Preconditioning human natural killer cells with chorionic villous mesenchymal stem cells stimulates their expression of inflammatory and anti-tumor molecules.

BACKGROUND: Mesenchymal stem cells derived from the chorionic villi of human placentae (pMSCs) produce a unique array of mediators that regulate the essential cellular functions of their target cells. These properties make pMSCs attractive candidates for cell-based therapy. Here, we examined the effects of culturing human natural killer (NK) cells with pMSCs on NK cell functions. METHODS: pMSCs were cultured with IL-2-activated and non-activated NK cells. NK cell proliferation and cytolytic activities were monitored. NK cell expression of receptors mediating their cytolytic activity against pMSCs, and the mechanisms underlying this effect on pMSCs, were also investigated. RESULTS: Our findings show that IL-2-activated NK cells, but not freshly isolated NK cells, efficiently lyse pMSCs and that this response might involve the activating NK cell receptor CD69. Interestingly, although pMSCs expressed HLA class I molecules, they were nevertheless lysed by NK cells, suggesting that HLA class I antigens do not play a significant role in protecting pMSCs from NK cell cytolytic activity. Co-culturing NK cells with pMSCs also inhibited NK cell expression of receptors, including CD69, NKpG2D, CD94, and NKp30, although these co-cultured NK cells were not inhibited in lysing cancer cells in vitro. Importantly, co-cultured NK cells significantly increased their production of molecules with anti-tumor effects. CONCLUSIONS: These findings suggest that pMSCs might have potential applications in cancer therapy.

ABO and Rh blood group genotypes in a cohort of Saudi stem cell donors.

The ABO and rhesus (Rh) blood group antigens are the most frequently studied genetic markers in a large group of people. Blood type frequencies vary in different racial/ethnic groups. Our objective was to investigate the distribution of the ABO and rhesus (Rh) blood groups by molecular typing method in a population of Saudi stem cell donors. Our data indicate that the most common blood group in our population is group O followed by group A then group B, and finally, the least common is group AB.

The prevalence of CCR5-Δ32 mutation in a cohort of Saudi stem cell donors.

CCR5 is a chemokine receptor that was found to be used by HIV as a co-receptor for entering target cells. A 32 bp deletion was described in certain people that rendered CCR5 non-functional. The mutant allele CCR5-Δ32 has been shown to prevent HIV infection. In addition, stem cell transplantation with the CCR5-Δ32 homozygous genotype can lead to clearance of HIV infection. In this study, our aim was to investigate the frequency of CCR5-Δ32 mutation in a cohort of stem cell donors from cord blood bank and stem cell donor registry. A total of 3025 samples were collected from healthy stem cell donors (2625) and from cord blood units (400). DNA was extracted and the CCR5 gene was amplified by polymerase chain reaction (PCR) in a light cycler system using SYBR Green dye. The mutated gene was further confirmed by direct gene sequencing. We found 38 heterozygous for CCR5-Δ32 and one homozygous CCR5 mutation (Δ32/Δ32) out of the 3025 tested individuals. We conclude that the protective CCR5-Δ32 allele appears to be rarely present in Saudi Arabia.

Phenotypic and Functional Characterization of Mesenchymal Stem/Multipotent Stromal Cells from Decidua Basalis of Human Term Placenta.

Mesenchymal stem cell (MSC) therapies for the treatment of diseases associated with inflammation and oxidative stress employ primarily bone marrow MSCs (BMMSCs) and other MSC types such as MSC from the chorionic villi of human term placentae (pMSCs). These MSCs are not derived from microenvironments associated with inflammation and oxidative stress, unlike MSCs from the decidua basalis of the human term placenta (DBMSCs). DBMSCs were isolated and then extensively characterized. Differentiation of DBMSCs into three mesenchymal lineages (adipocytes, osteocytes, and chondrocytes) was performed. Real-time polymerase chain reaction (PCR) and flow cytometry techniques were also used to characterize the gene and protein expression profiles of DBMSCs, respectively. In addition, sandwich enzyme-linked immunosorbent assay (ELISA) was performed to detect proteins secreted by DBMSCs. Finally, the migration and proliferation abilities of DBMSCs were also determined. DBMSCs were positive for MSC markers and HLA-ABC. DBMSCs were negative for hematopoietic and endothelial markers, costimulatory molecules, and HLA-DR. Functionally, DBMSCs differentiated into three mesenchymal lineages, proliferated, and migrated in response to a number of stimuli. Most importantly, these cells express and secrete a distinct combination of cytokines, growth factors, and immune molecules that reflect their unique microenvironment. Therefore, DBMSCs could be attractive, alternative candidates for MSC-based therapies that treat diseases associated with inflammation and oxidative stress.

Three new HLA-C alleles (HLA-C*14:02:13, HLA-C*15:72 and HLA-C*15:74) in Saudi bone marrow donors.

Three new HLA-C alleles were identified by sequence-based typing method (SBT) in donors for the Saudi Bone Marrow Donor Registry (SBMDR). HLA-C*14:02:13 differs from HLA-C*14:02:01 by a silent G to A substitution at nucleotide position 400 in exon 2, where lysine at position 66 remains unchanged. HLA-C*15:72 differs from HLA-C*15:22 by a nonsynonymous C to A substitution at nucleotide position 796 in exon 3, resulting in an amino acid change from phenylalanine to leucine at position 116. HLA-C*15:74 differs from HLA-C*15:08 by a nonsynonymous C to T substitution at nucleotide position 914 in exon 3, resulting in an amino acid change from arginine to tryptophan at position 156.

Human Chorionic Villous Mesenchymal Stem Cells Modify the Functions of Human Dendritic Cells, and Induce an Anti-Inflammatory Phenotype in CD1+ Dendritic Cells.

BACKGROUND: Mesenchymal stem cells derived from the chorionic villi of human term placenta (pMSCs) have drawn considerable interest because of their multipotent differentiation potential and their immunomodulatory capacity. These properties are the foundation for their clinical application in the fields of stem cell transplantation and regenerative medicine. Previously, we showed that pMSCs induce an anti-inflammatory phenotype in human macrophages. In this study, we determined whether pMSCs modify the differentiation and maturation of human monocytes into dendritic cells (DCs). The consequences on dendritic function and on T cell proliferation were also investigated. METHODS: Interleukin-4 (IL-4) and granulocyte-macrophage colony stimulating factor (GM-CSF) were used to stimulate the differentiation of monocytes into immature dendritic cells (iDCs), which were subsequently co-cultured with pMSCs. Lipopolysaccharide (LPS) was used to induce maturation of iDCs into mature dendritic cells (mDCs). Flow cytometry and enzyme-linked immunosorbent assays (ELISA) were used to quantify the effect pMSC co-culturing on DC differentiation using CD1a, a distinctive marker of DCs, as well as other molecules important in the immune functions of DCs. The phagocytic activity of iDCs co-cultured with pMSCs, and the effects of iDCs and mDC stimulation on T cell proliferation, were also investigated. RESULTS: Monocyte differentiation into iDCs was inhibited when co-cultured with pMSCs and maturation of iDCs by LPS treatment was also prevented in the presence of pMSCs as demonstrated by reduced expression of CD1a and CD83, respectively. The inhibitory effect of pMSCs on iDC differentiation was dose dependent. In addition, pMSC co-culture with iDCs and mDCs resulted in both phenotypic and functional changes as shown by reduced expression of costimulatory molecules (CD40, CD80, CD83 and CD86) and reduced capacity to stimulate CD4(+) T cell proliferation. In addition, pMSC co-culture increased the surface expression of major histocompatibility complex (MHC-II) molecules on iDCs but decreased MHC-II expression on mDCs. Moreover, pMSC co-culture with iDCs or mDCs increased the expression of immunosuppressive molecules [B7H3, B7H4, CD273, CD274 and indoleamine-pyrrole 2,3-dioxygenase (IDO). Additionally, the secretion of IL-12 and IL-23 by iDCs and mDCs co-cultured with pMSCs was decreased. Furthermore, pMSC co-culture with mDCs decreased the secretion of IL-12 and INF-γ whilst increasing the secretion of IL-10 in a T cell proliferation experiment. Finally, pMSC co-culture with iDCs induced the phagocytic activity of iDCs. CONCLUSIONS: We have shown that pMSCs have an inhibitory effect on the differentiation, maturation and function of DCs, as well as on the proliferation of T cells, suggesting that pMSCs can control the immune responses at multiple levels.

Two novel alleles HLA-A*02:433 and HLA-A*02:434 identified in Saudi bone marrow donors using sequence-based typing.

In this report, we present two novel HLA-A alleles: HLA-A*02:433 and HLA-A*02:434. These alleles were identified by sequence-based typing method (SBT), in two donors for the Saudi Bone Marrow Donor Registry (SBMDR). Allele A*02:433 is identical to A*02:05:01G except for a G to A substitution at nucleotide position 449 in exon 2. This substitution results in glycine to serine substitution at position 83. Whereas, allele A*02:434 is identical to A*02:01:01G except for a C to A substitution at nucleotide position 245 in exon 2, which results in phenylalanine to threonine substitution at position 15. The generation of both alleles appears to be the result of nucleotide point mutation involving 02:01:01 and 02:05:01.

Two novel alleles HLA-DRB1*11:150 and HLA-DRB1*14:145 identified in Saudi individuals.

Two new HLA- DRB1 alleles were identified by sequence-based typing method (SBT) in 1100 participants in the Saudi Stem Cell Donor Registry. HLA-DRB1*11:150 differs from HLA-DRB1*11:01:01G by a single C to A substitution at nucleotide position 5580 in exon 2, resulting in an amino acid change from alanine to glutamic acid at position 74. HLA-DRB1*14:145 differs from HLA-DRB1*14:04 by a C to G substitution at nucleotide position 5511 in exon 2, resulting in an amino acid change from threonine to arginine at position 51.

HLA-B50 polymorphism in the Saudi population.

The HLA-B50 serologic family is very frequent in people of Arabic origin. In Saudi Arabia, HLA-B50 is the most frequent HLA-B allele. The aim of this study was to investigate the distribution of HLA-B50 alleles in healthy Saudi individuals. A total of 162 healthy Saudi individuals were selected based on low-resolution HLA typing. DNA samples were typed by sequence-based typing method for exons 2, 3 and 4 of the HLA-B locus (Genome Diagnostics B.V.). The HLA-B*50 alleles were analysed using SBT engine software. HLA-B*50:01:01 was found in 161 of 162 individuals (99.4%), while HLA-B*50:09 was found in one individual (0.6%). HLA-B*50:01:01 is the most common HLA-B50 allele in Saudi Arabia.

Human placental mesenchymal stem cells (pMSCs) play a role as immune suppressive cells by shifting macrophage differentiation from inflammatory M1 to anti-inflammatory M2 macrophages.

BACKGROUND: Mesenchymal stem cells (MSCs) have a therapeutic potential in tissue repair because of capacity for multipotent differentiation and their ability to modulate the immune response. In this study, we examined the ability of human placental MSCs (pMSCs) to modify the differentiation of human monocytes into macrophages and assessed the influence of pMSCs on important macrophage functions. METHODS: We used GM-CSF to stimulate the differentiation of monocytes into the M1 macrophage pathway and then co-cultured these cells with pMSCs in the early stages of macrophage differentiation. We then evaluated the effect on differentiation by microscopic examination and by quantification of molecules important in the differentiation and immune functions of macrophages using flow cytometry and ELISA. The mechanism by which pMSCs could mediate their effects on macrophage differentiation was also studied. RESULTS: The co-culture of pMSCs with monocytes stimulated to follow the inflammatory M1 macrophage differentiation pathway resulted in a shift to anti-inflammatory M2-like macrophage differentiation. This transition was characterized by morphological of changes typical of M2 macrophages, and by changes in cell surface marker expression including CD14, CD36, CD163, CD204, CD206, B7-H4 and CD11b, which are distinctive of M2 macrophages. Co-culture with pMSCs reduced the expression of the costimulatory molecules (CD40, CD80 and CD86) and increased the expression of co-inhibitory molecules (CD273, CD274 and B7-H4) as well as the surface expression of major histocompatibility complex (MHC-II) molecules. Furthermore, the secretion of IL-10 was increased while the secretion of IL-1ß, IL-12 (p70) and MIP-1α was decreased; a profile typical of M2 macrophages. Finally, pMSCs induced the phagocytic activity and the phagocytosis of apoptotic cells associated with M2- like macrophages; again a profile typical of M2 macrophages. We found that the immunoregulatory effect of pMSCs on macrophage differentiation was mediated by soluble molecules acting partially via glucocorticoid and progesterone receptors. CONCLUSIONS: We have shown that pMSCs can transition macrophages from an inflammatory M1 into an anti-inflammatory M2 phenotype. Our findings suggest a new immunosuppressive property of pMSCs that may be employed in the resolution of inflammation associated with inflammatory diseases and in tissue repair.

Phenotypic and functional characterization of mesenchymal stem cells from chorionic villi of human term placenta.

BACKGROUND: Bone marrow derived mesenchymal stem cells (BM-MSCs) are used extensively in transplantation but their use is associated with many problems including low abundance in BM, low overall number, decreased differentiation potential with age and the invasive isolation procedures needed to obtain BM. We report a novel method of isolating placental MSCs (pMSCs) from chorionic villi, which exhibit the phenotypic and functional characteristics that will make them an attractive source of MSCs for cell-based therapy. METHODS: A novel explant approach was used to isolate pMSCs from chorionic villi of human placentae. These pMSCs were characterized by flow cytometry and were differentiated into adipocytes, osteocytes and chondrocytes using differentiation medium as demonstrated by cytochemical staining. The gene and protein expression profiles of pMSCs were also characterized using real time polymerase chain reaction (PCR) and flow cytometry, respectively. In addition, cytokine secretion by pMSCs was also analysed using sandwich enzyme-linked immunosorbent assay (ELISA) technique. Moreover, the migration and proliferation potentials of pMSCs were also determined. RESULTS: pMSCs were isolated from fetal part of the chorionic villi and these pMSCs expressed CD44, CD90, CD105, CD146, CD166 and HLA-ABC but not CD14, CD19, CD40, CD45, CD80, CD83, CD86 and HLA-DR. In addition, these pMSCs differentiated into osteocytes, chondrocytes and adipocytes and they also expressed several adhesion molecules, chemokines/receptors, growth factor receptors and cytokines/receptors. Moreover, they secreted many cytokines (IL-1Ra, IL6, IL8, IL10, IL11 and IL15) and they were able to proliferate. Furthermore, they migrated in response to chemotactic factors including stromal cell-derived factor-1 (SDF-1), platelet derived growth factor (PDGF), hepatocyte growth factor (HGF), and monocyte chemotactic protein-1 (MCP-1). CONCLUSIONS: We devised a novel explant method of isolating pMSCs that expressed many biological factors responsible for mediating cellular processes such as migration/homing, immune modulation and angiogenesis. Therefore, we suggest that pMSCs prepared from human term placental chorionic villous explants are an attractive source of MSCs for cell therapy.

Prevalence of autoantibodies in children newly diagnosed with type 1 diabetes mellitus.

Type 1 diabetes mellitus (T1DM) is an increasingly encountered chronic illness in Saudi Arabia. It is known to have an immune-mediated pathogenesis, which results in the loss of insulin-secreting beta-cells responsible for maintaining normal blood glucose levels. The three main autoantibodies identified to play a role in the pathogenesis are islet cell antibodies (ICA), insulin autoantibodies (IAA) and glutamic acid decarboxylase antibodies (GAD). This study aims to determine at what age during childhood the autoantibodies ICA, IAA and GAD are most prevalent, and identify any correlation between their presence and the severity of the initial clinical presentation. Medical records of children diagnosed with T1DM in Riyadh in 2000-2007 were reviewed, and a total of 98 patients were included in the study (age range: 1-12 years, mean: 6.6 years, equal numbers by gender), of which 49% presented with diabetic ketoacidosis (DKA). Results showed that 67% were positive for ICA, 36% for IAA and 84.4% for GAD. The presence of ICA was predominant in children aged under six years. The presence of ICA and GAD in the absence of IAA was associated with more severe clinical presentation.