Cross-Talk between Human Neural Stem/Progenitor Cells and Peripheral Blood Mononuclear Cells in an Allogeneic Co-Culture Model

Abstract Background: Vibrio cholerae, the causative agent of cholera, as a Gram-negative pathogen tend to colonize the small intestine. The intestinal mucus layer forms mucin physical barrier, consisted from high molecular weight proteins. Regarding the role of toxin–coregulated pilus (TCP) as one of the most important colonization factors of V. cholerae, this experimental study was designed to determine the role of TcpA in induction of mucin production and its regulatory effect on innate immunity molecules including TLRs and NODs using Caco2- PBMC cocultures as an interactive model.Materials and methods: The rTcp protein was expressed in pET-28a-tcpA construct and purified using Ni-column chromatography. The identity of rTcp was confirmed by western immunoblotting analysis using anti-poly-histidine antibody. Nontoxic doses of rTcpA was determined on Caco-2 cell lines. The effects of different concentrations of rTcpA (1, 5, 10 and 50 µg/mL) on the expression of mucin 1,3, 4, toll-like receptors (TLR1, 4), and Nucleotide-binding oligomerization domain-containing proteins (NOD1, 2) genes were evaluated in a co-culture model of human colon carcinoma cell line (Caco-2) and Peripheral Blood Mononuclear cells (PBMCs).Results:The rTcpA protein of V. choleraewas expressed in BL21 E. coli and confirmed by western blotting. The rTcpA showed a statistically significant effect on the expression of muc genes (MUC3 and MUC4) in a dose-dependent manner.This finding is supposed to facilitate physical adhesion and colonization of V. cholerae in intestinal lumen. The rTcpA moderately stimulated the expression of tlr4 and overexpressed tlr1, both of which are supposed to induce a mucosal protective response against bacterial infection and would help a promising protection in prophylaxis applications. No change in NOD2 expression might be attributed to the non-invasive nature of V. cholerae as an intestinal pathogen.Conclusion: In conclusion, the rTcpA protein of V. cholerae showed a statistically significant modulatory effect on the human gut epithelium gene expression which would help promising protection in prophylaxis applications.

Download Full-text

Human peripheral blood mononuclear cells enriched in endothelial progenitor cells via quality and quantity controlled culture accelerate vascularization and wound healing in a porcine wound model

Cell Transplantation ◽

10.1177/0963689718780307 ◽

2018 ◽

Vol 27 (7) ◽

pp. 1068-1079 ◽

Cited By ~ 7

Author(s):

Makiko Kado ◽

Rica Tanaka ◽

Kayo Arita ◽

Kayoko Okada ◽

Rie Ito-Hirano ◽

...

Keyword(s):

Wound Healing ◽

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Peripheral Blood Mononuclear Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Skin Defect ◽

Endothelial Progenitor ◽

Peripheral Blood Mononuclear ◽

Blood Mononuclear Cells

The transplantation of endothelial progenitor cells (EPCs) is used to promote wound angiogenesis. In patients with chronic wounds and accompanying morbidities, EPCs are often compromised in number and function. To overcome these limitations, we previously developed a quality and quantity controlled (QQ) culture system to enrich peripheral blood mononuclear cells (PBMNCs) in EPCs. To evaluate the wound healing efficacy of mononuclear cells (MNCs) harvested after QQ culture (QQMNCs), preclinical studies were performed on large animals. MNCs harvested from the blood of healthy human subjects were cultured in the presence of angiogenic cytokines and growth factors in a serum-free medium for 7 days. A total of 5 × 106 QQMNCs per full-thickness skin defect or control saline was injected into wounds induced in cyclosporine-immunosuppressed pigs. EPC colony-forming assays revealed a significantly higher number of definitive (partially differentiated) EPC colony-forming units in QQMNCs. Flow cytometry evaluation of QQMNC surface markers showed enrichment of CD34+ and CD133+ stem cell populations, significant reduction in CCR2+ cell percentages, and a greater than 10-fold increase in the percentage of anti-inflammatory M2-type macrophages (CD206+ cells) compared with PBMNCs. Wounds treated with QQMNCs had a significantly higher closure rate. Wounds were harvested, frozen, and sectioned at day 21 postoperatively. Hematoxylin and eosin staining revealed that the epithelization of QQMNC-treated wounds was more advanced than in controls. Treated wounds developed granulation tissue with more mature collagen and larger capillary networks. CD31 and human mitochondrial co-staining confirmed the presence of differentiated human cells within newly formed vessels. Real-time polymerase chain reaction (PCR) showed upregulation of interleukin 6 (IL-6), IL-10, and IL-4 in the wound bed, suggesting paracrine activity of the transplanted QQMNCs. Our data demonstrate for the first time that QQ culture of MNCs obtained from a small amount of peripheral blood yields vasculogenic and therapeutic cells effective in wound healing.

Download Full-text

Comparison of Whole Serum-Deprived Media for Ex Vivo Expansion of Hematopoietic Progenitor Cells from Cord Blood and Mobilized Peripheral Blood Mononuclear Cells

Journal of Hematotherapy ◽

10.1089/scd.1.1996.5.461 ◽

1996 ◽

Vol 5 (5) ◽

pp. 461-473 ◽

Cited By ~ 16

Author(s):

CRAIG E. SANDSTROM ◽

PAUL C. COLLINS ◽

TODD A. McADAMS ◽

JAMES G. BENDER ◽

E. TERRY PAPOUTSAKIS ◽

...

Keyword(s):

Progenitor Cells ◽

Peripheral Blood Mononuclear Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Ex Vivo ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor ◽

Peripheral Blood Mononuclear ◽

Blood Mononuclear Cells ◽

Mobilized Peripheral Blood

Download Full-text

Inhibitory effects of Lactobacillus casei subsp. rhamnosus on Salmonella lipopolysaccharide-induced inflammation and epithelial barrier dysfunction in a co-culture model using Caco-2/peripheral blood mononuclear cells

Journal of Medical Microbiology ◽

10.1099/jmm.0.009662-0 ◽

2010 ◽

Vol 59 (5) ◽

pp. 573-579 ◽

Cited By ~ 37

Author(s):

Hsu-Wei Fang ◽

Shiuh-Bin Fang ◽

Jen-Shiu Chiang Chiau ◽

Chun-Yan Yeung ◽

Wai-Tao Chan ◽

...

Keyword(s):

Mrna Expression ◽

Peripheral Blood Mononuclear Cells ◽

Peripheral Blood ◽

Lactobacillus Casei ◽

Mononuclear Cells ◽

Barrier Dysfunction ◽

Peripheral Blood Mononuclear ◽

Cell Monolayers ◽

Culture Model ◽

Blood Mononuclear Cells

In this study, we investigated the anti-inflammatory and reinforcing barrier effects of Lactobacillus casei subsp. rhamnosus (Lcr35) on Caco-2 intestinal epithelial cells already exposed to Salmonella LPS. Using the Transwell co-culture model, Salmonella LPS was apically added to polarized Caco-2 cells co-cultured with peripheral blood mononuclear cells (PBMCs) in the basolateral compartment. LPS-stimulated Caco-2 cells were incubated with Lcr35 for 1, 6, 24 or 48 h. Apical inoculation of Lcr35 after 48 h significantly inhibited the basolateral secretion of interleukin-8 (IL-8) in the Caco-2/PBMC co-culture. The PCR analysis showed that Lcr35 significantly downregulated mRNA expression of monocyte chemoattractant protein 1 (MCP-1) (P<0.05) and had a trend of decreasing mRNA expression of IL-8 (P=0.05), but did not alter mRNA expression of transforming growth factor-β1 in LPS-stimulated Caco-2 cells at 48 h after addition of Lcr35. Compared to non-LPS-pretreated controls, transepithelial electrical resistance (TEER) of the polarized Caco-2 cell monolayers pretreated with LPS for 48 h was decreased by 9.9 % (P<0.05). Additionally, compared to those cells only treated with LPS, apical co-incubation with Lcr35 showed biphasic TEER levels increased by 12.1 % (P<0.001), 5.7 % (P<0.05) and 86.8 % (P<0.001) in the Caco-2 cell monolayers compared to those without Lcr35 treatment after 1, 6 and 48 h, respectively. In conclusion, Lcr35 can exert anti-inflammatory effects and ameliorate barrier dysfunction in the Salmonella LPS-pretreated inflamed intestinal epithelium in vitro.

Download Full-text