Human umbilical cord blood-derived mesenchymal stem cells (MSCs) are known to possess the potential for multiple differentiations abilities in vitro and in vivo. In canine system, studying stem cell therapy is important, but so far, stem cells from canine were not identified and characterized. In this study, we successfully isolated and characterized MSCs from the canine umbilical cord and its fetal blood. Canine MSCs (cMSCs) were grown in medium containing low glucose DMEM with 20% FBS. The cMSCs have stem cells expression patterns which are concerned with MSCs surface markers by fluorescence-activated cell sorter analysis. The cMSCs had multipotent abilities. In the neuronal differentiation study, the cMSCs expressed the neuronal markers glial fibrillary acidic protein (GFAP), neuronal class III beta tubulin (Tuj-1), neurofilament M (NF160) in the basal culture media. After neuronal differentiation, the cMSCs expressed the neuronal markers Nestin, GFAP, Tuj-1, microtubule-associated protein 2, NF160. In the osteogenic & chondrogenic differentiation studies, cMSCs were stained with alizarin red and toluidine blue staining, respectively. With osteogenic differentiation, the cMSCs presented osteoblastic differentiation genes by RT-PCR. This finding also suggests that cMSCs might have the ability to differentiate multipotentially. It was concluded that isolated MSCs from canine cord blood have multipotential differentiation abilities. Therefore, it is suggested that cMSCs may represent a be a good model system for stem cell biology and could be useful as a therapeutic modality for canine incurable or intractable diseases, including spinal cord injuries in future regenerative medicine studies.
Animals ; *Cell Differentiation ; Chondrogenesis ; Dogs/blood/*physiology ; Fetal Blood/*cytology ; Mesenchymal Stem Cells/*cytology ; Neurons/cytology ; Osteogenesis

To investigate the effect of mycobacterium phlei F.U.36 suspended liquor (Utilin"s", U) on the culture and proliferation of dendritic cells (DCs) derived from human umbilical cord blood in vitro, the mononuclear cells (MNCs) were isolated from human umbilical cord blood and cultured with RPMI 1640 in the control group. Test groups consisted of Utilin"s" group (only Utilin"s"), GTI group (GM-CSF, TNF-alpha, IL-4) and GTIU group (GM-CSF, TNF-alpha, IL-4 and Utilin"s"). MNCs in all test groups were cultured with RPMI-1640. The growth of DCs was observed by the light microscopy, the phenotypes of DCs were determined by flow cytometry on the 10th day of culture, and some harvest cells were stained with Wright-Giemsa, then observed and photographed under the oil immersion objective. The results showed that the test groups all displayed some number of typical DCs; both CD1a positive cell rate and HLA-DR positive cell rate of the Utilin"s" group were higher than those of the control; HLA-DR positive cell rate of GTIU group increased most significantly and much higher than that of the GTI group. It is concluded that mycobacterium phlei F.U.36 not only promotes the proliferation of DCs derived from human umbilical cord blood in vitro, but also co-operates with rhGM-CSF, rhTNF-alpha and rhIL-4 in promoting the maturity of DCs.
Cell Proliferation ; Cells, Cultured ; Dendritic Cells ; cytology ; Fetal Blood ; cytology ; Humans ; Mycobacterium phlei ; physiology

OBJECTIVEThis study was designed to investigate the effect of human cytomegalovirus (HCMV) on the proliferation of colony forming unit granulocyte-macrophage (CFU-GM), CFU-erythroid (CFU-E), burst forming unit-erythroid (BFU-E), CFU-multipotential (CFU-Mix) and CFU-megakaryocytic (CFU-Mk) progenitor cells of cord blood in vitro as well as the possible mechanism.

METHODSTwenty cord blood specimens were collected from the umbilical vein of normal full-term neonates delivered spontaneously. This study consisted of five groups: 3 Infection groups in which 0.1 mL 10(3), 10(4) and 10(5) plague forming unit (PFU) HCMV-AD169 virus solution was added to the culture system, an Inactivated control group in which the equal volume of inactivated virus solution was added, and a Blank control group (normal progenitor cells culture system without HCMV virus infection). Colony forming unit-assay was applied to detect the effects of HCMV-AD169 strain on the colony formation, inhibition rate and colony-maintaining duration of CFU- GM, CFU-E, BFU-E, CFU-Mix and CFU-Mk of cord blood. PCR technique was used to demonstrate the existence of HCMV-DNA in the colony cells of cultured CFU-GM, CFU-E, CFU-Mix and CFU-Mk.

RESULTSHCMV-AD169 (10(3)PFU) in low concentration had inhibition effects on colony formation of the CFU-Mix and CFU-Mk (P < 0.05), whereas 10(5) PFU and 10(4) PFU HCMV-AD169 lead to decreased colonies in CFU-GM, CFU-E, BFU-E, CFU-Mix and CFU-Mk compared with the Blank control and the Inactivated control groups (P < 0.05). The suppression effect of HCMV on the colony formation was dose-dependent. The colony-maintaining duration of the CFU-GM, CFU-E, BFU-E, CFU-Mix and CFU-Mk in the 10(5) PFU and 10(4) PFU HCMV infection groups was significantly shorter than that in the two control groups (P < 0.01). The low concentration of HCMV-AD169 (10(3)PFU) infection resulted in a shortened colony-maintaining duration of the CFU-Mix and CFU-Mk (P < 0.01), but had no effects on the colony-maintaining duration of CFU-GM, CFU-E and BFU-E. PCR amplification demonstrated the existence of HCMV-AD169 DNA in the colony cells of the three Infection groups.

CONCLUSIONSHCMV-AD169 strain can infect hematopoietic progenitors of cord blood and inhibit the proliferation of hematopoietic progenitors, associated with anemia, neutropenia and thrombocytopenia in HCMV patients.

Cell Proliferation ; Cytomegalovirus ; pathogenicity ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; Humans

In order to study the influence of different gelatin concentrations, and lymphocyte isolation liquid on primary culture of umbilical cord blood-derived adhesive cells (hCBACs), the red blood cells of umbilical cord blood was separated by 3% and 6 % gelatin for detecting the effectiveness of sedimentation, then the adhesion rate at 48 hours, the day of initial expansion and the rate of culture success were detected for hCBACs cultured with CD34(+) cells after the mononuclear cells were separated by 6% gelatin followed by Ficoll and Percoll, and the morphological characteristics and growth status were observed by invert microscopy. Cytochemistry stain for nonspecific esterase stain (NSE), peroxidase (POX), periodic acid Schiff reaction (PAS) and alkali phosphatase (ALP) and immunocytochemistry labeling for CD31, CD45, CD68 and fibronectin (Fn) were detected. The results showed that 6 % gelatin was better than that 3% gelatin for red blood sedimentation. The Percoll was predominant over Ficoll in adhesion rate at 48 hours, the day of initial expansion, the time of initial formation of adhesive cell colony units, the time of maximal numbers of adhesive cell colony units, the the cell fusion time and ratio of culture success. 60% fibroblast-liked cells, 36% macrophage liked cells and 4% small-round cells were observed in cells isolated by both isolated methods. The cytochemistry stain for NSE, POX, PAS and ALP was similar in two groups, the difference was not statistically significant between these two groups. The immunocytochemistry labeling for CD31, CD45, CD68 and Fn was also similar in both groups and the difference was also not statistically significant between these two groups. It is concluded that the combination of 6% gelatin with Percoll is an ideal separation method for primary culture of hCBACs, which provides basic information for clinical application.
Cell Separation ; methods ; Cells, Cultured ; Fetal Blood ; cytology ; Gelatin ; administration & dosage ; pharmacology ; Humans ; Lymphocytes ; cytology

OBJECTIVETo investigate the mRNA expression profiles of megakaryocytes (MKs) from human cord blood CD34+ cells ex vivo expanded using Solexa technique.

METHODSCD34+ Cells were isolated using density gradient centrifugation and magnetic activated cell sorting. Cultures were stimulated with recombinant human thrombopoietin (100 ng/ml). After 12 days, the MKs fraction was separated from the non-MKs fraction using an anti-CD41 monoclonal antibody by immunomagnetic sorting. The mRNA expression of MKs and non-MKs was detected by Solexa sequencing.

RESULTSWe obtained 3 773 147 and 3 533 805 Tags from MKs and non-MKs, respectively. The amounts of unambiguous tags were 3 291 132 and 2 967 947 and those of distinct tags were 197 769 and 245 318. The expression of 1161 genes was up-regulated and that of 902 genes down-regulated. The expression of 2717 tags was up-regulated and that of 1519 tags down-regulated.

CONCLUSIONSMKs and non-MKs have remarkably different mRNA expression profiles. The differential gene-encoded products may be involved in cellular development, adhesion, apoptosis metabolism, intra- and intercellular signal transduction, and immune response. Further studies on this topic may clarify the expression mechanism, signal transduction, and regulation mechanisms.

Antigens, CD34 ; Cells, Cultured ; Fetal Blood ; cytology ; Humans ; Megakaryocytes ; cytology ; metabolism ; RNA, Messenger ; genetics ; Transcriptome

To optimize the culture environment and protocol of hematopoietic cells' expansion, avoiding the fluctuation caused by medium changing in stirred culture and concentration gradient in static culture, the hematopoietic cells from cord blood (CB) were cultured in a stirred bioreactor connected with a cell retention system, which is a gravity sedimentation settler designed for hematopoietic cell. Total cells expanded 11.5 and 18.6 fold respectively in the twice perfusion stirred cultures, in which CFU-Mix was expanded 23.2 and 20.4 fold, CFU-GM 13.9 fold and 21.5 fold, BFU-E 8.0 fold and 6.9 fold, CD34+ cells 17.1 fold and 15.4 fold. After 12-day culture, it was obtained that 1082 x 10(6) total cells, 6.31 x 10(6) CFU-GM, 6.2 x 10(6) CFU-Mix and 23 x 10(6) CD34+ cells from 267 x 10(6) CB mononuclear cells (MNC) in the first culture, and 1080 x 10(6) total cells, 4.65 x 10(6) CFU-GM, 11.0 x 10(6) CFU-Mix, and 25.0 x 10(6) CD34+ cells from 180 x 10(6) CB MNC. These two cultures met to the clinical scale. Due to the optimized dissolved oxygen (DO) and stable culture environment, the rate of stem/progenitor cells to total cells in the perfusion culture was higher than that in T-flask cell-retention feeding culture. But the cell growth was inhibited in the later phase of perfusion culture, when the cell density is high. The inhibition should be attribute to the high cell density itself. The perfusion culture environment in bioreactor with optimal DO and pH controlling is more favorable for stem/progenitor cells' maintenance and expansion, and the expanded cells' number has reached a clinical scale. But the high cell density in the later phase of perfusion culture caused inhibition to mature hematopoietic cell's growth.
Bioreactors ; Cell Culture Techniques ; methods ; Cells, Cultured ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; Humans

AIMTo provide experimental basis for clinical use of cryopreserved dendritic cells (DCs) by investigating the biological properties of cryopreserved DCs derived from cord blood.

METHODSThe biological discrepancies between unfrozen DCs (UFDC) derived from unfrozen cord blood (UFCB) and DCs from cryopreserved cord blood (CPCB) or cryopreserved DCs (CPDCs) were explored by morphological observation and immunophenotype analysis. Moreover, the stimulating index (SI) in mixed lymphocyte culture and cytotoxic eliminating rate (ER) were measured by MTT.

RESULTSThe TBR of CPCB and CPDCs were 95.8% and 88.7% respectively. Compared to UFCB, CPCB differentiated toward DCs in a delayed and decreased mode, displayed lower expression of CD1a, CD83 and HLA-DR, and had reduced SI and ER. Similarly, the CPDCs became adherent DCs later and grew less in number than UFDCs. After culture, the expression of CD1a, CD83 and HLA-DR as well as SI and ER were lower in CPDCs than those in UFDCs.

CONCLUSIONThough the biological properties of CPCB and CPDCs were injured after cryopreservation, they still had relatively complete basic function. Their recoveries rate were > 85%.

Cell Proliferation ; Cells, Cultured ; Cryopreservation ; Dendritic Cells ; cytology ; Fetal Blood ; cytology ; Humans

This study was aimed to investigate a beneficial approach for resolving the deficiency of blood source, preventing the infection resulting from blood transfusion and overcoming the knotty match of patients with rare blood group by using massive expansion of erythroid cells from cord blood CD34(+) cells in vitro. The CD34(+) cells from human cord blood were cultured in serum-free medium supplemented with stem cell factor (SCF), interleukin-3 (IL-3) and erythropoietin (EPO) for 1 week, then expansion and differentiation of CD34(+) cells into erythroid cells were supported by co-culture with human mesenchymal stem cells (MSCs) derived from bone marrow for 2 weeks. The results indicated that after culture for 23 days, the expansion multiple of total cell number reached 2.52 x 10(5), and over 95% of these cells were erythroid cells as compared with less than 1% of myelomonocytic (CD14(+) or CD15(+)) cells and megakaryocytic (CD41(+)) cells. However, the culture system without MSC support was significantly disadvantaged both in expansion ability and ratio of erythroid cells when compared with MSC supporting system. It is concluded that the erythroid cells can be produced from CD34(+) cells in large scale by culturing in the system comprised of cytokine sets and MSC feeders, in which MSCs can support the proliferation and differentiation of erythroid cells.
Antigens, CD34 ; Cell Culture Techniques ; methods ; Cell Differentiation ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; Humans

This study was purposed to isolate human embryonic AGM derived HSPCs and investigate the effect of AGM stromal cells on AGM-derived HSPCs. Immunohistochemical sections of human AGM tissue were investigated for CD34, Flk-1 and VEGF expression. Human AGM-derived single cells were isolated and seeded onto pre-treated feeder of human AGM stromal cells (hAGMS3 and hAGMS4) by direct contact and non-contact co-culture in Transwell culture system. Growth characteristics of HSPCs with cobblestone area-forming cells (CAFCs) were observed and number of cobblestone area (CA) was counted. Indirect immunofluorescent assay was used to detect CD34 and Flk-1 expression on the surface of suspended cells as well as CAFCs in contact co-culture system. The cells after culture for 2 weeks were collected from both contact and non-contact co-culture systems for CFU assay. The result showed that hematopoietic cells in AGM tissue expressed CD34 and Flk-1. Both of the hematopoietic culture systems could produce CFCs. Nevertheless, direct contact co-culture produced CD34(+)Flk-1(+) CAFC and more CFUs than those from indirect non-contact culture (hAGMS3 system: 1647 +/- 194 vs 389 +/- 31, p < 0.05; hAGMS4 system: 1586 +/- 75 vs 432 +/- 35, p < 0.05). It is concluded that there were CD34(+)Flk-1(+) HSCs in human embryonic AGM region. The hematopoietic co-culture systems composed of AGM-derived HSPCs and AGM stromal cells are successfully established, both direct contact and Transwell non-contact co-culture can expand AGM-derived definitive HSPCs. Cell-cell contact between AGM-derived HSPCs and AGM stromal cells are of most importance to maintain and expand AGM-HSPCs.
Aorta ; cytology ; Cell Culture Techniques ; methods ; Cell Separation ; Cells, Cultured ; Coculture Techniques ; Fetal Blood ; cytology ; Gonads ; cytology ; Hematopoietic Stem Cells ; cytology ; Humans ; Mesonephros ; cytology ; Stromal Cells ; cytology ; physiology

BACKGROUND: In the public cord blood (CB) banks, only safe CB units with adequate cell doses are processed and stored. Complete blood count (CBC) of CB is crucial for estimating total nucleated cells (TNC) and screening suitable CB units without hematologic abnormalities. We analyzed CBC parameters of the donated CB from healthy Korean neonates to establish CBC reference values. METHODS: A total of 2,129 Korean CB units, donated and processed during the period from August 2007 to December 2007, were enrolled. We measured hemoglobin (Hb), white blood cell (WBC) count, differential count of WBC, platelets and nucleated red blood cell (nRBC) count by XE-2100 automated hematology analyzer (Sysmex, Japan), and estimated reference value of each parameter by using parametric (Mean+/-2SD) and/or non-parametric methods (2.5-97.5 percentile). And also, we compared the result of each parameter in relation to sex of neonates and delivery method. RESULTS: Because the differences of CBC values among different subgroups were not remarkable, we established the reference intervals as follows without subgroup division: Hb, 9.0-14.4 g/dL; WBC count, 5.6-18.5x103/microL; differential count of WBC (neutrophils, 40.8-72.4%; lymphocytes, 17.2-46.7%; monocytes, 4.9-12.8%; eosinophils, 0.7-7.0%; basophils, 0.0-1.6%); platelet, 130-287x103/microL; nRBCs, 0.0-13.1/100 WBC. CONCLUSIONS: We established cord blood CBC reference values of healthy Korean neonates using a large-scale CB units. The established CBC reference values from our study will be useful as basic data for CBC interpretation and assessment of transplant suitability of donated CB.
Blood Cell Count/*standards ; Female ; Fetal Blood/*cytology ; Humans ; Infant, Newborn ; Korea ; Male ; Reference Values