Human lipocalin 6 (hLCN6) is an epididymis-specific secretory protein. It binds to sperm and plays important role in sperm maturation. To explore the feasibility for isolating spermatozoa from mixed cells using anti-hLCN6 monoclonal antibody-conjugated immunomagnetic beads (anti-hLCN6 IMBs) and establish a new method for the separation of sperms from mixed stains, 2 sets of 30 cases of cell mixture suspensions and stains containing different proportions of sperm and epithelial cells were prepared. Biotin-labeled anti-hLCN6 monoclonal antibody (mAb) was incubated with the cell mixtures, and the spermatozoa were then isolated with avidin-coated IMBs. Sperm DNA was extracted and analyzed by PCR-STR typing. Differential lysis was also conducted to compare the effect of the two different isolation methods. The dissociation constant (Kd) of anti-hLCN6 mAb was 3.47×10⁻⁹ mol/L measured by ELISA. Western blotting and immunofluorescence assays showed that hLCN6 was detectable on sperm cells and mainly located on the post-acrosomal region of the sperm head, but not in epithelial cells. Anti-hLCN6 IMBs could capture and separate the sperm cells successfully. Microscopic observation showed that the IMBs could bind to the head of sperm specifically. The success rate of STR typing (more than 13 STR loci, RFU>200) was 90% when the number of sperm cells was 10³/mL and 100% when the sperm cells number was equal to or more than 10⁴/mL. When the number of sperm cells was 10³/mL, 10⁴/mL and 10⁵/mL in mixed stain samples, the success rate of STR typing were 40%, 90% and 100%, respectively. Taken together, the anti-hLCN6 immunomagnetic beads (IMB) method described here could be effective for the isolation of sperm from mixed cells, and the success rate was higher than that of the traditional differential lysis strategy. IMB sorting is a simple and efficient method for the separation of sperms from sperm and epithelial cell mixture, and can be utilized as a supplementary method for forensic mixture samples analysis in sexual assault cases.
Cell Separation ; DNA ; Humans ; Immunomagnetic Separation ; Lipocalins ; Male ; Polymerase Chain Reaction ; Spermatozoa

OBJECTIVETo isolate and identify the cancer stem cells from primary human ovarian cancer tissues.

METHODSFresh tumor tissues from five cases of pathologically diagnosed ovarian cancers were taken, minced and then digested with collagenase and hyaluronidase to obtain single cell suspension. The erythrocytes were removed with ACK Lysis buffer. The suspensions were sorted by magnetic activated cell sorting (MACS) using CD133-binding microbeads. Then the sorted CD133(+) cells were verified by flow cytometry. The cells were cultured in serum-free medium supplemented with EGF, bFGF, insulin and BSA, and grew into spheroids. Immunofluorescence, differentiation and tumor formation tests of the cells were performed to characterize the properties of cancer stem cells.

RESULTSThe ovarian cancer stem cells were successfully isolated from primary human ovarian tumors, which formed typical spheroids in serum-free medium and had stronger ability of tumorigenesis. The results of related experiments verified that CD133 positive cells owned the properties of cancer stem cells.

CONCLUSIONSThe ovarian cancer stem cells presenting the characteristics of stemness in vitro and in vivo, have been successfully isolated from primary human ovarian tumor tissues by MACS. The isolated ovarian cancer stem cells could be used in future researches of their biological functions.

AC133 Antigen ; Animals ; Antigens, CD ; metabolism ; Cell Differentiation ; Cell Separation ; methods ; Female ; Flow Cytometry ; methods ; Glycoproteins ; metabolism ; Humans ; Immunomagnetic Separation ; methods ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Neoplasm Transplantation ; Neoplastic Stem Cells ; metabolism ; pathology ; Ovarian Neoplasms ; metabolism ; pathology ; Peptides ; metabolism

Hematopoietic stem cells (HSC), the well-characterized adult stem cells both in the markers and function, show tremendous therapeutic potential. However, the level of hematopoietic stem cells in bone marrow is very low, which makes it difficult to work with. Based on cell surface markers and dye staining, HSC can be isolated from bone marrow and peripheral blood by using magnetic-activated cell separation and fluorescence-activated cell sorting. Over 40 years of research, many surface markers have been identified to purify mouse HSC, and CD34(-)LSK cells are regarded as the mostly used enrichment of HSC in mouse bone marrow. The purpose of this review is to provide insight into the advance of these markers, and isolation and purification of HSC.
Animals ; Cell Separation ; Flow Cytometry ; Hematopoietic Stem Cells ; cytology ; Mice

Since flow cytometry was not feasible for sorting a huge amount of cells for clinical use, the method of double immunomagnetic positive sorting was used for selection of CD34(+)CD59(+) cells from bone marrow mononuclear cells in patients with paroxysmal nocturnal hemoglobinuria (PNH), which laid the groundwork for clinical ABMT/APBSCT of patients with PNH. Immunomagnetic positive selection was used for two times, the microbeads were removed from the CD34(+) cells selected firstly by means of overnight culture, then the sufficient CD34(+)CD59(+) cells were used for ex vivo expansion. The results showed that the survival, proliferation and colony-forming units of the selected CD34(+)CD59(+) cells by double immunomagnetic positive sorting had no significant difference as compared with that of CD34(+)CD59(+) cells selected by flow cytometry technique. It is suggested that the double immunomagnetic positive sorting promotes the use for separation and purification hematopoietic stem/progenitor cells and other cells with double or multiple markers cells for autologous hematopoietic stem cell transplantation in PNH patients.
Antigens, CD34 ; analysis ; Bone Marrow Cells ; cytology ; CD59 Antigens ; analysis ; Flow Cytometry ; Hematopoietic Stem Cell Transplantation ; Hemoglobinuria, Paroxysmal ; therapy ; Humans ; Immunomagnetic Separation ; Transplantation, Autologous

Using a fluorochrome Calcein-AM, leukemia cells were labeled and seeded into cell lines or bone marrow cells to establish three cell-models of grafts with leukemia. These cell-models were engaged with CD34 immunomagnetic beads and the purging efficacy was evaluated using both fluorescence microscopy and flow cytometry. The results showed that the cell-models established in this study could be evaluated successfully not only with fluorescence microscopy but also flow cytometry. After CD34 positive selection, KG1a cells were removed by (0.98 +/- 0.09) log in model II and NALM-6 cells were removed by (1.82 +/- 0.51) log in model III, respectively. It is concluded that the models established in this study are stable and direct with an excellent reproducibility and an accuracy, which can be used to evaluate purging efficacy of leukemia cells in model graft using immunomagnetic selection and the experimental studies on tumorcidal effect in vitro.
Bone Marrow Purging ; Flow Cytometry ; Hematopoietic Stem Cell Transplantation ; Humans ; Immunomagnetic Separation ; methods ; Leukemia ; pathology ; therapy ; Microscopy, Fluorescence ; Models, Biological ; Transplantation, Autologous

OBJECTIVETo isolate stem cells from human exfoliated deciduous teeth (SHEDs) and identify their phenotypes and multi-lineage differentiation potential.

METHODSHuman pulp tissue from exfoliated deciduous teeth were dissected and digested to obtain the single cell suspension. The SHEDs selected by magnetic activated cell sorting system (MACS) were identified by examination of the cell morphology and growth in vitro and detection of the expressions of the cell markers. Osteogenic and adipogenic induction was performed to test the multi-lineage differentiation potential of the cells.

RESULTSSHEDs were successfully isolated from human exfoliated deciduous teeth. SHEDs showed a lower growth rate than dental pulp cells and displayed high expressions of CD29 and CD105 but low expressions of CD34 and CD45 as shown by flow cytometry. Experiments of in vitro induction demonstrated a strong potential of the STRO-1+ SHEDs for osteogenic and adipogenic differentiation.

CONCLUSIONImmunomagnetic bead selection can be used to isolate and purify SHEDs, and the STRO-1+ SHEDs show the characteristics of stem cells with multipotent differentiation potentials.

Cell Separation ; Cells, Cultured ; Dental Pulp ; cytology ; Humans ; Immunomagnetic Separation ; methods ; Stem Cells ; cytology ; Tooth, Deciduous ; cytology

OBJECTIVETo study the isolation, culture and identification of CD133+ endothelial progenitor cells (EPCs) from human umbilical cord blood in vitro.

METHODSEPC separation was performed with density gradient centrifugation and MACS separation. Purity of EPCs was determined by flow cytometry. EPC was cultured with EBM-2 to study the cultivate features of EPC. Uptake test of Dil-LDL and FITC-Lectin and immunohistochemistry were performed.

RESULTSAccording to flow cytometry, (1.13 +/- 0.10)% of mono-nuclear cells were CD133+ and the purities of CD133+ EPCs were (91.45 +/- 1.04)% on average. CD133+ EPCs became adherent, spindle-shaped and formed cluster during culture. Uptake test of Dil-LDL and FITC-Lectin were positive. (95.83 +/- 1.72)% of CD133+ cells were found positive in both uptake tests. The positive rates of immunostaining of cell markers CD34 and factor VIII were (95.83 +/- 2.23)% and (95.92 +/- 1.43)% after cultured for one week, which showed no significant differences between CD133+ EPCs and human umbilical vein endothelial cells. Capillary structures were formed by CD133+ EPCs after cultured for 4 and 7 d in vitro.

CONCLUSIONSHigh purity of CD133+ EPCs can be obtained by MACS separation. CD133+ EPCs can differentiate into mature endothelial cells with the effects of stimulating factors.

AC133 Antigen ; Antigens, CD ; analysis ; blood ; Cell Culture Techniques ; Cell Differentiation ; Cells, Cultured ; Endothelial Cells ; cytology ; immunology ; Fetal Blood ; cytology ; immunology ; Flow Cytometry ; Glycoproteins ; analysis ; blood ; Humans ; Immunomagnetic Separation ; Peptides ; analysis ; blood ; Stem Cells ; cytology ; immunology

OBJECTIVEThe very nature of spermatogonial stem cells (SSC) is still poorly understood. The objective of this study is to explore the specific markers of human SSC and search for the suitable method for their isolation and functional identification.

METHODSAdults testicular cell suspensions were sorted by immunomagnetic beads method using alpha6,beta1 integrin and Thy-1 markers. The light-scattering properties and DNA Ploidy of the resultant subpopulations were analyzed by flow cytometry. The efficacies of the sorting on human SSC were evaluated by germ cell transplantation.

RESULTS(1) The alpha6+ Thy-1+ c-kit- and beta1+ Thy-1+ c-kit- cells were relatively uniform subpopulations in size and morphology, which represented about 2%-3% and 0.5%-1% of the unsorted testis cells, respectively. The analysis of light-scattering properties showed that both of the subpopulations had low side light-scattering properties. The DNA Ploidy analysis showed significant changes of these two cell subpopulations in DNA Ploidy. The percentage of diploid cells in alpha6+ Thy-1+ c-kit- cell subpopulation significantly increased to 51.2% and synthesis phase and tetraploid cells disappeared. (2) The functional evaluation showed that the SSC in the alpha6+ Thy-1+ c-kit- cells were enriched 40 times and the SSC in the beta1+ Thy-1+ c-kit- cells 20 times that of the unsorted cells.

CONCLUSIONThe alpha6,beta1 integrin and Thy-1 may be used for the SSC isolation as positive markers. The immunomagnetic beads sorting using alpha6,beta1 integrin and Thy-1 markers can result in significant enrichment of human SSC. It will open up a wide prospect for the researches on the biology of human SSC and the treatment of male sterility.

Adult ; Animals ; Cells, Cultured ; Flow Cytometry ; Humans ; Immunomagnetic Separation ; methods ; Integrin alpha6 ; analysis ; Integrin beta1 ; analysis ; Male ; Mice ; Spermatogonia ; cytology ; Stem Cell Transplantation ; Stem Cells ; cytology ; Testis ; cytology ; Thy-1 Antigens ; analysis

BACKGROUND AND OBJECTIVES: Lack of understanding of the interplay between hematopoietic stem cells (HSCs) and the immune system has severely hampered stem cell research. Programmed death-1 (PD-L1) has been reported on parenchymal cells in patients with chronically inflamed livers and found to play an essential role in T cell homeostasis regulation. However, the bidirectional interaction between HSCs and lymphocytes remains elusive. Here, we aimed to get more insight into circulating CD34+ HSCs PD-L1 expression and T cell apoptosis in chronic HCV infected patients. METHODS: CD34+ HSCs were isolated and purified by immunomagnetic separation. PD-L1 expression was analyzed by quantitative PCR and flow cytometry. Furthermore, co-culture experiments between CD34+ HSCs and T-lymphocytes were established. T-cell lymphocyte apoptosis in peripheral blood and in cultures was detected. RESULTS: CD34+ HSCs constitutively express low levels of PD-L1. Its expression is up-regulated in chronic HCV infected patients. Moreover, PD-L1 expression on circulating CD34+ HSCs enhanced T cell apoptosis in peripheral blood and co-culture. CONCLUSION: Our results suggest novel bidirectional interplay between HSCs and lymphocytes mediated by PD-L1 expression on CD34+ HSCs. PD-L1 expression correlated with T-cell lymphocyte apoptosis. This may contribute to immunomodulatory properties of HSCs which improves its use for allogeneic transplantation.
Apoptosis ; Coculture Techniques ; Flow Cytometry ; Hematopoietic Stem Cells ; Hepatitis C, Chronic ; Hepatitis, Chronic ; Homeostasis ; Humans ; Immune System ; Immunomagnetic Separation ; Liver ; Lymphocytes ; Polymerase Chain Reaction ; Stem Cell Research ; T-Lymphocytes ; Transplantation, Homologous

OBJECTIVETo optimize the protocols for isolation and culture of mesenchymal stem cells from rat bone marrow (BMSCs).

METHODSBMSCs were isolated by adherence to plastic with frequent medium change and reduced trypsinization time. The cell growth curves were drawn and the surface markers of BMSCs were detected by flow cytometry. The cells were induced to differentiate into osteogenic, adipogenic, hepatic and cholic lineages.

RESULTSThe cells isolated using this method were positive for CD29, CD44, and CD90 and negative for the hematopoietic surface markers CD45. The osteogenic and adipogenic differentiation of the BMSCs was verified by alkaline phosphatase staining, Alizarin red staining and Oil red staining. The cell subcultures up to passage 10 maintained capacities of differentiation into osteogenic and adipogenic lineages. The BMSCs induced with sequential addition of growth factors, cytokines and hormones differentiated into cells expressing hepatocyte- and cholangiocyte-specific markers.

CONCLUSIONThe optimized method allows efficient isolation of homogenous populations of MSCs from rat bone marrow, which can be induced into multiple cell lineages.

Animals ; Cell Culture Techniques ; Cell Differentiation ; Cell Proliferation ; Cell Separation ; Flow Cytometry ; Mesenchymal Stromal Cells ; cytology ; Rats