Proteome analysis of human Wharton's jelly cells during in vitro expansion

Abstract

Background: The human umbilical cord contains mucoid connective tissue and fibroblast-like cells. These cells named Wharton's jelly cells, (WJCs) display properties similar to mesenchymal stem cells therefore representing a rich source of primitive cells to be potentially used in regenerative medicine.

Results: To better understand their self-renewal and potential in vitro expansion capacity, a reference 2D map was constructed as a proteomic data set. 158 unique proteins were identified. More than 30% of these proteins belong to cytoskeleton compartment. We also found that several proteins including Shootin1, Adenylate kinase 5 isoenzyme and Plasminogen activator-inhibitor 2 are no longer expressed after the 2nd passage of in vitro replication. This indicates that the proliferative potency of these cells is reduced after the initial stage of in vitro growing. At the end of cellular culturing, new synthesized proteins, including, ERO1-like protein alpha, Aspartyl-tRNA synthetase and Prolyl-4-hydroxylase were identified. It is suggested that these new synthesized proteins are involved in the impairment of cellular surviving during replication and differentiation time.

Conclusions: Our work represents an essential step towards gaining knowledge of the molecular properties of WJCs so as to better understand their possible use in the field of cell therapy and regenerative medicine.

Figure 1

Umbilical Cord Compartment. Digital photo of human umbilical cord tissue. Traverse section through an umbilical cord after birth. Scale bar = 2 mm. Wharton's Jelly is the connective tissue included between the subamnion and the perivascular regions.

Figure 2

WJCs characterization. A. Light microscopic micrographs of WJCs in monolayer culture. In monolayer culture, the cells assumed a polymorphic, fibrobroblast-like morphology, which was maintained throughout the passaging processes. 10 days (left panel), 15 days of culture (central panel) and cells at passage 1, prior to reach confluence (right panel). Original magnification ×100. B. Analyses of surface antigen marker by flow cytometry. Cell suspensions were stained with specific mouse anti-human monoclonal antibodies (Mabs) as indicated in filled histograms. The empty histogram is the respective IgG isotype control. C. Ability to differentiate into several lineages. Osteogenic differentiation (left panel) was indicated by the increase in alkaline phosphatase (magnification 200×). Adipogenic differentiation (central panel) is visually marked by accumulation of neutral lipid vacuoles in cultures (red oil staining) (magnification 200×). Chondrogenic differentiation (right panel) is visually marked by toluidine blue staining (magnification 100×). D. Flow cytometry of cells showing DNA stained with propidium iodide. G1 and G2/M indicate 2n and 4n cellular DNA content, respectively. S indicates cells undergoing DNA synthesis, intermediate in DNA content between 2n and 4n. In the phase contrast image, arrow indicates one mitotic event. E. Telomere length assay on WJCs at different culture passages. The maintenance of long telomeres is a key feature of stem cells, ensuring the capability to undergo several cell cycles of replication. Molecular weight marker (M), control DNA with short telomeres (S) and control DNA with long telomeres (L). p4, p8 and p12 DNA extracts from WJCs at different 4^th 8^th and 12^th passages showing long telomeric ends. F, G. Growth Features of WJCs during expansion period. Proliferation (F) and doubling time (G) at different passages of WJCs in vitro culture calculated by Trypan blue exclusion test.

Figure 3

WJCs 2D map. Silver-stained 2D gels (pH4-7, 9-16% acrylamide, 200 μg protein loading) from the 2^nd, 4^th, 8^th and 12^th in vitro expansion passages. The synthetic gel is the result of the comparative analysis between the master gels obtained from 3 gels for each culture passage.

Figure 4

WJCs shyntetic gel (A) and the functional grouping of identified proteins (B). In the Figure 4B values represent the percent distribution of proteins classified by biological process of gene ontology terms into functional categories.

Figure 5

Panel A and B indicate proteins no longer expressed after the 2^nd passage. Protein spots detected by image analysis (A) are indicated with circles, whereas the proteins assigned by PMF analysis are annotated with the abbreviation name; the magnified views and peptide mass spectrum of spots 2, 22, 34, 35, 36, 61, 67 and 75 are indicated (B). Panel C and D indicate proteins spots expressed at the 12^th passage. All detected spots are indicated with circles, whereas the identified protein spots are designed with the abbreviation name (C); the magnified views and peptide mass spectrum of spots 5, 16, 17,31 and 43 are indicated (D). Panel E and F indicate immunoblot analysis of the WJCs protein only expressed at the 2^nd and at the 12^th passages. 1-D gels probed with antibodies raised against KAD5, EIF31, PAI2, SHOT1, ERO1, PHA1 AND PHA2 (E). 2D blot analysis raised against ACTB and VIME at the 2^nd and 12^th passages (F).