Mesenchymal stem cells from human umbilical cord express preferentially secreted factors related to neuroprotection, neurogenesis, and angiogenesis

Abstract

Mesenchymal stem cells (MSCs) are promising tools for the treatment of diseases such as infarcted myocardia and strokes because of their ability to promote endogenous angiogenesis and neurogenesis via a variety of secreted factors. MSCs found in the Wharton's jelly of the human umbilical cord are easily obtained and are capable of transplantation without rejection. We isolated MSCs from Wharton's jelly and bone marrow (WJ-MSCs and BM-MSCs, respectively) and compared their secretomes. It was found that WJ-MSCs expressed more genes, especially secreted factors, involved in angiogenesis and neurogenesis. Functional validation showed that WJ-MSCs induced better neural differentiation and neural cell migration via a paracrine mechanism. Moreover, WJ-MSCs afforded better neuroprotection efficacy because they preferentially enhanced neuronal growth and reduced cell apoptotic death of primary cortical cells in an oxygen-glucose deprivation (OGD) culture model that mimics the acute ischemic stroke situation in humans. In terms of angiogenesis, WJ-MSCs induced better microvasculature formation and cell migration on co-cultured endothelial cells. Our results suggest that WJ-MSC, because of a unique secretome, is a better MSC source to promote in vivo neurorestoration and endothelium repair. This study provides a basis for the development of cell-based therapy and carrying out of follow-up mechanistic studies related to MSC biology.

Figure 1. Differential gene expression between WJ-MSCs and BM-MSCs.

(A) Flow cytometry analysis of WJ-MSCs and BM-MSCs. (B–C) A total of 1096 probe sets (q ≦ 10^-3) differentiating WJ-MSCs and BM-MSCs were filtered out and 762 WJ-MSC probe sets (B) and 334 BM-MSC probe sets (C) were subjected to DAVID (http://david.abcc.ncifcrf.gov/) analysis. These categories were selected using the Biological Process organizing principle via the Gene Ontology project (http://www.geneontology.org/). The number of genes, as well as p values, for categories that are significantly (p<0.05) over-represented are listed. The terms indicated by arrows are discussed in the text. Genes involved in nervous system development and blood vessel development are listed, and RT-qPCR verified genes are underlined. (D) RT-qPCR validation of the relative expression levels of the neurogenic-related and angiogenic-related genes in the two MSC subtypes. Mean expression levels of the target genes were compared to that of the GAPDH control. Each bar represents a different individual. Results were expressed as the mean ± standard deviation (SD). (E) A heatmap showing BM-MSC and WJ-MSC-specific secreted factors. (F–G) Validation of array data by RT-qPCR. ANGPT1 and PGF, which are up-regulated in BM-MSCs (F), as well as various WJ-MSC abundant genes (G), were examined. (H) The secretion levels of CXCL5 and PGF in the conditioned medium of BM-MSC and WJ-MSC were quantified using enzyme-linked immunosorbent assays. Each bar represents the protein concentration of independent donors.

Figure 2. Higher neural induction ability of WJ-MSCs.

(A) An interaction network of secreted factors. Factors that pertained to angiogenesis, vasculogenesis, neurite outgrowth, and neuron migration are connected. Genes in red are abundant in WJ-MSCs and genes in green are abundant in BM-MSCs. (B) Differential chemotaxis effects of WJ-MSCs and BM-MSCs. MSCs cultured in MesenCult® medium were seeded in the lower part of Tranwell plates, while N2a cells were placed in the upper chambers (illustrated in left panel). Migrated N2a cells to the other side of the membrane were stained with Hoechst 33342 and counted. Data are mean ± SD (right panel; *p<0.05, **p<0.01). (C–D) Induction of N2a neural differentiation by MSCs. N2a cells were cultured with MSC conditioned medium, medium only (negative control) or retinoic acid (RA; positive control) for 4 days before the cellular lysates were subjected to Western blotting analysis (C) or the cells were fixed for immunofluorescence staining (D). Neural markers TUBB3 and NEFL were analyzed. Cell nuclei were stained with DAPI. Scale bars: 50 μm.

Figure 3. Preferential neuroprotection effects of WJ-MSCs.

(A) Schematic representation of the transmembranous stem cell co-culture system using an oxygen-glucose deprivation (OGD) model. (B) Immunofluorescence staining of the rat primary cortical cells subjected to OGD alone (left), to co-culture with BM-MSCs (middle), or to co-culture with WJ-MSCs (right) at 72 hours post-OGD. Neuronal marker MAP2 is shown in red, the astroglial marker GFAP in green, and DAPI nuclear staining in blue. Scale bar: 20 µm. (C) Quantification of cell death and apoptosis rate using PI and TUNEL staining, respectively, at 72 hours post-OGD. *p<0.05, **p<0.01, ***p<0.001 (D) Quantification of total neurite length (left) and neurite branch point numbers (right). (E) Percentage of neuron number (left) and astrocyte number (right) after 72 hours co-cultured with BM-MSCs or WJ-MSCs post-OGD.

Figure 4. Preferential angiogenic induction ability of WJ-MSCs.

(A–B) HMEC1 endothelial cells in vitro motility. Migrated HMEC1 that were attracted by BM-MSCs and WJ-MSCs were counted. MSC culture medium was used as negative control (medium only). Representative images of migrated HMEC1 cells are shown (B). (C–D) HMEC1 cells in vitro tube formation using Matrigel at 4 hours incubation in conditioned medium from BM-MSCs or conditioned medium from WJ-MSCs. Representative images of the HMEC1 tube formation are shown (D). (*p<0.05, **p<0.01, ***p<0.001).