OBJECTIVEThough the rabbit is one of most widely used experimental animals for medical regenerative research, it remains difficult to culture mesenchymal stem cells (MSC) on a in large scale due to the extremely lower number and hematopoietic cell contamination. This study was aimed to establish a novel protocol to generate rabbit MSC by culturing bone marrow plugs instead of bone marrow cells so as to obtain a large amount of MSC with higher proliferation and self-renewal properties.

METHODSThe primary MSC were generated from collagenase digested bone marrow plugs and bone marrow cells, respectively. The surface antigen profile of MSC was analyzed with flow cytometry and the cells were induced to differentiate into osteoblasts and adipocytes. The proliferation capacity of MSC were assessed by CCK-8 method. To test their self-renewal property, the colony forming unit-fibroblast assay was performed. Moreover, the cell yields of passage 1, 2, 3 and 4 were calculated.

RESULTSThe bone marrow plug-derived MSC shared the typical fibroblast-like morphology same as bone marrow cells derived MSC. Moreover, the ratio of CD45 positive hematopoietic cells in bone marrow plug-derived MSCs was significantly lower than that of bone marrow cell-derived MSC. The results of multi-differentiation experiments showed that bone marrow-plug-derived MSC exhibited similar multi-potent property to their bone marrow counterparts. In addition, the results of CCK-8 and CFU-F assay demonstrated that bone marrow plug-derived MSC grew more robustly and more CFU-F were formed in the culture plates, which indicated that the cells possessed higher proliferation and self-renewal capacities. Promisingly, a larger amount of cells were harvested via using the new protocol.

CONCLUSIONThe purity and yields of the bone marrow plug-derived MSC are satisfactory compared with previous rabbit MSC isolation methods. The findings may be helpful for the research of regenerative medicine.

Adipocytes ; Animals ; Bone Marrow ; Bone Marrow Cells ; Cell Differentiation ; Cell Separation ; Colony-Forming Units Assay ; Mesenchymal Stromal Cells ; Osteoblasts ; Rabbits

Cell Proliferation ; Colony-Forming Units Assay ; Glioma ; pathology ; Hematopoietic Stem Cells ; pathology ; Humans ; Stem Cells ; cytology ; metabolism

The aim of this study was to investigate the best method to preserve human bone marrow cells and the effectiveness of long term cryopreservation at -80 degrees C. The human bone marrow cells in 20 samples were firstly frozen by a programmed freezer or -80 degrees C refrigerator, and then were preserved in liquid nitrogen with DMSO-AuP (10% dimethylsulfonamide, 10% autologous plasma) or DMSO-HES-HuA (5% dimethylsulfonamide, 6% hydroxyethyl starch, 4% human serum albumin) as cryoprotectant for 21 to 25 years. They were thawed in 38 degrees C. The cell sample frozen in -80 degrees C refrigerator was frozen at a low frozen speed of 1 degrees C/min which was the same as the programmed freezer before -30 degrees C. Before detection the bone marrow cells were taken from liquid nitrogen and were thawed in 38 degrees C, then the suspension of bone marrow cells was prepared for detection. The cell morphology and recovery rate of erythrocytes, nucleocytes and platelets; the recovery rate of hematopoietic stem progenitors cells, as well as mesenchymal stem cells were determined. The results showed that the protective effectiveness of DMSO-HES-HuA was better than DMSO-AuP. The mature erythrocytes were destroyed lightly [(3.5 +/- 1.5)% versus (12.6 +/- 4.8)%], the hemolysis rate was lower [(3.3 +/- 1.6)% versus (23.1 +/- 5.1)%]. Osmotic fragility of erythrocytes in the former was not changed, but was dropped in the latter. The recovery rates of red cell, platelet, granulocyte-macrophage colony forming units and long term culture-initiating cells were higher in the former than that in the latter [(96.1 +/- 1.8)%, (70.0 +/- 9.5)%, (49.2 +/- 10.9)%, (54.2 +/- 13.8)% versus (76.3 +/- 5.6)%, (52.7 +/- 8.1)%, (43.5 +/- 12.3)%, (47.2 +/- 13.6)% respectively]. With each kind of cryoprotectant or frozen method, the frozen MSC could keep the original growth properties. With the same cryoprotectant and different frozen method, the cryopreservative effectiveness was not different. The influence of the cryoprotectant prescriptions and the frozen methods on the cryopreservative effectiveness was little. It is concluded that the human bone marrow cells with DMSO-AuP or DMSO-HES-HuA as cryoprotectant, frozen by a programmed freezer or -80 degrees C refrigerator, could be then preserved in liquid nitrogen for long time. When the preserving time was as long as 21 to 25 years, the morphology, the recovery rate and the activity of various kinds of cells were still good. The method of freezing by -80 degrees C refrigerator with 5% DMSO-6% HES-4% HuA and preserving in liquid nitrogen would be convenient, cheap and easily-manipulated for preservation of the human bone marrow cells.
Bone Marrow Cells ; cytology ; Cell Survival ; Colony-Forming Units Assay ; Cryopreservation ; methods ; Cryoprotective Agents ; Humans ; Nitrogen ; Time Factors

The aim of this study was to detect DNA damage during expansion ex vivo of umbilical cord blood (UCB) hematopoietic cells and explore the optimal harvest time for culture of CB hematopoietic cells. Mononuclear cells (MNCs) separated from UCB were cultured in a serum-free system supplemented with cytokines and colony forming units were assessed by semisolid culture at the same time. On day 0, 7, 14 and 21 cells were collected for single cell gel electrophoresis (SCGE) analysis and CFUs were also assayed by SCGE, CD34+ cells and CD133+ cells were quantitated by fluorescence-activated cell sorting (FACS). The results showed that the percentage of CD34+ and CD133+ cells was found to be highest after short-term culture (<14 days) and the cord blood DNA damage rate was observed to be less than 5.0% at earlier time points, but at day 21 the DNA damage rate was 28.2%, which was higher than that at day 0 (p=0.000), the tail length of the DNA comet was longer than that at day 0 (p=0.000). The tail lengths of DNA damage on other time points were not significantly different from that at day 0. It is concluded that the DNA damage rate is less than 5.0% after short-term (<14 days) culture of UCB cells ex vivo by using this method. After 14 days DNA damage rate increases significantly. The optimal harvest time of cord blood cells after culture ex vivo would be within 14 days.
Cell Division ; Cells, Cultured ; Colony-Forming Units Assay ; DNA Damage ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; Humans

In order to investigate the influence of angiotensin II on hematopoietic system, CD34+ cells in cord blood were purified, and the effects of angiotensin II in combination with various cytokines on their growth and differentiation were studied by cell culture in vitro. It was found that angiotensin II in suspending medium could stimulate both BFU-E and CFU-GM expansion. The number of BFU-E and CFU-GM was increased with the increases of angiotensin II concentrations during a certain range. In addition, the expansion fold of CFU-GM was increased from 2.3 +/- 0.8 times to 7.8 +/- 2.3 times when angiotensin II was added in the presence of SCF+G-CSF+GM-CSF+IL3 cytokines mixture. Similarly, the expansion fold of BFU-E was increased from 3.1 +/- 1.8 times to 9. 2 +/- 2.3 times with angiotensin II in the presence of SCF+EPO+TPO+IL-3. In the semi-solid medium, angiotensin II could stimulate CFU-GM expansion but had no effect on the growth of BFU-E. In conclusion, angiotensin II had some stimulating effects on cord blood hematopoietic progenitors expansion in vitro in the presence of other cytokines.
Angiotensin II/*pharmacology ; Antigens, CD34/*metabolism ; Cells, Cultured ; Colony-Forming Units Assay ; Fetal Blood/*cytology ; Granulocyte-Macrophage Colony-Stimulating Factor ; Hematopoietic Stem Cells/*cytology

OBJECTIVE: To investigate the effect of hematopoietic stimulating factors on the expansion of mature megakaryocytes. METHODS: (2, 4, 6, 8, 10) x 10(5)/mL bone marrow single nucleus cells (BMNC) were added in the culture system of colony forming unit-megkaryocyte (CFU-Meg) to find out the relationship of the cultured BMNC with the output of CFU-Meg. rmSCF + rmTPO + rmIL-3 (3HSFs) and rmSCF + rmTPO + rmIL-3 + rmIL-6 (4HSFs) or F-CM were added in the liquid culture system of megkaryocytes respectively. The number of mature megakaryocytes were counted every other day. RESULTS: The number of CFU-Meg increased with the increase of the cultured BMNC. The CFU-Meg productivity of 1 x 10(6) BMNC/mL culture system was more than that of 2 x 10(5) BMNC/mL culture system. 3HSFs and 4HSFs or F-CM significantly promoted the expansion of mature megakaryocytes in the liquid culture system, but the effect was different. The peak time of the number of mature megakaryocytes in 3HSFs and 4HSFs or F-CM were 7 d, 7 d and 5 d respectively. CONCLUSION 3HSFs and 4 HSFs or F-CM had positive effect on the expansion of mature megakaryocytes. 4HSFs was better than 3HSFs and F-CM. 3HSFs was better than F-CM. The peak time of the number of mature megakaryocytes in different culture systems was different.
Animals ; Cells, Cultured ; Colony-Forming Units Assay ; Female ; Hematopoietic Cell Growth Factors ; pharmacology ; Interleukin-3 ; pharmacology ; Interleukin-6 ; pharmacology ; Macrophage Colony-Stimulating Factor ; pharmacology ; Male ; Megakaryocytes ; cytology ; Mice

The multipotent differentiation and immunosuppression capability of mesenchymal stem cells (MSCs) make it attractive source for stem cell therapy to treat serious diseases, including neural system diseases and immune disorders. For large scale clinical applications, MSCs have to be expanded to produce sufficient quantity for multiple treatments. While conventional passaging is not appropriate for such a task, bioreactor can be used to expand MSCs more efficiently. Yet the efficacy and biosafety of expanded MSCs must be properly assessed before the expanded MSCs can be implanted. This review presented state-of-the-art in expanding MSCs focusing on the progress on the assessment of the efficacy and biosafety of in vitro expanded MSCs. Current obstacles were discussed and future research directions were outlined.
Animals ; Bioreactors ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Colony-Forming Units Assay ; Humans ; Mesenchymal Stem Cell Transplantation ; adverse effects ; Mesenchymal Stromal Cells ; cytology

To achieve efficient peripheral blood stem cell harvest (PBSCH), a simple method to monitor peripheral blood stem/progenitor cells was evaluated. The Sysmex XE-2100 hematology analyzer with an immature information (IMI) channel was used to identify and count the hematopoietic progenitor cell (HPC). Twenty-five donors mobilized with G-CSF in allogeneic and 11 patients in autologous peripheral blood stem cell transplantation (allo-PBSCT and auto-PBSCT) were involved. The HPC, CD34(+) cell and CFU-GM in the peripheral blood and leukapheresis samples were detected during mobilization and harvest. The results showed that HPC amount had a positive correlation with both the CD34(+) cell and CFU-GM in the peripheral blood. The peripheral blood hematopoietic stem/progenitor cells in allo-PBSCT donors remarkably increased on day 5 of the mobilization, followed the leukocytes increased. However, a fast increase of hematopoietic stem/progenitor cells was earlier than leukocytes in the peripheral blood. The HPC positively correlated with the CD34(+) cell or CFU-GM in the PBSCH. On the days of collection, the count of HPC and CD34(+) cell in peripheral blood was highly correlated with the CD34(+) cell yield. It is concluded that HPC as an estimate of progenitor cells in collected blood sample could be used to determine the optimal time of PBSCH and minimize the risk of missing an adequate harvest.
Antigens, CD34 ; blood ; Blood Donors ; Cell Separation ; methods ; Colony-Forming Units Assay ; Hematology ; instrumentation ; methods ; Hematopoietic Stem Cell Mobilization ; Hematopoietic Stem Cells ; cytology ; Humans ; Leukocytes ; cytology ; immunology

Colony-Forming Units Assay ; Cryopreservation ; methods ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; Humans ; Immunotoxins ; immunology ; Leukocytes, Mononuclear ; cytology ; Ricin ; immunology ; T-Lymphocytes ; cytology ; immunology ; Time Factors

OBJECTIVETo investigate the mechanism and the suppression effect of human cytomegalovirus (HCMV) on hematopoietic system.

METHODSSemi-solid culture system was used to observe the effect of HCMV AD169 strain on colony forming unit granulocyte/macrophage (CFU-GM), CFU-erythroid (CFU-E), CFU-multipotent (CFU-Mix) and CFU-megakaryocyte (CFU-MK) growth. The techniques of in situ polymerase chain reaction (IS-PCR) and polymerase chain reaction (PCR) were used to demonstrate the existence of HCMV DNA in the colony cells of cultured CFU-GM, CFU-Mix, CFU-MK and CFU-E, respectively. The immediate early antigen (IEA) mRNA in CFU-MK and late antigen (LA) mRNA in CFU-E were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). HCMV early protein P52 was detected with immunohistochemical technique.

RESULTSHCMV AD169 suppressed the differentiation and proliferation of CFU-GM, CFU-E, CFU-Mix and CFU-MK in vitro significantly (P < 0.05). The suppression was dose-dependent. HCMV DNA was successfully detected in CFU-GM, CFU-Mix, CFU-MK colony cells from viral infection groups by IS-PCR, and was detected in CFU-E by PCR, while it was negative in blank control or mock control groups. CFU-MK colony cells expressed HCMV IEA mRNA with the size of 340 bp in virus infection groups of 10(3) plague forming unit (PFU), 10(4) PFU and 10(5) PFU, respectively. The HCMV LA mRNA was detected by RT-PCR and was 263 bp long in positive control group of HCMV-infected human embryonic fibroblasts. The expression of HCMV LA mRNA in CFU-E was negative. The early protein P52 of HCMV in 10(4) PFU group was also identified by immunohistochemical staining.

CONCLUSIONHCMV AD169 strains inhibited the differentiation and proliferation of CFU-GM, CFU-E, CFU-Mix and CFU-MK by the infection of the hematopoietic progenitors. HCMV might cause the suppression of hematopoiesis by direct infection, which is thought to be one of the reasons of HCMV infection associated with thrombocytopenia, neutropenia and anemia.

Colony-Forming Units Assay ; Cytomegalovirus ; genetics ; DNA, Viral ; genetics ; Erythrocytes ; virology ; Hematopoietic System ; cytology ; virology ; Humans ; Megakaryocytes ; virology ; Multipotent Stem Cells ; virology ; Polymerase Chain Reaction