Int J Stem Cells.  2017 Nov;10(2):193-217. 10.15283/ijsc17036.

Proteomic Analysis of Human Adipose Derived Stem Cells during Small Molecule Chemical Stimulated Pre-neuronal Differentiation

• Santos J
• Milthorpe
• Herbert BR
• Padula MP

• KMID: 2400874
• DOI: http://doi.org/10.15283/ijsc17036

Abstract

BACKGROUND
Adipose derived stem cells (ADSCs) are acquired from abdominal liposuction yielding a thousand fold more stem cells per millilitre than those from bone marrow. A large research void exists as to whether ADSCs are capable of transdermal differentiation toward neuronal phenotypes. Previous studies have investigated the use of chemical cocktails with varying inconclusive results.
METHODS
Human ADSCs were treated with a chemical stimulant, beta-mercaptoethanol, to direct them toward a neuronal-like lineage within 24 hours. Quantitative proteomics using iTRAQ was then performed to ascertain protein abundance differences between ADSCs, beta-mercaptoethanol treated ADSCs and a glioblastoma cell line.
RESULTS
The soluble proteome of ADSCs differentiated for 12 hours and 24 hours was significantly different from basal ADSCs and control cells, expressing a number of remodeling, neuroprotective and neuroproliferative proteins. However toward the later time point presented stress and shock related proteins were observed to be up regulated with a large down regulation of structural proteins. Cytokine profiles support a large cellular remodeling shift as well indicating cellular distress.
CONCLUSION
The earlier time point indicates an initiation of differentiation. At the latter time point there is a vast loss of cell population during treatment. At 24 hours drastically decreased cytokine profiles and overexpression of stress proteins reveal that exposure to beta-mercaptoethanol beyond 24 hours may not be suitable for clinical application as our results indicate that the cells are in trauma whilst producing neuronal-like morphologies. The shorter treatment time is promising, indicating a reducing agent has fast acting potential to initiate neuronal differentiation of ADSCs.

Keyword

Adult Stem Cells; Adipose; Neuronal; Proteomics; Cytokines

MeSH Terms

Adult Stem Cells
Bone Marrow
Cell Line
Cytokines
Down-Regulation
Glioblastoma
Heat-Shock Proteins
Humans*
Lipectomy
Neurons
Phenotype
Proteome
Proteomics
Shock
Stem Cells*
Cytokines
Heat-Shock Proteins
Proteome

Figure

• 

  Fig. 1 Morphological comparisons between non-induced ADSCs and chemically induced ADSCs. (A) Basal human ADSCs cultured as a control prior to differentiation presents standard flat fibroblastic morphology. (B~D) hADSCs post induction 3 hours, 12 hours and 24 hours with neurodifferentiation media with the progressive structural remodelling over the 24 hour time period. 10×Magnification.

• 

  Fig. 2 Volcano plots. (A~C) showing p-values versus protein fold change (log2) of ADSCs and comparisons. Quantitation criteria cutoff of statistically significant p-values <0.05 and log2fold change cutoff of <−0.2 or >0.2. The blue nodes represent the above >0 log fold change i.e. up-regulated proteins and the below <0 fold change down-regulated proteins. The grey nodes represent the not significantly changed proteins with a p-value >0.05 and within the cut off for fold change. D shows the ratio of statistically changed proteins across all samples compared to basal ADSCs. Blue bar presents non-statistically significant changed proteins, red bar is the statistically significant up regulated proteins and green is the statistically significant down regulated proteins.

• 

  Fig. 3 Three-way Venn diagrams of up and down regulated proteins. Diagrams include the 12 hour differentiated, 24 hour differentiated hADSCs and the GBCs showing unique and shared proteins. (A) shows up regulated proteins revealing there are 29, 26, 357 unique proteins with 11, 32 and 25 shared proteins between each of the corresponding tested cell lines as well as 16 shared proteins between all three relative to basal hADSCs. (B) shows down regulated proteins revealing there are 36, 26, 364 unique proteins with 19, 27 and 50 shared proteins between each of the corresponding tested cell lines as well as 66 shared proteins between all three relative to basal hADSCs.

• 

  Fig. 4 (A) Western blot of BT3 positive in ADSCs, BME differentiated and GBCs seen at 55 kDa. (B) GFAP positively detected in GBCs at 48 kDa. (C) NF200 positively identified at 200 kDa in GBCs and very weakly in ADSC. (D) NeuN a very low positive in BME differentiated and GBCs. −VE is a negative control of a whole cell lysate of an unrelated cell line.

• 

  Fig. 5 Average total cell count at each time point over the BME treatment of ADSCs with mean error bars. Average cell count shows the total number of cells at each time point with drastic decreases in the final two points.

• 

  Fig. 6 Bioplex 27-plex cytokine array of ADSCs treated with BME over time. Bioplex comparisons of interleukins and cytokine secretions from basal ADSCs and temporal differentiation with BME neuronal differentiation media. Hierarchical clustering software and Euclidean test Red: expression above median; Green: expression below the median; Black: median expression across sample.

Reference

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