The Treasury of Wharton's Jelly

Abstract

Background: Postnatal umbilical cord tissue contains valuable mesenchymal progenitor cells of various differentiation stages. While mesenchymal stem cells are plastic-adherent and tend to differentiate into myofibroblastic phenotypes, some round cells detach, float above the adherent cells, and build up cell aggregates, or form spheroids spontaneously. Very small luminescent cells are always involved as single cells or within collective forms and resemble the common well-known very small embryonic-like cells (VSELs). In this study, we investigated these VSELs-like cells in terms of their pluripotency phenotype and tri-lineage differentiation potential.

Methods: VSELs-like cells were isolated from cell-culture supernatants by a process that combines filtering, up concentration, and centrifugation. To determine their pluripotency character, we measured the expression of Nanog, Sox-2, Oct-4, SSEA-1, CXCR4, SSEA-4 on gene and protein level. In addition, the cultured cells derived from UC tissue were examined regarding their potential to differentiate into three germ layers.

Result: The VSELs-like cells express all of the pluripotency-associated markers we investigated and are able to differentiate into meso- endo- and ectodermal precursor cells.

Conclusions: Umbilical cord tissue hosts highly potent VSELs-like stem cells.

Keywords: Multipotency; Pluripotency; Regenerative medicine; Trilineage differentiation.; Umbilical cord tissue; Very small embryonic like stem cells; Wharton's jelly.

Fig. 1

Morphology of UC tissue cells in relation to cultivation time. Cells were obtained by cell outgrowth from WJ tissue pieces and displayed of different morphologies as long, elongated shape, large flat, small triangular/stellate, small round or tiny round (a). Scale In passage three (round about 6 weeks after outgrown) the cells were less heterogeneous and mostly the flat or elongated and tiny round cells were represented (b). After long-term cultivation of more than 12 weeks, only large elongated cells remain, with many single, dubletts or aggregates of tiny round cells lining the surface (c, left) or tiny cell developed spheroids, swimming in the supernatatns of large cells. (c, right). Scale bar for all represents 100 μm with the exception of (c, left) which is 50 μm

Fig. 2

Flow cytometry. A typical example of FACS based expression analysis for transcription factors (upper histograms) and surface receptors (lower histograms) of VSELs. Each histogram compares given antibodies (given in black numbers) and specifc isotype controls (given in red numbers). Percentage of positive VSELs and the median fluorescence were assessed

Fig. 3

Pluripotency associated marker gene expression of enriched VSELs. Relatively normalized gene expression of KLF4, NANOF, POU5F1 and SOX2 in VSELs seperated from UC-MSCs supernatants (dark grey bars) were compared to according gene expression of Jurkat cell line cells (light grey bars) (a). Relative normalized gene expression of KLF4, NANOG and POU5F1 of UC-MSCs with (dark, grey bars) or without (light grey bars) in supernatant existing VSELs (b). Data were normalized by means of the housekeeping genes GAPDH and B2M

Fig. 4

Expression of pluripotency associated marker proteins examined by Immunofluorescence staining. VSELs were separated from UC-MSCs mass population, the tiny cells are between 5 and 7 μm and surrounded with dark spots (a). VSEL suspension cells were stained extracellular with anti-bodies against SSEA-4, CD184, and CD133 followed by nuclear staining of transcription factors Nanog, Oct-4, and Sox-2. Expression of each marker was detected. The nuclei were stained with HOECHST. Weak background Fluorescence signals of isotype controls were subtracted. The scale bars represented 50 μm (b). The magnification for SSEA-4/Sox-2 and CD133/Oct-4 was 20-fold and for CD184/Nanog 10-fold

Fig. 5

Evaluation of ecto-, endo-and mesodermal differentiation of UC-MSCs spheroids. To evaluate the differentiability into three different germ UC tissue was let grown out on chitosan coated surfaces until UC-MP cells performed spheroids from adhering cells. For multiplication spheroids with a diameter between 80–100 μm or 100–200 μm were seeded on collagen I coated surface until confluence was reached. Adherent cells were trypsinized and seeded onto matrigel coated surface for tri-lineage-differentiation. Phase contrast microscopy images demonstrated the morphology of cells after 1, 4 and 7 days. (a) The scale bars represents 50 μm. For confirmation of early differentiation into three germ layers, cells were with stained with appropriate ecto- endo- and mesodermal specific marker Pax6, Sox-2, and Sox-17, CD184 and CD140, CD144b, (b). The scale bars represent 100 μm