The follicle-associated epithelium (FAE) of Peyer's patches (PPs) contains M cells that are important for reducing mucosal immune responses by transporting antigens into the underlying lymphoid tissue. We generated a monoclonal antibody (C6) that reacted with the FAE of calf ileal PPs, and analyzed the characteristics of C6 using immunohistochemistry and Western blotting. FAE of the ileal PP was stained with C6 during both late fetal developmental and postnatal stages. Neither the villous epithelial cell nor intestinal crypt basal cells were stained at any developmental stage. During the prenatal stages, FAE of the jejunal PP was C6-negative. However, a few C6-positive cells were distributed diffusely in some FAE of the jejunal PPs during the postnatal stages. The protein molecular weight of the antigen recognized by C6 was approximately 45 kDa. These data show that C6 is useful for identifying the FAE in ileal PPs and further suggest that differentiation of the FAE in these areas is independent of external antigens.
Animals ; Antibodies, Monoclonal/*immunology ; *Cattle ; Fetus ; Hybridomas ; Ileum/*ultrastructure ; Intestinal Mucosa/*immunology ; Peyer's Patches/*immunology/ultrastructure

The follicle-associated epithelium (FAE) of Peyer's patches (PPs) contains M cells that are important for reducing mucosal immune responses by transporting antigens into the underlying lymphoid tissue. We generated a monoclonal antibody (C6) that reacted with the FAE of calf ileal PPs, and analyzed the characteristics of C6 using immunohistochemistry and Western blotting. FAE of the ileal PP was stained with C6 during both late fetal developmental and postnatal stages. Neither the villous epithelial cell nor intestinal crypt basal cells were stained at any developmental stage. During the prenatal stages, FAE of the jejunal PP was C6-negative. However, a few C6-positive cells were distributed diffusely in some FAE of the jejunal PPs during the postnatal stages. The protein molecular weight of the antigen recognized by C6 was approximately 45 kDa. These data show that C6 is useful for identifying the FAE in ileal PPs and further suggest that differentiation of the FAE in these areas is independent of external antigens.
Animals ; Antibodies, Monoclonal/*immunology ; *Cattle ; Fetus ; Hybridomas ; Ileum/*ultrastructure ; Intestinal Mucosa/*immunology ; Peyer's Patches/*immunology/ultrastructure

This study was aimed to establish a genotyping method to detect ABO group gene of fetus from peripheral blood of pregnant women for prenatal diagnosis of hemolytic disease of newborn (HDN) resulting from ABO blood group incompatibility. 4 pairs of primers were designed according to ABO blood group gene DNA and mRNA sequences. 20 plasma DNA samples from healthy donors were extracted and amplified to explore the best conditions for plasma DNA extraction and PCR amplification. The O group plasma DNA was mixed with A group or B group plasmas by the ratios of 1:1, 2:1, 4:1, 8:1, 10:1, 20:1, 40:1, 100:1 to simulate the status of mixed ABO gene from pregnant maternal blood and to establish the mixed blood group ABO genotyping technology. The pregnant maternal blood samples with more than 30 weeks of gestation were selected for detecting the fetal ABO blood group genotype. The blood samples should be taken as possible as after birth for identification of ABO blood group and evaluation of sensitivity and accuracy of fetal ABO blood group genotyping technology through peripheral blood of pregnant women. The results indicated that the minimal amount of template DNA from single blood plasma for accuracy identification was at least about 0.625 ng, the DNA amount extracted from 500 microl of plasma could meet the requirement for PCR amplification. When the proportion of O group plasma DNA in mixed plasma DNA was ABO Blood-Group System ; genetics ; immunology ; Blood Group Antigens ; blood ; genetics ; Blood Grouping and Crossmatching ; methods ; Female ; Fetus ; immunology ; Genotype ; Humans ; Pregnancy

This study was aimed to investigate if human heart harbored a population of primitive undifferentiated cells with the characteristics of MPC. Cells were isolated from human fetal heart and were cultured under conditions appropriate for bone marrow-derived MPCs. Their morphology, phenotypes and functions were tested by methods developed for MPC from other sources. The results showed that morphologically, cells were spindle shaped and resembled fibroblasts. In their undifferentiated state, cells were CD73, CD105, CD29, CD44, HLA-ABC, CD166 positive and CD45, CD34, CD86, HLA-DR negative. When cultured in adipogenic, osteogenic or chondrogenic media, cells differentiated into adipocytes, osteocytes and chondrocytes respectively. They could be extensively expanded in vitro and exhibited very low immunogenicity as evaluated by T cell proliferation assays. It is concluded that cells isolated from fetal heart possess similarity to their adult and fetal bone marrow counterparts in morphologic, immunophenotypic, and functional characteristics.
Bone Marrow Cells ; cytology ; Cell Adhesion ; Cell Differentiation ; Cells, Cultured ; Fetal Heart ; cytology ; Fetus ; Humans ; Mesenchymal Stromal Cells ; cytology ; immunology ; Multipotent Stem Cells ; physiology

Endothelial progenitor cells (EPCs) have been reported to possess the capacity to colonize vascular grafts and hold promise for therapeutic neovascularization. However, limited quantities of EPCs have been the major factor impeding effective research on vasculoangiogenesis. In this study, cytokine and culture conditions necessary for the provision of large quantities of endothelial cells (ECs) were investigated. Cord blood was collected from 18 normal full-term deliveries and CD34+ cells were isolated by MACS system (Miltenyi Biotech, Bergish-Gladbach, Germany). To evaluate the effect of cytokines, CD34+ cells were cultured with various cytokine combinations, such as stem cell factor (SCF), flt3-ligand (FL), and thrombopoietin (TPO) with vascular endothelial growth factor (VEGF), interleukin-1beta, fibroblast growth factor-basic (FGF-b) as basic cytokines. The quantities of non-adherent and adherent cells were the greatest with SCF, FL and TPO. The addition of TPO to all other cytokines significantly increased the number of non-adherent and adherent cells (p< 0.05, Wilcoxon rank sum test). After four weeks of culture, adherent cells expressed endothelial specific markers such as KDR, CD31 and CD62E. Typical morphology of ECs was observed during culture, such as cord-like structure and cobblestone appearance, suggesting that the adherent cells were consistent with ECs. In this study, the experimental conditions that optimize the production of ECs for therapeutic neovascularization were described. And it was possibly suggested that TPO plays a major role in differentiation from EPCs to ECs.
Antigens, CD34/analysis ; Cell Differentiation/drug effects ; Cell Division/drug effects ; *Cell Separation ; Cells, Cultured ; Cytokines/pharmacology ; *Endothelial Cells/immunology ; Fetal Blood/*cytology ; Fetus ; Flow Cytometry ; Humans ; Research Support, Non-U.S. Gov't ; *Stem Cells/*immunology ; Thrombopoietin/pharmacology

The aim of this study was to investigate the biological characteristics of human bone marrow mesenchymal stem cells from bone marrow of different age donors. The experiments were divided into four groups by donors age, group A represented MSC derived from fetal bone marrow, group B represented MSC derived from bone marrow of 0-20 years old donors, group C represented MSC derived from bone marrow of 20-40 years old donors and group D represented MSC derived from bone marrow of donors older than 40. The growth, purification, proliferation and multipotential abilities of MSC in 4 groups were observed and their immunophenotypes were determined by flow-cytometry. The level of cytokines (IL-6, SCF, FLT-3L, SDF-1 and TGF-beta1) were assayed by ELISA method. Cell cycles were analyzed to show the proliferation index (PrI). MSCs derived from bone marrow of 4 groups were injected subcutaneous into NOD/SCID mouse to observe the safety. The results showed that different age donors bone marrow all gave rise to MSC. These cells were similar in morphology, antigenic phenotype, differentiation potential and cell cycle. The primary culture time of group B was shorter than other groups. The duration of passage 1 (P1) was 5.5 days, and the duration of P10 was 33 days, after P10 culture, (5.19 +/- 2.15) x 10(10) MSCs were obtained from 8 x 10(6) MNC of this group. The primary culture time of groups A, C, D were longer, the duration of P1 were 15, 7 and 13 days for group A, C and D respectively, and the duration of P10 was 50, 60 and 72 days for group A, C and D, respectively. After P10 culture, (4.98 +/- 2.08) x 10(10), (1.86 +/- 0.47) x 10(10), (0.64 +/- 0.22) x 10(10) MSCs were obtained from 8 x 10(6) MNC of group A, C and D respectively. The morphology of MSC of group A was longer and slender. The ability of expansion decreased after P15 for A group, P10 for B group and P8 for C and D groups. The levels of SCF, FLT3-L, IL-6 and SDF-1 in group B were higher than other groups. Karyotype analysis showed that MSCs from 4 groups were normal, and tumor-like tissues were not developed after cultured MSCs were inoculated in NOD/SCID mice. It is concluded that there was relationship between age and the biological characteristics of human bone marrow mesenchymal stem cells. For clinical use, especially in hematopoietic stem cell transplantation (HSCT), 0-20 years old donors were perfect MSCs donors who can provide sufficient MSCs in relatively short times. MSCs of group B can be used as stem cell source because the biological characteristics of MSCs of groups B are superior to that of other groups.
Adolescent ; Adult ; Age Factors ; Animals ; Blood Donors ; Bone Marrow Cells ; cytology ; immunology ; metabolism ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Child ; Child, Preschool ; Cytokines ; analysis ; Enzyme-Linked Immunosorbent Assay ; Fetus ; Flow Cytometry ; Humans ; Immunophenotyping ; Infant ; Mesenchymal Stem Cell Transplantation ; methods ; Mesenchymal Stromal Cells ; cytology ; immunology ; metabolism ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Transplantation, Heterologous

This study was aimed to explore the feasibility of transplanting human cord blood stem cells (HSC) into pre-immune fetal and neonatal pigs, and to investigate the self-renewal of HSC in the recipient pigs. The fetus and neonate were manipulated in sterile separated room and human donor cells were injected into fetus via fetus muscle or umbilical vein (dissectted womb) or into neonate via umbilical vein before cutting it. Human CD45(+) cells s were detected by labeling with human anti-CD45 antibody and analyzed by fluorescence activated cell sorting (FACS). The results showed that tested pigs developed as well as control and a definite proportion of human cells existed in peripheral blood of chimeric pig on day 60 after transplantation. In conclusion, the fetus and neonate pigs can tolerate a definite proportion of human antigens, and to establish the human/pig model of hematopoietic chimerism is possible.
Animals ; Animals, Newborn ; Cord Blood Stem Cell Transplantation ; methods ; Fetus ; Flow Cytometry ; Humans ; Leukocyte Common Antigens ; blood ; Models, Animal ; Pilot Projects ; Swine ; Transplantation Chimera ; blood ; immunology ; Transplantation, Heterologous

This study was aimed to explore the role of human fetal bone marrow stromal cells (FBMSC) in combination with exogenous cytokines in supporting the in vitro expansion of cord blood mononuclear cells and the expression of CXCR4(+) and CD49d(+) in CD34(+) cells. Mononuclear cells (MNC) separated from cord blood (CB) were cultured in a serum-free support culture system with FBMSC or exogenous cytokines or both of them. On day 0, 6, 10 and 14, total cells were counted, CD34(+), CD34(+)CXCR4(+) and CD34(+)CD49d(+) cells were quantitated by FACS, and hematopoietic progenitor cells were assessed by semisolid culture assay. The results showed that after culturing for 14 days, CD34(+) cells, CD34(+)CXCR4(+) cells, CD34(+) CD49d(+) cells and colony forming unit (CFU) were significantly increased (P < 0.05). Compared with other groups, expansion multiple of CD34(+), CD34(+)CXCR4(+), CD34(+)CD49d(+) cells and CFU were higher than that in FBMSC and cytokine group (P < 0.05). It is concluded that the culture system used in this study can not only support the expansion of CB MNCs but also increase the number of hematopoietic stem and progenitor cells which has chemokine and adhesion capacity. This culture system may be a feasible way for in vitro culture of cord blood cells.
Antigens, CD34 ; blood ; Bone Marrow Cells ; cytology ; immunology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Coculture Techniques ; Cytokines ; pharmacology ; Fetal Blood ; cytology ; Fetus ; Flow Cytometry ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Integrin alpha4 ; blood ; Interleukin-3 ; pharmacology ; Leukocytes, Mononuclear ; cytology ; immunology ; Receptors, CXCR4 ; blood ; Stromal Cells ; cytology ; immunology ; Time Factors

OBJECTIVETo establish human colorectal crypt cell line.

METHODSColorectal crypt cells were separated from human fetal gut by dispase I digestion, AKP-negative cells from fetal colorectal crypt were collected and cultured on Matrigel matrix. Subsequently the primary cultured cells were transfected with recombinant retrovirus containing human telomerase reverse transcriptase (hTERT) and simian virus 40 large T antigen (SV40 LT) in 48 h. The characterization of immortalized cells was identified after the transfection and cells were screened with antibiotics for 12 approximately 16 weeks and expanded.

RESULTSMucin, cytokeratin-pan, 8, 19 were presented in immortalized cells by immunohistochemical staining; ectopic expressions of both hTERT and SV40 LT were also found in immortalized cells by Western blotting. Agarose electrophoresis showed that the cells expressed Musashi-1 mRNA. No evidence of carcinogenesis was found in nude mouse experiment and soft-agarose cloning test.

CONCLUSIONThe immortalized human colorectal crypt cells were characterized and the established cell line may be an ideal target for carcinogenesis study in vitro.

Cell Line, Transformed ; Colon ; cytology ; DNA-Binding Proteins ; Fetus ; Humans ; RNA, Neoplasm ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Simian virus 40 ; immunology ; Stem Cells ; cytology ; Telomerase ; genetics ; metabolism ; Transcription, Genetic ; Transfection ; methods

Adult ; Child ; Diet ; Female ; Fetus ; immunology ; Humans ; Hypersensitivity ; embryology ; immunology ; Maternal Behavior ; Pregnancy ; immunology ; Risk Factors