivo expansion of pure populations of tissue stem cells has proven elusive. Neural progenitor cells are propagated in combination with differentiating progeny in floating clusters called neurospheres. The proportion of stem cells in neurospheres is low, however, and they cannot be directly observed or interrogated. Here we demonstrate that the complex neurosphere environment is dispensable for stem cell maintenance, and that the combination of fibroblast growth factor 2 (FGF-2) and epidermal growth factor (EGF) is sufficient for derivation and continuous expansion by symmetrical division of pure cultures of neural stem (NS) cells. NS cells were derived first from mouse ES cells. Neural lineage induction was followed by growth factor addition in basal culture media. In the presence of only EGF and FGF-2, resulting NS cells proliferate continuously, are diploid, and clonogenic. After prolonged expansion, they remain able to differentiate efficiently into neurons and astrocytes in vitro and upon transplantation into the adult brain. Colonies generated from single NS cells all produce neurons upon growth factor withdrawal. NS cells uniformly express morphological, cell biological, and molecular features of radial glia, developmental precursors of neurons and glia. Consistent with this profile, adherent NS cell lines can readily be established from foetal mouse brain. Similar NS cells can be generated from human ES cells and human foetal brain. The extrinsic factors EGF plus FGF-2 are sufficient to sustain pure symmetrical self-renewing divisions of NS cells. The resultant cultures constitute the first known example of tissue-specific stem cells that can be propagated without accompanying differentiation. These homogenous cultures will enable delineation of molecular mechanisms that define a tissue-specific stem cell and allow direct comparison with pluripotent ES cells.

Retrograde interferon‐gamma signaling induces major histocompatibility class I expression in human‐induced pluripotent stem cell‐derived neurons

“…Recently, techniques have been established for reliably generating monolayers of induced pluripotent stem cell (iPSC)‐derived NSCs using supplementation with basic fibroblast growth factor (FGFb) and epidermal growth factor (EGF) 54, 55, 56. However, such cultured monolayer NSCs are more transcriptionally homogeneous than in vivo NSCs, and it has been speculated that this lack of heterogeneity is responsible, at least in part, for the challenges of promoting the differentiation of cultured NSCs into diverse neuronal subtypes 56, 57, 58, 59…”

Generation of a defined and uniform population of CNS progenitors and neurons from mouse embryonic stem cells

“…Recent transplantation studies with progenitors generated by RA treatment indicate that they are not fated to generate exclusively caudal cells and can effectively and meaningfully populate the brain when transplanted in either the prospective ventral or dorsal chick telencephalon (Nikoletopoulou and Barde, manuscript in preparation). Note that our procedure avoids the use of exogenous growth factors following CA dissociation, and while fibroblast growth factor and epidermal growth factor have been successfully used to grow ES cell-derived progenitors with the characteristics of radial glial cells 10 , these progenitors generate heterogeneous cell populations 10 . Trying to achieve homogeneous differentiation of neuronal types other than glutamatergic neurons, such as spinal cord motoneurons or dopaminergic neurons, is also an important objective for the future.…”

Conditional Activation of Bmi1 Expression Regulates Self-renewal, Apoptosis, and Differentiation of Neural Stem/Progenitor Cells In Vitro and In Vivo

“…To further investigate functional effect of increased Bmi1 expression in embryonic NSC and NPC, two in vitro culture systems were used, the neurosphere assay [21] and the adherent NSC culture system [22,23].…”