Molecular and Functional Verification of Wharton's Jelly Mesenchymal Stem Cells (WJ-MSCs) Pluripotency

Aleksandra Musiał-Wysocka, Marta Kot, Maciej Sułkowski, Bogna Badyra, Marcin Majka, Aleksandra Musiał-Wysocka, Marta Kot, Maciej Sułkowski, Bogna Badyra, Marcin Majka

Abstract

The properties of mesenchymal stem cells (MSCs), especially their self-renewal and ability to differentiate into different cell lines, are widely discussed. Considering the fact that MSCs isolated from perinatal tissues reveal higher differentiation capacity than most adult MSCs, we examined mesenchymal stem cells isolated from Wharton's jelly of umbilical cord (WJ-MSCs) in terms of pluripotency markers expression. Our studies showed that WJ-MSCs express some pluripotency markers-such as NANOG, OCT-4, and SSEA-4-but in comparison to iPS cells expression level is significantly lower. The level of expression can be raised under hypoxic conditions. Despite their high proliferation potential and ability to differentiate into different cells type, WJ-MSCs do not form tumors in vivo, the major caveat of iPS cells. Owing to their biological properties, high plasticity, proliferation capacity, and ease of isolation and culture, WJ-MSCs are turning out to be a promising tool of modern regenerative medicine.

Keywords: Nanog; OCT-4; SSEA-4; WJ-MSC; iPS; mesenchymal stem cells; pluripotency.

Conflict of interest statement

The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
(A–C)—The cultures of WJ-MSCs, fifth passage. The cells grow in a monolayer, show adherent properties and fibroblast-like morphology; Morphology of cells cultured in (A–C)—normoxia—magnification 100×, white bars correspond to 50 µm. (D)—Flow cytometry analysis of surface markers expression of WJ-MSCs. The WJ-MSCs were labelled with anti-CD3-FITC and anti-CD45-FITC antibodies (negative markers) and anti-CD73-PE, anti-CD90-PE, anti-CD105-PE antibodies (positive markers), and analyzed by flow cytometry. (D)—Representative histograms showing expression of set of markers for WJ-MSCs (detailed analysis presented in the table—(E). (F)—a heatmap showing the transcription profile of investigated WJ-MSCs. (G)—transcripts of selected genes expressed in the WJ-MSCs. The colors of the circles correspond to the colors on the heatmap (F): yellow—high expression; orange—medium expression; red—low expression; triangle—genes with different expression level.
Figure 2
Figure 2
Pluripotency markers expression on mRNA (A,B) and protein level (C,D) determined with RT-qPCR and flow cytometry (intracellular staining) respectively. (A,B)—Comparison of NANOG and OCT-4 relative expression level by RT-qPCR in WJ-MSCs and iPS. iPS exhibits relatively very high expression compared to WJ-MSCs. Note that hypoxia promotes NANOG expression in WJ-MSCs. Results are presented as means ± standard error, * p ≤ 0.0001 (A); * p ≤ 0.0006 (B). (C,D)—expression of NANOG and OCT-4 examined using intracellular staining and flow cytometry analysis. The iPS show a high expression of NANOG and OCT-4. In the case of WJ-MSCs the visible higher expression of NANOG was noticed in hypoxic conditions. H—hypoxia, N—normoxia. (E)—Expression level of pluripotency markers: SSEA-3, SSEA-4, TRA 1-60 (antibody labelling and flow cytometric analysis). Note that iPS exhibit high expression all of examined pluripotency markers whereas on WJ-MSCs only SSEA-4 is expressed. H—hypoxia; N—normoxia.
Figure 3
Figure 3
Immunofluorescence staining demonstrate distribution of pluripotency markers: NANOG, OCT-4 (transcription factors) and SSEA-3, SSEA-4, TRA 1-60 (surface markers) in iPS (A) and WJ-MSCs (B,C). (D,E)—expression of NANOG and OCT-4 in WJ-MSC Luc+ (the modified cell line expressing luciferase gene). Nuclear staining–Hoechst 33342: blue; NANOG and OCT-4 –Alexa Fluor 555: red; SSEA-4-FITC: green; TRA 1-60-PE, SSEA-3-PE: red. (A–E)—Florescence microscope, (A) magnification ×150, insets- magnification ×100; (B–E)—magnification ×100, insets—magnification ×80.
Figure 4
Figure 4
NOD-SCID mice injected subcutaneously with cells transduced with the vector transferring the luciferase gene (Luc): WJ-MSCs Luc+ (A–H) and iPS Luc+ (I–L). The modified WJ-MSCs were cultured in two oxygen conditions: WJ-MSCs Luc+ normoxia (A–D) and WJ-MSCs Luc+ hypoxia (E–H). The presence of injected cells was observed after luciferin administration by measuring the bioluminescence signal. After 30 days, in mice with normoxia, WJ-MSCs Luc+ signal was not detectable (C), whereas in mice with WJ-MSCs Luc+ hypoxia signal persisted. In mice with iPS Luc+ luminescence signal increased as a result of tumor development (G,H). 30 days after transplantation the tumors were isolated (M,N) and subsequently analyzed with hematoxylin and eosin staining (O). (O)—Light microscopy, magnification ×100, white bar corresponds to 150µm. Black asterisks—control mice without injected cells.

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