Wharton's Jelly Mesenchymal Stromal Cells Support the Expansion of Cord Blood-derived CD34+ Cells Mimicking a Hematopoietic Niche in a Direct Cell-cell Contact Culture System

Abstract

Wharton's jelly mesenchymal stromal cells (WJ-MSCs) have been recently exploited as a feeder layer in coculture systems to expand umbilical cord blood-hematopoietic stem/progenitor cells (UCB-HSPCs). Here, we investigated the role of WJ-MSCs in supporting ex vivo UCB-HSPC expansion either when cultured in direct contact (DC) with WJ-MSCs or separated by a transwell system or in the presence of WJ-MSC-conditioned medium. We found, in short-term culture, a greater degree of expansion of UCB-CD34⁺ cells in a DC system (15.7 ± 4.1-fold increase) with respect to the other conditions. Moreover, in DC, we evidenced two different CD34⁺ cell populations (one floating and one adherent to WJ-MSCs) with different phenotypic and functional characteristics. Both multipotent CD34⁺/CD38^- and lineage-committed CD34⁺/CD38⁺ hematopoietic progenitors were expanded in a DC system. The former were significantly more represented in the adherent cell fraction than in the floating one (18.7 ± 11.2% vs. 9.7 ± 7.9% over the total CD34⁺ cells). Short-term colony forming unit (CFU) assays showed that HSPCs adherent to the stromal layer were able to generate a higher frequency of immature colonies (CFU-granulocyte/macrophage and burst-forming unit erythroid/large colonies) with respect to the floating cells. In the attempt to identify molecules that may play a role in supporting the observed ex vivo HSPC growth, we performed secretome analyses. We found a number of proteins involved in the HSPC homing, self-renewal, and differentiation in all tested conditions. It is important to note that a set of sixteen proteins, which are only in part reported to be expressed in any hematopoietic niche, were exclusively found in the DC system secretome. In conclusion, WJ-MSCs allowed a significant ex vivo expansion of multipotent as well as committed HSPCs. This may be relevant for future clinical applications.

Keywords: Wharton’s jelly mesenchymal stromal cells; extracellular matrix; hematopoietic niche; hematopoietic stem and progenitor cell expansion; secretome.

Fig. 1.

The fold increase of expanded umbilical cord blood–CD34⁺ cells after 5 d of culture in different conditions: direct contact (DC) system, Wharton’s jelly mesenchymal stromal cells–conditioned medium (WJ-CM), expansion medium (EM), and transwell system (TS). This graph also reports the frequency of CD34⁺ cells after 5 d of culture in all conditions. All results are presented as mean ± SD.

Fig. 2.

Microscopical demonstration of the multiple ways of interactions between Wharton’s jelly mesenchymal stromal cells (WJ-MSCs) and CD34⁺ cells in the direct contact system. CD34⁺ cells seeded on a layer of WJ-MSCs and cultured for 5 d expand their numbers and establish different interactions with feeder cells (A–D). Phase-contrast microscopy allows for detection of a floating population that constitutes about two-thirds of the total expanded cells (indicated by white arrows in all panels). An adherent population is represented by cells which adhere to the feeder cells or to the culture surface (indicated by black arrows in all panels). Magnification: 200×. Bar: 100 µm.

Fig. 3.

Microscopic demonstration of the expression of intracellular and extracellular molecules in the direct contact (DC) system. CD34⁺ cells belonging to the adherent fraction can be easily visualized in areas around Wharton’s jelly mesenchymal stromal cells (WJ-MSCs) as shown by their staining for anti-CD34 antibody (A). (B, C) Vimentin stains both WJ-MSCs and hematopoietic stem/progenitor cells (HSPCs). Higher magnification images (400×, C) show the close association between the WJ-MSC feeder layer and HSPCs. Apart from interacting with WJ-MSCs, HSPCs clearly show arrangements directed by the extracellular fibronectin polymerized by WJ-MSCs. (D) Fibronectin antibody shows cytoplasmic stain in WJ-MSCs but not in HSPCs. The protein is polymerized in an extracellular network with fibers localized both at the periphery of cells and on the culture surface. As indicated, HSPCs show pseudopodia-like protrusions (arrows) indicating a migratory activity over the vessel surface, which is covered with fibronectin filaments. The inset shows a higher magnification view of the cellular protrusions. (E) Extracellular fibronectin directs the adhesion of HSPCs (100×). (F) Higher magnification of the panel E (200×) showing nests of HSPCs localizing across fibronectin extracellular fibers and at the periphery of WJ-MSCs. (G) High-power view (400×) of panel F showing the interactions between HSPCs and the surrounding fibronectin-rich microenvironment. (H) The peripheral positive fibronectin stain in some HSPCs (with absence of cytoplasmic stain) suggesting a close receptor-mediated interaction. Magnifications: 100× (B, E), 200× (D, F), and 400× (A, C, G, H). Bar: 100 µm.