Molecular and cellular regulation of motoneuron differentiation of human neural stem cells



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One main goal of stem cell biologists is to efficiently direct stem cells into particular cell fates useful for the treatment or study of neurological disease. Towards that goal, our laboratory has previously developed an in vitro procedure consisting of basic fibroblast growth factor (bFGF), heparin and laminin (FHL), which directs human fetal neural stem cells (hNSCs) to differentiate into choline acetyltransferase (ChAT) motoneurons in vitro and after transplantation into the rodent spinal cord in vivo. However, the mechanisms surrounding FHL-induced motoneuron differentiation of hNSCs are unknown. Thus, the overall goal for this thesis was to further understand the molecular and cellular basis underlying FHL-primed hNSC differentiation. As a first step, we focused upon the growth factor bFGF. We demonstrate here that application of exogenous bFGF induced hNSCs towards the motoneuron lineage in vitro. In particular, FHL-primed hNSCs expressed motoneuron developmental transcription factors including Olig2, Ngn2, Islet1 and Hb9.\r\nvi.\r\nWe also found that the motoneuron determinant, Hb9, was induced in hNSCs in bFGF-dose and time-dependent manner and that blockade of bFGF signaling reduced Hb9 expression. Furthermore, bFGF treatment did not significantly enhance cell survival and bFGF dependent proliferation was not required for Hb9 expression.\r\nBecause astrocytes are a known producer of bFGF and are also part of the post-transplantation environment, we further strove to understand the role of astrocytes and astrocytic-secreted factors (including astrocytic-secreted bFGF) upon hNSC differentiation and cell survival. In co-culture studies we found that astrocytes or astrocyte conditioned media (ACM) enhanced hNSC viability, decreased cell death and in particular, decreased apoptotic cell death. Interestingly, addition of astrocytes did not affect the overall neuron:glia ratio in long-term (LT) differentiated hNSCs. However, LT survival of a specific neuronal subtype, cholinergic neurons, was maintained in cultures with ACM but was lost in cultures differentiated without astrocytic factors. Addition of a FGF-receptor antagonist to ACM eliminated hNSC derived motoneurons from LT cultures suggesting that astrocyte secreted FGFs might modulate hNSC-derived cholinergic neuronal survival.\r\nTogether these studies demonstrate for the first time that bFGF is an important modulator of motoneuron differentiation and long-term in vitro maintenance of motoneurons differentiated from hNSCs.



transcription factor activation, Hb9, co-culture, cellular differentiation, basic fibroblast growth factor, astrocytes