Atherosclerosis ; Blood Vessels ; cytology ; Humans ; Stem Cells

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

Hematopoietic stem cells (HSCs) are tissue specific stem cells that replenish all mature blood lineages during the lifetime of an individual. Hematopoietic cell clusters in the aorta of vertebrate embryos play a pivotal role in the formation of the adult blood system. Recently, people have learned a lot about the embryonic HSCs on their development and homing. During their differentiation, HSCs are regulated by the transcription factors, such as Runx1 and Notch signaling pathway, etc. MicroRNAs also regulate the self-renewal and differentiation of hematopoietic stem/progenitor cells on the post-transcriptional levels. Since the onset of circulation, the formation of HSCs and their differentiation into blood cells, especially red blood cells, are regulated by the hemodynamic forces. It would be of great significance if we could treat hematologic diseases with induced HSCs in vitro on the basis of fully understanding of hemotopoietic stem cell development. This review is focused on the advances in the research of HSCs' development and regulation.
Blood Cells ; cytology ; Cell Differentiation ; Embryonic Stem Cells ; cytology ; Hematopoietic Stem Cells ; cytology ; Humans ; Signal Transduction ; Transcription Factors ; physiology

This study was aimed to construct a two-dimensional culture system by using osteoblasts induced from human marrow mesenchymal stem cells (MSC) and to investigate its support effect on survival of hematopoietic stem/progenitor cells for umbilical cord blood (UCB) ex vivo. MSCs were isolated from adult human bone marrow and were cultured, the second generation of MSCs were induced into osteoblasts which were irradiated with 20 Gy gamma rays in a Cobalt 60 source and confluenced into a feeder layer. CD34(+) cells were selected from fresh umbilical cord blood samples by using Microbead Kit of MiniMACS and seeded into the two-dimensional culture system to culture ex vivo without exogenous cytokines. By using colony-forming assay, high proliferative potential colony-forming cell assay, and long-term culture initiating cell assay, the ability of the two-dimensional system to culture HSCs/HPCs was observed. The results showed that the osteoblasts induced from bone marrow MSC in constructed two-dimensional culture system displayed more significant support effect on survival of hematopoietic stem/progenitor cells from umbilical cord blood (UCB) ex vivo, compared with other culture systems, especially on long term HSCs survival ex vivo. It is concluded that the two-dimensional culture system constituted by osteoblasts induced from human MSCs has certain ability of supporting maintenance and multipotency of HSCs/HPCs from umbilical cord blood in vitro, especially sustaining survival of HSC in long-term culture. It has also been proved that osteoblasts play a crucial role in regulation of HSC growth.
Bone Marrow Cells ; cytology ; Cell Differentiation ; Coculture Techniques ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; Humans ; Mesenchymal Stromal Cells ; cytology ; Osteoblasts ; cytology ; physiology

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

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

Blood Cells ; cytology ; Bone Marrow Cells ; cytology ; China ; Hematologic Tests ; standards ; 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

Clinical evidence of hematopoietic restoration with umbilical cord blood (UCB) grafts indicates the UCB can be a useful source of hematopoietic stem cells for routine bone marrow reconstitution. Considering (10 +/- 5) x 10(8) nucleared cells per cord blood unit, there is a potential limitation for the use of cord blood in adults, which, however, can be overcome by ex vivo expansion of cells. A prerequisite for expansion is the significantly higher recovery of MNC, CD34+ cells and colony-forming cells (CFC) by thawing cryopreserved MNC. Cooling rate always acts as a critical factor that can affect the recovery of cells. Although the rate of - 1 degrees C/min is adopted in most of the cryopreservations, no data has been reported about the detailed effects of different cooling rates. The aim of the study was to reveal the different effects of cooling rates on cryopreservation of hematopoietic stem cells from cord blood. UCB samples were collected, and cryopreserved as mononuclear cells (MNC) with different cooling rates of - 0.5 degrees C/min, - 1 degrees C/min, - 5 degrees C/min, and the recovery and viability of MNC and CD34+ cells, the clonogenic capacity and the ex vivo expansion potential of UCB progenitor cells were evaluated after thawing. With - 1 degrees C/min cooling rate, the recovery of MNC reached 93.3% +/- 1.8% , viability 95.0% +/- 3.9% , recovery of CD34+ cells 80.0% +/- 17.9% , and clonogenic recovery were 87.1% +/- 5.5%, 88.5% +/- 8.9%, 86.2% +/- 7.4% for BFU-E CFU-GM CFU-MK, respectively. After 14 days of liquid culture, no significant difference was detected in CFC expansion between fresh and cryopreserved MNC cells with - 1 degrees C/min cooling rate, but this was not the case with - 0.5 degreesC/min and - 5 degrees C/min. In conclusion, it was demonstrated that controlling the rate at - 1 degrees C/min is more suitable for cryopreservation of hematopoietic stem cells than - 0.5 degrees C/min and - 5 degrees C/min.
Cell Survival ; physiology ; Cells, Cultured ; Cryopreservation ; methods ; Erythroid Precursor Cells ; cytology ; Fetal Blood ; cytology ; Flow Cytometry ; Granulocyte-Macrophage Progenitor Cells ; cytology ; Hematopoietic Stem Cells ; cytology ; Humans

OBJECTIVETo perform in vitro magnetic resonance imaging on magnetic iron oxide (Fe(2)O(3)-PLL) labeled rabbit peripheral blood endothelial progenitor cells (EPCs).

METHODSFe(2)O(3) was incubated with PLL for 2 hours to form Fe(2)O(3)-PLL. Rabbit peripheral blood mononuclear cells (MNCs) were isolated and EPCs were selected by adherence method, expanded and incubated with Fe(2)O(3)-PLL. Intracellular iron was detected by Prussian blue stain and under electron microscope. MTT assay was used to evaluate cell survival and proliferation of Fe(2)O(3)-PLL labeled EPCs. Flow cytometry was used to analysis cell cycle and apoptosis. The cells underwent in vitro MR imaging with various sequences.

RESULTSIron-containing intracytoplasmatic vesicles could be observed clearly with Prussian blue staining and electron microscope observation. Survival, life cycle and apoptosis values obtained by MTT and flow cytometry analysis were similar among unlabelled EPCs and EPCs labeled with various concentrations Fe(2)O(3)-PLL. The signal intensity on MRI was significantly decreased in labeled cells compared with that in unlabeled cells. The percentage change in signal intensity (DeltaSI) was most significant on T(2)*WI and DeltaSI was significantly lower in cells labeled for 7 days than that labeled for 1 day.

CONCLUSIONSThe rabbit peripheral blood EPCs can be labeled with Fe(2)O(3)-PLL without significant change in viability and proliferation. The labeled EPCs can be imaged with standard 1.5 T MR equipment. The degree of MR signal decreasing may indirectly reflect the cells count, growth state and division.

Animals ; Biomarkers ; Blood Cells ; Cells, Cultured ; Endothelial Cells ; cytology ; Ferric Compounds ; Magnetic Resonance Imaging ; methods ; Male ; Rabbits ; Stem Cells ; cytology