Transplanted Umbilical Cord Mesenchymal Stem Cells Modify the In Vivo Microenvironment Enhancing Angiogenesis and Leading to Bone Regeneration

Abstract

Umbilical cord mesenchymal stem cells (UC-MSCs) show properties similar to bone marrow mesenchymal stem cells (BM-MSCs), although controversial data exist regarding their osteogenic potential. We prepared clinical-grade UC-MSCs from Wharton's Jelly and we investigated if UC-MSCs could be used as substitutes for BM-MSCs in muscoloskeletal regeneration as a more readily available and functional source of MSCs. UC-MSCs were loaded onto scaffolds and implanted subcutaneously (ectopically) and in critical-sized calvarial defects (orthotopically) in mice. For live cell-tracking experiments, UC-MSCs were first transduced with the luciferase gene. Angiogenic properties of UC-MSCs were tested using the mouse metatarsal angiogenesis assay. Cell secretomes were screened for the presence of various cytokines using an array assay. Analysis of implanted scaffolds showed that UC-MSCs, contrary to BM-MSCs, remained detectable in the implants for 3 weeks at most and did not induce bone formation in an ectopic location. Instead, they induced a significant increase of blood vessel ingrowth. In agreement with these observations, UC-MSC-conditioned medium presented a distinct and stronger proinflammatory/chemotactic cytokine profile than BM-MSCs and a significantly enhanced angiogenic activity. When UC-MSCs were orthotopically transplanted in a calvarial defect, they promoted increased bone formation as well as BM-MSCs. However, at variance with BM-MSCs, the new bone was deposited through the activity of stimulated host cells, highlighting the importance of the microenvironment on determining cell commitment and response. Therefore, we propose, as therapy for bone lesions, the use of allogeneic UC-MSCs by not depositing bone matrix directly, but acting through the activation of endogenous repair mechanisms.

FIG. 1.

Histological images of ectopic implants after 2 months and stained with hematoxylin and eosin (A–F) and analyzed by polarized light (G–I). Black rectangles indicate the areas from which higher magnification images where taken. (A, D, G) noncell-seeded empty scaffold; (B, E, H) bone marrow mesenchymal stem cell (BM-MSC)-seeded scaffolds; (C, F, I) umbilical cord mesenchymal stem cell (UC-MSC)-seeded scaffolds. s, scaffold; ft, fibrous tissue; b, bone; cft, compact fibrous tissue; (↑), polarized light signal. Scale bars=600 μm (A–C) and 100 μm (D–I). (n=4 for both cell-seeded and not seeded scaffold groups). Color images available online at www.liebertpub.com/scd

FIG. 2.

Histological images of ectopic implants recovered after 2 months and stained with Masson's trichrome showing a high number of blood vessel-like structures in the UC-MSC-seeded scaffold (C) compared with the BM-MSC-seeded scaffold (B) and empty scaffold (A). bv, blood vessel; ft, fibrous tissue; b, bone; (*), blood vessel count. Scale bar=100 μm. (n=4 for both cell-seeded and not seeded scaffold groups). In (D) a quantification of the number of blood vessels present in 2-month implants of the empty scaffold (SK) and BM-MSC and UC-MSC-seeded scaffolds (SK+BM-MSC and SK+UC-MSC) is presented. (****P<0.0001). Color images available online at www.liebertpub.com/scd

FIG. 3.

Histological images of ectopic implants recovered after 2 months and stained with hematoxylin and eosin (A, C, E, G) and analyzed by in situ hybridization for the human ALU repeat sequence (B, D, F, H). Empty scaffold (SK) (A, B); BM-MSC-seeded scaffold osteocytes and osteoblasts of human origin are present. Moreover, human cells are detectable and also found in the fibrous tissue as fibroblasts (C, D); UC-MSC-seeded scaffold. No human cells present in the compact fibrous tissue (E, F) and in the blood vessel endothelium (G, H). s, scaffold; F, fibroblast; ft, fibrous tissue; cft, compact fibrous tissue; Oc, osteocyte; Ob, osteoblast; b, bone; Ec, endothelial cell; bv, blood vessel; (↑), human cell. Scale bar=100 μm. (n=4). Color images available online at www.liebertpub.com/scd

FIG. 4.

In vivo bioluminescence imaging, at different time points after surgery, of mice ectopically implanted with scaffolds seeded with cells (s+c) or not seeded (s). A decreasing signal is noticeable for UC-MSC-seeded scaffolds (B), whereas the signal for the BM-MSC-seeded scaffold remains stable throughout the whole observation time (A). No signal is detected in empty scaffolds (A, B). (n=6 for both UC-MSC-seeded and not seeded scaffold groups, and n=4 for the BM-MSC-seeded scaffold group). Color images available online at www.liebertpub.com/scd

FIG. 5.

Blood vessel sprouting in explants of mouse fetal metatarsal bones maintained in different culture conditions. Vessels were stained with antibodies against PECAM-1/CD31. Explants were cultured in the presence of (A) control medium (CTR); (B) BM-MSC-conditioned medium (BM-MSC CM); and (C) UC-MSC-conditioned medium (UC-MSC CM). Scale bar=1,000 μm (A–C and C insert). Quantification of the number of PECAM-1/CD31-positive pixels and the area of vessel outgrowth are shown in (D, E), respectively. (*P<0.05) (n=3 for both cell CM and control medium).

FIG. 6.

Cytokine array membrane assay of MSC CM; (A) BM-MSC CM; (B) UC-MSC CM; (C) quantification of the positive pixels for each identified cytokine in BM-MSC and UC-MSC CM. rs, reference spots represent an internal control.

FIG. 7.

Histological images of orthotopic implants in mouse calvaria recovered after 3 months, stained with hematoxylin and eosin, and analyzed by polarized light at low magnification (A–C) and stained with hematoxylin and eosin and analyzed by in situ hybridization for human ALU repeat sequences at high magnification (E–H). Black rectangles indicate the areas from which higher magnification images where taken. (A, E) Empty scaffold (PU-HA); (B, F) BM-MSC-seeded scaffold (PU-HA+BM-MSC); (C, G, H) UC-MSC-seeded scaffold (PU-HA+UC-MSC). (n=3 for both cell-seeded and not seeded scaffold groups.) A quantification of the new bone formed in each condition determinated by measuring the surface area of new bone is shown in (D). s, scaffold; b, bone; (↑), polarized light signal; Oc, osteocyte; (*), human cell. Scale bar=1,000 and 50 μm for (A–C) and (E–H), respectively. ****(mHA/PU vs mHA/PU+UC-MSC P<0.0001 and vs mHA/PU+BM-MSC P<0.001). Color images available online at www.liebertpub.com/scd