Influence of the extracellular matrix on the in vitro differentiation of mouse embryonic stem cells into neurons.
thesisposted on 23.02.2017, 04:13 by Gulati, Pankaj
Stem cell maintenance and differentiation are regulated by local inductive cues; one potential source of inductive cues is the extracellular matrix. The fundamental hypothesis of the thesis was that differentiation of embryonic stem cells can be manipulated by substrates onto which cells are plated thus, our first experimental chapter explores the impact of the extracellular matrix proteins, laminin, fibronectin and type IV collagen on neural induction of E14Tg2a mouse embryonic stem cells (mESCs) plated as adherent monolayers. In-cell Western assays were carried out to determine coating efficiency of selected matrix proteins. Cell viability and proliferation was estimated using the MTT assay. In addition, the effect of matrix proteins on neurite outgrowth of mESCs-derived neurons was evaluated by counting the number of primary neurites (using βIII tubulin labelling) and neural progenitor (nestin). Total neurite length per neuron was quantified on neurons derived from the differentiated mESCs. Immunocytochemistry against tyrosine hydroxylase and βIII tubulin was used to observe the progression of neuronal maturation of cells plated onto matrix proteins. The MTT assay was used to determine the effect of each matrix on adhesion of mESCs, 24 hours after replating. The results showed that laminin and fibronectin were associated with increase in cell adhesion and they also enhanced the proliferation rate of mESCs when compared to type IV collagen. Laminin significantly increased the number of βIII tubulin positive cells 96 hours after re-plating, but failed to result in a larger numbers of positive catecholaminergic neurons. Second experimental chapter focused on effect of the immobilization of growth factors [sonic hedgehog (Shh), fibroblast growth factor 8b (FGF8) and fibroblast growth factor basic (FGF2)] on the generation of midbrain dopaminergic neurons from mESCs. A combination of matrix proteins components was produced by incubating laminin, fibronectin, type IV collagen and heparan sulphate in a ratio of 1:1:1:0.1. This mixture was used to immobilize growth factors within matrix proteins via non-covalent interactions. In-cell Western assays were carried out to identify stable immobilization of growth factors onto the matrix proteins. The effect of matrix proteins with and without growth factors on neural differentiation was investigated by determining the expression of the AMPR gene (encoding beta-lactamase) in Lmx1a-AMP mESCs. Immunocytochemistry was used to observe the progression of neuronal maturation under different conditions. FGF2 and FGF8b were successfully immobilized on a combination of selected matrix proteins but the immobilized factors failed to demonstrate any significant effect on the proportion of cells expressing Lmx1a. This is likely to be explained by the unexpected high proportion of Lmx1a expressing cells produced even in the absence of specific growth factors. In the last experimental chapter the focus was on investigation of the signaling pathways that regulate differentiations. New tools to promote the differentiation of stem cells into particular cell types can be generated by identifying specific cues in the microenvironments and deciphering how neighbouring cells and the extracellular matrix control developmental fate. It is well established that complex interactions between soluble and extracellular matrix molecules regulate intracellular signaling and differentiation. Many of these signaling events involve one or more kinases including; focal adhesion kinase (FAK), phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB/Akt, hereafter called Akt), and mitogen-activated protein kinases (MAPK) – such as those also known as Extracellular regulated kinases (ERKs). However, the relevance of a given signaling molecule in mediating the pro-survival signaling induced by extracellular matrix appears to be cell–type specific. Therefore, it is desirable to determine which of these intracellular signaling pathways might be involved in the effects of extracellular matrix on survival and differentiation of mouse embryonic stem cells. This chapter explores the role of small molecule inhibitors of FAK, PI3K, Akt and MAPK-ERK on adhesion and proliferation of cells produced by neural differentiation of embryonic stem cells. The focus of the chapter is on early events in neural induction making use of Sox1-eGFP reporter ESCs. This cell line expresses the eGFP reporter under control of the Sox1 promoter, one allele expressing eGFP and the other Sox1. The work showed that plating onto laminin (but not gelatin or poly-D-lysine) activated PI3K-Akt and MAPK-ERK survival signaling pathways. Plating of cells on laminin in the presence of CHIR99021, or in combination with PF562271, resulted in significant conversion of viable embryonic stem cells to Sox1+ neural stem/progenitor cells, indicating the role of laminin in the generation and survival of neurons.