Temporal analysis of neural differentiation using quantitative proteomics

Abstract

The ability to derive neural progenitors, differentiated neurons and glial cells from human embryonic stem cells (hESCs) with high efficiency holds promise for a number of clinical applications. However, investigating the temporal events is crucial for defining the underlying mechanisms that drive this process of differentiation along different lineages. We carried out quantitative proteomic profiling using a multiplexed approach capable of analyzing eight different samples simultaneously to monitor the temporal dynamics of protein abundance as human embryonic stem cells differentiate into motor neurons or astrocytes. With this approach, a catalog of approximately 1200 proteins along with their relative quantitative expression patterns was generated. The differential expression of the large majority of these proteins has not previously been reported or studied in the context of neural differentiation. As expected, two of the widely used markers of pluripotency, alkaline phosphatase (ALPL) and LIN28, were found to be downregulated during differentiation, while S-100 and tenascin C were upregulated in astrocytes. Neurofilament 3 protein, doublecortin and CAM kinase-like 1 and nestin proteins were upregulated during motor neuron differentiation. We identified a number of proteins whose expression was largely confined to specific cell types, embryonic stem cells, embryoid bodies and differentiating motor neurons. For example, glycogen phosphorylase (PYGL) and fatty acid binding protein 5 (FABP5) were enriched in ESCs, while beta spectrin (SPTBN5) was highly expressed in embryoid bodies. Karyopherin, heat shock 27 kDa protein 1 and cellular retinoic acid binding protein 2 (CRABP2) were upregulated in differentiating motor neurons but were downregulated in mature motor neurons. We validated some of the novel markers of the differentiation process using immunoblotting and immunocytochemical labeling. To our knowledge, this is the first large-scale temporal proteomic profiling of human stem cell differentiation into neural cell types highlighting proteins with limited or undefined roles in neural fate.

Figure 1. Experimental strategy for quantitative proteomics analysis

A. Outline of the protocol for differentiating hES cells along neural lineages. The cells were grown in N2B27 media on tissue culture plastic coated with matrigel. B. A schematic of the quantitative proteomic analysis by labeling with 8-plex iTRAQ reagents followed by strong cation exchange (SCX) based fractionation and liquid chromatography tandem mass spectrometry (LC-MS/MS)

Figure 2. Immunocytochemical characterization of the differentiation process

Indirect immunofluorescence labeling of different cell types was carried out using Alexa Fluor 594 or Alexa Fluor 488 conjugated secondary antibodies. A. Oct4 expression in undifferentiated hESC colony. B. Nestin positive staining of a cryosection through 10-day differentiating embryoid body (EB) cultured under neural inducing culture conditions. C. Neural rosette formation of nestin-positive cells cultured in monolayer from EBs shown in B. D. Neuronal marker staining of tubulin beta 3 (TUJ1) in 2-wk derived motor neuron cultures and E. HB9 expression in postmitotic motor neurons derived from 3-wk culture conditions favoring neuronal derivation. F. GFAP staining of astrocytes.

Figure 3. MS/MS spectra of iTRAQ labeled peptides

MS/MS spectra of peptide from representative differentially expressed proteins identified in this study. A. Karyopherin alpha 4 (KPNA4) B. Heat shock protein binding 1 (HSPB1) C. A kinase (PRKA) anchor protein (gravin) 12 (AKAP12); and, D. Neurofilament-3. The inset shows the reporter ions obtained during MS/MS that were used for quantitation.

Figure 4. Temporal patterns of differentially expressed proteins

Proteins were selected based on the temporal patterns of expression during differentiation. A. Proteins that show a high expression in early stages of motor neuron differentiation, B. Proteins showing progressive increase during motor neuron differentiation, C. Proteins showing progressive decrease during motor neuron differentiation. The fold-changes from the iTRAQ experiments are indicated in the corresponding tables.

Figure 5. Proteins showing high expression in specific cell types

Sets of proteins showing high expression levels in embryoid bodies (EBs) (A), astrocytes (B) or embryonic stem cells (ES) (C) are shown A. The fold-changes from the iTRAQ experiments are indicated in the corresponding tables.

Figure 6. Western blot validation

Cell lysates from different stages of differentiation as indicated were tested for expression of a select set of molecules against which antibodies were commercially available.

Figure 7. Immunocytochemical analysis of differentially expressed proteins

Panel A shows an increase in expression of five proteins in hESC-derived motor neurons as compared to neural progenitors. Panel B shows a decrease in expression of three proteins in hESC-derived motor neurons compared to neural progenitors. The nuclei in each case are visualized using DAPI (blue).